Utilizing Bimolecular Fluorescence Complementation (BiFC) to Assay ProteinProtein Interaction in Plants

Protein function is often mediated by the formation of stable or transient complexes. Here we present a method for testing protein–protein interactions in plants designated bimolecular fluorescence complementation (BiFC). The advantages of BiFC are its simplicity, reliability, and the ability to observe protein–protein interactions in different cellular compartments including membranes. BiFC is based on splitting the yellow fluorescent protein (YFP) into two nonoverlapping N -terminal (YN) and C-terminal (YC) fragments. Each fragment is cloned in-frame with a gene of interest, enabling expression of a fusion protein. Reconstitution of the fluorescing YFP chromophore takes place upon interaction of protein pairs that are coexpressed in the same cells.