臺灣博碩士論文加值系統

P. syringae hrp/hrc 基因編譯一個第三型蛋白質分泌系統，該系統可以轉移Avr和Hop等蛋白質至植物細胞內。在Pseudomonas syringae pv. syringae (Pss) 61、Pss B728a和P. syringae pv. tomato (Pto) DC3000的hrp/hrc DNA序列分析顯示Hrp pathogenicity island (Pais) 是一個三組基因群組成的鑲崁結構。hrp/hrc 基因群在該三種菌株中具有保留性，並在基因群兩側具有可交換作用區(EEL)和一個保留作用區(CEL)。在本研究中由來自Pss 61菌株的9.6 kb片段的pNCHU127選殖株進行大約4.1 kb CEL區域的次選殖。將該片段進行DNA定序並以NCBI Blast分析發現該片段包含兩個open reading frames (ORFs)，並顯示和Pto DC3000的avrE、orf1基因有同源性，其中avrE基因為一個不完整的基因而orf1基因編譯出的蛋白具有transglycosylase motif並可能具有該活性。利用nptII基因對這兩個基因作非極性突變，所產生的突變株為Pss61-N703(Δorf1:: nptⅡ)和Pss61-N707(avrE:: nptⅡ)。這兩株突變株接種在煙草葉上都能延遲過敏性反應的發生。顯示這兩個基因在誘發過敏性反應的過程中扮演重要的角色。特別是突變的orf1基因可能會影響Hrp系統的表現及組成。由orf1突變株的互補試驗及利用anti-harpin血清進行的西方點墨法試驗結果，顯示要恢復orf1突變株的Hrp分泌系統則可能需要除了orf1基因之外的其他hrp基因存在。而其機制有待進一步探討。另推測ORF1蛋白可能可以幫助Hrp組成蛋白的組合或形成通道來輸送一些蛋白如過敏蛋白或毒/無毒蛋白至細胞外，在Hrp系統中扮演一個輔助的角色。
另一方面便是試著建立一個利用第三型分泌系統來分泌外源蛋白質。我們以放線菌的glucanase為目標蛋白。依據之前的文獻，將第三型訊號序列Pss 61的hrpZ接至去除第二型訊號序列glucanase的5’端，將該重組體選殖至表現載體pT7 —7並在BL21表現。另外也將該重組體構築在數種廣泛宿主載體pML122、pRK415、 pCPP33送至Pss 61菌株測試表現。在BL21表現的結果顯示glucanase以內涵體型式產生，該形式並不具活性。而選殖至pML122、pRK415和pCPP33等表現載體，在Pss 61菌株則無法表現該基因。

The hrp/hrc cluster of Pseudomonas syringae pathovars encodes a type Ⅲ protein secretion system that appears to translocate Avr and Hop effector proteins into plant cell. DNA sequence analysis of the hrp/hrc regions in Pss 61, Pss B728a, and P. syringae pv. tomato (Pto) DC3000 has revealed a Hrp pathogenicity island (Pais) with a tripartite mosaic structure. The hrp/hrc cluster is conserved in all three strains and is flanked by a unique exchangeable effector locus (EEL) and a conserved effector locus (CEL). In this study, we sequenced a ca. 4.1 kb fragment localized at the right of hrpRS genes in Pss 61. Based on sequence analysis, this sequenced region contained two open reading frames, which showed homology with the avrE and orf1 genes of Pto DC3000 respectively. The data showed that the homology avrE gene is a incomplete clone and the ORF1 protein has a transglycosylase motif and might have corresponding activity. The mutants Pss61-N703 (Δorf1::nptⅡ) and Pss61-N707 (avrE::nptⅡ) caused a delay HR (hypersensitive response) on tobacco leaves, showing that both genes play an important role in HR-eliciting process, especially the orf1 gene. It influenced the expression and assembling of the Hrp system components. According to complementation assay and Western Blot assay with anti-harpin serum of orf1 mutant, the results revealed that additional hrp genes were required to restore Hrp system of orf1 mutant. It suggests that the ORF1 protein could help the assembling of components of Hrp system or to form a channel that could export some sensor proteins or Vir/Avr protein out of the cell. It plays an ancillary role in Hrp system. The other subject in my study is attempt to build a model that a foreign protein can be secreted out by the type Ⅲ secretion system. We choose the glucanase from Streptomyces sp. to be the target protein. The type Ⅲ signal sequence of Pss 61 hrpZ gene was fused with 5’ -terminal glucanase gene, which the predicted type Ⅱ signal sequence was excised, and then this recombinant construct was cloned into an overexpression vector pT7 —7 and expressed in E. coli BL21. Also this construct was cloned to several broad host range vectors pML122, pRK415 and pCPP33 respectively to test for expression in Pss 61. The result of expression in BL21 was already known to produce the form of inclusion body without enzyme activity. And the expression of this recombinant protein in Pss 61 with using pML122, pRK415 and pCPP33 vectors were not detectable.

I. Abstract (Chinese)----------------- --------------------1
II. Abstract (English)--------------------------------------3
III. Introduction -------------------------------------------5
IV. Materials and Methods ----------------------------------11
V. Results ------------------------------------------------21
VI. Discussion ---------------------------------------------26
VII. Tables and Figures -------------------------------------32
VIII.References ---------------------------------------------58

Alfano, J. R., Bauer, D. W., Milos, T. M. and Collmer, A. 1996. Analysis of the role of the Pseudomonas syringae pv. syringae HrpZ harpin in elicitation of the hypersensitive response in tobacco using functionally non-polar hrpZ deletion mutations, truncated HrpZ fragments, and hrmA mutations. Mol. Microbiol. 19: 715-728.
Alfano, J. R., Charkowski, A. O., Deng, W.-L., Badel, J. L., Petnicki-Ocwieja, T., van Dijk, K. and Collmer, A. 2000. The Pseudomonas syringae Hrp pathogenicity island has a tripartite mosaic structure composed of a cluster of type III secretion genes bounded by exchangeable effector and conserved effector loci that contribute to parasitic fitness and pathogenicity in plants. Proc. Natl Acad. Sci. USA 97: 4856-4861.
Alfano, J. R. and Collmer, A. 1997. The type III (Hrp) secretion pathway of plant pathogenic bacteria: trafficking harpins, Avr proteins, and death. J. Bacteriol. 179: 5655-5662.
Allaoui, A., Ménard, R., Sansonetti, P. J. and Parsot, C. 1993. Characterization of the Shigella flexneri ipgD and ipgF genes, which are located in the proximal part of the mxi locus. Infect. Immun. 61: 1707-1714.
Anderson, D. M., Fouts, D. E., Collmer, A. and Schneewind, O. 1999. Reciprocal secretion of proteins by the bacterial type III machines of plant and animal pathogens suggests universal recognition of mRNA targeting signals. Proc. Natl. Acad. Sci. USA 96: 12839-12843.
Anderson, D. M. and Schneewind, O. 1997. A mRNA signal for the type III secretion of Yop proteins by Yersinia enterocolitica. Science 278: 1140-1143.
Anderson, D. M. and Schneewind, O. 1999. Yersinia enterocolitica type III secretion: an mRNA signal that couples translation and secretion of YopQ. Mol. Microbiol. 31: 1139-1148.
Arlat, M., Van Gijsegem, F., 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.
Baker, C. J., Atkison, M. M. and Collmer, A. 1987. Concurrent loss in Tn5 mutants of Pseudomonas syringae pv. syringae of the ability to induce the hypersensitive response and host plasma memebrane K+/H+ exchange in tobacco. Phytopathology 77: 1268-1272.
Beck, E., Ludwig, G., Auerswald, E. A., Reiss, B. and Schaller, H. 1982. Nucleotide sequence and exact localization of the neomycin phosphotransferase gene from transposon Tn5. Gene 19: 327-336.
Betzner, A. S., Ferreira, L. C. S., Holtje, J.-V. and Keck, W. 1990. Control of the activity of the soluble lytic transglycosylase by the stringent response in Escherichia coli. FEMS Microbiol. Lett 67: 161-164.
Bogdanove, A. J., Beer, S. V., Bonas, U., Boucher, C. A., Collmer, A., Coplin, D. L., Cornelis, G. R., Huang, H.-C., Hutcheson, S. W., Panopoulos, N. J. and Van Gijsegem, F. 1996. Unified nomenclature for broadly conserved hrp genes of phytopathogenic bacteria. Mol. Microbiol. 20: 681-683.
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.
Charkowski, A. O., Huang, H.-C. and Collmer, A. 1997. Altered localization of HrpZ in Pseudomonas syringae pv. syringae hrp mutants suggests that different components of the type III secretion pathway control protein translocation across the inner and outer membranes of gram-negative bacteria. J. Bacteriol. 179: 3866-3874.
Cheng, L. W., Anderson, D. M. and Schneewind, O. 1997. Two independent type III secretion mechanisms for YopE in Yersinia enterocolitica. Mol. Microbiol. 24: 757-765.
Cheng, L. W. and Schneewind, O. 2000. Type III machines of Gram-negative bacteria: delivering the goods. Trends Microbiol. 8: 214-220.
Cornelis, G. R., Boland, A., Boyd, A. P., Geuijen, C., Iriarte, M., Neyt, C., Sory, M. P. and Stainier, I. 1998. The virulence plasmid of Yersinia, an antihost genome. Microbiol. Mol. Biol. Rev. 62: 1315-1352.
Deng, W.-L., Preston, G., Collmer, A., Chang, C.-J. and Huang, H.-C 1998. Characterization of the hrpC and hrpRS operons of Pseudomonas syringae pathovars syringae, tomato, and glycinea and analysis of the ability of hrpF, hrpG, hrcC, hrpT, and hrpV mutants to elicit the hypersensitive response and disease in plants. J. Bacteriol. 180: 4523-4531.
Dijkstra, A. J. and Keck, W. 1996. Peptidoglycan as a barrier to transenvelope transport. J. Bacteriol. 178: 5555-5562.
Ditta, G., Stanfield, S., Corbin, D. and Helinski, D. R. 1980. Broad host range DNA cloning system for Grame-negative Bacteria: construction of a gene bank of Rhizobium meliloti. Proc. Natl. Acad. Sci. USA 77: 7347-7351.
Engel, H., Kazemier, B. and Keck, W. 1991. Murein-metabolizing enzymes from Escherichia coli: sequence analysis and controlled overexpression of the slt gene, which encodes the soluble lytic transglycosylase. J. Bacteriol. 173: 6773-6782.
Fellay, R., Rahme, L. G., Mindrinos, M. N., Frederick, R. D., Pisi, A. and Panopoulos, N. J. 1989. Genes and signals controlling the Pseudomonas syringae pv. phaseolicola-plant interaction. In H. Hennecke and D. P. S. Verma (ed.), Advances in molecular genetic of plant-microbe interaction, vol. 1. Kluwer Academic Publisher, Dordrecht, The Netherlands, 45-52.
Flor, H. H. 1955. Host-parasite interactions in flax rust-its genetics and other implications. Phytopathology 45: 680-685.
Galan, J. E. and Collmer, A. 1999. Type III secretion machines: bacterial devices for protein delivery into host cells. Science 284: 1322-1328.
Gaudriault, S., Brisset, M. N. and Barny, M. A. 1998. HrpW of Erwinia amylovora, a new Hrp-secreted protein. FEBS Lett. 284: 1322.
Genin, S., Gough, C. L., Zischek, C. and Boucher, C. A. 1992. Evidence that the hrpB gene encodes a positive regulator of pathogenicity genes from Pseudomonas solanacearum. Mol. Microbiol. 6: 3065-3076.
Grimm, C., Aufsatz, W. and Panopoulos, N. J. 1995. The hrpRS locus of Pseudomonas syringae pv. phaseolicola constitutes a complex regulatory unit. Mol. Microbiol. 15: 155-165.
Grimm, C. and Panopoulos, N. J. 1989. The predicted protein product of a pathogenicity locus from Pseudomonas syringae pv. phaseolicola is homologous to a highly conserved domain of several procaryotic regulatory proteins. J. Bacteriol. 171: 5031-5038.
Gold, L., Pribnow, D., Schneider, T., Shinedling, S., Singer, B. S. and Stormo, G. 1981. Translational initiation in prokaryotes. Annu. Rev. Microbiol. 35: 365-403.
Goodell, E. W. 1985. Recycling of murein by Escherichia coli. J. Bacteriol. 163: 305-310.
Hacker, J., Blum-Oehler, G., Mühldorfer, I. and Tschäpe, H. 1997. Pathogenicity islands of virulent bacteria: structure, function and impact on microbial evolution. Mol. Microbiol. 23: 1089-1097.
He, S. Y. 1998. Type III protein secretion system in plant and animal pathogenic bacteria. Annu. Rev. Phytopathol. 36: 363-392.
He, S. Y., Huang, H.-C. and Collmer, A. 1993. Pseudomonas syringae pv. syringae harpinPss: a protein that is secreted via the Hrp pathway and elicits the hypersensitive response in plants. Cell 73: 1255-1266.
Hirano, S. S. and Upper, C. D. 1990. Population biology and epidemiology of Pseudomonas syringae. Annu. Rev. Phytopathol. 28: 155-177.
Huang, H.-C., Lin, R.-W., Chang, C.-J., Collmer, A. and Deng, W-L.. 1995. The complete hrp gene cluster of Pseudomonas syringae pv. syringae 61 includes two blocks of genes required for harpinPss secretion that are arranged colinearly with Yersinia ysc homologues. Mol. Plant-Microbe Interact. 8: 733-746.
Huang, H.-C., Schuurink, R., Denny, T. P., Atkinson, M. M., Baker, C. J., Yucel, I. I., Hutcheson, S. W. and Collmer, A. 1988. Molecular cloning of a Pseudomonas syringae pv. syringae gene cluster that enables Pseudomonas fluorescens to elicit the hypersensitive response in tobacco plants. J. Bacteriol. 170: 4748-4756.
Hueck, C. J. 1998. Type III protein secretion systems in bacterial pathogens of animals and plants. Microbiol. Mol. Biol. Rev. 62: 379-433.
Huynh, T. V., Dahlbeck, D. and Staskawicz, B. J. 1989. Bacterial blight of soybean: regulation of a pathogen gene determining host cultivar specificity. Science 245: 1374-1377.
Kearney, B. and Staskawicz, B. J. 1990. Widespread distribution and fitness contribution of Xanthomonas campestris avirulence gene avrBs2. Nature 346: 385-386.
Keen, N. T. 1990. Gene-for-gene complementarity in plant-pathogen interaction. Annu. Rev. Genet. 24: 447-463.
Keen, N. T., Tamaki, S., Kobayashi, D. and Trollinger, D. 1988. Improved broad-host-range plasmids for DNA cloning in gram-negative bacteria. Gene 70: 191-197.
Kim, J. F. and Beer, S. V. 1998. HrpW of Erwinia amylovora, a new harpin that contains a domain homologous to pectate of a distinct class. Mol. Plant-Microbe Interact. 27: 349-367.
King, E. O., Ward, M. K. and Raney, D. E. 1954. Two simple media for demonstration of pyocyanin and fluorescein., J. Lab. Clin. Med. 44:301-307.
Klement, Z. 1982. Hypersensitivity. In Phytopathogenic Prokaryotes, Volume 2, M. S. Mount and G. H. Lacy, eds. (New York: Academic Press), 149-177.
Koonin, E. V. and Rudd, K. E. 1994. A conserved domain in putative bacterial and bacteriophage transglycosylases. Trends Biochem. Sci. 19: 106-107.
Kubori, T., Matsushima, Y., Nakamura, D., Uralil, J., Lara-Tejero, M., Sukhan, A., GalÃin, J. E. and Aizawa, S. I. 1998. Supramolecular structure of the Salmonella typhimurium type III protein secretion system. Science 280: 602-605.
Kyte, J. and Doolittle, R. F. 1984. A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157:105-132.
Labes, M. Puhler, A. and Simon, R. 1990. A new family of RSF1010-derived expression and lac-fusion broad-host-range vectors for gram-negative bacteria. Gene 89:37-46
Lindgren, P. B., Peet, R. C. and Panopoulos, N. J. 1986. Gene cluster of Pseudomonas syringae pv. "phaseolicola" controls pathogenicity of bean plants and hypersensitivity of nonhost plants. J. Bacteriol. 168: 512-522.
Lonetto, M. A., Brown, K. L., Rudd, K. E. and Buttner, M. J. 1994. Analysis of the Streptomyces coelicolor sigE gene reveals the existence of a subfamily of eubacterial RNA polymerase sigma factors involved in the regulation of extracytoplasmic functions. Proc. Natl. Acad. Sci. USA 91: 7573-7577.
Lorang, J. M. and Keen, N. T. 1995. Characterization of avrE from Pseudomonas syringae pv. tomato: a hrp-linked avirulence locus consisting of at least two transcriptional units. Mol. Plant. Microbe. Interact. 8: 49-57.
Lorang, J. L., Shen, H., Kobayashi, D., Cooksey, D. and Keen, N. T. 1994. avrA and avrE in Pseudomonas syringae pv. tomato play a role in virulence on tomato plants. Mol. Plant-Microbe Interact. 7: 508-515.
Mudgett, M. B. and Staskawicz, B. J. 1999. Characterization of the Pseudomonas syringae pv. tomato AvrRpt2 protein: demonstration of secretion and processing during bacterial pathogenesis. Mol. Microbiol. 32: 927-941.
Mushegian, A. R., Fullner, K. J., Koonin, E. V. and Nester, E. W. 1996. A family of lysozyme-like virulence factors in bacterial pathogens of plants and animals. Proc. Natl. Acad. Sci. USA 93: 7321-7326.
Niepold, F., Anderson, D. and Mills, D. 1985. Cloning determinants of pathogenesis from Pseudomonas pathovar syringae. Proc. Natl. Acad. Sci. USA 82: 406-410.
Oku, T., Alvarez, A. M. and Kado, C. I. 1995. Conservation of the hypersensitivity-pathogenicith regulatory gene hrpX of Xanthomonas campestris and X. oryzae. DNA Sequence 5: 245-249.
Preston, G., Deng, W.-L., Huang, H.-C. and Collmer, A. 1998. Negative regulation of hrp genes in Pseudomonas syringae by HrpV. J. Bacteriol. 180: 4532-4537.
Rahme, L. G., Mindrinos, M. N. and Panopoulos, N. J. 1992. Plant and environmental sensory signals control the expression of hrp genes in Pseudomonas syringae pv. phaseolicola. J. Bacteriol. 174: 3499-3507.
Roine, E., Wei, W., Yuan, J., Nurmiaho-Lassila, E. L., Kalkkinen, N., Romantschuk, M. and He, S. Y. 1997. Hrp pilus: an hrp-dependent bacterial surface appendage produced by Pseudomonas syringae pv. tomato DC3000. Proc. Natl. Acad. Sci. USA 94: 3459-3464.
Sambrook, J., Nicklen, S. and Maniatis, T. 1989. A Laboratory Manual, 2nd ed. Cold Spring Habor Laboratory Press, Cold Spring Habor, NY.
Sory, M. P., Boland, A., Lambermont, I. and Cornelis, G. R. 1995. Identification of the YopE and YopH domains required for secretion and internalization into the cytosol of macrophages, using the cyaA gene fusion approach. Proc. Natl. Acad. Sci. USA 92: 11998-2002.
Stover, C. K., Pham, X. Q., Erwin, A. L., Mizoguchi, S. D., Warrener, P., Hickey, M. J., Brinkman, F. S., Hufnagle, W. O., Kowalik, D. J., Lagrou, M., Garber, R. L., Goltry, L., Tolentino, E., Westbrock-Wadman, S., Yuan, Y., Brody, L. L., Coulter, S. N., Folger, K. R., Kas, A. A., Larbig, K., Lim, R., Smith, K., Spencer, D., Wong, G. K., Wu, Z. and Paulsen, I. T. 2000. Complete genome sequence of Pseudomonas aeruginosa PA01, an opportunistic pathogen. Nature 406: 959-964.
Swarup, S., Yang, Y., Kingsley, M. T. and Gabriel, D. W. 1992. An Xanthomonas citri pathogenicity gene, pthA, pleiotropically encodes gratuitous avirulence on nonhosts. Mol. Plant Microbe Interact. 5: 204-213.
Tamano, K., Aizawa, S., Katayama, E., Nonaka, T., Imajoh-Ohmi, S., Kuwae, A., Nagai, S. and Sasakawa, C. 2000. Supramolecular structure of the Shigella type III secretion machinery: the needle part is changeable in length and essential for delivery of effectors. EMBO J. 19: 3876-3887.
Thompson, J. D., Higgins, D. G. and Gibson, T. J. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22: 4673-4680.
Thunnissen, A. M., Dijkstra, A. J., Kalk, K. H., Rozeboom, H. J., Engel, H., Keck, W. and Dijkstra, B. W. 1994. Doughnut-shaped structure of a bacterial muramidase revealed by X-ray crystallography. Nature 367: 750-753.
Van, D. K., Fouts, D. E., Rehm, A. H., Hill, A. R., Collmer, A. and Alfano, J. R. 1999. The Avr (effector) proteins HrmA (HopPsyA) and AvrPto are secreted in culture from Pseudomonas syringae pathovars via the Hrp (type III) protein secretion system in a temperature- and pH-sensitive manner. J. Bacteriol. 181: 4790-4797.
Wattiau, P., Woestyn, S. and Cornelis, G. R. 1996. Customized secretion chaperones in pathogenic bacteria. Mol. Microbiol. 20: 255-62.
Wei, W., Plovanich-Jones, A., Deng, W.-L., Jin, Q. L., Collmer, A., Huang, H.-C. and He, S. Y. 2000. The gene coding for the Hrp pilus structural protein is required for type III secretion of Hrp and Avr proteins in Pseudomonas syringae pv. tomato. Proc. Natl. Acad. Sci. USA 97: 2247-2252.
Wei, Z. M., Laby, R. J., Zumoff, C. H., Bauer, D. W. and He, S. Y. 1992. Harpin, elicitor of the hypersensitive response produced by the plant pathogen Erwinia amylovora. Science 257: 85-88.
Wengelnik, K. and Bonas, U. 1996. HrpXv, an AraC-type regulator, activates expression of five of the six loci in the hrp cluster of Xanthomonas campestris pv. vesicatoria. J. Bacteriol. 178: 3462-3469.
Wengelnik, K., Marie, C., Russel, M. and Bonas, U. 1996. Expression and localization of HrpA1, a protein of Xanthomonas campestris pv. vesicatoria essential for pathogenicity and induction of the hypersenitive response. J. Bacteriol. 178: 1061-1069.
Willis, D. K., Rich, J. J. and Hrabak, E. M. 1991. hrp genes of Phytopathogenic bacteria. Mol. Plant Microbe Interac. 4: 132-138.
Xiao, Y., Heu, S., Yi, J., Lu, Y. and Hutcheson, S. W. 1994. Identification of a putative alternate sigma factor and characterization of a multicomponent regulatory cascade controlling the expression of Pseudomonas syringae pv. syringae Pss 61 hrp and hrmA genes. J. Bacteriol. 176: 1025-1036.
Xiao, Y. and Hutcheson, S. W. 1994. A single promoter sequence recognized by a newly identified alternate sigma factor directs expression of pathogenicity and host range determinants in Pseudomonas syringae. J. Bacteriol. 176: 3089-3091.
Xiao, Y., Lu, Y., Heu, S. and Hutcheson, S. W. 1992. Organization and environmental regulation of the Pseudomonas syringae pv. syringae 61 hrp cluster. J. Bacteriol. 174: 1734-1741.
Yuan, J. and He, S. Y. 1996. The Pseudomonas syringae Hrp regulation and secretion system controls the production and secretion of multiple extracellular proteins. J. Bacteriol. 178: 6399-6402.
Zuker, M. 1989. On finding all suboptimal foldings of an RNA molecule. Science 244: 48-52.