Archives of Microbiology

, Volume 153, Issue 1, pp 85–89 | Cite as

Ti plasmid containing Rhizobium meliloti are non-tumorigenic on plants, despite proper virulence gene induction and T-strand formation

  • R. J. M. van Veen
  • H. den Dulk-Ras
  • R. A. Schilperoort
  • P. J. J. Hooykaas
Original Papers


We examined the expression of the vir genes of the Agrobacterium tumefaciens Ti plasmid in Rhizobium meliloti, which remains non-tumorigenic on plants after introduction of a Ti- or Ri-plasmid. Both the levels of virulence (vir) gene expression, induced by the plant phenolic compound acetosyringone, and of subsequent T-strand formation were comparable to what is observed in Agrobacterium. In contrast to the situation in Agrobacterium, though, vir induction in R. meliloti did not require a low pH (5.3) of the induction medium and the optimum temperature for induction in R. meliloti was significantly lower than in Agrobacterium. At 37°C no induction of the vir genes was found both in Agrobacterium and R. meliloti. We postulate that the lack of tumorigenicity of Ti carrying R. meliloti strains is due either to a lack of proper attachment of the bacteria to plant cells, or to an improper assembly of a virB-determined essential structure in the cell wall of R. meliloti.

Key words

Agrobacterium tumefaciens Rhizobium meliloti Vir gene expression T-strands Chromosomal virulence genes 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Alt-MoerbeJ, NeddermannP, LintigJvon, WeilerEW, SchröderJ (1988) Temperature sensitive step in Ti plasmid vir-region induction and correlation with cytokinin secretion by Agrobacterium. Mol Gen Genet 213: 1–8Google Scholar
  2. BeringerJE, BeynonJL (1978) Transfer of the drug-resistance transposon Tn5 to Rhizobium. Nature 276: 633–634Google Scholar
  3. Caetano AnollesG, FavelukesG (1986) Host-symbiont specificity expressed during early adsorption of Rhizobium meliloti to the root surface of alfalfa. Appl Environm Microbiol 52: 377–383Google Scholar
  4. DouglasC, HarperinW, NesterEW (1982) Agrobacterium tumefaciens mutants affected in attachment to plant cells. J Bacteriol 152: 1265–1275Google Scholar
  5. DraperJ, MackensieIA, DaveyMR, FreemanJP (1983) Attachment of Agrobacterium tumefaciens to mechanically isolated Asparagus cells. Plant Science Lett 29: 227–236Google Scholar
  6. GietlC, Koukolikova-NicolaZ, HohnB (1987) Mobilization of T-DNA from Agrobacterium to plant cells involves a protein that binds single-stranded DNA. Proc Natl Acad Sci USA 84: 9006–9010Google Scholar
  7. HooykaasPJJ, SchilperoortRA (1984) The molecular genetics of crown-gall tumorigenesis. In: ScandaliosHG (ed) Molecular genetics of plants (Advances in genetics vol 22). Academic Press, Orlando, USA, pp 209–283Google Scholar
  8. HooykaasPJJ, KlapwijkPM, NutiMP, SchilperoortRA, RörschA (1977) Transfer of the Agrobacterium tumefaciens Ti plasmid to avirulent agrobacteria and to Rhizobium ex planta. J Gen Microbiol 98: 477–484Google Scholar
  9. HooykaasPJJ, RoobolC, SchilperoortRA (1979) Regulation of the transfer of Ti plasmids of Agrobacterium tumefaciens. J Gen Microbiol 110: 99–109Google Scholar
  10. HooykaasPJJ, SnijdewindtFGM, SchilperoortRA (1982) Identification of the Sym plasmid of Rhizobium leguminosarum strain 1001 and its transfer to and expression in other rhizobia and Agrobacterium tumefaciens. Plasmid 8: 73–82Google Scholar
  11. KadoCI, LiuST (1981) Rapid procedure for detection and isolation of large and small plasmids. J Bacteriol 145: 1365–1373Google Scholar
  12. Koukolikova-NicolaZ, AlbrightL, HohnB (1987) The mechanism of T-DNA transfer from Agrobacterium tumefaciens to plant cells. In: HohnT, SchellJ (eds) Plant DNA infectious agents. Springer, Wien, pp 109–148Google Scholar
  13. KrensFA, MolendijkL, WullemsGJ, SchilperoortRA (1985) The role of bacterial attachment in the transformation of cell-wall-regenerating tobacco protoplasts by Agrobacterium tumefaciens. Planta 166: 300–308Google Scholar
  14. LafreniereC, BordeleauLM, AmargerN, AntainH (1984) Effect of heterologous bacteria and combined nitrogen on the adsorption of Rhizobium meliloti to lucerne seedling roots. Plant Soil 82: 223–229Google Scholar
  15. LerouxB, YanofskyMF, WinansSC, WardJE, ZieglerSF, NesterEW (1987) Characterization of the virA locus of Agrobacterium tumefaciens: a transcriptional regulator and host-range determinant. EMBO J 6: 849–856Google Scholar
  16. MatthysseAG (1986) Initial interactions of Agrobacterium tumefaciens with plant host cells. CRC Crit Rev in Microbiol 13: 281–307Google Scholar
  17. MatthysseAG (1987) Characterization of non-attaching mutants of Agrobacterium tumefaciens. J Bacteriol 169: 313–323Google Scholar
  18. Melchers LS, Hooykaas PJJ (1987) Virulence of Agrobacterium. In: Oxford surveys of plant molecular and cell biology, vol 4, pp 167–220Google Scholar
  19. MelchersLS, ThompsonDV, IdlerKB, SchilperoortRA, HooykaasPJJ (1986) Nucleotide sequence of the virulence gene virG of the Agrobacterium tumefaciens octopine Ti plasmid: significant homology between virG and the regulatory genes ompR, phoB and dye of E. coli. Nucl Acids Res 14: 9933–9942Google Scholar
  20. MelchersLS, ThompsonDV, IdlerKB, NeuteboomSTC, deMaagdRA, SchilperoortRA, HooykaasPJJ (1987) Molecular characterization of the virulence gene virA of the Agrobacterium tumefaciens octopine Ti plasmid. Plant Mol Biol 11: 227–237Google Scholar
  21. Melchers LS, Regenburg-Tuïnk AJG, Schilperoort RA, Hooykaas PJJ (1989) Specificity of signal molecules in the activation of Agrobacterium virulence gene expression. Mol Microbiol (in press)Google Scholar
  22. MemelinkJ, dePaterBS, HogeJHC, SchilperoortRA (1987) T-DNA hormone biosynthetic genes: phytohormones and gene expression in plants. Devel Genet 8: 321–337Google Scholar
  23. MillerJH (1972) In: Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., pp 352–355Google Scholar
  24. OomsG, HooykaasPJJ, MoolenaarG, SchilperoortRA (1981) Crown gall plant tumors of abnormal morphology, induced by Agrobacterium tumefaciens carrying mutated octopine Ti plasmids; analysis of T-DNA functions. Gene 14: 33–50Google Scholar
  25. VanSchieBJ, DijkenvanJP, KuenenJG (1984) Non-coordinated synthesis of glucose dehydrogenase and its prostethic group PQQ in Acinetobacter and Pseudomonas species. FEMS Microbiol Lett 24: 133–138Google Scholar
  26. Smit G (1988) Adhesins from Rhizobiaceae and their role in plant-bacterium interactions. PhD Thesis, Leiden UniversityGoogle Scholar
  27. StachelSE, MessensE, VanMontaguM, ZambryskiP (1985) Identification of the signal molecules produced by wounded plant cells that activate T-DNA transfer in Agrobacterium tumefaciens. Nature 318: 624–629Google Scholar
  28. StachelSE, TimmermanB, ZambryskiP (1986) Generation of single-stranded T-DNA molecules during the initial stages of T-DNA transfer from Agrobacterium tumefaciens to plant cells. Nature 322: 706–712Google Scholar
  29. StachelSE, TimmermanB, ZambryskiP (1987) 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 6: 857–863Google Scholar
  30. ThomashowMF, KarlinseyJE, MarksJR, HurlbertRE (1987) Identification of a new virulence locus in Agrobacterium tumefaciens that affects polysaccharide composition and plant cell attachment. J Bacteriol 169: 3209–3216Google Scholar
  31. ThompsonDV, MelchersLS, IdlerKB, SchilperpoortRA, hooykaasPJJ (1988) Analysis of the complete nucleotide sequence of the Agrobacterium tumefaciens virB operon. Nucl Acids Res 16: 4621–4636Google Scholar
  32. VanHaarenMJJ, SedeeNJA, SchilperoortRA, HooykaasPJJ (1987) Overdrive is a T-region transfer enhancer which stimulates T-strand production in Agrobacterium tumefaciens. Nucl Acids Res 15: 8983–8997Google Scholar
  33. VeenRJMvan, HooykaasPJJ, SchilperoortRA (1988a) Mechanisms of tumorigenesis by Agrobacterium tumefaciens. In: KeenNT, WallingU (eds) Physiology and biochemistry of plant microbial interactions. Waverly Press Inc, Baltimore, USA, pp 19–30Google Scholar
  34. VeenRJMvan, Dulk-RasHden, BisselingT, SchilperoortRA, HooykaasPJJ (1988b) Crown gall tumor and root nodule formation by the bacterium Phyllobacterium myrsinacearum after the introduction of an Agrobacterium Ti plasmid or a Rhizobium Sym plasmid. Molec. Plant-Microbe Interactions 1: 231–234Google Scholar
  35. WardJE, AkiyoshiDE, RegiersD, DattaA, GordonM, NesterEW (1988) Characterization of the virB operon from an Agrobacterium tumefaciens Ti plasmid. J Biol Chem 263: 5804–5814Google Scholar
  36. WinansSC, EbertPR, StachelSE, GordonMP, NesterEW (1986) A gene essential for Agrobacterium virulence is homologous to a family of positive regulatory loci. Proc Natl Acad Sci USA 83: 8278–8282Google Scholar
  37. YanofskyMF, PorterSG, YoungC, AlbrightLM, GordonMP, NesterEW (1986) The virD operon of Agrobacterium tumefaciens encodes a site-specific endonuclease. Cell 47: 471–477Google Scholar

Copyright information

© Springer-Verlag 1989

Authors and Affiliations

  • R. J. M. van Veen
    • 1
  • H. den Dulk-Ras
    • 1
  • R. A. Schilperoort
    • 1
  • P. J. J. Hooykaas
    • 1
  1. 1.Department of Plant Molecular Biology, Biochemical LaboratoryLeiden UniversityLeidenThe Netherlands

Personalised recommendations