BIOTECHNOLOGY INTELLIGENCE UNIT 3
Dominique Michaud
Recombinant Protease Inhibitors in Plants
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BIOTECHNOLOGY INTELLIGENCE UNIT 3
Dominique Michaud
Recombinant Protease Inhibitors in Plants
BIOTECHNOLOGY INTELLIGENCE UNIT 3
Recombinant Protease Inhibitors in Plants Dominique Michaud, Agr., Ph.D. Département de Phytologie Centre de Recherche en Horticulture Université Laval Sainte-Foy, Québec, Canada
LANDES BIOSCIENCE GEORGETOWN, TEXAS U.S.A.
EUREKAH.COM AUSTIN, TEXAS U.S.A.
RECOMBINANT PROTEASE INHIBITORS IN PLANTS Biotechnology Intelligence Unit EUREKAH.COM Designed by Kimberly Mitchell Georgetown, Texas, U.S.A. Copyright ©2000 Eurekah.com All rights reserved. No part of this book 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. Printed in the U.S.A. Please address all inquiries to the Publishers: Eurekah.com, 810 South Church Street, Georgetown, Texas, U.S.A. 78626 Phone: 512/ 863 7762; FAX: 512/ 863 0081 -ISBN: 1-58706-007-8
Library of Congress Cataloging-in-Publication Data Recombinant protease inhibitors in plants / [edited by] Dominique Michaud. p. cm. -- (Biotechnology intelligence unit) Includes bibliographical references and index ISBN 1-58706-007-8(alk. paper) 1. Plants--Disease and pest resistance. 2. Recombinant protease inhibitors. I. Michaud, Dominique. II. Series. [DNLM: 1. Protease Inhibitors--metabolism. 2. Plants--metabolism. 3. Recombinant Proteins--metabolism. QU 136 R311 1999] SB750.R43 1999 632' .96--dc21 DNLM/DLC for Library of Congress
CONTENTS 1. Protease/Inhibitor Interactions in Plant-Pest Systems: A Brief Overview .................................................................................... 1 Dominique Michaud 1.1. Introduction ................................................................................... 1 1.2. The Inhibition of Pest Extracellular Proteases ................................. 2 1.3. Plant Pest Control with PIs: After the Hits... the Misses ................. 3 1.4. Future Perspectives ......................................................................... 5 2. Control of Phytophagous Insect Pests Using Serine Proteinase Inhibitors ............................................................................................... 9 John A. Gatehouse, Angharad M.R. Gatehouse and David P. Bown 2.1. Serine PIs and Their Role in Plant Protection ................................ 9 2.2. Effects of Serine PIs on Insect Digestive Proteolysis ...................... 11 2.3. The Wounding Response in Plant Defsense ................................. 14 2.4. “First-Phase” Use of Foreign Serine PI Genes ............................... 16 2.5. Insect Responses to Dietary Serine PIs .......................................... 19 2.6. Conclusions .................................................................................. 23 3. Cystatin-Based Control of Insects, with Special Reference to Oryzacystatin ................................................................................... 27 Soichi Arai and Keiko Abe 3.1. Introduction ................................................................................. 27 3.2. Oryzacystatin and Other Plant Cystatins ...................................... 28 3.3. Cysteine Proteinases as Targets of Plant Cystatins ........................ 30 3.4. Control of Insects with Cystatins .................................................. 33 3.5. Regeneration of Cystatin-Expressing Transgenic Plants ................ 36 3.6. Conclusion ................................................................................... 38 4. Recombinant Protease Inhibitors as Management Tools to Suppress Parasitic Nematodes .......................................................... 43 Thierry C. Vrain 4.1. Introduction ................................................................................. 43 4.2. Proteases in Nematodes ................................................................ 44 4.3. Effects of Recombinant PIs .......................................................... 46 4.4. Outlook ....................................................................................... 48 5. The Control of Plant Pathogens with Protease Inhibitors: A Realistic Approach? ........................................................................... 53 Stephen Gleddie and Dominique Michaud 5.1. Introduction ................................................................................. 53 5.2. Extracellular Proteases in Plant Pathogens .................................... 54 5.3. Induction of PIs in Infected Plants ............................................... 57 5.4. Prospects for PI-Expressing Transgenic Plants .............................. 59 5.5. Conclusion ................................................................................... 62
6. Regulation of Plant Defense Against Herbivorous Pests ....................... 65 Hisashi Koiwa, Ray A. Bressan and Paul M. Hasegawa 6.1. Introduction ................................................................................. 65 6.2. The Plant Response ...................................................................... 68 6.3. Future Perspectives ....................................................................... 73 7. The Response of Insects to Dietary Protease Inhibitors ........................ 80 Roxanne M. Broadway 7.1. Introduction ................................................................................. 80 7.2. Evaluation of PI Biological Activity .............................................. 80 7.3. Resistance of Insects to Dietary PIs ............................................... 81 7.4. Summary ...................................................................................... 84 8. Interference of Protease Inhibitors on Non-Target Organisms ............. 89 Louise A. Malone and Elisabeth P.J. Burgess 8.1. Introduction ................................................................................. 89 8.2. Pollinators .................................................................................... 89 8.3. Natural Enemies ........................................................................... 99 8.4. Soil Fauna and Microorganisms ................................................. 102 8.5. Summary .................................................................................... 103 9. Multiple Protease/Inhibitor Interactions in Plant-Pest Systems ......... 107 Savita Visal-Shah, France Brunelle and Dominique Michaud 9.1. Introduction ............................................................................... 107 9.2. First-Level Interactions: The Plant-Pest Continuum .................. 107 9.3. Multi-level Interactions: The Ecosystem ..................................... 108 9.4. Future Perspectives ..................................................................... 111 10. Using Natural and Modified Protease Inhibitors ................................ 114 Dominique Michaud and Binh Nguyen-Quoc 10.1. Introduction ............................................................................. 114 10.2.The Choice of Effective Inhibitors ............................................. 114 10.3. The Design of Hybrid Inhibitors .............................................. 119 10.3.1. Site-Directed Mutagenesis of Natural PIs .............................. 120 10.4. Conclusion ............................................................................... 124 11. Engineering Protease Inhibitors by Phage Display ................................................................................ 128 Jules Beekwilder and Maarten Jongsma 11.1. Introduction ............................................................................. 128 11.2. Phage Display ........................................................................... 128 11.3. Phage Display to Identify Natural Inhibitors ............................ 136 11.4. From Phage to Plant ................................................................. 137 11.5. Conclusion ............................................................................... 137
12. Using Protease Proregions as Regulators of Insect Digestive Proteinases ......................................................................................... 141 Mark A.J. Taylor 12.1. Introduction ............................................................................. 141 12.2. Proteinase Precursors ................................................................ 141 12.3. Precursor Processing ................................................................. 142 12.4. Protease Proregions as Selective Inhibitors ................................ 142 12.5. Discussion ................................................................................ 145 13. Expression of Protease Inhibitors in Potato ........................................ 147 Conrad Cloutier and Dominique Michaud 13.1. Introduction ............................................................................. 147 13.2. Rationale for a Low-Dose Approach with PI-Based Resistance ................................................................................. 150 13.3. The CPB Digestive Proteolytic System ..................................... 152 13.4. Fitness Consequences of PI Ingestion in CPB ........................... 156 13.5. OC-I-Expressing Transgenic Potato and CPB Natural Predators .................................................................................. 158 13.6. Conclusions and Future Perspectives ........................................ 159 14. Expression of Protease Inhibitors in Sweetpotato ............................... 166 Dapeng Zhang, Giselle Cipriani, Isabelle Rety, Ali Golmirzae, Nicole Smit and Dominique Michaud 14.1. Introduction ............................................................................. 166 14.2. The Insect Pests of Sweet Potato .............................................. 166 14.3. Integrated Pest Management for SPWs— The Success and the Lesson ...................................................... 167 14.4. Digestive Proteinases in SPW (Cylas) Species ............................ 168 14.5. Expression of Recombinant PIs in Sweet Potato ....................... 169 14.6. PIs in Sweet Potato, and Their Nutritional Impact ................... 172 14.7. Discussion ................................................................................ 174 15. Expression of Protease Inhibitors in Rapeseed .................................... 178 Lise Jouanin, Michel Bonadé Bottino, Cécile Girard, Jacques Lerin and Minh Hà Pham Delègue 15.1. Introduction ............................................................................. 178 15.2. Digestive Protease Types in Pests of B. napus ........................... 179 15.3. Expression of Recombinant PIs in B. napus .............................. 181 15.4. Discussion ................................................................................ 187
16. Production of Useful Protease Inhibitors in Plants ............................ 190 Dominique Michaud and Serge Yelle 16.1. Introduction ............................................................................. 190 16.2. High-level Accumulation of PIs in Plant Cells .......................... 191 16.3. Extraction of Recombinant PIs from Plant Tissues ................... 192 16.4. Purification of Recombinant PIs from Crude Extracts .............. 193 16.5. Conclusions .............................................................................. 197 17. Proteinase INhibitors in Health and Disease Control—Medical and Industrial Aspects ........................................................................ 202 Michiel F.J. Blankenvoorde, Henk S. Brand, Yvonne M.C. Henskens, Enno C.I. Veerman and Arie V. Nieuw Amerongen 17.1. Introduction ............................................................................. 202 17.2. Anti-microbial Properties of Cystatins ...................................... 204 17.3. Cystatins in the Control of Tumor Growth and Metastasis ............................................. 205 17.4. Protection Against Tissue Destruction ...................................... 206 17.5. Industrial Aspects ..................................................................... 208 18. Protease Inhibitors in Food Processing............................................... 214 Fernando L. García-Carreño, Haejung An and Norman F. Haard 18.1. Introduction ............................................................................. 214 18.2. Controlled Proteolysis in Food ................................................. 215 18.3. Safety of PIs in Food and Feed ................................................. 215 18.4. Plant PIs in Foodstuff ............................................................... 216 18.5. Conclusion ............................................................................... 219 Appendix I. Substrates and Inhibitors Useful in Protease Characterization ............................................................... 223 France Brunelle and Dominique Michaud Appendix II. Plant Protease Inhibitors: Available mRNA Sequences ......... 227 Binh Nguyen-Quoc Appendix III. Tertiary Structures of Proteases and Protease Inhibitors Available in the Brookhaven National Laboratory Protein Data Bank ................................................................................................... 230 France Brunelle and Dominique Michaud Index .................................................................................................. 238
EDITOR Dominique Michaud, Agr., Ph.D. Département de Phytologie, Centre de Recherche en Horticulture, Université Laval Sainte-Foy, Québec, Canada Chapters 1, 5, 9, 10, 13, 14 and 16 Appendices I and III
CONTRIBUTORS Keiko Abe Department of Applied Biological Chemistry Graduate School of Agricultural and Life Sciences The University of Tokyo Tokyo, Japan
Michel Bonadé Bottino Laboratoire de Biologie Cellulaire Institut National de la Recherche Agronomique Versailles, France
Chapter 3
David P. Bown Department of Biological Sciences University of Durham Durham, U.K
Haejung An Seafood Laboratory, Oregon State University Astoria, Oregon,U.S.A. Chapter 18
Soichi Arai Department of Nutritional Science, Faculty of Applied Biological Sciences Tokyo University of Agriculture Tokyo, Japan Chapter 3
Jules Beekwilder Centre for Plant Breeding and Reproduction Research (CPRO-DLO), Wageningen,The Netherlands Chapter 11
Michiel F.J. Blankenvoorde Department of Oral Biochemistry Academic Centre for Dentistry Amsterdam (ACTA) Amsterdam, The Netherlands Chapter 17
Chapter 15
Chapter 2
Henk S. Brand Department of Oral Biochemistry Academic Centre for Dentistry Amsterdam (ACTA) Amsterdam, The Netherlands Chapter 17
Ray A. Bressan Center for Plant Environmental Stress Physiology Purdue University West Lafayette, Indiana, U.S.A. Chapter 6
Roxanne M. Broadway Department of Entomology, New York State Agricultural Experiment Station, Cornell University Geneva, New York, U.S.A. Chapter 7
France Brunelle Département de Phytologie, Centre de Recherche en Horticulture, Université Laval Québec, Canada Chapter 9, Appendices I and III
Stephen Gleddie Eastern Cereal and Oilseed Research Centre, Central Experimental Farm Agriculture and Agri-Food Canada Ottawa, Canada Chapter 5
Elizabeth P.J. Burgess The Horticulture and Food Research Institute of New Zealand Ltd Auckland, New Zealand
Ali Golmirzae Centro Internacional de la Papa Lima, Peru
Chapter 8
Chapter 14
Giselle Cipriani Centro Internacional de la Papa Lima, Peru
Norman F. Haard Department of Food Science and Technology University of Califormia Davis, California, U.S.A.
Chapter 14
Conrad Cloutier Département de Biologie Centre de Recherche en Horticulture Université Laval Québec, Canada Chapter 13
Fernando L. García-Carreño Centro de Investigaciones Biologicas del Noroeste, S.C., La Paz, Mexico Chapter 18
Angharad M. R. Gatehouse Department of Biological Sciences University of Durham Durham, U.K. Chapter 2
John A. Gatehouse Department of Biological Sciences University of Durham Durham, U.K. Chapter 2
Cécile Girard Laboratoire de Biologie Cellulaire Institut National de la Recherche Scientifique Versailles, France Chapter 15
Chapter 18
Paul M. Hasegawa Center for Plant Environmental Stress Physiology, Purdue University West Lafayette, Indiana, U.S.A. Chapter 6
Yvonne M.C. Henskens Laboratory for Clinical Chemistry and Hematology Diagnostic Center SSDZ Delft, The Netherlands Chapter 17
Maarten A. Jongsma Centre for Plant Breeding and Reproduction Research (CPRO-DLO) Wageningen, The Netherlands Chapter 11
Lise Jouanin Laboratoire de Biologie Cellulaire Institut National de la Recherche Agronomique Versailles, France Chapter 15
Hisashi Koiwa Center for Plant Environmental Stress Physiology, Purdue University West Lafayette, Indiana, U.S.A. Chapter 6 Jacques Lerin Laboratoire de Zoologie Institut National de la Recherche Agronomique Luzignan, France Chapter 15
Louise A. Malone The Horticulture and Food Research Institute of New Zealand Ltd Auckland, New Zealand Chapter 8 Binh Nguyen-Quoc Département de Phytologie Centre de Recherche en Horticulture Université Laval Québec, Canada Chapter 10, Appendix II
Arie V. Nieuw Amerongen Department of Oral Biochemistry Academic Centre for Dentistry Amsterdam (ACTA) Amsterdam, The Netherlands Chapter 17
Minh-Ha Pham Delegue Laboratoire de Neurobiologie Comparée des Invertébrés Institut National de la Recherche Agronomique Bures sur Yvette, France Chapter 15
Isabelle Rety Département de Phytologie Centre de Recherche en Horticulture Université Laval Québec, Canada Chapter 14
Nicole Smit Centro Internacional de la Papa Kampala, Uganda Chapter 14
Mark A.J. Taylor Institute of Food Research Reading, U.K. Chapter 12
Enno C.I. Veerman Department of Oral Biochemistry Academic Centre for Dentistry Amsterdam (ACTA) Amsterdam, The Netherlands Chapter 17
Savita Visal-Shah Département de Phytologie Centre de Recherche en Horticulture Université Laval Québec, Canada Chapter 9 Thierry C. Vrain Pacific Agri-Food Research Centre Agriculture and Agri-Food Canada Summerland, Canada Chapter 4
Serge Yelle Département de Phytologie Centre de Recherche en Horticulture Université Laval Québec, Canada Chapter 16 Dapeng Zhang Centro Internacional de la Papa Lima, Peru Chapter 14
PREFACE
A
survey of the current scientific literature clearly shows the implication of proteolytic enzymes and their inhibitors in various biological systems, in volving processes as diverse and important as the degradation of dietary proteins, the regulation of cellular protein catabolism and the inhibition of pathogen extracellular proteases during infection. Together, the rapidly growing body of literature in the broad research field of proteolysis —about 15 papers are published every day, and the many patents issued each year regarding the control of specific protease-related events in biological systems demonstrate the importance of proteolysis in biological processes and the increasing interest for this research field in the scientific community. While gene regulation is important to control the synthesis of proteins in living cells, proteolysis is important to mediate both their processing and their degradation. From a practical point of view, proteases may be seen as interesting targets for the control proteins after they are synthesized, as genes represent interesting targets to control proteins before they accumulate in the cell. In this context protease inhibitors (PIs), the ‘natural regulators’ of proteases, represent a useful tool to regulate these enzymes in biological systems. The inactivation of proteases with specific inhibitors may prove useful, for instance to control several protease-related physiological disorders in humans, to stop pathogen invasion by hindering the hydrolysis of the host tissue proteins, to protect defenserelated proteins produced by the host to counteract predation or infection, or to interfere with the digestion of dietary proteins in plant pests and pathogens. The use of PIs in plant protection, in particular, represents an attractive way to protect economically-important plants from predation or infection. With the growing concerns worldwide about the negative effects of chemical pesticides in the environment, several alternative approaches for pest control are now currently devised. The design of genetically-modified plants expressing recombinant PIs, notably, has been proposed as a way to protect plants from various organisms. It is now increasingly evident that most herbivorous insects and pathogens secrete extracellular proteases to mediate important processes like the digestion of plant proteins or the invasion of host tissues. In this perspective, altering proteolytic processes in target pests by allowing the plant to produce appropriate PIs certainly represents a potentially useful way to reduce the fitness of several target pest organisms, and thus help minimize the use of potentially harmful synthetic pesticides in the field. In the last ten years PI-encoding cDNA sequences have been integrated into the genome of plants as important as cereals, rapeseed, potato and sweetpotato, and convincing protective effects have been noted in some cases, especially against lepidopteran insects and parasitic nematodes. The general usefulness of protein PIs in plant protection, however still remains to be clearly established. During the course of their evolution, pests and pathogens
have been in contact with a variety of PIs produced naturally by their host, and they developed efficient strategies to elude their inhibitory effects, including the use of complex proteolytic systems composed of proteases differentially inhibited by PIs, compensation of inhibited proteolytic functions by the production of ‘insensitive’ proteases, and degradation of the inhibitors by nontarget proteases. As a result of these coevolutive processes protease/inhibitor interactions in plantpest systems became remarkably complex, and anyone wishing to alter specific proteolytic functions in a given target organism has to take into account not only the inhibitory effect of the PI against the target proteases, but also several additional factors associated with the release of such a novel biologically active compound in the whole system. The capacity of the target pest to elude the effect of the introduced inhibitor, or the possible negative effects of this inhibitor against nontarget organisms at the ecosystem level are just a few issues that should be considered to ensure the successful implementation of recombinant PIs in the field. With the aim of summarizing our current knowledge on the use of recombinant PIs in plant protection, this book presents a collection of articles written by experts actively involved in the field worldwide. After a brief introduction on some basic concepts related to protease/inhibitor interactions in plant-pest systems (Chapter 1), the first part of the book summarizes recent advances on the use of recombinant PIs to protect plants from predation by herbivorous insects (Chapters 2 and 3) and from infection by parasitic nematodes (Chapter 4) and pathogens (Chapter 5). The second part of the book deals with some basic aspects of the biological systems assessed, including the response of plants to herbivorous pests (Chapter 6), the response of herbivorous insects to dietary PIs (Chapter 7), the risks associated with the release of recombinant PIs in the environment (Chapter 8), and how all these elements interact with each other in complex biological systems (Chapter 9). Based on these considerations, strategies are then proposed for the choice and the development of effective PIs: Chapter 10 discusses the main biochemical criteria to consider for the choice and the design of appropriate inhibitors; Chapter 11 describes the potential of molecular phage display as a mean to improve the binding characteristics of PIs; and Chapter 12 discusses the potential of protease proregions as regulators of exogenous proteases in biological systems. The third section of the book, finally, deals with the practical and industrial aspects of recombinant PI expression in plants. The first three chapters assess the usefulness of PIs in the protection of three model plants of major economic importance: potato (Chapter 13), sweetpotato (Chapter 14) and rapeseed (Chapter 15). The following three chapters deal with alternative uses of recombinant PIs expressed in plants: Chapter 16 addresses the basic principles to consider when planning the use of plants as bioreactors to produce useful PIs, while Chapter 17 and Chapter 18 discuss the potential of these recombinant inhibitors in two fields of industrial importance, medicine and food science. Dominique Michaud Puétec, January 1999
CHAPTER 1
Protease/Inhibitor Interactions in Plant-Pest Systems A Brief Overview Dominique Michaud
1.1. Introduction
T
he metabolism of any living cell depends on proteolysis. Proteases, which form a diverse group of enzymes capable of cleaving peptide bonds, are implicated in various essential processes ranging from the fine control of protein catabolism and the selective degradation of damaged proteins to the bulk hydrolysis of dietary proteins. Considering the recent developments in the broad research field of proteolysis, it now appears obvious that proteases are essential not only in providing cells with simple metabolites essential for growth and development, but also in mediating a variety of key processes like the cleavage of specific peptide bonds in immature proteins or the removal of targeting signals in preproteins after their translocation to the appropriate cell compartment. Whereas proteolytic enzymes were previously seen as ‘destroying catalysts’ mostly involved in the bulk hydrolysis of dietary proteins, they are now considered as central control elements in the development of most living organisms.1 During the ‘life cycle’ of a protein, proteolysis is essential in regulating maturation and catabolism, as gene control is essential to regulate anabolism. Simply put, proteolytic enzymes mediate two types of hydrolytic processes:
1. limited proteolysis, in which only one or a few peptide bonds are cleaved to release a biologically active protein, and 2. extensive proteolysis, in which the protein is completely degraded through the hydrolysis of most of its peptide bonds.
Limited proteolysis is generally associated with the control of specific metabolic events; extensive proteolysis usually results in complete elimination of the protein, either to regulate its intracellular level or to recycle the resulting amino acids (Fig.1.1). Together, these processes contribute to the overall metabolism of living organisms by mediating important cellular functions like the final processing of pre- or proproteins before they play their role in the cell, the selective removal of these proteins when they are no longer useful, or the recycling of amino acids needed to synthesize novel proteins. Besides these endogenous processes, proteases mediate several molecular interactions taking place between the different organisms of a given environment. Intracellular parasites, for instance secrete proteases implicated in both their interactions with the host, their survival into the host cell environment and the processing of host
Recombinant Protease Inhibitors in Plants, edited by Dominique Michaud. ©2000 Eurekah.com
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Recombinant Protease Inhibitors in Plants
Fig. 1.1. The ‘life cycle’ of a protein. Protein metabolism in living cells may be seen as a two-step process in which the amino acids used as building blocks for protein synthesis are recycled as a result of protein turnover. While gene control appears of crucial importance to regulate and direct the synthesis of proteins (grey area), proteolysis is essential in mediating their posttranslational cleavage, either to remove targeting signals after the translocation of preproteins in the cell, to activate immature proproteins by removal of their regulatory propeptide, to hydrolyze a protein no longer useful in the cell, to remove ‘abnormal’—including foreign—proteins from the cell, or to digest dietary proteins obtained from food to provide the organism with the amino acids needed for growth and development (white area). Proteolysis is generally restricted to one or a few peptide bonds during preprotein or proprotein processing, while extensive hydrolysis of the polypeptide chain takes place during protein turnover. proteins for nutritional purposes.2,3 Likewise, processes like the hydrolysis of plant dietary proteins in the midgut of herbivorous insects, the digestion of these same proteins by our own digestive proteases, or the degradation of host tissue proteins during microbial infection all represent extracellular proteolytic processes implicating the different living entities of a biological system. In the course of their evolution, living organisms have acquired complex protease systems efficient
in hydrolyzing exogenous proteins, a process essential both to obtain the nutrients needed for growth, and to compete with the other organisms of the surrounding environment.
1.2. The Inhibition of Pest Extracellular Proteases In reaction to these hydrolytic processes, several organisms also developed strategies to counteract the adverse effects of exogenous
Protease/Inhibitor Interactions in Plant-Pest Systems
proteases. Plants challenged by a pathogen or a predator, for instance actively produce proteinaceous protease inhibitors (PIs), which react with proteases as pseudo-substrates to hinder their activity. It is now well established that PIs are used by a wide variety of organisms not only to direct the control of endogenous proteolytic functions, but also to ensure their protection against predation or infection.4-9 Like extracellular proteases, PIs actively contribute to the complex array of molecular interactions taking place between the different organisms of an ecosystem, by acting as regulators of extracellular proteolytic events. In this context, it is not surprising that PIs were proposed as a tool for the control of various pests and pathogens. Assuming that every protease or group of proteases in given target organisms mediates at least one useful process, one can speculate that altering this process by inactivating the protease(s) involved may represent a way to affect the development of these organisms, and thus eventually protect their host from predation or infection. Until now pest control strategies based on the inhibition of proteolytic enzymes with selective inhibitors have been devised to control pests as diverse as viruses, herbivorous insects, parasitic nematodes and microbial pathogens.6,10,11 Although the exact metabolic functions altered by the inhibitors remain to be elucidated in most cases, the importance of extracellular proteolysis in the target organisms appears obvious. Repressive effects of dietary PIs on growth and fecundity of herbivorous insects, for instance have been described for several species (see Chapters 2 and 3, this volume), and the implication of extracellular proteases in pathogenic processes has been documented in several instances (see Chapters 4, 5 and 17). Based on these findings, the use of protein PIs expressed in transgenic systems or delivered by any other appropriate mean has been proposed as a way to protect a wide variety of hosts from their natural enemies. The genetic transformation of plants with PI-encoding cDNA sequences, in particular, represents an attractive way to control a variety of herbivorous pests and pathogens attacking
3
plants (see Chapters 2-5).10-15 Many studies have demonstrated the ability of protein PIs to efficiently inhibit the extracellular proteases of various herbivorous insects, root-parasitic nematodes and fungal pathogens, and deleterious effects of several proteinaceous inhibitors included in artificial diets or culture media were reported for several insects16-20 and pathogens.21-23 Based on these observations, the use of recombinant PIs was proposed as an effective means of protecting plants from their natural enemies, and several plants of economic importance were genetically modified with PI-encoding cDNA sequences. In the last ten years the genes of several different PIs were integrated into the genome of almost fifteen different plants,12 clearly showing the growing interest of scientists for this control approach.
1.3. Plant Pest Control with PIs: After the Hits... the Misses Despite these promising developments, the general usefulness of recombinant PIs in plant protection still remains to demonstrate. The inhibitory spectrum of protein PIs is usually limited to proteases in one of several mechanistic classes, leaving free proteases in the surrounding medium after inhibition (see Figure 1.2 for a classification of proteases and PIs).24 Probably due to a progressive adaptation of plant pests to the continuous occurrence of PIs in the diet, the inhibitory spectrum of protein inhibitors against the extracellular proteases of several pests is even more limited, being often restricted to the family level.19,25-34 Nontarget proteases, that may allow metabolic compensation of inhibited proteolytic functions,35 may also challenge the structural integrity of several PIs and thus potentially affect their effectiveness in vivo.36 At this point, understanding the dynamic interactions implicating protein PIs and pest extracellular proteases appears important to correctly assess the actual usefulness of extracellular protease inhibition in plant protection (see Chapters 6, 7 and 9). Until now the adverse effects observed with PIs against several insects and pathogens were
4
Recombinant Protease Inhibitors in Plants
Fig. 1.2. Classification of proteases and PIs—a simple overview. According to the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology, proteases belong to the fourth subgroup of Group 3 enzymes, which includes hydrolytic enzymes. Given their huge structural and functional diversity, proteases may be classified based on three major criteria: the type of reaction catalyzed, the chemical nature of the catalytic site, and the structural evolutionary relationship between the different proteases.24 For practical reasons the classification of proteases based on the catalytic site appears quite useful since protein PIs, which act in several cases as pseudo-substrates on their target proteases, may be classified according to the same scheme. Based on this approach proteases are categorized into four main groups: serine proteases, which possess a catalytic triad composed an aspartate, a serine and a histidine; cysteine proteases, which possess a catalytic triad composed of a cysteine, a histidine and an aspartate; aspartate proteases, which rely on two aspartate residues for activity; and metalloproteases, which rely on the presence of a metallic ion, usually Zn2+. Protein PIs, which as a general rule show affinity for proteases from one of these groups, may be classified into four groups: serine PIs, cysteine PIs, aspartate PIs and metallo-PIs, thereby providing an easy way to rapidly determine candidate PIs potentially useful in the control of specific groups of proteases. presumably due to a drastic alteration of their protein digestive functions, but alternative or parallel effects cannot be excluded. Proteases excreted by fungi or parasitic nematodes, for instance may be associated not only with the uptake of dietary amino acids, but also with specific pathogenic processes like the activation of zymogens, the penetration of host tissues or the irreversible inactivation of certain defense-related proteins in the host cells. Similarly, inhibiting the midgut proteases of herbivorous insects or the
proteases released by microbial pathogens in the surrounding environment may not only alter dietary protein hydrolysis, but also protect the integrity of host defense proteins by preventing their degradation.36 While major progress has been made in the last several years regarding our knowledge on the roles of protein PIs in plant defense, their induction in plant tissues following predation or infection, and the complex physiological processes triggered in target pests by the inhibition of their extracellular proteases,4,5,35,37 our current
Protease/Inhibitor Interactions in Plant-Pest Systems
understanding of protease/inhibitor interactions in plant-pest systems still appears limited. Extensive studies remain necessary, for instance to understand basic processes like the sequential degradation of dietary proteins in the midgut of herbivorous insects, or the degradation of cell wall proteins by pathogenic fungi or bacteria when they infect plant tissues. While many important questions remain for a correct assessment of protease/PI interactions, however recent progress in the field suggests that several successful control strategies based on recombinant PIs should be available in the near future. The processes involved in the hydrolysis of plant proteins by pest extracellular proteases have not yet been elucidated in detail, but one can already assume that a PI-based control strategy will be effective if compensatory and PI- degradation processes in the target organism are avoided.35,36,38 From a practical point of view, recombinant PIs could be effective, for instance if they strongly inhibit the target proteases,31,36,39-42 and if the number of insensitive proteases is kept to a minimum.36,38 Several successful strategies have been devised recently to improve the efficiency of protein PIs (see Chapters 10-12), including the improvement of their binding capacity by site-directssis,40,43 the design of hybrid PIs with an extended inhibitory spectrum,44,45 the isolation of effective inhibitor variants by phage display,46,47 the identification from plant tissues of stress-induced PIs with a broad inhibitory spectrum,48,49 the use of insect PIs exhibiting high affinity for their cognate protease,50 and the use of protease regulatory propeptides as a complement to the currently used protein PIs.34,51 When expressed in transgenic plants these PIs, tailored for the inhibition of specific protease systems, should contribute significantly to the development of effective PI-based control strategies, especially in those systems where complex protease systems are found.
1.4. Future Perspectives With the recent developments in plant molecular biotechnology, DNA sequences encoding a variety of useful defense proteins
5
can now be easily integrated into the genome of plants to complement the effect of naturally-occurring defense proteins, and thus improve their ability to remain healthy and productive in commercial agricultural systems. 12 Transgenic plants expressing δ-endotoxins from the soil bacterium Bacillus thuringiensis (Bt toxins), for instance appear particularly useful for the control of various insect and nematode pests.52 Bt toxins interact with specific receptors in the digestive tract of these organisms, causing major physiological disorders and subsequent death. This antibiotic effect confers to Bt toxin-expressing plants a remarkable resistance status, making them already suitable for commercialization. Effective biocidal proteins like Bt toxins, however are single chemical compounds like any other pesticide, and the large-scale use of Bt toxin-expressing plants in the coming years could exert a strong selection pressure on the target pests and favor the buildup of resistant populations.53 Several recent studies have demonstrated the ability of insects to develop resistance to Bt toxins,54 and eventual resistance to other biocidal proteins expressed in transgenic plants appears plausible. To minimize the development of such resistance in pest populations and to ensure their effectiveness over a long period, transgenic plants expressing a single resistance factor should be seen as one component of more elaborated, multicomponent control systems, representing one in several means to control the pests targeted.53 In this context, recombinant PIs appear of particular interest. Unlike toxic proteins, PIs do not kill the target pests. In most cases the ingestion of high doses of PI causes growth delays and alters reproductive functions, thereby reducing the fitness of the target organism. In contrast with antibiotic compounds, recombinant PIs usually provide the plant with only partial resistance, making this control mean particularly suitable in control strategies based on the deployment of several control means (see Chapters 13-15). Herbivorous pests showing reduced fitness after ingestion of PIs could be more suscep-
6
tible, for instance to natural predators implemented in the field as biological control agents. Moreover, the inhibitory effect of PIs could improve the efficiency of defense proteins like Bt toxins or like the plant’s own defense proteins by preventing their degradation by the target pest proteases.36 Several PI-expressing plants have been developed in the last ten years, leading in some cases to promising developments. Considering the rapid developments in the field, it also appears likely that it is just a question of time before highly effective PIs are identified or designed for the inhibition of various pest protease systems (see for instance ref. 55). At this point continued efforts will still be needed, however to thoroughly understand protease/ PI interactions in the systems assessed. As noted above, extending the range of proteases susceptible to the action of recombinant PIs should allow the development of efficient PI-expressing plants, but this approach could have some drawbacks in the field. Maximizing the inhibitory spectrum of PIs should help improve their effect against target organisms, but it will at the same time decrease their specificity. Whereas biocidal compounds such as Bt toxins are interesting because of their high specificity toward the target pest, PIs with an extended inhibitory range could eventually interfere with nontarget organisms in the ecosystem (see Chapter 8). Alternatively, blocking a key process in the complex cascade of reactions leading to the degradation of proteins in specific biological systems could be sufficient to elude compensatory or PI-hydrolytic processes. The challenge, then, will be to define an ‘inhibitory equilibrium’ in the target environment allowing to alter specific physiological functions in the pest without affecting nontarget organisms. While at present considerable effort is made to maximize the binding capacity of recombinant PIs—a step which is essential to establish the usefulness of recombinant PIs in pest control, the next step could rather consist to optimize their inhibitory effects in the ecosystem.
Recombinant Protease Inhibitors in Plants
Acknowledgments I thank Line Cantin for helpful comments on the manuscript. This work was supported by an operating grant from the Natural Science and Engineering Research Council of Canada.
References 1. Wolf DH. Proteases as biological regulators— introductory remarks. Experientia 1992; 48:117-118. 2. North MJ. Comparative biochemistry of the proteinases of eucaryotic microorganisms. Microbiol Rev 1982; 46:308-340. 3. Branquinha MH, Vermelho AB, Goldenberg S et al. Ubiquity of cysteine- and metalloproteinase activities in a wide range of trypanosomatids. J Euk Microbiol 1996; 43:131-135. 4. Ryan CA. Proteinase inhibitors in plants: Genes for improving defenses against insects and pathogens. Annu Rev Phytopathol 1990; 28:425-449. 5. Koiwa H, Bressan RA, Hasegawa PM. Regulation of protease inhibitors and plant defense. Trends Plant Sci 1997; 2:379-384. 6. Henskens YMC, Veerman ECI, Nieuw Amerongen AV. Cystatins in health and disease. Biol Chem 1996; 377:71-86. 7. Vilcinskas A, Wedde M. Inhibition of Beauvaria bassiana proteases and fungal development by inducible protease inhibitors in the haemolymph of Galleria mellonella larvae. Biocontrol Sci Technol 1997; 7:591-601. 8. Dieguez-Uribeondo J, Cerenius L. The inhibition of extracellular proteinases from Aphanomyces spp. by three different proteinase inhibitors from crayfish blood. Mycol Res 1998; 102:820-824. 9. Polanowski A, Blum MS, Whitman DW et al. Proteinase inhibitors in the nonvenomous defensive secretion of grasshoppers: Antiproteolytic range and possible significance. Comp Biochem Physiol B 1997; 117:525-529. 10. Hilder VA, Gatehouse AMR, Boulter D. Proteinase inhibitor approach. In: Kung S-D, Wu R, eds. Transgenic Plants: Engineering and utilization, Vol. 1. New York: Academic Press, 1993:317-338. 11. Atkinson HJ, Urwin PE, Hansen PE et al. Designs for engineered resistance to rootparasitic nematodes. Trends Biotechnol 1995; 13:369-374. 12. Schuler TH, Poppy GM, Kerry BR et al. Insect-resistant transgenic plants. Trends Biotechnol 1998; 16:168-175.
Protease/Inhibitor Interactions in Plant-Pest Systems 13. Gatehouse AMR, Gatehouse JA. Identifying proteins with insecticidal activity: Use of encoding genes to produce insect-resistant transgenic crops. Pestic Sci 1998; 52:165-175. 14. Jouanin L, Bonadé-Bottino M, Girard C et al. Transgenic plants for insect resistance. Plant Sci 1998; 131:1-11. 15. Michaud D, Vrain TC. Expression of recombinant proteinase inhibitors in plants. In: Cunningham C, Porter AJR, eds. Recombinant proteins from plants: Production and isolation of clinically useful compounds. Totowa NJ:Humana Press, 1998:49-64. 16. Burgess EPJ, Stevens PS, Keen GK et al. Effects of protease inhibitors and dietary protein level on the black field cricket Teleogryllus commodus. Entomol Exp Appl 1991; 61:123-130. 17. Chen M-S, Johnson B, Wen L et al. Rice cystatin: Bacterial expression, purification, cysteine proteinase inhibitory activity, and insect growth suppressing activity of a truncated form of the protein. Protein Expr Purif 1992; 3:41-49. 18. Oppert B, Morgan TD, Culbertson C et al. Dietary mixtures of cysteine and serine proteinase inhibitors exhibit synergistic toxicity toward the red flour beetle, Tribolium castaneum. Comp Biochem Physiol C 1993; 105:379-385. 19. Michaud D, Bernier-Vadnais N, Overney S et al. Constitutive expression of digestive cysteine proteinase forms during development of the Colorado potato beetle, Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae). Insect Biochem Mol Biol 1995; 25:1041-1048. 20. Markwick NP, Reid SJ, Laing WA et al. Effects of dietary protein and protease inhibitors on codling moth (Lepidoptera: Tortricidae). J Econ Entomol 1995; 88:33-39. 21. Lorito M, Broadway RM, Hayes C et al. Proteinase inhibitors from plants as a novel class of fungicides. Mol Plant Microbe Interact 1994; 7:525-527. 22. Dunaevskii Y, Pavlyukova E, Belyakova G et al. Anionic trypsin inhibitors from dry buckwheat seeds: Isolation, specificity of action, and effect on growth of micromycetes. Biochemistry (Moscow) 1994; 59:739-743. 23. Joshi B, Sainani M, Bastawade K et al. Cysteine protease inhibitor from pearl millet: A new class of antifungal protein. Biochem Biophys Res Commun 1998; 246:382-387. 24. Barrett AJ. Classification of peptidases. Methods Enzymol 1994; 244:1-15. 25. Michaud D, Nguyen-Quoc B, Yelle S. Selective inhibition of Colorado potato beetle cathepsin H by oryzacystatins I and II. FEBS Lett 1993; 331:173-176.
7 26. Broadway RM. Are insects resistant to plant proteinase inhibitors? J Insect Physiol 1995; 41:107-116. 27. Jongsma MA, Bakker PL, Peters J et al. Adaptation of Spodoptera exigua larvae to plant proteinase inhibitors by induction of gut proteinase activity insensitive to inhibition. Proc Natl Acad Sci USA 1995; 92:8041-8045. 28. Bolter CJ, Jongsma MA. Colorado potato beetles adapt to proteinase inhibitors induced in potato leaves by methyl jasmonate. J Insect Physiol 1995; 41:1071-1078. 29. Michaud D, Cantin L, Vrain TC. Carboxyterminal truncation of oryzacystatin II by oryzacystatin-insensitive insect digestive proteinases. Arch Biochem Biophys 1995; 322:469-474. 30. Michaud D, Nguyen-Quoc B, Vrain TC et al. Response of digestive cysteine proteinases from the Colorado potato beetle (Leptinotarsa decemlineata) and the black vine weevil (Otiorynchus sulcatus) to a recombinant form of human stefin A. Arch Insect Biochem Physiol 1996; 31:451-464. 31. Michaud D, Cantin L, Raworth DA et al. Assessing the stability of cystatin/cysteine proteinase complexes using mildly-denaturing gelatin/polyacrylamide gel electrophoresis. Electrophoresis 1996; 17:74-79. 32. Broadway RM. Dietary proteinase inhibitors alter complement of midgut proteases. Arch Insect Biochem Physiol 1996; 32:39-53. 33. Broadway RM. Dietary regulation of serine proteinases that are resistant to serine proteinase inhibitors. J Insect Physiol 1997; 43:855-874. 34. Visal S, Taylor MAJ, Michaud D. The proregion of papaya proteinase IV inhibits Colorado potato beetle digestive cysteine proteinases. FEBS Lett 1998; 434:401-405. 35. Jongsma MA, Bolter C. The adaptation of insects to plant protease inhibitors. J Insect Physiol 1997; 43:885-895. 36. Michaud D. Avoiding protease-mediated resistance in herbivorous pests. Trends Biotechnol 1997; 15:4-6. 37. Bown DP, Wilkinson HS, Gatehouse JA. Differentially regulated inhibitor-sensitive and insensitive protease genes from the phytophagous insect pest, Helicoverpa armigera, are members of complex multigene families. Insect Biochem Mol Biol 1997; 27:625-638. 38. Jongsma MA, Stiekema WJ, Bosch D. Combatting inhibitor-insensitive proteases of insect pests. Trends Biotechnol 1996; 14:331-333. 39. Christeller JT, Shaw BD. The interaction of a range of serine proteinase inhibitors with bovine trypsin and Costelytra zealandica trypsin. Insect Biochem 1989; 19:233-241.
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8 40. Urwin PE, Atkinson HJ, Waller DA et al. Engineered oryzacystatin-I expressed in transgenic hairy roots confers resistance to Globodera pallida. Plant J 1995; 8:121-131. 41. Michaud D, Cantin L, Bonade Bottino M et al. Identification of stable plant cystatin/nematode proteinase complexes using mildlydenaturing gelatin/polyacrylamide gel electrophoresis. Electrophoresis 1996; 17:1373-1379. 42. Girard C, Bonadé-Bottino M, Pham-Delegue M-H et al. Two strains of cabbage seed weevil (Coleoptera: Curculionidae) exhibit differential susceptibility to a transgenic oilseed rape expressin oryzacystatin I. J Insect Physiol 1998; 44:569-577. 43. Szardenings M, Vasel B, Hecht H-J et al. Highly effective protease inhibitors from variants of human pancreatic secretory trypsin inhibitor (hPSTI): An assessment of 3-D structure-based protein design. Protein Eng 1995; 8:45-52. 44. Urwin PE, McPherson MJ, Atkinson HJ. Enhanced transgenic plant resistance to nematodes by dual proteinase inhibitor constructs. Planta 1998; 204:472-479. 45. Le-Nguyen D, Mattras H, Coletti-Previero MA et al. Design and chemical synthesis of a 32 residues chimeric microprotein inhibiting both trypsin and carboxypeptidase A. Biochem Biophys Res Commun 1989; 162:1426-1430. 46. Jongsma MA, Bakker PL, Stiekema WJ et al. Phage display of a double-headed proteinase inhibitor: Analysis of the binding domains of potato proteinase inhibitor II. Mol Breeding 1995; 1:181-191. 47. Koiwa K, Shade RE, Zhu-Salzman K et al. Phage display selection can differentiate insecticidal activity of soybean cystatins. Plant J 1998; 14:371-379.
48. Zhao Y, Botella MA, Subramanian L et al. Two wound-inducible soybean cysteine proteinase inhibitors have greater insect digestive s inhibitory activities than a constitutive homolog. Plant Physiol 1996; 111:1299-1306. 49. Visal S, Michaud D, Yelle S. Identification of a gamma-linolenic acid-induced tomato leaf cystatin-like protein with potential for biocontrol of the phytophagous pest Colorado beetle. Plant Physiol 1996; 111s:40. 50. Thomas JC, Adams DG, Keppenne VD et al. Manduca sexta encoded protease inhibitors expressed in Nicotiana tabacum provide protection against insects. Plant Physiol Biochem 1995; 33:611-614. 51. Taylor MAJ, Lee MJ. Trypsin isolated from the midgut of the tobacco budworm, Manduca sexta, is inhibited by synthetic pro-peptides in vitro. Biochem Biophys Res Commun 1997; 235:606-609. 52. Peferoen M. Progress and prospects for field use of Bt genes in crops. Trends Biotechnol 1997; 15:173-177. 53. Brattsen LB. Bioengineering of crop plants and resistant biotype evolution in insects: Counteracting coevolution. Arch Insect Biochem Physiol 1991; 17:253-267. 54. Tabashnik BE. Evolution of resistance to Bacillus thuringiensis. Annu Rev Entomol 1994; 39:47-79. 55. Gruden K, Strukelj B, Popovic T et al. The cysteine protease activity of Colorado potato beetle (Leptinotarsa decemlineata Say) guts, which is insensitive to potato protease inhibitors, is inhibited by thyroglobulin type-1 domain inhibitors. Insect Biochem Mol Biol 1998; 28:549-560.
CHAPTER 2
Control of Phytophagous Insect Pests Using Serine Proteinase Inhibitors John A. Gatehouse, Angharad M.R. Gatehouse and David P. Bown
2.1. Serine PIs and Their Role in Plant Protection 2.1.1. Introduction
T
he role of proteinase inhibitors (PIs) in plant defense against predators and pathogens is now well established. Although diverse endogenous functions for these proteins have been proposed, ranging from regulators of endogenous proteinases to storage proteins, evidence for many of the roles is partial, or confined to isolated examples. On the other hand, many PIs have been shown to act as defensive compounds by direct assay or by expression in transgenic plants, and a body of evidence consistent with their role in plant defense has been accumulated. The case for serine PIs as defensive compounds against predators is particularly clear-cut, since the major proteinases present in plants, used for processes such as protein mobilization in storage tissues, contain a cysteine residue as the catalytically active nucleophile in the enzyme active site. Serine proteinases are apparently not used by plants in processes involving large-scale protein digestion, and thus the presence of significant quantities of inhibitors with specificity towards these enzymes in plants cannot be for the purposes of regulating endogenous proteinase activity. In contrast, a major role for serine PIs in animals seems to be to block
the activity of endogenous proteinases in tissues where this activity would be harmful, as is the case with the pancreatic trypsin inhibitors found in mammals. The presence of significant amounts of serine PIs in plant tissues therefore suggests not an endogenous role, either protective or regulatory, but instead suggests that the targets of these inhibitors are the digestive proteinases of phytophagous animals. Further evidence for a role of serine PIs in plant defense is provided by considering the sites of synthesis and accumulation of these proteins. They are normally accumulated in storage tissues, both in seeds and vegetative storage tissues such as potato tubers, and can reach concentrations as high as 2% of total protein. Since plant survival depends on the protection of storage tissues against predators, this pattern of accumulation supports the defensive role. There is little evidence that PIs accumulated in these tissues function as a storage reserve by being broken down on germination or sprouting. Direct evidence for a defensive role is shown by the synthesis of serine PIs in wounded tissue, as described below.
Recombinant Protease Inhibitors in Plants, edited by Dominique Michaud. ©2000 Eurekah.com.
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2.1.2. Inhibitors of Serine Proteinases 2.1.2.1. Inhibitors from Plant Sources The serine proteinases trypsin, chymotrypsin and elastase, which belong to a common protein superfamily, are responsible for the initial digestion of proteins in the gut of most higher animals. In vivo they are used to cleave long, essentially intact polypeptide chains into short peptides which are then acted on by exopeptidases to generate amino acids, which are the end-point of protein digestion. The three types of digestive serine proteinases are distinguished by their specificity, trypsin specifically cleaving C-terminal to residues carrying a basic side chain (lys, arg), chymotrypsin showing a preference for cleaving C-terminal to residues carrying a large hydrophobic side chain (phe, tyr, leu), and elastase showing a preference for cleaving C-terminal to residues carrying a small neutral side chain (ala, gly). Inhibitors of these serine proteinases have been described in many plant species, and are probably universal throughout the plant kingdom, with inhibitors of trypsin by far the most common type. At least part of this bias can be accounted for by the fact that (mammalian) trypsin is readily available, is the easiest of the proteinases to assay using synthetic substrates, and thus is used in screening procedures. At least seven distinct families of serine PIs have been described in plants. These proteins have a common mechanism of action: the inhibitor binds to the active site on the enzyme to form a complex with a very low dissociation constant (10-7 to 10-14 M at neutral pH values), thus effectively blocking the active site. A binding loop on the inhibitor, usually “locked” into conformation by a disulphide bond, projects from the surface of the molecule and contains a peptide bond (“reactive site”) cleavable by the enzyme. This peptide bond may be cleaved in the enzymeinhibitor complex, but cleavage does not affect the interaction, so that a hydrolyzed inhibitor molecule is bound just as well as an unhydrolyzed one. The inhibitor thus directly mimics a normal substrate for the
Recombinant Protease Inhibitors in Plants
enzyme, but does not allow the normal enzyme mechanism of peptide bond cleavage to proceed to completion (dissociation of the product). Specificity of the inhibitor-enzyme interaction is primarily determined by the specificity of proteolysis determined by the enzyme. For example, trypsin cleaves C-terminally to a basic amino acid residue, so the reactive site in a trypsin inhibitor will comprise an arg-X or lys-X dipeptide. However, the strength of interaction, and thus the effectiveness of the inhibitor is not solely determined by the reactive site, since other residues in the reactive site region of the inhibitor are also important in stabilizing the enzyme-inhibitor complex. Extensive structurefunction studies have been carried out on certain inhibitors. The most widely studied types of serine PIs in plants are both typified by inhibitors isolated from soybean seeds, and named after their discoverers: the Kunitz and BowmanBirk families of inhibitors. Kunitz inhibitors are typically monomeric proteins containing a polypeptide of approximately 190 amino acid residues, with two intra-chain disulphide bridges. Each molecule contains a single binding site which interacts strongly with the proteinase against which the inhibitor is directed (usually trypsin or chymotrypsin). Generally the distribution of proteins of this type seems to be sporadic across a range of plant families (legumes and cereals), with certain plant species such as winged bean (Psophocarpus tetragonolobus) accumulating Kunitz-type inhibitors in their seeds to a significant proportion of total protein, whereas many others contain only small amounts or no detectable proteins of this type. The Kunitz inhibitors themselves belong to a superfamily which includes proteins such as the sweet-tasting protein thaumatin and proteins induced by pathogens (PR proteins), which have sequence homology, but are functionally distinct since they do not inhibit serine proteases. Bowman-Birk inhibitors are common in seeds of legume species, and are found in other families (such as cereals) also. These PIs are proteins based on a polypeptide of 70-80
Control of Phytophagous Insect Pests Using Serine Proteinase Inhibitors
amino acids, which can form oligomers. The basic polypeptide unit contains a high proportion of cysteine residues, and forms multiple intra-chain disulphide bridges (typically 7 per monomer), leading to a very tightly folded and rigid conformation. The monomeric unit contains two binding loops with reactive sites, and can thus inhibit two molecules of proteinase per molecule of inhibitor. The two binding loops can have similar or different inhibitory specificities. These proteins can be quite abundant in seeds, making up as much as 1% of total seed protein. A further family of serine PIs is found in seeds of barley and other cereals, and is termed the barley trypsin inhibitor family. These inhibitors are proteins of 11-14,000 Mr which contain a single enzyme inhibitory site. Like Bowman-Birk inhibitors, they have a high cysteine content. Potato and other members of the Solanaceae contain two families of PIs, which are described as the potato inhibitors I and II families (PI-I, PI-II), both being originally characterized from potato tubers, where they are accumulated. These are again based on relatively small polypeptides. PI-I is an oligomeric protein containing subunits of approx. Mr 8,000; the major form is a tetramer, with an indicated Mr of 39,000.1 PI-I has a low cysteine content, and, unusually for serine PIs, disulphide bonds are not essential for activity. PI-II is a dimer, with a polypeptide size of 12 kDa; it has two reactive sites and five intra-chain disulphide bonds per monomer.2 Finally, members of the family Cucurbitaceae contain a further group of PIs in their seeds, noteworthy for their very small size (29-32 amino acid residues); these are again relatively cysteine-rich. It seems likely that further families of PIs will be identified as more plant sources are screened for inhibitory activity, and the proteins responsible are purified and characterized. PIs in plants appear to form an example of what has been termed “convergent evolution,” where proteins with similar functions have arisen from different precursors, but share a similar functional mechanism. Further details of these inhibitors can be found in review articles 3-5.
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2.1.2.2. Inhibitors from Other Sources Inhibitors of serine proteinases are also found in animal and bacterial sources, and are often involved in the regulation of endogenous proteinases. For example, the pancreas in mammals produces large quantities of a trypsin inhibitor which prevents activation of the inactive zymogen precursors of digestive enzymes, synthesized in the pancreas, until they have passed into the gut. Insect haemolymph also contains significant amounts of PIs, which belong to a superfamily of proteins designated serpins, and may regulate the activities of proteinases involved in moulting although they also inhibit digestive proteinases. A summary of these inhibitor families is provided in a recent review by Reeck and coworkers.3
2.2. Effects of Serine PIs on Insect Digestive Proteolysis 2.2.1. Introduction Four main classes of peptidases have been distinguished on the basis of the catalytically active amino acid residue or other functional group they contain: serine proteases, cysteine proteases, aspartyl proteases and metalloproteases. All four classes have been identified as playing roles in protein digestion in different species within the insect kingdom,6 and inhibitors of all four classes have been identified in different plant species. The roles these different protease types play can be divided into primary digestion (by endopeptidases, or proteinases) and secondary digestion (by exopeptidases). Metalloproteases, such as carboxypeptidases and Aminoaeptidases, are employed as exopeptidases, and thus are usually not important in the initial stages of digestion. The other classes of enzyme all function as endopeptidases. By analogy with higher animals, and on the basis of some early characterization work in species such as locust, it was assumed that serine proteinases were the major digestive enzymes in all insects. However, many coleopteran species and some Hemiptera contain both cysteine and aspartyl proteinases,
12
with little or no detectable serine proteinase activity. It is tempting to view this switch in the nature of the major digestive enzymes as an adaptation by the insect to a diet of plant tissues rich in serine PIs,6 and the fact that many phytophagous Coleoptera, which feed on seeds or other storage tissues, contain cysteine proteinases supports the hypothesis. However, cysteine proteinases are also found as major digestive enzymes in carnivorous coleopterans such as ladybirds7 and blood- sucking hemipterans such as Rhodnius prolixus,8 which are not directly exposed to plant serine PIs, and thus the causal connection cannot be proved. Even in those insects which do use serine proteinases as their major digestive enzymes, the insect enzymes differ from their mammalian counterparts in some biochemical properties. The best-studied systems are the digestive enzymes of lepidopteran larvae, particularly some of the more significant crop pests. In these insects, the gut pH is highly alkaline9 and serine proteases have correspondingly high pH optima (typically 10.5-11, as compared to approx. 9 for the mammalian enzymes). The use of substrates and chemical inhibitors specific for the different proteinases has allowed the identification of enzymes with activities typical of trypsins, chymotrypsins and elastases in lepidopteran larvae,10 although their substrate specificities and sensitivity towards PIs may show differences from the mammalian enzymes. For example, the chymotrypsins in several noctuid larvae show very low activity towards chymotrypsin substrates or inhibitors which contain only a single amino acid residue (e.g., N-benzoyl-tyrosine p-nitroanilide, BTpNA; N-tosyl- phenylalanine chloroketone, TPCK), but readily hydrolyze substrates containing short peptides such as succinylalaninealanine-proline-phenylalanine-p-nitroanilide (SAAPFpNA), and are strongly inhibited by the specific peptide inhibitor chymostatin.10 The relative contributions of the trypsin, chymotrypsin and elastase activities to overall protein digestion vary from one species to another, with trypsin and chymotrypsin being normally the major contributors (Fig. 2.1).
Recombinant Protease Inhibitors in Plants
2.2.2. Effects of Purified Serine PIs on Insects and Insect Digestion Many studies have been carried out, both in vivo and in vitro, to determine the effects of PIs on insect survival and development. In general, assays carried out in vivo have used artificial diets, into which inhibitors can be incorporated at known concentration, as the basis for bioassays which follow insect development and survival. Relative to a control diet with no added inhibitor, an “effective” PI can be expected to lower insect survival, to decrease insect biomass, and to decrease the rate of insect development in terms of time taken to pass through defined stages of the life cycle (Fig. 2.2). PIs do not usually show toxic effects, which would produce high levels of insect mortality in a short period of assay. Instead, their effects are produced in the long term, and are thus termed antinutritional, antinutritive or antimetabolic rather than toxic. Unfortunately, some bioassays have been carried out with protein fractions of questionable purity. For example, in an early investigation reported by Lipke,11 it was shown that purified soybean Kunitz and Bowman-Birk trypsin inhibitors were not toxic to larvae of the flour beetle Tribolium confusum, but a partially purified protein fraction which inhibited proteolytic activity of both insect and mammalian gut extracts was toxic. The undefined nature of such fractions has led to questioning the role of PIs in the effects observed, although in the example given the failure of soybean Kunitz and Bowman-Birk inhibitors to affect Tribolium larvae is not surprising since these insects use cysteine proteinases for digestion.12 The inhibition of proteolysis observed was possibly due to an inhibitor of cysteine proteases also present in soybean, albeit at low level.13 In general, there can now be no doubt that PIs are antimetabolic to insects, but there is a specificity in the effect observed, both from inhibitor to inhibitor and from insect species to insect species. The effects of PIs on insect digestive enzymes can be measured directly, by means of enzyme assays carried out in vitro.14 Most workers have measured the effects of PIs on
Control of Phytophagous Insect Pests Using Serine Proteinase Inhibitors
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Fig. 2.1. Proportion of proteolytic activity (azoalbumin hydrolysis) attributable to different serine proteinases in gut extracts from two lepidopteran larvae, shown by inhibition of proteolysis with chemical inhibitors specific to different proteinase activities. the proteases present in insect gut contents, which usually contain mixtures of digestive enzymes. However a few assays have employed purified enzymes. Provided that the synthetic substrates used are specific for particular enzymes, the use of gut contents for enzyme assays does not cause problems in interpretation of data, and allows parameters measuring the effectiveness of different inhibitors (e.g., I50, the concentration of inhibitor to cause a 50% decrease in enzyme activity, or Ki, the
Michaelis-Menten Inhibition constant) to be determined. The use of gut contents is also advantageous when the effects of inhibitors on total proteolytic activity in the insect gut are measured. However, for detailed study of the parameters of interaction between inhibitors and proteolytic enzymes, purified insect enzymes are necessary. In vitro inhibition assays have shown that plant PIs are as effective against insect digestive proteases as against those of higher animals.
14
Recombinant Protease Inhibitors in Plants
Fig. 2.2. Typical effects of a serine PI (soybean Kunitz trypsin inhibitor, SKTI) on survival, growth and development of lepidopteran larvae (Lacanobia oleracea, tomato moth) when delivered in artificial diet (2.0% of total s). Slower growth is shown by the inhibitor causing a significant and progressive decrease in mean larval weight, and retarded development by slower progression through the larval instars, as shown by the instar distributions at three time points. See Gatehouse et al56 for complementary data. However, the strengths of interaction (i.e., the effectiveness of inhibition) for a given inhibitor with insect and mammalian enzymes of similar substrate specificities differ. Further, when different insect species are compared, a given inhibitor will be found to be more effective at inhibiting proteolysis in some species than others. Serine PIs in many phytophagous insect species will inhibit the enzymes of primary protein digestion, and might be thought to be antimetabolic simply by virtue of preventing efficient utilization of protein in the diet. In support of this idea, supplementation of
limiting amino acids in the diet can overcome the antimetabolic effects of inhibitors.15 Prevention of efficient recycling of the amino acids used to synthesize proteinases may be a further significant factor in the antimetabolic effects observed, since the digestive proteinases in animals are normally subject to autodigestion, and this can be blocked by PIs.
2.3. The Wounding Response in Plant Defense The clearest evidence for the role of serine PIs in the defense of plants against insect pests is the induced synthesis of these
Control of Phytophagous Insect Pests Using Serine Proteinase Inhibitors
proteins that occurs when many plant species are wounded, which can be caused by insect feeding, or mimicked by mechanical damage. The wound response in plants has been extensively investigated over recent years. Wounding has been shown to result in a variety of changes in the physiological state of the tissue, and can either result in a local reaction when it is restricted to a defined region in the near vicinity of the original wound such as in the case of the production of phytoalexins, or a systemic reaction, when the response occurs in tissues some distance from the wound site. One of the best studied cases of a systemic reaction induced in higher plants was the demonstration that wounding of the leaves of either potato or tomato plants by adult Colorado potato beetles or their larvae induced a rapid accumulation of serine PIs.16 This accumulation was not confined to just wounded leaves, but was also found to be present in leaves which had not been attacked. Mechanical damage was also found to cause an accumulation of these inducible PIs.17 Interestingly, it has recently been shown that the timing of transcript accumulation of several wound-induced genes in insectdamaged leaves is different to that in mechanically damaged leaves, with transcripts for the inhibitor PI-II accumulating more rapidly in potato leaves which had been damaged by insects than those damaged mechanically. It would appear that insect regurgitant, in this case from Manduca sexta larvae, causes the transcript accumulation profiles to shift to parallel those in insect damaged tissue. On the basis of their findings, Korth and Dixon18 suggested the presence of a heat-stable, insect derived elicitor which functions to induce the rapid accumulation of transcripts that may be involved in plant defense against herbivores. A factor, or wound hormone, called the PI-inducing factor (PIIF) was found to be released from the damaged leaves and transported to other leaves within 2-3 hours after wounding where it initiates synthesis and accumulation of the two potato serine PIs, PI-I and PI-II.17 Transport appears to take place in the phloem and is directed predomi-
15
nantly upwards to younger leaves,19 although evidence for hydraulic signals in the xylem and electrical signals have also been presented. The levels of PIIF released in response to wounding depends upon both the severity and location of the wound. Subsequent work revealed that the PIIF was a highly methylated polysaccharide containing galacturonic acid, rhamnose, galactose, arabinose and fucose, and that it was probably a fragment of the plant cell wall, at least in tomato.20 Within two to three days after attack the inhibitors can account for over 10% of the soluble proteins in leaves throughout the plant, where they can remain for long periods of time stored in the central vacuoles of the cells.21,22 The accumulation requires light and is temperature-dependent. These wound-induced inhibitors have been well characterized. Both PI-I and PI-II are potent inhibitors of chymotrypsin and subtilisin, and both inhibit trypsin, but less strongly. More recently, a wound-induced trypsin inhibitor has been isolated and characterized from alfalfa leaves. This particular inhibitor was identified as a member of the Bowman-Birk inhibitor family.23 Following these initial reports, genes encoding the wound-inducible inhibitors from both potato24 and tomato25 have been isolated and characterized. Transformation of tobacco plants with a gene encoding potato PI-II resulted in a systemic induction of the transgene expression after wounding, 26 showing that the signal inducing PI gene expression was similar in the two species. Several plant-derived chemicals, including methyl jasmonate, jasmonic acid4 and an 18-amino acid polypeptide called systemin were found to regulate the expression of wound-inducible PI genes. Growth regulators such as abscisic acid and auxin were similarly shown to regulate expression of these genes. A currently accepted model for the expression of wound-inducible PIs is that systemin is released by wounding and activates a membrane-derived lipid signal. This signal is thought to be linolenic acid, which is subsequently converted to jasmonic acid4 via the octadecanoid pathway.27 Further informa-
16
tion can be found in a recent review by Bergey et al.28 A signal cascade then leads to the activation of jasmonate-responsive genes (jrgs), and their products are responsible for the physiological response. The involvement of systemin in the wound response and subsequent accumulation of wound-inducible PIs has been elegantly demonstrated by exposing transgenic tomato plants expressing an antisense prosystemin gene to lepidopteran larvae. The plants containing the antisense construct were significantly more attacked than the corresponding control plants, with the insects on the transgenic plants being significantly larger, due to suppression of the normal wounding response.29 An alternative model for jasmonate signalling in barley has been put forward recently, whereby there are several distinct signal-transduction pathways. Contrary to what occurs in tomato and potato, in barley the abscisic acid signal is not mediated through a jasmonate signal cascade, but is independent of it. In the model put forward by Lobler and Lee,30 induction of jrgs 9 (a “wounding-induced” gene) is caused by the presence of products resulting from activation of expression of gene families, some of which are individually induced by either abscisic acid or jasmonate. Jasmonate signalling is mediated through two pathways, one sensing extracellular jasmonate and the other sensing intracellular jasmonate. It has also been suggested that two distinct signal transduction pathways that can distinguish between insect damage and abiotic damage may be involved in the wound-induced responses in potato.18
2.4. “First-Phase” Use of Foreign Serine PI Genes Although the possibility that plant PIs were insecticidal compounds had been suggested as early as 1947,31 a clear demonstration that these proteins could be significant factors in protecting plants against insect attack was not put forward until 1972, when Ryan and coworkers16 demonstrated their accumulation in the leaves of certain species of solanaceous plants as a result of wounding
Recombinant Protease Inhibitors in Plants
(see above). These authors suggested that insect damage might act as a trigger for the synthesis of PIs, which would then act as defensive proteins by blocking insect digestion, leading to starvation and death. Evidence that this protective role could be observed in plant tissues which had accumulated PIs was first provided by our group,32 after we observed a correlation between high levels of trypsin inhibitor and resistance to a seed storage pest, the bruchid Callosobruchus maculatus, in seeds of cowpea (Vigna unguiculata), and showed that addition of the cowpea PI to a semiartificial diet inhibited development and decreased survival of larvae of this insect. Although subsequent work has shown that other factors are also involved in resistance of cowpea varieties to the seed weevil,33 our initial finding supported the hypothesis put forward by Ryan and colleagues, and opened the way for the subsequent exploitation of such compounds in crop protection by genetic manipulation.
2.4.1. Identification of Antimetabolic PIs In order to identify suitable PIs and their encoding genes for transfer to plants, systematic studies of a wide range of inhibitors have been carried in an attempt to find a kinetic parameter which could be useful in predicting the potential of any inhibitor to act as a resistance factor to a given pest. The dissociation constant of the enzyme/inhibitor complex was suggested as such a parameter (see Chapter 10, this volume).34 Although this strategy has been successfully adopted in specific cases, there are in fact limitations on relying solely upon information obtained from in vitro tests, as will be discussed in detail elsewhere (see Section 2.5). To this end, testing the inhibitor in artificial diet against the target insects is more reliable, although this too has its limitations, as was found to be the case for SKTI when tested against the tomato moth Lacanobia oleracea in artificial diet and on SKTI-expressing transgenic potato plants (see Section 2.5).
Control of Phytophagous Insect Pests Using Serine Proteinase Inhibitors
2.4.2. Insect-Resistant Transgenic Plants Expressing Serine PIs The first gene of plant origin to be transferred to another plant species to produce enhanced insect resistance was isolated from cowpea, and encoded a Bowman-Birk type serine PI with two inhibitory sites active against bovine trypsin (CpTI). 35 We produced a full-length cDNA clone of CpTI, and placed its coding sequence under the control of the constitutive cauliflower mosaic virus (CaMV) 35S promoter in the final construct prepared for transfer to plants. Transgenic tobacco plants were produced by a standard Agrobacterium tumefaciensmediated transformation protocol, using a binary vector system. Transformants were screened for CpTI expression, which showed that many of the resulting plants expressed CpTI at levels greater than 0.1% of total soluble protein, a general observation for expression of proteins of this type in transgenic plants. Plants expressing CpTI at the highest levels (approximately 1% of total soluble protein) were clonally propagated, and used for insect bioassays. Initially bioassays were carried out using first-instar larvae of the tobacco budworm, Heliothis virescens, a pest of major economic importance attacking tobacco, cotton and maize. With these clonal plants and subsequent generations derived from their self-set seed, the CpTI-expressing plants showed reduced damage by up to 50% compared to the control plants, and reduced insect survival and biomass, again by as much as 50%. The antimetabolic effects of CpTI expressed in transgenic tobacco have also been observed with other lepidopteran pests including H. zea, Spodoptera littoralis and Manduca sexta. Subsequent trials carried out in California showed that the expression of CpTI in tobacco afforded significant protection in the field against H. zea.36 Following on from the study using tobacco as a model system, the gene encoding CpTI has been expressed in a range of different crops (Table 2.1). Constitutive expression of CpTI in potato showed significant antimetabolic effects on larvae of the tomato moth (Lacanobia oleracea), with
17
mean larval weight being decreased by nearly 50% in plants expressing CpTI at approx. 0.5% of total soluble protein in the leaves,37 although little effect on larval survival was observed and plant damage was also little reduced (Fig. 2.3). A better example is the use of this gene to protect strawbery against damage by vine weevil Otiorhynchus sulcatus, which is the major pest of strawbery in Europe, where it is endemic. The larvae damage root systems resulting in severe loss of yield or plant death, and recent reports suggest that damage is no longer confined to the root systems but is also occurring on the vegetative tissues. Since the withdrawal of persistent organochlorines, at least in parts of Europe, the control of adults and larvae has become difficult as few of the currently available products are effective under field conditions and no genetic resistance in commercial germplasm has been identified. The CpTI construct described above has been inserted into strawbery cultivars by Agrobacterium-mediated gene transfer for control of this pest.38,39 Results from two independent glasshouse trials showed that expression of the CpTI gene in transgenic strawbery plants was effective in protecting roots from vine weevil larval feeding.40 Control plants inoculated with vine weevil eggs had significantly less root mass than equivalent non-inoculated plants (see Fig. 2.4), and showed a substantial and significant reduction in weight compared with all but one of the transgenic lines; similar results have been obtained in recent field trials (Graham, pers. comm.). Although this inhibitor is at present being constitutively expressed, work is underway to identify a promoter which will not be active in the fruit of this plant. Similarly, encouraging results have also been obtained in rice where the constitutive expression of CpTI conferred significantly enhanced levels of resistance in the field towards two species of rice stem borer, Sesamia inferens and Chilo suppressalis.41 Despite CpTI being insecticidal against a wide spectrum of insect pests, mammalian feeding trials incorporating the purified protein at levels of 10% of total protein content in the diet showed no acute toxic effects,42 possibly
Recombinant Protease Inhibitors in Plants
18
Table 2.1. Transgenic plants expressing plant serine PIs with enhanced resistance towards insects Inhibitor
Plant
Pest
CpTIa
tobacco rice
Heliothis virescens(L) Chilo suppressalis (L) Sesamia inferens (L) Lacanobia oleracea (L) Otiorynchus sulcatus (C)
35 41 41 37 40
Manduca sexta (L) Chrysodeixis eriosoma (L) Chilo suppressalis (L) Sesamia inferens (L)
43 45 46 46
potato strawbery PI-II
tobacco rice
Reference
Barley TI
tobacco
AgrotisIpsilon (L) Spodoptera littoralis (L)
59 59
Na PI
tobacco
Helicoverpa punctigera (L)
60
aAbbreviations: CpTI, cowpea trypsin inhibitor; PI-II, potato wound inducible protease inhibitor II;
NA PI, NIcotiana alata multi-functional proteinase inhibitor; Barley TI, barley trypsin inhibitor; L, lepidoptera; C, Coleoptera
reflecting differences in the organization of insect and mammalian digestive systems. Other serine PI-encoding genes have also been tested as protective agents for crops. For example, the tomato PI-II gene (which encodes a trypsin inhibitor with some chymotrypsin inhibitory activity), when expressed in tobacco, was shown to confer insect resistance43 when expressed constitutively using the CaMV 35S promoter, but, interestingly, not when expressed with a wound-inducible promoter. The bioassays showed that the decrease in larval weight in insects reared on transgenic plants was roughly proportional to the level of PI-II being expressed. Several of the transgenic plants were shown to contain inhibitor levels over 200 µg/g tissue, which is within the range that is routinely induced by wounding leaves of either tomato or potato plants.44 However, tobacco plants expressing tomato PI-I at similar levels had no deleterious effects upon larval development, showing the specificity
of interactions between inhibitors and insect species. McManus et al45 obtained similar results with potato PI-II expressed in tobacco against the noctuid lepidopteran Chrysodeixis eriosoma, the green looper. The woundinducible potato PIs (PI-I and PI-II) have now been constitutively expressed in a range of crops where they have been shown to confer resistance (Table 2.1). As with CpTI, expression of PI-II in rice conferred significant levels of protection in the field towards rice stem borers.46 Serine PIs from animal sources have also been tested as potential protective agents. For example, the serpin-type serine PIs from insect haemolymph have been identified as possibly being targeted against insect proteinases, since they may play an endogenous regulatory role. Transgenic alfalfa, cotton and tobacco expressing insect haemolymph inhibitors have been produced,47-49 and some protection against insect pests has been observed. The most interesting result has been
Control of Phytophagous Insect Pests Using Serine Proteinase Inhibitors
19
Fig. 2.3. Effects of the serine PI cowpea trypsin inhibitor (CpTI) on larvae of tomato moth (Lacanobia oleracea) when expressed in transgenic potato plants at approx. 0.5% of total soluble leaf protein. Eight neonate larvae were placed onto each of 12 control and 19 transgenic plants, and allowed to develop for 22 days, when the most developed larvae were in the final instar. The transgenic plants showed marginally reduced survival (but not significant at p<0.05), and a reduction in mean larval weight by approx. 45% (significant at p<0.0001), but leaf damage was not reduced in this assay. the production of transgenic tobacco with enhanced resistance to the whitefly, Bemeisia tabaci.48 Although the range of serine PIs used and the range of crops transformed is ever increasing, the commercial viability of this strategy has yet to be proven. The level of protection afforded by the expression of these inhibitors in transgenic plants is often better than 50%, in terms of reduction in plant damage, decrease in insect survival and biomass, but these levels are not considered to be high enough to be commercially viable, where the benchmark is often protection levels of >95%, i.e., comparable with the results of chemical pesticides or tacillus thuringiensis endotoxins. Several laboratories are actively addressing ways in which to increase the efficacy of serine PIs as protective agents against insect pests. However, a greater understanding of the mechanism of action of
PIs in insects, both at the biochemical and molecular levels, will be necessary before the technology can be fully exploited.
2.5. Insect Responses to Dietary Serine PIs Despite successes in enhancing the resistance of transgenic plants towards insects by the expression of foreign PIs, other examples of this technology have given disappointing results, with little or no protection observed. For example Jongsma et al 50 produced transgenic tobacco plants expressing the chymotrypsin/trypsin-specific potato inhibitor II (PI-II) constitutively. In contrast to results reported above in Section 2.4, the growth of Spodoptera exigua larvae fed with detached leaves of plants expressing PI-II was not affected. These results, with other observations, enabled the authors to show that the insect could respond to the
20
Recombinant Protease Inhibitors in Plants
Fig. 2.4. Protection of strawbery plants expressing the trypsin inhibitor from cowpea (CpTI) against larvae of vine weevil (Otiorhyncus sulcatus). The plant on the left is a transgenic strawbery expressing CpTI; the plant on the right is a control. Both plants were exposed to vine weevil larvae in a glasshouse trial. (Photo courtesy of Julie Graham, Scottish Crop Research Institute, Invergowrie, Dundee, Scotland.) presence of PIs in its diet by inducing novel proteinase activities which are insensitive to inhibition. In the example quoted, larvae exposed to plants constitutively expressing PI-II exhibited a change in the nature of their protease enzymes. They contained lower levels of proteinase activity sensitive to inhibition by PI-II than larvae raised on control plants (by 4-fold), but had higher levels of proteinase activity which was insensitive to inhibition by PI-II (by 2.5-fold). The insects reared on transgenic plants thus contained protease activity which had a greatly reduced sensitivity to inhibition by the transgene product, explaining why no antimetabolic effect was observed. The authors also observed induction of proteinase activity insensitive to the endogenous wound-induced proteinase inhibitors of tobacco, and suggested that this was a general mechanism through which insects were able to overcome the accumulation of PIs caused by the wounding response in plants. Similar induction of inhibitor-insensitive
proteinase activity was also observed by Broadway51 in two other lepidopteran larvae as a result of chronic ingestion of cabbage PIs in artificial diet. Results recently reported by our group52 have clarified the molecular mechanisms involved in the response of lepidopteran larvae to PIs. The trypsin-like activity in Helicoverpa armigera larvae has been shown to be sensitive to inhibition by soybean Kunitz trypsin inhibitor (SKTI) in vitro, but preliminary assays had suggested that transgenic plants expressing this inhibitor showed little or no antimetabolic effects. Whereas insects fed on control artificial diet for 7 days contained trypsin-like activity which could be inhibited almost totally (94%) by SKTI, insects fed on diet containing SKTI showed adaptation and contained trypsin-like activity which was relatively insensitive to inhibition by SKTI (25% inhibition). The insects were shown to contain relatively large gene families encoding serine proteinases by Southern blot analysis
Control of Phytophagous Insect Pests Using Serine Proteinase Inhibitors
of genomic DNA, and by direct sequencing of cDNA clones; at least 28 genes encoding trypsin- and chymotrypsin-like proteins were identified. Differences in sequences were observed when different “trypsins” and “chymotrypsins” were compared. Whereas the catalytically active residues were conserved, as were residues in the substrate binding pocket which determined the specificity of cleavage, residues in the areas of contact between the proteinases and protein PIs were found to be variable. This variability was suggested to lead to differing interactions between different trypsins or chymotrypsins and PIs. The expression of different genes was differentially affected by exposure of the insects to SKTI. Chymotrypsin-encoding mRNAs generally increased in level whereas some trypsinencoding mRNAs decreased, while others increased or showed little change. We suggested that the presence of families of trypsin and chymotrypsin genes encoding proteinases with different sensitivities to PIs allows the insect to adapt to the presence of PIs in its diet. Thus, exposure to an inhibitor causes the synthesis of “sensitive” proteinases to be repressed, while the synthesis of “insensitive” proteinases is up-regulated. Time course experiments showed that the response of mRNA levels to dietary inhibitor is relatively rapid, and occurs over a timescale of hours (Fig. 2.5). This response is significantly faster than the wound-induced accumulation of PIs in plants (timescale approx. 48 h), and thus would be able to protect the insect from the plant’s response to wounding, in agreement with the observations of Jongsma et al50 Although these experiments have provided a basis to explain the poor performance of some transgenic crops expressing PIs when attacked by lepidopteran pests, the data leave many questions unanswered. H. armigera is a highly polyphagous pest, which must be able to adapt to a wide range of foodstuffs containing different PIs, and thus may be atypical of insects generally in possessing the capacity to induce a range of proteases with differing sensitivities to inhibitors. However, large families of genes encoding serine proteinases have been reported in dipteran insects, and
21
other Lepidoptera like Manduca sexta also have more than one gene encoding these enzymes, since several cDNA clones have been isolated, or multiple isoforms of the enzyme are present. In contrast the spruce budworm, Choristoneura fumiferana has been reported to contain only a single trypsin-encoding gene by Southern blotting of genomic DNA.53 The extent to which different PIs cause different changes in gene expression, and thus give rise to different mixtures of dietary proteinases is unknown. The response may be an “all-ornothing” switch, or it may be finely tuned to adjust the complement of dietary proteinases to the composition of the diet. Finally, the mechanism by which the insect “senses” the presence of PIs and adjusts gene expression accordingly remains unknown for phytophagous insects. Results obtained with bloodfeeding insects such as mosquito and blackfly (reviewed in Lehane et al54) show a strong induction of activity of trypsin-encoding genes by a blood meal, and also suggest that the activity of some proteinase genes (the so-called “late” trypsin genes) is controlled by the products of others (“early” trypsin genes). In this scheme, peptides produced by the action of the “early” trypsin enzymes (and other proteases) act as signals to induce the expression of the “late” trypsin genes. 55 Interestingly, in the mosquito, addition of a PI, SKTI, to the diet blocks the activation of “late” trypsin genes, providing another example of inhibitors affecting insect proteinase gene expression. The relevance of these results to lepidopteran larvae, which feed continuously, is difficult to assess, but it seems likely that similar control mechanisms are present and allow the expression of proteinase genes to be controlled by dietary components.
2.5.1. Implications for the Production of Insect-Resistant Transgenic Plants The responses of insects to dietary PIs pose significant obstacles to be overcome if genes encoding serine PIs are to be used for the protection of transgenic plants against polyphagous lepidopteran insect pests. A major problem is the removal of a logical link between the effectiveness of an inhibitor
22
Recombinant Protease Inhibitors in Plants
Fig. 2.5. Response of genes encoding serine proteases to dietary PI in larvae of the lepidopteran, Helicoverpa armigera (Bown et al, unpublished). mRNA levels for four different serine protease genes were assessed as a proportion of total RNA by Northern blotting of total RNA isolated from larval guts, followed by densitometry of the autoradiographs. Larvae were taken at the start of the fourth instar, and exposed to diet containing soybean Kunitz trypsin inhibitor (SKTI) at 2% of total protein, or control diet, for the stated times before RNA extraction. The differential expression of different protease mRNAs is shown by the strong up-regulation of sequences detected by cDNAs SR28 (trypsin-like) and SR42 (chymotrypsin-like) in the presence of SKTI, whereas sequences detected by cDNA SR40 (trypsin-like) are little affected, and sequences detected by HaTC16 (trypsin-like) are down-regulated relative to the control. determined in vitro, by enzyme assays carried out on gut extracts from the target insect pest, and the effectiveness of the inhibitor as an antimetabolite when expressed in transgenic plants. This point is well illustrated by a work we carried out with larvae of the tomato moth, Lacanobia oleracea, a noctuid lepidopteran pest.56 In this insect the serine proteases trypsin, chymotrypsin and elastase account for over 90% of measured gut proteolysis (see Fig. 2.1). Several plant protein PIs were assayed in vitro to determine their relative
effectiveness, both in terms of the concentration of inhibitor necessary to decrease proteinase activity against synthetic substrates by 50%, and in terms of estimated Ki values. SKTI was judged the most effective inhibitor tested, being the only one to inhibit all three proteinase activities. When tested in a semi-artificial diet based on freeze-dried potato leaves, the protein showed significant antimetabolic activity, causing enhanced larval mortality (especially during the initial stages of development), decreased larval biomass, and a retardation of development as estimated
Control of Phytophagous Insect Pests Using Serine Proteinase Inhibitors
by mean time to successive instars. However, when expressed in transgenic potatoes, SKTI had only a marginal effect on L. oleracea larvae. Small decreases in survival and larval biomass, and a slight retardation of development were noted, but in contrast to the artificial diet assay, where the decrease in larval biomass relative to controls was progressive as development proceeded, the difference between larvae on control and SKTI- expressing plants decreased as development proceeded. In this insect, there was little evidence for the production of SKTI-insensitive trypsin-like enzymes. Instead, trypsin-like activity was increased by 2-4 fold in insects exposed to SKTI in the artificial diet bioassay, but not in insects exposed to SKTI expressed in transgenic plants. We speculated that the over-production of trypsin-like enzymes as a response to SKTI leads to deleterious effects in artificial diet, but the over-production does not occur in transgenic plants because it is over-ridden by a response to the woundinduced PIs present in both control and transgenic plants. In contrast to the situation where PIs have no protective effects in transgenic plants when assays in vitro would predict antimetabolic effects, a number of examples have been reported where the expression of PIs in transgenic plants have insecticidal effects which might not have been expected on the basis of their activity in vitro. The most striking example is the resistance towards vine weevil larvae in strawberry40 produced by expression of the trypsin (serine) PI from cowpea, CpTI; see Section 2.4 above. Vine weevil larvae use cysteine proteinases as their major digestive enzymes, with serine proteases being either absent, or making a small contribution,57 and assays show that CpTI has very little or no effect on proteinase activity against synthetic substrates in vine weevil larval gut extracts (see note added in proof ). This result reflects the original observation that CpTI decreases survival and retards development of bruchid larvae, which also use cysteine proteinases as major digestive enzymes.32,58 CpTi is also more effective than SKTI as an antimetabolite towards
23
lepidopteran larvae when expressed in transgenic plants, despite being less effective as an inhibitor in vitro.37,56
2.6. Conclusions Without a detailed understanding of the responses of insects towards PIs at the molecular level, it is not possible to explain the results outlined above, and to rationalize why inhibitors are more or less effective against insect pests than would be predicted on the basis of in vitro enzyme inhibition when expressed in transgenic plants. However, such a detailed understanding is now achievable by applying molecular biological techniques to the characterization and expression of proteinase genes in phytophagous insects. In addition, such understanding will lead to identification of the mechanisms which control proteinase gene expression, and to the possibility of interfering with that control. Alternatively, through the techniques of in vitro mutagenesis and heterologous expression inhibitors can be designed and produced which will inhibit the “insensitive” proteases which insects produce as a response to PIs. These approaches will lead to a “second phase” of PI-based strategies for plant protection, which will form a valuable resource in the continuing need to counter the adaptation of insects to existing strategies of crop protection, both conventional and transgenic.
Addendum A Note added in proof Recent results in our laboratory show that vine weevil larvae do contain serine proteases similar to mammalian trypsin, and have significant levels of protease activity in gut extracts inhibitable by protein inhibitors of serine proteases.
Acknowledgments We thank the Scottish Office and BBSRC for current funding, Pestax Ltd. (Axis Genetics) for previous funding for work with CpTI, and Dr. Julie Graham for the photograph used in Fig. 2.4.
24
References 1. Melville JC, Ryan, CA. Chymotrypsin inhibitor from potatoes. J Biol Chem 1972; 247:3445-3453. 2. Bryant J, Green TR, Gurusaddaiah T et al. Proteinase inhibitor II from potatoes: Isolation and characterization of its protomer components. Biochemistry 1976; 15:3418-3423. 3. Reeck GR, Kramer KJ, Baker JE et al. Proteinase inhibitors and resistance of transgenic plants to insects. In: Carozzi N, Koziel M, eds. Advances in Insect Control: The Role of Transgenic Plants. London: Taylor and Francis, 1997:157-183. 4. Ryan CA. Proteinase inhibitors. In: Marcus A, ed. The Biochemistry of Plants. New York: Academic Press, 1981:351-370. 5. Garcia-Olmedo F, Salcedo G, SanchezMonge R et al. Plant proteinaceous inhibitors of proteinases and α-amylases. Oxford Surv Plant Mol Cell Biol 1987; 4:275-334. 6. Terra WR, Ferreira C, Jordao BP et al. Digestive enzymes. In: Lehane MJ, Billin London: Chapman and Hall, 1996:153-194. 7. Walker AJ, Ford L, Majerus MEN et al. Characterisation of the midgut digestive proteinase activity of the two-spot ladybird (Adalia bipunctata L.) and its sensitivity to proteinase inhibitors. Insect Biochem Mol Biol 1998; 28:173-180. 8. Houseman JG, Downe AER. Endoproteinase activity in the posterior midgut of Rhodnius prolixus Stal (Hemiptera: Reduviidae). Insect Biochem 1980;10:363-366. 9. Dow JAT. Insect midgut function. Adv Insect Physiol 1986; 19:187-238. 10. Johnston KA, Lee MJ, Brough C et al. Protease activities in the larval midgut of Heliothis virescens; identification of a chymotrypsin-like enzyme. Insect Biochem Mol Biol 1995; 25:375-383. 11. Lipke H, Fraenkel GS, Liener IE. Effect of soybean inhibitors on growth of Tribolium confusum. J Agric Food Chem 1954; 2:410-414. 12. Blancolabra A, Martinezgallardo NA, Sandovalcardoso L et al. Purification and characterization of a digestive cathepsin-D proteinase isolated from Tribolium castaneum larvae (Herbst). Insect Biochem Mol Biol 1996; 26:95-100. 13. Hines ME, Osuala CI, Nielsen SS. Isolation and partial characterization of a soybean cystatins of coleopteran digestive proteolytic activity. J Agric Food Chem 1991; 39:1515-1520. 14. Christeller JT, Laing WA, Markwick NP et al. Midgut protease activities in 12 phytophagous lepidopteran larvae: Dietary and protease inhibitor interactions. Insect Biochem Mol Biol 1992; 22:735-746.
Recombinant Protease Inhibitors in Plants 15. Broadway RM, Duffey SS. The effect of plant protein quality on insect digestive physiology and the toxicity of plant proteinase inhibitors. J Insect Physiol 1988; 34:1111-1117. 16. Green TR, Ryan CA. Wound-induced proteinase inhibitor in plant leaves: A possible defence mechanism against insects. Science 1972; 175:776-777. 17. Ryan CA. Defense responses of plants. In: Verma DPS, Hohn T, eds. Plant Gene Research: Genes Involved in Microbe-Plant Interactions. New York: Springer-Verlag, 1984:375-386. 18. Korth KL, Dixon RA. Evidence for chewing insect-specific molecular events distinct from a general wound response in leaves. Plant Physiol 1997; 115:1299-1305. 19. Makus D, Zuroske G, Ryan CA. The direction and rate of transport of the proteinase inhibitor inducing factor out of wounded tomato leaves. Plant Physiol 1980; 65:Suppl 150. 20. Ryan CA, Bishop P, Pearce G et al. A sycamore cell wall polysaccharide and a chemically related tomato leaf polysaccharide possess similar proteinase inhibitor-inducing activities. Plant Physiol 1982; 68:616-618. 21. Shumway K, Yang VV, Ryan CA. Evidence for the presence of proteinase inhibitor I in vacuolar bodies of plant cells. Planta 1976; 129:161-165. 22. Walker-Simmons M, Ryan CA. Immunological identification of proteinase inhibitors I and II in isolated tomato leaf vacuoles. Plant Physiol 1977; 60:61-63. 23. Brown WE, Takio K, Titani K et al. Woundinduced trypsin inhibitor in alfalfa leaves: Identity as a member of the Bowman-Birk inhibitor family. Biochemistry 1985; 24:2105-2108. 24. Cleveland TE, Thornburg RW, Ryan CA. Molecular characterization of a woundinducible inhibitor 1 gene from potato and the processing of its mRNA and protein. Plant Mol Biol 1987; 8:199-207. 25. Lee JS, Brown WE, Graham JS et al. Molecular characterisation and phylogenetic studies of a wound inducible proteinase inhibitor I gene in Lycopersicon species. Proc Natl Acad Sci USA 1986; 83:7277-7281. 26. Sanchez-Serrano JJ, Keil M, O’Connor A et al. Wound-induced expression of a potato proteinase inhibitor II gene in transgenic tobacco plants. EMBO J 1987; 6:303-306. 27. Farmer EE, Ryan CA. Octadecanoid precursors of jasmonic acid activate the synthesis of wound-inducible proteinase inhibitors. Plant Cell 1992; 4:129-134.
Control of Phytophagous Insect Pests Using Serine Proteinase Inhibitors 28. Bergey DR, Howe GA, Ryan CA. Polypeptide signaling for plant defensive genes exhibits analogies to defense signaling in animals. Proc Natl Acad Sci USA 1996; 93:12051-12058. 29. Orozco-Cardenas M, McGurl B, Ryan CA. Expression of an antisense prosystemin gene in tomato plants reduces resistance toward Manduca sexta larvae. Proc Natl Acad Sci USA 1993; 90:8273-8276. 30. Lobler M, Lee J. Jasmonate signalling in barley. Trends Plant Sci 1998; 3:8-9. 31. Mickel CE, Standish J. Susceptibility of processed soy flour and soy grits in storage to attack by Tribolium castaneum (Herbst). Univ Minnesota Agric Exp Stat Bull 1947; 178:1-20. 32. Gatehouse AMR, Gatehouse JA, Dobie P et al. Biochemical basis of insect resistance in Vigna unguiculata. J Sci Food Agric 1979; 30:948-958. 33. Xavier-Filho J, Campos FAP, Ary MB et al. Poor correlation between levels of proteinase inhibitors found in seeds of different cultivars of cowpea (Vigna unguiculata) and the resistance/susceptibility to predation by Callosobruchus maculatus. J Agric Food Chem 1989; 37:1139-1143. 34. Christeller JT, Shaw BD. The interaction of a range of serine proteinase inhibitors with bovine trypsin and Costelytra zealandica trypsin. Insect Biochem 1989; 19:233-241. 35. Hilder VA, Gatehouse AMR, Sherman SE et al. A novel mechanism for insect resistance engineered into tobacco. Nature 1987; 330:160-163. 36. Hoffman MP, Zalom FG, Smilanick JM et al. Field evaluation of transgenic tobacco containing genes encoding Bacillus thuringiensis δ-endotoxin or cowpea trypsin inhibitor: Efficacy against Helicoverpa zea (Lepiodptera: Noctuidae). J Econ Entomol 1991; 85:2516-2522. 37. Gatehouse AMR, Davison GM, Newell CA et al. Transgenic potato plants with enhanced resistance to the tomato moth Lacanobia oleracea; growthroom trials. Mol Breeding 1997; 3:49-63. 38. Graham J, McNicol RJ, Greig K. Towards genetic based insect resistance in strawberry using the cowpea trypsin inhibitor. Ann Appl Biol 1995; 127:163. 39. Graham J, Gordon SC, Williamson B. Progress towards the use of transgenic plants as an aid to control soft fruit pests and diseases. In: Brighton Crop Protection Conference—Pests and Diseases. Brighton, UK, 1996:777-782.
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40. Graham J, Gordon SC, McNicol RJ. The effect of the CpTI gene in strawberry against attack by vine weevil (Otiorhynchus sulcatus F. Coleoptera: Curculionidae). Ann Appl Biol 1997; 131:133-139. 41. Xu DP, Xue QZ, McElroy D et al. Constitutive expression of a cowpea trypsininhibitor gene, CpTI, in transgenic rice plants confers resistance to 2 major rice insect pests. Mol Breeding 1996; 2:167-173. 42. Pusztai A, Grant G, Stewart JC et al. Nutritional evaluation of the trypsin inhibitor from cowpea. Br J Nutr 1992; 68:783-791. 43. Johnson R, Narvaez J, An G et al. Expression of proteinase inhibitors I and II in transgenic tobacco plants: Effects on natural defense against Manduca sexta larvae. Proc Natl Acad Sci USA 1989; 86:9871-9875. 44. Graham JS, Hall G, Ryan CA. Regulation of synthesis of proteinase inhibitors l and ll mRNAs in leaves of wounded tomato plants. Planta 1986; 169:399-405. 45. McManus MT, White DWR, McGregor PG. Accumulation of a chymotrypsin inhibitor in transgenic tobacco can affect the growth of insect pests. Transgenic Res 1994; 3:50-58. 46. Duan X, Li X, Xue Q et al. Transgenic rice plants harboring an introduced potato proteinase inhibitor II gene are insect resistant. Nat Biotechnol 1996; 14:494-496. 47. Thomas JC, Wasmann CC, Echt C et al. Introduction and expression of an insect proteinase inhibitor in alfalfa (Medicago sativa L.). Plant Cell Rep 1994; 14:31-36. 48. Thomas JC, Adams DG, Keppenne VD et al. Manduca sexta encoded protease inhibitors expressed in Nicotiana tabacum provide protection against insects. Plant Physiol Biochem 1995; 33:611-614. 49. Thomas JC, Adams DG, Keppenne VD et al. Protease inhibitors of Manduca sexta expressed in transgenic cotton. Plant Cell Rep 1995; 14:758-762. 50. Jongsma MA, Bakker PL, Peters J et al. Adaptation of Spodoptera exigua larvae to plant proteinase inhibitors by induction of gut proteinase activity insensitive to inhibition. Proc Natl Acad Sci USA 1995; 92:8041-8045. 51. Broadway RM. Are insects resistant to plant proteinase inhibitors? J Insect Physiol 1995; 41:107-116. 52. Bown DP, Wilkinson HS, Gatehouse JA. Differentially regulated inhibitor-sensitive and insensitive protease genes from the phytophagous insect pest, Helicoverpa armigera, are members of complex multigene families. Insect Biochem Mol Biol 1997; 27:625-638.
26 53. Wang S, Young F, Hickey DA. Genomic organization and expression of a trypsin gene from the spruce budworm, Choristoneura fumiferana. Insect Biochem Mol Biol 1995; 25:899-908. 54. Lehane MJ, Müller HM, Crisanti A. Mechanisms controlling the synthesis and secretion of digestive enzymes in insects. In: Lehane MJ, Billingsley PF, eds. Biology of the Insect Midgut. London: Chapman and Hall, 1996:195-205. 55. Barillas-Mury C, Noriega FG, Wells MA. Early trypsin activity is part of the signal transduction system that activates transcription of the late trypsin gene in the midgut of the mosquito, Aedes aegypti. Insect Biochem Mol Biol 1995; 25:241-246. 56. Gatehouse AMR, Norton E, Davison GM et al. Digestive proteolytic activity in larvae of tomato moth, Lacanobia oleracea; effects of plant protease inhibitors in vitro and in vivo. J Insect Physiol 1999; 45:545-558.
Recombinant Protease Inhibitors in Plants 57. Michaud D, Nguyen-Quoc B, Vrain TC et al. Response of digestive cysteine proteinases from the Colorado potato beetle (Leptinotarsa decemlineata) and the black vine weevil (Otiorhynchus sulcatus) to a recombinant form of human stefin A. Arch Insect Biochem Physiol 1996; 31:451-464. 58. Gatehouse AMR, Butler KJ, Fenton KA et al. Presence and partial characterisation of a major proteolytic enzyme in the larval gut of Callosobruchus maculatus. Entomol Exp Appl 1985; 39:279-286. 59. Carbonero P, Royo J, Diaz I et al. Cereal inhibitors of insect hydolases (α-amylases and trypsin): Genetic control, transgenic expression and insect tests. In: Third International Workshop on Pathogenesis-Related Proteins in Plants. Arolla, Switzerland 1992. 60. Heath Rs, Christeller JT et al. Proteinase inhibitors from Nicotiana alata enhance plant resistance to insect pests. J Insect Physiol 1997; 9:833-842.
CHAPTER 3
Cystatin-Based Control of Insects, with Special Reference to Oryzacystatin Soichi Arai and Keiko Abe
3.1. Introduction
C
ystatins were initially defined as proteins that specifically inhibit cysteine proteinases in general,1 but this understanding has recently undergone certain changes. Recent advances in enzymology revealed the existence of a variety of cysteine proteinases, resulting in their classification into several families, namely the papain, the calpain, the caspase and the asparagine-specific processing enzyme families. It is now understood that cystatins are specific inhibitors of enzymes belonging to the papain family, which includes several animal catheptic enzymes and a number of plant enzymes, including bromelain, ficin, actinidin and papain. Conventionally, cystatins were classified into three families forming the cystatin superfamily.2 This classification, however was based exclusively on animal cystatins, as many studies had been conducted until then to clarify the physiological and medical functions of cystatins in animal tissues and products. In particular, modern DNA technologies had greatly contribued to the molecular cloning and structural dissection of various animal cystatins, while in contrast almost no information was available on plant cystatins until about a dozen years ago. In the meantime, a Tokyo group conducted extensive studies on the pastein reaction, a reversal of enzymatic proteolysis, and found that this reaction was best catalyzed by papain.3
We then attempted to apply the papaincatalyzed reaction for improving the functional and nutritive properties of food proteins, but to this end it was critical to use a safe inhibitor which would control the activity of the reaction under mild conditions. The need for such an inhibitor motivated us to search for cystatins in food plants, for which safety and constant supply are required. With this as a background, we began our work to find a cystatin in rice grain, with the result that we found it to contain a potent inhibitor of papain, then named oryzacystatin. We continued a series of dissections of oryzacystatin at both the DNA and protein levels, and this protein is now notable as the first well-defined cystatin of plant origin. Our further work then disclosed that oryzacystatin has endogenous target enzymes (oryzains) in rice. Several research groups have also found that this inhibitor, like other plant cystatins, has potential in the inhibition of exogenous target enzymes like those of herbivorous insect pests. After a discussion about oryzacystatin and related cystatins of plant origin, this chapter deals with cysteine proteinases as endogenous and exogenous target enzymes for these inhibitors. Our discussion then pinpoints the potential of cystatins in the control of insects, with emphasis on the usefulness of regenerating transgenic plants expressing recombinant cystatins. These
Recombinant Protease Inhibitors in Plants, edited by Dominique Michaud. ©2000 Eurekah.com.
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discussions highlight the importance of the model inhibitor oryzacystatin, a protein that has already been internationally studied and applied in various biotechnological systems. For additional references related in whole or in part to the present article, see references 4-6.
3.2. Oryzacystatin and Other Plant Cystatins The rice grain contains a cysteine proteinase inhibitor (PI) that is protein in nature.7 Extensive studies carried out by our group confirmed that this protein inhibits papain in a stoichiometric manner and that, interestingly, it is very heat-stable, even under cooking conditions.8 The use of an antibody raised against the inhibitor showed that it occurs in a number of rice cultivars at average concentrations of 2-3 mg/kg seed.9,10 Of possible physiological significance is the fact that oryzacystatin is synthesized in the ear of rice as soon as flowering begins with its quantity reaching a maximum a few weeks after,11 suggesting that it is produced in the seed for defense against herbivorous insects or some other invaders. Molecular cloning of oryzacystatin was conducted using a cDNA library constructed from immature seeds of rice, Oryza sativa L. japonica (cv. Nipponbare). Using this approach, a full-length cDNA clone encoding a 102-amino acid residues protein was isolated. The amino acid sequence deduced from the corresponding nucleotide sequence (Fig. 3.1) had a significant similarity to those of representative cystatins of animal origin,4 suggesting that it belongs to the cystatin superfamily.2 This inhibitor was subsequently renamed oryzacystatin I (OC-I), after a second oryzacystatin, OC-II, was identified in the grain. OC-I, like animal cystatins, was shown to possess the conserved central pentapeptide motif ‘gln-X-val-X-gly’ (gln-val-val-ala-gly) as a probable target enzyme-binding site,2 but no disulfide bonds were present (Fig. 3.1).12 After probing the same rice seed library with the cDNA of OC-I, a second clone was obtained that encoded a protein with 55% amino acid sequence identity to OC-I. Like OC-I, the deduced primary structure of this
Recombinant Protease Inhibitors in Plants
second oryzacystatin, OC-II, is characteristic of the cystatin superfamily in that its probable target enzyme binding site is in the form of ‘gln-X-val-X-gly’ (gln-val-val-gly-gly; see Fig. 3.1).13 Of enzymological interest is the finding that OC-I and OC-II show different spectra of inhibition against cysteine proteinases. While OC-I inhibits papain with a Ki value of 10-8 M, a value as large as 10-6 M is observed for the inhibition of cathepsin H. OC-II, in contrast gives a Ki value of 10-6 M against papain and a Ki value as small as 10-8 M against cathepsin H,13 the inhibitory process taking place non-covalently in all cases. Genomic DNA dissection was then carried out for both OC-I and OC-II. A genomic DNA clone encoding OC-I was isolated from a λ EMBL3 phage library constructed with partial digests of rice seed chromosomal DNA. The restriction map of the isolated DNA fragment showed that this clone derived from a single-copy gene. The gene, found on chromosome 1, was shown to include two introns: the first, 1.4 kbp in size, located between ala-38 and asn-39, and the second, 372 bp, in the 3'-noncoding region at the G nucleotide residue next to the stop codon TAA. 14,16 The same intron positioning was found in the gene of OC-II, present on chromosome 5.15,16 A characteristic feature of the OC-I and OC-II genes is that their intron boundaries are quite different from those of animal cystatin genes. Comparing the protein and DNA structures of OC-I and OC-II with those of animal cystatins, we can notice that both rice cystatins are chimera of the family 1 and family 2 cystatins, an observation which prompted us to suggest the existence of a new cystatin family including plant cystatins.4 The new term ‘phytocystatin’ was proposed, and then used in a review on the cystatin superfamily written by an independent group.2 We continued cloning studies with the expectation that members of the phytocystatin family would be present in most plants including corn, wheat and soybean. An OC-I-encoding cDNA sequence was used as a probe to clone a cystatin in corn kernel, corn
Fig. 3.1. Amino acid sequence of oryzacystatin and other cystatins of plant origin. Asterisks show putative target binding site to cysteine proteinases. White letters on black show the conserved amino acid residues. OC-I, oryzacystatin-I; OC-II, oryzacystatin II; CC-I, corn cystatin I; CC-II, corn cystatin II; WC, wheat cystatin; SC, soyacystatin; WSC, wound-inducible soybean cystatin; COC, cowpea cystatin; AVC, avocado cystatin; PAC, s cystatin.
Cystatin-Based Control of Insects, with Special Reference to Oryzacystatin 29
30
cystatin I (CC-I).17,18 Using the CC-I cDNA as a probe, a second cystatin, corn cystatin II (CC-II) was found in the kernel.19,20 CC-I and CC-II were apparently different from the cysteine PIs initially found in the endosperm of corn.21,22 Interestingly, both CC-I and CC-II are synthesized in the form of preproteins bearing a signal peptide (see Fig. 3.1), suggesting that both PIs are secreted proteins. A homologous cystatin was also cloned from the wheat grain, also in the form of a preprotein (Fig. 3.1). Recently we extended our cloning studies to dicotyledonous plants and obtained from soybean seeds a cDNA clone encoding a cystatin named soyacystatin.23 Unlike cereal cystatins, this inhibitor is characterized by a very large N-terminal extension (Fig. 3.1), but it still remains to be clarified whether such an attachment is a characteristic of cystatins from dicots. In parallel studies, other groups have found and characterized cystatins from potato,24 ragweed,25 cowpea,26 avocado27 and papaya,28 of which the potato cystatin is exceptionally large in molecular size. There also is a soybean cystatin whose expression takes place only in wounded tissues, 29 suggesting a defensive role for this inhibitor. Interestingly, homology searches show that plant cystatins, except for the potato cystatin, resemble family 2 cystatins of animal origin, but lack disulfide bonds like family 1 cystatins. Furthermore, genomic DNA dissection demonstrated that intron positions in the CC-I, CC-II and soyacystatin genes resemble those of the OC-I and OC-II genes, supporting the idea that plant cystatins constitute a distinct family in the cystatin superfamily.4 The similarities noted among plant cystatins may also indicate that they have similar target enzymes, both endogenous and exogenous.
Recombinant Protease Inhibitors in Plants
3.3. Cysteine Proteinases as Targets of Plant Cystatins 3.3.1. Endogenous Targets The existence of cystatins in plants suggests the occurrence of endogenous target enzymes. However, no detailed studies have been conducted so far to confirm this suggestion from a physiological point of view, although a number of papers were published concerning the isolation and characterization of plant cysteine proteinases of the papain family. Historically, papain has been most frequently used as a model target enzyme for cystatins in enzymological experiments, being essentially seen as an experimental target. Actually, insight into the chemical structure of papain has greatly contributed to establishing the so-called subsite theory, and to elucidating the catalytic triad, cys-25/his-159/asn-175,30 that is often used as a reference to categorize enzymes as cysteine proteinases. In parallel close attention has been given to aleurain, a cysteine proteinase in barley whose expression is induced by gibberellin.31 While investigating gibberellin-mediated expression of cysteine proteinases in rice grains, we found these enzymes to be possible target enzymes of OC-I and OC-II.32 To clone the corresponding genes, we used a cDNA clone encoding aleurain as a probe.31 A cDNA library was constructed from germinating rice 2 days after moisture imbibition. Screening the library with this probe yielded three distinct clones, each encoding a protein with a catalytic triad resembling those of papain and aleurain. The deduced amino acid sequence of the three enzymes, named oryzain α, β and γ (OZ-α, β and γ), are aligned in Fig. 3.2. Homology search showed that OZ-α and OZ-β are closely related to papain, while OZ-γ most resembles cathepsin H. Considering the differential inhibition spectra of OC-I and OC-II against papain and cathepsin H (see above), it is plausible that OZ-α and OZ-αβ are endogenous target enzymes of OC-I, while OZ-γ is targeted in planta by OC-II.
Fig. 3.2. Amino acid sequence of oryzains and other recently found cysteine proteinases, aligned with papain for reference. The arrows point out the possible N- and C- termini of mature enzymes to be formed from precursors. The asterisks show the cysteine, histidine and asparagine residues constituting the enzymes’ catalytic triad. White letters on black show the conserved amino acid residues. OZα, oryzain α; OZ β, oryzain β; OZγ, oryzain γ; CCP1, corn cysteine proteinase 1; CCP2, corn cysteine proteinase 2; DCP1, Drosophila cysteine proteinase 1; SCP1, Sitophilus cysteine s 1; PA, papain.
Cystatin-Based Control of Insects, with Special Reference to Oryzacystatin 31
32
OZ-α and OZ-β are highly homologous to each other, but were found to be quite different with respect to their developmental, stage-specific expression. In mature seeds no OZ-α mRNA is detected and the message only appears during germination, with a maximum level reached 5 days after the start of germination. In contrast, OZ-β mRNA already exists in mature seeds and increases remarkably upon germination. As expected, the expression of these mRNA species and the accumulation of OZ-α and OZ-β are induced by gibberellin in vitro. In the presence of added gibberellic acid at proper concentration in the medium, dipped rice seeds were shown to express OZ-α mRNA and OZ-β mRNA, with maximal accumulation within 1 day and within as short as 4 h, respectively. Gene cloning experiments were then carried out, yielding a 4.8 kbp gene encoding OZ-α, and a 5.7 kbp gene coding for OZ-β.33 Nucleotide sequence analysis showed that the OZ-α genomic DNA clone contains in its promoter region a gibberellin-responsive element (GRE) known to exist in the gene of a gibberellin-inducible α -amylase in barley.34 However no such sequence was found in the OZ-β clone, indicating the possible existence of some new GRE. Northern blotting and immunostaining showed that OZ-α occurs in both the aleurone and the endosperm, whereas OZ- β is localized only in the aleurone. Considering that OZ-β could exist over a long period of time from maturation to germination, it is likely that this cysteine proteinase plays a housekeeping role in rice aleurone layers, just as some catheptic enzymes do in lysosomes. On the other hand, OZ- γ resembles aleurain in amino acid sequence—having similar N-glycosylation sites and a vacuole-sorting signal—and shares very high structural similarity with the lysosomal enzyme, cathepsin H. Based on these observations the following putative functions can be inferred for the three OZ species: OZ-α being involved in proteolysis of seed storage proteins, OZ- β in the proteolysis of some functional proteins in the aleurone, and OZ- γ in the turnover of intracelullar proteins.
Recombinant Protease Inhibitors in Plants
Recently our group identified several other cysteine proteinases in cereals, including two species in corn kernels (CCP-1 and CCP-2),35 and three species in wheat grains (triticains α, β and γ). Other groups also have assessed the general occurrence of cysteine proteinases in plants,36-42 and their possible physiological functions. Some plant cystatins, by regulating their activities, may probably regulate these processes, either in a specific or nonspecific manner.
3.3.2. Exogenous Targets Along with their role in the regulation of endogenous proteolysis, plant cystatins may have exogenous targets originating from invaders such as viruses, bacteria, and insects. In recent years the potential of plant cystatins in inhibiting cysteine proteinases of insect origin has been evaluated and the importance of studying pest digestive proteases, in particular, has been stressed. Historically, a paper by Houseman43 is probably the first describing the existence of a cysteine proteinase (hemoglobin- hydrolyzing) activity in insects, more precisely in the heteropteran insect Rhodnius prolixus. A number of studies followed identifying insect cysteine proteinases and assessing their physiological roles. However, little was known for a long time concerning cysteine proteinases as digestive enzymes in the gut and related organs of insects. Murdock et al44 started pioneering work on such digestive enzymes, but until recently no detailed information was available about their molecular biology. To gain basic knowledge on the molecular structure of insect cysteine proteinases, we studied a gene in Drosophila encoding a putative digestive cysteine proteinase.45 The mature enzyme, termed Drosophila cysteine proteinase-1 (or DCP-1), consists in a 218-amino acids protein showing significant structural similarities with cysteine proteinases of animal origin such as cathepsins H and L, and with those of plant origin such as OZ-α and OZ-β (Fig. 3.2). In situ hybridization studies with embryos showed that DCP-1 mRNA is predominantly expressed in the
Cystatin-Based Control of Insects, with Special Reference to Oryzacystatin
midgut, although larval alimentary organs, such as the salivary gland and the midgut including the caeca, also express this mRNA at significant levels. Using the DCP-1 DNA clone as a probe, we then identified and characterized a gene family comprising at least four genes encoding cysteine proteinases, SCPs, in the Coleopteran insect, Sitophilus zeamais.46,47 A cDNA clone corresponding to a major mRNA species in adult insects was shown to encode an SCP of 338 amino acid residues (Fig. 2), while two other clones were encoding similar 331-amino acids proteins in which the COOH-terminal structure is seven amino acids shorter. The three SCPs have high sequence similarities to each other as well as to other insect and mammalian cathepsin L-like enzymes. A polyclonal antibody raised against a bacterially expressed SCP was used as a probe to examine the molecular forms and distribution of the enzyme, showing it to exist in both proenzyme and mature forms in larvae, pupae and adults, with the proenzyme converted in vitro into the mature form at acidic pH. Immunohistochemical analyses showed that a SCP is present in several tissues including alimentary organs and germ cells. In alimentary organs the SCP is distributed in the gastric caeca, but not in the midgut. It is also present in genital organs, especially in oocytes and nurse cells, suggesting that this SCP is implicated in a variety of physiological functions including food digestion, and that it could represent an interesting exogenous target for plant cystatins.
3.4. Control of Insects with Cystatins 3.4.1. Antifeedant Effects of Cysteine PIs While some pests invade plant seeds to consume their storage proteins, plant seeds have acquired the ability to defend themselves against insect invasion, notably by producing PIs active against pest digestive proteinases. In fact, it is thought that seeds and insects have been co-evolving over a long period of
33
time, and that when an insect adapts to the defenses of a given plant, it has the potential to become a pest. 6 Actually, there are a number of insect pests that inflict great damage on agricultural products, especially on nutritious grains, despite the occurrence of antifeedant molecules—including large amounts of PIs—in these products. Until now, many papers have been presented dealing with the efficacy of ‘exogenous’ serine PIs such as trypsin inhibitors in controlling insect growth (see Chapter 2, this volume). Besides serine PIs, cysteine PIs such as cystatins occur in plants with possible target enzymes in insects (see above), suggesting that they could be used as effective inhibitors of these enzymes. Indeed, the inhibitory and/or antifeedant effects of trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane (E-64), a low molecular-weight inhibitor of cysteine proteinases, have been demonstrated by several groups. For instance: 1. Thie and Houseman48 found that the midgut of the yellow mealworm, Tenebrio molitor contains serine and cysteine (E-64-sensitive) proteolytic activities, the latter occuring in the anterior portion of the midgut. 2. According to Zhao et al,29 there are wound-inducible cysteine PIs in soybean (see Fig. 3.1) that would function in host plant defense against predation by the Western corn rootworm. The third-instar larvae of this insect have digestive cysteine proteinases in their guts that are inhibited by E-64. Hines et al49 found that one of these soybean cystatins (Fig. 3.1) is similar in potency on a molar basis to chicken egg-white cystatin and E-64 when evaluated on extracts containing digestive proteases of the cowpea weevil, Callosobruchus chinensis and the red flour beetle, Tribolium castaneum. 3. In a study by Purcell et al,50 midgut juice was isolated from six insects of economic importance: the black cutworm (Agrotis ipsilon), the corn earworm (Heliothis zea), the tobacco budworm (H. virescens), the boll
Recombinant Protease Inhibitors in Plants
34 weevil (Anthonomus grandis), the Colorado potato beetle (Leptinotarsa decemlineata) and the Southern corn rootworm (Diabrotica undecimpunctata) in order to measure their digestive proteolytic activities. While the other species were shown to use serine proteinases, E-64 significantly inhibited the midgut proteinase activities of Southern corn rootworm and Colorado potato beetle, demonstrating that the major activities in these species derive from cysteine proteinases. 4. As shown by Oppert et al,51 combinations of cysteine and serine PIs in wheat germ diets are toxic to larvae of the red flour beetle, Tribolium castaneum when tested at levels where the individual inhibitors are nontoxic. Mixtures of 0.1% (w/w) E-64 plus 1% (w/w) of either Kunitz trypsin inhibitor, soybean Bowman-Birk trypsin-chymotrypsin inhibitor, or lima bean trypsin inhibitor all inhibited growth of the insect, leading to an 82-97% reduction in larval weight gain 17 days after hatching, and later to 40-60% mortality. Supplementation of the diet containing 0.1% E-64 plus 1% soybean Kunitz trypsin inhibitor with a mixture of amino acids led to partial reversal of the growth inhibition, with 91% of the larvae surviving. The synergism between different classes of PIs in the diet, that enhances growth inhibition and toxicity, demonstrates the potential of pest management strategies involving the coordinated use of two or more types of digestive enzyme inhibitors in plants. 5. In a recent study, Bolter and LatoszekGreen52 found that chronic ingestion of E-64 has a profound inhibitory effect on Colorado potato beetle larval growth, development and survival, as well as on adult fecundity. However, the number of insects surviving to the adult stage did not fall below 26% despite increasing the E-64 concentration above 1.5 µg/cm2 leaf surface. The time for development to the pupal stage
increased from 13 days for larvae reared on control leaves to 21 days at a concentration of 1.5 µg E-64/cm2. The most significant effect of dietary E-64 was on adult fecundity, with mated females reared on untreated leaves laying an average of 62 ± 5.7 eggs daily in the first 10 days, while those maintained on 0.5 µg E-64/cm2 laid 16 ± 2.4 eggs daily. Noteworthy, females given 1 µg E-64/cm2 laid few if any eggs, but started producing egg masses as large as control insects about 5 days after being switched to control leaves. These effects on the insect life cycle were directly related to the degree of inhibition of cysteine proteinase activity in gut extracts. The general proteinase activity in control extracts was 6.5 ± 0.16 units min-1/µg gut, a value which decreased to 1.9 ± 0.16 in the guts of insects reared on leaves coated with 1 µg E-64/cm2. The proportion of proteinase activity inhibitable by E-64 decreased from 66% in control guts to 10-15% in guts from larvae reared on 1 µg E-64/cm2. Interestingly, the aspartic proteinase inhibitor, pepstatin caused a decrease in proteinase activity in response to the inhibition by E-64, while no inhibition of gut enzyme activity by soybean trypsin inhibitor was measured in larvae fed any concentration of E-64. This study demonstrates that proteinase levels must be significantly reduced to have a pronounced inhibitory effect on larval growth and survival, while the fecundity of mated females is affected by lower concentrations of inhibitor. It also suggests that the Colorado potato beetle may be difficult to control with PIs unless efficient inhibitors are used. 6. In another study, Elden53 developed an in vivo laboratory bioassay to screen PIs for their ability to inhibit growth and development of the alfalfa weevil, Hypera postica. Larval foliar feeding, pupation and adult emergence were significantly decreased by E-64, p-chloro-
Cystatin-Based Control of Insects, with Special Reference to Oryzacystatin mercuribenzoate and leupeptin, all these inhibitors showing affinity for cysteine proteinases. Orr et al54 also found an insect-controlling efficacy for E-64, suggesting, along with the observations described above, the potential of using proteinaceous cysteine PIs such as oryzacystatin for controlling insects relying on cysteine proteinases for dietary protein digestion.
3.4.2. The Use of Oryzacystatin in Pest Control Several groups have tried to use oryzacystatin for pest control. Michaud et al55 found that OC-I, when fed to Colorado potato beetle whose digestive proteolytic system throughout development is apparently unique, has the same basic inhibitory effect on extracts of each developmental stage. However, in contrast to early stages, the growth of third and fourth instars is not significantly affected by feeding with OC-I, suggesting the potential of the rice cystatin for inhibition of Colorado potato beetle growth but also pointing out a stage-related limit to this control strategy, independent of the nature of proteinase forms used by the insect. In order to explain this limited effect of OC-I the same authors then analyzed the in vitro inhibitory effect of recombinant OC-I and OC-II expressed in E. coli against the beetle digestive proteinases.55,56 Both inhibitors had a significant effect on total proteolytic activity, but the maximal inhibitions ranged from 20% to 80% for pH values varying from 5.0 to 7.0, respectively. As demonstrated by the use of inhibitors with different specificities toward cathepsin B and cathepsin H, this pH-dependent efficiency was due to the selective inactivation of potato beetle cathepsin H and to their inability to inhibit the insect cathepsin B, showing the importance to have an adequate knowledge of the insect proteinases specifically recognized by the inhibitors to be used in pest control. The same group also assessed the effects of the cystatins OC-I and human stefin A on digestive cysteine proteinases of the Colorado
35
potato beetle and the black vine weevil, Otiorynchus sulcatus, using complementary inhibition assays, cystatin-affinity chromatography and recombinant forms of the two inhibitors.57 For both insects, either stefin A or OC-I used in excess caused partial but stable inhibition of total proteolytic activity, but unlike for OC-I the stefin A-mediated inhibition was significantly increased when the inhibitor was used in larger excess. As shown by complementary inhibition assays, this two-step inhibition of the insect proteinases by stefin A was due to the differential inactivation of two distinct cysteine proteinase populations in either insects, with the rapidly inhibited population corresponding to the OC-Isensitive fraction. After removing cystatinsensitive proteinases from Colorado potato beetle and black vine weevil midgut extracts using OC-I- (or stefin A-) affinity chromatography, the effects of the insect ‘non-target’ proteinases on the structural integrity of the two cystatins were assessed. While OC-I remained essentially stable, stefin A was subjected to hydrolysis without the accumulation of detectable stable intermediates, which suggested the presence of multiple exposed cleavage sites sensitive to the action of the insect proteases on this cystatin. This apparent susceptibility of stefin A to proteolytic cleavage was proposed to partially explain its low efficacy to inactivate the insect OC-I-insensitive cysteine proteinases when not used in large excess. Such instability problems could be of major concern when planning the use of cystatin-expressing transgenic plants for the control of coleopteran pests. In another study by Michaud et al58 the biochemical interactions between digestive proteinases of the black vine weevil and the two rice cystatins, OC-I and OC-II were assessed using gelatin-polyacrylamide gel electrophoresis, OC-I-affinity chromatography and recombinant forms of the two inhibitors. The insect proteinases were resolved in gelatin-containing polyacrylamide gels as five major bands, only three of which were totally or partially inactivated by OC-I and OC-II. The maximal inhibitory effect of both
36
oryzacystatins at pH 5.0 was estimated at 40% and the inhibition was stable with time despite the presence of oryzacystatin-insensitive proteases, indicating the stability of the OC-I and OC-II inhibitory effects. After removing oryzacystatin-sensitive proteinases from the insect crude extract by OC-I-affinity chromatography, the effects of the insensitive proteases on the structural integrity of the free inhibitors were analyzed. While OC-I remained stable, OC-II underwent limited proteolysis to a 10.5-kDa active, but unstable intermediate. As shown by the degradation pattern of a glutathione S-transferase/OC-II fusion protein, the appearance of this intermediate resulted from the C-terminal truncation of OC-II. Although these observations indicate the high conformational stability of OC-II near its active (inhibitory) site, they also suggest a general destabilization of this inhibitor following its initial cleavage, leading subsequently to its complete hydrolysis. As for stefin A, this apparent susceptibility of OC-II to proteolytic cleavage by the insect insensitive proteinases could have major implications when using it in insect control. Nevertheless, despite these complex interactions between cystatins and insect digestive proteinases, significant adverse effects have been observed in some cases. Chen et al59 isolated a cDNA clone of OC-I which they expressed in E. coli under the control of the T7 RNA polymerase promoter. The resulting construct encoded a fusion protein containing 11 amino acid residues from the NH2- terminus of the bacterially produced protein and 79 residues of oryzacystatin lacking the 23 NH2-terminal residues of the wild-type protein. Caseinolytic activity in midgut homogenates from seven species of stored product insects was inhibited in vitro by 18 to 85% with a sample of this modified inhibitor, showing the presence of OC-Isensitive proteases in these insects. When provided to the red flour beetle Tribolium castanum, an artificial diet containing the OC-I mutant at 10% (w/w) concentration suppressed growth of the insect by approximately 35%, as compared to control insects.
Recombinant Protease Inhibitors in Plants
Edmonds et al60 and Kuroda et al61 also have observed insect-controlling effects with recombinant oryzacystatins. The latter authors61 notably found that bacterially produced OC-I and OC-II cause growth retardation of two bean insect pests, Callosobruchus chinensis (Coleoptera) and Riptortus clavatus (Hemiptera) when added to their diets at concentrations of 0.3 to 0.5% (w/w). At a higher concentration, nearly all insects die (Fig. 3.3), again suggesting the usefulness of plant cystatins for insect control and the critical role of cysteine proteinases in digestive processes in herbivorous insects.
3.5. Regeneration of Cystatin-Expressing Transgenic Plants The discussion above confirms the potential of incorporating PI-encoding genes in plants for insect control. To this end, Duan et al62 carried out experiments to introduce the gene of the serine-type inhibitor potato PI-II into several Japonica rice varieties, and regenerated a large number of transgenic rice plants. Wound-inducible expression of this gene driven by its own promoter resulted in high-level accumulation of the inhibitor protein in transgenic plants. The introduced gene was stably inherited in the second, third, and fourth generations. Based on molecular analyses, several homozygous transgenic lines were obtained. Bioassays for insect resistance using plants of the fifth generation showed increased resistance to a major rice insect pest, the pink stem borer (Sesamia inferens), further suggesting that the introduction of a PI gene into cereal plants (and other plants) can be used as a general strategy to control insect pests. While the transformation of plants with exogenous PI genes represents an attractive pest control strategy, however it may necessitate a thorough characterization of the plant endogenous proteinases, which represent potential targets for the exogenous inhibitors expressed. In a study by Michaud et al,63 changes in general endoproteolytic activity were monitored during sprouting of the
Cystatin-Based Control of Insects, with Special Reference to Oryzacystatin
37
Fig. 3.3. Effect of oryzacystatins on the bean insects, Riptortus clavatus (A) and Callosobruchus chilensis A: Six groups of five second-instar nymphs were reared on artificial diets containing different doses of cystatin. Thirty days later, the number of living insects remaining (%) was determined. B: OC-I and OC-II were added to artificial diets at various concentrations. Thirty days after oviposition, the number of living insects was counted and compared to the number of hatched eggs.
Recombinant Protease Inhibitors in Plants
38
Table 3.1. Inhibition of Sitophilus zeamais midgut proteinases by a protein extract of transgenic rice expressing corn cystatin Ia µg) Amount of protein added (µ
Relative protease activity (%) Nontransformed
Transgenic (CC-expressing)
0
100
100
1
100
80
2
100
52
3
100
39
5
100
11
10
79
6
aFor each assay, the inhibitory activity was evaluated using a 30µU portion of gut proteinase
activity (see ref. 72), in the presence of various amounts of rice protein. data represent the activity remaining, as compared toa control in which no inhibitor was added.
potato (Solanum tuberosum L., cv. Kennebec) tuber. Quantitative data obtained by standard enzymatic assays showed an increase in cysteine proteinase activity during sprouting, resulting from the gradual appearance of new cysteine proteinase forms. While only one proteinase form was present during early sprouting, at least six new active forms of the same class were shown to appear gradually after the mature tuber was sown, suggesting the involvement of a complex cysteine proteolytic system in the last stages of tuber protein breakdown. Interestingly, the rice cystatins OC-I and OC-II had no measurable effect on any of the tuber proteinases detected. Similar results were obtained with leaf, stem and stolon proteinases, which suggests the eventual absence of direct metabolic interference by OC-I and OC-II in transgenic potato and supports the potential of these PIs for the development of healthy insect-resistant potato plants. The transformation of potato with cystatin genes, for instance, represents a
potential way of controlling the major insect pest Colorado potato beetle. Benchekroun et al64 described the Agrobacterium-mediated transformation of potato (cv. Kennebec) with an OC-I cDNA clone linked to the cauliflower mosaic virus 35S promoter. The transgenic plants accumulated active OC-I in their leaves, as demonstrated by the papain inhibitory activity of leaf extracts. In addition to their anti-papain activity, the extracts caused partial but significant inhibition of Colorado potato beetle digestive proteinases, similar to the inhibition observed with the pure inhibitors. Recombinant OC-I did not alter activity of the major potato leaf endogenous proteinases—which seemed to be of the serine-type—suggesting that the OC-I cDNA could be used to protect potato from predation or infection without interfering with the endogenous proteinases. Other plants expressing recombinant OC-I, including tobacco and poplar have been successfully developed. 65,66 After establising systems for producing recombi-
Cystatin-Based Control of Insects, with Special Reference to Oryzacystatin
nant oryzacystatin in microorganisms,67-69 we also conducted experiments to regenerate rice genetically transformed with endogenous genes, including that of OC-I.70,71 However there is sometimes the problem that regenerated rice plants transformed with isogenic genes become infertile, although the physiological reason for this remains obscure. As an alternative we used recombinant CC-I, which shows a wide inhibitory spectrum against cysteine proteinases. The rationale behind this attempt was based on the observations 1) that many insect pests, especially coleopteran insects, use cysteine proteinases for dietary protein hydrolysis, 2) that OC-I shows a narrow inhibitory spectrum against cysteine proteinases as compared to CC-I, and 3) that OC-I is found in the rice grain at very low levels. The transgenic plants generated contained high levels of CC-I mRNA and CC-I protein in both seeds and leaves, with the CC-I protein content in the seed reaching up to 2% of the total heat soluble protein fraction. Recombinant CC-I recovered from the seeds was found to efficiently inhibit papain, cathepsin H, and cysteine proteinases isolated from the gut of the insect pest, Sitophilus zeamais (Table 3.1), thereby suggesting the potential of heterologous cystatins in the protection of rice.
3.6. Conclusion While pointing out the complex interactions taking place between insect digestive proteinases and cysteine PIs, the data discussed in this chapter also demonstrate the potential of using cystatins in insect control, and justify work aimed at regenerating crops genetically engineered with cystatin cDNA sequences. Further research along these lines should facilitate the development of efficient cystatin-based control strategies, and help us understand the role of cystatins in plant defense.
Acknowledgments We express our sincere thanks to Dr. Masaharu Kuroda, Department of Rice Research, Hokuriku National Agricultural Experiment Station, Japan Ministry of
39
Agriculture, Forestry and Fisheries, and to Dr. Ichiro Matsumoto, Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, for their pertinent contribution to the writing of this article.
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40 12. Arai S, Watanabe H, Kondo H et al. Papain-inhibitory activity of oryzacystatin, a rice seed cysteine proteinase inhibitor, depends on the central Gln-Val-Val-Ala-Gly region conserved among cystatin superfamily members. J Biochem 1991; 109:294-298. 13. Kondo H, Abe K, Nishimura I et al. Two distinct cystatin species in rice seeds with different specificities against cysteine proteinases. Molecular cloning, expression, and biochemical studies on oryzacystatin-II. J Biol Chem 1990; 265:15832-15837. 14. Kondo H, Emori Y, Abe K et al. Cloning and sequence analysis of the genomic DNA fragment encoding oryzacystatin. Gene 1989; 81:259-265. 15. Kondo H, Abe K, Emori Y et al. Gene organization of oryzacystatin-II, a new cystatin superfamily member of plant origin, is closely related to that of oryzacystatin-I but different from those of animal cystatins. FEBS Lett 1991; 278:87-90. 16. Kishimoto N, Higo H, Abe K et al. Identification of the duplicated segments in rice chromosomes 1 and 5 by linkage analysis of cDNA markers of known functions. Theor Appl Genet 1994; 88:722-726. 17. Abe M, Abe K, Kuroda M et al. Corn kernel cysteine proteinase inhibitor as a novel cystatin superfamily member of plant origin: molecular cloning and expression studies. Eur J Biochem 1992; 209:933-937. 18. Abe M, Abe K, Iwabuchi K et al. Corn cystatin I expressed in Escherichia coli: investigation of its inhibitory profile and occurrence in corn kernels. J Biochem 1994; 116:488-492. 19. Abe M, Abe K, Domoto C et al. Two distinct species of corn cystatin in corn kernels. Biosci Biotechnol Biochem 1995; 59:756-758. 20. Abe M, Domoto C, Watanabe H et al. Structural organization of the gene encoding corn cystatin. Biosci Biotechnol Biochem 1996; 60:1173-1175. 21. Abe M, Arai S, Kato H et al. Thiol-protease inhibitors occurring in endosperm of corn. Agric Biol Chem 1980; 44:685-686. 22. Abe M, Arai S. Some properties of a cysteine proteinase inhibitor from corn endosperm. Agric Biol Chem 1991; 55:2417-2418. 23. Misaka T, Kuroda M, Iwabuchi K et al. Soyacystatin, a novel cysteine proteinase inhibitor in soybean, is distinct in protein structure and gene organization from other cystatins of animal and plant origin. Eur J Biochem 1996; 240:609-614. 24. Waldron C, Wegrich LM, Merlo PAO et al. Characterization of a genomic sequence coding for potato multicystatin, an eight-domain cysteine proteinase inhibitor. Plant Mol Biol 1993; 23:801-812.
Recombinant Protease Inhibitors in Plants 25. Rogers BL, Pollock J, Klapper DG et al. Sequence of the proteinase-inhibitor cystatin homolog from the pollen of Ambrosia artemisiifolia (short ragweed). Gene 1993; 133:219-221. 26. Fernandes KVS, Sabelli PA, Barratt DHP et al. The resistance of cowpea seeds to bruchid beetles is not related to level of cysteine proteinase inhibitors. Plant Mol Biol 1993; 23:215-219. 27. Kimura M, Ikeda T, Fukumoto D et al. Primary structure of a cysteine proteinase inhibitor from the fruit of avocado (Persea americana Mill). Biosci Biotechnol Biochem 1995; 59:2328-2329. 28. Song I, Taylor M, Baker K et al. Inhibition of cysteine proteinases by Carica papaya cystatin produced in Escherichia coli. Gene 1995; 162:221-224. 29. Zhao Y, Botella MA, Subramanian L et al. Two wound-inducible soybean cysteine proteinase inhibitors have greater insect digestive proteinase inhibitory activities than a constitutive homolog. Plant Physiol 1996; 111:1299-1306. 30. Garavito RM, Rossmann MG, Argos P et al. Convergence of active center geometries. Biochemistry 1977; 16:5065-5071. 31. Rogers JC, Dean D, Heck GR. Aleurain: A barley thiol protease closely related to mammalian cathepsin H. Proc Natl Acad Sci USA 1985; 82:6512-6516. 32. Watanabe H, Abe K, Emori Y et al. Molecular cloning and gibberellin-induced expression of multiple cysteine proteinases of rice seeds (oryzains). J Biol Chem 1991; 266:16897-16902. 33. Watanabe H, Abe K, Arai S. Gibberellin-responsive gene expression taking place with oryzains α and γ as cysteine proteinases of rice seeds. Biosci Biotechnol Biochem 1992; 56:1145-1155. 34. Skriver K, Olsen FL, Rogers JC et al. Cis-acting DNA elements responsive to gibberellin and its antagonist abscisic acid. Proc Natl Acad Sci USA 1991; 88:7266-7270. 35. Domoto C, Watanabe H, Abe M et al. Isolation and characterization of two distinct cDNA clones encoding corn seed cysteine proteinases. Biochim Biophys Acta 1995; 1263:241-244. 36. Koehler SM, Ho T-HD. Hormonal regulation, processing, and secretion of cysteine proteinases in barley aleurone layers. Plant Cell 1990; 2:769-783. 37. Guerrero FD, Jones JT, Mullet JE. Turgor-responsive gene transcription and RNA levels increase rapidly when pea shoots are wilted. Sequence and expression of three inducible genes. Plant Mol Biol 1990; 15:11-26.
Cystatin-Based Control of Insects, with Special Reference to Oryzacystatin 38. Schaffer MA, Fischer RL. Analysis of mRNAs that accumulate in response to low temperature identifies a thiol protease gene in tomato. Plant Physiol 1988; 87:431-436. 39. Kalinski A, Weisemann JM, Matthews BF et al. Molecular cloning of a protein associated with soybean seed oil bodies that is similar to thiol proteases of the papain family. J Biol Chem 1990; 265:13843-13848. 40. Dietrich RA, Maslar DJ, Heupel RC et al. Spatial patterns of gene expression in Brassica napus seedlings: identification of a cortex-specific gene and localization of mRNAs encoding isocitrate lyase and a polypeptide homolog to proteinases. Plant Cell 1989; 1:73-80. 41. Akasofu H, Yamauchi D, Mitsuhashi W et al. Nucleotide sequence of cDNA for sulfhydryl-endopeptidase (SH-EP) from cotyledons of germinating Vigna mungo seeds. Nucl Acids Res 1989; 17:6733. 42. Tanaka T, Yamauchi D, Minamikawa T. Nucleotide sequence of cDNA for an endopeptidase (EP-C1) from pods of maturing Phaseolus vulgaris fruits. Plant Mol Biol 1991; 16:1083-1084. 43. Houseman J. A thiol-activated digestive proteinase from adults of Rhodnius prolixus Stål (Hemiptera: Reduviidae). Can J Zool 1978; 56:1140-1143. 44. Murdock LL, Brookhart G, Dunn PE et al. Cysteine digestive proteinases in Coleoptera. Comp Biochem Physiol 1987; 87B:783-787. 45. Matsumoto I, Watanabe H, Abe K et al. A putative digestive cysteine proteinase from Drosophila melanogaster is predominantly expressed in the embryonic and larval midgut. Eur J Biochem 1995; 227:582-587. 46. Matsumoto I, Emori Y, Abe K et al. Characterization of a gene family encoding cysteine proteinases of Sitophilus zeamais (maize weevil), and analysis of the protein distribution in various tissues including alimentary tracts and germ cells. J Biochem 1997; 121:464-476. 47. Matsumoto I, Abe K, Arai S et al. Functional expression and enzymatic properties of two Sitophilus zeamais cysteine proteinases showing different autolytic processing profiles in vitro. J Biochem 1998; 123:693-700. 48. Thie NMR, Houseman JG. Cysteine and serine proteolytic activities in larval midgut of yellow mealworm, Tenebrio molitor L. (Coleoptera: Tenebrionidae). Insect Biochem 1990; 20:741-744. 49. Hines ME, Osuala CI, Nielsen SS. Isolation and partial characterization of a soybean cystatin cysteine proteinase inhibitor of coleopteran digestive proteolytic activity. J Agric Food Chem 1991; 39:1515-1520.
41
50. Purcell JP, Greenplate JT, Sammons RD. Examination of midgut luminal proteinase activities in six economically important insects. Insect Biochem Mol Biol 1992; 22:41-47. 51. Oppert B, Morgan TD, Culbertson C et al. Dietary mixtures of cysteine and serine proteinase inhibitors exhibit synergistic toxicity toward the red flour beetle, Tribolium castaneum. Comp Biochem Physiol 1993; 105C:379-385. 52. Bolter CJ, Latoszek-Green M. Effect of chronic ingestion of the cysteine proteinase inhibitor, E-64, on Colorado potato beetle gut proteinases. Entomol Exp Appl 1997; 83:295-303. 53. Elden TC. Selected proteinase inhibitor effects on alfalfa weevil (Coleoptera: Curculionidae) growth and development. J Econ Entomol 1995; 88:1586-1590. 54. Orr GL, Strickland JA, Walsh TA. Inhibition of Diabrotica larval growth by a multicystatin from potato tubers. J Insect Physiol 1994; 40:893-900. 55. Michaud D, Bernier-Vadnais N, Overney S et al. Constitutive expression of digestive cysteine proteinase forms during development of the Colorado potato beetle, Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae). Insect Biochem Mol Biol 1995; 25:1041-1048. 56. Michaud D, Nguyen-Quoc B, Yelle S. Selective inhibition of Colorado potato beetle cathepsin H by oryzacystatins I and II. FEBS Lett 1993; 331:173-176. 57. Michaud D, Bernier-Vadnais N, Vrain TC et al. Response of digestive cysteine proteinases from the Colorado potato beetle, (Leptinotarsa decemlineata) and the black vine weevil (Otiorynchus sulcatus) to a recombinant form of human stefin A. Arch Insect Biochem Physiol 1996; 31:451-464. 58. Michaud D, Cantin L, Vrain TC. Carboxyterminal truncation of oryzacystatin II by oryzacystatin-insensitive insect digestive proteinases. Arch Biochem Biophys 1995; 322:469-474. 59. Chen M-S, Johnson B, Wen L et al. Rice cystatin: bacterial expression, purification, cysteine proteinase inhibitory activity, and insect growth suppressing activity of a truncated form of the protein. Protein Express Purif 1992; 3:41-49. 60. Edmonds HS, Gatehouse LN, Hilder VA et al. The inhibitory effects of the cysteine protease inhibitor, oryzacystatin, on digestive proteases and on larval survival and development of the southern corn rootworm (Diabrotica undecimpunctata howardi). Entomol Exp Appl 1996; 78:83-94.
42 61. Kuroda M, Ishimoto M, Suzuki K et al. Oryzacystatins exhibit growth-inhibitory and lethal effects on different species of bean insect pests, Callosobruchus chinensis (Coleoptera) and Riptortus clavatus (Hemiptera). Biosci Biotechnol Biochem 1996; 60:209-212. 62. Duan X, Li X, Xue Q et al. Transgenic rice plants harboring an introduced potato proteinase inhibitor II gene are insect resistant. Nat Biotechnol 1996; 14:494-498. 63. Michaud D, Nguyen-Quoc B, BernierVadnais N et al. Cysteine proteinase forms in sprouting potato tuber. Physiol Plant 1994; 90:497-503. 64. Benchekroun A, Michaud D, Nguyen-Quoc B et al. Synthesis of active oryzacystatin I in transgenic potato plants. Plant Cell Rep 1995; 14:585-588. 65. Masoud SA, Johnson LB, White FF et al. Expression of a cysteine proteinase inhibitor (oryzacystatin-I) in transgenic tobacco plants. Plant Mol Biol 1993; 21:655-663. 66. Leplé JC, Bonadé-Bottino M, Augustin S et al. Toxicity to Chrysomela tremulae (Coleoptera: Chrysomelidae) of transgenic poplars expressing a cysteine proteinase inhibitor. Mol Breeding 1993; 1:319-328. 67. Abe K, Emori Y, Kondo H et al. The NH2-terminal 21 amino acid residues are not essential for the papain-inhibitory activity of oryzacystatin, a member of the cystatin superfamily. Expression of oryzacystatin cDNA and its truncated fragments in Escherichia coli. J Biol Chem 1988; 263:7655-7659.
Recombinant Protease Inhibitors in Plants 68. Emori Y, Ono N, Saigo K et al. Expression of oryzacystatin cDNA using yeast artificial chromosome under ADH promoter in baker’s yeast. Biochem Mol Biol Int 1993; 30:499-504. 69. Kondo H, Ijiri S, Abe K et al. Inhibitory effect of oryzacystatins and a truncation mutant on the replication of poliovirus in infected Vero cells. FEBS Lett 1992; 299:48-50. 70. Hosoyama H, Irie K, Abe K et al. Oryzacystatin exogenously introduced into protoplasts and regeneration of transgenic rice. Biosci Biotechnol Biochem 1994; 58:1500-1505. 71. Hosoyama H, Irie K, Abe K et al. Introduction of a chimeric gene encoding an oryzacystatin-β-glucuronidase fusion protein into rice protoplasts and regeneration of transformed plants. Plant Cell Rep 1995; 15:174-177. 72. Irie K, Hosoyama H, Takeuchi T et al. Transgenic rice established to express corn cystatin exhibits strong inhibitory activity against insect gut proteinases. Plant Mol Biol 1996; 30:149-157.
CHAPTER 4
Recombinant Protease Inhibitors as Management Tools to Suppress Parasitic Nematodes Thierry C. Vrain
4.1. Introduction
4.1.1. The Worm
ematodes are the most successful multicellular form of animal life on earth. They colonize all trophic milieux with enough water to sustain their aquatic origin, the bottom of the seas, rivers and lakes, all continents, the deserts and the toundra included, usually in billions per acre. Nematodes are important pests of global agriculture, debilitating crops and farm animals. They cause many crippling human diseases such as river blindness or elephantiasis in tropical areas of Africa, and many other diseases on all continents.1 However most nematodes are the good guys in our environment. Tens of thousands of species are essential to the proper balance of many ecological soil processes and they must be protected, especially in fragile agricultural systems.2 Nematodes feeding on bacteria that are extremely virulent in insects transmit these deadly diseases and keep insect epidemics under control.3 These biological control agents are now used on a large scale to guard many horticultural crops from insect pests, and are a boon to organic growers.
One bacterial feeding nematode, Caenorhabditis elegans, has achieved uncommon fame as the subject of extensive studies in cell biology, neurophysiology, genetics, gerontology and other fields of medical research,4,5 as well as nematology. Genome sequencing of this nematode was completed in 1998, and there are already obvious and immediate benefits flowing from this project to the study of parasitism, especially with human, animal and plant parasitic nematodes.6,7 Techniques for in situ hybridization, transformation and gene disruption by transposon, that were developed for C. elegans are now used to show similarities in molecular make up and development processes with other organisms.
N
4.1.2. Plant Parasitic Nematodes in Agriculture Several thousand species of nematodes are herbivory and a few species have become global pests of all agricultural crops, causing economic losses upward of $100 billion each year. 8 Most of these nematodes are soil dwellers and it has become an accepted practice to control these pests by injecting very large volumes of extremely toxic pesticides
Recombinant Protease Inhibitors in Plants, edited by Dominique Michaud. ©2000 Eurekah.com.
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such as methyl bromide, methylisothiocyanate or acetylcholinesterase inhibitors into agricultural soils before planting a susceptible crop. This practice often results in a noticeable decrease of many soil biological processes, notably by suppressing predators and parasites of these nematodes,9 and in the contamination of aquifers with the nematicides or their degradation products.10 In contrast to insect pests or fungal pathogens, that become resistant to ever more sophisticated pesticides, there have been no documented cases of plant parasitic nematode populations developing resistance against nematicides. Yet, most nematicides have been banned in the last 20 years because of their extreme toxicity towards birds or mammals and too many instances of human health risks through contamination of food, water and the atmosphere, or because of their effect in the ozone layer of the upper atmosphere.11 Impending regulations against the remaining few nematicides available12 make it even more urgent to develop new management tools against the major species of plant parasitic nematodes. Breeding for genetic resistance has been relatively successful and a number of cultivars of a few horticultural crops have been backcrossed with resistance against one or a few nematode species.13 But with the pressure that the absence of nematicides places on agronomists and breeders to greatly increase the number of crops and cultivars with durable resistance, the need to develop genetic resistance against nematodes becomes widespread, while the standard breeding techniques remain slow and limited in scope due to the extreme difficulties of breaking the species barriers. Other integrated management practices against nematodes are successful at maintaining yields but they still require the frequent or occasional intervention of nematicides.14 The advent of plant genetic engineering and the study of plant-nematode interactions at the molecular level offer many new opportunities to manage plant parasitic nematodes. The ability of the nematodes to establish functional feeding sites in the roots of their host can be thwarted by expressing single chain antibodies— plantibodies—in the plant, by expressing
Recombinant Protease Inhibitors in Plants
toxic molecules in the feeding site to interfere with its development or its function, or by expressing antifeedant molecules to interfere with the development of the nematodes. These genetic engineering strategies have been recently reviewed.15,16 Many related physiological processes of plant parasitic nematodes and of insect pests have traditionally been targeted with the same or similar molecules as active ingredients of nematicides and insecticides, or even parallel field management practices. It was natural that the first recombinant proteinase inhibitor (PI) in a plant used against an insect pest17 would be tested against plant parasitic nematodes.18 This first test demonstrated that recombinant PIs could help suppress root- knot and cyst nematodes, enough to warrant a full exploration of this strategy.
4.2. Proteases in Nematodes The efficient use of recombinant PIs against plant parasitic nematodes stems from our knowledge of their proteinases. Few proteinases of plant parasitic nematodes have been isolated or characterized, but inferences can be made from the study of proteinases from other nematodes.
4.2.1. Bacterial Feeding Nematodes In their genetic analysis of protease function in specific mutants of C. elegans with altered activity of single proteolytic enzymes, Sarkis et al 19 showed the activities of a cathepsin D, a carboxyl protease which is inhibited by pepstatin, two cathepsins which are inhibited by leupeptin, and a leupeptininsensitive, thiol-independent protease. A cysteine proteinase was subsequently characterized by homology with Haemonchus contortus, an animal parasitic nematode, with its expression localized into the intestine by in situ hybridization. 20 Nowadays the catalogue of proteases of C. elegans is growing fast thanks to the genome sequencing project.21 Other bacterial feeding nematodes are used as biological control agents of insect
Recombinant Protease Inhibitors as Management Tools to Suppress Parasitic Nematodes
pests. The nematodes enter their hosts through natural openings, penetrate the internal walls, and then enter the hemocoel using mechanical means and proteolytic enzymes. 22 The possible involvement of excreted proteinases was suggested by treating these nematodes with PIs.23 Untreated nematodes (Steinernema glaseri) killed 75 % of susceptible insects (Galleria mellonella larvae) in 18 hours, whereas nematodes incubated for 2 hours in a solution of iodoacetamide or 1,10-phenantroline killed only 17 and 30% of their host, respectively. The reduction in insect mortality suggested that the PIs had inactivated proteases from nematode secretions and delayed their penetration into the hemocoel.
4.2.2 Animal Parasitic Nematodes A considerable body of information has been recently developed about the nature and the role of animal parasitic nematode proteases. Potent zinc proteinases are excreted by the infective larvae of Strong yloides stercoralis, a human parasitic nematode, and by Trichuris suis, a nematode parasite of swine, to digest host tissue membrane components.24,25 There are several variants of an aspartic proteinase with the same proteolytic function in various tissues of Dirofilaria immitis.26 Haemonchus contortus excretes a cysteine proteinase27 that facilitates penetration of the abomasum, prevents blood clotting, degrades lost tissues and blood components, while Trichinella spiralis excretes a serine, a cysteine, and an aspartyl proteinases to degrade fibrinogen and plasminogen.28 Many other proteinases of human and animal parasitic nematodes have been identified and characterized in the last decade but it is only recently that enzyme inhibitors are considered as potential tools to overcome nematode infection.29
45
4.2.3 Plant Parasitic Nematodes 4.2.3.1. Role of Proteinases Plant parasitic nematodes procure their food by drawing in the content of individual cells of many plant tissues. Secretions from the esophageal glands are injected through the stylet in the cytoplasm presumably to achieve an enzymatic partial liquefaction of cytoplasmic and organelle proteins, before the nematode draws in the digested content through its stylet.30,31 The root-knot and cyst nematode species stimulate the formation of a dedicated feeding site. The juvenile nematodes penetrate the root and migrate through the cortical tissue usually to a specific group of cells near the protophloem at the tip of the root. The consequences of their first feed on these root cells is dramatic. Within a few hours of reaching the appropriate root tissue, the root-knot nematode injects its esophageal secretions into 4 to 6 cells. The current paradigm is that one or several of the proteins in these secretions act as transcription factors with a role in the gene deregulation that follows.32 The cells become polynucleated, enlarge considerably, and become the equivalent of transfer cells with a much increased metabolism. The cyst nematodes induce a syncitium with a similar function. Several proteins from the esophageal glands and their secretions have recently been isolated33,34 and a cellulase has been characterized.35 Dasgupta and Ganguly’s early study36 of a root-knot nematode, Meloidogyne incognita, demonstrated a trypsin-like proteinase activity in extracts of preparasitic juveniles. The tobacco cyst nematode Globodera tabacum, the potato cyst nematode Globodera pallida and M. incognita were nutritionally stressed in roots of transgenic tobacco and potato expressing a serine PI from cowpea (CpTI),18,32 suggesting that all three nematode species rely heavily on serine proteinases for digestive proteolysis. The presence of serine proteinases in their digestive system was later confirmed from sections of potato cyst and soybean cyst nematodes incubated in naphtyl amidelinked specific peptidic substrates.37,38 Two
46
serine proteinases were subsequently characterized from the soybean cyst nematodes, and their activity was shown to be inhibited by cowpea trypsin inhibitor (CpTI).38 Cysteine proteinase activity was also identified in the potato cyst nematode while a cathepsin L-like cysteine proteinase was identified in the intestine of the soybean cyst nematode.39 The cysteine proteinase activity fluoresced in sections of the preparasitic juveniles and of the mature females of soybean cyst nematodes, after incubation in naphtylamide-linked specific peptide substrates. A cathepsin S-like cysteine proteinase was also screened from a cDNA library of that nematode.40 Interestingly, the potato cyst nematode secretes a cathepsin B-like cysteine proteinase, and two unrelated cathepsin L- and B-like cysteine proteinases each excreted by the soybean cyst nematode and the potato cyst nematode are very susceptible to inhibition by the same oryzacystatin. 39,41,42
4.2.3.2. PIs in Nematodes Like proteases PIs are probably common in nematodes,43,44 and as with insect PIs they could be overexpressed in transgenic plants to suppress the digestive proteases they are normally supposed to regulate.45 Recombinant PIs from insects may also have a wide spectrum of action and be useful against nematodes. Brunke et al46 isolated a cysteine PI from corn rootworm (Diabrotica virgifera) and suggested that it could be effective against digestive proteinases of insects and nematodes when expressed in transgenic plants. Now that several PIs have been identified in C. elegans, PIs can be isolated by homology from plant parasitic nematodes and this approach can be tested.21
4.2.3.3. Specificity of Nematode Proteinases Preparasitic and parasitic stages of the root-knot nematode, Meloidogyne hapla, contain and secrete the same complex of cysteine proteinases.47,48 The major form, Mhp1, was practically obliterated by the rice cystatin oryzacystatin I (OC-I), but not so much by a
Recombinant Protease Inhibitors in Plants
second rice cystatin, oryzacystatin II (OC-II). The protease activity of freshly hatched nematodes (preparasitic), of young females and of mature, parasitic females was shown as one major and two minor proteins (Mhps) following gelatin/PAGE. The specificities of OC-I and OC-II for Mhps were similar, but their degree of affinity for Mhp1 differed, as suggested by their differential ability to maintain inhibition of the nematode proteinase during mildly-denaturing gelatin/PAGE. In these conditions the OC-I/Mhp1 complex remains stable, preventing restoration of Mhp1 activity following electrophoresis and suggesting the high stability of this inhibitorenzyme complex. Conversely, the major cysteine proteinases of M. incognita (Mips) and M. javanica (Mjps) are strongly inhibited by OC-II while the activity of OC-I-inhibited proteinases is significantly restored during electrophoresis. Ki values for the electrophoresisstable complexes OC-I/Mhp1, OC-II/Mip1 and OC-II/Mjp1 were estimated in the nanomolar range, while those of the electrophoresis-unstable complexes OC-II/Mhp1, OC-I/Mip1 and OC-I/Mjp1 were in the micromolar range. 47,48 These estimates allowed us to predict that OC-II would be much more effective than OC-I at suppressing the activity of M. incognita and M. javanica digestive proteinases in vivo.47 Despite this specificity, plant parasitic nematodes, like insects,49 may prove to have related proteinases that bind tightly to the same inhibitor.
4.3. Effects of Recombinant PIs 4.3.1. C. elegans
As shown by Urwin et al41 the effect of a cowpea cystatin or of a wild (OC-I) and an engineered (OC-I∆D86) oryzacystatins on C. elegans is anything but subtle. Only 2% of the larvae developed to adulthood on media containing 2.5 ppm of OC-I or OC-I∆D86, while 95% developed to adulthood on BSA medium without cystatins. A separate experiment showed that larvae hatched on media without cystatins, but transferred 6, 12 or 24 hours later to plates containing 2.5 ppm
Recombinant Protease Inhibitors as Management Tools to Suppress Parasitic Nematodes
of cystatin, were also sensitive to the effects of the PIs, all C. elegans juveniles becoming moribund on the media supplemented with either of three cystatins. In contrast, 76% of larvae that were transferred to the same supplemented media after 30 hours on a non supplemented medium survived and developed into adults.41 Obviously, some as yet unreported, fundamental and precise processes of C. elegans were still perturbed 24 hours after hatching but not after 30 hours. At this point there is as yet no evidence, however that early stages of plant parasitic nematodes are more susceptible to PIs than later stages. It is difficult to suggest that a differential effect on digestive proteolysis can have such quick and drastic effects, especially if we assume that C. elegans, like many insects,50-52 could possibly switch some of its digestive proteolytic activity to potentially inhibitorinsensitive proteinases,19,20 or synthesize de novo the affected enzyme.
4.3.2. Plant Parasitic Nematodes There is currently no method to reliably predict the tertiary structure of a protein from amino acid sequences. However, when a PI is relatively homologous to other proteins of known structure, a model can identify favorable and unfavorable interactions between the inhibitor and the proteinase receptor, and maximize the beneficial interactions to increase the binding affinity.53,54 While the OC-I protein has been engineered to study its biochemistry,55 Urwin et al41 engineered its conformation with the practical goal of controlling plant parasitic nematodes. A stereochemistry model, proposed from the amino acid sequences of twenty-eight plant and animal cystatins yielded consensus conformations and sequences that suggested the alterations that might make the oryzacystatin fit better when docked into a model of the active site of papain. Variant proteins were generated by changing the residues pro-83, trp-84 and asp-86 with 12 different amino acids in place of the original amino acid in OC-I. These substitutions and deletions of amino acids at various sites of the protein brought a range of variation of Ki
47
values in the variants, as determined against the model enzyme papain. Most of the mutations led to an increase in Ki when tested with papain or the C. elegans cysteine proteinase, gcp-1.20 One mutation maintained a similar Ki value to that of the wild type OC-I with a deletion of met-85 which is adjacent to asp-86. In contrast, when the residue asp-86 was deleted (OC-IDD86) the Ki against papain became 7% of the Ki of the wild protein, 41 strongly suggesting the usefulness of site-directed mutagenesis to design PIs effective against nematode extracellular proteases. That nematode proteinases are differentially inhibited by plant PIs also suggests that screening a large number of plants will yield highly efficient PIs with wide specificity against plant parasitic nematodes, but the cost of such a strategy may be prohibitive. Instead, potent inhibitors binding ‘irreversibly’ to nematode digestive proteinases could be selected using phage display libraries (see Chapter 11, this volume). Phages carrying PI genes expressing inhibitors of varying affinity to a particular recombinant nematode or insect proteinase can be selected by chromatography using the proteinase as an immobilized affinity ligand.56,57 The multitude of PI genes that can be generated using this technique, with mutations at the active site of the protein, will potentially yield inhibitors with extremely low Ki against previously isolated nematode proteinases.
4.3.3. Effects of Recombinant PIs on Nematodes in Plants Natural PIs are an important element of the plant defense response to herbivory,58,59 and they may also act to restrict infection by nematodes. Methodologies to express recombinant PIs in plants were reviewed recently.60 Serine and cysteine recombinant PIs expressed in plants may effectively suppress plant parasitic nematodes.
4.3.3.1 Serine-Type Inhibitors CpTI, a serine PI from an insect-resistant breeding line of cowpea, was shown to suppress growth of the tobacco budworm
48
Heliothis virescens feeding on transgenic tobacco leaves,17 of potato cyst nematode and root-knot nematodes feeding on transgenic potato roots,18 and of tobacco cyst nematode feeding on transgenic tobacco roots,67 expressing the protein. CpTI also affected the sexual development of tobacco and potato cyst nematode juveniles, a demonstrated sign of poor nutrition. Unexpectedly, the root-knot nematodes feeding on potato root giant cells expressing CpTI, although they were obviously stressed nutritionally and severely delayed in their development, did not show any sign of reversal from female to male.18 This is an unexpected response since sex reversal from female to male is common in root-knot nematodes under nutritional or other environmental stress.61
4.3.3.2. Cysteine-Type Inhibitors The development of cyst and root-knot nematodes can also be suppressed by a cysteine PI. Potato cyst and sugar beet cyst nematodes were not killed but they were nutritionally stressed, and their development and reproduction rates were severely depressed (Fig. 4.1) when feeding in transformed tomato hairy roots or Arabidopsis thaliana roots expressing OC-I∆D86.41 Their growth was measured by image analysis of cross sectional area and correlated to the number of eggs produced by female nematodes in untransformed roots. Female nematode size was significantly reduced compared to the controls. The females stopped growing prematurely, and produced very few eggs compared to the 200 eggs produced by the average female feeding on syncitia in non transformed roots of tomato or A. thaliana. The size of root-knot nematode females was also reduced, and similarly to the root-knot nematode feeding in roots of transgenic potato expressing CpTI, the suppression of digestive cysteine proteinase activity in the root-knot nematode and the sugar beet cyst nematode in transgenic Arabidopsis, and in the potato cyst nematode in transgenic hairy roots of tomato,41 did not induce the shift in gender determination that the potato cyst nematode displayed when feeding in potato
Recombinant Protease Inhibitors in Plants
roots engineered to express CpTI.18 Expression of OC-I∆D86 in A. thaliana, while it suppressed drastically the growth of sugar beet cyst nematodes (Fig. 4.1), did not affect its gender determination42 although a protein deficiency in the diet of the nematode would be expected to induce a significant shift in sex determination.62 These subtly different nutritional effects of two separate classes of PIs on these nematodes raise interesting possibilities of causality, and imply relatively different effects on various physiological processes in the nematodes.
4.3.3.3. Dual Cysteine/Serine PIs
In a recent study, Urwin et al63 expressed two inhibitors in A. thaliana as a single translation product. The coding regions of CpTI and of OC-I∆D86 were joined by peptide linkers refractory or susceptible to proteolytic cleavage. Cleavage was not essential for the activity of either inhibitors since papain and trypsin were inhibited by the fusion proteins. Surprisingly, the inhibitors from the cleavable fusion protein were not ingested by the sugar beet cyst nematode. The authors suggested that the linker may have caused sequestering of both inhibitors into cell membranes and away from the cytosol where they would have been available to the nematodes.63 The inhibitors from the other fusion protein, a 23-kDa non-cleavable protein, were also not taken up by the sugar beet cyst nematode, possibly because the size of the fusion protein was too large. However both fusion proteins still had very profound effects on female development and fecundity.63 This strategy could have obvious advantages for stacking distinct gene products active against various pests.
4.3.3.4. Lectins Lectins, which bind with high specificity to certain carbohydrate residues, are considered as defense-related proteins in plants.64,65 The pea lectin has no effect on the potato cyst nematode G. pallida feeding on transgenic potato plants expressing the protein,18 but a mannose-binding lectin (GNA) is proving to be an ideal candidate
Recombinant Protease Inhibitors as Management Tools to Suppress Parasitic Nematodes
49
Fig. 4.1. Growth of cyst nematode females in transgenic roots expressing OC-I∆D86: A. potato cyst nematode in transgenic tomato (in white), and in untransformed tomato (in black); B. sugar beet cyst nematode in transgenic Arabidopsis (in white) and in untransformed Arabidopsis (in black). Cross sectional area is expressed in mm2. Fecundity was estimated by the relationship between female size and the number of eggs produced. Redrawn with permission from: Urwin PE, Atkinson HJ, Waller DA et al. Plant J 8:121-131. © 1997 Blackwell Science Ltd., and from Urwin PE, Lilley CJ, McPherson MJ et al. Plant J 12:455-461. © 1997 Blackwell Science Ltd. for stacking with a PI to provide durable resistance against nematodes. Whereas the development of the tobacco budworm H. virescens is equally inhibited by the expression of CpTI or GNA,17,66 the development of the potato cyst nematode is affected by both CpTI, OC-I and GNA.18,41,67 GNA also suppresses the development or reproduction of root-lesion nematodes, Pratylenchus bolivianus and P. neglectus, and of the sugar beet cyst nematode H. schachtii,67 although no adverse effects were noted for Meloidogybe arenaria.68
4.4. Outlook The plant PIs that have been shown to inhibit plant parasitic nematode proteinases (e.g. OC-I, OC-II, OC-I∆D86 and CpTI), a number of even more potent inhibitors isolated from phage display libraries, and the lectin GNA will undoubtedly be engineered into a range of crops in the near future. There is no indication that soil and root dwelling parasitic nematodes can adapt anywhere as quickly as many insects. For instance, there have been no reports of field resistance developing in plant parasitic nematodes
against the acetylcholinesterase inhibitors after 30 years of extended use in agriculture, but there are many reports of species, notably the soybean cyst nematode, adapting quickly to major resistance genes.69 Current information is too sparse, however to predict to what extent plant parasitic nematodes will or will not be able to overcome recombinant PI-based genetic resistance. The fate and persistence of recombinant plant PIs in the environment is also an important issue because of possible direct and indirect effects on organisms other than plant parasitic nematodes and other pests (see Chapter 8, this volume).70 Soil arthropods and other beneficial organisms, in particular are of special concern.71 OC-I and OC-II, for instance inhibit strongly the proteinases of extracts of Amblyseius fallacis, a native predator of the major pest Tetranychus urticae, the two spotted spider mite.48 Nematode populations in the soil surrounding litterbags containing tobacco leaves expressing tomato proteinase inhibitor I72 were also greater than those in the soil surrounding non transformed tobacco litterbags.70 A different trophic group composition was also observed in this case,
50
including a significantly lower ratio of bacterial feeding nematodes to fungal feeding nematodes,70 strongly stressing the need to assess the effects of the recombinant PIs not only against the target pests, but also against the other organisms in the environment.
Acknowledgments Contribution #2064, Pacific Agri-Food Research Centre, Agriculture and Agri-Food Canada
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42. Urwin PE, Lilley CJ, McPherson MJ et al. Resistance to both cyst and root-knot nematodes conferred by transgenic Arabidopsis expressing a modified plant cystatin. Plant J 1997; 12:455-461. 43. Hawley JH, Peanasky RJ. Ascaris suum: Are trypsin inhibitors involved in species specificity of Ascarid nematodes? Exp Parasitol 1992; 75: 112-118. 44. Lustigman S, Brotman B, Huima T et al. Molecular cloning and characterization of onchocystatin, a cysteine proteinase inhibitor of Onchocerca volvulus. J Biol Chem 1992; 267: 17339-17346. 45. Thomas JC, Adams DG, Kepenne VD et al. Manduca sexta encoded protease inhibitors expressed in Nicotiana tabacum provide protection against insects. Plant Physiol Biochem 1995; 33:611-614. 46. Brunke KJ, Chan VJ, Deluca-Flaherty CR et al. Thiol proteinase inhibitor peptide, virgiferin, and its DNA—used to generate transformed plants with decreased susceptibility to damage by insect pests and nematodes. International Patent Application 1995; WO 9524479 A. 47. Michaud D, Cantin L, Bonade Bottino M et al. Identification of stable plant cystatin/nematode proteinase complexes using mildlydenaturing gelatin/polyacrylamide gel electrophoresis. Electrophoresis 1996; 17:1373-1379. 48. Michaud D, Cantin L, Raworth DA et al. Assessing the stability of cystatin/cysteine proteinase complexes using mildly-denaturing gelatin/polyacrylamide gel electrophoresis. Electrophoresis 1996; 17:74-79. 49. Kuroda M, Ishimoto M, Suzuki K et al. Oryzacystatins exhibit growth-inhibitory and lethal effects on different species of bean insect pests, Callosobruchus chinensis (Coleoptera) and Riptortus clavatus (Hemiptera). Biosci Biotechnol Biochem 1996; 60:209-212. 50. Bolter CJ, Jongsma MA. Colorado potato beetles (Leptinotarsa decemlineata) adapt to proteinase inhibitors induced in potato leaves by methyl jasmonate. J Insect Physiol 1995; 41:1071-1078. 51. Broadway RM. Are insects resistant to plant proteinase inhibitors? J Insect Physiol 1995; 41:107-116. 52. Jongsma MA, Bakker PL, Peters J et al. Adaptation of Spodoptera exigua larvae to plant proteinase inhibitors by induction of gut proteinase activity insensitive to inhibition. Proc Natl Acad Sci USA 1995; 92:8041-8045. 53. Cohen FE, Gregoret LM, Amiri P et al. Arresting tissue invasion of a parasite by protease inhibitors chosen with the aid of computer modeling. Biochemistry 1991; 30:11221-11229.
52 54. Li R, Chen X, Gong B et al. Structure-based design of parasitic protease inhibitors. Bioorgan Medicin Chem 1996; 4:1421-1427. 55. Arai S, Watanabe H, Kondo H et al. Papain-inhibitory activity of oryzacystatin, a rice seed cysteine protease inhibitor depends on the central Gln-Val-Val-Ala-Gly region conserved among cystatin superfamily members. J Biochem 1991; 109: 294-298. 56. Jespers LS, Messens JH, De Keyser A et al. Surface expression and ligand-based selection of cDNAs fused to filamentous phage gene IV. Bio/Technology 1995; 13:378-382. 57. Jongsma MA, Bakker PL, Stiekema WJ et al. Phage display of a double headed protease inhibitor: Analysis of the binding domains of potato proteinase inhibitor II. Mol Breeding 1995; 1:181-191. 58. Koiwa H, Bressan RA, Hasegawa PM. Regulation of protease inhibitors and plant defense. Trends Plant Sci 1997; 10:379-384. 59. Ryan CA. Proteinase inhibitors in plants: Genes for improving defenses against insects and pathogens. Annu Rev Phytopathol 1990; 28:425-449. 60. Michaud D, Vrain TC. Expression of recombinant proteinase inhibitors in plants. In: Cunningham C, Porter AJR, eds. Recombinant proteins from plants: Production and isolation of clinically useful compounds. Totowa NJ:Humana Press Inc 1998; 49-64. 61. Triantaphyllou AC. Sex determination in Meloidogyne incognita Chitwood 1949 and intersexuality in M. javanica (Treub 1885) Chitwood 1949. Ann Inst Phytopath Benaki NS 1960; 3:12-31. 62. Grundler F, Betka M, Wyss U. Influence of changes in the nurse cell system (syncitium) on sex determination and development of the cyst nematode Heterodera schachtii: Total amounts of proteins and amino acids. Phytopathology 1991; 81: 70-74. 63. Urwin PE, McPherson MJ, Atkinson HJ. Enhanced transgenic plant resistance to nematodes by dual proteinase inhibitor constructs. Planta 1998; 204:472-479.
Recombinant Protease Inhibitors in Plants 64. Chrispeels MJ, Raikhel NV. Lectins, lectin genes, and their role in plant defense. Plant Cell 1991; 3:1-9. 65. Peumans WJ, van Damme EJ. Lectins as plant defence proteins. In Pusztai A, Bardocz S, ed. Lectins, biomedical perspectives, London: Taylor and Francis 1995:1-21. 66. Gatehouse AMR, Boulter D, Hilder VA. Potential of plant-derived genes in the genetic manipulation of crops for insect resistance. In Gatehouse AMR, Hilder VA, Boulter D, eds. Plant genetic manipulation for crop protection. Wallingford, UK:CAB International 1993:155-181. 67. Burrows PR, Barker ADP, Newell CA et al. Plant-derived enzyme inhibitors and lectins for resistance against plant-parasitic nematodes in transgenic crops. Pesticide Sci 1998; 52: 176-183. 68. Anwar SA, McKenry MV. Field performance of two genetically transformed grape rootstocks against two root-knot nematode populations. St. Louis MO:Society of Nematologists 1998; 24 (abstr.). 69. Trudgill DL. Resistance to and tolerance of plant parasitic nematodes in plants. Annu Rev Phytopathol 1991; 29:167-192. 70. Donegan KK, Seidler RJ, Fieland VJ et al. Decomposition of genetically engineered tobacco under field conditions: Persistence of the proteinase inhibitor I product and effects on soil microbial respiration and protozoa, nematode and microarthropod populations. J Appl Ecol 1997; 34:767-777. 71. Malone LA, Giacon HA, Burgess EPJ et al. Toxicity of trypsin endopeptidase inhibitors to honey bees (Hymenoptera:Apidae). J Econ Entomol 1995; 88:46-50. 72. Narvaez-Vasquez J, Orozco-Cardenas ML, Ryan CA. Differential expression of a chimeric CaMV-tomato proteinase inhibitor I gene in leaves of transformed nightshade, tobacco and alfalfa plants. Plant Mol Biol 1992; 20:1149-1157.
CHAPTER 5
The Control of Plant Pathogens with Protease Inhibitors A Realistic Approach? Stephen Gleddie and Dominique Michaud
5.1. Introduction
P
lant pathogens contribute significantly to the yield losses suffered by producers and growers of numerous horticultural and field crops worldwide. Controlling plant pathogens by the use of pesticides and fungicides is a common procedure when the costs associated with the disease in question are high. In contrast many diseases occur sporadically since they are highly dependent upon environmental conditions such as temperature, humidity, rainfall or wind, and are therefore not a serious problem in each growing season or region. Predictions about disease outbreaks and crop damage in any season are sometimes very difficult, leading to the loss of yield in situations where an optimistic outlook is taken. Conversely, an overly negative outlook generally results in the unnecessary use of pesticides, with significant economic and environmental costs. As a consequence, the manipulation of host plant resistance to the major plant pathogens is an increasingly important objective of plant breeders. Host resistance may be modified by the use of interspecific and intergeneric hybridization to incorporate resistance traits from resistant germplasm. This approach, however is often time-consuming and involves the identification of resistant germplasm from plant introductions and seed banks, followed by wide hybridization which
may be achieved through conventional crosses and screening. Moreover, intergeneric and even interspecific crosses can be very difficult to achieve, and they may require more complicated manipulations such as embryo rescue and in vitro culture, or somatic hybridization, in which the fusion of somatic protoplasts is used to circumvent extreme sexual incompatibilities (see ref. 1 for a review). In each of these instances the selection process for hybrids and backcross progeny which carry the resistance traits often limits the successful transfer of resistance into commercial germplasm. The screening for resistance in vitro is not feasible, while the screening of whole plants in disease nurseries by artificial or natural infection is often difficult. Molecular genetic approaches aimed at identifying resistance traits or at directly modifying the host plant's resistance status offer an interesting alternative. The search for molecular markers linked or associated with disease and pathogen resistance traits, for instance is a key goal of many plant pathologists since such markers greatly aid in the indirect selection of resistant germplasm and tracking of resistant progeny. In parallel, the advent of plant genetic transformation now offers the possibility of manipulating host plant resistance to pathogens by the use of appropriate resistance genes.2 In this case
Recombinant Protease Inhibitors in Plants, edited by Dominique Michaud. ©2000 Eurekah.com.
Recombinant Protease Inhibitors in Plants
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plants with ‘built-in’ resistance may be designed by the addition of defense-related proteins which interact with specific receptors or interfere with some metabolic functions in the target organism. Two kinds of resistance may be obtained by this approach: 1. complete resistance, where the trait introduced kills the target pest, and 2. relative resistance or tolerance, where the novel trait only partly protects the plant.
Complete resistance is seen where the genes encoding toxic (pesticidal) molecules are integrated into the plant genome. This approach forms a resistance mechanism based upon antibiosis—the pathogen recognizes the host plant, attacks or invades, and is prevented from further invasion by some toxic metabolite. Relative resistance or tolerance also suggests that the pathogen recognizes the host, invades and attacks, but is prevented from causing a serious or full-scale host infection due to factors which interfere with specific metabolic processes. Although until now most emphasis has been put on the design of ‘killer plants,’ partial resistance shows increasing potential. Like any other pesticide, transgenic plants expressing toxic proteins will exert a strong selection pressure on the target pest population, which may result in the rapid development of resistant populations in the field and thus decrease the effectiveness of the modified plants when they are used as a sole control method.3 The effect of plants expressing antimetabolic proteins, in contrast, would be more diffuse, leading to reduced fitness of the target pest without exerting a high selection pressure on the target population. As a result, these plants would not provide complete control of the target pests, but instead alter their overall fitness in the environment. Such plants could prove to be useful, especially when used in combination with other control approaches. Pathogens with reduced fitness could be more susceptible, for instance, to biological control agents used in the field, thereby contributing to the improvement of their efficacy.
5.2. Extracellular Proteases in Plant Pathogens In this perspective, the use of protease inhibitor (PI)-encoding cDNA sequences may appear of particular interest in the development of plants which are partly resistant to pathogens. Because they can inhibit a wide variety of proteolytic enzymes, PIs were proposed as a tool to control pest and pathogenic organisms as diverse as herbivorous insects, parasitic nematodes and human microbial pathogens (see Chapters 2, 3, 4 and 17, this volume).4-6 Until now little is known about the potential of PIs in the control of plant pathogens, but one can speculate that blocking the activity of plant fungal or bacterial pathogen proteases could eventually decrease their fitness in planta by the alteration of some yet uncharacterized, but useful physiological functions. Extracellular proteases may be important in many pathogens for basic processes like dietary protein hydrolysis, penetration of host tissues or zymogen activation. While their role during the infection of plant tissues still remains to be elucidated, the secreted proteases of plant pathogens represent potential target molecules for the design of pathogen-resistant transgenic plants. Several studies examined the nature and the role of extracellular proteases in plant pathogenic fungi, bacteria and viruses.
5.2.1. Fungal Proteases Several soil-borne fungi secrete digestive proteases into the culture medium when grown in vitro.7,8 The pathogenic fungus Verticillium dahliae, for instance produces an extracellular trypsin-like protease in all media tested, including those containing a defined medium with glucose and ammonium or nitrogen salts.8 The total amount of protease in the cultures does however increase in the presence of animal or plant proteins, and appears to be regulated by carbon metabolite repression, as also reported for the extracellular proteases of two other plant pathogenic fungi, Cladosporium cucumerinum and Cochliobolus carbonum.9,10
The Control of Plant Pathogens with Protease Inhibitors
The basidiomycete Chondrostereum purpureum, the causal agent of silverleaf of fruit trees, also secretes proteases. 11 In particular, secreted metalloproteases can be found throughout the infection zone of silverleaf-susceptible plants while they could only be found at the wound site of resistant plants, thereby establishing a link between fungal growth and the secretion of digestive proteases. The authors proposed that this pathogen secretes the metalloproteases in the presence of limited nitrogen sources, for instance during natural infection of woody tissues or when grown on agar plates. Under artificial conditions of high nitrogen availability (i.e. when the fungus is grown in a liquid nitrogen-rich medium) the amount of secreted proteases is reduced, suggesting that these enzymes could be involved in amino acid uptake necessary for normal growth and development. A similar role may be associated with the proteases secreted by Glomerella cingulata, the causal agent of anthracnose in a variety of crops worldwide.12 The infection process involves the attachment of a fungal spore to the leaf surface, followed by differentiation to form an appressorium which penetrates the cuticle and the epidermal cells. After invasion, continued fungal development relies heavily upon the secretion of a multitude of enzymes, including an aspartic protease. Interestingly, fungal cultures grown in the presence of protein (BSA) secrete this protease into the medium within three days of culture, whereas those grown in the presence of ammonium salts do not secrete any protease. Moreover, the presence of ammonium ions in the medium totally represses BSA-induced expression of the protease at the transcriptional level, suggesting that it serves to provide the fungus with a nitrogen source necessary for growth. The recent molecular cloning of the gene encoding this secretory protease should permit researchers to disrupt synthesis of the enzyme, and allow an evaluation of its role in pathogenicity.12 While the exact roles of extracellular proteases in plant pathogens still remain unknown, their importance during the infection of plant tissues appears obvious in some
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cases. Several pathogenic fungi which are protease-deficient were shown to be nonpathogenic, suggesting a role for secreted proteases during the infection, colonization and pathogenesis of susceptible plants. A good example is that of the foliar pathogen Pyrenopeziza brassicae, the causal agent of light leaf spot in crucifers.13 This pathogen initially infects the subcuticular space of leaves without entering the epidermal or the mesophyll cells, and at this point very few symptoms are visible on the leaves of crops such as canola. By mutating pathogenic strains of P. brassicae, Ball et al13 demonstrated that several independent mutants deficient in the production of an extracellular protease had lost the ability to induce lesions either in vitro or in planta. Interestingly, fungal transformation with cosmid clones from wild-type fungal DNA resulted in a transformant with a single insertion of cosmid DNA, which was shown to have a restored pathogenicity and the ability to secrete the protease. Since sexual crosses between the mutant and wild-type fungal strains did not produce any strain in which the linkage between pathogenicity and protease secretion was broken, it is assumed that the two traits were either very tightly linked, or that indeed the protease was a pathogenicity determinant. A role in pathogenicity was also suggested both in vivo and in vitro for an aspartic protease secreted by Botrytis cinerea.14 In this study the authors suggested that the cytotoxic effects of the secreted protease were due to the digestion of plant cell walls, the production of the protease being observed in eight different fungal isolates which colonized carrot, strawbery, raspberry, cabbage, grape and broadbean. Interestingly, pre-treatment of fungal spores with the aspartic PI pepstatin markedly reduced the level of fungal infection and disease development in these host plants but did not affect the rate of spore germination, strongly suggesting a role for this secreted protease during pathogenesis.
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5.2.2. Bacterial Proteases Proteases are also secreted by plant pathogenic bacteria. Erwinia chrysanthemi, for instance secretes several extracellular enzymes to macerate plant tissues, including pectinases, cellulases, phospholipases, xylanases and proteases.15 Investigations on the pathogenicity of this organism, however suggested that the secreted proteases were not essential during the maceration process. “Knockout” strains were created by the deletion of certain genes, such that the relative contribution of the different enzymes could be studied independently. Conversely, “addbacks” or strains which had the gene replaced were also studied and the most significant enzyme contributing to the ability of E. chrysanthemi to macerate potato tuber tissue in vitro was shown to be pectin lyase E, with proteases contributing very little to the overall process. The situation was different, however with the pathogen Pseudomonas syringae pv. syringae, which secretes an EDTA-sensitive metallopeptidase active over a broad pH range.16 This enzyme is found in the periplasmic space of bacterial cells, where it presumably serves several important functions critical to the pathogenicity and survival of the pathogen. In particular, it would direct the processing of the dipeptide tabtoxin, which is an inactive precursor of tabtoxine-β-lactam. This monocyclic β-lactam antibiotic is unique in that it does not affect bacterial cell wall synthesis but rather is a potent inhibitor of glutamine synthase (GS), an enzyme found in many fungal, plant and bacterial species. This toxin was also studied in the pathogen P. syringae pv. tabaci, the causal agent of wildfire of tobacco. In this case a characteristic pale halo surrounds the necrotic infection sites on leaves due to the inhibition of the host GS, an effect that can be mimicked by application of the purified toxin. Presumably the evolutionary advantage to this bacterial pathogen of producing such a broad-spectrum antibiotic compound includes growth suppression of competitor pathogens and reduction of the host plant cells’ fitness. A defensive strategy based upon the extracellular expression in leaves of a metallo-PI might
Recombinant Protease Inhibitors in Plants
result in decreased pathogenicity by affecting the rate of toxin precursor processing once the pathogen invades the host. Inhibition of extracellular proteases could also be interesting to control Xanthomonas campestris pv. campestris, a pathogen of cruciferous plants responsible for black rot disease which produces a range of secreted extracellular enzymes including polygalacturonate lyase, endoglucanase, amylase and proteases. An important role for the proteases in this bacterium was confirmed by the use of a leaf-nicking bioassay, which clearly showed that mutants lacking the secreted proteases were less virulent than the wild-type strain.17 Moreover, when wild-type strains were cultured on a medium rich in protein (skim milk) they were found to secrete higher amounts of proteases than when they were cultured on medium supplemented with turnip cell wall fragments. Conversely, when cultured in a medium rich in casamino acids very little protease activity was secreted into the medium, strongly suggesting that the secretion of digestive proteases in this bacterium depends on the nature of the nitrogen source available.
5.2.3. Viral Proteases Unlike fungi and bacteria, viruses do not depend on amino acid intake for their ‘development’, but they often rely upon proteases, which mediate the processing of their constituent polyproteins. As noted by Babé and Craik,18 this mode of post- translational modification serves several purposes: the attached proteins can travel together to the proper assembly site, enzyme activation is delayed until assembly begins, and regulation of proteolysis can help control the concentration of key viral proteins. The processing protease also would contribute to mediate virus assembly/disassembly by converting uncleaved polyproteins into mature capsids capable of disassembling upon virus entry into a newly infected cell.18 Proteases have been found in a wide variety of viruses, including several ssRNA and dsDNA viruses. A papain-like processing enzyme, for instance has been identified within
The Control of Plant Pathogens with Protease Inhibitors
the large 206-kDa replicase polyprotein of turnip yellow mosaic virus.19 In this virus the protease is essential to process the polypeptide into two smaller peptides, a large C-terminal portion, and a smaller N-terminal portion. By mutation and deletion analysis it was possible to identify the catalytic amino acid residues responsible for activity, and it was noted that when amino acid substitutions were made to abolish proteolytic activity, a large molar excess of purified wild-type protease supplied in trans could not cleave these protease-deficient mutants. 19 This observation suggests that the protease must autoprocess or self-cleave, and that disruption of its activity (for example by a PI) would prevent polyprotein processing. The practical potential of inhibiting viral proteases was also recently suggested for the polyprotein of the rice tungro spherical virus, which is processed by a serine protease-like domain located in the C-terminal region of the polyprotein.20 In this case, mutation of putative cleavage sites on the polyprotein resulted in an altered processing, again suggesting that altering the interaction between the processing enzyme and the viral polyprotein could allow the prevention of replication of several viruses in planta. Although the experimental expression of PIs in transgenic plants coupled with viral inoculation experiments still remain necessary for the establishment of definitive conclusions, the use of protein PIs to block virus polyprotein processing clearly appears as a potential way of protecting plants from several viral pathogens. Similar to the animal Sindbis-like viruses, for instance, the replicase polyprotein of many plant counterparts such as the tymo-, carlo- and capilloviruses possess papain-like proteases, which represent possible targets for the design of viral control strategies. At present the usefulness of such a strategy to control plant viruses remains to be assessed, but the current use of PIs in the treatment of viral diseases in humans6,21,22 (see Chapter 17, this volume) strongly suggests that the expression of appropriate recombinant PIs in the cytoplasm of plant cells could represent an effective means of
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protecting plants from viral infections (see note added in proof ).
5.3. Induction of PIs in Infected Plants The fact that protein PIs are found in seeds, leaves and tubers of numerous plants, and that they are induced by wounding in many other tissues suggests that they are in some way defense-related compounds. This combination of wound-inducibility and biological activity against herbivorous insects has led to theories suggesting that they serve as natural phytochemical compounds.23,24 Although most studies carried out until now have dealt with plant-insect systems, it is well-known that pathogens also trigger the accumulation of protein PIs in plant tissues. The pathogen Phytophthora infestans, for example, induces the synthesis of three chymotrypsin inhibitors in infected potato tubers.25 These serine PIs were purified from infected tissues, and subsequently shown to inhibit fungal zoospore germination and hyphae development in vitro, suggesting an antifungal role for these PIs in potato tubers. Other pathogens were shown to induce protein PIs in plants. Cell wall elicitors from the fungus Phytophthora parisitica var. nicotianae, for instance were shown to induce a transient increase in ethylene and lipoxygenase activity followed by the production of protein PIs, strongly suggesting an activation of the octadecanoid /jasmonate defense signalling pathway—also induced by insects—by the fungal elicitors. 26 The phytotoxin coronatine, a virulence factor of the bacterial pathogen Pseudomonas syringae pv. tomato,27 may also induce the accumulation of PIs in plant tissues via a jasmonate-related pathway. This toxin shows structural homology with methyl jasmonate, a volatile form of jasmonate, and the biological effects of these two compounds on plant tissues are very similar.28 In particular coronatine, like methyl jasmonate, promotes the accumulation of large multicystatin crystals within the mesophyll of plant leaves (Fig. 5.1), suggesting the ability of the host plant to produce defensive PIs in reaction to the bacterial pathogen.
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A
B
Fig. 5.1. Effects of the bacterial toxin coronatine on tobacco and tomato plants. A) The morphology of a coronatine-treated tobacco plant. The leaves were spotted with four separate drops of 5 µl each containing coronatine solution (2.5 ng/µl in water) and photographed five days later. The visible symptoms are small white lesions absent on the leaves of nontreated plants. B) Expression of the cysteine PI multicystatin in tomato leaf cells treated with coronatine. A polyclonal antiserum directed against potato multicystatin was used to bind the cystatin, followed by gold-conjugated secondary antibody detection. The black grains (gold particles) observed on the electron micrograph are reacting heavily with the large multicystatin crystal induced in the cytoplasm of coronatine-treated leaf cells.
The Control of Plant Pathogens with Protease Inhibitors
The growth of P. syringae, however, is not affected when on coronatine-treated tissues despite the rapid accumulation of multicystatin in the leaves.28 The fact that some PIs induced during infection show very little adverse effects against pathogens still remains to be understood, but one can speculate that these PIs may not be accumulated at the proper location in the host cells (see for instance Fig. 5.1), and that the plant response to infection may be more general than specific. Such a lack of specificity of the plant defense apparatus was suggested, notably by the response of tomato plants to the citrus exocortis viroid.29 This viroid, which is simply composed of nude, circular RNA can induce the host plant to secrete extracellular serine-type proteases of the subtilisin family. These proteases are synthesized as inactive precursors including a signal peptide, and accumulate in the extracellular compartment of infected tissues, presumably to degrade exogenous proteins. Although a clearer understanding of the role of these proteases will require more data, it clearly appears that the defense response of plants may be in some cases an unspecific response aimed at counteracting infectious processes in general. Another reason explaining the limited effect of the host PIs could be that the pathogens have acquired the ability to secrete proteases insensitive to the host plant PIs, as is known to occur in herbivorous insects.30,31 Plant pathogens, like insects coevolved over a long period of time with their host plant, and therefore it is plausible that every defensive barrier developed by the plant to counteract infection resulted in the gradual adaptation of the pathogen to this specific barrier. As a consequence it appears obvious that the development of any pathogen control strategy based on recombinant PIs must be adapted to each particular plant-pathogen system, and that the PIs must be chosen with care for each particular protease or group of proteases to be regulated (see Chapter 10, this volume). Considering the recent literature in the field, it also appears that the importance of proteases during infection may vary with the specific pathogen studied. For instance, while
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protease-deficient mutants of the foliar pathogen Pyrenopeziza brassicae are not able to infect their host plant tissues,13 proteasedeficient mutants of other fungal species retain their pathogenicity.9,10 It is not clear at this point, however whether such proteasedeficient mutant strains can produce alternate proteases to compensate for the lack of some protease(s), or if they are indeed able to grow normally and infect the plant in the complete absence of extracellular proteases.
5.4. Prospects for PI-Expressing Transgenic Plants At present very little information is available about the actual potential of recombinant PIs in the control of plant pathogens, but recent data tend to justify the need for further investigations along this line. In particular, recent articles reported adverse effects of purified or semi-purified plant protein PIs against various fungal pathogens. Inhibitors of trypsin and chymotrypsin purified from cabbage leaves, for instance were shown to inhibit spore germination and germ tube elongation of Botrytis and Fusarium species when added to fungal cultures in vitro, while spores of the cabbage pathogen Alternaria brassicicola were unaffected.32 In another study Dunaevskii et al33 observed that trypsin/ chymotrypsin inhibitors purified from buckwheat (Fagopyrum esculentum) seeds could inhibit spore germination and mycelium growth of Alternaria alternata, while a cysteine PI purified from the pearl millet (Pennisetum glaucum) was recently shown to affect fungal spore and mycelium growth of several pathogenic species including Trichoderma reesei.34 Although the exact mechanisms altered by the PIs remain entirely unknown, these data suggest the potential of overexpressing PI-encoding cDNA sequences in transgenic plants as a strategy to control a variety of pathogens, provided that efficient inhibitors are used. The sedentary nature of most higher plants makes them easy targets for wind and soil-borne pathogens, and therefore the use of protein PIs as defensive molecules certainly is an attractive option when considering the
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array of possible pathogens which may be encountered by any single plant throughout a growing year. Several caveates should be addressed, however at the outset of such a strategy.
5.4.1. Intracellular Targeting Firstly, not only must the expression of the defensive inhibitor be targeted to the organs and tissues attacked by the pathogen, but the PI must also be translocated to the appropriate cellular location, usually to the extracellular compartment for fungi and bacteria, or to the cytoplasm for viruses. Considering factors like the variety of cellular intrinsic targeting signals in PIs, the posttranslational modifications needed for the maturation of certain protein PIs, and the ability of living cells to recognize ‘abnormal’ (including foreign) proteins and to degrade them, it may be a challenge for plant scientists to express adequate levels of certain PIs in specific cell compartments where they do not accumulate naturally (see Chapter 16, this volume).
5.4.2. Stability of the PIs A second point to consider is the choice of the PIs. Based on the information available about protease/inhibitor interactions in plant-insect systems, several criteria should be assessed when choosing a PI or combination of PIs for pest control, including the overall inhibitory spectrum of the PI(s), the degree of affinity between the PIs and the pest target proteases, and the stability of the inhibitors in the presence of insensitive proteases (see Chapter 10, this volume).35 It has been proposed that a good PI should possess a broad spectrum of activity against the pest proteases, that it should inhibit strongly the target proteases, and that it should retain its structural and functional integrity in the presence of insensitive, nontarget proteases.35,36 As recently shown for the proregion of papaya proteinase IV, a regulatory propeptide capable of inhibiting several cysteine proteases of the papain family, the susceptibility of a given inhibitor to the action of insect nontarget proteases may have a profound influence on its
Recombinant Protease Inhibitors in Plants
inhibitory activity against target proteases.37 As shown here for the interactions between soybean Kunitz trypsin inhibitor and the extracellular proteases of the pathogenic fungus Pythium ultimum (Fig. 5.2), the stability of protein PIs should also be assessed for plant pathogens.
5.4.3. Compensatory Processes A third factor to be considered is the ability of plant pathogens to adapt their digestive protease system to the presence of protein PIs in the surrounding environment. It has been recently demonstrated that a general adaptation to the host tissues occurs among saprophytic, phyto-, and entomopathogenic fungi whereby the pathogens aquire the specialized enzymatic machinery to degrade macromolecules, including proteins present in the cells of their hosts.39 This apparent plasticity of proteolytic complements in fungi, also observed for herbivorous insects38 suggests that plant pathogens, like herbivorous insects, possess the ability to elude the inhibitory effects of PIs by using complex proteolytic complements composed of multiple protease species differentially inhibited by protein PIs,40 or by producing ‘insensitive’ proteases to compensate for inhibited proteolytic functions.41,42 Although an empirical assessment is usually needed to understand such compensatory responses in the target pests, this factor should be taken into consideration when identifying protein PIs to control specific pathogens. To this end the use of protease system variants induced in the target organism by various means (see, for instance, refserences 37 and 43) may be of particular interest to assess the relative efficiency of different candidate PIs to remain active against the variety of extracellular proteases potentially used by this organism.
5.4.4. Resistance to PIs Finally a fourth factor to consider is the risk that the target organism can develop resistance to the inhibitor. As noted above (see Section 5.1.), recombinant PIs should not exert a selection pressure on the target pathogens as high as that exerted by toxic
The Control of Plant Pathogens with Protease Inhibitors
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Fig. 5.2. Interactions between soybean Kunitz trypsin inhibitor (SBTI) and the extracellular proteases of the plant fungal pathogen Pythium ultimum. The fungus was grown at room temperature in a liquid medium containing 0.35% (w/v) casein. After three weeks in the dark, the fungal mycelium was removed by filtration, and the proteins secreted in the medium were concentrated by ammonium sulfate precipitation at 90% saturation. A) Inhibition of P. ultimum extracellular proteases by PMSF and SBTI. Total protease activity in the resulting preparations was measured at pH 9.0, with azocasein as a substrate.38 Results are presented as the activity remaining in the extract after inhibition ± SE, as compared to a control sample in which no inhibitor was added (100% activity; n=3). B) Stability of SBTI challenged with P. ultimum extracellular proteases. The stability assay was performed by incubating 2 µg of the soybean inhibitor (in 5 µl water) with 5 µl of the fungal extracellular protein preparation for 60 min at 25˚C, before submitting the complete mixture to standard 10% (w/v) SDS-PAGE. The data are presented as the relative amount of trypsin inhibitor remaining after incubation ± SE, as compared to the amount added before incubation (2 µg = 100%; n=3). The amounts of protein were estimated by densitometry of the Coomassie Blue-stained bands. Abbreviations: 0', before incubation; 60', after incubation; PMSF, phenylmethylsulfonyl fluoride. compounds, but the eventual evolution of resistance cannot be excluded, especially when protease inhibition has indirect lethal effects. This problem of resistance may also be important when using PIs as viral control agents.44,45 In this case PIs usually show an ‘antibiotic-like’ effect, where inhibition of polyprotein maturation completely blocks the replication process. It has been reported that in vivo treatment of human immunodeficiency virus type 1 (HIV) with PIs for a 24-week period could be suffient to select multiple inhibitor-resistant strains in which the target maturation proteases are no longer sensitive to inhibition. 44 Some of these
variants displayed resistance to a set of six structurally unrelated inhibitors, which suggests that the selection pressure applied by PIs to viral pathogens such as HIV may be very high and lead to cross-resistance processes. Considering the numerous similarities observed between animal and plant pathogens, one can thus assume that the appearance of plant pathogens with novel mutant proteases is a real possibility, and that appropriate measures should be taken to ensure the long-term efficiency of PI-expressing ‘pesticidal’ transgenic plants. Using these plants in integrated pest management systems is an approach that could help minimize the selection pressure;
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Recombinant Protease Inhibitors in Plants
Fig. 5.3. Coronatine-induced expression of recombinant potato PI-II in transgenic tobacco. The plant was transformed with the potato PI-II gene (provided by Dr. C.A. Ryan), under the control of a wound-inducible promoter, the promoter of soybean vegetative storage protein (provided from Dr. P. Staswick). The transgene was induced by treating the plant with coronatine (see Figure 5.1), and the potato recombinant inhibitor was immunodetected with anti-potato PI-II rabbit polyclonal antibodies. It is evident from the blot that coronatine may induce the expression of the transgene within 1 day in the transgenic line (T7), while no reaction is observed for the control (non transformed) line (Delgold). the use of promoters directing the accumulation of recombinant PIs only in infected tissues could also be valuable. For example, using a promoter activated by coronatine could allow the plant to produce an inhibitor useful in controlling the bacterium P. syringae only when this particular pathogen infects the plant (see Figure 5.3), thereby preventing constitutive, energy-consuming expression of the transgene when it is not needed, and useless selection pressure on both the target and nontarget organisms when the plant is healthy.
5.5. Conclusion In summary, the use of recombinant PIs may represent an attractive way to protect plants from a variety of fungal, bacterial and viral pathogens. At this point the protease/ inhibitor interactions taking place in vivo between plant pathogens and their hosts remain poorly understood, but it clearly appears that proteolytic events may play a critical role in several key plant pathogenic processes. It also appears plausible, however that pathogens, like herbivorous insects
possess the metabolic machinery needed to grow and develop normally despite the presence of protein PIs in the surrounding environment, making the identification of effective PIs challenging. Nevertheless, while the specific processes targeted in pathogens could differ from those targeted in insect pests, some basic processes such as the hydrolysis of dietary proteins are universal, and the digestive enzyme systems in these organisms probably act in a similar way. In this perspective, the best way to develop effective PI-based control strategies against plant pathogens may be to take advantage of our current knowledge on protease/inhibitor interactions in plant-insect systems. The design of inhibitory strategies based on the criteria defined for herbivorous insects could prove quite useful in developing effective pathogen control tools.35,36 After clearly demonstrating the potential of recombinant PIs in pathogen control, the next step could be then to devise strategies adapted to the inhibition of proteases involved in more specific events like the breakdown of host cell wall protein components, the activation of enzymes implicated in fungal cell elongation,
The Control of Plant Pathogens with Protease Inhibitors
or the hydrolysis of defense proteins released from the host plant following infection.
Note added in proof A recent article was published demonstrating the actual usefullness of recombinant PIs expressed intransgenic plants as a way to effectively control plant viruses (see GutierrezCampos R, Torress-Acosta JA, Saucedo-Arias L et al. The use of cysteine proteinase inhibitors to engineer resistance against potyviruses in transgenic tobacco plants. Nat Biotechnol 1999; 17:1223-1226). Transgenic tobacco lines expressing the rice cystatin OC-I were shown to be resistant to two important potyviruses, tobacco etch virus and potato virus Y, which rely on cysteine proteinase activity for the processing of their polyprotein.
Acknowledgments We thank Line Cantin for helpful comments on the manuscript, and A.F. Yang for technical assistance with the immunoelectron microscopy. This work was supported in part by an operating grant from the Natural Science and Engineering Research Council of Canada to D.M. E.C.O.R.C contribution no. 991377 (S.G.)
References 1. Gleddie S, Keller WA. Protoplast fusion technology. J Tissue Culture Meth 1989; 12:157-162. 2. Shah DM, Rommens CMT, Beachy RN. Resistance to diseases and insects in transgenic plants: Progress and applications to agriculture. Trends Biotechnol 1995; 13:362-368. 3. Brattsen LB. Bioengineering of crop plants and resistant biotype evolution in insects: Counteracting coevolution. Arch Insect Biochem Physiol 1991; 17:253-267. 4. Schuler TH, Poppy GM, Kerry BR et al. Insect-resistant transgenic plants. Trends Biotechnol 1998; 16:168-175. 5. Atkinson HJ, Urwin PE, Hansen PE et al. Designs for engineered resistance to rootparasitic nematodes. Trends Biotechnol 1995; 13:369-374. 6. Henskens YMC, Veerman ECI, Nieuw Amerongen AV. Cystatins in health and disease. Biol Chem 1996; 377:71-86. 7. El-Shanshoury A, El-Sayed M, Sammour R et al. Purification and partial characterization
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of two extracellular alkaline proteases from Streptomyces corchorusii ST36. Can J Microbiol 1995; 41:99-104. 8. Dobinson K, Lecomte N, Lazarovits G. Production of an extracellular trypsin-like protease by the fungal plant pathogen Verticillium dahliae. Can J Microbiol 1997; 43:227-233. 9. Robertsen B. An alkaline extracellular protease produced by Cladosporium cucumerinum and its possible importance in the development of scab disease of cucumber seedlings. Physiol Plant Pathol 1984; 24:83-92. 10. Murphy JM, Walton JD. Three extracellular proteases from Cochliobolus carbonum: Cloning and targeted disruption of ALP1. Mol Plant-Microbe Interact 1996; 9:290-297. 11. McHenry J, Christeller JT, Shade E et al. The major extracellular proteinases of the silverleaf fungus, Chondrostereum purpureum, are metalloproteinases. Plant Pathol 1996; 45:552-563. 12. Clark S, Templeton MD, Sullivan PA. A secreted aspartic proteinase from Glomerella cingulata: Purification of the enzyme and molecular cloning of the cDNA. Microbiology 1997; 143:1395-1403. 13. Ball A, Ashby A, Daniels MJ et al. Evidence for the requirement of extracellular protease in the pathogenic interaction of Pyrenopeziza brassicae with oilseed rape. Physiol Mol Plant Pathol 1991; 38:147-161. 14. Movahedi S, Heale J. The roles of aspartic proteinase and endo-pectin lyase enzymes in the primary stages of infection and pathogenesis of various host tissues by different isolates of Botrytis cinerea Pers ex. Pers. Physiol Mol Plant Pathol 1990; 36:303-324. 15. Collmer A, Keen N. The role of pectic enzymes in plant pathogenesis. Annu Rev Phytopathol 1986; 24:383-409. 16. Levi C, Durbin RD. The isolation and properties of α-tabtoxin-hydrolysing aminopeptidase from the periplasm of Pseudomonas syringae pv. syringae. Physiol Mol Plant Pathol 1986; 28:345-352. 17. Dow JM, Clarke B, Milligan D et al. Extracellular proteases from Xanthomonas campestris pv. campestris, the black rot pathogen. Appl Environ Microbiol 1990; 56:2994-2998. 18. Babé LM, Craik CS. Viral proteases: Evolution of diverse structural motifs to optimize function. Cell 1997; 91:427-430. 19. Rozanov M, Drugeon G, Haenni A-L. Papain-like proteinase of turnip yellow mosaic virus: A new prototype of a new s group. Arch Virol 1995; 140:273-288. 20. Thole V, Hull R. Rice tungro spherical virus polyprotein processing: Identification of a virus-encoded protease and mutational analy-
64 sis of putative cleavage sites. Virology 1998; 247:106-114. 21. Kay J, Dunn BM. Viral proteinases: Weakness and strength. Biochim Biophys Acta 1990; 1048:1-18. 22. Gorbalenya AE, Snijder EJ. Viral cysteine proteinases. Perspect Drug Discov Design 1996; 6:64-86. 23. Green TR and Ryan CA. Wound-induced proteinase inhibitor in plant leaves: A possible defense mechanism against insects. Science 1972; 175:776-777. 24. Ryan CA. Proteinase inhibitors in plants: Genes for improving defenses against insects and pathogens. Annu Rev Phytopathol 1990; 28:425-449. 25. Valueva T, Revina T, Kladnitskaya G et al. Kunitz-type proteinase inhibitors from intact and Phytophthora-infected potato tubers. FEBS Lett 1998; 426:131-134. 26. Rickauer M, Bottin A, Esquerre-Tugayé M-T. Regulation of proteinase inhibitor production in tobacco cells by fungal elicitors, hormonal factors and methyl jasmonate. Plant Physiol Biochem 1992; 30:579-584. 27. Mittal S, Davis K. Role of the phytotoxin coronatine in the infection of Arabidopsis thaliana by Pseudomonas syringae pv. tomato. Mol. Plant Microbe Interact 1995; 8:165-171. 28. Palmer D, Bender C. Ultrastructure of tomato leaf tissue treated with the Pseudomonad phytotoxin coronatine and comparison with methyl jasmonate. Mol Plant Microbe Interact 1995; 8:683-692. 29. Tornero P, Conejero V, Vera P. Primary structure and expression of a pathogeninduced protease (PR-P69) in tomato plants: Similarity of functional domains to subtilisinlike endoproteases. Proc Natl Acad Sci USA 1996; 93:6332-6337. 30. Broadway RM. Dietary proteinase inhibitors alter complement of midgut proteases. Arch Insect Biochem Physiol 1996; 32:39-53. 31. Jongsma MA, Bolter C. The adaptation of insects to plant protease inhibitors. J Insect Physiol 1997; 43:885-895. 32. Lorito M, Broadway RM, Hayes C et al. Proteinase inhibitors from plants as a novel class of fungicides. Mol Plant Microbe Interact 1994; 7:525-527. 33. Dunaevskii Y, Pavlyukova E, Belyakova G et al. Anionic trypsin inhibitors from dry buckwheat seeds: Isolation, specificity of action, and effect on growth of micromycetes. Biochemistry (Moscow) 1994; 59:739-743. 34. Joshi B, Sainani M, Bastawade K et al. Cysteine protease inhibitor from pearl millet: A
Recombinant Protease Inhibitors in Plants new class of antifungal protein. Biochem Biophys Res Commun 1998; 246:382-387. 35. Michaud D. Avoiding protease-mediated resistance in herbivorous pests. Trends Biotechnol 1997; 15:4-6. 36. Jongsma MA, Stiekema WJ, Bosch D. Combatting inhibitor-insensitive proteases of insect pests. Trends Biotechnol 1996; 14:331-333. 37. Visal S, Taylor MAJ, Michaud D. The proregion of papaya proteinase IV inhibits Colorado potato beetle digestive cysteine proteinases. FEBS Lett 1998; 434:401-405. 38. Overney S, Fawe A, Yelle S et al. Dietrelated plasticity of the digestive proteolytic system in larvae of the Colorado potato beetle (Leptinotarsa decemlineata Say). Arch Insect Biochem Physiol 1997; 36:241-250. 39. St Leger R, Joshi L, Roberts D. Adaptation of proteases and carbohydrases of saprophytic, phytopathogenic and entomopathogenic fungi to the requriements of their ecological niches. Microbiology 1997; 143:1983-1992. 40. Michaud D, Bernier-Vadnais N, Overney S et al. Constitutive expression of digestive cysteine proteinase forms during development of the Colorado potato beetle, Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae). Insect Biochem Mol Biol 1995; 25:1041-1048. 41. Jongsma MA, Bakker PL, Peters J et al. Adaptation of Spodoptera exigua larvae to plant proteinase inhibitors by induction of gut proteinase activity insensitive to inhibition. Proc Natl Acad Sci USA 1995; 92:8041-8045. 42. Bolter C, Jongsma MA. Colorado potato beetles (Leptinotarsa decemlineata) adapt to proteinase inhibitors induced in potato leaves by methyl jasmonate. J Insect Physiol 1995; 41:1071-1078. 43. Gruden K, Strukelj B, Popovic T et al. The cysteine protease activity of Colorado potato beetle (Leptinotarsa decemlineata Say) guts, which is insensitive to potato protease inhibitors, is inhibited by thyroglobulin type-1 domain inhibitors. Insect Biochem Mol Biol 1998; 28:549-560. 44. Condra J, Schlelf W, Blahy O et al. In vivo emergence of HIV-1 variants resistant to multiple protease inhibitors. Nature 1995; 374:569-570. 45. Baer GS, Dermody TS. Mutations in reovirus outer-capsid sw σ3 selected during persistent infections of L cells confer resistance to protease inhibitor E-64. J Virol 1997; 4921-4928.
CHAPTER 6
Regulation of Plant Defense Against Herbivorous Pests Hisashi Koiwa, Ray A. Bressan and Paul M. Hasegawa
6.1. Introduction
I
t is estimated that potential crop yield is reduced by about 40% because of dam age or competition from arthropods, nematodes, diseases and weeds. Herbivorous pests such as insects, mites and nematodes are major contributors to yield loss either directly through consumption of plant biomass or indirectly as vectors for pathogens. Consequently, introgression of pest resistance into crops has been one of the major priorities for plant breeders. Resistant cultivars reduce dependence on pesticides and need for crop rotation, providing an economic benefit without compromise to environmental stewardship. Research on host plant resistance to herbivores has established that plants have evolved natural defensive strategies against pests, including the production of compounds that function either directly or indirectly to protect plants against invasion. Protease inhibitor (PI) proteins are among the defensive chemicals in plant tissues that affect growth and development of pests, presumably by attenuating enzyme function necessary for metabolic processes such as protein turnover or proteolytic digestion required for nutrient assimilation. The major digestive proteases utilized by herbivorous pests are serine, cysteine, and aspartyl proteases, that have been classified based on the active site residue. Inhibitors against each class of protease have been identified in plants,1 and shown to
inhibit proteolytic activity and growth of pests (Table 6.1). Confirmation that PIs have a defensive function was first provided by results showing that overexpression of cowpea trypsin inhibitor increases resistance of transgenic tobacco plants to Heliothis virescens.2 This demonstration of biological function implicated the potential for biotechnology in pest control.1,3 However, the genetic durability of a plant defense that is based on the activity of a single PI gene or protein is likely to be finite since insects have the genetic capacity for adaptation to PIs induced in planta or overexpressed in transgenic plants.4-6 Presumably, this adaptive capacity of the insect is based on the differential regulation of genes of a multigene family encoding proteinases that are less sensitive to the plant defensive PIs.5,7,8 Furthermore, resistant biotypes of insects may evolve after prolonged exposure to selection pressure that is mediated by an insecticidal protein or plant resistance gene.9 Unless a biotechnology strategy is designed and implemented to overcome these problems, it will become ineffective in due course like any pesticide based management strategy. Plant defense against herbivores involves the regulation of effectors, like PIs, either developmentally or in response to pest attack. It is presumed that developmental regulation is an enhanced protection mechanism for
Recombinant Protease Inhibitors in Plants, edited by Dominique Michaud. ©2000 Eurekah.com.
Recombinant Protease Inhibitors in Plants
66
Table 6.1. Pesticidal activity of plant protease inhibitors Inhibitor
Assay
Pest
References
Kunitz
in vitro in vitro in vitro in vitro in vitro in vivo in vitro
Helicoverpa armigera Teleogryllus commodus Spodoptera litura Spodoptera exigua Ostrinia nubilalis Manduca sexta Triolium castaneum
95 96,97 98 99 100 101 102a
Bowman-Birk
in vitro in vitro
Teleogryllus commodus Tribolium castaneum
96,97 102a
Potato inhibitor I
in vitro
Teleogryllus commodus
96,97
Potato inhibitor II
in vivo in vivo in vitro in vivo in vitro in vitro
Sesamia inferens Manduca sexta Teleogryllus commodus Chrysodeixis erisoma Heliothis zea Spodoptera exigua
103 104 96,97 105 99 99
Cowpea trypsin inhibitor
in vivo in vivo in vivo in vitro
Chilo suppressalis s inferens Heliothis virescens Callosobruchus maculatus
106 106 2 107
Squash trypsin inhibitor
in vivob
Heliothis virescens
108
in vitro
Trichoplusia ni
109
in vivo in vivo
Bemisia tabaci Frankliniella spp.
110 111
Oryzacystatin I
in vitro in vitro in vitro in vitro in vitro in vitro in vivo in vivo in vivo in vivo in vitro
Callosobruchus chinensis Riptortus clavatus Tribolium castaneum Diabrotica undecimpunctata Leptinotarsa decemlineata Caenorhabditis elegans Globodera pallida Heterodera schachtii Meloidogyne incognita Chrysomela tremula Anthonomus gradis
112 112 113 114 115 116 116 117 117,118 119 120
Oryzacystatin II
in vitro
Callosobruchus chinensis
112
Soyacystatin n
in vitro
Callosobruchus maculatus
84
Potato multicystatin
in vitro in vitro
Diabrotica virgifera Diabrotica undecimpunctata
121 121
Soybean trypsin inhibitors
Cabbage protease inhibitor Manduca sexta inhibitor
c
a Synergistic with E-64; b fused to Bacillus thuringiensis toxin; c PI of insect origin
Regulation of Plant Defense Against Herbivorous Pests
essential plant organs, such as those involved in reproduction. Regulated expression of defensive genes may allow the plant to allocate carbon and nitrogen resources to mount a specific and high impact defense at the site of invasion. Induced expression in response to pest attack may have important advantages. The constitutive expression of defensive molecules or other defensive traits intuitively would represent a substantial yield drag.10 Pest-resistant varieties often have lower harvest indices than susceptible varieties, implicating that there is a major resource allocation compromise for defense versus growth.11 Transgenic plants overexpressing systemin, a potent inducer of the synthesis of PIs and other defense proteins, exhibited a severe stunted phenotype that was linked directly to the overexpression of defensive proteins.12 Constitutive expression of defensive effectors could also compromise the genetic durability of the plant defense phenotype since it increases selection pressure on pests. On the other hand, abrogation of inducible plant defense capacity resulted in susceptibility to herbivores,13,14 and thus precise control of an inducible defensive system is essential for maximal fitness of the host plant. In this chapter we summarize the information available about regulatory cascades that are presumed to control plant defensive responses against herbivores, including plant recognition of pest attack to initiate defensive responses, and local and systemic regulation of defensive effectors. The tritrophic interaction among host plants, herbivores and natural enemies of herbivores will also be described. It is now known that plants respond to herbivore feeding activity by producing volatile compounds that attract carnivorous/parasitic natural predators of the plant pest.15,16 An understanding of the underlying mechanisms responsible for plant defensive response regulation will provide the knowledge base required for engineering host plant defense through biotechnology. Specifically, this information can facilitate the development of a genetic engineering strategy that is designed either to modulate the natural inducible defensive mechanism, or to regulate
67
the ectopic expression of defensive genes. The goal is maximal pest management efficacy and minimal resistant biotype evolution and plant yield reduction. It may be possible to engineer more genetically durable host plant resistance by modulating natural biocontrol processes such as those involved in plant signalling of predatory insects to attack herbivore invaders. Since the signal transduction pathway of the plant wound response, particularly the octadecanoid (OD) pathway, and function of jasmonic acid4 (JA) have been reviewed recently,17-19 this review will focus on the plant-herbivorous pest interaction.
6.2. The Plant Response 6.2.1. Kinetics of the Plant Response Mechanical injury (or wounding) and specific pest recognition processes trigger the plant defensive response to herbivore attack (Fig. 6.1). Chemicals in oral secretions of chewing and sucking insects/pests are also stimulants of the plant response to pest attack.16 Indeed, chewing insect regurgitates elicit the expression of plant defensive proteins or the emission of carnivore attractants.20,21 Currently, it is postulated that the rudiments of the signal pathway(s) induced by wounding or pest attack are similar.22 This signal cascade is commonly referred to as the OD pathway because of its key intermediate, α-linolenic acid (LA, 18:3) and the pivotal metabolite oxylipin, JA.23 Plant oxylipins are structural analogs of animal prostaglandin hormones that are produced from arachidonic acid in response to various signals to stimulate various reactions such as inflammation.24 Injury to plants caused by phytophagous insects or mechanical damage induces the accumulation of PIs both locally at the site of injury, and systematically in other organs distal to the primary wound site. Woundinduced JA production may result from injury that compromises the integrity of organelles. Consequently, enzymes and substrates that are normally compartmentalized within the cell interact to synthesize JA and its precursors.25
68
Interestingly, accumulation of the transcript encoding proteinase inhibitor II (PI-II) in potato occurs more rapidly in leaves damaged by insects relative to those subjected to mechanical injury.21 One mechanism by which plants differentiate pest attack from wounding involves signalling by prostaglandinlike molecules. Attacking pests are a source of compounds that trigger JA synthesis, including substrates of the OD pathway.20,26,27 Lipidderived substrates from pests, and perhaps other bioactive molecules in insect oral secretions mimic JA or potentiate the action of the hormone to elicit plant defense. Also, cell wall hydrolytic enzymes in oral secretions may catabolize plant polymers, resulting in the production of oligosaccharide inducers of the defensive response. Emission of a volatile carnivore attractant that is elicited by beet armyworm (BAW) infestation of maize or cotton leaves could not be stimulated by wounding.28,29 However, application of BAW oral secretion to the site of mechanical injury induces emission of the volatile compound.28 There is a high degree of specificity to the signalling that occurs in this interaction, which presumably is conditioned by the composition and the concentration of the volatile elicitor in the pest oral secretion. The predatory mite, Phytoseiulus persimilis, responds to volatile compounds emitted by apple leaves infested with the spider mite Tetranychus urticae, whereas Amblyseius finlandicus and A. andersoni are attracted by volatiles released from leaves infested by Panonychus ulmi (see Table 6.2 for a list of plant volatiles attracting carnivores).30 Apparently plants also have evolved the capacity to signal parasitic organisms at early stages of herbivore development when parasitism is the most beneficial to the host. The parasitic wasp, Cotesia kariyai, is attracted to corn plants infested with early instar larvae of Pieris separata, or treated with regurgitate of these larvae but not to plants attacked by larvae of later instars.31 Among the insect oral secretions that induce the defensive response of plants are hydrolytic enzymes like β-glucosidase, and volicitin. The process by which these
Recombinant Protease Inhibitors in Plants
molecules elicit the host plant defense has not been defined clearly, but evidence indicates that these compounds potentiate the OD pathway. Treatment of lima bean leaves with cellulysin (crude cellulase from Trichoderma viride) increased endogenous JA levels, and inhibitors of JA biosynthesis blocked cellulysininduced volatile production.32 β-glucosidase activity in caterpillar (Pieris brassicae) oral secretion triggered the emission from cabbage of a volatile carnivore attractant.27 Pectic oligosaccharide fragments, that are breakdown products of the plant cell wall, induced the accumulation of PIs via the OD pathway, implicating glucosidases in the release of oligosaccharide elicitors.33 Plant β-glucosidases can catalyze reactions that result in the release of phytohormones or defensive chemicals, such as auxin,34 cytokinin,35 salicylic acid,36 cyanide37 and, possibly, terpenoids38 from their respective inert β-glucoside conjugate. Since these glucosidases are highly conserved in plants and animals, and since they are rather nonspecific, the insect enzyme may also catalyze the activation of these chemicals to promote a defensive reaction. Structurally, volicitin is a derivative of LA and is presumed to be a precursor of JA derivatives.20
6.2.2. Molecules that Activate the OD Pathway Both PI accumulation and volatile emission are induced systemically in leaves distal to the site of injury. The nature of the systemic signal(s) is the subject of extensive examination and several different candidates have been proposed. Among them, the 18-mer peptide systemin is the best characterized. Systemin was isolated from wounded tomato leaves and shown to induce the expression of PI genes.39 Systemin is synthesized as a 200amino acid precursor protein, prosystemin.40 Overexpression of prosystemin in tomato plants resulted in constitutive accumulation of defensive proteins, including PIs of different classes.12 Furthermore, plants expressing antisense prosystemin exhibited substantial reduction in PI synthesis and were susceptible to insect attack,40 demonstrating the central role of the peptide hormone in inducing pest
Regulation of Plant Defense Against Herbivorous Pests
69
Fig. 6.1. Initiation and potentiation of a wound signal transduction pathway by plant and insect-derived elicitors. Mechanical injury facilitates the capacity of chemicals and enzymes in insect oral secretions to elicit a plant defensive response. β-glucosidase releases plant cell wall oligosaccharides that bind to a glucan elicitor binding protein (GEBP)126 and triggers membrane lipid degradation. Linolenic acid (LA), a product of membrane degradation, and LA derivatives of insect origin are converted to jasmonic acid (JA) via the octadecanoid (OD) pathway. JA production is enhanced in injured cells because partitioning into intracellular compartments of enzymes and substrates of the OD pathway has been compromised. The initial pulse of JA activates the expression of genes such as those that encode lipoxygenase (LOX) and systemin, that feedback to the OD pathway to facilitate JA biosynthesis, both locally and systemically. The function of systemin involves an interaction with a w binding protein (SBP50) that resembles a peptide hormone processing protease. JA is a pivotal intermediate in a signal pathway that regulates the production of defensive proteins and carnivore attractants. Some JA is converted to Me-JA, or perhaps amino acid conjugates (JA-AA), that function also as transducers of the defensive signal.
Recombinant Protease Inhibitors in Plants
70
Table 6.2. Plant volatiles attracting carnivores Plant
Herbivore (elicitor)
Volatile
Carnivore attracted
Lima bean
Tetranychus urticae
(E)-β-ocinmene (3E)-4,8-dimethyl 1,3,7-nonatriene methyl salicylate
Phytoseiulus persimilis Phytoseiulus persimilis
81 81
(β-glucosidase) (jasmonate) (coronatin) (cellulysin)
3,7-dimethyl1,6-octadien-3-ol mixture of above mixture of above mixture of above mixture of above
Phytoseiulus persimilis Amblyseius potentillae Phytoseiulus persimilis Amblyseius potentillae not tested not tested not tested not tested
81 81 81 81 122 122 57 32
Brussel
Pieris brassicae (β-glucosidase)
mixture mixture
Cotesia glomerata not tested
122 27
Maize
Spodoptera exigua (volicitin) Pseudaletia separata
mixture mixture mixture
Cotesia marginiventris Cotesia marginiventris Cotesia kariyai
28 20 31
Cotton
Helicoverpa zea
mixture
Microplitis croceipes
123
Bean
Tetranychus urticae
mixture
Metaseiulus occidentalis Phytoseiulus persimilis
124 124
Apple
Panonychus ulmi
mixture
Amblyseius finlandicus
124
Cucumber
Tetranychus urticae
mixture
Phytoseiulus persimilis
125
resistance. A 50-kDa plasma membrane systemin binding protein (SBP50) has been shown to be a kex2p-like protease. These proteases are involved in the processing of peptide hormones. Whether or not SBP50 plays a role as the receptor for, or in the turnover of systemin remains to be elucidated. The prosystemin gene was expressed in cells associated with the vascular bundles, consistent with its role as a systemic signal molecule.41 Application of systemin or oligosaccharide, or insect damage activated myelin basic protein kinase (MBPK) activity in local and systemic leaves in wild type and OD pathwaydeficient def1 mutant tomato plants. 42 Although the molecular identity of tomato MBPK is yet to be determined, tobacco wound-induced MAP kinase (WIPK) is
Reference
perhaps a functional homologue. WIPK transcript rapidly accumulated in tobacco plants after wounding,43 and co-suppression of WIPK in transgenic tobacco plants abolished wound-induced JA synthesis and PI expression, indicating that this kinase regulates the early response of plants leading to activation of the OD pathway. In parallel, mutant tomato and potato plants that are deficient in ABA biosynthesis were shown to do not express PIs in response to wounding or systemin treatment. 44 However, application of LA or JA resulted in strong induction of PI gene expression even in these mutant plants. Exogenous application of ABA resulted in either strong or no (or very weak) induction of PIs.45,46 ABA apparently functions as a modulator of the wound signal at some point after systemin action but prior
Regulation of Plant Defense Against Herbivorous Pests
to the OD pathway. ABA-induced PI expression requires the function of a staurosporinsensitive protein kinase while JA-induced defensive gene expression requires phosphatase activity that is inhibited by okadaic acid.47 These results implicate a JA-independent ABA signal transduction pathway for PI induction.
6.2.3. The OD Pathway Wounding, or the application of β-glucosidase (cellulysin), oligosaccharide, systemin or ABA causes the accumulation of JA. JA is synthesized from LA through a series of enzymatic steps that is initiated by lipoxygenase to produce 13-hydroperoxylinolenic acid. The 13-hydroperoxylinolenic acid molecule is converted to 12-oxo-phytodienoic acid (12-oxo-PDA) by the sequential action of allene oxide synthase and allene oxide cyclase. JA is formed from 12-oxo-PDA by reduction and three steps of β-oxidation.17 JA is then converted to Me-JA or amino-acid conjugates.48 The structure and activity of volicitin indicate that amino acid conjugates of JA are involved in the elicitation of pest-specific responses.20 Antisense expression of a chloroplast lipoxygenase in Arabidopsis suppressed wound-induced JA accumulation.25 It is suggested that JA is sequestered presumably in the chloroplasts.17,49 Interestingly, plants treated with β-glucosidase or JA emitted different volatile compounds. On the other hand, β-glucosidase-induced volatile production was abrogated by OD pathway inhibitors, indicating that a functional OD pathway is required for volatile production, but that JA is not the only functional end-product of the pathway that elicits specific defensive responses.32
6.2.4. The OD Pathway Regulates Host Plant Defense Against Herbivores Inhibitors of the OD pathway were shown to block wound-induced PI synthesis and cellulysin/β-glucosidase-induced volatile emission.32,50,51 Both Arabidopsis fad3-2/ fad7-2/fad8 triple mutant plants, that are
71
deficient in LA synthesis, or tomato def1 mutant plants, that are defective for the OD pathway were susceptible to insect attack.14,52 Exogenously supplied JA restored resistance of these mutants. Transgenic plants overexpressing allene oxide synthase constitutively accumulated JA but not defensive proteins. 49 These plants responded to wounding with further increased JA levels, and PI accumulation. Three genetic loci that mediate JA signal transduction have been identified in Arabidopsis. The jar1, jin1, jin4 mutants were selected based on suppression of root growth inhibition that is elicited by Me-JA.53,54 Genetic analysis indicated that jar1 and jin4 are allelic. The coi1 mutant was isolated based on insensitivity to the chlorosis-inducing (Pseudomonas syringae) toxin, coronatin, that mimics the action of JA.55.56 coi1 encodes a peptide containing a leucine-rich repeat and an F-box motif that appear to function in targeting repressor proteins for removal by ubiquitination.57 Analysis of defensive gene expression pattern in these mutants indicated that each locus modulates responsiveness to Me-JA.53 The coi1 plant did not express JA-inducible vegetative storage protein (VSP) in response to coronatin or Me-JA treatment and was male sterile, indicating a general role for the Coi1 product in the regulation of plant growth and gene expression. Neither woundnor JA-induced PI accumulation has been reported in Arabidopsis. However JA treatment increased resistance of Arabidopsis plants to insects,52 and so it is possible that these genetic loci will reveal conserved intermediates of signal transduction leading to plant defense. Since the accumulation of PIs and enzymes involved in terpenoid biosynthesis58 is transcriptionally regulated, signals induced or produced by herbivores may be transduced to control transcription of plant defensive genes. An AG-box motif (CACGTGG) in the promoter of PI and other JA-inducible genes has been implicated in JA activation of gene expression.59 The tobacco G-box-binding protein TFHP-1 binds to and regulates the promoter of wound-inducible horseradish
72
peroxidase (HRP), implicating its regulatory function in defense gene activation. TFHP-1 may be posttranslationally modified to activate wound-induced gene expression, since it was expressed in each organ and not induced by wounding.60 Inhibition of cytosolic protein synthesis by cycloheximide treatment abolished expression of the PI-II gene in potato.61 Wounding treatment induced expression of the tomato systemin gene to a maximum immediately after the peak of initial JA accumulation, and this preceded PI induction.40,62 Similarly, chloroplast lipoxygenase was upregulated by systemin, or JA application.63,64 Together, these findings indicate that the initial wound signal, that results in a pulse of JA, is amplified by the expression of systemin, that further induces OD pathway-mediated gene expression.
6.2.5. Resistance (R) Gene-Mediated Plant Defensive Response Tomato plants that contain the Meu1 locus are resistant to the potato aphid, Macrosiphum euphorbiae (Thomas). Meu1 is tightly linked to the root-knot nematode resistance Mi locus and recently these genes were determined to be allelic.65 The Mi/Meu1 gene product contains leucine zipper, nucleotide binding, and leucine-rich repeat domains.66 Another nematode resistance gene, Hs1pro-1 confers cyst-nematode resistance to sugerbeet.67 The plants carrying Hs1pro-1 responded to nematode invasion by localized destruction of cells before fully functional feeding structures were formed. This reaction resembled the hypersensitive response (HR) induced by pathogen invasion (see refs. 68 and 69 for a review), and presumably functioned to reduce nutrient flow from the host plant to the parasite. It is not clear if aphid or nematode infestation caused HR in host plants carrying Meu1 or Hs1pro-1, but the domain structure of MI/ MEU1 and HS1pro-1 proteins are indeed highly homologous to various pathogen resistance gene products that elicit HR.66,67 These similarities implicate an HR-like signal recognition and transduction pathway(s) in
Recombinant Protease Inhibitors in Plants
host plant resistance to insects and nematodes. HR is conditioned by a specific interaction between the products of a resistance (R) gene in the host and an avirulence (Avr) gene in the pathogen. Local necrotic lesions at the site of invasion characterize this so-called incompatible interaction. By analogy, some of the compounds in insect/nematode oral secretion may be equivalent to Avr product of pathogens.
6.2.6. Cross-Talk of Herbivore (OD) and Pathogen (Ethylene/SA) Signal Pathways The signaling pathways initiated by wounding and disease infection are considered to be predominantly independent because wounding generally does not induce systemic acquired resistance (SAR) of plants against pathogens.70 However it is evident that wound induction and pathogen defense pathways overlap considerably. Expression of wound- and JA-inducible genes can be positively and negatively regulated by ethylene or SA, both of which are components of the pathogen-induced signalling pathway.71,72 Similarly, intermediates of the wound signaling pathway can regulate the expression of plant genes involved in resistance to pathogens.73,74 Ethylene was required for JA induction of PI gene expression in tomato and soybean.75,76 Conversely, the expression of thionins in Arabidopsis74 and lectin II in Griffonia simplicifolia77 was elicited by JA but suppressed by ethylene. Local expression of the lectin II gene occurred upon wounding only if norbornardiene, an ethylene action inhibitor, or exogenous JA was applied. Apparently, expression of this latter class of defensive genes requires higher levels of JA in order to neutralize the inhibitory effects of endogenous ethylene. Induction of volatile attractants follows a response pattern similar to the expression of the latter class of defensive genes, since insect regurgitate/volicitin/ JA is required, in addition to wounding.20,26 The potentiating effects of JA were dramatically apparent with the wound-inducible antifungal osmotin gene in tobacco, where JA did not induce expression at all but greatly
Regulation of Plant Defense Against Herbivorous Pests
potentiated induction by ethylene.73 Lipoxygenase activity was also induced by pathogen infection and was required for disease resistance of tobacco plants, implicating a role for the OD signal pathway in plant defense against pathogens.78 It seems that plants sometimes specifically forego one type of defense response for another. Salicylic acid (SA) and its methyl ester (Me-SA) are both defense compounds that potently induce systemic acquired resistance of plants against pathogenic microorganisms.79,80 However, in response to spider mite infestation lima bean plants release Me-SA which functions as a volatile attractant of the predatory mite Amblyseius potentillae.81 At the same time, SA itself negatively regulates the OD pathway through inhibition of SA biosynthesis and action,51 indicating that SA may suppress the plant defensive response through attenuation of the OD pathway, but its methyl ester positively affects plant defense through another defense mechanism involving tritrophic plant-herbivore interaction.81
6.3. Future Perspectives Currently, two principal biotechnology strategies are proposed to engineer effective pest control in plants: ectopic expression of pesticidal proteins, and induction or potentiation of the plant natural defensive response. At present, screening gene pools without taxonomic constraint can help identify novel insecticidal determinants, but in the future this approach will be augmented by directed in vitro molecular evolution (see Chapter 11, this volume). Phage display has been reported for the plant defense protein ricin B chain, potato inhibitor II and soyacystatin,82-84 and biopanning selection techniques selectively identified the most insecticidal soyacystatin.84 Given the number of pesticidal proteins that are involved in host plant defense, it is presumed that effective pest control by this strategy will result from the co-expression of numerous determinants, each of which could be custom-engineered by directed molecular evolution to maximize its effectiveness against specific pests.
73
Ectopic expression of pesticidal proteins presumably will be controlled by inducible promoters, such as those of PI-II 85 and TobRB7, that are activated at the site of invasion by pests and nematodes, respectively.86 An ideal promoter should be highly responsive to invasion of the host plant by a pest, or regulated by inducers just prior to pest attack. The promoter should be sufficiently active to mediate a substantial defense, specially localized to the site of pest invasion. Suitable promoters such as those regulated in response to pest invasion can be identified using promoter trapping techniques.87 Recent demonstrations that tetracycline- and glucocorticoid-inducible systems can be modulated in plant cells 88,89 are indicative of how transgenes may be activated ectopically by chemical treatment to facilitate pest control. Insect resistance of transgenic tobacco plants was modulated by conditional expression of the Bt toxin gene.90 In this instance, the Bt gene was fused to the SA-responsive PR-1a promoter, and toxin production and resistance was conditioned by SA treatment. There is potential for engineering a synthetic molecular interaction system where a sensor protein recognizes a specific pest ligand to activate a signal cascade that regulates either natural or ectopic host plant defense. It has been shown that one can chemically induce pathogen resistance by activating the natural defense system. Application of SA and its nontoxic functional analog, benzothiadiazole (BTH), induced SAR in uninfected plants. 91 JA derivatives or coronatin may be good candidates as compounds that are not phytotoxic, wound signal activators. Alternatively, host plant defense might be achieved by modulating the signal pathway through the overexpression of activated forms of regulatory intermediates. Transgenic Arabidopsis plants expressing NPR1 exhibited resistance against a broad range of pathogens by constitutive activation of SAR.92 The high levels of pest defense protein accumulation in transgenic plants overexpressing prosystemin is indicative that herbivore resistance can be acquired by activating a signal transduction cascade.
74
The homology between R genes that control defensive responses against insects and nematodes and those that effect resistance to pathogens, leads naturally to the prediction that host plant defense against herbivorous pests involves a signal transduction pathway that is initiated by a molecular interaction between a compound produced by the pest and the plant R gene product. Heterologous expression of a R gene for insect/nematode resistance or an intermediary of the R gene-mediated defensive pathway may effect host plant defense against herbivorous pests, as it has for pathogen resistance.93 Furthermore, Avr- and R-gene interaction has been reconstituted across plant species and results in the activation of host resistance to pathogens.94 To date, the efficacy of this defensive response activation has been restricted taxonomically to related species. This is presumably limited by the ability of the R gene product to transduce the signal through the remainder of the pathway leading to the activation of the defense response. Intuitively, one critical mechanism may be direct interaction between the R gene product and the molecular substrates that transduce the signal. Signal strength might be affected by the affinity of the molecular interaction. It is conceivable that a heterologous R gene could be engineered by in vitro molecular evolution techniques to interact more effectively with the substrate in the non-host plant. Then the Avr-like and R gene interaction could be reconstituted in the heterologous species to initiate the signal, and the engineered R gene product would facilitate sufficient signal transduction for induction of the host plant defensive response. The capacity to condition the entire defensive response should maximize the fitness of the defense strategy in the context of the plant’s ecology.
Acknowledgments We acknowledge support from USDA/ CSRS/NRI (grant #91-37303-6443 and 9835503-6384) and the Indiana Soybean Board (grant ISB #98-210).
Recombinant Protease Inhibitors in Plants
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78 96. Burgess EP, Main CA, Stevens PS et al. Effects of proteinase inhibitor concentration and combinations on survival, growth and gut enzyme activities of the black field cricket, Teleogryllus commodus. J Insect Physiol 1994; 40:803-811. 97. Burgess EP, Stevens PS, Keen GK et al. Effects of proteinase inhibitors and dietary protein level on the black field cricket Teleogryllus commodus. Entomol Exp Appl 1991; 61:123-130. 98. McManus MT, Burgess EPJ. Effects of the soybean (Kunitz) trypsin inhibitor on growth and digestive proteases of larvae of Spodoptera litura. J Insect Physiol 1995; 41:731-738. 99. Broadway RM, Duffey SS. Plant proteinase inhibitors: Mechanism of action and effect on the growth and digestive physiology of larval Heliothis zea and Spodoptera exigua. J Insect Physiol 1986; 32:827-833. 100. Steffens R, Fox FR, Kassell B. Effect of trypsin inhibitors on growth and metamorphosis of corn borer larvae Ostrinia nubilalis (Hübner). J Agric Food Chem 1978; 26:170-174. 101. Shukle RH, Murdock LL. Lipoxygenase, trypsin inhibitor, and lectin from soybeans: Effects on larval growth of Menduca sexta (Lepidoptera: Sphingidae). Environ Entomol 1983; 12:787-791. 102. Oppert B, Morgan TD, Culbertson C et al. Dietary mixture of cysteine and serine proteinase inhibitors exhibit synergistic toxicity toward the red flour beetle, Tribolium castaneum. Comp Biochem Physiol C 1993; 105:379-385. 103. Duan X, Li X, Xue Q et al. Transgenic rice plants harbouring an introduced potato proteinase inhibitor II gene are insect resistant. Nat Biotechnol 1996; 14:494-498. 104. Johnson R, Narvaez J, An G et al. Expression of proteinase inhibitors I and II in transgenic tobacco plants: Effects on natural defense against Manduca sexta larvae. Proc Natl Acad Sci USA 1989; 86:9871-9875. 105. McManus MT, White DWR, McGregor PG. Accumulation of chymotrypsin inhibitor in transgenic tobacco can affect the growth of insect pests. Transgenic Res 1994; 3:50-58. 106. Xu D, Xue Q, McElroy D et al. Constitutive expression of a cowpea trypsin inhibitor gene, CpTi, in transgenic rice plants confers resistance to two major rice insect pests. Mol Breed 1996; 2:167-173. 107. Gatehouse AMR, Boulter D. Assessment of the antimetabolic effects of trypsin inhibitors from cowpea (Vigna unguiculata) and other legumes on development of the bruchid beetle Callosobruchus maculatus. J Sci Food Agric 1983; 34:345-350.
Recombinant Protease Inhibitors in Plants 108. MacIntosh SC, Kishore GM, Perlak FJ et al. Potentiation of Bacillus thuringiensis insecticidal activity by serine protease inhibitors. J Agric Food Chem 1990; 38:1145-1152. 109. Broadway RM. Are insects resistant to plant proteinase inhibitors? J Insect Physiol 1995; 41:107-116. 110. Thomas JC, Adams DG, Keppenne VD et al. Protease inhibitors of Manduca sexta expressed in transgenic cotton. Plant Cell Rep 1995; 14:758-762. 111. Thomas JC, Wasmann CC, Echt C et al. Introduction of an insect proteinase inhibitor in alfalfa (Medicago sativa L.). Plant Cell Rep 1994; 14:31-36. 112. Kuroda M, Ishimoto M, Suzuki K et al. Oryzacystatins exibit growth-inhibitory and lethal effects on different species of bean insect pests, Callosobruchus chinensis (Coleoptera) and Riptortus clavatus (Hemiptera). Biosci Biotechnol Biochem 1996; 60:209-212. 113. Chen M-S, Johnson B, Wen L et al. Rice cystatin: bacterial expression, purification, cysteine proteinase inhibitory activity, and insect growth suppressing activity of a truncated form of the protein. Protein Expres Purif 1992; 3:41-49. 114. Edmonds HS, Gatehouse LN, Hilder VA et al. The inhibitory activity of the cysteine protease inhibitor, oryzacystatin, on digestive proteases and on larval survival and development of the southern corn rootworm (Diabrotica undecimpunctata howardi). Entomol Exp Appl 1996; 78:83-94. 115. Michaud D, Bernier-Vadnais N, Overney S et al. Constitutive expression of digestive cysteine proteinase forms during development of the Colorado potato beetle, Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae). Insect Biochem Mol Biol 1995; 25:1041-1048. 116. Urwin PE, Atkinson HJ, Waller DA et al. Engineered oryzacystatin-I expressed in transgenic hairy roots confers resistance to Globodera pallida. Plant J 1995; 8:121-131. 117. Urwin PE, Lilley CJ, McPherson MJ et al. Resistance to both cyst and root-not nematodes conferred by transgenic Arabidopsis expressing a modified plant cystatin. Plant J 1997; 12:455-461. 118. Vain P, Worland B, Clarke MC et al. Expression of an engineered cysteine proteinase inhibitor (Oryzacystatin-ID86) for nematode resistance in transgenic rice plants. Theor Appl Genet 1998; 96:266-271. 119. Leplé JC, Bonadé-Bottino M, Augustin S et al. Toxicity to Chrysomela tremulae (Coleoptera: Chrysomelidae) of transgenic poplars expressing a cysteine proteinase inhibitor. Mol Breed 1995; 1:319-328.
Regulation of Plant Defense Against Herbivorous Pests 120. Pannetier C, Giband M, Couzi P et al. Introduction of new traits into cotton through genetic engineering: Insect resistance as example. Euphytica 1997; 96:163-166. 121. Orr GL, Strickland JA, Walsh TA. Inhibition of Diabrotica larval growth by a multicystatin from potato tubers. J Insect Physiol 1994; 40:893-900. 122. Mattiacci L, Dicke M, Posthumus MA. Induction of parasitoid attracting synomone in brussels sprouts plants by feeding of Pieris brassicae larvae: role of mechanical damage and herbivore elicitor. J Chem Ecol 1994; 20:2229-2247. 123. McCall PJ, Turlings TCJ, Lewis WJ et al. Role of plant volatiles in host location by the specialist parasitoid Micropilitis croceipes Cresson (Braconidae: Hymenoptera). J Insect Behav 1993; 6:625-639.
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124. Sabelis MW, Baan HEvd. Location of distant spider mite colonies by phytosiied predators: Demonstration of specific kairomones emitted by Tetranychus urticae and Panonychus ulmi. Entomol Exp Appl 1983; 33:303-314. 125. Takabayashi J, Dicke M, Takabayashi S et al. Within-plant differences in herbivore-induced volatile synomones. J Chem Ecol 1994; 20:373-386. 126. Umemoto N, Kakitani M, Yoshikawa M et al. The structure and function of a soybean β-glucan-elicitor-binding protein. Proc Natl Acad Sci USA 1997; 94:1029-1034.
CHAPTER 7
The Response of Insects to Dietary Protease Inhibitors Roxanne M. Broadway
7.1. Introduction
O
ne of the most important determinants for the successful use of recombinant proteinase inhibitors (PIs) against herbivorous insects is the selection of appropriate PIs. There are four classes of proteolytic enzymes responsible for the digestion of dietary proteins. These classes are based on the amino acid residue found at the active site of the enzyme (i.e. serine, cysteine—or thiol, carboxyl—or acidic, and metallo-proteases). PIs, in general, are competitive inhibitors—i.e., they bind to the active site of the enzyme— that interact with enzymes within a single mechanistic class. For instance serine PIs bind to serine proteases, which include trypsins, chymotrypsins, elastases, subtilisin, thrombin, and acetylcholinesterase. Evidence suggests that PIs in plants function as defensive agents against herbivores by interfering with the hydrolysis of dietary protein.1,2
7.2. Evaluation of PI Biological Activity In general, three approaches have been used to evaluate the biological activity of PIs against herbivorous insects. The most direct and usually the first approach is to determine the ability of selected PIs to inhibit enzyme activity in vitro. This procedure involves: 1. collecting fluid (including enzymes) from the midgut of the target species,
2. mixing enzymes from the midgut with inhibitors, in vitro, and 3. evaluating the mixture for enzyme activity.
Results provide an indication of the classes of proteinases in the midgut, and the strength of the interaction between the inhibitors and the enzymes. Numerous reports indicate that PIs from plants will significantly inhibit, in vitro, the proteolytic activity from insect midguts,3-14 suggesting their potential as effective phytochemical defenses against herbivorous insects. The value of in vitro inhibition studies is that they directly evaluate the interaction between the inhibitor and the target proteinases. However the limitation of this type of study is that it does not allow for examination of the insect’s physiological response to the treatment, which is critical in determining the biological activity of PIs against insects. A second approach, which addresses the limitation of in vitro inhibition assays, focuses on evaluation of the effect of PI ingestion on the growth, development and survival of test species. Artificial diets containing PIs were used to demonstrate that ingestion of inhibitors will reduce the growth, development and/or survival of Diptera, Coleoptera, Hymenoptera, and Lepidoptera.10,15-33 The value of this approach is that the treatment is well defined. However some limitations are that it can only
Recombinant Protease Inhibitors in Plants, edited by Dominique Michaud. ©2000 Eurekah.com.
The Response of Insects to Dietary Protease Inhibitors
be used to study species of insects for which artificial diets have been developed, and that an artificial diet is not equivalent to plant material. Artificial diet lacks numerous phytochemicals that have the potential to alter the toxicity of PIs to insects.17,24,34 In addition, the experiments often include PIs at concentrations higher than those found in plant tissues. Other diet-based assays use plant tissue containing PIs. Age, environmental stress and breeding practices modify PI levels in plants,35-37 and tissues containing high vs low PI levels have been used to evaluate the effect of PIs on herbivorous insects.37-43 However, these studies only are able to demonstrate a correlation between concentration of foliar PIs and the growth or development of herbivorous insects. These studies do not conclusively indicate that foliar PIs cause the effect, because in addition to PIs the quantity and the quality of other phytochemical constituents change with developmental stage, environmental stress and breeding practices. Consequently the insects’ physiological responses in this type of study may be due to factors other than PIs. Given the inherent limitations of the approaches discussed above, and the new advances in molecular biology that enable genetic introduction and/or enhancement of a single gene, we now are able to use plants that have been genetically engineered for enhanced levels of PIs to determine their effects on herbivorous insects. Hilder et al44 were the first authors to report the transgenic introduction of a PI-encoding gene in a plant. Tobacco genetically transformed with the gene for cowpea trypsin inhibitor was significantly more resistant to the tobacco budworm (Heliothis virescens) than plants without the gene encoding the inhibitor. In addition there was a significant decrease in survival of insects feeding on transformed plants. Since this report there have been a number of successful transgenic insertions of PIs into plants that have resulted in resistance of the plants to some species of herbivorous insects.10,45-57 All these studies support the hypothesis that plant PIs have the potential to inhibit growth and development of herbivorous
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insects. However, plant PIs do not significantly impact all species of herviborous pests,14,30,50,58,59 which is not surprising since these organisms feed and thrive on plant tissue that contains PIs. Insects must have developed adaptive strategies to overcome the PIs in their host plants, but until recently the mechanisms of resistance to plant PIs in insects were unknown and largely unexplored. Understanding the mechanisms of resistance of insects to plant PIs may enable us to use PIs more effectively as a strategy to regulate populations of herbivorous insects.
7.3. Resistance of Insects to Dietary PIs Insects have evolved at least two strategies to deal with dietary PIs. One strategy involves the secretion of enzymes that hydrolyze and thus inactivate the inhibitors.60-63 The other strategy involves the secretion of proteinases that are not susceptible to inhibition by those PIs.37,62,64-66 This second strategy may involve a number of physiological conditions, including the secretion of more than one protein with prote(in)ase activity, differential susceptibility of these proteinases to dietary PIs, and enhanced secretion of inhibitor-resistant enzymes following ingestion of PI. The susceptibility of an insect to a given PI appears to be directly related to the proportion of proteolytic enzyme activity in the midgut that can be suppressed by that inhibitor, and conversely resistance of insects to PIs is based, at least in part, on their ability to enhance the proportion of inhibitor-resistant enzymes in the midgut. This second strategy has been clearly established for insects feeding on artificial diets containing PIs from their host plants, and when feeding on their host plants. However this approach has an interesting twist, particularly for insects that feed on multiple families of plants. Insects appear to have multiple suites of proteinases that are selectively secreted in response to the quality of the diet.37,67,68 Thus, larvae may secrete one suite of enzymes following ingestion of tissue from one species of host plant, and may
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secrete other enzymes following ingestion of a different species of plant or PI. Based on these findings, it appears that the best approach for selecting PIs to control herbivorous insects may be to focus on PIs from non-host plants. However, this is not always successful.69 Lymantria dispar, the gypsy moth, is a pest of shade trees and does not feed on cabbage. The larval midgut contains serine proteinases that are susceptible to inhibition by cabbage PIs. However, the ingestion of cabbage PIs has no effect on larval growth or development, apparently as a result of induced secretion of inhibitor-resistant proteinases.37 This same response occurs in larval Pieris rapae, a crucifer specialist that is resistant to cabbage PIs (from their host plant) and is also resistant to the Kunitz trypsin inhibitor from soybean (a non-host plant). However, the Kunitz soybean trypsin inhibitor is in the same family of PIs as the cabbage PIs, suggesting that insects may be pre- adapted to specific PIs following adaptation to a PI from the same family. 69,70 Perhaps the best approach for selecting PIs for phytochemical defense against herbivorous insects is to focus on families of PIs that do not occur in the insect’s host plant. It is possible that PIs limit the range of hosts that are suitable for a species of insect. However recent studies suggest that the insect midgut contains a number of isozymes that differ in their susceptibility to a specific PI, that the level of activity of inhibitor-resistant enzymes in the midgut is induced in some cases by dietary PIs, and that insects may be pre-adapted to PIs from non-host plants. Therefore, if PIs are to be incorporated into an integrated pest management program to control populations of herbivorous insects, they must be carefully selected for each species of insect, and multiple inhibitors may be required for significant reduction of enzyme activity in situ.
7.3.1. Improving the Binding Capacity of PIs Our ability to select appropriate PIs may be enhanced by understanding the factors that
Recombinant Protease Inhibitors in Plants
influence the binding capacity of PIs. The active site of an enzyme forms a pocket where catalysis occurs, and determines its specificity. For example, trypsins have histidine, serine, and aspartic acid at the active site. The acidic amino acid, aspartic acid, at the bottom of the binding pocket determines trypsin’s specificity for the basic amino acids arginine and lysine. For chymotrypsins, the aspartic acid is replaced by serine, creating a hydrophobic site which accounts for the specificity of chymotrypsin for tryptophan, phenylalanine and tyrosine. The active site of elastase is constricted by the side chains of valine and threonin, which explains the specificity of elastase for the small amino acid residues alanine and valine. However the specificity of serine proteinases is not solely determined by the steric and electrostatic properties of their active sites, but also by surface loops outside the active site.71 These surface loops do not contact the substrate, but determine the configuration of the enzyme and thus the accessability of the active site.72,73 Minor changes in the amino acid residues that surround the active site of the proteinase may reduce the binding capacity between the proteinase and the inhibitor. This is exemplified by human trypsin that is resistant to the action of soybean trypsin inhibitor, pancreatic secretory trypsin inhibitor and aprotinin as a result of minor changes in the amino acid sequence surrounding the active site of the proteinase, that significantly reduced the binding capacity of the inhibitors for the enzyme.74 A similar phenomenon occurred in PI-resistant aspartyl proteases of human immunodeficiency virus type 1 (HIV-1), in which a few site mutations in the residues in and around the active site reduced the binding capacity for the PI, resulting in proteinases that were not susceptible to inhibition.75-79 One approach that may overcome this problem of incompatibility between the inhibitor and the enzyme is to use PIs that are very small, or PI domains which could easily reach the enzyme active site, such that they would interact with the proteinase primarily at the active site. This approach may help improve the binding capacity between
The Response of Insects to Dietary Protease Inhibitors
PI and proteinases, reduce the number of mutant enzymes that are successfully resistant to the inhibitor, and expand the array of proteinases within a single subclass (e.g. trypsins) that are susceptible to inhibition by the inhibitor.
7.3.2. Reducing the Insects’ Ability to Induce PI-Resistant Enzymes In addition to enhancing the inhibitory activity of PIs, it may be possible to disrupt the factors that regulate the induction of inhibitor-resistant enzymes, thus preventing or reducing the insect’s ability to physiologically respond to dietary PIs. However, an understanding of enzyme regulation in the midgut of herbivorous insects is required to accomplish this goal. Currently, very little information is available about the regulation of enzymes in the midgut of herbivorous insects. Recent data suggest that there are two types of regulation in herbivorous insects: a constitutive regulation of enzymes that are secreted at the time of ingestion, and an induced regulation that responds to the quantity and/or quality of dietary constitutents (e.g. PIs). However the factors responsible for this regulation are still unknown. This same dual regulatory system has been established for hematophagous Diptera. The juvenile hormone regulates transcription of the genes that encode the constitutive enzymes.80,81 The mRNA for the constitutive enzymes is either translated following ingestion of diet,82 or translated immediately following transcription with the enzymes stored as zymogens in the midgut epithelium,80,83,84 and secreted into the lumen following abdominal distention.85,86 The factors that regulate the induced enzymes are unknown for hematophagous Diptera, but the induction is influenced by the quantity and/or quality of dietary protein. 82,86-91 Secretion of the induced enzymes continues until digestion is complete, then the enzymes are excreted, and the level of constitutive enzymes is restored before the next meal is taken. Mammals also have a dual regulatory system for digestive enzymes, which has been
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extensively studied.92 Cholecystokinin (CCK) is a hormone secreted into the blood upon ingestion of food. It stimulates generalized pancreatic secretion and contraction of the gallbladder, regulates gastric emptyting, and induces satiety. The exact mechanisms by which foods cause CCK secretion have yet to be elucidated, but it is likely that multiple factors are involved. At least five endogenous factors regulate CCK secretion: 1. cholecystokinin releasing factor (CCK-RF) is secreted by the intestinal mucosa and is present during basal conditions, 2. monitor peptide (also known as pancreatic secretory trypsin inhibitor-I) is secreted by the pancreas (following pancreatic stimulation) into pancreatic juice where, upon reaching the intestine, it reinforces or perpetuates further secretion of enzymes by stimulating CCK release, 3. luminal cholecystokinin releasing factor (LCRF) is believed to stimulate pancreatic secretion and CCK release resulting from nutrient ingestion, 4. bombesin, is a neuropeptide, directly stimulates CCK release, and 5. Diazepam-binding inhibitor has CCKreleasing activity. 92-97 In addition, dietary factors including PIs98 and lectins99 directly stimulate CCK release.
The current theory regarding the regulation of intestinal digestive proteinases in mammals suggests that, in the absence of food in the intestine, a low background level of trypsin activity in the intestine digests the regulatory factors. When a protein (or a PI) enters the intestine, trypsin binds to this dietary protein, allowing undigested CCKregulatory factors, monitor peptide, and/or dietary factors to interact with cell surface receptors in the intestine, and to initiate a cascade of events that trigger CCK release into the blood stream, which stimulates the pancreas to secrete digestive enzymes into the intestine. However, some evidence suggests that CCK is responsible for a generalized regulation of digestive enzymes, while the monitor peptide is responsible for the
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Recombinant Protease Inhibitors in Plants
regulation of a specific enzyme or suite of enzymes. This idea is supported by results indicating that ingestion of soybean trypsin/ chymotrypsin inhibitors or extract from raw soybean meal significantly increases the level of inhibitor-resistant trypsin in humans,100 while there is no concomitant elevation in CCK level.101,102 To further complicate the story, there are factors other than CCK that stimulate pancreatic exocrine secretion, including secretin and gastrin releasing factor.103-107 Insects may also have numerous regulatory factors, each factor being associated with a unique suite of proteinases.68 Identification of these factors in insects may enable us to inhibit their activity and thus prevent enzymes induction.
3. identifying and enhancing factors that down-regulate the expression of digestive enzymes in the midgut of herbivorous insects.
7.3.3. Down-Regulating Enzyme Production A third approach that might improve the susceptibility of herbivorous insects to PIs is to identify factors that down-regulate the synthesis of proteinases. Borovsky et al discovered a peptide (TMOF, trypsinmodulating oostatic factor)108-112 in the mosquito, Aedes aegypti, and the fleshfly, Neobellieria bullata, that terminates translation of trypsin mRNA in the digestive tract.113 TMOF-like factors may be present in other species of insects that, if identified, could be used to prevent the production of proteinases.
7.4. Summary In conclusion, it is quite possible that some PIs in certain species of plants do limit herbivory by some insects. However insects have evolved mechanisms to neutralize or avoid the effects of plant PIs. We may be able to improve our success in using PIs to manage populations of herbivorous insects by: 1. isolating or designing small PIs or PI domains that may function against a broad array of enzymes within a single subclass, 2. identifying and inactivating factors that induce proteinases in the midgut, and
References 1. Foard DE, Murdock LL, Dunn PE. Engineering of crop plants with resistance to herbivores and pathogens: An approach using primary gene products. Plant Mol Biol 1983; 2:223-233. 2. Ryan CA. Proteinase Inhibitors. In: Stumpf PK, Conn EE, eds. The Biochemistry of Plants—A comprehensive treatise. New York:Academic Press 1981:351-370. 3. Alfonso J, Ortego F, Sanchez-Monge R et al. Wheat and barley inhibitors active toward alpha-amylase and trypsin-like activities from Spodoptera frugiperda. J Chem Ecol 1997; 23:1729-1741. 4. Bian X, Shaw BD, Han Y et al. Midgut proteinase activities in larvae of Anoplophora glabripennis (Coleoptera: Cerambycidae) and their interaction with proteinase inhibitors. Arch Insect Biochem Physiol 1996; 31:23-37. 5. Ceciliani F, Tava A, Iori R et al. A trypsin inhibitor from snail medic seeds active against pest proteases. Phytochemistry 1997; 44:393-398. 6. Christeller JT, Shaw BD. The interaction of a range of serine proteinase inhibitors with bovine trypsin and Costelytra zealandica trypsin. Insect Biochem 1989; 19:233-241. 7. Christeller JT, Laing WA, Markwick NP et al. Midgut protease activities in 12 phytophagous lepidopteran larvae: Dietary and protease inhibitor interactions. Insect Biochem Mol Biol 1992; 22:735-746. 8. Christeller JT, Gatehouse AMR, Laing WA. The interaction of the elastase inhibitor, Eglin c, with insect digestive endopeptidases: effect of pH on the dissociation constants. Insect Biochem Mol Biol 1994; 24:103-109. 9. Christeller JT, Markwick NP, Burgess EPJ. Midgut proteinase activities of three keratinolytic larvae, Hofmannophila pseudospretella, Tineola bisselliella, and Anthrenocerus australis, and the effect of proteinase inhibitors on proteolysis. Arch Insect Biochem Physiol 1994; 25:159-173. 10. Heath RL, McDonald G, Christeller JT et al. Proteinase inhibitors from Nicotiana alata enhance plant resistance to insect pests. J Insect Physiol 1997; 43:833-842. 11. Hines ME, Osuala CI, Nielsen SS. Isolation and partial characterization of a soybean cystatin cysteine proteinase inhibitor of coleopteran digestive proteolytic activity. J Agric Food Chem 1991; 39:1515-1520.
The Response of Insects to Dietary Protease Inhibitors 12. McGhie TK, Christeller JT, Ford R et al. Characterization of midgut proteinase activities of white grubs: Lepidiota noxia, Lepidiota negatoria, and Antiotrogus consanguineus (Scarabaeidae: Melolonthini). Arch Insect Biochem Physiol 1995; 28:351-363. 13. Novillo C, Castanera P, Ortego F. Inhibition of digestive trypsin-like proteases from larvae ofseveral lepidopteran species by diagnostic cysteine protease inhibitor E-64. Insect Biochem Mol Biol 1997; 27:247-254. 14. Purcell JP, Greenplate JT, Sammons RD. Examination of midgut luminal proteinase activities in six economically important insects. Insect Biochem Mol Biol 1992; 22:41-47. 15. Birk Y, Applebaum SW. Effect of soybean trypsin inhibitors on the development and midgut proteolytic activity of Tribolium castaneum larvae. Enzymologia 1960; 22:318-326. 16. Broadway RM, Duffey SS. Plant proteinase inhibitors: Mechanism of action and effect on the growth and digestive physiology of larval Heliothis zea and Spodoptera exigua. J Insect Physiol 1986; 32:827-833. 17. Broadway RM, Duffey SS. The effect of plant protein quality on insect digestive physiology and the toxicity of plant proteinase inhibitors. J Insect Physiol 1988; 34:1111-1117. 18. Burgess EPJ, Malone LA, Christeller JT. Effects of two proteinase inhibitors on the digestive enzymes and survival of honeybees (Apis mellifera ). J Insect Physiol 1996; 42:823-828. 19. Burgess EPJ, Stevens PS, Keen GK et al. Effects of protease inhibitors and dietary protein level on the black field cricket Teleogryllus commodus. Entomol exp appl 1991; 61:123-130. 20. Burgess EPJ, Main CA, Stevens PS et al. Effects of protease inhibitor concentration and combinations on the survival, growth and gut enzyme activities of the black field cricket, Teleogryllus commodus. J Insect Physiol 1994; 40:803-811. 21. Deloach JR, Spates GE. Effect of soybean trypsin inhibitor-loaded erythrocytes on fecundity and midgut protease and hemolysis activity of stable flies. J Econ Entomol 1980; 73:590-594. 22. Dymock JJ, Laing WA, Shaw BD et al. Behavioural and physiological responses of grass grub larvae (Costelytra zealandica) feeding on protease inhibitors. New Zealand J Zool 1992; 19:123-131. 23. Gatehouse AMR, Boulter D. Assessment of the antimetabolic effects of trypsin inhibitors from cowpea (Vigna unguiculata) and other legumes on development of the bruchid beetle Callosobruchus maculatus. J Sci Food Agric 1983; 34:345-350.
85 24. Hinks CF, Hupka D. The effects of feeding leaf sap from oats and wheat, with and without soybean trypsin inhibitor, on feeding behavior and digestive physiology of adult males of Melanoplus sanguinipes. J Insect Physiol 1995; 41:1007-1015. 25. Johnston KA, Gatehouse JA, Anstee JH. Effects of soybean protease inhibitors on the growth and development of larval Helicoverpa armigera. J Insect Physiol 1993; 39:657-664. 26. Larocque AM, Houseman JG. The effect of ingested soybean, ovomucoid and corn trypsin inhibitor on digestive processes of the European corn borer. J Insect Physiol 1990; 36:691-697. 27. Markwick NP, Reid SJ, Laing WA et al. Effects of dietary protein and protease inhibitors on codling moth (Lepidoptera: Tortricidae). J Econ Entomol 1995; 88:33-39. 28. McManus MT, Burgess EPJ. Effects of the soybean (Kunitz) trypsin inhibitor on growth and digestive proteases of larvae of Spodoptera litura. J Insect Physiol 1995; 41:731-738. 29. Oppert B, Morgan TD, Culbertson C et al. Dietary mixtures of cysteine and serine proteinase inhibitors exhibit synergistic toxicity toward the red flour beetle, Tribolium castaneum. Comp Biochem Physiol 1993; 105C:379-385. 30. Shade RE, Murdock LL, Foard DE et al. Artificial seed system for bioassay of cowpea weevil (Coleoptera: Bruchidae) growth and development. Environ Entomol 1986; 15:1286-1291. 31. Shukle RH, Murdock LL. Lipoxygenase, trypsin inhibitor, and lectin from soybeans: Effects on larval growth of Manduca sexta (Lepidoptera: Sphingidae). Environ Entomol 1983; 12:787-791. 32. Spates G. Fecundity of the stable fly: Effect of soybean trypsin inhibitor and phospholipase A inhibitor on the fecundity. Ann Entomol Soc Am 1979; 72:845-849. 33. Steffens R, Fox FR, Kassell B. Effect of trypsin inhibitors on growth and metamorphosis of corn borer larvae Ostrinia nubilalis (Hubner). J Agric Food Chem 1978; 26:170-174. 34. Felton GW, Broadway RM, Duffey SS. Inactivation of proteinase inhibitor activity by plant-derived quinones: Complications for host-plant resistance against noctuid herbivores. J Insect Physiol 1989; 35:981-990. 35. Broadway RM. Tryptic inhibitory activity in wild and cultivated crucifers. Phytochemistry 1989; 28:755-758. 36. Broadway RM, Missurelli DL. Regulatory mechanisms of tryptic inhibitory activity in cabbage plants. Phytochemistry 1990; 29:3721-3725.
86 37. Broadway RM. Are insects resistant to plant proteinase inhibitors? J Insect Physiol 1995; 41:107-116. 38. Seldal T, Dybwad E, Andersen K-J et al. Wound-induced proteinase inhibitors in grey alder (Alnus incana): A defense mechanism against attacking insects. Oikos 1994; 71:239-245. 39. Broadway RM, Duffey SS, Pearce G et al. Plant proteinase inhibitors: A defense against herbivorous insects? Entomol exp appl 1986; 41:33-38. 40. Broadway RM, Colvin AA. The influence of cabbage proteinase inhibitors, in situ, on the growth of larval Trichoplusia ni and Pieris rapae. J Chem Ecol 1992; 18:1009-1024. 41. Piergiovanni AR, Sonnante G, Della Gatta C et al. Digestive enzyme inhibitors and storage pest resistance in cowpea (Vigna unguiculata) seeds. Euphytica 1991; 54:191-194. 42. Weiel J, Hapner KD. Barley proteinase inhibitors: A possible role in grasshopper control? Phytochemistry 1976; 15:1885-1887. 43. Wolfson JL, Murdock LL. Growth of Manduca sexta on wounded tomato plants: Role of induced proteinase inhibitors. Entomol Exp Appl 1990; 54:257-264. 44. Hilder VA, Gatehouse AMR, Sheerman SE et al. A novel mechanism of insect resistance engineered into tobacco. Nature 1987; 330:160-163. 45. Duan X, Li X, Xue Q et al. Transgenic rice plants harboring an introduced potato proteinase inhibitor II gene are insect resistant. Nat Biotechnol 1996; 14:494-498. 46. Hoffmann MP, Zalom FG, Wilson LT et al. Field evaluation of transgenic tobacco containing genes encoding Bacillus thuringiensis delta-endotoxin or cowpea trypsin inhibitor: Efficacy against Helicoverpa zea (Lepidoptera: Noctuidae). J Econ Entomol 1992; 85:2516-2522. 47. Johnson R, Narvaez J, An G et al. Expression of proteinase inhibitors I and II in transgenic tobacco plants: Effects on natural defense against Manduca sexta larvae. Proc Natl Acad Sci USA 1989; 86:9871-9875. 48. Klopfenstein NB, Allen KK, Avila FJ et al. Proteinase inhibitor II gene in transgenic poplar: Chemical and biological assays. Biomass Bioenergy 1997; 12:299-311. 49. Leplé JC, Bonadé-Bottino M, Augustin S et al. Toxicity to Chrysomela tremulae (Coleoptera: Chrysomelidae) of transgenic poplars expressing a cysteine proteinase inhibitor. Mol Breeding 1995; 1:319-328. 50. McManus MT, White DWR, McGregor PG. Accumulation of a chymotrypsin inhibitor in transgenic tobacco can affect the growth of insect pests. Transgenic Res 1994; 3:50-58.
Recombinant Protease Inhibitors in Plants 51. Orozco-Cardenas M, McGurl B, Ryan CA. Expression of an antisense prosystemin gene in tomato plants reduces resistance toward Manduca sexta larvae. Proc Natl Acad Sci USA 1993; 90:8273-8276. 52. Santos MO, Adang MJ, All JN et al. Testing transgenes for insect resistance using Arabidopsis. Mol Breeding 1997; 3:183-194. 53. Thomas JC, Adams GG, Keppenne VD et al. Manduca sexta encoded protease inhibitors expressed in Nicotiana tabacum provide protection against insects. Plant Physiol Biochem 1995; 33:611-614. 54. Thomas JC, Adams DG, Keppenne VD et al. Protease inhibitors of Manduca sexta expressed in transgenic cotton. Plant Cell Rep 1995; 14:758-762. 55. Thomas JC, Wasmann CC, Echt C et al. Introduction and expression of an insect proteinase inhibitor in alfalfa (Medicago sativa L.). Plant Cell Rep 1994; 14:31-36. 56. Xu D, Xue Z, McElroy D et al. Constitutive expression of a cowpea trypsin inhibitor gene, CpTi, in transgenic rice plants confers resistance to two major rice insect pests. Mol Breeding 1996; 2:167-173. 57. Yeh K-W, Lin M-I, Tuan S-J et al. Sweet potato (Ipomoea batatas) trypsin inhibitors expressed in transgenic tobacco plants confer resistance against Spodoptera litura. Plant Cell Rep 1997; 16:696-699. 58. Fernandes KVS, Sabelli PA, Barratt DHP et al. The resistance of cowpea seeds to bruchid beetles is not related to levels of cysteine proteinase inhibitors. Plant Mol Biol 1993; 23:215-219. 59. Zhu K, Huesing JE, Shade RE et al. Cowpea trypsin inhibitor and resistance to cowpea weevil (Coleoptera: Bruchidae) in cowpea variety TVu2027. Environ Entomol 1994; 23:987-991. 60. Ishimoto M, Chrispeels MJ. Protective mechanism of the Mexican bean weevil against high levels of alpha-amylase inhibitor in the common bean. Plant Physiol 1996; 111:393-401. 61. Michaud D, Nguyen-Quoc B, Vrain TC et al. Response of digestive cysteine proteinases from the Colorado potato beetle (Leptinotarsa decemlineata) and the black vine weevil (Otiorynchus sulcatus) to a recombinant form of human stefin A. Arch Insect Biochem Physiol 1996; 31:451-464. 62. Michaud D, Cantin L, Vrain TC. Carboxyterminal truncation of oryzacystatin II by oryzacystatin-insensitive insect digestive proteinases. Arch Biochem Biophys 1995; 322:469-474.
The Response of Insects to Dietary Protease Inhibitors 63. Giri AP, Harsulkar AM, Deshpande VV et al. Chickpea defensive proteinase inhibitors can be inactivated by podborer gut proteinases. Plant Physiol 1998; 116:393-401. 64. Jongsma MA, Bakker PL, Peters J et al. Adaptation of Spodoptera exigua larvae to plant proteinase inhibitors by induction of gut proteinase activity insensitive to inhibition. Proc Natl Acad Sci USA 1995; 92:8041-8045. 65. Bown DP, Wilkinson HS, Gatehouse JA. Differentially regulated inhibitor-sensitive and insensitive protease genes from the phytophagous insect pest, Helicoverpa armigera, are members of complex multigene families. Insect Biochem Mol Biol 1997; 27:625-638. 66. Wu Y, Llewellyn D, Mathews A et al. Adaptation of Helicoverpa armigera (Lepidoptera: Noctuidae) to a proteinase inhibitor expressed in transgenic tobacco. Mol Breeding 1997; 3:371-380. 67. Overney S, Fawe A, Yelle S et al. Diet- related plasticity of the digestive proteolytic system in larvae of the Colorado potato beetle (Leptinotarsa decemlineata Say). Arch Insect Biochem Physiol 1997; 36:241-250. 68. Broadway RM. Dietary regulation of serine proteinases that are resistant to serine proteinase inhibitors. J Insect Physiol 1997; 9:855-874. 69. Broadway RM, Villani MG. Does host range influence susceptibility of herbivorous insects to novel plant proteinase inhibitors? Entomol Exp Appl 1995; 76:303-313. 70. Broadway RM. Dietary proteinase inhibitors alter complement of midgut proteases. Arch Insect Biochem Physiol 1996; 32:39-53. 71. Hedstrom L. Trypsin: A case study in the structural determinants of enzyme specificity. Biol Chem 1996; 377:465-470. 72. Hedstrom L, Farr-Jones S, Kettner CA et al. Converting trypsin to chymotrypsin: Ground-state binding does not determine substrate specificity. Biochemistry 1994; 33:8764-8769. 73. Kurth T, Ullmann D, Jakubke H-D et al. Converting trypsin to chymotrypsin: Structural determinants of S1' specificity. Biochemistry 1997; 36:10098-10104. 74. Nyaruhucha CNM, Kito M, Fukuoka S-I. Identification and expression of the cDNAencoding human mesotrypsin(ogen), an isoform of trypsin with inhibitor resistance. J Biol Chem 1997; 272:10573-10578. 75. Zhang Y-M, Imamichi H, Imamichi T et al. Drug resistance during indinavir therapy is caused by mutations in the protease gene and its gag substrate cleavage sites. J Virol 1997; 71:6662-6670.
87 76. Smidt ML, Potts KE, Tucker SP et al. A mutation in human immunodeficiency virus type 1 protease at position 88, located outside the active site, confers resistance to the hydroxyethylurea inhibitor SC-55389A. Antimicrob Agents Chemother 1997; 41:515-522. 77. Pazhanisamy S, Stuver CM, Cullinan AB et al. Kinetic characterization of human immunodeficiency virus type-1 proteaseresistant variants. J Biol Chem 1996; 271:17979-17985. 78. Schock HB, Garsky VM, Kuo LC. Mutational anatomy of an HIV-1 protease variant conferring cross-resistance to protease inhibitors in clinical trials. J Biol Chem 1996; 271:31957-31963. 79. Eberle J, Bechowsky B, Rose D et al. Resistance of HIV type 1 to proteinase inhibitor Ro 31-8959. AIDS Research & Human Retroviruses 1995; 11:671-676. 80. Horler E, Briegel H. Proteolytic enzymes of female Anopheles: Biphasic synthesis, regulation, and multiple feeding. Arch Insect Biochem Physiol 1995; 28:189-205. 81. Noriega FG, Shah DK, Wells MA. Juvenile hormone controls early trypsin gene transcription in the midgut of Aedes aegypti. Insect Mol Biol 1997; 6:63-66. 82. Felix CR, Betschart B, Billingsley PF et al. Post-feeding induction of trypsin in the midgut of Aedes aegypti L. (Diptera: Culicidae) is separable into two cellular phases. Insect Biochem 1991; 21:197-203. 83. Muller H-M, Catteruccia F, Vizioli J et al. Constitutive and blood meal-induced trypsin genes in Anopheles gambiae. Exp Parasitol 1995; 81:371-385. 84. Moffatt MR, Lehane MJ. Trypsin is stored as an inactive zymogen in the midgut of Stomoxys calcitrans. Insect Biochem 1990; 20:719-723. 85. Graf R, Briegel H. The synthetic pathway of trypsin in the mosquito Aedes aegypti L. (Diptera: Culicidae) and in vitro stimulation in isolated midguts. Insect Biochem 1989; 19:129-137. 86. Lemos FJA, Cornel AJ, Jacobs-Lorena M. Trypsin and aminopeptidase gene expression is affected by age and food composition in Anopheles gambiae. Insect Biochem Mol Biol 1996; 26:651-658. 87. Muller H-M, Crampton JM, Torre AD et al. Members of a trypsin gene family in Anopheles gambiae are induced in the gut by blood meal. EMBO J 1993; 12:2891-2900. 88. Noriega FG, Barillas-Mury C, Wells MA. Dietary control of late trypsin gene transcription in Aedes aegypti. Insect Biochem Mol Biol 1994; 24:627-631.
88 89. Barillas-Mury CV, Noriega FG, Wells MA. Early trypsin activity is part of the signal transduction system that activates transcription of the late trypsin gene in the midgut of the mosquito, Aedes aegypti. Insect Biochem Mol Biol 1995; 25:241-246. 90. Blakemore D, Williams S, Lehane MJ. Protein stimulation of trypsin secretion from the opaque zone midgut cells of Stomoxys calcitrans. Comp Biochem Physiol 1995; 110B:301-307. 91. Moffatt MR, Blakemore D, Lehane MJ. Studies on the synthesis and secretion of trypsin in the midgut of Stomoxys calcitrans. Comp Biochem Physiol 1995; 110B:291-300. 92. Liddle RA. Cholecystokinin cells. Annu. Rev. Physiol. 1997; 59:221-242. 93. Liddle RA. Regulation of cholecystokinin secretion by intraluminal releasing factors. Am J Physiol 1995; 269:G319-G327. 94. Herzig K-H, Schon I, Tatemoto K et al. Diazepam binding inhibitor is a potent cholecystokinin-releasing peptide in the intestine. Proc Natl Acad Sci USA 1996; 93:7927-7932. 95. Spannagel AW, Green GM, Guan D et al. Purification and characterization of a luminal cholecystokinin-releasing factor from rat intestinal secretion. Proc Natl Acad Sci USA 1996; 93:4415-4420. 96. Yamanishi R, Kotera J, Fushiki T et al. A specific binding of the cholecystokininreleasing peptide (monitor peptide) to isolated rat small-intestinal cells. Biochem J 1993; 291:57-63. 97. Tsuzuki S, Miura Y, Fushiki T et al. Molecular cloning and characterization of genes encoding rat pancreatic cholecystokinin (CCK)-releasing peptide (monitor peptide) and pancreatic secretory trypsin inhibitor (PSTI). Biochim Biophys Acta 1992; 1132:199-202. 98. Pusztai A, Grant G, Bardocz S et al. Both free and complexed trypsin inhibitors stimulate pancreatic secretion and change duodenal enzyme levels. Am J Physiol (Gastointest Liver Physiol) 1997; 272:G340-G350. 99. Jordinson M, Deprez PH, Playford RJ et al. Soybean lectin stimulates pancreatic exocrine secretion via CCK-A receptors in rats. Am J Physiol (Gastrointest Liver Physiol) 1996; 270:G653-G659. 100. Holm H, Krogdahl A, Hanssen LE. High and low inhibitor soybean meals affect human duodenal proteinase activity differently: In vitro comparison of proteinase inhibitor. J Nutr 1988; 118:521-525.
Recombinant Protease Inhibitors in Plants 101. Holm H, Hanssen LE, Krogdahl A et al. High and low inhibitor soybean meals affect human duodenal proteinase activity differently: In vivo comparison with bovine serum albumin. J Nutr 1988; 118:515-520. 102. Holm H, Reseland JE, Thorsen LI et al. Raw soybeans stimulate human pancreatic proteinase secretion. J Nutr 1992; 122:1407-1416. 103. Kim CD, Lee KY, Chang TM et al. Negative feedback regulation of pancreatic exocrine secretion in guinea pigs. Pancreas 1995; 10:173-179. 104. Miyasaka K, Funakoshi A. Involvement of gene expressions of cholecystokinin and secretin in luminal feedback regulation in conscious rats. Pancreas 1995; 10:200-203. 105. Jin HO, Song CW, Chang TM et al. Roles of gut hormones in negative-feedback regulation of pancreatic exocrine secretion in humans. Gastroenterology 1994; 107:1828-1834. 106. Shiratori K, Watanabe S-I, Moriyoshi Y et al. Pancreatic juice-diversion augments oleic acid-stimulated pancreatic secretion and release of secretin and CCK in rats. Biomed Res 1994; 15:387-389. 107. Imamura M, Lee KY, Song Y et al. Role of secretin in negative feedback regulation of postprandial pancreatic secretion in dogs. Gastroenterology 1993; 105:548-553. 108. Bylemans D, Verhaert P, Janssen I et al. Immunolocalization of the oostatic and prothoracicostatic peptide, Neb-TMOF, in adults of the fleshfly, Neobellieria bullata. Gen Comp Endocrin 1996; 103:273-280. 109. Borovsky D, Mahmood F. Feeding the mosquito Aedes aegypti with TMOF and its analogs; effect on trypsin biosynthesis and egg development. Regul Peptides 1995; 57:273-281. 110. Borovsky D, Powell CA, Nayar JK et al. Characterization and localization of mosquito-gut receptors for trypsin modulating oostatic factor using a complementary peptide and immunocytochemistry. FASEB J 1994; 8:350-355. 111. Borovsky D, Carlson DA, Griffin PR et al. Mosquito oostatic factor: A novel decapeptide modulating trypsin-like enzyme biosynthesis in the midgut. FASEB J 1990; 4:3015-3020. 112. Borovsky D. Oostatic hormone inhibits biosynthesis of midgut proteolytic enzymes and egg development in mosquitoes. Arch Insect Biochem Physiol 1988; 7:187-210. 113. Borovsky D, Janssen I, Vanden Broeck J et al. Molecular sequencing and modeling of Neobellieria bullata trypsin. Evidence for translational control by Neobellieria trypsinmodulating oostatic factor. Eur J Biochem 1996; 237:279-287.
CHAPTER 8
Interference of Protease Inhibitors on Non-Target Organisms Louise A. Malone and Elisabeth P.J. Burgess
8.1. Introduction
W
hen plants are genetically modified with proteinase inhibitor (PI)-encoding genes, the intended targets for these PIs are either herbivorous pests or pathogenic microorganisms attacking the plant (see Chapters 2-5, this volume). In any agricultural system, a number of non-target organisms will also be exposed to the PI. The most obvious of these are the stock animals or humans that feed on the crop. Less obvious, but no less important are insects which pollinate the crop, natural enemies of the pest, symbionts living in association with the plant and decomposers which utilize dead material from both the plant and the target herbivore population (Fig. 8.1). These providers of “ecological services” may be affected by PI-transgenic plants either directly or indirectly. Direct effects may arise when non-target organisms such as bees, symbiotic bacteria or saprophytic fungi ingest or absorb plant material which now contains a “foreign” PI. Indirect effects may arise in two ways. Firstly, genetic modification of the plant may result in inadvertent changes to its phenotype that could affect pollinators, decomposers or symbionts. These changes may be the result of insertional mutagenesis, whereby the positioning of the new gene within the plant’s genome alters the plant in some unexpected way. This will vary with each transformation event so that different lines of plants may have different phenotypic
changes. Other changes may result from pleiotropic effects, whereby the expression of the new gene unexpectedly alters a biochemical pathway with phenotypic consequences. Pleiotropic effects would thus be expected to occur in all lines of the transformed plant. Examples of phenotypic changes that might affect non-target organisms would be an alteration in the attractiveness of flowers to pollinators, biochemical changes that might inhibit decomposition, or changes in compounds important in plant-symbiont signalling. A second route whereby plants expressing recombinant PIs could indirectly affect non-target organisms is via changes in the target herbivore consuming the plant. Where this insect serves as prey, changes in its size, movement, nutritional quality, location or abundance will have an impact on natural enemies such as predators, parasitoids and entomopathogens. Similar effects could be expected for the decomposers once these target organisms have died. In the following sections we consider the likely effects of PI-expressing transgenic plants on four different groups of non-target organisms: pollinators, natural enemies, decomposers and symbionts. Current research is reviewed and future research needs are identified.
Recombinant Protease Inhibitors in Plants, edited by Dominique Michaud. ©2000 Eurekah.com.
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Recombinant Protease Inhibitors in Plants
Fig. 8.1. Relationships between PI-expressing transgenic plants, target organisms (shaded) and non-target organisms (unshaded).
8.2. Pollinators Table 8.1 lists the crop plants that have thus far been transformed with PI genes1 and the means by which they are pollinated.2 Clearly, honeybees and bumblebees are the most important insect pollinators of these crops, as they are for seed production in many of the major crop species being transformed with other genes,3 including cotton, melons, soybean, alfalfa, flax, squash and sunflower.
8.2.1. Risks Associated with Pollen and Honeybees Figure 8.2 shows the changes in PIexpressing plants that may affect pollinating honeybees, the possible fate of pollen expressing PIs and the risks that may be associated with it. A similar scheme may be applied to other bee species, with some minor variations depending on the degree of sociality and obviously without the risks to humans from ingestion of pollen or honey.
A transgenic plant expressing a PI may affect the bee indirectly via changes in flower attractiveness due to pleiotropic or insertional mutagenesis effects (e.g., nectar volume or sucrose concentration, flower morphology), or directly via ingestion of pollen expressing the inhibitor. Nectar, as a plant secretion containing only a few amino acids,4 is unlikely to be a source of foreign proteins in transgenic plants. Of course the direct effects may be avoided altogether if the PI-gene construct has a promoter which does not allow expression in pollen. However a recent study suggests that promoters may perform differently in different plant species.5 This investigation of the activity in pollen of marker genes controlled by the CaMV 35S and nos promoters showed that both constructs were inactive in Arabidopsis but active, albeit in a highly variable fashion in tobacco. Risks to humans from the consumption of PI-containing pollen have not been investigated, but Eady and co-workers6 have shown that pollen that has been transformed
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Table 8.1. Pollination of crop plants genetically modified with proteinase inhibitor genes Plant
Pollination method(s)2
Gene(s)1
Alfalfa
Honeybees, alfalfa leafcutter bee, alkali bee
tomato PI-I,a anti-elastase from Manduca sexta
Apple
Honeybees, bumblebees, wild bees
CpTI
55
Birch
Self-pollination
potato PI-II
Cotton
Self-pollination, honeybees (widely used but not very effective)
anti-chymotrypsin, antielastase anti-trypsin from M. sexta
Lettuce
Self-pollination, honeybees PI-II, CpTI
BPTI, Potato PI-I, Potato
Oilseed rape
Self-pollination, wind, honeybees, bumblebees, beetles, flies
CpTI, OC-I, C-II, PI-I (unspecified)
Petunia
Self-pollination56
potato PI-I, BPTI
Poplar
Wind
OC-I, C-II
Potato
Self-pollination, wind, honeybees, bumblebees, beetles
CpTI, BPTI, SI, α1AT, C-II, PI-IV, SBTI
Rice
Self-pollination, wind
potato PI-II, CpTI
Strawberry
Honeybees, flies, mechanical methods
CpTI
Sunflower
Honeybees, bumblebees, flies
CpTI
Sweetpotato
Honeybees
CpTI
Tobacco
Self-pollination, honeybees, other insects, hummingbirds
CpTI, SBTI, potato PI-I, potato PI-II, BPTI, C-II, CMe, CMTI, 14K-CI, MTI-2, OC-I, PI-IV, tomato PI-I, tomato PI-II, anti-chymotrypsin, anti-elastase, anti-trypsin from M. sexta
Tomato
Self-pollination, mechanical methods, bumblebees, honeybees, other bees, thrips
CpTI, Potato PI-I (J.T. Christeller, pers. comm.), potato PI-II (J.T. Christeller, pers. comm.), tomato PI-I, tomato PI-II
White clover
Honeybees
BPTI
a Abbreviations: α AT, α-antitrypsin inhibitor; BPTI, bovine pancreatic trypsin inhibitor; C-II, 1
soybean serine proteinase inhibitor C-II; CMe, barley trypsin inhibitor; CMTI, squash trypsin inhibitor; CpTI, cowpea trypsin inhibitor; 14K-CI, bifunctional cereal inhibitor of serine proteinases and α-amylases; MTI-2, mustard serine proteinase inhibitor; OC-I, oryzacystatin I; PI, proteinase inhibitor; PI-IV, soybean serine proteinase inhibitor IV; potato PI-I, potato proteinase inhibitor I; potato PI-II, potato proteinase inhibitor II; SBTI (or SKTI), Kunitz soybean trypsin inhibitor; SI, spleen inhibitor.
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Fig. 8.2. Effects of a PI-expressing transgenic plant on honeybees. Inadvertant changes to flowers may affect foraging decisions by bees (top left hand circle). The rest of the diagram tracks the possible fate and consequences of pollen expressing a PI (circles). Outcomes that may constitute ecological risks are shown in boxes. biolistically with marker genes is capable of transient expression of these genes after storage for 6 weeks in honey. The quantities of pollen likely to be ingested with honey are so low that they pose allergenic rather than toxic risks to humans. However the risks may be greater for people consuming quantities of pollen, especially if it has been collected exclusively from transgenic crop plants. The risks of PI genes “escaping” into the environment via cross-pollination of the transgenic crop with related weedy plant species are probably not different from those associated with other classes of genes. This whole area has been the subject of a significant number of investigations and is of continuing research interest,7,8 but will not be discussed further here. The recently reported technique of chloroplast genome transformation may well provide a means of
transforming plants without altering pollen, thus avoiding gene flow from transgenic crops.9
8.2.2. Direct Effects on Honeybees Pollen provides bees with their only source of protein. Because the honeybee has a complex social structure, changes in nutrition may have far-reaching effects (see Fig. 8.2), and any study on the impact of transgenic pollen on honeybees should thus extend beyond simple tests of toxicity. Pollen is collected from flowers by foraging adult worker bees, who display obvious preferences for pollen from particular plant species.10 The basis for these foraging decisions is unclear, but in bumblebees it appears that protein availability versus foraging costs is important,11 and so one may speculate that alterations in the quality of protein in transgenic pollen may influence bee foraging behavior.
Interference of Protease Inhibitors on Non-Target Organisms
Pollen collected by these foragers is deposited in empty cells in the hive where it is further processed by other worker bees, known as “house bees.” They press the pollen into the cells and add a little honey to it, creating what is known as “bee-bread.”12 Newly-emerged adult worker bees have a particular need for this protein food in order to complete the development of their hypopharyngeal glands and their fat bodies.13 Well-developed fat bodies are thought to be necessary for winter survival of workers, but it is the hypopharyngeal glands that have particular significance. These glands secrete “larval jelly”, a protein-rich substance with which the workers feed the larvae of the colony. It may be hypothesized that worker bees that receive insufficient bee bread and fail to develop these glands would be incapable of feeding larvae, would precociously take on foraging duties and then die early. These glands also produce sugar-digesting enzymes that the bee needs for the carbohydrate metabolism necessary for flight. Larger larvae, drones and queen bees are also fed pollen directly and so the impact of PI-containing pollen on the survival and development of these stages also needs to be addressed. Effects on queen bee fertility and pheromone production may be particularly important because of their critical role in the life of the colony. Effects on drones may be less significant as these bees are generally produced “in surplus”, but a change in the quality of protein in pollen collected by bees could have consequences for the functioning of the entire colony.
8.2.3. Research on Direct Effects of PIs on Bees A number of studies have now been published describing the effects of PI ingestion on adult worker honeybees in terms of toxicity and changes in bee gut protease activity levels.14-17 There has also been some unpublished work carried out on the effects of PI ingestion on olfactory learning behavior, which is a significant component of foraging behavior in adult bees.18,19 However there
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have been no published studies of the direct effects of PIs on larvae or reproductive adult bees, or on their effects on other aspects of adult bee performance such as development or foraging activity in the field. Published studies on PI effects at the whole colony level are also lacking. Belzunces et al14 found that the BowmanBirk soybean trypsin inhibitor (BBI) fed to foraging (older) honeybees at dose levels of 1, 0.1, 0.01 or 0.001 mg/g of sugar syrup had no effect on bee survival over 4 days. However trypsin activity levels in foraging bees fed three different doses of BBI in syrup for 3.5 days were significantly different from those in control bees. The lowest BBI dose (0.001mg/ g) resulted in a slight but significant increase in trypsin activity, while the two other doses (0.1 and 1 mg/g) resulted in significant reductions in activity. In vitro tests in which enzyme extracts from control bee guts were incubated with BBI at a range of concentrations showed an 80% reduction in non-specific protease activity and a 100% reduction in trypsin activity. In our own studies on the direct effects of PIs on bees we have used newly-emerged adult bees,15-17 because ingestion of a PI expressed in pollen is most likely to affect an adult bee via a disruption of protein digestion, and because it is only during these first few days of adulthood that honeybees consume and need to digest significant amounts of this food.20 We found that, when fed to these young bees, four different serine endopeptidase inhibitors have a dose-dependent effect on bee longevity and that protease activity levels in the midgut of these bees are significantly altered by many of the PI treatments.15-17 We have found that bovine pancreatic trypsin inhibitor (BPTI) and soybean Kunitz trypsin inhibitor (SBTI, or SKTI) significantly reduce the longevity of bees fed these PIs ad lib in sugar syrup at 10, 5 or 1 mg/ml, but not at 0.1 or 0.01 mg/ml.15,16 We also determined the in vivo activity levels of three midgut endopeptidases (trypsin, chymotrypsin and elastase) and the exopeptidase leucine aminopeptidase (LAP) at two time points: the 8th day after emergence and when
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75% of bees had died. LAP activity levels increased significantly in bees fed with either inhibitor at all concentrations. At day 8, bees fed BPTI at all concentrations had significantly reduced levels of trypsin, chymotrypsin and elastase. At the time of 75% mortality, bees fed BPTI at each concentration had reduced trypsin levels, but only those fed the inhibitor at the highest dose had reduced chymotrypsin or elastase activity. At both time points, only bees fed SBTI at the highest concentration had lowered trypsin, chymotrypsin and elastase activities. These results suggest that the observed reductions in bee longevity at the higher PI dose levels are in fact the result of a disruption in their ability to digest protein. We may also speculate that the increased levels of LAP represent some kind of compensatory mechanism to make up for the loss of proteolytic function in the gut. We obtained very similar results with bees fed potato proteinase inhibitor I (PI-I) and potato proteinase inhibitor II (PI-II).17 For these experiments newly-emerged bees were fed different doses of the PIs in either sugar syrup (2 or 0.1 mg/ml) administered ad lib, or in a pollen-based food (10 or 2 mg/ g) which was replaced with control food 8 days from the start of the experiment. As in our previous experiments we determined the in vivo activities of trypsin, chymotrypsin, elastase and LAP at two time points: at day 3 (Fig. 8.3) and at day 8. Enzyme activities were significantly lower at day 8 than at day 3, except for elastase which did not change. Potato PI-II significantly reduced the activity of all endopeptidases at both time points, regardless of the dose level or the medium in which the inhibitor was administered. Potato PI-I acted in a similar manner, except that 0.01% potato PI-I in syrup had no effect on bees. There was no consistent trend in changes in LAP activity. Bees fed either inhibitor at 1% in pollen or at 0.2% in syrup had significantly reduced lifespans, with the effect of the pollen treatment being greater than the syrup treatment (Fig. 8.4). Survival of bees fed potato PI-I or potato PI-II at 0.2% in pollen or 0.01% in syrup did not differ from the controls.
Recombinant Protease Inhibitors in Plants
Girard et al21 have conducted tests of short- and long-term toxicity of BBI, oryzacystatin I (OC-I) and chicken egg white cystatin to honeybees. In the short-term test, 15-day-old worker bees were supplied with 11 µg of PI each over a period of 24 hours, and then given control syrup. None of the treatments resulted in significant bee mortality at 24, 48 or 96 hours. In the long-term test, 2-day-old bees were given a continuous supply of syrup with 26 µg/ml PI added and their longevity recorded. There was considerable variability in bee longevity in this test, but no significant effects could be attributed to the ingestion of these PIs at this low concentration and bees taken from the long-term test at 15-16 days had levels of midgut proteolytic activity that did not differ from the controls. Sandoz19 conducted further long-term tests with SBTI, OC-I, BBI and a mixture of OC-I and BBI fed continuously to 2-day-old bees at concentrations of 1, 0.1 or 0.01 mg/ml. Significant bee mortality occurred only for bees fed SBTI, BBI or the OC-I/BBI mixture at the highest dose level. We conclude from these studies that different PIs have similar effects on adult honey bees and that low doses of PIs in pollen will probably not cause mortality in bees, but may cause changes in proteolytic enzyme activity in the gut. Transgenic rice plants with a potato PI-II gene directed by a woundinducible promoter have been shown to be effective against the pink stem borer when the gene is expressed at levels equivalent to 0.5 to 2% of total soluble leaf protein.22 Furthermore, transgenic tobacco leaves expressing potato PI-II as 0.22 to 0.65% of total soluble protein significantly affected growth rates of larval green loopers.23 Levels of inhibitor expression in pollen have not yet been measured for pest-resistant transgenic PI-pla detailed studies of gene expression in transgenic pollen have involved only reporter genes such as GUS and luciferase thus far, with expression levels being recorded in terms of enzyme activity rather than as a percentage of total protein (see for instance references 24-27). Therefore, the potential exposure of bees to PIs in the field cannot yet be estimated
Interference of Protease Inhibitors on Non-Target Organisms
A)
B)
C)
D)
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Fig. 8.3. Effects of two potato PIs (potato PI-I and potato PI-II), administered at different doses in either sugar syrup or pollen-food, on honeybee digestive peptidase activities after 3 days. A) Trypsin activity levels; B) chymotrypsin activity levels; C) elastase activity levels; D) leucine aminopeptidase (LAP) activity levels. accurately. However, if it is assumed that PI expression levels in pollen may be similar to leaf levels (i.e. 0.22 to 2%), and given that bees died when fed potato PI-I or potato PI-II17 at a level equivalent to 4% of total protein (the 1% pollen treatment) but survived when fed these PIs as 0.81% of their total protein (the 0.2% pollen treatment), it would appear that bees are not likely to be exposed to lethal levels of inhibitor in the pollen of pest-resistant PI-transgenic plants. The impact of exposure to sub-lethal doses of PIs on adult honeybees is not yet known, but some studies of one component of foraging behavior, olfactory learning, have
been carried out with bees that have consumed PIs. Addition of cowpea trypsin inhibitor (CpTI) at 1, 5 or 10 µg/ml to the reward syrup offered in a conditioned proboscis extension assay significantly reduced the ability of bees to learn this response.18 In contrast, addition of BBI or cystatin at the same concentrations did not affect short- or long-term learning ability in 15-day-old bees.21 Furthermore, the learning performances of bees that had been fed ad lib with syrup containing 26 mg/ml of either OC-I or BBI for about 13 days prior to the proboscis extension assay were unaltered by this treatment.21 When bees were fed with SBTI, OC-I, BBI or a OC-I/BBI
Recombinant Protease Inhibitors in Plants
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A)
B)
Fig. 8.4. Effects of two potato PIs, PI-I and PI-II, administered at different doses in either sugar syrup or pollen-food, on survival of adult honeybees. A) Survival of bees fed potato PI-I; B) survival of bees fed potato PI-II. mixture at 1, 0.1 or 0.01 mg/ml for 15 days prior to testing, their learning ability was significantly impaired only with the 1 mg/ml BBI treatment.19
Recent work in our laboratory with bumblebees has shown that their responses to PIs may differ somewhat from those of honeybees. We fed worker adult bumblebees
Interference of Protease Inhibitors on Non-Target Organisms
with BPTI, SBTI, potato PI-I and potato PI-II at 5 different dosage levels (10, 5, 1, 0.1 and 0.01 mg/g in pollen-food). As bumblebee adults consume pollen throughout their lives, they were provided with these diets ad lib. Even with this high consumption of PI, only potato PI-I had a significant, dose-dependent effect on bee longevity (Table 8.2). Bumblebees fed with potato PI-II also displayed a dosage effect, with bees receiving high doses having significantly shorter lifespans than those receiving the lower doses. However, poor survival of the control bees in the PI-II trial meant that none of the PI-treated bees had significantly shortened lifespans. BPTI and SBTI apparently had no effect on bumblebee survival.
8.2.4. Indirect Effects on Honeybees There have been surprisingly few published studies examining the interactions between transgenic plants and pollinating insects. Most of these have been concerned with pollination as a means of gene dispersal rather than with any effects that the plants may have had on the insects themselves. Paul et al28 observed insects visiting a field plot of tobacco transformed with a kanamycin resistance marker. They found no difference in the range of animals or the frequency of visits between modified and non-modified tobacco plants. Insects observed included thrips, aphids, pollen beetles, hoverflies, butterflies, honeybees and several species of bumblebees. Scheffler et al29 evaluated pollen dispersal in oilseed rape engineered to contain a herbicide resistance (bar) gene. To ensure effective pollination, honeybee hives were placed near the field site for this study, which consisted of a 1 m-circle of non-transgenic plants located in the centre of a 9-m circle of transgenics, surrounded by 1.1 ha of non-transgenic plants. Sampling involved recording numbers of flowering plants, recording numbers and types of insect visitors and collecting seed at various distances, radiating out from the transgenic circle. Interactions between the pollinating insects and the transgenic plants were not specifically examined, but the authors noted
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that bees were sighted among the transgenic plants. Cresswell30 also studied herbicideresistant transgenic oilseed rape and quantified gene flow mediated by bumblebees. Obviously these bees were able to successfully pollinate these plants. Skogsmyr’s31 field trial examined cross-pollination as a means of gene dispersal between transgenic potatoes carrying the marker genes Npt II and GUS, and non-transgenic potatoes. This study showed no differences in the numbers of pollen beetles, flies or bumblebees observed on either type of potato. Taking a different approach, PicardNizou et al32 focused directly on the interaction between foraging honeybees and flowering transgenic oilseed rape plants. Four sets of plants were used: two different control lines and two transgenic lines derived from these, which contained a chitinase gene to confer resistance to fungal pathogens. These plants were used in experiments conducted in an indoor flight room and in an outdoor flight cage. Small colonies of honeybees were introduced into the room or cage and their foraging behavior observed in terms of general bee behavior (total number of flower visits), and individual bee behavior was recorded by videotape. The quality and quantity of nectar produced by the plants used in these experiments was also determined. No significant differences were found between the transgenic and non-transgenic plants in: the number of bee visits per 5 flowers per line, the number of flowers visited per trip, the mean times spent on the plant, the first flower or the second flower visited, or the mean number of nectar collection trials (extension of the proboscis towards the nectary) per plant. However there were significant differences among the lines in the nectar characteristics. One of the transgenic lines produced a greater volume of nectar with a higher sugar content than the others. That these phenomena were observed in only one of the transgenic lines suggests that this may have been due to an insertional mutagenesis, rather than a pleiotropic effect. A similar study has also been carried out with oilseed rape engineered with the
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Table 8.2. Median survival times in days for bumblebees fed protease inhibitors added to pollen food at different dosage levelsa PI
10 mg/g
5 mg/g
1 mg/g
0.1 mg/g
0.01 mg/g
Control
BPTIb
32 (25-76)a
82 (57-100)a
43 (34-101)a
48 (25-83)a
66.5 (25-90)a
57 (41-73)a
SBTI
18 (15-25)b
27 (25-41)ab
29 (27-73)ab
39 (32-73)ab
39 (32-101)a
38 (32-69)ab
pot PI-I
20 (18-22)d
29 (27-57)cd
61 (46-78)bc
83 (53-109)ab
62 (29-84)bc
88 (75-129)a
pot PI-II
25 (22-43)d
35 (29-50)cd
40 (29-92)bc
62 (48-114)ab
87.5 (62-115)a
36 (29-73)cd
a
Confidence intervals (at 95%) are given in parentheses. Treatments without a letter in common (across a row) differ at the 5% level (log-rank test to compare survival curves). b Abbreviations: BPTI, bovine pancreatic trypsin inhibitor; PI, proteinase inhibitor; pot PI-I, potato proteinase inhibitor I; pot PI-II, potato proteinase inhibitor II; SBTI (or SKTI), Kunitiz soybean trypsin inhibitor.
cysteine-type inhibitor OC-I (M. H. PhamDelegue, personal communication). As serine proteinases predominate in the digestive tracts of honeybees, one would not expect any direct effects of this PI on bees. One control plant line (Drakkar), a low-expressing transgenic line (70 OCI) and a highexpressing transgenic line (W OCI) were examined. Both the volume and sugar content of the nectar of the W OC-I plants were significantly higher than those of the controls. However there were no significant differences among the lines in the numbers of bee visits to flowers or the times spent by individual bees engaging in a range of component activities that constitute foraging behavior.
8.2.5. Future Research Needs Clearly there is scope for further work on PI-expressing plants and pollinating insects, in the development of both laboratory-based tests with different dosages of PIs and field- or glasshouse-based tests with the plants themselves. This research will make a valuable contribution to the formulation of meaningful protocols for testing particular
lines of plants intended for field release. The effects of PI-containing pollen ingestion on the development and behavior of worker honeybees in field hives require further examination, and the effects of PIs on the growth and survival of honeybee larvae, on the survival, fecundity and pheromone production capability of queen bees, and on the survival, fertility and mating success of drones need to be established (see Fig. 8.2). As recombinant PI-expressing plants become available, further studies on their impact on foraging honeybees will be required. If the characteristics of the pollen or flowers of these plants are altered in such a way as to alter the bee’s foraging decisions or behavior, then research will be needed to establish whether these characteristics have arisen as a consequence of a particular transformation event, or whether they will occur in every plant line created. It may be that these effects will vary depending on the plant species and PI in question, and that tests with foraging bees will need to be conducted on a case-by-case basis. However there may be some features of PI-expressing plants that
Interference of Protease Inhibitors on Non-Target Organisms
will be common to all. Further studies are needed in order to establish some guiding principles in this area. Tests with PIs and PI-expressing plants should also be extended to other species of pollinators, particularly bumblebees.
8.3. Natural Enemies There are a number of mechanisms by which transgenic plants expressing PIs may affect the natural enemies of an herbivorous target pest (Fig. 8.5). PI ingestion could induce behavioral changes in the herbivore such as increased or slower movement, that would affect the probability of attack by a predator or a parasitoid. Changes in movement could also affect the likelihood that a pest will become infected with an entomopathogen. This will vary depending on whether the pathogen is transmitted via ingestion or simple contact. PI ingestion may also prompt other behavioral changes, such as altered patterns of movement, feeding and oviposition. It is also conceivable, although perhaps not very likely, that wound responses may be altered in PI-expressing transgenic plants (endogenous PIs are often expressed in plants in response to wounding) and that this could disrupt the production of volatile chemical cues used by some natural enemies to find their prey. Physiological changes such as slower development or lower weight resulting from PI ingestion will also have an impact on natural enemy/herbivore interactions. For example, longer development times may enlarge the herbivore’s “window of vulnerability” to parasitoids or predators, or increase its exposure to pathogens. Lower herbivore weights may make them more or less attractive as prey, depending on the hunting strategy of the predator or parasitoid in question. Insects that are of smaller size because they have ingested a PI may be subject to a higher mortality rate when infected with an entomopathogen than normal-sized insects. A third mechanism by which PI-expressing plants may exert tri-trophic effects is at the population level. Increased mortality or
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reduced fecundity of the target insect will result in lower pest populations, and this in turn will affect the sizes of populations of predators, parasitoids or pathogens. The nature of any extended ecological effects will depend on the characteristics of the species involved. For example specialist predators or parasitoids may starve and generalists may move onto an alternative prey.
8.3.1. Research on the Tri-Trophic Effects of PIs Research on the impacts of PIs and PI-expressing plants on natural enemies is still at a very early stage, with only a few studies using tri-trophic systems underway. One field study with CpTI-expressing tobacco plots infested with Helicoverpa zea surveyed the prevalence of predators of the families Nabidae, Aphididae and Chrysomelidae, and found no significant differences between plots of control and transgenic plants.33 Some preliminary work has also been carried out to monitor numbers of coccinellids (ladybeetles) preying on aphids on apple. Observations in a field trial involving CpTI-expressing apple trees, control non-transgenic trees and trees expressing marker genes only suggested that ladybeetles were not affected, but low numbers limited statistical analysis (T.H. Schuler and G.M. Poppy, personal communication). A laboratory study using artificial diet with 1% BPTI added to mimic a PI-expressing transgenic plant, the herbivore Helicoverpa armigera and the generalist predator Harpalus affinis (a ground beetle) has shown that predators offered PI-fed prey for 24 hours are less and gained less weight than controls during this time. When subsequently offered control food for a further 24 hours, these beetles also ate less and lost more weight than beetles that had fed on control prey only (G. L. Lövei, personal communication). Similar studies with OC-I, which is being engineered into potatoes for L. decemlineata (Colorado potato beetle) control, and the predatory stinkbug, Perillus bioculatus have shown that the fertility of female
100
Recombinant Protease Inhibitors in Plants
Fig. 8.5. Possible effects of a PI-expressing transgenic plant on natural enemies. stinkbugs is reduced by up to 50% when they are fed chronically with potato beetles loaded with 1-16 mg OC-I/day. These stinkbugs have negligible mortality, but there are other reproductive consequences, including delayed oviposition, reduced fecundity, lower egg mass size and reduced egg hatch. The effects on oviposition could be reversed in bugs receiving low doses of OC-I in their prey by restoring an OC-I-free food supply. Although these results suggest that OC-I-expressing potatoes may have a negative effect on this predator of Colorado potato beetle, the observation that stinkbugs feeding on OC-I appeared consistently hungrier than controls suggests that predation rates would be higher than in plots of non-transgenic potatoes.34,35 In a third study, digestive proteinase activities of the two-spot ladybird Adalia bipunctata, which is an important predator of aphids, scale insects and woolly aphids were
determined and their sensitivity to a range of PIs measured.36 Cysteine proteinases were found in both adult and larval ladybirds, and the adults also possessed metalloproteinase activity. As would be expected from these results, serine PIs (SBTI, CpTI and lima bean trypsin inhibitor) had little effect on ladybird proteolytic activity, but cysteine PIs (recombinant OC-I, papaya cystatin, chicken egg-white cystatin and cowpea cysteine proteinase inhibitor) were significant inhibitors of this activity. This suggests that cysteine PI-expressing transgenic plants may have the potential to impact on this predator, either through direct ingestion of plant products (ladybirds are known to ingest pollen when prey is scarce) or indirectly, via ingestion of their aphid prey. Some work is also underway with transgenic oilseed rape plants expressing OC-I, the peach potato aphid (Myzuspersicae)
Interference of Protease Inhibitors on Non-Target Organisms
and the hymenopteran parasitoid Diaeretiella rapae in a tri-trophic system (T.H. Schuler, personal communication). A first experiment suggests that there were no significant differences in percentage parasitism of aphids, rates of parasitoid emergence, or in the sex ratios of parasitoids emerging from aphids feeding on either transgenic or control plants.
8.3.2. Research on Tri-Trophic Effects of Transgenic Plants Expressing Other Resistance Proteins Although the mechanisms by which other resistance proteins such as Bacillus thuringiensis toxins operate are different from those of PIs, there are some similarities in their effects on target insects. Thus, it may be instructive to consider natural enemy research that has been conducted with these proteins as this may suggest useful experimental approaches that could be taken with PIs. The development of Bt toxin-expressing plants is well-advanced, with commercial varieties of some plants now available. There are a number of published field studies that compare the prevalence of beneficial insect species in Bt toxin-transgenic and nontransgenic plots. For example, no significant differences were found in the numbers of six beneficial insect species (Chrysoperla carnea, Hippodamia convergans, Geocoris punctipes, Orius tristicolor, Collops vittatus and Nabis spp.) sampled from Bt toxin-cotton and control cotton plants.37 Similarly, predation and parasitism rates in the pest Ostrinia nubilalis were similar in Bt toxin-transgenic and non-transgenic corn field plots.38 A second study with Bt toxin-expressing corn confirms that the field abundance of O. nubilalis predators, Coleomegilla maculata, Orius insidiosus and C. carnea, is not altered by the transgenic crop, and shows that these insects develop and survive normally in the lab when fed corn pollen expressing Bt toxin.39 Riggin-Bucci and Gould40 found no significant differences in percentage parasitism of diamondback moth (Plutella xylostella) by Diadegma insulare or in the incidence of
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the four most abundant predators of this insect on mixed and non-mixed plots of collards (Brassica oleracea). In this study the Bt toxin insecticide was applied to some of the plants to mimic Bt toxin-expressing plants. Other studies have used more complex experimental designs to look at the interactions between Bt toxin-transgenic plants, their herbivores and various organisms operating at the third trophic level. Mascarenhas and Luttrell41 looked at the combined effects of sublethal Bt toxin-cotton and natural enemies on Helicoverpa zea. Sub-lethal doses of the toxin were administered by exposing larvae to Bt toxin-cotton for 1, 2 or 4 days before placing them in field plots comprising control cotton plants harbouring four different densities of natural enemies. H. zea survival was then checked at 24, 48 and 96 hours post-infestation. H. zea mortality increased significantly with an increase in natural enemy density, and larvae conditioned on Bt toxin-cotton had lower survival than those exposed to control cotton. Where there were no natural enemies present in the field plots larval survival was related to length of exposure to Bt-toxin cotton, in that larvae exposed for 4 days had better survival than those exposed for 1 or 2 days. In a low-density natural enemy environment, there were significant interactions between the effects of tissue genotype (Bt toxin-cotton or control plant conditioning) and length of exposure on larval survival at 48 or 96 hours postinfestation. These data show that H. zea larvae exposed to sublethal doses of Bt toxin may have slower development and be smaller than larvae provided with control cotton, and suggests that they would be more susceptible to natural enemies. Interactions between two species of parasitoid wasps (Campoletis sonorensis and Cardiochiles nigriceps), Heliothis virescens and transgenic tobacco expressing low levels of Bt toxin were also examined in field tests.42 One parasitoid, C. sonorensis, acted in synergy with the Bt toxin-expressing plants to decrease larval survival at six days; the other species, C. nigriceps, did not cause a decrease in larval
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survival and did not interact with the Bt toxin-plants. Behavioral effects were also important. The positioning of larvae on the plants affected parasitism by C. nigriceps but not by C. sonorensis. First-day parasitism of neonates by C. sonorensis was greater on control plants than on Bt toxin-tobacco, suggesting that increased movement of larvae and decreased feeding on the Bt toxin-plants resulted in larvae dispersing, and also in less volatiles being produced from plant damage sites to act as cues for these parasitoids. A number of studies have focused on the influence of natural enemies on the rate at which pest insects may adapt to Bt toxins. This is of particular concern with Bt toxin-transgenic plants, as some insects such as diamondback moth have evolved resistance to Bt biopesticides applied in field situations.43 Furthermore, lines of insects resistant to purified Bt toxins similar to the gene products expressed by Bt toxin-transgenic plants have been successfully selected in laboratory trials (e.g., ref. 44). There are no records yet of insects developing a geneticallybased resistance to PIs, but this possibility exists for any factor which causes insect mortality and thus applies selection pressure for the evolution of resistance. A form of adaptation to the effects of PI ingestion has been demonstrated in some insects where alternative digestive proteinases, resistant to the PI, are produced.45-47 It has not yet been clearly established whether these are generalized responses that occur in all insects of a particular species when exposed to a particular PI, or a trait present in only some individuals with a certain genotype. If the latter proves to be the case, then questions concerning the impact of natural enemies on the evolution of resistance to PIs will need to be addressed. Studies of this sort conducted with herbivores, natural enemies and Bt toxintransgenic plants have suggested that the effects of the enemies on resistance evolution will vary depending on the organisms involved. Heliothis virescens exposed to the fungal entomopathogen Nomurea rileyi adapted faster to Bt toxin-tobacco in three out of four replicates in one study.48 On the
Recombinant Protease Inhibitors in Plants
toxic Bt toxin-plants the non-adapted insects moved more and thus were more likely to encounter the pathogen. They also grew more slowly and so were exposed to the pathogen for longer. Thus the higher mortality induced by the pathogen in the non-adapted larvae would increase selection pressure on them and would be expected to accelerate adaptation. In contrast to this, similar experiments with the wasp parasitoid, C. sonorensis demonstrated that this natural enemy would be expected to delay the evolution of resistance to Bt toxinexpressing plants. Non-adapted insects were less likely to be parasitized than adapted insects, probably because the non-adapted larvae fed less on the toxic Bt toxin-plants, thus depriving the parasitoids of cues they use to locate them.49 Similarly, greenhouse and field studies with Bt toxin-potatoes, L. decemlineata and its predator C. maculata have suggested that this natural enemy would reduce the rate of L. decemlineata adaptation to Bt toxin if mixed plantings were used.50
8.3.3. Future Research Needs Clearly there is much scope for further research with PIs and PI-expressing transgenic plants in tri-trophic systems. It is possible that the effects of PI-fed prey on natural enemies will vary depending on the organisms involved, as studies with Bt toxins are beginning to suggest for Bt toxin-transgenic plants. A promising start has been made using laboratory-based experiments to study the effects of PI-fed prey on predators. There is also an urgent need for research on how PI ingestion by a herbivore may alter its interactions with parasitoids and entomopathogens. Further work to elucidate the mode of action of PIs may help to determine how likely the evolution of PI-resistant pest insects will be and how this may impact on the effectiveness of natural enemies as pest control agents.
8.4. Soil Fauna and Microorganisms Many plants produce endogenous PIs in response not only to wounding, but also to
Interference of Protease Inhibitors on Non-Target Organisms
plant pathogens or microbial attack (e.g., alfalfa trypsin inhibitor mRNA is thought to be synthesized in roots in response to soil microorganisms51), suggesting that in plants engineered to express exogenous PIs, interactions between the plant and beneficial microorganisms in the soil may be altered. Symbiotic microorganisms represent one group of beneficials that may be affected by the expression of PIs in transgenic plants. There are no published reports of such studies, although the availability of transformable legume species such as Lotus and white clover should facilitate these. Endogenous PIs apparently play a role in legume nodule senescence52 and thus one may suppose that exogenous PIs might disrupt or alter important processes occurring in nitrogen-fixing root nodules. Saprophytic microorganisms and soildwelling arthropods are another group of beneficial organisms which might be affected by transgenic plants expressing PIs or other transgenes.53 Research on this important topic in relation to PI-expressing plants is at an early stage. Donegan et al54 have examined the decomposition over 147 days of tobacco engineered with tomato proteinase inhibitor I by burying transgenic and control leaves in litterbags in field plots. After 57 days in the soil, PI concentration in the transgenic leaf litter was 0.05% of its original value at the beginning of the experiment. The inhibitor was undetectable in the litter after that time. Decomposition rates were similar within the transgenic and control litterbags. The carbon content of the transgenic litter was significantly lower than that of the control plant litter. Numbers of nematodes in the soil around the bags containing transgenic leaf litter were significantly higher than those around control bags, and there was also a higher proportion of fungal-feeding nematodes in the transgenic samples. There were low numbers of insects in all plots and of the microarthropods, only the Collembola showed any differences. Collembola numbers in the soil around the transgenic bags were significantly lower than those around the control bags. Protozoan populations were not
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significantly different and neither were microbial respiration rates. Even though decomposition rates were not altered by the presence of this PI in leaf litter, its persistence in the soil for 57 days may cause concern if one considers the likelihood of widespread, long-term and repeated use of transgenic crop plants. Changes in nematode communities and in Collembola populations may also have a wider ecological impact. As exogenous PIs could well be released into the soil as roots are wounded by microorganisms, or as they senesce or cells slough off, there is clearly a need for further studies on the impact of recombinant PI-expressing plants on soil biological processes.
8.5. Summary PIs are natural products found in many plants and animals, and a number of them have been shown to provide effective protection against pest attack when engineered into transgenic plants. Because of their natural abundance and diversity, PIs represent a rich source of pest- or disease-resistance genes for use in genetically engineered crops. As with any such crops, however the risks to nontarget organisms first need to be estimated with as much accuracy as possible. Experimental data on which to base such risk assessments is relatively scant at present and systems with which to test potential impacts on non-targets are only just beginning to be developed. Laboratory-based experiments with adult honeybees and bumblebees suggest that PIs, expressed at levels at which they would be effective against pests, are unlikely to cause mortality in these pollinators. However sub-lethal and colonylevel effects need to be investigated. As with any transgenic plant line pollinated by insects, variations in flower form or function that inadvertently arise during the transformation process must be checked to ensure that they do not impair pollinator performance. Natural enemy work with PI-expressing plants is less advanced and the wide array of organisms included in this category may mean that generalizations could be difficult to make. No drastic population effects were observed
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in the only field study of natural enemy abundance made so far with recombinant PI-expressing plants,33 but laboratory studies have suggested that more subtle biological effects may occur. More work with tri-trophic systems incorporating predators, parasitoids and entomopathogens is needed. Muchneeded research on the interactions between PI-expressing plants and soil fauna has also only just begun, leaving plenty of scope for further work in this ecologically important area.
Acknowledgments We thank Heather Gatehouse, Bruce Philip and Dragana Stefanovic, all of the Horticulture and Food Research Institute of New Zealand Ltd, for valuable scientific and technical input. This work was supported by grants from the New Zealand Foundation for Research, Science and Technology (contract numbers C06536 and C10639).
References 1. Schuler TH, Poppy GM, Kerry BR et al. Insect-resistant transgenic plants. Trends Biotechnol 1998; 16:168-175. 2. Crane E, Walker P. Pollination Directory for World Crops. London: International Bee Research Association, 1984. 3. James C, Krattiger AF. Global review of the field testing and commercialization of transgenic plants: 1986 to 1995—The First Decade of Crop Biotechniology. ISAAA Briefs No. 1. Ithaca NY: ISAAA 1996. 4. Baker HG, Baker I. Intraspecific constancy of floral nectar amino acid complements. Bot Gaz 1977; 138:183-191. 5. Wilkinson JE, Twell D, Lindsey K. Activities of CaMV 35S and nos promoters in pollen: Implications for field release of transgenic plants. J Exp Bot 1997; 48:265-275. 6. Eady C, Twell D, Lindsey K. Pollen viability and transgene expression following storage in honey. Transgenic Res 1995; 4:226-231. 7. Raybould AF, Gray AJ. Genetically modified crops and hybridization with wild relatives: A UK perspective. J Appl Ecol 1993; 30:199-219. 8. Hancock JF, Grumet R, Hokanson SC. The opportunity for escape of engineered genes from transgenic crops. HortScience 1996; 31:1080-1085.
Recombinant Protease Inhibitors in Plants 9. Daniell H, Datta R, Varma S et al. Genetic engineering of herbicide resistance through the chloroplast genome and prevention of outcross to weedy relatives. Nat Biotechnol 1998; 16:345-348. 10. Schmidt LS, Schmidt JO, Rao H et al. Feeding preference and survival of young worker honey bees (Hymenoptera: Apidae) fed rape, sesame, and sunflower pollen. J Econ Entomol 1995; 88:1591-1595. 11. Rasheed SA, Harder LD. Economic motivation for plant species preferences of pollencollecting bumble bees. Ecol Entomol 1997; 22:209-219. 12. Stanley RG, Linskens HF. Pollen: Biochemistry Biology and Management. Berlin: Springer-Verlag, 1974. 13. Winston ML. The Biology of the Honey Bee. Cambridge: Harvard University Press, 1987. 14. Belzunces LP, Lenfant C, Pasquale SD et al. In vivo and in vitro effects of wheat germ agglutinin and Bowman-Birk trypsin inhibitor, two potential transgene products, on midgut esterase activities from Apis mellifera. Comp Biochem Physiol 1994; 109B:63-69. 15. Malone LA, Giacon HA, Burgess EPJ et al. Toxicity of trypsin endopeptidase inhibitors to honey bees (Hymenoptera: Apidae). J Econ Entomol 1995; 88:46-50. 16. Burgess EPJ, Malone LA, Christeller JT. Effects of two proteinase inhibitors on the digestive enzymes and survival of honey bees (Apis mellifera). J Insect Physiol 1996; 42:823-828. 17. Malone LA, Burgess EPJ, Christeller JT et al. In vivo responses of honey bee midgut proteases to two protease inhibitors from potato. J Insect Physiol 1998; 44:141-147. 18. Picard-Nizou AL. Etudes comportementales des interactions chimiques abeille-plante: Application à l’évaluation de l’impact de colzas transgéniques (Brassica napus var. oleifera) sur l’abeille domestique (Apis mellifera L.). Ph.D. thesis. Paris: Université de Paris-Sud, 1992. 19. Sandoz G. Etude des effets d’inhibiteurs de protéases sur un insecte pollinisateur, l’abeille domestique Apis mellifera L. Institut National Agronomique Paris-Grignon: Diplôme d’Agronomie Approfondie thesis, 1996. 20. Crailsheim K, Stolberg E. Influence of diet, age and colony condition upon intestinal proteolytic activity and size of the hypopharyngeal glands in the honeybee (Apis mellifera L.). J Insect Physiol 1989; 35:595-602. 21. Girard C, Picard-Nizou AL, Grallien E et al. Effects of proteinase inhibitor ingestion on survival, learning abilities and digestive proteinases of the honeybee. Transgenic Res 1998; 7:239-246.
Interference of Protease Inhibitors on Non-Target Organisms 22. Duan X, Li X, Xue Q et al. Transgenic rice plants harboring an introduced potato proteinase inhibitor II gene are insect resistant. Nat Biotechnol 1996; 14:494-498. 23. McManus MT, White DWR, McGregor PG. Accumulation of a chymotrypsin inhibitor in transgenic tobacco can affect the growth of insect pests. Transgenic Res 1994; 3:50-58. 24. Twell D. Use of a nuclear-targeted β-glucuronidase fusion protein to demonstrate vegetative cell-specific gene expression in developing pollen. Plant Cell 1992; 2:887-892. 25. Twell D, Patel S, Sorensen A et al. Activation and developmental regulation of an Arabidopsis anther-specific promoter in microspores and pollen of Nicotiana tabacum. Sex Plant Reprod 1993; 6:217-224. 26. Eady C, Lindsey K, Twell D. Differential activation and conserved vegetative cellspecific activity of a late pollen promoter in species with bicellular and tricellular pollen. Plant Cell 1994; 5:543-550. 27. Hirano H, Tabayashi N, Matsumura T et al. Tissue-dependent expression of the rice wx+ gene promoter in transgenic rice and petunia. Plant Cell Physiol 1995; 36:37-44. 28. Paul EM, Lewis GB, Dunwell JM. The pollination of genetically modified plants. Acta Hort 1991; 288:425-429. 29. Scheffler JA, Parkinson R, Dale PJ. Frequency and distance of pollen dispersal from transgenic oilseed rape (Brassica napus). Transgenic Res 1993; 2:356-364. 30. Cresswell JE. A method for quantifying the gene flow that results from a single bumblebee visit using transgenic oilseed rape, Brassica napus L. cv. Westar. Transgenic Res 1994; 3:134-137. 31. Skogsmyr I. Gene dispersal from transgenic potatoes to conspecifics: A field trial. Theor Appl Genet 1994; 88:770-774. 32. Picard-Nizou AL, Pham-Delegue MH, Kerguelen V et al. Foraging behaviour of honey bees (Apis mellifera L.) on transgenic oilseed rape (Brassica napus L. var. oleifera). Transgenic Res 1995; 4:270-276. 33. Hoffmann MP, Zalom FG, Wilson LT et al. Field evaluation of transgenic tobacco containing genes encoding Bacillus thuringiensis δ-endotoxin or cowpea trypsin inhibitor: Efficacy against Helicoverpa zea (Lepidoptera: Noctuidae). J Econ Entomol 1992; 85:2516-2522. 34. Overney S, Yelle S, Cloutier C. Occurrence of cysteine digestive proteases in Perillus bioculatus, a natural predator of the Colorado potato beetle. Comp Biochem Physiol B 1998: 120:191-195.
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35. Ashouri A, Overney S, Michaud D et al. Fitness and feeding are affected in the two-spotted stinkbug, Perillus bioculatus, by the cysteine proteinase inhibitor, oryzacystatin I. Arch Insect Biochem Physiol 1998; 38:74-83. 36. Walker AJ, Ford L, Majerus MEN et al. Characterisation of the mid-gut digestive proteinase activity of the two-spot ladybird (Adalia bipunctata L.) and its sensitivity to proteinase inhibitors. Insect Biochem Mol Biol 1998; in press. 37. Flint HM, Henneberry TJ, Wilson FD et al. The effects of transgenic cotton, Gossypium hirsutum L., containing Bacillus thuringiensis toxin genes for the control of the pink bollworm, Pectinophora gossypiella (Saunders) and other arthropods. Southwest Entomol 1995; 20:281-292. 38. Orr DB, Landis DA. Oviposition of European corn borer (Lepidoptera: Pyralidae) and impact of natural enemy populations in transgenic versus isogenic corn. J Econ Entomol 1997; 90:905-909. 39. Pilcher CD, Obrycki JJ, Rice ME et al. Preimaginal development, survival, and field abundance of insect predators on transgenic Bacillus thuringiensis corn. Environ Entomol 1997; 26:446-454. 40. Riggin-Bucci TM, Gould F. Impact of intraplot mixtures of toxic and nontoxic plants on population dynamics of diamondback moth (Lepidoptera: Plutellidae) and its natural enemies. J Econ Entomol 1997; 90:241-251. 41. Mascarenhas VJ, Luttrell RG. Combined effect of sublethal exposure to cotton expressing the endotoxin protein of Bacillus thuringiensis and natural enemies on survival of bollworm (Lepidoptera: Noctuidae) larvae. Environ Entomol 1997; 26:939-945. 42. Johnson MT. Interaction of resistant plants and wasp parasitoids of tobacco budworm (Lepidoptera: Noctuidae). Environ Entomol 1997; 26:207-214. 43. Tabashnik BE. Genetics of resistance to Bacillus thuringiensis. Annu Rev Entomol 1994; 36:47-79. 44. Gould F, Anderson A, Reynolds A et al. Selection and genetic analysis of a Heliothis virescens (Lepidoptera: Noctuidae) strain with high levels of resistance to Bacillus thuringiensis toxins. J Econ Entomol 1995; 88:1545-1559. 45. Jongsma MA, Bakker PL, Peters J et al. Adaptation of Spodoptera exigua larvae to plant proteinase inhibitors by induction of gut proteinase activity insensitive to inhibition. Proc Natl Acad Sci USA 1995; 92:8041-8045.
106 46. Broadway RM. Dietary regulation of serine proteinases that are resistant to serine proteinase inhibitors. J Insect Physiol 1997; 43:855-874. 47. Wu Y, Llewellyn D, Mathews A et al. Adaptation of Helicoverpa armigera (Lepidoptera: Noctuidae) to a proteinase inhibitor expressed in transgenic tobacco. Mol Breed 1997; 3:371-380. 48. Johnson MT, Gould F, Kennedy GG. Effect of an entomopathogen on adaptation of Heliothis virescens populations to transgenic host plants. Entomol Exp Appl 1997; 83:121-135. 49. Johnson MT, Gould F, Kennedy GG. Effects of natural enemies on relative fitness of Heliothis virescens genotypes adapted and not adapted to resistant host plants. Entomol Exp Appl 1997; 82:219-230. 50. Arpaia S, Gould F, Kennedy G. Potential impact of Coleomegilla maculata predation on adaptation of Leptinotarsa decemlineata to Bt-transgenic potatoes. Entomol Exp Appl 1997; 82:91-100. 51. McGurl B, Mukherjee S, Kahn M et al. Characterization of two proteinase inhibitor (ATI) cDNAs from alfalfa leaves (Medicago sativa var. Vernema): the expression of ATI genes in response to wounding and soil microorganisms. Plant Mol Biol 1995; 27:995-1001.
Recombinant Protease Inhibitors in Plants 52. Manen J-F, Simon P, Van Slooten J-C et al. A nodulin specifically expressed in senescent nodules of winged bean is a protease inhibitor. Plant Cell 1991; 3:259-270. 53. Glandorf DCM, Bakker PAHM, Van Loon LC. Influence of the production of antibacterial and antifungal proteins by transgenic plants on the saprophytic soil microflora. Acta Bot Neerl 1997; 46:85-104. 54. Donegan KK, Seidler RJ, Fieland VJ et al. Decomposition of genetically engineered tobacco under field conditions: Persistence of the proteinase inhibitor I product and effects on soil microbial respiration and protozoa, nematode and microarthropod populations. J Appl Ecol 1997; 34:767-777. 55. Everett TH. The New York Botanical Garden Illustrated Encylopedia of Horticulture. New York and London: Garland Publishing Inc., 1981:411. 56. Huxley A, Griffiths M, Levy M. The New Royal Horticultural Society Dictionary of Gardening. London: MacMillan Press Ltd. 1992:533.
CHAPTER 9
Multiple Protease/Inhibitor Interactions in Plant-Pest Systems Savita Visal-Shah, France Brunelle and Dominique Michaud
9.1. Introduction
R
ecent advances in the field of protease inhibitor (PI)-based control of pests and pathogens revealed the high complexity of protease/inhibitor interactions in host-pest systems.1-5 Such interactions have been thoroughly studied, in particular in plant-pest systems, where dynamic processes take place between the two ‘opponents’.1,6-9 On one side the pests excrete a complex set of proteolytic enzymes with various affinities and specificities toward peptide chains to hydrolyze dietary proteins, to invade the host plant tissues or to elude the effects of defense-related proteins produced by the plant to counteract predation or infection (see Chapters 1 and 7, this volume).3,10 On the other side the plants produce a large array of defensive molecules, including a variety of proteinaceous PIs active against several types of proteases (see Chapter 6).1,11 As exemplified by several recent studies, these complex interactions are not static, but very dynamic processes where the organisms implicated rapidly adjust their metabolic machinery to the ‘molecular reactions’ of their opponent. It is now well known that predation and infection activate defense-related gene induction pathways in the host, leading to the accumulation of various defense PIs exhibiting affinity for some proteases of the invading organisms (see Chapters 2-5).1,11 To elude the effects of these defensive compounds,
the invaders have acquired the ability to produce ‘novel’ proteases or protease variants insensitive to the inhibitors expressed,2,6,7 and to overcome the inhibitory effects of PIs by altering their structural integrity.3,12,13 As a result of these coevolutive processes the interactions between proteases and PIs in plant-pest systems are very complex, and the design of effective pest control strategies based on extracellular protease inhibition is in most cases anything but simple. While only a few years ago the choice of a PI to control a given organism was still mainly based on kinetic considerations about the affinity of candidate PIs toward proteases of the target organism,14-16 it is now obvious that several additional factors like compensation to dietary protein PIs or the hydrolysis of these inhibitors by nontarget proteases contribute to determine the success of a PI-based control approach.
9.2. First-Level Interactions: The Plant-Pest Continuum In this perspective it is also evident that a correct assessment of protease/inhibitor interactions in a given plant-pest system must take into account not only the proteases sensitive to the introduced PI, but also the insensitive proteases, which retain their hydrolytic activity following the primary inhibitory reaction. Taking as a model the
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interactions between a hypothetical target pest proteolytic system composed of three extracellular proteases, P1a, P1b and P2 (where P1a and P1b belong to two different families of a same protease mechanistic class) and a candidate inhibitor, PI(1a) (which shows affinity for P1a, but not for P1b and P2), one can assume that the resulting in vivo effect of the PI against the target pest will depend not only on the inhibition of P1a, but also on the potential of P1b and P2 to compensate for the loss of P1a activity, on their ability to use PI(1a) as a substrate and thus possibly protect P1a from inhibition,3,17 and on the capacity of the insect to compensate the inhibitory effect by the production of novel proteases, sensitive (e.g., P1a) or insensitive (e.g., P1a’, P3, etc.) to PI(1a).6-8,10 While the inhibition of P1a by PI(1a) could have in some cases negative effects on the target pest, it could induce compensatory and/ or hydrolytic processes in other cases, allowing the pest to develop normally despite the inhibition of a significant fraction of its extracellular proteases. On a broader basis, the effects of the recombinant PI should even be assessed for the whole plant-pest continuum. Poorlyspecific proteases are present in plant cells, particularly in the vacuole,18,19 and interactions of these enzymes with the introduced PI after tissue alteration and cell breakage by pests or pathogens cannot be excluded. We recently observed, for instance that recombinant oryzacystatin-I (OC-I) could be accumulated under a stable form in the cytoplasm of potato leaf cells, but that it could rapidly be degraded after breakage of the foliar tissues (see Chapter 16, this volume), suggesting possible hydrolytic processes implicating potato leaf proteases during predation or infection. 20 The general occurrence of poorly-specific cysteine and aspartate proteases in the vacuole of plant cells21 could also have some influence on the recombinant PI in planta.22 Some protein PIs naturally accumulated in the vacuolar compartment of plant cells (e.g., ref. 23) could interact with the plant endogenous proteases either as substrate or as inhibitor, thereby leading to a loss of
Recombinant Protease Inhibitors in Plants
protease inhibitory potential or to a reduced fitness of the host plant. In this context, while the identification or the development of an effective PI must take into consideration the degree of affinity between this inhibitor and the pest target proteases,14,15 it also appears important to assess the interactions between the PI and the various proteases found along the plant-pest continuum, which could drastically influence the effects actually observed in vivo (see Fig. 9.1).
9.3. Multi-level Interactions: The Ecosystem A strategy recently proposed to improve the effect of recombinant PIs in pest control consists in using inhibitors with a broad spectrum of activity against the target pest proteases, thereby limiting the number of insensitive proteases available to the pest to hydrolyze the inhibitor or to compensate for inhibited proteolytic functions.3,10 In such a case an effective PI or combination of PIs could inhibit, for instance both P1a and P1b (see the model above), thus leaving to the pest only P2 plus any other induced insensitive protease(s) for hydrolyzing the host plant proteins. Based on this principle Gruden et al24 recently showed that thyroglobulin type-1 domain inhibitors, which exhibit affinity for most cysteine protease activity in the midgut of Colorado potato beetle may strongly retard the growth of newly-hatched larvae, whereas we observed that OC-I, which exhibits a limited inhibitory spectrum against the digestive proteases of this insect,25 induces compensatory processes in the larvae without causing measurable growth delays.26 While clearly establishing the potential of protein PIs in plant protection, however this control strategy based on the use of broad-spectrum inhibitors could have some drawbacks in natural conditions. Extracellular proteases are produced by a wide variety of organisms to hydrolyze dietary proteins and to compete with the other organisms in the environment (see Chapter 1). Inhibiting the digestive proteases of beneficial organisms like natural predators, parasitoids or pollinators,
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Fig. 9.1. Protease/inhibitor interactions along the plant-pest continuum. See text for details. Abbreviations: P’s: proteases; P1a, P1b, P2: proteases 1a, 1b and 2; rec PI(1a): recombinant protease inhibitor (1a), which shows affinity for P1a. the proteases released in the soil by the resident fauna and microorganisms, or those found in our own digestive system could lead to unwanted inhibitory effects susceptible to affect natural equilibria in the ecosystem, or to alter the nutritive quality of plant food products (see Fig. 9.2; also see Chapters 8, 14 and 18). Transgenic potato plants expressing OC-I, for instance induce compensatory processes in the natural predator Perillus bioculatus via its prey, the Colorado potato beetle (E. Bouchard et al, unpublished observations), in agreement with the occurrence of digestive cysteine proteinases sensitive to this poorly-efficient cystatin in the predator.27 These compensatory responses did not cause developmental delays in the predator, but it cannot be excluded that using PIs more efficient than OC-I could eventually have negative effects,28 and thus affect the effectiveness of this useful biological control mean in the field.
In this context an alternative approach could be to block proteases implicated in specific key processes, or at key steps of more general processes. For instance, while an effective way of altering dietary protein digestion in the potato beetle consists in inactivating most proteases in the midgut,24 an alternative could be to block the protease(s) that initiate the digestive process. At present, little is known about the exact sequence of reactions mediating the complete breakdown of dietary proteins in herbivorous insects, but it is assumed that the process involves different endoproteases acting sequentially on the incoming proteins, and the subsequent hydrolysis of smaller peptides by exopeptidases (see Chapter 13).29 Based on this scheme, one could speculate that inhibiting an endoprotease implicated in the first steps of dietary protein hydrolysis would have more impact against the target pest than inhibiting an exopeptidase acting in the last steps of the process, i.e., when some amino
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Recombinant Protease Inhibitors in Plants
Fig. 9.2. Protease/inhibitor interactions at the ecosystem level. See text for details. Abbreviations: P’s: proteases; P1a, P1b, P2: proteases 1a, 1b and 2; rec PI(1a): recombinant protease inhibitor (1a), which shows affinity for P1a. acids and small peptides have already been released in the midgut. Blocking the OC-Isensitive cysteine protease fraction of Colorado potato beetle digestive proteases, for instance induces compensation in the insect,26 but the overall breakdown of dietary proteins is only slightly affected,30 which suggests that the OC-I- insensitive proteases can direct dietary protein hydrolysis in the absence of the OC-I target proteases, and that OC-I interferes with an enzyme involved at the end of the process. In contrast, we recently observed that the proregion of papaya proteinase IV, a 13-kDa polypeptide showing affinity for cysteine proteases of the papain
family17,31 is not degraded by the insect digestive proteases when the cathepsin D-like protease is inhibited by pepstatin A.17 This observation, while showing the importance of the insect aspartate protease in initiating degradation of the proregion, also suggests that the inhibition of a single protease in the midgut of this insect could be sufficient to prevent the hydrolysis of at least some dietary proteins. Another example of effective ‘restricted inhibition’ was recently provided for the coleopteran insect black vine weevil (Otiorynchus sulcatus), which like the potato beetle uses OC-I-sensitive and -insensitive cysteine
Multiple Protease/Inhibitor Interactions in Plant-Pest Systems
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Fig. 9.3. Differential inhibition of Colorado potato beetle digestive cysteine proteases by cysteine PIs. Different PIs may be used to inhibit digestive cysteine proteases of the potato beetle, including protein PIs showing different inhibitory spectra against the insect cysteine (E-64-sensitive) cysteine proteases. The lines represent the inhibitory spectrum of the PI against the insect cysteine proteases extracted from insects reared on healthy potato plants (cv. Kennebec), as compared to the spectrum observed for the model inhibitor OC-I.25 All inhibition assays (except for N2) were done in reduced conditions at pH 6, with azocasein as a substrate (see ref. 32). * This study: PMC was kindly provided by Dr. Stephen Gleddie, Agriculture and Agri-Food Canada, Ottawa, Canada. CC-II was purified as a glutathione S-transferase fusion protein in Escherichia coli,37 after isolating a cc-ii cDNA by PCR from a cDNA bank prepared from immature corn grains (unpubl. data).38 Abbreviations: CC-II, corn cystatin II; E-64, trans-epoxysuccinyl- L-leucylamido-4-(guanidino) butane; HSA, human stefin A; N2, wound-induced cystatin, from soybean; OC-I, oryzacystatin I; OC-II, oryzacystatin II; PMC, potato multicystatin; TCPI, tomato cysteine proteinase inhibitor, induced by a gamma-linolenic acid treatment. proteases for dietary protein hydrolysis.32 In this case, the use of broad-spectrum cysteine PIs such as thyroglobulin type-1 domain inhibitors24 could represent an effective way to control the pest, but the inhibition of more specific proteolytic processes may also be valuable. Transgenic strawberry plants expressing the serine PI cowpea trypsin inhibitor (CpTI), for instance show adverse effects against the insect (see Chapter 2),33 despite the negligible importance of serine- type protease activity in midgut extracts of this insect.32 At this stage the exact mode of action of CpTI against the vine weevil remains to be elucidated, but it appears plausible that the target serine protease recognized by this PI
mediates a key regulatory process which, when affected, may drastically alter other metabolic processes in the target insect.
9.4. Future Perspectives In brief, the actual usefulness of a recombinant PI in plant protection depends on the complex interactions taking place between this inhibitor and the various extracellular proteases found in the biological system assessed, which in turn determine the adverse effects actually observed against the target pest and the (usually unwanted) effects that the introduced inhibitor(s) could have against the nontarget organisms. While the
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use of broad-spectrum PIs may help improve the effectiveness of this control approach in plant protection,3,10,24 it must be kept in mind that other organisms in the surrounding environment also rely on proteases for the extracellular breakdown of proteins, and that the low specificity of broad-spectrum PIs could result in adverse inhibitory effects at the ecosystem level. In this context, the use of model systems integrating not only the host plant and the pest to control, but also the other organisms found in the environment could prove of interest to assess the actual potential of protein PIs in plant protection, and to develop control strategies which are both effective and safe (see Fig. 9.2). The use of model PIs exhibiting different inhibitory spectra against the target pest proteases may also appear useful in assessing the risks associated with the use of broad-spectrum PIs. Transformation of potato with the cDNA sequence of PIs showing different inhibitory spectra against the digestive cysteine proteases of Colorado potato beetle (Fig. 9.3), for instance could help us understand complex interactions implicating proteases and their inhibitors in this particular system, and devise strategies to optimize the inhibitory effect of cysteine PI(s) in natural conditions. The potential of recombinant PIs to protect plants from pests and pathogens appears increasingly obvious; the challenge now will be to implement this approach in agricultural systems in such a way that it does not negatively destabilizes the environment.
Acknowledgments We thank Line Cantin for helpful comments on the manuscript. This work was supported by a grant from the Natural Science and Engineering Research Council of Canada.
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Recombinant Protease Inhibitors in Plants 3. Michaud D. Avoiding protease-mediated resistance in herbivorous pests. Trends Biotechnol 1997; 15:4-6. 4. Henskens YMC, Veerman ECI, Nieuw Amerongen AV. Cystatins in health and disease. Biol Chem 1996; 377:71-86. 5. Potempa J, Pavloff N, Travis J. Porphyromonas gingivalis: A proteinase/gene accounting audit. Trends Microbiol 1995; 3:430-434. 6. Broadway RM. Are insects resistant to plant proteinase inhibitors? J Insect Physiol 1995; 41:107-116. 7. Broadway RM. Dietary proteinase inhibitors alter complement of midgut proteases. Arch Insect Biochem Physiol 1996; 32:39-53. 8. Jongsma MA, Bakker PL, Peters J et al. Adaptation of Spodoptera exigua larvae to plant proteinase inhibitors by induction of gut proteinase activity insensitive to inhibition. Proc Natl Acad Sci USA 1995; 92:8041-8045. 9. Bolter CJ, Jongsma MA. Colorado potato beetles adapt to proteinase inhibitors induced in potato leaves by methyl jasmonate. J Insect Physiol 1995; 41:1071-1078. 10. Jongsma MA, Stiekema WJ, Bosch D. Combatting inhibitor-insensitive proteases of insect pests. Trends Biotechnol 1996; 14:331-333. 11. Koiwa H, Bressan RA, Hasegawa PM. Regulation of protease inhibitors and plant defense. Trends Plant Sci 1997; 2:379-384. 12. Giri AP, Harsulkar AM, Deshpande VV et al. Chickpea defensive proteinase inhibitors can be inactivated by podborer gut proteinases. Plant Physiol 1998; 116:393-401. 13. Girard C, Le Métayer M, Bonadé-Bottino M et al. High level of resistance to proteinase inhibitors may be conferred by proteolytic cleavage in beetle larvae. Insect Biochem Mol Biol 1998; 28:229-237. 14. Christeller JT, Shaw BD. The interaction of a range of serine proteinase inhibitors with bovine trypsin and Costelytra zealandica trypsin. Insect Biochem 1989; 19:233-241. 15. Urwin PE, Atkinson HJ, Waller DA et al. Engineered oryzacystatin-I expressed in transgenic hairy roots confers resistance to Globodera pallida. Plant J 1995; 8:121-131. 16. Michaud D, Cantin L, Bonade Bottino M et al. Identification of stable plant cystatin/nematode proteinase complexes using mildlydenaturing gelatin/polyacrylamide gel electrophoresis. Electrophoresis 1996; 17:1373-1379. 17. Visal S, Taylor MAJ, Michaud D. The proregion of papaya proteinase IV inhibits Colorado potato beetle digestive cysteine proteinases. FEBS Lett 1998; 434:401-405. 18. Canut H, Dupré M, Carrasco A et al. Proteases of Melilotus alba mesophyll protoplasts. Planta 1987; 170:541-549.
Multiple Protease/Inhibitor Interactions in Plant-Pest Systems 19. Storey RD. Plant endopeptidases. In: Dalling M, ed. Plant Proteolytic enzymes. Boca Raton:CRC Press, 1986:119-135. 20. Michaud D, Cantin L, Visal S et al. Differential susceptibility of oryzacystatin I and oryzacystatin II to proteolytic cleavage. Plant Physiol 1996; 111s:103. 21. Callis J. Regulation of protein degradation. Plant Cell 1995; 7:845-857. 22. Michaud D, Vrain TC, Gomord V et al. Stability of recombinant proteins in plants. In: Cunningham C, Porter AJR, eds. Recombinant proteins from plants. Production and isolation of clinically useful compounds. Totowa NJ:Humana Press 1998:177-188. 23. Wingate VPM, Franceschi VR, Ryan CA. Tissue and cellular localization of proteinase inhibitors I and II in the fruit of the wild tomato, Lycopersicon peruvianum (L.) Mill. Plant Physiol 1991; 97:490-495. 24. Gruden K, Strukelj B, Popovic T et al. The cysteine protease activity of Colorado potato beetle (Leptinotarsa decemlineata Say) guts, which is insensitive to potato protease inhibitors, is inhibited by thyroglobulin type-1 domain inhibitors. Insect Biochem Mol Biol 1998; 28:549-560. 25. Michaud D, Nguyen-Quoc B, Yelle S. Selective inhibition of Colorado potato beetle cathepsin H by oryzacystatins I and II. FEBS Lett 1993; 331:173-176. 26. Cloutier C, Fournier M, Jean C et al. Growth compensation and faster development of Colorado potato beetle (Coleoptera: Chrysomelidae) feeding on potato foliage expressing oryzacystatin I. Arch Insect Biochem Physiol 1998; 1999; 40:69-79. 27. Overney S, Yelle S, Cloutier C. Occurrence of cysteine digestive proteases in Perillus bioculatus, a natural predator of the Colorado potato beetle. Comp Biochem Physiol B 1998; 120:191-196. 28. Ashouri A, Overney S, Michaud D et al. Fitness and feeding are affected in the twospotted stinkbug, Perillus bioculatus, by the cysteine proteinase inhibitor, oryzacystatin I. Arch Insect Biochem Physiol 1998; 38: 74-83. 29. Terra WR, Ferreira C. Insect digestive enzymes: Properties, compartmentalization and function. Comp Biochem Physiol B 1994; 109:1-62.
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30. Brunelle F, Nguyen-Quoc B, Cloutier C et al. Protein hydrolysis by Colorado potato beetle, leptinotarsa decemlineata, digestive proteases: The catalytic role of cathepsin D. Arch Insect Biochem Physiol 1999; 42:88-98. 31. Taylor MAJ, Baker KC, Briggs GS et al. Recombinant proregions from papain and papaya proteinase IV are selective high affinity inhibitors of the mature papaya enzymes. Protein Eng 1995; 8:59-62. 32. Michaud D, Cantin L, Vrain TC. Carboxy-terminal truncation of oryzacystatin II by oryzacystatin-insensitive insect digestive proteinases. Arch Biochem Biophys 1995; 322:469-474. 33. Graham J, Gordon SC, McNicol RJ. The effect of the CpTI gene in strawberry against attack by vine weevil (Otiorhynchus sulcatus F. Coleoptera: Curculionidae). Ann Appl Biol 1997; 131:133-139. 34. Zhao Y, Botella MA, Subramanian L et al. Two wound-inducible soybean cysteine proteinase inhibitors have greater insect digestive proteinase inhibitory activities than a constitutive homolog. Plant Physiol 1996; 111:1299-1306. 35. Michaud D, Nguyen-Quoc B, Vrain TC et al. Response of digestive cysteine proteinases from the Colorado potato beetle (Leptinotarsa decemlineata) and the black vine weevil (Otiorynchus sulcatus) to a recombinant form of human stefin A. Arch Insect Biochem Physiol 1996; 31:451-464. 36. Visal S, Michaud D, Yelle S. Identification of a gamma-linolenic acid-induced tomato leaf cystatin-like protein with potential for biocontrol of the phytophagous pest Colorado potato beetle. Plant Physiol 1996; 111s:40. 37. Michaud D, Nguyen-Quoc B, Yelle S. Production of oryzacystatins I and II in Escherichia coli using the glutathione S-transferase gene fusion system. Biotechnol Progr 1994; 10:155-159. 38. Abe M, Abe K, Domoto C et al. Two distinct species of corn cystatin in corn kernels. Biosci Biotechnol Biochem 1995; 59:756-758.
CHAPTER 10
Using Natural and Modified Protease Inhibitors Dominique Michaud and Binh Nguyen-Quoc
10.1. Introduction
C
onsidering the high complexity of protease/inhibitor interactions in host-pest systems and the diversity of proteolytic enzymes used by pests and pathogens to hydrolyze dietary proteins or to cleave peptide bonds in more specific processes,1-5 the choice of an appropriate proteinase inhibitor (PI) or set of PIs represents a primary determinant in the success or failure of any pest control strategy relying on protease inhibition. Ideally, the choice of suitable PIs should be based on a detailed understanding of the biological system assessed. Based on our current knowledge about the use of specific inhibitors in the study and control of various metabolic pathways, one could speculate that altering the activity of a protease involved in the first steps of a protein hydrolytic process will have more impact than inhibiting a protease implicated in the last steps of this process, i.e., when the immediate intermediates of the final products are present, and/or when these intermediates may replace, at least in part, the final products (Fig. 10.1). It is well known, for instance that dietary protein hydrolysis in many organisms implicates a suite of endo- and exopeptidases acting on the proteins in a coordinated manner, resulting in the efficient release and assimilation of small peptides and amino acids essential for normal growth and development.
Logically, altering this process as soon as protein cleavage begins would probably lead to a negligible accumulation of small peptides in the surrounding medium, and thus to an efficient disruption of digestive proteolytic functions in the target pest. However, little is known until now in most systems about the exact spatio-temporal sequence of events leading to the hydrolysis of a given protein to small peptides and free amino acids. In particular, while it is well accepted that the hydrolysis of proteins into amino acids in insect midguts involves the sequential action of endo- and exopeptidases,6 the relative functions of proteinase families within these two groups of peptidases remain obscure in most cases. For instance, the aminoendopeptidase cathepsin H and the endopeptidases cathepsin B, cathepsin D and chymotrypsin are present in the midgut of Colorado potato beetle presumably to initiate dietary protein hydrolysis,7-9 but their relative importance and their specific role in the hydrolysis of dietary proteins is not yet elucidated.
10.2.The Choice of Effective Inhibitors At this point, the choice of a PI to control a given organism is based mainly on biochemical (in vitro) data allowing the identification of protease classes and families in the
Recombinant Protease Inhibitors in Plants, edited by Dominique Michaud. ©2000 Eureka.com.
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Fig. 10.1. Accumulation of the final product of a hypothetical 6-step metabolic pathway, in the presence or absence of inhibitors acting at specific levels along the pathway. Inhibition at an early step generally results in a very low accumulaton of the final product. In contrast, since the efficiency of the inhibitor is usually less than 100%, inhibition at a subsequent step leads to a certain accumulation of the product.
digestive tract of target organisms, or the preliminary characterization of proteases excreted in artificial media by bacterial or fungal pathogens. In general, quantitative assays are first carried out with specific syntetic substrates and low-molecular-weight PIs, giving a general idea of the protease types present. This approach, however, while allowing to simply assess the relative importance of the different proteases present under the conditions of the assay, is not instructive about their relative importance in vivo, and does not always take into account the dynamic nature of digestive proteolytic processes in pests and pathogens.2,10-12 In most cases, the data obtained by in vitro assays give a static image of the enzyme system targetted, useful for preliminary analyses but somewhat approximative. Our increasing knowledge on protease systems in pests submitted to various diet or stress conditions now makes possible to predict in vitro the eventual efficiency (or loss of efficiency) of PIs in target insects after they adapt their digestive proteolytic complement to the diet ingested,12 but the conclusions obtained always need to be confirmed in vivo.
Intrinsic biases associated with in vitro procedures also have to be taken into account. The experimental procedures and substrates used to measure proteolytic activities in crude extracts are in some cases not suitable for the detection of specific proteases. Caseinolytic and gelatinolytic assays with crude extracts from the coleopteran insect Otiorynchus sulcatus and the root-knot nematode Meloidogyne incognita, for instance, allowed the detection of cysteine proteinase activity in both cases, but no serine-type activity could be detected13,14 despite the reported adverse effects of serine PI (cowpea trypsin inhibitor)-expressing transgenic plants against these two pests.15,16 Nevertheless, although in vivo assays appear essential for the definitive assessment of PIs’ efficacy in real conditions, in vitro assays with diagnostic substrates and inhibitors still represent a simple and useful approach for the preliminary characterization of proteases in crude extracts of target pests. When used in combination with complementary procedures like gel electrophoresis17 and protease/PI affinity chromatography, 13,18
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these assays may also prove very useful in the study of protease-inhibitor interactions, and in the identification of proteinaceous PIs with potential in pest control. On a practical point of view, three main points should be considered when using in vitro assays to identify an inhibitor useful in pest control: 1. the specificity of the inhibitor, 2. its affinity for the target proteases, and 3. its stability in the presence of nontarget proteases.
10.2.1. Specificity The first point to consider when planning the use of PIs is the nature of proteolytic enzymes used by the target pest, which determines the type of inhibitor to be used. A wide variety of syntetic substrates and diagnostic inhibitors have been devised for the functional characterization of proteases in biological samples (see Appendix I, this volume). Some substrates are relatively non-specific and allow the detection of proteases at the class level (e.g., D-val-leu-arg-NHMec for the detection of serine proteinases,19 or Z-phe-arg-NHMec and Suc-ala-phe-lys-NHMec for cysteine proteinases20), while others allow the detection of specific proteases or protease families and appear useful in identifying particular protease species in crude extracts. In any case, it is important to keep in mind that the actual specificity of a substrate must be confirmed by complementary means for each particular biological extract. The diversity of proteolytic enzymes in living organisms and the large number of proteases still uncharacterized strongly point out the importance of adapting the experimental procedures to each system assessed. To this end, the use of low-molecularweight diagnostic PIs as a complement to syntetic substrates appears useful in the primary characterization of protease classes and families,17 and in the identification of protein PIs showing potential for their regulation (see Appendix I). Also, although little is known until now regarding the characteristics of pest and pathogen proteases, the main protease types in these organisms appear to belong to a limited number of protease families, thereby facilitating the
primary identification of potentially useful protein PIs (e.g., trypsin inhibitors against trypsin, cystatins against cathepsins B, H and L, etc.). Again, it must be kept in mind that some target proteases in pests may represent distinct sub-groups differentially affected by PIs, and that some specific inhibitors could not easily recognize variants of a well-characterized family of proteases, or the members of certain protease families within a protease class or subclass. For instance, while most cystatins exhibit affinity for cathepsin B,21 the rice PI oryzacystatin I (OC-I), which shows potential in the control of various pests, 22-25 is ineffective in inhibiting cathepsin B-like enzymes.8,26 In contrast, other plant cystatins like corn cystatin I (CC-I) and corn cystatin II (CC-II), which act on cysteine proteinases in a similar way, may exhibit measurable affinity for these proteins,27 showing the importance of assessing the effect of several PIs for each enzyme or group of enzymes to control.
10.2.2. Affinity A second point to consider when searching for effective PIs is the strength of the enzyme/PI complex formed. In vivo, the efficacy of a given PI will be ensured by its ability to rapidly and ‘irreversibly’ react with the target protease. Conversely, it is likely that a low affinity between the inhibitor and the enzyme, coupled with the presence of nontarget proteases in the surrounding medium will help the pest to easily elude the detrimental effects of the inhibitor. Considering that PIs show considerable variation in their effectiveness as inhibitors of proteases from a same family,28-30 one can also speculate that the relative potential of distinct (even related) PIs will vary, according to the ability of each inhibitor to rapidly inhibit the target proteases, and to the inherent ability of the enzyme/PI complex to remain stable in biological conditions. Based on such considerations, Christeller and Shaw28 hypothesized that the identification of protease/PI complexes with a low dissociation constant (Kd, or Ki value) may
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be a good indicator of the effectiveness of protein PIs as resistance factors in plants. Simply put, the Ki value for an enzyme/ inhibitor complex is defined by the following equation: Ki = [E] [I] [EI] where [E] represents the concentration of free enzyme in the medium, [I] the concentration of free inhibitor, and [EI] the concentration of enzyme/inhibitor complex. In competitive inhibitory reactions—like in most protease/ protein PI interactions, a low Ki value indicates a high tendency for an inhibitor and its target protease to bind rapidly and strongly to each other, while a high Ki value indicates a weak interaction between the two molecules. As shown by two recent studies using OC-I as a model inhibitor, the Ki value indeed represents a good indicator for the effectiveness of PIs in vivo. A first demonstration was provided by Urwin et al,23 who modified the cystatin by site-directed mutagenesis to obtain inhibitor variants with increased affinity for papain and related proteinases. One mutant, OC-I_D86, showed a Ki value for papain 13-fold lower than the Ki value measured for the unmodified papain/OC-I complex. When expressed in transgenic tomato plants, both recombinant OC-I and OC-I∆D86 showed adverse effects against the root parasitic nematode Globodera pallida, but the effect was more significant for the mutant form of the cystatin. In a second report, Girard et al31 assessed the interactions between recombinant OC-I and the midgut cysteine proteinases from two strains of the coleopteran insect Ceutorhynchus assimilis differentially affected by a transgenic line of rapeseed expressing OC-I. Although they showed a similar pattern of proteolytic activity and a similar level of OC-I-sensitive cysteine proteinase activity in vitro, the two strains were differentially susceptible to recombinant OC-I expressed in the plant. As shown by complex dissociation studies in mild denaturing conditions,32 the non-affected strain possessed a cysteine proteinase weakly inhibited by OC-I. Conversely, the corresponding
proteinase in the susceptible strain was strongly inhibited by the same cystatin, thus confirming the effectiveness of PIs forming strong protease/PI complexes in vivo, and the usefulness of Ki values obtained in vitro as an indicator of PI effectiveness in transgenic systems. These observations also suggest the importance of measuring the Ki value for each particular protease/PI complex to be studied. The ‘general’ inhibitory effect of Kunitz-type serine PIs against trypsin-like proteinases, or the effect of cystatins against papain-like enzymes, for instance are well established, but important variations are noted between Ki values of the various complexes.14,26-29,32 Ki values measured for the interaction between various proteinaceous serine PIs and bovine trypsin, notably, drastically differ from those observed with the same inhibitors against a trypsin-like digestive enzyme of the grass grub Costelytra zealandica, with up to 1000-fold differences in Ki values measured for PIs of a same family.28 Several experimental procedures may be used to estimate Ki values, provided that the target proteases and candidate PIs are available under a purified (or semi-purified) form (see for instance refs. 33 and 34; also see: ). To simply obtain an idea about the relative strength of different protease/PI complexes without the need to purify the target proteases, one can also take advantage of the ability of strong protease/inhibitor noncovalent complexes to remain stable in mildly-denaturing (SDS-containing) electrophoretic systems.14,31,32,35,36 This approach may prove particularly useful, notably, in assessing the potential of disulfide bondlacking cystatins to strongly inhibit cysteine proteinases.14,32 In the presence of SDS, weak proteinase/cystatin complexes (with Ki values in the micromolar range or higher) are completely dissociated following gel electrophoresis, while complexes with lower Ki values remain partially or completely stable under the same conditions (see Fig. 10.2 and Table 10.1).
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Fig. 10.2. Rough estimation of Ki values for cysteine proteinase/cystatin complexes using mildly-denaturing gelatin-PAGE.32 PIs and target proteases are first incubated in mild conditions to allow formation of the enzyme/inhibitor complex, and then resolved by mildly-denaturing gelatin/SDS-PAGE. Strong protease/PI complexes remain stable during the process, while the activity of the proteases is completely or partly restored for weak or moderately-strong complexes, respectively. See ref. 32 for experimental details.
10.2.3. Stability While the ability of a PI to inhibit target proteases of the pest to control obviously appears essential, it does not ensure its actual efficacy in vivo. When proposing the use of Ki values as a key indicator for the potential of PIs in pest control, Christeller and Shaw also assessed the importance of PI stability during the inhibitory process.28 By using in vitro assays and appropriate detection procedures, they clearly showed that the PIs tested were hydrolyzed by nontarget proteases present in the surrounding medium, but that the initial levels of inhibition were maintained throughout the incubation period. Considering these facts, the authors suggested that the degradation of free PI molecules in insect midguts would have little impact on the efficiency of the whole inhibitory process. Although similar observations were made recently with midgut extracts of other insects,13,18 it must be kept in mind that protease/PI interactions in plant-pest systems are very dynamic processes involving complex compensatory processes (see chapter 9, this volume).2,10,37-39 In particular, it is now well established that herbivorous pests may easily adapt their digestive proteolytic system to the diet ingested.2,10-12,38,39 In such conditions, it appears plausible that the primary inhibition of specific target proteinases in the
midgut of a given pest will activate compensatory processes leading to the synthesis of novel proteases (e.g., refs. 37 and 39). As a consequence, an inhibitor susceptible to the action of nontarget proteases will rapidly lose its inhibitory potency in real conditions, as its concentration in the midgut will tend to decrease.3 The first study clearly showing the occurrence of such protease-mediated resistance to PIs in herbivorous pests was reported by Orr et al,40 who assessed the potential of cystatins in controlling the southern corn rootworm, Diabrotica undecimpunctata. Inclusion of different cystatins in the diet resulted in quite varying effects on growth of the insect, although all inhibitors caused significant inhibition of the insect proteases in vitro. Interestingly, the addition of a carboxypeptidase inhibitor from potato in the diet restored the inhibitory activity of the less effective cystatins and the ability of these inhibitors to alter the growth of the target pest.40 Such a differential susceptibility of cystatins to degradation by insect nontarget proteases was also reported for two other coleopteran insects, Otiorynchus sulcatus and Leptinotarsa decemlineata. 13,18 Nontarget midgut proteases of O. sulcatus were shown
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Table 10.1. Estimating the strength of cysteine proteinase-OC-I and -OC-II complexes using mildly-denaturing gelatin/SDS-PAGE: Some recent examples Source
Protease
Affinitya
Reference
OC-I OC-II Insects
Ceutorhynchus assimilis
P22 (strain CaP) P22 (strain CaR)
strong n.d. weak n.d.
31 31
Leptinotarsa decelineata
—
high
n.d.
12
Otiorynchus sulcatus
Osp4 Osp5
moderatemoderate high high
13 13
Tetranychus urticae
Tup2
strong weak
32
Amblyseius fallacis
—
strong strong
32
Meloidogyne hapla
Mhp1
strong weak
14
M. incognita
Mip1
weak
strong
14
M. javanica
Mjp1
weak
strong
14
Acarians
Nematodes
a A weak affinity is for high K values (e.g. higher than 1 µM); a strong affinity is for low K values i i (e.g. 1 nM or lower); a moderate affinity is for intermediate Ki values (e.g. between 10 nM and 1
µM), as estimated by gelatin-PAGE (see Fig. 10.2 and ref. 32); n.d., not determined.
to differentially affect the structure of the two rice cystatins OC-I and oryzacystatin II (OC-II).13 While OC-I was insensitive to the action of these proteases, OC-II was subjected to limited proteolysis at about 1.5 kDa of its carboxy-terminal extremity, leading to the rapid accumulation of a ~10.5-kDa truncation fragment, active but subsequently hydrolyzed by the insect proteases.13 Similarly, human stefin A and the proregion of papaya proteinase IV, two inhibitors of cysteine proteinases, appeared susceptible to the action of L. decemlineata nontarget proteases.12,18 In contrast with OC-II, no detectable intermediate was observed during the hydrolysis of HSA, suggesting the presence of multiple
cleavage sites at the surface of this PI. Along with other recent articles reporting the degradation of proteins PIs by the proteases of insects and pathogens,41-45 these observations strongly suggest that protease-mediated resistance to protein PIs is widespread among pests, and confirm the importance of finding PIs able to keep their structural integrity in biological environments where complex proteolysis events occur.3
10.3. The Design of Hybrid Inhibitors In brief, a good PI in pest control should be stable, possess an adequate inhibitory spectrum against the pest protease system, and
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bind tightly to the target proteases.3 Given the large diversity of PIs in nature, the rapid developments in our understanding of protease/PI interactions in biological systems and the increasing number of PI-encoding cDNA sequences available in gene databanks (see Appendix II, this volume), it appears obvious that plants and living organisms in general represent a quite valuable source of effective PIs useful in designing strategies to regulate almost any target protease. With the recent methodological developments in genetic and protein engineering, it is now also feasible to ‘adapt’ natural PIs for the inhibition of specific protease systems. In particular, the recent developments in protein engineering make possible the development of inhibitors with improved binding properties, with an altered specificity, or with a broader inhibitory spectrum against specific proteases or protease complements. Currently, two main approaches are used to engineer protein PIs. One approach consists to create a phage combinatorial library in which a wide variety of mutant inhibitors with varying kinetic characteristics are expressed at the surface of phage particles propagated in Escherichia coli, and then selected by appropriate means (see Chapter 11, this volume).46-48 The second approach consists in the creation of hybrid (or chimeric) PIs by ‘directed’ mutagenesis, based on the intrinsic structural and functional characteristics of natural PIs. By analogy with the term ‘hybrid enzyme,’ which is defined as an enzyme integrating structural elements of more than one enzyme,49 a hybrid PI may be defined as an inhibitor integrating structural elements of a second inhibitor or of a distinct defense-related protein. The bifunctional inhibitor from Ragi (Eleusine coracana), for instance, might be seen as a natural hybrid PI, as it possesses two completely independent inhibitory binding sites, one for trypsin, the other for α-amylase.50 In the lab, hybrid PIs can be generated following three main schemes:
mutagenesis, based on the characteristics of other inhibitors; 2. secondary-structure elements or whole functional domains of a PI can be grafted to a second inhibitor, these polypeptide chains serving as functional modules to give the ‘receptor’ protein an additional function; or 3. distinct PIs can be fused to each other or to a different defense protein, directly or with the addition of an appropriate amino acid spacer (see Fig. 10.3).
1. the primary structure of an existing PI can be slightly altered by one or several single point mutations by site-directed
10.3.1. Site-Directed Mutagenesis of Natural PIs Usually, protein PIs of a given class share a common mechanism of interaction with their cognate proteases. For instance, the P1 residue of serine PIs interacts with serine proteinases as a pseudo-substrate, its side chain becoming imbedded in the S1 cavity of the target enzyme. The nature of the P1 residue in serine PIs—also called the primary specificity residue—strongly determines the specificity of the inhibitor and its affinity for serine proteinases.51 Natural protein inhibitors of trypsin, for instance, possess in most cases an arginine or a lysine residue at the P1 position, while chymotrypsin inhibitors possess a leucine, a methionine, or an aromatic residue. Noteworthy, while P1 and the six or seven surrounding residues form a chain with conserved geometry among serine PIs,52 the remainder part of the molecule, or scaffold, is variable. Protein serine PIs consist in several different families, each characterized by a scaffold conserved within the family, but distinct from those of the other families. The main function of the scaffold would be to serve as a template for supporting the residues that interact with the target enzyme in the proper orientation.53 Based on these facts, early works by Michael Laskowski, Jr. and his colleagues showed that changing the residue arg-63 of soybean Kunitz trypsin inhibitor (i.e., the P1 residue) by a lysine residue does not affect the specificity of this inhibitor toward trypsin, while switching the same residue to tryptophan,
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Fig. 10.3. The design of hybrid PIs. 1) Site-directed mutagenesis: the primary structure of an existing PI can be slightly altered by one or several single point mutations by site-directed mutagenesis, based on the characteristics of a second other inhibitor; 2) grafting of functional domains: a secondary-structure element or a whole functional domain of a PI can be grafted to a second inhibitor, serving as a functional module to give the ‘receptor’ protein an additional function; or 3) distinct PIs can be fused to each other (or to a different defense protein), directly or with the addition of an appropriate amino acid spacer. an aromatic amino acid, renders the inhibitor active against chymotrypsin (reviewed in ref. 51). In a recent study, Jiang et al 54 changed the P1 (ala) residue of serpin-1B, a serine PI found in the hemolymph of the lepidopteran insect Manduca sexta, to lysine or to phenylalanine, and showed that changing P1 to lysine results in increased affinity for trypsin, while the switch to phenylalanine makes the inhibitor active against chymotrypsin. In agreement with the current paradigm stating the importance of P1 in serine PIs’ specificity, recent studies also demonstrated the importance of this residue for other serine-type inhibitors (e.g., refs. 55 and 56), suggesting the potential of sitedirected mutagenesis to easily modulate the specificity of serine PIs implicated in various biological systems. In contrast, changing more subtle characteristics may appear complex at this point. In most cases the formation of a complex between two proteins requires a large number of small rearrangements in both
structures, and minor changes in the sequence of a PI or its target protease may have a significant influence on the resulting inhibitory effect. Using human pancreatic secretory trypsin inhibitor as a model, Szardenings et al57 showed that the fine-tuning improvement of strong interactions between this inhibitor and serine proteinases may be difficult without the availability of a structural model for the protease/PI complex assessed. Primary sequence-based considerations allowed these authors to produce inhibitors with increased affinity for human leukocyte elastase, but the best inhibitor variants, developed with as little alterations as possible in the primary sequence, could only be designed using a 3-D structural model. In a similar attempt, Urwin et al 23 improved the binding capacity of OC-I toward papain and gcp-1, a cysteine proteinase of the free-living nematode Caenorhabditis elegans, by generating several variants of the inhibitor by site-directed mutagenesis. To assist in the design of their experiments, they first
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developed a structural model for OC-I, based on cystatin primary sequence alignments and on structural data available about the stefin B/papain complex.58 This hypothetical model was then used to predict changes that could be made in the inhibitor to alter the orientation of its amino acid side chains within the active site of cysteine proteinases. While most of the variants exhibited unchanged or decreased affinity for papain, one mutant, OC-I_D86 (i.e., OC-I lacking the asp-86 residue) showed increased affinity both for this enzyme and for gcp-1. Interestingly, OC-I_D86 expressed in tomato caused detrimental effects against the cyst nematode Globodera pallida greater than those observed with OC-I, tending to confirm the importance of using PIs with strong inhibitory activity in biological conditions (see Section 10.2.2). Despite these developments, however the actual potential of site-directed mutagenesis still appears limited for the improvement of protein PIs, due to the lack of structural data about most protein PIs and proteases implicated in biological processes (see Appendix III for a list of available structural data). The folding mechanisms giving natural proteins their structure remain poorly understood, and a generally-applicable strategy allowing to effectively predict complex folding events based on polypeptide sequences still appears out of reach.59 The complex mode of action of certain PIs against proteases also makes difficult the rationale design of effective mutagenesis strategies. Cystatins, for instance, possess three conserved regions essential for their activity.58,60,61 The first region consists in a loop including the sequence gln-X-val-X-gly (residues gln-53 to gly-57 in chicken egg cystatin), where X may be any amino acid; the second region corresponds to an N-terminal glycine residue (gly-9 in egg cystatin), which helps this part of the protein interact efficiently with the target proteinase; finally the third region consists in a hairpin loop with two conserved residues, pro-103 and trp-104 (egg cystatin-numbering), present at the C-terminal extremity of the inhibitor. During the inhibitory process, these three regions form
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a tripartite wedge which enters the active site of the target enzyme and causes inhibition.61,62 In contrast with serine PIs, several residues present on three different domains thus interact with each other to determine the specificity and the affinity of cystatins toward cysteine proteinases. As exemplified with OC-I,23,63,64 relatively small changes in the primary sequence of cystatins may significantly alter their affinity for cysteine proteinases, but our current knowledge on the structure of most cystatin/proteinase complexes renders difficult the rationale use of directed mutagenesis to tailor the activity of cystatins—and protein PIs in general—toward specific, in most cases poorly characterized target proteases. For instance, one cannot be sure that improving the affinity of a given PI (e.g., OC-I) toward a given proteinase (e.g., papain) based on hypothetical models will necessarily result in an increased affinity of this inhibitor for every other proteinase of the same family. In particular, hypothetical models based on model protease/PI complexes do not explain the differential affinities noted between related PIs (e.g., the rice inhibitors OC-I and OC-II) and the cysteine proteinases of closely-related pests (e.g., those of the root-knot nematodes Meloidogyne hapla, M. incognita and M. javanica).14 At this stage, simply screening for efficient natural PIs14,32 (see above, Section 10.2) or for highly efficient variants generated by empirical approaches like phage display48,65 (see Chapter 11) may prove appropriate for one wishing to identify PIs with strong affinity toward specific proteinases. For instance, while OC-I shows limited affinity for Mip1, the major extracellular cysteine proteinase of M. incognita, OC-II appears highly effective in inhibiting this particular proteinase (with a Ki value ~100-fold lower than with OC-I), leading to the formation of a complex stable in the presence of SDS.14 Similarly, variants of a soybean cystatin showing strong activity against papain have been easily identified and isolated by phage display using this particular proteinase as an affinity ligand.65
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10.3.2. The Transfer of Functional Domains to Natural PIs As a complement to site-directed mutagenesis, the design of novel proteins incorporating an appropriate scaffold (or template) and useful structural/functional motifs appears promising.59,66 As recently pointed out by Imperiali and Ottesen,67 the architecture of proteins shows remarkable structural and functional versatility. Despite the large number of amino acids present in proteins, only a limited number of residues are responsible for their biological activity. Like for protein PIs (see above, Section 10.3.1), most proteins consist in one or a few functional domains maintained in the proper orientation by the remainder part of the protein, or scaffold. As suggested by several recent studies,49,59,67,68 ‘grafting’ functional domains to small scaffolds allows the creation of small proteins (or miniproteins) reproducing the functions of much larger proteins.68 In this perspective, protein domains and scaffolds may be seen as building blocks useful in engineering chimeric proteins with desirable characteristics. A variety of proteins can serve as scaffolds, provided that they allow appropriate spatial orientation of the attachment sites for the functional domains.59 Artificial peptide templates with built-in devices for intramolecular folding have been developed and used to stabilize specific secondary structures;59,69 natural scaffolds, for instance small proteins containing several disulfide bonds may also be quite useful (see ref. 68 for a review). Interestingly, protein PIs may be useful either as templates and as ‘grafted domains’. By fusing the four C-terminal residues of potato carboxypeptidase inhibitor, which enter the active site of carboxypeptidase A to cause inhibition, to the squash trypsin inhibitor EETI-II, Le-Nguyen et al70 obtained a bifunctional inhibitor showing affinity for both trypsin and carboxypeptidase A. Similarly, inserting an elastase-binding loop into interleukin-1ß resulted in a mutant protein with anti-elastase activity,71 showing the potential of PI inhibitory loops as building blocks in miniprotein design. As suggested by
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these studies, the use of protein PIs as stable scaffolds for the introduction of additional PI functions, or for the grafting of other useful domains to natural PIs might prove particularly suitable for the creation of novel proteins adapted to specific biotechnological applications. In particular, the design of multifunctional PIs may prove particularly useful to extend the inhibitory spectrum of PIs against given pest protease complements, or to stack useful defensive protein functions within a single polypeptide chain.
10.3.3. The Construction of PI-Containing Fusion Proteins The simple fusion of distinct defense proteins (including PIs) appears interesting for the same reasons. Several authors suggested that two or more distinct defense proteins used in combination could have more impact on target pests than the use of only one of these proteins (see ref. 72). Oppert et al,73 for instance showed that a combination of serine and cysteine PIs in the diet causes important detrimental effects to larvae of the coleopteran insect, Tribolium castaneum when tested at levels where the individual inhibitors were nontoxic. Similarly, Orr et al40 reported that the adverse effects of certain cystatins against the southern corn rootworm, D. undecimpunctata are observed only if a ‘companion’ PI is included in the diet to protect their structural integrity (see Section 10.2.3). Based on such observations, it is now suggested that pest control strategies relying on extracellular protease inhibition will be effective only if compensatory and PI hydrolysis processes in the target pest are minimized, for instance by using broad-spectrum inhibitors (see Chapter 9, this volume).3,74 In this context, any attempt to create fusion PIs with an inhibitory spectrum broader than the spectrum of the individual PIs may appear interesting. As described above (see Section 10.3.2) the functional domains of protein PIs can be used as template or scaffold for the design of miniproteins, showing the rigidity of their structural/functional elements. As shown notably for OC-I and cystatins in general,18,75-78 PIs expressed
124
as fusion proteins should also keep in most cases their inhibitory activity despite the presence of a large polypeptide chain at their N- or C-terminal extremity. With the aim of developing a bi-functional PI useful in nematode pest control, Urwin et al79 recently reported the design of dual serine/cysteinetype PIs including cowpea trypsin inhibitor (CpTI) and the OC-I mutant, OC-I-∆D86, separated by two different polypeptide linkers, one susceptible to proteolysis, the other presumably resistant. When delivered to the plant parasitic nematode Heterodera schachtii via transgenic plants, the recombinant fusion showed additive detrimental effects against the nematode, as compared to the effects observed for the inhibitors expressed alone in the plant. Although several questions remain about the actual structural characteristics of these CpTI/ OC-I∆D86 fusions and about their exact mode of action against the nematodes, this study provided a first evidence supporting the potential of PI fusions in plant protection.
10.4. Conclusion In summary, it appears that several criteria must be taken into account to identify PIs effective in the control of specific pests. As a result of co-evolutive processes, pests and pathogens have aquired the ability to elude, at least partially, the adverse effects of PIs found in their hosts by producing ‘insensitive’ proteinases after PI ingestion,2 by using proteinases poorly-sensitive to the host PIs,31 or by hydrolyzing potentially ‘harmful’ inhibitors.3 Although PIs with improved binding characteristics are now isolated or developed on a regular basis, any strategy aimed at controlling pests and pathogens using these molecules will be successful and durable to the extent that the complex protease/PI interactions taking place in these systems are at least partially elucidated. Several approaches are currently being considered for the improvement of PI-based control strategies, including the identification of natural PIs with strong inhibitory activity against pest proteinases, 80-82 the use of protease pro-regions as a complement to ‘real’ PIs,12,83 the design of improved mutant PIs
Recombinant Protease Inhibitors in Plants
by site-directed mutagenesis,23 and the isolation of effective inhibitor variants by phage display.48,65 Considering the major recent developments in the elucidation of defenserelated signaling pathways in living cells, and the rapid progress in our understanding of protein structure-function relationships, one can expect that efficient protein PIs will soon be available for the inhibition of several specific proteases, and that the time needed to design strategies for the effective inhibition of newly-identified target proteases will tend to decrease. Despite these promising developments, however, it should be kept in mind that our actual knowledge on protein folding behavior and our comprehension of protein-protein interactions are still rudimentary. As suggested by Corey and Corey,84 the most fruitful way of designing useful protein variants at this stage may be to incorporate both iterative selection strategies and rationally chosen small perturbations, superimposed on frameworks designed by nature.
Acknowledgments We thank Line Cantin for helpful comments on the manuscript. This work was supported by an operating grant from the Natural Science and Engineering Research Council of Canada.
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7. Thie NM, Houseman JG. Identification of cathepsin B, D and H in the larval midgut of Colorado potato beetle, Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae). Insect Biochem 1990; 20:313-318. 8. Michaud D, Nguyen-Quoc B, Yelle S. Selective inhibition of Colorado potato beetle cathepsin H by oryzacystatins I and II. FEBS Lett 1993; 331:173-176. 9. Novillo C, Castanera P, Ortego F. Characterization and distribution of chymotrypsinlike and other digestive proteases in Colorado potato beetle larvae. Arch Insect Biochem Physiol 1997; 36:181-201. 10. Broadway RM. Are insects resistant to plant proteinase inhibitors? J Insect Physiol 1995; 41:107-116. 11. Overney S, Fawe A, Yelle S et al. Diet-related plasticity of the digestive proteolytic system in larvae of the Colorado potato beetle (Leptinotarsa decemlineata Say). Arch Insect Biochem Physiol 1997; 36:241-250. 12. Visal S, Taylor MAJ, Michaud D. The proregion of papaya proteinase IV inhibits Colorado potato beetle digestive cysteine proteinases. FEBS Lett 1998; 434:401-405. 13. Michaud D, Cantin L, Vrain TC. Carboxyterminal truncation of oryzacystatin II by oryzacystatin-insensitive insect digestive proteinases. Arch Biochem Biophys 1995; 322:469-474. 14. Michaud D, Cantin L, Bonade Bottino M et al. Identification of stable plant cystatin/nematode proteinase complexes using mildlydenaturing gelatin/polyacrylamide gel electrophoresis. Electrophoresis 1996; 17:1373-1379. 15. Graham J, Gordon SC, McNicol RJ. The effect of the CpTI gene in strawberry against attack by vine weevil Otiorhynchus sulcatus F. (Coleoptera: Curculionidae). Ann Appl Biol 1997; 131:133-139. 16. Hepher A, Atkinson HJ. Nematode control with proteinase inhibitors. European Patent Application 92301890.7, 1992; Pub 0502730 A1. 17. Michaud D. Gel electrophoresis of proteolytic enzymes. Anal Chim Acta 1998; 372:173-185. 18. Michaud D, Nguyen-Quoc B, Vrain TC et al. Response of digestive cysteine proteinases from the Colorado potato beetle (Leptinotarsa decemlineata) and the black vine weevil (Otiorynchus sulcatus) to a recombinant form of human stefin A. Arch Insect Biochem Physiol 1996; 31:451-464. 19. Clarke GA, Proctor GB, Garrett JR et al. Effects of inhibitors on proteinase activities in gels following isoelectric focusing. Appl Theoret Electrophor 1990: 1:201-204.
20. North MJ, Robertson CD, Coombs GH. The specificity of trichomonad cysteine proteinases analyzed using fluorogenic substrates and specific inhibitors. Mol Biochem Parasitol 1990; 39:183-194. 21. Barrett AJ, Rawlings ND, Davies ME et al. Cysteine proteinase inhibitors of the cystatin superfamily. In: Barrett AJ, Salvesen G, eds. Proteinase inhibitors. Amsterdam: Elsevier 1986;515-569. 22. Leplé JC, Bonadé-Bottino M, Augustin S et al. Toxicity to Chrysomela tremulae (Coleoptera: Chrysomelidae) of transgenic poplars expressing a cysteine proteinase inhibitor. Mol Breeding 1995; 1:319-328. 23. Urwin PE, Atkinson HJ, Waller DA et al. Engineered oryzacystatin-I expressed in transgenic hairy roots confers resistance to Globodera pallida. Plant J 1995; 8:121-131. 24. Kuroda M, Ishimoto M, Suzuki K et al. Oryzacystatins exhibit growth-inhibitory and lethal effects on different species of bean insect pests, Callosobruchus chinensis (Coleoptera) and Riptortus clavatus (Hemiptera). Biosci Biotechnol Biochem 1996; 60:209-212. 25. Michaud D, Vrain TC. Expression of recombinant proteinase inhibitors in plants. In: Cunningham C, Porter AJR, eds. Recombinant proteins from plants: Production and isolation of clinically useful compounds. Totowa NJ:Humana Press Inc 1998; 49-64. 26. Kondo H, Abe K, Nishimura I et al. Two distinct cystatin species in rice seeds with different specificities against cysteine proteinases. Molecular cloning, expression, and biochemical studies on oryzacystatin-II. J Biol Chem 1990; 265:15832-15837. 27. Abe M, Abe K, Domoto C et al. Two distinct species of corn cystatin in corn kernels. Biosci Biotechnol Biochem 1995; 59:756-758. 28. Christeller JT, Shaw BD. The interaction of a range of serine proteinase inhibitors with bovine trypsin and Costelytra zealandica trypsin. Insect Biochem 1989; 19:233-241. 29. Christeller JT, Gatehouse AMR, Laing WA. The interaction of the elastase inhibitor, eglin c, with insect digestive endopeptidases: Effect of pH on the dissociation constants. Insect Biochem Mol Biol 1994; 24:103-109. 30. Laskowski M, Jr, Kato I, Ardelt W et al. Ovomucoid third domains from 100 avian species: Isolation, sequences, and hypervariability of enzyme-inhibitor contact residues. Biochemistry 1987; 26:202-221. 31. Girard C, Bonadé-Bottino M, Pham-Delegue M-H et al. Two strains of cabbage seed weevil (Coleoptera: Curculionidae) exhibit differential susceptibility to a transgenic oilseed rape expressing oryzacystatin I. J Insect Physiol 1998; 44:569-577.
126 32. Michaud D, Cantin L, Raworth DA et al. Assessing the stability of cystatin/cysteine proteinase complexes using mildly-denaturing gelatin/polyacrylamide gel electrophoresis. Electrophoresis 1996; 17:74-79. 33. Salvesen G, Nagase H. Inhibition of proteolytic enzymes. In: Beynon RJ, Bond JS, eds. Proteolytic enzymes—a practical approach. New York: IRL Press 1989:83-104. 34. Dixon M, Webb EC. Enzyme inhibition and activation. In: Dixon M, Webb EC, eds. Enzymes. London: Longmans Co. 1979:332-380. 35. Nawata S, Nakamura K, Tanaka T et al. Electrophoretic analysis of the ‘cross-class’ interaction between novel inhibitory serpin, squamous cell carcinoma antigen-1 and cysteine proteinases. Electrophoresis 1997; 18:784-789. 36. Ware JH, Wan XS, Rubin H et al. Soybean Bowman-Birk protease inhibitor is a highly effective inhibitor of human mast cell chymase. Arch Biochem Biophys 1997; 344:133-138. 37. Broadway RM, Duffey SS. Plant proteinase inhibitors: Mechanism of action and effect on the growth and digestive physiology of larval Heliothis zea and Spodoptera exigua. J Insect Physiol 1986; 32:827-833. 38. Broadway RM. Dietary proteinase inhibitors alter complement of midgut proteases. Arch Insect Biochem Physiol 1996; 32:39-53. 39. Jongsma MA, Bakker PL, Peters J et al. Adaptation of Spodoptera exigua larvae to plant proteinase inhibitors by induction of gut proteinase activity insensitive to inhibition. Proc Natl Acad Sci USA 1995; 92:8041-8045. 40. Orr GL, Strickland JA, Walsh TA. Inhibition of Diabrotica larval growth by a multicystatin from potato tubers. J Insect Physiol 1994; 40:893-900. 41. Grenier D. Degradation of host protease inhibitors and activation of plasminogen by proteolytric enzymes from Porphyromonas gingivalis and Treponema denticola. Microbiology 1996; 142:955-961. 42. Abrahamson M, Wikström M, Potempa J et al. Modification of cystatin C activity by bacterial proteinases and neutrophil elastase in periodontitis. J Clin Pathol Mol Pathol 1997; 50:291-297. 43. Blankenvoorde MFJ, Henskens YMC, van’t Hof W et al. Inhibition of the growth and cysteine proteinase activity of Porphyromonas gingivalis by human salivary cystatin S and chicken cystatin. Biol Chem 1996; 377:847-850. 44. Giri AP, Harsulkar AM, Deshpande VV et al. Chickpea defensive proteinase inhibitors can be inactivated by podborer gut proteinases. Plant Physiol 1998; 116:393-401.
Recombinant Protease Inhibitors in Plants 45. Girard C, Le Métayer M, Bonadé-Bottino M et al. High level of resistance to proteinase inhibitors may be conferred by proteolytic cleavage in beetle larvae. Insect Biochem Mol Biol 1998; 28:229-237. 46. Scott JK, Smith GP. Searching for peptide ligands with an epitope library. Science 1990; 249:386-390. 47. Schultz JS, Schultz JS. The combinatorial library: A multifunctional resource. Biotechnol Progr 1996; 12:729-743. 48. Jongsma MA, Bakker PL, Stiekema WJ et al. Phage display of a double-headed proteinase inhibitor: Analysis of the binding domains of potato proteinase inhibitor II. Molecular Breeding 1995; 1:181-191. 49. Nixon AE, Ostermeier M, Benkovic SJ. Hybrid enzymes: Manipulating enzyme design. Trends Biotechnol 1998: 16:258-264. 50. Maskos K, Huber-Wunderlich M, Glockshuber R. RBI, a one-domain α-amylase/ trypsin inhibitor with completely independent binding sites. FEBS Lett 1996; 397:11-16. 51. Laskowski M, Jr, Kato I. Protein inhibitors of proteinases. Annu Rev Biochem 1980; 49:593-626. 52. Apostoluk W, Otlewski J. Variability of the canonical loop conformations in serine proteinase inhibitors and other proteins. Protein Structure Funct Genet 1998; 32:459-474. 53. Qasim MA, Ganz PJ, Saunders CW et al. Interscaffolding additivity. Association of P1 variants of eglin c and of turkey ovomucoid third domain with serine proteinases. Biochemistry 1997; 36:1598-1607. 54. Jiang H, Mulnix AB, Kanost MR. Expression and characterization of recombinant Manduca sexta serpin-1B and site-directed mutants that change its inhibitory selectivity. Insect Biochem Mol Biol 1995; 25:1093-1100. 55. Hasan Z, Leatherbarrow RJ. A study of the specificity of barley chymotrypsin inhibitor 2 by cysteine engineering of the P1 residue. Biochim Biophys Acta 1998; 1384:325-334. 56. Pal G, Sprengel G, Patthy A et al. Alteration of the specificity of ecotin, an E. coli serine proteinase inhibitor, by site directed mutagenesis. FEBS Lett 1994; 342:57-60. 57. Szardenings M, Vasel B, Hecht H-J et al. Highly effective protease inhibitors from variants of human pancreatic secretory trypsin inhibitor (hPSTI): An assessment of 3-D structure-based protein design. Protein Eng 1995; 8:45-52. 58. Stubbs MT, Laber B, Bode W et al. The refined 2.4 Å X-ray crystal structure of recombinant human stefin B in complex with the cysteine proteinase papain: A novel type of proteinase inhibitor interaction. EMBO J 1990; 9:1939-1947.
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59. Tuchscherer G, Scheibler L, Dumy P et al. Protein design: On the threshold of functional properties. Biopolymers 1998; 47:63-73. 60. Turk B, Turk V, Turk D. Structural and functional aspects of papain-like cysteine proteinases and their protein inhibitors. Biol Chem 1997; 378:141-150. 61. Bode W, Engh R, Musil D et al. The 2.0 Å crystal structure of chicken egg white cystatin and its possible mode of interaction with cysteine proteinases. EMBO J 1988; 7:2593-2599. 62. Machleidt W, Thiele U, Laber B et al. Mechanism of inhibition of papain by chicken egg white cystatin. FEBS Lett 1989; 243:234-238. 63. Arai S, Watanabe H, Kondo H et al. Papain-inhibitory activity of oryzacystatin, a rice seed cysteine proteinase inhibitor, depends on the central gln-val-val-ala-gly region conserved among cystatin superfamily members. J Biochem 1991; 109:294-298. 64. Urwin PE, Atkinson HJ, McPherson MJ. Involvement of the NH2-terminal region of oryzacystatin-I in cysteine proteinase inhibition. Protein Eng 1995; 8:1303-1307. 65. Koiwa K, Shade RE, Zhu-Salzman K et al. Phage display selection can differentiate insecticidal activity of soybean cystatins. Plant J 1998; 14:371-379. 66. Liu L-P, Deber CM. Guidelines for membrane protein engineering derived from de novo designed model peptides. Biopolymers 1998; 47:41-62. 67. Imperiali B, Ottesen JJ. Design strategies for the construction of independently folded polypeptide motifs. Biopolymers 1998; 47:23-29. 68. Vita C, Vizzavona J, Drakopoulou E et al. Novel miniproteins engineered by the transfer of active sites to small natural scaffolds. Biopolymers 1998; 47:93-100. 69. Tuchscherer G, Doemen B, Silla U et al. The TASP concept: Mimetics of peptide ligands, protein surfaces and folding units. Tetrahedron 1993; 49:3559-3575. 70. Le-Nguyen D, Mattras H, Coletti-Previero MA et al. Design and chemical synthesis of a 32 residues chimeric microprotein inhibiting both trypsin and carboxypeptidase A. Biochem Biophys Res Commun 1989; 162:1426-1430. 71. Wolfson AJ, Kanaoka M, Lau FTK et al. Insertion of an elastase-binding loop into interleukin-1β Protein Eng 1991; 4:313-317. 72. Gatehouse AMR, Gatehouse JA. Identifying proteins with insecticidal activity: Use of encoding genes to produce insect-resistant transgenic crops. Pestic Sci 1998; 52:165-175.
73. Oppert B, Morgan TD, Culbertson C et al. Dietary mixtures of cysteine and serine proteinase inhibitors exhibit synergistic toxicity toward the red flour beetle, Tribolium castaneum. Comp Biochem Physiol 1993; 105C:379-385. 74. Jongsma MA, Stiekema WJ, Bosch D. Combatting inhibitor-insensitive proteases of insect pests. Trends Biotechnol 1996; 14:331-333. 75. Chen M-S, Johnson B, Wen L et al. Rice cystatin: Bacterial expression, purification, cysteine proteinase inhibitory activity, and insect growth suppressing activity of a truncated form of the protein. Protein Expr Purif 1992; 3:41-49. 76. Michaud D, Nguyen-Quoc B, Yelle S. Production of oryzacystatin I and II in Escherichia coli using the glutathione S-transferase gene fusion system. Biotechnol Progr 1994; 10:155-159. 77. Hosoyama H, Irie K, Abe K et al. Introduction of a chimeric gene encoding an oryzacystatin-β-glucuronidase fusion protein into rice protoplasts and regeneration of transformed plants. Plant Cell Rep 1995; 15:174-177. 78. Tudyka T, Skerra A. Glutathione S-transferase can be used as a C-terminal, enzymatically active dimerization module for a recombinant protease inhibitor, and functionally secreted into the periplasm of Escherichia coli. Protein Sci 1997; 6:2180-2187. 79. Urwin PE, McPherson MJ, Atkinson HJ. Enhanced transgenic plant resistance to nematodes by dual proteinase inhibitor constructs. Planta 1998; 204:472-479. 80. Zhao Y, Botella MA, Subramanian L et al. Two wound-inducible soybean cysteine proteinase inhibitors have greater insect digestive proteinase inhibitory activities than a constitutive homolog. Plant Physiol 1996; 111:1299-1306. 81. Visal S, Michaud D, Yelle S. Identification of a gamma-linolenic acid-induced tomato leaf cystatin-like protein with potential for biocontrol of the phytophagous pest Colorado potato beetle. Plant Physiol 1996; 111(suppl.):40. 82. Thomas JC, Adams DG, Keppenne VD et al. Manduca sexta encoded protease inhibitors expressed in Nicotiana tabacum provide protection against insects. Plant Physiol Biochem 1995; 33:611-614. 83. Taylor MAJ, Lee MJ. Trypsin isolated from the midgut of the tobacco budworm, Manduca sexta, is inhibited by synthetic pro-peptides in vitro. Biochem Biophys Res Commun 1997; 235:606-609. 84. Corey MJ, Corey E. On the failure of de novo-designed peptides as biocatalysts. Proc Natl Acad Sci USA 1996; 93:11428-11434.
CHAPTER 11
Engineering Protease Inhibitors by Phage Display Jules Beekwilder and Maarten Jongsma
11.1. Introduction
R
ecent literature on the use of protease inhibitors (PIs) for pest control has made it clear that insects are well adapted to cope with a large range of these inhibitors. They have evolved proteases which are insensitive to the action of host plant inhibitors and are able to activate these genes in the presence of PIs in their diet.1-6 As a consequence, pest control using PIs in transgenic plants requires the isolation of inhibitors that are active towards the insensitive proteases.7 One can search for active inhibitors among naturally-occurring peptides.8 Alternatively, one can engineer inhibitors in such a way that they will acquire activity against the “PI-insensitive” protease. Engineering of inhibitors can be performed in two fundamentally different fashions. Based on structures of the inhibitor-protease complex, predictions can be made on mutations that will enhance binding.9 Lack of data on these complexes for insect proteases makes this procedure rather tough, however. It may be more appropriate to simply generate large arrays of mutants in the region of the inhibitor protein contacting the protease. The powerful method of phage display can subsequently be used to select the strongly binding mutants. This chapter deals with the general principles behind this method, some pitfalls, and the application to select improved PIs for pest control by means of phage display.
11.2. Phage Display 11.2.1. The Principle of Phage Display Phage display is a way of protein engineering based on an evolutionary strategy. Darwinian evolution comprises alternating rounds of mutation, selection and reproduction. Phage display deploys large scale mutation in vitro, followed by alternating rounds of selection in vitro and reproduction in vivo. In this scheme millions of variants of a protein can be tested for their fitness to bind to a target ligand. Phage display has first been applied to PIs by Ladner and coworkers to engineer inhibitors of potential pharmaceutical importance. This group has published a number of highly instructive papers and patent-applications on both practical and theoretical aspects.10-12 A considerable number of PIs have been subjected to phage display (Table 11.1). As a result, inhibitors which initially had a poor (meaning millimolar to micromolar) inhibiton constant (Ki) for a target protease have been dramatically improved. From pools of Kunitz-domain variants, extremely potent inhibitors of human neutrophil elastase (Ki = 1 pM),14 plasmin (Ki = 87 pM),18 plasma kallikrein (Ki = 40 pM)19 and tissue factorfactor VIIa (Ki = 10 nM)15 have been selected. Proof that the concept also works for other types of inhibitors has been provided by Wang et al.22 From a limited number of variants of
Recombinant Protease Inhibitors in Plants, edited by Dominique Michaud. ©2000 Eurekah.com.
Engineering Protease Inhibitors by Phage Display
129
Table 11.1. Functional display of protease inhibitors on phage Class
Family
Inhibitor
serine PIs
Kunitz
BPTIa
cysteine PIs
Library selection
Source
Ref.
+
cow
13,14
APPI
+
human
15-17
LACI
+
human
11,18,19
serpin
PAI-1
+
human
20
Kazal
PSTI
+
human
21
ecotin
ecotin
+
E. coli
22
PI-II
PI-II
(+)
potato
23
Ascaris inh.
Ancylostoma PI
-
nematode
24
eglin-c
eglin-c
-
leech
24
hirudin
hirudin
-
leech
25
type II cystatin
chicken cystatin
-
chicken
26
phytocystatin
soyacystatin
-
soybean
27
a Abbreviations: APPI, Alzheimer amyloid β-protein precursor inhibitor; BPTI, bovine pancreatic
trypsin inhibitor; ecotin, E. coli trypsin inhibitor; LACI, lipoprotein-associated coagulation inhibitor; PAI, plasminogen activator inhibitor; PI, proteinase inhibitor; PI-II, potato proteinase inhibitor II; PSTI, pancreatic secretory trypsin inhibitor.
the Escherichia coli trypsin inhibitor ecotin, an inhibitor with Ki = 1 nM for urokinasetype plasminogen activator was selected. The engineering of a PI by phage display involves five steps, shown in Figure 11.1: 1. Gene fusion. The inhibitor gene is fused to one of the coat-protein genes of bacteriophage fd. 2. Creating a pool. The DNA encoding the part of the inhibitor that is expected to bind proteases is randomized, resulting in a pool of mutant DNAs which are expressed in E. coli together with the other bacteriophage proteins. A
pool of phage particles is then being synthesized. These particles display a variant of the PI, and carry the corresponding variant gene. 3. Selection. The pool of variant particles is exposed to immobilized target proteases. Phages carrying a variant PI that does bind to the protease remain bound firmly, while weakly-binding phages are washed off. In this simple and rapid way variants are selected that are candidates to inhibit the protease. Usually, the selected phages are used to generate a new pool of phages by
130
Recombinant Protease Inhibitors in Plants
Fig. 11.1. Schematic representation of the protocol for phage display. The five stages are described in more detail in section 11.2.1.
Engineering Protease Inhibitors by Phage Display re-infecting and propagating them in E. coli. The enriched pool of phages can again be applied to the immobilized protease, and the whole process of selection and amplification can be repeated several times. After three to four cycles, one can assume to be left with phages that carry strongly-binding PI variants. 4. DNA analysis. The identity of selected variants is analyzed. One of the attractive features of phage display is that to each variant inhibitor the DNA that encodes it is attached in the phage particle, which can conveniently be sequenced. 5. Kinetic analysis. Selected variant PIs are produced on a large scale in order to test their inhibitory properties.
Each of these steps requires particular experimentation and should be fine-tuned. Not all of these experiments have as yet been described for insecticidal plant PIs. In those cases we discuss what experiments have been reported for mammalian PI display.
11.2.2. Choice of Vector For the PI to be displayed on bacteriophage fd, its gene is usually inserted between the signal peptide and the mature N-terminus of the gIII coat protein. There are basically two vector systems in which this fusion gene can be expressed: in the complete DNA of a bacteriophage, or in a phagemid system (reviewed in ref. 28). A phagemid is a pUC-like plasmid which carries the packaging signal of phage fd, its origin of synthesis of singlestranded DNA, and the PI-gIII fusion gene. The phagemid can be maintained as a plasmid in bacteria by selection for antibiotic resistance. When these cells are infected with a helperphage the system is complemented and the production of phage particles starts. The helperphage provides the phage-assembly machinery, but is itself poorly packaged as it carries a mutated packaging signal. As a result, phage particles are synthesized which carry in majority the phagemid DNA and a mixture of wild-type and fusion coat-proteins. The
131
phagemid system is often preferred over the phage system, as it offers a better manipulatable DNA which can be more efficiently transformed.29 Also the use of a phagemid vector presumably reduces the incidence of “polyvalent” phage particles, i.e., particles that carry more than one copy of the encoded inhibitor. 30 A polyvalent particle of a low-affinity gene could have a high apparent affinity (“avidity”), and thus confuse the selection system. On the other hand, several reports indicate that isolation of high-affinity inhibitors using a polyvalent phage system is very well possible.14,18
11.2.3. Choice of PI
Ladner12 has formulated a number of criteria which should be considered when choosing an inhibitor for phage display, or deciding whether or not to apply phage display for inhibitor engineering. In brief, it is recommended to choose a small-sized inhibitor that has already some affinity for the target protease, and can be produced in considerable amounts as a correctly folded protein in E. coli. Knowledge of its threedimensional structure, the position of its binding site and the amino-acid sequence of related inhibitors is also quite valuable. In addition to these general considerations there are special considerations when the PIs are planned for use in transgenic plants against insect pests. Such inhibitors should be stable in the insect gut, and should be expressed to high levels in plants. The insect midgut contains proteases to a concentration of 10 µM. For stoichiometrical inhibition of insect proteases, PIs should be expressed to 0.2-1% of total soluble plant protein. 1 Expression in plants of heterologous proteins to these levels proves to be difficult in some cases (see refs. 31 and 32), and therefore it may be preferable to use parent PIs which are known to be expressed to high levels in the plant. This does not always coincide with high expression in E. coli and may require specific precautions, as will be discussed in section 11.2.9.
132
11.2.4. Testing Display and Selection of PIs Once a PI has been picked and fused to gIII it is important to establish that the construct is able to direct the inhibitor to the surface of the phage particles, and that the fusion protein is reasonably stable and functional. Its presence and stability are tested for by Western blotting purified phage particles. The fusion protein is detected by an antiserum directed against the inhibitor,27 against the gIII protein 21 or against a tag sequence inserted between the inhibitor gene and gIII.23 The latter method shows a clear band of the expected size for particles carrying a gene fusion of potato inhibitor PI-II and gIII, while a PI-I-gIII fusion produces a diffuse smear (P. Bakker & M.A. Jongsma, unpubl. results; Figure 11.2). The observation that PI-I-gIII fusion proteins cannot be stably produced matches with that of Jespers et al,24 who reported that eglin-c, which is structurally similar to PI-I, is poorly displayed from gIII-fusions. This is ascribed to the structural role of the C-terminus of this inhibitor, which might be hampered by the fusion to the gIII protein. The solution presented by the authors is to fuse eglin-c at its N-terminus to another coat-protein of fd, gVI (see Section 11.3). Apart from being stable on the surface of the phage, the inhibitor should prove to be functional when displayed on a phage. The attached phage particle should not affect the ability of an inhibitor to bind protease, for instance by shielding its binding-site. Functional display can be demonstated by selective binding of inhibitor phages to immobilized protease. In such experiments, it is important to incorporate proper controls to distinguish true inhibitory properties from a background of aspecific protein-protein interactions. One should be aware of the fact that bacteriophages have a high aspecific affinity for surfaces. Functional display and selection is therefore tested by comparing inhibitor particles to weak- or non-inhibitor particles. Koiwa et al27 directly tested the inhibitory activity of phage particles towards protease. They constructed three different types of par-
Recombinant Protease Inhibitors in Plants
ticles: particles that do not carry any inhibitor, particles that carry a poor papain inhibitor from soybean, and particles that carry a strong papain inhibitor. Indeed, particles carrying the strong cystatin inhibited papain at a much lower concentration than the other phages, meaning that the particles were carrying a functional cystatin. For PI-II, the binding of strong-inhibitor and weak-inhibitor particles to immobilized trypsin was compared. First, weak-inhibitor particles were created by converting the P2, P1 and P1’ of both inhibitory loops of PI-II into alanines.23 Also phages were made that have both an alanine loop and a loop that is unaltered. Such an inhibitor was shown to strongly inhibit trypsin. Comparable amounts of strong-inhibitor and weak-inhibitor phages were added to immobilized trypsin, and loosely bound phages were washed off. A ratio of 111 to 1 was found for the inhibitor and inactive-inhibitor phages that were subsequently eluted from trypsin. Apparently, PI-II on phage particles can bind to immobilized trypsin. Furthermore, this experiment suggests that high-affinity PI-II phages can be segregated from low-affinity mutants by binding. To establish a selection system, it should be directly tested if a small fraction of strong-inhibitor phages can be efficiently selected from weak-inhibitor phages. To this end, model pools have been made which consist of a small amount of inhibitor phage, mixed with a large background of weakinhibitor phage. The model pool is passed through several selection cycles which involve binding to immobilized protease, elution and re-amplification of the eluted phages. After each selection cycle, the ratio of stronginhibitor to weak-inhibitor phages is established in the eluted phages (by plating and colony hybridization), and compared to the initial ratio in the model pool.23,27 In both reported cases, three cycles of selection were sufficient to reduce the background of weak-inhibitor phages from large excess to hardly detectable numbers. These results clearly demonstrate the feasibility of selecting plant inhibitors with high affinity for a target protease.
Engineering Protease Inhibitors by Phage Display
133
Fig. 11.2. Western blot analysis of recombinant phage particles carrying myc-gIII fusions (lane 1), PI-II-myc-gIII fusions (lanes 2 and 3) or PI-I-myc-gIII fusions (lanes 4 and 5). The myc-gIII fusion migrates at 67kDa, the PI-II-myc-gIII at 81 kDa and the PI-I-myc-gIII at 75 kDa. The myc sequence is an 18 amino acid-long linker between PI sequences and gIII. The fusion protein was detected using the anti-myc monoclonal antibody 9E10.
11.2.5. Identifying Positions for Randomization In order to adapt the specificity of an inhibitor, the regions of the inhibitor that determine this specificity should be identified. These can conveniently be found in a 3D structure model from the inhibitor itself or a related molecule. If such a model is not available, mutational data and sequence alignments can be used to identify specificity determinants. If one is to improve an inhibitor whose substrate binding mechanism is not fully understood, a more random approach may be invoked. The number of residues that contribute strongly to the specificity of an inhibitor is often very limited, as can notably be concluded from the observations that the dramatically altered specificities of Kunitz domains (see Section 11.2.1) have been
achieved by only two to five amino-acid substitutions. However in most cases it is not exactly known at which positions these determinants reside, and a larger region spanning up to 10 amino acids must be challenged by randomization. For serine PIs, the main region of contact is located at a linear sequence, the inhibitory loop or P1 region.33 This loop has therefore been the primary target of randomization in phage display experiments. Amino acids around the P1 residue of the inhibitor (adjacent to the pseudo-substrate scissile bond) have been randomized from P6 to P’6. Outside this loop, secondary regions in the inhibitor, like the 31-39 loop of Kunitz domains, may provide additional contacts with the protease. For cystatins and stefins, the inhibitory part is composed of a hydrophobic “wedge”
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composed of 3 surface loops.33 These loops, 3 to 4 amino acids in size, are each some 50 amino acids apart in the amino acid sequence. In addition, analysis of a cystatin from the Japanese quail indicates that residues well outside the inhibitory loops may contribute to the specificity of the inhibitor,34 meaning that targets for randomization are quite dispersed through the cystatin gene, and complicating the design of a randomization strategy.
11.2.6. Designing and Introducing Variability Generally, inhibitors have about 12 amino acid positions that may contribute to the binding of the protease, and are thus candidates for randomization. The most straightforward way to synthesize a variant pool would be to completely randomize the 36 base pairs encoding these 12 residues. If every nucleotide is allowed at every one of the 36 positions, such a pool would require at least 436 (= 4.7x1021) different phages. This number reaches beyond the physical limits of phage display: for instance, the normal titer of bacteriophages in a growth medium is 1012 particles per ml, meaning that over one million liters of culture would be needed to generate this amount of phages. The diversity of pools is further limited by the efficiency of bacterial transformation, which is the true bottleneck of this system. At best, about 109 independent transformants can be obtained after series of large-scale electroporations.18 Recently, in vivo recombination has been applied to produce larger phage pools of 1011 individual clones.35 Due to the limits of transformation, mutation pools cover mostly up to six codons. There are several ways to introduce cassettes of randomized codons into the inhibitor gene.36 Most commonly used is the insertion of oligonucleotides. First, one should create unique restriction sites in the DNA flanking the target region by introducing silent mutations. The DNA fragment between the restriction sites can then be replaced by a duplex of oligonucleotides containing variegated triplets.11 Often, the
Recombinant Protease Inhibitors in Plants
triplet NNK (where K is G or T) is used, which encodes all 20 amino acids by 32 variants, instead of 64 for NNN triplets.12 When one wishes to probe a region larger than six codons, an iterative selection can be applied.18,19 In a first step, a pool covering only five randomized residues is generated, and very poor binders are eliminated by a single round of selection. Next, the DNA of the enriched pool of phages is isolated, and used to randomize a second set of amino acids. In this way, a very large number of proteins can be tested by selection, but as these are produced in stages, each round of randomization is to yield pools of manageable size. Especially for inhibitors with a complex active site like cystatins, it can be envisaged that more than one region should be challenged by randomization (see above). For that purpose, the iterative method of randomization is also appropriate. In addition, it is conceivable that some positions that contact each other in the 3D structure of the inhibitor are codependent. To observe covariations at these positions, they should preferably be randomized at the same time. For this purpose Kunkel mutagenesis can be used,15 which prevents the need to introduce restriction sites, but permits a high incidence of unaltered wild-type inhibitors in the mutant pool. Alternatively two oligonucleotides containing randomized cassettes can be fused by PCR, and introduced as a double-cassette PCR fragment between unique restriction sites.12 If the inhibitory activity of a protein is poorly understood, or has not been confined to particular regions of the molecule as is for instance the case for equistatin,37 it may nevertheless be improved by phage display. In such a case the mutations may be randomly dispersed over the molecule by error prone PCR.38 If alignment of related inhibitors shows variation in loop size like in the case of cystatins,39,40 combining parts of related genes into hybrid genes may result in improved inhibitors.41 Such hybrid genes can be generated on large scale by DNA shuffling, as demonstrated for antibioticresistance genes by Stemmer.42,43 A combination of gene- shuffling and the selective
Engineering Protease Inhibitors by Phage Display
properties of phage display may prove very valuable.44
11.2.7. Circumventing Reproductive Biases The protocol of phage display involves several stages in which the pool-DNA or selected DNA is amplified in E. coli, which constitutes a considerable risk. During these amplifications, phage DNA that carries “toxic” DNA may become underrepresented in the populations. Such “toxicity” is not confined to known bactericidal proteins such as lysozyme.45 Growth is also retarded by the production of slowly or aberrantly folded proteins, which in their native fold may be harmless.46 In extreme cases, only dysfunctional mutants are selected from pools of toxic proteins. We encountered this problem when trying to re-engineer trypsin inhibiting variants of PI-II (Beekwilder, Rakonjac and Bosch, in prep.). We constructed a pool of PI-II phages, in which the P2-P1-P'1 positions of the trypsin-inhibitory loop had been randomized. Three rounds of selection were performed on bovine chymotrypsin and trypsin, and DNA of the selected phages was analyzed. Surprisingly, nearly all of the selected phages encoded either deletions or amber mutations in the PI-II gene. The reason why we found such mutants is probably that the PI-II inhibitor constitutes a major disadvantage in growth. Indeed, we observed that bacteria harboring wild-type PI-II DNA grow much slower than bacteria with amber mutation or deletion plasmids. Deletion mutants obviously do not produce the inhibitor. The amber mutants may allow some expression of the fusion protein, since host bacteria usually carry a supE amber-suppressor gene. Depending on the codon context of the amber mutations found, it can be deduced that expression in the amber mutants was effectively reduced to 2% of an uninterrupted gene.47,48 Altogether, the dominance of both deletion and amber mutants indicated a strong selection pressure against PI-II expression in our experiments.
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A related observation was described by Dennis and Lazarus,15 who engineered by directed mutations a Kunitz inhibitor of TF-FVIIa that is more potent than variants selected by phage display. It appeared that the potent inhibitor is poorly expressed in E. coli, which explains why it could be underrepresented in the phage library. A high incidence of deletion variants has also been reported for antibody phage pools.49,50 The growth disadvantage of bacteria carrying the PI-II gene is observed even in the presence of glucose to suppress the lacZ promoter, suggesting that suppression is insufficient. Indeed, improving control of the promoter reduces growth disadvantages of bacteria harboring toxic genes.45,51 Although growth differences cannot be avoided during phage synthesis, prior steps, like plating of transformants, should preferably be executed with a completely shut promoter. When using a phagemid system, the promoter of the psp-operon of E. coli may provide such control, since it only switches on after infection by the helperphage.52
11.2.8. Selection Targets The proteases for which inhibitors have been selected are commercially available and purchased in pure form. Insect proteases are only available in crude mixtures like midgut content for large insects, or whole-insect homogenates if midguts are too small to be isolated. Midgut contents appear to be highly complex mixtures, in which over 10 different proteases are represented, alongside with other enzymes.5,6,53,54 The selection of specific inhibitors may require that individual proteases are isolated from these mixtures, or expressed as recombinant proteins in insect cells or other expression systems. On the other hand, ligands to complex mixtures like whole mammalian organs have been selected,55 which suggests that gut contents could also serve as a target. There are several ways to immobilize proteases, including coating on wells of microtiter plates17,21-23 or beads.13,14,18,19 The concentration of target molecules may affect the affinity of the selected clones.56 At a low
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concentration of target, higher-affinity phages theoretically bind more exclusively, as illustrated in Table 11.2. The concentration of proteases can be controlled best when offered to the inhibitor in soluble form. To preferentially select inhibitors with Ki’s in the nanomolar range, one should for example perform selection at a concentration of 1 pmol protease per ml. After the inhibitors have contacted the protease, they can be immobilized by biotinylating the proteases prior to the binding of the phages, and catching the biotin-protease-PI-phage complexes with streptavidin-coated beads.56,57 An additional advantage of this strategy is that the required quantities and concentrations of proteases are reduced.
11.2.9. Analysis of the Selected Inhibitors The performance of the phage display procedure can be monitored by comparing the phage titers of input and output samples for each selection cycle. Generally after the first cycle, out of 1012 input phages, at most 106 are retained. After the third selection cycle, 108 to 1010 phages should be eluted. If such is the case, the DNA sequence of a representative number of phages that are eluted in the last step is determined. If a single protease is the target, the encoded amino acids display consensus at several positions. For instance, for serine PIs, the residue at the P1 position should be identical in most phages. If a mix of proteases is targeted, families of sequences should arise. The affinity of selected inhibitors can be roughly assessed by making phage stocks of the isolated clones, and quantifying their binding to coated target-protease, as described in section 11.2.4 for PI-II. As a rule of thumb, a higher affinity inhibitor is accompanied by a higher fraction of bound phages, although this relationship is non-linear.11 A quantitative measurement of on-rates, off-rates and inhibitor constants requires the production of the inhibitors as free molecules. One may need to shift to different expression systems for this purpose, since expression of heterologous inhibitors in the periplasm of
Recombinant Protease Inhibitors in Plants
Table 11.2. Dependence of the fraction and composition of bound phage on the equilibrium dissociation constant (Kd) and the protease concentrationa high protease concentration µM) (1µ
low protease concentration (1nM)
high affinity phage (Kd=1 nM) 99.9%
50%
low affinity phage (Kd=1µM)
0.1%
50%
ratio high-affinity phage 2:1 low-affinity phage
500:1
a Percentages of bound inhibitor-phages have been calculated from the equation [P] / Kd = ratio of bound to unbound inhibitor-phage, where [P] is the known protease concentration, and Kd is the proposed dissociation constant. Calculations do not take into account aspecific binding of phages to the target proteins.
E. coli is often poor,58 and measurements may easily be confused by the presence of endogenous E. coli inhibitors.59 This is the reason why often a Pichia pastoris expression system is used.60 Quantities generated by this system are generally sufficient both to study the inhibition of insect gut proteases, and to perform bio-assays.
11.3. Phage Display to Identify Natural Inhibitors The technology of phage display may serve other purposes than protein engineering. To identify natural inhibitors from plants that perform well in the inhibition of insect proteases, one would like to apply a functional selection to cDNA libraries. The N-terminus of the gIII protein is not a convenient site to fuse cDNAs to, however. cDNAs usually extend beyond the C-terminus of a reading
Engineering Protease Inhibitors by Phage Display
frame, into the poly-A tail, and thereby do not permit gene fusions. Hence, only fragments of cDNAs can be used with classical phage display vectors.61 For small proteins like PIs, this may not be satisfactory. Jespers et al24 described a system that allows the surface expression of cDNAs. The authors fused the 5´ regions of cDNAs to the 3´terminus of gVI. Fusion to the C-terminus of the gVI protein allows the allocation of a protein to the surface of the phage, albeit with poor efficiency. Nevertheless this method was used successfully to isolate a novel inhibitor of factor Xa from a cDNA library of the nematode Ancylostoma caninum. Alternatively, cDNAs can be fused indirectly to gIII. Crameri & Blaser62 made a construct encoding a fusion of gIII and the jun-protein, which contains a leucine zipper. On the same plasmid, the fos-gene is fused upstream of a library of cDNAs. The cDNA-protein is then connected to gIII through the jun-fos leucine zipper. This system allowed selective binding of allergenencoding cDNAs from Aspergillus fumigatus to immobilized IgE’s.
11.4. From Phage to Plant Effective pest control with PIs may require that most proteolytic activity in the insect gut is inhibited,7 meaning that a plant may need several additional inhibitors. Insect midguts contain an estimated number of 10-20 different proteases6 which are differentially regulated in response to different inhibitors.5 Several of these proteases cannot be inhibited by the plant’s own PIs, and may require several inhibitors generated by phage display. Moreover if different insect pests are targeted in one plant, even more different inhibitors may be needed. Therefore, to achieve an effective level of pest control it is important to achieve multi-inhibitor expression in a simple manner. For reasons yet unknown native plant PIs are often expressed as multidomain or multiheaded proteins, combining up to eight specificities in a single molecule.63 Analogously, inhibitor genes selected by phage display could be fused to encode a single multidomain molecule,
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greatly improving the ease of handling. The multidomain gene could then be expressed from a single promoter, thus reducing tedious plant transformations to a minimum.
11.5. Conclusion The technique of phage display is rapidly developing, and has proved to be a powerful tool in drug design. Its application for the design of insecticidal PIs is still in its young stages, and has not yet reached its final aim. Some particular problems associated with plant PI display may be encountered, and require additional experimentation. For instance, the toxicity of PI-II for E.coli proved to be a major hurdle in this regard, but it can be overcome. Another challenge will probably be the use of insect proteases as a selection target. We anticipate to tackle this problem in the coming year, and to engineer valuable inhibitors of the insect digestive proteolytic system.
Acknowledgments This work was funded by the Dutch Technology Foundation STW.
References 1. Jongsma MA, Bolter C. The adaptation of insects to plant protease inhibitors. J Insect Physiol 1997; 43:885-895. 2. Bolter C, Jongsma MA. Colorado potato beetles (Leptinotarsa decemlineata) adapt to proteinase inhibitors induced in potato leaves by methyl jasmonate. J Insect Physiol 1995; 41:1071-1078. 3. Jongsma MA, Bakker PL, Peters J et al. Adaptation of Spodoptera exigua larvae to plant proteinase inhibitors by induction of proteinase activity insensitive of inhibition. Proc Natl Acad Sci USA 1995; 92:8041-8045. 4. Broadway RM. Dietary proteinase inhibitors alter complement of midgut proteases. Arch Insect Biochem Physiol 1996; 32:39-53. 5. Broadway RM. Dietary regulation of serine proteinases that are resistant to serine proteinase inhibitors. Insect Biochem Mol Biol 1997; 43:855-874. 6. Bown DP, Wilkinson HS, Gatehouse JA. Differentially regulated inhibitor-sensitive and insensitive protease genes from the phytophagous insect pest, Helicoverpa armigera, are members of complex multigene families. Insect Biochem Mol Biol 1997; 27:625-638.
138 7. Jongsma MA, Stiekema WJ, Bosch D. Combatting inhibitor-insensitive proteases of insect pests. Trends Biotechnol 1996; 14:331-333. 8. Gruden K, Strukelj B, Popovic T et al. The cysteine protease activity of Colorado potato beetle (Leptinotarsa decemlineata say) guts, which is insensitive to potato protease inhibitors, is inhibited by thyroglobulin type-1 domain inhibitors. Insect Biochem Mol Biol 1998; 28:549-560. 9. Urwin PE, Atkinson HJ, Waller DA et al. Engineered oryzacystatin-I expressed in hairy roots confers resistance to Globodera pallida. Plant J 1995; 8:121-131. 10. Ladner RC, Guterman SK. Generation and selection of recombinant varied binding proteins. PCT patent application WO90/02809. 11. Markland W, Roberts BL, Ladner RC. Selection for protease inhibitors using bacteriophage display. Methods Enzymol 1996; 267:28-51. 12. Ladner RC. Display and selection of proteins on genetic packages. In: Kay BK, Winter J, McCafferty J, eds. Phage display of peptides and proteins. San Diego: Academic Press 1996:151-193. 13. Roberts BL, Markland W, Siranosian K et al. Protease inhibitor display M13 phage: Selection of high-affinity neutrophil elastase inhibitors. Gene 1992; 121:9-15. 14. Roberts BL, Markland W, Ley AC et al. Directed evolution of a protein: Selection of potent neutrophil elastase inhibitors displayed on M13 fusion phage. Proc Natl Acad Sci USA 1992; 89:2429-2433. 15. Dennis MS, Lazarus RA. Kunitz domain inhibitors of tissue factor-factor VIIa. I. Potent inhibitors selected from libraries by phage display. J Biol Chem 1994; 269: 22129-22136. 16. Dennis MS, Lazarus RA. Kunitz domain inhibitors of tissue factor-factor VIIa. II. Potent and specific inhibitors by competitive phage selection. J Biol Chem 1994; 269:22137-22144. 17. Dennis MS, Herzka A, Lazarus RA. Potent and selective Kunitz domain inhibitors of plasma kallikrein designed by phage display. J Biol Chem 1995; 270:25411-25417. 18. Markland W, Ley AC, Lee SW et al. Iterative optimization of high-affinity proteases inhibitors using phage display. 1. Plasmin. Biochemistry 1996; 35:8045-8057. 19. Markland W, Ley AC, Ladner RC. Iterative optimization of high-affinity protease inhibitors using phage display. 2. Plasma kallikrein and thrombin. Biochemistry. 1996; 35:8058-8067.
Recombinant Protease Inhibitors in Plants 20. Pannekoek H, van Meijer M, Schleef RR et al. Functional display of human plasminogenactivator inhibitor 1 (PAI-1) on phages: Novel perspectives for structure-function analysis by error-prone DNA synthesis. Gene 1993; 128:135-140. 21. Rottgen P, Collins J. A human pancreatic secretory trypsin inhibitor presenting a hypervariable highly constrained epitope via monovalent phagemid display. Gene 1995; 164:243-250. 22. Wang CI, Yang Q, Craik CS. Isolation of a high affinity inhibitor of urokinase-type plasminogen activator by phage display of ecotin. J Biol Chem 1995; 270:12250-12256. 23. Jongsma MA, Bakker PL, Stiekema WJ et al. Phage display of a double-headed proteinase inhibitor: Analysis of the binding domains of potato proteinase inhibitor II. Mol Breeding 1995; 1:181-191. 24. Jespers LS, Messens JH, De Keyser A et al. Surface expression and ligand-based selection of cDNAs fused to filamentous phage gene VI. Bio/Technology 1995; 13:378-382. 25. Wirsching F, Opitz T, Dietrich R et al. Display of functional hirudin on the surface of phage M13. Gene 1997; 204:177-184. 26. Tanaka AS, Sampaio CA, Fritz H et al. Functional display and expression of chicken cystatin using a phagemid system. Biochem Biophys Res Commun 1995; 214:389-395. 27. Koiwa K, Shade RE, Zhu-Salzman K et al. Phage display selection can differentiate insecticidal activity of soybean cystatins. Plant J 1998; 14:371-379. 28. Armstrong N, Adey NB, McConnell SJ et al. Vectors for phage display. In: Kay BK, Winter J, McCafferty J, eds. Phage display of peptides and proteins. San Diego: Academic Press 1996:35-53. 29. Hoogenboom HR, Griffiths AD, Johnson K et al. Multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (Fab) heavy and light chains. Nucl Acids Res 1991; 19:4133-4137. 30. Cwirla SE, Peters EA, Barrett RW et al. Peptides on phage: A vast library of peptides for identifying ligands. Proc Natl Acad Sci USA 1990; 87:6378-6382. 31. Thomas JC, Wasmann CC, Echt C et al. Introduction and expression of an insect proteinase inhibitor in alfalfa (Medicago sativa L.) Plant Cell Rep 1994; 14:31-36. 32. Strizhov N, Keller M, Mathur J et al. A synthetic cryIC gene, encoding a Bacillus thuringiensis delta-endotoxin, confers Spodoptera resistance in alfalfa and tobacco. Proc Natl Acad Sci USA 1996; 93:15012-15017.
Engineering Protease Inhibitors by Phage Display 33. Bode W, Huber R. Natural protein proteinase inhibitors and their interaction with proteinases. Eur J Biochem 1992; 204:433-451. 34. Gerhartz B, Engh RA, Mentele R et al. Quail cystatin: Isolation and characterisation of a new member of the cystatin family and its hypothetical interaction with cathepsin B. FEBS Lett 1997; 412:551-558. 35. Fisch I, Kontermann RE, Finnern R et al. A strategy of exon shuffling for making large peptide repertoires displayed on filamentous bacteriophage. Proc Natl Acad Sci USA 1996; 93:7761-7766. 36. Adey NB, Sparks AB, Beasley J et al. Construction of random peptide libraries in bacteriophage M13. In: Kay BK, Winter J, McCafferty J, eds. Phage display of peptides and proteins. San Diego: Academic Press 1996:67-78. 37. Lenarcic B, Ritonja A, Struklj B et al. Equistatin, a new inhibitor of cysteine proteinases from Actinia equina, is structurally related to thyroglobulin type-1 domain. J Biol Chem 1997; 272:13899-13903. 38. van Meijer M, Roelofs Y, Neels J et al. Selective screening of a large phage display library of plasminogen activator inhibitor 1 mutants to localize interaction sites with either thrombin or the variable region 1 of tissue-type plasminogen activator. J Biol Chem 1996; 271:7423-7428. 39. Turk V, Bode W. The cystatins: Protein inhibitors of cysteine proteinases. FEBS Lett 1991; 285:213-219. 40. Brown WM, Dziegielewska KM. Friends and relations of the cystatin superfamily—New members and their evolution. Protein Sci 1997; 6:5-12. 41. Auerswald EA, Naegler DK, Gross S et al. Hybrids of chicken cystatin with human kininogen domain 2 sequences exhibit novel inhibiton of calpain, improved inhibition of actinidin and impaired inhibition of papain, cathepsin L and cathepsin B. Eur J Biochem 1996; 235:534-542. 42. Stemmer WPC. Rapid evolution of a protein in vitro by DNA shuffling. Nature 1994; 370:389-391. 43. Patten PA, Howard RJ, Stemmer WPC. Applications of DNA shuffling to pharmaceuticals and vaccines. Curr Op Biotechnol 1997; 8:724-733. 44. Adey NB, Stemmer WPC, Kay BK. Preparation of second-generation phage libraries. In: Kay BK, Winter J, McCafferty J, eds. Phage display of peptides and proteins. San Diego: Academic Press 1996:277-291.
139 45. Maenaka K, Furuta M, Tsumoto K et al. A stable phage-display system using a phagemid vector: phage display of hen egg-white lysozyme (HEL), Escherichia coli alkaline phosphatase, and anti-HEL monoclonal antibody, HyHEL10. Biochem Biophys Res Commun 1996; 218:682-687. 46. Knappik A, Pluckthun A. Engineered turns of a recombinant antibody improve its in vivo folding. Protein Eng 1995; 8:81-89. 47. Miller JH, Albertini AM. Effects of surrounding sequence on the suppression of nonsense codons. J Mol Biol 1983; 164:59-71. 48. Bossi L. Context effects: Translation of UAG codon by suppressor tRNA is affected by the sequence following UAG in the message. J Mol Biol 1983; 164:73-87. 49. Fischer P, Leu S-JC, Yang Y-Y et al. Rapid simultaneous screening for DNA integrity and antigen specificity of clones selected by phage display. BioTechniques 1995; 16:828-830. 50. Smiley JA, Benkovic SJ. Selection of catalytic antibodies for a biosynthetic reaction from a combinatorial cDNA library by complementation of an auxotrophic Escherichia coli: Antibodies for orotate decarboxylation. Proc Natl Acad Sci USA 1994; 91:8319-8323. 51. Krebber A, Burmester J, Pluckthun A. Inclusion of an upstream transcriptional terminator in phage display vectors abolishes background expression of toxic fusions with coat protein g3p. Gene 1996; 178:71-74. 52. Rakonjac J, Jovanovic G, Model P. Filamentous phage infection-mediated gene expression: Construction and propagation of the gIII deletion mutant helper phage R408d3. Gene 1997; 198:99-103. 53. Terra WR, Ferreira C. Insect digestive enzymes: Properties, compartmentalization and function. Comp Biochem Physiol 1994; 109B:1-62. 54. Jongsma MA, Peters J, Stiekema WJ et al. Characterization and partial purification of gut proteinases of Spodoptera exigua Hübner. Insect Biochem Mol Biol 1996; 26:185-193. 55. Pasqualini R, Ruoslahti E. Organ targeting in vivo using phage display peptide libraries. Nature 1996; 380:364-366. 56. McCafferty J. Phage display: Factors affecting panning efficiency. In: Kay BK, Winter J, McCafferty J, eds. Phage display of peptides and proteins. San Diego: Academic Press 1996:261-276. 57. Hawkins RE, Russell SJ, Winter GP. Selection of phage antibodies by binding affinitymimicking affinity maturation. J Mol Biol 1992; 226:889-896.
140 58. Dimasi N, Martin F, Volpari C et al. Characterization of engineered hepatitis C virus NS3 protease inhibitors affinity selected from human pancreatic secretory trypsin inhibitor and minibody repertoires. J Virol 1997; 71:7461-7469. 59. Chung CH, Ives HE, Almeda S et al. Purification from Escherichia coli of a peri-plasmic protein that is a potent inhibitor of pancreatic proteases. J Biol Chem 1983; 258:11032-11038. 60. Cregg JM, Vedvick TS, Raschke WC. Recent advances in the expression of foreign genes in Pichia pastoris. Bio/Technology 1993; 11:905-910.
Recombinant Protease Inhibitors in Plants 61. Lang IM, Chuang TL, Barbas CF 3rd et al. Purification of storage granule protein-23. A novel protein identified by phage display technology and interaction with type I plasminogen activator inhibitor. J Biol Chem 1996; 271:30126-30135. 62. Crameri R, Blaser K. Cloning Aspergillus fumigatus allergens by the pJuFo filamentous phage display system. Int Arch Allergy Immunol 1996; 110:41-45. 63. Richardson M. Seed storage proteins: The enzyme inhibitors. In: Dey PM, Harborne JB. Methods in plant biochemistry 5. New York:Academic Press 1991;259-306.
CHAPTER 12
Using Protease Proregions as Regulators of Insect Digestive Proteinases Mark A.J. Taylor
12.1. Introduction
I
n order to maintain their rapid growth, the larvae of many insect pests need to feed nearly continuously and have very efficient digestive enzymes. Insect digestive proteinases are often targetted as potential candidates for pest control as they are easily accessible to ingested plant material. Transgenic plants expressing proteins that can selectively inhibit the midgut digestive enzymes of particular phytophagous pests but not those of other arthropods or human/mammalian consumers might provide a route to reducing the reliance on chemical pesticides in crop production and storage systems (see Chapters 1-5, this volume). Perhaps the most studied molecules for control of proteinases are the class-specific proteinases inhibitors (PIs), serine PIs and cystatins, which respectively inhibit serine or cysteine proteinases. There are, however, other physiological mechanisms that restrict the activity of proteinases. For example many digestive proteinases are initially present in the gut as inactive zymogens (e.g. chymotrypsinogen, pepsinogen and trypsinogen), which to become active require the cleavage and release of a peptide prosequence. The mechanisms for this have been revealed by DNA sequence analysis, which shows that almost all proteinases are synthesized as inactive pre-pro-enzymes. Peptide extensions to
the N- or C-termini of mature enzymes are important for conferring targetting information which directs proteins to specific storage, secretion, cellular or extracellular compartments, or adds functionality to the enzyme itself. There are examples from all four classes of proteinases of recombinant or synthetic N-terminal proregions acting as inhibitors of their mature enzymes.1-4 In some cases they can exhibit quite high potency, with Ki values in the nanomolar range.1-4 In this chapter I will discuss the potential of N-terminal proteinase proregions to act as selective inhibitors of pest digestive enzymes.
12.2. Proteinase Precursors Most proteins that are destined for functional roles on the extracytosolic side of the endoplasmic reticulum, Golgi complex, lysosomes and nuclear or plasma membranes bear N-terminal signal sequences that direct the ribosomes engaged in their synthesis to the rough endoplasmic reticulum.5 This signal peptide, often termed the preregion, is mainly composed of hydrophobic amino acids and is typically 15-30 residues long. It forms a complex with a signal recognition particle,6 which then binds to a receptor on the surface of the rough endoplasmic reticulum and allows the nascent protein chain to be translocated
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across, or to be integrated into the lipid bilayer. Most soluble proteins have the signal peptide component of their N-terminal extension cleaved before synthesis of their polypeptide chain is completed. For the other part of the N-terminal extension, the proregion, it is generally accepted that it has two roles.7 First, it may adopt a native-like conformation enabling it to guide the correct folding of the attached mature proteinase, effectively acting as an intramolecular chaperone. Second, it can interact with the mature enzyme and occlude the active site, thus acting as an inhibitor, with each proregion being cognate to its own mature proteinase. The length of the proregion is not always the same or seemingly important. For example, the proregion activation peptide of the serine proteinase human cathepsin G is only two residues long (gly-glu), but this is enough to enable procathepsin G to be transported and sorted within the cell, without the danger of unwanted proteolysis taking place.8 Other proteinase proregions may be much longer, with some up to several hundred amino acid residues.9
12.3. Precursor Processing The generation of active proteinases is and for physiological reasons needs to be, tightly regulated so as to prevent uncontrolled proteolysis. Processing of the inactive precursor into an active moiety may be an intra-molecular (unimolecular) event, or an intermolecular (bimolecular) process. The proaspartic acid proteinase pepsinogen is activated at pH values below 5.0. In this case the 44-residues propeptide moiety is not liberated intact; instead, several shorter peptides are liberated, suggesting multiple proteolytic cleavages.10 At neutral pH the highly positively charged proregion of pepsin is bound to pepsin via ionic interactions involving negatively charged carboxylate groups.10 Residues ser-11-leu-44 occupy the substrate binding cleft. At low pH a conformational change in the position of the inhibitory propeptide is induced which exposes the active site cleft including the two catalytic aspartic acid residues, and frees the
Recombinant Protease Inhibitors in Plants
substrate binding sites. Synthetic peptides homologous to the proregion peptide sequence have been shown to inhibit the mature enzyme when added to a reaction mixture.11 Mammalian trypsinogen is converted to trypsin by the removal of an N-terminal peptide, by the specific enzyme enterokinase.12 Enterokinase recognizes the (asp)4-lys consensus sequence at the C-terminus of the N-terminal extension. The subsequent formation of N-terminal ile-16 (precursor numbering) permits the formation of a salt bridge to asp-194, that triggers a conformational change in the ‘activation domain’ of trypsin, creating the S1 binding site and the oxyanion hole necessary for this enzyme’s activity.13 Some insect trypsins do not have this enterokinase recognition sequence, but possess the highly conserved ile-val-gly-gly motif as their N-terminal amino acid sequence.14
12.4. Protease Proregions as Selective Inhibitors In many cases the interaction of proteinase proregions with their cognate mature enzyme is highly specific, as found with the cysteine proteinases papain and cathepsin B.9 Karrer et al9 showed that the papain subfamily of enzymes, including the mammalian cathepsins H, L and S, all have proregions longer than 90 amino acid residues, whereas the cathepsin B-like enzymes have proregions about 60 amino acid residues long. Mature papain and cathepsin B both have very similar 3-D structures, but the proregions show about three orders of magnitude difference in the inhibition of their cognate enzyme, in comparison to the other enzyme (Table 12.1).15 It may be that the occluding loop of cathepsin B, which imparts this enzyme's exopeptidase activity has interactions with its cognate proregion that cannot take place with papain, which does not have this occluding loop. 16 Further selectivity of inhibition within the papain-like cysteine proteinase subfamily has been shown.17 The cathepsin L propeptide, for instance, inhibits cathepsin L with a Ki of 0.88 nM, whereas the Ki is 44.6 nM for cathepsin S and >1000 nM for
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Table 12.1. Comparison of Ki values for the inhibition of papain and cathepsin B by their respective propeptides Ki of papain propeptide a
Ki of cathepsin B propeptide b
Papain
1.89 X 10-9 M
6.9 X 10-6 M
Cathepsin B
5.6 X 10-6 M
0.4 X 10-9 M
a Taylor, unpublished. b Reproduced with permission from: Fox T, Demiguel E, Mort JS et al. Biochemistry 31:12571-12576. © 1992 American Chemical Society
papain.To determine which segments of the cathepsin B proregion confer inhibitory activity, Chagas et al18 made overlapping peptides homologous to the sequence of this proregion. Chen et al19 also made a series of synthetic peptides homologous to the same proregion but deleted successive 5 amino acid residues in order to determine the regions important for inhibitory activity. Both groups found that relatively small regions of less than 10 amino acids were responsible for inhibition. The X-ray crystal structures of some cysteine proteinase precursors graphically illustrate the close contact that proregions have with the mature enzyme. 15,20,21 An extended polypeptide chain in the proregion of caricain runs through the substrate binding cleft of caricain in the opposite direction to that of the substrate (Fig. 12.1). Gly-84p lies between the active site cysteine, cys-25, and the solvent (Fig. 12.2), effectively preventing any proteolytic activity. This is an example of the exquisite interactions between a proregion and its cognate mature enzyme. Any significant changes to either of the two structures, the proregion or the mature enzyme, would prevent this interaction. It is the specificity and selectivity of these interactions between proregions and mature enzymes that could be made use of in the design of selective inhibitors of specific enzymes.
12.4.1. Cysteine Proteinase Inhibition: Papaya Proteinase IV Proregion We previously showed that the propeptides of two Carica papaya cysteine proteinases are efficient inhibitors of all four papaya cysteine proteinases.1 To test the potential of exogenous inhibition of insect digestive proteinases by protease proregions, we added a recombinant form of the propeptide of papaya proteinase IV (PPIV) to different preparations of Colorado potato beetle (Leptinotarsa decemlineata Say) digestive enzymes induced by distinct diet treatments:22 1. feeding on control potato leaves; 2. feeding on transgenic potato expressing the model protease inhibitor oryzacystatin I (OC-I);23 and 3. starvation under similar conditions.
It had been observed that feeding potato foliage expressing OC-I to beetle larvae reduced the activity of one of their major digestive proteinase,22,23 demonstrating the actual inhibitory effect of this inhibitor in the plant. As shown by complementary inhibition assays with OC-I, the recombinant propeptide of PPIV was also found to inhibit the OC-I- sensitive proteinase of CPB larvae,22 showing the ability of this polypeptide to act as an inhibitor of herbivorous insect digestive proteinases, and suggesting that expressing both OC-I-sensitive and OC-Iresistant proteinase propeptides in transgenic plants may prove useful to reduce crop losses.
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Fig. 12.1. Ribbon representation of the secondary structure elements of procaricain. The three helices of the proregion (upper part of the model) pack onto the mainly β sheet C-terminal domain of the mature caricain (lower part). An extended polypeptide chain of the proregion runs through the substrate binding cleft between the C-terminal and the N-terminal domains of the mature caricain. Reprinted with permission from: Groves MR, Taylor MAJ, Scott M et al. Structure 1996; 4;1193-1203. © 1996 Current Biology Ltd.
12.4.2. Serine Proteinase Inhibition: Manduca Sexta Trypsin Propeptides To investigate the inhibitory potency of synthetic propeptides towards trypsin activity from the midgut of the lepidopteran insect
Manduca sexta, we chemically synthesized two seven residue peptides, val-pro-ala-tyrpro-gln-arg (peptide 1) and val-pro-ala-asn-progln-arg (peptide 2).24 These peptides are identical to the proregions of M. sexta midgut trypsins.14 The activity of purified M. sexta trypsin using benzoyl-arg-p-nitroaniline as a
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Fig. 12.2. A diagram showing the proregion of caricain as it passes through the substrate binding cleft. The proregion is shown in ball and stick representation and mature caricain is depicted as a molecular surface representation. The position of gly-84p (proregion numbering) is indicated by the black arrow. Reprinted with permission from: Groves MR, Taylor MAJ, Scott M et al. Structure 1996; 4;1193-1203. © 1996 Current Biology Ltd. substrate was measured in the absence or presence of each peptide, at both pH 7.0 and pH 10.0. Both inhibitors were significantly more potent at neutral pH (Ki = 6.1 µM for peptide 1; Ki = 8.5 µM for peptide 2) than at pH 10.0 (315 µM for peptide 1; 352 µM for peptide 2), representing a decrease in potency of around 50-fold for each inhibitor at pH 10.0. Interestingly, neither peptide inhibited porcine pancreatic trypsin also using benzoylarg-p-nitroaniline as the substrate at pH 8.0, even at a 350-fold molar excess of peptide over enzyme, and both peptides were resistant to
proteolysis by M. sexta trypsin at pH 10.0 after incubation for 1 h at room temperature.
12.5. Discussion The digestive proteolytic enzymes in different Orders of commercially important herbivorous pests tend to be predominantly of one of the different major classes of proteinases. Coleopteran and hemipteran species tend to utilize cysteine proteinases25 while lepidopteran, hymenopteran, orthopteran and dipteran species mainly use serine proteinases.26,27 Examples from both of these
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classes of proteinases have been shown to be inhibited by their cognate proregions.1,2 Whilst zymogens of insect gut proteinases have not as yet been isolated, cDNA analysis indicates that the genes of proteinases in insects code for these proteinase precursors.14 Targetting mechanisms similar to those found in mammalian systems are likely to be in place but the precise method of processing inactive zymogens to active mature enzymes is still unknown. The effect of plant class-specific inhibitors on pest digestive enzymes is not always a simple reduction in proteolytic activity. In fact the reverse may happen. It would appear that there are often two populations of digestive enzymes in target pests, those that are susceptible to inhibition and those that are resistant,28,29 and that some insects respond to ingestion of plant PIs such as soybean trypsin inhibitor28 and oryzacystatin29 by hyperproducing inhibitor-resistant enzymes. It would also appear that insect digestive trypsins do not fall into the classic classification of peptidase hydrolases, as defined by inhibition spectra. It has been shown, notably, that the trypsin-like digestive proteases of several lepidopteran species are inhibited by (L-3-transcarboxiran-2-carbonyl)- L -leu-agmatin (E-64),30,31 an inhibitor generally considered to be specific to cysteine proteinases.32 Thus it will only be when we have protein crystals and X-ray diffraction data for the structure of insect enzyme/inhibitor complexes that the true interactions will become clear. The cloning and expression of both inhibitor-sensitive and inhibitor-resistant proteinase propeptides in transgenic crops may be a method of overcoming the resistance built up to individual class-specific plant inhibitors. It would be possible to synthesize genes coding for multiple propeptides, joined together by proteolytically susceptible bait regions, which when hydrolyzed would release the individual propeptides. If these propeptides are shown to be specific for pest digestive enzymes and also shown not to affect non-target digestive tract proteinases, this strategy may offer interesting advantages over the exclusive reliance on chemical pesticides.
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Acknowledgments I thank Niki Cummings for expressing the propeptide of PPIV in E. coli. This work was funded by the UK Biotechnology and Biological Sciences Research Council.
References 1. Taylor MAJ, Baker KC, Briggs GS et al. Recombinant proregions from papain and papaya proteinase IV are selective high affinity inhibitors of the mature papaya enzymes. Protein Eng 1995; 8:59-62. 2. Ohta Y, Hojo H, Aimoto S et al. Propeptide as an intermolecular chaperone: Renaturation of denatured subtilisin E with a synthetic propeptide. Mol Microbiol 1991; 5:1507-1510. 3. Fusek M, Mares M, Vagner J et al. Inhibition of aspartic proteinases by propart peptides of human procathepsin-D and chicken pepsinogen. FEBS Lett 1991; 287:160-162. 4. Fotouhi N, Lugo A, Visnick M et al. Potent peptide inhibitors of stromelysin based on the prodomain region of matrix metalloproteinases. J Biol Chem 1994; 269:30227-30231. 5. von Figura K, Hasilik A. Lysosomal enzymes and their receptors. Annu Rev Biochem 1986; 55:167-193. 6. Walter P, Blobel G. Signal recognition particle contains a 7S RNA essential for protein translocation across the endoplasmic reticulum. Nature 1982; 299:691-698. 7. Ikemura H, Inouye M. In vitro processing of prosubtilisin produced in Escherichia coli. J Biol Chem 1988; 262:12959-12963. 8. Salvesen G, Farley D, Shuman J et al. Molecular cloning of human cathepsin G: Structural similarity to mast cell and cytotoxic T lymphocyte proteinases. Biochemistry 1987; 26:2289-2293. 9. Karrer KM, Peiffer SL, DiTomas ME. Two distinct gene subfamilies within the family of cysteine protease genes. Proc Natl Acad Sci USA 1993; 90:3063-3067. 10. James MG, Sielecki AR. Molecular structure of an aspartic proteinase zymogen, porcine pepsinogen, at 1.8Å resolution. Nature 1986; 319:33-38. 11. Dunn BM, Lewitt M, Pham C. Inhibition of pepsin by analogues of pepsinogen-(1-12)peptide with substitutions in the 4-7 sequence region. Biochem J 1983; 209:355-362. 12. Grant DAW, Hermon-Taylor. Hydrolysis of artificial substrates by enterokinase and trypsin and the development of a sensitive specific assay for enterokinase in serum. Biochim Biophys Acta 1979; 567:207-215.
Using Protease Proregions as Regulators of Insect Digestive Proteinases 13. Hedstrom L, Lin TV, Fast W. Hydrophobic interactions control zymogen activation in the trypsin family of serine proteases. Biochemistry 1996; 35: 4515-4523. 14. Peterson AM, Barillas-Mury CV, Wells MA. Sequence of the three cDNA’s encoding an alkaline midgut trypsin from Manduca sexta. Insect Biochem Mol Biol 1994; 24:463-471. 15. Fox T, Demiguel E, Mort JS et al. Potent slow-binding inhibition of cathepsin B by its propeptide. Biochemistry 1992; 31:12571-12576. 16. Cygler M, Sivaraman J, Grochulski P et al. Structure of rat procathepsin B: Model for inhibition of cysteine protease activity by the proregion. Structure 1996; 4:405-416. 17. Carmona E, Dufour E, Plouffe C et al. Potency and selectivity of the cathepsin L propeptide as an inhibitor of cysteine proteases. Biochemistry 1996; 35:8149-8157. 18. Chagas JR, Ferrer-Di Martino M, Gauthier F et al. Inhibition of cathepsin B by its propeptide: Use of overlapping peptides to identify a critical segment. FEBS Lett 1996; 392: 233-236. 19. Chen Y, Plouffe C, Ménard R et al. Delineating functionally important regions and residues in the cathepsin B propeptide for inhibitory activity. FEBS Lett 1996; 393:24-26. 20. Turk D, Podobnik M, Kuhelj R et al. Crystal structures of human procathepsin B at 3.2 and 3,3 angstrom resolution reveal an interaction motif between a papain-like cysteine protease and its propeptide. FEBS Lett 1996; 384:211-214. 21. Groves MR, Taylor MAJ, Scott M et al. The prosequence of procaricain forms an α-helical domain which prevents substrate access to the substrate binding cleft. Structure 1996; 4;1193-1203. 22. Visal S, Taylor MAJ, Michaud D. The proregion of papaya proteinase IV inhibits Colorado potato beetle digestive proteinases. FEBS Lett 1998; 434:401-405.
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23. Benchekroun A, Michaud D, Nguyen-Quoc B et al. Synthesis of active oryzacystatin 1 in transgenic potato plants. Plant Cell Rep 1995; 14:585-588. 24. Taylor MAJ, Lee M. Trypsin isolated from the midgut of the tobacco hornworm, Manduca sexta, is inhibited by synthetic propeptides in vitro. Biochem Biophys Res Commun 1997; 235:606-609. 25. Murdock LL, Brookhart G, Dunn PE et al. Cysteine digestive proteinases in Coleoptera. Comp Biochem Physiol 1987; 87B:783-787. 26. Wolfson JL, Murdock LL. Diversity in digestive proteinase activity among insects. J Chem Ecology 1990; 16:1089-1102. 27. Ryan CA. Protease inhibitors in plants: Genes for improving defenses against insects and pathogens. Annu Rev Phytopathol 1990; 28: 425-449. 28. Broadway RM, Duffey SS. Plant proteinase inhibitors: Mechanism of action and effect on the growth and digestive physiology of larval Heliothis zea and Spodoptera exigua. J Insect Physiol 1986; 32 827-833. 29. Michaud D, Nguyen-Quoc B, Vrain TC et al. Response of digestive cysteine proteinases from the Colorado potato beetle (Leptinotarsa decemlineata) and the black vine weevil (Otiorynchus sulcatus) to a recombinant form of human stefin A. Arch Insect Biochem Physiol 1996; 31:451-464. 30. Lee MJ, Anstee JH. Endoproteases from the midgut of larval Spodoptera littoralis include a chymotrypsin-like enzyme with an extended binding site. Insect Biochem Mol Biol 1995; 25:49-61. 31. Novillo C, Castañera P, Ortego F. Inhibition of digestive trypsin-like proteases from larvae of several lepidopteran species by the diagnostic cysteine protease inhibitor E-64. Insect Biochem Mol Biol 1997; 27:247-254. 32. Dunn BM. Determination of protease mechanism. In: Benyon RJ, Bond JS, eds. Proteolytic enzymes: A practical approach. Oxford: IRL Press, 1989:57-81.
CHAPTER 13
Expression of Protease Inhibitors in Potato Conrad Cloutier and Dominique Michaud
13.1. Introduction
P
otato crops are produced worldwide with an estimated production of 18 M ha in 148 countries (FAO, http://www.fao.org). Potato ranks number 4 as a plant cultivated for human food production, with approximately 1 billion consumers and over 295 M metric tons (mT) produced in 1997. Annual production in the US (3rd world producer), is about 20.2 M mT compared to 4.2 M mT in Canada (13th world producer). In Canada, potato is the most important commercial vegetable crop with over 145,000 ha. Potato production in Canada and the US is a high-risk business. Potato is sensitive to about 50 infectious diseases, several species of nematodes, and dozens of sucking and chewing insect pests. Insect pests are a major concern to potato producers as they can reduce yields by 30%.1,2 The most important insect pest of potato is the Colorado potato beetle (CPB), Leptinotarsa decemlineata (Say). The CPB originated in Central America but spread to most agroecosystems in North America with the expansion of potatoes as a ubiquitous human food source. It is now also established in most parts of western Eurasia and is still expanding its range and status as a major potato pest. The CPB is the main target of pest control programmes for potato in Canada and the US, followed in importance by the aphids Myzuspersicae Sulzer (the green peach aphid) and Macrosiphum euphorbiae (Thomas) (the
potato aphid). Other insect pests including leafhoppers, plant bugs, flea beetles, June beetles and wireworms are locally important, but they have not had as much influence on research and pest control programmes as the CPB and aphid pests.
13.1.1. Current Approaches for CPB Control Pest control practices for the CPB (as well as other insect pests of potato) is still mainly based on chemical insecticides.3,4 A wide variety of contact and systemic insecticides belonging to four major chemical classes have been used and are still available. Insecticide resistance in CPB populations is a major concern in potato production. Organochlorine insecticide resistance first appeared 40 years ago and CPB resistance to chemicals from other classes is now widespread (reviewed in refs. 5-7). Multiple resistance has led to the recent commercial introduction of several new (or newly rediscovered) chemicals. Of the newly introduced products, the chloronicotinyl imida- chloprid has enjoyed the most recent use despite high cost, but the potential for genetic resistance in CPB populations is already a concern.8 The CPB is the focus of intense research for non-chemical pest control. Pest control specialists have realized that for CPB and other major pests with high evolutionary potential, control based on single tactics such
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as insecticides is not sustainable. Even the pesticide industry recognizes that widely applicable alternative control tactics for the CPB and similar pests would diminish pressure for finding ‘magic bullets’ within diminishing pools of synthetic molecules. Such pests require a dynamic multiheaded approach such as defined by Integrated pest management or IPM (see ref. 9 for a recent review). Also, broad-spectrum chemical insecticides do not fit well into environmentally-sensitive food production systems. Recent research on non-chemical potato pest control has mainly emphasized biological,10 physical,7 and semiochemical11 alternatives (see Fig. 13.1). Among the most advanced alternatives are the classically formulated bioinsecticides based on delta-endotoxins of the bacterium Bacillus thuringiensis (Bt). Several Bt insecticides are commercially available for controlling CPB (serovar B.t. tenebrionis or Btt) and lepidopterous (toxins from B.t. kurstaki) potato pests. As with conventional insecticides, the risk of resistance evolution to Btt in CPB populations is real (e.g., ref. 12), but Btt insecticides are currently effective against the CPB, despite stage-limited toxicity to small larvae.13 Their widespread use is slowed by their relatively high cost and by the continued availability of effective chemical insecticides. Key advantages of Btt biopesticides over synthetic chemicals include wider compatibility with biological control agents and synergistic action with them. We recently showed that in addition to direct toxicity, delayed mortality can also result from sublethal effects of Btt on larval feeding, vigor and development, through synergism with mortality inflicted by natural predators.14 Similar effects can be expected from interaction of Btt with parasitoids15 and pathogens (Ellie Groden, pers. comm.). On non-crop plants in its original habitats, the CPB is attacked by a diverse guild of natural enemies, but the CPB has escaped most of them through its massive geographical expansion. Many natural enemies of the CPB are not suitable candidates for classical biological control (i.e., the inoculation of exotic natural enemies) because they are
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maladapted to the climatic conditions in countries of adoption such as Canada. The beetle can exceptionally survive the harsh Canadian climate through its use of the soil microhabitat as a refuge.16,17 Unfortunately, digging deep into the soil to diapause is a specific trait of the CPB that is not shared by most of its natural enemies. Experimental releases of predators such as stinkbugs and coccinellids (feeding on potato aphids as well as the CPB) have shown control potential of these pests (e.g., reviewed in ref. 10), but the predators are not commercially available for large scale application. On the other hand, the entomophagous fungus Beauveria bassiana is a common disease agent of CPB and other insect pests. The application of B. bassiana conidia to potato foliage and to the soil (used by CPB for pupation and overwintering) has clear potential to control CPB (see ref. 18 for a recent report). However research on fungus virulence and tolerance to physical extremes, formulation as bioinsecticides, and application strategies is still needed before effective B. bassiana biopesticides become available for potato pest control. Physical control machines have been developed for CPB control based on heat killing or injuring by flaming, and air flow removal.7 However the relatively high cost of application, low efficacy and side effects have limited their use. Trapping with fieldperipheral shallow trenches lined with plastic film on which the beetle’s foot pads cannot adhere is currently the only physical control tactic that is used commercially in Canada and the US. This inexpensive control tactic can work because adult movement to and from overwintering habitats located inside and outside potato fields is an important life strategy of the CPB.7,104 The early-season migratory potential of overwintered adult CPB seems to be relatively slow to develop as it probably interferes with reproduction. This allows potato producers to benefit from field rotation as a way to delay spring infestation. Because field rotation distance should be at least 1 km to be effective, farming
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Fig. 13.1. Current approaches for CPB control. See text for details. specialisation generally limits the possiblity for wide use of field rotation. Pheromones remain undescribed for the CPB despite evidence for male attraction to females.103 In aphids including green peach aphid, intraspecific semiochemicals have been shown to be essential for sex19-21 and colony defense.22 Interestingly, the sesquiterpene (E)-β-farnesene, an alarm pheromone causing quick dispersal and colony disintegration in several aphids is naturally released by the type B trichomes of Solanum berthaultii. This close relative of cutivated potato is currently used as a source of potato resistance to insects (see below). Many technical problems are still unresolved with respect to developing aphid pheromones for application in potato crops. Antifeedants against CPB and potato infesting aphids have also been intensely researched. Glycoalcaloids from potatoes and related Solanaceae are known to be both deterrent and toxic to the CPB.2,23 Limonoids, the source of bitterness in citrus plants,24,25 and azadirachtin from the neem tree,26,27 are both antifeedant and toxic to CPB larvae.
Antifeedants could be integrated into IPM as stressors and synergists to insecticides including bioinsecticides based on Bt toxin.11,24 Among many alternatives to chemicals, some are thus available for IPM in potato production, and ways to integrate them into IPM both horizontally (within one season) or vertically (across seasons) have been defined (e.g., ref. 4). As with other crops, IPM system design for potato still depends to a large extent on empiricism and field experience, however, it could be guided more by ecological theory in the areas of plant community organisation, competition and symbiosis. For example, synergistic interactions between pest fitness reducing factors and compatible biocides (including conventional insecticides) remain virtually unexploited. However, for such potential to become really significant in potato production, a wider variety of fitness reduction tactics must be available for insect pests. A potential pest fitness reduction tactic that has not yet been significantly exploited in potato pest control is plant resistance.
Expression of Protease Inhibitors in Potato
13.1.2. Pest Fitness Reduction Based on Genetically-Resistant Potato No commercial variety of potatoes resistant to potato insect pests have been produced by classical plant breeding, despite the fact that considerable resistance to insects has been found in various Solanum species.28 Both S. chacoense and S. berthaultii are currently used as sources of potato breeding lines that exhibit relative resistance to the CPB and other potato pests.29 Hybrids of potato x S. berthaultii developed at Cornell University express a broad resistance to insect pests, based apparently on trichome exudate chemicals.30 In parallel, the genetic transformation of germplasm with DNA encoding protein factors of resistance has enormous potential for developing specific pest resistance in crops.31,32 In recent years rapid progress in plant genetic transformation has markedly accelerated the development of recombinant proteins from various organisms as herbivorous pest control molecules. Chewing insects, especially coleopteran and lepidoteran pests, have been the main target of this approach based on manipulating Bt toxin and protease inhibitors (PI) genes (see below). For sucking insects such as aphids and leafhoppers, different proteins are needed because currently known Bt toxins are ineffective, and their is little evidence that proteolysis plays a significant role in homopteran digestion.33 However there is evidence that various proteins, especially lectins and some PIs have negative effects on aphids when ingested in artificial diets.34-36 Tobacco plants made transgenic for snowdrop lectin acquired protection from aphids,37 while citokinin expressed in leaves of Nicotiana plumbaginifolia transformed with the isopentenyl transferase gene (ipt) fused to the gene of potato proteinase inhibitor II (PI-II) was shown to retard development in Myzuspersicae.38 A few years ago the successful transfer of the Btt Cry-IIIA toxin gene to several commercial lines of potato has produced the first cultivated potato plants that were highly resistant to the CPB. 39 More recently
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eggplants, which also have the CPB as a major pest, have also been transformed with Btt.40 Currently the complete resistance of Btt potatoes to the CPB is a striking success, but given the established fact that the CPB can evolve resistance to Bt toxins 12,41-43 the durability of CPB-resistant Btt-potatoes is the subject of much preoccupation, despite genuine concern for the problem by industry and phytoprotection regulating agencies (e.g., refs. 44 and 45). High-dose toxins including recombinant Cry IIIA in transgenic potato plants cause acute mortality of even the only partly susceptible CPB individuals (heterozygous for ‘resistance’ or R genes).39,43,46 Such plants can act as powerful selectors of toxin resistance genes in genetically variable CPB populations comprised of more or less mobile individuals. The high-dose approach followed by designers of Btt-potatoes requires complex strategies of Btt resistance prevention, including the provision of mimimum-size susceptibleplant refuges that can act as sources of non-resistant CPB individuals.42,45,47 These strategies are based on simulation models with stringent assumptions about the genetic basis of resistance to Bt toxins in pests as well as their sex and movement habits. They may be also very difficult to implement in actual farming systems because of economic and practical limitations.
13.2. Rationale for a Low-Dose Approach with PI-Based Resistance Compared to Bt toxins, protease inhibitors (PIs) are not inherently biocidal. In all living organisms self-made PIs are essential macromolecules. Herbivores frequently are exposed to extraneous PIs through normal feeding, since there is evidence that plants produce and stock PIs in storage organs and seeds for defense.48,49 Insect feeding on living plant tissue causes local and systemic responses leading to the synthesis of inducible PIs in damaged tissues. Potato leaves damaged by mechanical or insect wounding accumulate high levels of PI.50,51
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In herbivore food, PIs would act primarily by making food protein more difficult to use for amino acid extraction. PI action would cause protein wastage, thus reducing overall food quality and eventually leading to nutritional deprivation or imbalance. Quantitative or qualitive nutritional deficiency reduces metabolic efficiency and can impair normal functions. Experimental evidence based on feeding PIs to insect herbivores indicate that PIs can reduce rates of growth, development and reproduction (reviewed in ref. 48), i.e., they effect relative rather than absolute fitness reduction. With a greater variety of negative effects over a larger range of effective doses, PIs do offer more options as plant resistance factors than toxins such as the Cry proteins of Bt. Also a high-dose approach with PIs may be undesirable or ineffective for various reasons. Extraneous PIs could interfere with normal plant metabolism and growth, or be unstable and subject to high turnover if recognized as nonself protein by plant cells. Significant amounts of relatively unspecific PIs in harvestable plant tissue may not be compatible with human or animal food safety requirements. Finally the production of large amounts of PIs by plant cells competes for the allocation of resources that are also essential to plant metabolism or are part of harvestable yield. On the other hand, low-dose expression of PIs (e.g., 0.5-1.0% of total soluble proteins) in plants may be especially suitable to designing crops with partial resistance, which has some advantages over full resistance. Relative resistance is more likely to integrate smoothly into biologically-oriented IPM systems. Compatibility with pollinators and other beneficial organisms that live within plant canopies and sometimes feed on transgenic pollen and nectar should be considered (see Chapter 8, this volume).52-56 When plant resistance is partial, positive interaction with various biocides and especially with the natural enemies of target pests can be exploited for enhanced pest control. Reduced foodplant qualilty causing lower herbivore fitness (e.g., small body size, slow
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development, low vigor, small egg clutches) is generally favorable to predators and disease agents. Factors of fitness reduction can provide for greater pest control when combined with natural control than when used as a single control tactic. These generalizations are supported by theory,57-59 as well as empirical evidence from diverse crop-pest systems.60-63 As with any effective pest control approach, we should be concerned with the potential in target pests for resistance evolution to a PI or PI-complex. This potential should be proportional to PI impact on pest fitness. Since PIs do not kill, the rate of resistance evolution in pest populations should be less than for a quick action biocide. In the worst situation, a pest might become resistant to a PI-expressing plant with partial resistance just as quickly as with a fully resistant plant (e.g., the current Btt-potato). This could occur if mortality factors acting jointly with plant resistance (e.g., natural enemies) in fully reducing pest fitness (i.e., controlling the pest) would do so in a highly specific and systematic way. A population genetics model by Gould et al64 indeed predicts that if control of a pest is effected by systematic natural mortality complementing partial crop resistance, its field resistance under pressure from a pest population with rare genes of virulence should persist just as long as that of a fully resistant plant. Nevertheless a rationale for PI-expressing transgenic plants with relative pest resistance does exist in IPM-based pest control. The slowing of pest development or reproduction may in itself be sufficient to prevent plant damage, and may also augment natural pest mortality through extended pest exposure. This qualitatively different potential with PIs should not be ignored with potato crops given their growing importance on a worldwide basis, and the parallel increasing importance of pests such as the CPB. However many questions must be answered before that potential could be exploited. It is well known that highly effective chemical insecticides initially were obtained empirically, with little knowledge of
Expression of Protease Inhibitors in Potato
their precise mode of action at the cellular level. By contrast, designing effective PIs will likely require very specific knowledge of target proteolytic enzyme systems, digestive proteolysis regulation in insect herbivores, and characteristics of defensive PIs used by plants. A problem emerges from the very fact that PI-based plant resistance does not involve gross toxicity. Herbivores feeding on such plants do survive at least in the short term. Meanwhile they can use all the resources of their complex life support systems to escape, tolerate or compensate negative effects of PIs. Therefore the nature of the biochemical and physiological interactions involved in PI activity are much more critical than for acutely toxic biocides.
13.3. The CPB Digestive Proteolytic System As with various insects of different food relationships, 65 food digestion in CPB involves multiple proteolytic enzymes. Wolfson and Murdock 66 first presented evidence of cysteine and aspartic proteinase activity in the midgut of CPB larvae. Proteolytic activity measured with methaemogobin as a substrate was maximum at mildly acidic pH, activated by the reducing agent L-cysteine, and inhibited by E-64, a fungal PI specific to cysteine proteinases of the papain family. The occurrence of aspartyl proteinase activity was indicated by inhibition with pepstatin, a specific inhibitor of aspartyl proteinases. The study suggested that at midgut pH, cysteine proteinases were mainly responsible for proteolysis, whereas aspartic activity could become effective at lower pH. Using syntetic substrates, protease activators and class-specific inhibitors at various pH, Thie and Houseman67 then indentified cathepsin B-, cathepsin D- and cathepsin H-like activities in the midgut of CPB larvae. Cathepsins B and H are cysteine proteinases active mostly under mildly alcaline pH and sensitive to inhibition by E-64. Cathepsin D is an aspartic proteinase active under acidic pH and sensitive to pepstatin. Without presenting direct evidence, Houseman et al65 also reported cathepsin L activity in CPB.
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13.3.1. Inhibition of CPB Digestive Proteases More recently we used various PIs and azocasein as a substrate to document potential sensitivity of the larval CPB proteolytic system to the protein PIs oryzacystatins I an II, OC-I and OC-II. 68 The tripeptidic inhibitor leupeptin—which discriminates cathepsins B and H69—and the OCs caused only partial inhibition when used singly, but E-64 or a combination of leupeptin plus either OC-I or OC-II almost completely inhibited proteolysis. When tested on purified animal cathepsins B, H and L, both rice cystatins were also shown to inhibit cathepsin H, but not cathepsins B and L. Noteworthy, the inhibition by OCs increased linearly between pH 5.0 and pH 7.0. As cathepsin B- and cathepsin H-like enzymes in CPB show differential pH optima for activity (i.e., slighltly-acidic pH for cathepsin B, and neutral pH for cathepsin H),67 this observation was strongly suggesting that the rice cystatins are specific to only one major catheptic form of CPB larvae, presumably a cathepsin H-like enzyme. Developmental variation of azocaseinolytic activity in extracts of CPB larvae of all stages and adults was then investigated.70 In all cases the activity was maximal in the pH range 4.5-6.5, with an optimual value of 5.5. Proteolytic activity increased exponentially with larval stage, as expected from growth. However on a weight specific basis, azocaseinase activity was similar among larval stages, being 3 to 3.5 times higher in larvae than in adults. Depending on stage, E-64 was inhibitory to 86-91% of total activity at pH 5.5, confirming the predominance of cysteine proteinases. Pepstatin caused 9-14% inhibition under the conditions used, confirming the presence of aspartyl protease activity, presumably the previously identified cathepsin D.67 We also attempted to separate proteolytic forms in extracts of 3rd-instar and adult CPB using mildly-denaturing gelatin/SDS-PAGE.70 The resolved proteins were tested for gelatinase activity in gel over a broad pH range after protease renaturation. All nine bands detected in adults and larvae were sensitive to E-64 but none to pepstatin, suggesting the loss of
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the expected aspartyl protease activity in the process. Interestingly PMSF activated gelatinase activity under reducing conditions, suggesting the existence of serine proteinase activity (this PI is specific to serine proteinases under such conditions). In another assay, cysteine proteolytic activity in extracts from 3rd instars and adults was fully sensitive to E-64 and to the combination leupeptinOC-I, but only partly sensitive to either inhibitor (e.g., maximal inhibition of 30% by OC-I at pH 5.5), confirming the previously reported specificity of OC-I against the CPB cathepsin H-like enzyme.68 This study further confirmed the complexity of the CPB protease system, and added to our knowledge by establishing that the same pattern of enzymes was available to all CPB stages with no qualitative variation. The specificity of OC-I and OC-II toward only one of two major proteinases of the CPB was also confirmed. Thus even PIs that evolved as actual plant defense systems (as is likely the case with OC-I: see Chapter 3, this volume) can exhibit narrow specificity, limiting their efficacy at challenging insect systems based on multiple proteases. Concern for OC specificity and particularly for immunity of the insect cathepsin B-like proteinase to OC-I led us to investigate the possibility of complementary inhibition by recombinant human stefin A (HSA), a cystatin showing affinity toward human cathepsin B.71 Inhibition of azocaseinase activity by extracts of adult CPB at pH 6.0 was 87% for E-64, 43-44% for OC-I, and 43-76% for HSA. The wider HSA inhibition suggested that at low concentration, HSA behaved similar to OC-I with respect to E-64 sensitive proteinases, but was markedly more inhibitory than OC-I when used in large excess. In beetle extracts cleared from either OC-I- or HSA-sensitive proteinases by affinity chromatography, there remained virtually no residual OC-I-sensitive activity, but significant HSA-sensitive activity. The stability of these relatively specific PIs to the non-target enzymes in the system was also examined in this study. When incubated with extracts cleared of sensitive
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enzymes, recombinant OC-I remained unaffected (as visualized on SDS-PAGE), but HSA was subject to multiple-site proteolysis. Overall, it appeared that HSA was little efficient at complementing the inhibitory spectrum of OC-I, partly due to its high susceptibility to the insect insensitive proteinases. To gain knowledge about the nature of the insect OC-sensitive and -insensitive cathepsins, our group recently purified and partially characterized CPB cysteine proteinases extracted from 3rd-instar larvae (Visal et al unpubl.). The putative cathepsin H- and B-like proteinases were first isolated by affinity chromatography using GST/OC-Iagarose and Divicell ε-aminocaproic acidPhe-Phe-CH3 affinity gels for cathepsin H and cathepsin B, respectively. Two cysteinetype proteinases were isolated, and their weight estimated at about ~28 kDa (cathepsin H-like enzyme) and ~67 kDa (cathepsin B-like enzyme) by SDS-PAGE. We also developed a two-step electroblotting gelatin/PAGE system72 to visually discriminate the two enzymes. In agreement with the chromatographic data, two proteins with molecular weights of 28 kDa and 67 kDa were detected using this approach. As expected, both enzymes were strongly inhibited in gel by E-64 (85-95%), while only the 28-kDa protein was sensitive to OC-I, OC-II and HSA. In a recent study, Novillo et al73 added a chymotrypsin-like enzyme with a pH optimum of 5.5-6.5 range to the list of proteinases secreted by CPB larvae feeding on potato foliage. Evidence was based on lysis of a protein (azocasein) and two different peptide substrates, combined with inhibition by several serine PIs, especially PMSF. Gelatin/ SDS-PAGE suggested that the chymotrypsin consisted of a major 63-kDa and a minor 100-kDa fractions, although such molecular weight estimations using the gelatin/PAGE system may be erroneous and would need to be confirmed by other means.72 The study also reported the occurrence of two exopeptidases, leucine aminopeptidase and carboxypeptidase A in CPB larvae. Activity zymograms of endoperitrophic midgut contents revealed 9 proteinase forms. This is similar to our
Expression of Protease Inhibitors in Potato
previous study,70 although only 5 bands were inhibited by the cysteine PI E-64—the 4 other bands were inhibited by PMSF—and only one E-64-sensitive band was activated by PMSF. L-Cysteine was used to examine the influence of reducing agents on the detection of chymotrypsin activity, which was reported to be null or negligible in previous studies (e.g., ref. 74). With L-cysteine added to the medium, azocaseinolysis attributable to cysteine, aspartic and chymotrypsin activity followed a 2:1:0 ratio, compared to 1:1:1 without L-cysteine. The addition of thiol compounds seemed to mask chymotrypsin proteolytic activity in assay buffers, possibly reflecting the low intrinsic reducing power of gut extracts from CPB larvae. Segregation of chymotrypsin and other proteolytic activity among the various compartments of the larval gut was also examined by Novillo et al.73 Chymotrypsin and cathepsin B, D and H activities were mainly located in the endoperitrophic and exoperitrophic spaces, with no clear differentiation along the midgut. Interestingly, nearly 17% of cathepsin H-like activity was associated with the epithelium tissue, suggesting a role for this aminoendopeptidase in the last steps of dietary protein breakdown in CPB. Leucine aminopeptidase activity was highest in the midgut epithelium, but traces were also present in the lumen. Based on these findings, proteolysis in the CPB larval midgut would start in the endoperitrophic space of the midgut lumen with chymotrypsin, cysteine (cathepsins B and H), and aspartyl (cathepsin D) proteinases. The resulting peptides would then be hydrolysed to amino acids in the ectoperitrophic space by aminopeptidases (including cathepsin H) and carboxypeptidase A in the midgut wall before amino acid absorption (see Fig. 13.2). Along with the presence of cathepsin D, chymotrypsin activity in CPB73 provided direct evidence for the production of midgut proteinases that are immune not only to OC-I but to cysteine PIs in general. This observation suggests that in order to challenge CPB digestion it may be suitable, in parallel to the use of cysteine PIs, to use chymotrypsin: or
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aspartic-type inhibitors, or a combination of these different inhibitors. Novillo et al.73 also stressed out the importance of assessing the role of chymotrypsin in the activation (cleavage) of the Btt pro-toxin, and thus in the insect susceptibiity to this biopesticide.
13.3.2. Plasticity and Adaptability of CPB Proteolytic Enzymes In a chronic PI ingestion study, Bolter and Latoszek-Green75 used specific PIs to characterize changes in the caseinolytic activity extracted from midguts of 4th-instar CPB larvae fed potato foliage. The E-64-sensitive fraction (i.e., the cysteine proteinase activity) accounted for ~65% of total proteolysis, whereas the pepstatin-sensitive fraction (aspartyl proteinase activity) accounted for the remaining ~35%. At high E-64 concentration in the food, the inhibitory power of pepstatin decreased, with the larvae feeding chronically on E-64 producing little aspartic proteinase. Curiously, these larvae seemed to function with proteinases that were sensitive in vitro to neither E-64, pepstatin or trypsin proteinase inhibitors. These results were rather discouraging regarding the potential for effective inhibition of the CPB proteolytic system, which momentarily appeared untracktably plastic. However the inhibitors E-64 and pepstatin (like HSA: see above) may simply be poorly suited to sustained in vivo inhibition of target cathepsins in the CPB. It is possible that these PIs do not exhibit sustained efficacy in vivo because their functionality as PIs has not been selected for in the right evolutionary context, in this case the CPB midgut. Plasticity of CPB midgut enzymes is also expressed in response to diet. To examine food plant effects, our group investigated the azocaseinolytic activity in midguts of 4th-instar CPB larvae that were continuously fed foliage from potato (standard diet), or larvae that had been switched 3 days before the test onto tomato or eggplant foliage.76 Compared to control larvae on potato, total protease activity of switched larvae was reduced to 40% on the eggplant diet, while being increased to 150% on tomato. Sensitivity to
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Recombinant Protease Inhibitors in Plants
Fig. 13.2. Two-step degradation of dietary proteins in the midgut of CPB. After entering the endoperitrophic space of the midgut lumen, the ingested proteins are first cleaved to polypeptides and smaller peptides by the endopeptidases cathepsin D (cD), cathepsin B (cB) and chymotrypsin (C), and probably by the aminoendopeptidase cathepsin H (cH). The resulting peptides are then processed to smaller peptides and amino acids in the ectoperitrophic space by the insect digestive exopeptidases leucine aminopeptidase (LA), carboxypeptidase A (CA) and cathepsin H, which are absorbed through the midgut epithelium and integrated to the insect endogenous metabolism. E-64 also varied with diet, with an inhibitory rate of 80 to 90% in extracts from potatoand tomato-fed larvae, but with a rate limited to 55% in the eggplant-fed larvae. Gelatinolytic activity detected on polyacrylamide gels indicated changes in midgut proteolytic forms that could be correlated with diet and its effects on overall protease activity. In eggplant-fed larvae compared to those fed potato or tomato, fewer proteinase forms and lower activity of some forms common to other diets became clear. Among a total of 9 bands detected in tomato-fed larvae, at least one was specific to each host plant. The overall diversity of enzyme forms that can be produced on natural food is possibly even larger than observed here with gelatin as a substrate. The experiment showed that even though CPB larvae are unlikely to experience a succession of food plants during development, they can quickly adapt their proteolytic system to a different host plant. Specific
factors in each diet were evidently triggering the changes, but their nature was not investigated. Starvation effects on enzyme patterns in the CPB represent another facet of plasticity, as revealed recently.77 In this study we used gelatin/PAGE to compare the proteolytic forms found in starved 3rd-instar CPB larvae to those found in similar larvae fed foliage of untransformed ‘Kennebec’ potato, or fed OC-I-transgenic potato. As expected from previous work with recombinant OC-I, the activity of a major proteinase form on gels was lowered in larvae fed OC-I foliage. Extracts of starved larvae produced gel patterns that were modified in various ways. Starved larvae overexpressed the OC-Isensitive proteinase, but did not express the insensitive forms. Most interestingly, they expressed a novel gelatinase form not seen in other extracts. The role of this starvationspecific enzyme as well as the physiological
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significance of starvation-induced changes in the ratio of OC-I-sensitive to -insensitive forms were not easily interpretable, but they clearly added a further dimension to the problem of digestive plasticity in the CPB.
available proteolytic potential, top layer compensatory processes could eventually come into action, possibly allowing continued use of food loaded with PI with little fitness reduction.
13.4. Fitness Consequences of PI Ingestion in CPB
13.4.1. Biossays with Purified Inhibitors
Many conditions must be fulfilled for a PI engineered in the host plant of an insect to reduce proteolysis to the extent that it could not sustain itself, and eventually adapt. PIs may have significant inhitory power on the in vitro proteolytic activity of whole-midgut extracts of insects, but they must function in structured pests rather than in crude extracts. Potential PI operational conditions in vivo are variable, including the multi-chambered insect midgut lumen, the endo- and ectoperitrophic spaces, and the intracellular midgut epithelium where final peptide digestion may occur.65,78 When foodstuff is being ingested and initially digested by a chewing insect, the inhibitory potential of a PI on target enzymes could also be affected by food plant proteins, as well as by other native biochemicals of plant cells that can interfere directly.79 As has become clear for the CPB, biochemical redundancy of the target system can be considerable at any time, and it is potentially dynamic. Midgut secreta may be variable in the short-term on a mealwise basis, with possible regulation by the food protein itself (ref. 65 and references therein). Also it is known that feeding on a diet loaded with PI can cause the secretion of insensitive midgut proteases (e.g., ref. 80). Higher order organismic reactions of the herbivore are also possible. When forced to depend on digesta (assimilable foodstuff) that are suboptimal because of PI content, insect performance could still be maintained by physiological and behavioral compensation. Homeostatic mechanisms have evolved in phytophagous insects as natural adaptations to allow sustained growth or reproductive output under a range of natural foods and feeding conditions. Unless a PI or PI-complex acts by quick and stable inhibition of most
With all these considerations it is difficult to predict the fitness consequences of feeding on a diet with PI, and thus empirical demonstration is essential. Several experiments have examined CPB fitness reduction by PIs, ranging from short-term feeding assays using purified or recombinant class-specific PIs coated on plant foliage to longer term trials extending several life stages. These trials have involved either purified recombinant PIs, recombinant PIs expressed in transgenic plants, or native PIs or PI complexes induced in live plants. Feeding assays with pure PIs at high concentrations in diet have biological significance only insofar as the PI level in the ingested food matches the target protease levels and binding affinity in the insect midgut.48 Given the historical development of current knowledge of the CPB proteolytic system, it is not surprising that experiments with this insect have mainly used cysteine PIs. Wolsfon and Murdock 66 fed newly hatched CPB larvae for up to 8 days with potato leaves coated with gelatin containing the inhibitors E-64 and pepstatin. E-64 had a dose-dependent effect on short-term weight gain. At high concentration (1 mg E-64/ml gelatin), 8 d-old larvae only weighed about 15% as much as controls. Pepstatin was much less effective, as similarly aged larvae weighed about 60% as much as controls, even at high PI concentration. Our group,70 in a 3-d feeding assay of growth inhibitory effects of recombinant OC-I coated on potato leaves (at 400 µg/cm2), observed that weight gain was reduced only 20-30% in 1st- and 2nd-instar larvae of CPB, while 3rd- and 4th-instars grew normally. Bolter and Latozek-Green75 reared CPB larvae and adults by ad libitum feeding on
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potato leaves coated with E-64-containing gelatin (0-2 µg E-64/cm2). Their experiment specifically aimed at mimicking the chronic aspect of feeding on a PI-resistant plant. The fitness indices measured were the survival to adulthood, development time, growth rate and female fecundity. All indices were affected in a more or less dose-dependent manner by E-64. At highest effective doses, pre-adult mortality reached 76%, development was prolonged by 61%, and egg production was prevented (reversible on untreated foliage). Hatching of F1 progeny was not affected, but their fecundity was more reduced by E-64 than that of the parent generation. Decreased caseinolytic activity in extracts of dissected midguts from 4th-instars correlated well with E-64 dose in the food, suggesting links between PI feeding, ability to digest food and fitness reduction. Overall the results were encouraging because it was apparent that CPB larvae adapted only with great difficulty to high doses of E-64. Strong fitness reduction even including significant mortality was observed, despite the adaptive responses that were also manifest.
13.4.2. Biossays with PI-Expressing Transgenic Plants Recently we studied the performance of CPB larvae feeding on OC-I-expressing potato,105 transformed with an OC-I cDNA sequence linked to the CaMV 35S promoter using the Agrobacterium tumefasciens vector.81 After confirming the inhibitory activity of recombinant OC-I against CPB digestive proteinases, transgenic potato clone K52 expressing OC-I in foliage at about 1% of total soluble protein was chhronically fed to newly hatched larvae. Larval performance was continuously monitored by daily measurement of food consumption, growth as weight gain, and development until early adulthood. During early larval stages (L1 to L3) the larvae consumed OC-I transgenic foliage 14 % faster and accumulated 28% more body mass than controls fed foliage from the same untransformed potato line. Improved performance of late larvae feeding on OC-I foliage was expressed as faster development rather
Recombinant Protease Inhibitors in Plants
than faster growth, leading to early emergence of full size adults significantly earlier than controls. Thus contrary to expectation, fitness indices were positively affected on OC-I foliage. Evident benefits of faster feeding on OC-I foliage were allocated initially to faster biomass accumulation, but late-stage larvae instead priorized faster development. Despite the presence of PI in the foliage and faster consumption by larvae, there was no evidence that more foliage was wasted as might be expected from inefficient protein digestion. The percentage conversion of foliage to body tissue during larval development averaged 22-24% for both control and OC-I-fed larvae. In a parallel study, young CPB adults were chronically fed K52 foliage expressing two levels of inhibitor on fully developed plants.106 In our transgenic line the PI level varies with leaf age, increasing initially and then decreasing in senescing leaves, so that the OC-I level is maximal in fully expanded mature leaves (≥1 order of magnitude higher than in youngest and oldest leaves). In females, performance was measured by pre-parturition weight gain during postemergence ovogenesis, as body mass increases approximately 50% from the time of emergence, and rate of egg laying. As with larvae, complex and unexpected responses to OC-I foliage were observed. Beetles consumed mature foliage with the highest OC-I level 3-4 times faster than mature untransformed foliage. They treated mature OC-I- containing leaves similar to untransformed immature foliage. This is normally consumed faster than mature foliage, indicating that it is less nutritious. Despite strong effects on consumption, maximum weight attained by gravid females and the rate of egg laying were similar on all foliage treatments, with no obvious diet-related variation of female ability to obtain nutrition for reproduction. However female productivity in terms of eggs laid per unit of food consumed was only about 35% on OC-I-rich foliage (70 vs. 187 eggs/g wet), suggesting that OC-I decreased foliage quality for ovarian growth, as expected with a PI. Thus there is evidence that OC-I expression in K52 can affect its nutritional value to the CPB in
Expression of Protease Inhibitors in Potato
the expected way, but females can easily compensate by being hypertrophic, thus achieving full fitness. Following these observations we also examined female CPB oviposition behavior on OC-I foliage, both in the field and the laboratory. In the laboratory, females were allowed to feed and lay eggs for 7 days in a simple choice situation with both K52 and control ‘Kennebec’ foliage. Clear evidence of female preference for OC-I foliage appeared within 24-48 hours of contact and feeding on both kinds of plants. In the field, data on egg laying during peak oviposition in replicated plots of both OC-I transgenic and control potato also indicated oviposition preference by overwintered adults. The laboratory data failed to indicate female ability to discriminate OC-I and control foliage on simple contact. Instead, it seems that female preference for oviposition on OC-I foliage might involve nutritional feedback, possibly in relation to the fact that they feed more voraciously on the OC-I foliage (see above).
13.5. OC-I-Expressing Transgenic Potato and CPB Natural Predators As indicated previously, attention must be given to potentially undesirable side effects of developing PIs as plant resistance factors (also see Chapter 8, this volume). Negative effects of PI-expressing transgenic plants on pest natural enemies could be counterproductive in IPM emphasizing plant resistance and limited reliance on harsh chemicals. Natural enemy guilds of particular pests comprise several taxonomic categories frequently overlapping that of the pest itself. It is likely that natural enemies could be affected by PIs of any level of specificity, especially by broad- spectrum PIs (see Chapter 9). This is particularly likely considering the emerging developments in the design of highly-efficient PIs by protein engineering (see Chapters 10 and 11). Of the natural predators and disease organisms currently investigated for biological control of the CPB, the stinkbug Perillus
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bioculatus is probably the most specialized and efficacious, having shown clear potential for mass releases as a predator of CPB eggs and larvae.82,83 This stinkbug is also a natural predator of the CPB in potato fields over large parts of North America, especially in western US. Its potential prompted us to study P. bioculatus as a model for investigating PI-predator interactions. Stinkbug predators could be exposed to PIs from transgenic host plants of their prey mostly through ingesting unbound PI in the gut of recently killed larval—or exceptionally adult—CPB prey. Stinkbugs could also ingest small amounts of plant juice obtained by superficial stinging of plant tissue using their haustellate mouthparts, in order to survive temporarily when hunting on plants at low prey density. We characterized the azocaseinolytic activity of P. bioculatus midgut and whole body extracts using class-specific PIs.55 Similar to CPB prey, proteolytic activity was maximum at mildly-acidic pH (pH 4-6). Between pH 3 and pH 8, inhibition by E-64 and PMSF ranged from 25 to 92%, and from 12 to 54%, respectively. On a weight specific basis, proteolytic activity was low in 1st- and 2nd-instar nymphs compared to 3rd-5thinstars and adults. Proteolytic activity was much more sensitive to E-64 than PMSF inhibition in stages 3 to adult, but it was about equally sensitive to these inhibitors in stage 1 and 2 stinkbugs. Gelatin/SDS-PAGE revealed 5 proteinase forms in 5th-instar nymphs, in addition to minor bands attributable to traces of CPB prey fed to the predators. Recombinant OC-I, OC-II and HSA all strongly inhibited proteolysis in extracts of 5th instars (>70% inhibition), revealing that in P. bioculatus up to 90% of proteolysis may be affected by cysteine proteinases, i.e., perhaps even more than in the CPB prey. The relatively high sensitivity to PMSF in P. bioculatus, especially in young nymphs, suggests a clear role for serine proteinases. Compared to older stages, digestion in small P. bioculatus nymphs may be more adapted to yolk protein digestion, either when feeding on prey egg masses such as those of the CPB or, in non-feeding 1st instars, when using maternal yolk still
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remaining in the gut after hatching. Also stinkbug predators must start digesting prey tissues extracorporally by injecting saliva into prey carcasses, a process which has been shown to involve serine proteinases in other predaceous Hemiptera.84 We also documented the potential for interference of OC-I with P. bioculatus female reproduction.53 Females were chronically fed for 18 d on 3rd-instar CPB larvae injected with 1-16 µg of recombinant OC-I, and monitored for fitness reduction using various indices for 28 d (up to 10 d after cessation of PI treatment). Mortality was negligible but at high doses, 50% of the females were sterile. In fertile females, egg laying was delayed in a dose-dependent manner. Dose-dependent effects also included lower fecundity, and reduced egg clutch size and egg eclosion incidence, indicating reduced oogenesis in OC-I-fed females. Azocaseinolytic activity in OC-I-fed females 3 days after the initial treatment had increased about two-fold, indicating partial compensation, but proteolysis was still sensitive to OC-I inhibition. Interestingly, females chronically fed OC-I tended to attack prey placed in front of them more promptly, suggesting consistently low satiation despite equal feeding frequency as controls. Hungrier stinkbugs in natural conditions should have more impact on CPB prey numbers, but whether sufficiently high field exposure to OC-I is attainable through normal feeding (i.e., from prey that ingested the PI from foliage) is unlikely (Brouchard et al, unpublished). In summary these studies are consistent in showing that cysteine-type proteolysis is very important to P. bioculatus, perhaps more important than for the CPB target pest which appeared better able to compensate OC-I ingestion. Fitness effects of OC-I on P. bioculatus seem more akin to those expected from its ‘unchallenged’ proteolytic profile, than for that of the CPB prey. Proteolytic redundancy and adaptability is probably lower in this predator than in its prey. Compared to living plants which generally can react to small herbivores feeding on them, prey are quickly killed and so prey defenses against
Recombinant Protease Inhibitors in Plants
predators may be generally less likely to involve PIs. Thus predators would be relatively more sensitive to PIs and less versatile when challenged with PIs. However more data are needed to support these contentions.
13.6. Conclusions and Future Perspectives Despite early evidence in our studies that the potential of OC-I for bioctechnological development of potato with useful resistance to the CPB was limited (see ref. 107 for data from a different source and another viewpoint), this inhibitor has been a useful tool in developing understanding of the CPB digestive proteolytic system, as well as potential problems with developing plant resistance based on the expression of relatively specific PIs. The potential of OC-I clearly is too limited in the face of the CPB digestive plasticity. CPB could be an especially difficult target, and thus specific PIs such as OC-I could still find applicability in less versatile pests. However, plant PIs may in general tend to be more specific than PIs from other organisms, as a result of a coevolutionary ‘arms race’ with proteolytic enzymes of insect herbivores. If so, PIs with useful specificity to herbivorous pests and immunity to their adaptive responses would most likely be found among plant defensive PIs, as fine tuned products of coevolutive adaptation to specific pest guilds. Thus the best potential source of PIs with these characteristics might be a plant that, under selective pressure from herbivory or microbial infection, has evolved effective PI-based protection against cognate pests. In some cases, herbivores may be sensitive to defense-related PIs that have evolved in plants unrelated to their hosts, as shown for example by the efficacy of potato PI-II against a lepidopterous pest of rice.85 However, despite potential for immediate efficacy even on extraguild pests, plant PIs may not generally be able to resist the modifiable proteolytic systems of versatile pests that are not cognate to their own evolutionary history. Our experimentation with OC-I in potato foliage has produced quite unexpected
Expression of Protease Inhibitors in Potato
results. Instead of being resistant to the CPB, the clone K52 was especially susceptible to the target pest. However, this situation has revealed important facts about the extent of compensation. Of course K52 is an ‘imperfect’ transgenic, one reason being the strongly heterogenous expression of the PI in leaves, allowing the insect to access both PI-rich and PI-free foliage for feeding. By contrast with results from our feeding assays using detached leaves of known OC-I content, those concerning behavior and performance on whole plants are not so easy to interpret. Since it was isolated from rice,86 OC-I was transferred to several plants87 including tobacco,88 rapeseed,89 poplar90 and potato.81 Although convincing results were obtained in some specific cases,90,91 in several cases relatively low levels of resistance to pests were conferred to OC-I-expressing plants. Considering the limited potential of narrow-spectrum PIs like OC-I alternative approaches are now being examined, including the improvement of PI binding capacities by protein engineering,80,91,92 the use of insect PIs specific to their cognate proteinases,93 the use of protease proregions as regulators of pest proteases77,94 and the isolation of stressinduced PIs in plants with broad-spectrum affinity for specific classes of proteinases.95,96 Recently we assessed the potency of γ-linolenic acid (GLA), a polyunsaturated fatty acid structurally homolog to α-linolenic acid (ALA), in inducing the synthesis of novel PIs in tomato. Polyunsaturated fatty acids in general are potent inducers of systemic resistance to pathogens in plants.97 For instance, arachidonic acid, which is produced naturally by the late blight fungus Phytophtora infestans, activates defense-related genes in potato via an ALA/JA-independent pathway.98,99 By using papain as an affinity ligand we isolated a 55-kDa protein named TCPI (for tomato cysteine proteinase inhibitor) from GLA- treated foliage. As shown on Western blots, TCPI is immunologically distinct from tomato multicystatin, an 88-kDa cysteine PI induced in tomato leaves via the ALA/JA pathway.100 When compared to OC-I, OC-II and HSA, TCPI was more
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broadly inhibitory to azocaseinolytic activity in extracts from 3rd-instar CPB which were either starved or fed diets of potato, tomato or eggplant foliage (see Chapter 9, this volume). On potato- fed larvae, for instance, relative inhibition by TCPI was 81% compared to 23% for OC-I, indicating the broad inhibitory spectrum of the new PI. Interestingly, TCPI retained a high level of efficacy in extracts from larvae which were fed OC-I-expressing potato foliage for 5 days.101 These larvae had recovered overall protease activity similar to controls, but they were no longer as sensitive to OC-I (10% inhibition vs. 30%). By constrast TCPI still inhibited 65-70% of the activity in OC-I-fed larvae, compared to 75-80% in OC-I-naive insects. This sustained inhibitory power of TCPI was similar to that observed for E-64, which is currently, with thyroglobulin type-1 domain inhibitors102 one of the broadest inhibitors of CPB digestive proteases investigated so far. These results with TCPI revealed new potential for plant proteinaceous cysteine PIs against the CPB. Further studies on both the inhibitor itself and its in vitro and in vivo effects on insects are now needed to confirm that potential, and to provide additional understanding of the plasticity of the target system. As discussed above, Bolter and Latoszek-Green75 found intriguing adaptation to E-64 in their chronic feeding study with CPB larvae.
Acknowledgments This work was supported by operating and strategic grants from the Natural Science and Engineering Research Council of Canada.
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Recombinant Protease Inhibitors in Plants 12. Whalon ME, Miller DL, Hollingworth RM et al. Selection of a Colorado potato beetle (Coleoptera: Chrysomelidae) strain resistant to Bacillus thuringiensis. J Econ Entomol 1993; 86:226-233. 13. Zehnder GW, Gelernter WD. Activity of the M-One formulation of a new strain of Bacillus thuringiensis against the Colorado potato beetle (Coleoptera: Chrysomelidae): Relationship between susceptibility and insect life stage. J Econ Entomol 1989; 82:756-761. 14. Cloutier C, Jean C. Synergism betweeen natural enemies and biopesticides: A test case using the stinkbug Perillus bioculatus (Hemiptera: Pentatomidae) and Bacillus thuringiensis tenebrionis against Colorado potato beetle (Coleoptera: Chrysomelidae). J Econ Entomol 1998; 91:1096-1098. 15. Lopez R, Ferro DN. Larviposition response of Myiopharus doryphorae (Diptera: Tachinidae) to Colorado potato beetle (Coleoptera: Chrysomelidae) larvae treated with lethal and sublethal doses of Bacillus thuringiensis Berliner subsp. tenebrionis. J Econ Entomol 1995; 88:870-874. 16. Boiteau G, Coleman W. Cold tolerance in the Colorado potato beetle, Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae). Can Entomol 1997; 128:1087-1099. 17. Noronha C, Cloutier C. Effect of soil conditions and body size on digging by prediapause Colorado potato beetle (Coleoptera: Chrysomelidae). Can J Zool 1998: 76:1705-1713. 18. Poprawski TJ, Carruthers RI,Speese III J et al. Early-season applications of the fungus Beauveria bassiana and introduction of the hemipteran predator Perillus bioculatus for control of Colorado potato beetle. Biol Control 1997; 10:48-57. 19. Hardie J, Nottingham SF, Powell W et al. Synthetic aphid sex pheromone lures female parasitoids. Entomol Exp Appl 1991; 61:97-99. 20. Hardie J, Hick AJ, Höller C et al. The responses of Praon spp. parasitoids to aphid sex pheromone components in the field. Entomol Exp Appl 1994; 71:95-99. 21. Powell W, Pennacchio F, Poppy GM et al. Strategies involved in the location of hosts by the parasitoid Aphidius ervi Haliday (Hymenoptera: Braconidae: Aphidiinae). Biol Control 1998; 11:104-112. 22. Pickett JA, Wadhams LJ, Woodcock CM et al. The chemical ecology of aphids. Annu Rev Entomol 1992; 37:67-90. 23. Hsiao TH. Chemical influence on feeding behavior of Leptinotarsa decemlineata (Say) beetles. In: Barton-Browne L, ed. Toxins: Animal, plant and microbial. Oxford: Pergamon Press 1974:675-688.
Expression of Protease Inhibitors in Potato 24. Murray KD, Alford AR, Groden E et al. Interactive effects of an antifeedant used with Bacillus thuringiensis var. san diego delta endotoxin on Colorado potato beetle (Coleoptera: Chrysomelidae). J Econ Entomol 1993; 86:1793-1801. 25. Murray KD, Groden E, Drummond FA et al. Citrus limonoid effects on Colorado potato beetle (Coleoptera: Chrysomelidae) colonization and oviposition. Environ Entomol 1995; 24:1275-1283. 26. Zehnder GW, Warthen JD. Feeding inhibition and mortality effects of neem-seed extract on the Colorado potato beetle (Coleoptera: Chrysomelidae). J Econ Entomol 1988; 81:1040-1044. 27. Kaethner M. Fitness reduction and mortality effects of neem-based pesticides on the Colorado potato beetle Leptinotarsa decemlineata (Say) (Col., Chrysomelidae). J Appl Entomol 1992: 113:456-465. 28. Flanders KL, Hawkes JG, Radcliffe EB et al. Insect resistance in potatoes: sources, evolutionary relationship, morphological and chemical defenses, and ecogeographical associations. Euphytica 1992; 61:83-111. 29. Tingey WM, Yencho GC. Insect resistance in potato: A decade of progress. In: Zehnder GW, Powelson ML, Jannson RK et al., eds. Advances in potato pest biology and management. St. Paul MN:APS Press 1994:405-425. 30. Pelletier Y, Smilowitz Z. Effect of trichome B exudate of Solanum berthaultii Hawkes on consumption by the Colorado potato beetle, Leptinotarsa decemlineata. J Chem Ecol 1990; 16:1547-1555. 31. Gatehouse AMR, Shi Y, Powell KS et al. Approaches to insect resistance using transgenic plants. Phil Trans Roy Soc Lond Ser B 1993; 342:279-286. 32. Estruch JJ, Carozzi NB, Desai N et al. Transgenic plants: An emerging approach to pest control. Nat Biotechnol 1997; 15:137-141. 33. Gatehouse AMR, Hilder VA, Powell KS et al. Insect-resistant transgenic plants: Choosing the gene to do the « job ». Biochem Soc Trans 1994; 22:944-949. 34. Rahbé Y, Febvay G. Protein toxicity to aphids: An in vitro test on Acyrthosiphon pisum. Entomol Exp Appl 1993; 67: 149-160. 35. Rahbé Y, Sauvion N, Febvay G et al. Toxicity of lectins and processing of ingested proteins in the pea aphid Acyrthosiphon pisum. Entomol Exp Appl 1995; 76:143-155. 36. Tran P, Cheesbrough TM, Keickhefer RW. Plant proteinase inhibitors are potential anticereal aphid compounds. J Econ Entomol 1997; 90:1672-1677. 37. Hilder VA, Powell KS, Gatehouse AMR et al. Expression of snowdrop lectin in transgenic tobacco plants results in added protection against aphids. Transgenic Res 1995; 4:18-25.
163 38. Smigocki A, Neal JW Jr, McCanna I et al. Cytokinin-mediated insect resistance in Nicotiana plants transformed with the ipt gene. Plant Mol Biol 1993; 23:325-335. 39. Perlak FJ, Stone TB, Muskopf YM et al. Genetically improved potatoes: Protection from damage from Colorado potato beetles. Plant Mol Biol 1993; 22: 313-321. 40. Jelenkovic G, Billings S, Chen Q et al. Transformation of eggplant with synthetic cryIIIA gene produces a high level of resistance to the Colorado potato beetle. J Am Soc Hort Sci 1998; 123:19-25. 41. Gill SS, Cowles EA, Pietrantonio PV. The mode of action of Bacillus thuringiensis endotoxins. Annu Rev Entomol 1992; 37:615-636. 42. Tabashnik BE. Evolution of resistance to Bacillus thuringiensis. Annu Rev Entomol 1994; 39:47-79. 43. Wierenga JM, Norris DL, Whalon ME. Stage-specific mortality of Colorado potato beetle (Coleoptera: Chrysomelidae) feeding on transgenic potatoes. J Econ Entomol 1996; 89:1047-1052. 44. Feldman J. New-Leaf™ Colorado potato beetle-resistant potatoes: A foundation for sustainable potato pest management. In: Duchesne RM, Boiteau G, eds. Proceedings for the Symposium: Insect Pest Control on Potato: Development of a Sustainable Approach. Québec, Canada: Ministère de l’Agriculture, des Pêcheries et de l’Alimentation du Québec 1995:192-196. 45. Gould F. Sustainability of transgenic insecticidal cultivars: Integrating pest genetics and ecology. Annu Rev Entomol 1998; 43:701-726. 46. Perlak FJ, Fuchs RL, Dean DA et al. Modification of the coding sequence enhances plant expression of insect control protein genes. Proc Natl Acad Sci USA 1991; 88:3324-3328. 47. Gould F. Evolutionary biology and genetically engineered crops. BioScience 1988; 38:26-33. 48. Jongsma MA, Bolter C. The adaptation of insects to plant protease inhibitors. J Insect Physiol 1997; 43:885-895. 49. Koiwa H, Bressan RA, Hasegawa PM. Regulation of protease inhibitors and plant defense. Trends Plant Sci 1997; 2: 379-384. 50. Green TR, Ryan CA. Wound-induced proteinase inhibitor in plant leaves: A possible defense mechanism against insects. Science 1972; 175: 776-777. 51. Peña-Cortés H, Fisahn J, Willmitzer L. Signals involved in wound-induced proteinase inhibitor II gene expression in tomato and potato plants. Proc Nat Acad Sci USA 1995; 92:4106-4113.
164 52. Malone LA, Giacon HA, Burgess EPJ et al. Toxicity of endopeptidase inhibitors to honey bees (Hymenoptera: Apidae). J Econ Entomol 1995; 88:46-50. 53. Ashouri A, Overney S, Michaud D et al. Fitness and feeding are affected in the twospotted stinkbug, Perillus bioculatus, by the cysteine proteinase inhibitor, oryzacystatin I. Arch Insect Biochem Physiol 1998; 38: 74-83. 54. Girard C, Picard-Nizou AL, Grallien E et al. Effects of proteinase inhibitor ingestion on survival, learning abilities and digestive proteinases of the honeybee. Transgenic Res 1998; 7:1-8. 55. Overney S, Yelle S, Cloutier C. Occurrence of cysteine digestive proteases in Perillus bioculatus, a natural predator of the Colorado potato beetle. Comp Biochem Physiol B 1998; 120:191-196. 56. Walker AJ, Ford L, Majerus MEN et al. Characterisation of the mid-gut digestive proteinase activity of the two-spot ladybird (Adalia bipunctata L.) and its sensitivity to proteinase inhibitors. Insect Biochem Mol Biol 1998; 28:173-180. 57. Hassell MP, Anderson RM. Host susceptibility as a component in host-parasitoid systems. J Anim Ecol 1984; 53:611-621. 58. van Emden HF. The interaction of plant resistance and natural enemies: Effects on populations of sucking insects. In: Boethel DJ, Eikenbary RD, eds. Interactions of plant resistance and parasitoids and predators of insects. Chichester, West Sussex: Ellis Horwood, 1986:138-150. 59. van Emden HF. Host plant—Aphidophaga interactions. Agric Ecosystems Environ 1995; 52:3-11. 60. Starks KJ, Muniappan R, Eikenbary RD. Interaction between plant resistance and parasitism against the greenbug on barley and sorghum. Ann Entomol Soc Am 1972; 65:650-655. 61. van Emden HF, Wearing CH. The role of the aphid host plant in delaying economic damage levels in crops. Ann Appl Biol 1965; 56:323-324. 62. Isenhour DJ, Wiseman BR, Layton RC. Enhanced predation by Orius insidiosus (Hemiptera:Anthocoridae) on larvae of Heliothis zea and Spodoptera frugiperda (Lepidoptera:Noctuidae) caused by prey feeding on resistant corn genotypes. Environ Entomol 1989; 18:418-422. 63. Wiseman BR. Plant resistance to insects in integrated pest management. Plant Dis 1994; 78:927-932. 64. Gould F, Kennedy GG, Johnson MT. Effects of natural enemies on the rate of herbivore adaptation to resistant host plants. Entomol Exp Appl 1991; 58:1-14.
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Expression of Protease Inhibitors in Potato 78. Terra WR, Ferreira C. Insect digestive enzymes: Properties, compartmentalization and function. Comp Biochem Physiol B 1994; 109:1-62. 79. Duffey SS, Stout M. Antinutritive and toxic components of plant defense against insects. Arch Insect Biochem Physiol 1996; 32:3-37. 80. Jongsma MA, Stiekema WJ, Bosch D. Combatting inhibitor-insensitive proteases of insect pests. Trends Biotechnol 1996; 14:331-333. 81. Benchekroun A, Michaud D, Nguyen-Quoc B et al. Production of active oryzacystatin I in transgenic potato plants. Plant Cell Rep 1995; 14:585-588. 82. Hough-Goldstein JA, Whalen J. Inundative release of predatory stink bugs for control of Colorado potato beetles. Biol Control 1993; 3:343-347. 83. Cloutier C, Bauduin F. Biological control of the Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae) in Québec by augmentative releases of the two-spotted stinkbug Perillus bioculatus (Hemiptera: Pentatomidae). Can Entomol 1995; 127:195-212. 84. Cohen AC. Solid-to-liquid feeding: The inside(s) story of extra-oral digestion in predaceous Arthropoda. Am Entomol 1998; 44:103-117. 85. Duan X, Li X, Xue Q et al. Transgenic rice harboring an introduced potato proteinase inhibitor II gene are insect resistant. Nat Biotechnol 1996; 14: 494-498. 86. Abe K, Hiroto K, Arai S. Molecular cloning of a cysteine proteinase inhibitor of rice (oryzacystatin). J Biol Chem 1987; 262:16793-16797. 87. Michaud D, Vrain TC. Expression of recombinant proteinase inhibitors in plants. In: Cunningham C, Porter AJR, eds. Methods in Biotechnology, Vol. 3. Recombinant proteins from plants: Production and isolation of clinically useful compounds. Totowa NJ:Humana Press 1998:49-64. 88. Masoud SA, Johnson LB, White FF et al. Expression of cysteine proteinase inhibitor (oryzacystatin I) in transgenic tobacco plants. Plant Mol Biol 1993; 21:655-661. 89. Bonadé-Bottino M. Défense du colza contre les insectes phytophages déprédateurs: Étude d’une stratégie basée sur l’expression d’inhibiteurs de protéases dans la plante: PhD Thesis. Paris:Université Paris-Sud, Centre d’Orsay 1993. 90. Leplé JC, Bonadé-Bottino M, Augustin S et al. Toxicity to Chrysomela tremulae (Coleoptera: Chrysomelidae) of transgenic poplars expressing a cysteine proteinase inhibitor. Mol Breeding 1995; 1:319-326.
165 91. Urwin PE, Atkinson HJ, Keppenne VD et al. Engineered oryzacystatin-I expressed in transgenic hairy roots confers resistance to Globodera pallida. Plant J 1995; 8:121-131. 92. Koiwa H, Shade RE, Zhu-Salzman K et al. Phage-display selection can differentiate insecticidal activity of soybean cystatins. Plant J 1998; 14:371-380. 93. Thomas JC, Adams CJ, Keppenne VD et al. Manduca sexta encoded protease inhibitors expressed in Nicotiana tabacum provide protection against insects. Plant Physiol Biochem 1995; 33:611-614. 94. Taylor MAJ, Lee M. Trypsin isolated from the midgut of the tobacco hornworm, Manduca sexta, is inhibited by synthetic propeptides in vitro. Biochem Biophys Res Commun 1997; 235:606-609. 95. Zhao Y, Botella MA, Subramanian L et al. Two wound-inducible soybean cysteine proteinase inhibitors have greater insect digestive proteinase inhibitory activities than a constitutive homolog. Plant Physiol 1996; 111:1299-1306. 96. Visal S, Michaud D, Yelle S. Identification of a gamma-linolenic acid-induced tomato leaf cystatin-like protein with potential for biocontrol of the phytophagous pest Colorado potato beetle. Plant Physiol 1996; 111s:40. 97. Cohen Y, Ghisi U, Mosinger E. Systemic resistance of potato plants against Phytophtora infestans by unsaturated fatty acids. Physiol Mol Plant Pathol 1991; 38:255-263. 98. Choi D, Bostock RM, Avdiushko S et al. Lipid-derived signals that discriminate woundand pathogen-responsive isoprenoid pathways in plants: methyl jasmonate and the fungal elicitor arachidonic acid induce different HMG-CoA reductase genes and antimicrobial isoprenoids in Solanum tuberosum L. Proc Natl Acad Sci USA 1994; 91:2329-2333. 99. Fidantsef AL, Bostock RM. Characterization of potato tuber lipoxygenase cDNAs and lipoxygenase expression in potato tubers and leaves. Physiol Plant 1998; 102: 257-271. 100. Bolter CJ. Methyl jasmonates induces papain inhibitor(s) in tomato leaves. Plant Physiol 1993; 103:1347-1353. 101. Visal S, Yelle S, Michaud D. ‘Cross-compensation’ to cysteine proteinase inhibitors in Colorado potato beetles feeding on oryzacystatin-I expressing potato plants. Plant Physiol 1997; 114s:220. 102. Gruden K, Strukelj B, Popovic T et al. The cysteine protease activity of Colorado potato beetle (Leptinotarsa decemlineata Say) guts, which is insensitive to potato protease inhibitors, is inhibited by thyroglobulin type-1 domain inhibitors. Insect Biochem Mol Biol 1998; 28:549-560.
166 103. Edwards MA, Seabrook WD. Evidence for an airborne sex pheromone in the Colorado potato beetle, Leptinotarsa decemlineata. Can Entomol 1997; 129:667-672. 104. Noronha C, Cloutier C. Ground and aerial movement of adult Colorado potato beetle (Coleoptera: Chrysomelidae) in a univoltine population. Can Entomol 1999; 131:521-538. 105. Cloutier C, Fournier M, Jean C et al. Growth compensation and faster development of Colorado potato beetle (Coleptera: Chrysomelidae) feeding on potato foliage expressing oryzacystatin I. Arch Insect Biochem Physiol 1999; 40:69-79.
Recombinant Protease Inhibitors in Plants 106. Cloutier C, Jean C, Fournier M et al. Adult Colorado potato beetles compensate nutritional stress on oryzacystatin I-transgenic potato by hypertrophic behaviour and overproduction of insensitive proteases. Arch Insect Biochem Physiol (in press). 107. Lecardonnel A, Chauvin L, Jouanin L et al. Effects of rice cystatin I in transgenic potato on Colorado potato beetle larvae. Plant Sci 1999; 140:87-98.
CHAPTER 14
Expression of Protease Inhibitors in Sweetpotato Dapeng Zhang, Giselle Cipriani, Isabelle Rety, Ali Golmirzae, Nicole Smit and Dominique Michaud
14.1. Introduction
S
weetpotato [Ipomoea batatas L. (Lam.)] is a dicotyledenous plant belonging to the family Convolvulaceae, the morning glory family.1 Among food crops of the world, sweetpotato ranks seventh in production, with a total global growing area of 9.2 million ha and an annual production around 124 million tons.2 About 95% of sweetpotato is grown in developing countries where it is cultivated as an annual, producing a harvestable crop of fleshy roots in as few as 90 days. In many tropical areas, there is no fixed harvest time. The roots may continue to grow or can be stored in the ground until needed.3 In general, sweetpotato fits into marginal cropping systems characterized by limited use of chemical inputs. It can grow and produce edible storage roots in marginal environments, where many food crops fail. Sweetpotato in Asia and Africa is very important in subsistence farming, especially in areas at high risk for food insecurity. The storage roots, together with its foliage rich in protein also are increasingly used in Asia as animal feed.4 Sweetpotato is also a regionally important crop in the southern U.S., where it is produced for consumption as a supplemental vegetable.5
14.2. The Insect Pests of Sweetpotato Major constraints in sweetpotato production are post-harvest quality and marketing problems. Insect damage accounts for increased post-harvest quality problems and a decline in the quality of new planting material. On a global basis, the most important insect pests are sweetpotato weevils (SPWs) of the genus Cylas,6 while other insects are important on a restricted regional or local basis.7 Although several genera and species of sweetpotato weevil exist, the term usually refers to Cylas species, in particular C. formicarius F., C. brunneus F. and C. puncticollis Bohe. The species C. brunneus and C. puncticollis are known to occur only in Africa, whereas C. formicarius is globally distributed, being the single most important insect pest of sweetpotato.7 SPWs attack stems, crowns and roots of sweetptoato plants, which significantly reduces the quality and marketable yield of storage roots. The SPW problem has been consistently ranked the single most important constraint to sweetpotato production.8 SPW larvae tunnel through storage roots, which results in major damage and yield loss.
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Weevil damage imparts a characteristic furanoterpenoid to the roots, which renders even slightly damaged roots unfit for human consumption. Production losses due to weevil feeding may often reach 60% to 100%.9 In field conditions, the cryptic feeding habit of the larvae and the nocturnal activity of the adults make it difficult to detect infestations. Moreover, approximately 80 to 90% of the weevil population within vines and roots is distributed below the soil surface, further limiting the effectiveness of chemical insecticides applied for weevil management. Second to the Cylas weevils, the West Indian SPW, Euscepes postfasciatus Fairmaire, is important in South Pacific countries, in South America and in the Caribbeans. The presence of E. postfasciatus in the South Pacific islands has resulted in strict quarantine regulations banning the export of sweetpotato from these islands, thereby limiting the economic value of sweetpotato as an export commodity.10 Other insects affect sweetpotato in some particular environments, but the magnitude of damage resulting from these minor insect infestations on an annual and global basis is not significant.7
14.3. Integrated Pest Management for SPWs— The Success and the Lesson In the last decade, concepts and practices of sweetpotato integrated pest management (IPM) have become mature, and good success has been achieved in certain important producing areas. The IPM program developed in Cuba through the joint efforts of the International Potato Center (Lima, Peru) and the Cuban agricultural research institutions is a good example for the development of a successful IPM strategy. 11 Sweetpotato is a staple food in Cuba, with about 60,000 ha planted annually.12 Until recently, Cuban farmers controlled the SPW with an average use of 12 insecticide sprays per season. Since the early 1990s, however, insecticides from the former Soviet Union are no longer available, and the sudden lack of insecticides caused a rapid increase in weevil
Recombinant Protease Inhibitors in Plants
damage in all provinces, which forced farmers to turn to IPM.12 The IPM technological package in Cuba includes four components: predatory ants, the fungus Beauveria bassiana, the planting of short-season cultivars and, most important, the mass capture of adult male weevils using sex pheromone traps. Added to these control approaches were cultural practices common to most other IPM programs, such as the use of healthy planting materials, crop rotation and destruction of crop residues. Only 3 years after implementing this IPM program, weevil damage decreased from an initial level of 40-50% to a level of 4-8%. The number of insecticide sprays was reduced from 10-12 per season in 1991/92 to zero in 1996, and the IPM program will cover 30,000 ha in 1998, representing about 50% of Cuba’s entire sweetpotato production.11,12 This successful experience in Cuba was recently transferred to East Africa, but the results were not as expected, because of two key factors. The first is related to the specific cultural practices for sweetpotato in East Africa. A common practice in this area is that farmers use in-ground storage and piecemeal harvesting. The crops are left in the ground for 7 months to even more than a year, and the roots are removed as necessary for family meals. This practice guarantees that fresh roots are available for consumption during a large part of the year, but it also means that sweetpotato crops are in the field throughout the year. Cultural practices, such as early harvesting and field sanitation become meaningless under such a situation.7 The second factor explaining the failure of the IPM strategy is the fact that the two SPW species found in East Africa, C. puncticollis and C. brunneus are unique to the continent, while their ‘universal’ counterpart, C. formicarius, is found in Asia, in the United States, and in the Caribbeans. Basic studies revealed differences in the biology of the two African species, notably the presence of species-specific sex pheromones. The pheromones synthesized so far cannot efficiently attract the male adults, which leaves enough males to ensure female fertilization and allow
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169
large populations to be maintained in the field.7 For most plants, genetic resistance represents a key anchor in IPM programs. For sweetpotato, the search for weevil resistance started fifty years ago, but no significant source of resistance in sweetpotato and its wild relatives has yet been found.13 Large genotype by environment interaction and low heritability often make conventional breeding efforts in vain. In this context, the use of biotechnological approaches to introduce ‘resistance genes’ holds the greatest promise to protect sweetpotato against SPWs. One approach, which consists to develop sweetpotato lines expressing recombinant protease inhibitors (PIs) active against the digestive proteases of SPWs and other pests, has been considered by our group in the last few years. The use of PI-expressing transgenic plants has been proposed as a mean of protecting crops from their natural enemies,14 and several plants of economic importance have been genetically engineered with inhibitor-encoding cDNA sequences during the last ten years.15 In the following sections we summarize recent progress made about the use of recombinant PIs in sweetpotato protection.
in larvae of C. formicarius,18 the proteinases detected were inhibited by serine-type inhibitors, including phenylmethylsulfonyl fluoride (PMSF), 4-(amidinophenyl) methylsulfonyl fluoride (APMSF), soybean BowmanBirk trypsin/chymotrypsin inhibitor (SBBI) and soybean Kunitz trypsin inhibitor (SBTI), regardless of the species or developmental stage.17 Noteworthy, PMSF exhibited affinity for most proteinases detected in gel, but the inhibitory spectrum of protein inhibitors like SBBI and SBTI was limited, 17 as also reported for other plant proteinaceous PIs. 19,20 Moreover, whereas PMSF and APMSF strongly inactivated the Rf82 proteinase in all extracts, SBBI and SBTI exhibited species- and stage-dependent affinities for this proteinase (Table 14.1), suggesting the existence of different variants of this enzyme and eventual differential effects of recombinant SBBI and SBTI against the different Cylas species. Despite this apparent complexity of the digestive protease complements in SPWs, quantitative measurements allowed us to show that in most cases SBTI and SBBI, two protein PIs of plant origin, are relatively good inhibitors of SPW digestive proteases (see Fig. 14.1 for SBTI), showing an affinity spectrum for these enzymes larger than that of the endogenous PIs in sweetpotato. 18,21 An additional evidence suggesting the potential of both soybean PIs in SPW control is their apparent stability in the presence of the insect insensitive proteinases. Despite the occurrence of SBBI- and SBTI-insensitive proteases in all extracts analyzed, both PIs remain essentially stable and active when incubated with the insect non-target proteases, 17 suggesting that they could effectively inhibit the Rf82 proteinase in the midgut of both larvae and adults. Although several questions remain concerning the ability of SPWs to compensate for any loss of digestive proteinase activity using their insensitive proteinases,22 or concerning the actual effect of plant protein PIs in vivo, these results suggest that plant trypsin inhibitors such as SBTI, SBBI or the cowpea trypsin inhibitor (CpTI) may represent good candidate PIs for the production
14.4. Digestive Proteinases in SPW (Cylas) Species Digestive proteinases of the three most important weevil species, C. formicarius, C. brunneus and C. puncticollis were characterized by qualitative and quantitative (in vitro) assessment of their midgut proteolytic activities. As shown on gelatin-containing polyacrylamide gels,16 Cylas insects use various digestive protease complements composed of several proteinase forms active in the alkaline pH range. 17 Although interspecific and developmental variations in digestive proteinase patterns were easily noted, a major form with a relative mobility of 0.82 (Rf82) was found in all extracts analyzed, suggesting the importance of this particular proteinase for dietary protein hydrolysis in SPWs. In agreement with a previous study reporting the occurrence of trypsin-like proteinase activity
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170
Table 14.1. Inhibition of sweetpotato weevils’ ‘Rf82’ proteinase by PMSF, APMSF, soybean Bowman-Birk inhibitor and soybean Kunitz trypsin inhibitora Species
Cylas brunneus
C. formicarius
C. puncticollis
Stage
Relative inhibition (%) PMSF
APMSF
SBBI
SBTI
Lb
88 ± 8
85 ± 5
42 ± 12
33 ± 5
A
87 ± 14
80 ± 9
37 ± 13
37 ± 6
L
93 ± 4
82 ± 3
27 ± 9
22 ± 6
A
99 ± 1
88 ± 11
67 ± 12
78 ± 8
L
98 ± 3
95 ± 5
93 ± 8
90 ± 10
A
99 ± 1
97 ± 3
77 ± 8
78 ± 2
a Data represent relative inhibitory rates, as compared to a control in which no inhibitor was
added (0% inhibition). The proteases were submitted to mildly-denaturing gelatin/SDS-PAGE, and the lysis (proteolysis) zones in the gel were quantified by densitometry, using the image analysis software NIH Image 1.6 (NIH, Bethesda MD).16,25 b Abbreviations: APMSF, 4-(amidinophenyl) methylsulfonyl fluoride; PMSF, phenylmethylsulfonyl fluoride; SBBI, soybean Bowman-Birk inhibitor; SBTI, soybean Kunitz trypsin inhibitor; A, adults; L, larvae.
of sweetpotato lines with measurable resistance to SPWs. The same inhibitors could also prove useful to control E. postfasciatus, a SPW also using serine-type digestive proteinases sensitive to plant serine PIs like SBBI and SBTI (D. Michaud et al., unpubl.).
14.5. Expression of Recombinant PIs in Sweet Potato 14.5.1. Expression of PIs for SPW Control
Newell et al 23 developed the first transgenic lines of PI-expressing sweetpotato, by transforming the plant with a cDNA sequence encoding the serine-type PI, CpTI. Two vectors were used in these experiments: a first vector, pCTI5, containing the CpTI gene under the control of the CaMV 35S promoter, and a second vector, pPCG6, carrying two genes for insect resistance, the CpTI gene and a DNA sequence encoding
GNA, a mannose-specific lectin from snowdrop, Galanthus nivalis L. (see Fig 14.2). In this second vector the two genes were oriented as a ‘head-to-head’ inverted repeat to limit deletion events due to eventual recombination. Either vectors were introduced into sweetpotato cv. ‘Jewel’ via Agrobacterium tumefasciens-mediated gene transfer, and transgenic plants expressing the recombinant proteins were regenerated by somatic embryogenesis.23 Feeding tests with the Indian SPW, E. postfasciatus were carried out at the International Potato Center by using a no-choice test in a screenhouse-based replicated bioassay.10 The resistance status of the transformed plants was assessed 60 days after infestation by estimating the percentage of internal damage and the weevil population in the storage roots using a five-grade damage index. Out of ten transgenic clones, seven were found resistant or moderately resistant (Table 14.2; A. Golmirzaie et al, unpubl.). In particular, the clones CTI-13 and CTI-7, with CpTI alone,
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171
Fig. 14.1. Inhibition of Cylas spp. midgut proteinases by class-specific PIs. Protease activity was measured with larval extracts at pH 10.5 using azocasein as a substrate. Data represent relative residual protease activity (%), as compared with a control for which no inhibitor was added (100% activity). CONT, control (no inhibitor); HEC, hen egg cystatin; OC-I, oryzacystatin I; PEP, pepstatin A; PMSF, phenylmethylsulfonyl fluoride; SBTI, soybean trypsin inhibitor (Kunitz-type). Adapted with permission from: Zhang D, Golmirzae A, Cipriani G et al. International Potato Center Program Report 1995-1996:205-210 © 1997 CIP. and the clones PCG-5 and PCG-7, with both CpTI and the snowdrop lectin, exhibited a good degree of resistance to the target pest, demonstrating the actual usefulness of the trypsin inhibitor in controlling the Indian SPW. Although no definitive results are available at this point concerning the Cylas weevils, the above discussion about the inhibiton of the Rf82 proteinase in these weevils renders plausible the existence of a similar protective effect of CpTI against these three species of the
same family. Given their general effect on the weevil proteinases, SBTI and SBBI also appear as promising antifeedant proteins useful in broad-spectrum protection of sweetpotato. SBTI-expressing lines of sweetpotato (cvs. Jewel, Jonathan and Maria Angola) were recently developed at the International Potato Center using the plasmid pKTI-4 (from Pestax Ltd., UK), and are currently assessed for their resistance status against the different SPW species.
172
Recombinant Protease Inhibitors in Plants
Fig. 14.2. T-DNA region of the plasmids pCTI5 and pPCG6. The coding regions of ß-glucuronidase (GUS), neomycin phosphotransferase II (NPTII), cowpea trypsin inhibitor (CpTI) and snowdrop lectin (GNA) were inserted between TR 2' (2') or CaMV 35S (35S) promoter sequences, and nopaline synthase (NOS) or octopine synthase (OCS) terminator sequences. Direction of transcription is shown by the arrowheads. LB and RB: left border and right border sequences of the T-DNA. Relevant restriction enzyme sites are indicated: B, BamHI; Bc, Bc/I; Bg, Bg/II; E, EcoRI; H, HindIII; X, XbaI. Adapted with permission from: Newell CA, Lowe JM, Merryweather A et al. Plant Sci 1995; 107:215-227 © 1995 Elsevier Science Ltd.
14.5.2. Expression of PIs for Nematode Control Besides their potential in SPW control, recombinant PIs also show potential in the control of parasitic nematodes attacking sweetpotato, in particular those of the genera Meloidogyne (root-knot nematodes; e.g. M. incognita and M. javanica) and Rotylenchulus, which affect sweetpotato cultures worldwide. Root-knot nematodes cause galls on the fibrous roots, cracking of fleshy roots and yield reduction. Injury to the flesh roots also decreases quality and provides wounds to pathogens through which they can invade the plant and cause further damage. Rotylenchulus nematodes usually cause severe root pruning, flesh root cracking and yield losses. While chemical control to reduce initial population levels is taken as a major control measure, genetic resistance also plays a very important role for nematode control in sweetpotato. High-level resistance has been
found in the sweetpotato germplasm, and nematode resistance is commonly evaluated in sweetpotato breeding programs. Nematode resistance can also be obtained by transgenic plant approaches, including the use of PI-expressing sweetpotato lines. In the case of sweetpotato, the transgenic approach may help render resistant cultivars which otherwise may lack resistance to the specific nematode to be controlled. Recombinant PIs have been proposed as a way to confer nematode resistance to plants. Inhibitors of serine and cysteine proteinases, in particular, were shown to inhibit the extracellular proteinases of root-knot nematodes,24,25 and adverse effects of PI-expressing plants against various nematode species were recently reported (see Chapter 4, this volume).26-28 To assess the usefulness of trypsin inhibitors in controlling nematodes in sweetpotato, ten transgenic clones expressing CpTI alone or CpTI+snowdrop lectin
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173
Table 14.2. Feeding tests with transgenic sweetpotato clones (cv. ‘Jewel’) expressing cowpea trypsin inhibitor and/or snowdrop lectin, against the West Indian sweetpotato weevil (Euscepes postfasciatus) Clone
Gene constructa
Jewel PCG 5
CpTI/GNA
R
Jewel PCG 7
CpTI/GNA
R
Jewel CTI 1
CpTI
MR
Jewel CTI 5
CpTI
MR
Jewel CTI 7
CpTI
R
Jewel CTI 8
CpTI
MR
Jewel CTI 13
CpTI
R
Jewel control
Effectb
HS
a CpTI encodes cowpea trypsin inhibitor from Vigna unguiculata; GNA encodes snowdrop lectin from Galanthus nivalis. b HS, S, MR, R mean highly susceptible, susceptible, moderately resistant and resistant to the weevil, respectively. The experiment was conducted in 1996, in Lima, Peru.
(see Section 14.5.1) were evaluated for their resistance against M. incognita. Seven days after transplanting, 4000 eggs and second instars of the nematode were inoculated to the plants, which were kept in screenhouses in Lima and San Ramon, Peru for 60 days. Root necrosis and root galling were then evaluated using a ‘5-grade standard’ resistance scale. Three transgenic clones, one with CpTI alone and two with CpTI+snowdrop lectin showed consistant resistance in both environments, whereas the non-transformed control plants were moderately resistant, with the same susceptibility to galling but with more root necrosis (Table 14.3; D. Zhang et al, unpubl. data). This observation, in agreement with a previous study proposing the use of CpTI for root-knot nematode control, shows the potential of this inhibitor as a tool for sweetpotato IPM. The potential of cysteine PIs in protecting sweetpotato from nematodes is also currently being assessed using lines of
the cultivars Morada Inta and Maria Angola transformed with the plasmid pBinh-OC-I, which includes a cDNA sequence encoding OC-I under the control of the CaMV 35S promoter.29
14.6. PIs In Sweetpotato, and Their Nutritional Impact Sweetpotato storage roots contain substantial amounts of trypsin inhibitors,30,31 which share significant amino acid sequence identity with sporamin, the major storage protein in the roots of sweetpotato.32 The nucleotide sequence of sporamin cDNA is homologous to those of SBTI and win-3, a wound-responsive gene in poplar.33 Recently Yeh et al32,34 isolated a full-length cDNA of the sweetpotato trypsin inhibitor, spTi-1, which they introduced into tobacco by genetic transformation. As shown by bioassays with the modified plants, larval growth of the
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Table 14.3. Resistance of transgenic sweetpotato clones expressing cowpea trypsin inhibitor and/or snowdrop lectin to the root-knot nematode, Meloidogyne incognita Degree of resistanceb Transgenic clones
Gene constructsa
San Ramón
Lima
Jewel PCG 5
CpTI/GNA
R
R
Jewel PCG 7
CpTI/GNA
R
R
Jewel CIT 7
CpTI
R
R
Control (non-transformed Jewel)
MR
MR
Maria Angola (susceptible check)
S
S
a CpTI encodes cowpea trypsin inhibitor from Vigna unguiculata; GNA encodes snowdrop lectin from Galanthus nivalis. b S, M and R mean susceptible, moderately resistant and resistant, respectively. The assays were carried out in 1997 in two different locations (San Ramon and Lima, Peru).
tobacco cutworm, Spodoptera litura (F.) was severely retarded as compared to larvae fed control plants.34 The level of trypsin inhibitor activity (or TIA) in sweetpotato, which varies among cultivars,30,31 also is affected by the location where the crop is grown,30,35 by the accumulated rainfall in the environment36 and by the application of fertilizers.31 In a survey of nine US sweetpoato clones, we have found that the storage roots contain 3.5-7 mg.g-1 (dmb.) of trypsin inhibitors in average, which is about one quarter the TIA level found in soybean seeds.31 Interestingly, sweetpotato PIs are sensitive to heat treatment. For instance, the TIA value is reduced below 10% of its original activity when the roots are boiled for 15 min.37 In another case the TIA was fully inactivated when the boiled or baked roots were soft enough to eat.38 As sweetpotato is almost always cooked before consumption by humans, the presence of natural trypsin inhibitors in sweetpotato roots present little risk to human consumers.
Such limited effects for recombinant SBTI and SBBI in sweetpotato were also recently proposed based on observations made about the stability of the inhibitors in the presence of human pepsin.17 In this experiment, human gastric conditions (acidic pH and pepsin treatment at 37˚C) were simulated in vitro, assuming that the soybean trypsin inhibitors expressed in sweetpotato could represent a risk for humans in blocking intestinal serine proteinases, in particular trypsin. In brief, our tests indicated that human pepsin in acidic conditions (unlike acidic conditions alone) may rapidly inactivate a significant fraction of SBTI and SBBI, causing an irreversible loss of their activity at pH 8.0 (Table 14.4). Although this destabilizing effect of pepsin needs to be confirmed in vivo— and although the effects of the non-inactivated molecules remain unclear at this point—these observations tend to indicate that under human gastrointestinal conditions a significant fraction of either SBTI and SBBI would be rapidly inactivated in the stomach, decreasing the risks of ‘harmful’ inhibitory
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Table 14.4. Antitrypsin activity of soybean Bowman-Birk and Kunitz inhibitors after acid/pepsin treatments a Treatment
Activity (U)
Inhibition (%)
Trypsin (T) alone (ctrl)
0.851
T + SBTI b
0.246
72
T + (SBTI + pepsin)
0.652
25
T + SBBI
0.230
74
T + (SBBI + pepsin)
0.741
15
a Trypsin activity was measured at pH 8.0 with azocasein as a substrate (see ref. 25 for protocol) in
the presence or absence (ctrl) of inhibitor (SBTI or SBBI), after an acid treatment at pH 2.0 (including or not pepsin). The pepsin treatment was performed in acidic conditions (pH 2.0 stomachal conditions); antitrypsin activity of the SBTI and SBBI truncation products was then assayed at pH 8.0 (intestinal conditions). No pepsin activity was detected at pH 8.0 under the assay conditions used. b Abbreviations: SBBI, soybean Bowman-Birk inhibitor; SBTI, soybean Kunitz trypsin inhibitor.
reactions in the intestine. The relatively low amount of recombinant proteins expressed in plants (between 0.2 and ~1% of soluble proteins in most cases), and the fact that cooking could alter their structural integrity as it is altering the integrity of sweetpotato inhibitors also suggest somewhat limited direct antinutritive effects for the soybean trypsin PIs expressed as recombinant proteins in sweetpotato. On the other hand, endogenous sweetpotato trypsin inhibitors and recombinant serine PIs from other plants expressed in sweetpotato could represent a risk to animals because uncooked storage roots are often used to feed animal in subsistence farming systems.3 Batterham et al39 found that growing pigs could tolerate up to 4.7 mg.g-1 of trypsin inhibitors in the diet. This threshold level would be lower for piglets because their digestive system is immature and thus more sensitive to the effects of digestive inhibitors.39 This threshold level might be exceeded when a sweetpotato-based feed, if not processed, is used in the animal diets. Reports have shown poor feeding efficiency when uncooked
sweetpotato was used in animal feeding trials conducted in Taiwan, although the actual effects of the trypsin inhibitors in these experiments were not clear.40
14.7. Discussion The use of transgenic plants expressing foreign biocidal or antimetabolic proteins is a promising approach in pest control.41,42 Transgenic plants expressing different kinds of defense-related proteins have been developed, and pesticidal effects have been demonstrated in several cases.42 Geneticallyengineered plants expressing δ-endotoxins from the soil bacterium Bacillus thuringiensis (Bt toxins), for instance, may show high-level resistance to insects and nematodes in plants, making this approach already suitable for commercialization. The forthcoming large-scale use of biocidal transgenic plants, however could drastically contribute to alter the high potential of the biocidal proteins expressed.43 Effective defense proteins like Bt toxins or lectins are single chemical compounds, as are the commercial synthetic pesticides currently
176
used. Given their high efficiency, it is plausible—and even probable—that these proteins will exert a strong selection pressure on the target pests and favor the build-up of resistant populations in the field. To minimize such a loss of usefulness, strategies must be developed to deploy the modified plants in such a way that their effect will be maintained in the long-term. Using these plants in IPM systems, notably, should contribute to ensure a certain durability since they would then represent one component of a more elaborate control system. A second way to decrease selection pressure in natural conditions is to use promoters directing the accumulation of the recombinant proteins in certain tissues or during specific developmental stages. For instance, using root periderm-specific promoters44 to direct the accumulation of recombinant proteins could represent an effective way to protect sweetpotato without exposing the target pest to the biocidal molecule throughout its development. The use of such promoters would allow the plant to express the introduced proteins preferentially in the skin of sweetpotato roots, avoiding constitutive, energy-consuming expression of the transgene. As SPWs complete their life cycle on foliage, this approach could help maintaining a low selection pressure on the population, and thus contribute to slow-down the appearence of resistant populations in the field. In this perspective, the use of transgenic plants expressing antinutritive or antifeedant proteins such as recombinant PIs also appears interesting. Unlike biocidal proteins like Bt toxins or lectins, PIs do not cause mortality directly. In the most successful cases, ingestion of PIs by an insect or a nematode causes growth delays and important problems of fecundity, which may finally result in the death of some individuals. Most importantly, reduction in fitness and extended life cycle of target pest would give PI-expressing plants relative (instead of absolute) resistance, making this control mean particularly suitable in IPM systems (see Chapter 13, this volume). Field trials carried out by the International
Recombinant Protease Inhibitors in Plants
Potato Center in Cuba and Dominica Republic showed that even a less susceptible variety could contribute significantly to the overall effectiveness of the IPM practice.12 As a result of fitness reduction following recombinant PI ingestion, for instance, some herbivorous pests could be more susceptible to natural predators or parasitoids implemented in the field as biocontrol agents. In addition to their potential as control molecules acting on specific metabolic processes in the pest, PIs might also be useful indirectly in protecting other (‘companion’) defense proteins of commercial interest including Bt toxins, lectins or even other PIs, thus extending their usefulness in IPM systems.20,45 Several PI-expressing sweetpotato lines developed by our group in the last few years have already shown potential in reducing the fitness of specific pests, but the actual usefulness of recombinant PIs in sweetpotato protection still remains to be clearly demonstrated. Continued efforts are needed for finding the most appropriate combination of PIs, for enhancing the expression of plant defense proteins in sweetpotato, and for designing strategies aimed at implementing PI-expressing plants in IPM systems. Finally, while the use of recombinant PIs increasingly appears promising in plant protection against insect predation and nematode infection, sweetpotato represents a good model for testing the efficiency of PIs against specific pests in real situations, as in several regions SPWs are the only insects attacking the plant.
Acknowledgments This work was supported by a Linkage Grant from the Canadian International Development Agency. The ‘Jewel’ transgenic sweetpotato lines were kindly supplied by Dr. Christine Newell, of Pestax Ltd., U.K. We thank Dr. M. Jusurf of Lembang Root & Legume Research Institute of Indonesia, the Crop Institute of Guangdong Agricultural Academy of China, Mr. Paul Kakande from the Biochemistry Department of Makerere University, and Mr. Justine Nantez, of the Namulonge Agricultural Research Station of
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Uganda for their help in collecting weevil samples.
13. Collins WW, Mendoza HA. Breeding sweetpotato for weevil resistance: Future outlook. In: Jansson RK, Raman KV, eds. Sweetpotato pest management: A global perspective. Boulder CO: Westview Press, 1991:399-406. 14. Hilder VA, Gatehouse AMR, Sheerman SE et al. A novel mechanism of insect resistance engineered into tobacco. Nature 1987; 330:161-163. 15. Michaud D, Vrain TC. Expression of recombinant proteinase inhibitors in plants. In: Cunningham C, Porter AJR, eds. Recombinant proteins from plants. Production and isolation of clinically useful compounds. Totowa NJ: Humana Press, 1998:49-64. 16. Michaud D, Faye L, Yelle S. Elecrophoretic analysis of plant cysteine and serine proteinases using gelatin-containing polyacrylamide gels and class-specific proteinase inhibitors. Electrophoresis 1993; 14: 94-98. 17. Rety I, Cipriani G, Zhang DP et al. Soybean Kunitz and Bowman-Birk inhibitors strongly inactivate the major digestive serine proteinase of sweetpotato weevils. Annual Meeting of the American Society of Plant Physiologists. Madison WI, June 27-July 1, 1998. 18. Baker JE, Woo SM, Mullen MA. Distribution of proteinases and carbohydrates in the midgut of larvae of the sweet potato weevil Cylas formicarius elegantulus and response of proteinases to inhibitors from sweetpotato. Entomol Exp Appl 1984; 36:97-105. 19. Michaud D, Bernier-Vadnais N, Overney S et al. Constitutive expression of digestive cysteine proteinase forms during development of the Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae). Insect Biochem Mol Biol 1995; 25:1041-1048 20. Michaud D. Avoiding protease-mediated resistance in herbivorous pests. Trends Biotechnol 1997; 14:331-333. 21. Zhang DP, Golmirzaie A, Cipriani G et al. Developing weevil resistance in sweetpotato with genetic transformation. In: International Potato Center 1995-1996 Program Report. Lima, Peru:CIP, 1997:205-210. 22. Jongsma MA, Bolter C. The adaptation of insects to plant protease inhibitors. J Insect Physiol 1997;43:885-895. 23. Newell CA, Lowe JM, Merryweather A et al. Transformation of sweetpotato (Ipomoea batatas (L.) Lam.) with Agrobacterium tumefaciens and regeneration of plants expressing cowpea trypsin inhibitor and snowdrop lectin. Plant Sci 1995; 107:215-227. 24. Hepher A, Atkinson HJ. Nematode control with proteinase inhibitors. European Patent Application 92301890.7, 1992; Pub 0502730 A1.
References 1. Austin DF. The taxonomy, evolution and genetic diversity of sweetpotatoes and related wild species, In: Gregory P, ed. Exploration, maintenance, and utilization of sweetpotato genetic resources. Lima, Peru:International Potato Center 1988:27-60. 2. International Potato Center. Sweetpotato facts. Lima, Peru: CIP, 1996. 3. Woolfe JA. Sweetpotato, an untapped food resource, Cambridge: Cambridge University Press 1992:24-40. 4. Scott GJ, Wheatly C. Recent advances in CIP’s strategy for collaborative postharvest research on sweetpotato. In: International Potato Center 1995-1996 program report. Lima, Peru:CIP 1997:264-269. 5. Wilson LG. Growing and marketing quality sweetpotatoes. North Carolina Agric Ext Serv Bull AG-09, North Carolina State University Agricultural Extension Service, Raleigh NC: NCSUAE, 1989:28. 6. Carey EE, Chujoy E, Dayal T et al Helping meet varietal needs of the developing world: The International Potato Center’s strategic approach for sweetpotato breeding. In: Hill WA, Bonsi CK, Loretan PA, eds. Sweetpotato Technology for the 21st Century, Tsukegee: Tuskegee University, 1992:521. 7. Smit N, Odongo B. Integrated pest management for sweetpotato in East Africa. In: International Potato Center 1995-1996 Program Report. Lima, Peru:CIP 1997:191-197. 8. Horton DE, Ewell PT. Sweetpotato pest management: a social science perspective. In: Jansson RK, Raman KV, eds. Sweetpotato pest management: a global perspective. Boulder CO: Westview Press, 1991:407-427. 9. Chalfant RB, Jansson RK, Seal DR et al. Ecology and management of sweetpotato insects. Annu Rev Entomol 1990; 35:157-180. 10. Raman KV, Alleyne EH. Biology and management of the West Indian sweetpotato weevil, Euscepes postfaciatus. In: Jansson RK, Raman KV, eds. Sweetpotato pest management: A global perspective. Boulder CO: Westview Press, 1991:263-282. 11. Anon. International Potato Center 1997 Annual Report. Lima, Peru: CIP 1998:26-27. 12. Alcazar J, Cisceros F, Morales A. Large-scale implementation of IPM for sweetpotato weevil in Cuba: A collaborative effort, In: International Potato Center 1995-1996 Program Report, Lima, Peru: CIP, 1997:185-190.
178 25. Michaud D, Cantin L, Bonadé-Bottino M et al. Identification of stable plant cystatin/ nematode proteinase complexes using mildlydenaturing gelatin-polyacrylamide gel electrophoresis. Electrophoresis 1996; 17:1373-1379. 26. Urwin PE, Atkinson HJ, Waller DA et al. Engineered oryzacystatin-I expressed in transgenic hairy roots confers resistance to Globodera pallida. Plant J 1995; 8:121-131. 27. Urwin PE, McPherson MJ, Atkinson HJ. Enhanced transgenic plant resistance to nematodes by dual proteinase inhibitor constructs. Planta 1998; 204:472-479. 28. Atkinson HJ, Urwin PE, Hansen PE et al. Designs for engineered resistance to rootparasitic nematodes. Trends Biotechnol 1995; 13:369-374. 29. Benchekroun A, Michaud D, Nguyen-Quoc B et al. Synthesis of active oryzacystatin I in transgenic potato plants. Plant Cell Rep. 1995;14:585-588. 30. Bradbury JH, Hammer BC, Nguyen T et al. Protein quantity and quality and trypsin inhibitor content of sweet potato cultivars from the highlands of Papua New Guinea. J Agric Food Chem 1985; 33:281-285. 31. Zhang DP, Collins WW, Andrade M. Genotype and fertilization effects on trypsin inhibitor activity in sweetpotato. HortScience 1998; 33:225-228. 32. Yeh KW, Chen JC, Lin MI et al. Functional activity of sporamin from sweetpotato (Ipomoea batatas Lam.): A tuber storage protein with trypsin inhibitory activity. Plant Mol Biol 1997; 33:565-570. 33. Bradshaw Jr. HD, Hollick JB, Parsons TJ et al. Systemically wound-responsive genes in poplar trees encode proteins similar to sweet potato sporamins and legume Kunitz trypsin inhibitors. Plant Mol Biol 1989; 14:51-59. 34. Yeh KW, Lin MI, Tuan SJ et al. Sweetpotato (Ipomoea batatas) trypsin inhibitors expressed in transgenic tobacco plants confer resistance against Spodoptera litura. Plant Cell Rep 1997; 16:696-699.
Recombinant Protease Inhibitors in Plants 35. Bouwkamp JC, Tsou SCS, Lin SSM. Genotype and environment effects on the relationship between protein concentration and trypsin inhibitor levels in sweet potatoes. HortScience 1985; 20:886-889. 36. Lin YH. Relationship between trypsininhibitor activity and water-soluble protein and cumulative rainfall in sweetpotatoes. J Amer Soc Hort Sci 1989; 114:814-818. 37. Dickey LF, Collins WW, Young CT et al. Root protein quantity and quality in a seedling population of sweet potatoes. HortScience 1984;19:689-692. 38. Bradbury JH, Hammer BC, Sugani I. Heat stability of trypsin inhibitors in tropical root crops and rice and its significance for nutrition. J Agric Food Chem 1992; 58:95-100. 39. Batterham ES, Harpal S, Lynette MA et al. Tolerance of growing pigs to trypsin and chymotrypsin inhibitors in chickpeas (Cicearietinum) and pigeonpeas (Cajanus cajan). J Sci Food Agric 1993; 61:211-216. 40. Yeh TP, Bouwkamp JC. Roots and vines as animal feed. In: Bouwkamp JC, ed. Sweet potato products: A natural resource for the tropics, Boca Raton: CRC Press, 1985:235-253. 41. Estruch JJ, Carozzi NB, Desai N et al. Transgenic plants: An emerging approach to pest control. Nat Biotechnol 1997; 15:137-141. 42. Schuler TH, Poppy GM, Kerry BR et al. Insect-resistant transgenic plants. Trends Biotechnol 1998; 16:168-175. 43. Brattsen LB. Bioengineering of crop plants and resistant biotypes evolution in insects: Counteracting coevolution. Arch Insect Biochem Physiol 1991; 17:253-267. 44. Scott D, Clark W, Deahl KL et al. Isolation of functional RNA from periderm tissue of potato tubers and sweetpotato storage roots. Plant Mol Biol Rep 1998; 16:3-8. 45. Visal S, Taylor MAJ, Michaud D. The proregion of papaya IV inhibits Colorado potato beetle digestive cysteine proteinases. FEBS Lett 1998; 434:401-405.
CHAPTER 15
Expression of Protease Inhibitors in Rapeseed Lise Jouanin, Michel Bonadé Bottino, Cécile Girard, Jacques Lerin and Minh Hà Pham Delègue
15.1. Introduction
B
rassica napus L., also known as rapeseed, oilseed rape and for some cultivars canola, is a crop grown primarily for its seed which yields oil and a high-protein animal food. Recent breeding has concentrated on cultivars with low erucic acid and low glucosinolate content (00 cultivars). B. napus, belonging to the Brassicaceae family (Cruciferae), is an allotetraploid species (n = 19) derived from ancient crosses between B. oleracea and B. campestris (USDA/APHIS Biotechnology Permit database). The world production of rapeseed oil in 1997 was 34 million metric tons, ranking it number four behind soybean, palm and sunflower. China, India, Canada and Europe are the top world producers (FAO database). Table 15.1 summarizes the main characteristics of world B. napus production. After crushing and extraction, Brassica oilseeds yield approximately 40-42% oil on a dry weight basis. The residual meal or ‘cake’ contains 38-42% protein and is widely used in livestock and poultry diets. In addition to these uses, industrial uses have recently emerged (e.g., fuel additive, detergents, lubricants),1 with biotechnology being greatly involved in these new breeding programs. Rapeseed has been among the first major agricultural crops to benefit from the application of modern biotechnological methods. Field trials of transgenic rapeseed were initiated in
the late 1980s and early 1990s. The major reasons for the rapid spreading of genetransfer technology in rapeseed include the early development of relatively simple transformation systems.2-5 Also, many important traits involved in the regulation of seed-oil and protein composition are determined by a small number of genes. Finally, rapeseed biotechnology has benefited from extensive research on the model plant Arabidopsis thaliana, which belongs to the same family. In the field, B. napus cultures require many insecticide treatments, while no insect resistance genes from wild-type genotypes have been found by traditional breeding. Many programs of transgenic crop design (maize, cotton, etc.) are related to insect resistance, the main strategy being the expression of γ-endotoxins from the soil bacterium Bacillus thuringiensis (Bt toxins).6 The pest complex in B. napus differs vastly between geographical areas (see Table 15.2 for Europe and Canada). Most of the pests are crucifer specialists, along with a few generalist herbivores. They feed on different parts of the plant such as cotyledons and leaves (Psylliodes chrysocephala L.), stems (Ceutorhynchus napi Gyll), flower buds (Melighethes aeneus F.) and pods (Ceutorhynchus assimilis Payk). Most of these pests are Coleoptera and their larvae are frequently borers. In Europe, none of the
Recombinant Protease Inhibitors in Plants, edited by Dominique Michaud. ©2000 Eurekah.com.
Recombinant Protease Inhibitors in Plants
180
Table 15.1. Progression in the production of B. napus worldwide (according to the FAO) 1990
1994
1997
Area harvested (million ha)
17,59
22,85
23,80
Yield (hg/ha)
13,89
13,05
14,30
Production (million MT)
24,44
29,82
34,05
Seed (MT)
388,43
459,66
404,52
major pests is known to be susceptible to any of the Bt toxins available, although some of them may not have been tested properly due to their mode of feeding. Of the minor pests, only Plutella xylostella L. (diamondback moth, a Lepidoptera) is susceptible to CryIA Bt toxins. The situation is quite different in Canada, where P. xylostella is an important pest. In addition, Mamestra configurata Walker (bertha armyworn, a Lepidoptera) is sensitive to new Bt toxins (e.g., CryIH).7 Transgenic canola expressing the CryIA toxin was obtained,8 and shown to be toxic to the diamondback moth. This system was used as a model to estimate the ecological risks associated with the field release of certain transgene/crop combinations with insecticidal activity.9 However since most of the main insect pests cannot be controlled with Bt toxins it is necessary to test other strategies. The best known alternative to obtain insect-resistant plants is the expression of recombinant proteinase inhibitors (PI).10 This chapter summarizes the works carried out along this line on B. napus.
15.2. Digestive Protease Types in Pests of B. Napus The digestive proteinases of three important pests of canola (two Lepidoptera: Mamestra configurata and Plutella xylostella, and one Coleoptera: Phyllotreta cruciferae) in
western Canada were characterized by assessing the proteolytic activity of midgut extracts against azocasein or azoalbumin at various pH, in the presence of different PIs.11 The larval midgut of M. configurata had maximum activity at pH 10.5, inhibited by 45-60 % with serine PIs. The maximum proteolytic activity of larval midgut in P. xylostella was measured at pH 10 and could be inhibited by 56-75 % with serine PIs. The midgut of adults of P. cruciferae, a flea beetle, exhibited maximum proteolytic activity at pH 5, activated by reducing agents and inhibited by cysteine PIs (33-61 % inhibition) and aspartate PIs (21-50% inhibition). When we initiated our work on PI-based control of B. napus pests, no report was available on midgut proteases of the major insect pests feeding on this plant in Europe. As a first step we characterized the midgut proteases of several B. napus coleopteran pests. Digestive proteolytic activity of larval midgut extracts was measured with azocasein as substrate for pH values ranging from 4.0 to 11.0 under reducing or non-reducing conditions, allowing to determine activity peaks. Activation of proteolytic activity at acidic pH in reducing conditions (in the presence of ß-mercaptoethanol) was suggesting the presence of cysteine proteinase s in the extracts, while the activity detected at neutral/ basic pH suggested the presence of serine proteinases. Qualitative detection and character-
Expression of Protease Inhibitors in Rapeseed
181
Table 15.2. Insect pests of B. napus in Europe and Canadaa Insect
Latin name
Family b
Europe Major pests Pollen beetle
Meligethes aeneus
Nitidulidae (Co)
Pod midge
Dasineura brassicae
Cecidomyiidae (Co)
Pod weevil
Ceutorhynchus assimilis
Curculionidae (Co)
Stem flea beetle
Psylliodes chrysocephala
Chrysomelidae (Co)
Cabbage aphid
Brevicoryne brassicae
Aphididae (Ho)
Rape stem weevil
Ceutorhynchus napi
Curculionidae (Co)
Delia radicum
Anthomyidae (Di)
D. brassicae
Anthomyidae (Di)
Cabbage stem weevil
Ceutorynchus pallidactylus
Curculionidae (Co)
Rape winter stem weevil
C. picitarsis
Curculionidae (Co)
Baris
Baris coerulescens
Curculionidae (Co)
Diamondback moth
Plutella xylostella
Plutellidae (Le)
Cabbage sawfly
Athalia orsae
Tenthredinidae (Hy)
Minor pests Cabbage root fly
Canada Major pests Bertha armyworn
Mamestra configurata
Noctuidae (Le)
Diamondback moth
Plutella xylostella
Plutellidae (Le)
Flea beetles
Phyllotreta cruciferae
Chrysomelidae (Co)
P. striolata
Chrysomelidae (Co)
Lygus elisus
Miridae (He)
L. lineolaris
Miridae (He)
Red turnip beetle
Entomoceslis americana
Chrysomelidae (Co)
Cabbage root fly
Delia radicum
Anthomyidae (Di)
Minor pests
a See ref. 7 for a review b Abbreviations: Co, Coleoptera; Ho, Homoptera; He, Heteroptera; Hy, Hymenoptera; Di, Diptera;
Le, Lepidoptera
182
ization of midgut proteases on gelatin-containing activity gels12 was also performed in parallel. The addition of specific PIs during azocasein tests and gel activity staining allowed to determine the types of proteases observed. Oryzacystatin I (OC-I) produced in E. coli (Lê Tân, unpublished results) and commercial soybean Bowman Birk inhibitor (BBI) were used as candidate PIs which could be expressed in transgenic plants to block the activity of cysteine and serine proteases, respectively. Figure 15.1 presents the different experiments performed for one Coleoptera, Ceutorhynchus napi. Peaks of activity were observed at pH 6 and pH 9. OC-I inhibited most of the activity observed at pH 6 while BBI completely inhibited the activity measured at pH 9. Several proteases were detected on activity gels. Similar results were found for the other Coleoptera tested (Table 15.3). Peaks of activity were observed at pH 6-6.5 and at pH 10 for Psylliodes chrysocephala, the stem flea beetle. Several proteases were detected on activity gels performed at the two pH optima, and the proteases active at pH 6 could be inhibited by OC-I.13 Two peaks of activity (pH 5 and pH 9) were also observed for Ceutorhynchus assimilis Scop., the pod weevil14 and for Baris coerulescens.37 For these two insects, cysteine protease s active at pH 5 were inhibited by OC-I. The Diptera, Delia radicum L. (cabbage root fly), mainly possessed serine-type gut proteases.15
15.3. Expression of Recombinant PIs in B. Napus 15.3.1. Expression of Serine PIs Lepidoptera mainly use serine proteinases for protein digestion,11,16 and serine PIs have been shown to be deleterious when ingested by these insects. Therefore these PIs have been regarded as suitable for the protection of plants from lepidopteran pests.17,18 Several transgenic lines of B. napus expressing serine PIs were obtained and tested against different pests (Table 15.4). The Bowman-Birk inhibitor cowpea trypsin inhibitor (CpTI) was introduced in oilseed rape under the control
Recombinant Protease Inhibitors in Plants
of the CaMV 35S promoter (Pestax Ltd, pers. comm.). A few bioassays with the pollen beetle Meligethes aeneus and the seed weevil Ceuthorynchus assimilis were performed in greenhouse conditions. No statistical difference was observed between normal and transgenic plants for these two insect pests. A vector containing a cDNA encoding the soybean serine PI, CII19 under the control of the CaMV 35S promoter with a double enhancer sequence was designed in our laboratory (Fig. 15.2). CII is a member of the Bowman-Birk family of PIs, which inhibit both trypsin and chymotrypsin. The PI-encoding vector was introduced via Agrobacterium tumefaciens-mediated gene transfer in the spring genotype Drakkar (Serasem, France), using a protocol derived from Moloney et al4 Several lines were selected according to the level of mRNA expression observed in Northern blot tests, and to their low T-DNA copy number.20,21 Western blots could not be performed since no antiserum was available against CII, which could not be expressed in E. coli. A low inhibitory activity against trypsin was detected in the selected lines, but serine PI activity was also detected in non-transformed plants, as already reported for the same cultivar by Visentin et al.22 Growth inhibition of the Egyptian cotton worm Spodoptera littoralis, a lepidopteran insect used as a model, was observed when providing these modified lines to the larvae (L. Jouanin, unpubl.). Feeding tests were also performed with the Diptera Delia radicum, but no effect on larval growth and development was observed (E. Brunel, pers. comm.). An experiment was also performed in growth chamber conditions with the Coleoptera Baris coerulescens. Adults were allowed to lay eggs for 5 days on the plants, and larval development was monitored by counting and weighing the larvae at different times. For all stages, except for L4 and pupae, there were no significant difference in developmental rates. The development was a little faster on CII-expressing plants: L4 were slightly heavier at the time of sampling but pupae were lighter, indicating a more
Expression of Protease Inhibitors in Rapeseed
183
A
B
C
Fig. 15.1. Digestive protease activity in the midgut of Ceutorhynchus napi. A: Total protease activity as a function of pH. «ß-mercaptoethanol» refers to the activity observed when this reducing agent was added at a 5 mM final concentration, as opposed to «basal», for which no ß-mercaptoethanol was added. B: Effects of OC-I and BBI on the overall protease activity at pH 6, in the presence of ß-mercaptoethanol, and at pH 9. C: Detection of C. napi protease activities resolved by gelatin/SDS-PAGE, as detected at pH 6 and pH 9. c, cysteine proteinase activity; s, serine proteinase activity.
Recombinant Protease Inhibitors in Plants
184
Table 15.3. Digestive proteinases in insect pests of B. napus Sensitivity to PIs a (in vitro tests)
Digestive Insect
proteases
serine PIs
cysteine PIs
aspartic PIs
Reference
P. xylostella (Le) b
serine
56-75%
0
0.5 %
11
M. configurata (Le)
serine
45-60 %
0
1%
11
P. cruciferae (Co)
cysteine + aspartic
1-6 %
33-61 %
21-50 %
11
C. assimilis (Co)
cysteine + serine
25% (BBI)
50% (OC-I)
ND
14
B. coerulescens (Co)
cysteine + serine
100 % (BBI)
100 % (OC-I)
ND
37
P. chrysocephala (Le)
cysteine + serine
ND
100 % (OC-I)
ND
13
C. napi (Co)
cysteine + serine
100 % (BBI)
100 % (OC-I)
ND
D. radicum (Di)
serine
83 % (BBI)
0%
ND
this work
15
a The inhibition rate (%) was determined at the pH considered as the optimum for serine or
cysteine protease activity (determined as shown for C. napi in Fig. 1A,B), using in most cases oryzacystatin I (OC-I) and soybean Bowman Birk inhibitor (BBI) as model inhibitors. b Abbreviations: Co, Coleoptera; Di, Diptera; Le, Lepidoptera; ND, not determined.
advanced state of pupation (Table 15.5). This phenomenon was due either to an effect of CII, or to an effect of temperature heterogeneity in the growth chamber, as suggested by temperature measurements made during larval development. In conclusion, the expression of CII in oilseed rape did not have a deleterious effect on this Coleoptera, which was shown to possess both serine and cysteine digestive proteases.
15.3.2. Expression of Cysteine PIs Until now, only one cysteine PI, the rice cystatin OC-I23 was introduced into plants on a regular basis to test the possibility of obtaining transgenic plants resistant to
Coleoptera.18 Poplars expressing OC-I were shown to induce high larval mortality of Chrysomela tremulae.24 This inhibitor was also introduced into B. napus (Table 15.4). Zaplachinski et al25 used an original strategy to express large quantities of OC-I in the first 4-10 days after germination, in order to inactivate flea beetle gut proteases when this insect feeds on the canola seedlings. OC-I was expressed in transgenic B. napus as a fusion with an 18-kDa seed oleosin.26 The fusion site between oleosin and OC-I contained a recognition sequence for the specific endopeptidase collagenase. Plant expressing high levels of OC-I were crossed with a second transgenic line
Expression of Protease Inhibitors in Rapeseed
185
Table 15.4. Recombinant PI-expressing lines of B. napus, and their effects against target pests in laboratory or greenhouse conditions Inhibitor (promoter)
Level of expression (% soluble proteins)
Target insect
Effects
Reference
CpTI (p35S) a
Unknown
M. aeneus (Co)
None
Pestax Ltd, pers. comm.
C. assimilis (Co)
None
Pestax Ltd, pers. comm.
S. littoralis (Le)
Growth inhibition
Jouanin, unpublished
D. radicum (Di)
None
15
P. cruciferae (Co)
Not 25 determined
0.1% in root crowns
B. coerulescens (Co)
None
0.3% in leaves
P. chrysocephala (Co) Enhanced growth
13
0.05% in pods
C. assimilis (Co)
Enhanced growth
14
0.1% in stems
C. napi (Co)
None
This work
0.1% in sap
M. persicae (Ho)
Reduced fecundity
Rahbé et al, unpublished
CII (p35S2)
Very low
OC-I (p oleosin)
2% (fusion protein) 0.2% free OC-I
2
(p35S )
37
a Abbreviations: 35S, cauliflower mosaic virus promoter 35S; CII, serine proteinase inhibitor CII,
from soybean; CpTI, cowpea trypsin inhibitor; Co, Coleoptera; Di, Diptera; Ho, Homoptera; Le, Lepidoptera; OC-I, oryzacystatin I; p, promoter.
expressing the collagenase gene under the control of the B. napus isocitrate lyase promoter, which is germination-specific. In the double trans- formants, cleavage of OC-I from oleosin was observed at the time of germination. The presence of OC-I is expected to provide protection to seedlings from flea beetle (Phyllotreta cruciferae) attack. Assuming that OC-I has a deleterious effect against P. cruciferae (insect bioassays are underway: Zaplachinski, pers. comm.), this strategy may
help decreasing selection for resistant insects since PI expression is highly regulated. As in Europe the main pests of B. napus are Coleoptera possessing cysteine proteinase s, we expressed OC-I under the control of the CaMV 35S promoter with a double enhancer in the spring cv. Drakkar (see Fig. 15.2). The protocols used to obtain and characterize the CII-expressing transgenic plants (see above) were used, except that the availability of an antiserum helped selecting lines expressing high levels of OC-I.20,21 The con-
186
Recombinant Protease Inhibitors in Plants
Fig. 15.2. Schematic representation of the T-regions of the binary plasmids used to express CII or OC-I in transgenic plants. The cDNA PI sequences, either for the Bowman-Birk CII (cII; ref. 19) or for the cysteine PI OC-I (ocI, ref. 23) were placed under the control of the CaMV 35S promoter with a double enhancer sequence (P35S2) and the pea Rubisco terminator (Trbcs). The TMV _ leader sequence (_) was added upstream of the coding sequences to enhance translation. The presence of the neomycin phosphotransferase gene (nptII) under the control of the nopaline synthase gene regulatory regions (Pnos, Tnos) allowed to select the transformed cells on kanamycin-containing medium. The ß-glucuronidase gene containing an intron (gus, ref. 36), under the control of the CaMV 35S promoter and terminator sequences (P35S, T35S) was added in the constructions to help selecting the transformed plants. tent in OC-I varied in different tissues (0.1 to 0.3 % of soluble proteins in young leaves, 0.05 % in seeds), and the inhibitor was not detected in pollen and nectar. Bioassays were conducted on entire plants in growth chamber or greenhouse conditions for several Coleopteran pests. Neonate larvae of P. chrysocephala were deposited at the crown of transgenic and control plants, collected 14 days after infestation, counted and weighed. No difference in the number of live larvae was observed. Larvae feeding on OC-Iexpressing plants were bigger (1.1 ± 0.34 mg) than larvae feeding on control plants (0.64 ± 0.25 mg). An increase of digestive protease activity was observed (67% for cysteine-type proteinases, and 140% for serine-type proteinases), but no new protease was detected.13 Adults of the cabbage seed weevil C. assimilis were collected in the field at two different locations. Flowering plants bearing young pods were infested with 5 females, that were allowed to lay eggs for 24 hours. The larvae were collected from the infested pods 21 days after egg-laying, weighed and measured. The two strains showed the same pattern of proteolytic activity and similar levels of OC-I-sensitive proteinase activity in vitro. However the larvae showed a differential sensitivity to the transgenic plants. One strain showed an increased growth rate when reared on transgenic OC-I plants (4.9 mg
compared to 2 mg on controls), while the growth of the other strain remained unaffected (3 mg compared to 3 mg on controls). Protease activity measured at pH 6 in larvae exhibiting increased weight was reduced by 60%, with the nearly complete disappearance of one specific cysteine proteinase . In contrast no modification of the total protease activity was observed for larvae of the other strain reared on either plants.14 In another study, adults of Baris coerulescens were allowed to lay eggs for 5 days on transgenic and control plants before being removed. After 36-39 days, the plants were dissected and the larvae were collected in roots, counted and weighed. No difference in the number or the weight of L4 larvae was observed for the two types of plants (9.8 ± 0.55 mg on OC-I-expressing plants, as compared to 10.6 ± 0.53 mg on controls). Cysteine protease activity at pH 6 was highly reduced (76%), but this was partly compensated by a 2-fold increase (227%) of the serine-type activity at pH 9.37 Experiments were also recently performed with the rape stem weevil Ceutorhynchus napi and the aphid Myzus persicae. For C. napi, no difference in larval development was observed (see Table 15.6), but the spring oilseed rape cultivar used for genetic transformation seemed to present natural partial resistance to infestation by this insect (J. Lerin, unpubl.). A reduction in fecundity (population reduced by about one half after 2 weeks on transgenic plants, as compared to control plants) was observed for the M. persicae reared on OC-I-expressing plants (Rahbé et al, unpubl.). Aphids are not thought to use
Expression of Protease Inhibitors in Rapeseed
187
Table 15.5. Weight of larvae, pupae and adults of Baris coerulescens fed CIIexpressing and control B. napus lines (cv. Drakkar) a L1 CII-
L2
L3
L4
Pupa
Adult
0.14 ± 0.01 (43)
0.43 ± 0.03 (56)
1.84 ± 0.16 (60)
9.20 ± 0.03 (114)
7.21 ± 0.28 (28)
5.26 ± 0.13 (4)
Control line
0.13 ± 0.01 (41)
0.43 ± 0.03 (49)
1.68 ± 0.14 (40)
8.31 ± 0.32 (77)
8.93 ± 0.33 (7)
5.40 ± 0.35 (2)
T-test [P]
1.76 [0.08]
0.07 [0.95]
0.80 [0.43]
1.95 [0.05]
-2.92 [0.006]
0.46 [0.66]
expressing line
a Data represent means (mg) ± standard errors. A t-test was performed to compare the data; the
numbers in parentheses indicate the number of insects used for each treatment; [P] is the level of significance for the statistical test.
digestive proteases, since they feed on phloem sap. The exact mode of action of OC-I against these insects is under investigation.
15.3.3. Double Transformants Using fixed homozygous lines, double transformants were obtained by crossing OC-I- and CII-expressing lines. Bioassays were performed with different Coleoptera, but no significant difference was observed when compared with the results obtained with OC-I-expressing plants. As a result of these crosses the level of OC-I expression was reduced by 50%, and the low level of CII did not confer an advantage to these lines. A double transformant line was also obtained by haploidization of a cross between OC-I and CII lines (M. Renard, unpubl.). This new line will be tested in the near future.
15.3.4. Field Trials with PI-Expressing Transgenic Plants Several winter lines of B. napus expressing CpTI were sown in UK (Pestax Ltd, pers. comm.). The rape winter stem weevil Ceutorhynchus picitarsis and the cabbage stem flea beetle Psylliodes chrysocephala were introduced both artificially and by natural infestation. No significant effects against these insects arose from the presence of CpTI. The
plants were not allowed to flower, so there were no data on flowering or yield characteristics. For some lines, variations were detected on the stand establishment, but it is difficult to relate this to PI expression. Several spring B. napus lines expressing OC-I were also sown in France, where no resistance to natural infestation of pests was observed (M. Renard, pers. comm.). A detailed characterization of the phenotype of OC-I-expressing and control plants was performed. No modification in size, flowering time and seed yield were noticed, suggesting that the expression of cysteine PIs in transgenic plants is not deleterious for their growth and yield. Moreover, bee frequentation was similar in the two types of plants (J. Pierre, pers. comm.).
15.3.5. Effects of PIs on Honeybees Honeybees (Apis mellifera L.) at larval and adult stages possess midgut serine proteinases,27 which stresses the importance of evaluating the impact of serine PI expression in oilseed plants, which are highly attractive to these insects. When expressed under the control of the CaMV 35S promoter, no PI was detected in the pollen, in accordance with results published on Arabidopsis thaliana.28 In addition, no PI was found in
Recombinant Protease Inhibitors in Plants
188
Table 15.6. Weights of L2 and L3 larvae of Ceutorhynchus napi fed OC-I-expressing and control B. napus (cv. Drakkar) a L2
L3
OC-I-expressing line
1.18 ± 0.17 (9)
13.73 ± 0.58 (26)
Control line
1.35 ± 0.15 (11)
13.71 ± 0.53 (26)
T-test
0.76
-0.03
[P]
[0.45]
[0.98]
a Data represent means (mg) ± standard errors. A t-test was performed to compare the data; the
numbers in parentheses indicate the number of insects used for each treatment; [P] is the level of significance for the statistical test.
the nectar, and no difference in bee behaviour was observed during pollen gathering on PI-expressing plants in greenhouse or field conditions (A.-L. Picard-Nizou and J. Pierre, unpubl.). Short- and long-term effects of ingestion of BBI- and OC-I-containing artificial diets by adult bees were investigated.29 A toxicity threshold dose of serine PI (50-100 mg/g pollen) was established, which is much higher than the expected level of expression in transgenic plants. Nevertheless these observations were demonstrating the need to evaluate the level of expression of transgenes showing potential risks in the pollen.
15.4. Discussion The results obtained until now regarding the control of B. napus insect pests using recombinant PIs expressed in plants are disappointing. Several reasons can explain these observations. Most of the published results on protection were obtained with Lepidoptera possessing mainly serine proteases, but the toxicity reported was not always sufficient to be used to protect plants in natural conditions.18,30 In B. napus, the pest complex is very large and a single pest cannot be considered as the major danger. Moreover, at least in Europe, Lepidoptera are minor pests, and at this point very little work has been devoted to the expression of serine PIs in this plant, CpTI and CII being the
only two inhibitors introduced. The level of CpTI in transgenic plants was not determined (C. Newell, pers. comm.), and the level of CII was very low. Finally, to our knowledge, no feeding test was performed with the diamondback moth, an insect which could be more sensitive to serine PIs. On the other hand, Coleoptera are major pests for B. napus in Europe. These insects possess a complex digestive system including at least serine and cysteine proteinase s, the latter group being in general more important (see Table 15.3). The cysteine PI OC-I was expressed in B. napus, but the expression level of this inhibitor was, even in the best lines, lower than the level obtained in transgenic poplars transformed with the same vector. In the latter case, OC-I was expressed at about 1% of total soluble protein in the leaves,24 while it was expressed at only 0.3% in B. napus leaves (and in lower amounts in the other tissues consumed by the insects). This low level could explain why delayed growth was observed in poplar for Chrysomela tremulae, but not in Coleoptera feeding on oilseed rape. In accordance with this hypothesis, a tobacco line expressing a serine PI isolated from mustard (MTI-2) was toxic to the lepidopteran insect Spodoptera littoralis only when expressed at a high dose in transgenic plants; a low MTI-2 expression level even induced better growth of the larvae and increased damage to
Expression of Protease Inhibitors in Rapeseed
the leaves.31 It is possible that the level of PI expression in the B. napus lines we analyzed corresponded to the level found in this latter group of transgenic tobacco lines, since in several bioassays the larvae feeding on OC-I plants were bigger than those fed control plants. It seems that a threshold in the expression level must be reached to observe a deleterious effect on insects provided with the PI-expressing plants. The easiest way to explain these results is that in a first stage, proteases can be overproduced or new proteases can be induced (depending on the insect) to compensate for the inactivation of a part of the midgut proteases. In a second stage, when the PI is present at a higher dose, the compensation cannot work, which would lead to developmental delays and increased mortality. Other mechanisms could be involved, such as the rapid degradation of PIs in the larval midgut, observed notably for the Coleoptera Phaedon cochleariae Fabricius.32 In addition, attention should be given to differences in susceptibility to PIs in different insect populations, as observed by Girard et al for C. assimilis.14 These observations demonstrate that more efficient PIs (i.e. possessing a higher affinity for insect proteases) or several different PIs used in combination should be expressed in transgenic plants to obtain efficient protection against insect pests.30,33,34 Nevertheless, even if oilseed rape expressing PIs is not ready for the market, this plant is a good candidate to test this strategy with Coleoptera. Also, oilseed rape is a melliferous plant visited by beneficial insects, therefore appearing as a good model to test the ‘non-toxicity’ of recombinant PI expression against honeybees. When expressed under the control of the CaMV 35S promoter, no PI was found in the pollen, which is very rich in proteins, and no PI could be detected in the nectar. However, given the diversified pest complex of B. napus, we do not find it realistic to hope achieving complete protection of the plant using PI-expressing transgenic lines. Instead, these plants should be considered as part of integrated pest man-
189
agement strategies, as previously exposed by Evans and Scarisbrick35.
Acknowledgments This work was supported by grants from the LIMAGRAIN Foundation, a “Saut technologique” between the French Ministry of Research and Technology and RUSTICA PROGRAIN GENETIQUE, and the European Union (BIOTECH program PL960365). The authors thank Dr. Mark Tepfer for critical reading of the manuscript.
References 1. Murphy DJ. Engineering oil production in rapeseed and other oil crops. Trends Biotechnol 1996; 14:206-213. 2. Pua E-C, Mehra-Palta A, Nagy F et al. Transgenic plants of Brassica napus L. Bio/ Technology 1987; 5:815-817. 3. Fry J, Barnason A, Horsch RB. Transformation of Brassica napus with Agrobacterium tumefasciens based vectors. Plant Cell Rep 1987; 6:321-325. 4. Moloney MM, Walker JM, Sharma KK. High efficiency transformation of Brassica napus using Agrobacterium vectors. Plant Cell Rep 1989; 8:238-242. 5. Boulter ME, Croy E, Simpson P et al. Transformation of Brassica napus L. (oilseed rape) using Agrobacterium tumefasciens and Agrobacterium rhizogenes—A comparison. Plant Sci 1990; 70:91-99. 6. Mazier M, Pannetier C, Tourneur J et al. The expression of Bacillus thuringiensis toxin genes in plant cells. Biotechol Annu Rev 1997; 3:313-347. 7. Hokkanen HMT, Wearing CH. Assessing the risk of pest resistance evolution to Bacillus thuringiensis engineered into crop plants: A case study of oilseed rape. Field Crops Res 1995; 45:171-179. 8. Stewart CN, Adang MJ, All JN et al. Insect control and dosage effects in transgenic canola containing a synthetic Bacillus thuringiensis cryIAc gene. Plant Physiol 1996; 112:115-120. 9. Stewart CN, All JN, Raymer A et al. Increased fitness of transgenic insecticidal rapeseed under selection pressure. Mol Ecol 1997; 6: 773-779. 10. Reeck GR, Kramer KJ, Baker JE et al. Proteinase inhibitors and resistance of transgenic plants to insects. In: Carozzi N Koziel M, eds. Advance in insect control: The role of transgenic plants. Taylor, Francis Publishers 1997:157-183.
190 11. Rymerson RT, Bodnaryk RP. Gut proteinase activity in insect pests of Canola. Can Entomol 1995; 127:41-48. 12. Michaud D. Gel electrophoresis of proteolytic enzymes. Anal Chim Acta 1998; 372:173-185. 13. Girard C, Le Métayer M, Zaccomer B et al. Growth stimulation of beetle reared on a transgenic oilseed rape expressing a cysteine proteinase inhibitor. J Insect Physiol 1998; 44:263-270. 14. Girard C, Bonadé-Bottino M, Pham-Delègue M-H et al. Two strains of cabbage seed weevil (Coleoptera: Curculionidae) exhibit differential susceptibility to a transgenic oilseed rape expressing oryzacystatin I. J Insect Physiol 1998; 44:569-577. 15. Brunel E, Bonadé M, Renoult TL et al. Estimate of rape genetically modified by protease inhibitors on Delia Radicum L. (Diptera: Anthomyidae). Brassica 97—10th crucifer genetic workshop. Rennes, France 1997:41 (abstr.). 16. Terra WR, Ferreira C. Insect digestive enzymes: Properties, compartimentalization and function. Comp Biochem Physiol 1994; 109B:1-62. 17. Hilder VA, Gatehouse AMR, Sherman SE et al. A novel mechanism of insect resistance engineered into tobacco. Nature 1987; 330:160-163. 18. Jouanin L, Bonadé-Bottino M, Girard C et al. Transgenic plants for insect resistance. Plant Sci 1998; 131:1-11. 19. Joudrier PE, Foard DE, Floener LA et al. Isolation and sequence of cDNA encoding the soybean protease inhibitors PI IV and CII. Plant Mol Biol 1987; 10:35-42. 20. Bonadé Bottino M. Défence du colza contre les insectes phytophages déprédateurs: Etude d’une stratégie basée sur l’expression d’inhibiteurs de protéases dans la plante. PhD thesis. Paris: Université Paris XI 1993. 21. Bonadé Bottino M, Girard C, Le Métayer M et al. Effects of transgenic oilseed rape expressing proteinase inhibitors on pests and beneficial insects. Acta Hort 1998; 459:235-239. 22. Visentin M, Iori R, Valdicelli L et al. Trypsin inhibitory activity in some rapeseed genotypes. Phytochemistry 1992; 31:3677-3680. 23. Abe K, Emori Y, Kondo H et al. Molecular cloning of a cysteine proteinase inhibitor of rice (oryzacystatin). J Biol Chem 1987; 262:16793-16797. 24. Leplé JC, Bonadé-Bottino M, Augustin S et al. Toxicity to Chrysomela tremulae (Coleoptera: Chrysomelidae) of transgenic poplars expressing a cysteine proteinase inhibitor. Mol Breeding 1995; 1:319-328.
Recombinant Protease Inhibitors in Plants 25. Zaplachinski S, Rymerson RT, Goll JM et al. Pulsed-release of flea beetle deterrence proteins in transgenic Brassica napus. 5th International Congress of Plant Molecular Biology. Singapore 1997:abstract 1317. 26. Van Rooijen GJH, Moloney MM. Plant seed oil-bodies as carriers for foreign proteins. Bio/Technology 1995; 13:72-77. 27. Moritz B, Crailsheim K. Physiology of protein digestion in the midgut of the honeybee (Apis mellifera). J Insect Physiol 1987; 33:923-931. 28. Wilkinson JE, Twell D, Lindsey K. Activities of CaMV 35S and nos promoters in pollen: Implications for field release of transgenic plants. J Exp Bot 1997; 48:265-275. 29. Girard C, Picard-Nizou AL, Grallien E et al. Effects of proteinase inhibitor ingestion on survival, learning abilities and digestive proteinases of the honeybee. Transgenic Res 1998; 7:239-246. 30. Jongsma MA, Bolter C. The adaptation of insects to plant protease inhibitors. J Insect Physiol 1997; 43:885-895. 31. De Leo F, Bonadé Bottino M, Ceci LR et al. Opposite effects on Spodoptera littoralis larvae of low and high expression level of a trypsin proteinase inhibitor in transgenic plants. Plant Physiol 1998; 118:997-1004. 32. Girard C, Le Métayer M, Bonadé-Bottino M et al. High resistance to proteinase inhibitor may be conferred by proteolytic cleavage in beetle larvae. Insect Biochem Mol Biol 1998; 28:229-237. 33. Jongsma MA, Stiekema WJ, Bosch D. Combatting inhibitor-insensitive proteases of insect pests. Trends Biotechnol 1996; 14:331-333. 34. Michaud D. Avoiding protease-mediated resistance in herbivorous pests. Trends Biotechnol 1996; 15:4-6. 35. Evans KA, Scarisbrick DH. Integrated insect pest management in oilseed rape crops in Europe. Crop Protect 1994; 13:403-412. 36. Vancanneyt G, Schmidt R, O’ConnorSanchez A et al. Construction of an introncontaining marker gene: Splicing of the intron in transgenic plants and its use in monitoring early events in Agrobacteriummediated plant transformation. Mol Gen Genet 1990; 220:245-250. 37. Bonadé-Bottino M, Lerin J, Zaccomer B et al. Physiological adaptaton explains the insensitivity of Baris coerulescens to transgenic oilseed rape expressing oryzacystatin I. Insect Biochem Mol Biol 1999; 29:131-138.
CHAPTER 16
Production of Useful Protease Inhibitors in Plants Dominique Michaud and Serge Yelle
16.1. Introduction
W
h i l e recombinant protease inhibitors(PIs) appear useful in plant protection (see Chapters 1-15, this volume),1-4 they also show potential for the regulation of proteases implicated in a variety of alternative processes, including processes of medical and industrial interest (see Chapters 17 and 18).5-7 In medicine, for instance, protein PIs may prove useful in the treatment of various infectious diseases and metabolic disorders. The production of extracellular proteinases by human pathogens during the infection process has been documented in many instances,5,8,9 and the therapeutic use of cystatins has notably been proposed for the treatment of several viral,10-15 bacterial16-20 and protozoan21,22 infections. Similarly, while in humans proteases are involved in a variety of important physiological and cellular processes including food digestion, complement activation, blood coagulation, apoptosis, immunity, prohormone processing and extracellular matrix remodeling important to bone development, 5,23 they also are associated with metabolic disorders like tumor metastasis,24,25 rheumatoid arthritis,26 Alzheimer’s disease,27,28 emphysema29 and pancreatitis.30 In this context, the transformation of plant genomes with PI-encoding cDNA clones appears attractive not only for the control of plant pests and pathogens, but also as a mean to produce PIs useful in alternative systems. Simpler organisms like Escherichia
coli have successfully been employed for the high-yield production of several protein PIs,31-35 but the use of plants as factories for the production of heterologous proteins offers several advantages. The costs associated with plant production are lower than those associated with fermentation devices or cell culture facilities,36,37 the production scale of plants may be easily increased without major logistic problems, and health risks for humans due to contamination with pathogens or toxins in recombinant products derived from plants appear negligible.38 Finally, in contrast with most microorganisms, protein post-translational modifications in plant cells, in particular protein glycosylation, are similar to those in animal cells,39 allowing the production of proteins submitted to complex posttranslational modifications. Like for any heterologous expression system, however the yields in recombinant protein are closely associated with the stability of this protein during the whole expression/ recovery process. 40-42 Plant cells possess proteases that may alter drastically the stability of foreign proteins either in planta before extraction, or ex planta during the recovery process. In this chapter we describe some basic strategies for the high-yield recovery of recombinant PIs from plants. After briefly discussing the potential of ‘controlled’ intracellular targeting of PIs in plant cells, we address the main points to consider for their extraction from plant tissues and for their
Recombinant Protease Inhibitors in Plants, edited by Dominique Michaud. ©2000 Eurekah.com.
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purification from crude extracts. Procedures for plant genetic transformation with PI-encoding cDNA sequences, and for the analysis of recombinant PIs expressed in plant tissues were reviewed elsewhere (see refs. 41 and 42).
16.2. High-level Accumulation of PIs in Plant Cells Despite the rapid developments in our understanding of protein catabolism in living cells, in vivo proteolysis still represents one of the most significant barriers to recombinant gene expression in heterologous systems.40 Some peptidases found in E. coli43 or yeast,44 for instance may rapidly cleave recombinant proteins, stressing out the importance of developing efficient strategies to minimize unwanted hydrolytic processes in these organisms. 45,46 Similarly, the post-translational ubiquitination of foreign proteins recognized as ‘abnormal’ in eukaryotic cells may lead to their rapid degradation via the well-characterized ubiquitin-mediated proteolysis pathway,47 showing the importance to detect any ubiquitin conjugation process taking place or likely to take place in the cytoplasm of yeast or animal cells when the proteins are to be expressed and accumulated in this cellular compartment.48 In plants, little is known about the interactions between recombinant proteins and the intracellular proteases, but the occurrence of hydrolytic processes similar to those observed in bacterial and yeast cells appears likely. Poorly-specific proteases reside in the vacuole of plant cells,49-55 and most components of the ubiquitin pathway have been identified in plants.53,55 Proteases of plant cells, in particular leaf vacuolar proteases active in the mildly-acidic pH range, may significantly alter the integrity of recombinant proteins, with significant negative effects on protein yields.42 At this stage, however the specific mechanisms underlying the action of plant proteases against heterologous proteins remain unknown. In contrast with bacteria43,46 and yeast,44,45 the resident proteases of plant cells have not been thoroughly characterized, and mutants lacking proteases potentially
Recombinant Protease Inhibitors in Plants
damaging to recombinant proteins are not available. Future progress in identifying and characterizing the main vacuolar proteases of plant cells should help us develop specific tests for each particular group of enzymes and devise strategies for specifically counteracting their effect, as has been the case for yeast,44,45 but at present the main strategy to avoid unwanted proteolysis in planta consists in directing the accumulation of recombinant polypeptides in alternative cellular locations using appropriate targeting signals. While several reports suggested that most ‘non-specific’ proteases found in the vegetative organs of plants are vacuolar cysteine and aspartate proteinases active in the mildlyacidic pH range,50,51,54 it is also well established that the ubiquitination of “abnormal”—including foreign—proteins takes place primarily in the cytoplasm.53 Given these facts, adding peptide signals to the primary sequence of recombinant proteins to direct their accumulation in extracellular compartments or in the endoplasmic reticulum (ER) through the secretory pathway represents an interesting alternative to “natural”, nonmodified targeting (see Fig. 16.1). Several peptide sequences directing the translocation of proteins in particular cellular compartments of plant cells have been elucidated (see refs. 56 and 57), and their fusion to recombinant proteins using appropriate gene constructs has proven functional to specifically control their final destination in transgenic plant cells.58-63 In particular, the addition of an ER-retention (K/H-DEL) signal at the C-terminus of vacuolar proteins may prove useful to prevent their further progression through the secretory pathway,64-66 and thus minimize unwanted proteolysis by leaf vacuolar proteases in vivo.42,61 Accumulation of the pea vacuolar storage protein vicilin, for instance, was increased by 100-fold in transgenic mesophyll cells of alfalfa when the tetrapeptidic signal lys-asp-glu-leu (KDEL) was fused to its carboxy-terminus.61 This increase was associated with retention of the protein in the ER, preventing its further progression through the secretory pathway and its subsequent degradation into the vacuole.56,61 Although important
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Fig. 16.1. The translocation of proteins in plant cells via the secretory pathway. Proteins including in their primary sequence a enter the secretion pathway via the endoplasmic reticulum (ER), while those with no such signal are accumulated in the cytoplasm (C) or subsequently directed to other organelles via a cytoplasmic route. Depending on the presence or not of additional signals in their primary structure, the secreted proteins are either retained in the ER (proteins with a KDEL or a HDEL sequence at their C-terminal end) or tranferred to the Golgi apparatus (G), from which they are transported to the vacuole (V) or secreted to the cell wall (CW) via a default pathway. See refs. 57 and 62 for reviews. questions remain regarding the exact role of the KDEL sequence in plant cells67 and the actual efficiency of this signal to retain proteins in the ER when vacuolar signals also are present,63,68 this approach appears attracting for the production of recombinant PIs. Some PIs in plants are naturally deposited in cell vacuoles,69-71 and thus possess intrinsic signals directing their accumulation in the vacuolar compartment. As reported for proteinase inhibitors I and II in tomato leaves, some of these inhibitors are very stable and can accumulate to high levels in plant cell vacuoles,71,72 but the occurrence of hydrolytic processes mediated by ‘foreign’ (non-natural) proteases in transgenic plants cannot be excluded. High-level production of animal PIs, in particular, could be compromised by the action of the plant resident proteases. To overcome this problem, fusing an ER retention signal to naturally-excreted PIs, or removing the N-terminal signal peptide that initiates their translocation through the secretory pathway could represent a simple and useful way to avoid their accumulation in the vacuole. In a recent study Gomord et al63 fused the HDEL sequence to the C-terminus
of either a vacuolar form and an extracellular form of sporamin, a storage protein of sweetpotato showing anti-trypsin activity and potential in plant protection.73,74 In both cases the protein was efficiently retained in the ER of transgenic tobacco leaf cells, showing the actual potential of this approach to prevent the progression of recombinant PIs along the secretory pathway.
16.3. Extraction of Recombinant PIs from Plant Tissues Along with the development of strategies aimed at achieving high-level expression and accumulation of recombinant PIs, procedures must be devised to ensure their stability during the extraction and purification processes. Until now, several procedures have been described for the recovery of recombinant PIs expressed in simple expression systems like E. coli,32-35 but no general procedure is available for the large-scale preparation of these proteins from plant tissues. In fact, given the diverse characteristics of PIs found in living cells and the various interactions susceptible to take place between these proteins and the resident
194
proteases of plant cells in crude extracts, adapted procedures must be developed for each particular combination of PI and plant tissue, keeping in mind some basic principles. Ideally, a good recovery strategy should ensure the stability of the inhibitor throughout the extraction process, and allow its purification by a simple, single-step purification scheme.42 General guidelines have been proposed to protect proteins during sample preparation from plant tissues.75,76 When extracting proteins from any cell or tissue, especially when intracellular proteins are recovered from intact cells, various compounds released in the extraction medium after cell breakage may alter their integrity. Proteases and phenolic compounds present in green tissues, in particular may readily and irreversibly affect the structural integrity of several proteins77-79 and thus drastically decrease protein yields. As noted above adequate protection strategies must be devised for each particular system, but some general rules have been defined to ensure protection of proteins during extraction from various sources, including the breakage of cells and tissues at 4˚C, the inclusion of compounds neutralizing phenolics in the extraction buffer,79,80 and the use of lowmolecular-weight PIs active against the plant resident proteases (see refs. 75, 76 and 81 for reviews). In particular, inhibition of plant endogenous proteases appears useful in most extraction procedures aimed at isolating recombinant PIs under a stable form (see Appendix I, this volume, for a list of useful low-molecular- weight PIs). Like any other protein, protein PIs may be recognized as substrates by poorly specific nontarget proteases, causing in certain cases their rapid disappearance from crude extracts. Oryzacystatin I (OC-I), for instance is rapidly degraded when incubated with a vacuole preparation of strawberry leaf cells. 42 Similarly, while this same inhibitor is accumulated under a single and stable form in the cytoplasm of transgenic potato leaf cells, it is partly degraded by proteases from the other cell compartments when crude extracts are prepared from the leaf tissues.82 In this
Recombinant Protease Inhibitors in Plants
specific case, the addition of phenylmethylsulfonyl fluoride to the extraction buffer, which inhibits most ‘gelatinases’ present in crude extracts of potato leaves,83 provides a simple way to stabilize the cystatin and most proteins present in the extract (Fig. 16.2). Provided that some information is available about the proteases present in host tissues, low-molecular-weight PIs can prove useful for the protection of protein PIs prepared from a large variety of cells and tissues.
16.4. Purification of Recombinant PIs from Crude Extracts For most medical and industrial applications, the inhibitors expressed in plant tissues must be recovered under a pure and active form, preferably by using an efficient single-step procedure. In practice, every step used to purify a given protein from complex biological extracts results in the enrichment of this protein, but also contributes to decrease the final yields. The original scheme reported for the purification OC-I from rice seed extracts, for instance, required multiple chromatographic steps leading to a final recovery yield of only 28% of OC-I activity, as compared with the activity found initially in the crude extract.84 Provided that the protein remains biologically active, the easiest way to efficiently recover a given PI from a crude extract consists in fusing an “affinity handle” (or affinity partner) to one or both of its extremities. Chen et al,31 for instance fused a basic peptidic tail to the N-terminus of OC-I, allowing purification of this inhibitor from E. coli crude extracts using a one-step anionexchange chromatography procedure. Similarly, adding the 26-kDa glutathione S-transferase (GST) from Schistosoma japonicum to the N-terminus of OC-I, 33 oryzacystatin II (OC-II),33 human stefin A34 and corn cystatin II (see Chapter 9, this volume) allowed us to purify these inhibitors from E. coli extracts by a single-step affinity purification procedure, using agarose beads embedded with glutathione, a cofactor of GST.
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Fig. 16.2. Degradation of recombinant OC-I in leaf crude extracts of a transgenic potato line expressing OC-I. The proteins were recovered from the fifth leaf in a 50 mM sodium phosphate buffer, pH 6.8. The incubation was carried out for 0 min (control) or 60 min at room temperature, in the presence (+) or absence (-) of 1 mM phenylmethylsulfonyl fluoride (PMSF). The data correspond to the relative amount of OC-I detected by immunoblotting using an anti-OC-I polyclonal antibody, as compared to the control sample (0 min incubation). Each datum is the mean of three values ± SE.
16.4.1. Affinity Handles Until now, several different systems have been described for the purification of recombinant proteins with fusion partners.85,86 Different kinds of fusion partners are currently used, from peptides to relatively large proteins. As a general rule, the purification procedure consists to express the fusion protein in a given host, and to extract the (soluble) proteins under appropriate conditions (see above, Section 16.3). The crude protein
preparation is then passed through a column coupled to a high-affinity ligand for the affinity partner, and the fusion is finally eluted by chemical competition or by a change in pH (Table 16.1). Although most gene fusion systems have been initially developed with E. coli as a host,86,87 they also are usually functional in eucaryotic systems. In yeast, animal and plant systems, some of the most useful fusion partners to purify heterologous proteins are polyhistidine tags, the FLAG
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Table 16.1. Affinity fusion partners frequently used for the purification of recombinant proteins (see text for details) Fusion partner
Affinity ligand
Elution
Poly-histidine
metal2+ (usually Ni2+)
low pH or imidazole
FLAG peptide
monoclonal antibody M1
chelating agent
Glutathione S-transferase
glutathione
reduced glutathione
Protein A
human IgG
low pH
Albumin-binding protein
human serum albumin
low or high pH
peptide, glutathione S-transferases, the Staphylococcal protein A and the albuminbinding protein.86 The use of polyhistidine tags is based on the interaction between the side chains of histidine residues present at the surface of a protein and metal ions immobilized by a chelating agent capable to present the metallic ions for binding to the protein.86 This method, known as immobilized metal ion affinity chromatography (or IMAC), simply consists in passing the crude extract containing the polyhistidine-tagged protein through a column containing immobilized Ni2+ ions, and then eluting the fusion protein by decreasing the pH, or by competition with imidazole. 88,89 IMAC allows single-step recovery of the fusion with a purity higher than 90%. Although a recent study reported the unexpected influence of a C-terminal polyhistidine tag on the maturation of a β-lactamase from the thermophilic bacterium Bacillus licheniformis, 90 this approach is generally thought to have little impact on the structure and the activity of the protein, preventing the need to cleave the fusion for restoring the affinity of the attached protein. The FLAG peptide system is based on the N-terminal fusion of an 8-amino-acid peptide (asp-tyr-lys-asp-asp-asp-asp-lys) to the recombinant protein. The usual affinity ligand of the FLAG peptide is a monoclonal antibody called M1, which reacts with the
peptide in a calcium-dependent manner. The simple addition of a chelating agent prevents the interaction between the two molecules, allowing elution of the fusion protein in very mild conditions. 91,92 Noteworthy, the C-terminal part of the FLAG peptide, asp-asp-asp-asp-lys corresponds to the cleavage recognition sequence of enterokinase, and allows recovery of the intact protein by a treatment with this protease.91 Glutathione S-transferases (or GSTs) form a family of enzymes naturally involved in the detoxication of nitrogenated and halogenated compounds. GSTs can easily be purified with the cofactor glutathione as an affinity ligand, followed by competitive elution with reduced glutathione.93 A wide variety of fusion proteins have been expressed fused to GST from the nematode Schistosoma japonicum as a fusion partner,94 with the recombinant protein usually located at the C-terminus. This system appears quite useful for the expression of active cystatins,33-35 but may not be appropriate for serine PIs containing disulfide bonds, as the elution step is performed in strongly reducing conditions.95 The staphylococcal protein A (or SPA) is an immunoglobulin-binding receptor found at the surface of the bacterium Staphylococcus aureus. This protein binds tightly to the Fc part of immunoglobulins in non-acidic conditions, and thus represents a quite useful tool for the purification of SPA-tagged
Production of Useful Protease Inhibitors in Plants
proteins.96 As noted by Nilsson et al,86 SPA possesses several interesting features as a fusion partner, including a poor susceptibility to proteolysis97 and the absence of cysteine residues in its primary structure, which could interfere with the formation of disulphide bonds in the tagged protein.98 SPA represents a quite versatile system for the single-step purification of proteins, having been used with various expression systems including bacteria, yeast, plant cells, mammalian ovary cells and insect cells.97,99 Finally the albumin-binding protein (or ABP) is a part of the streptococcal protein G showing strong affinity for human serum albumin.100 ABP-tagged proteins are easily eluted from serum albumin-affinity columns in acidic86 or basic101 conditions. Like SPA, ABP is poorly susceptible to proteolysis, suggesting its potential to stabilize fragile PIs. Interestingly, the very high stability of ABP once bound to serum albumins is associated with a parallel increased stability of the tagged proteins in vivo,101,102 suggesting the potential of this polypeptide as a fusion partner for the efficient delivery of peptidic therapeutics.86 Such an application could appear of particular interest to preserve the antimicrobial or antiviral properties of certain protein PIs found in ‘functional foods’ (see ref. 7).
16.4.2. Site-Specific Proteinases In many instances, proteins linked to a fusion partner retain their biological activity. For instance we observed that OC-I, OC-II, corn cystatin II and human stefin A remain fully active when GST is attached to their N-terminal end,33,34 and the same fusion partner attached to the C-terminus of OC-I does not affect the inhibitory activity of this inhibitor.35 Similarly, OC-I retains its inhibitory activity when a basic amino acid string is linked to its N-terminus,31 suggesting that this PI would remain stable and active when attached to a variety of fusion partners (also see Chapter 10, this volume). For applications in which the presence of a companion peptide does not cause problem, this remarkable characteristic of certain PIs to keep their activity when expressed as fusion proteins
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appears of particular interest to develop efficient one-step purification strategies, and thus obtain large amounts of pure inhibitor at low cost. In some cases, however the fusion partner may interfere with the activity of the protein or cause unwanted immune responses when the protein is used as a therapeutic agent or as a vaccine.86 Several procedures have been described for the removal of fusion partners, including chemical and enzymatic treatments. Although more expensive, enzymatic systems usually appear more appropriate as they allow to cleave the fusion protein at specific locations in mild conditions. In general, enzymatic procedures consist to include the specific cleavage site of a given protease between the protein and the fusion partner. After purification, the fusion protein is incubated under appropriate conditions with the protease, and then recovered in the eluate fraction following chromatography with an affinity ligand for the fusion partner. To avoid contamination of the final product, the cleavage step may be performed with the protease immobilized onto a solid matrix.103 Several proteases are currently employed to cleave fusion proteins, including the widely used proteases factor Xa and thrombin (see Table 16.2). In general, enzymes with long recognition sequences are used to avoid unspecific cleavage of either the recombinant protein and its fusion partner. Poorly-specific proteases may be used in certain cases, notably when both the fusion partner and the protein are not affected by this protease, or when the proteolytic fragments generated are active. Cleavage of GST-OC-I and GST-OC-II fusion proteins with bovine trypsin, for instance, results in the accumulation of OC-I and OC-II active truncated fragments (D. Michaud, unpubl. data), despite the presence of multiple basic residues in the primary sequence of these cystatins.42
16.5. Conclusions While until now recombinant PIs have been generally produced in E. coli, plant ‘molecular farming’ appears attractive for the large-scale production of these proteins.
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Table 16.2. Chemicals and proteases useful to cleave fusion proteinsa Cleavage agent
Cleavage specificity
Cleavage conditions
CNBr
-XM † X-
70% (v/v) formic acid, 20˚C
hydroxylamine
-XN † GX-
pH 9.0, 45˚C
enterokinase
-XDDDDK † X-
pH 8.0, 37˚C
factor Xa
-XIEGR † X-
pH 7.2, 20˚C
thrombin
-XLVPR † GSX-
pH 8.0, 25˚C
subtilisin (H64A)
-XFAHY † X-
pH 8.6, 37˚C
Chemicals
Proteinases
a See ref. 86 for a review; † indicates the site of cleavage; X is any amino acid.
Several peptidic signals controlling the intracellular targetting of proteins in plant cells have been elucidated, and the potential of some of these signals for the stabilization of heterologous proteins has been clearly established. In parallel several procedures were proposed to ensure the stability of proteins in plant crude extracts, and most biochemical strategies devised for the high-yield purification of recombinant proteins were adapted to plant systems. Fusion protein technologies, in particular, appear very suitable in the purification of protein PIs, which in several cases remain active when attached to a fusion partner. On a ‘content per cell’ basis, plants may seem poorly efficient in expressing recombinant proteins, with accumulation levels ranging between 1 and 2% in the best cases, as compared to yields of 10% and even more for E. coli and yeast. However the huge amounts of biomass accumulated in the field during the growing season makes plant molecular farming very attractive from an industrial point of view. In particular, the expression of recombinant proteins in plant leaves may give a significant added value to plants from which only fruits or tubers are
usually harvested. The recovery of recombinant PIs from potato leaves, for instance could prove of interest, as it would allow concommitant production of tubers and proteins in the same plant. Alternatively recombinant PIs could be expressed in tubers, which could then serve either as a source of recombinant protein for additional downstream processing, or as a shuttle for direct delivery of protein PIs useful in human health.7,104 The challenge, here will not be to purify the inhibitor, but to ensure its stability in the system targetted.
Acknowledgments We thank Line Cantin for helpful comments on the manuscript. This work was supported by operating and strategic grants of the Natural Science and Engineering Research Council of Canada.
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Production of Useful Protease Inhibitors in Plants 2. Hilder VA, Gatehouse AMR, Boulter D. Proteinase inhibitor approach. In: Kung S-D, Wu R, eds. Transgenic Plants: Engineering and utilization, Vol. 1. New York: Academic Press, 1993:317-338. 3. Lorito M, Broadway RM, Hayes CK et al. Proteinase inhibitors from plants as a novel class of fungicides. Mol Plant Microbe Interact 1994; 7:525-527. 4. Atkinson HJ, Urwin PE, Hansen PE et al. Designs for engineered resistance to rootparasitic nematodes. Trends Biotechnol 1995; 13:369-374. 5. Henskens YMC, Veerman ECI, Nieuw Amerongen AV. Cystatins in health and disease. Biol Chem 1996; 377:71-86. 6. Garcia-Carreno FL. Proteinase inhibitors. Trends Food Sci 1996; 7:197-204. 7. Arai S. Studies on functional foods in Japan—state of the art. Biosci Biotechnol Biochem 1996; 60:9-15. 8. McKerrow JH. Parasite proteases. Exp Parasitol 1989; 68:111-115. 9. Potempa J, Pavloff N, Travis J. Porphyromonas gingivalis: a proteinase/gene accounting audit. Trends Microbiol 1995; 3:430-434. 10. Kondo H, Ijiri S, Abe K et al. Inhibitory effect of oryzacystatins and a truncation mutant on the replication of poliovirus in infected Vero cells. FEBS Lett 1992; 299:48-50. 11. Aoki H, Akaike T, Abe K et al. Antiviral effects of oryzacystatin, a proteinase inhibitor in rice, against herpes simplex virus type 1 in vitro and in vivo. Antimicrob Agents Chemother 1995; 39:846-849. 12. Korant BD, Brzin J, Turk V. Cystatin, a protein inhibitor of cysteine proteinases alters viral protein cleavages in infected human cells. Biochem Biophys Res Commun 1985; 127:1072-1076. 13. Korant B, Towatari T, Kelley M et al. Interactions between viral protease and cystatins. Biol Chem Hoppe-Seyler 1988; 369:281-286. 14. Björck L, Grubb A, Kjellen L. Cystatin C, a proteinase inhibitor, blocks replication of herpes simplex virus. J Virol 1990; 64:385-386. 15. Collins AR, Grubb A. Inhibitory effects of recombinant human cystatin C on human coronaviruses. Antimicrob Agents Chemother 1991; 35:2444-2446. 16. Blankenvoorde MJF, Henskens YMC, Van’t Hof W et al. Antibacterial activity against Porphyromonas gingivalis by cystatins. In: Hopsu-Havu VK, ed. Proteolysis in Cell Functions. Amsterdam: IOS Press, 1997:532-539. 17. Blankenvoorde MFJ, Henskens YMC, Van ‘t Hof W et al. Inhibition of the growth and cysteine proteinase activity of Porphyromonas gingivalis by human salivary cystatin S and chicken cystatin. Biol Chem 1996; 377:847-850.
199 18. Grenier D. Effect of protease inhibitors on in vitro growth of Porphyromonas gingivalis. Microb Ecol Health Dis 1992; 5:133-138. 19. Naito Y, Sasaki M, Umemoto T et al. Bactericidal effect of rat cystatin S on an oral bacterium, Porphyromonas gingivalis. Comp Biochem Physiol 1995; 110C:71-75. 20. Takahashi M, Tezuka T, Katunuma N. Inhibition of growth and cysteine proteinase activity of Staphylococcus aureus V8 by phosphorylated cystatin A in skin cornified envelope. FEBS Lett 1994; 355:275-278. 21. Luaces AL, Barrett AJ. Affinity purification and biochemical characterization of histolysin, the major cysteine proteinase of Entamoeba histolytica. Biochem J 1988; 250:903-909. 22. Serveau C, Lalmanach G, Juliano MA et al. Investigation of the substrate specificity of cruzipain, the major cysteine proteinase of Trypanosoma cruzi, through the use of cystatin-derived substrates and inhibitors. Biochem J 1996; 313:951-956. 23. Chapman HA, Riese RJ, Shi G-P. Emerging roles for cysteine proteases in human biology. Annu Rev Physiol 1997; 59:63-88. 24. Sloane BF, Roxhin J, Lah TT et al. Tumor cathepsin B and its endogenous inhibitors in metastasis. Adv Exp Med Biol 1988; 233P:259-268. 25. Ren W-P, Sloane BF. Cathepsins D and B in breast cancer. In: Dickson R, Lippman M, eds. Mammary tumor cell cycle, differentiation and metastasis. Amsterdam: Kluwer Academic Publishers, 1996:325-352. 26. Trabandt A, Gay RE, Fassbender H-G et al. Cathepsin B in synovial cells at the site of joint destruction in rheumatoid arthritis. Arthritis Rheum 1991; 34:1444-1451. 27. Eriksson S, Janciauskiene S, Lannfelt L. α 1-antichymotrypsin regulates Alzheimer β-amyloid peptide fibril formation. Proc Natl Acad Sci USA 1995; 92:2313-2317. 28. Chen M. Alzheimer’s α-secretase may be a calcium-dependent protease. FEBS Lett 1997; 417:163-167. 29. Chapman HA, Jr, Stone OL. Comparison of live human neutrophil and alveolar macrophage elastolytic activity in vitro. Relative resistance of macrophage elastolytic activity to serum and alveolar proteinase inhibitors. J Clin Invest 1991; 74:1693-1700. 30. Steer ML, Meldonlesi J, Figarella C. Pancreatitis. The role of lysosomes. Dig Dis Sci 1984; 29:934-938. 31. Chen M-S, Johnson B, Wen L et al. Rice cystatin: Bacterial expression, purification, cysteine proteinase inhibitory activity, and insect growth suppressing activity of a truncated form of the protein. Protein Express Purif 1992; 3:41-49.
200 32. Sancho E, Tonge DW, Hockney RC et al. Purification and charactrerization of active and stable recombinant plasminogen-activator inhibitor accumulated at high levels in Escherichia coli. Eur J Biochem 1994; 224:125-134. 33. Michaud D, Nguyen-Quoc B, Yelle S. Production of oryzacystatins I and II in Escherichia coli using the glutathione S-transferase gene fusion system. Biotechnol Progr 1994; 10:155-159. 34. Michaud D, Bernier-Vadnais N, Vrain TC et al. Response of digestive cysteine proteinases from the Colorado potato beetle, (Leptinotarsa decemlineata) and the black vine weevil (Otiorynchus sulcatus) to a recombinant form of human stefin A. Arch Insect Biochem Physiol 1996; 31:451-464. 35. Tudyka T, Skerra A. Glutathione S-transferase can be used as a C-terminal, enzymatically active dimerization module for a recombinant protease inhibitor, and functionally secreted into the periplasm of Escherichia coli. Protein Sci 1997; 6:2180-2187. 36. Rudolph NS. Regularoty issues relating to protein production in transgenic systems. Genet Eng News 1995; 15:16-18. 37. Borwer V. PPL floats IPO as companies consider transgenic switch. Nat Biotechnol 1996; 14:692. 38. Sardana RK, Ganz PR, Dudani A et al. Synthesis of recombinant human cytokine GM-CSF in the seeds of transgenic tobacco plants. In: Cunningham C, Porter AJR, eds. Recombinant proteins from plants. Production and isolation of clinically useful compounds. Totowa NJ: Humana Press 1998:77-87. 39. Chrispeels MJ. Sorting of proteins in the secretory system. Annu Rev Plant Physiol Plant Mol Biol 1991; 42:21-53. 40. Emr SD. Heterologous gene expression in yeast. Meth Enzymol 1990; 185:231-233. 41. Michaud D, Vrain TC. Expression of recombinant proteinase inhibitors in plants. In: Cunningham C, Porter AJR, eds. Recombinant proteins from plants. Production and isolation of clinically useful compounds. Totowa NJ:Humana Press 1998:49-64. 42. Michaud D, Vrain TC, Gomord V et al. Stability of recombinant proteins in plants. In: Cunningham C, Porter AJR, eds. Recombinant proteins from plants. Production and isolation of clinically useful compounds. Totowa NJ:Humana Press 1998:177-188. 43. Maurizi MR. Proteases and protein degradation in Escherichia coli. Experientia 1992; 48:178-201. 44. Jones EW. Three proteolytic systems in the yeast Saccharomyces cerevisiae. J Biol Chem 1991; 266:7963-7966.
Recombinant Protease Inhibitors in Plants 45. Jones EW. Vacuolar proteases in yeast Saccharomyces cerevisiae. Methods Enzymol 1990; 185:372-386. 46. Gottesman S. Minimizing proteolysis in Escherichia coli: genetic solutions. Methods Enzymol 1990; 185:119-129. 47. Ciechanover A. The ubiquitin-proteasome proteolytic pathway. Cell 1994; 79:13-21. 48. Wilkinson KD. Detection and inhibition of ubiquitin-dependent proteolysis. Methods Enzymol 1990; 185:387-397. 49. Wittenbach VA, Lin W, Hebert RR. Vacuolar localization of proteases and degradation of chloroplasts in mesophyll protoplasts from senescing primary wheat leaves. Plant Physiol 1982; 69:98-102. 50. Storey RD. Plant endopeptidases. In: Dalling M, ed. Plant Proteolytic enzymes. Boca Raton:CRC Press, 1986:119-135. 51. Canut H, Dupré M, Carrasco A et al. Proteases of Melilotus alba mesophyll protoplasts. Planta 1987; 170:541-549. 52. Chrispeels MJ, Raikhel NV. Short peptide domains target proteins to plant vacuoles. Cell 1992; 68:613-616. 53. Vierstra RD. Protein degradation in plants. Annu Rev Plant Physiol Plant Mol Biol 1993; 44:385-410. 54. Callis J. Regulation of protein degradation. Plant Cell 1995; 7:845-857. 55. Vierstra RD. Proteolysis in plants: Mechanisms and functions. Plant Mol Biol 1996; 32:275-302. 56. Chrispeels MJ. Sorting of proteins in the secretory system. Annu Rev Plant Physiol Plant Mol Biol 1991; 42:21-53. 57. Bar-Peled M, Bassham DC, Raikhel NV. Transport of proteins in eukaryotic cells: More questions ahead. Plant Mol Biol 1996; 32:223-249. 58. Bednarek SY, Wilkins TA, Dombrowski JE et al. A carboxy-terminal propeptide is necessary for proper sorting of barley lectin to vacuoles of tobacco. Plant Cell 1990; 2:1145-1155. 59. Wilkins TA, Bednarek SY, Raikhel NV. Role of propeptide glycan in post-translational processing and transport of barley lectin to vacuoles in transgenic tobacco. Plant Cell 1990; 2:301-313. 60. Neuhaus J-M, Sticher L, Meins F, Jr et al. A short C-terminal sequence is necessary and sufficient for the targeting of chitinase to the plant vacuole. Proc Natl Acad Sci USA 1991; 88:10362-10366. 61. Wandelt CI, Khan MRI, Craig S et al. Vicilin with carboxy-terminal KDEL is retained in the endoplasmic-reticulum and accumulates to high-levels in the leaves of transgenic plants. Plant J 1992; 2:181-192.
Production of Useful Protease Inhibitors in Plants 62. Gomord V, Faye L. Signals and mechanisms involved in intracellular transport of secreted proteins in plants. Plant Physiol Biochem 1996; 34:165-181. 63. Gomord V, Denmat L-A, Fitchette-Lainé A-C et al. The C-terminal HDEL sequence is sufficient for retention of secretory proteins in the endoplasmic reticulum (ER) but promotes vacuolar targeting of proteins that escape the ER. Plant J 1997; 11:313-325. 64. Munro S, Pelham HRB. A C-terminal signal prevents secretion of luminal ER proteins. Cell 1987; 48:899-907. 65. Pidoux AL, Armstrong J. Analysis of the BiP gene and identification of an ER retention signal in Schizosaccharomyces pombe. EMBO J 1992; 11:1583-1591. 66. Denecke J, Goldman MHS, Demolder J et al. The tobacco luminal binding protein is encoded by a multigene family. Plant Cell 1991; 3:1025-1035. 67. Jones AM, Herman EM. A KDEL-containing auxin-binding protein is secreted to the plasma membrane and cell wall. Plant Physiol 1993; 101:595-606. 68. Herman EM, Tague BW, Hoffman LM et al. Retention of phytohemagglutinin with carboxyterminal tetrapeptide KDEL in the nuclear envelope and the endoplasmic reticulum. Planta 1990; 182:305-312. 69. Shumway LK, Yang VV, Ryan CA. Evidence for the presence of proteinase inhibitor I in vacuolar protein bodies of plant cells. Planta 1976; 129:161-165. 70. Walker-Simmons M, Ryan CA. Immunological identification of proteinase inhibitors I and II in isolated tomato leaf vacuoles. Plant Physiol 1977; 60:61-63. 71. Wingate VPM, Franceschi VR, Ryan CA. Tissue and cellular localization of proteinase inhibitors I and II in the fruit of the wild tomato, Lycopersicon peruvianum (L.) Mill. Plant Physiol 1991; 97:490-495. 72. Gustafson G, Ryan CA. Specificity of turnover in tomato leaves. Accumulation of proteinase inhibitors, induced with the wound hormone PIIF. J Biol Chem 1976; 251:7004-7110. 73. Yeh KW, Chen JC, Lin MI et al. Functional activity of sporamin from sweetpotato (Ipomoea batatas Lam.): A tuber storage protein with trypsin inhibitory activity. Plant Mol Biol 1997; 33:565-570. 74. Yeh KW, Lin MI, Tuan SJ et al. Sweetpotato (Ipomoea batatas) trypsin inhibitors expressed in transgenic tobacco plants confer resistance against Spodoptera litura. Plant Cell Rep 1997; 16:696-699.
201 75. Michaud D, Asselin A. Application to plant proteins of gel electrophoretic methods. J Chromatogr A 1995; 698:263-279. 76. Michaud D. Gel electrophoresis of proteolytic enzymes. Anal Chim Acta 1998; 372:173-185. 77. Granier F. Extraction of plant proteins for two-dimensional electrophoresis. Electrophoresis 1988; 9:712-718. 78. Wu F-S, Wang M-Y. Extraction of proteins for sodium dodecyl sulfate-polyacrylamide gel electrophoresis from protease-rich plant tissues. Anal Biochem 1984; 139:100-103. 79. Cremer F, van de Walle C. Method for extraction of proteins from green plant tissues for two-dimensional polyacrylamide gel electrophoresis. Anal Biochem 1985; 147:22-26. 80. van Driessche E, Beeckmans S, Dejaegere R et al. Thiourea: the antioxidant of choice for the purification of proteins from phenol-rich tissues. Anal Biochem 1984; 114:184-. 81. North MJ. Prevention of unwanted proteolysis. In: Beynon RJ, Bond JS, eds. Proteolytic enzymes. A practical approach. New York: IRL Press, 1989:105-124. 82. Michaud D, Cantin L, Visal S et al. Differential susceptibility of oryzacystatin I and oryzacystatin II to proteolytic cleavage. Plant Physiol 1996; 111s:103. 83. Benchekroun A, Michaud D, Nguyen-Quoc B et al. Synthesis of active oryzacystatin I in transgenic potato plants. Plant Cell Rep 1995; 14:585-588. 84. Abe K, Kondo H, Arai S. Purification and characterization of a rice cysteine proteinase inhibitor. Agric Biol Chem 1987; 51:2763-2768. 85. Uhlén M, Forsberg G, Moks T. Fusion proteins in biotechnology. Curr Opin Biotechnol 1992; 3:363-369. 86. Nilsson J, Stahl S, Lundeberg J et al. Affinity fusion strategies for detection, purification, and immobilization of recombinant proteins. Protein Expr Purif 1997; 11:1-16. 87. Makrides SC. Strategies for achieving highlevel expression of genes in Escherichia coli. Microbiol Rev 1996; 60:512-538. 88. Hochuli E, Bannwarth W, Döbeli H et al. Genetic approach to facilitate purification of recombinant proteins with a novel metal chelate adsorbent. Bio/Technology 1988; 6:1321-1325. 89. Hochuli E. Purification of recombinant proteins with metal chelate adsorbent. Genet Eng 1990; 12:87-98. 90. Ledent P, Duez C, Vanhove M et al. Unexpected influence of a C-terminal-fused His-tag on the processing of an enzyme and on the kinetic and folding parameters. FEBS Lett 1997; 413:194-196.
202 91. Hopp T, Prickett KS, Price VL et al. A short polypeptide marker sequence useful for recombinant protein identification and purification. Bio/Technology 1988; 6:1204-1210. 92. Prickett KS, Amberg DC, Hopp T. A calcium-dependent antibody for identification and purification of recombinant proteins. BioTechniques 1989; 7:580-589. 93. Simons PC, Vander Jagt DL. Purification of glutathione S-transferases by glutathioneaffinity chromatography. Methods Enzymol 1981; 235-237. 94. Smith DB, Johnson KS. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene 1988; 67:31-40. 95. Sassenfeld HM. Engineering proteins for purification. Trends Biotechnol 1990; 8:88-93. 96. Stahl S, Nygren P-A. The use of gene fusions to protein A and protein G in immunology and biotechnology. Pathol Biol 1997; 45:66-76. 97. Stahl S, Nygren P-A, Uhlén M. Detection and isolation of recombinant proteins based on binding affinity of reporter: Protein A. In: Tuan RS, ed. Recombinant proteins protocols: detection and isolation—Methods in Molecular Biology series, Vol. 63. Totowa NJ: Humana Press, 1997;103-118.
Recombinant Protease Inhibitors in Plants 98. Löfdahl S, Guss B, Uhlen M et al. Gene for staphylococcal protein A. Proc Natl Acad Sci USA 1983; 80:697-701. 99. Nygren P-A, Stahl S, Uhlén M. Engineering proteins to facilitate bioprocessing. Trends Biotechnol 1994; 12:184-188. 100. Nygren P-A, Ljungquist C, Tromborg H et al. Species-dependent binding of serum albumins to the streptococcal receptor protein G. Eur J Biochem 1990; 193:143-148. 101. Makrides SC, Nygren P-A, Andrews B et al. Extended in vivo half-life of human soluble complement receptor type I fused to a serum albumin-binding receptor. J Pharm Exp Ther 1996; 277:534-539. 102. Nygren P-A, Flodby P, Andersson R et al. In vivo stabilization of a human recombinant CD4 derivative by fusion to a serum albumin-binding receptor. In: Chanock RM, ed. Vaccines 91—Modern approaches to vaccine development. New York:Cold Spring Harbor Laboratory Press, 1991;363-368. 103. Assouline Z, Graham R, Miller RC, Jr. Processing of fusion proteins with immobilized factor Xa. Biotechnol Progr 1995; 11:45-49. 104. Mason HS, Arntzen CJ. Transgenic plants as vaccine production systems. Trends Biotechnol 1995; 13:388-392.
CHAPTER 17
Protease Inhibitors in Health and Disease Control—Medical and Industrial Aspects Michiel F.J. Blankenvoorde, Henk S. Brand, Yvonne M.C. Henskens, Enno C.I. Veerman and Arie V. Nieuw Amerongen
17.1. Introduction
P
roteolytic enzymes are involved in numerous physiological processes in man including food digestion, tissue remodelling, host defense, blood coagulation and the activation of proenzymes and prohormones, and proteinase inhibitors (PIs) play an important physiological role in the regulation of these enzymes. There are many examples of pathological conditions in which uncontrolled proteolytic activity of host enzymes leads to irreversible tissue destruction (e.g., in inflammatory processes, including rheumatoid arthritis and periodontitis), or to tumor growth and metastasis. In addition to host proteinases, exogenous proteinases derived from infectious agents such as bacteria, viruses or protozoa play a role in the onset and perpetuation of infection, making PIs potentially applicable therapeutic agents in the battle against these diseases. A necessary first step in the development of any PI-like drug is to identify the molecular target protease that is critical to the disease process or to the survival of the infectious agent. For instance, the selective inhibition of angiotensin converting-enzyme by PIs results in a decrease of the blood pressure and a prolonged survival of patients after myocardial infarction.1,2 Similarly, PIs are
used to treat diseases like AIDS, for which the major HIV target aspartyl protease has been well characterized.3 In this review we will focus on the role of proteinases and their inhibitors in human diseases, and on the possible application of proteinaceous PIs as drugs. As a case study, emphasis will be placed on cysteine proteinase s and cystatins, which are major proteinaceous inhibitors of cysteine proteinase s in man, animals and plants. We also will discuss the several criteria to be met before such drugs are applicable to clinical trials.
17.1.1. Cysteine Proteinases Cysteine proteinases are widely distributed among living organisms. The most important cysteine proteinase s in humans belong to the papain and calpain families.4,5 In the papain family, cysteine proteinase s are represented by proteinases such as the lysosomal cathepsins B, C, H, L and S. With the exception of cathepsin S, these enzymes are ubiquitous in the lysosomes of nearly all organs and tissues.6 Several other lysosomal cysteine proteinase s have been identified including the cathepsins K, N and T,6 but these are less well characterized. In general, cathepsins are relatively small proteins with a
Recombinant Protease Inhibitors in Plants, edited by Dominique Michaud. ©2000 Eurekah.com.
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molecular mass varying from 20 to 35 kDa,5,6 except for cathepsin C which is an oligomeric enzyme of 200 kDa.7 All cathepsin species are optimally active under reducing and slightly acidic conditions. In vivo, lysosomal cathepsins are mainly involved in the degradation of proteins into dipeptides and amino acids, which become again available for protein synthesis. 5,6 Lysosomal cysteine proteinase s have also been implicated in processes such as antigen presentation,8 extracellular matrix remodelling in bone 9 and prohormone activation of thyroglobulin and prorenin.10,11 To prevent unwanted and untimely proteolysis, cathepsin activity is rigorously regulated in various ways, including by endogenous inhibitors, pH, redox potential, synthesis as a proenzyme, compartmentalization and degradation.4 A disturbed balance between active enzymes and physiological inhibitors has been implicated in several pathological processes such as Alzheimer disease,12,13 muscular disorders,14,15 inflammation,16-19 and tumor growth and metastasis.20-22 Cysteine proteinases have also been identified in viral, bacterial and protozoan infectious agents. For example, viral cysteine proteinases are present in poliovirus, rhinovirus, human herpes simplexvirus and human immunodeficiency-virus.23-25 Bacterial cysteine proteinase s are produced, for instance, by Clostridium histolyticum, 26 Streptococcus pyogenes27,130 and Porphyromonas gingivalis.28 A number of protozoan parasites, including Leishmania tropica and Trypanosoma cruzi (the causative agent of American trypanosomiasis) also produce cysteine proteinase s.29 Most of these proteinases play a role in the replication or growth of the pathogen.24,30,31 They also have been considered as virulence factors, as they are able to degrade tissues of the host to obtain nutrients or to facilitate host tissue invasion. For example, invasion of host cells and tissues by the protozoan T. cruzi or by the oral pathogen P. gingivalis is mediated by their major cysteine proteinase s, cruzipain and gingipains, respectively.30,31 Human malarial parasites also produce cysteine proteinase s which are
Recombinant Protease Inhibitors in Plants
involved in the invasion and rupture of erythrocytes, resulting in anaemia.29 Several microorganisms were shown to proteolytically degrade host proteins including immunoglobulins, plasma PIs and components of the complement system, potentially impeding the host defence system. 29-32 Cysteine proteinases can also activate zymogens like procollagenase and proenzymes of the clotting system, and thus accelerate host tissue degeneration during the pathogenic process.28,32,33,127
17.1.2. Cystatins: Inhibitors of Cysteine Proteinases Cystatins comprise a superfamily of proteins, which on the basis of sequence homology have been subdivided into three main families.34-36 Family-1 cystatins (cystatins A and B, also known as stefins A and B) are homologous, single-chain intracellular proteins of ~11 kDa with no disulfide bond or carbohydrate chain. Family-2 cystatins are single-chain proteins of approximately 14 kDa containing two intramolecular disulfide bridges. These cystatins are usually nonglycosylated and mainly found in secretory fluids such as saliva, tears and semen. Family-3 cystatins (or kininogens) are glycosylated, multifunctional intravascular proteins with a single disulfide bridge. In man, kininogens are represented by low-molecular weight (50-68 kDa) and high-molecular weight (88-114 kDa) inhibitors. These PIs can be cleaved to release the vasoactive kinin peptide, which leaves a two-chain molecule consisting of a heavy chain and a light chain, the heavy chain being composed of three tandemly repeated cystatin-like regions. In brief, most cystatins are reversible, tight- binding competitive inhibitors of cysteine proteinase s37 which form equimolar complexes with their target enzymes.38,39 They contain three highly-conserved domains that are essential for inhibitory activity.5,38,39 The first domain is the N-terminal gly-9 (chicken egg white cystatin-numbering), which is important for the optimal orientation of the N-terminal region towards the cysteine proteinase . The second and third inhibitory domains of
Protease Inhibitors in Health and Disease Control—Medical and Industrial Aspects
cystatin encompass two hairpin loops. The first loop consists in the highly conserved motif QXVXG (residues gln-53 to gly-57 in egg white cystatin), while the second hairpin loop contains the two conserved residues, pro-103 and trp-104. The 3D-dimensional structure of egg white cystatin, determined by X-ray crystallography, showed that these three domains form a tripartite wedge which penetrates and covers the active site of papain and other cysteine proteinase s, thus causing the inhibition.38,40
17.2. Anti-Microbial Properties of Cystatins 17.2.1. Viruses Since many viruses require proteolytic cleavage to become mature and infectious, much attention has been paid to the antiviral effects of PIs.24,41,128 Several studies indicated that cystatins possess activity against a variety of viruses including poliovirus, rhinovirus, coronavirus and herpes simplexvirus. Both egg white cystatin and cystatin C inhibit virus production of poliovirus-infected cells without any cytopathological effects on the cells.23,42 Noteworthy, exposure of the cells to egg white cystatin prior to infection results in the absence of viral protein synthesis.42 The replication of rhinovirus type 1A (the common cold virus) is also inhibited by egg white cystatin,23 while the replication of coronavirus and herpes simplexvirus is blocked by cystatin C43,44 and that of the herpes simplexvirus type I by cystatin SN.45 Oryzacystatin I (OC-I) and oryzacystatin II (OC-II) also inhibit poliovirus replication in infected Vero cells in vitro, whereas soybean trypsin inhibitor, pepstatin and α2-macroglobulin do not show any effect.46 Recently it has been demonstrated that, in a mouse model of herpes simplexvirus keratitis, OC-I exhibits antiher- petic activity without any toxicity in the therapeutic dose range.47 The administration of OC-I in eyedrops to mice infected with herpes simplexvirus type I results in a remarkable reduction of lethality, similar to the potent antiviral agent acyclovir.
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Although it is generally suggested that the antiviral activity of cystatins is due to their proteinase inhibitory properties, the viral target enzymes have not been identified yet. Egg white cystatin and cystatin C, for instance inhibit weakly or not at all the major 3C cysteine proteinase of poliovirus.24,48,129 Similarly, the neutralization of rhinovirus by egg white cystatin cannot be attributed to inhibition of this proteinase,24 indicating that other viral cysteine proteinase s would be inhibited. Interestingly, cystatin B,23 rat cystatin S49 as well as the low-molecular weight cysteine PIs, E-64 and loxistatin46 hardly have antiviral effects, which suggests that the antiviral activity of cystatins could occur through a different, unknown mechanism.
17.2.2. Bacteria Cystatins also show antibacterial activity. For example, the tripeptide derivative N-benzyloxycarbonyl-leucyl-valyl-glycyldiazomethylketone (Z-LVG-CHN2), that mimics the proteinase binding site of cystatin C, displays specific antibacterial activity against group A streptococci.27 In addition, mice treated with this cystatin-like peptide were protected against injection of lethal doses of these pathogens. The susceptibility of group A streptococci to Z-LVG-CHN2 was shown to be comparable to that of well-established anti-streptococcal antibiotics such as tetracycline and bacitracin. Similarly, the growth of a major oral pathogen, Porphyromonas gingivalis is inhibited by several cystatins, whereas other inhibitors such as E-64 and leupeptin do not.49-52 Finally, phosphorylated rat cystatin α inhibits the growth of Staphylococcus aureus.53 Since cystatin α is present in the epidermis, it is suggested that it has a protective role against infectious agents on the skin surface. For some pathogens the antibacterial activity of cystatins was clearly shown to occur through the inhibition of cysteine proteinase s. For example, the cysteine proteinase exotoxin B, a main virulence factor of S. pyogenes,130 is inhibited by Z-LVG-CHN2, but the addition of this proteinase in excess relieves the growth inhibition.27 An alterna-
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tive mechanism involving membrane disturbance was suggested for a basic cystatin isolated from a horseshoe crab,54 which displays antimicrobial activity against the Gram-negative bacteria Salmonella typhimurium, Escherichia coli and Klebsiella pneumoniae.
17.2.3. Parasites Cysteine PIs have also been proposed as chemotherapeutic agents against parasite infections including schistosomiasis, malaria, amoebiasis and trypanosomiasis.29,30,55,131,132 For example, the trophozoites of Plasmodium falciparum obtain free amino acids for protein synthesis via the action of a cysteine proteinase , which intracellularly degrades the host erythrocyte hemoglobin. Inhibition of this proteinase by fluoromethylketone PIs correlates with blocking of the hemoglobin degradation and killing of cultured parasites.56 Similarly, cruzipain is essential for the development and survival of T. cruzi within host cells,30 which makes this enzyme a potential target for trypanocidal drugs. Indeed, several studies demonstrated that cystatins and cystatin-like peptides inhibit cruzipain,30,57 and that inhibitors of this enzyme, such as fluoromethylketones and diazomethylketones, inhibit growth and differentiation of T. cruzi parasites.30,58,59 Preliminary results indicated that fluoromethylketones also protect mice from a lethal infection with this pathogen.60 Other parasitic cysteine proteinase s are inhibited by cystatins, including that of Entamoeba histolytica,61 which is thought to play an important role in tissue invasion. Taken together, these studies demonstrate that cysteine PIs exert antiparasitic effects and suggest that cystatins may play a protective role against various parasitic infections.
17.3. Cystatins in the Control of Tumor Growth and Metastasis Cysteine proteinases present in the plasma membrane protrusions of cancer cells may facilitate tumor outgrowth through the degradation of stromal tissues and basement membranes.62,63 Also, cysteine proteinases
Recombinant Protease Inhibitors in Plants
have been implicated in cancer malignancy by activating proproteinases like prourokinase and the precursors of metallo-proteinases.62,64 Cysteine proteinases can also interfere with chemotherapy through inactivation of the antitumor drug bleomycin.65 Contradictory data have been presented on cystatin activity in malignant tumors. This activity has been found higher, similar or lower compared to the activity found in normal tissues. It has been proposed that the progression of malignant tumor cells is associated with an altered ratio of inhibitor to cysteine proteinase , resulting in increased effective activities of the cysteine proteinase s.20-22 This has been illustrated for a large number of carcinomas. Human breast carcinomas exhibit a decreased total cystatin activity compared to normal breast tissue, and the net increase of cathepsin B and cathepsin L activity in these breast carcinomas can at least in part be ascribed to a decrease in cystatin activity.66,67 Human lung tumors display a similar imbalance between cathepsin B levels and cystatin activity. Although cystatin activity is increased in tumor tissues, cathepsin B activity is increased to a higher extent. 68 Similarly, human amelanotic melanoma contains an excess of cathepsin B but not sufficient cystatins to achieve complete inhibition.69 Cystatin A has been associated frequently with malignant progression. In general reduced activity or concentration of cystatin a correlates inversely with tumor malignancy. For example, prostatic adenocarcinomas express no cystatin A in contrast to normal prostatic tissue.70 Cystatin A is less abundant in neoplastic lesions of the uterine cervix than in normal squamous epithelium. 71 The mRNA level of cystatin A is decreased during the progression of murine skin papillomas to carcinomas.72 The degree of malignancy and the differentiation of human breast carcinomas show a negative correlation with the expression of cystatin A.66,67 In addition, cystatin A purified from human sarcoma has a reduced inhibitory activity against papain and cathepsins B, H and L.73 Collectively, these studies suggest that cystatin A is
Protease Inhibitors in Health and Disease Control—Medical and Industrial Aspects
important for the maintenance of cell differentiation and prevention of tumor growth. In this context it is interesting to note that an increase in cystatin A mRNA expression is also observed in keratinocytes upon UV exposure.74 Recently it was also shown that increased expression of another cystatin, cystatin C, inhibits motility and in vitro invasiveness of B16 melanoma cells, 133 supporting the hypothesis that cystatins play a role in the maintenance of cell differentation. However, additional studies in which the expression of cystatins is experimentally altered are needed to provide further evidence whether an imbalance between active enzyme and cystatin is a key factor in tumor growth and progression. It has been suggested that several PIs which are present in cereals such as rice and maize can prevent certain types of cancer.75,76 This idea is supported by studies in which different cysteine PIs displayed antitumor activity both by reducing selectively the growth of transformed cells and by decreasing the occurrence of cancer in animal models. For example, E-64 arrests human epidermoid carcinoma cells at the mitotic metaphase77 and inhibits the invasion of malignant melanoma cells.78 Colon neoplasms in rats, chemically induced by 1,2-dimethylhydrazine, are inhibited by the administration of leupeptin. In the tumor-bearing mucosa of leupeptin-treated animals the cathepsin B activity levels are decreased compared to those in the non-treated group, suggesting that the inhibition of cathepsin B suppresses tumor growth. 79 In addition, haematogenous metastasis of hepatoma cells in lungs of rats is inhibited by leupeptin.80 Cystatin A inhibits the stimulated motility of both human melanoma cells and W256 carcinosarcoma cells as well as the adherence of melanoma cells.81 In contrast, egg white cystatin has no significant effect on mice bearing Lewis lung carcinoma subcutaneously82 and stimulates the proliferation of 3T3 fibroblasts,83 which may reflect essential differences in tumor tissues.
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The above review, while not exhaustive, points out a role for cystatins in the control of tumor growth and the possible application of cysteine PIs as cancer chemo-preventive agents. Since proteinases other than cysteine proteinase s may also play a role in tumor growth, 84,85 one can speculate on the potential application of combinations of PIs. For example in breast cancer the aspartic proteinase, cathepsin D is thought to promote tumor growth.86 Inhibitors of this proteinase may not only prevent tumor outgrowth but also prevent the possible inactivation of cystatins by cathepsin D,87 resulting in an accumulative inhibition of tumor growth.
17.4. Protection Against Tissue Destruction Cysteine proteinases play an important role in inflammatory diseases like periodontitis, psoriasis, multiple organ failure, bronchiectasis, emphysema and arthritis. Their involvement is either direct by tissue degradation or indirect by activation of proinflammatory mediators and other proteolytic enzymes.16-19,88-91 The most intensively studied inflammatory diseases implicating proteinases are periodontitis and rheumatoid arthritis.
17.4.1. Periodontitis In periodontal inflammatory diseases, high levels of cathepsins B, H, and L have been detected in gingival tissues and gingival crevicular fluids.92-96 These cathepsins originate most probably from monocytes, macrophages and fibroblasts. Since they may play an essential role in the pathogenesis of periodontitis, cystatins in gingival tissue, saliva and crevicular fluids have been investigated as possible protective proteins against tissue and bone destruction. Homogenates of inflamed gingival tissue contain mainly cystatin A and kininogen, and to a lesser extent cystatin C,97 the concentration of this PI being negatively correlated to pocket depth.98 Despite the relatively low levels of cystatin C in gingival tissues, this PI might inhibit cathepsin B under physiological conditions.98 Recently we
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have also shown that gingival crevicular fluid of periodontal patients contains cystatin A, whereas cystatins C, S and SN could not be detected.99 It has been postulated that inflammation induces the synthesis of cystatins. For example, cystatin C concentration and total cystatin activity are higher in saliva of periodontal patients than in healthy subjects.100-103 After periodontal treatment, the salivary cystatin activity and cystatin C concentration are diminished,104 but no differences are found in the saliva concentration of cystatins S, SN and SA between a healthy and a periodontitis group.105 The induction of cystatins has also been described in the rat salivary glands, which under normal conditions hardly secrete cystatin S. After treatment with the β-adrenergic agonist isoproterenol,106,107 after inflammatory stimuli108 or after surgical trauma,109 cystatin S is induced and secreted by the salivary glands. In addition, oral administration of the protozoan T. cruzi or of the cysteine proteinase papain to rats causes a dramatic increase in the salivary level of cystatin S,110-111 and therefore it is tempting to suggest that salivary cystatins play a protective role towards cysteine proteinase s from both endogenous and exogenous sources. Indeed, proteinases secreted from the oral pathogen P. gingivalis are partially inhibited by cystatin S.51 A protective role of cystatins on bone resorption has also been demonstrated. Cystatin C inhibits bone resorption, as measured by the release of 45Ca and 3H-hydroxyproline from mouse calvariae in vitro, upon different stimuli including parathyroid hormone, 1,25(OH)2-vitamin D3, interleukin-1 and prostaglandin E2. This effect is probably the result of inhibition of the activity of osteoclastic proteolytic enzymes which are released into the resorption lacuna.112,113 Comparable inhibitory effects on bone resorption have been observed with cystatin A,114 demonstrating that cystatins may be effective in preventing and treating bone destruction during periodontitis.
Recombinant Protease Inhibitors in Plants
17.4.2. Rheumatoid Arthritis Rheumatoid arthritis is a chronic inflammatory disease characterized by the progressive destruction of joints. Cathepsins B and L are strongly associated with this disease.18,89,115 Levels of cathepsins, which can degrade cartilage components such as collagens and proteoglycans, are elevated in synovial tissues and fluids of patients with arthritis and in animals with experimentallyinduced arthritis. Cathepsin B may be involved in an early stage of a cascade of proteolytic events by activating enzymes including prostromelysins and procollagenases.18 Proteinases like cathepsin B seem to be good targets for pharmacological intervention, as this may inhibit tissue destruction either directly or indirectly by preventing the activation of a cascade of other proteolytic enzymes. Several studies have demonstrated that cysteine PIs can limit tissue destruction in rheumatoid arthritis. For example the effect of a synthetic cysteine PI, Z-Phe-Alafluoromethylketone was studied in rats with antigen-induced arthritis. Treatment of the animals with this inhibitor, both intravenously and orally, resulted in reduced cathepsin B activity, reduced inflammation and decreased cartilage damage. 116 In addition, fluoroketone treatment of animals with adjuvant-induced arthritis was shown to significantly reduce the severity of clinical joint disease, to decrease the destruction of articular cartilage and to inhibit the activity of both cathepsin B117-119 and cathepsin L.119 Furthermore, interleukin-1-induced proteoglycan loss from cartilage was reduced by inhibitors of cathepsins B and L.120,121 Anti-inflammatory and anti-rheumatic drugs like pyrazolone derivatives and indomethacin also inhibit cathepsin B.122,123 Although the inhibition of cysteine proteinase s has been associated with a reduced destruction of cartilage and bone, further studies are needed to provide more evidence for the role of cystatins in this disease process.
Protease Inhibitors in Health and Disease Control—Medical and Industrial Aspects
17.5. Industrial Aspects The data described above show the great potential of proteinaceous PIs, in particular cystatins, as drugs in the treatment of several diseases. Patients with infectious diseases such as gingivostomatitis (HSV-I), poliomyelitis (poliovirus), atopic dermatitis (S. aureus), pharyngitis (S. pyogenes), and Chagas disease (T. cruzi) may benefit from the pharmacological use of these drugs. In addition, cystatins may affect pathological processes like arthritis, periodontitis, osteoporosis and cancer. Cystatins may also be used to prevent infection and inflammation. For example they can be applied by coating of materials such as catheters and implants. Finally, cystatins may be applied in tooth paste and mouth rinses to prevent gingivitis and periodontitis. However, before such proteinaceous PIs can be applied, several criteria have to be met with regard to toxicity, synthesis and biological stability. One problem that may occur is that exogenously applied PIs can inhibit other host enzymes with essential metabolic functions. To come to an acceptable therapeutic index, several cystatins should be compared with regard to their specificity for the enzymes targeted. Engineered recombinant cystatins may be applied with improved specificity and effective therapeutic concentration. Local application of cystatins to the affected tissue may also restrict side effects. In periodontitis, the local delivery of cystatins in the periodontal pocket seems attractive since it can reduce the growth of P. gingivalis, bone resorption and inflammation. A potential hazard associated with the use of cystatins, however, is that they might provoke an unwanted immune response in the host. To avoid this problem, cystatinlike peptides could be designed which could exert less antigenic activity and thus impede the immune response or hypersensitivity reactions. Cystatins have been successfully expressed as recombinant proteins in plants like rice, corn and potatoes, which may appear as suitable sources for large-scale purification (see Chapter 16, this volume). In addition, several cystatin genes have been cloned and expressed in Escherichia coli. These cystatins,
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for instance OC-I and OC-II, can easily be purified in large quantities and exert biological activity.124 In this context it is worth to underline that cystatins are very stable under conditions of extreme pH and high temperatures. Recently we observed that egg white cystatin remains stable and biologically active in a commercial mouth rinse over a period of several months at room temperature (Blankenvoorde, unpublished results). In vivo, however, the degradation of cystatins could be important. For example cystatins can be inactivated by an aspartic proteinase 87 while human PMN elastase rapidly cleaves the N-terminal region of cystatin C, resulting in a decreased affinity of the truncated cystatin C for cathepsins B, H and L.125 On the other hand, cystatins truncated by proteolytic enzymes of P. gingivalis were shown to remain biologically active,51 and other proteinases like cathepsin G and proteinase 4 failed to hydrolyze peptide bonds in cystatin C.126 In any case, to bypass in vivo degradation of natural cystatins, recombinant engineered cystatins could be developed which are more resistant to proteolytic degradation. In conclusion, before cystatins (or any other proteinaceous PI showing potential in medicine) can be administered to patients in clinical trials, studies in animal models are needed to determine their effectiveness, host resistance and toxicity. In addition, hemodynamic parameters, pharmacokinetics and optimal formulation have to be investigated. Nevertheless it is clear that cystatins and protein PIs in general are promising therapeutical compounds that can open a PI-based area of therapy and treatment.
Acknowledgments This project was supported by the Dutch Prevention Foundation, project no 28-2488, and by the Netherlands Institute for Dental Sciences. We thank Dr Wim van t' Hof for critically reading the manuscript.
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Protease Inhibitors in Health and Disease Control—Medical and Industrial Aspects 89. Müller-Ladner U, Gay RE, Gay S. Cysteine proteinases in arthritis and inflammation. Perspect Drug Discov Design 1996; 6:87-98. 90. Takahashi H, Ishidoh K, Muno D et al. Cathepsin L activity is increased in alveolar macrophages and bronchoalveolar lavage fluid of smokers. Am Rev Respir Dis 1993; 147:1562-1568. 91. Buttle DJ, Burnett D, Abrahamson M. Levels of neutrophil elastase and cathepsin B activities, and cystatins in human sputum: Relationship to inflammation. Scand J Clin Lab Invest 1990; 50:509-516. 92. Eisenhower DA, Hutchinson R, Javed T et al. Identification of a cathepsin B-like protease in the crevicular fluid of gingivitis patients. J Dent Res 1983; 62:917-921. 93. Eley BM, Cox SW. Cathepsin B/L-, elastase-, tryptase-, trypsin- and dipeptidyl peptidase IV-like activities in gingival crevicular fluid: Correlation with clinical parameters in untreated chronic periodontitis patients. J Periodontal Res 1992; 27:62-69. 94. Eley BM, Cox SW. Cathepsin B/L-, elastase-, tryptase-, trypsin- and dipeptidyl peptidase IV-like activities in gingival crevicular fluid: A comparison of levels before and after periodontal surgery in chronic periodontitis patients. J Periodontol 1992; 63:412-417. 95. Cox SW, Eley BM. Preliminary studies on cysteine and serine proteinase activities in inflamed human gingiva using different 7-amino-4-trifluoromethyl coumarin substrates and protease inhibitors. Arch Oral Biol 1987; 32:599-605. 96. Cox SW, Eley BM. Detection of cathepsin B- and L-, elastase-, tryptase-, trypsin- and dipeptidyl peptidase IV-like activities in crevicular fluid from gingivitis and periodontitis patients with peptidyl derivatives of 7-amino-4-trifluoromethyl coumarin. J Periodontal Res 1989; 24:353-361. 97. Babnik J, Curin V, Lah T et al. Cysteine proteinase inhibitors in inflamed human gingiva. Biol Chem 1988; 369:271-276. 98. Skaleric U, Babnik J, Curin V et al. Immunochemical quantitation of cysteine proteinase inhibitor cystatin C of inflamed human gingiva. Arch Oral Biol 1989; 34:301-305. 99. Blankenvoorde MFJ, Henskens YMC, Van der Weijden GA et al. Cystatin A in gingival crevicular fluid of periodontal patients. J Periodontal Res 1997; 32:583-588. 100. Henskens YMC, Van der Velden U, Veerman ECI et al. Protein, albumin and cystatin concentrations in saliva of healthy subjects and of patients with gingivitis or periodontitis. J Periodontal Res 1993; 28:43-48.
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101. Henskens YMC, Van der Velden U, Veerman ECI et al. Cystatins S and C in human whole saliva and in glandular salivas in periodontal health and disease. J Dent Res 1994; 73:1606-1614. 102. Henskens YMC, Van den Keijbus PAM, Veerman ECI et al. Protein composition of whole and parotid saliva in healthy and periodontitis subjects. J Periodontal Res 1996; 31:57-65. 103. Henskens YMC, Veerman ECI, Nieuw Amerongen AV. Cystatins in health and disease. Biol Chem 1996; 377:71-86. 104. Henskens YMC, Van der Weijden GA, Van den Keijbus PAM et al. Effect of periodontal treatment on the protein composition of whole and parotid saliva. J Periodontol 1996; 67:205-212. 105. Aguirre A, Testa-Weintraub LA, Banderas A et al. Levels of salivary cystatins in human parotid and submandibular-sublingual salivas. J Dent Res 1990; 69: 166 (abstr.). 106. Bedi GS. Purification and characterization of an inducible cysteine proteinase inhibitor from submandibular glands of isoproterenol- treated rats. Arch Biochem Biophys 1989; 270:335-343. 107. Shaw PA, Cox JL, Barka T et al. Cloning and sequencing of cDNA encoding a rat salivary cysteine proteinase inhibitor inducible by beta-adrenergic agonists. J Biol Chem 1988; 263:18133-18137. 108. Cohen RE, Bedi GS, Neiders ME. Induction of type 2 cystatin with systemically administered drugs. J Dent Res 1989; 69:167 (abstr.). 109. Yagil C, Barka T. Induction of a specific (LM) protein in the submandibular gland of the rat by repeated amputation of the lower incisor teeth. Am J Anat 1986; 177:513-518. 110. Naito Y, Suzuki I, Hasegawa S. Induction of cystatin S in rat submandibular glands by papain. Comp Biochem Physiol 1992; 102B:861-865. 111. Silvia GAB, Alves JB, Alves MSD. Cystatin S in secretory granules fractions isolated from submandibular gland of infected rats by Trypanosoma cruzi. Tissue Cell 1995; 27:167-172. 112. Lerner UH, Grubb A. Human cystatin C, a cysteine proteinase inhibitor, inhibits bone resorption in vitro stimulated by parathyroid hormone and parathyroid hormone-related peptide of malignancy. J Bone Miner Res 1992; 7:433-440. 113. Lerner UH, Johansson L, Ransjo M et al. Cystatin C, an inhibitor of bone resorption produced by osteoblast. Acta Physiol Scand 1997; 161:81-92.
214 114. Kakegawa H, Nikawa T, Tagami K et al. Participation of cathepsin L on bone resorption. FEBS Lett 1993; 321:247-250. 115. Lenarcic B, Gabrijelcic, D, Rozman B et al. Human cathepsin B and cysteine proteinase inhibitors (CPIs) in inflammatory and metabolic joint diseases. Biol Chem 1988; 369:S257-S261. 116. Van Noorden CJ, Smith RE, Rasnick D. Cysteine proteinase activity in arthritic rat knee joints and the effects of a selective systemic inhibitor, Z-Phe-AlaCH 2 F. J Rheumatol 1988; 15:1525-1535. 117. Ahmed NK, Martin LA, Watts LM et al. Peptidyl fluoromethyl ketones as inhibitors of cathepsin B. Implication for treatment of rheumatoid arthritis. Biochem Pharmacol 1992; 44:1201-1207. 118. Esser RE, Watts LM, Angelo RA et al. The effects of fluoromethyl ketone inhibitors of cathepsin B on adjuvant induced arthritis. J Rheumatol 1993; 20:1176-1183. 119. Esser RE, Angelo RA, Murphey MD et al. Cysteine proteinase inhibitors decrease articular cartilage and bone destruction in chronic inflammatory arthritis. Arthritis Rheum 1994; 37:236-247. 120. Buttle DJ, Saklatvala J. Lysosomal cysteine endopeptidases mediate interleukin-1 stimulated cartilage proteoglycan degradation. Biochem J 1992; 287:657-661. 121. Buttle DJ, Saklatvala J, Barrett AJ. The inhibition of interleukin-1 stimulated cartilage proteoglycan degradation by cysteine endopeptidase inactivators. Agents Actions Suppl 1993; 39:161-165. 122. Kruze K, Fehr K, Boni A. Effect of antirheumatic drugs on cathepsin B1 from bovine spleen. Z Rheumatol 1976; 35:95-102. 123. Yamamoto K, Kamata O, Kato Y. Differential effects of anti-inflammatory agents on lysosomal cysteine proteinases cathepsins B and H from rat spleen. Jpn J Pharmacol 1984; 35:253-258.
Recombinant Protease Inhibitors in Plants 124. Michaud D, Nguyen-Quoc B, Yelle S. Production of oryzacystatins I and II in Escherichia coli using the glutathione S-transferase gene fusion system. Biotechnol Progr 1994; 10:155-159. 125. Abrahamson M, Mason RW, Hansson H et al. Human cystatin C. Role of the N-terminal segment in the inhibition of human cysteine proteinases and its inactivation by leucocyte elastase. Biochem J 1991; 273:621-626. 126. Abrahamson M, Buttle DJ, Mason RW et al. Regulation of cystatin C activity by serine proteinases. Biomed Biochim Acta 1991; 50:587-593. 127. DeCarlo AA, Windsor LJ, Bodden MK et al. Activation and novel processing of matrix metalloproteinases by a thiol-proteinase from the oral anaerobe Porphyromonas gingivalis. J Dent Res 1997; 76:1260-1270. 128. Gorbalenya AE, Snijder EJ. Viral cysteine proteinases. Perspect Drug Discov Design 1996; 6:64-86. 129. Baum EZ, Bebernitz GA, Palant O et al. Purification, properties, and mutagenesis of poliovirus 3C protease. Virology 1991; 185:140-150. 130. Lukomski S, Sreevatsan S, Amberg C et al. Inactivation of Streptococcus pyogenes extracellular cysteine protease significantly decreases mouse lethality of serotype M3 and M49 strains. J Clin Invest 1997; 99:2574-2580. 131. Brindley PJ, Kalinna BH, Dalton JP et al. Proteolytic degradation of host hemoglobin by schistosomes. Mol Biochem Parasitol 1997; 89:1-9. 132. Robertson CD, Coombs GH, North MJ et al. Parasite cysteine proteinases. Perspect Drug Discov Design 1996; 6:99-118. 133. Sexton PS, Cox JL. Inhibition of motility and invasion of B16 melanoma by the overexpression of cystatin C. Melanoma Res 1997; 7:97-101.
CHAPTER 18
Protease Inhibitors in Food Processing Fernando L. García-Carreño, Haejung An and Norman F. Haard
18.1. Introduction
F
ood technology is a market-driven activity. The current generation of food technologists is looking for added value for the consumer, better profit margins, and more efficient utilization of resources. Enzymatic modification of food proteins has an important role in the food industry with respect to both traditional and high technology food processing as well as food spoilage. The study of proteolysis in foodstuffs by food scientists and nutritionists has been a major area of research activity in food technology. Ancient traditional arts such as brewing, cheese making, meat tenderization with papaya leaves and condiment preparation (e.g., soy sauce and fish sauce) rely on proteolysis, albeit the methods were developed prior to our knowledge of enzymes. Early food processes involving proteolysis were normally the inadvertent consequence of endogenous or microbial enzyme activity in the foodstuff. The idea of adding exogenous enzymes to improve existing reactions or to create new products dates to early in the 20th century, and became a significant part of food processing in the 1960’s.1 Protein modification by enzymes yields products with improved nutritional, functional and organoleptic properties, and aids a variety of processing operations. Proteinases are used by the food industry to control viscosity, elasticity, cohesion, emulsification, foam stability and whipability, flavor development, texture
modification, nutritional quality, solubility, digestibility and extractability. Applications include processes for meat flavor development and tenderization, continuous bread making and modification of cracker and cookie texture, malt supplementation and chillproofing in the brewing industry, and hydrolysis of protein gels to lower viscosity for concentration or filtration.2 The desired degree of hydrolysis (DH), or percentage of peptide bonds hydrolyzed, varies considerably with the different food processing operations. Some proteolytic processes, such as for bouillon from soy protein or fish sauce from whole fish, require a DH close to 100%. In contrast, in many food processing operations there is a balancing act in which just enough, but not too much protein hydrolysis must be achieved.3-5 Among enzymatic food protein modifications, “limited hydrolysis” is a technique receiving considerable attention because it can yield products with improved properties and added value. Protein hydrolysis is achieved by enzymes collectively called proteases. Enzymes hydrolyzing peptide bonds in the interior of the amino acid chain are called proteinases (or endopeptidases, or endoproteases) and belong to the IUB groups EC 3.4.21-24. Proteases hydrolyzing peptide bonds at either the amino- or carboxy-terminal end of the protein are called exopeptidases (or exopro- teases, or simply peptidases) and belong to the groups
Recombinant Protease Inhibitors in Plants, edited by Dominique Michaud. ©2000 Eurekah.com.
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EC 3.4.11-17. Products of proteinase-catalyzed hydrolysis are mostly intermediate molecular weight peptides, while those of exopeptidase hydrolysis are free amino acids, dipeptides or tripeptides.6 Proteases also are classified based on the amino acid or metal present at their active site, i.e., aspartyl-, serine, cysteine and metallo-proteases. A limited or controlled hydrolysis of protein may give rise to products having particularly interesting functional and organoleptic properties.7,8 Hydrolysis is allowed to proceed to a calculated end point until a given DH yields optimal functional properties.
18.2. Controlled Proteolysis in Food Uncontrolled or prolonged protein hydrolysis often results in the formation of bitter peptides, loss in desired structural and functional properties, and decreased feed value.4,9 For example, adding proteinases to Cheddar cheese can accelerate aging, but prolonged hydrolysis results in bitterness and texture defects.10 Excessive proteolysis during “chill proofing” of beer leads to a loss of “head” since small peptides cannot retain CO2.8 Controlled proteolysis of crustacean offal recovers about 90% of both protein and astaxanthin as a carotenoprotein complex,11 but the yield of product progressively decreases as the DH of the process exceeds the optimum. Limited hydrolysis of wheat proteins leads to improved gluten development and bread loaf volume, but the product has poor texture and a collapsed structure with too much proteolysis.8 Likewise, increased whippability of soy proteins or meltability of cheese occurs best under conditions of controlled proteolysis.12,13 Sometimes in food technology, as in the lab, proteinases are pervasive, perplexing, persistent and pernicious for proteins, but with proper precautions, preventable (the nine Ps rule). Enzymatic protein hydrolysis is controlled in several ways, including the choice of enzyme(s) with appropriate specificity, and inactivation of the enzyme at an appropriate time with heat, pH, ionic strength or by the addition of specific enzyme inhibitors. An enzyme inhibitor is any substance
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that reduces the rate of an enzyme-catalyzed reaction.14 Usually, proteinase inhibitors (PIs) mimic the protein substrate by binding to the active site of the enzyme, and are specific for the active site of a given class of proteinases. Enzyme inhibitors differ from enzyme inactivators, that act by removing cations from metal-dependent proteinases (e.g., chelators), or from protein denaturants, that act by altering the structure of catalytic sites. Most natural inhibitors react reversibly with the enzyme. Because PIs are naturally present in several food and feed ingredients, food technologists should consider their impact on food processing operations as well as on food quality and nutrition. Proteinase control by endogenous inhibitors is a ubiquitous phenomenon in nature, but it has not been extensively exploited by food technologists. Proteinase/ inhibitor interactions are found in processes as diverse as protein digestion, cell physiology (e.g., coagulation, fibrinolysis, complement activation and phagocytosis), developmental biology, pathology (e.g., hypertension), and infection (e.g., AIDS and invasive parasites).15 The large concentration of PIs in mammalian blood plasma—where they account for more than 10 % of total protein, or in plant seeds such as legumes, illustrate the importance of protease/inhibitor interactions in nature and the potential of these inhibitors to regulate proteases in various biological systems.15
18.3. Safety of PIs in Food and Feed On the other hand PIs are, like other enzyme inhibitors or like lectins, phytin and phenol derivatives, endogenous antinutritive factors. The presence of PIs in food decreases the apparent nutritional quality of protein in both vertebrates and invertebrates. For instance, exposure to diets containing 0.1% of soybean or potato trypsin inhibitor was shown to cause pancreatic pathology in rodents, the coadministration of food protein with PIs reducing degradation of the proteins.16 Likewise, when Atlantic salmons were fed diets containing soybean trypsin inhibitor, a dose-dependent reduction in
Protease Inhibitors in Food Processing
digestibility of proteins and fat, weight gain, and trypsin activity was observed. The fish was able to compensate for a certain concentration of inhibitor by increasing trypsin secretion, but higher levels of the inhibitor exhausted pancreatic synthesis of the enzyme. 17 Trouts and salmons fed diets containing trypsin inhibitor, however were shown to secrete another trypsin isoform that was less sensitive to the trypsin inhibitor, showing a certain ability of these organisms to elude the antinutritive effects of dietary PIs.18 Similarly in humans, ingestion of raw soybean products was shown to reduce chymotrypsin and trypsin activity, but a sindependent, neuraldependent increase in trypsin and chymotrypsin secretion was found ten minutes after the challenge.19 Despite this compensatory process, and despite the fact that the inhibitory effect of PIs on protein digestion could be advantageous in certain situations to improve the stability and the absorption of therapeutic proteins such as orally-delivered insulin,20 these observations illustrate the potential adverse effects of dietary PIs. Several approaches have been proposed to control the adverse effects of proteinaceous PIs, including physical treatments. Inhibitor activity can be adversely affected by heat, albeit they are often relatively heat-stable proteins. Excessive heat treatment, however can impair the nutritional value of the food by lowering amino acid availability. As heat treatment increases, the nondegradable protein intake increases while protein degradation rates and total and available lysine decrease. As an alternative, soybean intended for animal feed is treated with hexane to extract the oil, which allows to eliminate most of the PIs. Another treatment less damaging to nutritional value is the extrusion of diets containing inhibitors at 104 to 120°C for 30 to 60 sec. When this treatment was used to prepare a feed for chicks, the growth performance was greater and pancreas weight was lower.21 The thermostability of plant PIs is species-specific. Six legume seed meals intended for shrimp feed inhibited mammal
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trypsin, chymotrypsin, papain and shrimp hepatopancreas proteinases. When the meals were heated for 60 min at 85°C, the DH of the meal protein by the shrimp hepatopancreas extract increased by 110 to 370%. An interesting result of this study was that the protein of the untreated seed meals was hydrolyzed by the hepatopancreas extract (with a DH of 6 to 32%), allowing to explain why animals fed with raw seed meals can survive but with poor growth performance. In such cases a mild-heat denaturation of the inhibitors may help improve the digestibility of the protein and allow better foodconversion ratios,22 while many PIs found in edible plants may also be specific for unique digestive proteases of plant pests.23,24
18.4. Plant PIs in Foodstuff Plant PIs, that are normally protein in nature, are found throughout the plant kingdom.25 Most storage organs used as foodstuff, including seeds, fruits and tubers contain as much as 1 to 10% of their proteins as PIs. Plants contain inhibitors for all four classes of proteolytic enzymes, with some fruits, for instance containing up to 50% of their proteins as serine PIs.26 The functions of PIs in plants include the regulation of protein turnover and plant development, and protection from insects and microorganisms.25,27-29 Through agricultural breeding programs, cultivars selected for pest resistance were developed with a fortified ‘immune system’ which includes PIs. For instance, Californiagrown rice cultivars contain inhibitors of cysteine and serine (trypsin, chymotrypsin and subtilisin) proteases,30 the cysteine-type, trypsin and chymotrypsin inhibitors being concentrated in the germ, while most of the subtilisin inhibitor is found in the endosperm. Although leaf tissues do not normally contain high levels of PIs, mechanical injury to the leaves of various species including tomato, tobacco, potato and poplar elicits a massive accumulation of serine and cysteine PIs.31-33 The wounding response would be mediated by a peptide hormone called systemin, and would involve other hormone signals such as jasmonic acid, abscisic acid and auxin.33-35
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18.4.1. Phytocystatins Despite the negative effects of PIs on food nutritional quality (see Section 18.3), and despite the fact that these molecules also can adversely affect intended enzymatic modifications of ingredients during food processing and thus decrease the benefits of commercially processed foods,15 considerable attention has been given to the use of cystatins, notably both as a tool to protect crops and as a food processing aid.28 Cystatins are inhibitors of the papain family of enzymes. They form reversible, tight-binding complexes that mask the reactive site of cysteine proteinase s, making it inaccessible to protein substrates. Members of the cystatin superfamily, which occur ubiquitously among animals and plants are categorized based on primary sequence similarities into four families: family-I cystatins, or stefins; family-II cystatins, or cystatins; family-III cystatins, or kininogens; and family IV-cystatins, or phytocystatins.28,36,37 Despite structural differences between these families, the physiological functions of cystatins are common to all members of the superfamily.38 The N-terminal 11 amino acid residues of cystatin C were shown to be important for its high affinity binding to papain, and it has been suggested that the polypeptide chain region around the gly-11 residue might serve as a substrate-like binding region for cysteine proteinase s.39 Peptides with amino acid sequences identical to those of the N-terminal segment of cystatin C are good substrates for papain, being cleaved at the bond following residue gly-11 after incubation with the enzyme.40 Abrahamson et al41 showed that a serine proteinase, leukocyte elastase, could act as a regulator of cysteine PI activity in human biological fluids by cleaving a single N- terminal cystatin C bond. The removal of an N-terminal decapeptide from human cystatin C decreased the inhibition of human cathepsins B and L by over 1000-fold. Even though it is still uncertain which parts of the cystatin molecule bind the target enzymes, highly-conserved sequences are found in cystatins between residues 53 and 57 (QxVxG, where x is any amino acid) in family-I cystatins and segments 2 and 3 of
Recombinant Protease Inhibitors in Plants
kininogens. However, after mutating this sequence to KxVxG or QxTxG and expressing the mutant inhibitors under the control of the tac promoter in Escherichia coli, Nikawa et al 42 found the purified recombinant proteins to show Ki values similar to wild-type cystatin A, suggesting that the QxVxG region may be less important than the N-terminal region for inhibitory activity against cysteine proteinase s. Phytocystatins have been isolated from rice, corn, wheat, soybean, barley, injured tomato leaves and potato tuber. 28 Rice contains two cystatins, oryzacystatin I (OC-I) and oryzacystatin II (OC-II), that target three different gibberellin-inducible endogenous enzymes: oryzains α, β and γ. OC-II has an affinity for cathepsin H (Ki=10-8M) higher than for papain (Ki=10-6M), while the reverse in true for OC-I. This is of interest since different food processing applications involving the control of cathepsins and papain have been proposed.30,42
18.4.2. Applications When a proteinase causes the loss of some desirable food attribute, the reduction of this activity should be sought. For instance, the main quality problem with beef is tough texture. Papain has long been used as a meat tenderizer, but due to its broad specificity the tenderized meat structure is excessively degraded, giving a mushy texture uncharacteristic of high-quality tender cuts of beef. Researchers in Japan have shown that OC-I can be effectively used to arrest the action of papain after the meat has been partially tenderized.43 Unlike myosystems from land animals, post mortem muscle softening in seafood (fish, mollusks and crustaceans) is a serious problem in food storage and processing due to endogenous proteinases.44 Muscle softening or mushiness is caused by a wide range of muscle proteinases, such as cathepsins, as well as by digestive proteases that seep into the muscle post mortem. Some fish proteinases are activated at the moment of cooking, causing myosin degradation and subsequent textural destruction. While most fish protein-
Protease Inhibitors in Food Processing
ases are activated at 60°C, some squid enzymes are activated at temperatures as low as 35°C,45 which explains why some seafood harvest is not intended for direct human consumption but only aimed for industrialization, where proteolytic activity can be controlled. An important example of the deleterious effect of endogenous proteinases is the gel weakening phenomenon observed during cooking of surimi. Surimi is a washed, minced fish muscle which forms a thermo-irreversible elastic gel upon heating. The gel-forming ability, bland taste and color of this product has made it possible to use it as a functional ingredient for many different food products.46 For instance, surimi has been largely used as a main ingredient in formulating seafood analog products such as crab legs, scallops and shrimp. In surimi gelation, myosin plays an important role in forming the gel matrix in final products. When Pacific whiting surimi was tested with slow cooking by incubation at 60°C for 30 min prior to heat-setting at 90°C, most myosin heavy chain was degraded, and surimi did not undergo gelation even with heat setting. The resultant gel strength was nondetectable due to proteolysis of myosin.47 The most detrimental effect of autolysis was found at 55°C during heating of Pacific whiting muscle,47,48 and degradation of the myofibrillar proteins by a cysteine proteinase was also most severe at this temperature (approximately 90% of myosin molecules were hydrolyzed within 5 min).49,50 Proteinase activity in the muscle was reduced to less than 3% of the original activity by using the specific inhibitor of cysteine proteinase s, E-64, indicating that the resident protease complex is mainly made up of cysteine proteinase s.48 Although numerous cysteine proteinase s, including cathepsins B, H and L exist in fish muscle, only cathepsin L activity was detected in surimi.49 The activity was found to be only 15% of the original level in fish muscle, since the washing step eliminated a large portion of the proteinase from the muscle.50,51 This remaining activity, however was enough to rapidly degrade myosin and alter the rheological properties of final products during the subsequent heat process.
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Protein additives have been widely used to control proteinase activity in surimi.52,53 The most commonly used food-grade inhibitors in commercial processing are beef plasma proteins (BPP), egg white and potato powder. These additives exert various degrees of inhibition towards proteinases.52,54,55 Among food-grade inhibitors used in surimi, BPP is the most effective in both inhibiting proteolytic activity and enhancing gel strength of surimi.47,56 Although the mode of action of this additive has not yet been clearly elucidated, it is generally believed that 2-macroglobulin (2-M) plays a major role in the process.57 2-M is a large, 725-kDa proteinasebinding plasma protein composed of tetrameric subunits. It is found in many vertebrates including mammals, birds, reptiles and amphibians, where it may inhibit all four main classes of proteases, i.e., serine, cysteine, aspartic and metallo-proteases.58 The use of this PI at 1% (w/w) concentration was shown to inhibit 78% of proteolytic activity in Pacific whiting surimi.47 Egg white contains of at least two PIs, cystatin and ovomucoid. As noted above, cystatins form a family of cysteine PIs in which the conserved NH 2-terminal segment is responsible for the competitive interaction with the active site of the enzyme.59 Egg cystatin Ki values for the inhibition of cysteine proteinase s are: 1 X 10-11 M for papain, 8 X 10-10 M for cathepsin B, 2 X 10-8 M for cathepsin H, and 3 X 10-12 M for cathepsin L.60 Ovomucoids are glycoproteins about 28 kDa in size, containing ~25% carbohydrate, no tryptophan and a high cysteine content. In general, these PIs have three complexforming domains which react with trypsinlike and chymotrypsin-like enzymes independently.61 Although food grade PIs have been widely used in surimi production, unwanted side-effects have been noticed, including modified color and/or taste not inherent to surimi.54 The limitations of commercially available PIs has prompted effort to find alternative sources of inhibitors for surimi processing. Rice, whey protein concentrate, tomato leaves and legume seed meals have been under study as sources of cystatins.5,15,30,33,62
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Izquierdo-Pulido and coworkers 30 partially purified OC-I from rice bran and reported that 0.14 IU of isolated OC-I per mg protein inhibited amidase activity of arrowtooth flounder by approximately 50%. Oryzacystatins isolated from different rice cultivars showed 5 to 10-fold higher proteinase inhibitory efficiency than bovine plasma proteins (on a weight basis) and prevented gel weakening during cooking of surimi. 5 El-Shamei et al32 studied the effect of wound injury in tomato, which enhanced accumulation of serine and, to a lesser degree, cysteine PIs in the leaves. On the other hand, application of methyl jasmonate to tomato resulted in massive accumulation of an 88-kDa cystatin.33 A PI-containing extract of tomato applied to Pacific whiting surimi was highly efficient in inhibiting autolytic activity during cooking.33 In parallel we tested various types of legume seed extracts for proteinase inhibition of Pacific whiting and Merluza, and reported that 6 out of 12 legume seed extracts tested were capable of reducing azocaseinolytic activities in fish extracts by more than 50%.63 Squids from the Atlantic and the Pacific Oceans are underutilized marine resources, partially because the flesh contains active proteinases. A cathepsin C (cysteine proteinase ) from the Atlantic squid, Illex illecebrosus was characterized, and shown to depend on the presence of Cl- and sulfide activators for activity.64 The spear squid Loligo bleekeri has several muscle proteinases, and one of them, a trypsin-like enzyme which can degrade myosin to a great extent when activated at 35°C, is inhibited by serine PIs.45 Some fresh-water and marine crustaceans such as krill (mostly Euphausia superba) from the Antarctic Ocean, langostilla (Pleuroncodes planipes) from the Pacific shoreline of the Baja California peninsula, and crayfish from European and North American rivers are large resources. Wild and cultivated crustaceans for human consumption, such as shrimp, prawns, crayfish and American lobster are becoming important because of their demand by consumers and their high commercial value. Crustaceans possess highly-active proteinases in the hepatopancreas, including trypsin,
Recombinant Protease Inhibitors in Plants
chymotrypsin and collagenase.44,65-67 These enzymes are released during post mortem processes, leading to meat autolysis. Several proteinases from krill have been reported, the most important during autolysis being a trypsin-like enzyme.67,68 The production of a protein hydrolysate from the cephalothorax of crayfish has been reported.69 The cephalothorax is a waste, after recovery of the tail for human consumption. Proteinase activity is significantly reduced during the 7-min boiling used to facilitate peeling of crayfish. However, in langostilla elimination of the enzyme activity was not as simple, showing important species-related differences in the heat stability of proteinases among crustaceans.66 Finally, blackspot development in crustaceans depends on proteolytic activation of a phenolase that transforms tyrosine into the dark pigment melanin. Sulfite is used to reduce post mortem blackspot development, but some potential hazard is associated with this practice and alternative methods of control need to be developed. Three proteinases have been isolated from the Norway lobster Nephrops norvegicus, two of them being thiol-proteinases, and the third being a metal-dependent serine protease.70 Whereas inhibition of the thiol proteinases had negligible effect on blackspot development, inhibition of the serine proteinase was shown to reduce phenolase activation.71
18.5. Conclusion The use of PIs as food processing aids has not received much attention, but there is a well-documented need to control protein hydrolysis in food processing. Protein hydrolysis is useful in extracting proteins from food processing wastes and other foodstuffs, but the hydrolysates typically lose functional properties such as foaming and gelation, and develop bitter taste due to the formation of low-molecular-weight peptides. In applying proteases in baked goods, meats and dairy products, excessive proteolysis also gives rise to texture and taste defects. The specific type of inhibitor needed in a given food processing application depends on the types of proteinases involved in deteriorative reactions or used
Protease Inhibitors in Food Processing
as processing aid. There are needs for aspartyl-, cysteine, serine and metallo - PIs in different food processing operations. To date, most attention has been given to the application of cysteine PIs in the food industry since it is probable that, unlike trypsin inhibitors, cystatins have no anti-nutritional effect on the human digestive system, which does not contain cysteine proteinase s. Further research to identify new inhibitors, to assess the influence of PIs on human digestive physiology, and to understand the modes of action and the kinetic properties of proteases in food products will expand the possibilities of using plant PIs in practical food applications. The search for unique inhibitors from plants and organisms from extreme environments could notably provide us with molecules that work at high or low temperatures, with the possibility of decreasing their activity by mild heating or specific pH conditions. Agricultural by-products such as cereal bran and injured tomato leaves are potential sources of PIs. Transgenic plants expressing different kinds of natural or modified PIs useful in plant protection or in alternative applications (see Chapters 1-17, this volume) also represent an interesting source of active inhibitors potentially useful in food processing. In parallel, methods to evaluate the activity of proteinases and PIs in food products are needed. The design of rational selection and artificial neural-network methods, in particular should be useful to select suitable inhibitors from several sources. Considering that PIs can impact a variety of food quality attributes, methods such as multi-response optimization techniques should help us identify appropriate inhibitors for a given application.
Acknowledgments This work was partially supported by Grant 5735-CG, from the US Department of Agriculture to NFH. FLGC thanks Angeles Navarrete for her assistance.
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References 1. Wolnak B. Status of the US Enzyme Industry. In: Danehy J, Wolnak B, eds. The interface between technology and Economics. New York: Marcel Dekker, 1980:33-40. 2. Mittal S. Food Biotechnology. Techniques and Applications. Lancaster PA: Technomic Publishing Co, 1992. 3. Kay J. Proteolysis—a degrading business but food for thought. Biochem Soc Trans 1982; 10:277-280. 4. Ericksen S. Controlled proteolysis of food protein ingredients. Biochem Soc Trans 1982; 10, 285-287. 5. Haard N. Plant protease inhibitors as food processing aids. In: Shahidi F, Jones Y, Kitts D, eds. Seafood Safety, Processing, and Biotechnology. Lancaster PA: Technomic Publishing Co 1997:213-224. 6. García-Carreño F, Navarrete A. Classification of proteases without tears. Biochem Educ 1997; 25:161-167. 7. Adler-Nissen J. Limited enzymic degradation of proteins: a new approach in the industrial application of hydrolases. J Chem Technol Biotechnol 1982; 32:138-156. 8. Chobert J, Sitohy M, Whitaker J. Proteolytic degradation of proteins. In: Flick G, Martin R, eds. Advances in Seafood Biochemistry. Lancaster PA:Technomic Co 1992:291-304. 9. Haard N, Kariel N, Herzberg G et al. Stabilization of protein and oil in fish silage for use as a ruminant feed supplement. J Sci Food Agric 1985; 36: 229-241. 10. Shamsuzzaman K, Haard N. Evaluation of harp seal gastric proteases as a rennet substitute for Cheddar cheese. J Food Sci 1983; 48:179-182. 11. Simpson B, Haard N. The use of proteolytic enzymes to extract carotenoproteins from shrimp wastes. J Appl Biochem 1985; 7:212-222. 12. Horiuchi T, Fukushima D, Sugimoto H et al. Studies on enzyme-modified proteins as foaming agents: Effect of structure on foam stability. Food Chem 1978; 3:35-42. 13. Lazaridis N, Rosenau J, Mahoney R. Enzymatic control of meltability in direct acidified cheese product. J Food Sci 1981; 46:332-335,339. 14. Whitaker J. Principles of Enzymology for the Food Sciences, 2nd Edition. New York: Marcel Dekker, 1994. 15. García-Carreño F. Proteinase inhibitors. Trends Food Sci Technol. 1996; 7:197-204. 16. Olli J, Hjelmeland K, Krodahl A. Soybean trypsin inhibitor in diets for Atlantic salmon (Salmo salar, L): Effects on nutrients digestibilities and trypsin in pyloric ceca homogenate and intestinal content. Comp Biochem Physiol A 1994; 109:923-928.
222 17. Gumbmann M, Dugan G, Spangler W et al. Pancreatic response in rats and mice to trypsin inhibitors from soy and potato after short- and long-term dietary exposure. J Nutr 1989; 119:1598-1609. 18. Haard N, Arndt R, Dong F. Estimation of protein digestibility. IV. Properties of the pyloric caeca enzymes from Coho salmon (Oncorhynchus kisutch) fed diets containing soybean meal. Comp Biochem Physiol B 1996; 115:533-540. 19. Holm H, Reseland J, Thorsen L et al. Raw soybean stimulates human pancreatic proteinase secretion. J Nutr 1992; 122:1407-1416. 20. Yamamoto A, Taniguchi T, Rikyuu K et al. Effects of various protease inhibitors on the intestinal absorption and degradation of insulin in rats. Pharmacol Res 1994; 11:1496-1500. 21. Zhang Y, Parsons C. Effects of extrusion and expelling on the nutritional quality of conventional and Kunitz trypsin-free soybean. Poultry Sci 1993; 7:2299-2308. 22. García-Carreño F, Navarrete del Toro A, Ezquerra M. Digestive shrimp proteinases for the evaluation of protein digestibility. I. The effect of proteinase inhibitors in protein ingredients. J Marine Biotechnol 1997; 5:36-40. 23. Belew M, Porath J, Sundberg L. The trypsin and chymotrypsin inhibitors in chick peas (Cicer arietinum L.). Eur J Biochem 1975; 60:247-258. 24. Liang C, Brookhart G, Feng G. Inhibition of digestive proteinases of stored grain coleoptera by oryzain, a cysteine proteinase inhibitor from rice seed. FEBS Lett 1991; 278:139-142. 25. Ryan CA. Proteinase inhibitors in plants: Genes for improving defenses against insects and pathogens. Annu Rev Phytopathol 1990; 28:425-449. 26. Pearce G, Liljegren D, Ryan C. Proteinase inhibitors in wild tomato species. In: Nevins D, Jones R, eds. Tomato Biotechnology. New York:Alan R Liss 1987;139-144. 27. Prat S, Frommer W, Hofgene R et al. Gene expression during tuber development in potato plants. FEBS Lett 1990; 268:334-338. 28. Arai S, Matsumoto I, Abe K. Phytocystatins and their target enzymes: from molecular biology to practical application. J Food Biochem 1998; 22:287-299. 29. Ohtsuba K, Richardson M. The amino acid sequence of a 20-kDa bifunctional subtilisin/ alpha-amylase inhibitor from bran of rice (Oryza sativa L) seeds. FEBS Lett 1992; 309:68-72. 30. Izquierdo-Pulido M, Haard T, Hung J. Oryzacystatin and other proteinase inhibitors in rice grain: potential use as fish processing aid. J Agric Food Chem 1994; 42:616-622.
Recombinant Protease Inhibitors in Plants 31. Pearce G, Strydom D, Johnson S et al. A polypeptide from tomato leaves induces wound-inducible proteinase inhibitor proteins. Science 1991; 253:895-898. 32. El-Shamei Z, Wu J, Haard N. Influence of wound injury on accumulation of proteinase inhibitors in leaf and stem tissues of two processing tomato cultivars. J Food Biochem 1996; 20:155-171. 33. Wu J, Haard N. Use of cysteine proteinase inhibitor from injured tomato leaves in whiting surimi. J Food Biochem 1998; 22:383-398. 34. Lightner J, Pearce G, Ryan C. Isolation of signaling mutants of tomato (Lycopersicum esculentum). Mol Gen Genet 1993; 241:595-601. 35. Lorberth R, Dammann C, Ebneth M. Promoter elements involved in environmental and developmental control of potato proteinase inhibitor II expression. Plant J 1992; 2:477-486. 36. Rawlings N, Barrett A. Evolution of protein of the cystatin superfamily. J Mol Evol 1990; 30:60-71. 37. Turk B, Turk V, Turk D. Structural and functional aspects of papain-like cysteine proteinases and their protein inhibitors. Biol Chem Hoppe-Seyler 1997; 378:141-150. 38. Saitoh E, Isemura S, Sanada K. Human cysteine proteinase inhibitors—nucleotide sequence analysis of 3 members of the cystatin gene family. Biol Chem Hoppe-Seyler 1988; 369s:191-197. 39. Abrahamson M, Ritonja A, Brown M et al. Identification of the probable inhibitory reactive sites of the cysteine proteinase inhibitors human cystatin C and chicken cystatin. J Biol Chem 1987; 262:9688-9694. 40. Grubb A, Abrahamson M, Olafsson I et al. Synthesis of cysteine proteinase inhibitors structurally based on the proteinase interacting N-terminal region of human cystatin-C. Biol Chem Hoppe-Seyler 1990;371s:137-144. 41. Abrahamson M, Mason R, Hansson H. Human cystatin C: role of the N-terminal segment in the inhibition of human cysteine proteinases and in its inactivation by leukocyte elastase. Biochem J 1991; 273:621-626. 42. Nikawa T, Towatari T, Ike Y et al. Studies on the reactive site of the cystatin superfamily using recombinant cystatin A mutants. FEBS Lett 1989; 255:309-314. 43. Funaki J, Abe K, Hayabuchi H et al. Modulating the conditioning of meat by the use of oryzacystatin, a cysteine proteinase inhibitor of rice seed origin. J Food Biochem 1991; 15:253-262. 44. Haard N. Protein hydrolysis in seafoods. In: Shahidi F, Botta R, eds. Seafoods-Chemistry, Processing technology and Quality. Galsgo: Blackie Academic & Professional, 1994:10-33.
Protease Inhibitors in Food Processing 45. Ebina H, Nagashima Y, Ishizaki S et al. Myosin heavy chain-degrading proteinase from spear squid muscle. Food Res Int 1995; 28:31-36. 46. Lee C. Surimi process technology. Food Technol 1984; 38:69-80. 47. Morrissey M, Wu J, Lin D et al. Effect of food grade protease inhibitors on autolysis and gel strength of surimi. J Food Sci 1993; 58:1050-1054. 48. An H, Seymour T, Wu J et al. Assay systems and characterization of Pacific whiting (Merluccius productus) protease. J Food Sci 1994; 59:277-281. 49. An H, Weerashinghe V, Seymour T et al. Degradation of Pacific whiting surimi proteins by cathepsins. J Food Sci 1994; 59:1013-1017,1033. 50. Chang-Lee M, Pacheco-Aguilar R, Crawford L et al. Proteolytic activity of surimi from Pacific whiting (Merluccius productus) and heat-set gel texture. J Food Sci 1989; 54:1116-1119,1124. 51. Morrissey M, Hartley P, An H. Proteolytic activity in Pacific whiting and effect of surimi processing. J Aquat Food Prod Technol 1995; 4:5-18. 52. Reppond K, Babbitt J. Protease inhibitors affect physical properties of arrowtooth flounder and walleye pollock surimi. J Food Sci 1993; 58:96-98. 53. Wasson D, Reppond K, Babbitt J et al. Effects of additives on proteolytic and functional properties of arrowtooth flounder surimi. J Aquat Food Prod Technol 1992; 1:147-165. 54. Akazawa H, Miyauchi Y, Sakurada K et al. Evaluation of protease inhibitors in Pacific whiting surimi. J Aquat Food Prod Technol 1993; 2:79-95. 55. Porter R, Koury B, Kudo G. Inhibition of protease activity in muscle extracts and surimi from Pacific whiting, Merluccius productus, and arrowtooth flounder, Atheresthes stomias. Marine Fish Rev 1993; 55:10-15. 56. Weerasinghe V, Morrissey M, An H. Characterization of active components in food-grade protease inhibitors for surimi manufacture. J Agric Food Chem 1996; 44:2584-2590. 57. Hamman D, Amato P, Wu M et al. Inhibition of modori (gel weakening) in surimi by plasma hydrolysate and egg white. J Food Sci 1990; 55:665-669,795. 58. Starkey P, Barrett A. Alpha-2-macroglobulin, a physiological regulator of proteinase activity. In: Barrett A, ed. Proteinases in Mammalian Cells and Tissues. New York: North- Holland Publishing, 1977.
223 59. Takeda A, Iwasawa A, Nakamura Y et al. Monoclonal antibodies as probes to detect conformational changes in the rat cysteine proteinase inhibitor cystatin A. J Immunol Meth 1994; 168:69-78. 60. Anastasi A, Brown M, Kembhavi A. Cystatin, a protein inhibitor of cysteine proteinases. Biochem J 1983; 211:129-138. 61. Feeney R, Osuga D. Egg-white and bloodserum proteins functioning by noncovalent interactions: studies by chemical modification and comparative biochemistry. J Protein Chem 1988; 7:667-687. 62. Weerasinghe V, Morrissey M, Chung Y et al. Whey protein concentrate as a proteinase inhibitor in Pacific whiting surimi. J Food Sci 1996; 61:367-371. 63. García-Carreño F, Navarrete del Toro A, Díaz-López M et al. Proteinase inhibition of fish muscle enzymes using legume seed extracts. J Food Protect 1996; 59:312-318. 64. Hameed K, Haard N. Isolation and characterization of cathepsin C from Atlantic short finned squid Illex illecebrosus. Comp Biochem Physiol B 1985; 82:241-246. 65. García-Carreño F, Haard N. Characterization of proteinase classes in langostilla (Pleuroncodes planipes) and crayfish (Pacific astacus) extracts. J Food Biochem 1993; 17:97-113. 66. García-Carreño F, Hernández-Cortés M, Haard N. Enzymes with peptidase and proteinase activity from digestive system of fresh water and marine decapods. J Agric Food Chem 1994; 42:1442-1456. 67. Osnes K, Mohr V. Peptide hydrolases of Antarctic krill, Euphasia superba. Comp Biochem Physiol 1985; 82:599-606. 68. Kawamura Y, Nishimura K, Matoba T et al. Effects of protease inhibitors on the autolysis and protease activities of Antarctic krill. Agric Biol Chem 1984; 48:923-930. 69. Baek H, Cadwallader K. Enzymatic hydrolysis of crayfish by-products. J Food Sci 1995; 60:929-935. 70. Wang Z, Taylor A, Yan X. Studies on the protease activities in Norway lobster (Nephrops norvegicus) and their role in the phenolase activation process. Food Chem 1992; 45:111-116. 71. Wang Z, Taylor A, Yan X. Further studies on the role of proteases in the activation of phenolase from Norway lobster (Nephrops norvegicus). Food Chem 1994; 51:99-103.
APPENDIX I
Substrates and Inhibitors Useful in Protease Characterization France Brunelle and Dominique Michaud
T
he first point to consider when initiating protease characterization studies is the choice of the substrate. When proteases are isolated from extracts poorly characterized, the choice of a protein substrate appears appropriate, since proteins generally contain multiple susceptible bonds that can be recognized as substrates by a large variety of proteases. When the proteases assayed are well characterized or when emphasis is to be placed on the detection of specific proteases, synthetic substrates allowing their specific detection under well-defined conditions should be employed.1 The most widely used protein substrates for the detection of proteases are inexpensive, commercially available proteins like casein, azocasein, bovine serum albumin, azoalbumin, hemoglobin and gelatin. Although proteins like gelatin or casein are not natural substrates for most proteases, they have proved useful in the characterization of protease-related processes in a large variety of biological samples.1 In contrast with protein substrates, synthetic substrates are useful in the detection of specific types of proteases and in the assessment of their functional, mechanistic characteristics. Synthetic substrates are usually synthesized by fusing specific substituents to either the -NH2 and/or the -COOH groups of an amino acid string. The amino acid sequence of the substrate and the position of its substituents generally determine its specificity, while the nature of one of the substituents, in several cases the carboxy-terminal substituent, gives the
substrate a chromogenic or fluorogenic nature useful in subsequent quantitation of hydrolytic activity. Based on the position of the substituent(s), synthetic substrates may be classified in two groups: endoprotease (or proteinase) substrates, in which both the amino- and carboxy- termini are blocked and thereby not available to exoproteases; and exoprotease substrates, in which only one extremity is blocked, the carboxy-terminal end for aminopeptidases or the aminoterminal end for carboxypeptidases. The most common substituents added to block -NH2 extremities are the benzoyl (Bz), benzoyloxycarbonyl (Z), acetyl (Ac) and succinyl (Suc) functional groups, while those added to -COOH extremities, which give in most instances the substrate its chromogenic or fluorogenic potential, are 4-nitroanilide, a chromogenic substituent, 7-amido-4-methylcoumarin, a fluorogenic substituent, and 2-naphtylamide, a substituent exhibiting either chromogenic or fluorogenic potential, depending on the detection procedure used to visualize the activity.2 The chromogenic detection of proteases primarily depends on the difference in molar absorptivity between the substrate and its hydrolytic products while fluorogenic substrates, generally more sensitive, contain a substituent group exhibiting measurable fluorescence when separated from the intact substrate.2,3 Given the extreme diversity of proteolytic enzymes in living cells and the large number of biological samples still poorly characterized, the specificity of a substrate
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Appendix I
225
Table AI.1. Synthetic substrates commonly used for protease detection and characterization in vitro a Protease
Substrates
Cathepsin B
Bz-arg-pNAb Pyr-phe-leu-pNA Z-arg-arg-pNA (or MCA) Z-phe-arg-pNA (or MCA)
Cathepsin H
H-arg-pNA (or MCA) Bz-arg-pNA
Cathepsin L
Z-phe-arg-pNA (or MCA)
Chymotrypsin
Ac-ala-ala-pro-phe-pNA H-ala-ala-phe-MCA Suc-ala-ala-pro-phe-pNA (or MCA) Suc-val-pro-phe-pNA Suc-ala-ala-phe-MCA Suc-leu-tyr-MCA
Elastase
Ac- (or Suc) ala-ala-ala-pNA Ac-ala-ala-pro-ala-pNA (or MCA) Ac-ala-pro-ala-pNA Suc-ala-ala-pro-leu-pNA Suc-ala-ala-pro-phe-pNA (or MCA)
Substilisin
Suc-ala-ala-ala-pNA (or MCA) Z-gly-gly-leu-pNA (or MCA)
Thermolysin
Suc-ala-ala-phe-MCA
Trypsin
Z-phe-arg-pNA (or MCA) Z-arg-MCA
a The amino acid strings appear in bold; see refs. 2 and 3 for reviews. b Abbreviations: Ac, acetyl; Bz, benzoyl; MCA, 4-methyl-7-aminocoumarin;
pNA, para-nitroanilide; Pyr, pyroglutamic acid; Suc, succinyl; Z, benzyloxycarbonyl.
for a given protease should be confirmed for each particular biological system assessed using appropriate complementary approaches. Low-molecular-weight protease inhibitors (PIs), in particular represent a good complement to synthetic substrates, as they allow in several cases the inhibition of specific protease classes or families. When used in combination, synthetic substrates and low molecular-weight
PIs appear quite useful in the primary characterization of protease activities in crude biological extracts. Table AI.1 lists synthetic substrates commonly used to detect proteases found in a variety of biological samples; Table AI.2 lists low- molecular-weight PIs useful in protease characterization.
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226
Table AI.2. Low-molecular-weight protease inhibitors useful in protease characterization a Inhibitor
Target proteases
Remarks
Mode
AEBSF b
Serine
Amastatin
Metallo
Aminopeptidases
Reversible
Antipain
Serine (also Cysteine)
Trypsin-like
Reversible
APMSF
Serine
Trypsin-like
Irreversible
Bestatin
Metallo
Aminopeptidases
Reversible
Chymostatin
Serine (also Cysteine)
Chymotrypsin-like
Reversible
3,4-DCI
Serine
Diprotin A
Metallo
Aminopeptidases
Reversible
Diprotin B
Metallo
Aminopeptidases
Reversible
E-64
Cysteine
EDTA
Metallo
Chelator
Reversible
Elastatinal
Serine
Elastase-like
Reversible
Iodoacetamide
Cysteine
May block other enzymes
Irreversible
Leupeptin
Serine (also Cysteine)
Trypsin-like, cathepsin B
Reversible
Pepstatin
Aspartate
1,10-Phenanthroline
Metallo
Chelator
Reversible
Phosphoramidon
Metallo
Bacterial endopeptidases
Reversible
PMSF
Serine
Also cysteine protease s in nonreducing conditions
Irreversible
TLCK
Serine
Trypsin-like
Irreversible
TPCK
Serine
Chymotrypsin-like
Irreversible
Z-Phe-Ala-CHN2
Cysteine
Irreversible
Irreversible
Irreversible
Reversible
Irreversible
a See refs. 1 and 4 for more details about most of these PIs. b Abbreviations: AEBSF, 4-(2-aminoethyl)-benzenesulfonyl fluoride, hydrochloride; 3,4-DCI, 3,4-
dichloroisocoumarin; E-64, trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane; EDTA, ethylenediaminetetraacetic acid; PMSF, phenylmethylsulfonyl fluoride; TLCK, tosyl lysyl chloromethyl ketone; TPCK, tosyl phenylalanyl chloromethyl ketone.
Appendix I
References 1. Michaud D. Gel electrophoresis of proteolytic enzymes. Anal Chim Acta 1998; 372:173-185. 2. Sarath G, de la Motte RS, Wagner FW. Protease assay methods. In: Beynon RJ, Bond JS, eds. Proteolytic enzymes. A practical approach. New York:IRL Press, 1989:25-55.
227 3. Weder JKP, Kaiser K-P. Fluorogenic substrates for hydrolase detection following electrophoresis. J Chromatogr A 1995; 698:181-201. 4. Beynon RJ, Salvesen G. Commercially available protease inhibitors. In: Beynon RJ, Bond JS, eds. Proteolytic enzymes. A practical approach. New York: IRL Press, 1989:241-249.
APPENDIX II
Plant Protease Inhibitors: Available mRNA Sequences Binh Nguyen-Quoc
T
he GenBank Database was searched with the Wisconsin Package, Version 9.1 (Madison Genetics Computer Group, Madison WI), using the following keywords: protease inhibitor, proteinase inhibitor, cystatin, trypsin inhibitor, chymotrypsin inhibitor, Kunitz, Bowman-Birk and serpin. Several hundred sequences were found, including about 275 mRNA sequences. Table AII.1 lists most plant protease inhibitor-encoding mRNA sequences available in the bank.
Table AII.1. Plant protease inhibitor-encoding mRNA sequences Accession N° a
Source / Inhibitor Serine PIs
Acacia confusa, trypsin inhibitor ‘acti’ (Kunitz)
M92852
Arabidopsis thaliana, protease inhibitor II
X69139
Brassica rapa, protease inhibitor II
L31937
Cucurbita maxima, fruit trypsin inhibitor
X81647
Cucurbita maxima, chymotrypsin inhibitor (Bowman-Birk)
X81447
Glycine max, Essex protease inhibitor(Kunitz)
U12150
G. max, ‘Kti3’ Kunitz trypsin inhibitor
S45092
G. max, ‘Ti-a’ (Kunitz trypsin inhibitor subtype A)
X64447
G. max, ‘Ti-b’ (Kunitz trypsin inhibitor subtype B)
X64448
G. max, Bowman-Birk proteinase inhibitors
Z13956 X68704
G. max, Provar Bowman-Birk protease inhibitor
U11260
G. max, protease inhibitor CII (Bowman-Birk)
M20732
Hordeum vulgare, chymotrypsin inhibitor CI-1A (Bowman-Birk)
M21400
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229
Table AII.1. Plant protease inhibitor-encoding mRNA sequences (cont'd) H. vulgare, chymotrypsin inhibitor CI-1B
M21401
H. vulgare, chymotrypsin inhibitor-2 (CI-2A)
X05404
H. vulgare, alpha-amylase/trypsin inhibitor
X13443
Luffa cylindrica, trypsin inhibitor (Kunitz)
M98055
Lycopersicon esculentum, leaf wound-induced proteinase inhibitor I
K03290
L. esculentum, leaf wound-induced proteinase inhibitor II
K03291
L. esculentum, proteinase inhibitor ‘ARPI’ (PI-II type)
L21194
L. esculentum, fruit-ripening protein (ethylene responsive (ER1))
J04099
Lycopersicon peruvianum, proteinase inhibitor I
J05094
Nicotiana sp., tumor-related proteinase inhibitor I
D13662
Nicotiana sylvestris, serine proteinase inhibitor I
M74102
Oryza sativa, proteinase inhibitor RPI (Kunitz)
U72942
O. sativa, Bowman-Birk proteinase inhibitor
U76004
Pisum sativum, trypsin/chymotrypsin inhibitor (clone TI12-36)
X83211
P. sativum, trypsin/chymotrypsin inhibitor (clone TI5-72)
X83210
Sinapsis alba, trypsin inhibitor 2 (Kunitz)
Y16190
Solanum tuberosum, proteinase inhibitor I
X67675 X67950 L06137 L06606 L06985 U30861
S. tuberosum, proteinase inhibitor II
M29965 X03778 X03779 L37519
S. tuberosum, tuber proteinase inhibitor (type II)
X13180
S. tuberosum, proteinase inhibitor - Kunitz-type
D17328 D17329 D17330 X56874
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230
Recombinant Protease Inhibitors in Plants
Table AII.1. Plant protease inhibitor-encoding mRNA sequences (cont'd) Vigna unguiculata, trypsin inhibitor
X51618
V. unguiculata, trypsin inhibitor fIV
X51617
Zea mays, trypsin inhibitor Z. mays, subtilisin-chymotrypsin inhibitor
AF057184 X69972
Cysteine PIs
Ambrosia artemisiifolia, cystatin
L16624
Brassica campestris, cysteine proteinase inhibitor (clone BCPI-1)
L41355
B. campestris, cysteine proteinase inhibitor (clone BIF 172)
L42819
B. campestris, cysteine proteinase inhibitor (BCPI-2)
U51119
B. campestris pekinensis, cysteine proteinase inhibitor
L48182
Carica papaya, cysteine proteinase inhibitor
X71124
Cucumis sativus, cysteine proteinase inhibitor
AB014760
Cyprinus carpio, cystatin
L23572
Daucus carota, extracellular insoluble cystatin
D85623
Glycine max, cysteine proteinase inhibitor
D31700
Oryza sativa, oryzacystatin I
J03469
O. sativa, oryzacystatin II
05595
Solanum tuberosum, multicystatin (gene)
L16450
Vigna unguiculata, cysteine proteinase inhibitor
Z21954
Zea mays, corn cystatin I
D10622
Z. mays, corn cystatin II
D38130
Aspartate PIs
Cucurbita maxima, aspartic proteinase inhibitor (API-1)
AF038166
C. maxima, aspartic proteinase inhibitor (API-2)
AF038167
Solanum tuberosum, aspartic proteinase inhibitor a Sequences available in the GenBank and EMBL databases.
X62095
APPENDIX III
Tertiary Structures of Proteases and Protease Inhibitors Available in the Brookhaven National Laboratory Protein Data Bank France Brunelle and Dominique Michaud Table AIII.1. Protease, protease inhibitor and protease/inhibitor complex structures available in the Brookhaven National Laboratory Protein Data Bank Source
Resolution (Å)
PDB #a
α-chymotrypsin A
Cow (Bos taurus)
1.67
5CHA
γ-chymotrypsin A
Bovine (Bos taurus) pancreas
1.60
1GCT
Bovine (Bos taurus) pancreas (orthorhombic) Bovine (Bos taurus) pancreas (trigonal) Streptomyces griseus
1.55
2PTN
1.70
3PTN
1.70
1SGT
1.65
3EST
Proteases, Inhibitors 1. Proteases A. Serine Proteinases Chymotrypsin (E.C 3.4.21.1)
Trypsin (E.C 3.4.21.4) Trypsin
Pancreatic elastase (E.C 3.4.21.36) Elastase (native)
Porcine (Sus scrofa) pancreas
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232
Table AIII.1. Protease, protease inhibitor and protease/inhibitor complex structures available in the Brookhaven National Laboratory Protein Data Bank (cont'd) Source
Resolution (Å)
PDB #a
Subtilisin
Bacillus lentus
——
1ST3
Subtilisin Carlsberg
Bacillus subtilis Bacillus licheniformis
2.50 2.00
1SBC 1SCA
Subtilisin BPN prime
Bacillus amyloliquefaciens (expressed in Bacillus subtilis)
1.80
2ST1
Human (Homo sapiens)
2.10
1HUC
Cathepsin B (mutant Ser115Ala) Rat (Rattus norvegicus), expressed in yeast
1.90
1THE
Papaya (Carica papaya) fruit latex
1.61
1PPN
Kiwifruit (Actinidia chinensis)
1.70
2ACT
Human (Homo sapiens) liver
2.50
1LYA
Pig (Sus scrofa) Mucor pusillus
1.80 2.00
4PEP 1MPP
Bacillus thermoproteolyticus
1.60
3TLN
Proteases, Inhibitors Subtilisin (E.C 3.4.21.62)
B. Cysteine proteinases Cathepsin B (E.C 3.4.22.1) Cathepsin B
Papain (E.C 3.4.22.2) Papain
Actinidin (E.C 3.4.22.14) Actinidin C. Aspartic proteinases Cathepsin D (E.C 3.4.23.5) Cathepsin D Pepsin (E.C 3.4.23.1) Pepsin
D. Metallo proteinases Thermolysin (E.C 3.4.24.27) Thermolysin
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Appendix III
233
Table AIII.1. Protease, protease inhibitor and protease/inhibitor complex structures available in the Brookhaven National Laboratory Protein Data Bank (cont'd) Proteases, Inhibitors
Source
Resolution (Å)
PDB #a
1.60
1LAM
1.80
1AMP
E. Aminopeptidases Cytosol aminopeptidase (E.C 3.4.11.1) Leucine aminopeptidase
Bovine (Bos taurus) lens
Bacterial leucyl aminopeptidase (E.C 3.4.11.10) Leucyl aminopeptidase
Aeromonas proteolytica
F. Carboxypeptidases Serine carboxypeptidase (E.C 3.4.16.1) Serine Carboxypeptidase II (CPDW-II)
Wheat germ (Triticum vulgaris) 2.20
3SC2
Carboxypeptidase A (E.C 3.4.17.1) Carboxypeptidase A
Bovine (Bos taurus) pancreas
1.50
2CTB
Bovine (Bos taurus) pancreas
2.80
1CPB
Bovine Pancreatic Trypsin Inhibitor
Bovine (Bos taurus) pancreas
1.80
5PTI
Trypsin Inhibitor
Sea anemone (Stichodactyla helianthus)
n/a
1SHP
1.90
1OVO
1.50
2OVO
n/a
1TUR
Carboxypeptidase B (E.C 3.4.17.3) Carboxypeptidase B fraction II 2. Protease Inhibitors A. Serine protease Inhibitors Kunitz - Kunin family
Kazal family Ovomucoid Third Domain
Japanese quail (Coturnix coturnix japonica) Silver pheasant (Lophura nycthemera) Turkey (Meleagris gallopavo)
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Recombinant Protease Inhibitors in Plants
234
Table AIII.1. Protease, protease inhibitor and protease/inhibitor complex structures available in the Brookhaven National Laboratory Protein Data Bank (cont'd) Resolution (Å)
PDB #a
Proteases, Inhibitors
Source
Human Pancreatic Secretory Trypsin Inhibitor (variant 3)
Human (Homo sapiens), 2.30 purified from PMAMPF-PSTI3, transduced E. coli
1HPT
Proteinase inhibitor IIA
Bovine (Bos taurus) seminal plasma
n/a
1BUS
Chymotrypsin Inhibitor 2 (CI-2)
Barley (Hordeum vulgare) seeds
2.00
2CI2
Eglin C
Leech (Hirudo medicinalis): synthetic gene expressed in E. coli
n/a
1EGL
Soybean (Glycine max)
n/a
1BBI
2.50
1TIE
Potato-Inhibitor-I family
Bowman-Birk family Trypsin/chymotrypsin Bowman Birk inhibitor Kunitz family
Erythrina Trypsin Inhibitor DE-3 Erythrina caffra seeds
Bifunctional proteinase inhibitor Ragi seeds (Eleusine coracana) n/a trypsin/α-amylase RBI
1BIP
Streptomyces Subtilisin Inhibitor family Streptomyces Subtilisin Inhibitor Streptomyces albogriseolus
2.60
2SSI
Squash seed Inhibitors Trypsin Inhibitor II EETI-II
Ecballium elaterium
n/a
2ETI
Trypsin Inhibitor CMTI-I
Squash seeds (Cucurbita maxima)
n/a
1CTI
Cystatin A (stefin A)
Human (Homo sapiens), expressed in E.coli
n/a
1CYV
Chicken cystatin
Chicken (Gallus gallus) egg white
2.00
1CEW
B. Cystatins
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Appendix III
235
Table AIII.1. Protease, protease inhibitor and protease/inhibitor complex structures available in the Brookhaven National Laboratory Protein Data Bank (cont'd) Proteases, Inhibitors
Source
Resolution (Å)
PDB #a
3. Protease-Inhibitor Complexes A. Complexes with small inhibitors Alpha-chymotrypsin A / PEBAb
Cow (Bos taurus)
1.80
6CHA
Anionic trypsin (Asp102Asn)/ benzamidine
Rat (Rattus rattus)
2.30
1TRM
Cathepsin D/pepstatin
Human (Homo sapiens) liver
2.50
1LYB
Elastase/TFAP
Porcine (Sus scrofa) pancreas
1.80
7EST
Leucine aminopeptidase/ amastatin
Bovine (Bos taurus) lens
2.40
1BLL
Papain/E-64C (form II)
Papaya (Carica papaya) fruit latex
1.90
1PPP
Papain/leupeptin
Papaya (Carica papaya) fruit latex
2.10
1POP
Pepsin/A62095
Pig (Sus scrofa)
2.90
1PSA
Serine carboxypeptidase II/ antipain and arginine
Wheat germ (Triticum vulgaris) 2.50
1BCR
Serine carboxypeptidase II/ L-benzylsuccinate
Wheat germ (Triticum vulgaris) 2.00
1WHT
Serine carboxypeptidase II/ chymostatin and arginine
Wheat germ (Triticum vulgaris) 2.10
1BCS
Thermolysin/benzylsuccinic acid
Bacillus thermoproteolyticus
1.70
1HYT
Thermolysin/phosphoramidon
Bacillus thermoproteolyticus
2.30
1TLP
Trypsin/benzamidine
Atlantic salmon (Salmo salar)
1.80
2TBS
2.00
1ACB
B. Complexes with protein inhibitors Alpha-chymotrypsin/eglin c
Oxen (Bos taurus)/ leech (Hirudo medicinalis)
(continued on next page)
Recombinant Protease Inhibitors in Plants
236
Table AIII.1. Protease, protease inhibitor and protease/inhibitor complex structures available in the Brookhaven National Laboratory Protein Data Bank (cont'd) Proteases, Inhibitors
Source
Resolution (Å)
PDB #a
α-chymotrypsin/Bovine third domain (OMTKY3)
(Bos taurus) pancreas/ turkey (Meleagris gallopavo)
1.80
1CHO
Carboxypeptidase A/potato carboxypeptidase A inhibitor
Bovine (Bos taurus) pancreas/ potato (Solanum tuberosum)
2.50
4CPA
Human neutrophil elastase Human (Homo sapiens) (HNE)/ third domain of leukocyte/turkey ovomucoid inhibitor (OMTKY3) (Meleagris gallopavo)
1.80
1PPF
Papain/Cystatin B (stefin B) (mutant Cys8Ser)
Human (Homo sapiens), expressed in E. coli
2.37
1STF
Subtilisin Carlsberg/N-acetyl eglin C
Bacillus subtilis / leech (Hirudo 1.80 (medicinalis)
2SEC
Subtilisin Carlsberg/Eglin C
Commercial product from 1.20 Serra/leech (Hirudo medicinalis)
1CSE
Subtilisin BPN prime/ Streptomyces Subtilisin Inhibitor (SSI mutant Met73Lys)
Bacillus amyloliquefaciens/ Streptomyces lividans
1.80
3SIC
Subtilisin BPN prime (mutant Glu251Gln)/Chymotrypsin Inhibitor 2 (CI-2)
Bacillus amyloliquefaciens/ Barley seeds (Hordeum vulgare 2.10
2SNI
Trypsin/bitter gourd inhibitor
Porcine (Sus scrofa) pancreas/ gourd (Momordica charantia)
——
1MCT
Trypsin/Bovine Pancreatic Trypsin Inhibitor
Bovine (Bos taurus) pancreas
1.90
2PTC
Trypsin/Cucurbita maxima inhibitor (CMTI)
Bovine (Bos taurus) pancreas/ Cucurbita maxima
2.00
1PPE
Trypsin/Bowman Birk inhibitor
Bovine (Bos taurus) pancreas/ Synthetic peptide from Mung Bean Trypsin Inhibitor
——
1SMF
Trypsin / Bowman-Birk Inhibitor (AB-I)
Bovine (Bos taurus) pancreas/ Adzuki beans (Phaseolus angularis)
——
1TAB
(continued on next page)
Appendix III
237
Table AIII.1. Protease, protease inhibitor and protease/inhibitor complex structures available in the Brookhaven National Laboratory Protein Data Bank (cont'd) Proteases, Inhibitors
Source
Trypsin / Streptomyces Subtilisin Bovine (Bos taurus) pancreas/ Inhibitor (SSI mutant Met70Gly, Streptomyces lividans Met 73Lys)
Resolution (Å)
PDB #a
——
2TLD
a For structural data, see the Brookhaven National Laboratory website: . b Abbreviations: E-64, trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane; PEBA,
phenylethane boronic acid; TFAP, trifluoroacetyl-L-leucyl-L-alanyl-p-trifluoromethylphenylanilide; n/a: not applicable (NMR spectroscopy).
Index δ-endotoxin (see Bt toxin) 5, 19
A Abscisic acid 15, 16, 40 Affinity handle 194 Agrotis sp. 18 AIDS 203, 216 Albumin-binding protein 196, 197 Aminopeptidase 11, 93, 95, 154, 155, 157, 224, 233, 235 Aphid 72, 76, 99, 100, 101, 148-151, 162, 181, 186 Apis mellifera (honeybee) 187 Arabidopsis thaliana 48 Aspartate protease 4, 108, 110 Aspartate proteinase 192 Aspartyl protease 153, 154, 203 Aspartyl protein 11, 45 Aspartyl proteinase 11, 45, 153, 155 Avr gene 72
B Bacillus thuringiensis 5, 8, 66, 101, 149, 175, 179 Bacterial protease 56 Baris sp. 181, 182, 186, 187 Barley trypsin inhibitor 11, 18, 91 Biocidal protein 5 Botrytis 55, 59 Bowman-Birk inhibitor 170, 175,182, 184, 234, 236 Bt toxin 5, 6, 73, 101, 102, 150, 151, 179, 180 Bumblebee 97
C Caenorhabditis elegans 43, 44, 46, 47, 66, 121 Carboxypeptidase 11, 123, 154, 157, 233, 235, 236 Cathepsin 28, 30, 32, 35, 38, 40, 44, 46, 50, 110, 114, 116, 125, 142, 143, 146, 153-155, 157, 203, 204, 206-209, 218-220, 226 Cathepsin B 35, 46, 114, 116, 142, 143, 153-155, 157, 206-208, 210, 219, 226
Cathepsin D 44, 110, 114, 153, 155, 157, 207 Cathepsin H 28, 30, 32, 35, 38, 114, 125, 153-155, 157, 218, 219 Cathepsin L 32, 46, 142, 153, 206, 208, 219 Ceutorhynchus 117, 118 Chicken egg white cystatin 94, 204 Cholecystokinin 83 Choristoneura sp. 21 Chymotrypsin 10, 12, 15, 18, 19, 21, 22, 33, 57, 59, 80, 82, 84, 93-95, 114, 120, 121, 135, 154, 155, 182, 217, 219, 220, 226, 228-231, 234-236 Clostridium sp. 204 Cochliobolus sp. 54 Coleoptera 12, 25, 32, 35, 36, 38, 78, 80, 179-182, 184-189 Colorado potato beetle 15, 33-36, 99, 100, 108-112, 114, 125, 143, 148 Compensation [to prote(in)ase inhibitors] 3, 107, 110, 157, 160, 161, 189 Compensatory process 60, 217 Controlled proteolysis 216 Corn 28, 29, 30, 32, 33, 46, 68, 101, 111, 116, 118, 123, 194, 197, 209, 218 Coronatine 57-59, 62 Costelytra sp. 117 Cowpea trypsin inhibitor 19, 46, 65, 81, 91, 95, 111, 115, 124, 169, 173, 174, 182, 185 Crustaceans 218, 220 Cylas sp. 167-171 Cyst nematode 45, 46, 48, 49, 122 Cystatin 27-30, 33-39, 46, 47, 58, 63, 94, 95, 100, 109, 111, 116, 117, 119, 122, 125, 129, 132, 134, 138, 154, 170, 184, 194, 197, 204-209, 218-220, 222, 230, 234, 236 Cystatin α 205, 206 Cystatin C 205-209, 211, 218, 222 Cystatin S 205, 208, 211, 213 Cystatin SA 208 Cystatin SN 205 Cysteine protease 4, 11, 12, 60, 108, 110, 182, 184, 226
Index Cysteine proteinase 28, 30-32, 34-36, 39, 44, 46-48, 51, 63, 100, 111, 115, 117-119, 121, 122, 142, 143, 155, 161, 180, 183, 185, 186, 188, 203-208, 210, 218-221, 230 Cysteine proteinase inhibitor 28, 100, 105, 111, 161, 230
D Degree of hydrolysis 107, 109, 110, 111, 114, 119, 169, 215, 216, 220 Delia sp. 181, 182 Diabrotica sp. 118 Diagnostic prote[in]ase inhibitor 115, 116, 147 Digestive protease 169, 184, 186, 187, 217, 218 Digestive proteinase 9, 11, 14, 33-36, 38, 40, 46, 47, 83, 100, 169, 180 Diptera 21, 80, 83 Dissociation constant (Kd) 10, 16, 136 Drosophila 31, 32
E E-64 66, 226, 235, 237 Elastase 10, 12, 22, 80, 82, 91, 93-95, 121, 123, 125, 128, 209, 218, 225, 226, 231, 235, 236 Enzyme 10-13, 21-23, 27, 28, 30, 32, 34, 45, 46, 47, 56, 57, 62, 65, 68, 80-84, 93, 94, 110, 115-117, 119, 120, 122, 125, 126, 140-143, 145-147, 153, 154, 156, 173, 203, 204, 206, 207, 215, 216, 220 affinity 107-109, 110, 113, 115-118, 120-123, 131, 132, 135, 136, 138, 146, 154, 157, 161, 169 specificity 87 Erwinia sp. 56 Escherichia coli 111, 120, 129, 191, 206, 209, 218 Euscepes sp.168, 173, 177 Exopeptidase 10, 11, 93, 109, 142, 215, 216 Exoprotease 224 Extensive proteolysis 1
239 Fungal protease 54 Fusion protein 35, 48, 111, 132, 135, 185, 195-198
G Gel weakening 219, 220 Globodera sp. 117, 122 Glutathione 35, 111, 194, 196
H Helicoverpa sp. 99, 101 Heliothis sp. 101, 102 Hemiptera 11, 12, 36, 160 Herpes simplex virus 204, 205 Honeybee 92, 95, 97, 98 Hybrid enzyme 120 Hybrid PI 5, 120, 121 Hymenoptera 80, 181
I I50 13 IMAC 196 Inhibition constant 13 Inhibitor interactions 107, 109, 110, 114, 116 Inhibitory spectrum 3, 5, 6, 38, 60, 108, 111, 119, 120, 123, 154, 161, 169 Integrated pest management 61, 82, 149, 168, 189
J Jasmonic acid 4, 15, 67, 217 Juvenile hormone 83
K Kd value 116, 136 Ki value 22, 28, 46, 47, 116-119, 122, 128, 129, 141-143, 145, 218, 219 Kunitz trypsin inhibitor 14, 20, 22, 33, 60, 61, 82, 93, 120, 169, 170, 175
L F Factor Xa 137, 197, 198 FLAG peptide 195, 196 Food safety 152
Lacanobia sp. 14, 16-19, 22 Lepidoptera 12-14, 16-18, 20-23, 180-182, 184, 185, 188
240 Leptinotarsa decemlineata 125, 143, 147, 148, 163, 200 Leucine aminopeptidase 93, 95, 154, 155, 157, 233, 235 Limited proteolysis 1, 35, 119 Lobster 220 Lymantria sp. 82
M Macrosiphum sp. 148 Malaria 204, 206 Mamestra sp. 180, 181 Manduca sexta 121, 126, 144, 147 Mechanical injury 68, 217 Meligethes sp. 181, 182 Metalloprotease 4, 55 Metalloproteinase 100 Metastasis 191, 203, 204, 207 Methyl jasmonate 15, 51, 57, 64, 220 Molecular farming 197, 198 Monitor peptide 83 Multicystatin 57-59, 111, 161, 230 Myzus sp. 100, 148, 151, 186
N Natural enemies 3, 67, 89, 99-102, 149, 152, 159, 169 Natural predators 6, 67, 108, 149, 159, 176 Nematode control 172, 173
O Octadecanoid (OD) pathway 15, 57, 67-73 Oryzacystatin 27-29, 34-38, 46, 47, 51, 91, 94, 108, 116, 119, 146, 147, 153, 182, 184, 185, 190, 194, 205, 218, 220, 222, 230 Oryzain 27, 30, 31, 222 Otiorynchus sp. 110, 115, 118, 125, 147, 200 Ovomucoid 219, 233, 236
P Pacific whiting 219, 220 Papaya 29,30, 60, 100, 110, 113, 119, 143, 146, 147, 164, 178 Parasitic nematodes 3, 4, 44-47, 49, 54, 172 Parasitoids 89, 99, 101, 102, 104, 105, 108, 149, 176 Pepsinogen 141, 142
Recombinant Protease Inhibitors in Plants Perillus bioculatus 99, 109, 159 Periodontitis 203, 207-209 Phaedon sp. 189 Phage display 5, 47, 49, 122, 124, 128-131, 133-137 Phyllotreta sp. 180, 181, 185 Phytocystatin 129, 218 Plasmodium sp. 206 Pleiotropic effects 89, 90, 97 Plutella sp. 180, 181 Poliovirus 204, 205, 209, 211 Pollen 90, 92-98, 100, 101, 152, 182, 186-189 Pollinators 89, 90, 99, 103, 108, 152 Polyhistidine tags 195, 196 Poplar 38, 173, 184, 188 Porphyromonas sp. 204, 205 Post mortem muscle softening 218 Potato 9, 11, 15-19, 22-24, 30, 33, 35, 36, 91, 94-100, 108-110, 112, 169, 194, 198, 200, 216-219, 234, 236 Protease 2, 3, 5, 6, 11, 20, 22-24, 44, 46, 50, 54-57, 59-61, 63, 65 Protease inhibitor 109, 110, 228 Protease inhibitor II 18, 228 Protein 1-5, 9-12, 14, 17, 19, 21-23, 27, 28, 30, 32, 34-36, 38, 39, 47, 48, 54, 56, 57, 59-62, 64, 65, 68-73, 80, 81, 83, 85 digestion 9-12, 34, 93, 109, 158, 159, 182, 216, 217 intracellular targetting 191 kinase 70, 71 preregion 141 proregion 142-145 secretory pathway 192, 193 substrates 115, 116, 153, 154, 218, 224 turnover 2, 65, 217 Proteinase 9-14, 18, 20-23, 27-36, 38, 44-49, 80-84, 89, 91, 94, 98, 100, 103, 105, 106, 111-115, 117-119, 121, 122, 124-127, 129, 137, 138, 142, 143, 146, 147, 153-156, 159, 161, 163-165, 169-171, 177, 178 Proteinase inhibitor 9, 20, 28, 34, 44, 49, 63, 68, 89, 91, 94, 98, 100, 103, 185, 190, 229, 230, 234 Proteinase inhibitor I 201, 229 Proteinase inhibitor II 68, 229 Pseudomonas sp. 56, 57, 71 Psylliodes sp. 179, 181, 182, 187 Pyrenopeziza sp. 55, 59 Pythium sp. 60, 61
Index
241
R
T
R gene 74 Rapeseed 117, 161, 179 Rheumatoid arthritis 191, 199, 203, 207, 208 Rhinovirus type 1A 205 Rhodnius sp. 12, 32 Rice 17, 18, 27, 28, 30, 31, 34-36, 38, 39, 46, 57, 63, 91, 94, 116, 119, 122, 125, 127, 153, 160, 161, 184, 190, 194, 199, 201, 207, 209, 211, 217-220, 222 Riptortus sp. 125 Root-knot nematodes 174 Rotylenchus sp. 172
Tetranychus sp. 49, 68, 70, 118 Thermostability (of protease inhibitors) 217 Thrombin 80, 197, 198 Tobacco 15-20, 33, 38, 45, 47-49, 56, 58, 62, 63, 70-73, 81, 90, 91, 94, 97, 99, 101-103, 151, 161, 174, 188, 189, 193 Tribolium sp. 123 Trichoderma sp. 59, 68 Tri-trophic interactions 99, 101, 102, 104 Trypanosoma sp. 204 Trypsin 9-12, 14-26, 33, 34, 45, 46, 48, 54, 59, 60, 61, 65, 66, 78, 81-84, 91, 93-95, 98, 100, 103, 111, 115-117, 120, 121, 123, 124, 129, 132, 135, 142, 144-146, 155, 169-175, 182, 205, 216, 217, 219-221, 225, 226, 228-231, 233-236 Trypsinogen 141, 142 Tumor growth 203, 204, 206, 207
S Salmon 235 Schistosoma sp. 194, 196 Serine protease 22, 57, 111, 220, 233 Serine proteinase 12, 91, 116, 120, 121, 145, 154, 159, 160, 174, 183, 185, 218, 220, 229 Serine proteinase inhibitor 91, 185, 229 Signal peptide 30, 59, 131, 141, 142, 193 Site-directed mutagenesis 47, 117, 120-124 Soil fauna 104 Soybean 12, 20, 22, 28, 30, 33, 34, 45, 46, 49, 51, 61, 62, 72, 74, 82, 84, 90, 91, 93, 98, 111, 120, 122, 129, 132, 146, 169, 179, 182, 185, 190, 205, 216-218 Spodoptera sp. 174, 182, 188 Squash trypsin inhibitor 66, 91, 123 Squid 219, 220 Stability [of prote(in)ase inhibitor] 116, 118, 132, 154, 169, 174, 191, 194, 197, 198, 209, 215, 217 Staphylococcal protein A 196 Stefin A 35, 111, 119, 154, 197, 200 Strawberry 17, 18, 20, 55, 91, 111, 194 Streptococcus sp. 204 Subtilisin 15, 59, 80, 217, 230, 232, 234, 236 Sweetpotato 91, 167-177 Sweetpotato weevil (SPW) 167, 170 Synthetic substrates 10, 13, 22, 23, 15, 116, 153, 224, 225 Systemin 15, 16, 67, 68, 69, 70, 71, 72, 75, 217
U Ubiquitin 192, 200
V Vacuolar signal 192, 193 Verticillium 54 Volatiles 68, 70, 102
W Wounding response 14, 16, 20, 217
X Xanthomonas sp. 56