Studies of the role and function of barley stripe mosaic virus encoded proteins in replication and movement using GFP fusions

Sophie Haupt, Angelika Ziegler, Graham Cowan, Lesley Torrance*

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingChapter

3 Citations (Scopus)

Abstract

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.

Original languageEnglish
Title of host publicationViral Applications of Green Fluorescent Protein
Subtitle of host publicationMethods and Protocols
PublisherHumana Press Inc.
Pages287-297
Number of pages11
ISBN (Print)9781934115879
DOIs
Publication statusPublished - 1 Jan 2009

Publication series

NameMethods in Molecular Biology
Volume515
ISSN (Print)1064-3745

Keywords

  • Barley stripe mosaic virus
  • Binary vectors
  • Chloroplasts
  • Confocal laser scanning microscopy
  • In vivo imaging
  • Microprojectile bombardment
  • Run-off transcripts
  • Virus protein function
  • Virus replication
  • Virus-based vectors

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