Functional Analysis Of VpWRKY75 In Response To Downy Mildew In Chinese Wild Grape Vitis Piasezkii

Grape downy mildew caused by Plasmopara viticola is one of the most devastating diseases for grape production.Vitis vinifera is a dominant grape variety with good quality and commercial value,but is resistant to downy mildew.Compared with European grapes,Chinese wild grapes are highly resistant to downy mildew.China is rich in wild grape resources.It is of great significance to exploit the inherent resistance genes of wild grape germplasm resources to improve the pathogen resistance of Vitis vinifera.VpWRKY75 was differently expressed in Chinese wild grape Vitis piasezkii liuba-8 and Vitis vinifera Pinot Noir after infected by Plasmopara viticola.In this study,VpWRKY75 was cloned from the above two kinds of grapes,and the related expression vectors were constructed to identify the disease resistance by the model plants Nicotiana benthamiana and Arabidopsis.Pathogenesis-related proteins（PRP/PRs）were predicted by co-expression network analysis and the interactions were verified by yeast one-hybrid technique.Upstream sequence of VpWRKY75 was used as a bait to screen the yeast c DNA library which was built from Vitis piasezkii Liuba-8 after PV infection.And the target protein interacting with the VpWRKY75 promoter was obtained.The main results were obtained as following:1.VpWRKY75 has a WRKY domain and transcriptional activation function and localized in the nucleus,whereas WRKY conserved domain has no activation function.Transient overexpression of VpWRKY75 in tobacco leaves caused cell death and increased the level of hydrogen peroxide（H₂O₂）.The At WRKY75 mutant was sensitive to pathogenic bacteria.2.Three PR proteins were predicted by co-expression network analysis,which may be regulated by VpWRKY75,namely VpPR10.8,VpPR-like and VpPR4.These PR proteins’promoters were cloned.By yeast one-hybrid assay,VpWRKY75 was found to bind the promoter of VpPR10.8 and VpPR-like.Transient overexpression of VpWRKY75 in grape leaves caused up-regulation expression of VpPR10.8 and VpPR-like.3.The VpWRKY75 promoter was cloned and its cis-acting elements were analyzed.The upstream regulatory factor of VpWRKY75 was obtained by screening the yeast expression c DNA library of Chinese wild grape Vitis piasezkii liuba-8 induced by downy mildew with VpWRKY75 promoter was used as bait.