Skip to main content
SpringerLink
Log in

The VirD2 protein of Agrobacterlum tumefaciens carries nuclear localization signals important for transfer of T-DNA to plants

  • Published:
Molecular and General Genetics MGG Aims and scope Submit manuscript

Abstract

Agrobacterium tumefaciens is able to transfer a piece of DNA, the T-DNA, to the nucleus of the plant cell. The VirD2 protein is required for the production of the T-DNA, it is tightly linked to the T-DNA and it is thought to direct it to the plant genome. Two nuclear localization signals (NLS), one in the N-terminal part and one in the C-terminal part of the VirD2 protein, have been shown to be able to target marker proteins to the plant nucleus. Here we analyze nuclear entry of the T-DNA complex using a new and very sensitive assay for T-DNA transfer. We show that optimal T-DNA transfer requires the VirD2 NLS located in the C-terminal part of the protein, whereas mutations in the N-terminal NLS coding sequence seem to have no effect on T-DNA transfer.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Bonnard G, Tinland B, Paulus F, Szegedi E, Otten L (1989) 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 216:428–438

    Google Scholar 

  • Bradford M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein-dye binding. Annal Biochem 72:248–254

    Google Scholar 

  • Castle LA, Morris RO (1990) A method for early detection of T-DNA transfer. Plant Mot Biol Rep 8:28–39

    Google Scholar 

  • Chelsky D, Ralph R, Jonak G (1989) Sequence requirements for synthetic peptide-mediated translocation to the nucleus. Mol Cell Biol 9:2487–2492

    Google Scholar 

  • Citovsky V, Wong ML, Zambryski P (1989) Cooperative interaction of Agrobacterium VirE2 protein with single stranded DNA: implication for the T-DNA transfer process. Proc Natl Acad Sci USA 86:1193–1197

    Google Scholar 

  • De Vos G, Zambryski P (1989) Expression of Agrobacterium nopaline specific VirD1, VirD2 and VirC1 proteins and their requirement for T-strand production in E. coli. Mol Plant Microbe Interact 2:42–52

    Google Scholar 

  • Dingwall C, Laskey RA (1991) Nuclear targeting sequences — a consensus? Trends Biochem Sci 16:478–481

    Google Scholar 

  • Garcia Bustos J, Heitman J, Hall MN (1991) Nuclear protein localization. Biochim Biophys Acta Rev 1071:83–101

    Google Scholar 

  • Henkel T, Zabel U, van Zee K, Fanning E, Baeuerle PA (1992) Intramolecular masking of the nuclear location signal and dimerisation domain in the precursor for the p50 NF-kB subunit. Cell 68:1121–1133

    Google Scholar 

  • Herrera-Estrella A, Van Montagu M, Wang K (1990) A bacterial peptide acting as a plant nuclear targeting signal: the amino-terminal portion of Agrobacterium VirD2 protein directs a β-galactosidase fusion protein into tobacco nuclei. Proc Natl Acad Sci USA 87:9534–9537

    Google Scholar 

  • Hooykaas PJJ, Schilperoort RA (1992) Agrobacterium and plant genetic enginering. Plant Mol Biol 19:15–38

    Google Scholar 

  • Howard EA, Zupan JR, Citovsky V, Zambryski PC (1992) The VirD2 protein of A. tumefaciens contains a C-terminal bipartite nuclear localisation signal: implication for nuclear uptake of DNA in plant cells. Cell 68:109–118

    Google Scholar 

  • Jansen BJ, Gardner RC (1989) Localised transient expression of GUS in leaf discs following cocultivation with Agrobacterium. Plant Mol Biol 14:61–72

    Google Scholar 

  • Jefferson RA (1987) Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol Biol Rep 5:387–405

    Google Scholar 

  • Kado CI (1991) Molecular mechanisms of crown gall tumorigenesis. Crit Rev Plant Sci 10:1–32

    Google Scholar 

  • Kalderon D, Richardson WD, Markham AF, Smith AE (1984) Sequence requirements for nuclear location of simian virus 40 large-T antigen. Nature 311:33–38

    Google Scholar 

  • Koukolíková-Nicola Z, Raineri D, Stephens K, Ramos C, Tinland B, Nester EW, Hohn B (1993) Genetic analysis of the virD operon of Agrobacterium tumefaciens: a search for functions involved in the transport of T-DNA into the plant cell nucleus and in T-DNA integration. J Bacteriol 175:723–731

    Google Scholar 

  • Van der Krol AR, Chua NH (1991) The basic domain of plant B-ZIP proteins facilitates import of a reporter protein into the plant nuclei. Plant Cell 3:667–675

    Google Scholar 

  • Lanford RE, Butel JS (1984) Construction and characterisation of an SV40 mutant defective in nuclear transport of T antigen. Cell 37:801–813

    Google Scholar 

  • Machida Y, Shimoda N, Yamamoto-Toyoda A, Takahashi Y, Nishihama R, Aoki S, Matsuoka K, Nakamura K, Yoshioka Y, Ohba T, Obata RT (1992) Molecular interactions between Agrobacterium and plant cells. In: Nester EW (ed) Advances in Molecular Genetics of Plant-Microbe Interactions, vol 2. Kluwer, Dordrecht, pp 85–96

    Google Scholar 

  • Mattanovitch D, Ruker F, da Câmara Machado A, Laimer M, Regner F, Steinkellner H, Himmler G, Katinger H (1989) Efficient transformation of Agrobacterium spp. by electroporation. Nucleic Acids Res 17:6747

    Google Scholar 

  • McBride KE, Summerfelt KR (1990) Improved binary vectors for Agrobacterium-mediated transformation. Plant Mol Biol 14:269–276

    Google Scholar 

  • Mozo T, Hooykaas PJJ (1992) Factors affecting the rate of T-DNA transfer from Agrobacterium tumefaciens to Nicotiana glauca plant cells. Plant Mol Biol 19:1019–1030

    Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–493

    Google Scholar 

  • Parke D (1990) Construction of mobilisable vectors derived from plasmids RP4, pUC18 and pUC19. Gene 93:135–137

    Google Scholar 

  • Puchta H, Hohn B (1991) A transient assay in plant cells reveals a positive correlation between extrachromosomal recombination rates and length of homologous overlap. Nucleic Acids Res 19:2693–2700

    Google Scholar 

  • Ream W (1989) Agrobacterium tumefaciens and interkingdom genetic exchange. Annu Rev Phytopathol 27:583–618

    Google Scholar 

  • Ribs HP, Jans DA, Fan H, Peters R (1991) The rate of nuclear cytoplasmic protein transport is determined by the casein kinase II site flanking the nuclear localization sequence of the SV40 T-antigen. EMBO J 10:633–639

    Google Scholar 

  • Robbins J, Dilworth SM, Laskey RA, Dingwald C (1991) Two interdependent basic domains in nucleoplasmin nuclear targeting sequence: identification of a class of bipartite nuclear targeting sequence. Cell 64:615–623

    Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: A laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York

    Google Scholar 

  • Schultze M, Hohn T, Jiricny J (1990) The reverse transcriptase gene of CaMV is translated separately from the capsid gene. EMBO J 9:1177–1185

    Google Scholar 

  • Shurvinton CE, Hodges L, Ream W (1992) 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 USA 89:11837–11841

    Google Scholar 

  • Silver PA (1991) How proteins enter the nucleus. Cell 64:489–497

    Google Scholar 

  • Simon R, Priefer U, Pühler A (1983) A broad host range mobilisation system for in vivo genetic engineering: transposon mutagenesis in gram negative bacteria. Biotechnology 1:784–791

    Google Scholar 

  • Stachel SE, Nester EW (1986) The genetic and transcriptional organization of the vir region of the A6 Ti plasmid of Agrobacterium tumefaciens. EMBO J 5:1445–1454

    Google Scholar 

  • Steck TR, Morel P, Kado CI (1988) Vir box sequence in Agrobacterium tumefaciens pTiC58 and A6. Nucleic Acids Res 16:8736

    Google Scholar 

  • Steck TR, Close TJ, Kado CI (1989) High level of double-stranded transferred DNA (T-DNA) processing from an intact nopaline Ti plasmid. Proc Natl Acad Sci USA 86:2133–2137

    Google Scholar 

  • Steck TR, Lin TS, Kado CI (1990) VirD2 gene product from the nopaline plasmid pTiC58 has at least two activities required for virulence. Nucleic Acids Res 18:6953–6958

    Google Scholar 

  • Tinland B, Koukoliková-Nicola Z, Hall MN, Hohn B (1992) The T-DNA linked VirD2 contains two distinct functional nuclear localization signals. Proc Natl Acad Sci USA 89:7442–7446

    Google Scholar 

  • Tinland B, Rossi L, Hohn B (1993) The T-DNA transfer from Agrobacterium to the plant cell nucleus. In: Kado C (ed) Molecular mechanisms of bacterial virulence. Kluwer, Dordrecht (in press)

    Google Scholar 

  • Vogel AM, Das A (1992) Mutational analysis of Agrobacterium tumefaciens VirD2: tyrosine-29 is essential for endonuclease activity. J Bacteriol 174:303–312

    Google Scholar 

  • Ward ER, Barnes WM (1988) VirD2 protein of Agrobacterium tumefaciens is very tightly linked to the 5′ end of T-strand DNA. Science 242:927–930

    Google Scholar 

  • Yanofsky MF, Porter SG, Young C, Albright LM, Gordon MP, Nester EW (1986) The virD2 operon of Agrobacterium tumefaciens encodes a site-specific endonuclease. Cell 47:471–477

    Google Scholar 

  • Zambryski PC (1992) Chronicles from the Agrobacterium-plant cell DNA transfer story. Annu Rev Plant Physiol Plant Mol Biol 43:465–490

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Friedrich Miescher-Institut, PO Box 2543, CH-4002, Basel, Switzerland

    L. Rossi, B. Hohn & B. Tinland

Authors
  1. L. Rossi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. B. Hohn
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. B. Tinland
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

Communicated by H. Hennecke

Rights and permissions

Reprints and permissions

About this article

Cite this article

Rossi, L., Hohn, B. & Tinland, B. The VirD2 protein of Agrobacterlum tumefaciens carries nuclear localization signals important for transfer of T-DNA to plants. Molec. Gen. Genet. 239, 345–353 (1993). https://doi.org/10.1007/BF00276932

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00276932

Key words

Search

Navigation