Gateway Vectors for Simultaneous Detection of Multiple Protein−Protein Interactions in Plant Cells Using Bimolecular Fluorescence Complementation

Bimolecular fluorescence complementation (BiFC) is widely used to detect protein-protein interactions, because it is technically simple, convenient, and can be adapted for use with conventional fluorescence microscopy. We previously constructed enhanced yellow fluorescent protein (EYFP)-based Gateway cloning technology-compatible vectors. In the current study, we generated new Gateway cloning technology-compatible vectors to detect BiFC-based multiple protein-protein interactions using N- and C-terminal fragments of enhanced cyan fluorescent protein (ECFP), enhanced green fluorescent protein (EGFP), and monomeric red fluorescent protein (mRFP1). Using a combination of N- and C-terminal fragments from ECFP, EGFP and EYFP, we observed a shift in the emission wavelength, enabling the simultaneous detection of multiple protein-protein interactions. Moreover, we developed these vectors as binary vectors for use in Agrobacterium infiltration and for the generate transgenic plants. We verified that the binary vectors functioned well in tobacco cells. The results demonstrate that the BiFC vectors facilitate the design of various constructions and are convenient for the detection of multiple protein-protein interactions simultaneously in plant cells.