Skip to main content
SpringerLink
Log in

Site-specific mutagenesis in the TR-DNA region of octopine-type Ti plasmids

  • Published:
Plant Molecular Biology Aims and scope Submit manuscript

Abstract

Site-specific insertion and deletion mutations affecting all six of the eukaryotic-like genes in the TR-DNA region of the octopine-type Ti plasmids pTil5955 or pTiA6 have been generated. None of the mutations affected virulence or tumor morphology on sunflower. Mutations in the coding regions of two of the genes resulted in tumors without any detectable mannopine, mannopinic acid or agropine, and mutations in either the coding region or in the 3′ untranslated region of a third gene eliminated biosynthesis of agropine, but not mannopine or mannopinic acid. Detection of two previously unobserved silver nitrate-positive substance in tumors incited by one of the mutant strains, together with data on the presence of opines in tumors incited by coinoculation with mixtures of different mutant strains, allowed us to propose the functional order of all three genes involved in the biosynthesis of mannopine, mannopinic acid and agropine. TR-DNA was absent in tumors incited by anAgrobacterium tumefaciens strain harboring a Ti plasmid in which the right border of the TR-DNA region was deleted.

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

Access this article

Log in via an institution

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

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Appleyard RK: Segregation of new lysogenic types during growth of a doubly lysogenic strain derived fromEscherichia coli K12. Genetics 39:440, 1954.

    PubMed  CAS  Google Scholar 

  2. Barker RF, Idler KB, Thompson DV, Kemp JD: Nucleotide sequence of the T-DNA region from theAgrobacterium tumefaciens octopine Ti plasmid pTil5955. Plant Molecular Biology 2:335–350, 1983.

    Article  CAS  Google Scholar 

  3. Boyer HW, Roulland-Dussoix D: A complementation analysis of the restriction and modification of DNA inEscherichia coli. J Mol Biol 41:459, 1969.

    Article  CAS  PubMed  Google Scholar 

  4. Coxon DT, Davies AMC, Fenwick GR, Self R, Firmin JL, Lipkin D, Janes NF: Agropine, a new amino acid derivative from crown gall tumours. Tetrahedron Lett 21:495–498, 1980.

    Article  CAS  Google Scholar 

  5. Dahl GA, Guyon P, Petit A, Temé J: Silver nitrate-positive opines in crown gall tumors. Plant Sci Lett 32:193–203, 1983.

    Article  CAS  Google Scholar 

  6. De Beuckeleer M, Lemmers M, de Vos G, Willmitzer L, Van Montagu M, Schell J: Further insight on the transferred-DNA of octopine crown gall. Mol Gen Genet 183:283–288, 1981.

    Article  PubMed  Google Scholar 

  7. Ditta G, Stanfield S, Corbin D, Helinski DR: Broad host range DNA cloning system for gram-negative bacteria: Construction of a gene bank ofRhizobium meliloti. Proc Natl Acad Sci USA 77:7347–7351, 1980.

    CAS  PubMed  Google Scholar 

  8. Figurski D, Meyer R, Miller DS, Helinski DK: Generationin vitro of deletions in the broad host range plasmid RK2 using phage Mu insertions and a restriction endonuclease. Gene 1: 107–119, 1976.

    Article  CAS  PubMed  Google Scholar 

  9. Firmin JL, Fenwick GR: Agropine — a major new plasmid-determined metabolite in crown gall tumors. Nature (London) 276:842–844, 1978.

    Article  CAS  Google Scholar 

  10. Garfinkel DJ, Simpson RB, Ream LW, White FF, Gordon MP, Nester EW: Genetic analysis of crown gall: fine structure map of the T-DNA by site-directed mutagenesis. Cell 27:143–153, 1981.

    Article  CAS  PubMed  Google Scholar 

  11. Gelvin, SB, Thomashow MF, McPherson JC, Gordon MP, Nester EW: Sizes and map positions of several plasmid-DNA-encoded transcripts in octopine-type crown gall tumors. Proc Natl Acad Sci USA 79:76–80, 1982.

    CAS  PubMed  Google Scholar 

  12. Goldsbrough PB, Karcher SJ, Gelvin SB, Larkins BA: Introduction of a zein gene into the Ti plasmid ofAgrobacterium tumefaciens. In: Goldberg RB (ed) UCLA Symposia on Molecular and Cellular Biology New Series Volume 12 Plant Molecular Biology. Alan R. Liss, Inc., New York, 1983, pp. 35–43.

    Google Scholar 

  13. Gurley WB, Kemp JD, Albert MJ, Sutton DW, Callis J: Transcription of Ti-plasmid-derived sequences in three octopine type crown gall tumor lines. Proc Natl Acad Sci, USA 76:2828–2832, 1979.

    CAS  PubMed  Google Scholar 

  14. Hirsch PR, Beringer JE: A physical map of pPH1JI and pJB4JI. Plasmid 12:139–141, 1984.

    Article  CAS  PubMed  Google Scholar 

  15. Holsters M, De Waele D, Depicker A, Messens E, Van Montagu M, Schell J: Transfection and transformation ofAgrobacterium tumefaciens. Mol Gen Genet 163:181–187, 1978.

    Article  CAS  PubMed  Google Scholar 

  16. Jorgensen RA, Rothstein SJ, Reznikoff WS: A restriction enzyme cleavage map of Tn5 and location of a region encoding neomycin resistance. Mol Gen Genet 177:65–72, 1979.

    Article  CAS  PubMed  Google Scholar 

  17. Kahl G, Schell J: Molecular Biology of Plant Tumors. Academic Press, 1982.

  18. Karcher SJ, DiRita VJ, Gelvin SB: Transcript analysis of TR-DNA in octopine-type crown gall tumors. Mol Gen Genet 194:159–164, 1984.

    Article  CAS  Google Scholar 

  19. Leemans J, Deblaere R, Willmitzer L, de Greve H, Hernalsteens JP, Van Montagu M, Schell J: Genetic identification of functions of TL-DNA transcripts in octopine crown galls. EMBO J 1:147–152, 1982.

    CAS  PubMed  Google Scholar 

  20. Linsmaier EM, Skoog F: Organic growth factor requirements of tobacco tissue cultures. Physiol Plant 18:100–101. 1965.

    Article  CAS  Google Scholar 

  21. Messing J: New M13 vectors for cloning. In: Wu R, Grossman L, Moldave K (eds) Methods in Enzymology Volume 101 Recombinant DNA Part C. Academic Press, 1983, pp 20–78.

  22. Murai N, Kemp JD: T-DNA of pTil5955 fromAgrobacterium tumefaciens is transcribed into a minimum of seven polyadenylated RNAs in a sunflower crown gall tumor. Nucl Acids Res 10:1679–1689, 1982.

    CAS  PubMed  Google Scholar 

  23. Murai N, Sutton DW, Murray MG, Slightom JL, Merlo DJ, Reichert NA, Sengupta-Gopalan C, Stock CA, Barker RF, Kemp JD, Hall TC: Phaseolin gene from bean is expressed after transfer to sunflower via tumor-inducing plasmid vectors. Science 222:476–482, 1983.

    CAS  PubMed  Google Scholar 

  24. Oka A, Nomura N, Sugimoto K, Sugisaki H, Takanami M: Nucleotide sequence at the insertion sites of a kanamycin transposon. Nature (London) 276:845–847, 1978.

    Article  CAS  Google Scholar 

  25. Ooms G, Hooykaas PJJ, Moolenaar G, Schilperoort RA: Crown gall plant tumors of abnormal morphology, induced byAgrobacterium tumefaciens carrying mutated octopine Ti plasmids; analysis of T-DNA function. Gene 14:33–50, 1981.

    Article  CAS  PubMed  Google Scholar 

  26. Petit A, David C, Dahl GA, Ellis GJ, Guyon P, Casse-Delbart F, Tempé J: Further extension of the opine concept: plasmids inAgrobacterium rhizogenes cooperate for opine degredation. Mol Gen Genet 190:204–214, 1983.

    Article  CAS  Google Scholar 

  27. Ruvkun GB, Ausubel FM: A general method for site-directed mutagenesis in prokaryotes. Nature (London) 289:85–88, 1981.

    Article  CAS  Google Scholar 

  28. Salomon F, Deblaere R, Leemans J, Hernalsteens J-P, Van Montagu M, Schell J: Genetic identification of functions of TR-DNA transcripts in octopine crown galls. EMBO J 3:141–146, 1984.

    CAS  PubMed  Google Scholar 

  29. Southern EM: Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503–517, 1975.

    CAS  PubMed  Google Scholar 

  30. Tate ME, Ellis JG, Kerr A, Tempé J, Murray KE, Shaw KJ: Agropine: a revised structure. Carbohydrate Res 104:105–120, 1982.

    Article  CAS  Google Scholar 

  31. Thomashow MF, Nutter R, Montoya AL, Gordon MP, Nester EW: Integration and organization of Ti-plasmid sequences in crown gall tumors. Cell 19:729–739, 1980.

    Article  CAS  PubMed  Google Scholar 

  32. Vogelstein B, Gillespie D: Preparative and analytical purification of DNA from agarose. Proc Natl Acad Sci USA 76:615–619, 1979.

    CAS  PubMed  Google Scholar 

  33. Willmitzer L, Dhaese P, Schreier PH, Schmalenbach W, Van Montagu M, Schell J: Size, location and polarity of T-DNA encoded transcripts in nopaline crown gall tumors; common transcripts in octopine and nopaline tumors. Cell 32:1045–1056, 1983.

    Article  CAS  PubMed  Google Scholar 

  34. Winter JA, Wright RL, Gurley WB: Map locations of five transcripts homologous to TR-DNA in tobacco and sunflower crown gall tumors. Nucl Acids Res 12:2391–2406, 1984.

    CAS  PubMed  Google Scholar 

  35. Wood WB: Host specificity of DNA produced byEscherichia coli: Bacterial mutations affected the restriction and modification of DNA. J Mol Biol 16:118, 1966.

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Molecular Biology and Plant Pathology, University of Wisconsin, 53706, Madison, WI, USA

    C. T. Komro

  2. Department of Biological Sciences, Purdue University, 47907, West Lafayette, IN, USA

    V. J. DiRita & S. B. Gelvin

  3. Agrigenetics Advanced Research Division, 5649 E. Buckeye Road, 53716, Madison, WI, USA

    J. D. Kemp

Authors
  1. C. T. Komro
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. V. J. DiRita
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. S. B. Gelvin
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. J. D. Kemp
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Komro, C.T., DiRita, V.J., Gelvin, S.B. et al. Site-specific mutagenesis in the TR-DNA region of octopine-type Ti plasmids. Plant Mol Biol 4, 253–263 (1985). https://doi.org/10.1007/BF02418244

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF02418244

Keywords

Search

Navigation