MdRLKT1-MdRAX2-MdMKS1 Module Positively Regulating Resistance to Cytospora mali in Apple

Valsa canker (caused by Cytospora mali = Valsa mali. C. mali) is one of the most destructive diseases affecting apple cultivation. The scarcity of natural germplasm resources with high resistance and immunity underscores the importance of exploring plant immune regulation factors of disease-resistant breeding. Protein post-translational modifications, particularly phosphorylation, are critical regulatory mechanisms in plant immunity. This study investigates how the apple receptor-like kinase MdRLKT1 modulates resistance to Valsa canker through the phosphorylation of the transcription factor MdRAX2. We found that MdRLKT1-interference (RNAi) transgenic lines exhibit increased susceptibility to C. mali infection compared to wild-type controls, indicating that MdRLKT1 positively regulates apple immune responses. Notably, MdRLKT1 interacts with the MYB transcription factor MdRAX2, facilitating its translocation into the nucleus. In vitro phosphorylation assays identified serine 147 (Ser147) as the phosphorylation site of MdRAX2 by MdRLKT1. Mutant MdRAX2^S147A, with this phosphorylation site inactivated, demonstrated reduced resistance to C. mali. Further analysis revealed that MdRAX2 binds to the promoter region of MdMKS1, transcriptionally repressingits expression, whereas MdRAX2^S147A failed to regulate MdMKS1 transcriptionally. Overexpression of MdMKS1 in apple resulted in reduced resistance to C. mali, suggesting that MdMKS1 negatively regulates apple immunity. These findings establish that the MdRLKT1-MdRAX2-MdMKS1 module plays a positive regulatory role in enhancing apple resistance to C. mali. In conclusion, MdRLKT1 activates the transcriptional repressor function of MdRAX2 through phosphorylation, thereby alleviating the negative regulatory effect of MdMKS1 on disease resistance and ultimately boosting the defensive capabilities of apple against pathogens.