A non-canonical RNAi pathway controls virulence and genome stability in Mucorales

Abstract

AbstractEpimutations in fungal pathogens are emerging as novel phenomena that could explain the fast-developing resistance to antifungal drugs and other stresses. These epimutations are generated by RNA interference (RNAi) mechanisms that transiently silence specific genes to overcome stressful stimuli. The early-diverging fungus Mucor circinelloides exercises a fine control over two interacting RNAi pathways to produce epimutants: the canonical RNAi pathway and a new RNAi degradative pathway. The latter is considered a non-canonical RNAi pathway (NCRIP) because it relies on RNA-dependent RNA polymerases (RdRPs) and a novel ribonuclease III-like named R3B2 to degrade target transcripts. Here in this work, we uncovered the role of NCRIP in regulating virulence processes and transposon movements through key components of the pathway, RdRP1, and R3B2. Mutants in these genes are unable to launch a proper virulence response to macrophage phagocytosis, resulting in a decreased virulence potential. The transcriptomic profile of rdrp1Δ and r3b2Δ mutants revealed a pre-exposure adaptation to the stressful phagosomal environment even when the strains are not confronted by macrophages. These results suggest that NCRIP represses key targets during regular growth and release its control when the fungus is challenged by a stressful environment. NCRIP interacts with the RNAi canonical core to protect genome stability by controlling the expression of centromeric retrotransposable elements. In the absence of NCRIP, these retrotransposons are robustly repressed by the canonical RNAi machinery; thus, supporting the antagonistic role of NCRIP in containing the epimutational pathway. Both interacting RNAi pathways might be essential to govern host-pathogen interactions through transient adaptations, contributing to the unique traits of the emerging infection mucormycosis.Author summaryMucormycosis is an emergent and lethal infectious disease caused by Mucorales, a fungal group resistant to most antifungal drugs. Mucor circinelloides, a genetic model to characterize this infection, can develop drug resistance via RNAi epimutations. This epimutational RNAi mechanism interacts with a novel non-canonical RNAi pathway (NCRIP), where the ribonuclease III-like R3B2 and the RNA-dependent RNA polymerase RdRP1 are essential. The analysis of the transcriptomic response to phagocytosis by macrophage in rdrp1Δ and r3b2Δ mutants revealed that NCRIP might control virulence in M. circinelloides. These mutants showed constitutive activation of the response to phagocytosis and a reduction in virulence in a mouse model, probably caused by a disorganized execution of the genetic program to overcome host defense mechanisms. The antagonistic role of the NCRIP and the RNAi canonical core is evident during post-transcriptional regulation of centromeric retrotransposons. These retrotransposons are silenced by the canonical RNAi pathway, but this regulation is restrained by NCRIP, proven by an overproduction of small RNAs targeting these loci in NCRIP mutants. These new insights into the initial phase of mucormycosis and transposable element regulation point to NCRIP as a crucial genetic regulator of pathogenesis-related molecular processes that could serve as pharmacological targets.