Swapping two K/N residues in N88/N89 do not affect their compatibility with Prf.

(A) Left panel, the polymorphisms in ancestral NRC3 variants at the 6 positions identified. Right panels, cell death assays testing two lysine-asparagine changes in both N88 and N89 backgrounds. All the variants functioned with Prf and none of them were auto-active. The variants were co-expressed with Pto/AvrPto in nrc2/3/4_KO N. benthamiana. Auto-activity analysis was done by expressing NRC3 variants alone in WT N. benthamiana. The dot plots represent cell death intensity quantified by UVP ChemStudio PLUS at 6 dpi. The line in the boxplots represents the medium, the box edges represent the 25th and 75th percentiles, and the whiskers extend to the most extreme data points no more than 1.5x of the interquartile range. Statistical differences were examined using Dunn’s test (p < 0.05). (B) Protein accumulation of NRC3 variants tested in (A). The proteins were extracted from leaf samples at 2 dpi and the NRC3 protein accumulations were detected by α-myc antibody. SimplyBlue SafeStain-staining of Rubisco was used as the loading control.
(JPF)