Clubroot is a devastating soil-borne disease that parasitizes cruciferous crops, posing a severe threat to rapeseed production. To date, no clubroot-resistant (CR) genes have been successfully cloned in cabbage (Brassica oleracea). This study aimed to identify CR genes and elucidate the molecular mechanisms underlying clubroot resistance in B. oleracea. A BC₁ mapping population was developed from a cross between CR cabbage W12 and clubroot-susceptible cabbage Z5. A major CR locus, Bol.CR7.1, was identified on chromosome C07 by Bulk Segregant Analysis. Subsequently, the Bol.CR7.1 was fine-mapped to a 170.2-kb interval using linkage analysis. Two candidate genes, Bol.TNL.2 and Bol.TNL.3, exhibiting sequence variations between the parents were induced upon Plasmodiophora brassicae infection. Overexpression of Bol.TNL.2W (CR cabbage W12) in Arabidopsis and rapeseed significantly reduced the disease index compared to the wild type (WT) after P. brassicae inoculation. In contrast, plants overexpressing Bol.TNL.2Z (the susceptible cabbage Z5), Bol.TNL.3W, and Bol.TNL.3Z exhibited symptoms comparable to those of WT, indicating that Bol.TNL.2 is a CR gene. RNA-seq analysis revealed that Bol.TNL.2 may mediate resistance to P. brassicae by modulating pathways related to reactive oxygen species, cell wall metabolism and modification, as well as secondary metabolite synthesis. In addition, long noncoding RNAs were found to play a significant role in regulating gene expression associated with P. brassicae interaction. This study broadens the repertoire of CR genes, offering a solid foundation for breeding CR cruciferous crops. Additionally, it provides novel insights into resistance mechanisms in response to P. brassicae infection in B. oleracea.

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