Glavincheska, I.; Lorrain, C.

Three-dimensional (3D) genome organization plays a central role in gene regulation and genome stability, yet its functional relevance in fungal pathogens remains largely uncharacterized. Here, we present a high-resolution Hi-C analysis of the wheat pathogen Zymoseptoria tritici, revealing a hierarchical genome organization. The genome adopts a Rabl-like conformation characterized by centromere clustering and the spatial segregation of accessory chromosomes, which remain tethered to core pericentromeres, supporting their mitotic stability. At finer resolution, we identify homotypic interactions among B compartments and self-interacting domains demarcated by distinct histone modifications and insulator-like sequence motifs at their boundaries. A subset of highly insulated domains enriched in heterochromatin and transposable elements engages in strong inter-domain interactions. During wheat infection, several self-interacting domains exhibit coordinated transcriptional activity, linking spatial genome folding to dynamic gene regulation. These findings uncover multi-scale principles of nuclear organization in a major fungal plant pathogen and establish a framework for investigating 3D genome function in filamentous fungi.