Functional Analyses On The Mechanisms Underlying The Pathogenesis Of The Rice Stripe Virus Gene NS2

Rice stripe disease is one of the most significant rice viral diseases, endangeringrice production of China. This disease is caused by Rice stripe virus (RSV), a typicalmember of the genus Tenuivirus. With the improvement of molecular biotechnologyin recent years, researches on RSV have been gradually advanced. Especially, afterdiscovery that two model plants, Nicotiana benthamiana (N. benthamiana) andArabidopsis thaliana (A. thaliana) can be infected by RSV, some difficulties andobstacles of researches in this field, such as long time requirment for growing rice andconstructing transgenic plants, were overcome. It has been reported that NS2, encodedby RSV, is a weak gene silencing suppressor, localizing in nucleus and interactingwith OsSGS3. SGS3, a type of relatively conservative gene, is a gene silencingsuppressor encoded by plant itself, and SGS3mutant A.thaliana is more susceptible tovirus. However, the functional mechanism of NS2during RSV infection on host plantsand the role of OsSGS3in RSV resistance are still unclear. In this PhD research, N.benthamiana and virus inducing gene silencing (VIGS) method were used to analyzethe pathogenicity functions of NS2and disease resistance conferred by OsSGS3toRSV; OsSGS3-RNAi transgenic plants were also constructed, laying the foundationfor further dissection of role of OsSGS3in rice resistance to RSV.This dissertation comprises three parts of researches. In the first part,co-localization and BiFC analyses were conducted to verify the interaction of NS2with cajal body and nucleolus proteins in three different hosts, A. thaliana, N.benthamiana and rice. In the second part, the NbFib2and NS2genes were silenced byVIGS, then the silenced plants were infected with RSV, thereby the function of thesetwo genes in RSV infection was analyzed. In the last part, the function of SGS3toRSV was analyzed by silencing NbSGS3, a homolog of OsSGS3, in N. benthamianaand challenging the silenced plants with wild type RSV using the same approache asthe second part; OsSGS3-RNAi transgenic rice was constructed for further studies.Following is the summary of the research:1. The subcellular localization of NS2varied over time.The protein first localizesin the nucleus and then migrates outside the nucleus. The subcellular localization ofNS2and the interaction between NS2and the cajal body and nuclelous proteins fromthree different hosts were identified. The co-localization results showed that NS2 localized in the cajal body and nucleolus. The BiFC assay revealed that NS2interacted with Atcoilin, NbFib2and OsFib2from three hosts.2. The roles of NS2and NbFib2in RSV infection were identified. RSV can bedetected in the leaves of NS2-silenced plants or NbFib2-silenced plants inoculatedwith the virus, but not in uninoculated leaves of the same silenced plants. Theaccumulation of RSV in inoculated leaves of silenced plants was less than in theun-silenced plants (CK) that were the inoculated only with Agrobacterium containingempty VIGS vector, suggesting that NS2and NbFib2play important roles in RSVreplication and infection. NbFib2-silenced also altered the subcellular localization ofNS2in the earlier stage. Granules induced by NS2localized not only in nucleus, butalso in other organelles. These results indicate that both NS2and NbFib2are involvedin RSV systemic infection, NS2migrates into nucleus with assistance to completeRSV replication and systemic infection.3. In comparison with the CK plants, the NbSGS3-silenced plants were moresusceptible to RSV infection as indicated by both latent period and diseased severity.Based on this result, OsSGS3-RNAi transgenic T0rice was constructed, laying afoundation for further characterization of the OsSGS3-conferred disease-resistance.