University of Kentucky UKnowledge University of Kentucky Doctoral Dissertations Graduate School 2011 COMPARISON OF PLANT‐ADAPTED RHABDOVIRUS PROTEIN LOCALIZATION AND INTERACTIONS Kathleen Marie Martin University of Kentucky, [email protected] Right click to open a feedback form in a new tab to let us know how this document benefits ou.y Recommended Citation Martin, Kathleen Marie, "COMPARISON OF PLANT‐ADAPTED RHABDOVIRUS PROTEIN LOCALIZATION AND INTERACTIONS" (2011). University of Kentucky Doctoral Dissertations. 172. https://uknowledge.uky.edu/gradschool_diss/172 This Dissertation is brought to you for free and open access by the Graduate School at UKnowledge. It has been accepted for inclusion in University of Kentucky Doctoral Dissertations by an authorized administrator of UKnowledge. For more information, please contact [email protected]. ABSTRACT OF DISSERTATION Kathleen Marie Martin The Graduate School University of Kentucky 2011 COMPARISON OF PLANT‐ADAPTED RHABDOVIRUS PROTEIN LOCALIZATION AND INTERACTIONS ABSTRACT OF DISSERTATION A dissertation submitted in partial fulfillment of the requirements for the Degree of Doctor of Philosophy in the College of Agriculture at the University of Kentucky By Kathleen Marie Martin Lexington, Kentucky Director: Dr. Michael M Goodin, Associate Professor of Plant Pathology Lexington, Kentucky 2011 Copyright © Kathleen Marie Martin 2011 ABSTRACT OF DISSERTATION COMPARISON OF PLANT‐ADAPTED RHABDOVIRUS PROTEIN LOCALIZATION AND INTERACTIONS Sonchus yellow net virus (SYNV), Potato yellow dwarf virus (PYDV) and Lettuce Necrotic yellows virus (LNYV) are members of the Rhabdoviridae family that infect plants. SYNV and PYDV are Nucleorhabdoviruses that replicate in the nuclei of infected cells and LNYV is a Cytorhabdovirus that replicates in the cytoplasm. LNYV and SYNV share a similar genome organization with a gene order of Nucleoprotein (N), Phosphoprotein (P), putative movement protein (Mv), Matrix protein (M), Glycoprotein (G) and Polymerase protein (L). PYDV contains an additional predicted gene between N and P, denoted as X, that has an unknown function. In order to gain insight into the associations of viral proteins and the mechanisms by which they may function, we constructed protein localization and interaction maps using novel plant expression vectors. Sub‐cellular localization was determined by expressing the viral proteins fused to green fluorescent protein in leaf epidermal cells of Nicotiana benthamiana. Protein interactions were tested in planta using bimolecular fluorescence complementation (BiFC). All three viruses showed Mv to be localized to the cell periphery and the G protein to be membrane associated. Comparing the interaction maps revealed that only the N‐P and M‐M interactions are common to all three viruses. Associations unique to only one virus include G‐Mv for SYNV, M‐Mv, M‐G, and N‐M for PYDV and P‐M for LNYV. The cognate N‐P proteins of all three viruses exhibit changes in localization when co‐expressed. To complement the mapping data, we also mapped the functional domains in the glycoproteins of SYNV and LNYV. The truncation of the carboxy terminus has no effect on localization compared to the full‐length protein; the nuclear localization signals (NLSs) present in SYNV‐G do not interact with known importins. These data suggest that although the interactions of the three viruses differ, each protein may have similar functional domains. KEYWORDS: rhabdovirus, Nicotiana benthamiana, BiFC, interaction, localization Kathleen M Martin Student Signature April 28, 2011 Date COMPARISON OF PLANT‐ADAPTED RHABDOVIRUS PROTEIN LOCALIZATION AND INTERACTIONS By Kathleen Marie Martin Dr. Michael M Goodin Director of Dissertation Dr. Lisa J. Vaillancourt Director of Graduate Studies April 28 , 2011 Date RULES FOR THE USE OF DISSERTATIONS Unpublished dissertations submitted for the Doctor's degree and deposited in the University of Kentucky Library are as a rule open for inspection, but are to be used only with due regard to the rights of the authors. Bibliographical references may be noted, but quotations or summaries of parts may be published only with the permission of the author, and with the usual scholarly acknowledgments. Extensive copying or publication of the dissertation in whole or in part also requires the consent of the Dean of the Graduate School of the University of Kentucky. A library that borrows this dissertation for use by its patrons is expected to secure the signature of each user. Name Date DISSERTATION Kathleen Marie Martin The Graduate School University of Kentucky 2011 COMPARISON OF PLANT‐ADAPTED RHABDOVIRUS PROTEIN LOCALIZATION AND INTERACTIONS DISSERTATION A dissertation submitted in partial fulfillment of the requirements for the Degree of Doctor of Philosophy in the College of Agriculture at the University of Kentucky By Kathleen Marie Martin Lexington, Kentucky Director: Dr. Michael M Goodin, Associate Professor of Plant Pathology Lexington, Kentucky 2011 Copyright © Kathleen Marie Martin 2011 TABLE OF CONTENTS LIST OF TABLES ......................................................................................................................................... v LIST OF FIGURES ....................................................................................................................................... vi CHAPTER I .................................................................................................................................................... 1 A review of Agrobacterium‐mediated plant vector technologies. ........................................ 1 History of Agrobacterium ............................................................................................................. 1 History of the development of Agrobacterium Vectors .................................................. 2 EXPRESSION VECTORS....................................................................................................................... 4 SILENCING VECTORS ....................................................................................................................... 11 CONCLUDING REMARKS ................................................................................................................ 13 CHAPTER II* .............................................................................................................................................. 40 Transient expression in Nicotiana benthamiana fluorescent marker lines provides enhanced definition of protein localization, movement and interactions in planta. 4 0 METHODS .............................................................................................................................................. 41 RESULTS ................................................................................................................................................ 44 DISCUSSION .......................................................................................................................................... 48 CHAPTER III* ............................................................................................................................................ 70 Membrane and protein dynamics in live plant nuclei infected with Sonchus yellow net virus, a plant‐adapted rhabdovirus. ........................................................................................ 70 METHODS .............................................................................................................................................. 72 RESULTS ................................................................................................................................................ 74 DISCUSSION .......................................................................................................................................... 77 CHAPTER IV .............................................................................................................................................. 90 Comparison of the functional domains of Sonchus yellow net virus and Lettuce necrotic yellows virus glycoproteins. ............................................................................................ 90 METHODS .............................................................................................................................................. 91 RESULTS ................................................................................................................................................ 92 DISCUSSION .......................................................................................................................................... 94 CHAPTER V .............................................................................................................................................. 105 Lettuce necrotic yellows virus protein localization and interaction map and comparison to two nucleorhabdoviruses, Sonchus yellow net virus and Potato yellow dwarf virus. ............................................................................................................................... 105 METHODS ............................................................................................................................................ 106 RESULTS .............................................................................................................................................. 106 DISCUSSION .......................................................................................................................................