Agrobacterium Tumefaciens-Mediated Transformation: Patterns of T-Dna Integration Into the Host Genome

  • Pieter Windels
  • Sylvie De Buck
  • Ann Depicker

The Agrobacterium tumefaciens-mediated transformation system is widely used to introduce genes into plants and is based on the conjugative transfer of the T-DNA to the plant nucleus. In this process, T-DNA formation, T-DNA transfer, and T-DNA integration via illegitimate recombination can be distinguished. In addition to some transformants with one T-DNA copy, transformants with multicopy T-DNA loci are also often found. In these multicopy loci, the T-DNAs often occur as inverted repeats about the right or left border. The T-DNA plant junctions frequently contain insertions of filler DNA, short regions of microhomology, small deletions of both T-DNA ends and target sequences, and integration of vector backbone sequences. To date, extensive scientific research has paved the way for a better understanding of the bacterial and plant host-driven molecular mechanisms that underlie the different steps in the Agrobacterium-mediated plant cell transformation process. The aim of this chapter is to discuss the final stage and outcome of the T-DNA transformation process, i.e. to focus on the molecular mechanism that integrates the T-DNA and, in addition, to describe the various patterns documented in the literature.


Left Border Illegitimate Recombination Vector Backbone Sequence VirD2 Protein Citovsky Versus 
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5 References

  1. Albright LM, Yanofsky MF, Leroux B, Ma DQ, Nester EW (1987) Processing of the T-DNA of Agrobacterium tumefaciens generates border nicks and linear, single-stranded T-DNA. J Bacteriol 169: 1046-1055PubMedGoogle Scholar
  2. Alonso JM, Stepanova AN, Leisse TJ, Kim CJ, Chen H, Shinn P, Stevenson DK, Zimmerman J, Barajas P, Cheuk R, Gadrinab C, Heller C, Jeske A, Koesema E, Meyers CC, Parker H, Prednis L, Ansari Y, Choy N, Deen H, Geralt M, Hazari N, Hom E, Karnes M, Mulholland C, Ndubaku R, Schmidt I, Guzman P, Agui-lar-Henonin L, Schmid M, Weigel D, Carter DE, Marchand T, Risseeuw E, Brogden D, Zeko A, Crosby WL, Berry CC, Ecker JR (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301: 653-657PubMedGoogle Scholar
  3. Azpiroz-Leehan R, Feldmann KA (1997) T-DNA insertion mutagenesis in Arabi-dopsis: going back and forth. Trends Genet 13: 152-156PubMedGoogle Scholar
  4. Bakó L, Umeda M, Tiburcio AF, Schell J, Koncz C (2003) The VirD2 pilot pro-tein of Agrobacterium-transferred DNA interacts with the TATA box-binding protein and a nuclear protein kinase in plants. Proc Natl Acad Sci USA 100: 10108-10113PubMedGoogle Scholar
  5. Ballas N, Citovsky V (1997) Nuclear localization signal binding protein from Arabidopsis mediates nuclear import of Agrobacterium VirD2 protein. Proc Natl Acad Sci USA 94: 10723-10728PubMedGoogle Scholar
  6. Barakat A, Gallois P, Raynal M, Mestre-Ortega D, Sallaud C, Guiderdoni E, Delseny M, Bernardi G (2000) The distribution of T-DNA in the genomes of transgenic Arabidopsis and rice. FEBS Lett 471: 161-164PubMedGoogle Scholar
  7. Boulton SJ, Jackson SP (1996) Saccharomyces cerevisiae Ku70 potentiates ille-gitimate DNA double-strand break repair and serves as a barrier to error-prone DNA repair pathways. EMBO J 15: 5093-5103PubMedGoogle Scholar
  8. Braun AC (1958) A physiological basis for autonomous growth of the crown-gall tumor cell. Proc Natl Acad USA 44: 344-349Google Scholar
  9. Britt AB (1999) Molecular genetics of DNA repair in higher plants. Trends Plant Sci 4: 20-25PubMedGoogle Scholar
  10. Brunaud V, Balzergue S, Dubreucq B, Aubourg S, Samson F, Chauvin S, Bechtold N, Cruaud C, DeRose R, Pelletier G, Lepiniec L, Caboche M, Lecharny A (2002) T-DNA integration into the Arabidopsis genome depends on sequences of pre-insertion sites. EMBO Rep 3: 1152-1157PubMedGoogle Scholar
  11. Bundock P, Hooykaas PJJ (1996) Integration of Agrobacterium tumefaciens T-DNA in the Saccharomyces cerevisiae genome by illegitimate recombination. Proc Natl Acad Sci USA 93: 15272-15275PubMedGoogle Scholar
  12. Castle LA, Errampalli D, Atherton TL, Franzmann LH, Yoon ES, Meinke DW (1993) Genetic and molecular characterization of embryonic mutants identi-fied following seed transformation in Arabidopsis. Mol Gen Genet 241: 504-514PubMedGoogle Scholar
  13. Chateau S, Sangwan RS, Sangwan-Norreel BS (2000) Competence of Arabidopsis thaliana genotypes and mutants for Agrobacterium tumefaciens-mediated gene transfer: role of phytohormones. J Exp Bot 51: 1961-1968PubMedGoogle Scholar
  14. Chen S, Jin W, Wang M, Zhang F, Zhou J, Jia Q, Wu Y, Liu F, Wu P (2003) Dis-tribution and characterization of over 1000 T-DNA tags in rice genome. Plant J 36: 105-113PubMedGoogle Scholar
  15. Chilton M-D, Drummond MH, Merio DJ, Sciaky D, Montoya AL, Gordon MP, Nester EW (1977) Stable incorporation of plasmid DNA into higher plant cells: the molecular basis of crown gall tumorigenesis. Cell 11: 263-271PubMedGoogle Scholar
  16. Chilton M-D, Que Q (2003) Targeted integration of T-DNA into the tobacco ge-nome at double-strand breaks: new insights on the mechanism of T-DNA in-tegration. Plant Physiol 133: 956-965PubMedGoogle Scholar
  17. 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
  18. Citovsky V, Zupan J, Warnick D, Zambryski PC (1992) Nuclear localization of Agrobacterium VirE2 protein in plant cells. Science 256: 1802-1805PubMedGoogle Scholar
  19. Cluster PD, O’Dell M, Metzlaff M, Flavell RB (1996) Details of T-DNA structural organization from a transgenic Petunia population exhibiting co-suppression. Plant Mol Biol 32: 1197-1203PubMedGoogle Scholar
  20. Coin F, Frit P, Viollet B, Salles B, Egly JM (1998) TATA binding protein dis-criminates between different lesions on DNA, resulting in a transcription de-crease. Mol Cell Biol 18: 3907-3914PubMedGoogle Scholar
  21. Critchlow SE, Jackson SP (1998) DNA end-joining: from yeast to man. Trends Biochem Sci 23: 394-398PubMedGoogle Scholar
  22. De Block M, Debrouwer D (1991) Two T-DNA’s co-transformed into Brassica napus by a double Agrobacterium tumefaciens infection are mainly integrated at the same locus. Theor Appl Genet 82: 257-263Google Scholar
  23. De Buck S, De Wilde C, Van Montagu M, Depicker A (2000a) Determination of the T-DNA transfer and the T-DNA integration frequencies upon cocultiva-tion of Arabidopsis thaliana root explants. Mol Plant-Microbe Interact 13: 658-665PubMedGoogle Scholar
  24. De Buck S, De Wilde C, Van Montagu M, Depicker A (2000b) T DNA vector backbone sequences are frequently integrated into the genome of transgenic plants obtained by Agrobacterium-mediated transformation. Mol Breed 6: 459-468Google Scholar
  25. De Buck S, Jacobs A, Van Montagu M, Depicker A (1998) Agrobacterium tume-faciens transformation and cotransformation frequencies of Arabidopsis thaliana root explants and tobacco protoplasts. Mol Plant-Microbe Interact 11: 449-457PubMedGoogle Scholar
  26. De Buck S, Jacobs A, Van Montagu M, Depicker A (1999) The DNA sequences of T-DNA junctions suggest that complex T-DNA loci are formed by a re-combination process resembling T-DNA integration. Plant J 20: 295-304PubMedGoogle Scholar
  27. De Buck S, Windels P, De Loose M, Depicker A (2004) Single-copy T-DNAs in-tegrated at different positions in the Arabidopsis genome display uniform and comparableȕ-glucuronidase accumulation levels. Cell Mol Life Sci 61: 2632-2645PubMedGoogle Scholar
  28. de Kathen A, Jacobsen HJ (1995) Cell competence for Agrobacterium-mediated DNA transfer in Pisum sativum L. Transgenic Res 4: 184-191Google Scholar
  29. De Neve M, De Buck S, Jacobs A, Van Montagu M, Depicker A (1997) T-DNA integration patterns in co-transformed plant cells suggest that T-DNA repeats originate from co-integration of separate T-DNAs. Plant J 11: 15-29PubMedGoogle Scholar
  30. Deng W, Chen L, Wood DW, Metcalfe T, Liang X, Gordon MP, Comai L, Nester EW (1998) Agrobacterium VirD2 protein interacts with plant host cyclophil-ins. Proc Natl Acad Sci USA 95: 7040-7045PubMedGoogle Scholar
  31. Depicker A, Herman L, Jacobs A, Schell J, Van Montagu M (1985) Frequencies of simultaneous transformation with different T-DNAs and their relevance to the Agrobacterium/plant cell interaction. Mol Gen Genet 201: 477-484Google Scholar
  32. Deroles SC, Gardner RC (1988) Analysis of the T DNA structure in a large num-ber of transgenic petunias generated by Agrobacterium-mediated transforma-tion. Plant Mol Biol 11: 365-377Google Scholar
  33. Dillen W, De Clercq J, Kapila J, Zambre M, Van Montagu M, Angenon G (1997) The effect of temperature on Agrobacterium tumefaciens-mediated gene trans-fer to plants. Plant J 12: 1459-1463Google Scholar
  34. Ditt RF, Nester EW, Comai L (2001) Plant gene expression response to Agrobac-terium tumefaciens. Proc Natl Acad Sci USA: 10954-10959Google Scholar
  35. Dong J, Kharb P, Teng W, Hall TC (2001) Characterization of rice transformed via an Agrobacterium-mediated inflorescence approach. Mol Breed 7: 187-194Google Scholar
  36. Dürrenberger F, Crameri A, Hohn B, Koukolíková-Nicola Z (1989) Covalently bound VirD2 protein of Agrobacterium tumefaciens protects the T-DNA from exonucleolytic degradation. Proc Natl Acad Sci USA 86: 9154-9158PubMedGoogle Scholar
  37. Dynan WS, Yoo S (1998) Interaction of Ku protein and DNA-dependent protein kinase catalytic subunit with nucleic acids. Nucleic Acids Res 26: 1551-1559PubMedGoogle Scholar
  38. Eamens AL, Blanchard CL, Dennis ES, Upadhyaya NM (2004) A bidirectional gene trap construct suitable for T-DNA and Ds-mediated insertional mutagenesis in rice (Oryza sativa L.). Plant Biotechnol J 2: 367-380PubMedGoogle Scholar
  39. Escobar MA, Dandekar AM (2003) Agrobacterium tumefaciens as an agent of disease. Trends Plant Sci 8: 380-386PubMedGoogle Scholar
  40. Fladung M (1999) Gene stability in transgenic aspen (Populus). I. Flanking DNA sequences and T-DNA structure. Mol Gen Genet 260: 574-581PubMedGoogle Scholar
  41. Forsbach A, Schubert D, Lechtenberg B, Gils M, Schmidt R (2003) A comprehen-sive characterization of single-copy T-DNA insertions in the Arabidopsis thaliana genome. Plant Mol Biol 52: 161-176PubMedGoogle Scholar
  42. Francis KE, Spiker S (2005) Identification of Arabidopsis thaliana transformants without selection reveals a high occurrence of silenced T-DNA integrations. Plant J 41: 464-477PubMedGoogle Scholar
  43. Friesner J, Britt AB (2003) Ku80- and DNA ligase IV-deficient plants are sensitive to ionizing radiation and defective in T-DNA integration. Plant J 34: 427-440PubMedGoogle Scholar
  44. Gallego ME, Bleuyard JY, Daoudal-Cotterell S, Jallut N, White CI (2003) Ku80 plays a role in non-homologous recombination but is not required for T-DNA integration in Arabidopsis. Plant J 35: 557-565PubMedGoogle Scholar
  45. Gelvin SB (2000) Agrobacterium and plant genes involved in T-DNA transfer and integration. Annu Rev Plant Physiol Plant Mol Biol 51: 223-256PubMedGoogle Scholar
  46. Gelvin SB (2003) Agrobacterium-mediated plant transformation: the biology be-hind the “gene-jockeying” tool. Microbiol Mol Biol Rev 67: 16-37PubMedGoogle Scholar
  47. Gheysen G, Angenon G, Van Montagu M (1998) Agrobacterium-mediated plant transformation: a scientifically intriguing story with significant applications. In K Lindsey, ed, Transgenic Plant Research. Harwood Academic Publishers, Amsterdam, pp 1-33Google Scholar
  48. 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 94: 155-163PubMedGoogle Scholar
  49. Gheysen G, Van Montagu M, Zambryski P (1987) Integration of Agrobacterium tumefaciens transfer DNA (T-DNA) involves rearrangements of target plant DNA. Proc Natl Acad Sci USA 84: 6169-6173PubMedGoogle Scholar
  50. Gheysen G, Villarroel R, Van Montagu M (1991) Illegitimate recombination in plants: a model for T-DNA integration. Genes Dev 5: 287-297PubMedGoogle Scholar
  51. Goedecke W, Eijpe M, Offenberg HH, van Aalderen M, Heyting C (1999) Mre11 and Ku70 interact in somatic cells, but are differentially expressed in early meiosis. Nat Genet 23: 194-198PubMedGoogle Scholar
  52. Gorbunova V, Levy AA (1997) Non-homologous DNA end joining in plant cells is associated with deletions and filler DNA insertions. Nucleic Acids Res 25: 4650-4657.PubMedGoogle Scholar
  53. Gorbunova VV, Levy AA (1999) How plants make ends meet: DNA double-strand break repair. Trends Plant Sci 4: 263-269PubMedGoogle Scholar
  54. Grevelding C, Fantes V, Kemper E, Schell J, Masterson R (1993) Single-copy T-DNA insertions in Arabidopsis are the predominant form of integration in root-derived transgenics, whereas multiple insertions are found in leaf discs. Plant Mol Biol 23: 847-860PubMedGoogle Scholar
  55. Grimsley N, Hohn T, Davies JW, Hohn B (1987) Agrobacterium-mediated deliv-ery of infectious maize streak virus into maize plants. Nature 325: 177-179Google Scholar
  56. Grimsley NH, Ramos C, Hein T, Hohn B (1988) Meristematic tissues of maize plants are most susceptible to agroinfection with maize streak virus. Bio/Technology 6: 185-189Google Scholar
  57. Grunstein M (1997) Histone acetylation in chromatin structure and transcription. Nature 389: 349-352PubMedGoogle Scholar
  58. Haber JE (2000) Lucky breaks: analysis of recombination in Saccharomyces. Mutat Res 451: 53-69PubMedGoogle Scholar
  59. Hanson B, Engler D, Moy Y, Newman B, Ralston E, Gutterson N (1999) A simple method to enrich an Agrobacterium-transformed population for plants con-taining only T-DNA sequences. Plant J 19: 727-734PubMedGoogle Scholar
  60. Herman L, Jacobs A, Van Montagu M, Depicker A (1990) Plant chromo-some/marker gene fusion assay for study of normal and truncated T-DNA in-tegration events. Mol Gen Genet 224: 248-256PubMedGoogle Scholar
  61. Herrera-Estrella A, Chen Z, 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: 4055-4062PubMedGoogle Scholar
  62. Herrera-Estrella A, Van Montagu M, Wang K (1990) A bacterial peptide acting as a plant nuclear targeting signal: the amino-terminal portion of Agrobacterium VirD2 protein directs a ȕ -galactosidase fusion protein into tobacco nuclei. Proc Natl Acad Sci USA 87: 9534-9537PubMedGoogle Scholar
  63. Howard EA, Zupan JR, Citovsky V, Zambryski PC (1992) The VirD2 protein of tumefaciens contains a C-terminal bipartite nuclear localization signal: im-plications for nuclear uptake of DNA in plant cells. Cell 68: 109-118PubMedGoogle Scholar
  64. Jackson SP, Jeggo PA (1995) DNA double-strand break repair and V(D)J recom-bination: involvement of DNA-PK. Trends Biochem Sci 20: 412-415PubMedGoogle Scholar
  65. Janssen B-J, Gardner RC (1990) Localized transient expression of GUS in leaf discs following cocultivation with Agrobacterium. Plant Mol Biol 14: 61-72PubMedGoogle Scholar
  66. Jorgensen R, Snyder C, Jones JDG (1987) T-DNA is organized predominantly in inverted repeat structures in plants transformed with Agrobacterium tumefa-ciens C58 derivatives. Mol Gen Genet 207: 471-477Google Scholar
  67. Kaya H, Sato S, Tabata S, Kobayashi Y, Iwabuchi M, Araki T (2000) hosoba toge toge, a syndrome caused by a large chromosomal deletion associated with a T-DNA insertion in Arabidopsis. Plant Cell Physiol 41: 1055-1066PubMedGoogle Scholar
  68. Kertbundit S, Linacero R, Rouzé P, Galis I, Macas J, Deboeck F, Renckens S, Hernalsteens J-P, De Greve H (1998) Analysis of T-DNA-mediated transla-tional ß-glucuronidase gene fusions. Plant Mol Biol 36: 205-217PubMedGoogle Scholar
  69. Khrustaleva LI, Kik C (2001) Localization of single-copy T-DNA insertion in transgenic shallots (Allium cepa) by using ultra-sensitive FISH with tyramide signal amplification. Plant J 25: 699-707PubMedGoogle Scholar
  70. Kim S-R, Lee J, Jun S-H, Park S, Kang H-G, Kwon S, An G (2003) Transgene structures in T-DNA-inserted rice plants. Plant Mol Biol 52: 761-773PubMedGoogle Scholar
  71. Kirik A, Salomon S, Puchta H (2000) Species-specific double-strand break repair and genome evolution in plants. EMBO J 19: 5562-5566PubMedGoogle Scholar
  72. Köhler F, Cardon G, Pöhlman M, Gill R, Schieder O (1989) Enhancement of transformation rates in higher plants by low-dose irradiation: are DNA repair systems involved in the incorporation of exogenous DNA into the plant genome? Plant Mol Biol 12: 189-199Google Scholar
  73. Kohli A, Twyman RM, Abranches R, Wegel E, Stoger E, Christou P (2003) Transgene integration, organization and interaction in plants. Plant Mol Biol 52: 247-258PubMedGoogle Scholar
  74. Komari T, Ishida Y, Hiei Y (2004) Plant transformation technology: Agrobacte-rium-mediated transformation. In P Christou, H Klee, eds, Handbook of Plant Biotechnology, Vol 1. John Wiley and Sons, Chichester, pp. 233-261Google Scholar
  75. 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: 8467-8471PubMedGoogle Scholar
  76. Koncz C, Schell J (1986) The promoter of TL-DNA gene 5 controls the tissue-specific expression of chimaeric genes carried by a novel type of Agrobacte-rium binary vector. Mol Gen Genet 204: 383-396Google Scholar
  77. Kononov ME, Bassuner B, Gelvin SB (1997) Integration of T-DNA binary vector ‘backbone’ sequences into the tobacco genome: evidence for multiple com-plex patterns of integration. Plant J 11: 945-957PubMedGoogle Scholar
  78. Krizkova L, Hrouda M (1998) Direct repeats of T-DNA integrated in tobacco chromosome: characterization of junction regions. Plant J 16: 673-680PubMedGoogle Scholar
  79. Krysan PJ, Young JC, Jester PJ, Monson S, Copenhaver G, Preuss D, Sussman MR (2002) Characterization of T-DNA insertion sites in Arabidopsis thaliana and the implications for saturation mutagenesis. OMICS 6: 163-174PubMedGoogle Scholar
  80. Kumar S, Fladung M (2000) Transgene repeats in aspen: molecular characterisa-tion suggests simultaneous integration of independent T-DNAs into receptive hotspots in the host genome. Mol Gen Genet 264: 20-28PubMedGoogle Scholar
  81. Kumar S, Fladung M (2002) Transgene integration in aspen: structures of integra-tion sites and mechanism of T-DNA integration. Plant J 31: 543-551PubMedGoogle Scholar
  82. Kumar SV, Rajam MV (2005) Polyamines enhance Agrobacterium tumefaciens vir gene induction and T-DNA transfer. Plant Sci 168: 475-480Google Scholar
  83. Kuraya Y, Ohta S, Fukuda M, Hiei Y, Murai N, Hamada K, Ueki J, Imaseki H, Komari T (2004) Suppression of transfer of non-T-DNA vector backbone’ se-quences by multiple left border repeats for transformation of higher plant mediated by Agrobacterium tumefaciens. Mol Breed 14: 309-320Google Scholar
  84. Laufs P, Autran D, Traas J (1999) A chromosomal paracentric inversion associ-ated with T-DNA integration in Arabidopsis. Plant J 18: 131-139PubMedGoogle Scholar
  85. Lechtenberg B, Schubert D, Forsbach A, Gils M, Schmidt R (2003) Neither in-verted repeat T-DNA configurations nor arrangements of tandemly repeated transgenes are sufficient to trigger transgene silencing. Plant J 34: 507-517PubMedGoogle Scholar
  86. Li J, Krichevsky A, Vaidya M, Tzfira T, Citovsky V (2005) Uncoupling of the functions of the Arabidopsis VIP1 protein in transient and stable plant genetic transformation by Agrobacterium. Proc Natl Acad Sci USA 102: 5733-5738PubMedGoogle Scholar
  87. Loyter A, Rosenbluh J, Zakai N, Li J, Kozlovsky SV, Tzfira T, Citovsky V (2005) The plant VirE2 interacting protein 1. A molecular link between the Agrobac-terium T-complex and the host cell chromatin? Plant Physiol 138: 1318-1321PubMedGoogle Scholar
  88. Martineau B, Voelker TA, Sanders RA (1994) On defining T-DNA. Plant Cell 6: 1032-1033PubMedGoogle Scholar
  89. Maximova SN, Dandekar AM, Guiltinan MJ (1998) Investigation of Agrobacte-rium-mediated transformation of apple using green fluorescent protein: high transient expression and low stable transformation suggest that factors other than T-DNA transfer are rate-limiting. Plant Mol Biol 37: 549-559PubMedGoogle Scholar
  90. Mayerhofer R, Koncz-Kalman Z, Nawrath C, Bakkeren G, Crameri A, Angelis K, Redei GP, Schell J, Hohn B, Koncz C (1991) T-DNA integration: a mode of illegitimate recombination in plants. EMBO J 10: 697-704PubMedGoogle Scholar
  91. Meza TJ, Stangeland B, Mercy IS, Skårn M, Nymoen DA, Berg A, Butenko MA, Håkelien AM, Haslekås C, Meza-Zepeda LA, Aalen RB (2002) Analyses of single-copy Arabidopsis T-DNA-transformed lines show that the presence of vector backbone sequences, short inverted repeats and DNA methylation is not sufficient or necessary for the induction of transgene silencing. Nucleic Acids Res 30: 4556-4566PubMedGoogle Scholar
  92. Milne GT, Jin S, Shannon KB, Weaver DT (1996) Mutations in two Ku homologs define a DNA end-joining repair pathway in Saccharomyces cerevisiae. Mol Cell Biol 16: 4189-4198PubMedGoogle Scholar
  93. Mysore KS, Bassuner B, Deng X-B, Darbinian NS, Motchoulski A, Ream LW, Gelvin SB (1998) Role of the Agrobacterium tumefaciens VirD2 protein in T-DNA transfer and integration. Mol Plant-Microbe Interact 11: 668-683PubMedGoogle Scholar
  94. Mysore KS, Nam J, Gelvin SB (2000) An Arabidopsis histone H2A mutant is de-ficient in Agrobacterium T-DNA integration. Proc Natl Acad Sci USA 97: 948-953PubMedGoogle Scholar
  95. Nacry P, Camilleri C, Courtial B, Caboche M, Bouchez D (1998) Major chromo-somal rearrangements induced by T-DNA transformation in Arabidopsis. Genetics 149: 641-650PubMedGoogle Scholar
  96. Nam J, Matthysse AG, Gelvin SB (1997) Differences in susceptibility of Arabi-dopsis ecotypes to crown gall disease may result from a deficiency in T-DNA integration. Plant Cell 9: 317-333PubMedGoogle Scholar
  97. Nam J, Mysore KS, Zheng C, Knue MK, Matthysse AG, Gelvin SB (1999) Identi-fication of T-DNA tagged Arabidopsis mutants that are resistant to transfor-mation by Agrobacterium. Mol Gen Genet 261: 429-438PubMedGoogle Scholar
  98. Narasimhulu SB, Deng X-B, Sarria R, Gelvin SB (1996) Early transcription of Agrobacterium T-DNA genes in tobacco and maize. Plant Cell 8: 873-886PubMedGoogle Scholar
  99. Negruk V, Eisner G, Lemieux B (1996) Addition-deletion mutations in transgenic Arabidopsis thaliana generated by the seed co-cultivation method. Genome 39: 1117-1122PubMedGoogle Scholar
  100. Offringa R, de Groot MJA, Haagsman HJ, Does MP, van den Elzen PJM, Hooykaas PJJ (1990) Extrachromosomal homologous recombination and gene targeting in plant cells after Agrobacterium mediated transformation. EMBO J 9: 3077-3084PubMedGoogle Scholar
  101. Ohba T, Yoshioka Y, Machida C, Machida Y (1995) DNA rearrangement associ-ated with the integration of T-DNA in tobacco: an example for multiple dupli-cations of DNA around the integration target. Plant J 7: 157-164PubMedGoogle Scholar
  102. Orel N, Puchta H (2003) Differences in the processing of DNA ends in Arabidop-sis thaliana and tobacco: possible implications for genome evolution. Plant Mol Biol 51: 523-531PubMedGoogle Scholar
  103. Ortega D, Raynal M, Laudié M, Llauro C, Cooke R, Devic M, Genestier S, Picard G, Abad P, Contard P, Sarrobert C, Nussaume L, Bechtold N, Horlow C, Pelletier G, Delseny M (2002) Flanking sequence tags in Arabidopsis thaliana T-DNA insertion lines: a pilot study. C R Biol 325: 773-780PubMedGoogle Scholar
  104. Pâques F, Haber JE (1999) Multiple pathways of recombination induced by dou-ble-strand breaks in Saccharomyces cerevisiae. Microbiol Mol Biol Rev 63: 349-404PubMedGoogle Scholar
  105. Podevin N, De Buck S, De Wilde C, Depicker A (2006) Insight into recognition of the T-DNA border repeats as termination sites for T-strand synthesis by Agro-bacterium tumefaciens. Transgenic Res, in pressGoogle Scholar
  106. Ramanathan V, Veluthambi K (1995) Transfer of non-T-DNA portions of the Agrobacterium tumefaciens Ti plasmid pTiA6 from the left terminus of TL-DNA. Plant Mol Biol 28: 1149-1154PubMedGoogle Scholar
  107. Reliü B, Andjelkoviü M, Rossi L, Nagamine Y, Hohn B (1998) Interaction of the DNA modifying proteins VirD1 and VirD2 of Agrobacterium tumefaciens: analysis by subcellular localization in mammalian cells. Proc Natl Acad Sci USA 95: 9105-9110Google Scholar
  108. Rinehart TA, Dean C, Weil CF (1997) Comparative analysis of non-random DNA repair following Ac transposon excision in maize and Arabidopsis. Plant J 12: 1419-1427PubMedGoogle Scholar
  109. Rossi L, Hohn B, Tinland B (1996) Integration of complete transferred DNA units is dependent on the activity of virulence E2 protein of Agrobacterium tumefa-ciens. Proc Natl Acad Sci USA 93: 126-130PubMedGoogle Scholar
  110. Sallaud C, Gay C, Larmande P, Bès M, Piffanelli P, Piégu B, Droc G, Regad F, Bourgeois E, Meynard D, Périn C, Sabau X, Ghesquière A, Glaszmann JC, Delseny M, Guiderdoni E (2004) High throughput T-DNA insertion mutagenesis in rice: a first step towards in silico reverse genetics. Plant J 39: 450-464PubMedGoogle Scholar
  111. Sallaud C, Meynard D, van Boxtel J, Gay C, Bès M, Brizard JP, Larmande P, Ortega D, Raynal M, Portefaix M, Ouwerkerk PBF, Rueb S, Delseny M, Guiderdoni E (2003) Highly efficient production and characterization of T-DNA plants for rice (Oryza sativa L.) functional genomics. Theor Appl Genet 106: 1396-1408PubMedGoogle Scholar
  112. Salomon S, Puchta H (1998) Capture of genomic and T-DNA sequences during double-strand break repair in somatic plant cells. EMBO J 17: 6086-6095PubMedGoogle Scholar
  113. Scheiffele P, Pansegrau W, Lanka E (1995) Initiation of Agrobacterium tumefa-ciens T-DNA processing. Purified proteins VirD1 and VirD2 catalyze site-and strand-specific cleavage of superhelical T-border DNA in vitro. J Biol Chem 270: 1269-1276PubMedGoogle Scholar
  114. Sha Y, Li S, Pei Z, Luo L, Tian Y, He C (2004) Generation and flanking sequence analysis of a rice T-DNA tagged population. Theor Appl Genet 108: 306-314PubMedGoogle Scholar
  115. Shurvinton CE, Hodges L, Ream LW (1992) A nuclear localization signal and the C-terminal omega sequence in the Agrobacterium tumefaciens VirD2 endonu-clease are important for tumor formation. Proc Natl Acad Sci USA 89: 11837-11841PubMedGoogle Scholar
  116. Smith EF, Townsend CO (1907) A plant tumor of bacterial origin. Science 25: 671-673PubMedGoogle Scholar
  117. Sonti RV, Chiurazzi M, Wong D, Davies CS, Harlow GR, Mount DW, Signer ER (1995) Arabidopsis mutants deficient in T-DNA integration. Proc Natl Acad Sci USA 92: 11786-11790PubMedGoogle Scholar
  118. Szabados L, Kovács I, Oberschall A, Ábrahám E, Kerekes I, Zsigmond L, Nagy R, Alvarado M, Krasovskaja I, Gál M, Berente A, Rédei GP, Haim AB, Koncz C (2002) Distribution of 1000 sequenced T-DNA tags in the Arabidop-sis genome. Plant J 32: 233-242PubMedGoogle Scholar
  119. Tamura K, Adachi Y, Chiba K, Oguchi K, Takahashi H (2002) Identification of Ku70 and Ku80 homologues in Arabidopsis thaliana: evidence for a role in the repair of DNA double-strand breaks. Plant J 29: 771-781PubMedGoogle Scholar
  120. Tax FE, Vernon DM (2001) T-DNA-associated duplication/translocations in Arabidopsis. Implications for mutant analysis and functional genomics. Plant Physiol 126: 1527-1538PubMedGoogle Scholar
  121. ten Hoopen R, Robbins TP, Fransz PF, Montijn BM, Oud O, Gerats AGM, Nanninga N (1996) Localization of T-DNA insertions in Petunia by fluore-scence in-situ hybridization: physical evidence for suppression of recombination. Plant Cell 8: 823-830PubMedGoogle Scholar
  122. Tinland B (1996) The integration of T-DNA into plant genomes. Trends Plant Sci 1: 178-184Google Scholar
  123. Tinland B, Hohn B, Puchta H (1994) Agrobacterium tumefaciens transfers single-stranded transferred DNA (T-DNA) into the plant cell nucleus. Proc Natl Acad Sci USA 91: 8000-8004PubMedGoogle Scholar
  124. Tinland B, Koukolíková-Nicola Z, Hall MN, Hohn B (1992) The T-DNA-linked VirD2 protein contains two distinct nuclear localization signals. Proc Natl Acad Sci USA 89: 7442-7446PubMedGoogle Scholar
  125. Tinland B, Schoumacher F, Gloeckler V, Bravo-Angel AM, Hohn B (1995) The Agrobacterium tumefaciens virulence D2 protein is responsible for precise in-tegration of T-DNA into the plant genome. EMBO J 14: 3585-3595PubMedGoogle Scholar
  126. Topping JF, Wei W, Clarke MC, Muskett P, Lindsey K (1995) Agrobacterium-mediated transformation of Arabidopsis thaliana: application in T DNA tag-ging. In H Jones, ed, Plant Gene Transfer and Expression Protocols, Methods in Molecular Biology, Vol 49. Humana Press, Totowa, pp 63-76Google Scholar
  127. Tsukamoto Y, Ikeda H (1998) Double-strand break repair mediated by DNA end-joining. Genes Cells 3: 135-144PubMedGoogle Scholar
  128. Tsukamoto Y, Kato J-I, Ikeda H (1997) Silencing factors participate in DNA re-pair and recombination in Saccharomyces cerevisiae. Nature 388: 900-903PubMedGoogle Scholar
  129. Tzfira T, Citovsky V (2002) Partners-in-infection: host proteins involved in the transformation of plant cells by Agrobacterium. Trends Cell Biol 12: 121-129PubMedGoogle Scholar
  130. Tzfira T, Li J, Lacroix B, Citovsky V (2004a) Agrobacterium T-DNA integration: molecules and models. Trends Genet 20: 375-383PubMedGoogle Scholar
  131. Tzfira T, Rhee Y, Chen M-H, Kunik T, Citovsky V (2000) Nucleic acid transport in plant-microbe interactions: the molecules that walk through the walls. Annu Rev Microbiol 54: 187-219PubMedGoogle Scholar
  132. Tzfira T, Vaidya M, Citovsky V (2002) Increasing plant susceptibility to Agrobac-terium infection by overexpression of the Arabidopsis nuclear protein VIP1. Proc Natl Acad Sci USA 99: 10435-10440PubMedGoogle Scholar
  133. Tzfira T, Vaidya M, Citovsky V (2004b) Involvement of targeted proteolysis in plant genetic transformation by Agrobacterium. Nature 431: 87-92PubMedGoogle Scholar
  134. Vain P, James VA, Worland B, Snape JW (2003) Transgene behaviour across two generations in a large random population of transgenic rice plants produced by particle bombardment. Theor Appl Genet 105: 878-889Google Scholar
  135. Valentine L (2003) Agrobacterium tumefaciens: the David and Goliath of modern genetics. Plant Physiol 133: 948-955PubMedGoogle Scholar
  136. van Attikum H, Bundock P, Hooykaas PJJ (2001) Non-homologous end-joining proteins are required for Agrobacterium T-DNA integration. EMBO J 20: 6550-6558PubMedGoogle Scholar
  137. van Attikum H, Bundock P, Overmeer RM, Lee LY, Gelvin SB, Hooykaas PJJ (2003) The Arabidopsis AtLIG4 gene is required for the repair of DNA dam-age, but not for the integration of Agrobacterium T-DNA. Nucleic Acids Res 31: 4247-4255PubMedGoogle Scholar
  138. van der Graaff E, den Dulk-Ras A, Hooykaas PJJ (1996) Deviating T-DNA trans-fer from Agrobacterium tumefaciens to plants. Plant Mol Biol 31: 677-681PubMedGoogle Scholar
  139. Van Haaren MJJ, Pronk JT, Schilperoort RA, Hooykaas PJJ (1987) Functional analysis of the Agrobacterium tumefaciens octopine Ti-plasmid left and right T-region border fragments. Plant Mol Biol 8: 95-104Google Scholar
  140. Van Lijsebettens M, Inzé D, Schell J, Van Montagu M (1986) Transformed cell clones as a tool to study T DNA integration mediated by Agrobacterium tume-faciens. J Mol Biol 188: 129-145PubMedGoogle Scholar
  141. Veena, Jiang H, Doerge RW, Gelvin SB (2003) Transfer of T-DNA and Vir pro-teins to plant cells by Agrobacterium tumefaciens induces expression of host genes involved in mediating transformation and suppresses host defense gene expression. Plant J 35: 219-236PubMedGoogle Scholar
  142. Vergunst AC, van Lier MCM, den Dulk-Ras A, Grosse Stüve TA, Ouwehand A, Hooykaas PJJ (2005) Positive charge is an important feature of the C-terminal transport signal of the VirB/D4-translocated proteins of Agrobacterium. Proc Natl Acad Sci USA 102: 832-837PubMedGoogle Scholar
  143. Vichi P, Coin F, Renaud J-P, Vermeulen W, Hoeijmakers JHJ, Moras D, Egly J-M (1997) Cisplatin- and UV-damaged DNA lure the basal transcription factor TFIID/TBP. EMBO J 16: 7444-7456PubMedGoogle Scholar
  144. Villemont E, Dubois F, Sangwan RS, Vasseur G, Bourgeois Y, Sangwan-Norreel BS (1997) Role of the host cell cycle in the Agrobacterium-mediated genetic transformation of Petunia: evidence of an S-phase control mechanism for T-DNA transfer. Planta 201: 160-172Google Scholar
  145. Wallroth M, Gerats AGM, Rogers SG, Fraley RT, Horsch RB (1986) Chromosomal localization of foreign genes in Petunia hybrida. Mol Gen Genet 202: 6-15Google Scholar
  146. Wang J, Lewis ME, Whallon JH, Sink KC (1995) Chromosomal mapping of T-DNA inserts in transgenic Petunia by in situ hybridization. Transgenic Res 4: 241-246Google Scholar
  147. Wenck A, Czakó M, Kanevski I, Márton L (1997) Frequent collinear long transfer of DNA inclusive of the whole binary vector during Agrobacterium-mediated transformation. Plant Mol Biol 34: 913-922PubMedGoogle Scholar
  148. West CE, Waterworth WM, Jiang Q, Bray CM (2000) Arabidopsis DNA ligase IV is induced by ϑ-irradiation and interacts with an Arabidopsis homologue of the double strand break repair protein XRCC4. Plant J 24: 67-78PubMedGoogle Scholar
  149. West CE, Waterworth WM, Story GW, Sunderland PA, Jiang Q, Bray CM (2002) Disruption of the Arabidopsis AtKu80 gene demonstrates an essential role for AtKu80 protein in efficient repair of DNA double-strand breaks in vivo. Plant J 31: 517-528PubMedGoogle Scholar
  150. Windels P, De Buck S, Van Bockstaele E, De Loose M, Depicker A (2003) T-DNA integration in Arabidopsis chromosomes. Presence and origin of filler DNA sequences. Plant Physiol 133: 2061-2068PubMedGoogle Scholar
  151. Wolters A-MA, Trindade LM, Jacobsen E, Visser RGF (1998) Fluorescence in situ hybridization on extended DNA fibres as a tool to analyse complex T-DNA loci in potato. Plant J 13: 837-847Google Scholar
  152. Zaenen I, Van Larebeke N, Teuchy H, Van Montagu M, Schell J (1974) Super-coiled circular DNA in crown gall inducing Agrobacterium strains. J Mol Biol 86: 109-127PubMedGoogle Scholar
  153. Zambre M, Terryn N, De Clercq J, De Buck S, Dillen W, Van Montagu M, Van Der Straeten D, Angenon G (2003) Light strongly promotes gene transfer from Agrobacterium tumefaciens to plant cells. Planta 216: 580-586PubMedGoogle Scholar
  154. Zambryski PC, Depicker A, Kruger K, Goodman HM (1982) Tumor induction by Agrobacterium tumefaciens: analysis of the boundaries of T-DNA. J Mol Appl Genet 1: 361-370PubMedGoogle Scholar
  155. Zheng SJ, Henken B, Sofiari E, Jacobsen E, Krens FA, Kik C (2001) Molecular characterization of transgenic shallots (Allium cepa L.) by adaptor ligation PCR (AL-PCR) and sequencing of genomic DNA flanking T-DNA borders. Transgenic Res 10: 237-245PubMedGoogle Scholar
  156. Ziemienowicz A, Tinland B, Bryant J, Gloeckler V, Hohn B (2000) Plant enzymes but not Agrobacterium VirD2 mediate T-DNA ligation in vitro. Mol Cell Biol 20: 6317-6322PubMedGoogle Scholar
  157. Zupan J, Muth TR, Draper O, Zambryski PC (2000) The transfer of DNA from Agrobacterium tumefaciens into plants: a feast of fundamental insights. Plant J 23: 11-28PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Pieter Windels
    • 1
    • 2
  • Sylvie De Buck
    • 3
  • Ann Depicker
    • 3
  1. 1.Pioneer Hi-Bred InternationalBrusselsBelgium
  2. 2.Department of Plant Genetics and BreedingCentre for Agricultural ResearchMelleBelgium
  3. 3.Department of Plant Systems Biology, Flanders Interuniversity Institute for Biotechnology (VIB)Ghent UniversityGentBelgium

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