Fig. 7. The RGA5m1m2 mutant recognizes AVR-Pia and AVR-PikD in N. benthamiana.

a The indicated combinations of constructs were transiently expressed in N. benthamiana leaves. The AVR-PikD (without signal peptide) and RGA4 constructs carry an HA tag at their C-termini, while RGA5 and RGA5m1m2 are YFP-tagged at their N-termini and AVR-Pia (without signal peptide) is untagged. The auto-active RGA4 construct was used as a positive control for cell death induction. Cell death was visualized 5 days after infiltration. Greyscale pictures were taken using a fluorescence scanner with settings allowing visualization of the disappearance of red fluorescence due to cell death (white patches of dead cells). A picture of corresponding leaves is shown below. b Cell death was quantified by measuring fluorescence levels (arbitrary unit, AU) in the infiltrated areas using ImageJ³⁴. The resulting data were plotted. The boxes represent the first quartile, median, and third quartile. The difference in fluorescence levels was assessed by an ANOVA followed by a Tukey HSD test. Groups with the same letter (a–c) are not significantly different at level 0.05. For each combination of constructs, all of the measurements are represented as dots with a distinct color (red, green, and blue) for each of the three biological replicates.