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

July 2007 , Volume 20 , Number  7
Pages  740 - 750

Romit Chakrabarty , 1 Rituparna Banerjee , 1 Sang-Min Chung , 2 Mark Farman , 1 Vitaly Citovsky , 3 Saskia A. Hogenhout , 4 Tzvi Tzfira , 5 and Michael Goodin 1

1Department of Plant Pathology, University of Kentucky, Lexington 40546, U.S.A.; 2Department of Life Science, Dongguk University, South Korea; 3Department of Biochemistry and Cell Biology, State University of New York, Stony Brook 11794-5215, U.S.A.; 4Department of Entomology, The Ohio State University-OARDC, Wooster 44691, U.S.A.; 5Department of Molecular, Cellular, & Developmental Biology, The University of Michigan, Ann Arbor 48109-1048, U.S.A.

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Accepted 14 February 2007.

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 virus-infected cells, which should facilitate high-throughput characterization of protein dynamics in host-virus interactions.

Additional keywords: agroinfiltration , confocal microscopy , nucleolus , SYNV , transformation .

© 2007 The American Phytopathological Society