Molecular Mechanism Of Enhanced Powdery Mildew Resistance By VqWRKY56-VqbZIPC22 Molecular Module In Chinese Wild Vitis Quinquangularis

Grapes are one of the economically significant fruit crops.Currently,the world’s predominant grape variety is the European grape(Vitis vinifera L.),known for its excellent fruit quality but highly susceptible to infestation by powdery mildew(Erysiphe necator).China is one of the most important centers of origin for grapevine species globally,boasting rich genetic resources of disease-resistant wild grape varieties.Therefore,the utilization of disease-resistant wild grape germplasm resources for identifying resistance genes against powdery mildew,uncovering their mechanisms of action,and using genetic engineering to enhance disease resistance in grape varieties holds significant theoretical and practical value.In recent years,an increasing body of research has highlighted the critical role of WRKY transcription factors in regulating plant immune responses.In the preliminary work conducted by our research group,grape WRKY gene family identification,combined with transcriptome analysis following Erysiphe necator treatment,led to the identification of WRKY56 as a participant in regulating grapevine resistance to powdery mildew.Building upon this foundation,this study cloned VqWRKY56 from the Chinese wild grape V.quinquangularis‘Shang-24’.Through genetic transformation in Arabidopsis and grapevine,we elucidated its role and regulatory mechanisms in conferring resistance to powdery mildew.The key research findings are as follows:1.VqWRKY56 from Chinese wild Vitis quinquangularis positively regulates grapevine resistance to powdery mildew.VqWRKY56 exhibits the highest expression levels in young leaves and is strongly induced by powdery mildew and salicylic acid(SA).Particularly,its expression levels are significantly higher in the disease-resistant ‘Shang-24’ grape variety compared to the susceptible ‘Thompson Seedless’ variety.Heterologous transformation experiments in Arabidopsis and stable transformation experiments in grapevine confirmed that VqWRKY56 positively regulates grapevine resistance to powdery mildew.It enhances leaf hypersensitive cell death,promotes the accumulation of reactive oxygen species(ROS)and endogenous SA,and increases the expression levels of SA biosynthetic genes and SA pathway-related defense genes(PR1 and PR5).Furthermore,transient overexpression or silencing of VqWRKY56 in ‘Thompson Seedless’ and ‘Shang-24’ leaves revealed that overexpression of VqWRKY56 enhances resistance to powdery mildew,while silencing VqWRKY56 reduces resistance to powdery mildew.These results suggest that VqWRKY56 may activate the SA defense signaling pathway,promote ROS burst,and thereby enhance resistance to grapevine powdery mildew.2.Chinese wild Vitis quinquangularis VqWRKY56 regulates powdery mildew resistance by promoting the accumulation of proanthocyanidins.Overexpression of VqWRKY56 in‘Thompson Seedless’ grapes increased E.necator-induced proanthocyanin accumulation in transgenic grape leaves,while transient interference with VqWRKY56 reduced E.necatorinduced proanthocyanin accumulation.In V.quinquangularis ‘Shang-24’ grape leaves,transient interference with VqWRKY56 reduced E.necator-induced proanthocyanin accumulation as well.Moreover,after E.necator infection,VqWRKY56-overexpressing lines significantly increased the expression of proanthocyanin biosynthetic genes.Proanthocyanidins have been widely proven to have antifungal activity in vitro and are considered to be an important part of plant antimicrobial defense.It was revealed that VqWRKY56 was involved in the regulation of grape powdery mildew resistance by promoting the accumulation of proanthocyanidins.3.The interaction between VqWRKY56 and Vqb ZIPC22 positively regulates the resistance to powdery mildew and the accumulation of proanthocyanidins induced by E.necator.Through yeast two-hybrid screening,Vqb ZIPC22 was identified as an interacting partner of VqWRKY56.This interaction was confirmed both in planta and in vitro through bimolecular fluorescence complementation(Bi FC),Split-LUC,and immunoprecipitation assays(Co-IP).Heterologous overexpression of Vqb ZIPC22 in Arabidopsis enhanced resistance to powdery mildew.Transient expression of Vqb ZIPC22 in the susceptible‘Thompson Seedless’ grape leaves strengthened resistance to powdery mildew and promoted the accumulation of E.necator-induced proanthocyanins.In the disease-resistant ‘Shang-24’grape leaves,transient silencing of Vqb ZIPC22 reduced resistance to powdery mildew and lowered the accumulation of E.necator-induced proanthocyanins.These results indicate that Vqb ZIPC22 acts as a positive regulator of powdery mildew resistance and that the two proteins may collaborate to enhance grapevine resistance to powdery mildew by promoting proanthocyanin accumulation.4.VqWRKY56 and Vqb ZIPC22 cooperate to activate the expression of proanthocyanin biosynthesis genes Vv CHS3,Vv LAR1,and Vv ANR.Analysis using the JASPAR online tool revealed potential binding sites for both VqWRKY56 and Vqb ZIPC22 in the promoter sequences of proanthocyanin pathway genes(Vv CHS3,Vv LAR,Vv ANR,Vv CHI,and Vv FLS).These promoter sequences contained multiple WRKY transcription factor binding sites(Wbox: TTGAC(C/T))and b ZIP transcription factor binding sites(G-box: ACGTG).Sequence alignment analysis of these gene promoters in ‘Shang-24’ and ‘Thompson Seedless’ grape varieties showed complete conservation of the W-box and G-box motifs in both varieties.Y1 H assays confirmed that VqWRKY56 can bind to the promoters of proanthocyanin biosynthesis genes Vv CHS3,Vv LAR1,and Vv ANR but not to the promoters of Vv CHI and Vv FLS.On the other hand,Vqb ZIPC22 directly interacts with the promoter of the proanthocyanin biosynthesis gene Vv ANR but cannot bind to the promoters of Vv CHS3,Vv LAR1,Vv CHI,and Vv FLS genes.Additionally,dual-luciferase reporter gene assays demonstrated that both VqWRKY56 and Vqb ZIPC22 individually significantly enhanced LUC activity driven by the promoters of Vv CHS3,Vv LAR1,and Vv ANR genes.When VqWRKY56 and Vqb ZIPC22 were co-expressed,the LUC activity of Vv CHS3,Vv LAR1,and Vv ANR reporters was higher than that observed when VqWRKY56 or Vqb ZIPC22 was expressed individually.These results indicate that VqWRKY56 and Vqb ZIPC22 cooperatively activate the expression of proanthocyanin biosynthesis genes,thereby promoting the accumulation of proanthocyanins.In summary,this study proposed the mechanism of powdery mildew resistance mediated by the promotion of proanthocyanidin accumulation by VqWRKY56-Vqb ZIPC22 molecular module.The mechanism can be outlined as follows: the invasion of grapevine by E.necator triggers PTI or ETI responses in the plant.These responses activate the expression of VqWRKY56 and Vqb ZIPC22.VqWRKY56 and Vqb ZIPC22 can interact with each other.They could synergistically activate the expression of proanthocyanin biosynthesis genes,including Vv CHS3,Vv LAR1,and Vv ANR,leading to the accumulation of proanthocyanins.The accumulation of proanthocyanins enhances grapevine resistance to powdery mildew.