Molecular interactions between Maize fine streak virus and insect vector, Graminella nigrifrons DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Yuting Chen, M.S. Graduate Program in Entomology The Ohio State University 2013 Dissertation Committee: Dr. Andrew P. Michel, Advisor Dr. David L. Denlinger Dr. Omprakash Mittapalli Dr. Margaret G. Redinbaugh Copyrighted by Yuting Chen 2013 Abstract Phytophagous hemipteran insects are suitable vectors for plant viruses, such as plant-infecting rhabdoviruses, which have caused severe yield loss. These single-stranded negative RNA viruses are specifically transmitted by hemipteran insects in a circulative propagative manner. My research investigated the vector-virus interactions using the black-faced leafhopper Graminella nigrifrons and an emerging plant-infecting rhabdovirus, Maize fine streak virus (MFSV) from molecular, genetic and ecological perspectives. G. nigrifrons is the only identified vector for MFSV, and vector competence varies within laboratory populations. G. nigrifrons can be experimentally separated into three types based on their ability to transmit MFSV: transmitters, which can transmit MFSV to new host plants; acquirers, which are positive MFSV, but do not transmit the virus; and non-acquirers, which are neither positive for nor able to transmit the virus. My research focused on how antiviral immunity responds to MFSV challenge among different types of G. nigrifrons, which was proposed to associate with vector competence. The transcriptome of G. nigrifrons was first characterized, and a significant similarity of immune response transcripts was discovered with other well-characterized insects. Expression of ten transcripts that putatively functioned in insect RNAi and humoral pathways was evaluated among three types of G. nigrifrons using RT-qPCR: Ars-2, Dcr-2, Ago-2, four PGRPs (SB1, SD, LB and LC), Toll, ii spaetzle and defensin. Overall down regulation was seen in MFSV challenged leafhoppers. In particular, Ars-2, Dcr-2 and Ago-2 were significantly suppressed in acquirers and non-acquirers compared to transmitters or control (MFSV unchallenged) leafhoppers. Expression of three PGRPs (SB1, SD, and LC) and Toll were similar in all MFSV challenged leafhoppers but was significantly suppressed compared to control. Genetic variation of RT-qPCR evaluated transcirpts was analyzed among three types of G. nigrifrons. A non-synonymous SNP in the MFSV-N gene was identified only in transmitters. However, there was no correlation between differentially expressed immune transcripts and the presence of putative synonymous/non-synonymous SNPs. RNAi was successfully developed to investigate the functions of PGRP-LC and Dcr-2 related to virus acquisition and transmission. Expression of PGRP-LC and Dcr-2 was reduced to 30% and 20% of levels in control leafhoppers respectively, after 14 and 22 dpi of dsRNAs. The reduction in expression was equally effective for both nymphs and adults. There was no effect on MFSV transmission or acquisition between non-injected or dsRNAs for GFP and PGRP-LC or Dcr-2 injected leafhoppers, however, acquisition was slightly higher in Dcr-2 silenced leafhoppers. Silencing PGRP-LC resulted in 90% mortality before MFSV could be transmitted. A significantly higher number of ‘abnormally molted’ leafhoppers were observed after silencing PGRP-LC. Higher temperature and light intensity tended to increase MFSV transmission during 1-week inoculation access period. There was no difference of transmission between insect genders under different environmental treatments. To conclude, my research revealed complex vector-virus-plant interactions at the molecular, genetic and environmental levels. Successful use of RNAi to decrease G. iii nigrifrons transcript levels may provide possible targets of RNAi-based pest management. iv Acknowledgments I especially would like to thank my advisor, Andy Michel, for his endless support and help with my study and research. Any growth, improvement or award from my research and even my English, could not have been achieved without his patient and kind guidance. I appreciate his allowing me do the research that I am really interested in. He is a truly wonderful advisor, and I count myself lucky to have been his student! I would like to thank my advisory committee members, David Denlinger, Omprakash Mittapalli and Margaret (Peg) Redinbaugh for their advice and support to this project. I really enjoyed our discussion about this project during every meeting. I appreciate the helpful and professional advice from Luis Canas regarding my statistical analysis. I appreciate Dan Herms and Larry Phelan for teaching the classes “Nature and Practice of Science” and “Journal Club”, from which I learned some of the most important things in my academic career. I appreciate Peter Piermarini and Nuris Acosta for helping me develop the RNAi technique using micro-injection. I also appreciate the helpful bioinformatics expertise of Bryan Cassone, Asela Wijeratne, Saranga Wijeratne and Xiaodong Bai. I also wish to thank Feng Qu and Lucy Stewart for their constructive advice during our group meeting. I am very grateful to Jane Todd for teaching me, from the very beginning, how to rear and maintain the G. nigrifrons colony in the laboratory and transfer MFSV using G. nigrifrons. Without these basic techniques, my project could not have succeeded. I enjoyed our discussion about research, and I also appreciate that she shared her v limited space in the growth chamber with me. I appreciate Kristen Willie for providing me MFSV antiserum and other reagents for my virus experiment. I also wish to thank Lee Wilson, Bob James and Judy Smith for arranging the greenhouse and growth chamber space for me in Selby and Thorne Hall. I thank Brenda Franks, Lori Jones and Shirley Holmes for their administrative support. I also thank my colleagues in both Andy and Peg’s lab for their technical help and friendship. Finally, I deeply appreciate the OSU Entomology Department for providing me this opportunity to study here. This department is such a wonderful family, which has made me feel at home in the US. The endless support received from other students and faculty helped make a significant improvement in not only my English, but also my study and research, and their friendship helped me get through every difficulty. I consider myself extremely fortunate to have been a part of this department. This project was partially funded by The OARDC Research Enhancement Competitive Grants Program. vi Vita 2001 − 2005……………………………… .. B.S. Plant Protection, China Agricultural University 2005 − 2008………………………………...M.S. Plant Pathology, China Agricultural University 2009 to present .............................................Entomology, The Ohio State University Publications Chen, Y., Cassone, B.J., Bai, X., Redinbaugh, M.G., Michel, A.P., 2012. Transcriptome of the plant virus vector Graminella nigrifrons, and the molecular interactions of Maize fine streak rhabdovirus transmission. Plos One 7, e40613 Cheng, Y.-Q., Liu, Z.-M., Xu, J., Zhou, T., Wang, M., Chen, Y.-T., Li, H.-F., Fan, Z.-F., 2008. HC-Pro protein of sugar cane mosaic virus interacts specifically with maize ferredoxin-5 in vitro and in planta. Journal of General Virology 89, 2046-2054. Fields of Study Major Field: Entomology vii Table of Contents Abstract ......................................................................................................................... ii Acknowledgments..........................................................................................................v Vita .............................................................................................................................. vii Table of Contents ....................................................................................................... viii List of Figures ................................................................................................................x List of Tables ............................................................................................................... xi Chapters: 1. Introduction ..............................................................................................................1 Biology of G. nigrifrons ........................................................................................ 2 Biology of plant-infecting rhabdoviruses .............................................................. 4 Interaction between plant-infecting rhabdoviruses and insect vector .................... 6 Interaction between MFSV and Graminella nigrifrons ......................................... 8 References ............................................................................................................ 10 2. Transcriptome of the plant virus vector Graminella nigrifrons, and the molecular interactions of Maize fine streak virus transmission ....................................................18 Abstract ................................................................................................................ 18 Introduction .......................................................................................................... 19 Methods and Materials ......................................................................................... 23 Results and Discussion .......................................................................................