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Competing Interests: The authors have declared that no competing interests exist.

Members of the ribonuclease III (RNase III) family regulate gene expression by processing double-stranded RNA (dsRNA). This family includes eukaryotic Dicer and Drosha enzymes that generate small dsRNAs in the RNA interference (RNAi) pathway. The fungus Mucor lusitanicus, which causes the deadly infection mucormycosis, has a complex RNAi system encompassing a non-canonical RNAi pathway (NCRIP) that regulates virulence by degrading specific mRNAs. In this pathway, Dicer function is replaced by R3B2, an atypical class I RNase III, and small single-stranded RNAs (ssRNAs) are produced instead of small dsRNA as Dicer-dependent RNAi pathways. Here, we show that R3B2 forms a homodimer that binds to ssRNA and dsRNA molecules, but exclusively cuts ssRNA, in contrast to all known RNase III. The dsRNA cleavage inability stems from its unusual RNase III domain (RIIID) because its replacement by a canonical RIIID allows dsRNA processing. A crystal structure of R3B2 RIIID resembles canonical RIIIDs, despite the low sequence conservation. However, the groove that accommodates dsRNA in canonical RNases III is narrower in the R3B2 homodimer, suggesting that this feature could be responsible for the cleavage specificity for ssRNA. Conservation of this activity in R3B2 proteins from other mucormycosis-causing Mucorales fungi indicates an early evolutionary acquisition., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Centromeres are rapidly evolving across eukaryotes, despite performing a conserved function to ensure high-fidelity chromosome segregation. CENP-A chromatin is a hallmark of a functional centromere in most organisms. Due to its critical role in kinetochore architecture, the loss of CENP-A is tolerated in only a few organisms, many of which possess holocentric chromosomes. Here, we characterize the consequence of the loss of CENP-A in the fungal kingdom. Mucor circinelloides, an opportunistic human pathogen, lacks CENP-A along with the evolutionarily conserved CENP-C but assembles a monocentric chromosome with a localized kinetochore complex throughout the cell cycle. Mis12 and Dsn1, two conserved kinetochore proteins, were found to co-localize to a short region, one in each of nine large scaffolds, composed of an ∼200-bp AT-rich sequence followed by a centromere-specific conserved motif that echoes the structure of budding yeast point centromeres. Resembling fungal regional centromeres, these core centromere regions are embedded in large genomic expanses devoid of genes yet marked by Grem-LINE1s, a novel retrotransposable element silenced by the Dicer-dependent RNAi pathway. Our results suggest that these hybrid features of point and regional centromeres arose from the absence of CENP-A, thus defining novel mosaic centromeres in this early-diverging fungus., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Mucormycosis is an emerging fungal infection that is often lethal due to the ineffectiveness of current therapies. Here, we have studied the first stage of this infection-the germination of Mucor circinelloides spores inside phagocytic cells-from an integrated transcriptomic and functional perspective. A relevant fungal gene network is remodeled in response to phagocytosis, being enriched in crucial functions to survive and germinate inside the phagosome, such as nutritional adaptation and response to oxidative stress. Correspondingly, the phagocytic cells induced a specific proinflammatory and apoptotic response to the pathogenic strain. Deletion of fungal genes encoding putative transcription factors ( atf1 , atf2 , and gcn4 ), extracellular proteins ( chi1 and pps1 ), and an aquaporin ( aqp1 ) revealed that these genes perform important roles in survival following phagocytosis, germination inside the phagosome, and virulence in mice. atf1 and atf2 play a major role in these pathogenic processes, since their mutants showed the strongest phenotypes and both genes control a complex gene network of secondarily regulated genes, including chi1 and aqp1 These new insights into the initial phase of mucormycosis define genetic regulators and molecular processes that could serve as pharmacological targets. IMPORTANCE Mucorales are a group of ancient saprophytic fungi that cause neglected infectious diseases collectively known as mucormycoses. The molecular processes underlying the establishment and progression of this disease are largely unknown. Our work presents a transcriptomic study to unveil the Mucor circinelloides genetic network triggered in fungal spores in response to phagocytosis by macrophages and the transcriptional response of the host cells. Functional characterization of differentially expressed fungal genes revealed three transcription factors and three extracellular proteins essential for the fungus to survive and germinate inside the phagosome and to cause disease in mice. Two of the transcription factors, highly similar to activating transcription factors (ATFs), coordinate a complex secondary gene response involved in pathogenesis. The significance of our research is in characterizing the initial stages that lead to evasion of the host innate immune response and, in consequence, the dissemination of the infection. This genetic study offers possible targets for novel antifungal drugs against these opportunistic human pathogens., (Copyright © 2019 Pérez-Arques et al.)

The increasing number of infections by species of Mucorales and their high mortality constitute an important concern for public health. This study aims to decipher the genetic basis of Mucor circinelloides pathogenicity, which displays virulence in a strain dependent manner. Assuming that genetic differences between strains may be linked to different pathotypes, we have conducted a study to explore genes responsible for virulence in M. circinelloides by whole genome sequencing of the avirulent strain NRRL3631 and comparison with the virulent strain CBS277.49. This genome analysis revealed 773 truncated, discontiguous and absent genes in the NRRL3631 strain. We also examined phenotypic traits resulting in reduced heat stress tolerance, chitosan content and lower susceptibility to toxic compounds (calcofluor white and sodium dodecyl sulphate) in the virulent strain, suggesting the influence of cell wall on pathogenesis. Based on these results, we focused on studying extracellular protein-coding genes by gene deletion and further pathotype characterization of mutants in murine models of pulmonary and systemic infection. Deletion of gene ID112092, which codes for a hypothetical extracellular protein of unknown function, resulted in significant reduction of virulence. Although pathogenesis is a multifactorial process, these findings highlight the crucial role of surface and secreted proteins in M. circinelloides virulence and should promote further studies of other differential genes.

The carotene producer Mucor circinelloides is the fungus within the Mucoromycota phylum with the widest repertoire of molecular tools to manipulate its genome. The initial development of an effective procedure for genetic transformation and later improvements have resulted in an expansion of available tools, which include gene replacement, inactivation of gene expression by RNA silencing, gene overexpression, and functional genomics. Moreover, sequencing of its genome has given a definitive boost to these techniques making attainable the study of genes involved in many physiological or developmental processes, including carotenoid biosynthesis. Here, we describe in detail the latest molecular techniques currently used in M. circinelloides that have made it a valuable model for studying gene function within its phylum.

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.

Mucorales are an emerging group of human pathogens that are responsible for the lethal disease mucormycosis. Unfortunately, functional studies on the genetic factors behind the virulence of these organisms are hampered by their limited genetic tractability, since they are reluctant to classical genetic tools like transposable elements or gene mapping. Here, we describe an RNAi-based functional genomic platform that allows the identification of new virulence factors through a forward genetic approach firstly described in Mucorales. This platform contains a whole-genome collection of Mucor circinelloides silenced transformants that presented a broad assortment of phenotypes related to the main physiological processes in fungi, including virulence, hyphae morphology, mycelial and yeast growth, carotenogenesis and asexual sporulation. Selection of transformants with reduced virulence allowed the identification of mcplD, which encodes a Phospholipase D, and mcmyo5, encoding a probably essential cargo transporter of the Myosin V family, as required for a fully virulent phenotype of M. circinelloides. Knock-out mutants for those genes showed reduced virulence in both Galleria mellonella and Mus musculus models, probably due to a delayed germination and polarized growth within macrophages. This study provides a robust approach to study virulence in Mucorales and as a proof of concept identified new virulence determinants in M. circinelloides that could represent promising targets for future antifungal therapies., Competing Interests: "The authors have declared that no competing interests exist."

The increasing knowledge on the functional relevance of endogenous small RNAs (esRNAs) as riboregulators has stimulated the identification and characterization of these molecules in numerous eukaryotes. In the basal fungus Mucor circinelloides, an emerging opportunistic human pathogen, esRNAs that regulate the expression of many protein coding genes have been described. These esRNAs share common machinery for their biogenesis consisting of an RNase III endonuclease Dicer, a single Argonaute protein and two RNA-dependent RNA polymerases. We show in this study that, besides participating in this canonical dicer-dependent RNA interference (RNAi) pathway, the rdrp genes are involved in a novel dicer-independent degradation process of endogenous mRNAs. The analysis of esRNAs accumulated in wild type and silencing mutants demonstrates that this new rdrp-dependent dicer-independent regulatory pathway, which does not produce sRNA molecules of discrete sizes, controls the expression of target genes promoting the specific degradation of mRNAs by a previously unknown RNase. This pathway mainly regulates conserved genes involved in metabolism and cellular processes and signaling, such as those required for heme biosynthesis, and controls responses to specific environmental signals. Searching the Mucor genome for candidate RNases to participate in this pathway, and functional analysis of the corresponding knockout mutants, identified a new protein, R3B2. This RNase III-like protein presents unique domain architecture, it is specifically found in basal fungi and, besides its relevant role in the rdrp-dependent dicer-independent pathway, it is also involved in the canonical dicer-dependent RNAi pathway, highlighting its crucial role in the biogenesis and function of regulatory esRNAs. The involvement of RdRPs in RNA degradation could represent the first evolutionary step towards the development of an RNAi mechanism and constitutes a genetic link between mRNA degradation and post-transcriptional gene silencing.

Background: RNA interference (RNAi) is a conserved mechanism of genome defence that can also have a role in the regulation of endogenous functions through endogenous small RNAs (esRNAs). In fungi, knowledge of the functions regulated by esRNAs has been hampered by lack of clear phenotypes in most mutants affected in the RNAi machinery. Mutants of Mucor circinelloides affected in RNAi genes show defects in physiological and developmental processes, thus making Mucor an outstanding fungal model for studying endogenous functions regulated by RNAi. Some classes of Mucor esRNAs map to exons (ex-siRNAs) and regulate expression of the genes from which they derive. To have a broad picture of genes regulated by the silencing machinery during vegetative growth, we have sequenced and compared the mRNA profiles of mutants in the main RNAi genes by using RNA-seq. In addition, we have achieved a more complete phenotypic characterization of silencing mutants., Results: Deletion of any main RNAi gene provoked a deep impact in mRNA accumulation at exponential and stationary growth. Genes showing increased mRNA levels, as expected for direct ex-siRNAs targets, but also genes with decreased expression were detected, suggesting that, most probably, the initial ex-siRNA targets regulate the expression of other genes, which can be up- or down-regulated. Expression of 50% of the genes was dependent on more than one RNAi gene in agreement with the existence of several classes of ex-siRNAs produced by different combinations of RNAi proteins. These combinations of proteins have also been involved in the regulation of different cellular processes. Besides genes regulated by the canonical RNAi pathway, this analysis identified processes, such as growth at low pH and sexual interaction that are regulated by a dicer-independent non-canonical RNAi pathway., Conclusion: This work shows that the RNAi pathways play a relevant role in the regulation of a significant number of endogenous genes in M. circinelloides during exponential and stationary growth phases and opens up an important avenue for in-depth study of genes involved in the regulation of physiological and developmental processes in this fungal model.

The basal fungus Mucor circinelloides has become, in recent years, a valuable model to study RNA-mediated gene silencing or RNA interference (RNAi). Serendipitously discovered in the late 1900s, the gene silencing in M. circinelloides is a landscape of consensus and dissents. Although similar to other classical fungal models in the basic design of the essential machinery that is responsible for silencing of gene expression, the existence of small RNA molecules of different sizes generated during this process and the presence of a mechanism that amplifies the silencing signal, give it a unique identity. In addition, M. circinelloides combines the components of RNAi machinery to carry out functions that not only limit themselves to the defense against foreign genetic material, but it uses some of these elements to regulate the expression of its own genes. Thus, different combinations of RNAi elements produce distinct classes of endogenous small RNAs (esRNAs) that regulate different physiological and developmental processes in response to environmental signals. The recent discovery of a new RNAi pathway involved in the specific degradation of endogenous mRNAs, using a novel RNase protein, adds one more element to the exciting puzzle of the gene silencing in M. circinelloides, in addition to providing hints about the evolutionary origin of the RNAi mechanism., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Microorganisms evolve via a range of mechanisms that may include or involve sexual/parasexual reproduction, mutators, aneuploidy, Hsp90 and even prions. Mechanisms that may seem detrimental can be repurposed to generate diversity. Here we show that the human fungal pathogen Mucor circinelloides develops spontaneous resistance to the antifungal drug FK506 (tacrolimus) via two distinct mechanisms. One involves Mendelian mutations that confer stable drug resistance; the other occurs via an epigenetic RNA interference (RNAi)-mediated pathway resulting in unstable drug resistance. The peptidylprolyl isomerase FKBP12 interacts with FK506 forming a complex that inhibits the protein phosphatase calcineurin. Calcineurin inhibition by FK506 blocks M. circinelloides transition to hyphae and enforces yeast growth. Mutations in the fkbA gene encoding FKBP12 or the calcineurin cnbR or cnaA genes confer FK506 resistance and restore hyphal growth. In parallel, RNAi is spontaneously triggered to silence the fkbA gene, giving rise to drug-resistant epimutants. FK506-resistant epimutants readily reverted to the drug-sensitive wild-type phenotype when grown without exposure to the drug. The establishment of these epimutants is accompanied by generation of abundant fkbA small RNAs and requires the RNAi pathway as well as other factors that constrain or reverse the epimutant state. Silencing involves the generation of a double-stranded RNA trigger intermediate using the fkbA mature mRNA as a template to produce antisense fkbA RNA. This study uncovers a novel epigenetic RNAi-based epimutation mechanism controlling phenotypic plasticity, with possible implications for antimicrobial drug resistance and RNAi-regulatory mechanisms in fungi and other eukaryotes.

The mechanism of RNAi is well described in metazoans where it plays a role in diverse cellular functions. However, although different classes of endogenous small RNAs (esRNAs) have been identified in fungi, their biological roles are poorly described due, in part, to the lack of phenotype of mutants affected in the biogenesis of these esRNAs. Argonaute proteins are one of the key components of the RNAi pathways, in which different members of this protein family participate in the biogenesis of a wide repertoire of esRNAs molecules. Here we identified three argonaute genes of the fungus Mucor circinelloides and investigated their participation in exogenous and endogenous RNAi. We found that only one of the ago genes, ago-1, is involved in RNAi during vegetative growth and is required for both transgene-induced RNA silencing and the accumulation of distinct classes of esRNAs derived from exons (ex-siRNAs). Classes I and II ex-siRNAs bind to Ago-1 to control mRNA accumulation of the target protein coding genes. Class III ex-siRNAs do not specifically bind to Ago-1, but requires this protein for their production, revealing the complexity of the biogenesis pathways of ex-siRNAs. We also show that ago-1 is involved in the response to environmental signals, since vegetative development and autolysis induced by nutritional stress are affected in ago-1(-) M. circinelloides mutants. Our results demonstrate that a single Ago protein participates in the production of different classes of esRNAs that are generated through different pathways. They also highlight the role of ex-siRNAs in the regulation of endogenous genes in fungi and expand the range of biological functions modulated by RNAi.

RNA-dependent RNA polymerases (RdRPs) play key roles in the RNA silencing pathway in a number of organisms. They have been involved in the production of double-stranded RNA (dsRNA) molecules that initiate the silencing mechanism as well as in the amplification of the silencing signal. The roles of RdRPs from fungi in these processes are poorly described compared with other eukaryotes. RNA silencing in the zygomycete Mucor circinelloides exhibits uncommon features, such as induction by self-replicative sense transgenes and an amplification process associated with two size classes of antisense small interfering RNAs (siRNAs). To investigate the function of fungal RdRP proteins in initiation and amplification of silencing we have cloned and characterized two different rdrp genes in M. circinelloides. Functional analysis of rdrp(-) disruption mutants indicates that rdrp-1 is essential for initiation of silencing by sense transgenes by producing antisense RNA transcripts derived from the transgene, but it is not necessary for amplification of the silencing signal, whereas rdrp-2 is required for efficient accumulation of the two different classes of secondary siRNAs regardless the nature of the trigger. Our results provide evidence for a functional diversification of M. circinelloides rdrp genes in different steps of the same RNA silencing pathway., (© 2011 Blackwell Publishing Ltd.)

RNA silencing in the zygomycete Mucor circinelloides exhibits uncommon features, such as induction by self-replicative sense transgenes and the accumulation of two size classes of antisense small interfering RNAs (siRNAs). To investigate whether this silencing phenomenon follows the rules of a canonical RNA-silencing mechanism, we used hairpin RNA (hpRNA)-producing constructs as silencing triggers and analyzed the efficiency and stability of silencing in different genetic backgrounds. We show here that the dsRNA-induced silencing mechanism is also associated with the accumulation of two sizes of antisense siRNAs and that this mechanism is not mediated by the previously known dcl-1 (dicer-like) gene, which implies the existence of an additional dicer gene. An M. circinelloides dcl-2 gene was cloned and characterized, and the corresponding null mutant was generated by gene replacement. This mutant is severely impaired in the silencing mechanism induced by self-replicative sense or inverted-repeat transgenes, providing the first genetic evidence of a canonical silencing mechanism in this class of fungus and pointing to a role for dcl-2 in the mechanism. Moreover, a functional dcl-2 gene is required for the normal accumulation of the two sizes of antisense RNAs, as deduced from the analysis of dcl-2(-) transformants containing hpRNA-expressing plasmids. In addition to its critical role in transgene-induced silencing, the dcl-2 gene seems to play a role in the control of vegetative development, since the dcl-2 null mutants showed a significant decrease in their production of asexual spores.

Initiation and maintenance of the RNA silencing mechanism was investigated after transcriptional activation of the transgene carB in Mucor circinelloides. Light induced transcription of the reporter gene carB specifically increased the 21-nt siRNA compared with the 25-nt siRNA and rose the silencing maintenance from 48% up to 93% of the descendent colonies. In accordance, induced transcription of the gene carB through disruption of the gene crgA increased the initial silencing frequency up to 30-fold, when compared with the frequency of silencing obtained using a crgA(+) genetic background.

Mucor circinelloides responds to blue light by activating the biosynthesis of carotenoids and bending its sporangiophores towards the light source. The CrgA protein product acts as a repressor of carotene biosynthesis, as its inactivation leads to the overaccumulation of carotenoids in both the dark and the light. We show here that asexual sporulation in Mucor is also stimulated by light and that the crgA gene is involved in sporulation, given that lack of crgA function affects both carotenogenesis and the normal production of spores. A small interference RNA (siRNA) gene silencing approach was used to block the biosynthesis of carotenoids and to demonstrate that abnormal sporulation in crgA mutants is not a consequence of a defective production of carotenes. These results reveal an active role for the predicted CrgA product, a RING-finger protein, in the control of cellular light-regulated processes in Mucor.

Dicer proteins are ribonuclease III enzymes that process double stranded RNA precursors into small RNAs categorized as small interfering RNAs (siRNAs) or microRNAs (miRNAs), which suppress gene expression through the RNA silencing mechanism. We have isolated a dicer-like gene (dcl-1) of Mucor circinelloides, the first gene of this family to be identified in zygomycetes. The dcl-1 mRNA occurred in multiple forms, including the truncated molecules that result from premature polyadenylation. Null dcl-1 mutants were not impaired as regards transgene-induced gene silencing, since they exhibited the same silencing frequency as the wild-type strain and accumulated the two size classes of siRNA associated with RNA silencing in M. circinelloides. However, dcl-1 mutants showed a reduced growth rate and a hyphal growth alteration, which suggests that the dcl-1 gene has some role in the control of endogenous functions.

Transformation of Mucor circinelloides with self-replicative plasmids containing a wild-type copy of the carotenogenic gene carB causes silencing of the carB function in 3% of transformants. Genomic analyses revealed a relationship between silenced phenotype and number of copies of plasmids. This phenotype results from a reduction of the steady-state levels of carB mRNA, a reduction that is not due to differences in the level of transcription, indicating that silencing is post-transcriptional. Small sense and antisense RNAs have been found to be associated with gene silencing in M. circinelloides. Two size classes of small antisense RNAs, differentially accumulated during the vegetative growth of silenced transformants, have been detected: a long 25-nucleotide RNA and a short 21-nucleotide RNA. Secondary sense and antisense RNAs corresponding to sequences of the endogenous gene downstream of the initial triggering molecule have also been detected, revealing the existence of spreading of RNA targeting in fungi. These findings, together with the self-replicative nature of the triggering molecules, make M. circinelloides a suitable organism for investigating some unresolved questions in RNA silencing.

Mucor circinelloides responds to blue light by activating carotene biosynthesis. Wild-type strains grown in darkness contain minimal amounts of beta-carotene because of the low levels of transcription of the structural genes for carotenogenesis. When exposed to a light pulse, the level of transcription of these genes increases strongly, leading to the formation of high concentrations of beta-carotene. The crgA gene is involved in the regulation of light-induced carotenoid biosynthesis. This gene, originally identified as a 3'-truncated ORF which causes carotene over-accumulation in the dark, encodes a protein with a cysteine-rich, zinc-binding, RING-finger motif, as found in diverse groups of regulatory proteins. The expression of the crgA gene is activated by a light pulse, with a time course similar to that of the structural genes for carotenogenesis. To understand the regulatory role of the crgA gene in carotenogenesis, we have used a genetic approach based on the construction of crgA null mutants by gene replacement. Lack of the crgA function provokes the over-accumulation of carotenoids both in the dark and the light. Introduction of the wild-type crgA allele into these mutants restores the wild-type phenotype for carotenogenesis. The high levels of carotenoid accumulation shown by the null crgA mutants are correlated with an increase in the expression of carotenogenic structural genes. These results strongly indicate that crgA acts as a negative regulator of light-inducible carotenogenesis in M. circinelloides.