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Kafe 바로가기주관연구기관 | 고려대학교 Korea University |
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보고서유형 | 최종보고서 |
발행국가 | 대한민국 |
언어 | 한국어 |
발행년월 | 2016-01 |
과제시작연도 | 2014 |
주관부처 | 농촌진흥청 Rural Development Administration(RDA) |
등록번호 | TRKO201600003060 |
과제고유번호 | 1395039547 |
사업명 | 국책기술개발 |
DB 구축일자 | 2016-06-25 |
DOI | https://doi.org/10.23000/TRKO201600003060 |
Ⅳ. 연구개발결과
○ 가지과 작물 면역 관련 신호전달 인자의 발굴 및 우량인자 선발을 위해Genomics 및 생물정보학 분석 방법을 통해 전사 인자로 CaWRKYb, CaWRKYd, CaWRKYe 유전자, 병 저항성 신호전달인자로 MAP kinase인 CaMK1, CaMK2 유전자, 바이러스 저항성 인식에 관여하는 인자로 CaRLK 유전자를 확보하고 이들의 가지과 병 저항성에서의 역할과 기능을 규명하였다.
○ 식물면역 관련 식물 방어기능 유전자, 단백질 및 유전자원 확보 및 기능연구를 통한 방어조절 mechanism 규명
Ⅳ. 연구개발결과
○ 가지과 작물 면역 관련 신호전달 인자의 발굴 및 우량인자 선발을 위해Genomics 및 생물정보학 분석 방법을 통해 전사 인자로 CaWRKYb, CaWRKYd, CaWRKYe 유전자, 병 저항성 신호전달인자로 MAP kinase인 CaMK1, CaMK2 유전자, 바이러스 저항성 인식에 관여하는 인자로 CaRLK 유전자를 확보하고 이들의 가지과 병 저항성에서의 역할과 기능을 규명하였다.
○ 식물면역 관련 식물 방어기능 유전자, 단백질 및 유전자원 확보 및 기능연구를 통한 방어조절 mechanism 규명 연구를 위해 CaRLK의 병 저항성 발현에서의 역할, RNA 결합 단백질인 APUM5의 바이러스 직접 결합 및 분해, 다수의 고추 유래 WRKY 전사인자의 target 유전자 결합 및 CaMK1과 CaMK2를 통한 순차전 인산화 과정의 역할을 규명하였다.
○ 식물의 병저항성에 대한 심층적 이해 및 기작 규명으로 타식물체 및 병원균에 대한 기술확산 효과와 병 저항성에 대한 전반적 이해를 증진시켰고, 세계 최고수준의 그룹, 국내 최고수준의 연구진과의 공동연구를 통하여 병저항성 기작을 이해함으로써 그 결과를 세계최고의 학술지에 발표하고 동시에, 진흥청 연구진의 연구역량을 배양시키는 효과를 유발하였다.
○ 새로운 식물면역 관련 식물방어 신호전달 인자의 발굴 및 우량인자 선발을 위해 Proteomics, Genomics 및 생물정보학 분석 방법을 통해 단백질의 열안정성조절 관련 인자,분자샤페론, 병저항성, 소포체스트레스 반응, 환경스트레스 반응 관련 인자들을 발굴 조사하였다. 식물병저항성 관련 우량인자로 AtPLT5와 AtPLT6를 선발하였다.
○ 선별된 식물방어신호전달인자의 세포 내 생화학적, 생리적 기능을 샤페론 활성연구, 레독스조절 연구, 소포체스트레스반응 연구, 프로테아좀체계 연구, 세포 사멸체계 연구 등을 통해 연구하여 규명하였다. 우량인자로 선발된 AtPLT5와 AtPLT6의 구조적 특성, 열안정성, 샤페론 활성, 레독스 특성 등의 생화학적 특성과 유전자발현분석을 통해 AtPLT5와 AtPLT6의 유전자가 SA, JA 등의 병저항성 관련 식물호르몬이나 병원균 감염에 의해 증가됨을 확인하였다. 이를 통해 선발된 AtPLT5와 AtPLT6가 식물방어신호전달인자들로서 레독스조절 및 샤페론 활성이 있음을 확인하였다.
○ Arabidopsis의 식물 면역반응에서의 chromatin remodeling 분석과 ChIP-seq analysis 통해 히스톤 탈메칠효소 (LDL1/ENI1, LDL2/ENL1)와 Nonsense-mediated mRNA decay(NMD)에 의한 돌연변이체 들을 통해 이 들 유전자의 식물 면역반응 동안의 역할을 분석하였다.
○ 병·해충 저항성을 부여하는 식물 면역반응에서 생리활성물질 분리 및 역할 분석을 위해 ALD1의 식물 저항성 반응에서의 역할, ALD1 조절 대사물질 분석 및 기초저항성과 effector triggered-immunity에서의 역할을 분석하였다.
○ 유용유전자원을 이용한 콩/벼 형질전환 식물체 제조와 특성 분석을 위해 야생벼유래 PATHOGENESIS-RELATED1 유전자, 벼 ALD1 유전자, 애기장대 A vacuolar calcium- binding protein-like gene, 애기장대 leucine-rich repeat receptor-like kinase, immune-defective3 돌연변이체 등에서 병 저항성 및 감수성 조절 기작을 규명하고 형질전환체를 제작하여 분석하였다.
○ 벼흰잎마름병저항성 신호전달경로규명을 위해 PR10a promoter 조절 전사 인자 선발, OsWRKY6 기능 분석 및 벼 면역에서 ubiquitination의 역할을 규명하였다.
○ 벼흰잎마름병균의 AVIRULENCE 규명을 위해 TAL effector의 분리 및 target gene분석 및 애기장대와 무름병과의 상호작용 연구를 통해 RTP1/ W75/RPE1과 STP1/W55 유전자들의 기능을 분석하였다.
○ RRS1에 의한 AvrRps4와 PopP2의 인식 기작을 규명하기 위해 AvrRps4의 단백질 3차구조의 규명, PopP2에 의한 RRS1의 acetyl화 기작 및 RRS1에 의한 자가면역 반응의 유전적 기작을 규명하였다.
○ 면역 수용체의 구체적 인식 현상을 규명하기 위한 연구에서 병원균의 effector인 AvrRps4와 PopP2는 RRS1의 WRKY도메인과 상호작용을 하고, PopP2는 그 부분을 아세틸화 하여 DNA결합작용을 억제함으로써 면역 반응을 방해하는 것을 입증하였다.
The title of this project is “Exploitation of Plant Immune Genes for Crop Improvement”. In nature, plants are exposed to many kinds of microorganisms. However, only some microorganisms can successfully infect the plants. That is, plant disease is the exception rather than the rule; the majority of p
The title of this project is “Exploitation of Plant Immune Genes for Crop Improvement”. In nature, plants are exposed to many kinds of microorganisms. However, only some microorganisms can successfully infect the plants. That is, plant disease is the exception rather than the rule; the majority of plants are resistant to infection by the majority of microbes. The whole purpose of this project is dissection and understanding of plant disease resistance mechanism. With this in mind, the renowned research groups were teamed for superb publication, one of the important plans of Woojangchun Project.
< 1세부과제: 고려대 백경희: 가지과 작물의 병저항성 인식현상 및 방어신호전달 기작 규명연구 >
The detailed assignment project 1; Pathogen-induced programmed cell death (PCD) is one of the crucial events of disease resistance response. In this project, the factors involved in immune response of Solaneceae, especially hot pepper plants against Tobaccomosaic virus (TMV) infection were studied. (1) One of the Capsicum annuum receptor-like kinases (CaRLKs), CaLecRK-S.5, was studied. By using virus-induced gene silencing(VIGS), when the gene was knocked-down, the hypersensitive response (HR) was diminished. This gene also seems to be involved in HR induced by INF1, one of the elicitins of Phytophthora infestans. Transient over-expression of the CaLecRK-S.5 promoted more rapid and stronger INF1-mediated HR, ROS production, MAPK activation, marker genes expression compared to empty vector control. Based on these results, we suggest that the CaLecRK-S.5 functions as a positive regulator in plant immunity. (2) The hot pepper heat shock transcription factors (CaHsfs) and others associated with disruption of the immune response to TMV at ambient high temperature were investigated. Plants should balance growth and energy-requiring defense system. At ambient high temperature, hot pepper plants exhibit reduced TMV resistance including much reduced HR lesion numbers. However, it remains largely unknown how plants lose defense mechanism at elevated temperature. At first CaHsfs were screened through VIGS, and CaHsf012 and CaHsf023 showed reduced HR phenotype and PR gene expression upon TMV-P0 treatment at 26°C for 72 h when the expression of genes were down-regulated in hot pepper plants.These results suggest that some Hsfs could be involved with disruption of the hot pepper immune response to TMV-P0 at high temperature. Furthermore, RNA sequencing was carried out at several different conditions and systemic and massive factors can be searched and analyzed in terms of innate immune response.
< 1협동과제: 경상대 이상열: 식물면역 반응의 이해와 신호전달 과정 연구 >
Worldwide, major economic losses for farmers are caused by plant diseases. Control of plant diseases is crucial to the reliable production of crop, and it is reasonably successful for most crops. Development of good diseases resistance plants is essential to achieve the aim of disease control. Typically, diseases resistance plants were developed by breeding technic; however, recent advance in science made apply molecular biology efficiently to confer plants new traits of improving their immunity. In this research, we performed a systemic approach to identify novel components involved in plant immunity, and selected AtPLT5 among the candidates for the deeper study. The deduced amino acid sequence suggested that AtPLT5 is an Arabidopsis oxidoreductase which has been poorly studied, and the AtPLT5 gene was cloned by polymerase chain reaction (PCR)-based amplification and the in vitro biochemical properties and the in vivo physiological roles of the protein were intensively studied in this research. For the biochemical studies, the recombinant protein of AtPLT5 was bacterially produced and homogeneously purified. According to our results from enzyme assays, oxidoreductase function of the recombinant AtPLT5 was confirmed by assessing both disulfide reductase and oxidase activities. Interestingly,AtPLT5 also showed protein disulfide isomerase activity by correcting disulfide bonds of misfolded substrates. Both oxidoreductase and protein disulfide isomerase activities of AtPLT5 were eliminated by replacing the six Cys residues at the active site with Ser residues simultaneously. Our Real Time (RT)-PCR results showed that the gene expression of AtPLT5 is specifically induced by treatments of bacterial or fungal pathogens to the wild type plants, implying possible roles of AtPLT5 in the plant defense system. Consequently, when Pseudomonas syringae, a bacterial pathogen were treated to the wild type (Col-0), mutant (plt5) and overexpressing (AtPLT5OE) Arabidopsis plants, the plt5 plants were more resistant while the AtPLT5OE plants were more susceptible against the pathogen attack comparing to the wild type plants. Accordingly, accumulation of the reactive oxygen species (ROS) such as superoxide anion and hydrogen peroxide was higher in the plt5 plants whereas less in the AtPLT5OE plants upon the pathogen treatment comparing to the wild type plants. Also, the plt5 plants showed faster hypersensitive response (HR) than the wild type plants upon the pathogen attack. Phenotypes of the plt5 plants were balanced by introduction of the wild type AtPLT5 gene under a constitutive expression promoter; however introduction of AtPLT5C72,75,339,342,403,406S couldn’t complement the mutant phenotype. Conclusively, our data suggest that AtPLT5 negatively regulates the plant defense system by delaying the ROS generation during the pathogen invasion and the enzymatic function of the protein as a sulfhydryl oxidase is essential for its in vivo physiological roles.
< 2협동과제: 동아대 정호원: 병·해충 저항성 관련 식물면역 조절 생리활성물질 및 전신획득저항성 조절 유전자 연구 >
Tremendous alternation of transcriptome was generally observed in the infected plants by pathogens. In order to unveil the molecular mechanisms leading to the transcriptional reprogramming during immune response, we are studying the modification of histone tails correlated with gene expression. For the last few years, we carried out reverse genetic screening to identify ENHANCED-IMMUNE (ENI ) and/or IMMUNE-DEFECTIVE(IMD) genes involved in plant immune responses against the infection with Pseudomonas syringae. The studies enabled us to isolate enhanced-immune1 (eni1) mutants showing disease resistance response against the infection with Pseudomonas syringae. ENI1 gene encodes a histone demethylase, which has three different homologs in Arabidopsis. As a result, we clearly confirmed that the eni1enl1(eni1-like1) double mutant was hyper-resistant against virulent P. syringae infection, compared with wild-type and each single mutant. Interestingly, primary immunization with an avirulent pathogen in local leaves could not enhance systemic resistance in eni1enl1 mutants, in which bacterial growth was already dramatically inhibited against secondary infection. RNA-seq analysis revealed that expression of hundreds of genes was robustly changed in the eni1enl1 mutants, as compared with those in wild-type plants. Taken together, these results strongly suggest that the two histone demethylases have a specific role in plant immune response.
Arabidopsis ALD1 is an aminotransferase that participates in several aspects of defense regulation via the production of signaling metabolites. Previous studies implicated ALD1 in a regulatory relationship with salicylic acid (SA), a major small molecule regulator produced via the ICS1/SID2-mediated biosynthesis pathway and transduced by NPR1. Defense regulatory circuitries can vary depending on the infecting pathogen and the repertoire of plant receptors. To probe this variation in the context of ALD1, phenotypes of single and double mutants between ald1 and sid2 or npr1 were compared after infection with different Pseudomonas syringae strains. Under the conditions that employed an attenuated P. syringae strain showed that during basal defense, ALD1 does not have a synergistic effect with NPR1 and SID2 to suppress pathogen growth, but additively activate MAPK signaling at early infection phase. In contrast, after an infection that leads to strong pathogen recognition via a cytoplasmic immune receptor, ALD1 acts additively with both SID2 and NPR1 to suppress pathogen growth. The additive effect was dependent on the recognition pathway assayed, indicating a diversity of roles for ALD1 in the defense regulatory network.
In a given environment, plants are exposed to attacks of various pathogens. When pathogens invade plants, plants activate MAMP (microbe-associated molecular pattern)-triggered immunity (PTI), such as transient activation of MAPK cascade, rapid and transient production of reactive oxygen species (ROS), reprogramming of hormone signaling, ion fluxes and so on. One of mechanisms is calcium influx. Not only is calcium ion a key messenger to regulate many growth and developmental processes, but also plays a crucial role in defense responses against biotic stresses. Here we introduce a loss of function mutant whose corresponding gene encodes a vacuolar calcium binding protein-like(VCBL) protein, showing enhanced disease susceptibility against Pseudomonas infection. The VCBL gene has three homologous genes in Arabidopsis genome. Interestingly, the vcbl mutant that we isolated via reverse genetic screening was only susceptible to Pseudomonas infection. Transcription of the VCBL gene was down-regulated by Pseudomonas infection. The vcbl mutant was deficient in accumulation of salicylic acid during infection. We also checked the level of basal defense responses, including transient activation of MPK3 and MPK6, and oxidative burst at early infection phase as well as transcriptional changes of MAMP-responsive genes in vcbl mutant during infection. Taken together we suggest a possibility that VCBL might be one of components to regulate Ca2+-dependent defense responses in Arabidopsis.
Plants are continuously attacked from plant pathogenic bacteria. Plants are immotile organisms unlike animals. Therefore plants have evolved unique immune mechanisms to protect themselves from microbial attacks. Microbe (or pathogen)-associated molecular patterns (MAMPs), which are derived from pathogens, are perceived by pattern recognition receptors (PRRs). The recognition initiates patterns-triggered immunity (PTI), such as accumulation of reactive oxygen species (ROS), transient phosphorylation of mitogen-activated protein kinase (MAPK), and callose deposition. Most of PRRs of plants are composed of an extracellular leucine-rich repeat domain (LRR), a transmembrane domain and an intracellular kinase domain. RNA-seq analysis to see transcriptional reprogramming in immune defective3 (imd3) mutant revealed that tens of genes were differentially expressed in imd3 mutant compared with those in wild-type Arabidopsis(ecotype Nössen). Among these genes, we found that expression of a gene encoding LRR-receptor like kinase (LRR-RLK) was diminished in the imd3 mutant. A dozen LRR-RLK genes were clustered in chromosome 1 of Arabidopsis genome. In order to isolate genes important for immune responses, we checked mRNA expression of the LRR-RLK genes in the infected leaves with virulent and avirulent Pseudomonas syringae.As well, we have identified a mutant showing disease susceptibility against pathogen infection. Taken together, the LRR-RLK that we identified in this study may participate in basal defense response as a key player.
Plants have many mechanisms to adapt to unfavorable environments for survival.Basically, plant immunity is initiated through recognition of pathogen-derived molecules, such as microbe-associated molecular patterns (MAMPs) and pathogen-derived effectors by plant immune receptors. The recognition activates plant’s built-in immune responses including hormone-dependent defenses, accumulation of antimicrobial compounds, and transcriptional reprogramming. Effector proteins, which were injected into plant cells from pathogen, can manipulate defense responses for successful colonization of pathogen in their host plants. Thus it has been thought that effectors are responsible for determining host range and virulence of pathogenic microorganisms. Here we introduce a pathovar-specific susceptibility of immune-defective3 (imd3) mutant against Pseudomonas syringae. Arabidopsis imd3 mutant, whose corresponding gene was still unidentified, was hyper-susceptible to the infection by P. syringae pv. tomato DC3000 (Pst DC3000), but not P. syringae pv. maculicola ES4326 carrying an empty vector (Psm DG3), as compared with wild-type Arabidopsis (ecotype Nössen). These results showed Pst DC3000-specific effectors might be causal agents for the race-specific susceptibility in imd3 mutant. Thus we are checking roles of 8 different Pst DC3000-specific effectors during pathogenesis to find out what kind of Pst DC3000-specific effectors are involved in the disease susceptibility of imd3 mutant.
During race-specific resistance, also called effector-triggered immunity (ETI), recognition of pathogen-derived effector by cytoplasmic immune receptors (resistance (R) proteins) is the forefront of the battle between plant and pathogen. Even if it has been reported that small RNAs regulated mRNA expression of the R gene in plants, we did not fully understand how R genes were transcribed in plants against pathogen infection. Here we briefly introduce role of nonsense-mediated mRNA decay (NMD) in transcriptional regulation of R gene during bacterial infection. As one of post-transcriptional events, NMD eliminates aberrant mRNAs to avoid accumulation of truncated polypeptides. RNA-seq analysis of NMD-compromised mutant plants revealed that most of TIR-NBS-LRR(TNL)-type R genes were NMD-targets. Thus the NMD-compromised mutant plants exhibited aberrant mRNA accumulation of R genes that should have been degraded by NMD machinery. Interestingly the NMD-compromised mutants showed hypersensitive response (HR)-like cell death in infected leaves with virulent Pseudomonas syringae. Taken together we propose that transcription of R gene was post-transcriptionally regulated by NMD during infection in Arabidopsis.
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