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Host-Bacteria Interactions pp 185–208Cite as

Bimolecular Fluorescence Complementation for Imaging Protein Interactions in Plant Hosts of Microbial Pathogens

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1197))

Abstract

Protein–protein interactions mediate many aspects of cellular function. Scientists have developed numerous techniques to investigate these interactions, both in vitro and in vivo. Among these, the peptide complementation assay Bimolecular Fluorescence Complementation (BiFC) allows visualization of the subcellular sites of protein–protein interactions in living cells. BiFC comprises a “split GFP” system: GFP protein (or its derivatives) is split into two fragments, neither of which fluoresces on its own. Interacting proteins linked to these peptide fragments may bring them into proximity, allowing them to refold and restore fluorescence. Although this system was first exploited for use in animal cells, we have developed BiFC for use in plants. Pathogens transfer numerous effector proteins into eukaryotic cells and manipulate host cellular processes through interactions between effector and host proteins. BiFC can therefore facilitate studies of host–bacterial interactions. In this chapter, we describe the numerous BiFC vectors we have constructed, their uses, and their limitations.

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References

  1. Hu CD, Chinenov Y, Kerppola TK (2002) Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation. Mol Cell 9:789–798

    Article  CAS  PubMed  Google Scholar 

  2. Hu C-D, Grinberg A, Kerppola T (2005) Visualization of protein interaction in living cells using bimolecular fluorescence complementation (BiFC) analysis. Curr Protoc Cell Biol 21:3.1–3.2

    Google Scholar 

  3. Jach G, Pesch M, Richter K et al (2006) An improved mRFP1 adds red to bimolecular fluorescence complementation. Nat Methods 3:597–600

    Article  CAS  PubMed  Google Scholar 

  4. Fujikawa Y, Kato N (2007) Split luciferase complementation assay to study protein–protein interactions in Arabidopsis protoplasts. Plant J 52:185–195

    Article  CAS  PubMed  Google Scholar 

  5. Chen H, Zou Y, Shang Y et al (2008) Firefly luciferase complementation imaging assay for protein-protein interactions in plants. Plant Physiol 146:368–376

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  6. Fan J-Y, Cui Z-Q, Wei H-P et al (2008) Split mCherry as a new red bimolecular fluorescence complementation system for visualizing protein-protein interactions in living cells. Biochem Biophys Res Commun 367:47–53

    Article  CAS  PubMed  Google Scholar 

  7. Chu J, Zhang Z, Zheng Y et al (2009) A novel far-red bimolecular fluorescence complementation system that allows for efficient visualization of protein interactions under physiological conditions. Biosens Bioelectron 25:234–239

    Article  CAS  PubMed  Google Scholar 

  8. Kodama Y, Wada M (2009) Simultaneous visualization of two protein complexes in a single plant cell using multicolor fluorescence complementation analysis. Plant Mol Biol 70:211–217

    Article  CAS  PubMed  Google Scholar 

  9. Grinberg AV, Hu C-D, Kerppola TK (2004) Visualization of Myc/Max/Mad family dimers and the competition for dimerization in living cells. Mol Cell Biol 24:4294–4308

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  10. Atmakuri K, Ding Z, Christie PJ (2003) VirE2, a Type IV secretion substrate, interacts with the VirD4 transfer protein at cell poles of Agrobacterium tumefaciens. Mol Microbiol 49:1699–1713

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  11. Wilson CGM, Magliery TJ, Regan L (2004) Detecting protein-protein interactions with GFP-fragment reassembly. Nat Methods 1:255–262

    Article  CAS  PubMed  Google Scholar 

  12. Bracha-Drori K, Shichrur K, Katz A et al (2004) Detection of protein–protein interactions in plants using bimolecular fluorescence complementation. Plant J 40:41–427

    Article  Google Scholar 

  13. Walter M, Chaban C, Schutze K et al (2004) Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation. Plant J 40:428–438

    Article  CAS  PubMed  Google Scholar 

  14. Citovsky V, Lee L-Y, Vyas S et al (2006) Subcellular localization of interacting proteins by bimolecular fluorescence complementation in planta. J Mol Biol 362:1120–1131

    Article  CAS  PubMed  Google Scholar 

  15. Gehl C, Waadt R, Kudla J et al (2009) New GATEWAY vectors for high throughput analyses of protein–protein interactions by bimolecular fluorescence complementation. Mol Plant 2:1051–1058

    Article  CAS  PubMed  Google Scholar 

  16. Citovsky V, Gafni Y, Tzfira T (2008) Localizing protein–protein interactions by bimolecular fluorescence complementation in planta. Methods 45:196–206

    Article  CAS  PubMed  Google Scholar 

  17. Lee L-Y, Fang M-J, Kuang L-Y et al (2008) Vectors for multi-color bimolecular fluorescence complementation to investigate protein-protein interactions in living plant cells. Plant Methods 4:24. doi:10.1186/1746-4811-4-24

    Article  PubMed Central  PubMed  Google Scholar 

  18. Nelson BK, Cai X, Nebenfuhr A (2007) A multicolored set of in vivo organelle markers for co-localization studies in Arabidopsis and other plants. Plant J 51:1126–1136

    Article  CAS  PubMed  Google Scholar 

  19. Bhat RA, Lahaye G, Panstruga R (2006) The visible touch: in planta visualization of protein-protein interactions by fluorophore-based methods. Plant Methods 2:12. doi:10.1186/1746-4811-2-12

    Article  PubMed Central  PubMed  Google Scholar 

  20. Ohad N, Shichrur K, Yalovsky S (2007) The analysis of protein-protein interactions in plants by bimolecular fluorescence complementation. Plant Physiol 145:1090–1099

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  21. Kerppola TK (2008) Bimolecular fluorescence complementation (BiFC) analysis as a probe of protein interactions in living cells. Annu Rev Biophys 37:465–87

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  22. Shyu YJ, Hu C-D (2008) Fluorescence complementation: An emerging tool for biological research. Trends Biotechnol 26:622–630

    Article  CAS  PubMed  Google Scholar 

  23. Weinthal D, Tzfira T (2009) Imaging protein–protein interactions in plant cells by bimolecular fluorescence complementation assay. Trends Plant Sci 14:59–63

    Article  CAS  PubMed  Google Scholar 

  24. Nagai T, Ibata K, Park ES et al (2002) A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat Biotechnol 20:87–90

    Article  CAS  PubMed  Google Scholar 

  25. Shyu Y, Liu H, Deng X et al (2006) Identification of new fluorescent fragments for BiFC analysis under physiological conditions. Biotechniques 40:61–66

    Article  CAS  PubMed  Google Scholar 

  26. Bayle V, Nussaume L, Bhat RA (2008) Combination of novel green fluorescent protein mutant TSapphire and DsRed variant mOrange to set up a versatile in planta FRET-FLIM assay. Plant Physiol 148:51–60

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  27. Waadt R, Schmidt LK, Lohse M et al (2008) Multicolor bimolecular fluorescence complementation reveals simultaneous formation of alternative CBL/CIPK complexes in planta. Plant J 56:505–516

    Article  CAS  PubMed  Google Scholar 

  28. Chung S-M, Frankman EL, Tzfira T (2005) A versatile vector system for multiple gene expression in plants. Trends Plant Sci 10:357–361

    Article  CAS  PubMed  Google Scholar 

  29. Tzfira T, Tian G-W, Lacroix B et al (2005) pSAT vectors: A modular series of plasmids for autoautofluorescent protein tagging and expression of multiple genes in plants. Plant Mol Biol 57:503–516

    Article  CAS  PubMed  Google Scholar 

  30. Goderis IJWM, De Bolle MFC, François IEJA et al (2002) A set of modular plant transformation vectors allowing flexible insertion of up to six expression units. Plant Mol Biol 50:17–20

    Article  CAS  PubMed  Google Scholar 

  31. Kodama Y, Hu C-D (2010) An improved bimolecular fluorescence complementation assay with a high signal-to-noise ratio. Biotechniques 49:793–803

    Article  CAS  PubMed  Google Scholar 

  32. 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

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  33. Shyu YJ, Suarez CD, Hu C-D (2008) Visualization of AP-1–NF-kB ternary complexes in living cells by using a BiFC-based FRET. Proc Natl Acad Sci U S A 105:151–156

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  34. Kwaaitaal M, Keinath NF, Pajonk S et al (2010) Combined bimolecular fluorescence complementation and Forster resonance energy transfer reveals ternary SNARE complex formation in living plant cells. Plant Physiol 152:1135–1147

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  35. Zaltsman A, Krichevsky A, Loyter A et al (2010) Agrobacterium induces expression of a host F-Box protein required for tumorigenicity. Cell Host Microbe 7:197–209

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  36. Lee L-Y, Wu F-H, Hsu C-T et al (2012) Screening a cDNA library for protein-protein interactions directly in planta. Plant Cell 24:1746–1759

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  37. Shaner NC, Steinbach PA, Tsien RY (2005) A guide to choosing fluorescent proteins. Nat Methods 2:90–909

    Article  Google Scholar 

  38. Campbell RE, Tour O, Palmer AE et al (2002) A monomeric red fluorescent protein. Proc Natl Acad Sci U S A 99:7877–7882

    Article  CAS  PubMed Central  PubMed  Google Scholar 

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Acknowledgments

The authors thank Yanjun Yu, Solomon Bisangwa, Yu-Chen Yen, Shengjie Xu, and Nathan Hood for help in constructing several of the plasmids. Research in the authors’ laboratory is funded by the US National Science Foundation, the US Department of Energy, the Corporation for Plant Biotechnology Research, the Biotechnology Research and Development Corporation, and Dow AgroSciences.

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Authors and Affiliations

  1. Department of Biological Sciences, Purdue University, 1392 Lilly Hall, West Lafayette, IN, 47907-1392, USA

    Lan-Ying Lee & Stanton B. Gelvin Ph.D.

Authors
  1. Lan-Ying Lee
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  2. Stanton B. Gelvin Ph.D.
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Corresponding author

Correspondence to Stanton B. Gelvin Ph.D. .

Editor information

Editors and Affiliations

  1. U1047, UFR Médecine, INSERM, Université Montpellier 1, Nîmes, France

    Annette C. Vergunst

  2. U1047, UFR Médecine, INSERM, Université Montpellier 1, Nîmes, France

    David O'Callaghan

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Lee, LY., Gelvin, S.B. (2014). Bimolecular Fluorescence Complementation for Imaging Protein Interactions in Plant Hosts of Microbial Pathogens. In: Vergunst, A., O'Callaghan, D. (eds) Host-Bacteria Interactions. Methods in Molecular Biology, vol 1197. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1261-2_11

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  • DOI: https://doi.org/10.1007/978-1-4939-1261-2_11

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-1260-5

  • Online ISBN: 978-1-4939-1261-2

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