Brassica napus genes Rlm4 and Rlm7, conferring resistance to Leptosphaeria maculans, are alleles of the Rlm9 wall-associated kinase-like resistance locus

Abstract

Brassica napus (canola/rapeseed) race specific resistance genes against blackleg disease, caused by the ascomycete fungus Leptosphaeria maculans, have been commonly used in canola breeding. To date; LepR3, Rlm2 and Rlm9 R genes against L. maculans have been cloned from B. napus. LepR3 and Rlm2 are Receptor Like Proteins (RLP) and the recently reported Rlm9 is a Wall Associated Kinase-Like (WAKL) protein. Rlm9 located on chromosome A07 is closely linked with Rlm3, Rlm4, RLm7 genes. Recognition of AvrLm5-9 and AvrLm3 by their corresponding Rlm9 and Rlm3 proteins is masked in the presence of AvrLm4-7. Here we report cloning of Rlm4 and Rlm7 by generating genome sequence of the doubled haploid (DH) B. napus cv Topas DH16516 introgression lines Topas-Rlm4 and Topas-Rlm7. Candidate Rlm4 and Rlm7 genes were identified form the genome sequence and gene structures were determined by mapping RNA-sequence reads, generated from infected cotyledon tissues, to the genome of Topas-Rlm4 and Topas-Rlm7. Rlm4 and Rlm7 genomic constructs with their native promoters were transferred into the blackleg susceptible B. napus cv Westar N-o-1. Complementation of resistance response in the transgenic Westar:Rlm4 and Westar:Rlm7 that were inoculated with L. maculans transgenic isolates 2367:AvrRlm4-7 or 2367:AvrLm7 confirmed the function of Rlm4 and Rlm7 genes. Wild type L. maculans isolate 2367 that does not contain AvrLm4-7 or AvrLm7, and transgenic 2367:AvrLm3 and 2367:AvrLm5-9 did not induce resistance proving the specificity of Rlm4 and Rlm7 response. Rlm4 and Rlm7 alleles are also allelic to Rlm9. Rlm4 and Rlm7 genes encode WAKL proteins. Comparison of highly homologous sequences of Rlm4 and Rlm7 with each other and with the sequence of additional alleles, using whole genome sequencing of additional 128 lines, identified a limited number of point mutation located within the predicted extracellular receptor domains.