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. 1990 Aug;172(8):4432–4440. doi: 10.1128/jb.172.8.4432-4440.1990

Overexpression of virD1 and virD2 genes in Agrobacterium tumefaciens enhances T-complex formation and plant transformation.

K Wang 1, A Herrera-Estrella 1, M Van Montagu 1
PMCID: PMC213272  PMID: 2165478

Abstract

The VirD1 and VirD2 proteins encoded by an inducible locus of the virulence (vir) region of the Agrobacterium tumefaciens Ti plasmid are required for site-specific nicking at T-DNA border sites. We have determined the nucleotide sequence of a 3.6-kilobase-pair fragment carrying the virD locus from nopaline Ti plasmid pTiC58. In contrast to the previous report (Hagiya et al., Proc. Natl. Acad. Sci. USA 82:2669-2673, 1985), we found that the first three open reading frames were capable of encoding polypeptides of 16.1, 49.7, and 21.4 kilodaltons. Deletion analysis demonstrated that the N-terminal conserved domain of VirD2 was absolutely essential for its endonuclease activity. When extra copies of the virD1 and virD2 genes were present in an A. tumefaciens strain carrying a Ti plasmid, increased amounts of T-strand and nicked molecules could be detected at early stages of vir induction. Such strains possessed the ability to transform plants with higher efficiency.

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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. An G., Watson B. D., Stachel S., Gordon M. P., Nester E. W. New cloning vehicles for transformation of higher plants. EMBO J. 1985 Feb;4(2):277–284. doi: 10.1002/j.1460-2075.1985.tb03626.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bolton G. W., Nester E. W., Gordon M. P. Plant phenolic compounds induce expression of the Agrobacterium tumefaciens loci needed for virulence. Science. 1986 May 23;232(4753):983–985. doi: 10.1126/science.3085219. [DOI] [PubMed] [Google Scholar]
  4. Casadaban M. J., Cohen S. N. Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J Mol Biol. 1980 Apr;138(2):179–207. doi: 10.1016/0022-2836(80)90283-1. [DOI] [PubMed] [Google Scholar]
  5. Chelsky D., Ralph R., Jonak G. Sequence requirements for synthetic peptide-mediated translocation to the nucleus. Mol Cell Biol. 1989 Jun;9(6):2487–2492. doi: 10.1128/mcb.9.6.2487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. 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]
  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. De Vos G., Zambryski P. Expression of Agrobacterium nopaline-specific VirD1, VirD2, and VirC1 proteins and their requirement for T-strand production in E. coli. Mol Plant Microbe Interact. 1989 Mar-Apr;2(2):43–52. doi: 10.1094/mpmi-2-043. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Dworetzky S. I., Lanford R. E., Feldherr C. M. The effects of variations in the number and sequence of targeting signals on nuclear uptake. J Cell Biol. 1988 Oct;107(4):1279–1287. doi: 10.1083/jcb.107.4.1279. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ghai J., Das A. The virD operon of Agrobacterium tumefaciens Ti plasmid encodes a DNA-relaxing enzyme. Proc Natl Acad Sci U S A. 1989 May;86(9):3109–3113. doi: 10.1073/pnas.86.9.3109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Hagiya M., Close T. J., Tait R. C., Kado C. I. Identification of pTiC58 plasmid-encoded proteins for virulence in Agrobacterium tumefaciens. Proc Natl Acad Sci U S A. 1985 May;82(9):2669–2673. doi: 10.1073/pnas.82.9.2669. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Herrera-Estrella A., Chen Z. M., Van Montagu M., Wang K. 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. 1988 Dec 20;7(13):4055–4062. doi: 10.1002/j.1460-2075.1988.tb03299.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hirayama T., Muranaka T., Ohkawa H., Oka A. Organization and characterization of the virCD genes from Agrobacterium rhizogenes. Mol Gen Genet. 1988 Aug;213(2-3):229–237. doi: 10.1007/BF00339586. [DOI] [PubMed] [Google Scholar]
  17. Howard E. A., Winsor B. A., De Vos G., Zambryski P. Activation of the T-DNA transfer process in Agrobacterium results in the generation of a T-strand-protein complex: Tight association of VirD2 with the 5' ends of T-strands. Proc Natl Acad Sci U S A. 1989 Jun;86(11):4017–4021. doi: 10.1073/pnas.86.11.4017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Itoh Y., Watson J. M., Haas D., Leisinger T. Genetic and molecular characterization of the Pseudomonas plasmid pVS1. Plasmid. 1984 May;11(3):206–220. doi: 10.1016/0147-619x(84)90027-1. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Joos H., Timmerman B., Montagu M. V., Schell J. Genetic analysis of transfer and stabilization of Agrobacterium DNA in plant cells. EMBO J. 1983;2(12):2151–2160. doi: 10.1002/j.1460-2075.1983.tb01716.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Peralta E. G., Hellmiss R., Ream W. Overdrive, a T-DNA transmission enhancer on the A. tumefaciens tumour-inducing plasmid. EMBO J. 1986 Jun;5(6):1137–1142. doi: 10.1002/j.1460-2075.1986.tb04338.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Peralta E. G., Ream L. W. T-DNA border sequences required for crown gall tumorigenesis. Proc Natl Acad Sci U S A. 1985 Aug;82(15):5112–5116. doi: 10.1073/pnas.82.15.5112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Platt S. G., Yang N. S. Dot assay for neomycin phosphotransferase activity in crude cell extracts. Anal Biochem. 1987 May 1;162(2):529–535. doi: 10.1016/0003-2697(87)90429-5. [DOI] [PubMed] [Google Scholar]
  24. Porter S. G., Yanofsky M. F., Nester E. W. Molecular characterization of the virD operon from Agrobacterium tumefaciens. Nucleic Acids Res. 1987 Sep 25;15(18):7503–7517. doi: 10.1093/nar/15.18.7503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Reiss B., Sprengel R., Schaller H. Protein fusions with the kanamycin resistance gene from transposon Tn5. EMBO J. 1984 Dec 20;3(13):3317–3322. doi: 10.1002/j.1460-2075.1984.tb02297.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sanger F. Determination of nucleotide sequences in DNA. Science. 1981 Dec 11;214(4526):1205–1210. doi: 10.1126/science.7302589. [DOI] [PubMed] [Google Scholar]
  27. Sen P., Pazour G. J., Anderson D., Das A. Cooperative binding of Agrobacterium tumefaciens VirE2 protein to single-stranded DNA. J Bacteriol. 1989 May;171(5):2573–2580. doi: 10.1128/jb.171.5.2573-2580.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Stachel S. E., Nester E. W. The genetic and transcriptional organization of the vir region of the A6 Ti plasmid of Agrobacterium tumefaciens. EMBO J. 1986 Jul;5(7):1445–1454. doi: 10.1002/j.1460-2075.1986.tb04381.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. Stachel S. E., Zambryski P. C. Agrobacterium tumefaciens and the susceptible plant cell: a novel adaptation of extracellular recognition and DNA conjugation. Cell. 1986 Oct 24;47(2):155–157. doi: 10.1016/0092-8674(86)90437-x. [DOI] [PubMed] [Google Scholar]
  31. Stachel S. E., Zambryski P. C. Bacteria-yeast conjugation. Generic trans-kingdom sex? Nature. 1989 Jul 20;340(6230):190–191. doi: 10.1038/340190a0. [DOI] [PubMed] [Google Scholar]
  32. Tabor S., Richardson C. C. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074–1078. doi: 10.1073/pnas.82.4.1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Teeri T. H., Lehväslaiho H., Franck M., Uotila J., Heino P., Palva E. T., Van Montagu M., Herrera-Estrella L. Gene fusions to lacZ reveal new expression patterns of chimeric genes in transgenic plants. EMBO J. 1989 Feb;8(2):343–350. doi: 10.1002/j.1460-2075.1989.tb03383.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Van den Broeck G., Timko M. P., Kausch A. P., Cashmore A. R., Van Montagu M., Herrera-Estrella L. Targeting of a foreign protein to chloroplasts by fusion to the transit peptide from the small subunit of ribulose 1,5-bisphosphate carboxylase. 1985 Jan 31-Feb 6Nature. 313(6001):358–363. doi: 10.1038/313358a0. [DOI] [PubMed] [Google Scholar]
  35. Wang K., Herrera-Estrella L., Van Montagu M., Zambryski P. 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. 1984 Sep;38(2):455–462. doi: 10.1016/0092-8674(84)90500-2. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. 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]
  40. 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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