Comprehensive analysis of Brassica napus nonexpressor of pathogenesis-related genes family and functional analysis of BnaNPR3.A02 in clubroot resistance

Background Nonexpressor of pathogenesis-related genes (NPR) are key regulators of salicylic acid (SA)-mediated plant immunity, acting as transcription co‑regulators. However, a comprehensive genome-wide analysis of the NPR family in Brassica napus has not been reported. Therefore, this study aims to comprehensively identify NPR family members in B. napus and its diploid progenitors, analyze their evolutionary characteristics and expression patterns, and functionally characterize the role of BnaNPR3.A02 in clubroot resistance. Results In this study, we identified 13, 7, and 7 NPR genes in B. napus , B. oleracea , and B. rapa , respectively, which were phylogenetically divided into four conserved subfamilies (Ⅰ–Ⅳ). Structural characterization revealed conserved domain architectures and variable exon-intron organizations, while spatiotemporal expression profiling indicated distinct tissue-specific patterns. Promoter cis‑element analysis and transcriptomic responses to phytohormones and abiotic stresses suggested broad involvement of BnaNPRs in environmental adaptation. Notably, BnaNPR3.A02 displayed striking down-regulation following Plasmodiophora brassicae infection. Functional assays in Arabidopsis demonstrated that overexpression of BnaNPR3.A02 significantly increased clubroot susceptibility, whereas AtNPR3 knockout enhanced resistance, confirming its negative regulatory role. Protein-protein interaction network analysis predicted that BnaNPR3.A02 interacts with multiple proteins including BnaNPR3.C02, and co-expression analysis of its Arabidopsis homolog AtNPR3(AT5G45110) identified 300 co-expressed genes significantly enriched in defense response and response to stress GO terms, further supporting its participation in immune responses. Conclusions These findings provide a foundation for understanding the evolutionary and functional divergence of the NPR family in B. napus and identify BnaNPR3.A02 as a potential target for breeding clubroot‑resistant B. napus varieties.