RESUMO
A fungus causing false smut in the flowers of Murdannia keisak (Commelinaceae, Commelinales, Monocots) in Japan was morphologically identical to Ustilago aneilematis. The fungus infected ovaries of most flowers of host plants. Infected flowers were filled with yellow to orange thick-walled conidia that became olivaceous green at maturity. However, multilocus phylogenetic analysis of DNA sequences (18S, 28S, translation elongation factor 1α [TEF], the largest [RPB1] and the second largest [RPB2] subunit of RNA polymerase II) showed that the fungus belonged to the tribe Ustilaginoideae (Clavicipitaceae, Hypocreales, Ascomycota). Microscopic examination showed that the fungus developed conidia at the apex of conidiogenous cells, in contrast to other species in the Ustilaginoideae that develop conidia pleurogenously. A new genus, Commelinaceomyces, is formally proposed in the Ustilaginoideae to accommodate this fungus. Four species previously misplaced in Ustilago (Ustilaginales, Basidiomycota) are transferred to Commelinaceomyces, including the type of the genus, C. aneilematis, on Murdannia keisak. This is the first report of a clavicipitaceous species infecting host plants in the Commelinaceae.
Assuntos
Commelinaceae/parasitologia , Hypocreales/classificação , Hypocreales/citologia , Hypocreales/genética , Ustilago/classificação , Ustilago/citologia , Ustilago/genética , DNA Fúngico , Japão , Filogenia , Análise de Sequência de DNARESUMO
In the present manuscript, we describe the mechanisms involved in the yeast-to-hypha dimorphic transition of the plant pathogenic Basidiomycota fungus Ustilago maydis. During its life cycle, U. maydis presents two stages: one in the form of haploid saprophytic yeasts that divide by budding and the other that is the product of the mating of sexually compatible yeast cells (sporidia), in the form of mycelial dikaryons that invade the plant host. The occurrence of the involved dimorphic transition is controlled by the two mating loci a and b. In addition, the dimorphic event can be obtained in vitro by different stimuli: change in the pH of the growth medium, use of different carbon sources, and by nitrogen depletion. The presence of other factors and mechanisms may affect this phenomenon; among these, we may cite the PKA and MAPK signal transduction pathways, polyamines, and factors that affect the structure of the nucleosomes. Some of these factors and conditions may affect all these dimorphic events, or they may be specific for only one or more but not all the processes involved. The conclusion reached by these experiments is that U. maydis has constituted a useful model for the analysis of the mechanisms involved in cell differentiation of fungi in general.
Assuntos
Transdução de Sinais , Ustilago/citologia , Ustilago/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Metilação de DNA , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Histidina Quinase/metabolismo , Histona Acetiltransferases/metabolismo , Homeostase , Concentração de Íons de Hidrogênio , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Poliaminas/metabolismoRESUMO
Itaconic acid (IA) is a naturally occurring dicarboxylic acid with applications in the manufacture of polymers. IA can be produced by fermentation using the fungi Aspergillus terreus or Ustilago maydis as biocatalysts. Indirect evidence has suggested that the mitochondrial carriers U. maydis Um_Mtt1 and A. terreus At_MttA export mitochondrially synthesized cis-aconitate to the cytosol for IA synthesis using malate as a countersubstrate. Here, by assaying the transport features of recombinant Um_Mtt1 and At_MttA in reconstituted liposomes, we find that both proteins efficiently transport cis-aconitate, but malate is well transported only by Um_Mtt1 and 2-oxoglutarate only by At_MttA. Bioinformatic analysis shows that Um_Mtt1 and At_MttA form a distinctive mitochondrial carrier subfamily. Our data show that although fulfilling the same physiological task, Um_Mtt1 and At_MttA have different biochemical features.
Assuntos
Aspergillus/citologia , Mitocôndrias/metabolismo , Succinatos/metabolismo , Ustilago/citologia , Sequência de Aminoácidos , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , CinéticaRESUMO
The ESCRT machinery (endosomal sorting complex required for transport) is an evolutionarily highly conserved multiprotein complex involved in numerous cellular processes like endocytosis, membrane repair, or endosomal long-distance transport. In fungal hyphae, endocytosis and long-distance mRNA transport are tightly linked, as endocytotic vesicles are also the key carrier vehicles for mRNAs. Studying the regulatory component Did2 (CHMP1) in the plant pathogen Ustilago maydis revealed that loss of Did2 resulted in disturbed endosomal maturation, thereby causing defects in microtubule-dependent transport of early endosomes. Here, we describe methods and protocols that allow studying the role of ESCRT components during endosomal transport. We present experimental strategies to analyze U. maydis ESCRT mutant phenotypes and test complementation with heterologous components, such as ESCRT regulators from Drosophila melanogaster.
Assuntos
Proteínas de Drosophila/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Proteínas Fúngicas/metabolismo , Microscopia Intravital/métodos , Ustilago/metabolismo , Proteínas de Drosophila/genética , Expressão Ectópica do Gene , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Endossomos/metabolismo , Proteínas Fúngicas/genética , Hifas/citologia , Hifas/genética , Hifas/metabolismo , Microscopia de Fluorescência/métodos , Deleção de Sequência , Ustilago/citologia , Ustilago/genéticaRESUMO
Protein export in eukaryotes can either occur via the classical pathway traversing the endomembrane system or exploit alternative routes summarized as unconventional secretion. Besides multiple examples in higher eukaryotes, unconventional secretion has also been described for fungal proteins with diverse functions in important processes such as development or virulence. Accumulating molecular insights into the different export pathways suggest that unconventional secretion in fungal microorganisms does not follow a common scheme but has evolved multiple times independently. In this study, we review the most prominent examples with a focus on the chitinase Cts1 from the corn smut Ustilago maydis. Cts1 participates in cell separation during budding growth. Recent evidence indicates that the enzyme might be actively translocated into the fragmentation zone connecting dividing mother and daughter cells, where it supports cell division by the degradation of remnant chitin. Importantly, a functional fragmentation zone is prerequisite for Cts1 release. We summarize in detail what is currently known about this potential lock-type mechanism of Cts1 secretion and its connection to the complex regulation of fragmentation zone assembly and cell separation.
Assuntos
Ustilago/citologia , Ustilago/metabolismo , Divisão Celular/genética , Divisão Celular/fisiologia , Separação Celular , Quitinases/genética , Quitinases/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Ustilago/genéticaRESUMO
The most important signal transduction mechanism related to environmental pH responses in fungi is the Pal/Rim pathway. Our knowledge of this pathway came initially from studies on Ascomycota species where it is made by seven members divided into two complexes, one located at the plasma membrane, and other at the endosomal membrane. In Basidiomycota sepecies only the homologs of the endosomal membrane complex (genes PalA/Rim20, PalB/ Rim13, and PalC/ Rim23), plus the transcription factor PacC/Rim101 have been identified. In this study, we describe the identification in Ustilago maydis of a gene encoding a Rho-like protein (tentatively named RHO4) as a novel member of this pathway. The RHO4 gene possibly plays, among other functions, a role in the second proteolytic cleavage that leads to the activation of the transcription factor PacC/Rim101. Mutants in this gene showed a pleiotropic phenotype, displaying similar characteristics to the Pal/Rim mutants, such as a lower growth rate at alkaline pH, high sensitivity to ionic and osmotic stresses, and impairment in protease secretion, but no alteration of the yeast-to-mycelium dimorphic transition induced by acid pH whereas it has a function in the dimorphic transition induced by fatty acids.
Assuntos
Proteínas Fúngicas/genética , Genes Fúngicos/genética , Ustilago/genética , Ustilago/metabolismo , Ácidos Graxos/metabolismo , Regulação Fúngica da Expressão Gênica , Concentração de Íons de Hidrogênio , Mutação , Micélio/metabolismo , Pressão Osmótica , Peptídeo Hidrolases/metabolismo , Fenótipo , Proteínas Repressoras/genética , Estresse Fisiológico , Fatores de Transcrição , Ustilago/citologia , Ustilago/crescimento & desenvolvimento , Proteínas rho de Ligação ao GTP/genéticaRESUMO
Ustilago maydis causes common smut disease in maize. Although pathogenic diploid strains of the fungus have been known for many years, the normal life cycle was thought to involve an extended dikaryotic stage, with nuclear fusion occurring in immature teliospores. However, microscopic examination of both living and fixed tumor material showed that nuclei fuse long before sporulation begins and that tumors are filled with uninucleate cells undergoing mitosis. Quantification of DNA in the nuclei confirmed these observations. Additionally, fungal cells from tumor material placed on nutrient agar produced colonies of diploid budding cells. Time-lapse observations showed that at least some of these colonies arose from thin-walled fungal cells rather than from immature spores. Ultrastructural examination of developing teliospores from tumors confirmed that they were uninucleate. Condensed chromatin and other structures characteristic of nuclei in prophase I of meiosis were observed. These observations support revising the U. maydis life cycle to include a diploid mitotic stage that corresponds with rapid tumor enlargement and conversion of plant to fungal biomass. Because mitotic division of diploid nuclei is so unusual as a life cycle feature in the fungi, it will be interesting to explore the consequences of its presence in U. maydis.
Assuntos
Diploide , Mitose , Ustilago/citologia , Ustilago/crescimento & desenvolvimento , Microscopia , Doenças das Plantas/microbiologia , Ustilago/genética , Zea mays/microbiologiaRESUMO
Ustilago maydis is an obligate biotrophic fungal pathogen which causes common smut disease of corn. To proliferate in host tissue, U. maydis must gain access to nutrients and overcome plant defence responses, such as the production of reactive oxygen species. The elucidation of the mechanisms by which U. maydis meets these challenges is critical for the development of strategies to combat smut disease. In this study, we focused on the contributions of phospholipases (PLs) to the pathogenesis of corn smut disease. We identified 11 genes encoding putative PLs and characterized the transcript levels for these genes in the fungus grown in culture and during infection of corn tissue. To assess the contributions of specific PLs, we focused on two genes, lip1 and lip2, which encode putative phospholipase A2 (PLA2 ) enzymes with similarity to platelet-activating factor acetylhydrolases. PLA2 enzymes are known to counteract oxidative damage to lipids in other organisms. Consistent with a role in the mitigation of oxidative damage, lip2 mutants were sensitive to oxidative stress provoked by hydrogen peroxide and by increased production of reactive oxygen species caused by inhibitors of mitochondrial functions. Importantly, mutants defective in lip2, but not lip1, were attenuated for virulence in corn seedlings. Finally, a comparative analysis of fatty acid and cardiolipin profiles in the wild-type strain and a lip2 mutant revealed differences consistent with a protective role for Lip2 in maintaining lipid homeostasis and mitochondrial health during proliferation in the hostile host environment.
Assuntos
Proteínas Fúngicas/metabolismo , Estresse Oxidativo , Fosfolipases/metabolismo , Ustilago/enzimologia , Ustilago/patogenicidade , Cardiolipinas/metabolismo , Respiração Celular , Proteínas Fúngicas/genética , Deleção de Genes , Genoma Fúngico , Haploidia , Mutação/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ustilago/citologia , Ustilago/genética , Virulência/genéticaRESUMO
Ustilago maydis is a well-established model system for biotrophic fungal plant pathogens. The fungus has a dimorphic life cycle with a yeast-like saprophytic phase switching to filamentous, pathogenic growth upon hyphal fusion. Due to its highly differentiated development and the amenability for reverse-genetics U. maydis provides a model system for both fungal cell biology as well as the study of biotrophic plant interaction. The present article highlights key findings in different aspects of cell biology on the corn smut disease and provides an outlook on the most intriguing open questions.
Assuntos
Doenças das Plantas/microbiologia , Ustilago/citologia , Ustilago/fisiologia , Proteínas Fúngicas/metabolismo , Hifas/metabolismo , Ustilago/genética , Ustilago/ultraestrutura , Zea mays/microbiologiaRESUMO
DNA damage response (DDR) leads to DNA repair, and depending on the extent of the damage, to further events, including cell death. Evidence suggests that cell differentiation may also be a consequence of the DDR. During the formation of the infective hypha in the phytopathogenic fungus Ustilago maydis, two DDR kinases, Atr1 and Chk1, are required to induce a G2 cell cycle arrest, which in turn is essential to display the virulence program. However, the triggering factor of DDR in this process has remained elusive. In this report we provide data suggesting that no DNA damage is associated with the activation of the DDR during the formation of the infective filament in U. maydis. We have analyzed bulk DNA replication during the formation of the infective filament, and we found no signs of impaired DNA replication. Furthermore, using RPA-GFP fusion as a surrogate marker of the presence of DNA damage, we were unable to detect any sign of DNA damage at the cellular level. In addition, neither MRN nor 9-1-1 complexes, both instrumental to transmit the DNA damage signal, are required for the induction of the above mentioned cell cycle arrest, as well as for virulence. In contrast, we have found that the claspin-like protein Mrc1, which in other systems serves as scaffold for Atr1 and Chk1, was required for both processes. We discuss possible alternative ways to trigger the DDR, independent of DNA damage, in U. maydis during virulence program activation.
Assuntos
Dano ao DNA , Reparo do DNA , Proteínas Fúngicas/metabolismo , Proteínas Quinases/metabolismo , Ustilago/metabolismo , Citoesqueleto de Actina/metabolismo , Quinase 1 do Ponto de Checagem , Replicação do DNA , Pontos de Checagem da Fase G2 do Ciclo Celular , Plantas/microbiologia , Transdução de Sinais , Ustilago/citologia , Ustilago/patogenicidade , VirulênciaRESUMO
Here we report identification of the lkh1 gene encoding a LAMMER kinase homolog (Lkh1) from a screen for DNA repair-deficient mutants in Ustilago maydis. The mutant allele isolated results from a mutation at glutamine codon 488 to a stop codon that would be predicted to lead to truncation of the carboxy-terminal kinase domain of the protein. This mutant (lkh1(Q488*)) is highly sensitive to ultraviolet light, methyl methanesulfonate, and hydroxyurea. In contrast, a null mutant (lkh1Δ) deleted of the entire lkh1 gene has a less severe phenotype. No epistasis was observed when an lkh1(Q488*)rad51Δ double mutant was tested for genotoxin sensitivity. However, overexpressing the gene for Rad51, its regulator Brh2, or the Brh2 regulator Dss1 partially restored genotoxin resistance of the lkh1Δ and lkh1(Q488*) mutants. Deletion of lkh1 in a chk1Δ mutant enabled these double mutant cells to continue to cycle when challenged with hydroxyurea. lkh1Δ and lkh1(Q488*) mutants were able to complete the meiotic process but exhibited reduced heteroallelic recombination and aberrant chromosome segregation. The observations suggest that Lkh1 serves in some aspect of cell cycle regulation after DNA damage or replication stress and that it also contributes to proper chromosome segregation in meiosis.
Assuntos
Instabilidade Genômica , Proteínas Quinases/metabolismo , Ustilago/enzimologia , Ustilago/genética , Ciclo Celular/efeitos dos fármacos , Segregação de Cromossomos/efeitos dos fármacos , Clonagem Molecular , Reparo do DNA , Epistasia Genética/efeitos dos fármacos , Proteínas Fúngicas , Teste de Complementação Genética , Testes Genéticos , Hidroxiureia/farmacologia , Meiose/efeitos dos fármacos , Metanossulfonato de Metila/farmacologia , Mutação/genética , Fenótipo , Recombinação Genética/efeitos dos fármacos , Recombinação Genética/genética , Raios Ultravioleta , Ustilago/citologia , Ustilago/efeitos dos fármacosRESUMO
Chromatin in the eukaryotic nucleus is highly organized in the form of nucleosomes where histones wrap DNA. This structure may be altered by some chemical modifications of histones, one of them, acetylation by histone acetyltransferases (HATs) that originates relaxation of the nucleosome structure, providing access to different transcription factors and other effectors. In this way, HATs regulate cellular processes including DNA replication, and gene transcription. Previously, we isolated Ustilago maydis mutants deficient in the GCN5 HAT that are avirulent, and grow constitutively as mycelium. In this work, we proceeded to identify the genes differentially regulated by GCN5, comparing the transcriptomes of the mutant and the wild type using microarrays, to analyse the epigenetic control of virulence and morphogenesis. We identified 1203 genes, 574 positively and 629 negatively regulated in the wild type. We found that genes belonging to different categories involved in pathogenesis were downregulated in the mutant, and that genes involved in mycelial growth were negatively regulated in the wild type, offering a working hypothesis on the epigenetic control of virulence and morphogenesis of U. maydis. Interestingly, several differentially regulated genes appeared in clusters, suggesting a common regulation. Some of these belonged to pathogenesis or secondary metabolism.
Assuntos
Epigênese Genética , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Histona Acetiltransferases/biossíntese , Ustilago/genética , Deleção de Genes , Histona Acetiltransferases/genética , Hifas/citologia , Hifas/crescimento & desenvolvimento , Análise em Microsséries , Ustilago/citologia , Ustilago/crescimento & desenvolvimento , VirulênciaRESUMO
Chitin is an essential component of the fungal cell wall, providing rigidity and stability. Its degradation is mediated by chitinases and supposedly ensures the dynamic plasticity of the cell wall during growth and morphogenesis. Hence, chitinases should be particularly important for fungi with dramatic morphological changes, such as Ustilago maydis. This smut fungus switches from yeast to filamentous growth for plant infection, proliferates as a mycelium in planta, and forms teliospores for spreading. Here, we investigate the contribution of its four chitinolytic enzymes to the different morphological changes during the complete life cycle in a comprehensive study of deletion strains combined with biochemical and cell biological approaches. Interestingly, two chitinases act redundantly in cell separation during yeast growth. They mediate the degradation of remnant chitin in the fragmentation zone between mother and daughter cell. In contrast, even the complete lack of chitinolytic activity does not affect formation of the infectious filament, infection, biotrophic growth, or teliospore germination. Thus, unexpectedly we can exclude a major role for chitinolytic enzymes in morphogenesis or pathogenicity of U. maydis. Nevertheless, redundant activity of even two chitinases is essential for cell separation during saprophytic growth, possibly to improve nutrient access or spreading of yeast cells by wind or rain.
Assuntos
Divisão Celular , Quitinases/metabolismo , Proteínas Fúngicas/metabolismo , Ustilago/enzimologia , Sequência de Aminoácidos , Quitinases/química , Quitinases/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Dados de Sequência Molecular , Ustilago/citologia , Ustilago/genéticaRESUMO
Many of the most important plant diseases are caused by fungal pathogens that form specialized cell structures to breach the leaf surface as well as to proliferate inside the plant. To initiate pathogenic development, the fungus responds to a set of inductive cues. Some of them are of extracellular nature (environmental signals) while others respond to intracellular conditions (developmental signals). These signals have to be integrated into a single response that has as a major outcome changes in the morphogenesis of the fungus. The cell cycle regulation is pivotal during these cellular differentiations, and we hypothesized that cell cycle regulation would be likely to provide control points for infection development by fungal pathogens. Although efforts have been done in various fungal systems, there is still limited information available regarding the relationship of these processes with the induction of the virulence programs. Hence, the role of fungal cell cycle regulators -which are wide conserved elements- as true virulence factors, has yet to be defined. Here we discuss the recent finding that the formation of the appressorium, a structure required for plant penetration, in the corn smut fungus Ustilago maydis seems to be incompatible with an active cell cycle and, therefore genetic circuits evolved in this fungus to arrest the cell cycle during the growth of this fungus on plant surface, before the appressorium-mediated penetration into the plant tissue.
Assuntos
Pontos de Checagem da Fase G2 do Ciclo Celular , Ustilago/citologia , Ustilago/fisiologia , Zea mays/microbiologia , Regulação para Baixo , Proteínas Fúngicas/metabolismoRESUMO
BACKGROUND: Maternal or uniparental inheritance (UPI) of mitochondria is generally observed in sexual eukaryotes, however, the underlying mechanisms are diverse and largely unknown. Recently, based on the use of mutants blocked in autophagy, it has been demonstrated that autophagy is required for strict maternal inheritance in the nematode Caenorhabditis elegans. Uniparental mitochondrial DNA (mtDNA) inheritance has been well documented for numerous fungal species, and in particular, has been shown to be genetically governed by the mating-type loci in the isogamous species Cryptococcus neoformans, Phycomyces blakesleeanus and Ustilago maydis. Previously, we have shown that the a2 mating-type locus gene lga2 is decisive for UPI during sexual development of U. maydis. In axenic culture, conditional overexpression of lga2 triggers efficient loss of mtDNA as well as mitophagy. To assess a functional relationship, we have investigated UPI in U. maydis Δatg11 mutants, which are blocked in mitophagy. RESULTS: This study has revealed that Δatg11 mutants are not affected in pathogenic development and this has allowed us to analyse UPI under comparable developmental conditions between mating-compatible wild-type and mutant strain combinations. Explicitly, we have examined two independent strain combinations that gave rise to different efficiencies of UPI. We demonstrate that in both cases UPI is atg11-independent, providing evidence that mitophagy is not critical for UPI in U. maydis, even under conditions of strict UPI. CONCLUSIONS: Until now, analysis of a role of mitophagy in UPI has not been reported for microbial species. Our study suggests that selective autophagy does not contribute to UPI in U. maydis, but is rather a consequence of selective mtDNA elimination in response to mitochondrial damage.
Assuntos
DNA Mitocondrial , Deleção de Genes , Mitofagia , Ustilago/fisiologia , Proteínas de Transporte Vesicular/genética , Testamentos , Genes Fúngicos Tipo Acasalamento , Ustilago/citologia , Ustilago/genéticaRESUMO
Ustilago maydis, causal agent of corn smut, can proliferate saprobically in a yeast form but its infectious filamentous form is an obligate parasite. Previously, we showed that Ust1, the first APSES (Asm1p, Phd1p, Sok2p, Efg1p, and StuAp) transcription factor functionally characterized in the phylum Basidiomycota, controlled morphogenesis and virulence in this species. Here, we further analyzed Ust1 function using multiple experimental approaches and determined that i) Ust1 activity was able to partially reverse stuA− conidiophore defects in Aspergillus nidulans; ii) in U. maydis, normal development and virulence were strongly dependent on precise induction or repression of Ust1 activity; iii) consistent with its role as a transcription factor regulating multiple processes, Ust1 accumulated in the nucleus at various stages of the life cycle; iv) however, it was undetectable at specific stages of pathogenic growth, indicating that Ust1 repression is part of normal development in planta; v) StuA response elements upstream of the ust1 open reading frame exhibited affinity for U. maydis DNA-binding proteins; vi) however, loss of regulated ust1 transcription had minor phenotypic effects; and vii) Ust1 was subject to post-translational phosphorylation but is not a target of cAMP signaling. Thus, the broad functional conservation between Ust1 and Ascomycota APSES proteins does not extend to the mechanisms regulating their activity.
Assuntos
Doenças das Plantas/microbiologia , Processamento de Proteína Pós-Traducional , Transdução de Sinais , Fatores de Transcrição/metabolismo , Ustilago/genética , Zea mays/microbiologia , Aspergillus nidulans/citologia , Aspergillus nidulans/genética , Aspergillus nidulans/crescimento & desenvolvimento , Aspergillus nidulans/metabolismo , Ciclo Celular , Núcleo Celular/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genes Reporter , Modelos Biológicos , Mutação , Fosforilação , Transporte Proteico , Esporos Fúngicos , Fatores de Transcrição/genética , Ustilago/citologia , Ustilago/crescimento & desenvolvimento , Ustilago/patogenicidade , VirulênciaRESUMO
Positional cues localized to distinct cell domains are critical for the generation of cell polarity and cell morphogenesis. These cues lead to assembly of protein complexes that organize the cytoskeleton resulting in delivery of vesicles to sites of polarized growth. Tea4, an SH3 domain protein, was first identified in fission yeast, and is a critical determinant of the axis of polarized growth, a role conserved among ascomycete fungi. Ustilago maydis is a badiomycete fungus that exhibits a yeast-like form that is nonpathogenic and a filamentous form that is pathogenic on maize and teozintle. We are interested in understanding how positional cues contribute to generation and maintenance of these two forms, and their role in pathogenicity. We identified a homologue of fission yeast tea4 in a genetic screen for mutants with altered colony and cell morphology and present here analysis of Tea4 for the first time in a basidiomycete fungus. We demonstrate that Tea4 is an important positional marker for polarized growth and septum location in both forms. We uncover roles for Tea4 in maintenance of cell and neck width, cell separation, and cell wall deposition in the yeast-like form, and in growth rate, formation of retraction septa, growth reversal, and inhibition of budding in the filamentous form. We show that Tea4::GFP localizes to sites of polarized or potential polarized growth in both forms, as observed in ascomycete fungi. We demonstrate an essential role of Tea4 in pathogencity in the absence of cell fusion. Basidiomycete and ascomycete Tea4 homologues share SH3 and Glc7 domains. Tea4 in basidiomycetes has additional domains, which has led us to hypothesize that Tea4 has novel functions in this group of fungi.
Assuntos
Polaridade Celular/fisiologia , Proteínas Fúngicas/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Ustilago/fisiologia , Ustilago/patogenicidade , Biomarcadores/metabolismo , Parede Celular/metabolismo , Proteínas Fúngicas/genética , Pontos de Checagem da Fase G2 do Ciclo Celular , Proteínas Associadas aos Microtúbulos/genética , Morfogênese , Mutação , Micélio/fisiologia , Ustilago/citologia , VirulênciaRESUMO
Early endosomes (EEs) mediate protein sorting, and their cytoskeleton-dependent motility supports long-distance signaling in neurons. Here, we report an unexpected role of EE motility in distributing the translation machinery in a fungal model system. We visualize ribosomal subunit proteins and show that the large subunits diffused slowly throughout the cytoplasm (Dc,60S = 0.311 µm(2)/s), whereas entire polysomes underwent long-range motility along microtubules. This movement was mediated by "hitchhiking" on kinesin-3 and dynein-driven EEs, where the polysomes appeared to translate EE-associated mRNA into proteins. Modeling indicates that this motor-driven transport is required for even cellular distribution of newly formed ribosomes. Indeed, impaired EE motility in motor mutants, or their inability to bind EEs in mutants lacking the RNA-binding protein Rrm4, reduced ribosome transport and induced ribosome aggregation near the nucleus. As a consequence, cell growth was severely restricted. Collectively, our results indicate that polysomes associate with moving EEs and that "off- and reloading" distributes the protein translation machinery.
Assuntos
Endossomos/metabolismo , Polirribossomos/metabolismo , Ustilago/metabolismo , Transporte Biológico Ativo/efeitos dos fármacos , Difusão/efeitos dos fármacos , Dineínas/metabolismo , Endossomos/efeitos dos fármacos , Endossomos/ultraestrutura , Proteínas Fúngicas/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Hifas/metabolismo , Hifas/ultraestrutura , Cinesinas/metabolismo , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Modelos Biológicos , Mutação/genética , Polirribossomos/efeitos dos fármacos , Polirribossomos/ultraestrutura , Inibidores da Síntese de Proteínas/farmacologia , Transporte Proteico/efeitos dos fármacos , Proteínas de Ligação a RNA/metabolismo , Estresse Fisiológico/efeitos dos fármacos , Ustilago/citologia , Ustilago/efeitos dos fármacosRESUMO
Intracellular cargos that are transported by groups of molecular motors often display bidirectional movement. This can be seen experimentally by tracking the trajectories of individual cargos in vivo. Typically, the cargo trajectories display many turning events that result from the stochastic nature of the involved motor processes. In this paper, we simulate cargo trajectories for different binding mechanisms. We introduce a series of statistical tools to analyze and quantitatively characterize these trajectories. As we demonstrate for specified single-motor properties, the novel statistical methods allow us to quantitatively distinguish between different models for bidirectional transport. In this way, the tools provide a quantitative connection between the statistical properties of the cargo trajectories and the molecular properties of the motor proteins. Such methods are also applicable to experimentally measured cargo trajectories and should be helpful in elucidating the mechanisms that lead to bidirectional transport.
Assuntos
Transporte Biológico Ativo , Proteínas Fúngicas/metabolismo , Modelos Biológicos , Ustilago/metabolismo , Proteínas Fúngicas/química , Microtúbulos/metabolismo , Proteínas Motores Moleculares/química , Proteínas Motores Moleculares/metabolismo , Ustilago/citologiaRESUMO
The cell wall is the structure that provides the shape to fungal cells and protects them from the difference in osmotic pressure existing between the cytosol and the external medium. Accordingly, changes in structure and composition of the fungal wall must occur during cell differentiation, including the dimorphic transition of fungi. We analyzed, by use of microarrays, the transcriptional regulation of the 639 genes identified to be involved in cell wall synthesis and structure plus the secretome of the Basidiomycota species Ustilago maydis during its dimorphic transition induced by a change in pH. Of these, 189 were differentially expressed during the process, and using as control two monomorphic mutants, one yeast like and the other mycelium constitutive, 66 genes specific of dimorphism were identified. Most of these genes were up-regulated in the mycelial phase. These included CHS genes, genes involved in ß-1,6-glucan synthesis, N-glycosylation, and proteins containing a residue of glycosylphosphatidylinositol, and a number of genes from the secretome. The possible significance of these data on cell wall plasticity is discussed.