Abstract
Non-invasive fluorophore-based protein interaction assays like fluorescence resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC, also referred to as "split YFP") have been proven invaluable tools to study protein-protein interactions in living cells. Both methods are now frequently used in the plant sciences and are likely to develop into standard techniques for the identification, verification and in-depth analysis of polypeptide interactions. In this review, we address the individual strengths and weaknesses of both approaches and provide an outlook about new directions and possible future developments for both techniques.
Highlights
Having the first completed plant genomes of the monocotyledonous and dicotyledonous reference species rice (Oryza sativa) and thale cress (Arabidopsis thaliana) in hand [1,2,3], the analysis of protein function(s) represents a major scientific challenge of the post-genomic era
Co-immunoprecipiatation ("pull-down"; [5]), in vitro association studies, blue native gel electrophoresis [8], bioluminescence resonance energy transfer (BRET) [9] and fluorescent protein-based methods [1012] are nowadays commonly used to achieve this goal. We focus on the latter, non-invasive, microscopy-based approaches with a particular emphasis on fluorescence resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC) both of which allow monitoring protein-protein interactions in vivo and in real time
Systematic analysis of twelve combinations of different green fluorescent proteins (GFPs), yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP) sub-fragments resulted in the identification of twelve bimolecular fluorescent complexes with seven distinct spectra [85] that provide an ideal basis for multicolor BiFC
Summary
The visible touch: in planta visualization of protein-protein interactions by fluorophore-based methods. Address: 1Department of Plant-Microbe Interactions, Max-Planck-Institute for Plant Breeding Research, Carl-von-Linné-Weg 10, D-50829 Köln, Germany and 2Institute of Genetics, Martin-Luther University, Weinbergweg 10, D-06099 Halle, Germany
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