Skip to main content
The EMBO Journal logoLink to The EMBO Journal
. 1995 Jul 17;14(14):3585–3595. doi: 10.1002/j.1460-2075.1995.tb07364.x

The Agrobacterium tumefaciens virulence D2 protein is responsible for precise integration of T-DNA into the plant genome.

B Tinland 1, F Schoumacher 1, V Gloeckler 1, A M Bravo-Angel 1, B Hohn 1
PMCID: PMC394426  PMID: 7628458

Abstract

The VirD2 protein of Agrobacterium tumefaciens was shown to pilot T-DNA during its transfer to the plant cell nucleus. We analyze here its participation in the integration of T-DNA by using a virD2 mutant. This mutation reduces the efficiency of T-DNA transfer, but the efficiency of integration of T-DNA per se is unaffected. Southern and sequence analyses of integration events obtained with the mutated VirD2 protein revealed an aberrant pattern of integration. These results indicate that the wild-type VirD2 protein participates in ligation of the 5'-end of the T-strand to plant DNA and that this ligation step is not rate limiting for T-DNA integration.

Full text

PDF
3585

Images in this article

Selected References

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

  1. Argos P., Landy A., Abremski K., Egan J. B., Haggard-Ljungquist E., Hoess R. H., Kahn M. L., Kalionis B., Narayana S. V., Pierson L. S., 3rd The integrase family of site-specific recombinases: regional similarities and global diversity. EMBO J. 1986 Feb;5(2):433–440. doi: 10.1002/j.1460-2075.1986.tb04229.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. 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]
  4. 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]
  5. Earp D. J., Lowe B., Baker B. Amplification of genomic sequences flanking transposable elements in host and heterologous plants: a tool for transposon tagging and genome characterization. Nucleic Acids Res. 1990 Jun 11;18(11):3271–3279. doi: 10.1093/nar/18.11.3271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Filichkin S. A., Gelvin S. B. Formation of a putative relaxation intermediate during T-DNA processing directed by the Agrobacterium tumefaciens VirD1,D2 endonuclease. Mol Microbiol. 1993 May;8(5):915–926. doi: 10.1111/j.1365-2958.1993.tb01637.x. [DOI] [PubMed] [Google Scholar]
  7. Gasser C. S., Fraley R. T. Genetically engineering plants for crop improvement. Science. 1989 Jun 16;244(4910):1293–1299. doi: 10.1126/science.244.4910.1293. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Guyon P., Chilton M. D., Petit A., Tempé J. Agropine in "null-type" crown gall tumors: Evidence for generality of the opine concept. Proc Natl Acad Sci U S A. 1980 May;77(5):2693–2697. doi: 10.1073/pnas.77.5.2693. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Hooykaas P. J., Schilperoort R. A. Agrobacterium and plant genetic engineering. Plant Mol Biol. 1992 May;19(1):15–38. doi: 10.1007/BF00015604. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. 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]
  14. Janssen B. J., Gardner R. C. Localized transient expression of GUS in leaf discs following cocultivation with Agrobacterium. Plant Mol Biol. 1990 Jan;14(1):61–72. doi: 10.1007/BF00015655. [DOI] [PubMed] [Google Scholar]
  15. Jasper F., Koncz C., Schell J., Steinbiss H. H. Agrobacterium T-strand production in vitro: sequence-specific cleavage and 5' protection of single-stranded DNA templates by purified VirD2 protein. Proc Natl Acad Sci U S A. 1994 Jan 18;91(2):694–698. doi: 10.1073/pnas.91.2.694. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Jayaram M. Phosphoryl transfer in Flp recombination: a template for strand transfer mechanisms. Trends Biochem Sci. 1994 Feb;19(2):78–82. doi: 10.1016/0968-0004(94)90039-6. [DOI] [PubMed] [Google Scholar]
  17. Lee K. Y., Lund P., Lowe K., Dunsmuir P. Homologous recombination in plant cells after Agrobacterium-mediated transformation. Plant Cell. 1990 May;2(5):415–425. doi: 10.1105/tpc.2.5.415. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lehman C. W., Trautman J. K., Carroll D. Illegitimate recombination in Xenopus: characterization of end-joined junctions. Nucleic Acids Res. 1994 Feb 11;22(3):434–442. doi: 10.1093/nar/22.3.434. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lessl M., Lanka E. Common mechanisms in bacterial conjugation and Ti-mediated T-DNA transfer to plant cells. Cell. 1994 May 6;77(3):321–324. doi: 10.1016/0092-8674(94)90146-5. [DOI] [PubMed] [Google Scholar]
  20. Matsumoto S., Ito Y., Hosoi T., Takahashi Y., Machida Y. Integration of Agrobacterium T-DNA into a tobacco chromosome: possible involvement of DNA homology between T-DNA and plant DNA. Mol Gen Genet. 1990 Dec;224(3):309–316. doi: 10.1007/BF00262423. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. McBride K. E., Summerfelt K. R. Improved binary vectors for Agrobacterium-mediated plant transformation. Plant Mol Biol. 1990 Feb;14(2):269–276. doi: 10.1007/BF00018567. [DOI] [PubMed] [Google Scholar]
  23. Miranda A., Janssen G., Hodges L., Peralta E. G., Ream W. Agrobacterium tumefaciens transfers extremely long T-DNAs by a unidirectional mechanism. J Bacteriol. 1992 Apr;174(7):2288–2297. doi: 10.1128/jb.174.7.2288-2297.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Offringa R., de Groot M. J., Haagsman H. J., Does M. P., van den Elzen P. J., Hooykaas P. J. Extrachromosomal homologous recombination and gene targeting in plant cells after Agrobacterium mediated transformation. EMBO J. 1990 Oct;9(10):3077–3084. doi: 10.1002/j.1460-2075.1990.tb07504.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Pansegrau W., Schoumacher F., Hohn B., Lanka E. Site-specific cleavage and joining of single-stranded DNA by VirD2 protein of Agrobacterium tumefaciens Ti plasmids: analogy to bacterial conjugation. Proc Natl Acad Sci U S A. 1993 Dec 15;90(24):11538–11542. doi: 10.1073/pnas.90.24.11538. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Pansegrau W., Schröder W., Lanka E. Concerted action of three distinct domains in the DNA cleaving-joining reaction catalyzed by relaxase (TraI) of conjugative plasmid RP4. J Biol Chem. 1994 Jan 28;269(4):2782–2789. [PubMed] [Google Scholar]
  27. Paszkowski J., Shillito R. D., Saul M., Mandák V., Hohn T., Hohn B., Potrykus I. Direct gene transfer to plants. EMBO J. 1984 Dec 1;3(12):2717–2722. doi: 10.1002/j.1460-2075.1984.tb02201.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Puchta H., Hohn B. A transient assay in plant cells reveals a positive correlation between extrachromosomal recombination rates and length of homologous overlap. Nucleic Acids Res. 1991 May 25;19(10):2693–2700. doi: 10.1093/nar/19.10.2693. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. Scheiffele P., Pansegrau W., Lanka E. Initiation of Agrobacterium tumefaciens T-DNA processing. Purified proteins VirD1 and VirD2 catalyze site- and strand-specific cleavage of superhelical T-border DNA in vitro. J Biol Chem. 1995 Jan 20;270(3):1269–1276. doi: 10.1074/jbc.270.3.1269. [DOI] [PubMed] [Google Scholar]
  31. Schultze M., Hohn T., Jiricny J. The reverse transcriptase gene of cauliflower mosaic virus is translated separately from the capsid gene. EMBO J. 1990 Apr;9(4):1177–1185. doi: 10.1002/j.1460-2075.1990.tb08225.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. 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]
  36. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Vogel A. M., Das A. Mutational analysis of Agrobacterium tumefaciens virD2: tyrosine 29 is essential for endonuclease activity. J Bacteriol. 1992 Jan;174(1):303–308. doi: 10.1128/jb.174.1.303-308.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. 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]
  39. Waters V. L., Guiney D. G. Processes at the nick region link conjugation, T-DNA transfer and rolling circle replication. Mol Microbiol. 1993 Sep;9(6):1123–1130. doi: 10.1111/j.1365-2958.1993.tb01242.x. [DOI] [PubMed] [Google Scholar]
  40. 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]
  41. 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 The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES