RhETR3 interacts with RhTSPO in vivo and in planta. (A) Co-localization of RhTSPO-GFP and RhETR3-mCherry in the leaves of N. benthamiana plants infiltrated with the indicated constructs. ER-mCherry was used as an endoplasmic reticulum (ER) localization marker. Scale bar, 50 μm. (B) Split-ubiquitin membrane-based yeast two-hybrid (MYTH) assay for the interaction of RhETR3 and RhTSPO. Top, diagram of RhETR3 and two truncated variants used in the assay. Bottom, MYTH assay for RhETR3 and RhTSPO. pPR3N with pBT3-STE-RhETR3 were used as negative control. (C) Bimolecular fluorescence complementation (BiFC) assay of the RhETR3–RhTSPO interaction in N. benthamiana leaves. The RhETR3-cYFP and nYFP-RhTSPO vectors were infiltrated into N. benthamiana leaves via Agrobacterium-mediated infiltration. RhETR3-nYFP and cYFP were used as negative control. Scale bars, 50 μm. (D) Co-immunoprecipitation (Co-IP) analysis of RhETR3 with RhTSPO in vivo. Total protein extracts (input) from N. benthamiana leaves infiltrated with the construcs RhETR3-Flag and RhTSPO-GFP were incubated with anti-GFP magnetic beads. Immunoblot analysis was performed using anti-GFP and anti-Flag antibodies. The detected proteins are indicated by arrowheads.