Please use this identifier to cite or link to this item:
標題: Xanthomonas axonopodis pv. citri二元反應調控蛋白BfdR藉由rpf/DSF系統調控生物膜形成及毒力
A two component regulator BfdR regulates biofilm formation and virulence through the rpf/DSF system in Xanthomonas axonopodis pv. citri
作者: 陳郁璇
Chen, Yu-Hsuan
關鍵字: 柑桔潰瘍病;Xanthomonas axonopodis pv. citri;二元反應調控蛋白;DSF;two component regulatory protein;DSF
出版社: 植物病理學系所
引用: Andrade, M. O., M. C. Alegria, C. R. Guzzo, C. Docena, M. C. P. Rosa, C. H. I. Ramos & C. S. Farah (2006) The HD-GYP domain of RpfG mediates a direct linkage between the Rpf quorum-sensing pathway and a subset of diguanylate cyclase proteins in the phytopathogen Xanthomonas axonopodis pv citri. Mol. Microbiol. 62: 537-551. Bornke, F., J. Kirchberg, D. Buttner, B. Thiemer & R. G. Sawers (2012) Aconitase B is required for optimal growth of Xanthomonas campestris pv. vesicatoria in pepper plants. PLoS ONE 7: e34941. Barber, C. E., J. L. Tang, J. X. Feng, M. Q. Pan, T. J. Wilson, H. Slater, J. M. Dow, P. Williams & M. J. Daniels (1997) A novel regulatory system required for pathogenicity of Xanthomonas campestris is mediated by a small diffusible signal molecule. Mol. Microbiol. 24: 555-566. Beinert, H., M. C. Kennedy & C. D. Stout (1996) Aconitase as iron-sulfur protein, enzyme, and iron-regulatory protein. Chem. Rev. 96: 2335-2374. Boon, C., Y. Deng, L.-H. Wang, Y. He, J.-L. Xu, Y. Fan, S. Q. Pan & L.-H. Zhang (2007) A novel DSF-like signal from Burkholderia cenocepacia interferes with Candida albicans morphological transition. ISME J. 2: 27-36. Chatterjee, A., Y. Cui, H. Yang, A. Collmer, J. R. Alfano & A. K. Chatterjee (2003) GacA, the response regulator of a two-component system, acts as a master regulator in Pseudomonas syringae pv. tomato DC3000 by controlling regulatory RNA, transcriptional activators, and alternate sigma factors. Mol. Plant-Microbe Interact. 16: 1106-1117. Chatterjee, S. & R. V. Sonti (2002) rpfF mutants of Xanthomonas oryzae pv. oryzae are deficient for virulence and groth inder low iron conditions. Mol. Plant-Microbe Interact. 15: 463-471. Colnaghi, S., V. d. S. Ana, S. Denise, M. R. Lambais & E. Carrilho (2007) Characterization of a putative Xylella fastidiosa diffusible signal factor by HRGC-EI-MS. J Mass Spectrom 42: 490-496. Costerton, J. W., K. J. Cheng, G. G. Geesey, T. I. Ladd, J. Nickel, M. Dasgupta & T. J. Marrie (1987) Bacterial biofilms in nature and disease. Annu. Rev. Microbiol. 41: 435-464. Costerton, J. W., P. S. Stewart & E. P. Greenberg (1999) Bacterial biofilms: a common cause of persistent infections. Science 284: 1318-1322. Deng, Y., C. Boon, L. Eberl & L.-H. Zhang (2009) Differential Modulation of Burkholderia cenocepacia virulence and energy metabolism by the quorum-sensing signal BDSF and its synthase. J. Bacteriol. 191: 7270-7278. Deng, Y., N. Schmid, C. Wang, J. Wang, G. Pessi, D. Wu, J. Lee, C. Aguilar, C. H. Ahrens, C. Chang, H. Song, L. Eberl & L. H. Zhang (2012) Cis-2-dodecenoic acid receptor RpfR links quorum-sensing signal perception with regulation of virulence through cyclic dimeric guanosine monophosphate turnover. Proc. Natl. Acad. Sci. U S A. 109: 15479-15484. Dong, Y.-H., L.-H. Wang, J.-L. Xu, H.-B. Zhang, X.-F. Zhang & L.-H. Zhang (2001) Quenching quorum-sensing-dependent bacterial infection by an N-acyl homoserine lactonase. Nature 411: 813-817. Dong, Y. H., A. R. Gusti, Q. Zhang, J. L. Xu & L. H. Zhang (2002) Identification of quorum-quenching N-Acyl homoserine lactonases from Bacillus species. Appl. Environ. Microbiol. 68: 1754-1759. Dow, J. M., J.-X. Fenga, C. E. Barber, J.-L. Tanga & M. J. Daniels (2000) Novel genes involved in the regulation of pathogenicity factor production within the rpf gene cluster of Xanthomonas campestris. Microbiology 146: 885-891. Dow, M. (2008) Diversification of the function of cell-to-cell signaling in regulation of virulence within plant pathogenic xanthomonads. Sci Signal 1: pe23. Dunger, G., V. M. Relling, M. L. Tondo, M. Barreras, L. Ielpi, E. G. Orellano & J. Ottado (2007) Xanthan is not essential for pathogenicity in citrus canker but contributes to Xanthomonas epiphytic survival. Arch. Microbiol. 188: 127-135. Flavier, A. B., S. J. Clough, M. A. Schell & T. P. Denny (1997) Identification of 3‐hydroxypalmitic acid methyl ester as a novel autoregulator controlling virulence in Ralstonia solanacearum. Mol. Microbiol. 26: 251-259. Fuqua, W. C., S. C. Winans & E. P. Greenberg (1994) Quorum sensing in bacteria: the LuxR-LuxI family of cell density-responsive transcriptional regulators. J. Bacteriol. 176: 269. Guo, Y., Y. Zhang, J.-L. Li & N. Wang (2012) Diffusible signal factor-mediated quorum sensing plays a central role in coordinating gene expression of Xanthomonas citri subsp. citri. Mol. Plant Microbe Interact. 25: 165-179. Gustavo, A.-M., F.-A. Juliana, B. Gisele, R. Juliana, A. C. Sergio & A. M. Marcos (2005) Expression profiling of virulence and pathogenicity genes of Xanthomonas axonopodis pv. citri. J. Bacteriol. 187: 1201-1205. He, Y.-W., J. e. Wu, J.-S. Cha & L.-H. Zhang (2010) Rice bacterial blight pathogen Xanthomonas oryzae pv. oryzae produces multiple DSF-family signals in regulation of virulence factor production. BMC Microbiol. 10: 187. Hornby, J. M., E. C. Jensen, A. D. Lisec, J. J. Tasto, B. Jahnke, R. Shoemaker, P. Dussault & K. W. Nickerson (2001) Quorum sensing in the dimorphic fungus Candida albicans is mediated by farnesol. Appl. Environ. Microbiol. 67: 2982-2992. Huang, T.-P. & A. C. Lee Wong (2007) Extracellular fatty acids facilitate flagella-independent translocation by Stenotrophomonas maltophilia. Res. Microbiol. 158: 702-711. Huang, T.-P., K.-M. Lu & Y.-H. Chen (2013) A novel two-component response regulator links rpf with biofilm formation and virulence of Xanthomonas axonopodis pv. citri. PLoS ONE 8: e62824. Huang, T. P. & A. C. L. Wong (2007) A cyclic AMP receptor protein-regulated cell-cell communication system mediates expression of a fecA Homologue in Stenotrophomonas maltophilia. Appl. Environ. Microbiol. 73: 5034-5040. Hyytiainen, H., S. Sjoblom, T. Palomaki, A. Tuikkala & P. E. Tapio (2003) The PmrA‐PmrB two‐component system responding to acidic pH and iron controls virulence in the plant pathogen Erwinia carotovora subsp. carotovora. Mol. Microbiol. 50: 795-807. Kleerebezem, M., L. E. Quadri, O. P. Kuipers & W. M. De Vos (1997) Quorum sensing by peptide pheromones and two‐component signal‐transduction systems in gram‐positive bacteria. Mol. Microbiol. 24: 895-904. Korem, M., Y. Gov, M. D. Kiran & N. Balaban (2005) Transcriptional profiling of target of RNAIII-activating protein, a master regulator of Staphylococcal virulence. Infect Immun 73: 6220-6228. Kwaga, J. K., B. J. Allan, J. V. V. D. Hurk, H. Seida & A. Potter (1994) A carAB mutant of avian pathogenic Escherichia coli serogroup O2 is attenuated and effective as a live oral vaccine against colibacillosis in turkeys. Infect Immun 62: 3766-3772. Kwon, D. H., C. D. Lu, D. A. Walthall, T. M. Brown, J. E. Houghton & A. T. Abdelal (1994) Structure and regulation of the carAB operon in Pseudomonas aeruginosa and Pseudomonas stutzeri: no untranslated region exists. J. Bacteriol. 176: 2532-2542. Lin, H., S.-T. Hsu, A.-S. Hwang & K.-C. Tzeng (2005) Phenotypic and genetic characterization of novel strains of Xanthomonas axonopodis pv. citri which induce atypical symptoms on citrus leaves in Taiwan. Plant Pathol. Bull. 14: 227-238. Lin, Y.-H., J.-L. Xu, J. Hu, L.-H. Wang, S. L. Ong, J. R. Leadbetter & L.-H. Zhang (2003) Acyl-homoserine lactone acylase from Ralstonia strain XJ12B represents a novel and potent class of quorum-quenching enzymes. Mol. Microbiol. 47: 849-860. Mahan, M. J., J. M. Slauch & J. J. Mekalanis (1993) Selection of bacterial virulence genes that are specifically induced in host tissues. Science 259: 686-688. Malamud, F., V. P. Conforte, L. A. Rigano, A. P. Castagnaro, M. R. Marano, A. Morais do Amaral & A. A. Vojnov (2012) HrpM is involved in glucan biosynthesis, biofilm formation and pathogenicity in Xanthomonas citri subsp. citri. Mol. Plant Pathol. 13: 1010-1018. Malamud, F., P. S. Torres, R. Roeschlin, L. A. Rigano, R. Enrique, H. R. Bonomi, A. P. Castagnaro, M. R. Marano & A. A. Vojnov (2011) The Xanthomonas axonopodis pv. citri flagellum is required for mature biofilm and canker development. Microbiology 157: 819-829. Mergeay, M., D. Gigot, J. Beckmann, N. Glansdorff & A. Pierard (1974) Physiology and genetics of carbamoylphosphate synthesis in Escherichia coli K12. Mol. Gen. Genet. 133: 299-316. Miller, M. B. & B. L. Bassler (2001) Quorum sensing in bacteria. Annu. Rev. Microbiol. 55: 165-199. Newman, K. L., S. Chatterjee, K. A. Ho & S. E. Lindow (2008) Virulence of plant pathogenic bacteria attenuated by degradation of fatty acid cell-to-cell signaling factors. Mol. Plant-Microbe Interact. 21: 326-334. Parkinson, J. S. & E. C. Kofoid (1992) Communication modules in bacterial signaling proteins. Annu. Rev. Genet. 26: 71-112. Pirtte, J., H. Nyunoya, C. J. Lusty, R. Cunin, G. Weyens, M. Carabeel, D. Charlier, N. Glansdrff & A. Pierad (1983) DNA sequence of the carA gene and the control region of carAB : tandem promoters, respectively controlled by arginine and the pyrimidines, regulates the synthesis od carbamoyl-phosphate synthetase in Escherichia coli K-12. Proc. Natl. Acad. Sci. U.S.A. 81: 4134-4138. Rigano, L. A., F. Siciliano, R. Enrique, L. Sendin, P. Filippone, P. S. Torres, J. Questa, J. M. Dow, A. P. Castagnaro, A. A. Vojnov & M. R. Marano (2007) Biofilm formation, epiphytic fitness, and canker development in Xanthomonas axonopodis pv. citri. Mol. Plant-Microbe Interact. 20: 1222-1230. Ryan, R. P., Y. Fouhy, J. F. Lucey, L. C. Crossman, S. Spiro, Y. W. He, L. H. Zhang, S. Heeb, M. Camara, P. Williams & J. M. Dow (2006) Cell-cell signaling in Xanthomonas campestris involves an HD-GYP domain protein that functions in cyclic di-GMP turnover. Proc. Natl. Acad. Sci. U.S.A. 103: 6712-6717. Shaw, P. D., G. Ping, S. L. Daly, C. Cha, J. E. Cronan, K. L. Rinehart & S. K. Farrand (1997) Detecting and characterizing N-acyl-homoserine lactone signal molecules by thin-layer chromatography. Proc. Natl. Acad. Sci. U.S.A. 94: 6036-6041. Slater, H., A. Alvarez-Morales, C. Barber, M. Daniels & J. Dow (2000) A two-component system involving an HD-GYP domain protein links cell-cell signalling to pathogenicity gene expression in Xanthomonas campestris. Mol. Microbiol. 38: 986-1003. Stock, A. M., V. L. Robinson & P. N. Goudreau (2000) Two-component signal transduction. Annu. Rev. Biochem. 69: 183-215. Tang, J. L., Y. N. Liu, C. E. Barber, J. M. Dow, J. C. Wootton & M. J. Daniels (1991) Genetic and molecular analysis of a cluster of rpf genes involved in positive regulation and polysaccharide in Xanthomonas campestris pathovar campestris. Mol. Gen. Genet. 226: 409-417. Torres, P. S., F. Malamud, L. A. Rigano, D. M. Russo, M. R. Marano, A. P. Castagnaro, A. Zorreguieta, K. Bouarab, J. M. Dow & A. A. Vojnov (2007) Controlled synthesis of the DSF cell-cell signal is required for biofilm formation and virulence in Xanthomonas campestris. Environ. Microbiol. 9: 2101-2109. Vilchez, R., A. Lemme, B. Ballhausen, V. Thiel, S. Schulz, R. Jansen, H. Sztajer & I. Wagner‐Dobler (2010) Streptococcus mutans inhibits Candida albicans hyphal formation by the fatty acid signaling molecule trans‐2‐decenoic acid (SDSF). Chembiochem 11: 1552-1562. Wang, L.-H., Y. He, Y. Gao, J. E. Wu, Y.-H. Dong, C. He, S. X. Wang, L.-X. Weng, J.-L. Xu, L. Tay, R. X. Fang & L.-H. Zhang (2004) A bacterial cell-cell communication signal with cross-kingdom structural analogues. Mol. Microbiol. 51: 903-912. Waters, C. M. & B. L. Bassler (2005) Quorum sensing:cell-to-cell communication in bacteria. Annu. Rev.Cell Dev. Biol. 21: 319-346. Wilson, T., N. Bertrand, J. L. Tang, J. X. Feng, M. Q. Pan, C. Barber, J. Dow & M. Daniels (1998) The rpfA gene of Xanthomonas campestris pathovar campestris, which is involved in the regulation of pathogenicity factor production, encodes an aconitase. Mol Microbiol 28: 961-970. Wood, D. W., F. Gong, M. M. Daykin, P. Williams & L. S. Pierson (1997) N-acyl-homoserine lactone-mediated regulation of phenazine gene expression by Pseudomonas aureofaciens 30-84 in the wheat rhizosphere. J. Bacteriol. 179: 7663-7670. Yan, Q., X. Hu & N. Wang (2012) The novel virulence‐related gene nlxA in the lipopolysaccharide cluster of Xanthomonas citri subsp. citri is involved in the production of lipopolysaccharide and extracellular polysaccharide, motility, biofilm formation and stress resistance. Mol. Plant Pathol. 13: 923-934. Yan, Q. & N. Wang (2011) The ColR/ColS two-component system plays multiple roles in the pathogenicity of the citrus canker pathogen Xanthomonas citri subsp. citri. J. Bacteriol. 193: 1590-1599.
由Xanthomonas axonopodis pv. citri (syn. X. citri subsp. citri) 引起的細菌性柑橘潰瘍病 (citrus bacteria canker) 為影響全世界柑桔生產最嚴重之細菌性病害。本實驗室前期研究為瞭解柑桔潰瘍病菌之生物膜形成機制,利用跳躍子進行X. axonopodis pv. citri XW19菌株之隨機突變,並篩選生物膜生成能力缺失菌株作為後續研究試驗,其中一突變菌株跳躍子插入基因功能為一二元反應調控蛋白,並將其命名為bfdR (for biofilm formation defective, regulator)。bfdR突變株於葉面上形成生物膜、葉面殘存與形成潰瘍病徵之能力,及擴散性訊息傳遞分子DSF (for diffusible signal factors) 生合成基因rpfF表現於XVM2培養基中明顯較野生型 (wild type) 菌株低。此研究結果證實X. axonopodis pv. citri於柑桔葉表之生物膜形成能力、殘存能力與病原菌的毒力相關,且由BfdR藉由調控rpfF表現控制。本研究目的即在釐清X. axonopodis pv. citri XW19之BfdR是否藉由rpf/DSF系統調控生物膜形成及其期間調控機制。試驗中利用相對定量反轉錄聚合酶連鎖反應 (semi-quantitative reverse-transcription polymerase chain reaction) 分析比較野生型、bfdR突變菌株及其互補菌株之rpf (for regulation of pathogenicity factors) 基因群組中基因及carAB之表現量,結果顯示bfdR突變菌株之rpfB、rpfF與carA基因表現量明顯低於野生型及互補菌株;而rpfA、rpfC、rpfD及rpfG之表現量則高於野生型及互補菌株;而carB基因於三菌株中則無明顯差異。另將bfdR突變菌株培養於添加cis-11-methyl-2-dodecenoic acid之胰化大豆酪蛋白培養液 (tryptic soy broth),可使其於聚苯乙烯 (polystyrene) 表面及墨西哥萊姆葉面上形成生物膜的能力回復至與野生型及互補菌株相近的程度。另利用逆相高效液相層析儀 (reverse phase high performance liquid chromatograghy) 分析轉殖有X. axonopodis pv. citri XW19 rpfF質體YHC1菌株之胞外萃取物,可在滯留時間為10 -11 min,以波長210 nm偵測一吸收波峰,此波峰滯留時間近似於人工合成之cis-11-methyl-2-dodecenoic acid,此結果顯示X. axonopodis pv. citri XW19 rpfF生合成產物可能結構近似於cis-11-methyl- 2-dodecenoic acid。綜合上述結果顯示X. axonopodis pv. citri XW19 BfdR可經由rpf/DSF系統中基因表現而影響生物膜形成。

Citrus bacteria canker, which is caused by Xanthomonas axonopodis pv. citri (syn. X. citri subsp. citri) is the most devastating bacterial disease of citrus plants worldwide. In our previous study, to understand the mechanisms of biofilm formation by X. axonopodis pv. citri, strain XW19 was subjected for transposon mutagenesis and biofilm formation defective mutants for further analysis. The mutant with a mutation of two-component response regulator gene bfdR (for biofilm formation defective, regulator) which showed reduction in the development of biofilm on the leaf surfaces of citrus plants and canker lesions as well as epiphytic growth was isolated. BfdR was also found to positively control the rpfF gene expression in XVM2 medium, the medium which mimics the cytoplasmic fluid in plants. The results indicated that biofilm formation and bacterial survival of X. axonopodis pv. citri on leaf surfaces of citrus are associated with bacterial virulence and is controlled by BfdR through regulation of rpfF. Here, we investigate whether X. axonopodis pv. citri BfdR controls biofilm formation via rpf/DSF system and its regulatory mechanisms. The transcript levels of genes in the rpf (for regulation of pathogenicity factors) gene cluster and carAB in the wild type, bfdR mutant and the complemented strain were assessed. The results from semi-quantitative reverse-transcription polymerase chain reaction showed that the transcript levels of rpfB, rpfF and carA were reduced ; while those of rpfA, rpfC, rpfD and rpfG were regulated in the bfdR mutant compared to the wild type and the complemented strain. The transcript levels of carB in all three strains were similar. The extracellular fatty acids of X. axonopodis pv. citri XW19 harboring rpfF in trans, strain YHC1, were analyzed using reverse-phase high-performance liquid chromatography. A peak at retention time 10-11 mins and UV absorption at 210 nm was found in samples from strain YHC1 similar to the synthetic cis-11-methyl-2-dodecenoic acid. The data suggested that the products synthesized by rpfF of X. axonopodis pv. citri XW19 may be close derivatives of cis-11-methyl-2- dodecenoic acid. In conclusion, our results suggested that BfdR of X. axonopodis pv. citri strain XW19 controls biofilm formation through regulation of gene expression in rpf/DSF system. Addition of synthetic DSF in the tryptic soy broth restored biofilm formation by the bfdR mutant on polystyrene and leaf surfaces of citrus that by the wild type.
其他識別: U0005-2908201312092800
Appears in Collections:植物病理學系

Show full item record

Google ScholarTM


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.