Function Analysis Of Two Viral Suppressors Of Rna Silencing In Hosts

RNA silencing is a natural defense system against viral infection in plant by degrading the viral genomic RNAs. However, nearly all plant viruses can encode the viral suppressors of RNA silencing (VSR) to inhibit or escape this defense mechanism. Commonly, each VSR has the different mode to suppress RNA silencing. For example p19, the suppressor of Tomato bushy stunt virus (TBSV) functions by binding siRNA to prevents them into RISC, a core complex of RNA silencing mechanism. p69, the suppressor of Turnip yellow mosaic virus (TYMV) can bind double-stranded (ds)RNA to inhibit the initiation of RNA silencing.2b, the suppressor of Cucumber mosaic virus (CMV) shows the functions by targeting Argonaute (AGO) protein that is a core component of RISC. The previous reports have been shown that the ectopic expression of suppressors in plants disturbs the internal siRNA and miRNA-mediated RNA silencing, and as well as the expression profile of mRNAs, and causes the abnomal growth and development of plant. Meanwhile, some VSRs have no effect on plant. Here, we analyzed the interaction of p25, the suppressor of Potato virus X (PVX) and a host protein in Nicotiana benthamiana, and detected the effect of expression of p3, the suppressor of Rice stripe virus (RSV) to plant.p25of PVX is a multifunction protein. As the movement protein, p25interacts with other two movement proteins TGBp2and TGBp3, and three proteins together are responsible for PVX movement. As the suppressor of PVX, p25is proposed to be able to interact with AGO to inhibit the formation of RISC, and hence suppresses RNA silencing. Here, a gene encoding a potato type I serine PI in Nicotiana benthamiana, named NbPI, was identified to interact with p25by yeast two hybridization, Biomolecular fluorescence complementary (BiFC). Silencing of NbPI by PVX-induced gene silencing resulted in the serious viral symptom, and viral RNAs were accumulated in N. benthamiana. Meanwhile, plants overexpressing NbPI showed resistance to PVX infection. Further research revealed that expression of NbPI inhibited p25function as suppressor of RNA silencing and restricted its ability to help virus movement. We also found that PVX infection reduced the expression of NbPI. The results outline a model that NbPI participates plant defense against PVX infection by inhibiting the functions of p25, meanwhile, PVX could reduced NbPI expression by an unknown mode to antagonize the NbPI-mediated defense.Rice stripe virus (RSV), belonging to the genus Tenuivirus, contains four single stranded genomic RNAs (RNA1-4). Except RNA1that encodes the RNA-dependent RNA polymerase (RdRp) in the viral complementary strand, other three segments have ambisense polarity and each of them has two non-overlapping ORFs on opposite strands, separated by a noncoding intergenic region (IR). p3protein of RSV, encoded by viral strand of RNA3, is a viral suppressor of RNA silencing (VSR). In this study, we got transgenic rice plants by agrobacterium-mediated transformation, and showed that rice expressing p3had a normal pHenotype, was initially sensitive to RSV but recovered well at the later stages of infection. RSV accumulated slightly more in transgenic than in wild type plants at the early stage of infection, but accumulation was similar later. The results of array and real-time PCR detection showed the expression of microRNA and mRNA were affected in p3transgenic rice. The defense system mediated by NBS-LRR and PRs genes may be induced when p3expresses in rice. Transgenic rice expressing p3showed enhanced resistance to the bacterial blight (Xanthomonas oryzae) and rice blast disease (Magnaporthe oryzae). The defense system mediated by NBS-LRR and PR-4b genes may be induced in p3transgenic rice plants. However, the expressional levels of genes related to the salicylic acid (SA) and jasmonic acid (JA) pathways were not significantly altered in p3transgenic rice. So it was showed that p3transgenic rice contributes to the enhanced resistance against and Magnaporthe oryzae by a manner maybe independent of the SA and JA pathways. p3appears to exhibit dual functions, facilitating viral infection as a VSR and inhibiting pathogenic development as an inducer of host defense. We also obtained the p3transgenic Nicotiana benthamiana and used it for challenge of RSV inoculation, and found that the viral symptoms on transgenic plants were weak. And the incidence of disease and the accumulation of virus were reduced in p3transgenic Nicotiana benthamiana.RSV, has four genomic RNAs. RNAs2-4have an ambisense coding strategy and the noncoding intergenic regions (IRs) separating the two ORFs are thought to function in termination of transcription. Sequence the3’-untranslated region of transcripts from RNA1to RNA4in virus-infected Oryza sativa, Nicotiana benthamiana and LaodelpHax striatellus. The results showed that the sequences of p2and pc2transcripts on RNA2, and p4and pc4transcripts on RNA4terminated with high frequency at a palindromic sequence AUCCGGAU that was located in a region predicted to form a hairpin secondary structure. The AUCCGGAU sequence is highly conserved in RNA2and RNA4of different RSV isolates and is also conserved among the corresponding genomic RNAs of other tenuiviruses. p3transcripts from the there hosts all had the same dominant termination site, while pc3transcripts from different hosts terminated at different sites. All pc13’-UTR sequences ended at the3’-end of the viral complementary strand of RNA1, indicating that the pc1transcript may be synthesized by runoff of viral polymerase, but had no characteristic termination sequence. This is the first experimental report determining the exact transcription termination sites of a plant ambisense virus, and has implications for understanding the transcription of RSV as well as other plant viruses with an ambisense coding strategy.