Mucor circinelloides is a zygomycete fungus and an emerging opportunistic pathogen in immunocompromised patients, especially transplant recipients and in some cases otherwise healthy individuals. We have discovered a novel example of size dimorphism linked to virulence. M. circinelloides is a heterothallic fungus: (+) sex allele encodes SexP and (−) sex allele SexM, both of which are HMG domain protein sex determinants. M. circinelloides f. lusitanicus (Mcl) (−) mating type isolates produce larger asexual sporangiospores that are more virulent in the wax moth host compared to (+) isolates that produce smaller less virulent sporangiospores. The larger sporangiospores germinate inside and lyse macrophages, whereas the smaller sporangiospores do not. sexMΔ mutants are sterile and still produce larger virulent sporangiospores, suggesting that either the sex locus is not involved in virulence/spore size or the sexP allele plays an inhibitory role. Phylogenetic analysis supports that at least three extant subspecies populate the M. circinelloides complex in nature: Mcl, M. circinelloides f. griseocyanus, and M. circinelloides f. circinelloides (Mcc). Mcc was found to be more prevalent among clinical Mucor isolates, and more virulent than Mcl in a diabetic murine model in contrast to the wax moth host. The M. circinelloides sex locus encodes an HMG domain protein (SexP for plus and SexM for minus mating types) flanked by genes encoding triose phosphate transporter (TPT) and RNA helicase homologs. The borders of the sex locus between the three subspecies differ: the Mcg sex locus includes the promoters of both the TPT and the RNA helicase genes, whereas the Mcl and Mcc sex locus includes only the TPT gene promoter. Mating between subspecies was restricted compared to mating within subspecies. These findings demonstrate that spore size dimorphism is linked to virulence of M. circinelloides species and that plasticity of the sex locus and adaptations in pathogenicity have occurred during speciation of the M. circinelloides complex.

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.

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 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.RNA-mediated gene silencing (RNA silencing) acts through transcriptional and posttranscriptional mechanisms to suppress gene expression in a sequence-specific manner. Widespread among plants, animals, and fungi, these mechanisms are involved in a wide variety of biological processes, including defense against viruses and transposons, the regulation of endogenous gene expression, and heterochromatin formation (reviewed in references 2, 5, 31, and 35). Although a number of specialized RNA-silencing pathways have been reported, a common mechanism has emerged, with homologous genes and proteins acting in different organisms. A central feature in all of these silencing phenomena is the processing of doublestranded RNA (dsRNA) (long dsRNA and pre-microRNAs [miRNAs]) into small duplexes of 21 to 25 nucleotides (nt) (small interfering RNAs [siRNAs] and miRNAs) by the RNase III enzyme Dicer. The small RNAs generated by Dicer are subsequently incorporated into the RNA-induced silencing complex (RISC), where they are used as a guide for the sequence-specific degradation of the target mRNA, the repression of its translation, or the inhibition of its transcription.The Dicer endonucleases are multidomain proteins evolutionarily conserved in eukaryotes. In metazoans and plants, in addition to two copies of the RNase III domain and a dsRNAbinding domain at the carboxyl terminus, the Dicer proteins include an amino-terminal ATP-dependent RNA helicas...

A light-inducible promoter (P B ) drives the carB operon (carotenoid genes) of the bacterium Myxococcus xanthus. A gene encoding a regulator of carotenoid biosynthesis was identified by studying mutant strains carrying a transcriptional fusion to P B and deletions in three candidate genes. Our results prove that the identified gene, named carA, codes for a repressor of the P B promoter in the dark. They also show that the carA gene product does not participate in the light activation of two other promoters connected with carotenoid synthesis or its regulation in M. xanthus. CarA is a novel protein consisting of a DNA-binding domain of the family of MerR helix-turn-helix transcriptional regulators, directly joined to a cobalamin-binding domain. In support of this, we report here that the presence of vitamin B 12 or some other cobalamin derivatives is absolutely required for activation of the P B promoter by light.Cells of the gram-negative bacterium Myxococcus xanthus respond to blue light by accumulating significant amounts of carotenoids. This response relies on the transcriptional activation of the structural genes for carotenoid synthesis (Fig. 1A) (see reference 17 for a review). All but one of the carotenoid biosynthesis genes are clustered together at the carB operon. The exception is gene crtI, unlinked to carB. Regulatory genes participating in the activation of the crtI promoter (P I ) by blue light have been identified. Two of them, carQ and carR, form part of the carQRS operon, the promoter of which (P QRS ) is also regulated by blue light. Protein CarQ is a member of the extracytoplasmic function (ECF) subfamily of factors. CarR, a membrane-spanning protein, acts as an anti-factor, sequestering protein CarQ to the membrane in the dark. Illumination of the cells somehow results in the loss of CarR, leaving CarQ free to activate the P QRS and P I promoters. These two promoters share two DNA segments, centered at the Ϫ31 and Ϫ10 positions, which correspond to the binding sites of CarQ. Another protein, CarD, which contains a DNA-binding domain similar to that of eukaryotic high-mobility group A proteins, is required independently for light activation of the P I and P QRS promoters. The latter also requires the normal action of integration host factor (10,12,23,27).Less is known on the regulation of the carB promoter (P B ), but previous lines of evidence indicated that the mechanism of the action of light is different from the one involved in the activation of P QRS and P I . On the one hand, the P B promoter lacks the binding sites for the ECF-factor CarQ, showing instead a Ϫ35 sequence that perfectly matches the Ϫ35 consensus for the major bacterial factor (6). On the other hand, the P B promoter, but not P QRS nor P I , is affected by mutations at two unlinked genes. One is carS, the third gene of the carQRS operon mentioned above. The first known carS mutation (carS1) was identified because it caused constitutive expression from the normally light-inducible P B promoter (4). Mutation carS1 is, howev...