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(MFSV) Gene Expression and Protein Interaction Maize fine streak virus (MFSV) Gene Expression and Protein Interaction DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Fiorella Melina Cisneros Delgadillo Graduate Program in Plant Pathology The Ohio State University 2013 Dissertation Committee: Professor Feng Qu, Adviser Professor Margaret Redinbaugh, Co-adviser Professor Omprakash Mittapalli Professor Christopher Taylor Copyrighted by Fiorella Melina Cisneros Delgadillo 2013 Abstract Maize fine streak virus (MFSV) belongs to the family Rhabdoviridae, and is transmitted by the leafhopper Graminella nigrifrons. The MFSV genome is a 13,782 nucleotide, non- segmented, negative-sense RNA that encodes five core structural proteins: the nucleocapsid protein (N), the phosphoprotein (P), the polymerase (L), the matrix protein (M) and the glycoprotein (G) and two non-structural proteins, MFSV 3 and 4. The genes follow the order 3´-N-P-3-4-M-G-L-5’. Functions for the MFSV N, P, M, G, and L genes have been assigned based on sequence homologies to other Rhabdoviridae, but functions for the non-structural MFSV 3 and 4 genes remain unknown. To begin to define the roles of the genes and proteins encoded by the MFSV 3 and 4 in replication and systemic infection, we analyzed the accumulation of their corresponding transcripts in maize and G. nigrifrons using RT-qPCR assays. We hypothesized that the expression pattern for the MFSV 3 and 4 would provide an indication of their relative importance in virus replication and movement in animal and plant host. To determine whether the MFSV 3 and 4 protein function requires interaction with other MFSV proteins (e.g., for virus movement in plants), a protein-protein interaction map was generated by means of yeast two-hybrid (YTH) and bimolecular fluorescence complementation (BiFC). To determine the potential of MFSV to be engineered as gene expression system, we examined the expression of viral and reporter components required to produce infectious ii MFSV in Drosophila S2 cells. The reporter, driven by a T7 promoter, was designed to contain MFSV sequences important for replication. Expression of the MFSV N and P genes, and the T7 DdRp was detected by western blot in S2 cells over a period of 4 days and only under inducible conditions, indicating that these proteins were produced in S2 cells. Also, co-transfection experiments indicated than more than one protein could be produced in S2 cells at the same time. However, the epression of the MFSV L gene, the other protein component required for replication of rhabdoviruses, could not be detected in S2 cells. We developed a robust and reproducible RT-qPCR assay for the specific quantification of each of the seven MFSV genes using oligo(dT) primers for cDNA synthesis and primers designed to have high amplification efficiency and specificity for each of the seven MFSV genes. We used the RT-qPCR assay to determine the abundance of MFSV P, 3, 4, M, G and L transcripts relative to the MFSV N transcripts in infected maize and G. nigrifrons. CT values for each gene were normalized to the reference 18S RNA in maize plants and to the Ribosomal protein S13 gene RPS13 in insects and the accumulation of transcripts was analyzed by the comparative CT method. Higher levels of the MFSV P and 3 transcripts were found relative to N transcripts at each week, whereas temporal changes in the accumulation of the MFSV M, G and L transcripts were found from week two to week four of infection. Interestingly, the accumulation of MFSV 4 transcripts remained similar to the N transcripts from week two to four. In insects, the accumulation of MFSV P, 3, M and G transcript levels were significantly higher than those for N iii transcripts, whereas MFSV 4 and L transcripts accumulated at lower levels. Our results indicate that the regulation of MFSV gene transcription is different from that of animal- infecting rhabdoviruses and that MFSV has alternative means for regulating gene expression in insects and plants. To shed lights into the associations between MFSV proteins, a protein-protein interaction map for MFSV 3 and 4 proteins was generated by means YTH and BiFC assays. The MFSV 3 and 4 proteins strongly interacted with each other in both assays, suggesting that this interaction is likely to occur in nature. No interaction of the MFSV 3 or 4 proteins was detected with the MFSV N, P, M or G proteins in either assay, suggesting that these interactions are not important during infection. Based on the location of MFSV 4 in the genome, the size of the encoded protein and higher accumulation of transcripts in the plant compared to the insect host, MFSV 4 may be a movement protein, and its strong interaction with MFSV 3 may be important during infection of the plant host. In addition, the interaction of the MFSV N and P proteins was detected by BiFC, but not by YTH assays. Because N-P interaction is conserved across the Mononegavirales, we expect that the MFSV N – MFSV P interaction occurs in nature. iv To my parents, Guillermo and Mercedes, and my husband, Jeffrey, for their unconditional love and support v Acknowledgments I would like to thank to the Department of Plant Pathology for giving me the opportunity to pursue my academic goals. I would also like to thank to my adviser, Dr. Margaret Redinbaugh, for her intellectual guidance and constant support on my path to become a scientist. I am very grateful for her encouragement and assistance on every step of my graduate studies. I am also grateful to my co-adviser, Dr. Feng Qu, and my advisory committee members, Dr. Omprakash Mittapalli and Christopher Taylor for their advice and support. Finally, I would like to thank to the current and past members of my lab Kristen Willie, Jane Todd, Chris Nacci, Braden Conn, Jose Luis Zambrano, Dr. Valdir Correa, Dr. Chi Wei Tsai, Dr. Lucy Stewart and Dr. Bryan Cassone for their help and friendship. vi Vita 2003................................................................B.S. Biology, San Marcos Major National University, Peru 2011................................................................M.A. Science, The Ohio State University 2008 to present ..............................................Graduate Research Associate, Department of Plant Pathology, The Ohio State University Fields of Study Major Field: Plant Pathology vii Table of Contents Abstract ............................................................................................................................... ii Acknowledgments.............................................................................................................. vi Vita .................................................................................................................................... vii List of Tables .......................................................................................................................x List of Figures .................................................................................................................... xi Chapter 1: Literature Review ..............................................................................................1 1.1. Introduction .......................................................................................................... 1 1.2. Classification ........................................................................................................ 2 1.3. MFSV transmission .............................................................................................. 2 1.4. Virion morphology ............................................................................................... 3 1.5. MFSV genome organization ................................................................................ 4 1.6. MFSV transcription and replication ..................................................................... 5 1.7. MFSV structural proteins ..................................................................................... 6 1.7.1. The nucleocapsid protein (N)........................................................................ 6 1.7.2. The phosphoprotein (P)................................................................................. 8 1.7.3. The matrix protein (M) ................................................................................. 9 1.7.4. The glycoprotein (G)................................................................................... 10 1.7.5. The polymerase protein (L) ........................................................................ 10 1.7.6. MFSV non-structural proteins .................................................................... 11 1.8. MFSV-insect-plant interaction ........................................................................... 14 1.9. Plant viruses as gene expression systems ........................................................... 17 1.10. References ............................................................................................................ 19 Chapter 2: Development of a reverse genetics system for Maize fine streak virus (MFSV)…………………………………………………………………………………..26 2.1. Abstract .............................................................................................................. 26 2.2. Introduction ........................................................................................................ 26 2.3. Material and Methods........................................................................................
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