Figure 5

In vivo protein interaction between GmTIP2;1, GmTIP1;7 and GmTIP1;8. (a) Yeast two-hybrid assay analysis identified that GmTIP2;1 can form homodimers and heterodimers in yeast cells. The full-length ORF sequences of GmTIP2;1, GmTIP1;7 and GmTIP1;8 were cloned and ligated into pGADT7 and pGBKT7 vectors. After co-transformation of the baits and preys, equal amounts of yeast clones were plated on SD-Leu-Trp and SD-Leu-Trp-His-Ade + X--gal selective plates, and the plates were incubated at 30 °C until formation of the colonies. Yeast cells carrying pGBKT7–53 and pGADT7-SV40 plasmids were used as positive controls, and those with pGBKT7-Lam and pGADT7-SV40 were used as negative controls. (b) Bimolecular fluorescence complementation (BiFC) analysis for conducting the interaction studies in tobacco leaves. The assay was performed to confirm the results of the yeast two-hybrid assays. GmTIP2;1, was fused to the N-terminal (YN) and GmTIP2;1, GmTIP1;7 and GmTIP1;8 were fused to the C-terminal (YC) halves of YFP and were imaged using a confocal microscope after incubation at room temperature for over 18 h. The images are shown as YFP, merged YFP and bright field. The last panel shows the negative control.