Study On The Mechanism Of Powdery Mildew Resistance Of New Members Of Stilbene Synthase Family Of Chinese Wild Vitis Quinquangularis

Cultivated worldwide,Vitis L.is a fruit tree with a long history of cultivation and high economic value.Most high-quality grape varieties used for wine cultivation,such as European grape varieties,are affected by a series of pathogens,resulting in serious yield losses.The most significant diseases of grape are gray mold,downy mildew,and powdery mildew.The accumulation of resveratrol—a major phytoalexin—is part of the constitutive and induced defense reaction.In addition to playing a crucial role in plants as phytoalexins,stilbenes have great benefits for human health.China is one of the main grape-producing areas,with rich germplasm resources.The Chinese wild grape(V.quinquangularis)Danfeng-2 has strong resistance to Erysiphe necator and accumulates more stilbene substances.Our research team identified six new stilbene synthase transcripts,VqNSTS1–VqNSTS6,by analyzing the transcriptome data of Danfeng-2.However,it is unclear how other VqNSTS genes regulate the production of stilbene substances,the function of E.necator resistance,and the regulatory mechanism under the infection of E.necator.In this study,VqNSTS3 and VqNSTS6 genes were cloned by homologous cloning.DNA and amino-acid sequences were analyzed to identify the two transcripts were new stilbene synthase genes.Additionally,the resistance expression of the two transcripts to E.necator was examined.Through Agrobacterium-mediated genetic transformation,the two new transcripts were transferred into the diseased European grape“Thompson Seedless” for gene function analysis and disease resistance identification.The transcription factors and the functions regulating the new transcripts were screened and studied.After transforming both VqNSTS3 and VqNSTS6 with their own promoters into Arabidopsis thaliana,a model plant without a stilbene synthase gene,the interaction between VqNSTS3 and VqNSTS6 and Golovinomyces cichoracerum was observed by laser confocal microscope.This procedure provides a basis for disease resistance breeding.The main results are presented as follows.1.Two new members of the Chinese wild grape Danfeng-2 stilbene synthase family,VqNSTS3 and VqNSTS6,were cloned and obtained.The VqNSTS3 and VqNSTS6 genes were 1179 bp long and encoded 393 amino acids.The nucleotide sequences of VqNSTS3 and VqNSTS6 are most similar to those of Vv STS4 in“Pinot Noir,” with 91% and 98% homology,respectively.Through cluster analysis,we found that VqNSTS3,VqNSTS6,and VqSTS33 have a high homology of 99.24% and 93.89%,respectively.Both VqNSTS3 and VqNSTS6 have the conserved domain of the stilbene synthase family,but VqNSTS6 has two amino-acid mutations in the conserved sequence of IPNSAGAIAGN.Real-time quantitative polymerase-chain-reaction(q PCR)analysis of the VqNSTS3 and VqNSTS6 gene expression in Danfeng-2 after inoculation showed that both genes could significantly respond to the induction of E.necator.2.VqNSTS3 and VqNSTS6 were identified to participate in the resistance response to E.necator.VqNSTS3 and VqNSTS6 of Chinese wild grape Danfeng-2 were transferred into European grape “Thompson Seedless” by an Agrobacterium-mediated method and overexpressed.The transgenic plants were identified by q PCR and Western blot assays.The identified positive plants were artificially inoculated with E.necator.By observing the plant phenotype,trypan blue staining,counting the spore number,aniline blue staining,scanning electron microscopy,q PCR,and high-performance liquid chromatography(HPLC)analysis,it was found that the content of stilbene in the overexpressed transgenic plants significantly increased after E.necator infection.Moreover,hypersensitive response(HR)cell death increased,callose accumulation rose,and resistance-related genes such as PR1,PR5,RBOHD,and CHIT4 C were induced to enhance the resistance to E.necator.However,RNAi interference plants showed opposite phenotypes.3.Select transcription factors VqWRKY33,VqGT3,and VqGT33 that regulate the expression of VqNSTS3 and VqNSTS6 genes.The promoter sequence of VqNSTS3 was obtained,and its cis-acting elements were analyzed.The WRKY transcription factor represents a significant family of transcription factors that have vital functions in plant disease resistance.It was found that there were three WRKY transcription factor binding cis-acting elements.W-box elements were identified on the promoter of VqNSTS3.According to the quantitative data analysis of the WRKY transcription factor family gene artificially inoculated with E.necator in Danfeng-2 in the early stage of the laboratory,eight WRKY transcription factors that significantly responded to the induction of E.necator were selected.Among them,VqWRKY33 has the greatest activation activity on the VqNSTS3 promoter.Through Y1 H,dual luciferase,and chromatin immunoprecipitation(Ch IP)-q PCR assays,the results showed that VqWRKY33 by combining TTGACC on VqNSTS3 promoter directly regulate the expression of VqNSTS3,thus promoting the synthesis of stilbene.The promoter of VqNSTS6 was analyzed to identify the transcription factors that regulate the expression of the VqNSTS6 gene.It was found in addition to hormoneresponsive elements,there are also four GT-boxes as binding sites for GT transcription factors and one MYB binding site.The GT transcription factor VqGT3 induced by E.necator can bind directly to the GT-box(GGTAAT)in the VqNSTS6 promoter and activate gene expression.VqGT33 belongs to the GT-1 subfamily and can directly bind with the GT-box element in the VqNSTSs promoter and activate gene expression.4.Discovered protein kinases VqMAPK3,VqMAPK4,and VqMAPK6 interact with and phosphorylate transcription factors,regulating the expression of VqNSTS3 and VqNSTS6,forming a disease resistance cascade signaling pathway,regulating the accumulation of stilbene and disease resistance.VqMAPK3 and VqMAPK6 directly interact with VqWRKY33,which was proven by yeast two-hybrid,bimolecular fluorescence complementation(Bi FC)and co-immunoprecipitation(Co-IP)assays.A phos-tag phosphorylation test showed that VqMAPK3 and VqMAPK6 can phosphorylate VqWRKY33 to enhance the transcriptional regulation activity of VqWRKY33 on the VqNSTS3 promoter.Under infection with E.necator,VqMAPK3/6 sense the stimulation of the pathogen and release phosphorylation signals,which cause downstream transcription factors(TF)VqWRKY33 to start the positive regulation of target gene VqNSTS3,which expresses and accumulates a large number of stilbenes,enhancing disease resistance.We also found that in the early stage of E.necator infection,VqGT3 promoted the accumulation of stilbene through positive regulation of VqNSTS6.VqMAPK4 and VqMAPK6 interact with VqGT3 through yeast two-hybrid,Bi FC and Co-IP assays.Additionally,a phos-tag phosphorylation test proved that VqMAPK4 can phosphorylate VqGT3,but VqMAPK6 cannot.When there are too many stilbene substances accumulated,phosphorylated VqMAPK4 promotes the degradation of VqGT3 and reduces its regulation of VqNSTS6,thus reducing the generation of stilbene.5.Pro VqNSTS3::VqNSTS3-GFP and Pro VqNSTS6::VqNSTS6-GFP moves to and wraps the haustoria to prevent pathogen invasion in transgenic A.thaliana.Resveratrol is a crucial phytoalexin produced by grapes,which is generated in large quantities and aggregates at spore invasion sites when grapes are infected by pathogens.However,there is currently no direct evidence to prove this phenomenon.Stilbene synthase(STS)is a rate-limiting enzyme used for the synthesis of resveratrol.In this study,VqNSTS3 and VqNSTS6 were respectively linked to their own promoters and stably transferred into the model plant A.thaliana(Col-0)without the stilbene synthase gene through an Agrobacteriummediated flower dipping method.After artificial inoculation with G.cichoracerum,the interaction between VqNSTS3,VqNSTS6,and G.cichoracerum was observed through laser confocal microscopy.Results showed that before spore germination,the fluorescence of VqNSTS3 and VqNSTS6 was localized on the plasma membrane.As the infection time of G.cichoracerum increases and the spores germinate,the green fluorescence of VqNSTS3 and VqNSTS6-GFP is transported to the spore germination site by the multivesicle structures.Finally,the green fluorescence envelops the haustorium of G.cichoracerum to form a haustorium encasement,forming a barrier to inhibit the growth of powdery mildew.In conclusion,two new stilbene synthase genes,VqNSTS3 and VqNSTS6,were cloned from Chinese wild grape Danfeng-2.VqNSTS3 is positively regulated by VqWRKY33.VqMAPK3 and VqMAPK6 can phosphorylate VqWRKY33 to enhance the transcriptional regulation activity of VqWRKY33 on the VqNSTS3 promoter.VqNSTS6 is positively regulated by VqGT3 and VqGT33.Phosphorylated MAPK4 degrades VqGT3 to negatively regulates VqNSTS6 gene expression.After inoculation with Golovinomyces cichoracerum,Pro VqNSTS3::VqNSTS3-GFP and Pro VqNSTS6::VqNSTS6-GFP transgenic actively gathered in the haustorium encasements and papilla structure to inhibit the growth of Golovinomyces cichoracerum in Arabidopsis thaliana plants.This study further analyzed the mechanism of grape stilbene substances participating in the resistance to Erysiphe necator and proved that the Chinese wild grape Danfeng-2 is a vital germplasm resource for disease resistance breeding.