[보고서]고추에서 병저항성관련유전자의 분리와 기능분석

황병국

고추에서 병저항성관련유전자의 분리와 기능분석
Isolation and Functional Analyses of Disease Resistance-Related Genes in Pepper 원문보기

보고서 정보
주관연구기관	고려대학교 Korea University
연구책임자	황병국
참여연구자	황인선 , 최두석 , 김대성 , 김낙헌 , 이동혁 , 김정도
보고서유형	최종보고서
발행국가	대한민국
언어	한국어
발행년월	2015-02
과제시작연도	2014
주관부처	농촌진흥청
사업 관리 기관	농촌진흥청 Rural Development Administration
등록번호	TRKO201500010168
과제고유번호	1395035364
DB 구축일자	2015-07-11
키워드	Capsicum annuum, Xanthomonas campestris pv. vesicatoria, Functional genomics, Disease resistance-related genes, Defense response

초록 ▼

Ⅳ. 연구개발결과
고추 (Capsicum annuum L.)는 한국에서 경제적으로 중요한 채소작물의 하나이며 병원미생물에 대한 식물병저항성을 조절하는 분자적 기작과 기능유전체학(functional genomics)를 연구하기 위한 좋은 실험시스템을 제공해 주고 있다. 비병원성 세균성점무늬병균 (Xanthomonas campestris pv. vesicatoria, Xcv) 균주 Bv5-4a로 고추잎을 감염시키면 과민성 세포사멸 (hypersensitive response (HR) cell death)반응과 방어반응을 유도하였다. 이들 고추병저항성반응은 병저항성관련유전자의 발현이 조정하였다. 본 연구개발에서는 고추 병저항성관련 유전자 E3 ubiquitin ligase RING1 (CaRING1), asparagine synthetase1 (CaAS1), pathogenesis-related protein10 (CaPR10), RNA-binding protein (CaRBP1), arginine decarboxylase1(CaADC1), pathogenesis-related protein 4b (CaPR4c), glycine-rich RNA-binding protein1 (CaGRP1), phenylalanine ammonia-lyase (CaPAL1) 등을 Xcv감염된 고추잎에서 분자적으로 크로닝하여 동정(identification)하였다. Virus-induced gene silencing (VIGS) 기술을 이용한 고추 식물에서 병저항성관련 유전자의 기능을 분석하고 solubly modified green fluorescent protein (smGFP) tagging 시스템을 이용한 병저항성관련 단백질의 세포내위치 (subcellular localization)를 분석하였다. Xcv 감염된 잎에서 관찰된 세포사멸 및 방어반응에서 병저항성관련유전자가 발현되었고 reactive oxygen species (ROS)과 nitric oxide (NO) bursts, 그리고 ion leakage와 callose deposition이 유도되었다. 이들 몇가지 병저항성관련 유전자를 이용하여 애기장대 (Arabidopsis thaliana)형질전환 식물체를 제작, 이를 이용한 병저항성을 검정한 결과 이들 병저항성관련유전자의 과발현(overexpression)은 병원미생물에 대해 병저항성을 증가시켰다. 또한 Bimoleucular Fluorescent Complementation (BiFC) 및 Co-immunoprecipitation을 통하여 고추 식물에서 CaLRR1 (leucine rich-repeat protein1)-CaPR10 및 CaGRP1-CaPIK1 (receptor-like cytoplasmic kinase1) 단백질 복합체를 검증, 기능분석을 하였다. 이들 연구 결과를 토대로 형질전환 병저항성 식물의 개발 기술을 분석하고 고추병저항성관련 유전자의 분자유전육종자료로서의 활용가능성을 제시하였다.

Abstract ▼

Pepper (Capsicum annuum L.) is one of the economically important vegetable crops growing in Korea. Pepper provides a good experimental system for studying the molecular mechanisms and functional genomics underlying the plant resistance to microbial pathogens. Infection of pepper leaves with the avirulent strain Bv5-4a of Xanthomonas campestris pv. vesicatoria (Xcv) induced hypersensitive response (HR) cell death and defense responses.
These responses were controlled by expression of the disease resistance-related genes. The pepper disease resistance-related genes encoding pepper E3 ubiquitin ligase RING1 (CaRING1), asparagine synthetase1 (CaAS1), pathogenesis-related protein10 (CaPR10), RNA-binding protein (CaRBP1), arginine decarboxylase1 (CaADC1), pathogenesis-related protein 4b (CaPR4c), glycine-rich RNA-binding protein1 (CaGRP1), phenylalanine ammonia-lyase (CaPAL1), etc. were isolated and identified from Xcv-infected pepper leaves. We analyzed loss-of-functions and subcellular localization of these genes using virus-induced gene silencing (VIGS) and smGFP-tagging systems, respectively. The cell death and defense responses were accompanied by the expression of the disease resistance-related genes, reactive oxygen species (ROS) and nitric oxide (NO) bursts, and the induction of both ion leakage and callose deposition in the Xcv-inoculated leaves. Ectopic expression of some disease resistance-related genes in Arabidopsis also conferred enhanced resistance to microbial pathogens. The CaLRR1 (leucine rich-repeat protein1)-CaPR10 and CaGRP1-CaPIK1 (receptor-like cytoplasmic kinase1) protein complexes were identified using Bimoleucular Fluorescent Complementation (BiFC) and co-immunoprecipitation. Taken together, development techniques of transgenic disease resistant plants were evaluated and the possibility of use of the disease resistance-related genes for plant molecular breeding as gene sources was also suggested.

목차 Contents

완결과제 최종보고서 ... 1
제출문 ... 2
요약문 ... 3
SUMMARY ... 6
목 차 ... 7
제 1 장 서 론 ... 8
제 2 장 국내외 기술개발 현황 ... 10
제 3 장 연구개발수행 내용 및 결과 ... 13
제1절 연구개발 수행방법 ... 13
제 2 절 연구 결과 ... 18
제 4 장 연구개발목표 달성도 및 대외 기여도 ... 100
제 1 절 목표대비 대외 달성도 ... 100
제 2 절 정량적 성과 ... 101
제 5 장 연구개발결과의 활용계획 ... 108
제 6 장 연구개발과정에서 수집한 해외과학기술정보 ... 109
제 7 장 기타 중요 변동사항 ... 110
제 8 장 국가과학기술종합정보시스템에 등록한 연구장비 현황 ... 110
제 9 장 참고문헌 ... 111
끝페이지 ... 120

표/그림 (70)

표 Amino acid sequence analysis of pepper CaRING1 and the RING-H2 finger domain.
표 RNA gel-blot analysis of CaRING1 expression in pepper plants. Samples were hybridized with a [32P]dCTP-labeled CaRING1 probe.
표 E3 ubiquitin ligase activity of CaRING1. A, Hydrophobicity index and expression constructs used for the in vitro ubiquitination assay.
표 Subcellular localization of CaRING1 and its mutants using transient expression in onion epidermal cells.
표 Induction of cell death by transient expression of CaRING1 in pepper leaves.
표 Enhanced susceptibility of CaRING1-silenced pepper plants to infection with the virulent Xcv strain Ds1 and the avirulent Xcv strain Bv5-4a.
표 Expression of pepper defense-related maker genes and SA accumulation in CaRING1-silenced pepper leaves infected with Xcv.
표 CaRING1-OX transgenic Arabidopsis plants exhibit enhanced resistance to Pst DC3000 infection.
표 CaRING1-OX transgenic Arabidopsis plants show enhanced resistance to H. arabidopsidis isolate Noco2 infection.
표 RNA gel blot analyses of CaAS1 and CaBPR1 expression in pepper. RNA gel blots were hybridized with a 32P-labeled probe against the CaAS1 3′-UTR or the full-length cDNA clone of CaBPR1.
표 Enhanced susceptibility of CaAS1-silenced (TRV:CaAS1) pepper leaves to Xanthomonas campestris pv. vesicatoria (Xcv).
표 Effects of CaAS1 silencing on gene expression and SA accumulation in pepper leaves infected by Xanthomonas campestris pv. vesicatoria (Xcv).
표 Effect of CaAS1 silencing on amino acid levels in pepper leaves.
표 Enhanced resistance of CaAS1-OX Arabidopsis transgenic lines to Pseudomonas syringae pv. tomato (Pst) infection.
표 Expression of Arabidopsis defense-related genes in wild-type (WT) and CaAS1-OX transgenic plants (#11) inoculated with Pseudomonas syringae pv. tomato (Pst) DC3000 or DC3000 (avrRpm1) (106 cfu ml−1).
표 Quantification of infection phenotypes on cotyledons of wild-type (WT) and CaAS1-OX Arabidopsis transgenic plants (#1) inoculated with Hyaloperonospora arabidopsidis isolate Noco2.
표 Effect of CaAS1 overexpression on amino acid levels in Arabidopsis. (a) Amino acid contents in leaves of wild-type (WT) and CaAS1-OX transgenic plants (lines #1, #11 and #12).
표 Interaction between LRR1 and PR10 in yeast and N. benthamiana.
표 Transient expression of LRR1, PR10, and LRR1/PR10 in pepper leaves.
표 Transient expression of LRR1, PR10, LRR1/PR10, avrPto/Pto, and Bax in N. benthamiana leaves.
표 Synergistic effect of LRR1 on RNase activity and phosphorylation of PR10.
표 Cytoplasmic localization of the LRR1/PR10 complex is essential for cell death induction.
표 Silencing of LRR1/PR10 compromises defense gene expression and SA accumulation in pepper leaves infected by Xcv.
표 Enhanced resistance of PR10- and LRR1/PR10-OX transgenic Arabidopsis plants to Pst infection.
표 Real-time RT-PCR analysis of defense marker gene expression in LRR1-, PR10-, and LRR1/PR10-OX transgenic Arabidopsis plants.
표 Enhanced resistance of LRR1-, PR10-, and LRR1/PR10-OX transgenic Arabidopsis plants to H. arabidopsidis infection.
표 RT-PCR analysis of the expression of CaRBP1, CaPR1 and CaDEF1 in pepper leaves infected with the virulent (compatible) Ds1 strain and the avirulent (incompatible) Bv5-4a strain of Xanthomonas campestris pv. vesicatoria (107 cfu ml-1).
표 RNA- and DNA-binding activity of CaRBP1.
표 Subcellular localization of CaRBP1 and its mutants in Nicotiana benthamiana leaves. (a) Confocal images of subcellular localization of CaRBP1 and its mutants. Empty vector (35S:00) expressing cells are shown throughout the cell as a control [subsection
표 Induction of cell death in pepper leaves by Agrobacterium-mediated transient expression of CaRBP1.
표 CaRBP1-induced cell death is dependent on the CaRBP1 N-terminal region and cytoplasmic localization.
표 Enhanced susceptibility and reduced HR of CaRBP1-silenced pepper plants infected with Xanthomonas campestris pv. vesicatoria (Xcv).
표 Reduction of early defense responses in CaRBP1-silenced pepper leaves infected with the Xcv avirulent strain.
표 Altered regulation of defense-related gene expression and signaling hormones in CaRBP1-silenced plants.
표 Enhanced resistance of CaRBP1 over-expressing Arabidopsis plants to infection with Hyaloperonospora (Hpa) isolate Noco2.
표 Interactions of CaADC1 with AvrBsT. A, Yeast two-hybrid assay of CaADC1 and AvrBsT.
표 Transient expression of CaADC1 promotes AvrBsT-triggered cell death.
표 Effect of transient expression of CaADC1, CaADC1 K154A, and avrBsT on polyamine levels in Nicotiana benthamiana leaves.
표 Quantification of the nitric oxide (NO) burst in Nicotiana benthamiana leaves 24 h after infiltration with either polyamines or Agrobacterium harboring the indicated constructs.
표 Quantification of the H2O2 burst in Nicotiana benthamiana leaves 24 h after infiltration with either polyamines or Agrobacterium harboring the indicated constructs.
표 Effect of polyamine treatment on cell death response in Nicotiana benthamiana leaves.
표 Spermine promotes AvrBsT-triggered cell death.
표 Enhanced susceptibility of CaADC1-silenced pepper plants to avirulent Xanthomonas campestris pv. vesicatoria (avrBsT) infection.
표 Levels of putrescine, spermidine, and spermine in leaves of empty vector control (TRV:00) and CaADC1-silenced (TRV: CaADC1) pepper plants infected with virulent Ds1 (EV) and avirulent Ds1 (avrBsT) strains of Xanthomonas campestris pv. vesicatoria (Xcv) (107 cfu mL-1). Data are means ± standard deviations from three independent experiments. Asterisks indicate statistically significant differences (Student’s t-test; P < 0.05).
표 CaADC1 silencing compromises SA-dependent defense gene expression and SA accumulation in pepper leaves infected with Xanthomonas campestris pv. vesicatoria (Xcv).
표 γ-Aminobutyric acid (GABA) suppresses Xanthomonas campestris pv. vesicatoria (Xcv) growth in pepper leaves.
표 RNA gel blot analyses of CaPR4c gene expression in pepper leaves infected with virulent (compatible) Ds1 or avirulent Ds1(avrBsT) (incompatible) Xanthomonas campestris pv. vesicatoria strains.
표 CaPR4c has cysteine protease inhibitor activity.
표 Subcellular localization of CaPR4c.
표 Agrobacterium- mediated transient expression of CaPR4c and CaPR4c ΔSP in pepper leaves.
표 Enhanced susceptibility of CaPR4c -silenced pepper plants to Xanthomonas campestris pv. vesicatoria (Xcv) infection.
표 Real-time RT-PCR analyses of expression of CaPR4c and defense response genes in empty vector control (TRV:00) and CaPR4c-silenced (TRV:CaPR4c) pepper leaves infected with Xanthomonas campestris pv. vesicatoria (Xcv). CaPR4c, protease inhibitor; CaPR1, pathogenesis-related protein 1; CaDEF1, defensin; CaSAR82A, SAR 8.2; CaPR10, PR-10; CaPO2, peroxidase 2. Capsicum annuum18S RNA was used as an internal control gene.
표 Enhanced resistance of CaPR4c-OX transgenic Arabidopsis plants to Pseudomonas syringae pv. tomato (Pst).
표 Enhanced resistance of CaPR4c-OX transgenic Arabidopsis to Hyaloperonospora arabidopsidis Noco2 infection.
표 Capsicum annuum RECEPTOR-LIKE CYTOPLASMIC PROTEIN KINASE1 (CaPIK1) interacts with GLYCINE-RICH RNA-BINDING PROTEIN1 (CaGRP1) in planta.
표 Capsicum annuum GLYCINE-RICH RNA-BINDING PROTEIN1 (CaGRP1) has both RNA- and DNA-binding activities in vitro.
표 CaGRP1 is phosphorylated by CaPIK1 in vitro.
표 v
표 CaGRP1 coexpression with CaPIK1 suppresses CaPIK1-triggered reactive oxygen species (ROS) burst, but not nitric oxide (NO) burst, in pepper leaves.
표 Enhanced resistance of CaGRP1-silenced pepper plants to Xanthomonas campestris pv. vesicatoria infection.
표 Quantitative real-time RT-PCR analysis of the expression of CaGRP1, CaPIK1 and defense-related genes in empty-vector control, and CaGRP1-, CaPIK1- and CaGRP1/CaPIK1-silenced pepper plants infected with virulent strain Ds1 (compatible) and avirulent strain Bv5-4a (incompatible) of Xanthomonas campestris pv vesicatoria.
표 Increased susceptibility of CaPAL1-silenced pepper plants to infection by Xcv.
표 Reduced ROS burst and cell death response in leaves of CaPAL1-silenced pepper plants infected with Xcv.
표 Real-time RT-PCR analysis of CaPAL1 expression and defence related genes in empty-vector control and CaPAL1-silenced leaves infected with Xcv. H, healthy leaves; hai, hours after infection; CaPR1, pathogenesisrelated protein; CaDEF1, defensin.
표 Levels of (A) free and (B) total SA in empty-vector control (TRV:00) and CaPAL1-silenced (TRV:CaPAL1) pepper leaves infected with virulent (compatible) Ds1 and avirulent (incompatible) Bv5-4a strains of Xcv (107 cfu ml–1).
표 Increased resistance of CaPAL1-OX Arabidopsis plants to Pst DC3000 and DC3000 (avrRpm1) infection.
표 Increased ROS burst and cell death in leaves of CaPAL1-OX transgenic Arabidopsis plants infected with Pst DC3000 and DC3000 (avrRpm1).
표 Real-time RT-PCR analyses of the expression of CaPAL1 and defence-related genes in WT and CaPAL1-OX Arabidopsis plants infected with Pst DC3000 and Pst DC3000 (avrRpm1).
표 Levels of free (A) and total (B) SA in WT and CaPAL1-OX Arabidopsis plants infected with virulent Pst DC3000 and avirulent Pst DC3000 (avrRPM1) strains (107 cfu ml–1).
표 Increased resistance of Arabidopsis CaPAL1-OX plants to Hpa infection.

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연합인증

고추에서 병저항성관련유전자의 분리와 기능분석
Isolation and Functional Analyses of Disease Resistance-Related Genes in Pepper 원문보기