Fundamentals of Viroid Biology

Fundamentals of Viroid Biology provides a comprehensive introduction to emerging research on viroids and their biology. The book is organized into three parts, with sections that discuss historical perspectives as well as information on classifications, structure, life cycle and replication and viroid movement. The book goes on to discuss viroid diseases and their geographic distributions across Asia, Europe, Africa, Australia, North and South America. Viroid pathogenesis and viroid-host interaction rounds out the coverage which includes viroid associated disease symptoms and viroid regions associated and viroid-host protein and translations. Detection and disease control strategies are also covered.

Contributed by an international group of renown contributors in viroid research, this book is a useful introductory reference to advanced undergraduates and graduate and postgraduate students. It is also ideal for early career researchers and scientists engaged in the study of viroid biology, virology, plant virology and microbiology.

• Covers introductory to advanced level of information in viroid research
• Supported by relevant flow chart, figures and graphics to enhance understanding of the concepts and protocols
• Offers contributions by an international array of experts who are authorities in viroid research

• Language: English
• Publisher: Academic Press
• Release date: Nov 16, 2023
• ISBN: 9780323996891

Book preview

9780323996891_FC

Fundamentals of Viroid Biology

First Edition

Charith Raj Adkar-Purushothama

RNA Group, Department of Biochemistry and Functional Genomics, Faculty of Medicine and Health Sciences, Applied Cancer Research Pavilion, University of Sherbrooke, Sherbrooke, QC, Canada

Teruo Sano

Faculty of Agriculture and Life Science, Hirosaki University, Hirosaki, Japan

Jean-Pierre Perreault

RNA Group, Department of Biochemistry and Functional Genomics, Faculty of Medicine and Health Sciences, Applied Cancer Research Pavilion, University of Sherbrooke, Sherbrooke, QC, Canada

Sreenivasa Marikunte Yanjarappa

Department of Studies in Microbiology, University of Mysore, Mysuru, India

Francesco Di Serio

Consiglio Nazionale delle Ricerche (CNR), Istituto per la Protezione Sostenibile delle Piante (IPSP), Bari, Italy

José-Antonio Daròs

Instituto de Biología Molecular y Celular de Plantas (Consejo Superior de Investigaciones Científicas-Universitat Politècnica de València), Valencia, Spain

Image 1

Table of Contents

Cover image

Title page

Copyright

Dedication

Contributors

Foreword

Preface

Introduction

Section A: Introduction

Chapter 1 Milestones in viroid research

Abstract

Graphical representation

Acknowledgments

Definitions

Learning objectives

Fundamental introduction

Viroid conformation(s)

Replication

Pathogenicity

Movement

Origin/evolution

Epidemiology/control

Questions for the reader

Further reading

References

Chapter 2 Viroid taxonomy

Graphical representation

Abstract

Definitions

Chapter outline

Learning objectives

Fundamental introduction

History of viroid classification

Demarcation criteria of viroid families

Demarcation criteria of viroid genera

Species demarcation criteria

Current viroid classification

Protocols/procedures/methods

Assigning a name to novel viroid species

Prospective and future implications

Chapter summary

Study question

References

Chapter 3 Structure of viroids

Abstract

Graphical representation

Acknowledgments

Definitions

Chapter outline

Learning objectives

Fundamental introduction

Avsunviroidae

Pospiviroidae

Perspective

Questions for the reader

References

Chapter 4 Replication and movement of viroids in host plants

Abstract

Graphical representation

Definitions

Chapter outline

Learning objectives

Fundamental introduction

Viroid replication occurs through a rolling circle mechanism

Replication of nuclear viroids

Replication of chloroplastic viroids

How viroid RNAs reach the organelles where they replicate

Intercellular and vascular movement

Prospective and future implications

Chapter summary

Study question

References

Section B: Prospecting for viroid

Chapter 5 Viroids diseases and its distribution in Asia

Graphical representation

Abstract

Definitions

Chapter outline

Learning objectives

Fundamental introduction

Viroid diseases of vegetable crops

Viroids infecting ornamental plants

Viroids infecting pome fruits

Viroids infecting avocado

Viroids infecting citrus

Viroid infecting coconut palm

Viroids infecting grapevine

Viroids of hop

Viroids infecting other fruit trees and weeds

Prospective

Future implications

Chapter summary

Study questions

Further reading

References

Chapter 6 Viroid-associated plant diseases in Europe

Abstract

Graphical representation

Definitions

Chapter outline

Learning objectives

Fundamental introduction

Viroids of herbaceous plants

Viroids of specialized crops

Viroids of woody plants

Procedures

Prospective

Future implications

Chapter summary

Study questions

Further reading

References

Chapter 7 Naturally occurring viroid diseases of economically important plants in Africa

Abstract

Graphical representation

Acknowledgment

Definitions

Chapter outline

Learning objectives

Introduction

Occurrence of Avsunviroidae members in fruits trees in Africa

Occurrence of Pospiviroidae members in fruits trees in Africa

Occurrence of viroids in herbaceous plants

Procedure

Prospective

Future implications

Chapter summary

Study questions

Further reading

References

Chapter 8 Finding the coconut cadang-cadang and tinangaja viroids, naturally occurring pathogens of tropical monocotyledons of Oceania

Graphical representation

Abstract

Acknowledgments

Definitions

Chapter outline

Learning objectives

Fundamental introduction

A short history of cadang-cadang disease

Discovery of coconut cadang-cadang viroid in the Philippines

CCCVd beyond the Philippines

Prospective: Are there viroid sequences in progenitors of the palms?

Chapter summary

Study question

Further reading

References

Chapter 9 Viroids and their distribution in North America

Graphical representation

Abstract

Definitions

Chapter outline

Learning objectives

Introduction

Viroids of solanaceous plants

Viroids of ornamentals

Viroids of Humulus plants

Viroids of fruit trees

Viroids in pome and stone fruit trees

Viroids in citrus

Viroids in grapevine

Chapter summary

Study questions

Further reading

References

Chapter 10 Viroid-associated plant diseases in South America

Graphical representation

Abstract

Definitions

Chapter outline

Learning objectives

Fundamental introduction

Herbaceous ornamentals

Horticultural plants

Fruit trees and grapevine

Prospective

Future implications

Chapter summary

Study questions

Further reading

References

Section C: Viroid pathogenesis and viroid-host interaction

Chapter 11 Viroid pathogenicity

Abstract

Graphical representation

Definitions

Chapter outline

Learning objectives

Introduction

Viroid-associated symptoms in host plants

Structural motifs and nucleotides modulating viroid pathogenicity

Role of the host components in viroid pathogenicity

Viroid-induced biochemical changes

Effect of viroids on host gene expression and the host response to viroid infection

Role of gene silencing in viroid pathogenesis

Prospective

Future implications

Chapter summary

Protocols/procedures/methods

Study questions

Further reading

References

Chapter 12 Viroids and protein translation

Abstract

Graphical representation

Definitions

Chapter outline

Learning objectives

Fundamental introduction

Non-coding nature of viroids

Changes in protein expression associated with viroid infection

Interaction of viroids with host proteins

Alterations in the translational machinery caused by viroids

Revisiting viroid translation capacity: are negative results conclusive?

Chapter summary

Study questions

Further reading

References

Chapter 13 Viroid infection and host epigenetic alterations

Graphical representation

Abstract

Definitions

Chapter outline

Learning objectives

Fundamental introduction

Discovery of RNA directed DNA methylation

Viroid infection and host epigenetic changes

Potential ways of promoting viroid-associated host epigenetic changes

Remarks and future perspectives

Simplified strategy to analyze epigenetic changes induced by viroid infection*

Study questions

Further reading

References

Chapter 14 Transcriptomic analyses provide insights into plant-viroid interactions

Graphical representation

Abstract

Acknowledgment

Chapter outline

Learning objectives

Fundamental introduction

Transcriptomic studies of viroid-infected samples

Gene expression profiles underlying plant-viroid interactions

Methods for transcriptomic analyses

Prospective

Future implications

Chapter summary

Study questions

Further reading

References

Chapter 15 Viroid-induced RNA silencing and its secondary effect on the host transcriptome

Graphical representation

Abstract

Definitions

Chapter outline

Learning objectives

Introduction

RNA silencing

Viroid induced RNA silencing

Direct effect of viroid derived small RNAs on the host transcriptome

Generation of secondary siRNAs triggered by viroid infection and their global effect on both the transcriptome and the phenotype—A working hypothesis

Protocols/procedures/methods

Perspective

Future implications

Chapter summary

Study questions

Further reading

References

Chapter 16 Detection of viroids

Abstract

Graphical representation

Definitions

Chapter outline

Learning objectives

Fundamental introduction

Sample collection and preparation

Detection methods

Method selection

Prospective

Chapter summary

Study questions

Further reading

References

Chapter 17 Viroid disease control and strategies

Abstract

Graphical representation

Definitions

Chapter outline

Learning objectives

Fundamental introduction

Viroid replication and spread—Control of viroid diseases

Conventional methods of control

Transgenic methods of control

Perspective/future implications

Questions for the reader

References

Chapter 18 Policies, regulations, and production of viroid-free propagative plant materials for sustainable agriculture

Graphical representation

Abstract

Definitions

Chapter outline

Learning objectives

Fundamental introduction

Chapter

Protocols/procedures/methods

Prospective

Future implications

Chapter summary

Study questions

References

Chapter 19 Bioinformatic approaches for the identification and discovery of viroid-like genomes

Abstract

Graphical representation

Definitions

Chapter outline

Learning objectives

Introduction

The discovery of viroid-like RNAs

Initial approaches to identify viroid-like RNAs through bioinformatics

De novo discovery of viroid-like genomes

Perspective

Study questions

Further reading

References

Chapter 20 Contributions of viroid research to methods for RNA purification, diagnostics, and secondary structure prediction

Abstract

Graphical abstract

Definitions

Chapter outline

Learning objectives

Fundamental introduction

Chapter

Protocols/procedures/methods

Future implications

Study questions

Further reading

References

Chapter 21 Future perspectives in viroid research

Abstract

Graphical representation

Acknowledgments

Chapter outline

Learning objectives

Fundamental introduction

Do viroids infect hosts other than flowering plants?

How do viroid RNAs survive in the hostile environment of an infected cell?

Do viroids contain auto-catalytic motifs other than hammerhead?

May viroids still encode proteins?

Are viroids useful for biotechnology?

Questions for the reader

References

Index

Copyright

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Dedication

Francesco Di Serio; José-Antonio Daròs; Vicente Pallas

Unlabelled Image

The book Fundamentals of Viroid Biology is dedicated to the memory of Prof. Ricardo Flores, who passed away on December 20, 2020. During his outstanding scientific career, Ricardo contributed enormously to the advancement of knowledge on these small, nonprotein-coding RNAs that infect plants. After graduating in both agricultural sciences (Polytechnic University of Valencia) and chemistry (University of Valencia), Ricardo carried out a doctoral thesis in plant virology, developing and applying new biochemical approaches to characterize a virus causing severe diseases in citrus called citrus tristeza virus. Then, he decided to use his strong background in agronomy and biochemistry to study viroids. After a postdoctoral period spent in the laboratory of Prof. Joseph S. Semancik (Riverside, California), where Ricardo worked on the citrus exocortis viroid, he was back to his city, Valencia, where he established his own laboratory. Ricardo focused his research activity on many aspects related to viroids, including their subcellular localization, replication, self-cleaving activity, structure, pathogenesis, detection, and evolution, always generating outstanding results that contributed enormously to the current knowledge of biological, structural, and functional features of viroids. In recent years, Ricardo focused on the role of RNA silencing in plant-viroid interaction, contributing to showing the role of this sequence-specific degradation mechanism in both plant antiviroid defense and in viroid pathogenesis.

Ricardo was a fantastic teacher. In his laboratory, many researchers of different nationalities, currently affirmed scientists, had the opportunity of being formed. Ricardo used to remind his young students that what we write in science is the part of our efforts to increase knowledge that actually remains and is transferred to society. Therefore, time and energy must be dedicated to correctly design and perform the experiments, but also to communicate our working hypotheses and results. In moments of discouragement accompanying the failure of some experiments, Ricardo used to remind his students that doing science is like running a marathon, not a sprint. With this metaphor, he wanted to underline the need to proceed by successive steps, including all possible controls, to identify potential technical errors and consequently to correct them, or to conclude that the starting hypothesis was wrong. In fact, a well-designed experiment, although it requires time and effort, allows us to obtain clear and conclusive results, which help science to progress more rapidly. But, above all, Ricardo leaves to his students and to all who reads his publications a brilliant example of how to formulate fascinating scientific hypotheses and design experiments capable of validating or denying them.

The deep passion for science, cultivated with rigor, competence, and seriousness, already evident in all Ricardo’s writings, was captured even better by the audience during his seminars and lectures. People like us who were lucky enough to chat with him outside the laboratory, at dinner, at lunch, or walking through Valencia, will also remember his astonishing culture, especially in history, and his fantastic ability to transfer his knowledge. A generous and helpful friend, Ricardo placed the progress of knowledge as the inspiration of his life and followed this path with constancy and dedication until the end of his days. Ricardo received important distinctions, although he never boasted of any of them, including being an honorary member of the Hungarian Academy of Sciences, Vice President of the Spanish Society of Virology and recipient of its biannual distinction, and the Plaque of Honor from the Spanish Association of Scientists. However, without a doubt, his best prize was enjoying a model family that loved and admired him and of which he felt very proud. His wife Marita is an excellent chemistry teacher, his daughter María is a magnificent architect, and his son Ricky is an associate professor of economics.

Under the kind suggestion of the editors, many of his colleagues were delighted to participate in this book, inspired by the idea of dedicating it to Ricardo’s memory. Although this provides only minimal relief to the great loss caused by his sudden death, it reassures us to know that many of Ricardo’s discoveries, collected in this book as a relevant part of the current knowledge on viroids, will be preserved as an example of how to generate new and exciting scientific novelties by the young generation of researchers.

Contributors

Charith Raj Adkar-Purushothama     RNA Group, Department of Biochemistry and Functional Genomics, Faculty of Medicine and Health Sciences, Applied Cancer Research Pavilion, University of Sherbrooke, Sherbrooke, QC, Canada

Irene Bardani     Department of Biology, University of Crete, Heraklion, Greece

M. Francisca Beltrán     University of Chile, Faculty of Agricultural Sciences, Department of Plant Health, Santiago, Chile

François Bolduc     RNA Group, Department of Biochemistry and Functional Genomics, Faculty of Medicine and Health Sciences, Applied Cancer Research Pavilion, University of Sherbrooke, Sherbrooke, QC, Canada

Michela Chiumenti     Consiglio Nazionale delle Ricerche (CNR), Istituto per la Protezione Sostenibile delle Piante (IPSP), Bari, Italy

C.A. Cueto     Philippine Coconut Authority-Albay Research Center, Guinobatan, Albay, Philippines

José-Antonio Daròs     Instituto de Biología Molecular y Celular de Plantas (Consejo Superior de Investigaciones Científicas-Universitat Politècnica de València), Valencia, Spain

Marcos de la Peña     Institute of Molecular and Cellular Biology of Plants, Politechnic University of Valencia-CSIC, Valencia, Spain

Francesco Di Serio     Consiglio Nazionale delle Ricerche (CNR), Istituto per la Protezione Sostenibile delle Piante (IPSP), Bari, Italy

Amine Elleuch     Laboratoire de Biotechnologie Végétale Appliquée à l’amélioration des plantes, Faculté des Sciences de Sfax, Université de Sfax, Sfax, Tunisia

Nicola Fiore     University of Chile, Faculty of Agricultural Sciences, Department of Plant Health, Santiago, Chile

Gustavo Gómez     Institute for Integrative Systems Biology (I2SysBio), Consejo Superior de Investigaciones Científicas (CSIC)—Universitat de València (UV), Paterna, Spain

Imen Hamdi     Laboratoire de Protection des Végétaux, Institut National de la Recherche Agronomique de Tunis, Université de Carthage, Tunis, Tunisia

Rosemarie W. Hammond     USDA ARS Molecular Plant Pathology Laboratory, Beltsville, MD, United States

D. Hanold     School of Agriculture, Food and Wine, The University of Adelaide, Adelaide, SA, Australia

Scott Harper     Clean Plant Center Northwest, Washington State University, Prosser, WA, United States

Y. Iftikhar     Department of Plant Pathology, College of Agriculture, University of Sargodha, Sargodha, Pakistan

Jernej Jakše     Department of Agronomy, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia

Kriton Kalantidis

Department of Biology, University of Crete

Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology, Heraklion, Greece

Robert Krueger     USDA-ARS National Clonal Germplasm Repository for Citrus and Dates, Riverside, CA, United States

Nikoleta Kryovrysanaki     Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology, Heraklion, Greece

Irene Lavagi-Craddock     Citrus Clonal Protection Program and the University of California, Riverside, CA, United States

Shifang Li     Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China

Purificación Lisón     Institute for Plant Molecular and Cellular Biology, Universitat Politècnica de València-Consejo Superior de Investigaciones Científicas, Valencia, Spain

Maria José López-Galiano

Institute of Molecular and Cellular Biology of Plants, Politechnic University of Valencia-CSIC

Department of Genetics, University of Valencia, Burjassot, Valencia, Spain

Joan Marquez-Molins

Institute for Integrative Systems Biology (I2SysBio), Consejo Superior de Investigaciones Científicas (CSIC)—Universitat de València (UV), Paterna, Spain

Department of Plant Biology, Uppsala BioCenter, Swedish University of Agricultural Sciences and Linnean Center for Plant Biology, Uppsala, Sweden

German Martinez     Department of Plant Biology, Uppsala BioCenter, Swedish University of Agricultural Sciences and Linnean Center for Plant Biology, Uppsala, Sweden

Jaroslav Matoušek     Biology Centre of the Czech Academy of Sciences, Department of Molecular Genetics, Institute of Plant Molecular Biology, České Budějovice, Czech Republic

Beatriz Navarro     Institute for Sustainable Plant Protection, National Research Council, Bari, Italy

Xianzhou Nie     Fredericton Research and Development Centre, Agriculture and Agri-Food Canada, Fredericton, NB, Canada

Robert A. Owens     Molecular Plant Pathology Laboratory, Beltsville Agricultural Research Center (retired), Beltsville, MD, United States

Vicente Pallás     Instituto de Biología Molecular y Celular de Plantas (IBMCP), Consejo Superior de Investigaciones Científicas (CSIC)—Universitat Politècnica de València (UPV), Valencia, Spain

Jean-Pierre Perreault     RNA Group, Department of Biochemistry and Functional Genomics, Faculty of Medicine and Health Sciences, Applied Cancer Research Pavilion, University of Sherbrooke, Sherbrooke, QC, Canada

Paulina Quijia-Lamiña     Citrus Clonal Protection Program and the University of California, Riverside, CA, United States

J.W. Randles     School of Agriculture, Food and Wine, The University of Adelaide, Adelaide, SA, Australia

Detlev Riesner     Institut für Physikalische Biologie, Heinrich Heine University Düsseldorf, Universitätsstraße 1, Düsseldorf, Germany

Ismael Rodrigo     Institute for Plant Molecular and Cellular Biology, Universitat Politècnica de València-Consejo Superior de Investigaciones Científicas, Valencia, Spain

Teruo Sano     Faculty of Agriculture and Life Science, Hirosaki University, Hirosaki, Japan

B. Shruthi     Department of Studies in Microbiology, University of Mysore, Mysuru, India

Dijana Škorić     University of Zagreb, Faculty of Science, Department of Biology, Zagreb, Croatia

Gerhard Steger     Institut für Physikalische Biologie, Heinrich Heine University Düsseldorf, Universitätsstraße 1, Düsseldorf, Germany

S.S. Thanarajoo     CABI South East Asia, Serdang, Selangor, Malaysia

G. Vadamalai     Department of Plant Protection, Faculty of Agriculture, Institute of Plantation Studies, Universiti Putra Malaysia, Serdang, Selangor, Malaysia

Francisco Vázquez-Prol     Institute for Plant Molecular and Cellular Biology, Universitat Politècnica de València-Consejo Superior de Investigaciones Científicas, Valencia, Spain

Georgios Vidalakis     Citrus Clonal Protection Program and the University of California, Riverside, CA, United States

Ying Wang     Plant Pathology Department, University of Florida, Gainesville, FL, United States

Sreenivasa Marikunte Yanjarappa     Department of Studies in Microbiology, University of Mysore, Mysuru, India

Zhixiang Zhang     Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China

Foreword

An oft-cited quotation from physicist Erwin Schrodinger reminds scientists that The task is not to see what has never been seen before, but to think what has never been thought before about what you see everyday. Diseases associated with potato spindle tuber and citrus exocortis viroids were long believed to be caused by conventional plant viruses, and it was only the creative application of then state-of-the-art technology that, in 1971, identified the true nature of their causal agents. The ability of these small, circular, noncoding RNA molecules to replicate autonomously in susceptible plant hosts immediately focused attention on their biological, biochemical, and evolutionary significance.

Beginning in 1979 with T.O. Diener’s Viroids and Viroid Diseases, a series of monographs have documented advances in our understanding of how viroids replicate, move from cell to cell, and induce disease. This publication is the first book in the field designed to be used as both course material for students and a reference manual for researchers and the scientific community. Each chapter starts with a graphical summary of the chapter followed by an introduction and main content. To help researchers and students, every chapter contains an experimental design and study questions. The prospective and future implications sections in each chapter provide readers with an idea of the research questions that need to be addressed and the corresponding effects or consequences. All chapters end with a Further reading section that references sources the author has deemed valuable to readers seeking additional information or context about the research problem.

My own introduction to viroid research, in contrast, owed much to serendipity. As a graduate student in early 1972, I happened to pick up the issue of Virology in which T.O. Diener reported the discovery of potato spindle tuber viroid. The significance of this discovery was immediately apparent, and little more than 3 years later, I found myself in Beltsville working with Dr. Diener just as viroid research began to expand rapidly. New thoughts were seemingly everywhere in those early days of viroid research. For those now entering the field at a time when the knowledge base is so much larger and the experimental tools so much more powerful, this book will be particularly helpful in choosing the most promising questions to investigate.

As this volume was going to press, I received word that Dr. Diener had recently passed away at the age of 102. Ted was a truly remarkable scientist whose many achievements are widely recognized. Those not fortunate enough to have met him or heard him speak need only read his 1971 description of the discovery of potato spindle tuber viroid to appreciate the breadth of knowledge and creative vision that he brought to viroid research.

R.A. Owens, (Retired), Molecular Plant Pathology Laboratory, Beltsville Agricultural Research Center, Beltsville, MD, United States

Preface

Theodor O. Diener, in the year 1971, coined the term viroid when he identified the causal agent of potato spindle tuber disease, the smallest RNA replicon with links between living and nonliving. Since the discovery of the first viroid, many researchers, including master’s and PhD students, across the globe have contributed to the field of viroid biology, including viroid diseases, classification, pathogenicity, interaction with hosts, crop protection, biotechnology, and molecular evolution. However, most of this research has been and is being performed in a few laboratories, indicating viroid research has not yet achieved a worldwide reach. When we started to think more and more about this problem, we realized we had failed to attract young researchers to the field of viroids. Recent observations of the presence of long circular nonprotein-coding RNAs in mammalian cells and their role in modulating microRNAs, as well as the identification of viroid-like RNAs in fungi, may further widen the scope of the viroid host range. At this point, we thought of bringing out a student-friendly handbook that presents the fundamentals of viroid biology. Hence, we contacted the researchers who have made substantial contributions to the field of viroids and requested their contribution to their favorite topic. Therefore, as you read this book, you will understand that each chapter is written by authors who have dedicated themselves to viroid research for many years. We are wholeheartedly thankful to all the contributors for their patience, cooperation, and collaboration and for sharing their vast and excellent scientific expertise on viroids, especially in the midst of the uncertainty surrounding the SARS-CoV-2/COVID-19 pandemic. We are also indebted to the Elsevier staff for supporting the publication of this book. We hope this book serves as a one-stop resource for researchers, teachers, and students who want to know more about the biology of viroids and nonprotein-coding RNAs.

Charith Raj Adkar-Purushothama

Teruo Sano

Jean-Pierre Perreault

Sreenivasa Marikunte Yanjarappa

Francesco Di Serio

José-Antonio Daròs

Introduction

John W. Randles, School of Agriculture, Food and Wine, The University of Adelaide, SA, Australia

A new entrant into the field of viroidology might be a scientist reading a research paper describes a new viroid. The host plant, infectivity, and pathogenicity of the viroid; its primary and secondary structure; and its classification based on a comparison of its nucleotide sequence with known viroids may be described. Each of these aspects is often a complex undertaking, and the authors of the chapters in this comprehensive book have been asked to explain their topic in sufficient detail for newcomers to understand the history, recent advances in this field, and the background to current thinking on the evolution of these autonomously replicating RNA molecules.

In his Foreword to Viroids and Satellites (2017), Theodor O. Diener stated that the discovery of viroids in 1971 ushered in the third major extension in the history of the biosphere. His pioneering experiments shocked both virologists and molecular biologists. The science of virology had developed from the discovery in the 1890s that some diseases could be induced by agents smaller than bacteria. Viruses followed the central dogma of the flow of genetic information, competing with and parasitizing the host’s transcription and translation processes for virus-directed replication. The ability of naked small RNA without a translatable genome to induce disease proved the existence of a subviral world where the host’s RNA metabolism was parasitized for viroid replication. More than 30 viroids have been shown to cause plant diseases that previously were of unknown etiology. Although the causal prions of animal subacute spongiform encephalopathies have also been described as subviral pathogens, there are no similarities in the pathologies or biologies of the viroids and prions.

Viroids concern both the agricultural and the biochemical sciences. Plant pathology originated from the need to control major epidemics of plant disease, even famine, due to any of a wide range of microscopic plant parasites. While pathologists take the holistic view that plant disease results from interactions between a pathogen, the host, the environment, and mode of spread, diagnosis of the cause is the first requirement for achieving control. For plant virus research, the rules of causation attributed to Henle, Koch, and Loeffler in the 19th century for human diseases have been adapted to include symptomatology, host range, and bioassays as the first steps in characterizing unknown viruses by various differential centrifugation techniques and electron microscopy. When the spindle tuber disease of the potato was subjected to standard virus purification procedures without any virions being found, it was thought in 1967 that a naked virus nucleic acid could be the cause. It was Diener’s skills in basic plant virus and nucleic acid research that led to his first publication recognizing that the pathogen was a small unencapsidated autonomously replicating circular RNA, the potato spindle tuber viroid (PSTVd). His dedicated research uncovered a number of biological and morphological properties of this viroid. It was the first viroid to be sequenced by molecular biologists in Germany, opening up an era of structure-function analyses related to biology and viroid classification.

Diener’s discovery of the cause of the economically important potato spindle tuber disease led rapidly to the identification of the causes of the economically damaging citrus exocortis, coconut cadang-cadang, hop stunt, and avocado sunblotch diseases. Viroid diseases are now known to affect orchards, plantations, greenhouses, and plant nurseries around the world, and advanced molecular diagnostic methods are used for the application of quarantine procedures to international plant germplasm movement. Little is known about the distribution of viroids in natural ecosystems, including those which have provided germplasm for commercial plantings, but the latest molecular diagnostic methods have the potential to find viroids and viroid-like molecules even where they do not cause overt disease. The signatures of viroids are now being found in some species without symptoms of disease. Relationships between nucleotide sequence variation and disease severity are being explored in attempts to discover how viroids interact with the host. Due to the high mutability of viroids and the accumulation of quasi-species during infection, populations of variants change during the infection cycle. High-throughput sequencing is the most recent tool for studying viroid population dynamics.

This volume emphasizes the biology of viroids, including their history, taxonomy, structure, replication, global distribution, host-viroid interactions, perceptions of their origins, and how they may have evolved. Future directions for viroid research are suggested, showing that a multitude of questions are yet to be addressed in the field and laboratory. Readers are introduced to a team of authors representing many of the laboratories that have collaborated in viroid research over the last five decades.

Section A

Introduction

Chapter 1 Milestones in viroid research

Robert A. Owens    Molecular Plant Pathology Laboratory, Beltsville Agricultural Research Center (retired), Beltsville, MD, United States

Abstract

When announcing the discovery of the first viroid in 1971 T.O. Diener suggested that the spindle tuber disease caused by potato spindle tuber viroid (PSTVd) might reflect its ability to act as an abnormal regulatory RNA. Fifty years later, much evidence has accumulated to support this hypothesis. This historical overview briefly describes the most significant findings, grouping them according to the research area. PSTVd was the first pathogen infecting a eukaryotic host to have its genome completely sequenced. Later advances include demonstrations of (i) the importance of viroid tertiary structure for replication, (ii) the role of RNA silencing in both pathogenicity and replication, and (iii) the role of specific sequence motifs in regulating intracellular viroid movement as well as movement across specific tissue boundaries. The possibility that viroids originated in the precellular RNA world remains a viable hypothesis, and new information about all aspects of viroid-host interaction continues to accumulate.

Graphical representation

Unlabelled Image

Viroid research milestones. For clarity, major advances in different areas of research (indicated by vertical arrows) have been arranged along separate timelines. Dates for the first reports of selected viroid diseases and identification of representative members of the eight currently recognized viroid genera are indicated at the top of the figure.

Keywords

Abnormal regulatory RNA; Disease induction; Molecular signals; Systemic infection; Rolling circle replication; RNA world; Indicator host; Molecular diagnostics

Abbreviations

HDV hepatitis delta virus

HP I secondary hairpin I

HP II secondary hairpin II

PFOR progressive filtering of overlapping small RNAs

RT-PCR reverse transcription polymerase chain reaction

SHAPE selective 2′-hydroxyl acylation analyzed by primer extension

Viroid species

ADFVd apple dimple fruit viroid

ASBVd avocado sunblotch viroid

ASSVd apple scar skin viroid

CbVd coleus blumei viroid

CCCVd coconut cadang-cadang viroid

CChMVd chrysanthemum chlorotic mottle viroid

CDVd citrus dwarfing viroid

CEVd citrus exocortis viroid

CLVd columnea latent viroid

ELVd eggplant latent viroid

GHVd grapevine hammerhead viroid

HSVd hop stunt viroid

PLMVd peach latent mosaic viroid

PSTVd potato spindle tuber viroid

Pospiviroid domains

C central domain contains CCR (central conserved region)

P pathogenicity domain contains VM (virulence-modulating) region

TL terminal left

TR terminal right

V variable domain

Acknowledgments

I thank the editors for this opportunity to revisit many memorable events from a career in viroid research. My first exposure to viroids came about by chance one evening when, as a graduate student at University of California, Davis, I picked up a recent issue of Virology in the departmental library. As I began to scan the article by T.O. Diener announcing his discovery of potato spindle tuber viroid, its significance was immediately obvious. Three years later I found myself at Beltsville working with Dr. Diener, and the ensuing 35 years saw a series of deeply satisfying collaborations with many other members of the viroid research community including Biao Ding, Luis Salazar, Teruo Sano, and Gerhard Steger.

Definitions

Agroinoculation: introduction of a potentially infectious viroid or viral cDNA into plant cells via the Ti plasmid of A. tumefaciens.

Cross protection: process in which exposure to a mild virus or viroid isolates induces resistance against a closely related, more virulent isolate.

Dot blot hybridization: a diagnostic technique in which total RNA extracted from the plant to be tested is spotted on a nylon or nitrocellulose membrane and then hybridized with a viroid-specific RNA or DNA probe.

Indicator host: plants specifically chosen for experimental studies because they either support high levels of viroid replication or exhibit strong symptoms.

RNA structure

Primary: linear sequence of ribonucleotides (A, C, G, and U) linked by phosphodiester bonds.

Secondary: RNA molecules contain both single- and double-stranded regions. Base pairing between complementary regions creates helices, bulged nucleotides, internal loops, and junctions.

Tertiary: three-dimensional arrangement of helical duplexes and other secondary structural components that is stabilized by long-range interactions.

Isosteric base pairs: base pairs in which the relative positions and distances between the two C1′ carbon atoms are very similar. Watson-Crick pairs are only one of 12 different families of base pairs containing at least two H bonds between atoms located on the three edges of nucleotide bases.

Loop E motif: type of internal loop first identified in 5S rRNA that contains several non-Watson-Crick base pairs.

Tetraloops: hairpin loops containing four unpaired nucleotides that cap many RNA hélices.

Ribozymes: RNA molecules able to catalyze specific biochemical reactions (e.g., self-cleavage) similar to the action of protein enzymes.

Rolling circle replication: the process of unidirectional nucleic acid replication that can rapidly synthesize multiple copies of a circular RNA or DNA molecule.

Temperature gradient gel electrophoresis: a specialized form of polyacrylamide gel electrophoresis used to study specific structural features of nucleic acids or nucleic acid-protein complexes.

Viroid chimera: viroid genomes portions of which are similar/identical to sequences in two or more other viroids.

Learning objectives

•Objective 1: Understand the fundamental differences between viroids and plant RNA viruses

•Objective 2: Understand how viroids have been used to examine the relationship between RNA structure and biological function(s)

•Objective 3: Understand the role of common agricultural practices such as vegetative propagation in the origin/spread of viroid diseases

Fundamental introduction

When announcing the discovery of the first viroid in 1971 T.O. Diener suggested that the spindle tuber disease caused by potato spindle tuber viroid (PSTVd) might reflect its ability to act as an abnormal regulatory RNA. Fifty years later, much evidence has accumulated to support this hypothesis. This historical overview briefly describes the most significant findings, grouping them according to the research area. PSTVd was the first pathogen infecting a eukaryotic host to have its genome completely sequenced. Later advances include demonstrations of (i) the importance of viroid tertiary structure for replication, (ii) the role of RNA silencing in both pathogenicity and replication, and (iii) the role of specific sequence motifs in regulating intracellular viroid movement as well as movement across specific tissue boundaries. The possibility that viroids originated in the precellular RNA world remains a viable hypothesis, and new information about all aspects of viroid-host interaction continues to accumulate.

Viruses (especially bacteriophages) played a crucial role in the development of modern molecular biology. For early plant molecular biologists the small RNA genomes of tobacco mosaic virus (TMV), brome mosaic virus (BMV), and cowpea mosaic virus (CPMV) provided tools to probe many aspects of plant gene expression. The 1971 discovery of potato spindle tuber viroid (PSTVd) by T.O. Diener revealed a more extreme form of parasitism and, therefore, an even more powerful tool with which to examine the biochemical capacities of their host cells.

Initial characterization of PSTVd (Diener, 1971; Singh and Clark, 1971) and CEVd (Semancik and Weathers, 1972) revealed that their genomes were approximately 10-fold smaller than those of the smallest known RNA viruses. Furthermore, the lack of a protective capsid frees their genomes from the need to encode the corresponding structural protein(s). In announcing the discovery of viroids Diener noted that The demonstration of a low molecular weight RNA that replicates in a variety of host species apparently uninfected by another agent entails a number of important implications for virology, molecular biology, and genetics. These implications include (i) the nature of the molecular signals allowing host enzymes to accept viroids as templates for replication, (ii) the possibility that such pathways might also be operative in uninfected cells, (iii) the mechanism(s) by which viroids induce disease in the infected host plant, (iv) the factors limiting viroids to higher plants, and (v) the question of viroid origin (Diener, 1987). Studies carried out over the past 50 years have addressed each of these questions.

Fig. 1 presents a timeline of viroid research milestones that begins with descriptions of the first two diseases later shown to be viroid-induced; namely, potato spindle tuber disease in the early 1920s (Schultz and Folsom, 1923) and citrus exocortis disease in the late 1940s (Fawcett and Klotz, 1948). At a time when sucrose density gradient centrifugation and polyacrylamide gel electrophoresis were the states of the art in plant virology neither potato nor citrus was particularly well-suited for molecular studies; thus, identification of more-amenable indicator hosts like tomato (PSTVd), Etrog citron, or Gynura aurantiaca (CEVd) in the 1960s played a key role in establishing the true nature of the causal agent. Shortly after the discoveries of PSTVd (Diener, 1971) and CEVd (Semancik and Weathers, 1972) several other diseases affecting vegetatively propagated crops like chrysanthemum and hops were also shown to be caused by viroids. More than 30 different species of viroids are now known (10th ICTV Report; https://talk.ictvonline.org/ictv-reports/ictv_online_report/subviral-agents/w/viroids), and a combination of modern high throughput RNA sequencing and bioinformatic analysis has revealed the presence of several novel viroids in asymptomatic plants (e.g., Wu et al., 2012).

Fig. 1

Fig. 1 Viroid research milestones. For clarity, major advances in different areas of research (indicated by vertical arrows ) have been arranged along separate timelines. Dates for the first reports of selected viroid diseases and identification of representative members of the eight currently recognized viroid genera are indicated at the top of the figure.

By the early 1980s viroid genomes were known to be covalently closed, circular molecules (the first such RNAs to be described), the complete nucleotide sequences of PSTVd and several other viroids had been determined, and physical-chemical studies exploring the unusual structural properties of PSTVd were underway. Studies from several laboratories had shown that viroids replicate via a rolling circle mechanism, and demonstration of the ability of a greater-than-full-length cDNA copy of PSTVd to initiate infection in tomato seedlings (Cress et al., 1983) allowed recombinant DNA technology to be applied to studies of viroid replication, movement within the host, and disease induction.

As the molecular details of these processes became better understood, viroids were seen to form two natural groupings (or taxonomic families). PSTVd, CEVd, and other members of the more numerous group have an unbranched, rod-like structure with several conserved sequence motifs and replicate in the nucleus; members of the second group which includes avocado sunblotch viroid (ASBVd) are structurally more diverse and replicate in the chloroplast. Importantly, multimeric forms of ASBVd of both polarities cleave spontaneously due to the presence of novel hammerhead ribozymes. In 1989 Diener proposed that viroids and viroid-like satellite RNAs may represent relics of precellular evolution with origins in the RNA world. Two years later additional support for this hypothesis was presented by Elena et al. (1991); namely, the results of the phylogenetic analysis which indicated a monophyletic origin for viroids, viroid-like satellite RNAs, and the viroid-like domain of hepatitis delta virus (HDV) RNA.

Even before the publication of the first complete viroid nucleotide sequence (Gross et al., 1978), RNA fingerprinting of selected PSTVd isolates had shown that small changes in sequence could lead to dramatic differences in symptom expression. In 1985 Sänger's laboratory proposed the first molecular model for viroid pathogenicity. Nucleotides within the so-called virulence modulating region of PSTVd were proposed to modulate the binding- and hence the competition potential of the genomic RNA for an unknown host factor(s). Genetic analysis of a series of novel viroid chimeras (Sano et al., 1992) revealed the presence of multiple (i.e., three) pathogenicity determinants. Following several later reports that viroid infection induces RNA silencing (e.g., Itaya et al., 2001) there was a pronounced shift in the focus of studies of viroid pathogenicity. Rather than continuing to search for direct interactions between viroid genomic RNAs and host protein(s) that could explain symptom induction, several groups began to look for possible indirect interactions between the viroid and host genomes via viroid small RNA-mediated RNA silencing.

Interest in the molecular details of viroid movement developed more slowly. An early study (Palukaitis, 1987) used dot-blot hybridization to demonstrate that PSTVd (like most plant viruses) moves rapidly from inoculated leaf (source) to actively growing tissues (sink) via the phloem. More recently, site-directed mutagenesis identified a number of PSTVd variants that are able to replicate in single cells but unable to spread systemically in whole plants. In situ hybridization studies have shown that several naturally-occurring PSTVd variants are unable to cross specific cellular boundaries in the leaf. Other studies have identified structural motifs that allow viroids to leave the cytoplasm and enter either the nucleus (PSTVd) or chloroplast (ELVd) before replication.

When the first viroids were discovered in the early 1970s, bioassays using suitable indicator hosts provided the primary tool available to detect new viroids. The introduction of polyacrylamide gel electrophoresis and, later, dot-blot hybridization assays greatly facilitated efforts to control the increasing number of viroid diseases affecting crop species. RT-PCR analysis and high throughput RNA sequencing have now largely replaced these first-generation molecular techniques. In this process much has been learned about the epidemiology and, in some cases, the origin of the viroid diseases affecting economically important crop species (Fig. 2).

Fig. 2

Fig. 2 Participants in a 1983 symposium on Subviral pathogens of plants and animals: viroids and prions held in Bellagio, Italy, and sponsored by the Rockefeller Foundation. From the left in the front row are HJ Gross, E Shikata, and HL Sänger. Beginning third from the left in the rear row are D Peters, RIB Francki, TO Diener, L Salazar, F Solymosy, G Boccardo, JW Randles, RA Owens, RH Symons, Tien Po, and A Branch. DC Gajdusek (second from left, rear row) and SB Prusiner (second from the right, front row) were awarded the Nobel Prize in Physiology and Medicine in 1976 and 1997 respectively for their studies of prion diseases.

Viroid conformation(s)

Fifty years ago, sucrose density gradient centrifugation and polyacrylamide gel electrophoresis were the methods most commonly used to fractionate RNAs extracted from diseased plants, and bioassays on a suitable indicator host were the most sensitive method available to detect new viroids. Diener (2003) vividly describes how these techniques were combined to define the essential properties of the first viroid (PSTVd) without directly visualizing its small RNA genome. Soon thereafter, the viroid-specific band on the polyacrylamide gel had been identified, and processing kilogram amounts of infected leaf tissue yielded the milligram amounts of purified viroid required for more detailed molecular studies. Thus, PSTVd was shown to lack mRNA activity in a cell-free wheat germ translation system (Davies et al., 1971), and electron microscopy of formaldehyde-denatured PSTVd revealed the unexpected presence of circular molecules (Sogo et al., 1973). Also, a comparison of their structural properties revealed that PSTVd, CEVd, and cucumber pale fruit viroid (CPFVd) were all single-stranded, covalently closed circular molecules with a highly base-paired, rod-like secondary structure (Sänger et al., 1976).

The next advance in viroid molecular biology was featured on the cover of the journal Nature. Determination of the complete nucleotide sequence and secondary structure of PSTVd by Gross et al. (1978) was a landmark event, the first genome of a eukaryotic pathogen to be characterized in such detail. DNA sequencing was still in its infancy in the mid-late 1970s, and PSTVd had to be directly sequenced using fragments of the genomic RNA that were produced by nuclease digestion in vitro and then radioactively-labeled. Shortly thereafter, Riesner et al. (1979) used a variety of biophysical techniques to show that all the base pairs in the rod-like conformation dissociate in one highly cooperative main transition, thereby forming three very stable secondary hairpins. Two of these hairpins can also be formed by other pospiviroids, and the roles of HP I and HP II in replication are discussed in the next section.

Over the next several years complete nucleotide sequences were determined for a number of additional viroids. Comparison of seven different viroid genomes led Keese and Symons (1985) to propose that PSTVd and related viroids contain five structural/functional domains. These include (i) a central domain whose conserved central region (CCR) is capable of forming alternative structures that may regulate the viroid replication cycle, (ii) a domain associated with pathogenicity, (iii) a variable domain with high sequence variability, and (iv and v) two terminal domains that are interchangeable between viroids. These authors also point to the partial duplications that appear de novo in the genome of coconut cadang cadang viroid (CCCVd) during the infection process (Haseloff et al., 1982) as evidence for the importance of RNA rearrangements in viroid evolution. The possibility that similar rearrangements may have led to the repeated exchange of information between RNA pathogens and other RNA molecules in the host cell has attracted widespread attention (see the final section on Origin/evolution). ASBVd, the one viroid whose rod-like secondary structure lacks identifiable domains, was later shown to replicate in the chloroplasts rather than the nucleus of infected cells.

RNA folding programs (e.g., Zuker and Stiegler, 1981) have been widely used to investigate viroid structure/function relationships. In most cases, the lowest-free-energy secondary structure predicted in silico is reasonably consistent with the results of chemical or enzymatic probing in vitro. Probing results obtained using a technique known as SHAPE (selective 2′-hydroxyl acylation analyzed by primer extension) which interrogates the RNA backbone at single nucleotide resolution are of particular interest. For example, López-Carrasco and Flores (2017) report that certain nucleotides in the conserved central region of PSTVd (including several within its so-called loop E motif) are more SHAPE-reactive in vitro than in vivo. Such localized differences could reflect interaction with proteins involved in replication (see next section). The overall similarity in SHAPE reactivities, however, supports the long-standing view that PSTVd accumulates in planta as a naked RNA rather than in a tight complex with host proteins.

First identified by Branch et al. (1985) on the basis of its susceptibility to UV crosslinking, several lines of evidence now indicate that the loop E motif plays a key role in regulating PSTVd replication (see Replication section). Many other RNAs including eukaryotic 5S rRNA and hepatitis delta virus (HDV) genomic RNA contain similar loop E motif(s), and their tertiary structures have been studied in great detail. The comprehensive mutational analysis of the PSTVd loop E motif carried out by Zhong et al. (2006) using isostericity matrix analysis of non-Watson-Crick base pairing to rationalize mutagenesis followed by systematic in vitro and in vivo functional assays of the resulting mutants illustrates the ability of such studies to elucidate complex structure-function relationships.

Unlike the rod-like secondary structure of PSTVd, the computer-predicted conformations of peach latent mosaic viroid (PLMVd) and chrysanthemum chlorotic mottle viroid (CChMVd) are branched. Co-variation analysis of naturally-occurring CChMVd variants indicates a very similar conformation in vivo. Furthermore, this pattern of natural variability also suggests that the branched conformation of CChMVd may be stabilized by a kissing-loop interaction similar to one previously proposed for PLMVd (Gago et al., 2005). Using a combination of site-directed mutagenesis, infectivity assays, and PAGE analysis under denaturing and nondenaturing conditions these authors showed that the kissing-loop interaction determines proper folding of CChMVd RNA in vitro. The presence of similar kissing-loop interactions in two hammerhead viroids exhibiting a low overall degree of sequence similarity suggests that this tertiary interaction facilitates the adoption and stabilization of a compact structure critical for viroid viability in vivo. Temperature gradient gel electrophoresis (Riesner et al., 1989) provides a powerful tool with which to detect just these sorts of structural changes in co-existing RNA structures.

Replication

All viroids replicate via a rolling circle mechanism, either an asymmetric mechanism for PSTVd and related viroids that replicate in the nucleus or a symmetric mechanism for ribozyme-containing viroids like PLMVd that replicate in the chloroplast. While an early study (Mühlbach and Sänger, 1979) revealed the involvement of α-amanitin sensitive RNA polymerase II in pospiviroid replication, the identity of the nuclear-encoded polymerase that catalyzes the replication of ASBVd and related viroids (Navarro et al., 2000) and the role of a type III RNase in the in vivo cleavage of multimeric (+)-strand pospiviroid RNAs (Gas et al., 2007) were established much more recently. Many important questions await future investigation.

In the early 1980s publications from several different laboratories described the characterization of replicative intermediates isolated from viroid-infected leaf tissue. The Robertson lab at Rockefeller University was the first to arrange various PSTVd-related RNAs into a rolling circle mechanism (Branch et al., 1981) and later proposed a symmetric mechanism to account for the replication of viroids and other small infectious RNAs (Branch and Robertson, 1984). The presence of comparatively large amounts of (+)-strand genomic RNA makes detection of low levels of a putative monomeric circular form of viroid (−)-strand RNA technically difficult (Hutchins et al., 1985), and fingerprint analysis of purified PSTVd replication intermediates failed to yield evidence for the presence of the monomeric circular (−)-strand RNA required by a symmetric mechanism (Branch et al., 1988).

Initial evidence that at least one viroid (ASBVd) replicates via a symmetric rolling circle mechanism was provided by Hutchins et al. (1986) who reported the ability of both (+) and (−)-ASBVd RNA transcripts synthesized from cloned dimeric cDNA templates to spontaneously self-cleave at two sites in each transcript, thereby giving rise to the corresponding monomeric (+)- and (−)-RNA. Circularization of the linear (−)-ASBVd monomer would then yield the second circular template required for replication via a symmetric (rather than asymmetric) mechanism. Many prokaryotic and eukaryotic genomes contain multiple copies of DNA sequences encoding similar hammerhead ribozymes (the name subsequently given to the RNA motif responsible for ASBVd self-cleavage).

Even as the mechanism(s) of viroid replication were being clarified, other studies sought to develop new tools with which to study viroid replication, which include the construction of the first infectious cloned PSTVd cDNA (Cress et al., 1983), development of an in vitro transcriptional system for in vitro synthesis of infectious viroid RNAs (Tabler and Sänger, 1985), and the use of A. tumefaciens to introduce infectious PSTVd cDNAs into potential host cells (Gardner et al., 1986). Similar agroinoculation strategies quickly became the method of choice to transmit a number of conventional plant viruses; for example, geminiviruses.

In the early-mid 1980s, the first generation of low copy number plasmid vectors each containing only a limited number of cloning sites was being replaced by high copy number plasmids whose more versatile multiple cloning sites were flanked by promoters for SP6 and T7 RNA polymerase. Completely by chance, the sequence of a portion of the PSTVd upper conserved central region (i.e., …GGATCCCCGGG…) turns out to be identical to that of the overlapping BamHI/SmaI recognition sites present in several polylinkers. While cloned PSTVd cDNAs (or their corresponding RNA transcripts) which contain this 11-nt sequence duplication are highly infectious, those containing only a shorter 6-nt GGATCC duplication are essentially noninfectious (Tabler and Sänger, 1985). This single observation had several important consequences: First, an immediate focus on secondary hairpin I (or related structures) as the probable site of pospiviroid cleavage/ligation during normal replication (e.g., Diener, 1986); second, a dramatic increase in the ease of generating viroid variants containing precisely targeted mutations to be screened for their effects on infectivity or other biological properties. One particularly informative mutagenesis study compared the effect of systematically closing each of the predicted interior loops or bulges in the secondary structure of PSTVd genomic RNA on the viroid's ability to replicate in single cells (protoplasts) with the effect on its ability to spread systemically in whole Nicotiana benthamiana plants (Zhong et al., 2008). The results of this study provided the framework for later high-throughput studies of the role of RNA tertiary structures in regulating viroid replication and trafficking (see Pathogenicity and Movement sections).

Two different mechanisms have been proposed to explain the processing of pospivirioid replicative intermediates to release monomeric linear (+)-strand progeny. Both involve cleavage/ligation between nucleotides G95 and G96 in the upper portion of the conserved central region but differ in the type of nuclease involved. As described by Baumstark et al. (1997), efficient cleavage/ligation of a longer-than-unit-length PSTVd RNA transcript in a potato nuclear extract requires that the central conserved region folds into a multihelix junction containing at least one GNRA tetraloop hairpin. The first cleavage occurs within the stem of the GNRA tetraloop, and a local conformational change then converts the tetraloop into a loop E motif, thereby stabilizing its base-paired end (5′-hydroxyl). The second cleavage yields a unit-length linear intermediate whose base-paired 3′ end (2′, 3′-cyclic phosphate) can then be ligated to form mature circular progeny. In a separate series of experiments, Gas et al. (2007) examined the in vivo processing of dimeric CEVd RNA transcripts expressed in transgenic A. thaliana. In this case, the monomeric linear processing product was shown to contain 5′-Phosphate and 3′-Hydroxyl termini. Such termini are the hallmarks of a type III RNase activity, and the authors propose that the necessary dsRNA substrate is formed by a kissing loop interaction between two copies of secondary hairpin I.

Finally, baker's yeast (S. cerevisiae) provides a variety of powerful genetic and molecular tools that could find many applications in studies of viroid replication. Unlike higher