pSITE Vectors for Stable Integration or Transient Expression of Autofluorescent Protein Fusions in Plants: Probing Nicotiana benthamiana-Virus Interactions
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 integrati...
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| Published in | Molecular plant-microbe interactions Vol. 20; no. 7; pp. 740 - 750 |
|---|---|
| Main Authors | , , , , , , , |
| Format | Journal Article |
| Language | English |
| Published |
St Paul, MN
APS Press
01.07.2007
The American Phytopathological Society |
| Subjects | |
| Online Access | Get full text |
| ISSN | 0894-0282 1943-7706 |
| DOI | 10.1094/MPMI-20-7-0740 |
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| Abstract | 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. |
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| AbstractList | 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. 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.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. 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. |
| Author | Citovsky, V Chakrabarty, R Tzfira, T Goodin, M Hogenhout, S.A Banerjee, R Chung, S.M Farman, M |
| Author_xml | – sequence: 1 fullname: Chakrabarty, R – sequence: 2 fullname: Banerjee, R – sequence: 3 fullname: Chung, S.M – sequence: 4 fullname: Farman, M – sequence: 5 fullname: Citovsky, V – sequence: 6 fullname: Hogenhout, S.A – sequence: 7 fullname: Tzfira, T – sequence: 8 fullname: Goodin, M |
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| Keywords | 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 |
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| SubjectTerms | 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 |
| Title | pSITE Vectors for Stable Integration or Transient Expression of Autofluorescent Protein Fusions in Plants: Probing Nicotiana benthamiana-Virus Interactions |
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