(A) Yeast-two-hybrid assay of Pikp-HMA and Pikp-HMANK-KE with AVR-Pik alleles. Control plates for yeast growth are on the left, with quadruple dropout media supplemented with X-α-gal and increasing concentrations of Aureobasidin A on the right for each combination of HMA/AVR-Pik. The unrelated M. oryzae effector AVR-Pii was used as a negative control. Growth and the development of blue coloration in the selection plate are both indicative of protein–protein interaction. HMA domains were fused to the GAL4 DNA binding domain, and AVR-Pik alleles to the GAL4 activator domain. Each experiment was repeated a minimum of three times, with similar results. (B) Box plots showing %Rmax, as measured by surface plasmon resonance, for Pikp-HMA and Pikp-HMANK-KE with the AVR-Pik effectors alleles at an HMA concentration of 40 nM. Pikp-HMA and Pikp-HMANK-KE are represented by blue and purple boxes, respectively. The centre line within each box represents the median, the box limits are the upper and lower quartiles, the whiskers extend to the largest value within Q1 – 1.5 × the interquartile range (IQR) and the smallest value within Q3 + 1.5 × IQR. All of the data points are represented as dots with distinct colors for each biological replicate. For each experiment, three biological replicates with three internal repeats were performed. ‘p’ is the p-value obtained from statistical analysis and Tukey’s HSD. For results of experiments with 4 nM and 100 nM HMA protein concentration, see Figure 2—figure supplement 2. (C) Co-immunoprecipitation of full length Pikp-1 and Pikp-1NK-KE with AVR-Pik variants. N-terminally 4xMyc tagged AVR-Pik effectors were transiently co-expressed with Pikp-1:6xHis3xFLAG (left) or with Pikp-1NK-KE:6xHis3xFLAG (right) in N. benthamiana. Immunoprecipitates (IPs) obtained with anti-FLAG antiserum, and total protein extracts, were probed with appropriate antisera. The dashed line indicates a crop site on the same blot used to compose the figure. Each experiment was repeated at least three times, with similar results. The asterisks mark the Pik-1 band. PS = Ponceau Stain.