Advances in
BOTANICAL RESEARCH incorporating Advances in Plant Pathology
VOLUME 24
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BOTANICAL RESEARCH ...
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Advances in
BOTANICAL RESEARCH incorporating Advances in Plant Pathology
VOLUME 24
Advances in
BOTANICAL RESEARCH incorporating Advances in Plant Pathology Editor-in-Chief J. A. CALLOW
School of Biological Sciences, University of Birmingham, Birmingham, UK
Editorial Board J. H. ANDREWS
H. G. DICKINSON M. KREIS R. M. LEECH R. A. LEIGH E. LORD D. J. READ I. C. TOMMERUP
University of Wisconsin-Madison, Madison, USA University of Oxford, Oxford, UK Universitb de Park-Sud, Orsay, France University of York, York, UK Rothamsted Experimental Station, Harpenden, U K University of California, Riverside, USA University of Shefield, Shefield, UK CSIRO, Perth, Australia
Advances in
BOTANICAL RESEARCH incorporating Advances in Plant Pathology edited by
J. H. Andrews
I. C. Tommerup
and
Department of Plant Pathology, The University of Wisconsin-Madison Madison, USA
CSIRO Forestry and Forest Products, CSIRO Centre for Mediterranean Agricultural Research, Perth, PO Wembley 6014, Australia
Series editor
J. A. CALLOW School of Biological Sciences, University of Birmingham, Birmingham, UK
VOLUME 24
1997
ACADEMIC PRESS San Diego London Boston New York Sydney Tokyo Toronto
This book is printed on acid-free paper Copyright
0 1997 by ACADEMIC PRESS
All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. Academic Press, Inc. 525 B Street, Suite 1900, San Diego, California 92101-4495, USA http://www. apnet .com Academic Press Limited 24-28 Oval Road, London NW1 7DX, UK http://www.hbuk.co.uk/ap/ A catalogue record for this book is available from the British Library ISBN 0-12-005924-X Typeset by Keyset Composition, Colchester, Essex Printed in Great Britain by Hartnolls Limited, Bodmin, Cornwall 97 98 99 00 01 02 EB 9 8 7 6 5 4 3 2 1
CONTENTS
CONTRIBUTORS TO VOLUME 24 ..................................... CONTENTS OF VOLUMES 13-23
xv
....................... ,............ .... xvii
SERIES PREFACE ................................. .......................... ....... xxiii PREFACE .................................................................................
xxv
Contributions of Population Genetics to Plant Disease Epidemiology and Management M. G. MILGROOM and W. E. FRY I.
Introduction _........._................................_...................,...... ..... ..
1
11.
Population Genetics and Plant Pathology ..... ................... .............. A. What is (and what isn’t) Population Genetics? ........................ B. Brief history of Population Genetics in Plant Pathology ............
3 3 4
111. What can Population Genetics Contribute to Epidemiology and Plant Disease Management? ....... .................................. . . . ......... .......... A. The Concept of a Population ............................................... B. Genetic Variation in Populations .....................,............... ...,. C. The Use of Neutral Genetic Markers to Estimate Ecologically Important Variation ........................... ...................,........,.. .. D. The Interface between Population Genetics and Epidemiology ... E. Population Characteristics vary among Populations ....... ..,. ... . ...
10 13 18
IV. Examples of the Integration of Population Genetics and Epidemiology A. Fungicide Resistance in Pyrenophora teres ........................... ... B. Epidemiology and Population Genetics of Phytophthora infestans
19 21
... ....
24
V.
Future Contributions of Population Genetics to Plant Pathology
5 5 8
19
Acknowledgements ....................................................................
24
...............................................................................
25
References
vi
CONTENTS
A Molecular View Through the Looking Glass: the Pyrenopeziza brassicae-Brassica Interaction A . M . ASHBY I . Introduction I1. The A. B. C. D.
...........................................................................
P . brassicae-Brassica Interaction ......................................... The Fungus ..................................................................... Pathogenesis .................................................................... Sexual Morphogenesis ....................................................... Disease Epidemiology .......................................................
32 32 32 33 33 34
111. Molecular Techniques in the Analysis of the P. brassicae-Brassica Interaction .............................................................................
38 .
IV. Molecular Analysis of Pathogenesis ........................................... A . Surface Growth and Penetration: the Role of Cutinase ........... B . Subcuticular Growth: the Role of Protease ........................... C . Using Reporter Genes to Measure Fungal Biomass In Planta . . D . Proposed Role of Extracellular Protease in Pathogenicity ........ E . Implications for Disease Control .........................................
40 41 42 45 47 47
V. Analysis of the Hemibiotrophic Phase: the Role of Cytokinins ....... A . Biochemical Analysis of Cytokinin Production by P. brassicae . B . Molecular Analysis of P. brassicae Cytokinins ....................... C . The Role of Cytokinins in Pathogenicity .............................. D . Implications for Disease Control .........................................
47 49 49 51 51
VI . Analysis of Sexual Morphogenesis ............................................. A . Biochemical Analysis: Identification of a Post-Mating Factor ... B . Molecular Analysis ........................................................... C . Sexual Morphogenesis in P . brassicae: a Speculative Summary . D. Implications .....................................................................
51 52 53 58 60
VII . A Molecular View through the Looking Glass: the P. brassicaeBrassica Interaction ....................................................................
60
...............................................................
65
.................................................................
65
............................................................................
65
VIII . Concluding Remarks Acknowledgements References
vii
CONTENTS
The Balance and Interplay Between Asexual and Sexual Reproduction in Fungi M . CHAMBERLAIN and D . S. INGRAM
.............................................................................
I . Introduction
71
..............
72
I11. Comparisons of Genetic Variation. Physiological Costs and Fitness between Asexual and Sexual Systems ........................................... A . Does the Sexual System Generate more Variants? ................... B . Is the Sexual System Physiologically more Costly? ................... C . Are Sexual Progeny more “Fit”? .............................................
73 73 74 74
I1. Initiation of Asexual Sporulation and Sexual Reproduction
IV. Maintaining and Changing the Balance between Reproductive Processes .................................................................................... A . Species-determined Equilibria ............................................... B . Genotype-determined Equilibria ........................................... C. Seasonally Maintained Equilibria .......................................... D . Physical and Nutritional Factors ........................................... E . Density and Competition ..................................................... F. The Effect of Mycelial Extracts and Specific Morphogens .........
80
...............................................................................
81
....................................................................
82
Acknowledgements References
76 77 77 77 78
......................
V. Trade-off between Asexual and Sexual Reproduction VI . Conclusion
74 75
...............................................................................
82
The Role of Leucine-Rich Repeat Proteins in Plant Defences D . A . JONES and J . D . G . JONES I . Introduction
...........................................................................
I1. Resistance Genes Encoding Proteins with Extracytoplasmic LRRs .. A . Resistance Genes Encoding Membrane-anchored Proteins with Extracytoplasmic LRRs and no Kinase Domain .................... B . A Resistance Gene Encoding a Membrane-anchored Protein with Extracytoplasmic LRRs and a Cytoplasmic Kinase Domain ..... C . Avirulence Determinants that Interact with Resistance Proteins Containing Extracytoplasmic LRRs ..................................... D . Activation of Plant Defences by Resistance Genes Encoding Proteins Containing Extracytoplasmic LRRs ............................
90 91 91 95 97 99
I11. Resistance Genes Encoding Proteins with Cytoplasmic LRRs ......... 101 A . Resistance Genes Encoding Proteins with Cytoplasmic LRRs and 102 Potential Leucine Zippers ..................................................
...
CONTENTS
Vlll
B . Resistance Genes Encoding Proteins with Cytoplasmic LRRs and Homology to the Cytoplasmic Domains of Toll and the Interleukin-1 Receptor ........................................................... C . A Gene Encoding a Protein with Cytoplasmic LRRs that is Required for a Resistance Gene to Function ........................ D . Avirulence Determinants that Interact with Resistance Proteins Containing Cytoplasmic LRRs ............................................ E . Activation of Plant Defences by Resistance Proteins Containing Cytoplasmic LRRs ........................................................... IV . Defence-related Genes Encoding Proteins with Extracytoplasmic LRRs ................................................................................... A . Polygalacturonase-inhibiting Proteins ................................... B . LRR Extensins ................................................................ C . A Viroid-induced LRR Protein ..........................................
108 109 113 114 119 120 127 131
V. Genes Encoding Proteins of Unknown Function with Extra131 cytoplasmic LRRs ................................................................... 131 A . The AWJL Proteins of Wheat ............................................ 135 B . Receptor-like Protein Kinases ............................................ VI . A Gene Encoding a Protein of Unknown Function with Cytoplasmic 137 LRRs ...................................................................................
VII . The A. B. C.
Evolution of Plant LRR Proteins ........................................ The Evolution of LRR Proteins in the Eukaryotes ................ Evolutionary Clues Provided by Intron Arrangements ............ Evolution of Different Specificities in LRR Proteins ..............
138 138 141 143
VIII . The Structure and Molecular Specificity of Plant LRR Proteins ...... A . Inferences about the Structure of Plant LRRs by Comparison with the Known Structure of Porcine Ribonuclease Inhibitor ... B . Inferences about the Structure and Interactions of Extracellular Plant LRR Proteins Based on their Potential Patterns of Glycosylation .................................................................. C . Inferences about the Interactions between Plant LRR Proteins and their Ligands Based on Comparisons with the Interactions between Ribonuclease Inhibitors and Ribonucleases .............. XI . Concluding Remarks ...............................................................
144 144 147 150 153
.................................................................
156
References ............................................................................
156
Acknowledgements
ix
CONTENTS
Fungal Life-styles and Ecosystem Dynamics: Biological Aspects of Plant Pathogens. Plant Endophytes and Saprophytes R . J . RODRIGUEZ and R . S . REDMAN I . Introduction
...........................................................................
I1. Plant Pathogens
........
.......................................................
171
...................................................................
174
...........................................................................
176
...............................................................
179
..........................................
182
I11. Plant Endophytes IV. Saprophytes
V . Life-style Crossroads
VI . Life-styles and Ecosystem Dynamics
VII . Fungal Biology in Agricultural Versus Natural Ecosystems VIII . The Evolution of Agriculture IX . Conclusion
............
...................................................
............................................................................
.........................
183 184 186
....................................
187
............................................................................
187
Acknowledgements References
169
Cellular Interactions oetween Plants and Biotrophic Fungal Parasites M . C . HEATH and D . SKALAMERA I . Introduction
.............................................................................
I1 . Why do Fungal and Oomycetous Parasites form Intracellular Structures? A . General Characteristics of Plant-Haustorium Interfaces ............. B . Maintenance of a Differentiated Extrahaustorial Membrane ...... C . Solute Transport Across the Plant-Parasite Interface ................ D . Other Roles of the Haustorium ............................................ E . Concluding Remarks ........................................................... 111. Of what Significance are the Plant Cellular Rearrangements that Accompany Parasite Invasion? .................................................... A . Defensive Responses to Parasite Invasion ............................... B . Parasite-Induced Changes in the Plant’s Endomembrane System C . Associations of Intracellular Fungal Structures with the Plant Nucleus ............................................................................ D . Changes in the Plant Cytoskeleton ........................................ E . Concluding Remarks ........................ ..............................
196 198 198 199 202 204 205 206 206 207 209 210 210
CONTENTS
X
IV. Why do Biotroph-Invaded Cells Die in Resistant Plants? ................ A . Is Cell Death the “Default State” following Cell Penetration? ... B . Do Invaded Cells Die in Host and Non-host Plants for the Same Reason? ........................................................................... C. Arguments for Cell Death in Resistant Plants being a Form of Programmed Cell Death ...................................................... D . Arguments against Cell Death in Resistant Plants being a Form of Programmed Cell Death ...................................................... E . Cellular Mechanisms of Cell Death ....................................... F. Concluding Remarks ........................................................... V . Conclusions References
210 211 212 214 215 216 218
..............................................................................
219
...............................................................................
219
Symbiology of Mouse-Ear Cress (Arubidopsis thalhna) and Oomycetes E . B . HOLUB and J . L . BEYNON I . Introduction
............................................................................
I1. Defining a New Research Arena of Plant Biology ......................... A . The Dawn of Arabidopsis ................................................... B . Relevant Trends in Modern Biology ..................................... C . A Rebirth in Plant Pathology .............................................. D . A Copernican Perspective ...................................................
228 229 229 230 231 232
I11. Symbionts from the Wild .......................................................... A . The Phytobiont: Arabidopsis thaliana ................................... B . The Biotrophs: Peronospora parasitica and Albugo candida ...... C . Three Rs of Symbiosis ....................................................... D . The Phenotypes of Interactions: Consequences of Recognition ..
233 233 235 238 240
IV. Molecular Genetics of Natural Variation ...................................... A . Building Models: Predicting the Host Genotype ..................... B . Nonallelism: Juggling with Apples and Oranges ..................... C . Major Complexes of Recognition Genes: How Big is a Cluster? D . A Natural Anomaly of Susceptible Origin .............................
243 246 248 254 258
V . Mutations: Revealing Complexity from Black and White ................ 259 260 A . A Myriad of Columbia Mutants ........................................... 261 B . Surprising Extremes in Wassilewskija ................................... VI . Avenues of Future Research
......................................................
262
................................................................ ...................................................................
268 269
..............................................................................
269
VII . Concluding Remarks Acknowledgements References
xi
CONTENTS
Use of Monoclonal Antibodies to Detect. Quantify and Visualize Fungi in Soils F. M . DEWEY. C . R . THORNTON and C . A . GILLIGAN I . Introduction
...........................................................................
I1. Production of Species-specific and Genus-specific Monoclonal Antibodies ................................................................................... A . Selection and Preparation of Immunogens ............................ B . Selection of Hybridoma Cell Lines Secreting Specific Antibodies C . Choice of Antibody Subclass .............................................
276 278 278 279 279
I11. Assay Formats ....................................................................... 280 A . Enzyme-linked lmmunosorbent Assays (ELISAs) .................. 281 B . Membrane Assays ............................................................ 282 IV. Sample Preparation ................................................................. 283 A . Extraction of Antigens from Soil ........................................ 283 B. Elimination and Reduction of Interference from Soil Components .......................................................................... 286 V. Detection
..............................................................................
VI . Quantification ........................................................................ A . Estimation of Biomass ...................................................... B . Immunological Estimation of Colony-forming Units ............... C. Setting of Thresholds for Detection and Quanta1 Assay Systems D . False-negative Results ....................................................... VII . Visualization
..........................................................................
VIII . Concluding Remarks
289 289 293 296 298 300 302
.................................................................
304
............................................................................
304
Acknowledgements References
...............................................................
287
Function of Fungal Haustoria in Epiphytic and Endophytic Infections P. T. N . SPENCER-PHILLIPS I . Introduction
.............................................................................
I1. Strategies for Nutrition and Biotrophy: an Overview
309
.......................
311
I11. The Challenge of Biotrophic Nutrient Accumulation ....................... A . The Apoplastic Environment ................................................
313 314
xii
CONTENTS
IV. The Role of Haustoria and Intercellular Hyphae in Transfer Intercept A . Vascular Association: General Considerations ......................... B . Minor Vein Type. Mechanisms of Phloem Loading and Interception Strategy ................................................................. C . Evolutionary Aspects ..........................................................
316 317 318 324
..............................................
325
................................................
327
....................................................................
328
References ...............................................................................
328
V . Alternative Functions of Haustoria VI . Summary and Research Priorities Acknowledgements
Towards an Understanding of the Population Genetics of Plant-Colonizing Bacteria B . HAUBOLD and P. B . RAINEY I . Introduction
.............................................................................
335
I1. Quantifying Genetic Variation: the Indirect Approach ....................
337
I11. The Neutral Theory: Historical Background to the Study of Bacterial 338 Population Genetics .................................................................. IV. Population Structure .................................................................. A . Genetic Structure ........................................... B . Phylogenetic Structure ........................................................ C. Spatial and Temporal Structure ............................................
340 340 342 343
......................................................
345
VI . Conclusions ..............................................................................
347
V . The Metapopulation Concept
Acknowledgements
....................................................................
References ...............................................................................
347 347
Asexual Sporulation in the Oomycetes A . R . HARDHAM and G . J . HYDE I . Introduction
.............................................................................
I1. Sporangiogenesis ....................................................................... A . Induction .......................................................................... B . Morphological Development ..................................................
353 357 357 359
...
CONTENTS
Xlll
C. Synthesis of Zoospore-specific Components during Sporangio361 genesis ............................................................................. 111. Zoosporogenesis
....................................................................... 377 A . Induction .......................................................................... 377 B . The Process of Cleavage ...................................................... 378 C. Synthesis of Zoospore-specific Components during Zoosporo384 genesis ............................................................................. D . Polarization of Zoosporic Organelles ..................................... 386 E . Zoospore Discharge ............................................................ 386 F. Conclusions ....................................................................... 389
....................................................................
390
...............................................................................
390
Acknowledgements References
Horizontal Gene Transfer in the Rhizosphere: a Curiosity or a Driving Force in Evolution? J . WOSTEMEYER. A . WOSTEMEYER and K . VOIGT I . Horizontal or Lateral Gene Transfer: where does it Occur?
.............
399
I1. Bacteria as Recipients of Foreign DNA ........................................
401
I11. Fungi as Recipients of Foreign DNA ...........................................
410
IV . Plants as Recipients of Foreign DNA ........................................... A . Agrobacteriurn turnefaciens ................................................... B . Rhizobiurn Interactions with Legumes ...................................
416 416 418
.......................................................
420
V. VI .
Interkingdom Gene Transfer
.........................
422
...............................................................................
424
Refevance of Lateral Gene Transfer for Evolution References
The Origins of Phytophthora Species Attacking Legumes in Australia J . A . G . IRWIN. A . R . CRAWFORD and A . DRENTH I . Introduction
...........................................................................
432
I1. Legumes in Australia ..............................................................
432
111. Phytophthora Species Attacking Legumes in Australia
A . Taxonomy
.................. 433 ....................................................................... 433
xiv
CONTENTS
B . Asexual Life Cycle ........................................................... C . Sexual Life Cycle ............................................................. IV. Genetic Variation and Possible Origins of Phytophthora Species Attacking Legumes ................................................................. A . Phytophthora sojae .......................................................... B . Phytophthora medicaginis ................................................... C . Phytophthora vignae .......................................................... D . Phytophthora macrochlamydospora ......................................
436 437 437 438 439 439 440
V . Evolutionary Relationships between Phytophthora Species Attacking 440 Legumes ............................................................................... VI . Evolution of Species and Host Specificity in Phytophthora Species attacking Legumes .................................................................. A . Biological Species and Speciation ........................................ B . Evolution of Host Specificity in Phytophthora .......................
442 442 443
VII . Evolution of Cultivar Specificity in Phytophthora sojae ....................
448
..............................................................
450
.................................................................
451
VIII . Concluding Remarks Acknowledgements
............................................................................
451
INDEX ...............................................................................
457
References
CONTRIBUTORS TO VOLUME 24
A. M. ASHBY, Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge CB2 3EA, UK J . L. BEYNON, Department of Biological Sciences, Wye College, University of London, Wye, Ashford, Kent TN25 5AH, UK M. CHAMBERLAIN, (previously known as M. Jurand) Royal Botanic Garden, 20A Inverleith Row, Edinburgh EH3 5LR, UK A. R. CRAWFORD, CRC for Tropical Plant Pathology, The University of Queensland, Brisbane 4072, Australia F. M. DEWEY, Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 3RB, U K A. DRENTH, CRC for Tropical Plant Pathology, The University of Queensland, Brisbane 4072, Australia W. E. FRY, Department of Plant Pathology, Cornell University, Ithaca, NY 14853, USA C. A. GILLIGAN, Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge CB2 3EA, UK A. R. HARDHAM, Plant Cell Biology Group, The Research School of Biological Sciences, The Australian National University, P. 0. Box 475, Canberra 2601, Australia B. HAUBOLD, Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 3RB, UK M. C . HEATH, Department of Botany, University of Toronto, Toronto, Ontario M5S lA1, Canada E. B. HOLUB, Plant Pathology and Weed Science Department, Horticulture Research International- Wellesbou me, Warwickshire CV35 9EF, UK G. J . HYDE, School of Biological Science, University of New South Wales, Kensington, NSW 2033, Australia D. S . INGRAM, Royal Botanic Garden, 20A Inverleith Row, Edinburgh EH3 5LR, UK J . A. G. IRWIN, CRC for Tropical Plant Pathology, The University of Queensland, Brisbane 4072, Australia D. A. JONES, The Sainsbury Laboratory, John Innes Centre, Colney Lane, Norwich NR4 7UH, UK J . D. G. JONES, The Sainsbury Laboratory, John Innes Centre, Colney Lane, Norwich NR4 7UH, U K M. G. MILGROOM, Department of Plant Pathology, Cornell University, Ithaca, NY 14853, USA
xvi
CONTRIBUTORS TO VOLUME 24
P. B. RAINEY, Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 3RB, U K R. S . REDMAN, National Biological Service, NW Biological Science Center, Seattle, W A 98115, U S A R. J. RODRIGUEZ, National Biological Service, NW Biological Science Center, Seattle, W A 98115, U S A D. SKALAMERA, Department of Botany, University of Toronto, Toronto, Ontario M5S lA1, Canada P. T. N. SPENCER-PHILLIPS, Department of Biological Sciences, University of the West of England, Bristol BS16 l Q Y , UK C. R. THORNTON, Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge CB2 3EA, U K K . VOIGT, Friedrich-Schiller-Universitat Jena, Lehrstuhl fur Allgemeine Mikrobiologie and Mikrobengenetik, Neugasse 24, 0-07743 Jena, Germany A. WOSTEMEYER, Friedrich-Schiller-Universitat Jena, Lehrstuhl fur Allgemeine Mikrobiologie und Mikrobengenetik, Neugasse 24, 0-07743 Jena, Germany J. WOSTEMEYER, Friedrich-Schiller-Universitat Jena, Lehrstuhl fur Allgemeine Mikrobiologie und Mikrobengenetik, Neugasse 24, 0-07743 Jena, Germany
xvii
CONTENTS TO VOLUMES 1 S 2 3
Contents of Volume 13 Interactions Between Photosystems N. R. BAKER and A. N. WEBBER
Cyanobacterial Water-Blooms C. S. REYNOLDS
Determinants of Yield of Secondary Products in Plant Tissue Cultures H. A. COLLIN
Contents to Volume 14 Protein Targeting R. J. ELLIS and C. ROBINSON
Control of Isoprenoid Biosynthesis in Higher Plants J. C. GRAY
Dunaliella: A Green Alga Adapter to Salt M. GINZBURG
Contents to Volume 15 Perception of Gravity by Plants T. BJORKMAN
Crassulacean Acid Metabolism: a Re-Appraisal of Physiological Plasticity in Form and Function H. GRIFFITHS
xviii
CONTENTS TO VOLUMES 13-23
Potassium Transport in Roots L. V. KOCHIAN and W. J. LUCAS
Sporogenesis in Conifers R. I. PENNELL
Contents of Volume 16 Lipid Metabolism in Algae J. L. HARWOOD and A . L. JONES
The Alteration of Generations P. R. BELL
The Formation and Interpretation of Plant Fossil Assemblages R. A . SPICER
Primary Productivity in the Shelf of North-West Europe P. M. HOLLIGAN
Contents of Volume 17 Plant Evolution and Ecology During the Early Cainozoic Diversification M. E. COLLINSON
Origin and Evolution of Angiosperm Flowers E. M. FRIIS and P. K . ENDRESS
Bacterial Leaf Nodule Symbiosis I. M. MILLER
Fracture Properties of Plants J. F. V. VINCENT
CONTENTS TO VOLUMES 13-23
xix
Contents of Volume 18 Photosynthesis and Stomatal Responses to Polluted Air, and the Use of Physiological and Bacterial Responses for Early Detection and Diagnostic Tools H. SAXE
Transport and Metabolism of Carbon and Nitrogen in Legume Nodu1es J. G. STREETER
Plants and Wind P. VAN GARDINGEN and J. GRACE
Fibre Optic Microprobes and Measurement of the Light Microenvironment within Plant Tissues T. C. VOGELMANN, G. MARTIN, G. CHEN and D. BUTI'RY
Contents of Volume 19 Oligosaccharins S. ALDINGTON and S. C. FRY
Are Plant Hormones Involved in Root to Shoot Communication? M. B. JACKSON
Second-Hand Chloroplasts: Evolution of Cryptomonad Algae G. I. McFADDEN
The Gametophytdporophyte Junction in Land Plants R. LIGRONE, J. G. DUCKETT and K. S. RENZAGLIA
Contents of Volume 20 Global Photosynthesis and Stomatal Conductance: Modelling the Controls by Soil and Climate F. I. WOODWARD and T. M. SMITH
In vivo NMR Studies of Higher Plants and Algae R. G. RATCLIFFE
xx
CONTENTS TO VOLUMES 13-23
Vegetative and Gametic Development in the Green Alga Chlamydomonas H. VAN DEN ENDE
Salicylic Acid and its Derivatives in Plants: Medianes, Metabolites and Messenger Molecules W. S. PIERPOINT
Contents of Volume 21 Defense Responses of Plants to Pathogens E. KOMBRINK and I. E. SOMSSICH
On the Nature and Genetic Basis for Resistance and Tolerance to Fungal Wilt Diseases of Plants C. H. BECKMAN and E. M. ROBERTS
Implication of Population Pressure on Agriculture and Ecosystems A. H. EHRLICH
Plant Virus infection: Another Point of View G. A. DE ZOETEN
The Pathogens and Pests of Chestnuts S. L. ANAGNOSTAKIS
Fungal Avirulence Genes and Plant Resistance Genes: Unraveling the Molecular Basis of Gene-for-Gene Interactions P. J. G. M. DE WIT
Phytoplasmas: Can Phylogeny Provide the Means to Understand Pathogenicity B. C. KIRKPATRICK and C. D. SMART
Use of Categorical Information and Correspondence Analysis in Plant Disease Epidemiology S. SAVARY, L. V. MADDEN, J. C. ZADOKS and H. W. KLEIN-GEBBINCK
CONTENTS TO VOLUMES 13-23
xxi
Contents of Volume 22 Mutualism and Parasitism: Diversity in Function and Structure in the “Arbuscular” (VA) Mycorrhizal Symbiosis F. A. SMITH and S. E. SMITH
Calcium Ions as Intracellular Second Messengers in Higher Plants A. A. R. WEBB, M. R. McAINSH, J. E. TAYLOR and I. M. HETHERINGTON
The Effects of Ultraviolet-B Radiation on Plants: A Molecular Perspective B. R. JORDAN
Rapid, Long-Distance Signal Transmission in Higher Plants M. MALONE
Keeping in Touch: Responses of the Whole Plant to Deficits in Water and Nitrogen Supply A. J. S. McDONALD and W. J. DAVIES
Contents of Volume 23 The Value of Indexing for Disease Control Strategies D. E. STEAD, D. L. EBBELS and A. W. PEMBERTON
Detecting Latent Bacterial Infections S. H. DE BOER, D. A. CUPPELS and R. GITAITIS
Sensitivity of Indexing Procedures for Viruses and Viroides H. HUTTINGA
Detecting Propagules of Plant Pathogenic Fungi S. A. MILLER
Assessing Plant-Nematode Infestations and Infections K. K. BARKER and E. L. DAVIS
xxii
CONTENTS TO VOLUMES 13-23
Potential of Pathogen Detection Technology for Management of Diseases in Glasshouse Ornamental Crops I. G. DINESEN and A. VAN ZAAYEN
Indexing Seeds for Pathogens J. LANGERAK, R. W. VAN DEN BULK and A. A. J. M. FRANKEN
A Role for Pathogen Indexing Procedures in Potato Certification S. H. DE BOER, S. A. SLACK, G. VAN DEN BOVENKAMP and I. MASTENBROEK
A Decision Modelling Approach for Quantifying Risk in Pathogen Indexing C. A. LBVESQUE and D. M. EAVES
Quality Control and Cost Effectiveness of Indexing Procedures C. SUTULAR
SERIES PREFACE
Advances in Botanical Research is one of Academic Press’ longest standing serials, and has established an excellent reputation over more than 30 years. Advances in Plant Pathology, although somewhat younger, has also succeeded in attracting a highly respected name for itself over a period of more than a decade. The decision has now been made to bring the two serials together under the title of Advances in Botanical Research incorporating Advances in Plant Pathology. The resulting synergy of the merging of these two serials is intended to greatly benefit the plant science community by providing a more comprehensive resource under one “roof”. John Andrews and Inez Tommerup, the previous editors of Advances in Plant Pathology, are now on the editorial board of the new series. Our joint aim is to continue to include the very best articles, thereby maintaining the status of a high impact factor review series.
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PREFACE
All plants, whether in the wild or cultivation, co-exist and interact with numerous phylogenetically and ecologically diverse microbes. Some are potential plant pathogens. Plant pathology is a complex field of study; the future is an exciting one with rapidly evolving scientific approaches to research, many of them at the cusp of development. Articles in this volume analyse developments in plant defence mechanisms, pathogen reproduction, growth, survival and population genetics and dynamics. The recognition of disease resistance in plants is ancient. Plant resistance genes were recognized a century ago, pathogen virulence and avirulence genes 50 years ago, the first pathogen avirulence genes were isolated in the last decade and the first plant resistance genes in the past 4 years. Jones and Jones focus on key resistance genes that encode proteins containing leucine-rich repeats or that require a protein with leucine-rich repeats to function. They discuss evolution, molecular specificity and the role in plant defence mechanisms of leucine-rich proteins, a protein class involved in specific protein-protein interactions. Precision and efficiency in analysing disease phenotype variation and bridging the conceptual gap between genetical and functional aspects of symbiosis is underpinned by basic principles and modern research tools. Holub and Beynon examine the concepts and approaches involved in associating host resistance specificity with a single locus, determining whether the phenotype is actually due to single o r multiple genes and unravelling the interacting network of genes involved in disease expression. Ashby shows how, by employing a multidisciplinary approach using biochemical, molecular biological and classical plant pathology to dissect a host-pathogen interaction into its component parts, new leads towards developing novel control strategies are emerging. Heath and Skalamera examine the hypotheses and supporting evidence about cellular interactions between plants and biotrophic fungal parasites. They question why fungal biotrophic parasites form intracellular structures, the significance of cellular rearrangements that accompany invasion and why invaded resistant cells die. Their synthesis throws new interpretative light on the complex associations of haustoria and cells in compatible and incompatible interactions. Spencer-Phillips has taken on the challenge of exploring the roles of intercellular hyphae and haustoria in intercepting and transferring organic and inorganic nutrients from host tissue in epiphytic and endophytic infections. To persist, pathogens must produce propagules. Chamberlain and Ingram consider asexual and sexual reproduction of fungal pathogens for their ability
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to generate genetic variation, the physiological and fitness costs of the processes, how the balance is maintained and what are the trade-offs between the two reproductive processes. Hardham and Hyde synthesize the current state of knowledge of sporangiogenesis and zoospore production in oomycetes. Adoption of function-oriented approaches and new techniques reveal similarities in the process of sporulation across the taxon where previously diversity was thought to be the case. The importance of capitalizing on recent technical advances to develop accurate and reliable methods for detecting pathogens in soil, a microbially , chemically and physically complex environment, is the theme of Dewey, Thornton and Gilligan’s chapter. They emphasize the need to couple this with new approaches to sampling techniques and methods of statistical analysis to quantify soil pathogens. Evolution of plants is influenced by their pathogens, and vice versa. Evolution of pathogens may also be influenced by other microbes. That plant rhizosphere and soil communities provide opportunities for horizontal gene transfer amongst microbes and plants is the thesis of Wostemeyer, Wostemeyer and Voigt. They highlight the significance of foreign DNA transfers for recipient organism evolution. Legumes, like most agricultural crop species, have been relocated amongst continents, in some cases with their pathogens. Irwin, Crawford and Drenth discuss approaches to unravelling the possible evolutionary origins and genetic variation of Phytophthoru species associated with the pasture legumes in Australia. That information is used in searches for sources of host resistance. Can a pathogen life-style be defined? Rodrigues and Redman point out deficiencies in current understanding of fungal biology related to cross-overs in behaviour by organisms amongst the categories plant pathogen, endophyte or saprophyte. Prominent pathogens move between categories and they may influence natural and agricultural ecosystem community structure and dynamics. Haubold and Rainey challenge microbial pathologists and phytopathologists to consider questions relating to the extent and significance of genetic variation within plant-colonizing bacterial pathogens. If effective biological control strategies are to be developed, then temporal and spatial variation must be understood. They also stress that understanding the mechanisms, rates and extent of recombination in natural populations are essential if genetically engineered bacteria are to be safely exploited in agriculture. Milgroom and Fry illustrate how the practical need to understand pathogen variation and evolution is the most significant application of population genetics to epidemiology and disease management. Sufficient knowledge exists for this critical step in integration to be taken in a few diseases, and they highlight the experimental structures needed for others to follow that route. John H Andrews Inez C Tommerup
Contributions of Population Genetics to Plant Disease Epidemiology and Management
M. G. MILGROOM and W. E. FRY
Department of Plant Pathology, Cornell University, Ithaca, NY 14853, USA
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11. Population Genetics and Plant Pathology .....,,..............,................ A. What is (and what isn’t) Population Genetics? ........................ B. Brief history of Population Genetics in Plant Pathology .............
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I. Introduction
111. What can Population Genetics Contribute to Epidemiology and Plant Disease Management? ........................................................ A. The Concept of a Population ................................................ B. Genetic Variation in Populations ........................................... C. The Use of Neutral Genetic Markers to Estimate Ecologically Important Variation ................................................,............ D. The Interface between Population Genetics and Epidemiology . . . E. Population Characteristics vary among Populations ............... .... IV. Examples of the Integration of Population Genetics and Epidemiology ......................................................... ........ .... A. Fungicide Resistance in Pyrenophora teres .............................. B. Epidemiology and Population Genetics of Phytophthora infestans ............................................................................ V. Future Contributions of Population Genetics to Plant Pathology . ... ... Acknowledgements .................................................................... References ...............................................................................
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I. INTRODUCTION Population genetics and genetic variation in plant pathogens are subjects that have generated much interest since the late 1980s. Almost every recent issue Advances in Botanical Research Vol. 24 incorporating Advances in Plant Pathology
ISBN 0-12-005924-X
Copyright 0 1997 Academic Press Limited All rights of reproduction in any form reserved
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of major plant pathological and mycological journals has at least one article on genetic variation of a plant pathogen species. The visibility of this area in plant pathology is also evident from the number of recent reviews of concepts and methods appropriate to population genetic studies with plant pathogens (Burdon, 1992; Leung et al., 1993; McDermott and McDonald, 1993; McDonald and McDermott, 1993; Anderson and Kohn, 1995; Brasier, 1995; Milgroom, 1995a, 1996), as well as recent reviews of population genetic studies of specific organisms (Fry et al., 1992; Wolfe and McDermott, 1994; Kohn, 1995; Leslie, 1995; McDonald et al., 1995; Milgroom, 1995b). Although there is an exciting potential for population genetics to contribute to plant pathology, it is difficult to predict whether it will become an indispensable part of plant pathology, or fade away as fashions change. The study of population genetics of plant pathogens is still in its infancy, with some of the requisite growing pains associated with development into a mature discipline within plant pathology. Its rapid emergence has been made possible, in part, by technological advances that have increased the ease with which one can find polymorphic genetic markers; further fueling this rise is the apparent fashion of using molecular biological techniques. As a result, researchers are undertaking numerous studies in which genetic variation is described and quantified on various scales. The future of this field, from the perspective of population genetics, will be to progress beyond the description of genetic variation, towards an emphasis on evolutionary processes. Its future with respect to epidemiology is an open question, and the subject of this chapter. Whether population genetics becomes an integral discipline within plant pathology depends, in part, on whether it can be integrated with epidemiology and disease management. Evolutionary biology and population genetics have the potential to deliver much basic information about plant pathogens. Two books in the 1980s (Wolfe and Caten, 1987; Leonard and Fry, 1989) initiated the integration, but much has happened in this field since they were published. Useful integration of these fields will require mutual understanding and better communication between geneticists and epidemiologists. The value of applying population genetics in plant pathology is not always obvious, and is sometimes impeded by biological and conceptual obstacles. We will try to highlight some of the ways population genetic studies relate to epidemiology and disease management, and identify the various obstacles. We begin with an overview of population genetics and a brief account of its history in plant pathology. This is followed by some of the general principles that unite population genetics and epidemiology. Finally, we finish by describing research on two pathosystems in which links between population genetics and epidemiology are relatively strong.
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11. POPULATION GENETICS IN PLANT PATHOLOGY A . WHAT IS (AND WHAT ISN’T) POPULATION GENETICS?
Population genetics is a field concerned with determining the extent and pattern of genetic variation in populations with the goal of understanding the evolutionary processes affecting the origin and maintenance of genetic variation. The conceptual framework is based on evolutionary biology and on the processes affecting the genetic composition of populations: selection, mutation, gene flow, genetic drift and mating systems. With an understanding of the relative importance of different evolutionary processes, predictions can be made about changes in the genetic composition of populations. Sometimes predictions can be made for responses of pathogen populations to selection by various management practices. The term “population genetics” has sometimes been interpreted broadly among plant pathologists to include any study on genetic variation. This interpretation is perhaps one reason why population genetics is not fully understood in plant pathology as a discipline distinct from systematics and diagnostics. Although knowledge of genetic variation is key to population genetic studies, it is but the raw material from which inferences are made. In population genetics, evolutionary inferences are made based on the dynamics of gene and genotype frequencies within and between populations. Systematics is fundamentally different: phylogenetic inferences are made at the species level (or higher) from morphological and genetic differences among taxa. Although there is overlap of concepts and techniques between population genetics and systematics, the scale is different, in both time and space. Systematics focuses on differences between species or higher taxa, which have evolved over relatively long time scales, and often in different locations. Most emphasis in population genetics is on microevolutionary events within populations of single species, and on time scales that are generally shorter than those required for speciation. However, this distinction is not always clear, especially when taxonomic complexity is revealed by closer inspection of genetic variation within species complexes (e.g. Vilgalys and Cubeta, 1994; Leslie, 1995). Furthermore, the distinction between microevolution and macroevolution is somewhat arbitrary because microevolutionary events may lead eventually to speciation (Brasier, 1995). Population genetics is sometimes confused with those studies of genetic variation in which the goal is to find diagnostic genetic markers among species or among different phenotypes within species. The search for diagnostic markers and studies of population genetics share the need for genetic variation as the basic empirical observation, but the objectives are very different. Genetic differences exploited for diagnostic purposes are more akin to systematics than to population genetics. Neither the population concept nor that of evolution, however, is considered rigorously in the search for
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diagnostic tools. Similarly, the use of molecular genetic markers in population genetics must be distinguished from molecular biology, where questions are addressed at a subcellular as opposed to a population level of organization. Clearly, a discipline is defined by the questions asked, not the tools used. B. BRIEF HISTORY OF POPULATION GENETICS IN PLANT PATHOLOGY
Plant pathologists began their studies of population genetics when they discovered variation in physiological races (now often referred to as “pathotypes”) within populations of plant pathogenic fungi. Pathotypes are defined solely by their abilities to cause disease on host plants with different resistance genes. From a management perspective, deployment of resistance genes in host plant populations has become a fundamental concept in plant pathology (Vanderplank, 1963). Successful deployment of resistance genes depends on knowledge of the genetic composition of pathogen populations, at least with respect to the relative frequencies of different pathotypes. Pathotype surveys have been conducted on numerous pathogens, and are still part of the strategies for managing diseases with host plant resistance (e.g. Kolmer, 1989). Population genetic concepts received greater attention in relation to variation in pathotypes when pathologists began asking questions such as “Why is there more pathotype variation in some populations than others?”, or “Will complex pathotypes (super-races) evolve in response to resistance gene deployment?” Questions like these grew out of the need to manage disease, but could be addressed adequately only within a population genetics framework; in other words, there was a natural integration of epidemiology and population genetics. These questions generated studies on fitness, host specialization, sexuality and recombination, population structure, etc., in relation to pathotype evolution (reviewed in Wolfe and Caten, 1987). As a consequence, an awareness was established of the link between evolutionary processes and disease management. In the late 1970s to mid-l980s, a different perspective emerged as attention was turned towards variation in genetic markers besides pathotypes. Some of the earlier work was done with enzyme polymorphisms, vegetative incompatibility and mating types (e.g. Leonard, 1978; Burdon et al., 1982; Puhalla, 1985; Stenlid, 1985; Tooley et. al., 1985; Leung and Williams, 1986). Plant pathologists were not only applying well-known laboratory techniques to plant pathogen populations, but also began asking questions about evolutionary processes beyond selection for pathotypes by host plant resistance. By the late 1980s and early 1990s studies of molecular variation in plant pathogens all but eclipsed those of pathogenic variation in plant pathology. These recent studies were made possible by technological innovations in molecular biology that made genetic markers (e.g. RFLPs and RAPDs) more accessible.
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The advances in technology also brought about a marked change in emphasis in population genetics of plant pathogens. The biology of pathogens at the population level and processes other than selection have been emphasized. For example, studies have been conducted on population structure to make inferences on gene flow, genetic drift and recombination (reviewed in Fry et al., 1992; Leung et al., 1993; McDermott and McDonald, 1993; McDonald and McDermott, 1993; Anderson and Kohn, 1995; Milgroom, 1995a, 1996). This field has been exciting, resulting in new knowledge about the biology of pathogens. However, with few exceptions, little effort has yet been made to apply this new knowledge to epidemiology and disease management.
111. WHAT CAN POPULATION GENETICS CONTRIBUTE
TO EPIDEMIOLOGY AND PLANT DISEASE MANAGEMENT? The answer to this question is not simple. At present, there are few generalities about the contributions that population genetics can make. The obstacles to applying population genetics to epidemiological questions are both historical and conceptual. For some plant pathologists, the concepts of population genetics and evolutionary biology are foreign ideas, and conversely, few population geneticists have an understanding of plant pathology. Population genetics derives from general biology, in which organisms are considered primarily in natural environments (or experimental cages in the laboratory). The separate developments of population genetics in biology and in plant pathology have inhibited effective communication among researchers and delayed the integration of these disciplines. There are several fundamental concepts from population genetics that can be applied profitably to epidemiology and disease management. In the following sections we identify some concepts that seem most likely to integrate these fields. We also discuss some of the obstacles that need to be recognized in order to apply population genetics appropriately to epidemiological problems. Each concept is illustrated with relevant plant pathological examples. A . THE CONCEPT OF A POPULATION
A population is not simply a haphazard collection of individuals, but has biological and practical significance that is important from a research perspective. A fundamental concept in any research is that the samples or experiments are so structured that the inferences made can be applied to more general phenomena beyond the objects being observed. The key question is to what populations will inferences be made? Although this is
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often done implicitly, a more careful examination of this question is crucial in studies of population genetics - and indeed, this may be one of the defining features of population biology. The challenge is to direct research efforts towards an appropriate target population. A precise definition of a population is somewhat elusive. A restrictive definition of a population that has sometimes been used by ecologists is: a collection of individuals that are actually or potentially interbreeding (Pianka, 1988). For most microorganisms, such as many plant pathogens, the dependence of this definition on “interbreeding” makes it irrelevant because of their largely asexual life histories. As an alternative, a population may be viewed as “a group of organisms of the same species occupying a particular space at a particular time” (Krebs, 1985, p. 157). Although the potential to interbreed is not required in this definition, and is therefore more appropriate for most plant pathogen species, it is somewhat unsatisfying from a genetic perspective. The missing concept in genetic and evolutionary terms is that a population is a pool of individuals from which the next generation will be drawn. In addition, the concept of a population has to be extended further to include genetic composition: the spatial and temporal limits of a population are defined by uniform allele frequencies. Populations may sometimes have underlying genetic structure in which subpopulations have differences in allele frequencies. In this case, a population comprises multiple subpopulations, some or all of which have differences in allele frequencies. Accurate population definition is essential so that sampling is done in a manner which will enable inferences to be made about the population of interest. There are two practical problems that commonly limit the inferences that can be made appropriately to the population level; neither is unique to plant pathology. The first problem is that of recognizing and sampling populations. The second problem is the study of laboratory strains that are not representative of individuals present in field populations. Unfortunately, it is frequently difficult to define the target population, and therefore appropriate sampling strategies are not always clear. Because of these practical difficulties, analyses are sometimes done on laboratory strains. Each of these problems will be discussed in more detail. From an operational perspective, recognition of population structure is very important when estimating population genetic parameters. Wolfe and Knott (1982) discussed this problem in relation to pathotype surveys, but many of the same caveats apply to any population sampling. The most important caveat is to avoid pooling individuals belonging to genetically distinct subpopulations. This problem can arise by sampling over diverse areas or at different times. Knowledge of the biology of an organism can be useful for rationally delimiting the scale of a population. A population may comprise individuals within a given geographic region on a continental scale, e.g. Puccinia graminis f. sp. tritici, which migrates hundreds of kilometres in North America (Roelfs, 1985), or as small as a soil core, e.g.
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Fusarium oxysporum, where populations 10 m apart in undisturbed soil may be genetically distinct (Gordon et al., 1992). The defining feature is the subdivision of the population into genetically distinct subpopulations. In addition, the size of genetic individuals (genets) also varies greatly (see Brasier, 1992; Anderson and Kohn, 1995), which, in turn, influences the sampling strategy to be adopted. Although studies on characterized laboratory strains can be valuable for revealing biological phenomena, inferences made from them to natural populations may be misleading or inappropriate. For example, an important question for the management of fungicide resistance is the fitness of resistant individuals (Milgroom et al., 1989). Some studies have shown that laboratoryselected mutants resistant to fungicides have reduced fitness compared to wild type strains (e.g. De Waard and Van Nistelrooy, 1990). To apply this finding to management in the field, however, one needs to ask whether it is appropriate to make inferences from laboratory strains. Surveys of field populations have sometimes shown quite different results from those in the laboratory. For example, there were only two variable amino acid sites in the p-tubulin gene that conferred benomyl resistance in Venturia inaequalis and other fungal plant pathogens collected from field populations; in contrast, seven additional variable amino acid sites were found among resistant laboratory mutants (Koenraadt et al., 1992). A possible explanation for this discrepancy is that most mutants are at such a selective disadvantage that they are not likely to be found anywhere but in a laboratory. Inferences made about fitness of laboratory strains in this case would be very misleading. The problem of defining a population on which to make inferences is perhaps best exemplified by studies in which genetic variation of culture collections is described. These studies may be valuable for systematics or biogeography, or finding diagnostic markers, but they are less useful for population genetics because the target “population” is artificial, being a collection of disparate individuals which would normally be separated both in time and space. One example of different inferences from different samples is found with the chestnut blight fungus, Cryphonectriaparasitica. The effects of vegetative incompatibility (vic) genes on transmission of fungal viruses (hypoviruses), which cause hypovirulence in C. parasitica, were studied independently in two laboratories. One approach was to start with laboratory strains from various populations, but with characterized vic genes (Huber and Fulbright, 1994). The other approach was to sample a large number of uncharacterized isolates randomly from a single natural population (Liu and Milgroom, 1996). Although both studies found similar results, the choice of strains made a fundamental difference to the inferences that could be made. The strength of the Huber and Fulbright (1994) study was the valuable insight into genetic control and cellular-level interactions affecting virus transmission. However,
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inferences to the population level were not possible because of lack of knowledge of vic allele frequencies in specific populations. In contrast, Liu and Milgroom (1996) estimated the average effects of vic genes in a natural population, but without an understanding of the effects of each individual gene. The inference from this study was the average transmission of hypoviruses and the potential success of biological control with hypovirulence in a specific population. The weakness was that inferences could not be made to other populations without assuming that vic allele frequencies were similar to those in the sampled population. The important lesson is that the population to which inferences can be made depends on the population that is sampled for study. For epidemiology and management, where it is essential to make inferences beyond laboratory strains, the best approach is to sample directly from the target population of interest. Wolfe and Knott (1982) and Milgroom (1995a) discussed some of the practical constraints on sampling populations for population genetic studies; this issue will not be discussed further here. B. GENETIC VARIATION IN POPULATIONS
Almost every population has some degree of genetic diversity; the few exceptions are usually where single clones have colonized new areas (e.g. Correll et al., 1992; Goodwin et al., 1994b). There are two basic types of genetic variation: ecologically important variation and selectively neutral variation. Ecologically important variation refers to traits that affect fitness and, therefore, may be under selection. Selectively neutral variation refers to variation in traits that do not (or are assumed not to) affect fitness and are not under selection. Changes in selectively neutral traits are affected by evolutionary forces such as mutation, genetic drift and gene flow (although they may be affected indirectly by selection on linked genes). The different inferences that can be made from each type of genetic variation will become evident below. The challenge is to understand how each type of genetic variation relates to epidemiology and disease management. In many situations, ecologically important variation is the type of variation that is more relevant for disease management. Variation in pathogen populations in response to a management practice may have implications for the durability of that practice, since individuals may be selected for or against. The best-known examples in which genetic variation, and therefore selection, has a direct effect on disease management are pathotypes and fungicide resistance. However, variation in other traits may also be relevant to management and may help to explain some failures of bialogical or cultural controls, although these latter areas have been studied little to date. Similarly, variation in response to environmental conditions has also been hypothesized to affect the genetic composition of fungal populations (e.g. Koller et al., 1995).
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An understanding of variation in ecologically important traits is relevant to epidemiology and management because it helps predict how a population will respond to selection. The strength of selection (or more formally, the rate of change of the mean fitness in a population) is proportional to the variance in fitness in the population; this is known as the fundamental theorem of natural selection (Fisher, 1930). For example, the variance in fitness in a fungal population when a fungicide is applied is roughly equivalent to the variance in fungicide resistance phenotypes (Milgroom et af., 1989). Therefore, surveys of pathogen populations may be used to estimate this variance and to predict the rate of selection. If a pathogen population is uniformly sensitive to a fungicide, i.e. it exhibits no variation in fungicide resistance, then resistance cannot be selected and the use of that fungicide may provide effective disease management. Conversely, once there is variation for response to fungicide, selection will favour the resistant individuals, whose fitness will be greater than sensitive individuals when fungicide is applied; the resistant individuals will then increase in frequency. Estimates of variance need to be made from adequate samples, from defined target populations (e.g. Smith et af., 1991; Peever and Milgroom, 1993) in order to make inferences to the population level that are relevant to management. A culture collection is unlikely to represent a target population accurately. Successful breeding and deployment of durable host-plant resistance depends on an understanding of variation in pathogen populations. Proper resistance screening requires first defining the pathogen populations against which resistance is being bred, and then sampling the target populations adequately. In some cases a target population has been well sampled. For example, a collection of the most common clones of Sclerotinia sclerotzorurn that occur across the canola-growing regions of Canada (Kohli et af., 1992) is available for screening for resistance in Canada (L. M. Kohn, personal communication). The goal in screening for resistance against large samples of the pathogen is not only to find the best resistance, but also to assess the range of variation in the pathogen population for virulence on particular breeding lines. In other words, successful deployment of resistance relies on both breeding and screening against appropriate target populations of pathogens. From an evolutionary perspective, there are two main outcomes of a comprehensive resistance screening programme. First, interactions between breeding lines and pathogen isolates can be identified, which indicate specialization of pathogen genotypes to resistant lines. The magnitude of the interaction and variation on individual hosts are important features in interpreting the significance of interactions, Second, the variance in virulence and fitness among isolates on particular host lines can also be estimated, allowing the rate at which a population will shift to higher levels of virulence to be predicted.
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As biological control is used increasingly against plant pathogens, failures are likely to occur because target organisms evolve resistance to the introduced agents. There are few examples of plant pathogens in which resistance to biological control agents has been documented. The potential for resistance to some hypoviruses in C. parasitica was recently demonstrated in the laboratory (Polashock el al., 1994). The evolutionary processes leading to failures of biological control are similar to those for the evolution of fungicide resistance or pathotypes, but are rarely considered when biocontrol agents are deployed. Surveys of existing vaiiation (as with fungicide resistance and virulence) would be logical steps in estimating the evolutionary potential for resistance in target populations. An equally challenging evolutionary problem in biological control is the potential for declining effectiveness of introduced agents because of selection against more effective individuals. An outstanding example is myxoma viruses released in Australia to control rabbits (reviewed in Fenner and Myers, 1978). The myxoma virus caused nearly 100% mortality in the rabbit population when it was first introduced. However, several years later, moderately virulent strains increased in frequency, and the most virulent viruses all but disappeared. Simultaneously, viruses placed such intense selection pressure on rabbit populations that the average level of virus resistance increased. Needless to say, the effectiveness of biological control decreased dramatically. Comparable phenomena have yet to be described in plant pathology, but the example of the myxoma virus should not be ignored. The primary lesson from these examples is that an understanding of ecologically important variation is needed in order to predict the durability of any particular management practice. The evolutionary process most pertinent to management is selection, and direct measurement of the phenotype of interest is usually the best approach for studying ecologically important variation. C. THE USE OF NEUTRAL GENETIC MARKERS TO ESTIMATE ECOLOGICALLY IMPORTANT VARIATION
As selectively neutral genetic markers have become more accessible, there have been numerous attempts to substitute them for direct assessment of ecologically important characters. The motivation is quite reasonable: it is generally simpler to determine an allozyme or RAPD profile of a pathogen isolate than it is to inoculate a series of differential cultivars to determine its pathotype. If neutral genetic markers are good predictors of pathotypes, then their use could save valuablettime and expense. However, this approach must be undertaken cautiously; there is no general rule that variation in one marker can predict variation in another. The success of this approach depends on the type of marker used, or knowledge of the population structure of the
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pathogen. This issue will be discussed at the level of both individuals and populations. 1. Predicting variation among individuals Genetic markers can sometimes identify an ecologically important trait. A direct assay is sometimes possible by detecting a particular gene responsible for the trait of interest. For example, benzimidazole resistance can be assayed directly by selective polymerase chain reaction (PCR) amplification of alleles of the P-tubulin gene that confer resistance (Koenraadt and Jones, 1992). Alternatively, when a gene of interest cannot be assayed directly, neutral genetic markers tightly linked to it may be used if the association between loci is strong. Finding linked markers is becoming increasingly possible given the efforts in numerous laboratories to develop genetic maps of various pathogens, or the use of techniques such as bulked segregant analysis (Michelmore et al., 1991). For example, RAPD markers tightly linked to avirulence loci in barley powdery mildew, Erysiphe graminis f. sp. hordei, have been found to have strong non-random associations (cited in Wolfe and McDermott, 1994), but the association is not perfect and estimation of avirulence allele frequencies from these markers would include a measurable amount of error. Exploiting non-random associations is an approach that has been used successfully to predict ecological traits with neutral markers in some organisms. There are a number of examples of plant pathogens for which there are almost perfect associations between neutral markers and pathotypes or vegetative compatibility groups (e.g. Burdon and Roelfs, 1985; Kistler et at., 1991; Kohn et al., 1991; Goodwin et al., 1992b; Anderson and Kohn, 1995). Non-random association among alleles at different loci is referred to as “gametic disequilibrium”, or “gametic phase disequilibrium”, and is caused primarily by two different factors. First, simultaneous selection for different traits can result in non-random associations; this is exemplified by selection for pathotypes with combinations of avirulence genes compatible to host-plant resistance with two or more corresponding specific resistance genes (Wolfe and Knott, 1982; Hovmoller and 0sterglrd, 1991). Second, and more relevant to the use of neutral markers, gametic disequilibrium often occurs between tightly linked loci. An extreme case of gametic disequilibrium is when organisms are asexual such that the entire genome is effectively linked; all the organisms cited above for strong non-random associations among different markers are asexual for much or all of their life cycles (the exception above is S. sclerotiorum, which is homothallic and, therefore, no recombination occurs [Kohn et al., 19911). Conversely, gametic disequilibrium is minimal in sexually reproducing organisms because of recombination (e.g. Milgroom et al., 1992a; McDonald et al., 1994; Liu et al., 1996; Milgroom, 1996). Therefore, population structure and reproductive biology should be known before attempting to use neutral markers to predict other traits.
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Prediction of ecologically important variation from neutral markers has several serious drawbacks. First, different mutation rates among loci can result in uncorrelated variation. For example, in the rice blast fungus, Magnaporthe grisea, a single clonal lineage, defined by a multilocus RFLP fingerprint, may comprise many diverse pathotypes because the mutation rate for avirulence genes is apparently greater than for the RFLP marker (Levy et al., 1993; Zeigler et al., 1995). Therefore, in M . grisea, even though the range of pathotypes within a lineage may be limited, a lineage does not necessarily identify pathotype. A similar situation occurs with Phytophthora infestans, in which there is much pathotype variation within some clonal lineages (Goodwin et al., 1995~).As a contrasting example, there was no significant variation in virulence among clones of S . sclerotiorum from canola in Canada that differed in RFLP genotypes and mycelial compatibility (L. M. Kohn, personal communication), The problem of differential mutation rates is further highlighted in pathogens such as F. oxysporum in which host specialization is only sometimes associated with vegetative compatibility groups (Correll, 1991; Appel and Gordon, 1994). These discrepancies can be explained simply by variations in mutation rates between different loci. The restrictions on using neutral genetic markers to predict ecologically important variation should be considered carefully before attempting such a project. First, a decision has to be made whether a genetic marker is simpler than a direct assay of the phenotype of interest. For example, it may be simpler in some organisms to assay a fungicide resistance phenotype than it is to probe for a particular gene: compare a simple cultural technique (Lalancette et al., 1984) to amplifying P-tubulin genes (Koenraadt and Jones, 1992) to assay for benomyl resistance. On the other hand, the determination of a pathotype may require considerably more effort compared to determining simple molecular markers. However, a random search for markers associated with pathotypes may be fruitless unless the target population is clonal and markers can be found with appropriate levels of variation. Alternatively, finding tightly linked markers with predictive value in sexual populations may require extraordinary effort unless a genetic map has already been constructed. In addition, it may be necessary to verify an association between markers for each population to be studied because different degrees of association may be found within different populations. For example, the simple relationship between pathotypes and clonal lineages found in US populations of M . grisea (Levy et al., 1991) was more complex in populations in Colombia and the Philippines (Levy et al., 1993; Zeigler et al., 1995) (see also section IIIE, p. 18). When all these factors are considered, it may be more accurate, and less effort, to survey variation in the ecologically important trait of interest than it is to find reliable diagnostic genetic markers.
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2. Predicting variation among populations An important misconception about the use of neutral genetic markers is that knowledge of their variation can be used to predict variation in ecologically important traits. This misconception may derive from generalization of the special cases described above (clonal populations, use of linked markers, similar mutation rates, etc.). Unfortunately, there are surprisingly few studies comparing diversity of both types of markers among different populations. Diversity in pathotypes and allozymes showed a rough correlation in the asexual barley scald fungus, Rhyncosporium secalis (Goodwin et al., 1993); however, the correlation was not strong, and sample sizes were sometimes small. In contrast, variations in RAPD markers were poor predictors of variation in avirulence alleles in E . gruminis f. sp. hordei (Wolfe and McDermott, 1994), and in fungicide resistance in Pyrenophora teres (Peever and Milgroom, 1994b; see also section IVA). Conceptually, any attempt to correlate diversity of different markers is fraught with problems. First, as mentioned above, differential mutation rates confound any correlation. Second, and more important, is the effect of selection on ecologically important variation but not on unlinked neutral markers. For example, a population that is highiy diverse for both pathotypes and neutral markers could lose much of its pathotype diversity if exposed to highly selective host-plant resistance such that few pathotypes survive. This same population could maintain its diversity for neutral markers if the population is not highly clonal. Claims that knowledge of variation of neutral markers can predict variation in pathotypes or fungicide resistance should, therefore, be interpreted with caution. D. THE INTERFACE BETWEEN POPULATION GENETICS AND EPIDEMIOLOGY
We will discuss two entirely different ways in which neutral genetic variation can be applied to epidemiological questions. First, selectively neutral markers can be exploited directly for addressing epidemiological questions in which specific strains are tracked in the environment. Second, concepts of population genetics allow inferences to be made about evolutionary processes, which in turn may affect interpretations of epidemiology. Examples of how evolutionary inferences can be made to plant pathogen populations have been reviewed recently (see section 1, p. 1 for references) and will not be repeated here. In the following sections, we give examples of how genetic variation and population genetic concepts can be integrated with epidemiology and disease management. 1 . Direct tracking o j genotypes One of the simplest applications of genetic variation and genetic markers in an epidemiological context is the direct tracking of specific genotypes. This
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may be useful for determining the source of inoculum or for identifying different genotypes in competition studies. The ability to discriminate among genotypes can facilitate competition experiments (Ennos and McConnell, 1995; Legard et al., 1995), and may be used even for organisms that have regular sexual cycles if tracking is done during the asexual phase (Sierotzki et al., 1994). An excellent example of tracking specific genotypes to address epidemiological questions is the work summarized by Webber et al. (1987) on the saprophytic phase of the Dutch elm disease fungus, Ophiostoma novo-ulmi. Using benomyl-resistant isolates, Webber and colleagues showed that there were two distinct sources of inoculum contributing to the colonization of elm bark: the fungus present in the xylem from the pathogenic phase and spores introduced by bark beetle vectors from the saprophytic phase in dead elm trees. In addition, after inoculating trees with benomyl-resistant isolates of known vegetative compatibility types, they found recombinant genotypes on the emerging beetles. This result demonstrated that at least some ascospores contributed to bark colonization and that recombination maintained genotype diversity in the population. In another example of determining the source of inoculum, Shah et al. (1995) tested the hypothesis that a major source of Stagonospora nodorum on wheat in New York state was infected seed. They used two complementary approaches, one using classical epidemiological methods, the other taking advantage of genetic variation in S. nodorum populations. They infected wheat seeds with S. nodorum isolates that had known RFLP genotypes and then mixed these with clean seed to create seed lots with varying proportions of infected seed, which were then planted in the field. The severity of the ensuing foliar epidemics correlated to the proportions of infected seeds planted. In addition, the majority of the isolates recovered from foliage and seed in the next generation were the same genotypes as those used to infect seeds, although these same genotypes were not found in the resident background population. Results from both approaches clearly demonstrated a significant role of seed-borne inoculum in this pathosystem. As a source of inoculum, long-distance dispersal, or gene flow, is usually quantitatively insignificant, although there are exceptions. For example, the Puccinia pathway in North America has been described in detail (Roelfs, 1985) in which Puccinia graminis f. sp. tritici overwinters in Texas and Mexico and migrates annually throughout the midwestern USA and into Canada; migration occurs in the reverse direction in autumn. Another pathogen that overwinters far from an agricultural host population in the USA is the tobacco blue mould pathogen, Peronospora tabacina. Each year inoculum must be reintroduced into tobacco-growing regions in the USA from overwintering areas. However, the exact locations of the inoculum sources are not known despite attempts to identify them by trajectory studies (Davis and Main, 1988). Tobacco blue mould is an example where direct tracking of genotypes
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could potentially be applied to answer a basic epidemiological problem. An essential requirement would be to develop a set of polymorphic genetic markers and to characterize genetic variation in all possible source populations. This approach has been used successfully for tracking the sources of immigrant genotypes of Phytophthora infestans in the USA and Canada (Goodwin et a f . , 1994a,b; see also section IVB). Although long-distance dispersal is generally not important quantitatively as a source of inoculum, it can sometimes have significant qualitative effects, especially the introduction of novel pathotypes. A good example of this is barley powdery mildew in Europe. Analyses of neutral genetic markers made it possible to demonstrate that some genotypes have dispersed on a continental scale, introducing pathotypes compatible with host resistance genes that otherwise were effective against the resident mildew population (Brown et af., 1991; Wolfe and McDermott, 1994). By surveying genetic variation throughout many possible source populations and matching genotypes, the origin of dispersing inoculum could be identified confidently. 2.
Evolutionary inferences f r o m population genetic studies
Gene pow. The analysis of selectively neutral genetic variation among subpopulations by gene-diversity analyses provides another approach to studying gene flow and sources of inoculum (for methods see Leung et al., 1993; McDermott and McDonald, 1993; Milgroom, 1995a). Populations that are genetically subdivided are likely to have restricted gene flow among subpopulations, and to some degree may be managed as separate entities (Leung et a f . , 1993; Milgroom, 1995a). In contrast, a population that is genetically uniform over a large area may be experiencing widespread dispersal among subpopulations such that, in theory, the whole population must be considered as a management unit. Disease management for subdivided pathogen populations with host-plant resistance has been recently summarized (Leung et al., 1993) and will not be discussed further here. Management of fungicide resistance in subdivided populations is discussed below (section IV). Population structure due to various levels of gene flow may be a factor that can be exploited to a greater extent in future disease-management efforts. Estimates of gene flow from analyses of gene diversity have some inherent weaknesses, especially in agricultural systems that are highly disturbed by episodic introductions of plant pathogens. When populations are subdivided, it is an indication that gene flow is restricted to some degree. However, lack of genetic differentiation among populations is more problematic because it can be explained in at least two ways: by high levels of current gene flow, and/or by gene flow that occurred in the past (Slatkin, 1987). It is possible that populations with little genetic subdivision because of past gene flow
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actually experience low levels of current gene flow among subpopulations (although gene-diversity analyses would not show this). An example of historical gene flow is when a pathogen is introduced into a new area along with an agricultural crop. If the introduction occurred relatively recently it is unlikely that populations would have reached an equilibrium between gene flow and drift. To estimate current gene flow from gene-diversity data, one must assume that populations are in equilibrium for gene flow and genetic drift (Slatkin, 1987). Therefore, estimates of gene flow are likely to be fairly reliable when populations are highly subdivided, but claims of high levels of gene flow may be overestimated when subdivision is weak, and need to be supported by more information about the biology of an organism, especially its ability to disperse or be transported long distances. This problem was discussed in detail in relation to population subdivision in C. parasitica in North America (Milgroom and Lipari, 1995). Recombination. There are numerous plant-pathogenic fungi that reproduce sexually. Sexual reproduction not only contributes to recombination and the potential to generate new genotypic diversity, but equally important from an epidemiological perspective, it often involves structures that differ in dormancy, survival or dispersal characteristics from asexual propagules (see also section IVB). As an example, primary inoculum of Mycosphaerellu graminicola on wheat could be either sexual or asexual. Asexual pycnidiospores produced on crop debris are generally dispersed over short distances. However, ascospores of M. gruminicola can disperse over longer distances (Shaw and Royle, 1989). Identification of sexual or asexual inoculum sources for M . graminicola has been investigated both with epidemiological techniques and population genetics. The importance of airborne inoculum of M . graminicola, most likely ascospores, as primary inoculum was demonstrated by Shaw and Royle (1989) by excluding other sources of inoculum. A complementary population genetic approach was used by Chen et al. (1994) in the same system. They showed that, although allele frequencies did not change over 3 years, there was no carryover of multilocus genotypes from year to year; this finding is consistent with frequent recombination and annual recolonization by ascospores. Both approaches led to similar conclusions about the role of ascospores: one observed the effects of airborne spores on disease progress, while the other made inferences from patterns of genetic variation. In contrast to the above example, where airborne inoculum could be assayed directly, studies of genetic variation are sometimes the only approach available for determining if sexually produced inoculum is present. For example, until recently only the A1 mating type of Phytophthora infestans was found outside of central Mexico (Goodwin et al., 1994b). The discovery of the A2 mating type in Europe in the 1980s (Hohl and Iselin, 1984; Fry et al., 1993) signalled an important potential for oospore formation. Oospores
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would allow P. infestuns to survive in the soil between potato crops, potentially altering the source of inoculum and epidemiology of late blight. The increase in genotypic diversity of P. infestuns populations in Europe in recent years has been attributed to immigration of new genotypes and the occurrence of sexual reproduction (Drenth et uf., 1994; Sujkowski et uf., 1994). Using genetic markers in an epidemiological context, Drenth et uf. (1995) confirmed that oospores survived in soil and that recombinant genotypes infected potato crops the following year. Recombination also has significant effects on the diversity of multilocus genotypes (reviewed in Milgroom, 1996), although recombination per se does not affect gene diversity, which is a function of the number and frequencies of different alleles. The importance of recombination to the diversity of pathotypes is well documented for Pucciniu gruminis f. sp. tritici in North America (Roelfs and Groth, 1980). Following the eradication of barberry, on which P. graminis reproduces sexually and produces overwintering teliospores, the diversity of pathotypes decreased significantly. Similarly, the diversity of pathotypes west of the Rocky Mountains in the USA, where the barberry was not eradicated, was significantly greater than in areas to the east where barberries were eradicated (Roelfs and Groth, 1980). The population structure of this pathogen was significantly altered such that it is now almost exclusively asexual with few clones (Burdon and Roelfs, 1985). Recombination and multilocus population structure are highly relevant to resistance gene deployment. For example, non-random associations among avirulence alleles in P. gruminis f. sp. tritici sparked a debate about the causes and significance of gametic disequilibrium. Vanderplank (1982) speculated that non-random associations were caused by selection against particular combinations of avirulence alleles. The implication of such selection is that certain combinations of resistance genes would be durable because compatible pathotypes would have reduced fitness. However, a simpler explanation for the observed gametic disequilibrium in populations of P. gruminis f. sp. tritici is clonal population structure (Knott, 1986). This explanation has fundamentally different implications for resistance gene deployment, since cultivars with particular resistance gene combinations would be vulnerable to compatible pathotypes if they occurred in the population, for example by immigration or mutation. The importance of gametic disequilibrium and gene deployment has been studied in much detail for barley powdery mildew, Erysiphe gruminis f. sp. hordei. Hovmcdller and 0stergbrd (1991) and Hovmdler et uf. (1993) demonstrated both positive and negative associations among avirulence alleles, depending on which resistance genes occurred together in common barley cultivars; their findings were similar to theoretical predictions for gametic disequilibrium (0stergird and Hovm~ller,1991). However, they also found that selection was not entirely predictable because of hitch-hiking selection, in which the frequency of avirulence alleles not under selection
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increased (or decreased) as a result of selection for other avirulence alleles in clonal populations. Brown (1995) modelled gametic disequilibrium among alleles at different avirulence loci in relation to the extent of recombination occurring and initial genotype distributions, and showed that hitch-hiking selection is difficult to predict in asexually reproducing populations. The dynamics of gametic disequilibria among avirulence alleles offers promise for understanding and improving resistance gene deployment in response to knowledge of pathotype distributions in populations. A unique example of disease management that required a thorough understanding of recombination was described by Crute (1989) for lettuce downy mildew, caused by Bremia Zacfucae. During the 1980s, resistance to the fungicide metalaxyl occurred in B. lucfucae in the UK. However, metalaxyl resistance initially arose only in a single clone, with the B2 mating type, which was avirulent on cultivars with the D m l l resistance gene. In order for metalaxyl resistance genes to be recombined into pathotypes virulent on Dmll cultivars, resistant and sensitive isolates had to occur on the same cultivar to mate successfully. Mating between resistant and sensitive isolates could only occur on cultivars that lacked D m l l (to allow metalaxylresistant isolates to colonize) and were not treated with metalaxyl (to allow metalaxyl-sensitive isolates to colonize). Therefore, to prevent recombination, growers were recommended to apply metalaxyl to cultivars lacking D m l l , even though control against the resistant isolates would be ineffective. Although this strategy was short lived due to the appearance of metalaxyl resistance in other pathotypes (Crute, 1992), it provides insight into the direct integration of population genetics and disease management.
E. POPULATION CHARACTERISTICS VARY AMONG POPULATIONS
It is important to recognize that different populations of the same species sometimes have completely different population structures. This concept is important because management strategies applicable to one population may not apply to other populations. Unfortunately, this means that studies need to be replicated in several different populations in order to understand the range of variation that occurs within a species. Populations may vary in allele frequencies, or their overall genetic structures may be completely different. Differences in allele frequencies may result from restricted gene flow and genetic drift (Goodwin et al., 1993; Peever and Milgroom, 1994b; Milgroom and Lipari, 1995), founder effects (Milgroom et uZ., 1992b) or selection (Kolmer, 1989; Wolfe and McDermott, 1994). The two case studies discussed below (section IV) demonstrate some of the management implications of different allele frequencies among populations. Populations may also differ in the extent to which recombination occurs,
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affecting the multilocus structure of populations. There are several striking examples of both clonal and sexual population structure in the same species: Puccinia graminis f. sp. tritici (Roelfs and Groth, 1980), Magnaporthe grisea (Kumar et al., 1995), Ophiostoma novo-ulmi (Brasier, 1988), Phytophthora infestans (Goodwin et al., 1992b; Drenth et al., 1994) and Cryphonectria parasitica (Liu et a f . , 1996). Other examples are likely to emerge as more investigations are done.
IV. EXAMPLES OF THE INTEGRATION OF POPULATION GENETICS AND EPIDEMIOLOGY A. FUNGICIDE RESISTANCE IN PYRENOPHORA TERES
Population genetics has made a significant contribution to managing resistance to sterol biosynthesis-inhibiting fungicides (SBIs) in Pyrenophora teres, which causes net blotch of barley (Peever and Milgroom, 1992, 1993, 1994a,b). The questions addressed in this system were familiar in fungicide resistance management: (1) is there cross-resistance among chemically related fungicides, and (2) are there fitness costs associated with fungicide resistance? Approaching these questions from the perspective of population and ecological genetics yielded some useful information about population structure and disease management. The approach used to address questions of cross-resistance and fitness costs was to sample discrete populations of P. teres. Samples of 22-35 isolates were taken from each of four populations in North America and one in Germany; SBIs had been used previously for disease control in only two of the five populations. The growth rate of each isolate was measured in vitro in the presence of five different SBIs. There was significant variation in SBI resistance within populations, indicating that SBI applications would potentially result in selection for higher frequencies of resistant phenotypes. Cross-resistance among different SBIs was analysed in terms of genetic correlations, to determine whether the same genes controlled resistance to different SBIs (Peever and Milgroom, 1993). Resistance to many pairs of SBIs was correlated in some populations, but not in others. In other words, there were different cross-resistance relationships in different populations. A similar approach to estimating genetic correlations was used to determine if genes that control SBI resistance also affect fitness (Peever and Milgroom, 1994a). Latent period and sporulation were estimated in isolates from two populations. However, there was no correlation between SBI resistance phenotype and fitness. These results must be interpreted somewhat cautiously, as it is difficult to make inferences from greenhouse studies to fitness in natural populations. In addition to studying SBI resistance and fitness, Peever and Milgroom
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(1994b) also analysed population structure using RAPD markers in the same samples. The diversity of RAPDs was not correlated with variation in SBI resistance between populations, i.e. variation in neutral genetic markers was not a good predictor of ecologically important variation for these populations. In addition, there was no discernible association between RAPDs and SBI resistance within populations. Analysis of the multilocus genetic structure revealed that four of five populations did not deviate from what was expected under random mating. Therefore, recombination may be sufficient to uncouple selection for SBI resistance from neutral genetic markers (contrast this with the Phytophthoru infestans example below; section IVB). The analysis of population structure among Pyrenophora teres populations using RAPDs showed moderately high genetic subdivision, with 33-46% of the variation attributable to differences among populations (Peever and Milgroom, 1994b). Genetic subdivision is most likely a result of restricted gene flow among populations; therefore, each population may have to be considered individually in terms of fungicide resistance management. Interestingly, when phenotypic variation in fungicide resistance was partitioned within and among populations, the result was similar to that found with RAPDs: 38% of the variation was due to differences among populations. The similar degree of genetic differentiation for both ecologically important and selectively neutral traits suggests that selection has not strongly affected population structure (Spitze, 1993), even though SBIs had been applied occasionally in two of the populations studied. A result that merits closer examination is the finding that different populations had different cross-resistance relationships (Peever and Milgroom, 1993). The cause of these differences is not understood entirely, but the simplest explanations are that there are different alleles conferring SBI resistance, or allele frequencies are different in each population. An alternate hypothesis is that fungicide resistance genes are non-randomly associated. However, the lack of gametic disequilibrium among RAPD loci suggests that populations may experience a significant amount of recombination. In addition, selection from fungicide use appears minimal since only two populations had experienced these fungicides prior to sampling, and, as shown above, differentiation among populations was similar for both RAPDs and resistance phenotypes. These studies have some clear lessons for SBI resistance management in P. teres. First, because of significant phenotypic variation in SBI resistance in each population, it can be predicted that resistance would evolve if fungicides were applied intensely; and, because there are no detectable fitness costs associated with SBI resistance in P. teres, the frequencies of resistant phenotypes would not necessarily decline if fungicide use were subsequently discontinued. Second, differences in cross-resistance among populations mean that SBI resistance has to be managed independently in each population; this conclusion is further supported by strong genetic differentia-
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tion among subpopulations. Third, by assaying for cross-resistance in different populations, we can predict which combinations of fungicides would be effective in each population. Fourth, differences in allele frequencies for SBI resistance may strongly affect genetic correlations for cross-resistance. Therefore, no single model of cross-resistance among SBIs is appropriate for P. teres. Whether similar conclusions apply to other populations of P. teres, or to different species, awaits further studies of this type. An important feature of this research is the sampling and, therefore, the populations to which inferences can be made. Inferences that can be made from the population approach are simple to understand and relevant for management. B.
EPIDEMIOLOGY AND POPULATIONS GENETICS OF PHYTOPHTHORA INFESTAN S
Recent studies of Phytophthora infestans have revealed unexpected population structure that in turn has led to hypotheses concerning migrations, and insights into management of late blight. The initial motivation for studying the population genetics of P. infestans came from disease management problems in Europe in the early 1980s. At that time, there were two factors that signalled some unexpected variation in the pathogen population: disease control failures caused by metalaxyl resistance (Davidse et al., 1981), and the detection of A2 mating types (Hohl and Iselin, 1984). In addition, there was a profound lack of knowledge concerning geographic distribution, population structure, and the evolutionary processes shaping populations of f. infestam. Population genetic studies in North America have provided the bases for short-term management strategies, and may eventually lead to long-term management strategies. 1. Population structure
Prior to the 1980s there were probably three geographically defined populations around the world. First, the source population in central'Mexico is unique among P. infestans populations. This population has characteristics of a randomly mating sexual population (Tooley ef al., 1985; Goodwin et al., 1992b): approximately equal frequencies of A1 and A2 mating types (Tooley et ul., 1985), allozyme genotypes in Hardy-Weinberg equilibrium (Goodwin et al., 1992b), and a huge diversity of multilocus genotypes in any small geographic area (e.g. a single field) (Matuszak et al., 1990). Second, populations in the USA and Canada were derived from the population in Mexico, and were asexual, but with several clonal lineages. Third, on other continents, populations were dominated by a single clonal lineage (termed US-l), with most genetic diversity caused by mutation or mitotic recombination within that lineage (Goodwin et al., 1994b). The hypothesized pathways of migrations of f. infestans were recently summarized (Fry et al., 1993).
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Recent immigration of P. infestans into the USA and Canada illustrates the importance of population genetics to epidemiology and management. A significant migration occurred into the USA and Canada, probably from northern Mexico beginning in the late 1970s, but became most noticeable in the early 1990s (Goodwin et al., 1994a, 1995b). As in Europe, these migrations were first signalled by metalaxyl resistance (Deahl et al., 1993) and then by detection of individuals with the A2 mating type (Deahl et al., 1995; Goodwin et al., 1995b). In the early 1990s, populations in the USA and Canada were dominated by four clonal lineages: the resident lineage (US-l), and three recent immigrant lineages (US-6, US-7 and US-8) (Goodwin et al., 1994a, 1995b). The recent immigrant lineages represented A1 (US-6) and A2 (US-7 and US-8) mating types; all were resistant to metalaxyl (Fry et al., 1993). In addition, in most locations, the individuals in a single field and often over a vast region were in the same lineage (Goodwin et al., 1994a, 1995b). Analysis of pathotypes has generally not been as useful as analysis of neutral markers in deciphering relationships among populations. Individuals sampled in Mexico generally represent complex pathotypes, which were virulent on cultivars with multiple specific resistance genes (Tooley et al., 1985; Rivera-Pena and Molina-Galan, 1989). In addition, clonal lineages found in the USA and Canada that were recently derived from Mexico had greater pathotype complexity than resident pathotypes (Goodwin et al., 199%). Selection for complex pathotypes may be important in some locations, e.g. Mexico, but in the USA and Canada most potato cultivars lack specific resistance. In the absence of selection, rare pathotypes (and virulence factors) are likely to be lost because of genetic drift: relatively small numbers of individuals survive over winter in asexual populations, i.e. there are annual bottlenecks (Goodwin et al., 1995~).Further confounding our understanding of pathotypes, it has also been shown that pathotype diversity can develop rapidly within a clonal lineage (Goodwin et al., 199%). 2. Late blight management The strong clonal population structure of P. infestans in the USA and Canada during the early 1990s has led to some interesting management options. Knowledge of the widespread occurrence of immigrant genotypes in 1994 stimulated plant pathologists to launch an intensive effort to educate growers and extension personnel concerning the attributes of immigrant genotypes. At the same time, plant pathologists began to petition for the use of fungicides that otherwise would not have been available. Convincing documentation that the significant late blight problems were caused by immigrant genotypes was instrumental in convincing growers and regulatory personnel that new measures were needed. These efforts helped avert major epidemics in 1995. Knowledge of genetic variation and population structure made it possible
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to develop rapid diagnostic tests to identify P. infestuns lineages. Because field populations usually consisted of a single clonal lineage, and because the four major lineages in the USA and Canada could be distinguished via allozymes (Gpi), a rapid assessment of the allozyme genotype (Goodwin et al., 1995a) enabled practitioners to learn (sometimes within hours) which lineage was causing the problem in a particular field. This was especially important in predicting the efficacy of metalaxyl. Metalaxyl was extremely effective against US-1, but was much less effective against the immigrant lineages US-6, US-7 and US-8. The observation that certain lineages appeared to be associated mainly with potatoes and that other lineages were associated with both potatoes and tomatoes motivated investigations of host specialization. It became clear that US-6 and US-7 were pathogenic on both tomatoes and potatoes, whereas US-8 was primarily a pathogen of potatoes (Legard et ul., 1995). Host specialization was polymorphic in US-1 (Legard et al., 1995). Because P. infestuns is aerially dispersed, tomato growers needed to know if late blight in nearby potato fields was caused by US-6 or US-7. If so, then the fungus in neighbouring fields posed a serious threat to tomatoes, and management efforts were needed. Variation in aggressiveness was another characteristic that has been important to late blight management. Because clonal lineages could be easily distinguished, it became possible to investigate whether the immigrant lineages posed a more significant threat than did the resident US-1. During 1994 and 1995, several investigators began testing the hypothesis that US-8 was more aggressive than US-1 on potatoes. If the immigrant lineages are more aggressive, an overall intensification of management efforts will be needed. In contrast to earlier populations where only A1 mating type individuals were present, the occurrence of both A1 and A2 individuals in a few fields signals the possibility of sexual reproduction. The presence of oospores could alter the life history of P. infestans in the USA and Canada, consequently altering the epidemiology of the disease. The potential for sexual reproduction has two significant implications for late blight management. First, oospores survive as dormant propagules in soil and may serve as a significant additional source of inoculum. Previously, the soil had not been a source of inoculum for this fungus; in its asexual phase P. infestuns is essentially an obligate parasite and cannot persist in the absence of living hosts. Second, recombinant progeny could result in greater genotypic diversity in the population of P. infestuns in the USA and Canada, destroying the simple clonal structure that exists. Recombination will eliminate the association between allozyme genotype and pathogenic traits, and destroy the potential for using neutral genetic markers to predict epidemiologically important traits. Knowledge that populations of P. infestuns in the USA and Canada are in flux signals the need for significant new investigations in epidemiology and
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management. Probably most urgent is the need to investigate the role of oospores in the epidemiology of potato and tomato late blight in the USA and Canada; whether oospores will contribute significantly to initial inoculum is now unknown. Quantification of the greater aggressiveness of recent immigrant genotypes should lead to appropriate intensifications of late blight management strategies. Some opportunities for adjustments in late blight management include development and use of more resistant plants and new chemistries of fungicides. Knowledge that a single clone can be broadly and rapidly dispersed emphasizes the need to determine factors affecting dispersal of P. infesruns and to determine if all genotypes are similarly capable of such dispersal.
V. FUTURE CONTRIBUTIONS OF POPULATION GENETICS TO PLANT PATHOLOGY Population genetics can enhance our understanding of pathogen biology and, therefore, has much to offer plant pathology. Although much of the recent research on population genetics of plant pathogens has involved the description of genetic variation at various scales, some research has progressed to asking questions about evolutionary processes. This is a critical step beyond the descriptive phase because it allows greater insight into pathogen evolution and biology. Like any basic science, the utility of population genetics in plant pathology may at first seem negligible. Rather than letting its fundamental nature deter us, we need to broaden our perspectives to examine how this information can be applied. The most significant applications of population genetics to epidemiology and disease management are likely to derive from practical needs to understand pathogen variation and evolution. Descriptive studies of variation without questions focused on evolutionary processes are not sufficient. Similarly, elegant evolutionary inferences made about pathogen populations for which there are few associated management problems contribute little to the integration of population genetics and disease management. To apply population genetics to epidemiology and disease management, the burden is on plant pathologists to increase their understanding of population genetics and evolution. This educational development is essential, and fortunately is well underway. Education on evolutionary concepts will enhance communication with geneticists and lead to a better integration of population genetics into plant pathology in the future.
ACKNOWLEDGEMENTS We thank Jim Anderson, Clive Brasier, Tom Gordon and Bob Marra for making helpful suggestions on an earlier draft of this chapter.
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Drenth, A., Tas, I. C. Q . and Govers, F. (1994). DNA fingerprinting uncovers a new sexually reproducing population of Phytophthora infestans in the Netherlands. European Journal of Plant Pathology 100, 97-107. Drenth, A., Janssen, E. M. and Govers, F. (1995). Formation and survival of oospores of Phytophthora infestans under natural conditions. Plant Pathology 44, 86-94. Ennos, R. A. and McConnell, K. C. (1995). Using genetic markers to investigate natural selection in fungal populations. Canadian Journal of Botany 73(Suppl. l), S302-S310. Fenner, F. and Myers, K. (1978). Myxoma virus and myxomatosis in retrospect: the first quarter century of a new disease. I n “Viruses and Environment”, 3rd International Conference on Comparative Virology, Mont Gabriel, Quebec (E. Kurstak and K. Maramorosch, eds), pp. 539-570. Academic Press, New York. Fisher, R. A. (1930). “The Genetical Theory of Natural Selection.” Clarendon Press, Oxford. Fry, W. E., Goodwin, S. B., Matuszak, J. M., Spielman, L. J., Milgroom, M. G. and Drenth, A. (1992). Population genetics and intercontinental migrations of Phytophthora infestans. Annual Review of Phytopathology 30, 107-129. Fry, W. E., Goodwin, S. B., Dyer, A. T., Matuszak, J. M., Drenth, A., Tooley, P. W., Sujkowski, L. S . , Koh, Y. J., Cohen, B. A., Spielman, L. J., Deahl, K. L., Inglis, D. A. and Sandlan, K. P. (1993). Historical and recent migrations of Phytophthora infestans: chronoIogy, pathways, and implications. Plant Disease 17, 653-661. Goodwin, S. B., Allard, R. W., Hardy, S. A. and Webster, R. K. (1992a). Hierarchical structure of pathogenic variation among Rhynchosporium secalis populations in Idaho and Oregon. Canadian Journal of Botany 70, 810-817. Goodwin, S. B., Spielman, L. J., Matuszak, J. M., Bergeron, S. N. and Fry, W. E. (1992b). Clonal diversity and genetic differentiation of Phytophthora infesrans populations in northern and central Mexico. Phytopathology 82, 955-961. Goodwin, S. B., Saghai-Maroof, M. A., Allard, R. W. and Webster, R. K. (1993). Isozyme variation within and among populations of Rhynchosporium secalis in Europe, Australia and the United States. Mycological Research 97, 49-58. Goodwin, S. B., Cohen, B. A., Deahl, K. L. and Fry, W. E. (1994a). Migration from northern Mexico was the probable cause of recent genetic changes in populations of Phytophthora infestans in the United States and Canada. Phytopathology 84, 553-558. Goodwin, S. B., Cohen, B. A. and Fry, W. E. (1994b). Panglobal distribution of a single clonal lineage of the Irish potato famine fungus. Proceedings of the National Academy of Sciences, USA 91, 11591-11595. Goodwin, S. B., Schneider, R. E. and Fry, W. W. (1995a). Cellulose-acetate electrophoresis provides rapid identification of allozyme genotypes of Phytophthora infestans. Plant Disease 79, 1181-1185. Goodwin, S. B., Sujkowski, L. S., Dyer, A. T., Fry, B. A. and Fry, W. E. (1995b). Direct detection of gene flow and probable sexual reproduction of Phytophthora infestans in northern North America. Phytopathology 85, 473-479. Goodwin, S. B., Sujkowski, L. S. and Fry, W. E. (1995~).Rapid evolution of pathogenicity within clonal lineages of the potato late blight disease fungus. Phytopathology 85, 669-676. Gordon, T. R., Okamoto, D. and Milgroom, M. G. (1992). The structure and interrelationship of fungal populations in native and cultivated soils. Molecular Ecology 1, 241-249.
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A Molecular View Through the Looking Glass: the Pyrenopeziza brassicae-Brasska Interaction
A . M . ASHBY
Department of Plant Sciences. University of Cambridge. Downing Street. Cambridge CB2 3EA. UK
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I1 . The P. brassicae-Brassica Interaction ......................................... A . The Fungus ..................................................................... B . Pathogenesis .................................................................... C . Sexual Morphogenesis ....................................................... D . Disease Epidemiology .......................................................
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111. Molecular Techniques in the Analysis of the P . brassicae-Brassica Interaction .............................................................................
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I . Introduction
IV . Molecular Analysis of Pathogenesis ........................................... A . Surface Growth and Penetration: the Role of Cutinase .......... B . Subcuticular Growth: the Role of Protease ........................... C . Using Reporter Genes to Measure Fungal Biomass In Pfanra . . D . Proposed Role of Extracellular Protease in Pathogenicity ....... E . Implications for Disease Control .........................................
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V. Analysis of the Hemibiotrophic Phase: the Role of Cytokinins ....... A . Biochemical Analysis of Cytokinin Production by P . brassicae . B . Molecular Analysis of P . brassicae Cytokinins ....................... C . The Role of Cytokinins in Pathogenicity .............................. D . Implications for Disease Control .........................................
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VI . Analysis of Sexual Morphogenesis ............................................. A . Biochemical Analysis: Identification of a Post-Mating Factor ... B . Molecular Analysis ........................................................... C . Sexual Morphogenesis in P . brassicae: a Speculative Summary . D . Implications ....................................................................
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VII. A Molecular View through the Looking-glass: the P. brassicaeBrassica Interaction .................................................................
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VIII. Concluding Remarks ............................................................... Acknowledgements ................................................................. References ............................................................................
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I. INTRODUCTION Molecular biology has had a profound influence on all of the biological sciences. Over the last decade the use of molecular techniques in plant pathology has advanced to such a degree that we are now able to analyse complex plant-microbe interactions at the molecular level and to establish the role of certain genes and gene products in these interactions. This approach, encompassing biochemistry, molecular biology and conventional plant pathology, involves the dissection of an interaction into its component parts and investigates how the manipulation of these component parts may alter the interaction as “a whole”. This type of multidisciplinary approach will lead to great advances in our understanding of plant pathology in the decade to come. We are using such an approach to address three key questions concerning the Pyrenopeziza brassicae-Brassica interaction: 1. How does P. brassicae pathogenize Brassica species? 2. How does P. brassicae derive nutrients from host tissue during its biotrophic phase of growth? 3. What are the mechanisms controlling sexual morphogenesis in P. brassicae? By addressing and beginning to obtain answers to these questions through molecular analysis, a greater understanding of this fungus-host interaction will be realized, which will ultimately allow the development of novel strategies for controlling light leaf spot disease of brassicas. This review focuses initially on our understanding of the P. brassicae-Brassica interaction from a conventional plant pathological standpoint. Current progress on the use of molecular and biochemical techniques to analyse pathogenicity and sexual morphogenesis are then discussed and, finally, using the information gained from our molecular analyses together with a little speculation, a view of the interaction “through the molecular looking-glass’’ is proposed.
11. THE P. BRASSICAE-BRASSZCA INTERACTION A. THEFUNGUS
Pyrenopeziza brassicae Sutton and Rawlinson (anamorph Cylindrosporium concentricum) is a haploid, heterothallic discomycete and is the cause of light
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leaf spot disease of brassicas, which is considered one of the most damaging diseases of winter oilseed rape (Brassica napus L. subsp. ofeifera (Metzger) sink) in the UK (Lacey et al., 1987; Yarman and Giltrap, 1989; Hardwick et al., 1991). B . PATHOGENESIS
During pathogenesis the fungus displays a mode of nutrition characteristic of the hemibiotrophic plant pathogens within the Ascomycotina. After spore germination and initial infection (Fig. lA), a biotrophic phase of growth within the host is established. Here a relatively balanced physiological relationship exists between host and pathogen, and only limited visible signs of infection are evident (Courtice et a f . , 1988). These include thickening and stiffening of infected tissue and the formation of “green islands”, which result from the redirection of host nutrients to the site of infection leading to preferential retention of chlorophyll around the lesion as a consequence of growth substance imbalance (Maddock, 1979). During this early phase of development, the fungus obtains all its nutritional requirements from the plant, but little is known about how this is achieved. The first and most characteristic visible symptom of the disease is the formation of minute, snow-white spots erupting through the leaf surface. Each spot or conidiomata consists of numerous, unicellular, cylindrical spores, formed within the host in an acervulus beneath the cuticle (Fig. 1B,C). Sporulation ultimately results in rupture of the cuticle, with the lesion enlarging in a concentric fashion (Fig. 1D). The central chlorotic region becomes cracked and blistered, possibly as a result of toxin production by the fungus or simply as a result of separation of the cuticular layer from the upper epidermal membrane, rendering the lower surface susceptible to desiccation. In cases of severe infection the lesions will coalesce and infected areas may eventually wither and die. Asexual conidia are then dispersed to other susceptible hosts primarily by rain-splash, although atmospheric dispersal of conidia is possible (McCartney el al., 1986). C. SEXUAL MORPHOGENESIS
P. brussicae is heterothallic, having two mating types designated MAT 1-1 and MAT 1-2 (Mot et al., 1984). Reproduction occurs by two mutually exclusive pathways of development (Fig. 2). In the absence of a compatible mating type, spores germinate and differentiate by a process of enteroblastic conidiation to form asexual conidia (Fig. 1C). Conversely, in the presence of the opposite mating-type, an early interaction of opposite mating type hyphae results in suppression of asexual sporulation and initiation of a complex, co-ordinated, pathway of development culminating in the formation
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of apothecia (Fig. 3A-F). The fungus is competent to undergo sexual development during the first 6 days of conidial growth. After this period commitment to asexual sporulation is irreversibly established. Early interaction between opposite mating types is therefore required for successful induction of the sexual cycle, after which hyphae are destined to reproduce asexually (Illot, 1984; Siddiq, 1989; Ashby, unpublished observations). The production of fertile ascocarps in vitro was first described by Maddock and Ingram (1981), and subsequently the sexual system has been used to analyse fungicide-resistant, auxotrophic and pathogenicity mutants (Illot et al., 1987; Courtice et al., 1988; Ball et al., 1991a,b). The clear delineation of this state is now an important marker for a molecular analysis of sexual morphogenesis and pathogenicity in the fungus (Ball et al., 1991b; Ashby and Johnstone, 1994). The teleomorph of the fungus has been shown to occur naturally on oilseed rape in the UK and Ireland (Staunton and Kavanagh, 1966; Lacey et al., 1987; McCartney and Lacey, 1990) and is commonly found on vegetable brassicas in New Zealand (Cheah and Hartill, 1985). A survey of isolates of P . brassicae from 27 winter oilseed rape crops in East Anglia in 1987 revealed that both mating types were represented amongst the isolates from 74% of the fields tested, suggesting that there is a possibility that the teleomorphic stage may appear regularly (Ball et al., 1990). D. DISEASE EPIDEMIOLOGY
Oilseed rape is now a well-established and important “break crop”, and the increased acreage under cultivation may potentiaIly act as a source of wind-borne inoculum of light leaf spot disease of other brassica crops (Gladders, 1984). Most damage occurs after severe infection of new oilseed rape crops by P. brassicae in the autumn, although visible symptoms often go unnoticed until the following spring (Jeffery et al., 1989). Recent work by Figueroa et al. (1995) using a number of isolates of P. brassicae and a range of double-low (low erucic acid and low glucosinolate) cultivars of Brassica napus, demonstrated that low-temperature regimes increased both the incubation period (the time from inoculation until 50% of the lesions were produced) and the latent period (the time until the first lesion with
Fig. 1. Scanning electron micrographs illustrating the pathogenesis of P. brassicae. (A) Hyphae of P.brassicae JH26 (MAT 1-2) germinatingon a leaf surface and growing subcuticularly within B. napus (L. ssp. oleifera cv. Shogun). (B) An acervular conidioma rupturing the epidermis. (C) Enteroblastic conidiation (scale bar represents 10prn). (D) Typical symptoms induced in planta and signifying the late stages of infection; concentric rings of erumpant conidiornata surrounding a central necrotic lesion (scale bar represents 12mm). S. Batish, K . Johnstone and A . Ashby, unpublished observations.
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Fig. 2. Mutually exclusive pathways of development in P. brussicue and the proposed role of sex factor (SF) in controlling these pathways. Open and closed conidia depict different mating types of the fungus. The sex factor inhibits asexual sporulation, resulting in all available resources being diverted into fuelling sexual morphogenesis. From Ashby and Johnstone (1994).
acervuli appeared) of infection and also decreased the rate at which conidia were able to germinate. The low temperatures prevailing during the winter months may therefore account for the decrease in the rate of progression of epidemics of light leaf spot disease on oilseed rape. However, it is equally probable that the incidence and severity of the disease during the winter months of some years may be sufficiently low to go unnoticed (Figueroa et al., 1995). The significance of light leaf spot is such that substantial reductions in green leaf area and plant dry weight at flowering were observed when fungicide applications were delayed, resulting in a 46% loss of seed yield (Jeffery et al., 1994).
Fig. 3. Sexual morphogenesis in P. brussicue. (A) Scanning electron micrograph (SEM) showing the interaction between JH26 (MATl-2) and NHlO (MAT1-1) after 4 days (scale bar represents 100pm). T. Cole and A. Ashby, unpublished observations. (B) Mycelia of a MAT 1-1GUS isolate (NHlOpNOM102/18) interacting with wild type MAT 1-2 (JH26) mycelia after 4 days on CMM medium stained with X-Gluc (scale bar represents 1 mm). Reproduced from Ashby and Johnstone (1994). (C) SEM showing a mature fertile apothecium from a cross of JH26 and NHlO (scale bar represents 100pm). T. Cole and A. Ashby, unpublished observations. (D) Crushed apothecium from a cross of a GUS transformant of P. brassicae MAT 1-2 (JH26pNOM102/4) and a wild-type MAT 1-1 isolate (NH10). The apothecia were stained with X-Gluc to show the differential contribution made by each mating type to fruiting body formation (scale bar represents 25 pm). Reproduced from Ashby and Johnstone (1993). (E, F) SEMs showing magnification of the apothecium to reveal the ectal explicium (E), paraphyses (P) and asci (A) (scale bar represents 10 pm). T. Cole and A. Ashby, unpublished observations.
P YRENOPEZIZA BRASSICAE-BRASSICA INTERACTION
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38
A. M. ASHBY
Annual crop losses in the UK have recently been estimated at over 25 million (Thomas and Walker, 1994). The disease has greatest impact in oilseed rape crops sown in consecutive years (Figueroa et al., 1994), which suggests that inoculum may be carried over from one season to the next on infected rape stubble as well as volunteer plants, probably as a result of overwintering by the teleomorphic stage (McCartney and Lacey, 1990). It has also been suggested that the release of ascospores from apothecia may provide an important inoculum source later in the season, reducing the efficacy of fungicide application (Figueroa et al., 1994). The occurrence of wind-borne ascospores sheds new light on disease dissemination, and, in particular, the fact that P. brassicae has, by this route, the potential to spread beyond the immediate vicinity of oilseed rape into other brassica crops, thus providing an effective mechanism for continuing infection from season to season. Occurrence of the sexual stage provides the opportunity for enhagced genetic variability which may affect both pathogenicity and the development of tolerance to fungicides. Such factors are likely to become more predominant as oilseed rape is exploited by more intensive agricultural practice (Maddock and Ingram, 1981). A survey carried out by Maddock et al. (1981) revealed considerable heterogeneity of resistance between a range of brassica varieties and subspecies. This was the case both between cultivars within these groups and between individuals of a single cultivar. Although differential interactions between cultivars and isolates were shown, they were not sufficiently defined to establish clear physiological races of the pathogen. It has been suggested that as agricultural exploitation of the host proceeds, as a result of breeding for resistance, P. brassicae may face more selection pressure than ever before and that by monitoring changes in the population of P. brassicae it may be possible to follow the evolution of a host-pathogen relationship from a state of relatively balanced coexistence (Maddock et al., 1981). To this end, Simons and Skidmore were able to demonstrate that a differential interaction between several host genotypes of Brassica oleracea and P. brassicae isolates can occur and that resistance to P. brassicae appears, in most cases, to be expressed as a dominant character (Simons and Skidmore, 1988). A survey of host-pathogen interactions is required, allowing a differential set of host cultivars to be defined for the identification of key virulence factors in P. brassicae.
111. MOLECULAR TECHNIQUES IN THE ANALYSIS OF THE P. BRASSICA E-BRASSICA INTERACTION Over the last decade a range of molecular techniques has been developed for analysing the P. brassicae interaction (Table I; Skidmore et al., 1984; Ashby and Johnstone, 1994). The extraction of high-molecular-weight DNA is an essential prerequisite for the cloning of fungal genes. Using the method of Raeder and Broda (1985) up to 1pg DNA per milligram of fungal tissue
TABLE I Molecular techniques developed to analyse the P. brassicae-Brassica interaction Molecular technique Generation of mutants DNA extraction Fungal transformation Co-transformation Reporter genes GUS Luciferase Genomic library complementation cDNA library construction Differential display PCR AFLP analysis
Status in P. brassicae research Auxotrophic Pathogenicity Developmental 1 p g per mg dry weight 2-200 transformants per pg DNA 70% co-transformation with pAN7-1 Mitotically stable GUS transformants
4 luciferase vectors for expression in fungi have been constructed Auxotrophic mutants Pathogenicity mutants Developmental mutants Sex factor induced in preparation Casein induced Technique currently being developed for P. brassicae Analysis of P. brassicae population substructure
Reference Courtice and Ingram (1987) Ball et al. (1991b) Siddiq et nl. (1992) Ball et al. (1991a) Ball et al. (1991a) Ashby and Johnstone (1993)
0
2
5 !2 ta
a
R
Ashby and Johnstone (1993) 7 M. Chadwick and A. Ashby (unpublished observations) Ball et al. (1991a) Ball et al. (1991b) Ashby and Johnstone (1994) M. Robb (unpublished observations) A. J. Clark (unpublished observations) ga A. J. Clark (unpublished observations) @
z R
3 2
Majer et a1 (1995)
E
40
A. M. ASHBY
with a molecular weight >50 kDa is obtained (Ball et al., 1991a). There are several vectors available for fungal transformation, but those most suitable for this system are the PAN series of vectors (Punt et al., 1987). Both PAN 7-1 and PAN 7-2 are shuttle vectors which encode ampicillin resistance for expression in Escherichia coli and carry the hygromycin B-phosphotransferase gene flanked by the gpd promoter and trp C terminator sequences from Aspergillus for expression in the fungus. Since P. brassicae is highly sensitive to hygromycin (growth is inhibited at concentrations above 5 pg ml-’) these vectors were useful in establishing a transformation system for this fungus. A third vector, pNOM102 (Roberts et al., 1989) based on the PAN vectors but containing a P-glucuronidase gene under the control of a constitutive promoter, has been used as a reporter gene in P. brassicae (Ashby and Johnstone, 1993). A second reporter gene vector has been constructed based on “click beetle” luciferases (Chadwick and Ashby, unpublished observations). There are four luciferases, each emitting light at a different wavelength (Wood et al., 1989). Each of the four click beetle cDNAs was cloned into pAN52-1 (Punt et al., 1987) and they are currently being evaluated as reporter genes in P. brassicae and other fungi. Using such reporter genes in combination will enable both spatial and temporal expression of a number of fungal genes to be analysed at any one time. Fungal protoplasts can be prepared by a method based on that of Yelton et al. (1984), and are transformed using a modified method of Vollmer and Yanofsky (1986), as described by Ball et al. (1991a). Transformation frequencies in the region of 50 transformants per microgram of transforming DNA are routinely achieved. A genomic library has been constructed in pAN7-2 and used successfully in transformation experiments to complement auxotrophic mutants to restored prototrophy (Ball et al., 1991a), a protease positive pathogenic phenotype to a protease negative non-pathogenic m:itant (Ball et al., 1991b; see section IVB) and a sex factor responsive fertile phenotype to a sexual development mutant (Ashby and Johnstone, 1994; see section VIB). cDNA libraries have also been constructed (M. Robb and A. Clark, unpublished observations; Table I). Recently, the technique of amplified fragment length polymorphism (AFLP) analysis has been developed to study both P. brassicae population substructure and the evolution of a relationship between P. brassicae and B . napus (Majer et al., 1995).
IV. MOLECULAR ANALYSIS OF PATHOGENESIS With a view to understanding the complexities of plant-microbe interactions, the “molecular dialogue” between pathogens and their hosts has been intensively studied over the last decade. For biotrophic and hemibiotrophic plant pathogens, a number of criteria have to be met to establish successful pathogenesis. Pathogenicity factors must be expressed to enable penetration of host tissue by mechanical or enzymatic degradation and to allow the
PYRENOPEZIZA BRASSICAE-BRASSICA INTERACTION
41
ingressing pathogen to access nutrients from the host without initiating any visible disease symptoms. The successful biotrophic pathogen must also resist activating host defence mechanisms which may result in a hypersensitive reaction (HR), thereby culminating in localized cell death and the consequent localization of the pathogen. Since P. brassicae lacks a clear race structure and no differential interaction with the host is evident, the fungus does not appear to elicit an H R response. The fungus does, however, appear enzymatically to degrade the cuticle prior to ingress (Maddock, 1979), and recently, and in view of the controversy surrounding the role of cutinase in the pathogenicity of Fusarium solani f. sp. pisi on pea (Stahl and Schifer, 1992; Rogers et al., 1994), we have begun to analyse the role of an extracellular cutinase in penetration of B. napus by P. brassicae. The importance of cell-wall-degrading enzymes in pathogenesis is well documented (Cooper, 1984). An initial strategy to look for such determinants in the P. brassicae interaction involved screening UV mutants of P. brassicae for deficiencies in cell-wall-degrading enzyme production. Mutants that were deficient in endopolygalacturonase (EndoPG), pectin methyl esterase (PME) and protease production in vitro were also found to be non-pathogenic in detached cotyledon tests (Ball et al., 1991b). Our research has focused primarily on the role of the protease in pathogenicity (see sections IVB and IVD).
A . SURFACE GROWTH AND PENETRATION: THE ROLE OF CUTINASE
Suspensions of conidia of P. brassicae artificially inoculated onto a leaf surface tend to be deposited preferentially over the anticlinal walls of epidermal cells (Rawlinson et al. , 1978). Upon germination conidia become septate forming relatively short germ tubes, which may swell slightly at the apex prior to penetration. Appressoria are never formed and hyphae d o not enter through stomata but directly penetrate through the cuticle. From microscopical analysis showing sites of pathogen ingress it appears that enzymatic degradation of the cuticle may be important in facilitating penetration (Maddock, 1979). P. brassicae has been shown to produce methyl esterase activity (Ball, 1989) and quantification of enzyme production was obtained by hydrolysis of p-nitrophenylbutyrate. Degradation of tritiated cutin confirmed that this activity was consistent with that of a cutinase (K. Davies, unpublished observations). The cutinase was also inhibited by PMSF, and ebelactones A and B (A. Jones and K. Davies, unpublished observations). Identification of the cutinase gene(s) by heterologous probing and polymerase chain reaction (PCR) is currently ongoing within the laboratory and our long-term aim is to perform gene knockout experiments on the P. brassicaecutinase(s) to assess the role played by cutinase in fungal penetration of brassica species.
42
A . M. ASHBY B. SUBCUTICULAR GROWTH: THE ROLE OF PROTEASE
Upon penetration, the fungus forms a hypomycelium of long thin septate infection hyphae which extend through the subcuticular space between the cuticle and walls of the epidermal cells (Rawlinson et a f . , 1978; Maddock, 1979). The hyphae then branch and proliferate below the cuticle to form a mycelial plate, beneath which hyphae begin to penetrate into leaf tissue along anticlinal walls and between cells of the upper mesophyll, but never through into cell lumina (Rawlinson et al., 1978; Maddock, 1979). The mechanisms by which the fungus is able to occupy space beneath the cuticle and to derive nutrients from within the plant are still unknown, and are therefore of great scientific interest. A UV mutant, designated NHlO 247, was found to be deficient in its ability to produce an extracellular protease and was also found to be nonpathogenic in a cotyledon-based pathogenicity test (Ball et al., 1991b, 1992). When crossed with the opposite wild type mating type, both protease and pathogenicity phenotypes co-segregated, suggesting that protease is a determinant of pathogenicity or that both protease and pathogenicity genes are closely linked. The resulting protease minus pathogenicity minus progeny from the cross (NH10 247:JH26; Ball et al., 1991b) were functionally complemented by a single integration event from a bulk library transformation (Fig. 4) and cloned sequences resulting in the acquired phenotype were recovered by the technique of cosmid rescue (Perucho et al., 1980). Approximately 4.5 kb of genomic DNA which flanked the original cosmid insert in NHlO 247:JH26 T was rescued in pAN7-2 and designated pPROTl (Fig. 5A). PCR using a primer reverse translated from the N-terminal amino acid sequence and a primer flanking the 4.5 kb insert in pPROTl gave a unique product (Fig. 5B,C), suggesting that the N-terminus of protease was located on the 4.5 kb fragment from pPROT1. Sequencing identified the unique ClaI site; however, analysis of open-reading frames (ORFs) on either side revealed no ORF with significant sequence homology to proteases. When pPROTl was transformed into P. brassicae, 40% of the resulting transformants displayed a loss of protease and pathogenicity phenotypes. Fig. 4. Complementation of the protease minus pathogenicity minus mutant by bulk cosmid library transformation. (A) Proteolytic transformant recovered from transformation of P. brussicue NH10247:JH26 (MAT 1-2) with a bulk cosmid library. A cleared zone of enzyme activity is evident around the complemented transformant (arrowed) (scale bar represents 2 cm). (B) Southern blot of NH10247:JH26T (MAT 1-2). Genomic DNA restricted with ClaI (which does not cut within the vector pAN7-2, track 1) and restricted with EcoRV (which linearizes pAN7-2, track 2) was probed with radiolabelled pAN7-2. (C) Symptoms induced by the protease transformant NH10247:JH26T (MAT 1-2) in a detached cotyledon test. Small white conidiomata are visible on the cotyledon (scale bar represents 2 mm). Reproduced with permission from Ball et al. (1991b).
PYRENOPEZIZA BRASSICAE-BRASSICA INTERACTION
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44
A.
M. ASHBY
Barn HI
Fig. 5. Molecular analysis of the functionally complemented transformant, NH10247:JH26T which displayed a restored protease and pathogenicity phenotype. (A) pAN7-2 with 4.5 kb of rescued flanking sequence extending to the first ClaI site on either side of the original insert in NH10247:JH26T and designated pPROTl. (B) The unique product derived from a PCR reaction using the N-terminal protease primer and a primer flanking the 4.5kb insert in pPROTl. A. Ashby, unpublished observations.
PYRENOPEZIZA BRASSICAE-BRASSICA INTERACTION
45
Similarly, all subcloned fragments were able to elicit some effect on protease production by the fungus. It is possible that the pPROTl fragments rescued from the transformant contain regulatory sequences, rather than encoding the protease structural gene. Fragments from either side of the ClaI site are currently being used to chromosome walk through the genomic library of P. brussicue and 10 putative positive genomic clones are currently being analysed (A. Hunter, A. Clark and A. Ashby, unpublished observations). The extracellular protease was purified by FPLC separation and a biochemical profile of the protease obtained as well as sequence from its N-terminus (Batish, 1992). Biochemical analysis of the purified protease showed a pH optimum of 8.0, temperature optimum of 40°C and 100% inhibition with HgC12 confirming the findings of Ball et ul. (1991b) using a crude preparation. However, further biochemical analysis and information from the N-terminal amino acid sequence suggests that the protease is likely to belong to the serine family of proteases rather than cysteine (S. Batish, A. Clark, A. Hunter, A. Ashby and K. Johnstone, unpublished observations). A parallel approach has involved the construction of a cDNA library to casein-induced mycelium. The primer derived from the N-terminus was used in a PCR reaction against messenger RNA from induced mycelium to generate a unique RT-PCR product as a probe for library screening. cDNA clones are currently being analysed (A. Clark and A. Hunter, unpublished observations). C . USING REPORTER GENES TO MEASURE FUNGAL BIOMASS IN PLANTA
Reporter genes such as P-galactosidase, P-glucuronidase (GUS) and luciferase are widely used in plant and bacterial systems, but are relatively new tools for use in fungi. The enzymes encoded by the reporter genes hydrolyse specific chromogenic and fluorogenic substrates, liberating a specific colour or fluorescence indicative of reporter gene activity. Co-transformation of P. brussicae isolates NHlO (MAT 1-1)and JH26 (MAT 1-2) with pAN7-1 (for hygromycin selection; Punt et ul., 1987) and the constitutively expressing GUS vector pNOM102 (Roberts et ul., 1989) resulted in a range of transformants of both mating types expressing GUS activity (Ashby and Johnstone, 1993). One transformant, designated JH26pNOM102/13, was also found to be non-pathogenic and deficient in its ability to produce extracellular protease in vitro. In pluntu studies using the GUS expressing, protease minus, pathogenicity minus mutant and a wild-type GUS expressing isolate revealed that, although penetration and growth of infection hyphae were achieved in both cases, the mutant was unable to build up sufficient biomass to elicit the formation of acervular conidiomata and, therefore, visual symptoms of disease progression (Fig. 6).
C
0
2
4
6
8
10
12
Days after inoarlaton
Fig. 6. Thin sections through detached cotyledons of B . napus L. ssp. oleifera cv. Shogun showing the extent of fungal biomass after 15 days. (A) A MAT1-1 GUS expressing wild-type P. brassicae (NH10 pNOM102/18) (scale bar represents 150 Wm). (B) The MAT 1-2 protease minus pathogenicity minus GUS expressing isolate (JH26 pNOM102/13) (scale bar represents 150 pm). (C) Quantification of fungal biomass from NHlO pNOM 102/18 (MAT 1-1) and JH26 pNOM102/13 (MAT 1-2) using GUS expression mutant JH26 pNOM102/13. S. Batish, K . Johnstone and as a direct measurement: (0)wild-type NHlO pNOM102/18; (.)protease A. Ashby, unpublished observations.
14
16
PYRENOPEZIZA BRASSICAE-BRASSICA INTERACTION
47
D. PROPOSED ROLE OF EXTRACELLULAR PROTEASE IN PATHOGENICITY
From our findings one might expect that a key role for protease is the degradation of matrix glycoproteins which decrease the association between cells of the epidermis, facilitating growth of fungal mycelium both subcuticularly and, eventually, between the cells of the mesophyll. In support of this hypothesis, we have recently identified a brassica glycoprotein that is preferentially degraded by the protease ( A . Clark, A. Hunter, A . Ashby and K. Johnstone, unpublished observations). Secondly, localized degradation may result in release of nutrients from the apoplast, thus allowing the fungus to establish sufficient biomass prior to sporulation. There are other possible roles for a protease, however, including degradation of host cell matrices during the necrotrophic and saprophytic phase of growth and the degradation of host pathogenesis-related proteins. E.
IMPLICATIONS FOR DISEASE CONTROL
Establishing that cutinase is required for penetration of the cuticle of brassicas may allow the formulation of a number of strategies to effect disease control. These may include the spraying of ebelactones or other cutinase inhibitors on brassica crops at the beginning of the growing season or the use of genetically manipulated brassicas which express a cutinase inhibitor constitutively throughout their developmental cycle. Equally, cultivars with thicker cuticles could be generated transgenically or by conventional plant-breeding methods. Subcuticular growth of the fungus may be limited by expression of protease inhibitors in plants. Equally, once the natural substrate of the protease is established, subtle modification may prevent proteolytic cleavage, thereby reducing the space available for fungal growth in pluntu. Both strategies might be easily implemented through brassica transformation.
V. ANALYSIS OF THE HEMIBIOTROPHIC PHASE: THE ROLE OF CYTOKININS One of the major gaps in our current knowledge of plant pathology is an understanding of the physiological mechanisms of biotrophy and hemibiotrophy in fungal plant pathogens (Brian, 1967). In particular, several key questions remain unanswered, namely: how this delicate state of co-existence is maintained, what differentiates it from the necrotrophic mode of nutrition and, in a broader context, how these interactions relate to host specificity and disease resistance. There is substantial evidence that plant-growth regulators play a role in bacterial and fungal diseases of plants, including abnormal plant growth as
48
A . M. ASHBY
a result of infection, for example in formation of galls, hypertrophies, stem elongation and premature senescence. In addition, it has been established that many plant pathogenic micro-organisms are able to synthesize plantgrowth regulators including auxins and cytokinins (Greene, 1980). Cytokinins are N-6 substituted derivatives of adenine and in healthy plants are synthesized in the roots and transported to other regions of the plant through the xylem. They are responsible for stimulating metabolism and transportation of nutrients concomitant with a general stimulation of metabolic activity and cell division. In pathogenic interactions where hormonal imbalance does not result in prolific cell division in the host, but where the host is metabolically responsive to changes in endogenous levels of growth regulators, fungal cytokinins could function as key determinants of pathogenicity by increasing host metabolism, diverting nutrients and retaining chlorophyll to establish localized “metabolic sinks”, thus reducing the expression of senescence symptoms (Elstner, 1983). Fungal cytokinins may by themselves, or through the activation of enzymes such as superoxide dismutase, catalase and peroxidase, defend against the deleterious effects of free radicals, scavenging these highly active oxygen species, and therefore limit the processes which collectively contribute to the HR (Elstner, 1983; Beckman, 1990). A classic symptom of biotrophic rust infections is the presence of “green islands” around the point of infection which have been attributed to an imbalance in plant-growth regulators, caused directly or indirectly by the pathogen. In addition, aqueous extracts have been taken from spores of several fungi and have been shown to induce “green island” effects, suggesting that cytokinin-like molecules can be produced by both host and pathogen (Sequeira, 1973; Moore, 1979; Skoog, 1980). In the P. brassicae-Brussica interaction, “green islands” are often observed during the early stages of infection, indicative of plant-growth regulator imbalance (Maddock, 1979). This effect may be a result of either overproduction of cytokinins by the host in response to pathogen ingress, or synthesis of fungal cytokinins. There are a number of disparate reports in the literature on the ability of plant pathogenic fungi and mycorrhiza to synthesize cytokinins in vitro (Laloue and Hall, 1973; Miura and Hall, 1973; Mills and Van Staden, 1978; Kraigher et al., 1991), but definitive evidence for a role for fungalderived cytokinins in these interactions is yet to be established. Since both plant and pathogen may be capable of synthesizing plant-growth regulators and can influence each other’s ability to produce plant-growth regulators, it is experimentally difficult to identify the contribution of each partner to this phenomenon. In the cases of Agrobacterium turnefuciens and Pseudomonas savastanoi there is direct molecular evidence that bacterial genes are required for the synthesis of plant-growth regulators, and mutation of these genes either causes loss of or a change in disease symptoms (Nester and Gordon, 1991; Surico and Iacobellis, 1992). In order for the role of pathogen-derived plant-growth regulators to be
PYRENOPEZIZA BRASSICAE-BRASSICA INTERACTION
49
definitively established in a plant-microbe interaction, it is essential to produce direct molecular evidence. This is best provided by insertional inactivation of gene(s) essential for biosynthesis of the pathogen-derived plant-growth regulators and comparing disease symptoms of mutants with near-isogenic counterparts. To date, no such molecular evidence has been produced for a fungal plant pathogen. One of the major difficulties associated with the analyses of most obligate biotrophs is their dependence on the host for growth and development. Conveniently, P. brussicue is a hemibiotroph and can be cultured in vitro on nutrient medium. We are therefore well placed to test the hypothesis that cytokinins of fungal origin are responsible for maintaining the physiological balance between host and pathogen during the biotrophic phase of growth. This hypothesis draws together the ideas of early workers (Brian, 1967; Lewis, 1973) who, frustrated by the complexity of the interactions displayed by obligate parasites, were unable to investigate this phenomenon further. This source of control over plant metabolism maintained by the pathogen, if a widespread phenomenon, may be a fundamental determinant of biotrophy in plant-pathogen interactions and may be a significant contributing factor towards host specificity and disease resistance. A.
BIOCHEMICAL ANALYSIS OF CYTOKININ PRODUCTION BY P. BRASSICAE
Initial studies have shown that cytokinins are synthesized and exuded by P. brussicae during the early stages of its development, and cytokinin presence can be qualitatively visualized using a detached barley cotyledon assay (Fig. 7). A high-performance liquid chromatography (HPLC) enzyme-linked immunosorbent assay (ELISA) for cytokinins has been established (Huntley , 1995) and has been used both to analyse levels of cytokinins from aqueous spore washes of P. brassicae and to quantify the levels of cytokinins found in extracts of mycelium and culture filtrate from. P. brassicae grown under a range of different culture conditions. Cytokinins with cross-reactivity to zeatin riboside (ZR) antibodies were identified in spore washes from P. brussicae, and when grown under different culture conditions the fungus produced differing levels of predominantly ZR cross-reactive cytokinins, with most ZR being produced under conditions comparable to those experienced by the fungus in planta (Table 11; A. Murphy, K . Johnstone and A. Ashby, unpublished observations). B. MOLECULAR ANALYSIS OF P. BRASSICAE CYTOKININS
From comparison of deduced amino acid sequence homologies between the p t z gene from Pseudomonas savastanoi and the ipt and tzs genes from A .
50
A . M. ASHBY
Fig. 7. Detached barley leaf assay. “Green island” inducing activity from 5 PI of aqueous spore extracts from: (A) P. brassicae JH26 (MAT 1-2); (B) P. brassicae NHlO (MAT 1-1); (C) Venturin inaeqclalis (positive control); (D) Erysiphe graminis (positive control); (E, F) Water (control), spotted onto detached barley leaves. 5/11 of concentrated extract was equivalent to approximately lo5 spores (A. Murphy, K. Johnstone and A. Ashby, unpublished observations). (Scale bar represents 1 cm.)
TABLE 11 Cytokinin production (pmol gfwt-’) by P. brassicae isolate JH26 (MAT 1-2) Culture filtrate Medium Malt extract Murshige and Skoog
Mycelium
z type
IPA type
z type
IPA type
9.6 13.4
ND 19.4
1.8 118.0
Z, zeatin; IPA, isopentyl adenosine; ND, none detected.
tumefuciens (Powell and Morris, 1986), two degenerate oligonucleotide primers from regions of these sequences showing high homology and which flanked a central 400 bp region of these genes were generated. The two oligonucleotides were used as primers in PCR reactions using P. brussicue genomic DNA as template. In the control PCR reaction with pGV0201 (De Vos et ul., 1981; Engler
PYRENOPEZIZA BRASSICAE-BRASSICA INTERACTION
51
et ul., 1981) containing the tmr locus from the T-DNA of A . tumefuciens and harbouring the ipt gene, a PCR product of 400 bp was produced, as expected. PCR against fungal DNA revealed two unique PCR products. Both products were sequenced but failed to show significant sequence homology with the bacterial genes. However, they are currently being used to perform gene knockout experiments by transformation (A. Murphy, K. Johnstone and A. Ashby, unpublished observations). To analyse further the effects of cytokinin imbalance on pathogenicity, a fungal expression vector allowing constitutive expression of the ipt gene from A . tumefuciens in P. brussicue has been constructed and transformants overproducing cytokinins are being screened in pathogenicity tests (A. Murphy, K. Johnstone and A. Ashby, unpublished observations). C. THE ROLE OF CYTOKININS IN PATHOGENICITY
We have demonstrated that P. brussicue synthesizes both Z R and IPA cross-reactive cytokinins in vitro and research is underway to clone cytokinin biosynthetic genes. It is proposed that fungal cytokinin production enables re-direction of nutrients to sites of pathogen ingress, increasing the availability of nutrients for pathogen consumption. We are now able to test if cytokinins of fungal origin are produced in plunta and if gene knockout of fungal cytokinin biosynthetic genes affects the ability of P. brussicue to infect brassica species. The molecular dissection of cytokinin biosynthesis in fungal biotrophic and hemibiotrophic plant pathogens may highlight a possible global importance of fungal-derived cytokinins in facilitating obligate parasitic interactions and the mutualistic mycorrhizal associations. D. IMPLICATIONS FOR DISEASE CONTROL
At present this research is still in its infancy and a more detailed analysis of fungal cytokinin expression both in vitro and in plunta is required. However, should we discover in the future that fungal-derived cytokinins are regulated and synthesized via an alternative biosynthetic pathway or by different enzymes to those of plant-derived cytokinins, one could envisage the development of a targeted approach for disease control whereby biosynthesis of the fungal cytokinin was preferentially inhibited. This would prevent re-direction of nutrients to the site of pathogen ingress and therefore reduce the available nutrients for establishment of fungal biomass.
VI. ANALYSIS OF SEXUAL MORPHOGENESIS Understanding development is currently one of the major challenges throughout biology. Fungal development is relatively simple, with hyphal
52
A. M. ASHBY
Fig. 8. Parallel approaches adopted for the analysis of fungal sexual morphogenesis involving both biochemical and molecular analyses. (Reproduced from Ashby and Johnstone, 1994.)
tubes differentiating into asexual, sexual, resting or specialized structures, depending on the perceived environmental stimulus. Physiological studies have revealed that chemical sex factors have important regulatory roles in sexual development in fungi (Machlis, 1966), with evidence available that hormones are likely to trigger a switch between asexual and sexual reproduction (Ashby and Dyer, 1992; Dyer et al., 1992). Molecular studies have focused primarily on the cloning and characterization of mating type idiomorphs (Giasson et al., 1989; Kronstad and Leong, 1989; Glass et al., 1990; Mutasa et al., 1990; Staben and Yanofsky, 1990; Froelinger and Leong, 1991; Piccard et al., 1991; Bolker et al., 1992); however, cloning of mating type loci from plant pathogenic ascomycetes is still in its infancy, with Cochliobolus heterostrophus being the only example other than P. brassicae (Turgeon et al., 1993). We have adopted a two-pronged approach to the analysis of sexual morphogenesis in P. brassicae, involving a biochemical analysis of pre- and post-mating factors and a molecular approach involving the complementation of mutants, cloning and characterization of the mating-type loci, analysis of protein profile changes concurrent with sexual morphogenesis and the use of reporter genes to study the relative contributions made by each mating type to sexual morphogenesis (Fig. 8). A. BIOCHEMICAL ANALYSIS: IDENTIFICATION OF A POST-MATING FACTOR
The biochemical analysis of sexual morphogenesis in P. brassicae has focused on the identification, characterization and partial purification of a lipoidal sex factor termed SF (Illot et al., 1986; Siddiq et al., 1989, 1992). Preliminary analysis demonstrated that crude SF was lipophilic, with the active component having a molecular weight of less than 5000 kDa and was present as a minor proportion of the crude extract (Siddiq, 1989; Siddiq et al., 1989).
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SF behaves as a sex morphogen and analysis of the physiological mechanisms involved with fruit-body formation has revealed that SF, which is extracted from mated cultures only, has the following effects on controlling development of P. brassicae (Fig. 9A,B; Siddiq, 1989; Siddiq et al., 1989, 1992): (i) when added to single mating-type isolates of the fungus, SF inhibits asexual development and stimulates immature ascocarp formation; and (ii) when added to mated cultures, SF stimulates an increase in the rate of formation and number of mature ascocarps. Methanolic extracts from single mating-type extractions had no effect on sexual morphogenesis. Partial purification of SF by HPLC (Fig. 9C) and thin layer chromatography (TLC) indicates that the active component is likely to be a modified hydroxy fatty acid (A. Ashby, S. Ertz and K. Johnstone, unpublished observations). SF therefore acts as a morphological trigger molecule, suppressing asexual sporulation and stimulating ascocarp development (see Fig. 2). SF functions over a defined time window of between 3 and 6 days post-incubation with spores of P. brassicae (Siddiq, 1989). Removal of cultures from the influence of SF before 3 days or addition of SF to 7-day-old mycelium results in normal levels of asexual sporulation with no stimulation of immature ascocarp development. Similarly, reciprocal spermatization experiments where mycelium of one mating type was inoculated with spores of the opposite mating type, and vice versa, revealed that P. brassicae was able to undergo sexual morphogenesis providing that both mating types were present and that germination of individual mating-type isolates had not been allowed to proceed further than approximately 7 days prior to mixing (A. Ashby, unpublished observations). Illot (1984) showed that an early interaction of opposite mating types was a prerequisite for sexual morphogenesis and that ascocarps did not result when mycelial blocks of opposite mating type were allowed to grow towards one another and interact in the centre of a petri dish. It appears from this information that P. brassicae is able to initiate sexual development only within a defined time window of between 3 and 7 days post-germination. Within this phase, the fungus appears to be highly perceptive to environmental stimuli such as the presence of its opposite mating type or the presence of sex factor. Such tight control over sexual morphogenesis may be of significance to the life cycle of the fungus in planta. SF (both partially purified and crude) has been used as a morphological trigger for the molecular analysis of sexual morphogenesis in P. brassicae (Ashby and Johnstone, 1994).
B. MOLECULAR ANALYSIS
I . Complementation of developmental mutants A range of sexuality mutants was generated by ultraviolet (UV) mutagenesis and partially characterized by Siddiq (Siddiq et al., 1989, 1992). One such
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Fig. 9. Bioassay and purification of SF. (A) Numbers of conidia produced by single-mating-type isolate JH26 (MAT 1-2) of P. brassicue after 15 days’ incubation on 3% MA supplemented with different concentrations of SF. (B) Total numbers of mature apothecia (solid columns) and numbers of mature apothecia per cm2 (hatched columns) produced in mated cultures of P. brassicue (NHIO x JH26) after 30 days’ incubation on 3% MA supplemented with different concentrations of SF. Each column is based on nine replicate counts of three individual experiments. The bars represent standard errors of the means. (Reproduced with permission from Siddiq et ul. (1992).) (C) Results of a bioassay of sequential 1 -min fractions of crude SF separated by normal phase HPLC. (Reproduced from Ashby and Johnstone, 1994.)
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Fig. 10. Complementation of the P. brassicae developmental mutant JH26 973 with a wild-type genomic library which resulted in transformant JH26 973(T) with restored fertility and responsiveness to SF. (A) Light micrograph of JH26 973 (MAT 1-2) crossed with wild-type NHlO (MAT 1-1) after 14 days on CMM medium in which development of asexual spores, but not apothecia, has occurred (scale bar represents 5 mm). (B) Light micrograph of JH26 973(T) crossed with wild-type NHlO after 14 days on CMM medium in which very little asexual sporulation and abundant fertile apothecia are evident (scale bar represents Smm). (C) Southern blot of sex transformant JH26 973(T). Genomic DNA restricted with ClaI (which does not cut within the vector pAN7-2, track 1) and restricted with Sac1 and NcoI (which linearize pAN7-2, tracks 2 and 3) was probed with radiolabelled pAN7-2. (Reproduced from Ashby and Johnstone, 1994.)
mutant, designated JH26 973, was unable to respond to SF and was infertile when crossed with a wild-type isolate of compatible mating type. This mutation was fully complemented by genomic library transformation and the resulting transformant designated JH26 973(T) (Fig. 10A,B).Southern hybridization of genomic DNA from the transformant revealed that functional complementation resulted from a single integration event (Fig. 1OC). Cosmid rescue is currently being performed in order to rescue flanking DNA of interest. One may propose that such flanking DNA will contain gene(s) involved in
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“upstream” sexual development and may well encode for the SF receptor, an SF transport protein or even gene products at the MAT 1-2 locus itself.
2. Cloning of the mating-type loci Mating-type loci in lower eukaryotes appear to be “master regulatory switches” controlling pathways of cell specialization and sexual morphogenesis. Using heterologous probing we have identified the MAT 1-2 locus of P. brassicae which is the first identification of a MAT locus from a discomycete member of the Ascomycotina. A southern blot of Hind I11 digested genomic DNA from a MAT 1-2 isolate was probed at relatively high stringency with the MAT A probe from Neurospora crassa and a single hybridizing band at 3 kb was observed, with no cross-hybridization of the probe to MAT 1-1genomic DNA (Fig. 11; P. Venkatasurbramanian and A. Ashby, unpublished observations). The 3 kb fragment was subcloned into pBluescript and sequence analysis is currently ongoing. Flanking sequences surrounding the MAT 1-2 locus will be used to identify the MAT 1-1 locus. Once unique MAT sequences are available, it will be possible to generate primers for a PCR-based diagnostic kit, allowing plant pathologists to identify and assign mating type to P. brassicae isolates harvested from infected leaves very early in the disease cycle. It will therefore be possible to inform farmers of potential epidemics of light leaf spot well in advance, which will enable them to administer a suitable fungicide regime. This is currently difficult as there are no visible signs of infection in the early stages of the disease, and identification of the fungus involves a laborious culturing procedure that can take up to a month to complete. PCR offers the information within a single day and will therefore reduce the need to spray crops with fungicides as a precautionary measure. Unique primers specific for the P. brassicae mating types will also eliminate the difficulties often associated with distinguishing between ascospores of P. brassicae and the morphologically similar saprophytic fungus, Unguicularia cfr. raripila (Inman et al., 1992). Mating-type loci and pathogenicity. In the badisiomycete genus Ustilago, whose members cause smut disease of cereals, there appears to be an association between mating type and pathogenicity. In U. maydis, a pathogen of corn, the mating-type genes consist of two idiomorphs at the “a” locus and at least 25 alleles at an unlinked “b” locus. It is the “b” locus that determines both pathogenicity of the dikaryon and sexual morphogenesis in planta. In U. hordei, a pathogen of barley, the mating system is bipolar. Successful mating results in the formation of an infectious dikaryon which is able to complete its sexual cycle in planta. Therefore in this basidiomycete pathogen, a single locus controls both pathogenicity and sexual morphogenesis (Thomas, 1988). At present there are no reports showing an association between mating type and pathogenicity in ascomycete plant pathogens. Until recently, however, MAT loci had not been cloned from
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Fig. 11. Southern blot of wild-type P. brussicae JH26 (MAT 1-2) showing hybridization with a 3 kb fragment of genomic DNA. Genomic DNA was restricted with Hind I11 and probed with a digoxygenin (DIG) labelled 5.5 kb NdeI/NsiI fragment from p2A4, containing the MAT A locus from Neurospora crassa (kindly provided by Dr B. Metzenberg). Track M, lambda Hind I11 markers (DIG labelled); track 1, restricted genomic DNA of MAT 1-2 (JH26); track 2, restricted genomic DNA of MAT 1-1 (NH10). Arrow highlights the 3 kb hybridizing band. (P. Venkatasurbramanian and A. Ashby, unpublished observations.)
plant pathogenic ascomycetes, and therefore there has been no realistic way of specifically mutating MAT genes so that this question could be addressed directly. Although the sexual cycle in P . brussicae is not a prerequisite for pathogenicity and no evidence for the involvement of the MAT genes in pathogenicity is available, we will now be able to test this directly. 3. Protein profile analysis of developmentally regulated genes As part of a programme of research focusing on the molecular analysis of sexual morphogenesis, SF has been used as a morphological trigger and resulting changes in protein profiles have been analysed. Protein profile analysis of single mating-type isolates of P. brussicae both alone and induced with SF, as well as profiles of mature fertile ascocarps from crosses revealed the presence of a major protein in SF-induced single mating-type isolate extracts and in extracts from mature ascocarps. This protein is designated sex factor-induced (SFI1) protein (Fig. 12A). The
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protein has a molecular weight of 35 kDa, a pl of 7.3 and has hydrophobic characteristics. Data base searches on internal amino acid sequences from the purified SFIl protein revealed no homology with the hydrophobins, but did reveal partial homology with a number of membrane-bound proteins, suggesting that SFIl may be membrane associated (A. Ashby and M. Robb, unpublished observations). SFIl was found in extracts from SF-induced mycelium of a range of field isolates of both mating types of P. brassicae, suggesting that the protein is neither isolate nor mating type specific. SFIl separated as a single peak on FPLC hydrophobic interaction chromatography (Fig. 12B) and internal amino acid sequence data from the purified protein was obtained. Reverse translation allowed oligonucleotides to be generated for PCR, and a unique PCR product of 700 bp was generated. Southern blotting of both MAT 1-1 and MAT 1-2 genomic DNA using the 700 bp PCR probe revealed a different banding pattern between mating-type isolates, but a consistent banding pattern within isolates of the same mating type, suggesting close linkage with the MAT idiomorphs. This is currently being tested by the analysis of progeny from sexual crosses in which co-segregation of hybridizing band pattern and mating type will be analysed. 4. Use of GUS as a tool to study sexual morphogenesis The reporter gene GUS was used to study developmental interactions (Ashby and Johnstone, 1993) with a view to using the technique to study the spatial and temporal expression of developmentally regulated genes during apothecia1 formation in P. brassicae. Both MAT 1-1 and MAT 1-2 isolates were transformed with pNOM102 and reciprocal crosses performed on GUS MAT 1-1 x wt MAT 1-2 and GUS MAT 1-2 x wt MAT 1-1. An analysis of the contribution made by each mating type to fruiting body development was assessed and preliminary results suggested that each mating type differentially contributes to apothecial morphology (Ashby and Johnstone, 1993). The results, in parallel with SEM work, showed that an early interaction after germination of opposite mating-type mycelium is a prerequisite for sexual morphogenesis (see Fig. 3). C. SEXUAL MORPHOGENESIS IN P. BRASSZCAE: A SPECULATIVE SUMMARY
Interaction between newly germinating hyphae is a prerequisite for sexual morphogenesis, with attraction of opposite mating-type hyphae towards one another clearly evident. This implies a role for some hitherto unidentified sex factor in pre-mating. Such a factor may be proteinaceous, and pheromone like in its mode of action. Anastomosis of germinating hyphae involves recognition of "self" and "non-self" with hyphae of opposite mating type, forming dikaryotic hyphae which generate into pseudoapothecia. The post-mating lipoidal SF is produced once sexual development is initiated and
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Fig. 12. Identification and purification of SFIl. (A) Sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis (PAGE) of total soluble protein from asexually reproducing culture of P. brassicue JH26 (MAT 1-2) after 15 days incubation (track l), JH26 in the presence of 1.25 mgml-' SF after 15 days incubation (track 2), and individually picked mature fertile apothecia (track 3). Track M corresponds to low-molecular-weight PHAST protein markers (Pharmacia). The arrow highlights the SFIl protein. (B) FPLC profiles of total soluble protein from asexually reproducing cultures of P. brussicae JH26 and JH26 in the presence of 1.25 mg ml-' SF after 15 days' incubation. The large peak from SF-induced extracts corresponds to SFIl. (A. Ashby and M. Robb, unpublished observations.)
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stimulates apothecial development at the expense of asexual morphogenesis. At present it is unclear at what stage of development karyogamy and meiosis or “true sex” occurs. The gene(s) at the mating-type locus clearly will play a role in downstream processes that culminate in apothecial development. These will involve the regulation of downstream structural genes such as SFZl and genes involved in the SF biosynthetic pathway. Ultimately, the signal transduction pathways triggered in response to SF will be elucidated and a greater understanding of the cellular processes that facilitate apothecial development obtained. D. IMPLICATIONS
A greater understanding of the processes controlling sexual development in plant pathogenic fungi will ultimately lead to new strategies for disease control. Once SF is purified and a structure obtained, analogues can be generated and screened for their ability to inhibit asexual reproduction while failing to stimulate ascocarp formation, thus preventing both reproduction and dissemination of P. brassicae. Information gained from insertional inactivation experiments will allow us to ascertain whether or not MAT functions are required for pathogenicity and to analyse the role of MAT genes in sexual morphogenesis. In the long term, the role of MAT loci in controlling downstream cascades that lead to fruit-body formation will be established and genes involved in the downstream processes characterized, Such genes may control “shape determination”, and sequence comparison of such genes throughout the Ascomycotina may shed new light on the apparent morphological differences between ascocarps within this division. Equally, in combination with the traditional taxonomic analysis, a comparison of DNA sequences at the MAT loci may provide a unique approach for the classification of members within the Ascomycotina as well as providing useful sequence for the design of PCR-based diagnostic kits. In addition, by understanding the control of fruit-body formation, there may be implications for fermentative biotechnology, allowing control over fungal secondary metabolism, that may give rise to new products. Implications are also evident in the food industries where a greater understanding of the controlling elements of sexual reproduction may lead to the artificial induction of fruit-body formation in edible fungi.
VII. A MOLECULAR VIEW THROUGH THE LOOKING-GLASS: THE P. BRASSICAE-BRASSICA INTERACTION Like any plant-microbe interaction the P. brassicae-Brassica interaction is a dynamic process, and therefore our interpretation of the disease has relied
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on observed sequential changes in development of both the host and pathogen. Traditionally this has been by visual examination, for example, green islands and leaf curl are often observed as early stages of infection and this is followed by the formation of concentric rings of erumpant conidiomata indicating that sporulation and rupture of the cuticular layer has occurred. With the application of molecular biological techniques we are now able to “stop the clock” and analyse component parts of an interaction by in vifro analysis involving gene cloning techniques, as well as looking at the effects of gene knockouts on the ability of the fungus to undergo its normal life cycle in pfanta. By relating molecular findings to visual observations of pathogenesis we are now beginning to build a “molecular picture” of the life cycle of the fungus. This picture is far from complete and will require many more years of dedicated research to unravel. However, as a framework for future research and discussion, I will look through the “molecular looking glass” and try to speculate on some of the cellular processes that may play a role in establishing the life cycle of P. brussicue in planfa. From our current state of knowledge such speculation highlights some of the key questions regarding the P. brassicue-Brussicu interaction that have yet to be addressed. The molecular life cycle of P. brussicae (Fig. 13) begins when either ascospores disseminated by wind currents, which act as primary inoculum in the early spring, or asexual conidia, dispersed primarily by rain splash and acting as secondary inoculum, land on a brassica leaf surface. Adhesion molecules such as hydrophobins may be important, as is the case with the rice pathogen, Mugnoporthe griseu (Talbot et ul., 1993). However, to date, there is no evidence for such involvement in P. brassicue. Spores of P. brussicae germinate in the presence of water droplets and relatively short hyphae are produced prior to penetration, which curiously is never through stomata. Penetration does not seem to be primarily mechanical and one can assume therefore that the process involves degradation of the host cuticle. This could perhaps be mediated by an extracellular cutinase, although there is much controversy surrounding the role of such enzymes as pathogenicity determinants in other fungi (Stahl and Schifer, 1992; Rogers el al., 1994). Non- or weakly pathogenic isolates are poor penetrators, tending to produce longer hyphae on the leaf surface which may be indicative of a decreased level of expression of cutinase or insufficient build-up of pressure at the hyphal tips prior to penetration. Alternatively, some hitherto unknown recognition event may be a prerequisite for successful penetration. P. brussicue has been shown to infect a wide range of brassica hosts, however, with the pressures afforded by modern agricultural practices such as the breeding of resistant cultivars of brassicas and the widespread use of systemic fungicides, a greater selection pressure for more virulent isolates will prevail. Differential interactions between P. brussicue and Brussica sp. may become
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Fig. 13. A molecular view through the looking-glass at the life cycle of P. brassicae. The proposed molecular interpretation of the life cycle of the fungus based on recent experimental data combined with a certain degree of speculation. Figures are not drawn to scale.
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more evident with gene-for-gene associations playing a role in early interaction events. Further investigation may initiate a programme of research focused on the identification of resistance genes and their corresponding avirulence gene counterparts, as well as the mechanisms controlling host specificity. Once penetration has occurred, the fungus is protected from the hostile environment of the phylloplane and is likely to have little competition, in stark contrast with most necrotrophic fungi which have to compete with resident microbes. However, as a biotroph, the fungus has to establish biomass within the host, but with little or no disturbance to the host. P. brussicue rarely penetrates the cells of the mesophyll and is localized subcuticularly. The fungus is localized within the tissue in which it initially penetrated and never advances in a systemic fashion throughout the plant, underlining the tight controls that enable the physiological balance of host and pathogen to be maintained. Presumably, because of its biotrophic mode of growth during the early stages of the interaction, biomass must be established gradually and by delicate regulation of gene expression. This process involves tight regulation of extracellular enzyme production and it is probably limited to localized expression of an extracellular protease that allows the fungus to create space for itself by degradation of host glycoproteins such as the hydroxy proline-rich glycoproteins. This subtle breakdown of the host intracellular matrices may also cause localized leakage of nutrients from the apoplast. Fungal-derived cytokinins, which are likely to be produced in response to the stressful environment experienced by the fungus during the biotrophic phase of growth in pluntu, may alter the host cytokinin balance and cause re-direction of nutrients to the site of pathogen ingress, as well as cause retention of chlorophyll which is manifested as green islands on the leaf surface. Changes in the levels of cytokinin production as a result of pathogen ingress mimic the normal plant physiology and therefore give the plant a “false sense of security”, whilst providing localized regions of high metabolic activity within the host which will encourage an increase in fungal biomass. Sporulation may be biomass and space dependent, with space and/or nutrient supply becoming limited, resulting in a stress response by the fungus which presumably triggers asexual sporulation. Rupture of the cuticle causes water loss and desiccation stimulating stress responses in both plant and pathogen. At this point, the in pfuntu environment is dramatically altered, and the pathogen must switch to a different mode of nutrition. This switch to necrotrophy, and ultimately saprotrophy, is essential to survival and involves production, by the fungus, of a battery of extracellular cell wall degrading enzymes including endo-PG, PME, cellulase and protease. Sexual morphogenesis may occur on senescing leaves if germinating spores of both mating types are present. There may be environmental indicators such as polyphenolics associated with lignin degradation, that signal whether or not
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the spore is germinating on live or decaying host material. Longer germ tubes may be produced as the fungus searches for living host tissue and during this phase of growth the fungus may display increased susceptibility to environmental stimuli, such as the presence of a compatible mating-type pheromone or the presence of hyphae of the opposite mating type. Since an early interaction of germinating mycelium is required for sexual morphogenesis, this may be more easily achieved once the fungus is survivingas a saprophyte on the decaying leaf surface rather than during early development within the host. The defined time window in which sexual morphogenesis can occur is of significance here since it culminates in the formation of a protective resting structure, the apothecium. The fruiting body provides protection from environmental extremes and allows the pathogen to lay dormant until favourable conditions prevail. The extreme competition experienced by most saprophytes surviving on decaying matter would then be avoided. The nutritional status of the fungus, as well as environmental factors such as temperature, may therefore be important in establishing sexual development in preference to asexual development. The formation of a protective resting structure may also be in response to the development of the host, such as growth, development of flowers, pod formation and crop harvest. Once opposite mating types have interacted, SF triggers a developmental cascade that ultimately culminates in fruiting body formation. From GUS analysis it appears that hyphae exist in a dikaryotic state prior to karyogamy and meiosis, both processes being initiated within the developing apothecium. Regulatory genes at the mating-type locus presumably control SF biosynthesis. SF, in turn, presumably inhibits asexual sporulation, and fungal resource is then targeted towards fuelling sexual morphogenesis. SFIl, a protein that is produced in large amounts and within the first 7 days of development in response to sex factor, is hydrophobic in nature but is not itself a hydrophobin, and probably contributes to the structure of the apothecium. The melanized fruiting bodies act as survival structures which are able to resist desiccation, extreme temperature regimes and the effects of W irradiation. Sexual morphogenesis is an important developmental process for P. brussicue as it provides a means of survival from one growing season to the next. Occurrence of the sexual stage also offers the potential for genetic variability which may affect both pathogenicity and the development of tolerance to fungicides, both factors becoming more predominant as oilseed rape is exploited by agricultural practice. It has also been suggested that the release of ascospores from apothecia may provide an important inoculum source later in the season, reducing the efficacy of fungicide application. Dissemination from the apothecium is by wind currents, spreading inoculum over wider distances once conditions are favourable, either later in the growing season or early in the spring of the following season. This dynamic process continues from season to season, providing that a suitable brassica host is found.
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VIII. CONCLUDING REMARKS Within the last decade a number of molecular tools have been developed for the molecular analysis of filamentous fungi. Such tools will allow scientists to analyse how differential gene expression governs both pathogenesis and sexual morphogenesis in plant pathogenic fungi. With continued scientific input into P. brassicae research, from our group and from others, it is hoped that a true molecular picture of the life cycle of this important plant pathogen will be established. This multidisciplinary approach involving an integration of both conventional plant pathology and molecular and biochemical techniques will yield most progress towards a greater understanding of the biology of plant-microbe interactions and will ultimately lead to the development of novel and sophisticated strategies for disease control.
ACKNOWLEDGEMENTS I am indebted to my colleague Keith Johnstone, for jointly co-ordinating the P. brussicue research in Cambridge, for detailed discussions, for critically reading the manuscript and for helping with the figures. I would like to thank the Royal Society for a University Research Fellowship, past and present members of the Cambridge P. brussicue group, without whose efforts this review would not have been possible, and particularly Alex Murphy, Anthony Clark and Amy Hunter for commenting on the manuscript. I thank Rebecca Stratford and Robert Shields (Plant Breeding International) for sequencing pPROTl and providing pPROTl subclones, Chris Sidebottom for help with internal amino acid sequencing of SFI1, Stefan Drury-Morris for preparing the photographs and Barry Goddard for help with Fig. 13. Finally, I would like to thank my husband, Andy, for critically reading the manuscript, helpful discussions, drawing Fig. 13 and much encouragement, and my daughter, Victoria, for sleeping so blissfully when required. The research carried out in Cambridge was performed under provisions of licence number PHF 17419191 (28) issued by the Ministry of Agriculture, Fisheries and Food under the Plant Health (Great Britain) Order, 1987.
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leaf spot (Pyrenopeziza brassicae), seed yield and quality of winter oilseed rape (Brassica napus). Annals of Applied Biology 124, 221-239. Figueroa, L., Fitt, B. D., Shaw, M. W., McCartney, H. A. and Welham, S. J. (1995). Effects of temperature on the development of tight leaf spot (Pyrenopeziza brassicae) on oilseed rape (Brassica napus). Plant Pathology 44, 51-62. Froelinger, E. H. and Leong, S. A. (1991). The a mating type genes of Ustilago maydis are idiomorphs. Gene 100, 113-122. Giasson, L., Specht, C. A., Milgrim, C., Novotny, C. P. and Ullrich, R. C. (1989). Cloning and comparison of Aa mating-type alleles of the Basidiomycete Schizopyllum commune. Molecular and General Genetics 218, 72-77. Gladders, P. (1984). Present and potential disease interaction between oilseed rape and vegetable brassicas. Proceedings of the 1984 British Crop Protection Conference - Pests and Diseuse, pp. 799-806. Farnham: British Crop Protection Council. Glass, N. L., Grotelueschen, J. and Metzenberg, R. L. (1990). Neurospora crassa A mating-type region. Proceedings of the National Academy of Sciences, USA 87, 49124916. Greene, E. M. (1980). Cytokinin production by microorganisms. The Botanical Review 46, 25-78. Hardwick, N. V., Fitt, B. D. L., Wale, S. J. and Sweet, J. B. (1991). Oilseed rape diseases. Home Grown Cereals Authority Research Review OS4, London: Home Grown Cereals Authority. Huntley, R. (1995). Cytokinins and giberellins in oil palm sex determination. Ph.D. Thesis, University of Cambridge. Mot, T. W. (1984). Studies on the reproduction of Pyrenopeziza brassicae. Ph.D. Thesis, University of Cambridge. Mot, T. W., Ingram, D. S. and Rawlinson, C. J. (1984). Heterothallism in Pyrenopeziza brassicae, cause of light leaf spot of brassicas. Transactions of the Brifish Mycological Society 82(3), 477483. Illot, T. W., Ingram, D. S. and Rawlinson, C. J . (1986). Evidence of a chemical factor involved in the control of sexual development in the light leaf spot fungus, Pyrenopeziza brassicae (Ascomycotina). Transactions of the British Mycological Society 87(2), 303-308. Mot, T. W., Ingram, D. S. and Rawlinson, C. J. (1987). Studies of fungicide resistance in Pyrenopeziza brassicae, cause of light leaf spot disease of oilseed rape and other brassicas. Transactions of the British Mycological Society 88(4), 515523. Inman, A. J., Fitt, B. D. L. and Evans, R. L. (1992). A species of Unguiculuria on oilseed rape, and its importance in studies of the epidemiology of light leaf spot (Pyrenopeziza brassicae). Plant Pathology 41, 646-652. Jeffery, D. C., Jones, G. D. and Jenkins, P. D. (1989). Effects of early infections of light leaf spot (Pyrenopezia brassicae) on oilseed rape (Brassica napus L.). Aspects of Applied Biology 23, 409-415. Jeffery, D. C., Jenkins, P. D. and Gareth Jones, D. (1994). Comparative studies of light leaf spot (Pyrenopeziza brassicae) epidemics on the growth and yield of winter oilseed rape. Annals of Applied Biology 124, 19-25. Kraigher, H., Grayling, A., Wang, T. L. and Hanke, D. E. (1991). Cytokinin production by two ectomycorrizal fungi in liquid culture. Phytochemistry 30, 2249-2254. Kronstad, J. W. and Leong, S . A. (1989). Isolation of two alleles of the b locus of Ustilago maydis. Proceedings of the National Academy of Sciences USA 86, 978-982.
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Lacey, M. E., Rawlinson, C. J. and McCartney, H. A. (1987). First record of the natural occurrence in England of the teleomorph of Pyrenopeziza brassicae on oilseed rape. Transactions of the British Mycological Society 89( l ) , 135-140. Laloue, M. and Hall, R. H. (1973). Cytokinins in Rhizopogon roseolus. Plant Physiology 51, 559-562. Lewis, D. H. (1973). Concepts of fungal nutrition and the origin of biotrophy. Biological Reviews 48, 261-278. Machlis, L. (1966). Sex hormones in fungi. In “The Fungi’’, Vol. 2 (G. C. Ainsworth and A. S. Sussmann, eds), pp. 415433. Academic Press, London. Maddock, S. E. (1979). Studies of the biology of the light leaf spot disease of oilseed rape and other brassicas. Ph.D. Thesis, University of Cambridge. Maddock, S. E. and Ingram, D. S. (1981). Studies of the perfect stage of the light leaf spot pathogen of brassicas, Pyrenopeziza brassicae. Transactions of the British Mycological Society 77, 207-210. Maddock, S. E., Ingram, D. S. and Gilligan, C. A. (1981). Resistance of cultivated brassicas to Pyrenopeziza brassicae. Transactions of the British Mycological Society 76, 371-382. Majer, D., Mithen, R., Lewis, B. G., Oliver, R. P., deVos, P. and Zabeau, M. (1995). Detecting genetic variation in a fungal population using amplified length polymorphism (AFLP). Presented at the Eighteenth Fungal Genetics Conference, Asilomar, CA (Posters 111, abstract number 40). McCartney, H. A. and Lacey, M. E. (1990). The production and release of ascospores of Pyrenopeziza brassicae on oilseed rape. Plant Pathology 39, 17-32. McCartney, H. A., Lacey, M. E. and Rawlinson, C. J. (1986). Dispersal of Pyrenopeziza brassicae spores from an oilseed rape crop. Agricultural Science, Cambridge 107, 299-305. Mills, L. J. and Van Staden, J. (1978). Extraction of cytokinins from maize, smut tumours of maize and Ustilago maydis cultures. Physiological Plant Pathology 13, 73-80. Miura, G. and Hall, R. H. (1973). trans-Ribosylzeatin. Plant Physiology 51, 563-569. Moore, T. C. (1979) “Biochemistry and Physiology of Plant Hormones”, Springer Verlag, Berlin. Mutasa, E. S., Tymon, A. M., Gottgens, B., Mellon, F. M., Little, P. F. R . and Casselton, L. A. (1990). Molecular organisation of an A-mating type factor of the basidiomycete fungus Coprinus cinereus. Current Genetics 18, 223-229. Nester, E . W. and Gordon, M. P. (1991). Molecular strategies in the interaction between Agrobacterium and its hosts. In “Advances in Molecular Genetics of Plant-Microbe Interactions”, Vol. 1 (H. Hennecke and D. P. S. Vermal, eds), pp. 3-9 Kluwer, Dordrecht. Perucho, M., Hanahan, D., Lipsich, L. and Wigler, M. (1980). Isolation of the chicken thymidine kinase gene by plasmid rescue. Nature 285, 207-210. Picard, M., Debuchy, R. and Coppin, E. (1991). Cloning the mating types of the heterothaliic fungus Podospora anserina, developmental features of haploid transformants carrying both mating types. Genetics 128, 539-547. Powell, G. K. and Morris, R. 0. (1986). Nucleotide sequence and expression of a Pseudomonas savastanoi cytokinin biosynthetic gene: homology with Agrobacterium tumefaciens tmr and tzs loci. Nucleic Acids Research 14: 25552565. Punt, P. J., Oliver, R. P., Dingemanse, M. A., Pouwels, P. H. and Van Den Hondel, C. A. M. J . J. (1987). Transformation of Aspergillus based on the hygromycin resistance marker from E. coli. Gene 56, 117-124.
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Raeder, U. and Broda, P. (1985). Rapid preparation of DNA from filamentous fungi. Letters in Applied Microbiology I , 17-20. Rawlinson, C. J., Sutton, B. C. and Muthyalu, G. (1978). Taxonomy and biology of Pyrenopeziza brassicae sp. nov. (Cylindrosporium concentricum), a pathogen of winter oilseed rape (Brassica napus ssp. Oleifera). Transactions of the British Mycological Society 71(3), 425439. Roberts, I. N., Oliver, R. P., Punt, P. J. and van den Hondel, C . A. M. J. J . (1989). Expression of the E. coli P-glucuronidase gene in industrial and phytopathogenic filamentous fungi. Current Genetics 15, 177-180. Rogers, L. M., Flaishman, M. A. and Kolattukudy, P. E. (1994). Cutinase gene disruption in Fusarium solani f. sp. pisi decreases its virulence on pea. The Plant Cell 6 , 935-945. Sequeira, L. (1973). Hormone metabolism in diseased plants. Annual Reviews of Plant Physiology 24, 353-380. Siddiq, A. A. (1989). Sexual morphogenesis in P. brassicae. Ph.D. Thesis, University of Cambridge. Siddiq, A.A., Ingram, D. S., Johnstone, K . , Friend, J. and Ashby, A. M. (1989). The control of asexual and sexual development by morphogens in fungal pathogens. Aspects of Applied Biology 23, 417426. Siddiq, A. A., Johnstone, K. and Ingram, D. S. (1992). Evidence for the production during mating of factors involved in suppression of asexual sporulation and the induction of ascocarp formation in Pyrenopeziza brassicae. Mycological Research 96, 757-765. Simons, A. J. and Skidmore, D. I . (1988). Race-specific resistance to light leaf spot in Brassica oleracea. Transactions of the British Mycological Society 90, 43 1435. Skidmore, D. I., Ingram, D. S. and Mot, T. W. (1984). Genetic studies with Pyrenopeziza brassicae. In “Proceedings of Better Brassicas ’84 Conference”, pp. 139-143. Dundee: Scottish Crop Research Institute. Skoog, F. (Ed.) (1980). “Plant Growth Substances.” Springer Verlag, Berlin. Staben, C. and Yanofsky, C. (1990). Neurospora crassa A mating type region. Proceedings of the National Academy of Sciences, USA 87, 49174921. Stahl, D. J. and Schifer, W. (1992). Cutinase is not required for fungal pathogenicity on pea. The Plant Cell 4, 621-629. Staunton, W. P. and Kavanagh, T. (1966). Natural occurrence of the perfect stage of Gleosporium concentricum Grev. Berk. et Br. Irish Journal of Agricultural Research 5 , 140-141. Surico, G. and Iacobellis, N. S. (1992). Phytohormones and olive knot disease. In “Molecular Signals in Plant-Microbe Interactions” (D. P. S. Verma, ed.), pp. 209-227. CRC Press, Boca Raton, FL. Talbot, N. J., Ebbole, D. J. and Hamer, J . E. (1993). Identification and characterisation of MPG1, a gene involved in pathogenicity from the rice blast fungus Magnaporthe grisea. Plant Cell 5( 1l), 1575-1590. Thomas, J. E. and Walker, K. C. (1994). Exploitation of disease resistance in oilseed rape cultivars. Aspects of Applied Biology 40, 237-243. Thomas, P. L. (1988). Ustilago hordei, covered smut of Barley and Ustilago nigra, false loose smut of Barley. Advances in Plant Pathology 6 , 415425. Turgeon, B. G., Bohlmann, H . , Ciuffetti, L. M., Christiansen, S. K., Yang, G., Schafer, W. and Yoder, 0. C. (1993). Cloning and analysis of the mating type genes from Cochliobolus heterostrophus. Molecular and General Genetics 238, 270-284. Vollmer, S. J. and Yanofsky, C. (1986). Efficient cloning of genes of Neurospora
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crassa. Proceedings of the National Academy of Sciences, USA 83, 48694873. Wood, K. V., Lam, Y. A., Seliger, H. H. and McElroy, W. D. (1989). Complementary DNA coding click beetle luciferases can elicit bioluminescence of different colours. Science 244, 700-702. Yarman, D. J. and Giltrap, N. J. (1989). Crop diseases in a changing agriculture. Arable crops - a review. Plant Pathology 38, 459-477. Yelton, M. M., Hamer, J. E . and Timberlake, W. E. (1984). Transformation of Aspergillus nidulans by using a Trp C plasmid. Proceedings of the National Academy of Sciences, USA 81, 147G-1474.
The Balance and Interplay Between Asexual and Sexual Reproduction in Fungi
M. CHAMBERLAIN* and D . S. INGRAM
Royal Botanic Garden. 20A Inverleith Row. Edinburgh EH3 5LR. UK
I . Introduction
.............................................................................
I1. Initiation of Asexual Sporulation and Sexual Reproduction ..............
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111. Comparisons of Genetic Variation. Physiological Costs and Fitness
between Asexual and Sexual Systems ........................................... A . Does the Sexual System Generate more Variants? ................... B . Is the Sexual System Physiologically more Costly? ................... C . Are Sexual Progeny more “Fit”? ..........................................
IV. Maintaining and Changing the Balance between Reproductive Processes ......................... .................................................... A . Species-determined Equilibria ............................................... B . Genotype-determined Equilibria ............................................ C . Seasonally Maintained Equilibria ........................................... D . Physical and Nutritional Factors ............................................ E . Density and Competition ..................................................... F. The Effect of Mycelial Extracts and Specific Morphogens .......... V. Trade-off between Asexual and Sexual Reproduction
73 73 74 74 74 75 76 77 77 77 78
......................
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VI . Conclusion ............................................................................... Acknowledgements .................................................................... References ...............................................................................
81 82 82
I . INTRODUCTION All mycelial fungi grow vegetatively. most reproduce asexually. some reproduce only asexually. some only sexually. but most undergo all three *Previously known as M . Jurand Advances in Botanical Research Vol . 24 incorporating Advances in Plant Pathology ISBN 0-12405924-X
Copyright 0 1997 Academic Press Limited All rights of reproduction in any form reserved
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processes in the course of their life cycle. An actively growing fungal mycelium therefore may have a number of possible courses of development open to it. It may continue with purely vegetative growth, it may concentrate on asexual sporulation or it may initiate sexual development, provided that mating has taken place or that the strain is self-compatible. Cochrane (1958) recognized that a major barrier to generalizing about reproduction in fungi is the repeated failure of experimental work to discriminate adequately between growth and sporulation. This still remains a problem, except that in some species there are three courses of action: vegetative growth, asexual sporulation and sexual development. Whether the three courses of action are in fact alternatives or occur in sequence one after another, the balance between them may be a feature of the individual species, population o r genotype and is also fine tuned by environmental factors. The situation is further complicated by the fact that the adaptability to environmental change is itself under genetic control. According to Andrews (1984, 1992), life history theory must account for input and output, both of which reflect allocation of limited time and resources among competing demands. In fungi, which are totipotent and modular, the demarcation between growth and reproduction is not absolute, except that reproduction tends to be associated with dispersal. Spores, both asexual and sexual, tend to be uninucleate and haploid. The dissemination of single-celled propagules allows each generation to start with what Crow (1988) calls “tabula rasa” , allowing deleterious mutations to be eliminated quickly. Dawkins (1982) differentiates two life-history traits, G (growth) and R (reproduction), and discusses the advantages of cycling between the singlecell and multicell states. However, he does not mention the cycling between asexual and sexual phases, which must surely affect both the ecological requirements (Elliott, 1994) and the uniformity-variability equilibrium. While the asexual cycle is mitotic, the sexual cycle consists of two variation-generating events: fertilization and meiosis. There are, therefore, commonly held beliefs that asexual reproduction generates uniform progeny, whereas the sexual process generates genetically different individuals, provided that the mates coming together at fertilization are genetically different from one another. The sexual structures and sometimes the sexual spores themselves are more durable, allowing the organism to resist unfavourable conditions. In some fungal groups the sexual structures also allow for better dispersal.
11. INITIATION OF ASEXUAL SPORULATION AND SEXUAL DEVELOPMENT Whether produced mitotically o r meiotically, the spores of most fungi germinate to give rise to a vegetatively growing mat, each hyphal cell dividing
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mitotically to produce a network of hyphae or mycelium. At some stage in the. course of the vegetative growth the mycelium becomes ready for asexual sporulation, a state given the term “competence” (Champe et al., 1981). The sporulation schedule has been worked out for the homothallic species Aspergillus niduluns (reviewed by Champe et al., 1981). A single conidium placed on nutrient agar surface grows vegetatively to form a mycelium for a fixed period of 24 h at 37OC. Then comes a sudden onset of conidiation, which is remarkably synchronous from colony to colony (Axelrod, 1972) and is unaffected by medium composition (Pastushok and Axelrod, 1976). The time of conidiation is, however, temperature dependent and varies inversely with colony density. The induction to conidiate has been worked out to occur 20 h after inoculation, after which follows a fixed maturation period of 4 h. These times appear to be controlled endogenously. Whereas conidiation is a precisely timed event, the decision to sporulate sexually depends on the genetic background and cultural conditions. This may happen several days to more than a week after conidiation is complete. The sexual spores, or ascospores, form following meiotic division within developing asci in closed spherical ascocarps or cleistothecia. Clearly, the later that sexual reproduction is induced, the greater is the ratio of asexual to sexual reproduction. In homothallic species, that is those in which each mycelium can complete the sexual cycle on its own, sexual reproduction is triggered without fertilization between two different strains. In heterothallic species, that is those that require the interaction of two different strains to complete the life cycle, sexual reproduction is initially dependent on the availability of compatible nuclei. It is controlled by two separate systems. The first operates at the population level and regulates the level between inbreeding and outbreeding, and the second is concerned with the sequence leading to sexual morphogenesis. In heterothallic ascomycetes the act of mating does not by itself trigger morphogenesis. Turian (1978) believes that morphogenesis of reproductive structures is a process which is typically under the control of factors other than those which determine sexual compatibility. It may include up to five phases: sexual differentiation, plasmogamy , karyogamy, meiosis and sporogenesis.
111. COMPARISONS OF GENETIC VARIATION, PHYSIOLOGICAL COSTS AND FITNESS BETWEEN ASEXUAL AND SEXUAL SYSTEMS A. DOES THE SEXUAL SYSTEM GENERATE MORE VARIANTS?
The totipotent nature of fungal cells, and hence the opportunity for the propagation of spontaneous mutations and the existence of parasexual systems (Pontecorvo, 1956), have raised the question of whether the
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assumption that the sexual system generates more variants is in fact rigorously justified. One study which confirms this elegantly is a comparison of sexual and asexual populations of Puccinia graminis f.sp. tritici in different parts of North America (Burdon and Roelfs, 1985). The origin of genetic diversity exhibited by the largely asexually reproducing genus Fusarium is hotly debated, with some authors favouring the asexual origin (Gordon, 1993) and others favouring origin from inconspicuous sexual events (literature reviewed by Anderson et al., 1992). B. IS THE SEXUAL SYSTEM PHYSIOLOGICALLY MORE COSTLY?
Another commonly held assumption is that the sexual system is physiologically more costly than the asexual one. To our knowledge there have been no studies in fungi to show how dry matter and energy are apportioned at different stages in the life cycle that are comparable to those carried out in higher plants, for example in Senecio vulgaris (Harper and Ogden, 1970). Studies on how organisms budget their resources in response to the competing demands of growth and reproduction have been carried out in lizards and higher plants (literature reviewed by Giesel, 1976). Composites, such as Tussilago farfura, devote up to 30% of their assimilated energy to reproduction, of which 3-8% is devoted to seeds and >23% to asexual reproduction (Ogden, 1974). Similar studies are needed in the fungi. C. ARE SEXUAL PROGENY MORE “FIT”?
Studies in the grass Anthoxanthum odoratum have shown short-term and immediate fitness advantages for sexually produced progeny (Kelley et al., 1988). Although the mathematical models proposed by Nauta (1994) have predicted that this is also likely to be true in the fungi, there is no experimental proof available to confirm this. Hastings (1992), also on the basis of mathematical models, but in diploid organisms, showed that a large number of asexual divisions in between occasional sexual ones offers an ideal system, since it inhibits the spread of deleterious cytoplasmic genomes (DCGs), which can invade and dramatically reduce the fitness of a sexual population. Although fungi lend themselves well to addressing the above three questions, there is surprisingly little evidence available to provide rigorous answers.
IV. MAINTAINING AND CHANGING THE BALANCE BETWEEN REPRODUCTIVE PROCESSES In the following sections we summarize how the balance between different reproductive processes is maintained in nature and adjusted by genetic and environmental factors.
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A . SPECIES-DETERMINED EQUILIBRIA
The strategy theory of Dobzhansky (1950) has been adapted for fungi by Pugh (1980) and Cooke and Whipps (1993) and for fungal pathogens by Andrews and Rouse (1982). The strategy model proposes a spectrum of strategies for survival, at the opposite ends of which are two types of organisms: the r-selected and the K-selected. The former have a short life expectancy and rapidly commit all their available resources to reproduction. The latter have a long life expectancy and either devote only a small proportion of available resources to reproduction at any one time or only commit themselves to reproduction at the end of their life span. Since a diversity of strategies has been found in phylogenetic lineages, one might argue that this points to the fixation of a life-history trait in a given species (Reynolds, 1993). However, as taxonomists often base their classifications on life-history traits, one must beware of circular thinking. There are, nevertheless, several examples of related species apportioning differing ratios of their resources to the two methods of reproduction. In Eupenicillium the balance between asexual and sexual phases of the life cycle varies from species to species. The balance of resources devoted to the two phases depends on the length of the life cycle and hence the interval between consecutive ascospore generations. Those species which show rapid rates of ascostromatal ripening have fragmentary penicilli, and so clearly devote less of their resources to asexual sporulation. By contrast, those species which have ascostromata that are slow to ripen typically produce a larger penicillus with greater numbers of phialides (Pitt, 1979). This indicates that, as long as the sexual cycle generates a quick turnover of ascospores, conidia become less important. Only a few Aspergilli and Penicillia have been found to be heterothallic. Homothallism most likely confers the advantage of bypassing the hit-and-miss business of encountering a mate (also referred to as the “lonely spore hypothesis” by Herskowitz, 1988). The existence of a sexual phase in addition to the prolific asexual phase probably confers an advantage in an ephemeral environment. A homothallic organism with a resting stage in its life cycle would be able to overwinter adverse conditions or oversummer in the semi-arid tropics, like, for example, downy mildews in India. Anderson et al. (1992) question the premise of Pugh (1980) that life strategies are adaptive. Nevertheless, there are certain correlations to be made. For example, asexual reproduction tends to be favoured in fastreproducing Fusarium species, whereas some of the long-lived, slowly colonizing basidiomycetes invest more of their resources in sexual structures, thus achieving longer range dispersal and genetic variation (Anderson et al., 1992). Perhaps the old adage “Do not confine your children to your own learning, for they were born in another time” applies to fungal spores too.
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While reproductive strategy may become fixed in a given species, the existence within the same species of genetic polymorphism for different reproductive strategies, discussed by Giesel (1976) for higher plants, appears to be operating in the following fungal examples. Many species can exist as either anamorphic (mitotic), teleomorphic (meiotic) or pleomorphic (both mitotic and meiotic) strains. For example, the species Glomerella cingulata includes a large number of pathogenic forms that attack plants of different types. Strains from apple, grape and pepper usually produce both sexual and asexual spores, while those from wild Zpomoea sp., kudzu and okra produce fertile perithecia in abundance, but few or no conidia (Wheeler, 1956). Cultures which produce only conidia are occasionally obtained from all hosts, sometimes from the same lesions that yield cultures of one of the other types. One conidial strain was obtained from an ascospore derived from a predominantly perithecial strain. The progeny of crosses between the original perithecial form and the conidial variant were of the two parental types only, suggesting a simple Mendelian inheritance. A similar situation was found in Gleotinia temulenta (Griffiths, 1958). Mono-ascospore cultures grown from apothecia collected in the field varied genetically in their capacity to reproduce asexually. Some produced huge numbers of conidia, others were only mycelial, and a third category produced both conidia and mycelia. In Fusarium graminearum, Burgess et al. (1975) and Francis and Burgess (1975) found two population groups, one of which rarely forms perithecia while the other readily forms perithecia in culture. In this case the two populations of this species were found to be heterothallic and homothallic, respectively (Francis and Burgess, 1977). Perhaps further studies on this species would disclose a truly polymorphic situation. To date few examples of polymorphism between homothallic and heterothallic individuals in the same population have been found, although the mathematical models proposed by Nauta (1994) predicted that such a situation is to be expected. In Mugnaporthe grisea, the blast fungus of cereals and grasses, the sexual cycle can only be induced under laboratory conditions, but the degree of fertility is highly dependent on host origins (Yaegashi, 1977). The fungus is a heterothallic ascomycete and at least some of the isolates function only as males. Apparently the loss of hermaphroditic function, or maleness, appeared to be due to a single gene mutation (Leung and Williams, 1985). However, since sexual reproduction has not yet been found in nature and an active parasexual system is operating, mechanisms for generating variants in the field are thought to be of somatic origin (Leung and Taga, 1988).
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C. SEASONALLY MAINTAINED EQUILIBRIA
Seasonal factors are known to maintain prolific conidium production on living leaves, while the sexual telemorph stages are restricted to dead or dying stems in the autumn. This is the situation, for example, in apple scab Venturia inaequalis (Boone, 1988) and light leaf spot Pyrenopeziza brussicae (McCartney and Lacey, 1990). Similarly, rusts produce large quantities of asexual uredospores during the summer and teliospores only later in the year (Hawker, 1966). These seasonally maintained equilibria may be sustained by endogenous factors which control the natural senescence of the pathogen and its host. D . PHYSICAL AND NUTRITIONAL FACTORS
External factors which are known to affect the switch between the different reproductive processes include general physical and nutritional factors such as temperature, light, humidity and water availability, aeration and pH as well as the quality and quantity of nutrients. The influence of these factors on the reproductive development of fungi has been studied extensively (Hawker, 1966; Turian, 1966; Muller, 1979; Moore-Landecker, 1982; Cooke and Whipps, 1993; Griffin, 1994). However, many of these articles do not specify the type of reproduction involved. There are also review articles on the initiation of asexual reproduction (Clutterbuck, 1978; Smith, 1978) and sexual reproduction (Turian, 1978; van den Ende, 1978) in the excellent synthesis edited by Smith and Berry (1978), but the subjects are dealt with separately. A detailed review of the factors which affect the switch between the two types of sporulation is being prepared (Chamberlain, in preparation). E. DENSITY AND COMPETITION
It has already been mentioned that in Aspergillus nidulans the time of conidiation depends on the density of conidia sown (Champe et al., 1981). A confluent lawn of sown conidia initiates the conidiation process sooner than a single colony derived from one conidiurn. The same was observed in Pyrenopezizu brassicae (personal observation). Increasing the density of single spore inocula also enhances sexual reproduction. In the basidiomycete genus Psathyrella fruiting was promoted on dishes evenly sown with basidiospores compared with dishes inoculated with a single dikaryotic isolate (Jurand, 1975). Interactions between mycelia of different species of fungi have also been widely reported to induce sexual sporulation. For example, Trichoderrna
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strains release volatile antibiotics which stimulate the production of oospores in homothallic species of Phytophthora (Brasier, 1975).
F. THE EFFECT OF MYCELIAL EXTRACTS AND SPECIFIC MORPHOGENS
Given that competition within and between species affects the choice of developmental strategy, it is not surprising that mycelial extracts are also effective. Most evidence points to early stimulation of sexual reproduction or to a swing in the balance between asexual and sexual reproduction in favour of the latter. There are at least 20 independent reports of extracts of mycelia playing a role in switching the balance in favour of sexual development (Table I). Extracts of Pyrenopeziza brassicae, for example, were found to influence the reproductive development of P. brassicae itself (Ilott et al., 1986; Siddiq et al., 1990) and about 40 different species of fungi (Siddiq et al., 1989; Chamberlain et al., 1995). In those species of fungi which reproduce sexually, the effect of the extracts was to promote sexual development at the expense of asexual sporulation, while in those species, e.g. Deuteromycotina, which do not have a sex option, the effect of the extracts was to enhance asexual sporulation. It would appear that much the same pattern of response can be seen from all the other examples listed in Table I. In most of the studies with mycelial extracts the active ingredient has not been characterized, but in a few cases specific morphogens have been discovered. These include trisporic acid, linoleic acid, zearalenone, CAMP, mycosporines, cerebrocides and indole acetic acid (reviewed by Dyer et al., 1992). The effects of some chemicals, antibiotics and metabolites on the production of primordia in sclerotium-producing species are summarized by Cooke (1983). However, attempts to find a universal sex-inducing hormone have so far failed. Another possibility is that some mycelial extracts supply a negative effect in the form of substances antagonistic to growth, or even have a more drastic effect of causing mutilation, injury or other forms of physiological stress. It has been suggested that injured or moribund cells release substances which act on surviving cells and divert them into a new developmental path (Cochrane, 1958). Very early work by Rands (1917) and Kunkel (1918) showed that scraped or broken hyphae stimulate conidiation. This kind of evidence suggests that the primary activity of the extracts is on growth rather than reproduction. It is generally accepted that reproduction in fungi and also in higher plants is initiated by factors which check vegetative growth without drastically poisoning its metabolism. According to the Klebs principle (Klebs, 1898, 1899, 1900) reproduction is favoured by depriving an established mycelium of one or more nutrients (Cochrane, 1958). The edge effect is well known, both in the field situation and in petri
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TABLE 1 Effect of extracts of fungal mycelia on the reproductive development of the same or other species of fungi Extract of mycelia Of
Agaricus bisporus Aspergillus nidulans Fusarium spp.
Active on:
Marasmius sp.
Stimulation of fruiting Inhibition of conidium Same species formationlenhanced cleistothecium formation Increased sexual 40 different species reproduction 90% reduction Same species (conidial isolate) of conidiation
Glomerella cingulata (perithecial isolate) Hormodendrum Schizophyllum cladosporioides commune Lenticus edodes Marasmius sp. Neurospora crassa Neurospora crassa Mating type a Mating type A Mating types A'X a
Nature of activity
Same species (filtrate of both mating types) Same species (mating type A) Same species (mating type a) Same species (mating type
Induction of fruiting
Reference Uramaya (1969) Champe et al. (1987)
Nelson (1971) Wheeler (1956) Leonard and Dick (1968) Uramaya (1969)
Stimulation of fruiting Stimulation of perithecium formati on
It0 (1956)
Protoperithecium stimula tion
Islam and Weijer (1972)
N o effect
Islam and Weijer (1972)
Protoperithecium stimulation
Islam (1981)
Stimulation of perithecia Induction of fruiting
Wilson (1927)
Ascocarp stimulation No effect
Ilott et al. (1986)
A)
Venturia inaequalis Schizophyllum Penicillium commune funiculosum Pyrenopeziza brassicae Mated isolates Same species Penicillium spp.
Unmated isolates Unmated and mated isolates
Same species 30 different species
Reduction of asexual sporulation and stimulation of sexual reproduction
Kawai et al. (1985)
Ilott et al. (1986) Chamberlain ef al. (I 995)
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M. CHAMBERLAIN and D. S . INGRAM
TABLE 1 cont. Schizophyllum commune Sclerotinia sclerotiorum Verticillium sp. Microsclerotial isolate
Pyrenopeziza brassicae Sordaria brevicollis Same species
Stimulation of sexual reproduction Stimulation of sexual reproduction Enhancement of sclerotium formation
Same species
Stimulates production of microsclerotia/ inhibits sporulation Stimulation of perithecium formation Stimulation of apothecia
Several species
Thielavia bassicola
Several species
Pyronema domesticum
Siddiq (1989) Siddiq ( 1989) Bedi (1958)
Brandt and Reese (1964) McKormick (1925) Moore-Landecker (1988)
dishes. Macdonald and Bond (1976) showed in Sordaria brevicollis that protoperithecial production commenced all over the colony only after the mycelial front had reached the edge of the containing vessel. The presence of a growing mycelial front inhibited the differentiation of fruiting bodies. A similar effect has been found in Chaetomium globosum (Buston and Rickard, 1956) and Gelasinospora reficulospora (Inoue and Furuya, 1970).
V. TRADE-OFF BETWEEN ASEXUAL AND SEXUAL REPRODUCTION Most of the above studies seem to indicate that the asexual and sexual reproductive processes are in competition with each other for the availability of resources and that various factors can alter the balance of resource allocation. The same trade-off situation has been observed in the field. Michelmore and Ingram (1980) observed an inverse relationship between intensity of asexual sporulation and the incidence of sexual reproduction in the heterothallic oomycete Bremia lactucae. An “eithedor” situation with sexual organs and conidiophores borne on different parts of the onion foliage was evident in the onion mildew Peronospora schleideniana (McKay, 1939) and in graminaceous downy mildews (Safeeulla, 1976). Simultaneous sexual and asexual sporulation was, however, observed in Peronospora parasitica (McMeekin, 1960) and P. destructor (Berry and Davis, 1957). Michelmore and Ingram (1980) thought that in Bremia lactucae the sexual and asexual processes were antagonistic. However, at lower levels
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of sexual reproduction, they occasionally observed both sexual and asexual sporulation in close proximity, apparently involving the same vegetative hyphae. They concluded that at the onset of sporulation there may be a developmental switch which prevents asexual reproduction during sexual differentiation; alternatively asexual sporulation may be suppressed by greater nutritional demands of sexual reproduction which take priority. Observations of trade-off between asexual and sexual reproduction in the field are further confirmed by experimental evidence. In Aspergillus nidulans conidiation and cleistothecium formation are considered to be alternatives (Clutterbuck, 1977). It is evident from the behaviour of mutants and also from variants found in the wild that an excess of either inevitably means a reduction in the other. In mutants with reduced conidiation, resources which would otherwise be employed in asexual reproduction are diverted to cleistothecial production (Martinelli and Clutterbuck, 1971). A negative correlation between the numbers of conidia and ascospores was observed by Chamberlain et al. (1995). Studies in higher plant reproductive ecology have shown many examples of negative correlation between asexual and sexual reproduction. For example, in Pofygonum viviparum, Law et al. (1983) plotted the number of bulbils versus the number of flowers on single inflorescences and found evidence of a highly significant correlation between the two. They concluded that there was a trade-off, so that one could not be increased without reducing the other. In higher plants it is difficult to determine whether the balance between asexual and sexual reproduction is genetically determined or is the result of phenotypic responses to a changing environment. Differences in biomass allocation in Impatiens capensis from two different habitats are shown to be environmentally determined, since the differences are not evident when the plants are transplanted to uniform greenhouse conditions (Abrahamson and Hershey, 1977). The authors refer to the environmentally induced “sedasex” switch as a “plastic switch hitter strategy”. There is also evidence, reviewed by Willson (1983), that competitive pressure induces plants to put a greater proportion of their resources into seeds. It would be plausible to suggest that the situation is similar in the fungi.
VI. CONCLUSION It is clear that a trade-off operates between asexual and sexual reproduction in fungi. The balance of resources allocated to the competing demands of the two modes of reproduction can be determined genetically and can also be regulated by numerous environmental factors. The earlier the switch is “hit”, the greater is the ratio of sexually to asexually produced offspring. There is evidence that competition and stress induce an earlier switch. Mycelial extracts of the same, as well as different, species contain diffusible
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factors which may serve as a signal to neighbouring mycelia of the presence of a potential competitor. Studies on the factors which operate the switch are of considerable economic interest, whether the aim is to control a destructive pathogen or to propagate a beneficial species. They are also of academic interest as they have a bearing on the issue of the sexual process itself. Many fundamental questions still remain unanswered. Is sexual reproduction merely a response to changed circumstances (sensu Williams, 1975)? Are there enough correlations to support the view that sexual reproduction survives despite the costs, because it generates variation, or is its survival related to the fact that in most sexual species of fungi the sexual propagules serve as resting spores? Indeed, the combination of recombinant genomes within a resting structure must enhance considerably the chances of survival of a species faced with adverse environmental conditions. What are the costs? Does it in fact generate significantly more genetic variation than do asexual and parasexual systems combined? Are sexually produced fungal “individuals” more “fit”? Are the different observed life-history traits in related species merely correlates or have they resulted from selection pressures? All these questions urgently require investigation. The situation is probably most easily explained teleologically. Each fungal organism, in its particular niche, “writes” its own hypothetical balance sheet and then, weighing up a multitude of ecological, genetical and physiological factors, “decides” which method to select: asexual, sexual or indeed a carefully tuned balance between the two. All organisms proceed with life cycles in response to information received from the environment (Demetrius, 1975; Giesel, 1976), but some are more adaptable than others. In some cases the balance appears to be fixed in a given species or genotype; at the other end of the spectrum are organisms which adjust their life-history traits readily, the environment playing the part of a flexible “switch hitter”.
ACKNOWLEDGEMENTS We thank the Gatsby Charitable Foundation for financial support and numerous colleagues for stimulating discussions, in particular Drs A. M. Ashby, D. J. Bond, A. F. Dyer, K. Johnstone, K. Jong, R. F. 0. Kemp, J. Ratter and Professor J. Friend.
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Anderson, J. B., Kohn, L. M. and Leslie, J . F. (1992). Genetic mechanisms in fungal adaptation. In “The Fungal Community, Its Organisation and Role in the Ecosystem” (G. C. Carroll and D. T. Wicklow, eds). Marcel Dekker, New York. Andrews, J. H. (1984). Life history strategies in plant parasites. Advances in Plant Pathology 2 , 105-130. Andrews, J. H. (1992). Fungal life-history strategies. In “The Fungal Community, Its Organisation and Role in the Ecosystem” (G. C. Carroll and D. T. Wicklow, eds). Marcel Dekker, New York. Andrews, J . H. and Rouse, D. I. (1982). Plant pathogens and the theory of r- and K-selection. American Naturalist 120, 283-296. Axelrod, D. E. (1972). Kinetics of differentiation of conidiophores and conidia by colonies of Aspergillus nidulans. Journal of General Microbiology 73, 181184. Bedi, K. S. (1958). The role of stale products in the formation of sclerotia of Sclerotinia sclerotiorum (Lib.) De Bary. Indian Phytopathology 16, 29-36. Berry, S. Z. and Davis, G. N. (1957). Formation of oospores by Perenospora destructor (Bert.) Casp. and their possible relation to epiphytology. Plant Disease Reporter 41, 3-6. Boone, D. M. (1988). Venturia inaequalis, cause of scab of apples. Advances in Plant Pathology 6, 249-262. Brandt, W. H. and Reese, J. E. (1964). Morphogenesis in Verticillium: a selfproduced diffusable morphogenetic factor. American Journal of Botany 51, 922-927. Brasier, C. M. (1975). Stimulation of sex organ formation in Phytophthora by antagonistic species of Trichoderma. I. The effect in vitro. New Phytologist 74, 183-194. Burdon, J . J. and Roelfs, A. P. (1985). The effect of sexual and asexual reproduction on the isozyme structure of populations of Puccinia graminis. Phytopathology 75, 1098-1073. Burgess, L. W., Wearing, A. H. and Toussoun, T. A. (1975). Surveys of the fusaria associated with crown rot of wheat in Eastern Australia. Australian Journal of Agricultural Research 26, 791-799. Buston, H. W. and Rickard, B. (1956). The effect of a physical barrier on sporulation of Chaetomium globosum. Journal of General Microbiology 15, 194-197. Chamberlain, M., Ertz, S. and Ingram, D. S. (1995). Effect of extracts of Pyrenopeziza brassicae on other species of fungi. Mycologia 87, 846-856. Champe, S. P., Kurtz, K. B., Yager, L. N., Butnick, N. J. and Axelrod, D. E. (1981). Spore formation in Aspergillus nidulans: competence and other developmental processes. In “The Fungal Spore: Morphogenic Controls” (G. Turian and H. R. Hohl, eds). Academic Press, London. Champe, S. P., Rao, P. and Chang, A. (1987). An endogenous inducer of sexual development in Aspergillus nidulans. Journal of General Microbiology 133, 1383-1387. Clutterbuck, A. J . (1977). The genetics of conidiation of Aspergillus nidulans. In “The Physiology and Genetics of Aspergillus” (J. E. Smith and J . Pateman, eds), pp. 305-317. Academic Press, New York. Clutterbuck, A. J. (1978). Genetics of vegetative growth and asexual reproduction. In “The Filamentous Fungi, Vol. 111: Developmental Biology” (J. E. Smith and D. R. Berry, eds), pp. 47-73. Edward Arnold, London. Cochrane, V. W. (1958). “Physiology of Fungi.” Wiley, New York. Cooke, R. (1983). Morphogenesis of Sclerotia. In “Fungal Differentiation” (J. E .
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Smith, ed.), pp. 397-418. Marcel Dekker, USA. Cooke, R. C. and Whipps, J. M. (1993). “Ecophysiology of Fungi”, pp. 337 ff. Blackwell Scientific, Oxford. Crow, J. F. (1988). The importance of recombination. In “The Evolution of Sex: An Examination of Current Ideas” (R. E. Michod and B. R. Levin, eds), pp. 56-73. Sinauer Associates, Supderland, MA. Dawkins, R. (1982). “The Extended Phenotype: The Gene as the Unit of Selection.” Oxford University Press, Oxford. Demetrius, L. (1975). Reproductive strategies and natural selection. American Naturalist 109, 243-249. Dobzhansky, T. (1950). Evolution in the tropics. American Scientist 38, 209-221. Dyer, P. S., Ingram, D. S. and Johnstone, K. (1992). The control of sexual morphogenesis in the Ascomycotina. Biological Reviews 67, 421458. Elliott, C. G. (1994). “Reproduction in Fungi.” Chapman & Hall, London. Francis, R. G. and Burgess, L. W. (1975). Surveys of fusaria and other fungi associated with stalk rot in maize in eastern Australia. Australian Journal of Agricultural Research 26, 801-807. Francis, R. G. and Burgess, L. W. (1977). Characteristics of two populations of Fusarium roseurn “Graminearum” in Eastern Australia. Transactions of the British Mycological Society 68, 421-427. Giesel, J. T. (1976). Reproductive strategies as adaptations to life in temporarily hetergeneous environments. Annual Review of Ecology and Systematics 7 , 57-79. Gordon, T. R. (1993). Genetic variation and adaptive potential in an asexual soilborne fungus. In “The Fungal Holomorph: Mitotic, Meiotic and Pleomorphic Speciation in Fungal Systematics” (D. R. Reynolds and J, W. Taylor, eds). CAB International, Wallingford, UK. Griffin, D. H. (1994). “Fungal Physiology”, pp. 458 ff. Wiley-Liss, New York. Griffiths, E. (1958). Sexual reproduction and variation in Gleotinia temulenta (Prill. and Delacr.) Wilson and Gray. Transaction of the British Mycological Society 41, 461-482. Harper, J. L. and Ogden, J. (1970). The reproductive strategy of higher plants. I The concept of strategy with special reference to Senecio vulgaris L. Journal of Ecology 58, 681-698. Hastings, I. (1992). Why is sex so frequent? Trends in Ecology and Evolution 7 , 278-279. Hawker, L. E. (1966). Environmental influences on reproduction. In “The Fungi: An Advanced Treatise”, Vol. I1 (G. C. Ainsworth and A. S. Sussman, eds). Academic Press, London. Herskowitz, I. (1988). Life cycle of the budding yeast Saccharomyces cerevisiae. Microbiological Reviews 5 2 , 536-553. Illott, T. W., Ingram, D. S . and Rawlinson, C. J. (1986). Evidence of a chemical factor involved in the control of sexual development in the light leaf spot fungus Pyrenopeziza brassicae (Ascomycotina). Transactions of the British Mycological Society 87, 303-308. Inoue, Y. and Furuya, M. (1970). Perithecial formation in Gelasinospora reticulospora: effects of light at two different growth states. Development, Growth and Differentiation 12, 141-150. Islam, M. S. (1981). Sex pheremone in Neurospora crassa. In “Sexual Interaction in Eukaryotic Microbes” (D. H. O’Day and P. A. Horgen, eds), pp. 131-154. Academic Press, New York. Islam, M. S. and Weijer, J. (1972). Development of fertile fruit bodies (perithecia)
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in the single strain culture of (Em A) of Neurospora crassa. Folia Microbiologica, Prague 17, 316-319. Ito, T. (1956). Fruit body formation in red bread mould, Neurospora crassa. I. Effect of culture filtrate on perithecial production. Botanical Magazine, Tokyo 69, 369-372. Jurand, M. K. (1975). Breeding biology of the genus Psathyrella. Ph.D. Thesis, University of Edinburgh. Kawai, G., Ikeda, Y. and Tubaki, K. (1985). Fruiting of Schizophyllum commune induced by certain ceramides and cerebrosides from Penicillium funiculosum. Agricultural and Biological Chemistry 49, 2137-2146. Kelley, S. E., Antonovics, J. and Schmitt, J. (1988). A test for short-term advantage of sexual reproduction. Nature 331, 714-716. Klebs, G. (1898). Zur physiologie der Fortpflanzung einiger Pilzer. I: Sporodinia grandis. Jahrbuch fur Wissenschaftliche Botanik 32, 1-69. Klebs, G.(1899). Zur physiologie der Fortpflanzung einiger Pilzer. 11: Saprolegnia mixta. Jahrbuch f u r Wissenschaftliche Botanik 33, 513-539. Klebs, G. (1900). Zur physiologie der Fortpflanzung einiger Pilzer. 111: Allgemeine Betrachtungen. Jahrbuch fur Wissenschaftliche Botanik 35, 80-203. Kunkel, L. 0. (1918). A method of obtaining abundant sporulation in cultures of Macrosporium solani. E&M. Brooklyn Botanic Garden Memoirs 1, 306-312. Law, R., Cook, R. E. D. and Manlove, R. J. (1983). The ecology of flower and bulbil production in Polygonum viviparum. Nordic Journal of Botany 3, 559-565. Leonard, T.J. and Dick, S. (1968). Chemical induction of haploid fruiting bodies in Schizophyllum commune. Proceedings of the National Academy of Science, USA 59, 745-751. Leung, H. and Taga, M. (1988). Magnaporthe grisea (Pyricularia species), the blast fungus. Advances in Plant Pathology 6, 175-188. Leung, H. and Williams, P. H. (1985). Genetic analyses of electrophoretic enzyme variants, mating type and hermaphroditism in Pyricularia oryzae. Canadian Journal of Genetical Cytology 27, 697-704. MacDonald, D. J. and Bond, D. J. (1976). Genetic and environmental factors influencing the production and distribution of photperithecia in Sordaria brevicollis. Journal of General Microbiology 95, 375-380. Martinelli, S. D. and Clutterbuck, A. J. (1971). A quantitative survey of conidiation mutants in Aspergillus nidulans. Journal of General Microbiology 69, 261268. McCartney, H. A. and Lacey, M. E. (1990). The production and release of ascospores of Pyrenopeziza brassicae. Plant Pathology 39, 17-32. McKay, R. (1939). Observations on onion mildew caused by the fungus Peronospora schleideniana. Journal of the Royal Horticultural Society 64, 272-285. McKormick, F. A. (1925). Perithecia of Thielavia brassicola Zopf in culture and the stimulation of their production by extracts of other fungi. Connecticut Agricultural Experimentation Station Bulletin 269, 539-554. McMeekin, D. (1960). The role of oospores of Peronospora parasitica in downy mildew of crucifers. Phytopathology 50, 93-97. Michelmore, R. W. and Ingram, D. S. (1980). Heterothallism in Bremia lactucae. Transactions of the British Mycological Society 75 ( l ) , 47-58. Moore-Landecker, E. (1982). “Fundamental of the Fungi”, 2d edn. Prentice Hall, Englewood Cliffs, NJ. Moore-Landecker, E . (1988). Response of Pyronema domesticum to volatiles from microbes, seeds and natural substrata. Canadian Journal of Botany 66, 194-198.
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Muller, E. (1979). Factors inducing asexual and sexual sporulation in fungi (mainly Ascomycetes). In “The Whole Fungus: The Sexual Asexual Synthesis”, Vol. I (B. Kendrick, ed.), pp. 265-282. National Museums of Canada, Ottawa. Nauta, M. (1994). Evolution of genetic systems in filamentous ascomycetes. Ph.D. Thesis, Cip-Gegevens Koninklijke Bibliotheek, Den Haag. Nelson, R. R. (1971). Hormonal involvement in sexual reproduction in the fungi, with special reference to F-2, a fungal estrogen. In “Morphological and Biochemical Events in Plant-Parasite Interaction” (S. Akai and S.Ouchi, eds), pp. 181-205. Phytopathological Society of Japan, Tokyo. Ogden, J. (1974). The reproductive strategy of higher plants. 11: The reproductive strategy of Tussilago farfara L. Journal of Ecology 62, 291-324. Pastushok, M. and Axelrod, E. E. (1976). Effect of glucose, ammonia and media maintenance on the time of conidiophore initiation by surface colonies of Aspergillus nidulans. Journal of General Microbiology 94, 221-224. Pitt, J. I. (1979). “The genus Penicillium and its telemorphic states Eupenicillium and Talaromyces.” Academic Press, London. Pontecorvo, G. (1956). The parasexual cycle in fungi. Annual Review of Microbiology 10, 393-400. Pugh, G. J. F. (1980). Strategies in fungal ecology. Transactions of the British Mycological Society 7 5 , 1-14. Rands, R. D. (1917). The production of spores by Alternaria solani in pure culture. Phytopathology 7 , 316-317. Reynolds, D. R. (1993). The fungal holomorph: an overview. In “The Fungal Holomorph: Mitotic, Meiotic and Pleomorphic Speciation in Fungal Systematics” (D. R. Reynolds and J. W. Taylor, eds), pp. 15-25. CAB International, Wallingford, UK. Safeeula, K. M. (1976). “Biology and Control of the Downy Mildews of Pearl Millet, Sorghum and Finger Millet.” Wesley Press, Mysore. Siddiq, A. A. (1989). The control of sexual morphogenesis in Pyrenopeziza brassicae. Ph.D. Thesis, Cambridge University. Siddiq, A. A., Ingram, D. S., Johnstone, K., Friend, J. and Ashby, A. M. (1989). The control of asexual and sexual development by morphogens in fungal pathogens. Aspects of Applied Biology 23, 417425. Siddiq, A. A., Johnstone, K. and Ingram, D. S. (1990). Evidence of the production during mating of factors involved in uppression of asexual sporulation and the induction of ascocarp formation in Pyrenopeziza brassicae. Mycological Research 96, 757-765. Smith, J. E. (1978). Asexual sporulation in filamentous fungi. In “The Filamentous Fungi. Vol. 111: Developmental Biology ” (J. E. Smith and D. R. Berry, eds), pp. 214-239. Edward Arnold, London. Smith, J. E. and Berry, D. R. (1978). “The Filamentous Fungi. Vol. 111: Developmental Biology.” Edward Arnold, London. Turian, G. (1966). Morphogenesis in Ascomycetes. In “Fungi: An Advanced Treatise. Vol. 11: The Fungal Organism” (G. C. Ainsworth and A. S. Sussman, eds), pp. 339-369. Academic Press, London. Turian, G. (1978). Sexual morphogenesis in the ascomycetes. In “The Filamentous Fungi. Vol. 111: Developmental Biology” (J. E. Smith and D. R. Berry, eds), pp. 315-333. Edward Arnold, London. Uramaya, T. (1969). Stimulative effects of extract from fruit bodies of Agaricus bisporus and some hymenomycetes on primordia formation of Marasmius species. Transactions of the Mycological Society of Japan 10, 73-78. van den Ende, H. (1978). Sexual morphogenesis in the Phycomycetes. In “The
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The Role of Leucine-Rich Repeat Proteins in Plant Defences
D. A. JONES and J. D. G . JONES
The Sainsbury Laboratory, John Innes Centre, Colney Lane, Norwich, NR4 7UH, UK
I.
Introduction
...........................................................................
11. Resistance Genes Encoding Proteins with Extracytoplasmic LRRs. A. Resistance Genes Encoding Membrane-anchored Proteins with Extracytoplasmic LRRs and no Kinase Domain ............. B. A Resistance Gene Encoding a Membrane-anchored Protein with Extracytoplasmic LRRs and a Cytoplasmic Kinase Domain .......................................................................... C. Avirulence Determinants that Interact with Resistance Proteins Containing Extracytoplasmic LRRs ..................................... D. Activation of Plant Defences by Resistance Genes Encoding Proteins Containing Extracytoplasmic LRRs ......................... 111. Resistance Genes Encoding Proteins with Cytoplasmic LRRs ....... A. Resistance Genes Encoding Proteins with Cytoplasmic LRRs and Potential Leucine Zippers ................................................ B. Resistance Genes Encoding Proteins with Cytoplasmic LRRs and Homology to the Cytoplasmic Domains of Toll and the Interleukin-1 Receptor ...................................................... C. A Gene Encoding a Protein with Cytoplasmic LRRs that is Required for a Resistance Gene to Function ........................ D. Avirulence Determinants that Interact with Resistance Proteins Containing Cytoplasmic LRRs ............................................ E. Activation of Plant Defences by Resistance Proteins Containing Cytoplasmic LRRs ........................................................... IV.
Defence-related Genes Encoding Proteins with Extracytoplasmic LRRs ................................................................................... A. Polygalacturonase-inhibiting Proteins ................................... B. LRR Extensins ................................................................ C. A Viroid-induced LRR Protein ..........................................
Advances in Botanical Research Vol. 24 incorporating Advances in Plant Pathology ISBN &12405924-X
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Genes Encoding Proteins of Unknown Function with Extracytoplasmic LRRs ........................................................... A. The AWJL Proteins of Wheat ............................................ B. Receptor-like Protein Kinases ............................................
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A Gene Encoding a Protein of Unknown Function with Cytoplasmic LRRs ...................................................................................
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Evolution of Plant LRR Proteins ........................................ The Evolution of LRR Proteins in the Eukaryotes ................ Evolutionary Clues Provided by Intron Arrangements ............ Evolution of Different Specificities in LRR Proteins ..............
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VIII. The Structure and Molecular Specificity of Plant LRR Proteins ...... A. Inferences about the Structure of Plant LRRs by Comparison with the Known Structure of Porcine Ribonuclease Inhibitor ... B. Inferences about the Structure and Interactions of Extracellular Plant LRR Proteins Based on their Potential Patterns of Glycosylation .................................................................. C. Inferences about the Interactions between Plant LRR Proteins and their Ligands Based on Comparisons with the Interactions between Ribonuclease Inhibitors and Ribonucleases ..............
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IX.
The A. B. C.
Concluding Remarks ............................................................... Acknowledgements ................................................................. References ............................................................................
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147 150 153 156
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I. INTRODUCTION After a long drought in our understanding of disease resistance in plants, a flood of information has now been unleashed by the isolation and characterization of a number of disease-resistance and other genes involved in plant defences. Already a recurrent theme is emerging, with genes encoding proteins containing leucine-rich repeats (LRRs) taking centre stage. LRR proteins are involved in specific protein-protein interactions and are confined predominantly to eukaryotes. The existence of LRR proteins in animals and fungi has been known for some time, but has been described only recently and incompletely for plants (for reviews see Kobe and Deisenhofer, 1994, 1995b). The first plant LRR proteins described were receptor serinekhreonine kinases and polygalacturonase-inhibiting proteins (PGIPs) (see sections VB and IVA). In contrast, the existence of disease-resistance genes in plants has been known since the 1900s. These genes confer the capacity to recognize specific races of plant pathogens and to initiate a defence response that is often, but not always, characterized by rapid plant-cell death at the site of infection. Similarly, the existence of genes in plant pathogens, which confer the capacity to be recognized by plants carrying specific resistance genes, has been known
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since the 1940s. These genes have been designated avirulence genes. A precise genetic relationship (usually a one-to-one correspondence) exists between resistance genes in plants and avirulence genes in plant pathogens. Based on this relationship, the products of resistance genes and the products of avirulence genes are predicted to interact, resulting in the initiation and transduction of a defence activation signal. All this has been reviewed extensively previously (e.g. Gabriel and Rolfe, 1990; Keen, 1992), but the recent isolation of some avirulence genes, and the more recent isolation of some resistance genes, now allow a more critical assessment of this prediction at the molecular level. The first gene for race-specific resistance to a plant pathogen to be isolated and characterized was the tomato Pto gene for resistance to the bacterium Pseudomonas syringae pv. tomato which causes specking in tomato (Martin et al., 1993). Pro encodes a serinehhreonine kinase, but does not contain LRRs. However, it has been shown to require an LRR protein to function (see section JIIC). Subsequent to the isolation of Pto, several resistance genes, all encoding LRR proteins, were isolated in quick succession. These resistance genes fall into two clear classes: those encoding proteins containing extracytoplasmic LRRs with the 24 amino acid consensus LxxLxxLxxLxLxxNxLxGxIPxx (see section II), and those encoding proteins containing cytoplasmic LRRs with the 23 or 24 amino acid consensus LxxLxxLxxLxLxx(N/C/T)x(x)LxxIPxx(see section 111).
11. RESISTANCE GENES ENCODING PROTEINS WITH EXTRACYTOPLASMIC LRRS The first resistance genes to be isolated in this class were the tomato Cf-9 gene for resistance to the fungus Cladosporium fulvum (syn. Fulvia fulva) (Jones et al., 1994), and the rice Xa-21 gene for resistance to the bacterium Xanfhomonas oryzae pv. oryzae (Song et al., 1995). These genes represent two subclasses of membrane-anchored extracytoplasmic LRR resistance genes, distinguishable by the presence or absence of a cytoplasmic serinehhreonine kinase domain. A. RESISTANCE GENES ENCODING MEMBRANE-ANCHORED PROTEINS WITH EXTRACYTOPLASMIC LRRs AND NO KINASE DOMAIN
The tomato C’9 gene for resistance to the fungus C . fulvum (syn. Fulvia fulva), which causes leaf mould in tomato, encodes an 863 amino acid, membrane-anchored, extracytoplasmic, glycoprotein containing 27 imperfect LRRs (originally reported as 28) averaging 24 amino acids in length (Fig. 1) (Jones er al., 1994). The LRRs show a good match to the extracytoplasmic LRR consensus LxxLxxLxxLxLxxNxLxGxIPxx (Table I). However, the
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LRR domain is interrupted by a short region (domain C2 in Fig. l ) , designated a “loop out” domain, which was originally reported as an LRR, but which actually has little LRR homology. This domain divides the LRR domain into 23 amino terminal LRRs (domain C1) and 4 carboxyl terminal LRRs (domain C3). The short cytoplasmic carboxyl terminus of Cf-9 concludes with the motif KKxx, which as noted previously (Jones et al., 1994), may function to localize the protein to the endoplasmic reticulum. DNA gel blot analysis reveals Cf-9 to be a member of a multigene family with at least 10-12 members. Segregation analysis shows these to all be clustered on the short arm of chromosome 1 of tomato (M. Parniske, D. A. Jones, C. M. Thomas and J. D. G. Jones, unpublished). RNA gel blot analysis reveals only a single class of transcript which presumably contains mRNAs derived from several members of the gene family. Cf-4,located at the same complex locus on the short arm of chromosome 1 as Cf-9, encodes a protein that is very similar to that encoded by Cf-9 (C. M. Thomas, D.A. Jones, P. J. Balint-Kurti, K. Harrison and J. D. G. Jones, unpublished). Interestingly, the carboxyl terminal portion of Cf-4 is identical to Cf-9, suggesting that the specificity differentiating Cf-4 from Cf-9 must reside in the amino terminal portions of these proteins. If specificity of resistance gene function is determined by direct binding of the Avr ligand then the conserved carboxyl termini are presumably not involved in ligand binding. A third Cf gene, Cf-2, isolated from a complex resistance locus located near the centromere of chromosome 6 of tomato, comprises two closely linked, nearly identical genes (designated Cf-2. I and Cf-2.2) encoding 1112 amino acid proteins that differ by only three residues (Dixon et al., 1996). Both Cf-2 genes confer resistance independently. The Cf-2 proteins are membrane-anchored, extracytoplasmic glycoproteins similar to Cf-9, but lack the KKxx motif of Cf-9, suggesting that either Cf-9 functions in a different
Fig. 1. Primary structure of the predicted Cf-9 protein (Jones et al., 1994). The amino acid sequence, shown in single-letter code, has been divided into seven domains A to G. A, the predicted signal peptide; B, the predicted mature amino terminus; C, the LRR domain; D, a connecting domain; E, acidic domain; F, the predicted transmembrane domain; G, basic domain. Domains E, F and G are predicted to anchor and orient Cf-9 in the cell membrane, so that domains B , C and D are extracytoplasmic domains and domain G is a cytoplasmic domain. Domains B and D show homology to the polygalacturonase-inhibitingproteins (PGIPs) (see section IVA) and receptor-like protein kinases (RLPKs) (see section VC) (Jones et al., 1994). Domain C has been divided into three subdomains C1 to C3. C1 and C3, LRR domains; C2, a “loop-out” domain which departs from the LRR consensus. LRRs have been numbered sequentially from the amino terminus. Potential N glycosylation sites are underlined. Residues matching the LRR consensus LxxLxxLxxLxLxxNxLxGxIPxx,in which L and I tend to be substituted by each other and by M, V and F, are highlighted in bold. The vertical lines bracket the region predicted to form a solvent-exposed p sheet.
I
LEUCINE-RICH REPEAT PROTEINS IN PLANT DEFENCES
A
MDCVKLVFLMLYTFLCQLALSSS
B
LPHLCPEDQALSLLQFKNMFTINPWDYCYDIR TYVDIQSYPRTLSW&!&!TSCCSWDGVHCDETTGQ
c1
V LFQLSNL FGEFSE ICHLSKL LKNLTOL SHL VFHLSNL WSASL FSHLTSL LWJJJJNI FTIFEKL LSFNTQL SGLQNL IFSLPSL FKS KTL LLNQKNL ICNLKTL VERNEYL FSVGNIL MINCKYL LGYLFQL TNLFMGL LGNLQTW
IALDLRC KRLDLSF ZHLDLSH HVLRICD RELNLES TTLQLSG QSLHLSV MTLYVDS HELYMGR VFLHLGD KRLSLVN ERLDLSS ECLYLSS VELDLSK SAVTLKQ QLLLLSH ILLDLGS SHLDLSK RVISLHG TLLDLGN KILSLRS QILDLSS KEID
SQ NN SS QY
LQGKFHSE FTGSLISPK FTGLIPSE GLSLVPYNFELL VN ISSTIPSB TE LHGILPER NPQLTVRFPTTK VN IADRIPKS CN LSGPIPKP NH LEGPISH NN FDGGLEF NS LTGPIPSB NH L m I P s w FSGKIQE NK LKGRIPNS NN ISGHISSA NN LEGTIPQCV NR LSGTINTT NK LTGKVPRS NM LNmFPNW NK LHGPIKSSGN NG FSGNLPERI
93
signal peptide
LRRl 1rr2 1rr3 1rr4 1rr5 1rr6 1rr7 1rr8 1rr9 LRRlO 1rr11 1rr12 1rr13 1rr14 1rr15 1rr16 1rr17 1rr18 1rr19 LRR20 LRR21 LRR22 LRR23
c2 ESTGFPEYISDPYDIYYNYLTTISTKGQDYDS lOOpOUt c3 VRILDSN , M I I m KNR IGDLVGL R T L U H NV PQNLsVL 'ESLDLSSNK NH LASLTFL
HMLVEI
FEGHIPSI LEGHIPAS ISGEIPQQ LVGCIPKG
LRR24 LRR25 LRR26 LRR27
D
KQFDSFGmYQGNDGLRGFPLSKLCGG
E
EDQVTTPAELDQEEEEEDSPMISWQ
acidic domain
F
GVLVGYGCGLVIGLSVIYIMSTQ
hydrophobic domain
G
YPAWFSRMDLKLEHIITTKMKKHKKRY
basic domain
TABLE I LRR consensus sequences for plant proteins with extracytoplasmic LRRs and a known or possible role in plant defences Protein
Species
Repeatsa
Consensus sequenceb
Reference
L--L--L--LDLS-N-L-G-IP-a N a
Jones et al. (1994)
LGNL--L--L-L--N-L-GSIP-Y
Dixon et al. (1996)
Xa-21
Lycopersicon pimpinellifolium Lycopersicon pimpinellifolium Oryza sativa
L--L--L--L-L--N-L-G-IpsI I 1
Song et al. (1995)
PGIP
Actinidia deliciosa
Simpson er al. (1995)
PGIP
Antirrhinum majus
PGIP
Glycine max
PGIP PGIP
Lycopersicon esculentum Phaseolus vulgaris
PGIP
Pyrus communis
LRP
Lycopersicon esculentum Lycopersicon esculentum Zea mays
aG-L--L--L-as-NKLTG-IP-S N S L--L--L-SL-LSRNRLSG-IP-H N T a-SLK-L--LDLS-NNL-G-IP-aP L aG-LPNL--L-LSR"Ln;-IP- S S K H + A/G aA-LK-L--L-LS-NNL-G-IP-G P R F--LK-L--L-LS-NNLSG-IPP K T LG-L--L--LDL-NN-L-GTIP-E V I S L--L--L--L-LS-N-F-G-aP-a a N a L--L--L--1DaS-N-F-G-aP-a a a N
Cf-9
Cf-2
LRR extensin LRR extensin
\o P
-
Steinmayr et al. (1994)
P ?
Favaron et al. (1994) Stotz et al. (1994) Toubart et al. (1992) Stotz et al. (1993) Tornero et al. (1996)
D. A. Jones (unpublished) Rubinstein et al. (1995)
aThe number of full-length LRRs is indicated together with the number of incomplete LRRs in parentheses. bAmino acid residues are shown in single-letter code. Residues matching the LRR consensus LxxLxxLxxLxLxxNxLxGxIPxx are highlighted in bold. a, The aliphatic residues L, I, M, V or F; -, any residue; +, positively charged residues K or R. Where single residues are shown these comprise more than 50% of the residues at this position. Where two residues are shown, the two residues together comprise more than 50% of the residues at this position, with the upper of the two being more frequent.
c
P F
LEUCINE-RICH REPEAT PROTEINS IN PLANT DEFENCES
95
cellular location to Cf-2, which might account for the differences in the response to their respective avirulence peptides (Hammond-Kosack and Jones, 1994; Hammond-Kosack et a f . , 1996; May et al., 1996), or this motif has little bearing on the resistance function of these proteins. Each Cf-2 protein has 38 LRRs which show a good match to the extracytoplasmic LRR consensus LxxLxxLxxLxLxxNxLxGxIPxx (Table I). Of these, 34 (LRRs 2-31 and 35-38) are exactly 24 amino acids in length, and 20 (LRRs 6-24) show an alternating structure of two highly conserved repeats designated type A and B, reminiscent of the alternating LRRs of the porcine ribonuclease inhibitor (see section VIIIA). Type A repeats have the consensus EEIGYLRSL(T/N)xL(D/S/G)LSENALNGSIP and type B repeats have the consensus ASLGNLNNLS(M/F/R)L(Y/F/N)LYNNQLSGSIP. These repeats are arranged (AB)3AB4(AB)4A followed by the first five residues of a B repeat. Interestingly, the carboxyl terminal 360 amino acids of Cf-2 show high homology with the carboxyl terminal 352 amino acids of Cf-9, which includes the region that is identical between Cf-9 and Cf-4, suggesting that this region of conservation may play a similar and important role in all three proteins. This region includes the LRR domains C1 and C3, and domains D, E and F, but to a lesser extent the “loop out” domain C2 and domain G (Fig. 2). A “loop out” domain seems to be absent from most other extracytoplasmic LRR proteins and may play an important role unique to the Cf proteins, possibly as a molecular hinge connecting the two flanking LRR regions. It seems unlikely that the conserved flanking LRRs are involved in ligand binding, but it is possible that this region interacts with other protein components of a defence-activation, signal-transduction pathway. The conservation between Cf-9 and Cf-2 in this region suggests that they might interact with similar components, but the difference in the phenotypes conferred by Cf-9 and Cf-2 suggests they may be distinct. Alternatively, the differences in phenotype may be due to a difference in gene expression. Cf-5, located at the same complex locus on chromosome 6 as Cf-2, encodes a protein that is very similar to that encoded by Cf-2, but with fewer LRRs (K. Hatzixanthis, M. S. Dixon, K. Harrison, D. A.Jones and J. D. G. Jones, unpublished). B. A RESISTANCE GENE ENCODING A MEMBRANE-ANCHORED PROTEIN WITH EXTRACYTOPLASMIC LRRS AND A CYTOPLASMIC KINASE DOMAIN
The rice Xa-21 gene for resistance to the bacterium X. o. pv. oryzae, which causes leaf blight in rice, encodes a 1025 amino acid, glycoprotein with an extracytoplasmic receptor domain containing 23 LRRs linked by a single transmembrane domain to a cytoplasmic serinehhreonine kinase domain (Song et al., 1995). The LRRs of Xa-21 also match the extracytoplasmic LRR consensus LxxLxxLxxLxLxxNxLxGxIPxx (Table I). Unlike the Cf genes there is no conspicuous “loop out” within the LRR domain. D N A gel blot
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D. A. JONES and J . D. G. JONES
c1 C f -9 C f -2
VERNEYL SHLDLSK NRLSWINTT F G U S L EVFDMQN NKLSGTLPTN
LRRl8 LRR29
C f -9 C f -2
FSVGNIL RVISLHG NKLTGKVPRS mIGcsL ISLNLHG NELEDEIPRS
LRR19 LRR30
C f -9 C f -2
MINCKYL TLLDLGN -FPNW LDNCKKL QVLDLGD NQLmFPMW
LRR20 LRR31
Cf-9 C f -2
LGYLFQL KILSLRS NKLHGPIKSSGN LRR21 LGTLPEL RVLRLTS NICLHGPIRSSRA LRR32
C f -9 C f -2
TNLFMGL QILDLSS NGFSGNLPERI EIMFPDL RIIDLSR NAFSQDLPTSL
LRR22 LRR33
C f -9 C f -2
LGNLQTM KEID FEHLKGM RTVD
LRR23 LRR34
c2 C f - 9 C f -2
c3 C f -9 C f -2
ESTGFPEYISDPYDIYYNYLTTISTKGQDYDS KTMEEPS YSSYYDDSWWTKGLELEI VRILDSN M I I M K NRFEGHIPSI LRR24 LRR35 VRILSLY TVIDLSS NKFEGHIPSV
C f -9 C f -2
IGDLVGL R T L U H NVLEGHIPAS LGDLIAI R I L U H NALQGYIPSS
LRR25 LRR36
C f -9 C f -2
FQNLSVL ESLDLSS NKISGEIPQQ LGSLSIL ESLDLSF NQLSGEIPQQ
LRR26 LRR37
C f -9 C f -2
LASLTFL E V L m H NHLVGCIPKG LASLTFL E F L U H NYLQGCIPQG
LRR27 LRR38
Cf-9 C f -2
KQFDSFGmYQGNDGLRGFPLSKLCGG
E
C f -9 C f -2
EDQVTTPAELDQEEEEEDSPMISWQ DPVSEWSALEDQESNSEFFNDF WK
Fa
C f -9 C f -2
GVLVGYGCGLVIGLSVIYIMWSTQ AALMGYGSGLCIGISIIYILISTG
D
G
PQFRTFESNSYEGNDGLRGYPVSKGCGK
C f -9 YPAWFSRMDLKLEHIITTKMKKHKKRY* C f -2 NLRWLARIIEELEHKIIMQRRKKQRGQRNYRRFWNRF* Fig. 2. Comparison of the primary structures of the Cf-9 and Cf-2.1 proteins in the conserved carboxyl terminal portions of the proteins. Various features are indicated as described in Fig. 1, except that identical residues are highlighted in bold.
LEUCINE-RICH REPEAT PROTEINS IN PLANT DEFENCES
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analysis, with a probe that hybridizes to the Xu-22 coding sequence, revealed a gene family with eight members and segregation analysis showed that at least seven of these co-segregate with Xu-21, indicating a complex resistance locus. RNA gel blot analysis revealed a 3.1 kb band corresponding to the expected full-length Xa-21 transcript, and three additional smaller bands which may correspond to alternative splice products or to other members of the Xu-21 gene family which may have fewer LRRs. The Xa-21 protein shows homology with the receptor-like serinehhreonine kinases TMKl (Chang et al., 1992) and RLKS (Walker, 1993) from Arabidopsis, the receptor-like Cf-9 protein (Jones et a f . , 1994) and the Pto serinekhreonine kinase (Martin et al., 1993). Xu-21 provides a conceptual link between resistance genes that seem to encode a receptor but lack a signalling mechanism (i.e. the Cfgenes) and those that encode a signalling mechanism but lack a receptor (i.e. Pro), suggesting that the Cfgenes may in fact activate plant defences by interaction with a signalling component such as a protein kinase and that Pto may interact with and require activation by a receptor. It also raises the possibility that receptor-like protein kinases with no known function (see section VB) may in fact be resistance proteins analogous to Xa-21.
C. AVIRULENCE DETERMINANTS THAT INTERACT WITH RESISTANCE GENES ENCODING PROTEINS CONTAINING EXTRACYTOPLASMIC LRRs
The C. fulvum Avr9 gene encodes a mature secreted avirulence peptide of 28 amino acids (van den Ackerveken et ul., 1992). This peptide elicits a necrotic response in plants carrying Cf-9, but not in plants lacking Cf-9, and seems likely to be the ligand that interacts directly with the Cf-9 protein. However, binding studies, using 1251 labelled Avr9 peptide, show that membranes from tomato plants lacking Cf-9 bind Avr9 to the same extent as membranes from plants carrying Cf-9 (Kooman-Gersmann et ul., 1996). Membranes from other solanaceous species also bind Avr9. However, these plants although lacking Cf-9, carry Cf-9 homologues that might also bind Avr9. It is possible that only Cf-9 is able to trigger the activation of plant defences in response to Avr9 binding. Thus, the specificity of interaction between resistance and avirulence gene products may be a combination of the ability not only to bind the avirulence ligand but also to generate the appropriate signal. Alternatively, Avr9 may bind to other proteins and produce a high background of binding upon which a low amount of Cf-9-specific binding is superimposed. If Avr9 does bind to other proteins, it is possible that Cf-9 recognizes a conformational change in one of these proteins rather than recognizing Avr9 directly. The Avr9 peptide is folded into three anti-parallel p strands connected by loops and cross-linked by three disulphide bonds forming a compressed barrel-like structure (Vervoort et at., 1996). This structure is extremely small
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D. A. JONES and J . D. G . JONES
in relation to the Cf-9 protein, implying a very small area of contact. The presumed interactive face of the Avr9 peptide contains a number of positively charged residues. Cf-4 interacts specifically with the C. fufvum Avr4 gene which encodes a mature secreted peptide of 106 amino acids (Joosten et u f . , 1994). There is no amino acid sequence similarity between Avr4 and Avr9, but both have a similar proportion of charged residues (23.5% for Avr4 and 25% for Avr9) and a similar ratio of 2.5 times more basic residues than acidic residues, so Avr4 may also have a positively charged interactive face. The positively charged residues of Avr4 are composed entirely of lysine residues except for a single histidine. Avr4 is also proline rich (16%), and therefore differs significantly from Avr9 which has no proline residues. The Avr2 avirulence determinant, although not yet isolated, also seems to be a small secreted peptide (P. J. G. M. De Wit, unpublished). The uvrXu21 avirulence gene of X . 0. pv. oryzae has not been characterized, but a number of other Xunthornonas avirulence genes, including avrxa5, avrXa7 and avrXal0, form a homologous group of which uvrXu21 may be a member. The uvrXa10 gene has been sequenced and found to encode a protein with tandem-repeats of a highly conserved 34 amino acid motif LTPxQVVAIASxxGGKQALETVQRLLPVLCQxHG (Hopkins et u f . , 1992). These repeats suggest a structural rather than enzymatic function for these avrXa proteins, and therefore that they may interact directly with resistance gene proteins. A protein ligand would also be consistent with the LRR nature of the receptor domain in the Xa-21 protein. Xu-10 and Xu-21 are members of the same resistance gene cluster on chromosome 11 of rice (Ronald et af., 1992), so it seems likely that they may encode related receptor protein kinases that interact with similar avirulence ligands. Homologous avirulence genes from other Xunthomonas pathovars have also been isolated and sequenced. These include the avrBs3 and uvrBs3-2 genes from Xunthomonus cumpestris pv. vesicatoriu (Bonas et al., 1989, 1993), the avrb6 gene from Xunthomonus campestris pv. mulvucearum (De Feyter et af., 1993) and thepthA gene from Xanthomonus citri (Genbank Accession U28802). These genes show variation in the number of repeats of the 34 amino acid motif, with avrb6 having 13.5, avrXalO having 15.5, and avrBs3, avrBs3-2 and pthA having 17.5. These genes also show variation at four positions within the 34 amino acid repeat consensus with A, D, E, Q or V at position 4, N or H at position 12, D, G , I, N or S at position 13 and A or D at position 32. Each gene shows a distinct pattern of residues in these positions and these are thought to determine the avirulence specificity. Deletion analysis of the avrBs3 gene provides additional evidence to support this hypothesis (Herbers et af., 1992). Precise deletions of internal repeats in uvrBs3 has several different effects on pathogenicity towards near-isogenic lines of pepper either carrying Bs3 or lacking Bs3. Some retained avirulence on Bs3, some showed intermediate avirulence and the remainder showed no avirulence. Surprisingly, some of the latter had lost their avrBs3 specificity,
LEUCINE-RICH REPEAT PROTEINS IN PLANT DEFENCES
99
but gained new specificities, including avirulence to the recessive bs3 allele which hitherto did not confer resistance. Similar changes in specificity have also been observed among intragenic recombinants of the pthA gene of X . citri (Yang and Gabriel, 1995b). The generation of new specificity by artificially altering the number of repeats supports a model for the natural generation of new specificity by unequal cross-overs between repeats, which would increase or decrease their number and generate new permutations of the variable residues in the sequence. This may in part explain the different numbers of repeats seen not only for the 34 amino acid repeats of these avirulence genes, but also for the LRRs of the Cf genes of tomato and possibly for the Xu genes of rice. Despite the arguments in favour of a direct interaction between a cell surface receptor like Xa-21 and an avirulence protein like avrXal0, this hypothesis is not easily accommodated by observations that several of these avirulence proteins are only detectable in the bacterial cytoplasm (Brown et al., 1993; Young et al., 1994). Expression of these avirulence proteins is hrp independent (Knoop et at., 1991; De Feyter et al., 1993; Young et ul., 1994; Yang and Gabriel, 1995a), but their ability to cause avirulence is hrp dependent (Knoop et al., 1991; Yang and Gabriel, 1995a). The hrp genes (see review by Willis et al., 1991) are induced in planta and are required for the ability of incompatible bacteria to produce a hypersensitive response (HR) and for pathogenicity. Among other functions, they encode a signal-peptide-independent pathway for secretion of proteins homologous to that of a number of animal pathogens (Fenselau et al., 1992; Van Gijsegem et al., 1993). The cytoplasmic location of avirulence proteins is consistent with their constitutive expression, but their redistribution upon induction of the hrp-dependent secretory pathway may be subtle in comparison. It is conceivable that small quantities of avirulence protein are secreted into the apoplast and interact there with plant receptors like Xa-21. It is also conceivable that hrp-dependent translocation of proteins from bacterial to plant cytoplasm occurs, since this has been shown to occur in the homologous animal systems (Rosqvist et al., 1994, 1995). Avirulence proteins could then be presented to plant receptors at points of contact between bacterial and plant cells. Consistent with this hypothesis, Brown et al. (1993) observed that the first response of Bs3 pepper plants to either compatible or incompatible X . c. vesicutoria was localized convolution of the cell membranes adjacent to contacts between the plant and bacterial cell walls, prior to cytoplasmic vesiculation and HR in incompatible cells. D. ACTIVATION OF PLANT DEFENCES BY RESISTANCE GENES ENCODING PROTEINS CONTAINING EXTRACYTOPLASMIC LRRs
Two functions are required for the activation of plant defences by resistance proteins. A ligand must be recognized and, upon recognition, a signal
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initiated. For Xa-21, which has an LRR receptor domain and a protein kinase signalling domain, this would seem relatively straightforward, although downstream components of the signalling pathway still need to be identified. For the Cf proteins, which have LRR receptor domains, the recognition of a ligand is readily envisaged, but the initiation of a signal is enigmatic in the absence of any obvious signalling domains. A similar enigma exists for the structurally analogous RP105 protein of mouse, which is a membraneanchored protein with a large extracytoplasmic domain containing 22 LRRs and a short, basic, cytoplasmic tail of 11 amino acids (Miyake et af., 1995). Interestingly, binding of an anti-RPlO5 monoclonal antibody to RP105 leads to B cell proliferation and protection from dexamethasone- and irradiationinduced apoptosis. Thus RP105, like the Cf genes in tomato, is implicated in defence and cell death responses. There are two possible ways in which the Cf proteins may generate a defence activation signal. One is via their small cytoplasmic tails, which may interact with a cytoplasmic signalling component such as a Pto-like kinase. A precedent for such a mechanism has already been noted (Jones et af., 1994) and there are many others (Weiss and Littman, 1994). The other is via another transmembrane signalling protein such as an Xa-21-like receptor kinase. The comparison between Cf-9, Cf-4 and Cf-2, showing conservation between the carboxyl terminal 10 LRRs of these proteins (see section IIA), suggests that these regions are the most likely to interact with common or conserved signalling partners. Binding of Avr peptide to Cf protein might then cause a conformational change in these domains, causing the activation of a signalling partner (Fig. 3). The amino terminal 17 LRRs of Cf-9 and 28 LRRs of Cf-2 are likely to contain the regions that bind the Avr9 and Avr2 peptides, respectively. For Cf-9, it seems unlikely that all the LRRs participate in binding of the small Avr9 peptide. There are several possible explanations for the large number of LRRs. They may be required to provide a structural platform or for multiple avirulence peptide binding. The latter may be an explanation for the number of conserved alternating LRRs in Cf-2, but seems unlikely for Cf-9, because the LRRs are so different from one another. Alternatively, Cf-9 may be a multiple receptor able to bind different avirulence determinants at different locations. However, no such avirulence specificities have yet been identified. Another alternative is that the LRR domain binds to a large plant protein and Avr9 modulates this interaction, either by binding to Cf-9 or to the other protein. A precedent for the latter exists for the platelet glycoprotein complex (PGC) which is composed of four extracytoplasmic LRR proteins GP Iba, GP Ibp, GP V and GP IX (reviewed by Lopez, 1994). Vascular trauma activates von Willebrand factor (vWf) which then binds to the PGC and induces platelet aggregation to help staunch the flow of blood. Two snake-venom proteins, the one- and two-chain botrocetins (Fujimura et al., 1991; Usami ef af., 1993) produced by the South American pit viper,
LEUCINE-RICH REPEAT PROTEINS IN PLANT DEFENCES AvrS
101
Cf-9
44 SIGNAL
4 4
SIGNAL
Fig. 3. Models for the initiation of a plant defence activation signal following interaction between Cf-9 and Avr 9. Binding of Avr9 is presumed to induce a steric change in Cf-9 which activates a protein partner comprising either a membraneassociated, cytoplasmically located, protein kinase or a receptor-like protein kinase, which then initiates a cytoplasmic, defence-activation signal. Consistent with the mode of action of many mammalian receptor kinases (Heldin, 19951, the models incorporate a dimerization requirement for kinase activation. However, it should be noted that the signalling partner need not be a protein kinase.
Bofhrops jararaca, bind not to the PGC but to vWf, causing its inappropriate activation and binding to the PGC and the pathological aggregation of platelets. Clearly, further understanding of the Cf-protein-mediated activation of plant defences depends on the isolation of their signalling partners.
111. RESISTANCE GENES ENCODING PROTEINS WITH CYTOPLASMIC LRRs The first resistance genes to be isolated in this class were the Arubidopsis RPS2 gene for resistance to the bacterium P. s. pv. tomato, isolated simultaneously by Bent ef a f . (1994) and Mindrinos et al. (1994), and the Nicotiana glutinosa N gene for resistance to tobacco mosaic virus, isolated
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D. A. JONES and J . D. G . JONES
by Whitham et al. (1994). These genes represent two subclasses of resistance proteins with cytoplasmic LRRs, distinguishable by features in their amino termini. A third subclass, represented by a candidate for the wheat Cre3 gene for resistance to the cereal cyst nematode Heterodera avenae, also encodes a protein with cytoplasmic LRRs, but a novel amino terminus (E. S. Lagudah, S. Chandramohan, 0. Moullet, R. Eastwood, R. Appels, K. Anderson and S. Anderson, unpublished). This subclass is not discussed further. A fourth subclass is represented by the tomato Prf gene (Salmeron et al., 1996), which is not technically a resistance gene, but is required for the Pto gene for resistance to P. s. pv. tomato to function.
A. RESISTANCE GENES ENCODING PROTEINS WITH CYTOPLASMIC LRRs AND POTENTIAL LEUCINE ZIPPERS
The Arabidopsis RPS2 gene for resistance to the bacterium P. s. pv. tomato encodes a 909 amino acid protein with a hydrophobic amino terminal domain which may be a membrane anchor, a potential leucine zipper comprising four leucines with heptad spacing, a nucleotide binding site (NBS) comprising kinase l a (P-loop), 2 and 3a domains, an internal hydrophobic domain, and 14 imperfect LRRs (Bent er al., 1994; Mindrinos et al., 1994). The leucine zipper domain is predicted to form an amphipathic a helix able to form homo- or hetero-dimers. The NBS binds ATP/GTP in various kinases and kinase-activating proteins and, although RPS2 is clearly not a kinase, the presence of a NBS suggests that it may activate a kinase. The internal hydrophobic domain contains sequence matching the consensus motif GLPL(A/T)(L/I)xxaG(S/G)aa(where a is an aliphatic amino acid) found in all the resistance genes with cytoplasmic LRRs. The function of this motif is unknown, but its conservation suggests that it may be functionally important. The LLRs show a good match to the cytoplasmic LRR consensus LxxLxxLxxLxLxx(NICIT)x(x)LxxIPxx with five C, four T and two N at position 15 in the consensus and 10 of the 14 repeats showing the extra residue at position 17 (Table I1 and Fig. 4). RNA gel blot analysis of uninfected leaves revealed 3.1 and 3.8 kb transcripts which differ by an extension of the 3' untranslated region. DNA gel blot analysis carried out under high stringency reveals RPS2 to be a single copy gene, but additional bands were detected under low stringency, suggesting that there may be related sequences in the Arabidopsis genome. The Arabidopsis RPMl gene for resistance to the bacterium P. s. pv. maculicofa encodes a 926 amino acid protein which shows the same structural organization and significant homology with RPS2, except that it has no amino terminal hydrophobic domain (Grant et a f . , 1995). It contains a potential leucine zipper comprising five leucines and one isoleucine with
TABLE I1 L R R consensus sequences for plant proteins with cytoplasmic LRRs and a known or possible role in plant defences Protein
Species
Repeats
Consensus sequencea
Reference
RPS2 RPMl Myosin homologue N L6
"Amino acid residues are shown in single-letter code. Residues matching the LRR consensus LxxLxxLxxLxLxx(N/C/T)x(x)LxxIPxx are highlighted in bold. a, The aliphatic residues L, I, M, V or F; -, any residue; +, positively charged residues K or R. Where single residues are shown these comprise more than 50% of the residues at this position. Where two residues are shown, the two residues together comprise more than 50% of the residues at this position with the upper of the two being more frequent. Three consensus sequences are shown for L6 corresponding to the various lengths of its LRRs.
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D. A . JONES and J D. G . JONES
RPSP
RPM1
AENWRQ A LI CPK L FMHMPV L IKYLVE L LGNLRK L ICWLSK L LG FAD L FGALHKHI LTNHGRNL NDWLPS L QDCLRN I VQKLPK L PTLFPS L RFSFQK V
LVISLLD TTLMLQQ RVLDLSF YHLSMSG KHLDLQR EVLNLYY EYLENLT QHLHVEE RRLSIKS EVLTLHS RCINISH EVIELFD KTLRTRD ETLVITN
NR IQTL PEK NSSLKKI PTGF TS ITEI PLS TK ISVL PQE TQFLQTI PRDA SYAGWELQSFGEDEAE TLGITVLSLETLKTLFE CNELLYFNLPS CHDLEYLVTPADFE LHNLTRVWGNSVS CNKLKNVSW CREIEELISEHESPSVED LPELNSI LPS CPRVKKL PFQ
AETMENY I RATNL LPSLNLL LVTMFNL FHKLVNL MWKLKKL VPKIWQL LGCMTQL LNKIKRI L IATASI FNTLQNL IQTLPRL FQNLKIL MFEL IENLINL
GSRHLCI HSLLVCS RALDLED KYLNLSK ETLNTKH RYLITFR KDLQVMD TRISLVM RFLSL T EKLFL A TYLGLRG VWLSFYN EIVQMKH QKLYVRA QELHLIH
Q KEMTPDS SA KHKMEL SS ISKLPDC TQ VKELPKN SK IEELPLG RNEGHDSNWNYVLGTRV CFNA EDELIKN VRREHGRDLCDS SI DEEEPLEIDD GK LERVPSW SQ LQENAILS AY MGPRLRFAQG LT EWIEDGA CRG LEYVPRG VS NQLVERIRGEGSVDRS
Fig. 4. Comparison of the primary structure of the LRR domains of the cytoplasmic resistance proteins, RPS2, RPM1, N and L6,and the Arubidopsis myosin heavy-chain homologue (Genbank U19616) which is a potential resistance protein. The amino acid sequence is shown in single-letter code. Residues matching the L R R consensus LxxLxxLxxLxLxx(N/C/T)x(x)LxxIPxx, in which L and I tend to be substituted by each other and by M, V and F, are highlighted in bold. The individual consensus sequences are shown beneath each protein. a , The aliphatic residues L,
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myosin FTDCKYL RVL DISK SIFDAP LSE ILDE hom IASLQHL ACL SLSN THP LIQ FPRS MEDLHNL QIL DASY CQN LKQ LQPC IVLFKKL LVL DMTN CGS LEC FPKG IGSLVKL EVLLGFKP ARSNNGCKLSE VKN LTNLRKL GL SLTR GDQ IEEEELDS LINLSKL MSI SINC YDSYGDDLITK IDA LTPPHQL HEL SLQF YP GKS SPSW LSPHKLPML RYM TICS GN LVK MQEPFWGNENTHWR IEGLMLSSL SDL DMDW EV LQQS MPYL a--L--L
N
IDYLPNN TFELKML TKHLPSL FTGMPNL LGCCSKV V NVESL YGRMKPE FQYKTHV ICRLKSL IGDLDNL IIRLNKL AEGLHSL IGSLSSL IAQLGAL LP PEL VTKRKKL
LRCFVCT VHLQLRH RRIDLSW EYVNLYQ IGLYLND EYLGLRS IQIHMQG TKLLLWN VSLSVSG RVFDAS IILMFRG EYLNLSY KKLDLSR QSLDLKD NELHVDC HRVKLDD
NYP WE SFPS NS LR HLWTE SKR LT RTPD CSN LE EVHHS CKS LK RFPC CDS LE KLPEI SG IR ELPSSI MKN LV ALPSS CSK LE SLPEE DTL IL RPPSS FKDG VHFEFPPV CNL IDGGLPEE NN FE HLPSS CQR LT QLPE HMA LK FIHYL AHNDTMY NLFAY
I--L--L --L-L-- c-- L-- Lp-N a Fig. 4. (concd.) I. M, V or F; -, any residue, +, positively charged residues K or R. Where single residues are shown in the consensus, these comprise more than 50% of the residues at this position. Where two residues are shown, the two residues together comprise more than 50% of the residues at this position, with the upper of the two being more frequent. Three consensus sequences are shown for L6 corresponding to the various lengths of its LRRs. The duplicated LRRs of L6 are underlined. The vertical lines bracket the region predicted to form a solvent-exposed p sheet.
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FLNLSEL LPNL WKNL MKMAERL LSDCWRF IGELKKL FGMLKGL VADIGQL IPNLSQLLDL WWKVSKLKSL LPRYLLPTSL IENLENL LDGLQGL LARIKGL ITEC V O T W VPSM IRSLPKFPML LGAIGSLEEL IVSSSKLOKL IEGLEELKSL LEKL VOTWAVPSL IOSLPKFPML LEVLGSLEEL ISFLSKLOKL IEGLAFLKSG QQQLGSLKNL
RYLHARE KWLELPF IIVILEH KWRLAS PKSIEVLS KTLVLKF RELCLEF SSLKVLK EVLKWD QLEKTRI TYLKIYQ TSLEV RSLEIL KDLLCSS PDLIELL AELTIRD KKLDLAV VSLELEL TTLW QDLYLEG KELDIGG RGLTIRD NEL TLSm DSLELTI, TTLIV RILYLEG NVLDIQG
AMLTGDFNNL YKRGEDDPPLTNYT SHITADDWGGWRHM NYSLYGRRVR MTAIEMDEVD CP IQKISGGT NWGTNLREV TTGAKEVEINEFPLGLKELSTSSR CKDGFDMPPASPSEDESSV "VVDDASSGGH CTEPTWLPG NDIFQTLGGD RIRKVNG TCKLRKFY PCELG CPRLEVGPM UIITKEED DDTSSGIER duplication 1 KVPSLRE; CTSLGRQP CPDLTEL CPRLEVGPM
UITKEDE
DDTCSSIER duplication 2 EVPSLRE CTSLERLWPD CKSLSVDH
f--L--L--L --L-L-a a a--L--L --L-a-a--L --L-a-Fig. 4 (contd.)
heptad spacing, an NBS, an internal hydrophobic domain and 15 imperfect LRRs. The potential leucine zipper motif of RPMl overlaps but is out of phase with that of RPS2 and is therefore not strictly homologous. The leucine residues in the homologous positions are reciprocally substituted by other aliphatic amino acids, so that the consensus heptad motif is Lxxaxxx in RPMl and axxLxxx in RPS2 (where a is L, I, M, V or F). The internal hydrophobic domain of RPMl is homologous to that of RPS2 and matches the consensus motif GLPL(A/T)(L/I)xxaG(S/G)aa.The LRRs of RPMl are poorly conserved after residue 12 (underlined) of the consensus
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LxxLxxLxxLxLxx(N/C/T)x(x)LxxIPxxwith only two C , one T and no N at position 15 and with aliphatic amino acids and prolines comprising less than half the residues at positions 18 and 22, respectively (Table I1 and Fig. 4). Interestingly five of the residues at position 15 were serines, suggesting that this may function as an alternative to N, C or T in this position. Only one LRR showed the extra residue at position 17 of the consensus out of the 10 LRRs in which this could be assessed. The LRR domain of RPMl is therefore quite different from that of RPS2. A single 3 kb transcript was detected by gel blot analysis of RNA prepared from leaves of resistant lines. DNA gel blot analysis of DNA prepared from resistant ecotypes of Arabidopsis revealed only two hybridizing bands both corresponding to R P M l , under stringent hybridization conditions. Surprisingly, no homologue of RPMZ was detected in DNA from susceptible ecotypes of Arabidopsis. In all other cases so far described, resistance gene probes have detected homologous genes in susceptible lines of the host. Low stringency hybridization analysis of DNA from a resistant ecotype revealed one strongly hybridizing band in addition to R P M l , and several weakly hybridizing bands, suggesting there may be related sequences in the Arabidopsis genome. In fact, a homologous sequence is already represented in the current data base, described as a myosin heavy-chain homologue from Arabidopsis (Genbank Accession U19616). This sequence has an amino terminus more closely related to RPMl than RPS2, with no hydrophobic amino terminal domain, and a potential leucine zipper comprising six heptad repeats homologous to those of RPM1. It has an NBS, but the internal hydrophobic domain, although matching the consensus motif GLPL(A/T)(L/I)gaG(S/G)aa, differs from RPS2 and RPMl by a lysine residue at position 7 (underlined), which disrupts the hydrophobic domain and suggests that it is unlikely to associate with the membrane. By extrapolation, the hydrophobic domains of RPS2 and RPMl would also seem unlikely to be membrane associated. The LRR domain of the myosin homologue also differs from RPMl and RPS2 by having only 10 discernible LRRs. Like RPMl it has poor conservation of residues 15 and 22 in the consensus LxxLxxLxxLxLxx(N/C/T)x(x)LxxIPxxwith only two C, one T and no N (but only one S) at position 15 and only three prolines at position 22, but like RPS2 it has better conservation of the L at position 18 and tends to have the extra residue at position 17 (Table I1 and Fig. 4). Given that both of the published resistance genes in this subclass are genes for resistance to bacterial pathogens, it is tempting to speculate that U19616 is also a gene for resistance to a bacterial pathogen. However, the tomato Zz gene for resistance to the wilt fungus Fusarium oxysporurn f. sp. lycopersici (isolated by G. Simons, P. Vos, J. Groenendijk, J. Wijbrandi, M. Reijans, T. van der Lee, J. Groenen, P. Diergaarde, M. Bleeker andM. Zabeau, unpublished; and possibly also by R. Fluhr and co-workers, unpublished) also seems to fall into
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the leucine zipper class of resistance genes with cytoplasmic LRRs. This indicates that the subclass to which a resistance gene belongs cannot be predicted from the nature of the pathogen, or vice versa.
B. RESISTANCE GENES ENCODING PROTEINS WITH CYTOPLASMIC LRRs AND HOMOLOGY TO THE CYTOPLASMIC DOMAINS OF TOLL AND THE INTERLEUKIN-1 RECEPTOR
The N. glutinosa N gene for resistance to tobacco mosaic virus gives rise to two mRNAs arising from alternative splicing of the same transcript (Whitham et al., 1994;Dinesh-Kumar etal., 1995). One encodes a 1144 amino acid Nprotein (N) that contains an NBS followed by 16LRRs. The other encodes a 652 amino acid truncated protein (Ntr)which is identical to the amino terminal 616 amino acids of N, but has a novel sequence of 36 amino acids at the carboxyl terminus replacing most of the LRR domain of N. N is postulated to be the functional resistance protein and Nt' a dominant negative inhibitor which regulates the resistance response induced by N (Dinesh-Kumar et al., 1995). The N protein shows similar structural organization to RPS2, but there is no amino terminal hydrophobic domain and no leucine zipper. It contains an internal hydrophobic domain matching the consensus motif GLPL(A/T)(L/I)xxaG(S/G)aabut, like the myosin homologue, it is also disrupted by a lysine at position 7 (see section IIIA). The 16 LRRs of N show a reasonably good match to the consensus LxxLxxLxxLxLxx(N/C/T)x(x)LxxIPxx, with six C, three N and no T at position 15, and 11LRRs show the extra residue at position 17 (Table I1 and Fig. 4).The amino terminal domain of N is reported to have homology with the cytoplasmic domains of the Drosophila Toll protein and the mammalian interleukin-1 receptor (IL-1R) protein. The homology between the amino terminal domain of N and the cytoplasmic signal-activation domains of these receptors suggests that this portion of the N protein is involved in signal activation rather than ligand binding, and consequently that the LRR domain is involved in ligand binding rather than signal activation. Interestingly, the extracytoplasmic receptor domain of Toll contains 15 LRRs with the consensus LLxxLxxLxxLxLxxNxLxxIPxx characteristic of extracytoplasmic LRRs in animals. This provides an additional parallel, suggesting that the LRR domain of N is involved in ligand binding. Gel blot analysis of DNA from tobacco lines carrying the Ngene reveal it to be a member of a multigene family clustered near the N locus. The Linum usitatissimum L6 gene for resistance to flax rust (Melampsora h i ) , like N, gives rise to two mRNAs arising from alternative splicing of the same transcript (Lawrence et al., 1995). One encodes a 1294 amino acid protein (L6) that contains an NBS followed by 27 LRRs. The other encodes a 705 amino acid protein (L6") that is identical to the amino terminal 676 amino acids of L6
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but has a novel sequence of 29 amino acids at the carboxyl terminus replacing most of the LRR domain of L6. The alternative transcripts of L6 may play a similar role to that suggested for the alternative transcripts of N . The L6 protein shows similar structural organization to the N protein, with an amino terminal domain also showing some homology to the cytoplasmic domains of the Drosophifa Toll protein and the mammalian IL-1R protein, an NBS, a disrupted internal hydrophobic domain matching the consensus motif GLPL(A/T)(L/I)xxaG(S/G)aa with iysine at position 7, and an LRR domain. Like N, L6 contains no amino terminal leucine zipper, but like RPS2 is does contain an amino terminal hydrophobic domain. The amino terminus of L6 is extended in the amino terminal direction relative to N. This region shares some homology with the myosin heavy chain homologue (see section IIIA) which also extends in the amino terminal direction relative to RPS2 and RPM1. Lawrence et al. (1995) note a possible internal leucine zipper domain located downstream of the NBS, but in fact this appears to be part of the LRR domain. The LRRs of L6 show a reasonably good match to the LxxLxxLxL portion of the consensus LxxLxxLxxLxLxx(N/C/T)x(x)LxxIPxx, but are more variable in length with only eight LRRs approximating the above consensus and 15 approximating an extended 27 amino acid consensus LxxLxxLxxLxxLxLxx(N/C/T)x(x)LxxIPxxand four to a reduced 21 amino acid consensus LxxLxxLxLxx(N/C/T)x(x)LxxIPxx(Table I1 and Fig. 4). In the 27 LRRs, there are only nine C, four N and two T at position 15 relative to the 24 amino acid consensus, and the majority show the extra residue at position 17. However, they often lack residue 22, 23 or 24 (only six LRRs have a P at position 22) and often contain several extra residues between position 21 of one repeat and position 1 of the next. Interestingly, there are two tandem duplications of approximately 140 amino acids (five LRRs) towards the carboxyl terminus of the LRR region (Fig. 4). The L6 gene was found to hybridize to a single polymorphic gene located at the L locus, consistent with the multi-allelic nature of the L locus, and to a cluster of genes located at or near the unlinked M locus, consistent with the complex nature of the M locus (Ellis et a f . , 1995). At least one other resistance gene, the Arabidopsis RPPS gene for resistance to the downy mildew fungus Peronospora parasitica, also seems to fall into the TolUIL-1R class of resistance genes with cytoplasmic LRRs (J. E. Parker, M. J. Coleman, V. Szabo, R. Schmidt, L. N. Frost, T. Moores, C. Dean, M. J . Daniels and J . D. G. Jones, unpublished).
C. A GENE ENCODING A PROTEIN WITH CYTOPLASMIC LRRs THAT IS REQUIRED FOR A RESISTANCE GENE TO FUNCTION
The Pro gene for resistance to the bacterium P. s. pv. tomato, encodes a serinekhreonine protein kinase, and is a member of a gene family clustered
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on chromosome 5 of tomato (Martin et al., 1993). The Pto homologue Fen is a member of this gene family that confers sensitivity to the insecticide fenthion (Loh and Martin, 1995a,b; Rommens et al., 1995). Mutations of Pto affect resistance to races of P. syringae carrying avrPto, but do not affect sensitivity to fenthion (Salmeron et al., 1994). However, Salmeron et al. (1994) have isolated mutants of a gene they have designated Prf p s e u d o monas Eesistance and fenthion sensitivity) that affect both functions. Prf is located within the Pto cluster and, like Pto, has been isolated by positional cloning (Salmeron et al., 1996). Sequence analysis reveals that Prf encodes an LRR protein similar to those encoded by the RPS2, R P M I , N and L6 genes. It has an NBS and a carboxyl terminal domain containing 18 LRRs with a cytoplasmic LRR consensus (Table 11). This provides the first direct evidence that a cytoplasmic LRR protein participates with a protein kinase in the generation of a defence response signal. However, Prf has a distinct amino terminal domain, suggesting that it does so by a different means than a leucine zipper or a Toll-like domain. Either Prf or Pto could be the receptor for the avrPto ligand, but Prf is a single copy gene, unlike most resistance genes. If Prf is the receptor for the avrPto and fenthion ligands then it would require specific receptor and signalling domains to trigger the Pto and Fen kinases, respectively, and for each member of the Pto family. This seems unlikely for the product of a single gene. Alternatively, if the Pto and Fen kinases are the receptors for the avrPto and fenthion ligands then it seems likely that Prf acts as a common component in a signal transduction pathway either prior or subsequent to the kinases, or as part of a protein complex. Perhaps the Pto pathway is similar to those of RPS2, RPM1, N or L6, and Prf plays a similar role to RPS2, RPMl, N and L6. The difference may be that these pathways are contacted at different points by the respective avirulence determinants and this may dictate which component becomes the switch that activates plant defences and is elaborated genetically to enable changes in host specificity in response to changes in pathogen specificity. Unlike most resistance genes, which are members of multigene families, RPS2 and R P M l are single copy genes like Prf. It is possible that RPS2 and RPMl are actually monomorphic Prf-like components of defence activation pathways that involve other genetically more elaborated components as the receptors for their respective avirulence determinants. It has already been noted “that because Pto and Prf seem to have the same resistance specificities, Prf could just as easily have been designated the R gene - and would have been, had susceptible tomato cultivars lacked Prf rather than Pto activity” (Chasan, 1994). Perhaps this has happened for RPS2 and R P M l . This might explain why two dissimilar A v r genes, avrB and avrRpml (see section IIID), both seem to interact with R P M l . The avrB and avrRpml avirulence determinants might interact with other
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signalling components, analogous to the interaction of the avrPto avirulence determinant and fenthion with the Pto and Fen kinases, respectively. These components may require R P M l to function, in the same way that Pro and Fen require Prf. Other genes required for resistance genes to function have also been identified for several other plant-pathogen interactions. These include Rcr (Iequired for C . fulvum resistance) genes in tomato required for Cf-2 and Cf-9 to function (Hammond-Kosack et al., 1994; M. S . Dixon, K. E. Hammond-Kosack and J. D. G. Jones, unpublished), Rar (required for Mla resistance, formerly designated Nar) genes in barley required for Mialz t i function (Freialdenhoven et al., 1994) and the N D R l (non-specific disease resistance) gene in Arabidopsis required for RPS2, R P M l and several RPP genes for resistance to Peronospora parasitica to function (Century et al., 1995). LRR proteins corresponding to Cf-2, Cf-9, RPS2, R P M l and at least one RPP gene (RPP5) have been identified, and it seems plausible that the Mla genes might also encode LRR proteins. It is therefore unlikely that the genes required for these resistance genes to function will encode LRR proteins like Prf, but more likely that they encode other signal transduction components such as protein kinases such as Pto. The interaction between resistance proteins and the proteins required for them to function may therefore resemble that between Pto and Prf. However, it remains puzzling that different components of pathways which are probably similar confer specific recognition of the corresponding avirulence ligands. This might be resolved conceptually by considering the reasons why an avirulence ligand might interact with a protein kinase. It seems plausible that plants have evolved a means to recognize the products of pathogen genes involved in the pathogenic process and to trigger a defence response as a consequence of such recognition. Such pathogenicity genes would then function as avirulence genes. A prediction of such a hypothesis would be that avirulence genes may function as pathogenicity genes. In fact, there is mounting evidence that Avr genes do function as virulence or pathogenicity factors during compatible interactions with their hosts, e.g. avrBs2 of X . c. pv. vesicatoria on pepper (Kearney and Staskawicz, 1990), pthA of X . citri on grapefruit (Swarup et al., 1992), avrb6 and several other avr genes of X . c. pv. malvacearum on cotton (Yang et a f . , 1994, 1996), avrA and avrE of P. s. pv. tomato on tomato (Lorang et al., 1994), avrRpml of P. s. pv. maculicola on Arabidopsis (Ritter and Dangl, 1995), and nip1 of Rhynchosporium secalis on barley (Rohe et al., 1995). This role is even more conspicuous in the case of viral pathogens where the pathogenicity function of avirulence determinants such as viral replicases (Meshi et al., 1988; Padgett and Beachy, 1993), coat proteins (Culver and Dawson, 1989; Goulden et al., 1993) and movement proteins (Meshi et al., 1989; Calder and Palukaitis, 1992; Weber et al., 1993) is obvious. This role is also supported
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by the existence of durable resistance genes such as Xu-21 of rice, and Cf-9 and Mi of tomato (Roberts et af., 1992; Song et af., 1995; P. J. G. M. de Wit, unpublished) that continue to confer effective resistance despite the existence of virulent races of the pathogen, which appear to be less fit than avirulent races. However, in other cases the pathogenicity effect of avirulence genes may be subtle and so dispensable with only minor effect, or it may be redundant and so dispensable with no effect. It is also possible that, just as specificity exists for the interaction between resistance genes and avirulence genes, the same concept may be extended to avirulence genes in their potential role as pathogenicity genes, so that unless corresponding host and pathogen components are both present the pathogenicity function of avirulence genes will not be discernible. The roles of pathogenicity genes may be many and varied, but in general directed towards deriving nutrients from the plant and avoiding the activation of its defences. This objective may be achieved in part by manipulating various plant signal transduction pathways, e.g. to redirect the flow of nutrients, and might employ both extracytoplasmic and cytoplasmic signals. Plant defences may have evolved the capacity to recognize these signals and to respond appropriately to them, thereby converting pathogenicity determinants to avirulence determinants. LRR proteins seem well suited to recognizing such signals, but protein kinases are not intuitively obvious candidates for such a role. However, if a protein kinase were the target for the pathogenicity function of an avirulence gene, then it would not be surprising that a protein kinase involved in the activation of plant defences might also evolve the capacity to recognize this pathogenicity determinant. The defence activation pathway itself might even be the target for such manipulation, i.e. to avoid the activation of plant defences, so that a protein kinase may be involved directly as the target of a pathogenicity determinant, which might bind to the protein kinase and suppress signal generation. A mutation in the protein kinase which converts this binding from signal suppression to signal activation, would constitute a resistance gene and the pathogenicity gene would then become an avirulence gene. This scenario might explain why the genetic diversity and specificity of interaction resides in the Pto gene family rather than Prf. However, this does not explain why Prf is required for Pto to function. An obvious explanation might be that Prf and Pto form a signalling complex and that Pto cannot function alone. A less obvious explanation might be that binding of the avrPto avirulence determinant to Pto alters the conformation of Pto in a way that enables it to be recognized by the LRR domain of Prf, which then activates other unaffected members of the Pto family or a different signalling pathway. If this were true, then Prf would be predicted to recognize a similar conformational change in Fen in response to fenthion, and in other members of the Pto family in response to their avirulence ligands, if they exist.
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D. AVIRULENCE DETERMINANTS THAT INTERACT WITH RESISTANCE PROTEINS CONTAINING CYTOPLASMIC LRRs
At present, nothing is known about the avirulence determinant encoded by the A - L 6 gene of flax rust that interacts with the L6 gene of flax. The avirulence determinant of tobacco mosaic virus that interacts with the N protein of tobacco has not been identified definitively, but may be the viral-encoded replicase (Padgett and Beachy , 1993). The P. syringae avirulence genes avrRpt2 (which interacts with the RPS2 gene of Arabidopsis), avrB and avrRpml (which interact with the RPMl gene of Arabidopsis) and avrPto (which interacts with the PtolPrf genes of tomato) have all been isolated and sequenced (Tamaki et a f . , 1988; Dangl et a f . , 1992; Innes et a f . , 1993; Salmeron and Staskawicz, 1993), but their functions remain poorly understood. The only P . syringae avirulence determinant that has been characterized biochemically is that specified by avrD of P. s. pv. tomato. The avrD gene is thought to encode an enzyme involved in the synthesis of a race-specific syringolide elicitor (Smith et a f . , 1993). Thus, in at least one interaction involving P. syringae, the avirulence determinant is not a peptide elicitor. Likewise, fenthion which interacts with the Pto homologue Fen is also a non-peptide elicitor, Therefore, it is conceivable that avrPto may also encode the synthesis of a non-peptide elicitor, possibly one that is structurally analogous to fenthion. The observation that avrRpt2 and avrRpml, which encode dissimilar proteins and interact with different resistance genes, interfere with one another (Reuber and Ausubel, 1996; Ritter and Dangl, 1996), is also consistent with the enzymatic modification of an elicitor, since it suggests they may both modify the same elicitor. However, avrRpml and avrB, which both interact with R P M l , also encode dissimilar proteins, except for seven of the first eight amino acids, which are identical (Tamaki et al., 1988; Dangl et a f . , 1992). This sequence identity might reflect a domain required for interaction with and modification of a common elicitor, but also raises the possibility that these proteins, or amino terminal fragments derived from them, might interact with RPMl directly. Likewise, the interference between avrRpt2 and avrRpml could also arise because their products interact directly with the same host component(s). This interaction might even involve the pathogenicity function of one suppressing the defence activation signal generated by the other, as suggested might be possible in section IIIC. Also in section IIIC, a correlation was proposed between where an avirulence ligand acts and the genetic elaboration of the affected host component. It is possible that this correlation may be extended further to a correlation between the type of avirulence ligand and where it acts in a signalling pathway. If the pathogen produces a peptide avirulence ligand then it may interact with an LRR receptor, but if the avirulence determinant is
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not a peptide then it may interact with different components of the signal transduction pathway. It therefore follows that if the avrRpt2, avrB and avrRpml avirulence determinants are not peptides, they might interact with proteins other than RPS2 and RPMl, and these proteins may be encoded by members of multigene families. The identification of cytoplasmic resistance proteins implies that they interact with cytoplasmic avirulence ligands. Tobacco mosaic virus is essentially an intracellular pathogen, and therefore it is logical that the N gene encodes a cytoplasmic protein. However, the avirulence determinants of extracellular pathogens must either be membrane permeable, endocytosed (Horn ef al., 1989) or actively secreted from the pathogen into the plant cell. If the avrPto, avrRpt2, avrB and avrRpml avirulence determinants are small molecules like the syringolides encoded by avrD, then the plant-cell membrane may be permeable to them. However, avrPto (Salmeron and Staskawicz, 1993), avrRpf2 (Innes et al., 1993), avrB (Huynh et af., 1989), avrRpmZ (Ritter and Dangl, 1995) and avrD (Shen and Keen, 1993) are all hrp regulated and their ability to induce a defence response is presumed to be hrp dependent. It seems likely that the avirulence determinants are produced in the bacterial cytoplasm and exported to the extracellular environment by the hrp secretory mechanism, and then diffuse into or are taken up by host cells. Alternatively, as discussed in section IIC, it is possible that the avirulence determinants are secreted directly into host cells by hrpmediated translocation.
E. ACTIVATION OF PLANT DEFENCES BY RESISTANCE PROTEINS CONTAINING CYTOPLASMIC LRRs
Little is known yet about the way in which plant defences are activated by resistance genes encoding cytoplasmic LRR proteins. However, the homology of these proteins to proteins in other systems may provide clues and testable ideas about the way they might work. Interestingly, there is one human LRR protein, the major histocompatibility complex (MHC) class I1 transcriptional activator (CIITA) (Steimle et al., 1993), that shows a similar structure to the cytoplasmic resistance proteins. MHC class I1 molecules present antigens to T helper lymphocytes and are a critical component of the immune response. Mutations in CIITA result in a complete lack of MHC class I1 molecules and lead to severe immunodeficiency, multiple infections and early death in those affected. The CZZTA gene encodes a protein of 1130 amino acids with an NBS, comprising a kinase l a motif at position 420-426, kinase 2 motif at 492-501 and a possible kinase 3a motif at 539-550, and an LRR domain comprising five LRRs with the 28 amino acid consensus LPxaxSLxxLxLxxNxIxDxGAxxLAxx extending from position 972 to the carboxyl terminus. The amino terminus, which is rich in serine, threonine
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and proline, is suggested to be similar to a number of proteins that activate transcription by interaction with other proteins. The role of the LRRs in CIITA is unknown. Similarities between plant resistance and the MHC have been noted previously (Dangl, 1992), but this is the first evidence that such similarities may also exist at the molecular level. The amino termini of N and L6 show no significant homology to CIITA, but show some homology with the cytoplasmic domains of Toll (Hashimoto et al., 1988), a receptor protein of Drosophila, and the mammalian IL-1R (Sims et al., 1988, 1989) (see section IIIB) and other members of the Toll/IL-1R family (Fig. 5 ) . In the Drosophila embryo, Toll is activated by a proteolytically activated extracytoplasmic protein ligand, Spatzle (Schneider et al., 1994). Activation of Toll is postulated to activate a cytoplasmic protein, Tube (Letsou et al., 1991), which then activates a serinehhreonine kinase, Pelle (Shelton and Wasserman, 1993) (homologous to Pto), by recruiting it to the cell membrane (Galindo et al., 1995). Pelle, in turn, phosphorylates an inhibitory protein, Cactus (Geisler et al., 1992; Kidd, 1992), which is bound to a transcriptional activation factor, Dorsal (Steward, 1987). Phosphorylation-dependent degradation of Cactus allows the nuclear localization of Dorsal and the activation of genes responsible for ventralization of the embryo (Belvin et al., 1995). Dorsal is a member of the re1 family of proteins (reviewed by Rushlow and Warrior, 1992), of which another member, Dif (dorsal-related immunity factor) , is involved in the activation of immune responses in Drkophila (fp et al., 1993; Petersen et al., 1995). Following the formation of the dorsal-ventral axis Toll seems to play a role in cell adhesion (Keith and Gay, 1990) and motor neuron and muscle development (Halfon et al., 1995). Interestingly, it may also play a role in the nuclear localization of Dif (Ip et al., 1993). A similar chain of events involving a number of homologous proteins is postulated for the activation of inflammatory and immune responses by the related transcriptional activation factor NFKB following the activation of IL-1R by interleukin-1 (Cao et al., 1996; reviewed by Siebenlist et al., 1994). However, the cytoplasmic domain of IL-1R may function in a slightly different manner to the cytoplasmic domain of Toll. Based on sequence and structural comparisons, Hopp (1995) suggests that the cytoplasmic domains of IL-IR and ST2L (Fig. 5 ) may be GTPases related to ras. Toll and MyD88 (Fig. 5 ) , on the other hand, show no conservation of the specific sequence and structural motifs necessary for GTPase activity. Hopp speculates that the presence of negatively charged amino acids and tryptophan in the regions of Toll and MyD88 homologous to the putative triphosphate and guanine binding pockets, respectively, of IL-1R and ST2L, might fill these pockets in a manner analogous to GTP binding, so that these proteins no longer need to bind GTP to become active. The N and L6 proteins also lack the conserved GTPase motifs in their region of IL-1R and Toll homology, but seem to have acquired a separate NBS domain that may serve a similar regulatory function
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to the putative GTPase domain of IL-1R and ras. It could therefore be inferred that the amino terminal domains of the cytoplasmic resistance proteins are effector domains that activate protein kinases following their own activation by the binding of ATP/GTP at their NBS. The availability of the NBS for ATP/GTP binding may be determined by ligand binding to the LRR domain. Alternatively, ligand binding and ATP/GTP binding may occur independently, but both may be required for activation of the effector domain. The relationship between Prf and Pto suggests the possible activation of a serinehhreonine kinase by an ATP/GTP-binding protein, analogous to the activation of the raf serinehhreonine kinase (homologous to Pto) by ras in mammals. Ras contains kinase l a and kinase 2a domains like RPS2, R P M l , N, L6 and Prf, but contains a kinase 3b domain rather than 3a (Traut, 1994). The activity of ras is regulated by its NBS (reviewed by Boguski and McCormick, 1993). If GTP is bound, ras is active, but if G D P is bound, it is inactive. Guanine-nucleotide exchange factors (GEFs) activate ras by exchanging free GTP for bound GDP. GTPase activating proteins (GAPS) inactivate ras by stimulating the hydrolysis of bound GTP to GDP. Guanine nucleotide dissociation inhibitors (GDIs) prevent the exchange of GTP for GDP, the hydrolysis of GTP to GDP and the binding of ras-GDP to the cell membrane, so preventing a change of state from active to inactive, or vice versa. Analogous proteins may be involved in regulating the activity of the resistance proteins through their nucleotide binding sites. Binding of ras to the cell membrane is necessary for ras to activate raf by recruitment to the cell membrane (Leevers et al., 1994; Stokoe et al., 1994; Marais et af., 1995) and is achieved via carboxyl terminal farnesyl groups (Boguski and McCormick, 1993). Other ras-related proteins, such as rab, bind to the membrane via carboxyl terminal geranylgeranyl groups (Boguski and McCormick, 1993). Intriguingly, the a subunit of the rat rab-geranylgeranyltransferase (Genbank accession L10415) contains six carboxyl ter-
Fig. 5. Comparisons of the primary structures of the amino terminal domains of the N and L6 proteins with the carboxyl terminal cytoplasmic domains of various members of the Toll/IL-lR family of proteins. The amino acid sequences are shown in single-letter code and three or more identical residues are highlighted in bold. Conserved hydrophobic regions, corresponding to two of the core p-strand regions of the GTPase protein superfamily (Hopp, 1995),are overlined. Toll, The Toll protein of Drosophila (Hashimoto et nl., 1988); Wheeler = 18 wheeler, a Toll homologue from Drosophila (Eldon et a l . , 1994), which is also involved in embryonic development; Mo IL-lR, the interleukin-1 receptor from mouse (Sims etal., 1988);Ch IL-lR, the interleukin-1 receptor from chicken (Guida et al., 1992); MyD88, the entirely cytoplasmic, interleukin-6 induced, MyD88 myeloid differentiation primary response protein from mouse (Lord et a l . , 1990);ST2L, the receptor-like, ST2L primary growth response protein from mouse (Yanagisawa et a l . , 1993).
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N L6 Toll Wheeler MO IL-1R Ch IL-1R ST2L MyD88
YDVFL . . . . . . . . . . . . SFRGEDTRKTF.TSHLYEVLNDKG1KT YEVFL . . . . . . . . . . . .SFRGPDTREQF.TDFLYQSLRRYKIHT FDAFISY. . . . . . . . . . SHK..B..QSFIEDYLVPQL.EHGPQK YDAIILH . . . . : . . . . . S E K . . D . . Y E F V C R N I A A E L . E H G R P P YDAYILYPKTLGEG . . .SFSDLD...TFVFKLLPEVL.E.GQFG YDAYVLYPKN.RESC..LYSS.D..I.FALKILPEVL.E.RQCG YDAYIIYPRVFRGSAAGTHS.VE..Y.FVHHTLPDVL.E.NKCG FDAFICY. . . . . . . . . . CPN..D..IEFVQE.MIRQL.EQTDYR
N L6 Toll Wheeler MO IL-1R Ch IL-1R ST2L MyD88
F......QDDKRLEYGATIPGELCKAIEESQFAITA~SEIWATS F......RDDDELLKGKEIGPNLLRAIDQSKIYVPIISSGYADS FQLCVHERD...WLVGGHIPENIMRSVADSRRTIIVLSQNFIKS FRLCIQQRD....LPPQASHLQLVEGARASRKIILVLTRNLLAT YKLFIYGRDD. . .WGEDTIEVTNENVKKSRRLIIILVRDMGGF YNLFIFGRND...LAGEAVIDVTDEKIHQSRRVIIILVPEP..S YKLCIYGRD...LLPGQDAATVVESSIQNSRRQVFVLAPH?YMHS LKLCVSDRD. . .VLPGTCVWSIASELIEKRCRRMVWSDDYLQS
N L6 Toll Wheeler Mo IL-1R Ch IL-1R ST2L MyD88
RWCLNELVKIM.ECKTRFK........Q TVIPIFYDVDPSHVRN KWCLMELAEIVRRQEEDPR. . . . . . . .RIILPIFYMVDPSDVRH EWARLEF . . . . .RAAHRSAL.NEGR.SRIIV.1IYS.DIGDVEK EWNRIEF . . . .RNAFHES L..RGLAQrtLVI.IEETSVSAEAED SW..LGQSSEEQIAIY.NALIQEGI..KIVL.LELE.KIQDYEK CYGILEDASEKHLAVY.NALIQDGI..KIIL.IELE.KIEDYAN K....EFAYEQEIALH.SALIQNN..SKVIL.IEMEP.LGEASR K..ECDF...QTKA...LSL.SPGVQQKRLIPIKYKAMKKD...
N L6 Toll Wheeler MO IL-1R Ch IL-1R ST2L MyD88
.QKES FAKAFEEHETKYKDD. . . .VE.GIQ . . . . . . .RW.RI .QT.G.CYKKAFRK H A N X . . . . . . . . FDQTIQ . . . . . . .N D . LDEE . . . .LKAYL . . . .KMNTYLKW.GDP. . . . . . . . .WFWDKL VAELSP.YLKSVP..SNRL..LTC . . .DR . . . . . . . . .YFWEKL MPD.SIQFIK ......QK.HGVICWSGDFQERPQSAKTRFWK.NL MPE.SIKYVK......Q K.YGA1RWTGDFSERSHSASTRFWKKV
N L6 Toll Wheeler MO IL-1R Ch IL-1R ST2L MyD88
.................... ....................
..
LQV.GD..LQDSLQHLVKIQGTIKWREDHVADKQSLSSKF
FP . .S..ILR.FITICD..YTNPCT....... . . . .K S W F R L ALNEAA.NLKGSCDNR.DKTDAD ALKKVGDLKGWHIGKNDKQGAI RFA..LPH.RR PVGNIGNGALIKTA.LKGSTD...DKLELI RYA..IPI.ELSPRGN..NYTLDHHERFKQPVSPGMIFRQAP RYQ..MPAQRRSPLSKHRLLTLDPVRDTKEKLPAATHLPLG* RYH..MPS RKHGSSSGFHLSS* RYQ..MPVPERA..SKTASVAAPLSGKACLDLKHF* AKALSLP*
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minal LRRs with unknown function. Since the homologous a subunit of yeast rab-geranylgeranyltransferase (Genbank accession U14132) lacks this LRR domain, it would not seem necessary for catalysis, but it may be required to regulate activity or substrate specificity. It is also intriguing that another ras-related protein, rac, is involved in the activation of NADPH oxidase to generate an oxidative burst in activated phagocytes (reviewed by Segal and Abo, 1993). The activation of an NADPH oxidase to generate an oxidative burst is also thought to be a key component of plant defences (Tenhaken et al., 1995). The prenylation of rac also seems to be required for the binding of rac to the cell membrane and for the interaction between rac and rho GDT (Segal and Abo, 1993). Ras does not possess an LRR domain, but LRR proteins have been shown to interact with ras. In yeast (Saccharomyces cerevisiae), adenylate cyclase is activated by the binding of ras to an LRR domain (Kataoka et al., 1985; Suzuki et al., 1990). The mouse rsp-1 gene, encodes an LRR protein capable of suppressing cell transformation by ras (Cutler et al., 1992) and is believed to regulate ras signal transduction negatively. The LRR domains of rsp-1 and yeast adenylate cyclase contain regions of sequence identity and high similarity suggesting the recognition of similar features of ras, but they have different roles because ras does not activate adenylate cyclase in mammals. In trypanosomes, the ESAG8 gene, encoding a similar LRR protein (Revelard et al., 1990; Smiley et al., 1990; Ross et al., 1991), is co-ordinately transcribed in a polycistronic message (Kooter et al., 1987), together with the ESAG4 gene encoding an adenylate cyclase. Although there is no direct evidence for interaction between these two proteins, the circumstantial evidence suggests that a regulatory interaction between an LRR protein and adenylate cyclase may also exist in this system. If the NBSLRR resistance proteins do possess a ras-like function then it seems unlikely that their LRR domains would bind to this ras-like domain in the same way as the yeast or mammalian examples above. It seems likely that the LRR domain would positively regulate any ras-like activity by the propagation of liganddependent conformational changes, analogous to the activation of the catalytic domain of yeast adenylate cyclase by the binding of ras to its LRR domain. In each of these comparisons, it seems that the cell membrane is a critical location for signal activation. Ras and Tube seem to activate the raf and Pelle kinases, respectively, by recruitment to the cell membrane. Activated rac binds to the cell membrane to activate NADPH oxidase and the rasactivatable yeast adenylate cyclase is a membrane-anchored protein. The Pto kinase has a potential amino terminal myristoylation site for attachment to the cell membrane. Constitutive membrane anchoring might negate a requirement for activation by recruitment to the cell membrane, but this would suggest constitutive activation of plant defences, unless an additional mechanism was involved. Alternatively, myristoylation alone may be insuffi-
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cient to anchor the Pto kinase to the membrane constitutively, but may assist its anchoring in response to a membrane-recruiting partner. RSP2 and L6, but not RPM1, N or Prf, have amino terminal hydrophobic domains which may be involved in the membrane anchoring of resistance proteins and potentially the recruitment of interacting partners to the membrane. Perhaps the resistance proteins which lack potential membrane-anchoring domains activate kinases that are already associated with the membrane. Based on these considerations the activation of plant defences by the cytoplasmic LRR resistance proteins is likely to be a complex process involving some or all of the following components: (1) binding of the avirulence ligand to the LRR domain; (2) binding of ATP/GTP to the NBS; (3) activation of an amino terminal effector domain dependent on avirulence ligand and ATP/GTP binding; (4) interaction of the effector domain with a protein kinase either directly or via one or more intermediary proteins; ( 5 ) ligand-dependent dimerization may be involved, especially for the resistance proteins with amino terminal leucine zippers; (6) recruitment to the plasma membrane may be involved in the activation of the resistance protein o r the protein kinase that it activates; (7) other proteins may be involved in regulating resistance protein activity, e.g. by affecting the status of the NBS; and (8) other proteins may be involved in regulating the protein kinase activity, e.g. protein phosphatases (see section VB). The mechanism of plant defence activation for Prf/Pto may be similar, but may represent a special case where Pto itself interacts with the avirulence ligand, allowing Prf to activate Pto without any avirulence ligand binding to Prf. Alternatively, Prf may recognize a change in the conformation of Pto in response to binding of the avirulence ligand. Examples of models that incorporate some of these ideas are shown in Fig. 6.
IV. DEFENCE-RELATED GENES ENCODING PROTEINS WITH EXTRACYTOPLASMIC LRRs Defence-related genes may be defined loosely as genes whose transcription is induced by various offensive stimuli such as pathogen challenge, insect predation and wounding (reviewed by Bowles, 1990). Pathogenesis-related (PR) genes were initially defined as genes whose transcription is induced following incompatible pathogen challenge (see reviews by €301 et al., 1990; Bowles, 1990; Linthorst, 1991). More recently, the concept of PR genes has been modified to that of genes induced differentially in incompatible and compatible plant-pathogen interactions (see e.g. Ashfield et al., 1994). Systemic acquired resistance (SAR) genes are a subset of P R genes whose transcription is induced both locally at the site of incompatible plantpathogen interactions and systemically in unchallenged parts of the plant (Ward et al., 1991; and reviewed briefly by Ryals et al., 1994). PWSAR
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proteins are thought to be involved in restricting pathogen ingress and may even have a role in effecting resistance (Alexander et al., 1993). A. POLYGALACTURONASE-INHIBITINGPROTEINS
Polygalacturonase-inhibiting proteins (PGIPs) are soluble, extracytoplasmic glycoproteins, ionically bound to the extracellular matrix (ECM) of plant cells (Salvi et al., 1990; Johnston et al., 1994) and capable of inhibiting the endopolygalaturonases (PGs) of many fungal pathogens, but not those of bacterial pathogens or endogenous endopolygalacturonases (Abu-Goukh and Labavitch, 1983; Johnston et al., 1993). Fungal PGs function as pathogenicity factors that degrade the pectic component 'of the ECM to facilitate fungal colonization and nutrition. Each of the PGIPs isolated to date is almost entirely composed of 10 LRRs matching the extracytoplasmic LRR consensus LxxLxxLxxLxLxxNxLxGxIPxx (Table I). The first PGIP gene to be isolated was that of bean (Toubart et al., 1992), which encodes a 333 amino acid mature peptide (Fig. 7). DNA gel blot analysis (Frediani et al., 1993) reveals bean PGIP to be a member of a multigene family with at least five members and possibly as many as 12-15 members. All have been localized to the junction between the euchromatin and the pericentric heterochromatin of bean chromosome 10 by in situ hybridization (Frediani et al., 1993). RNA gel blot analysis reveals a single band under stringent hybridization conditions (Toubart et al., 1992), but a larger weakly hybridizing band has also been observed under lower stringency (G. De Lorenzo, unpublished). Western analysis has also revealed a larger, cross-reacting, membrane-bound protein (G. De Lorenzo, unpublished). It has been postulated that this cross-reacting protein may be a receptor PGIP linked to a signalling mechanism, perhaps a serinekhreonine kinase. This seems feasible, given that the PGIPs show some homology with
Fig. 6. Examples of models for the initiation of a plant defence activation signal following interaction between cytoplasmic resistance proteins and their respective avirulence determinants. (A) A model involving a resistance protein with cytoplasmic LRRs and a potential leucine zipper depicted here for RPS2 and a hypothetical intermediary protein. (B) A model involving a resistance protein with cytoplasmic LRRs and a TollIIL-1R-like domain depicted here for N. (C) A model for Pto kinase activation involving a Pto/Prf complex. (D) A model involving Prf recognition of Pto following binding of the avirulence determinant to Pto and activation of a different kinase. In this model the signalling protein may be another member of the Pto family, an unrelated kinase or even a different signalling molecule. AVR, The avirulence determinant; HD, hydrophobic domain; KAD, kinase activation domain; KINASE, serinekhreonine protein kinase; LZ, leucine zipper; NBS, nucleotide binding site; TOLL, domain with homology to the cytoplasmic domains of the Toll and interleukin-1 receptors.
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A
Cell
membrane
GTP-
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I
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NAL
c
C
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B
ELCNPQDKQALLQIKKDLGETLSS ELCNPQDKQTLLQIKKELGETLSS
Bean Soybean Pear Tomato Antirrhinum Kiwifruit Bean Soybean Pear Tomato Antirrhinum Kiwifruit
C
Bean Soybean Pear Tomato Antirrhinum Kiwifruit
DLCNPDDKKVLLQIKKAFGDPYVLAS VRCNPKDKKVLLQIKKDLGNPYHIAS ERCHPQDKRVLLKIKKAFNNPYHLAS DRCNPNDKKVLLRIKQALNNPYLLAS
WLPTTDCC-GVLCDTDTQTYR WHPKTDCCWWVGVSCDTPTYR WKSDTDCCD WYCVTCDS T TNR WJ3PNTDCCY WYVIKCDR K TNR WIPDTDCCS WYWECDR T TNR WNPDNDCCD WYNVDCDL T TNR V V I I I I
NNLDLSG DNLDLSE NSLTIFA NALTVFQ NDFHLFS IALTIFS
HNLPKPYPIPSS LNLRKPYPIPPS GQVSG QIPAL W G QIPAA ASVSG QIPET G W G QIPAA
LRRl
INNLVOPIPPA NPNIVOTIPTT QP-PIQPA VTLyLTOTIPPA I-IPHA LSmGQIPSA
LRRZ
Bean Soybean Pear Tomato Antirrhinum Kiwifruit
LANLPYL NFLYIGG VGSLPCL KFLYITN VGDLPYL ETLEFHK VGDLPYL ETLEFHH IAELPFL ESLMFRK VGDLPYL QTLIFRK
Bean Soybean Pear Tomato Ant irrhinum Kiwifruit
IAKLTQL ITKLTKL IAKLKGL IAKLTNL ITRLTRL ISKLSNL
HYLYITH RELNIRY KSLRLSW KMLRLSF RSLTISW KMVRLSW
TMGAIPDF aGQIPHF TWGSVPDF T W O P IPEF mGPVPAF TWGPVPSF
LRR3
Bean Soybean Pear Tomato Antirrhinum Kiwifruit
LSQIKTL LSQIKAL LSQLLSQLm LSELFSQL-
VTLDFSY GFLDLSN TFLDLSF TLLELNY TSLDLSF TFLDLSF
NALSOTLPPS NKLSGNLPSW W G A IPSS NQFTGTIPSS -0s IPPS NDLTGSIPSS
LRR4
Fig. 7. Comparison of the primary structures of the polygalacturonase inhibiting proteins from bean, soybean, pear, tomato, Antirrhinum and kiwifruit. Various features are indicated as described in Fig. 1, except that three or more identical residues are highlighted in bold.
Bean Soybean Pear Tomato Antirrhinum Kiwifruit
ISSLPNL LPSLPDL LSELPNL LSQLPNL LIQLRNL LSKLTNL
NRISGAIPDS NYISGPIPDL NKLTGHIPIS NKLTGTIPES NKLTGNIPES NKLTGPIPNS
LRR5
Bean Soybean Pear Tomato Antirrhinum Kiwifruit
NRLTGKIPPT NRLIGKIPSL NQLSGNIPTS NSLTGHVPAS NQLSG IPRA NQLTGSIPKT
LRR6
Bean Soybean Pear Tomato Antirrhinum Kiwifruit
NMLEGDASVL NMLEGDASVL NKLEGDASVI NKLEGDVSFL NRLEGDVSFM NMLSGDISFM
LRR7
GGITFDG YGISFDN GALRLDR LAMYLDR NDMRLDR DAIHLDR
Bean Soybean Pear Tomato Antirrhinum Kiwifruit
FGSDKNT FGSEKHT FGLETT FGm-TS FGm-TI FGSETI
KKIHLAK ERIYLAN QIVDLSR QVIDLSR QYADFSR QIVDFSR
NSLAFDLGK NLFAFDLGK NLLE-K NLLEFDISK NMLQFDLSH NKFQFDLSK
LRR8
Bean Soybean Pear Tomato Antirrhinum Kiwifruit
VGLSKNL VRLSKTL VEFPTSL SEFAESL VEFPDSL WFPQSL
NGLDLRN GVLDGGH TSLDINH ISLDLNH SSLDLNH TSLDLNH
NRIYGTLPQG NLIYGTLPKG NKIYGSIPVE NRIFGSLPPG NRITGSLPEG NKIYGSLPVG
LRR9
Bean Soybean Pear Tomato Antirrhinum Kiwifruit
LTQLKFL LTSLKDL FTQLN F LKDVP L LTKLESL LTKLD L
QSLWF YYLDVSY QFLWY QFFWY YNLWY QYLWY
"LCGEIPQG "LCGEIPRG NRLCGQIPVG NRLCGQIPQG NRLCGKIPVG NRLCGHIPTG
LRRlO
D
Bean Soybean Pear Tomato Antirrhinum Kiwifruit
GNLKRFDVSSYANNKCPCGSPLPSCT GKLQEFDASLYANNKCLCGSPLPSCT GKLQSFDEYSYFHNRCLCGAPLPSCK GTLQSFDIYSYLHNKCLCGSPLPKCK GKLQELDYTAYFHNRCLCGAPLPDCK GKLQGFDQTSYFHNRCLCGAPLPDCK
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both the Cf genes (see section IIA) and the receptor kinases (see section VB) (Jones et al., 1994). Extraction and bioassay of PGIP activity from various bean tissues revealed very low levels of PGIP activity in roots, higher levels in cotyledons, stems, leaves, seeds and embryos, and the highest levels in the vegetative apex and flowers (Salvi et al., 1990). Leaf activity remained fairly constant, but stem activity increased, peaked and then decreased in an acropetal wave during growth. Flower activity also increases to a maximum during development, and the highest activity was localized to the stamens and the stigmatic region of the style. PGIP purified from flowers had a slightly different molecular mass compared to that purified from stems, suggesting that different members of the PGIP family may be expressed in tissue-specific fashion. Bean PGIP is not involved in host cultivar/pathogen race specificity since PGs isolated from race a, p or y of Colletotrichum lindemuthianum and PGIPs isolated from four cultivars of bean showed no differences in PG or PGIP activity when assayed against one another in vitro, regardless of whether they were isolated from a compatible or an incompatible host/pathogen combination (De Lorenzo et al., 1990). However, immunolocalization studies indicate that bean PGIP is induced by inoculation with both compatible and incompatible C. lindemuthianum, and Western analysis indicates that bean PGIP is inducible by both wounding and salicylic acid (Bergmann et al., 1994). RNA gel blot analysis of PGIP induction following pathogen challenge shows that PGIP is induced 20-fold over the basal level of transcription by about 40 h after inoculation in the incompatible interaction and returns to the basal level by about 68 h (Nuss et al., 1996). By comparison, PGIP expression does not increase above the basal level before 68 h in the compatible interaction, but shows an eightfold induction by 84 h and returned to the basal level by 100 h. Therefore, it seems that bean PGIP behaves as a PR protein. Cervone et al. (1989) have shown that purified bean PGIP slows the kinetics of sodium polypectate degradation by purified Aspergillus niger or Fusarium moniliforme PG in vitro, allowing the production of oligogalacturonides, 10-15 residues in length, able to induce plant defences. This observation led these authors to propose the hypothesis that PGIPs not only interfere with fungal ingress by inhibiting the degradation of the ECM by fungal PGs, but also have a signalling role in plants, because they slow down the degradation of the ECM by fungal PGs, causing the production of oligogalacturonides which activate other plant defences against further fungal ingress. Sharrock and Labavitch (1994), in an attempt to test this hypothesis, reached the conclusion that elicitor active oligogalacturonides do not accumulate under more realistic circumstances, and therefore the PGIPs do not have a role in defence gene activation. In their experiments crude preparations of pear PGIP were found partially to inhibit pear cell wall degradation by crude preparations of PGs from Botrytis cinerea that had been grown on a pear
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cell wall substrate, but did not allow the accumulation of oligogalacturonides larger than dimers. They argue that the different outcomes to these two experiments may be explained in part by the multiple PGs present in B. cinerea, some of which were relatively unaffected by pear PGIP. The fruit-specific pear PGIP gene (Stotz et al., 1993) encodes a mature protein of 306 amino acids (Fig. 7). DNA gel blot analysis with low stringency washes reveals two homologous genes, consistent with the previous identification and purification of two PGIPs (Abu-Goukh et al., 1983). RNA gel blot analysis with high or low stringency washes revealed only a single hybridizing band expressed at a very low Ievel in leaves, sevenfold higher in flowers, but 1400-fold higher in fruit. In addition to a different pattern of expression compared to bean PGIP, pear PGIPs are competitive inhibitors of B. cinerea PG (Abu-Goukh et al., 1983), whereas bean PGIPs are non-competitive inhibitors of C. lindemuthianum P G (Lafitte et al., 1984), although this difference may depend on the source of the PG rather than the source of the PGIP. Purified pear PGIPs are inhibitory to PGs from A . niger, B. cinerea and Dothiorella gregaria, slightly inhibitory to PG from Penicillium expansurn and are not inhibitory to PG from F. oxysporum (Abu-Goukh and Labavitch, 1983). The fruit-specific tomato PGIP gene (Stotz et al., 1994) encodes a mature protein of 307 amino acids (Fig. 7). DNA gel blot analysis revealed two homologous genes, one hybridizing strongly and the other weakly. At least one of these maps to chromosome 7 of tomato (D. A. Jones, unpublished) and a second wound-inducible PGIP, detectable by a pear PGIP probe, maps to chromosome 2 (A. L. T. Powell, unpublished). RNA gel blot analysis revealed a single hybridizing band expressed in immature and ripening fruits. Tomato PGIP was found to inhibit B. cinerea PG 20-fold less effectively than pear PGIP. Consistent with this observation, ripe fruits of transgenic tomato plants carrying 35S:pear PGIP constructs, show a significant reduction in the amount of infection with B. cinerea following artificial inoculation (A. L. T. Powell, unpublished). In contrast, transgenic tomato plants overexpressing bean PGIP showed no increase in resistance to F. 0. f. sp. lycopersici, Afternaria solani or B. cinerea, and the bean PGIP recovered from the transgenic tomatoes did not inhibit F. rnoniliforme PG unlike the PGIPs purified from bean ( G . De Lorenzo, unpublished). This may reflect an alteration of the protein by the heterologous host, e.g. in glycosylation. Alternatively, this member of the bean PGIP gene family may not have possessed the appropriate inhibitory activity, but another member might. The soybean PGIP gene (Favaron et al., 1994) encodes a 313 amino acid mature peptide (Fig. 7). DNA gel blot analysis reveals a single gene, but other weakly hybridizing bands indicate the presence of related genes. Extraction and bioassay of PGIP activity from various soybean tissues revealed low levels of PGIP activity in roots and hypocotyls, higher activity in leaves and the highest levels in cotyledons. RNA gel blot analysis revealed
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that soybean PGIP, like bean PGIP, is wound inducible. Purified soybean PGIP inhibits A . niger PG and two PGs (PGs I1 and IV) from Sclerofinia sclerotiorum, but was found to inhibit one (PG 11) much less effectively than the other. By comparison bean PGIP was found to inhibit both to a similar extent. A fruit-specific raspberry PGIP has been purified and the amino terminal amino acid sequence determined (Johnston et al., 1993). Purified raspberry PGIP inhibits A . niger PG and two PGs (PGs I and 11) from B. cinerea. No PGIP activity was detected in flowers but high activity was detectable in immature green fruit and found to decrease with ripening. Like pear PGIP, raspberry PGIP was found to be a competitive inhibitor of B . cinerea PG . A fruit-specific apple PGIP has also been purified and the amino terminal amino acid sequence determined (Yao et al., 1995). Purified apple PGIP shows various degrees of inhibition against four B. cinerea PGs (PGs I-IV), being most effective against PGs I and 11, but is ineffective against a fifth (PG V) or against PG purified from apple fruit infected with B . cinerea. Apple PGIP was found to be a mixed-type inhibitor of B . cinerea PG 11, as opposed to a strictly competitive or non-competitive inhibitor. The cDNA for a PGIP has been isolated from kiwifruit and sequenced (Simpson ef al., 1995) (Fig. 7). DNA gel blots of genomic DNA from a diploid relative suggest there may be two PGIP genes in kiwifruit. The FIL2 gene of Anfirrhinum majus (Steinmayr et al., 1994), predicted to encode a mature protein of 307 amino acids (Fig. 7), was not isolated as a PGIP, but as a consequence of its expression in the filaments of young flowers. However, its strong homology with the PGIPs reveals that it almost certainly is a PGIP. RNA gel blot and Western analyses revealed that FZL2 is not expressed in roots, stems, leaves, flower bracts, sepals or petals, but only in stamens and weakly in carpels. In the latter, expression was found to be localized to the pistils and to increase with maturity to a level comparable with that in stamens, where expression remained constant throughout development. Tissue immunolocalization studies showed that FIL2 was expressed in the filament, the connective, the epidermis and the endothecium of the anther, but not the pollen. They also showed that FZL2 was expressed in the stigmatic and transmitting tissues of the pistils. Subcellular immunolocalization showed that FIL2 protein was localized to the middle lamella of the ECM and concentrated in fibrous structures surrounding intercellular spaces. The Arabidopsis thaliana DRTlOO gene (Pang et al., 1992), like FZL2, was not isolated as a PGIP, but curiously as a cDNA clone presumed to encode a chloroplast targeted LRR protein able to complement recA proteins in Escherichia coli. Sequence analysis of the published DR TI00 sequence revealed several anomalies prompting re-sequencing of the gene (D. A. Jones, unpublished). This has in turn revealed sequencing errors in the
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published sequence with significant effects on the predicted protein sequence because of reading-frame alterations. The revised DRT100 sequence (data not shown) almost certainly encodes a PGIP and shows no evidence for a chloroplast targeting signal. Nothing more is known about DRTlOO at present, or its improbable ability to complement recA mutations, which remains a complete mystery. Although the PGIPs form a group of homologous proteins (Fig. 7), different PGIPs show different inhibitory activities against various PGs from different fungi or from within a single species. There seems to be an inverse correlation between the effectiveness of the PGIP and fungal pathogenicity, i.e. the more effective the PGIP the less invasive the pathogen, and vice versa (Abu-Goukh and Labavitch, 1983). There may even be a degree of specificity involved, with a given PGIP being most effective against fungi that are pathogens of the plant from which it was derived. For example, bean PGIP is more effective against the PG of Colletotrichum lindemuthianum than against the PG of the related non-pathogen C . lugenurium which is a pathogen of cucurbits, and vice versa, with respect to cantaloupe PGIP (Lafitte et al., 1984). It also seems that within a given plant species there may be several PGIPs with different specificity and with different tissuespecific patterns of expression. Each of the PGIPs isolated to date probably represents only one component of the overall PGIP activity in each plant. Clearly the differences in PGIP specificity are determined by differences in sequence and these are confined mainly to residues interspersed between the consensus residues of the 10 LRRs (Fig. 7 ) .
B . LRR EXTENSINS
Extensins are hydroxyproline-rich glycoproteins (HPRGPs) which are thought to be a structural component of the plant cell wall that may also have a role in plant defences (reviewed by Showalter, 1993; Kieliszewski and Lamport, 1994). Hydroxyproline (0) is formed by the post-translational modification of prolines by prolyl hydroxylase. HPRGPs are typically characterized by the motif SO4 (hydroxlated SP4) often found within larger repeats. HPRGPs are glycosylated with 1-4 arabinose residues at most 0 residues and single galactose residues at some S residues. Extensins are often found as modular domains in chimeric proteins, including proteins with LRR domains. Two LRR extensin proteins have been described to date, but their LRR nature seems to have gone unnoticed. The first, a tomato L R R extensin, Tom-L4 (Zhou et al., 1992) probably went unnoticed because sequencing errors only allowed the reading-frame for the carboxyl terminal portion of the protein, containing the extensin domain, to be recognized. The second, a maize LRR extensin, Pexl (Rubenstein et al., 1995), was correctly
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sequenced, but the homology with other LRR proteins seems to have been missed. The LRR nature of these proteins was detected using the predicted amino acid sequence of the Cf-9 resistance gene to search the sequence databases for homologous proteins (D. A. Jones, unpublished). Tom-L4 was isolated using a carrot extensin clone, pDC5A, to screen a tomato genomic DNA library. A hybridizing subclone was sequenced and the conceptual translation of this sequence yielded a protein of 322 amino acids consistent with an extensin (Zhou et al., 1992). However, an openreading frame upstream and out of frame with the extensin domain contains an LRR domain extending beyond the available DNA sequence. This region has been isolated by PCR, and the sequences further upstream by IPCR (D. A. Jones, unpublished). The region joining the LRR domain to the extensin domain has been re-sequenced and reveals errors in the published sequence. The LRR domain is in fact fused to the extensin domain in a single ORF. The region upstream of the available DNA sequence has been sequenced beyond the LRR domain and reveals that Tom-L4 contains 10 LRRs matching the extracytoplasmic consensus LxxLxxLxxLxLxxNxLxGxIPxx (Fig. 8 and Table I). DNA gel blot analysis reveals a number of hybridizing bands with a probe covering the extensin domain and a small amount of the LRR domain (Zhou et al., 1992), but may be part of an extensin multigene family rather than an LRR multigene family. There are no DNA gel blot data for a probe covering the LRR domain alone. RNA gel blot analysis shows that Tom-L4 hybridizes to a 2.6 kb transcript and less intensely with both larger and smaller transcripts, and that the Tom-L4 transcript accumulates in response to wounding. The extensin domain of Tom-L4 contains six contiguous KP (lysineproline) repeats, followed by six dispersed SP4 repeats separated by various amino acids, followed by seven repeats with the consensus SP2-5TPSYEHPKTP, followed by several repeats with the consensus SSP4SPSP4TY1-3. Tom-L4 differs from other extensins in the nature of its extensin repeats which are not represented amongst other extensins. It also lacks the YxYK and VYK motifs characteristic of many other extensins. These motifs are thought to be involved in the formation of isodityrosine cross-links within and between extensins. The lack of these motifs implies that Tom-L4 is a non-cross-linking extensin like most other chimeric extensins. The other LRR extensin, Pexl, was isolated initially as a partial cDNA
Fig. 8. Comparison of the primary structures of the LRR extensins from maize and tomato, excluding the non-homologous, carboxyl terminal extensin domains. Various features are indicated as described in Fig. 1, except that identical residues are highlighted in bold.
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A
Maize MDPPLRLLPDGRLMAALLLLAACLSACS Tomato . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6
Maize QAVTSAEASYIAHRQLLAMKEA GGGEAGDLPADFEFDD RVGAAN Tomato .PPSRGVLISFFVITLLSYQISYWOQEOD SDIGLDDIK ANXKLS
signal peptide
Maize FPNPRLRRAYIALQAWHRAFYSDPKGYTA"VGEDVCKYNGV1CTEALDDPK Tomato FENSRIRDAY1ALQYWKTAMFSDPFNFTA"T'GPNVCSYGGVF'CAPSLMDDS
C
Maize Tomato
I T W AGIDLNG ADIAGYLPPE I R W AGIDLNH ADIAGSLVAE
LRRl
Maize Tomato
LGLLTDL AFFHINT NRFCGIIPKS LGLLTDL VLFHLNS NRFCGWPKT
LRR2
Maize Tomato
MSRLSLL HEFDVSN NRFVGVFPWC FSHLKLL RELDLSN NRFVGGFPKW
LRR3
Maize Tomato
LE MVSL KYLDLRF NDFEGELPPA LS LPSL KFLDLRF NDFEGPVPSG
LRR4
Maize Tomato
LFD KDL DAIFVNT NRFVGPIPEN LFD KDL DALFLND NRFRFGIPEN
LRR5
Maize Tomato
LON STA SVIVFAN NAFVGCIPKS LON SPV SVLVFAN NDLGGCIPAS
LRR6
Maize Tomato
IGRMVKTL DEIIFLN NKLDGCLPLE IGKMGKTL NELILMN DUGCLPME
LRR7
Maize Tomato
MGLLVN__TWTDVSG NMLVGTIPEQ IGLLNKL TVFDVSF NKIQGSLPST
LRR8
Maize Tomato
LSNIAKL EQLDVSR NVFTGIVHES VSKMRSV EELNVAH NKLTOVIPAS
LRR9
Maize Tomato
ICELPAL V F A F NFFNSEAAVC ISQLPRL QNFTYSF NYFTGEAPVC
LRRlO
Maize Tomato
MPSDKAL VNLDDRD NCLGALRPAQ AAT RSGDGQE NCIVGKKN Q
LRRl1
D
Maize KTALQCAPVLARPVDCSKHVC Tomato RSAKECSSDDAKPYDCRKSKC
E
extensin domains not shown
linking domain
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clone, pSF21, from a maize, immature-pollen cDNA library by differential screening (Rubenstein et al., 1995). Sequence analysis of pSF21 revealed coding sequence for SP4 repeats consistent with an extensin. A genomic clone, pZmPl, encompassing the entire gene, was isolated from a lambda genomic library using pSF21 as a probe. Sequencing of pZmPl revealed a single long open-reading-frame encoding a protein of 1188 amino acids with an amino terminal domain carrying a putative signal peptide and 11 LRRs matching the extracytoplasmic consensus (Fig. 8 and Table I), and a carboxyl terminal extensin domain. DNA gel blot analysis with a probe covering the LRR domain (described by Rubenstein et a!. as a globular domain) gave two strongly hybridizing bands, one of which also showed strong hybridization with a probe covering the extensin domain, while the other showed only weak hybridization, indicating that the LRR domains were conserved, but the extensin domains were divergent. The LRR probe also showed one weakly hybridizing band which did not hybridize to the extensin probe. A similar result was observed for RNA gel blot analysis where the LRR probe detected two transcripts, but the extensin probe detected only one of these, indicating the existence of another LRR gene that does not encode an extensin or at least not a similar extensin. It is not clear whether transcripts representing one or other or both extensin hybridizing bands on the DNA gel blot are contained in the single hybridizing band detected on the RNA gel blot. No transcript was detected by the extensin probe in any other tissue except maturing pollen, and transcription was not induced by wounding. A similar result was claimed for the LRR probe, although no data were given. The extensin domain of Pexl comprises 11 XP (any amino acid followed by a proline) repeats followed by 27 repeats alternating between the consensus sequences SP4(A/T)PV(NG/S) and SP4(E/V)K then 34 repeats alternating between SP3(A/T/V)PKS and SP3(A/TN)PVS and, finally, 16 repeats alternating between SP4APVS and SP4VK. The extensin domain is therefore quite different from that of Tom-L4. However, like Tom-L4, Pexl lacks YxYK and VYK motifs thought to be involved in the formation of isodityrosine cross-links, suggesting it is also a non-cross-linking extensin. The LRR domain of Pexl is highly homologous to that of Tom-L4 showing 56.6% amino acid sequence identity (Fig. 8), but, despite very similar LRR domains, Tom-L4 and Pexl have distinct extensin domains, different temporal and spatial patterns of expression and different responses to wounding. They would appear to have very different functions, yet the intriguing similarity of the LRR domains suggests they may be involved in the recognition of similar ligands. The extensins have been implicated in plant defence and the wound response of Tom-L4 suggests that it may be a defence-related protein, but the responses of these genes to pathogen challenge have not been examined, so it is not known if they are also pathogenesis-related genes.
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C. A VIROID-INDUCED LRR PROTEIN
A pathogen-inducible, secreted, leucine-rich protein (LRP) has been isolated by differential screening of a cDNA library made from tomato plants infected by citrus exocortis viroid (Tornero et af., 1996). LRP has five extracytoplasmic LRRs (Table I) preceded by four leucines with heptad spacing (a potential leucine zipper). Unlike the genes for many other LRR proteins, the gene for LRP does not appear to be a member of a multigene family, based on DNA gel blot analysis. The biological role of LRP has not yet been elucidated, but it seems to possess the properties of a PR protein, since RNA gel blot analysis indicates that transcription of the LRP gene, although constitutive at a background level, is significantly induced following viroid infection. Interestingly, Western blot analysis using anti-LRP antibodies shows that the protein is proteolytically cleaved in viroid infected tissues to release a 2 kDa fragment. This cleavage introduces a new dimension to the possible role of this protein. The cleavage product may be an antiviroid or antimicrobial peptide whose release enables it to help restrict pathogenesis. Alternatively LRP could be a signal-generating or regulating molecule whose effect on signalling may be activated or destroyed by the cleavage.
V. GENES ENCODING PROTEINS OF UNKNOWN FUNCTION WITH EXTRACYTOPLASMIC LRRs A . THE AWJL PROTEINS OF WHEAT
A wheat LRR gene with significant homology to the Cf-2, Cf-4 and Cf-9 resistance genes was found when an incomplete cDNA clone, AWJL3, was isolated from a wheat floret cDNA library probed with a maize cDNA clone, pZm9 (Ji and Langridge, 1994). pZm9 was isolated from an immature maize anther cDNA library probed with a lily cDNA clone of an expressed meiotic prophase repeat (EMPR) sequence. AWJL3 contains three LRRs and carboxyl terminal sequences homologous to domains C3 to G of Cf-9 (see Fig. l ) , suggesting that the AWJL3 gene encodes a membrane anchored extracytoplasmic glycoprotein very similar to that predicted for Cf-9. Gel blot analysis of wheat DNA with AWJL3 revealed a gene family with more than 20 members. Genomic DNA clones, AWJL172, 175, 218 and 236, corresponding to four members of this family, were isolated and sequenced, and their sequences deposited in the Genbank database (Accessions X81367X81370). Conceptual translation of the long open-reading frame (ORF) in AWJL172 yields a protein that appears incomplete at both the amino and carboxyl termini, but shows homology with domains C1 to D of Cf-9 (see Fig. 1). A contiguous downstream ORF located in a different reading-frame
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shows a continuation of the homology over domains D to G . AWJL175, 218 and 236 all contain long ORFs encoding proteins that are complete at the carboxyl termini and show homology with domains C1 to G of Cf-9(see Fig. l ) , but are incomplete at the amino termini. The conceptual protein sequences shown in the Genbank entries have been initiated from the first methionines in the long ORFs. These are probably not the initiation codons because conceptual translation of upstream sequences show these methionines to be embedded in contiguous LRRs. The LRRs continue upstream of these methionines and indeed upstream of the long ORFs in various reading-frames. In AWJL218, one of the most upstream ORFs has homology to domain B of Cf-9.Comparison of AWJL3 with these genomic AWJL sequences reveals it to also be incomplete at the carboxyl terminus. Homology to the G domain of the genomic AWJL sequences continues in a contiguous downstream ORF located in a different reading-frame. Clearly, all five AWJL sequences are incomplete and probably contain sequencing errors or sequence changes that reflect degeneration into pseudogenes. Nevertheless, these sequences indicate the existence in wheat of a family of genes very similar in nature to the Cf genes of tomato, encoding membraneanchored extracytoplasmic glycoproteins with large receptor domains composed of LRRs. The homology between these genes and both Cf-9 and Cf-2 is particularly striking in the penultimate LRR with the sequence LESLDLSxNKLxGEIPxxLxSLT tightly conserved. Interestingly, N-glycosylation sites, at the infrequently glycosylated position 11 of the LRR consensus (see section VIIIB), are also conserved in the flanking LRRs. The significance of these conserved features is not clear. They could be critical to the structure of these proteins or they may indicate a conserved interaction with another protein that transduces a signal triggered by the binding of a ligand to these LRR proteins. If the latter is true then it may be possible to use resistance/avirulence determinants from one interaction to trigger a defence response in a different host context, i.e. to engineer resistance in other species. Using aneuploid wheat lines, two members of the AWJL gene family were shown to map to chromosome 5A, three to 5B,five to 5D and four to 3D. Three of those on 3D were shown to be on the short arm and to be absent from deletion mutants lacking the Ph2 gene, a weak suppressor of homoeologous chromosome pairing in hexaploid wheat. No members of the gene family were absent from deletion mutants lacking the Phl gene, a strong suppressor of homoeologous pairing, located on the long arm of chromosome 5B. Using reverse transcriptase (RT) PCR, expression of AWJL3 in immature barley anthers was found to peak at the time pollen meiocytes entered pachytene and zygotene of meiosis. Unfortunately, controls for the amount of total RNA used and for quantitative yield of PCR products were not reported, nor were measurements of AWJL3 expression made for ovule meiocytes or for other tissues except young leaves
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and mature pollen. Based on the loss of three members of the AWJL gene family in deletion mutants of Ph2 and on the timing of AWJL3 expression in pollen meiocytes, Ji and Langridge speculate that Ph2 is a member of the AWJL gene family. However, the deletion of Ph2 may encompass a relatively large physical region that also includes a cluster of AWJL genes. Deletion of Phl did not result in the loss of any members of the AWJL family even though they mapped to the same chromosome. The AWJL genes should also be expressed during meiosis in ovule meiocytes but this prediction has not been tested nor has the possibility that the AWJL genes may be expressed in other parts of the plant, in particular other parts of the flower. The AWJL genes could be under the control of homoeotic genes involved in floral development analogous to the control of FZL2 expression by DEFICIENS in Anrirrhinum (Steinmayr et al., 1994). The evidence to support the speculation that Ph2 is a member of the AWJL gene family is only circumstantial. Ji and Langridge further suggest that, because the EMPR RNAs are the most abundant polyadenylated RNA species present in early meiosis in lily, they may play a structural role in the formation of the synaptonemal complex. They suggest that the AWJL proteins are members of the EMPR family with structural features (i.e. LRRs) consistent with such a role. However, the authors note and are puzzled by the observation that there is no sequence homology between AWJL3 and members of the EMPR family. They suggest that the bridging maize cDNA clone, pZm9, may have intermediate or dual homology. An extreme interpretation of the latter suggestion would be that the clone may have been chimeric. The pZm9 probe detects highly repetitive D N A sequences on gel blot blots of wheat DNA. Two of the wheat cDNA recovered using pZm9 also detect highly repetitive sequences, whereas the AWJL3 clone detects only moderately repetitive sequences. This observation also suggests that the AWJL gene family does not correspond to the EMPR gene family. Structurally the AWJL proteins bear all the hallmarks of extracytoplasmic membrane-anchored proteins and carry LRRs which match the consensus for extracytoplasmic LRRs rather than cytoplasmic or nuclear LRRs, so they do not bear the structural features consistent with a role in the assembly of the synaptonemal complex. Rather they bear the structural features consistent with a role in disease resistance like that of the Cf resistance genes. Floral tissues are the specific targets for a number of pathogens, e.g. nematodes (Stynes and Bird, 1982) and smuts (numerous examples may be found in the CMIIIMI Descriptions of Pathogenic Fungi and Bacteria) in the grasses, so it would not be surprising to find resistance genes with a correspondingly specific pattern of gene expression. It is therefore possible to rationalize the pattern of AWJL gene expression with a role in disease resistance. However, it is also possible that the AWJL proteins are receptors for peptide signals involved in the meiotic process.
TABLE I11 LRR consensus sequences for plant-receptor-like protein kinases Protein TMKl
Species
Arabidopsis thaliana
Repeatsa 11 ( 2 )
RLKS
Arabidopsis thaliana
21 (1)
TMKLl
Arabidopsis thaliana
6 (1)
PRKl
Petunia injlata
4-6
ERECTA
Arabidopsis thaliana
19 (1)
Consensus sequenceb L--L--LQ-L-L--NNLSGa--
K K T L--L--L--L-LS-N-LSGEIP-a A S I--a--L-SLDLSGN-LSGSIP-L N GS V
Reference Chang et al. (1992)
P ?
Walker (1993) Valon et al. (1993)
e,
a
---L--L--L-LS”-L-OIp--
Mu et a f . (1994)
V E F LG-L--L--LDLSNN-LSG-IP-I N +G T
Toni et al. (1996)
?
P
aThe number of full-length LRRs is indicated together with the number of incomplete LRRs in parentheses. bAmino acid residues are shown in single-letter code. Residues matching the LRR consensus LxxLxxLxxLxLxxNxLxGxIPxx are highlighted in bold. a, The aliphatic residues L, I, M, V or F; -, any residue; +, positively charged residues K or R. Where single residues are shown these comprise more than 50% of the residues at this position. Where two residues are shown, the two residues together comprise more than 50% of the residues at this position, with the upper of the two being more frequent.
F
E
E
LEUCINE-RICH REPEAT PROTEINS IN PLANT DEFENCES B.
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RECEPTOR-LIKE PROTEIN KINASES
The first gene isolated encoding a receptor-like serinehhreonine kinase with LRRs was the TMKl gene of Arabidopsis (Chang et al., 1992). TMKl contains 13 LRRs which show a good match to the extracytoplasmic LRR consensus LxxLxxLxxLxLxxNxLxGxIPxx (Table 111). Like the Cf genes, TMKl shows an interrupted LRR domain with 11 repeats on the amino terminal side of the interruption and two on the carboxyl terminal side. However, as noted previously (Jones et a f . , 1994), this is probably a consequence of a duplication of the amino terminus and so does not resemble the “loop-out’’ domain of the Cfgenes. DNA gel blot analysis indicates that TMKl is not a member of a gene family, although weakly hybridizing bands are seen if hybridizations are carried out under low stringency. Western analysis indicates that TMKl protein is expressed in roots, leaves, flowers and siliques but not stems. The RLKS gene of Arabidopsis (Walker, 1993) encodes a receptor-like serinehhreonine kinase with 22 LRRs that match the extracytoplasmic LRR consensus (Table 111). DNA gel blot analysis indicates that RLKS, like TMK1, is a single copy gene. RNA gel blot analysis indicates that RLK5 is expressed in rosettes of Arabidopsis and at a lower level in roots. RLKS has been shown to interact with a 582 amino acid, kinase associated protein phosphatase (KAPP) that contains a type-1 signal anchor for attachment to the cell membrane, a kinase interaction domain and a serinehhreonine phosphatase domain (Stone et al., 1994). The kinase interaction domain of KAPP binds to the phosphorylated kinase domain of RLK5 and is phosphorylated by RLKS. The effect of RLKS on KAPP, or vice versa, is not clear, but KAPP is likely to either propagate or clear the signal originating from RLKS (for a review of the interaction between protein kinases and protein phosphatases see Hunter, 1995). KAPP is also a single copy gene like RLK5, but DNA gel blots carried out at low stringency suggest that there may be related genes in Arabidopsis. KAPP is also expressed in rosettes, but, unlike RLKS, is expressed at a higher level in roots, suggesting that it may have functions additional to those involving RLK5. Because of the resemblance between RLKS and Xa-21 and Pto, a KAPP-like protein may also interact with Xa-21 and PtolPrf to regulate activation of plant defences. Although signal initiation involves a protein kinase in the case of Pto, Prf could conceivably regulate the signal by interaction with a protein phosphatase. In the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae, the serinekhreonine protein phosphatase PP1 is regulated by an LRR protein Sds22 (EGP1) (Ohkura and Yanagida, 1991; Hisamoto et a f . , 1995; MacKelvie et a f . , 1995), that alters the substrate affinity and specificity of PP1. The TMKLl gene of Arabidopsis (Valon et al., 1993) encodes a receptor protein with seven LRRs that match the extracytoplasmic LRR consensus
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(Table 111) and a kinase-like domain that has homology with serinekhreonine kinases but lacks five of the 11 subdomains conserved among all known protein kinases including those of the NBS. DNA gel blot analysis indicates that TMKLl, like TMKl and RLKS, is a single copy gene. RNA gel blot analysis indicates that a single 2.5 kb TMKLl transcript is expressed in all vegetative tissues, except siliques where expression decreases with maturation. These data indicate that TMKLI is not part of a gene family in which the defective kinase domain may be complemented by a functional homologue. The function of TMKLl is unknown, but the absence of a kinase function does not preclude a signalling role via a cryptic function or interaction with a heterologous transmembrane or cytoplasmic partner. A receptor protein with a cytoplasmic domain that has lost its kinase activity is functionally equivalent to a receptor protein that has lost its cytoplasmic kinase domain or never had one attached. Thus TMKLl effectively resembles the Cf proteins and may provide clues to the way in which they function. The PRKl gene of Petunia inflata (Mu et al., 1994) encodes a receptor protein kinase with 4-6 LRRs that show a poor match with the extracytoplasmic LRR consensus, but are definitely of the extracytoplasmic type (Table 111). The protein kinase activity of PRKl differs from that of TMKl and RLKS because it phosphorylates on serine and tyrosine, suggesting that it may be a dual specificity kinase. DNA gel blot analysis with either LRR or protein kinase domain probes indicates that PRKl is a single copy gene, but the protein kinase probe also hybridized weakly to another band, suggesting a related protein kinase gene in Petunia. Unlike TMKl, RLKS or TMKL1, RNA gel blot and Western analyses showed no expression of PRKl in roots, leaves or flowers, except in developing anthers, mature pollen and germinating pollen tubes. The function of PRKl is unknown, but the pattern of expression suggests a different role to that of TMKl, RLKS or TMKLl, possibly in the regulation of pollen development or pollen tube growth. The ERECTA gene of Arabidopsis (Torii et al., 1996) encodes a receptor serinekhreonine kinase with 20 LRRs that match the extracytoplasmic LRR consensus (Table 111). This gene is thought to function in apical meristem development because the erecta mutation confers round leaves with short petioles, compact plant stature, compact inflorescence and short blunt siliques but has no effects on roots. DNA gel blot analysis indicates that ERECTA is a single copy gene. RNA gel blot analysis indicated that ERECTA is expressed in all aerial parts of the plant especially the shoot apical meristem, but not in roots, consistent with the phenotypic effects observed for the erecta mutation. A candidate for the CLAVATAl gene of Arabidopsis has also been identified as a receptor serinehhreonine kinase with LRRs (S. E. Clark, R. W. Williams, S. E. Jacobsen and E. M. Meyerowitz, unpublished). CLAVATAl is also thought to function in apical meristem development, because the clavatal mutant accumulates large numbers of undifferentiated
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cells in the meristem leading to fasciation and increased numbers of whorls in the flowers and increased numbers of organs within the whorls (Clark et al., 1993). The involvement of LRR receptor kinases in plant development suggests that peptide ligands may play a role in intercellular signalling in plants and that other signals involving peptide ligands such as systemin, involved in the systemic activation of protease inhibitors in response to wounding or insect feeding (Pearce et al., 1991; Orozco-Cardenas et at., 1993), also might be perceived by LRR receptor kinases. In section IIB, it was suggested that the rice Xu-21 gene for resistance to X . oryzae might indicate a role in disease resistance for LRR receptor serinekhreonine kinases with unknown function. However, the fact that two such proteins seem to have a role in plant development suggests that there are other possible roles for TMK1, RLKS and TMKLl. The fact that, unlike most resistance genes, these genes are also single copy genes also argues against a role in disease resistance for these proteins.
VI. A GENE ENCODING A PROTEIN OF UNKNOWN FUNCTION WITH CYTOPLASMIC LRRs A sunflower LRR gene was isolated initially as a cDNA clone, SF17, by differential screening of a pollen cDNA library (Reddy et al., 1995). SF17 encodes a 540 amino acid protein with an amino terminal hydrophilic domain of approximately 210 amino acids, followed by 10 LRRs and two carboxyl terminal hydrophobic domains in tandem. The LRRs match the 23 amino acid LRR consensus LxxLxxLxxLxLxxNxLxxIPxx (rather than a 24 amino acid consensus as reported), which is a subset of the plant cytoplasmic LRR consensus LxxLxxLxxLxLxx(N/C/T)x(x)LxxIPxx that is characteristic of the cytoplasmic resistance proteins (see sections IIIA to IIIC; Table 11). The LRRs of SF17 show a more regular length than the LRRs of the cytoplasmic resistance proteins, no substitution of N by C or T, no extra residue at position 17 and good conservation of the LxxIP portion of the consensus. Interestingly, the consensus LxxLxxLxxLxLxxNxLxxIPxx also matches that of several animal cytoplasmic LRR proteins. The hydrophilic amino terminus is reported to begin with a relatively hydrophobic sequence from residues 7 to 23 that might function as a signal anchor. In fact this region contains five basic residues followed immediately by three more basic residues, suggesting that it is unlikely to function as a signal anchor. However, the two carboxyl terminal hydrophobic domains may both function as transmembrane domains. The LRR domain is reported to be linked to the first of these hydrophobic domains by an acidic region from residues 458 to 479 that contains seven acidic residues and three basic residues, which might suggest an extracytoplasmic location, thus orienting the protein in the membrane. However, the range of residues is somewhat
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arbitrary, and had it been from residues 449 to 479 it would have contained six basic residues and seven acidic residues suggesting nothing about the possible orientation of this protein. By the same token, if the range had been extended to the entire interval between the LRR domain and the first hydrophobic domain, i.e. residues 438 to 479, then there are 10 acidic residues and six basic residues, suggesting a slight excess of acidic residues. This is not compelling evidence for an extracytoplasmic location of this domain. The cytoplasmic nature of the LRR consensus provides much more compelling evidence about the location of this domain. This protein is most likely a predominantly cytoplasmic protein anchored to the membrane by two transmembranes connected by a very short extracytoplasmic loop and with both the amino and carboxyl termini exposed to the cytoplasm. This membrane topology resembles that of the rat p34 ribosome binding protein which has a large, cytoplasmic, amino terminal domain with 4.5 LRRs anchored to the endoplasmic reticulum membrane by a single transmembrane domain and has a small carboxyl terminal domain in the lumen of the endoplasmic reticulum (Ohsumi et al., 1993). However, unlike SF17, the LRRs of p34 are at the amino terminus and separated from the transmembrane domain by a hydrophilic domain rich in lysine, arginine and glutamic acid. DNA gel blot analysis reveals that SF17 is a member of a small gene family with at least two members. RNA gel blot analysis showed that expression of SF17 was limited to mature pollen, with no transcripts detected in roots, leaves, closed flowers, ovaries, corolla, styles and stigmas or immature seeds. The role of SF17 is unknown, but it is thought to be involved in pollen development or pollination.
VII. THE EVOLUTION OF PLANT LRR PROTEINS A. THE EVOLUTION OF LRR PROTEINS IN THE EUKARYOTES
Comparison of the consensus sequences for plant, animal and fungal cytoplasmic and extracytoplasmic LRRs suggests that the 24 amino acid LRR consensus sequences for plant versus animal extracytoplasmic LRRs and also some plant cytoplasmic LRRs may be derived by insertion of single residues at different locations within the 23 amino acid consensus sequence for cytoplasmic LRRs (Fig. 9). This suggests that extracytoplasmic LRRs may have evolved from a cytoplasmic precursor and that plant and animal extracytoplasmic LRRs may have arisen independently of one another. Both the extracytoplasmic and cytoplasmic LRRs of plants show a remarkable similarity to those of protozoan parasites. Two cyst-wall proteins (CWPI and CWP2) of Giardia larnblia (Mowatt et al., 1995) each have six LRRs that show a good fit to the plant extracytoplasmic LRR consensus
intracellular L x x L x x ~ L x L x x c ~ L x x I X xtrypanosome
LRRs
t
extracellular
ex an.. .._._. .. ._
intracellular
L x x L ~ L ~ L x L (-1 ~ ~ L ~ I-I--+
L
.
I DD-
c
C
T
Fig. 9. A dendrogram of minimal changes in the consensus amino acid sequences for LRR proteins, that may indicate the way in which they have evolved. The amino acid sequence is shown in single-letter code and changes in the consensus sequences are highlighted in bold. x , Any residue; L and I tend to be substituted by each other and by M, V and F.
8
TABLE IV LRR consensus sequences for protozoan proteins with extracytoplasmic or cytoplasmic LRRs Protein
Species
CWPl
Ciardia lamblia
CWP2
Ciardia Iamblia
PSA-2 tr-1 PSA-2 2.5 PSA-2 4.6 PSA-2 6.4 GP46M2
Leishmania major
ESAG8
Leishmania major Leishmania major Leishmania major Leishmania amazonensis Trypanosoma brucei
Repeatsa
Consensus sequenceb
Reference
a--L--L--L-LSDN-L-DIP-NC a--L--a--LYaS"-LTGPIPTC H NC D AC W--M--L--L-L-G--LSGSW-S I TL a WSSM--L--L-L---KaSGTIPPE L a T AQ WSSM--L--L-LNG-KLSGTIPPQ a a WSAM--L-SL-L-G-KaSGLe-S
Mowatt et al. (1995)
WSSM--L--L-L-G--LSG-LP-S E A a a L--L--LE-L-LSGC--L--L-a i a
Mowatt et al. (1995) Murray and Spithill (1991)
P ?
Murray and SpithiU (1991) Murray and Spithill (1991)
e,
1
n
Murray and Spithill (1991) Lohman et al. (1990) Revelard et al. (1990) Smiley et al. (1990)
aThe number of full-length LRRs is indicated together with the number of incomplete LRRs in parentheses. bAmino acid residues are shown in single-letter code. Residues matching the extracytoplasmic LRR consensus LxxLxxLxxLxLxxNxLxGxIPxx or the cytoplasmic LRR consensus LxxLxxLxxLxLxx(N/C/T)x(x)LxxIPxx are highlighted in bold. ESAG8 is the only sequence shown with cytoplasmic LRRs. a, The aliphatic residues L, I, M, V or F; -, any residue; positively charged residues K or R. Where single residues are shown these comprise more than 50% of the residues at this position. Where two residues are shown, the two residues together comprise more than 50% of the residues at this position, with the upper of the two being more frequent.
+,
?
P 0
LEUCINE-RICH REPEAT PROTEINS IN PLANT DEFENCES
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LxxLxxLxxLxLxxNxLxGxIPxx (Table IV). One complete and three incomplete sequences for promastigote surface antigens of Leishmania major also show a good fit to the plant extracytoplasmic LRR consensus, although the L at position 1 is replaced by W (Table IV). A trypanosome leucine repeat (T-LR or ESAG8) from Trypanosoma brucei and T. equiperdum (Revelard et af., 1990; Smiley et al., 1990; Ross et al., 1991) has 22 LRRs that show a good fit to the plant cytoplasmic LRR consensus LxxLxxLxxLxLxx(N/C/T)x(x)LxxIPxxwith C at position 15 and the extra residue at position 17, but lacking any proline residue at position 22, resembling some of the repeats of L6 in particular (Table IV). These comparisons suggest that the consensus sequences for the protozoan LRRs are either the same as those of plant LRRs or may be derived horn them by insertion or deletion of single residues at different locations within the plant consensus. These similarities between plant and protozoan LRRs are either a remarkable example of convergent evolution or they suggest that the plant and protozoan LRRs reflect an ancestral state and the animal and fungal LRRs reflect a derivative state. They also suggest that both cytoplasmic and extracytoplasmic LRRs existed before the radiation of the eukaryotes, since the protozoa represent some of the earliest branches of the eukaryotes. B. EVOLUTIONARY CLUES PROVIDED BY INTRON ARRANGEMENTS
Unlike the genes for most other LRR proteins, the tomato gene for LRP (Tornero et al., 1996) and the Arabidopsis ERECTA gene (Torii et af., 1996) contain introns in the LRR domain. Curiously, in four out of the five LRRs of LRP and in all 20 of the LRRs of ERECTA, these introns disrupt the coding sequence between the second and third base of the codon for leucine (or valine in the case of the first truncated LRR) at position 10 (underlined) in the consensus LxxLxxLxxLxLxxNxLxGxIPxx.A similar situation exists in the mammalian follitropinrthyrotropin and lutropin/gonadotropin receptor genes (Gross et al., 1991; Koo et a f . , 1991; Tsai-Morris et a f . , 1991; Heckert et al., 1992), where introns disrupt eight out of the 10 LRRs between the second and third base of the homologous leucine at position 11 (underlined) in the consensus AFxxLxxLxxLxLxxNx(x)LxxIPxx.The same is also true in the homologous G-protei&oupled receptor of the sea anemone (Nothacker and Grimmelikhuijzen, 1993), where introns disrupt two out of the 10 LRRs. These introns vary considerably in size within each protein. The follitropin, thyrotropin and lutropin/gonadotropin receptor genes encode homologous proteins, with LRRs having identifiable counterparts in the corresponding positions in each protein. The differences in specificity between these proteins are therefore not a consequence of exon shuffling that might be facilitated by the presence of introns. In contrast to
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D. A. JONES and J . D. G. JONES
the exons, the corresponding introns vary considerably in size between each protein. The intron sequences appear to have evolved rapidly and divergently, so that no inferences may be drawn from their size or sequence composition, but their consistent and repeated location, in LRP, ERECTA and the hormone receptor genes, has several evolutionary implications. Firstly, it suggests that the LRR domain in each of these proteins may have evolved by the amplification of an exon containing a single LRR. Secondly, it suggests that this LRR exon was ancestral to both plant and animal extracytoplasmic LRRs. The consensus sequence of both can be derived from that of cytoplasmic LRRs by the insertion of single amino acid residues at different locations (Fig. 9), so it is possible that the ancestral exon encoded a cytoplasmic LRR with the consensus sequence xLxxNxLxxIPxxLxxLxxLxxL.Thirdly, and almost paradoxically, it suggests that exon duplication occurred after the divergence of the consensus sequences for plant and animal extracytoplasmic LRRs (Fig. 9). Alternatively, the LRRs may have evolved in concert after exon duplication, although this seems less likely. The consensus sequences for the extracytoplasmic LRR exons, i.e. xLxxNxLxGxIPxxLxxLxxLxxLfor plants and xLxxNxLxxIPxxLLxxLxxLxxL for animals, represent circular permutations of the consensus sequences as they are normally and arbitrarily depicted in the literature. If the LRRs of Cf-9 were to be permutated in this manner, then the first block of 23 consecutive LRRs comprising 21 full-length and 2 truncated LRRs (Fig. 1) could be viewed as 22 full-length LRRs. Similarly, the first block of 34 consecutive LRRs in Cf-2, comprising 32 full-length and 2 truncated LRRs (Dixon et al., 1996), could be viewed as 33 full-length LRRs. By this criterion, the first apparently truncated LRR of several other extracytoplasmic proteins could also be viewed as full length. This would support the idea that LRR proteins lacking introns may also have arisen by amplification of a single exon of this nature. However, the last LRR of these proteins would then be considered truncated rather than complete. Interestingly, the last LRR exon of LRP (Tornero et al., 1996) lacks the LRR motif beyond xLxxNxLxGxIP, but the next intron occurs in exactly the right location expected for a full-length LRR exon. This suggests that the LRR motif beyond proline is not required structurally for the last LRR and selection has perhaps favoured alternative residues leading to a stable protein or domain terminating structure. Another view of the intron structure of LRP, ERECTA and the hormone receptor genes might be that the positions of the introns are an example of convergent evolution due to some selective advantage conferred by introns in these locations. This seems an unlikely explanation given that so many other LRR proteins possess no introns within their LRR domains and that the sea anemone receptor lacks, and has probably lost six of the eight introns present in the LRRs of mammalian receptors. However, the presence of introns provides the opportunity for variation in specificity through the precise deletion
LEUCINE-RICH REPEAT PROTEINS IN PLANT DEFENCES
143
of one or more LRRs by alternative splicing. Despite the extensive opportunity for alternative splicing in the LRR domains and the extensive occurrence of alternative splicing of introns outside the LRR domains, there is only limited alternative splicing of the introns within the LRR domains of the mammalian hormone receptor genes (Aatsinki et al., 1992). However, the more carboxyl terminal of the two introns in the sea anemone receptor remains unspliced in some transcripts to generate a truncated soluble receptor domain (Nothacker and Grimmelikhuijzen, 1993). There is no evidence for alternative splicing of either the LRP or ERECTA transcripts. An alternative view of this intron structure is that the retention of introns stabilizes these genes by reducing the likelihood of unequal crossing over between LRRs. Stability might be advantageous if the LRR domain binds an invariant endogenous ligand or a limited number of related variants. On the other hand, the genetic flexibility provided by a potential for unequal crossing over (see section VIIC) might be advantageous if the LRR domain binds various and varied exogenous ligands, such as those produced by plant pathogens. This might explain to some extent the general absence of introns from the LRR domains of many LRR proteins.
C. EVOLUTION OF DIFFERENT SPECIFICITIES IN LRR PROTEINS
There are several ways in which LRR proteins can evolve different specificity. One way would be unequal crossing over to create a cluster of genes which can mutate independently to give new specificities (reviewed by Pryor and Ellis, 1993). Further unequal cross-overs could even produce hybrid genes with novel specificity. Another way would be unequal crossing over between LRRs within a gene to increase the number of LRRs upon which mutation could act to create a variety of binding specificities. The conserved alternating A and B LRRs of Cf-2 (see section IIA) are consistent with an increase in LRR numbers by intragenic, unequal crossing over. The (AB)3AB4(AB)4A arrangement of these repeats suggests that there may be at least two units of recombination, one comprising two LRRs in the case of the AB repeats, and the other a single LRR in the case of the four consecutive B repeats. Some of the repeats are identical, suggesting recent duplication. Others have slight variations, suggesting less recent duplication. However, only residues at some positions within these repeats are varied, whereas residues at other positions are tightly conserved. This may indicate the selective retention of an advantageous structural arrangement conferred by the conserved residues and changes in specificity conferred by the vaned residues. It is interesting that the LRR consensus of the A and B repeats of Cf-2 are best represented by a circular permutation different from that of LRR exons (see section VIIB) or that used in the literature, i.e. xxLxxLxxLxLxxNxLxGxIP (Dixon et al., 1996). This is consistent with the
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D. A. JONES and J. D. G . JONES
idea that, after exon duplication, further amplification can occur by unequal crossing over which can occur anywhere within the consensus. However, it may also be argued that selection may only favour exchanges which maintain appropriate interactions between amino acid residues of adjacent LRRs (see section VIIIA). In L6 (section IIIB) there are two tandem duplications of approximately 140 amino acids towards the carboxyl terminus of the LRR region (see Fig. 4), showing that in cytoplasmic LRRs there is also potential for intragenic unequal crossing over between LRRs within a gene, despite the greater variation between repeats. Other alleles at the L locus seem to differ in the number of these duplications (Ellis et al., 1995). This suggests a slightly different mechanism for the generation of diversity, by increasing or decreasing the numbers of blocks of LRRs through intragenic recombination.
VIII. THE STRUCTURE AND MOLECULAR SPECIFICITY OF PLANT LRR PROTEINS A. INFERENCES ABOUT THE STRUCTURE OF PLANT LRRs BY COMPARISON WITH THE KNOWN STRUCTURE OF PORCINE RIBONUCLEASE INHIBITOR
The crystal structure of only one LRR protein, the porcine ribonuclease inhibitor (PRI), has been produced to date (Kobe and Deisenhofer, 1993). PRI is novel amongst LRR proteins because it has alternating repeats of 28 and 29 amino acids that differ from those identified so far in plants, which average 23 or 24 amino acids and, apart from Cf-2, do not appear to alternate. For this reason it is difficult to draw inferences about the structure of plant LRR proteins except for regions of identity between the consensus sequences of the plant LRRs and those of PRI (Fig. 10A). PRI is a cytoplasmic protein that contains eight repeats of 28 amino acids designated type A alternating with seven repeats of 29 amino acids designated type B (Fig. 10A). These are flanked by a truncated repeat of 23 amino acids at the amino terminus and one of five amino acids at the carboxyl terminus. Each repeat, apart from the latter, is capable of forming a short region of p strand, a loop (pa), a short region of a helix and another loop (ap) (Fig. 10B) leading into another repeat which forms a p strand parallel to the first, a Pa loop, an a helix parallel to the first, an ap loop leading into another repeat and so on, ending in a p strand at the carboxyl terminus. This structure is stabilized by interactions between residues in adjacent repeats which form ladders of buried hydrophobic residues and ladders of exposed hydrophilic residues of alternate charge or polarity. Thus a parallel p sheet is formed on one face of the protein and parallel a helices on the other. The greater steric diameter of the a helices compared to the p strands imparts curvature to the protein
145
LEUCINE-RICH REPEAT PROTEINS IN PLANT DEFENCES
A
Conserved consensus region T X X X C X X L,X X a L
p strand p strand Plant extracytoplasmic X L X
I
X X X
a helix
=
p strand
X
a helix
L X X N X L X G X I P X X L X X L X
1
Plant cytoplasmic X L X iFLx L X X N X L X X I P X X L X X L X p strand
B
C
;
Basic structure of a single LRR PRI LRRs
Plant LRRs
a helix
unknown
p strand
p strand
Structural arrangement of several LRRs Structure observed for PRI LRRs
Possible structure for plant LRRs
Fig. 10. Structural domains of plant LRRs based on comparisons with those of the porcine ribonuclease inhibitor (PRI). (A) Structural domains identified in PRI and inferred for plants, shown relative to the respective LRR consensus sequences. The amino acid sequence is shown in single-letter code: X, any residue; PRI A and B , consensus sequences for the alternating A and B LRRs of PRI. (B) Schematic representation of the basic structural unit identified for a single LRR of PRI and inferred for plants. (C) Schematic representation of the structural arrangement identified in PRI for several consecutive LRRs and inferred for plants: arrows represent p strands and the cylinders a helices; N, amino terminus; C, carboxy terminus.
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D. A. JONES and J. D. G. JONES
(Fig. lOC), resulting in a horseshoe-shaped protein with a p sheet lining the inner face (here designated the p face) and a helices lining the outer face (here designated the ct face). Interaction with ribonuclease A involves predominantly the p face and the Pa loop (Kobe and Deisenhofer, 1995a). This is the region which shows identity between the consensus sequences of PRI and those of the plant LRRs (Fig. 10A). This consensus LxxLxLxx(N/C)xL involves residues in the end of the ap loop, the p strand and the beginning of the Pa loop (see Fig. lOB). The conserved residues in this consensus are all buried in the hydrophobic core between the /3 and a faces of the protein. The peptide backbone of the XXLXLXX portion of the consensus, comprising the p strand (underlined) flankedby p turns, provides a relatively flat surface profile on the p face of the protein with the leucine side-chains buried and those of the interstitial residues exposed. Twenty of the 28 points of contact between PRI and ribonuclease A lie in this region. Nineteen of these are interstitial residues, and the twentieth, although located in a consensus leucine position, is contained in the truncated carboxyl terminal repeat and is therefore exposed. Overall, this suggests a curvilinear backbone, comprising parallel p sheet flanked by p turns, established by the conserved residues of the LRR, with an exposed face composed of the various interstitial residues, of which a large proportion mediate and determine the specificity of protein binding. Based on the identities in the consensus sequences, the interacting faces of plant LRR proteins are also likely to have curvilinear backbones of parallel p strands and flanking p turns (Fig. 1OC). The alternating repeat structure of Cf-2 (see section IIA) supports this model, as it suggests interactions between residues in adjacent repeats analogous to those observed for PRI. The conservation of residues outside of the XXLXLXX domain in these repeats is also consistent with the conservation of structural interactions between adjacent repeats. However, some of the residues (underlined) inside the _ XXLXLXX domain in these repeats seem to be varied, consistent with a role in specificity of ligand binding. A pattern of alternating repeats is not obvious in Cf-9, but there may be localized alternation of residues consistent with interactions between adjacent repeats. Plant LRR proteins may show curvilinear interacting faces, but they are unlikely to show the same curvature as PRI. The curved shape of the /3 face of PRI is largely determined by the arrangement of the @aand ctP loops and the steric diameter of the ct helices. Apart from a portion of the pa loop, there are no obvious counterparts to these features in the consensus sequences of the plant LRRs (Fig. 10). The shorter consensus sequence of plant LRRs and the presence of residues which often disrupt (Y helices, i.e. a glycine and a proline in the extracytoplasmic LRR consensus, and a proline in the cytoplasmic LRR consensus, would tend to argue against the formation of a helices. The presence of additional glycines and prolines at other positions in the individual consensus sequences for Cf-2, Xa-21 and the PGIPs (Table I) would also tend to argue against the formation of a helices.
LEUCINE-RICH REPEAT PROTEINS IN PLANT DEFENCES
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However, the first repeat of PRI is of comparable length to the plant LRRs, yet generates an a helix, and several LRRs in PRI contain proline kinks in their a helical regions, so that the shorter length and the presence of glycines and prolines are not necessarily incompatible with a structure similar to PRI. A structure with regular kinks might have a smaller steric diameter than the a helices of PRI and therefore generate a protein with shallower curvature than PRI. Several of the plant LRR proteins described so far contain more than 21 consecutive repeats. If a structure such as PRI were to contain more than 21 consecutive LRRs, then the termini would collide, although more repeats could be accommodated if the curvature was shallower or a twist is introduced into the P sheet to produce a spiral. Kajava et al. (1995) have modelled the LRR structure of the thyrotropin receptor and shown that structures with shallower curvature are feasible for proteins with shorter LRR consensus sequences containing prolines. However, Kobe and Deisenhofer (1994, 1995b) suggest that a different structure to that of PRI may be possible for LRR consensus sequences shorter than those of PRI. They suggest by analogy to the P roll of alkaline protease or the /3 helix of pectate lyase C that a repeating PIP or PIPIP arrangement may occur rather than a repeating @laarrangement, which would generate a rod-shaped structure rather than a curved structure. The extracytoplasmic plant LRR proteins have more regular and conserved repeats with a different consensus sequence compared to cytoplasmic plant LRR proteins. The most notable differences are the glycine at position 19 of the extracytoplasmic LRR consensus, and the frequent occurrence of an extra residue at position 17 and C or T in place of N at position 15 of the plant cytoplasmic LRR consensus. Extracytoplasmic proteins are also formed in a different environment compared to cytoplasmic proteins. They are formed at a different pH, which will confer different charge properties to some amino acid residues and so affect structure, and in an oxidizing versus a reducing environment, which enables cysteine residues to form disulphide bonds. The extracytoplasmic LRRs also contain numerous glycosylation sites which may affect structure. The plant extracytoplasmic LRR proteins are therefore likely to have a different, more ordered structure than plant cytoplasmic LRR proteins and may have different-shaped interacting faces. However, in the absence of structural data, the shape of the interacting face in either class of plant LRR protein is an open question.
B. INFERENCES ABOUT THE STRUCTURE AND INTERACTIONS OF EXTRACELLULAR PLANT LRR PROTEINS BASED ON THEIR POTENTIAL PATTERNS OF GLYCOSYLATION
The potential patterns of glycosylation for the extracytoplasmic LRR proteins may give some clues about their structure and function. The majority of the
TABLE V Locations of potential N-glycosylation sites in plant extracytoplasmic LRR proteinsa Plant extracytoplasmic LRR consensus
N-Glycosylation sites ~~
Protein Cf-9 Cf-2 Xa-21 BPGIP SPGIP PPGIP TPGIP FIL2 KPGIP LRP Tom-LA Pexl TMKl RLKS TMKLl ERECTA ~
Total 22 32 17 4 3 7 7 8 7 2 5 3 6 9
8 12
~
In LRRs L x x L x x L x x L x L x x N X L x G x I P x x 19 30 16 2 1 7 7 8 7 1 2 3 6 9 5 10
3 2 6 1 7 1 1 1 1 1 1 2 3
10 1
3 3 2 1
1 2 2 2
1 1 1 1
1
1 1 1
1 3
1 3
2 7
5 1 1 3 3 3 3 1 1
1 1 1 1 1 4
4
2 1
2 1 3 1
1
1
1 1
2 1
Reference Jones et al. (1994) Dixon et al. (1996) Song et al. (1995) Toubart et al. (1992) Favaron et al. (1994) Stotz et a1 (1993) Stotz et al. (1994) Steinmayr et al. (1994) Simpson et al. (1995) Tornero et al. (1996) D. A. Jones (unpublished) Rubenstein et al. (1995) Chang et al. (1992) Walker (1993) Valon et al. (1993) Toni et al. (1996)
__
aAmino acid residues are shown in single-letter code: x, any residue. PGIP prefixes: B, bean; S, soybean; P, pear; T, tomato; K, kiwifruit. FIL2, Antirrhinum PGIP; Pexl and Tom-LA, maize and tomato LRR extensins, respectively. In cases where ambiguities in position may have arisen because of departures from the LRR consensus, the glycosylation site has been positioned relative to the nearest LRR consensus residue.
P ?
W
1
a ' .
P 0
+ 0
Z
c1
LEUCINE-RICH REPEAT PROTEINS IN PLANT DEFENCES
149
predicted p faces of these proteins seldom contain potential glycosylation sites, consistent with this region being involved in ligand binding (Table V). Surprisingly, the variable residue at position 11, underlined in the consensus LxxLxxLxxLxLxxNxLxGxIPxx, located in the p strand region of the p face, does often contain potential glycosylation sites. However, these sites are not randomly distributed among the LRRs. They are located either in LRRs at the beginnings or ends of the LRR domains. Of the 18 potential glycosylation sites shown in position 11 in Table V, the three in Cf-9 are in the last four LRRs, one of the three in Cf-2 is in the first LRR and the remaining two are in the last three LRRs, the two in Xa-21 are in the last six LRRs, the one in each of the PGIPs (except soybean), RLKS and TMKLl is in the last LRR, and one of the four in ERECTA is in the first LRR and the remaining three are in the last six LRRs. This suggests that LRRs on the extremities of the LRR domain may not have p face amino acid residues available for protein-protein interaction, because these may be masked by glycosyl residues. These LRRs may not participate in ligand binding unless proteinglycosyl interactions are also involved. In the case of Cf-9 and Cf-2 these potential glycosylation sites occur in the conserved LRRs, LRRn and LRRn-2 (where n is the total number of LRRs), following the “loop-out” region (see Fig. 2). This is the region that may be involved in a conserved interaction with a protein partner, suggesting that such a protein might have glycosyl binding capacity if it binds to the p face. However, in LRRn and LRRn-2, the conservation between Cf-9 and Cf-2 is mostly outside of the p face region (see Fig. 2), suggesting that the other face and the connecting loops may be important for interaction with another protein. On the other hand, in LRRn-1, the conservation between Cf-9 and Cf-2 lies in the p face region (see Fig. 2). In the AWJL proteins (see section VA), only the potential glycosylation sites in LRRn and LRRn-2, and the amino acid sequence of the p face region of LRRn-1 are conserved, and in Xa-21 only the potential glycosylation site in LRRn-1 (corresponding to LRRn of the Cf genes) and the amino acid sequence of the p face region of LRRn-2 (corresponding to LRRn-1 of the Cf genes) are conserved (see section IIA). The more widespread conservation of the p face motif LE(SD’)LDLS in LRRn-1 (or LRRn-2 of Xa-21) and flanking potential glycosylation sites at position 11 in LRRn and LRRn-2 (or LRRn-1 of Xa-21) suggests that the /3 face region might be the most important for interaction with partner proteins. The potential glycosylation sites outside of the p face regions of the extracytoplasmic plant LRR proteins occur predominantly in the positions underlined in the consensus Lx~LxxLxxLxLxxNxLxGxIPxx, immediately prior to Ls (Table V). This is conistent with &e L being buried in a hydrophobic core and the preceding residues being exposed. The conserved N at position 15 is never glycosylated, consistent with it also being buried in a hydrophobic core. Glycosylation in this position would cause a size and charge change that would be structurally disruptive. Interestingly, Cf-2 has
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D . A . JONES and J . D. G . JONES
a quite different pattern of potential glycosylation to that of Cf-9, but this may be in part due to the repetition of potential glycosylation sites at positions 6 and 18 in the conserved alternating repeats of Cf-2. At present, the true pattern of glycosylation is not known for any of the plant LRR proteins and has only been studied in the case of FIL2 (Steinmayr et al., 1994), a putative PGIP (see section IVA). Surprisingly, of the eight potential N-glycosylation sites in FIL2 only five seem to be glycosylated. Interestingly, five of the glycosylation sites are NxT and three are NxS. Perhaps these motifs are differentially glycosylated. Alternatively, some of the N may be inaccessible to glycosyltransferases because of their sequence context. The role of N-glycosylation has been investigated for the LRR ligand-binding domain of the lutropin receptor (Zhang et al., 1995). Of six potential N-glycosylation sites in the lutropin receptor, three lie within the LRR domain. Of these, two are glycosylated and their glycosyl residues are involved in ligand binding, and the third is unglycosylated. The two glycosylated residues are located in regions of slight departure from the lutropin receptor LRR consensus AFxxLxxLxxLxLxxNxLxxIPxx, with one occurring as an extra residue between positions 6 and 7 of the consensus and the other at either position 3 or 4, depending on the positioning of a single residue deletion. The unglycosylated residue is located at position 7 (underlined) in the consensus AFxxLx~LxxLxLxxNxLxxIPxx, equivalent to position 6 (underlined) in the plant consensus LxxLxxLxxLxLxxNxLxGxIPxx. There are a significant number of potential N-glycosylation sites in plant LRR proteins at this position. These data indicate that glycosyl residues can have a role in ligand binding, but that it is probably not possible to predict the glycosylation pattern based on the location of the potential glycosylation sites in the LRR consensus sequences. C. INFERENCES ABOUT THE INTERACTIONS BETWEEN PLANT LRR PROTEINS AND THEIR LIGANDS BASED ON COMPARISONS WITH THE INTERACTIONS BETWEEN RIBONUCLEASE INHIBITORS AND RIBONUCLEASES
Although some inferences may be drawn about the possible structures of plant LRR proteins based on comparison of their LRR consensus sequences with those of PRI (see section VIIIA), it is even more difficult to draw inferences about the molecular interactions with their ligands, because the nature of these ligands is unknown in most cases. The only ligands known for plant LRR proteins with any certainty are the polygalacturonases (PGs) which are the ligands for the polygalacturonase inhibitor proteins (PGIPs). PGIPs are capable of inhibiting a range of PGs from a number of fungal sources. This capacity is analogous to that of ribonuclease inhibitors (RI) , including PRI and the homologous human placental ribonuclease inhibitor (HRI), which are able to inhibit a range of RNases including angiogenin and
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placental RNase in addition to RNase A, and whose points of contact with their ligands have been characterized (Kobe and Deisenhofer, 1995a; Shapiro et al., 1995). HRI has 77% sequence identity with PRI and 24 of the 28 RNase A contact residues in PRI are retained in HRI and three are conservative replacements. The 28 contact residues of PRI interact with 24 residues in RNase A contained within three structural domains (Fig. 11). One is the active centre which binds and cleaves the RNA substrate. Fifteen residues in the active site of PRI are contacted by 16 residues in the three carboxyl terminal LRRs (two complete and one incomplete) of PRI. These indude four contiguous residues, three in the pa loop and one at the beginning of the a helix, in each of the two complete LRRs (Fig. 11). The remaining eight are located on the /3 face. Of the 15 residues contacted in the active site of RNase A, seven are identical in angiogenin and placental RNase. These seven conserved residues are contacted by eight residues in the last complete LRR of PRI and one in the truncated carboxyl terminal LRR, of which four are on the p face (Fig. 11). The other two domains of RNase A bound by PRI comprise an a helix, in which four residues of RNase A are contacted by five residues of PRI on the p face of LRRs 1, 2, 5 and 6 of PRI, and a loop, in which five residues of RNase A are contacted by seven residues on the p face of LRRs 8-12 of PRI (Fig. 11). There is little conservation between RNase A, angiogenin and placental RNase for the residues contacted in the a helix of RNase A and only a few identities for those contacted in the loop. Overall, less than half of the contact residues are conserved between RNase A and angiogenin and, of those that are conserved, some are contained in different structural contexts. For example, one loop in the active site of RNase A contacted by the residues CVG in LRR 15 of PRI is completely absent from angiogenin. The loop domain of RNase A bound by PRI has a completely different structure from that in angiogenin, despite the conservation of two residues. Placental RNase has even fewer contact residues conserved and has other conformational differences compared to RNase A and differs even more extensively from angiogenin, yet HRI binds angiogenin and placental RNase even more tightly than RNase A. These considerations lead Shapiro et al. (1995) to suggest that both angiogenin and placental RNase have contacts with HRI that are not present in RNase A (and by extrapolation, in one another). Consistent with this idea, mutations directly or indirectly affecting the a helix and loop domain binding regions of HRI affect the binding of angiogenin much more than they affect the binding of RNase A. It seems that RIs and PGIPs have evolved a wide specificity enabling them to inhibit most RNases and fungal PGs, respectively. To do so, RIs seem to interact with key conserved features in the active site and with non-conserved features unique to each RNase. This provides a model for how plant LRR proteins may work, not only for PGIPs, but also for disease-resistance proteins,
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since it enables the apparent broad specificity of PGIPs to be accommodated at the molecular level with the narrow specificityof disease-resistance genes. PGs, because of their enzymatic nature, cannot vary the amino acids of their active site without constraint and so may contain features in common. Molecular recognition of these common features needs no more than a single binding domain, which, as seen for PRI, does not require a large number of LRRs. Consistent with this idea, the pear and raspberry PGIPs (see section IVA) show competitive inhibition of PGs, indicating that they are binding to the catalytic site of the PGs. However, recognition of these common features alone may be insufficient to achieve the affinity of binding required to inhibit PGs from different sources effectively. Other unique structural features might require unique binding sites. The extended @ face of the LRR proteins provides an ideal platform upon which these unique binding sites might be arrayed and the flexibility of the LRR backbone may be such that it can accommodate one fixed point of contact and one or more different points of contact elsewhere. Consistent with this idea, apple PGIP (see section IVA) shows mixed inhibition of PGs indicating that it may be binding to the catalytic site and at least one other site and that both are important for inhibition. At the other extreme, interaction between an LRR protein and its ligand may involve features unique to each variant of the ligand. Consistent with this idea, the bean PGIPs (see section IVA) show non-competitive inhibition of PGs, indicating that they are not binding to the catalytic site. The genetic elaboration of the bean PGIPs into a family of at least five and possibly as many as 15 members may reflect the need to generate many different binding specificities recognizing various different features to ensure inhibition of a wide range of PGs. In the case of the resistance genes there is no evidence for inhibition of enzymatic activities, but there is evidence that some avirulence determinants that interact with similar resistance genes, e.g. Avr4 andAvr9 (see section IIC) or avrRpml and avrB, have little in common with one another, so there may be no common features for these LRR proteins to interact with. Thus, the apparent anomaly of massively large LRR resistance proteins for the recognition of very small avirulence ligands might reflect a capacity of the resistance protein to contact many different avirulence ligands. A number of resistance genes have been proposed to have dual specificities,for example the Arabidopsis RPMl gene, which recognizes both the avrRpml and avrB determinants of P. syringae (see section IIID), would support this concept.
IX. CONCLUDING REMARKS Clearly, LRR proteins have a significant role in plant defences. Resistance to a diverse range of pathogens, including nematodes, fungi, bacteria and viruses, involves LRR proteins either as resistance proteins (see sections HA, IIB, IIIA and IIIB) or as proteins required for resistance proteins to function
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(see section IIIC). The limitation of pathogen ingress and aggressiveness may also involve LRR proteins which function as PR proteins (see sections IVA and IVC). However, the role of the LRR domains in most of these proteins remains unclear, largely due to ignorance about the nature of the ligands they bind. The only ligands known with certainty are those for the PGIPs (see section IVA). Some ligands may be inferred for the resistance proteins (see sections IIC and IIID), but the requisite proof is still lacking. Information about the structure of the LRR proteins (see section VIIIA), the nature and structure of their ligands and the nature of the interactions between the two (see section VIIIC) is required to understand how LRR proteins recognize their ligands and, where appropriate, convert recognition into action. As in animals and yeast, plant LRR domains are often linked to various other protein domains in modular fashion. In plants, these modules comprise membrane anchors (see sections IIA, VA and VI) , serinekhreonine kinases (see sections IIB and VB), nucleotide binding sites (see sections IIIA to IIIC), leucine zippers (see sections IIIA and IVC), TolVIL-1R-like cytoplasmic signalling domains (see section IIIB), extensins (see section IVB) and a hydrophilic cytoplasmic domain of unknown function (see section VI) (Fig. 12). These modules are likely to be responsible for the various actions initiated upon ligand recognition. Clearly, information about the nature of these modules, the signals they generate and the proteins they interact with, is also required to understand how LRR proteins convert recognition into action. From the similarities between the tomato Cf proteins (see section IIA) and the wheat AWJL proteins (see section VA), the Cf proteins and the rice Xa-21 proteins (see section IIB), the cytoplasmic LRR resistance proteins (see sections IIIA and IIIB) and the wheat Cre3 protein (see section II), it seems that the cereals probably possess the same kinds of resistance genes as the experimentally more tractable dicots. The knowledge gained from the behaviour of LRR resistance proteins in dicot model systems will therefore be applicable to the economically and sociologically more important cereals. At the present time, a greater diversity of plant LRR proteins is known for tomato compared to any other plant (Fig. 12), with only the receptor protein
Fig. 12. Schematic representation of examples of each kind of plant LRR protein identified to date. The plant species and name of the protein depicted are indicated. Proteins represented by Cf-2 and Cf-9 are discussed in sections IIA and VA; Xa-21 in sections IIB and VB;RPS2 in section IIIA; N in section IIIB; Prf in section IIIC; the PGIPs in section IVA; LRR extensins in section VB; LRP in section IVC; and SF17 in section VI. KINASE, serinelthreonine protein kinase; HD, hydrophobic domain; HYDRO, hydrophilic domain; LZ, leucine zipper; NBS, nucleotide binding site; TOLL, domain with homology to the cytoplasmic domains of the Toll and interleukin-1 receptors.
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Plant proteins with extracytoplasmic LRRs
Plant proteins with cytoplasmic LRRs
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kinases (see section VB) and a homologue of the sunflower SF17 protein (see section VI) unrepresented. This, combined with the diversity of pathogenic interactions characterized for tomato and potentially involving LRR proteins, makes tomato a model system of choice for the characterization of LRR proteins and their roles in plant defences. At least two plant LRR proteins, ERECTA and CLAVATAl (see section VB) are not involved in plant defences, and a number of others may not be (see sections V and VI). Several LRR proteins, such as FIL2 (see section IVA), the AWJL proteins (see section VA), Pexl (see section IVB), PRKl (see section VB) and SF17 (see section VI), seem to be expressed in reproductive tissues. These proteins might have roles in the defence of reproductive tissues from pathogens or roles in reproduction itself. Pollen self-incompatibility and species incompatibility may also be viewed as forms of defence. It would not be surprising if the parallels between resistance and pollen incompatibility extended to the involvement of LRR proteins in both processes. Clearly, much needs to be done yet to understand and appreciate fully the roles LRR proteins have in plant defence.
ACKNOWLEDGEMENTS We wish to thank Rudi Appels, Mark Coleman, Giulia De Lorenzo, Pierre De Wit, Mark Dixon, Robert Fluhr, Evans Lagudah, Elliot Meyerowitz, Anne Powell, Pam Ronald, Guus Simons, Brian Staskawicz, Colwyn Thomas, Keiko Torii, and Pablo Vera for sharing unpublished or prepublication data.
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Fungal Life-styles and Ecosystem Dynamics: Biological Aspects of Plant Pathogens, Plant Endophytes and Saprophytes
R. J. RODRIGUEZ and R. S. REDMAN
National Biological Service, h W Biological Science Center, Seattle, WA 98115, USA
I. Introduction
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11. Plant Pathogens
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111. Plant Endophytes IV. Saprophytes
VI . Life-styles and Ecosystem Dynamics
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VII . Fungal Biology in Agricultural Versus Natural Ecosystems VIII.
The Evolution of Agriculture
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IX. Conclusion ............................................................................ Acknowledgements ................................................................. References ............................................................................
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I. INTRODUCTION The extensive degradation of terrestrial and aquatic habitats over the last few decades has made the need to understand ecosystem dynamics and functions of paramount importance. Comprehension of complex ecosystems and the significance of discrete biological and chemical components of these systems are complicated by limited scientific understanding. Although a significant amount of ecological research has been performed, additional knowledge Advances in Botanical Research Vol. 24 incorporating Advances in Plant Pathology ISBN &12-5924-X
Copyright 0 1997 Academic Press Limited All rights of reproduction in any form reserved
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concerning ecosystem function must be accrued before a comprehensive understanding of such multifaceted systems can be achieved. Ecosystems may be viewed chemically as the flow of carbon and other elements through component systems, biologically as species diversity and interaction, or physically as defined by geography or hydrology. Regardless of how ecosystems are viewed, all terrestrial ecosystems involve complex food webs which begin underground with a large array of microscopic and macroscopic organisms. Fungi play a dominant role in these food webs. Fungi occupy positions near the bottom of food webs and carry out a tremendous diversity of ecosystem functions. Some of these functions include: (1) establishment of soil structure and composition (Metting, 1993); (2) decomposition of organic matter and nutrient cycling (Cromack and Caldwell, 1992); (3) formation of root associations that confer disease, heavy metal, and drought resistances, and extend root systems for greater nutrient foraging capacity (Bradley et al., 1982; Carroll, 1992); (4) causing disease on plants, animals and other fungi (Scott, 1988; Lumsden, 1992; Burdon, 1993); (5) formation of symbiotic associations with plants, green algae and cyanobacteria (Hudson, 1986); (6) providing nutrition for many arthropods, marsupials and small mammals (Maser et al., 1978; Lamont et al., 1985; Rabatin and Stinner, 1985); (7) providing the physical and nutritional components for insect life cycles (Shaw, 1992); and (8) exerting some effect on the structure of plant communities (Allen and Allen, 1992; Dickman, 1992; Burdon, 1993). Unfortunately, the difficulties associated with performing in vivo experiments have limited the understanding of these organisms such that the degree of functional specialization of most fungal species is poorly understood. Fungal life-styles may be categorized as either symbiotic, saprophytic, or a combination of the two (Hudson, 1986). Symbiotic associations can be either parasitic (one organism benefits at the expense of the other), mutualistic (both organisms benefit), or commensalistic (no apparent loss to either organism with or without an apparent gain to one). Saprophytic life-styles are considered more primitive than symbiotic life-styles and refer to fungi that live off dead organic matter (Cooke and Rayner, 1984). Although there are numerous possible permutations to these life-styles (Culver, 1992), the focus of this chapter is the difficulties associated with classifying fungi into discreet life-styles. For example, it is possible that in many or all cases of commensalism, the perceived lack of interorganismal impact reflects an incomplete understanding of the organisms involved. Although there have been biochemical studies on symbiotic associations (Read, 1987), the genetic basis of these life-styles remains enigmatic. Regardless of life-style, fungi employ similar biochemical mechanisms for the acquisition and conversion of nutrients into complex biomolecules which are necessary for vegetative growth, production and dissemination of progeny, organismal competition, and survival during periods of nutrient
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deprivation or environmental inclemency. In general, acquiring carbon and nitrogen based nutrients requires the synthesis and secretion of extracellular enzymes which convert biopolymers to subunit forms compatible with membrane transport and cellular metabolism. Here we focus on various biochemical, genetic, ecological, and evolutionary aspects of fungi that express either symbiotic (plant pathogens and endophytes) or saprophytic life-styles. The difficulties involved in discriminating between these two groups will be addressed, and information from experiments involving plant pathogenic species of Colletotrichurn will be incorporated to support the concepts presented here. Collectively, Cofletotrichum species represent a model system in fungal biology. These species have several attributes which facilitate their use as experimental systems. For example, mycelial cells and conidia are mononucleate haploids which can easily be mutated and cultured on defined media (Chilton and Wheeler, 1948, 1949; Markert, 1952; Tu, 1985; TeBeest et al., 1989). In addition, several species are amenable to genetic segregation analysis, biochemical analysis of extracellular enzymes, and molecular biological manipulations (see Rodriguez and Redman, 1992). Most importantly, Colfetotrichum species collectively express all the fungal life-styles discussed here. We begin with descriptions of plant pathogens, endophytes and saprophytes. These descriptions are followed by a series of discussions concerning the cross-over between life-styles, the influence of these fungi on ecosystem dynamics, differences in fungal biology between agricultural and natural ecosystems, and the potential involvement of fungi in the evolution of agriculture. We conclude with a brief discussion about the need for an appropriate classification system to accommodate fungal life-style capabilities. It is important to point out that this chapter is not intended to serve as a review, but rather to elucidate some deficiencies in our understanding of fungal biology. There are several excellent texts and reviews on plant pathogens, endophytes and saprophytes which are used as references throughout the text. It is also important to acknowledge the myriad of scientists who have invested their careers toward the understanding of fungal biology and ecology, without which, the concepts and questions raised in this chapter would not have been possible.
11. PLANT PATHOGENS An enormous pool of potential pathogens exists in both agricultural and natural ecosystems, and virtually all plant species are susceptible to one or more fungal pathogens. Despite these odds, plant resistance to fungal pathogens is the rule and susceptibility is the exception. Research performed by many laboratories over the past two decades has brought about a significant increase in our understanding of fungal pathology. However, the
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biochemical, genetic and evolutionary aspects of why a pathogen may cause disease in one host and not another are just beginning to be understood. Most of our understanding of fungal plant pathogens comes from studies of their impacts in agricultural ecosystems. There are relatively few studies on the role these fungi play in natural ecosystems (Cook et al., 1989; Jarosz and Burdon, 1991; Burdon, 1993). Fungal pathogens have the potential to impact on the genetic structure of populations of individual plant species, the composition of plant communities and the process of plant succession. These impacts can be imposed by various mechanisms, including modification of plant competitive vigour, fecundity and herbivory, or by selecting for disease resistance (Burdon, 1993). While most studies have focused on the detrimental aspects of plant disease, these fungi may also have several beneficial effects on plant community structure and succession (see section VI). Although plant pathogenic fungi comprise a very large and divergent group of genera and species, there are only a small number of known ultrastructural and biochemical mechanisms involved in the pathogenesis of plants. Plant pathogenic fungi are categorized as either necrotrophic, biotrophic or latent with regard to the mortality, morbidity and timing of the diseases they induce. These fungi can be either obligate or facultative pathogens, and the biochemical basis of disease can involve general or specific virulence mechanisms. General virulence mechanisms include vascular occlusions, a cytotoxic response by the host plant to fungal metabolites, and the production by fungi of plant cell wall degrading enzymes. Specific virulence mechanisms involve low-molecularweight toxins which have high levels of biochemical and host specificity. The ability of fungal pathogens to infect and cause disease in plants has been an area of extensive investigation for more than 100 years. The biochemical and/or genetic bases of pathogenicity have only been partially elucidated in a few host plant-fungal pathogen systems. For example, pathogenicity in Ustilago maydis has been shown to be dependent on the presence of specific mating type genes (Kronstad and Leong, 1989; Schulz et al., 1990). The virulence of Nectria haematococca on pea plants is associated with the presence of a pisatin demethylation gene (Van Etten and Kistler, 1988). The ability of Colletotrichum gloeosporioides to penetrate papaya fruit is dependent on the expression of a gene encoding a cutinase enzyme (Dickman and Patil, 1986; Pascholati et al., 1993). The pathogenicity of fungi and oomycetes in gene-for-gene interactions such as Cladosporium fulvum, Bremia lactucae, Phytophthora megasperma and Magnaporthe griseae is determined by genetic interaction between products of virulence genes in the pathogen and resistance genes in the host (Michelmore et al., 1984; Chumley et al., 1988; Leung et al., 1988; Van den Ackerveken et al., 1992). In Colletotrichum magna (teleomorph Glomerella magna), it appears that a single genetic locus is required to elicit disease (Freeman and Rodriguez, 1993). Although an extensive amount of data has been collected which demonstrate the dependence of pathogenicity on specific genes o r proteins,
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very little information has been obtained to explain the process by which most fungal pathogens cause disease in plants. All fungal plant pathogens have a limited number of compatible hosts which can range from one to dozens of plant species. Determining absolute host ranges of pathogens is not possible because all plants cannot be screened. More importantly, not all physiological states that plants and fungi express can be isolated for screening. Therefore, host ranges have been determined by the ability of a pathogen to induce disease symptoms either in natural or agricultural ecosystems, or during controlled cultivation. As a result, host range often reflects the disease range rather than the range of plant species susceptible to colonization by individual fungi. The genus Coffetotrichumcontains many filamentous fungal species which collectively infect virtually all the major agricultural plant species grown world-wide. Species from this genus vary tremendously in host range, with some causing disease on a single plant species while others cause disease in more than 50 hosts (Farr et al., 1989). In Colfetotrichum interactions, the plant disease response is an indication of fungal virulence and varies from virtually no disease symptoms to large sunken lesions on dead plant tissue covered with conidia. There is also a hypersensitive response in some incompatible interactions resulting in very small lesions with no sporulation. Intermediate disease reactions also occur and can vary from hypersensitivity to necrotic lesions with fungal sporulation. The physical process of pathogenesis has been studied extensively in several species of Coffetotrichum and is similar in many plant pathogens (Mercer et a f . , 1975; O’Connel et a f . , 1985; O’Connel and Bailey, 1986). Infection begins when a spore comes into contact with host tissue and adheres very tightly. The adhesion is sufficient to keep spores from being washed off a compatible substrate. We have found that in at least one species, Cofletotrichum musae (teleomorph Glomerella musae), this process involves an adhesion protein(s) on the fungal cell surface (Sela-Buurlage et af., 1992). After adhering to host tissue, the spore germinates to form a germ tube which, when stimulated to penetrate, differentiates into an infection structure called an “appressorium”. Prior to penetration of the first host cell, the appressorium becomes black as it produces the pigment melanin, which strengthens the appressorial wall. Next, an infection peg is formed which physically penetrates through the plant cell wall. Penetration may involve the assistance of extracellular enzymes which degrade plant cell walls. These enzymes, produced by the fungus, are hypothesized to be involved in the infection process (Karr and Albersheim, 1970; English et al., 1971, 1972; Anderson, 1978). After successful penetration by the fungus into the first plant cell, a compatible or incompatible interaction will ensue. In a compatible interaction, the fungus emerges from the first infected cell without activating the host defence system (Bailey etaf.,1992). After the fungus has begun disseminating through host tissue, the initially infected cells
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disintegrate and release several plant and fungal metabolites which initiate a defence response at that location (Bailey et al., 1992). The defence response leads to the collapse of additional host cells, resulting in necrotic lesions. As the fungus disseminates in the host it leaves behind a path of necrotic tissue, which further activates the defence system, but the growing tip of the fungal mycelium remains spatially and/or temporally ahead of the defence systems. In an incompatible interaction the fungus enters the first cell and activates a host defence response which results in the collapse of the plant cells surrounding the infection site, thereby "walling off" the pathogen (Skip and Deverall, 1972; Erb et al., 1973; Anderson, 1988; Bailey et al., 1992). Presumably, in the initial stages of an incompatible reaction, receptors on the plant cell surface recognize specific pathogen-generated molecules and this causes the activation of cellular transduction pathways leading to the rapid accumulation of specific gene transcripts within the plant (Kamoun et al., 1993; Dixon et al., 1994). Thereafter, the host activates complex biochemical pathways inducing a resistant reaction, usually accompanied by a hypersensitive response (HR) (Klement, 1982; Fernandez and Heath, 1985). For example, the rapid accumulation of phenylalanine ammonia lyase (PAL) and chalcone synthase (CHS) has been correlated with plant resistance in incompatible reactions (Yamada et al., 1989; Jakobek and Lindgren, 1993). In addition, various plant compounds have been identified which are associated with the HR and resistance, such as salicylic acid, jasmonate, phytoalexins, proteases, peroxidases, lignin, callose, and several pathogenesis-related proteins (Bell, 1981; Hammerschmit and Kuc, 1982a; Legrand et al., 1987; Kuc, 1990; Esquerre-Tugaye et al., 1992; Dixon et al., 1994; Kessman et al., 1994). One of the major differences between compatible and incompatible interactions appears to be the timing of defence system activation. In fact, Kuc and Strobe1 (1992) have implied that susceptible cultivars are capable of resisting pathogen attack by altering the timing and magnitude of the defence response. After extensive analyses of fungal pathogenesis, many correlations have been established between host resistance or susceptibility and the expression of specific pathogen and host enzymes or metabolites.
111. PLANT ENDOPHYTES Endophytic fungi exist for at least part of their life cycles within the tissues of a plant host (Clay, 1991). This group of fungi is distinguished from plant pathogens because they do not elicit significant disease symptoms. However, endophytes do maintain the genetic and biochemical mechanisms required for infection and colonization of plant hosts. Host specificity has also been observed in endophytic associations and can range from a few to many species. Because the genetic basis of host specificity is poorly understood and
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so few endophyte-plant systems have been studied in detail, it is premature to assign significance to this aspect of the association. The role of endophytes in ecosystems has been shown to involve: (1) inducing plant protection against pathogens (Carroll, 1992) and insects (Johnson et al., 1985); (2) altering plant community structure by impacting on the herbivory of specific plant species (Lyons et al., 1986); ( 3 ) increasing plant fitness (Hill et al., 1990); and (4) increasing the nutrient acquiring capacity of plant roots (Carroll, 1992). Although the list of endophytic fungi is continually increasing, it is important to distinguish between the different types of fungal life-styles that have been reported as endophytic. We would like to designate four classes of endophytic fungi as defined by their behaviour in plant tissues: (1) fungi that actively grow through host tissues, resulting in extensive colonization (White and Morrow, 1990); (2) fungi that actively grow through host tissues but only colonize a small percentage of host tissues (Suske and Acker, 1987; Cabral et al., 1993; Fisher el al., 1994); (3) fungi that are quickly “walled off” o r inhibited from colonization by plant defence responses or metabolic inhibitors, and remain metabolically quiescent until the host becomes senescent (Prusky and Plumbley, 1992); and (4) fungi that are quickly “walled off’ but remain metabolically active (Meyer, 1974; Anderson, 1992). The work of Cabral ef al. (1993) suggests that within these general classifications there may be several subdivisions of finer resolution. Identification of endophytic fungi routinely involves surface sterilization of host tissue and characterizing the fungi that grow out of those tissues. However, this does not define the extent of tissue colonization or the metabolic activity of the endophyte. Regardless of this, all endophytic fungi must maintain the genetic and biochemical mechanisms necessary for infecting host tissues. The major difference between these four groups of endophytes is that fungi which extensively colonize plant tissues either avoid activation of or are “immune” to the host defence systems. Endophytes that have restricted host colonization patterns may either be activating and are suppressed by host defence systems or are inhibited by preformed, compartmentalized plant metabolites. In essence, endophytes that colonize plant tissues are functionally equivalent to host-compatible fungal plant pathogens but do not elicit disease symptoms. In addition, the third and fourth classes of endophytes, described above, are functionally identical to host-incompatible fungal plant pathogens. The ultrastructural mechanisms involved in the infection process of well-studied endophytic fungi are similar to those of plant pathogens (Scannerini and Bonfante-Fasolo, 1983). Like fungal plant pathogens, fungal endophytes have a limited number of strategies for infecting plant tissues. For example, in the Rhabdocline-Pseudotsuga system, infections occur externally through the formation of appressoria by the fungus (SherwoodPike et al., 1986). In the Acremonium-Festuca (forage grass) system, the plants are susceptible to infection only during the embryonic o r young-
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seedling stages and infection can occur either externally o r by seed transmission (Kearney et al., 1991). However, the mechanism(s) of external infection of Festuca by Acremonium is not known. The interactions between endophytic fungi and plant hosts are dynamic throughout the host life cycle (Philipson, 1991). Timing of infection is critical to the establishment of the fungus within the plant and may affect the extent to which a fungus colonizes host tissues. The interaction between a fungus and host late in the host life cycle has not been studied widely. As plant tissues become senescent, an endophyte may enter into reproductive and/or survival developmental stages, become senescent along with the host, or enter into a saprophytic phase (Philipson, 1991). There are several species of Colletotrichum which are considered “latent pathogens” because they induce lesions on senescing plant tissues. However, the life-styles of these species are not significantly different from class 3 fungal endophytes (defined above). Isolates of Colletotrichum coccodes (Dillard, 1992), C. musae (Muirhead and Deverall, 1981), and Colletotrichum gloeosporioides (Prusky and Plumbley, 1992) infect tomato, banana, and avocado, respectively. All three of these “pathogens” are capable of infecting young plant tissues by the mechanisms described for other Colletotrichum species. Once they have infected plants, the fungi are capable of only limited tissue colonization and then remain quiescent until the plant becomes senescent. There are two proposed mechanisms for the limitation of host colonization. The fungi may induce the plant host defence system resulting in a hypersensitive reaction and the “walling off” of the fungus (Muirhead and Deverall, 1981). Alternatively, the non-senescent plant tissues may contain antifungal compounds that restrict fungal growth (Swinburne, 1978; Prusky and Plumbley, 1992). As the plants become senescent, the antifungal compounds break down and fungi are able to colonize host tissues extensively. It is possible that latent pathogens are actually in evolutionary transitional stages between virulent pathogens and either endophytes or saprophytes. Alternatively, latent pathogens may be sophisticated saprophytes that have evolved a mechanism to avoid soil competition by partially colonizing host tissues prior to host senescence. In fact, restricting host colonization until the host begins to senesce may have little or no impact on plant fitness. This type of “avoidance competition” may afford latent pathogens significant competitive advantages against saprophytic fungi which are unable to colonize non-senescent host tissues.
IV. SAPROPHYTES Fungi that obtain chemical nutrients from dead organic matter are known as saprophytes and are critical to the dynamics and resilience of ecosystems
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(Meyer, 1993). There are two modes of saprophytic growth: one in which biomolecules that are amenable to transport across cell walls and membranes are directly absorbed, and another in which fungi must actively convert complex biopolymers into subunit forms amenable to transportation into cells. Most fungi possess the genetic potential to produce extracellular enzymes capable of converting complex biopolymers into nutritional substrates. However, there is tremendous specificity with regard to the types of chemical structure that individual species are capable of degrading. It is likely that chemical specificity and the extracellular enzymatic capabilities of individual species play a major role in fungal community structure (see section V). Plants are the dominant primary producers in ecosystems, synthesizing nutrients from carbon dioxide via photosynthesis. As a result of seasonal dormancy, senescence, disease, fire and anthropogenic activities, plantgenerated organic matter continually accumulates at the soil surface. In the absence of saprophytic decomposition, many or most of the nutrients assimilated during photosynthesis would not be available for metabolic conversion by other organisms and would result in a decline in ecosystem productivity, soil structure and soil composition. The majority of carbon and nitrogen in plant organic matter is in the form of complex chemical polymers such as cellulose, lignin, proteins and nucleic acids, which require conversion into monomeric subunits before they are available for cell transport and metabolic conversion. Cellulose represents the majority of carbon in plant biomass and is readily converted into monomeric sugar molecules by extracellular cellulase enzymes produced by a variety of fungi (Meyer, 1993). Although lignin polymers are recalcitrant to biodegradation, many fungi produce extracellular ligninase enzymes which convert lignin into its monomeric subunits (Meyer, 1993). Fungi, like many micro-organisms, produce extracellular proteases and nucleases which convert proteins and nucleic acids into monomeric forms conducive to metabolic conversion. In addition to these biopolymers, there are a myriad of complex chemical polymers in plant organic matter. Collectively, fungi produce extracellular enzymes capable of degrading most, if not all, chemicals that occur in plant litter (Cooke and Rayner, 1984). The soil environment is complex, harbouring numerous species of microscopic bacteria, fungi, nematodes and microarthropods, all of which compete for nutrients. Competition can occur by several different mechanisms, including exploitation (Lockwood, 1992) and interference (Wicklow, 1992). As a result, saprophytic fungi have had to evolve mechanisms to compete in soil environments in order to complete their life cycles. Although temporal and spatial complexities make in vivo studies difficult, in uitro studies have suggested several potential strategies for successful competition. These include: (1) accelerated growth rates (see
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Lockwood, 1992); (2) overexpression of extracellular enzymes; (3) colonization of plant material prior to plant senescence; (4) adaptation to extreme environments (Hudson, 1986); (5) synthesis and secretion of chemicals that inhibit other fungi and/or bacteria (Fravel, 1988); (6) biochemical symbioses (Lewis, 1985; Culver, 1992); and (7) mycoparasitism (Lumsden, 1992). There are several potential pitfalls in interpreting data derived from in vitro experiments due to differences in fungal physiology that occur under controlled laboratory conditions versus natural and/or agricultural ecosystems. In particular, high mycelial densities and low carbotdnitrogen ratios may allow for the expression andfor repression of genes rarely induced or repressed in natural ecosystems. In addition, the understanding of these systems has been confused by incomplete experimentation. For example, the hypothesis pertaining to exploitation competition is largely based on biological control experiments involving the suppression of fungal diseases on plants by non-pathogenic fungi (see Lockwood, 1992). However, it is not possible to discriminate between the depletion of resources (exploitation competition) and the induction of plant defence systems (induced resistance) by non-pathogenic fungi unless the studies include plant genetic and/or biochemical analyses. This is best illustrated with experiments involving a non-pathogenic mutant (pathl) of C. magna (Freeman and Rodriguez, 1993). When cucurbit plants are colonized by path1 prior to challenge with pathogenic wild type isolates, disease is suppressed. This could be the result of physical exclusion, antibiosis or exploitation competition. However, a combination of microbiological, biochemical and genetic analyses has demonstrated that disease suppression is based on the interaction between pathl and the host defence system rather than any form of interorganismal competition (R. S . Redman, S. Freeman and R. J. Rodriguez, unpublished). Like most fungi, species of Colletotrichum are capable of growing on senescent plant organic matter and many species will undergo meiosis if they are grown on senescent plant tissue (Vaillancourt and Hanau, 1991; Rodriguez and Owen, 1992). Like all saprophytic fungi, Colletotrichum species produce a variety of extracellular enzymes such as cellulases, proteinases, pectinases and cutinases (Anderson, 1978). Because ColEetotrichum species are plant pathogens, to date most studies have concentrated on the role of these proteins in the disease process. Although cutinase has been shown to be necessary for plant colonization (Dickman and Patil, 1986), there are no studies involving enzyme expression on senescent plant material and the role of these enzymes in saprotrophy. Although the full arsenal of genes responsible for extracellular enzymes is not known for any fungal species, it is likely that the known biochemical diversity of fungi gives only a glimpse of their collective capabilities.
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V. LIFE-STYLE CROSSROADS Regardless of life-style, fungal growth and developmental patterns reflect, at a minimum, a balance between genetic composition, nutrient availability and acquisition, community structure, and environmental conditions. Nutrient availability is based on the type of organic matter present, which in turn controls to some extent the biochemical mechanisms of acquisition that are expressed. For example, if spores of a hypothetical huckleberry symbiont are transported to an environment devoid of huckleberry, the fungus has the option of dying, becoming dormant until huckleberry becomes established in the area, infecting alternate plant species, existing saprophytically, or existing as a microbial symbiont (see below). Regardless of which life-style is expressed, each of these options would require unique genetic and biochemical capabilities. Therefore, the life-style capabilities of any fungus are dependent on the genetic composition and gene expression, which are only partially understood in fungi. The biochemical diversity expressed by individuals in a microbial community has significant impact on life-style expression. For example, in a high density of leaf litter, fungi that secrete cellulases will degrade cellulose into monomeric forms which may then be available for organisms that do not express cellulase enzymes. Alternatively, the non-cellulolytic organisms may express ligninases and provide nutrients to organisms incapable of that biochemical activity. These types of interaction make it difficult to discriminate between true saprophytes and “microbial” symbionts (one organism lives off the extracellular enzymatic capabilities of another), as both would appear to be living off dead organic matter. Environmental factors such as temperature, moisture, pH, conductivity and soil structure also play a major role in biogeochemistry and life-style expression. If temperature is extreme or available moisture is low, pathogens and endophytes may be incapable of infecting plants due to altered host physiology and/or inhibition of fungal developmental cycles. For example, Stermer and Hammerschmitt (1987) reported that when cucumber plants were exposed to a brief heat shock, they became resistant to the cucurbit pathogen Cladosporium cucumberinum. Infection studies with Colletotrichum lindemuthianum on Phaseolus vulgaris demonstrated that the disease process can be abated anytime before or after initial infection by shifting the plants from permissive temperatures (23OC) to non-permissive temperatures (3OOC) (R. J. Rodriguez, unpublished data). The role of fungi in biogeochemistry is based predominantly on the production and activity of extracellular enzymes. Therefore, environmental pH and temperature are very important because all enzymes have pH and temperature optima for activity and can be inactive at conditions above or below those optima. Little is known about the effect of plant dormancy, which is controlled by environmental conditions, on fungal community
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dynamics. Extracellular fungal enzymes can be very stable in the soil environment (Ladd, 1978; Mulvaney and Bremner, 1981). Considering both enzyme stability and environmental fluctuations that occur in microhabitats, ecosystem dynamics may occur at many different temporal levels ranging from minutes to years. Because the completion of life cycles is dependent upon biological and environmental factors, fungi have had to evolve genetic and biochemical mechanisms to accommodate dynamic rather than static ecosystems. Two potential mechanisms that fungi have evolved to deal with this phenomenon are long-term survival or dormancy structures and genetic complexity allowing for life-style conversion. Fungi produce a variety of survival structures, including various spore types and sclerotia, which can allow them to survive environmental extremes for several years (Hudson, 1986). For example, sclerotia formed by C. coccodes on tomato skin were able to survive seasonal freezing and thawing under field conditions for more than 3 years (Dillard, 1992). Although many fungi have the genetic capabilities for life-style conversions, it is unclear how frequently this may occur in natural ecosystems. Characterization of fungi generally reflects the life-style that fungi most frequently express rather than the life-styles they are capable of expressing. It is possible that most or all fungi can express multiple life-styles, but experimental limitations make it very difficult to identify all the life-stylesof any one fungus. It has been suggested that there is no reason, at least theoretically, why fungi cannot express several or all life-styles under different conditions (Cooke and Rayner, 1984). Recent experimental data suggest that some fungi are able to express all the life-styles described here. For example, one wild type isolate of the plant pathogen C. rnagna is pathogenic on a variety of cucurbits, a saprophyte on dead plant matter, and has recently been observed to be a non-pathogenic endophyte in tomato (Freeman and Rodriguez, 1993). However, the type of symbiosis that occurs between C. rnagna and tomato plants has not yet been defined. It is tempting to speculate that, depending on the stage of ecosystem dynamics, and hence community structures, fungi like C. rnagna may commonly traverse the physiological bounds of pathogenesis, endophytism, saprotrophy, and long-term dormancy. If this is common, then the classification of these organisms into restricted life-style categories is unrealistic and reflects a lack of scientific understanding. Fungal life-styles are also dependent on genetic composition and gene expression patterns, both of which may be quite variable. Fungi are able to perpetuate genetic variation by a variety of mechanisms, including limited mutation of DNA sequences (Fincham ef al., 1979), transposon movement (Kinsey, 1993), modification of chromosome number and size (Kistler and Miao, 1992), modification of sequences during meiosis (Selker, 1990), meiotic exchange (Fincham et al., 1979), and mitotic exchange (Fincham et al., 1979). The impact of these genetic modifications on fungi may range from insignificant to lethal events.
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Phenotypic and genotypic variation in fungi has been observed for some time; however, the role of variation and its impact on fungal life-styles are poorly understood. Because the genetic basis of fungal life-styles is not defined, it is not possible to formulate an accurate hypothesis about the degree of genetic variation necessary to impact life-style. Until the genetic basis of symbioses and saprophytism are understood, it will be difficult to address questions concerning the genetic fluidity between life-styles, the impact of life-style change on ecosystem dynamics, or defining the genetic flexibility of different life-styles. Recently, we reported that the mutation of a single genetic locus was sufficient to convert a virulent pathogenic isolate of C. rnagna into a non-pathogenic endophyte of susceptible cucurbits. The mutant (pathl) retained the ability to infect and colonize host tissues, but did not elicit disease symptoms (Freeman and Rodriguez, 1993). In addition, plants that were colonized with pathl were resistant to disease caused by the virulent C. rnagna isolates as well as virulent Fusarium isolates. Preliminary experiments indicate that there is no detectable metabolic cost to plants colonized by pathl, as indicated by growth rates, flowering frequencies, and fruit production (R. S. Redman and R. J. Rodriguez, unpublished data). Life-style conversion of pathogens to saprophytes has been observed in many fungi, but the genetic basis of conversion remains unknown. The C. rnagna system demonstrates that, at least in some fungal systems, life-style conversions can involve fairly simple genetic modifications. The pathogen-to-endophyte conversion in C. rnagna also brings up several interesting and, most likely, contentious issues regarding the evolution of fungi. Did pathogens evolve from saprophytes by gaining function? Are endophytes evolutionarily advanced forms of pathogens? In other words, does the relationship between pathogens, endophytes and saprophytes involve traversing up, down, or across the evolutionary ladder of genetic complexity? It has been suggested that pathogens evolved from saprophytes by overcoming host resistance mechanisms (McNew, 1960) and that biotrophy evolved from necrotrophy by a perpetual decrease in virulence (Lewis, 1973,1974). It has also been suggested that saprotrophy evolved from biotrophy (Raper, 1968; Cooke and Whipps, 1980). Heath (1987) has indicated that there is very little evidence to support any hypothesis regarding the evolutionary development of fungal life-styles and that several scenarios are possible. However, recent studies with a non-pathogenic mutant of C. rnagna indicate that if the modification of a single genetic locus results in the conversion of a virulent pathogen into an endophytic mutualist then, most likely, the reverse may also occur. It is possible that throughout evolutionary time fungi have commonly converted between pathogenic and nonpathogenic endophytic or saprophytic forms to accommodate changes in plant community structure and/or fungal competition.
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VI. LIFE-STYLES AND ECOSYSTEM DYNAMICS Based on our limited understanding of fungal biology, it is conceivable that there are many fungal life-styles expressed in natural ecosystems which do not conform to either strict symbiotic or saprophytic classifications. It would be premature to attempt painting a comprehensive picture of fungal biology and ecology in ecosystems. However, as many researchers have shown, it is possible to begin constructing a temporal and spatial perspective concerning the role of fungi in ecosystem dynamics (see Frankland, 1992). The majority of information on fungal biology in natural ecosystems has been obtained from early to late successional stage, terrestrial ecosystems. Therefore, we shall concentrate our discussion on these types of ecosystem. Fungal activities are often dichotomized as either annual or perennial, based on the expression of specific developmental structures. As more information is obtained, exceptions to such established delineations will inevitably arise. It may be more accurate to describe fungal ecosystem dynamics according to metabolic activities rather than structural expression. For example, most basidiomycetes are considered seasonal because of the timing involved with sporocarp production. However, little is known about the metabolic activity of these fungi when they are not producing sporocarps. Do they become dormant after they fruit or do they exist saprophytically in a mycelial state? Recently, Griffiths et ul. (1990) demonstrated that fungal mycelia in mycorrhizal mats of old-growth Douglas fir forests are metabolically active all year round. An understanding of temporal aspects of metabolic activity will lead to a better understanding of fungal community structure as well as the role of fungi in ecosystem dynamics. While there is much literature describing the roles of endophytes and saprophytes in ecosystem dynamics, the role of plant pathogens in that process has received much less attention. Most discussions concerning plant pathogens in natural ecosystems are presented from the perspective of plant selection during periods of significant disease expression (Alexander, 1992; Dickman, 1992; Burdon, 1993). However, based on the fact that old-growth ecosystems and long-term plant succession are common, the occurrence of severe plant disease outbreaks in these natural ecosystems is probably infrequent. The occurrence of low to moderate levels of plant diseases is common in natural ecosystems, and the impact of plant pathogens on ecosystem dynamics under those conditions is poorly understood (Burdon, 1993). Perhaps the life-style capabilities of C. mugnu are common among fungal pathogens such that pathogens spend a significant amount of time in non-pathogenic, endophytic associations in natural ecosystems. If these associations are mutualistic, then pathogens may play a significant role in protecting plants against disease. In addition, pathogens may be protecting plants against serious disease problems in natural environments by eliciting low levels of necrosis, which have been shown to induce systemic
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resistance against pathogens in many plants (Kuc, 1990; Kuc and Strobel, 1992). Ecosystem dynamics, with regard to fungi, may be viewed in part as the flow of carbon assimilated during photosynthesis in and out of the fungal community (Wainwright, 1992). The conventional view is that carbon flows from plants to fungi and other elements such as phosphorus flow from fungi into plants. Symbiotic and saprophytic fungi obtain carbon from living and dead plants, respectively. While carbon flow to both occurs simultaneously, the amount of carbon going to symbionts versus saprophytes can vary significantly depending on environmental conditions and the metabolic activity of the respective life-style groups. In addition, there are reports which indicate that carbon flow also occurs from fungi to plants (see Meyer, 1974), but it is unclear how significant this phenomenon is to plant health and ecosystem dynamics.
VII. FUNGAL BIOLOGY IN AGRICULTURAL VERSUS NATURAL ECOSYSTEMS Much of the current understanding in fungal biology is based predominantly on observations from either agricultural ecosystems or laboratory, greenhouse or growth-chamber experiments. While the data collected thus far are extremely valuable for understanding fungal biology, it is important to point out some of the problems associated with extending the present body of knowledge to understanding the biology of fungi in natural ecosystems. We will discuss three aspects: (1) biochemical expression, (2) community structure and (3) genetic adaptation and evolution. Western agricultural practices result in homogeneity with regard to soil composition and plant genetics, which are rare phenomena in natural ecosystems. When a pathogen significantly decreases product quality and/or yield, single resistance genes from resistant plants are usually bred into desired varieties. Although this abates the disease problem for some period of time, eventually pathogens will overcome the single-gene resistance. The length of time for which single-gene resistance protects plants varies from one or two seasons to decades, depending on the basis of resistance and the pathogens involved. Given the present understanding of microbial evolution, it seems likely that this practice has created a biased form of genetic selection. In fact, it is possible that many pathogens have become hypervariable in virulence genes in order to overcome plant resistance genes with which they are confronted. Another major difference between agricultural and natural ecosystems lies below ground. Commonly, agricultural fields are chemically treated to eliminate pathogens and weeds, and the soils are extensively tilled. Unfortunately, many or all of the organisms which are beneficial to plants may
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also be eliminated. As a result, the community structure and biochemical activity in agricultural soils may be fairly simple and homogenous. It is not possible to determine the full impact of this on fungal adaptation and evolution, but the competitive dynamics of agricultural soils are very different from those of natural soils. Studies have indicated that natural soils appear to suppress the ability of pathogens to cause disease (Alabouvette et al., 1993; Larkin et af., 1993; Gilbert et af., 1994). These “suppressive soils” are based on specific biological and chemical interactions. The ability of suppressive soils to abate fungal infections has been attributed to antibiosis, organismal competition and chemical inhibition of fungal metabolism (Alabouvette et al., 1986; Young et al., 1991; Harrison et al., 1993). The suppressive aspects of soils can be eliminated by a variety of chemical and physical modifications, some of which are common agricultural practice (Amir and Alabouvette, 1993; Larkin et af., 1993). Once suppressive soils are converted to nonsuppressive soils, plant pathogens are able to infect susceptible hosts, and elicit disease symptoms. One very interesting aspect of fungal biology with regard to agricultural and natural ecosystems concerns the evolution of host range. Fundamentally, there are significant differences between these ecosystems with respect to plant and fungal diversity and environmental conditions. Natural ecosystems have very high levels of plant species and pathogen diversity compared with agricultural ecosystems. It is possible that a greater diversity of indigenous plant species gives pathogens more opportunity to expand their host range. As a result, natural ecosystems may act as repositories of plant pathogens which, by virtue of causing low levels of disease or having endophytic capabilities, may have greater potential for expanding host ranges. As natural areas around the world are becoming biological islands surrounded by either agriculture and/or development, it is important to address questions concerning the interface between agricultural and natural ecosystems. What are the dynamics of pathogen evolution and dispersal in these ecosystems, and what will the impact be on plants in both ecosystems? How much agrichemical contamination is occurring in natural ecosystems, and what are the resulting biological impacts? The long-term health of natural ecosystems and sustainable agriculture may depend on understanding the sensitive interfaces between these ecosystems.
VIII. THE EVOLUTION OF AGRICULTURE Agriculture in the western world has become sophisticated, and product distribution is global. In the USA, the last 100 years has brought about a change from low-efficiency, labour-intensive agriculture to a highly efficient and energy-intensive agriculture. Much of this conversion has been due to the production and application of energy-intensive agrichemicals for crop
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fertilization, weed control, manipulation of fruit development for mechanical harvest and shipping, post-harvest treatments for long-term storage, and combating insect, bacterial and fungal pests. Although there have been several problems associated with pesticide use, such as groundwater contamination, the generation of chemically resistant pathogens, and the elimination of beneficial microorganisms, the efficiency of these chemicals and resulting crop yields have maintained the appeal of agrichemicals. Our dependence on agrichemicals brings to light some very interesting questions concerning agriculture and ancient civilizations. Some ancient civilizations, such as the Aztecs, Mayans, Incas and Egyptians, had communities that ranged from 5000 to 200 000 residents (Butzer, 1976; Moriarty et al., 1983; Hodge, 1984; D’Altroy, 1992). How did those civilizations produce enough food and keep the agricultural lands productive without the aid of any known agrichemicals? Although there are insufficient data to formulate a tenable hypothesis, it is possible to speculate about some of the biological phenomena that may have contributed to the successful agricultural practices of ancient civilizations. It is important to keep a temporal, spatial, and functional perspective when speculating about ancient agriculture. The cultivation of plants for human consumption allegedly began more than 10 000 years ago in the Jordan river valley (Dimbleby, 1967). Although it is unclear which plants constituted the first agricultural crops, no major crop has been brought into cultivation from wild plants for the last 5000 years (Dimbleby, 1967). Archaeological excavations have indicated that the diet of the Aztecs and Mayans included corn, squash, beans and root crops (Moriarty et al., 1983; Hodge, 1984). It is conceivable that practices such as land terracing, crop rotation, mixed crop planting and tillage of the soil at the end of the growing season may have kept the land productive in the absence of chemical fertilizers. It is important to remember that the crop plants of those cultures were derived from indigenous species which had evolved for centuries with indigenous fungal pathogens, endophytes and saprophytes. Therefore, it is possible that natural selection resulted in plants which were resistant to fungal disease. Several mechanisms may have contributed to this phenomenon, including: (1) the evolution of resistance genes in the plants which would allow for resistance to specific fungal pathogens; (2) the evolution of symbiotic associations between endophytic fungi and plants may have conferred resistance to different fungal pathogens similar to that observed with C. mugna (Freeman and Rodriguez, 1993) or mycorrhiza (Carroll, 1992); (3) the occurrence of low levels of fungal disease throughout growing seasons may have induced systemic plant resistance; and (4) soils may have naturally evolved to be suppressive to indigenous virulent pathogens. It is also possible that planting crops involved seed derived from several plant varieties or from the most productive plants. Multiple varieties may have afforded a mixture of resistant and susceptible plants, so potentially only a portion of each crop would be
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lost in a single season. Practices such as selecting seed from the most productive plants may have led to the selection of varieties resistant to the fungal pathogens present at that time as well as increasing crop yields. Considering the estimated field sizes of Mayan farms ( 1 4 acres), it is difficult to understand how pathogen-induced crop losses could have been tolerated without frequent famine (Cook, 1919). Realistically, agricultural productivity of ancient civilizations probably involved several or all of these factors. It is possible that indigenous fungi were largely responsible for plant health in many ancient agricultural situations. Present-day understanding of fungal biology in natural ecosystems is predominantly descriptive. Although the importance of different fungal life-styles in ecosystem health is recognized, the physiological and environmental conditions conducive to life-style expression are poorly defined. More importantly, little is known about physiological or environmental sensitivities of symbiotic and saprophytic associations and how much conditions can change before those associations degenerate. Therefore, when plants began to be transported around the world by the historically venerated explorers, it is possible that plant transportation resulted in the disruption of existing co-evolved plant-fungus associations. The end result would have been the cultivation of plants in foreign habitats which had decreased protective capabilities and a concomitant increase in plant disease. The severity of this problem may have varied depending on the distance of transport and the extent of habitat change.
IX.
CONCLUSION
Fungal taxonomy has historically been based on the morphological analysis of developmental structures and cultural characteristics. Recently, DNA sequence analysis has been applied to fungi and has improved the taxonomic resolution of these organisms. However, taxonomic descriptions do not address either fungal life-style expression or life-style capabilities. In bacteriology, a life-style classification system has been established such that if a bacterium is designated a photoheterotroph it is defined as using light as an energy source and an organic compound as a carbon source. In fungi, there are four primary life-styles to consider for a classification system: (1) pathogenic, (2) mutualistic, (3) commensalistic, and (4) saprophytic. Each of these life-styles can be described in greater detail, for example: pathogens may be described as either necrotrophic, biotrophic, hemibiotrophic or latent; mutualists as class 1-4 endophytes; and saprophytes can be categorized by chemical specificity. In addition, the expression of each life-style would have to be characterized as facultative or obligate. We recognize that researchers will have different preferences with regard to life-style definitions and that there are many more permutations to life-style expression than
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described in this chapter. However, this does not diminish the need to enhance current taxonomic descriptions to incorporate life-style capabilities. The establishment of a life-style classification system would greatly increase our ability to design studies in order to understand better fungal community structure and the roles of fungi in ecosystem dynamics.
ACKNOWLEDGEMENTS We would like to thank Dr Tom Wolpert, Dr Jennifer Lorang, Dr Gael Kurath and Ms April Mills for critical reviews of this manuscript.
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Cellular Interactions Between Plants and Biotrophic Fungal Parasites
M . C . HEATH and D . SKALAMERA
Department of Botany. University of Toronto. Toronto. Ontario M5S I A l . Canada
I . Introduction
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I1. Why do Fungal and Oomycetous Parasites form Intracellular Structures? ............................................................................... A . General Characteristics of Plant-Haustorium Interfaces ............. B . Maintenance of a Differentiated Extrahaustorial Membrane ....... C . Solute Transport Across the Plant-Parasite Interface ................ D . Other Roles of the Haustorium ............................................ E . Concluding Remarks ...........................................................
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I11. Of what Significance are the Plant Cellular Rearrangements that 206 Accompany Parasite Invasion? ..................................................... A . Defensive Responses to Parasite Invasion ............................... 206 B. Parasite-Induced Changes in the Plant’s Endomembrane System . 207 C . Associations of Intracellular Fungal Structures with the Plant Nucleus ............................................................................. 209 D . Changes in the Plant Cytoskeleton ........................................ 210 E . Concluding Remarks ........................................................... 210 IV . Why do Biotroph-Invaded Cells Die in Resistant Plants? ................. A . Is Cell Death the “Default State” Following Cell Penetration? . . B . Do Invaded Cells Die in Host and Non-host Plants for the Same Reason? ............................................................................ C . Arguments for Cell Death in Resistant Plants being a Form of Programmed Cell Death ...................................................... D . Arguments Against Cell Death in Resistant Plants Being a Form of Programmed Cell Death .................................................. E . Cellular Mechanisms of Cell Death ....................................... F. Concluding Remarks ........................................................... Advances in Botanical Research Vol . 24 incorporating Advances in Plant Pathology ISBN C12-005924-X
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V. Conclusions
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References ...............................................................................
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I. INTRODUCTION Biotrophic fungi are defined as deriving their energy from living cells of other organisms (Lewis, 1973; Luttrell, 1974) and, most commonly, their hosts are vascular plants. The resulting plant-fungal associations range from being clearly mutualistic (e.g. mycorrhizal roots) to clearly parasitic. This chapter is concerned with those biotrophic fungi (and, for historical reasons, biotrophic oomycetes) that traditionally have been considered as parasites because they feed on the plant without providing any demonstrated benefit in return (Lewis, 1973). It should be mentioned, however, that although many of these biotrophs cause measurable yield losses in crop plants, they may appear to have little effect on host fitness in natural ecosystems. This has led some researchers to consider the plant-fungus interaction as “nearly commensalistic” (Jarosz and Davelos, 1995). However, such terminology seems to us to be misleading, not only because the common definition of parasitism (Morris, 1992) does not require that parasites demonstrably harm their hosts, but also because, at the cellular level, there is no doubt that these fungi have a significant, non-beneficial, effect on the plant. Organisms that traditionally have been considered as biotrophic fungal parasites include those that cause plant diseases such as leaf curl, rust, smut and powdery mildew; they also include oomycetes that cause downy mildew and white rust diseases (Lewis, 1973), which will be included in this chapter although the oomycetes are no longer considered to be closely related to the true fungi. Even within the true fungi, there is considerable taxonomic diversity among biotrophs and, not unexpectedly, enormous variation in the morphological features of their associations with their hosts and in the duration of the biotrophic relationship between individual fungal structures and host cells. This relationship is usually physically intimate, as most parasitic biotrophs form indeterminate hyphae o r determinate haustoria within host cells; only a few biotrophic fungal parasites, such as the tomato pathogen, Cladosporium fulvum, grow entirely intercellularly. At one end of the spectrum of those that form intracellular structures are rust and powdery mildew fungi with haustoria that may co-exist with living host cells for weeks and cause little macroscopic tissue damage. At the other end of the spectrum are haustoria of some oomycetes that cause the death of invaded susceptible cells within a few hours (Coffey and Wilson, 1983). These short-term, biotrophic relationships are also found among fungi that form intracellular hyphae, rather than haustoria, such as the rice blast fungus, Magnuporthe griseu (Heath et aE., 1990). They are also found in the so-called “hemibiotrophs” (Luttrell, 1974), such as Col-
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letotrichum lindemuthianum, which sequentially show distinctive biotrophic and necrotrophic or saprotrophic growth in their hosts (O’Connell et al., 1985). Examples of similar short-term biotrophic growth may be much more common among pathogens than is generally appreciated, since the existence and duration of biotrophy within individual cells cannot be detected without careful cytological studies of the early stages of infection. Many fungi and oomycetes that have long-term biotrophic associations with their hosts do not readily grow in culture, which has led to the commonly held view that they require some factor for growth that is only available in living cells. However, recent evidence for rust fungi suggests that the obligacy of parasitism in this group is more probably due to the tight linkage of fungal development to the requirement of signals from the plant than to any requirement for special nutrients (Heath, 1995). This conclusion, and observations that biotrophy is associated with a more restricted host range in fungal genera with hemibiotrophic and nonbiotrophic members (Pring et al., 1995), suggest that biotrophy requires a higher degree of specialization than other forms of parasitism. This raises the question of what advantage is there to the parasite to grow biotrophically? For short-term biotrophy, delaying plant cell death may sufficiently delay death-induced defence responses (Mansfield, 1984) to tip the plant-fungus interaction in favour of the parasite. For long-term biotrophy, the benefits may be even greater if the fungus can alter its host’s translocation patterns to provide access to the plant’s nutritional resources beyond the invaded tissue (Lewis, 1973). In either case, the mode of nutrition may not be the most critical factor that defines the plant-parasite interaction. From the fungus’ point of view, the most important fungal attribute related to biotrophy may be to prevent host cell death; as a result, the fungus may have no choice but to evolve ways to obtain its nutrients from living tissue (Heath, 1987). There is a wealth of literature to show that susceptible, living plant cells invaded by fungal and oomycetous biotrophic parasites respond with changes in gene expression, metabolism, and cellular organization that are ultimately reflected in changes in whole plant physiology. It has long been recognized, however, that knowledge of the cellular events that accompany intracellular parasite growth is essential for complete understanding of the plant-parasite interaction. The recent application of video-enhanced differential interference contrast light microscopy, confocal laser scanning microscopy, high-pressure freezing techniques for electron microscopy, and a variety of cytochemical techniques and probes has built upon earlier information from conventional light and electron microscopy to give us a fascinating glimpse of the complexities of these interactions at the cellular level. This chapter tries to provide an overview of this complexity and an appreciation of how much is still left to discover. Specifically, this chapter addresses three questions which we consider pivotal in understanding biotrophic, fungal (and oomycetous) parasitism.
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11. WHY DO FUNGAL AND OOMYCETOUS PARASITES FORM INTRACELLULAR STRUCTURES? Most biotrophic parasites grow primarily intracellularly (e.g. Heath and Bonde, 1983, 1988) or form a primarily extracellular mycelium from which form intracellular haustoria. None of these “intracellular” structures, however, is truly intracellular, as the only plant structure that they breach is the cell wall and they are separated from the plant cytoplasm by an extension of the plant plasma membrane. The obvious question, therefore, is what advantage is there to the fungus to being on one side of the plant cell wall rather than on the other? There are two possible answers: (1) the cell wall is a barrier to molecular exchange, indicating that the association requires the exchange of fairly large molecules that cannot cross a normal plant cell wall; or (2) there is an advantage to being in contact with the plant plasma membrane, The often striking modifications that have been documented by cytochemical techniques at all plant-haustorium interfaces that have been examined suggest the second possibility is most likely.
A. GENERAL CHARACTERISTICS OF PLANT-HAUSTORIUM INTERFACES
The majority of cytological and cytochemical studies of biotroph-plant interfaces have concentrated on haustoria. Currently available data for interfacial modifications associated with haustoria of a variety of fungal and oomycete groups are summarized in Fig. 1. Not all types of interface have been examined in the same detail or with the same techniques, but all show some differentiation of one or more of the haustorial wall, haustorial plasma membrane, or the invaginated portion of the plant plasma membrane known as the extrahaustorial membrane (EHM). Nevertheless, there are significant differences between different haustoria in the nature of this differentiation, as well as differences in the fluidity, staining characteristics, and composition of the extrahaustorial matrix which lies between the fungal wall and the EHM. As a striking example, the EHM surrounding powdery mildew haustoria generally appears unusually thick, has a high tensile strength and is unusually rich in carbohydrate (Aist and Bushnell, 1991) compared to other types of EHMs. Even this apparent cytological uniformity among powdery mildew EHMs may be accompanied by underlying diffxences in properties, since the EHM surrounding different Erysiphe species eems to have different permeabilities to water (Aist and Bushnell, 1991). Var,ation among rust fungi haustoria is also common. Dikaryotic (D-) haustoria produced by the rust fungus, Cronartium quercuum f. sp. fusiforme are associated with unique outgrowths of the EHM that sheath the haustorium neck (Gray et al., 1982) and which are not induced by other rust fungal species. Some, but not all, rust fungus-host cell interfaces are characterized by connections of the EHM
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to tubules in the cytoplasm which do not resemble endoplasmic reticulum (ER) (Littlefield and Heath, 1979; Mendgen et al., 1991). These, and other, observations suggest that many features of the interface between plant and parasite vary not only with the major taxonomic group to which the latter belongs, but also with the specific plant-parasite combination at the species level. One of the more consistently reported modifications of the EHM surrounding fungal and oomycetous haustoria is the lack of cytologically detectable ATPase activity (Fig. 1). For haustoria of the taxonomically distinct organisms that cause white rust (oomycetes) and powdery mildew (true fungi ascomycetes) diseases, and the dikaryotic stage of rust fungi (true fungi basidiomycetes), there also is a remarkable evolutionary convergence in the presence of one o r more neckbands along the haustorium which mark the junction between the normal plant plasma membrane and the EHM. This structure differs between groups in appearance and chemical composition (Woods and Gay, 1983; Heath and Allen, 1985; Stumpf and Gay, 1990). However, for rust fungi (Heath, 1976) and powdery mildew fungi (see references in Stumpf and Gay, 1990), these neckbands have been shown to provide a seal between the plant and fungal membranes, preventing apoplastic solute flow along the haustorial neck. Thus, in these systems, the haustorial body is surrounded by a sealed membrane compartment formed by the EHM and the fungal plasma membrane.
B. MAINTENANCE OF A DIFFERENTIATED EXTRAHAUSTORIAL MEMBRANE
The functional similarity of haustorial neckbands to tight junctions in animal cells suggests that they may be responsible for maintaining the EHM as a membrane domain separate from the rest of the plant plasma membrane (Spencer-Phillips and Gay, 1981). However, for haustoria without detectable neckbands, one might expect that lateral diffusion of membrane components would eventually eliminate any unique features of the EHM. Possibly, the EHM surrounding such haustoria is less fluid than normal plasma membrane (Woods et al., 1988), but it also may be significant that the neckband-lacking haustoria of some oomycetes and smut fungi (see references in Bauer ef al., 1995) appear, after conventional fixation for electron microscopy, to be surrounded by a particularly obvious and solid matrix between the EHM and the fungal wall. Conceivably, this matrix may interact with, and anchor, EHM components. In support of such a suggestion, the plant membrane that surrounds invading hyphae (not haustoria) of directly penetrating biotrophic parasites such as Phytophfhoru infesfuns (Nozue et al., 1979) and the monokaryotic stage of the rust fungus, Uromyces vignae (M. C. Heath, unpublished), adheres strongly to the fungus when the host cell is plasmolysed. Also of significance is the fact that the presence of haustoria of the
200 M. C. HEATH and D. SKALAMERA Fig. 1. Diagrammatic representation of the interfaces between susceptible plant cells and haustoria formed by fungi (c, e, f) or oomycetes (a, b, d). Long-term biotrophic relationships are formed in (c)-(€). Illustrated are the fungal and plant plasma membranes (dotted and/or dashed lines), the haustorial wall between them, and the penetration region through the plant cell wall (W). Any difference in shading or line characteristics represents a documented difference in appearance or some cytochemical feature.
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Fig. 1. (coat) C, Callose collar; PACP, electron microscope technique that specifically stains normal plasma membranes; *, data from U. appendiculutus, M, mononucleate; D, dinucleate. Information taken from Coffey and Wilson (1983) for (a), Hickey and Coffey (1977, 1978) for (b), Woods and Gay (1987) for (c), Coffey (1983) and Woods and Gay (1983) for (d), McKeen and Rimmer (1973) and Spencer-Phillips and Gay (1981) for (e), and Spencer-Phillips and Gay (1981) and Harder and Chong (1991) for (f).
h)
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powdery mildew fungus, Erysiphe graminis, in barley epidermal cells increases the adherence of the plasma membrane to the plant cell wall (Lee-Stadelmann et af., 1984). It is possible, therefore, that even in the absence of a detectable neckband there may be interactions between the fungal wall and/or the adjacent matrix and the plant membrane that limit lateral diffusion of membrane components.
C . SOLUTE TRANSPORT ACROSS THE PLANT-PARASITE INTERFACE
Because the thallus of powdery mildew fungi normally develops on the leaf surface, and haustoria are the only fungal structures within the tissue, it is reasonable to assume that haustoria of these fungi are intimately involved in nutrient uptake from the invaded plant cell. Gay and colleagues have argued that the sealed membrane compartment surrounding the haustorium, and the lack of ATPase activity on the EHM, tightly couples the transport systems of host and parasite (Spencer-Phillips and Gay, 1981). To support this hypothesis they have provided evidence for ionic coupling of the cytoplasm of the haustorium with that of the invaded cell (Gay et af., 1987). A steep concentration gradient for glucose and fructose which possibly drives the facilitated diffusion of sugars into the haustorium seems to be maintained by the rapid utilization of these sugars by the fungus (Aked and Hall, 1993a). In addition, solute efflux from the plant appears to be greatly stimulated at the plant-parasite interface, which Patrick (1989) has theorized could be the result of an increase in plant cytoplasmic solute concentration and/or changes in EHM properties. The non-photosynthetic epidermal cells, in which haustoria of powdery mildew fungi form, normally show a net influx of solutes controlled by proton symports. Thus, the absence of proton ATPase activity on the EHM could abolish the plant’s capacity to retrieve nutrients from the extrahaustorial compartment (Patrick, 1989) and favour uptake by the haustorium. Such a model is supported by the observation that, in uninfected soybean leaf discs, sucrose efflux is promoted by inhibition of plasma membrane ATPase activity and by an elevation in extracellular p H (Secor, 1987). Plant sugars lost to the haustorium are most likely replenished by increased influx through the plasma membrane adjacent to the epidermal cell wall caused by the demonstrated increase in apoplastic sugar concentration in the leaf (Aked and Hall, 1993b). Even in the powdery mildew system, however, there are many unanswered questions. For example, ATPase activity is cytochemically demonstrable on the haustorial plasma membrane (Spencer-Phillips and Gay, 1981), raising the possibility that the fungus may be generating elevated proton levels in the extrahaustorial compartment despite the lack of proton pumping by the EHM. Thus, plant proton symports involved in solute retrieval from the apoplast could still be active, if they are present. Moreover, even in the
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absence of ATPase activity, proton extrusion by the EHM could be effected by a process that is coupled to a transmembrane redox system and does not require the action of ATPase (Neufeld and Bown, 1987). Since this system was reported for mesophyll cells, it may be of particular relevance to biotrophs, such as rust fungi, that characteristically form haustoria in mesophyll, rather than epidermal, tissue. Redox activity surrounding developing D-haustoria of the rust fungus, U. vignae, has been demonstrated cytologically using nitroblue tetrazolium (M. C. Heath, unpublished); this activity is only partly related to the production of superoxide radicals (since much of it is not inhibited by superoxide dismutase) and could be involved in driving proton, or other ion, transport across the EHM. As photosynthetic leaf mesophyll cells might be expected to be net exporters, not importers, of solutes, inhibition of solute retrieval systems from the EHM in these cells may not be as important as it is in the epidermis. Since so much importance has been placed on the apparent absence of ATPase activity on the EHM in relation to nutrient uptake by the haustorium, it is worth considering the accuracy of the cytochemical data. Cytochemical studies of ATPase activity for biotrophs other than powdery mildew fungi often have not demonstrated any staining of fungal plasma membranes, even in extracellular structures (Spencer-Phillips and Gay, 1981; Woods and Gay, 1983, 1987). The complete absence of ATPase activity on these membranes seems highly unlikely, given the general importance of plasma membrane ATPase activity in membrane transport processes in fungi (Garrill, 1995). The reliability of the cytochemical ATPase assay has also been questioned for uninfected plant cells (Katz et al., 1988), and immunocytochemical studies with ATPase antibodies are urgently needed to demonstrate whether or not these proteins really are absent from the EHM and the fungal plasma membrane. Another factor to be considered in sugar transport into haustoria is the increased invertase activity commonly found in leaves infected with biotrophic parasites. This activity also may promote sucrose effluxes from plant cells if the enzyme is in the apoplast (Patrick, 1989). Apoplastic plant invertases tend to have a low optimal p H and might be expected to have low activity in the extrahaustorial compartment if proton pumps on plant and fungal membranes are inactive. However, fungal invertases tend to have a higher pH optimum and could play a role in sugar transport to the fungus if they were secreted by the haustorium (Tetlow and Farrar, 1993). Currently, however, there is no evidence that the elevated invertase activity resides in the extrahaustorial compartment. It is possible, therefore, that the increase in activity in biotroph-infected tissue is involved in phloem unloading and affects sugar translocation at the tissue level by converting infected tissue from a sugar source to a sugar sink (Roitsch et al., 1995). Biotrophic parasites must require substances other than sugars from their hosts. For example, the few rust fungi that will grow in culture generally
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require a number of specific amino acids (Fasters et al., 1993), and it seems likely that similar amino acids are required when the fungus grows parasitically. Indeed, there is some evidence that sugar uptake is not the main role of haustoria of biotrophic parasites that form a mycelium within the plant. For example, the intercellular mycelium of the oomycete, Peronospora viciae, seems more important than haustoria for sucrose uptake (Clark and Spencer-Phillips, 1993). Similarly, two rust fungi and one downy mildew fungus readily incorporated radioactive label from tritiated glucose prior to haustorium formation (Onoe et al., 1973; Andrews, 1975; Rey and Garnett, 1985). However, haustorium formation was required for the uptake of exogenous nucleosides by Puccinia coronata (Onoe et al., 1973) and leucine by Bremia lactucae (Andrews, 1975). This latter example is interesting since the amino acid apparently was not taken up by invading hyphae of the oomycete, even though both haustoria and invading hyphae are intracellular (Andrews, 1975); thus, the type of nutrients passed to the biotroph depends on the type of structure within the plant cell. Overall, the data suggest that for biotrophic parasites which form a mycelium within the plant, haustoria may only be involved in the uptake of specific nutrients. Simple sugars may be absorbed through intercellular or intracellular hyphae, aided perhaps by the increased permeability of infected and uninfected cells in infected tissue (discussed in Littlefield and Heath, 1979).
D. OTHER ROLES OF THE HAUSTORIUM
The possibility that haustoria may have functions in addition to nutrient acquisition is raised by the common observation that cells containing haustoria of rust or powdery mildew fungi are rendered “accessible” to fungi to which the plant is normally resistant (see references in Fernandez and Heath, 1991; Yamaoka et al., 1994). For powdery mildew fungi, this phenomenon has been suggested to be the result of sugar depletion in the epidermal cell (Yamaoka et al., 1994). However, for the rust fungus, Uromyces appendiculatus, induced accessibility occurs very soon after haustorium formation in photosynthetic mesophyll cells (Heath, 1983) and continues even while the chloroplasts are accumulating starch (M. C. Heath, unpublished). Therefore, it is difficult to believe that the cell has depleted sugar reserves. Nutrient depletion also seems unlikely to explain the unusual effects of haustoria of the rust fungus, Physopella zeae, which forms both intracellular hyphae and haustoria in the same cell. Haustoria seem to be necessary for intracellular hyphae to enter the cell (Heath and Bonde, 1988) and only haustoria reduce the incidence of the plant’s defensive responses (Heath and Bonde, 1983). The larger bulk of the intracellular hyphae in relation to that of the haustoria should make the former, not the latter, effective in influencing the cell if the cell’s responses were controlled purely
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by nutrient depletion. Moreover, the types of cell responses prevented by haustoria of this fungus, and by haustoria of U . appendiculatus, suggest that they specifically prevent certain defensive secretory activities of the cell (Heath and Bonde, 1983; Fernandez and Heath, 1991).
E. CONCLUDING REMARKS
Overall, the data suggest that the value to the parasite of being inside the plant cell wall is the potential to manipulate the plant-parasite interface in order to control nutrient efflux and/or other cellular activities of the plant. While there is some commonality between different plant-parasite systems, there also are striking differences, suggesting that the role of the haustorium may not be identical in every case. This is perhaps not surprising given the millions of years of restricted gene flow between, and independent evolution of, each major group of biotrophs. Although not discussed in detail here, the differing roles of some haustoria may also explain differences in their cytoplasmic features; some types of haustoria lack nuclei (Fig. l ) , those of the oomycete, Albugo, are packed with mitochondria (Coffey, 1983), and those of the rust fungus, U. viciae fabae, have wide, tubular, E R rich in proteins bearing the HDEL ER-retention signal (Bachem and Mendgen, 1995). For haustoria other than those of powdery mildew fungi, we still know relatively little about their roles. Why, for example, is one of the most complex forms of differentiation at the haustorium-plant interface exhibited by dikaryotic (D-) haustoria of rust fungi when monokaryotic (M-) haustoria have one of the simplest (Fig. l)? For autoecious species such as U. vignae, the different mycelial stages that produce these two types of haustoria develop equally well in mesophyll leaf tissue of the same host plant. Why, then, has such a complex interface evolved in one stage of the fungus, when it apparently is not needed in the other? In understanding the role of the haustorium it is significant that the types of cytochemical studies that have been performed on haustoria did not reveal any differentiation of the interface between the plant cell and primary hyphae of the non-haustorium-forming hemibiotroph, C . Zindemuthianum (O’Connell, 1987). Thus, it seems that the modifications currently detected at interfaces with haustoria are not mandatory responses of the plant cell to parasite invasion, but are haustorium induced and haustorium controlled. An important point to remember, however, is that the identification of interfacial modifications depends heavily on the types of cytochemical technique available. Recent cytochemical localization of monoclonal antibodies prepared against powdery mildew haustorial complexes have illustrated the potential of this technique for identifying proteins and glycoproteins specific for fungal or plant components of the interface and for
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investigating temporal changes during haustorium development (Roberts et al., 1993). Similar studies with C . lindemuthianum have revealed an epitope in the invading hyphal wall and surrounding matrix which is not present in extracellular parts of the mycelium (Pain et al., 1994), even though other cytochemical tests revealed no differentiation of this interface. Even in biotrophic systems that have not been studied cytochemically, it seems universal that intracellular hyphae in living cells are conspicuously wider than those growing non-biotrophically in the same plant. This change in morphology seems to be a mandatory part of biotrophy for M. grisea (Heath et al., 1990) and is even observed when the non-biotrophic maize pathogen, Cochliobolus heterostrophus, grows in living epidermal cells of detached cowpea leaves (M. C. Heath, unpublished). This “biotrophic” growth form may be a response to some physical feature of the cellular environment, since it is triggered in M . grisea when the fungus grows embedded in cellophane (Bourett and Howard, 1990). Nevertheless, the general conclusion from all of these data is that all intracellular structures of biotrophic fungi and oomycetes are modified in some way by intimate association with the host protoplast, with the most complex modifications being associated with haustoria of specific fungal groups.
111. OF WHAT SIGNIFICANCE ARE THE PLANT CELLULAR REARRANGEMENTS THAT ACCOMPANY PARASITE INVASION? One of the most interesting features of plant cells invaded by biotrophic fungi and oomycetes is their ability to position cellular components in a precise location with respect to the parasite. A striking example is the common aggregation of plant cell cytoplasm at the site of fungal penetration (e.g. Aist and Bushnell, 1991), but additional rearrangements of organelles and the plant endomembrane system have been reported. These observations lead to two important questions: (1) what is the purpose of these cellular rearrangements, and (2) how are these rearrangements initiated and directed? A.
DEFENSIVE RESPONSES TO PARASITE INVASION
In some systems, organelle rearrangements and other cell responses to invasion appear to be involved in the deposition of materials on and in the plant cell wall that are assumed to be defensive in purpose, although they commonly do not prevent the parasite from entering the cell. The extent to which they are elicited in susceptible plants by biotrophic fungi and oomycetes varies considerably between plant-parasite systems, and seems unrelated to the type of biotrophic relationship. For example,
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oomycete pathogens (Beakes et al., 1982; Coffey and Wilson, 1983) and the hemibiotroph C . Zindernuthianurn (O’Connell et al., 1985), which have a relatively short biotrophic relationship with each invaded cell, commonly trigger the deposition of electron-translucent material between the plasma membrane and the plant wall during the process of cell penetration. This material may form a collar around the invading fungus (Fig. 1). Among fungi with long-term biotrophic associations, powdery mildew fungi also trigger this response (Edwards and Allen, 1970), but rust fungi often do not (Littlefield and Heath, 1979). The deposited material is generally identified as callose (a carbohydrate containing p-1, 3-linked glucan) by its fluorescence under ultraviolet (UV) irradiation following treatment with aniline blue. Given that this polysaccharide is universally deposited following mechanical stresses that mimic the penetration process (Russo and Bushnell, 1989), it seems likely that rust fungi can suppress the response. Since the ultrastructural, cytoplasmic, signs of callose deposition are lacking in these situations (Skalamera and Heath, 1995), the fungus cannot be merely degrading the callose as it forms, but must be suppressing its induction. The mechanism of suppression is unknown, although it seems not to be plant specific since callose is often absent in cells of non-host species in the rare circumstance of the fungus avoiding prehaustorial defences and forming a haustorium (Littlefield and Heath, 1979). Callose deposition may not be the only defensive response to invasion to be suppressed in compatible interactions involving rust fungi. For example, the monokaryotic stage of U . vignae suppresses, or does not trigger, any autofluorescence of epidermal walls during penetration, regardless of whether the host cultivar is resistant or susceptible (Chen and Heath, 1991). Powdery mildew fungi and the oomycete, P. infestans, however, commonly trigger this response in host epidermal cells, as well as the activation of defence genes, and the deposition of other materials in the cell wall (Aist and Bushnell, 1991; Freytag et al., 1994; Clark et al., 1995).
B. PARASITE-INDUCED CHANGES IN THE PLANT’S ENDOMEMBRANE SYSTEM
The most obvious change induced in biotroph-invaded cells is the synthesis of the extension of the plasma membrane which surrounds the parasite. Why the cell synthesizes this membrane at all is an interesting question (Heath, 1995), and it is obvious from data discussed in section I1 that, around haustoria, this membrane (the EHM) is often quite different from the plasma membrane to which it is attached. Plasma membrane normally is synthesized in plants in association with wall synthesis, and involves membrane processed through the Golgi apparatus (Driouich et al., 1994). However, there seems to be no consistent association of the EHM with plant dictyosomes during
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haustorium formation in many systems and, therefore, no clear indication of Golgi involvement in EHM synthesis. Nevertheless, the presence of coated vesicles or tubules associated with the EHM around D-haustoria of some rust fungi indicates that parts of the EHM may be retrieved by endocytosis into the plant cytoplasm (Mendgen et al., 1991); such retrieval is usually found in situations in which excess membrane is being formed by the fusion of secretory vesicles. An apparently unique situation has been reported for the smut fungus, Ustucysris waldsteiniae, in which Golgi vesicles are suggested to fuse with the EHM to form coralloid invaginations towards the fungus, possibly stabilized by the thick extrahaustorial matrix (Bauer et al., 1995). These invaginations have been suggested to increase the surface area of the EHM to aid nutrient transfer in a manner analogous to the wall and membrane ingrowths typical of plant transfer cells (Bauer et al., 1995). One of the most commonly reported or illustrated changes in cell ultrastructure in response to biotrophic parasites involves the ER. E R commonly accumulates around the young haustorium and may extend into complex lattices in the plant cytoplasm (Hickey and Coffey, 1977; Littlefield and Heath, 1979; Harder and Chong, 1991; Leckie et a f . , 1995). For D-haustoria of rust fungi, this increase in ER is due to synthesis not redistribution (Skalamera and Heath, 1995) and the ER may show direct connections to the EHM (Harder and Chong, 1991). These observations raise the possibility that the EHM around rust fungal haustoria is formed, at least in part, from the ER, which may account for some of the differences between the EHM and the plasma membrane. The controlling role of the fungus in any change to plant membranes is illustrated by the fact that D-haustoria of different Puccinia species trigger morphologically different membrane complexes in the same host plant (Harder and Chong, 1991). It seems that haustoria induce greater changes in the cell’s membrane systems than do other intracellular structures, since primary hyphae of both C. findemuthianum (O’Connell, 1987) and the monokaryon of U. vignae seem to be less associated with ER than monokaryotic haustoria formed later by the last fungus (Heath, unpublished). If the ER is involved in forming the EHM, it may be less involved in forming the membrane around these primary hyphae. Additional roles of the ER surrounding haustoria are suggested by recent studies of uninfected plant cells that show the normal plant plasma membrane to be closely associated with a peripheral network of ER. This network has immobile fixed bridges to the plasma membrane (Knebel et al., 1990) and is associated with actin microfilaments (Lichtscheidl et a f . , 1990). The ER-actin complex extends to other parts of the cell and it has been suggested that membrane binding stabilizes the microfilaments and provides a fixed point against which to generate the forces that cause cytoplasmic streaming (Lichtscheidl et a f . , 1990). In addition, the network may control local
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intracellular calcium concentration and thereby regulate the fusion of secretory vesicles (Craig and Staehelin, 1988) and aid in signal transduction (Hepler etal., 1990). The contact points with the plasma membrane also raise the possibility of transient secretion sites such as has been suggested in animal neurons (Fesce et al., 1994). Thus, the association of E R with the EHM could be important in directing the flow of plant cytoplasm to the haustorium and in the secretion of solutes into the extrahaustorial compartment. It is also possible that the ER around some haustoria is involved in redirecting secretory pathways towards the EHM, rather than the plasma membrane, thereby inhibiting defensive secretory activities and causing the observed induced accessibility of infected plant cells.
C. ASSOCIATIONS OF INTRACELLULAR FUNGAL STRUCTURES WITH THE PLANT NUCLEUS
Although the plant nucleus does not consistently associate with powdery mildew haustoria once they are formed (Aist and Bushnell, 1991), close association of this organelle with invading biotrophic structures is common in other examples of short- and long-term biotrophy (e.g. Littlefield and Heath, 1979; Coffey and Wilson, 1983). It seems particularly prevalent for rust fungi and has been reported for M- and D-haustoria of rust fungi in mesophyll cells (Littlefield and Heath, 1979) as well as primary hyphae of U. vignae in epidermal cells (Chen and Heath, 1991; Xu and Mendgen, 1991). In the latter system, nuclear behaviour is particularly interesting as the nucleus migrates to the penetration site during an early stage of penetration peg formation, but leaves as the fungus enters the cell. Subsequent nuclear movements and cytoplasmic streaming seem unaffected by the fungus as it expands to form a spherical vesicle, but the fungus becomes a focus for cytoplasmic streaming as it resumes tip growth and the nucleus then migrates to, and stays at, the hyphal apex (M. C. Heath, unpublished). The importance of the spatial location of the nucleus in biotrophic systems is further emphasized by the fact that nuclei lie close to arbuscules, but not intercellular hyphae, of mutualistic, arbuscular mycorrhizal fungi (Balestrini ef at., 1992). The reasons why the nucleus needs to associate with the parasite, or not associate with it during U. vignae primary hypha penetration, is unknown. The most obvious reason for the association is that the nucleus provides a localized source of mRNA for protein synthesis, but treatments that inhibit plant gene transcription or protein synthesis generally have little effect on the early growth of biotrophic fungi in susceptible plants (Heath, 1979; Hazen and Bushnell, 1983; Chen and Heath, 1994; M. C. Heath, unpublished). Taken at face value, these results suggest that the cellular changes associated with the initial formation of haustoria of rust and powdery mildew fungi do not require new mRNA synthesis, which seems difficult to imagine. Another role for the plant
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nucleus is that its outer membrane could act as a source of ER, since it is often in continuity with the ER surrounding D-haustoria of rust fungi (M. C. Heath, unpublished). However, it is also possible that nuclear migration is the inevitable response to localized “irritations” caused by the parasite, and that the plant nucleus plays no role in the biotrophic relationship. D. CHANGES IN THE PLANT CYTOSKELETON
Immunofluorescence labelling studies of short- and long-term biotrophic parasites have shown dramatic changes in the distribution of plant microtubules and microfilaments before and during penetration of cells expressing host and non-host resistance (Kobayashi et af., 1992, 1994; Gross et al., 1993; BaluSka et al., 1995). In contrast, little or no obvious changes were detected in compatible interactions (Kobayashi et af., 1992, 1994; BaluSka et al., 1995). These observations suggest that the most obvious cytoskeletal rearrangements are involved in defence responses, such as callose deposition, rather than the cellular reorganization characteristic of successful biotrophy . Nevertheless, inhibitor studies suggested that nuclear movement towards the fungal penetration site in non-host, cultured, parsley cells infected with P. infestans depended on the presence of microfilaments and the localized depolymerization of microtubules (Gross et al., 1993). Therefore, it seems highly likely that changes in the cytoskeleton occur during nuclear migration in susceptible plants, but high-resolution cytochemical studies may be required to detect them. It still remains to be determined whether the cytoskeleton is involved only in the mechanistic execution of organelle movements in infected cells, or whether it also helps transmit the signals that lead to these movements (BaluSka et af., 1995). E. CONCLUDING REMARKS
The presence of a biotrophic fungus or oomycete in a susceptible plant cell can have a dramatic effect on the nature and location of cellular components. Although we are beginning to appreciate the extent of these changes, we have little idea of how the invading organism brings them about. However, it is clear from the differences between different plant-parasite combinations that the parasite has considerable control over these events.
IV. WHY DO BIOTROPH-INVADED CELLS DIE IN RESISTANT PLANTS? When a biotrophic fungus or oomycete enters a cell of a resistant plant, the most common expression of resistance is cell death, a phenomenon
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originally described for rust fungi as the hypersensitive response (HR) (Stakeman, 1915). The speed at which the invaded cell dies in different systems may vary from a few minutes in potato cultivars resistant to P. infestuns (Tomiyama, 1967), to a few hours or a few days after invasion for rust and mildew systems, but in all cases, death is accompanied by the cessation of fungal growth. This association of cell death with resistance is most obvious for long-term biotrophic parasites because of the absence of cell death in susceptible plants. However, cell death which is more rapid than that seen in the susceptible plant is also a characteristic of plant resistance to hemibiotrophic and non-biotrophic fungi as well as to other pathogenic micro-organisms (Goodman and Novacky, 1994). Generally, “hypersensitive” cell death is accompanied by more rapid browning of the cell than is seen during cell death in susceptible plants. This almost ubiquitous association of cell death with disease resistance has led to the common assumption that the hypersensitive response is a universal, single phenomenon which acts as a defence mechanism against biotrophs and non-biotrophs alike (Goodman and Novacky, 1994). This section primarily addresses the validity of such a hypothesis with respect to biotrophic parasites.
A . IS CELL DEATH THE “DEFAULT STATE’’ FOLLOWING CELL PENETRATION?
The available literature suggests that cell death inevitably follows fungal or oomycete penetration of cells of non-host species, regardless of whether or not the parasite has a biotrophic relationship with its host (e.g. Fernandez and Heath, 1989). However, plant cells that are penetrated with a needle do not normally die. Instead, the needle is encased in callose-containing material (Russo and Bushnell, 1989). Therefore, cell death is not an inevitable consequence of physical cell penetration. Likewise, when a rust fungus haustorium is killed, either in susceptible plants or in resistant ones prior to the onset of cell death, the typical plant response is haustorial encasement, not death (Heath, 1988). These observations strongly suggest that cell death in resistant plants is a response to some activity of the parasite, rather than any non-self-recognition process. The corollary of this conclusion is that successful biotrophy must involve suppressing this cell death (for which there is some direct evidence in the P. infestuns-potato system, Doke et al., 1994), or eliminating any activity that would otherwise cause this death, in the host species. One might expect, therefore, that fungi and oomycetes with long-term biotrophic relationships with their hosts do not secrete molecules with inherent toxicity to plant cells (Fig. 2) but, as discussed below, this may not be sufficient to prevent the death of the invaded cell.
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PARASITE SECRETION J
Nonspecific elicitor I
+
Elicitor-receptor binding and signal transduction
Molecules with inherent toxicity (e.9. degradative enzymes, cell toxins)
Specific elicitor (Avirulence gene product) I
i
initiation of processes that lead to compatibility in susceptible plants (applicable only to biotrophic interactions)
Elicitor-receptor binding and signal transduction
cellular activities Generation of active oxygen
Activation of ion pumps
Membrane
I
TRIGGERING OF SUICIDAL RESPONSE (PROGRAMMED CELL DEATH)
-
I
PLANT CELL DEATH
Fig. 2. Possible ways in which a parasite may cause cell death in resistant host or non-host plant cells. Fungi or oomycetes with long-term biotrophic relationships with their hosts presumably do not secrete molecules with inherent cellular toxicity; however, they may initiate processes that lead to compatibility in susceptible, but adverse effects in incompatible, cells.
B. DO INVADED CELLS DIE IN HOST AND NON-HOST PLANTS FOR THE SAME REASON?
The most obvious difference between the HR in host and non-host plants is that, for most biotrophic systems, rapid cell death in resistant host cultivars appears to require some form of parasite-specific recognition process. The genetic manifestation of this recognition is that resistance is expressed only if the parasite has a gene for avirulence that is “matched” by a parasitespecific gene for resistance in the plant (the so-called “gene-for-gene’’ interaction). The current molecular interpretation of gene-for-gene interactions is that the avirulence gene produces a “specific elicitor’’ which is recognized by the product of the resistance gene (Staskawicz et al., 1995).
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Only a few resistance genes have been cloned so far, but the structural similarity between those conferring resistance to a virus, a bacterium, the intercellular biotroph, Cludosporium fulvum, and the rust fungus, Melumpsoru lini, may be indicative of some common function (Staskawicz et ul., 1995). Nevertheless, of the two genes controlling resistance to fungal biotrophs, only the C. fulvum gene product has characteristics of a membrane-bound receptor (Staskawicz et al., 1995) and the function of the rust resistance gene awaits determination. Although the genetic basis of parasite resistance in non-host plant species is unknown, it makes no evolutionary sense for cell death to be the result of parasite-specific recognition processes (Heath, 1991). The fact that many fungal and oomycete components (“non-specific elicitors”), from both biotrophic and non-biotrophic parasites, can induce defence responses in host and non-host plants alike suggests that plants have evolved to respond defensively to a variety of components and activities of these organisms which may not be inherently damaging to the plant cell (Heath, 1991). Thus, cell death elicited in non-host plants by biotrophic parasites could be the result of the action of these non-specific elicitors that are not counteracted by activities that prevent such death in susceptible host cultivars. If cell death in host and non-host plants involves different types of elicitor and recognition events, the next question is, do the two modes of elicitation feed into the same pathway leading to cell death? For the rust fungi, U. vignue and U. appendiculutus, D-haustoria develop further in cells of some non-host plants, and with fewer signs of adverse plant-fungus interactions, than in resistant host cultivars (Littlefield and Heath, 1979; M. C. Heath, unpublished). Delaying death of the first-invaded cell with a pre-inoculation heat treatment will often allow the fungus to form a small colony in non-host plants but not in resistant cultivars of host plants (Elmhirst and Heath, 1989). These data suggest that cell death may be more important in rust resistance in non-host plants than in resistance involving parasite-specific recognition processes (i.e. gene-for-gene interactions). This conclusion is supported by examples of host resistance that involve abnormal development of the EHM but not cell death (Skalamera and Heath, 1995) and observations that haustoria of Puccinia gruminis tritici formed in wheat containing a temperature-sensitive resistance gene show abnormalities even at the permissive temperature at which cell death does not occur (Harder et al., 1979). The simplest conclusion from these observations is that the interaction between the specific elicitor and its receptor in rust-resistant cultivars results in adverse cellular effects in addition to cell death. Therefore, cell death could be (1) the direct consequence of these adverse effects, (2) a suicidal response that is triggered by these effects, or (3) a suicidal response triggered independently of these effects (Fig. 2). If options (1) or (2) are correct, then the lack of these adverse cellular effects in non-host cells raises the possibility that a non-specific elicitor, or some activity that the plant is programmed to respond
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to, triggers the death of invaded cells by a different process than that occurring in resistant host cultivars.
C. ARGUMENTS FOR CELL DEATH IN RESISTANT PLANTS BEING A FORM OF PROGRAMMED CELL DEATH
Despite the many theoretically possible ways in which fungi and oomycetes may cause cell death (Fig. 2), there is a recent popular trend to consider the HR in both host and non-host cells as a form of plant cell suicide, or programmed cell death (Greenberg et al., 1994; Tenhaken er al., 1995). Examples of programmed cell death do occur in healthy plants, an obvious example being the death of xylem cells during their differentiation. However, dying xylem cells, and other examples of programmed cell death in plants (see Mittler and Lam, 1995), do not show the characteristics of cell death e.g. coagulation of cytoplasm, browning, phytoalexin accumulation, etc.) elicited in resistant plants by microbial pathogens. Therefore, if the HR is a form of programmed cell death, it must be a specific form that has evolved primarily as a mode of defence against microbial invaders. The strongest evidence that the HR represents a form of programmed cell death is its almost ubiquitous association with disease resistance. Moreover, cell death appears to be a potent defence mechanism. Not only is cell death a defence against biotrophs that are reliant on processes of living cells, but, by virtue of cellular decompartmentalization, it also releases toxic molecules from the vacuole, causes the oxidation of phenolic materials, and releases endogenous elicitors that trigger phytoalexin accumulation in surrounding cells (Mansfield, 1984; Aist and Bushnell, 1991). Thus it would seem advantageous for plants to have a way of triggering cell death, even if the invading pathogen does not directly cause this response. Arguments that the HR is a homologous phenomenon in every plantmicrobe interaction in which it is triggered depend on how much homology in the triggering and execution of cell death is required to accept the hypothesis. As yet, there is no definitive reaction or change in gene expression that defines plant cell death as the HR. This is in contrast to a common form of programmed cell death in animal cells (apoptosis), which is identified by a distinctive set of cytological changes including the fragmentation of the cell into apoptotic bodies, and the activation of an endonuclease that cleaves nuclear DNA into oligonucleosome fragments (Raff, 1992). The primary purpose of these striking cellular events seems to be to allow the dead cell to be removed by macrophages without triggering an inflammation response (Raff, 1992), so there is no reason to expect a strictly homologous process in higher plants. Nevertheless, nuclear DNA cleavage has been detected cytochemically in differentiating xylem cells (Mittler et al., 1995; Mittler and Lam, 1995) and similar cleavage, as well
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as oligonucleosome fragments, has been detected in haustorium-containing dead cells in a resistant cowpea cultivar infected with the rust fungus, U. vignae (Ryerson and Heath, 1996). Since similar nuclear changes are not associated with all forms of chemically induced cell death (Ryerson and Heath, 1996), such observations are at least consistent with the hypothesis that the HR is a special form of cell death that may have some features in common with animal apoptosis. In addition, nuclear DNA cleavage also has been detected in lesions in tobacco caused by virus infection and in spontaneous patches of dead cells in transgenic plants expressing a bacterial proton pump (Mittler et al., 1995). Whether this nuclear DNA cleavage is an indicator of a unique form of cell death or a constant marker for the HR remains to be determined. Proof that the HR is a form’of programmed cell death depends heavily on the discovery of such markers and a link between the response and some endogenous plant activity.
D. ARGUMENTS AGAINST CELL DEATH IN RESISTANT PLANTS BEING A FORM OF PROGRAMMED CELL DEATH
For haustorial biotrophic parasites, most commonly it is the invaded cell that dies in resistant plants. As already described, these haustoria have such a profound influence on the invaded cell that it is not hard to imagine that the compatible relationship could be easily disrupted or disturbed. Such disruptions could result from a lack of metabolic “compatibility” between plant and parasite in non-host species, or from modifications to proteins that interact with parasite molecules in resistant host cultivars; the end result in both cases could be metabolic perturbances that kill the cell. As discussed earlier, each major group of parasitic fungal and oomycetous biotrophs appears to have unique features with respect to the plant-parasite interface, and may interact with their hosts in different ways. Therefore, resistance based on perturbations of the plant-parasite interaction could be different in each system. For rust fungi, such an argument is supported by the ultrastructural differences, and different effects on fungal growth, between different forms of cell death (Littlefield and Heath, 1979). The argument is also supported by apparent differences in the cellular mechanisms of different forms of cell death discussed in the next section. A currently popular argument for the existence of programmed cell death in plants is the existence of single gene mutants which show various forms of spontaneous or stress-induced cell death (Dietrich et al., 1994; Greenberg et al., 1994). The fact that some of these mutations are recessive has been interpreted to indicate that the wild-type gene functions to negatively regulate cell death. Thus, as has been suggested in animal cells (Raff, 1992), it could be argued that cell death is the default state in plant cells, and specific cellular activity is required to keep cells alive. However, in the absence of any clear
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marker for programmed cell death, or the HR, there is little hard evidence to prove that these mutations affect a suicide pathway or mimic the HR. It is possible that each mutation modifies one of a variety of cellular functions that, under certain conditions, lead to metabolic catastrophe and death. The fact that this cell death leads to the types of events typical of the HR, such as cell autofluorescence, callose deposition and the accumulation of defence gene transcripts (Dietrich et al., 1994; Greenberg et al., 1994), is not proof that the cell is killed in a manner identical to that seen in parasite-infected plants. If cellular decompartmentalization releases endogenous elicitors of defence processes (Mansfield, 1984), many forms of cell death are likely to trigger such responses. Indeed, the number of these HR-mimicking mutants could be interpreted as evidence that there are many different ways of triggering cell death. More relevant and informative mutants, with respect to understanding the HR, may prove to be those that are defective in genes that appear to be necessary for the expression of resistance-gene controlled cell death in the presence of the parasite (Hammond-Kosack et a f . , 1994).
E. CELLULAR MECHANISMS OF CELL DEATH
In general, ultrastructural studies, alone, have not been particularly informative about the cellular processes involved in cell death caused by biotrophic parasites. Adverse effects on cell membranes have been commonly reported (e.g. Littlefield and Heath, 1979; Meyer and Heath, 1988), but whether they are the cause or consequence of cell death is unknown. It is becoming obvious that meaningful information requires temporal studies of cell death involving detailed, correlative, cytological, cytochemical and biochemical investigations. Correlative light and electron microscope studies, for example, have shown that one of the first signs of cell death in cowpea epidermal cells infected with haustoria of the plantain powdery mildew fungus, Erysiphe cichoracearum, is the cessation of cytoplasmic streaming; this correlated with the disappearance of microtubules in the plant cytoplasm, even though they were still detectable when cytoplasmic streaming stopped in uninfected cells treated with copper chloride (Meyer and Heath, 1988, and references therein). Therefore, a rapid effect on the cytoskeleton appears to be a characteristic of this example of the HR. The disappearance of microtubules also has been reported in haustorium-containing cells of flax inoculated with incompatible strains of M . fini (Kobayashi et al., 1994), but the timing of this response relative to the cessation of cytoplasmic streaming is unknown. Correlative cytological and biochemical studies applied to potato cultivars infected with P. infesfans (Doke et al., 1994) have led to the conclusion that initial cell invasion by this oomycete triggers bursts of superoxide generation.
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One occurs prior to penetration in both susceptible and resistant cultivars, and the second is observed only after penetration of resistant cultivars. The latter burst is suggested to lead to the generation of other active oxygen species that could damage the plasma membrane, leading to electrolyte leakage and death (Doke et al., 1994, and references therein). Subsequent studies have revealed that similar oxidative bursts seem to be a ubiquitous response of cell suspension cultures to avirulent bacteria, non-specific elicitors, and a specific elicitor from C. fulvum (Vera-Estrella et al., 1994). These, and other, data have led to the hypothesis that all examples of the HR are the result of the generation of hydrogen peroxide by enzyme systems in the plasma membrane, which then elicits programmed cell death (Tenhaken et al., 1995). Consistent with the model of hydrogen peroxide playing a pivotal role in the HR to rust fungi, the cell autofluorescence caused by invasion of resistant host cultivars by the monokaryotic primary hypha of U. vignae, or by injection of a specific elicitor from this fungus into uninfected leaves, can be delayed by catalase treatment (Chen and Heath, 1994). However, more recent studies suggest that, although catalase delays the autofluorescence and browning of invaded cells, it does not affect the cessation of cytoplasmic streaming or protoplast collapse (M. C. Heath, unpublished). Therefore, although hydrogen peroxide may be involved in the later stages of cell death, there is no evidence that it triggers its initiation in this system. Moreover, cytological studies in cowpea using nitroblue tetrazolium, similar to those used to demonstrate superoxide formation prior to the HR in Phytophthorainfected potato (Doke et al., 1994), did not reveal any increase in superoxide dismutase-inhibitable staining prior to cell death. Similar results were observed with both monokaryotic infection hyphae and dikaryotic haustoria and, in fact, greater staining was seen around these structures in cells of susceptible plants than in resistant ones (M. C. Heath, unpublished). Therefore, the currently available data do not support an inducing role of superoxide or hydrogen peroxide in the HR in this system. Given the apparent differences between the HR caused by the short-term biotroph, P. infestans, and by the long-term biotroph, U. vignae, comparable data from powdery mildew systems would be of interest; unfortunately, redox changes associated with the HR in powdery-mildew-infected resistant cultivars have not been examined. The best temporal accounts of the cellular events that accompany cell death induced by biotrophs come from light microscope studies of infected, living, tissue. Three pathogens have been studied in some detail: the powdery mildew fungus, Erysiphe graminis f.sp. hordei, forming haustoria in resistant barley; the rust fungus, U. vignae, forming primary hyphae in resistant host and non-host plants (Chen and Heath, 1991; Xu and Mendgen, 1991); and the oomycete, P. infestans, invading resistant potato (Tomiyama, 1967; Doke et al., 1994; Freytag et al., 1994). Cell death in all these systems involves the
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cessation of cytoplasmic streaming, the granulation of the cytoplasm, and the subsequent collapse of the protoplast or cell. In each system, cell death can be delayed with a pre-inoculation heat treatment and metabolic inhibitors, suggesting that cell metabolism is required during the induction or execution phase. However, despite this similarity, there are subtle differences between systems. In the P. infestuns system, the plant cytoplasm and nucleus conglomerate around the fungus and both fungus and conglomerate show a sudden collapse (Freytag et ul., 1994). This HR may not be dependent on plant resistance genes because it occurred almost equally in both resistant and susceptible cultivars (Freytag et al., 1994). In resistance-gene-dependent cell death caused by powdery mildew haustoria, the host nucleus has no special spatial relationship with the haustorium, and it collapses a few minutes before the entire cell collapses; haustoria visibly collapse just before or after host cell collapse (Aist and Bushnell, 1991). Resistance-gene-dependent expression of the HR induced by U. vignue requires exogenous kinetin (Chen and Heath, 1991), a feature not reported in other systems. Primary hyphae of this rust fungus do not collapse but merely cease to grow and become vacuolated either at the time of, or a little while after, the cessation of cytoplasmic streaming in the plant cell (Chen and Heath, 1991; M. C. Heath, unpublished). Interestingly, the plant nucleus behaves differently in host and non-host examples of the HR to this fungus. In a non-host cell, the plant nucleus migrates to the primary hypha and becomes enlarged in comparison with nuclei similarly associated with primary hyphae in susceptible host cells; the nucleus then shrinks in size when the protoplast collapses (Xu and Mendgen, 1991). In the resistant host cultivar, however, the first sign of cellular incompatibility is the lack of nuclear migration to the primary hypha at a time when such migration normally takes place in susceptible cells; this lack of nuclear migration is associated with normal movements of the plant cytoplasm and other organelles (M. C. Heath, unpublished). A change in nuclear appearance commonly precedes the cessation of cytoplasmic streaming (Chen and Heath, 1991), making an effect on the nucleus the first observable sign of cell death in this system. Taken altogether, the studies described above suggest considerable diversity in the cellular features of cell death induced by different biotrophs, which supports differences in mode of induction if not execution. However, the data are still fragmentary, and we basically know as little about how biotrophs kill cells as how they keep them alive.
F. CONCLUDING REMARKS
Almost all proven eukaryotic examples of gene-for-gene systems involve biotrophic fungi or oomycetes (Heath, 1994), and it is commonly accepted that the HR in such systems (except, perhaps, for the P. infestuns system
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(Doke ef al., 1994)) is triggered by a specific elicitor. Therefore, it is remarkable that only one specific elicitor of cell death has been found for haustorium-producing organisms. This elicitor is secreted by extracellular parts of the monokaryon of U. vignue, but has not been detected in non-haustorial parts of the dikaryon, leading to the hypothesis that the dikaryon only secretes the elicitor at the haustorium-plant interface (Chen and Heath, 1992). If specific elicitors in other systems are similarly localized, they will be difficult to isolate. Without them, it will be a formidable challenge to understand how these intracellular parasites trigger cell death.
V.
CONCLUSIONS
The more we learn about the cellular interactions between plants and biotrophic fungal (and oomycetous) parasites, the more complex they seem to be. Although the degree of complexity varies between plant-parasite combinations, the most complex involve establishing membrane domains with specific properties that favour the parasite, rearrangements of organelles that may be accompanied by localized alterations in their functions, and changes in the way the cell responds to outside stimuli (i.e. other pathogens). In the case of long-term biotrophy, the parasite also has to keep the cell alive in the face of attributes that apparently universally cause the death of non-host cells, and that can trigger parasite-specific events which lead to cell death in resistant host cultivars. Knowledge of how biotrophic parasites achieve these feats will advance our knowledge, not only of this form of parasitism, but also of the normal control of many cellular functions.
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Elmhirst, J. F. and Heath, M. C. (1989). Interactions of the bean and cowpea rust fungi with species of the Phaseolus-Vigna plant complex. 11. Histological responses to infection in heat-treated and untreated leaves. Canadian Journal of Botany 67, 58-72. Fasters, M. K., Daniels, U. and Moerschbacher, B. M. (1993). A simple and reliable method for growing the wheat stem rust fungus, Puccinia graminis f s p . tritici, in liquid culture. Physiological and Molecular Plant Pathology 42, 259-263. Fernandez, M. R. and Heath, M. C. (1989). Interactions of the nonhost French bean plant (Phaseolus vulgaris) with parasitic and saprophytic fungi. 111. Cytologically-detectable responses. Canadian Journal of Botany 67, 676-686. Fernandez, M. R. and Heath, M. C. (1991). Interactions of the nonhost French bean plant (Phaseolus vulgaris) with parasitic and saprophytic fungi. IV. Effect of pre-inoculation with the bean rust fungus on growth of parasitic fungi nonpathogenic on beans. Canadian Journal of Botany 69, 1642-1646. Fesce, R., Grohovaz, F., Valtorta, F. and Meldolesi, J. (1994). Neurotransmitter release: fusion or “kiss-and-run”? Trends in Cell Biology 4, 1 4 . Freytag, S., Arabatzis, N., Hahlbrock, K. and Schmelzer, E. (1994). Reversible cytoplasmic rearrangements precede wall apposition, hypersensitive cell death and defense-related gene activation in potatolPhytophthora infestans interactions. Planta 194, 123-135. Garrill, A. (1995). Transport. In “The Growing Fungus” (N. A. R. Gow and G. M. Gadd, eds), pp. 163-181. Chapman and Hall, London. Gay, J. L., Salzberg, A. and Woods, A. M. (1987). Dynamic experimental evidence for the plasma membrane ATPase domain hypothesis of haustorial transport and for ionic coupling of the haustorium of Erysiphe graminis to the host cell (Hordeum vulgare). New Phytologist 107, 541-548. Goodman, R. N. and Novacky, A. J. (1994). “The Hypersensitive Reaction in Plants to Pathogens; A Resistance Phenomenon.” American Phytopathological Society Press, St Paul, MN. Gray, D. J., Amerson, H. V. and Van Dyke, C. G. (1982). An ultrastructural comparison of monokaryotic and dikaryotic haustoria formed by the fusiform rust fungus Cronartium quercuum f. sp. fusiforme. Canadian Journal of Botany 60, 2914-2922. Greenberg, J. T., Guo, A . , Klessig, D. F. and Ausubel, F. M. (1994). Programmed cell death in plants: a pathogen-triggered response activated coordinately with multiple defense functions. Cell 77, 551-563. Gross, P., Julius, C., Schmelzer, E. and Hahlbrock, K. (1993). Translocation of cytoplasm and nucleus to fungal penetration sites is associated with depolymerization of microtubules and defence gene activation in infected, cultured parsley cells. The EMBO Journal 12, 1735-1744. Hammond-Kosack, K. E., Jones, D. A. and Jones, J. D. G . (1994). Identification of two genes required in tomato for full Cf-9-dependent resistance to Cladosporium fulvum. Plant Cell 6, 361-374. Harder, D. E. and Chong, J. (1991). Rust haustoria. In “Electron Microscopy of Plant Pathogens” (K. Mendgen and D.-E. Lesemann, eds), pp. 235-250. Springer Verlag, Berlin. Harder, D. E., Rogringer, R., Samborski, D. J., Rimmer, S. R., Kim, W. K. and Chong, J. (1979). Electron microscopy of susceptible and resistant near-isogenic (sr6/Sr6) lines of wheat infected by Puccinia graminis tritici. 11. Expression of incompatibility in mesophyll and epidermal cells and the effects of temperature on host-parasite interactions in these cells. Canadian Journul of Botany 57, 2617-2625.
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Knebel, W., Quader, H. and Schnepf, E. (1990). Mobile and immobile endoplasmic reticulum in onion bulb epidermis cells: short- and long-term observations with a confocal laser scanning microscope. European Jouhal of Cell Biology 52, 328-340. Kobayashi, I., Kobayashi, Y. and Hardham, A. R. (1994). Dynamic reorganization of microtubules and microfilaments in flax cells during the resistance response to flax rust infection. Planta 195, 237-247. Kobayashi, I., Kobayashi, Y., Yamoaka, N. and Kunoh, H. (1992). Recognition of a pathogen and a nonpathogen by barley coleoptile cells. 111. Responses of microtubules and actin filaments in barley coleoptile cells to penetration attempts. Canadian Journal of Botany 70, 1815-1823. Leckie, C. P., Callow, J . A. and Green, J. R. (1995). Reorganisation of the endocytoplasmic reticulum in pea leaf epidermal cells infected by powdery mildew fungus Erysiphe pisi. New Phytologist 131, 211-222. Lee-Stadelmann, 0. Y., Bushnell, W. R. and Stadelmann, E. J. (1984). Changes of plasmolysis form in epidermal cells of Hordeum vulgare infected by Erysiphe graminis: evidence for increased membrane-wall adhesion. Canadian Journal of Botany 62, 1714-1723. Lewis, D. H. (1973). Concepts in fungal nutrition and the origin of biotrophy. Biological Reviews 48, 261-278. Lichtscheidl, I. K., Lancelle, S. A. and Hepler, P. K . (1990). Actin-endoplasmic reticulum complexes in Drosera. Their structural relationship with the plasmalemma, nucleus, and organelles in cells prepared by high pressure freezing. Protoplasma 155, 116-126. Littlefield, L. J. and Heath, M. C. (1979). “Ultrastructure of Rust Fungi.” Academic Press, New York. Luttrell, E. S. (1974). Parasitism of fungi on vascular plants. Mycofogia 66, 1-15. Mansfield, J. W. (1984). Plant cell death during infection by fungi. I n “Cell Ageing and Cell Death” (I. Davies and D. C. Sigee, eds), pp. 323-345. Cambridge University Press, Cambridge. McKeen, W. E. and Rimmer, S. R. (1973). Initial penetration process in powdery mildew infection of susceptible barley leaves. Phytopathology 63, 1049-1053. Mendgen, K., Welter, K., Scheffold, F. and Knauf-Beiter, G. (1991). High pressure freezing of rust infected plant leaves. In “Electron Microscopy of Plant Pathogens” (K. Mendgen and D.-E. Lesemann, eds), pp. 31-42. SpringerVerlag, Berlin. Meyer, S. L. F. and Heath, M. C. (1988). A comparison of the death induced by fungal invasion or toxic chemicals in cowpea epidermal cells. 11. Responses induced by Erysiphe cichoracearurn. Canadian Journal of Botany 66, 624634. Mittler, R. and Lam, E. (1995). In situ detection of nDNA fragmentation during the differentiation of tracheary elements in higher plants. Plant Physiology 108, 489493. Mittler, R., Shulaev, V. and Lam, E. (1995). Coordinated activation of programmed cell death and defense mechanisms in transgenic tobacco plants expressing a bacterial proton pump. Plant Cell 7 , 29-42. Morris, C. (ed.) (1992). “Academic Press Dictionary of Science and Technology.” Academic Press, San Diego. Neufeld, E . and Bown, A. W. (1987). A plasmamembrane redox system and proton transport in isolated mesophyll cells. Plant Physiology 83, 895-899. Nozue, M . , Tomiyama, K . and Doke, N. (1979). Evidence for adherence of host plasmalemma to infecting hyphae of both compatible and incompatible races
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of Phytophthora infestans. Physiological Plant Pathology 15, 111-115. O’Connell, R. J. (1987). Absence of a specialized interface between intracellular hyphae of Colletotrichum lindemuthianum and cells of Phaseolus vulgaris. New Phytologist 107, 725-734. O’Connell, R. J., Bailey, J. A. and Richmond, D. V. (1985). Cytology and physiology of infection of Phaseolus vulgaris by Colletotrichum lindemuthianum. Physiological Plant Pathology 27, 75-98. Onoe, T., Tani, T. and Naito, N. (1973). The uptake of labelled nucleosides by Puccinia coronata grown in susceptible oat leaves. Report of the Tottori Mycological Institute (Japan) 10, 303-312. Pain, N. A., OConnell, R. J., Mendgen, K. and Green, J. R. (1994). identification of glycoproteins specific to biotrophic intracellular hyphae formed in the Colletotrichum lindemuthianum-bean interaction. New Phytologist 127, 233242. Patrick, J. W. (1989). Solute efflux from the host at plant-microorganism interfaces. Australian Journal of Plant Physiology 16, 53-67. Pnng, R. J . , Nash, C., Zakaria, M. and Bailey, J. A . (1995). Infection process and host range of Colletotrichum capsici. Physiological and Molecular Plant Pathology 46, 137-152. Raff, M. C. (1992). Social controls on cell survival and cell death. Nature 356, 397400. Rey, M. E. C. and Garnett, H. M. (1985). Transfer of label from ’H-glucose in Digitaria eriantha leaves to the rust fungus Puccinia digitariae Pole Evans. The Journal of Histochemistry and Cytochemistry 33, 809-812. Roberts, A. M., Mackie, A. J., Hathaway, V., Callow, J. A. and Green, J. R. (1993). Molecular differentiation in the extrahaustorial membrane of pea powdery mildew haustoria at early and late stages of development. Physiological and Molecular Plant Pathology 43, 147-160. Roitsch, T., Bittner, M. and Godt, D. E. (1995). Induction of apoplastic invertase of Chenopodium rubrum by D-glucose and a glucose analog and tissue-specific expression suggest a role in sink-source regulation. Plant Physiology 108, 285-294. Russo, V. M. and Bushnell, W. R. (1989). Responses of barley cells to puncture by microneedles and to attempted penetration by Erysiphe graminis f. sp. hordei. Canadian Journal of Botany 67, 2912-2921. Ryerson, D. E. and Heath, M. C. (1996). Cleavage of nuclear DNA into oligonucleosomal fragments during cell death induced by fungal infection or abiotic treatments. Plant Cell 8, 393402. Secor, J. (1987). Regulation of sucrose efflux from soybean leaf discs. Plant Physiology 83, 143-148. Skalamera, D. and Heath, M. C. (1995). Changes in the plant endomembrane system associated with callose synthesis during the interaction between cowpea (Vigna unguicu~ata)and the cowpea rust fungus (Uromyces vignae). Canadian Journal of Botany 73, 1731-1738. Spencer-Phillips, P. T. N. and Gay, J. L. (1981). Domains of ATPase in plasma membranes and transport through infected plant cells. New Phytologist 89, 393-400. Stakman, E. C. (1915). Relations between Puccinia graminis and plants highly resistant to its attack. Journal of Agricultural Research 4, 193-199. Staskawicz, B. J., Ausubel, F. M., Baker, B. J., Ellis, J. G. and Jones, J. D. G. (1995). Molecular genetics of plant disease resistance. Science 268, 661-4367. Stumpf, M. A. and Gay, J. L. (1990). The composition of Erysiphe pisi haustorial
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complexes with special reference to the neckbands. Physiological and Molecular Plant Pathology 37, 125-143. Tenhaken, R., Levine, A., Brisson, L. F., Dixon, R. A. and Lamb, C. (1995). Function of the oxidative burst in hypersensitive disease resistance. Proceedings of the National Academy of Science, USA 92, 41584163. Tetlow, I. J. and Farrar. (1993). Apoplastic sugar concentration and pH in barley leaves infected with brown rust. Journal of Experimental Botany 44, 929936. Tomiyama, K. (1967). Further observations on the time requirement for hypersensitive cell death of potatoes infected by Phytophthora infestans and its relation to metabolic activity. Phytopathologische Zeitschrift 58, 367-378. Vera-Estrella, R . , Higgins, V. J. and Blumwald, E. (1994). Plant defense response to fungal pathogens. 11. G-Protein-mediated changes in host plasma membrane redox reactions. Plant Physiology 106, 97-102. Woods, A. M. and Gay, J. K. (1983). Evidence for a neckband delimiting structural and physiological regions of the host plasma membrane associated with haustoria of Albugo candida. Physiological Plant Pathology 23, 73-88. Woods, A. M. and Gay, J. L. (1987). The interface between haustoria of Puccinia poarum (monokaryon) and Tussilago farfara. Physiological and Molecular Plant Pathology 30, 167-185. Woods, A. M., Didehvar, F., Gay, J. L. and Mansfield, J . W. (1988). Modification of the host plasmalemma in haustorial infections of Lactuca sativa by Bremia lactucae. Physiological and Molecular Plant Pathology 33, 299-310. Xu, H. and Mendgen, K. (1991). Early events in living epidermal cells of cowpea and broad bean during infection with basidiospores of the cowpea rust fungus. Canadian Journal of Botany 69, 2279-2285. Yamaoka, N., Toyoda, K., Kobayashi, I. and Kunoh, H. (1994). Induced accessibility and enhanced inaccessibility at the cellular level in barley coleoptiles. XIII. Significance of haustorium formation by the pathogen Erysiphe graminis for induced accessibility to the non-pathogen E. pisi as assessed by nutritional manipulations. Physiological and Molecular Plant Pathology 44, 217-225.
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Symbiology of Mouse-Ear Cress (Arubidopsis thuliuna) and Oomycetes
E . B . HOLUB' and J . L . BEYNON2
'Plant Pathology and Weed Science Department. Horticulture Research International-Wellesbourne. Warwickshire CV35 9EF, UK. Department of Biological Sciences. Wye College. University of London. Wye. Ashford. Kent TN25 5AH. UK.
I . Introduction
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I1 . Defining a New Research Arena of Plant Biology ..........................
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A. B. C. D.
The Dawn of Arabidopsis .................................................... Relevant Trends in Modern Biology ....................................... A Rebirth in Plant Pathology ................................................ A Copernican Perspective ....................................................
I11 . Symbionts from the Wild ............................................................ A . The Phytobiont: Arabidopsis thaliana ..................................... B . The Biotrophs: Peronospora parasitica and Albugo candida ....... C . Three Rs of Symbiosis ......................................................... D . The Phenotypes of Interactions: Consequences of Recognition ...
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IV. Molecular Genetics of Natural Variation ....................................... A . Building Models: Predicting the Host Genotype ....................... B . Nonallelism: Juggling with Apples and Oranges ....................... C . Major Complexes of Recognition Genes: How Big is a Cluster? D . A Natural Anomaly of Susceptible Origin ...............................
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V. Mutations: Revealing Complexity from Black and White ................. 259 260 A . A Myriad of Columbia Mutants ............................................ B . Surprising Extremes in Wassilewskija ..................................... 261 VI . Avenues of Future Research
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VII . Concluding Remarks .................................................................. Acknowledgements .................................................................... References ............................................................................... Advances in Botanical Research Vol . 24 incorporating Advances in Plant Pathology ISBN (L12-005924X
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Copyright @ 1997 Academic Press Limited All rights of reproduction in any form reserved
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1. INTRODUCTION The common wild flower Arabidopsis thaliana (L.) Heynh. (referred to hereafter as Arabidopsis) has a story to tell, one of how its evolution has been influenced by symbiotic relationships with micro-organisms. Most of the story has yet to unfold, but research programmes already well established encompass several classes of parasite and pathogen (fungus, bacteria, virus, nematode and protoctist) (Crute et al., 1994; Sijmons et al., 1994; Simon, 1994). In most cases, the parasite isolates used have been collected from other host species and have been shown to infect certain genotypes of Arabidopsis. These interactions may be deemed artificial. They have nonetheless contributed, in less than 6 years of research, to the mainstream discoveries of polypeptides required for disease resistance (Bent ef al., 1994; Mindrinos et al., 1994; Grant et al., 1995). More importantly, they most likely foreshadow common themes in the genetic adaptation of herbaceous angiosperms to their biotic environment. In this chapter we highlight what has been learned to date from the natural symbioses of Arabidopsis with two biotrophs, Peronospora parasitica (Pers. ex Fr.) Fr. (downy mildew) and Albugo candida (Pers. ex Fr.) 0. Kuntze (white blister). We have used this opportunity to demonstrate the value of studying a common ephemeral wild flower. The methods used are familiar; modem methods of molecular biology have been developed simultaneously with traditional methods of descriptive biology and classical genetics from inception of the research. The traditional methods provide more than a stepping-stone for the basic molecular studies of gene structure; they have become integral in generating further insight and questions that perhaps extend beyond conventional thinking. This chapter has been written entirely for those who consider themselves students of biology, whether officially enrolled as such or continuing on from advanced degrees. It should appeal to those who were attracted to biology by an enthusiasm for studying organisms, but justifiably find themselves in the realm of molecular biology. Our own research interests represent a marriage of plant pathology (the discipline of the first author) and molecular biology (the discipline of the second author). The ideas we employ have been inspired and reinforced by the past experience of many within and outside our own respective disciplines; several general texts are recommended (Futuyma and Montgomery, 1983; Bohm and Peat, 1987; Burdon, 1987; Holub, 1990; Barlow, 1991; Sapp, 1994; Margulis and Sagan, 1995; Wilson, 1995). The exploration of Arabidopsis and biotrophic oomycetes commenced in 1989 with a clean slate of expectations and ideas. The overriding interest has been to be conscious of fundamental assumptions and to remain receptive to what the organisms themselves present for investigation. Although the subjective trappings of dogma cannot be avoided entirely, we have experi-
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mented with terminology and concepts within our research group with the intent of promoting greater objectivity. The play with ideas continues, but the data presented here will attest to whether progress is being made. The unfolding story of mouse-ear cress and oomycetes begins with an impression of the research arena in which we participate.
11. DEFINING A NEW RESEARCH ARENA OF PLANT BIOLOGY A . THE DAWN OF ARABIDOPSIS
Only since the mid-l980s, with the momentum generated from rediscovering Arubidopsis as a subject for genetic investigation (Meyerowitz, 1987), have biologists appreciated its abilities to interact with prokaryotic and eukaryotic micro-organisms. This interest forms the basis for an important contribution that Arubidopsis provides in understanding the mechanisms of disease resistance in plants (Dangl, 1993). Nonetheless, the natural history and evolutionary biology of this wild flower remains largely unexplored. It is an undeclared topic at international meetings focused on the species and may at present only be considered by a few research groups. In the minds of many, the only relevant existence of the species occurs in research facilities. Its primary virtue is the enormous flexibility it provides researchers for conducting genetical analyses of cell cycles, tissue development, sexual reproduction and how a plant otherwise functions in its environment. The research community fondly refers to it as a “weed”, with scant collective awareness that it struggles to compete in nature with every other plant species and is choice forage for slugs and insects. It usually grows so inconspicuously in the wild that most researchers would be hard-pressed to locate it except where seed has been unintentionally dispersed around the periphery of glasshouses known to harbour the species. The word “ecotype” is symbolic of the parochial impression most people have of Arubidopsis. This term has been widely adopted in the past few years for referring to germplasm sampled from different geographic locations, even though virtually nothing is known about characteristic adaptations to the original habitat. These shortcomings are perhaps symptomatic of rapid growth in a blossoming research arena, and fortunately will not deter what promises to be monumental progress in a reductionist dream of plant biology. The research of parasitic symbioses described here began with the same enthusiastic sense of exploration that gave birth to the current Arubidopsis research community. However, natural history is of paramount importance. Symbiosis research would not be possible without germplasm collections of the host and parasites, and some degree of knowledge of their origins.
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Collecting is an essential component of the research, not just for providing a few case studies for molecular investigation.
B. RELEVANT TRENDS IN MODERN BIOLOGY
Any differences that may exist in our approach to research of Arabidopsis emerge from a deliberate attempt to encourage lateral thinking, particularly by the Ph.D. students in our group who have contributed enormously to the research. This is in part being achieved by incorporating two important trends: rejuvenation of comparative biology, and a realization that symbiosis is a fundamental driving force of evolution. Comparative biology is turning full circle. Once the mainstay of biology at an organismal level, it was eventually eclipsed when molecular biology emerged as a powerful and essential means for revolutionizing biology (Wilson, 1995). Yet, the potential advances in molecular biology can themselves benefit greatly from comparative methods. This is clearly demonstrated by the realization that structurally similar molecules are utilized by plants from different genera to recognize bacteria, fungi and viruses (Staskawicz et al., 1995). This not only indicates that signal transduction plays an instrumental role in plant defensive strategies, but it now appears possible to reveal some common themes across the phylogenetic spectrum of parasites and pathogens. Symbiosis, or the process of “evolution of association”, has brought about a quiet shift of paradigm by providing an important corollary to the popular Darwinian notion of “survival of the fittest’’ (Sapp, 1994; Margulis and Sagan, 1995). The term is often used in a conclusive, static manner for pigeon-holing different life-styles, and used narrowly by some to imply co-operation and mutual benefit between symbionts. However, as originally coined by Frank in 1877 and defined by DeBary in 1879 (translation taken from Sapp, 1995), “parasitism, mutualism, lichenism etc., are each special cases of that one general association for which the term symbiosis is proposed as the collective name”. Symbiosis is impartial, suggesting only a mutual influence between symbionts on their development and evolutionary history that results from their intimate association. It is technically extremely difficult to define the costs and benefits if one considers a temporal dimension and environmental influence on the development of an intimate, physiological union between two organisms. Nonetheless, the processes by which organisms become coupled in their evolution is an important research frontier; one that will render transparent the boundaries that currently divide speciality disciplines such as plant pathology, lichenology and rhizobium biology. The study of symbiosis bridges such disciplines because it is fundamentally a subject of evolutionary biology. Perhaps it is itself an emerging discipline, namely symbiology (sensu Read, 1970).
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C . A REBIRTH IN PLANT PATHOLOGY
Theoretical concepts of plant parasitic symbiosis are rudimentary, but are on the verge of metamorphosis as recent advances in research begin to bridge the gap between genetics and physiology. The seminal papers by Flor (1971) have served well to stimulate debate about the genetics of plant-parasite interactions in the absence of sufficient physiological information. However, some aspects can still be quite confusing for someone uninitiated in the idiosyncrasies of the research field. A prime example is “avirulence”, a word now entrenched in the literature, which clearly implies a functional meaning of “lack of virulence”. It does, however, have a subtly different genetical meaning: a pathogen trait which segregates in pair-wise correspondence with a resistance trait of the plant. The resistance and avirulence traits are generally thought to be conferred by single genes, hence the colloquial phrase “gene for gene” first coined by Flor. Numerous so-called ‘‘avirulence genes” have been isolated and characterized, primarily from bacterial pathogens, but no common features have been determined, and it has proven conceptually difficult to define their role (reviewed by Dangl, 1995). Biochemists have coined “elicitor” as a similar term that bypasses the ambiguity of avirulence when discussing possible molecular interactions between the plant and parasite gene products. The concept of a resistance gene can itself be paradoxical. On the one hand, the phenomenon of resistance can be defined as the consequence of events initiated by the perception of a parasite gene product which in turn triggers host defence response. The host response effectively contains and/or has the effect of killing the intruder, thereby reducing or nullifying the reproductive potential of the parasite. This, in essence, is resistance to the parasite. Models have predicted that resistance results either from a single host gene product which in effect receives and destroys the essential gene product from the parasite (e.g. a detoxifying enzyme), or from a process involving a co-ordinated sequence of gene products (i.e. signal transduction leading to defence responses). Both mechanisms have been shown to be utilized by plants (reviewed by Briggs and Johal, 1994; Staskawicz et al., 1995). On the other hand, resistance is often defined on the basis of symptomology, assessed either on the basis of host productivity or cosmetic features of the crop. This would be disease resistance in the classical sense (i.e. lessening of the severity of symptoms). The pitting phenotype of Arabidupsis (described in section IIID) illustrates the dual nature of resistance. The phenotype is associated with effective containment of the parasite (colonization ceases within 48 h) but also with an expansive, apparently self-inflicted host cell necrosis which can severely stunt the seedling. In other words, the PN phenotype (see section IIID) is associated with strong resistance to the parasite, but, when the symptoms are severe, disease resistance overall appears to be weak.
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Like avirulence, a resistance has conventionally been defined genetically. The phenotype of resistance is determined relative to susceptibility, a host trait associated with unrestricted growth and reproduction of the parasite. Simple inheritance of resistance is common, so it is generally assumed that a disease resistance gene must be that which is correlated with the resistance phenotype. It logically follows that the cause of disease resistance can be revealed by determining the molecular basis of the segregating gene, as in the case of the Hml toxin reductase gene isolated from maize (Johal and Briggs, 1992). Several other investigations have revealed genes thought to be involved in the earliest steps of signal transduction (Staskawicz er al., 1995), but the mode of resistance in each of these examples has thus far remained elusive. Nevertheless, these genes are often referred to in research literature as resistance genes (or R-genes) in order to accommodate the gene-for-gene theory. Alternatively, one could argue that since resistance is the phenotype it actually refers to a whole process. It may be misleading to refer to something as “the resistance gene” if the phenotype in fact requires the co-ordinated action of several genes. What one observes is segregation of a single gene, but that gene would be unable to complete its function without the support of the other conserved genes. Any gene in the signal transduction pathway or network which is polymorphic within the species, could in fact be an R-gene as defined in the previous paragraph. The most common examples would be found in the least conserved steps of the pathway, presumably this would be in the earliest signalling events. Polymorphism in the latter steps may actually be very difficult to demonstrate because of the manner in which material has been chosen for experimentation. The evolution of signal transduction as a mechanism for disease resistance has yet to be debated. An important milestone of plant pathology has been achieved with the isolation of the first R-genes, and the stage is now set to bridge the gap between genetic and biochemical aspects of disease resistance.
D. A COPERNICAN PERSPECTIVE
The paradigm of parasitic symbiosis described above in essence revolves around or is strongly biased by an interest in the end result of a biochemical process, i.e. disease resistance. Obviously, this can be justified by the economic value of knowing how resistance actually functions on a molecular level to enable more sophisticated methods of improving crop species. A Copernican-like perspective of parasitic symbioses should be considered as well. It instead revolves around the earliest events of self- and non-selfrecognition between cells. The embryo is viewed in modern biology as the fundamental stage for understanding development of tissues, organs and whole organisms. By analogy, Wallin proposed in 1927 that the biological
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principle responsible €or the communication and organization of cells is fundamental to an understanding of how a parasitic relationship between two organisms is established and how the partner species evolve as a result. The downstream consequences of cellular recognition may be unpredictable without knowledge of earlier events. For example, the non-self-recognition which triggers the death of host cells may in some cases confer a resistance phenotype, but in other cases the dead cells may be exploited as a nutritional source by an invading pathogen. Plants appear to be resistant to most micro-organisms they encounter. If parasitism by a micro-organism is relatively uncommon in nature, then how compatibility has and continues to evolve is of central interest. For this reason, theory may be unrealistic if it is based on the assumption that compatibility is the starting-point for a metaphorical “arms race” of host-parasite evolution. Debate about the dominance of disease resistance remains unresolved. Semi-dominant and even recessive resistance is common (Hooker, 1967; Crute and Norwood, 1986; Roelfs, 1988; Kesseli et al., 1993; Hammond-Kosack and Jones, 1994; Holub et al., 1994,1996; Kolmer and Dyck, 1994; Lawrence et al., 1994) and should therefore stimulate questions about the molecular basis of dominance including whether susceptibility is also an active function. The pivotal role of cellular interaction in determining the outcome of a symbiotic association has been discussed since the beginning of this century (Sapp, 1994). The concept of cellular recognition playing a role in host defence was developed over a decade ago in anticipation of plant pathology being revolutionized by the techniques of molecular biology (Ellingboe, 1982; Keen, 1982; Callow, 1984), and attempts have been made recently to reformulate the concept in light of recent discoveries including a more substantive account of compatibility (see e.g. Dangl, 1992, 1995; Briggs and Johal, 1994). In reality, most researchers in plant pathology will exercise both the resistance-centred and recognition-centred perspectives. Unlike the astronomical models, the two perspectives of symbiosis are complementary. They are perhaps more akin to the metaphor of opposite three-dimensional impressions of a Necker cube, a two-dimensional line drawing of a cube (Dawkins, 1982).
111. SYMBIONTS FROM THE WILD A.
THE PHYTOBIONT: ARABIDOPSIS THALIANA
The useful qualities of Arabidopsis for genetic analyses are now widely known, but its most important advantage is the international, largely co-operative, research community. The history of Arabidopsis as a subject of classical genetics and the expansion of research into developmental, cellular and molecular biology has been documented in a recent book, edited
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by founders of the modern age of Arubidopsis research (Meyerowitz and Somerville, 1994). The rate of appearance of publications on Arubidopsis has increased enormously in the last few years, with at least one article now appearing in each issue of North American and Western European journals that publish fundamental plant research. An international electronic-mail network was established in 1990 serving as a forum for ideas and exchange of materials and information. The plant pathology community was introduced to Arubidopsis by a symposium at the 1991 meeting of the American Phytopathological Society (Davis and Hammerschmidt, 1993). The international Arubidopsis Pathology Network was organized by Jeff Dangl in 1991, and four spring workshops have since been held in Europe. There is now a growing awareness among Arubidopsis researchers that success by individuals is entirely dependent on co-operative effort (perhaps an example of cultural symbiosis). An internationally co-ordinated effort has enabled development of invaluable resources that will aid molecular genetic analyses of Arubidopsis. Several detailed genetic maps based on phenotypic and several types of molecular marker have been created (reviewed by Koornneef, 1994). The density of markers on these maps now allows the positioning of a new gene, or cloned sequence, within an average distance of 250 kbp of a known marker. Recombinant inbred lines (described below) have played an essential role as mapping populations in development of these maps. Genomic clone libraries have been constructed in a number of different vector systems. Most important of these have been the yeast artificial chromosome (YAC) libraries that carry large pieces of DNA (100-800 kb) allowing the physical DNA between two molecular map markers to be identified. Rapidly, the complete genome of Arubidopsis is being identified as a series of overlapping genomic clones, thereby aiding the analysis of any particular region in the genome. A further programme of genomic analysis involved the use of randomly sequenced cDNA. These sequences named expressed sequence tags (ESTs) will enable researchers efficiently to identify cDNA clones associated with genomic sequences or to search for particular motifs within polypeptide products that may be associated with gene functions under investigation. The final piece of the jigsaw is the ability to reinsert particular clones into the Arubidopsis genome to analyse their function. This step has been greatly simplified by a method of direct transformation based on vacuum infiltration of Agrobucterium tumefuciens into plant tissue (Chang et u f . , 1994), thus avoiding the need for tissue culture. A computer data base called AAtDB (an Arubidopsis thafiana data base), freely available on the Internet, has been established to co-ordinate the collection of the vast amount of data being generated. Also of great importance has been the establishment of international stock centres at the Ohio State University (USA) and Nottingham University (UK) for distributing seed and DNA materials to the research community.
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The ephemeral nature of Arabidopsis can be put to good use in the production of recombinant sets of inbred lines (Reiter et a f . ,1992). An inbred set is usually established from a randomly selected group of 100 or more F2 plants. A single F3 individual is grown from the seed of each F2 plant and self-pollinated. The cycle of single-seed descent to produce plants for the next generation is continued until at least the F8 generation. If rapid-cycling genotypes are used, this final generation can be produced in 18-24 months. By then, most genes are in a homozygous condition, and each of the inbred lines will have inherited a different set of genes due to recombination in a previous segregating generation. The resulting set of inbreds can then be used for purposes such as genetic mapping of loci, without the constraints imposed by segregating genes. The genotypes can also be maintained indefinitely. Two sets of inbreds were produced for general use by the research community (Reiter et a f . , 1992; Lister and Dean, 1993) from the crosses Ler-0 X Col-4 and Ws-1 x Ler-W100f. An extensive data base of molecular markers has been produced for each cross and released to the community in AAtDB. Ler-W100f also carries nine recessive, morphological markers on eight of the 10 chromosome arms. We have developed two inbred sets ourselves from the cross Col-0 x Nd-1 and Wei-1 x Ksk-1, and are continuing to produce additional inbred material which is being tailored to specific investigations of downy mildew and white blister.
B. THE BIOTROPHS: PERONOSPORA PARASITICA AND ALBUGO CANDIDA
The two natural parasites of Arabidopsis are both eukaryotes belonging to the phylum Oomycota in the kingdom Protoctista (Margulis and Schwartz, 1988). Although the oomycetes have a filamentous growth habit like that of true fungi (ascomycetes, basidiomycetes and deuteromycetes), they differ in many aspects of life cycle, cellular and molecular structure, and metabolism (reviewed by Michelmore el a f . , 1988; Lucas et a f . , 1995). Members of the phylum are diploid for much of their life cycle, and combine sex and long-term survival by producing thick-walled, lipid-rich sexual propagules called oospores. The phylum name refers to these egg-like spores. Most species reproduce asexually by means of biflagellate zoospores. They in fact appear to be more closely related to yellow-green algae than to fungi. The oomycetes exhibit a wide spectrum of trophic adaptations. There are numerous saprophytic species in terrestrial and aquatic environments and voracious necrotrophs causing damping-off of seedlings (e.g. Pythiurn spp.). The genus Aphanornyces includes tenacious root-rotting species and pathogens of freshwater and marine fish. Phytophthora infestans, known for having influenced human history as the microbial cause of the Irish potato famine in the mid-l800s, is hemibiotrophic while there are strictly biotrophic species such as Peronospora parasitica and Albugo candida.
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Fig. 1. Oospores of Peronospora parasitica and AIbugo candida in a rosette leaf of Arabidopsis thafiana. The oospores of A . candida (ca. 25 pm diameter) are ornamented and contain darker pigmentation than the transparent, smooth-walled oospores of P. parasitica (bottom left). As the leaf decomposes, the oospores are released into the soil. They germinate when in contact with the root of a host seedling, hyphae grow up the hypocotyl and the biotroph begins to reproduce asexually from cotyledon surfaces. A seedling infected with a P. parasitica oospore via the root is shown (bottom right) in a characteristic aetiolated posture shedding conidiosporangia above the canopy of neighbouring seedlings.
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Fig. 2. Birth of the Peronospora parasiticu isolate Emoy2: (A) the whole infected seedling of the accession Columbia, seven days after germination; (B) an oospore (arrow) of the biotroph that infected via the root; (C) at least two strands of hypha (arrows) grew up the hypocotyl producing bulbous haustoria at regular intervals; (D) the biotroph colonized both cotyledons producing extensive branched hyphae (left arrow), a profusion of haustoria and then re-emerged by producing a single sporangiophore (top right arrow) to release numerous, asexual conidiosporangia. Asexual inoculum of this isolate is incompatible with cotyledons of the original host, Columbia; therefore the culture must be maintained on other compatible accessions.
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P. parasifica and A. candida are in part defined physiologically as specialist biotrophs of the Cruciferae. Pathotypes within each oomycete species are usually only capable of completing their life cycle in genotypes of a single host genus. The life cycles in Arabidopsis have been documented by Koch and Slusarenko (1990) and Holub et al. (1996), and is further illustrated in Figs 1 and 2. The two parasites often occur as fellow travellers, infecting tissue of the same host plant and reproducing asexually and sexually in intimate contact (Fig. 1). The life cycles are initiated by oospores. Large numbers of these spores are produced in rosette leaves of Arabidopsis. When the host tissue disintegrates, the inoculum is released into the soil. The oospores will germinate when in contact with roots of a host seedling, hyphae quickly grow upwards and proliferate in the cotyledons of a compatible host (Fig. 2). In the case of P. parasitica, the biotroph emerges to reproduce asexually within 7-10 days from germination of the host seed. We routinely utilize this process of seedling infection from oospores to produce new isolates of P. parasifica for our collection. Once an isolate has been established, subsequent experimentation can be conducted using the asexual inoculum. The key macroscopic feature that distinguishes between the two oomycetes is their means of asexual reproduction. P. parasifica produces tree-like structures called “sporangiophores” (Fig. 2) which bear numerous conidiosporangia. These structures emerge through stomata and are produced profusely in a compatible host in a manner that characterizes the disease it causes, downy mildew. A . candida instead produces stout, barrel-shaped sporangiophores which bear chains of deciduous zoosporangia (Holub et al., 1996). These are produced in subepidermal masses that eventually appear as white blisters. The different prefixes for the two types of sporangia indicate another important difference between the species: sporangia of A . candida release infective zoospores when germinated in water, whereas P. parasitica has lost this ability so the sporangia germinate and infect the host directly. Germplasm of both biotrophs is abundant throughout many regions of Europe (Holub ef al., 1994,1996). In England and southern Scotland, nearly half of the Arabidopsis populations contain plants infected with either or both of the biotrophs. This suggests that parasitism could potentially be a significant evolutionary feature of natural Arabidopsis populations.
C. THREE Rs OF SYMBIOSIS
The task of describing phenotypic variation, the challenge of bridging the conceptual gap between the genetic and functional aspects of symbiosis, and the precision demanded by modern research have forced us to think clearly about basic principles: recognition, response and reproduction. Recognition
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and response by both the host and a parasite encompass the early and middle stages of communication and development leading to the final stage of reproduction by either or both organisms. The early and middle stages will obviously determine the capability that the host and parasite will have to reproduce. It should be possible to assess each of these principles objectively, and to keep the separate but related questions open for consideration: has non-self-recognition occurred, what is the host response, how does the parasite respond in turn, and is the host response effective at hindering the parasite? These questions are universal across the spectrum of symbioses. Some of these fundamentals have been developed more than others in genetical analyses of interactions between Arabidopsis and the two oomycetes. All the experimentation we describe here has been focused on interactions within cotyledons. We define recognition in an abstract manner as “recognition specificities” (described below). The host component of a specificity can be examined genetically; whereas the corresponding biotroph component will be entirely speculative until a procedure for genetic analyses has been developed. The subsequent responses by each organism to cellular recognition will determine the qualitative nature of a phenotype. The host response can be examined indirectly by the type of necrotic lesions that are visible macroscopically (see section IIID); however, the biotroph’s response is much less tangible for investigation. A more detailed cytological and histochemical examination of host and biotroph responses has been impractical given the large number of inoculated seedlings used for genetic analyses. On the other hand, asexual reproduction by the biotroph is clearly visible macroscopically and provides a useful measure of biotroph development and the degree of host resistance. Since we have only examined interactions in any detail during the seedling stage, host reproduction has so far not been assessed. Recognition specificity focuses attention on the earliest determinant of an interaction. It is drawn from the more encompassing concept of the “aegricorpus” proposed by Loegering in 1966, which acknowledges the inseparable role of both host and parasite in determining the observed phenotype of an interaction. Recognition specificities imply two classes: compatibility, in which the biotroph develops and reproduces without significant detection by the host; and incompatibility, in which detection is possible and usually triggers some process that impedes further development of the biotroph. A specificity is by nature imprecise compared with the individual genetic components of an interaction (host resistance genes and parasite avirulence genes) that can in practice be isolated for molecular investigation. Although a specificity can often be associated genetically with a single locus, it is usually unknown how many genes at that locus are required for the observed phenotype or whether genetic recombination within a single gene can alter the specificity. However, we are inevitably bound at the early stage of an investigation to the task of describing the
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phenotype of a host-parasite interaction. In doing so, we have frequently encountered examples in our own research which suggest that the interaction phenotype is not necessarily reliable for predicting the host genotype (see examples in section IVA). For this reason alone, recognition specificity is an essential concept for considering those events, perhaps the most important ones, that do not fit conveniently into a genetic model. Reciprocal examples of recognition specificities have been observed in downy mildew of Arabidopsis where one isolate of the biotroph is compatible but a second isolate is incompatible in the same host accession; and in a second accession, the compatibility is reversed for the two biotroph isolates (Fig. 3) (Holub et al., 1994). The specificity of interactions begins to appear increasingly more complex as different patterns of interaction are identified among numerous combinations of biotroph isolates and Arabidopsis accessions (Table I). Incompatibility has thus far appeared to be uncommon among interactions between Arabidopsis and A . candida (Holub er al., 1996). From genetic analyses of recognition specificities, we have postulated the existence of the corresponding host and biotroph genetic components of an interaction: host genes at RPP (recognition of P. parasitica) and R A C (recognition of A . candida) loci and biotroph genes ATR ( A . thaliana recognizable) loci. For recognition in this context, we are assuming that natural genetic variability will most likely occur as functional polymorphism in the early signalling events, not necessarily the receptor. Later regulatory events and downstream plant defence responses are assumed at this stage to be genetically conserved within the species. Given the increasing efficiency of gene isolation, the rapid pace of genome sequencing and the level of mutational analyses possible in Arabidopsis, the species is an ideal system for eventually testing the validity of this assumption. Once the primary defence-related host responses have been determined, a survey of natural variation in such responses could prove to be illuminating. In the meantime, there are practical constraints in investigating a biotroph, so we have concentrated genetic analyses first on the natural variation of RPP and R A C genes in Arabidopsis. The existence of ATR genes in both oomycetes is entirely speculative, but highly probable given what is known about plant and biotroph genetics of lettuce downy mildew (Michelmore et al., 1988; Lucas et al., 1995).
D. THE PHENOTYPES OF INTERACTIONS: CONSEQUENCES OF RECOGNITION
In addition to complex patterns of recognition specificity, we have observed phenotypic variation among incompatible interactions (Holub et al., 1994, 1996) (Fig. 3). A nomenclature of single-letter abbreviations has been developed. Host cell responses range from minute flecks (F) to expansive
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Fig. 3 . Interaction phenotypes of Arabidopsis thaliana accessions Wassilewskija (A, C, E) and Landsberg erecta (B, D, F) inoculated with Peronospora parasitica isolates Emwal ( A . B), Cala2 ( C , D) and Ernoy2 (E, F). The full spectrum of phenotypes (Holub et al., 1994) is illustrated including heavy sporulation in a compatible host (A, D). discrete flecking necrosis with low sporulation (B) (a single sporophore. lower left edge), flecking necrosis with no sporulation (F), expansive pitting necrosis with chlorotic halo and no sporulation (E), and intermediate necrotic cavities with no sporulation ( C ) .
TABLE I Recognition specificities (compatible (C) and incompatible (I)) of interactions between Arabidopsis accessions and isolates of Peronospora parasitica and Albugo candida Arabidopsis Accession Biotroph isolatea Aswal Ahco2 Cala2 Can& Edcol Edwal Emcol Emco2 EmcoS Emoy2 EmwaI Em wa2 GacoI Gowal Hiksl Hind2 Hind4 Madil Maks9 Noksl Wacol Wandl Wela3 Aceml Acksl
Nd-1
Col-5
Ler-1
Ws-3
I I C C I I I I I I C I I I I C
C C I I C I C C C I I C C C I I
I I
C I I I C C C I C I C C I C
C I
I I I I I I I
I I I I I C I
I I I C
Mop-1 Tsu-1 Oy-1 C C C C ?
C C I I C C I C I C C
? C C C I ?
C I I I C C
I I
1 1
C
I
c c
c c
? C ?
I I I
c
c
C C
I
Wei-1 C I I I I C C C C C C C I C I C
Ksk-1 Ema-1 Cnt-1
I I I C
?
?
I
I
c c
c c
? ?
C
C C I C
I I I I I
?
?
I C
I I I
I I I
?
?
?
?
C I I I I I I ?
?
?
Di-19
Rld-2
C24
I I I I I C I I I I I I C I I I
I I
I I
I
I
I I I I I I I I I I I I I
I I I I I I I
C C C I I
All biotroph isolates were collected from Arabidopsis. The last two are A . candida, and all others are P. parasitica.
I I I I I I I I I I I I
P
m
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243
pits (P), with an intermediate class described as cavities (C). These abbreviations are applicable to host responses following inoculations with either P. parasitica or A . candida. Asexual reproduction by P. parasitica varies in the amount of sporangiophores produced per cotyledon: heavy (H;>20), medium (M;10-20), low (L;
IV. MOLECULAR GENETICS OF NATURAL VARIATION Recombinant sets of inbred lines (see section IIIC) have revolutionized the genetic analyses of RPP and RAC loci in Arabidopsis. The F2 generation has been irreplaceable for developing hypotheses about the number of genes segregating in a population, the dominance of a phenotype ascribed to a single gene, and for serving as an inexhaustible supply of recombinants for fine-scale genetic mapping of RPP loci. F3 families were of critical importance during the early stages of mapping the first loci (Parker er al., 1993; Tor et al., 1994). At the same time, however, we began to appreciate the tremendous value of inbred material for rapidly mapping new loci including ones associated with a very weak or a recessive phenotype and for sorting out segregation of two or three genes in the same cross; the inbreds also began to reveal non-allelic effects between genes (Holub et al., 1994). Given the tremendous data base already available for the Ler X Col and Ws X LerWlOOf inbred sets, not only could we postulate the existence of an RPP locus, but also there was actual proof that defined its position within a narrow interval of flanking markers. As shown throughout this section, we have since refined the use of existing inbred material and have begun tailoring the development of new inbreds.
h)
P
P
TABLE I1 RPP loci mapped in four accessions of Arabidopsis thaliana associated with isolate-specific recognition of Peronospora parasitica
A. thaliana accession Col-0
Nd-0
P. parasitica isolate Hiksl Weld Hind4 Weld Hind2 Wandl Cala2 E m wal Emoy2 Hind4 Can& AhcoO Ahco2 Emoy2 Hiksl Wac05 Maks9 Aswal EmcoS Emcol Emco2 Emwd
IPa FR FR
FN FR FN FN FR FDL FDL
FN FN DM DM PN PN PN
FN FN FN FN FN
Iw
Flanking markers
Inbred cross'
B-7 B-6 C-19 c-20 C-Hind2 C-Wand1 H-2 H-4 H-Emoy2 H-18 ?-Can&
m21kn315 Near RPP7 Above GPAl Above GPAl Above GPAl Above GPAl AG-g3883 111226-AG m226-AG rn226-g3883 Not determined
1 2 1 1 1 1 1, 2 1, 3 1, 3
B-25 B-Ahc02 F-1 F-Hiksl F-26 F-13 F-16 F-17 F-Emcol F-Erne02 F-E m wa2
Near RPP7 Near RPP7 GLl-RPP13 cosegr. wIRPP1 cosegr. wlRPPl R PPl-m249 R PPl-m249 R PPl-m249 RPP1-m249 R PPl-m249 RPPI-m249
1 1 1 1 1 1 1 1 1 1 1
M R C-RP P ~
2 1
Ler-0
ws-0
Gocol Madil Edcol
FN FN FN
F-GOCO~ F-Madil F-Edcol
RPPI-m249 RPPI-m249 Below m249
Hiksl AhcoO Ernwal, Ernoy2 Noksl EmcoS, Emoy2 MaksY, Madil Aswal Gowal EdcoS AhcoO
FN
B- 7
FL FL
B-AhcoO H-4 H-5 J-8 J-21 J-22 J-23 J-24 J-AhcoO
m213-m315 Near RPP7 m226g3883 cosegr. w/Cl8a cosegr. wlagp6 nga129-m435 mi2-nga129 agp6-mi2 agp6mi2 RPP8-m435
3 2 3 2, 3 2, 3 2, 3 2, 3 2, 3 2, 3 2
F-I F-I0 F-Emco2 H-I2 H-Ahc02
GLI-RPP13 cosegr. wlRPPl RPPI-m249 m226-g3883 Near RPPl2
3 3 3 3 3
Emoy2, Hiksl, Maks9 Cala2 Ernco2 Weld Ahco2
FN FN FL FL FN
FN FL PN CN CL FN
FN
1 1 1
'Interaction phenotype (see Wolub et a l . , 1994). Host necrotic response: F, minute flecks; P, expansive pits; C, intermediate cavities. Parasite sporangiophore production: H, heavy (>20 per cotyledon); M, medium (5-20); L, low (4 per cotyledon); R, rare sporangiophore; N, none; D, delayed (5-7 days after inoculation). bRecognition specificity identified by location to region on one of the 10 chromosome arms @lajor _Recognition Complex identified by a letter A-J) and locus Becognition of Eeronospora parasitica identified by a number). Unnumbered RPP loci (identified by isolate name) have not been designated, but preliminary phenotypic evidence suggests that they are different specificities. 'Inbred crosses used for mapping recognition specificities: (1) F9 Col-0 x Nd-1, (2) Fg Ler-0 X col-4, (3) F8 Ws-1 X Ler-W100f. N P
VI
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The data are presented in a pictorial format which indicates recognition specificities that were uniform for either parental phenotype as solid grey or black blocks, segregating specificities as hatched blocks, and not determined as white blocks (Fig. 4). Abbreviations for the parental phenotype are shown in the rows for each parent; only non-parental phenotypes are shown for the inbred lines. Most of these data are unpublished; publications at various stages of preparation will present this information along with the essential data for molecular markers that confirm the position of RPP loci. A Ph.D. student, Canan Can, produced many of the new isolates shown in the figures as well as the data using the Col x Nd inbred set as part of her doctoral research. She was also instrumental in discovering the apparent anomaly described in section IVD.
A.
BUILDING MODELS: PREDICTING THE HOST GENOTYPE
The birth of Emoy2 (Fig. 2) changed the course of our research. It was the first of our standard isolates to be germinated from an oospore; every isolate since has been generated in the same manner as a means of refinement (bottle-necking). Most importantly, Emoy2 introduced the possibility that several regions of the Arubidopsis genome are involved in parasite recognition. A description of how this isolate has been used for research will in effect provide a synopsis of a genetic approach to investigating Arubidopsis downy mildew. At first, Emoy2 did not appear to be an informative isolate because the most popular laboratory accessions Landsberg erectu (Ler), Columbia (Col) , Wassilewskija (Ws) and Niederzenz (Nd) were all incompatible hosts. There was no phenotypic segregation observed in the Ler X Col inbred set, two independent genes were predicted from segregation in the Col x Nd inbred set, and three independent genes were predicted from segregation in the Ws X Ler inbred set (one in Ws and two in Ler). Compatible inbreds in the latter two sets were chosen as providing the most reliable evidence for mapping RPP loci. These inbreds would in theory carry compatibility alleles at all RPP loci, so narrow intervals of molecular markers that were non-random (i.e. they had inherited DNA from the same parent) could be predicted as being the regions that were most likely segregating for RPP genes in the cross. From the Ws x Ler cross, three regions were postulated using the Emoy2-compatible inbreds: one region on chromosome three that was mostly Ler DNA and two on chromosomes 4 and 5 that were mostly Ws DNA (Holub et ul., 1994). However, fine-scale mapping of RPP loci would require the ability to predict genotypes of incompatible inbreds. Emoy2 was clearly unsuitable for the task. On the other hand, this isolate was fascinating because of its complexity. Inbreds from the two segregating sets exhibited
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the full spectrum of interaction phenotypes (EH, DL, FR, FN and PN). The independent segregation of RPP loci suggested that at least three of the ten chromosome arms of Arabidopsis carry genes involved in recognition of P. parasitica. In addition, as a consequence of phenotypic variation, there were opportunities to investigate non-allelic effects between RPP genes. A decision was made to begin developing a library of P. parasitica isolates from natural oospore populations, rather than develop another host crosstailored for Emoy2. We expected to find isolates more suitable on one of three host inbred sets already available. In particular, we were seeking three new isolates of simple pathotype that could separately map each of the RPP loci that were suggested from the Ws X Ler inbred set following inoculation with Emoy2. Quite fortuitously, the next two isolates could each be used to map a single RPP locus in this inbred set (Holub et. al., 1994). Cala2 mapped a single locus (RPZO) to chromosome 3 that was associated with the CN phenotype inherited from Ws. Figure 4 illustrates the position of this locus in the interval between the glabrous locus GLI and the molecular marker m249. Similarly, Emwal mapped a locus (RPP4) to chromosome 4 associated with the FN phenotype inherited from Ler. It lies in the interval between mS80 and the waxless locus CER2. A segregation pattern began to emerge suggesting that the R P P loci mapped using Cala2 and Emwal were closely linked to, or perhaps even matched, two of the loci predicted using Emoy2. In addition, the third locus that was predicted using Emoy2 was closely linked to the transparent testa locus TT3 on chromosome 5 (Holub ef al., 1994). The 25 Emoy2-compatible inbreds were all compatible with Emwal and Cala2, and they all carried the Ws allele at TT3 (Fig. 4). There were 46 inbreds that exhibited a PN phenotype following inoculation with Emoy2; these were all incompatible (CN) with Cala2. From this, we ascribed the PN incompatibility of Ws and Emoy2; to the RPPl locus on chromosome 3 (previously mapped in Col x Nd; Tor et al., 1994) very near RPPZO identified with Cula2. Similarly, the two RPP loci identified in Ler using Emoy2 were tentatively mapped to chromosome 4 (linked to RPP4 identified by Emwal) and chromosome 5 (named RPP8; linked with TT3). A fourth P. parasitica isolate was still required that was compatible with Ws and could be used to map the RPP8 locus on chromosome 5 . This was eventually achieved using a strategy designed to selectively bait for the desired pathotype (Fig. 5 ) . The first place to search for such an isolate was the oospore population from East Malling, which was the source for Emoy2 and Emwal. The population appeared to contain a diversity of pathotypes. Ws-compatible isolates were common in this oospore population and therefore would not be a limiting factor. For other genetic analyses, Col and Ler appeared to share alleles at RPP4 associated with recognition of both Emwal and Emoy2, so Col was chosen as a baiting host that should have favoured selection of biotroph genotypes that lacked the A T R gene(s)
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corresponding to RPP4. Emcol was the first isolate selected which was compatible with Col and Ws. However, it was still recognized by two genes in Ler at RPP4 and RPPB; several additional isolates were tested but they were all similar pathotypes to Emcol. A mixture of asexual inoculum from several hundred Col-compatible isolates was then used to produce a subpopulation of oospores (i.e. enriched for a mixture of Col and Ws compatible pathotypes). A new baiting host, CL175, was chosen from the F8 Ler X Col inbred set which carried the Ler allele at RPP4 and the Col allele at RPP8. The first isolate selected was Emco.5 which proved to be the isolate we were seeking. It was compatible with Ws and Col and mapped a single gene in Ler to chromosome 5 using both the Ler x Col and Ws x Ler inbred sets. In fact, a co-segregating marker ugp6 was identified using the former inbred set. Among the 25 Emoy2-compatible inbreds from the W S XLer cross, all of them were compatible with Emco.5. This was preliminary evidence to suggest that Emoy2 and Emco.5 were recognized by a gene (or genes) in Ler at the same locus RPP8. B . NONALLELISM: JUGGLING WITH APPLES AND ORANGES
The puzzle of interpreting phenotypes of the Ws X Ler inbreds following inoculation with Emoy2 was not entirely solved. The Emoy2-compatible inbreds had been used for postulating R P P loci, and the PN class of inbreds further supported a position for the R P P l locus on chromosome 3. However, the genotype of three phenotypic classes had not been interpreted. One class exhibited the parental FN phenotype of Ler, and the other two classes exhibited non-parental phenotypes FL and CN (see Fig. 4). Genotype predictions of these three phenotypic classes required assumptions about non-allelic effects (cumulative and/or epistatic) between genes at R P P l , R P P l and RPP8. The simplest model to emerge is illustrated in Fig. 4: the FL class correlated with Culu2 and Emco.5 compatibility and Emwul incompatibility, so this phenotype was ascribed to the Ler allele at RPP4 (i.e. compatibility alleles must be present at R P P l and RPP8 from Ler and Ws, respectively); the FN class correlated well with Culu2 compatibility and Emco.5 incompatibility, so this phenotype was predicted to be a combination of Ler incompatibility at K'P8 and Ler compatibility
Fig. 4. RPP loci identified in the F8 Wsl x Ler-W100F inbred set (Reiter et a l . , 1992) following inoculations with four isolates of Peronosporu parasitica. Predicted genotype: R, functional parasite recognition; S, silent allele (permitting sporulation). (Black) female parent Wsl; (light grey) male parent Ler-WlOOf, (hatched) heterozygous, (white) not determined. Host cell necrosis: P, pitting; C, cavity; F, flecking. Parasite sporulation: H, heavy; L, low; N , none, H*, phenotype in the absence of the other two genes that recognize Ernoy2.
249
SYMBIOLOGY OF MOUSE-EAR CRESS AND OOMYCETES
Chromosome 3 Predicted No. genotype inbreds
RPPlO RPPI GLl C a b 2 Emoy2 m249
Chromosome 4
RPP4 m580 Emwal Emoy2 CER2
Chromosome 5 7T3
RPP8 Emco5 Emoy2
250
E. B. HOLUB and J. L. BEYNON
Populatioo of Perowspom wspores produced naturally in wild East Malling Arabidopsis
Fig. 5. Dangling for a big catch: the Peronospora parasitica isolate EmcoS.
at RPPZ; and the CN class correlated well with Culu2 and ErncoS incompatibility, so this phenotype was predicted to be the combined effect of Ler incompatibility at RPP8 and Ws incompatibility at RPPZ. A CN phenotype in effect was postulated as a cumulative effect of genes that separately would confer PN and FN phenotypes. In summary, the model was generated in three steps; compatible inbreds were used to determine the number and most likely locations for segregating RPP genes; an attempt was made to ascribe the PN, FL and FN phenotypes each to a single locus using flanking molecular markers as a guide (we assumed that each of these phenotypes could be explained by incompatibility at only one RPP locus); and by default we ascribed the CN phenotype to the combined effect of incompatibility at RPPZ (PN) and RPP8 (FN). It would be impossible to build such a model without a recombinant set of inbred Arabidopsis; three different phenotypes associated with each of the RPP loci also helped to simplify steps towards predicting genotypes of the inbreds. Building this genetic model required a thorough data base of molecular markers distributed across the entire genome as well as phenotypic data from the three simple-pathotype isolates. Close flanking markers were used to confirm the predicted genotypes, but markers elsewhere were necessary for excluding other possible locations. Arguing where a locus does not fit in the genome is as important as deciding where it does fit. The assumptions about non-allelic effects to phenotypic expression do not appear to apply universally. For the previous example, we assumed that the PN and FN phenotypes were cumulative when combined, and that each entirely masked the FL phenotype. For the Col X Nd inbred set, the isolates
SYMBIOLOGY OF MOUSE-EAR CRESS AND OOMYCETES Chromosome 1
No. inbreds
RPP7 Hiksl
Ahcol
Chromosome 3
GLl
Hiksl
RPPl RPP13 Emoy2 Maks9
25 1
Chromosome 4 RPP4 Emoys2 Emwal
Fig. 6. R P P loci identified in the F9 ColO x Nd inbred set following inoculations with five isolates of Peronosporu parusitica. (Light grey) female parent ColO, (black) male parent Ndl, (white) not determined. Host cell necrosis: P, pitting; F, flecking. Parasite sporulation: H, heavy; L, low; N, none, H * , phenotype in the absence of the other gene that recognizes Hiksl or Emoy2.
Emoy2 and Hiksl were incompatible with both accessions. The isolates shared recognition by an allele in Nd at locus RPPl on chromosome 3 associated with a PN phenotype (Fig. 6) (Holub et al., 1994). The second locus RPP4 on chromosome 4 for Emoy2-incompatibility with Col was associated with an FL phenotype. In this case, the PN inherited from Nd appeared to mask the FL phenotype from Col. The second locus RPP7 on chromosome 1 for HiksZ-incompatibility with Col was associated with an F N phenotype. In this case, the PN phenotype inherited from Nd appears to mask the FN phenotype from Col; this appears to contrast with the additive interaction between PN and FN phenotypes observed in the Ws x Ler inbred
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set. The isolates Ahcol and EmwaZ were used to predict the Col alleles on chromosomes 1 and 4, respectively, involved with recognition of HiksZ and Emoy2. Flanking molecular markers have also been used for additional confirmation (Can, C. and Beynon, J . , unpublished). The last seven inbred genotypes listed in Fig. 6 presented some interesting exceptions in which one isolate (either Emoy2 or Hiksl) caused the expression of a PN phenotype but the other isolate caused a definite FN phenotype. In some cases, recombination may have occurred within the RPPl locus which created different specificities for recognizing each isolate (e.g. inbreds B and C). However, this is less plausible for the other exceptional inbreds. These examples will be challenging to explain, but they illustrate the tentative nature of predicting genotype from the interaction phenotype. Two examples of dosage-dependent effects have been observed using inbred sets. The isolate AhcoO is compatible with Col and incompatible (FN) with Ler. When the Ler X Col inbreds were inoculated with this isolate, the non-parental FL phenotype emerged as a major class (Fig. 7). Using the AhcoO-compatible inbreds, R PP loci were postulated on chromosomes 1 and 5. These were very near to previously named loci RPP7 (identified in Col and in Ler with Hiksl) and RPP8 (identified in Ler with EmcoS), respectively. The FL inbred class appeared to divide into two classes, one that had inherited Ler incompatibility on chromosome 1 and the other that had inherited Ler incompatibility on chromosome 5. The F N phenotype appeared to be the combined effect of incompatibility at both loci. In the second example, three RPP loci are postulated (Table 111). The isolate Wac05 is compatible with Col and incompatible (PN) with Nd. When the Col x Nd inbreds were inoculated with this isolate, nearly half of the inbreds exhibited the PN phenotype. The other half exhibited different amounts of biotroph reproduction (H, M, L). A three, independent gene model would account for the non-parental phenotype observed, including two loci associated with moderate sporulation. Incompatibility at each of these loci would confer an FL phenotype. The PN phenotype cosegregated entirely with the RPPZ locus on chromosome 3 (identified in Nd with Emoy2); and appeared to mask the M and FL phenotypes. The examples provided in this section illustrate the complexity of phenotypic variation in the symbiosis between Arabidopsis and P. parasitica, but they also demonstrate that genetic models can be generated from the
Fig. 7. Additive dosage effect of genes at two R P P loci each associated with a low sporulation phenotype. Loci identified in the F, LerO X Co14 inbred set (Lister and Dean, 1993) following inoculation with Peronosporu parasitica isolate AhcoO. (Black) LerO, (light grey) Co14, (white) not determined. Parasite sporulation: H, heavy; L, low; N, none.
SYMBIOLOGY OF MOUSE-EAR CRESS AND OOMYCETES Chromosome 5
Chromosome I Predicted genotype
No. inbreds
g4026
RPP B-AhcoO
253
m315
RPP8 Ernco.5
RPP J-AhcoO
111435
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TABLE 111 Proposed three-gene model for explaining phenotypic segregation of F9 Col-0 x Nd-I inbreds of Arabidopsis thaliana following inoculation with Peronospora parasitica isolate Waco5. (2= 1.56; p = 0.67)
No. inbreds Interaction phenotypea
Predicted genotype(slb
I
Predicted
Observed
50
44
12.5
15
PN
AA, AA, AA, AA,
FL
aa, B B ,
M
aa, B B , cc aa, b b , CC
I
25
27
H
aa, b b , cc
}
12.5
14
B B , CC B B , cc b b , CC b b , cc
cc 1
aHost necrotic response: F, minute flecks; P, expansive pits. Parasite sporangiophore production: H, heavy (>20 per cotyledon); M, medium (5-20); L, low (<5 per cotyledon); N, none (see Holub et al., 1994). ’Upper-case letters refer to alleles from the incompatible parent Col-0; lower-case letters refer to the compatible Nd-1 alleles. A is associated with the PN phenotype; B and C are associated with the FL phenotype when combined and each with the M phenotype when separate. The phenotype of A masks the other phenotypes.
modest definition of phenotypic classes and a few simple rules for predicting genotypes. Furthermore, the non-allelic effects observed will be important to an understanding of cellular recognition and plant defence. We have intentionally avoided the term “epistasis” to describe the apparent masking of one phenotype by another. At such a gross level of phenotypic characterization, we were unable to conclude that the different component phenotypes (e.g. PN and FL) represent genes at different steps in the same biochemical process. It is still possible that analyses as basic as detailed microscopic examination will reveal cumulative effects as being more common than epistasis between any two incompatible phenotypes. C . MAJOR COMPLEXES OF RECOGNITION GENES: HOW BIG IS A CLUSTER?
Plant genes involved in the ability to recognize parasites provide a unique opportunity to investigate fundamental questions about how plant genome organization and evolution affect gene function. There are several classic examples in which a crop species contains extensive polymorphism in clusters
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of recognition loci or as an allelic series at a single locus as a means of coping with a genetically adaptable parasite (reviewed by Roelfs, 1988; Pryor and Ellis, 1993; Lucas et al., 1995). Models have been proposed for explaining how different modes of genetic recombination and rearrangement can give rise to genetic variability in non-self-recognition specificity, and how the generated polymorphism is maintained if a population has been allowed to evolve naturally (Hadwiger, 1988; Bennetzen and Hulbert, 1992: Lawrence et al., 1994). Wild pathosystems offer valuable opportunities for examining these evolutionary processes, unlike many crop systems in which natural selection has been disrupted by decades or centuries of human-directed selection. The organization of RPP loci in Arabidopsis is being revealed quite simply by adapting the library of P. parasitica isolates for further genetical analyses of recombinant inbred sets as described in the previous section. For example, we have continued to select isolates compatible with both Col and Ws to use for mapping RPP loci in the Ler x Col and Ws x Ler inbred sets. Many of these isolates identify two major loci (one on each of chromosomes 1 and 5), but we often find an isolate that maps a single locus. As in the case of Emco5 (see Fig. 5 ) , we have recently been choosing baiting hosts that will improve the chance of selecting simple pathotypes. Five such isolates map RPP loci between the transparent testa locus tt3 and molecular marker m435 (see Table 11). These loci span a region of about 15 cM, much greater in scale than a classical gene cluster such as R p l in maize of less than 1cM (Bennetzen and Hulbert, 1992). We are attempting to resolve what may be a much tighter cluster (1-5 cM) of RPP loci in Arabidopsis near RPP13 on chromosome 3. A majority of Col-compatible and Nd-incompatible isolates of P. parasitica have been used to inoculate the Col x Nd inbred set and most identify RPP loci on chromosome 3 (Fig. 8). A few inbreds were found which appear to separate RPP13 from one and probably two other loci ( R P P l 6 and R P P l 7 ) . F3 families are now being developed as a preliminary step towards identifying additional recombinants in this region. The families shown in Fig. 8 were produced from F2seedlings inoculated with Maks9 and subsequently selected as being recombinant between the glabrous locus gll and RPP13 (either FN and glabrous, or EH and glabrate). These families support the idea that several recognition specificities lie within the interval; some novel specificities may also have been generated. However, the actual number and order of loci are difficult to interpret from phenotypes of a segregating generation. Inbred lines are now being produced from the F3 families to create homozygous material that will be more reliable for predicting genotypes. Over 400 additional inbreds are being produced from F2 seedlings selected as gll-RPP recombinants using Emco.5, Aswal or Gowa2 as well as more selected using Maks9. Given the interesting patterns of recognition specificity already generated in the F3 families, the investment of time required to produce more inbreds is certainly worthwhile
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A summary of R P P loci that have been identified in four accessions from the three inbred sets is provided in Table 11. Loci have been identified on all five chromosomes of Arabidopsis, and four regions of about 15 cM have been defined which each contain at least three loci (Fig. 9). Although loci themselves are currently the primary targets for molecular genetic investigations, the much larger regions have increasingly become the focus of discussion and speculation. For this reason, it has been helpful to name them as M R C (major recognition complex) regions that are further distinguished with a letter to indicate location on one of the 10 chromosome arms (e.g. M R C - B refers to the region on the bottom arm of chromosome 1). Assignment of the growing number of named R P P loci to M R C regions is also of practical necessity to more readily identify linkage groups (e.g. MRC-RPP: F-1, F-13, F-16, F-17) (see Table 11). The M R C regions on the bottom arms of chromosomes 3 and 5 ( M R C - F and MRC-J) are of particular interest for investigating the effect of genome organization on gene function. MRC-F contains R P P loci, mapped using incompatibility of Nd and Ws, that are associated with the full spectrum of interaction phenotypes (see Table 11). In contrast, the phenotypes associated with loci in MRC-J show no obvious variation in host response, but they exhibit a range of dominance: R P P 8 is associated with complete dominance, RPP22 is associated with complete recessiveness, and the other loci are associated with semi-dominance (not shown). As described in section IVB, both regions are of interest because of non-allelism such as cumulative effects between with the PN ( R P P I ) and FN ( R P P 8 ) phenotypes. The M R C regions have been defined on the basis of mapping R P P loci; however, the same regions appear to be involved in other phenotypic attributes related to parasitic symbiosis. Loci associated with recognition of viruses have been mapped to M R C - B , M R C - H and MRC-J (reviewed by Kunkel, 1996). Loci for recognition of A. candida (RAC3) (Borhan, H. and Beynon, J., unpublished) and the bacterium Pseudomonas syringae ( R P S 4 ) have also been mapped to the MRC-J region (Kunkel, 1996). Loci of mutations that cause deficiency in phytoalexin production or else produce necrotic lesions that mimic a defence-related response have been mapped to MRC-F and M R C - H (Greenberg and Ausubel, 1993; Dietrich etal., 1994; Glazebrook and Ausubel, 1994; Greenberg et a f . ,1994). Several of these mutants are discussed further in section V with regard to altered phenotype in response to P. parasitica.
Fig. 8. Recombination in a region on chromosome three (MRC-F) containing a t least four RPP loci. Loci identified in F9 and F3 ColO x N d l . (Light grey) female parent ColO, (black) male parent N d l , (hatched) heterozygous, (white) not determined. Parasite sporulation: H, heavy; L, low; N, none; H*, phenotype in the absence of a ColO gene from chr. 4 at RPP4. Host cell necrosis: P, pitting; F, flecking.
257
SYMBIOLOGY OF MOUSE-EAR CRESS AND OOMYCETES RPP13
No. lines
GLL
Maks9
Edcol Gocol Madil
Emwa2
Em01
Em02
RPPl7
RPPl6
EmcoS
Aswal
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m226 g3883
1 MRC-F
nga280 m315
MRC-B
IMRC-H TT? m435
I
MRC-J
20 CMI
Fig. 9. Genetic map of Arabidopsis thaliana showing the position of major recognition complexes (MRC) relative to morphological (GLI and TT3) and molecular markers. The MRC regions each contain numerous RPP loci associated with recognition of Peronospora parasitica. The oval on each chromosome indicates the approximate location of the centromere.
D. A NATURAL ANOMALY OF SUSCEPTIBLE ORIGIN
Researchers of Arabidopsis downy mildew have benefited from one of the least dogmatic educators in the modern age of plant biology, Paul H. Williams from the University of Wisconsin-Madison. He searched for parasites of Arabidopsis in the late 1980s when common folklore suggested that the species grew too fast to be afflicted by parasites. Although not by design, his success at finding downy mildew and white blister in Europe gave spark to the research of Arabidopsis downy mildew that began shortly after in Switzerland and the UK (Koch and Slusarenko, 1990; Holub et al., 1993). His gift for sowing questions of a probing but seemingly unanswerable nature has directly affected the research described in this chapter. One question in particular was used to arouse discussion by a class of plant-breeding students in which the senior author was a fortunate participant: would it be possible to select disease resistance from a cross between two susceptible plants? There was no definitive student opinion, nor did Williams offer the slightest hint of his own opinion. The question, however, has lain dormant in the author’s mind until very recently. We have encountered several anomalies which suggest that the choice of compatible parent for cross-pollination can influence the expression of incompatibility and the mapping of a single major locus. The first example arose when we observed that the dominance of incompatibility with A . candida appeared to vary depending on the choice of compatible parent used for crosses with the same incompatible accession (Holub et al., 1996). The
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genetics of this pathosystem is less advanced than for downy mildew, so we merely concluded that the host genetic background somehow affected expression of the major recognition gene. Recombinant inbred lines would greatly aid in sorting out the genetic nature of this phenomenon, but this material is not currently available. Curious examples in Arubidopsis downy mildew first emerged from experiments with isolates that were compatible with both Ler and Nd but incompatible with Col. Following inoculation of the Ler x Col set with the isolate Welu3, a major locus RPP6 was mapped on chromosome 1 in the MRC-B region. Following inoculation of the Col X Nd set with the same isolate, segregation of a single RPP locus was independent of molecular markers that flank all of the known MRC region on chromosomes 1, 3, 4 and 5 . Recombination of less than 20% was eventually found between this locus (named RPP20) and m247 on the top arm of chromosome 2; an attempt to find closer markers on chromosome 2 is currently underway. A second isolate Hind4 was collected which could be used in the same manner. In this case, a single locus R P P l 9 was found closely linked to RPP20 following inoculation of the Col X Nd inbreds, but a locus RPP18 was mapped on chromosome 4 following inoculation of the Ler X Col inbreds. The next obvious experiment was to inoculate a segregating population derived from a cross between the two compatible parents Nd and Ler. We have postulated that Col carries two functional genes, both of which are required for incompatibility with Hind4 and with W e l d . Purely by chance we may have stumbled on compatible accessions, Nd and Ler, which each carry a single but different gene having functional homology with one of the genes from Col. Therefore, only one locus will exhibit functional dimorphism (or segregates) in each of the inbred sets. If this is true, then recombination of the respective genes in Nd and Ler should create an incompatible phenotype. FL and FN phenotypes have in fact been observed in an F2 Nd x Ler population following inoculation with Hind4. These seedlings have been saved for progeny testing to confirm the parental phenotype. Crosses have also been made between Hind4-compatible inbreds from the Ler x Col and Col x Nd inbred sets to determine whether incompatibility alleles from Col can be recombined.
V.
MUTATIONS: REVEALING COLOUR FROM BLACK AND WHITE
In the previous section we emphasized recognition specificity as a black and white interpretation of phenotypes for the purpose of simplifying genetic analyses. We effectively drew a line to separate natural phenotypic variation into compatible and incompatible classes. However, bridging the gap between genetic and physiological investigations will inevitably involve more complex appraisal of interaction phenotypes than a dichotomous classification. This
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will certainly become apparent as molecular genetic research of the so-called “R-genes” begins to link the early recognition and signalling events with mechanisms of host response. As shown in this section, the efforts of researchers to generate artificial mutations of Arabidopsis has begun to reveal interesting shifts within the phenotypic spectrum of incompatibility, including the possibility of mutating a compatible interaction. Several research groups have been selecting directly for alterations in mechanisms of host response (e .g. phytoalexin production, PR-protein expression) or indirectly for mutations that mimic necrotic lesions often associated with disease or that reduce incompatibility, in a non-specific manner, with different genotypes and even different species of parasites. A . A MYRIAD OF COLUMBIA MUTANTS
The accession Columbia has been a popular workhorse for generating artificial mutants of several different classes including: phytoalexin-deficient mutants (pad) (Glazebrook and Ausubel, 1994); accelerated cell death mutants (acd) (Greenberg and Ausubel, 1993); a non-expressor of PRprotein (npr) (Cao et al., 1994); a constitutive expressor of PR-protein (Bowling et al., 1994); and a non-specific disease resistance mutant (ndr) (Century et al., 1995). The current library of P. parasitica isolates has been adapted for use as a bioassay to compare these and future mutants of Columbia with the wild-type accession. Five Col-compatible isolates have been collected thus far which are useful for examining mutants that may exhibit an enhancement of incompatibility (e.g. acd), measured as a decrease in parasite reproduction (numbers of sporangiophores). At least nine Col-incompatible isolates have been collected which are useful for examining mutants that may exhibit a shift towards greater compatibility (e.g. p a d , npr and ndr), measured as an increase in parasitic reproduction. Inoculations with several of each type of P. parasitica isolate is important for assessing the specific nature of an altered phenotype. This is especially true for Col-incompatible isolates as demonstrated with the Col-ndrl (Century et al., 1995). This mutant exhibited increased susceptibility to strains of Pseudomonas syringae that carried different avirulence genes recognized by Col. Interestingly, Col-ndrl also permitted increased sporulation by several Col-incompatible isolates of P. parasitica; however, the shift towards compatibility was incremental (not complete) with most isolates. These isolates were used to identify several Col alleles at R P P loci on chromosomes 1, 2 and 4. More dramatic shifts to heavy sporulation were observed with Col-nprl and especially with Col-pad4 (Table IV). Glazebrook has also produced the three combinations of double mutants between Col-padl, pad2 and p a d . These support moderate to heavy sporulation by Col-incompatible isolates, but each combination exhibits a
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SYMBIOLOGY OF MOUSE-EAR CRESS AND OOMYCETES
TABLE IV Mutations of Arabidopsis thaliana (Columbia-0) affecting non-RPP specific changes in asexual reproduction by incompaible isolates of Peronospora parasitica
P. parasitica isolate Col-0 line” Wild type ndrl nprl pad1 Pad Pad pad4
Emwal R4
Ernoy2 R4
Hiksl R7
Hind4 R18, 19
Wela3 R6,20
NC
N
L L N
M M N N
L M M L M L H
N R
N N M N R R H
N L M N
Cala2 R2b
N N
H
N H
N N N N N
N N L
“ndr, Non-specific disease resistance (Century et al., 1995); npr, non-expressor of PR protein (Cao et al., 1994); pad, phytoalexin deficient (Glazebrook and Ausubel, 1994). bRPP locus identified in Col-0 following inoculation with the P. parasitica isolate. ‘Sporangiophore production: H, heavy (>20 per cotyledon); M, medium (5-20); L. low (4 per cotyledon); R , rare sporangiophore (1-2 in
different pattern of compatibility (Glazebrook, J. and Holub, E. B., unpublished). The Col-acd mutants exhibited no change in phenotype following inoculations with Col-compatible isolates. However, the cprl of Col did exhibit incompatibility, in some cases no sporulation was observed, following inoculations with several Col-compatible isolates (Holub, E. B. and Dong, X.,unpublished). The molecular basis for all these mutations should prove to be very informative about signalling pathways of networks coupled with recognition genes and perhaps pathways of biosynthesis. They may already suggest differences amongst R P P genes, those which are definitely coupled with the effect of a non-specific mutation and those which appear to be unaffected. Among those which are coupled, it will be important to determine whether recognition genes necessarily join at the same step of a pathway. B.
SURPRISING EXTREMES IN WASSILEWSKIJA
The accession Wassilewskija has so far been the source for the most extreme mutant phenotypes. Anticipated mutants of R P P loci have been found such as Ws-cog1 (altered recognition of P. parasitica) which has specifically lost the ability to recognize Cala2 and has effectively distinguished between two tightly linked loci, RPPI (locus for recognition of Emoy2) and RPPZO (locus
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for cogl) (Bittner-Eddy, P. and Beynon, J . L., unpublished). It has not been possible to separate these loci by genetic recombination. Jane Parker (Nonvich, UK) has selected an unexpected mutant named Ws-edsl (extreme downy mildew susceptibility) which appears to be supersusceptible to Ws-incompatible isolates of P. parasitica collected from Arubidopsis and also supports colonization and moderate sporulation on young, still-expanding cotyledons by isolates from Brussica oleracea and Capsella-bursa pastoris (Parker et al., in press). Plants inoculated at the seedling stage invariably become systemically infected, exhibiting downy mildew on all leaf and stem tissues and have difficulty producing seed. This occurs whether the isolate collected from Arabidopsis is compatible o r incompatible on wild-type Ws. Non-inoculated seedlings of Ws-edsZ will exhibit a high degree of susceptibility when they have subsequently been inoculated at the rosette stage. This enhancement of susceptibility to P. parasitica at any growth stage creates serious difficulties in handling the mutant because it is vulnerable to infection by airborne, contaminating P. parusitica. Wild-type accessions rarely exhibit systemic infection unless they are inoculated when very young, and we have rarely observed any downy mildew in wild-type plants that were exposed to P. parasitica at later growth stages. P. parasitica isolates from B. oleracea and C. pastoris are not entirely compatible in Ws-edsl; sporulation by these isolates is restricted in young cotyledons and inhibited entirely in true leaves. Nevertheless, any sporulation is of enormous significance. Damping-off by a biotroph appears to be the phenotype of another unusual mutant, Ws-lsdl (lesion-simulating disease) (Dietrich et al., 1994). Seedlings die within a few days after their cotyledons have been inoculated with Ws-incompatible isolates of P. parasitica (Fig. 10) (Holub, E. B., unpublished). The most rapid seedling death (24-48 h) is caused by isolates that exhibit a PN phenotype in wild-type Ws. Compatible isolates, on the other hand, colonize tissue and reproduce asexually the same as in wild type. However, the seedling subsequently dies within 14 days after inoculation. This contrasts with inoculations of true leaves in which the rapidly induced necrosis is sufficient to stop colonization by Ws-compatible isolates; hence, sporulation in true leaves has never been observed (Dietrich et al., 1994). Ws-compatible isolates of A. candida form blisters in the mutant the same as in wildtype Ws and do not appear to trigger the formation of lesions. No pathogen was previously reported that could infect Ws-Zsdl without inducing lesion formation.
VI. AVENUES OF FUTURE RESEARCH The prime motivation for molecular research of plant parasitic symbioses has been to reveal mechanisms of the fourth R of symbiosis (see section IIIC), host resistance to the parasite. A major hurdle was overcome by several research
SYMBIOLOGY OF MOUSE-EAR CRESS AND OOMYCETES
263
Fig. 10. LSDI (lesion-simulating disease) mutation of Arabidopsis thaliana accession Wassilewskija (Dietrich et al., 1994). This mutation is responsible for very rapid seedling death (24-48 h) when cotyledons are inoculated with incompatible isolates of feronospora parasitica (left panel) and Albugo candida. Compatible isolates of f . parasitica reproduce asexually (right panel) the same as in wild type, but the mutant seedling dies 10-14 days after inoculation. Wild-type seedlings usually outgrow infection and sporulation by a compatible isolate. A compatible isolate of A . candida reproduces asexually (not shown) the same as in wild type, and inoculated Ws-lsdl seedlings continue to grow the same as non-inoculated mutant seedlings.
groups when they isolated naturally polymorphic genes that define the genetic difference between a compatible and an incompatible host (Staskawicz et al. , 1995), the so-called “R-genes”. The research continues along two main avenues of research: the avenue of revealing causal relationships, and the avenue of practical implementation. In the former, the isolated R-gene is used as the anchor to begin dissecting the pathway or network of genes that somehow interact to bring about an incompatible phenotype. Tremendous challenges lie ahead in designing mutational and biochemical analyses that will in fact reveal the sequence of genes downstream from the first. In the avenue of practical implementation, the isolated R-genes will be exploited for the purposes of crop improvement. Molecular probes will be developed as plant-breeding tools for identifying homologous genes that are already present in crop species. The challenge here will be to demonstrate whether the homologues are functional, a task which is entirely dependent on the collection of parasite isolates. The isolated genes will also be examined for function upon transfer into the crop species. Appropriate genetic engineering will most likely be required, but this
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promises to be a means for greatly expanding the resource of useful genetic material. A third complementary avenue emerges from an interest in how biological systems have and continue to evolve: the avenue of comparative biology. Molecular biologists automatically use this approach when they combine results from the different case studies they have chosen to investigate such as the comparison of DNA sequence and structural similarity among the R-genes already isolated (Staskawicz et ul., 1995). However, a comparative method can also be used much more directly as a framework for conducting research. We suggest examples for how it will be possible to advance our knowledge of plant symbioses by applying a comparative method to research of Arabidopsis parasitism. A given plant species may have more than one mechanism of defence against the same parasite species. Since molecular analyses require enormous investment in labour and materials for each case study under investigation, a single research group cannot easily afford to search actively for different mechanisms. However, on the basis of interaction phenotype, one can choose contrasting examples of host response such as the PN and FN phenotypes of Arubidopsis downy mildew that may improve the chance of revealing different mechanisms of host defence. A non-specific mutation such as Ws-edsl or Col-nprl will also be useful for separating wildtype RPP genes into classes that are either affected or not affected by the mutation, thereby implying the existence of more than one pathway of host defence. A single defence mechanism may be dependent on the dosage of a recognition gene. This is suggested by the common occurrence of semidominant incompatibility, from a series of interaction phenotypes that vary in parasite reproduction (e.g. FN, FDL and DM), and from examples of apparent emergence of weak but additive recognition genes from a parental phenotype as shown in Table I11 and Fig. 7. The different phenotypes would have to share a similar molecular basis for recognition if dosage plays a role. Even examples in which a single locus is associated with non-sporulation could involve additivity of tightly linked genes. This will become apparent in transformation experiments when only a partial, low-sporulation phenotype is observed from a single copy of the putative recognition gene. Presumably, the full phenotype would be restored by increasing the copies of this gene in a transformed plant. Genes within a given MRC region may reveal some interesting comparisons with respect to several phenomena. Molecular investigation of the range of phenotypes (PN, FN, CN and CL) associated with MRC-Fmay reveal different mechanisms of host defence (see Table 11). Dosage dependence may be revealed by comparing RPP genes from Landsberg erectu associated with FN and FL phenotypes in MRC-J. The gene at RPP22 that recognizes Aswul is recessive and may be useful for revealing something about the nature of dominance when compared with other examples from the same region
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associated with dominant or semi-dominant phenotypes. Homology among recognition genes within the same M R C region will be useful for examining how such genes evolve and may provide clues for what determines the specificity of recognition. The M R C regions are actually quite large, so subclusters of less than 2 cM most likely exist within a region. It will therefore be interesting to compare recognition genes from different subclusters and from different chromosome arms to predict whether they are of common origin. The full extent of M R C regions in Arabidopsis is largely unexplored. For example, a given Arabidopsis accession appears to carry functional RPP genes in some M R C regions but not in others (see Table 11).As more parasite isolates are used to detect RPP loci, it may be possible to reveal that functional RPP genes do in fact exist in every known region in the same accession. Alternatively, the M R C regions may have independent origins, and it is therefore possible that some accessions have not inherited a full complement of functional recognition genes in every M R C region. If this is true then it is uncertain whether all regions containing functional recognition genes have been found; it may be necessary to examine additional host germplasm to detect other regions. It will also be interesting to compare the organization of RPP loci with that of genes that recognize other parasites. The parasites and respective loci of particular interest would be: Albugo candida, RAC; Pseudomonas syringae, RPS;Xanthomonas campetris, RXC; and Erysiphe cruciferarum, ECR. Libraries of these species could be developed for use on recombinant inbred sets as we have done for P. parasitica (see section IVC). Curiously, Shauna Somerville (Stanford University) and Richard Oliver (University of East Anglia) have been finding numerous ECR loci mostly on chromosome arms opposite to where RPP loci have been found (see review by Kunkel, 1996). Evidently a large portion of the Arabidopsis genome, much of it as yet unexplored, could be involved in parasite recognition. The close proximity in the same M R C region of recognition genes and non-specific genes such as those involved in phytoalexin production or lesion formation (see section IVC) may seem counterintuitive. Arabidopsis most likely maintains high recombination and rearrangement in regions that contain recognition genes enabling it to generate necessary polymorphism. However, the non-specific genes must presumably be conserved within the species. Therefore a comparison should eventually be considered of genome structure between closely adjacent regions that are highly variable or highly conserved. Collinearity of both regions in Arabidopsis with heterologous regions in a related species such as Brassica oleracea may provide an indication of the relative rates of evolution. Artificial mutagenesis of Arabidopsis offers worthwhile opportunities for comparative biology. Arabidopsis is an ideal species for large-scale screening of phenotypic variants (Redei and Koncz, 1992); with a simple procedure such as spray inoculation of cotyledons with parasite inoculum, more than
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10 000 seedlings can be processed. Several phenotypic variants would be expected from a population of this size. The main limitation is the researcher’s efficiency at sifting through the population for altered phenotypes such as increased parasite reproduction or altered host response. With little investment, it is worthwhile attempting a severalfold screen of every non-lethal mutation that can affect a given phenotype such as selecting for sporulation from a non-sporulating wild-type interaction. This is being attempted by several research groups using P. parasitica. Progeny from the mutant host selections will be tested with a collection of parasite isolates to classify them as either RPP-specific or non-specific mutations. Genetic complementation studies and biochemical analyses could further subdivide mutant classes. Biochemical analyses will be important to assess mutants for alterations observed in processes such as phytoalexin (Col-pad) or PR-protein production (Col-npr). With the ease of artificial mutagenesis, it is worthwhile attempting much more speculative screening. Crop protection could be revolutionized if it was possible to determine what it is that enables Arabidopsis to be incompatible with parasites from other host species. For example, the Ws-edsl mutant already supports modest colonization and reproduction by P. parasitica isolates from other crucifer hosts. A second mutagen treatment of this genotype may produce the next variant which is fully compatible with the same isolates in true leaves. P. parasitica from the crucifer Cardamine pratense, P. tabacina (tobacco blue mould) and Bremia iactucae (lettuce downy mildew) were all unable to reproduce on Ws-edsl (Holub, E. B . , unpublished). One could therefore attempt to screen for variants that permit reproduction by any of these more distantly related oomycetes; perhaps even Phytophthora infestans (potato late blight). Initially one could screen for single gene mutations; however, it may be necessary to “peel back” the layers of multigene, non-host resistance, one mutation at a time. Speculative mutagenesis could easily be discounted by theory, but the minimal investment and the potential rewards make them undeniably worthwhile. Can the process of signal transduction required for plant defence still be evolving? The interaction between single gene products of host and parasite is certainly evolving in most pathosystems. However, it is unknown whether any pair of interacting host molecules along a single transduction pathway can also be naturally polymorphic and, as a consequence, create phenotypic variation. It is inherently difficult to distinguish whether the parasite-host molecular interaction or a host-host molecular interaction determines the specificity of an observed phenotype. However, the anomalies described in section IVD may provide an important clue that the latter case can indeed affect incompatibility. From these observations, it is plausible that two naturally polymorphic components of the host determine an incompatible phenotype. We were fortunate to stumble upon the examples described. However, it is now worth continuing to examine crosses between two
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compatible accessions. In genetic analyses and transformation experiments, it will be worthwhile to compare the effect of several compatible genetic backgrounds on phenotypic expression of a putative recognition gene. Comparisons between different pathosystems of Arubidopsis could help to resolve the debate of whether plant-parasite interactions necessarily evolve as a molecular arms race between two species. Futuyma and Montgomery (1983) proposed an alternative that “the most prevalent pattern [of coevolution], in fact, seems to be adaptation of a species to a suite of often phylogenetically diverse species, to which it holds a similar ecological relationship”. They named this less-specific pattern “diffuse coevolution”. In Arabidopsis it should eventually be possible to determine whether a given recognition gene is capable of perceiving isolates of different parasite species. For example, the tremendous diversity of pathotypes evident in P. parasitica could be sampled to find an isolate which is recognized by a bacterial recognition gene such as RPS2. This could be attempted using a host accession that carries a mutation of the RPS2 gene as a baiting host for a P. parasitica isolate that is compatible with the mutant but incompatible with the wild-type accession. Artificial mutations such as Col-ndrl and Col-nprl already demonstrate that genes exist which are involved in defence against bacteria and oomycetes. Natural variants of these genes, if they exist, would support the possibility of diffuse coevolution. Further circumstantial evidence will be provided by identifying close linkage relationships between genes that recognize different parasite species. Another important debate described by Sapp (1994, pp. 131-147) is whether mutualism and parasitism emerge from the same pool of genetic variability. In Table V we illustrate that a wide spectrum of symbiotic relationships can be viewed conceptually as being related in their gross patterns of recognition and response. Conventional terms have been used to classify different microbial symbionts. The plant symbionts, on the other hand, are described in two parts according to general modes of microbiont recognition and types of cellular response. Molecular analyses will be essential to determine whether a common mechanistic basis does in fact exist between different types of association. Specific examples for comparison would be to determine whether the proposed receptor in maize for attaching to the HM toxin (Briggs and Johal, 1994) is similar in structure to the receptor-like molecules characterized from other pathosystems (Staskawicz et al., 1995). The same structural features could be sought in plant receptors in legume-rhizobium interactions, lichens or mycorrhizal associations. Characterization of the lsdl gene that appears to cause damping-off when the mutant is inoculated with P. parasitica may even provide useful clues for understanding the process of true damping-off by pathogens such as the oomycete Pythium. These are some of the obvious examples in which comparative biology can be used to expand our understanding of symbiotic relationships. With an
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TABLE V Hypothetical cellular inteructions of symbioses between a plant and a micro-organism Microbiont
Interaction phenotype
Necrotroph
Compatible Incompatible
Parasitic biotroph
Compatible Incompatible
Mutualistic biotroph
Compatible Incompatible
Non-troph
Compatible Incompatible
Phytobionta None; killedb Suppressed; killed Non-self; suicide‘ Non-self; biochemical defenced None; manipulatede Suppressed; manipulated Self; manipulated Non-self; suicide and/or biochemical defence None; co-operativee Self; co-operative Suppressed; co-operative (Extinct, rare or unknown combination) (Non-existent or unknown combination) None; none Foreign; suicide and/or biochemical defence
aCharacterized by the host’s mode of recognizing the microbiont; and by the host’s cellular response. For suppressed and self-recognition, the parasite must actively suppress the host’s ability to recognize an intruder or else mimic the factors used by the host to identify self. bNecrotroph requires extracellular enzymes and/or toxins to aggressively kill host cells before digestion. ‘Assumes that the necrotroph takes advantage of being recognized and the subsequent suicide of host cells. Some extracellular enzymes may enable passage through barriers (e.g. cuticle) but others are only used passively for digestion of material from dead cells. dSuicide of the penetrated host cell is assumed to have no impact by itself on a necrotroph so another means of defence is required. Biochemical defence could be antibiotic or structural (e.g. lignification, cell wall cross-linking). eManipulated and co-operative cellular responses refer to whether the host metabolism is circumvented entirely to benefit the parasite, or instead remains engaged in providing for both organisms, respectively.
open mind, it should be possible to think of other more original possibilities to explore.
VII. CONCLUDING REMARKS From one perspective, the storybook character Robin Hood was an underdog struggling to provide sustenance for lesser mortals; from another, he was
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nothing but a parasite. It is the former perspective that makes this character and those that lived in his world so appealing to the imagination. His relationships with the rich and the poor are unveiled; one begins to appreciate his morals and his motives, and to take interest in how he plots to acquire riches. There would scarcely be a story nor would lessons be learned if he was merely portrayed as a parasite. In a similar way, invaluable insight can be unveiled when Arabidopsis is viewed as a wild flower with a natural history instead of just a weed; and when biotrophs such as P. parasitica or A . candida are recast objectively as partners of a symbiosis (sensu Frank and DeBary) instead of just selfish parasites. An understanding of symbiosis in the modern age of biology will be advanced by at least two lines of research: biochemical analyses that reveal commonality, particularly on the part of the host, in the cellular communication and physiological co-ordination between two organisms; and genetic analyses that reveal the co-ordinated evolution of two organisms (e.g. genome organization, and mechanisms of generating and maintaining polymorphism). Great insight will arise from comparisons between species of host and species of parasite, and at the boundaries of different types of associations such as between mutualism and parasitism. The contrasts will be important, but a search for common themes will be most illuminating.
ACKNOWLEDGEMENTS This chapter was written as a farewell tribute to Grace M. Johnson, a one-room schoolhouse teacher, a naturalist and the boyhood mentor of E. B. Holub. Both authors are especially grateful to Ian R. Crute for establishing the research of Arabidopsis pathology in Britain and for his generous contribution of experience and ideas. Most of the research was conduced with financial support from the UK Biotechnology and Biological Sciences Research Council (formerly the Agricultural and Food Research Council).
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Use of Monoclonal Antibodies to Detect. Quantify and Visualize Fungi in Soils
F. M . DEWEY'. C . R . THORNTON' and C . A . GILLIGAN'
'Department of Plant Sciences. University of Oxford. South Parks Road. Oxford OX1 3RB. U K. 2Departrnent of Plant Sciences. University of Cambridge. Downing Street. Cambridge CB2 3EA. UK
I . Introduction
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I1 . Production of Species-specific and Genus-specific Monoclonal Antibodies ............................................................................. A . Selection and Preparation of Immunogens ............................ B . Selection of Hybridoma Cell Lines Secreting Specific Antibodies ....................................................................... C . Choice of Antibody Subclass ........ .................................
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111. Assay Formats
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IV. Sample Preparation ................................................................. A . Extraction of Antigens from Soil ......................................... B . Elimination and Reduction of Interference from Soil Components ....................................................................
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V. Detection
VI . Quantification ........................................................................ A . Estimation of Biomass ....................................................... B . Immunological Estimation of Colony-forming Units ................ C . Setting of Thresholds for Detection and Quanta1 Assay Systems ........................................................................... D . False-negative Results ....................................................... VII . Visualization
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VIII. Concluding Remarks
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Acknowledgements ................................................................. References ............................................................................
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I. INTRODUCTION Studies on fungi in soils have, until recently, been severely constrained by the lack of techniques that allow specific fungi to be detected and quantified in environments that are microbially, chemically and physically complex. Modern immunological techniques, particularly those employing monoclonal antibodies (MAbs), provide powerful tools that allow us to determine far more about the activities and interactions of specific soil fungi. Previously, the detection and quantification of soil-borne fungi have depended on the use of baiting or host infection techniques. Success of such techniques relies heavily on favourable soil conditions for colonization, penetration and infection and also on host responses. The formulation of selective chemical inhibitors has led to the development of a large number of relatively effective and useful selective growth media. These have been reviewed extensively by Tsao (1971), with considerable additions since. These selective media have been incorporated into methods designed for the isolation and enumeration of specific pathogens or a related group of fungi in soils such as soil plates (Warcup, 1950), immersion tubes (Chesters, 1940), buried slide techniques (e.g. Rossi, 1928; Cholodny, 1930), slide traps, hyphal isolation, baiting and flotation. The development of these methods and appropriate media have undoubtedly advanced our understanding of many soil-borne fungi, especially with respect to inoculum density, population dynamics, saprophytic behaviour and survival. However, they are severely flawed in a number of aspects. Dilution-plate counts are slow, time consuming, expensive and invariably require considerable taxonomic expertise to differentiate fungal species. Plate counts are frequently subject to bias because of the selectivity of the media. They often lead to overestimation of population densities because of fragmentation of hyphae during successive dilutions. Moreover, plate assays do not distinguish satisfactorily between populations of mixed species (for example, Trichoderma species will rapidly outgrow and often occlude colonies of Rhizoctonia, when present at high densities, even on Rhizoctonia-selective media). Plate counts do not distinguish between mycelial fragments and spores, yet, in the case of a hyperparasite such as Trichoderma, the two may have quite different epidemiological significance in the control of fungal hosts. Spores frequently remain dormant in the soil while mycelial fragments germinate, grow, infect a parasite and lyse. Hence the production of spores by a hyperparasitic population may swamp ecologically important changes in the density of mycelial fragments.
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The advent of hybridoma technology, pioneered by Kohler and Milstein (1975), has enabled unlimited quantities of highly specific monoclonal MAbs to be raised, thus allowing the development of sensitive and quick immunological assays that can be used to detect and quantify specific species or genera of fungi in mixed communities. However, exploitation of immunoassays for soil fungi has been slow because intrinsic factors such as the effects of soil components, the collection and concentration of propagules and their respective antigens, and sample preparation have proved difficult. Nevertheless, technical issues such as these can and have been overcome. Furthermore, because immunological assays are relatively quick, inexpensive and are easy to perform and replicate, compared with conventional assays, they allow a more detailed and thorough analysis of soil populations than can be achieved by plate counts. More thorough analyses of naturally infested field soils can be made and could, in principle, enable growers and consultants to make improved crop management decisions by providing information on the spatial and temporal variability of populations of potentially damaging pathogens and their response to crop rotations and often costly and unnecessary fumigation and fungicide applications. MAb-based assays have the potential to provide “risk assessment” analyses, but it should be made clear from the start that a considerable amount of further work needs to be performed before this becomes an absolute reality. The aim of this chapter is not to present a review of all the relevant literature (for a wider overview of fungal immunodiagnostics the reader is referred to Werres, 1994; Dewey and Thornton, 1995), but more to explore the development of soil-based immunoassays from concept through to application. In doing this, we will necessarily draw on experience from our own studies since the area of soil-based fungal immunodiagnostics is, at present, still very much in its infancy but is an area in which we and a number of other workers world-wide are actively involved. In attempting to write an account of the use of MAbs in the study of soil-borne fungi we have concentrated, predominantly, on pathogenic fungi of agriculturally important crop plants. In so doing we have specified three key areas: detection, quantification and visualization. Clearly, these three areas are not mutually exclusive. For example, detection of soil-borne fungi can fall into all three of these categories. Likewise, visual appraisal of fungal growth can be both qualitative and quantitative in nature. While most quantitative studies are performed using enzyme-linked immunosorbent assays (ELISA), an absorbance value obtained by immunoassay can be used not only as a means of quantifying fungal biomass but also as an indicator of the presence of a target organism within a test sample.
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11. PRODUCTION OF SPECIES-SPECIFIC AND GENUS-SPECIFIC MONOCLONAL ANTIBODIES A . SELECTION AND PREPARATION OF IMMUNOGENS
Relatively little is known about the nature or site of fungal antigens that are specific. We have routinely used surface washings from solid cultures or mycelial fragments as immunogens. Little benefit appears to be gained from using purified cell walls or protein precipitates from mycelial extracts as immunogens. When hybridoma cell lines are raised to hyphal fragments or surface washings of mycelial cultures a large percentage of the cell lines secrete non-specific MAbs. In our experience, approximately 10% are genus specific and only 1% are species specific (Dewey and Brasier, 1988). More recently, in order to reduce the number of hybridoma cell lines produced that secrete non-specific MAbs we have used co-immunization techniques after the method of Barclay and Smith (1986). We have used this technique to produce monoclonal antibodies specific to mycelial antigens from the take-all fungus Gaeumannomyces graminis ( C . R. Thornton and F. M. Dewey, unpublished) and MAbs specific to the aquatic hyphomycete Anguillospora longissima (Bermingham et al. , 1995). Co-immunization involves blocking the formation of non-specific antibodies by co-injecting antigens of the target fungus together with antisera or non-specific hybridoma supernatant, previously raised to the same or a related fungus or host. For technical details of immunization procedures and methods for hybridoma production see Harlow and Lane (1988) and Dewey (1992). Virtually no information is available about the proportion of molecules shared by the different life stages of a particular fungus. Relatively little similarity appears to exist between immunogenic molecules associated with spores and mycelium. Thus, we have found that it is possible to raise monoclonal antibodies of Trichoderma harzianum that are either mycelium or phialoconidia specific (Thornton et al., 1994; Thornton and Dewey, 1996, respectively) and these have been used to develop immunoassays that distinguish the two components in complex environments. Interestingly, both of these monoclonal antibodies recognize low-molecular-weight peptides. More commonly, fungal monoclonal antibodies recognize higher molecular weight glycoprotein molecules that appear to be abundant throughout the fungal kingdom. This is perhaps fortuitous since, as antigens, they are less prone to degradation by micro-organisms in soil, thereby rendering them more accessible to extraction and detection. Not so favourable, however, is the fact that many fungi share similar antigenic binding sites or epitopes. This is particularly true of the carbohydrate moieties of fungal glycopro teins. In developing assays for the detection of fungi that lack a mycelial phase
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or for which the mycelial stage of growth is restricted, spores have been used directly as the immunogen. Examples are: oospores of Phytophthora spp. (Miller et al., 1989), zoospores of Phytophthora spp. (Hardham et al., 1986; Jones and Shew, 1988; Klopmeyer et al., 1988), Pythium aphanidermatum (Mitchell et al., 1994) and Pythium ultimum (Yuen et al., 1993), cystosori of Spongospora subterranea (Wallace et al., 1995), resting spores of Plasmodiophora brassicae (White and Wakeham, 1995) and phialoconidia of T. harzianum (Thornton and Dewey, 1996). It should be noted that, in the case of P. brassicae, the immunogen was used to raise rabbit polyclonal antiserum and not MAbs. It seems from our own work, and that of others (for example, Hardham et al., 1986), that it is easier to raise and select out genus- and species-specific MAbs to spores than to mycelium.
B. SELECTION OF HYBRIDOMA CELL LINES SECRETING SPECIFIC ANTIBODIES
Screening of hybridoma cell line supernatants against a range of related and unrelated soil-borne organisms is an obvious and necessary prerequisite in the selection of cell lines secreting specific MAbs. Testing of hybridoma supernatants should be performed at the earliest possible stage to eliminate cell lines secreting non-specific antibodies. The number of fungal isolates or otherwise to be screened will vary according to the desired specificity, but will be governed also by variation within a genus, species or strain and the extent of relatedness to other organisms. Hybridomas isolated in a typical fusion secrete MAbs exhibiting a range of characteristics including specificity and affinity, immunoglobulin class and subclass. We have found, as have others, that although antibodies may perform well in one assay format, for example ELISA, they do not necessarily perform well in others, so careful consideration should be given to the assay format used in selecting cell lines to ensure that the antibodies will work well in the proposed soil-based tests.
C. CHOICE OF ANTIBODY SUBCLASS
Of the five major classes of immunoglobulins IgA, IgD, IgE, IgG and IgM, immunologists generally regard IgG immunoglobulins belonging to the subclasses 2a, 2b and 3 to be the most useful for the purposes of detection and immunolocalization. Immunoglobulins from the class IgG readily bind to protein A and protein G and can therefore be easily concentrated and purified using commercially produced sepharose beads to which protein A or G has been covalently bound. Furthermore, IgG antibodies can be readily
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conjugated to fluorescent markers, enzymes or biotin (e.g. Holtz et al., 1994). However, we have consistently found that when raising MAbs to surface washings of fungi or hyphal fragments, for diagnostic purposes, that almost all the hybridoma cell lines raised secrete IgM antibodies and that, of these, a small percentage is highly specific and useful. Increasing the number of immunizations or the time intervals between immunizations seems to make little difference to the ratio of cell lines secreting IgG to IgM antibodies. It appears that the carbohydrate moieties of fungal surface molecules are immunodominant. Many of the specific IgM MAbs that we have raised recognize heat-stable, periodate-sensitive, carbohydrate epitopes on glycoprotein molecules and the high level of sensitivity displayed by these MAbs suggests that they are recognizing multiple repeat epitopes on each molecule. We have also found that these IgM MAbs bind rapidly so that the incubation time for the primary antibody can be reduced substantially, thereby increasing the speed of the detection assay (Dewey et al., 1992; Thornton et al., in press). In certain instances this can also improve the specificity of the assay. Where possible, we have found that it is useful to select antibodies of two different classes because this allows greater flexibility in developing detection assays; one class can be used as the capture antibody and the other, in conjunction with a commercial subclass-specific enzyme conjugate, can be used as the detector antibody, for example in immunomagnetic bead assays (see section IIIA2).
111. ASSAY FORMATS Several different assay formats exist, but the most common are the simple direct or indirect ELISAs. The type of assay chosen invariably reflects its mode of application. ELISAs have many advantages: they are highly sensitive and can easily be replicated, automated and quantified. The only disadvantage is that they require laboratory facilities. A few “user-friendly”, membrane-bound dip-stick assays, which do not require laboratory facilities, have been developed and are being used increasingly as “on site” screening tests (see Miller et al., 1992; section V). Immunofluorescence (IMF) assays, which were developed for the immunolocalization of fungal antigens, are useful but they are never likely to become widely used for mass screening because they involve microscopy and an ultraviolet (UV) light source. Nonetheless, we have found them useful for the differentiation of mycelium of Rhizoctonia solani from other species of Rhizoctonia and contaminant organisms in culture plates (Thornton et al., 1993), whilst Holtz et al. (1994) developed an IMF assay for the visualization of Thieluviopsis busicola on cotton roots using the IgG fraction purified from polyclonal mouse ascites antibodies.
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A. ENZYME-LINKED IMMUNOSORBENT ASSAYS (ELISAS)
1 . Plate-trapped-antigen (PTA) and double-antibody-sandwich (DAS) assays Most of the ELISA tests developed for fungi are, with the exception of the commercial assays, all simple indirect plated trapped antigen (PTA) assays in which the microtitre wells are directly coated with the test sample or fungal antigens. Fungal antigens, particularly glycoproteins, which appear to be the immunodominant molecules, bind strongly to microtitre wells. We find that once the wells have been coated with fungal carbohydrates or glycoproteins, washed and dried, they can be stored dry at 4°C for several years (Dewey, 1992). The disadvantage of using antigen-coated wells is the relatively long binding step of 5 h or more (generally an overnight step of 16 h) needed to ensure maximum binding of the antigens to the wells, but the simplicity of such assays is attractive. Fungal carbohydrates and most glycoproteins are heat stable, which means that many of the cross-reactive proteinaceous antigens can be precipitated from the test sample by heat treatment before the antigen mixture is used to coat wells. Heat treatment of soil extracts has proved very useful in the detection of R. sofani antigens (Thornton et at., 1993). Most of the commercial enzyme immunoassays involve double-antibodysandwich (DAS)-ELISA tests in which one of the antibodies is generally a specific monoclonal antibody and the other polyclonal antisera. Sensitivity in these assays can be improved, as it can with antigen-coated wells, by the use of reporter molecules such as biotinylated secondary antibodies or biotinylated protein A conjugates, in conjunction with Streptavidin-enzyme conjugates. It is interesting that competition ELISAs which are now used extensively for the detection of pesticides in ground water and mycotoxins in food have not, apparently, been used for the detection of soil-borne fungi despite the increase in sensitivity of such assays. Exceptions are the competition assays developed by Lyons and White (1992) and Kitagawa et al. (1989) for the detection of Pythium sp. and Fusarium sp., respectively, in infected plant tissue. It should be noted that the assay developed by Lyons and White (1992), for the detection of Pythium violae and Pythium sulcatum in carrots, involved the use of rabbit polyclonal antiserum, not MAbs. 2. Magnetic bead assays Magnetic beads provide a promising alternative to solid support systems for capturing fungal antigens. Immunoassays incorporating magnetized beads have been used for some time in the fields of medicine, plant virology and pesticide detection (see e.g. Banttari et al., 1991) but their use in the detection and quantification of fungi has yet to be exploited fully. Enzyme immunoassays that use small beads have a number of advantages over conventional assays that use microtitre plates or membranes as the immunosorbent surface.
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They are less constrained by the slow binding kinetics, and the small surface areas of plates and membranes and are ideally suited to efficiently extracting antigens from samples that contain particulate debris. We have developed an indirect-magnetic microsphere-enzyme immunoassay (h4M-EIA) for the detection and quantification of R . solani in soils (Thornton, 1996a). Soil-sample extracts are incubated with a mixture of IgM and IgA murine MAbs specific to R . solani. Water-soluble antigens are bound by the antibodies and the complex is extracted using commercially available beads (Dynabeads, Dynal, Oslo, Norway) pre-coated with rat antibodies that specifically recognize mouse antibodies of the immunoglobulin class IgM. The beads plus bound complex are pulled out of suspension by placing in a magnetic field and the complex is detected by sequential exposure to commercial goat anti-mouse IgA (a-chain specific) alkaline phosphatase conjugate and the substrate p-nitrophenol. Using this method it has been possible to extract antigens for R . solani from soils and demonstrate a direct correlation between concentration of fresh hyphal fragments and absorbance values. It is possible that beads coated with spore-specific MAbs could be used to pull fungal spores out of soil suspensions, similar to the method developed by Wipat et al. (1994) for the capture of spores of Streptomyces Zividans from soils. It should be noted, however, that Wipat et al. (1994) were not able to achieve a recovery of more than 30% from sterilized soils and only 5% from non-sterilized soils.
B. MEMBRANE ASSAYS
I. Dot-blot and dip-stick assays The mechanism of the dot-blot or dip-stick assay is essentially the same as that in PTA-ELISA tests. Some test systems use nitrocellulose membranes or nitrocellulose coated plastic tags or cards and others use polyvinylidene difluoride (PVDF; Immobilon P, Millipore) as the support to which the target antigen is immobilized. The reporter molecule is generally an enzyme conjugate, but gold conjugates, which can be silver-enhanced, have been used by a number of workers and we find that they are more sensitive and give higher resolution. Cahill and Hardham (1994a,b) have developed a novel dip-stick immunoassay for the detection of Phytophthora cinnamomi in soils. By exploiting the phenomena of chemotaxis and electrotaxis they have developed an assay for the detection of zoospores of this fungus in aqueous suspensions. They found that zoospores exhibited strong electrotaxis to a positively charged membrane. As few as 40 zoospores per millilitre could be detected with the dip-stick assay in less than 45 min. Immunolabelled cysts attached to the membrane were observed with the naked eye or with low-power magnification after silver enhancement of a gold-labelled secondary probe or after an
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enzyme colour reaction. We have found, as have others, that the use of enzyme conjugates with coloured substrate products, for example purple or red, is often preferable because it allows detected antigens to be more clearly distinguished from stains on the membrane caused by soil particles. Arie et al. (1993) have developed a combined culture-immunoblot assay for the detection of Fusarium in soils. A membrane in a Petri dish is overlaid with a nutrient gel on which a dilute soil sample is spread. After 20 h, the gel is peeled off and the membrane is probed using a specific antibody. Fungal propagules in the soil, which have grown during the incubation period and released antigens which diffuse through the gel onto the membrane, can then be immunolabelled. We have developed a dip-stick immunoassay for the detection of R . solani in soil (Thornton et al., 1993). Water-soluble glycoprotein antigens released by actively growing mycelium are immobilized on a membrane of PVDF. Subsequent visualization is achieved by incubating the membrane sequentially with R. solani-specific IgM MAbs, commercial alkaline phosphatase conjugate and the chromogen nitroblue tetrazolium. An immunoblotting procedure was developed by Wright and Morton (1989) to overcome the difficulty in identifying root colonization by Glomus occultum, a vesicular-arbuscular mycorrhizal fungus. Whole roots are squashed onto a nitrocellulose membrane and, after inactivation of endogenous peroxidase, a MAb-based indirect ELISA is performed using an anti-mouse peroxidase conjugate as the secondary detector molecule. Antigens from spores and hyphae of G. occulturn, revealed by the precipitating chromogen 4-chloro-l-naphthol, are seen as purple dots on the membrane, which also retains an impression of the root.
IV. SAMPLE PREPARATION A. EXTRACTION OF ANTIGENS FROM SOIL
Efficient and simple extraction of fungal antigens from soil is one of the biggest hurdles in the development of quick and sensitive immunoassays for the detection and quantification of soil-borne fungi. Many of the soil-based immunoassays developed to date employ a period of biological amplification, which usually involves baiting with host tissue (Klopmeyer et al., 1988) or enrichment in solid or aqueous semi-selective media (MacDonald and Duniway, 1979; Thornton et al., 1993, 1994; Yuen et al., 1993) to allow germination and growth of viable propagules. Alternatively, lengthy extraction and concentration procedures involving sieving, flotation, centrifugation and filtration are employed (e.g. Wallace et al., 1995; White and Wakeham, 1995). At first glance, then, it would appear that little advantage is gained by the use of immunoassays over conventional techniques. However, as the field of soil immunodiagnostics advances, problems of antigen extraction and
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concentration are gradually being resolved. This is suitably illustrated by the elegant approach adopted by Cahill and Hardham (1994b) in the development of the dip-stick immunoassay described above for the detection of the soil-borne zoosporic pathogen P. cinnumomi. Antigen induction through nutrient enrichment is required, particularly in naturally infested soil samples, because of the small amounts and often quiescent nature of fungal propagules and their antigens in soil. This is not to say that simple and direct extraction procedures cannot be used (e.g. Thornton and Dewey, 1996), but depends to a large extent on amounts of antigen present in the test sample. Inability to detect fungal antigens within samples using simple buffer extraction procedures is not altogether disadvantageous, since antigen synthesis, induced by a period of amplification through nutrient enrichment, must be stimulated in propagules that are metabolically active and, therefore, potentially parasitic. Failure to detect antigens, without biological amplification, may also be due to other factors, both biological and chemical. For example, it might be that the antigen is not normally produced in soil or, if produced in the soil, is either destroyed by micro-organisms or is adsorbed onto soil colloids thereby rendering it inaccessible to extraction and detection. It is becoming increasingly apparent from work performed in our laboratories that both factors can play an extremely important role in antigen immobilization and degradation. Chemical effects depend to a great extent on soil type, with interference being less pronounced in soils with high sand contents. For example, experiments performed in the Cambridge laboratory, using soils spiked with antigen extracts from lyophilized mycelium of R. soluni, have shown that almost 100% of antigen introduced into a clay soil is immobilized onto the surface of soil particles immediately upon addition and that a proportion of immobilized antigen is degraded over time (W. Otten, personal communication). Little loss is experienced in whole sand, although slight reductions do nevertheless occur. The reason for degradation of antigen in soil is, at present, not fully resolved. Its importance, however, in terms of antigen loss is significantly less than immobilization of antigen onto soil colloids. This may be because the antigen in question is heavily glycosylated; in our experience fungal glycoproteins are extremely robust molecules that are less prone to rapid degradation than are low-molecular-weight peptides. Loss of antigen through immobilization on soil surfaces is more easily explained but the chemical reactions involved can be extremely complex. The removal of immobilized antigen from soil particles is difficult. In many cases simple extraction buffers such as Tris buffered saline (pH 8.2), bicarbonate (pH 9.6), acetate (pH 4.5) and, more commonly, phosphate buffered saline (pH 7.2) are inadequate. Addition of detergents or surfactants such as Tween 20 or Nonidet P40 can improve extraction but the presence of detergents in soil extracts limits their use to DAS-ELISA formats; the presence of a detergent inhibits the binding of fungal antigens to polyvinyl
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chloride (PVC) microtitre wells and hence cannot be used for PTA-ELISAs. The pH of the buffer extract is another important factor. Many soils that contain organic matter can efficiently buffer against changes in pH. This has important consequences in relation to the immunoassay itself. Acidic (pH 4.5) and alkaline (pH 9.6) buffers can be rendered neutral when mixed with certain types of soil, for example loams. However, the optimum pH for the assay buffer might be alkaline. For example, the immobilization of proteinaceous fungal antigens onto microtitre wells is most effectively performed at high pH (pH 9.6) using bicarbonate buffers. It is extremely important, therefore, that the pH of the antigen extract is adjusted accordingly. This can be done by dilution into fresh buffer, prior to antigen immobilization. Another important consideration in choice of extraction buffer pH is the solubility of humic substances in both dilute alkali and dilute acid. This is often evident from the colour of soil solutions after extraction in bicarbonate buffer for example. Solubilized humic acids will compete with fungal antigens for binding on a solid phase and further dilution of extracts is required to eliminate such effects. Humic substances make up about 6O-8O% of the soil organic matter. Humic substances are characterized by aromatic ring-type structures that include polyphenols and polyquinones and are classified into three chemical groupings, fulvic acid (FA), humic acid (HA) and humin. Non-humic substances (approximately 20-30% of organic matter in soil) are less complex than those of the humic group and are composed of specific organic compounds with definite physical and chemical properties. Included among the non-humic substances are polysaccharides and polyuronides which are synthesized by soil micro-organisms. Some simpler compounds are also part of the non-humic group, for example organic acids and some protein-like materials. Humic materials interact with minerals and a wide variety of organic compounds, including alkanes, fatty acids, pesticides, herbicides, carbohydrates, amino acids, peptides and proteins (Schnitzer, 1982). It is not surprising, therefore, that fungal carbohydrates, peptides and glycoproteins are readily immobilized on soil particles and that their extraction is difficult. Important characteristics of all humic materials are their ability to form water-soluble and water-insoluble complexes with metal ions and hydrous oxides. Recent work performed in the Cambridge laboratory (W. Otten and C. R. Thornton, unpublished) has shown that extraction of immobilized antigen can be significantly improved by the addition of certain cations to the extraction solution. It would appear that immobilization of antigen is a reversible reaction and that the formation of complexes between fungal antigens (peptides, carbohydrates, glycoproteins, etc.) and fulvic acids, for example, can be reversed by the preferential formation of organomineral complexes. Similar findings were reported by Jenkinson and Oades (1979), who found that sorption of adenosine 5’-triphosphate on soil colloids was
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minimized by the addition of the weedkiller paraquat to the extraction buffer. B. ELIMINATION AND REDUCTION OF INTERFERENCE FROM SOIL COMPONENTS
Soil extracts obtained by mixing samples with extraction buffer contain not only the target antigen but also a heterogeneous suspension of biological and chemical soil components. The simplest and most effective means of removing insoluble matter is to centrifuge suspensions at high speed; the length of centrifugation being largely dependent on the extent of contamination. In general, we centrifuge soil extracts at 12000g for at least 20min. We also found that by heating centrifuged soil extracts at 100°C for 5 min we were able to reduce significantly the amount of interference from heat-labile soil contaminants in detection assays for heat-stable mycelial glycoprotein antigens of R . solani (Thornton et at., 1993). The ability to heat soil extracts depends on the stability of the fungal antigen in question. We have observed that fungal carbohydrate antigens or antigens that contain a carbohydrate component are reasonably robust molecules that can often withstand periods at high temperatures and even autoclaving. Elimination of contaminant soil components is most important where the detection assay involves the immobilization of fungal antigen on to a solid support, for example a PVC microtitre well. Soluble soil components, for example high-molecular-weight humic substances, will compete during the binding process and in extreme circumstances can completely eliminate binding of the target antigen. In this respect, it is extremely important that sufficient dilution of the soil extract is made in order to minimize interference from soil components but not dilute the antigen beyond the level of detection. This must be determined for each soil type in order to optimize detection and quantification. Presence of soluble components presents less of a problem in DAS-ELISA formats since the target antigen is removed from a heterogeneous population of soluble molecules by a previously immobilized capture antibody. Once the target antigen has been isolated and immobilized onto a solid support (PVC microtitre well, PVDF membrane or otherwise) the wells, in the case of a PTA-ELISA, are washed with buffers to remove unbound antigen. The addition of detergents in the washing step contributes enormously to the reduction in interference by non-specific soil contaminants. Blocking of the wells prior to addition of a specific monoclonal antibody and throughout the assay is desirable, but not always necessary, to minimize non-specific binding interactions. For this purpose the non-ionic detergent Tween 20 is conventionally included in washing and incubation buffers. Tween 20 alone has not, however, proved to be an adequate blocking agent in all instances, and where this is the case addition of a non-reactive protein
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(commonly bovine serum albumin (BSA) or gelatin) to buffers has sometimes proved of value. We have found that, in the case of soil-based immunological assays, casein is a more efficient blocking agent than either BSA or gelatin. Casein is a mixture of milk proteins which differ in size, amino acid sequence, degree of glycosylation and sulphation, and also surface charge, which contrasts with the homogeneity of BSA and gelatin, the water-soluble form of collagen prepared by heat denaturation. This may explain, in our experience, casein’s superiority as a blocking agent, yielding significantly lower levels of non-specific binding than any other published strategy. Use of casein has several other merits. Casein buffers are inexpensive and easily prepared and, since a single buffer may be used for washing and as antibody diluent, the ELISA protocol is simplified. Assays using casein can be performed in the absence of detergent, which is advantageous if the antigen under study is detergent sensitive. While casein has proved of general value as a blocking agent in solid-phase assays, we found that it could not be used in the development of an immunomagnetic bead assay for the detection and quantification of R. solani in soil (Thornton, 1996a). An alternative strategy was adopted to reduce non-specific binding and involved the use of a cycle of high salt buffers during the washing process. Addition of BSA to one of the buffers also helped to reduce interference from non-specific soil components.
V. DETECTION Direct detection of fungi, in soils, is as difficult as is the direct detection of bacteria (see Schlotter et al., 1992) and other micro-organisms. This is due primarily to the problems encountered in extracting antigens from soil (see section IVA). It is not surprising, therefore, that only a few assays have been developed and most of these are for sporulating fungi, for example, zoospores of P. cinnurnorni (Cahill and Hardham, 1994b) and S. subterrunea (Harrison et al., 1993). In the case of Spongospora, Harrison et al. (1993), using polyclonal antisera, were able to achieve a higher recovery rate from spiked soils than were Wallace et al. (1995) using MAbs. Several assays have been developed for the detection of soil-borne pathogens in host plant tissues, particularly the roots and stem, or leaf tissues such as that developed by Priestley and Dewey (1993) for the detection of the eyespot pathogen of cereals, Pseudocercosporella herpotrichoides. CIBA Agrochemicals and DuPont have also developed assay kits for the detection of P. herpotrichoides on cereals. These kits are, at present, marketed only with their agrochemical products. Most of the commercially available kits are membrane-based assays that can be processed rapidly, as are the assays developed by Cahill and Hardham (1994b) for the detection of zoospores of P. cinnamomi in soils and by Priestley et al. (1994) for the detection and
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differentiation of mycelium of different species of Armillaria adherent to the inner bark tissue from infected trees. Neogen Corporation (Lansing, MI, USA) and ADGEN (Auchincruive, UK) now supply membrane-based ALERT “on-site” Crop Disease Detection Kits (originally developed by Agri-Diagnostics) for the diagnosis of Phytophthora, Pythium and Rhizoctonia spp. in root, stem and leaf samples (Miller et al. ,1992). These kits detect a wide range of important species within the target genus. These 10-min “on-site” assays that were specially designed for speed, simplicity, specificity and sensitivity were first introduced in 1988 (Rittenburg et al., 1988). The assays utilize a “flow-through” design in which the capture antibody is immobilized on the surface of a membrane. The sample extract, enzyme-conjugated secondary antibody, rinse solution, enzyme substrate and finishing solution are added sequentially from dropper bottles. The enzyme conjugated to the detector antibody is horseradish peroxidase, and the substrate is 4-chloronaphthol. As the substrate flows through the reaction area a blue precipitate forms if any bound peroxidase is encountered. Laboratory-based research assays are also available for detection of fungi in the genera Phytophthora, Pythium, Rhizoctonia and Sclerotinia (AgriDiagnostics Phytophthora E and Pythium C multiwell ELISA kits). These plate-based DAS-ELISA formats have been used by a number of workers for the detection of the pathogens in the soil or in irrigation water (Schmitthenner and Miller, 1988; Ali-Shtayeh et al., 1991; Dunsuncelli and Fox, 1992; Pscheidt et al., 1992; Timmer et al., 1993). In all assays, whether they be laboratory or field based, the incorporation of controls into the test system is imperative in order to eliminate both false-positive and false-negative results. The commercial “on-site” detection assays described above include both internal negative and positive controls. Setting of controls for soil-based immunoassays requires careful consideration because of the large number of components that might interfere with the specificity and sensitivity of assays. The best controls are extracts from comparable soils that are known to be free of the pathogen, but this may seldom be realized in practice. Alternative controls for labile antigens might include extracts from samples of test soil that have been exposed to a period of sterilization, for example steam treatment and microwaving, or soils that have been irradiated with y-rays or treated with chemicals or metabolic inhibitors such as fungicides, sodium azide or thimerosal. It should be remembered that while treatments such as these might be efficient in inhibiting or eliminating the target organisms, they might not be so effective in destroying their antigens. Moreover, the inherent variability encountered amongst soil samples means that appropriate measures should be taken in interpreting differences between test and control samples. This can be achieved by the use of simple statistical analysis, for example Student’s t-test, which allows for variation in the test sample and the negative control. However, more rigorous statistical analysis of data is often required. This
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is the topic of section VI, where the setting of thresholds for detection and the protection against false-positive and false-negative results is dealt with in relation to the broader context of quantification.
VI. QUANTIFICATION Quantification involves estimation of the amount of fungus in soil. It is different from “detection”, although the terms are often used interchangeably. Detection is concerned with determining presence or absence of the fungus. It yields a quantal variable which tells us where the fungus is but not how much is there. Detection merges into quantification only when estimates are derived from more than one sample. For example, concomitant analysis of quantal variables for presence or absence of a fungus in a large number of contiguous samples along a transect provides a profile of inoculum occurrence, from which patch size can be estimated (Gilligan et al., 1996). Two different forms of quantitative immunoassay are discussed below: one for estimating fungal biomass and one for estimating colony-forming units (cfus). These variables estimate different properties of a fungal population. Biomass is a measure of the amount of fungal material present. It may be a direct measurement of the mass of live fungal hyphae or, more rarely, fungal spores. More usually, estimation of biomass involves indirect measurement of fungal activity by the detection of a soluble antigen, whereas colony-forming units measure, indirectly, in the case of immunoassays, the number of discrete units of fungal inoculum (spores, sclerotia and/or fragments of hyphae) in soil. The choice of methods (biomass or cfus) depends on the objectives of an investigation as well as on logistical constraints. In general, conventional biomass measurements tend to be used for microbiological analyses of physiological and nutrient dynamics in soil, whereas colony-forming units have been used for epidemiological analyses where much work is centred on population dynamical changes in fungal inoculum over time and space (see e.g. Gilligan, 1994, 1995). This could, in principle, be done using measures of fungal biomass, but colony-forming units offer a convenient entity for tracking population changes, whereby each unit, though variable, is capable of initiating an infection. Colony-forming units are also more appropriate than biomass for relating the behaviour of single propagules in the rhizosphere (Gilligan, 1990) to overall epidemic behaviour (Jeger, 1987). Some approaches to the quantification and interpretation of biomass and cfus with MAbs are discussed below. A. ESTIMATION OF BIOMASS
Before using a MAb for biomass determination it is imperative that the distribution of its corresponding antigen within the fungus be known. For
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example, if a measure of total hyphal length is required then it is essential that the antigen be expressed uniformly along the length of the hypha, both old and new, and not just at the hyphal tips or on spore surfaces. It is also important to determine if the specific antigen is produced when the fungus is growing naturally in all the soil types to be investigated (see MAb-based determinations of biomass of specific-aquatic fungi in the presence of heavy metals, Bermingham et al., 1995). Estimation of biomass by ELISA requires comparison with a standard calibration curve. The calibration must be repeated each time that a sample is tested on an ELISA well, because of variability in ELISA response curves not only from day to day but also from plate to plate (Dewey et al., 1992; Bauske et al., 1994). Freshly harvested spores can be used for some fungi such as Trichodermu spp. For non-sporulating fungi, such as certain isolates of R . solani, extracts from freeze-dried mycelium are commonly used for calibration. We have also used disks of fresh mycelium of R . soluni punched from cultures growing on membranes placed over appropriate media (Thornton, 1996a). Alternatively, thawed aliquots of previously frozen surface washings of culture on solid slants or culture filtrates are sometimes preferred, especially if it is suspected that freeze drying reduces the amount of soluble antigen available. Selection of an appropriate standard for use in calibration raises numerous problems of biological interpretation in relating measures of sample activity to behaviour in the soil. Although it appears that most antigens are expressed in actively metabolizing mycelium, we do not yet know how ecological activity, disease thresholds or epidemiological activity can properly be related to equivalent quantities of pure mycelium, whether fresh or freeze dried. Nonetheless, inclusion of standard reference material, such as extracts from a stock of freeze-dried mycelium does allow comparative biomass measurements to be made. The use of spores suggests a simple interpretation of calibration curves, since the production of antigen, at least under certain carefully specified conditions, can be related to the density of spores. Problems may arise, however, when spores remain viable for long periods, but the antigen content changes with age. The problem is exacerbated when there are overlapping generations of spores of different ages, degree of antigen expression and viabilities. Clearly, more needs to be done to compare the biological activity of samples and standards. This will almost certainly have to be done using simplified microcosms to which known amounts of inoculum are added, retrieved and quantified (see below). Simplified microcosms offer the possibility of eliminating uncontrolled variation that might otherwise mask mechanisms of fungal activity and population growth in soil. Microcosms can be made progressively more complex until they resemble soil and the fate of added inoculum is monitored and analysed. At each stage of complexity, the activity of the test populations must be compared with standards by means of calibration curves.
USE OF MONOCLONAL ANTIBODIES
29 1
The construction of a calibration curve, once a standard is selected, involves the following steps:
1. Dilution of a certain aliquot of the stock suspension of spores or mycelial extract and determination of the absorbance. 2. Selection and fitting of an appropriate model to the data to provide the calibration curve. 3. Assay of the sample and estimation of the biomass by inverse prediction from the calibration curve. Most experimenters are familiar with this procedure, but surprisingly little attention is given to the statistical aspects of model fitting and use for calibration. Most calibration curves are sigmoidal, when absorbance is plotted against the logarithm of dilution. Common models which describe these curves are the logistic and the Klotz version of the Michaelis-Menten model. These models have upper and lower asymptotes and they can be used to summarize the calibration curve without necessarily inferring any mechanistic interpretation (about the mechanisms of binding). They simply describe the data efficiently. Full use of the data in fitting a model is vitally important if biased and erroneous results are to be avoided. Notwithstanding this, it is not unusual for experimenters using ELISA to work only with the “straight” portion of a calibration curve. In doing so, they are throwing away useful biological information about the behaviour of the calibration data, for even in this portion the curve is not really straight. Instead it changes from convex upwards to convex downwards and the degree of curvature in this so-called “straight” region is critically influenced by the upper and lower values that are so often ignored. It is better, therefore, to fit a model to all the data. This can easily be done for non-linear models, such as the logistic or Michaelis-Menten, using modern software packages including Genstat (Anonymous, 1987), MLP (Ross, 1987) and SAS (Anonymous, 1982). An example, for freeze-dried mycelium of R. solani, for which a logistic model was fitted using MLP, is given in Fig. 1A. The estimated biomass for two test samples are given in the figure. Estimation of biomass involves inverse prediction, that is estimation of x from y . This is contrary to conventional estimation using regression models for which y , the dependent variable, which is subject to error or uncontrolled variation, is usually predicted from the independent variable x , measured without error. Methods are described in Ross (1990) for the estimation of standard errors associated with inverse prediction. The smallest standard error occurs when y is known exactly. This is unlikely to occur in practice and is approached only when the number of identical samples to be tested is large. The degree of replication and of variation in absorbance between replicates affects the standard errors and hence the confidence intervals for the prediction of biomass. Confidence intervals for estimated biomass (in logarithmic units) are shown in Fig. 1B,C
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2.0
-
1.5
-
.E! 1.0
-
P
5: a 4
A
Calibration Curve
cv = 10%
310.
-::&J
X
. I
3 2
.5 4 0.5
8
-
3
2 -
G
0.0
c
u”
-*
0-
-------_______-----
- - - - - - _ _ _ _ _ _ _ _ _ - - - *
0.0
0.5
1.o Absorbance
1.5
Fig. 1. (A) Logistic calibration curve relating absorbance (450 nm) to log biomass of freeze-dried mycelium of R. soluni. The arrows show inverse estimation of biomass from absorbance values for two test samples. (B, C) 95% confidence intervals for estimated biomass (in logarithmic units) when absorbance is subject to variability amongst replicate samples and there are 2, 3 or 10 replicates per dilution compared with the erroneous, but commonly used, value (dashed line) when absorbance is assumed to be known exactly for a given biomass. The curves in (B) and (C) are shown for coefficients of variation of 10% and 20%, respectively. (The coefficients of variation are calculated for duplicate replications around the midpoint in the absorbance range.) Note that, for convenience, the confidence intervals are presented in logarithmic units. Back-transformation leads to asymmetrical confidence intervals.
for the more commonly used sample sizes corresponding to duplicate and triplicate samples as well as for samples with 10 replicates. The dashed line corresponds to the case when absorbance is known exactly. Graphs of confidence intervals are shown for two levels of variation expressed as coefficients of variation of the mean of duplicate samples at the midpoint in the absorbance range. A coefficient of variation of 10% represents moderate variation among test values, while 20% represents substantial
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variation. Variability between samples considerably inflates the standard error of the predicted biomass and hence the confidence interval for the population estimates (Fig. lB,C). The situation can be ameliorated by increasing sample size, but the advantage is small except at the extremes of the absorbance range. Prediction of biomass close to these extremes in absorbance is not robust. The confidence intervals are so wide as to be practically useless (Fig. 1B,C) yet detection and quantification of low levels of fungus may be very important in this range. More work needs to be done in improving sampling and assay protocols at low signals. Increasing the sensitivities of the assay systems now seems possible. For example, the use of sensitive chemiluminescence-based assays for the quantification of soil bacteria has increased the detection threshold for Azosporillum in soil extracts from lo4ml-' to 100 bacteria ml-' (Schlotter et al., 1992). Similarly, the incorporation of the lux operon of the marine vibrios into various bacterial strains has enabled the detection of a single genetically modified bioluminescent bacterial cell, in soil, by using charge-coupled deviceenhanced microscopy (see e.g. Silcock et al., 1992). B . IMMUNOLOGICAL ESTIMATION OF COLONY-FORMING UNITS
There are two reasons for using colony-forming units to quantify the behaviour of fungal pathogens in soil: one is epidemiological, and the other is procedural. The epidemiological advantages concern the desirability of measuring discrete countable units (see above). The procedural problem of using biomass estimates occurs when either there is little antigen present in soil samples or it is difficult to extract and a period of amplification is needed prior to testing by ELISA. Amplification usually involves incubation of the soil sample in nutrient solutions for 24-48 h in order to allow the fungus to increase and produce more antigen (Thornton et al., 1993). This clearly introduces bias by inflating the estimate of the initial amount of antigen and hence of fungus in the sample. Amplification does not, however, interfere with a quanta1 (presence or absence) assay for the fungus; incubation will lead to the detection of positives as the antigen titre rises in samples containing the fungus, but leave unaffected those negative samples from which the fungus is absent. Estimates of colony-forming units are derived in ELISA using most probable number (mpn) techniques, which are based on the presence or absence of a detectable signal in replicate samples (C. A. Gilligan, C. R. Thornton and D. J. Bailey, unpublished). The procedure involves:
1. Collection of replicated samples of test soil in which the density of a fungus is to be estimated. 2. Dilution of the test samples to provide replicated dilution series. 3. Determination of an absorbance threshold for positive response.
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TABLE 1 Computation of a most probable number estimate for density of R. solani in artificially infested soil, showing observed and expected numbers of positive responses under an assumption of random distribution of inoculum Dilution
Dilution volumea
(ml> 1116 1132 1164 11128 11256 11512 111024 112048
Total replicates
Observed positive replicates
2
5.00 4.89 4.26 3.08 1.90 1.07 0.56 0.29
0.06250 0.03125 0.01563 0.00781 0.00391 0.00195 0.00098 0.00049
Maximum likelihood estimate of most probable number: 95% Confidence limits Degree of freedom
Expected positive replicates
122.6 cfu mi-’ 67.2-212.7 cfu ml-’ 2.23 7
aThe dilution volume is expressed relative to a test sample of 1ml.
4. Scoring of replicates for the presence or absence of the fungus and construction of a table for the number of positives and total number of replicates at each dilution. 5 . Estimation of most probable number of colony-forming units in the parent sample using a maximum likelihood estimation. Much has been written on the number of replicates and number of dilutions to be used in quanta1 bioassays (see e.g. Finney, 1978). Adams and Welham (1995) discuss experimental conditions, replication and dilution factors in relation to the use of mpn techniques for conventional bioassays of soil-borne plant pathogens. It is important to select a range of dilutions that encompasses a gradual change from 100% positive to 100% negative responses. We have found that between five and 10 replicates per dilution is satisfactory for estimation of colony-forming units for R . soluni in sand and soil. An example of some data for R . solani is given in Table I, together with estimates of most probable number obtained using the “dilution” procedure in Genstat (Ridout and Welham, 1993). The latter is computed from the Poisson distribution under an assumption of a random distribution of colony-forming units in the samples. The assumption of a Poisson distribution is tested by comparing the expected and observed values by using the 2 statistic on 7 degrees of freedom (d.f.) (given by the number of dilutions less one). Note that the confidence intervals of 67.2 and 212.7 are markedly skewed around the mpn estimate of 122.6 cfu g-’. When there is evidence
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Fig. 2. Two dilution protocols for estimating the most probable number. Successive dilution of a single replicate can lead to autocorrelated errors. Independent dilution is effected by separating each replicate sample of soil (shown here as replicate A) into n subsamples corresponding to each of n dilutions, thereby avoiding autocorrelation amongst errors that can lead to biased estimates of colony density (see text for further details).
of clumping of inoculum, the Poisson model is unlikely to fit and the mpn should instead be estimated using a different distribution, such as the negative binomial. This can be done by writing the model explicitly in Genstat or SAS or by use of the routine in Fit Dilution in MLP (Ross, 1987). Successive replication of the same sample to provide a dilution series should be avoided whenever possible because this introduces autocorrelated errors into the dilution series (Fig. 2). Put simply, suppose that incomplete mixing of the first dilution results in a disproportionately small amount of the fungus, relative to the parent sample, in that aliquot. The aliquot will be biased and successive dilution of the aliquot compounds the bias leading to a dilution series with too many negative results giving an unrepresentatively low estimate of the density in the parent sample. The risk of autocorrelated errors can be reduced by splitting each replicate parent sample into as many subsamples as there are to be dilutions and then using a different subsample to obtain each dilution (Fig. 2). This avoids any bias by preventing disproportionate mixing from being carried through from one dilution to the next.
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C. SETTING OF THRESHOLDS FOR DETECTION AND QUANTAL ASSAY
SYSTEMS
The detection of a positive response in ELISA, whether for simple detection or for the estimation of colony-forming units, requires the setting of thresholds for absorbance. If the value for absorbance of a test sample is above the threshold the assay is recorded as positive, otherwise it is usually taken to be negative. The experimenter is confronted with two kinds of error: false positives, which inflate estimates of inoculum density; and false negatives, which deflate estimates. The problem of setting thresholds and minimizing the risk of false positives and negatives has been discussed by a number of authors, including Fenlon and Sopp (1991) and Sutulu et al. (1986). We have recently examined a range of methods for determining thresholds for the detection of R. solani in population studies. Thresholds are usually expressed relative to a negative control. Setting of a threshold, essentially, involves: 1. Selection of a negative control from which the fungus is absent. 2. Estimation of the population mean and variance for absorbance in the negative controls. 3. Estimation of the population mean and variance for the test sample. 4. Use of the estimates in (2) and (3) to compare an observed difference with some arbitrarily or theoretically defined thresholds.
Frequently used thresholds are multiples from two to four of the control mean, or the mean plus multiples of the standard error of the mean (Sutulu et al., 1986). The mean on its own should not be used since it does not take explicit account of the variability. Moreover, both the test-sample estimate and the negative control are subject to statistical error associated with natural variation and the critical difference ought to reflect this. It follows, therefore, that setting a threshold for ELISA based on a critical difference is equivalent to setting of least significant differences in classical t-tests for the comparison of two means. Three types of negative control can be used in setting thresholds: 1. Independent control samples such as pure buffer or samples from uninfested soils or, for labile antigens, samples of sterilized soil. 2. The lowest density in a dilution series. 3. The lower asymptotic value of a curve fitted to a dilution series.
Negative controls should replicate as closely as possible the solution containing the antigen. Problems arise when highly artificial controls, such as pure buffer, are used since these do not mimic exactly the non-fungal influences in the test sample. 3iased results may ensue due to unrepresentatively low signals in these controls, so that almost all samples appear to give positive signals. The problems can be reduced by the use of equivalent soil
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Most proboble number based on lowest dilutions
Time (days after infestation)
B
Most probable number based on lower asymptotes
0
5
10
I5
20
55
1
s
Tme (days after infestalion)
Fig. 3. Population dynamic curves showing the change in density of R. solani (cfu g-') in a microcosm over time. (A) Colony-forming units curve with a threshold for detection derived from negative controls based on the mean and variance of the lowest dilution in the dilution series at each time. (B) Colony-forming units curve with a threshold for detection derived from negative controls based on the expected value and variance of the asymptote of a Michaelis Menton model fitted to the dilution series at each time.
samples known to be free of the fungus or, in the case of labile antigens, soils from which the test and other fungi have been killed by sterilizing. Another alternative to circumvent bias is to use well-diluted test samples as negative controls. One possibility is to use the lowest densities in a dilution series. Alternatively, we can recognize that the signal associated with the lowest dilution occurs as part of an asymptotic (levelling-off) trend due to
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dilution (see e.g. Fig. 1A). The trend then becomes important and the asymptote, obtained by fitting a model to all the data, is a more reliable estimate of a negative control. This is so because the estimate is based on more data points, rather than on the mean at only one dilution as in the case of the lowest dilution. It follows that the degrees of freedom for the asymptotic estimate will also be larger, giving potentially smaller standard errors and reduced values for the least significant difference. In Fig. 3 we illustrate the effects of using an estimate based on asymptotes and one based on final dilutions for a population dynamic experiment involving R. solani. Inoculum of R. solani was incorporated into small sand microcosms and allowed to develop over time. Samples were removed at successive intervals. The figures show population trajectories for the density of R. soluni estimated as colony-forming units by most probable number using (a) the minimum dilution and (b) the asymptotic estimate of a Michaelis-Menten model fitted to the dilution series. It can be seen that, although the population profiles are similar, the magnitude of the population estimates differs markedly between the two estimates. The dotted lines show the confidence intervals for the most probable numbers. A preliminary account of this population work is given in Thornton (1996b). Fuller details of the comparison of the immunological with conventional methods of plate counting and of the statistical problems of estimation are given in Thornton et al., unpublished. The degree of replication of the negative controls influences the precision with which the means are estimated and hence the precision of the test. Although it is possible to improve precision of the test by increasing the replication of the negative controls on each plate, this can impose severe limitations on the numbers of wells available for test solutions on a plate, especially if the 36 outer wells are unused because of their tendency to yield atypical results (Fenlon and Sopp, 1991). Experimental designs for reducing uncontrolled error variation in ELISA plates are discussed by Bauske et al. (1994). Fenlon and Sopp (1991) discuss different sources of variation (between replicates, plates, sampling times populations) that may be used to obtain different estimates for the standard errors.
D. FALSE-NEGATIVE RESULTS
So far we have considered only protection against false-positive results. The problem of false-negative results is well known in statistical theory, where it is referred to as type I1 error, but its accommodation in practice is difficult. Suppose that a critical value for the difference between a test sample and a negative control is specified as C,with a probability of a false positive of 0.05. That is, if there is no difference between the test sample and the
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0: = 0.05
Negative
I
Negative
Positive
I Uncertain I
Positive
I
Fig. 4. Setting of critical differences to protect against false-positive and false-negative results when comparing test samples with negative controls. The sampling distributions represent the differences between the test samples and negative controls when: (A) there is no difference between the populations; (B) the populations differ by C and the critical difference is based only on a probability of false positives (a = 0.05), the null hypothesis that there is no difference is accepted when the difference between the test and negative control is greater than C and rejected when the difference is less than C, giving a probability of false negatives ( p = 0.50); (C) the populations differ by C + D and the critical difference is based on a probability of false positives (a= 0.05) and a probability of false negatives (p = 0.05). The null hypothesis is accepted when the difference between the means is greater than C + D and rejected when it is less than C with a region of uncertainty when the difference lies between C and D.
negative controls, we can expect falsely to “detect” a difference, on average, 5 times out of 100, i.e. 5% of the time (Fig. 4A). Yet the corresponding probability of false negatives may be as high as 50%. This occurs when the true difference between the test and standard is C units (Fig. 4B). Half of the samples will be below C and half above C. Hence 50% of samples will be erroneously misclassified as negatives. The probability of false negatives clearly diminishes as the true (but unknown) difference between test and control populations diverge. Given that the true difference is unknown, the a priori probability of false negatives can only be reduced by increasing the critical difference to C + D, say, where D is an additional least significant
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difference based on a t-value associated with a specified probability of false negatives, while C is a conventional least significant difference based on a t-value associated with a specified probability of false positives. Two cases are illustrated in Fig. 4 for probabilities of false negatives of 0.50 and 0.05, each with a probability of false positives of 0.05. Further details are given in Fenlon and Sopp (1991); details of the calculation of type I and type I1 errors can be found in many statistical texts. If the observed difference between a test sample and the negative controls is greater than C + D, the sample is said to be positive (Fig. 4B,C). When the observed difference is less than C, the sample is said to be negative (Fig. 4B,C). If, however, the difference lies between C and D, the result is inconclusive and, strictly, the sample should be discarded. Clearly this is not satisfactory from a practical perspective, and some compromise is likely to be found by classifying the sample as positive or negative, but this can only be done at the expense of inflating the probability of one or other type of error. We have found that attempts to reduce the probability of false negatives in assays for R. soluni from 0.5 to 0.2 substantially inflates the critical difference (C + D ) so that a large number of samples occurs in the uncertain region. These can be eliminated from estimates of the most probable number, thereby reducing the total number of replicates. This creates few problems with large numbers of replicates, but can cause serious problems in the estimation of most probable numbers when there are only five replicates to begin with. Of course, the consequences of errors involving false negatives and false positives differ. Underestimation of fungal density or failure to detect the presence of the fungus when it is present in a sample is likely to be more important in crop management than is overestimation. More work needs to be done on devising assay rubrics that give adequate protection against false negatives, without increasing the amount of replication unrealistically.
VII. VISUALIZATION Past attempts to visualize the temporal and spatial growth characteristics of soil-borne fungi in situ have inevitably resulted in the disturbance of mycelium. Early attempts at estimating fungal growth employed, amongst others, the buried slide technique described by Rossi et ul. (1936). In spite of a number of criticisms this technique has remained one of the most useful means of observing microbial growth in situ. More recently, static root windows (Egli and Kalin, 1990), minirhizotrons (Taylor, 1987) and observation chambers (e.g. Finlay and Read, 1986a) have been used to determine the basic kinetics of growth and development of the mycelia of some of the most important ectomycorrhizal fungi, and have proved extremely useful in studies of the translocation and accumulation of nutrients using radioactive
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Fig. 5. Diagrammatic representation of the irnmunoblotting assay developed for the non-destructive visualization of growth of R. solani mycelium in situ. For further details see section VII.
isotopes of carbon and phosphorus (Finlay and Read, 1986a,b). While these techniques have enabled the non-destructive observation of mycelial growth over time and space, they are hampered by their lack of flexibility. Moreover, discrimination between hyphae of individual fungal species is extremely difficult in mixed populations. The flexibility of hybridoma technology and specificity of monoclonal antibodies permit the development of techniques that allow the nondestructive visualization of mycelial growth in situ. To this end, we have developed a MAb-based immunoblotting technique (first described and
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illustrated in Thornton, 1996b) which allows the qualitative and quantitative analysis of the temporal and spatial dynamics of the saprophytic growth of R. solani. The technique is ideally suited to studying the effects of environmental variables, for example temperature and nutrient status, on fungal growth and development. Moreover, the specificity of the MAbs employed enables the efficacy of biological control agents, such as T. harzianum, to be assessed quantitatively. Moistened sand containing randomly dispersed soil-borne inoculum is placed in a plastic box and overlaid with nylon microfilament cloth with a pore diameter of 0.45 pm (Fig. 5 [l]). Alternatively, inoculum can be placed at known co-ordinates within the microcosm, using forceps. Mycelium that develops from individual units of inoculum and spreads across the surface of the microcosm is then visualized by immobilization of extracellular water-soluble antigens to a PVDF membrane [2] in a similar fashion to that described above for dip-stick and dot-blot assays. Activated PVDF membrane is placed on the microfilament cloth overnight (16 h) and then removed for processing by enzyme immunoassay. The membranes can be air dried and stored or processed immediately. Bound antigen is detected by incubating the membrane in R. solani-specific IgM MAb supernatant [3](a) followed by anti-mouse IgM gold conjugate [3](b) and subsequent silver enhancement [4]. After visualization, the membrane is air dried and can be stored indefinitely for subsequent image analysis. Figure 6 shows a photographic image of a typical R. soluni anastomosis group 2-1 colony visualized using the immunoblotting system described. To gain maximum assay sensitivity and resolution we use gold conjugates, but enzyme conjugates with coloured chromogen products, for example horseradish peroxidase and alkaline phosphatase, can also be used.
VIII.
CONCLUDING REMARKS
Remarkable progress has been made in the development and application of immunological methods for the detection, quantification and visualization of soil-borne fungi using MAbs. MAb-based methods can have a considerable advantage over nucleic acid-based procedures in detecting only live rather than total (living and dead) fungal material. We do not, however, advocate exclusive use of molecular and immunological techniques for investigation of soil fungi. Comparative studies are necessary to examine the merits, sensitivity, bias and logistic constraints of immunological and molecular methods. Species specificity can now easily be achieved with MAbs and it is possible to raise separate antibodies to distinguish between spores and mycelium of a single species. This enables quantification of the different components of the fungal population. Greater flexibility can be achieved by selecting two antibodies from different immunoglobulin classes or subclasses
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Fig. 6. Photographic image of an immunoblot of a 48-h-old mycelial colony of R. solani produced using the technique described in section VII and outlined in Fig. 5 (scale bar = 1.5cm).
that are suited to different assay formats, particularly for immunomagnetic bead assays, where one can be used as the capture and the other as the detector antibody. Much has been done to improve the sensitivity of assays. Many problems have yet to be resolved in the extraction of antigens from soil. This work can best be done by immunologists working closely with soil chemists and microbiologists, not only in the exploitation of MAbs, but also early in the screening and selection of hybridoma cell lines. The prospects for successful extraction of antigen are more promising if the immobilization of antigens in soils can be reversed, as we now think for certain can be done with some glycoprotein molecules. The problems of extraction from soil are closely related to amplification of the signal from the extractant and to converting the signal to measures of biomass. The inherent non-linearities in the production of antigen during incubation in a biological amplification step make the quantification of initial amounts in the test samples difficult. Double the signal does not reasonably imply double the initial amounts. This in turn has led to the development of immunological methods based on the presence
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or absence of a signal, giving most probable number estimates of colonyforming units. Other problems concern the selection of appropriate internal standards for the estimation of biomass. We anticipate that much profitable effort will be given to the development and testing of methods for the efficient sampling and quantification of soil-borne fungi using MAbs.
ACKNOWLEDGEMENTS The authors gratefully acknowledge support from the Biotechnology and Biological Sciences Research Council for some of the research work described in this chapter, and F. M. Dewey wishes to thank the Leverhulme Trust for continued support. We also thank Wilfred Otten and Douglas Bailey for permission to reproduce some of their data.
REFERENCES Adams. M. J. and Welham. S. J. (1995). Use of most vrobable number techniaues to quantify soil-borne plant pathogens. Annali of Applied Biology i26, 181-196. Ali-Shtayeh, M. S., McDonald, J. D. and Kabashima, J. (1991). A method for using commercial ELISA tests to detect zoospores of Phytophthora and Pythium species in irrigation water. Plant Disease 75, 305-311. Anonymous (1982). “SAS User’s Guide: Statistics.” SAS Institute, Cary, NC. Anonymous (1987). “Genstat 5: Reference Manual.” Oxford University Press, Oxford. Arie, T., Hayashi, S., Schimazaki, K., Yoneyama, I. and Yamaguchi, I. (1993). Novel diagnosis of Fusarium infestation of seedlings and soils by immunoassay methods. In “Proceedings of the Sixth International Congress of Plant Pathology”, Montreal, Canada, July-August 1993, p. 40, Abstract 2.1.15. Banttari, E. E., Clapper, D. L., Sheau-Ping, Hu., Daws, K. M. and Khurana, S. M. P. (1991). Rapid magnetic microsphere enzyme-linked immunoassay for potato virus X and potato leafroll virus. Phytopathology 81, 1039-1042. Barclay, S. L. and Smith, A. M. (1986). Rapid isolation of monoclonal antibodies specific for cell surface differentiation antigens. Proceedings of the National Academy of Sciences, USA 83, 4336-4340. Bauske, E. M., Hewings, A. D., Kolb, F. L. and Carmer, S. G. (1994). Variability in enzyme-linked immunosorbent assays and control of experimental error by use of experimental designs. Plant Disease 78, 1206-1210. Bermingham, S., Dewey, F. M. and Maltby, L. (1995). Development of a monoclonal antibody-based immunoassay for the detection and quantification of Anguillospora longissima colonizing leaf material. Applied and Environmental Microbiology 61, 2606-2613. Cahill, D. M. and Hardham, A. R. (1994a). Exploitation of zoospore taxis in the development of a novel dipstick immunoassay for the detection of Phytophthora cinnamomi. Phytopathology 84, 193-200. Cahill, D. M. and Hardham, A. R. (1994b). A Dipstick immunoassay for the specific detection of Phytophthora cinnamomi in soils. Phytopathology 84, 1 2 8 4 1292. .
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Harrison, J. G., Rees, E. A., Barker, H. and Lowe, R- (1993). Detection of spore balls of Spongospora subterranea on potato tubers by enzyme-linked immunosorbent assay. Plant Pathology 42, 181-186. Holtz, B. A., Alexander, E. K. and Weinhold, A. R. (1994). Enzyme-linked immunosorbent assay for detection of Thielaviopsis basicola. Phytopathology 84, 977-983. Jeger, M. J. (1987). The influence of root growth and inoculum density on dynamics of root disease epidemics: theoretical analysis. New Phytologist 107, 459-478. Jenkinson, D. S. and Oades, J. M. (1979). A method for measuring adenosine triphosphate in soil. Soil Biology and Biochemistry 11, 193-199. Jones, K. and Shew, H. D. (1988). Irnmunoassay procedure for the detection of Phytophthora parasitica var. nicotianae in soil. Phytopathology 78, 1577 (abstract). Kitagawa, T., Sakamoto, Y . , Furumi, K. and Ogwra, H. (1989). Novel enzyme immunoassays for specific detection of Fusarium oxysporum f s p . cucumerinum and for the general detection of various Fusarium species. Phytopathology 79, 162-1 65. Klopmeyer, M. J., Miller, S . A., Rittenburg, J . H., Petersen, F. P. and Grothaus, G. D. (1988). Detection of Phytophthora in soybean soil by immunoassay analysis of infected bait. Phytopathology 52, 1576 (Abs.). Kohler, G. and Milstein, C. (1975). Continuous culture of fused cells secreting antibodies of predefined specificity. Nature 256, 495497. Lyons, N. F. and White, J . G. (1992). Detection of Pythium violae and Pythium sulcatum in carrots with cavity spot using competition ELISA. Annals of Applied Biology 120, 235-244. MacDonald, J. D. and Duniway, J. M. (1979). Use of fluorescent antibodies to study the survival of Phytophthora megasperma and P. cinnamomi zoospores in soil. Phytopathology 69, 436-441. Miller, S. A,, Rittenburg, J. H., Petersen, F. P. and Grothaus, G. D. (1992). From the research bench to the market place: development of commercial diagnostic kits. I n “Techniques for the Rapid Detection of Plant Pathogens” (J. M. Duncan and L. Torrance, eds), pp. 208-221. Blackwell Scientific, Oxford. Miller, S. M., Petersen, F. P., Miller, S. A . , Rittenburg, J. H., Wood, S. C. and Grothaus, G. D. (1989). Development of a direct immunoassay to detect Phytophthora megasperma f s p . glycinea in soil. Phytopathology 79, 1139 (abs.). Mitchell, A. J., Mackie, A. J., Roberts, A. M., Hutchison, K. A., Estrada-Garcia, M. T., Callow, J. A. and Green, J. R. (1994). Specificity of monoclonal antibodies raised to Pythium aphanidermatum and Erysiphe pisi. In “Modern Assays for Plant Pathogenic Fungi” (A. Schots, F. M. Dewey and R. Oliver, eds). CAB International, Wallingford. Priestley, R. and Dewey, F. M. (1993) Development of a monoclonal antibody immunoassay for the eyespot pathogen of cereals, Pseudocercosporella herpotrichoides. Plant Pathology 42, 403412. Priestley, R., Mohammmed, C. and Dewey, F. M. (1994). The development of monoclonal antibody-based ELISA and dipstick assays for the detection and identification of Armillaria species in infected wood. In “Modern Assays for Plant Pathogenic Fungi” (A. Schots, F. M. Dewey and R. Oliver, eds). CAB International, Wallingford. Pscheidt, J. W., Burket, J. Z . , Fischer, S. L. and Hamm, P. B . (1992). Sensitivity and clinical use of Phytophthora-specific immunoassay kit. Plant Disease 76, 928-932.
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Rossi, G. M., Riccardo, S., Gesue, G., Stanganella, M. and Wang, T. K. (1936). Direct microscope and bacteriological examination of the soil. Soil Science 41, 53-66. Schlotter, M., Bode, W., Hartmann, A. and Beese, F. (1992). Sensitive chemiluminescence-based immunological quantification of bacteria in soil extracts with monoclonal antibodies. Soil Biology and Biochemistry 24, 399403. Schmitthenner, A. F. and Miller, S. (1988). ELISA detection of Phytophthora from soil. Phytopathology 78, 1576 (abs.). Schnitzer, M. (1982). Organic matter characterization. In “Methods of Soil Analysis: Part 2 - Chemical and Microbiological Properties” (A. L. Page, R. H. Miller and D. R. Keeney, eds), Agronomy 9(2). Soil Science Society of America, Madison, WI. Silcock, D. J., Waterhouse, R. N., Glover, L. A., Prosser, J. I. and Killham, K. (1992). Detection of a single genetically modified bacterial cell in soil by using charge coupled device-enhanced microscopy. Applied and Environmental Microbiology 58, 2444-2448. Sutulu, C. L., Gilett, J. M., Morrisey, S. M. and Ramsdell, D. C. (1986). Interpreting ELISA data and establishing the positive-negative threshold. Plant Disease 70, 722-726. Taylor, H. M. (1987). Minirhizotron observation tubes. Methods in application for measuring rhizosphere dynamics. American Society of Agronomy Special Publication 50. Thornton, C . R. (1996a). Detection and quantification of Rhizoctonia solani in soil by monoclonal antibody-based immuno-magnetic bead assay. Soil Biology and Biochemistry 28, 527-532. Thornton, C. R. (1996b). Development of monoclonal antibody-based immunoassays for the quantification of Rhizoctonia solani and Trichoderma harzianum in soil. In “Proceedings of the NORFA/OECD funded Workshop: Monitoring Antagonistic Fungi deliberately released into the Environment”. Royal Veterinary and Agricultural University, Copenhagen, Denmark, 1-3 April, 1995. pp. 147-153. Kluwer Academic Press, Dordrecht. Thornton, C. R. and Dewey, F. M. (1996). Detection of phialoconidia of Trichoderma harzianum in peat-bran using a monoclonal antibody-based enzyme-linked immunosorbent assay. Mycological Research 100, 217-222. Thornton, C. R., Dewey, F. M. and Gilligan, C. A. (1993). Development of monoclonal antibody-based immunological assays for the detection of live propagules of Rhizoctonia solani in soil. Plant Pathology 42, 763-773. Thornton, C. R., Dewey, F. M. and Gilligan, C. A . (1994). Development of a monoclonal antibody-based enzyme-linked immunosorbent assay for the detection of live propagules of Trichoderma harzianum in a peat-bran medium. Soil
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Function of Fungal Haustoria in Epiphytic and Endophytic Infections
P. T. N. SPENCER-PHILLIPS
Department of Biological Sciences, University of the West of England, Bristol, BSl6 I QY, UK
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......................... The Challenge of Biotrophic Nutrient Accumulation ....................... A. The Apoplastic Environment ................................................
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I. Introduction
11. Strategies for Nutrition and Biotropy: an Overview 111.
IV. The Role of Haustoria and Intercellular Hyphae in Transfer Intercept .................................................................................. A. Vascular Association: General Considerations ......................... B. Minor Vein Type, Mechanisms of Phloem Loading and Interception Strategy ........................................................... C. Evolutionary Aspects ..........................................................
............................................... Summary and Research Priorities ................................................. Acknowledgements .................................................................... References ...............................................................................
V. Alternative Functions of Haustoria VI.
313 314 316 317 318 324 325 327 328 328
I. INTRODUCTION “Rust, powdery mildew and downy mildew. . .fungi.. . are obligate parasites that extract their nutrients from living plant cells by means of specialized feeding structures (haustoria) formed within plant cells.” (Lawrence et al., 1994). Is this widely held belief about the function of haustoria true and supported by convincing evidence? The answer is “yes”, for some powdery mildew fungi Advances in Botanical Research Vol. 24 incorporating Advances in Plant Pathology ISBN 0-12-005924X
Copyright 0 1997 Academic Press Limited All rights of reproduction in any form reserved
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(and it is probably true for all epiphytic species), but the role of haustoria in downy mildew and rust infections remains to be proved. Haustoria provide the obvious route for nutrient transfer in epiphytic powdery mildew fungi (but see Myriogenosporu, section 11), but downy mildew and rust fungi also have endophytic mycelia which provide an extensive, additional interface for nutrient accumulation. Haustoria were defined by Bushnell(l972) as specialized structures formed inside living host cells as branches of intercellular hyphae, which terminate in host cells and probably have a role in the exchange of substances between the host and fungus. This remains a useful working definition, because it does not define a role restricted to nutrient accumulation by the pathogen, and encompasses the possibility of a two-way, molecular dialogue between haustorial and host cytoplasms. Importantly, it also does not imply that haustoria are the only route for nutrient uptake. Thus in a comparative review of microbial pathogens and parasitic plants, Ayres et al. (1996) emphasize that “apoplastic nutrients are sufficient qualitatively and quantitatively to support the growth of rust and downy mildew fungi . . . just as they are sufficient to support the growth of angiosperm parasites”. They conclude that “Clearly too much emphasis has previously been put on the haustorial route in fungal associations”. It is significant that heterotrophic, parasite angiosperms (holoparasites) are entirely dependent on their host for photoassimilates, and that some photosynthetic hemiparasites are able to reach maturity in darkness by satisfying all their carbon requirements from the apoplast of their host. The efficiency of this strategy is indicated by the fact that the ratio of parasite-to-host biomass may reach 80%, whilst in fungal-plant associations this is usually far less (Ayres et al., 1996). Clearly the apoplast can provide sufficient nutrients for extensive parasite growth, particularly if the host metabolism is manipulated via enzymes, hormones and other regulatory molecules. The intention of this chapter is not to provide an in-depth review of haustorial structure in relation to function (covered extensively elsewhere, e.g. Manners and Gay, 1983; Gay and Woods, 1987; Manners, 1989; Smith and Smith, 1990; Harder and Chong, 1991), but to focus on broader issues by challenging assumptions and stimulating fresh approaches based on recent developments in the understanding of solute regulation and transport in plant tissues. It is also hoped to convince plant pathologists of the need to devise experiments to determine the function of haustoria, especially in endophytic infections, and to focus equal attention on non-haustorial routes for nutrient accumulation and towards roles for haustoria beyond that of simply providing an interface for nutrient transfer. Indeed it is encouraging to note that this broader outlook has been adopted in some of the new-generation textbooks on fungi and plant pathology (e.g. Isaac, 1992; Carlile and Watkinson, 1994).
FUNGAL HAUSTORIA IN EPIPHYTIC AND ENDOPHYTIC INFECTIONS 31 1
11. STRATEGIES FOR NUTRITION AND BIOTROPHY: AN OVERVIEW The ability of plant parasites to accumulate photoassimilates and other nutrients from their hosts is a key requirement for success, with inability to obtain nutrients resulting in arrested growth in otherwise compatible interactions. For example, cucumber leaves with depleted sugar content do not support development of powdery mildew (Sphaerotheca fuliginea) colonies unless the tissue is supplemented with sucrose (Arimoto and Homma, 1995). Subsequent success of colony growth correlates directly with the concentration of sucrose supplied. Nutritional properties of plant tissues have also been shown to affect expression of pathogenesis and avirulence genes in bacterial and fungal infections (see Van den Ackerveken et al., 1994). Among the pathogenic fungi, two broad strategies have been adopted, namely biotrophy and necrotrophy (Lewis, 1973). The two strategies are not mutually exclusive, and it is possible that some fungi classed as necrotrophs obtain nutrients biotrophically for a limited period before host cell death occurs (Heath, 1987). The mechanisms employed may be similar to those described for the so-called hemibiotrophs such as Colletotrichum lindemuthianum (O’Connell and Bailey, 1991) and Phytophthora infestans (Hohl, 1991). Furthermore, there is no phylogenetic restriction to one strategy or another, with closely related groups of pathogens showing a gradation from extreme biotrophy to extreme necrotrophy. A good example is provided by the Peronosporales (Oomycetes) where Pythium, Phytophthoru and Peronospora spp. span the spectrum (Ingram, 1981). Concern here, however, is with the biotrophic strategy, where nutrients are obtained from living cells of the host over a prolonged period. Many biotrophic pathogens are ecologically obligate, and can only complete their life cycle by producing spores for dispersal or dormancy on living host tissue. This prolonged association has required adaptation to the extreme environments presented by inter- and intracellular locations (Cooke and Whipps, 1987), which have the potential to hamper fungal growth through production of mechanical barriers and biochemical defences, as well as compete for nutrients throughout the duration of the interaction. Thus biotrophic fungi tend to form specialized, host-specific associations, which involve manipulation of host structure, physiology and metabolism to ensure a continued supply of soluble nutrients such as carbohydrates, minerals, growth factors and vitamins. It is pertinent to note that Oomycete fungi evolved a hyphal, and hence haustorial, habit independent of other fungi. Therefore data relating to mechanisms of biotrophy may not be directly comparable between the different groups of biotrophic pathogens. However, these mechanisms have probabIy evolved in parallel, as phylogenetically diverse fungi have
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TABLE I The main growth habits of biotrophic plant pathogenic fungi, with examples ~~
~
Growth habit
Examples
Epiphytic Non-haustorial Haustorial
Myrogenospora atrarnentosa Erysiphe, Sphaerotheca, Uncinula spp.
Epiphytic and endophytic Intercellular hyphae, haustorial Endophytic Intercellular hyphae, non-haustorial Intercellular and intracellular hyphae Intercellular hyphae, haustorial Exclusively intracellular
Leveillula, Phyllactinia spp. Taphrina spp., Cladosporium fulvum Claviceps purpurea, Ustilago maydis Bremia, Peronospora, Puccinia, Uromyces spp. Olpidium spp., Plasmodiophora brassicae
responded to a similar range of stimuli and nutritional challenges within their host plants (evolutionary aspects are considered in section IVC). The fact that a variety of assimilate distribution systems (including mechanisms of phloem loading and unloading) are found in different plants, and indeed organs, was recognized by Hancock and Huisman (1981). The implications of this with regard to the need for a matching variety of strategies for pathogen nutrition are discussed in section IV. Thus biotrophic pathogens have adopted a wide range of growth habits, and include epiphytic and endophytic, haustorial and non-haustorial species (Table I). The powdery mildew fungi (Erysiphales) comprise the epiphytic haustorial group, where haustoria typically represent the only fungal structures in an endophytic location. These haustoria are restricted to epidermal cells, except in the genera Leveillulu and Phylluctiniu which, as well as forming extensive epiphytic mycelia, also invade via stomata to produce endophytic hyphae with haustoria in mesophyll cells (e.g. in Phylluctiniu guttutu infections of Corylus uvellunu; Spencer-Phillips, 1984). Not all epiphytic pathogens form haustoria, however. An interesting example is provided by the fungus Myrogenosporu utrurnentosu (Clavicipitiaceae) which causes tangletop symptoms on grasses. The disease is so named because epiphytic stromata bridge the leaves, and are formed in the folds presented by the adaxial surface, adjacent to the midrib. Whilst there is evidence that the shape and size of leaf epidermal cells are modified by infection, the appearance of their cuticles stained with Sudan IV is not altered, and they do not appear to be penetrated by the fungus (Smith et al., 1985). Thus haustoria are not essential even for epiphytic pathogens. The downy mildew (Peronosporaceae) and rust (Uredinales) fungi com-
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prise the main groups of endophytic pathogens which form haustoria. Smut fungi (Ustilaginales) and other biotrophs such as Claviceps purpurea produce intracellular hyphae during certain stages of the infection process (Snetselaar and Mims, 1994; Tudzynski et al., 1995), whilst others such as Taphrina spp. and Cladosporium fulvurn are exclusively non-haustorial (Syrop, 1975; Van den Ackerveken and De Wit, 1995). Other pathogens such as Plasmodiophora and Olpidium spp. are endophytic, but strictly intracellular with no intercellular phase (Bracker and Littlefield, 1973), although their status as obligate biotrophs needs revision in light of evidence that Plusmodiophora brassicue can grow saprotrophically (Arnold et al., 1996). The most intimate contact achieved between a biotrophic fungus and plant cell is in some chytrid infections where the fungal plasma membrane alone separates fungal and plant cytoplasms. Direct continuity of cytoplasms has not been reported as anything other than a freak occurrence in fungal infections (e.g. O’Connell et al., 1984), but is a pathway that has been exploited by mycoparasitic fungi (Jefferies and Young, 1994) and parasitic plants. For example, the primary haustorium of the non-photosynthetic holoparasite Orobanche crenata and infected Vicia narbonensis roots has phloem sieve elements in direct continuity (Dorr and Kollman, 1995). Their cytoplasms are connected via sieve pores derived from interspecific plasmodesmata, and it is assumed that this provides a direct route for sucrose accumulation by the parasite. Xylem-to-xylem contacts are more common, with solutes from both xylem and phloem typically transported apoplastically at the parasitic interface (Ayres et al., 1996). This is therefore directly analogous to most fungus-plant associations, where the interface usually comprises host and fungal plasma membranes separated by extracellular matrices.
111. THE CHALLENGE OF BIOTROPHIC NUTRIENT ACCUMULATION The challenges to biotrophic pathogens are, firstly, to be successful in establishing an interface with the host across which nutrients can be obtained (this includes pre-invasion events and avoiding, tolerating or suppressing defence responses), and secondly to outcompete the plant for the nutrients required to sustain growth and allow completion of the life cycle. Thus it is appropriate to focus attention on the positioning of the interface in relation to supply of nutrients, and to consider how this facilitates competition with the host and manipulation of nutrient supply. Mechanisms are required to divert some or all of the carbohydrate, up to 80% of the total product of photosynthesis in some plant species (Bush, 1993), that is normally exported from mature leaves. Clearly a key target must be the process of phloem loading, which may involve manipulation of primary active transport
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P. T. N. SPENCER-PHILLIPS SOURCES
SINKS Respiration and growth metabolism Storage
Photosynthesis 0
Translocation Normal host activities
HEALTHY
NUTRIENT POOL
.........
..~............ ... Pathogen-related activities
INFECTED
Pathogen.respirationand growth metabolism
Translocation
Pathogen-relatedresponses 0
Defence responses
Fig. 1. Carbon flow through the nutrient pool within mature tissues of healthy and biotroph-infected leaves. Sources of carbohydrate include carbon fixed locally by photosynthesis, together with translocates from uninfected regions. The nutrient pool comprises soluble translocates and metabolites as well as insoluble storage carbohydrates within the apoplastic and symplastic compartments. Within infected tissues, normal host activities are added to by pathogen-related sinks. (Reprinted from Ayres et al., 1996, by courtesy of Marcel Dekker Inc.)
processes (e.g. H + extrusion by plasma membrane ATPases) or secondary active transport (e.g. proton-coupled sucrose and amino acid transport). It is necessary to consider first, however, the apoplastic environment as a habitat for microbial growth. A. THE APOPLASTIC ENVIRONMENT
Essentially, there are two components of the nutrient pool (Fig. 1) within infected tissues, represented by the apoplast and symplast. The symplast, which is the living part of plant cells bounded by the plasma membrane, is a rich source of nutrients which will sustain microbial growth. Nutrients within the apoplast, which includes cell walls and xylem vessels, are likely to be more restricted. 1. Organic solutes
Hancock and Huisman (1981) drew attention to the fact that apoplastic concentrations of amino acids ( 2 - 3 m ~ ) , sugars (up to 1 0 m ~ and ) other solutes are both replenished from symplastic sources and sufficient to support
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prolonged growth of endophytic micro-organisms. The presence of organic solutes at these concentrations has been confirmed for apoplasts of a number of plant species. Whilst sucrose and glucose are almost universally present, fructose is apparently usually absent (see e.g. Delrot, 1989). Tetlow and Farrar (1993), however, showed that in the apoplast of barley leaves, fructose and glucose concentrations were similar, and the relative proportion of fructose increased following infection by Puccinia hordei. The proportion of total leaf sucrose in the apoplast was 0.5% and 0.3% for healthy and infected barley leaves, respectively, whilst infection increased apoplastic hexoses from 1.6% to 4.2% of the leaf content. The nutrient content of apoplastic solutions was also recognized by Durbin (1984), who likened them to “a very dilute microbial culture medium”. The apoplast also resembles a continuous culture system, because the apoplastic solution is not a stagnant pool, but a dynamic system involved in solute translocation and regulation. Thus the time taken for a solute such as glucose to equilibrate 1 mrn (approximate distance from the centre to outside of a P. hordei pustule) in the apoplast is estimated to be about 6 min, which is about three times the half-life of carbon in the apoplast of healthy leaves (Tetlow and Farrar, 1993). Walls of plant cells that are not lignified or suberized contain pores ranging from 3 to 8 nm diameter, which are large enough for free diffusion of solutes, including small proteins, although diffusion coefficients through the pores are at least one order of magnitude less than in free solution for ionic solutes (see Grignon and Sentenac, 1991). Farrar (1992) also reported that concentrations of apoplastic sugars are sufficient for hyphal growth in rust infections. Calculation of the potential hexose flux through haustoria showed it to be more than sufficient to support growth of rust colonies, but intercellular hyphae would allow a flux almost 14-fold greater (Farrar, 1984). The theoretical value for rust haustoria compares well with the vzlue obtained experimentally for Erysiphe pisi infections (see Ayres et al., 1996). Reference to Fig. 1 suggests two main locations in leaf tissue that are advantageous for intercepting host photoassimilates within the symplastic and apoplastic compartments of the nutrient pool. These are the photosynthetic cells themselves, typified by mesophyll, and the vascular tissue, where nutrients destined for export in the phloem (especially carbohydrates and nitrogen-containing organics) as well as those entering via the xylem can be intercepted. In infected tissue, as well as solutes and water within the transpiration steam, this may include organic solutes in the phloem, because leaves with heavy infections can become net importers of photoassimilates (Farrar and Lewis, 1987). 2. Inorganic solutes and water As inorganic ions account for about 25% of apoplastic solutes, they comprise an important element of the apoplastic environment (Cosgrove and Cleland,
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1983), although this has often been overlooked in relation to the apoplast as a habitat for endophytic micro-organisms. The ionic conditions in the apoplast have been reviewed recently by Grignon and Sentenac (1991). The external surfaces of plant cell walls have a particularly high cation binding capacity, and consequently have a concentrated ionic content (Starrach and Mayer, 1986). The electrostatic field of fixed charges in cell walls is thought to affect membrane transport, and this may be a means by which fungi (and perhaps host responses) can modulate transport processes. Changes in apoplastic pH, which occur following infection, provide an insight of how this may operate (see section IVB1). Whilst apoplastic pH ranges from about 4 to 7, in most plant tissues it lies between pH 5 and 6.5. Generally the pH is higher for dicotyledons than monocotyledons, and higher for angiosperms than gymnosperms (Pfanz and Dietz, 1987). However, a number of abiotic and biotic factors influence apoplast pH, which also varies in the same cell and between cells and tissues according to their function and physiological state (Grignon and Sentenac, 1991). The concentrations of other ions in the apoplast of leaves are variable, depending on the balance between import and export (xylem and phloem) and utilization by cells. A consequence is that the concentrations are generally lower in the apoplast than xylem sap (Jachetta et al., 1986). K+ is the major ion typically present in the apoplast at concentrations of between 2 and l o o m . Current theory on the transpiration stream in leaves (Canny, 1990) proposes that water enters the symplast in the parenchyma or bundle sheath cells very close to the xylem elements, with many solutes left in the apoplast. This is likely to produce gradients of osmolarity within leaf tissues, which may influence pathogen growth. As well as mineral elements, xylem sap may also contain organic solutes such as malate, amino acids, sucrose and hormones.
IV. THE ROLE OF HAUSTORIA AND INTERCELLULAR HYPHAE IN TRANSFER INTERCEPT Fungi are likely to have adopted strategies for intercepting nutrients moving through the apoplastic and symplastic compartments of host tissues. Gay (1984) referred to such fungi as causing “transfer intercept” infections. He used C. purpurea to illustrate this notion, which envisages the fungal mycelium outcompeting the developing embryo for nutrients within the apoplast of the endosperm cavity. This example, however, involves growth of the fungus at the interface of the ovary and the tip of the rachilla, the primary function of which was already to mediate nutrient transfer towards the site of fungal infection, which eventually replaces the developing ovary
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(Tudzynski et ul., 1995). Other fungi probably intercept translocates in host tissues specialized for unloading from the phloem, but the notion of a transfer intercept strategy can also be extended to foliar biotrophs such as downy mildew and rust fungi.
A. VASCULAR ASSOCIATION: GENERAL CONSIDERATIONS
It is not unusual for intercellular hyphae of biotrophic pathogens to grow in close association with vascular tissue. This may be because a lack of intercellular spaces has restricted growth through the tissue: for example, colonies of Peronospora viciae are sometimes bounded by larger veins, with hyphae forced to grow in parallel proximity with the vascular tissue, whilst minor veins are traversed more readily (Clark and Spencer-Phillips, 1994). Advantages of growth in close association with vascular tissue, especially the phloem, include potential access to the highest concentration of organic solutes within the colonized leaf, and to the nutrient pool of non-infected parts of the plant (Fig. 1). P. hordei forms hyphae which grow in parallel with veins in barley leaves (Farrar, 1992), and an increased frequency of this phenomenon has been correlated with increasing age of infection in other rusts (Larous and Losel, 1993). Invasion of the vascular tissue by the dikaryotic phase of rust fungi is restricted mostly to formation of haustoria in bundle sheath cells, although there are isolated reports of penetration to form intracellular hyphae or haustoria within xylem and phloem cells (e.g. Puccinia graminis; Andreev et al., 1982). The monokaryotic phases, however, seem to specialize towards vascular infection of both woody and herbaceous hosts (Larous and Losel, 1993). In these, hyphae penetrate vascular bundles to form haustoria in all cell types. Evidence for the direct accumulation of solutes by these hyphae in Pucciniu infections has been provided by experiments with tritiated glycerol fed to infected leaves via the vascular tissue (reported in Larous and Losel , 1993). Preferential colonization of vascular tissue also results in a very close association of pustules with veins, which becomes apparent when infected leaves are macerated enzymically (Farrar, 1992). As the transverse veins in graminaceous plants provide the major export and import routes for photoassimilates and xylem translocates respectively (Canny, 1990), fungal colonies may develop preferentially at these sites. Likewise, solute scavenging cells may be targeted. Thus the rust variants of the transfer intercept strategy are likely to be particularly useful for intercepting host translocates from uninfected tissues (Fig. 1; but see section IVBl for interception of exports), especially in later stages of infection. As the infected tissue becomes desiccated following rupture of the epidermis due to sporulation, and the photosynthetic activity
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of mesophyll cells has declined to an extent that they are net importers of photoassimilates, the only new solutes available to support further colony growth and sporulation will be within the vascular tissue. As Larous and Lose1 (1993) point out, it is unclear why in rust fungi vascular penetration appears to be characteristic of the monokaryotic phase alone. They suggest that it may be related to the relative inefficiency of monokaryotic, filamentous haustoria and a more restricted production and regulation of cell-wall degrading enzymes in the dikaryotic phase. However, it should be remembered that there is no evidence for the relative importance of intercellular hyphae versus haustoria in these vascular infections. All fungal structures within the vascular bundle are well placed to interfere with sieve element loading, which is distinct from phloem loading (Van Bel, 1993).
B . MINOR VEIN TYPE, MECHANISM OF PHLOEM LOADING AND INTERCEPTION STRATEGY
Another variant of the transfer intercept strategy is to intercept photoassimilates in transit from source cells (leaf mesophyll) to the sieve elementcompanion cell complex of the phloem. Knowledge of the transport pathway involved in phloem loading, which is probably different in different plant families (Van Bel, 1993), is clearly essential before consideration of the propriety of a particular strategy for a specific host-parasite combination. Indeed it is likely that different intercept strategies have been adopted by different biotrophs.
1. A diversity of strategies Following an extensive survey of 700 dicotyledonous, monocotyledonous and coniferous plant species, Gamalei (1989) classified minor veins of leaves to be of either the open type (type l ) , with numerous plasmodesmata connecting mesophyll and phloem intermediary cells (taken here to include companion cells, see Van Be1 and Gamalei, 1992) or the closed type (type 2), with few or no plasmodesrnata at the boundary between mesophyll and phloem. The closed type was subdivided into three subtypes according to whether intermediary cells were differentiated into transfer cells (type 2b; undifferentiated in 2a) with characteristic wall ingrowths, and whether plasmodesmata connected bundle-sheath and mesophyll cells, but not intermediary cells (type 2c). The open type of minor vein is typical in trees and shrubs, but also in some herbaceous genera such as Cucurbita, Ipomoea and Commelina (Gamalei, 1989). In these, the main pathway of phloem loading is symplastic (i.e. from cytoplasms of mesophyll to intermediary cells via plasmodesmata), and 20-80% of the translocated sugars within the phloem sap are oligosaccharides (e.g. raffinose, stachyose) and sugar alcohols (e.g. mannitol, sorbitol).
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The closed type is predominant amongst dicotyledonous, herbaceous genera, including Arachis, Beta, Brassica, Glycine, Helianthus, Lactuca, Phuseolus, Pisum, Senecio and Vicia, as well as the majority of monocotyledonous species. These hosts are often used in studies of biotrophic pathogens. Gamalei (1989) makes the important point that coincidence of vein type and growth form (woody o r herbaceous) within dicotyledons only occurs in about 80% of species examined, and so just because a species is herbaceous does not mean that it has type 2 minor veins. Species of this type translocate sucrose almost exclusively, with phloem loading by the apoplastic route. Vascular infection would seem to be particularly advantageous in woody hosts with type 1 veins, where mainstream products of photosynthesis remain symplastic between mesophyll and intermediary cells. Once within the vascular tissue, a pathogen would benefit from interacting intimately with protoplasts containing high concentrations of photoassimilates, and cause leakage that can be accumulated by haustoria and intercellular hyphae. Arguably, one role of haustoria may be to induce leakage, and so there is no need for neckbands (impermeable regions around necks of haustoria, which delimit the special apoplast of the extrahaustorial matrix: Heath, 1976; Gay and Manners, 1987); indeed they would restrict nutrient accumulation to haustoria and therefore not take advantage of the much more extensive hyphal plasma membrane which provides a large surface area for accumulation. Could this explain the tendency towards filamentous, so-called “unspecialized” haustoria in monokaryotic rust infections, where hosts are often woody species? The mechanism by which the pathogen causes leakage of sugars could be either by simply creating a concentration gradient and sink, or similar to that employed in “sugar feeding”, where invertases are implicated. Thus it may be instructive to compare activities of oligosaccharide hydrolases in veins of healthy and infected leaves. Alternatively, the mechanism may involve localized increases in the permeability of the host plasma membrane (e.g. in the extrahaustorial membrane, which is a portion of host plasma membrane surrounding the haustorium, modified in structure and molecular composition; see Green et al., 1992) for organic solutes. A gradual decrease in the permeability of the extrahaustorial matrix would be an effective host response to limit this leakage, and indeed age-related changes have been noted at the haustorial interface in downy mildew and rust infections (e.g. see Gay and Woods, 1987; Beale et al., 1990; Larous and Losel, 1993). Production of haustorial neckbands in powdery mildew and dikaryotic rust infections could be viewed as the pathogen successfully limiting this host response to a region where it is beneficial to the fungus (and host, especially in epidermal infections?). Lack of neckbands in rust haustoria formed in vitro (Heath, 1989, 1990) suggests that they may be at least partly of host origin, although the in vitro haustoria do not reach the developmental stage where neckbands form in plantu.
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In the closed types of vein 2a and 2b, interception of apoplastic photoassimilates could occur at any point between mesophyll and intermediary cells. Intercellular hyphae growing in close association with mesophyll cells and the surface of minor veins of leaves could achieve this relatively simply. Pathogens such as C. fulvum, the host of which (Lycopersicon esculenturn) has type 2b veins, clearly obtain sufficient nutrients in this location. Apoplastic solutions from leaves infected by a virulent race of C. fulvum had increased concentrations of sucrose, glucose and fructose (Joosten et al., 1990). It was suggested that this is due to fungal invertases which maintain a concentration gradient of sucrose and thus interfere with phloem loading. The significance of invertases in relation to symptoms and pathogen nutrition in biotrophic infections should not be underestimated (Hall et al., 1992; Scholes, 1992; Tetlow and Farrar, 1993; Lam et al., 1995; Ayres et al., 1996). The presence of transfer cells in type 2b plants (e.g. Brassica, Phaseolus, Pisum, Senecio and Vicia spp.) may allow the phloem to compete more effectively with intercellular pathogens for apoplastic solutes. In these hosts, haustoria may provide a necessary competitive edge, functioning as suggested for pathogens of hosts with type 1 veins. This strategy would be coupled effectively with induction of apoplastic invertases that convert the main translocate, sucrose, to glucose and fructose which cannot be loaded by transfer cells, but which may be accumulated by intercellular hyphae. An additional mechanism is to modify the apoplastic pH, as in P. hordei infections of barley (Tetlow and Farrar, 1993) where pH increases from 6.6 to 7.3, and Uromyces vicae-fabae infections of broad bean (Deising et al., 1995) where pH increases from 6.3 to 6.7. This would reduce active transport processes involved in scavenging apoplastic sugars by the phloem and intercellular hyphae, because both are likely to be energized by proton gradients (Slayman et al., 1990; Bush, 1993). Thus there are clear advantages for pathogens of type 2b hosts to manipulate mesophyll cells to unload photoassimilates into a special apoplast that can only be tapped by the pathogen, which would also remove photoassimilates from the phloem loading pathway altogether. This would be achieved via haustoria, with the extrahaustorial matrix sealed by neckbands providing the special apoplast, and may be particularly important in later stages of infection when the apoplastic environment (including pH) mitigates against accumulation by intercellular hyphae. Photoassimilates are unloaded through the extrahaustorial membrane by what has been termed the “ATPase deficiency coupled transport mechanism” (Gay and Woods, 1987; Gay et al., 1987), with haustorial neckbands delimiting the functionally distinct domains of host plasma membrane. Also, as significant amounts of photoassimilate (40% of the sucrose in barley; Farrar, 1992) may be stored in vacuoles before export from healthy leaves, haustoria are closer to this pool than intercellular hyphae. Often, only very thin layers of host cytoplasm
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separate the extrahaustorial membrane and tonoplast, so large concentration gradients of sugars maintained by the haustorium may facilitate transport from this pool, which appears to become less available for translocation and metabolism following infection (Farrar, 1992). Another important role of the neckbands may be to maintain a pH appropriate for active transport of nutrients across the extrahaustorial and haustorial plasma membranes. Indeed there is evidence that charasomes, which are specialized periplasmic apoplasts in cells of the alga Chara, play this role in relation to H+ co-transport of nutrients (Price et al., 1985). Sucrose efflux from mesophyll cells also requires an appropriate pH gradient across the plasma membrane (Aloni et al., 1988). Evidence for movement of protons from the haustorium and across the extrahaustorial membrane to the host cell cytoplasm has been obtained in Erysiphe graminis infections (Gay et at., 1987). In plants with vein type 2c, such as Zea mays, bundle sheath cells are connected to the mesophyll symplastically, but there is no symplastic continuity via plasmodesmata to the phloem. Therefore interception of photoassimilates would best be achieved by inserting haustoria into mesophyll and bundle sheath cells, or by intercellular growth within the vascular bundle in order to intercept sugars transferred via the apoplast to the phloem. In other plants, there is evidence for combined and parallel symplastic and apoplastic phloem loading, although the significance of this is not fully understood (Van Bel, 1993). Evidence for continuity between extrahaustorial membranes and endoplasmic reticulum (ER) in rust- and downy mildew-infected cells (Harder and Chong, 1991) is relevant here. In rusts, this phenomenon is prominent in dikaryotic infections, where smooth ER is mostly involved, whilst continuity with rough ER is characteristic of monokaryotic haustoria. It has been suggested that symplastic transfer of photoassimilates from mesophyll to phloem may be through the lumen of the ER, and from cell to cell via the desmotubules of plasmodesmata (see Van Bel, 1993). Continuity between the chloroplast envelope and ER membranes in Phaseolus vulgaris (Whatley et al., 1991), although only reported in early stages of plastid development, would provide a direct link with the source of photosynthates. Clearly there are great advantages for haustoria to link into this system, from both the nutritional and manipulatory (see section V) perspectives.
2. Implications of the apoplastic loading path way In plants with the apoplastic pathway of phloem loading (type 2 minor veins), proposals for transfer intercept mechanisms are restricted by the lack of consensus about where photoassimilates are released to the apoplast. Does this occur from each mesophyll cell, or only in close proximity to the sieve element-companion cell complex? Van Be1 (1993) described knowledge of the mechanism as “fragmentary at best”, and Bush (1993) identified
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elucidation of the putative sucrose efflux system in the mesophyll as a critical research goal. Nevertheless, if certain cells are specialized to release photosynthate, then these are likely to be targeted by pathogens. It would seem sensible for release to be close to sieve elements (Delrot, 1989), and therefore there is a clear advantage for pathogens of these hosts to grow close to the vascular tissue. A consequence of a localized release mechanism in the plant is that mesophyll cells distal from veins may be retrieving photosynthate actively from the apoplast, and may therefore be poor sources of apoplastic nutrients. This would encourage preferential colonization of release cells and veins, and insertion of haustoria into distal mesophyll cells to provide special export domains in plasma membranes that would otherwise be effecting net import of photoassimilates.
3. The interception strategy in downy mildew infections: a case study In downy mildew infections, hyphae are often adpressed to host cell walls, filling intercellular spaces and therefore in intimate and extensive continuity with the host apoplast. This characteristic was noted by Fraymouth (1956) to be associated particularly with species which formed few haustoria, in an extensive review of some 38 downy mildew fungi in over 70 different hosts. By cross-referencing her descriptions of haustorial frequency with Gamalei’s (1989) descriptions of vein types, some interesting correlations emerge. For example, infrequent haustoria were typical in Peronospora infections of plant families with type 2a veins (e.g. Ranunculaceae, Caryophyllaceae, Scrophulariaceae), whilst haustoria were frequent in Afbugo, Bremia and Peronospora infections of members of the Alliaceae, Asteriaceae, Cruciferae and Rubiaceae, which have type 2b veins. Haustoria were also frequent in hosts with type 1 veins infected by Peronospora (host: Lamium purpureum) and Plasmopara (host : Vitis winifera). Type 1 veins have a symplastic phloem loading pathway, and haustoria would be required to intercept photoassimilates by making intimate contact with the symplast, as described in section IVBl. In plants with type 2a veins, loading is apoplastic, but there are no specialized phloem transfer cells, and so intercellular hyphae may be sufficient to out-compete the phloem for assimilates in the loading pathway. Type 2b minor veins have transfer cells, and therefore the pathogen needs an additional strategy, deployment of haustoria, to compete successfully. There are inevitably exceptions to this generalization: for example, Fraymouth describes Peronospora effusa forming many haustoria in the type 2a host Chenopodiurn (although perhaps only in heavily infected tissue, where there is greater competition for apoplastic nutrients?; see p. 92 of Fraymouth’s paper), but few in type 2c Atriplex. Also, Plasmopara spp. form haustoria frequently in type 2c Anemone, as well as type 2b Geranium. The picture is particularly confused in P. viciae infections of the Leguminosae. Few or no haustoria were observed in Lathyrus and Vicia species, although
FUNGAL HAUSTORIA IN EPIPHYTIC AND ENDOPHYTIC INFECTIONS r 3
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li4c] in infected tissue, dprn x
In
:ilrllm
0
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Control
C] in hyphal
I
Transient
Continuous
PCMBS treatment
Fig. 2. Accumulation of insoluble label b hyphae of Peronosporu viciae and infected leaflets of Pisum sutivum exposed to [‘Clsucrose. Treatment with p-chloromercuribenzene sulphonic acid (PCMBS) significantly reduces incorporation by infected leaf tissue (scintillation counts, dpm) compared to control (minus PCMBS), but transient PCMBS increases accumulation by hyphae (autoradiography, silver grains). (Reproduced from data in Clark and Spencer-Phillips (1993).)
it may be significant that in Vicia sativa the few were “in the neighbourhood of a vein”, whilst haustoria are frequent in Pisum sativum (for detailed analysis, see Clark and Spencer-Phillips, 1994): all these hosts are type 2b. Haustorial frequency is also likely to depend on degree of compatibility (fewer haustoria in more resistant host, e.g. Clark and Spencer-Phillips, 1994) and on the physiological status of the host tissue. Thus Fraymouth (1956) notes that frequency and size seem to increase where hyphal growth is restricted (physically and nutritionally?), and that it varies significantly in different tissues of the same host plant. The ability of downy mildew hyphae to accumulate carbon from the apoplast has been shown by experiments with P. viciae infections of P. sativum (Clark and Spencer-Phillips, 1993). [‘4C]Sucrose was introduced to the apoplast of infected leaves, and the distribution of insoluble label subsequently quantified by autoradiography, following enzymic maceration to separate plant and fungal components (Clark and Spencer-Phillips, 1990). Transient treatment with p-chloromercuribenzene sulphonate (PCMBS), which inhibits sucrose transport across plant cell plasma membranes, reduced accumulation by plant cells, but not hyphae (Fig. 2). These results suggest that hyphae assimilate label from sucrose directly from the apoplast, and not from plant cells and haustoria (Fig. 3). This evidence is supported by ATPase cytochemistry which shows activity at the plasma membrane of intercellular hyphae (Beale et al., 1990). Viable mycelia have now been isolated (Ashton and Spencer-Phillips, 1993) and this provides an experimental system by which to investigate further the function of endophytic hyphae of haustorial biotrophs.
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Fig. 3. Pathways for accumulation of carbon from apoplastic sucrose by hyphae of Peronospora viciae and cells of Pisum sativum leaves. Transient treatment with PCMBS blocks uptake ( x ) by plant cells and hence to hyphae via haustoria, but not uptake via the direct route from apoplast to hyphae. (Reproduced from Clark and Spencer-Phillips (1993).) C. EVOLUTIONARY ASPECTS
The data reported by Gamalei (1989) are interpreted (see also Van Be1 and Gamalei, 1992; Van Bel, 1993) to suggest that symplastic loading is the most ancient mechanism, with an evolutionary progression of minor vein type in the order 1,2a, 2b and 2c. Thus primitive biotrophic fungi infecting type 1plants would have been subjected by selection pressure to devise means of infecting vascular tissue (containing the highest concentration of photoassimilate) and to produce haustoria in photosynthetic cells to intercept assimilates in transit. It is tempting to speculate that by inducing leakage to the apoplast (perhaps via unspecialized haustoria similar to those of monokaryotic rust and some downy mildew fungi), these primitive biotrophs encouraged plants to devise efficient mechanisms for scavenging the apoplast in order to out-compete endophytic parasites. Therefore this process may have played a role in the development of apoplastic mechanisms of phloem loading seen in the more modem plant families, alongside other selection pressures (Van Be1 and Gamalei, 1992). Perhaps haustoria also developed to give a competitive advantage over non-haustorial endophytes. This may be linked to the correlation between decreased soluble sugars in the apoplast and formation of haustoria in mesophyll cells, versus increased amounts of sugars when mesophyll cells are not infected (Ayres et al., 1996). Clearly, experiments are needed to explore this in more detail; for example, comparing downy mildew infections of hosts where haustoria are absent or infrequent, powdery mildews with endophytic mycelia and monokaryotic and dikaryotic rusts.
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The importance of the fossil record in substantiating co-evolutionary processes has been emphasized by Pirozynski and Malloch (1975), who suggest that the first land plants were mutualistic associations of semi-aquatic algae and aquatic Oomycetes. Cooke and Whipps (1987) reviewed arguments for evolutionary progress from saprotrophy through necrotrophy to biotrophy, and vice versa, and challenge the view that necrotrophy needs to be an intermediate step. Essentially, a saprotrophic fungus that enters the plant tissues via natural openings such as stomata, and avoids eliciting host defences, can grow as an intercellular endophyte by utilizing the apoplastic pool of nutrients. Many bacteria (Gunson and Spencer-Phillips, 1994) and non-haustorial fungi (Carroll, 1988) that do not cause disease symptoms have exploited this niche. There is no need to have gained, and then lost, the special abilities of neaotrophs in order to achieve this life-style. From an endophytic vantage point, development of mechanisms for manipulating host processes, perhaps by forming haustoria, would result in a more specialized association, although it is widely recognized (see section V) that incompatibility is often triggered by haustoria, and so their absence has advantages. Perhaps it is more helpful to think of necrotrophy, biotrophy and saprotrophy as strategies for nutrition and habitat acquisition which form three corners of a triangle. Thus any one strategy could have developed directly from any other, or via an intermediary. It is not unreasonable to speculate that, as the picture emerges from examination of fossil and gene records, different groups of biotrophic pathogens will have arrived at their present shared strategy by different routes.
V. ALTERNATIVE FUNCTIONS OF HAUSTORIA If haustoria are not essential for mass accumulation of photoassimilate, at least in some hosts and when apoplastic nutrients are not depleted (e.g. see section IVBJ), then they are likely to play other roles. There may also be additional roles for haustoria in infections which do rely on them for nutrient uptake (e.g. in epiphytic powdery mildews). It must be remembered that there is a major penalty for forming haustoria, because in all groups of haustorial pathogens recognition by the host has been correlated with penetration of host cells (e.g. Goodman and Novacky, 1994; Lennox and Rijkenberg, 1994). Successful formation of haustoria requires complex regulation of enzymes used for cell wall penetration (e.g. Rauscher et af., 1995) in order to maintain biotrophy, because these enzymes release elicitors of host defence. Thus fungi must gain a significant advantage from haustoria, although it is always possible that in some cases they are vestiges of ancestral function. Possible alternate or additional roles relate to either non-photosynthate
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nutrients, or establishment and maintenance of compatibility. Accumulation of specific nutrients that are not available in the apoplast (such as certain amino acids, sterols, growth factors and vitamins) has been suggested by Clark and Spencer-Phillips (1993) for pea downy mildew. Some factors involved in maintenance of compatibility are reviewed by Durbin (1984), and it is possible that these functions are mediated by haustoria. Certainly several investigators have suggested that suppressors of host defences are haustorium derived (see Harder and Chong, 1991). Furthermore, promoters of some plant genes appear to be up-regulated by about 10-fold in compatible interactions alone, and probably only in cells containing haustoria (Pryor, personal communication; Roberts and Pryor, 1995), which presumably results from molecular signalling between haustorial and host cytoplasms. The presence of capsid antigens of a dsRNA virus within the extrahaustorial matrix and cell wall of haustoria of infected Puccinia recondita (Zhang et al., 1994) indicates that proteins can be transferred from the fungal cytoplasm to the extrahaustorial matrix (but note specificity problems with immunogold labelling of the extrahaustorial matrix; Heller, 1995). Continuity with the ER lumen would allow movement of such proteins through the endomembrane system of infected and adjacent cells interconnected by plasmodesmata, and provide a means by which haustorial pathogens regulate host metabolic and transport processes. Direct effects on components of plant transport systems have been reported for necrotrophic and hemibiotrophic pathogens (see Clark and SpencerPhillips, 1993), and suggested recently for C. fulvum infections. Lowmolecular-weight, extracellular proteins (ECPs) in intercellular fluids from compatible interactions are thought to play a role in pathogenicity (De Wit et a!., 1989). Two ECPs have been purified and the genes isolated, but sequence data have not provided clues to their function (Van den Ackerveken et al., 1993). Thus, conclusive proof that ECPs are essential for pathogenesis is still awaited, but it has been suggested (Van den Ackerveken and De Wit, 1995) that they may interfere with host metabolism and solute transport. Confirmation of this effect would provide new information about regulation of photoassimilate transport within infected leaves, and useful tools for probing these processes in healthy tissues. Thus the main advantages of haustoria are that they can more effectively manipulate host metabolism and access intracellular pools of solutes, obtaining nutrients not available in the apoplast and exploiting host resources to the full at times of peak demand, such as during sporulation. Indeed there is evidence that haustorial function may change at different developmental stages (see Green et al., 1992). These functions may be aided by haustoria directing mass flow driven by turgor pressure and water loss from the epiphytic mycelium (Wyness and Ayres, 1987), or modifying fluxes in the apoplast and symplast due to water flow through intercellular hyphae.
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VI. SUMMARY A N D RESEARCH PRIORITIES The critical research goal of proving the functions of downy mildew and rust haustoria is inextricably linked with the need to answer three key questions about nutrient transfer in biotrophic infections. What are the translocated nutrients, in what quantities are they transferred, and by what mechanism (or mechanisms) is transfer effected? The pioneering work of Gay and colleagues provided answers to all these questions for photoassimilates acquired by the epiphytic powdery mildew fungus E. phi, but there is little conclusive evidence available for endophytic pathogens. Special attention should now be focused on haustoria in downy mildew and rust infections, both in relation to nutrient accumulation and molecular conversation between host and fungus. It should be expected that function will change at different developmental stages of individual haustoria and the fungal colony, and it is important to conduct parallel experiments to investigate the role of intercellular hyphae. The availability of techniques to isolate haustoria and hyphae in a viable condition should facilitate this research. A holistic approach which relates function of fungal structures to nutrient partitioning and transport in host tissues promises to be particularly rewarding. Thus, a comprehensive review of infection strategy in relation to minor vein type, mechanism of phloem loading and identity of translocate is now required. Absolute conformity between interception strategy and vein type should not be expected, because continuing evolution of strategies and host range is likely to be occurring in pathogens, just as in vein type and growth habit of plants (Gamalei, 1989). Furthermore, mechanisms of nutrient transfer may be modified in response to the marked changes in the apoplastic environment and nutrient status of host tissues which occur as infection proceeds. Fundamental knowledge about the apoplastic and symplastic pathways of solute movement in fungal infections is lacking. Use of fluorescent probes coupled with microinjection and optical sectioning by confocal microscopy provides an approach by which this could be remedied. Dynamic, real-time experiments with living tissues promise to reveal key components of transport pathways, such as: rates and routes of solute flux; molecular exclusion by apoplastic barriers associated with haustoria; and physiological continuity between extrahaustorial matrix and the lumen of endomembrane systems of host cells. Finally, the molecular mechanisms of transmembrane transport in plant tissues are likely to be key targets for manipulation by biotrophic pathogens. Recent advances in understanding the expression and regulation of membrane transport functions (Sussman, 1994) provide an exciting basis for exploring this possibility.
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Thus it is clear that fundamental structural and physiological studies are needed in conjunction with the application of contemporary technology in cell and molecular biology. The methods are now available to prove or disprove assumptions about haustorial function, and to explore a wider role. To adapt a quotation cited by Shoemaker (1981): what we need are plant pathologists who will combine their interest in how fungi copulate (taken to encompass molecular genetics and biology) with how they eat!
ACKNOWLEDGEMENTS This article is dedicated to Dr John Gay, pioneer in haustorial function. I am also indebted to Dr Brian Williamson for helpful comments on a draft version, to Dr Dawn Arnold for providing a copy of a paper in press and to Dr Tony Pryor for a summary of unpublished data.
REFERENCES Aloni, B., Daie, J. and Wyse, R. (1988). Regulation of apoplastic pH in source leaves of Vicia faba by gibberellic acid. Plant Physiology 88, 367-369. Andreev, L. N., Plotnikova, Y. M. and Serezhkina, G. V. (1982). Haustoria of Puccinia graminis Pers. f. sp. tritici Eriks. & Henn. in the vascular system of wheat. Mikologia i Fitopatologiya 16, 335-338. Arimoto, Y. and Homma, Y. (1995). Influence of sucrose supply on development of Sphaerotheca fuliginea (Schlecht.) Pollacci on carbohydrate free Cucumis sativus L. Annals of the Phytopathological Society of Japan 61, 82-87. Arnold, D. L., Blakesley, D. and Clarkson, J. M. (1996). Evidence for the growth of Plasmodiophora brassicae in vitro. Mycological Research 100, 535-540. Ashton, H. A. and Spencer-Phillips, P. T. N. (1993). Hydroethidine as a vital stain for Peronospora viciae spores, germlings and isolated hyphae. In “Abstracts, 6th International Congress of Plant Pathology”, p. 142. National Research Council of Canada, Ottawa. Ayres, P. G., Press, M. C. and Spencer-Phillips, P. T. N. (1996). Effects of pathogens and parasitic plants on source-sink relationships. In “Photoassimilate Distribution in Plants and Crops: SourceSink Relationships” (E. Zamski and A. A. Schaffer, eds), pp. 479-499. Marcel Dekker, New York. Beale, A. J., Clark, J. S. C. and Spencer-Phillips, P. T. N. (1990). Microscopy of endophytic hyphae facilitated by enzymic maceration and ATPase cytochemistry. In “EMAG-MICRO 89, 2, Biological” (H. Y. Elder and P. J. Goodhew, eds), pp. 711-714. Institute of Physics, Bristol. Bracker, C. E. and Littlefield, L. J. (1973). Structural concepts of host-pathogen interfaces. In “Fungal Pathogenicity and the Plant’s Response” (R. J. W. Byrde and C. V. Cutting, eds), pp. 159-318. Academic Press, London. Bush, D. R. (1993). Proton-coupled sugar and amino acid transporters in plants. Annual Review of Plant Physiology and Plant Molecular Biology 44, 513542. Bushnell, W. R. (1972). Physiology of fungal haustoria. Annual Review of Phytopathology 10, 151-176.
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Canny, M. J. (1990). What becomes of the transpiration stream? New Phytologist 114, 341-368. Carlile, M. J. and Watkinson, S. C. (1994). “The Fungi.” Academic Press, London. Carroll, G . C. (1988). Fungal endophytes in stems and leaves: from latent pathogen to mutualistic symbiont. Ecology 69, 2-9. Clark, J . S. C. and Spencer-Phillips, P. T. N. (1990). Isolation of endophytic mycelia by enzymic maceration of Peronospora infected leaves. Mycological Research 94, 283-287. Clark, J. S. C. and Spencer-Phillips, P. T. N. (1993). Accumulation of photoassimilate by Peronospora viciae (Berk.) Casp. and leaves of Pisum sativum L.: evidence for nutrient uptake via intercellular hyphae. New Phytologist 124, 107-1 19. Clark, J. S. C. and Spencer-Phillips, P. T. N. (1994). Resistance to Peronosporu viciue expressed as differential colony growth in two cultivars of Pisum sativum. Plant Pathology 43, 56-64. Cooke, R. C. and Whipps, J. M. (1987). Saprotrophy, stress and symbiosis. In “Evolutionary Biology of the Fungi” (A. D. M. Rayner, C. M. Brasier and D. Moore, eds), pp. 137-148. Cambridge University Press, Cambridge. Cosgrove, D. J. and Cleland, R. E. (1983). Solutes in the free space of growing stem. Plant Physiology 7 2 , 326-321. Deising, H., Frittrang, A. K., Kunz, S. and Mendgen, K. (1995). Regulation of pectin methylesterase and polygalacturonate lyase activity during differentiation of infection structures in Uromyces viciae-fubae. Microbiology 141, 561-571. Delrot, S. (1989). Loading of photoassimilates. In “Transport of Photoassimilates” (D. A. Baker and J. A. Milburn, eds), pp. 167-205. Longman, Harlow. De Wit, P. J. G. M., Van den Ackerveken, G. F. J. M., Joosten, M. H. A. J. and Van Kan, J. A. L. (1989). Apoplastic proteins involved in communication between tomato and the fungal pathogen Cladosporium fulvum. In “Signal Molecules in Plants and Plant-Microbe Interactions” (B. J. J. Lugtenberg, ed.), pp. 273-280. Springer-Verlag, Berlin. Dorr, I. and Kollman, R. (1995). Symplastic sieve element continuity between Orobanche and its host. Botanica Acta 108, 47-55. Durbin, R. D. (1984). Effects of rusts on plant development in relation to the translocation of inorganic and organic solutes. In “The Cereal Rusts. Vol. I. Origins, Specificity, Structure and Physiology” (W. R. Bushnell and A. P. Roelfs, eds), pp. 509-528. Academic Press, London. Farrar, J. F. (1984). Effects of pathogens on plant transport systems. In “Fungal Infections of Plants” (R. K . S. Wood and J. G. Jellis, eds), pp. 87-104. Blxckwell Scientific, Oxford. Farrar, J . F. (1992). Beyond photosynthesis: the translocation and respiration of diseased leaves. In “Pests and Pathogens. Plant Responses to Foliar Attack” (P. G. Ayres, ed.), pp. 107-127. Bios Scientific, Oxford. Farrar, J. F. and Lewis, D. H. (1987). Nutrient relations in biotrophic infections. In “Fungal Infection of Plants” (G. F. Pegg and P. G. Ayres, eds), pp, 92-132. Cambridge University Press, Cambridge. Fraymouth, J. (1956). Haustoria of the Peronosporales. Transactions of the British Mycological Society 39, 79-107. Gamalei, Y. (1989). Structure and function of leaf minor veins in trees and herbs: a taxonomic review. Trees 3, 96-110. Gay, J. L. (1984). Mechanisms of biotrophy in fungal pathogens. In “Fungal Infections of Plants” (R. K. S. Wood and J. G. Jellis, eds), pp. 49-59. Blackwell Scientific. Oxford.
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Towards an Understanding of the Population Genetics of Plant-Colonizing Bacteria
B. HAUBOLD and P. B. RAINEY
Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 3RB, UK
I. Introduction
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11. Quantifying Genetic Variation: the Indirect Approach
111. The Neutral Theory: Historical Background to the Study of Bacterial Population Genetics ...................................................................
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IV. Population Structure .................................................................. A . Genetic Structure ................................................................ B. Phylogenetic Structure ......................................................... C. Spatial and Temporal Structure .............................................
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VI. Conclusions .............................................................................. Acknowledgements .................................................................... References ...............................................................................
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V. The Metapopulation Concept
I. INTRODUCTION Population genetics is the study of evolutionary change. It is concerned with natural genetic diversity, its causes, its distribution and its biological significance. More specifically, population genetics is concerned with the basic forces of evolution, that is, mutation, recombination, migration, natural selection and genetic drift, and the contribution that these forces make to the nature and rate of evolutionary change. Knowledge of the effects of these forces on populations can provide information on likely future directions of Advances in Botanical Research Vol. 24 incorporating Advances in Plant Pathology ISBN 0-1245924-X
Copyright 0 1997 Academic Press Limited All rights of reproduction in any form reserved
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evolutionary change and, conversely, patterns of genetic variability can yield information about the processes that generated them. The link between the effects of specific evolutionary forces and patterns of variation is provided by the theory of population genetics which gives evolutionary biology its most significant and far-reaching body of theory. Why should we be concerned with the population biology of bacteria? The study of prokaryote populations represents a significant intellectual goal in itself (see Young, 1989; Maynard Smith, 1995), but, in addition, it has practical significance. The brief generation times and large population sizes of bacteria suggest that evolutionary change will be swift. This is clearly evident in the rapid spread of antibiotic resistance genes in infectious bacteria (Cohen, 1992; Bennett, 1995; Levin, 1995) and in the sudden outbreak of disease in plants (Burdon, 1993; McDermott and McDonald, 1993). An understanding of the genetic structure of microbial populations provides a framework within which epidemic outbreaks of pathogens can be monitored and traced, the spread of drug and pesticide resistance can be managed, the efficacy and safety of genetically engineered micro-organisms intended for environmental applications can be assessed and biological control strategies can be rationally designed (Istock, 1991; Lenski, 1993; Maynard Smith, 1995). In addition, population genetics has a positive contribution to make to the debate on the “species concept” in prokaryotes and provides a guide to the practical problems of bacterial classification. The value of population genetics as a means of understanding changes in patterns of virulence and drug resistance is widely recognized by those concerned with infectious human pathogens (see e.g. Anderson and May, 1991), and by a small number of plant pathologists, most notably those concerned with pathogenic fungi (Price, 1992; Burdon, 1993; McDermott and McDonald, 1993), but with the exception of studies on Rhizobium, Brudyrhizobium and Bacillus, the role and value of population genetics in the study of natural bacterial populations in the environment are often neglected. In the past, the field of bacterial population biology has tended to be divided between population geneticists, who apply the framework of ecological and evolutionary genetics to bacterial populations, and phytopathologists/microbial ecologists, who document phenotypic diversity, functional diversity, population dynamics, genetic diversity and the spread of disease, often without considering the underlying genetics, and frequently side-stepping issues relating to the causes and consequences of observed patterns of variation. Ultimately, the study of biological and functional diversity must be based on a firm understanding of the extent and significance of genetic variability within populations. In writing this chapter it has been our aim to draw the attention of those studying plant-colonizing bacteria (both pathogens and saprophytes) to the field of population genetics. We have purposely avoided a review format (especially as there is so little known about the population genetics of
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plant-colonizing bacteria), and recommend that for more detailed information on specific bacterial populations (Escherichia coli, Rhizobium, Bacillus and populations of medically significant bacteria including Salmonella, Neisseria and Haemophilus) the excellent review chapters in Baumberg et al. (1995) should be consulted (see also the comprehensive review by Young, 1989).
11. QUANTIFYING GENETIC VARIATION: THE INDIRECT APPROACH A variety of methods are used for detecting genetic variation in populations and include traditional markers such as specific virulence genes (in pathogens), morphological markers, electrophoretic markers (allozymes, DNA restriction fragment polymorphisms (RFLPs), polymerase chain reaction (PCR)-based markers), and direct DNA sequence analysis. The appropriateness of each method depends largely on the nature and scope of the questions being addressed (detailed descriptions of the various methods can be found in most standard textbooks and laboratory manuals). In considering approaches for the analysis of genetic variation in populations, a useful, although not rigid, distinction can be made between direct and indirect methods. The distinction is made not on the basis of the laboratory procedures, but on the amount of mathematical reasoning involved in drawing conclusions from the data. Direct methods, such as DNA fingerprinting, are commonly used in studies of soil- and plant-colonizing bacterial populations (see e.g. Boccara et al., 1991; Gillings and Fahy, 1993; Louws et al., 1994; Rainey et al., 1994) and with careful planning can provide useful information on genetic variation, epidemiology and the distribution of genotypes. Indirect methods, upon which we wish to focus attention, are less commonly used. They generally require more complex and thorough sampling strategies and also mathematical analysis, but they can provide quantitative assessments of population structure, estimates of gene flow and information on the spatial and temporal distribution of genetic variation. Indirect methods are based on the analysis of allele frequencies in populations and traditionally rely on data obtained from studies of allozyme variation using multilocus enzyme electrophoresis (MLEE). For the last 25 years MLEE has been one of the principal tools for detecting genetic variation in populations (Lewontin, 1991). It is relatively crude when compared with DNA sequencing, but it has the advantage of being comparatively rapid, inexpensive and allows a large number of isolates to be processed. MLEE reveals differences (polymorphism) in proteins caused by amino acid replacements that alter electrophoretic mobility. The power of MLEE lies in the fact that bands observed on gels are the primary products of known genes and therefore the observed phenotypic variation can be interpreted as allelic variation at those loci. Proteins selected for analysis by MLEE are typically enzymes encoded by house-keeping genes and involved
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in central cellular metabolism. A typical study would examine variation in multiple genetic markers (loci) from a collection of several hundred isolates, thus allowing a potentially large number of distinct genotypes (also called electrophoretic types (ETs)) to be identified (Selander et al., 1986).
111. THE NEUTRAL THEORY: HISTORICAL BACKGROUND TO THE STUDY OF BACTERIAL POPULATION GENETICS Application of MLEE in the 1960s revealed a much greater degree of genetic polymorphism than had been previously assumed (Selander, 1976). Moreover, the observed variation did not appear to have any phenotypic effect and was not correlated with environmental conditions (Kimura, 1968). These findings stimulated considerable debate as to the significance of the variation. On one side were those who argued that selection maintained the different allelic forms, while on the other side were those, most notably Motoo Kimura, who claimed that the differences between allelic forms were so small that they were selectively neutral. The neutral theory, or neutral mutation-random drift hypothesis (Kimura, 1968), which is now central to much population genetics theory, posits that the selection intensity involved in the process of evolution is so weak that mutant pressure and random drift largely determine the fate of molecular mutants. It also asserts that the majority of polymorphic alleles are maintained by the balance between mutational input and random extinction, rather than by selection (Kimura, 1983). In mathematical terms, the neutral hypothesis predicts that for haploid populations, at equilibrium (when mutational input and random extinction balance each other), the effective number of alleles (a,)' will be Ite = 2N,v + 1 (1) where N , is effective population size and v is the mutation rate (Crow and Kimura, 1970). This formula is more often expressed in terms of mean genetic diversity
(m2:
H=
2Nev 2Nev + 1
'The effective number of alleles (n,) can be estimated from allele frequency data using 1 Z a; where ne. is the effective number of alleles at the jth locus and aij is the frequency of the ith alleles at the jth locus. *The mean genetic diversity (If) can be estimated from allele frequency data using n,. =
1 "
H=-X(l
-z
a$)
n I= . 1 where n is the number of loci scored and aij is the frequency of the ith allele at the jth locus.
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The first study in bacterial population genetics was designed to test the predictions made by the neutral hypothesis. The organism selected for study was E. coli which, because of its immense population size and evolutionary history, was likely to have reached equilibrium (Milkman, 1973). For such a population the neutral theory predicts the effective number of alleles (n,) will be approximately 200 (obtained from Eq. (l), assuming an effective population size (N,) of 10” and a mutation rate ( v ) of The effective number of alleles actually observed by Milkman, following his analysis of allozyme variation at five enzyme loci in 829 E. coli strains, was 1.38 (H = 0.242). This was substantially less than ne = 200 (H = 0.99) predicted by the neutral hypothesis (Eq. (1)). This discrepancy, combined with the fact that the distribution of mobility classes was not normal at any of the loci surveyed - a further prediction of the neutral hypothesis, led Milkman to doubt the validity of the neutral theory (Milkman, 1973). Rejection of the neutral theory rested on the assumption that E. coli populations are in linkage equilibrium (Selander and Levin, 1980), i.e. that the probability of finding any particular genotype is determined solely by the allele frequencies in the gene pool. If the population was in linkage disequilibrium then Ne would probably be smaller than 10” and ne would be correspondingly affected. In 1980, Selander and Levin reported the results of a study which examined allelic variation in 20 loci from 109 E. coli isolates and showed that despite extensive allelic diversity there was much less genotypic diversity than expected from the neutral model, because certain alleles were commonly found in association, whereas other allele combinations were rare or absent. This suggested that recombination in E. coli was rare (i.e. that the population was not in linkage equilibrium) and that, despite an immense population size, the number of extant genotypes was limited to a relatively small number of separately evolving lineages in which random sampling of lines occurred frequently through periodic selection3 (Selander and Levin, 1980). Milkman may therefore have overestimated the genetically effective population size (Ne). Selander and Levin’s results led Maruyama and Kimura (1980) to construct a model of bacterial evolution based on the view that bacterial populations consist of a number of asexual (clonal) subpopulations in which extinction and subsequent recolonization events occur frequently. Using this model they showed that a hypothetical bacterial species comprising 16 lines would have an effective population size of about 5 X lo7 (even though each line could 3“Periodic” or “recurrent” selection can occur in clonal populations as a result of competition between an occasional favourable mutant and all less fit genotypes. In the absence of recombination an adaptive mutant can eventually replace all less fit genotypes, taking with it just those alleles which were linked to the mutant when it first arose. Periodic selection therefore effectively purges populations of neutral genetic variation and results in the fixation of near-neutral alleles by hitch-hiking(Atwood et al., 1951; Koch, 1974; Levin, 1981; Young, 1989; Maynard Smith, 1991).
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comprise a huge number of individuals). When this value for N , is placed into Eqs (1) and (2) the neutral hypothesis predicts n, = 2 and H = 0.5. These predictions were in agreement with the mean genetic diversity calculated by Selander and Levin ( H = 0.472) and fit reasonably well the empirical data of Milkman (n, = 1.38). The clonal model of bacterial populations became generally accepted during the 1980s (Selander et al., 1986), although an indication that all was not straightforward was provided by Dykhuizen and Green (1991) and Guttman and Dykhuizen (1994), who showed that phylogenies constructed for different chromosomal genes from the same set of E. coli isolates were significantly different from one another. The favoured interpretation of these data is that recombination between loci has been sufficiently common to disrupt linkage, but not sufficiently common to lead to linkage equilibrium. At the same time as the clonal model for bacterial populations was formulated, Dykhuizen and Hartl (1980) directly examined the selective neutrality of allozymes detected by MLEE. Alleles of 6-phosphogluconate dehydrogenase were transferred into isogenic strains and the relative rate of growth of the clones was measured. Within the limits of resolution the alleles were shown to be selectively neutral (Dykhuizen and Hartl, 1980). Three years later, Whittam et al. (1983) used a statistical approach to show that the polymorphism observed at all the 12 loci surveyed in their study of 1705 E. coli isolates was in agreement with the neutral theory. These studies largely resolved the question of neutrality of bacterial allozymes, but the debate about the extent of clonality in bacterial populations has continued.
IV. POPULATION STRUCTURE A. GENETIC STRUCTURE
Populations of strictly asexually reproducing organisms are, by definition, clonal. In such populations differences between individuals arise due to spontaneous mutation (point mutation, inversions, deletions, duplications). In a finite asexual population of constant size with a finite number of possible allelic states and without extinction or migration, the random nature of mutations would lead to linkage equilibrium. Such a population is, however, a purely hypothetical construct, because in nature rapid successions of bacterial populations occur as a result of periodic extinction and recolonization (Caugant et al., 1981). Linkage equilibrium in bacterial populations can therefore be directly attributed to recombination. Recombination in bacterial populations can be mediated by a variety of accessory elements, including plasmids, bacteriophages, transposons and
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insertion sequences. Some species, including Haemophilus influenzae and Neisseria gonorrhoeae, are naturally competent for transformation and are able to take up naked DNA directly from the environment. The potential for gene transfer among bacteria (even among distantly related species) is therefore considerable. The statistical investigation of linkage equilibrium constitutes a powerful indirect method of assessing the extent of recombination in bacterial populations. In 1980, Brown et al. suggested a convenient index of association as an overall measure of linkage. This can be illustrated by considering two bacterial isolates with the electrophoretic types AlB2Cl and A2BIC1 which are characterized by mismatches at two loci. If the number of mismatches is recorded for all pair-wise comparisons in a given sample, the variance4 of the observed number of mismatches (V,) can be compared to the expected variance in the absence of linkage (V,) to yield the index of association
For populations in linkage equilibrium Vo = VE and hence, ZA = 0. Whittam et al. (1983) were the first to apply this statistic to bacterial populations. Maynard Smith et al. (1993) have recently used the ZA as the basis for a survey of MLEE data sets from several bacterial groups and found population structures ranging from clonal to panmictic. Four population structures were specifically identified: (i) populations that are clonal at all levels of analysis, such as Salmonella (ZA = 3.11); (ii) populations in which recombination is sufficiently frequent to produce a random assortment of alleles, such as Neisseria gonorrhoea (IA = 0.04; these populations are also termed panmictic) ; (iii) populations which display an intermediate, “epidemic”, population structure such as Neisseria meningitidis (ZA = 1.96 for all isolates and 0.21 for ETs only), where the observed association between loci is a result of recent outbreaks of meningitis caused by rapid increases in the numbers of a few genotypes; and (iv) Rhizobium-like populations which are clonal on a world-wide scale (IA = 6.34) but closer to linkage equilibrium on a fine scale (ZA = 0.24). Sequence analysis has shown that recombination occurs even within clonal populations such as Salmonella and E. coli (Smith et al., 1990; Dykhuizen and Green, 1991; Milkman and McKane Bridges, 1993; Guttman and Dykhuizen, 1994; see also Milkman and McKane, 1995), but it appears not to occur frequently enough to disrupt the overall clonal structure. Milkman has considered this in detail (reviewed in Milkman and McKane, 1995) and 41t is important to note that the data used to calculate this variance are not independent, which
makes computer simulation the method of choice when testing statistical significance.
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proposed the term “meroclone” to describe a cluster of strains which through recombinational replacements are no longer strict clones, but which still share substantial recent ancestry. A strong correlation has been observed between the transformability of bacterial strains and their genetic structure. All “panmictic” populations discovered to date belong to bacterial groups that are naturally transformable. However, competency alone is not sufficient to render a population panmictic - Haemophilus influenzae is naturally transformable (Lorenz and Wackernagel, 1994), but possesses a clonal population structure (Maynard Smith et al., 1993). In principle, panmixis could also result from the activity of chromosome mobilizing plasmids or transducing phages, but to date this has not been shown. We are only aware of one study which has applied indirect methods to assess the genetic structure of a plant-colonizing bacterial population. Denny et al. (1988) investigated the genetic diversity of two pathovars of Pseudomonas syringae (P. s. tomato and P. s. syringae) using DNA-DNA hybridization, RFLP and MLEE. They found that the two pathovars differed widely in genetic diversity. The mean genetic diversity per locus among P. s. tomato isolates was 0.076, while among P. s. syringae isolates it was 0.479. The P. s. tomato sample had been collected from different parts of the world and the high degree of genetic similarity suggested the recent dissemination of a single lineage via contaminated seeds and infected plants. Analysis of the distribution of alleles showed that both populations were in linkage disequilibrium (Maynard Smith et al., 1993) and this is consistent with the knowledge that P. syringae is not naturally transformable (Carlson et al., 1983).
B. PHYLOGENETIC STRUCTURE
The extent of recombination within and between populations determines the pattern of evolution and this has implications for phylogenetic reconstruction and the identification of natural groupings of organisms. Two broad phylogenetic patterns can be distinguished based on the mode of reproduction. In a clonal population the genetic relatedness between members can be best described by a tree, but in a panmictic population, the relationship between individuals is more appropriately viewed as a multidimensional net (Maynard Smith, 1990). In this context, the evolutionary history of bacteria such as Salmonella and E. coli, which have predominantly clonal population structures, can be described by a “local continuum tree” in which genetic material is exchanged among similar members of the group. The precise structure of these trees can be found using the appropriate method of phylogenetic reconstruction (but see Swofford and Olsen (1990) for a discussion of the practical problems of phylogenetic reconstruction). For
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sexual populations, such as N. gonorrhoea, the relationship between individuals can be thought of as a multidimensional net, while Rhizobium, which is clonal world-wide but sexual on a fine scale, can be viewed as a “large-scale tree” in which the individual “branches” (local populations) have a net-like structure. Only in clonal (tree) populations can the number of genetic differences (mismatches) between pairs of strains be interpreted as evolutionary distances. In such populations it is possible to trace the evolutionary history of a group of organisms, as was elegantly done for the evolution of E. coli strains implicated in haemorrhagic colitis (Whittam, 1995). The reconstruction of trees for panmictic populations, such as N. gonorrhoeae, will not yield meaningful information and thus long-term epidemiological studies in such populations are unfeasible (Spratt et al., 1995). Phylogenetic reconstruction has also been used as a heuristic tool for identifying genetically distinct subgroups within traditionally defined bacterial species. For example, Eardly et al. (1990) discovered two distinct subclusters within a sample of Rhizobium meliloti isolates and subsequent analysis showed that these subclusters were near linkage equilibrium, while the sample as a whole was in strong linkage disequilibrium (Maynard Smith et al., 1993). Duncan et al. (1994) observed a similar subdivision within a group of Bacillus lichenifomis isolates and, finding no evidence of gene transfer between isolates of the two subgroups, suggested that they may represent two distinct species. Studies such as these are important in terms of helping to identify natural “species” boundaries and in the future may help to alleviate some of the confusion surrounding the taxonomy of the more heterogeneous groups of bacteria, such as the pseudomonads and xanthomonads.
C. SPATIAL AND TEMPORAL STRUCTURE
Given appropriate sampling, a collection of bacteria may reveal information on spatial and temporal structure, as well as genetic structure. Souza et al., (1992) examined allozyme variation in a global collection of Rhizobium strains and showed that the population as a whole was in linkage disequilibrium. However, a subsequent hierarchical analysis, which subdivided the population on the basis of ecologically relevant criteria, showed that bacteria sampled from the same plant were in linkage equilibrium. This result indicates that extensive recombination may take place on a local scale and emphasizes the importance of sampling local populations if questions relating to genetic exchange within or between species are to be adequately addressed (Duncan et al., 1994). The population structure of Rhizobium was also investigated by Young and colleagues (Young, 1985; Young et al., 1987; Young and Wexler, 1988)
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who examined spatial structure on a finer scale than that studied by Souza et al. (1992). Young et al. (1987) sampled Rhizobium leguminosarum bv. viceae isolates from 40 individual nodules from nine pea plants located along a single transect (20 nodules from each primary root and 20 nodules from each lateral root); they also collected isolates from two soil sites: one in which the original plants had been grown and the other from soil 20 m distant from the original site. MLEE analysis of more than 400 isolates revealed a population in strong linkage disequilibrium. It also showed that individual plants harboured genetically diverse populations and that significantly different populations (in terms of frequencies of ETs) occurred on plants as compared to in soil, and on primary roots as opposed to on lateral roots. Young et al. (1987) were also able to show that the domain size of an individual clone, while limited relative to an entire root system, was sufficiently large to ensure a significant chance of finding related ETs in adjacent nodules. Temporal variation within bacterial populations can also be determined from the analysis of MLEE data. In a study of genetic diversity in E. coli populations Caugant et al. (1981) collected 530 isolates from a single human host over an 11-month period. The sample contained 53 highly diverse genotypes and, since recombination in E. coli populations is limited, it was concluded that the diversity was generated by successive invasions of E. coli genotypes (Caugant et al., 1981). We are currently studying the genetic structure of a population of sugar beet leaf-colonizing fluorescent Pseudomonas using MLEE combined with biotyping (Haubold and Rainey, 1996). Our work aims to address two key questions: (i) does the population have an ecotypic structure, i.e. does it consist of locally adapted clones; and (ii) what is the extent of recombination among fluorescent pseudomonads? In order to address these questions, we sampled a field of sugar beet at the level of individual leaves, i.e. at the level of potential habitats. Fluorescent pseudomonads were collected from three leaf types (immature, mature and senescent), from three plants and from three plots, yielding a total of 108 isolates. We found that all plants harboured genetically distinct and diverse subpopulations, but individual leaves were frequently dominated by single ETs, suggesting “epidemic” outbreaks at the level of the individual leaf. The distribution of ETs among leaf types and plots was correlated with genetic distance, indicating that the population had an ecotypic structure. A small number of ETs were found on more than one plot. Repeated isolation of the same genotype from diverse ecotypes has been one of the hallmarks of clonal population structure (Selander and Levin, 1980) and statistical analysis showed that the fluorescent Pseudomonas population as a whole (including both pathogens and saprophytes) was in linkage disequilibrium and was therefore essentially asexual. However, two genetically defined subpopulations were in linkage equilibrium, leaving the possibility of frequent genetic exchange among their members.
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These results suggest that plant architecture leads to subdivision of the plant-colonizing Pseudomonas population, with each leaf harbouring a discrete population, apparently separated from that found on every other leaf. In reality, populations on different leaves are likely to be linked by migration events facilitated by a variety of biotic and abiotic factors and thus form part of a large regional population or metapopulation. Founder effects may be particularly important in determining the nature of the population colonizing each leaf, especially given the exposed habit of leaves. Rapid and frequent fluctuations in environmental parameters are likely to lead to “bottlenecks” and the potential for the establishment of new populations from bacteria carried long distances in the atmosphere on dust particles and distributed by rain droplets, insects, birds and small mammals, is considerable.
V. THE METAPOPULATION CONCEPT The metapopulation concept recognizes that the distribution of individuals of a population across a landscape is uneven (Levins, 1968) and that the “regional population”, or metapopulation, consists of a variety of apparently discrete subpopulations confined to particular patches and linked by patchto-patch migration events (Levins, 1969; Slatkin, 1977; Whitlock and McCauley, 1990; Hanski and Gilpin, 1991). Given the apparent patchiness of leaf-colonizing Pseudomonas populations, the metapopulation concept would appear to provide a valid conceptual and experimental framework. Thompson and Burdon have recently recognized the appropriateness of this concept for the study of plant pathogenic fungi (Thompson and Burdon, 1992; Burdon, 1993). Related concepts from the equilibrium theory of island biogeography (MacArthur and Wilson, 1967), and Wright’s island model (Wright, 1951) have been used in the study of filamentous fungi populations colonizing apple leaves (Kinkel et al., 1987) and to provide an estimate of migration between geographically separated subpopulations of Mycosphaerella graminicola (Boeger et al., 1993), respectively. In nature many species have patchy distributions with local populations subject to frequent extinction and recolonization events. These events are highly significant because they can result in a severe reduction in effective population size and lead to radical shifts in population structure (Wright, 1940). A number of workers have considered the effect of local extinction and recolonization of subpopulations on genetic variability within metapopulations. Slatkin’s early work (Slatkin, 1977) was fundamental in recognizing two principal consequences of the extinction and recolonization process: (i) the founder effect and (ii) the random mixing of individuals from several source populations to make up a new population. Slatkin realized that the source of genotypes founding a new population was the most important factor determin-
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ing the outcome of extinction and recolonization, and this led to the development of two extreme models ,designated the “propagule pool” model and “migrant pool” model. These models show that differentiation between local populations either increases, or decreases, depending upon whether the source of founding propagules comes from a single population (propagule pool), or from multiple populations (migrant pool), respectively. In the propagule pool model, variation increases, because genetic drift outweighs gene flow, whereas in the migrant pool model, sampling of variation from multiple populations (in which local mutation tends to increase local differentiation) decreases differences among populations (Slatkin, 1977). Maruyama and Kimura (1980) pursued these ideas further and developed a clonal model of bacterial populations in which frequent local extinction and recolonization not only drastically reduces the effective population size, but also largely prevents divergence of subpopulations. Cohan (1994) has also used mathematical models to investigate factors affecting evolutionary divergence in metapopulations. He examined the effects of recombination on evolutionary divergence in a metapopulation of bacterial populations inhabiting ecologically distinct niches. Cohan’s models show that for such ecotypically structured populations adaptive divergence is unlikely to be constrained by low rates of recombination and that divergence in adaptive characters can occur in populations with high rates of recombination, even though divergence in neutral characters is constrained. Significantly, the models suggest that sexual isolation may not be necessary for the maintenance of adaptive divergence between bacterial populations. If permanent adaptive divergence is to occur then it is essential for populations to become ecologically distinct, so that adaptive mutants can co-exist and are not extinguished by periodic selection (Maynard Smith, 1991; Cohan, 1994). We have recently documented ecological divergence within populations of Pseudornonusfluorescens (founded from single cells) inhabiting heterogeneous environments. The key to stable maintenance of adaptive mutants in these populations is niche diversity and this has been demonstrated by the absence of ecological divergence in populations inhabiting homogeneous environments (P. B. Rainey, unpublished). Clearly, many factors affect the degree of divergence between subpopulations and Cohan (1994) writes “. . . diversity within a metapopulation is determined by a complex interaction among the rate of genetic exchange, the rate and intensity of periodic selection, population size, and ecological divergence among populations”. To this list could be added frequency- and density-dependent selection, and the balance between DNA mismatch repair, and SOS-inducible repair and recombination. The opposing activities of RecBC-dependent SOS repair (which increases recombination through overproduction of RecA) nd mismatch repair (which acts as a major barrier to recombination between bacterial species) has recently been suggested to
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be a key component determining the degree of divergence between populations (Matic et al., 1995).
VI. CONCLUSIONS This chapter has attempted to provide a brief introduction to bacterial population genetics and in so doing provoke microbial ecologists and phytopathologists to consider questions relating to the extent and significance of genetic variation within plant-colonizing bacterial populations. If genetically engineered bacteria are to be safely and profitably exploited in agriculture, then it is essential that we develop an understanding of the mechanisms, rates and extent of recombination in natural populations. If effective biological control strategies are to be developed, then a sound knowledge of temporal and spatial variation of genotypes is essential, and if outbreaks of disease are to be effectively tracked and monitored we must understand gene flow as a force that shapes the evolution of pathogen populations. More specifically, we need to determine the extent of genetic diversity and understand how it is distributed. Do bacterial populations have ecotypic structure? That is, do individual plants contain similar populations, or do populations vary from plant to plant, or even from leaf to leaf, or root to root? Do different plant species harbour different populations, and how widely distributed are individual clones? How do environmental conditions affect population structure and is there a predictable succession of genotypes over a season? What are the principal barriers to gene flow? How clearly delineated are population boundaries and how do natural population subdivisions correlate with taxonomic subdivisions? This list of questions is by no means exhaustive and clearly there is much to learn, but powerful tools and a strong conceptual framework of ecological and evolutionary genetics have already provided substantial insight into the genetic structure and evolution of a small number of prokaryote populations. We hope for similar progress in our understanding of plant-colonizing bacterial populations.
ACKNOWLEDGEMENTS We are indebted to Peter Young for constructive criticism and helpful comments on a draft of this manuscript. This work is supported by Grants from the B.B.S.R.C., Oxford University “Pump-Priming” Scheme and The Royal Society. P.B.R. is a B.B.S.R.C. Research Fellow.
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Asexual Sporulation in the Oomycetes
A. R. HARDHAM’ and G. J. HYDE2
‘Plant Cell Biology Group, The Research School of Biological Sciences, The Australian National University, P. 0.Box 475, Canberra 2601, Australia. 2School of Biological Science, University of New South Wales, Kensington, NSW 2033, Australia
I. Introduction ....... .............,..................... .................... . .............. 353
....................................................................... 357 A. Induction ......,......,...........,................................................ . 357 B. Morphological Development ........................................ ....... .. 359 C. Synthesis of Zoospore-specific Components during 361 Sporangiogenesis .................................................................
11. Sporangiogenesis
111. Zoosporogenesis
........................................................................
A. Induction ........................................................................... B. The Process of Cleavage ...................................................... C. Synthesis of Zoospore-specific Components during Zoosporogenesis .................................................. . ...,. ......... D. Polarization of Zoosporic Organelles .. .................................... E. Zoospore Discharge ......................... ................ ................... F. Conclusions ........................................................................ Acknowledgements ..............,......................................... ............ References ................................................................... ....... .... .
377 377 378 384 386 386 389 390 390
I. INTRODUCTION The oomycetes are an important and distinct group of organisms. The class contains species that cause many of the world’s serious plant diseases, perhaps the most infamous being Phytophthoru infestuns (order Peronosporales), the late blight of potato (Agrios, 1988). Phytophthoru species cause root rots of a wide variety of crop, ornamental and forest plants; species of Pythium cause seed rot and seedling damping off; species of Advances in Botanical Research Vol. 24 incorporating Advances in Plant Pathology ISBN &12-005924-X
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A. R. HARDHAM and G . J. HYDE
Bremia, Plasmopara, Peronospora, Pseudoperonospora and Sclerospora cause downy mildew diseases of crops and ornamentals; species of Albugo cause white rusts of cruciferous plants. One large order, the Saprolegniales, contains species in the genera Saprolegnia, Aphanomyces, Achlya and Dictyuchus which are responsible for diseases in fish (Wilson, 1976). Oomycetes have been traditionally included within the fungal kingdom: they form aseptate hyphae, have a fungus-like growth habit and nutritional requirements similar to those of the true fungi (Eumycota: ascomycetes, basidiomycetes and zygomycetes and deuteromycetes). The oomycetes, however, differ from true fungi and are more plant-like in a range of structural and biochemical characters (see Beakes, 1987; Dick, 1990; Hardham et al., 1994). For example, they produce cellulosic cell walls (Bartnicki-Garcia, 1987), have a diploid somatic thallus (Sansome, 1987) and have tubular rather than plate-like mitochondria1 cristae (Cavalier-Smith, 1981). Comparisons of mtDNA and rRNA sequences indicate that oomycetes are more closely related to heterokont, chromophyte algae than to uniflagellate chytridiomycetes or to the higher fungi (Gunderson et al., 1987; Wolters and Erdmann, 1988; Forster et al., 1990; Sachay et al., 1993). Their unique complement of attributes has led Margulis et al. (1990) to place the oomycetes into a separate phylum, the Oomycota. Both the taxonomy and pathogenicity of oomycetes depend greatly upon the features of motile zoospores that are produced in large numbers by most of these organisms (Dick, 1990). Oomycete zoospores are uninucleate, biflagellate, membrane-bound cells that lack a cell wall. Some species of oomycetes are dimorphic and produce two types of zoospore: primary zoospores are pear shaped with the two flagella emerging from the apex of the cell; secondary zoospores are roughly ellipsoidal, with a longitudinal groove along a slightly flattened face (the ventral surface) giving the zoospores a kidney-shaped profile in transverse section. The flagella, an anterior tinsel flagellum and a posterior whiplash flagellum, emerge from the centre of the groove on the ventral surface. Monomorphic species may produce primary- or secondary-type zoospores, depending on the species. The size of zoospores of a given species and type is fairly uniform, although there may be some variation due to physiological conditions (Lange and Olson, 1983). Zoospore shape is thought to depend, at least in part, on microtubular arrays centred on the flagellar basal region; cortical actin may also be involved (Holloway and Heath, 1977a; Hardham, 1987b; Heath and Harold, 1992; S. L. Jackson and A. R. Hardham, unpublished observations). The structure of the flagellar apparatus and the configuration of basal bodies and associated flagellar roots are important characteristics for taxonomic studies (Barr, 1981; Barr and Allan, 1985). The flagellar apparatus is one of several morphological features that are critical for zoosporic pathogenicity. Flagellar activity makes the zoospores motile, allowing them to swim towards potential hosts, to which they are
ASEXUAL SPORULATION IN THE OOMYCETES
355
Figs 1 4 . Transmission electron micrographs of Phytophthora cinnamomi zoospores (Figs 1, 3 and 4) and sporangia (Fig. 2). Fig. 1: Transverse section of a zoospore of P. cinnamomi showing nucleus (n), water expulsion vacuole (wev), lipid (l), fingerprint vesicles (fp), large peripheral vesicles (L), dorsal vesicles (d), ventral vesicles (v), basal body (bb) and groove (8) (scale bar = 1pm). Figs 2-4: Fingerprint vesicles in sporangium (Fig. 2) and zoospores (Figs 3 and 4). Fig. 2: In sporangia, the fingerprint vesicle (arrows) contents usually form an electron-dense aggregate within the vesicle. This appearance gives rise to their appellation of dense-body vesicle (scale bar = 1pm). Fig. 3: In chemically-fixed zoospores the fingerprint vesicle contents usually have a lamellated appearance either throughout or in part of the vesicle (scale bar = 0.5 prn). Fig. 4: Freeze-etch preparations consistently show lamellated contents within the fingerprint vesicles (scale bar = 0.1 pm).
356
A. R. HARDHAM and G . J. HYDE
chemotactically attracted. The elongated and grooved shape of the zoospores probably contributes to hydrodynamic stability during motility. The flagella appear to facilitate accurate docking and encystment of the zoospore at the host surface. Also of crucual importance to the encystment process is the organization of various components with the zoospore. In general, zoospores show a high and consistent degree of internal spatial order (Fig. 1).The most significant organizational feature of both primary and secondary types is the existence of an axis of polarity running from the two flagellar basal bodies at the apex of the nucleus, back through the pear-shaped nucleus. Many components, especially in secondary zoospores, exhibit characteristic, nonrandom distributions about the polar axis, a situation most thoroughly studied in Phytophthora cinnumomi (e.g. Hardham and Gubler, 1990). These components include certain vesicles confined to the ventral surface of zoospores. In Phytophthora and Suprolegnia these vesicles appear to be the source of adhesive material that is secreted during encystment (Lehnen and Powell, 1989; Hardham and Gubler, 1990). Prior to encystment the zoospores orient the ventral surface so that it faces the host and the glue is secreted onto the host surface. The cyst subsequently germinates from this same surface and the germ tube grows directly towards the host. Because of the central importance of zoospores to oomycete research, much effort has been directed towards gaining an understanding of how they are formed, i.e. the process of sporulation. Several previous reviews (e.g. Heath, 1976; Olson et al., 1981; Beakes, 1995) have considered sporulation in the oomycetes together with that in other zoosporic organisms, but the present chapter focuses specifically on this group. Typically sporulation involves the subdivision of multinucleate sporangia which develop at the tips of hyphae. In some species, sporangia do not cleave to form zoospores but instead the sporangia are dehiscent, functioning like conidia of higher fungi. They germinate directly on the surface of a potential host, producing an invasive germ tube. In zoosporic species, sporulation has two aspects: sporangiogenesis, the formation of the sporangium, and zoosporogenesis, its subdivision into zoospores. Critical events in these processes include: (i) induction of sporangiogenesis; (ii) formation and differentiation of sporangia, including development of a basal septum and apical papillum; (iii) synthesis of spore-specific components; (iv) induction of zoosporogenesis; (v) formation of the cleavage membranes; (vi) development of polarity of zoospore components; and (vii) discharge of zoospores from sporangia. Understanding the requirements for and mechanisms involved in oomycete sporangiogenesis and zoosporogenesis promises to provide a basis for the development of novel disease control measures that will target and inhibit sporulation events. In addition, the production of oomycete sporangia and zoospores provides many valuable opportunities for studies of fundamental processes in plant and fungal cell biology. Sporulation must incorporate many examples of signal reception and transduction, differential gene expression,
ASEXUAL SPORULATION IN THE OOMYCETES
357
synthesis of new proteins and other molecules, and the spatial regulation of cell components. Zoosporogenesis is an example of cytokinesis and the development of cell polarity, and depends on the correct functioning of the cytoskeleton. Zoospores are examples of motile and chemotactic cells, and encystment is one of the few examples of regulated secretion in a non-animal system. Recent studies of sporulation in the oomycetes have both benefited from and contributed to advances in these broader areas, and while this review focuses on sporulation per se, its aim is also to place our understanding of oomycete sporulation in this wider biological context.
IT. SPORANGIOGENESIS A. INDUCTION
Asexual sporulation of oomycetes, like that of true fungi, is induced by conditions that restrict hyphal growth (Dahlberg and Van Etten, 1982; Ribeiro, 1983; Hohl, 1990; Griffin, 1994). This means that many environmental factors, such as temperature, light, aeration, humidity, carbon and nitrogen sources or inorganic ions, can influence the transition from vegetative to reproductive growth. Of these, nutritional status is recognized as one of the most important factors. In general, replacement of nutrient medium with a mineral salts solution is used to trigger sporulation in vitro, however, more subtle changes in nutrient conditions can be sufficient to induce sporulation. For example, P. cinnamomi mycelia will begin to form sporangia after 1 day in nutrient broth (A. R. Hardham, unpublished observations) or after 2 days on nutrient agar plates (Dearnaley et al., 1996). Although P. cinnamomi mycelia begin to produce sporangia about 7.5 h after transfer to mineral salts solution, germinated cysts grown in nutrient broth for 6 h require a further 24 h in mineral salts solution before the first sporangia appear (Dearnaley et al., 1996). This behaviour may indicate that oomycete spores require a certain period of vegetative growth before they are “competent” to be induced to sporulate, as has been observed in the higher fungi (see Griffin, 1994). In Aspergillus nidulans, for example, competence is acquired over a 20h period following spore germination (Axelrod et al., 1973). Conidiation subsequently takes 4 h. The Chytridiomycetes, Allomyces and Blastocladiella, on the other hand, are able to form sporangia almost immediately after zoospore encystment (Youatt , 1976; Barstow and Lovett, 1978). The repeated emergence of motile zoospores from encysted spores in the oomycetes has been taken to indicate that these organisms are also immediately competent (see Griffin, 1994). However, this is a specialized case where a single zoospore is released from a uninucleate cyst and does not involve development of a sporangium or cleavage of multinucleate sporangial cytoplasm.
358
A. R. HARDHAM and G . J. HYDE
The nature of receptors that recognize inductive factors and molecular details of the pathways that transduce these signals to bring about initiation of spore development are poorly understood even in the best-studied organisms. Most investigations have confirmed the expectation that sporulation involves changes in gene expression and the appearance of new, sporulation-specific proteins. Pharmacological treatments of Achlyu with actinomycin D, which inhibits RNA synthesis, and cycloheximide, which inhibits protein synthesis, have shown that both RNA and protein synthesis are needed for sporangium formation (Griffin and Breuker, 1969; Timberlake et al., 1973; Gwynne and Brandhorst, 1982); the need for protein synthesis has also been demonstrated in P. infestans (Clark et al., 1978). Novel mRNAs are synthesized specifically during sporulation (Gwynne and Brandhorst, 1982); many accumulate late in sporangium formation, suggesting that they may be stored for subsequent use during cleavage (Clark et al., 1978) or cyst germination (Gwynne and Brandhorst, 1982; Jaworski and Harrison, 1986). There has been little information on the nature of the proteins encoded by these sporulation-specific genes, however, recent studies of the appearance of zoospore vesicle proteins in Phytophthora (Dearnaley et al., 1996) and, in one case, the mRNAs that encode them (J. S. Marshall and A. R. Hardham, unpublished observations), are an initial contribution in this area. As the molecular biology of the induction of sporulation in the oomycetes is unravelled, it may be important to bear in mind a potential difference between the oomycetes and the higher fungi. In Phytophthoru, after initial induction, changing conditions can inhibit and reverse the production of sporangia, and spore-specific components that had appeared in staling nutrient medium, disappear on transfer to fresh medium (Dearnaley et al., 19%). In addition, oomycete sporangia can themselves act as dispersive propagules. They may be dehiscent, and they may, if conditions are suitable, germinate directly through the production of hyphae rather than indirectly through the production of zoospores (Hemmes and Hohl, 1973; Ribeiro, 1983). Thus in the oomycetes, sporangiogenesis is separated temporally from zoosporogenesis and is not by necessity followed by it. This situation contrasts with that in higher fungi. In A . nidulans, for example, the production of dispersive spores (conidia) is the end-product of a pathway that requires the prior differentiation of a number of cell types, such as the foot cell, stalk, vesicle, metula and phialide; the first genes to be activated encode transcription factors that regulate the expression of other genes, and may also autoregulate their own expression (Andrianopoulos and Timberlake, 1994). This means that once it is activated, an autoregulatory gene’s expression continues and ensures that genes further downstream in the expression pathway also remain in an active state. This has been interpreted as a mechanism for ensuring that the mycelium remains committed to the pathway of sporulation even in the absence of continued inductive conditions
ASEXUAL SPORULATION IN THE OOMYCETES
359
(Andrianopoulos and Timberlake, 1994). This situation may not prevail in the oomycetes. Although it is almost certain that there are genes that act as master switches and code for regulatory proteins such as transcription factors in the oomycetes, autoregulation and commitment to all steps of the developmental sporulation sequence may not be present.
B. MORPHOLOGICAL DEVELOPMENT
Oomycetes vary as to how they produce the sporangia in which zoospores form. In some species, the entire vegetative thallus may be converted into a single sporangium or, in other species, into a chain of sporangia (Dick, 1990). In most species, however, and especially those of pathogenic importance, sporangial development involves the differentiation of limited, terminal sections of the vegetative mycelium, from which the sporangia are eventually isolated by transverse septa. This review concentrates on these more typical cases, drawing upon studies of the Peronosporales, Saprolegniales, Lagenidiales and Leptomitales (sensu Barr, 1983). From a morphological standpoint, typical sporangiogenesis begins when extension of the subtending hypha ceases. When this occurs, the normal zonation of organelles in the growing hyphal tip is rapidly lost (Armbruster, 1982a). Cytoplasm, nuclei and other organelles flow into the incipient sporangium which often swells as a cylinder, ellipsoid or sphere. The next major development is delimitation of the sporangium from the hypha by a septum that may form as an annular ingrowth of the hyphal wall, as a cytoplasmic plug or as a combination of both. The trigger for septum formation is unknown: it is not sporangial in size, since this may vary considerably within a species. The sporangial wall may also differentiate. For example in Phytophthoru palmivora, secondary thickening of the wall occurs, and proceeds from base to apex (Christen and Hohl, 1972). Such changes may prepare sporangia for the internal forces generated during sporangial discharge. In many species, the wall at the sporangial apex differentiates as a specialized papillum which typically ruptures to allow zoospore release. The most important components of the developing sporangium are its nuclei. While it is commonly thought that all nuclei in the sporangium flow in from the hypha, Trigano and Spurr (1987) have provided evidence of mitosis within sporangia of one species of the Peronosporales. Nevertheless, even in this study, most mitoses occurred before septum formation, and it is likely that in most oomycetes few, if any, further divisions occur after delimitation. Regardless of how they arise, the nuclei of oomycete sporangia are typically arranged in a predictable fashion, with respect both to each other and to the sporangium as a whole (Fig. 5). Nuclei in mature sporangia are regularly spaced throughout the cytoplasm (Kevorkian, 1935; Hohl and Hamamoto, 1967; Hoch and Mitchell, 1975; Oertel and Jelke, 1986; Hyde
360
A . R. HARDHAM and G . J. HYDE
Fig. 5. Diagram showing the arrangement of microtubules (fine lines), nuclei (shaded bodies), cleavage vacuoles (black planes) and actin microfilaments (shaded lines) in P. cinnamomi sporangia before ( Aand B) and after ( C ) cleavage is induced. The pair of short lines represents the pair of basal bodies at the apex of the nucleus. (A) Surface view; (B and C) median sections.
et al.,
1991a), indicating that uninucleate, cytoplasmic domains corresponding to the future zoospores are established during sporangial maturation. Most or all nuclei are found near the sporangial wall (e.g. Kevorkian, 1935; Elsner et al., 1970; Armbruster, 1982a; Hyde el al., 1991a). This is particularly true of small sporangia or those with cylindrical geometry. In larger ellipsoidal sporangia of P. cinnamomi some nuclei lie centrally (Hyde, 1991). In oomycetes generally, those nuclei that are near the sporangial wall are invariably oriented with their narrow pole towards the wall (Williams and Webster, 1970; Elsner et al., 1970; Armbruster, 1982b; Oertel and Jelke, 1986; Hyde et al., 1991a). The significance of this arrangement is uncertain. The narrow poles of sporangial nuclei are characterized by the presence of two basal bodies (Chapman and Vujicic, 1965; Hohl and Hamamoto, 1967; King et al., 1968; Elsner et al., 1970; Gotelli, 1974b; Schnepf et al., 1978c; Armbruster, 1982b; Lange et al., 1984, 1989; Hyde et al., 1991a) and dictyosomes (King et al., 1968; Williams and Webster, 1970; Elsner et al., 1970; Gotelli, 1974b; Schnepf et al., 1978c; Lange et al., 1989; Hyde et al., 1991a). Similar arrangements occur in many protoctistan and animal cells. All the above-mentioned aspects of nuclear arrangement are likely to be microtubule dependent. In ultrastructural studies limited microtubule arrays are commonly reported to emanate from the region of cytoplasm surrounding the basal bodies (Williams and Webster, 1970; Hoch and Mitchell, 1972b; Gotelli, 1974b; Hoch and Mitchell, 1975; Schnepf et al., 1978c; Lange et al., 1984; Jelke et al., 1987; Lange et al., 1989; Hyde et al., 1991a,b). Immunofluorescence studies indicate that these arrays are sufficiently extensive to allow microtubules from adjacent nuclei to interact (Fig. 5 )
ASEXUAL SPORULATION IN THE OOMYCETES
361
(Hyde and Hardham, 1992,1993). The mechanisms by which the arrays space the nuclei apart and orient them towards the wall are uncertain, but that they do is supported by the irregular nuclear positioning seen in sporangia treated with antimicrotubule, but not anti-actin, drugs (Hyde and Hardham, 1993). Microtubular disruption also causes nuclei to lose their pyriform shape and round up. The regular nuclear spacing of the pre-cleavage sporangium could be seen as a preparatory development for future events. For example, in some terrestrial species which only produce zoospores after rain, the all-butcleaved nature of the sporangium allows for rapid zoospore formation, helping to ensure that the zoospores will emerge into a moist environment. However, regular microtubule-dependent nuclear spacing also exists in other plant, animal and fungal coenocytic structures, many of which do not become cleaved (e.g. McNaughton and Goff, 1990). In some situations, regular nuclear spacing may contribute to efficient utilization of the available total DNA (see McNaughton and Goff, 1990). However, in other cases, such efficiency must be of secondary importance; for example, nuclei in the subapical regions of Saprolegnia hyphae are highly concentrated and without any apparent spatial order. Considering that oomycete zoospores of a given species are typically of uniform size, and that no surplus cytoplasm remains after cleavage, the developing sporangium must somehow accurately gauge its nuclear/cytoplasmic ratio. One fine-tuning mechanism might involve degeneration of excess nuclei (Hemmes and Hohl, 1973; Armbruster, 1982a).
C. SYNTHESIS OF ZOOSPORE-SPECIFIC COMPONENTS DURING SPORANGIOGENESIS
Oomycete zoospores have, in addition to the usual repertoire of eukaryotic organelles, many specialized structures, most notably the flagellar apparatus and an assortment of vesicles. Some components such as lipid globules and carbohydrate stores (fingerprint or dense-body vesicles) are present in zoospores, sporangia and vegetative hyphae (Tables 1-111; Figs 1-4); other components, both cytoplasmic and on the cell surface, appear only after sporulation is induced. These sporulation-specific components include three types of vesicle that occur in the zoospore cortical cytoplasm and packets of tubular hairs (mastigonemes) that adorn the surface of the zoospore anterior flagellum. Early ultrastructural studies of a range of oomycete species gave rise to a diverse array of descriptive names for zoospore vesicles, such that by 1976, vesicles from four genera (Phytophthora, Pythium, Saprolegnia and Aphanomyces) had been given 24 different names (Lunney and Bland, 1976b). Over recent years, new information on vesicle structure, immunoreactivity, distribution, ontogeny and fate has become available,
w
m
N
TABLE I Occurrence of zoospore components throughout the asexual life cycle in the Peronosporalesa Vegetative hyphae
Sporulating Post-septum hyphae sporangia
Cleaving sporangia
Zoospores
Cysts
+
-I
Germinating cysts
Species
References
~
Dense bodylfingerprint vesicles
+
+
+
+ + +
+
+
+
+
+
+
t
+ + -
Lipid
+
+ + I
+
+
+
+
+ + +
+
+ +
+ + +
+
+ + + +
+
+
+>-
+
+ +
t
+
+
+>-
+
+
+
+
+
+
+
i
+
P. P. P. P. P.
rapsicr cinnomomi eryrhroseprica infesrans megaspermu var. sojae P. palmivora
P. parasirica Pyrh. aphanidermarum Pyrh. proliferum Bremia lacrucae Pwudopernnnspora ruhenric Sclerospora graminicola
Williams and Webster, 1970 Hardham. 1987a. Hyde er nl . 1WIa Chapman and Vujicic, 1965 King er a / . , 1968; Elsner er al. , 1Y70 Ho er a / . . 1968 Hohl and Hamamoto, 1967; Bimpong and Hickman, 1975; Barlnicki-Garcia and Hemmes. 1976 Reichle, 1969 Grove and Bracker, 1978 Lunney and Bland, 1976a,b Sargent and Payne. 1974 Lange el a / . . lYX9 Lange er a / . . 1984
Pyrh. proliferum Bremia lacmcae Pseudoperonospora cubensis Sclerospora graminrcob
Williams and Webster, 1970 Hardham, 1987a; Hyde e r a / . , 1991a Chapman and Vujicic, 1965 Ehrlich and Ehrlich. 1966; King er a / . , 1968; Elsner ec a l . , 1970 Hohl and Hamamoto, 1967; Bimpong and Hickman, 1975; Bartnicki-Garcia and Hemmes, 1976 Ehrlich and Ehrlich. 1966 Grove and Bracker, 1978 Lunney and Bland, 1976a, l976b Sargent and Payne, 1974 Lange el ol.. 1989 Lange er al.. 1984
P. capsici
Williams and Webster. 1970
P. P P. P. P.
cnppsici cinnamomi eryrhrosepnca infescans palmivora
P. parasirica 0 t h . aphanrdermarum
Fihrillar/large peripheral vesicles
+
-
+
+
+
+
+
+
+
+ +
+
+
+
+ +
+
+>-
P. cinnamomi
+
+>-
P. megasperma var. sojae P. palmrvora
+ +
+>-
P. parasirica Pyrh. aphonidermarum Pyrh. proliferum Pyrhium spp Sclerospora graminicolo
Hardham, 1987a; Gubler and Hardham, 1988. 1990: Hyde el a / . , 1991a; Hyde and Hardham. 1993; Deamaley and Hardham. 1994; Dearnaley el al.. 1996 Ho er o f . , 1968 Hemmes and Hohl, 1969; Bimpong and Hickman. 1975; Bartnicki-Garcia and Hemmes, 1976 Reichle. 1%9 Grove and Bracker, 1978; Estrada-Garcia er a / . 1990 Lunney and Bland. 1976a.b Cope el
01..
1996
Lange er al., 1984 (Fig. 5 )
Encystmentidorsal cyst coal vesicles -
+
+ +
I
L
-
-
+
P. megarperma var. sojae Pyrhrum spp.
+
K-hodiedventral vesicles -
+
+ +
+
+ +
+
-
~
L
+
Pyth. aphanidermanrm 0 t h . proliferum Pythium spp. Pseudopcronospora cubensh
t
+ Mastigonemes -
+
+
+ +
+
+
+
+
+
+ + +
+
P. capsici P. cinnamoni
P. infesfans P. megarperma var. sojae P. pdmivom
+ I
P. capsici P. cinnamomi
+
+
P. P. P. P.
cqsici
cinnamomi infesrans poraririca Pyrh. apJianidermatum Pyth. proliferum
Williams and Webster. 1970 (Fig. 26) Gubler and Hardham, 1988, 19w; Hardham and Gubler. 1990; Hyde er a/.. 1991a; Hyde and Hardham. 1993: Dearnaley and Hardham. 1994: Dearnaley er al., 1996 Ho el al.. 1%8 (Fig. 23) Cope er al., 1996 Williams and Webster, 1970 (Fig. 26) Gubler and Hardham, 1988, 1990; Hardham and Gubler. 1990: Hyde et a / . , IWIa; Hyde and Hardham. 1993; Dearnaley and Hardham, 1994; Dearnaley cr al.. IW6 King er al.. 1%8 Ho et al., 1964 (Figs 25 and 26) Hohl and Hamamoto, 1967; Hemmes and Hohl, 1969; Bimpong and Hickman, 1975 (Figs I, 3 and 4): Powell and Bracker, 1986 Grove and Bracker, 1978 (Figs 17-19) Lunney and Bland, 1976b (Fig. 1) Cope er al.. 19% Lange er a/., 1989
Williams and Webster,
1WO (Figs 25, 27 and 28)
Cope and Hardham, 1994 Elsner er al.. 1970
Reichle, 1%9 Grove and Bracker, 1978 Lunney and Bland, 1976a.b
w o\ w
TABLE I-continued Vegetative Sporulating Post-septum Cleaving hyphae hyphae sporangia sporangia Flagella
Zmspores
Cysts
+
+ -
Germinating cysts
Species
+
P. apsici P. c*mcunomi
+ +
P. infestm P. pdmivora Pyth. proliferum Pseudopronospara cubensis Sclerospora graminicola
+ + +
References
Williams and Webster, 1970 Hyde el d.,1991a; Cope and Hardham, 1994 C o l b U O , 1%; King er d.,1%. Elsner et d.,1970 HoM and Hamamoto, 1%7; Hernmes and HoM, 1969 Lunney and Bland, 1976a.b Lange et a/., 1989 Lange d d.,1%
Peripheral cisternae
P. cinluvnomi
Water expulsion vacuole
+
+ + +
+
+ +
+>-
P. palm'vora P. parmifica Pyth. aphonidcrmnrum Pyih. proliferum
Hardham, 1 W a ; Hyde er d.,1991b Bimpong and Hickman. 1975; Bartnicki-Garcia and Henunes, 1976 Reichle, 1969 Grove and Bracker, 1968 Lunney and Bland, 1976b
P. cinluvnom' P. mcgmperma P. patm'wra Pyrh. aphami&rmahun Pyth. prdifwum Sckrospom graminicola
Hardham, 1981, Ho d d.,1968 Bimpong and Hickman, 1975 Grove and Bracker, 1978 Lunney and Bland, 1976b Lange el d..1984
aData from individual papers have been combined for each species within a single line. In some cases the presence of a component is not mentioned in the text but evidence for its presence has been deduced from micrographs within the papers cited. + , Character present; - , character absent, +>-, character initially present but subsequently disappearing.
ASEXUAL SPORULATION IN THE OOMYCETES
365
allowing zoospore vesicles to be grouped into five main categories (Beakes, 1989, 1995; Hardham, 1995): (1) dense-body or fingerprint vesicles; (2) fibrillar or large peripheral vesicles; (3) encystment, cyst coat or dorsal vesicles; (4) K-bodies or ventral vesicles; and ( 5 ) peripheral cisternae. Justification for the interpretations of vesicle homologies basic to this categorization is given in the following section. Summaries of alternative names for these vesicles as used in the older literature can be found in earlier reviews (Lunney and Bland, 1976b; Holloway and Heath, 1977b; Hemmes, 1983; Hardham, 1987a; Beakes, 1989, 1995; Hardham, 1995). Detailed studies of P. cinnamomi (Cope and Hardham, 1994; Dearnaley et al., 1996) and collation of data from a diverse range of oomycete species (Tables 1-111) indicate that the five different categories of vesicles (Figs 1-4 and 6-18), mastigonemes (Figs 19-21), flagella (Fig. 22) and the water expulsion vacuole (Fig. 1) show three different temporal patterns of synthesis. Dense-body or fingerprint vesicles are present throughout the asexual life cycle; large peripheral or fibrillar vesicles, encystment or dorsal vesicles, K-bodies or ventral vesicles and packets of mastigonemes are synthesized after the onset of sporulation, and peripheral cisternae, the flagella (with two possible exceptions) and water expulsion vacuoles are synthesized during zoosporogenesis (summarized diagrammatically in Fig. 23). The reasons for the synthesis of different components at specific times are as yet unknown, however they may relate (as in the case of the precise arrangement of nuclei within newly formed sporangia) to the advantage gained by the ability of the organism to complete zoosporogenesis very rapidly. Some species can cleave within 10-15 min (P. cinnamomi: Byrt and Grant, 1979; P. nicotianae: Y. Gautam and A. R. Hardham, unpublished observations), and even if sporangia already contained the appropriate mRNA transcripts, it would be likely to take much longer than this to synthesize, process and package the contents of all three types of peripheral vesicle and the mastigonemes. Preformation might not be needed for the other three components that are produced only after cleavage has begun (the plasma membrane, peripheral cisternae and water expulsion vacuole) since their predominantly membranous nature probably allows them to be manufactured more quickly. I. Large peripheral vesicles or jibrillar vesicles Most ultrastructural studies of oomycete zoospores report the presence, in the cortical cytoplasm, of large ellipsoidal vesicles with a diameter of 0.2-0.5 wm in the plane parallel to the zoospore surface and approximately 0.8 p m in the plane perpendicular to the surface (Figs 1,6,7,13, 14) (Lunney and Bland, 1976b; Grove and Bracker, 1978; Hemmes, 1983; Cerenius et al., 1984; Beakes, 1987; Hardham et al., 1991a). Their contents appear electron lucent, granular or fibrillar, a difference in morphology which is likely to be due to variability in the preservation of their contents by different chemical
TABLE I1 Occurrence of zoospore components throughout the asexual life cycle in the Saprolegnialesa Vegetative hyphae
Sporulating Post-septum hyphae sporangia
Dense-bcdylfingerprint vesicles
+ + +
-
+
+
+ +
+
+ + +
Cleaving sporangia
Primary zoospores
Primary cysts
+
+ +
+
+
+ +
+
+
+
+
+
+
+>-
+
+
+
+ +
+ +
+
+
+
+ + +
Fibrillarnarge peripheral vesicles
+
-/+
+
EncystrnentldorsaVcyst coat vesicles
+
+
+
+ +
+
+ +
+
-/+
+
+
+ + +
Species
t
Beakes, 1987, 1989 Armbruster, 1982d Hoch and Mitchell, 197Za.b. 1975 Armbruster, 1982a,b Armbruster, 1982a Gay and Greenwood, 1966; Holloway and Heath, 1977b; Lehnen and Powell, 1989
A . pageUara A . debaryanwn Aphanomyces eufeiches
Beakes, 1987 Armbruster, 1982a Hoch and Mitchell. 1972a.b; Sadowski and Powell, 1990 Armbruster. 1982a.b Armbruster, 1982a Gay and Greenwood, 1966; Holloway and Heath, 1977b
A . pagellafa Aphanomyces eufezches
+
+
-
+
-I+
+
References
A . flageUala A . debaryanwn Aphanomyces eureiches Brevilegnia spp. Dictyuchlcr spp. Saprolegnu spp.
Brevilegnw spp. Dictyuchus spp. Saprolegnia spp
+ +
+
+
Secondary Germinating cysts cysts
+
Lipid
+
Secondary zoospores
Saprolegnia spp
A . pagellafa Aphanomyces eureiches Brevilegnia spp. Saprolegnia spp
Beakes. 1987 Hoch and Mitchell. 1972b; Sadowski and Powell. 1990 Heath and Greenwood, 1971; Holloway and Heath, 197%; Beakes, 1983; Lehnen and Powell, 1989; Burr and Beakes, 1994
Beakes, 1987 Hoch and Mitchell, 1972a.b: Sadowski and Powell, 19w Armbruster, 1982a,b Gay and Greenwood. 1966; Holloway and Heath, 3977b; Beakes, 1983; Lehnen and Powell, 1989; Burr and Beakes, 1994
K-hodiedventral vesicles
+ -
+
+
+ +
+
-/t
+
+
-/+
+ Mastigonemes
+ Flagella
-
+
+
+
+
+
+
+ +
+ -
-
+
+
+
+ +
+ Water expulsion vacuole
+ +
-
-
Brevilegnia spp. Saprolegnia spp.
Thrausrorheca
+
Peripheral vesicles
A . pugelluiu A . umbibr~e+uu/rs Aphanomyces eurerches
+
-
Beakes. 1987 Powell er ol.. 1985 Hoch and Mitchell. 1972a; Powell er u/ 1985; Sadowski and Powell, 1990 Armbruster, 1982b Holloway and Heath, 1977h; Beakes. 1983; Lehnen and Powell, 1989. 1991; Burr and Beakes, 1994 Powell er a / . , 1985
,
Aphunomym eureiches Brevilegniu Dicryuchus Saprolegnw spp
Hoch and Mitchell. 1972a.b Armbuster, 1982b Armbuster, 1982b Heath PI a / . , 1970; Holloway and Heath, 1977b
Achlyu spp. Aphanomyres eureiches Suprolegniu spp.
Cotner, 1930 (cited in Colhoun, 1966) Hoch and Mitchell, 1972a Heath and Greenwood, 1971
A . flagellorn Aphanomyres eureiches
Beakes. 1987 Hoch and Mitchell. 1972a.b; Sadowski and Powell, 1990 Beakes, 1983; Lehnen and Powell. 1989; Burr and Beakes, 1994
Saprolegnia spp.
+
-
A . flage/[otu Aphanomyces euieiches
Beakes, 1987 Powell,and Hoch 1990 Mitchell. 1972a; Sadowski and
+
-
Suprolegnia spp.
Holloway and Heath, 1977b; B u n and Beakes, 1994
aData from individual papers have been combined for each species within a single line. In some cases the presence of a component is not mentioned in the text but evidence for its presence has been deduced from micrographs within the papers cited. +, Character present; -, character absent, +>-, character initially present but subsequently disappearing; +/-, character reported in different studies to be present or absent.
'8 z
0
0
5
' 0
2
w
m
4
TABLE 111 Occurrence of zoospore components throughout the asexual life cycle in the Lagenidialesa Vegetative hyphae
Sporulating hyphae
Dense-bodylfingerprintvesicles
+
+
Post-septum sporangia
Cleaving sporangia
+
+
+ + Lipid
+ +
+
Zoospores
QSS
+
+ + + +
+
+
+
+
+
+
+
+ +
+
FibrillarAarge peripheral vesicles
+
+
+ + Encystment/dorsaVcyst coat vesicles
+
+
+
+
-
References
Species
Lagenidium callinectcs Lagenidium giganfeum Lagena radicola Olpidiopsir sapmlegniae Olpidwpsis varians Petersenia palmariae
Gotelli. 1')74a,b Domnas er 111.. 1986 Ban and Waulnien. 1987, 1990 Bortnick et PI., 1985 Martin and Miller. 1986 Pueschel and van der Meer, 1985
Lagenidium callinecfes Lagenidium giganfeum Lagena radicola Olpidiopsis saprolegniae Olpidwpsir v a b Perersenia palmariac Lagenisma coscinodisci
Gotelli, 1974a.b
Domnas er el., 1986 Barr and h u l n i e n . 1987, 1990 Bortnick cr al.. 1985 Martin and Miller, 1986 Pueschel and van der Meer,1985 Schnepf el al., 1978a
Lagenidium callinecres Lagenidium giganfeum Lagena radicola Olpidbpsis saprolegniae
Gotelli, 19741, Domnas er al., 1986 Barr and Desaulnien, 1987, 1990 Bortnick er al., 1985
Lagenirma coscinodisci
Schnepf el 01.. 1978b
?
P 3:
$U
3:
s J
n.
P ?
3: 4 U
rn
K-bodieslventral vesicles Lagenidiwn callinecres
Lngena radkoln OIpidiopsis saprolegniae Olpidiopsis varians Mastigonemes
+
+
+ +
+ Flagella
Peripheral vesicles
+
+
+
+ +
+ -
+ +
+
+ +
+
+
+
+
+ +
Water expulsion vacuole
+
+ +
Gotelli, 1974b Ban and Desauloien. 1987, 1990 Bortnick er a/., 1985 Martin and Miller, 1986
Lngenidiwn caUinecvs Lagenidium gigantcum Lagena radicoln Olpidiopsis saprolcgniae Olpidiopsis varians Petemenio palmariae Lagenisma coscinodisci
Gotelli, 1914a
Lagenidium callinecres Perersenia palmariae Lagenisma coscinodisci
Gotelli, 1974b Pueschel and van der Meer, 1985 Schnepf et al., 1978a
Lagenidiwn giganleum Olpidiopsis saprolegniae
Domnas er al., 1986 Bortnick er al., 1985
Lagenidium giganreum Olpidiopsir saprolegniae Lagena radicola
Domas et d.,1986 Bortnick er a/.,1985 Barr and Desaulniers, 1987, 1990
Domnas er d.,1986
b m X C cn
F
Barr and Dhaulnien, 1987, 1990 Bortnick et al., 1985 Martin and Miller, 1986 Pueschel and van der Meer, 1985 Schnepf er al., 1978a
aData from individual papers have been combined for each species within a single line. In some cases the presence of a component is not mentioned in the text but evidence for its presence has been deduced from micrographs within the papers cited. +, Character present; -, character absent.
0 Z
0 0
5 m
370
A. R. HARDHAM and G. J. HYDE
fixation regimes (Hardham, 1987a). They tend to be called large peripheral vesicles in Phytophthora and Pythium and fibrillar vesicles in Sapro legnia. Early studies of P. palmivora (Pinto da Silver and Noguiera, 1977; Sing and Bartnicki-Garcia, 1975a), Pythium aphanidermatum (Grove and Bracker, 1978) and Saprolegnia (Beakes, 1983), led to the suggestion that the contents of the large peripheral vesicles were secreted during zoospore encystment. However, it was later shown that this was an artefact arising during chemical fixation (Hardham, 1985). More recently, immunolabelling of Pyth. aphanidermatum zoospores was also interpreted as giving evidence of secretion of large peripheral vesicle contents (Estrada-Garcia et al., 1990). This interpretation has also been challenged, and it has been suggested that ambiguity may have arisen because the antibodies recognize an epitope common to large peripheral vesicles and to other vesicles whose contents are secreted (Cope et al., 1996). We believe that there is good evidence that the contents of the large peripheral vesicles are not secreted during encystment in species of any of these genera (Lunney and Bland, 1976b; Holloway and Heath, 1977b; Gubler and Hardham, 1988; Cope et al., 1996). Biochemical analyses of their contents have shown that they contain high-molecular-weight glycoproteins (Sing and Bartnicki-Garcia, 1975b; Gubler and Hardham, 1988; Estrada-Garcia et al., 1990). The large peripheral vesicles in Phytophthora and Pythium disappear from cysts (see Table I) and it has been shown in P. cinnamomi that they are broken down several hours after cyst germination (Gubler and Hardham, 1990). This observation has led to the suggestion that their glycoprotein contents form protein stores for use during germling growth before the pathogen has established access to an exogenous source of nutrients (Gubler and Hardham, 1990).
Figs 6-12. Transmission electron micrographs showing vesicles in zoospores of Phytophthora (Figs 6 and 7) and Saprolegnia (Figs 8-12) species. Fig. 6: Zoospore of P.megasperma showing groove (g), large peripheral vesicles (L) and ventral vesicles (v). From morphology alone, it is not possible to identify with certainty the two vesicles that are unlabelled. Peripheral cisternae (arrowheads) lie beneath the plasma membrane (scale bar = 0.5 pm). Fig. 7: Zoospores of P. cactorum showing large peripheral vesicles (L), a dorsal vesicle (d) and ventral vesicles (v) (scale bar = 0.5 pm). Fig. 8: Primary encystment vesicle in sporangium of Saprolegnia parasitica (scale bar = 0.1 pm). Fig. 9: Primary encystment vesicle in sporangium of S. ferax (scale bar = 0.1 pm). Fig. 10: Secondary encystment vesicle in primary cyst of S. diclinu-parusitica (scale bar 0.5 pm). Fig. 11: K,-bodies and vesicles similar to primary bars in primary zoospores of Saprolegnia (scale bar = 0.5 pm). Fig. 12: K2-body in Saprolegnia (scale bar = 0.5 pm). (Fig. 7 is reproduced with permission from Hardham et al. 1991a; Figs 8-10 are reproduced with permission from Beakes, 1983; and Figs 11 and 12 are reproduced with permission from Holloway and Heath, 1977b).
ASEXUAL SPORULATION IN THE OOMYCETES
371
372
A. R. HARDHAM and G . J. HYDE
Figs 13-18. Immunogold (Figs 13, 15 and 17) and immunofluorescence (Figs 14, 16 and 18) labelling of the contents of large peripheral vesicles (Figs 13 and 14), dorsal vesicles (Figs 15 and 16) and ventral vesicles (Figs 17 and 18) in zoospores or P. cinnamomi. Large peripheral and dorsal vesicles occur on the dorsal surface and tend to avoid the groove (g) on the ventral surface. Ventral vesicles occur predominantly close to the ridges of the ventral groove (scale bar in Figs 13, 15 and 17 = 0.5 pm. Figs 14, 16 and 18 are at the same magnification. Scale bar in Fig. 18 = 10pm) (Figs 13 and 15 courtesy of Dr Frank Gubler. Figs 14 and 17 reproduced with permission from Hardham et al., 1991a).
Large peripheraYfibrillar vesicles are absent from vegetative hyphae (Dearnaley et al., 1996), but have been described in sporulating hyphae in Phyrophrhora species (Dearnaley et al., 1996) and in sporangia of species of Phytophthoru (Hemmes and Hohl, 1969; Williams and Webster, 1970; Bartnicki-Garcia and Hemmes, 1976; Dearnaley et al., 1996), Pythium (Lunney and Bland, 1976a), Lagena (Barr and DCsaulniers, 1990) and
ASEXUAL SPORULATION IN THE OOMYCETES
373
Figs 19-22. The flagellar apparatus of zoospores of P. cinnamomi. Fig. 19: Transmission electron micrograph of a packet of tubular mastigonemes (scale bar = 0.5 pm). Fig. 20: Immunofluorescent labelling of packets of mastigonemes within a zoospore. (Reproduced with permission from Hardham et al., 1994.) Fig. 21: Immunofluorescent labelling of mastigonemes distributed in two rows on the surface of the anterior flagellum of a zoospore. Fig. 22: Immunofluorescent labelling of microtubules in the flagellar axonemes, in the flagella roots and cytoplasm of a zoospore. (Scale bar in Figs 20-22 = 10pm.)
Saprolegnia (Beakes, 1983). Immunocytochemical studies of P. cinnamomi have shown that they begin to form about 5 h after the induction of sporulation by transfer of mycelia from nutrient broth to mineral salts solution (Dearnaley et al., 1996). Immunogold labelling with a monoclonal antibody specific for the contents of large peripheral vesicles in P. cinnamomi has demonstrated the appearance of these antigens within Golgi cisternae, indicating that the vesicles are formed by the Golgi apparatus (Dearnaley and Hardham, 1994). 2. K-bodies or ventral vesicles Zoospores of members of the Saprolegniales, Leptomitales and Lagenidiales contain a distinctive type of vesicle in the groove region on the ventral surface close to the basal bodies (kinetosomes) (Figs 11 and 12) (Hoch and Mitchell, 1972b; Holloway and Heath, 1977b; Powell et al., 1985; Bortnick et al., 1985; Gotelli and Hanson, 1987; Lehnen and Powell, 1989, 1991). The structure of these kinetosome-associated organelles, or K-bodies, differs in primary and secondary zoospores: K1-bodies are about 0.4 pm in diameter and have
374
A . R. HARDHAM and G. J. HYDE
Fig. 23. Diagram summarizing data on the occurrence of zoospore components in cells throughout the life cycle of oomycete species. WEV, Water expulsion vacuole.
ASEXUAL SPORULATION IN THE OOMYCETES
375
a granular matrix and a narrow osmiophilic cortex (Fig. 11); K2-bodies are about 0.8 pm in diameter and (except in Thraustotheca; Powell et al., 1985) have a complex internal structure including a hemispherical cavity containing tubules, a granular matrix and cortical shell (Fig. 12) (Lehnen and Powell, 1991). There are typically one to six K-bodies per zoospore (Holloway and Heath, 1977b; Powell et al., 1985). Vesicles of similar morphology have not been observed in members of the Peronosporales; however, the occurrence of a special type of organelle near the ventral region (Figs 6 and 7) was noted in early studies (Ho et al., 1968; Lunney and Bland, 1976b; Grove and Bracker, 1978). Immunolabelling of Phytophthora (Figs 17 and 18) (Hardham and Gubler, 1990; Hardham et al., 1991a; Hardham, 1995), Pythium (Cope et al., 1996) and Plasmopara species (A. R. Hardham and L. Lange, unpublished observations) has now confirmed the presence of a distinct type of vesicle that is confined predominantly to the groove on the ventral surface of zoospores. They are about 0.3 pm in diameter and usually contain electron-dense plate-like structures. They have been called ventral vesicles. A similar distribution within the zoospore is not the only feature shared by K-bodies and ventral vesicles. The contents of both K2-bodies and ventral vesicles are secreted during encystment, and in both cases there is evidence that these ventrally located vesicles give rise to an adhesive pad that sticks the cysts to the adjacent substratum (Lehnen and Powell, 1989; Hardham and Gubler, 1990). We thus propose that K-bodies and ventral vesicles are equivalent organelles serving homologous functions. Lectin labelling of Saprolegnia zoospores indicates that the tubular portion of K2-bodies consists of glycoconjugates containing N-acetylglucosaminosyl residues (Lehnen and Powell, 1989). Ventral vesicles in P. cinnamomi contain proteins over 220 kDa in molecular weight (Hardham and Gubler, 1990). K-bodies and ventral vesicles are formed after the onset of sporulation (Tables 1-111). K1-bodies have been observed in differentiating sporangia; K2-bodies develop in primary cysts (Holloway and Heath, 1977b; Lehnen and Powell, 1991) and may arise from a smooth endomembrane system, possibly the endoplasmic reticulum (Lehnen and Powell, 1991). Ventral vesicles in P. cinnamomi first appear in hyphae about 5 h after the induction of sporulation (Dearnaley et al., 1996). They have been observed in developing sporangia and are thought to be formed in the Golgi apparatus (Dearnaley and Hardham, 1994). 3. Encystment vesicles, cyst coat vesicles or dorsal vesicles The peripheral cytoplasm of oomycete zoospores contains a second population of vesicles that are secreted during encystment. In species of Phytophthora these vesicles are spherical and approximately 0.3 pm in diameter (Fig. 7); in species of Saprolegnia, they are cylindrical in shape and 0.2-0.5pm in diameter and 0.3-1.5pm in length (Figs 8-10) (Beakes, 1983). In
376
A. R. HARDHAM and G. J. HYDE
Saprolegnia, they have been recognized by their inclusions of spines and hairs, and have been described as barbodies, encystment vesicles or cyst coat vesicles (Beakes, 1983, 1987, 1989). In Phytophthora they have a marbled appearance and have been identified by labelling with monoclonal antibodies (Figs 15 and 16) (Gubler and Hardham, 1988; Hardham et al., 1991b). Immunofluorescence microscopy reveals that they occur predominantly on the dorsal surface of the zoospores, and avoid the ventral groove region (Fig. 16). They have been called small peripheral vesicles or dorsal vesicles (Hardham, 1987a; Hardham and Gubler, 1990). The morphological and immunological markers have allowed the fate of the vesicles to be followed during encystment and it is clear that in both Phytophthora and Pythium species they are secreted, their contents giving rise to a layer of material that coats the surface of the cyst (Beakes, 1983, 1987; Gubler and Hardham, 1988; Beakes, 1989; Hardham and Suzaki, 1990; Hardham et at., 1994). The cyst wall subsequently forms between the cyst coat and the plasma membrane. Thus, while their morphology varies, their similar role in the formation of the cyst coat during encystment suggests to us that cyst coat vesicles (first suggested by Beakes, 1987) would be the most appropriate name for these vesicles. The function of the cyst coat is not altogether clear, although the spines and boathooks that adorn it in species of Saprolegnia may aid attachment to the host surface (Beakes, 1983). In Phytophthora the cyst coat first forms on the side of the cyst facing away from the host surface (Hardham and Gubler, 1990), but subsequently spreads to cover surrounding surfaces (Hardham et al., 1994). In P. cinnamomi the cyst vesicles contain high-molecular-weight glycoproteins that possess N-acetylgalactosaminosyl residues (Gubler and Hardham, 1988). The ontogeny of cyst coat vesicles has been studied in Lugenisma (Schnepf et al., 1978a), Saprolegnia (Holloway and Heath, 1977b; Beakes, 1983) and P. cinnamomi (Dearnaley and Hardham, 1994). Ultrastructural observations have been interpreted as indicating an origin in the endoplasmic reticulum in Saprolegnia (Beakes, 1983) and in the Golgi apparatus in Lagenisma coscinodisci (Schnepf et al., 1978a). Immunogold labelling has unambiguously demonstrated that cyst coat vesicles form in the Golgi apparatus in P. cinnamomi (Dearnaley and Hardham, 1994). In fact, individual Golgi cisternae process cyst coat vesicle and large peripheral vesicle glycoproteins simultaneously, sorting and packaging them into separate categories of vesicles. Cyst coat vesicles have been observed in sporulating hyphae of P. cinnamomi (Dearnaley et al., 1996), and in developing sporangia of Lagenisma coscinodisci (Schnepf et al., 1978a) and Saprolegniacean species (Armbruster, 1982a; Beakes, 1983) (Tables 1-111). In P. cinnamomi, they are not present in vegetative hyphae, and appear about 5.5-6h after the induction of sporulation; this is about 30-60 min after the appearance of large peripheral and ventral vesicles (Dearnaley et al., 1996).
ASEXUAL SPORULATION IN THE OOMYCETES
377
4. Mmtigonemes Mastigonemes are tripartite, tubular hairs about 1.5 pm in length that are attached in two rows to the anterior flagellum of zoospores of the oomycete and heterokont algae (Fig. 21) (Heath et al., 1970; Bouck, 1972; Holwill, 1982; Hardham, 1987b; Inouye, 1996). It was proposed from theoretical considerations that mastigonemes reverse the thrust of flagellar beat (Jahn et al., 1964), an hypothesis that has recently received experimental verification (Cahill el af., 1996). Mastigonemes form in rectangular, dilated regions of the endoplasmic reticulum or nuclear envelope (Fig. 19) (Bouck, 1969; Reichle, 1969; Heath et al., 1970; Leedale et al., 1970; Deason, 1971; Hoch and Mitchell, 1972a; Loiseaux, 1973; Hill and Outka, 1974; Hardham, 1987b). They are arranged within the packets in an antiparallel fashion with the base of each hair attached to the membrane at one end of the packet (Heath et al., 1970). Packets of mastigonemes are easily recognized (Figs 19 and 20) and have been observed in the cytoplasm of zoospores in members of the Peronosporales, Lagenidiales and Saprolegniales (Tables 1-111). They become associated with the flagella surface in late cleavage (Heath and Greenwood, 1971; Cope and Hardham, 1994). There is little information on the biochemical composition of oomycete mastigonemes, although one mastigoneme antigen in P. cinnamomi is a glycoprotein (Cope and Hardham, 1994). Evidence that mastigonemes are synthesized during sporangiogenesis comes from ultrastructural observations of their presence in developing sporangia in Lagenisma coscinodisci (Schnepf et al., 1978a) and in post-septum (but before cleavage) sporangia in species of Phytophthora (King et al., 1968; Hemmes, 1983), Pythium (Lunney and Bland, 1976a) and Saprolegnia (Heath et al. , 1970). Immunolabelling with monoclonal antibodies specific for mastigonemes in P. cinnamomi have shown that mastigonemes form in hyphae and developing sporangia after sporulation is induced (Cope and Hardham, 1994).
111. ZOOSPOROGENESIS A. INDUCTION
A reduction in temperature causes multinucleate sporangia to cleave into uninucleate zoospores. Nothing is known about the molecules that perceive this signal but, by analogy with other plant and animal systems, it is likely that within minutes the receptors initiate a signal transduction cascade which leads to changes in gene expression and cellular organization. Two common components of this cascade are Ca2+ and H+. Recently, changes in the cytoplasmic concentration of these ions has been investigated during cleavage of P. cinnamomi sporangia. The concentration of Ca2+ and H + in the cytoplasm has been measured
378
A. R. HARDHAM and G. J. HYDE
by microinjecting fluorescent indicator dyes into sporangia before cleavage is induced by a cold shock treatment (Suzaki et al., 1996; Jackson and Hardham, 1996). Fura-2 is a ratiometric dye whose characteristics of fluorescence are indicative of [Ca2+] (Tsien, 1989); BCECF (2’,7’-bis(2carboxethyl)-5(6)-carboxyfluorescein) is a ratiometric dye indicative of pH. Dextran-conjugated dyes were used because they are unable to cross cell membranes and thus remain in the cytosol and are not sequestered into organelles. Microinjection of fura-Zdextran revealed that there are two rises in [Ca2+] during cleavage (Jackson and Hardham, 1996). The first is rapid and transient: the [Ca2+]increases by 25-130% from a resting level of 104 f 54 nM in the first minutes of the cold shock treatment. The concentration of Ca2+ returns to near resting levels and then rises slowly during the course of cleavage. If dibromo-BAPTA, a Ca2+ buffer, is microinjected into sporangia before the cold shock, cleavage is inhibited; if it is injected after cold shock, most sporangia cleaved. Artificially raising the concentration of Ca2+ by incubating sporangia in the Ca2+ ionophore (A23187) in the presence of 10 mM Ca2+ induced cleavage in about 37% of sporangia in the absence of a cold shock. Both internal and external sources of Ca2+ contribute to the initial rise in [ ~ a * + ] . Injection of sporangia with BCECF-dextran revealed that cytoplasmic pH increases rapidly by about 0.2 pH units from a resting level of 6.84f0.05 during an inductive cold shock treatment (Suzaki et al., 1996). If this rise in pH is inhibited by injection of a pH buffer such as HEPES, the sporangia failed to cleave. These results indicate that a rise in the cytoplasmic concentration of Ca2+ and H+ not only accompanies cleavage, but is also required for cleavage to occur. It seems likely that these factors are part of the signal transduction pathway operating during the onset of cleavage, but additional details of their role and of other molecules involved await further studies.
B . THE PROCESS OF CLEAVAGE
1. Patterns of cleavage membrane formation: a reappraisal The sporangium is subdivided into uninucleate domains by the elaboration of partitioning membranes that give rise to the plasma membrane of the zoospore. The formation of these membranes has been much studied and many different developmental patterns have been proposed. In other eukaryotes, differing patterns of cleavage, and cytokinesis in general, have long been recognized and are typically considered as evolutionary variations (e.g. Pickett-Heaps, 1972). Thus, until recently, there had been no suggestion that the various patterns of oomycete cleavage represented anything more than a set of possibly useful taxonomic characters. However, as will be
ASEXUAL SPORULATION IN THE OOMYCETES
379
Fig. 24. Diagram showing models proposed to explain cleavage of oomycete sporangia. The upper portion of the diagram shows, clockwise, vesicle pre-alignment, expansion of a large central vacuole, invagination of the plasma membrane and expansion of a cytoplasmic vacuole. In the lower portion of the diagram, which is based on studies of cleavage in Phytophthora species, the cleavage vacuoles are shown to develop as a ramifying network in the sporangial cortex and between nuclei.
described in detail below, recent rapid-freezing and freeze-substitution (RF-FS) studies have now raised the possibility that much of the diversity reported for oomycete zoosporogenesis may be artefactual and derive from inadequate fixation of the developing partitioning membranes (Hyde et al., 1991b,c). It is therefore worthwhile to now reconsider all the existing data on cleavage in this taxon in the light of the recent RF-FS studies of oomycete and other sporogenic fungi. Five main patterns of zoosporogenic membrane development have been described in the oomycetes (see Fig. 24). For any one species, several of these processes may have been proposed.
Vesicles. Vesicles of one or more types, either pre-existent within the sporangium or induced to form by some event, redistribute in planes around the nuclei; after alignment the vesicles fuse, thus forming the cleavage vacuoles (Fig. 24). Images of arrays of aligned vesicles are found in studies of most of the Phytophthora species examined so far (Hohl and Hamamoto, 1967; Elsner et al., 1970; Williams and Webster, 1970; Hemmes, 1983; Hyde et al., 1991a) and of Pythium (Lunney and Bland, 1976a), Pseudoperonospora (Lange et al., 1989), Dictyuchus and Brevilegnia (Armbruster, 1982b). All these studies have, however, relied upon chemical fixation, and when P. cinnamomi and P. palmivora were re-examined with RF-FS no vesicular arrays were apparent. Instead, cleavage appeared to follow from the extension of partitioning membranes in a progressive fashion (Fig. 24; Hyde et al., 1991b). It is likely that the RF-FS results represent more
380
A. R. HARDHAM and G . J. HYDE
faithfully the events that take place in the living cell: numerous studies have indicated that elongated membranous systems are preserved in material prepared by RF-FS, but become highly vesiculated by chemical fixation (e.g. McCully and Canny, 1985; Shepherd et al., 1993). This indicates that any images showing vesicles lined up neatly along “future” planes of cleavage are to be treated with suspicion. In hindsight it is easy to understand the misinterpretation of these arrays. In the final stages of cleavage, as the zoospores round-up, the interzoosporic spaces widen so much that they are not prone to the chemical fixation-induced vesiculation typical of the narrow, early stage vacuoles. Hence, in chemically fixed material a plausible, but incorrect, story suggests itself: the vesicular network that forms from the disrupted early vacuoles is an intermediate stage of a process completed by fusion of the vesicles. Some reports of vesicular fusion in zoosporogenesis in the Saprolegniales require further mention because they involve the fusion of dense body vesicles with (Armbruster, 1982b) or without (Gay and Greenwood, 1966; Gay et al., 1971) the participation of other vesicle types. Dense-body, or fingerprint, vesicles are common in other oomycete sporangia but are not involved in the cleavage process, and it is possible that their apparent involvement in the cases above also results from misinterpretation of poorly preserved, chemically fixed material. In chemically fixed sporangia of Phytophthora, not only cleavage vesicles but also other vesicles at the periphery of developing zoospores (e.g. large peripheral vesicles) are prone to rupture, and often appear fused with the cleavage membrane (G. 1. Hyde and A. R. Hardham, unpublished observations; see also section IICl). It should be noted that chemical fixation is not intrinsically incapable of preserving progressively extending cleavage vacuoles: the basic form of ascosporogenic membranes, for example, is very similar in material preserved by chemical fixation or RF-FS, although the latter technique has revealed some previously undetected features (Mims et al., 1990; Van Wyk et al., 1991). Why the cleavage vacuoles of some taxa are more resistant to vesiculation than others is unknown; it is not their size, since early cleavage vacuoles of ascomycetes (Mims et al., 1990) are as narrow as those of P. cinnamomi. Work with P. cinnamomi has, however, indicated that the fixation protocol itself can affect the degree and type of vesiculation. When fixative solutions were made up with high concentrations of buffer, the cleavage system was less vesiculated and those vesicles that did form were large (Hyde et al., 1991a). In the “preserved” sections of the cleavage system, the paired membranes were much further apart than in RF-FS material. This suggests that contraction of the sporangial domains (a likely effect of the more concentrated, highly-buffered solutions) may have pulled sections of the cleavage membranes too far apart to permit vesiculation. As discussed by Hyde et al. (1991b) it is also probable that fixation problems have led to misinterpretations of cytokinesis in the many other
ASEXUAL SPORULATION IN THE OOMYCETES
381
eukaryotic organisms for which fusion of pre-aligned vesicles has been reported. Indeed it is possible that this process does not occur at all: to our knowledge not one RF-FS study has yet confirmed its existence and the first RF-FS study of plant cytokinesis has now indicated that membrane formation in these organisms also involves progressive elongation of partitioning membranes (Samuels et al., 1995). The use of variations in the cytokinetic process as taxonomic indicators is now open to question.
Involvement of a large central vacuole. Some studies of oomycete zoosporogenesis report the incorporation or expansion of a large central vacuole (Fig. 24) in cleavage (Gay and Greenwood, 1966; Hohl and Hamamoto, 1967; Williams and Webster, 1970; Gay et al., 1971; Beakes, 1995). Mature sporangia often contain one or more large central vacuoles which disappear during zoospore formation, but recent use of monoclonal antibody markers in P. cinnamomi and P. palmivora has indicated that in these species the vacuoles do not contribute to the cleavage process (Hyde et al., 1991a,b). Their role in cleavage is uncertain. In S . ferax, numerous smaller vacuoles in the sporangial cortex fuse to form a central vacuole which then expands centrifugally, and preferentially, between the nuclei (Gay and Greenwood, 1966). The mature Saprolegnia sporangium also sometimes contains a pre-existing central vacuole, but micrographs indicate that these have different contents to the newly forming ones, and that the two types often abut each other over large areas without signs of fusion (Gay and Greenwood, 1966; Gay et al., 1971). It is possible that as in Phytophthora, the pre-existing vacuoles play no part in cleavage, but disappear coincidentally. Involvement of the sporangial plasma membrane. Centripetal invaginations of the plasma membrane to form cleavage vacuoles between nuclei (Fig. 24) have only been reported in Pyth. middletoni (Heintz, 1970; Beakes, 1995). More common are reports in which other parts of the developing cleavage system fuse with the plasma membrane as in Saprolegnia ferax, Pyth. proliferum and P. cinnamomi (Gay and Greenwood, 1966; Gay et al., 1971; Lunney and Bland, 1976a; Hyde et al., 1991b). Cortical cleavage vacuole. A cortical cleavage vacuole forms parallel to the sporangial wall (Fig. 24; Elsner et al., 1970; Williams and Webster, 1970; Armbruster, 1982b; Hyde et al., 1991b). Apart from their peripheral location such vacuoles, reported in some Phytophthora and Saprolegnia species, appear to behave similarly to the central vacuole of S . ferax. For example, in Phytophthora, once having formed, the cortical vacuole expands preferentially between the nuclei, and fuses with the sporangial plasma membrane (Hyde et al., 1991b). The cortical vacuole itself forms by the fusion of smaller vacuoles initiated at the narrow poles of the cortical nuclei. The relationship between the cortical and internal planes of the Saprolegnia species is perhaps obscured by
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chemical fixation artefacts (Armbruster, 1982b). Other cases of cortical cleavage plane formation in the oomycetes may have also been missed or misinterpreted because of poor preservation and/or insufficient sampling.
Progressive extension of cleavage vacuoles around or between nuclei. This process (Fig. 24) is mostly clearly seen in the two Phytophthora species studied by RF-FS and is also described as one aspect of cleavage in S. ferax and Pyth. middletoni (Gay and Greenwood, 1966; Heintz, 1970; Gay et al., 1971; Hyde et al., 1991b; Beakes, 1995). For nuclei in the sporangial cortex of ~ ~ y t ~ ~ h tthese ~ o rcleavage u , vacuoles are mostly, or completely, composed of the cortical vacuole and its ingrowths. If a sporangium is large enough to have non-cortical nuclei, then vacuoles initiated near their narrow poles also contribute to the cleavage network (Fig. 24). Progressive extension of cleavage vacuoles is likely to be a cleavage process occurring in most oomycete, and indeed fungal, sporogenesis. In support of this, two ongoing RF-FS studies of chytrid zoosporogenesis report that cleavage occurs by progressive extension, and not by vesicular fusion as thought previously (D. Lowry and J. Shields, personal communications); also, RF-FS and chemical fixation studies of ascosporogenesis describe a comparable process to that seen in Phytophthora (Mims et al., 1990; Van Wyk et al., 1991; Van Wyk and Wingfield, 1991a,b). There are two accounts of sporogenesis in the oomycetes which do not fit into our schema. One, that of zoosporogenesis in L . coscinodisci (Schnepf et al., 1978a), will not be discussed here because we feel that the model proposed needs verification. The other, that of spore (not zoospore) formation in Aphanomyces euteiches (Hoch and Mitchell, 1972b, 1975), involves a possibly more primitive mechanism than that seen in true zoosporogenesis. Spores form within a non-swollen hypha, not by active subdivision, but by the aggregation of originally dispersed cytoplasm around regularly spaced nuclei, and the concomitant passive redistribution of a central vacuole between the spore units (Hoch and Mitchell, 1972b, 1975). If it is accepted that organisms that were thought to cleave by the fusion of aligned vesicles actually do so by vacuolar extension, then a number of topological commonalities in the cleavage processes of oomycete sporangia emerge. The first, most basic feature, shared possibly by all species, is that the development of the paired cleavage membranes and the interzoosporic space they enclose proceeds in a progressively ramificatory fashion. Second, the “outside” space between the membranes establishes a continuity with the true extracellular space of the sporangium, either immediately (in the case of plasma membrane invaginations) or when internally generated cleavage planes fuse with the sporangial plasma membrane. Third, many of the ramifications derive, in a systematic fashion, from one communal “pool” of future outside space, either a central or cortical vacuole, or the
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extracellular space between the sporangial plasma membrane and wall. The remainder of the cleavage system arises from discrete, often paranuclear, sites of initiation and these may originally be the source of the larger communal pools. A final shared feature, and one which distinguishes cleavage in the oomycetes from that in other taxa (e.g. ascosporogenesis), is the degree of “co-operation” between adjacent domains: they do not cleave independently but utilize common furrows. The ramificatory sporangial cleavage process described above can usefully be likened to dynamic branching systems in which energy becomes channelled along paths of least resistance (Rayner et al., 1994). As suggested previously (Olson et al., 1981; Heath and Harold, 1992), the driving energy for sporangial cleavage development probably derives mainly from osmotic expansion of the cleavage vacuoles. RF-FS has shown that the vacuoles are filled with a very dense matrix material and that the leading edges of cleavage vacuoles are swollen (Hyde et al., 1991b), both of which are consistent with this idea. Another aspect of vacuolar expansion is the addition of the new membrane and matrix material required by the developing network. With an osmotically driven, interconnected system these elements would not need to be transferred to the developing edges of the cleavage system. Thus, without any change in the end result, new material could be added at any point in the network, for example near the narrow nuclear poles where the dictyosomes are concentrated.
2. Spatial regulation of cleavage If the developing cleavage network is a dynamic branching system, then how are the paths of least resistance defined? That is, what causes the ramifications to occur along such neatly prescribed courses? Considerable evidence indicates that both microtubules and actin microfilaments are involved. Treatment of sporangia with antimicrotubule or antimicrofilament drugs results in grossly abnormal cleavage (Schnepf et al., 1978a; Oertel and Jelke, 1986; Heath and Harold, 1992; Hyde and Hardham, 1993). Structural studies have shown that both microtubules and microfilaments are present during the cleavage process. In P. cinnamomi microtubule arrays persist in similar form to that seen in the uncleaved sporangium. Microfilament arrangements in cleaving sporangia have recently been studied, using fluorescence microscopy, in Saprofegnia, Achlya and P. cinnarnomi (Heath and Harold, 1992; S. L. Jackson and A. R. Hardham, unpublished observations). In each species microfilament arrays show a similar pattern of development to that of the cleavage vacuoles, but unfortunately the data do not clarify whether the actin arrays precede o r follow after the cleavage membranes with which they finally associate. As yet we do not understand how microtubules or microfilaments control cleavage, but there are at least two broad possibilities. In the first model, microtubules act indirectly, determining where the actin arrays will form. The
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actin arrays, which would need to form prior to the cleavage system, would then be directly responsible for ensuring the extension of the network along the correct lines. The actin arrays might, for example, form the equivalent of canal banks that direct the expansive “flow” of the system between and around the zoosporic nuclei. In the second model, microtubules are the more important element, stabilizing domains of cytoplasm around the nuclei: the paths of least resistance for vacuolar expansion lie between, and around the domains, where there are the least number of microtubules. This role for microtubules was first proposed by Heath (Heath and Greenwood, 1971). In this model the role of actin might not be to guide the vacuoles into new regions but to brace in situ those that have already formed. They would thus be the equivalent of levee banks added to rivers in danger of flooding. In their absence the microtubules may not be sufficient to hold back the formation of inappropriate ramifications, leading to abnormal cleavage. One of these two models will be eliminated when it is known which comes first: actin or the vacuoles. C. SYNTHESIS OF ZOOSPORE-SPECIFIC COMPONENTS DURING ZOOSPOROGENESIS
Two cell components, the water expulsion vacuole and peripheral cisternae, are consistently reported to develop during zoosporogenesis. In most, but not all, cases flagella are also reported to form at this time (Tables 1-111). I . Water expulsion vacuoles The water expulsion vacuole complex consists of a central vacuole surrounded by numerous vesicles and tubular cisternae. The whole complex is flanked by stacks of Golgi cisternae. The water expulsion vacuole is believed to be responsible for zoospore osmoregulation, although the molecular basis of its operation is not known. Its cycle of dilation and contraction can be observed in living cells and takes about 4-6s (Grove and Bracker, 1978; A. R. Hardham, unpublished observations). Ultrastructural examination of fixed material (Lange et al., 1984) and observations of living material (A. R. Hardham, unpublished observations) indicate that the complex forms and begins operating during late cleavage. After zoospore encystment, the pulsation cycle time increases (A. R. Hardham, unpublished observations) and within about 10 min the water expulsion vacuole disappears (Holloway and Heath, 1977b; Grove and Bracker, 1978; Hemmes, 1983). 2. Peripheral cisternae The plasma membrane of oomycete zoospores is lined by a system of flattened membranous discs, the peripheral cisternae. In chemically fixed material, the
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disks tend to be dilated at the edges (e.g. Hemmes, 1983; Hardham et al., 1991a) but in RF-FS material the cisternae are of uniform thickness throughout their length (Cho and Fuller, 1989). The cisternae line the plasma membrane except in the groove region (see Grove and Bracker, 1978; Hemmes, 1983). The membrane of the peripheral cisternae is more similar to that of the plasma membrane than to the endoplasmic reticulum (Grove and Bracker, 1978; Hardham, 1987a), and during encystment, peripheral cisternae vesiculate and are believed to fuse with the plasma membrane of the zoospore. Their function is not known, although a reaction with an antibody that also labels the cyst wall has led to the suggestion that they play a role in cyst wall formation (Hardham et al., 1991a). Whether or not the possession of this common epitope is a true indication of a functional relationship remains to be determined, but one thing is clear: their fusion with the plasma membrane during encystment has the potential to bring about rapid and dramatic changes in plasma membrane properties. Peripheral cisternae first appear during cleavage plane formation (Hyde el al., 1991b). Short segments of cisternae become aligned next to the new plasma membrane of the zoospores even before cleavage is complete. Their site and mode of formation are unknown, although the Golgi apparatus is a likely candidate. 3. Flagellar axonema Oomycete zoospores are biflagellate, the two flagella arising at basal bodies near the centre of the groove on the ventral surface. Microtubule flagellar roots and cytoplasmic arrays are associated with the flagellar apparatus (see Fig. 22) (Barr, 1981; Barr and Allan, 1985; Hardham, 1987b). Flagella are responsible for zoospore motility, and some components of the flagellar apparatus may also maintain zoospore shape. During encystment the flagella are detached at the level of the terminal plate (Hardham, 1987b). The basal body acts as a template for axoneme formation and nine microtubule doublets assemble by addition of tubulin dimers to two microtubules in each of the nine triplets in the basal bodies. The central pair of microtubules has the opposite polarity and apparently assembles by tubulin addition at the proximal end (Lefebvre and Rosenbaum, 1986). In oomycete sporangia, in all but two cases, flagella have been observed to form during cleavage (Heath, 1976; Lange et a f . , 1984, 1989; Cope and Hardham, 1994). However, in P. infestam (King et al., 1968; Elsner et al., 1970) and Lagenidium callinectes (Gotelli, 1974b), flagella are reported to be present in sporangia that have not been induced to cleave. Although flagella formation during direct germination in P. parasitica (Hemmes and Hohl, 1969) also suggests that flagella assembly may be independent of cleavage events, sporangia are very sensitive to changes in environmental conditions and cleavage may be induced by mild manipulations, for example, by a 5°C decrease in temperature (Byrt and Grant, 1979; Suzaki et al., 1996). It may
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be worthwhile to re-examine the timing of flagella formation using immunofluorescence microscopy with anti-tubulin antibodies. This technique allows the examination of large numbers of cells and can thus produce more accurate assessment of modes of development. D. POLARIZATION OF ZOOSPORIC ORGANELLES
Oomycete zoospores are highly polarized cells. Many of the components described in previous sections are more concentrated at one or the other end of an axis running from the centrioles back through the broad pole of the nucleus (see Figs 1,14,16,18,25A).This organellar polarity is highly defined in the secondary zoospore which is also morphologically polarized about the same axis, with grooved ventral, and convex dorsal, surfaces. While a great deal is known about the arrangement of zoosporic components, only recently has any work addressed how it develops. With the aid of monoclonal antibodies raised against various vesicles of P. cinnamomi, Hyde et al. (1991a) showed that three of the vesicles attained their characteristic distributions along the zoospore periphery in a sequential manner. If abnormal cleavage were induced by elimination of the sporangial microtubule arrays (by use of the drug oryzalin), the three vesicles still managed to reach the periphery of the disrupted cleavage vacuoles, but were not concentrated along any particular portions of it, as occurs normally (Fig. 25B) (Hyde and Hardham, 1993). Abnormal cleavage per se was not responsible for these effects, since this could also be induced with the anti-actin drug cytochalasin D without inhibiting either vesicle migration to the cortex or localized concentration along it (Fig. 25C). It is therefore likely that, under normal conditions, vesicles do not require either microtubules or microfilaments to reach the zoospore periphery, but that microtubules somehow regulate where along the periphery the vesicles will concentrate. A possible model is that vesicles reach the zoospore membrane by Brownian motion and then dock with plasma membrane receptors with polarized distributions maintained by microtubules. Mitochondria1 redistribution was also examined in this study, and both mitochondria1 movements and polarization were inhibited by the antimicrotubule drug. E. ZOOSPORE DISCHARGE
Zoospores may be released directly into the surrounding medium by rupture of the papilla at the apex of the sporangium (as in Saprolegnia and Aphanomyces: Gay and Greenwood, 1966; Hoch and Mitchell, 1972a), or they may be temporarily confined within an evanescent vesicle derived from papillar material (as in Phytophthoru, Pythium and Lagenidium: Webster and Dennis, 1967; Gotelli, 1974b; Gisi et al., 1979). In those species in which
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Fig. 25. Diagram showing (A) the distribution of (a) microtubules, (b) nuclei, (c) mitochondria, (d) ventral vesicles, (e) dorsal vesicles and (f) large peripheral vesicles in zoospores within a recently cleaved sporangium of Phyfophthoru. The effects of (B) microtubule and ( C ) microfilament inhibitors on cleavage planes and on the distribution of the components during cleavage are shown. In (B), loss of microtubules causes abnormal cleavage and misalignment and rounding up of the nuclei; mitochondria remain randomly distributed; peripheral vesicles concentrate along the abnormal cleavage planes but do not become segregated into discrete cortical domains. In (C), loss of microfilaments causes abnormal cleavage, however mitochondria and peripheral vesicles are both transported to the cortex and segregated as normal. (Reproduced with permission from Hyde and Hardham, 1993.)
a vesicle forms, zoospores or sporangial cytoplasm rapidly flow into the vesicle as it expands. The time at which this occurs relative to the cleavage process varies, a feature that is used to distinguish taxa of Phytophthora and Pythium (de Bary, 1887, cited in Dick, 1990). In Phytophthora, movement into the vesicle occurs after cleavage is more or less complete. Thirty to seventy per cent of the zoospores move into the vesicle before it ruptures and the zoospores swim away (Gisi et u l . , 1979). Zoospores remaining in the sporangium swim out through the activity of their flagella. In Pythium, the uncleaved mass of cytoplasm flows into the vesicle where cleavage subsequently occurs (Webster and Dennis, 1967). In Lagenidium an apparently intermediate situation exists whereby partially cleaved cytoplasm is expelled into the vesicles (Gotelli, 1974b). Interpretations of the extent of cleavage in electron microscopic images of discharging sporangia may, however, be confused by fixation artefacts. Studies of the release of zoospores or cytoplasm from sporangia indicate that a difference in the osmotic pressure between the inside of the sporangium and the external solution is required for discharge (Gisi, 1983). Reduction of this difference by depression of external water potential retards or inhibits spore release in Aphanomyces euteiches, and Phytophthora species (Hoch and Mitchell, 1973; Gisi et al., 1979). There are two current theories as to how
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this pressure differential could be utilized to bring about papillar expansion and release of sporangial contents. One theory suggests that extracellular material that surrounds the zoospores (or sporangial protoplast) acts as a gel that takes up water and swells, thereby building up pressure within the sporangium (Lunney and Bland, 1976a; MacDonald and Duniway, 1978; Gisi and Zentmyer, 1980). The other theory suggests that extracellular solutes in the medium surrounding the spores act as an osmoticum to build up turgor pressure within the sporangium (Gisi, 1983; Money and Webster, 1985; Money et al., 1988). Until recently, one of the main shortcomings of the swelling gel theory was the fact that there was little evidence of extracellular material surrounding spores or uncleaved cytoplasm. However, in freeze-substituted sporangia of P. cinnamomi and P. palmivora the extracellular space was found to be filled with electron-dense material that was labelled by an antibody that reacted with the contents of the cleavage vacuoles in pre-cleavage sporangia (Hyde et al., 1991b). It seems likely that cryofixation studies could reveal the presence of similar material in sporangia of other species of oomycetes. One of the requirements of the osmotic pressure theory is that the permeability of the wall of the sporangium is such that osmotically active solutes remain inside the sporangium while water enters along the osmotic potential gradient. Plant and fungal cell walls are semipermeable structures which allow the passage only of molecules whose molecular dimensions are less than the sizes of the smallest pores within the wall (Nobel, 1991). Sporangial walls could thus retain osmotically active molecules of an appropriate size within the sporangial extracellular space. Studies of Achlya and Saprolegnia sporangia have demonstrated that the pore size of the walls is of similar magnitude to that of higher plants and fungi (Money and Webster, 1985; Money et al., 1988). The pore size for Achlya is of the order of about 2 nm (Money et al., 1988). This compares with a value of 4-5 nm for walls of a range of higher plant cells (Carpita et al., 1979). A pore size of 2nm would mean that molecules with a molecular weight above about 1000 Da would be trapped inside the sporangium. Dense-body/fingerprint vesicles were reported to fuse with developing cleavage furrows thus transferring their p-1,3-glucan contents, the mycolaminarans, into the extracellular space (Gay and Greenwood, 1966; Gay et al., 1971; Annbruster, 1982b) and it has thus been suggested that these molecules could act as the osmoticum (Money et al., 1988). However, it now seems unlikely that dense-body vesicle contents are secreted at any stage of zoosporogenesis, and therefore could not play this role. Western blotting with a monoclonal antibody that reacts with the extracellular material within Phytophthora sporangia indicates that these polypeptides have a range of apparent molecular weights between 60 and 330 kDa (Hyde et al., 1991a). Molecules of this size would not be able to move across a wall with a porosity
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of the order of 2 nm, but it remains to be determined if they could accumulate to sufficient concentration to generate the required osmotic potential difference. To date there have been no definitive experiments that demonstrate which of the two proposed mechanisms operates during release of zoospores or protoplast from oomycete sporangia. Increasing the osmotic potential of the external solution would inhibit the operation of either mechanism. Similarly, the extracellular material visualized in Phytophthoru sporangia could play a role in either mechanism. One possible approach might be to examine if the extracellular material remains within or near the sporangia after spore release. A gel-like material could be expected to persist at least temporarily, while solutes would be expected to quickly disperse. Immunolabelling of the extracellular material in Phytophthora sporangia could give evidence of the longevity of the material after zoospore release. In some respects, it is easier to envisage either of these two mechanisms operating in sporangia in which cleavage is complete within the sporangia before release. In this case, spaces (in which the gel or the solute can accumulate) are observed between the spores and between spores and the sporangial wall (e.g. Hyde et al., 1991b). But in Pythium the cytoplasmic mass appears to be appressed to the sporangial wall right up to the time of release (see Webster and Dennis, 1967, Plate 11). There thus seems to be little space for the extracellular gel or solute to accumulate before release begins. Perhaps in Pythium the secretion of gel-like material or solutes is very rapid and coincides with the onset of release.
F. CONCLUSIONS
Ten years ago sporulation in the oomycetes might have been cited as a telling example of biological diversity. For the sporangium and zoospores of each species, there seemed to be a unique assortment of vesicles; for each species, a different way for how the sporangium cleaved to produce the zoospores. But as the recent research reviewed in this chapter demonstrates, what is emerging now are similarities in the processes of sporulation across this taxon. By following the fate of vesicles through into encystment, with the help of immunolocalization techniques, it has become apparent that sporangial vesicles that vary morphologically from species to species have homologous future functions. The adoption of this more function-oriented approach has reduced the perplexing multitude of zoospore vesicles down to a manageable handful of five types. Likewise, use of RF-FS to study development of the partitioning membranes has indicated that progressive ramification of cleavage vacuoles may be a common feature of the cleavage process throughout the oomycetes. Such clarifications indicate that, as long as adequate methods of study are used, observations of sporulation in
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different oomycete species can be compared and contrasted in order to discover the underlying principles in operation. The principles that emerge may have direct relevance for our understanding of biological processes fundamental to the growth and development of other eukaryotic organisms.
ACKNOWLEDGEMENTS We would like to thank David Lowry, John Shields and Gordon Beakes for providing unpublished results and helpful comments during the preparation of the manuscript. We would also like to thank Frank Gubler for allowing us to use some of his unpublished micrographs and Gordon Beakes and Brent Heath for supplying copies of their previously published micrographs.
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Horizontal Gene Transfer in the Rhizosphere: a Curiosity or a Driving Force in Evolution?
J. WOSTEMEYER, A WOSTEMEYER and K. VOIGT
Friedrich-Schiller-UniversistatJena, Lehrstuhl fur Allgemeine Mikrobiologie und Mikrobengenetik, Neugasse 24, 0-07743 Jena, Germany
I. Horizontal or Lateral Gene Transfer: where does it Occur?
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VI . Relevance of Lateral Gene Transfer for Evolution .......................... References ...............................................................................
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11. Bacteria as Recipients of Foreign DNA 111. Fungi as Recipients of Foreign DNA
V. Interkingdom Gene Transfer
I. HORIZONTAL OR LATERAL GENE TRANSFER: WHERE DOES IT OCCUR? Since modern biotechnology enables the construction of defined genotypes of prokaryotic and eukaryotic micro-organisms, plants and animals, the question of uncontrolled spread of artificially manipulated genes over species boundaries has provoked the interest of many ecologists, geneticists and the general public. Very often, artificial gene transfer by in vitro techniques is regarded as a taboo, forbidden either due to religious considerations or the argument that gene exchange in nature is limited to sexual o r parasexual Advances in Botanical Research Vol. 24 incorporating Advances in Plant Pathology ISBN &12-005924-X
Copyright 0 1997 Academic Press Limited All rights of reproduction in any form reserved
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recombination systems within a single defined species. Geneticists know that there is no such strict confinement to intraspecific gene transfer. Initially, bacterial conjugation based on the F-factor was detected as a parasexual system within the single species Escherichia cofi (Lederberg and Tatum, 1946). However, as soon as the nature of F as a DNA molecule was detected, it was observed that F can be easily transferred from E. coli to Serratia marcescens (Marmur et al., 1961). Both species belong to the enteric bacteria and are phylogenetically related, but certainly no bacteriologist will ever doubt that these species are distinct enough to be assigned to different genera. Apart from F, a huge variety of different plasmids, especially colicinogenic factors (Bhaskaran, 1958) and resistance transfer factors (RTFs) (Mitsuhashi et a f . , 1960) were described, many of which were transmissible among many different species of enterobacteria. Very early during the development of bacterial genetics, it became evident that interspecies gene transfer is more the rule than the exception. Later it was found that conjugation is also encountered in bacteria which are often found in soil: Gram-positive bacteria (Clewell and Flannagan, 1993) and streptomycetes (Hopwood and Kieser, 1993). We discuss the bacterial systems in more detail in section 11. When looking at organismic communities in soil and, in the immediate vicinity of plant roots, at rhizosphere and rhizoplane systems, we have to take into account fungi, bacteria, plants and also animals. Relationships between soil organisms may be of different kinds: there is in any case a competition for nutrients. The least-fit organisms will die and their DNA becomes available. Close cell-to-cell contact is a starting point for most symbioses. In these, genetic exchange may be facilitated. Genetic exchange is less probable in parasitic o r predator-prey relationships, but even here gene transfer is possible. In this chapter we will not consider animal systems, although we suspect that at least protozoa are good candidates for incorporating foreign DNA into their chromosomal complement. We should not forget that even in vertebrates DNA fragments survive the intestinal tract to some degree and can be found in the bloodstream by using sensitive polymerase chain reaction (PCR) methods (Schubert et al., 1994). Analyses of this type do not prove a true gene transfer. But, on an evolutionary scale, the occasional appearance of foreign DNA molecules outside the organs or, in the case of protozoa, the organelles for food processing may represent the starting point for recombination events. Evolution does not need frequent events. The incorporation of a single highly recombinogenic or replicative molecule into a pre-existing genome may subsequently lead to considerable rearrangements. On the whole, very little research has been done in animals with regard to naturally occurring horizontal gene transfer. In fungi, the situation is somewhat different. Although actual genetic investigations on interspecies gene transfer are rare, appropriate mechanisms are known. Many fungi have the ability to form anastomoses; we will discuss the possibilities for genetic hybridization via this mechanism. Apart from this
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more conventional and widespread option, several mycoparasitic systems are known which involve the formation of plasma bridges between host and parasite. Such parasites are named “fusion biotrophs” (Jeffries and Young, 1994). For two of these mycoparasitic systems, experimental evidence has been obtained that the cytoplasmic fusion is accompanied by the invasion of genetic material of the parasite into the host (Kellner et a f . , 1993; A . Wostemeyer, unpublished). We discuss fungal gene transfer in section 111. Possibly the best-described gene transfer system in the rhizosphere involves the soil bacterium Agrobacterium turnefaciens, which transfers a special region of its Ti-plasmid to many different plant species. This interkingdom system is based on a conjugational mechanism (see section IV). Are there other interkingdom gene transfers in nature? The answer is “possibly”. We have some evidence that DNA from decaying plants may be introduced into soil fungi (Hoffmann et a f . , 1994). We also have an idea that host DNA may show up in a plant pathogen, at least transiently (Bryngelsson et al., 1988). Taking into account that conjugational gene transfer systems have also been observed between E. coli and yeasts (Sprague, 1991), we should perhaps address the questions of frequency and importance of genetic exchange between kingdoms more seriously (see section V). Other questions of major importance for the evolutionary impact of horizontal gene transfer are addressed in section VI: Is horizontal gene transfer a rare event, limited to rather specialized organismic associations, or do certain biotopes represent a genetic continuum, where gene pools are shared by many different species? Is there a reasonable probability for permanent gene acquisition via interspecies gene transfer? Are there mechanisms that remove foreign DNAs or suppress their activity? By necessity, due to sparse experimental information, the attempts to answer these questions are speculative.
11. BACTERIA AS RECIPIENTS OF FOREIGN DNA During evolution several types of mechanisms for horizontal gene transfer in bacteria have been evolved. Diverse independent lines of research have provided convincing evidence that these processes occur in the environment (Cresswell and Wellington, 1992; Pickup, 1992; Saunders, 1992). All of them are essentially infection processes, in which DNA passes from a donor to a recipient, integrates into the prokaryotic chromosome by recombination and expresses its genetic information in the phenotype. Unlike the common case in eukaryotes, this recombination followed by natural gene flow is not linked to reproduction (Young, 1992). It is not the intention of this general overview to give a fully detailed compilation of the biology of the three main mechanisms and their genetic regulation, although general aspects are discussed. Details may be found in a number of excellent reviews (Schmidt,
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J. WOSTEMEYER, A WOSTEMEYER and K. VOIGT
1992; Stewart, 1992; Dreiseikelmann, 1994; Lorenz and Wackernagel, 1994). The aim of this overview is to give a general picture of bacterial gene flux based on recent experimental concepts and its putative role in long-term evolution. Natural genetic transformation seems to be the easiest way for genetic exchange in the environment. Bacteria enter a physiologically regulated state of competence, bind, and take up actively extracellular DNA (plasmid and chromosomal) from the surrounding medium and express this exogeneous DNA after heritable incorporation. This process depends on the function of several genes located on the bacterial chromosome and involves many not easily recognizable regulation mechanisms (for a review see Lorenz and Wackernagel, 1994). For a systematic experimental investigation and for development of a general model for natural transformation, it was previously proposed to divide the complex process of gene transfer by free DNA into separate steps which are common to all bacteria. Lorenz and Wackernagel (1994) distinguish various phases between the release of donor DNA, development of competence and the final recombination event into the recipient’s chromosomal complement. Many conditions have to be fulfilled for a natural transformation in the rhizosphere to occur. The first question is: Is a pool of free DNA available for soil micro-organisms in interacting in situ systems? One reason for the presence of free DNA in the environment may be the continuous production of DNA by micro-organisms. Indeed, naked DNA of microbial origin can be released during growth of Streptomyces in nature by autolysis following cell death. In certain phases of their development, Streptornyces vegetative hyphae could lyse, supplying not only nutrients but also free DNA to the environment (Schmidt, 1992). The main cause for the pickup of external DNA might be of a trophic nature. Like other biopolymers, nucleic acids serve as nutrients for many degradative micro-organisms. DNA-degrading bacteria can be readily isolated from environmental samples. In fact, nucleases which are ubiquitously present in the environment attack free DNA. The action of such dissolved, not cell-associated DNA-degrading enzymes may cause a decrease in stability and availability of naked DNA in soil habitats. On the other hand, several factors have been identified that contribute to the persistence and protection of extracellular DNA (Lorenz and Wackernagel, 1994). It has been demonstrated that DNA forms complexes with several particulate constituents of soils and sediments, such as quarz, feldspar and clay minerals, which also have sorptive capacities for other organic material. This adsorption on soil particles decreases the fluctuation of DNA in soil caused by free nucleases and reduces or abolishes its immediate utilization by soil micro-organisms. Thus, the degree of exogenous nucleolytic degradation is an important factor in determining the relative efficiency of transformation in the environment. The relative concentration and the heterogeneity of free DNA will also influence natural
HORIZONTAL GENE TRANSFER IN THE RHIZOSPHERE
403
transformation. In addition, distribution of free DNA and local contact between transforming DNA and bacterial cells will influence natural transformation in nature. Moreover, the cells must be transformable and this is determined by competence. So the second important question is: Are soil bacteria able to take up and process foreign DNA? Competence is a regulated state in transformable bacteria, the development of which requires the influence of certain physiological stress factors (e.g. nutrient limitation, varying pH, temperature, fluctuation of available water in soil, oxygen radicals and other cytotoxic agents, ionic strength). The ability of Azotobacter vinelandii, and similarily of the cyanobacterium Anacystis nidulans, to become competent in soil has been estimated to be influenced by the mobilization of iron from soil minerals (for a review see Lorenz and Wackernagel, 1994). The production of siderophores coincided with a decreased appearance of transformants. Interestingly, the rhizosphere of plants is an iron-limited habitat (O’Sullivan and O’Gara, 1992). Consequently, the competence development may be increased in this case. Competence is internally regulated among most transformable bacteria (for a review see Stewart, 1992). Moreover, in Bacillus subtilis and in some species of Streptococcus the induction of competehce is controlled by the concentration of a competence factor, a low-molecular-weight polypeptide which is synthesized constitutively and secreted in the surrounding medium. At a critical concentration the exogenous protein stimulates the expression of genes involved in competence induction. Bacteria grow in soil by formation of colonies (Creswell and Wellington, 1992). This kind of growth provides the high densities of bacterial cells required for accumulation of competence factors for competence induction. Competent cells of B. subtilis and Streptococcus pneumoniae bind any kind of double-stranded DNA noncovalently and without base-sequence specificity, although only homologous DNA can be integrated into the genome by homologous recombination. The purity of the extracellular DNA may not affect its transforming activity. The DNA may be complexed with material from lysed cells or other environmental substances, which can lead to reduction of transforming activity (Romanowski et al., 1992). These data suggest that in maintenance of transforming activity of stable DNA, there is no simple rule which is common to all variations of bacteria-DNA interaction. The efficiency of transformation also depends on the molecular size of the transforming DNA molecule. In B. subtilis, the shearing of DNA from 28.5 to 4.5kb decreased the transformation by 100-fold. Further shearing to 2 kb reduced it a further 100-fold. DNA of about 1kb mean molecular size was inactive (Morrison and Guild, 1972). Table I indicates similarities and differences between Gram-positive and Gram-negative bacteria in some aspects of the mechanism of transformation (according to data reviewed by Dreiseikelmann, 1994). Despite the differences in the cell surface of Gram-positive and Gram-negative organisms
TABLE I Comparison of natural transformation in Gram-positive and Gram-negative bacteria (adapted from Dreiseikelmann, 1994) ~
~~
Gram-positive
~~~~
~
Gram-negative L.
Model systems Nature of transforming DNA Sensitivity of free DNA to extracellular nucleases Sequence-specific DNA uptake Existence of transformasomes Receptor proteins for DNA binding Existence of transmembrane channeldpores Place of resistance development of DNA against nucleolytic degradation, including cellular restriction enzymes Conversion of ds DNA to single-strand DNA with the help of membrane-bound translocase (endonuclease I) probably by degradation of the 5'-strand
Polarity of DNA entry: 3'-end ahead (exceptions might be possible) Specialized pores or channels formed by membrane proteins involved in the transformation mechanism Existence of polyhydroxybutyrate (PHB) channels Protection of single-strand DNA by a competence-specific SSB protein Existence of DNA-containing membrane vesicles (blebs)
Bacillus subtilis Streptococcus pneumoniae Double strand Yes No No At the cell surface Yes Transmembrane channel Yes
Haemophilus spp. Neisseria spp. Double strand Yes Yes Yes At the surface of transformasomes Yes Transformasome
Yes Yes
Yes (hypothetical; not shown for Haemophilus or Neisseria, but shown for Acinetobacter calcoaceticus) Yes Not identified
Yes (hypothetical) Yes No
No Probably Yes
Gg ;1
54
m
m > G
8: ;;f
6.f
m
me,
a 7;:
HORIZONTAL GENE TRANSFER IN THE RHIZOSPHERE
405
and the differences in the early steps of the transformation processes of the model organisms, which have been manifested in DNA binding, and early DNA passage through the different membrane systems, the DNA transformation machineries seem to be similar. Modifications occur in specific details of transformation steps in dependence on differences in components of the bacterial cell surfaces. These modifications suggest that they follow separate mechanisms in some aspects and therefore have unique characteristics. So Gram-negative bacteria such as Neisseria gonorrhoeae take up DNA into membrane vesicles called “blebs” (Dorward and Garon, 1990), which are a kind of mobile transformasomes analogous to the situation in Haemophilus. These membrane vesicles may be transport carriers to other cells and reach the cytoplasm of the recipient cell by fusion. This special case of transformation would not include DNA transfer across membranes, and may be advantageous in overcoming competence problems. Speculatively, the range of recipient cells may be expanded into interspecific, intergeneric or perhaps transkingdom regions, just in case the DNA uptake is not homospecific (sequence-specific DNA binding see Table I). The third important question concerning DNA exchange between soil bacteria is: Do recipient bacteria express the acquired foreign DNA? Model experiments have shown that resistance against mercury and the ability to degrade a herbicide are expressed after plasmid transfer from Alcaligenes eutrophus to Variovorax paradoxus in soil (Neilson et al., 1994). So the third main prerequisite for a successful gene transfer may be fulfilled. Besides transformation there are two more mechanisms of DNA transfer: conjugation and transduction. Conjugation is the only form of bacterial sex that involves direct contact, the donor producing a pilus to which the recipient becomes attached and transfers a copy of its F-plasmid as a single strand with the 5‘-end ahead. The whole conjugative machinery, including the pilus, is normally plasmid encoded. The transferred DNA is, in some cases, not only that of the plasmid. DNA regions of chromosomal origin integrated into the plasmid flanking regions by incorrect recombinational excision from the genome (F’-plasmids) have also been observed. In this way conjugative plasmids can assist in the transfer of both chromosomal DNA and other plasmids mediated by formation of a co-integrate between the plasmid and part or all of the other replicon. Streptomycetes are widely distributed among rhizosphere bacteria. They contain different types of plasmids: normal closed circular double-stranded DNA, single-stranded DNA and integrative elements. The conjugative plasmid PIG 101 is especially interesting. It is a closed circle of doublestranded DNA with a size of 8.8 kb. A Streptomyces bacterium can carry up to 300 copies of this plasmid. It can be transferred in vitro into Micromonospora, Thermonospora, Saccharopolyspora and Amycolatopsis, so perhaps it can also experience this wide host range in vivo (Hopwood and Kieser, 1993). The DNA consists of 73% GC, which is normal for
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J. WOSTEMEYER, A WOSTEMEYER and K. VOIGT
Streptomyces DNA, and 9 ORFs can be deduced from the DNA sequence. A search for homologies to corresponding protein sequences in a data base showed no obvious similarities with known proteins. Where does this plasmid DNA come from? Did it travel through many organisms until it came to an intermediate stop in Streptomyces? The plasmid carries genes for its own replication (rep coding for a 456 amino acid protein) and a transfer protein (tru coding for a protein of about 621 amino acids). A regulatory gene on this plasmid is Kor B which limits plasmid copy number. The second regulatory gene, Kor A , represses the promoter of the tra gene and three other genes belonging to the same operon. These three other genes code for hydrophobic proteins which may be localized at the cell membrane. During plasmid transfer chromosomal genetic markers are mobilized to a small percentage. In vitro insertion of the tru region of the plasmid into a Streptomyces chromosome mobilizes this chromosome. The plasmid pIJ 101 proves to be a good carrier for a horizontal gene transfer between bacteria. Another type of DNA element which can move and rearrange the DNA of the recipient is a transposon. Transposable elements, which may have several copies per genome scattered across the chromosome and plasmids, frequently provide homology that can lead to co-integrate formation by recombination. Transposons occur in Gram-negative and Gram-positive bacteria, as well as in eukaryotes. Transposons can integrate into chromosomes and plasmids, their integration and excision creating short duplications at the respective sites. They carry their own enzyme genes for integration and excision, and in prokaryotes usually antibiotic resistance genes as well. The frequency of transposition varies from element to element, and for some eukaryotic elements there is no clear evidence for their “jumping”. Generally, plasmids and transposons appear to be restricted in host range. Thus, for example, each type of a Sym plasmid is found only in a restricted range of chromosomal genotypes within the species Rhizobium leguminosurum. Likewise the insertion sequence Rml is widespread in Rhizobium meliloti but is not found in other species. Also the insertion sequences in E. coli, which tend to be confined to a part of the genotypic range of this species, provide evidence for rather limited chromosomal gene exchange (for reviews see Schmidt, 1992; Young, 1992). At first sight the accessory element-mediated genetic traffic seems to be intraspecific or even narrower. But some accessory elements have been transferred across much greater taxonomic distances. The best known examples are the antibiotic resistance transposons and the plasmids that carry them (for a review see van Elsas, 1992). Inc P-group plasmids, for example, can be transferred between and maintained in bacteria from both the y (enterics, pseudomonads) and the a (rhizobia) subdivisions of proteobacteria, which are by molecular sequence divergence as distant from each other as whales are from worms (Young, 1992).
HORIZONTAL GENE TRANSFER IN THE RHIZOSPHERE
407
Several in vitro studies show conjugational gene transfer which permits genetic elements to cross the Gram barrier. Bertram et al. (1991) demonstrate the genetic transfer of the conjugative streptococcal transposon Tn 916 from the Gram-positive Enterobacter faecalis to the Gram-negative Alcaligenes eutrophus, Citrobacter freundii and E. coli and from E. coli to the Gram-positive B . subtilis, Clostridium acetobutylicum, Enterobacter faecalis and Streptococcus lactis. Such heterogramic transfers have been shown to occur in vitro. However, data about their occurrence under natural conditions are lacking. Kreps et al. (1990) conjugatively transferred an Inc Q plasmid from E . coli to the cyanobacterium Synechocystis PCC 6830. Transduction is the transfer of bacterial genes between bacteria by bacteriophages. Since most phages have a narrow host range, this reduces the probability of extensive gene transfer within a mixed bacterial population. Another group of virus-like elements may play a bigger role in genetic exchange processes, these are retrotransposons and retrons (Garfinkel, 1992). Retrotransposons occur in yeast and other fungi, like Ty in Saccharomyces cerevisiae and Tf in Schizosaccharomyces pombe. Besides their passive replication with the chromosome, these elements can replicate via an RNA intermediate and integrate at a new site. At the target site they cause short duplications. In a few bacteria a multicopy element with resemblance to retroviruses is found: the retron. It consists of a single strand DNA covalently bound to RNA forming a stem-loop structure. In E . coli the retron Ec 67 has been demonstrated to generate a 26 nt target-site duplication and to carry 34 kb novel sequences into the target site. So this retron serves as carrier for a gene transfer. Another retron, Ec 73, is part of an E . coli phage. Transposons may assist in increasing the probability of survival and dispersal in new ecological niches. Class I1 transposons are widespread and contain systems for gene integration and dispersal among bacteria. They might be based originally on drug-producing (antibiotics) micro-organisms like streptomycetes and fungi, and may have been evolved for self-protection from autotoxicity (Schmidt, 1992). Table I1 gives a short overview of the differences between several forms of horizontal gene transfer in bacteria. However, with new answers from investigations and increased understanding the boundaries which distinguish the transfer processes become less defined and fuse to a supermechanism containing different stages, steps and machineries of genetic exchange pathways. In addition, like sequence comparisons in search of homologies at the nucleic acid or protein level, retrospective studies could be generalized and summarized to a more complete picture of the main pathways of DNA transfer. The requirements for homology by the recombination machinery will ensure that the great majority of successful genetic exchange events are intraspecific. Interspecific and intergeneric gene transfer may thus be one of the reasons for
P 0
00
TABLE I1 Comparison of the four main mechanisms (transformation, transduction, transposition, conjugation) belonging to horizontal gene transfer (adapted from reviews by Pickup, 1992; Lorenz and Wackernagel, 1994; Dreiseikelmann, 1994) Conjugative processes involving direct contact Transformation Cell-cell contact Carrier of transversing DNA Nature of DNA to be transversed
Nature of DNA passing the cytoplasm of the recipient In vitro sensibility of the process to DNases Transmembrane channels formed by proteins encoded by genes dispersed over the genome Limiting factor for long-term evolutionary steps Interbacterial genetic exchange
No Free DNA (associated with organic and inorganic particles) Double strand (linear)
Single strand
Transduction No Phages
Transposition Yes Transposons
Conjugation Yes Plasmids
<8; $
w2
;d
P
Double/single strand (linear)
Doublekingle strand
e8;
Double strand (probably covalently closed circular intermediates)
Double strand (circular)
?
m
7: e
Yes
No
NO
Single strand No
Yes
Yes
Yes
Yes
Specific competence development, DNAbinding receptor proteins
Host range of phage
Sexual compatibility of the mating partners
;1
w
4
;d e, '3
P
Increasing intensity
2
2
HORIZONTAL GENE TRANSFER IN THE RHIZOSPHERE
409
major evolutionary transitions breaking the balance of genetic constancy. Transposons and plasmids not only carry D N A to other organisms, but also integrate and deliver their D N A to the chromosomal complement of the recipient. Consequently, high similarities are detected between antibiotic resistance genes in a variety of bacteria at the nucleic acid sequence and amino acid level. A gene encoding an aminoglycoside phosphotransferase (aphA-3) of Enterococcus fuecalis located on a transposon showed 100% sequence homology to a corresponding gene of Staphylococcus uureus, providing evidence for a recent gene transfer event. However, convincing evidence for transfer of aph genes from the antibiotic producing soil streptomycetes and bacilli to clinical bacteria, which would be indicative of horizontal gene transfer over a long evolutionary period, is lacking. Similarly, low homologies between erythromycin resistance genes among Gram-positive antibiotic-producing and Gram-negative clinical bacteria have been obtained at the levels both of nucleic acids and amino acids. These sequence data do not support the hypothesis of recent transfer of these resistance genes. If the antibiotic-producing organisms have been the origin of the different antibiotic-resistance genes, these probably diverged early during evolution of their various hosts. A possible exception might be a fosfomycin resistance gene from Streptornycesfradiae which showed strong sequence homology to a transposable fosfomycin resistance gene in Serrutiu rnurcescens. But do sequence data provide sufficient evidence to prove that horizontal gene transfer in soil communities occurs? Is it enough to compare sequence similarities between genes from different organisms in relation to similarities between their house keeping genes? How can similarity due to gene transfer or parallel evolution be distinguished? With our present knowledge we have no conclusive answers. The potential of soil bacteria to pick up heterologous D N A in laboratory and microcosm experiments under selection pressure by the appropriate antibiotics or xenobiotics makes them good candidates for D N A uptake and transfer to other organisms in the rhizosphere. The physical variability and complexity of soils makes comparable studies of horizontal gene transfer between micro-organisms living in the rhizosphere of plants difficult (Cresswell and Wellington, 1992). It has been estimated that the cell densities in soil are very variable. Paradoxically, a single soil particle can have many thousands of sites for microbial colonization but the entire bacterial population of 1 g could be located on just a few particles. Perhaps less than 0.001% of the soil surface is available. Generally, 1 g of fertile soil contains approximately 105-108 bacterial, 106-107 actinomycete and 105-106 fungal colony-forming units (cfus) (Gottlieb, 1976). Bacteria grow either in homospecific colonies or in associations of related species. This fact is certainly a limiting factor in gene dispersal through putative genetic barriers. Transformation within these bacteria sharing the same ecological niches may be the transfer mechanism of choice. Limitation in the availability of nutrients and other environmental stress factors would limit overall gene
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J. WOSTEMEYER, A WOSTEMEYER and K. VOIGT
transfer, but speculatively permit higher frequencies of gene transfer at specific sites. In the case of a disadvantageous genetic exchange the microbial fitness will be reduced. If the genetic change is neutral or advantageous for the individual, the organism survives with similar or increased fitness against physical and ecological challenges. Those DNA transfer mechanisms requiring large DNA homologies to have a chance for recombinational integration into the genome and subsequent expression may primarily serve to repair mutational damage and retain constancy and stability. Mechanisms meant to conserve the status quo in individuals may serve as driving forces of variability during evolution. The acquisition of genes by horizontal gene transfer could cause a change in the regulation of phase variation of some pathogenic bacteria (Lorenz and Wackernagel, 1994). The protection of cells against bacteriophages and the exploitation of new nutrients would also be possible after gene transfer.
111. FUNGI AS RECIPIENTS OF FOREIGN DNA Although many fungi from different taxonomic groups can be transformed in vitro, either by polyethylene glycol mediated DNA uptake of protoplasts, by electroporation or by means of a particle gun, possibilities for fungi to incorporate foreign DNA in vivo appear to be rather limited. Transformation under natural conditions has never been described. This may, however, be rather a matter of a lack of scientific interest. We are not aware of a single systematic investigation in this direction. Parasexual systems which are analogous to the widespread bacterial conjugational systems are also not known in fungi. The only phenomena where one might suspect a reasonably high frequency of natural gene transfer are: (i) mycoparasitic interactions, which involve close contact between two different fungal organisms; and (ii) the formation of hyphal anastomoses between different species. At first glance, anastomosis formation may appear to be an efficient mechanism for the exchange of nuclei. This would lead first to heterokaryons, followed eventually by occasional nuclear fusions and the formation of recombinants. A closer look, however, at the significance of this process may give rise to doubts. Normally, anastomoses are formed between hyphae of the same individual. Genetically, this is without consequence. The parasexual cycle, based on anastomosis formation, which has been introduced into modern genetics for Aspergillus nidulans (Pontecorvo and Kafer, 1958) by Pontecorvo, is no exception. With the exception of a very limited number of defined mutations, the partners of a fusion experiment are normally isogenic. The consequences of such events for speciation and evolution are evidently low. Even fusions between genetically different individuals belonging to the same species seem to be rare. Incompatibility of mycelia seems to be the rule and compatibility an exception. These considerations have been
HORIZONTAL GENE TRANSFER IN THE RHIZOSPHERE
41 1
discussed carefully (Caten and Jinks, 1966; Hoekstra, 1994). Nevertheless, no mechanism in nature works with perfect efficiency. Therefore, we suspect that some fusions between non-related fungal hyphae do occur. We can only speculate about the fate of newly acquired non-related nuclei in a foreign genetic background. Probably, due to the lack of proper communication between nuclei and cytoplasm, the invading nuclei will undergo lysis. Most of the DNA will presumably be hydrolysed to the level of nucleotides, which will finally be used as building blocks for nucleic acid synthesis. Sometimes, however, DNA molecules will enter the nucleus of the host organism. Most of these fragments will be lost in subsequent nuclear division cycles, but sometimes such DNA will be added to the genetic complement of the recipient. With our present knowledge, it is not possible to calculate the statistical probability for such events. It will certainly be very low and presumably below the level of experimental detection, at least as long as there is no selective pressure on the potential recombinants. We wonder, however, about the outcome of such experiments if genetic traits are selected for. It would be highly relevant to our understanding of fungal evolution if model experiments are performed where at least one of the partners carries a gene that can be selected for in the recipient. Under these conditions even the consequences of very rare anastomosis formation could be made visible. Again, as for natural transformation of fungi, we are not aware of any report on a gene transfer event that is based on anastomosis bridge formation between different fungal species. Let us now look at the interface between the two partners of a mycoparasitic association. Hawksworth (1994), in the foreword of a recent book on mycoparasitism, speaks of roughly 1200 fungicolous fungi and perhaps 2000 fungi that are associated with algae in lichens. In the majority of these associations host and parasite remain separated by walls and membranes. Thus, the probability of genetic exchange is certainly low and remains restricted to those rare events, where lysis events occur between the partners due to biochemical deviations during the formation of the specialized host-parasite interphase. Until now, no experimental evidence has been obtained for the transfer of genetic information in haustorial interactions. Several years ago we proposed that the chance for genetic exchange should be higher between fungi which form a cytoplasmic continuum between host and parasite during the formation of infection structures (Kellner et al., 1991). These mycoparasites are termed “fusion biotrophs”; they are characterized by local lysis of adjacent cell walls of the host and the parasite. Very often, the fusion zones are rather small and it is generally believed that the cytoplasms stay separated. The infection of the soil and rhizosphere fungi from the genus Rhizoctonia by Tetragoniomyces uliginosus includes the formation of micropores with a diameter in the range 14-19 nm (Bauer and Oberwinkler, 1990). This is even less than the diameter of plasmodesmata (3&60 nm) in plants, and it seems impossible that genetic material in nuclei
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J . WOSTEMEYER, A WOSTEMEYER and K. VOIGT
or mitochondria will be exchanged through these channels. A larger pore (0.2-1.0 pm) is formed at the interphase between Dicyma parasitica and its hosts (Hock, 1977). Pores of this size suggest that cytoplasm, and even nuclei, are exchanged between the partners. Unfortunately, no cytological studies have been performed with this goal in mind, and there has been no analysis of whether genetic markers pass the interface between the partners of this mycoparasitic association. Today, with the methods that we have available the transformation of many filamentous fungi, it should not be too complicated to introduce dominantly selectable resistance markers into either host or parasite. Following an infection cycle, the marker can be looked for in the vegetative offspring of the partner. We think that looking at genetic interactions between hosts and their fusion-biotrophic parasites may open u p a rewarding area of evolutionary research. A special case of fusion biotrophs is represented by some members of Mucor-like zygomycetes. As early as 1924 Burgeff recognized that the parasitism of Chaetocladium brefeldii and Parasitella parasitica (syn. simplex) on their hosts is accompanied by cytoplasmatic fusion. It is a pity that no detailed ultrastructural analysis has been performed using all the techniques of modern electron microscopy. In several respects these interactions resemble sexual reactions. We will discuss this aspect in detail. In contrast to the minute cytoplasmic connections in fusion biotrophs belonging to imperfect fungi, ascomycetes or basidiomycetes, C . brefeldii, P. parasitica and Absidia parricida form fusion zones, which are extended enough to allow nuclear migration. At the level of genetic marker transfer the consequences of the fusion between host and parasite have been analysed in some detail (Kellner et al., 1993) for P. parasitica with Absidia glauca as host organism. Similar results have been obtained for A. parricida, again grown on the host A . glauca. We are not aware of a genetic analysis concerning the interaction between C. brefeldii and its hosts. The chances are high that this interaction, too, involves the transfer of genetic information from the parasite’s nuclei into the host organism. According to our knowledge the genetic transfer in the system P. parasitica-A. glauca is strictly unidirectional, from the parasite to the host. A closer look at the morphological peculiarities of the infection pathway provides us with a plausible explanation for this asymmetry (Fig. 1). After anchoring of a parasite’s hyphal tip to a host hypha, Parasitella forms a septum in the otherwise essentially unseptate mycelium close to the contact site. Only after this compartmentalization do the organisms fuse and the nuclei of Parasitella’s distal compartment, the primary sikyotic cell, become able to invade the host’s mycelium. Following the fusion event, the infection site differentiates to a complicated morphological structure. Short hyphal branches of the host form a gall-like structure, which surrounds the so-called “secondary sikyotic cell” of the parasite. The Greek word sikyotic was coined by Hans Burgeff (1924) and
HORIZONTAL GENE TRANSFER IN THE RHIZOSPHERE
413
Absidia
D
E
)k sikyospore
gal I
Fig. 1. Schematic illustration of the infection pathway of P. simplex. P. simplex grows towards the host (A) and forms the primary sikyotic cell (SI) and a septum (B). The primary sikyotic cell fuses with the host (C). A secondary sikyotic cell is formed (D), which is surrounded by a gall from the host (E). Finally, P. simplex forms a sikyospore (E).
means “cupping glass”, suggesting that this swollen bulb-like, terminal region works as a resorption organ for nutrients. This is plausible, but has never been proven by physiologica1 uptake measurements. A t the end of the parasitic differentiation pathway the secondary sikyotic cell forms a dark, resting structure, the sikyospore, which may germinate under favourable conditions. Genetically, these sikyospores represent presumably pure Parasitella material. With regard to the flux of genes in soil fungi it is worth questioning the fate of the parasite’s nuclei which have entered the host’s mycelium. Morphologically, we have no conclusive answer. We simply do not know how long the nuclear envelope stays intact, nor do we have the faintest idea if these nuclei play a designated role during gall formation. We do know, however, that Parasitella genes that have entered the host can be found in the spores of Absidia one vegetative sporulation cycle later. What are the experiments which prove that genetic chimerae are generated as a consequence of sikyotic parasitism? Obtaining experimental proof is not complicated. We have infected a whole variety of different auxotrophic mutants of A . glauca with a wild type strain of P. parasitica. As the host
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does not suffer too much from the infection, it still forms sporangiospores, in normal frequency, that are also in the vicinity of infection structures. These spores can be checked easily for their auxotrophic markers. Among these vegetative Absidia offspring we found recombinant prototrophic derivatives in the range 0.1-1.5%. The mean value for nine independent experiments was 0.42% (Kellner et al., 1993). For a naturally occurring parasexual system this frequency is enormously high. The difference between the formation of prototrophic derivatives and reversion rates of the recipient mutants was larger than lo4 for any individual experiment. In most cases we were not able to obtain revertants from our mutant strains. Thus, the statistical difference between recombinant formation and reversion events is much more pronounced than lo4 in most cases. In genetic analyses of this type we seek the transfer of chromosomal markers. In zygomycetes it is also possible to work with markers residing on autonomously replicating plasmids. P. parasitica can be transformed with a plasmid harbouring the Tn 5-derived neomycin resistance gene under the control of one of the TEF-promoters from A . gluuca (Burmester, 1992). This hybrid gene is expressed in both the donor and the recipient organisms. Infection of A . glauca by a transformed Parasitella donor opens the possibifity for transfer of the plasmid. Indeed, we could find neomycin resistant derivatives of A . gluuca following an infection experiment. We were also able to detect the plasmid in the recipients by Southern-hybridization experiments (Kellner et al., 1993). Taking both the genetic and the physical evidence together, we were able to prove a high frequency of marker transfer from a mycoparasite to its host. These experimental results were, of course, obtained in carefully controlled laboratory model systems. The possibility of detecting the recombinants depends completely on the selection of dominant markers, prototrophies in the first type of experiment, an antibiotic resistance in the second one. The experimental set-up used for proving physical plasmid transfer is highly artificial, and auxotrophic derivatives of A . glauca should be very rare in nature, if they ever exist long enough to be of any ecological o r evolutionary relevance. Thus, the question has to be addressed of whether this highly efficient parasexual transfer system between two soil- and leaf-litter inhabiting fungi plays an important role under natural conditions? A conclusive answer is not yet possible. With our present knowledge we can only provide you with some considerations. One important question is: How frequent are these organisms in nature and do they really play an important part in soil ecosystems? For the potential recipients of genetic information the question can be answered clearly. They are widespread in nature, and members from the single host genus Absidia alone may represent roughly 1% of the total spores in forest soils (unpublished). The homothallic species Absidia spinosa has been found with a frequency of occurrence of 42% in the rhizosphere of Phaseolus vutgaris (Dix and Webster, 1995). For the donor, P. parasitica,
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the answer is more difficult. The species has been reported from only very few locations. However, it is not easy to recognize if it does not grow parasitically. So we wonder, if it is not sometimes identified as one of the many Mucor species. At any rate, in comparison with other zygomycetous mycoparasites (Chaetocladium and Piptocephalis spp.), P. parasitica appears to be less abundant. However, we should bear in mind that, in contrast to the haustorial parasites from the genus Piptocephafis, Chaetocladium is a fusion biotroph. It would be highly interesting to measure the ability of this species for transferring genes. We would not be too surprised if interspecies parasexuality is more the rule than the exception in Mucor-like fungi of soil and rhizosphere. That this idea may be close to reality is perhaps documented by our recent observation that the facultatively parasitic species A . parricida is also able to transfer genetic material to its host during infection. Overall, the abundance of fusion biotrophs in soil and rhizosphere appears to be high enough to play a role in fungal evolution. However, another important aspect has to be considered, i.e. the stability of genetic material acquired via parasitic interactions. The impact of foreign genetic material on the evolution of the recipient genome is higher if the DNA manages to establish itself in the new genetic background either by integration into the genomic complement or by efficient autonomous replication and by a balanced partitioning during subsequent mitotic divisions. We have measured the stability of chromosomal markers in recombinants (“pararecombinants”) between A . gfauca and P. parasitica through several sporulation cycles. We were surprised by the results of such analyses. Most of the pararecombinants are somewhat unstable and tend to lose their prototrophy (Kellner et af., 1993; Wostemeyer et a f . , 1995). Loss rates vary considerably, between 20% and more than 90% if we start with a single uninucleate recombinant spore and measure the amount of prototrophic offspring after a complete growth and sporulation cycle. These data are not comparable with the instability rates given for unicellular organisms such as bacteria or yeasts. As A . glauca grows coenocytically, we cannot measure the stability of individual markers in single cells during mycelial growth. Meaningful data are only obtained if a single spore is allowed to grow out and sporulate again. The spore progeny can then be analysed for prototrophy. Starting with a single spore on a Petri dish, A . gfauca produces roughly lo9 progeny spores. Assuming that nuclear divisions take place in a logarithmic manner, the 109-fold increase corresponds to approximately 30 nuclear division cycles ( lo9 = 229.4.Consequently, the 90% loss of a marker after one sporulation cycle means that 3% (0.90/30) of the nuclei lose the foreign gene in every nuclear division. Compared with the loss rates of several autonomously replicating plasmids in yeast this is rather stable, but compared with the normal stability of chromosomal genes this is incredibly high. We do not know what this means. It cannot be excluded that the Parasitefla genes in an Absidia background are inactivated by some kind of DNA modification.
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It is also possible that the invading DNA is established as a form of autonomously replicating DNA, which, due to inefficient partitioning abilities, may fail to be included in daughter nuclei.
IV. PLANTS AS RECIPIENTS OF FOREIGN DNA A . AGROBACTERIUM TUMEFACIENS
Bacteria in the rhizosphere are a mixture of saprophytic, plant-pathogenic and beneficial ones. They can mobilize their DNA for a transfer between bacteria, and one special bacterium, Agrobacterium tumefaciens, has even found ways to transfer its DNA to plants (Schell et al., 1979). Everyone would expect something weird coming out of the interactions between bacterial and plant DNA, and the result is indeed the formation of a tumour. These crown galls are predominantly callus-like outgrowths but, with certain bacterial strains or under changed physiological conditions, transformed shoots can emerge from these galls (Braun, 1948). Agrobacteria have evolved a special system of DNA transfer. They carry large tumour-inducing plasmids (van Larebeke et al., 1974; Watson et al., 1975), and at the beginning of molecular research on crown gall it was speculated that the whole plasmid would be transferred to the plant. However, the bacteria synthesize a single-strand copy of only the tumour-inducing DNA segment, the T-strand. This T-strand is covered with proteins which are the product of a Ti-plasmid encoded virE gene, and the ends are secured by the product of the virD gene (Stachel and Nester, 1986). Chromosomal vir genes are also necessary to accomplish the mobilization of the T-strand and the transfer to the plant (Klee et al., 1983; Hill et al., 1984). Direct cell contact, for example in a plant wound, is needed for the transfer. Plant wound factors, like acetosyringone, attract the bacteria and induce virulence gene functions (Stachel et al., 1985). After the bacteria have reached the wound they bind to plant cell surfaces and enter broken cells (Lippincott el al., 1977). Avirulent strains can also bind, so there is competition for binding sites between different bacteria. On the bacterial side, two chromosomal loci are needed for attachment. One binding factor, which may be the product of one gene, seems to be LPS. There has to be a chromosomal gene for LPS formation, as avirulent strains without a Ti-plasmid also produce this binding factor (Whatley et al., 1976). On the other hand, there may be an extra LPS gene on the Ti-plasmid, since an Agrobacterium radiobacter strain which does not bind plant cells itself can produce LPS and bind to plant cells after having received a Ti-plasmid. So the Ti-plasmid confers a selective advantage on its carriers. A second binding factor is a P-2-glucan which is produced when the chromosomal chvB gene is active. This P-2-glucan is a cyclic molecule with features similar to LPS which leads to copurification (Puvanesarajah et al., 1985). Thus it seems to
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be easier to separate the two types of binding factor by genetic means, by making mutants in the synthesis genes, than by physical means. The binding factor on the plant side seems to be pectin (Rao et al., 1982). While bacteria have to be active for binding, the plant cells still bind if they are inactivated, e.g. heat-killed. After binding to the plant cell surface the bacteria synthesize cellulose microfibrils to anchor themselves to the plant cells (Matthysse, 1983). The gene for this synthesis resides on the bacterial chromosome and is not essential for virulence. The cellulose production is increased during the infection process, so there seems to be a stimulation by plant factors in addition to the constitutive synthesis. The bacterial part of the interaction may end at this point; crown galls need not contain bacteria to grow. Bacteria can be eliminated by heat treatment after the tumours have been formed (White, 1945; Braun and Mandle, 1948) or by antibiotic treatment in vitro, and tumours still proliferate. How the plant channels the T-strand into its nucleus and integrates it into its genome is still a mystery. Integration seems to take place at random, several copies may be integrated, sometimes in tandem arrays (Chilton et al., 1977; Willmitzer et al., 1980). In the junction area a repeat of 158 bp is generated. Different types of crown gall tumour contain different T-DNA. For the nopaline type one stretch of T-DNA suffices for the neoplastic transformation. In octopine type tumours two different T-DNAs are found. The root forming tumours incited by Agrobacterium rhizogenes also contain two different RT-DNAs (Chilton et al., 1982). This transferable DNA has a chimaeric nature. Three such genes which code for enzymes active in plant hormone synthesis seem to be of prokaryotic origin. But for these genes to function in the plant eukaryotic 5’-expression signals are needed, and indeed they precede these oncogenes. Did these regulatory sequences arise by mutation in the bacteria, or were they picked up from a plant via some gene transfer? Two of these three genes, iaaH and iaaM (tmsl and tms2), are involved in auxin synthesis and one gene, iptZ (tmr),is responsible for cytokinin synthesis (Garfinkel et al., 1981; Inze et al., 1984). They are transcribed in the plant and their expression leads to perturbation of the plant’s hormone balance and to the callus-like growth of crown galls. Two more genes, gene 5 and 6b (tml)do not code directly for enzymes involved in hormone synthesis, but they are thought to have a modifying effect on hormone action. Gene 5 modulates the effects on the T-DNA auxin synthesis genes iaaH and iaaM (Koncz and Schell, 1986). Gene 6b reduces the cytokinin action. Some effects of gene 6b hint at a reduction of auxin sensitivity in cells carrying this gene (Tinland et a/., 1990). Like the hormone synthesis genes, these two genes have eukaryotic signals for their transcription. A third set of genes are those which code for enzymes that produce new plant products, the opines (Bomhoff et al., 1976). These metabolites are made from an amino acid (e.g. lysine or arginine) and a carbonyl compound (Zketoacid or glucose). The close linkage of the corresponding genes with plant hormone genes ensures that the cells producing opines are actively
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dividing. Opines can easily pass through the membrane and diffuse within the intercellular spaces. As long as tumours contain agrobacteria, these bacteria are found in the intercellular space. These bacteria possess not only the genes for opine synthesis on their Ti-plasmid, but also the genes for opine catabolism. Thus they profit within the plant from these metabolites. Their relatives in the rhizosphere also profit, as opines induce the transfer functions needed for the Ti-plasmid transfer between bacteria (Hooykaass et al., 1979). Guyon et al. (1993) showed that growth of opine-utilizing bacteria was increased in an association with transformed plants. So this gene transfer from a bacterium to a plant is not accidental, but has evolved mechanisms which favour and stabilize the gene transfer. Plant signals induce bacterial genes to start the T-strand transfer. The T-DNA, once integrated, switches on active proliferation of the transformed tissue and initiates production of the opines which the bacteria can utilize for their growth. In vitro this DNA segment can be used as a carrier for foreign genes. Whether it sometimes carries foreign genes in vivo is not known. There could be mechanisms of gene acquisition, such as involvement of transposons or IS sequences or recombinations with parts of the bacterial chromosome.
B. RHIZOBIUM INTERACTIONS WITH LEGUMES
The association of agrobacteria with plants is a pathogenic one; in many cases the crown gall disease leads to reduced growth, vigour or even death of the plants. Another type of bacteria which belongs in the same family, the rhizobia, have managed to set up a more mutualistic association with plants (Vance, 1983; Long, 1989). In the nodule-forming system no gene transfer is necessary, as the bacteria stay in the plant. Nevertheless, there could be genetic interaction which has not yet been elucidated. One species of Rhizobium usually only interacts with one species of legume. Binding of the bacteria to plant root hairs has to be specific. The plant binds rhizobia via a surface lectin (Bohlool and Schmidt, 1974). If lectins are saturated by their corresponding hapten, no rhizobia can bind and no nodules are formed. When one plant species (white clover) was engineered to synthesize the lectin of another plant species (pea), the species of Rhizobium binding to it changed from the original one to the one belonging to the lectin producer (Diaz et al., 1989). However, the binding of plant lectins to sugar moities on the bacterial surface cannot be the only mechanism of attachment, as plant mutants without the respective lectin can still bind rhizobia and be nodulated. A second contact mechanism may use the bacterial EPS (exopolysaccharide) as a binding factor (Leigh et al., 1985). FITC-labelled EPS binds preferentially to the root hair tip, maybe to specific receptors. Bacterial mutants in EPS production fail to bind to legumes and to nodulate.
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After the bacteria have been bound to root hairs they induce cell divisions and root hair curling. The inducing factors are secreted EPS and plant hormones produced by the bacteria. Only young root hairs are usually susceptible to this stimulation. Bacterial genes which are necessary for the initial steps in nodule formation are the genes nod A , B and C which are located on the megaplasmids that symbiotic rhizobia contain, called Sym plasmids (Casse et al., 1978). These three genes are conserved among rhizobia. They function in root hair curling and perhaps interfere with cytokinin production. Other modulation genes on the Sym plasmid are nod F , E , G , H and L M N ; these are not interchangeable between rhizobia and deal with host specificity (Kondorosi et al., 1984). The whole set of nodulation genes is under the control of the nod D product, a protein of 33 kDa which binds to a 50 bp so-called “nod box” in nod gene promoters (Rossen et al., 1985). Plant substances, especially flavonoids such as luteollin in alfalfa, stimulate nodulation gene expression, so that in these early steps a rather co-ordinate interaction between plant and bacteria takes place (Peters et al., 1986). The next step in the infection process is the development of the infection thread, a structure which the plant produces to capture the bacteria and release them one by one into cortical cells (Callaham and Torrey, 1981). During the release the bacteria are wrapped by a plant membrane, the peribacteroid membrane. The bacterial LPS is a signal which leads to infection thread formation. Once the bacteria have been enclosed in the plant membrane, they develop into bacteroids (Paau et al., 1978). These bacteroids have the task of fixing nitrogen for the plant. They use two different sets of genes for this task: the nifand thefix genes (Corbin et al., 1983;Earl et al., 1987).The nifgenes also function in free-living bacteria and are homologous to nif genes of other free-livingbacteria such as Klebsiella pneumoniae, whereasfix genes only work in the symbiosis with the plant. A regulator for the expression of nifgenes is encoded in n i f A (Gussin et al., 1986). The genes for nitrogen fixation are located on the large Sym plasmids in close vicinity to the nod genes. A region of 32 kb carries all the genes that are essential for establishment of a functional symbiosis (Innes et al., 1988). Why these bacteria keep Sym plasmids of about 184 kb or larger is not known. The legume plants need extra genes which only act in the symbiosis. Some of the needed proteins are called “nodulins” (Delauney and Verma, 1988). They are responsible for the synthesis of the infection thread and the peribacteroid membrane, Another important nodule-specific plant product is leghaemoglobin, a protein which is necessary to keep oxygen concentration low (Ostergaard Jensen et al., 1981). The genes for leghaemoglobins are a multigene family containing even truncated and pseudogenes (Brisson and Verma, 1982). They are eukaryotic genes, usually having three introns and eukaryotic signal sequences, and so they cannot be acquired from bacteria by a gene transfer, at least not in a functional form.
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A recent observation may lead to new speculation about gene transfer: rhizobia possess on their Sym plasmid genes for the production and the catabolism of a special metabolite which can be called a “rhizopine” (Murphy et ai., 1987, 1988). So there is an exciting parallel to the Agrobacterium system. As agrobacteria and rhizobia are related to each other, in vitro methods could be devised to test the effect of plasmids in the other genus. Hooykaas et al. (1977) managed to transfer the Agrobacterium Ti-plasmid into a Rhizobium trifolii strain. Rhizobium gained the ability to induce tumours on plants, and to catabolize the respective opine. It was still able to nodulate its host clover. Interestingly, nodules did not produce opines, which may show that the Ti-plasmid stayed in the bacteroid and did not reach a plant cell nucleus where it could be transcribed. The recipient Rhizobium had lost the ability to nodulate a second host, Trifolium parviporum; perhaps rearrangements in the Sym plasmid had occurred leading to a deletion of genes for recognition of T. parviflorum. In another type of experiment an Agrobacterium tumefaciens strain, which was cured of its Ti-plasmid, received a Sym plasmid from R. trifoZii (Hooykaas et al., 1981). This plasmid endowed Agrobacterium with the capability of nodulating clover. Host recognition was followed by root-hair curling and infection-thread formation. Even the release into the cytoplasm in an intact peribacteroid membrane worked. However, later stages of bacteroid formation and nitrogen fixation were not reached. It is not very probable that this type of genetic exchange occurs in vivo. Nevertheless, such experiments show that DNA can find ways to move between rhizosphere bacteria and from bacteria to plants. Organisms build up barriers against foreign DNA, but every system makes mistakes, and only time can tell whether the escaped DNA will be detrimental or beneficial.
V. INTERKINGDOM GENE TRANSFER In several biological systems members belonging to different kingdoms live in close contact. Bacteria can live within special compartments in fungi, in plants, in protozoa (Finlay et af., 1993), and in animals or humans. Investigations on genetic transfer in these specialized systems very often measured the exchange of DNA between bacteria in the same compartment (Walton, 1966; Anderson, 1975). A direct measurement of interkingdom gene flow in such systems has not been successful as far as we know. A lot of indirect evidence for gene exchange in evolution has been accumulating in recent years. The methodological approach is sequence comparison for conserved and variable genes between different organisms. A genetic transfer event is claimed if unexpectedly high similarity between non-related taxa is observed for individual genes within otherwise completely
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unrelated genomes. Some examples are given here, but the list is far from being complete. In a review Smith and Doolittle (1992) provided evidence for unexpected sequence similarities between a glyceraldehyde-3-phosphate dehydrogenase from E. coli and eukaryotes. Another example is the glucose-6-phosphate isomerase of E. coli, which has a highly similar sequence to the one in the plant Clurkia ungulatu. In these two examples an interkingdom gene transfer may have taken place from a eukaryote to a prokaryote. Indirect evidence which may suggest a prokaryotic origin of an eukaryotic gene is obtained by the observation that the isopenicillin N-synthetase genes from Streptornyces lipmanii and Aspergillus nidulans show a relatively high degree of homology (54-57%), well above that of the most conserved genes between prokaryotes and eukaryotes (e.g. glyceraldehyde 3-phosphate dehydrogenase; Weigel et ul., 1988). Gene flow from prokaryotes to eukaryotes may be the reason for sequence similarity of a ferric superoxide dismutase between bacteria and Entumoebu histolyticu. These systems cannot prove in a rigorous sense that genetic transfer has happened, they only offer a plausible explanation. It is not easy to decide between gene transfer as a cause of illegitimate genetic mixing or parallel mutations. As the curiosity of scientists rises to find out whether horizontal gene transfer still occurs today, experiments are being designed to trace such a transfer in laboratory systems. Investigations on Plusmodiophoru brussicue, a fungus pathogenic for Brussica nupus, led to the conclusion that the fungus takes up high-molecular-weight DNA of its host (Bryngelsson et ul., 1988). DNA of purified zoospores of this fungus showed a repetitive DNA pattern characteristic of its host. Changing the host to Sinupis albu also changed the repetitive DNA pattern to the one of S . ulba. Similar results were obtained after probing the fungal DNA with rDNA or a single-copy gene. Treatment of the fungi with DNase did not abolish the plant pattern in the fungal DNA. Another reason for studying genetic transfer is to assess risk before genetically engineered organisms are released. An interkingdom gene transfer was followed in a study on transgenic plants and soil fungi. The resistance to hygromycin which had been introduced in transgenic plants of different species could be found in colonies of Aspergillus niger co-cultivated with these plants in microcosm experiments (Hoffmann et ul., 1994). The demonstration of the resistance gene in fungal recipients showed that, in many cases, the acquisition of foreign DNA was transient. However, a stable fungal colony could be isolated which even contained a rearranged copy of the original vector plasmid used to transform the plant. These experiments show that genetic exchange between different members of a soil community occur in nature. It is not to be expected that the foreign DNA is always stably integrated into the chromosomal complement of the recipient, but it offers a choice to the recipient to use it in case of need, e.g. to cope with an unexpected stress.
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Experiments of this latter type represent specially designed set-ups that are meant to reveal a putative interkingdom gene transfer. In the BrassicaAspergiffussystem, a strong selection on the hygromycin resistance could be exerted. This was made possible by putting the dominantly selectable marker artificially under the control of the CAMV 35S-promoter, which is known to promote transcription in both Brassica and aspergilli. Similar considerations hold true for the genetic transfer from E. cofi to the yeast Saccharomyces cerevisiae (Sprague, 1991). Heinemann and Sprague (1989) constructed a special vector plasmid that contained selectable markers for E. coli and yeast. It also harboured an ori T and appropriate tra and mob functions. With this special constellation it was shown that the conjugation system of the bacterium is able to promote the transfer of genes to an eukaryote. The frequency is somewhat lower than between compatible E. cofi cells, but it is reasonably high to obtain plasmid transfer reliably under experimental conditions. It was also shown that the F-plasmid promotes conjugation with eukaryotic cells (Buchanan-Wollaston et a f ., 1987). At least that part of the bacterial conjugation system which is responsible for contact formation seems to work within a wide phylogenetic range. In addition, another yeast, Schizosaccharomyces pombe, was shown to act as a recipient in conjugation experiments (Sikorski et af., 1990). The problems for permanent gene transfer to occur seem to lie more at the level of establishing the gene in the new genetic background. Consequently, we know from specially designed laboratory experiments that genetic transfer between fairly different species, even between kingdoms, does occur. However, we have very little information about the frequency of these events under natural conditions. Due to their completely different environmental requirements, conjugative enterobacteria and Saccharomyces yeasts are not very likely to undergo frequent genetic exchange. In this respect, the Aspergillus-Brassica experiment seems more intriguing. These organisms live in close contact, in the rhizosphere. Unfortunately, we have no idea about the mechanism behind this gene transfer. Perhaps the DNA from decaying plant material is simply taken up by processes which resemble transformation. If this is the case, we could imagine that many more fungi in the rhizosphere will participate in gene exchange processes. But, strictly speaking, we simply don’t know and there are ample opportunities here for research.
VI. RELEVANCE OF LATERAL GENE TRANSFER FOR EVOLUTION We have seen that intertaxonomic gene transfer can be shown experimentally in various systems. Sometimes, especially if filamentous fungi are involved, genetic fluxes have been shown to occur in the rhizosphere. This biotope
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represents a complex community of plants, animals, fungi and bacteria. Members from all these kingdoms have been shown to be involved in parasexual systems across species barriers. Thus, the question arises of whether the rhizosphere community may be interpreted as a genetic continuum rather than as a collection of genetically strictly separate species. There is clearly a contradiction between the abundance of intertaxon gene transfer that has been demonstrated in model experiments and the constancy of species that we recognize. This discrepancy must be interpreted. Obviously, mechanisms have been established during evolution which tend towards streamlining of genomes rather than to acquiring new information. Phenomena such as restriction in prokaryotes o r RIP in filamentous fungi will certainly decrease the frequency of successful gene establishment. We do not discuss these mechanisms in detail here, but try to look at the fate of a foreign DNA fragment in a new genetic background. In most cases the problem will not be the uptake of DNA through the cell wall and through the membranes separating exterior DNA from the compartments of the recipient harbouring their genomes. But following the invasion into the nucleus the DNA fragment has to find a way to establish itself. This can be achieved by various routes. If there is sufficient homology between donor and recipient DNA a homologous recombination event may be expected to occur. Such events will lead to either gene replacement, gene disruption or to the formation of chimeric genes depending on the architecture of the DNA fragment. If single-copy genes are involved the recipient will have to use the foreign information in most cases. It is highly improbable that the foreign DNA will be transcribed and regulated as efficiently as the original one. Consequently, there will be a selection against the interspecies-recombinant in subsequent generations. The situation may be different if the foreign DNA is integrated at non-homologous sites, especially if it goes into repetitive regions which are not essential for the reproductive success of the recipient. Such events may indeed supply a recipient with sparse DNA which can be expected to represent the raw material for the development of new functions during evolution. Another method for establishing foreign genetic material is to carry an efficient replicator function. We do not know how frequently such events occur, but we have seen at least one example. After protoplast fusion between A . gluuca and P. parasiticu, a DNA element which occurred in a low copy number in Parasitelfa was amplified tremendously in the recombinants, possibly by efficient transposition. It is not known if such events lead to organisms that prove fit enough to survive the selective pressure exerted by their natural environment, but there is some evidence that acquiring transposable elements from different species may be more frequent than expected: the P elements of the fruit-fly Drosophila rnefanogaster may have been introduced by mites only some 40 years ago (Marx, 1991).
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The Origins of Phytophthora Species Attacking Legumes in Australia
J . A . G . IRWIN. A . R . CRAWFORD and A . DRENTH
CRC for Tropical Plant Pathology. The University of Queensland. Brisbane 4072. Australia
I . Introduction
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I1 . Legumes in Australia
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I11. Phytophthora Species Attacking Legumes in Australia .................. .................... A . Taxonomy ................ ................................................. B . Asexual Life Cycle ..... C . Sexual Life Cycle .............................................................
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IV. Genetic Variation and Possible Origins of Phytophthora Species attacking Legumes .................................................................. A . Phytophthora sojae ........................................................... B . Phytophthora medicaginis ................................................... C . Phytophthora vignae ................... .................................. D . Phytophthora macrochlamydospora ..................................
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V . Evolutionary Relationships between Phytophthora Species Attacking Legumes ............................................................................... VI . Evolution of Species and Host Specificity in Phytophthora Species attacking Legumes .................................................................. A . Biological Species and Speciation ............................. B . Evolution of Host Specificity in Ph ra ....................... VII . Evolution of Cultivar Specificity in Phytophthora sojae
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VIII . Concluding Remarks ... ........................................................ Acknowledgements ................................................................. References ............................................................................ Advances in Botanical Research Vol . 24 incorporating Advances in Plant Pathology ISBN 0-12-00.5924-X
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Copyright 0 1997 Academic Press Limited All rights of reproduction in any form reserved
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I. INTRODUCTION Phytophthora is one of the world’s most economically significant genera of plant pathogens. In Australia alone, it is estimated to cause in excess of $200 million in lost production annually (Irwin et al., 1995). This does not include damage in natural ecosystems, and subsequent losses to associated industries such as tourism. Due to the importance of legumes to Australian agriculture, both as pasture plants and as grain crops in rotation with cereals, it is not surprising that several of the first records of new Phytophthora spp. attacking legumes were published in Australia (Purss, 1957; Taylor et al., 1985; Irwin, 1991). It is of much more than academic interest to deduce the geographic centres of origin of Phytophthora spp. attacking legumes and to gain a better understanding of evolutionary relationships between taxa of Phytophthora. Such an understanding identifies possible areas to search for sources of host resistance. This chapter considers the genetic variation within and the evolutionary relationships among Phytophthora pathogens of legumes in Australia, leading to a discussion of their origins, and the evolutionary basis of host and cultivar specificity. Emphasis has been placed on identifying areas of deficiency in our understanding of the biology of Phytophthora which are needed to control Phytophthora-incited diseases on legumes.
11. LEGUMES IN AUSTRALIA Of the 16 OOG19 000 legume species known world-wide, less than 2000 are native to Australia (Davidson and Davidson, 1993). When legumes first appeared on the Australian continent remains a puzzle. Truswell (1987) indicated that legumes very likely arrived in Australia by island hopping from southern Asia. Many indigenous Australian leguminous taxa have links with Asian forms. Eighty-two legume genera are native (indigenous to Australia, but also found outside Australia), while another 42 are endemic (found only in Australia) (Davidson and Davidson, 1993). The following families have endemic and native representatives in Australia: Papilionaceae, Mimosaceae and Caesalpiniceae. The Papilionaceae contains several genera which have, or could have, economic significance in Australian agriculture: Desmodiurn, Crotalaria, Lotus, Trigonella, Psoralea, Indigofera, Vigna and Glycine. Pasture legumes have for a long time played a major role in Australian agriculture. At the beginning of this century it was found that the accidentally introduced subterranean clover would flourish in regions with a winter rainfall and a short growing season. These initial introductions were improved by additional selection and after the introduction of appropriate Rhizobium spp., they contributed significantly to Australian agriculture. Until recently,
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crop legumes have generally played a relatively minor role in Australian agriculture compared to cereals, due to the lack of varieties adapted to Australian conditions, and the absence of processing and marketing facilities, in combination with economic constraints (Davidson and Davidson, 1993). Pasture legumes were grown in alternative years with wheat, and in some regions have recently been replaced by crop legumes to provide a major source of nitrogen for the wheat crop. There is now increasing awareness of their value in crop rotations, and their use is increasing, particularly in southern Australia where the combined lupin and field pea area exceeds 1 million ha annually (Davidson and Davidson, 1993).
111. PHYTOPHTHORA SPECIES ATTACKING LEGUMES IN AUSTRALIA A.
TAXONOMY
There are over 50 species of Phytophthoru, of which almost all are plant pathogens (Brasier, 1992; Erwin and Ribeiro, 1996). The current classification is heavily based on morphological characters, in particular whether sporangia are papillate (Waterhouse groups I and 11), semipapillate (Waterhouse groups I11 and IV) or non-papillate (Waterhouse groups V and VI) (Waterhouse, 1963). A distinguishing characteristic for the genus Phytophthoru is that zoospores differentiate within a sporangium which is produced on either a determinate or indeterminate sporangiophore. Meiosis in Phytophthoru is gametangial, giving rise to a diploid (or higher ploidy level) somatic phase (Shaw and Khaki, 1971; Sansome and Brasier, 1973; Shaw, 1983). There are several biological features of Phytophthoru spp. that make them unique as plant pathogens and provide a significant body of evidence, which indicates that Phytophthoru has evolved from unicellular algae, in particular chrysophytes and diatoms (Gunderson et ul., 1987; Wainright et ul., 1993) and are only distantly related to the higher fungi. Approximately half of the 50 morphological species are heterothallic (Brasier, 1992), exhibiting two mating types (A, and A2). This is coupled with the capacity to self-fertilize under certain conditions, thus providing substantial reproductive flexibility (Brasier, 1972). Homothallism can provide a sound mechanism for species isolation, as suggested by Brasier (1987). However, formation of forced heterokaryons of the homothallic Phytophthoru sojue by Long and Keen (1977a,b) and Bhat and Schmitthenner (1993) were indicative of possible outbreeding mechanisms. Whisson et ul. (1994) were the first to demonstrate that outbreeding occurs in P. sojue using unambiguous genetic markers. The species of Phytophthoru recorded from legumes in Australia are listed in Table I. Two species which were first described in Australia (Phytophthoru
TABLE I Species of Phytophthora recorded on legumes in Australia Species
Morphological group (Waterhouse, 1963)
Phytophthora cinnamomi
VI
Phytophthora clandestina
I
Phytophthora cryptogea
VI
Phytophthora drechsleri
VI
Host and country where originally described Cinnamomi burmanni, Indonesia (Meded. Inst. PI Ziekt., Buitenz. 54:41) Trifolium subterraneum, Australia (Mycotaxon 22:77-85) Lycopersicon esculentum, Ireland (Scient. Proc. R. Dubl. SOC.,N.S. 15:498) Solunum tuberosum, USA (Res. Bull. Mo. Agric. Exp. Stn. 153:188)
Centre of diversity of host from which originally described
Legume hosts in Australia
Indonesia
Lupinus digitatus (Simmonds, 1966)
Mediterranean countries
Trifolium subterraneum (Taylor et al., 1985)
South America
Cyamopsis tetragonoloba (Stirling and Irwin, 1986)
South America
Stizolobium deeringianum (Simmonds, 1966), Cajanus cajan (J. L. Alcorn, Queensland Department of Primary Industries, unpublished records)
Phytophthora macrochlamydospora
VI
Phytophthora medicaginis (syn. P. megasperma f. sp. medicaginis)
V
Phytophthora megasperma
V
Phytophthora sojae (syn. P. megasperma f. sp. glycinea) Phytophthora vignae
V
VI
Glycine m a , Australia (Mycologia 8 3 5 17-5 19) Medicago sativa, USA (Mycologia 83:376-381) Althea rosea, USA (J. Wash. Acad. Sci. 21 5 2 5 ) Glycine m a , USA (Mycologia 83:376-381) Vigna unguiculata, Australia (Qd. J . Agric. Sci. 14:141)
China
Glycine m a (Irwin, 1991)
Transcaucasia
Medicago sativa (Simmonds, 1966), Cicer arietinum (Irwin and Dale, 1982) Trifolium repens (Williams and Pascoe, 1994)
Europe
China
Glycine max (Pegg et al., 1980)
Japan
Vigna unguiculata (Purss, 1957)
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Fig. 1. Life cycle of Phytophthora sojae.
vignae and Phytophthora mucrochlamydosporu) are in groups V and VI, and the third, Phytophthoru clundestinu is in group I. The remaining taxa are in either group V or group VI. B. ASEXUAL LIFE CYCLE
The life cycle of P. sojue is illustrated in Fig. 1 and demonstrates many of the features discussed below. Sporangia can either germinate directly by forming a germtube or, most commonly, differentiate into up to 50 biflagellate zoospores. The flagella enable the zoospores to move actively in water for short distances before they encyst. Zoospores preferentially accumulate on plant surfaces at the region of elongation immediately behind the root tip (Zentmyer, 1961), the junctions of tap and lateral roots (Irwin, 1976), around subterranean stomata (Dale and Irwin, 1991), or on the surface of saturated soils, resulting in hypocotyl infection at the soil line (Pfender et ul., 1977). These different infection courts result in lesions being produced at a range of sites. After encystment of the zoospores, a germ tube emerges. In the case of many root-infecting species, such as P. sojue and P. rnedicuginis, an appressorium is not produced, but penetration occurs directly from the germ tube (Miller and Maxwell, 1984) (Fig. 1). Following penetration, hyphal structures are formed in the epidermal cell layer from which the mycelium grows, initially intercellularly while intracellular haustoria are formed in the mesophyll or cortical cell layers. In a compatible
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interaction between a virulent isolate and a susceptible host plant for a hemibiotroph such as Phytophthora infestans, the first macroscopic sign of lesion development is often the appearance of water-soaked areas (Cooke, 1977). As the pathogen grows, the centre of the spreading lesion becomes necrotic and appears as a dark, sunken external lesion on either the roots or stems. Sporangiophores are produced at the periphery of the lesions on roots and stems. The asexual life cycle of Phytophthora involving sporangium production and release of motile zoospores, is very closely tied to an aquatic environment. Many asexual generations may be produced in one growing season, which explains the tremendous potential for spread and epidemic development of Phytophthora-incited diseases. C. SEXUAL LIFE CYCLE
Phytophthora species may either be homothallic (selfing) or heterothallic (outbreeding). The sexual spores, called “oospores” (Fig. l), arise from fusion of two morphologically different gametangia, the oogonium and the antheridium. Since meiosis occurs in the multinucleate gametangia (Sansome and Brasier, 1973; Shaw, 1983), shortly before gamete fusion and zygote formation, the somatic hyphae of Oomycetes are, in general, diploid (or polyploid), resulting in a life cycle with a long diplophase and a short haplophase. Oospores can survive for many years in soil (Duncan, 1980; Duncan and Cowan, 1980; Drenth et al., 1995). Oospore germination involves solubilization of the lipid bodies in the ooplast vacuole, dissolution of the inner oospore wall and the formation of one or more germ tubes. These germ tubes can either initiate mycelial growth directly or terminate in a sporangium which can germinate directly or produce zoospores (Shaw, 1983, 1988). Despite many investigations, limited information is available concerning the dormancy of oospores and stimuli which trigger their germination.
IV. GENETIC VARIATION AND POSSIBLE ORIGINS OF PHYTOPHTHORA SPECIES ATTACKING LEGUMES In studies on the origins of pathogens, much can be learned from the work that has been done to determine the origins of crop species. Vavilov (1949) looked at total genetic diversity of individual crop species and their wild relatives world-wide, and from this work deduced centres of diversity, which he also believed were centres of origin. The rationale behind Vavilov’s reasoning was that the longer a plant existed in a region, the more genetically diverse it would become, and the greater would be its distribution. Zhukovsky (1961) further developed Vavilov’s concepts to include primary gene microcentres (restricted areas where cultivated forms first originated)
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and the secondary megacentres into which the cultivated forms subsequently spread. A consideration of the origins of Phytophthora spp. attacking legumes in Australia cannot be attempted without a consideration of the origins of their hosts. Selection-neutral and co-dominant markers such as isozymes and restriction fragment length polymorphisms (RFLP) (Southern, 1975), which can identify different alleles at one locus, are powerful tools in population genetics. Dominant markers such as random amplified polymorphic DNA (RAPD) (Williams et al., 1990) are less powerful but have the advantages of speed and simplicity. Such markers are typically used to obtain DNA fingerprints or multilocus phenotypes which enables identification of clonal isolates in fungal populations.
A . PHYTOPHTHORA SOJAE
P. sojae, causing root and stem rot on soybean (Fig. l ) , was first recorded in the USA in 1951 (Sukovesky and Schmitthenner, 1955), and was not recorded in Australia until 1979 (Pegg et al., 1980). Population genetic studies, using RFLP markers in the USA and Australia revealed higher levels of genetic variation in the USA than in the Australian P. sojue populations (Forster et al., 1994; Drenth et al., 1996). Initial research using RFLPs revealed that the Australian P. sojae population appeared as a subset of the USA population (Whisson et al., 1992, 1993), indicating that the Australian population was introduced from the USA. More recently, intensive sampling of Australian populations has confirmed this earlier hypothesis, and has also demonstrated convergent evolution in which new pathogenic races in Australia are arising by mutation into a common genetic background (Drenth et al., 1996). P. sojae is host specific to soybean and the genetics of resistance to P. sojae has been studied extensively in soybean. Thirteen dominant resistance (Rps) genes against Phytophthora root rot in soybean have been identified at seven loci and it is hypothesized that P. sojae has a long history of co-evolution with its soybean host, giving rise to a gene-for-gene system (Keen, 1982). However, the genetics of virulence (the ability to overcome resistance genes) in the pathogen was largely unknown due to the inability to perform genetic manipulations of the pathogen. Recent breakthroughs in the genetics of P. sojae have enabled detailed analysis of the Mendelian genetics of seven avirulence genes which confirmed the gene-for-gene hypothesis with its host soybean (Whisson et al., 1994, 1995). Soybeans are believed to originate in China (Vavilov, 1949). Forster et al. (1994) indicated that P. sojae might be native to the USA, “perhaps as a pathogen of native legumes, as the disease was identified for the first time shortly after soybeans were introduced on a large scale into the Midwest”. They further indicated that its origin had not been systematically investigated.
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From the evidence to date, extremely low diversity in Australia (Drenth et al., 1996) and relatively low diversity in the USA (Forster et al., 1994; Drenth et al., 1996) it is apparent that P. sojae is a relatively recent introduction into Australia (Drenth et al., 1996). However, the question of whether it is native to China and originated there along with its host, or was introduced to the USA can only be resolved by population genetic studies on collections from those continents. B. PHYTOPHTHORA MEDICAGINIS
P. medicaginis is host specific and causes root rot of chickpea and lucerne. Population genetic studies using RAPD markers on P. medicaginis collections from Australia and the USA revealed low levels of genetic variation (E. C. Y. Liew, personal communication), indicating this pathogen is probably a recent introduction to both continents. Lucerne and chickpea both have centres of diversity in Asia Minor (Vavilov, 1949). This, in conjunction with its high levels of host specificity towards these plant species (Erwin, 1954; Irwin and Dale, 1982), indicates that P. rnedicaginis has most likely co-evolved with either one or maybe even both hosts in Asia Minor. Its interaction with chickpea may be more recent than with lucerne, because high levels of resistance in wild chickpea have not been found, despite extensive screening of the world collection (Brinsmead et al., 1985). Chickpea also has a centre of diversity in Abyssinia, from which lucerne is absent (Vavilov, 1949). It is possible that chickpea originated in Abyssinia in the absence of the pathogen, and Asia Minor is a secondary megacentre to which it spread, thus explaining its higher level of susceptibility. C . PHYTOPHTHORA VIGNAE
P. vignae, causing root rot on cowpea and adzuki bean is host specific. RFLP analysis conducted on Australian and Japanese isolates of P. vignae from cowpea and adzuki bean have shown a similar situation to P. sojae and P. medicaginis; low levels of genetic variation (Liew et al., 1991). Neither of these host species are native to Australia, and this combined with the low level of genetic variation, indicate that P. vignae has most likely been introduced to Australia. Adzuki bean has a centre of diversity in China, and cowpea has independent centres in Abyssinia and Indo-China, although it is thought to have been first domesticated in West Africa (Steele et al. ,1985), making these locations the most likely centres of diversity of P. vignae. Since P. vignae was first reported on cowpea in Australia in 1952 (Purss, 1957), it has subsequently been reported from Japan (Kitazawa et al., 1979), India (Nirwan and Uphadyaya, 1972), Tanzania (Singh et al., 1981) and Sri Lanka (Fernando and Linderman, 1993). The presence of cultivar specificity of
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P. vignue towards three cowpea genotypes (Purss, 1972) indicates it also has a long history of co-evolution with its Vignu hosts. Population genetic studies on collections from centres of origin of its hosts may reveal the centre of origin of P. vignue. D. PHYTOPHTHORA MACROCHLAMYDOSPORA
The only records for this organism are from Australia on soybean and Desmodium intortum. It is pathogenic, in glasshouse tests, to some native legumes (Stovold et ul. , 1996). A relatively small number of isolates collected from Queensland and New South Wales have been analysed with molecular markers, and very low levels of genetic variation were detected (A. Crawford, unpublished data). Both P. mucrochlumydosporu and P. sojue are pathogenic to Lupinus ungustifolius (A. Crawford, unpublished data), confirming earlier findings of Jones and Johnson (1969) for P. sojue. From the limited number of isolates available it is unclear whether or not P. mucrochlumydosporu is an endemic pathogen of native legumes in Australia or has been introduced.
V. EVOLUTIONARY RELATIONSHIPS BETWEEN PHYTOPHTHORA SPECIES ATTACKING LEGUMES Evolutionary relationships between species can be deduced from DNA sequences of conserved genes. Ribosomal DNA (rDNA) genes contain highly conserved regions that enable comparisons between families and genera, and variable internal transcribed spacer (ITS) regions that allow investigations of relationships among closely related species (Hibbet, 1992). There are potential problems when using rDNA sequence data to deduce evolutionary relationships between species. Even within the same individual, diversity may exist between individual repeat units, of which over 200 copies can be present per haploid genome of Oomycetes (Martin, 1990). Nevertheless, rDNA sequence analysis has great potential to clarify evolutionary relationships among species. Analysis of ITS I and ITS I1 regions of the rDNA of four Phytophthora spp. by Lee and Taylor (1992) revealed a common lineage for P. pulmivoru and P. megukuryu, and close evolutionary relationships between cocoa isolates of P. cupsici and P. citrophthoru. Evolutionary relationships deduced from rDNA sequence analysis of the ITS I and ITS I1 regions from one or a few isolates of each of a large number of Phytophthoru species demonstrated that morphological groups based on sporangial papillation have phylogenetic significance, with the three groups (papillate, semipapillate and non-papillate) each forming separate clusters (Fig. 2) (Crawford et ul., 1996). The three clusters formed in the non-papillate group compared to the uniformity of the papillate and the semipapillate
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P
P.iranica
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P.nicotianae P P.pa/mivora P.citriwla
I
Lpsyringae
P SP SP
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P.megasperma (Spain) NP L P .medicaginis
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P.macrochlamydospora
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P,macrochlamydospora DAR522BO NP'
Fig. 2. Single most parsimonious phylogram generated from branch and bound algorithm in Paup 3.1.1. The percentages are the frequencies with which a given branch appeared in 500 bootstrap replications. P, SP and NP, represent the sporangial classes of papillate, semipapillate and non-papillate, respectively. NP* indicates non-papillate, previously classified as semipapillate. A-D indicate the four main branches. Horizontal separation between nodes of the trees is proportional to phylogenetic distance. The tree was rooted by making Achlya bisexualis the outgroup. (Reprinted with permission from Crawford et al., 1996.)
groups reflects the larger number of different non-papillate Phyrophrhora spp. analysed from Waterhouse groups V and VI (Crawford et al., 1996). The most closely related non-papillate species to those in the papillate group were P. cinnammi, P. sojae, P. vignae and the P. megasperma spp. from asparagus and Douglas fir. P. cinnarnorni is heterothallic and shows high
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levels of genotypic diversity, whereas P. vignue and P. sojue are homothallic with relatively low levels of genotypic diversity as measured with RAPD markers. Sequence analysis by Crawford et ul. (1996) of the rDNA indicates a relatively close relationship between the homothallic and host-specific species, P. vignae and P. sojue, and the heterothallic and wide-host-range species, P. cinnumomi. These results contrast with the hypothesis of Hansen (1987), who proposed that host specific taxa which formerly belonged to the P. megaspermu complex (e.g. P. sojue and P. medicuginis) shared a common ancestry with broad-host-range isolates of P. megaspermu. While these findings are based entirely on sequence homologies of the rDNA gene, greater confidence would be obtained by also comparing sequence homologies of conserved single copy nuclear genes such as actin. It is obvious that more comprehensive studies are needed to deduce evolutionary relationships among taxa of Phytophthoru that attack legumes. In the next cluster both P. drechsleri and P. cryptogeu grouped together, indicating their close affinity, which has been previously suggested from morphological data (Ho and Jong, 1986). P. medicuginis, P. trifolii and P. meguspermu (Spain) also appeared to share a relatively recent common ancestry with the heterothallic P. drechsleri-P. cryptogeu complex. P. mucrochlumydosporu formed a separate cluster, showing its distant relatedness to the other non-papillate species discussed above (Fig. 2). Gaumann and Wynd (1952) proposed that within the Peronosporales, the downy mildews have evolved from soil-borne Pythium spp., with Phytophthoru being a reflection of an intermediate stage in the evolutionary advance. The findings of Crawford et al. (1996) indicate the importance of sporangial morphology (papillation) as an indication of evolutionary relationships within Phytophthoru which, if extended to Pythium and the downy mildews, could allow testing of Gaumann and Wynd’s hypothesis.
VI. EVOLUTION OF SPECIES AND HOST SPECIFICITY IN PHYTOPHTHORA SPECIES ATTACKING LEGUMES A. BIOLOGICAL SPECIES AND SPECIATION
Biological species are the operational units of evolution and are often defined as groups of interbreeding populations reproductively isolated from other such populations. However, this zoologically orientated biological species concept does not adequately accommodate fungi, which may reproduce uniparentally and asexually in addition to sexual reproduction. Hence, a broader species definition is needed such as the one suggested by Brasier and Hansen (1992), which indicates that species are groups of populations that share a common lineage and have maintained genetic similarity in
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morphology, physiology and ecological behaviour. Biological species and speciation processes in fungi are not always obvious as morphological differences between apparently closely related but distinct species might be minimal (i.e. so-called “sibling species”), due to the fact that fungi are not highly differentiated and structured organisms. In addition, among closely related taxa, and within taxa, there can be considerable variation in morphological characteristics such as spore dimensions, leading to misidentification. The key element in speciation is reproductive isolation, or mechanisms that restrict or prevent gene flow between populations. There are different forms of speciation and different elements contributing to it. The best-documented form is allopatric speciation in which different species develop through differentiation of populations in geographic isolation (Mayr, 1963, 1970). Over time, gradual changes and polymorphisms will develop among such populations. In contrast, sympatric speciation is a process by which populations inhabiting the same geographic range become reproductively isolated (Maynard Smith, 1966; Carson and Templeton, 1984). This requires a stable polymorphism between the divergent species. Less definite forms of speciation include parapatric and peripatric speciation. In parapatric speciation a new species arises from populations which maintain genetic contact during the entire process by a narrow zone of overlap and in peripatric speciation gene frequencies shift under the influence of genetic drift in small subpopulations on the periphery of the distribution of the parental population (White, 1968, 1978). Peripatric speciation is most likely to occur when new populations arise from a few founder individuals and no gene flow occurs between the isolates and the main population. The main forces contributing to speciation in plant pathogenic fungi are: (i) the level of genetic variation in the original population; (ii) natural selection through changes in the host species, leading to host specificity or even resistance gene specificity (e.g. gene-for-gene co-evolution) , or through environmental changes; (iii) reproductive isolation, which can either be geographically based (allopatric speciation) or genetically based, either preor post-zygotic, typically leading to sympatric speciation; and (iv) the genetic system, as reproduction in fungi can either be heterothallic, homothallic, asexual or combinations of these.
B. EVOLUTION OF HOST SPECIFICITY IN PHYTOPHTHORA
An interesting area of research concerns the evolution and origin of different Phytophthora species. Some Phytophthora species are host specific while other species have an extremely wide host range (Ribeiro, 1978; Erwin and Ribeiro, 1996). The evolutionary relationships presented in Fig. 2 provide some insights into the possible origins of the different Phytophthora spp.
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under discussion in this review. Questions that need to be considered are as follows. (i) Did hemibiotrophic Phytophthoru spp. evolve from necrotrophic species? (ii) Did host-specific Phyfophfhoru spp. evolve from the broad-host-range species? (iii) Did the host-specific Phytophthoru spp. co-evolve with their host plants or did sympatric speciation occur at a much later stage in the evolution of the pathogen? (iv) Did host specificity in Phyfophfhoru towards plant roots and foliage evolve from each other or independently? (v) Did homothallic Phytophfhoru spp. recently evolve from genetically diverse heterothallic species? (vi) Did hybridization between different Phyfophthoru spp. lead to new species? Each of these questions is discussed in the following paragraphs. 1. Did hemibiotrophic Phytophthora spp. evolve from necrotrophic species? Most plants are resistant to most pathogens (Agrios, 1988), which immediately draws attention to the fact that plants have developed a multitude of quite effective defence mechanisms. Necrotrophic fungi utilizing nutrients from plant cells they have killed usually grow intercellularly followed by intracellular invasion through dissolution of the host cell wall. They do not actively interact with their host and as a result do not generally show host specificity (Tippet et ul., 1976). Examples of this include P. cinnumomi, P. drechsleri, P. cryptogea and broad-host-range strains of P. meguspermu (Malajczuk and McComb, 1977). However, in the case of hemibiotrophic pathogens, growth is both inter- and intracellular, with intimate cell to cell interactions occurring between the plant and the pathogen. Most biotrophic and hemibiotrophic pathogens are specialized plant pathogens which have profound interactions with their hosts, and P. infesfuns is a good example of this type of pathogen (Cooke, 1977). Phytophthoru spp. which attack legumes, produce rudimentary haustoria and show host specificity include P. sojue, P. vignue and P. medicuginis (Stossel et ul., 1980; Miller and Maxwell, 1984; Ralton et ul., 1988). In order to become a hemibiotrophic or biotrophic pathogen, a fungus must contend with and avoid defence mechanisms in the host plants. In contrast to necrotrophs, biotrophic and hemibiotrophic pathogens must keep the plant alive while avoiding the plant’s defence mechanisms. This requires specific adaptations. Hence, biotrophic pathogens may require a higher level of specialization than broad-host-range pathogens. In order to cope with different defence mechanisms possessed by the different groups of plants, it could be argued that some pathogens have developed adaptations to avoid the defence mechanisms present in only particular groups of plants, thus leading to host specificity. Comparing sequence information from genes with major roles in pathogenicity and host specificity among a large number of Phytophthoru spp. can be used to gain a better understanding of the evolution of hemibiotrophic pathogens.
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2. Did host-specific Phytophthora spp. evolve from the broad-host-range species? In plant pathology it is generally believed that host-specific pathogens have co-evolved with their hosts in their centres of origin. Gene-for-gene co-evolution, leading to cultivar specificity, might also occur at species level, as suggested by Thompson (1990) who called this at the species level, “escape-and-radiation co-evolution”. Speciation in most plant species is considered allopatric. Speciation in the pathogen may follow that in the host plant, leading to specialization. Alternatively, the pathogen may maintain its ability to attack the plant’s progenitor and its descendants, thus broadening its host range (e.g. P. infestans which can infect many plant species of the Solanaceae, closely related genera and a few species belonging to rather distantly related genera) (Turkensteen, 1973). In both cases the evolution of pathogen species specialized towards a certain family of plants would be similar to evolution in the corresponding host-plant family. If speciation in the pathogen follows speciation in the host, and accepting Savile’s (1955) first principle of evolution, which states that new major groups do not arise from specialized climax groups but rather from unspecialized groups of greater genetic plasticity, then we would expect to be able to trace host specific Phytophthoru spp. back to a common ancestor which in the case of Phytophthora would probably be heterothallic with a broad host range. Modern examples of such species may be P. cinnamomi or P. drechsleri. rDNA based sequence information of a large number of Phytophthora spp. (Crawford et al., 1996) (Fig. 2 ) supports the hypothesis that host-specific Phytophthora spp. attacking legumes have evolved independently from different broad-host-range species as they do not form a separate evolutionary line as suggested by Hansen (1987). 3. Did the host-specific Phytophthora spp. co-evolve with their host plants or did sympatric speciation occur at a much later stage in the evolution of the pathogen? Sympatric speciation might occur frequently in host specific fungal pathogens where the mating occurs in the host and altered host specificity leads to reproductive isolation. This is supported by the fact that different Phytophthora spp. host specific towards distinct host plants can hybridize in the laboratory (Goodwin and Fry, 1994). Sympatric speciation may also arise when polyploidy occurs ldading to postzygotic reproductive isolation. With sympatric speciation a new species may arise almost instantaneously and may differ from the parental species in only a few genes. In this case the speciation in the host and in the pathogen would be following different evolutionary pathways. Once evolutionary relationships among host-plant species and pathogen species have been elucidated, they can be compared to test such a hypothesis.
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Many broad-host-range pathogens attack host plants with which they have not co-evolved. Australia, a relatively isolated island continent, has many excellent examples of this. For instance, P. cinnamomi, which is at least introduced to southern Australia, is pathogenic on many native species in this country (Weste, 1994). In this case, an introduced pathogen attacks many host plants, although they have evolved separately. Similarly, crop species which originated, evolved and were domesticated outside Australia are attacked by pathogens thought to be native to Australia. For example, P. macrochlamydospora which is only known to occur in Australia, is pathogenic on lupin and soybean, which have evolved in North America and China, respectively. 4. Did host specificity in Phytophthora towards roots and foliage evolve from each other or independently? Roots and leaves of plants have evolved under different physical and biological environmental conditions. Thus roots may differ from aerial plant parts in the ways in which pathogenic associations with fungi have evolved, and in the fungal adaptations required for successful pathogenicity (Pirozynski, 1983). Some species of Phytophthora are specialized towards attacking foliar plant parts, e.g. P. infestans vs P. medicaginis which is specialized towards attacking roots. Generally, foliage-infecting species of Phytophthora are more likely to produce papillate or semipapillate sporangia (e.g. P. infestans and P. colocasiae), whereas root-infecting species are generally non-papillate (e.g. P. cinnamomi and P. cryptogea). Foliage-infecting Phytophthora species (e.g. P. infestans) produce appressoria, but root-infecting ones such as P. medicaginis do not produce conspicuous appressoria (Miller and Maxwell, 1984; Dale and Irwin, 1991). Did the foliage attacking Phytophthora spp. have a common evolutionary lineage or did they evolve from different root attacking species on separate occasions? Alternatively, root-attacking Phytophthora spp. could have evolved from foliage attacking species. Once genes for appressorium formation have been cloned and characterized, remnants of their presence can be investigated in the root-attacking species pathogens thought to be native to Australia. For example, P. macrochlamydospora, which is only known to occur in Australia, is pathogenic on lupin and soybean, which have evolved in North America and China, respectively.
5. Did homothallic Phytophthora spp. recently evolve from genetically diverse heterothallic species? Breeding systems are important in speciation as they can determine the nature and extent of reproductive isolation. This is especially important in Phytophthora spp., as well as in higher fungi in general, where mating takes place inside the host plant. If an individual of one mating type of a
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heterothallic Phytophthora spp. mutates so that it can colonize a new host plant, it will lack the presence of the opposite mating type in this host plant to form sexual spores (i.e. oospores). Hence, there may be strong selection for asexual reproduction and selfing, ultimately leading to homothallism and genetic separation from the original heterothallic population. If a homothallic or asexually reproducing fungus finds a new host, it can almost immediately be reproductively isolated from the population from which it originated. Both these cases would implicate the occurrence of sympatric speciation. This immediately raises questions concerning the role of sexuality in the different Phytophthora species. Homo- and heterothallism in Phytophfhora spp. is not as absolute as commonly believed. Recent research has shown that homothallic species occasionally outbreed (Whisson et al., 1994), while heterothallic species show low levels of inbreeding (Brasier, 1972; Campbell et al., 1985; Shattock et al., 1986). The homothallic and host-specific P. medicaginis, P. megasperma and P. trifolii appear to be closely related to the P. cryptogea-P. drechsleri complex which is genetically diverse, heterothallic and non-host specific (Fig. 2). Along similar lines the genetically diverse and heterothallic P. cinnamomi has a broad host range, but is closely related to the homothallic and host-specific P. vignae and P. sojae. Most host specific homothallic Phytophthora species do not form a separate evolutionary cluster; instead, they appear to be individually more closely related to several different non-host specific heterothallic species. This provides evidence for the occurrence of sympatric speciation. DNA sequence data are rapidly becoming available to test if homothallic Phytophthora species have evolved from a few heterothallic and genetically diverse Phytophthora species. 6. Did hybridization between different Phytophthora spp. lead to new species? Different heterothallic Phytophfhora spp. can colonize the same host plant and thus interact with each other (e.g. P. palmivora and P. megakarya on cocoa). Oospores are readily produced when pairing different heterothallic Phytophthora spp. such as P. cinnamomi with P. drechsleri (Barrett, 1948), P. cryptogea (Waterhouse, 1950; Stamps, 1953) and P. palmivora (Cohen, 1950). Crosses between different Phytophthora spp. and analysis of the progeny has resulted in the identification of interspecific hybrids between P. capsici and P, palmivora (Boccas, 1981) and between P. mirabilis and P. infestans (Goodwin and Fry, 1994). Claims of interspecific hybridization not confirmed with unambiguous markers are ignored in this review. There is evidence to support the hypothesis that P. meadii, a pathogen o n rubber in India and Sri Lanka is an interspecific hybrid of P. palmivora and P. heveae (Sansome et al., 1991). Tetraploids of P. meadii are sexually fertile while the diploids are infertile, suggesting an allopolyploid (Sansome et al., 1991). Many native Australian plants, as well as some economically
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important crop species are susceptible to more than one Phyfophfhoraspp. For example, P. cinnamomi, P. drechsleri and P. nicofianaeare all pathogenic on Banksia, Eucalyptus and Ananas spp. (Simmonds, 1966; J. L. Alcorn, Queensland Department of Primary Industries, unpublished records). Hence, interspecific hybridization in the susceptible Australian flora might play a greater role than is commonly believed. With the advent of molecular taxonomy and recent breakthroughs in the genetics of Phyfophthora spp. it will become possible to study and compare the (co-)evolution of host and pathogen, evolutionary relationships between homothallic and heterothallic species, centres of origin, and genetics of host-specificity of Phytophfhora spp. This will undoubtedly lead to an increasing insight into the evolution and origin of Phytophfhora spp. in the near future.
VII. EVOLUTION OF CULTIVAR SPECIFICITY IN PHY TOPH THORA SOJAE Basic compatibility, the capacity of a pathogen to grow and cause disease in its host (Keen, 1982) has presumably evolved ahead of the development of gene-for-gene relationships conditioning cultivar specificity. It is reasonable to hypothesize that once basic compatibility has been established and a Phytophfhoru sp. has become a highly specialized hemibiotroph, it has abandoned its necrotrophic phase by relinquishing some of its general elicitors and extracellular enzymes so as to prevent or suppress defence mechanisms by the plant. The presence of resistance genes in the host plant with the ability to recognize specific elicitors of the pathogen resulting in the expression of a hypersensitive response (HR) (rapid cell death and restriction of pathogen colonization) in the plant are highly favourable for the host plant. Such plants will be subjected to positive selection pressure. In response, the pathogen with none or mutated corresponding specific elicitors, which trigger the HR in the host plant, will remain virulent and be subjected to strong positive selection pressure. This scenario forms the basis of the co-evolution of a gene-for-gene system between the plant and the pathogen, akin to an armaments race. In this situation, resistance genes in the plant and avirulence genes in the pathogen are acquired, fixed or lost in the population, and at any point in time in their co-evolution there will be a number of such genes actively and passively (“fossil genes”) present in the host and pathogen populations. Genetic evidence for a “gene-for-gene” correspondence came from Flor’s work on flax and its rust, Melampsora lini (Flor, 1942), where host and pathogen show complementary genic systems (Flor, 1956). The gene-for-gene interaction implies that incompatibility results from the interaction between the products of the dominant genes for resistance and avirulence in the host
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and pathogen, respectively (Day, 1974). Since Flor's pioneering work (Flor, 1942) this gene-for-gene correspondence, which requires parallel genetic studies in the host and the pathogen, has been reported, but not always tested, for a range of host-pathogen interactions, including endemic species in natural ecosystems (Thompson and Burdon, 1992). Modern agricultural practices, utilizing major genes with specific resistance, have allowed the ready detection of gene-for-gene relationships. In contrast to the suggestion by Hansen (1987), it seems unlikely that the genes themselves have been recently (in the 10000 years of agricultural practice) acquired. In haploid pathogens only a one base pair mutation is needed to change a dominant avirulent phenotype to a virulent one, as was recently demonstrated in Cladosporium fulvum (Joosten et al. , 1994). In fungi, which produce immense numbers of spores, such mutations rapidly give rise to new virulent races which can overcome resistance genes in the host plant. New virulent races in oomycetes can also occur through convergent evolution in different parts of the world, as recently demonstrated for P. sojae (Drenth et al., 1996). In the interaction between P. sojae and soybean, a strong gene-for-gene relationship exists for the resistance/avirulence loci studied to date (Whisson et ul., 1994, 1995). The question which one immediately asks concerns the origin of these genes. The simplest explanation is that, once basic compatibility had been established between soybean and P. sojue, this gene-forgene recognition system came into play. One can also speculate that the resistance genes and the avirulence genes, were present in progenitors of soybean and P. sojae, respectively, and may well have had, and still have, an additional function(s) to specific host-pathogen recognition. While both host and cultivar specificity are suggestive of long periods of co-evolution between host and pathogen, for P. sojue, and for that matter all other Phytophthora spp., there are still many unanswered questions. For example, the host specificity is not absolute, with P. sojue able to attack lupins, native to North America, in addition to soybean native to China (Jones and Johnson, 1969). One possible explanation is that it has, in fact, evolved with either soybean or lupins in either China or North America respectively, then transferred to the other host when the opportunity arose. This hypothesis can be tested by studying genetic variation of the pathogen in China, if the organism does in fact exist there. Irrespective of its centre of origin, we can speculate that considerable homology exists between genes in soybean and lupin which recognize P. sojae. Analogous situations exist in P. vignue, which is host specific to cowpea and adzuki bean, with apparent centres of origin in Abyssinia and Indo-China respectively, and P. medicaginis, with specificity towards alfalfa and chickpea. Functional processes leading to recognition of pathogens by plants are still not fully understood for any host-pathogen system involving Phytophthoru spp. The evolutionary relationships and origin of genes conditioning host specificity, and those conditioning cultivar specificity in different Phytoph-
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thoru spp. are, as yet, unknown. Answers will come when genes conditioning cultivar specificity have been cloned and characterized, and functions found for them.
VIII. CONCLUDING REMARKS Understanding the origins of Phytophthoru spp. has importance from a number of perspectives. It provides insights into speciation processes, as well as applied relevance with respect to sources of resistance, particularly if a long history of co-evolution is expected. On the basis of present evidence, it is not unreasonable to suspect that long periods of co-evolution between plants and host specific Phytophthoru spp. are common. Use of molecular markers and DNA sequence information has contributed substantially to our understanding of population genetic structure and evolutionary relationships between Phytophthoru spp. We can conclude that P. sojue, P. medicuginis and P. vignae have been introduced into Australia, probably on very few occasions for each organism. The technology is available to allow us to accurately deduce the geographic origins of most Phytophthoru spp. and this has the potential to lead to improved levels of disease control through identifying new sources of host resistance from these regions. For hosts with monogenic resistance to Phytophthoru, such as soybean and cowpea, more extensive searches for sources of host resistance should be done in China and Abyssinia, respectively. Further population genetic research is needed on other Phytophthoru spp. to identify centres of diversity. For P. clundestina a closer look in the Mediterranean areas (e.g. Sardinia) where subterranean clover has evolved is needed. Recent breakthroughs, made possible by using molecular markers, in making controlled crosses between different races of homothallic Phyrophthoru spp. will lead to an increased understanding of Phytophthora genetics. Map based cloning of cultivar specificity genes, assisted by the relatively small genome size (62 Mbp for P. sojue, Mao and Tyler, 1991) is now feasible. When genes involved in essential recognition events, in particular, appressorium formation, host and cultivar specificity, are cloned and characterized, they will provide vital clues concerning the origin and evolutionary relationships between the different Phytophthoru spp. DNA sequence data and genetic crosses between different heterothallic Phytophthoru spp. will determine whether the current morphologically based classification system accurately reflects the underlying evolutionary relationships between the different species. We have earlier indicated the possibility that papillate Phytophthoru spp. are more evolved and closer to the downy mildews than the non-papillate species. Cloning of genes encoding sporangium papillation and searching for homologous sequences in the downy mildews and Pythiums could provide a means for testing Gaumann and Wynd’s (1952) hypothesis that the downy mildews had evolved from Pythium.
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The evolution and significance of different modes of reproduction in fungal evolution is not clear. Did host-specific and homothallic Phytophthoru spp. evolve from genetically diverse heterothallic species through sympatric speciation? Once the evolutionary relationships between the different Phytophthoru spp. have been elucidated such questions can be answered. Sporangial papillation is of greater evolutionary significance in Phytophthoru than amphigyny or paragyny of the antheridium (Crawford et ul., 1996). Currently in plant pathology a large research effort is directed towards cloning and characterization of virulence and resistance genes. This will undoubtedly lead to an understanding of how virulence and resistance genes work and how they evolved. Only when the interactions at the molecular level between host and pathogen have been elucidated can hypotheses concerning the evolution of cultivar specificity be rigorously tested. Current questions, such as whether cultivar specificity in different Phytophthoru spp. is conditioned by functionally different or similar genes, can then be answered. The next logical step would be to study the mechanisms and evolution of non-host resistance in plants and host-species specificity in pathogens. Could it be that host-species specificity in some cases is conditioned by gene-for-gene interactions, as suggested by Thompson (1990), similar to co-evolution of cultivar specificity? As the molecular and biochemical basis of host-species specificity is unknown, strategies for cloning genes with uncharacterized products must be utilized, such as chromosome walking or transposon mutagenesis. The cloning of cultivar and host-species specificity genes will allow investigations on the biochemical basis of specificity. This may lead to novel and more durable strategies for disease control. Gene-for-gene interactions in plant pathogens are the most rigorous demonstrations of co-evolution between antagonistic species. Hence, plant pathology has the potential to significantly contribute to the understanding of co-evolution and speciation in evolutionary biology.
ACKNOWLEDGEMENTS We thank S. C. Whisson for comments on earlier drafts of this manuscript, Ms G. Wagels for technical assistance and M. E. Riedlinger for help with construction of the life cycle diagram.
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INDEX
A AAtDB database, 234-5 Absidia glauca, 412-15, 423 Absidiu parricida, 412, 415 Absidia spinosa, 414 Achlya, 354, 358, 383, 388 Achlya bisexualis, 441 Acremonium, 176 Acremonium-Festuca system, 175 Actinomycin D, 358 Adenylate cyclase, 118 Agrichemicals, 185 Agricultural systems, fungal biology in, 1834 Agriculture, evolution, 184-6 Agrobacterium radiobacter, 416 Agrobacterium rhizogenes, 417 Agrobacterium tumefaciens, 48-51, 234, 401, 416-18, 420 Albugo, 205, 322. 354 Afbugo candida, 228, 235-8, 240-3, 265 Alcaligenes eutrophus, 405, 407 Allomyces, 357 Allozymes, 337, 340 Alternaria solani, 125 Amycolatopsis, 405 Anacystk nidulans, 403 Ananas, 448 Anastomoses, 400 Anenome, 322 Anguillospora longissima, 278 Anthoxanthum odoratum, 74 Antirrhinum, 133 Antirrhinum majus, 126 Aphanomyces, 235, 354, 361, 386 Aphanomyces euteiches, 382, 387 Apoplastic environment, 314-16 Apoplastic loading, pathway implications, 321-2
Arabidopsis, 101, 102, 107, 109, 111, 135, 136, 141, 153 Arabidopsis thaliana, 126, 227-73 accession Columbia, 260 accession Wassilewskija, 261-2, 263 downy mildew, 258-9 future research, 262-8 genetic analyses, 233-5 genetic analyses of RPP and RAC loci, 243-6 genetic map, 258 interaction phenotypes, 241 MRC regions, 265 mutations, 259-62 rebirth in plant pathology, 231-2 relevant trends in modem biology, 230 research arena, 229-33 symbiotic relationships with micro-organisms, 228 Arachis, 319 Asexual life cycle of Peronosporales, 362-4 of Phytophthora, 436-7 of Saprolegniales, 366-9 Asexual reproduction in fungi, 80-1 Asexual sporulation in fungi,72-3 of oomycetes, 353-98 AspergiUus, 40, 75, 422 Aspergillus nidulans, 73, 77, 81, 357, 358, 410, 421 Aspergillus niger, 124-6, 421 ATPIGTP, 116, 119 ATPase activity, 199, 202-3 ATPase deficiency coupled transport mechanism, 320 Atriplex, 322 Avirulence, 231 Avirulence determinants
458
INDEX
interaaing with resistance genes encoding proteins mntaining extracytoplasmic LRRs, 97-9 interacting with resistamx proteins containing cytopiasmic LRRs, 113-14 AWJL proteins, 131-3 Azosporillum, 293 Azotoboacr vinflandii, 403
B BaciUw, 336,337 Bacillus lichmifrmis, 343 B a d u s subtih, 403,407 Bacteria as recipients of foreign DNA,
401-10 Banksia, 448 Beta, 319 Bogeochemistry, 179 Biomass estimation, 289-93 Biomass measurement in planta, 45-7 Biotroph-invaded cells, 210-18 Biotrophic fungal parasites, 195-225 Biotrophic pathogens, 312 Biotrophic plant pathogenic fungi, growth habits, 312 Biotrophy, strategy for, 311-13 Blnstoc&dieUa, 357 Bodrrops jararaca, 101 BotryL cinema, 124-5, 126 Bmdyrhizobium, 336 Bmica, 319, 320, 422 B m k a napus, 40-1, 421 Brassica napus L.subsp. oleifera, 33, 46 BrassiCa oleracea, 38, 262, 265 Bremia, 322,354 Bremia lactucae, 18, 80, 172, 266 C
CAMP 78 Cardatnine pratense, 266 Cell death, 210-18 cellular mechanisms, 216-18 following cell penetration, 211 in host and non-host plants, 212-14 programmed, 214-16 Cellular interactions between plants and biotrophic fungal parasites, 195-225 in symbiotic association, 233 Cellular rearrangements in parasite invasion,
206-10 Cerebrocides, 78
Chaetocladium, 415 Chaetocladium brefedii, 412 Chaetomium globosum, 80 Chalcone synthase (CHS), 174 Chenopodium, 322 p-Chloromercuribenzene sulphonate (PCMBS), 323, 324 Chromosomal DNA, 405 Chromosome number, 180 Citrobacrer freundii, 407 Cladosporium cucwnberinum, 179 Cladosporium fulvum, 91, 97, 172, 196, 213,
217, 313, 320, 326, 449 Clarkia ungulata, 421 Claviceps purpurea, 313,316 Clonal populations, 34&3 Clostridium acetobutylicum, 407 Clusters, 254-6 Cochliobolus heterostrophus, 52, 206 Colicinogenic factors 400 Colletotrichum, 171, 173 Colletotrichum coccodes, 176, 180 Colletotrichum gloeosporioides, 172, 176 Colletotrichurn lagenarium, 127 Colletotrichum lindemuthianum, 124, 127,
179, 196-7, 205-8, 311 Colletotrichum magna, 172, 178, 180, 181,
182, 185 Colletotrichum musae, 173, 176 Colony-forming units, 293, 296 Commelina, 318 Conjugation, 408 Cronartium quercuum f. sp. fusiforme, 198 Crotalaria, 432 Cruciferae, 238 Cryphonectria parasitica, 7, 10, 16, 19 Cucumber plants, 179 Cucurbita, 318 Cutinase, 41, 47 Cycloheximide, 358 Cyst coat vesicles, 375-6 Cyst-wall proteins, 138 Cytokinesis, 357 Cytokinins, 47-51 biochemical analysis, 49 fungal-derived, 63 implications for disease control, 51 molecular analysis, 49-51 role in pathogenicity, 51 Cytoplasmic LRRs, 155 activation of plant defences by resistance proteins containing, 114-19
459
INDEX avirulence determinants interacting with resistance proteins containing, 113-14 gene encoding protein of unknown function with, 137-8 required for resistance gene to function, 109-12 resistance genes encoding proteins with, 101-19 Cytoskeieton, changes in, 210
D DEFICIENS, 133 Deleterious cytoplasmic genomes (DCGs), 74 Desmodium, 432 Desmodium intortum, 440 Deuteromycotina, 78 Dictyuchus, 354 Dicyma parasitica, 412 Dikaryotic rust infections, 319 Dip-stick assays, 282-3 D m l l resistance gene, 18 DNA-degrading bacteria, 402 DNA-degrading enzymes, 402 DNA-DNA hybridization, 342 DNA fingerprinting, 337, 438 DNA fragments, 400,423 DNA modification, 415 DNA molecules, 400,411 DNA restriction fragment polymorphisms (RFLPs), 337 DNA sequence, 180, 450 DNA sequence analysis, 186, 337 DNA transfer, 405, 410, 416 Dorsal vesicles, 375-6 Dot-blot assays, 282-3 Dothiorella gregaria, 125 Double-antibody-sandwich (DAS) assays, 281 Double-stranded DNA, 403 Downy mildew infections, 258-9, 319 interception strategy, 322-3 Drosophila, 115 Drosophila melanogaster, 423 Drosophila Toll protein, 108-9 DRTIOO gene, 126-7
E Ebelactones A and B, 41 Ecosystem dynamics, 182-3 Ecotype, 229 Electromorphs (ETs), 338, 344
Electrophoretic markers, 337 Em05,250 Emoy2, 244-8 Encystment vesicles, 375-6 Endomembrane system, parasite-induced changes in, 20'7-9 Endophytic pathogens, 313 Endoplasmic reticulum (ER), 199, 208-10, 321
Endopolygalacturonase (E&W), 41 Endopolygalacturonam (PGs), 12U E n m o e b a histolycica, 421 En@robacter f a e d i s , 407, 409 Environmental factors, 179 Enzyme-linked immunosorbent assays (ELISA), 277, 280-2, 290-3, 2% Epidemiology and population genetics, 13-24 EPS (exopolysaccharide), 418-19 Erysiphe, 198-202 Erysiphe cichoracearum, 216 Erysiphe cruciferarum, 265 Erysiphe graminis, 50, 2.02, 321 Erysiphe graminis f. sp. hordei, 11, 13, 17, 217 Erysiphe p h i , 315 ESAG4 gene, 118 Escherichia coli,40, 126, 337, 341, 342, 400, 401, 406,407, 421, 422 Eucalyptus, 448 Eukaryotes, evolution of LRR proteins, 138-41 Eupenicillium, 75 Expressed meiotic prophase- repeat (EMPR) sequence, 131, 133 Extracellular DNA, 402,403 Extracellular fungal enzymes, 180 Extracellular matrix (ECM), 120, 126 ExtraceIlular proteins (ECPs), 326 Extracytoplasmic LRRs avirulence determinants interacting with resistance genes encoding proteins containing, 97-9 defence-related genes encoding proteins with, 119-31 genes encoding proteins of unknown function with, 131-7 potential patterns of glycosylation, 147-50 resistance genes encoding proteins with, 91-101 structure and interactions, 147-50 Extrahaustorial matrix, 319
460
INDEX
Extrahaustorial membrane (EHM), 198-202,
207-9, 213, 321 maintenance, 199-202
F False-negative results, 298-9 False-positive results, 298 Festuca, 176 F-factor, 400 FZL2 gene, 126, 133 Flagellar axonemes, 385-6 Foreign DNA bacteria as recipients of, 401-10 fungi as recipients of, 4 1 N 6 plants as recipients of, 416-20 Free DNA, 402,403 Fulvic acid (FA), 285 Fungal antigens, extraction from soil, 283-6 Fungal life-styles, 169-93 crossroads, 179-83 Fungal plant endophytes, 1745 Fungal plant pathogens, 171-4 Fungi asexual and sexual reproduction, 71-87 comparisons of genetic variation, physiological costs and fitness between asexual and sexual systems, 73-4 density and competition, 77-8 effect of mycelial extracts and specific morphogens, 78-80 genotype-determined equilibria, 76 in soils, 275-308 initiation of asexual sporulation and sexual development, 72-3 maintaining and changing balance between reproductive processes,
74-80 physical and nutritional factors, 77 seasonally maintained equilibria, 77 species-determined equilibria, 75 trade-off between asexual and sexual reproduction, 80 Fungicide resistance, 19-21 Fusarium, 74, 75, 181, 281, 283 Fusarium graminearum, 76 Fusarium moniliforme, 124, 125 Fusarium oxysporum, 7, 12, 125 Fusarium oxysporum f. sp. lycopersici, 107,
125 Fusarium solani f. sp. piri, 41 Fusion biotrophs, 401
G Gaeumannomyces graminir, 278 /3-Galactosidase, 45 Gametic disequilibrium, 11, 17-18 Gametic phase disequilibrium, 11 GDP, 116 Gelasinospora reticulospora, 80 Gene deployment, 17 Gene flow, 15-16 Gene transfer by free DNA, 402 conjugational, 401, 407 horizontal, 399-401, 408, 409 interkingdom, 420-2 lateral, 399401, 422-3 Genetic markers, 10-13 predicting variation among individuals, 11 restrictions on using, 12 Genetic modifications, 180 Genetic structure, 340-2 Genotypes, direct tracking, 13-15 Genotypic variation, 181 Geranium, 322 Giardia lamblia, 138 Gleotinia temulenta, 76 Glomerella cingulata, 76 Glomerella magna, 172 Glomerella musae, 173 Glomus occultum, 283 P-Glucuronidase (GUS), 45, 58 Glycine, 319, 432 Gram-negative bacteria, 402-5 Gram-positive bacteria, 400, 403-5 “Green islands”, 48, 50 GTPase, 115, 116 GTPase activating proteins (GAPS), 116 Guanine-nucleotide dissociation inhibitors (GDIs), 116 Guanine-nucleotide exchange factors (GEFs), 116
H Haemophilus, 337 Haemophilus infiuenzae, 341, 342 Haustoria, 309-33 alternative functions, 325-6 evolutionary aspects, 324-5 research priorities, 327-8 roles of, 2045 in transfer intercept, 316-25 Helianthus, 319 Hemibiotrophs, 196-7, 205, 207, 311
461
INDEX Heterodera avenae, 102 Homologous DNA, 403 Human placental ribonuclease inhibitor (HRI), 1.50-1 Humic acid (HA), 285 Humic substances, 285 Humin, 285 Hybridoma cell lines secreting specific antibodies, 279 Hybridoma technology, 277, 301 Hydrophilic amino terminus, 137 Hydroxyproline-rich glycoproteins (HPRGPs), 127 Hypersensitive response (HR), 99, 174, 211, 214-18, 448 I Immunoblotting technique, 301, 302 Immunofluorescence (IMF) assays, 280 Immunogens, selection and preparations, 278-9 Immunoglobulins, IgA, IgD, I@, IgG and IgM, 279-80 Impatiens capensis, 81 Inbred sets, 235 Index of association, 341 Indigofera, 432 Indirect-magnetic microsphere-enzyme irnmunoassay (MM-EIA), 282 Indole acetic acid, 78 Inoculum sources, 14-15 Inorganic solutes, 315-16 Intercellular hyphae role in transfer intercept, 316-25 vascular association, 317-18 Interleukin-1 receptor ( I L l R ) , 108-9, 115 Intracellular structures, 198-206 associations with plant nucleus, 209-10 Intron arrangements, 141-3 Ipomea, 318 Ipomoea, 76
K Kinase associated protein phosphatase (KAPP), 135 Klebsiella pneumoniae, 419 Kor A , 406 Kor B, 406
L Lactuca, 319
Lagenidiales, zoospore components throughout asexual life cycle, 368-9 Lagenidium, 386, 387 Lagenidium callinectes, 385 Lagenisma coscinodisci, 376, 377 Lamium purpureum, 322 Lathyrus, 322 Legumes, 431-56 in Australia, 432-3 Rhizobium interactions with, 418 Leishmania major, 141 Leucine-rich repeat (LRR) proteins. See LRR proteins Leucine-rich repeat proteins (LRP), 131 Leveillula, 312 Linoleic acid, 78 Linum usitatissimum, 108 Lotus, 432 LPS, 419 LPS formation, 416 LRR extensins, 127-30 LRR proteins, 89-167 activation of plant defences by resistance proteins containing cytoplasmic, 114-19 consensus amino acid sequences, 139 evolution, 138-45 evolution of different specificities, 1 4 3 4 interactions with ligands, 150-3 resistance genes encoding proteins with cytoplasmic, 101-19 resistance genes encoding proteins with extracytoplasmic, 91-101, 99-101 and molecular specificity, 144-53 see also Cytoplastic LRRs; Extracytoplasmic LRRs Luciferase, 45 Lupinus angustifoius, 440 Lycopersicon esculentum, 320
M Magnaporthe grisea, 12, 19, 61, 76, 172, 196, 206 Magnetic bead assays, 281-2 Major histocompatibility complex (MHC) class I1 transcriptional activator (CIITA), 114 Mastigonemes, 377 Meiosis, 180 Melampsora lini, 108, 213, 216, 448 Membrane assays, 282-3 Mesophyll cells, 318
462
INDEX
Metapopulation concept, 345-7 Methyl esterase activity, 41 Michaelis Menton model, 297 Micromonospora, 405 Minor vein closed type, 319, 320 open type, 318 Molecular analysis of Pyrenopeziza brassicae cytokinins, 4S51 Molecular biology, 32 Molecular genetics of natural variation, 243-59 Monoclonal antibodies, 275-308 assay formats, 280-3 choice of subclasses, 279-80 immunological estimation of colony-forming units, 293-5 interference from soil components, 286 production of species-specificand genus-specific, 278-80 sample preparation, 283-7 thresholds for detection and quanta1 assay systems, 296-8 use for biomass determination, 289-93 use in detection of fungi in soils, 287-9 use in quantification of fungi in soil, 289-300 visualization of temporal and spatial growth characteristics of soilborne fungi, 300-2 Morphological markers, 337 Mouse-ear cress, 227-73 MRC regions, 256, 264-5 Mucor, 415 Mucor-like zygomycetes, 412 Multilocus enzyme electrophoresis (MLEE), 337, w 2 , 344 Multilocus population structure, 17, 19 Mycosphaerella graminicola, 16, 345 Mycosporines, 78 Myrogenospora atramentosa, 312 Myxoma virus, 10
N NADPH oxidase, 118 Natural ecosystems, fungal biology in, 1 8 H Natural variation, molecular genetics of, 243-59 Nectria haematococca, 172 Neisseria, 337 Neisseria gonorrhoeae, 341, 343, 405 Neisseria meningitidis, 341
Neurospora crassa, 56 Neutral theory, 338-40 Nicotiana glutinosa N , 101, 108 p-Nitrophenylbutyrate, 41 Nonallelism, 248-54 Nuclear DNA cleavage, 214-15 Nutrients, biotrophic accumulation, 313-16 Nutrition, strategy for, 311-13 0
Olpidium, 313 Oomycetes, 227-73 asexual sporulation, 353-98 flagellar apparatus, 354 pathogenicity, 354 taxonomy, 354 zoospores, 354-7 see also Sporangiogenesis; Zoosporogenesis Oomycetous parasites, 198-206 Oospores, 23 formation, 16-17 Open-reading frames (ORFs), 42, 131-2 Ophiosfoma novo-ulmi, 14, 19 Organic solutes, 114-15 Orobanche crenata, 313
P pAN7-1, 45 Parasite-induced changes in endomembrane system, 207-9 Parasite invasion, defensive responses, 206-7 Parasitella, 415 Parasitella parasitica. See Parasitella simplex Parasitella simplex, 412, 413 Parasitic symbiosis, Copernican-like perspective, 232-3 Pathogenesis-related (PR)genes, 119 PCMBS, 323, 324 Pectin methyl esterase (PME), 41 Penicillia, 75 Penicillium expansum, 125 Peripheral cisternae, 384-5 Peronospora, 311, 322, 354 Peronospora destructor, 80 Peronospora effusa, 322 Peronospora parasitica, 80, 109, 111, 228, 235-8, 24&5, 247, 250, 252, 254, 255, 258, 260, 262, 266, 267, 412-15, 423 Peronospora pisi, 200 Peronospora schleideniana, 80
INDEX Peronospora tabacina, 14, 266 Peronospora viciae, 204, 317, 322-4 Peronosporales, occurrence of zoospore components throughout asexual life cycle, 362-4 Petunia infiata, 136 pH effects, 316, 320, 321 Phaseolus, 319, 320 Phaseolus vulgaris, 179, 321, 414 PHAST protein markers, 59 Phenotypic variation, 181 Phenotypic variation among incompatible interactions, 2@3 Phenylalanine ammonia lyase (PAL), 174 Phloem loading and interception strategy, 318-25 Phosphotransferase, 409 Phyllactinia, 312 Phyllactinia guttata, 312 Phylogenetic structure, 342-3 Physopella zeae, 204 Phytophthora 78, 217, 279, 288, 311, 358, 361, 370, 372, 375, 376, 377, 379, 380, 386, 387, 388, 389, 431-56 asexual life cycle, 43&7 biological species and speciation, 442-3 evolution of host specificity, 443-8 evolution of species, 442-3 evolutionary relationships between species attacking legumes, 440-2 genetic variation and possible origins of species attacking legumes, 437-40 sexual life cycle, 437 taxonomy, 433-6 Phytophthora cactorum, 370 Phytophthora capsici, 447 Phytophthora cinnamomi, 282,284,287,355, 356,357,360,365,370,372,373,375, 376,377,379,380,381,383,386,388,
441-2,444,445,446,447,448 Phytophthora cryptogea, 442, 444,447 Phytophthora drechsleri, 442, 444, 445, 447, 448 Phytophthora heveae, 447 Phytophthora infestam, 12, 15, 16-17, 19, 199, 200, 207, 210, 211, 216-18, 235, 266, 311, 353, 385, 444,446, 447 epidemiology and populations genetics, 214 genotypic diversity, 23 late blight management, 22-4 lineages, 23
463
population structure, 21-2 Phytophthora macrochlamydospora, 440, 442,446 Phytophthora meadii, 447 Phytophthora medicaginis, 439, 444,446, 447, 449, 450 Phytophthora megakarya, 440, 447 Phytophthora megasperma, 172, 370, 441-2, 444,447 Phytophthora mirabilis, 447 Phytophthora nicotianae, 365, 448 Phytophthora palmivora, 359, 370, 379, 381, 388, 440, 447 Phytophthora parasitica, 385 Phytophthora sojae, 438-9, 440, 441-2, 444, 449, 450 evolution of cultivar specificity, 448-50 life cycle, 436 Phytophthora trifolii, 447 Phytophthora vignae, 439-40, 441-2, 444, 450 Piptocephalis, 415 Pisum, 319, 320 Pisum sativum, 323, 324 Plant defence activation signal, 120 Plant-haustorium interfaces, 198-9 Plant nucleus, 209-10 Plant-parasite interface, solute transport, 202-4 Plasma bridges, 401 Plasmodiophora, 313 Plasmodiophora brassicae, 279, 421 Plasrnopara, 322, 354, 375 Plate-trapped-antigen (PTA) assays, 281 PMSF, 41 Polygalacturonase-inhibitingproteins (PGIPs), 90,120-7, 150-1 Polygalacturonases (PGs), 150-1 Polygonum viviparum, 81 Polymerase chain reaction (PCR), 11, 41, 337, 400 Polyvinylidene dilluoride (PVDF), 282-3 Polyvinyl chloride (PVC), 2846 Population characteristics varying among,l8-19 concept of, 5-8 definition of, 6 genetic variation, 8-10 predicting variation among, 13 Population genetics and epidemiology, 13-18, 19-24 bacterial, 335-51
464
INDEX
Population genetics and epidemiology cont’d historical background, 338-40 neutral theory, 338-40 contribution to epidemiology and plant disease management, 5-19 evolutionary inferences, 1S18 fundamental concepts, 5-19 history, 4 use of term, 3-4 variation in, 337-8 Population structure, 340-5 multilocus, 17, 19 Porcine ribonuclease inhibitor (PRI), 144-7 Potato, 211, 216, 217 Potential leucine zippers, 102-8 PPROTl, 42-5 Protease, 42-5, 47 Protease production, 41 Protein profile analysis of developmentally regulated genes, 57-8 Protein synthesis, 358 Protozoan proteins, LRR consensus sequences, 140 Psathyrella, 77 Pseudocercosporella herpotrichoides, 287 Pseudomonas, 344, 345 Pseudornonas Puorescens, 346 Pseudomonas savastanoi, 48, 49 Pseudomonas syringae, 110, 113, 153, 260, 265, 342 Pseudomonas syringae pv. maculicola, 102, 111 Pseudomonas syringae pv. tomato, 91, 101, 102, 109, 111, 342 Pseudoperonospora, 354, 379 Psoralea, 432 Puccinia, 208 Puccinia graminis, 317 Puccinia graminis f. sp. tritici, 6, 14, 17, 19, 74 Puccinia graminis tritici, 213 Puccinia hordei, 315, 317, 320 Puccinia poarum, 200 Puccinia recondita, 326 PVDF, 302 Pyrenopeziza brassicae, 32-3, 77, 78 analysis of sexual morphogenesis, 5 1 4 0 biochemical analysis of cytokinin production, 49 biochemical analysis of sexual morphogenesis, 52-3 cloning of mating-type loci, 56
complementation of developmental mutants, 5 3 4 disease dissemination, 38 disease epidemiology, 34-8 heterogeneity of resistance, 38 life cycle in planta, 61 mating-type loci and pathogenicity, 56-7 molecular analysis of cytokinins, 49-51 molecular analysis of sexual morphogenesis, 5 3 4 0 molecular life cycle, 61 pathogenesis, 33 pathways of development, 36 protein profile analysis of developmentally regulated genes, 57-8 sexual morphogenesis in, 36, 58-60, 63-4 Pyrenopeziza brassicae-Brassica interaction, 31-70 analysis of hemibiotrophic phase, 47-51 implications for disease control, 47 molecular analysis of pathogenesis, 40-1 molecular techniques in analysis, 38-9 molecular view, 604 proposed role of extracellular protease in pathogenicity, 47 role of cutinase, 41 role of cytokinins, 47-51 role of protease, 42-5 sexual morphogenesis, 33-4 subcuticular growth, 42-5 surface growth and penetration 41 Pyrenophora teres, 19-21 Pythium, 267, 288, 311, 353, 361, 370, 372, 375, 376, 377, 379, 386, 387, 389, 442 Pythium aphanidermatum, 279, 370 Pythium middtetoni, 381, 382 Pythium profiferum, 381 Pythium sulcatum, 281 Pythium ultimum, 279 Pythium violae, 281
R R-genes, 263-4 Random amplified polymorphic DNA (RAPD), 10, 11, 13, 20, 438 Receptor-like protein kinases,l35 Receptor serinelthreonine kinases, 90 Recognition specificities, 239-40, 242 Recombinant sets of inbred lines, 235 Recombination, 16-18, 20 in bacterial populations, 340 within and between populations, 342
INDEX Replicating DNA, 416 Reporter genes, 45 Resistance, dual nature, 231-2 Resistance genes, 232 concept of, 231 encoding protein with cytoplasmic LRRs required for resistance gene to function, 109-12 encoding proteins containing extracytoplasmic LRRs, 99-101 encoding proteins with cytoplasmic LRRs, 101-19 encoding proteins with cytoplasmic LRRs and homology to the cytoplasmic domains of Toll and interleukin-1 receptor, 108-9 encoding proteins with extracytoplasmic LRRs, 91-101 Resistance proteins containing cytoplasmic LRRs activation of plant defences by, 114-19 avirulence determinants interacting with, 113-14 Resistance transfer factors (RTFs) 400 Restriction fragment length polymorphisms (RFLP), 12, 14, 342, 438 Reverse transcriptase (RT) PCR 132 Rhabdocline-Pseudotsuga system, 175 Rhizobium, 336, 337, 341, 343, 432 interactions with legumes, 418-20 Rhizobium leguminosarum, 344, 406 Rhizobium meliloti, 343, 406 Rhizobium trijolii, 420 Rhizoctonia, 276, 288, 411 Rhizoaonia solani, 280, 281, 282, 286, 291, 292, 294, 296, 297, 298, 300, 301, 302, 303 Rhizosphere, horizontal gene transfer, 399-429 Rhynchosporium seculb, 13, 111 Ribonuclease A (RNase A), 151, 152 Ribonuclease inhibitors (RI), 150-3 Ribonucleases, 150-3 RNA synthesis, 358 S Saccharomyces cerevkiae, 118, 135, 407, 422 Saccharopolyspora , 405 Salmonella, 337, 341, 342 Saprolegnia, 354, 356, 361, 370, 373, 375, 376, 383, 386, 388 Saprolegnia diclina-parasitica, 370
465
Saprolegnia ferax, 370, 381 Saprolegnia parasitica, 370 Saprolegniales, occurrence of zoospore components throughout asexual life cycle, 366-9 Saprophytes, 1 7 6 8 Schizosaccharomyces pombe, 135, 407, 422 Sclerospora, 354 Sclerotinia, 288 Sclerotinia sclerotiorum, 9, 11, 12, 126 Senecw, 319, 320 Senecio vulgaris, 74 Serinehhreonine protein kinase, 109 Serinehhreonine protein phosphatase, 135 Serratia marcescens, 400,409 Sexual development in fungi, 72-3 Sexual life cycle, Phytophthora, 437 Sexual morphogenesis analysis of, 51-60 GUS as tool to study, 58 MAT genes in, 60 P. brassicae, 36, 51-64 P. brassicae-Brassica interaction, 33-4 Sexual reproduction, 16, 23, 80-1 SF17, 137 Sinapb alba, 421 Soil organisms, relationships between, 400 Soils extraction of antigens, 283-6 fungi in, 275-308 Sordaria brevicollis, 80 Spatial structure, 343-5 Sphaerotheca fuliginea, 311 Spongospora subterranea, 279, 287 Sporangiogenesis cyst coat vesicles, 375-6 dorsal vesicles, 375-6 encystment vesicles, 375-6 induction, 357-9 K-bodies or ventral vesicles, 373-5 large peripheral vesicles or fibrillar vesicles, 365-73 mastigonemes, 377 morphological development, 359-61 synthesis of zoospore-specific components during, 361-77 Stagonospora nodorum, 14 Staphylococcus aureus, 409 Sterol biosynthesis-inhibiting fungicides (SBIs), 19-21 Streptococcus, 403 Streptococcus lactis, 407
466
INDEX
Streptococcus pneumoniae, 403 Streptomyces, 402,405,4&i Streptomyes fiadiae, 409 Streptomyces lYpmnii, 421 Streptomyes l i v i h , 282 Streptomycetes, 400,405 Symbiosis, 230 three Rs (recognition, response and reproduction), 238-40 Synechocysh PCC 6830, 407 Systemic acquired resistance ( S A R ) genes, 119
Venturia inaequalis, 7, 50 Vicia, 319, 320, 322 Vicia narbonensis, 313 Vicia sativa, 323 Vigna, 432 Viroid-induced LRR protein 131 Virulence genes 337
W Water expulsion vacuoles, 384 Wheat, 131-3
T Taphrina, 313 T-DNA, 417 Temporal structure, 343-5 Tmagonwmyces uliginosus, 411 Thermonospora, 405 Thielavwpsis baricola, 280 Thraustotheca, 375 Ti-plasmid, 416, 418, 420 Tobacco mosaic virus, 108 Tomato plants, 180 Transduction, 408 Transfer intercept infections, 316-25 Transformation, 408 Transforming DNA, 403 Transposition, 408 Trichoderma, 77,276 Trichoderma harzianum, 278, 279 Trifolium parvijlorum, 420 Trigonella, 432 Trisporic acid, 78 Trypanosoma brucei, 141 Trypanosoma equiperdum, 141 Twilago farfara, 74 Type I1 error, 298 U
Unguicularia dr. raripila, 56 Uromyces appendiculatus, 204, 213 Uromyces vicae-fabae, 205, 320 Uromyces vignae, 199, 207-9, 213, 217-19 Ustacystis waldsteiniae, 208 Ustilago maydis, 172
V Variovorar paradoxus, 405 Vascular infection, 319 Vascular tissue, intercellular hyphae in association with, 317-18
X Xanthomonas, 98 Xanthomonas campestris pv. malvacearum, 98, 111 Xanthomonas campestris pv. vesicatoria, 98, 111 Xanthomonas campetris, 265 Xanthomonas citri, 98, 111 Xanthomonas oryzae, 137 Xylem-to-xylem contacts, 313
Y Yeasts 401 2 Zea mays, 321 Zearalenone, 78 Zeatin riboside (ZR), 49 Zoospore discharge, 386-9 Zoosporogenesis, 357 cortical cleavage vacuole, 381-2 flagellar axonemes, 385-6 induction, 377-8 involvement of large central vacuole, 381 involvement of sporangial plasma membrane, 381 patterns of cleavage membrane formation, 37883 peripheral cisternae, 384-5 polarization of zoosporic organelles, 386 progressive extension of cleavage vacuoles around or between nuclei, 382-3 spatial regulation of cleavage, 383-4 synthesis of zoospore-specific components during, 384-6 vesicles, 379-81 water expulsion vacuoles, 384