Molecular, Proteomics and Cytochemical Analyses of the PR 10-LRR Complex, Abscisic Acid Responsive Protein 1, Osmotin-like Protein, Formate Dehydrogenase and Phosphoenolpyruvate Carboxykinase Genes for Cell Death and Defense Responses to Microbial Pathogens in Pepper and Arabidopsis = 고추와 애기장대에서 병원미생물에 대한 식물 세포 사멸과 방어 반응에 관여하는 PR 10-LRR Complex, Abscisic Acid Responsive Protein 1, Osmotin-like Protein, Formate Dehydrogenase, Phosphoenolpyruvate Carboxykinase 유전자의 분자적, 단백질체학적, 세포화학적 분석

다국어 초록 (Multilingual Abstract)

Plants have evolved a multitude of defense strategies to combat an abundance of microbial pathogens. Recently, molecular research of plant innate immunity has advanced toward the plant molecular breeding to generate the genetically modified, disease resistant plants. In this study, molecular and biochemical mechanisms underlying plant defense immune responses were analyzed using the pepper (Capsicum annuuum)-Xanthomonas campestris pv. vesicatoria (Xcv) and Arabidopsis (Arabidopsis thaliana)-Pseudomonas syringae pv. tomato (Pst) systems. The pepper defense response genes including Pathogenesis-related protein 10 (CaPR10), Abscisic acid-responsive protein 1 (CaABR1), Osmotin-like protein 1 (CaOSM1), Formate dehydrogenase 1 (CaFDH1), and Phosphoenolpyruvate carboxykinase 1 (CaPEPCK1) have been isolated and identified from the pepper leaves infected with both virulent (Ds1) and avirulent (Bv5-4a) strains of Xcv using the differential hybridization screening, yeast-two-hybrid assay as well as proteomics approach. In addition, Arabidopsis Formate dehydrogenase 1 (AtFDH1) has been isolated from the transgenic Arabidopsis plants overexpressing pepper Pathogen-induced membrane protein 1 (CaPIMP1). Virus-induced gene silencing (VIGS), ectopic gene overexpression, transient in planta expression, and T-DNA insertional mutation were used to investigate the gain-of- and loss-of-functions of the defense response genes in pepper, Arabidopsis and Nicotiana benthamiana plants. CaPR10 triggered hypersensitive cell death response (HR), which was promoted by the formation of the protein complex with a leucine-rich repeat protein (CaLRR1) as a positive regulator. CaABR1, a GRAM (for Glucosyltransferases, Rab-like GTPase activators, and Myotubularins) domain-containing protein, functioned in cell death regulation and abscisic acid (ABA)-salicylic acid (SA) antagonism. Notably, the specific subcellular localization of the CaABR1 protein and the CaPR10-CaLRR1 complex to the nucleus and the cytoplasm, respectively, was essential for their function to induce HR. CaOSM1 was required for the induction of HR and reactive oxygen species (ROS) burst in plant cells. CaFDH1, which catalyzes the oxidation of formate into carbon dioxide in the mitochondria in a NAD+-dependent manner, acted as a positive regulator of cell death response. AtFDH1 also played a distinct role for defense and cell death responses to microbial pathogens. CaPEPCK1 positively regulated plant innate immunity against the hemibiotrophic bacterial Pst and obligate biotrophic oomycete Hyaloperonospora arabidopsidis pathogens. Taken together, these results presented in this study suggest that these defense-related genes in pepper and Arabidopsis are responsible for plant cell death and immunity against microbial pathogens.

국문 초록 (Abstract)

식물은 다수의 방어 전략을 진화시켜 수많은 병원미생물들에 대항해오고 있다. 최근에 식물의 선천적 면역성에 대한 분자적 연구가 식물 분자 육종으로 발전하여 유전적으로 변형된 병저항성 식물을 제조하기에 이르렀다. 본 연구에서는 고추식물 (Capsicum annuuum)과 Xanthomonas campestris pv. vesicatoria (Xcv), 애기장대 (Arabidopsis thaliana)와 Pseudomonas syringae pv. tomato (Pst) 시스템을 이용하여 식물 방어 면역 반응에 기초하는 분자적•생화학적 기작을 분석하였다. Proteomics 접근방법뿐만 아니라 differential hybridization screening, yeast-two-hybrid assay를 이용하여 Xcv 의 병원성 (Ds1) 균주와 비병원성 (Bv5-4a)균주에 감염된 고추 잎에서 Pathogenesis-related protein 10 (CaPR10), Osmotin-like protein 1 (CaOSM1), Abscisic acid-responsive protein 1 (CaABR1), Formate dehydrogenase 1 (CaFDH1), Phosphoenolpyruvate carboxykinase 1 (CaPEPCK1) 유전자를 분리•동정하였다. 또한 고추의 Pathogen-induced membrane protein 1 (CaPIMP1) 유전자를 과발현하는 형질전환 애기장대식물에서 Arabidopsis Formate dehydrogenase 1 (AtFDH1)를 분리하였다. Virus-induced gene silencing (VIGS), 외부 유전자 과발현, 일시적 in planta 유전자 발현, 그리고 T-DNA 삽입 돌연변이를 이용하여 고추, 애기장대, Nicotiana benthamiana 식물에서 이들 방어반응 유전자의 기능 획득(gain-of-function)과 기능 손실(loss-of-function)을 연구하였다. CaPR10은 과민성 세포사멸을 일으키며 CaLRR1과 단백질 복합체를 형성하여 긍정적 조정자로서 세포사멸을 촉진시켰다. CaABR1은 GRAM (for Glucosyltransferases, Rab-like GTPase activators, and Myotubularins) 도메인을 함유하는 단백질이며 세포사멸조절과 앱시스산-살리실산의 길항작용의 기능을 하였다. 특히, CaABR1와 CaPR10-CaLRR1 복합체가 각각 세포내의 핵(nucleus)과 세포질(cytoplasm)로 특이적으로 위치하는 것이 과민성 세포사멸을 유도하기 위해 필수적이었다. CaOSM1은 식물세포에서 과민성 세포사멸의 유도와 활성산소(ROS) 생성에 필요하였다. 미토콘드리아에서 NAD+ 의존적인 방법으로 formate를 산화시켜 이산화탄소를 만드는 반응을 촉매하는 CaFDH1은 세포 사멸 반응의 긍정적 조절자로서 기능을 하였다. 애기장대의 AtFDH1 역시 병원미생물에 대한 방어와 세포 사멸 반응을 위해 상이한 역할을 하였다. CaPEPCK1은 반활물 세균성 병원균 Pst와 절대기생성 난균류 Hyaloperonospora arabidopsidis 에 대한 식물의 선천적 면역성을 긍정적으로 조절하였다. 종합하면, 본 연구에서 제시된 실험결과들은 고추와 애기장대의 이들 방어관련 유전자들이 병원미생물에 대한 식물의 세포 사멸과 면역성에 관여한다는 것을 시사하고 있다.

목차 (Table of Contents)

• 1. Introduction 1
• 1.1. Plant cell death and defense signaling pathways 3
• 1.2. Hypersensitive cell death response and reactive oxygen species (ROS) burst for plant disease resistance 6
• 1.3. Defense-related proteins for plant disease resistance 7
• 1.4. Roles of salicylic acid and abscisic acid in plant defense response 9
• 1.5. Formate and pyruvate metabolism during plant defense response 12
• 1.6. Objectives of the study 14
• 2. Requirement of the Cytosolic Interaction between PR10 and Leucine-rich Repeat Protein 1 for Cell Death and Defense Signaling in Pepper 17
• 2.1. Abstract 18
• 2.2. Introduction 18
• 2.3. Materials and methods 23
• 2.3.1. Plant materials and pathogen inoculation 23
• 2.3.2. RNA blot and quantitative real-time RT-PCR analyses 24
• 2.3.3. Yeast two-hybrid assay 26
• 2.3.4. Bimolecular fluorescence complementation (BiFC) analysis 26
• 2.3.5. Co-immunoprecipitation (Co-IP) 27
• 2.3.6. Antibody production and immunoblot analysis 28
• 2.3.7. RNase Activity Assay 29
• 2.3.8. Phosphorylation Assay 30
• 2.3.9. Agro-mediated transient expression and subcellular localization assays 31
• 2.3.10. Engineering of nuclear expression 32
• 2.3.11. Virus-induced gene silencing (VIGS) 33
• 2.3.12. Arabidopsis tansformation 33
• 2.3.13. Measurement of ion conductivity 34
• 2.3.14. SA quantification 34
• 2.3.15. Histochemical assay 35
• 2.4. Results 36
• 2.4.1. Expression of CaLRR1 and CaPR10 in pepper 36
• 2.4.2. Pathogenesis-related protein 10 interacts with leucine-rich repeat protein 1 36
• 2.4.3. Transient coexpression of CaLRR1 and CaPR10 induces cell death and defense responses 41
• 2.4.4. LRR1 Promotes the Ribonuclease Activity and Phosphorylation of PR10 48
• 2.4.5. Cytoplasmic localization of the CaLRR1-CaPR10 complex is essential for cell death induction 49
• 2.4.6. Silencing of CaPR10 attenuates disease resistance and compromises the hypersensitive cell death response (HR) 56
• 2.4.7. Enhanced resistance of PR10- and LRR1/PR10-OX transgenic Arabidopsis to bacterial and oomycete infection 62
• 2.5. Discussion 74
• 2.5.1. The Involvement of PR10 in Disease Resistance 74
• 2.5.2. PR10 Interacts with LRR1 to Activate Cell Death and Defense Responses 75
• 2.5.3. LRR1 Promotes the Ribonuclease Activity and Phosphorylation of PR10 76
• 2.5.4. Cytoplasmic Localization of the LRR1-PR10 Complex is Essential for the Induction of the Cell Death Response 77
• 2.5.5. Silencing of LRR1 and PR10 compromises R gene-mediated resistance, the cell death response and ROS accumulation 79
• 2.5.6. Overexpression of LRR1 and PR10 in Arabidopsis confers enhanced resistance against pathogen infection 81
• 2.5.7. The pepper PR10 and LRR1 complex in the cytoplasm is essential for cell death-mediated defense signaling 83
• 3. Proteomics and Functional Analyses of the Pepper GRAM Domain-Containing Abscisic Acid-responsive Protein (ABR1) Gene Involved in Cell Death and Defense Signaling 99
• 3.1. Abstract 100
• 3.2. Introduction 100
• 3.3. Materials and methods 104
• 3.3.1. Plant growth and pathogen inoculation 104
• 3.3.2. 2-dimensional electrophoresis and protein staining 105
• 3.3.3. Identification of proteins by LC/MS-MS or MALDI-TOF MS 107
• 3.3.4. Isolation and sequence analysis of pathogen-induced ABR1 cDNA 108
• 3.3.5. Bombardment assays of onion epidermal cells with GFP constructs 109
• 3.3.6. RNA isolation, RNA blot analysis and quantitative real-time RT-PCR 109
• 3.3.7. Immunoblot analysis 110
• 3.3.8. Virus-induced gene silencing (VIGS) in pepper 110
• 3.3.9. Chemical treatment 111
• 3.3.10. Arabidopsis transformation 111
• 3.3.11. Agrobacterium-mediated transient expression 112
• 3.3.12. Measurement of endogenous ABA and SA levels 113
• 3.3.13. DAB staining and H2O2 measurement 114
• 3.3.14. Cell death assay 114
• 3.3.15. Aniline blue staining 115
• 3.4. Results 115
• 3.4.1. Proteomics Analysis of Pepper Leaves Infected by Xcv 115
• 3.4.2. Pepper and bacterial proteins differentially expressed following Xcv infection 116
• 3.4.3. Isolation and sequence analysis of pathogen-induced ABR1 cDNA 118
• 3.4.4. Expression pattern of the ABR1 gene 119
• 3.4.5. Agrobacteruim-mediated transient expression of the ABR1 gene induces cell death response 124
• 3.4.6. The GRAM domain is required to initiate the cell death response and to localize ABR1 to the nucleus 125
• 3.4.7. Nuclear localization of ABR1 is essential for cell death induction and hormone regulation 128
• 3.4.8. Virus-induced gene silencing (VIGS) of the ABR1 gene in pepper plants 134
• 3.4.9. Enhanced disease resistance of ABR1-OX transgenic Arabidopsis 140
• 3.4.10. Enhanced disease susceptibility of the Arabidopsis T-DNA insertion mutant abr1 144
• 3.5. Discussion 148
• 3.5.1. Proteomics Analysis and Identification of Pepper Defense-Related Proteins 148
• 3.5.2. The pepper ABA-responsive protein ABR1 contains the GRAM domain required for nuclear localization and cell death response 148
• 3.5.3. ABR1 plays a role in cell death and defense responses in pepper and Arabidopsis 149
• 3.5.4. Crosstalk between ABA- and SA-mediated signalings in pepper 151
• 4. Role of Pepper Osmotin-like Protein 1 (CaOSM1) for Hypersensitive Cell Death and Defense Response to Microbial Pathogens 165
• 4.1. Abstract 166
• 4.2. Introduction 166
• 4.3. Materials and methods 171
• 4.3.1. Plant materials and growth conditions 171
• 4.3.2. Generation of recombinant plasmids 171
• 4.3.3. Transient in Planta expression assays 172
• 4.3.4. Confocal microscopy 173
• 4.3.5. Cell death assay 173
• 4.3.6. Arabidopsis transformation 173
• 4.3.7. Virus-induced gene silencing 174
• 4.3.8. Pathogen inoculation and disease ratings 174
• 4.3.9. RNA isolation and RT-PCR 175
• 4.3.10. DAB staining and H2O2 assay 176
• 4.4. Results 176
• 4.4.1. Subcellular localization of CaOSM1 176
• 4.4.2. Transient expression of CaOSM1 triggers cell death and oxidative burst in pepper leaves 178
• 4.4.3. Virus-induced gene silencing (VIGS) of CaOSM1 in pepper compromises resistance to Xcv infection 179
• 4.4.4. CaOSM1 overexpression in Arabidopsis confers enhanced resistance to Pseudomonas syringae pv. tomato and Hyaloperonospora arabidopsidis 186
• 4.5. Discussion 187
• 5. Pepper Mitochondrial FORMATE DEHYDROGENASE1 (CaFDH1) Regulates Hypersensitive Cell Death and Disease Response against Microbial Pathogens 195
• 5.1. Abstract 196
• 5.2. Introduction 196
• 5.3. Materials and methods 200
• 5.3.1. Plant, pathogen inoculation, and chemical treatment 200
• 5.3.2. Quantitative real-time PCR and RNA gel blotting 201
• 5.3.3. Virus-induced gene silencing (VIGS) in pepper 202
• 5.3.4. Construction of FDH1 mutants and transgenic lines 202
• 5.3.5. Immunoblot analysis 203
• 5.3.6. Cell death assay 204
• 5.3.7. DAB staining and H2O2 measurement 204
• 5.3.8. Enzyme Activity Assay 205
• 5.3.9. SA measurement 205
• 5.3.10. Confocal microscopy 206
• 5.4. Results 207
• 5.4.1. Isolation and sequence analysis of CaFDH1 207
• 5.4.2. Activation of CaFDH1 by biotic and abiotic stress 210
• 5.4.3. CaFDH1 is required for induction of hypersensitive response (HR)-like cell death 212
• 5.4.4. Subcellular localization of CaFDH1 217
• 5.4.5. D-isomer specific 2-hydroxyacid dehydrogenase signatures are essential for triggering HR-like cell death 220
• 5.4.6. CaFDH1 silencing compromises R gene-mediated resistance in pepper leaves 226
• 5.4.7. Enhanced disease resistance of CaFDH1-OX transgenic Arabidopsis 232
• 5.5. Discussion 232
• 5.5.1. CaFDH1 is induced in pepper leaves by Xanthomonas campestris pv. vesicatoria infection 234
• 5.5.2. CaFDH1, especially the catalytic domain of 2-hydroxyacid dehydrogenase, is required for initiation of cell death response 235
• 5.5.3. Mitochondrial localization of CaFDH1 235
• 5.5.4. The role of FDH1 in disease resistance 236
• 6. Role of Arabidopsis Formate Dehydrogenase in Defense and Cell Death Responses to Microbial Pathogens 239
• 6.1. Abstract 240
• 6.2. Introduction 240
• 6.3. Materials and methods 242
• 6.3.1. Plant materials and pathogen inoculation 242
• 6.3.2. Protein extraction 243
• 6.3.3. Two-dimensional electrophoresis 244
• 6.3.4. Image analysis and protein identification 245
• 6.3.5. Isolation and complementation of T-DNA insertion fdh1 mutants 246
• 6.3.6. Measurement of electrolyte leakage 246
• 6.3.7. Histochemical assays 247
• 6.4. Results 247
• 6.4.1. Identification of the proteome differentially expressed in CaPIMP1-overexpression (OX) Arabidopsis 227
• 6.4.2. Newly induced protein in CaPIMP1-OX plants 250
• 6.4.3. Proteins associated with metabolism and energy 250
• 6.4.4. Proteins associated with cell rescue and defense 254
• 6.4.5. Candidate proteins as defense response regulators 258
• 6.4.6. Distinct roles of formate dehygdrogenase (FDH) in response to Pst and Hpa infection 258
• 6.5. Discussion 264
• 6.5.1. Heterogeneous CaPIMP1 expression contributes to the disease resistance in transgenic Arabidopsis plants 265
• 6.5.2. Proteome alteration in CaPIM1-OX transgenic Arabidopsis 266
• 6.5.3. Formate dehydrogenase is required for defense response to microbial pathogens 267
• 7. The Pepper Phosphoenolpyruvate Carboxykinase CaPEPCK1 Is a Positive Regulator of Plant Innate Immunity against the Bacterial and Oomycete Pathogens 269
• 7.1. Abstract 270
• 7.2. Introduction 270
• 7.3. Materials and methods 274
• 7.3.1. Plant growth and pathogen inoculation 274
• 7.3.2. Isolation and sequence analysis of CaPEPCK1 275
• 7.3.3. Abiotic elicitor and stress treatment 275
• 7.3.4. Phosphoenolpyruvate carboxykinase activity assay 276
• 7.3.5. RNA isolation, RNA gel blotting and RT-PCR analysis 277
• 7.3.6. Virus-induced gene silencing in pepper 277
• 7.3.7. Arabidopsis transformation 278
• 7.4. Results 279
• 7.4.1. Isolation and sequence analysis of CaPEPCK1 279
• 7.4.2. Expression of CaPEPCK1 in response to biotic and abiotic stresses 282
• 7.4.3. Phosphoenolpyruvate carboxykinase (PEPCK) activity during Xcv infection 284
• 7.4.4. CaPEPCK1 is essential for innate immunity against Xcv infection 284
• 7.4.5. CaPEPCK1 overexpression confers enhanced resistance to Pseudomonas syringae pv. tomato and Hyaloperonospora arabidopsidis in Arabidopsis 286
• 7.4.6. The Arabidopsis Ortholog mutant pck1 is more susceptible to Pseudomonas syringae pv. tomato and Hyaloperonospora arabidopsidis in Arabidopsis 290
• 7.5. Discussion 292
• 7.5.1. CaPEPCK1 share sequence homology with other PEPCKs 292
• 7.5.2. CaPEPCK1 was induced by biotic and aboitic stress 296
• 7.5.3. The loss-of- and gain-of-function of CaPEPCK1 for the functional analysis of CaPECK1 297
• 8. Conclusion 301
• 9. Literature Cited 307