Molecular characterization, differential movement and construction of infectious cDNA clones of an Ohio isolate of Hosta virus X Thesis Presented in Partial Fulfillment of the Requirements for The Degree Master of Science in the Graduate School of The Ohio State University By Carola De La Torre Cuba, BSc. The Ohio State University 2009 Thesis Committee: Dr. Dennis Lewandowski, Advisor Dr. Pierluigi Bonello Dr. Margaret G. Redinbaugh Copyright by Carola De La Torre 2009 Abstract Hostas (Hosta Tratt.) are the second most popular herbaceous perennial plant in the USA with sales exceeding $32 million in 2008 (USDA Floriculture crops 2008 Summary, 2009). Despite its relatively recent discovery in 1996, Hosta virus X (HVX) has already had a significant economic impact on hosta growers and producers. HVX is easily mechanically transmitted and can survive in the infected plants for years without showing symptoms. HVX has been reported in many states including Ohio. Very little is known about HVX biology with respect to diversity, symptom determinants and mechanisms of host resistance. The objectives of this research were to 1) Screen for resistance against HVX in a number of hosta cultivars, 2) Examine coat protein (CP) variability among Ohio isolates, 3) Molecular characterize an Ohio HVX isolate and 4) Construct an infectious cDNA clone of HVX. An Ohio HVX isolate, HVX-37, was selected for resistance screening, construction of an infectious cDNA clone and phylogenetic comparisons. Local and long-distance movement and accumulation of HVX-37 were determined for twenty-four hosta cultivars over three growing seasons resulting in five types of responses. Four cultivars (H. sieboldiana ‘Elegans’, H. sieboldiana ‘Northern Exposure’, H. ‘Nightlife’ and H. ‘Olive Bailey Langdon’) exhibited only infection of inoculated leaves. These cultivars are possible sources of resistance for future breeding programs for resistance to HVX. The CP gene of ten Ohio ii HVX isolates were found to be highly similar to all HVX isolates available in GenBank. Molecular characterization of HVX-37 showed a typical potexvirus genome organization but with some intriguing differences between the replicase proteins of an isolate collected in Korea (Accession No. AJ620114). Full-length cDNA clones of HVX-37 (pHVX) were constructed downstream of the T7 promoter sequence. In vitro RNA transcripts derived from pHVX were infectious to hostas and Nicotiana benthamiana Domin. plants, representing the first infectious clone for HVX. iii Dedicated to Rosa Postigo, my grandmother, and to all hosta lovers iv Acknowledgements I especially would like to thank my advisor, Dr. Dennis Lewandowski, whose guidance, excellent intellectual support and enthusiastic encouragement over the past two years made this thesis possible. I am also grateful for all his efforts in reviewing this text. I am very grateful to my SAC Committee members, Dr. Peg Redinbaugh and Dr. Enrico Bonello for their useful inputs and accurate revision of this thesis. I would like to thank Dr. Tea Meulia for all her help with the TEM and her helpful advice with the experiments. Thank you to Dr. J. Rob Fisher, Ohio Department of Agriculture, for kindly providing me with magnetic bead-conjugated anti-HVX antiserum. I am very grateful to all my teachers in the Plant Pathology Department for giving me an excellent education and the firm basis that I will need to succeed in my career. A special thank you to my lab partners: Mike Kelly (thank you for all the grinding and watering!), Chris Woltjen, Matt Wallhead, Jessica Schaffer, Amanda Hayes. I am indebted to all my friends who have made Kottman Hall a very special place over these years: Nun, Gautam, Fiorella, Oscar, Chan Ho, Songbiao, Miguel, Anne Marie, Madge, Dr. Graham, Dr. Rhodes, Barry, Clara, Monica Lewandowski, Niqui, Ramona, Duan, Xenxi, Jinnan, Michelle. Without any doubt, I would like to enormously thank my partner and best friend, Antonio Cabrera, for his invaluable help and for being there when I most needed. Lastly, but very importantly, I wish to thank my parents Carlos and Amalia, my sister, Amanda, and my brother, Juan Pablo, who are a constant inspiration to me. Thank you for always being there for me every step of the way. The research was supported in part by grants from the Ohio Plant Biotechnology Consortium and the OARDC SEEDS grant program. v Vita June 17th, 1982 …………………………………………….………Born – Lima, Peru 2005 …………………………………………………………....Bsc. Biology, Universidad Nacional Agraria La Molina 2005 – 2007 …………………………………………………………...Research Assistant, International Potato Center, Lima, Peru 2007 – present ………………………………………...Graduate Research Associate, Department of Plant Pathology, OSU Publications De La Torre C, Lewandowski D. In vitro transcripts of a full-length cDNA clone of Hosta virus X are infectious to Hosta and Nicotiana benthamiana plants. 2009. Phytopathology 99:S27. De La Torre C, Lewandowski D. Sequence comparisons between Hosta virus X (HVX) isolates and differential infection of hosta cultivars. 2008. Phytopathology 98:S45 Fields of Study Major Field: Plant Pathology vi Table of Contents Abstract ………………………………………………………………………………...ii Dedication ………………………………………………………………………………..iv Acknowledgements ………………………………………………………………………...v Vita ………………………………………………………………………………………..vi List of Tables ………………………………………………………………………………..ix List of Figures ………………………………………………………………………………..xi Chapter 1: Literature Review ………………………………………………………...1 Hosta ………………………………………………………………………...1 Hosta diversity ………………………………………………………...2 Pest and diseases of hosta ………………………………………………...3 Viral diseases of hostas ………………………………………………...5 Hosta virus X ………………………………………………………………...6 Chapter 2: Differential responses of hosta cultivars to Hosta virus X infection .............10 Introduction ……………………………………………………….............10 Materials and methods ……………………………………….............11 Preparation of inoculum ……………………………….............12 Hosta cultivars ……………………………………….............12 Inoculation of hosta cultivars ………………………….....................12 DAS-ELISA ……………………………………………….............13 IC-RT-PCR ……………………………………………….............14 Results ……………………………………………………….............15 Discussion ……………………………………………………….............17 Chapter 3: Sequence analysis of Hosta virus X isolates ……………………….............32 Introduction ……………………………………………………….............32 Materials and methods ……………………………………….............35 Ohio HVX isolates ……………………………………….............35 CP cDNA synthesis and RT-PCR amplification ……….............35 Sequencing and assembly of CP consensus ……………….............36 Multiple alignments of CP sequences ……….........................36 RT-PCR amplification of overlapping 5’ and 3’ terminal fragments of HVX-37 ……...………………………………………..37 Sequencing, assembly and annotation of the HVX-37 genome .........38 vii Results………..…………………………………………………….……….39 CP sequence comparisons of Ohio HVX isolates ..……………..39 CP sequence comparisons of all available HVX isolates……...........40 HVX- 37 genome sequence analysis ………………………………40 Sequence comparison between HVX-37 and HVX-Kr……………..41 HVX replicase protein sequence comparison of HVX isolates with other potexviruses ……………………………………................42 Discussion ………………………………………………………………43 Chapter 4: In-vitro transcripts of full-length cDNA clones of Hosta virus X are infectious to hosta and Nicotiana benthamiana plants ………………………61 Introduction ………………………………………………………………61 Materials and methods ………………………………………………63 RNA extraction ………………………………………………63 cDNA synthesis ………………………………………………63 Long fusion PCR ………………………………………………65 Construction of pHVX ………………………………………65 In vitro transcription and plant inoculation ………………………67 DAS-ELISA ………………………………………………………68 IC-RT-PCR ………………………………………………………68 Plant-to-plant passage of in vitro RNA transcript-derived progeny 69 Sequencing of pHVX ………………………………………………69 Sequencing of progeny derived from pHVX ………………………70 Transmission electron microscopy ………………………………70 Results ………………………………………………………………………71 Construction of a full-length cDNA clone of HVX ………………71 Infectivity of transcripts derived from pHVX in hosta and N. benthamiana ……………………………………………....71 Sequence comparisons between pHVX and HVX-37 ………72 Sequence comparisons between progeny derived from pHVX and HVX-37 ………………………………………………………73 Discussion ………………………………………………………………73 References ………………………………………………………………………………85 viii List of Tables Table 1.1. Potexvirus species isolated from ornamental hosts …………………….….9 Table 2.1. Infectivity and accumulation of HVX in inoculated and upper non-inoculated leaves of 24 different hosta cultivars and species. Type 1: Systemic infection both years; Type 2: Systemic infection second year only; Type 3:Localized infection year one and no systemic infection either year; Type 4:Systemic infection year one; no detectable infection year two; Type 5: No detectable infection year one; systemic infection year two ….…..…….......….21 Table 2.2 Detection of HVX by DAS-ELISA of inoculated and upper non-inoculated leaves of individual hosta plants from cultivars classified as Type 1……....23 Table 2.3 Detection of HVX by DAS-ELISA of inoculated and upper non-inoculated leaves of individual hosta plants from cultivars classified as Type 2....……25 Table 2.4 Detection of HVX by DAS-ELISA of inoculated and upper non-inoculated leaves of individual hosta plants from cultivars classified as Type 3………27 Table 2.5 Detection of HVX by DAS-ELISA of inoculated and upper non-inoculated leaves of individual hosta plants from cultivars classified as Type 4…...….28 Table 2.6