CsMLO8/11 are required for full susceptibility of cucumber stem to powdery mildew and interact with CsCRK2 and CsRbohD

Abstract

Powdery mildew (PM) is one of the most destructive diseases that threaten cucumber production globally. Efficient breeding of novel PM-resistant cultivars will require a robust understanding of the molecular mechanisms of cucumber resistance against PM. Using a genome-wide association study, we detected a locus significantly correlated with PM resistance in cucumber stem, pm-s5.1. A 1449-bp insertion in the CsMLO8 coding region at the pm-s5.1 locus resulted in enhanced stem PM resistance. Knockout mutants of CsMLO8 and CsMLO11 generated by CRISPR/Cas9 both showed improved PM resistance in the stem, hypocotyl, and leaves, and the double mutant mlo8mlo11 displayed even stronger resistance. We found that reactive oxygen species (ROS) accumulation was higher in the stem of these mutants. Protein interaction assays suggested that CsMLO8 and CsMLO11 could physically interact with CsRbohD and CsCRK2, respectively. Further, we showed that CsMLO8 and CsCRK2 competitively interact with the C-terminus of CsRbohD to affect CsCRK2-CsRbohD module-mediated ROS production during PM defense. These findings provide new insights into the understanding of CsMLO proteins during PM defense responses.

Acknowledgements

This research was supported by the National Natural Science Foundation of China (32202487), the Beijing Municipal Science and Technology Commission Program (Z231100003723005), the Key-Area Research and Development Program of Guangdong Province (2020B020220001), the Key-Area Research and Development Program of Shandong Province (2021LZGC016), the Beijing Joint Research Program for Germplasm Innovation and New Variety Breeding (G20220628003-03), the Earmarked Fund for Modern Agro-industry Technology Research System (CARS-23), and the Science and Technology Innovation Program of the Chinese Academy of Agricultural Science (CAAS-ASTIP-IVFCAAS).

Author contributions

S.D. drafted the manuscript; S.D., X.L., J.H., and H.M. conducted the experiments and analyzed the data; D.M.B., Y.B., J.G., X.P.L., R.Y., and X.G. helped collect the data; J.S., X.Y., and S.Z. conceived and designed the study.

Data availability

All data that support the findings of this study are available in the article and in the supplementary materials published online.

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Supplementary data

Supplementary data is available at Horticulture Research online.

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