Fungal spatial organization predominates over grapevine host effects in shaping transcriptional programs of Rhizophagus irregularis.

Arbuscular mycorrhizal fungi (AMF) exhibit strong spatial organization, with the intraradical mycelium (IRM) interacting with host roots and the extraradical mycelium (ERM) exploring soil. However, the putative contributions of fungal spatial organization and/or host genotype to transcriptional regulation remain poorly understood. Here, we analysed the transcriptomic profiles of Rhizophagus irregularis in the intraradical mycelium (IRM) of ten grapevine rootstocks, while the extraradical mycelium (ERM) transcriptome was obtained from a pooled sample representing all rootstocks. By integrating RNA-seq datasets, we observed a clear and consistent separation between IRM and ERM transcriptomes, indicating that the spatial organization of the mycelium is the dominant driver of fungal gene expression. Across all rootstocks, 719 genes were consistently upregulated in the IRM (including fatty acid, sugar and general transporters) and 510 in the ERM (mainly associated with environmental sensing and metabolic processes), defining robust compartment-specific transcriptional signatures. Host genotype significantly modulated IRM transcriptional profiles, but this effect remained secondary to spatial differentiation. Importantly, no correlation was detected between host phylogenetic relatedness and fungal transcriptional profiles, indicating that transcriptional variation in R. irregularis is independent of host genetic relatedness. Taken together, our results support a model where transcriptional variation in R. irregularis is primarily structured by spatial organization, with host genotype only weakly tuning gene expression. These findings highlight the predominance of fungal spatial organization over host effects in shaping AMF transcriptional programs.