RNA network maps fungal spore-to-hypha transition

The globally distributed zoophilic dermatophyte Trichophyton mentagrophytes infects keratinized tissues in humans and diverse mammalian hosts, causing conditions including tinea manuum, onychomycosis, tinea capitis and tinea pedis. Recurrent infections and increasing antifungal resistance have made this organism an important public-health concern, while the regulatory architecture controlling its germination remains incompletely understood. A multi-RNA study integrated messenger RNA, long non-coding RNA and microRNA expression profiles to portray the transition from dormant spores to hyphae as a coordinated developmental event.

The researchers compared spores with hyphae, identified thousands of stage-specific transcripts and assembled candidate competing endogenous RNA networks in which non-coding RNAs may help determine which fungal genes are switched on or off as the organism changes form. Microscopy established a clear timetable for the morphological transition. After inoculation into liquid medium, dormant spores began swelling within six hours, approximately doubling their diameter while establishing a polarized growth site.

By 12 hours, a single germ tube had emerged, and at 18 hours multiple germ tubes had developed into hyphae. Germination therefore involves more than enlargement: the fungus must remodel its cell wall, reorganize its cytoskeleton, transport cellular materials, enter the cell cycle and alter its metabolism while committing to polarized growth. The researchers prepared ribosomal-RNA-depleted long-RNA libraries to retain coding and non-coding transcripts and separately constructed small-RNA libraries enriched for molecules in the microRNA size range.

Three biological replicates were analyzed for each of the two sampled stages, spores and hyphae. The coding-RNA analysis identified 8,059 messenger RNAs, including 505 previously unannotated transcripts, and detected 3,193 differentially expressed mRNAs between spores and hyphae. Of these, 692 were more abundant in hyphae and 2,501 were more abundant in spores, using a false-discovery-rate threshold below 0.05 and a magnitude of change greater than twofold.

The hyphal-upregulated set was enriched for mitotic processes, including spindle-midzone organization, microtubule polymerization and sister-chromatid separation. Gene Set Enrichment Analysis also associated DNA replication, the cell cycle and spore germination or early outgrowth with the hyphal state. In contrast, pathways for amino-acid biosynthesis—including methionine, cysteine, arginine, proline and branched-chain amino-acid production—were enriched among transcripts declining during germination.

Other enriched functions included mitochondrial activity, lipid and carbohydrate metabolism, pigment production and fungal-type cell-wall organization, placing the transcriptional response at the intersection of metabolism and the physical changes required for polarized growth. Long non-coding RNAs formed a substantial part of the stage-specific response. This procedure identified 3,961 high-confidence lncRNAs, of which 34 were previously known and 3,927 were novel.

Their genomic positions included 2,367 antisense transcripts, 732 bidirectional transcripts, 236 intergenic transcripts, 436 categorized as other, 17 intronic transcripts and 173 sense-overlapping transcripts. Overall lncRNA abundance was significantly greater in hyphae than in spores, and 409 lncRNAs were differentially expressed: 213 increased and 196 decreased. Their number and broad distribution across genomic contexts indicated that non-coding RNA expression was an important feature of the transition, although the computational annotations did not establish the function of each transcript.

Several analyses suggested that lncRNAs may influence nearby or distant genes through different routes. The researchers identified 99 high-confidence antisense associations between 97 differentially expressed antisense lncRNAs and their candidate target mRNAs. Targets of these associations were enriched for functions including regulation of the G2-to-M transition, establishment of cell polarity and differentiation-related morphogenesis.

Enriched pathways included the yeast mitogen-activated protein kinase signaling pathway, oxidative phosphorylation, ribosome biogenesis, cell-cycle regulation and secondary-metabolite biosynthesis. A separate analysis searched for local regulatory relationships within a 10-kilobase genomic window and found 1,158 candidate cis interactions involving 389 lncRNAs and 950 mRNAs. Their targets included cell-wall disassembly during cellular fusion, fructose metabolism and cell-cycle control, making regulation of genes needed during hyphal development one plausible function.

Broader co-expression analysis produced 247,586 candidate trans relationships between 409 differentially expressed lncRNAs and 3,138 target mRNAs. Associated functions included amino-acid transport, pigment biosynthesis and adhesion of a symbiont to a host, while enriched pathways covered several amino-acid biosynthetic routes, carbohydrate and lipid metabolism, and cofactor production. One example was MSTRG.8251.1, a transcript positioned upstream of the cyclin gene CDC2.

Both were upregulated in hyphae and showed correlated expression, making local regulation of cell-cycle entry a plausible interpretation. The authors also linked lncRNAs to genes encoding cytoskeletal and transport components, including microtubule-associated proteins and vesicle SNAREs. Such results are consistent with non-coding RNAs helping coordinate the physical and metabolic demands of germ-tube formation, but correlation and enrichment analysis alone cannot prove a molecular mechanism or direct regulatory interaction.

Small-RNA sequencing added a predicted post-transcriptional regulatory layer. The analysis recovered 733 known microRNAs and predicted 52 novel candidates from genomic position and hairpin-forming potential. Both groups exhibited a characteristic 5-prime uracil bias and the expected 18–25-nucleotide size range.

A total of 119 miRNAs differed significantly between spores and hyphae, with 56 more abundant in hyphae and 63 more abundant in spores. Target prediction connected the miRNAs to 8,662 potential mRNAs through 626,128 target sites. Predicted target functions included the plasma membrane, transferase and oxidoreductase activity, growth, reproduction and mitotic cell-cycle regulation.

The scale of this targeting landscape suggests extensive potential for post-transcriptional tuning during germination, particularly involving cell-cycle regulators and metabolic enzymes, but the study did not directly demonstrate repression by every candidate miRNA. The researchers combined the three RNA classes using a competing endogenous RNA model, in which an lncRNA and an mRNA sharing a microRNA response element can compete for the same miRNA. Increased lncRNA abundance may consequently free an mRNA from miRNA-mediated suppression, whereas increased miRNA abundance could reduce the availability of both competitors.

Expression correlations and target predictions ultimately yielded a candidate network of 14,360 statistically selected pairs containing 2,627 mRNAs, 107 miRNAs and 329 lncRNAs. Highly connected nodes included the mRNAs glt1, dtxS1, g3795, SPBC20F10.07 and MSTRG.440, together with five lncRNAs designated MSTRG.9453.1, MSTRG.2311.1, MSTRG.2710.1, MSTRG.3534.1 and MSTRG.8002.1. One proposed axis, MSTRG.10182.6, miR-1388-y and the growth-related gene ro-4, was examined with quantitative real-time PCR.

The lncRNA and ro-4 were both more abundant in hyphae, which is compatible with the proposed sequestration model, although the experiment did not independently show that miR-1388-y regulated both targets. The resulting atlas offers a systems-level view of fungal developmental control, but its conclusions are constrained by the experimental design. Sequencing compared only spores and mature hyphae, leaving RNA changes during the observed 6-, 12- and 18-hour transitions unresolved.

The long-RNA pipeline deliberately excluded single-exon candidates, and the final hypergeometric screening of ceRNA pairs used an uncorrected P-value below 0.05 without a false-discovery-rate adjustment, so some interactions may be false positives. More fundamentally, correlation, sequence matching and enrichment analysis can nominate regulatory relationships without establishing causality. Additional sampling, direct RNA–RNA binding measurements, target-repression tests and reporter assays would be needed to validate the candidate circuits, while protein-interaction studies could reveal whether lncRNAs recruit chromatin regulators or RNA-binding complexes.

Even with these limitations, the study identifies stage-specific molecules and pathways that may help explain how T. mentagrophytes coordinates wall remodeling, cell division, cytoskeletal change and metabolic adaptation during its spore-to-hypha transition.

Subject of Research: Stage-specific mRNA, lncRNA and miRNA regulation during fungal spore-to-hypha transition

Article Title: Stage-specific RNA regulomes of Trichophyton mentagrophytes : mRNA-lncRNA-miRNA interplay in spore-hypha transition

Article References: Xiao, W., Wu, Z., Zhang, J., Wan, J., Zhang, R., Xiang, X., Yu, Y., Fu, L., Yang, K., Chen, Y., Xiao, Z., Wang, Z., He, L., You, J., & Zhang, C. (2025). Stage-specific RNA regulomes of Trichophyton mentagrophytes: mRNA-lncRNA-miRNA interplay in spore-hypha transition. IMA Fungus, 16, Article e166433. https://doi.org/10.3897/imafungus.16.166433

Image Credits: AI Generated

DOI: 10.3897/imafungus.16.166433

Keywords: Trichophyton mentagrophytes, fungal germination, dermatophytes, long non-coding RNA, microRNA, competing endogenous RNA, RNA sequencing, hyphal morphogenesis, transcriptomics, cell-wall remodeling, Stage-specific, regulomes