Mucorales are a group of basal fungi that includes the casual agents of the human emerging disease mucormycosis. Recent studies revealed that these pathogens activate an RNAi-based pathway to rapidly generate drug-resistant epimutant strains when exposed to stressful compounds such as the antifungal drug FK506. To elucidate the molecular mechanism of this epimutation pathway, we performed a genetic analysis in Mucor circinelloides that revealed an inhibitory role for the non-canonical RdRP-dependent Dicer-independent silencing pathway, which is an RNAi-based mechanism involved in mRNA degradation that was recently identified. Thus, mutations that specifically block the mRNA degradation pathway, such as those in the genes r3b2 and rdrp3 , enhance the production of drug resistant epimutants, similar to the phenotype previously described for mutation of the gene rdrp1 . Our genetic analysis also revealed two new specific components of the epimutation pathway related to the quelling induced protein (qip) and a Sad-3-like helicase ( rnhA ), as mutations in these genes prevented formation of drug-resistant epimutants. Remarkably, drug-resistant epimutant production was notably increased in M . circinelloides f . circinelloides isolates from humans or other animal hosts. The host-pathogen interaction could be a stressful environment in which the phenotypic plasticity provided by the epimutant pathway might provide an advantage for these strains. These results evoke a model whereby balanced regulation of two different RNAi pathways is determined by the activation of the RNAi-dependent epimutant pathway under stress conditions, or its repression when the regular maintenance of the mRNA degradation pathway operates under non-stress conditions. Author summary Mucormycosis is a fungal infection that is attracting the attention of both clinical and research communities because of the lack of effective antifungal treatments and its often fatal prognosis. Our previous studies revealed an RNAi-mediated epimutation mechanism that operates in the casual human fungal pathogens (Mucorales species) and which might underlie the lack of efficacy of some antifungal treatments. This epimutation mechanism represses the expression of antifungal drug target genes and thereby generates antifungal drug resistant strains. Here, we studied the regulation and identified new components of the epimutation pathway. We found that a newly identified mRNA degradation pathway, named the non-canonical RdRP-dependent Dicer-independent silencing pathway, exerts an inhibitory effect on the RNAi-mediated epimutation mechanism and operates during growth under non-stressful conditions. Interestingly, the RNAi-based epimutation mechanism is more active in M . circinelloides f . circinelloides isolates from human and other animal sources, suggesting that this mechanism may influence host-pathogen interactions. These results further our understanding of the mechanisms deployed by fungal pathogens to survive and adapt under stressful environmental conditions that may include the host niche.
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