The ZAR1 α1 helix can functionally replace the NRC2 and NRC3 α1 helix for Cf-4/Avr4-triggered hypersensitive cell death.
A) Structure of the ZAR1 resistosome with the N-terminal α1 helix highlighted. B) Schematic representation of ZAR1, NRC2, NRC3, and the respective NRC2ZAR1α1 and NRC3 ZAR1α1 chimaeras in which residues 1–17 and 1–21 are replaced by residues 1–17 from ZAR1, respectively. C) NRC ZAR1 α1 helix chimaeras accumulate to similar levels as wild-type NRC proteins. Immunoblot analysis of transient NRC-6xHA accumulation 5 days after agroinfiltration in wild-type N. benthamiana plants. D) NRC ZAR1 α1 helix chimaeras can complement Cf-4/Avr4 and Pto/AvrPto-triggered hypersensitive cell death in the N. benthamiana nrc2/3/4 CRISPR lines. Representative N. benthamiana leaves infiltrated with appropriate constructs were photographed 7–10 days after infiltration. The receptor/effector pair tested, Cf-4/Avr4 and Prf (Pto/AvrPto), are labelled above the leaf of NRC CRISPR line nrc2/3/4-18.104.22.168. The NRC tested, NRC2 and NRC3, are labelled on the leaf image. To ensure the NRC ZAR1 α1 helix chimaeras were not autoactive when expressed with either Cf-4 or Pto an EV control was taken along. A representative leaf of the independent nrc2/3/4-22.214.171.124 CRISPR line is shown in S8E Fig) 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 (*). Details of statistical analysis are presented in S8 Fig.