University of Massachusetts Amherst ScholarWorks@UMass Amherst Doctoral Dissertations 1896 - February 2014 1-1-1991 Turnip crinkle virus (TCV) and its sub-viral RNAs. Xiao Hua Li University of Massachusetts Amherst Follow this and additional works at: https://scholarworks.umass.edu/dissertations_1 Recommended Citation Li, Xiao Hua, "Turnip crinkle virus (TCV) and its sub-viral RNAs." (1991). Doctoral Dissertations 1896 - February 2014. 6111. https://scholarworks.umass.edu/dissertations_1/6111 This Open Access Dissertation is brought to you for free and open access by ScholarWorks@UMass Amherst. It has been accepted for inclusion in Doctoral Dissertations 1896 - February 2014 by an authorized administrator of ScholarWorks@UMass Amherst. For more information, please contact [email protected]. TURNIP CRINKLE VIRUS (TCV) AND ITS SUB-VIRAL RNAs A Dissertation Presented by XIAO HUA LI Submitted to the Graduate School of the University of Massachusetts in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY May 1991 Department of Plant Pathology © Copyright by Xiao Hua Li 1991 All Rights Reserved TURNIP CRINKLE VIRUS (TCV) AND ITS SUB-VIRAL RNAs A Dissertation Presented by XIAO HUA LI Approved as to style and content by: Mark S. Mount, Member Mark S. Mount, Department Head Department of Plant Pathology ACKNOWLEDGMENT This thesis is the result of my hard work and many people's contributions. First of all, I like to express my deep gratitude to my advisor Dr. Anne Simon for her inspiring attitude and enthusiasm towards science, her close involvement in the research, her innovative way in solving research problems, and, of course, the detailed advice in my daily research. Without this, I can't imagine completing my research. I have considered myself most fortunate for being given the opportunity to work in her lab. I would like thank other members on my thesis committee. Dr. Bert Zuckerman, Dr. Mark Mount and Dr. Robert Bernatzky, for their constructive suggestions and patience. I feel indebted to Dr. Clifford Carpenter, for sharing his tremendous experience in my daily experiments and his generous help in research. I would like to mention his great sense of humor which made the lab a fun place to work in. My sincere thanks go to Dr. Mary Polacco, for her cooporation and generous help in the research covered in Chapter III. I want to extent my thanks to my colleague Pamela Cascone. Besides her help in research, her energy and attitude towards life are all inspiring- I would also like to thank Angela Valinsky, Anne, Pam and Cliff for helping with the Arabidopsis inoculations. Thanks also go to Chun-xia for her friendship. I am very fortunate to have worked with such a wonderful group of people. I am very grateful to the Department of Plant Pathology for giving me the opportunity to study here, to all the faculty, staff members and students for their caring, encouragement and help. I specially appreciate their patience during my first year here when I had many difficulties in both language and culture. I would like to use this opportunity to express my gratitude to my parents. Their ever-lasting love, care and morale support are the constant source of my energy. To Hao-jin, my husband and best friend, I own special thanks for his understanding, patience and love which have enriched my life in many ways. Finally, I want to say thanks, with love, to my little son, Andrew, who has almost grown up with this thesis and has given me so much joy in life. V ABSTRACT TURNIP CRINKLE VIRUS (TCV) AND ITS SUB-VIRAL RNAs MAY, 1991 XIAO HUA LI, B.A., NORTHWEST UNIVERSITY, XIAN, CHINA M.S., UNIVERSITY OF MASSACHUSETTS Ph.D., UNIVERSITY OF MASSACHUSETTS Directed by: Professor Anne E. Simon TCV isolate, TCV-M, supports a family of satellite (sat-) RNAs. The virulent sat-RNA C intensifies TCV symptoms on turnip cultivar Just Right. In this thesis, I report that sat-RNA C (356 b) exacerbates symptoms on all hosts where TCV produces visible symptoms including cultivars of Brassica rapa and Arabidopsis thaliana. This finding has led to studies of TCV-resistance using A. thaliana, a small plant with a well characterized genome, as a host. After screening over 6,000 M2 A. thaliana plants from EMS treated Columbia cultivar seeds and 22 ecotypes of A. thaliana, ecotype Dijon was found to be resistant to TCV. A second isolate, TCV-B, supports an RNA species with a size similar to that of sat-RNA C. Northern hybridization and cDNA cloning and sequencing demonstrate that this RNA is actually a defective interfering (DI) RNA, denoted DI RNA G. Infection of turnip with virus derived from cloned transcripts of TCV-B resulted in de novo generation of a DI vi RNA, Dll RNA. Unlike DI RNAs associated with other plant viruses (or animal viruses), TCV DI RNAs intensify TCV symptoms. To understand sequences required for DI RNA infectivity, a series of mutation have been qenerated in a full lenqth cDNA copy of DI RNA G. Stepwise deletions at base 98 of DI RNA G, at which an Apa I linker had been inserted, has shown that DI RNA GA (DI RNA G with a 8-base insertion at base 98) harborinq deletions of bases 74-98 or less are infectious. However, deletion of bases 73-98 or more abolishes RNA infectivity. DI RNA GA with a deletion of bases 107-124 is infectious. However, DI RNA GA harborinq a deletion of bases 107-138 is not infectious. I have found that infectivity of RNA harborinq 31- or 32-base deletions can be restored by inserting foreign sequences into deletion sites. This implies that at least 71 bases (including the 8-base insertion) in DI RNA GA near the 5' end are not specifically required for RNA infectivity. By combining deletions of infectious clones to generate larger deletions (40 or 43 bases deleted), I demonstrate that the infectivity of the RNA is abolished. This result suggests that size of the DI RNA is important in maintaining RNA infectivity. TABLE OF CONTENTS Eage ACKNOWLEDGMENT . iv ABSTRACT . vi LIST OF TABLES . xii LIST OF FIGURES .xiii Chapter I. INTRODUCTION . 1 Turnip Crinkle Virus (TCV) . 2 Satellite (Sat-) RNAs . 4 Defective Interfering RNAs (DI RNAs) . 17 Plant Resistance to Viral Infection . 23 Thesis Plan . 29 II. SYMPTOM INTENSIFICATION ON CRUCIFEROUS HOSTS BY THE VIRULENT SATELLITE RNA OF TURNIP CRINKLE VIRUS .... 31 Introduction . 31 Materials and Methods . 32 Inocula and Inoculations . 32 Plant Materials and Growth Conditions . 33 RNA Extraction and Analysis . 34 Results . 35 Effect of Sat-RNA C on Different Cruciferous Plants . 36 Symptoms Induced by TCV on A. Thaliana Are Affected by Age of the Plant at Inoculation .. 43 TCV-m Genomic RNA and Sat-RNA Levels Are Not Reduced in TCV Tolerant Plants . 49 Sat-RNA F Is Not Responsible for Symptom Intensification . 52 Discussion . 52 III. PROTEIN AND RNA ACCUMULATION IN ARABIDOPSIS THALIANA UPON TCV INFECTION AND TCV-RESISTANCE IN A. THALIANA . 60 Introduction . 60 Materials and Methods . 64 Plant Materials and Inoculations . 64 Total Protein Extraction . 65 Total RNA Extraction and In Vitro Translation .. 66 Two-dimensional (2-D) SDS-polyacrylamide Gel Electrophoresis . 67 Results . 70 Early Accumulation of TCV Viral RNAs in Infected Susceptible A. Thaliana Plants . 70 Differential Expression of Cellular Proteins in Mock and TCV Infected Susceptible A. Thaliana Plants . 70 Differential Accumulation of Cellular RNA in Mock and TCV-M Infected Susceptible A. Thaliana Plants . 78 Screening of TCV-resistance in A. Thaliana Plants . 79 Discussion . 86 IV. TURNIP CRINKLE VIRUS DEFECTIVE INTERFERING RNAs INTENSIFY VIRAL SYMPTOMS AND ARE GENERATED DE NOVO . 95 Introduction . 95 Materials and Methods . 97 Virus Strains and Plant Inoculations . 97 Northern Hybridizations . 99 Cloning and Sequencing of DI-RNA G and Dll RNA . 99 Primer-extension Sequencing . 100 Results . 101 Identification of Low Molecular Weight RNAs Associated with the Berkeley Isolate of TCV .. 101 RNA G Is Composed of Sequence Derived from TCV Genomic RNA. 106 ix DI RNA G Intensifies the Symptoms of TCV . 110 De Novo Generation of a DI RNA and Its Correlation with a Severe Symptom Expression . 115 Discussion . 122 V. TURNIP CRINKLE VIRUS DEFECTIVE INTERFERING RNA INFECTIVITY AND GENERATION STUDIES . 131 Introduction . 131 Materials and Methods . 133 Virus Isolates and Plant Inoculations . 133 Artificially Synthesizing DI RNAs from a Full Length cDNA Clone, pTCV-Tldl, of the Berkeley TCV Strain, TCV-B . 134 Cloning of a Full Length Infectious DI RNA G Molecule . 138 Insertion of a Unique Restriction Site into a Full Length DI RNA G cDNA at Base 98 . 143 Gapped Duplex Mutagenesis . 144 Sub-cloning of DI RNA G cDNA into pT7E19 Vector . 150 Deletion Mutations in the 5' Region of DI RNA G . 150 Insertion of Foreign Sequences into the Deletion Mutants . 156 Combination of Deletions Upstream and Downstream of the Apa I Site . 158 Generation of Mutations in the Central and the 3' Portions of DI RNA G . 161 In Vitro RNA Synthesis . 161 Results . 162 Synthetic DIs Derived from TCV-B Genomic RNA Were Not Infectious . 162 Ten Nucleotides at the 5' Ends of DI RNA G and Sat-RNA F Are Identical . 163 In Vitro Transcripts of pT7-G, a Full Length cDNA Clone of DI RNA G, and Its Derivative, pT7-GA, Were Infectious On Turnip cv. Just Right . 168 Deletions Greater than 26 Nucleotides in the 5' Region of DI RNA GA Abolish RNA Infectivity .. 171 X At Least 71 Bases in DI RNA GA (Bases 68-138, Including the 8-base Apa I Linker) Are Not Required for RNA Infectivity - Revealed by Insertion of Foreign Sequences into DI RNA GA Deletion Mutants .