Construction of a bimolecular fluorescence complementation (BiFC) platform for protein interaction assays in plants

Abstract

Protein–protein interactions are essential for signal transduction in cells. Bimolecular fluorescence complementation (BiFC) is a novel technology that utilises green fluorescent proteins to visualize protein–protein interactions and subcellular protein localisation. BiFC based on pSATN vectors are a good system for high-level expression of fused protein. A series of pCAMBIA vectors were most widely used in plant transgene and transient expression. To provide multiple options in the study of protein interactions that utilise BiFC, we reconstructed a new pair of BiFC vectors, pCAMBIA1301-nEYFP and pCAMBIA1301-cEYFP. These vectors were generated by eliminating restriction enzyme cutting sites (BanII, SacI, KpnI, SmaI, BamHI, SalI, PstI and SbfI) at the multiple cloning sites (MCSs) of pCAMBIA1301 (p1301), and introducing cEYFP/nEYFP cassettes containing MCSs generated from pSATN medium. Fluorescence can be imaged when AtCBL1 and AtCIPK23 are co-injected, but imaging cannot be done when co-injecting AtCBL1 and AtCIPK23-NAF-deleted (AtCIPK23m), suggesting that the proposed modified vector system is effective for the study of protein interactions.

Keywords: Protein–protein, bimolecular fluorescence complementation (BiFC), vector reconstruction