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RISS 인기검색어
- 검색키워드
- 저자 : Chae, Jong Moon (검색결과 753 건)
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Studies in molecular interaction between 2,4-Diacetylphloroglucinol and plants
채대한 Gyeongsang National University 2020 국내석사
2,4-Diacetylphloroglucinol (DAPG)는 생물적 방제 미생물인 Pseudomonas spp.가 생산하는 많은 연구가 진행된 유명한 2차 대사산물이다. DAPG는 다양한 병원균에 대하여 광범위한 독성을 지니고 있으며, 이는 식물 병원성을 가진 세균, 곰팡이, 선충등으로 알려져 있다. DAPG는 병원균에 복합적인 세포적 경로에 공격을 하는 것으로 알려져 있으며, 이러한 특성으로 병원균이 이 항생제에 대한 저항성을 갖기는 어렵다는 것으로 알려져 있다. 이러한 특성을 가진 DAPG는 식물에게 양면의 효과를 지니는 것으로 알려져 있다. 긍정적 효과로서는 DAPG가 식물에게 전신 유도 저항성 (ISR)을 유도하여 면역 체계를 형성화 한다고 알려져 있으며, 이러한 영향으로 식물은 병원균에 대한 저항성을 얻게 된다. 이러한 연구는 npr1-1, jar1, etr1 등 호르몬에 관련한 애기장대 돌연변이체로 증명을 하였다. 하지만 호르몬은 다양한 역할을 하기에, 돌연변이체를 이용한 연구는 다른 기능의 상실로 인해서 나타나는 표현형인지 알 수 없다. 이 실험에서는 DAPG의 분해 유전자 phlG를 형질전환하여 식물체인 D16과 H2를 선발하였다. 이는 Botrytis cinerea 와 Pseudomonas syringae pv. tomato DC3000에 대하여 DAPG ISR 활성을 잃었으며, 이는 JA/ET 신호에 의존하는 방어 시스템의 유전자가 발현 기능을 잃는 것을 확인할 수 있었다. 반면에 부정적 효과로서는 고농도의 DAPG는 식물에게 독성을 가지며, 이러한 활성은 다양한 작물의 씨앗의 발아와 중장의 식물체 생장을 방해하는 것으로 알려져 있다. 이전 실험에서는 DAPG의 독성은 원형질막의 전자 전달을 방해하고 옥신 신호 전달을 방해하는 것으로 알려져 있었다. 하지만 이 효과에 대한 메커니즘은 구체적이지 않으며, 많은 분자적 상호관계와 관계가 있을 것으로 추정하였다. 이를 구체화하기 위하여, 강력한 게놈 연구의 도구인 과 발현 애기장대 식물체를 스크리닝을 진행하여 DAPG에 대하여 식물이 반응하는 세포적 기능을 알아보고자 하였다. 이는 5가지 기능 (활성 산소 조절, 세포막의 기능, 유기 화합물 대사 과정, 액포 수송 전달, 아미노산 생합성) 증가는 DAPG 독성에 대하여 덜 민감해지는 것을 확인할 수 있었다. 이는 식물이 DAPG 독성을 피하기 위해서 복합적인 메커니즘의 역할이 필요하다는 것을 알 수 있다. 이러한 연구는 DAPG와 식물간의 분자적 상호 관계를 더욱 구체화하는데 의의가 있다. 2,4-Diacetylphloroglucinol (DAPG), produced by certain benefical Pseudomonas spp., acts as an induced systemic resistance (ISR) elicitor. DAPG has a wide range of antibiotic activity against various plant pathogenic bacteria, fungi, and nematodes. Antibiotic mechanism of DAPG was proposed that DAPG may attack multiple basic cellular pathways and the pathogens may have difficult to develop resistance against DAPG. On the other hand, DAPG also has both negative or positive effect to plants. As a positive effect, DAPG can induces ISR, which allows plants to resistant to pathogens. Previous studies of ISR by DAPG have demonstrated with hormone-related Arabidopsis mutants such as npr1-1, jar1, etr1. However, plant hormones play various roles and the mutants may have function not only in resistance mechanism, but also unexpected physiological roles in plant defense system. In this study, DAPG hydrolase gene (phlG), which hydrolase toxic DAPG to non-toxic MAPG, was introduced into Arabidopsis. After screen the transformation lines, D16 and H2 was selected as DAPG insensitive plants. Wild-type Arabidopsis was response to DAPG priming and the wild-type plant showed strong ISR activity against both necrotrophic fungal pathogen, B. cinerea and hemi-biotrophic bacterial pathogen, P. syringae. However, the DAPG insensitive D16 and H2 plants fail to induce ISR against the both pathogens. Additionally, JA/ET defense systems were not activated in both D16 and H2 plants. As a negative effect, high concentrations of DAPG has phytotoxicity, and this activity is known to inhibit seed germination and growth in various plants. In previous studies, DAPG was known to interfere with the electron transport of the plasma membrane and the auxin signaling in plants. However, the mechanism for DAPG phytotoxicity is not clear and remain to unveil. To define plant response to DAPG, 62,000 activation tagging Arabidopsis lines were screened. Among the lines, total 16 independent lines were confirmed as DAPG tolerance lines, and 14 of 16 lines was identified activated genes and the genes were classified five functional group; regulation of active oxygen, membrane function, organic compound metabolism process, vacuole transport, amino acid biosynthesis. These finding suggested that plant may has multiple mechanism to avoid DAPG phytotoxicity. This study is critical to investigate the molecular interaction between DAPG and plants. Plants immune responses are trigged by chemicals, microbes, pathogens, insects, and abiotic stresses. Specially, induced systemic resistance (ISR) refers to activation of the immune system due to interaction with beneficial microorganisms. 2,4-diacetylphloroglucinol (DAPG), produced by Pseudomonas spp., acts as an ISR elicitor, which activated the plant defense system through JA/ET signal pathways. Mechanism of ISR studies have been demonstrated with npr1-1, jar1 and etr1 deletion Arabidopsis. However, JA and ET related genes are not involved only in ISR, but also activated by pathogens infection. In this study, phlG (DAPG hydrolase) transgenic Arabidopsis thaliana lines were created to study role of DAPG in ISR. DAPG (10, 100, 200 μM) were treated on 3-weeks-old wild-type A. thaliana Col-0 and the primed plants shown resistance against Botrytis cinerea and Pseudomonas syringae pv. tomato DC3000 (Pst DC 3000). However, phlG transgenic A. thaliana (D16, H2) failed to trigger ISR mediated resistance against the pathogens. The transgenic D16, H2 showed no difference in the disease severity and incidence as water or methanol (DAPG solvent) treatments. Relative gene expression level of the ISR regulated genes were analyzed, A. thaliana Col-0 showed an increase of pdf 1.2 gene expression but not in D16 and H2 transgenic lines. The finding suggested that D16 and H2 lines failed to activate JA/ET signal dependent defense system. We anticipate that D16, H2 may contribute to elucidate DAPG mediated ISR mechanism. 2,4-diacetylphloroglucinol (DAPG) is a known secondary metabolite produced by a biocontrol agent Pseudomonas spp. DAPG displays remarkably broad spectrum of toxic activity against the various of plant pathogens. However, high concentrations of DAPG also presents a negative effect on plants, but the phytotoxicity mechanisms of DAPG is not understood clearly. Therefore, in this study, Arabidopsis thaliana genome-wide activation tagging population was employed to screen plant responsible genes to DAPG. Among 62,000 activation tagging lines, 16 DAPG-tolerance lines were obtained. Flanking gene insertion of the 14 selected lines was identified by TAIL-PCR and reveal the genes that are related tolerance mechanism to DAPG. Based on gene ontology, five biological functional groups were related to tolerance to DAPG including regulation of active oxygen burst, membrane function, organic compound metabolism process, vacuole transport, and amino acid biosynthesis. These finding suggested that plant may has multiple mechanisms to avoid DAPG phytotoxicity.
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