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Multicolor bimolecular fluorescence complementation reveals simultaneous formation of alternative CBL/CIPK complexes in planta
Author(s) -
Waadt Rainer,
Schmidt Lena K.,
Lohse Marc,
Hashimoto Kenji,
Bock Ralph,
Kudla Jörg
Publication year - 2008
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1111/j.1365-313x.2008.03612.x
Subject(s) - bimolecular fluorescence complementation , protein–protein interaction , subcellular localization , microbiology and biotechnology , biology , green fluorescent protein , fusion protein , complementation , computational biology , signal transduction , gene , biochemistry , cytoplasm , phenotype , recombinant dna
Summary The specificity of intracellular signaling and developmental patterning in biological systems relies on selective interactions between different proteins in specific cellular compartments. The identification of such protein–protein interactions is essential for unraveling complex signaling and regulatory networks. Recently, bimolecular fluorescence complementation (BiFC) has emerged as a powerful technique for the efficient detection of protein interactions in their native subcellular localization. Here we report significant technical advances in the methodology of plant BiFC. We describe a series of versatile BiFC vector sets that are fully compatible with previously generated vectors. The new vectors enable the generation of both C‐terminal and N‐terminal fusion proteins and carry optimized fluorescent protein genes that considerably improve the sensitivity of BiFC. Using these vectors, we describe a multicolor BiFC (mcBiFC) approach for the simultaneous visualization of multiple protein interactions in the same cell. Application to a protein interaction network acting in calcium‐mediated signal transduction revealed the concurrent interaction of the protein kinase CIPK24 with the calcium sensors CBL1 and CBL10 at the plasma membrane and tonoplast, respectively. We have also visualized by mcBiFC the simultaneous formation of CBL1/CIPK1 and CBL9/CIPK1 protein complexes at the plasma membrane. Thus, mcBiFC provides a useful new tool for exploring complex regulatory networks in plants.