pGD vectors: versatile tools for the expression of green and red fluorescent protein fusions in agroinfiltrated plant leaves

• Published in: The Plant journal : for cell and molecular biology Vol. 31; no. 3; pp. 375 - 383
• Main Authors: Goodin, Michael M., Dietzgen, Ralf G., Schichnes, Denise, Ruzin, Steven, Jackson, Andrew O.
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Science Ltd 01.08.2002; Blackwell Science
• Subjects: agroinfiltration; bacteria; Biological and medical sciences; cell nucleolus; Diverse techniques; DsRed; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation, Plant; genes; Genes, Reporter - genetics; Genetic Vectors - genetics; GFP; green fluorescent protein; Green Fluorescent Proteins; hosts; leaves; Luminescent Proteins - genetics; Molecular and cellular biology; Nicotiana - genetics; Nicotiana benthamiana; Plant Leaves - genetics; Plant Proteins - genetics; Plant Viruses - genetics; Plants, Genetically Modified; protein expression plasmids; protein synthesis; Recombinant Fusion Proteins - genetics; Red Fluorescent Protein; Sonchus yellow net virus; Transgenes - genetics; transient gene expression; viral proteins; Nucleorhabdovirus; Molecular interaction; Plant leaf; Gene expression; Experimental protocol; Virus; Plasmid; Mononegavirales; Gene; Rhabdoviridae; Dicotyledones; Angiospermae; Green fluorescent protein; Spermatophyta; Infiltration; Solanaceae; Experimental plant; Localization; Fusion protein
• ISSN: 0960-7412; 1365-313X
• DOI: 10.1046/j.1365-313X.2002.01360.x

Summary We have constructed a matched set of binary vectors designated pGD, pGDG and pGDR for the expression and co‐localization of native proteins and GFP or DsRed fusions in large numbers of plant cells. The utility of these vectors following agroinfiltration into leaves has been demonstrated with four genes from Sonchus yellow net virus, a plant nucleorhabdovirus, and with a nucleolar marker protein. Of the three SYNV proteins tested, sc4 gave identical localization patterns at the cell wall and nucleus when fused to GFP or DsRed. However, some differences in expression patterns were observed depending on whether DsRed or GFP was the fusion partner. In this regard, the DsRed:P fusion showed a similar pattern of localization to GFP:P, but localized foci appeared in the nucleus and near the periphery of the nucleus. Nevertheless, the viral nucleocapsid protein, expressed as a GFP:N fusion, co‐localized with DsRed:P in a subnuclear locale in agreement with our previous observations (Goodin et al., 2001). This locale appears to be distinct from the nucleolus as indicated by co‐expression of the N protein, DsRed:P and a nucleolar marker AtFib1 fused to GFP. The SYNV M protein, which is believed to be particularly prone to oligomerization, was detectable only as a GFP fusion. Our results indicate that agroinfiltration with bacteria containing the pGD vectors is extremely useful for transient expression of several proteins in a high proportion of the cells of Nicotiana benthamiana leaves. The GFP and DsRed elements incorporated into the pGD system should greatly increase the ease of visualizing co‐localization and interactions of proteins in a variety of experimental dicotyledonous hosts.