Target leaf spot (TLS), caused by Corynespora cassiicola, is a prevalent leaf disease that significantly impacts cucumber yield and quality. Breeding disease-resistant cucumber varieties is a key strategy for managing this disease, and identifying critical resistance genes is essential for genetic improvement. In this study, we identified a highly susceptible mutant to TLS in the Tnt1 retrotransposon mutant library. Bulked segregation analysis sequencing (BSA-seq) further pinpointed a candidate gene for TLS resistance, encoding the Mildew Resistance Locus O (MLO) protein, CsMLO4. Expression analysis revealed that CsMLO4 is strongly induced by C. cassiicola infection. Functional analyses revealed that loss of function and silencing of CsMLO4 attenuated resistance to TLS and exhibited reduced reactive oxygen species (ROS) accumulation, while transient overexpression of CsMLO4 enhanced both disease resistance and ROS levels. These findings suggest that CsMLO4 mediates cucumber defense against C. cassiicola by modulating ROS levels. Additionally, transcriptome analysis identified multiple disease-resistance-related pathways affected by the loss of function of CsMLO4. Overexpression of CsMYB, a potential candidate gene regulated by CsMLO4, showed enhanced resistance to C. cassiicola. This study expands insights into the functional role of MLO family beyond their association with powdery mildew resistance and offers new perspectives on the mechanisms underlying TLS resistance in cucumber.

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