Tomato and pepper NRC3 complement Cf-4/Avr4-triggered hypersensitive cell death in the <i>nrc2/3/4</i> knock-out lines.

A) Cf-4/Avr4-triggered hypersensitive cell death is complemented in the nrc2/3/4 lines with tomato and pepper NRC3 (SlNRC3 and CaNRC3), but not tomato or pepper NRC1 (SlNRC1 and CaNRC1), or NRC2 (SlNRC2 and CaNRC2). Representative N. benthamiana leaves infiltrated with appropriate constructs were photographed 7–10 days after agroinfiltration. The receptor/effector pair tested, Cf-4/Avr4 or Cf-4/Avr2 as a negative control, and the NRC used for complementation are labelled on the leaf image. Hypersensitive cell-death is indicated by magenta circles, whereas combinations not displaying hypersensitive cell-death are marked with cyan circles. B) SlNRC1, SlNRC2, and SlNRC3 and CaNRC1, CaNRC2, and CaNRC3 protein accumulation. Immunoblot analysis of transiently expressed C-terminally EGFP-tagged NRCs together with the p19 silencing inhibitor 5 days after agroinfiltration in wild-type N. benthamiana plants detected using anti-GFP antibody. P19 alone was used as an empty vector control. C) Quantification of hypersensitive cell death. Cell death was scored based on a 0–7 scale 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 (*). Some of the codon-optimized NRC constructs display weak autoactivity. D) Statistical analysis was conducted using besthr R package [72]. The dots represent the ranked data and their corresponding means (dashed lines), with the size of each dot proportional to the number of observations for each specific value (count key below each panel). The panels on the right show the distribution of 100 bootstrap sample rank means, where the blue areas under the curve illustrate the 0.025 and 0.975 percentiles of the distribution. A difference is considered significant if the ranked mean for a given condition falls within or beyond the blue percentile of the mean distribution of the wild-type control. (TIF)