Skip to main content
Log in

Sequence context of the T-DNA border repeat element determines its relative activity during T-DNA transfer to plant cells

  • Published:
Molecular and General Genetics MGG Aims and scope Submit manuscript

Summary

We present a detailed analysis of the function of the right and left T-DNA border regions of the nopaline Ti plasmid of Agrobacterium tumefaciens. An avirulent deletion of the right border of the nopaline Ti plasmid (pGV3852) was used as an acceptor for 14 different T-DNA border constructs. The functional activities of these constructs were assayed by their ability to restore virulence, i.e. transformation on inoculated plants. Tumorigenicities were measured in several independent experiments over a 2 year period and the statistical significance of their relative levels was evaluated. The data indicate: (i) the entire sequence of the 25 bp direct repeat of the T-DNA is required to provide an efficient substrate for mediating T-DNA transfer events; deletion derivatives of either the conserved or the vaiable domain of the repeat are defective in T-DNA transfer; (ii) while the 25 bp direct repeat alone can promote the T-DNA transfer, the flanking sequences of the repeats enhance (on the right) or attenuate (on the left) their activity; and (iii) tumorigenicity measurements vary depending on the plant assay system: potato discs are more sensitive than wounded tobacco leaves in detecting differences in T-DNA border activity.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Similar content being viewed by others

References

  • Albright LM, Yanofsky MF, Leroux B, Ma S, Nester EW (1987) Processing of the T-DNA of Agrobacterium tumefaciens generates border nicks and linear, single-stranded T-DNA. J Bacteriol 169:1046–1055

    Google Scholar 

  • Barker RF, Idler KB, Thompson DV, Kemp JD (1983) Nucleotide sequence of the T-DNA region from the Agrobacterium tumefaciens octopine Ti plasmid pTi15955. Plant Mol Biol 2:335–350

    Google Scholar 

  • Bolivar F, Rodriguez R, Greene P, Betlach M, Heynecker H, Boyer H, Crosa J, Falkow S (1977) Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene 2:95–113

    Google Scholar 

  • Casadaban MJ, Cohen SN (1980) Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J Mol Biol 138:179–207

    Google Scholar 

  • Chilton M-D, Currier TC, Farrand SK, Bendich AJ, Gordon MP, Nester EW (1974) Agrobacterium tumefaciens DNA and PS8 bacteriophage DNA not detected in crown gall tumors. Proc Natl Acad Sci USA 71:3672–3676

    Google Scholar 

  • Depicker A, De Wilde M, De Vos G, De Vos R, Van Montagu M, Schell J (1980) Molecular cloning of overlapping segments of the nopaline Ti-plasmid pTiC58 as a means to restriction endonuclease mapping. Plasmid 3:193–211

    Google Scholar 

  • De Vos G, De Beuckeleer M, Van Montagu M, Schell J (1981) Restriction endonuclease mapping of the octopine tumor inducing pTiAch5 of Agrobacterium tumefaciens. Plasmid 6:249–253

    Google Scholar 

  • Douglas CJ, Staneloni RJ, Rubin RA, Nester EW (1985) Identification and genetic analysis of an Agrobacterium tumefaciens chromosomal virulence region. J Bacteriol 161:850–860

    Google Scholar 

  • Gheysen G, Dhaese P, Van Montagu M, Schell J (1985) DNA flux across genetic barriers: the crown gall phenomenon. In: Hohn B, Dennis ES (eds) Genetic flux in plants. Advances in plant gene research, vol. 2. Springer, Wien, Heidelberg, Berlin, New York, pp 11–47

    Google Scholar 

  • Gielen J, De Beuceleer M, Seurinck J, Deboeck F, De Greve H, Lemmers M, Van Montagu M, Schell J (1984) The complete nucleotide sequence of the TL-DNA of the Agrobacterium tumefaciens plasmid pTiAch5. EMBO J 3:835–846

    Google Scholar 

  • Holsters M, Villarroel R, Gielen J, Seurinck J, De Greve H, De Greve H, Schell J (1983) An analysis of the boundaries of the octopine TL-DNA in tumors induced by Agrobacterium tumefaciens. Mol Gen Genet 190:35–41

    Google Scholar 

  • Jen GC, Chilton M-D (1986) The right border region of pTiT37 T-DNA is intrinsically more active than the left border region in promoting T-DNA transformation. Proc Natl Acad Sci USA 83:3895–3899

    Google Scholar 

  • Joos H, Inzé D, Caplan A, Sormann M, Van Montagu M, Schell J (1983) Genetic analysis of T-DNA transcripts in nopaline crown galls. Cell 32:1057–1067

    Google Scholar 

  • Leong SA, Ditta GS, Helinski DR (1982) Heme biosynthesis in Rhizobium: identification of a cloned gene coding for δ-aminolevulinic acid synthetase from Rhizobium meliloti. J Biol Chem 257:8724–8730

    Google Scholar 

  • Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning, a laboratory manual. Cold Spring Harbor Laboratory, New York

    Google Scholar 

  • Maxam A, Gilbert W (1977) A new method for sequencing DNA. Proc Natl Acad Sci USA 74:560–564

    Google Scholar 

  • Nagy JI, Maliga P (1976) Callus induction and plant regeneration from mesophyll protoplasts of Nicotiana sylvestris. Z Pflanzenphysiol 78:453–455

    Google Scholar 

  • Nomura N, Yamagishi H, Oka A (1978) Isolation and characterization of transducing coliphage fd carrying a kanamycin resistance gene. Gene 3:39–51

    Google Scholar 

  • Peralta EG, Ream LW (1985) T-DNA border sequences required for crown gall tumorigenesis. Proc Natl Acad Sci USA 82:5112–5116

    Google Scholar 

  • Peralta EG, Hellmiss R, Ream W (1986) Overdrive, a T-DNA transmission enhancer on the A. tumefaciens tumour-inducing plasmid. EMBO J 5:1137–1142

    Google Scholar 

  • Rao RN, Rogers SG (1979) Plasmid pKC7: a vector containing ten restriction endonuclease sites suitable for cloning DNA fragments. Gene 7:79–82

    Google Scholar 

  • Rubin RA (1986) Genetic studies on the role of octopine T-DNA border regions in crown gall tumor formation. Mol Gen Genet 202:312–320

    Google Scholar 

  • Simpson RB, O'Hara PJ, Kwok W, Montoya AL, Lichtenstein C, Gordon MP, Nester EW (1982) DNA from the A6S/2 crown gall tumor contains scrambled Ti-plasmid sequences near its junctions with the plant DNA. Cell 29:1005–1014

    Google Scholar 

  • Slightom JL, Jouanin L, Leach F, Drong RF, Tepfer D (1985) Isolation and identification of TL-DNA/plant junctions in Convolvulus arvensis transformed by Agrobacterium rhizogenes strain A4. EMBO J 4:3069–3077

    Google Scholar 

  • Stachel SE, Nester EW (1986) The genetic and transcriptional organization of the vir region of the A6 Ti plasmid of Agrobacterium tumefaciens. EMBO J 5:1445–1454

    Google Scholar 

  • Stachel SE, Zambryski P (1986) Agrobacterium tumefaciens and the susceptible plant cell: a novel adaptation of extracellular recognition and DNA conjugation. Cell 47:155–157

    Google Scholar 

  • Stachel SE, Messens E, Van Montagu M, Zambryski P (1985) Identification of the signal molecules produced by wounded plant cells that activate T-DNA transfer in Agrobacterium tumefaciens. Nature 318:624–629

    Google Scholar 

  • Stachel SE, Timmerman B, Zambryski P (1986) Generation of single-stranded T-DNA molecules during the initial stages of T-DNA transfer from Agrobacterium tumefaciens to plant cells. Nature 322:706–712

    Google Scholar 

  • Stachel SE, Timmerman B, Zambryski P (1987) Activation of Agrobacterium tumefaciens vir gene expression generates multiple single-stranded T-strand molecules from the pTiA6 T-region: requirements for 5′ virD gene products. EMBO J 6:857–863

    Google Scholar 

  • Van Lijsebettens M, Inzé D, Van Montagu M, Schell J (1986) Transformed cell clones as a tool to study T-DNA integration mediated by Agrobacterium tumefaciens. J Mol Biol 188:129–145

    Google Scholar 

  • Vieira J, Messing J (1982) The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19:259–268

    Google Scholar 

  • Wang K, Herrera-Estrella L, Van Montagu M, Zambryski P (1984) Right 25-bp terminus sequences of the nopaline T-DNA is essential for and determines direction of DNA transfer from Agrobacterium to the plant genome. Cell 38:455–462

    Google Scholar 

  • Wang K, Stachel S, Timmerman B, Van Montagu M, Zambryski P (1987) Site-specific nick in the T-DNA border sequence following vir gene expression in Agrobacterium. Science 235:587–591

    Google Scholar 

  • Yadav NS, Vanderleyden J, Bennett DR, Barnes WM, Chilton M-D (1982) Short direct repeats flank the T-DNA on a nopaline Ti plasmid. Proc Natl Acad Sci USA 79:6322–6326

    Google Scholar 

  • Yanofsky MF, Porter SG, Young C, Albright LM, Gordon MP, Nester EW (1986) The virD operon of Agrobacterium tumefaciens encodes a site-specific endonuclease. Cell 47:471–477

    Google Scholar 

  • Zambryski P, Depicker A, Kruger K, Goodman H (1982) Tumor induction by Agrobacterium tumefaciens: analysis of the boundaries of T-DNA. J Mol Appl Genet 1:361–370

    Google Scholar 

  • Zambryski P, Joos H, Genetello C, Leemans J, Van Montagu M, Schell J (1983) Ti plasmid vector for the introduction of DNA into plant cells without alteration of their normal regeneration capacity. EMBO J 2:2143–2150

    Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Additional information

Communicated by J. Schell

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wang, K., Genetello, C., Van Montagu, M. et al. Sequence context of the T-DNA border repeat element determines its relative activity during T-DNA transfer to plant cells. Molec Gen Genet 210, 338–346 (1987). https://doi.org/10.1007/BF00325703

Download citation

  • Received:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00325703

Key words

Navigation