The NRC2 α1 helix can functionally replace the NRC3 α1 helix for Cf-4/Avr4-triggered hypersensitive cell death.
A) Residues 1–17 of NRC2 and 1–21 of NRC3 were replaced with residues 1–21 of NRC3 and 1–17 of NRC2 to generate the NRC2NRC3α1 and NRC3NRC2α1 chimaeras. NRC3 with the NRC2 α1 helix (NRC3NRC2α1) can complement Cf-4/Avr4 and Pto/AvrPto-triggered hypersensitive cell death in the N. benthamiana nrc2/3/4 CRISPR lines, while NRC2 with the NRC3 α1 helix (NRC2NRC3α1) cannot. Representative N. benthamiana leaves infiltrated with appropriate constructs were photographed 7–10 days after infiltration. The receptor tested, Cf-4 and Prf (Pto), is labelled above the leaf of NRC CRISPR line nrc2/3/4-184.108.40.206. The NRC and NRC chimaeras tested as well as the effectors are labelled on the leaf image. To ensure the NRC α1 helix chimaeras were not autoactive when expressed with either Cf-4 or Pto and an Avr2 or EV control was taken along, respectively. B) 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. Significant differences between the conditions are indicated with an asterisk (*). C) Statistical analysis was conducted using besthr R package . 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.