The Viroids THE VmUSES

Series Editors HEINZ FRAENKEL-CONRAT, University of California Berkeley, California

ROBERT R. WAGNER, University of Virginia School of Medicine Charlottesville, Virginia

THE VIRUSES: Catalogue, Characterization, and Classification Heinz Fraenkel-Conrat

THE ADENOVIRUSES Edited by Harold S. Ginsberg

THE HERPESVIRUSES Volumes 1-3 • Edited by Bernard Roizman Volume 4 • Edited by Bernard Roizman and Carlos Lopez

THE PAPOVAVIRIDAE Volume 1 • Edited by Norman P. Salzman Volume 2 • Edited by Norman P. Salzman and Peter M. Howley

THE PARVOVIRUSES Edited by Kenneth I. Berns

THE PLANT VIRUSES Volume 1 • Edited by R. I. B. Francki Volume 2. Edited by M. H. V. Van Regenmortel and Heinz Fraenkel-Conrat Volume 3 • Edited by Renate Koenig

THE REOVIRIDAE Edited by Wolfgang K. Joklik

THE RHABDOVIRUSES Edited by Robert R. Wagner

THE TOGAVIRIDAE AND FLA VIVIRIDAE Edited by Sondra Schlesinger and Milton J. Schlesinger

THE VIROIDS Edited by T. O. Diener The Viroids

Edited by T. o. DIENER Microbiology and Plant Pathology Laboratory Agricultural Research Service U.S. Department of Agriculture Beltsville, Maryland

PLENUM PRESS • NEW YORK AND LONDON Library of Congress Cataloging in Publication Data The Viroids. (The Viruses) Includes bibliographies and index. 1. Viroids. I. Diener, T. O. (Theodor Otto), 1921- . II. Series. QR500.V568 1987 576'.6483 87-13017 ISBN-13: 978-1-4612-9035-3 e-ISBN-13: 978-1-4613-1855-2 DOl: 10.1007/978-1-4613-1855-2

© 1987 Plenum Press, New York Softcover reprint of the hardcover 1st edition 1987 A Division of Plenum Publishing Corporation 233 Spring Street, New York, N.Y. 10013 All rights reserved No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise, without written permission from the Publisher Contribu tors

Patricia Broadbent, Biological and Chemical Research Institute, New South Wales Department of Agriculture, Rydalmere, 2116, Australia Paul R. Desjardins, Department of Plant Pathology, University of Cali• fornia, Riverside, California 92521 T. O. Diener, Microbiology and Plant Pathology Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, Mary• land 20705 Gail Dinter-Gottlieb, Department of Bioscience and Biotechnology, Drexel University, Philadelphia, Pennsylvania 19104 R. I. B. Francki, Department of Plant Pathology, Waite Agricultural Re• search Institute, University of Adelaide, Glen Osmond, 5064, South Australia Jorge Galindo A., Centro de Fitopatologia, Colegio de Postgraduados, Montecillos, Texcoco, Mexico S. M. Garnsey, Agricultural Research Service, U.S. Department of Agri• culture, Orlando, Florida 32803 Rosemarie W. Hammond, Microbiology and Plant Pathology Laboratory, Plant Protection Institute, Beltsville Agricultural Research Center, U.S. Department of Agriculture, Beltsville, Maryland 20705 R. Kenneth Horst, Department of Plant Pathology, Cornell University, Ithaca, New York 14853 Paul Keese, Department of Biochemistry, University of Adelaide, Ade• laide, 5000, South Australia; Present address: Research School of Biological Sciences, Australian National University, Canberra, A.C.T. 2600, Australia Roger H. Lawson, Florist and Nursery Crops Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, Mary• land 20705 v vi CONTRIBUTORS

Robert A. Owens, Microbiology and Plant Pathology Laboratory, Plant Protection Institute, Beltsville Agricultural Research Center, U.S. Department of Agriculture, Beltsville, Maryland 20705 Tien Po, Institute of Microbiology, Academia Sinica, Beijing, China John W. Randles, Department of Plant Pathology, Waite Agricultural Research Institute, University of Adelaide, Glen Osmond, 5064, South Australia Detlev Riesner, Institut fur Physikalische Biologie, Universitat Dussel• dorf, D-4000 Dusseldorf, Federal Republic of Germany Heinz L. Sanger, Max-Planck-Institut fur Biochemie, Abteilung Viroid• forschung, D-8033 Planegg-Martinsried, Federal Republic of Ger• many Eishiro Shikata, Department of Botany, Faculty of Agriculture, Hokkaido University, Sapporo, Japan Robert H. Symons, Department of Biochemistry, University of Adelaide, Adelaide, 5000, South Australia B. Walter, INRA, Station de Pathologie Vegetale, B.P. 507, Colmar, France Chen Wei, Institute of Microbiology, Academia Sinica, Beijing, China Preface

More than seven years have passed since the first monograph on viroids was published. At that time, the existence of viroids as a novel type of pathogen far smaller than viruses had been amply demonstrated and some of their unusual molecular properties had been elucidated, but the entry of molecular biology into viroid research was still in its infancy. Since that time, our knowledge of the molecular properties of viroids has increased exponentially and viroids have become even more fasci• nating than was the case seven years ago. Today, aside from transfer RNA, viroids are probably the best known type of RNA-at least from a struc• tural standpoint. Much less is known of the mechanisms of viroid func• tion, such as the exact pathway and enzymology of viroid replication and the biochemistry of viroid pathogenesis. Recently, however, emphasis in viroid research has shifted from structural to functional themes and im• portant beginnings have been made in the elucidation of viroid struc• ture-function relationships. With the discovery of viroidlike RNAs within the capsids of certain plant viruses and the finding of surprising structural similarities between viroids and plant satellite RNAs, the conceptual gap between viroids and conventional viruses has significantly narrowed. Even beyond virology, connecting links with cellular RNAs have come to light and the long isolation of viroids land "viroidologists"J has come to an end. It is hoped not only that the present volume brings the reader up to date on our present knowledge of viroids and viroid diseases, but that it also reflects some of the excitement and enthusiasm that motivates viroid investigators and permeates the field. It has been pointed out before that, in contrast to many other areas of virology or plant pathology, our knowledge of the molecular aspects of viroids is far greater than that of more traditional aspects, such as the mechanisms of viroid spread in the field, possible vector transmission of viroids, and all aspects of disease epidemiology.

vii viii PREFACE

It is for this reason that this volume is divided into two parts: a general section that is mostly dedicated to the physical-chemical and biological properties that viroids have in common and a special section that treats each viroid in tum with emphasis on those properties that are peculiar to each and concentrating on the plant pathological aspects of the diseases that each viroid causes. I wish to thank all the authors who contributed to the volume for their excellent and up-to-date treatments, and I hope that the book will prove useful not only to viroid investigators but also to scientists in other fields who wish to learn of the newest results regarding viroids and viroid diseases.

T. O. Diener Beltsville, Maryland Contents

Viroid Abbreviations ...... , xxi

Introduction T. O. Diener I. Organisms, Viruses, and Viroids ...... 2 II. The Biochemical Significance of Viroids ...... 3 III. The Possible Evolutionary Significance of Viroids...... 4 IV. References...... 5

I. General Section

Chapter 1 Biological Properties T. O. Diener I. Introduction ...... 9 II. Symptomatology...... 10 A. Macroscopic Symptoms ...... 10 B. Cytopathic Effects...... 11 C. Biochemical Disturbances...... 13 III. Ecology and Epidemiology...... 16 A. Origin of Viroid Diseases...... 16 B. Environmental Factors...... 19 C. Natural Transmission...... 19 D. Economic Importance and Control Measures...... 21 IV. Experimental Biology...... 22 A. Experimental Transmission ..... , ...... 22 B. Experimental Host Range ...... 22 ix x CONTENTS

C. Propagation ...... 23 D. Identification...... 24 E. Quantitation...... 25 F. Purification ...... 25 G. Diagnostic Procedures ...... 25 H. Interaction with Other Pathogens...... 28 V. References ...... 29

Chapter 2 Physical-Chemical Properties: Molecular Structure (Primary and Secondary) Paul Keese and Robert H. Symons I. Introduction ...... 37 II. Purification of Viroids ...... 37 A. Preparation of Plant Extracts ...... 38 B. Purification...... 39 III. Sequence Determination of Viroids ...... 40 A. Fingerprint Analysis...... 40 B. Direct RNA Sequencing...... 41 C. Primer-Directed Sequence Analysis...... 42 D. Sequencing of Viroids Using Cloned cDNA...... 43 IV. Structure of Viroids ...... 45 A. Circular Structure of Viroids ...... 45 B. Sequence and Structure of Viroids ...... 45 C. Structural Domains of Viroids ...... 49 D. Structure of ASBV ...... 55 V. Conclusion ...... 58 VI. References...... 59

Chapter 3 Physical-Chemical Properties: Structure Formation Detlev Riesner I. Introduction ...... 63 II. Experimental Analysis...... 64 A. Optical Melting Curves ...... 64 B. Calorimetry...... 67 C. Electron Microscopy ...... 69 D. Gel Electrophoresis...... 70 E. Kinetics ...... 71 F. Influence of Ionic Strength and Other Solvent Conditions...... 74 CONTENTS n

III. Theoretical Analysis ...... 76 A. Elementary Parameters...... 76 B. Mechanistic Models ...... 80 IV. Mechanism...... 82 A. Premelting Regions ...... 82 B. Main Transition and Formation of Stable Hairpins ...... 83 C. Reversibility ...... 86 D. Experiment and Theory...... 88 V. Structure Formation and Function...... 89 A. Viroids: A Dynamic Principle...... 90 B. Premelting and Virulence ...... 91 C. Relevance of Stable Hairpins ...... 93 VI. Addendum...... 94 VII. References...... 96

Chapter 4 Viroid Function: Subcellular Location and in Situ Association with Cellular Components Detlev Riesner I. Introduction ...... 99 II. Subcellular Location...... 100 A. Nuclei ...... 100 B. Other Organelles ...... 103 III. Subnuclear Location...... 103 A. Chromosomal Network...... 103 B. Nucleoli...... 103 C. Nucleolar Nucleosomes ...... 104 IV. Nature of Viroid Complexes ...... 106 A. Ionic Strength Dependence...... 107 B. Viroid-Binding Proteins...... 108 V. Reconstitution of Viroid-Host Interactions...... III A. Nuclei ...... III B. Nucleoli...... III VI. Location of Viroid Intermediates ...... 112 VII. Location and Function...... 113 VIII. References...... 114

Chapter 5 Viroid Function: Viroid Replication Heinz L. Sanger I. Introduction ...... 117 II. The Question of Viroid Translation ...... " 118 xii CONTENTS

III. Potentially Possible Pathways of Viroid Replication...... 120 A. Presumed DNA-Directed Viroid Replication...... 120 B. RNA-Directed Viroid Replication...... 121 IV. Intermediates and Products of Viroid Replication...... 122 A. Monomeric Circular Viroid (+) RNA, the "Mature" Viroid Proper...... 122 B. Oligomeric Linear Viroid ( - ) RNA ...... 123 C. Oligomeric Linear Viroid ( + ) RNA ...... 124 D. Viroid (+) . (-) RNA Hybrid Molecules...... 125 V. Studies on Viroid Replication in Vivo...... 126 A. Replication in Intact Plants...... 127 B. Replication in Plant Protoplasts and Cultured Cells. . . .. 129 C. Replication in Isolated Plant Cell Nuclei...... 130 VI. Inhibition Studies on Viroid Replication ...... 132 VII. Replication Models...... 135 A. The Model of Owens and Diener...... 136 B. The Models of Branch and Robertson ...... 137 C. The Model of Ishikawa etal...... 139 D. The Model of Hutchins et al...... 140 VIII. In Vitro Transcription of Viroid RNA with Purified Polymerases ...... 143 A. Transcription with DNA-Dependent Plant RNA Polymerases...... 143 B. Transcription with RNA-Dependent Plant RNA Polymerases...... 144 IX. Viroid Replication as Initiated by Cloned Viroid DNA...... 145 A. Infectivity of Cloned Double-Stranded Viroid DNA. . . .. 146 B. Infectivity of Cloned Single-Stranded Viroid DNA ...... 147 C. Viroid-Related RNA Transcripts in E. coli...... 149 X. In Vitro Synthesis of Viroid RNA with RNA Transcription Systems...... 150 A. Viroid Synthesis with Bacterial RNA Polymerase/Promoter Systems...... 151 B. Viroid Synthesis with Bacteriophage RNA Polymerase/Promoter Systems...... 151 XI. The Processing of Viroid RNAs ...... 153 A. Interconversion of Monomeric Circular and Linear Viroid Molecules in Vitro...... 153 B. Cleavage of Oligomeric Viroids into Monomers...... 154 C. Perspectives ...... 155 XII. Structural Requirements for the Replicability of Viroids ...... 156 XIII. Structural Requirements for the Processing of Viroid RNA...... 157 XIV. Conclusions...... 160 XV. References...... 161 CONTENTS xiii

Chapter 6 Viroid Function: Molecular Biology of Viroid-Host Interactions Robert A. Owens and Rosemarie W. Hammond I. Viroid Pathogenesis-A Structure/Function Paradigm...... 167 A. Current Models for Viroid Pathogenesis...... 168 B. Possible Mechanisms for Disease Induction ...... 171 C. Possible Relationships between Viroid Structure and Function...... 173 II. Site-Specific Mutagenesis of Infectious cDNAs ...... 174 A. Minimal Requirements for cDNA Infectivity...... 174 B. Site-Specific Mutagenesis of PSTV and CEV cDNAs. . . .. 176 C. Construction of Chimeric Viroid cDNAs ...... 177 D. Characterization of Mutant Viroids ...... 180 III. Viroid Transcripts as Substrates for RNA Recombination .,. 182 IV. Concluding Remarks ...... " 184 V. References...... 186

Chapter 7 Possible Viroid Origin: Viroids, Virusoids, and Group I Introns Gail Dinter-Gottlieb I. Introduction ...... 189 II. Types of Introns ...... 189 III. Viroids as Introns ...... 191 IV. Viroids and the Self-Splicing Intron of Tetrahymena ...... 196 V. Discussion and Digressions...... 198 VI. References...... 200

Chapter 8 Possible Viroid Origin: Encapsidated Viroidlike RNA R. I. B. Francki I. Introduction ...... 205 II. Viruses That Encapsidate Viroidlike RNAs ...... 205 III. Are Viroidlike RNAs Satellite RNAs? ...... 206 IV. The Dependence of Viroidlike RNA on Viral RNA 1 ...... 209 V. Structure of Viroidlike RNA...... 210 VI. Replication of Viroidlike RNA...... 212 A. In Vivo Studies...... 213 B. In Vitro Studies ...... 213 C. How Is Viroidlike RNA Synthesized? ...... 214 xiv CONTENTS

VII. Possible Relationships of Viroidlike RNAs to Viroids ...... 215 VIII. References ...... 216

II. Special Section

Chapter 9 Potato Spindle Tuber T. O. Diener I. Historical ...... 221 II. Geographic Distribution...... 222 III. Symptomatology...... 222 IV. Field Transmission and Spread ...... 224 V. Economic Losses...... 225 VI. HostRange ...... 226 VII. Strains ...... 227 A. Unmottled Curly Dwarf Strain ...... 227 B. Mild Strains...... 227 VIII. Experimental Transmission ...... 228 IX. Diagnosis...... 228 A. Bioassay ...... 228 B. Polyacrylamide Gel Electrophoresis...... 230 C. Molecular Probes...... 230 X. Interaction with Other Pathogens ...... , . . . .. 231 XI. Purification...... 231 XII. Physical-Chemical Properties...... 232 XIII. References...... 232

Chapter 10 Citrus Exocortis Patricia Broadbent and S. M. Garnsey I. Field Symptomatology...... 235 II. Geographic Distribution...... 236 III. Host Range ...... 236 IV. Importance of Exocortis ...... 237 V. Strains ...... 237 VI. Transmission ...... 238 A. Transmission by Grafting ...... 238 B. Mechanical Transmission ...... 238 C. Transmission by Vectors...... 238 D. Transmission through Seed...... 239 E. Transmission by Dodder ...... 239 CONTENTS xv

VII. Detection...... 239 A. Indicator Plants ...... 239 B. Biochemical Methods of Detection...... 239 VIII. Cytopathic Effects in Host...... 240 IX. Control...... 240 X. Physical-Chemical Properties...... 241 XI. References...... 241

Chapter 11 Chrysanthemum Stunt Roger H. Lawson I. Introduction ...... 247 II. Symptomatology...... 248 A. CSV in Chrysanthemum ...... 248 B. Other Viroids in Chrysanthemum ...... 249 III. Transmission and Spread ...... 249 IV. Host Range...... 249 V. Diagnosis...... 251 A. Bioassay ...... 251 B. Polyacrylamide Gel Electrophoresis...... 253 C. Nucleic Acid Hybridization ...... , 255 VI. Purification...... 255 VII. Physical-Chemical Properties...... 256 VIII. Control Measures ...... , 257 A. Elimination by Heat Treatment and Meristem-Tip Culture ...... 257 B. Cross-Protection...... 257 IX. References...... 258

Chapter 12 Cucumber Pale Fruit T. O. Diener I. Historical...... 261 II. Geographic Distribution ...... 261 III. Symptomatology ...... 261 IV. Field Transmission and Spread ...... 262 V. Economic Losses...... 262 VI. Control...... 262 VII. Purification...... 262 VIII. Host Range ...... 263 IX. Strains ...... 263 X. References...... 263 xvi CONTENTS

Chapter 13 Coconut Cadang-Cadang fohn W. Randles I. Introduction ...... 265 II. History...... 265 III. Distribution ...... 267 IV. Symptoms and Host Range ...... 268 V. Epidemiology ...... 269 VI. Economic Losses...... 269 VII. Diagnosis...... 270 VIII. Strains ...... 271 IX. Experimental Transmission ...... 271 X. Purification...... 272 XI. Physical Properties...... 272 XII. Nucleotide Sequence...... 274 XIII. Relationship between Viroid Structure and Disease Progress 274 XIV. References ...... 275

Chapter 14 Hop Stunt Eishiro Shikata I. Disease Occurrence ...... 279 II. Symptoms and Damage to Hop Plants ...... 280 III. Host Range and Symptomatology...... 281 IV. Transmission and Distribution...... 283 V. Etiology and Purification ...... ,... 284 VI. Structure and Function ...... 285 VII. Cucumber and Grapevine Isolates ...... 285 VIII. Diagnosis and Control...... 287 IX. References ...... 288

Chapter 15 Chrysanthemum Chlorotic Mottle R. Kenneth Horst I. Introduction...... 291 II. Symptomatology...... 292 III. Etiology...... 293 IV. Physical Characteristics ...... 293 CONTENTS xvii

V. Control...... 294 VI. References...... 294

Chapter 16 Columnea Latent ...... 297 T. O. Diener

Chapter 17 Avocado Sunblotch Paul R. Desjardins I. Introduction ...... 299 II. Geographic Distribution and Economic Importance...... 300 III. Host Range ...... 300 IV. Symptomatology...... 300 A. Symptomatic Trees...... 300 B. Symptomless Carrier Trees...... 302 C. Effect of Light and Temperature ...... 302 V. Strains ...... 303 VI. Recovery Phenomenon ...... 303 A. Development of Symptomless Condition...... 303 B. Consequences of the Symptomless Carrier Condition ... 304 VII. Transmission ...... 304 A. Graft Transmission ...... 304 B. Mechanical Transmission ...... 304 C. Seed Transmission...... 305 D. Pollen Transmission...... 305 VIII. Mixed Infections with Viruses ...... 305 A. Viruses Involved...... 305 B. Problems for ASBV Research ...... 305 IX. Isolation and Characterization ...... 306 A. Methods of Isolation and Purification ...... 306 B. Multiple RNA Species ...... 307 C. Nucleotide Sequence ...... 307 X. Ultrastructural Cytology...... 307 XI. Detection and Control...... 309 A. Diagnosis...... 309 B. Viroid Inactivation on Tools...... 310 C. Use of Symptomless Trees ...... 310 XII. Discussion...... 310 XIII. References...... 311 xviii CONTENTS

Chapter 18 Tomato Plattta Macho Jorge Galindo A. I. HistoricaL...... 315 II. Geographic Distribution ...... 315 III. Symptomatology ...... 316 IV. Epidemiology ...... 316 V. Economic Losses ...... 318 VI. Diagnosis ...... I • • • • • • • • • • • • • • • • • • • • • • • • • • •• 318 VII. Strains ...... 318 VIII. Experimental Transmission...... 319 IX. Purification ...... 319 X. Physical Properties...... 319 XI. Nucleotide Sequence...... 319 XII. References...... 320

Chapter 19 Tomato Apical Stunt B. Walter I. Historicalj Geographic Distribution ...... 321 II. Symptomatology, Epidemiology, and Strains ...... 322 III. Experimental Transmission ...... ; ...... 323 A. Mechanical Inoculation ...... 323 B. Aphid Transmission ...... 324 IV. Purification...... 325 A. Polyethylene Glycol Method " ...... 325 B. Rapid Method...... 325 V. Physical-Chemical Properties...... 326 VI. Nucleotide Sequence ... ; ...... 326 VII. References...... 326

Chapter 20 Tomato Bunchy Top T. O. Diener I. Symptomatology...... 329 II. Host Range ...... 330 III. Strains ...... 330 IV. References ...... 331 CONTENTS xix

Chapter 21 Burdock Stunt Tien Po and Chen Wei I. Historical...... 333 II. Geographic Distribution ...... 333 III. Symptomatology...... 333 IV. Transmission ...... 334 V. Viroid Etiology...... 335 VI. Purification...... 335 VII. Some Physical Properties of BSV ...... 335 VIII. The Occurrence of BSV RNA-l and RNA-2 in Separate Plants ...... 337 IX. Ultrastructure of Diseased Leaf Tissues ...... 338 X. Propagation of BSV RNA-l in Tissue Culture ...... 338 XI. Conclusions ...... 338 XII. References...... 339

Index ...... 341 Viroid Abbreviations

ASBV Avocado sunblotch viroid BSV Burdock stunt viroid CCCV Coconut cadang-cadang viroid CCMV Chrysanthemum chlorotic mottle viroid CEV Citrus exocortis viroid CLV Columnea latent viroid CPFV Cucumber pale fruit viroid CSV Chrysanthemum stunt viroid HSV Hop stunt viroid PSTV Potato spindle tuber viroid TASV Tomato apical stunt viroid TBTV Tomato bunchy top viroid TPMV Tomato planta macho viroid

xxi