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Molecular and Physiological Characterization of the Effect of Ralstonia solanacearum NLS-containing Type III Effectors on Immunity and Development in Nicotiana benthamiana and Arabidopsis thaliana
Get PDF학위논문 (석사)-- 서울대학교 대학원 : 농업생명과학대학 식물생산과학부, 2019. 2. Helene, Segonzac Cecile Marie.Ralstonia solanacearum is a soil-borne phytopathogen that causes lethal bacterial wilt in a wide range of food crops. Pathogenicity of R. solanacearum is mediated by the type III secretion system (T3SS) that injects type III effectors (T3Es) directly into host cells. T3Es are known to modulate not only plant immunity, but also various host cell processes for the bacterial infection. However, because of the complexity of R. solanacearum T3E repertoire, only few of them have been characterized for their mode of action. Here, I used heterologous expression of individual nuclear localization sequence (NLS)-containing R. solanacearum T3Es in Nicotiana benthamiana and Arabidopsis thaliana to screen the effectors that have effect on plant immunity and development. Transient expression of the effectors in N. benthamiana revealed several effectors that induce cell death. Also, some of the candidate effectors disturbed reactive oxygen species (ROS) production and the subsequent induction of defense gene expression, which are early pattern-triggered immunity (PTI) responses. For stable expression of the effectors, effector-expressing Arabidopsis homozygous lines (EELs) were generated, and the ability of the effectors to interfere with PTI was examined. Finally, the effect of the effectors on Arabidopsis developmental processes was assessed in EELs, and one of the effectors, RipD, was shown to impair seed germination and root development. Selected effectors that target immune system and root development will be further characterized for their molecular targets and contribution to the pathogen virulence.Ralstonia solanacearum 은 토양을 통해 전염되는 병원균으로 광 범위한 식량 작물들에 풋마름병을 일으킨다. 이 병원균의 병원성 은 기주의 세포 내로 이펙터 (type III effector, T3E) 단백질을 직접 주입하는
type III secretion system (T3SS)을 매개로 한 다. T3E는 세균의 감염을 촉진하기 위해 식물의 면역 뿐만 아니 라, 다양한 기주의 세포 과정들을 조절한다. 그러나 R. solanacearum 의 T3E 구성의 복잡성 때문에, 그들이 어떻게 작용 하는 지는 아직 잘 규명되지 않았다. 본 연구에서는 이펙터들이 식물에 미치는 영향을 알아내기 위해, 핵 위치 신호 서열을 가지 는 것으로 추정되는 R. solanacearum 의 T3E들을 각각 담배( Nicotiana benthamiana )와 애기장대( Arabidopsis thaliana )에 발현시켰다. 이펙터를 담배에서 일시적으로 발현 시킴으로써 (transient expression), 일부 이 펙터들이 세포 사멸(cell death)을 유도하고 또 다른 이펙터들은 pattern-triggered immunity (PTI) 반응인 활성산소종(ROS) 생 성과 뒤 이은 방어 관련 유전자들의 발현을 억제함이 밝혀졌다. 이 펙터를 과다 발현하는 형질 전환 애기장대 식물체(EELs)를 생산하 여 이들이 PTI를 억제할 수 있는지 또한 확인되었다. 마지막으로 EELs에서 이펙터들이 애기장대의 발달에 미치는 영향을 분석한 결과, 선별된 이펙터들 중 하나인 RipD가 종자 발아와 뿌리 발달 을 방해함이 밝혀졌다. 면역 시스템과 뿌리 발달에 영향을 주는 것으로 밝혀진 이펙터들은 그들의 분자적 타겟과 병원성에 대한 기여도를 구명하기 위해 이후 더 연구될 것이다.ABSTRACT········································································· ⅰ
CONTENTS ····································································· ⅲ
LIST OF TABLES ························································· ⅵ
LIST OF FIGURES ······················································· ⅶ
LIST OF ABBREVIATIONS········································ ⅷ
INTRODUCTION ······························································· 1
MATERIALS AND METHODS········································ 8
Plant materials··································································8
Construction of Ralstonia solanacearum NLS-containing effector library··················································································8
A. tumefaciens-mediated transient expression assays···9
Ion leakage assay·····························································9
Measurement of ROS production···································9
RT-PCR···········································································10
PAMP-triggered growth inhibition assay·····················11
Root growth assay·························································11
Seed germination assay·················································11
Analysis of leaf development·······································12
RESULTS··············································································14
Sequence analysis of R. solanacearum type III effectors to identify NLS-containing effectors·····························14
Three of the selected effectors elicited cell death in N. benthamiana·································································17
RipAD, RipD, and RipAF1 disturbed flg22-induced ROS production in N. benthamiana·······································19
RipD, RipAO, and RipAD disturbed flg22-induced gene expression in N. benthamiana···········································21
Obtention of Arabidopsis transgenic lines overexpressing NLS-containing effectors··············································· 23
Preliminary characterization of PAMP-induced immune responses in EELs····························································29
Some of EELs showed reduced sensitivity to elf18-induced seedling growth inhibition···············································31
Some of EELs exhibited disturbed root elongation and lateral root development···················································34
RipD inhibited seed germination···································37
EELs exhibited impaired germination and vegetative growth on soil·································································40
DISCUSSION·······································································44
REFERENCES······································································50
ABSTRACT IN KOREAN·················································60Maste
세포 분비 경로 단백질과 Ralstonia solanacearum 이펙터 단백질의 상호작용 구명
Get PDF학위논문 (석사) -- 서울대학교 대학원 : 농업생명과학대학 농림생물자원학부, 2021. 2. Segonzac Cecile Marie Helene.Plants have innate immune responses against pathogens. Many bacterial pathogens secrete type III effectors (T3Es) to promote their growth. Thus, many T3Es target host defense-related components. Therefore, it is important to identify and characterize T3E interactors in the host plant. One bacterial pathogen, Ralstonia solanacearum (R. solanacearum) causes severe wilt in a wide range of horticultural crops. One of R. solanacearum T3E, RipAO, suppresses a defense response, namely flg22-triggered ROS production in Nicotiana benthamiana. Here, I screened Arabidopsis thaliana total cDNA library using RipAO to identify its interactors and test whether candidate interactors are required for the RipAO role to suppress flg22-ROS production. Using yeast two-hybrid screening, three candidate interactors, exocyst complex component SEC3A (SEC3A), SNF1 related protein kinase (KIN10), and Arabidopsis thaliana homolog of yeast oxidase assembly 1 (OXA1) were identified. Interestingly, SEC3A could not accumulate in the presence of RipAO. Moreover, RipAO could not or not fully suppress flg22-triggered ROS production in N. benthamiana plants silenced for SEC3A homologs. Taken together, these results suggest that RipAO targets the exocyst component SEC3A and that SEC3A is required for RipAO suppression of flg22-ROS production. Hence, this study uncovers a possible role of the vesicle trafficking machinery as the target of pathogen effectors to impair the host defense response.식물은 병원균에 대항하여 내재된 면역체계를 가지고 있다. 그래서, 많은 세균성 병원균들은 증식을 위해 이펙터(type III effectors, T3E) 단백질을 주입하며, 이펙터는 기주식물의 방어와 관련된 요소들을 타겟으로 한다. 이러한 이유로, 기주 식물에서 이펙터 단백질과 상호작용하는 단백질을 규명하는 것은 중요하다. 세균성 병원균의 하나인 R. solanacearum은 다양한 원예작물에 심각한 세균성 풋마름병을 일으킨다. R. solanacearum의 이펙터 중 하나인 RipAO는 담배 (Nicotiana benthamiana) 에서 식물의 방어 작용의 하나인 활성산소종 (reactive oxygen species) 생성을 억제한다. 본 연구에서는 애기장대(A. thaliana) cDNA 라이브러리에서 RipAO와 상호작용하는 단백질을 선별하여 이 단백질들이 RipAO의 담배의 활성산소종 생성을 억제하는 것과 연관성이 있는지 실험하였다. 효모 단백질 잡종법(yeast two-hybrid) 스크리닝 결과, 세가지의 단백질, exocyst complex component SEC3A (SEC3A), SNF1 related protein kinase (KIN10) 그리고 Arabidopsis thaliana homolog of yeast oxidase assembly 1 (OXA1)들이 RipAO와 상호작용하는 후보 단백질들로 발견되었다. 흥미롭게도, 담배에서 RipAO 존재 하에 SEC3A 단백질의 축적이 감소하는 것이 발견되었다. 또한 애기장대의 SEC3A의 homolog 단백질(NbSEC3A)들의 발현이 억제된 담배에서 RipAO가 활성산소종의 생성을 억제하지 못하는 것으로 확인 되었다. 이러한 결과들을 종합해 볼 때 RipAO는 SEC3A 단백질을 타겟으로 하며, SEC3A 단백질이 RipAO가 활성산소종을 억제하는데 필요한 요소로 밝혀졌다. 그러므로 본 연구에서는 기주 식물의 방어 작용을 억제하기 위해 막 수송 조직을 타겟으로 하는 이펙터 단백질을 규명하였다.CONTENTS
ABSTRACT i
CONTENTS iii
LIST OF TABLES vi
LIST OF FIGURES vii
LIST OF ABBREVIATIONS x
INTRODUCTION 1
MATERIAL AND METHODS 5
Plant and yeast strain culture 5
Molecular cloning 5
Yeast two-hybrid system 7
Agrobacterium tumefaciens-mediated transient expression in N. benthamiana 9
Immunoprecipitation and immunoblotting 9
Virus induced gene silencing (VIGS) assay 11
Measurement of ROS production 11
Semi-quantitative RT-PCR 11
RESULTS 15
Selection of NLS-containing type III effectors 15
Cloning, expression and activity of the three selected effectors in yeast 16
Some Arabidopsis thaliana genes can activate MEL1 gene with GAL4 DNA-binding domain 20
Yeast two-hybrid screening of A. thaliana cDNA library for RipAO interactors 23
Re-transformation of the original fished-out plasmids 27
Cloning of full-length and truncated cDNA for candidate interactors of RipAO 29
Full-length and truncated candidate genes cannot activate MEL1 gene with GAL4 DNA-activation domain 30
RipAO fused with GAL4 DNA binding-domain interacts with the full-length candidate fused with GAL4 DNA-activation domain 32
SEC3A-YFP accumulates less in the presence of RipAO-FLAG in N. benthamiana 35
Co-expression and co-IP assay of RipAO with KIN10 and OXA1 37
RipAO suppresses flg22-triggered ROS production in N. benthamiana 39
Design silencing fragment to knock-down candidate interactor homolog in N. benthamiana 41
ROS measurement in N. benthamiana silenced for SEC3A homolog and confirmation of silencing efficiency by semi-quantitative RT-PCR 45
Reactive oxygen species (ROS) measurement in N. benthamiana silenced for KIN10 homolog and confirmation of silencing efficiency by semi-quantitative RT-PCR 48
DISCUSSION 52
REFERENCES 59
ABSTRACT IN KOREAN 68Maste
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