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PMID: 15821977 Published · ppublish English Comparative Study Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S. Research Support, U.S. Gov't, P.H.S.

pSAT vectors: a modular series of plasmids for autofluorescent protein tagging and expression of multiple genes in plants.

Plant molecular biology ·Vol. 57 ·No. 4 ·2005-03-00 ·Pages 503-16

Tzfira T, Tian GW, Lacroix B, Vyas S, Li J, Leitner-Dagan Y, Krichevsky A, Taylor T, Vainstein A, Citovsky V

Abstract

Autofluorescent protein tags represent one of the major and, perhaps, most powerful tools in modern cell biology for visualization of various cellular processes in vivo. In addition, advances in confocal microscopy and the development of autofluorescent proteins with different excitation and emission spectra allowed their simultaneous use for detection of multiple events in the same cell. Nevertheless, while autofluorescent tags are widely used in plant research, the need for a versatile and comprehensive set of vectors specifically designed for fluorescent tagging and transient and stable expression of multiple proteins in plant cells from a single plasmid has not been met by either the industrial or the academic communities. Here, we describe a new modular satellite (SAT) vector system that supports N- and C-terminal fusions to five different autofluorescent tags, EGFP, EYFP, Citrine-YFP, ECFP, and DsRed2. These vectors carry an expanded multiple cloning site that allows easy exchange of the target genes between different autofluorescence tags, and expression of the tagged proteins is controlled by constitutive promoters, which can be easily replaced with virtually any other promoter of interest. In addition, a series of SAT vectors has been adapted for high throughput Gateway recombination cloning. Furthermore, individual expression cassettes can be assembled into Agrobacterium binary plasmids, allowing efficient transient and stable expression of multiple autofluorescently tagged proteins from a single vector following its biolistic delivery or Agrobacterium-mediated genetic transformation.

MeSH Terms
Base Sequence Cloning, Molecular/methods Gene Expression Genetic Vectors/chemistry,genetics Green Fluorescent Proteins/genetics,metabolism Luminescent Proteins/genetics,metabolism Microscopy, Confocal Molecular Sequence Data Mutation Plant Proteins/genetics,metabolism Plants, Genetically Modified Plasmids/chemistry,genetics Recombinant Fusion Proteins/genetics,metabolism Rhizobium/genetics Sequence Analysis, DNA Sequence Homology, Nucleic Acid
Chemicals
Luminescent Proteins Plant Proteins Recombinant Fusion Proteins Green Fluorescent Proteins
Authors & Affiliations
10 authors, click to expand affiliations / ORCID
Tzfira Tzvi
Department of Biochemistry and Cell Biology, State University of New York, Stony Brook, NY 11794-5215, USA. [email protected]
Tian Guo-Wei
Lacroix Benoît
Vyas Shachi
Li Jianxiong
Leitner-Dagan Yael
Krichevsky Alexander
Taylor Tamir
Vainstein Alexander
Citovsky Vitaly
References (57)
57 references, click to expand
  1. Imaging protein-protein interactions in living cells.
    Plant Mol Biol. 2002 Dec;50(6):871-83 PMID: 12516859
  2. Reducing the environmental sensitivity of yellow fluorescent protein. Mechanism and applications.
    J Biol Chem. 2001 Aug 3;276(31):29188-94 PMID: 11387331
  3. Biotechnological applications of green fluorescent protein.
    Appl Microbiol Biotechnol. 2003 Sep;62(4):303-15 PMID: 12768245
  4. Fluorescence resonance energy transfer microscopy of localized protein interactions in the living cell nucleus.
    Methods. 2001 Sep;25(1):4-18 PMID: 11558993
  5. GATEWAY recombinational cloning: application to the cloning of large numbers of open reading frames or ORFeomes.
    Methods Enzymol. 2000;328:575-92 PMID: 11075367
  6. GFP illuminates the cytoskeleton.
    Trends Cell Biol. 1998 Feb;8(2):72-7 PMID: 9695812
  7. Visualization of Rab5 activity in living cells by FRET microscopy and influence of plasma-membrane-targeted Rab5 on clathrin-dependent endocytosis.
    J Cell Sci. 2003 Dec 1;116(Pt 23):4799-810 PMID: 14600265
  8. GFP is the way to glow: bioimaging of the plant endomembrane system.
    J Microsc. 2004 May;214(Pt 2):138-58 PMID: 15102062
  9. Seeing the machinery of live cells.
    Science. 1998 Jun 19;280(5371):1954-5 PMID: 9669950
  10. GFP imaging: methodology and application to investigate cellular compartmentation in plants.
    J Exp Bot. 2001 Apr;52(356):529-39 PMID: 11373302
  11. Increasing plant susceptibility to Agrobacterium infection by overexpression of the Arabidopsis nuclear protein VIP1.
    Proc Natl Acad Sci U S A. 2002 Aug 6;99(16):10435-40 PMID: 12124400
  12. The VirE3 protein of Agrobacterium mimics a host cell function required for plant genetic transformation.
    EMBO J. 2005 Jan 26;24(2):428-37 PMID: 15616576
  13. Green fluorescent protein as a marker for gene expression.
    Science. 1994 Feb 11;263(5148):802-5 PMID: 8303295
  14. Carotenoid sequestration in plants: the role of carotenoid-associated proteins.
    Trends Plant Sci. 1999 Jun;4(6):232-235 PMID: 10366880
  15. Confocal optics microscopy for biochemical and cellular high-throughput screening.
    Drug Discov Today. 2003 Dec 1;8(23):1085-93 PMID: 14693468
  16. High-throughput viral expression of cDNA-green fluorescent protein fusions reveals novel subcellular addresses and identifies unique proteins that interact with plasmodesmata.
    Plant Cell. 2003 Jul;15(7):1507-23 PMID: 12837943
  17. Autofluorescent proteins in single-molecule research: applications to live cell imaging microscopy.
    Biophys J. 2001 May;80(5):2396-408 PMID: 11325739
  18. Quantitative motion analysis and visualization of cellular structures.
    Methods. 2003 Jan;29(1):3-13 PMID: 12543067
  19. Biochemical, immunological, and immunocytochemical evidence for the association of chalcone synthase with endoplasmic reticulum membranes.
    Proc Natl Acad Sci U S A. 1987 Dec;84(24):8966-70 PMID: 3321063
  20. Molecular cloning of a carotenoid-associated protein from Cucumis sativus corollas: homologous genes involved in carotenoid sequestration in chromoplasts.
    Plant J. 1996 Dec;10(6):1111-8 PMID: 9011091
  21. The green fluorescent protein.
    Annu Rev Biochem. 1998;67:509-44 PMID: 9759496
  22. Analysis of flavanone 3-hydroxylase in Arabidopsis seedlings. Coordinate regulation with chalcone synthase and chalcone isomerase.
    Plant Physiol. 1996 May;111(1):339-45 PMID: 8685272
  23. Targeting of rough endoplasmic reticulum membrane proteins and ribosomes in invertebrate neurons.
    Mol Biol Cell. 2002 May;13(5):1778-91 PMID: 12006669
  24. Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation.
    Plant J. 2004 Nov;40(3):428-38 PMID: 15469500
  25. Fluorescent proteins from nonbioluminescent Anthozoa species.
    Nat Biotechnol. 1999 Oct;17(10):969-73 PMID: 10504696
  26. Spectral imaging and its applications in live cell microscopy.
    FEBS Lett. 2003 Jul 3;546(1):87-92 PMID: 12829241
  27. GATEWAY vectors for Agrobacterium-mediated plant transformation.
    Trends Plant Sci. 2002 May;7(5):193-5 PMID: 11992820
  28. Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer.
    Curr Biol. 1996 Feb 1;6(2):178-82 PMID: 8673464
  29. In vivo dynamics and differential microtubule-binding activities of MAP65 proteins.
    Plant Physiol. 2004 Dec;136(4):3956-67 PMID: 15557096
  30. A gateway cloning vector set for high-throughput functional analysis of genes in planta.
    Plant Physiol. 2003 Oct;133(2):462-9 PMID: 14555774
  31. Improved green fluorescence.
    Nature. 1995 Feb 23;373(6516):663-4 PMID: 7854443
  32. Real-time single cell analysis of Smac/DIABLO release during apoptosis.
    J Cell Biol. 2003 Sep 15;162(6):1031-43 PMID: 12975347
  33. Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral.
    Proc Natl Acad Sci U S A. 2000 Oct 24;97(22):11984-9 PMID: 11050229
  34. Interactions and nuclear import of the N and P proteins of sonchus yellow net virus, a plant nucleorhabdovirus.
    J Virol. 2001 Oct;75(19):9393-406 PMID: 11533202
  35. Dual-color fluorescence imaging distinguishes tumor cells from induced host angiogenic vessels and stromal cells.
    Proc Natl Acad Sci U S A. 2003 Nov 25;100(24):14259-62 PMID: 14614130
  36. Nuclear transport of plant potyviral proteins.
    Plant Cell. 1990 Oct;2(10):987-98 PMID: 2136629
  37. A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications.
    Nat Biotechnol. 2002 Jan;20(1):87-90 PMID: 11753368
  38. Fluorescence imaging of physiological activity in complex systems using GFP-based probes.
    Curr Opin Neurobiol. 2003 Oct;13(5):591-6 PMID: 14630223
  39. The plant Spc98p homologue colocalizes with gamma-tubulin at microtubule nucleation sites and is required for microtubule nucleation.
    J Cell Sci. 2002 Jun 1;115(Pt 11):2423-31 PMID: 12006626
  40. A set of modular plant transformation vectors allowing flexible insertion of up to six expression units.
    Plant Mol Biol. 2002 Sep;50(1):17-27 PMID: 12139006
  41. A glucocorticoid-mediated transcriptional induction system in transgenic plants.
    Plant J. 1997 Mar;11(3):605-12 PMID: 9107046
  42. pGD vectors: versatile tools for the expression of green and red fluorescent protein fusions in agroinfiltrated plant leaves.
    Plant J. 2002 Aug;31(3):375-83 PMID: 12164816
  43. A simple and general method for transferring genes into plants.
    Science. 1985 Mar 8;227(4691):1229-31 PMID: 17757866
  44. Three-color imaging using fluorescent proteins in living zebrafish embryos.
    Biotechniques. 2001 Jul;31(1):66-70, 72 PMID: 11464522
  45. The utility of green fluorescent protein in transgenic plants.
    Plant Cell Rep. 2001 Jul;20(5):376-82 PMID: 12448420
  46. Using GFP in FRET-based applications.
    Trends Cell Biol. 1999 Feb;9(2):57-60 PMID: 10087619
  47. Localization of flavonoid enzymes in Arabidopsis roots.
    Plant J. 2001 Jul;27(1):37-48 PMID: 11489181
  48. The power and prospects of fluorescence microscopies and spectroscopies.
    Annu Rev Biophys Biomol Struct. 2003;32:161-82 PMID: 12598370
  49. DsRed as a potential FRET partner with CFP and GFP.
    Biophys J. 2003 Jul;85(1):599-611 PMID: 12829514
  50. VIP1, an Arabidopsis protein that interacts with Agrobacterium VirE2, is involved in VirE2 nuclear import and Agrobacterium infectivity.
    EMBO J. 2001 Jul 2;20(13):3596-607 PMID: 11432846
  51. A monomeric red fluorescent protein.
    Proc Natl Acad Sci U S A. 2002 Jun 11;99(12):7877-82 PMID: 12060735
  52. Detection of protein-protein interactions in plants using bimolecular fluorescence complementation.
    Plant J. 2004 Nov;40(3):419-27 PMID: 15469499
  53. FRET-based in vivo screening for protein folding and increased protein stability.
    J Mol Biol. 2003 Mar 14;327(1):239-49 PMID: 12614622
  54. Evaluation of spectral imaging for plant cell analysis.
    J Microsc. 2004 May;214(Pt 2):174-81 PMID: 15102064
  55. Involvement of targeted proteolysis in plant genetic transformation by Agrobacterium.
    Nature. 2004 Sep 2;431(7004):87-92 PMID: 15343337
  56. High-throughput fluorescent tagging of full-length Arabidopsis gene products in planta.
    Plant Physiol. 2004 May;135(1):25-38 PMID: 15141064
  57. Optimized codon usage and chromophore mutations provide enhanced sensitivity with the green fluorescent protein.
    Nucleic Acids Res. 1996 Nov 15;24(22):4592-3 PMID: 8948654
Article Info
Journal
Plant molecular biology
Abbr.
Plant Mol Biol
ISSN
0167-4412
DOI
10.1007/s11103-005-0340-5
Email
[email protected]
Published
2005-03-00
Pages
503-16
Language
English
Region
Netherlands
NLM ID
9106343
Subset
IM
Databases
GENBANK
AY818363, AY818364, AY818365, AY818366, AY818367, AY818368, AY818369, AY818370, AY818371, AY818372, AY818373, AY818374, AY818375, AY818376, AY818377, AY818378, AY818379, AY818380, AY818381, AY818382, AY818383, AY818384, AY819771
External Links
PubMed source DOI: 10.1007/s11103-005-0340-5 View journal details
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