Skip to main content

Localization of Viral Proteins in Plant Cells: Protein Tagging

  • Protocol
Plant Virology Protocols

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 451))

  • 2920 Accesses

Summary

This chapter describes techniques for in vivo imaging of fluorescent fusion proteins in living cells by confocal laser scanning microscopy (CLSM). Methods are provided for (i) producing the constructs for transient expression from plasmids or virus-based vectors, (ii) introduction of constructs to plant epidermal cells; (iii) imaging of the expressed proteins by CLSM and image processing, and (iv) studying the expression in the presence of agents that affect the integrity or function of cytoskeletal elements. Notes are provided to aid comprehension and indicate problems.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. 1. Crawford, K.M. and Zambryski, P.C. (2001) Non-targeted and targeted protein movement through plasmodesmata in leaves in different developmental and physiological states. Plant Phys. 125, 1802–1812.

    Article  CAS  Google Scholar 

  2. 2. Oparka, K.J., Prior, D.A., Santa Cruz, S., Padgett, H.S. and Beachy, R.N. (1997) Gating of epidermal plasmodesmata is restricted to the leading edge of expanding infection sites of tobacco mosaic virus (TMV). Plant J. 12, 781–789.

    Article  PubMed  CAS  Google Scholar 

  3. 3. Solovyev, A.G., Stroganova, T.A., Zamyatnin, A.A., Fedorkin, O.N., Schiemann, J. and Morozov, S.Y. (2000) Subcellular sorting of small membrane-associated triple gene block proteins: TGBp3-assisted targeting of TGBp2. Virology. 269, 113–127.

    Article  PubMed  CAS  Google Scholar 

  4. 4. Zamyatnin, A.A., Solovyev, A.G., Sablina, A.A., Agranovsky, A.A., Katul, L., Vetten, H.J., Schiemann, J., Hinkkanen, A.E., Lehto, K. and Morozov, S.Y. (2002). Dual-colour imaging of membrane protein targeting directed by poa semilatent virus movement protein TGBp3 in plant and mammalian cells. J. Gen. Virol. 83, 651–662.

    PubMed  Google Scholar 

  5. 5. Zamyatnin, A.A. Jr., Solovyev, A.G., Savenkov, E.I., Germundsson, A., Sandgren, M., Valkonen, J.P.T. and Morozov, S.Y. (2004). Transient coexpression of individual genes encoded by the triple gene block of potato mop-top virus reveals requirements for TGBp1 trafficking. Mol. Plant Microbe Interact. 17, 921–930.

    Article  Google Scholar 

  6. 6. Haupt, S., Cowan, G.H., Ziegler, A., Roberts, A.G., Oparka, K.J. and Torrance, L. (2004) Two plant-viral movement proteins traffic in the endocytic recycling pathway. Plant Cell. 17: 164–181.

    Article  PubMed  Google Scholar 

  7. 7. Tamai, A. and Meshi, T. (2001). Cell-to-cell movement of potato virus X: The role of p12 and p8 encoded by the second and third open reading frames of the triple gene block. Mol. Plant Microbe Interact. 14, 1158–1167.

    Article  PubMed  CAS  Google Scholar 

  8. 8. Ryabov, E.V., Oparka, K.J., Santa Cruz, S., Robinson, D.J. and Taliansky, M.E. (1998) Intracellular location of two groundnut rosette umbravirus proteins delivered by PVX and TMV vectors. Virology. 242, 303–313.

    Article  PubMed  CAS  Google Scholar 

  9. 9. Ryabov, E.V., Kim, S.H., Taliansky, M. (2004) Identification of a nuclear localization signal and nuclear export signal of the umbraviral long-distance RNA movement protein. J. Gen. Virol. 85, 1329–1333.

    Article  PubMed  CAS  Google Scholar 

  10. 10. Escobar N.M., Haupt, S., Thow, G., Boevink, P., Chapman, S. and Oparka, K. (2003) High-throughput 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.

    Article  PubMed  CAS  Google Scholar 

  11. 11. Gillespie, T., Boevink, P., Haupt, S., Roberts, A.G., Toth, R., Valentine, T., Chapman, S. and Oparka, K.J. (2002) Functional analysis of a DNA-shuffled movement protein reveals that microtubules are dispensable for the cell-to-cell movement of tobacco mosaic virus. Plant Cell. 14: 1207–1222.

    Article  PubMed  CAS  Google Scholar 

  12. 12. Waigmann, E., Ueki, S., Trutuyeva, K. and Citovsky, V. (2004) The ins and outs of nondestructive cell-to-cell and systemic movement of plant viruses. Crit. Rev. Plants. 236, 195–250.

    Article  Google Scholar 

  13. 13. Horton, R.M., Hunt, H.D., Ho, S.N., Pullen, J.K. and Pease, L.R. (1989) Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene. 77, 61–68.

    Article  PubMed  CAS  Google Scholar 

  14. 14. Restrepo, M.A., Freed, D.D. and Carrington, J.C. (1990) Nuclear transport of plant potyviral proteins. Plant Cell. 2, 987–998.

    Article  PubMed  CAS  Google Scholar 

  15. 15. Shivprasad, S., Pogue, G.P. and Lewandowski, D.J. (1999) Heterologous sequences greatly affect foreign gene expression in tobacco mosaic virus-based vectors. Virology. 255, 312–323.

    Article  PubMed  CAS  Google Scholar 

  16. 16. Hellens R.P., Edward, 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.

    Article  PubMed  CAS  Google Scholar 

  17. 17. Gal-On, A., Meiri, E., Elman, C., Gray, D.J. and Gaba, V. (1997) Simple hand-held devices for the efficient infection of plants with viral-encoding constructs by particle bombardment. J. Virol. Methods. 64, 103–110.

    Article  PubMed  CAS  Google Scholar 

  18. QuikChange XL Site Directed Mutagenesis Kit. Instruction manual. Stratagene

    Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2008 Humana Press, a part of Springer Science + Business Media, LLC

About this protocol

Cite this protocol

Haupt, S., Ziegler, A., Torrance, L. (2008). Localization of Viral Proteins in Plant Cells: Protein Tagging. In: Foster, G.D., Johansen, I.E., Hong, Y., Nagy, P.D. (eds) Plant Virology Protocols. Methods in Molecular Biology™, vol 451. Humana Press. https://doi.org/10.1007/978-1-59745-102-4_31

Download citation

  • DOI: https://doi.org/10.1007/978-1-59745-102-4_31

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-827-0

  • Online ISBN: 978-1-59745-102-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics