Histological Responses To Plasmopara Viticola In Chinese Wild Grape And Study Of The Pathogenic Mechanism Of A P. Viticola Effector RXLR31154

Grape downy mildew,caused by Plasmopara viticola（Berk.&M.A.Curtis Berl.&De Toni）,caused serious damage to the quality and yield of grapes,especially in relatively warm and humid summer.Since the cultivated varieties V.vinifera are susceptible to P.viticola,requiring to repeat application of chemical fungicides.China is one of the most important origins of Vitis,and it has abundant wild grape germplasm resources.Therefore,studying the disease resistance of these wild species and identifying the disease-resistant species and strains is of great significance for grape resistance breeding.Pathogens can successfully infect host plants by secreting effectors to alter host plant cell processes and immune responses.Therefore,it is of great significance to identify pathogenic effectors of P.viticola for studying the molecular mechanism of grapevine-P.viticola interaction.The results of this study are as follows:1.According to statistical disease incidence,disease severity,and disease index,V.piasezkii accession Liuba-8 was classified as highly resistant to P.viticola,V.pseudoreticulata accession Baihe-35-1 and V.davidii var.Cyanocarpa accession Langao-5as resistant to P.viticola,and V.vinifera cv.Pinot Noir as susceptible to P.viticola.Histological study suggested that callose deposition was observed in V.pseudoreticulata accession Baihe-35-1,V.davidii var.Cyanocarpa accession Langao-5,and V.piasezkii accession Liuba-8.H₂O₂accumulation was observed at 12 h,24 h,72 h,and 96 h in V.piasezkii accession Liuba-8,V.davidii var.Cyanocarpa accession Langao-5,V.pseudoreticulata accession Baihe-35-1,and V.vinifera cv.Pinot Noir,respectively.2.The transcriptomic changes were investigated at 0 h,12 h,24 h,48 h,96 h,and 120 h of V.pseudoreticulata accession Baihe-35-1 leaves inoculated by P.viticola.Transcriptome analysis identified a total of 175 differentially expressed genes.Most of them were found to be associated with transciption factor（b HLH1）,oxidative stress（GSTU17,CRL1,and ce QORH）,cell wall modification（F5H,PRX52,PME17,BXL1,and CESA6）and protein modification（FKBP17-2,RHA2A,UGT86A1,and UGT88A1）,terpene synthesis（TPS04 and TPS21）and hormone synthesis（SAUR39）.3.By the transcriptome sequencing of the sporangia and germinated spores of P.viticola,425 effectors were predicted.28 genes encoding 18 RXLRs,2 Crinklers and 4 NPPs,and 4elicitin-likes were cloned from P.viticola genome DNA.All of the 28 effectors could not cause cell death in Nicotiana benthamiana by Agrobacterium-mediated transient transformation.Moreover,RXLR31154,RXLR31197,CRN61994,and CRN55318 could suppress cell death triggered by BAX.Moreover,RXLR31154 and RXLR31197 could suppress cell death triggered by PsCRN63.Subsequently,a semi-quantitative analysis suggested that their expression patterns were different and most of the effectors were highly expressed during grapevine-P.viticola interaction,indicating that they could play an important role in grapevine-P.viticola interaction.4.In this study,an RXLR effector RXLR31154 was up-regulated in the early stage of P.viticola infection.The transgenic lines overexpressing RXLR31154 promoted the colonization of P.viticola and Phytophthora capsici in grapes and Nicotiana benthamiana leaves,respectively.Then,using RXLR31154 as a bait,an interaction protein VpOEE2（oxygen evolving enhancer protein 2）was screened from the yeast library of V.piasezkii accession Liuba-8 inoculated by P.viticola.And,the interaction of RXLR31154 and VpOEE2 protein was confirmed by yeast two-hybrid,bimolecular fluorescence complementation,GST pull down and co-immunoprecipitation.VpOEE2,encoded by a nuclear gene Psb P1,is an extrinsic subunits of the photosystem II in eukaryotic photosynthetic organisms.Western blotting experiment suggested OEE2 was significantly up-regulated compared with mock in V.vinifera cv.Pinot Noir and V.piasezkii accession Liuba-8 following inoculated by P.viticola using anti-OEE2.Moreover,the transgenic lines overexpressing VpOEE2 enhanced grapes and N.benthamiana leaves susceptibility to P.viticola and P.capsici,respectively.Subcellular localization and chloroplast separation experiments indicated that RXLR31154 partially with VpOEE2 co-localized to the chloroplasts.Next,the chlorophyll fluorescence parameters of the transgenic lines overexpressing RXLR31154 and VpOEE2 were not significantly altered compared with the wild type.RNAi of Nb Psb Ps reduced the chlorophyll fluorescence in N.benthamiana.Protein degradation experiments in vivo showed that RXLR31154 stablized VpOEE2.qRT-PCR experiments suggested that the transgenic grape lines overexpressing RXLR31154 and VpOEE2 up-regulated the expression of antioxidant enzymes genes and genes involving in singlet oxygen ¹O₂signaling.DAB staining showed that H₂O₂accumulation in transgenic lines overexpressing RXLR31154 and VpOEE2 was reduced compared to wild type.Thus,it is hypothesized that RXLR31154 inhibits plant immunity through reducing H₂O₂ accumulation and activating ¹O₂signaling pathway in grape by stabilizing OEE2.