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Characterization of virulence and avirulence activity of Ralstonia solanacearum effector RipB in Nicotiana benthamiana : Nicotiana benthamiana에서의 Ralstonia solanacearum 이펙터 RipB의 병원성 및 비병원성 특성 연구

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dc.contributor.advisorKang Byoung Cheorl-
dc.contributor.advisorCecile Segonzac-
dc.contributor.advisorHuh Jin Hoe-
dc.contributor.authorLlanes Melgoza Laura Cristina-
dc.date.accessioned2021-11-30T06:26:29Z-
dc.date.available2021-11-30T06:26:29Z-
dc.date.issued2021-02-
dc.identifier.other000000164669-
dc.identifier.urihttps://hdl.handle.net/10371/176501-
dc.identifier.urihttps://dcollection.snu.ac.kr/common/orgView/000000164669ko_KR
dc.description학위논문 (석사) -- 서울대학교 대학원 : 농업생명과학대학 식물생산과학부(원예과학전공), 2021. 2. Kang Byoung Cheorl-
dc.descriptionCecile Segonzac-
dc.descriptionHuh Jin Hoe.-
dc.description.abstract식물은 내재된 면역체계를 가지고 병원균을 감지하거나 대응한다. Pattern
recognition receptor 에 의한 pathogen-associated molecular pattern 의 인식은
PAMP-triggered immunity(PTI)를 유발한다. 해당 면역반응은 빠르고 강력한
활성산소종의 생성과 다양한 유전자의 발현변화를 포함한다. 병원균은
식물의 이러한 면역반응을 피하기 위해 여러 가지 병원성 인자들을 생산한다.
대표적으로 type III secretion system 을 이용하여 세포 내로 직접 주입되는
이펙터 단백질(type-III effector, T3E)이 있다. 이 단백질은 식물에서 effectortriggered susceptibility 를 야기한다. 하지만 식물이 가진 저항성 단백질은 특정
이펙터를 인식하여 effector-triggered immunity 반응을 가져온다. 이 강한 면역
반응은 세포 사멸의 형태로 나타나는 과민성 반응을 일으킨다. Ralstonia
solanacearum(Rso)은 가지과 식물에 심각한 풋마름병을 일으키는 병원균이다.
Rso 는 Ralstonia-injected protein(Rip)이라는 다양한 이펙터들을 식물 내로
주입하며 감염을 촉진한다. 본 연구에서는 선행연구에서 클로닝한 50 개의
이펙터 중 세포사멸을 일으키지 않는 36 개의 이펙터를 선별하여 식물에 PTI
signaling 에 미치는 영향을 연구하기 위하여 담배(Nicotiana benthamiana)에
일시적으로 발현(transient expression)시켰다. 이 이펙터들 중 일부는 flg22
단백질이 유도하는 활성산소종의 생산을 저해하는 것으로 나타났다.
흥미롭게도 이펙터 중 하나인 RipB 의 4 개의 allele 들 중 3 개는 활성산소종의
생성을 거의 완전히 억제하였다. 유전자 서열분석과 키메라를 이용하여 RipB 의 활성산소종 생성 억제가 이 3 가지의 RipB allele 들의 C-terminal
domain 과 관련이 있음을 확인하였다. RipB 는 다른 식물 세균인 Xantomonas
와 Pseudomonas 의 이펙터인 XopQ 와 HopQ1 의 homolog 단백질이다. 이 두
이펙터를 인식하는 저항성 단백질인 Roq1 의 발현이 억제된 식물에서는 RipB
allele 들의 C-terminal 부분이 인식이 되지 않았지만, 여전히 활성산소종의
생성이 억제되었다. 이러한 결과들을 종합해 볼 때, RipB 는 식물의
면역반응을 조절할 수 있는 것으로 보이며 그들이 어떤 경로를 통해
기능하는지를 밝히기 위한 추가적인 연구가 필요할 것이다.
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dc.description.abstractPlants rely on innate immunity to perceive and ward off
potential pathogens. The perception of pathogen-associated molecular
patterns (PAMPs) by pattern recognition receptors leads to PAMPtriggered immunity (PTI) and involves a rapid and strong production
of reactive oxygen species (ROS) and massive gene expression change.
To circumvent PTI, bacteria produce different classes of virulence
factors, in particular type-III secreted effectors (T3E) which are
injected into the host cell, thereby providing effector-triggered
susceptibility. However, plants possess resistance proteins (R) that
recognize the T3Es and initiate the effector-triggered immunity. This
triggers a strong defense response which can lead to localized cell
death termed hypersensitive response. Ralstonia solanacearum (Rso),
the causal agent of bacterial wilt in solanaceous crops, is one of the
most destructive plant bacterial pathogens. Rso injects an array of
effector proteins called Rips (Ralstonia-injected proteins) via its type
III secretion system to promote infection. However, the function of
these effectors in plant cells remains mostly unknown.
In this study, I selected 36 Rips from 50 previously cloned that
are not causing cell death when expressed in Nicotiana benthamiana to
test their effect on PTI signaling. Transient expression of the selected
Rips in N. benthamiana shows that several Rips could disturb ROS
production triggered by the bacterial PAMP flg22. Interestingly among
the four RipB alleles present in this screen, three could almost
completely suppress flg22-triggered ROS, whereas one could not.
Sequence comparison and design of chimeric constructs revealed that
the differential effect of RipB on flg22-triggered ROS production
might be due to the C-terminal domain, present in the three RipB
alleles suppressing flg22-triggered ROS. RipB shares close homology
with the Xantomonas and Pseudomonas effector protein XopQ and
HopQ1, respectively, which are recognized by the plant resistance
protein Roq1. In Roq1 silenced plants, the RipB allele with the Cterminal region could not be recognized although it could still suppress
flg22-triggered ROS production. Together these results give a glimpse
of how RipB alleles are modulating the immune responses in N.
benthamiana, however, more studies are needed to understand the
pathway it follows.
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dc.description.tableofcontentsABSTRACT·········································i
CONTENTS ········································ iv
LIST OF TABLES ···································vi
LIST OF FIGURES ··································vii
LIST OF ABBREVIATIONS ·························· ix
INTRODUCTION ··································· 1
MATERIAL AND METHODS··························6
Plant materials ··································· 6
Agrobacterium tumeraciences-mediated transient expression
assays (agroinfiltration) ··························· 6
Measurement of ROS production ····················7
Semiquantitative RT-PCR/ quantitative PCR ··········· 8
Western blotting ································· 9
Virus-induced Gene Silencing (VIGS) ··············· 10
Cell death quantification ·························· 10
RESULTS ········································ 13
Screening for Ralstonia solanacearum effectors that
modulate plant PTI responses ·····················13
RipB alleles modulate flg22 but not chitin-triggered ROS
burst ·········································18
RipB alleles are differentially recognized in N.
benthamiana···································19
Determination of the RipB domain required for flg22-
induced ROS burst suppression and/or recognition in N.
benthamiana···································26
Roq1 silencing in N.benthamiana to assess in Roq1
recognition of RipB effectors is the reason for ROS burst
suppression····································33
DISCUSSION······································40
REFERENCES·····································46
ABSTRACT IN KOREAN···························65
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dc.format.extentx, 66-
dc.language.isoeng-
dc.publisher서울대학교 대학원-
dc.subjectreactive oxygen species-
dc.subjectRalstonia solanacearum-
dc.subjectNicotiana benthamiana-
dc.subjecteffectors-
dc.subjectPTI-
dc.subjectETI-
dc.subjectRipB-
dc.subjectPlant immunity-
dc.subject.ddc635-
dc.titleCharacterization of virulence and avirulence activity of Ralstonia solanacearum effector RipB in Nicotiana benthamiana-
dc.title.alternativeNicotiana benthamiana에서의 Ralstonia solanacearum 이펙터 RipB의 병원성 및 비병원성 특성 연구-
dc.typeThesis-
dc.typeDissertation-
dc.contributor.AlternativeAuthor라우라-
dc.contributor.department농업생명과학대학 식물생산과학부(원예과학전공)-
dc.description.degreeMaster-
dc.date.awarded2021-02-
dc.identifier.uciI804:11032-000000164669-
dc.identifier.holdings000000000044▲000000000050▲000000164669▲-
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