Function Analysis Of Domains Of Downy Mildew Resistance Gene MrRPV1and Effectors Of Plasmopara Viticola

Grape downy mildew, caused by the pathogen Plasmopara viticola, is a destructive oomycete disease for viticulture. MrRPVl gene which encoded a typical TIR-NBS-LRR protein is the first downy mildew resistance gene cloned from grapevine. Researches on MrRPVl domain functions, interacting proteins and effectors secreted during the compatible interaction with the host are of great significance to determine the downy mildew resistance mechanism and the pathogenesis mechanism of P. viticola.Boundary definition and function studies of MrRPVl-TIR were performed by protein truncation and site-directed mutagenesis. Transient expression results showed that MrRPVl-TIR1-189is the minimum fragment with activity. The first22amino acids from exon2and complete a-helix structure were included in this fragment. Site-directed mutagenesis revealed that the mutations of the highly conserved regions blocked the signal transduction induced by TIR domain.In order to define the function of LRR domain in specific recognition to P. viticola, we swapped the LRR domains of MrRPVl and MrRUNl and made recombined constructs in this study. Then the resistance of transgenic lines to different disease was observed. The inoculation result in the lab showed that transgenic line RGA10-8-13exhibited similar necrotic spots to MrRPVl-transgenic plant S8-1. And the numbers of sporangia per infected leaf disc for all the positive RGA10-8transgenic lines were decreased significantly comparing with Shiraz control. Powdery mildew resistance was observed for two transgenic lines, RGA8-10-3and RGA8-10-28. It suggest that the LRR domain play a role in specific recognition to different pathogens.Bombardment of MrRPVl-GFP fusion protein in onion cells showed that MrRPVl was localized in cytoplasm and nucleus. Downy mildew resistance of R23plants was compromised after treatment with GDA, an inhibitor of HSP90. Bimolecular fluorescence complementation experiment in onion cells showed that MrRPVl can interact with EDS1. So we speculate that HSP90and EDS1were involved in the downy mildew resistance induced by MrRPVl.Forty-three RXLR effectors and five CRN effectors were indentified from de novo assembled P. viticola transcriptome by bioinformatic analysis based on their conserved module structure. Pfam domain analysis of the secretome also identified a diverse range of putative pathogenesis factors including elicitin, NLPs, ubiquitin ligase, glycosyl hydrolases, etc.PvR_XLR18was selected to be further studied based on its expression profiling during infection. Transient expression of PvRXLR18in Nicotiana benthamiana demonstrated that it was capable of suppressing programmed cell death triggered by the mouse BAX protein or the PAMP INF1. Y2H screening the cDNA library of downy mildew early-infected cabernet sauvignon leaves using PvRXLR18as bait found that it can interact with carbonic anhydrase and reactive oxygen species enhancer protein OEE2.