Ethylene signaling negatively regulates rapeseed resistance to Plasmodiophora brassicae

Clubroot, caused by Plasmodiophora brassicae, poses a serious threat to the rapeseed (Brassica napus) industry. Due to B. napus being an allopolyploid with a complex genome and the current scarcity of available resistance gene resources, the molecular basis of rapeseed resistance to P. brassicae remains poorly understood. Here, we performed a functional characterization of BnEIN2 (ethylene-insensitive protein) to explore the role of ethylene signaling in rapeseed resistance to P. brassicae. The Bnein2 mutants generated through CRISPR/Cas9 technology exhibited enhanced resistance to P. brassicae, along with reduced 1-aminocyclopropane-1-carboxylic acid (ACC)/S-adenosyl-L-methionine (SAM) accumulation and ethylene insensitivity. Pharmacological assays demonstrated that inhibitors of ethylene biosynthesis or signaling improved the resistance of Bnein2 mutant plants to P. brassicae. Transcriptome analysis revealed that loss-of-function of BnEIN2 affected the expression of ethylene-, auxin-, and cytokinin-related genes. Moreover, the increased resistance of Bnein2 mutants to P. brassicae was accompanied by a reduction in auxin (indole-3-acetic acid, IAA) biosynthesis and degradation of cytokinin (trans-zeatin, TZ). Collectively, these findings establish the negative regulatory role of ethylene signaling in rapeseed resistance to P. brassicae. This study represents the first effort to elucidate rapeseed resistance to P. brassicae by directly obtaining rapeseed genetic material and offer novel insights into the hormonal regulatory network underlying disease resistance and valuable resources for breeding clubroot-resistant varieties.