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. 1990 Dec 11;18(23):6953–6958. doi: 10.1093/nar/18.23.6953

VirD2 gene product from the nopaline plasmid pTiC58 has at least two activities required for virulence.

T R Steck 1, T S Lin 1, C I Kado 1
PMCID: PMC332755  PMID: 2263456

Abstract

Virulence genes virD1 and virD2 are required for T-DNA processing in Agrobacterium tumefaciens. The regions within virD2 contributing to T-DNA processing and virulence were investigated. Some insertional mutations in virD2 prevented T-DNA border endonucleolytic cleavage and produced an avirulent phenotype. However, a non-polar insertion immediately after bp 684 of the 1344 bp open reading frame of virD2 did not inhibit endonucleolytic cleavage but still caused a loss of virulence. This suggested that in addition to T-DNA border cleaving activity, the VirD2 protein has another virulence function which resides in the C-terminal half of the protein. Comparative nucleotide sequence analyses of virD2 showed that the first 684 bp were 81% homologous to virD2 of an octopine Ti plasmid whereas the remaining 660 bp were only 44% homologous. A plasmid containing the virD region from octopine Ti plasmid could restore both virulence and processing to a nopaline virD2 mutant. No complementation resulted when a nopaline virD2 clone containing a region similar to eukaryotic nuclear envelope transport sequences was deleted from the 3' end. These results suggest that virD1 and only the first half of virD2 are required to encode for the T-DNA processing endonuclease, and that the 3'-half of virD2 encodes a function separate from endonuclease activity that is required for virulence.

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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. Close T. J., Zaitlin D., Kado C. I. Design and development of amplifiable broad-host-range cloning vectors: analysis of the vir region of Agrobacterium tumefaciens plasmid pTiC58. Plasmid. 1984 Sep;12(2):111–118. doi: 10.1016/0147-619x(84)90057-x. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. 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]
  5. Ditta G., Stanfield S., Corbin D., Helinski D. R. Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7347–7351. doi: 10.1073/pnas.77.12.7347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  8. Friedman A. M., Long S. R., Brown S. E., Buikema W. J., Ausubel F. M. Construction of a broad host range cosmid cloning vector and its use in the genetic analysis of Rhizobium mutants. Gene. 1982 Jun;18(3):289–296. doi: 10.1016/0378-1119(82)90167-6. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. 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]
  11. 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]
  12. 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]
  13. 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]
  14. 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]
  15. Kamoun S., Cooley M. B., Rogowsky P. M., Kado C. I. Two chromosomal loci involved in production of exopolysaccharide in Agrobacterium tumefaciens. J Bacteriol. 1989 Mar;171(3):1755–1759. doi: 10.1128/jb.171.3.1755-1759.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kao J. C., Perry K. L., Kado C. I. Indoleacetic acid complementation and its relation to host range specifying genes on the Ti plasmid of Agrobacterium tumefaciens. Mol Gen Genet. 1982;188(3):425–432. doi: 10.1007/BF00330044. [DOI] [PubMed] [Google Scholar]
  17. Klapwijk P. M., van Beelen P., Schilperoort R. A. Isolation of a recombination deficient Agrobacterium tumefaciens mutant. Mol Gen Genet. 1979 Jun 7;173(2):171–175. doi: 10.1007/BF00330307. [DOI] [PubMed] [Google Scholar]
  18. Morel P., Powell B. S., Rogowsky P. M., Kado C. I. Characterization of the virA virulence gene of the nopaline plasmid, pTiC58, of Agrobacterium tumefaciens. Mol Microbiol. 1989 Sep;3(9):1237–1246. doi: 10.1111/j.1365-2958.1989.tb00274.x. [DOI] [PubMed] [Google Scholar]
  19. Rogowsky P. M., Close T. J., Chimera J. A., Shaw J. J., Kado C. I. Regulation of the vir genes of Agrobacterium tumefaciens plasmid pTiC58. J Bacteriol. 1987 Nov;169(11):5101–5112. doi: 10.1128/jb.169.11.5101-5112.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Rogowsky P. M., Powell B. S., Shirasu K., Lin T. S., Morel P., Zyprian E. M., Steck T. R., Kado C. I. Molecular characterization of the vir regulon of Agrobacterium tumefaciens: complete nucleotide sequence and gene organization of the 28.63-kbp regulon cloned as a single unit. Plasmid. 1990 Mar;23(2):85–106. doi: 10.1016/0147-619x(90)90028-b. [DOI] [PubMed] [Google Scholar]
  21. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. 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]
  24. Steck T. R., Close T. J., Kado C. I. High levels of double-stranded transferred DNA (T-DNA) processing from an intact nopaline Ti plasmid. Proc Natl Acad Sci U S A. 1989 Apr;86(7):2133–2137. doi: 10.1073/pnas.86.7.2133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. 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]
  27. Wang K., Herrera-Estrella A., Van Montagu M. Overexpression of virD1 and virD2 genes in Agrobacterium tumefaciens enhances T-complex formation and plant transformation. J Bacteriol. 1990 Aug;172(8):4432–4440. doi: 10.1128/jb.172.8.4432-4440.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. 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]
  30. 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]
  31. Zyprian E., Kado C. I. Agrobacterium-mediated plant transformation by novel mini-T vectors in conjunction with a high-copy vir region helper plasmid. Plant Mol Biol. 1990 Aug;15(2):245–256. doi: 10.1007/BF00036911. [DOI] [PubMed] [Google Scholar]

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