Idenfidication Of Key Factor For SRBSDV Multiplication In Sogatella Furcifera With RNA Interference Strategy

Southern rice black-streaked dwarf virus (SRBSDV), a recent identified member of the genus Fijivirus in the family Reoviridae, is transmitted by the white-backed planthopper (WBPH) (Sogatella furcifera Horvath) in a persistent-propagative manner and is not tansovarially transmitted. The Southern rice black-streaked dwarf disease caused by Southern rice black-streaked dwarf virus (SRBSDV) is one of the important rice virus diseases and spread rapidly throughout southern China, northern Vietnam, and western Japan. The non-structure viral proteins are considered to be important to viral replication, assemble and spreading. The non-structure proteins P5, P6 and P9-1 are the constituents of the matrix of viroplasm, which was the site for replication and assemble of progeny virions. However, specific function of them in the formation of viroplasm and viral replication and package is not identified. RNA interference induces the degradation of target mRNA by the introduction of long dsRNA, which plays a fundamental role in antiviral defense in insect, including plant reovirus. to reduce the expression of some genes of virus based on RNAi mechanism is mechanism to reduce it expression can offer a new method to control the viral disease. In this study, with RNA interference strategy, we aim to find an important gene from SRBSDV which can idenfidicate origin of viral replication in bodies of WBPH, to provide a theoretical basis for controlling the disease.This study firstly amplify the gene P5-1 N-terminal 1-750 bp by RT-PCR, and construct a Gateway technology-based prokaryotic expression vector, then we express the specific protein to inject New Zealand white rabbit for polyclonal antibody. Protein of P5-1 was detected in infected rice plants using western blot and viruliferous WBPH using immunofluorescence. confirming the specificity of polyclonal antibody against P5-1. This study choose P5, P6 and P9-1 protein as object, we synthesized double RNA in vitro with T7 reversetranscripatase. At 2-day post-first access to diseased plants, WBPH were microinjected with 0.5μg/μL dsRNA or dsRNA targeting GFP gene (dsGFP).6 days later, stained with related fluorescence antibody. Confocal microscopy revealed that inhibiting the expression of P5-1, P6 and P9-1 could inhibit the expression related of genes P5-1, P6, P9-1, P7-1 and P10 in Sogatella furcifera, and the virus is limited to primary infection in intestinal epithelial cells. After inhibiting expression of P5-1 protein, P6 and P9-1 protein still can be expressed, but just in intestinal epithelial cells. P7-1 and P10 protein were significantly inhibited. We considered that P5-1 protein was related with assembly of viral particle, and was involved in the expression of coat protein and diffusion-related protein, which could block viral assembly and spread. If P6 protein is inhibited, P6 was not detected in 84% of insects, and P5-1, P9-1, P7-1 and P10 protein were not also detected. While P6 protein was found in 16% of intestinal epithelial cells, P5-1, P9-1, P7-1 and P10 protein were also found. This indicated that.P6 protein might start viral multiplication, and if a little of P6 protein was expressed, virus could multiplicate. After P9-1 protein was inhibited, P6 was still expressed in intestinal epithelial cells, but P7-1 and P10 protein were also inhibited, while P5-1 was inhibited. This indicated that P9-1 protein, involved in viral genome replication, was inhibited, then P5-1 and P10 protein which were related with viral assembly, were inhibited. At the same time, because virus can not be assembled, P7-1 protein, component of tubular structure, was not expressed. Furthermore, in this study, we aimed to clear that dsRNA have an effect on viral multiplication. The results showed that inhibiting the expression of P5-1, P6 and P9-1 could reduce the copynumber of gene PIO encoding viral coat protein. This indicadited dsRNA block viral replication and multiplication. Analysis of Western blot also found inhibiting respectively expression of P5-1, P6 and P9-1 protein can obviously inhibited expression of other related protein. After inhibiting expression of P5-1 protein, P6 and P9-1 protein were little detected, and P10 protein was not almost detected. Other related proteins were not detected after inhibiting expression of P9-1 protein. Only P6 was detectd marginally except other related protein.In conlusion, respectively inhibiting the expression of some gene can significantly inhibited viral replication and diffusion in vector insects with RNAi induced by dsRNA, suggesting P5-1, P6 and P9-1 were important protein for SRBSDV multiplication in insects. We suggested that P5-1 protein was place of viral assembly, and inhibiting its expression regulated and controled expression of coat protein and tubular-structural protein P7-1. P9-1 protein may be place of viral genome replication, and inhibiting its expression could regulate indirectly viral assembly-related protein, not expressed. P6 protein, another component of viroplasm, played a vital role on viral multiplication, and start the viral primary infection in epithelial cell. Inhibiting expression of P6 protein could block that of all viral protein. Therefore we suggested P6 protein was the most key-factor of viral multiplication, and play a role on starting formation of viroplasm, which could serve as a proper target to block viral preventation and control.