Molecular and General Genetics MGG

, Volume 204, Issue 3, pp 383–396 | Cite as

The promoter of TL-DNA gene 5 controls the tissue-specific expression of chimaeric genes carried by a novel type of Agrobacterium binary vector

  • Csaba Koncz
  • Jeff Schell


A “plant gene vector cassette” to be used in combination with various Escherichia coli gene-cloning vectors was constructed. This cassette contains a replication and mobilization unit which allows it to be maintained and to be transferred back and forth between E. coli and Agrobacterium tumefaciens hosts provided these hosts contain plasmid RK2 replication and mobilization helper functions. The cassette also harbors a transferable DNA unit with plant selectable marker genes and cloning sites which can be combined with different bacterial replicons, thus facilitating the reisolation of transferred DNA from transformed plants in E. coli. The vector cassette contains two different promoters derived from the T-DNA-encoded genes 5 and nopaline synthase (NOS). By comparing the levels of expression of the marker enzymes linked to each of these promoter sequences, it was found that the gene 5 promoter is active in a tissue-specific fashion whereas this is not the case for the NOS promoter. This observation provides the first documented instance of a gene derived from a procaryotic host the expression of which is apparently regulated by plant growth factors.

Key words

Agrobacterium binary vector system Plant transformation Tissue specificity T-DNA-encoded genes 



octopine synthase (gene)


nopaline synthase (gene)


neomycin phosphotransferase (gene) of transposon Tn5


Ti-plasmid region encoding virulence functions


















basis of mobilization


origin of conjugational plasmid transfer

Tra, Mob

functions required for conjugational transfer of plasmids




α-naphthaleneacetic acid


N-cetyl-N,N,N-trimethyl-ammonium bromide


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  1. An G, Watson BD, Stachel S, Gordon MP, Nester EW (1985) New cloning vehicles for transformation of higher plants. EMBO J 4:277–284Google Scholar
  2. Barker RF, Idler KB, Thompson DV, Kemp PJD (1983) Nucleotide sequence of the T-DNA region from Agrobacterium tumefaciens octopine Ti plasmid pTi 15955. Plant Mol Biol 2:335–350Google Scholar
  3. Beck E, Ludwig G, Auerswald EA, Reiss B, Schaller H (1982) Nucleotide sequence and exact localization of the neomycin phototransferase gene from transposon Tn5. Gene 19:327–336Google Scholar
  4. Bedbrook J (1981) A plant nuclear DNa preparation procedure. Plant Mol Biol News Lett 2:3–4Google Scholar
  5. Bevan M (1984) Binary Agrobacterium vectors for plant transformation. Nucleic Acids Res 12:8711–8721Google Scholar
  6. Bolivar F, Rodriguez R, Greene PJ, Betlach M, Heynecker HL, Boyer HW, Crosa J, Falkow S (1977) Construction and characterization of new cloning vehicles II. A multipurpose cloning system. Gene 2:95–113Google Scholar
  7. Bolivar F (1978) Construction and characterization of new cloning vehicles III. Derivatives of plasmid pBR322 carrying unique EcoRI sites for selection of EcoRI generated recombinant molecules. Gene 4:121–136Google Scholar
  8. Boyer HW, Roulland-Dussoix D (1969) A complementation analysis of the restriction and modification of DNA in E. coli. J Mol Biol 41:459–468Google Scholar
  9. Bradford M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–251Google Scholar
  10. Caplan A, Herrera-Estrella L, Inze D, Van Haute E, Van Montagu M, Schell J, Zambryski P (1983) Introduction of genetic material into plant cells. Science 222:815–821Google Scholar
  11. Deblaere R, Bytebier B, De Greve H, Deboeck F, Schell J, Montagu M, Leemans J (1985) Efficient octopine Ti plasmid-derived vectors for Agrobacterium-mediated gene transfer in plants. Nucleic Acids Res 13:4777–4788Google Scholar
  12. De Block M, Herrera-Estrella L, Van Montagu M, Schell J, Zambryski P (1984) Expression of foreign genes in regenerated plants and in their progeny. EMBO J 3:1681–1690Google Scholar
  13. De Framond A, Barton KA, Chilton M-D (1983) Mini-Ti: A new vector strategy for plant genetic engineering. Bio/technology 1:262–272Google Scholar
  14. Dellaporta SL, Wood J, Hicks JB (1983) A plant DNA minipreparation: version II. Plant Mol Biol Rep 1:19–21Google Scholar
  15. Depicker A, De Block M, Inze D, Van Montagu M, Schell J (1980) IS-like element IS8 in RP4 plasmid and its involvement in cointegration. Gene 10:329–338Google Scholar
  16. De Vos G, De Beuckeleer M, Van Montagu M, Schell J (1981) Restriction endonuclease mapping of the octopine tumor-inducing plasmid pTiAch5 of Agrobacterium tumefaciens. Plasmid 6:249–253Google Scholar
  17. Ditta G, Stanfield S, Corbin D, Helinski DR (1980) Broad host range DNA cloning system for Gram-negative bacteria: Construction of gene bank of Rhizobium meliloti. Proc Natl Acad Sci USA 77:7347–7351Google Scholar
  18. Eckes P, Schell J, Willmitzer L (1985) Organ-specific expression of three leaf/stem specific cDNAs from potato is regulated by light and correlated with chloroplast development. Mol Gen Genet 199:216–224Google Scholar
  19. Engler G, Depicker A, Maehaut R, Villarroel-Mandiola R, Van Montagu M, Schell J (1981) Physical mapping of DNA sequence homologies between an octopine and a nopaline Ti plasmid of Agrobacterium tumefaciens. J Mol Biol 152:183–208Google Scholar
  20. Facciotti D, O'Neal KJ, Lee S, Shewmaker CK (1985) Light-inducible expression of a chimeric gene in soybean tissue transformed with Agrobacterium. Bio/technology 3:241–246Google Scholar
  21. Figurski DH, Helinski DR (1979) Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci USA 76:1648–1652Google Scholar
  22. Finnegan J, Sherratt D (1982) Plasmid ColE1 conjugal mobility: the nature of bom, a region required in cis for transfer. Mol Gen Genet 185:344–351Google Scholar
  23. Friedman AM, Long ShR, Brown SE, Buikema WJ, Ausubel FM (1982) Construction of a broad host range cosmid cloning vector and its use in the genetic analysis of Rhizobium mutants. Gene 18:289–296Google Scholar
  24. Gielen J, De Beuckeleer 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 3:835–846Google Scholar
  25. Herrera-Estrella L, Depicker A, Van Montagu M, Schell J (1983a) Expression of chimeric genes transferred into plant cells using a Ti-plasmid derived vector. Nature 303:209–213Google Scholar
  26. Herrera-Estrella L, De Block M, Messens E, Hernalsteens JP, Van Montagu M, Schell J (1983b) Chimeric genes as dominant selectable markers in plant cells. EMBO J 2:987–995Google Scholar
  27. Hille J, Van Kan J, Schilperoort R (1984) Trans-Acting virulence functions of the octopine Ti plasmid from Agrobacterium tumefaciens. J Bacteriol 158:754–756Google Scholar
  28. Hoekema A, Hirsch PR, Hooykaas PJJ, Schilperoort RA (1983) A binary plant vector strategy based on separation of Vir- and T-region of the Agrobacterium tumefaciens Ti-plasmid. Nature 303:179–180Google Scholar
  29. Hoekema A, Roelvink PW, Hooykaas PJJ, Schilperoort RA (1984) Delivery of T-DNA from the Agrobacterium tumefaciens chromosome into plant cells. EMBO J 3:2485–2490Google Scholar
  30. Holsters M, Silva B, Van Vliet F, Genetello C, De Block M, Dhaese P, Depicker A, Inze D, Engler G, Villarroel R, Van Montagu M, Schell J (1980) The functional organization of the nopaline A. tumefaciens plasmid pTiC58. Plasmid 3:212–230Google Scholar
  31. Hohn B, Collins J (1980) A small cosmid for efficient cloning of large DNA fragments. Gene 11:291–298Google Scholar
  32. Horsch RB, Fry JE, Hoffman NL, Eichholz D, Rogers SG, Fraley RT (1985) A simple and general method for transferring genes into plants. Science 227:1229–1231Google Scholar
  33. Joos H, Inze D, Caplan A, Sormann M, Van Montagu M, Schell J (1983) Genetic analysis of T-DNA transcripts in nopaline crown galls. Cell 32:1057–1067Google Scholar
  34. Kahl G, Schell J (1982) Molecular biology of plant tumours. Academic Press, New YorkGoogle Scholar
  35. Kaulen H, Schell J, Kreuzaler F (1986) Light-induced expression of the chimeric chalcone synthase-NPTII gene in tobacco cells. EMBO J 5:1–8Google Scholar
  36. Kemper E, Waffenschmidt S, Weiler EW, Rausch I, Schröder J (1985) T-DNA encoded auxin formation in crown gall cells. Planta 163:257–263Google Scholar
  37. Klapwijk PM, Van Beelen P, Schilperoort RA (1979) Isolation of a recombination deficient Agrobacterium tumefacient. Mol Gen Genet 173:171–175Google Scholar
  38. Klee HJ, White FF, Iyer VN, Gordon MP, Nester EW (1983) Mutational analysis of the virulence region of an Agrobacterium tumefaciens Ti plasmid. J Bacteriol 153:878–882Google Scholar
  39. Klee HJ, Yanofsky MF, Nester EW (1985) Vectors for transformation of higher plants. Bio/technology 3:637–642Google Scholar
  40. Koncz C, De Greve H, Andre D, Deboeck F, Van Montagu M, Schell J (1983) The opine synthase genes carried by Ti plasmids contain all signals necessary for expression in plants. EMBO J 2:1597–1603Google Scholar
  41. Koncz C, Kreuzaler F, Kálmán Zs, Schell J (1984) A simple method to transfer, integrate and study expression of foreign genes, such as chicken ovalbumin and α-actin in plant tumors. EMBO J 3:1929–1937Google Scholar
  42. Koulikova-Nicola Z, Shillito R, Hohn B, Wang K, Van Montagu M, Zambryski P (1985) Involvement of circular intermediates in the transfer of T-DNA from Agrobacterium tumefaciens to plant cells. Nature 313:191–196Google Scholar
  43. Lamppa G, Nagy F, Chua NH (1985) Light-regulated and organspecific expression of a wheat Cab gene in transgenic tobacco. Nature 316:750–752Google Scholar
  44. Leemans J, Shaw Ch, Deblaere R, De Greve H, Hernalsteens JP, Maes M, Van Montagu M, Schell J (1982a) Site-specific mutagenesis of Agrobacterium Ti plasmids and transfer of genes to plant cells. J Mol Appl Genet 1:149–164Google Scholar
  45. Leemans J, Deblaere R, Willmitzer L, De Greve H, Hernalsteens JP, Van Montagu M, Schell J (1982b) Genetic identification of functions of TL-DNA transcripts in octopine crown galls. EMBO J 1:147–152Google Scholar
  46. Leemans J, Langenakens J, De Greve H, Deblaere R, Van Montagu M, Schell J (1982c) Broad-host-range cloning vectors derived from the W-plasmid Sa. Gene 19:361–363Google Scholar
  47. Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning, a laboratory manual. Cold Spring Harbor Laboratory, NYGoogle Scholar
  48. Márton L, Wullems GJ, Molendjik L, Schilperoort RA (1982) In vitro transformation of cultured cells from Nicotiana tabacum by Agrobacterium tumefaciens. Nature 277:129–131Google Scholar
  49. Márton L (1984) Transformation of tobacco cells by coculture with Agrobacterium tumefaciens. In: Vasil IK (ed) Cell culture and somatic cell genetics of plants, vol 1. Academic Press, New York, p 514–521Google Scholar
  50. Murai N, Sutton DW, Murray MG, Slightom JL, Merlo DJ, Reichert NA, Sengupta-Gopalan C, Stock CA, Barker RF, Kemp JD, Hall TC (1983) Phaseolin gene from bean is expressed after transfer to sunflower via tumor-inducing plasmid vectors. Science 222:476–482Google Scholar
  51. Odell JT, Nagy F, Chua NM (1985) Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter. Nature 313:810–812Google Scholar
  52. Otten LA, Schilperoort RA (1978) A rapid microscale method for the detection of lysopine- and nopaline dehydrogenase activities. Biochim Biophys Acta 527:497–500Google Scholar
  53. Otten L (1982) Lysopine dehydrogenase as an early marker in crown gall formation. Plant Sci Lett 25:15–27Google Scholar
  54. Rao NR, Rogers SG (1979) Plasmid pKC7: A vector containing ten restriction endonuclease sites suitable for cloning DNA sequences. Gene 7:79–82Google Scholar
  55. Reiss B (1982) Das Neomycin-Phosphotransferase-Gen aus Transposon Tn5. Inaugural-Dissertation, Ruprecht-Karls-Universität, HeidelbergGoogle Scholar
  56. Schell J, Van Montagu M, Willmitzer L, Leemans J, Deblaere R, Joos H, Inze D, Wöstemeyer A, Otten L, Zambryski P (1984) Transfer of foreign genes to plants and its use to study developmental processes. In: Beers RF, Bassett EG (eds) Cell fusion: gene transfer and transformation. Raven Press, New YorkGoogle Scholar
  57. Schmidhauser TJ, Filutowicz M, Helinski DR (1983) Replication of derivatives of the broad host range plasmid RK2 in two distantly related bacteria. Plasmid 9:325–330Google Scholar
  58. Schmidhauser TJ, Helinski DR (1985) Regions of broad-host-range plasmid RK2 involved in replication and stable maintenance in nine species of Gram-negative bacteria. J Bacteriol 164:446–455Google Scholar
  59. Schreier P, Seftor EA, Schell J, Bohnert HJ (1985) The use of nuclear-encoded sequences to direct the light-regulated synthesis and transport of a foreign protein into plant chloroplast. EMBO J 4:25–32Google Scholar
  60. Schröder G, Waffenschmidt S, Weiler EW, Schröder J (1984) The T-region of Ti plasmids codes for an enzyme synthesizing indole-3-acetic acid. Eur J Biochem 138:387–391Google Scholar
  61. Seed B (1983) Purification of genomic sequences from bacteriophage libraries by recombination and selection in vivo. Nucleic Acids Res 11:2427–2445Google Scholar
  62. Simon R, Preifer U, Pühler A (1983) A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram-negative bacteria. Bio/technology 1:784–791Google Scholar
  63. Simpson J, Timko M, Cashmore AR, Schell J, Van Montagu M, Herrera-Estrella L (1985) Light-inducible and tissue specific expression of a chimeric gene under control of a pea chlorophyll a/b-binding protein gene. EMBO J 4:2723–2729Google Scholar
  64. Spena A, Hain R, Ziervogel U, Saedler H, Schell J (1985) Construction of a heat-inducible gene for plants. Demonstration of heatinducible activity of the Drosophila hsp70 promoter in plants. EMBO J 4:2739–2743Google Scholar
  65. Stachel SE, An G, Flores C, Nester EW (1985a) A Tn3 lacZ transposon for the random generation of β-galactosidase gene fusions: application to the analysis of gene expression in Agrobacterium. EMBO J 4:891–898Google Scholar
  66. Stachel SE, Messens E, Van Montagu M, Zambryski P (1985b) Identification of the signal molecules produced by wounded plant cells that activate T-DNA transfer in Agrobacterium tumefaciens. Nature 318:624–630Google Scholar
  67. Taylor B, Powell A (1983) Isolation of plant DNA and RNA. BRL Focus 4:4–6Google Scholar
  68. Thomas ChM (1981) Complementation analysis of replication of maintenance functions of broad host range plasmids RK2 and RP1. Plasmid 5:277–291Google Scholar
  69. Thomas ChM (1983) Instability of a high-copy number mutant of a miniplasmid derived from broad host range IncP plasmid RK2. Plasmid 10:184–195Google Scholar
  70. Van den Elzen P, Lee KY, Townsend J, Bedbrook J (1985) Simple binary vectors for DNA transfer to plant cells. Plant Mol Biol 5:149–154Google Scholar
  71. Van Haute E, Joos H, Maes M, Warren G, Van Montagu M, Schell J (1983) Intergeneric transfer and exchange recombination of restriction fragments cloned in pBR322: a novel strategy for the reversed genetics of the Ti plasmids of Agrobacterium tumefaciens. EMBO J 2:411–417Google Scholar
  72. Van Larebeke N, Engler G, Holsters M, Van den Elsacker S, Zaenen J, Schilperoort RA, Schell J (1974) Large plasmid in Agrobacterium tumefaciens essential for crown gall-inducing ability. Nature 252:169–170Google Scholar
  73. Verma PDS, Hohn TH (1984) Genes involved in microbe-plant interaction. Springer, Berlin Heidelberg New York TokyoGoogle Scholar
  74. Wang K, Herrera-Estrella L, Van Montagu M, Zambryski P (1984) Right 25 bp terminus sequence 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
  75. Willmitzer L, Simons G, Schell J (1982) The TL-DNA in octopine crown-gall tumors codes for seven well-defined polyadenylated transcripts. EMBO J 1:139–146Google Scholar
  76. Willmitzer L, Dhaese P, Schreier PM, Schmalenbach W, Van Montagu M, Schell J (1983) Size, location and polarity of T-DNA-encoded transcripts in nopaline crown gall tumors; common transcripts in octopine and nopaline tumors. Cell 32:1045–1056Google Scholar

Copyright information

© Springer-Verlag 1986

Authors and Affiliations

  • Csaba Koncz
    • 1
    • 2
  • Jeff Schell
    • 2
  1. 1.Institute of GeneticsBiological Research Center of Hungarian Academy of SciencesSzegedHungary
  2. 2.Max-Planck-Institut für ZüchtungsforschungKöln 30Federal Republic of Germany

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