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


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.

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


  1. 1

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

    CAS  Article  Google Scholar 

  2. 2

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

    CAS  Article  Google Scholar 

  3. 3

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

    CAS  Article  Google Scholar 

  4. 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. 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).

    CAS  Article  Google Scholar 

  6. 6

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

    Article  Google Scholar 

  7. 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).

    CAS  Article  Google Scholar 

  8. 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).

    CAS  Article  Google Scholar 

  9. 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).

    CAS  Article  Google Scholar 

  10. 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).

    CAS  Article  Google Scholar 

  11. 11

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

  12. 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).

    CAS  Article  Google Scholar 

  13. 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).

    CAS  Article  Google Scholar 

  14. 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).

    CAS  Article  Google Scholar 

  15. 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).

    CAS  Article  Google Scholar 

  16. 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).

    CAS  Article  Google Scholar 

  17. 17

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

    CAS  Article  Google Scholar 

  18. 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. 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).

    CAS  Article  Google Scholar 

  20. 20

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

    CAS  Article  Google Scholar 

  21. 21

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

    Google Scholar 

  22. 22

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

    CAS  Article  Google Scholar 

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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.

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Correspondence to Imogen A Sparkes.

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The authors declare no competing financial interests.

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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).

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