A single NLR gene confers resistance to leaf and stripe rust in wheat

Abstract

Nucleotide-binding leucine-rich repeat (NLR) disease resistance genes typically confer resistance against races of a single pathogen. Here, we report that Yr87/Lr85, an NLR gene from Aegilops sharonensis and Aegilops longissima, confers resistance against both P. striiformis tritici (Pst) and Puccinia triticina (Pt) that cause stripe and leaf rust, respectively. Yr87/Lr85 confers resistance against Pst and Pt in wheat introgression as well as transgenic lines. Comparative analysis of Yr87/Lr85 and the cloned Triticeae NLR disease resistance genes shows that Yr87/Lr85 contains two distinct LRR domains and that the gene is only found in Ae. sharonensis and Ae. longissima. Allele mining and phylogenetic analysis indicate multiple events of Yr87/Lr85 gene flow between the two species and presence/absence variation explaining the majority of resistance to wheat leaf rust in both species. The confinement of Yr87/Lr85 to Ae. sharonensis and Ae. longissima and the resistance in wheat against Pst and Pt highlight the potential of these species as valuable sources of disease resistance genes for wheat improvement.

Fig. 1. Cloning and functional validation of Ae. sharonensis Yr87/Lr85.

a Genetic (cM) and physical (Mb) maps of the Yr/Lr locus in the D42 introgression line^,: black, wheat background; gray, Aegilops background; genes in the interval delimiting the presence of Yr87/Lr85: HK histidine kinase; LRR PK LRR protein kinase, CRK cysteine-rich receptor kinase, RPP13 disease resistance protein RPP13, Serpin serine protease inhibitors, GR glutamate receptor. Predicted gene structure and protein domains according to NLRscape: black lines denote introns, shaded rectangles denote untranslated regions (UTRs), lilac rectangles are exons, closed arrows denote EMS point mutations, open arrows denote locations of sequences targeted by VIGS (for details see Supplementary Data 4), double slash denotes aproximately 3kb of intronic region not shown, asterisk denotes the Rx-CC domain. The number below the nucleotide in parentheses represents the mutant lines. The number below the nucleotide in parentheses represents the mutant line numbers. b Yr87/Lr85 expression levels in D42 following VIGS. Data are mean ± s.e.m. Significant differences were evaluated using a two-tailed Student’s t-test. n = 4 biological replicates. Black dots represent individual data points. P values are shown on the graph. c Reaction of D42 to leaf and stripe rust isolates after VIGS targeting the 5′ UTR and exon 2 of Yr87/Lr85. Wheat Phytoene deacetylase (PDS) was used as a control to monitor the effectiveness of gene silencing. Scale bars, 2 mm. d AlphaFold2 predicted YR87/LR85 protein structure, where the Rx-CC (blue) and NB-ARC (green) domains mostly comprise α-helixes and the LRR (yellow and pink) domains comprise a β-helical “horseshoe” structures. Magenta spheres denote EMS point mutations. Source data are provided as a Source Data file.

Fig. 2. Cloning and functional validation of Ae. longissima Yr87/Lr85.

a Distribution of leaf rust resistance across 137 Ae. longissima accessions. Infection types range from 1 (complete resistance) to 9 (fully susceptible). Pearson's correlation coefficient was calculated to assess the relationship between infection types of the two isolates, revealing a strong correlation (r = 0.91, P = 5.97e⁻⁵³). A two-tailed test was used, and no adjustments were made for multiple comparisons. b Identification of Yr87/Lr85 using Pt isolate #12337. The x-axis shows the genomic position on the AEG-6782-2 genome assembly, the y-axis represents the score of the k-mers associated with resistance across the diversity panel. The association score is defined as the negative log of the P value obtained using the general linear model (GLM). The dotted red line is the significance threshold at P = 0.001 and is adjusted for multiple comparisons using “Bonferroni’ correction,* denotes Rx-CC domain. c Diagrams of the gene constructs used to transform wheat cv. Fielder. Shaded rectangles denote promoter and terminator regions, lilac rectangles denote exons, black lines (bottom scheme) denote partial (first and second introns, where 4 kb and 5.5 kb were excluded respectively from the construct) or full (third) intron sequences, ^ denotes Exon 4. d Reaction of exemplary transgenic lines to infection with Pt and Pst isolates. gDNA (T-g) and cDNA (T-c) transgenic lines. Scale bars, 2 mm. e Relative transcript levels of the Yr87/Lr85 gene in gDNA (T-g) and cDNA (T-c) transgenic lines vs. wild-type Fielder and in Ae. longissima AEG-6782-2 at the seedling stage. f Relative transcript levels of the Yr87/Lr85 gene in uninfected (green) or post-infected (blue) leaves, at the seedling stage, after infection with Pt isolate #12460. Data are mean ± s.e.m. Significant differences were evaluated using a two-tailed Student’s t-test. n = 3 biological replicates. Black dots represent individual data points. P values are shown on the graph. The D6 protein kinase (D6PK) like gene (XM_044524929) was used as an internal control. Source data are provided as a Source Data file.

Fig. 3. Yr87/Lr85 confers resistance in seedlings and adult plants.

a Pustule development on seedling (Pt, 7 days post-inoculation [dpi]; Pst, 14 dpi) and adult (Pt, 18–20 dpi; Pst, 17–20 dpi) leaves of wheat cv. Fielder and transgenic plants expressing Ae. longissima Yr87/Lr85. Scale bars, 2 mm. Light (b) and fluorescent (c) micrographs of leaf segments of wheat seedlings inoculated with Pt or Pst. b Yr87/Lr85 transgenic seedlings developed chlorotic halos that occasionally produced micropustules (Pt, 7 dpi; Pst, 14 dpi). Scale bars, 200 µm. c Leaves were stained with WGA-FITC. Spores of Pt and Pst germinated and penetrated the epidermis of Yr87/Lr85 transgenic plants but progressed slower and developed smaller colonies within the leaf compared with infection of cv. Fielder plants. The arrows mark the edges of the colonies. Scale bars, 200 µm. The experiments were independently repeated three times, yielding consistent results.

Fig. 4. Yr87/Lr85 accounts for the majority of resistance to P. triticina in Ae. sharonensis and Ae. longissima populations and underwent horizontal gene transfer.

a Phenotype by allele analysis of Ae. longissima accessions carrying identical LR85, from one to four amino acid variations (LR85 Δ1-4), interrupted open reading frame or large InDel (lr85), and absence of Lr85. b Phenotype by allele analysis of Ae. sharonensis accessions carrying identical LR85, from one to four amino acid variations (LR85 Δ1-4), interrupted open reading frame or large InDel (lr85), and absence of Lr85 with P. triticina isolate THBJ infection type (IT), phenotypic data from Olivera et al.^,. c ML tree based on alleles’ sequences, color – average latitude of the origin of accessions that have the allele, shape-species. Map shows distribution of the Ae. sharonesis (blue) and Ae. longissima (red) samples used.