Identification and characterization of <i>Bol.TNL.2,</i> a key clubroot resistance gene from cabbage, in <i>Arabidopsis</i> and <i>Brassica napus</i> L.

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 (<i>Brassica oleracea</i>). This study aimed to identify CR genes and elucidate the molecular mechanisms underlying clubroot resistance in <i>B. oleracea</i>. A BC₁ mapping population was developed from a cross between CR cabbage W12 and clubroot-susceptible cabbage Z5. A major CR locus, <i>Bol.CR7.1</i>, was identified on chromosome C07 by Bulk Segregant Analysis. Subsequently, the <i>Bol.CR7.1</i> was fine-mapped to a 170.2-kb interval using linkage analysis. Two candidate genes, <i>Bol.TNL.2</i> and <i>Bol.TNL.3</i>, exhibiting sequence variations between the parents were induced upon <i>Plasmodiophora brassicae</i> infection. Overexpression of <i>Bol.TNL.2^W</i> (CR cabbage W12) in <i>Arabidopsis</i> and rapeseed significantly reduced the disease index compared to the wild type (WT) after <i>P. brassicae</i> inoculation. In contrast, plants overexpressing <i>Bol.TNL.2^Z</i> (the susceptible cabbage Z5), <i>Bol.TNL.3^W</i> , and <i>Bol.TNL.3^Z</i> exhibited symptoms comparable to those of WT, indicating that <i>Bol.TNL.2</i> is a CR gene. RNA-seq analysis revealed that <i>Bol.TNL.2</i> may mediate resistance to <i>P. brassicae</i> 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 <i>P. brassicae</i> 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 <i>P. brassicae</i> infection in <i>B. oleracea</i>.