Exploration of Host Factors Governing Fusarium graminearum virus 2 Accumulation and Antiviral RNAi Response in Fusarium graminearum = 붉은곰팡이 바이러스 2의 축적과 항바이러스 RNA 간섭 반응을 조절하는 붉은곰팡이의 기주인자들에 관한 연구

다국어 초록 (Multilingual Abstract)

Mycovirus–fungal interactions often lead to remarkable physiological changes in the host, occasionally resulting in hypovirulence, which highlights the potential of mycoviruses as biocontrol agents. However, the molecular basis of fungus–mycovirus interactions including antiviral responses remains poorly understood. To elucidate the regulatory mechanisms underlying antiviral RNAi and host–virus interplay, I investigated host factors affecting viral accumulation and RNAi induction in Fusarium graminearum infected with Fusarium graminearum virus 2 (FgV2).
FgV2 was horizontally transferred into a transcription factor (TF) gene deletion mutant library of F. graminearum, and comparative analyses of mycelial growth and viral accumulation revealed an inverse correlation between them. Defective interfering RNAs mainly derived from FgV2 RNA3 were detected in wild type and several TF mutants. In ΔGzGATA007, FgV2 infection attenuated resistance to hydroxyurea, while ΔGzWING020 and ΔGzZC121 exhibited altered ROS accumulation accompanied by transcriptional changes in ROS-related genes. These suggest that the F. graminearum–FgV2 system is a valuable model for dissecting fungus–virus interactions.
Further analysis identified GzC2H056 as a key regulator of antiviral RNAi. Expression of FgDICER-2 and FgAGO-1 was not properly induced in ΔGzC2H056 during FgV2 infection. In wild-type, GzC2H056 was significantly induced prior to FgDICER-2 and FgAGO-1 during FgV2 infection. Domain prediction and in vitro biochemical assays revealed that GzC2H056 is an RNA-binding E3 ubiquitin ligase. GzC2H056 co-localized with Pab1 and Xrn1, indicating its presence in stress granules and processing bodies. Transcriptome analysis revealed alterations of mRNA turnover–related genes and putative TFs linked to FgDICER-2 and FgAGO-1 expression in ΔGzC2H056 during FgV2 infection.
Moreover, FgDICER-2, FgAGO-1, and GzC2H056 were up-regulated in virus-free ΔFgSmt3 and ΔFgUbc9 mutants, implicating SUMOylation in the transcriptional repression of antiviral RNAi. Deletion of GzC2H056 abolished this up-regulation, confirming that GzC2H056 mediates the derepression of FgDICER-2 and FgAGO-1 in SUMOylation-defective backgrounds. Promoter–GFP reporter assays further demonstrated that this SUMOylation-dependent regulation occurs at the promoter level for FgDICER-2. I further identified GzZC098 as a conserved transcription factor that functions independently of GzC2H056 to repress antiviral RNAi, and whose RNAi-repressive activity is suggested by bioinformatic and genetic evidence to be modulated by SUMOylation.
Collectively, this study elucidates host factors related to FgV2 accumulation and transcriptional regulatory mechanism governing antiviral RNAi in F. graminearum, providing new insights into fungal antiviral defense and host–virus interactions.

국문 초록 (Abstract)

곰팡이바이러스와 균류의 상호작용은 기주에서 현저한 생리적 변화를 일으키며, 때로는 병원성 약화(hypovirulence)를 초래하여 곰팡이바이러스가 생물학적 방제제로 활용될 가능성을 보여준다. 그러나 항바이러스 반응을 포함한 균류–곰팡이바이러스 상호작용의 분자적 기작은 아직 충분히 규명되지 않았다. 본 연구에서는 항바이러스 RNA 간섭 (RNAi) 및 기주–바이러스 상호작용의 전사 조절 메커니즘을 규명하기 위해, 붉은곰팡이와 붉은곰팡이 바이러스 2 (FgV2)를 이용하여 바이러스 축적과 항바이러스 RNAi 유도에 관여하는 기주 인자를 탐색하였다.
FgV2를 붉은곰팡이 전사인자 유전자 결실 돌연변이 라이브러리에 전이시킨 결과, 균사 생장과 바이러스 축적 수준 사이에 역상관 관계가 확인되었다. FgV2 RNA3에서 유래한 결손 간섭 RNA가 야생형과 여러 전사인자 결실 돌연변이에서 검출되었다. ΔGzGATA007에서는 FgV2 감염 시 하이드록시유레아 저항성이 감소하였으며, ΔGzWING020과 ΔGzZC121에서는 활성산소종 조절 유전자의 전사 변화와 함께 활성산소종의 축적 수준이 변하였다. 이러한 결과는 붉은곰팡이–FgV2 시스템이 균류–곰팡이바이러스 상호작용을 규명하기 위한 유용한 모델임을 시사한다.
또한, GzC2H056이 항바이러스 RNAi의 핵심 조절인자임을 확인하였다. ΔGzC2H056에서는 FgV2 감염 중 FgDICER-2와 FgAGO-1의 발현이 정상적으로 유도되지 않았으며, 야생형에서는 FgV2 감염 시 GzC2H056이 두 유전자보다 앞서 강하게 유도되었다. 도메인 예측과 in vitro 생화학적 분석 결과, GzC2H056은 RNA 결합 능력을 가진 E3 유비퀴틴 리가아제임이 밝혀졌다. 또한 GzC2H056은 Pab1 및 Xrn1과 공존하는 것이 관찰되어 stress granule과 processing body에 위치함을 확인하였다. 전사체 분석 결과, ΔGzC2H056에서는 mRNA 대사 관련 유전자 및 FgDICER-2 및 FgAGO-1 발현과 연관된 TF 유전자의 발현 변화가 관찰되었다.
더 나아가, 바이러스가 없는 ΔFgSmt3 및 ΔFgUbc9 변이주에서 FgDICER-2, FgAGO-1, GzC2H056 발현이 증가하는 것이 관찰되었고, 이는 수모화(SUMOylation)가 항바이러스 RNAi의 전사적 억제에 관여함을 시사한다. ΔFgSmt3 및 ΔFgUbc9에 GzC2H056을 추가로 결실시켰을 때 FgDICER-2 및 FgAGO-1 발현 증가가 소실되어, GzC2H056이 수모화 결함 조건에서 FgDICER-2 및 FgAGO-1의 탈억제를 매개함을 확인하였다. 추가적으로 프로모터–GFP 리포터 분석을 통해, 수모화에 의한 FgDICER-2의 발현 억제가 프로모터 수준에서 이루어짐을 입증하였다. 또한 GzZC098이 GzC2H056과 독립적으로 항바이러스 RNA 간섭을 억제하는 기능을 하는 보존된 전사조절인자임을 규명하였으며, 생물정보학적 및 유전학적 증거를 통해 그 RNAi 억제 기능이 수모화에 의해 조절될 가능성이 있음을 제시하였다.
종합적으로, 본 연구는 FgV2 축적에 영향을 미치는 기주 인자와 항바이러스 RNAi의 전사 조절 메커니즘을 규명하였으며, 이는 균류의 항바이러스 방어 및 기주–바이러스 상호작용에 대한 새로운 통찰을 제공한다.

목차 (Table of Contents)

• ABSTRACT i
• CONTENTS iv
• LIST OF TABLES viii
• LIST OF FIGURES ix
• CHAPTER I. The Evolutionary Arms Race: Fungal Antiviral Defense Mechanisms and Mycoviral Counterstrategies 1
• ABSTRACT 2
• INTRODUCTION 3
• I. Physical barrier restricting mycovirus transmission 5
• Ⅱ. Sequence-specific antiviral responses targeting viral genomes 9
• Ⅲ. Other defense mechanisms and counterstrategies 17
• CONCLUSION 20
• LITERATURE CITED 21
• CHAPTER Ⅱ. Identifying transcription factors associated with Fusarium graminearum virus 2 accumulation in Fusarium graminearum by phenome-based investigation 35
• ABSTRACT 36
• INTRODUCTION 37
• MATERIALS AND METHODS 40
• I. Fungal strains and culture conditions 40
• Ⅱ. Virus transmission 40
• Ⅲ. Measurement of fungal isolates growth 41
• Ⅳ. Total RNA extraction and cDNA synthesis 42
• V. Estimation of FgV2 dsRNA accumulation 42
• Ⅵ. RT-qPCR and semi-quantitative RT-PCR 43
• Ⅶ. Defective interfering RNA extraction and sequence analysis 49
• Ⅷ. Vertical transmission of FgV2 in the fungal host through asexual reproduction 49
• Ⅸ. ROS detection 50
• Ⅹ. Protein-protein interaction (PPI) network of transcription factors associated with the FgV2 infection 50
• RESULTS 52
• I. Phenotypic analysis of FgV2-infected TF gene deletion mutant library 52
• Ⅱ. Relationship between mycelial growth and FgV2 accumulation in TF mutants 81
• Ⅲ. Defective interfering RNAs in FgV2-infected TF gene deletion mutants 84
• Ⅳ. Association of TFs to transcription of FgDICER-2 and FgAGO-1 during FgV2 infection 89
• Ⅴ. Association of TFs involved in DNA damage response or oxidative stress response with FgV2 infection 91
• Ⅵ. Protein-protein interactions of TFs related to FgV2 infection 98
• DISCUSSION 104
• LITERATURE CITED 114
• CHAPTER Ⅲ. RNA granule–associated regulation of antiviral RNAi by the RNA-binding E3 ubiquitin ligase in Fusarium graminearum 123
• ABSTRACT 124
• INTRODUCTION 125
• MATERIALS AND METHODS 129
• I. Fungal materials and culture conditions 129
• Ⅱ. Construction of gene construct for complementation and over-expression fungal mutants 129
• Ⅲ. Generation of fungal transformations 131
• Ⅳ. Total RNA extraction, cDNA synthesis, RT-qPCR, and semi-quantitative PCR analysis 140
• V. Measurement of relative FgV2 dsRNA accumulation and radial colony growth 140
• Ⅵ. Observation of subcellular localization 141
• Ⅶ. Phylogenetic analysis 141
• Ⅷ. Recombinant protein preparation 142
• Ⅸ. In vitro transcription and electrophoretic mobility shift assay (EMSA) 143
• Ⅹ. E3 ubiquitin ligase activity test 144
• Ⅺ. RNA-seq and GO term analysis 144
• Ⅻ. Quantification and statistical analysis 145
• RESULTS 147
• I. GzC2H056 transcriptionally activates antiviral RNAi components in Fusarium graminearum during FgV2 infection 147
• Ⅱ. GzC2H056 represents an uncharacterized class of RNA-binding E3 ubiquitin ligase 157
• Ⅲ. Domain dissection reveals the dual RNA-binding and ubiquitin ligase activities of GzC2H056 160
• Ⅳ. The N-terminal RNA-binding domain of GzC2H056 drives the transcriptional induction of antiviral RNAi genes 164
• V. GzC2H056 localizes to cytoplasmic SGs and PBs during FgV2 infection 168
• Ⅵ. GzC2H056 deletion alters RBP expression and global gene regulation during FgV2 infection 172
• Ⅶ. GzC2H056 transcriptionally regulates downstream TFs that control FgDICER-2 and FgAGO-1 expression 180
• DISCUSSION 186
• LITERATURE CITED 192
• CHAPTER Ⅳ. SUMOylation pathway represses antiviral RNAi components under virus-free conditions in Fusarium graminearum 203
• ABSTRACT 204
• INTRODUCTION 205
• MATERIALS AND METHODS 207
• I. Culture conditions of fungal strains 207
• Ⅱ. Construction of gene construct for fungal mutants 207
• Ⅲ. Generation of fungal transformants 211
• Ⅳ. Total RNA extraction, cDNA synthesis, and RT-qPCR 213
• V. Statistical analysis 213
• Ⅵ. Phylogenetic analysis 213
• RESULTS 215
• I. SUMOylation pathway represses antiviral RNAi components in Fusarium graminearum under virus-free condition 215
• Ⅱ. SUMOylation negatively regulates antiviral RNAi through transcriptional repression of GzC2H056 217
• Ⅲ. SUMOylation represses FgDICER-2 transcription through promoter-dependent regulation 220
• Ⅳ. Identification of GzZC098 as a putative SUMOylation-associated negative regulator of antiviral RNAi 222
• DISCUSSION 227
• LITERATURE CITED 230
• ABSTRACT (in Korean) 233