Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1983;2(12):2151–2160. doi: 10.1002/j.1460-2075.1983.tb01716.x

Genetic analysis of transfer and stabilization of Agrobacterium DNA in plant cells

H Joos 1, B Timmerman 1, M Van Montagu 1,*, J Schell 1,2,*
PMCID: PMC555427  PMID: 16453483

Abstract

In an attempt to elucidate the transfer and integration mechanism of Agrobacterium DNA upon crown gall induction, we translocated a borderless T-DNA to different sites of the C58 Ti plasmid. As a result of the physical linkage of the T-DNA onc genes with other Ti plasmid functions, the concerned strain retained tumor-inducing capacity. However, when the borderless T-DNA is separated on an independent replicon while all other pTi functions are provided in trans, the strain can no longer induce tumors on plants. We provide evidence that the right T-DNA border region harbors one or more in cis active functions essential in the transfer and/or stabilization of the T-DNA into plant cells. The strains used in these experiments allowed us to conclude that some function(s) of the Ti plasmid can induce plant cell proliferations independently of the T-DNA transformation event. The results described here indicate that other Ti plasmid sequences than solely the T-region can be transferred to plant cells.

Keywords: Agrobacterium, vir-region, T-DNA integration, translocation

Full text

PDF
2160

Images in this article

2153Fig. 2.
on p.2153
2154Fig. 3.
on p.2154
2155Fig. 4.
on p.2155

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Barton K. A., Binns A. N., Matzke A. J., Chilton M. D. Regeneration of intact tobacco plants containing full length copies of genetically engineered T-DNA, and transmission of T-DNA to R1 progeny. Cell. 1983 Apr;32(4):1033–1043. doi: 10.1016/0092-8674(83)90288-x. [DOI] [PubMed] [Google Scholar]
  2. Bomhoff G., Klapwijk P. M., Kester H. C., Schilperoort R. A., Hernalsteens J. P., Schell J. Octopine and nopaline synthesis and breakdown genetically controlled by a plasmid of Agrobacterium tumefaciens. Mol Gen Genet. 1976 May 7;145(2):177–181. doi: 10.1007/BF00269591. [DOI] [PubMed] [Google Scholar]
  3. CLARK A. J., ADELBERG E. A. Bacterial conjugation. Annu Rev Microbiol. 1962;16:289–319. doi: 10.1146/annurev.mi.16.100162.001445. [DOI] [PubMed] [Google Scholar]
  4. Chilton M. D., Drummond M. H., Merio D. J., Sciaky D., Montoya A. L., Gordon M. P., Nester E. W. Stable incorporation of plasmid DNA into higher plant cells: the molecular basis of crown gall tumorigenesis. Cell. 1977 Jun;11(2):263–271. doi: 10.1016/0092-8674(77)90043-5. [DOI] [PubMed] [Google Scholar]
  5. De Greve H., Decraemer H., Seurinck J., Van Montagu M., Schell J. The functional organization of the octopine Agrobacterium tumefaciens plasmid pTiB6s3. Plasmid. 1981 Sep;6(2):235–248. doi: 10.1016/0147-619x(81)90069-x. [DOI] [PubMed] [Google Scholar]
  6. Depicker A., De Wilde M., De Vos G., De Vos R., Van Montagu M., Schell J. Molecular cloning of overlapping segments of the nopaline Ti-plasmid pTiC58 as a means to restriction endonuclease mapping. Plasmid. 1980 Mar;3(2):193–211. doi: 10.1016/0147-619x(80)90109-2. [DOI] [PubMed] [Google Scholar]
  7. Dhaese P., De Greve H., Decraemer H., Schell J., Van Montagu M. Rapid mapping of transposon insertion and deletion mutations in the large Ti-plasmids of Agrobacterium tumefaciens. Nucleic Acids Res. 1979 Dec 11;7(7):1837–1849. doi: 10.1093/nar/7.7.1837. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Douglas C. J., Halperin W., Nester E. W. Agrobacterium tumefaciens mutants affected in attachment to plant cells. J Bacteriol. 1982 Dec;152(3):1265–1275. doi: 10.1128/jb.152.3.1265-1275.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Engler G., Depicker A., Maenhaut R., Villarroel R., Van Montagu M., Schell J. Physical mapping of DNA base sequence homologies between an octopine and a nopaline Ti plasmid of Agrobacterium tumefaciens. J Mol Biol. 1981 Oct 25;152(2):183–208. doi: 10.1016/0022-2836(81)90239-4. [DOI] [PubMed] [Google Scholar]
  10. Garfinkel D. J., Nester E. W. Agrobacterium tumefaciens mutants affected in crown gall tumorigenesis and octopine catabolism. J Bacteriol. 1980 Nov;144(2):732–743. doi: 10.1128/jb.144.2.732-743.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Garfinkel D. J., Simpson R. B., Ream L. W., White F. F., Gordon M. P., Nester E. W. Genetic analysis of crown gall: fine structure map of the T-DNA by site-directed mutagenesis. Cell. 1981 Nov;27(1 Pt 2):143–153. doi: 10.1016/0092-8674(81)90368-8. [DOI] [PubMed] [Google Scholar]
  12. Guyon P., Chilton M. D., Petit A., Tempé J. Agropine in "null-type" crown gall tumors: Evidence for generality of the opine concept. Proc Natl Acad Sci U S A. 1980 May;77(5):2693–2697. doi: 10.1073/pnas.77.5.2693. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Holsters M., Silva B., Van Vliet F., Genetello C., De Block M., Dhaese P., Depicker A., Inzé D., Engler G., Villarroel R. The functional organization of the nopaline A. tumefaciens plasmid pTiC58. Plasmid. 1980 Mar;3(2):212–230. doi: 10.1016/0147-619x(80)90110-9. [DOI] [PubMed] [Google Scholar]
  14. Iyer V. N., Klee H. J., Nester E. W. Units of genetic expression in the virulence region of a plant tumor-inducing plasmid of Agrobacterium tumefaciens. Mol Gen Genet. 1982;188(3):418–424. doi: 10.1007/BF00330043. [DOI] [PubMed] [Google Scholar]
  15. Joos H., Inzé D., Caplan A., Sormann M., Van Montagu M., Schell J. Genetic analysis of T-DNA transcripts in nopaline crown galls. Cell. 1983 Apr;32(4):1057–1067. doi: 10.1016/0092-8674(83)90290-8. [DOI] [PubMed] [Google Scholar]
  16. Klee H. J., White F. F., Iyer V. N., Gordon M. P., Nester E. W. Mutational analysis of the virulence region of an Agrobacterium tumefaciens Ti plasmid. J Bacteriol. 1983 Feb;153(2):878–883. doi: 10.1128/jb.153.2.878-883.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Leemans J., Deblaere R., Willmitzer L., De Greve H., Hernalsteens J. P., Van Montagu M., Schell J. Genetic Identification of functions of TL-DNA transcripts in octopine crown galls. EMBO J. 1982;1(1):147–152. doi: 10.1002/j.1460-2075.1982.tb01138.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Leemans J., Langenakens J., De Greve H., Deblaere R., Van Montagu M., Schell J. Broad-host-range cloning vectors derived from the W-plasmid Sa. Gene. 1982 Oct;19(3):361–364. doi: 10.1016/0378-1119(82)90027-0. [DOI] [PubMed] [Google Scholar]
  19. Leemans J., Shaw C., Deblaere R., De Greve H., Hernalsteens J. P., Maes M., Van Montagu M., Schell J. Site-specific mutagenesis of Agrobacterium Ti plasmids and transfer of genes to plant cells. J Mol Appl Genet. 1981;1(2):149–164. [PubMed] [Google Scholar]
  20. Lemmers M., De Beuckeleer M., Holsters M., Zambryski P., Depicker A., Hernalsteens J. P., Van Montagu M., Schell J. Internal organization, boundaries and integration of Ti-plasmid DNA in nopaline grown gall tumours. J Mol Biol. 1980 Dec 15;144(3):353–376. doi: 10.1016/0022-2836(80)90095-9. [DOI] [PubMed] [Google Scholar]
  21. O'Farrell P. H., Kutter E., Nakanishi M. A restriction map of the bacteriophage T4 genome. Mol Gen Genet. 1980;179(2):421–435. doi: 10.1007/BF00425473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Ooms G., Klapwijk P. M., Poulis J. A., Schilperoort R. A. Characterization of Tn904 insertions in octopine Ti plasmid mutants of Agrobacterium tumefaciens. J Bacteriol. 1980 Oct;144(1):82–91. doi: 10.1128/jb.144.1.82-91.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Schell J., Van Montagu M., De Beuckeleer M., De Block M., Depicker A., De Wilde M., Engler G., Genetello C., Hernalsteens J. P., Holsters M. Interactions and DNA transfer between Agrobacterium tumefaciens, the Ti-plasmid and the plant host. Proc R Soc Lond B Biol Sci. 1979 Apr 11;204(1155):251–266. doi: 10.1098/rspb.1979.0026. [DOI] [PubMed] [Google Scholar]
  24. Simpson R. B., O'Hara P. J., Kwok W., Montoya A. L., Lichtenstein C., Gordon M. P., Nester E. W. DNA from the A6S/2 crown gall tumor contains scrambled Ti-plasmid sequences near its junctions with plant DNA. Cell. 1982 Jul;29(3):1005–1014. doi: 10.1016/0092-8674(82)90464-0. [DOI] [PubMed] [Google Scholar]
  25. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  26. Thomashow M. F., Nutter R., Postle K., Chilton M. D., Blattner F. R., Powell A., Gordon M. P., Nester E. W. Recombination between higher plant DNA and the Ti plasmid of Agrobacterium tumefaciens. Proc Natl Acad Sci U S A. 1980 Nov;77(11):6448–6452. doi: 10.1073/pnas.77.11.6448. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Van Larebeke N., Genetello C., Schell J., Schilperoort R. A., Hermans A. K., Van Montagu M., Hernalsteens J. P. Acquisition of tumour-inducing ability by non-oncogenic agrobacteria as a result of plasmid transfer. Nature. 1975 Jun 26;255(5511):742–743. doi: 10.1038/255742a0. [DOI] [PubMed] [Google Scholar]
  28. Watson B., Currier T. C., Gordon M. P., Chilton M. D., Nester E. W. Plasmid required for virulence of Agrobacterium tumefaciens. J Bacteriol. 1975 Jul;123(1):255–264. doi: 10.1128/jb.123.1.255-264.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Willmitzer L., Dhaese P., Schreier P. H., 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. 1983 Apr;32(4):1045–1056. doi: 10.1016/0092-8674(83)90289-1. [DOI] [PubMed] [Google Scholar]
  30. Willmitzer L., Simons G., Schell J. The TL-DNA in octopine crown-gall tumours codes for seven well-defined polyadenylated transcripts. EMBO J. 1982;1(1):139–146. doi: 10.1002/j.1460-2075.1982.tb01137.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Yadav N. S., Vanderleyden J., Bennett D. R., Barnes W. M., Chilton M. D. Short direct repeats flank the T-DNA on a nopaline Ti plasmid. Proc Natl Acad Sci U S A. 1982 Oct;79(20):6322–6326. doi: 10.1073/pnas.79.20.6322. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Zaenen I., Van Larebeke N., Van Montagu M., Schell J. Supercoiled circular DNA in crown-gall inducing Agrobacterium strains. J Mol Biol. 1974 Jun 15;86(1):109–127. doi: 10.1016/s0022-2836(74)80011-2. [DOI] [PubMed] [Google Scholar]
  33. Zambryski P., Depicker A., Kruger K., Goodman H. M. Tumor induction by Agrobacterium tumefaciens: analysis of the boundaries of T-DNA. J Mol Appl Genet. 1982;1(4):361–370. [PubMed] [Google Scholar]
  34. Zambryski P., Holsters M., Kruger K., Depicker A., Schell J., Van Montagu M., Goodman H. M. Tumor DNA structure in plant cells transformed by A. tumefaciens. Science. 1980 Sep 19;209(4463):1385–1391. doi: 10.1126/science.6251546. [DOI] [PubMed] [Google Scholar]
  35. Zambryski P., Joos H., Genetello C., Leemans J., Montagu M. V., Schell J. Ti plasmid vector for the introduction of DNA into plant cells without alteration of their normal regeneration capacity. EMBO J. 1983;2(12):2143–2150. doi: 10.1002/j.1460-2075.1983.tb01715.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES