Fig. 4

AvrPm8 haplovariant mining in a global B.g. tritici population reveals 17 gain-of-virulence mutations. a Schematic gene model (top) and phenotypes (bottom) of isolates with the identified gain-of-virulence mutations in the AvrPm8 effector gene. The two exons of AvrPm8 are depicted as yellow boxes with gene orientation indicated by an arrow. Start codon, splice sites, and stop codon are indicated. Mutations leading to an amino acid change (blue) or a premature stop codon (red) are indicated by a colored line. For each mutation, the virulence phenotype of a representative isolate on the Pm8 transgenic line Pm8#34 is depicted. Susceptible cultivar “Bobwhite” serves as a control. The phenotype of the avirulent isolate ISR_7 is shown as comparison (middle). Additional phenotyping data for each isolate including phenotypes on the near-isogenic line “Kavkaz/4*Federation” and an independent Pm8 transgenic line Pm8#12 are depicted in Additional file 1: Fig. S5. For two mutations (K28STOP and spliceAT-AG), we were unable to find a representative isolate in our living B.g. tritici collection. b Agrobacterium mediated co-expression of genes encoding AvrPm8-FLAG, AvrPm8_F43Y-FLAG, AvrPm8_F43L-FLAG, AvrPm8-R96STOP-FLAG, or AvrPm8-L77STOP-FLAG with Pm8-HA in N. benthamiana. Leaves were imaged at 5 days post inoculation using a Fusion FX imager system (top panel). Co-infiltrations were performed with a ratio of 4 (effector): 1 (Pm8-HA). Middle panel: quantification of HR intensity in co-expression assay depicted in top panel. Individual datapoints are color coded based on four independent experiments with n = 4 leaves per experiment (total n = 16). Detection of N. benthamiana expressed AvrPm8 constructs by anti-FLAG western blotting (bottom panel) and total protein Ponceau S staining as a loading control. Black arrows indicate positions of protein size markers. c Worldwide distribution of identified AvrPm8 haplovariants. Haplovariants are depicted in different colors according to the legend above the world map. Size of pie charts are scaled as log10(n) where n is the number of isolates per region