Studies of the role and function of barley stripe mosaic virus encoded proteins in replication and movement using GFP fusions.
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 gammab 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.
Scottish Crop Research Institute, University of Dundee, Dundee, UK.
Sophie
S
Haupt
Angelika
A
Ziegler
Graham
G
Cowan
Lesley
L
Torrance
D002736:Chloroplasts / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D016679:Genome, Viral / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D018515:Plant Leaves / Q000821:virology; D012328:RNA Viruses / Q000235:genetics / Q000378:metabolism*; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D012231:Rhizobium / Q000235:genetics / Q000378:metabolism; D014026:Nicotiana / Q000821:virology; D019076:Transgenes / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D018406:Virus Physiological Phenomena
D018076:DNA, Complementary; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D049452:Green Fluorescent Proteins