Vectors for multi-color bimolecular fluorescence complementation to investigate protein-protein interactions in living plant cells

Background: The investigation of protein-protein interactions is important for characterizing protein function. Bimolecular fluorescence complementation (BiFC) has recently gained interest as a relatively easy and inexpensive method to visualize protein-protein interactions in living cells. BiFC uses split YFP tags on proteins to detect interactions: If the tagged proteins interact, they may bring the two split fluorophore components together such that they can fold and reconstitute fluorescence. The sites of interaction can be monitored using epifluorescence or confocal microscopy. However, conventional BiFC can investigate interactions only between two proteins at a time. There are instances when one may wish to offer a particular bait protein to several prey proteins simultaneously. Preferential interaction of the bait protein with one of the prey proteins, or different sites of interaction between the bait protein and multiple prey proteins, may thus be observed. Results: We have constructed a series of gene expression vectors, based upon the pSAT series of vectors, to facilitate the practice of multi-color BiFC. The bait protein is tagged with the C-terminal portion of CFP (cCFP), and prey proteins are tagged with the N-terminal portions of either Venus (nVenus) or Cerulean (nCerulean). Interaction of cCFP-tagged proteins with nVenus-tagged proteins generates yellow fluorescence, whereas interaction of cCFP-tagged proteins with nCerulean-tagged proteins generates blue fluorescence. Additional expression of mCherry indicates transfected cells and sub-cellular structures. Using this system, we have determined in both tobacco BY-2 protoplasts and in onion epidermal cells that Agrobacterium VirE2 protein interacts with the Arabidopsis nuclear transport adapter protein importin alpha-I in the cytoplasm, whereas interaction of VirE2 with a different importin alpha isoform, importin alpha-4, occurs predominantly in the nucleus. Conclusion: Multi-color BiFC is a useful technique to determine interactions simultaneously between a given bait protein and multiple prey proteins in living plant cells. The vectors we have constructed and tested will facilitate the study of protein-protein interactions in many different plant systems.