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The Molecular Basis of the Interactions of Rhabdoviruses with Their Insect and Plant Hosts

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The Molecular Basis of the Interactions of Rhabdoviruses with Their Insect and Plant Hosts THE MOLECULAR BASIS OF THE INTERACTIONS OF RHABDOVIRUSES WITH THEIR INSECT AND PLANT HOSTS DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Chi-Wei Tsai, M.S. * * * * * The Ohio State University 2006 Dissertation Committee: Professor Saskia A. Hogenhout, Adviser Approved by Professor David M. Francis Professor Margaret G. Redinbaugh ________________________________ Professor Thomas G. Wilson Adviser Graduate Program in Entomology ABSTRACT Maize fine streak virus (MFSV) and Maize mosaic virus (MMV) are insect transmitted plant rhabdoviruses. Many rhabdoviruses are economically important pathogens of human, livestock, and crops worldwide. Insects transmit many animal and all plant rhabdoviruses. These viruses invade and replicate in cells of various tissues in insects, vertebrates, and plants. Therefore, insects are not only vectors but also replication hosts for rhabdoviruses. Sigma rhabdovirus only infects Drosophila flies and is transmitted to the progeny of flies through germinal cells. D. melanogaster is extensively used as a model organism because of its traceable genetics and fully sequenced genome. MFSV, MMV, and Sigma virus, and interactions with their hosts provide unique systems for characterization of molecular factors determining rhabdovirus host ranges. First, I completed the genome sequences of two maize-infecting rhabdoviruses, MFSV and MMV. The MFSV genome encodes seven genes on the antigenomic strand, whereas the MMV genome encodes six genes. More information about possible functions of MFVS and MMV proteins was obtained through in planta cellular localization of fluorescent-protein fusions. The results showed that the MFSV N, P4, M, and MMV P proteins target the nuclei of plant cells, whereas the MFSV P, P3, and MMV N, P3, M proteins do not. These findings are consistent with the presence of nuclear localization ii signals (NLSs) only in nuclear targeting proteins. Co-introductions of rhabdovirus proteins revealed that the rhabdovirus N and P proteins interact in which the P proteins follow the distribution of the N proteins, and that the N and P protein interaction is specific to cognate proteins of each virus. MFVS and MMV replicate in the nucleus and assemble at the inner nuclear membrane in insect and plant cells, and therefore nuclear import of viral proteins is critical to complete virus morphogenesis. Using virus-induced gene silencing (VIGS) combined with in planta cellular localization experiments, I discovered that silencing of Importin αs in plant cells inhibits the nuclear localization of the MFSV N protein and the MFSV N-P complex in plant cells, suggesting that the MFSV N protein and the MFSV N-P complex are dependent on Importin αs for nuclear import in plants. In addition, the MFSV N protein and the MFSV N-P complex also targeted the nuclei of insect cells, consistent with the hypothesis that the MFSV proteins interact with conserved nuclear import pathways of plants and insects. Studies to determine whether the Importin αs are involved in nuclear import of the MFSV N protein and the MFSV N-P complex into the nuclei of drosophila cells are ongoing. To investigate the drosophila response to Sigma virus infection, hybridization experiments with RNAs from virus-infected and virus-free drosophila adults and cDNA microarrays that contain whole drosophila genome were conducted. The Imd signaling pathway was identified as the main component of the drosophila anti-Sigma virus iii response, whereas there were no indications of activation of the Toll pathway. This is in contrast with Drosophila C virus (DCV) and Drosophila X virus (DXV), which mainly activate the Jak-STAT and Toll pathways, respectively. This is the first comparative study showing that viruses can induce different immune pathways in drosophila, similarly to Gram-negative bacteria and Gram-positive bacteria, which predominantly activate the Imd and Toll pathways, respectively. These findings and future studies of Sigma virus-drosophila interactions with will help to elucidate innate immune response pathways against enveloped viruses in vertebrates. A comparison of how different insects, including drosophila, leafhoppers and planthoppers, respond to rhabdovirus infections should prove interesting. iv ACKNOWLEDGMENTS I am grateful to my advisor, Dr. Saskia A. Hogenhout, for her intellectual guidance, encouragement, and constant support for this project, and for her assistance in editing this dissertation. I would like to thank my advisory committee members, Drs. David M. Francis, Margaret G. Redinbaugh, and Thomas G. Wilson, for their advice and support of my graduate study. I am also grateful for the assistance of Drs. El-Desouky Ammar, Xiaodong Bai, Ralf G. Dietzgen, Roy E. Gingery, Michael M. Goodin, Sophien Kamoun, John A. Lindbo, Elizabeth A. McGraw, Tea Meulia, Scott L. O’Neill, Markus Riegler, Anna E. Whitfield, and Joe Win. I also thank John J. Abt, Valdir R. Correa, Tatiana Fazzolari, Dave E. Fulton, Mark W. Jones, Roger Mitchell, Sharon E. Reed, Angela D. Strock, William E. Styer, Jane C. Todd, Tania Y. Toruno, and Kristen J. Willie for their help and friendship. This research was supported by the OARDC Graduate Research Enhancement Grant Program, the National Research Initiative of the USDA Cooperative State Research, Education and Extension Service Grant 2002-35302-12653, and the Australian Research Council Linkages International Grant LX0452397. v VITA 1995 ........................................................... B.S. Entomology, National Taiwan University 1997 ........................................................... M.S. Entomology, National Taiwan University 1997-1999 .................................................. Second lieutenant, Army Reserve Officers' Training Corps (ROTC), Taiwan 1999-2001 .................................................. Research assistant, Department of Entomology, National Taiwan University 2001-present ............................................... Graduate Research Associate, The Ohio State University PUBLICATIONS 1. Tsai CW, Redinbaugh MG, Willie KJ, Reed S, Goodin M, and Hogenhout SA. 2005. Complete genome sequence and in planta subcellular localization of Maize fine streak virus proteins. J. Virol. 79:5304-5314. 2. Reed SE, Tsai CW, Willie K, Redinbaugh MG, and Hogenhout SA. 2005. Shotgun sequencing of the negative-sense RNA genome of the rhabdovirus Maize mosaic virus. J. Virol. Methods 129:91-96. 3. Tsai CW and Lee HJ. 2001. Analysis of specific adaptation to a domicile habitat: a comparative study of two closely related cockroach species. J. Medical Entomol. 38:245-252. vi 4. Tsai CW and Lee HJ. 2000. Circadian locomotor rhythm masked by the female reproduction cycle in cockroaches. Physiol. Entomol. 25:63-73. 5. Tsai CW and Lee HJ. 1997. Volatile pheromone detection and calling behavior exhibition: secondary mate-finding strategy of the German cockroach, Blattella germanica (L.). Zoological Studies 36:325-332. FIELDS OF STUDY Major Field: Entomology vii TABLE OF CONTENTS Page Abstract ………………………………………..……….…………….…………... ii Acknowledgements …………………………..……….………………….………. v Vita …………………………………………..……….…………….…………….. vi List of Tables ………………………………………………………………….….. x List of Figures ………………...……………………………………………...…… xi Chapters: 1. Literature Review …………………………………………………...…….. 1 1.1 Abstract ...………………………………………………………...…… 2 1.2 Introduction ……………………………………………………..……. 3 1.3 MFSV and MMV genome organization ……………...……........……. 6 1.4 Gene expression and genome replication ……………...…...…….…... 7 1.5 Protein function ………………………………………......…………... 9 1.6 Virus-insect interactions ………………………………...….…...……. 15 1.7 Virus-plant interactions ………………………………...……....…….. 22 1.8 Research objectives ……………………………….……….......……... 23 1.9 References …………………………………………………......……... 25 2. Complete genome sequence and in planta subcellular localization of Maize fine streak virus proteins …………..…….………………………… 36 2.1 Abstract ………………………..……………………………………… 37 2.2 Introduction …………………………………………………………… 38 2.3 Materials and methods ………………………………………………... 40 2.4 Results ………………………………………………………………… 48 2.5 Discussion …………………………………………………………….. 54 2.6 Acknowledgments …………………………………………………….. 58 2.7 References……………………………………………………………... 60 viii 3. Shotgun sequencing of the negative-sense RNA genome of the rhabdovirus Maize mosaic virus …………..……………………………… 74 3.1 Abstract ………………………..……………………………………… 75 3.2 Introduction …………………………………………………………… 76 3.3 Materials and methods ………………………………………………... 77 3.4 Results and discussion ……….……………………..………………… 80 3.5 Acknowledgments …………………………………………………….. 87 3.6 References……………………………………………………………... 88 4. Subcellular localization and nuclear import of Maize fine streak virus and Maize mosaic virus proteins …………..……………………….…………. 96 4.1 Abstract ………………………..……………………………………… 97 4.2 Introduction …………………………………………………………… 98 4.3 Materials and methods ………………………………………………... 100 4.4 Results ………………………………………………………………… 105 4.5 Discussion …………………………………………………………….. 110 4.6 Acknowledgments …………………………………………………….. 113 4.7 References……………………………………………………………... 114 5. Sigma rhabdovirus activates the innate immune response of drosophila …. 123 5.1 Abstract ………………………..……………………………………… 124 5.2 Introduction …………………………………………………………… 125 5.3 Materials and methods ………………………………………………... 128 5.4 Results ………………………………………………………………… 132 5.5 Discussion …………………………………………………………….. 138 5.6 Acknowledgments …………………………………………………….. 143 5.7 References……………………………………………………………..
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