CRISPR/Cas9-mediated mutagenesis of results in enhanced resistance to powdery mildew in grapevine ().

Grapevine (), one of the most economically important fruit crops in the world, suffers significant yield losses from powdery mildew, a major fungal disease caused by . In addition to suppressing host immunity, phytopathogens modulate host proteins termed susceptibility (S) factors to promote their proliferation in plants. In this study, CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) technology was used to enable the targeted mutagenesis of (mildew resistance Locus O) family genes that are thought to serve as factors for powdery mildew fungi. Small deletions or insertions were induced in one or both alleles of two grapevine genes, and , in the transgenic plantlets of the powdery mildew-susceptible cultivar Thompson Seedless. The editing efficiency achieved with different CRISPR/Cas9 constructs varied from 0 to 38.5%. Among the 20 -edited lines obtained, one was homozygous for a single mutation, three harbored biallelic mutations, seven were heterozygous for the mutations, and nine were chimeric, as indicated by the presence of more than two mutated alleles in each line. Six of the 20 -edited grapevine lines showed normal growth, while the remaining lines exhibited senescence-like chlorosis and necrosis. Importantly, four -edited lines showed enhanced resistance to powdery mildew, which was associated with host cell death, cell wall apposition (CWA) and HO accumulation. Taken together, our results demonstrate that CRISPR/Cas9 genome-editing technology can be successfully used to induce targeted mutations in genes of interest to improve traits of economic importance, such as disease resistance in grapevines.