Identification Of Pathogenicity Determinant And RNA Silencing Suppressor Of Wheat Dwarf Virus (WDV)

Wheat dwarf virus (WDV), transmitted by leafhopper (Psammotettix striatus L.), is a member of the genus Mastrevirus in the family Geminiviridae. In2007, WDV was reported to cause severe damage in wheat in Hancheng of Shaanxi province. In order to elucidate the mechanisms of pathogenicity of WDV, functions of Rep gene of WDV were studied in this dissertation.The V1、 V2、 Rep A and Rep genes were cloned into the Potato virus X (PVX) vector PGR106, respectively. Agroinfiltration showed N.benthamiana leaves infiltrated with the PVX vector expressing Rep (PVX-Rep) produced veinal necrosis, yellowing, crinkle and necrotic symptoms while PVX expressing the other three genes only produced symptoms similar as induced by PVX vector. This result indicates that WDV Rep protein is a pathogenicity determinant in the PVX heterogenous expression system. ELISA and Western blot analysis indicated that the concentrations of PVX protein were lower in the N. benthamiana plants infiltrated with the PVX-Rep than these in the N. benthamiana plants infiltrated with the PGR106vector at9days post infiltration (dpi),12dpi and15dpi, indicating necrosis in N. benthamiana plants infiltrated with PVX-Rep is caused by WDV Rep itself. Deletion mutations indicate that the N-terminal of Rep is important for its pathogenicity while the C-terminal of Rep is not necessary for its pathogenicity.To determine which genes of WDV is involved in suppression of RNA silencing, co-infiltration assays in GFP transgenic Nicotiana benthamiana line16c were carried out and Rep protein is found to suppress GFP local silencing.GFP green fluorescence could be observed at3dpi in the16c N. benthamiana leaves co-infiltrated with the Agrobacterium tumefaciens harboring the GFP gene and the Rep genes, and GFP mRNA accumulation was increased significantly when tested by Northern blot. Co-infiltration of a series of deletion mutants of Rep with GFP show the N-terminal and C-terminal of Rep are not necessary for suppressing RNA silencing. In addition, GFP green fluorescence could be observed in the systemic leaves of16c N. benthamiana plants co-infiltrated with the GFP and Rep genes at30dpi indicating that WDV Rep protein can inhibit systemic RNA silencing of GFP gene. Meanwhile, WDV Rep protein could interfere with the spread of systemic RNA silencing signal of GFP gene when tested by Agrobacterium-mediated infiltration assay. The above results indicate that Rep protein is a RNA silencing suppressor.The RNA binding ability of recombinant Rep protein was determined by electrophoresis mobility shift assay (EMSA). The results showed that Rep protein could bind21nt and24nt single-stranded siRNA and siRNA duplex with preferences for single-stranded siRNA, and siRNA binding ability was co-related with protein concentrations, These results show that Rep inhibits RNA silencing by preventing formation of siRNA RISC complex.