Fig. 3: SsPEIE1 interacts with Arabidopsis hypersensitive induced reaction 4 (AtHIR4) in the plasma membrane.

a SsPEIE1-GFP was localized to the membrane and cytoplasm of N. benthamiana. The fluorescence of GFP was monitored at 3 days post-agroinfiltration using confocal laser scanning microscopy. N. benthamiana cells were treated with 0.5 M NaCl for 5 min to observe plasmolysis. Bars, 20 μm. b Yeast two-hybrid (Y2H) assays showing that SsPEIE1 interacted with AtHIR4. LW, SD–Leu/–Trp. LWHU+AbA+X-gal, SD–Ade/–His/–Leu/–Trp containing 125 ng · mL⁻¹ Aureobasidin A (AbA) and 40 μg · mL⁻¹ X-α-Gal. c SsPEIE1 and AtHIR4 showed strong interaction in the split-LUC and reverse direction split-LUC assay. SsPEIE1-nLUC and AtHIR4-cLUC or SsPEIE1-cLUC and AtHIR4-nLUC were co-expressed in N. benthamiana leaves and luciferase activity was assayed 2 days later. d Co-immunoprecipitation (Co-IP) and reverse direction Co-IP assays showed that SsPEIE1 was physically associated with AtHIR4. AtHIR4-FLAG and SsPEIE1-GFP or AtHIR4-GFP and SsPEIE1-FLAG fusion proteins were co-expressed in N. benthamiana leaves. Immunoprecipitation with anti-GFP agarose (GFP IP) was performed, and AtHIR4 was detected in the post-immunoprecipitation product with an anti-Flag antibody. The experiments were performed three times independently, and similar results were obtained. The red dots indicate the expected size of the associated proteins, respectively. e Subcellular localization of SsPEIE1 and AtHIR4 in N. benthamiana epidermal cells showed that both SsPEIE1-mCherry and AtHIR4-GFP were localized at the cell membrane. Source data are provided as a Source Data file.