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

Abstract

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.2^W (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.2^Z (the susceptible cabbage Z5), Bol.TNL.3^W, and Bol.TNL.3^Z 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.

Acknowledgments

This work was supported by Science Technology Innovation 2030-Key Project: Identification of Novel Pest-Resistant Genes and Breeding Value Assessment (2023ZD04070), the National Key Research and Development Program (2022YFD1200400), the National Natural Science Foundation of China (32441051), the Shaanxi Province postdoctoral research project (2023BSHYDZZ66), the Shaanxi Provincial Natural Science Basic Research Program (2023-JC-YB-191), the Key Research and Development Project of Shaanxi Province (2024NC-ZDCYL-01-06), the Key Research and Development Plan of Shaanxi Province (2023-YBNY-020).

Author contributions

Y.S., S.L., and Z.H. conceived the study. Y.S., S.B., A.X., H.S., and K.W. prepared the tissue samples for sequencing. K.X., L.Z., T.Z., and M.L. performed the bioinformatics analyses. Y.S., F.Z., W.S., J.W., and X.L. performed the genetic transformation of A. thaliana and B. napus. Y.S. and K.L. drafted the manuscript. S.L. and Z.H. reviewed and edited the manuscript.

Data availability

All sequencing data have been deposited in the NCBI Sequence Read Archive (SRA). The BSA resequencing data are available under accession number PRJNA1172340, the ssRNA-seq data under PRJNA1172278, and the RNA-seq of OE B. napus Bol.TNL.2^W-OE data under PRJNA1191364.

Conflict of interest statement

The authors declare no competing interests.

Supplementary data

Supplementary data is available at Horticulture Research online.

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