pSITE Vectors for Stable Integration or Transient Expression of Autofluorescent Protein Fusions in Plants: Probing Nicotiana benthamiana-Virus Interactions

• Published in: Molecular plant-microbe interactions Vol. 20; no. 7; pp. 740 - 750
• Main Authors: Chakrabarty, R, Banerjee, R, Chung, S.M, Farman, M, Citovsky, V, Hogenhout, S.A, Tzfira, T, Goodin, M
• Format: Journal Article
• Language: English
• Published: St Paul, MN APS Press 01.07.2007; The American Phytopathological Society
• Subjects: Agrobacterium; agroinfiltration; Bacterial plant pathogens; Biological and medical sciences; Blotting, Western; confocal microscopy; Fluorescence Recovery After Photobleaching; Fundamental and applied biological sciences. Psychology; Gene Expression; genetic markers; Genetic Vectors; Genetic Vectors - genetics; genetics; Green Fluorescent Proteins; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; growth & development; infection; Luminescent Proteins; Luminescent Proteins - genetics; Luminescent Proteins - metabolism; metabolism; Microscopy, Confocal; new methods; Nicotiana - genetics; Nicotiana - metabolism; Nicotiana - virology; Nicotiana benthamiana; nucleolus; Phytopathology. Animal pests. Plant and forest protection; Plant Leaves; Plant Leaves - genetics; Plant Leaves - metabolism; Plant Leaves - virology; plant viruses; Plants, Genetically Modified; plasmid vectors; plasmids; proteomics; recombinant fusion proteins; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Red Fluorescent Protein; reporter genes; SYNV; Tobacco; transformation; transgenic plants; viral fusion proteins; virology; Viruses; Viruses - growth & development; High throughput screening; Chromatin; Recombination; Nicotiana benthamiana; SYNV; Cell fusion; Agrobacterium; Actin; Dicotyledones; Angiospermae; nucleolus; Bacteria; Transgenic plant; Solanaceae; Infected cell; Fusion protein; Vector; Hybrid gene; Gram negative bacteria; agroinfiltration; Gene expression; transformation; Plasmid; Virus; confocal microscopy; Green fluorescent protein; Proteomics; Spermatophyta; Cyan fluorescent protein; Rhizobiaceae; Experimental plant; Endoplasmic reticulum
• ISSN: 0894-0282; 1943-7706
• DOI: 10.1094/MPMI-20-7-0740

Plant functional proteomics research is increasingly dependent upon vectors that facilitate high-throughput gene cloning and expression of fusions to autofluorescent proteins. Here, we describe the pSITE family of plasmids, a new set of Agrobacterium binary vectors, suitable for the stable integration or transient expression of various autofluorescent protein fusions in plant cells. The pSITE vectors permit single-step Gateway-mediated recombination cloning for construction of binary vectors that can be used directly in transient expression studies or for the selection of transgenic plants on media containing kanamycin. These vectors can be used to express native proteins or fusions to monmeric red fluorescent protein or the enhanced green fluorescent protein and its cyan and yellow-shifted spectral variants. We have validated the vectors for use in transient expression assays and for the generation of transgenic plants. Additionally, we have generated markers for fluorescent highlighting of actin filaments, chromatin, endoplasmic reticulum, and nucleoli. Finally, we show that pSITE vectors can be used for targeted gene expression in virusinfected cells, which should facilitate high-throughput characterization of protein dynamics in host-virus interactions.