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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 鄭秋萍(Chiu-Ping Cheng) | |
dc.contributor.author | Tai-Hsiang Chu | en |
dc.contributor.author | 鞠泰翔 | zh_TW |
dc.date.accessioned | 2021-05-15T17:53:21Z | - |
dc.date.available | 2016-08-05 | |
dc.date.available | 2021-05-15T17:53:21Z | - |
dc.date.copyright | 2014-08-05 | |
dc.date.issued | 2014 | |
dc.date.submitted | 2014-08-04 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/5196 | - |
dc.description.abstract | 青枯病菌(Ralstonia solanacearum)引發全球許多作物之致死萎凋病害,常造成全球作物巨大經濟損失。隸屬於phylotype I的青枯病菌菌株可根據在抗感病品系番茄上引起的致死率,進一步定義其毒力高低,但目前關於決定青枯病菌致病力之分子機制所知極少。為瞭解青枯病菌之致病力因子,我們已完整定序四個不同毒力的青枯病菌株,發現效應蛋白(effector)RSp0213只存在中毒力菌株(如Pss4)與低毒力菌株(如Pss216)中,而高毒力菌株(如Pss190)則無此effector。本研究之研究目標為探討RSp0213在青枯病菌致病力及與寄主交互作用中之角色。結果發現在具有RSp0213的中毒力菌株Pss4中將RSp0213剔除會提高Pss4在番茄之致病力,而在無RSp0213的高毒力菌株Pss190中大量表現此effector基因則可降低Pss190在番茄之致病力,故RSp0213確實是決定青枯病菌致病力之關鍵因子。此外,在菸草葉片中表現RSp0213會造成細胞死亡的現象;在番茄中過量表現RSp0213亦導致細胞死亡與植株矮化,且會誘導水楊酸、乙烯及過敏性反應訊息傳導相關基因之表現,進而增強番茄對高毒力青枯病菌菌株Pss190之抗性。另外,序列分析預測與GFP融合蛋白檢測確認RSp0213座落在菸草細胞膜上,且此座落特性為執行其功能之必要條件。以上結果顯示,RSp0213可能被番茄細胞辨認並進而誘發防禦機制,導致含有此效應蛋白之青枯病菌菌株的致病力降低,而高毒力菌株則因無RSp0213而較不會誘發寄主之強烈抗病機制。 論文的第二部分由於源自蕃茄的Pss190菌株具由極高毒力,根據其基因體序列顯示 Pss190特有效應蛋白Pseudomonas outer protein P 3 (PopP3),故本研究旨在探討PopP3在青枯病菌致病力之功能與其相關機制。本研究室的先前研究證實此T3E會受T3SS調控,研究結果亦顯示基因轉移表現至中毒力菌株Pss4可增強其在番茄上的致病力,而將PopP3表現於菸草葉片無病徵產生但有利於青枯病菌之生長,並推測其在植物粒線體的座落位置與其功能有關,可能藉此影響番茄PTI指標之一的癒傷葡聚糖累積 (callose deposition),以抑制植物PTI反應。本研究旨在進一步證實PopP3粒線體座落位置對其功能之重要性,並檢測此T3E在基因轉移表現下,青枯病菌在番茄上的致病力表現,和其功能與HR之相關性,以闡明PopP3如何抑制植物的相關防禦反應機制。 | zh_TW |
dc.description.abstract | Ralstonia solanacearum, the causal bacterium of bacteria wilt (BW), causes severe production losses on many important crops worldwide. R. solanacearum phylotype I strains confer varied degrees of virulence/aggressiveness on tomato plants; however, genetic information on pathogen virulence-related factors is still very limited. To uncover virulence-related factors, genomes of four R. solanacearum phylotype I strains with different virulence were sequenced. A putative effector protein RSp0213 is present in medium-virulence and low-virulence strain such as Pss4 and Pss216, but absent in high-virulnece strain such as Pss190. This study aimed to elucidate roles of RSp0213 in R. solanacearum virulence and plant-bacterium interaction. Diruption of RSp0213 in medium-virulence strain Pss4 resulted in increased bacterial virulence in tomato, while RSp0213 overexpression in high-virulence strain Pss190 leads to decreased bacterial virulence, evidencing a decisive role of RSp0213 in R. solanacearum virulence. Additionally, transient local overexpression of RSp0213 in Nicotiana benthamiana leaves led to cell death. Virus-mediated overexpression of RSp0213 in tomato also caused cell death, reduced plant growth, increased expression of marker genes involved in defense hormones and hypersensitive-response signaling pathways, and enhanced resistance to the high-virulence strain Pss190. Furthermore, GFP-tagged RSp0213 colocalizes with cell membrane of N. benthamiana and membrane localization is required for its functions. These results suggest that RSp0213 can be recognized by host cell, leading to induced defense response and thus reduced virulence of RSp0213-containing R. solanacearum strains. The absence of this effector enables high-virulence strains escape from host recognition, leading to the high virulence. The second part of this thesis aimed to uncover a YopJ-type effector protein Pseudomonas outer protein P 3 (PopP3) unique to a high-virulence strain Pss190. Our previous study showed that overexpression of popP3 in Rs medium-virulence strain Pss4 led to increased bacterial virulence in tomato. Transient overexpression of popP3 in tomato suppresses pathogen-associated molecular patterns (PAMPs)-triggered immunity (PTI) related callose deposition. popP3 transcripts was positively regulated by the type III secretion system (T3SS) regulator HrpG. Furthemore, This study proved the mitochondria localization of PopP3 in plant cell is critical to it function on tomato. These results together suggest that PopP3 is a T3SS effector and contributes to the virulence of R. solanacearum by suppressing plant defense response which related to mitochondrial functions. | en |
dc.description.provenance | Made available in DSpace on 2021-05-15T17:53:21Z (GMT). No. of bitstreams: 1 ntu-103-R01B42034-1.pdf: 6679019 bytes, checksum: aba23db8c4883acead9dd65621cd22ad (MD5) Previous issue date: 2014 | en |
dc.description.tableofcontents | 口試委員審定書I
謝誌II 中文摘要III 英文摘要IV 常用縮寫與全名對照表VI 目錄VII 第一章 前言 1 1. 第三型蛋白分泌系統 1 1.1 調控蛋白 1 1.2 構造蛋白 2 1.3 效應蛋白 2 2. 青枯病與青枯病菌 3 3. 青枯病菌的分子致病機制 4 4. 青枯病菌第三型分泌蛋白T3Es 4 4.1 Harpin 6 4.2 GALA 家族 6 4.3 AvrA 7 4.4 YopJ superfamily 7 5. 植物病害防禦反應 8 5.1 植物PTI 防禦反應 8 5.2 植物ETI 防禦反應 9 5.3 植物細胞膜構造蛋白參與防禦反應 9 6. 病原細菌效應蛋白對植物之作用 10 6.1 作用於細胞膜之效應蛋白 10 6.2 作用於粒線體之效應蛋白 11 6.3 作用於葉綠體之效應蛋白 11 6.4 作用於囊泡運輸之效應蛋白 12 6.5 作用於MAPK 訊息傳遞之效應蛋白 12 6.6 作用於細胞核之效應蛋白 12 6.7 攻擊轉譯之效應蛋白 13 7. 前人研究與研究動機 13 第二章 材料與方法 15 1. 供試菌株、質體及菌株培養條件 15 2. 供試植物培育條件 15 3. 生物統計分析 15 4. 重組質體構築與popP3 過量表現菌株建構 15 4.1. DNA 瓊脂糖凝膠電泳 15 4.1.1. 外染瓊脂糖凝膠電泳分析 15 4.1.2. 內染瓊脂糖凝膠電泳純化 16 4.2. 聚合酶連鎖反應 16 4.2.1. Taq PCR (Violet) 16 4.2.2. Q5R Taq PCR (Invitrogen) 16 4.3. 質體萃取 17 4.3.1. 大腸桿菌質體萃取 17 4.3.2.青枯病菌與農桿菌質體萃取 17 4.4. DNA 純化 18 4.5. DNA 限制酶消化水解 18 4.6. DNA 片段接合 18 4.7. TOPOR 質體構築 18 4.8. LR 重組互換反應 19 4.9. 大腸桿菌勝任細胞熱休克轉型作用 19 4.10. 電穿孔轉型作用之勝任細胞置備 19 4.10.1.青枯病菌、農桿菌 20 4.10.2. 大腸桿菌 20 4.11. 電穿孔轉型作用 20 4.12. 青枯病菌染色體DNA 萃取 21 5. 青枯病菌效應蛋白功能研究 21 5.1. 菸草原生質體萃取與蛋白座落位置檢測 21 5.2. 效應蛋白於菸草葉片細胞座落位置檢測 22 5.3. 青枯病菌致病力測試 22 6. 植物體短暫表現效應蛋白 與其功能性研究 23 6.1. 菸草葉部基因短暫表現 23 6.2. 番茄短暫性病毒誘導基因過量表現 23 6.3. 植物體短暫表現基因檢測 24 6.3.1 植物RNA 萃取 24 6.3.2 反轉錄聚合酶連鎖反應 24 6.3.3.半定量RT-PCR 24 6.3.4. 即時定量Real-time PCR 25 第三章 結果 27 I. 低/中毒力菌株特有之RSp0213與RSc3174在青枯病菌致病力之功能研究 27 1. RSp0213與RSc3174在已完全解序之青枯病菌菌株中之分佈 27 2. RSp0213和RSc3174對青枯病菌致病力之影響 27 3. RSp0213蛋白在植物細胞中之座落位置 28 4. RSp0213過量表現對植物之影響 28 5. 在番茄表現RSp0213對病害反應之影響 29 6. 在番茄表現RSp0213對防禦反應相關標誌基因表現之影響 29 7. 決定RSp0213蛋白在植物細胞中之座落位置的重要區段分析 29 8. RSp0213座落於植物細胞膜對其功能之重要性 29 II. 高毒力菌株特有之效應蛋白PopP3在青枯病菌致病力之功能研究 30 1. PopP家族成員在青枯病菌菌株之分佈與序列相似性 30 2. 在高毒力菌株PopP3之分佈 31 3. PopP2對青枯病菌在番茄上致病力之影響 31 4. PopP3對病菌誘導之菸草過敏性反應的抑制能力 31 5. 在番茄表現popP3對病害反應之影響 32 6. 決定PopP3蛋白在植物細胞中之座落位置的重要區段分析 32 7. 片段刪除後之PopP3對青枯病菌致病力及對番茄病害反應之影響 33 第四章 討論 34 一、青枯病菌RSp0213 之功能研究 34 1. RSp0213降低青枯病菌對番茄之致病力 34 2. 在植物表現RSp0213會誘導防禦反應 35 3. RSp0213座落於植物細胞膜對其功能至關重要 36 4. RSp0213誘導植物ETI反應的可能機制與未來展望 37 二、青枯病菌PopP3 之功能研究 37 1. 高毒力菌株Pss190特有之PopP3為青枯病菌重要的致病力因子 37 2. PopP3會抑制植物PTI反應 38 3. PopP3座落於植物粒線體對其功能至關重要 39 4. PopP3抑制植物PTI反應的可能機制與未來展望 40 參考文獻 42 表 50 圖 52 附表 75 附圖 90 | |
dc.language.iso | zh-TW | |
dc.title | 效應蛋白RSp0213與PopP3在青枯病菌致病力之功能研究 | zh_TW |
dc.title | Roles of Effectors RSp0213 and PopP3 in Virulence of Ralstonia solanacearum | en |
dc.type | Thesis | |
dc.date.schoolyear | 102-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 劉瑞芬(Ruei-Fen Liou),賴爾?(Erh-Min Lai),林乃君(Nai-Chun Lin),劉啟德(Chi-Te Liu) | |
dc.subject.keyword | 青枯病菌,效應蛋白,RSp0213,PopP3,致病力,effector-triggered immunity,effector-triggered susceptibility, | zh_TW |
dc.subject.keyword | Ralstonia solanacearum,effector,RSp0213,PopP3,virulence,effector-triggered immunity,effector-triggered susceptibility, | en |
dc.relation.page | 107 | |
dc.rights.note | 同意授權(全球公開) | |
dc.date.accepted | 2014-08-04 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 植物科學研究所 | zh_TW |
顯示於系所單位: | 植物科學研究所 |
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