The 3D genome of Gigaspora margarita unveils stable chromatin and nucleolar organization and symbiont-dependent genome dynamics.

Arbuscular mycorrhizal fungi (AMF) are widespread plant symbionts that enhance nutrient acquisition and influence ecosystem productivity. Previous chromosome-level assemblies of the model species Rhizophagus irregularis revealed a two-compartment genome architecture (active A and repressed B chromatin compartments), yet its conservation across evolutionarily distant AMF lineages remains unresolved. Here, we present a chromosome-scale and 3D genome assembly of Gigaspora margarita isolate BEG34 - the largest and most repeat-rich AMF genome to date - alongside that of its obligate endobacterium, Candidatus Glomerobacter gigasporarum (CaGg), using PacBio HiFi and Hi-C sequencing. The G. margarita genome comprises 43 chromosomes (792 Mb) organized into A/B compartments and Topologically Associating Domains, structures that are conserved across two AMF orders and remain stable irrespective of the presence of endobacteria in germinating spores. We uncover 21 divergent rDNA operons distributed across six chromosomes and show that these physically interact, suggesting conserved nucleolar organization. We also reveal that the CaGg genome is tripartite and mobilome-rich, encoding prophages, an orphan CRISPR array, and complete pathways for many novel and essential cofactors, including heme, which may enhance host bioenergetics. We also find that the endobacterium's presence modulates transposable elements expression in G. margarita. These findings reveal conserved principles of chromatin architecture in AMF symbionts and highlight the tight molecular interplay between fungal hosts and their endosymbionts, offering new insights into genome evolution and symbiotic adaptation.