Multicolor bimolecular fluorescence complementation reveals simultaneous formation of alternative CBL/CIPK complexes in planta
Article first published online: 4 JUL 2008
© 2008 The Authors. Journal compilation © 2008 Blackwell Publishing Ltd
The Plant Journal
Volume 56, Issue 3, pages 505–516, November 2008
How to Cite
Waadt, R., Schmidt, L. K., Lohse, M., Hashimoto, K., Bock, R. and Kudla, J. (2008), Multicolor bimolecular fluorescence complementation reveals simultaneous formation of alternative CBL/CIPK complexes in planta. The Plant Journal, 56: 505–516. doi: 10.1111/j.1365-313X.2008.03612.x
- Issue published online: 22 OCT 2008
- Article first published online: 4 JUL 2008
- Received 9 May 2008; revised 11 June 2008; accepted 16 June 2008; published online 29 July 2008.
- protein–protein interaction;
- bimolecular fluorescence complementation;
- multicolor bimolecular fluorescence complementation;
- calcium signaling;
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.