Skip to main content
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1996 Jan 9;93(1):126–130. doi: 10.1073/pnas.93.1.126

Integration of complete transferred DNA units is dependent on the activity of virulence E2 protein of Agrobacterium tumefaciens.

L Rossi 1, B Hohn 1, B Tinland 1
PMCID: PMC40191  PMID: 8552588

Abstract

Agrobacterium tumefaciens transfers transferred DNA (T-DNA), a single-stranded segment of its tumor-inducing (Ti) plasmid, to the plant cell nucleus. The Ti-plasmid-encoded virulence E2 (VirE2) protein expressed in the bacterium has single-stranded DNA (ssDNA)-binding properties and has been reported to act in the plant cell. This protein is thought to exert its influence on transfer efficiency by coating and accompanying the single-stranded T-DNA (ss-T-DNA) to the plant cell genome. Here, we analyze different putative roles of the VirE2 protein in the plant cell. In the absence of VirE2 protein, mainly truncated versions of the T-DNA are integrated. We infer that VirE2 protects the ss-T-DNA against nucleolytic attack during the transfer process and that it is interacting with the ss-T-DNA on its way to the plant cell nucleus. Furthermore, the VirE2 protein was found not to be involved in directing the ss-T-DNA to the plant cell nucleus in a manner dependent on a nuclear localization signal, a function which is carried by the NLS of VirD2. In addition, the efficiency of T-DNA integration into the plant genome was found to be VirE2 independent. We conclude that the VirE2 protein of A. tumefaciens is required to preserve the integrity of the T-DNA but does not contribute to the efficiency of the integration step per se.

Full text

PDF
126

Images in this article

Selected References

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

  1. Benson F. E., Stasiak A., West S. C. Purification and characterization of the human Rad51 protein, an analogue of E. coli RecA. EMBO J. 1994 Dec 1;13(23):5764–5771. doi: 10.1002/j.1460-2075.1994.tb06914.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. Bonnard G., Tinland B., Paulus F., Szegedi E., Otten L. Nucleotide sequence, evolutionary origin and biological role of a rearranged cytokinin gene isolated from a wide host range biotype III Agrobacterium strain. Mol Gen Genet. 1989 Apr;216(2-3):428–438. doi: 10.1007/BF00334387. [DOI] [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. 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]
  7. 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]
  8. 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]
  9. 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]
  10. 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]
  11. Gheysen G., Villarroel R., Van Montagu M. Illegitimate recombination in plants: a model for T-DNA integration. Genes Dev. 1991 Feb;5(2):287–297. doi: 10.1101/gad.5.2.287. [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. Grimsley N., Hohn B., Ramos C., Kado C., Rogowsky P. DNA transfer from Agrobacterium to Zea mays or Brassica by agroinfection is dependent on bacterial virulence functions. Mol Gen Genet. 1989 Jun;217(2-3):309–316. doi: 10.1007/BF02464898. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Hirooka T., Kado C. I. Location of the right boundary of the virulence region on Agrobacterium tumefaciens plasmid pTiC58 and a host-specifying gene next to the boundary. J Bacteriol. 1986 Oct;168(1):237–243. doi: 10.1128/jb.168.1.237-243.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. Mayerhofer R., Koncz-Kalman Z., Nawrath C., Bakkeren G., Crameri A., Angelis K., Redei G. P., Schell J., Hohn B., Koncz C. T-DNA integration: a mode of illegitimate recombination in plants. EMBO J. 1991 Mar;10(3):697–704. doi: 10.1002/j.1460-2075.1991.tb07999.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Mehlin H., Daneholt B., Skoglund U. Translocation of a specific premessenger ribonucleoprotein particle through the nuclear pore studied with electron microscope tomography. Cell. 1992 May 15;69(4):605–613. doi: 10.1016/0092-8674(92)90224-z. [DOI] [PubMed] [Google Scholar]
  19. Richardson W. D., Mills A. D., Dilworth S. M., Laskey R. A., Dingwall C. Nuclear protein migration involves two steps: rapid binding at the nuclear envelope followed by slower translocation through nuclear pores. Cell. 1988 Mar 11;52(5):655–664. doi: 10.1016/0092-8674(88)90403-5. [DOI] [PubMed] [Google Scholar]
  20. Rossi L., Hohn B., Tinland B. The VirD2 protein of Agrobacterium tumefaciens carries nuclear localization signals important for transfer of T-DNA to plant. Mol Gen Genet. 1993 Jun;239(3):345–353. doi: 10.1007/BF00276932. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. 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]
  23. 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]
  24. 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]
  25. 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]
  26. Tinland B., Hohn B., Puchta H. Agrobacterium tumefaciens transfers single-stranded transferred DNA (T-DNA) into the plant cell nucleus. Proc Natl Acad Sci U S A. 1994 Aug 16;91(17):8000–8004. doi: 10.1073/pnas.91.17.8000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. 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]
  29. 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]
  30. 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]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES