Fig. 5: SsPEIE1 competitively binds to AtHIR4 and disrupts its oligomerization capacity and oligomerization-mediated disease resistance.
a SsPEIE1 inhibits homo-oligomerization of AtHIR4. AtHIR4-Flag and SsPEIE1-GFP fusion proteins were co-expressed in N. benthamiana leaves. b Yeast tree-hybrid (Y3H) assay to confirm that SsPEIE1 competitively binds to AtHIR4. c In vitro traction assays demonstrated that SsPEIE1 competitively bind to AtHIR4 and that the AtHIR4 self-interaction weakened with increasing SsPEIE1 protein. AtHIR4-His or GST-AtHIR4, MBP and MBP-SsPEIE1 fusion proteins were dissolved in PBS (PH = 7.4), MBP and MBP-SsPEIE1 with different concentration gradients were incubated with AtHIR4-His and GST-AtHIR4 mixed proteins and immunoprecipitated with Glutathione Resin. d Co-immunoprecipitation (Co-IP) assays show that SsPEIE1 physically binds to AtHIRs in vivo to block the self-interaction of AtHIRs. AtHIRs-Flag, AtHIRs-Myc, and SsPEIE1-GFP fusion proteins were co-expressed in N. benthamiana leaves. e Co-immunoprecipitation (Co-IP) assay showed that the self-interaction of AtHIR4v157a was significantly weakened. f Co-immunoprecipitation (Co-IP) experiments showed that SsPEIE1 interacts with AtHIR4v157a. g Symptoms on N. benthamiana leaves transiently expressing different proteins 36 h after inoculation with wildtype S. sclerotiorum. h Western blot analysis for expression of AtHIR4 and AtHIR4v157a proteins in infiltrated N. benthamiana leaves. i Lesion area by the cross over method in (g), n = 6 biologically independent samples. j Relative biomass in (g) analyzed by qPCR with equal area samples from infected sites used for DNA extraction (n = 3 biologically repetitions). Data represent means ± SD. Different letters on the same graph indicate statistical significance at p < 0.05 in one-way ANOVA. The red dots indicate the expected size of the associated proteins, respectively. Source data are provided as a Source Data file.
