Genome-wide association study and evolutionary analysis of the CrRLK1L family reveal BnCrRLK1L1_5 as a positive regulator of Sclerotinia sclerotiorum resistance in Brassica napus

BACKGROUND: Sclerotinia stem rot, caused by the necrotrophic pathogen Sclerotinia sclerotiorum (S. sclerotiorum), poses a significant threat to rapeseed (Brassica napus), resulting in substantial yield losses and economic damage worldwide. While receptor-like kinases, particularly the Catharanthus roseus RLK1-like (CrRLK1L) family, are known to play vital roles in plant immunity and development, their specific functions in B. napus defense against S. sclerotiorum and their evolutionary dynamics remain largely unexplored. RESULTS: Through a GWAS, we identified BnCrRLK1L1_5, a CrRLK1L family member, as a candidate gene conferring resistance to S. sclerotiorum. To understand its evolutionary context, genome-wide and phylogenetic analyses of the CrRLK1L family across three Brassica species were performed, revealing significant diversification driven by whole-genome duplications and gene loss. Nevertheless, key functional domains, such as the malectin-like and kinase domains, remained highly conserved. Further analysis of cis-elements and the identification of six BnCrRLK1L1 paralogs in B. napus suggested potential functional redundancy and broad roles in mediating stress responses. Transcriptomic analysis revealed that the expression of BnCrRLK1L1_5 was strongly induced in resistant genotypes upon inoculation. Functional characterization using fer-4, a mutant of the Arabidopsis ortholog FERONIA (FER), showed enhanced susceptibility to S. sclerotiorum. Furthermore, complementation with BnCrRLK1L1_5 in the fer-4 background restored resistance to levels exceeding the wild-type, indicating functional conservation between BnCrRLK1L1_5 and FER. Co-expression of BnCrRLK1L1_5 with BAX in N. benthamiana leaves significantly reduced necrosis and ion leakage, suggesting that BnCrRLK1L1_5 functions as a suppressor of cell death. Moreover, protein-protein interaction network analysis predicted interactions between BnCrRLK1L1_5 and key partners, including BnCrRLK1L1_4, PTM9, RALF23, and Ferritin 4. These findings suggest that BnCrRLK1L1_5 modulates resistance through regulating cell wall integrity, oxidative stress, and immune signaling. CONCLUSIONS: Together, this study elucidates the dynamic evolution of the CrRLK1L family and establishes BnCrRLK1L1_5 as a key regulator of resistance against S. sclerotiorum. The potential mechanisms involving immune signaling and cell death regulation provide valuable insights for improving disease resistance in rapeseed and other crops.