The Effect Of DsRNA Synthesized On Infection Of Rice Dwarf Virus In Cultured Cells Of Recilia Dorsalis

Rice dwarf disease is a common rice disease, caused by Rice dwarf virus (RDV) which belongs to Phytoreovius of Reoiridae, that affects the secure yield of rice seriously. Under natural conditions, RDV is transmitted through leafhoppers in a persistent-propagative manner, and can be transovarially transmitted. Recilia dorsalis, as an intermediary insect of RDV, has a low rate of infection in the field, while its cultured cells line formed by the embryo is compatible with RDV.RNAi (RNA interference) is an emerging technology, which can effectively reduce the amount of target gene expression and is widely used in antiphytoviral research. The RDV induces the formation of viroplasm matrix (mainly consisting of non-structural protein Pns6 and Pns12) related to viral proliferation and tubular structure (consisting of non-structural protein Pns1O) related to viral transportation. This study firstly establishes the infection system of RDV on R. dorsalis, and observes the fixed position of RDV and its encoding Pns6, Pns10 and Pns12 in cells by immunofluorescence. Then dsRNAs of RDV fragments S6, S10 and S12 synthesized in vitro will be transfected into cells, and induce RNAi to affect the infection of RDV on cells. Through the technology of immunofluorescence observation, purification of viral dsRNA and western blot analysis, the effect of virus infect with cells transfected dsRNAs will be measured. The immunofluorescence results show that the three kinds of dsRNAs transfection can reduce RDV infection on cells and viral proteins expression. In addition, treatment on dsPns6 will slightly affects Pnsl2 expression, but the treatment on dsPns12 will completely affect Pns6 expression, illustrating that these two play a different role in the formation of viroplasm matrix. The Pns12 relates to the formation of viroplasm matrix, while Pns6 to the maturation. The result of western blot analysis also shows that dsPnslO can reduce the Pns10 tubular protein of RDV in cells, but the detection of viral dsRNAs shows no significant decreasing.Through this study, we can conclude that dsPns10 restrains the formation of tubule induced by RDV in cells of R. dorsalis to affect the viral transportation, thus confining the virus in the primarily infectious location. Both dsPns6 and dsPns12 can reduce the formation of viroplasm induced by RDV inside cells, thereby affecting the proliferation of the virus. In addition, Pnsl2 is related to the formation of viroplasm matrix, while Pns6 promotes viroplasm maturation. This study lays the foundation for further study of the interaction between the RDV with its mediator leafhoppers, and provides reference for disease prevention through polypide control with the established antiviral system of cultured cell RNAi.