Bimolecular Fluorescence Complementation in Plants

by Nir Ohad & Shaul Yalovsky

Department of Plant Sciences, Tel Aviv University, Tel Aviv 69978, ISRAEL.

Bimolecular Fluorescent Complementation (BiFC) is a non-invasive fluorescent-based 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 non-fluorescent GFP variants, primarily 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 (CLSM). BiFC has several major advantages. The assay is simple and does not require sophisticated dedicated equipment. There is either no or low background signal because a fluorescing YFP would only form after interaction between proteins fused to split fragments. BiFC enables determination of the subcellular localization of interacting proteins complexes as well as the mutual affect of interacting partners on the subcellular localization of the complex. BiFC is a sensitive assay, enabling detection of weak and transient interactions, primarily due to the stability of the reconstituted YFP complexes (Hu et al., 2002).
The principles and development of BiFC
The BiFC principle. BiFC is based upon tethering split YFP or other GFP variants to form a functional fluorophore. The association of the split YFP/GFP/CFP molecule does not occur spontaneously and requires interaction between proteins or peptides that are fused to each of the fluorophore fragments (Fig. 1). Upon interaction of these fused proteins/peptides, the split fluorophore fragments can interact to form a fluorescent protein that has the same spectral properties as the un-split YFP (or other GFP variants). If the proteins that are fused to the split fluorophore fragments do not interact, reconstitution of the YFP/GFP/CFP usually does not take place and no fluorescence is detected.

Figure 1. The principle of BiFC. Under physiological conditions reconstitution of a fluorescent YFP molecule can only take place following interaction between proteins or peptides that are fused to YN and YC fragments.

Basic design of BiFC vectors. Proteins under study can be expressed as either N-terminal or C-terminal fusions with the split YFP fragments, often referred to as YN and YC, respectively (Hu et al., 2002; Bracha-Drori et al., 2004; Citovsky et al., 2006; Kerppola, 2006a). Using different combinations of YN and YC fusion pairs is advisable since the orientation of the fusion can greatly affect YFP complex formation (Bracha-Drori et al., 2004). It is also recommended to place a flexible spacer between the split YFP fragment and the proteins under investigation, to alleviate structural constraints that might compromise YFP complex formation (Hu et al., 2002; Kerppola, 2006a). A series of BiFC system vectors can be obtained from the Arabidopsis Biological Resource Center (ABRC) at the University of Ohio (http://www.biosci.ohio-state.edu/~plantbio/Facilities/abrc/abrchome.htm).
Equipment required for BiFC experiments. A foremost advantage of the BiFC assay is its simplicity and the ability to carry out experiments with either a regular epifluorescence microscope equipped with the relevant filter sets and a Charged Coupled Device (CCD) camera, or with a confocal laser scanning microscope (CLSM) (Bracha-Drori et al., 2004).
BiFC in plants
Adaptation of BiFC to plants was described in several studies (Bracha-Drori et al., 2004; Walter et al., 2004; Citovsky et al., 2006; Ohad et al., 2007). These studies demonstrated the usefulness of BiFC to detect protein-protein interaction in plant cells. Furthermore, BiFC has been used to demonstrate the mutual effect of interacting proteins on their subcellular localization (Fig. 2). An interaction between a ROP GTPase and an effector protein was detected in the plasma membrane (Fig. 2 left panel). Interaction of a mutant non-prenylated ROP with the same effector was detected in the cytoplasm and nuclei (Fig. 2 right panel). BiFC together with a CFP-tagged protein has been used to demonstrate co-localization of three proteins (Lavy et al., 2007). BiFC has been used to demonstrate the requirement for a third factor as in the case of the interaction between the gibberellin (GA) receptor GID1 and the DELLA transcriptional regulators, which require the presence of GA (Ueguchi-Tanaka et al., 2007).

Figure 2. Detection of subcellular localization of protein complexes by BiFC. Left panel 'A' of a BiFC assay showing that complexes of ROP GTPase and an effector protein are localized in the plasma membrane of N. Benthamiana leaf epidermal cells. The Right panel 'B' showing that the complexes between the non-prenylated AtropmS mutant, in which the prenyl-acceptor cysteine was changed to serine, and the same ROP effector are localized in the cytoplasm and nuclei.

References

1. Aniento, 1. Bracha-Drori, K., Shichrur, K., Katz, A., Oliva, M., Angelovici, R., Yalovsky, S., and Ohad, N. (2004). Detection of protein-protein interactions in plants using bimolecular fluorescence complementation. Plant J 40, 419-427.

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5. Kerppola, T.K. (2006a). Design and implementation of bimolecular fluorescence complementation (BiFC) assays for the visualization of protein interactions in living cells. Nat Protoc 1, 1278-1286.

6. Lavy, M., Bloch, D., Hazak, O., Gutman, I., Poraty, L., Sorek, N., Sternberg, H., and Yalovsky, S. (2007). A Novel ROP/RAC effector links cell polarity, root-meristem maintenance, and vesicle trafficking. Curr Biol 17, 947-952.

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