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Biology of RPV Barley Yellow Dwarf Virus Satellite RNA Lada Rasochova Iowa State University

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Biology of RPV Barley Yellow Dwarf Virus Satellite RNA Lada Rasochova Iowa State University Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 1996 Biology of RPV barley yellow dwarf virus satellite RNA Lada Rasochova Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Molecular Biology Commons, and the Plant Pathology Commons Recommended Citation Rasochova, Lada, "Biology of RPV barley yellow dwarf virus satellite RNA " (1996). Retrospective Theses and Dissertations. 11563. https://lib.dr.iastate.edu/rtd/11563 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. INFORMATION TO USERS This manuscript has been reproduced from the microfihn master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely a£fect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be remove4 a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand comer and continuing from left to right in equal sections with small overlaps. Each original is also photographed in one exposure and is included in reduced form at the back of the book. Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6" x 9" black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. UMI A Bell & Howell Information Company 300 North Zeeb Road, Ann Arbor MI 48106-1346 USA 313/761-4700 800/521-0600 Biology of RPV barley yellow dwarf virus satellite RNA by Lada Rasochova A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Major Molecular, Cellular and Developmental Biology Major Professor W. Allen Miller Iowa State University Ames, Iowa 1996 XJMI Number: 9712592 UMI Microform 9712592 Copyright 1997, by UMI Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. UMI 300 North Zeeb Road Ann Arbor, MI 48103 a Graduate College Iowa State University This is to certify that the doctoral dissertation of Lada Rasochova has met the dissertation requirements of Iowa State University Signature was redacted for privacy. Major Professor Signature was redacted for privacy. For the Major Program Signature was redacted for privacy. iii TABLE OF CONTENTS ABSTRACT v CHAPTER I. GENERAL INTRODUCTION 1 Dissertation Organization 1 Introduction 1 Research Goals 8 CHAPTER! LITERATURE REVIEW 9 Luteoviruses 9 Qassification 9 Virion properties 9 Symptoms 12 Cytopathological effects 13 Tissue specificity 13 Host range 14 Serological relationships 15 Luteovirus transmission 15 Genome organization and replication 17 RNA agents associated with luteoviruses 21 Satellites 23 Classification 23 Requirements for replication of plant satellites 28 Helper virus specificity and host range of plant virus satellites 31 B type mRNA satellites 32 C ty^ linear satRNAs 33 D type circular satRNAs 35 Effects of satellites on disease symptoms 37 Possible mechanisms 37 Satellite viruses 41 B type mRNA satellites 41 C ty^ linear satRNAs 42 D ty^ circular satRNAs 46 CHAPTER 3. SATELLITE RNA OF BARLEY YELLOW DWARF-RPV VIRUS REDUCES ACCUMULATION OF RPV HELPER VIRUS RNA AND ATTENUATES RPV SYMPTOMS IN OATS 49 Abstract 49 Introduction 50 Results 52 Lack of mechanical transmission of satRPV RNA 52 Aphid transmission of satRPV RNA-containing virus from protoplasts to plants 53 SatRPV RNA affects helper virus RNA accumulation in oats but has no effect on symptoms caused by mixed infection of RPV andPAV 54 SatRPV RNA attenuates symptoms and decreases helper virus RNA accumulation in oats infected with pure RPV 55 SatRPV RNA decreases helper virus RNA accumulation in oat protoplasts 57 Discussion 56 Lack of mechanical transmission of satRPV RNA 57 iv Helper vims titer is a limiting factor for satRPV RNA transmission by aphids from infected protoplasts extract 58 Lack of effect of satllPV I^A on symptoms caused by RPV+PAV 59 Possible mechanisms of reduction of virus symptoms severity and RNA accumulation 60 Acknowledgments 62 Materials and Methods 62 In vitro transcription 62 Whole plants: inoculation, virus propagation and purification, and extraction of virion and total RNAs 63 Protoplasts: inoculation, partial virus extraction, and total RNA isolation 63 RNA analysis 64 Aphid feeding on artificial membranes and inoculation of plants 64 References 65 CHAPTER 4. SPECLFICrrY OF RPV BARLEY YELLOW DWARF VIRUS SATELLrTE RNA IS ASSOCIATED WrrH POLYMERASE GENE OF SUBGROUP n LUTEOVIRUSES 76 Abstract 76 Introduction 77 Materials and Methods 79 In vitro transcription 79 Virus propagation, purification, and extraction of virion RNA 80 Electroporation of protoplasts and extraction of total RNA 80 Encapsidation assay 81 Purification of virions from tobacco protoplasts 82 Aphid feeding on artificial membranes, inoculation of plants, and isolation of total leaf RNA 82 RNA analysis 82 Results 83 Subgroup n BWYV supports satRPV RNA replication in tobacco cells 83 Subgroup I ST9a RNA failed to replicate satRPV RNA in tobacco protoplasts 84 SatRPV RNA is encapsidated in mostly circular form in BWYV capsids but as a linear form in B YDV particles 85 Aphid transmission of satRPV RNA from protoplasts to shepherd's purse plants required both BWYV and ST9a RNA 86 Effect of satRPV RNA on accumulation of BWYV and ST9a iRNA and disease symptoms in shepherd's purse plants 87 Discussion 88 Specificity of satRPV RNA - helper virus polymerase interaction 88 Effect of satRPV RNA on symptoms and viral RNA accumulation in plants 90 Host plant requirements of satRPV RNA 91 Acknowledgments 92 References 92 CHAPTER 5. GENERAL CONCLUSIONS 109 Conclusions 109 Recommendation for Future Research 111 REFERENCES CrrED 117 V ABSTRACT Satellite RNAs (satRNAs) depend on their helper viruses for replication, encapsidation and spread. The goal of the research was to determine helper virus specificity and host range of the RPV barley yellow dwarf luteovirus (B YDV-RPV) satRNA (satRPV RNA) and assay satRPV RNA for the ability to affect the accumulation and modulate disease symptoms of its helper viruses in protoplasts and plants. Although similar in structural genes, subgroup I and n luteoviruses have very different polymerases. BYDV-RPV and beet western yellows virus (BWYV), members of subgroup II luteoviruses, supported satRPV RNA replication m monocotyledonous and dicotyledonous hosts, respectively. In contrast, BYDV-PAV and ST9 associated RNA (ST9a RNA), subgroup I luteoviruses, failed to replicate satRPV RNA. However, the stimulation of BWYV by ST9a RNA resulted in an increased accumulation of satRPV RNA progeny in BWYV + ST9a RNA + satRPV RNA inoculated tobacco protoplasts. BWYV encapsidated satRPV RNA, but in a form different from that found in BYDV (RPV + PAV) particles. SatRPV RNA was transmitted to plants by aphids that acquired virus from infected protoplasts. The concentration of encapsidated helper and satRPV RNA had to be above a threshold level to facilitate aphid transmission. Oat plants infected with BYDV-RPV and satRPV RNA had milder symptoms than those infected with BYDV-RPV alone. SatRPV RNA reduced BYDV-RPV helper RNA accumulation in oat plants and protoplasts. However, it did not affect symptoms caused by the severe mixed infection of RPV and PAV BYDVs and had no effect on PAV RNA accumulation in oats. In contrast, satRPV RNA reduced the accumulation of both BWYV helper RNA and nonhelper ST9a RNA in shepherd's purse plants but attenuated BWYV and ST9a RNA symptoms only slightly. SatRPV RNA symptom modulation seems to be determined by competition between the satRPV RNA and its helper virus for replication and encapsidation. The results showed that satRPV RNA can replicate in both monocotyledonous and dicotyledonous protoplasts and plants and suggested that the vi specificity determinants of satRPV RNA replication are contained within the polymerase genes of supporting viruses rather than in structural genes or host plants. 1 CHAPTER 1. GENERAL INTRODUCTION Dissertation Organization This dissertation contains two manuscripts. The manuscript "Satellite RNA of barley yellow dwarf RPV virus reduces accumulation of RPV helper virus RNA and attenuates RPV symptoms in oats" is formatted for publication in Molecular Plant-Microbe Interactions and was published in a shorter version. The manuscript "Specificity of RPV barley yellow dwarf virus satellite RNA replication is associated with polymerase gene of subgroup n luteoviruses" is formatted and will be submitted for publication in Virology. I have been principally involved
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