MdHXK1 interacts with MdNHX1 in vivo and in vitro. A, Split-ubiquitin membrane-based yeast two-hybrid assays for interaction between MdHXK1 and MdNHX1. The hexokinase_2 domain of MdHXK1 and Na_H_Exchanger domain of MdNHX1 are crucial for their interaction. B, Interaction between MdHXK1 and MdNHX1 proteins in vivo. Total protein extracted from 35S::MdHXK1-GUS + 35S::MdNHX1-Myc or 35S::MdHXK1-GUS + 35S::Myc double transgenic apple calli were immunoprecipitated with anti-Myc. The proteins from crude lysates (input) were detected with anti-GUS antibody and anti-Myc antibody. The immunoprecipitated proteins (output) were detected with anti-GUS antibody. C, Interaction between MdHXK1 and MdNHX1 proteins in vitro. MdNHX1-His proteins were incubated with immobilized MdHXK1-GST or GST, and the proteins immunoprecipitated with His-beads were detected using anti-GST antibody. D, BiFC analysis of the interaction between MdHXK1 and MdNHX1 in N. benthamiana. Arabidopsis CBL3 was fused to RFP and used as a vacuole-located marker. Scale bars represent 50 μm.