CRISPR/Cas9 based mlo -mediated resistance against Podosphaera xanthii in cucumber ( Cucumis sativus L.)

Powdery mildews (PM) are common and severe pathogen groups that threaten plants, and PM resistance is complex and polygenic in cucumbers. Previously -based resistance was reported in various plants, including cucumber, with generated loss-of function mutants. However, -based resistance in cucumber is also complex and involves additional mechanisms such as hypersensitive response (HR) and papillae formation. For this reason, we focused on determining the -based powdery mildew resistance mechanism in cucumber. CRISPR/Cas9 was used in the present study to generate loss-of-function mutants for , , and of PM susceptible ADR27 cucumber inbred lines and mutants were obtained and validated. Trypan Blue and DAB staining were performed to detect germination/penetration rates and accumulation of Reactive Oxygen Species (ROS). Our results indicate that PM-susceptibility associated s in cucumber are negative regulators in different defense mechanisms against powdery mildew at early and late stages of infection. Further, the experiment results indicated that mutation-based resistance was associated with the pre-invasive response, while and could be negative regulators in the post-invasive defense response in cucumber against . . Although the loss-of function confers the highest penetration resistance, and double mutations could be potential candidates for HR-based resistance against PM pathogen in cucumber. These results highlighted the crucial role of CRISPR/Cas9 to develop PM resistant cucumber cultivars, possessing strong pre-invasive defense with or post-invasive with / mutations.