Fig. 6: Soybean NPR1 (GmNPR1) is sumoylated by GmSUMO5.
From: A plant RNA virus inhibits NPR1 sumoylation and subverts NPR1-mediated plant immunity
a Multiple alignment of partial amino acid sequences of NPR1 homologs of different plants. GenBank accession numbers are indicated at the front, and SIM3 is highlighted by a black box with a conserved aa pattern on the bottom. b Evaluation of the interaction between GmNPR1 and GmSUMO homologs by Y2H assay. c Evaluation of the interaction between SMV NIb (SMVNIb) and GmNPR1 and Gmsim3 by split-luciferase assay. d Analysis of the sumoylation of GmNPR1 by Co-IP assay. Proteins were expressed in N. benthamiana leaves by agroinfiltration, analyzed by western blotting (left panel) or immunoprecipitated with anti-FLAG M2 affinity gel and then detected with anti-Myc and anti-GFP-N antibodies (right panel) at 2 dpi. Sumoylated GmNPR1 is indicated. To avoid overexposure, samples that were detected with anti-Myc antibodies were diluted 20 times for SDS‒PAGE. The experiment was independently repeated twice with similar results. e Western blot analysis of the influence of SMVNIb on GmNPR1 sumoylation. Proteins were expressed in N. benthamiana leaves and analyzed at 2 dpi by Western blotting with anti-Myc, anti-GFP-N, and anti-mRFP polyclonal antibodies (left panel). GmSUMO5 was immunoprecipitated by anti-FLAG M2 affinity gel and then analyzed by anti-Myc or anti-GFP antibodies. Nonmodified and posttranslationally modified GmNPR1 are indicated by an arrow and square bracket, respectively. Similar results were observed in three independent experiments.
