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標題: | 植物 RNase L Inhibitor (RLI) 蛋白質為逆境反應之負調控者與青枯病菌 FruR 基因調控胞外多醣體之合成 Plant RNase L Inhibitors (RLIs) Play a Negative Role in Stress Responses, and FruR Regulates the Production of Extracellular Polysaccharide in Ralstonia solanacearum |
作者: | Yu-Mei Lin 林玉梅 |
指導教授: | 鄭秋萍 |
關鍵字: | 青枯病菌,細菌性軟腐病菌,核醣核酸?L抑制因子,粒線體,氧化逆境,胞外多醣體,FruR轉錄因子,醣類代謝, Ralstonia solanacearum,Pectobacterium carotovorum subsp. carotovorum,RNase L inhibitor protein,Mitochondrion,Oxidative stress,Exopolysaccharides,FruR transcription factor,Carbohydrate metabolism, |
出版年 : | 2014 |
學位: | 博士 |
摘要: | 第一章 Plant RNase L Inhibitors (RLIs) Play a Negative Role in Stress Responses
核醣核酸靜默在調控動植物發育與抗病機制上扮演重要角色。核醣核酸酶L抑制因子(RNase L inhibitor proteins,簡稱 RLIs)可抑制內生性核醣核酸靜默機制,且在真核生物與古細菌中具有高度保守性。前人研究發現RLIs能調控生長發育與病毒防禦之相關基因,然而,RLIs是否參與植物抵抗病害與非生物逆境相關調控機制卻仍未知。透過序列比對分析本研究發現番茄RLI(SlRLI)的蛋白質序列與人類、酵母菌與植物的同源基因保守性極高,SlRLI-GFP重組蛋白質位於阿拉伯芥葉原體之粒線體,SlRLI在番茄各組織中為持續表現,且多種逆境賀爾蒙與青枯病菌(Ralstonia solanacearum)處理不會明顯改變其表現。在番茄與菸草(Nicotiana benthamiana)中將其RLI靜默後,雖會影響其生長發育,但對氧化逆境與乾旱的抗性卻增加。另外,RLI被靜默後可增加菸草對青枯病與細菌性軟腐病之抗性,但相反地卻會降低番茄對這兩種病害之抗性,推估可能因RLI被靜默後造成番茄植株生長發育極不正常以致於影響病害試驗之正確性。進一步在阿拉伯芥atrli2剔減株與過量表現SlRLI植株中之試驗證實RLIs在對抗氧化逆境、青枯病及細菌性軟腐病確實是扮演負調控角色,而轉錄體分析(transcriptomic analyses)也顯示RLI可藉由調控氧化逆境相關基因之表現而影響植物之逆境反應。綜合目前結果顯示,RLIs為植物逆境防禦反應之關鍵負向調控者。 第二章 FruR Regulates the Production of Extracellular Polysaccharide in Ralstonia solanacearum 青枯病菌(Ralstonia solanacearum)為土壤傳播性病原細菌,會造成植物萎凋致死,造成許多經濟作物之嚴重損失,exopolysaccharides(EPS)為 R. solanacearum 的重要致病因子,在感染初期參與biofilm生成以協助病菌在植物根部與莖基部附著與纏繞,對病菌之增生與系統性感染能力更是關鍵,且在感染後期大量 EPS 的合成更會阻塞維管束而導致植物缺水萎凋。合成為關鍵致病因子之一。本研究室前先以本土 R. solanacearum 高毒力菌株 Pss190 為材料,建立跳躍子(Tn5)隨機插入突變菌種庫(Lin et al., 2008),篩選出在番茄與阿拉伯芥上致病力皆減弱之突變菌株 fruR::Tn5。本論文之研究目標為深入探討 FruR 影響致病機制的可能原因。FruR(舊稱 Cra)為廣泛存在之 GlaR-LacI 轉錄調控因子家族成員。R. solanacearum FruR 與其相關 gene cluster 構造與其他 Gram-negative 細菌具保守性,且 R. solanacearum 的 fruR::Tn5 突變株可有效地使用 fructose 當做碳源,而其 gluconeogenesis 與 TCA 循環重要酵素基因之表現則可能受到抑制,故 R. solanacearum FruR 與大多細菌之 FruR 一樣具抑制 fructose 之代謝分解與活化 gluconeogenesis 系統等功能。此外,野生株在外加 fructose 的條件下可誘發較多的 EPS 的合成,且 fruR::Tn5 產生exopolysaccharides(EPS)能力有缺陷,並降低 biofilm 的形成。本研究利用啟動子活性分析發現:(1)fructose 與 sucrose 皆會誘導 fruR 高表現量;(2)FruR 會正調控 EPS 合成之關鍵轉錄因子 epsA 與 xpsR 之表現;(3)以 fructose 為碳源時,FruR 會自我回饋誘導 fruR 表現,而以 glucose 或 fructose 為碳源時,XpsR 也會回饋誘導 fruR 表現。故 R. solanacearum FruR 會藉由抑制 fructose之使用、使醣類代謝途徑導向 gluconeogenesis 及活化 EPS 合成基因之表現,同時因 FruR 之自身與被 XpsR 的正向回饋控制,促進病菌生成 EPS,以利達到系統性感染。 第一章 Plant RNase L Inhibitors (RLIs) Play a Negative Role in Stress Responses RNase L inhibitor proteins (RLIs), an endogenous suppressor of RNA silencing, are highly conserved in eukaryotes and archaea. Their function in mammal defense against viruses has been shown; however, whether RLIs are involved in plant stress defense remains undetermined. Our study shows that the tomato RLI (SlRLI) shares highly significant sequence homology with orthologs in eukaryotes. SlRLI-GFP protein co-localizes with mitochondria under normal condition. SlRLI is constitutively expressed in various tissues, and its expression is not significantly altered after treatments of various plant stress hormones and Ralstonia solanacearum, the causal agent of bacterial wilt (BW). Silencing of RLIs in tomato and Nicotiana benthamiana causes defective plant growth and increased tolerance to paraquat (MV). RLI silencing in N. benthamiana also leads to enhanced tolerance to BW and bacterial soft rot (BSR), but RLI silencing in tomato reduces the tolerance to these two diseases. This inconsistency might be due to the severe growth and development defects in RLI-silenced tomato plants. Further characterization of Arabidopsis transgenic lines with reduced expression of AtRLI2 or ectopic expression of SlRLI confirms the negative role of RLIs in plant defense against MV, BW and BSR. Transcriptomic analyses further show that RLIs function in modulating oxidative stress response at the transcriptional level. These results together evidence that plant RLIs are important negative regulators in stress defense responses. 第二章 FruR Regulates the Production of Extracellular Polysaccharide in Ralstonia solanacearum Ralstonia solanacearum, a soil-borne Gram-negative bacterium, causes deadly wilting diseases on many enconomically important crops. Production of exopolysaccharides (EPS) is essential for establishing systemic infection of this pathogen. Previously, we identified an avirulent R. solanacearum Pss190 fruR::Tn5 mutant. This study aimed to elucidate how FruR modulates R. solanacearum pathogenesis. FruR (Cra) is a member of GlaR-LacI transcriptional factor family. R. solanacearum FruR shares conserved protein sequence and gene cluster organization with the orthologous systems in most Gram-negative bacteria. fruR::Tn5 utilized fructose much more efficiently than the wild-type strain and the expression of enzyme genes involved in gluconeogenesis and TCA cycle was repressed in this mutant. Therefore, R. solanacearum FruR may have functions in inhibiting fructose utilization and promoting gluconeogenesis, similar to its well-studied orthologs in Gram-negative bacteria. In addition, fructose enhances EPS production of the wild-type strain and fruR::Tn5 was defective in EPS production and biofilm formation. Furthermore, promoter activity assays reveal that: (1) fructose and sucrose significantly induce fruR expression, (2) FruR activates EPS biosynthesis key genes epsA and xpsR, (3) fruR expression is activated by FruR when fructose is the carbon source and by XpsR when fructose or glucose are the carbon sources. Together these data show that by inhibiting fructose utilization, promoting gluconeogenesis, activating EPS biosynthesis key genes and being feedback regulated, R. solanacearum FruR plays an essential positive role in EPS production, leading to successful systemic infection. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/55823 |
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顯示於系所單位: | 植物科學研究所 |
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