Summary
This chapter describes techniques to investigate the localisation and function of virus-encoded proteins in plants using green fluorescent protein (GFP) transiently expressed from plasmids or infectious cDNA reporter clones of barley stripe mosaic virus. Virus movement and the localisation of GFP-tagged proteins in living cells were monitored by confocal laser scanning microscopy (CLSM). In addition, GFP expression was imaged in transgenic plants where specific organelles or subcellular structures such as endoplasmic reticulum were labelled with another fluorophore (e.g., monomeric red fluorescent protein). Using these approaches we discovered evidence for additional roles played by virus encoded movement protein TGB2 and γb protein in virus replication. Methods are described for clone construction and mutagenesis, and for transient expression (biolistic bombardment or agrobacterium infiltration) in the epidermal cells of Nicotiana benthamiana or barley. In addition, techniques for chloroplast isolation and imaging of the different fluorescent proteins, and the avoidance of interference from autofluorescence, are described.
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References
Harrison, B. D., and Robinson, D. J. (2005) Another quarter century of great progress in understanding the biological properties of plant viruses. Ann Appl Biol. 146, 15–37.
Donald, R. G. K., and Jackson, A. O. (1996) RNA-binding activities of barley stripe mosaic virus &ggr;b fusion proteins. J Gen Virol. 77, 879–888.
Bragg, J. N., and Jackson, A. O. (2004) The C-terminal region of the Barley stripe mosaic virus &ggr;b protein participates in homologous interactions and is required for suppression of RNA silencing. Mol Plant Pathol. 5, 465–481.
Haupt, S., Cowan, G. H., Ziegler, A., Roberts, A. G., Oparka, K. J., and Torrance, L. (2005) Two plant–viral movement proteins traffic in the endocytic recycling pathway. Plant Cell. 17, 164–181.
Torrance, L., Cowan, G. H., Gillespie, T., Ziegler, A., and Lacomme, C. (2006) Barley stripe mosaic virus-encoded proteins triple-gene block 2 and gb localize to chloroplasts in virus-infected monocot and dicot plants, revealing hitherto-unknown roles in virus replication. J Gen Virol. 87, 2403–2411.
Lawrence, D. M., and Jackson, A. O. (2001) Interactions of the TGB1 protein during cell-to-cell movement of Barley stripe mosaic virus. J Virol. 75, 8712–8723.
Petty, I. T. D., Hunter, B. G., and Jackson, A. O. (1988) A novel strategy for one-step cloning of full-length cDNA and its application to the genome of barley stripe mosaic virus. Gene. 74, 423–432.
Ryan, M. D., King, A. M. O., and Thomas, G. P. (1991) Cleavage of foot-and-mouth-disease virus polyprotein is mediated by residues located within a 19 amino acid sequence. J Gen Virol. 72, 2727–2732.
Horton, R. M., Hunt, H. D., Ho, S. N., Pullen, J. K., and Pease, L. R. (1989) Enginee-ring hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene. 77, 61–68.
Restrepo, M. A., Freed, D. D., and Carrington, J. C. (1990) Nuclear transport of plant potyviral proteins. Plant Cell. 2, 987–998.
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.
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. PMB. 42, 819–832.
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.
Nivison, H. T., Fish, L. E., and Jagendorf A.T. (1986) Translation by isolated pea chloroplasts. Methods Enzymol. 118, 282–295.
Acknowledgement
We thank Andy Jackson, Greg Pogue and Merete Albrechtsen for providing BSMV clones.
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© 2009 Humana Press, a part of Springer Science+Business Media, LLC
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Haupt, S., Ziegler, A., Cowan, G., Torrance, L. (2009). Studies of the Role and Function of Barley Stripe Mosaic Virus Encoded Proteins in Replication and Movement Using GFP Fusions. In: Hicks, B.W. (eds) Viral Applications of Green Fluorescent Protein. Methods in Molecular Biology™, vol 515. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-59745-559-6_20
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DOI: https://doi.org/10.1007/978-1-59745-559-6_20
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-934115-87-9
Online ISBN: 978-1-59745-559-6
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