RESUMO
Mucormycosis is a life-threatening fungal infection caused by various ubiquitous filamentous fungi of the Mucorales order, although Rhizopus spp. and Mucor spp. are the most prevalent causal agents. The limited therapeutic options available together with a rapid progression of the infection and a difficult early diagnosis produce high mortality. Here, we developed an adult zebrafish model of Mucor circinelloides infection which allowed us to confirm the link between sporangiospore size and virulence. Transcriptomic studies revealed a local, strong inflammatory response of the host elicited after sporangiospore germination and mycelial tissue invasion, while avirulent and UV-killed sporangiospores failed to induce inflammation and were rapidly cleared. Of the 857 genes modulated by the infection, those encoding cytokines, complement factors, peptidoglycan recognition proteins, and iron acquisition are particularly interesting. Furthermore, neutrophils and macrophages were similarly recruited independently of sporangiospore virulence and viability, which results in a robust depletion of both cell types in the hematopoietic compartment. Strikingly, our model also reveals for the first time the ability of mucormycosis to induce the apoptosis of recruited macrophages but not neutrophils. The induction of macrophage apoptosis, therefore, might represent a key virulence mechanism of these fungal pathogens, providing novel targets for therapeutic intervention in this lethal infection.
Assuntos
Apoptose , Macrófagos/microbiologia , Mucormicose/microbiologia , Mucormicose/patologia , Peixe-Zebra/fisiologia , Animais , Biomarcadores/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Rim Cefálico/microbiologia , Rim Cefálico/patologia , Inflamação/patologia , Camundongos , Mucorales/patogenicidade , Mucormicose/genética , Células Mieloides/metabolismo , Neutrófilos/metabolismo , Esporos Fúngicos/citologia , Peixe-Zebra/genéticaRESUMO
RNA interference (RNAi) is a conserved eukaryotic mechanism that uses small RNA molecules to suppress gene expression through sequence-specific messenger RNA degradation, translational repression, or transcriptional inhibition. In filamentous fungi, the protective function of RNAi in the maintenance of genome integrity is well known. However, knowledge of the regulatory role of RNAi in fungi has had to wait until the recent identification of different endogenous small RNA classes, which are generated by distinct RNAi pathways. In addition, RNAi research on new fungal models has uncovered the role of small RNAs and RNAi pathways in the regulation of diverse biological functions. In this review, we give an up-to-date overview of the different classes of small RNAs and RNAi pathways in fungi and their roles in the defense of genome integrity and regulation of fungal physiology and development, as well as in the interaction of fungi with biotic and abiotic environments.
Assuntos
Fungos/genética , Regulação Fúngica da Expressão Gênica , Interferência de RNA , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/metabolismoRESUMO
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.
Assuntos
Mucor/genética , Mutação , Interferência de RNA , RNA Fúngico/genética , Sequência de Aminoácidos , Farmacorresistência Fúngica/efeitos dos fármacos , Farmacorresistência Fúngica/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , Imunossupressores/farmacologia , Modelos Genéticos , Mucormicose/microbiologia , Estabilidade de RNA , RNA Fúngico/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/genética , Tacrolimo/farmacologiaRESUMO
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.
Assuntos
Proteínas Fúngicas/genética , Larva/microbiologia , Mariposas/microbiologia , Mucor/patogenicidade , Mucormicose/patologia , Miosina Tipo V/genética , Fosfolipase D/genética , Fatores de Virulência/genética , Animais , Antifúngicos/farmacologia , Farmacorresistência Fúngica Múltipla , Macrófagos/microbiologia , Masculino , Camundongos , Mucor/genética , Mucormicose/virologia , Interferência de RNA , RNA Interferente Pequeno/genéticaRESUMO
Plants and fungi use light and other signals to regulate development, growth, and metabolism. The fruiting bodies of the fungus Phycomyces blakesleeanus are single cells that react to environmental cues, including light, but the mechanisms are largely unknown [1]. The related fungus Mucor circinelloides is an opportunistic human pathogen that changes its mode of growth upon receipt of signals from the environment to facilitate pathogenesis [2]. Understanding how these organisms respond to environmental cues should provide insights into the mechanisms of sensory perception and signal transduction by a single eukaryotic cell, and their role in pathogenesis. We sequenced the genomes of P. blakesleeanus and M. circinelloides and show that they have been shaped by an extensive genome duplication or, most likely, a whole-genome duplication (WGD), which is rarely observed in fungi [3-6]. We show that the genome duplication has expanded gene families, including those involved in signal transduction, and that duplicated genes have specialized, as evidenced by differences in their regulation by light. The transcriptional response to light varies with the developmental stage and is still observed in a photoreceptor mutant of P. blakesleeanus. A phototropic mutant of P. blakesleeanus with a heterozygous mutation in the photoreceptor gene madA demonstrates that photosensor dosage is important for the magnitude of signal transduction. We conclude that the genome duplication provided the means to improve signal transduction for enhanced perception of environmental signals. Our results will help to understand the role of genome dynamics in the evolution of sensory perception in eukaryotes.
Assuntos
Evolução Molecular , Duplicação Gênica , Genoma Fúngico , Mucor/genética , Phycomyces/genética , Transdução de Sinais/genética , Luz , Mucor/efeitos da radiação , Família Multigênica , Percepção , Phycomyces/efeitos da radiação , Transcrição Gênica/efeitos da radiaçãoRESUMO
The existence of an RNA-mediated silencing mechanism in the opportunistic fungal pathogen Mucor circinelloides was first described in the early 2000. Since then, Mucor has reached an outstanding position within the fungal kingdom as a model system to achieve a deeper understanding of regulation of endogenous functions by the RNA interference (RNAi) machinery. M. circinelloides combines diverse components of its RNAi machinery to carry out functions not only limited to the defense against invasive nucleic acids, but also to regulate expression of its own genes by producing different classes of endogenous small RNA molecules (esRNAs). The recent discovery of a novel RNase that participates in a new RNA degradation pathway adds more elements to the gene silencing-mediated regulation. This review focuses on esRNAs in M. circinelloides, the different pathways involved in their biogenesis, and their roles in regulating specific physiological and developmental processes in response to environmental signals, highlighting the complexity of silencing-mediated regulation in fungi.
Assuntos
Proteínas Fúngicas/metabolismo , Mucor/fisiologia , Interferência de RNA/fisiologia , RNA Fúngico/metabolismo , RNA Citoplasmático Pequeno/metabolismo , Animais , Proteínas Fúngicas/genética , Humanos , Redes e Vias Metabólicas , Mucor/enzimologia , Mucor/genética , Mucor/metabolismo , RNA Fúngico/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Citoplasmático Pequeno/genéticaRESUMO
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.
Assuntos
Mucor/crescimento & desenvolvimento , Mucor/fisiologia , Interferência de RNA , Inativação Gênica , Mucor/genética , Estresse Oxidativo , Ribonuclease III/metabolismo , Esporos Fúngicos/metabolismoRESUMO
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.
Assuntos
Regulação Fúngica da Expressão Gênica , Inativação Gênica , Mucor/genética , Estabilidade de RNA , RNA Mensageiro/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Mucor/enzimologia , Mucor/metabolismo , RNA Mensageiro/genética , Ribonuclease III/química , Ribonuclease III/genética , Ribonuclease III/metabolismoRESUMO
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.
Assuntos
Mucor/genética , Interferência de RNA , Proteínas Fúngicas/antagonistas & inibidores , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Concentração de Íons de Hidrogênio , Modelos Biológicos , Mucor/metabolismo , Mutação , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Esporos Fúngicos/crescimento & desenvolvimento , Esporos Fúngicos/metabolismoRESUMO
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.
Assuntos
Farmacorresistência Fúngica/genética , Epigênese Genética/genética , Mucor/efeitos dos fármacos , Mucor/genética , Mutação/genética , Interferência de RNA , Tacrolimo/farmacologia , Calcineurina/genética , Calcineurina/metabolismo , Inibidores de Calcineurina , Humanos , Hifas/efeitos dos fármacos , Hifas/genética , Hifas/crescimento & desenvolvimento , Dados de Sequência Molecular , Mucor/crescimento & desenvolvimento , Mucormicose/tratamento farmacológico , Mucormicose/microbiologia , Fenótipo , Tacrolimo/metabolismo , Proteína 1A de Ligação a Tacrolimo/deficiência , Proteína 1A de Ligação a Tacrolimo/genética , Proteína 1A de Ligação a Tacrolimo/metabolismoRESUMO
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.
Assuntos
Proteínas Argonautas/genética , Genes Fúngicos/genética , Mucor/citologia , Mucor/genética , Interferência de RNA , Sequência de Aminoácidos , Proteínas Argonautas/química , Proteínas Argonautas/metabolismo , Autólise , Regulação Fúngica da Expressão Gênica , Técnicas de Inativação de Genes , Dados de Sequência Molecular , Mutação/genética , Ligação Proteica , Estrutura Terciária de Proteína , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Reprodução Assexuada/genética , Esporos Fúngicos/fisiologia , Transgenes/genéticaRESUMO
Yeast and filamentous fungi have been essential model systems for unveiling the secrets of RNA interference (RNAi). Research on these organisms has contributed to identifying general mechanisms and conserved eukaryotic RNAi machinery that can be found from fungi to mammals. The development of deep sequencing technologies has brought on the last wave of studies on RNAi in fungi, which has been focused on the identification of new types of functional small RNAs (sRNAs). These studies have discovered an unexpected diversity of sRNA, biogenesis pathways and new functions that are the focus of this review.
Assuntos
Neurospora crassa/genética , RNA Interferente Pequeno/genética , Leveduras/genética , Animais , Caenorhabditis elegans/genética , Regulação Fúngica da Expressão Gênica , Interferência de RNA , RNA Polimerase Dependente de RNA/genéticaAssuntos
Fungos/genética , Regulação Fúngica da Expressão Gênica , Inativação Gênica , Parasitos/genética , Interferência de RNA , Animais , Exossomos/genética , Exossomos/metabolismo , Fungos/virologia , Regulação Viral da Expressão Gênica , Vigilância Imunológica , MicroRNAs/metabolismo , Parasitos/virologia , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Interferente Pequeno , Vírus/metabolismoRESUMO
The carotene producer fungus Mucor circinelloides is the zygomycete more amenable to genetic manipulations by using molecular tools. Since the initial development of an effective procedure of genetic transformation, more than two decades ago, the availability of new molecular approaches such as gene replacement techniques and gene expression inactivation by RNA silencing, in addition to the sequencing of its genome, has made Mucor a valuable organism for the study of a number of processes. Here we describe in detail the main techniques and methods currently used to manipulate M. circinelloides, including transformation, gene replacement, gene silencing, RNAi, and immunoprecipitation.
Assuntos
Carotenoides/biossíntese , Técnicas Genéticas , Mucor/genética , Mucor/metabolismo , Western Blotting , Eletroforese , Inativação Gênica , Genes Fúngicos/genética , Vetores Genéticos/genética , Imunoprecipitação , Peso Molecular , Mucor/citologia , Hibridização de Ácido Nucleico , Protoplastos/metabolismo , Sondas RNA/química , RNA Fúngico/química , RNA Fúngico/isolamento & purificação , Transformação GenéticaRESUMO
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.
Assuntos
Regulação Fúngica da Expressão Gênica , Mucor/enzimologia , Mucor/metabolismo , Interferência de RNA , RNA Fúngico/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , Clonagem Molecular , DNA Fúngico/genética , Deleção de Genes , Mucor/genética , RNA Antissenso/metabolismoRESUMO
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.
Assuntos
Mucor/patogenicidade , Esporos Fúngicos/citologia , Virulência/fisiologia , Processos de Crescimento Celular/genética , Processos de Crescimento Celular/fisiologia , Tamanho Celular , Individualidade , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Mucor/citologia , Mucor/genética , Mucor/fisiologia , Tamanho das Organelas/fisiologia , Filogenia , Reprodução/genética , Reprodução/fisiologia , Esporângios/citologia , Esporos Fúngicos/genética , Esporos Fúngicos/fisiologia , Esporos Fúngicos/ultraestrutura , Virulência/genéticaRESUMO
Endogenous short RNAs (esRNAs) play diverse roles in eukaryotes and usually are produced from double-stranded RNA (dsRNA) by Dicer. esRNAs are grouped into different classes based on biogenesis and function but not all classes are present in all three eukaryotic kingdoms. The esRNA register of fungi is poorly described compared to other eukaryotes and it is not clear what esRNA classes are present in this kingdom and whether they regulate the expression of protein coding genes. However, evidence that some dicer mutant fungi display altered phenotypes suggests that esRNAs play an important role in fungi. Here, we show that the basal fungus Mucor circinelloides produces new classes of esRNAs that map to exons and regulate the expression of many protein coding genes. The largest class of these exonic-siRNAs (ex-siRNAs) are generated by RNA-dependent RNA Polymerase 1 (RdRP1) and dicer-like 2 (DCL2) and target the mRNAs of protein coding genes from which they were produced. Our results expand the range of esRNAs in eukaryotes and reveal a new role for esRNAs in fungi.
Assuntos
Regulação Fúngica da Expressão Gênica , Mucor/genética , RNA Fúngico/metabolismo , RNA Mensageiro/metabolismo , RNA não Traduzido/metabolismo , Éxons , Sequências Repetitivas Dispersas , MicroRNAs/genética , Mucor/enzimologia , Mutação , RNA Fúngico/biossíntese , RNA Fúngico/genética , RNA Interferente Pequeno/biossíntese , RNA não Traduzido/biossíntese , RNA não Traduzido/genética , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Ribonuclease III/genética , Ribonuclease III/metabolismoRESUMO
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.
Assuntos
Proteínas Fúngicas/genética , Mucor/genética , Interferência de RNA , RNA Antissenso/metabolismo , RNA Fúngico/metabolismo , RNA Interferente Pequeno/metabolismo , Ribonuclease III/genética , Proteínas Fúngicas/metabolismo , Mucor/enzimologia , Mucor/metabolismo , Mutação , Ribonuclease III/metabolismoRESUMO
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.
Assuntos
Mucor/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ativação Transcricional , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Luz , Mucor/metabolismo , Mutação , Oxirredutases/genética , Oxirredutases/metabolismo , RNA Interferente Pequeno/genética , Transcrição GênicaRESUMO
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.