Advertisement

Fluorescent Protein Fusions for Protein Localization in Plants

  • John Runions
  • Chis Hawes
  • Smita Kurup
Protocol
Part of the Methods in Molecular Biology™ book series (MIMB, volume 390)

Protein localization in living plant cells is commonly studied using fluorescent protein fusions. Stable transformation of plant cells requires the use of binary vectors, which are larger and not as amenable to genetic manipulation as animal cell transfection vectors. Binary vectors containing fluorescent protein fusion constructs are prepared using standard molecular biological techniques. Fusion genes as well as promoters and selection markers are stably incorporated into the plant cell genome via Agrobacterium-mediated transfer. Presented here are a series of protocols that detail binary vector construction, bacterial transformation, and a rapid transient assay technique that can be used to evaluate fusion protein fluorescence in leaves.

Key Words

Fluorescent protein green fluorescent protein binary vector Agrobacterium Nicotiana tobacco Arabidopsis

References

  1. 1.
    Chiu, W., Niwa, Y., Zeng, W., Hirano, T., Kobayashi, H., and Sheen, J. (1996) Engineered GFP as a vital reporter in plants. Curr. Biol. 6, 325–330.CrossRefPubMedGoogle Scholar
  2. 2.
    Brandizzi, F., Irons, S., Johanson, J., Kotzer, A., and Neumann, U. (2004) GFP is the way to glow: bioimaging of the plant endomembrane system. J. Microsc. 214, 138–158.CrossRefPubMedGoogle Scholar
  3. 3.
    Dixit, R., Cyr, R., and Gilroy, S. (2006) Using intrisically fluorescent proteins for plant cell imaging. Plant J. 45, 599–615.CrossRefPubMedGoogle Scholar
  4. 4.
    Niedz, R. P., Sussman, M. R., and Satterlee, J. S. (1995) Green fluorescent protein—an in-vivo reporter of plant gene- expression. Plant Cell Rep. 14, 403–406.CrossRefGoogle Scholar
  5. 5.
    Grebenok, R. J., Pierson, E., Lambert, G. M., et al. (1997) Green fluorescent protein fusions for efficient characterization of nuclear targeting. Plant J. 11, 573–586.CrossRefPubMedGoogle Scholar
  6. 6.
    Haseloff, J., Siemering, K. R., Prasher, D. C., and Hodge, S. (1997) Removal of a cryptic intron and subcellular localisation of green fluorescent protein are required to mark transgenic Arabidopsisplants brightly. Proc. Natl. Acad. Sci. USA 94, 2122–2127.CrossRefPubMedGoogle Scholar
  7. 7.
    Cutler, S. R., Ehhardt, D. W., Griffitts, J. S., and Somerville, C. R. (2000) Random GFP::cDNA fusions enable visualization of subcellular structures in cells of Arabidopsis at a high frequency. Proc. Natl Acad. Sci. USA 97, 3718–3723.CrossRefPubMedGoogle Scholar
  8. 8.
    Escobar, N. M., Haupt, S., Thow, G., Boevink, P., Chapman, S., and Oparka, K. (2003) High-thoughput viral expression of cDNA-green fluorescent protein fusions reveals novel subcellular addresses and identifies unique proteins that interact with plasmodesmata. Plant Cell 15, 1507–1523.CrossRefPubMedGoogle Scholar
  9. 9.
    Tian, G.W., Mohanty, A., Chary, S.N., et al. (2004) High-thoughput fluorescent tagging of full-length Arabidopsis gene products in planta. Plant Physiol. 135, 25–38.CrossRefPubMedGoogle Scholar
  10. 10.
    Koroleva, O. A., TomLinson, M. L., Leader, D., Shaw, P., and Doonan, J. H. (2005) High-thoughput protein localization in Arabidopsis using Agrobacterium-mediated transient expression of GFP-ORF fusions. Plant J. 41, 162–174.CrossRefPubMedGoogle Scholar
  11. 11.
    Ambard-Bretteville, F., Small, I., Grandjean, O., and Colas des Francs-Smal, C. (2003) Discrete mutations in the presequence of potato formate dehydrogenase inhibit the in vivo targeting of GFP fusions into mitochondria. Biochem. Biophys. Res. Commun. 311, 966–971.CrossRefPubMedGoogle Scholar
  12. 12.
    Mackenzie, S. A. (2005) Plant organellar protein targeting: a traffic plan still under construction. Trends Cell Biol. 15, 548–554.CrossRefPubMedGoogle Scholar
  13. 13.
    Draper, J., Scott, R., Armitage, P., and Walden, R. (1988) Plant Genetic Transformation and Gene Expression, A Laboratory Manual, Blackwell Scientific.Google Scholar
  14. 14.
    Gelvin, S.B. and Schilperoort, R. (eds.) (1995) Plant Molecular Biology Manual, 2nd ed., Kluwer Academic Publishers, Dordrecht.Google Scholar
  15. 15.
    Veluthambi, K., Gupta, A. K., and Sharma, A. (2003) The current status of plant transformation technologies. Curr. Sci. 84, 368–380.Google Scholar
  16. 16.
    Wroblewski, T., Tomczak, A., and Michelmore, R. (2005) Optimization of Agrobacterium-mediated transient assays of gene expression in lettuce, tomato and Arabidopsis. Plant Biotechnol. J. 3, 259–273.CrossRefPubMedGoogle Scholar
  17. 17.
    Earley, K. W., Haag, J. R., Pontes, O., et al. (2006) Gateway-compatible vectors for plant functional genomics and proteomics. Plant J. 45, 616–629.CrossRefPubMedGoogle Scholar
  18. Old, R.W. and Primrose, S. B. (1994) Principles of Gene Manipulation: An Introduction to Genetic Engineering, 5th ed. Blackwell Scientific.Google Scholar
  19. Brown, T. A. (ed.) (1998) Molecular Biology Labfax. Academic Press Inc., London.Google Scholar
  20. Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. E., Smith J. A., and Struhl, K. (1999) Short Protocols in Molecular Biology, 4th ed., John Wiley, New York.Google Scholar
  21. Sambrook, J. and Russell, D.W. (2001) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, New York.Google Scholar
  22. 22.
    Clough, S. J. and Bent, A. F. (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735–743.CrossRefPubMedGoogle Scholar
  23. Martinez-Zapater, J. M. and Salinas, J. (eds.) (1998) Arabidopsis Protocols. Humana Press, Totowa, NJ.Google Scholar
  24. 24.
    An, G., Ebert, P. R., Mitra, A., and Ha, S. B. (1988) Binary Vectors, in Plant Molecular Biology Manua. (Gelvin, S. B. and Schilperoort, R. A., eds.), Kluwer Academic Publishers, Dordrecht, The Netherlands, A3, pp. 1–19Google Scholar
  25. 25.
    Haseloff, J. and Siemering, K. (1998) The uses of GFP in plants, in Green Fluorescent Protein: Properties, Applications and Protocols (Chalfie, M. and Kain, S., eds.) John Wiley, New York.Google Scholar
  26. 26.
    Batoko, H., Zheng, H.-Q., Hawes, C., and Moore, I. (2000) A Rab1 GTPase is required for transport between the endoplasmic reticulum and Golgi apparatus and for normal Golgi movement in plants. Plant Cell 12, 2201–2218.CrossRefPubMedGoogle Scholar
  27. 27.
    Hellens, R. P., Edwards, E. A., Leyland, N. R., Bean, S., and Mullineaux, P. M. (2000) pGreen: a versatile and flexible binary Ti Vector for Agrobacterium-mediated plant transformation. Plant Mol. Biol. 42, 819–832.CrossRefPubMedGoogle Scholar
  28. 28.
    Hajdukiewicz, P., Svab, Z. and Maliga, P. (1994) The small versatile pPZP family of Agrobacterium binary vectors for plant transformation. Plant Mol. Biol. 25, 989–994.CrossRefPubMedGoogle Scholar
  29. 29.
    Bevan, M. (1984) Binary Agrobacterium vectors for plant transformation. Nucl. Acids Res. 12, 8711–8721.CrossRefPubMedGoogle Scholar
  30. 30.
    Gleave, A.P. (1992) A versatile binary vector system with a T-DNA organisational structure conducive to efficient integration of cloned DNA into the plant genome. Plant Mol. Biol. 20, 1203–1207.CrossRefPubMedGoogle Scholar
  31. 31.
    Curtis, M. D. and Grossniklaus, U. (2003) A gateway cloning vector set for high-thoughput functional analysis of genes in planta. Plant Physiol. 133, 462–469.CrossRefPubMedGoogle Scholar
  32. 32.
    Kurup, S., Runions, J., Köhler, U., Laplaze, L., Hodge, S., and Haseloff, J. (2005) Marking cell lineages in living tissues. Plant J. 42, 444–453.CrossRefPubMedGoogle Scholar
  33. 33.
    Boisnard-Lorig, C., Colon-Carmona, A., Bauch, M., et al. (2001) Dynamic analyses of the expression of the HISTONE::YFP fusion protein in Arabidopsis show that syncytial endosperm is divided in mitotic domains. Plant Cell, 13, 495–509.CrossRefPubMedGoogle Scholar
  34. 34.
    Runions, J., Thorsten, B., Kühner, S., and Hawes, C. (2005) Photoactivation of GFP reveals protein dynamics within the endoplasmic reticulum membrane, J Exp. Bot. 57, 43–50.CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press Inc. 2007

Authors and Affiliations

  • John Runions
    • 1
  • Chis Hawes
    • 1
  • Smita Kurup
    • 2
  1. 1.School of Life SciencesOxford Brookes UniversityOxfordUK
  2. 2.CPI DivisionRothamsted ResearchHertfordshireUK

Personalised recommendations