Epitranscriptomic RNA editing resolves Mus81 DNA repair tradeoffs in heat tolerance and meiosis

Domain: biology
Published: 2026-03-30
Editorial outcome: major_revision

Abstract
Maintaining genome integrity is essential, yet how DNA repair is balanced across life stages remains poorly understood. Here we uncover an epitranscriptomic mechanism in the fungal pathogen Fusarium graminearum that alleviates a trade-off between heat-stress adaptation and sexual reproduction. We show that FgMus81 acts independently of its nuclease activity and canonical partner FgMms4, and has dosage-dependent, stage-specific functions: restrained levels support meiosis, whereas elevated levels promote heat-stress survival. We identify a sexual stage-specific A-to-I RNA editing event that recodes FgMus81 (N420D) and tunes its abundance to meet meiotic demands without compromising stress resilience. Notably, both pre-editing and post-editing isoforms support meiotic interhomolog crossovers, but the post-editing isoform impairs mitotic recombination. Conservation of this editing across Sordariomycetes suggests evolutionary selection for stage-specific control. Together, these findings reveal an epitranscriptomic switch that partitions Mus81 functions across life stages and identify adaptive RNA editing as a regulator of homologous recombination in fungal pathogens.

Introduction
Maintaining genome integrity is a universal requirement, yet the optimal deployment of DNA repair activities is not constant across life stages and environments. Double-strand breaks (DSBs) and replication-associated lesions arise from endogenous metabolism as well as external stresses, and their misrepair threatens viability and genome stability 1, 2. In eukaryotes, homologous recombination (HR) provides a high-fidelity route to repair such lesions using homologous DNA templates, but the extent to which HR components are required—and how their activities are tuned—can differ between vegetative growth, stress conditions, and sexual reproduction 3, 4, 5.

Programmed DSBs generated by Spo11 initiate meiotic recombination and are indispensable for faithful chromosome segregation and gamete formation 6, 7, 8. Mitotic HR primarily repairs DSBs and replication-associated lesions using the sister chromatid via noncrossover pathway, preserving genomic stability and preventing chromosomal rearrangements 9. In contrast, meiotic HR promotes genetic diversity through crossovers between homologous chromosomes during meiosis I 8, 9. In both mitotic and meiotic contexts, a central step in HR is catalyzed by the Rad51 recombinase, which promotes homology search and strand invasion to form joint-molecule intermediates 10. These intermediates must then be processed through competing outcomes, including noncrossovers and crossovers. In many eukaryotes, a major class of meiotic crossovers is produced through a pathway involving ZMM proteins and the MutL\gamma complex Mlh1–Mlh3, which promotes resolution of recombination intermediates toward crossovers with interference 7, 11, 12. In parallel, additional structure-selective endonucleases can process persistent or atypical joint molecules, providing alternative routes to resolve recombination and replication-associated DNA structures. These include Mus81–Mms4, Slx1–Slx4, and Yen1, which can cleave branched intermediates (e.g., nicked junctions or related structures) to yield crossover or noncrossover products depending on substrate configuration and cellular context 13, 14. Importantly, the relative contributions of Mlh1–Mlh3 versus Mus81–Mms4/Slx1–Slx4/Yen1 are known to vary across organisms and conditions, implying that pathway choice and the division of labor among these factors are evolutionarily and physiologically plastic 7, 11, 12.

Mus81 is best characterized as functioning together with a regulatory partner—Mms4 (Eme1 in Schizosaccharomyces pombe; Eme1/Eme2 in mammals)—to form a structure-selective endonuclease complex whose activity depends on this partnership 15, 16. The activity of the Mus81–Mms4 complex is critical for processing replication and recombination intermediates, especially under replication stress 17, 18. It resolves toxic DNA structures such as stalled replication forks, nicked Holliday junctions, and under-replicated DNA at common fragile sites 18, 19, 20, which, if left unresolved, would disrupt chromosome segregation and cause genetic instability. Because inappropriate cleavage of HR intermediates can be harmful, Mus81–Mms4 activity is typically subject to tight regulation, including cell-cycle-dependent control and regulated access to DNA substrates, ensuring that cleavage occurs at the appropriate time and place 21, 22, 23, 24. Nevertheless, how the Mus81–Mms4 complex–centered processing is coordinated with other joint-molecule resolution pathways, and how this coordination is modulated by environmental stress versus sexual development, remains insufficiently understood in filamentous fungi. This knowledge gap is particularly relevant for species in which growth-stage stress adaptation and meiosis impose potentially conflicting constraints on genome maintenance activities.

Fusarium graminearum is the main causal agent of Fusarium head blight (FHB), a devastating disease that severely threatens global cereal crop yields 25, 26. Sexual reproduction is central to its life cycle, as forcibly discharged ascospores from sexual fruiting bodies (perithecia) are the primary inoculum for FHB epidemics 27. Meiotic recombination during sexual development greatly increases genetic diversity, enabling the rapid emergence of diverse genotypes that can adapt to environmental changes and host defenses 28, 29. This high-frequency genetic reshuffling accelerates the evolution of fungicide resistance and enhanced virulence, sustaining aggressive field populations 30, 31, 32.

As a homothallic fungus, F. graminearum can self-fertilize and readily forms perithecia under laboratory conditions, making it a suitable model for studying the molecular basis of sexual development 27. Recent research has revealed that adenosine-to-inosine (A-to-I) mRNA editing acts as a key post-transcriptional regulatory mechanism during the sexual stage in F. graminearum and related filamentous ascomycetes 33, 34, 35. This epitranscriptomic modification is catalyzed by the Tad2-Tad3-Ame1 ternary complex, which is conserved within Sordariomycetes 36, 37. While Tad2-Tad3 is responsible for canonical tRNA editing at the wobble position (A34), the fungal-specific factor Ame1—expressed only during sexual development—enables the complex to target mRNAs via its interaction with Tad3 36. Although the editing machinery differs, A-to-I mRNA editing is also a widespread phenomenon in animals and bacteria 37, 38, 39, 40.

Functionally, inosine is interpreted as guanosine by the cellular translation machinery, so A-to-I editing can alter codons and recode proteins, similar to A-to-G substitutions at the RNA level. Genome-wide studies have identified tens of thousands of A-to-I editing sites in the perithecia of F. graminearum and Neurospora crassa, mainly within coding regions 41, 42. While the crucial roles of Ame1 and some specific A-to-I editing events in fungal sexual development have been established 36, 40, 43, 44, the functions of most editing sites remain unclear. This raises key questions about why RNA editing is maintained as a regulatory mechanism instead of fixing adaptive changes in DNA, and what advantages temporary RNA modifications offer over permanent genomic mutations 45, 46.

In this work, we dissect key HR components in F. graminearum and uncover an epitranscriptomic mechanism that resolves a trade-off between heat-stress growth adaptation and sexual reproduction. We show that FgRad51-dependent HR is dispensable for asexual development under standard conditions but essential for sexual reproduction. Among downstream factors, FgMus81 emerges as a pivotal node linking development and environment: loss of FgMUS81 compromises ascospore formation and causes pronounced heat-stress defects in growth and pigmentation. Strikingly, these roles are uncoupled from the canonical Mus81 partner FgMms4 and from nuclease catalytic activity, indicating a noncanonical mode of action. We further identify a sexually induced, site-specific A-to-I editing event in FgMUS81 (N420D) that is required for normal sexual development. Mechanistically, the post-editing isoform exhibits reduced protein stability, attenuates a FgMus81-dependent mitotic HR readout, and supports normal meiotic progression. In contrast, forcing expression of the pre-editing isoform elevates FgMus81 output and disrupts meiotic nuclear division in a dosage-sensitive manner. A fixed genomic A-to-G substitution that mimics the edited codon phenocopies the meiotic benefits but incurs a cost to heat-stress survival, highlighting an antagonism in optimal FgMus81 dosage across different contexts and illustrating the adaptive advantage of flexible RNA editing over irreversible mutation. Comparative analyses reveal both conservation and dynamic retention of the N420D editing site across Sordariomycetes, consistent with evolutionary selection for stage-specific attenuation of Mus81 function. Together, our results establish N420D RNA editing as a single-nucleotide rheostat that fine-tunes Mus81 dosage to reconcile the competing demands of meiosis and thermal adaptation. This highlights RNA editing as an adaptive mechanism for life-cycle stage–specific optimization of genome maintenance and identifies it as a potential regulatory vulnerability for disease control.

Results
FgRad51 HR is required for sexual but not asexual development
To define the contribution of HR to development in F. graminearum, we identified homologs of core and downstream HR factors, including the recombinase gene FgRAD51 (FG1G17440) and representative meiotic crossover-associated genes FgMLH1 (FG4G25380), FgMLH3 (FG1G24760), FgMUS81 (FG1G17120), FgMMS4 (FG1G25820), FgSLX1 (FG3G17370), and FgYEN1 (FG2G38190) (Fig. 1a; Supplementary Table 1). Targeted deletion mutants were generated using the split-marker strategy 47. For each gene, at least two independent transformants with comparable phenotypes were obtained (Supplementary Table 2), and a representative strain is presented.

Across all mutants, vegetative colony morphology and radial growth on potato dextrose agar (PDA), as well as conidiation in carboxymethyl cellulose (CMC) medium, were indistinguishable from the wild type (Fig. 1b; Supplementary Fig. 1a), indicating that these HR factors are not required for asexual development under the conditions tested. By contrast, at 8 days post-fertilization (dpf), Fgrad51 exhibited a severe defect in perithecium formation. Moreover, the few perithecia that developed contained only a small number of asci, most of which were empty and lacked ascospores (Fig. 1b). Together, these data indicate that FgRad51-dependent HR is largely dispensable for vegetative growth and asexual reproduction but is essential for sexual reproduction in F. graminearum.

FgRad51, not crossover factors, confers DNA stress resistance
To test whether HR contributes to genome maintenance during vegetative growth, we challenged the indicated mutants with multiple genotoxic agents, including methyl methanesulfonate (MMS), camptothecin (CPT), hydrogen peroxide (H2O2), and hydroxyurea (HU). MMS induces DNA alkylation, CPT stabilizes topoisomerase I–DNA cleavage complexes and generates replication-dependent DSBs, H2O2 triggers oxidative damage and strand breaks, and HU causes replication stress by depleting dNTP pools 48.

Fgrad51 was hypersensitive to all four agents (Fig. 1c), consistent with a central role of FgRAD51 in damage tolerance. In contrast, mutants lacking crossover-associated downstream factors (Fgmus81, Fgmms4, Fgslx1, Fgyen1, Fgmlh1, and Fgmlh3) showed no detectable changes in sensitivity under any tested condition (Fig. 1c). These results indicate that FgRad51-dependent HR is a major determinant of resistance to diverse DNA-damaging and replication-stress conditions during vegetative growth in F. graminearum, whereas the tested crossover-associated factors are dispensable in these assays. Alternatively, the absence of an observable sensitivity phenotype could reflect functional redundancy among structure-selective endonucleases and/or crossover-associated pathways, whereby loss of any single component is compensated by others.

FgMus81 is required for sexual development and heat tolerance
Among the crossover-associated factors examined, only Fgmus81 displayed an overt defect in sexual development (Fig. 1b; Supplementary Fig. 1a). Specifically, Fgmus81 showed reduced ascospore production, with 15.6% of asci containing fewer ascospores than the wild type (Fig. 1d). Notably, although the MutL\gamma pathway (Mlh1–Mlh3) is a principal meiotic crossover mechanism in many eukaryotes 9, 12, Fgmlh1 and Fgmlh3 did not exhibit discernible sexual defects in F. graminearum, supporting a species-specific requirement for FgMUS81 during sexual development.

Despite wild-type sensitivity to MMS, CPT, H2O2, and HU (Fig. 1c), Fgmus81 was markedly hypersensitive to elevated temperature. At 30°C, Fgmus81 showed reduced vegetative growth and diminished pigment accumulation relative to the wild type (Fig. 1e), a phenotype not observed in other crossover-associated mutants (Supplementary Fig. 1b). Fgrad51 also exhibited heat-associated growth defects, but did not show the pigmentation defect (Fig. 1e). Collectively, these data identify FgMUS81 as an important determinant of cellular fitness under heat stress. The pigmentation defect specific to Fgmus81 is consistent with the possibility that FgMus81 may prevent the build-up of downstream toxic recombination/repair intermediates under heat stress, thereby indirectly supporting pigment production.

FgMus81 acts independently of FgMms4 and nuclease activity
Unexpectedly, deletion of FgMMS4, which encodes the conserved regulatory partner of Mus81, did not recapitulate the sexual-development or heat-stress phenotypes of Fgmus81 (Fig. 1b, e; Supplementary Fig. 1), despite a physical interaction between FgMms4 and FgMus81 (Supplementary Fig. 2). These results suggest that the contributions of FgMus81 to sexual development and heat-stress tolerance are largely independent of FgMms4 in F. graminearum.

To determine whether FgMus81 nuclease activity is required for these functions, we generated two putative catalytic-dead alleles: FgMUS812AE (D362A, D363A), targeting residues conserved in Saccharomyces cerevisiae 49, and FgMUS815A (D294A, E297A, D327A, D362A, D363A), additionally targeting residues implicated in human Mus81 catalysis 16. Quantitative reverse-transcription (RT)-PCR indicated that transcript levels of both alleles were comparable to the wild type (Fig. 1f), arguing against altered mRNA stability. Notably, both alleles displayed wild-type growth and pigmentation at 30°C and normal ascospore formation (Fig. 1f), indicating that FgMus81-dependent heat-stress tolerance and sexual development do not require canonical nuclease activity. This nuclease-independent function provides a parsimonious explanation for the absence of heat-stress and sexual development phenotypes in Fgmms4 and uncouples the roles of FgMus81 in heat-stress adaptation and sexual reproduction from its DNA cleavage activity.

Site-specific N420D editing enables FgMus81 sexual roles
Notably, with the exception of FgRAD51, all crossover-associated factor genes examined in F. graminearum harbored nonsynonymous A-to-I mRNA editing sites that resulted in amino acid substitutions (Supplementary Fig. 3). Among these genes, only FgMUS81 contained nonsynonymous sites with editing levels exceeding 50%. Specifically, FgMUS81 carried three highly edited sites (each >50%) (Fig. 2a).

To examine the functional importance of these sites, we generated a multi-site uneditable mutant (FgMUS81UE) by simultaneously mutating the −1 thymine (T) base required for editing specificity 41, 44 to guanine (G) at all three highly edited sites (Fig. 2a). Because these T-to-G substitutions were synonymous, they were expected to abolish editing without altering the encoded amino acid sequence. Complete loss of A-to-I editing in FgMUS81UE was confirmed by sequencing RT–PCR products (Fig. 2b). Notably, FgMUS81UE exhibited ascospore development defects comparable to those of Fgmus81 (Fig. 2c), indicating that RNA editing is required for FgMUS81 function during sexual development.

To further test whether the post-editing isoform is sufficient for normal sexual reproduction, we generated a post-editing mutant (FgMUS81PE) in which the editing adenosines were replaced with guanosines in the corresponding genomic positions, thereby constitutively encoding the edited amino acids (Fig. 2a). FgMUS81PE displayed normal sexual development (Fig. 2c), supporting the conclusion that the post-editing isoform—rather than the pre-editing isoform—is required for normal sexual reproduction.

To pinpoint the functionally critical editing event(s), we next constructed single-site uneditable mutants for each of the three highly edited sites. While the other two single-site uneditable mutants exhibited wild-type phenotypes, unediting at position 420 (FgMUS81N420)—corresponding to the Asn-to-Asp substitution (N420D)—caused ascospore formation defects similar to those observed in FgMUS81UE (Fig. 2c). However, the single-site post-editing mutant FgMUS81420D displayed normal sexual development. These results indicate that site-specific N420D RNA editing is essential for FgMUS81 function in sexual development.

Edited FgMus81 supports meiotic but not mitotic recombination
To delineate the respective contributions of the pre-editing and post-editing FgMus81 isoforms to recombination, we first quantified mitotic HR using transformation-based gene-targeting assays, in which plasmids carrying homology to a chromosomal locus integrate via HR. We transformed the indicated strains with pKNTG carrying the FG1G36140 fragment and scored HR by PCR-confirmed targeted integration. The uneditable mutant (FgMUS81N420) exhibited an HR rate comparable to that of the wild type, whereas the post-editing mutant (FgMUS81420D) showed a significantly reduced HR rate, similar to Fgmus81 (Fig. 3a). Consistently, an independent assay at the FgPKS12 locus—using the frequency of white colonies as a readout of HR-mediated disruption—revealed a significant reduction in HR in FgMUS81420D and Fgmus81 but not in FgMUS81N420, relative to the wild type (Fig. 3a). Together, these results indicate that the post-editing FgMus81 isoform is less proficient in promoting mitotic HR during vegetative growth than the pre-editing isoform.

We next tested whether the two isoforms differentially affect meiotic interhomolog recombination. Post-editing and uneditable FgMUS81 mutants were generated in the F. graminearum Gz3639 background. To quantify interhomolog crossovers, selectable markers (hph and zeo) were inserted near opposite ends of chromosome 2 in the PH-1 and GZ3639 backgrounds, respectively (Fig. 3b). Among double-resistant single-ascospore progeny derived from crosses between these backgrounds, the apparent interhomolog crossover rate in FgMUS81420D was indistinguishable from that in FgMUS81N420 (Fig. 3b). Moreover, whole-genome sequencing of 10 randomly selected single-ascospore isolates (intraspecific F1 hybrids) enabled reconstruction of crossover maps using SNP markers, revealing no significant differences in either the number or genomic distribution of interhomolog crossovers among FgMUS81420D, FgMUS81N420, and the wild type (Fig. 3c; Supplementary Fig. 4). Collectively, these data suggest that although the post-editing FgMus81 isoform is compromised in mitotic HR, both isoforms are competent to support meiotic interhomolog crossover recombination in F. graminearum.

Finally, to assess whether this mitotic HR function requires the canonical Mus81 partner, we measured HR-mediated transformation efficiency at the FgPKS12 locus in Fgmms4. In contrast to Fgmus81 and FgMUS81420D, Fgmms4 showed no detectable reduction in HR relative to the wild type (Supplementary Fig. 5). This genetic dissociation suggests that FgMus81 can promote mitotic HR events in F. graminearum independently of FgMms4.

N420D editing attenuates FgMus81 to ensure normal meiosis
In the wild type, most asci at 6 dpf completed meiosis followed by post-meiotic mitosis, yielding eight nuclei that were each encapsulated by a forespore membrane and ultimately producing eight morphologically well-defined ascospores per ascus. By contrast, Fgmus81 displayed pronounced nuclear division defects: the proportion of asci containing eight nuclei was reduced by 59%, accompanied by a marked increase in asci containing only one to four nuclei (Fig. 4a). These observations indicate that FgMUS81 is required for proper meiotic nuclear division and subsequent ascospore formation.

Consistent with this requirement, the uneditable mutant (FgMUS81N420) exhibited nuclei division defects in asci resembling those of Fgmus81, albeit less severe, whereas the post-editing mutant (FgMUS81420D) was indistinguishable from the wild type (Fig. 4a). Given that FgMUS81N420 promoted higher mitotic HR than FgMUS81420D during vegetative growth (Fig. 4a), we hypothesized that although FgMUS81 is essential for sexual development, excessive activity/dosage of the pre-editing isoform can be detrimental to meiotic progression, thereby contributing to the defects observed in FgMUS81N420.

To test this idea, we introduced an additional copy of FgMUS81N420 into the FG1G36140 locus in the FgMUS81N420 background, generating a two-copy strain (d-FgMUS81N420). d-FgMUS81N420 exhibited more severe ascospore formation defects than the original FgMUS81N420 mutant (Fig. 4b–c). Similarly, introducing an extra copy of FgMUS81420D into the FgMUS81420D background (d-FgMUS81420D) also impaired ascospore formation, but the defect was weaker than that observed in d-FgMUS81N420 (Fig. 4b–c). Together, these data support a model in which excessive FgMus81 dosage compromises ascospore development, and N420D editing helps restrain FgMus81 function during meiosis to promote proper nuclear division and ascospore formation.

N420D editing reduces FgMus81 protein stability
Quantitative RT-PCR analysis showed that FgMUS81 transcript levels were comparable between FgMUS81N420 and FgMUS81420D (Fig. 4c), suggesting that N420D editing does not measurably alter FgMUS81 mRNA abundance.

To evaluate protein stability, we generated strains expressing Flag-tagged pre-editing or post-editing proteins (FgMus81N420-Flag and FgMus81420D-Flag). Although the corresponding mRNA levels were similar (Supplementary Fig. 6a), cycloheximide (CHX) chase assays during vegetative growth revealed a weaker FgMus81420D-Flag signal than FgMus81N420-Flag after 5 h of CHX treatment (Fig. 4d), consistent with faster turnover of the post-editing isoform in vivo. After multiple rigorous attempts, we were unable to extract sufficient FgMus81 protein from sexual tissues for reliable Western blot analysis (Supplementary Fig. 6b). However, purified FgMus81420D-Flag protein showed more extensive degradation than purified FgMus81N420-Flag in an in vitro stability assay (Fig. 4d). These results were reproducible across independent experiments (Supplementary Fig. 6c). Since this instability is observed in both vegetative cells and cell-free systems, it suggests that the increased degradation is an intrinsic property of the edited protein rather than a stage-specific regulatory event. Collectively, these data indicate that N420D editing decreases FgMus81 protein stability and thereby reduces FgMus81 dosage.

N420D editing resolves FgMus81 trade-offs in heat tolerance and meiosis
These results raise an important question: why is the edited residue (D) not constitutively encoded in the genome, but instead generated via stage-specific RNA editing? Given that FgMUS81 contributes to heat-stress tolerance, we compared heat-stress phenotypes among FgMUS81420D, the wild type, and FgMUS81N420. At 30°C, FgMUS81420D exhibited reduced radial growth and diminished pigmentation, closely resembling Fgmus81, whereas FgMUS81N420 displayed wild-type-like growth (Fig. 4b). These data suggest that the post-editing isoform alone is insufficient to support normal vegetative performance under heat stress, consistent with its reduced protein stability and its impaired proficiency in mitotic HR assays (Fig. 3a, Fig. 4d).

Interestingly, both two-copy strains (d-FgMUS81N420 and d-FgMUS81420D) displayed wild-type-like growth under heat stress (Fig. 4b). This observation suggests that increasing FgMUS81 dosage can compensate for the reduced stability of FgMus81420D with respect to heat-stress adaptation, and that elevated FgMus81 dosage per se does not compromise heat tolerance. Together with the sexual-development phenotypes, these results support a trade-off model in which lower FgMus81 dosage/function favors normal sexual development, whereas higher dosage/function is beneficial for growth adaptation under heat stress.

Consistent with our earlier observations that Fgmus81 did not alter sensitivity to MMS, CPT, H2O2, or HU during vegetative growth (Fig. 1c), neither FgMUS81N420 nor FgMUS81420D displayed detectable differences from the wild type in these genotoxin assays (Supplementary Fig. 7). Thus, the mitotic function of FgMus81 revealed by HR-based transformation appears to be particularly relevant to heat-stress adaptation rather than to the tested DNA damage responses. Notably, the reduced mitotic HR observed in Fgmus81 and FgMUS81420D, but not in Fgmms4 (Fig. 3a; Supplementary Fig. 5), further supports a specific contribution of FgMus81 to this vegetative recombination-associated phenotype that is at least partially separable from FgMms4.

Evolutionary dynamics of N420D editing in Sordariomycetes
To examine whether N420D editing extends beyond F. graminearum, we analyzed publicly available RNA-seq datasets from the sexual stages of 10 Sordariomycetes species that retain an editable adenosine at the corresponding site. A-to-I editing at this position, with variable editing levels, was detected in three additional Fusarium species and in two more distantly related taxa (Chaetomium globosum and Colletotrichum cliviicola) (Fig. 5a; Supplementary Fig. 8). In contrast, no evidence of editing at this site was detected in Neurospora, Pyricularia oryzae, or Sordaria macrospora, consistent with the absence of the preferred local sequence context and/or predicted hairpin-like RNA structures surrounding this site in these species (Fig. 5a). Notably, the N420D site was not predicted to reside in a hairpin-like structure in Fusarium neocosmosporiellum or C. cliviicola, suggesting that such structures may be transient, condition-dependent, or not reliably captured by in silico prediction. By comparison, editing at two other sites without known functional importance (K90E and K221E) is less conserved and detected only within Fusarium (Supplementary Fig. 9). Together, these analyses suggest that N420D editing is conserved in a subset of Sordariomycetes but remains evolutionarily flexible.

To further investigate the evolutionary history of this site, we identified MUS81 orthologs from 581 representative ascomycete species, including 343 Sordariomycetes and 154 Fusarium species (Supplementary Data 1). MUS81 orthologs were broadly conserved across most ascomycete lineages but were not detected in Dothideomycetes, consistent with a lineage-specific loss of MUS81 in this class (Fig. 5b). Parsimony analysis suggests that the pre-editing residue N represents the ancestral state across ascomycetes, as it predominates at this position in most examined lineages except Saccharomycotina (Fig. 5b). The genomically encoded D state was also observed in multiple lineages, with higher frequencies in Leotiomycetes, Lecanoromycetes, and Pezizomycetes. In contrast, both the N residue and the corresponding editable adenosine are relatively conserved in Sordariomycetes (Fig. 5b), a clade in which A-to-I RNA editing is prevalent.

Although core sequence preferences for A-to-I editing are broadly conserved among filamentous ascomycetes, predicted hairpin-like RNA structures encompassing the N420D site were enriched in Sordariomycetes (Fig. 5b), suggesting that acquisition of local RNA secondary structure may represent a derived feature that facilitates editing in this clade. Among Sordariomycetes lineages, the fraction of species with predicted hairpin-like structures varied substantially (Fig. 5c), consistent with either multiple independent gains or an ancestral origin followed by repeated loss. Within Fusarium, most species retained predicted hairpin-like structures despite apparent losses in some lineages (Fig. 5d), suggesting overall conservation of the editing potential during Fusarium evolution. Moreover, although cytosine (C) is inferred to be the ancestral −1 base in Fusarium, independent substitutions to uracil (U)—the preferred −1 base for efficient editing—occurred in several lineages, including the Sambucinum species complex (which includes F. graminearum). Conversely, in the Incarnatum–equiseti species complex, this position was replaced by G, a strongly disfavored −1 base for A-to-I editing 41, 44. These observations suggest that N420D editing is likely ancestral within Fusarium but has been independently optimized or attenuated across lineages.

Collectively, the recurrent gain and loss of local RNA structural features—and thus the capacity for editing—highlights an evolutionary landscape in which some lineages favor flexible, editing-mediated regulation, whereas others adopt fixed genomic encoding, potentially reflecting differences in ecology and life history.

Methods
Strain and culture conditions
We routinely cultured wild-type Fusarium graminearum strains PH-1 and Gz3639 25, along with their derived mutants and transformants, on potato dextrose agar (PDA) plates (20% potato, 2% dextrose, 1.5% agar) at 25°C. We assessed colony growth rate and morphology on PDA plates after 3 days of incubation. To test strain sensitivity to various stresses, we supplemented PDA plates with one of the following agents: 0.05% methyl methanesulfonate (MMS), 50 nM camptothecin (CPT), 0.02% hydrogen peroxide (H2O2), or 1 mM hydroxyurea (HU). We then incubated the strains for 3 days. We harvested conidia from 5-day-old liquid cultures grown in carboxymethyl cellulose (CMC) medium (1.5% carboxymethylcellulose, 0.1% NH4NO3, 0.1% KH2PO4, 0.05% MgSO4·7H2O, and 0.1% yeast extract).

For sexual reproduction assays, we gently pressed down aerial mycelia from 7-day-old cultures grown on carrot agar plates (1.5% agar, 250 g/L carrot) using 1 mL of sterile 0.1% Tween-20. Subsequently, we incubated the plates under black light at 25°C. We examined perithecium formation using an Olympus SZX16 stereoscope. The morphology of asci and ascospores was observed with an Olympus BX-51 microscope.

Gene deletion and site-directed mutagenesis
We performed gene deletions of DSB repair pathway genes using the split-marker approach 47. We amplified approximately 1.0 kb fragments upstream and downstream of each target gene. We then fused these fragments, via overlapping PCR, to the 5’- and 3’-termini of a recyclable marker module that conferred hygromycin resistance and sensitivity to the nucleoside analog 5-fluoro-2’-deoxyuridine 41. Following transformation of PH-1 protoplasts, we selected transformants using hygromycin B (H005, MDbio, China) at a final concentration of 300 μg/ml.

For site-directed mutagenesis to generate specific uneditable and post-editing mutants, we constructed allelic fragments containing the desired mutations by overlapping PCR. Each allelic fragment included approximately 1.0 kb upstream and downstream homologous arms. We then transformed these fragments into the corresponding gene deletion mutant protoplasts. We screened transformants resistant to 25 μg/mL floxuridine (HY-B0097, MCE, USA) by PCR, and desired mutations were confirmed by DNA sequencing. We used quantitative PCR to verify the absence of unintended integration of allelic fragments in the transformants. For each mutation, we identified at least two independent mutants exhibiting similar phenotypes. All generated transformants and primers used are listed in Supplementary Table 2 and Supplementary Data 2, respectively.

Reverse-transcription (RT)-PCR
We extracted total RNA from 12-hour hyphae or 7-day post-fertilization (dpf) perithecia of each strain. We performed cDNA synthesis using the RevertAid Master Mix (Thermo Scientific) according to the manufacturer’s instructions. We gel-purified RT-PCR products and directly sequenced them. We visualized Sanger sequencing traces using SnapGene Viewer 4.3 (https://www.snapgene.com/snapgene-viewer/) 44. We quantified relative expression levels by real-time RT-PCR using the 2⁻ΔΔCT method 57, with the GzUBH gene serving as an internal control. The primers used are listed in Supplementary Data 2.

Overexpression of FgMUS81N420 and FgMUS81420D alleles
To achieve ectopic overexpression of the FgMUS81N420 and FgMUS81420D alleles at a defined genomic locus, we utilized the FG1G36140 locus, as its deletion does not alter the wild-type phenotype 36. First, in the FgMUS81N420 and FgMUS81420D mutant backgrounds, we replaced the FG1G36140 gene with a recyclable marker module. This generated recipient strains such as FgMUS81N420ΔFG1G36140.

Next, approximately 1.0 kb fragments upstream and downstream of the FG1G36140 gene were amplified. These fragments were then fused, via overlapping PCR, to the FgMUS81N420 or FgMUS81420D allele (including its native promoter), creating the overexpression constructs. The resulting constructs were transformed into the protoplasts of the FgMUS81N420ΔFG1G36140 or FgMUS81420DΔFG1G36140 mutants, respectively. Transformants resistant to floxuridine were screened by PCR. RT-PCR was performed to confirm that the transformants overexpressed the FgMUS81 alleles in 7 dpf perithecia.

Single ascospore isolation
To facilitate outcrosses, we replaced the key MAT gene MAT1-2-1 in the Gz3639 strain with a GFP-G418 resistance marker, thereby generating a Gz3639 mat1-2-1 strain that exhibited GFP fluorescence. We also constructed uneditable and post-editing mutants of FgMUS81 in the Gz3639 mat1-2-1 background.

For outcrosses, we inoculated 7-day-old cultures of Gz3639 mat1-2-1, Gz3639 mat1-2-1 FgMUS81N420, and Gz3639 mat1-2-1 FgMUS81420D strains (grown on carrot agar plates) with 1 mL of conidial suspension (1×106 conidia/mL). These conidial suspensions were derived from PH-1, FgMUS81N420, and FgMUS81420D strains in the PH-1 background, respectively.

Fifty perithecia were randomly collected from each outcross near a flame under a dissecting microscope. These were then crushed with fine forceps and transferred into 100 μL of sterile water in a centrifuge tube. The tube was immediately sealed and vortexed to obtain an ascospore suspension. We transferred 10 μL of this suspension onto a 2% agar plate. By adjusting the focal plane of the dissecting microscope to the medium surface, we located individual ascospores and transferred a single ascospore to the plate edge using a capillary tube. The ascospore’s position was marked on the bottom of the plate. Plates were sealed with Parafilm and incubated at 25°C for 8 hours. Subsequently, we excised a small piece of medium containing the germinating ascospore using a sterile needle and transferred it to a PDA plate, where single ascospore isolates were obtained after further incubation at 25°C.

DNA-seq and whole-genome meiotic crossover profiling
For each outcross (wild-type, uneditable mutants, and post-editing mutants), we randomly selected ten single ascospore isolates for whole-genome DNA sequencing. The Gz3639 mat1-2-1 strain was also sequenced as a reference. DNA was extracted using the CTAB method. Sequencing libraries were prepared and subsequently sequenced on an Illumina NovaSeq 6000 system (2 × 150 bp paired-end) at Novogene Bioinformatics Institute (Tianjin, China). Low-quality reads and adapter sequences were removed using Trimmomatic 58 with default settings. The raw DNA-seq data have been deposited in the Genome Sequence Archive at the National Genomics Data Center, China National Center for Bioinformation, under the accession numbers provided in Supplementary Table 3.

We downloaded the latest genome assembly (YL1) of F. graminearum PH-1 59 from FgBase (http://fgbase.wheatscab.com/) to serve as the reference genome. DNA-seq reads from each strain were mapped to the PH-1 reference genome using Bowtie 2 v2.5.1 60 with default parameters. SAM files were then converted to BAM format and indexed using SAMtools v1.17 61. To avoid biases in SNP calling caused by amplification artifacts, duplicated reads were removed using the MarkDuplicates tool from the Picard package v3.1.0 (https://broadinstitute.github.io/picard/). SNPs were identified using BCFtools v1.17 61, and variant calls were filtered based on sequencing depth and quality scores. We utilized 76,117 high-quality SNP sites, identified between Gz3639 mat1-2-1 and PH-1, as genetic markers to detect crossover events in each single ascospore isolate using custom scripts (https://github.com/qinhuwang/FG.crossover). Crossover points were defined as the midpoint between two adjacent markers flanking a crossover event.

Genetic assays of meiotic recombination rates
To evaluate meiotic recombination rates, we inserted two selectable markers into intergenic regions at opposite ends of chromosome 2 in both PH-1 and Gz3639 background strains. Specifically, in the PH-1 background, a hygromycin resistance cassette (hph) was integrated into both FgMUS81N420 and FgMUS81420D strains at the 5’-terminus of chromosome 2 (69040-70803 bp). Concurrently, in the Gz3639 background, a zeocin resistance cassette (zeo) was inserted into both Gz3639 mat1-2-1 FgMUS81N420 and Gz3639 mat1-2-1 FgMUS81420D strains at the 3’-terminus of chromosome 2 (8938050-8939537 bp).

For outcrosses, Gz3639 mat1-2-1 FgMUS81N420 zeo or Gz3639 mat1-2-1 FgMUS81420D zeo strains were used as the female parent, and these were outcrossed with the male parent strains FgMUS81N420 hph or FgMUS81420D hph, respectively. We randomly collected ascospores from each outcross and plated 100 ascospores per plate on PDA medium supplemented with 300 μg/mL hygromycin B and 700 μg/mL zeocin (SL4140, Coolaber, China). Crossover recombination efficiency was quantified by counting the number of dual-resistant colonies.

Genetic assays of mitotic recombination rates
To evaluate mitotic recombination rates, we employed transformation-based assays. For the first assay, the FG1G36140 gene was cloned into the pKNTG plasmid 62, and the resulting pKNTG-FG1G36140 construct was transformed into protoplasts of PH-1, Fgmus81, FgMUS81N420, and FgMUS81420D mutants. Transformants were selected on TOP plates (0.3% yeast extract, 0.3% casein acid hydrolysate, 1.5% agar, and 20% dextrose) supplemented with 400 μg/mL G418 (HY-17561, MCE). Plasmid integration was confirmed by PCR using primers targeting the G418 resistance cassette (G418-F and G418-R). To detect recombination events, a forward primer (up-F, approximately 1.0 kb upstream of FG1G36140) and a reverse primer (down-R, approximately 1.0 kb downstream) were designed. If the plasmid integrated into the FG1G36140 locus via homologous recombination, a PCR product was amplified using the up-F and G418-R primer pair. Conversely, if no integration occurred, no product was detected with this primer pair, but another product was detected using the up-F and down-R primer pair. As a control, wild-type PH-1 genomic DNA yielded no product with the up-F/G418-R pair but produced an expected band with the up-F/down-R pair, confirming the integrity of the locus in the absence of plasmid integration.

A similar strategy was applied to assess recombination at the FgPKS12 locus. This locus was chosen because its disruption leads to a readily observable phenotype: white colony 63. The 5’-part of the FgPKS12 coding region was cloned into the pUC19 vector to generate pUC19-FgPKS12, and this construct was transformed into protoplasts of PH-1, Fgmus81, Fgmms4, FgMUS81N420, and FgMUS81420D mutants. Integration of pUC19-FgPKS12 into the endogenous FgPKS12 locus via homologous recombination disrupted gene function. This disruption resulted in the formation of two tandemly repeated FgPKS12 copies, with one copy lacking the 3’-part of the coding region and the other lacking its promoter, leading to white colony transformants.

Yeast two-hybrid assays
To investigate protein-protein interactions between FgMus81 and FgMms4, yeast two-hybrid assays were performed. The open reading frames (ORFs) of FgMUS81 and FgMMS4 were amplified and subsequently cloned into the pGBKT7 (bait) and pGADT7 (prey) vectors (TaKaRa Bio, Tokyo, Japan), respectively. The resulting constructs were then co-transformed pairwise into the yeast strain AH109. Interactions were assessed by evaluating growth on SD-His-Leu-Trp plates, as previously described 36. Additionally, the activity of Mel1 α-galactosidase was evaluated on SD-His-Leu-Trp plates supplemented with X-α-gal.

Western blotting
To facilitate protein detection, FgMUS81N420-3×FLAG and FgMUS81420D-3×FLAG constructs were generated and transformed into PH-1 protoplasts. Transformants were selected on TOP plates supplemented with G418, and their integration was confirmed by PCR.

To assess protein stability in vivo, conidia from the generated transformants were harvested and inoculated into YEPD medium (0.3% yeast extract, 1% peptone, 2% dextrose), followed by 12 hours of shaking at 25°C. Subsequently, 25 μg/mL cycloheximide (CHX; Merck, USA) was added. Hyphae were collected after 0, 5, and 10 hours of incubation at 30°C. Sexual tissues (perithecia) were collected at 7 days post-fertilization (7 dpf) from carrot agar plates. For in vitro protein stability assessment, conidia were inoculated in YEPD medium and shaken for 12 hours. The resulting hyphae were harvested, total proteins extracted, and then incubated at 30°C for 0, 4, and 8 hours prior to Western blotting.

Total proteins were isolated and separated on 10% SDS-PAGE gels, then transferred to nitrocellulose membranes. To prevent non-specific antibody binding, the membranes were incubated in 5% skim milk for 1 hour. Subsequently, proteins were incubated overnight at 4°C with either anti-FLAG (dilution:1:5000, Merck, F3165, Germany) or anti-GAPDH (dilution:1:5000, Sangon Biotech, D110016-0200, China) antibodies. Following three washes with 1×TBST (100 mM Tris-HCl, pH 7.5; 136 mM NaCl; 0.1% Tween-20), the membranes were incubated with corresponding secondary antibodies at room temperature for 1 hour. After additional washes with 1×TBST, a chemiluminescent substrate was applied for 5 minutes, and signals were detected using a chemiluminescence imaging system.

Bioinformatic analysis
To identify genes encoding core and downstream homologous recombination factors in F. graminearum, we employed a robust reciprocal BLASTp search strategy using protein sequences from S. cerevisiae and S. pombe as reference queries. The A-to-I RNA editing sites in F. graminearum used in this study were obtained from our previous work 34 (see online Supplemental Material). To identify MUS81 orthologs and analyze the N420D editing site, genome sequences of 581 representative ascomycete species were downloaded from the NCBI Genome database. MUS81 orthologs, along with their corresponding amino acid states at the N420D editing site and flanking DNA sequence context, were identified using tBLASTn. Only hits with e-values < 1×10-5 were considered as homologous sequences.

For RNA editing analysis, RNA-seq data from the sexual stage of 10 Sordariomycetes species were obtained from the NCBI SRA database (details regarding these datasets are documented in Supplementary Table 3). RNA-seq reads were mapped to their respective genomes using HISAT2 v2.2.1 64, as previously described 36. Subsequently, N420D editing events were manually inspected using IGV (https://igv.org/).

The phylogenetic tree for the genus Fusarium was constructed based on our prior research 43. Evolutionary relationships among major Sordariomycetes and Ascomycota groups were inferred from the MycoCosm tree (https://mycocosm.jgi.doe.gov/mycocosm/home).

Sequence logos flanking the editing sites were visualized using ggseqlogo v0.1 65. RNA secondary structures, encompassing 30 nucleotides upstream and downstream of each editing site, were predicted using RNAFold 66. The distributions of secondary structure types were analyzed with forgi v2.0.3 67, and the predicted structures were visualized using RNAstructure v6.5 68.

Statistics & reproducibility
The investigators were not blinded to group allocation during data collection and analysis, as blinding was not feasible due to the nature of the experiments. However, objective and standardized methods were employed to minimize bias. No formal statistical method was used to predetermine sample sizes. Instead, sample sizes were selected based on standard practices in the field and prior experience to ensure sufficient biological replicates for reliable and reproducible results. All statistical analyses were performed in R. P-values were calculated using two-sided t-tests with adjustments for multiple comparisons or one-way ANOVA followed by Tukey’s multiple comparisons test. To ensure reproducibility, each experiment was independently repeated at least twice, yielding consistent results. All blot images presented represent consistent findings from independent experiments. For phenotype observations, at least two independent mutants or transformants with three biological replicates each were used per experiment, although only representative images are shown.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript, the authors clarified the evolutionary significance of A-to-I N420D recoding site in nuclease gene Mus81 of Fusarium graminearum. The unedited version benefits vegetative stage especially under heat stress, while the edited version benefits the sexual stage, aligning with the fact that mRNA editing in this fungus is sexual stage-specific. The methodology is sound and the results support the conclusions. This is a perfect story that continues to discover adaptive RNA editing sites in fungi, based on the previous fruitful findings of this research group. I support its publication pending some minor corrections.\n(1) Background messages from lines 30-96 are too detailed, and many of which might not be needed for understanding the story of RNA editing? I encourage the authors to shorten this part. Particularly, from the beginning, the authors need to concisely and rapidly establish a logical link between the up/down of Mus81 activity and the fitness change of the fungus under vegetative or sexual stage. Such logic flow will allow readers to understand the main results regarding this trade-off. The final model is represented in Figure 7, but the background narration should be friendlier to non-microbiologists. Minor: lines 84-85, “other species” needs further clarification.\n(2) It is not immediately clear to me that how the results in Figure 2 will influence the subsequent story? It seems that the only purpose is to find out Mus81?\n(3) Lines 251-253, nearly all DSB repair genes have nonsynonymous A-to-I editing sites. How unexpected it is? Do other pathway genes less frequently have nonsynonymous RNA editing sites? A simple example of some other pathways will be helpful.\n(4) Line 259, please clarify if the -1 mutation is synonymous.\n(5) Line 287, from this section, why do the authors only focus on N420D editing in FgMUS81 but not K551E editing in FgEXO1 identified in the previous section?\n(6) Figure 6A, what is the conservation level of other nonsynonymous editing sites in FgMUS81?\n(7) Line 403, it would be better to say “the evolution of the genomic sequence corresponding to N420D editing site”.", "reviewer_2": "In this paper, the authors comprehensively identified double-strand break (DSB) repair–related genes in the filamentous fungus Fusarium graminearum. Notably, they discovered that FgMus81 undergoes epitranscriptomic regulation through A-to-I mRNA editing, resulting in distinct isoforms that perform stage-specific functions. This study provides valuable and original insights into the role of A-to-I editing in filamentous fungi, particularly in the context of DNA repair and developmental regulation.\nOverall, the manuscript is well-conceived and the findings are compelling. The experimental design is solid, and the results significantly advance our understanding of RNA editing–mediated regulation in eukaryotic pathogens. Below, I list a few suggestions and questions that could further strengthen the manuscript by clarifying specific points and providing additional experimental context.\n1) L158–159: The authors identified several DSB repair–related genes. While this is an important foundation for the study, the description could be expanded slightly to clarify how these genes were identified. Providing a brief explanation of the gene selection criteria or sequence homology approach—such as the model organisms used as references and their conservation percentages—would make the identification process clearer and more convincing.\n2) L178–179: The statement that “DNA damage detection is essential for the initiation of sexual development” is intriguing. However, since Figure 2a mainly shows phenotypic consequences of gene deletions, it would be valuable to include a short discussion or supporting data that directly link DNA damage sensing to the onset of sexual reproduction. Clarifying this relationship would further strengthen the authors’ interpretation.\n3) The observation of reduced ascospore numbers is noteworthy and potentially important. The authors could enhance the impact of this result by briefly discussing the physiological or pathogenic implications of this reduction.\n4) In Figure 2e, the authors primarily assess DNA damage responses during vegetative growth. If feasible, it would further strengthen the study to examine DNA damage responses during meiotic recombination in the sexual stage (e.g., on carrot agar following fertilization). Even a limited analysis — such as monitoring sensitivity of perithecia formation or ascospore development to the same genotoxic stress conditions — could help clarify whether the relevant DNA repair pathways are regulated similarly (or differently) during meiosis.\n5) L243–248: The authors present convincing data showing that the functions of FgMus81 in heat-stress tolerance and sexual development are independent of FgMms4. To further expand this interesting finding, the authors could consider examining whether FgMus81 and FgMms4 interact in a stage-specific manner through a co-immunoprecipitation (co-IP) assay. Such an analysis might provide additional mechanistic insight into how FgMus81 functions independently of its canonical partner.", "reviewer_3": "Epitranscriptomic resolution of 1 a DNA repair trade-off: RNA editing 2 splits Mus81 roles in heat-stress survival and meiosis\nMus81 is the catalytic subunit of a heterodimeric structure-selective endonuclease that is required to maintain genomic stability in somatic (mitotically growing) cells and for the class II crossover pathway in meiosis which is predominant in fission yeast and Tetrahymena. Fusarium graminearum causes Fusarium head blight affecting global cereal crop yields and produces a mycotoxin that poses a health risk to humans and livestock. This manuscript reports that the Mus81 encoding mRNA in Fusarium graminearum is subject to adenosine to inosine mRNA editing, where a single editing site (N420D) differentiates its function in meiosis (post-editing) and heat-stress response in mitotic cells (pre-editing). This point is well documented by creating pre- and post-editing mutants that clearly separate the functions of Mus81.\nThe manuscript is in need of major revisions, additions, and streamlining to better work out the central points about Mus81. There are important points that remain unaddressed (see below), and several statements in the manuscript are not supported by the evidence provided.\nMajor points:\n1) What is meant by the first half sentence in the abstract “Eukaryotic pathogens balance genome stability for stress tolerance with genetic variability”? I do not recognize this as a factual statement, as it sounds Lamarckian suggesting some undefined mechanism of adaptive evolution.\n2) Figure 1: It is unclear how the RNA editing sites were identified.\n2) The manuscript would benefit from focusing on the main points of RNA editing in MUS81 rather than adding the entire screen and the Exo1 data.\n3) It is unclear what role Mus81 plays in the heat-stress response. What is the mechanism? What causes the phenotype shown in Figure 2f? Is the nuclease activity needed for this? Figure 2e shows that mus81 (or mms4) mutants are not sensitive to MMS, CPT, H2O2 or HU. This is very surprising, and may suggest that Fusarium employs these genes in a vastly different way than other fungi.\n4) The claim that Mus81 acts independently of Mms4 is very surprising and unprecedented. This requires further elaboration. Do mutants in mms4 have any phenotype, for example in the assay in Figure 4? Is there potentially a duplicated gene?\n5) The claim that the N420D edit attenuates Mus81 function in meiosis requires more mechanistic insight. Is the meiotic or mitotic function dependent on the Mus81 nuclease activity? The protein stability would have to be analyzed in meiotic cells to be meaningful.\nAdditional points:\n6) The introduction is replete with inaccurate statements and requires careful editing. A few points are listed below:\nLine 38: One of the key NHEJ proteins is ligase 4, and ligase mutants are typically used to define the NHEJ pathway.\nLines 42/43: I am not sure that filamentous fungi are commonly referred to as complex eukaryotes.\nLine 49: RAD54 is not a mediator protein and not involved in nucleating the Rad51 filament on RPA-coated ssDNA.\nLine 56: Mus81 is not a nuclease by itself, it is the catalytic subunit of the heterodimeric Mus81-Mms4, MUS81-EME1 nuclease. This nuclease is not structure-specific, as it cleaves multiple substrates, it is structure-selective, as it is correctly described later in the manuscript.\nLine 64: Decapitalize programmed.\nLines 72, 78: Replace Mus81 with Mus81-Mms4 or Mus81-Eme1/2.\n7) Lines 176-179: The Rad50-Mre11-Xrs2 complex is required for meiotic DSB formation in budding yeast, which could be the reason for the meiotic requirement rather than its signaling role.\n8) Lines 212ff: The section heading that non-crossover DSA is necessary for mitotic recombination is not supported by the data. Srs2, Mph1 and Sgs1 play multiple roles in genome maintenance which all relate to the functions in the DNA damage response. The sensitivity phenotypes are not the sole consequence of an SDSA defect.\n9) Line 440: mitotic"}, "author_response": "Point-by-point response to reviewers' comments\nReviewer #1:\nIn this manuscript, the authors clarified the evolutionary significance of A-to-I N420D recoding\nsite in nuclease gene Mus81 of Fusarium graminearum. The unedited version benefits\nvegetative stage especially under heat stress, while the edited version benefits the sexual stage,\naligning with the fact that mRNA editing in this fungus is sexual stage-specific. The\nmethodology is sound and the results support the conclusions. This is a perfect story that\ncontinues to discover adaptive RNA editing sites in fungi, based on the previous fruitful\nfindings of this research group. I support its publication pending some minor corrections.\nResponse: We are deeply grateful to you for your highly encouraging and positive assessment\nof our manuscript. We particularly appreciate your characterization of our work as a “perfect\nstory” and your recognition of our ongoing efforts to uncover adaptive RNA editing sites in\nfungi.\nIt is rewarding to see that the evolutionary significance of the FgMUS81 N420D recoding site—\nbalancing vegetative heat tolerance with sexual development—resonated with your expertise.\nWe have carefully addressed the minor corrections you suggested, which have further improved\nthe clarity and precision of our manuscript.\n(1) Background messages from lines 30-96 are too detailed, and many of which might not be\nneeded for understanding the story of RNA editing? I encourage the authors to shorten this part.\nParticularly, from the beginning, the authors need to concisely and rapidly establish a logical\nlink between the up/down of Mus81 activity and the fitness change of the fungus under\nvegetative or sexual stage. Such logic flow will allow readers to understand the main results\nregarding this trade-off. The final model is represented in Figure 7, but the background narration\nshould be friendlier to non-microbiologists.\nResponse: We sincerely appreciate your point regarding the logical flow of the Introduction.\nWe agree that the background should be a focused narrative leading directly to the core\nbiological question of our study. Following your and Reviewer 3’s suggestions, we have\nsignificantly condensed and restructured the Introduction to improve accessibility. By removing\nredundant technical details and focusing on the \"one protein, two stages, one editing site\" puzzle,\nthe text is now much more accessible to non-microbiologists and more effectively sets the stage\nfor our discovery of the functional trade-off.\nMinor: lines 84-85, “other species” needs further clarification.\nResponse: We thank you for pointing this out. We have changed the phrasing to “Eme1 in\nSchizosaccharomyce pombe; Eme1/Eme2 in mammals”\n(2) It is not immediately clear to me that how the results in Figure 2 will influence the\nsubsequent story? It seems that the only purpose is to find out Mus81?\nResponse: We sincerely thank you for this critical and constructive observation. While the\noriginal manuscript included a broader characterization of various DSB repair-related genes to\nprovide context, we completely agree that the study is far more impactful when focused strictly\non its most novel discovery. The findings regarding MUS81 RNA editing represent the core\ncontribution and the most compelling narrative of our work.\nTherefore, in the revised manuscript, we have followed the strategic advice of both you and\nReviewer 3 to prioritize focus over breadth. We have removed the preliminary screening data\n(including the general recombination pathways and EXO1) to allow the manuscript to center\nexclusively on the mechanisms and biological significance of A-to-I mRNA editing in MUS81.\nWe believe this deliberate refocusing has significantly enhanced the clarity and cohesion of the\npaper, and we appreciate your help in sharpening the final story.\n(3) Lines 251-253, nearly all DSB repair genes have nonsynonymous A-to-I editing sites. How\nunexpected it is? Do other pathway genes less frequently have nonsynonymous RNA editing\nsites? A simple example of some other pathways will be helpful.\nResponse: This is an excellent question that highlights the remarkable selectivity of the fungal\nepitranscriptome. The enrichment of nonsynonymous A-to-I editing sites within DSB repair\ngenes is indeed a striking feature of F. graminearum.\nOur analysis revealed that while DNA repair machinery is heavily targeted for recoding, many\nother essential biological pathways show little to no editing. For instance, functional groups\nsuch as transmembrane transporter activity (GO:0022857, 41 genes), vacuole (GO:0005773,\n35 genes), and antibiotic catabolic processes (GO:0017001, 9 genes) lacked any detectable\nediting sites in our editing site dataset (Liu et al, Genome Research 2016). This contrast\nunderscores that A-to-I editing is not a random occurrence across the transcriptome but is likely\na specialized regulatory mechanism evolved to fine-tune specific cellular processes—like\ngenomic integrity—during the sexual stage.\nHowever, as you and Reviewer 3 suggested to prioritize focus on the most impactful finding,\nwe have removed the extensive descriptions of these broader pathways from the revised\nmanuscript. We have instead concentrated the narrative on the FgMUS81 N420D recoding site,\nwhich serves as a definitive example of how this specific enrichment translates into a significant\nfunctional and evolutionary advantage.\n(4) Line 259, please clarify if the -1 mutation is synonymous.\nResponse: We thank you for this careful reading. This is a critical point for clarity. In the revised\nmanuscript, we have specified this by adding the term “synonymous,” clarifying that the -1\nmutation does not result in an amino acid change.\n(5) Line 287, from this section, why do the authors only focus on N420D editing in FgMUS81\nbut not K551E editing in FgEXO1 identified in the previous section?\nResponse: This is an excellent and insightful question. In our initial study, we characterized\nboth the N420D editing in FgMUS81 and the K551E editing in FgEXO1. While our preliminary\ndata indicated that K551E editing is important for FgEXO1 function during sexual development,\nthe precise molecular mechanism remains elusive and warrants a separate, dedicated\ninvestigation.\nElucidating how a single recoding event modulates protein function—especially during\ncomplex fungal developmental stages—is highly challenging. We were successful in\nuncovering the sophisticated regulatory mechanism of the FgMUS81 N420D site, making it the\nmost complete and impactful story of our research.\nInitially, we included FgEXO1 as part of a broader screening narrative. However, following the\nstrategic advice from you and Reviewer 3 to prioritize focus on the most novel and fully\nresolved finding, we have removed the sections regarding FgEXO1 and other general repair\ngenes. This allows the revised manuscript to center exclusively on the FgMUS81 N420D\nrecoding site as a definitive and mechanistic example of how epitranscriptomic regulation\nconfers a significant functional and evolutionary advantage.\n(6) Figure 6A, what is the conservation level of other nonsynonymous editing sites in\nFgMUS81?\nResponse: We thank you for this insightful comment. During revision, we analyzed editing at\ntwo additional sites of FgMUS81 (K90E and K221E) that have no known functional\nsignificance, using publicly available RNA-seq datasets from the sexual stages of the 10\nSordariomycetes species previously examined for N420D editing. We found that editing at\nthese two sites is less conserved and detected only within Fusarium. We have added a new\nfigure (Supplementary Fig. 9) and the corresponding description (lines 361–363) in the revised\nmanuscript.\n(7) Line 403, it would be better to say, “the evolution of the genomic sequence corresponding\nto N420D editing site”.\nResponse: We thank you for this insightful correction. We agree that this phrasing is more\nscientifically precise. In the revised manuscript, we have substituted “the evolution of the\nN420D editing site” with “the evolution of the genomic sequence corresponding to the N420D\nediting site”.\nFurthermore, we have conducted a comprehensive review of the entire manuscript to ensure\nthat similar descriptions of genomic and epitranscriptomic evolution are consistently and\naccurately articulated throughout.\nReviewer #2:\nIn this manuscript, the authors comprehensively identified double-strand break (DSB) repair–\nrelated genes in the filamentous fungus Fusarium graminearum. Notably, they discovered that\nFgMus81 undergoes epitranscriptomic regulation through A-to-I mRNA editing, resulting in\ndistinct isoforms that perform stage-specific functions. This study provides valuable and\noriginal insights into the role of A-to-I editing in filamentous fungi, particularly in the context\nof DNA repair and developmental regulation.\nOverall, the manuscript is well-conceived and the findings are compelling. The experimental\ndesign is solid, and the results significantly advance our understanding of RNA editing–\nmediated regulation in eukaryotic pathogens. Below, I list a few suggestions and questions that\ncould further strengthen the manuscript by clarifying specific points and providing additional\nexperimental context.\nResponse: We sincerely thank you for the positive and encouraging assessment of our work.\nWe are gratified that you found our discovery of A-to-I mRNA editing in MUS81 to be a\nvaluable and original contribution to the field of fungal epitranscriptomics and DNA repair.\nYour insightful comments have been instrumental in helping us refine the manuscript. We have\ncarefully addressed each of your suggestions and questions in the following point-by-point\nresponse, which we believe has significantly strengthened the experimental context and\nclarified the key mechanisms of our study.\nSpecific comments.\n1) L158–159: The authors identified several DSB repair–related genes. While this is an\nimportant foundation for the study, the description could be expanded slightly to clarify how\nthese genes were identified. Providing a brief explanation of the gene selection criteria or\nsequence homology approach—such as the model organisms used as references and their\nconservation percentages—would make the identification process clearer and more convincing.\nResponse: We thank you for this constructive suggestion. To clarify the identification process\nof DSB repair-related genes in F. graminearum, we employed a robust reciprocal BLASTp\nsearch strategy using protein sequences from well-characterized model organisms, including\nSaccharomyces cerevisiae, Schizosaccharomyces pombe, and Homo sapiens, as reference\nqueries.\nIn the revised manuscript, we have added a detailed description of this approach in the Methods\nsection (lines 723-725). Furthermore, we have provided a table (Supplementary Table 1), which\nincludes comprehensive information for the identified genes, such as the reference model\norganisms used, accession numbers, and sequence alignment percentages (identity/similarity).\nIt is important to note that, following the recommendation of Reviewer 3 to focus the\nmanuscript more tightly on MUS81, we have removed the descriptive sections for several\nbroader DSB repair pathways. However, we have retained and thoroughly documented the\ninformation for the core recombinase gene FgRAD51 and representative meiotic crossover-\nassociated genes (FgMLH1, FgMLH3, FgMUS81, FgMMS4, FgSLX1, and FgYEN1), as these\nare central to the core findings of our study.\n2) L178–179: The statement that “DNA damage detection is essential for the initiation of sexual\ndevelopment” is intriguing. However, since Figure 2a mainly shows phenotypic consequences\nof gene deletions, it would be valuable to include a short discussion or supporting data that\ndirectly link DNA damage sensing to the onset of sexual reproduction. Clarifying this\nrelationship would further strengthen the authors’ interpretation.\nResponse: We sincerely appreciate your interest in this intriguing observation. The potential\nlink between DNA damage sensing and the transition to sexual reproduction represents a\nsophisticated regulatory interface.\nAs you noted, our data indicate that the MRN complex is indispensable for perithecia initiation.\nThis observation aligns with the \"Abandon-ship hypothesis,\" which suggests that organisms\nunder environmental stress—often accompanied by endogenous DNA damage—may trigger\nsexual reproduction as an evolutionary strategy to reshuffle genotypes and escape low-fitness\nstates through recombination (Griffiths & Bonser, Am Nat. 2013). While it remains unknown\nhow DNA damage sensing directly signals the onset of sexual reproduction, our ongoing\nresearch has identified specific proteins that promote the transition from vegetative growth to\nreproduction by modulating DNA repair efficiency (in preparation).\nHowever, to comply with the strategic suggestion from Reviewer 3 to focus the manuscript\nexclusively on the RNA editing of MUS81, we have removed the extensive sections regarding\nthe MRN complex and general DNA repair genes from the revised manuscript. We believe this\nensures the narrative remains tightly centered on our most novel finding regarding\nepitranscriptomic regulation.\n3) The observation of reduced ascospore numbers is noteworthy and potentially important. The\nauthors could enhance the impact of this result by briefly discussing the physiological or\npathogenic implications of this reduction.\nResponse: We sincerely thank you for this constructive suggestion. We agree that the reduction\nin ascospore numbers has significant implications for both the biology of the fungus and its\nfield-scale impact. In the revised manuscript, we have expanded our Discussion to address these\nimplications. Specifically, we highlight that in F. graminearum, sexual reproduction is a vital\nphase of the life cycle, as forcibly discharged ascospores from perithecia serve as the primary\ninoculum for Fusarium Head Blight (FHB) epidemics. Therefore, the defect in ascospore\nformation observed in our FgMUS81 editing-deficient mutants leads to a substantial reduction\nin primary inoculum potential, which would logically decrease the severity and spread of FHB\noutbreaks. This finding underscores the critical role of A-to-I mRNA editing in maintaining the\nepidemiological fitness of this significant fungal pathogen.\n4) In Figure 2e, the authors primarily assess DNA damage responses during vegetative growth.\nIf feasible, it would further strengthen the study to examine DNA damage responses during\nmeiotic recombination in the sexual stage (e.g., on carrot agar following fertilization). Even a\nlimited analysis — such as monitoring sensitivity of perithecia formation or ascospore\ndevelopment to the same genotoxic stress conditions — could help clarify whether the relevant\nDNA repair pathways are regulated similarly (or differently) during meiosis.\nResponse: We are grateful to you for this insightful suggestion. We agree that examining DNA\ndamage responses specifically during the sexual stage would provide a more comprehensive\nunderstanding of these pathways.\nDuring the revision, we conducted experiments to monitor the sensitivity of perithecia\nformation on carrot agar following fertilization. We applied the same genotoxic stress\nconditions (MMS, CPT, and HU at concentrations used for vegetative growth, mixed with\nTween-20 to enhance permeability) to all DSB repair-related gene mutants. However, we did\nnot observe discernible differences or obvious inhibitory effects on perithecia formation under\nthese specific stress conditions (data summarized in the table below).\nWe speculate that several factors may have contributed to this lack of observed phenotype,\nincluding:\n• Bioavailability and Penetration: The complex, multicellular structure of the perithecium\nmight act as a physical barrier, preventing genotoxic agents from reaching the internal\nmeiotic tissues at effective concentrations.\n• Timing and Dosage: The sensitivity threshold during the morphogenetic transition of\nsexual development may differ significantly from that of vegetative hyphal growth.\nHowever, to comply with the strategic suggestion from Reviewer 3 to streamline the manuscript\nand focus exclusively on the RNA editing of MUS81, we have removed the extensive sections\nregarding general DNA repair genes from the revised manuscript. This ensures the narrative\nremains tightly centered on our most novel finding regarding epitranscriptomic regulation.\nConsequently, these sexual-stage stress assays are no longer central to the revised scope of the\npaper.\nNo. of perithecia / microscopic field\nStrain\nMock MMS CPT HU\nFgmre11 0±0 0±0 0±0 0±0\nFgrad50 0±0 0±0 0±0 0±0\nFgnbs1 0±0 0±0 0±0 0±0\nFgrad51 3±2 3±1 3±1 3±1\nFgrad52 3±1 3±1 4±2 5±2\nFgrad54 19±4 18±4 20±4 19±5\nInhibition rate (%) for perithecia formation\nStrain\nMMS CPT HU\nWT 0.02±0.17a 0.02±0.13a -0.02±0.28a\nFgmec1 0.07±0.2a 0.01±0.13a -0.05±0.07a\nInhibition rate (%) for perithecia formation\nStrain\nMMS CPT HU\nFgrad53 0.02±0.28a -0.04±0.09a 0.04±0.06a\nFgexo1 -0.03±0.14a 0.03±0.15a 0.08±0.1a\nFgsrs2 0.03±0.1a 0.03±0.13a 0.01±0.19a\nFgmph1 -0.04±0.2a 0.1±0.15a -0.11±0.2a\nFgsgs1 -0.03±0.1a -0.04±0.04a 0.06±0.05a\nFgmus81 0.02±0.08a -0.08±0.11a 0.05±0.11a\nFgmms4 -0.04±0.14a 0.06±0.06a 0.03±0.15a\nFgslx1 0.09±0.06a 0.05±0.06a -0.03±0.11a\nFgyen1 0.04±0.06a -0.06±0.14a 0.05±0.02a\nFgmlh1 0.02±0.09a -0.03±0.18a -0.08±0.09a\nFgmlh3 -0.01±0.21a 0.01±0.18a 0.09±0.09a\nFgku70 0.02±0.11a 0.01±0.09a 0.05±0.2a\nFgku80 0.03±0.17a -0.07±0.13a 0.05±0.15a\nIdentical letters denote no significant differences (one-way ANOVA, Tukey’s test, P < 0.05).\n5) L243–248: The authors present convincing data showing that the functions of FgMus81 in\nheat-stress tolerance and sexual development are independent of FgMms4. To further expand\nthis interesting finding, the authors could consider examining whether FgMus81 and FgMms4\ninteract in a stage-specific manner through a co-immunoprecipitation (co-IP) assay. Such an\nanalysis might provide additional mechanistic insight into how FgMus81 functions\nindependently of its canonical partner.\nResponse: We thank you for this insightful suggestion. We agree that investigating the stage-\nspecific interaction between FgMus81 and its canonical partner FgMms4 could provide\nvaluable mechanistic depth.\nRegarding the stage-specific co-IP assay, we encountered significant technical challenges.\nDespite multiple rigorous attempts, we were unable to extract sufficient total protein from\nmeiotic cells for a reliable Western blot or co-IP analysis (please see Supplementary Fig. 6b in\nthe revised manuscript). In F. graminearum, meiotic cells (asci) constitute only a minute\nfraction of the highly heterogeneous tissues within the perithecia (fruiting bodies), making\nprotein-level detection in this specific stage extremely difficult. However, we have confirmed\nthe physical interaction between FgMus81 and FgMms4 using yeast two-hybrid (Y2H) assays,\nsuggesting that their ability to form a complex is conserved.\nTo address the mechanistic core of your question—how FgMus81 functions independently of\nits canonical nuclease partner—we generated and analyzed two catalytic-dead mutants:\nFgMUS812A (double-point mutant) and FgMUS815A (quintuple-point mutant).\nOur results revealed that both nuclease-inactive mutants:\n• Exhibited no sensitivity to heat stress.\n• Displayed normal ascospore formation and development, mirroring the wild-type\nphenotype.\nThese findings provide a clear, alternative mechanistic insight: the roles of FgMus81 in thermal\nstress tolerance and sexual development are non-canonical and nuclease-independent. Since\nMms4 primarily functions as an activator of Mus81 nuclease activity, its absence (in the\nFgmms4 mutant) does not affect these specific processes. This explains the independent nature\nof FgMus81 in these stages and significantly strengthens our original interpretation.\nReviewer #3:\nMus81 is the catalytic subunit of a heterodimeric structure-selective endonuclease that is\nrequired to maintain genomic stability in somatic (mitotically growing) cells and for the class\nII crossover pathway in meiosis which is predominant in fission yeast and Tetrahymena.\nFusarium graminearum causes Fusarium head blight affecting global cereal crop yields and\nproduces a mycotoxin that poses a health risk to humans and livestock. This manuscript reports\nthat the Mus81 encoding mRNA in Fusarium graminearum is subject to adenosine to inosine\nmRNA editing, where a single editing site (N420D) differentiates its function in meiosis (post-\nediting) and heat-stress response in mitotic cells (pre-editing). This point is well documented\nby creating pre- and post-editing mutants that clearly separate the functions of Mus81.\nThe manuscript is in need of major revisions, additions, and streamlining to better work out the\ncentral points about Mus81. There are important points that remain unaddressed (see below),\nand several statements in the manuscript are not supported by the evidence provided.\nResponse: We greatly appreciate your critical comments and suggestions, as they have\nsignificantly contributed to the improvement of our manuscript.\nMajor points:\n1) What is meant by the first half sentence in the abstract “Eukaryotic pathogens balance\ngenome stability for stress tolerance with genetic variability”? I do not recognize this as a\nfactual statement, as it sounds Lamarckian suggesting some undefined mechanism of adaptive\nevolution.\nResponse: We thank you for this insightful comment. We agree that the original phrasing could\nbe misinterpreted as implying teleological or Lamarckian mechanisms. Our original choice of\nwords employed “agential language,” which is often used as a heuristic framework in biology\nto understand adaptation by treating organisms as if they were agents maximizing their fitness.\nHowever, we recognize that such language can be imprecise and lead to misunderstandings\nregarding the underlying evolutionary processes. Our intention was to describe the evolutionary\ntrade-off between the maintenance of genome integrity and the generation of adaptive genetic\ndiversity, both of which are shaped by natural selection.\nThe revised sentence now reads as follows: \" Maintaining genome integrity is essential, yet\nhow DNA repair is balanced across life stages remains poorly understood.\"\n2) Figure 1: It is unclear how the RNA editing sites were identified.\nResponse: We thank you for pointing this out. The RNA editing sites depicted in Figure 1 were\nidentified and validated in our previously published study (Liu et al., Genome Research 2016).\nWe have now clarified the origin of these data in the Methods section of the revised manuscript\n(lines 725-727).\n2) The manuscript would benefit from focusing on the main points of RNA editing in MUS81\nrather than adding the entire screen and the Exo1 data.\nResponse: We thank you for this valuable and constructive suggestion. Our original manuscript\nwas designed to cover two areas: a broad characterization of genes in the homologous\nrecombination and non-homologous end-joining pathways, and a specific focus on the RNA\nediting of MUS81. We completely agree with your assessment that the MUS81 findings\nrepresent the core novelty and most significant contribution of our work.\nIn the revised manuscript, we have followed your advice and removed the data related to the\ninitial screen and Exo1. This allows the narrative to focus squarely on the mechanisms and\nimpact of RNA editing in MUS81. We believe this change has significantly improved the clarity\nand cohesion of our study, and we thank you for helping us sharpen the focus of this paper.\n3) It is unclear what role Mus81 plays in the heat-stress response. What is the mechanism?\nWhat causes the phenotype shown in Figure 2f? Is the nuclease activity needed for this? Figure\n2e shows that mus81 (or mms4) mutants are not sensitive to MMS, CPT, H2O2 or HU. This is\nvery surprising, and may suggest that Fusarium employs these genes in a vastly different way\nthan other fungi.\nResponse: We thank you for these insightful and challenging questions. They have led us to\nperform additional experiments that significantly clarify the role of Mus81 in F. graminearum.\n1. Regarding the mechanism and nuclease activity under heat stress: To determine whether\nthe nuclease activity of Mus81 is required for heat stress tolerance, we generated two sets of\ncatalytic-dead mutants:\n• FgMUS812A: A double-point mutant (D362A, D363A) targeting residues conserved in S.\ncerevisiae.\n• FgMUS815A: A quintuple-point mutant (D294A, E297A, D327A, D362A, D363A)\ntargeting additional catalytic residues reported in human Mus81.\nInterestingly, both FgMUS812A and FgMUS815A showed no sensitivity to heat stress, mirroring\nthe wild-type phenotype. This clearly demonstrates that Mus81’s role in the heat stress response\nis independent of its canonical nuclease activity. This finding also explains why its activator,\nFgMms4, is not involved in the heat stress response. While the precise non-canonical\nmechanism—perhaps acting as a structural scaffold or in a regulatory capacity—remains to be\nfully elucidated in future studies, our results establish a clear distinction between its role in heat\ntolerance and its DNA-cleaving function.\n2. Regarding the lack of sensitivity to genotoxic agents (MMS, CPT, etc.): We agree that\nthe lack of sensitivity in Fgmus81 and Fgmms4 mutants to standard genotoxic agents is\nsurprising compared to other fungal models. Our data suggests that F. graminearum prioritizes\nthe Synthesis-Dependent Strand Annealing (SDSA) pathway, which produces non-crossovers,\nover the Double-Strand Break Repair (DSBR) pathway (responsible for crossovers) during\nvegetative growth. This may represent an evolved mechanism to prevent genome instability\ncaused by mitotic crossovers.\nHowever, following your earlier suggestion to focus the manuscript strictly on the RNA editing\nof MUS81, we have removed the genotoxic stress data and the Exo1 related content from the\nrevised manuscript. This ensures the paper remains centered on the most novel finding: the\nfunctional impact of RNA editing on MUS81 during environmental adaptation.\n4) The claim that Mus81 acts independently of Mms4 is very surprising and unprecedented.\nThis requires further elaboration. Do mutants in mms4 have any phenotype, for example in the\nassay in Figure 4? Is there potentially a duplicated gene?\nResponse: We thank you for this critical and valuable comment. We completely agree that the\nindependent role of FgMus81 from FgMms4 is an unconventional finding. To address your\nconcerns regarding the mechanism and the possibility of gene duplication, we have performed\nthe following validations:\n1. Verification of FgMMS4 Single Copy and Deletion: We re-performed exhaustive BLAST\nanalyses of the F. graminearum genome, confirming that only a single MMS4 homolog\n(FG1G25820) exists. We further validated the Fgmms4 knockout strains using Whole-Genome\nSequencing (WGS). The IGV image (please see Supplementary Fig. 12 in the revised\nmanuscript) showing zero read coverage at the FgMMS4 locus provides unambiguous evidence\nof successful gene deletion.\n2. FgMus81 Acts Independently of FgMms4 in Homologous Recombination (HR): As you\nsuggested, we compared the HR-mediated plasmid transformation efficiency. While the\nFgmus81 mutant showed a significant reduction in HR efficiency (as shown in Figure 4A), the\nFgmms4 mutant exhibited no significant difference compared to the wild type. This dissociation\nof FgMus81 and FgMms4 in vegetative HR is indeed striking and suggests that Mus81 can\nfacilitate certain recombination events in F. graminearum without its canonical partner.\n3. FgMus81 Acts Independently of its Nuclease Activity in Heat Stress and Ascospore\nFormation: To further explore the \"unprecedented\" nature of FgMus81 in this species, we\ntested whether its nuclease activity is required for heat stress tolerance and ascospore formation.\nWe generated two catalytic-dead mutants, FgMUS812A (double-point mutant) and FgMUS815A\n(quintuple-point mutant). Both mutants showed no sensitivity to heat stress and normal\nascospore formation, mirroring the wild-type phenotype.\nTaken together, these results provide a clearer mechanistic picture: The role of FgMus81 in heat\nstress and ascospore formation is non-canonical (nuclease-independent), which explains the\nlack of Fgmms4 phenotype in that assay. The role of FgMus81 in vegetative HR (plasmid\ntransformation) is also independent of FgMms4, representing a specialized evolutionary\ndivergence in F. graminearum.\nWe have included these experimental results and the PCR and IGV validation in the revised\nmanuscript.\n5) The claim that the N420D edit attenuates Mus81 function in meiosis requires more\nmechanistic insight. Is the meiotic or mitotic function dependent on the Mus81 nuclease activity?\nThe protein stability would have to be analyzed in meiotic cells to be meaningful.\nResponse: We thank you for this critical and constructive comment. To address the mechanism\nof FgMus81 and the impact of the N420D edit, we have performed additional experiments and\nprovided a detailed technical explanation regarding protein stability.\n1. Mechanism: Meiotic and mitotic functions are independent of nuclease activity.\nTo investigate whether FgMus81 function depends on its canonical nuclease activity, we\ngenerated two catalytic-dead mutants: FgMUS812A (double-point mutant) and FgMUS815A\n(quintuple-point mutant). Our results show that both mutants exhibited no sensitivity to heat\nstress and, importantly, displayed normal ascospore formation (meiosis), mirroring the wild-\ntype phenotype. These findings provide a clear mechanistic insight: the roles of FgMus81 in\nboth environmental stress tolerance and meiotic development are non-canonical (nuclease-\nindependent). This also explains why the Fgmms4 mutant lacks a phenotype in these processes,\nas Mms4 is primarily a nuclease activator.\n2. Protein Stability: Technical constraints and multi-dimensional evidence.\nWe agree that analyzing protein stability directly in meiotic cells would provide the most direct\nevidence. However, after multiple rigorous attempts, we were unable to extract sufficient\nprotein from meiotic cells for reliable Western blot analysis (please see Supplementary Fig. 6b\nin the revised manuscript). In F. graminearum, meiotic cells (asci) constitute only a very small\nfraction of the complex tissues within the perithecia (fruiting bodies), making stage-specific\nprotein detection extremely challenging.\nTo ensure the reliability of our conclusion that N420D editing reduces FgMus81 protein\nstability, we have relied on a combination of evidence:\n• In vivo (vegetative hyphae): Western blots clearly show reduced stability of the post-edited\nFgMus81420D compared to the pre-edited FgMus81N420.\n• In vitro: Our in vitro assays confirm that the FgMus81420D protein is inherently less stable.\nSince this instability is observed in both vegetative cells and in vitro systems, it strongly\nsuggests that the degradation is an intrinsic property of the edited protein rather than a stage-\nspecific regulatory event. We believe this multi-faceted evidence supports our conclusion that\nN420D editing attenuates FgMus81 function during meiosis by reducing its protein levels.\nWe have added these results to the revised manuscript.\nAdditional points:\n6) The introduction is replete with inaccurate statements and requires careful editing. A few\npoints are listed below:\nLine 38: One of the key NHEJ proteins is ligase 4, and ligase mutants are typically used to\ndefine the NHEJ pathway.\nResponse: We appreciate your emphasis on DNA Ligase IV, which is indeed the definitive\ncatalytic enzyme for the final ligation step of the NHEJ pathway. While we acknowledge that\nDNA Ligase IV represents the essential catalytic core, XRCC4 was prioritized in our original\nfunctional screen as it serves as the obligatory regulatory linchpin for pathway assembly.\nIn the original manuscript, we highlighted XRCC4 because it confers NHEJ specificity by\nacting as a molecular scaffold: it not only stabilizes DNA Ligase IV against proteasomal\ndegradation but also recruits the ligation complex specifically to DNA double-strand break\n(DSB) ends. Thus, while Ligase IV executes the enzymatic reaction, XRCC4 provides the\nnecessary regulatory framework that directs this activity to the NHEJ site.\nHowever, in accordance with your earlier strategic suggestion to focus the manuscript\nexclusively on the RNA editing of MUS81, we have removed the NHEJ-related content,\nincluding the description on XRCC4 and other auxiliary components. We believe this revision\nsignificantly streamlines the narrative, ensuring the paper remains centered on our most novel\nfinding: the functional impact of MUS81 RNA editing on environmental adaptation and stress\ntolerance.\nLines 42/43: I am not sure that filamentous fungi are commonly referred to as complex\neukaryotes.\nResponse: We thank you for this thoughtful point. We recognize that the term \"complex\neukaryotes\" is traditionally and most frequently associated with metazoans and land plants.\nOur use of the term was informed by recent evolutionary frameworks (e.g., Nagy et al., Biol.\nRev. 2018 and Knoll, Annu. Rev. Earth Planet. Sci. 2011), which classify fungi among the few\nlineages that have independently evolved complex multicellularity. This is exemplified by the\ndevelopment of sophisticated structures such as sexual fruiting bodies (perithecia),\nrhizomorphs,\nand sclerotia.\nHowever, we recognize that this term can be ambiguous or unconventional in certain contexts.\nIn accordance with your earlier suggestion to streamline the manuscript and focus specifically\non the RNA editing of MUS81, we have removed this introductory section. Consequently, this\nspecific phrasing no longer appears in the revised manuscript.\nLine 49: RAD54 is not a mediator protein and not involved in nucleating the Rad51 filament\non RPA-coated ssDNA.\nResponse: We thank you for this technical clarification. While our original phrase \"aided by\nRad52 and Rad54\" was intended as a broad description of their involvement in the overall HR\nprocess, we recognize that placing them together in the context of Rad51 loading could be\nmisleading. Rad54 functions downstream of Rad51 filament nucleation, primarily facilitating\nhomology search and chromatin remodeling rather than acting as a mediator for Rad51\nrecruitment.\nHowever, as you suggested that the manuscript would benefit from a tighter focus on MUS81\nRNA editing, we have removed this introductory overview of HR sub-pathways and their\nenzymatic factors. Therefore, this specific sentence has been excised to ensure the revised\nmanuscript remains streamlined and focused on our core findings.\nLine 56: Mus81 is not a nuclease by itself, it is the catalytic subunit of the heterodimeric Mus81-\nMms4, MUS81-EME1 nuclease. This nuclease is not structure-specific, as it cleaves multiple\nsubstrates, it is structure-selective, as it is correctly described later in the manuscript.\nResponse: We thank you for this rigorous clarification of the nomenclature. We agree that in\nthe canonical model, Mus81 functions as the catalytic subunit within a heterodimeric complex\n(Mus81-Mms4/Eme1) and that \"structure-selective\" is the more precise term to describe its\nenzymatic preference for specific DNA branched structures.\nTo address your concern, we have revised the text to ensure terminological consistency:\n• We have replaced \"structure-specific\" with \"structure-selective\" throughout the manuscript.\n• We have clarified that while Mus81 is the catalytic subunit of the Mus81-Mms4 complex\nin most systems, our findings in F. graminearum suggest a more nuanced role where\nFgMus81 can function independently of FgMms4 in certain contexts.\nLine 64: Decapitalize programmed.\nLines 72, 78: Replace Mus81 with Mus81-Mms4 or Mus81-Eme1/2.\nResponse: We thank you for your meticulous attention to these details. We have made the\nfollowing corrections in the revised manuscript:\n• \"Programmed\" has been decapitalized.\n• \"Mus81\" has been replaced with \"Mus81-Mms4\" (Lines 72, 78) to accurately reflect the\nfunctional nuclease complex in the context of the general background.\nWe appreciate your thorough review, which has helped ensure the terminological accuracy of\nour manuscript.\n7) Lines 176-179: The Rad50-Mre11-Xrs2 complex is required for meiotic DSB formation in\nbudding yeast, which could be the reason for the meiotic requirement rather than its signaling\nrole.\nResponse: We thank you for this insightful observation based on the classical model in S.\ncerevisiae. We agree that in budding yeast, the MRX complex is indispensable for Spo11-\ndependent DSB formation during meiosis.\nHowever, in F. graminearum, the initiation of sexual development—specifically the formation\nof perithecia (the sexual fruiting bodies)—occurs significantly prior to the onset of meiotic DSB\nformation. Our data show that the MRN complex mutants completely fail to initiate perithecia\nformation on carrot agar. Since this developmental block occurs at the very earliest stages of\nfruiting body morphogenesis, it suggests that the MRN complex is required for the initiation of\nthe sexual program itself, likely through its DNA damage sensing or signaling roles, rather than\na failure in subsequent meiotic recombination.\nThat being said, following your earlier strategic suggestion to focus the manuscript exclusively\non the RNA editing of MUS81, we have removed the sections regarding the MRN complex and\nthe broader functional characterization of DNA repair genes. This ensures the revised\nmanuscript remains tightly centered on the core findings related to MUS81.\n8) Lines 212ff: The section heading that non-crossover SDSA is necessary for mitotic\nrecombination is not supported by the data. Srs2, Mph1 and Sgs1 play multiple roles in genome\nmaintenance which all relate to the functions in the DNA damage response. The sensitivity\nphenotypes are not the sole consequence of an SDSA defect.\nResponse: We thank you for this insightful mechanistic critique. We agree that Srs2, Mph1,\nand Sgs1 are multifunctional proteins involved in various aspects of genome maintenance,\nincluding replication fork stabilization and DNA damage signaling, in addition to their roles in\nthe SDSA pathway. We acknowledge that our original heading was oversimplified and did not\nfully account for the pleiotropic effects of these genes.\nHowever, as you suggested to focus the manuscript strictly on the RNA editing of MUS81, we\nhave entirely removed this section and the associated data for the SDSA pathway genes in the\nrevised manuscript. This allows the paper to remain focused on the most novel finding: the\nfunctional impact of MUS81 RNA editing. By removing this broad characterization, we have\nalso eliminated the potential for over-interpretation regarding the specific roles of Srs2, Mph1,\nand Sgs1 in mitotic recombination.\n9) Line 440: mitotic\nResponse: We thank you for catching this typographical error. We have corrected the word to\n\"mitotic\" in the revised manuscript. We truly appreciate your meticulous review and attention\nto detail, which has been instrumental in ensuring the accuracy of our paper."}, {"round": 2, "reviewer_comments": {"reviewer_1": "I congratulate the authors for addressing all the reviewers' comments. I believe this paper is ready for publication and will significantly advance our understanding of transcriptomic plasticity and adaptive evolution.", "reviewer_2": "The authors carried out a substantial amount of additional work that address the majority of my concerns. They also explained the limitations that are technically not feasible to overcome. In this context, I do not have any further questions and recommend accepting the paper for publication.", "reviewer_3": "Epitranscriptomic RNA editing resolves Mus81 DNA repair tradeoffs in heat and meiosis\nThe revision greatly improved the manuscript by focusing on the major novelty of RNA editing of the Mus81 mRNA and its functional consequences. Addition of the analysis of two catalytic-deficient Mus81 mutations revealed the surprising finding that Mus81 acts independent of its nuclease activity. More analysis of the Mms4 mutant and genome analysis clarified some concerns and led to the surprising conclusion that Mus81 acts independent of Mms4, despite their physical interaction. The RNA editing finding is well documented and novel. The findings raise many questions about Mus81-Mms4 functions in this organism that will require future experimentation."}, "author_response": "Point-by-point response to the reviewers' comments\nReviewer #1 (Remarks to the Author):\nI congratulate the authors for addressing all the reviewers' comments. I believe this\npaper is ready for publication and will significantly advance our understanding of\ntranscriptomic plasticity and adaptive evolution.\nResponse: Thank you very much for your positive comments and support. We are glad\nthat our work is considered a valuable contribution to the understanding of\ntranscriptomic plasticity and adaptive evolution.\nReviewer #2 (Remarks to the Author):\nThe authors carried out a substantial amount of additional work that address the\nmajority of my concerns. They also explained the limitations that are technically not\nfeasible to overcome. In this context, I do not have any further questions and\nrecommend accepting the paper for publication.\nResponse: We appreciate your recognition of our additional experiments and\nexplanations. Thank you for your constructive feedback and recommendation for\npublication.\nReviewer #3 (Remarks to the Author):\nEpitranscriptomic RNA editing resolves Mus81 DNA repair tradeoffs in heat and\nmeiosis\nThe revision greatly improved the manuscript by focusing on the major novelty of RNA\nediting of the Mus81 mRNA and its functional consequences. Addition of the analysis\nof two catalytic-deficient Mus81 mutations revealed the surprising finding that Mus81\nacts independent of its nuclease activity. More analysis of the Mms4 mutant and\ngenome analysis clarified some concerns and led to the surprising conclusion that\nMus81 acts independent of Mms4, despite their physical interaction. The RNA editing\nfinding is well documented and novel. The findings raise many questions about Mus81-\nMms4 functions in this organism that will require future experimentation.\nResponse: We sincerely thank you for your insightful comments and appreciation of the\nmajor novelties in our revision. We agree that the findings raise important questions\nabout Mus81-Mms4 functions in F. graminearum and potentially other filamentous\nascomycetous fungi, which we look forward to investigating in future studies. Your\nfeedback has been invaluable in improving the manuscript."}]

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