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. 1988 Jun;170(6):2659–2667. doi: 10.1128/jb.170.6.2659-2667.1988

The Agrobacterium tumefaciens virE2 gene product is a single-stranded-DNA-binding protein that associates with T-DNA.

P J Christie 1, J E Ward 1, S C Winans 1, E W Nester 1
PMCID: PMC211185  PMID: 2836366

Abstract

Agrobacterium tumefaciens transfers T-DNA into the plant genome by a process mediated by Ti plasmid-encoded vir genes. Cleavage at T-DNA border sequences by the VirD endonuclease generates linear, single-stranded T-DNA molecules. In the work described in this report, we used electrophoretic mobility shift assays to show that the purified virE2 gene product binds to single-stranded DNA. VirE2 protein associates with T-DNA as shown by immunoprecipitation studies with VirE2-specific antiserum. The VirE2 protein was detected primarily in the cytoplasm, but also in the inner and outer membrane and periplasmic fractions. Virulence of a virE2 mutant was restored by mixed infection with strains carrying an intact vir region, but not with virA, virB, virD, virE, or virG mutants or chvA, chvB, or exoC mutants. We propose that the VirE2 protein is involved in the processing of T-DNA and in T-strand protection during transfer to the plant cell.

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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. 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. 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: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  5. Bruist M. F., Simon M. I. Phase variation and the Hin protein: in vivo activity measurements, protein overproduction, and purification. J Bacteriol. 1984 Jul;159(1):71–79. doi: 10.1128/jb.159.1.71-79.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cangelosi G. A., Hung L., Puvanesarajah V., Stacey G., Ozga D. A., Leigh J. A., Nester E. W. Common loci for Agrobacterium tumefaciens and Rhizobium meliloti exopolysaccharide synthesis and their roles in plant interactions. J Bacteriol. 1987 May;169(5):2086–2091. doi: 10.1128/jb.169.5.2086-2091.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chase J. W., Williams K. R. Single-stranded DNA binding proteins required for DNA replication. Annu Rev Biochem. 1986;55:103–136. doi: 10.1146/annurev.bi.55.070186.000535. [DOI] [PubMed] [Google Scholar]
  8. Chilton M. D., Currier T. C., Farrand S. K., Bendich A. J., Gordon M. P., Nester E. W. Agrobacterium tumefaciens DNA and PS8 bacteriophage DNA not detected in crown gall tumors. Proc Natl Acad Sci U S A. 1974 Sep;71(9):3672–3676. doi: 10.1073/pnas.71.9.3672. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Douglas C. J., Staneloni R. J., Rubin R. A., Nester E. W. Identification and genetic analysis of an Agrobacterium tumefaciens chromosomal virulence region. J Bacteriol. 1985 Mar;161(3):850–860. doi: 10.1128/jb.161.3.850-860.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Engström P., Zambryski P., Van Montagu M., Stachel S. Characterization of Agrobacterium tumefaciens virulence proteins induced by the plant factor acetosyringone. J Mol Biol. 1987 Oct 20;197(4):635–645. doi: 10.1016/0022-2836(87)90470-0. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. 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]
  14. 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]
  15. Gaylo P. J., Turjman N., Bastia D. DnaA protein is required for replication of the minimal replicon of the broad-host-range plasmid RK2 in Escherichia coli. J Bacteriol. 1987 Oct;169(10):4703–4709. doi: 10.1128/jb.169.10.4703-4709.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. Golub E. I., Low K. B. Conjugative plasmids of enteric bacteria from many different incompatibility groups have similar genes for single-stranded DNA-binding proteins. J Bacteriol. 1985 Apr;162(1):235–241. doi: 10.1128/jb.162.1.235-241.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hager D. A., Burgess R. R. Elution of proteins from sodium dodecyl sulfate-polyacrylamide gels, removal of sodium dodecyl sulfate, and renaturation of enzymatic activity: results with sigma subunit of Escherichia coli RNA polymerase, wheat germ DNA topoisomerase, and other enzymes. Anal Biochem. 1980 Nov 15;109(1):76–86. doi: 10.1016/0003-2697(80)90013-5. [DOI] [PubMed] [Google Scholar]
  19. Hendrickson W., Schleif R. F. Regulation of the Escherichia coli L-arabinose operon studied by gel electrophoresis DNA binding assay. J Mol Biol. 1984 Sep 25;178(3):611–628. doi: 10.1016/0022-2836(84)90241-9. [DOI] [PubMed] [Google Scholar]
  20. Horsch R. B., Klee H. J., Stachel S., Winans S. C., Nester E. W., Rogers S. G., Fraley R. T. Analysis of Agrobacterium tumefaciens virulence mutants in leaf discs. Proc Natl Acad Sci U S A. 1986 Apr;83(8):2571–2575. doi: 10.1073/pnas.83.8.2571. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ippen-Ihler K. A., Minkley E. G., Jr The conjugation system of F, the fertility factor of Escherichia coli. Annu Rev Genet. 1986;20:593–624. doi: 10.1146/annurev.ge.20.120186.003113. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Jin S. G., Komari T., Gordon M. P., Nester E. W. Genes responsible for the supervirulence phenotype of Agrobacterium tumefaciens A281. J Bacteriol. 1987 Oct;169(10):4417–4425. doi: 10.1128/jb.169.10.4417-4425.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kadonaga J. T., Tjian R. Affinity purification of sequence-specific DNA binding proteins. Proc Natl Acad Sci U S A. 1986 Aug;83(16):5889–5893. doi: 10.1073/pnas.83.16.5889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Knauf V. C., Nester E. W. Wide host range cloning vectors: a cosmid clone bank of an Agrobacterium Ti plasmid. Plasmid. 1982 Jul;8(1):45–54. doi: 10.1016/0147-619x(82)90040-3. [DOI] [PubMed] [Google Scholar]
  27. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  28. Leroux B., Yanofsky M. F., Winans S. C., Ward J. E., Ziegler S. F., Nester E. W. Characterization of the virA locus of Agrobacterium tumefaciens: a transcriptional regulator and host range determinant. EMBO J. 1987 Apr;6(4):849–856. doi: 10.1002/j.1460-2075.1987.tb04830.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. Oliver D. Protein secretion in Escherichia coli. Annu Rev Microbiol. 1985;39:615–648. doi: 10.1146/annurev.mi.39.100185.003151. [DOI] [PubMed] [Google Scholar]
  31. Osborn M. J., Gander J. E., Parisi E., Carson J. Mechanism of assembly of the outer membrane of Salmonella typhimurium. Isolation and characterization of cytoplasmic and outer membrane. J Biol Chem. 1972 Jun 25;247(12):3962–3972. [PubMed] [Google Scholar]
  32. 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]
  33. 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]
  34. 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]
  35. 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]
  36. 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]
  37. 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]
  38. Towbin H., Gordon J. Immunoblotting and dot immunobinding--current status and outlook. J Immunol Methods. 1984 Sep 4;72(2):313–340. doi: 10.1016/0022-1759(84)90001-2. [DOI] [PubMed] [Google Scholar]
  39. 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]
  40. Willetts N., Wilkins B. Processing of plasmid DNA during bacterial conjugation. Microbiol Rev. 1984 Mar;48(1):24–41. doi: 10.1128/mr.48.1.24-41.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. 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]
  42. Winans S. C., Ebert P. R., Stachel S. E., Gordon M. P., Nester E. W. A gene essential for Agrobacterium virulence is homologous to a family of positive regulatory loci. Proc Natl Acad Sci U S A. 1986 Nov;83(21):8278–8282. doi: 10.1073/pnas.83.21.8278. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. 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]
  44. Yamamoto A., Iwahashi M., Yanofsky M. F., Nester E. W., Takebe I., Machida Y. The promoter proximal region in the virD locus of Agrobacterium tumefaciens is necessary for the plant-inducible circularization of T-DNA. Mol Gen Genet. 1987 Jan;206(1):174–177. doi: 10.1007/BF00326554. [DOI] [PubMed] [Google Scholar]
  45. Yanofsky M. F., Nester E. W. Molecular characterization of a host-range-determining locus from Agrobacterium tumefaciens. J Bacteriol. 1986 Oct;168(1):244–250. doi: 10.1128/jb.168.1.244-250.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. 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]
  47. de Maagd R. A., Lugtenberg B. Fractionation of Rhizobium leguminosarum cells into outer membrane, cytoplasmic membrane, periplasmic, and cytoplasmic components. J Bacteriol. 1986 Sep;167(3):1083–1085. doi: 10.1128/jb.167.3.1083-1085.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]

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