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標題: | 植物青枯病菌 tol-pal 基因群突變株的特性研究與噬菌體感受性相關的基因篩選 Characterization of tol-pal gene cluster mutants and identification of determinants in phage infection in Ralstonia solanacearum |
作者: | Yu-Ju Chu 朱昱如 |
指導教授: | 鄭秋萍(Chiu-Ping Cheng) |
關鍵字: | 青枯病菌,tol-pal 基因群,寄主適應性,噬菌體感染, Ralstonia solanacearum,tol-pal gene cluster,host adaptation,phage infection, |
出版年 : | 2008 |
學位: | 碩士 |
摘要: | 青枯病菌是土壤傳播性的植物病原細菌,能夠入侵寄主植物維管束系統導致嚴重萎凋及造成許多重要的經濟作物的損失,若能對此病原菌的致病機制作全盤性的瞭解,預期將可有助於研發有效防治此病害的策略。本研究分為兩大部分,第一部份是關於青枯病菌 tol-pal 基因組的研究。tol-pal 基因組是一群在葛蘭氏陰性菌中相當廣泛存在的保守基因組,主要負責維持細胞膜的完整和穩定性,並且也和接受線狀噬菌體 DNA 輸入寄主細胞有關。本實驗室先前的研究發現,四株青枯病菌tol-pal跳躍子突變株在番茄的致病力並無顯著影響,但在阿拉伯芥中上卻幾乎不具有致病力。為進一步釐清青枯病菌 tol-pal 基因組在寄主適應性上所扮演的角色,本研究利用allelic mutants驗證此突變株在番茄與阿拉伯芥的致病力確實有差異,這些突變株可在茄科作物菸草及番茄體內纏據並且具有和野生型菌株相當的族群量,但在阿拉伯芥中的族群量及在維管束系統中的移動卻顯著變差。此外,這些突變株所的游動性的下降、細胞膜結構較為脆弱、對於碳源吸收或氧化能力的改變、以及細胞形態的改變,這些因子也許可能和它們在兩種寄主植物中的致病力不同有關,值得未來進行進一步研究來驗證。因此,除了互補實驗已經驗證tol-pal 基因組在青枯病菌和維持膜的穩定性有關之外,本研究更發現這群基因在寄主適應性上亦可能也具有一定的功能。此外,本研究也是首次針對系統性感染的植物病原菌探討 tol-pal 的操縱子結構進行分析,並發現 tol-pal 基因組有三個轉錄單位,且其表現會受到生長時期的調控。由於 tol-pal 基因組和噬菌體感染有關,加上噬菌體可能用於進行青枯病菌的生物防治,也引發我們對於噬菌體和青枯病菌之間交互作用的研究興趣。在本研究第二部份的工作中,利用跳躍子隨機插入青枯病菌突變株的研究策略,自約兩千株突變株中已篩選出12株不會被由本土所分離之噬菌體ΦRS29感染之突變菌株,共計找到了7個和此噬菌體相關的青枯病菌基因。這些基因依其功能大致可分為兩群:參與細胞壁及相關成份合成(ampG, wecA, hldD, RSc2201) 與運輸蛋白 (RSc2958和RSc2962) 的基因。本研究是第一篇全面性研究青枯病菌參與和噬菌體交互作用的基因群的研究報告。從本研究目前所獲得的資訊,我們推測噬菌體成功感染青枯病菌的重要關鍵可能包括,辨識與吸附細胞壁相關組成物質,並進一步利用寄主運輸蛋白將其基因組注入宿主細胞:未來值得進一步進行更全面之基因搜尋及個別基因之深入功能研究。 Ralstonia solanacearum is a soil-borne bacterium infecting the vascular system and causing deadly wilting symptoms in many economically important crops. Gaining insight into pathogenesis mechanism of this devastating pathogen may provide information for future disease control. This study contains two main themes. First, the tol-pal gene cluster is widely conserved in many Gram-negative bacteria, involving in membrane integrity and filamentous phage DNA transport. Interestingly, R. solanacearum mutants of strain Pss190 possessing transposon insertions in this gene cluster displayed normal pathogenesis on tomato plants but reduced virulence on Arabidopsis, suggesting the plausible involvement of this gene cluster in host adaptation. Allelic mutants displayed consistent differential pathogenesis on the two host plants, confirming the transopon insertions are responsible for the altered phenotype. Proliferation and colonization of these mutants in solanaceous host Nicotiana benthamiana and tomato plants were not significantly different from the wild-type strain, while their colonization in Arabidopsis was significantly reduced. The tol-pal mutants also conferred certain phenotypic alterations which may possibly correlate to the differential pathogenesis, including reduced motility, altered capability in uptake and oxidation of carbon sources, lability of membrane structure and altered cell morphology (blebs-formation). Complementation test have further confirmed that tol-pal gene cluster involves in membrane stability. These results together suggest a role of tol-pal gene cluster in the adaptation to different host environments. Furthermore, promoter assays revealed that R. solanacearum tol-pal gene cluster could be organized as three transcription units and their activity is moderated throughout the growth phase. This is the first report investigating the function and organization of the tol-pal gene cluster in a vascular phytopathogenic bacterium. Secondly, because the tol-pal gene cluster has been reported to involve in phage infection, and R. solanacearum-infecting bacteriophages can be a putative agent for control of bacterial wilt, we are interested in studying phage-R. solanacearum interaction. To systematically identify R. solanacearum determinants for phage infection, a transposon–insertional mutant screening approach was employed to isolate phage-resistant mutants of R. solanacearum strain Pss4. Twelve mutants were identified to be resistant to a locally isolated phage ΦRS29 from a preliminary screen of approximately 2,000 mutants, and seven loci were identified. These loci are predicted to be involved two main functional groups: (a) Biogenesis of cell-wall and related components (ampG, wecA, hldD, rfaD, RSc2201); (b) Transporters (RSc2958, RSc2962). This is the first study reporting determinants of R. solanacearum involved in interaction with phages. Current results suggest that key steps for successful phage infection include recognition and adsorption of R. solanacearum cell wall and related components by the phages and injection of phage genomic DNA into the host cell via host transporters. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/40734 |
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顯示於系所單位: | 植物科學研究所 |
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