전기 영동에 의한 Leishmania major의 단백질 성분 분석

학위논문(석사)--서울대학교 대학원 :의학과 기생충학전공,1995.Maste

Flowcytometric quantification of toxoplasma gondii tachyzoite infection in mice and in vitro lymphocyte cultures

학위논문(박사)--서울대학교 대학원 :의학과 기생충학전공,2001.Docto

비만이 유도된 생쥐에서 유산균의 경구투여가 미치는 항 비만효과와 장내 세균총에 미치는 영향

학위논문(석사) --서울대학교 대학원 :생명과학부,2008.2.Maste

기주 면역을 조절하는 벼 도열병균의 핵 이펙터 기능 구명

학위논문 (박사) -- 서울대학교 대학원 : 농업생명과학대학 농생명공학부, 2021. 2. 이용환.Plant pathogens threat human being for devastating crop loss in worldwide. Two-layered plant immune systems, PAMP- and effector-triggered immunity, eliminate most of invaders. Despite of robust immune systems of plants, compatible pathogen effectors neutralize hostile environment during infection via versatile strategies. Spatial compartments emerged during the plant infection classify the effectors such as apoplastic effector and cytoplasmic effector. Translocated cytoplasmic effectors move to specific organelles and modify immune responses. A group of effectors located in host nuclei are classified as nuclear effector, facilitating efficient colonization of plant tissues. Numerous nuclear effectors are discovered in bacterial and oomycete pathogens, and some of them target the same immune responses in terms of biological processes such as transcriptional regulation, hormonal signaling, programmed cell death. However, functional mechanism of fungal nuclear effectors remains to be revealed. Magnaporthe oryze, rice blast pathogen, causes serious yield loss for rice production. Rice-M. oryzae pathosystem is used for model of plant-microbe interaction owing to plenty of genome/transcriptome information and robustness of experimental system. M. oryzae penetrates rice cell wall using appressoria, and invaginates plant plasma membrane using invasive hyphae. Apoplastic effectors are secreted into extracellular matrix, interfering host defense responses. Cytoplasmic effectors are translocated via a specialized secretory structure, biotrophic interfacial complex (BIC), and moved to cellular organelles. In this study, two nuclear effectors of rice blast fungus named as MoHTR1 and MoHTR2 are characterized. Both nuclear effectors are translocated via BIC, and transferred into the nuclei of initially penetrated and surrounding cells. Theses effectors bind effector binding elements in target gene promoters and reprogram the expression of immunity-associated genes in rice. Transgenic rice expressing each MoHTR showed ambivalent response to pathogens with different lifestyles: increased susceptibility to M. oryzae and Xanthomonas oryzae pv. oryzae, hemibiotrophic pathogens, but enhanced resistance to Cochliobolus miyabeanus, a necrotrophic pathogen. The opposite effect of the transcriptional reprogramming by these effectors on defense against hemibiotrophic pathogen vs. necrotrophic pathogens, a phenomenon defined as ambivalent immunity, suggest that they target genes and processes involved in defense against all types of pathogens, not just M. oryzae. Findings in this study help understanding functional role of DNA-targeting nuclear effectors in fungal pathogen, and solve the question underlying how pathogens manipulate plant immunity.CHAPTER I. Nuclear effectors of plant pathogens Crop plants provides essential foods to mankind, but numerous plant pathogens destroy substantial amount of the products worldwide. Among the microbial pathogens, bacteria, oomycete, and fungi are major causal agent. They invade different strategies according to their lifestyle such as biotrophy, necrotrophy, and hemibiotrophy. Host plants protect themselves via two-layered immune systems. PAMP- and effector-triggered immunity efficiently block the offense of the pathogens, but compatible pathogens evade the immune systems using effectors. Effectors, molecular weapons of pathogens, are secreted and change the infection environment. According to the effector localization, they classified as apoplastic effectors and cytoplasmic effectors. Some effectors are transferred to host nuclei, essential organelle for immunity regulation, interrupting host defense responses. This type of effectors is called nuclear effector, and several effectors are discovered in the bacteria, oomycete, and fungi. In this review, nuclear effectors that have been discovered in the plant microbial pathogens are described. How these effectors modulate plant immunity is also highlighted in terms of biological processes and molecular functions.CHAPTER II. Two nuclear effectors of the rice blast fungus modulate host immunity via transcriptional reprogramming Pathogens utilize multiple types of effectors to modulate plant immunity. Although many apoplastic and cytoplasmic effectors have been reported, nuclear effectors have not been well characterized in fungal pathogens. Here, two nuclear effectors are characterized in the rice blast pathogen Magnaporthe oryzae. Both nuclear effectors are secreted via the biotrophic interfacial complex, translocated into the nuclei of initially penetrated and surrounding cells, and reprogram the expression of immunity-associated genes by binding on effector binding elements in rice. Their expression in transgenic rice caused ambivalent immunity: increased susceptibility to M. oryzae and Xanthomonas oryzae pv. oryzae, hemibiotrophic pathogens, but enhanced resistance to Cochliobolus miyabeanus, a necrotrophic pathogen. Findings in this study help remedy a significant knowledge deficiency in the mechanism of M. oryzae-rice interactions and underscore how effector-mediated manipulation of plant immunity by one pathogen may also affect the disease severity by other pathogens.식물 병원체는 전세계 곡물 생산에 큰 손실을 일으켜 식량 문제를 일으킨다. 식물의 면역체계는 대부분의 외부 침입을 막아내지만 병원체는 다양한 종류의 이펙터(effector)를 활용하여 기주의 방어반응을 무력화한다. 병원체가 식물에 접촉한 후 식물침입이 이루어지는 과정에서 공간적 분리가 일어나는데, 이러한 공간적 분리에 따라 병원체 이펙터는 세포 간극 이펙터(apoplastic effector)와 세포질 이펙터(cytoplasmic effector)로 나누어진다. 식물 침입과정에서 기주의 핵으로 이동하여 작동하는 이펙터를 핵 이펙터(nuclear effector)로 분류하며, 병원체가 병 발생환경을 효과적으로 조작하는데 큰 영향을 준다. 핵 이펙터는 주로 식물병원성 세균과 난균에서 기능이 분석되었으며 공통적으로 방어 유전자 발현, 호르몬 신호전달체계, 세포사멸 등을 조절하는 것으로 알려져 있다. 하지만 식물병원성 곰팡이의 핵 이펙터에 대한 기능분석은 상대적으로 부족한 상황이다. 본 연구에서는 벼 수확량에 큰 손실을 일으키는 벼 도열병균 (Magnaporthe oryzae)의 핵 이펙터 기능을 분석하였다. 벼 도열병균의 침입과정을 살펴보면, 포자가 벼 표면에 부착, 발아한 후 부착기를 통해 물리적으로 식물 세포를 침입한다. 침입 후 벼 도열병균은 기주 면역을 조절하기 위해 이펙터를 분비하는데, 세포 간극 이펙터는 세포 외 기질(extracellular matrix)로 분비되어 기주 방어반응을 방해한다. 세포질 이펙터는 활물기생 계면 복합체(biotrophic interfacial complex)를 통해 기주 세포 내부로 이동하며 특정 소기관에서 작동한다. 벼 도열병균의 핵 이펙터로 동정된 유전자들 중 MoHTR1과 MoHTR2는 침입균사로부터 분비된 후 식물 세포 내부로 이동하고, 핵에 도달한다. 그리고 이펙터 결합 인자(effector binding element)를 지니고 있는 벼 유전자의 프로모터(promoter)에 결합하여 목표 유전자 발현을 억제하고, 간접적인 효과로 식물 면역 유전자의 발현을 재 프로그래밍한다. MoHTR1과 MoHTR2를 발현하도록 설계한 유전자 도입 식물체는 반활물기생 병원체인 벼 도열병균과 벼 흰잎마름병균(Xanthomonas oryzae pv. oryzae)에 대한 감수성이 증가하며, 반대로 사물기생 병원체인 벼 깨씨무늬병균(Cochliobolus miyabeanus)에 대한 저항성이 증가한다. 병원체의 생활사에 따라 다르게 나타나는 병 저항성은 양가 면역의 한 사례로 볼 수 있으며, MoHTR의 목표 유전자들이 벼 도열병 저항성에 관여할 뿐만 아니라 다른 병원체의 저항성에도 관여할 수 있다는 것을 나타낸다. 본 연구 결과는 벼와 벼 도열병균 상호 작용 기작에 대한 새로운 정보를 제공하며, 병원체 핵 이펙터에 의한 식물 면역 조절이 다른 병원체에 대한 저항성에 어떻게 영향을 미칠 수 있는지를 제시한다.CHAPTER I. Nuclear effectors of plant pathogens 1 ABSTRACT 2 INTRODUCTION 3 I. Nuclear effectors of plant pathogenic bacteria 7 II. Nuclear effectors of plant pathogenic oomycetes 14 III. Nuclear effectors of plant pathogenic nematodes 15 IV. Nuclear effectors of plant pathogenic fungi 16 V. Multiple nuclear effectors target the same biological processes 18 VI. Molecular mechanisms of nuclear effectors 20 VII. Effects of nuclear effectors on multiple microbe interactions 21 PERSPECTIVES 23 LITERATURE CITED 25 CHAPTER II. Two nuclear effectors of the rice blast fungus modulate host immunity via transcriptional reprogramming 45 ABSTRACT 46 INTRODUCTION 47 MATERIALS AND METHODS 50 I. Genome mining to identify nuclear effector candidates and identification of their target genes in rice 50 II. Assays for subcellular location of MoHTR proteins 51 III. Protein binding microarray to identify MoHTR-binding DNA sequences 52 IV. Validation of MoHTR-DNA interaction in vivo 53 V. Transcriptional activity assay in rice protoplasts 55 VI. Genetic manipulation of M. oryzae 56 VII. Production of transgenic rice plants expressing each MoHTR gene without the signal peptide 57 VIII. Gene expression analysis via quantitative RT-PCR and RNA-Seq 58 IX. Growth and developmental characteristics of MoHTR mutants 59 X. Infection assays for fungal and bacterial pathogens 60 RESULTS 66 I. Identification of nuclear effectors of M. oryzae 66 II. Expression patterns of MoHTRs, their distribution, and structural features 70 III. In planta localization of MoHTR1 and MoHTR2 77 IV. Identification of effector binding elements (EBEs) for MoHTR1 and MoHTR2 84 V. Identification of the rice genes targeted by MoHTR1 and MoHTR2 91 VI. MoHTR1 and MoHTR2 regulate transcription in rice protoplasts 110 VII. MoHTR1 and MoHTR2 reprogram the expression of many immunity-associated genes in rice 114 VIII. Effect of MoHTR deletion on fungal development and virulence 124 IX. Expression of MoHTR1 and MoHTR2 in rice affects susceptibility to multiple pathogens 129 DISCUSSION 132 LITERATURE CITED 139 ABSTRACT IN KOREAN 149Docto

Digital Twin for Prediction of the Steady State Temperature Field of a Natural Gas Boiler

In this study, a simulation-based digital twin is developed for large-scale industrial natural gas boiler. The digital twin can predict temperature distribution under unexplored operating conditions by a regression model and a limited number of sensor data. The former is generally known as a surrogate model and the latter is known as sparse reconstruction. Proper orthogonal decomposition method is employed together with a kriging regression model for the surrogate model and gappy proper orthogonal decomposition for sparse reconstruction. Three-dimensional simulation data of the natural gas boiler are collected in the parameter space, which consists of excess air rate and swirl vane angle of the device. The surrogate model provides prediction performance within 5% least squared error. With sparse reconstruction, more than 32 measuring points provides prediction performance within 10% least squared error. The results show that the proper orthogonal decomposition has high potential as an analysis method for digital twin and prediction of reacting scalars in industrial combustion devices.22Nkc