Fungal Genome Guard Turns Out to Be a Secret Engine of Gene Evolution

In the bread mold Neurospora crassa, a genomic defense system long viewed as a blunt weapon against invading DNA may be doing far more than scientists ever suspected. A new study published in BMC Biology reports that repeat-induced point mutation, or RIP, a hypermutation mechanism that fungi use to shred transposable elements, leaves its fingerprints across hundreds of single-copy functional genes in natural populations. The findings, from a team led by Long Wang, Sihai Yang, and Ju Huang at Nanjing University and Nanjing Agricultural University, suggest that this genome-defense machinery is not merely a destroyer of repetitive DNA but an active, population-wide force shaping the evolution of ordinary, essential genes.

RIP works during the sexual cycle of certain filamentous fungi. When the fungus detects duplicated sequences in its genome, it floods them with C-to-T mutations, effectively scrambling and disabling repetitive elements such as transposons before they can spread. The process is famously efficient at silencing genome parasites, but it has a well-known drawback: mutations do not always stay confined to the repeats. Genes located near repetitive DNA, or genes that themselves arose through duplication, can be caught in the crossfire. What has remained unclear is just how much collateral damage natural populations actually tolerate, and whether genes can survive, and even function, under such heavy mutational bombardment.

To answer that question, the researchers took an unusually broad view. Rather than examining a single laboratory strain, they analyzed 134 strains of N. crassa collected across natural populations, and they sequenced and assembled a second, highly divergent reference genome from the strain FGSC2225 to complement the classic FGSC2489 reference. Having two distantly related references matters because RIP leaves a distinctive mutational signature: a strong excess of transition mutations, particularly changes at CpA dinucleotides, which produces an elevated ratio of transitions to transversions and a characteristic depletion of the dinucleotide CpA. Using these signatures, the team could scan protein-coding genes across the population and identify those that had been hit by RIP-like hypermutation with far greater accuracy than a single-reference approach would allow.

The scale of what they found was striking. On average, each strain carried roughly 259 to 293 genes classified as highly RIP-affected genes, or HRAGs, and across the entire population the team cataloged 3,188 non-redundant HRAGs. Most of these genes carried RIP-like mutations that were specific to individual lineages rather than shared across strains, a pattern pointing to independent, repeated origins of the mutational hits. The researchers confirmed this interpretation through parent-progeny sequencing: by examining the offspring of 117 tetrads produced during sexual reproduction, they showed that new RIP-like mutations arise independently in each sexual cycle rather than being inherited as a common ancestral burden.

Perhaps the most surprising aspect of the data concerns which genes get hit. Intuition might suggest that RIP would preferentially strike genes with duplicated ancestors or genes sitting close to transposable element nests, since proximity to repeats is what draws the mutation machinery in the first place. Instead, the analysis showed the opposite: RIP-like mutations significantly affected protein-coding genes, and the effect was strongest in single-copy genes that lack ancestral paralogs and are not located near repetitive sequences. In other words, even the most protected, evolutionarily solitary genes in the genome are routinely exposed to RIP-associated hypermutation, and they persist in the population despite carrying heavy mutational loads.

That persistence raises an obvious question: can genes riddled with RIP mutations still work? The team addressed this experimentally using gene knockouts, complementation assays, and fitness evaluations. The results demonstrated that highly RIP-affected genes can be essential for fitness or even for survival, meaning that N. crassa strains are living with functional genes that have been extensively rewritten by the defense system. In one particularly vivid example, a highly RIP-affected gene was linked to resistance to sorbic acid, a preservative widely used in food and industrial applications. A gene shaped by a genome-defense mutation system turns out to have consequences for how the fungus copes with a human-made chemical stress.

The study also used comparative genomics to place RIP in a broader evolutionary context. By comparing transition-to-transversion ratios and the proportion of highly RIP-affected genes across 27 species of Ascomycete fungi, the team found that fungi capable of RIP show elevated signatures of this mutational process relative to relatives that lack it. Simulation experiments, in which RIP-like mutations were artificially introduced into the genome of the related fungus Sordaria macrospora, helped validate the detection pipeline and rule out the possibility that the observed signals were artifacts of sequencing or annotation. Phylogenetic analyses of individual HRAGs further revealed inconsistencies with the species tree, consistent with lineage-specific mutational histories rather than shared ancestry.

For decades, RIP has been framed primarily as a gatekeeper against genome inflation, a system that suppresses gene duplication by destroying duplicated sequences before they can diverge into new functions. The new findings complicate that picture. If hundreds of single-copy genes in every natural strain carry RIP-like mutations, and if many of those genes remain functional and even essential, then RIP is not simply an evolutionary dead end for the genes it touches. It may instead act as an ongoing source of genetic variation, generating novel allelic diversity on which natural selection can act. The authors suggest that RIP, beyond suppressing gene duplication, may contribute directly to the evolution of functional genes, including those that exist in only a single copy per genome.

There is also a practical payoff. Because RIP leaves a recognizable and computable mutational signature, tracing RIP-associated marks across fungal genomes offers a rapid way to flag genes that matter functionally, without waiting for slow knockout screens. The authors highlight this as a tool for gene mining in fungal pathology, where identifying functionally important genes in pathogenic fungi is a persistent challenge. If a hypermutation system marks the genes under its influence, those marks can serve as a map, guiding researchers toward genes involved in stress tolerance, host interaction, or survival, including traits such as preservative resistance that have direct industrial relevance.

The work reframes one of biology’s classic genome-defense systems as a double-edged evolutionary instrument. N. crassa has long served as the model organism in which RIP was discovered, and this population-scale analysis shows that the mechanism’s reach extends deep into the functional core of the genome. A system evolved to silence selfish DNA appears, in practice, to be continuously rewriting the fungus’s own genes, sometimes with lethal consequences, sometimes with no effect, and occasionally in ways that change how the organism survives in the world. For evolutionary biologists, the message is that genome defense and genome innovation are not separate processes; in fungi at least, they may be two faces of the same mutational machinery.

Subject of Research: Repeat-induced point mutation and its evolutionary effects on single-copy functional genes in Neurospora crassa

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

Article References: Tan, H., Chen, J., Ji, R., Sun, Y., He, Z., Xue, L., Zhang, X., Yang, S., Huang, J., & Wang, L. (2026). Pervasive influences of repeat-induced point mutation on single-copy functional genes reveal its evolutionary importance in Neurospora crassa. BMC Biology. https://doi.org/10.1186/s12915-026-02756-8

Image Credits: AI Generated

DOI: 10.1186/s12915-026-02756-8

Keywords: Neurospora crassa, repeat-induced point mutation, RIP, transposable elements, genome defense, hypermutation, single-copy genes, gene duplication, fungal genetics, population genomics, sorbic acid resistance, evolutionary biology