Molecular and General Genetics MGG

, Volume 224, Issue 2, pp 248–256 | Cite as

Plant chromosome/marker gene fusion assay for study of normal and truncated T-DNA integration events

  • L. Herman
  • A. Jacobs
  • M. Van Montagu
  • A. Depicker


During Agrobacterium tumefaciens infection, the T-DNA flanked by 24 by imperfect direct repeats is transferred and stably integrated into the plant chromosome at random positions. Here we measured the frequency with which a promoterless reporter gene is activated after insertion into the Nicotiana tabacum SR1 genome. When adjacent to the right or left T-DNA border sequences, at least 35% of the transformants express the marker gene, suggesting preferential T-DNA insertion (>70%) in transcriptionally active regions of the plant genome. When the promoterless neomycin phosphotransferase II (nptII) gene is located internally in the T-DNA, the activation frequency drops to 1% since gene activation requires T-DNA truncation. These truncation events in the nptII upstream region occur independently of the nature of the upstream sequence and of the T-DNA length. Deletion of the right border region prevents the detection of activated marker genes. Therefore, T-DNA truncation probably occurs after synthesis of a normal T-DNA intermediate during the transfer and/or integration process. In the absence of border regions, expression of the nptII selectable marker directed by the nopaline synthase promoter was detected in 1 out of 105 regenerated calli, suggesting the possibility that any DNA sequence from the Ti plasmid can be transformed into the plant genome, albeit at a low frequency.

Key words

Agrobacterium tumefaciens T-DNA borders Intergeneric gene transfer pseudoborder Insertional activation Aberrant T-DNAs 


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  1. 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 69:1046–1055Google Scholar
  2. Ambros PF, Matzke AJM, Matzke MA (1986) Localization of Agrobacterium rhizogenes T-DNA in plant chromosomes by in situ hybridization. EMBO J 5:2073–2077Google Scholar
  3. André D, Colau D, Schell J, Van Montagu M, Hernalsteens J-P (1986) Gene tagging in plants by a T-DNA insertion mutagen that generates APH(3′) II-plant gene fusions. Mol Gen Genet 204:512–518Google Scholar
  4. Bakkeren G, Koukolíková-Nicola Z, Grimsley N, Hohn B (1989) Recovery of Agrobacterium tumefaciens T-DNA molecules from whole plants early after transfer. Cell 57:847–857Google Scholar
  5. Buchanan-Wollaston V, Passiatore JE, Cannon F (1987) The mob and oriT mobilization functions of a bacterial plasmid promote its transfer to plants. Nature 328:172–175Google Scholar
  6. Caplan AB, Van Montagu M, Schell J (1985) Genetic analysis of integration mediated by single T-DNA borders. J Bacteriol 161:655–664Google Scholar
  7. Casadaban MJ, Cohen SN (1980) Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J Mol Biol 138:179–207Google Scholar
  8. Christie PJ, Ward JE, Winans SC, Nester EW (1988) The Agrobacterium tumefaciens virE2 gene product is a single-stranded-DNA-binding protein that associates with T-DNA. J Bacteriol 170:2659–2667Google Scholar
  9. Chyi YS, Jorgensen RA, Goldstein D, Tanksley SD, Loaiza-Figueroa F (1986) Locations and stability of Agrobacterium-mediated T-DNA insertions in the Lycopersicon genome. Mol Gen Genet 204:64–69Google Scholar
  10. Citovsky V, De Vos G, Zambryski P (1988) Single-stranded DNA binding protein encoded by the virE locus of Agrobacterium tumefaciens. Science 240:501–504Google Scholar
  11. Das A (1988) Agrobacterium tumefaciens virE operon encodes a single-stranded DNA-binding protein. Proc Natl Acad Sci USA 85:2909–2913Google Scholar
  12. 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 6:249–253Google Scholar
  13. Deblaere R, Bytebier B, De Greve H, Deboeck F, Schell J, Van Montagu M, Leemans J (1985) Efficient octopine Ti plasmid-derived vectors for Agrobacterium-mediated gene transfer to plants. Nucleic Acids Res 13:4777–4788Google Scholar
  14. Depicker A, Herman L, Jacobs A, Schell J, Van Montagu M (1985) Frequencies of simultaneous transformation with different TDNAs and their relevance to the Agrobacterium/plant cell interaction. Mol Gen Genet 201:477–484Google Scholar
  15. Deroles SC, Gardner RC (1988) Analysis of the T-DNA structure in a large number of transgenic petunias generated by Agrobacterium-mediated transformation. Plant Mol Biol 11:365–377Google Scholar
  16. Dhaese P, De Greve H, Gielen J, Seurinck J, Van Montagu M, Schell J (1983) Identification of sequences involved in the polyadenylation of higher plant nuclear transcripts using Agrobacterium T-DNA genes as models. EMBO J 2:419–426Google Scholar
  17. Dürrenberger F, Crameri A, Hohn B, Koukolíková-Nicola Z (1989) Covalently bound VirD2 protein of Arabidopsis thaliana protects the T-DNA from exonucleolytic degradation. Proc Natl Acad Sci USA 86:9154–9158Google Scholar
  18. Gheysen G, Herman L, Breyne P, Van Montagu M, Depicker A (1989) Agrobacterium tumefaciens as a tool for the genetic transformation of plants. In: Butler LO, Harwood C, Moseley BEB (eds) Genetic transformation and expression. Intercept, Andover, pp 151–174Google Scholar
  19. Gheysen G, Herman L, Breyne P, Gielen J, Van Montagu M, Depicker A (1990) Cloning and sequence analysis of truncated T-DNA inserts from Nicotiana tabacum. Gene, in pressGoogle Scholar
  20. Hain R, Stahel P, Czernilofsky AP, Steinbiss H-H, Herrera-Estrella L, Schell J (1985) Uptake, integration, expression and genetic transmission of a selectable chimaeric gene to plant protoplasts. Mol Gen Genet 199:161–168Google Scholar
  21. Heinemann JA, Sprague GF Jr (1989) Bacterial conjugative plasmids mobilize DNA transfer between bacteria and yeast. Nature 340:205–209Google Scholar
  22. Hepburn AG, White J (1985) The effect of the right terminal repeat deletion on the oncogenicity of the T-region of pTiT37. Plant Mol Biol 5:3–11Google Scholar
  23. Herman LMF, Van Montagu M, Depicker AG (1986) Isolation of tobacco DNA segments with plant promoter activity. Mol Cell Biol 6:4486–4492Google Scholar
  24. Herrera-Estrella A, Chen Z-M, Van Montagu M, Wang K (1988) VirD proteins of Agrobacterium tumefaciens are required for the formation of a covalent DNA-protein complex at the 5′ terminus of T-strand molecules. EMBO J 7:4055–4062Google Scholar
  25. Herrera-Estrella A, Van Montagu M, Wang K (1990) A bacterial peptide acting as a plant nuclear-targeting signal: the Agrobacterium VirD2 directs β-galactosidase into tobacco nuclei. Proc Natl Acad Sci USA, 87: in pressGoogle Scholar
  26. 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 Nall Acad Sci USA 83:3895–3899Google Scholar
  27. Joos H, Timmerman B, Van Montagu M, Schell J (1983) Genetic analysis of transfer and stabilization of Agrobacterium DNA in plant cells. EMBO J 2:2151–2160Google Scholar
  28. Koncz C, Martini N, Mayerhofer R, Koncz-Kalman Z, Körber H, Redei GP, Schell J (1989) High-frequency T-DNA-mediated gene tagging in plants. Proc Natl Acad Sci USA 86:8467–8471Google Scholar
  29. Lemmers M, De Beuckeleer M, Holsters M, Zambryski P, Depicker A, Hernalsteens J-P, Van Montagu M, Schell J (1980) Internal organization, boundaries and integration of Ti-plasmid DNA in nopaline crown gall tumours. J Mol Biol 144:353–376Google Scholar
  30. Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning, a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New YorkGoogle Scholar
  31. Mooslehner K, Karls U, Harbers K (1990) Retroviral integration sites in transgenic Mov mice frequently map in the vicinity of transcribed DNA regions. J Virol 64:3056–3058Google Scholar
  32. Peralta EG, Hellmiss R, Ream W (1986) Overdrive, a T-DNA transmission enhancer on the A. tumefaciens tumour-inducing plasmid. EMBO J 5:1137–1142Google Scholar
  33. Simpson RB, Spielmann A, Margossian L, McKnight TD (1986) A disarmed binary vector from Agrobacterium tumefaciens functions in Agrobacterium rhizogenes. Plant Mol Biol 6:403–415Google Scholar
  34. Spielmann A, Simpson RB (1986) T-DNA structure in transgenic tobacco plants with multiple independent integration sites. Mol Gen Genet 205:34–41Google Scholar
  35. 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 318:624–629Google Scholar
  36. Stachel SE, Timmerman B, Zambryski P (1986) Generation of single-stranded T-DNA molecules during the initial stages of TDNA transfer from Agrobacterium tumefaciens to plant cells. Nature 322:706–712Google Scholar
  37. 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 6:857–863Google Scholar
  38. Teeri T, Herrera-Estrella L, Depicker A, Van Montagu M, Palva ET (1986) Identification of plant promoters in situ by T-DNA-mediated transcriptional fusions to the npt-II gene. EMBO J 5:1755–1760Google Scholar
  39. 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 188:129–145Google Scholar
  40. Wallroth M, Gerats AGM, Rogers SG, Fraley RT, Horsch RB (1986) Chromosomal localization of foreign genes in Petunia hybrida. Mol Gen Genet 202:6–15Google Scholar
  41. 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 38:455–462Google Scholar
  42. 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 235:587–591Google Scholar
  43. 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 47:471–477Google Scholar
  44. Young C, Nester EW (1988) Association of the VirD2 protein with the 5′ end of T strands in Agrobacterium tumefaciens. J Bacteriol 170:3367–3374Google Scholar

Copyright information

© Springer-Verlag 1990

Authors and Affiliations

  • L. Herman
    • 1
  • A. Jacobs
    • 1
  • M. Van Montagu
    • 1
  • A. Depicker
    • 1
  1. 1.Laboratorium voor GeneticaGentBelgium

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