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Pakistan Journal of Biological Sciences

Year: 2005 | Volume: 8 | Issue: 10 | Page No.: 1368-1375
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Research Article

Characterization of the Hypersensitive Response Eliciting Protein, HarpinEs, of Erwinia (Pantoea) stewartii and its Role in Determining the Pathogenicity of the Bacterium on Corn

Musharaf Ahmad, Doris R. Majerczak and David L. Coplin

ABSTRACT

Using hrpN of Erwinia amylovora as a radio-active probe, hrpN of Erwinia stewartii was located within its wts cluster. This gene was cloned and mutagenized with Tn5 to get mutants MA1 and MA2. These hrpN mutants were verified by Southern blotting, Western blotting, sequencing and hypersensitive response to make sure that the Tn5 insertions were in the open reading frame of the hrpNEs gene and no truncated harpinEs was produced by these mutants. In order to assess the role of the hrpNEs gene product i.e., harpinEs in the pathogenicity of the bacterium, mutants MA1 hrpN 123 :: Tn5, MA2 hrpN 254 :: Tn5 and DM760 hrpN 189 :: Tn5 were compared to the wild-type strain DC 283 using ED50, disease severity, growth rate in planta and response time as pathogenicity parameters. The ED50 of the mutants was not significantly different (p=0.05) from that of the wild-type. The differences between the response times for mutants and the wild-type were not statistically significant (p=0.05) either. The mutants grew and caused as severe disease in the corn seedlings as the wild-type did. The results indicated that the mutations in the hrpN did not affect the pathogenicity of E. stewartii in any way i.e. the mutants were neither qualitatively nor quantitatively different from the wild-type controls.
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Musharaf Ahmad, Doris R. Majerczak and David L. Coplin, 2005. Characterization of the Hypersensitive Response Eliciting Protein, HarpinEs, of Erwinia (Pantoea) stewartii and its Role in Determining the Pathogenicity of the Bacterium on Corn. Pakistan Journal of Biological Sciences, 8: 1368-1375.

DOI: 10.3923/pjbs.2005.1368.1375

URL: https://scialert.net/abstract/?doi=pjbs.2005.1368.1375

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REFERENCES

  1. Lindgren, P.B., R.C. Peet and N.J. Panopoulos, 1986. Gene cluster of Pseudomonas syringae pv. Phaseolicola controls pathogenicity of bean plants and HR on non hosts. J. Bacteriol., 168: 512-522.
    Direct Link
  2. Willis, D.K., J.J. Rich and E.M. Hrabak, 1991. Hrp genes of phytopathogenic bacteria. Mol. Plant Microbiol. Interact., 4: 132-138.
  3. Wei, Z.M., R.J. Laby, C.H. Zumoff, D.W. Bauer, S.Y. He, A. Collmer and S.V. Beer, 1992. Harpin elicitor of the hypersensitive response produced by the plant pathogen Erwinia amylovora. Science, 257: 85-88.
    Direct Link
  4. Bonas, U., 1994. Hrp Genes of Phytopahogenic Bacteria. In: Current Topics in Micribiology and Immunology. Bacterial Pathogenesis of Plants and Animals: Molecular and Cellular Mechanisms, Dangle, J.L (Ed.). Springer- Berlin, pp: 79-98.
  5. Buttner, D. and U. Bonas, 2002. Getting across bacterial type III effector proteins on their way to the plant cell. EMBO J., 21: 5313-5322.
    CrossRef
  6. Buttner, D. and U. Bonas, 2002. Port of entry the type III secretion translocon. Trends Microbiol., 10: 186-192.
  7. Cornelis, G.R. and F.V. Gijsegem, 2000. Assembly and function of type III secretory systems. Ann. Rev. Microbiol., 54: 735-774.
  8. Hueck, C.J., 1998. Type III protein secretion systems in bacterial pathogens of animals and plants. Microbiol. Mol. Biol. Rev., 62: 379-433.
    PubMedDirect Link
  9. Fenselau, S., I. Balbo and U. Bonas, 1992. Determinants of pathogenicity in Xanthomonas campestris pv. Vesicatoria are related to proteins involved in secretion in bacterial pathogens of animals. Mol. Plant Microbiol. Interact., 5: 390-396.
  10. Gough, C.L., S. Genin, C. Zischeck and C.A. Boucher, 1992. Hrp genes of Pseudomonas solanacearum are homologous to pathogenicity determinants of animal pathogenic bacteria and are conserved among plant pathogenic bacteria. Mol. Plant Microbiol. Interact., 5: 384-389.
    Direct Link
  11. Rahme, L.G., E.J. Stevens, S.F. Wolfort, J. Shao, R.G. Tompkins and F.M. Ausubel, 1995. Common virulence factors for bacterial pathogenicity in plants and animals. Science, 268: 1899-1902.
    CrossRef
  12. He, S.Y., H.C. Huang and A. Collmer, 1993. Pseudomonas syringae pv. Syringae harpin Pss a protein that is secreted via the Hrp pathway and elicits the hypersensitive response in plants. Cell, 73: 1255-1266.
    Direct Link
  13. Preston, G., H.C. Huang, S.Y. He and A. Collmer, 1995. The HrpZ proteins of Pseudomonas syringae pvs. Syringae, glycinea and Tomato are encoded by an operon containing Yersinia ysc homologs and elicit the hypersensitive response in tomato but not soybean irrespective of bacterial host range. Mol. Plant Microbiol. Interact., 8: 717-732.
  14. Bauer, D.W., A.J. Bogdanove, S.V. Beer and A. Collmer, 1994. Erwinia chrysanthemi hrp genes and their involvement in soft rot pathogenesis and elicitation of the hypersensitive response. Mol. Plant Microbiol. Interact, 7: 573-581.
  15. Arlat, M., F.V. Gijsegem, J.C. Huet, J.C. Pernolet and C.A. Boucher, 1994. PopA a protein which induces a hypersensitive like response on specific petunia genotypes is secreted via the Hrp pathway of Pseudomonas solanacearum. EMBO J., 13: 543-553.
    Direct Link
  16. Abedi, M.J. and P. Najafi, 1994. Structural and functional analysis of Erwinia amylovora harpins. Proceedings of the International Symposium Molecular Plant Microbe Interactions, (ISMPMI'94), Edinburgh, Scotland, pp: 66-66.
  17. Alfano, J.R., D.W. Bauer, A.O. Loniello and A. Collmer, 1994. Hypersensitive response elicitor activity of Pseudomonas syringae pv. Syringae harpin is not confined to a single region. Proceedings of the International Symposium Molecular Plant Microbe Interact, (ISMPMI'94), Edinburgh, Scotland, pp: 102-102.
  18. Bauer, D.W., Z.M. Wei, S.V. Beer and A. Collmer, 1995. Erwinia chrysanthemi harpinEch an elicitor of the hypersensitive response that contributes to soft rot pathogenesis. Mol. Plant Microbiol. Interact., 8: 484-491.
    Direct Link
  19. Coplin, D.L., R.D. Frederick and E.S. Haas, 1986. Molecular cloning of virulence genes from Erwinia stewartii. J. Bacteriol., 168: 619-623.
    Direct Link
  20. Frederick, R.D., M. Ahmad, D.R. Majerczak, A.S.A. Rodriguez, S. Manulis and D.L. Coplin, 2001. Genetic organization of Pantoea stewartii ssp. Stewartii hrp gene cluster and sequence analysis of the HrpA, hrpC, hrpN and WtsE operons. Mol. Plant Microbiol. Interact., 14: 1213-1222.
  21. Laby, R.J. and S.V. Beer, 1992. Hybridization and functional complementation of the Hrp gene cluster from Erwinia amylovora strain Ea 321 with DNA of other bacteria. Mol. Plant Microbiol. Interact., 5: 412-419.
  22. Bogdanove, A.J., Z.M. Wei, L. Zhao and S.V. Beer, 1996. Erwinia amylovora secretes harpin via a type III pathway and contains a homolog of YopN of Yersinia. J. Bacteriol., 178: 1720-1730.
    Direct Link
  23. Coplin, D.L., R.G. Rowan, D.A. Chisholm and R.E. Whitmoyer, 1981. Characterization of plasmids in Erwinia stewartii. Applied Environ. Microbiol., 42: 599-604.
    Direct Link
  24. Coplin, D.L., 1978. Properties of F and P group plasmids in Erwinia stewartii. Phytopathology, 68: 1637-1643.
  25. Dolph, P.J., D.R. Majerczak and D.L. Coplin, 1988. Characterization of a gene cluster for exopolysaccharide biosynthesis and virulence in Erwinia stewartii. J. Bacteriol., 170: 865-871.
    Direct Link
  26. Sambrook, J., E.F. Fritsch and T. Maniatis, 1989. Molecular Cloning a Aboratory Manual. 2nd Edn., Cold Spring Harbor Laboratory, Cold Spring Harbor, New Yark.
  27. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227: 680-685.
    CrossRefDirect Link
  28. Hager, D.A. and R.R. Burgees, 1980. Elution of proteins from sodium dodecyl sulfate polyacrylamide gels removal of SDS and renaturation of enzymatic activity results with sigma subunit of E. Coli RNA polymerase, wheat germ DNA topoisomerase and other enzymes. Ann. Biochem., 109: 76-86.
    CrossRef
  29. Ausubel, F.M., R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith and K. Struhl, 1992. Current Protocols in Molecular Biology. Vol. 2, John Wiley and Sons, Inc., New Yark.
  30. Ahmad, M., D.R. Majerczak, S. Pike, M.E. Hoyos, A. Novacky and D.L. Coplin, 2001. Biological activity of harpin produced by Pantoea stewartii subsp. stewartii. Mol. Plant Microbiol. Interact, 14: 1223-1234.
    Direct Link
  31. Studier, F.W. and B.A. Moffatt, 1986. A bacteriophage T7 RNA polymerase to direct selective high level expression of cloned genes. J. Mol. Biol., 189: 113-130.
  32. Loniello, A.O., J.R. Alfano, D.W. Bauer and A. Collmer, 1995. Analysis of pathogenicity of a Pseudomonas syringae pv. Syringae B728a ΔHrpZ, Npt II mutant on bean. Phytopathology, 85: 1148-1148.
  33. Parsot, C., R. Menard, P. Gounon and P.J. Sansonetti, 1995. Enhanced secretion through the Shigella flexnerri Mxi Spa translocon leads to assembly of extracellular proteins into macromolecular structures. Mol. Micribiol., 16: 291-300.
  34. Poos, F.W., 1940. Host plants harboring Aplanobacter stewartii without showing external symptoms after inoculation by Chaetocnema pulicaria. Econ. Entomol., 16: 291-300.
  35. Staskawicz, B.J., F.M. Ausubel, B.J. Baker, J.G. Ellis and J.D.G. Jones, 1995. Molecular genetics of plant disease resistance. Science, 268: 661-667.
    CrossRef
  36. Gijsegem, F.V., C. Gough, C. Zischeh, E. Niqueux and C. Boucher et al., 1995. The Hrp gene locus of Pseudomonas salanacearum which controls the production of Type III secretion system, encodes eight proteins related to components of the bacterialflagellar biosynthesis complex. Mol. Microbiol., 15: 1095-1114.
  37. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72: 248-254.
    CrossRefPubMedDirect Link

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