Summary
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.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
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 69:1046–1055
Ambros PF, Matzke AJM, Matzke MA (1986) Localization of Agrobacterium rhizogenes T-DNA in plant chromosomes by in situ hybridization. EMBO J 5:2073–2077
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 204:512–518
Bakkeren G, Koukolíková-Nicola Z, Grimsley N, Hohn B (1989) Recovery of Agrobacterium tumefaciens T-DNA molecules from whole plants early after transfer. Cell 57:847–857
Buchanan-Wollaston V, Passiatore JE, Cannon F (1987) The mob and oriT mobilization functions of a bacterial plasmid promote its transfer to plants. Nature 328:172–175
Caplan AB, Van Montagu M, Schell J (1985) Genetic analysis of integration mediated by single T-DNA borders. J Bacteriol 161:655–664
Casadaban MJ, Cohen SN (1980) Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J Mol Biol 138:179–207
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 170:2659–2667
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 204:64–69
Citovsky V, De Vos G, Zambryski P (1988) Single-stranded DNA binding protein encoded by the virE locus of Agrobacterium tumefaciens. Science 240:501–504
Das A (1988) Agrobacterium tumefaciens virE operon encodes a single-stranded DNA-binding protein. Proc Natl Acad Sci USA 85:2909–2913
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
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 13:4777–4788
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 201:477–484
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 11:365–377
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 2:419–426
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 86:9154–9158
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 151–174
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 press
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 199:161–168
Heinemann JA, Sprague GF Jr (1989) Bacterial conjugative plasmids mobilize DNA transfer between bacteria and yeast. Nature 340:205–209
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 5:3–11
Herman LMF, Van Montagu M, Depicker AG (1986) Isolation of tobacco DNA segments with plant promoter activity. Mol Cell Biol 6:4486–4492
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 7:4055–4062
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 press
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 83:3895–3899
Joos H, Timmerman B, Van Montagu M, Schell J (1983) Genetic analysis of transfer and stabilization of Agrobacterium DNA in plant cells. EMBO J 2:2151–2160
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 86:8467–8471
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 144:353–376
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning, a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York
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 64:3056–3058
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
Simpson RB, Spielmann A, Margossian L, McKnight TD (1986) A disarmed binary vector from Agrobacterium tumefaciens functions in Agrobacterium rhizogenes. Plant Mol Biol 6:403–415
Spielmann A, Simpson RB (1986) T-DNA structure in transgenic tobacco plants with multiple independent integration sites. Mol Gen Genet 205:34–41
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
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 322:706–712
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
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 5:1755–1760
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
Wallroth M, Gerats AGM, Rogers SG, Fraley RT, Horsch RB (1986) Chromosomal localization of foreign genes in Petunia hybrida. Mol Gen Genet 202:6–15
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
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
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
Young C, Nester EW (1988) Association of the VirD2 protein with the 5′ end of T strands in Agrobacterium tumefaciens. J Bacteriol 170:3367–3374
Author information
Authors and Affiliations
Additional information
Communicated by J. Schell
Rights and permissions
About this article
Cite this article
Herman, L., Jacobs, A., Van Montagu, M. et al. Plant chromosome/marker gene fusion assay for study of normal and truncated T-DNA integration events. Molec. Gen. Genet. 224, 248–256 (1990). https://doi.org/10.1007/BF00271558
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00271558