tRNA hypomodification facilitates 5-fluorocytosine resistance via cross-pathway control system activation in Aspergillus fumigatus

Increasing antifungal drug resistance is a major concern associated with human fungal pathogens like Aspergillus fumigatus . Genetic mutation and epimutation mechanisms clearly drive resistance, yet the epitranscriptome remains relatively untested. Here, deletion of the A. fumigatus tRNA-modifying isopentenyl transferase ortholog, Mod5, led to altered stress response and unexpected resistance against the antifungal drug 5-fluorocytosine (5-FC). After confirming the canonical isopentenylation activity of Mod5 by LC-MS/MS and Nano-tRNAseq, we performed simultaneous profiling of transcriptomes and proteomes to reveal a comparable overall response to 5-FC stress; however, a premature activation of cross-pathway control (CPC) genes in the knockout was further increased after antifungal treatment. We identified several orthologues of the A. nidulans Major Facilitator Superfamily (MFS) transporter nmeA as specific CPC-client genes in A. fumigatus . Overexpression of Mod5-target tRNA ^Tyr _GΨA in the Δ mod5 strain rescued select phenotypes but failed to reverse 5-FC resistance, whereas deletion of nmeA largely, but incompletely, reverted the resistance phenotype, implying additional relevant exporters. In conclusion, 5-FC resistance in the absence of Mod5 and i ⁶ A likely originates from multifaceted transcriptional and translational changes that skew the fungus towards premature CPC-dependent activation of antifungal toxic-intermediate exporter nmeA , offering a potential mechanism reliant on RNA modification to facilitate transient antifungal resistance.