Molecular mechanism of the CmDSK2b-CmMLO5 module in regulating powdery mildew resistance in melon

• The melon powdery mildew resistance gene CmMLO5 is localized in the plasma membrane and its expression is up-regulated during powdery mildew infection. In addition, CmDSK2b was identified as a direct binding protein of CmMLO5. • CmDSK2b is a pathogen-inducible gene with elevated expression during powdery mildew infection and positively regulates disease susceptibility. • Co-expression experiments showed that CmDSK2b reduced CmMLO5 protein stability through the ubiquitin-proteasome pathway. Powdery mildew (PM), caused by Podosphaera xanthii , is a significant threat to melon cultivation globally. The MLO ( Mildew Locus O ) gene family is known to play broad spectrum resistance to PM. However, the precise molecular mechanisms responsible for the MLO resistance phenotype in melon have yet to be fully elucidated. In this study, we found the CmMLO5 protein localizes to plasma membrane. Virus-induced gene silencing (VIGS) analysis revealed that CmMLO5 expression is transcriptionally upregulated in response to PM infection. Through yeast two-hybrid screening of a melon cDNA library, CmDSK2b-annotated as a ubiquitin receptor protein-was identified as the binding partner of CmMLO5. This interaction was further validated by luciferase complementation assay (LCA) and bimolecular fluorescence complementation (BiFC), confirming a direct physical association between the two proteins. Expression profiling, coupled with VIGS assays, revealed that CmDSK2b is a pathogen-inducible gene whose transcript levels increase upon PM infection and that it positively regulates disease susceptibility in melon. Transient co-expression assays in Nicotiana benthamiana demonstrated a significant reduction in CmMLO5-GFP protein abundance when co-expressed with CmDSK2b. These results support a model in which CmDSK2b contributes to fungal susceptibility by promoting ubiquitin-mediated proteasomal degradation of the CmMLO5 protein. Collectively, our findings define a CmMLO5-CmDSK2b regulatory axis that provides new insight into the molecular determinants underlying melon-PM resistance. This discovery not only deepens our understanding of DSK (Dominant Suppressor of KAR2) -family gene function in plant-pathogen interactions but also highlights their potential as targets for biotechnological approaches to disease resistance breeding.