Development of Powdery Mildew Resistance in Cucumber Using CRISPR/Cas9-Mediated Mutagenesis of CsaMLO8

Powdery mildew (PM) diseases may severely limit the production of various crops, including members of the family Cucurbitaceae. Successful PM infection relies on the Mildew Resistance Locus O (MLO) plant gene family, which encodes susceptibility factors essential for fungus penetration into the host cell. In cucumber (Cucumis sativus), natural mutations in CsaMLO8 confer resistance to the PM pathogen Podosphaera xanthii. Here, we used CRISPR/Cas9-mediated mutagenesis to generate PM resistance in the susceptible cucumber cultivar Ilan. Two transgene-free Csamlo8 CRISPR mutant lines (Csamlo-cr-1 and Csamlo-cr-2) were isolated, the first with a 5-bp deletion in exon 1, and the second harboring a 1,280-bp deletion and 10-bp insertion between exons 1 and 5. Both lines showed high resistance to PM under semicommercial growth conditions in the summer growing seasons of 2019 and 2021. These results provide the basis for generating transgene-free powdery mildew resistance in cucumber in any genetic background. This method can directly be employed on commercial cultivars and hybrid parental lines, and thereby substantially shorten and simplify the breeding process for PM resistance in cucumber.