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. 1989 Apr;86(7):2133–2137. doi: 10.1073/pnas.86.7.2133

High levels of double-stranded transferred DNA (T-DNA) processing from an intact nopaline Ti plasmid.

T R Steck 1, T J Close 1, C I Kado 1
PMCID: PMC286865  PMID: 2928322

Abstract

To obtain bacterial-mediated oncogenic transformation of plants, the transferred DNA (T-DNA) of the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens is transferred to its plant host cells during infection. The initial phases of transformation involve the processing of the T-DNA in the bacterial cell after induction of the vir genes located on the Ti plasmid. The kinetics and conditions of this processing were examined and upon induction with acetosyringone up to 40% of the left and right borders of the T-DNA were cleaved. This cleavage was dependent upon virA, virG, and VirD and was rec-independent. Processed T-DNA was observed within 30 min after induction and was delayed by an increased concentration of phosphate in the induction medium. When DNA was isolated in the absence of protease treatment, the DNA fragment corresponding to the left side of the cut at both the left and right border region exhibited gel retardation, suggesting one or more "pilot" proteins may be involved in T-DNA transfer. Although the relative abundance of a processed product does not necessarily imply relative importance, the preponderance of double-stranded cleavage products suggests that double-stranded T-DNA should be considered as a possible intermediate in T-DNA transfer.

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Selected References

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

  1. Albright L. M., Yanofsky M. F., Leroux B., Ma D. Q., Nester E. W. Processing of the T-DNA of Agrobacterium tumefaciens generates border nicks and linear, single-stranded T-DNA. J Bacteriol. 1987 Mar;169(3):1046–1055. doi: 10.1128/jb.169.3.1046-1055.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Alt-Moerbe J., Rak B., Schröder J. A 3.6-kbp segment from the vir region of Ti plasmids contains genes responsible for border-sequence-directed production of T region circles in E. coli. EMBO J. 1986 Jun;5(6):1129–1135. doi: 10.1002/j.1460-2075.1986.tb04337.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chilton M. D., Saiki R. K., Yadav N., Gordon M. P., Quetier F. T-DNA from Agrobacterium Ti plasmid is in the nuclear DNA fraction of crown gall tumor cells. Proc Natl Acad Sci U S A. 1980 Jul;77(7):4060–4064. doi: 10.1073/pnas.77.7.4060. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Christie P. J., Ward J. E., Winans S. C., Nester E. W. The Agrobacterium tumefaciens virE2 gene product is a single-stranded-DNA-binding protein that associates with T-DNA. J Bacteriol. 1988 Jun;170(6):2659–2667. doi: 10.1128/jb.170.6.2659-2667.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Citovsky V., DE Vos G., Zambryski P. Single-Stranded DNA Binding Protein Encoded by the virE Locus of Agrobacterium tumefaciens. Science. 1988 Apr 22;240(4851):501–504. doi: 10.1126/science.240.4851.501. [DOI] [PubMed] [Google Scholar]
  6. Close T. J., Rogowsky P. M., Kado C. I., Winans S. C., Yanofsky M. F., Nester E. W. Dual control of Agrobacterium tumefaciens Ti plasmid virulence genes. J Bacteriol. 1987 Nov;169(11):5113–5118. doi: 10.1128/jb.169.11.5113-5118.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Close T. J., Tait R. C., Kado C. I. Regulation of Ti plasmid virulence genes by a chromosomal locus of Agrobacterium tumefaciens. J Bacteriol. 1985 Nov;164(2):774–781. doi: 10.1128/jb.164.2.774-781.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Das A. Agrobacterium tumefaciens virE operon encodes a single-stranded DNA-binding protein. Proc Natl Acad Sci U S A. 1988 May;85(9):2909–2913. doi: 10.1073/pnas.85.9.2909. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. 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]
  11. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  12. Gietl C., Koukolíková-Nicola Z., Hohn B. Mobilization of T-DNA from Agrobacterium to plant cells involves a protein that binds single-stranded DNA. Proc Natl Acad Sci U S A. 1987 Dec;84(24):9006–9010. doi: 10.1073/pnas.84.24.9006. [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. Jayaswal R. K., Veluthambi K., Gelvin S. B., Slightom J. L. Double-stranded cleavage of T-DNA and generation of single-stranded T-DNA molecules in Escherichia coli by a virD-encoded border-specific endonuclease from Agrobacterium tumefaciens. J Bacteriol. 1987 Nov;169(11):5035–5045. doi: 10.1128/jb.169.11.5035-5045.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kao J. C., Perry K. L., Kado C. I. Indoleacetic acid complementation and its relation to host range specifying genes on the Ti plasmid of Agrobacterium tumefaciens. Mol Gen Genet. 1982;188(3):425–432. doi: 10.1007/BF00330044. [DOI] [PubMed] [Google Scholar]
  16. Klapwijk P. M., van Beelen P., Schilperoort R. A. Isolation of a recombination deficient Agrobacterium tumefaciens mutant. Mol Gen Genet. 1979 Jun 7;173(2):171–175. doi: 10.1007/BF00330307. [DOI] [PubMed] [Google Scholar]
  17. Lundquist R. C., Close T. J., Kado C. I. Genetic complementation of Agrobacterium tumefaciens Ti plasmid mutants in the virulence region. Mol Gen Genet. 1984;193(1):1–7. doi: 10.1007/BF00327406. [DOI] [PubMed] [Google Scholar]
  18. Reed K. C., Mann D. A. Rapid transfer of DNA from agarose gels to nylon membranes. Nucleic Acids Res. 1985 Oct 25;13(20):7207–7221. doi: 10.1093/nar/13.20.7207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Rogowsky P. M., Close T. J., Chimera J. A., Shaw J. J., Kado C. I. Regulation of the vir genes of Agrobacterium tumefaciens plasmid pTiC58. J Bacteriol. 1987 Nov;169(11):5101–5112. doi: 10.1128/jb.169.11.5101-5112.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Stachel S. E., Nester E. W., Zambryski P. C. A plant cell factor induces Agrobacterium tumefaciens vir gene expression. Proc Natl Acad Sci U S A. 1986 Jan;83(2):379–383. doi: 10.1073/pnas.83.2.379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Stachel S. E., Timmerman B., Zambryski P. Activation of Agrobacterium tumefaciens vir gene expression generates multiple single-stranded T-strand molecules from the pTiA6 T-region: requirement for 5' virD gene products. EMBO J. 1987 Apr;6(4):857–863. doi: 10.1002/j.1460-2075.1987.tb04831.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Stachel S. E., Zambryski P. C. virA and virG control the plant-induced activation of the T-DNA transfer process of A. tumefaciens. Cell. 1986 Aug 1;46(3):325–333. doi: 10.1016/0092-8674(86)90653-7. [DOI] [PubMed] [Google Scholar]
  24. Usami S, Morikawa S, Takebe I, Machida Y. Absence in monocotyledonous plants of the diffusible plant factors inducing T-DNA circularization and vir gene expression in Agrobacterium. Mol Gen Genet. 1987 Sep;209(2):221–226. doi: 10.1007/BF00329646. [DOI] [PubMed] [Google Scholar]
  25. Veluthambi K., Jayaswal R. K., Gelvin S. B. Virulence genes A, G, and D mediate the double-stranded border cleavage of T-DNA from the Agrobacterium Ti plasmid. Proc Natl Acad Sci U S A. 1987 Apr;84(7):1881–1885. doi: 10.1073/pnas.84.7.1881. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Veluthambi K., Ream W., Gelvin S. B. Virulence genes, borders, and overdrive generate single-stranded T-DNA molecules from the A6 Ti plasmid of Agrobacterium tumefaciens. J Bacteriol. 1988 Apr;170(4):1523–1532. doi: 10.1128/jb.170.4.1523-1532.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Wang K., Stachel S. E., Timmerman B., VAN Montagu M., Zambryski P. C. Site-Specific Nick in the T-DNA Border Sequence as a Result of Agrobacterium vir Gene Expression. Science. 1987 Jan 30;235(4788):587–591. doi: 10.1126/science.235.4788.587. [DOI] [PubMed] [Google Scholar]
  28. Winans S. C., Kerstetter R. A., Nester E. W. Transcriptional regulation of the virA and virG genes of Agrobacterium tumefaciens. J Bacteriol. 1988 Sep;170(9):4047–4054. doi: 10.1128/jb.170.9.4047-4054.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. Yanofsky M. F., Porter S. G., Young C., Albright L. M., Gordon M. P., Nester E. W. The virD operon of Agrobacterium tumefaciens encodes a site-specific endonuclease. Cell. 1986 Nov 7;47(3):471–477. doi: 10.1016/0092-8674(86)90604-5. [DOI] [PubMed] [Google Scholar]
  31. Young C., Nester E. W. Association of the virD2 protein with the 5' end of T strands in Agrobacterium tumefaciens. J Bacteriol. 1988 Aug;170(8):3367–3374. doi: 10.1128/jb.170.8.3367-3374.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]

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