Screening Of VqWRKY56 Interaction Protein And Study On Its Powdery Mildew Resistance From Chinese Wild Vitis Quinquangularis

Grape is widely grown and is used for fresh eating,wine and other processed products around the world.Powdery mildew is an obligate parasitic biotrophic fungus,which is very common in grape growing areas all over the world.Widely cultivated European grapes(Vitis vinifera L.)have very low resistance to this pathogen.Powdery mildew affects all green tissues of grapes,especially young organs,and seriously affects the yield and quality of grapes.The Chinese Wild Vitis quinquangularis ‘Shang-24’ has strong resistance to powdery mildew and is a very important powdery mildew resistance resource.It is particularly important to study and use its key resistance genes and to explore their resistance molecular mechanisms to enhance the disease resistance of grape.In the early stage,VqWRKY56 gene was stably transformed into ‘Thompson Seedless’ grape for phenotype observation and disease resistance detection,and it was found that VqWRKY56 gene had good resistance to powdery mildew.In this study,VqWRKY56 was selected as bait protein by yeast two-hybrid system and its interaction factors were verified.After that,transient transformation of interacting protein into grape leaves was studied,and the main results were as follows:1.Functional validation of VqWRKY56 from Chinese Wild Vitis quinquangularis.Bioinformatics analysis showed that VqWRKY56 is located on chromosome 19.Homology analysis showed that VqWRKY56 is closely related to Pyrus x bretschneideri(Gen Bank accession no.NW＿008988394)and Malus domestica(Gen Bank accession no.XP＿028955967).VqWRKY56 is localized on the nucleus of tobacco epidermis by subcellular localization.VqWRKY56 overexpression and interference vector were transiently transformed into ‘Thompson Seedless’ grape leaves and ‘Shang-24’ grape leaves by Agrobacterium-mediated vacuum osmosis.After 144 h of inoculation with powdery mildew,phenotypic observation and trypan blue staining showed that compared with the control,the‘ Thompson Seedless ’ leaves with overexpressed VqWRKY56 gene showed stronger resistance to powdery mildew,and the ‘Shang-24’ leaves with silenced VqWRKY56 gene showed higher sensitivity to powdery mildew.2.Screening of VqWRKY56 interacting proteins from Chinese Wild Vitis quinquangularis.The interaction protein VqbZIPC22 of VqWRKY56 was screened from yeast library induced by powdery mildew by yeast two-hybrid system.The interaction between VqWRKY56 and VqbZIPC22 was further verified by response verification,luciferase reporter system(Split-Luc),bimolecular fluorescence complementation technology(Bi FC)and co-immunoprecipitation technology(Co-IP).Bioinformatics analysis showed that the open reading frame of VqbZIPC22 gene was 438 bp,encoding 145 amino acids.The similarity to the CDS sequence of European grape Vvb ZIPC22 in grape genome database was 98.63%,and the similarity of amino acid sequence was 100%.The VqbZIPC22 gene is located on chromosome 7 and contains only one exon.Phylogenetic tree analysis showed that grape VqbZIPC22 had a high homology with Arabidopsis Atb ZIP53,with a similarity of 51.37%,followed by Soybean Gmb ZIP53 A,with a similarity of 49.33%.3.Study on the function of VqbZIPC22 in powdery mildew from Chinese Wild Vitis quinquangularis.The VqbZIPC22 overexpression and interference vector were transiently transferred into ‘Thompson Seedless’ grape leaves and ‘Shang-24’ grape leaves by Agrobacterium-mediated vacuum osmosis.After 144 h inoculation with powdery mildew,phenotype observation and trypan blue staining showed that the incidence of disease of overexpressed VqbZIPC22 gene in ‘Thompson Seedless’ leaves was significantly lighter than that of empty vector control,they showed stronger resistance to powdery mildew.Silenced VqbZIPC22 gene in ‘Shang-24’ leaves can promote the growth of powdery mildew,showing higher sensitivity than the wild type.After inoculation with powdery mildew,the expression of defense-related genes in SA pathway were significantly up-regulated in transient overexpression leaves compared with empty vector,while the expression of defense-related genes in SA pathway were significantly down-regulated in interference leaves compared with wild-type.These results suggested that VqbZIPC22 may be involved in powdery mildew resistance by mediating the expression of SA signaling pathway defense-related genes.