3D genome architecture of Zynoseptoria tritici IPO323

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 forms an inter-domain interaction network. 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. Overall design: The reference strain Z. tritici IPO32326 was preserved in 50% glycerol at -80°C. Pre-cultures were prepared with inoculations of ~100uL of glycerol stock spore suspension onto solid YMS medium (4 g/L yeast extract, 4 g/L malt extract, 4 g/L sucrose, and 12 g/L of agar) and grown for 4 days at 18°C. Spores were scraped from plates and re-suspended into PBS buffer (8g/L NaCl, 1.44g/L Na2HPO4, 0.2g/L KCl, 0.24g/L KH2PO4) for sample preparation for epigenomics sequencing as described in Möller et al. 202332. Spores were fixed with formaldehyde according to PhaseGenomics instructions (Seattle, Washington, USA). Hi-C experiments were performed with the Phase Genomics Proximo Hi-C Kit (Fungal), which combines four restriction enzymes to ensure homogeneous digestion at AT-rich genomic regions. The resulting libraries were sequenced at Biomarker Technologies GmbH (BMKgene, Münster, Germany). The sequencing was performed with Illumina NovaSeq X, paired-end 150bp reads. Sample preparation, Hi-C experiment, and sequencing were performed for two independent replicates.