Plant Molecular Biology

, Volume 59, Issue 5, pp 771–780 | Cite as

Expression of the Hypersensitive Response-assisting Protein in Arabidopsis Results in Harpin-dependent Hypersensitive Cell Death in Response to Erwinia carotovora

  • Ajay-Kumar Pandey
  • Mang-Jye Ger
  • Hsiang-En Huang
  • Mei-Kuen Yip
  • Jiqing Zeng
  • Teng-Yung Feng


Active defense mechanisms of plants against pathogens often include a rapid plant cell death known as the hypersensitive cell death (HCD). Hypersensitive response-assisting protein (HRAP) isolated from sweet pepper intensifies the harpinPss-mediated HCD. Here we demonstrate that constitutive expression of the hrap gene in Arabidopsis results in an enhanced disease resistance towards soft rot pathogen, E. carotovora subsp. carotovora. This resistance was due to the induction of HCD since different HCD markers viz. Athsr3, Athsr4, ion leakage, H2O2 and protein kinase were induced. One of the elicitor harpin proteins, HrpN, from Erwinia carotovora subsp. carotovora was able to induce a stronger HCD in hrap-Arabidopsis than non-transgenic controls. To elucidate the role of HrpN, we used E. carotovora subsp. carotovora defective in HrpN production. The hrpN mutant did not induce disease resistance or HCD markers in hrap-Arabidopsis. These results imply that the disease resistance of hrap-Arabidopsis against a virulent pathogen is harpin dependent.


Arabidopsisthaliana Erwinia carotovora harpin HRAP hypersensitive response 



adenosine tri-phosphate




Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid


hypersensitive response-assisting protein


hypersensitive cell death


mitogen activated protein kinase


polymerase chain reaction


sodium dodecyl sulphate-polyacrylamide gel electrophoresis


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  1. Baker, C.J., Orlandi, E.W., Mock, N.M. 1993Harpin, an elicitor of the hypersensitive response in tobacco caused by Erwinia amylovora, elicits active oxygen production in suspension cellsPlant Physiol.10213411344PubMedGoogle Scholar
  2. Barras, F., Gijsegem, F., Chatterjee, A. 1994Extracellular enzymes and pathogenesis of soft rot ErwiniaAnnu. Rev. Phytopathol.32201234Google Scholar
  3. Bauer, D.W., Bogdanove, A.J., Beer, S.V., Collmer, A. 1994Erwinia chrysanthemi hrp genes and their involvement in soft rot pathogenesis and elicitation of the hypersensitive responseMol. Plant–Microbe Interact.7573581PubMedGoogle Scholar
  4. Bauer, D.W., Wei, Z.-M., Beer, S.V., Collmer, A. 1995Erwinia chrysanthemi Harpin Ech: an elicitor of the hypersensitive response that contributes to soft-rot pathogenesisMol. Plant–Microbe Interact.8484491PubMedGoogle Scholar
  5. Bechtold, N., Ellis, J., Pellettier, G. 1993In planta Agrobacterium-mediated gene transfer by infiltration of adult Arabidopsis thaliana plantsCR Acad. Sci.31611941199Google Scholar
  6. Charkowski, A.O., Alfano, J.R., Preston, G., Yuan, J., He, S.Y., Collmer, A. 1998The Pseudomonas syringae pv. tomato HrpW protein has domains similar to harpins and pectate lyases and can elicit the plant hypersensitive response and bind to pectateJ. Bacteriol.18052115217PubMedGoogle Scholar
  7. Chen, C.H., Lin, H.J., Feng, T.Y. 1998An amphipathic protein from sweet pepper can dissociate harpinPss multimeric forms and intensify the harpinPss-mediated hypersensitive responsePhysiol. Mol. Plant Pathol.52139149CrossRefGoogle Scholar
  8. Chen, C.H., Lin, H.J., Ger, M.J., Chow, D., Feng, T.Y. 2000cDNA cloning and characterization of a plant protein that may be associated with the harpinPSS-mediated hypersensitive responsePlant Mol. Biol.43429438CrossRefPubMedGoogle Scholar
  9. Cornelis, G.R., Gijsegem, F. 2000Assembly and function of type III secretory systemsAnnu. Rev. Microbiol.54735774CrossRefPubMedGoogle Scholar
  10. Cui, Y., Madi, L., Mukherjee, A., Dumenyo, C.K., Chatterjee, A. 1996The RsmA-mutants of Erwinia carotovora subsp. carotovora strain Ecc71 overexpress hrpN Ecc and elicit a hypersensitive reaction-like response in tobacco leavesMol. Plant–Microbe Interact.9565573PubMedGoogle Scholar
  11. Dangl, J.L., Jones, J.D. 2001Plant pathogens and integrated defence response to infectionNature411826833PubMedGoogle Scholar
  12. Dangl, J.L., Diretrich, R.A., Richberg, M.H. 1996Death don’t have no mercy: cell death programs in plant–microbe interactionsPlant Cell817931807CrossRefPubMedGoogle Scholar
  13. Desikan, R., Clarke, A., Atherfold, P., Hancock, J.T., Neill, S.J. 1999Harpin induces mitogen-activated protein kinase activity during defence responses in Arabidopsis thaliana suspension culturesPlanta21097103CrossRefPubMedGoogle Scholar
  14. Desikan, R., Hancock, J.T., Ichimura, K., Shinozaki, K., Neill, S.J. 2001Harpin induces activation of the Arabidopsis mitogen-activated protein kinases AtMPK4 and AtMPK6Plant Physiol.12615791587CrossRefPubMedGoogle Scholar
  15. Dixon, R.A., Harrison, M.J. 1990Activation, structure and organization of genes involved in microbial defense in plantsAdv. Genet.28165234PubMedGoogle Scholar
  16. Galan, J.E., Collmer, A. 1999Type III secretion machines: bacterial devices for protein delivery into host cellsScience2113221328Google Scholar
  17. Gaudriault, S., Brisset, M.N., Barny, M.A. 1998HrpW of Erwinia amylovora, a new Hrp-secreted proteinFEBS Lett.428224228CrossRefPubMedGoogle Scholar
  18. Ger, M.J., Chen, C.H., Hwang, S.Y., Huang, H.E., Podile, A.R., Dayakar, B.V., Feng, T.Y. 2002Constitutive expression of hrap gene in transgenic tobacco plant enhances resistance against virulent bacterial pathogens by induction of a hypersensitive responseMol. Plant–Microbe Interact.15764773PubMedGoogle Scholar
  19. Gopalan, S., Bauer, D.W., Alfano, J.R., Loniello, A.O., He, S.Y., Collmer, A. 1996Expression of the Pseudomonas syringae avirulence protein AvrB in plant cells alleviates its dependence on the hypersensitive response and pathogenicity (Hrp) secretion system in eliciting genotype-specific hypersensitive cell deathPlant Cell810951105CrossRefPubMedGoogle Scholar
  20. He, S.Y., Bauer, D.W., Collmer, A., Beer, S.V. 1994Hypersensitive response elicited by Erwinia amylovora harpin requires active plant metabolismMol. Plant–Microbe Interact.7289292Google Scholar
  21. Holsters, M., Waele, D., Depicker, A., Messens, E., Montagu, M., Schell, J. 1978Transfection and transformation of Agrobacterium tumefaciensMol. Gen. Genet.163182187Google Scholar
  22. Jana, S., Choudhuri, M.A. 1982Glycolate metabolism of three submerged aquatic angiosperm during agingAquat. Bot.12345354CrossRefGoogle Scholar
  23. Kawano, T. 2003Roles of reactive oxygen species-generating peroxidase reaction in plant defense and growth inductionPlant Cell Rep.21829837PubMedGoogle Scholar
  24. Keller, H., Pamboukdjian, N., Ponchet, M., Poupet, A., Delon, R., Verrier, J.L., Roby, D., Ricci, P. 1999Pathogen-induced elicitin production in transgenic tobacco generates a hypersensitivity response and nonspecific disease resistancePlant Cell11223235CrossRefPubMedGoogle Scholar
  25. Kim, J.F., Beer, S.V. 1998HrpW of Erwinia amylovora, a new harpin that contains a domain homologous to pectate lyases of a distinct classJ. Bacteriol.18052035210PubMedGoogle Scholar
  26. Kotoujansky, A. 1987Molecular genetics of pathogenesis by soft-rot ErwiniasAnnu. Rev. Phytopathol.25405430CrossRefGoogle Scholar
  27. Lacomme, C., Roby, D. 1999Identification of new early markers of the hypersensitive response in Arabidopsis thalianaFEBS Lett.459149153CrossRefPubMedGoogle Scholar
  28. Lamb, C., Dixon, R.A. 1997The oxidative burst in plant disease resistanceAnnu. Rev. Plant Physiol. Plant Mol. Biol.48223250CrossRefPubMedGoogle Scholar
  29. Menke, F.L., Pelt, J.A., Pieterse, C.M., Klessig, D.F. 2004Silencing of the mitogen-activated protein kinase MPK6 compromises disease resistance in ArabidopsisPlant Cell16897907CrossRefPubMedGoogle Scholar
  30. Mukherjee, A., Cui, Y., Liu, Y., Chatterjee, A.K. 1997Molecular characterization and expression of the Erwinia carotovora hrpN Ecc gene, which encodes an elicitor of the hypersensitive reactionMol. Plant–Microbe Interact.10462471PubMedGoogle Scholar
  31. Pontier, D., Godiard, L., Marco, Y., Roby, D. 1994hsr203J, a tobacco gene whose activation is rapid, highly localized and specific for incompatible plant/pathogen interactionsPlant J.5507521PubMedGoogle Scholar
  32. Pontier, D., Tronchet, M., Rogowsky, P., Lam, E., Roby, D. 1998Activation of hsr203, a plant gene expressed during incompatible plant pathogen interaction, is correlated with program cell deathMol. Plant–Microbe Interact.11544554PubMedGoogle Scholar
  33. Sambrook, J., Fritsch, E.F., Maniatis, T. 1989Molecular Cloning: A Laboratory Manual2ndCold Spring Harbor Laboratory Press Cold Spring HarborNY, USA931957Google Scholar
  34. Shen, S., Li, Q., He, S.Y., Barker, K.R., Li, D., Hunt, A.G. 2000Conversion of compatible plant–pathogen interactions into incompatible interactions by expression of the Pseudomonas syringae pv. syringae 61 hrmA gene in transgenic tobacco plantsPlant J.23205213CrossRefPubMedGoogle Scholar
  35. Staskawicz, B.J. 2001Genetics of plant–pathogen interactions specifying plant disease resistancePlant Physiol.1257376CrossRefPubMedGoogle Scholar
  36. Strobel, R.N., Gopalan, J.S., Kuc, J.A., He, S.Y. 1996Induction of systemic acquired resistance in cucumber by Pseudomonas syringae pv. syringae 61 HrpZPss proteinPlant J.9431439CrossRefGoogle Scholar
  37. Vailleau, F., Daniel, X., Tronchet, M., Montillet, J.L., Triantaphylides, C., Roby, D. 2002A R2R3-MYB gene, AtMYB30, acts as a positive regulator of the hypersensitive cell death program in plants in response to pathogen attackProc. Natl. Acad. Sci. USA991017910184CrossRefPubMedGoogle Scholar
  38. Verberne, M.C., Verpoorte, R., Bol, J.F., Mercado-Blanco, J., Linthorst, H.J.M. 2000Overproduction of salicylic acid in plants by bacterial transgenes enhances pathogen resistanceNat. Biotechnol.18779783CrossRefPubMedGoogle Scholar
  39. Wei, Z.-M., Beer, S.V. 1996Harpin from Erwinia amylovora induces plant resistanceActa Hortic.411223225Google Scholar
  40. Zhang, S., Jin, C.D., Roux, S.J. 1993Casein kinase II-type protein kinase from pea cytoplasm and its inactivation by alkaline phosphatase in vitroPlant Physiol.103955962CrossRefPubMedGoogle Scholar
  41. Zhou, J., Loh, Y.T., Bressan, R.A., Martin, G.B. 1995The tomato gene Pti1 encodes a serine/threonine kinase that is phosphorylated by Pto and is involved in the hypersensitive responseCell15925935CrossRefGoogle Scholar

Copyright information

© Springer 2005

Authors and Affiliations

  • Ajay-Kumar Pandey
    • 1
  • Mang-Jye Ger
    • 2
  • Hsiang-En Huang
    • 1
  • Mei-Kuen Yip
    • 1
  • Jiqing Zeng
    • 1
  • Teng-Yung Feng
    • 1
  1. 1.Institute of Plant and Microbial BiologyTaipeiTaiwan
  2. 2.Department of Life ScienceNational University of KaohsiungKaohsiungTaiwan

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