指導教授：劉瑞芬臺灣大學：植物病理與微生物學研究所黃俊慈Huang, Chun-TzuChun-TzuHuang2014-11-292018-06-292014-11-292018-06-292014http://ntur.lib.ntu.edu.tw//handle/246246/263049植物藉由細胞膜上之pattern recognition receptor (PRRs)辨識病原保守性構造pathogen/microbe-associated molecular patterns (PAMPs/MAMPs)以啟動PAMP-triggered immunity (PTI)。受到PAMPs活化之PRRs通常會與其他leucine-rich repeat (LRR)-receptor-like kinases (RLKs)交互作用，如BRASSINOSTEROID INSENSITIVE1-ASSOCIATED RECEPTOR KINASE1 (BAK1)及SUPPRESSOR OF BIR1-1/EVERSHED (SOBIR1)，進而啟動PTI下游防禦反應。Elicitins為疫病菌及腐黴菌特有的外泌性elicitor蛋白，能誘發部分雙子葉植物產生過敏性反應 (hypersensitive response, HR)，引起細胞壞疽性死亡。本實驗室近期發現番茄SlSOBIR1與SlSOBIR1-like參與Phytophthora parasitica elicitin ParA1引發壞疽的反應途徑，為能進一步瞭解SlSOBIR1在植物接收ParA1後相關的反應途徑，本研究以Nicotiana benthamiana為研究系統，透過共免疫沉澱與質譜儀分析，分析處理與未處理ParA1時之SlSOBIR1交互作用蛋白體，總共發現157個SlSOBIR1免疫共沉澱蛋白，其中25個蛋白只專一性的出現於ParA1處理組。基因註解分析發現三個biological process Gene ontology (GO) terms在ParA1處理時顯著提升，包含cell communication, response to endogenous stimulus以及 response to stress。為驗證這些基因是否參與ParA1誘導之過敏性反應，進一步以TRV-mediated gene silencing靜默其中11個基因，並於菸草葉片短暫表現ParA1，結果發現降低NbArcA2及NbGBLP (兩者皆為guanosine nucleotide-binding proteins, G protein)，NbPDR1 [ATP-binding cassette tranporter (ABC) transporter]、NbPR10 [pathogenesis-related (PR) protein]，NbOligoA (Oligopeptidase A)與NbrbohB (NADPH oxidase)的表現量皆延遲及減少ParA1引發的細胞壞疽現象，其中尤以NbPDR1的作用效果最為顯著。Recognition of microbe/pathogen-associated molecular patterns (MAMPs/PAMPs) by plasma membrane-localized pattern recognition receptors (PRRs) is the initial step to activate PAMP-triggered immunity (PTI) in plants. In addition to PRRs, other leucine-rich repeat receptor-like kinases are known to play important roles in PTI, including BRASSINOSTEROID INSENSITIVE1-ASSOCIATED RECEPTOR KINASE1 (BAK1), BAK1-INTERACTING RECEPTOR-LIKE KINASE1 (BIR1), and SUPPRESSOR OF BIR1-1/EVERSHED (SOBIR1/EVR), which interact with the receptors to regulate downstream defense responses. Recently, tomato homologs of SOBIR1 (known as SlSOBIR1 and SlSOBIR1-like) are known to mediate plant basal defense against Phytophthora parasitica. Furthermore, they are involved in the signaling of ParA1, an elicitin of P. parasitica. In this study, coimmunoprecipitation (co-IP) and mass spectrometry were performed to identify SlSOBIR1-interacting proteins to find out how SlSOBIR1 mediates ParA1 signaling in Nicotiana benthamiana. A total of 157 SlSOBIR1-interacting proteins were identified, of which 25 of them are associated with SlSOBIR1 only in the presence of ParA1. GO term analysis indicated that three biological process terms were specifically enriched in response to ParA1 treatment, including cell communication, response to endogenous stimulus, and response to stress. To uncover their roles, we silenced 11 of the 25 genes by TRV-mediated gene silencing, and then expressed ParA1 by agroinfiltration. ParA1-induced necrosis is compromised upon silencing of NbArcA2 and NbGBLP (guanosine nucleotide-binding proteins, G protein), NbPDR1 [ATP-binding cassette tranporter (ABC) transporter], NbPR10 [pathogenesis-related (PR) protein], NbOligoA (Oligopeptidase A), and NbrbohB (NADPH oxidase), with silencing of NbPDR1 showing the most prominent effect.誌謝 i 中文摘要 ii ABSTRACT iii 目錄 iv 表目錄 vii 圖目錄 viii 縮寫對照表 ix 壹、 前言 1 1. 植物防禦反應 1 1.1 植物先天免疫 (Plant innate immunity) 1 1.2 PTI防禦反應 1 1.3 PRRs (植物辨識PAMPs/MAMPs之受器) 2 1.4 BAK1在PTI扮演重要角色 3 1.5 SOBIR1參與LRR-RLP所調控之PTI訊息傳導途徑 4 2. 植物病原卵菌 5 2.1 卵菌的特性及生活史 5 2.2 疫病菌Phytophthora 6 3. Elicitin 8 3.1 Elicitin之特性及分類 8 3.2 Elicitin誘發之植物防禦反應及反應途徑 9 4. SOBIR1參與ParA1誘導之訊息傳導途徑 9 5. 研究動機 11 貳、 材料與方法 12 1. 植物材料與生長條件 12 2. 分析SlSOBIR1-GFP與SlSOBIR-like-GFP之分佈位置 12 2.1 農桿菌注射法 (Agroinfiltration) 12 2.2 以雷射掃描式共軛焦顯微鏡 (Laser Scanning Confocal Microscope)觀察螢光蛋白分布位置 12 3. 純化SlSOBIR1-GFP之交互作用蛋白 13 3.1 抽取植物全蛋白 13 3.2 免疫沉澱 13 3.3 西方轉漬法 (western blot) 14 4. 以質譜儀分析SlSOBIR1共免疫沉澱蛋白 14 4.1 蛋白質萃取與免疫沉澱 14 4.2 質譜儀樣品前處理 14 4.3 質譜儀數據分析 16 5. 生物資訊學分析SlSOBIR1共免疫沉澱蛋白 17 5.1 基因註解 17 6. SlSOBIR1共免疫沉澱蛋白功能性分析 17 6.1 TRV-mediated基因靜默 17 7. RNA表現量分析 18 7.1 抽取植物RNA 18 7.2 製備cDNA 19 7.3 即時定量聚合酶鏈鎖反應 (quantitative real-time polymerase chain reaction, qPCR) 19 7.4 ParA1處理 19 參、 結果 20 1. 共表現ParA1導致SlSOBIR1-GFP及SlSOBIR1-like-GFP向植物細胞質移動 20 2. 以免疫沉澱分離SlSOBIR1-GFP及SlSOBIR1-like-GFP在植物體內的交互作用蛋白 20 3. 利用LC-MS/MS辨識SlSOBIR1免疫共沉澱蛋白 22 4. SlSOBIR1免疫共沉澱蛋白結合Gene ontology (GO)之功能性分析 23 5. ParA1處理時SlSOBIR1免疫共沉澱蛋白之GO term enrichment分析 25 6. 靜默NbPDR1, NbPR10, NbOligoA, NbAraA2, NbGBLP，或NbrbohB緩降ParA1誘導之細胞壞死 25 6.1 供試基因的挑選與命名 26 6.2 興趣基因靜默及ParA1誘導過敏性反應之功能性測試 27 肆、 討論 30 1. 以菸草分析SlSOBIR1-GFP及SlSOBIR1-like-GFP之免疫共沉澱蛋白 30 2. 以質譜儀辨識SlSOBIR1之交互作用蛋白 31 3. ParA1處理時SlSOBIR1免疫共沉澱蛋白的GO terms分布變化 34 3.1 ParA1處理時SlSOBIR1免疫共沉澱蛋白的GO terms enrichment分析 35 4. 供試基因在植物防禦反應中的生物意義 36 4.1 G proteins 36 4.2 Pleiotropic drug resistance protein 38 4.3 Reactive oxygen species (ROS)生合成相關蛋白 39 4.4 防禦相關蛋白及其他 40 5. 結語 43 伍、 參考文獻 44 附表 64 附圖 77 附錄 891732902 bytesapplication/pdf論文公開時間：2024/05/13論文使用權限：同意無償授權PAMP-triggered immunityreceptor-like kinase共免疫沉澱交互作用蛋白體elicitin疫病菌thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/263049/1/ntu-103-R01633005-1.pdf