Transcriptomic characterization of Coccidioides morphological states using RiboMarker-enhanced RNA sequencing.

Coccidioides is a dimorphic fungal pathogen responsible for the mammalian disease commonly known as Valley fever. While previous studies have characterized transcriptomic changes associated with its life stage transitions, the contribution of small RNAs (sRNAs)-key regulators of virulence in other pathogenic fungi-remains unexplored. Here, we profile sRNA expression across three Coccidioides posadasii morphologies-arthroconidia, mycelia, and spherules-analyzing both intracellular and extracellular RNA fractions. Utilizing RiboMarker sRNA and RNA fragment library preparation, we achieved enhanced transcriptome coverage by incorporating RNA species that are typically underrepresented or incompatible with standard sequencing workflows. We observed pronounced transcriptomic remodeling during the transition from arthroconidia to mycelia or spherules, driven primarily by changes in protein-coding transcripts, tRNA, and unannotated loci. These data identify sRNA- and fragment-producing loci that may be required for progression between the saprobic and parasitic life cycles of Coccidioides. In addition, distinct RNA fragmentation patterns were associated with each morphological state. Notably, we detected evidence of RNA export to the extracellular space, particularly snRNAs and tRNA-derived fragments, during these transitions, suggesting potential roles in cell-cell or host-pathogen communication. Together, this intra- and extracellular Coccidioides sRNA atlas provides a resource for biomarker discovery and advances our understanding of the molecular basis of fungal virulence.