MoEnd3 interacts with MoArk1 and F-actin.

(A) Yeast two-hybrid assay for examining the interaction between MoEnd3 and MoArk1. The yeast transformants were isolated from SD-Leu-Trp plates. Their β-galactosidase activity was assayed on SD-Leu-Trp-His-Ade plates containing X-Gal. The transformants expressing AD-MoEnd3 and empty BD, empty AD and BD-MoArk1, and empty AD and BD were used as negative control. (B) In vitro protein binding assay for the MoEnd3-MoArk1 interaction. Ni-NTA beads were used to bind His protein (6 kDa) as a negative control and His-tagged MoArk1 protein (115 kDa), respectively, and incubated with the GST-tagged MoEnd3 protein (69 kDa). The total proteins eluted from beads (output) were separated by 12% SDS-PAGE and immunoblotted with GST and His antibodies. (C) BiFC assay for the MoEnd3-MoArk1 interaction in vivo. Conidia and 24 h appressoria were examined by DIC and fluorescence microscopy. The strains expressing the MoArk1-YFP^C and empty YFP^N, MoEnd3-YFP^N and empty YFP^C, empty YFP^N and empty YFP^C constructs were used as negative controls, Bars = 10 μm. (D) MoEnd3:GFP is co-localized with F-actin. The localization pattern of MoEnd3:GFP was displayed in 6 h and 24 h-appressoria, conidium and hyphal tip region. F-actin was labeled with Lifeact:RFP. Bars = 10 μm. (E) Yeast two-hybrid assay was used to examine the interaction between MoEnd3 and actin. Transformants were isolated from SD-Leu-Trp plates and their β-galactosidase activity was assayed on SD-Leu-Trp-His-Ade plates containing X-Gal. Transformants expressing AD and BD, BD-MoAct1 and AD, and BD and AD-MoEnd3 were used as negative controls. (F) Protein binding assay for MoEnd3-MoAct1 interaction in vitro. GST-beads were used to bind GST protein (24 kDa) or GST-tagged MoEnd3 protein (68 kDa), respectively, and incubated with His-tagged MoAct1 protein (42 kDa). Total eluted fractions from the beads (output) were immunoblotted with the His and GST antibodies. MoEnd3 is important for sexual reproduction and normal endocytosis.