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1.
mBio ; 10(2)2019 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-30967462

RESUMO

The filamentous fungus Alternaria alternata is a common postharvest contaminant of food and feed, and some strains are plant pathogens. Many processes in A. alternata are triggered by light. Interestingly, blue light inhibits sporulation, and red light reverses the effect, suggesting interactions between light-sensing systems. The genome encodes a phytochrome (FphA), a white collar 1 (WC-1) orthologue (LreA), an opsin (NopA), and a cryptochrome (CryA) as putative photoreceptors. Here, we investigated the role of FphA and LreA and the interplay with the high-osmolarity glycerol (HOG) mitogen-activated protein (MAP) kinase pathway. We created loss-of function mutations for fphA, lreA, and hogA using CRISPR-Cas9 technology. Sporulation was reduced in all three mutant strains already in the dark, suggesting functions of the photoreceptors FphA and LreA independent of light perception. Germination of conidia was delayed in red, blue, green, and far-red light. We found that light induction of ccgA (clock-controlled gene in Neurospora crassa and light-induced gene in Aspergillus nidulans) and the catalase gene catA depended on FphA, LreA, and HogA. Light induction of ferA (a putative ferrochelatase gene) and bliC (bli-3, light regulated, unknown function) required LreA and HogA but not FphA. Blue- and green-light stimulation of alternariol formation depended on LreA. A lack of FphA or LreA led to enhanced resistance toward oxidative stress due to the upregulation of catalases and superoxide dismutases. Light activation of FphA resulted in increased phosphorylation and nuclear accumulation of HogA. Our results show that germination, sporulation, and secondary metabolism are light regulated in A. alternata with distinct and overlapping roles of blue- and red-light photosensors.IMPORTANCE Light controls many processes in filamentous fungi. The study of light regulation in a number of model organisms revealed an unexpected complexity. Although the molecular components for light sensing appear to be widely conserved in fungal genomes, the regulatory circuits and the sensitivity of certain species toward specific wavelengths seem different. In N. crassa, most light responses are triggered by blue light, whereas in A. nidulans, red light plays a dominant role. In Alternaria alternata, both blue and red light appear to be important. In A. alternata, photoreceptors control morphogenetic pathways, the homeostasis of reactive oxygen species, and the production of secondary metabolites. On the other hand, high-osmolarity sensing required FphA and LreA, indicating a sophisticated cross talk between light and stress signaling.


Assuntos
Alternaria/crescimento & desenvolvimento , Alternaria/efeitos da radiação , Luz , Fotorreceptores Microbianos/metabolismo , Fitocromo/metabolismo , Esporos Fúngicos/crescimento & desenvolvimento , Esporos Fúngicos/efeitos da radiação , Alternaria/genética , Alternaria/metabolismo , Regulação Fúngica da Expressão Gênica/efeitos da radiação , Técnicas de Inativação de Genes , Redes Reguladoras de Genes , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Transdução de Sinais , Esporos Fúngicos/genética , Esporos Fúngicos/metabolismo
2.
J Biotechnol ; 296: 7-13, 2019 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-30853634

RESUMO

Xylitol is a sugar alcohol that is used as a sweetener in food and confections. Industrially, xylitol is manufactured by chemical hydrogenation of d-xylose, which requires expensive separation and purification steps as well as high pressure and temperature. The microbial production of xylitol has been examined as an alternative to the chemical process. In this study, a xylitol over-producing strain is breeded by mutagenesis of a newly isolated yeast Candida tropicalis with a new mutation breeding system named atmospheric and room temperature plasma. The highest yield strain T31 was screened among more than 200 mutants with a xylitol yield of 0.61 g/g, which represents a yield increase of 22%. Furthermore, a two-stage dissolved oxygen supply strategy was used in a fermentation process resulting the maximum xylitol yield 0.79 g/g, which makes it a promising candidate for xylitol production. Further biochemical analysis indicating the relative gene expression and the enzyme activity of xylose reductase were higher in mutants than those in the original strain, which partly explained the high yield of xylitol. Thus, this study provides a new strategy to breed the over-producing strains for the xylitol industry.


Assuntos
Candida tropicalis/genética , Mutagênese/efeitos da radiação , Gases em Plasma , Xilitol/biossíntese , Aldeído Redutase/genética , Candida tropicalis/efeitos dos fármacos , Fermentação , Regulação Fúngica da Expressão Gênica/efeitos da radiação , Temperatura Ambiente , Xilitol/química , Xilose/química , Xilose/genética
3.
BMC Genomics ; 20(1): 67, 2019 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-30665350

RESUMO

BACKGROUND: The orange pigmentation of the agar cultures of many Fusarium species is due to the production of carotenoids, terpenoid pigments whose synthesis is stimulated by light. The genes of the carotenoid pathway and their regulation have been investigated in detail in Fusarium fujikuroi. In this and other Fusarium species, such as F. oxysporum, deep-pigmented mutants affected in the gene carS, which encodes a protein of the RING-finger family, overproduce carotenoids irrespective of light. The induction of carotenogenesis by light and its deregulation in carS mutants are achieved on the transcription of the structural genes of the pathway. We have carried out global RNA-seq transcriptomics analyses to investigate the relationship between the regulatory role of CarS and the control by light in these fungi. RESULTS: The absence of a functional carS gene or the illumination exert wide effects on the transcriptome of F. fujikuroi, with predominance of genes activated over repressed and a greater functional diversity in the case of genes induced by light. The number of the latter decreases drastically in a carS mutant (1.1% vs. 4.8% in the wild-type), indicating that the deregulation produced by the carS mutation affects the light response of many genes. Moreover, approximately 27% of the genes activated at least 2-fold by light or by the carS mutation are coincident, raising to 40% for an 8-fold activation threshold. As expected, the genes with the highest changes under both regulatory conditions include those involved in carotenoid metabolism. In addition, light and CarS strongly influence the expression of some genes associated with stress responses, including three genes with catalase domains, consistent with roles in the control of oxidative stress. The effects of the CarS mutation or light in the transcriptome of F. oxysporum were partially coincident with those of F. fujikuroi, indicating the conservation of the objectives of their regulatory mechanisms. CONCLUSIONS: The CarS RING finger protein down-regulates many genes whose expression is up-regulated by light in wild strains of the two investigated Fusarium species, indicating a regulatory interplay between the mechanism of action of the CarS protein and the control by light.


Assuntos
Proteínas Fúngicas/fisiologia , Fusarium/genética , Regulação Fúngica da Expressão Gênica/efeitos da radiação , Luz , Proteínas Fúngicas/genética , Fusarium/metabolismo , Fusarium/efeitos da radiação , Perfilação da Expressão Gênica , Mutação , Ativação Transcricional , Transcriptoma/efeitos da radiação
4.
Microbiol Res ; 217: 81-90, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30384911

RESUMO

Light and nutrients are crucial environmental factors influencing fungal sexual reproduction. Blue light induces simultaneous hyphal knot formation in Coprinopsis cinerea mycelia grown on low-glucose media but not in mycelia grown on high-glucose media. Many hyphal knots are visible in the arc near the edge of the colony one day after 15 min of blue light stimulation. These findings collectively suggest that blue light accelerates hyphal knot induction in nutrient-limited conditions. Transcriptome analysis revealed that gene expression after light exposure is divided into at least two major stages. In the first stage, genes coding for fasciclin (fas1), cyclopropane-fatty-acyl-phospholipid synthases (cfs1 and cfs2), and putative lipid exporter (nod1) are highly expressed after 1 h of light exposure in the mycelial region where the hyphal knot will be developed. These genes are upregulated by blue light and not influenced by glucose condition and mating. These results suggest that although some of the genes are critical for induction of the hyphal knots, they are not sufficient for hyphal knot development. In the second gene expression stage, genes encoding galectins (cgl1-3), farnesyl cysteine-carboxyl methyltransferases, mating pheromone-containing protein, nucleus protein (ich1), and laccase (lcc1) are specifically upregulated at 10-16 h after blue light exposure when the mycelia are cultivated on low-glucose media. These genes might be involved in the architecture of hyphal knots or signal transduction for further fruiting body development. These results contribute to the understanding of the effect of environmental factors on sexual reproduction in basidiomycetous fungi.


Assuntos
Coprinus/genética , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica/efeitos da radiação , Hifas/genética , Luz , Nutrientes/farmacologia , Coprinus/efeitos dos fármacos , Coprinus/crescimento & desenvolvimento , Coprinus/efeitos da radiação , Carpóforos/efeitos dos fármacos , Carpóforos/genética , Carpóforos/crescimento & desenvolvimento , Carpóforos/efeitos da radiação , Proteínas Fúngicas/genética , Galectinas/genética , Genes Fúngicos/efeitos dos fármacos , Genes Fúngicos/efeitos da radiação , Glucose/metabolismo , Hifas/efeitos dos fármacos , Hifas/crescimento & desenvolvimento , Hifas/efeitos da radiação , Lacase/genética , Proteínas de Membrana/genética , Metiltransferases/genética , Micélio/efeitos dos fármacos , Micélio/crescimento & desenvolvimento , Micélio/efeitos da radiação , Proteínas Nucleares/genética , Feromônios/genética
5.
mBio ; 9(4)2018 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-30065085

RESUMO

Optogenetic switches permit accurate control of gene expression upon light stimulation. These synthetic switches have become a powerful tool for gene regulation, allowing modulation of customized phenotypes, overcoming the obstacles of chemical inducers, and replacing their use by an inexpensive resource: light. In this work, we implemented FUN-LOV, an optogenetic switch based on the photon-regulated interaction of WC-1 and VVD, two LOV (light-oxygen-voltage) blue-light photoreceptors from the fungus Neurospora crassa When tested in yeast, FUN-LOV yields light-controlled gene expression with exquisite temporal resolution and a broad dynamic range of over 1,300-fold, as measured by a luciferase reporter. We also tested the FUN-LOV switch for heterologous protein expression in Saccharomyces cerevisiae, where Western blot analysis confirmed strong induction upon light stimulation, surpassing by 2.5 times the levels achieved with a classic GAL4/galactose chemical-inducible system. Additionally, we utilized FUN-LOV to control the ability of yeast cells to flocculate. Light-controlled expression of the flocculin-encoding gene FLO1, by the FUN-LOV switch, yielded flocculation in light (FIL), whereas the light-controlled expression of the corepressor TUP1 provided flocculation in darkness (FID). Altogether, the results reveal the potential of the FUN-LOV optogenetic switch to control two biotechnologically relevant phenotypes such as heterologous protein expression and flocculation, paving the road for the engineering of new yeast strains for industrial applications. Importantly, FUN-LOV's ability to accurately manipulate gene expression, with a high temporal dynamic range, can be exploited in the analysis of diverse biological processes in various organisms.IMPORTANCE Optogenetic switches are molecular devices which allow the control of different cellular processes by light, such as gene expression, providing a versatile alternative to chemical inducers. Here, we report a novel optogenetic switch (FUN-LOV) based on the LOV domain interaction of two blue-light photoreceptors (WC-1 and VVD) from the fungus N. crassa In yeast cells, FUN-LOV allowed tight regulation of gene expression, with low background in darkness and a highly dynamic and potent control by light. We used FUN-LOV to optogenetically manipulate, in yeast, two biotechnologically relevant phenotypes, heterologous protein expression and flocculation, resulting in strains with potential industrial applications. Importantly, FUN-LOV can be implemented in diverse biological platforms to orthogonally control a multitude of cellular processes.


Assuntos
Adesão Celular , Regulação Fúngica da Expressão Gênica/efeitos da radiação , Optogenética/métodos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiologia , Estimulação Luminosa , Saccharomyces cerevisiae/efeitos da radiação
6.
Yeast ; 35(9): 531-541, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29727488

RESUMO

Targeted induced gene expression for industrial fermentation processes in food and beverage production could fulfill future demands. To avoid metabolic burden and disturbances owing to the fermentation procedure, induced gene expression is necessary for combating stress, such as that caused by temperature shifts that occur during the transition from fermentation to maturation in the brewing process. The aim of this study was to target gene expression in industrial yeast using stress-responsive promoters and homologues of the selection marker SMR1. Self-cloning strains of the industrial brewing yeast Saccharomyces pastorianus TUM 34/70 were constructed to overexpress the alcohol acetyltransferase (ATF1) gene under the control of inducible promoters P SSA3, P HSP104 and P UBI4. Transcription analysis shows the highest induction after 72 h of shock situation for P HSP104 with 1.3-fold and P UBI4 with 2.2-fold. Further, at the end of shock situation the concentrations of ethyl acetate were 1.2- and 1.3-fold higher than the wild type for P HSP104 and P UBI4, respectively. In addition, the influence of the final temperature and temporal sequence of temperature shock to 4°C had a major impact on expression patterns. Therefore, these data show that temperature-induced gene expression of self-cloning industrial yeast could be an option for optimization of the beverage fermentation.


Assuntos
Regulação Fúngica da Expressão Gênica/efeitos da radiação , Engenharia Metabólica/métodos , Proteínas/metabolismo , Saccharomyces/enzimologia , Saccharomyces/efeitos da radiação , Ativação Transcricional/efeitos da radiação , Clonagem Molecular , Perfilação da Expressão Gênica , Microbiologia Industrial/métodos , Regiões Promotoras Genéticas , Proteínas/genética , Saccharomyces/genética , Saccharomyces/crescimento & desenvolvimento , Temperatura Ambiente
7.
Fungal Biol ; 122(6): 497-504, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29801794

RESUMO

Light regulates fungal gene transcription transiently leading to photoadaptation. In the ascomycete Neurospora crassa photoadaptation is mediated by interactions between a light-regulated transcription factor complex, the white collar complex, and the small photoreceptor VVD. Other proteins, like the RCO-1/RCM-1 repressor complex participate indirectly in photoadaptation. We show that RCO-3, a protein with high similarity to glucose transporters, is needed for photoadaptation. The mutation in rco-3 modifies the transcriptional response to light of several genes and leads to changes in photoadaptation without significantly changing the amount and regulation of WC-1. The mutation in rco-3, however, does not modify the capacity of the circadian clock to be reset by light. Our results add support to the proposal that there is a connection between glucose sensing and light regulation in Neurospora and that the fungus integrates different environmental signals to regulate transcription.


Assuntos
Adaptação Fisiológica/genética , Proteínas Fúngicas/fisiologia , Proteínas Facilitadoras de Transporte de Glucose/fisiologia , Glucose/metabolismo , Neurospora crassa/efeitos da radiação , Relógios Circadianos/genética , Relógios Circadianos/fisiologia , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica/efeitos da radiação , Genes Fúngicos/efeitos da radiação , Proteínas Facilitadoras de Transporte de Glucose/genética , Luz , Mutação , Neurospora crassa/genética , Neurospora crassa/fisiologia , Fatores de Transcrição/genética , Transcrição Genética
8.
J Photochem Photobiol B ; 182: 100-107, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29656218

RESUMO

Hypocrellin A (HA) is a major bioactive perylenequinone from the fruiting body of Shiraia bambusicola used for the treatment of skin diseases and developed as a photodynamic therapy (PDT) agent against cancers and viruses. The mycelial culture of S. bambusicola under dark is a biotechnological alternative for HA production but with low yield. In this study, light and dark conditions were investigated to develop effective elicitation on HA production in the cultures. Our results showed the constant light at 200 lx stimulated HA production without any growth retardation of mycelia. A light/dark shift (24: 24 h) not only increased HA content in mycelia by 65%, but stimulated HA release into the medium with the highest total HA production 181.67 mg/L on day 8, about 73% increase over the dark control. Moreover, light/dark shifting induced the formation of smaller and more compact fungal pellets, suggesting a new effective strategy for large-scale production of HA in mycelium cultures. The light/dark shift up-regulated the expression levels of two reactive oxygen species (ROS) related genes including superoxide-generating NADPH oxidase (Nox) and cytochrome c peroxidase (CCP), and induced the generation of ROS. With the treatment of vitamin C, we found that ROS was involved in the up-regulated expression of key biosynthetical genes for hypocrellins and improved HA production. These results provide a basis for understanding the influence of light/dark shift on fungal metabolism and the application of a novel strategy for enhancing HA production in submerged Shiraia cultures.


Assuntos
Ascomicetos/química , Regulação Fúngica da Expressão Gênica/efeitos da radiação , Microbiologia Industrial/métodos , Luz , Perileno/análogos & derivados , Quinonas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Ascomicetos/metabolismo , Ascomicetos/efeitos da radiação , Micélio/crescimento & desenvolvimento , Micélio/metabolismo , Micélio/efeitos da radiação , Perileno/química , Perileno/metabolismo , Fotoperíodo , Quinonas/química , Reação em Cadeia da Polimerase em Tempo Real
9.
Appl Microbiol Biotechnol ; 102(9): 3849-3863, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29569180

RESUMO

Regulatable promoters are important genetic tools, particularly for assigning function to essential and redundant genes. They can also be used to control the expression of enzymes that influence metabolic flux or protein secretion, thereby optimizing product yield in bioindustry. This review will focus on regulatable systems for use in filamentous fungi, an important group of organisms whose members include key research models, devastating pathogens of plants and animals, and exploitable cell factories. Though we will begin by cataloging those promoters that are controlled by nutritional or chemical means, our primary focus will rest on those who can be controlled by a literal flip-of-the-switch: promoters of light-regulated genes. The vvd promoter of Neurospora will first serve as a paradigm for how light-driven systems can provide tight, robust, tunable, and temporal control of either autologous or heterologous fungal proteins. We will then discuss a theoretical approach to, and practical considerations for, the development of such promoters in other species. To this end, we have compiled genes from six previously published light-regulated transcriptomic studies to guide the search for suitable photoregulatable promoters in your fungus of interest.


Assuntos
Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica/efeitos da radiação , Genes Fúngicos/genética , Luz , Neurospora crassa/genética , Neurospora crassa/efeitos da radiação , Regiões Promotoras Genéticas/genética
10.
Biotechnol Bioeng ; 115(5): 1321-1330, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29315481

RESUMO

Conflict between cell growth and product accumulation is frequently encountered in biosynthesis of secondary metabolites. Herein, a temperature-dependent dynamic control strategy was developed by modifying the GAL regulation system to facilitate two-stage fermentation in yeast. A temperature-sensitive Gal4 mutant Gal4M9 was created by directed evolution, and used as a protein switch in ΔGAL80 yeast. After EGFP-reported validation of its temperature-responsive induction capability, the sensitivity and stringency of this system in multi-gene pathway regulation was tested, using lycopene as an example product. When Gal4M9 was used to control the expression of PGAL -driven pathway genes, growth and production was successfully decoupled upon temperature shift during fermentation, accumulating 44% higher biomass and 177% more lycopene than the control strain with wild-type Gal4. This is the first example of adopting temperature as an input signal for metabolic pathway regulation in yeast cell factories.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação Fúngica da Expressão Gênica/efeitos da radiação , Engenharia Metabólica/métodos , Metabolismo/efeitos da radiação , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Anti-Inflamatórios/metabolismo , Proteínas de Ligação a DNA/genética , Licopeno/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Temperatura Ambiente , Fatores de Transcrição/genética
11.
Res Microbiol ; 169(2): 78-89, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29203212

RESUMO

Stimulation by light of carotenoid biosynthesis in the mycelia of the fungus Neurospora crassa starts with transient transcriptional induction of the structural genes of the pathway triggered by the White Collar photoreceptor complex. Most studies on this process were carried out under standard growth conditions, but photoinduced carotenoid accumulation is more efficient if the fungus is incubated at low temperatures, from 6 to 12 °C. We have investigated the transcriptional photoresponse at 8 °C of the genes for proteins that participate in the carotenoid pathway. Exposure to light pulses of different light intensities revealed higher sensitivity if the mycelia were subsequently incubated at 8 °C compared to 30 °C. Illumination of precooled mycelia resulted in delayed kinetics of mRNA accumulation for the structural genes, and high mRNA accumulation for a longer time. Additionally, after a light pulse, stronger reduction in mRNAs for carotenoid genes was observed at 30 °C compared to 8 °C. A similar pattern was found for mRNAs of the photoreceptor genes wc-1 and vvd, the latter involved in photoadaptation. These results suggest that the increased efficiency in carotenoid photoinduction at low temperature is due to the higher mRNA levels of the structural genes under these conditions.


Assuntos
Carotenoides/biossíntese , Neurospora crassa/metabolismo , Transcrição Genética , Temperatura Baixa , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica/efeitos da radiação , Luz , Neurospora crassa/genética , Neurospora crassa/efeitos da radiação , Transcrição Genética/efeitos da radiação
12.
Sci Rep ; 7(1): 17586, 2017 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-29242620

RESUMO

An isolated black yeast-like strain was obtained from radiation-polluted soil collected from Xinjiang province in northwest China. On the basis of ITS and LSU rDNA sequence analysis, in combination with the colony morphology and phenotypic properties, the isolated strain was revealed to represent a novel variety of Aureobasidium subglaciale, designated as A. subglaciale F134. Compared to other yeasts and bacteria, this isolate displayed superior resistance to gamma irradiation, UV light, and heavy metal ions. It was discovered that the resistance of the isolate was correlated with the stress protector trehalose. Through the overexpression of the trehalose-6-phosphate synthase gene tps1 and the deletion of acid trehalase gene ath1, the APT∆A double mutant exhibited a survival rate of 1% under 20 kGy of gamma-radiation, 2% survival rate at a UV dosage of 250 J/m2, and tolerance towards Pb2+ as high as 1500 mg/L, which was in agreement with the high accumulation of intracellular trehalose compared to the wild-type strain. Finally, the protective effects and the mechanism of trehalose accumulation in A. subglaciale F134 were investigated, revealing a significant activation of the expression of many of the stress tolerance genes, offering new perspectives on the adaptations of radioresistant microorganisms.


Assuntos
Ascomicetos/efeitos dos fármacos , Ascomicetos/efeitos da radiação , Metais Pesados/toxicidade , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/efeitos da radiação , Trealose/metabolismo , Adaptação Fisiológica/efeitos dos fármacos , Adaptação Fisiológica/efeitos da radiação , Ascomicetos/metabolismo , Ascomicetos/fisiologia , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica/efeitos da radiação , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/efeitos da radiação , Filogenia , Tolerância a Radiação/efeitos dos fármacos , Poluentes do Solo/toxicidade
13.
Sci Rep ; 7(1): 17346, 2017 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-29229981

RESUMO

Cellulase production in the model cellulolytic fungus Trichoderma reesei is subject to a variety of environmental and physiological conditions involving an intricate regulatory network with multiple transcription factors. Here, we identified the mating type locus protein MAT1-2-1 as an interacting partner for the key transcriptional activator Xyr1 of T. reesei cellulase genes. Yeast two-hybrid and GST pulldown analyses revealed that MAT1-2-1 directly interacted with the putative transcription activation domain (AD, 767~940 aa) and the middle homology region (MHR2, 314~632 aa) of Xyr1. Disruption of the mat1-2-1 gene compromised the induced expression of cellulase genes with Avicel in response to light or with lactose. Chromatin immunoprecipitation (ChIP) demonstrated that MAT1-2-1 was recruited to the cbh1 (cellobiohydrolase 1-encoding) gene promoter in a Xyr1-dependent manner. These results strongly support an important role of MAT1-2-1 as a physiological cofactor of Xyr1, and suggest that MAT1-2-1 represents another regulatory node that integrates the light response with carbon source signaling to fine tune cellulase gene transcription.


Assuntos
Carbono/metabolismo , Celulase/metabolismo , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica/efeitos da radiação , Luz , Domínios e Motivos de Interação entre Proteínas/efeitos da radiação , Trichoderma/metabolismo , Celulase/genética , Imunoprecipitação da Cromatina , Proteínas Fúngicas/genética , Regiões Promotoras Genéticas , Trichoderma/genética , Trichoderma/efeitos da radiação
14.
PLoS One ; 12(8): e0182530, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28809958

RESUMO

Changing light conditions, caused by the rotation of earth resulting in day and night or growth on the surface or within a substrate, result in considerably altered physiological processes in fungi. For the biotechnological workhorse Trichoderma reesei, regulation of glycoside hydrolase gene expression, especially cellulase expression was shown to be a target of light dependent gene regulation. Analysis of regulatory targets of the carbon catabolite repressor CRE1 under cellulase inducing conditions revealed a secondary metabolite cluster to be differentially regulated in light and darkness and by photoreceptors. We found that this cluster is involved in production of trichodimerol and that the two polyketide synthases of the cluster are essential for biosynthesis of dihydrotrichotetronine (syn. bislongiquinolide or bisorbibutenolide). Additionally, an indirect influence on production of the peptaibol antibiotic paracelsin was observed. The two polyketide synthetase genes as well as the monooxygenase gene of the cluster were found to be connected at the level of transcription in a positive feedback cycle in darkness, but negative feedback in light, indicating a cellular sensing and response mechanism for the products of these enzymes. The transcription factor TR_102497/YPR2 residing within the cluster regulates the cluster genes in a light dependent manner. Additionally, an interrelationship of this cluster with regulation of cellulase gene expression was detected. Hence the regulatory connection between primary and secondary metabolism appears more widespread than previously assumed, indicating a sophisticated distribution of resources either to degradation of substrate (feed) or to antagonism of competitors (fight), which is influenced by light.


Assuntos
Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica/efeitos da radiação , Luz , Trichoderma/metabolismo , Trichoderma/efeitos da radiação , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Trichoderma/genética
15.
Appl Environ Microbiol ; 83(17)2017 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-28667107

RESUMO

The plant-pathogenic leotiomycete Botrytis cinerea is known for the strict regulation of its asexual differentiation programs by environmental light conditions. Sclerotia are formed in constant darkness; black/near-UV (NUV) light induces conidiation; and blue light represses both differentiation programs. Sensing of black/NUV light is attributed to proteins of the cryptochrome/photolyase family (CPF). To elucidate the molecular basis of the photoinduction of conidiation, we functionally characterized the two CPF proteins encoded in the genome of B. cinerea as putative positive-acting components. B. cinerea CRY1 (BcCRY1), a cyclobutane pyrimidine dimer (CPD) photolyase, acts as the major enzyme of light-driven DNA repair (photoreactivation) and has no obvious role in signaling. In contrast, BcCRY2, belonging to the cry-DASH proteins, is dispensable for photorepair but performs regulatory functions by repressing conidiation in white and especially black/NUV light. The transcription of bccry1 and bccry2 is induced by light in a White Collar complex (WCC)-dependent manner, but neither light nor the WCC is essential for the repression of conidiation through BcCRY2 when bccry2 is constitutively expressed. Further, BcCRY2 affects the transcript levels of both WCC-induced and WCC-repressed genes, suggesting a signaling function downstream of the WCC. Since both CPF proteins are dispensable for photoinduction by black/NUV light, the origin of this effect remains elusive and may be connected to a yet unknown UV-light-responsive system.IMPORTANCEBotrytis cinerea is an economically important plant pathogen that causes gray mold diseases in a wide variety of plant species, including high-value crops and ornamental flowers. The spread of disease in the field relies on the formation of conidia, a process that is regulated by different light qualities. While this feature has been known for a long time, we are just starting to understand the underlying molecular mechanisms. Conidiation in B. cinerea is induced by black/near-UV light, whose sensing is attributed to the action of cryptochrome/photolyase family (CPF) proteins. Here we report on the distinct functions of two CPF proteins in the photoresponse of B. cinerea While BcCRY1 acts as the major photolyase in photoprotection, BcCRY2 acts as a cryptochrome with a signaling function in regulating photomorphogenesis (repression of conidiation).


Assuntos
Botrytis/enzimologia , Botrytis/efeitos da radiação , Criptocromos/metabolismo , Reparo do DNA/efeitos da radiação , Desoxirribodipirimidina Fotoliase/metabolismo , Proteínas Fúngicas/metabolismo , Botrytis/crescimento & desenvolvimento , Botrytis/metabolismo , Criptocromos/genética , Desoxirribodipirimidina Fotoliase/genética , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica/efeitos da radiação , Luz , Esporos Fúngicos/enzimologia , Esporos Fúngicos/crescimento & desenvolvimento , Esporos Fúngicos/metabolismo , Esporos Fúngicos/efeitos da radiação
16.
Autophagy ; 13(8): 1318-1330, 2017 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-28594263

RESUMO

Magnaporthe oryzae, the ascomycete fungus that causes rice blast disease, initiates conidiation in response to light when grown on Prune-Agar medium containing both carbon and nitrogen sources. Macroautophagy/autophagy was shown to be essential for M. oryzae conidiation and induced specifically upon exposure to light but is undetectable in the dark. Therefore, it is inferred that autophagy is naturally induced by light, rather than by starvation during M. oryzae conidiation. However, the signaling pathway(s) involved in such phototropic induction of autophagy remains unknown. We identified an M. oryzae ortholog of GCN5 (MGG_03677), encoding a histone acetyltransferase (HAT) that negatively regulates light- and nitrogen-starvation-induced autophagy, by acetylating the autophagy protein Atg7. Furthermore, we unveiled novel regulatory mechanisms on Gcn5 at both transcriptional and post-translational levels, governing its function associated with the unique phototropic response of autophagy in this pathogenic fungus. Thus, our study depicts a signaling network and regulatory mechanism underlying the autophagy induction by important environmental clues such as light and nutrients.


Assuntos
Autofagia , Biocatálise , Proteínas Fúngicas/metabolismo , Magnaporthe/citologia , Magnaporthe/metabolismo , Processos Fototróficos , Acetilação , Autofagia/efeitos da radiação , Regulação Fúngica da Expressão Gênica/efeitos da radiação , Genes Fúngicos , Luz , Magnaporthe/genética , Magnaporthe/efeitos da radiação , Processos Fototróficos/efeitos da radiação , Ligação Proteica , Processamento de Proteína Pós-Traducional/efeitos da radiação , Esporos Fúngicos/metabolismo , Esporos Fúngicos/efeitos da radiação , Transcrição Genética/efeitos da radiação
17.
World J Microbiol Biotechnol ; 33(5): 99, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28429279

RESUMO

Oleaginous yeasts are considered a promising alternative lipid source for biodiesel fuel production. In this study, we attempted to improve the lipid productivity of the oleaginous yeast Rhodosporidium toruloides through UV irradiation mutagenesis and selection based on ethanol and H2O2 tolerance or cerulenin, a fatty acid synthetase inhibitor. Glucose consumption, cell growth, and lipid production of mutants were evaluated. The transcription level of genes involved in lipid production was also evaluated in mutants. The ethanol and H2O2 tolerant strain 8766 2-31M and the cerulenin resistant strain 8766 3-11C were generated by UV mutagenesis. The 8766 2-31M mutant showed a higher lipid production rate, and the 8766 3-11C mutant produced a larger amount of lipid and had a higher lipid production rate than the wild type strain. Transcriptional analysis revealed that, similar to the wild type strain, the ACL1 and GND1 genes were expressed at significantly low levels, whereas IDP1 and ME1 were highly expressed. In conclusion, lipid productivity in the oleaginous yeast R. toruloides was successfully improved via UV mutagenesis and selection. The study also identified target genes for improving lipid productivity through gene recombination.


Assuntos
Basidiomycota/genética , Metabolismo dos Lipídeos/efeitos da radiação , Mutagênese , Basidiomycota/efeitos dos fármacos , Basidiomycota/crescimento & desenvolvimento , Tolerância a Medicamentos , Etanol/farmacologia , Proteínas Fúngicas/efeitos da radiação , Regulação Fúngica da Expressão Gênica/efeitos da radiação , Peróxido de Hidrogênio/farmacologia , Transcriptoma
18.
J Biotechnol ; 258: 117-125, 2017 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-28455204

RESUMO

The manipulation of cellular function, such as the regulation of gene expression, is of great interest to many biotechnological applications and often achieved by the addition of small effector molecules. By combining effector molecules with photolabile protecting groups that mask their biological activity until they are activated by light, precise, yet minimally invasive, photocontrol is enabled. However, applications of this trendsetting technology are limited by the small number of established caged compound-based expression systems. Supported by computational chemistry, we used the versatile photolabile chelator DMNP-EDTA, long-established in neurobiology for photolytic Ca2+ release, to control Cu2+ release upon specific UV-A irradiation. This permits light-mediated control over the widely used Cu2+-inducible pCUP1 promoter from S. cerevisiae and thus constitutes the first example of a caged metal ion to regulate recombinant gene expression. We screened our novel DMNP-EDTA-Cu system for best induction time and expression level of eYFP with a high-throughput online monitoring system equipped with an LED array for individual illumination of every single well. Thereby, we realized a minimally invasive, easy-to-control, parallel and automated optical expression regulation via caged Cu2+ allowing temporal and quantitative control as a beneficial alternative to conventional induction via pipetting CuCl2 as effector molecule.


Assuntos
Cobre/metabolismo , Regulação Fúngica da Expressão Gênica/efeitos da radiação , Optogenética/métodos , Saccharomyces cerevisiae/efeitos da radiação , Cálcio/metabolismo , Quelantes/química , Quelantes/metabolismo , Cobre/química , Ácido Edético/análogos & derivados , Ácido Edético/química , Ácido Edético/metabolismo , Regulação Fúngica da Expressão Gênica/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
19.
Curr Genet ; 63(5): 931-949, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28382431

RESUMO

Botrytis cinerea is a plant pathogenic fungus with a broad host range. Due to its rapid growth and reproduction by asexual spores (conidia), which increases the inoculum pressure, the fungus is a serious problem in different fields of agriculture. The formation of the conidia is promoted by light, whereas the formation of sclerotia as survival structures occurs in its absence. Based on this observation, putative transcription factors (TFs) whose expression is induced upon light exposure have been considered as candidates for activating conidiation and/or repressing sclerotial development. Previous studies reported on the identification of six light-responsive TFs (LTFs), and two of them have been confirmed as crucial developmental regulators: BcLTF2 is the positive regulator of conidiation, whose expression is negatively regulated by BcLTF1. Here, the functional characterization of the four remaining LTFs is reported. BcLTF3 has a dual function, as it represses conidiophore development by repressing bcltf2 in light and darkness, and is moreover essential for conidiogenesis. In bcltf3 deletion mutants conidium initials grow out to hyphae, which develop secondary conidiophores. In contrast, no obvious functions could be assigned to BcLTF4, BcLTF5 and BcLTF6 in these experiments. BcREG1, previously reported to be required for virulence and conidiogenesis, has been re-identified as light-responsive transcriptional regulator. Studies with bcreg1 overexpression strains indicated that BcREG1 differentially affects conidiation by acting as a repressor of BcLTF2-induced conidiation in the light and as an activator of a BcLTF2-independent conidiation program in the dark.


Assuntos
Botrytis/fisiologia , Botrytis/efeitos da radiação , Regulação Fúngica da Expressão Gênica/efeitos da radiação , Luz , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Botrytis/classificação , Núcleo Celular/genética , Núcleo Celular/metabolismo , Biologia Computacional/métodos , Mutação , Fenótipo , Filogenia , Fatores de Transcrição/química , Fatores de Transcrição/genética , Virulência/genética
20.
Sci Rep ; 7: 44790, 2017 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-28322269

RESUMO

Light is an environmental signal perceived by most eukaryotic organisms and that can have major impacts on their growth and development. The MadC protein in the fungus Phycomyces blakesleeanus (Mucoromycotina) has been postulated to form part of the photosensory input for phototropism of the fruiting body sporangiophores, but the madC gene has remained unidentified since the 1960s when madC mutants were first isolated. In this study the madC gene was identified by positional cloning. All madC mutant strains contain loss-of-function point mutations within a gene predicted to encode a GTPase activating protein (GAP) for Ras. The madC gene complements the Saccharomyces cerevisiae Ras-GAP ira1 mutant and the encoded MadC protein interacts with P. blakesleeanus Ras homologs in yeast two-hybrid assays, indicating that MadC is a regulator of Ras signaling. Deletion of the homolog in the filamentous ascomycete Neurospora crassa affects the circadian clock output, yielding a pattern of asexual conidiation similar to a ras-1 mutant that is used in circadian studies in N. crassa. Thus, MadC is unlikely to be a photosensor, yet is a fundamental link in the photoresponses from blue light perceived by the conserved White Collar complex with Ras signaling in two distantly-related filamentous fungal species.


Assuntos
Ritmo Circadiano/fisiologia , Fotobiologia , Fototropismo/fisiologia , Phycomyces/metabolismo , Phycomyces/fisiologia , Proteínas ras/metabolismo , Alelos , Sequência de Bases , Mapeamento Cromossômico , Ritmo Circadiano/efeitos da radiação , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica/efeitos da radiação , Genes Fúngicos , Teste de Complementação Genética , Luz , Mutação com Perda de Função/genética , Fenótipo , Fototropismo/efeitos da radiação , Phycomyces/genética , Phycomyces/efeitos da radiação , Homologia de Sequência do Ácido Nucleico , Transdução de Sinais/efeitos da radiação , Transcrição Genética/efeitos da radiação
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