Fig 1.
GWAS identifies a candidate gene controlling avirulence on the cultivar ‘Soissons’.
(a) Manhattan plot representing the association of multiple SNPs with the percentage of leaf area covered by necrosis (PLACN) on cultivar ‘Soissons’. The X and Y axes indicate positions on chromosome 1 and −log10 (p-value) for associations, while the red line represents the Bonferroni threshold (α = 0.05). (b) Top: Gene annotations (black arrows) and transposable elements (grey arrow) on the reference genome IPO323. The candidate gene is highlighted in red. Bottom: Pairwise linkage disequilibrium plot around the lead significant SNP. The association peak is associated with several polymorphisms in the gene Zt_1_693. (c) The difference in PLACN of isolates carrying the virulent and avirulent alleles (based on the lead SNP). Significance difference from the mean was computed using a two-sided Mann-Whitney test (****, p<0.0001). (d) Gene expression profile of Zt_1_693 measured as fragments per kilobase of exon per million mapped fragments (FPKM) throughout the time course of Z. tritici infection on a susceptible wheat cultivar [23].
Fig 2.
Zt_1_693 encodes for the avirulence gene AvrStb9 and triggers Stb9 resistance.
(a) Percentage of leaf area covered by pycnidia (PLACP) and percentage of leaf area covered by necrosis (PLACN) at 26 dpi measured after inoculation with the avirulent isolate IPO09593, the virulent isolate IPO323 and IPO323 transformants carrying the avirulent allele (IPO323Avrstb9) or the virulent allele (IPO323avrstb9) on cultivars ‘Courtot’ (Stb9), a ‘Courtot’ mutant lacking Stb9 and ‘Soissons’ (details on the phenotype of each mutant is provided in S5 Fig; S1 Table). Letter above the boxplots represent statistical groups based on a post-hoc Tukey’s HSD test (α = 0.05) performed independently for each wheat background. (b) Genetic mapping of Stb9 resistance gene using linkage mapping. Top panel: Linkage map and marker position in cM. Bottom panel: Logarithm of odds (LOD) score plot of the QTL analysis (Relative AUDPC for sporulation and necrosis in black and grey, respectively) possibly containing the Stb9 locus mapped to the long arm of the chromosome 2B. The horizontal dashed line indicates a suggestive linkage threshold (LOD = 3). The position of the marker in the QTL peak is highlighted in red. (c) Manhattan plots representing association mapping results of relative AUDPC sporulation of the avirulent (top) and virulent (bottom) isolates in the bread wheat panel. Multiple SNPs in the telomeric region of chromosome 2B associated with the avirulent isolate are located in the Stb9 genomic region.
Table 1.
Polymorphisms in AvrStb9 protein sequences between the wild type and transgenic mutant strains.
Difference in amino acid residues between the virulent (avrstb9) and avirulent (Avrstb9) phenotypes are highlighted in orange while residues potentially involved in virulence are highlighted in red.
Fig 3.
AvrStb9 diversity and phylogeny in the global Z. tritici population.
(a) The top panel shows the AvrStb9 gene structure. Middle and low panels are sliding window analyses for nucleotide diversity (π) and Tajima’s D across the AvrStb9 locus, respectively, with a window size of 200 bp and a step size of 20 bp. The plots show a reduced nucleotide diversity and a drop in Tajima’s D values, likely corresponding to a selective sweep spanning the region. (b) Schematic diagram of AvrStb9 protein sequence with an annotation of amino acid substitutions between virulent and avirulent alleles. Substitutions potentially involved in virulence are highlighted in red. SP = Signal peptide. (c) A phylogenetic tree of AvrStb9 protein sequence generated from a global Z. tritici population and the reference isolate IPO323.
Fig 4.
Phylogenetic tree of selected AvrStb9 orthologs/paralogs belonging to five distinct classes of Ascomycota lineages.
Leaf labels are color-coded according to class and represent abbreviated names of the fungal species (S5 Table). AvrStb9 is indicated with a black arrow.
Fig 5.
Three-dimensional structure of the protein AvrStb9 predicted with the ColabFold v1.5.2: AlphaFold2.
(a) An overlay of the 3D structures of the virulent avrStb9 protein (isolate IPO323 in red) and of the avirulent AvrStb9 protein (isolate IPO09593 in blue). (b) The 3D structure of the virulent avrStb9 protein with the localization of the five residues potentially involved in virulence (highlighted in yellow). The (c) virulent avrStb9 and (d) avirulent AvrStb9 proteins with Top panel: zoom-in on the five residues in the 3D structure, and Bottom panel: distances (in Å) between the five residues.