Abstract
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.
Highlights
Bimolecular fluorescence complementation (BiFC) is used to detect protein—protein interactions through visualization in living cells
We applied our pUGW-based Gateway cloning technology-compatible vector construction system, which uses vectors derived from pUC119 [14, 21, 22], to generate destination vectors for BiFC containing each split fluorescent fragment
The N- and C-terminal fragments of enhanced cyan fluorescent protein (ECFP) and enhanced green fluorescent protein (EGFP) were amplified using the same primers, which were used for amplification of N- and C-terminal fragments of enhanced yellow fluorescent protein (EYFP), since the nucleotide sequences at the positions used to split a fluorescent protein into two fragments in ECFP, EGFP and EYFP are identical
Summary
BiFC is used to detect protein—protein interactions through visualization in living cells. This technique uses two non-fluorescent fragments derived from a split fluorescent protein, such as GFP or its derivatives. The non-fluorescent fragments are fused to genes encoding proteins or peptides of interest, and the fusion genes are expressed simultaneously in the same cell. Reconstitution of fluorescence takes place only when the two proteins or peptides of interest interact (see reviews; [1,2,3,4,5]). The protein—protein interaction is revealed by the appearance of a reconstituted fluorescence signal. The BiFC technique has become one of the most popular techniques in plant biology [6,7,8,9,10,11,12,13,14,15], due to its simplicity, ease of use, and its compatibility with conventional fluorescence microscopy
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