Molecules and Cells

  • 제25권1호
  • /
  • Pages.30-42
  • /
  • 2008
  • /
  • 1016-8478(pISSN)
  • /
  • 0219-1032(eISSN)
한국분자세포생물학회 (Korean Society for Molecular and Cellular Biology)
권호 표지

Genetic Organization of the hrp Genes Cluster in Erwinia pyrifoliae and Characterization of HR Active Domains in HrpNEp Protein by Mutational Analysis

  • Shrestha, Rosemary (Laboratory of Bacterial Genetics and Biotechnology, Division of Bio-Resources Technology, College of Agriculture and Life Sciences, Kangwon National University) ;
  • Park, Duck Hwan (Department of Plant Pathology, Cornell University) ;
  • Cho, Jun Mo (Pioneer Co. Ltd., Kangwon National University) ;
  • Cho, Saeyoull (Division of Bio-resources Technology, College of Agriculture and Life Sciences, Kangwon National University) ;
  • Wilson, Calum (Department of Agricultural Sciences, School of Agricultural Sciences, University of Tasmania) ;
  • Hwang, Ingyu (School of Agricultural Biotechnology, Seoul National University) ;
  • Hur, Jang Hyun (Department of Biological Environment, College of Agriculture and Life Sciences, Kangwon National University) ;
  • Lim, Chun Keun (Laboratory of Bacterial Genetics and Biotechnology, Division of Bio-Resources Technology, College of Agriculture and Life Sciences, Kangwon National University)
  • 투고 : 2007.03.30
  • 심사 : 2007.07.23
  • 발행 : 2008.02.29
⟨ 이전 논문 다음 논문 ⟩

초록

The disease-specific (dsp) region and the hypersensitive response and pathogenicity (hrp) genes, including the hrpW, $hrpN_{Ep}$, and hrpC operons have previously been sequenced in Erwinia pyrifoliae WT3 [Shrestha et al. (2005a)]. In this study, the remaining hrp genes, including the hrpC, hrpA, hrpS, hrpXY, hrpL and hrpJ operons, were determined. The hrp genes cluster (ca. 38 kb) was comprised of eight transcriptional units and contained nine hrc (hrp conserved) genes. The genetic organization of the hrp/hrc genes and their orientation for the transcriptions were also similar to and collinear with those of E. amylovora, showing ${\geq}80%$ homologies. However, ORFU1 and ORFU2 of unknown functions, present between the hrpA and hrpS operons of E. amylovora, were absent in E. pyrifoliae. To determine the HR active domains, several proteins were prepared from truncated fragments of the N-terminal and the C-terminal regions of $HrpN_{Ep}$ protein of E. pyrifoliae. The proteins prepared from the N-terminal region elicited HR, but not from those of the C-terminal region indicating that HR active domains are located in only N-terminal region of the $HrpN_{Ep}$ protein. Two synthetic oligopeptides produced HR on tobacco confirming presence of two HR active domains in the $HrpN_{Ep}$. The HR positive N-terminal fragment ($HN{\Delta}C187$) was further narrowed down by deleting C-terminal amino acids and internal amino acids to investigate whether amino acid insertion region have role in faster and stronger HR activity in $HrpN_{Ep}$ than $HrpN_{Ea}$. The $HrpN_{Ep}$ mutant proteins $HN{\Delta}C187$ (D1AIR), $HN{\Delta}C187$ (D2AIR) and $HN{\Delta}C187$ (DM41) retained similar HR activation to that of wild-type $HrpN_{Ep}$. However, the $HrpN_{Ep}$ mutant protein $HN{\Delta}C187$ (D3AIR) lacking third amino acid insertion region (102 to 113 aa) reduced HR when compared to that of wild-type $HrpN_{Ep}$. Reduction in HR elicitation could not be observed when single amino acids at different positions were substituted at third amino acids insertion region. But, substitution of amino acids at L103R, L106K and L110R showed reduction in HR activity on tobacco suggesting their importance in activation of HR faster in the $HrpN_{Ep}$ although it requires further detailed analysis.

키워드

과제정보

연구 과제 주관 기관 : Ministry of Agriculture-Forestry

참고문헌

  1. Ahmad, M., Majerczak, D.R., Pike, S., Hoyos, M.E., Novacky, A., and Coplin, D.L. (2001). Biological activity of harpin produced by Pantoea stewartii subsp. stewartii. Mol. Plant-Microbe Interact. 14, 1223-1234 https://doi.org/10.1094/MPMI.2001.14.10.1223
  2. Arlat, M., Gijsegem, F., Van, Huet, J.C., Pernollet, J.C., and Boucher, C.A. (1994). PopA1, a protein which induces a hypersensitivity-like response on specific Petunia genotypes, is secreted via the Hrp pathway of Pseudomonas solanacearum. EMBO J. 13, 543-553
  3. Bauer, D.W., Wei, Z.-M., Beer, S.V., and Collmer, A. (1995). Erwinia chrysanthemi harpinEch: an elicitor of the hypersensitive response that contributes to soft-rot pathogenesis. Mol. Plant-Microbe Interact. 8, 484-491 https://doi.org/10.1094/MPMI-8-0484
  4. Bell, K.S., Sebaihia, M., Pritchard, L., Holden, M.T., Hyman, L.J., Holeva, M.C., Thomson, N.R., Bentley, S.D., Churcher, L.J.C., Mungall, K., et al. (2004). Genome sequence of the enterobacterial phytopathogen Erwinia carotovora subsp. Atroseptica and characterization of virulence factors. Proc. Natl. Acad. Sci. USA 101, 11105-11110
  5. Bogdanove, A.J., Wei, Z.-M., Zhao, L., and Beer, S.V. (1996). Erwinia amylovora secretes Harpin via a type III pathway and contains a homolog of yopN of Yersinia spp. J. Bacteriol. 178, 1720-1730 https://doi.org/10.1128/jb.178.6.1720-1730.1996
  6. Bonn, W.G. and van der Zwet, T. (2000). Distribution and economic importance of fire blight. In Fire Blight: The Disease and Its Causative Agent, Erwinia amylovora, J.L. Vanneste, ed. (UK: CABI Publishing), pp. 37-53
  7. Charkowski, A.O., Alfano, J.R., Preston, G., Yuan, J., He, S.Y., and Collmer, A. (1998). The Pseudomonas syringae pv. tomato HrpW protein has domains similar to harpins and pectate lyases and can elicit the plant hypersensitive response and bind to pectate. J. Bacteriol. 180, 5211-5217
  8. Charkowski, A.O., Huang, H.-C., and Collmer, A. (1997). Altered localization of the HrpZ harpin in Pseudomonas syringae pv. syringae hrp mutants suggests that different components of the type III secretion pathway control secretion across the inner and outer membranes of gram-negative bacteria. J. Bacteriol. 179, 3866-3874 https://doi.org/10.1128/jb.179.12.3866-3874.1997
  9. Combet, C., Blanchet, C., Geourjon, C., and Deleage, G. (2000). NPS@: Network protein sequence analysis. Trends Bichem. Sci. 25, 147-150 https://doi.org/10.1016/S0968-0004(99)01540-6
  10. Ehrenfeld, N., Canon P., Stange C., Medina C., and Arce-Johnson, P. (2005). Tobamovirus coat protein CPCg induces an HR-like response in sensitive tobacco plants. Mol. Cells 19, 418-427
  11. Frederick, R.D., Ahmad, M., Majerczak, D.R., Arroyo-Rodriguez, A.S., Manulis, S., and Coplin, D.L. (2001). Genetic organization of the Pantoea stewartii subsp. stewartii hrp gene cluster and sequence analysis of the hrpA, hrpC, hrpN, and wtsE operons. Mol. Plant-Microbe Interact. 14, 1213-1222 https://doi.org/10.1094/MPMI.2001.14.10.1213
  12. Hacker, J. and Kaper, J.B. (2000). Pathogenicity islands and the evolution of microbes. Annu. Rev. Microbiol. 54, 641-679 https://doi.org/10.1146/annurev.micro.54.1.641
  13. He, S.Y. (1998). Type III secretion systems in animal and plant pathogenic bacteria. Annu. Rev. Phytopathol. 36, 363-392 https://doi.org/10.1146/annurev.phyto.36.1.363
  14. He, S.Y., Huang, H.-C., and Collmer, A. (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 https://doi.org/10.1016/0092-8674(93)90354-S
  15. Huang, H.-C., Lin, R., Chang, C., Collmer, A., and Deng, W. (1995). The complete hrp gene cluster of Pseudomonas syringae pv. syringae 61 includes two blocks of genes required for $harpin_{Pss}$ secretion that are arranged colinearly with Yersinia ysc homologs. Mol. Plant-Microbe Interact. 8, 733-746 https://doi.org/10.1094/MPMI-8-0733
  16. Jock, S., Jacob, T., Kim, W.-S., Hilgebrand, M., Vosberg, H.P., and Geider, K. (2003). Instability of short-sequence DNA repeats of pear pathogenic Erwinia strains from Japan and Erwinia amylovora fruit tree and raspberry strains. Mol. Genet. Genomics 268, 739-749
  17. Kim, J.H. and Beer, S.V. (1998). HrpW of Erwinia amylovora, a new harpin that contains a domain homologous to pectate lyases of a distinct class. J. Bacteriol. 180, 5203-5210
  18. Kim, W.-S. and Geider, K. (2000). Characterization of a viral EPS-depolymerase, a potential tool for control of fire blight. Phytopathol. 90, 1263-1268 https://doi.org/10.1094/PHYTO.2000.90.11.1263
  19. Kim, J.F., Wei, Z.-M., and Beer, S.V. (1997). The hrpA and hrpC operons of Erwinia amylovora encode components of a type III pathway that secretes harpin. J. Bacteriol. 179, 1690-1697 https://doi.org/10.1128/jb.179.5.1690-1697.1997
  20. Kim, W.-S., Garden, L., Rhim, S.-L., and Geider, K. (1999). Erwinia pyrifoliae sp. nov., a novel pathogen that affects Asian pear trees (Pyrus pyrifolia Nakai). Intl. J. Sys. Bacteriol. 49, 899-906 https://doi.org/10.1099/00207713-49-2-899
  21. Kim, W.-S., Hildebrand, M., Jock, S., and Geider, K. (2001a). Molecular comparison of pathogenic bacteria from pear trees in Japan and the fire blight pathogen Erwinia amylovora. Microbiology. 147, 2951-2959 https://doi.org/10.1099/00221287-147-11-2951
  22. Kim, W.-S., Jock, S., Paulin, J.-P., Rhim, S.-L., and Geider, K. (2001b). Molecular detection and differentiation of Erwinia pyrifoliae and host range analysis of the Asian pear pathogen. Plant Dis. 85, 1183-1188 https://doi.org/10.1094/PDIS.2001.85.11.1183
  23. Kim, J.-G., Park, B.K., Yoo, C.-H., Jeon, E., Oh, J., and Hwang, I. (2003). Characterization of the Xanthomonas axonopodis pv. glycines Hrp pathogenicity island. J. Bacteriol. 185, 3155-3166 https://doi.org/10.1128/JB.185.10.3155-3166.2003
  24. Kim, J.-G., Jeon, E., Oh, J., Moon, J.S., and Hwang, I. (2004). Mutational analysis of Xanthomonas harpin HpaG identifies a key functional region that elicits the hypersensitive response in nonhost plants. J. Bacteriol. 186, 6239-6247 https://doi.org/10.1128/JB.186.18.6239-6247.2004
  25. Lee, J., Lee, H.-Y., Shin, M.-K., and Ryu, W.-S. (2004). Versatile PCR-mediated insertion or deletion mutagenesis. BioTechniques 36, 398-400 https://doi.org/10.2144/04363BM04
  26. Lingren, P.B. (1997). The role of hrp genes during plantbacterial interactions. Anun. Rev. Phytopathol. 35, 129-152 https://doi.org/10.1146/annurev.phyto.35.1.129
  27. Makarova, O., Kamberov, E., and Margolis, B. (2000). Generation of deletion and point mutations with one primer in a single cloning step. BioTechniques 29, 970-972
  28. McGhee, G.C., Schnabel, E.L., Maxson-Stein, K., Jones, B., Stromberg, V.K., Lacy, G.H., and Jones, A.L. (2002). Relatedness of chromosomal and plasmid DNAs of Erwinia pyrifoliae and Erwinia amylovora. Appl. Environ. Microbiol. 68, 6182-6192 https://doi.org/10.1128/AEM.68.12.6182-6192.2002
  29. Mor, H., Manulis, S., Zuck, M., Nizan, R., Coplin, D.L., and Barash, I. (2001). Genetic organization of the hrp gene cluster and dspAE/BF operon in Erwinia herbicola pv. gypsophilae. Mol. Plant-Microbe Interact. 14, 431-436 https://doi.org/10.1094/MPMI.2001.14.3.431
  30. Mukherjee, A., Cui, Y., Liu, Y., and Chatterjee, A.K. (1997). Molecular characterization and expression of the Erwinia carotovora $hrpN_{Ecc}$ gene, which encodes an elicitor of the hypersensitive reaction. Mol. Plant-Microbe Interact. 10, 462-471 https://doi.org/10.1094/MPMI.1997.10.4.462
  31. Nakada, H., Tahara, A., Hayashi, M., Shimazu, R., Tanaka, R., Ichinose, Y., Shiraishi, T., and Yamada, T. (1999). Cloning and structural characterization of hrp locus of Pseudomonas syringae pv. pisi. Ann. Phytopathol. Soc. Jpn. 65, 147-152 https://doi.org/10.3186/jjphytopath.65.147
  32. Nizan, R., Barash, I., Valinsky, L., Lichter, A., and Manulis, S. (1997). The presence of hrp genes on the pathogenicity-associated plasmid of tumorigenic bacterium Erwinia herbicola pv. gypsophilae. Mol. Plant-Microbe Interact. 10, 677-682 https://doi.org/10.1094/MPMI.1997.10.5.677
  33. Oh, C.-S., Kim, J.F., and Beer, S. (2005). The Hrp pathogenicity island of Erwinia amylovora and identification of three novel genes required for systemic infection. Mol. Plant Pathol. 6, 125-138 https://doi.org/10.1111/j.1364-3703.2005.00269.x
  34. Rhim, S.-L., Voelkisch, B., Gardan, L., Paulin, J.-P., Langlotz, C., Kim, W.-S., and Geider, K. (1999). An Erwinia species, different from E. amylovora cause a necrotic disease of Asian pear trees. Plant Pathol. 48, 514-520 https://doi.org/10.1046/j.1365-3059.1999.00376.x
  35. Rojas, C.M., Ham, J.H., Deng, W.L., Doyle, J.J., and Collmer, A. (2002). HecA, a member of a class of adhesins produced by diverse pathogenic bacteria, contributes to the attachment, aggregation, epidermal cell killing, and virulence phenotypes of Erwinia chrysanthemi EC16 on Nicotiana clevelandii seedlings. Proc. Natl. Acad. Sci. USA 99, 13142-13147
  36. Sambrook, J. and Russel, D.W. (2001) Molecular Cloning: A Laboratory Manual, 3rd ed. (New York Cold Spring Harbor Press, Cold Spring Harbor)
  37. Shrestha, R., Koo, J.H., Park, D.H., Hwang, I., Hur, J.H., and Lim, C.K. (2003). Erwinia pyrifoliae, a causal endemic pathogen of shoot blight of Asian pear tree in Korea. Plant Pathol. J. 19, 294-300 https://doi.org/10.5423/PPJ.2003.19.6.294
  38. Shrestha, R., Tsuchiya, K., Baek, S.J., Bae, H.N., Hwang, I., Hur, J.H., and Lim, C.K. (2005a). Identification of dspEF, hrpW, and hrpN loci and characterization of the $hrpN_{Ep}$ gene in Erwinia pyrifoliae. J. Gen. Plant Pathol. 71, 211-220 https://doi.org/10.1007/s10327-005-0191-6
  39. Shrestha, R., Lee S. H., Hur, J.H., and Lim, C.K. (2005b). The effects of temperature, pH and bactericides on the growth of Erwinia pyrifoliae and Erwinia amylovora. Plant Pathol. J. 21, 127-131 https://doi.org/10.5423/PPJ.2005.21.2.127
  40. Staskawicz, B., Dahlbeck, D., Keen, N., and Napoli, C. (1987). Molecular characterization of cloned avirulence genes from race 0 and race 1 of Pseudomonas syringae pv. glycinea. J. Bacteriol. 169, 5789-5794 https://doi.org/10.1128/jb.169.12.5789-5794.1987
  41. Steinberger, E.M. and Beer, S.V. (1988). Creation and complementation of pathogenicity mutants of Erwinia amylovora. Mol. Plant-Microbe Interact. 1, 135-144 https://doi.org/10.1094/MPMI-1-135
  42. Strobel, R.N., Gopalan, J.S., Kuc, J.A., and He, S.Y. (1996). Induction of systemic acquired resistance in cucumber by Pseudomonas syringae pv. syringae 61 $HrpZ_{Pss}$ protein. Plant J. 9, 431-439 https://doi.org/10.1046/j.1365-313X.1996.09040431.x
  43. Van der Zwet, T. and Keil, H.L. (1979). Fire blight - A bacterial disease of rosaceous plants (Washington, United States Department of Agriculture Handbook 510)
  44. Van Gijsegem, F., Gough, C., Zischek, C., Niqueux, E., Arlat, M., Genin, S., Barberis, P., German, S., Castello, P., and Boucher, C. (1995). The hrp gene locus of Pseudomonas solanacearum, which controls the production of type III secretion system, encodes eight proteins related to components of the bacterial flagellar biogenesis complex. Mol. Microbiol. 15, 1095-1114 https://doi.org/10.1111/j.1365-2958.1995.tb02284.x
  45. Wei, Z.-M. and Beer, S.V. (1995). hrpL activates Erwinia amylovora hrp gene transcription and is a member of the ECF sub family of factors. J. Bacteriol. 175, 6201-6210
  46. Wei, Z.-M. and Beer, S.V. (1996). Harpin from Erwinia amylovora induces plant disease resistance. Acta Hortic. 411, 223-225
  47. Wei, Z.-M., Laby, R.J., Zumoff, C.H., Bauer, D.W., He, S.Y., Collmer, A., and Beer, S.V. (1992). Harpin, elicitor of the hypersensitive response produced by the plant pathogen Erwinia amylovora. Science 257, 85-88 https://doi.org/10.1126/science.1621099
  48. Wei, Z.-M., Kim, F.J., and Beer, S.V. (2000). Regulation of hrp genes and type III protein secretion in Erwinia amylovora by HrpX/HrpY, a novel two component system, and HrpS. Mol. Plant-Microbe Interact. 13, 1251-1262 https://doi.org/10.1094/MPMI.2000.13.11.1251