Pervasive influences of repeat-induced point mutation on single-copy functional genes reveal its evolutionary importance in Neurospora crassa

Background Repeat-induced point mutation (RIP) is a hypermutation mechanism that targets repetitive sequences to control transposable element proliferation but also impacts single-copy genes through off-target effects. The extent to which genes can tolerate high mutational loads from RIP remains unclear. Results Here, we examine protein-coding genes that have been affected by RIP-associated hypermutagenesis across 134 Neurospora crassa strains, leveraging two highly divergent reference genomes to enhance detection accuracy. Our data reveal that RIP-like mutations significantly affect protein-coding genes, especially single-copy genes lacking ancestral paralogs or proximity to repetitive sequences. We identified hundreds of Highly RIP-affected gene (HRAGs) per strain (average ~ 259–293), with a total of 3188 non-redundant HRAGs across the population, most with lineage-specific RIP-like mutations. RIP-like mutations are generally not shared across different strains within a single HRAG, suggesting independent origins of these mutations, as further confirmed by parent-progeny sequencing. Experimental validation, including gene knockout, complementation assays, and fitness evaluations, demonstrated that HRAGs can even be essential for fitness or survival. Notably, one HRAG was associated with resistance to sorbic acid, a commonly used preservative. Conclusions Our findings suggest that RIP, beyond suppressing gene duplication, may contribute to the evolution of functional genes, including single-copy ones. This study also highlights how tracing RIP-associated signatures can help rapidly identify functionally important genes, enhancing our ability for gene mining in fungal pathologies.