SummaryHere, we describe the rapid cloning of a plant gene, Leptosphaeria maculans 3 (RLM3 Col ), which encodes a putative Toll interleukin-1 receptor-nucleotide binding (TIR-NB) class protein, which is involved in defence against the fungal pathogen L. maculans and against three other necrotrophic fungi. We have, through microarray-based case control bulk segregant comparisons of transcriptomes in pools of Col-0 · An-1 progeny, identified the absence of a locus that causes susceptibility in An-1. The significance of this locus on chromosome 4 for L. maculans resistance was supported by PCR-based mapping, and denoted resistance to RLM3 Col . Differential susceptible phenotypes in four independent T-DNA insertion lines support the hypothesis that At4g16990 is required for RLM3 Col function. The mutants in RLM3 Col also exhibited an enhanced susceptibility to Botrytis cinerea, Alternaria brassicicola and Alternaria brassicae. Complementations of An-1 and T-DNA mutants using overexpression of a short transcript lacking the NB-ARC domain, or a genomic clone, restored resistance to all necrotrophic fungi. The elevated expression of RLM3 Col on B. cinerea-susceptible mutants further suggested convergence in signalling and gene regulation between defence against B. cinerea and L. maculans. In the case of L. maculans, RLM3 Col is required for efficient callose deposition downstream of RLM1 Col .