Development of powdery mildew (Podosphaera xanthii) resistance in cucumber (Cucumis sativus) using CRISPR/Cas9-mediated mutagenesis of CsaMLO8.

Abstract

Powdery mildew (PM) diseases may severely limit the production of various crops, including members of to the Cucurbitaceae family. Successful PM infection relies on the Mildew Resistance Locus O (MLO) plant gene family, which encodes susceptibility (S) factors essential for fungus penetration into the host cell. In cucumber, 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 cv. '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 1280 bp deletion and 10 bp insertion between exons 1 and 5. Both lines showed high resistance to PM under semi-commercial 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.