Browse > Article

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)
Publication Information
Molecules and Cells / v.25, no.1, 2008 , pp. 30-42 More about this Journal
Abstract
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.
Keywords
dsp/hrp Genes; Harpin; Hypersensitive Response; Pyrus pyrifolia; Shoot Blight; Site-directed Mutagenesis;
Citations & Related Records
Times Cited By KSCI : 4  (Citation Analysis)
Times Cited By Web Of Science : 6  (Related Records In Web of Science)
연도 인용수 순위
1 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
2 Combet, C., Blanchet, C., Geourjon, C., and Deleage, G. (2000). NPS@: Network protein sequence analysis. Trends Bichem. Sci. 25, 147-150   DOI   ScienceOn
3 Hacker, J. and Kaper, J.B. (2000). Pathogenicity islands and the evolution of microbes. Annu. Rev. Microbiol. 54, 641-679   DOI   ScienceOn
4 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
5 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   DOI
6 Lee, J., Lee, H.-Y., Shin, M.-K., and Ryu, W.-S. (2004). Versatile PCR-mediated insertion or deletion mutagenesis. BioTechniques 36, 398-400   DOI
7 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
8 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
9 Sambrook, J. and Russel, D.W. (2001) Molecular Cloning: A Laboratory Manual, 3rd ed. (New York Cold Spring Harbor Press, Cold Spring Harbor)
10 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   DOI   ScienceOn
11 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   DOI   ScienceOn
12 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
13 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   DOI
14 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   DOI
15 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   과학기술학회마을   DOI
16 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
17 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   DOI   ScienceOn
18 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   DOI
19 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   DOI   ScienceOn
20 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   DOI
21 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   DOI   ScienceOn
22 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
23 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   DOI   ScienceOn
24 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   DOI
25 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
26 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   DOI
27 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   DOI   ScienceOn
28 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   DOI   ScienceOn
29 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   DOI
30 Steinberger, E.M. and Beer, S.V. (1988). Creation and complementation of pathogenicity mutants of Erwinia amylovora. Mol. Plant-Microbe Interact. 1, 135-144   DOI
31 He, S.Y. (1998). Type III secretion systems in animal and plant pathogenic bacteria. Annu. Rev. Phytopathol. 36, 363-392   DOI   ScienceOn
32 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
33 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   DOI   ScienceOn
34 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   DOI
35 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   DOI   ScienceOn
36 Lingren, P.B. (1997). The role of hrp genes during plantbacterial interactions. Anun. Rev. Phytopathol. 35, 129-152   DOI   ScienceOn
37 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   DOI
38 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   DOI
39 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   DOI
40 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   DOI   ScienceOn
41 Wei, Z.-M. and Beer, S.V. (1996). Harpin from Erwinia amylovora induces plant disease resistance. Acta Hortic. 411, 223-225
42 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   DOI
43 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   DOI
44 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   DOI
45 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   DOI   ScienceOn
46 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)
47 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
48 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   DOI   ScienceOn