Methylation-sensitive binding of BraBPC2.A08 to the BraCYP707A3.A02 promoter regulates Brassica clubroot resistance by ABA catabolism

Clubroot disease, caused by Plasmodiophora brassicae, seriously threatens global production of cruciferous crops. Epigenetic regulation, particularly DNA methylation, is increasingly recognized as important in plant–pathogen interactions, but its role in clubroot resistance remains poorly understood. Here, we integrated whole-genome bisulfite sequencing and transcriptome profiling of resistant and susceptible Brassica rapa lines following P. brassicae infection. The resistant line exhibited global hypomethylation, predominantly in the CHH context, whereas the susceptible line showed progressive hypermethylation. A key abscisic acid (ABA) catabolic gene, BraCYP707A3.A02, was downregulated and its promoter hypermethylated in susceptible plants, leading to ABA accumulation and increased susceptibility. Chemical demethylation with 5-azacytidine or overexpression of the DNA demethylase gene DME reduced promoter methylation, restored BraCYP707A3.A02 expression, lowered ABA levels, and enhanced clubroot resistance in both B. rapa and B. napus. Furthermore, the transcription factor BraBPC2.A08 specifically bound to the GA-rich motif in the BraCYP707A3.A02 promoter to activate its expression, and this binding was abolished by cytosine methylation. Our findings reveal a novel epigenetic regulatory module where DNA methylation fine-tunes ABA homeostasis by modulating BraCYP707A3.A02 expression, thereby determining B. rapa resistance to clubroot. Our findings uncover a methylation-sensitive transcriptional activation mechanism and provide a potential target for epigenome-assisted breeding of clubroot-resistant Brassica crops.