The analysis of protein-protein interactions in plants by bimolecular fluorescence complementation.

Following the complete genome sequencing of different plant species such as Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Physcomitrella (Physcomitrella patens), as well as advances toward deciphering entire proteomes, the need for a reliable way to identify protein-protein interactions is becoming a major task for the future. Bimolecular fluorescent complementation (BiFC) is a noninvasive fluorescentbased technique that allows detection of protein-protein interactions in living cells, and furthermore can be used to determine subcellular localization of the interacting proteins, and if it changes over time, without requiring addition of external agents. BiFC is based upon reconstitution of split nonfluorescent GFP variants, primarily yellow fluorescent protein (YFP), to form a fluorescent fluorophore (Ghosh et al., 2000; Hu et al., 2002). The technique has become increasingly popular due to its simplicity, ease of use, and the capability to carry out experiments with regular epifluorescence or confocal laser scanning microscopes (CLSMs). In this Update, we first discuss the principles of BiFC and its major advantages and disadvantages. We then describe the adaptation of BiFC to plant systems, provide practical suggestions for its use, and review protein-protein interactions that have been identified and confirmed in plants using this technique. Finally, additional potential exploitations of BiFC are discussed. Due to lack of space we did not discuss other fluorescent-based techniques for detection of proteinprotein interactions, such as fluorescent resonance energy transfer, and refer the readers to a recent review on fluorescent resonance energy transfer and BiFC (Bhat et al., 2006). Discussion of additional protein fragment complementation assays techniques can be found in a recent review (Remy and Michnick, 2007). We also apologize to those colleagues whose work we have not cited due to lack of space. THE PRINCIPLES AND DEVELOPMENT OF BiFC