RNA Interference Pathway Regulate Persistent Infection Of Rice Ragged Stunt Virus In Cultured Cells Of Nilaparvata Lugens

Rice ragged stunt virus (RRSV), a typical member of Oryzavirus in Spinareovirinae of Reoviridae, is transmitted by brown planthopper (BPH; Nilaparvata lugens) in a persistent propagative manner with nontranovarial transmission.Previous studies confirm the nonstructural protein Pns6 and Pns10 of RRSV participate viral replication using cultured cells of Nilaparvata lugens, and Pns7 play a significant role in viral spread. The sequential infection of RRSV in insect vector of BPH is also revealed. In these processes, RRSV not only overcome the tissue and membrane barriers in insect vector, but also escape from the various mechanisms of antivirus, such as RNA interference (RNAi). RNAi is a transcriptional gene silencing phenomena caused by mRNA degradation of the homologous gene by the endogenous or exogenous double-stranded RNA, which is an efficient form of antiviral defense for organism, and a way of protecting genome of organism from exogenous gene.In order to address whether replication and proliferation of RRSV was regulated by the RNAi in insect vector of BPH, the influence of genes expression in RNAi antiviral pathway was analyzed by real-time fluorescent quantitative PCR (RT-qPCR) assay. The results demonstrated that the infection of RRSV induced the relative expression of related genes in RNAi antiviral pathway were up-regulated, such as argonaute 1 (Agol), argonaute 2 (Ago2), argonaute 3 (Ago3), Dicer 1, Dicer 2, Eri-like-a (Eri-la), Eri-like-b (Eri-lb), Piwi, and systemic RNA interference defective-1 (SID-1). Then cultured cells of BPH were transfected dsRNAs synthesized in vitro, which were specific for RNAi pathway, then infected by RRSV. Immunofluorescence assay showed that knockdown genes of Dicer2 and Ago2 in siRNA pathway significantly improved the infection of RRSV in cultured cells of BPH, but knockdown genes of Dicer 1 and Agol in miRNA pathway, Piwi and Ago3 in piRNA pathway, Eri-la gene and Eri-lb had no effect on infection of RRSV. These results revealed that the siRNA pathways of BPH was possible to involve in regulation of RRSV infection and replication in BPH culture cell, but miRNA pathways and piRNA pathways may have no relationship with RRSV infection. RT-qPCR results also confirmed that dsRNA transfection derived from Dicer2 and Ago2 significantly increased the accumulation of RRSV P8 gene, while transfection other dsRNA had no significant effect on the accumulation of RRSV P8 gene, which are dsAgo1, dsAgo3, dsDicerl, dsEri-la, dsEri-lb, dsPiwi and dsSID-1.This study initially investigated antiviral immune response in BPH, and proved that siRNA was an efficient way of regulating infection replication and proliferation of RRSV in BPH cultured cells, and Dicer2 and Ago2 were the key factors of this pathway. While the miRNA pathways and piRNA pathways may not be involved in regulating infection, replication and proliferation of RRSV. These results will base for confirming the mechanism of RNAi subjected by plant virus in insect vector, analyzing the mechanism of co-evolution of virus and its vector insect, and controlling rice ragged stunt virus disease caused by RRSV in the method of blocking viral transmission by BPH.