Genome-Wide Identification and Analysis of Brassica napus Nonexpressor of Pathogenesis-Related Genes Family and Functional Analysis of BnaNPR3.A02 in Clubroot Resistance

Abstract

Nonexpressor of pathogenesis-related (NPR) proteins are key regulators of salicylic acid (SA)-mediated plant immunity, functioning as transcriptional co-regulators. In this study, we aimed to comprehensively identify NPR family members in Brassica napus and its diploid progenitors, investigate their evolutionary dynamics and expression profiles, and functionally characterize the role of BnaNPR3.A02 in clubroot resistance. We identified 23, 12, and 12 NPR genes in B. napus, B. rapa, and B. oleracea, respectively, which were phylogenetically divided into six conserved subfamilies (I–VI). Structural characterization showed that these genes possess conserved ankyrin repeats, BTB-POZ domains or the DUF3420 domains, and exhibit diversified exon–intron structures. Spatiotemporal expression profiling uncovered tissue-specific expression patterns of NPRs under normal conditions. Promoter cis-element analysis and transcriptomic profiling in response to phytohormones and abiotic stresses suggested broad involvement of BnaNPRs in environmental adaptation. GO annotation revealed that NPR1–4 members are enriched in jasmonic acid/salicylic acid-mediated defense pathways, whereas NPR5/6 members are associated with floral development. qRT-PCR analysis of 11 expressed BnaNPR1–4 genes was performed in resistant (R) and susceptible (S) rapeseed plants upon P. brassicae infection. BnaNPR3.A02 showed the greatest expression divergence, with significant induction in R plants but consistent suppression in S plants. Because NPR3 is a negative feedback regulator of SA signaling, the induction of BnaNPR3.A02 in R plants likely reflects feedback attenuation after immune activation, whereas its suppression in S plants may indicate a defective SA signaling node. Heterologous overexpression of BnaNPR3.A02 in Arabidopsis significantly increased disease severity and pathogen biomass, whereas Arabidopsis npr3 knockout mutants showed enhanced resistance, supporting a conserved negative regulatory role of NPR3 in this heterologous system. Protein–protein interaction and co-expression analyses further revealed that BnaNPR3.A02 is embedded in a stress- and defense-related network. These findings provide a foundation for understanding the evolutionary and functional divergence of the NPR family and identify BnaNPR3.A02 as a candidate target for future functional validation and breeding of clubroot-resistant B. napus varieties.