指導教授：賴爾珉臺灣大學：植物病理與微生物學研究所吳竑毅Wu, Hung-YiHung-YiWu2014-11-292018-06-292014-11-292018-06-292014http://ntur.lib.ntu.edu.tw//handle/246246/263039農桿菌為一種革蘭氏陰性的植物病原細菌，其能藉由演化上具保守性的細菌第四型蛋白質分泌系統(type IV secretion system, T4SS)將T-DNA傳送並嵌入植物基因體中。T4SS主要由VirD4與11個VirB蛋白質(VirB1至VirB11)所組成。T線毛(T-pilus)主要由VirB2與次要之VirB5蛋白質所組成，所有的VirB 蛋白質都為產生T線毛所必需。VirB2為一具保守性之T4SS必需組成元件，但是VirB2蛋白質及其所組成的T線毛對於農桿菌的毒性與T-DNA傳送的過程中所扮演的角色仍然未知。因此在此篇論文當中，我們利用產生34個VirB2胺基酸替換的突變株，來研究VirB2蛋白質對於VirB2的穩定度，細胞外VirB2/T線毛的產生以及農桿菌的毒力所扮演的功能。藉由分析細胞外VirB2的產生(ExB2+ 或 ExB2-)與在蕃茄的莖上產生腫瘤的能力(Vir+ 或Vir-)，所有的突變株可以分為三種：ExB2-/Vir-，ExB2-/Vir+與ExB2+/Vir+。並且我們也藉由電子顯微鏡確認了五種ExB2-/Vir+的突變株為具有野生株程度的毒力但失去產生T線毛的能力，因此稱其為T-pilus-/Vir+突變株。雖然這些T-pilus-/Vir+的突變株在蕃茄莖上或馬鈴薯塊莖上仍保有野生株程度的毒力，但是在阿拉伯芥的小苗上，報導基因的短暫表現效率卻大幅下降。因此，我們提供了T線毛在農桿菌轉型過程上扮演的一個角色，並且鑑定出對於VirB2蛋白質的穩定度、T線毛的產生、腫瘤產生的能力與短暫表現效率有嚴重影響的區域或胺基酸。有鑑於在阿拉伯芥上使用短暫基因表現的方法來進行植物功能性研究的潛力，因此我們更進一步在阿拉伯芥的小苗上分析不同生物因子與生長條件，並確定一種組合可以得到最佳短暫轉型效率與短暫基因表現的方法。在使用β-glucuronidase (GUS)作為報導基因下，我們發現使用一種特定的農桿菌菌株並且配合毒性基因的預先誘導表現，能於所有被感染的阿拉伯芥小苗中達到高效率的基因短暫表現。我們也發現在感染的培養液中添加AB 鹽類與pH 5.5的緩衝液能明顯地提高短暫表現效率，並且較目前已發表的兩種方法更佳。更重要地，這樣高效率的短暫表現並不僅限於阿拉伯芥免疫受器突變株efr-1，在阿拉伯芥野生株Col-0也能達到。最後，這種高效率的基因表達方法也證實得以應用在不同的研究當中，如檢視轉錄因子的動態表現情形、觀察生物時鐘基因的調控情形、蛋白質於細胞內的分佈位置與蛋白質與蛋白質之間的交互作用等等。因此我們命名此方法為AGROBEST (Agrobacterium-mediated enhanced seedling transformation)。這種簡單、快速又可靠的短暫基因表達方法在阿拉伯芥小苗上達到了可以進行功能性分析的層級，並且也提供了一個新的系統來剖析農桿菌傳送DNA的分子機制。Agrobacterium tumefaciens is a Gram-negative plant pathogenic bacterium with the unique ability to transfer DNA into plant genomes. This transfer is mediated by an evolutionarily conserved type IV secretion system (T4SS) comprising VirD4 coupling protein and 11 VirB proteins (VirB1 to VirB11). All VirB proteins are required for the production of T-pilus, which consists of processed VirB2 (T-pilin) and VirB5 as major and minor subunits, respectively. VirB2 is an essential component of T4SS, but the roles of VirB2 and the assembled T-pilus in Agrobacterium virulence and the T-DNA transfer process remain unknown. In this study, we generated 34 VirB2 amino acid substitution variants to study the functions of VirB2 involved in VirB2 stability, extracellular VirB2/T-pilus production and virulence of A. tumefaciens. From the capacity for extracellular VirB2 production (ExB2+ or ExB2-) and tumorigenesis on tomato stems (Vir+ or Vir-), the mutants could be classified into three groups: ExB2-/Vir-, ExB2-/Vir+, and ExB2+/Vir+. We also confirmed by electron microscopy that five ExB2-/Vir+ mutants exhibited a wild-type level of virulence with their deficiency in T-pilus formation and therefore renamed as T-pilus-/Vir+ mutants. Interestingly, although the five T-pilus-/Vir+ uncoupling mutants retained a wild-type level of tumorigenesis efficiency on tomato stems and/or potato tuber discs, their transient transformation efficiency in Arabidopsis seedlings was highly attenuated. In conclusion, we have provided evidence for a role of T-pilus in Agrobacterium transformation process and have identified the domains and amino acid residues critical for VirB2 stability, T-pilus biogenesis, tumorigenesis, and transient transformation efficiency. In view of the potential applications of the transient transformation assay using the Arabidopsis seedlings in gene functional studies, further systematic investigations of various biological factors and growth variances were performed to define a combination of key factors that maximize the high transient transformation and reporter gene expression efficiency in Arabidopsis seedlings. Using β-glucuronidase (GUS) as a reporter for Agrobacterium transient transformation assay, we found that the use of a specific disarmed Agrobacterium strain with vir gene pre-induction resulted in high transient expression levels in all infected young Arabidopsis seedlings. The presence of AB salts in plant culture medium buffered with acidic pH 5.5 during Agrobacterium infection greatly enhanced transient expression levels, which are significantly higher than two existing methods. Importantly, the highly increased transient expression efficiency is not only achieved in the immune receptor mutant efr-1 but also in wild type Col-0 seedlings. Finally, this high transformation efficiency enabled the versatile applicability of the method for examining transcription factor actions and circadian reporter-gene regulation, as well as protein subcellular localization and protein–protein interactions in physiological contexts. This simple, fast, reliable, and robust transient expression system is named as AGROBEST (Agrobacterium-mediated enhanced seedling transformation), which elevates the transient expression technology to the level of functional studies in Arabidopsis seedlings and offers a new system to dissect the molecular mechanisms involved in Agrobacterium-mediated DNA transfer.口試委員會審定書 I 中文摘要 II ABSTRACT IV TABLE OF CONTENTS VI LIST OF FIGURES XII LIST OF TABLES XVI LIST OF ABBREVIATIONS XVIII CHAPTER 1 GENERAL INTRODUCTION - 1 - 1.1 GENERAL INTRODUCTION OF AGROBACTERIUM TUMEFACIENS 2 1.1.1 Taxonomy and pathogenesis 2 1.1.2 Brief historical review of Agrobacterium 3 1.1.3 Agrobacterium virulence genes 3 1.2 MOLECULAR MECHANISMS OF AGROBACTERIUM-PLANT INTERACTIONS 4 1.2.1 Signals sensing, virulence genes activation and attachment to the plant cell 4 1.2.2 Production of substrates and formation of VirB-complex 7 1.2.3 Substrate transport through T4SS and into the plant cell 9 1.2.4 T-complex assembly and trafficking, nuclear import and T-DNA integration inside the plant cell 10 1.3 PLANT IMMUNITY AND HOST RESPONSES 13 1.3.1 Pattern-triggered immunity (PTI) 13 1.3.2 Effector-triggered immunity (ETI) 14 1.4 AGROBACTERIUM PROTEIN SECRETION SYSTEMS 15 1.4.1 General introduction of bacterial protein secretion systems 15 1.4.2 General introduction of bacterial T4SS 17 1.4.3 Energetic components - VirD4, VirB4 and VirB11 18 1.4.4 Core subcomplex components - VirB6, VirB7, VirB8, VirB9 and VirB10 19 1.4.5 T-pilus components - VirB2 and VirB5 22 1.4.6 VirB1 and VirB3 23 1.5 BACTERIAL PILI 24 1.5.1 General introduction of bacterial pili 24 1.5.2 Type III secretion pili/needle 25 1.5.3 Type IV secretion pili 26 1.5.4 Agrobacterium T-pilus 27 1.6 FIGURES 29 1.7 REFERENCE 35 CHAPTER 2 EXPRESSION AND FUNCTIONAL CHARACTERIZATION OF THE AGROBACTERIUM VIRB2 AMINO ACID SUBSTITUTION VARIANTS IN T-PILUS BIOGENESIS, VIRULENCE, AND TRANSIENT TRANSFORMATION EFFICIENCY 55 2.1 ABSTRACT - 56 - 2.2 INTRODUCTION - 57 - 2.3 MATERIALS AND METHODS - 60 - 2.3.1 Bacterial strains, growth and T-pilus induction conditions - 60 - 2.3.2 Construction of mutant strains and complementing plasmids - 61 - 2.3.3 Isolation of intracellular and extracellular fractions - 62 - 2.3.5 SDS-PAGE and western blot analysis - 64 - 2.3.6 Electron microscopy - 64 - 2.3.7 Transient transformation assay in Arabidopsis seedlings - 65 - 2.3.8 Potato tumor assay - 66 - 2.3.9 RNA extraction and quantitative PCR - 67 - 2.4 RESULTS - 68 - 2.4.1 VirB2 family proteins comprise variable N-terminal signal peptides and conserved C-terminal processing products - 68 - 2.4.2 Identification of domains and amino acid residues critical for VirB2 stability, processing, and extracellular VirB2 production - 68 - 2.4.3 Identification of VirB2 variants uncoupling virulence and T-pilus biogenesis phenotypes - 70 - 2.4.4 T-pilus-/Vir+ uncoupling mutants show highly attenuated transient transformation efficiency in Arabidopsis seedlings - 72 - 2.4.5 Transient transformation assays of Arabidopsis seedlings with wounded cotyledons - 73 - 2.4.5 Phenotypes comparison of pTiC58 G119A and G119C - 73 - 2.4.7 Measuring plant defense marker gene FRK1 expression infected with uncoupling mutants - 75 - 2.5 DISCUSSION - 76 - 2.6 FIGURES - 81 - 2.7 TABLES - 99 - 2.8 REFERENCE 113 CHAPTER 3 AGROBEST: AN EFFICIENT AGROBACTERIUM-MEDIATED TRANSIENT EXPRESSION METHOD FOR VERSATILE GENE FUNCTION ANALYSES IN ARABIDOPSIS SEEDLINGS 119 3.1 ABSTRACT 120 3.2 INTRODUCTION 121 3.3 MATERIALS AND METHODS 124 3.3.1 Bacterial growth conditions and constructions 124 3.3.2 Plant material and growth conditions 126 3.3.3 Agrobacterium infection in Arabidopsis seedlings 127 3.3.4 Infection procedures by Li et al. (FAST method) 128 3.3.5 Infection procedures by Marion et al. 128 3.3.6 Plant RNA extraction and quantitative RT-PCR 129 3.3.7 GUS staining and activity assays 130 3.3.8 Confocal microscopy 130 3.3.9 Transient expression of MYB75 and anthocyanin content assay 130 3.3.10 Transient expression of GI::LUC2 and bioluminescence measurement 131 3.3.11 Luciferase activity assay 132 3.4 RESULTS 133 3.4.1 Cotyledons of young Arabidopsis EF-TU receptor mutant is highly susceptible to Agrobacterium-mediated transient transformation 133 3.4.2 Buffered medium at pH 5.5 with AB salts is critical for high transient expression efficiency 134 3.4.3 Disarmed Agrobacterium strain C58C1(pTiB6S3ΔT)H enables highly efficient AGROBEST-mediated transient expression in Col-0 seedlings 135 3.4.4 AGROBEST achieves higher transient expression efficiency than existing methods in both efr-1 and Col-0 seedlings 137 3.4.5 Impact of seedling age and infection time on transient expression efficiency of AGROBEST in efr-1 seedlings 138 3.4.6 Widespread transient transformation events in different organs and cell types 139 3.4.7 Studies of protein subcellular localization and protein–protein interactions 140 3.4.8 AGROBEST for the expression analysis of a circadian clock reporter gene 141 3.4.9 AGROBEST for functional assays of transcription factor MYB75 142 3.4.10 Measuring plant defense marker gene FRK1 expression with ABM200 and AGROBEST methods 143 3.4.10 Analysis of VirB2 protein levels of A. tumefaciens cells cultured in ABM200 and AGROBEST methods 144 3.5 DISCUSSION 145 3.5.1 AGROBEST enables high transient transformation and expression efficiency in intact Arabidopsis young seedlings 145 3.5.2 Widespread and differential transient transformation events in different organs and cell types 147 3.5.3 Key factors for high transient transformation/expression efficiency 148 3.6 FIGURES 153 3.7 TABLES 171 3.8 REFERENCE 181 CHAPTER 4 CONCLUSION REMARKS AND FUTURE PROSPECTS 1875224529 bytesapplication/pdf論文公開時間：2014/08/08論文使用權限：同意有償授權(權利金給回饋學校)農桿菌VirB2毒性T線毛阿拉伯芥短暫表現基因表現功能性分析thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/263039/1/ntu-103-D96633001-1.pdf