A systematic screen for genetic factors underpinning transposon defense systems across the fungal kingdom

Fungal genome sizes exhibit more than a 100-fold variation, largely driven by the expansion of repetitive sequences such as transposable elements (TEs). Genomic defenses have evolved to counteract TE proliferation through silencing mechanisms operating at the epigenetic or transcript level. In fungi, repeat-induced point mutation (RIP) targets TEs by recognizing repetitive sequences and inducing mutagenesis. However, the prevalence of RIP across the fungal kingdom and the fidelity of the canonical C-to-T mutation signatures remain unclear. In this study, we address these gaps by tracking shifts in genome architecture across the fungal kingdom. We show that the loss of RIP components is associated with an 80% (∼30 Mb) increase in genome size in ascomycetes (class of Leotiomycetes). To track the impact of genome defenses, we designed a quantitative screen for RIP-like mutation signatures. The phylum of ascomycetes was unique in showing enrichment in mutation signatures in non-coding and repetitive sequences, consistent with a phylogenetically restricted occurrence of RIP-like genome defense systems. Then, we gene functions associated in a phylogenetic context with RIP-like mutation signatures. We identified a zinc-finger protein as the strongest candidate underpinning a novel mechanism of genome defenses. Our findings reveal the multifaceted drivers of genome defense systems and their close ties to genome size evolution in fungi, highlighting how proximate molecular mechanisms can shape genome evolution on deep phylogenetic scales.