Multi-Omics Analysis Uncovers Synergistic Phenylpropanoid-Glucosinolate Metabolism Regulating Clubroot Resistance in Brassica napus L.

Clubroot caused by Plasmodiophora brassicae threatens rapeseed production. Metabolic networks mediate defense, yet cross‐pathway synergy remains elusive. We performed GWAS on a resistant–susceptible segregating population (T13) and identified 51 resistance‐associated SNPs, with four loci on C04 co‐localizing with known QTLs/genes. Time‐series root transcriptomes of resistant vs. susceptible genotypes after inoculation were analyzed. WGCNA resolved six co‐expression modules tightly linked to resistance and identified 29 hub genes among 2,859 GWAS‐annotated candidates. Functional enrichment and centrality analyses converged on a defense network centered on flavonoid biosynthesis and glucosinolate metabolism, with this subnetwork being the top contributor for clubroot resistance. Genome‐wide identification of ADT, FLS, and upstream MYB regulators across Brassica species yielded 47, 494, and 497 members (6, 12, 12 subfamilies), expanded by segmental duplication. Expression profiling and RT‐qPCR confirmed significant upregulation of multiple members in resistant genotypes at late infection. PPI networks revealed hub genes form a synergistic module with enzymes in phenylpropanoid, lignin, glucosinolate, amino‐acid metabolism and development (BnaPAL1, BnaAOP3, BnaBCAT3/4). Collectively, we elucidate the genetic loci and transcriptional architecture, and propose a multi‐layered synergistic model wherein flavonoid and glucosinolate pathways act as core defenses, functionally integrated via the MYB28–ADT5–FLS3 axis and key nodes PAL1, AOP3, BCAT3/4, with upstream primary metabolism, immune signaling, and development to shape a systemic network at late infection. This model provides a theoretical framework and concrete candidate genes for breeding.