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Abstract 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. 

Summary Calcium‐dependent protein kinases (CDPKs) play vital roles in metabolic regulations and stimuli responses in plants. However, little is known about their function in grapevine.Here, we report thatVpCDPK9andVpCDPK13, two paralogousCDPKsfromVitis pseudoreticulataaccession Baihe‐35‐1, appear to positively regulate powdery mildew resistance. The transcription of them in leaves of ‘Baihe‐35‐1’ were differentially induced upon powdery mildew infection. Overexpression ofVpCDPK9‐YFPorVpCDPK13‐YFPin theV. viniferasusceptible cultivar Thompson Seedless resulted in enhanced resistance to powdery mildew (YFP, yellow fluorescent protein). This might be due to elevation of SA and ethylene production, and excess accumulation of H₂O₂and callose in penetrated epidermal cells and/or the mesophyll cells underneath.Ectopic expression ofVpCDPK9‐YFPin Arabidopsis resulted in varied degrees of reduced stature, pre‐mature senescence and enhanced powdery mildew resistance. However, these phenotypes were abolished inVpCDPK9‐YFPtransgenic lines impaired in SA signaling (pad4sid2) or ethylene signaling (ein2). Moreover, both of VpCDPK9 and VpCDPK13 were found to interact with and potentially phosphorylate VpMAPK3, VpMAPK6, VpACS1 and VpACS2in vivo(ACS, 1‐aminocyclopropane‐1‐carboxylic acid (ACC) synthase; MAPK, mitogen‐activated protein kinase).These results suggest thatVpCDPK9andVpCDPK13contribute to powdery mildew resistance via positively regulating SA and ethylene signaling in grapevine.