A unique wheat disease resistance-like gene governs effector-triggered susceptibility to necrotrophic pathogens

Abstract

Plant disease resistance is often conferred by genes with nucleotide binding site (NBS) and leucine-rich repeat (LRR) or serine/threonine protein kinase (S/TPK) domains. Much less is known about mechanisms of susceptibility, particularly to necrotrophic fungal pathogens. The pathogens that cause the diseases tan spot and Stagonospora nodorum blotch on wheat produce effectors (host-selective toxins) that induce susceptibility in wheat lines harboring corresponding toxin sensitivity genes. The effector ToxA is produced by both pathogens, and sensitivity to ToxA is governed by the Tsn1 gene on wheat chromosome arm 5BL. Here, we report the cloning of Tsn1, which was found to have disease resistance gene-like features, including S/TPK and NBS-LRR domains. Mutagenesis revealed that all three domains are required for ToxA sensitivity, and hence disease susceptibility. Tsn1 is unique to ToxA-sensitive genotypes, and insensitive genotypes are null. Sequencing and phylogenetic analysis indicated that Tsn1 arose in the B-genome diploid progenitor of polyploid wheat through a gene-fusion event that gave rise to its unique structure. Although Tsn1 is necessary to mediate ToxA recognition, yeast two-hybrid experiments suggested that the Tsn1 protein does not interact directly with ToxA. Tsn1 transcription is tightly regulated by the circadian clock and light, providing further evidence that Tsn1-ToxA interactions are associated with photosynthesis pathways. This work suggests that these necrotrophic pathogens may thrive by subverting the resistance mechanisms acquired by plants to combat other pathogens.

Fig. 1.

Map-based cloning of the Tsn1 gene. (A) Genomic region containing the Tsn1 gene on the long arm of wheat chromosome 5B is shown in red. (B) Genetic linkage map of the Tsn1 region. (C) BAC-based physical maps of the Tsn1 region anchored to the genetic map. LDN BACs previously described (14) are shown in gray. The 5B and 5A BACs reported in the current work are shown in green and orange, respectively. (D) Predicted genes (ovals with names in dark red) and markers (names beginning with an “X”). Markers used for association mapping of the 386 Triticum accessions are indicated in blue. The blue and red lines indicate the candidate gene regions as defined by recombination in the mapping population and association mapping of the 386 Triticum accessions, respectively. (E) Exons and UTRs of Tsn1 are shown in purple and gray, respectively.

Fig. 2.

Leaves of Kulm (Tsn1) (A and C) and Kems103 (Tsn1 mutant) (B and D) inoculated with S. nodorum (A and B) and infiltrated with ToxA (C and D).

Fig. 3.

Southern and PCR analysis of 24 selected wheat lines. (Top panel) Southern hybridization of DNA digested with restriction enzyme XbaI and probed with FCG34, which is derived from the NBS region of Tsn1. (Bottom panel) PCR amplification with primers for marker Xfcp623(Tsn1) derived from intron five of Tsn1. The wheat genotypes labeled in bold and not bold are sensitive and insensitive to ToxA, respectively.

Fig. 4.

Colinearity at the Tsn1 locus. (A) Colinearity of genes (colored ovals) within the Tsn1 region of wheat chromosome 5B, the homologous region of wheat chromosome 5A, Brachypodium chromosome 4, and rice chromosome 9. Gene descriptions are presented in Table S4. Note that Tsn1 is present only in wheat 5B. (B) Comparisons of the Tsn1 domains with homologous domains/genes on rice chromosome 11 and Brachypodium chromosome 2. The NBS-LRR region of Tsn1 corresponds to the full coding regions of the predicted rice and Brachypodium NBS-LRR genes, but the genes containing homologous S/TPK domains in rice and Brachypodium harbor additional domains not found in Tsn1.

Fig. 5.

Transcriptional expression of Tsn1. (A) Tsn1 expression survey by RT-PCR with GAPDH as an endogenous control. (B) Tsn1 expression levels in 2-wk-old plants entrained with a 12-h light/dark cycle evaluated every 3 h over a 72-h period (orange) and in plants subjected to continuous dark for the same time periods (dark green) using RQ-PCR. (C) RQ-PCR evaluation of Tsn1 expression in 2-wk-old plants entrained with a 12-h light/dark cycle (control; blue bars) and plants subjected to 3 h of dark followed by 2 h of light (red bars). (D) RQ-PCR evaluation of Tsn1 expression in ToxA-challenged plants (blue bars, ToxA infiltrated; red bars, H₂O infiltrated; yellow bars, no infiltration).