VviWRKY10 and VviWRKY30 co-regulate powdery mildew resistance through modulating SA and ET-based defenses in grapevine

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 by Erysiphe necator. In this study, CRISPR-Cas9 was used to simultaneously knock out VviWRKY10 and VviWRKY30 encoding two transcription factors reported to be implicated in defense regulation. Fifty-three wrky10 (Vviwrky10 single mutant) transgenic plants and fifteen wrky10wrky30 (Vviwrky10Vviwrky30 double mutant) transgenic plants were generated. In a 2-year field evaluation of resistance to powdery mildew, the wrky10 showed strong resistance, while the wrky10wrky30 showed moderate resistance. Further analyses revealed that salicylic acid (SA) and reactive oxygen species (ROS) contents in the leaves of wrky10 and wrky10wrky30 were significantly increased, so did the ethylene (ET) content in the leaves of wrky10. 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 of VviEDS5-2, VviPR1, VviPR5 and VviRBOHD2 and 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 of VviACS3 and VviACS3L and promote their transcription, playing a positive role in ET production and ET-dependent defense. Moreover, VviWRKY10 and VviWRKY30 can bind to each others promoters and mutually inhibit each others 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 summaryVviWRKY10 and VviWRKY30 play different roles in powdery mildew resistance through the salicylic acid and ethylene, respectively, and there is mutual inhibition between VviWRKY10 and VviWRKY30.