RESUMO
The aim of this work was to evaluate the effect of the addition of huitlacoche paste to nixtamalized blue-corn flours (NBCF) on the physicochemical, thermal, and rheological properties of masas. Raw blue maize was nixtamalized (hydrothermal alkalinized process), then was wet-milled in a stone mill, masa was dehydrated, pulverized and sieved to obtain NBCF; commercial nixtamalized blue-corn flour (CNBCF) was used as a control. Huitlacoche paste in concentrations of 3, 6, 9, 12, 15, and 18% was added to nixtamalized flours. Characteristics of the blue grain showed its great effects on water absorption, viscosity, and masa cohesiveness; the addition of huitlacoche significantly influenced adhesiveness, water-absorption, color, and the rheological properties (pâ¯<â¯0.05). Values between 0.03 and 0.083â¯kg-force resulted in masas with optimal adhesiveness. The inclusion of huitlacoche paste can be achieved with a maximal addition of 9% in NBCF for an industrial process and could comprise a new industrialization alternative.
Assuntos
Farinha/análise , Ustilago/crescimento & desenvolvimento , Zea mays/química , Cor , Reologia , Solubilidade , Viscosidade , Água/química , Zea mays/metabolismoRESUMO
Previously, we demonstrated that when Ustilago maydis (DC) Cda., a phytopathogenic basidiomycete and the causal agent of corn smut, is grown in the vicinity of maize embryogenic calli in a medium supplemented with the herbicide Dicamba, it developed gastroid-like basidiocarps. To elucidate the molecular mechanisms involved in the basidiocarp development by the fungus, we proceeded to analyze the transcriptome of the process, identifying a total of 2002 and 1064 differentially expressed genes at two developmental stages, young and mature basidiocarps, respectively. Function of these genes was analyzed with the use of different databases. MIPS analysis revealed that in the stage of young basidiocarp, among the ca. two thousand differentially expressed genes, there were some previously described for basidiocarp development in other fungal species. Additional elements that operated at this stage included, among others, genes encoding the transcription factors FOXO3, MIG3, PRO1, TEC1, copper and MFS transporters, and cytochromes P450. During mature basidiocarp development, important up-regulated genes included those encoding hydrophobins, laccases, and ferric reductase (FRE/NOX). The demonstration that a mapkk mutant was unable to form basidiocarps, indicated the importance of the MAPK signaling pathway in this developmental process.
Assuntos
Dicamba/farmacologia , Carpóforos/genética , Transcriptoma/efeitos dos fármacos , Ustilago/genética , Carpóforos/efeitos dos fármacos , Carpóforos/crescimento & desenvolvimento , Proteínas Fúngicas/biossíntese , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Doenças das Plantas/microbiologia , Ustilago/efeitos dos fármacos , Ustilago/crescimento & desenvolvimento , Ustilago/patogenicidade , Zea mays/microbiologiaRESUMO
In this study, we investigated the reverse transcriptase subunit of telomerase in the dimorphic fungus Ustilago maydis. This protein (Trt1) contains 1371 amino acids and all of the characteristic TERT motifs. Mutants created by disrupting trt1 had senescent traits, such as delayed growth, low replicative potential, and reduced survival, that were reminiscent of the traits observed in est2 budding yeast mutants. Telomerase activity was observed in wild-type fungus sporidia but not those of the disruption mutant. The introduction of a self-replicating plasmid expressing Trt1 into the mutant strain restored growth proficiency and replicative potential. Analyses of trt1 crosses in planta suggested that Trt1 is necessary for teliospore formation in homozygous disrupted diploids and that telomerase is haploinsufficient in heterozygous diploids. Additionally, terminal restriction fragment analysis in the progeny hinted at alternative survival mechanisms similar to those of budding yeast.
Assuntos
Telomerase/biossíntese , Telomerase/genética , Ustilago/enzimologia , Sequência de Aminoácidos , Replicação do DNA/genética , Diploide , Regulação Fúngica da Expressão Gênica , Esporos/genética , Telomerase/isolamento & purificação , Ustilago/genética , Ustilago/crescimento & desenvolvimentoRESUMO
Dimorphism is the property of fungi to grow as budding yeasts or mycelium, depending on the environmental conditions. This phenomenon is important as a model of differentiation in eukaryotic organisms, and since a large number of fungal diseases are caused by dimorphic fungi, its study is important for practical reasons. In this work, we examined the transcriptome during the dimorphic transition of the basidiomycota phytopathogenic fungus Ustilago maydis using microarrays, utilizing yeast and mycelium monomorphic mutants as controls. This way, we thereby identified 154 genes of the fungus that are specifically involved in the dimorphic transition induced by a pH change. Of these, 82 genes were up-regulated, and 72 were down-regulated. Differential categorization of these genes revealed that they mostly belonged to the classes of metabolism, cell cycle and DNA processing, transcription and protein fate, transport and cellular communication, stress, cell differentiation and biogenesis of cellular components, while a significant number of them corresponded to unclassified proteins. The data reported in this work are important for our understanding of the molecular bases of dimorphism in U. maydis, and possibly of other fungi.
Assuntos
Proteínas Fúngicas/genética , Transcrição Gênica , Ustilago/crescimento & desenvolvimento , Ustilago/genética , Proteínas Fúngicas/metabolismo , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Concentração de Íons de Hidrogênio , Micélio/genética , Micélio/crescimento & desenvolvimento , Micélio/metabolismo , Ustilago/química , Ustilago/metabolismoRESUMO
Ustilago maydis is a dimorphic corn pathogenic basidiomycota whose haploid cells grow in yeast form at pH7, while at pH3 they grow in the mycelial form. Two-dimensional gel electrophoresis (2-DE) coupled with LC-ESI/MS-MS was used to analyze the differential accumulation of proteins in yeast against mycelial morphologies. 2-DE maps were obtained in the pH range of 5-8 and 404 total protein spots were separated. From these, 43 were differentially accumulated when comparing strains FB2wt, constitutive yeast CL211, and constitutive mycelial GP25 growing at pH7 against pH3. Differentially accumulated proteins in response to pH are related with defense against reactive oxygen species or toxic compounds. Up-accumulation of CipC and down-accumulation of Hmp1 were specifically related with mycelial growth. Changes in proteins that were affected by mutation in the gene encoding the adaptor of a MAPK pathway (CL211 strain) were UM521* and transcription factors Btf3, Sol1 and Sti1. Mutation of GCN5 (GP25 strain) affected the accumulation of Rps19-ribosomal protein, Mge1-heath shock protein, and Lpd1-dihydrolipoamide dehydrogenase. Our results complement the information about the genes and proteins related with the dimorphic transition in U. maydis and changes in proteins affected by mutations in a MAPK pathway and GCN5 gene.
Assuntos
Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Genes Fúngicos/fisiologia , Histona Acetiltransferases/genética , Sistema de Sinalização das MAP Quinases/genética , Ustilago/genética , Concentração de Íons de Hidrogênio , Proteoma/genética , Ustilago/crescimento & desenvolvimento , Ustilago/metabolismoRESUMO
Ustilago maydis (DC) Cda., a phytopathogenic Basidiomycota, is the causal agent of corn smut. During its life cycle U. maydis alternates between a yeast-like, haploid nonpathogenic stage, and a filamentous, dikaryotic pathogenic form that invades the plant and induces tumor formation. As all the members of the Subphylum Ustilaginomycotina, U. maydis is unable to form basidiocarps, instead it produces teliospores within the tumors that germinate forming a septate basidium (phragmobasidium). We have now established conditions allowing a completely different developmental program of U. maydis when grown on solid medium containing auxins in dual cultures with maize embryogenic calli. Under these conditions U. maydis forms large hemi-spheroidal structures with all the morphological and structural characteristics of gastroid-type basidiocarps. These basidiocarps are made of three distinct hyphal layers, the most internal of which (hymenium) contains non-septate basidia (holobasidia) from which four basidiospores develop. In basidiocarps meiosis and genetic recombination occur, and meiotic products (basidiospores) segregate in a Mendelian fashion. These results are evidence of sexual cycle completion of an Ustilaginomycotina in vitro, and the demonstration that, besides its quasi-obligate biotrophic pathogenic mode of life, U. maydis possesses the genetic program to form basidiocarps as occurs in saprophytic Basidiomycota species.
Assuntos
Carpóforos/crescimento & desenvolvimento , Doenças das Plantas/microbiologia , Reguladores de Crescimento de Plantas/farmacologia , Ustilago/crescimento & desenvolvimento , Zea mays/microbiologia , Citocininas/farmacologia , DNA Fúngico/genética , Diploide , Carpóforos/citologia , Carpóforos/genética , Giberelinas/farmacologia , Haploidia , Hifas/citologia , Hifas/efeitos dos fármacos , Hifas/genética , Hifas/crescimento & desenvolvimento , Ácidos Indolacéticos/farmacologia , Meiose , Metamorfose Biológica , Recombinação Genética , Esporos Fúngicos/citologia , Esporos Fúngicos/efeitos dos fármacos , Esporos Fúngicos/genética , Esporos Fúngicos/crescimento & desenvolvimento , Ustilago/citologia , Ustilago/efeitos dos fármacos , Ustilago/genética , Virulência , Leveduras/citologia , Leveduras/efeitos dos fármacos , Leveduras/genética , Leveduras/crescimento & desenvolvimento , Zea mays/citologia , Zea mays/embriologiaRESUMO
Ustilago maydis displays dimorphic growth, alternating between a saprophytic haploid yeast form and a filamentous dikaryon, generated by mating of haploid cells and which is an obligate parasite. Induction of the dimorphic transition of haploid strains in vitro by change in ambient pH has been used to understand the mechanisms governing this differentiation process. In this study we used suppression subtractive hybridization to generate a cDNA library of U. maydis genes up-regulated in the filamentous form induced in vitro at acid pH. Expression analysis using quantitative RT-PCR showed that the induction of two unigenes identified in this library coincided with the establishment of filamentous growth in the acid pH medium. This expression pattern suggested that they were specifically associated to hyphal development rather than merely acid pH-induced genes. One of these genes, UmRrm75, encodes a protein containing three RNA recognition motifs and glycine-rich repeats and was selected for further study. The UmRrm75 gene contains 4 introns, and produces a splicing variant by a 3'-alternative splicing site within the third exon. Mutants deleted for UmRrm75 showed a slower growth rate than wild type strains in liquid and solid media, and their colonies showed a donut-like morphology on solid medium. Interestingly, although ΔUmRrm75 strains were not affected in filamentous growth induced by acid pH and oleic acid, they exhibited reduced mating, post-mating filamentous growth and virulence. Our data suggest that UmRrm75 is probably involved in cell growth, morphogenesis, and pathogenicity in U. maydis.
Assuntos
Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Doenças das Plantas/microbiologia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ustilago/crescimento & desenvolvimento , Ustilago/patogenicidade , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas Fúngicas/química , Regulação Fúngica da Expressão Gênica , Genes Fúngicos Tipo Acasalamento , Hifas/genética , Hifas/crescimento & desenvolvimento , Hifas/metabolismo , Dados de Sequência Molecular , Proteínas de Ligação a RNA/química , Alinhamento de Sequência , Ustilago/genética , Ustilago/metabolismo , Virulência , Zea mays/microbiologiaRESUMO
In previous communications the essential role of spermidine in Ustilago maydis was demonstrated by means of the disruption of the genes encoding ornithine decarboxylase (ODC) and spermidine synthase (SPE). However, the assignation of specific roles to each polyamine in different cellular functions was not possible because the spermidine added to satisfy the auxotrophic requirement of odc/spe double mutants is partly back converted into putrescine. In this study, we have approached this problem through the disruption of the gene-encoding polyamine oxidase (PAO), required for the conversion of spermidine into putrescine, and the construction of odc/pao double mutants that were unable to synthesize putrescine by either ornithine decarboxylation or retroconversion from spermidine. Phenotypic analysis of the mutants provided evidence that putrescine is only an intermediary in spermidine biosynthesis, and has no direct role in cell growth, dimorphic transition, or any other vital function of U. maydis. Nevertheless, our results show that putrescine may play a role in the protection of U. maydis against salt and osmotic stress, and possibly virulence. Evidence was also obtained that the retroconversion of spermidine into putrescine is not essential for U. maydis growth but may be important for its survival under natural conditions.
Assuntos
Técnicas de Inativação de Genes , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/deficiência , Putrescina/metabolismo , Ustilago/fisiologia , Genes Fúngicos , Viabilidade Microbiana , Mutagênese Insercional , Ornitina/metabolismo , Pressão Osmótica , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Espermidina/metabolismo , Estresse Fisiológico , Ustilago/genética , Ustilago/crescimento & desenvolvimento , Ustilago/metabolismo , Virulência , Poliamina OxidaseRESUMO
The effects of octyl gallate on Ustilago maydis yeast cells were analysed in relation to its capacity to oxidize compounds (pro-oxidant actions). All phenolic compounds tested inhibited the alternative oxidase (AOX). However, only octyl gallate induced a morphological change in yeast cells and collapsed the mitochondrial membrane potential. In contrast to octyl gallate, propyl gallate and nordihydroguaiaretic acid caused only a negligible cell change and the membrane potential was not affected. Our findings show that structurally related phenolic compounds do not necessarily exert similar actions on target cells. Preincubation of U. maydis cells with trolox inhibited the change to pseudohyphal growth produced by octyl gallate. These results suggest that in addition to the inhibitory action of octyl gallate on the AOX, this compound induces a switch from yeast to a mycelium, probably through the formation of lipid peroxides.
Assuntos
Ácido Gálico/análogos & derivados , Ustilago/citologia , Ustilago/crescimento & desenvolvimento , Proteínas Fúngicas/metabolismo , Ácido Gálico/metabolismo , Potencial da Membrana Mitocondrial , Proteínas Mitocondriais , Oxirredutases/metabolismo , Proteínas de Plantas , Galato de Propila/metabolismo , Ustilago/metabolismoRESUMO
Alterations occurring in polyamine metabolism of maize in tumors formed during the interaction with the biotrophic pathogenic fungus Ustilago maydis were analyzed. During the process, a striking increase in maize polyamine biosynthesis, mainly free and conjugated putrescine occurred in the tumors induced by the fungus, and in the neighbor plant tissues. This increase correlated with an activation mainly of Adc, Samdc1, Zmsamdc2 and Zmsamdc3, but not of Zmodc, Zmspds1 and Zmspds2 genes, and an elevation in arginine decarboxylase activity, confirming a predominant role of this enzyme in the process. Evidences for a possible contribution of spermidine and spermine degradation by polyamine oxidase activity, probably related to cell wall stiffening or lignification during tumor growth, were also obtained. It is suggested that polyamines, mainly putrescine, might play an active role in the pathosystem maize-U. maydis.
Assuntos
Tumores de Planta/microbiologia , Putrescina/metabolismo , Ustilago/crescimento & desenvolvimento , Zea mays/metabolismo , Zea mays/microbiologia , Carboxiliases/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Ornitina Descarboxilase/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , RNA de Plantas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Zea mays/enzimologia , Zea mays/genética , Poliamina OxidaseRESUMO
The domestication of crops and the development of agricultural societies not only brought about major changes in human interactions with the environment but also in plants' interactions with the diseases that challenge them. We evaluated the impact of the domestication of maize from teosinte and the widespread cultivation of maize on the historical demography of Ustilago maydis, a fungal pathogen of maize. To determine the evolutionary response of the pathogen's populations, we obtained multilocus genotypes for 1088 U. maydis diploid individuals from two teosinte subspecies in Mexico and from maize in Mexico and throughout the Americas. Results identified five major U. maydis populations: two in Mexico; two in South America; and one in the United States. The two populations in Mexico diverged from the other populations at times comparable to those for the domestication of maize at 6000-10000 years before present. Maize domestication and agriculture enforced sweeping changes in U. maydis populations such that the standing variation in extant pathogen populations reflects evolution only since the time of the crop's domestication.
Assuntos
Ustilago/fisiologia , Zea mays/crescimento & desenvolvimento , Zea mays/microbiologia , América Central , Ecossistema , Variação Genética , Geografia , América do Norte , Doenças das Plantas/microbiologia , América do Sul , Ustilago/genética , Ustilago/crescimento & desenvolvimentoRESUMO
Intraspecies diversity within Ustilago scitaminea isolates from South Africa, Reunion Island, Hawaii and Guadeloupe was assessed by RAPDs, bE mating-type gene detection, rDNA sequence analysis, microscopy and germination and morphological studies. Except for sequence data, the other analyses yielded no differences in the isolates that could be used in a phylogenetic separation. Mycelial DNA of the SA isolate shared 100% sequence identity with that of mycelial DNA cultured from in vitro produced teliospores of the parent cultivar. Overall the ITS1 and ITS2 regions were found to have 96.1% and 96.9% sequence identity with a total of 17 and 21 base changes, respectively, amongst the isolates. The Reunion Island isolate was shown to be most distantly related by 3.6% to the other isolates, indicating a single clonal lineage. The lack of germination in teliospores from Guadeloupe may be attributed to changes in temperature and humidity during transportation.
Assuntos
Variação Genética , Ustilago/genética , Sequência de Bases , Impressões Digitais de DNA , DNA Fúngico/análise , DNA Espaçador Ribossômico/análise , Guadalupe , Havaí , Dados de Sequência Molecular , Filogenia , Técnica de Amplificação ao Acaso de DNA Polimórfico , Alinhamento de Sequência , África do Sul , Ustilago/crescimento & desenvolvimento , Ustilago/ultraestruturaRESUMO
We utilized our modification of the amplified fragment length polymorphism technique for the determination of changes occurring in the DNA methylation patterns during the dimorphic transition of the fungi Mucor rouxii, Yarrowia lipolytica, and Ustilago maydis. To determine the specificity of differential methylation in regards to dimorphism, we obtained the yeast-like form of the three fungi under conditions that induced mycelial growth, by addition of 1,4-diaminobutanone (DAB), an inhibitor of ornithine decarboxylase in the case of M. rouxii and Y. lipolytica. In an odc null mutant of U. maydis, repression of the dimorphic transition was brought about by limitation in the amounts of exogenous putrescine. Yeasts from the three fungi thus obtained conserved a significant number of the differential DNA fragments with the methylation pattern displayed by normal yeasts, indicating their true correlation with dimorphism. Our results also confirm a role of polyamines in differential DNA methylation and fungal dimorphic transition.
Assuntos
Metilação de DNA , DNA Fúngico/metabolismo , Fungos/crescimento & desenvolvimento , Fungos/metabolismo , Poliaminas/metabolismo , Putrescina/análogos & derivados , Impressões Digitais de DNA , DNA Fúngico/análise , DNA Fúngico/isolamento & purificação , Inibidores Enzimáticos/farmacologia , Fungos/genética , Mucor/genética , Mucor/crescimento & desenvolvimento , Mucor/metabolismo , Micélio/crescimento & desenvolvimento , Técnicas de Amplificação de Ácido Nucleico , Ornitina Descarboxilase/genética , Inibidores da Ornitina Descarboxilase , Putrescina/metabolismo , Putrescina/farmacologia , Ustilago/genética , Ustilago/crescimento & desenvolvimento , Ustilago/metabolismo , Yarrowia/genética , Yarrowia/crescimento & desenvolvimento , Yarrowia/metabolismoRESUMO
Ustilago maydis is an edible parasitic basidiomycete, which specifically infects corn (Zea mays) and teocintle (Z. diploperennis). To characterise the interaction between the basidiomycete and its host organism, we tested the effect of plant lectins with well-known sugar specificity on the growth and germination of U. maydis spores. Lectins specific for N-acetyl-D-galactosamine, such as those from Dolichos biflorus and Phaseolus lunatus, and the wheatgerm agglutinin specific for N-acetyl-D-glucosamine inhibited spore germination, but were ineffective in modifying U. maydis cell growth. The galactose-specific lectin from the corn coleoptyle inhibited both germination and cell growth, while the lectin concanavalin A (mannose/glucose specific) activated spore germination and growth. Our results suggest that specific saccharide-containing receptors participate in regulating the growth and maturation of U. maydis spores.
Assuntos
Lectinas/farmacologia , Proteínas de Plantas/farmacologia , Ustilago/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Concanavalina A/farmacologia , Cinética , Lectinas de Plantas , Esporos Fúngicos/citologia , Esporos Fúngicos/efeitos dos fármacos , Esporos Fúngicos/crescimento & desenvolvimento , Ustilago/citologia , Ustilago/crescimento & desenvolvimento , Zea maysRESUMO
Ustilago maydis is a plant pathogen fungus responsible for corn smut. It has a complex life cycle. In its saprophitic stage, it grows as haploid yeast cells, while in the invasive stage it grows as a mycelium formed by diploid cells. Thus, a correlation exists between genetic ploidy, pathogenicity and morphogenesis. Dimorphism can be modulated in vitro by changing environmental parameters such as pH. Studies with auxotrophic mutants have shown that polyamines play a central role in regulating dimorphism. Molecular biology approaches are being employed for the analysis of fundamental aspects of the biology of this fungus, such as mating type regulation, dimorphism or cell wall biogenesis.
Assuntos
Ustilago , Parede Celular/fisiologia , Parede Celular/ultraestrutura , Culinária , Metilação de DNA , DNA Fúngico/metabolismo , Indígenas Norte-Americanos , México , Mutação , Pesquisa , Ustilago/genética , Ustilago/crescimento & desenvolvimentoRESUMO
PCR was used to amplify fragments corresponding to CHS genes from Ustilago maydis, utilizing as primers oligonucleotides devised according to the conserved regions of fungal CHS genes. The PCR product was employed as a probe to screen a genomic library of the fungus. Two different CHS genes (Umchs1 and Umchs2) were thus identified in the positive clones recovered. Their sequence revealed high similarity with the CHS genes previously cloned from other fungi, especially in their central region. Alignment with the deduced protein sequences of all CHS genes reported up to date showed the existence of seven conserved domains. Transcripts from both genes were detected in the yeast and mycelial forms. In general, the transcripts from the Umchs1 gene appeared to be present at a higher level than the transcripts from the Umchs2 gene; the transcripts from both genes appeared to be more abundant in the mycelial form. Gene replacement of either gene and analysis of the resulting phenotype demonstrated that they are non-essential. Nevertheless, growth, chitin synthase activity levels, and chitin content of mycelial cells induced by cultivation in acidic media were all reduced in chs1 and chs2 mutants. However, mating, virulence and dimorphic behaviour were unaffected. Overall, the results indicate that the CHS1 and CHS2 genes encode products with redundant functions in U. maydis.