Investigate Fungal Effectors and Their Contribution to Fusarium Oxysporum Virulence From the Genome Evolution, Transcription Regulation, and Protein Function Perspectives

The Fusarium oxysporum species complex (FOSC) comprises more than 100 host-specific fungal pathogens and ranked as the top ten most destructive fungal plant pathogens worldwide, causing severe diseases and substantial agricultural losses. Although FOSC members share a largely conserved core genome, their pathogenic diversity and host specificity are primarily driven by lineage-specific accessory chromosomes acquired through horizontal transfer. These accessory chromosomes encode strain-specific genes, many of which contribute to host adaptation and virulence. Among them, small-secreted effector proteins play central roles in manipulating host cellular processes and facilitating infection. While individual effectors have been studied extensively, a comprehensive and integrative understanding of how effector diversity and regulation underlie lineage specificity in FOSC remains limited. This dissertation investigates the biological basis of FOSC lineage specificity through an integrated, multi-scale framework consist of genomic diversity, transcriptional regulation, and protein-level functional characterization, with a central focus on fungal effectors. In Chapter 1, comparative genomics was applied to define the global effector repertoires (Effectomes) of FOSC in an evolutionary context, using a standardized pipeline to analyze effector profiles across 30 fungal genomes spanning basal ascomycetes, closely related Fusarium species, and FOSC members. This analysis reveals patterns of effector conservation, lineage-specific expansion, and evolutionary gain or loss, highlighting the role of accessory chromosomes in shaping host-adapted effector landscapes. In Chapter 2, functional characterization of selected effectors was performed at the protein level, with a focus on fungal-specific effectors containing the Common in Fungal Extracellular Membrane (CFEM) domain. Using heterologous expression in Nicotiana tabacum, these effectors were evaluated for their roles in host immune responses, subcellular localization, and interaction with host proteins. Chapter 3 further explores genome-wide transcription factor binding using DNA affinity purification sequencing (DAP-seq) to understand how these lineage-specific effectors are transcriptionally regulated, this work. By comparing the conserved transcription factors SGE1 across 5 FOSC strains and an outgroup Fusarium verticillioides, this study dissects regulatory divergence between core and accessory chromosomes to uncover how a conserved regulators acquire lineage-specific regulatory functions. Collectively, this work provides a systematic framework linking evolutionary dynamics of accessory chromosomes to effector diversity, regulation, and function in FOSC, offering broadly applicable insights into host–pathogen interactions and fungal pathogenicity.