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. 1987 Jul;169(7):3209–3216. doi: 10.1128/jb.169.7.3209-3216.1987

Identification of a new virulence locus in Agrobacterium tumefaciens that affects polysaccharide composition and plant cell attachment.

M F Thomashow, J E Karlinsey, J R Marks, R E Hurlbert
PMCID: PMC212371  PMID: 3597321

Abstract

We have identified a new virulence locus in Agrobacterium tumefaciens. Strains carrying Tn5 inserts at this locus could not incite tumors on Kalanchoe daigremontiana, Nicotiana rustica, tobacco, or sunflower and had severely attenuated virulence on carrot disks. We termed the locus pscA, because the mutants that defined the locus were initially isolated as having an altered polysaccharide composition; they were nonfluorescent on media containing Leucophor or Calcofluor, indicating a defect in the production of cellulose fibrils. Further analysis showed that the pscA mutants produced little, if any, of the four species of exopolysaccharide synthesized by the wild-type strain. DNA hybridization analysis and genetic complementation experiments indicated that the pscA locus is not encoded by the Ti plasmid and that it is distinct from the previously described chromosomal virulence loci chvA and chvB. However, like chvA and chvB mutants, the inability of the pscA mutants to form tumors is apparently due to a defect in plant cell attachment. Whereas we could demonstrate binding of the wild-type strain to tobacco suspension cells, attachment of the pscA mutants was drastically reduced or completely absent.

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Selected References

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  1. Akiyoshi D. E., Klee H., Amasino R. M., Nester E. W., Gordon M. P. T-DNA of Agrobacterium tumefaciens encodes an enzyme of cytokinin biosynthesis. Proc Natl Acad Sci U S A. 1984 Oct;81(19):5994–5998. doi: 10.1073/pnas.81.19.5994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Alt-Moerbe J., Rak B., Schröder J. A 3.6-kbp segment from the vir region of Ti plasmids contains genes responsible for border-sequence-directed production of T region circles in E. coli. EMBO J. 1986 Jun;5(6):1129–1135. doi: 10.1002/j.1460-2075.1986.tb04337.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barry G. F., Rogers S. G., Fraley R. T., Brand L. Identification of a cloned cytokinin biosynthetic gene. Proc Natl Acad Sci U S A. 1984 Aug;81(15):4776–4780. doi: 10.1073/pnas.81.15.4776. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
  5. Boyer H. W., Roulland-Dussoix D. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 1969 May 14;41(3):459–472. doi: 10.1016/0022-2836(69)90288-5. [DOI] [PubMed] [Google Scholar]
  6. Buchholz W. G., Thomashow M. F. Comparison of T-DNA oncogene complements of Agrobacterium tumefaciens tumor-inducing plasmids with limited and wide host ranges. J Bacteriol. 1984 Oct;160(1):319–326. doi: 10.1128/jb.160.1.319-326.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. Douglas C. J., Halperin W., Nester E. W. Agrobacterium tumefaciens mutants affected in attachment to plant cells. J Bacteriol. 1982 Dec;152(3):1265–1275. doi: 10.1128/jb.152.3.1265-1275.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Douglas C. J., Staneloni R. J., Rubin R. A., Nester E. W. Identification and genetic analysis of an Agrobacterium tumefaciens chromosomal virulence region. J Bacteriol. 1985 Mar;161(3):850–860. doi: 10.1128/jb.161.3.850-860.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dylan T., Ielpi L., Stanfield S., Kashyap L., Douglas C., Yanofsky M., Nester E., Helinski D. R., Ditta G. Rhizobium meliloti genes required for nodule development are related to chromosomal virulence genes in Agrobacterium tumefaciens. Proc Natl Acad Sci U S A. 1986 Jun;83(12):4403–4407. doi: 10.1073/pnas.83.12.4403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Finan T. M., Hirsch A. M., Leigh J. A., Johansen E., Kuldau G. A., Deegan S., Walker G. C., Signer E. R. Symbiotic mutants of Rhizobium meliloti that uncouple plant from bacterial differentiation. Cell. 1985 Apr;40(4):869–877. doi: 10.1016/0092-8674(85)90346-0. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Gallie D. R., Hagiya M., Kado C. I. Analysis of Agrobacterium tumefaciens plasmid pTiC58 replication region with a novel high-copy-number derivative. J Bacteriol. 1985 Mar;161(3):1034–1041. doi: 10.1128/jb.161.3.1034-1041.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Garfinkel D. J., Nester E. W. Agrobacterium tumefaciens mutants affected in crown gall tumorigenesis and octopine catabolism. J Bacteriol. 1980 Nov;144(2):732–743. doi: 10.1128/jb.144.2.732-743.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gill R., Heffron F., Dougan G., Falkow S. Analysis of sequences transposed by complementation of two classes of transposition-deficient mutants of Tn3. J Bacteriol. 1978 Nov;136(2):742–756. doi: 10.1128/jb.136.2.742-756.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Holsters M., de Waele D., Depicker A., Messens E., van Montagu M., Schell J. Transfection and transformation of Agrobacterium tumefaciens. Mol Gen Genet. 1978 Jul 11;163(2):181–187. doi: 10.1007/BF00267408. [DOI] [PubMed] [Google Scholar]
  17. Hooykaas P. J., Hofker M., den Dulk-Ras H., Schilperoort R. A. A comparison of virulence determinants in an octopine Ti plasmid, a nopaline Ti plasmid, and an Ri plasmid by complementation analysis of Agrobacterium tumefaciens mutants. Plasmid. 1984 May;11(3):195–205. doi: 10.1016/0147-619x(84)90026-x. [DOI] [PubMed] [Google Scholar]
  18. Hughes J., McCully M. E. The use of an optical brightener in the study of plant structure. Stain Technol. 1975 Sep;50(5):319–329. doi: 10.3109/10520297509117082. [DOI] [PubMed] [Google Scholar]
  19. Hurlbert R. E. Isolation and characterization of Chromatium vinosum membranes. Arch Microbiol. 1974;101(2):169–186. doi: 10.1007/BF00455937. [DOI] [PubMed] [Google Scholar]
  20. Klee H. J., White F. F., Iyer V. N., Gordon M. P., Nester E. W. Mutational analysis of the virulence region of an Agrobacterium tumefaciens Ti plasmid. J Bacteriol. 1983 Feb;153(2):878–883. doi: 10.1128/jb.153.2.878-883.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Leigh J. A., Signer E. R., Walker G. C. Exopolysaccharide-deficient mutants of Rhizobium meliloti that form ineffective nodules. Proc Natl Acad Sci U S A. 1985 Sep;82(18):6231–6235. doi: 10.1073/pnas.82.18.6231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Mandel M., Higa A. Calcium-dependent bacteriophage DNA infection. J Mol Biol. 1970 Oct 14;53(1):159–162. doi: 10.1016/0022-2836(70)90051-3. [DOI] [PubMed] [Google Scholar]
  23. Matthysse A. G., Holmes K. V., Gurlitz R. H. Elaboration of cellulose fibrils by Agrobacterium tumefaciens during attachment to carrot cells. J Bacteriol. 1981 Jan;145(1):583–595. doi: 10.1128/jb.145.1.583-595.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Matthysse A. G. Role of bacterial cellulose fibrils in Agrobacterium tumefaciens infection. J Bacteriol. 1983 May;154(2):906–915. doi: 10.1128/jb.154.2.906-915.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Puvanesarajah V., Schell F. M., Stacey G., Douglas C. J., Nester E. W. Role for 2-linked-beta-D-glucan in the virulence of Agrobacterium tumefaciens. J Bacteriol. 1985 Oct;164(1):102–106. doi: 10.1128/jb.164.1.102-106.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Schröder G., Waffenschmidt S., Weiler E. W., Schröder J. The T-region of Ti plasmids codes for an enzyme synthesizing indole-3-acetic acid. Eur J Biochem. 1984 Jan 16;138(2):387–391. doi: 10.1111/j.1432-1033.1984.tb07927.x. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Stachel S. E., An G., Flores C., Nester E. W. A Tn3 lacZ transposon for the random generation of beta-galactosidase gene fusions: application to the analysis of gene expression in Agrobacterium. EMBO J. 1985 Apr;4(4):891–898. doi: 10.1002/j.1460-2075.1985.tb03715.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. Sutherland I. W. Biosynthesis and composition of gram-negative bacterial extracellular and wall polysaccharides. Annu Rev Microbiol. 1985;39:243–270. doi: 10.1146/annurev.mi.39.100185.001331. [DOI] [PubMed] [Google Scholar]
  31. Thomashow L. S., Reeves S., Thomashow M. F. Crown gall oncogenesis: evidence that a T-DNA gene from the Agrobacterium Ti plasmid pTiA6 encodes an enzyme that catalyzes synthesis of indoleacetic acid. Proc Natl Acad Sci U S A. 1984 Aug;81(16):5071–5075. doi: 10.1073/pnas.81.16.5071. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Thomashow M. F., Hugly S., Buchholz W. G., Thomashow L. S. Molecular basis for the auxin-independent phenotype of crown gall tumor tissues. Science. 1986 Feb 7;231(4738):616–618. doi: 10.1126/science.3511528. [DOI] [PubMed] [Google Scholar]
  33. Watson B., Currier T. C., Gordon M. P., Chilton M. D., Nester E. W. Plasmid required for virulence of Agrobacterium tumefaciens. J Bacteriol. 1975 Jul;123(1):255–264. doi: 10.1128/jb.123.1.255-264.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]

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