Cf-4/Avr4-triggered hypersensitive cell death requires NRC2/3.

A) Cf-4/Avr4-triggered hypersensitive cell death is lost in the nrc2/3 and nrc2/3/4 knock-out lines, but not in the nrc4 line. Representative N. benthamiana leaves infiltrated with appropriate constructs photographed 7–10 days after infiltration. The NRC CRISPR lines, nrc2/3-209.1.3.1, nrc2/3/4-210.4.3.1, and nrc4-185.1.2.1, are labelled above the leaf and the receptor/effector pair tested, Cf-4/Avr4, Prf (Pto/AvrPto), Rpi-blb2/AVRblb2 or R3a/Avr3a, are labelled on the leaf image. Cf-4/EV and EV/Avr4 were also included as negative controls. A representative leaf of the independent nrc2/3-209.3.3.1, and nrc2/3/4-210.5.5.1 CRISPR line are shown in S1B Fig) Cf-4 protein accumulation is not affected in the nrc2/3/4 lines. For the immunoblot analysis, total protein was extracted 5 days after transient expression of Cf-4-EGFP by agroinfiltration in wild-type, nrc2/3/4-210.4.3.1 and nrc2/3/4-210.5.5.1 N. benthamiana leaves. Cf-4-EGFP accumulation was detected using anti-GFP antibody. C) Quantification of hypersensitive cell death. Cell death was scored based on a 0–7 scale (S1 Fig) between 7–10 days post infiltration. The results are presented as a dot plot, where the size of each dot is proportional to the count of the number of samples with the same score within each biological replicate. The experiment was independently repeated three times. The columns correspond to the different biological replicates. Significant differences between the conditions are indicated with an asterisk (*). Details of statistical analysis are presented in S1 Fig.