Transcription factors VviWRKY10 and VviWRKY30 co-regulate powdery mildew resistance in grapevine

Grapevine (Vitis vinifera) is an economically important fruit crop worldwide. The widely cultivated grapevine is susceptible to powdery mildew caused by Erysiphe necator. In this study, we used CRISPR-Cas9 to simultaneously knock out VviWRKY10 and VviWRKY30 encoding two transcription factors reported to be implicated in defense regulation. We generated 53 wrky10 single mutant transgenic plants and 15 wrky10 wrky30 double mutant transgenic plants. In a 2-yr field evaluation of powdery mildew resistance, the wrky10 mutants showed strong resistance, while the wrky10 wrky30 double mutants showed moderate resistance. Further analyses revealed that salicylic acid (SA) and reactive oxygen species contents in the leaves of wrky10 and wrky10 wrky30 were substantially increased, as was the ethylene (ET) content in the leaves of wrky10. The results from dual luciferase reporter assays, electrophoretic mobility shift assays and chromatin immunoprecipitation (ChIP) assays demonstrated that VviWRKY10 could directly bind to the W-boxes in the promoter of SA-related defense genes 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 ET-related defense genes and 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 reveal a complex mechanism of regulation by VviWRKY10 and VviWRKY30 for activation of measured and balanced defense responses against powdery mildew in grapevine.