NRC3 complements Cf-4/Avr4-triggered hypersensitive cell death in the nrc2/3/4 knock-out lines.
A) A schematic representation of the cell death complementation assay. NRCs and empty vector (EV) were transformed into A. tumefaciens, and transiently co-expressed in N. benthamiana plants with either Cf-4/Avr4 or Pto/AvrPto. Hypersensitive cell death was scored based on a modified 0–7 scale between 7–10 days post infiltration (S1A Fig). B) SlNRC1, NRC2, NRC3, and NRC4 protein accumulation. Immunoblot analysis of transiently expressed C-terminally 6xHA-tagged NRCs 5 days after agroinfiltration in wild-type N. benthamiana plants. C) Cf-4/Avr4-triggered hypersensitive cell death is complemented in the nrc2/3/4 lines with NRC3 and to a lesser extent NRC2, but not SlNRC1 or NRC4. Representative N. benthamiana leaves infiltrated with appropriate constructs were photographed 7–10 days after agroinfiltration. The receptor/effector pair tested, Cf-4/Avr4 and Prf (Pto/AvrPto), are labelled above the leaf of nrc2/3/4-188.8.131.52. The NRC used for complementation or EV control are labelled on the leaf image. A representative leaf of the independent nrc2/3/4-184.108.40.206 line is shown in S4D 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 S4 Fig.