RESUMO
Mannosylerythritol lipids (MELs) are surface active molecules produced by many basidiomycetous fungi. MELs consist of a mannosylerythritol disaccharide, which is acylated with short and medium chain fatty acids at the mannosyl moiety. A gene cluster composed of five genes is required for MEL biosynthesis. Here we show that the plant pathogenic fungus Ustilago hordei secretes these glycolipids under nitrogen starvation conditions. In contrast to MELs produced by the closely related fungus Ustilago maydis those secreted by U. hordei are mostly mono-acetylated and contain a different mixture of acyl groups. Cross-species complementation between these fungi revealed that these differences result from different catalytic activities of the acetyltransferase Mat1 and the acyltransferases Mac1 and Mac2. U. maydis mat1 mutants expressing the homologous mat1 gene from U. hordei produced mostly mono-acetylated variants and lack di-acetylated MELs normally produced by U. maydis. Furthermore, we determined that the acyltransferase Mac1 acylates the mannosylerythritol moiety at position C2 while Mac2 acylates C3. The identification of decorating enzymes with different substrate specificities will allow the tailor-made production of novel subsets of MELs.
Assuntos
Glicolipídeos/biossíntese , Ustilaginales/enzimologia , Ustilaginales/metabolismo , Acetilação , Acetiltransferases/genética , Acetiltransferases/metabolismo , Aciltransferases/genética , Aciltransferases/metabolismo , Ácidos Graxos/metabolismo , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Família Multigênica , Nitrogênio/metabolismo , Especificidade por Substrato , Transcriptoma , Ustilaginales/genéticaRESUMO
Many microorganisms secrete surface-active glycolipids. The basidiomycetous fungus Ustilago maydis produces two different classes of glycolipids, mannosylerythritol lipids (MEL) and ustilagic acids (UAs). Here we report that biosynthesis of MELs is partially localized in peroxisomes and coupled to peroxisomal fatty acid degradation. The acyltransferases, Mac1 and Mac2, which acylate mannosylerythritol with fatty acids of different length, contain a type 1 peroxisomal targeting signal (PTS1). We demonstrate that Mac1 and Mac2 are targeted to peroxisomes, while other enzymes involved in MEL production reside in different compartments. Mis-targeting of Mac1 and Mac2 to the cytosol did not block MEL synthesis but promoted production of MEL species with altered acylation pattern. This is in contrast to peroxisome deficient mutants that produced MELs similar to the wild type. We could show that cytosolic targeting of Mac1 and Mac2 reduces the amount of UA presumably due to competition for overlapping substrates. Interestingly, hydroxylated fatty acids characteristic for UAs appear in MELs corroborating cross-talk between both biosynthesis pathways. Therefore, peroxisomal localization of MEL biosynthesis is not only prerequisite for generation of the natural spectrum of MELs, but also facilitates simultaneous assembly of different glycolipids in a single cell.
Assuntos
Acetiltransferases/metabolismo , Glicolipídeos/biossíntese , Peroxissomos/metabolismo , Ustilago/enzimologia , Acetiltransferases/genética , Acilação , Motivos de Aminoácidos , Vias Biossintéticas , Citosol/metabolismo , Ácidos Graxos/metabolismo , Proteínas Fúngicas/metabolismo , Glicolipídeos/química , MutaçãoRESUMO
Differentiation of hyphae into specialized infection structures, known as appressoria, is a common feature of plant pathogenic fungi that penetrate the plant cuticle. Appressorium formation in U. maydis is triggered by environmental signals but the molecular mechanism of this hyphal differentiation is largely unknown. Infectious hyphae grow on the leaf surface by inserting regularly spaced retraction septa at the distal end of the tip cell leaving empty sections of collapsed hyphae behind. Here we show that formation of retraction septa is critical for appressorium formation and virulence in U. maydis. We demonstrate that the diaphanous-related formin Drf1 is necessary for actomyosin ring formation during septation of infectious hyphae. Drf1 acts as an effector of a Cdc42 GTPase signaling module, which also consists of the Cdc42-specific guanine nucleotide exchange factor Don1 and the Ste20-like kinase Don3. Deletion of drf1, don1 or don3 abolished formation of retraction septa resulting in reduced virulence. Appressorium formation in these mutants was not completely blocked but infection structures were found only at the tip of short filaments indicating that retraction septa are necessary for appressorium formation in extended infectious hyphae. In addition, appressoria of drf1 mutants penetrated the plant tissue less frequently.
Assuntos
Doenças das Plantas/microbiologia , Folhas de Planta/microbiologia , Ustilago/patogenicidade , Zea mays/microbiologia , Actomiosina/biossíntese , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Técnicas de Inativação de Genes , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Hifas/citologia , Hifas/genética , Hifas/fisiologia , Reação em Cadeia da Polimerase , Deleção de Sequência , Transdução de Sinais , Ustilago/genética , Ustilago/metabolismo , Proteína cdc42 de Ligação ao GTP/genética , Proteína cdc42 de Ligação ao GTP/metabolismoRESUMO
Ustilago maydis is a ubiquitous pathogen of maize and a well-established model organism for the study of plant-microbe interactions. This basidiomycete fungus does not use aggressive virulence strategies to kill its host. U. maydis belongs to the group of biotrophic parasites (the smuts) that depend on living tissue for proliferation and development. Here we report the genome sequence for a member of this economically important group of biotrophic fungi. The 20.5-million-base U. maydis genome assembly contains 6,902 predicted protein-encoding genes and lacks pathogenicity signatures found in the genomes of aggressive pathogenic fungi, for example a battery of cell-wall-degrading enzymes. However, we detected unexpected genomic features responsible for the pathogenicity of this organism. Specifically, we found 12 clusters of genes encoding small secreted proteins with unknown function. A significant fraction of these genes exists in small gene families. Expression analysis showed that most of the genes contained in these clusters are regulated together and induced in infected tissue. Deletion of individual clusters altered the virulence of U. maydis in five cases, ranging from a complete lack of symptoms to hypervirulence. Despite years of research into the mechanism of pathogenicity in U. maydis, no 'true' virulence factors had been previously identified. Thus, the discovery of the secreted protein gene clusters and the functional demonstration of their decisive role in the infection process illuminate previously unknown mechanisms of pathogenicity operating in biotrophic fungi. Genomic analysis is, similarly, likely to open up new avenues for the discovery of virulence determinants in other pathogens.
Assuntos
Genoma Fúngico/genética , Ustilago/genética , Ustilago/patogenicidade , Zea mays/microbiologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Genes Fúngicos/genética , Genômica , Família Multigênica/genética , Ustilago/crescimento & desenvolvimento , Virulência/genéticaRESUMO
Phosphorylation is a key event in many cellular processes like cell cycle, transformation of environmental signals to transcriptional activation or polar growth. The chemical genetics approach can be used to analyse the effect of highly specific inhibition in vivo and is a promising method to screen for kinase targets. We have used this approach to study the role of the germinal centre kinase Don3 during the cell division in the phytopathogenic fungus Ustilago maydis. Due to the easy determination of the don3 phenotype we have chosen this approach for a genetic course for M.Sc. students and for IMPRS (International Max-Planck research school) students. According to the principle of "problem-based learning" the aim of this two-week course is to transfer knowledge about the broad spectrum of kinases to the students and that the students acquire the ability to design their own analog-sensitive kinase of interest. In addition to these training goals, we benefit from these annual courses the synthesis of basic constructs for genetic modification of several kinases in our model system U. maydis.
Assuntos
Biologia Molecular/educação , Ustilago/genética , Currículo , Humanos , Ciência/educaçãoRESUMO
Upon nitrogen starvation, the basidiomycete Ustilago maydis, which causes smut disease on corn, secretes amphipathic glycolipids, including mannosylerythritol lipids (MELs). MELs consist of a carbohydrate core whose mannosyl moiety is both acylated with fatty acids of different lengths and acetylated. Here, we report the transport of MELs into and out of the cell depending on the transport protein Mmf1, which belongs to the major facilitator superfamily. Analysis of mmf1 mutants and mutants lacking the acetyltransferase Mat1 revealed that Mmf1 is necessary for the export of acetylated MELs, while MELs without an acetyl group are secreted independently of this transporter. Upon deletion of mmf1, we detected novel MEL species lacking the acyl side chain at C-3'. With the help of feeding experiments, we demonstrate that MELs are taken up by U. maydis in an mmf1-independent manner. This leads to catabolism or rearrangement of acetyl and acyl side groups and subsequent secretion. The catabolism of MELs involves the presence of Mac2, an enzyme required for MEL biosynthesis. In cocultivation experiments, mutual exchange of MELs between different mutants was observed. Thus, we propose a novel function for fungal glycolipids as an external carbon storage. IMPORTANCE Fungi produce and secrete various secondary metabolites that can act as weapons against competitors, help in accessing nutrients, or assist in development and communication. One group of secondary metabolites are surface-active glycolipids, which have significant biotechnological potential as biodegradable detergents. While the biosynthesis of several fungal biosurfactants is well characterized, their biological functions and transport routes are less understood. We developed a cocultivation assay to show that a class of glycolipids from Ustilago maydis called mannosylerythritol lipids (MELs) can be exchanged between cells and modified or even degraded by recipient cells. Feeding assays with purified MELs led to similar results. These data provide insight into the surprising biological role of MELs as putative external carbon sources. Applying feeding and cocultivation experiments on MEL biosynthesis mutants turned out to be a valuable strategy for systematically studying the import routes and degradation pathways of glycolipids. By using these assays, we demonstrate the function of the transport protein Mmf1 as a specific exporter of acetylated MELs. We propose that these assays may be applied more generally, thereby opening novel areas of research.
Assuntos
Detergentes , Ustilaginales , Detergentes/metabolismo , Glicolipídeos/metabolismo , Ustilaginales/genética , Ustilaginales/metabolismo , Acetiltransferases/metabolismo , Ácidos Graxos/metabolismo , Nitrogênio/metabolismo , Carbono/metabolismo , Proteínas de Transporte/metabolismo , Tensoativos/química , Tensoativos/metabolismoRESUMO
The phytopathogenic smut fungus Ustilago maydis is a versatile model organism to study plant pathology, fungal genetics, and molecular cell biology. Here, we report several strategies to manipulate the genome of U. maydis by the CRISPR/Cas9 technology. These include targeted gene deletion via homologous recombination of short double-stranded oligonucleotides, introduction of point mutations, heterologous complementation at the genomic locus, and endogenous N-terminal tagging with the fluorescent protein mCherry. All applications are independent of a permanent selectable marker and only require transient expression of the endonuclease Cas9hf and sgRNA. The techniques presented here are likely to accelerate research in the U. maydis community but can also act as a template for genome editing in other important fungi.
RESUMO
Mannosylerythritol lipids (MELs) are surface active glycolipids secreted by various fungi. MELs can be used as biosurfactants and are a biodegradable resource for the production of detergents or pharmaceuticals. Different fungal species synthesize a unique mixture of MELs differing in acetyl- and acyl-groups attached to the sugar moiety. Here, we report the construction of a toolbox for production of glycolipids with predictable fatty acid side chains in the basidiomycete Ustilago maydis. Genes coding for acyl-transferases involved in MEL production (Mac1 and Mac2) from different fungal species were combined to obtain altered MEL variants with distinct physical properties and altered antimicrobial activity. We also demonstrate that a U. maydis paralog of the acyltransferase Mac2 with a different substrate specificity can be employed for the biosynthesis of modified MEL variants. In summary, our data showcase how the fungal repertoire of Mac enzymes can be used to engineer tailor-made MELs according to specific biotechnological or pharmaceutical requirements.
RESUMO
Proteins of the 14-3-3 and Rho-GTPase families are functionally conserved eukaryotic proteins that participate in many important cellular processes such as signal transduction, cell cycle regulation, malignant transformation, stress response, and apoptosis. However, the exact role(s) of these proteins in these processes is not entirely understood. Using the fungal maize pathogen, Ustilago maydis, we were able to demonstrate a functional connection between Pdc1 and Rho1, the U. maydis homologues of 14-3-3epsilon and Rho1, respectively. Our experiments suggest that Pdc1 regulates viability, cytokinesis, chromosome condensation, and vacuole formation. Similarly, U. maydis Rho1 is also involved in these three essential processes and exerts an additional function during mating and filamentation. Intriguingly, yeast two-hybrid and epistasis experiments suggest that both Pdc1 and Rho1 could be constituents of the same regulatory cascade(s) controlling cell growth and filamentation in U. maydis. Overexpression of rho1 ameliorated the defects of cells depleted for Pdc1. Furthermore, we found that another small G protein, Rac1, was a suppressor of lethality for both Pdc1 and Rho1. In addition, deletion of cla4, encoding a Rac1 effector kinase, could also rescue cells with Pdc1 depleted. Inferring from these data, we propose a model for Rho1 and Pdc1 functions in U. maydis.
Assuntos
Proteínas 14-3-3/metabolismo , Divisão Celular/fisiologia , Polaridade Celular/fisiologia , Proteínas Fúngicas/metabolismo , Ustilago/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Proteínas 14-3-3/genética , Diferenciação Celular/fisiologia , Crescimento Celular , Sobrevivência Celular/fisiologia , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Piruvato Descarboxilase/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais/fisiologia , Ustilago/citologia , Ustilago/genética , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/genéticaRESUMO
The highly conserved GTP-binding proteins Cdc42 and Rac1 regulate cytokinesis, establishment of cell polarity and vesicular trafficking. In the dimorphic fungus Ustilago maydis, Rac1 is required for cell polarity and budding, while Cdc42 is essential for cell separation during cytokinesis. The same cell separation defect is also observed in mutants that lack Don1, a guanine nucleotide exchange factor (GEF) of the Dbl family. We have generated a series of chimeric GTP-binding proteins consisting of different portions of Cdc42 and Rac1. In vivo complementation analysis revealed that a short region encompassing amino acids 41-56 determines signalling specificity. Remarkably, substitution of a single amino acid at position 56 within this specificity domain is sufficient to confer Cdc42 function to Rac1 in vivo. Expression of Rac1(W56F) in Delta cdc42 mutant cells resulted in complementation of the cell separation defect. In vitro GDP/GTP exchange assays demonstrated that the Dbl family GEF Don1 is highly specific for Cdc42 and cannot activate Rac1. However, if Rac1(W56F) is used as a substrate, Don1 is able to stimulate GDP/GTP exchange. Together these data indicate that activation by the GEF Don1 is an important determinant of Cdc42-specific signalling in vivo.
Assuntos
Proteínas Fúngicas/metabolismo , Transdução de Sinais , Ustilago/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Sítios de Ligação , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Teste de Complementação Genética , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Dados de Sequência Molecular , Ligação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Ustilago/citologia , Proteína cdc42 de Ligação ao GTP/química , Proteína cdc42 de Ligação ao GTP/genética , Proteína cdc42 de Saccharomyces cerevisiae de Ligação ao GTP/genética , Proteína cdc42 de Saccharomyces cerevisiae de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/química , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Unconventional secretion has emerged as an increasingly important cellular process in eukaryotic cells. The underlying translocation mechanisms are diverse and often little understood. We study unconventional secretion of chitinase Cts1 in the corn smut fungus Ustilago maydis. This protein participates in the cytokinesis of yeast cells. During budding it localizes to the septated fragmentation zone where it presumably functions in the degradation of remnant chitin to allow separation of mother and daughter cell. However, the mechanistic details of Cts1 export remain unclear. Here we investigated the mechanism of unconventional Cts1 secretion with a focus on cytokinesis. Cell-cycle inhibition experiments supported the hypothesis that Cts1 export is connected to cytokinesis. To substantiate this finding we analysed gene deletion mutants impaired in cell separation and discovered that strains defective in secondary septum formation were affected in Cts1 export. The germinal centre kinase Don3 had a particularly strong influence on unconventional secretion. Using a synthetic switch, we unambiguously verified an essential role of Don3 for cytokinesis-dependent Cts1 export via the fragmentation zone. Thus, we gained novel insights into the mechanism of unconventional secretion and discovered the first regulatory component of this process.
Assuntos
Quitinases/metabolismo , Proteínas Fúngicas/metabolismo , Quinases do Centro Germinativo/metabolismo , Ustilago/metabolismo , Ciclo Celular , Transporte ProteicoRESUMO
XPD functions in transcription, DNA repair and in cell cycle control. Mutations in human XPD (also known as ERCC2) mainly cause three clinical phenotypes: xeroderma pigmentosum (XP), Cockayne syndrome (XP/CS) and trichothiodystrophy (TTD), and only XP patients have a high predisposition to developing cancer. Hence, we developed a fly model to obtain novel insights into the defects caused by individual hypomorphic alleles identified in human XP-D patients. This model revealed that the mutations that displayed the greatest in vivo UV sensitivity in Drosophila did not correlate with those that led to tumor formation in humans. Immunoprecipitations followed by targeted quantitative MS/MS analysis showed how different xpd mutations affected the formation or stability of different transcription factor IIH (TFIIH) subcomplexes. The XP mutants most clearly linked to high cancer risk, Xpd R683W and R601L, showed a reduced interaction with the core TFIIH and also an abnormal interaction with the Cdk-activating kinase (CAK) complex. Interestingly, these two XP alleles additionally displayed high levels of chromatin loss and free centrosomes during the rapid nuclear division phase of the Drosophila embryo. Finally, the xpd mutations showing defects in the coordination of cell cycle timing during the Drosophila embryonic divisions correlated with those human mutations that cause the neurodevelopmental abnormalities and developmental growth defects observed in XP/CS and TTD patients.
Assuntos
Neoplasias/metabolismo , Xeroderma Pigmentoso/genética , Alelos , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Ciclo Celular , Cromatina/metabolismo , Clonagem Molecular , Quinases Ciclina-Dependentes/metabolismo , Modelos Animais de Doenças , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Humanos , Dados de Sequência Molecular , Mutação , Neurônios/patologia , Fenótipo , Fatores de Risco , Homologia de Sequência de Aminoácidos , Fator de Transcrição TFIIH/metabolismo , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
Peroxisomes are nearly ubiquitous single-membrane organelles harboring multiple metabolic pathways beside their prominent role in the ß-oxidation of fatty acids. Here we review the diverse metabolic functions of peroxisomes in fungi. A variety of fungal metabolites are at least partially synthesized inside peroxisomes. These include the essential co-factor biotin but also different types of secondary metabolites. Peroxisomal metabolites are often derived from acyl-CoA esters for example ß-oxidation intermediates. In several ascomycetes a subtype of peroxisomes has been identified that is metabolically inactive but is required to plug the septal pores of wounded hyphae. Thus, peroxisomes are versatile organelles that can adapt their function to the life style of an organism. This remarkable variability suggests that the full extent of the biosynthetic capacity of peroxisomes is still elusive. Moreover, in fungi peroxisomes are non-essential under laboratory conditions making them attractive organelles for biotechnological approaches and the design of novel metabolic pathways in customized peroxisomes.
Assuntos
Vias Biossintéticas , Fungos/metabolismo , Peroxissomos/metabolismo , Biotecnologia , Fermentação , OxirreduçãoRESUMO
Dimorphic switching from budding to filamentous growth is a characteristic feature of many pathogenic fungi. In the fungal model organism Ustilago maydis polarized growth is induced by the multiallelic b mating type locus and requires the Rho family GTPase Rac1. Here we show that mating type-induced polarized growth involves negative feedback regulation of the Rac1-specific guanine nucleotide exchange factor (GEF) Cdc24. Although Cdc24 is essential for polarized growth, its concentration is drastically diminished during filament formation. Cdc24 is part of a protein complex that also contains the scaffold protein Bem1 and the PAK kinase Cla4. Activation of Rac1 results in Cla4-dependent degradation of the Rac1-GEF Cdc24, thus creating a regulatory negative feedback loop. We generated mutants of Cdc24 that are resistant to Cla4-dependent destruction. Expression of stable Cdc24 variants interfered with filament formation, indicating that negative feedback regulation of Cdc24 is critical for the establishment of polarized growth.
Assuntos
Proteínas Fúngicas/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Ustilago/enzimologia , Proteínas rac1 de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Regulação para Baixo , Ativação Enzimática , Retroalimentação Fisiológica , Fatores de Troca do Nucleotídeo Guanina/genética , Hifas/enzimologia , Hifas/crescimento & desenvolvimento , Dados de Sequência Molecular , Morfogênese , Complexos Multiproteicos/metabolismo , Fosforilação , Proteólise , Ustilago/crescimento & desenvolvimentoRESUMO
In the dimorphic fungus Ustilago maydis the Rho-family GTP-binding protein Cdc42 and the Ste20-like kinase Don3 are both essential for triggering cell separation during cytokinesis. Since Don3 does not contain a Cdc42/Rac interaction and binding domain (CRIB), it is unclear how Cdc42 and Don3 cooperate in the regulation of cytokinesis. To analyse the regulatory network we generated an analogue-sensitive Don3 variant (Don3-as) that allows specific inhibition in vivo. The engineered kinase Don3(M157A) is fully active in vivo and can be specifically inhibited by low concentrations of the ATP-analogue NA-PP1. Inhibition of the Don3-as kinase activity immediately blocked cell separation resulting in the formation of clusters of nonseparated cells. Covalent labelling of cell wall proteins showed that, upon release of inhibition, cytokinesis was resumed instantaneously in all cells. By sequentially activating Don3 and Cdc42 we were able to demonstrate that both proteins act independently of each other and that Don3 activity precedes that of Cdc42. We provide evidence that Don3 and Cdc42 are crucial for the assembly of a contractile actomyosin ring, which is a prerequisite for secondary septum formation. We propose, that Don3 is involved in establishing a landmark, at which the Cdc42-dependent actomyosin ring formation will occur.
Assuntos
Citocinese , Proteínas Serina-Treonina Quinases/fisiologia , Ustilago/fisiologia , Proteína cdc42 de Ligação ao GTP/fisiologia , Actomiosina/química , Trifosfato de Adenosina/química , Sequência de Aminoácidos , Membrana Celular/metabolismo , Inibidores Enzimáticos/farmacologia , Escherichia coli/metabolismo , Quinases do Centro Germinativo , Modelos Biológicos , Dados de Sequência Molecular , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Pirazóis/farmacologia , Pirimidinas/farmacologia , Homologia de Sequência de AminoácidosRESUMO
Septum formation is a crucial step of cytokinesis in fungi. In the basidiomycete Ustilago maydis, the germinal centre kinase Don3 triggers initiation of a secondary septum necessary for cell separation after cytokinesis. Here we show that oligomerization of Don3 via a putative coiled-coil domain is critical for secondary septum formation. Within the Don3 sequence we detected a characteristic C-terminal sequence motif (T-motif), which determines the subcellular localization of Don3 but is not required for regulation of cell separation. This motif defines a novel family of fungal protein kinases including Sid1p, an essential component of the septation initiation network (SIN) in Schizosaccharomyces pombe. Using the yeast two-hybrid system we isolated the Don3-interacting protein Dip1, which is similar to S. pombe Cdc14p, another member of the SIN. Remarkably, deletion of dip1 did not interfere with cytokinesis in U. maydis, but both dip1 and don3 mutants were affected in nuclear envelope breakdown (NEBD) during mitosis. This phenotype has already been described for mutants, which lack the small GTPase Ras3, the U. maydis homologue of the SIN component Spg1p. We propose that the Don3 kinase exerts a dual function in the regulation of cell separation and NEBD.
Assuntos
Citocinese , Mitose , Proteínas Serina-Treonina Quinases/metabolismo , Ustilago/enzimologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Quinases do Centro Germinativo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Dados de Sequência Molecular , Mutação , Membrana Nuclear/genética , Membrana Nuclear/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Estrutura Terciária de Proteína , Proteínas Tirosina Fosfatases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Técnicas do Sistema de Duplo-Híbrido , Ustilago/citologia , Ustilago/genética , Proteínas ras/genética , Proteínas ras/metabolismoRESUMO
Small GTP-binding proteins of the highly conserved Rho family act as molecular switches regulating cell signalling, cytoskeletal organization and vesicle trafficking in eukaryotic cells. Here we show that in the dimorphic plant pathogenic fungus Ustilago maydis deletion of either cdc42 or rac1 results in loss of virulence but does not interfere with viability. Cells deleted for cdc42 display a cell separation defect during budding. We have previously shown that the Rho-specific guanine nucleotide exchange factor (GEF) Don1 is required for cell separation in U. maydis. Expression of constitutive active Cdc42 rescues the phenotype of don1 mutant cells indicating that Don1 triggers cell separation by activating Cdc42. Deletion of rac1 affects cellular morphology and interferes with hyphal growth, whereas overexpression of wild-type Rac1 induces filament formation in haploid cells. This indicates that Rac1 is both necessary and sufficient for the dimorphic switch from budding to hyphal growth. Cdc42 and Rac1 share at least one common essential function because depletion of both Rac1 and Cdc42 is lethal. Expression of constitutively active Rac1(Q61L) is lethal and results in swollen cells with a large vacuole. The morphological phenotype, but not lethality is suppressed in cla4 mutant cells suggesting that the PAK family kinase Cla4 acts as a downstream effector of Rac1.
Assuntos
Ciclo Celular/fisiologia , Divisão Celular/fisiologia , Ustilago/citologia , Ustilago/crescimento & desenvolvimento , Proteína cdc42 de Ligação ao GTP/fisiologia , Proteínas rac1 de Ligação ao GTP/fisiologia , Genes Fúngicos , Genes Letais , Proteína cdc42 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/genéticaRESUMO
The phytopathogenic basidiomycete Ustilago maydis displays a dimorphic switch between budding growth of haploid cells and filamentous growth of the dikaryon. In a screen for mutants affected in morphogenesis and cytokinesis, we identified the serine/threonine protein kinase Cla4, a member of the family of p21-activated kinases (PAKs). Cells, in which cla4 has been deleted, are viable but they are unable to bud properly. Instead, cla4 mutant cells grow as branched septate hyphae and divide by contraction and fission at septal cross walls. Delocalized deposition of chitinous cell wall material along the cell surface is observed in cla4 mutant cells. Deletion of the Cdc42/Rac1 interaction domain (CRIB) results in a constitutive active Cla4 kinase, whose expression is lethal for the cell. cla4 mutant cells are unable to induce pathogenic development in plants and to display filamentous growth in a mating reaction, although they are still able to secrete pheromone and to undergo cell fusion with wild-type cells. We propose that Cla4 is involved in the regulation of cell polarity during budding and filamentation.
Assuntos
Citocinese , Proteínas Fúngicas/metabolismo , Morfogênese , Proteínas Serina-Treonina Quinases/metabolismo , Ustilago/enzimologia , Ustilago/fisiologia , Actinas/metabolismo , Sequência de Aminoácidos , Citocinese/genética , Proteínas Fúngicas/genética , GTP Fosfo-Hidrolases/metabolismo , Humanos , Dados de Sequência Molecular , Fenótipo , Proteínas Serina-Treonina Quinases/genética , Proteínas de Saccharomyces cerevisiae , Alinhamento de Sequência , Técnicas do Sistema de Duplo-Híbrido , Ustilago/ultraestrutura , Zea mays/microbiologiaRESUMO
Small interfering RNAs (siRNAs) represent RNA duplexes of 21 nucleotides in length that inhibit gene expression. We have used the human gene-external 7S K RNA promoter for synthesis of short hairpin RNAs (shRNAs) which efficiently target human lamin mRNA via RNA interference (RNAi). Here we demonstrate that orientation of the target sequence within the shRNA construct is important for interference. Furthermore, effective interference also depends on the length and/or structure of the shRNA. Evidence is presented that the human 7S K promoter is more active in vivo than other gene-external promoters, such as the human U6 small nuclear RNA (snRNA) gene promoter.