VviWRKY10andVviWRKY30co-regulate powdery mildew resistance through modulating SA and ET-based defenses in grapevine

Abstract

ABSRACTGrapevine is one of the most important economic fruit crops in the world. The widely cultivated grapevine (Vitis vinifera,Vvi) is susceptible to powdery mildew caused byErysiphe necator. In this study, CRISPR-Cas9 was used to simultaneously knock outVviWRKY10andVviWRKY30encoding two transcription factors reported to be implicated in defense regulation. Fifty-threewrky10(Vviwrky10single mutant) transgenic plants and fifteenwrky10wrky30(Vviwrky10Vviwrky30double mutant) transgenic plants were generated. In a 2-year field evaluation of resistance to powdery mildew, thewrky10showed strong resistance, while thewrky10wrky30showed moderate resistance. Further analyses revealed that salicylic acid (SA) and reactive oxygen species (ROS) contents in the leaves ofwrky10andwrky10wrky30were significantly increased, so did the ethylene (ET) content in the leaves ofwrky10. The results from dual luciferase (DLUC) reporter assays, electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP)-qPCR assays demonstrated that VviWRKY10 could directly bind to the W-boxes in the promoter ofVviEDS5-2,VviPR1,VviPR5andVviRBOHD2and inhibit their transcription, supporting its role as a negative regulator of SA-dependent defense. By contrast, VviWRKY30 could directly bind to the W-boxes in the promoter ofVviACS3andVviACS3Land promote their transcription, playing a positive role in ET production and ET-dependent defense. Moreover, VviWRKY10 and VviWRKY30 can bind to each other’s promoters and mutually inhibit each other’s transcription. Taken together, our results have revealed a complex mechanism of regulation by VviWRKY10 and VviWRKY30 for activation of measured and balanced defense responses against powdery mildew in grapevine.One-sentence summaryVviWRKY10andVviWRKY30play different roles in powdery mildew resistance through the salicylic acid and ethylene, respectively, and there is mutual inhibition betweenVviWRKY10andVviWRKY30.