Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1989 May;8(5):1291–1301. doi: 10.1002/j.1460-2075.1989.tb03508.x

An ice nucleation reporter gene system: identification of inducible pathogenicity genes in Pseudomonas syringae pv. phaseolicola.

P B Lindgren 1, R Frederick 1, A G Govindarajan 1, N J Panopoulos 1, B J Staskawicz 1, S E Lindow 1
PMCID: PMC400954  PMID: 2548841

Abstract

We have constructed derivatives of the transposon Tn3 that allow an ice nucleation gene (inaZ) to be used as 'reporter' of the transcriptional activity of genes into which it is inserted. In these derivatives (Tn3-Ice and Tn3-Spice), the lacZYA sequences of transposon Tn3-HoHo1 were replaced with inaZ lacking its native promoter. The ice nucleation activity of virB::inaZ fusions in the correct transcriptional orientation was inducible by acetosyringone, a plant metabolite which activates the vir operon of Agrobacterium tumefaciens Ti plasmids, while fusions in the opposite orientation were unresponsive to the inducer. Tn3-Spice was also used to investigate the expression of a cluster of genes (hrp) which control pathogenicity and hypersensitivity elicited by Pseudomonas syringae pv. phaseolicola. An inducible region was identified which is expressed at low levels in vitro but becomes activated when the bacteria come into contact with the susceptible host, bean. Activation of this region occurred within 2 h post-inoculation and was nearly complete by the time the bacteria began to multiply in the leaf tissue. The inaZ reporter appears to be at least 10(5)-fold more sensitive than lacZ in P.s.phaseolicola. Thus, the inaZ fusion system provides a sensitive, convenient and inexpensive tool for the study of bacterial gene expression, particularly during plant pathogenesis, and should be generally useful as a reporter gene system in Gram-negative bacteria.

Full text

PDF
1294

Selected References

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

  1. 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]
  2. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  3. Carmi O. A., Stewart G. S., Ulitzur S., Kuhn J. Use of bacterial luciferase to establish a promoter probe vehicle capable of nondestructive real-time analysis of gene expression in Bacillus spp. J Bacteriol. 1987 May;169(5):2165–2170. doi: 10.1128/jb.169.5.2165-2170.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Downie J. A., Johnston A. W. Nodulation of legumes by Rhizobium: the recognized root? Cell. 1986 Oct 24;47(2):153–154. doi: 10.1016/0092-8674(86)90436-8. [DOI] [PubMed] [Google Scholar]
  6. Govindarajan A. G., Lindow S. E. Size of bacterial ice-nucleation sites measured in situ by radiation inactivation analysis. Proc Natl Acad Sci U S A. 1988 Mar;85(5):1334–1338. doi: 10.1073/pnas.85.5.1334. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Ish-Horowicz D., Burke J. F. Rapid and efficient cosmid cloning. Nucleic Acids Res. 1981 Jul 10;9(13):2989–2998. doi: 10.1093/nar/9.13.2989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. KING E. O., WARD M. K., RANEY D. E. Two simple media for the demonstration of pyocyanin and fluorescin. J Lab Clin Med. 1954 Aug;44(2):301–307. [PubMed] [Google Scholar]
  9. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  10. Lindgren P. B., Peet R. C., Panopoulos N. J. Gene cluster of Pseudomonas syringae pv. "phaseolicola" controls pathogenicity of bean plants and hypersensitivity of nonhost plants. J Bacteriol. 1986 Nov;168(2):512–522. doi: 10.1128/jb.168.2.512-522.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Lindow S. E., Arny D. C., Upper C. D. Bacterial ice nucleation: a factor in frost injury to plants. Plant Physiol. 1982 Oct;70(4):1084–1089. doi: 10.1104/pp.70.4.1084. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Maki L. R., Galyan E. L., Chang-Chien M. M., Caldwell D. R. Ice nucleation induced by pseudomonas syringae. Appl Microbiol. 1974 Sep;28(3):456–459. doi: 10.1128/am.28.3.456-459.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Orser C., Staskawicz B. J., Panopoulos N. J., Dahlbeck D., Lindow S. E. Cloning and expression of bacterial ice nucleation genes in Escherichia coli. J Bacteriol. 1985 Oct;164(1):359–366. doi: 10.1128/jb.164.1.359-366.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Osbourn A. E., Barber C. E., Daniels M. J. Identification of plant-induced genes of the bacterial pathogen Xanthomonas campestris pathovar campestris using a promoter-probe plasmid. EMBO J. 1987 Jan;6(1):23–28. doi: 10.1002/j.1460-2075.1987.tb04713.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Peet R. C., Lindgren P. B., Willis D. K., Panopoulos N. J. Identification and cloning of genes involved in phaseolotoxin production by Pseudomonas syringae pv. "phaseolicola". J Bacteriol. 1986 Jun;166(3):1096–1105. doi: 10.1128/jb.166.3.1096-1105.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Phelps P., Giddings T. H., Prochoda M., Fall R. Release of cell-free ice nuclei by Erwinia herbicola. J Bacteriol. 1986 Aug;167(2):496–502. doi: 10.1128/jb.167.2.496-502.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Prentki P., Krisch H. M. In vitro insertional mutagenesis with a selectable DNA fragment. Gene. 1984 Sep;29(3):303–313. doi: 10.1016/0378-1119(84)90059-3. [DOI] [PubMed] [Google Scholar]
  18. Southworth M. W., Wolber P. K., Warren G. J. Nonlinear relationship between concentration and activity of a bacterial ice nucleation protein. J Biol Chem. 1988 Oct 15;263(29):15211–15216. [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. Stachel S. E., Nester E. W., Zambryski P. C. A plant cell factor induces Agrobacterium tumefaciens vir gene expression. Proc Natl Acad Sci U S A. 1986 Jan;83(2):379–383. doi: 10.1073/pnas.83.2.379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Stachel S. E., Zambryski P. C. virA and virG control the plant-induced activation of the T-DNA transfer process of A. tumefaciens. Cell. 1986 Aug 1;46(3):325–333. doi: 10.1016/0092-8674(86)90653-7. [DOI] [PubMed] [Google Scholar]
  23. Staskawicz B., Dahlbeck D., Keen N., Napoli C. Molecular characterization of cloned avirulence genes from race 0 and race 1 of Pseudomonas syringae pv. glycinea. J Bacteriol. 1987 Dec;169(12):5789–5794. doi: 10.1128/jb.169.12.5789-5794.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Struck D. K., Maratea D., Young R. Purification of hybrid beta-galactosidase proteins encoded by phi X174 E phi lacZ and Escherichia coli prlA phi lacZ: a general method for the isolation of lacZ fusion polypeptides produced in low amounts. J Mol Appl Genet. 1985;3(1):18–25. [PubMed] [Google Scholar]
  25. Tamaki S., Dahlbeck D., Staskawicz B., Keen N. T. Characterization and expression of two avirulence genes cloned from Pseudomonas syringae pv. glycinea. J Bacteriol. 1988 Oct;170(10):4846–4854. doi: 10.1128/jb.170.10.4846-4854.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Warren G., Corotto L., Wolber P. Conserved repeats in diverged ice nucleation structural genes from two species of Pseudomonas. Nucleic Acids Res. 1986 Oct 24;14(20):8047–8060. doi: 10.1093/nar/14.20.8047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Weinstock G. M., Berman M. L., Silhavy T. J. Chimeric genetics with beta-galactosidase. Gene Amplif Anal. 1983;3:27–64. [PubMed] [Google Scholar]
  28. Wolber P. K., Deininger C. A., Southworth M. W., Vandekerckhove J., van Montagu M., Warren G. J. Identification and purification of a bacterial ice-nucleation protein. Proc Natl Acad Sci U S A. 1986 Oct;83(19):7256–7260. doi: 10.1073/pnas.83.19.7256. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES