Studies On The Mechanisms Underlying The Pathogenesis Of RSV NSvc4

Since the 1960s, rice stripe disease found in Jiangsu province, has been endangering rice production in China, especially it broke out in rice growing areas of our country in the years 2004 and 2005. Although rice stripe disease incidence area decreased in recent years for the comprehensive prevention and control measures, it is likely to break out again due to some features of the pathogens, thus it is still threatening China’s grain production. Rice stripe virus (RSV), the member of Tenuivirus, is the pathogens of rice stripe disease. Over the years, by scholars long-term study defined the biological characteristics of RSV, such as host range, serological, pathogenic differentiation, and genetic diversity etc. At the molecular level, it is known the genome structure of RSV encoding 7 proteins, but its pathogenic mechanism has not been understood. Therefore, it has an important practical significance to find the causative agent and clarify the pathogenesis of RSV on the molecular level to control and prevent of Rice stripe virus, for the purpose of reducing the rice stripe disease threat to Chinese grain production.Firstly, this study made a series of experiments to identify the pathway of NSvc4 protein localizing from the cytoplasmic to plasmodesmata. We found that in the N. benthamiana cell the early secretory pathway and an actin-myosin VIII motility system were required for plasmodesmatal localization of the NSvc4 protein.For the pathogenesis, we mechanical inoculated N. benthamiana with crude extracts from leaves with typical symptom of rice stripe disease.12 dayap.i., we observed the symptoms mosaic, leaf malformation and multi-branch on inoculated N. benthamiana. With constructed plant localization vectors of NSvc4 NS2 NSvc2-N (the NSvc2 the N-terminal 380 amino acids) NS3 CP and SP protein of RSV, we found that NS2 protein localized in the cell nucleus and membrane, NSvc2-N protein in the cytoplasm as mobile particles, NS3 protein in the nucleus, CP protein in the cytoplasm as large mobile particles, SP protein in the cytoplasm, respectively. It is worth to memtion that NSvc4 protein targeted to plasmodesmata and chloroplast, which was the only one protein of RSV could target to the chloroplast. Through Bimolecular fluorescence complementation (BiFC) experiment, we found the NSvc4 protein could interact with CP protein and verified the result by membrane protein yeast two-hybrid screen (YTHS) test. We constructed NSvc4 CP and NSvc4CP (Chimeric protein, N-terminal was NSvc4) into PVX vector respectively and inoculated N. benthamiana by agrobacterium-mediated to studied the pathogenic function of them. It was shown that the PVX-RSV-NSvc4 caused mosaic symptoms; PVX-RSV-CP caused serious mosaic symptoms on inoculated leaves but no symptoms on system leaves; PVX-RSV-NSvc4&PVX-RSV-CP co-inoculated N. benthamiana and PVX-RSV-NSvc4CP inoculated N. benthamiana either inoculated leaves and system leaves developed into serious symptoms. Finally, the Real-time PCR experiments analysis and pathogenicity verification were carried out on the molecular level.Finally, we also used NSvc4 as bait protein to screen Arabidopsis library, which is proved to be one of the RSV host recently, in order to study the virus and plant host interaction Preliminary result of library screening was 35 yeast stains were obtained which could grow on select medium and turned the buffer into blue in colorful testing. Extracted the plasmids from transformed E. coli were digested by restriction enzyme, and then 33 eligible plasmids were sent to sequencing. Sequence analysis revealed three plasmids was the same plasmids, and 64.5% in these 30 possible interacting proteins were membrane-associated protein, we selected 12 proteins to design primers for further experiments. Constructed YTHS and BiFC vectors of the 10 protein and identified the interactions. The results showed there were NN-76, NN-80, NN-104, NN-133 and NN-135 proteins could interact with NSvc4 protein. Then we found the proteins corresponding to the 5 protein in rice and design primers for Real-time PCR to detect the difference expressions of the 5 proteins in of healthy and RSV infected rice. The results showed that their expression levels of these 5 proteins in RSV infected rice increased compared to healthy rice, which suggested that replication and spread process of RSV in rice might require the participation of these proteins.To sum up this study, for the first time, we identified the pathway of NSvc4 protein localization to the plasmodesmata, verified the interaction between NSvc4 protein and CP protein and the interaction contributed to the pathogenicity of RSV, and described the symptoms of RSV infected N. benthamiana.