A bright green-colored bimolecular fluorescence complementation assay in living plant cells

Protein-protein interactions are important for various biological and cellular processes. To analyze protein-protein interactions in living cells, the bimolecular fluorescence complementation (BiFC) assay, based on structural complementation of two non-fluorescent N- and C-terminal fragments from a fluorescent protein, has been developed and widely used in various research fields. Here I report a bright green-colored BiFC assay in living plant cells by using the N-terminal fragment (GN) of green fluorescent protein-S65T (GFP-S65T) and the C-terminal fragment (CC) of cyan fluorescent protein (CFP), but not GN and the C-terminal fragment (GC) of GFP. Fluorescence intensity of the GN/CC-based BiFC was 7-fold higher than that of the GN/GC-based BiFC. The emission spectrum of the GN/CC-based BiFC in planta was identical to that of full-length GFP-S65T. Ala163 residue within the CC fragment was found to be responsible for the improvement of the BiFC efficiency. These findings provide a BiFC method for in vivo protein-protein interaction studies to many GFP users in various research fields.