Molecular basis of early infection in plant pathogenic Fusarium species = 식물병원성 Fusarium 속 초기 감염의 분자적 기반 구명

다국어 초록 (Multilingual Abstract)

Plant pathogenic fungi have long posed a major threat to crop health and global food security. Plant diseases are initiated when pathogens attach to the host surface and penetrate its outer barriers to establish infection. Because penetration is a critical step in pathogenesis, a comprehensive understanding of the molecular mechanisms underlying this process is essential for developing effective control strategies against phytopathogenic fungi. Nevertheless, the genetic regulation of penetration in the plant pathogenic Fusarium species remains poorly understood. In this dissertation, I focused on virulence factors that govern the initial phase of host infection, with particular emphasis on single-gene contributions in Fusarium species. First, I characterized a key enzyme linked to carbon metabolism that is essential for penetrating and colonizing the host plant. Pyruvate carboxylase (PYC) plays an important anaplerotic role by catalyzing pyruvate carboxylation to form oxaloacetate. Surprisingly, the PYC1 knockout mutant of the vascular wilt fungus F. oxysporum exhibited pleiotropic defects in fungal development and early plant penetration, unlike F. graminearum, where PYC1 knockout did not significantly impact virulence. Through comprehensive metabolic profiling, distinct reprogramming in central carbon metabolism was identified between the two fungal pathogens. These findings highlight the species-specific role of PYC1-mediated anaplerosis in shaping fungal metabolism. Secondly, I identified a transcription factor (TF), FgIec1, that is essential for the early penetration process of the wheat head blight fungus F. graminearum. Using a membrane-based penetration assay to monitor fungal developmental dynamics, I integrated transcriptome and proteome analyses, revealing that ribosome biogenesis is tightly regulated and should be downregulated during penetration to optimize energy use. Chromatin immunoprecipitation sequencing (ChIP-seq) further revealed that the INO80 chromatin remodeling complex is a pivotal regulator of this process. These findings provide novel insights into the molecular mechanisms governing host penetration by plant pathogenic fungi. Taken together, this study not only deepens our mechanistic understanding of Fusarium infection biology but also provides genetic resources for innovative approaches to sustainable plant disease control.

국문 초록 (Abstract)

식물병은 병원균이 기주 식물 표면에 부착하고 침투한 후 감염을 성공함으로써 시작된다. 식물병원성곰팡이의 초기 감염 과정은 병원성의 핵심 단계이므로 해당 과정의 분자적 기전을 이해하는 것은 식물병원성곰팡이의 효과적인 방제 전략을 개발하는 데 필수적이다. 그러나 전세계적으로 널리 분포하고 경제적으로 막심한 피해를 야기하는 Fusarium 속 식물병원균에서의 초기 감염 과정에 대한 유전적 조절 메커니즘은 연구가 충분히 이루어지지 않았다. 따라서 본 학위 논문은 Fusarium 속 곰팡이의 초기 감염 과정에서 단일 유전자 수준에서 기여할 수 있는 병원성 인자를 찾고 그 기능을 연구하였다. 첫번째로 기주 식물의 초기 감염에 필수적인 탄소 대사 관련 핵심 효소를 구명하였다. 피루브산 카복실화효소(PYC)는 피루브산을 카복실화하여 옥살로아세트산을 보충해주는 역할을 한다. 시들음병균 F. oxysporum에서 PYC1 결손 돌연변이는 곰팡이 발달 과정 및 초기 감염 과정에 결함을 보였으나 F. graminearum에서는 PYC1 유전자의 결손이 병원성에 큰 영향을 주지 않았다. 비타겟 및 타겟 대사체 분석을 통해 두 병원균은 PYC1 결손 시 중심 탄소 대사 및 전반적인 대사체의 변화 양상이 달라지는 것을 확인하였다. 이 결과는 PYC1가 매개하는 탄소 대사가 종 특이적으로 곰팡이 대사 조절에 기여함을 밝혔다. 더 나아가 밀에 붉은곰팡이병을 일으키는 F. graminearum에서 초기 침투 과정에 필수적인 전사인자(FgIec1)를 확인하였다. 반투과성 막을 활용하여 초기 침투 과정에서 곰팡이의 감염 역학을 관찰하고, 통합된 전사체 및 단백질체 분석을 통해 초기 감염 과정 특이적으로 유도되거나 억제되는 유전자 군집을 구명하였다. 특히, 초기 감염 과정 특이적으로 리보솜 생합성이 조절되며 에너지 사용을 최적화하기 위해 리보솜 생합성이 침투 단계에 따라 억제되는 것을 밝혔다. 또한, 염색질 면역침강 시퀀싱(ChIP-seq)을 통해 INO80 염색질 리모델링 복합체가 FgIec1과 함께 리보솜 생합성을 핵심적으로 조절함을 구명하였다. 이러한 발견은 식물병원성 곰팡이의 초기 감염 과정을 조절하는 분자적 기전에 대해 새로운 통찰을 제공한다. 본 연구는 Fusarium 감염 생물학에 대한 분자적 이해를 심화시켰을 뿐만 아니라, 지속가능한 식물병 방제를 위한 유전적 자원을 제공한다.

목차 (Table of Contents)

• ABSTRACT i
• CONTENTS iii
• LIST OF TABLES vi
• LIST OF FIGURES vii
• CHAPTER I. Molecular strategies and regulatory networks in the early infection stages of plant pathogenic Fusarium species
• ABSTRACT 2
• INTRODUCTION 3
• Ⅰ. Early infection structures in Fusarium species 6
• Ⅱ. Key genetic factors involved in early plant penetration in Fusarium species 8
• ⅠⅡ. Translational research for disease control 21
• CONCLUSIONS AND PERSPECTIVES 24
• LITERATURE CITED 34
• CHAPTER II. Oxaloacetate anaplerosis differently contributes to pathogenicity in plant pathogenic fungi Fusarium graminearum and F. oxysporum
• ABSTRACT 56
• INTRODUCTION 57
• MATERIALS AND METHODS
• I. Fungal strains and growth conditions 60
• II. Vegetative growth, conidiation, and sexual development 60
• III. Nucleic acid manipulations, Southern blotting and PCR 61
• IV. Genetic manipulations and fungal transformations 61
• V. Microscopic observation 67
• Ⅵ. Conidia germination assay 67
• Ⅶ. Virulence tests 67
• Ⅷ. Mycotoxin analysis 68
• Ⅸ. PG plate assay and exo-polygalacturonase activity 69
• X. Quantitative PCR and RT-qPCR 69
• Ⅺ. Metabolic profiling of mycelia samples 70
• Ⅻ. Untargeted analysis using LC-orbitrap MS 72
• XIII. 3-Nitrophenylhydrazine derivatization for targeted analysis 73
• XIV. Data analysis 73
• RESULTS
• I. Identification and characterization of the pyruvate carboxylase-encoding gene (PYC1) in F. graminearum 75
• II. FoPYC1 is important for vegetative growth, conidiation, and virulence in F. oxysporum 80
• III. The deletion of PYC1 depletes TCA cycle metabolites in F. graminearum and F. oxysporum 85
• IV. The PYC1 deletion triggered a substantial impact on the global metabolome in both F. oxysporum and F. graminearum 90
• V. FoPYC1 contributes to penetration and root colonization 95
• Ⅵ. The metabolome was highly correlated with pathogenicity 98
• Ⅶ. FoPYC1 contributes to early infection process 101
• DISCUSSION 105
• LITERATURE CITED 110
• CHAPTER III. Energy trade-off via ribosome biogenesis control underlies early plant penetration in plant pathogenic fungi
• ABSTRACT 123
• INTRODUCTION 124
• MATERIALS AND METHODS
• I. Fungal strains and culture condition 127
• II. Vegetative growth, conidiation, and sexual development 127
• III. Nucleic acid manipulations, Southern blotting and PCR 128
• IV. Genetic manipulations and fungal transformations 128
• V. Microscopic observation 134
• Ⅵ. Virulence test 134
• Ⅶ. Mycotoxin analysis 134
• Ⅷ. Quantitative PCR and RT-qPCR 135
• Ⅸ. Penetration assay 136
• Ⅹ. ChIP-seq analysis and ChIP-qPCR 137
• ⅩI. Transcriptome analysis 138
• Ⅻ. Proteome analysis 139
• XIII. Data analysis 141
• RESULTS
• I. Identification of transcription factor (TF) knockout mutants with penetration defects in Fusarium graminearum 142
• II. Identification of C2H2 zinc finger transcription factor FgIec1 in F. graminearum 149
• III. FgIEC1 is important for the early plant penetration process 151
• IV. FgIec1 acts as key regulator of the chromatin remodeler INO80 complex 158
• V. Combined transcriptome and proteome analyses reveal a penetration-specific gene regulation 166
• Ⅵ. FgIEC1 orchestrates ribosome biogenesis via the TORC1 pathway 171
• Ⅶ. Energy metabolism is upregulated during the penetration process 175
• DISCUSSION 179
• LITERATURE CITED 183
• ABSTRACT (in Korean) 196