Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1997 Feb;179(4):1165–1173. doi: 10.1128/jb.179.4.1165-1173.1997

Functional domains of Agrobacterium tumefaciens single-stranded DNA-binding protein VirE2.

P Dombek 1, W Ream 1
PMCID: PMC178812  PMID: 9023198

Abstract

The transferred DNA (T-DNA) portion of the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid enters infected plant cells and integrates into plant nuclear DNA. Direct repeats define the T-DNA ends; transfer begins when the VirD2 endonuclease produces a site-specific nick in the right-hand border repeat and attaches to the 5' end of the nicked strand. Subsequent events liberate the lower strand of the T-DNA from the Ti plasmid, producing single-stranded DNA molecules (T strands) that are covalently linked to VirD2 at their 5' ends. A. tumefaciens appears to transfer T-DNA into plant cells as a T-strand-VirD2 complex. The bacterium also transports VirE2, a cooperative single-stranded DNA-binding protein, into plant cells during infection. Both VirD2 and VirE2 contain nuclear localization signals that may direct these proteins, and bound T strands, into plant nuclei. Here we report the locations of functional regions of VirE2 identified by eight insertions of XhoI linker oligonucleotides, and one deletion mutation, throughout virE2. We examined the effects of these mutations on virulence, single-stranded DNA (ssDNA) binding, and accumulation of VirE2 in A. tumefaciens. Two of the mutations in the C-terminal half of VirE2 eliminated ssDNA binding, whereas two insertions in the N-terminal half altered cooperativity. Four of the mutations, distributed throughout virE2, decreased the stability of VirE2 in A. tumefaciens. In addition, we isolated a mutation in the central region of VirE2 that decreased tumorigenicity but did not affect ssDNA binding or VirE2 accumulation. This mutation may affect export of VirE2 into plant cells or nuclear localization of VirE2, or it may affect an uncharacterized activity of VirE2.

Full Text

The Full Text of this article is available as a PDF (1.1 MB).

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. Amann E., Ochs B., Abel K. J. Tightly regulated tac promoter vectors useful for the expression of unfused and fused proteins in Escherichia coli. Gene. 1988 Sep 30;69(2):301–315. doi: 10.1016/0378-1119(88)90440-4. [DOI] [PubMed] [Google Scholar]
  3. Binns A. N., Beaupré C. E., Dale E. M. Inhibition of VirB-mediated transfer of diverse substrates from Agrobacterium tumefaciens by the IncQ plasmid RSF1010. J Bacteriol. 1995 Sep;177(17):4890–4899. doi: 10.1128/jb.177.17.4890-4899.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. 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]
  7. 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]
  8. 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]
  9. Citovsky V., Warnick D., Zambryski P. Nuclear import of Agrobacterium VirD2 and VirE2 proteins in maize and tobacco. Proc Natl Acad Sci U S A. 1994 Apr 12;91(8):3210–3214. doi: 10.1073/pnas.91.8.3210. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Citovsky V., Wong M. L., Zambryski P. Cooperative interaction of Agrobacterium VirE2 protein with single-stranded DNA: implications for the T-DNA transfer process. Proc Natl Acad Sci U S A. 1989 Feb;86(4):1193–1197. doi: 10.1073/pnas.86.4.1193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Citovsky V., Zupan J., Warnick D., Zambryski P. Nuclear localization of Agrobacterium VirE2 protein in plant cells. Science. 1992 Jun 26;256(5065):1802–1805. doi: 10.1126/science.1615325. [DOI] [PubMed] [Google Scholar]
  12. Covacci A., Rappuoli R. Pertussis toxin export requires accessory genes located downstream from the pertussis toxin operon. Mol Microbiol. 1993 May;8(3):429–434. doi: 10.1111/j.1365-2958.1993.tb01587.x. [DOI] [PubMed] [Google Scholar]
  13. Csonka L. N., Clark A. J. Deletions generated by the transposon Tn10 in the srl recA region of the Escherichia coli K-12 chromosome. Genetics. 1979 Oct;93(2):321–343. doi: 10.1093/genetics/93.2.321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Ditta G., Schmidhauser T., Yakobson E., Lu P., Liang X. W., Finlay D. R., Guiney D., Helinski D. R. Plasmids related to the broad host range vector, pRK290, useful for gene cloning and for monitoring gene expression. Plasmid. 1985 Mar;13(2):149–153. doi: 10.1016/0147-619x(85)90068-x. [DOI] [PubMed] [Google Scholar]
  16. Dürrenberger F., Crameri A., Hohn B., Koukolíková-Nicola Z. Covalently bound VirD2 protein of Agrobacterium tumefaciens protects the T-DNA from exonucleolytic degradation. Proc Natl Acad Sci U S A. 1989 Dec;86(23):9154–9158. doi: 10.1073/pnas.86.23.9154. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gardner R. C., Knauf V. C. Transfer of Agrobacterium DNA to Plants Requires a T-DNA Border But Not the virE Locus. Science. 1986 Feb 14;231(4739):725–727. doi: 10.1126/science.231.4739.725. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. 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]
  20. 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]
  21. 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]
  22. Herrera-Estrella A., Van Montagu M., Wang K. A bacterial peptide acting as a plant nuclear targeting signal: the amino-terminal portion of Agrobacterium VirD2 protein directs a beta-galactosidase fusion protein into tobacco nuclei. Proc Natl Acad Sci U S A. 1990 Dec;87(24):9534–9537. doi: 10.1073/pnas.87.24.9534. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Holsters M., de Waele D., Depicker A., Messens E., van Montagu M., Schell J. Transfection and transformation of Agrobacterium tumefaciens. Mol Gen Genet. 1978 Jul 11;163(2):181–187. doi: 10.1007/BF00267408. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Howard E. A., Zupan J. R., Citovsky V., Zambryski P. C. The VirD2 protein of A. tumefaciens contains a C-terminal bipartite nuclear localization signal: implications for nuclear uptake of DNA in plant cells. Cell. 1992 Jan 10;68(1):109–118. doi: 10.1016/0092-8674(92)90210-4. [DOI] [PubMed] [Google Scholar]
  26. Hurley J. M., Chervitz S. A., Jarvis T. C., Singer B. S., Gold L. Assembly of the bacteriophage T4 replication machine requires the acidic carboxy terminus of gene 32 protein. J Mol Biol. 1993 Jan 20;229(2):398–418. doi: 10.1006/jmbi.1993.1042. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Jorgensen R. A., Rothstein S. J., Reznikoff W. S. A restriction enzyme cleavage map of Tn5 and location of a region encoding neomycin resistance. Mol Gen Genet. 1979;177(1):65–72. doi: 10.1007/BF00267254. [DOI] [PubMed] [Google Scholar]
  29. Krassa K. B., Green L. S., Gold L. Protein-protein interactions with the acidic COOH terminus of the single-stranded DNA-binding protein of the bacteriophage T4. Proc Natl Acad Sci U S A. 1991 May 1;88(9):4010–4014. doi: 10.1073/pnas.88.9.4010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  31. Lessl M., Balzer D., Pansegrau W., Lanka E. Sequence similarities between the RP4 Tra2 and the Ti VirB region strongly support the conjugation model for T-DNA transfer. J Biol Chem. 1992 Oct 5;267(28):20471–20480. [PubMed] [Google Scholar]
  32. McBride K. E., Knauf V. C. Genetic analysis of the virE operon of the Agrobacterium Ti plasmid pTiA6. J Bacteriol. 1988 Apr;170(4):1430–1437. doi: 10.1128/jb.170.4.1430-1437.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Okamoto S., Toyoda-Yamamoto A., Ito K., Takebe I., Machida Y. Localization and orientation of the VirD4 protein of Agrobacterium tumefaciens in the cell membrane. Mol Gen Genet. 1991 Aug;228(1-2):24–32. doi: 10.1007/BF00282443. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. 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]
  36. Pohlman R. F., Genetti H. D., Winans S. C. Common ancestry between IncN conjugal transfer genes and macromolecular export systems of plant and animal pathogens. Mol Microbiol. 1994 Nov;14(4):655–668. doi: 10.1111/j.1365-2958.1994.tb01304.x. [DOI] [PubMed] [Google Scholar]
  37. Robbins J., Dilworth S. M., Laskey R. A., Dingwall C. Two interdependent basic domains in nucleoplasmin nuclear targeting sequence: identification of a class of bipartite nuclear targeting sequence. Cell. 1991 Feb 8;64(3):615–623. doi: 10.1016/0092-8674(91)90245-t. [DOI] [PubMed] [Google Scholar]
  38. Rossi L., Hohn B., Tinland B. Integration of complete transferred DNA units is dependent on the activity of virulence E2 protein of Agrobacterium tumefaciens. Proc Natl Acad Sci U S A. 1996 Jan 9;93(1):126–130. doi: 10.1073/pnas.93.1.126. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Ruvkun G. B., Ausubel F. M. A general method for site-directed mutagenesis in prokaryotes. Nature. 1981 Jan 1;289(5793):85–88. doi: 10.1038/289085a0. [DOI] [PubMed] [Google Scholar]
  40. 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]
  41. Shaw C. H., Watson M. D., Carter G. H., Shaw C. H. The right hand copy of the nopaline Ti-plasmid 25 bp repeat is required for tumour formation. Nucleic Acids Res. 1984 Aug 10;12(15):6031–6041. doi: 10.1093/nar/12.15.6031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Shurvinton C. E., Hodges L., Ream W. A nuclear localization signal and the C-terminal omega sequence in the Agrobacterium tumefaciens VirD2 endonuclease are important for tumor formation. Proc Natl Acad Sci U S A. 1992 Dec 15;89(24):11837–11841. doi: 10.1073/pnas.89.24.11837. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Shurvinton C. E., Ream W. Stimulation of Agrobacterium tumefaciens T-DNA transfer by overdrive depends on a flanking sequence but not on helical position with respect to the border repeat. J Bacteriol. 1991 Sep;173(17):5558–5563. doi: 10.1128/jb.173.17.5558-5563.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Silver P. A. How proteins enter the nucleus. Cell. 1991 Feb 8;64(3):489–497. doi: 10.1016/0092-8674(91)90233-o. [DOI] [PubMed] [Google Scholar]
  45. 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]
  46. 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]
  47. 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]
  48. Sundberg C., Meek L., Carroll K., Das A., Ream W. VirE1 protein mediates export of the single-stranded DNA-binding protein VirE2 from Agrobacterium tumefaciens into plant cells. J Bacteriol. 1996 Feb;178(4):1207–1212. doi: 10.1128/jb.178.4.1207-1212.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Tinland B., Koukolíková-Nicola Z., Hall M. N., Hohn B. The T-DNA-linked VirD2 protein contains two distinct functional nuclear localization signals. Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7442–7446. doi: 10.1073/pnas.89.16.7442. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Tinland B., Schoumacher F., Gloeckler V., Bravo-Angel A. M., Hohn B. The Agrobacterium tumefaciens virulence D2 protein is responsible for precise integration of T-DNA into the plant genome. EMBO J. 1995 Jul 17;14(14):3585–3595. doi: 10.1002/j.1460-2075.1995.tb07364.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Toro N., Datta A., Carmi O. A., Young C., Prusti R. K., Nester E. W. The Agrobacterium tumefaciens virC1 gene product binds to overdrive, a T-DNA transfer enhancer. J Bacteriol. 1989 Dec;171(12):6845–6849. doi: 10.1128/jb.171.12.6845-6849.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Van Larebeke N., Engler G., Holsters M., Van den Elsacker S., Zaenen I., Schilperoort R. A., Schell J. Large plasmid in Agrobacterium tumefaciens essential for crown gall-inducing ability. Nature. 1974 Nov 8;252(5479):169–170. doi: 10.1038/252169a0. [DOI] [PubMed] [Google Scholar]
  53. 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]
  54. 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]
  55. 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]
  56. Ward J. E., Jr, Dale E. M., Binns A. N. Activity of the Agrobacterium T-DNA transfer machinery is affected by virB gene products. Proc Natl Acad Sci U S A. 1991 Oct 15;88(20):9350–9354. doi: 10.1073/pnas.88.20.9350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. 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]
  58. Weiss A. A., Johnson F. D., Burns D. L. Molecular characterization of an operon required for pertussis toxin secretion. Proc Natl Acad Sci U S A. 1993 Apr 1;90(7):2970–2974. doi: 10.1073/pnas.90.7.2970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Winans S. C., Allenza P., Stachel S. E., McBride K. E., Nester E. W. Characterization of the virE operon of the Agrobacterium Ti plasmid pTiA6. Nucleic Acids Res. 1987 Jan 26;15(2):825–837. doi: 10.1093/nar/15.2.825. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Winans S. C. Two-way chemical signaling in Agrobacterium-plant interactions. Microbiol Rev. 1992 Mar;56(1):12–31. doi: 10.1128/mr.56.1.12-31.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. 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]
  62. 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]
  63. 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]
  64. Yusibov V. M., Steck T. R., Gupta V., Gelvin S. B. Association of single-stranded transferred DNA from Agrobacterium tumefaciens with tobacco cells. Proc Natl Acad Sci U S A. 1994 Apr 12;91(8):2994–2998. doi: 10.1073/pnas.91.8.2994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Zupan J. R., Citovsky V., Zambryski P. Agrobacterium VirE2 protein mediates nuclear uptake of single-stranded DNA in plant cells. Proc Natl Acad Sci U S A. 1996 Mar 19;93(6):2392–2397. doi: 10.1073/pnas.93.6.2392. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES