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Functional analysis of HEX1 gene in interaction between Fusarium graminearum and Fusarium graminearum virus 1 : 붉은 곰팡이 바이러스 1 과 상호작용하는 붉은 곰팡이의 HEX1 유전자에 대한 기능분석연구
| DC Field | Value | Language |
|---|---|---|
| dc.contributor.advisor | 김국형 | - |
| dc.contributor.author | 손문일 | - |
| dc.date.accessioned | 2017-07-13T08:22:43Z | - |
| dc.date.available | 2017-07-13T08:22:43Z | - |
| dc.date.issued | 2015-02 | - |
| dc.identifier.other | 000000025960 | - |
| dc.identifier.uri | https://hdl.handle.net/10371/119489 | - |
| dc.description | 학위논문 (박사)-- 서울대학교 대학원 : 농생명공학부, 2015. 2. 김국형. | - |
| dc.description.abstract | Fusarium graminearum virus 1 strain DK21 (FgV1) is fungal virus (mycovirus) isolated from the devastating plant pathogenic fungi, Fusarium graminearum. The FgV1 genome consists of 6,624 nucleotides, excluding the 3ʹ-terminal poly (A) tail. The viral genome has 53- and 46-nucleotide 5ʹ-and 3ʹ-untranslated regions (UTRs), respectively, and four putative open reading frames. The FgV1 infection perturbs in various aspects of host fungus biology such as morphology, growth, development, metabolism, and even virulence to plant. To identify a certain host factor interacting with FgV1 which involves in such alteration of host, proteome and transcriptome based approaches have been tried previously. Among them, the hexagonal peroxisome (Hex1) protein is fungal protein that is highly expressed when FgV1 infects its host. The Hex1 protein constitutes Woronin body (WB), which is peroxisome derived dense-core micro organelle for sealing the septal pore in response to hyphal damage. In the present study, cellular functions of Hex1 in F. graminearum and particular function for FgV1 were characterized. First, in order to investigate roles of Hex1, HEX1 gene deletion, over-expression and complementation mutants were generated and then infected by FgV1. Deletion and over-expression of Hex1 did not affect to vegetative growth in virus-free (VF) strains, while both changes resulted in reduced production of conidia and reduced virulence. Additionally, fungal cells could not maintain cellular integrity after hyphal wounding in the absence of HEX1 gene and even they showed cytoplasmic bleeding. Taken together, combined results suggested that HEX1 gene is required for asexual reproduction and pathogenesis and maintaining cellular integrity. Although deletion and over-expression did not affect vegetative growth at all in VF strains, both genetic alterations substantially affected vegetative growth in virus-infected (VI) strains. Vegetative growth was increased in deletion strains and decreased in over-expression strains comparing with WT-VI strain, while viral RNA accumulation was decreased in deletion strains and increased in over-expression strain. To clarify the cause of these different RNA accumulations in different HEX1 strains, FgV1 and Hex1 interaction studies were carried out. Homology based protein tertiary structure prediction analysis demonstrated that the structure of Hex1 is similar to that of eukaryotic translational initiation factor 5A (IF-5A) which have RNA-binding folds on its surface. Therefore, I carried out electrophoretic mobility shift assays (EMSAs) and the analysis revealed Hex1 protein bind to both 5ʹ-and 3ʹ-untranslated regions (UTRs) of plus-strand viral RNA. Strand specific Northern blot and qRT-PCR analyses were conducted to determine the effect of Hex1 on FgV1 (+)- and (-)-strand RNA accumulation. Both analyses exhibited that Hex1 protein affected both (+)- and (-)-strand RNA accumulations. | - |
| dc.description.tableofcontents | ABSTRACT i
CONTENTS iv LIST OF TABLES ix LIST OF FIGURES x GENERAL INTRODUCTION page I. Mycovirus 14 II. Hypovirulence associated mycoviruses 15 III. Isolation of mycoviruses from Fusarium graminearum 16 IV. Fusarium grmaniearum virus 1 17 V. Identification of fungal host factors in response to FgV1 18 VI. Fungal host factor Hex1 21 VII. Hex1 and FgV1 interaction studies in this work 22 LITERATURE CITED 23 CHAPTER I. The HEX1 gene of Fusarium graminearum is required for fungal asexual reproduction and pathogenesis and for efficient viral RNA accumulation of Fusarium graminearum virus 1 page ABSTRACT 28 INTRODUCTION 30 MATERIALS AND METHODS I. Fungal strains and culture condition 34 II. Computational analysis 34 III. Genomic DNA extraction, primers, and PCR conditions 35 IV. Fungal transformation for construction of targeted gene-deletion, over-expression, and complementation mutants 36 V. Radial growth, conidial production, and virulence test 37 VI. Observation of WB formation using electron microscopy 38 VII. Measurement of intercalary length 39 VIII. Fungal protein extraction and western blot analysis 40 IX. RNA preparation and RT-PCR 41 RESULTS I. Sequence analysis of F. graminearum HEX1 and homologs from other plant-pathogenic fungi 44 II. Targeted deletion, over-expression, and complementation of the HEX1 gene 47 III. Colony morphology and vegetative growth of HEX1 mutants 50 IV. Conidial production 52 V. Subcellular localization of WBs and western blot analysis 55 VI. Functions of the HEX1 gene with respect to the maintenance of cellular integrity and pathogenesis 55 VII. Quantification of HEX1 gene expression 58 VIII. Quantification of FgV1 viral RNA accumulation 62 DISCUSSION 68 LITERATURE CITED 77 CHAPTER II. Hex1 protein binds specifically to both plus-strand 5ʹ-and 3ʹ-untranslated regions of the Fusarium graminearum virus 1 and functions in virus RNA replication. page ABSTRACT 87 INTRODUCTION 88 MATERIALS AND METHODS I. Fungal strains and culture conditions 91 II. Computational analysis 91 III. Preparation of protoplast and total RNA extraction 92 IV. Plasmid construction and RNA transcription 93 V. Hex1 protein expression in E. coli 94 VI. Electrophoretic mobility shift assays 94 VII. Fungal protein extraction and western blot analysis 95 VIII. qRT-PCR and Northern blot analysis 96 RESULTS I. Structure of Hex1 protein and homology with eIF-5A 99 II. Purification of Hex1 protein from E. coil and western blot analysis 99 III. Plasmid construction and Hex1 protein manipulation 102 IV. Specific binding of Hex1 to both 5ʹ and 3ʹ UTR of FgV1 plus- strand RNA 104 V. Strand specific quantification of FgV1 viral RNA accumulation 107 VI. Effect of Hex1 on strand specific RNA synthesis in protoplast 111 DISCUSSION 115 LITERATURE CITED 122 ABSTRACT (in Korea) 129 ACKNOWLEDGEMENTS 133 LIST OF TALBLES CHAPTER I page Table 1. Fungal strains used in this study 43 Table 2. Average intercalary lengths (= cell length) of the HEX1 mutants 67 CHAPTER II Table 1. Fungal strains used in this study 98 LIST OF FIGURES GENERAL INTRODUCTION page Fig. 1. Genome organization of Fusarium graminearum virus 1 19 CHAPTER I page Fig. 1. Phylogenetic tree of predicted amino acid sequences of HEX1 homologs 45 Fig. 2. Deduced amino acid sequence alignment with closely related plant-pathogenic fungi 46 Fig. 3. Construction strategy of HEX1 gene-deletion, over-expression, and complementation mutants in F. graminearum 48 Fig. 4. Southern blot hybridization 49 Fig. 5. Colony morphology of mutants 51 Fig. 6. Radial growth after 5 days on PDA 53 Fig. 7. Conidial production after 5 days in CMC liquid medium 54 Fig. 8. Subcellular localization of Woronin bodies (WBs) 56 Fig. 9. Western blot analysis using Hex1 antibody in the HEX1 mutants 57 Fig. 10. Growth of hyphae of WT-VF, △hex1-VF, HEX OE-VF, and △hex1::HEX1-VF strains after amputation with a razor blade 59 Fig. 11. Cytoplasmic bleeding in the HEX1 deletion strain 60 Fig. 12. Virulence assay with HEX1 mutants and the wild type (WT) of F. graminearum that were infected with FgV1 (indicated by VI) or virus free (indicated by VF) 61 Fig. 13. qRT-PCR analysis of expression of HEX1 and its orthologs in F. graminearum and Cryphonectria parasitica as affected by mycovirus infection 63 Fig. 14. Quantification of FgV1 viral RNA accumulation in F. graminearum using qRT-PCR 64 Fig. 15. Quantification of FgV1 viral RNA accumulation in F. graminearum using sqRT-PCR 66 CHAPTER II page Fig. 1. Deduced amino acid sequence alignment of Hex1 from N. crassa (NcHex1), F. graminearum (FgHex1) and IF-5A from F. graminearum (FgIF5A), P. aerophilum (PaIF5A), M. jannaschii (MjIF5A) …100 Fig. 2. Predicted structures of Hex1 and IF-5A …101 Fig. 3. E. coli expression of Hex1 and western blot analysis 103 Fig. 4. Construction of plasmid and Hex1 protein manipulation 105 Fig. 5. Investigation of direct interaction using RNA-protein complexes formation …106 Fig. 6. Relative free RNA probes in EMSA experiments 108 Fig. 7. Electrophoretic mobility shift assay (EMSA) of FgV1 RNA-Hex1 protein complex 109 Fig. 8. Plus- and minus-strand RNA accumulation in all virus-infected strains 110 Fig. 9. Relative plus- and minus-strand viral RNA accumulation in all virus-infected strains of F.graminearum 112 Fig. 10. Time course mediated quantification of FgV1 viral RNA replication in virus-infected F. graminearum protoplasts 113 Fig. 11. Relative plus- and minus-strand viral RNA accumulation in all virus-infected protoplasts 114 | - |
| dc.format | application/pdf | - |
| dc.format.extent | 4485314 bytes | - |
| dc.format.medium | application/pdf | - |
| dc.language.iso | en | - |
| dc.publisher | 서울대학교 대학원 | - |
| dc.subject | Fusarium graminearum virus 1 | - |
| dc.subject | Fusarium graminearum | - |
| dc.subject | Hex1 protein | - |
| dc.subject | host factor | - |
| dc.subject | RNA-protein binding | - |
| dc.subject | viral RNA replication | - |
| dc.subject.ddc | 630 | - |
| dc.title | Functional analysis of HEX1 gene in interaction between Fusarium graminearum and Fusarium graminearum virus 1 | - |
| dc.title.alternative | 붉은 곰팡이 바이러스 1 과 상호작용하는 붉은 곰팡이의 HEX1 유전자에 대한 기능분석연구 | - |
| dc.type | Thesis | - |
| dc.contributor.AlternativeAuthor | Moonil Son | - |
| dc.description.degree | Doctor | - |
| dc.citation.pages | xiii, 132 | - |
| dc.contributor.affiliation | 농업생명과학대학 농생명공학부 | - |
| dc.date.awarded | 2015-02 | - |
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