Subcellular Localization of Interacting Proteins by Bimolecular Fluorescence Complementation in Planta

Vitaly Citovsky, Lan Ying Lee, Shachi Vyas, Efrat Glick, Min Huei Chen, Alexander Vainstein, Yedidya Gafni, Stanton B. Gelvin*, Tzvi Tzfira

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

305 Scopus citations

Abstract

Bimolecular fluorescence complementation (BiFC) represents one of the most advanced and powerful tools for studying and visualizing protein-protein interactions in living cells. In this method, putative interacting protein partners are fused to complementary non-fluorescent fragments of an autofluorescent protein, such as the yellow spectral variant of the green fluorescent protein. Interaction of the test proteins may result in reconstruction of fluorescence if the two portions of yellow spectral variant of the green fluorescent protein are brought together in such a way that they can fold properly. BiFC provides an assay for detection of protein-protein interactions, and for the subcellular localization of the interacting protein partners. To facilitate the application of BiFC to plant research, we designed a series of vectors for easy construction of N-terminal and C-terminal fusions of the target protein to the yellow spectral variant of the green fluorescent protein fragments. These vectors carry constitutive expression cassettes with an expanded multi-cloning site. In addition, these vectors facilitate the assembly of BiFC expression cassettes into Agrobacterium multi-gene expression binary plasmids for co-expression of interacting partners and additional autofluorescent proteins that may serve as internal transformation controls and markers of subcellular compartments. We demonstrate the utility of these vectors for the analysis of specific protein-protein interactions in various cellular compartments, including the nucleus, plasmodesmata, and chloroplasts of different plant species and cell types.

Original languageAmerican English
Pages (from-to)1120-1131
Number of pages12
JournalJournal of Molecular Biology
Volume362
Issue number5
DOIs
StatePublished - 6 Oct 2006

Bibliographical note

Funding Information:
The work in our laboratories is supported by grants from NIH, USDA, and BSF to V.C., from the NSF 2010 Program to S.B.G. and V.C. (MCB-0418709), from BARD to V.C. and Y.G., and from BARD to T.T. We thank Sergei Kraznyanski and Kelly Babb for technical assistance.

Keywords

  • BiFC
  • YFP
  • cellular localization
  • plasmids
  • protein-protein interactions

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