Plant Molecular Biology

, Volume 31, Issue 3, pp 677–681 | Cite as

Deviating T-DNA transfer fromAgrobacterium tumefaciens to plants

  • Eric van der Graaff
  • Amke den Dulk-Ras
  • Paul J. J. Hooykaas
Short Communication


We analyzed 29 T-DNA inserts in transgenicArabidopsis thaliana plants for the junction of the right border sequences and the flanking plant DNA. DNA sequencing showed that in most lines the right border sequences transferred had been preserved during integration, corroborating literature data. Surprisingly, in four independent transgenic lines a complete right border repeat was present followed by binary vector sequences. Cloning of two of these T-DNA inserts by plasmid rescue showed that in these lines the transferred DNA consisted of the complete binary vector sequences in addition to the T-region. On the basis of the structure of the transferred DNA we propose that in these lines T-DNA transfer started at the left-border repeat, continued through the vector part, passed the right border repeat, and ended only after reaching again this left-border repeat.

Key words

Agrobacterium tumefaciens Arabidopsis thaliana T-DNA transfer transformation 


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  1. 1.
    Bundock P, den Dulk-Ras A, Beijersbergen A, Hooykaas PJJ: Trans-kingdom T-DNA transfer fromAgrobacterium tumefaciens toSaccharomyces cerevisiae. EMBO J 14: 3206–3214 (1995).PubMedGoogle Scholar
  2. 2.
    Dürrenberger F, Crameri A, Hohn B, Koukolíková-Nicola Z: Covalently bound VirD2 protein ofAgrobacterium tumefaciens protects the T-DNA from exonucleolytic degradation. Proc Natl Acad Sci USA 86: 9154–9158 (1989).PubMedGoogle Scholar
  3. 3.
    Frisch DA, Harris-Haller LW, Yokubaitis NT, Lomas TL, Hardin SH, Hall TC: Complete sequence of the binary vector Bin 19. Plant Mol Biol 27: 405–409 (1995).PubMedGoogle Scholar
  4. 4.
    Hooykaas PJJ, Beijersbergen AGM: The virulence system ofAgrobacterium tumefaciens. Annu Rev Phytopath 32: 157–179 (1994).Google Scholar
  5. 5.
    Howard EA, Zupan JR, Citovsky V, Zambryski PC: The VirD2 protein ofAgrobacterium tumefaciens contains a C-terminal bipartite nuclear localization signal: implications for nuclear uptake of DNA in plant cells. Cell 68: 109–118 (1992).CrossRefPubMedGoogle Scholar
  6. 6.
    Martineau B, Voelker TA, Sanders RA: On defining T-DNA. Plant Cell 6: 1032–1033 (1994).CrossRefPubMedGoogle Scholar
  7. 7.
    Matsumoto S, Ito Y, Hosoi T, Takahashi Y, Machida Y: Integration ofAgrobacterium T-DNA into a tobacco chromosome: possible involvement of DNA homology between T-DNA and plant DNA. Mol Gen Genet 224: 309–316 (1990).CrossRefPubMedGoogle Scholar
  8. 8.
    Mayerhofer R, Koncz-Kalman Z, Nawrath C, Bakkeren G, Crameri A, Angelis K, Redei GP, Schell J, Hohn B, Koncz C: T-DNA integration: a mode of illegitimate recombination in plants. EMBO J 10: 697–704 (1991).PubMedGoogle Scholar
  9. 9.
    Pansegrau W, Lanka E: Common sequence motifs in DNA relaxases and nick regions from a variety of DNA transfer systems. Nucl Acids Res 19: 3455 (1991).PubMedGoogle Scholar
  10. 10.
    Peralta EG, Hellmiss R, Ream W: Overdrive, a T-DNA transmission enhancer on theAgrobacterium tumefaciens tumour-inducing plasmid. EMBO J 5: 1137–1142 (1986).Google Scholar
  11. 11.
    Ramanathan V, Veluthambi K: Transfer of non T-DNA portions of theAgrobacterium tumefaciens Ti plasmid pTiA6 from the left terminus of TL-DNA. Plant Mol Biol 28: 1149–1154 (1995).PubMedGoogle Scholar
  12. 12.
    Rommens CMT, Rudenko GN, Dijkwel PP, Van Haaren MJJ, Ouwerkerk PBF, Blok KM, Nijkamp HJJ, Hille J: Characterization of theAc/Ds behaviour in transgenic tomato plants using plasmid rescue. Plant Mol Biol 20: 61–70 (1992).PubMedGoogle Scholar
  13. 13.
    Stachel SE, Timmerman B, Zambryski P: Activation ofAgrobacterium tumefaciens vir gene expression generates multiple single-stranded T-strand molecules from the pTiA6 T-region: requirement for 5′virD gene products. EMBO J 6: 857–863 (1987).PubMedGoogle Scholar
  14. 14.
    Tinland B, Koukolíková-Nicola Z, Hall MN, Hohn B: The T-DNA linked VirD2 protein contains two distinct functional nuclear localization signals. Proc Natl Acad Sci USA 89: 7442–7446 (1992).PubMedGoogle Scholar
  15. 15.
    Tinland B, Hohn B: Recombination between prokaryotic and eukaryotic DNA: integration ofAgrobacterium tumefaciens T-DNA into the plant genome. In: Setlow JK (ed) Genetic Engineering vol 17, pp. 209–229 Plenum Press, New York (1995).Google Scholar
  16. 16.
    Tinland B, Schoumacher F, Gloeckler V, Bravo-Angel AM, Hohn B: TheAgrobacterium tumefaciens virulence D2 protein is responsible for precise integration of T-DNA into the plant genome. EMBO J 14: 3585–3595 (1995).PubMedGoogle Scholar
  17. 17.
    van der Graaff E, Hooykaas PJJ: Improvements in the transformation ofArabidopsis thaliana C24 leaf discs byAgrobacterium tumefaciens. Plant Cell Rep, in press (1996).Google Scholar
  18. 18.
    van Haaren MJJ, Sedee NJA, Schilperoort RA, Hooykaas PJJ: Overdrive is a T-region transfer enhancer which stimulates T-strand production inAgrobacterium tumefaciens. Nucl Acids Res 15: 8983–8997 (1987).PubMedGoogle Scholar
  19. 19.
    Ward ER, Barnes WM: VirD2 protein ofAgrobacterium tumefaciens very tightly linked to the 5′ end of T-strand DNA. Science 242: 927–930 (1988).Google Scholar
  20. 20.
    Zambryski PC: Chronicles from theAgrobacterium plant cell DNA transfer story. Annu Rev Plant Physiol Plant Mol Biol 43: 465–490 (1992).CrossRefGoogle Scholar

Copyright information

© Kluwer Academic Publishers 1996

Authors and Affiliations

  • Eric van der Graaff
    • 1
  • Amke den Dulk-Ras
    • 1
  • Paul J. J. Hooykaas
    • 1
  1. 1.Institute of Molecular Plant SciencesLeiden University, Clusius LaboratoryLeidenNetherlands

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