Integrative Analyses of Protein Glycosylation and Cell Wall Proteins to Determine Virulence Networks in Fusarium graminearum = 붉은곰팡이의 병원성 네트워크 구명을 위한 단백질 당화 및 세포벽 단백질의 통합적 분석

국문 초록 (Abstract)

식물 병원성 곰팡이는 농업과 인류 보건에 심각한 위협을 가하고 있으나, 그 분자적 병원성 기작은 아직 충분히 규명되지 않았다. 다양한 조절 기작 가운데, 단백질 당쇄화(glycosylation)는 곰팡이의 발달, 스트레스 적응, 숙주 상호작용을 조절하는 핵심적인 번역 후(post-translational) 단백질 수식으로 주목받고 있다. 당쇄화는 단백질의 접힘, 안정성, 세포 내 수송 및 면역 회피에 직접적으로 관여하여 병원성 과정 전반을 형성한다. 그러나 모델 생물에서 활발히 연구된 것과 달리, 식물 병원성 곰팡이에서의 당쇄화 연구는 단편적으로 이루어져 있으며, 발달과 병원성을 통합적으로 연결하는 체계적 연구는 부족하다. 본 학위논문에서는 우선 전사체학, 단백질체학, 당체학 연구를 종합하여 곰팡이 글리코바이올로지의 최근 성과를 정리하고, 글리코유전자(glycogene) 다양성, 계층적 조절 네트워크, 당단백질 기반 병원성 기작을 개괄하였다. 이어, 식물 병원성 곰팡이 F. graminearum을 대상으로 세포벽 단백질(cell wall proteins, CWPs)의 포괄적 단백질체 분석을 수행하였다. 총 1,373종의 CWPs가 동정되었으며, 이들은 비공유 결합, 이황화 결합, 알칼리-민감 결합, 글리코실포스파티딜이노시톨(GPI) 앵커 등 다양한 결합 형태를 보였다. 전사체 및 단백질체 데이터 통합 분석을 통해 발달 단계에 따른 CWPs의 차등적 발현을 확인하였고, FCA7과 같은 특정 단백질의 기능 연구를 통해 세포벽–숙주 접점에서의 국소화 및 병원성 기여도를 검증하였다.
마지막으로, F. graminearum의 단백질 당쇄화 유전자와 당 구조, 그리고 당단백질의 기능을 체계적으로 규명하였다. 총 65개의 당쇄화 관련 유전자를 분석하였으며, N- 및 O-당쇄 구조를 전반적으로 규명하였다. 특히 ALG3와 ALG12 유전자 결실은 코어 N-당쇄 구조의 절단을 유발하였고, 이는 전사인자, 인산화효소, 과산화효소 등 다양한 당단백질의 변화를 초래하여 발달 및 병원성에 광범위한 영향을 미쳤다. 종합하면, 본 연구는 F. graminearum의 병원성을 규정하는 당쇄화 중심 네트워크를 다층적으로 제시하였다. 글리코유전자, 당쇄 구조, 당단백질 기능을 곰팡이 발달과 병원성에 연결함으로써, 곰팡이 글리코바이올로지의 기초적 이해를 확장하였을 뿐 아니라, 당쇄화를 기반으로 한 새로운 항진균 전략의 가능성을 제시하였다.

다국어 초록 (Multilingual Abstract)

Pathogenic fungi pose severe threats to agriculture and human health, yet the molecular basis of their virulence remains incompletely understood. Among diverse regulatory mechanisms, protein glycosylation has emerged as a pivotal post-translational modification that governs fungal development, stress adaptation, and host interactions. Glycosylation influences protein folding, stability, trafficking, and immune evasion, thereby shaping infection processes. Despite its recognized importance in model organisms, research on fungal glycosylation remains fragmented, lacking a unified framework that links glycosylation to fungal development and pathogenicity. To address this gap, I first synthesized recent advances in fungal glycobiology, integrating transcriptomic, proteomic, and glycomic studies to highlight interspecific variation in glycogenes, hierarchical regulatory networks, and glycoprotein-mediated virulence mechanisms. Building on this conceptual framework, I performed a comprehensive proteomic analysis of cell wall proteins (CWPs) in the plant pathogenic fungus Fusarium graminearum. A total of 1,373 CWPs were identified, revealing diverse linkage types, including non-covalent interactions, disulfide bonds, alkali-sensitive linkages, and glycosylphosphatidylinositol (GPI) anchors. Integration of transcriptomic and proteomic datasets revealed dynamic stage-specific expression of CWPs, and functional studies of selected proteins, such as FCA7, confirmed their localization at the cell wall–host interface and demonstrated their contribution to virulence. Finally, I systematically investigated the genetic and structural basis of protein glycosylation in F. graminearum. Sixty-five putative glycogenes were functionally characterized, and N- and O-glycan structures were profiled. Disruption of ALG3 and ALG12 resulted in truncated core N-glycans, which in turn altered the glycoproteome landscape, affecting regulators such as transcription factors, kinases, and peroxidases. These glycosylation defects impaired multiple biological processes and significantly attenuated virulence. Together, this work establishes a multi-layered view of the glycosylation-centered virulence network in F. graminearum. By bridging glycogenes, glycan structures, and glycoprotein functions with fungal development and pathogenicity, this dissertation not only provides fundamental insights into fungal glycobiology but also identifies glycosylation as a promising frontier for antifungal strategies.

목차 (Table of Contents)

• ABSTRACT i
• CONTENTS iv
• LIST OF TABLES vii
• LIST OF FIGURES viii
• CHAPTER I. Integrative perspectives on glycosylation networks in fungi
• ABSTRACT 2
• INTRODUCTION 3
• I. Comparative analysis of glycogenes among fungal species 6
• II. Hierarchical functional roles of glycosylation in fungal development and pathogenesis 9
• III. Potential regulatory mechanisms of glycosylation in fungal virulence 44
• FUTURE PERSPECTIVES AND CONCLUSION 50
• LITERATURE CITED 53
• CHAPTER II. Proteomic analysis of cell wall proteins with various linkages in F. graminearum
• ABSTRACT 77
• INTRODUCTION 78
• MATERIALS AND METHODS
• I. Fungal strain and culture conditions 81
• II. Genetic manipulations and PCR primers 81
• III. Fungal transformations 91
• IV. Cell wall isolation and purification 95
• V. Sequential CWPs extraction 96
• Ⅵ. Preparation of cell wall glycoprotein samples 97
• Ⅶ. Tryptic digestion 97
• Ⅷ. LC-MS/MS analysis and protein identification 98
• Ⅸ. Bioinformatic analysis 100
• Ⅹ. Wheat coleoptile virulence assay 101
• RESULTS AND DISCUSSION
• I. Extraction of CWPs in F. graminearum 103
• II. Distinct linkages of CWPs in F. graminearum 110
• III. Functional divergence between typical and atypical CWPs 113
• IV. Functional variation of CWPs based on linkage types 116
• Ⅴ. Differential expression of CWPs across developmental stages 120
• Ⅵ. Functions and localizations of the selected CWPs 128
• LITERATURE CITED 133
• CHAPTER III. Integrative glycomic analysis reveals the crucial role of protein glycosylation in fungal pathogenesis
• ABSTRACT 144
• INTRODUCTION 145
• MATERIALS AND METHODS
• I. Strains and culture conditions 149
• II. Nucleic acid manipulations, Southern blotting, and PCR 149
• III. Genetic manipulations and fungal transformations 168
• IV. Vegetative growth, conidiation, and sexual development 168
• V. Cellophane membrane penetration assay 169
• Ⅵ. Stress response 169
• Ⅶ. Virulence test and mycotoxin analysis 170
• Ⅷ. HPLC analysis of N&O-linked glycans from cell wall mannoproteins 171
• Ⅸ. Microscopic observation 172
• Ⅹ. Western blotting 172
• Ⅺ. Cell wall components analysis 173
• Ⅻ. Preparation of glycoprotein samples for proteomic analysis 175
• ⅩⅢ. Tandem mass spectrometry analysis 175
• ⅩⅣ. Data analysis 176
• ⅩⅤ. Bioinformatic analysis 177
• RESULTS
• I. Identification of protein glycosylation-related genes in F. graminearum 178
• II. Construction of the F. graminearum mutant library involved in protein glycosylation 184
• III. Profiling the collective phenotypic traits of the F. graminearum mutant library 193
• IV. Glycan maturation does correlate with the various stress responses 209
• V. The composition of cell wall components and glycan structures showed notable differences between conidia and mycelia 228
• Ⅵ. ALG3 (Fg26583) and ALG12 (Fg03053) are implicated in various developmental processes and N-glycan biosynthesis 236
• Ⅶ. Deletion of ALG3 (Fg26583) and ALG12 (Fg03053) affect the glycosylation pattern of Cnb1, Gic1, and Glx, which are essential for the virulence of F. graminearum 241
• Ⅷ. Quantitative glycoproteomics profiling and functional classification of identified glycoproteins in F. graminearum 244
• Ⅸ. Glycosyltransferases involved in the early core N-glycosylation pathway influence global protein glycosylation across various biological processes 251
• Ⅹ. Identification of virulence-related glycoproteins affected by early core N-glycosylation in F. graminearum 255
• DISCUSSION 262
• LITERATURE CITED 273
• ABSTRACT (in Korean) 293
• LIST OF TABLES
• CHAPTER I
• page
• Table 1. TFs regulating glycogenes 9
• Table 2. N&O-Glycogenes and glycan structures discussed in this review 14
• Table 3. Glycoproteins in fungal virulence and development 37
• CHAPTER II
• Table 1. F. graminearum strains used in this study 83
• Table 2. Primers used in this study 84
• Table 3. The sources of all previously studied transcriptome and proteome data used in this study 101
• CHAPTER Ⅲ
• Table 1. Primers used in this study 151
• Table 2. Comparative analysis of protein glycosylation-related genes among fungal species 179
• Table 3. Collection of phenotypic analyses for 65 deletion mutants involved in protein glycosylation in F. graminearum 185
• Table 4. Monosaccharide components analysis in cell wall and glycoprotein in F. graminearum 230
• Table 5. Glycoproteins identified in previous studies 257
• LIST OF FIGURES
• CHAPTER I
• page
• Figure 1. Potential regulatory mechanisms of glycosylation in fungal virulence 45
• CHAPTER II
• page
• Figure 1. Targeted gene deletion 94
• Figure 2. Schematic representation of proteomic analysis of fungal cell wall proteins (CWPs) using liquid chromatography with tandem mass spectrometry (LC-MS/MS) 105
• Figure 3. Preparation of native CWPs 106
• Figure 4. The total number of CWPs decreases as the cutoff value for peptide counts increases 107
• Figure 5. Venn diagram showing the distribution of CWPs in the fungal pathogens 108
• Figure 6. Comparative BLAST matrix analysis of total CWPs 109
• Figure 7. Profile of F. graminearum CWPs isolated from cell walls using various extraction methods 111
• Figure 8. Comparison between typical and atypical CWPs 114
• Figure 9. Functional characterization of CWPs according to their types of linkages 117
• Figure 10. Protein-protein interaction network analysis for the CWPs having more than two types of linkages 119
• Figure 11. Differential expression and linkage distribution of CWP genes across developmental stages 122
• Figure 12. Comparison of functional enrichment of differentially expressed CWP genes across developmental stages 125
• Figure 13. Gene ontology enrichment analysis for the down-regulated cell wall protein genes 127
• Figure 14. Virulence test of selected CWP mutants on wheat coleoptiles 130
• Figure 15. Subcellular localization of Fca7 and Cpd1 131
• CHAPTER Ⅲ
• Figure 1. Comparative analysis of protein glycosylation-related genes among fungal species 181
• Figure 2. Confirmation of gene involved in protein glycosylation deletion mutants by Southern blot analysis 191
• Figure 3. Phenotypic traits of genes involved in protein N, O-glycosylation at the ER and Golgi 199
• Figure 4. Profiling overall protein N-glycosylation pathway genes in F. graminearum 201
• Figure 5. Profiling overall protein O-glycosylation pathway genes in F. graminearum 203
• Figure 6. Spearman's rank correlation among multiple phenotypes was calculated for each mutant phenotype 206
• Figure 7. Transcript levels of genes involved in protein glycosylation across these stages 207
• Figure 8. Characterizing in vitro phenotypic traits of genes involved in protein glycosylation 211
• Figure 9. Phenotype of deletion mutants under various stress conditions 225
• Figure 10. Spearman's rank correlation among multiple phenotypes under various stress conditions was calculated for each mutant phenotype 227
• Figure 11. The proportion of cell wall components and glycan structures differed between conidia and mycelia 232
• Figure 12. F. graminearum Fg03053 (Alg12) and Fg26583 (Alg3) are involved in N-glycan biosynthesis within the ER lumen 239
• Figure 13. Fg03053 and Fg26583 are involved in the N-glycosylation of glycoproteins Cnb1, Gic1, and Glx, which are required for the virulence of F. graminearum 242
• Figure 14. Sample preparation for glycoproteomic analysis 245
• Figure 15. PCA analysis for glycoproteomic samples 247
• Figure 16. Prediction of subcellular localization and functional classification of identified glycoproteins 249
• Figure 17. Pearson correlation analysis between whole-cell extracts and glycoprotein samples in the wild type, fg03053, and fg26583 deletion mutants 252
• Figure 18. Glycoproteomic analysis of wild type, Δfg03053 (ALG12), and Δfg26583 (ALG3) 254