請用此 Handle URI 來引用此文件: http://hdl.handle.net/11455/31904
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
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摘要: 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.
URI: http://hdl.handle.net/11455/31904
其他識別: U0005-2908201312092800
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