指導教授：金洛仁臺灣大學：植物科學研究所陳敬偉Chen, Ching-WeiChing-WeiChen2014-11-282018-07-062014-11-282018-07-062014http://ntur.lib.ntu.edu.tw//handle/246246/262851膜結合模式識別受體(PRR)例如鞭毛接收器(FLS2)和延伸因子接受器(EFR)，一旦辨識到微生物相關分子(Microbe-Associated Molecular Patterns; MAMP)即誘發先天性免疫作用(Pattern-Triggered Immunity; PTI)。本研究利用誘發劑BABA處理阿拉伯芥並藉由基因微陣列方法指出減弱卵菌敏感性1(IMPAIRED OOMYCETE SUSCEPTIBILITY1,IOS1)扮演一個誘發先天性免疫作用的關鍵角色。阿拉伯芥ios1突變株高度敏感於細菌性斑點病原菌(Pseudomonas syringae)。另外，研究證明ios1突變株在誘發先天性免疫作用產生缺陷,值得注意的是，突變株經由細菌或是微生物相關分子處理後會延遲誘發先天性免疫作用相關基因的表現,降低癒傷葡聚醣(callose)的沉澱並減弱絲裂原活化蛋白激酶mitogen-activated protein kinase (MAPK)的活性。反之，IOS1大量表現轉植株表現更抗細菌性斑點病原菌，並誘發更強的先天性免疫表現。另外，藉由胞外免疫沉澱法(pull-down)、雙分子螢光互補分析(BiFC)、共免疫沉澱法(Co-IP)及質譜分析(mass spectrometry analysis)結果證明IOS1與FLS2、EFR形成複合體。然而，IOS1也證實與油菜素內酯INSENSITIVE 1受體相關激酶1 (BRI1-Associated Kinase 1; BAK1)相互結合，並在微生物相關分子處理後正調控FLS2-BAK1結合。最後，IOS1突變株在BABA所誘發防禦過程中產生缺陷。整個研究顯示IOS1為一個新的調節蛋白參予在FLS2及EFR所首的訊息傳遞中並且在病菌攻擊時快速誘發先天性免疫反應。Plasma membrane-localized pattern recognition receptors (PRRs) such as FLAGELLIN SENSING2 (FLS2) and EF-TU RECEPTOR (EFR) recognize microbe-associated molecular patterns (MAMPs) to activate the first layer of plant immunity termed pattern-triggered immunity (PTI). A reverse genetics approach with genes responsive to the priming agent beta-aminobutyric acid (BABA) revealed IMPAIRED OOMYCETE SUSCEPTIBILITY1 (IOS1) as a critical PTI player. Arabidopsis thaliana ios1 mutants were hyper-susceptible to Pseudomonas syringae bacteria. Accordingly, ios1 mutants demonstrated defective PTI responses, notably delayed up-regulation of PTI-marker genes, lower callose deposition and mitogen-activated protein kinase activities upon bacterial infection or MAMP treatment. Moreover, Arabidopsis lines over-expressing IOS1 were more resistant to P. syringae and demonstrated a primed PTI response. In vitro pull-down, bimolecular fluorescence complementation, co-immunoprecipitation and mass spectrometry analyses supported the existence of complexes between the membrane-localized IOS1 and FLS2, and EFR. IOS1 also associated with BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1 (BAK1) in a ligand-independent manner, and positively regulated FLS2-BAK1 complex formation upon MAMP treatment. Finally, ios1 mutants were defective in BABA-induced resistance and priming. This work reveals IOS1 as a novel regulatory protein of FLS2- and EFR-mediated signaling that primes PTI activation upon bacterial elicitation.致謝 I 摘要 II ABSTRACT III ABBREVIATIONS IV CONTENTS V INTRODUCTION 1 Basal immunity response in plant 1 Receptor-Like Kinase / Leucine-Rich Repeat Receptor-Like Kinase 2 Malectin-Like / Receptor-Like Kinase 3 Pattern-triggered immunity in Arabidopsis: PRR (FLS2 and EFR) perception 3 Pattern-triggered immunity in Arabidopsis: Immune receptor complexes 4 Pattern-triggered immunity in Arabidopsis: The PTI Response 5 BABA-induced Priming 6 OBJECTIVE 8 MATERIALS and METHODS 9 Biological Materials and Growth Conditions 9 Pathogen Infection Assays 9 IOS1 Over-Expression Plants 10 BABA and MAMP Treatments 10 Callose Deposition 11 qRT-PCR 11 MAP Kinase Assay 12 ROS Burst 12 Stomatal Assay 13 Subcellular Localization in Protoplast and Root 13 BIK1 Phosphorylation 13 Cloning, Expression and Purification of Recombinant Proteins 14 In vitro Pull-Down Assay 15 BiFC Assay 15 Transient Expression in Nicotiana benthamiana 16 Protein Extraction and Immunoprecipitation in N. benthamiana 16 Protein Extraction and Immunoprecipitation in Arabidopsis 16 SDS-PAGE and Immunoblotting 17 Mass Spectrometry 17 In vitro Kinase Assay 18 Accession Numbers 18 RESULTS 18 IOS1 is Required for Resistance to Hemi-Biotrophic Bacteria 18 IOS1 is Critical for Late PTI Responses 20 IOS1 Modulates Several Early PTI Responses 21 IOS1 Localizes to the Plasma Membrane 22 IOS1 Associates with FLS2, EFR and BAK1 in a Ligand-Independent Manner 22 IOS1 is required for optimal flg22-induced FLS2-BAK1 association 25 IOS1 is Necessary for BABA-Induced Resistance and Priming 27 DISCUSSION 28 IOS1 is Necessary for Full Activation of Anti-Bacteria PTI in Arabidopsis 28 IOS1 Associates with FLS2, EFR and BAK1 in a Ligand-Independent Manner 30 IOS1 Positively Regulates FLS2-BAK1 Complex Formation 31 IOS1 Plays a Critical Role in Priming of PTI 33 CONCLUSION AND FUTURE PERSPECTIVES 35 FIGURES 36 Figure 2. Altered Late PTI Responses in ios1 Mutants and IOS1-OE Lines. 38 Figure 3. Early PTI Responses. 40 Figure 4. IOS1 Localizes to the Plasma Membrane. 42 Figure 5. Pull-Down and BiFC Analyses of IOS1 Interaction with PRRs. 43 Figure 6. IOS1 Associates with Unstimulated and Stimulated FLS2, EFR and BAK1. 45 Figure 7. IOS1 Regulates Ligand-Induced FLS2/BAK1 Association. 47 Figure 8. IOS1 Does Not Modulate flg22-Mediated BIK1 Phosphorylation. 48 Figure 9. IOS1 Function is BAK1 Dependent but BIK1 Independent. 49 Figure 10. BABA Action is Defective in ios1 Mutants. 50 Appdendix 52 Supplemental Figure 1. The mutant ios1-1 is a knock-out while ios1-2 and ios1-3 still produce some IOS1 transcripts. 52 Supplementarl Figure 2. Susceptibility phenotypes of ios1-2 and bak1-5 to Pst DC3000. 52 Supplementarl Figure 3. ios1 mutants demonstrate increased susceptibility to Pst DC3000 hrcC-. 53 Supplemental Figure 4. Resistance of ios1 mutants to B. cinerea. 53 Supplemental Figure 5. IOS1 mRNA expression levels in 3 IOS1 over-expression lines. 54 Supplemental Figure 6. Stomatal innate immunity in ios1 mutants. 54 Supplemental Figure 7. Expression of IOS1 is up-regulated by bacterial MAMPs. 55 Supplemental Figure 8. PTI-responsive CYP81F2 and WRKY53 up-regulation. 56 Supplemental Figure 9. Early PTI responses in ios1 mutants and IOS1-OE lines upon elf26 elicitation. 57 Supplemental Figure 10. Bimolecular fluorescence complementation analyses of IOS1 interactions with EFR and BAK1. 58 Supplemental Figure 11. IOS1-HA3 does not bind non-specifically to anti-GFP magnetic beads. 59 Supplemental Figure 12. FLS2-FLS2 and IOS1-IOS1 associations. 60 Supplemental Figure 13. IOS1 in vitro autophosphorylation. 61 Supplemental Figure 14. BABA does not regulate ligand-induced FLS2-BAK1 association. 62 Supplemental Figure 15. IOS1 hypothesis model in flagellin. 63 Table 1. Identification of IOS1 Tryptic Peptides by HPLC-ESI-MS/MS Analysis of EFR Immunoprecipitates. 64 Table 2. Primer sequences used in this study. 66 REFERENCES 673549679 bytesapplication/pdf論文公開時間：2014/07/29論文使用權限：同意有償授權(權利金給回饋學校)β-氨基丁酸阿拉伯芥細菌性斑點病原菌先天性免疫作用白胺酸重覆受體激酶減弱卵菌敏感性1thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/262851/1/ntu-103-D96b42014-1.pdf