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 fromBotrytis cinereaas a representative NLP inBrassica napusto 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 diverseB. napusaccessions and bulk segregant analysis (BSA) on DNA pools created from a bi-parental cross of NLP-responsive (Ningyou1) and non-responsive (Ningyou7) lines. In silicomapping 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-bzhreference 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 Ning1×7 population demonstrated a highly significant association between NLP-recognition and resistance toBotrytis cinerea. Also, the lines non-responsive to NLP had significantly greater response to the bacterial MAMP flg22. Additionally,BnaA01g02190D, a homologue of ArabidopsisAtBSK1(At4g35230)BR-SIGNALLING KINASE1,was associated with a high BcNEP2-induced ROS response phenotype. We show that in Arabidopsis,Atbsk1mutants had significantly lower response to BcNEP2 and increased susceptibility toB. cinerea(p-value=1.12e-14***). Overall, the results define the genomic location for NLP-recognition on theB. napusgenome and demonstrate that NLP recognition has a positive contribution to disease resistance which can have practical application in crop improvement.

Translational research is required to advance fundamental knowledge on plant immunity towards application in crop improvement. Recognition of microbe/pathogenassociated molecular patterns (MAMPs/PAMPs) triggers a first layer of immunity in plants. The broadly occurring family of necrosis-and ethylene-inducing peptide 1 (NEP1)-like proteins (NLPs) contains immunogenic peptide patterns that are recognized by a number of plant species. Arabidopsis can recognize NLPs by the pattern recognition receptor AtRLP23 and its co-receptors SOBIR1, BAK1, and BKK1, leading to induction of defence responses including the production of reactive oxygen species (ROS) and elevation of intracellular [Ca 2+ ]. However, little is known about NLP perception in Brassica crop species. Within 12 diverse accessions for each of six Brassica crop species, we demonstrate variation in response to Botrytis cinerea NLP BcNEP2, with Brassica oleracea (CC genome) being nonresponsive and only two Brassica napus cultivars responding to BcNEP2. Peptides derived from four fungal pathogens of these crop species elicited responses similar to BcNEP2 in B. napus and Arabidopsis. Induction of ROS by NLP peptides was strongly reduced in Atrlp23, Atsobir1 and Atbak1-5 Atbkk1-1 mutants, confirming that recognition of Brassica pathogen NLPs occurs in a similar manner to that of HaNLP3 from Hyaloperonospora arabidopsidis in Arabidopsis. In silico analysis of the genomes of two B. napus accessions showed similar presence of homologues for AtBAK1, AtBKK1 and AtSOBIR1 but variation in the organization of AtRLP23 homologues. We could not detect a strong correlation between the ability to respond to NLP peptides and resistance to B. cinerea.