Recognition of Necrosis and Ethylene-inducing like peptides confers disease resistance in Brassica napus and is modulated by BSK1 in Arabidopsis

Brassicas are important crops susceptible to significant losses caused by disease: thus, breeding resistant lines can mitigate the effects of pathogens. MAMPs (microbe-associated molecular patterns) are conserved molecules of pathogens that elicit host defence responses known as pattern-triggered immunity (PTI). Necrosis & Ethylene-inducing peptide 1-like proteins (NLPs) are MAMPs found in a wide range of phytopathogens, including major disease-causing fungal species. We studied the response to the BcNEP2 from Botrytis cinerea as a representative NLP in Brassica napus to improve our understanding of recognition mechanisms that could enable the development of disease-resistant crops. To genetically map regions responsible for NLP recognition, we used an associative transcriptomics (AT) approach using diverse B. napus accessions and bulk segregant analysis (BSA) on DNA pools created from a bi-parental cross of NLP-responsive (Ningyou1) and non-responsive (Ningyou7) lines. In silico mapping with AT identified two peaks associated with NLP recognition on chromosomes A04 and C05 whereas the BSA narrowed it down to a main peak on A04. BSA delimited the region associated with NLP-responsiveness to 3 Mbp, containing [~]245 genes on the Darmor-bzh reference genome. Variants detected in the region were used for KASP marker design and four KASP markers were identified co-segregating with the phenotype. The same pipeline was performed with the ZS11 genome, and the highest associated region was confirmed on chromosome A04. Comparative BLAST analysis revealed there were unannotated clusters of RLP homologs on ZS11 chromosome A04. To reduce the number of candidate genes responsible for NLP recognition, RNA-Seq data was used to detect the unannotated expressed putative genes. Screening the BSA Ning1x7 population demonstrated a highly significant association between NLP-recognition and resistance to Botrytis cinerea. Also, the lines non-responsive to NLP had significantly greater response to the bacterial MAMP flg22. Additionally, BnaA01g02190D, a homologue of Arabidopsis AtBSK1 (At4g35230) BR-SIGNALLING KINASE1, was associated with a high BcNEP2-induced ROS response phenotype. We show that in Arabidopsis, Atbsk1 mutants had significantly lower response to BcNEP2 and increased susceptibility to B. cinerea (p-value=1.12e-14***). Overall, the results define the genomic location for NLP-recognition on the B. napus genome and demonstrate that NLP recognition has a positive contribution to disease resistance which can have practical application in crop improvement.