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Geminivirus Al2 and L2 Proteins Interact with and Inactivate

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Geminivirus Al2 and L2 Proteins Interact with and Inactivate GEMINIVIRUS AL2 AND L2 PROTEINS INTERACT WITH AND INACTIVATE ADENOSINE KINASE DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Hui Wang, M.S. ***** The Ohio State University 2004 Dissertation Committee: Dr. David Bisaro, Adviser Dr. Biao Ding Approved by Dr. Erich Grotewold Dr. Deborah Parris _________________________________________ Adviser Molecular, Cellular, and Developmental Biology ABSTRACT AL2 and L2 are related proteins encoded by geminiviruses of the Begomovirus and Curtovirus genera, respectively. Both are pathogenicity determinants that cause enhanced susceptibility when expressed in transgenic plants. To understand how geminiviruses defeat host mechanisms that limit infectivity, we searched for cellular proteins that interact with AL2 and L2. Here, evidence is presented which indicates that the viral proteins interact with and inactivate adenosine kinase (ADK), a nucleoside kinase that catalyzes the salvage synthesis of 5'-AMP from adenosine and ATP. We show that the AL2 and L2 proteins inactivate ADK in vitro and following coexpression in E. coli and yeast. We also demonstrate that ADK activity is reduced in transgenic plants expressing the viral proteins and in geminivirus infected plant tissue. In contrast, ADK activity is increased following inoculation of plants with diverse RNA viruses or a geminivirus lacking a functional L2 gene. Consistent with its ability to interact with multiple cellular kinases, we also demonstrate that AL2 is present in both the nucleus and the cytoplasm of infected plant cells. To our knowledge this is the first evidence that ii ADK is targeted by viral pathogens, and the first evidence that this "housekeeping" enzyme might be a part of host defense responses. In previous work, we showed that AL2 and L2 also interact with and inactivate SNF1 kinase, a global regulator of metabolism that is activated by 5'-AMP. Together these observations suggest that metabolic alterations mediated by SNF1 are an important component of innate antiviral defenses and that inactivation of ADK and SNF1 by the geminivirus proteins represents a dual strategy for countering this defense. Another connection between ADK and viral pathogenesis relates to RNA silencing suppression, which is associated with methylation. By recycling adenosine, ADK plays a critical role in sustaining the methyl cycle and SAM-dependent methyltransferase activity. AL2 proteins have also been shown to act as suppressors of RNA silencing, an adaptive host defense response. In this thesis, we confirm that the geminiviruses TGMV and BCTV induce and are targeted by RNA silencing in the course of a normal infection, and that the AL2 and L2 proteins they encode are capable of suppressing RNA silencing in a transient three component system. Remarkably, we found that inhibiting ADK activity at the RNA level by expression of a dsRNA construct directed against ADK, or at the protein level by the use of an ADK inhibitor (the adenosine analogue RBI), also results in silencing suppression. Direct measurement of ADK activity in tissue showing silencing suppression following infiltration with AL2, L2, dsADK, or RBI revealed that in all cases ADK activity was significantly reduced iii (>50%). These data provide strong evidence that AL2 and L2 suppress silencing by inhibiting ADK activity. Taken together, the studies in this thesis first demonstrated that geminivirus AL2 and L2 proteins interact with and inactivate adenosine kinase and that this inactivation has two effects on pathogenesis: interference with plant innate defense and interference with plant adaptive defense. iv Dedicated to my parents v ACKNOWLEDGMENTS I express my deepest gratitude to my mentor Dr. David M. Bisaro for his patience and support. His enthusiasm and encouragement in pursuing novel ideas and insights were the key to the success of this project. I extend my appreciation to my committee members Dr. Biao Ding, Dr. Erich Grotewold and Dr. Deborah S. Parris for their time, support, guidance and advice. I would like to express my sincere thanks to Dr. Garry Sunter and Dr. Kenneth Buckley for their assistance in overcoming many technical difficulties and for all the useful suggestions and insightful discussions to improve my research. Especially, I would like to thank Dr. Garry Sunter for the work of AL2 protein localization. I would also like to acknowledge other members of the Bisaro lab: Janet Sunter, Dr. Linhui Hao, Duan Wang, Xiaojuan Yang, Cody Buchmann, Nick Green and Tim Cowley, and members in Dr. Biao Ding’s lab, Dr.Yijun Qi and Dr. Asuka Itaya for their help, friendship and stimulating discussions. vi I would also like to thank the staff of the biotechnology center: Melinda Parker, Diane Furtney, Dave Long, Scott Hines, Joe Takayama and MCDB secretary, Jan Zinich for all of their help. Finally, I am indebted to my parents and my family for their unconditional love, continual support and encouragement. To my father, Jiwen Wang, my sister Ai Wang and my brother Yun Wang, I cannot say thank you enough. Whatever happened, they were always there with me. To my wife Xiuping and daughter Emily, I am sorry for all the inconvenience I have brought home over all the challenging time. Thank you for your understanding, constant love and support. vii VITA 1983 - 1987…………… B.S, Plant Pathology, Plant Protection Department, Zhejiang Agricultural University, Hangzhou, PRC 1987 -1990…………… M.S, Phytobacteriology, Plant Pathology Department, Nanjing Agricultural University, Nanjing, PRC 1990 - 1996…………… Researcher, Institute of Virology, Zhejiang Academy of Agricultural Sciences, Hangzhou, PRC 1996 - 1998…………… Research associate, Molecular Virology, Plant Pathology Department, University of Hawaii at Manoa, Honolulu, HI, USA 1998 -present………… Research associate, MCDB, The Ohio State University PUBLICATIONS Research Publications: 1. Hui Wang, Linhui Hao, Garry Sunter and David Bisaro (2003) Adenosine Kinase Is Inactivated by Geminivirus AL2 and L2 Proteins. Plant Cell 15(12):3020-3032 (The research published in this paper is highlighted in “In This Issue” section of the journal, as “Viral Defense and Counterdefense: A Role for Adenosine Kinase in Innate Defense and RNA Silencing”.) viii 2. Linhui Hao, Hui Wang, Garry Sunter and David Bisaro (2003) Geminivirus AL2 and L2 Proteins Interact with and Inactivate SNF1 Kinase. Plant Cell 15(4):1034- 1048 3. Hui Wang and Ren Xinzheng (1993) Adsorption, penetration and multiplication of the pathogen (Pseudomonas solanacearum) in the roots of tomato plant. Acta Phytopathological Sinica 23 (2):143-150 4. Hui Wang and Yili Ruan (1993) Plant viral pinwheel inclusion bodies. Virol. Sinica 8(2):119-124 (review article) 5. Hui Wang and Yili Ruan (1993) Mature embryo culture of barley cultivars resistant or susceptible to Barley Yellow Mosaic Virus (BaYMV). Barley Science 23(2):17-22 6. Yili Ruan, Wanhe Zou, and Hui Wang (1995) Identification of resistance in barley varieties to BaYMV with ELISA. Acta Phytopathological Sinica 25(3):232-238 7. Yili Ruan, Wanhe Zou, and Hui Wang (1995) Evaluation of barley cultivars to BaYMV with ELISA method. Acta Agri. Zhejiangesesis 7(3):187-193 8. Yili Ruan, Wanhe Zou, Hui Wang, Chen Shi and He Chen (1994) Resistance and susceptibility reactions of barley varieties of Japan and West-Europe to Chinese BaYMV. Acta Phytophylac. Sinica 21(3):239-245 9. Yili Ruan, Wanhe Zou, Hui Wang, and Shuimiao Huang (1993) Pathogenicity of main isolates of BaYMV to barley cultivars in China. Acta Phytopathol. Sinica 23(4):348-354 10. Yili Ruan, Wanhe Zou, Hui Wang, Chen Shi and He Chen (1993) Pathogenicity of BaYMV in the provinces of Zhejiang, Jiangsu and Shanghai to parts of ix BaYMV-resistant cultivars of Japan, England and Germany. Acta Agri. Zhejiangesesis 5(3):182-187 11. Jianping Chen, Majia Dong, Jinfei Tao, Yili Ruan, Hui Wang and Shengxiang Chen (1993) Detection and identification of plant viruses with different morphology in leaf-saps by colloidal gold immunosorbent electron microscopy. Acta Phytopathol. Sinica 23(2):169-175 FIELDS OF STUDY Major Field: Molecular, Cellular, and Developmental Biology x TABLE OF CONTENTS Abstract ………………………………………………………………………………….ii Dedication……………………………………………………………………………… v Acknowledgments……………………………………………………………………....vi Vita………………………………………………………………………………….....viii List of Figures………………………………………………………………………….xvi Chapters: CHAPTER 1 ...................................................................................................................... 1 INTRODUCTION................................................................................................... 1 1.1 Geminiviruses........................................................................................................... 1 1.1.1 Geminivirus classification...................................................................... 2 1.1.2 Geminivirus nomenclature and gene organization ................................. 3 1.1.3 Geminivirus replication and cell cycle reprogramming.......................... 6 1.2 AL2 and L2 have multiple functions...................................................................... 12 1.2.1 AL2 is a transcriptional activator but L2 is not .................................... 12 1.2.2 AL2 and L2 act as pathogenicity determinants by interfering with global metabolism .............................................................................................................. 15 1.2.3 AL2 and L2
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