NRC3 variants with S202P, T203K, N221K, C824H, N832K, or V881I mutations quantitatively affected the ability of NRC3 to function with Rpi-blb2.

Cell death assays of chimeric NRC3 variants designed for pinpointing the polymorphisms in (A) NB-ARC domain region 3 which contains six amino acid differences, (B) LRR domain region 5b containing five amino acid differences, and (C) LRR domain region 5e contain a small insertion/indel (labeled as X) three amino acid (TIH) differences between NbNRC3c and SlNRC3a. The analysis was done in the NN3NT5be background by replacing each residue with the amino acid of NbNRC3c. The variants were co-expressed with Rpi-blb2/AVRblb2 or Pto/AvrPto in nrc2/3/4_KO N. benthamiana. Mutations at positions 202, 203, 221 (NB-ARC domain), 824, 832 (LRR domain region 5b), and 881 (LRR domain region 5e) affected the ability of NN3NT5be to function with Rpi-blb2. 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 between the NN3NT5be and tested groups were examined by paired Wilcoxon signed rank test (* = p < 0.01, ** = p < 0.001, *** = p < 0.0001). (D-F) Protein accumulation of chimeric NRC3 variants tested in (A-C). 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)