Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants

Abstract

Expression and tracking of fluorescent fusion proteins has revolutionized our understanding of basic concepts in cell biology. The protocol presented here has underpinned much of the in vivo results highlighting the dynamic nature of the plant secretory pathway. Transient transformation of tobacco leaf epidermal cells is a relatively fast technique to assess expression of genes of interest. These cells can be used to generate stable plant lines using a more time-consuming, cell culture technique. Transient expression takes from 2 to 4 days whereas stable lines are generated after approximately 2 to 4 months.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Expression of fluorescent fusion proteins in tobacco epidermal cells.
Figure 2
Figure 3: N. tabacum leaf infiltration.
Figure 4

Similar content being viewed by others

References

  1. Lippincott-Schwartz, J. & Patterson, G.H. Development and use of fluorescent protein markers in living cells. Science 300, 87–91 (2003).

    Article  CAS  Google Scholar 

  2. Chapman, S., Oparka, K.J. & Roberts, A.G. New tools for in vivo fluorescence tagging. Curr. Opin. Plant Biol. 8, 565–573 (2005).

    Article  CAS  Google Scholar 

  3. Lukyanov, K.A., Chudakov, D.M., Lukyanov, S. & Verkhusha, V.V. Photoactivatable fluorescent proteins. Nat. Rev. Mol. Cell Biol. 6, 885–890 (2005).

    Article  CAS  Google Scholar 

  4. Runions, J., Hawes, C. & Kurup, S. Fluorescent protein fusions for protein localization in plants. in Methods in Molecular Biology: Protein Targeting Protocols (ed. van der Giezen, M.) Humana Press, New Jersey, USA, in the press.

  5. Runions, J., Brach, T., Kühner, S. & Hawes, C. Photoactivation of GFP reveals protein dynamics within the endoplasmic reticulum. J. Exp. Bot. 57, 43–50 (2006).

    Article  CAS  Google Scholar 

  6. Hawes, C. Cell biology of the plant Golgi apparatus. New Phytol. 165, 29–44 (2005).

    Article  Google Scholar 

  7. Brandizzi, F., Irons, S.L., Johansen, J., Kotzer, A. & Neumann, U. GFP is the way to glow: bioimaging of the plant endomembrane system. J. Microsc. 214, 138–158 (2004).

    Article  CAS  Google Scholar 

  8. Kotzer, A.M. et al. AtRabF2b (Ara7) acts on the vacuolar trafficking pathway in tobacco leaf epidermal cells. J. Cell Sci. 117, 6377–6389 (2004).

    Article  CAS  Google Scholar 

  9. Sparkes, I.A., Hawes, C. & Baker, A. AtPEX2 and AtPEX10 are targeted to peroxisomes independently of known endoplasmic reticulum trafficking routes. Plant Physiol. 139, 690–700 (2005).

    Article  CAS  Google Scholar 

  10. Irons, S.L., Evans, D.E. & Brandizzi, F. The first 238 amino acids of the human lamin B receptor are targeted to the nuclear envelop in plants. J. Exp. Bot. 54, 943–950 (2003).

    Article  CAS  Google Scholar 

  11. Walter, M. et al. Visualisation of protein interactions in living plants cells using bimolecular fluorescence complementation. Plant J. 40, 428–438.

  12. daSilva, L.L.P. et al. ER export sites and Golgi bodies behave as single mobile secretory units in plant cells. Plant Cell 16, 1753–1771 (2004).

    Article  CAS  Google Scholar 

  13. Clough, S.J. & Bent, A.F. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana . Plant J. 16, 735–743 (1998).

    Article  CAS  Google Scholar 

  14. Zhang, X., Henriques, R., Lin, S.-S., Niu, Q.-W. & Chua, N.-H. Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method. Nat. Protocols 1, 641–646 (2006).

    Article  CAS  Google Scholar 

  15. Valvekens, D., van Montagu, M. & van Lijsebettens, M. Agrobacterium tumefaciens-mediated transformation of Arabidopsis thaliana root explants by using kanamycin selection. Proc. Natl. Acad. Sci. USA 85, 5536–5540 (1988).

    Article  CAS  Google Scholar 

  16. Sparkes, I.A. et al. An Arabidopsis pex10 null mutant is embryo lethal, implicating peroxisomes in an essential role during plant embryogenesis. Plant Physiol. 133, 1809–1819 (2003).

    Article  CAS  Google Scholar 

  17. Earley, K.W. et al. Gateway-compatible vectors for plant functional genomics and proteomics. Plant J. 45, 616–629 (2006).

    Article  CAS  Google Scholar 

  18. Hellen, R., Mullineaux, P. & Klee, H. Technical focus: a guide to Agrobacterium binary Ti vectors. Trends Plant Sci. 5, 446–451 (2000).

    Article  Google Scholar 

  19. Wroblewski, T., Tomczak, A. & Michelmore, R. Optimization of Agrobacterium-mediated transient assays of gene expression in lettuce, tomato and Arabidopsis . Plant Biotechnol. J. 3, 259–273 (2005).

    Article  CAS  Google Scholar 

  20. Hofgen, R. & Willmitzer, L. Storage of competent cells for Agrobacterium transformation. Nucleic Acids Res. 16, 9877 (1988).

    Article  CAS  Google Scholar 

  21. Evans, D.E., Coleman, J.O.D. & Kearns, A. Plant Cell culture. BIOS Scientific Publishers, London, (2003).

    Google Scholar 

  22. Boevink, P. et al. Stacks on tracks: the plant Golgi apparatus traffics on an actin/ER network. Plant J. 15, 441–447 (1998).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We are grateful to members of C. Hawes' and I. Moore's laboratories for fruitful discussions and members of the laboratories who have helped refine these techniques over the years. The work reported here was funded by various BBSRC grants.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Imogen A Sparkes.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Sparkes, I., Runions, J., Kearns, A. et al. Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants. Nat Protoc 1, 2019–2025 (2006). https://doi.org/10.1038/nprot.2006.286

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2006.286

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing