Abstract
Bimolecular fluorescence complementation (BiFC) is an approach used to analyze protein–protein interaction in vivo, in which non-fluorescent N-terminal and C-terminal fragments of a fluorescent protein are reconstituted to emit fluorescence only when they are brought together by interaction of two proteins to fuse both fragments. A method for simultaneous visualization of two protein complexes by multicolor BiFC with fragments from green fluorescent protein (GFP) and its variants such as cyan and yellow fluorescent proteins (CFP and YFP) was recently reported in animal cells. In this paper we describe a new strategy for simultaneous visualization of two protein complexes in plant cells using the multicolor BiFC with fragments from CFP, GFP, YFP and a red fluorescent protein variant (DsRed-Monomer). We identified nine different BiFC complexes using fragments of CFP, GFP and YFP, and one BiFC complex using fragments of DsRed-Monomer. Fluorescence complementation did not occur by combinations between fragments of GFP variants and DsRed-Monomer. Based on these findings, we achieved simultaneous visualization of two protein complexes in a single plant cell using two colored fluorescent complementation pairs (cyan/red, green/red or yellow/red).
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Blondel M, Bach S, Bamps S, Dobbelaere J, Wiget P, Longaretti C, Barral Y, Meijer L, Peter M (2005) Degradation of Hof1 by SCFGrr1 is important for actomyosin contraction during cytokinesis in yeast. EMBO J 24:1440–1452. doi:10.1038/sj.emboj.7600627
Bracha-Drori K, Shichrur K, Katz A, Oliva M, Angelovici R, Yalovsky S, Ohad N (2004) Detection of protein–protein interactions in plants using bimolecular fluorescence complementation. Plant J 40:419–427. doi:10.1111/j.1365-313X.2004.02206.x
Chiu W, Niwa Y, Zeng W, Hirano T, Kobayashi H, Sheen J (1996) Engineered GFP as a vital reporter in plants. Curr Biol 6:325–330. doi:10.1016/S0960-9822(02)00483-9
Ghosh I, Hamilton AD, Regan L (2000) Antiparallel leucine zipper-directed protein reassembly: application to the green fluorescent protein. J Am Chem Soc 122:5658–5659. doi:10.1021/ja994421w
Hoff B, Kuck U (2005) Use of bimolecular fluorescence complementation to demonstrate transcription factor interaction in nuclei of living cells from the filamentous fungus Acremonium chrysogenum. Curr Genet 47:132–138. doi:10.1007/s00294-004-0546-0
Hu C-D, Kerppola TK (2003) Simultaneous visualization of multiple protein interactions in living cells using multicolor fluorescence complementation analysis. Nat Biotechnol 21:539–545. doi:10.1038/nbt816
Hu C-D, Chinenov Y, Kerppola TK (2002) Visualization of interactions among bZIP and Rel proteins in living cells using bimolecular fluorescence complementation. Mol Cell 9:789–798. doi:10.1016/S1097-2765(02)00496-3
Jach G, Pesch M, Richter K, Frings S, Uhrig JF (2006) An improved mRFP1 adds red to bimolecular fluorescence complementation. Nat Methods 3:597–600. doi:10.1038/nmeth901
Kodama Y, Shinya T, Sano H (2008) Dimerization of N-methyltransferases involved in caffeine biosynthesis. Biochimie 90:547–551. doi:10.1016/j.biochi.2007.10.001
Matz MV, Fradkov AF, Labasl YA, Savitsky AP, Zaraisky AG, Markelov ML, Lukyanov SA (1999) Fluorescent proteins from nonbioluminescent Anthozoa species. Nat Biotechnol 17:969–973. doi:10.1038/13657
Mizuno H, Sawano A, Eli P, Hama H, Miyawaki A (2001) Red fluorescent protein from Discosoma as a fusion tag and a partner for fluorescence resonance energy transfer. Biochemistry 40:2502–2510. doi:10.1021/bi002263b
Shimomura O, Johnson FH, Saiga Y (1962) Extraction, purification and properties of Aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. J Cell Comp Physiol 59:223–239. doi:10.1002/jcp.1030590302
Shyu YJ, Suarez CD, Hu C-D (2008) Visualization of AP-1 NF-kappaB ternary complexes in living cells by using a BiFC-based FRET. Proc Natl Acad Sci USA 105:151–156. doi:10.1073/pnas.0705181105
Tisien RY (1998) The green fluorescent protein. Annu Rev Biochem 67:509–544. doi:10.1146/annurev.biochem.67.1.509
Uefuji H, Shinjiro O, Yamaguchi Y, Koizumi N, Sano H (2003) Molecular cloning and functional characterization of three distinct N-methyltransferases involved in the caffeine biosynthetic pathway in coffee plants. Plant Physiol 132:372–380. doi:10.1104/pp.102.019679
Waadt R, Schmidt LK, Lohse M, Hashimoto K, Bock R, Kudla J (2008) Multicolor bimolecular fluorescence complementation reveals simultaneous formation of alternative CBL/CIPK complexes in planta. Plant J 56:505–516. doi:10.1111/j.1365-313X.2008.03612.x
Walter M, Chaban C, Schütze K, Batistic O, Weckermann K, Näke C, Blazevic D, Grefen C, Schumacher K, Oecking C, Harter K, Kudla J (2004) Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation. Plant J 40:428–438. doi:10.1111/j.1365-313X.2004.02219.x
Acknowledgments
The authors thank Prof. Hiroshi Sano (Stockholm University, Sweden) and Mr. Tomotaka Shinya (Nippon Paper Group, Inc., Tokyo, Japan) for experimental collaboration on YFP-based BiFC using the CaMXMT gene. We also thank Dr. Nir Ohad (Tel Aviv University, Israel) and Dr. Yun-Soo Kim (Nara Institute of Science and Technology, Japan) for providing BiFC vectors (pSY735 and pSY736) and the CaDXMT cDNA clone, respectively.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Kodama, Y., Wada, M. Simultaneous visualization of two protein complexes in a single plant cell using multicolor fluorescence complementation analysis. Plant Mol Biol 70, 211–217 (2009). https://doi.org/10.1007/s11103-009-9467-0
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11103-009-9467-0