RESUMO
For the industrial-scale production of useful enzymes by microorganisms, technological development is required for overcoming a technical bottleneck represented by poor efficiency in the induction of enzyme gene expression and secretion. In this study, we evaluated the potential of a non-thermal atmospheric pressure plasma jet to improve the production efficiency of cellulolytic enzymes in Neurospora crassa, a filamentous fungus. The total activity of cellulolytic enzymes and protein concentration were significantly increased (1.1~1.2 times) in media containing Avicel 24-72 h after 2 and 5 min of plasma treatment. The mRNA levels of four cellulolytic enzymes in fungal hyphae grown in media with Avicel were significantly increased (1.3~17 times) 2-4 h after a 5 min of plasma treatment. The levels of intracellular NO and Ca2+ were increased in plasma-treated fungal hyphae grown in Avicel media after 48 h, and the removal of intracellular NO decreased the activity of cellulolytic enzymes in media and the level of vesicles in fungal hyphae. Our data suggest that plasma treatment can promote the transcription and secretion of cellulolytic enzymes into the culture media in the presence of Avicel (induction condition) by enhancing the intracellular level of NO and Ca2+.
Assuntos
Celulase , Neurospora crassa , Celulase/metabolismo , Celulose/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Neurospora crassa/genéticaRESUMO
BACKGROUND: During the disease cycle, plant pathogenic fungi exhibit a morphological transition between hyphal growth (the phase of active infection) and the production of long-term survival structures that remain dormant during "overwintering." Verticillium dahliae is a major plant pathogen that produces heavily melanized microsclerotia (MS) that survive in the soil for 14 or more years. These MS are multicellular structures produced during the necrotrophic phase of the disease cycle. Polyketide synthases (PKSs) are responsible for catalyzing production of many secondary metabolites including melanin. While MS contribute to long-term survival, hyphal growth is key for infection and virulence, but the signaling mechanisms by which the pathogen maintains hyphal growth are unclear. RESULTS: We analyzed the VdPKSs that contain at least one conserved domain potentially involved in secondary metabolism (SM), and screened the effect of VdPKS deletions in the virulent strain AT13. Among the five VdPKSs whose deletion affected virulence on cotton, we found that VdPKS9 acted epistatically to the VdPKS1-associated melanin pathway to promote hyphal growth. The decreased hyphal growth in VdPKS9 mutants was accompanied by the up-regulation of melanin biosynthesis and MS formation. Overexpression of VdPKS9 transformed melanized hyphal-type (MH-type) into the albinistic hyaline hyphal-type (AH-type), and VdPKS9 was upregulated in the AH-type population, which also exhibited higher virulence than the MH-type. CONCLUSIONS: We show that VdPKS9 is a powerful negative regulator of both melanin biosynthesis and MS formation in V. dahliae. These findings provide insight into the mechanism of how plant pathogens promote their virulence by the maintenance of vegetative hyphal growth during infection and colonization of plant hosts, and may provide novel targets for the control of melanin-producing filamentous fungi.
Assuntos
Policetídeo Sintases , Verticillium , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Melaninas/metabolismo , Policetídeo Sintases/genética , Policetídeo Sintases/metabolismo , Metabolismo Secundário , Verticillium/metabolismo , VirulênciaRESUMO
Understanding the adverse effects of genotoxic chemicals and identifying them effectively from non-genotoxic chemicals are of great worldwide concerns. Here, Saccharomyces cerevisiae (yeast) genome-wide single-gene knockout screening approach was conducted to assess two genotoxic chemicals (4-nitroquinoline-1-oxide (4-NQO) and formaldehyde (FA)) and environmental pollutant dichloroacetic acid (DCA, genotoxicity is controversial). DNA repair was significant enriched in the gene ontology (GO) biology process (BP) terms and KEGG pathways when exposed to low concentrations of 4-NQO and FA. Higher concentrations of 4-NQO and FA influenced some RNA metabolic and biosynthesis pathways. Moreover, replication and repair associated pathways were top ranked KEGG pathways with high fold-change for low concentrations of 4-NQO and FA. The similar gene profiles perturbed by DCA with three test concentrations identified, the common GO BP terms associated with aromatic amino acid family biosynthetic process and ubiquitin-dependent protein catabolic process via the multivesicular body sorting pathway. DCA has no obvious genotoxicity as there was no enriched DNA damage and repair pathways and fold-change of replication and repair KEGG pathways were very low. Five genes (RAD18, RAD59, MUS81, MMS4, and BEM4) could serve as candidate genes for genotoxic chemicals. Overall, the yeast functional genomic profiling showed great performance for assessing the signatures and potential molecular mechanisms of genotoxic chemicals.
Assuntos
Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Testes de Mutagenicidade/métodos , Saccharomyces cerevisiae/efeitos dos fármacos , 4-Nitroquinolina-1-Óxido/toxicidade , Dano ao DNA , Reparo do DNA/efeitos dos fármacos , DNA Fúngico , Ácido Dicloroacético/toxicidade , Formaldeído/toxicidade , Técnicas de Inativação de Genes/métodos , Mutagênicos , Saccharomyces cerevisiae/genéticaRESUMO
In fluctuating environments, switching between different growth strategies, such as those affecting cell size and proliferation, can be advantageous to an organism. Trade-offs arise, however. Mechanisms that aberrantly increase cell size or proliferation-such as mutations or chemicals that interfere with growth regulatory pathways-can also shorten lifespan. Here we report a natural example of how the interplay between growth and lifespan can be epigenetically controlled. We find that a highly conserved RNA-modifying enzyme, the pseudouridine synthase Pus4/TruB, can act as a prion, endowing yeast with greater proliferation rates at the cost of a shortened lifespan. Cells harboring the prion grow larger and exhibit altered protein synthesis. This epigenetic state, [BIG+] (better in growth), allows cells to heritably yet reversibly alter their translational program, leading to the differential synthesis of dozens of proteins, including many that regulate proliferation and aging. Our data reveal a new role for prion-based control of an RNA-modifying enzyme in driving heritable epigenetic states that transform cell growth and survival.
Cells make different proteins to perform different tasks. Each protein is a long chain of building blocks called amino acids that must fold into a particular shape before it can be useful. Some proteins can fold in more than one way, a normal form and a 'prion' form. Prions are unusual in that they can force normally folded proteins with the same amino acid sequence as them to refold into new prions. This means that a single prion can make many more that are inherited when cells divide. Some prions can cause disease, but others may be beneficial. Pus4 is a yeast protein that is typically involved in modifying ribonucleic acids, molecules that help translate genetic information into new proteins. Sometimes Pus4 can adopt a beneficial prion conformation called [BIG+]. When yeast cells have access to plenty of nutrients, [BIG+] helps them grow faster and larger, but this comes at the cost of a shorter lifespan. Garcia, Campbell et al. combined computational modeling and experiments in baker's yeast (Saccharomyces cerevisiae) to investigate the role of [BIG+]. They found that the prion accelerated protein production, leading to both faster growth and a shorter lifespan in these cells, even without any changes in gene sequence. Garcia, Campbell et al.'s findings explain the beneficial activity of prion proteins in baker's yeast cells. The results also describe how cells balance a tradeoff between growth and lifespan without any changes in the genome. This helps to highlight that genetics do not always explain the behaviors of cells, and further methods are needed to better understand cell biology.
Assuntos
Proliferação de Células , Transferases Intramoleculares/metabolismo , Meiose , Proteínas Priônicas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Crescimento Celular , Epigênese Genética , Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Transferases Intramoleculares/genética , Longevidade , Proteínas Priônicas/genética , Biossíntese de Proteínas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Fatores de TempoRESUMO
All living cells, including yeast cells, are challenged by different types of stresses in their environments and must cope with challenges such as heat, chemical stress, or oxidative damage. By reversibly adjusting the physiology while maintaining structural and genetic integrity, cells can achieve a competitive advantage and adapt environmental fluctuations. The yeast Saccharomyces cerevisiae has been extensively used as a model for study of stress responses due to the strong conservation of many essential cellular processes between yeast and human cells. We focused here on developing a tool to detect and quantify early responses using specific transcriptional responses. We analyzed the published transcriptional data on S. cerevisiae DBY strain responses to 10 different stresses in different time points. The principal component analysis (PCA) and the Pearson analysis were used to assess the stress response genes that are highly expressed in each individual stress condition. Except for these stress response genes, we also identified the reference genes in each stress condition, which would not be induced under stress condition and show stable transcriptional expression over time. We then tested our candidates experimentally in the CEN.PK strain. After data analysis, we identified two stress response genes (UBI4 and RRP) and two reference genes (MEX67 and SSY1) under heat shock (HS) condition. These genes were further verified by real-time PCR at mild (42°C), severe (46°C), to lethal temperature (50°C), respectively.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Regulação Fúngica da Expressão Gênica , Resposta ao Choque Térmico/genética , Humanos , Proteínas Nucleares , Proteínas de Transporte Nucleocitoplasmático , Estresse Oxidativo , Proteínas de Ligação a RNA , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
The present study examines the trend in distribution of Candida species and their antifungal resistance patterns in hospitals across Haryana, a North Indian state with poorly addressed epidemiology of fungal infections. In our collection of 228 Candida isolates, Candida albicans dominated in both high vaginal swab (HVS) and urine samples while Candida glabrata and Candida tropicalis were the second-highest non-albicans Candida species (NAC), respectively. Of note, in blood samples, C. tropicalis and C. albicans were present in equal numbers. All 228 isolates were subjected to antifungal susceptibility tests, whereby 51% of C. albicans recovered from HVS samples displayed fluconazole resistance. To understand its mechanistic basis, expression profiling of efflux pump genes CDR1, CDR2, MDR1 and azole drug target, ERG11 was performed in 20 randomly selected resistant isolates, wherein many isolates elicited higher expression. Further, ERG11 gene sequencing suggested that most of the isolates harbored mutations, which are not reported with azole resistance. However, one isolate, RPCA9 (MIC 64 µg/mL) harbored triple mutation (Y132C, F145L, A114V), wherein Y132 and F145 sites were previously implicated in azole resistance. Interestingly, one isolate, (RPCA61) having MIC > 128 µg/mL harbored a novel mutation, G129R. Of note, HVS isolates RPCA 21, RPCA 22, and RPCA 44 (MICs 64 to > 128 µg/mL) did not show any change in alteration in ERG11 or overexpression of efflux pump genes. Together, this study presents a first report of Candida infections in selected hospitals of Haryana State.
Assuntos
Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Candida albicans/isolamento & purificação , Farmacorresistência Fúngica/genética , Azóis/farmacologia , Candida/classificação , Candida/efeitos dos fármacos , Candida/genética , Candida/isolamento & purificação , Candida albicans/genética , Candidíase/epidemiologia , Candidíase/microbiologia , Sistema Enzimático do Citocromo P-450/genética , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Genes MDR/genética , Hospitais , Humanos , Índia/epidemiologia , Testes de Sensibilidade Microbiana , Mutação , Estudos RetrospectivosRESUMO
Using Saccharomyces cerevisiae as an experimental model, the potential toxicological effects of Fe3O4 nanoparticles (Fe3O4-NPs) were investigated following exposure to 0-600â¯mg/L for 24â¯h. Results revealed that cell proliferation was significantly inhibited by Fe3O4-NPs with an IC50 value of 326.66â¯mg/L. Mortality showed a concentration-dependent increase, and the highest concentration in this study (600â¯mg/L) resulted in 22.30% mortality. In addition, Effects on proliferation and mortality were accounted for Fe3O4-NPs rather than iron ion released from Fe3O4-NPs. Scanning and transmission electron microscope observation showed that Fe3O4-NPs extensively attached on the cell surfaces, causing cells to deform and shrink. Moreover, Fe3O4-NPs could be internalized in S. cerevisiae cells via endocytosis and then be distributed in cytoplasm and vesicles. The data of uptake kinetics demonstrated that the maximal accumulation (4.898â¯mg/g) was reached at 15â¯h. Besides, percentage of late apoptosis/necrosis was observably increased (pâ¯<â¯0.01) at 600â¯mg/L (15.80%), and the expression levels of apoptosis-related genes (SOD, Yca1 and Nuc1) were dramatically increased following exposure to Fe3O4-NPs for 24â¯h. As expected, mitochondrial transmembrane potential was significantly decreased (pâ¯<â¯0.01) at 50-600â¯mg/L, and biomarkers of oxidative stress (ROS, CAT and SOD) were also markedly changed following exposure. Altogether, the combined results so far indicated Fe3O4-NPs could induce S. cerevisiae cell apoptosis that mediated by mitochondrial impairment and oxidative stress.
Assuntos
Nanopartículas de Magnetita/efeitos adversos , Saccharomyces cerevisiae/efeitos dos fármacos , Animais , Materiais Biocompatíveis , Sobrevivência Celular/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Camundongos , Células RAW 264.7 , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Sclerotinia homoeocarpa causes dollar spot disease on turfgrass and is a serious problem on many species worldwide. Fludioxonil, a phenylpyrrole fungicide, is not currently registered for dollar spot control in China. In this study, the baseline sensitivity to fludioxonil was established using an in vitro assay for 105 isolates of S. homoeocarpa collected from 10 locations in different regions of China. Results indicate that the frequency distribution of effective concentration for 50% inhibition of mycelial growth (EC50) values of the S. homoeocarpa isolates was unimodal (Wâ¯=â¯0.9847, Pâ¯=â¯.2730). The mean EC50 value was 0.0020⯱â¯0.0006⯵g/ml with a range from 0.0003 to 0.0035⯵g/ml. A total of 7 fludioxonil-resistant mutants were obtained in laboratory, the mutants were stable in fludioxonil sensitivity after the 10th transfer, with resistance factor (RF) ranging from 4.320 to >13,901.4. The mutants showed a positive cross-resistance between fludioxonil and the dicarboximide fungicide iprodione, but not propiconazole, fluazinam, and thiophanate-methyl. When mycelial growth rate, pathogenicity and osmotic sensitivity were assessed, the mutants decreased in the fitness compared with their parental isolates. Sequence alignment of the histidine kinase gene Shos1 revealed a 13-bp fragment deletion only in one mutant, no mutations were observed on Shos1 in the rest resistant mutants.
Assuntos
Ascomicetos/efeitos dos fármacos , Ascomicetos/metabolismo , Dioxóis/farmacologia , Fungicidas Industriais/farmacologia , Pirróis/farmacologia , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Aminopiridinas/farmacologia , Ascomicetos/genética , China , Farmacorresistência Fúngica/genética , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica/genética , Hidantoínas/farmacologia , Mutação/genética , Tiofanato/farmacologia , Triazóis/farmacologiaRESUMO
Zinc is an essential cofactor for many proteins. A key mechanism of zinc homeostasis during deficiency is "zinc sparing" in which specific zinc-binding proteins are repressed to reduce the cellular requirement. In this report, we evaluated zinc sparing across the zinc proteome of Saccharomyces cerevisiae. The yeast zinc proteome of 582 known or potential zinc-binding proteins was identified using a bioinformatics analysis that combined global domain searches with local motif searches. Protein abundance was determined by mass spectrometry. In zinc-replete cells, we detected over 2500 proteins among which 229 were zinc proteins. Based on copy number estimates and binding stoichiometries, a replete cell contains â¼9 million zinc-binding sites on proteins. During zinc deficiency, many zinc proteins decreased in abundance and the zinc-binding requirement decreased to â¼5 million zinc atoms per cell. Many of these effects were due at least in part to changes in mRNA levels rather than simply protein degradation. Measurements of cellular zinc content showed that the level of zinc atoms per cell dropped from over 20 million in replete cells to only 1.7 million in deficient cells. These results confirmed the ability of replete cells to store excess zinc and suggested that the majority of zinc-binding sites on proteins in deficient cells are either unmetalated or mismetalated. Our analysis of two abundant zinc proteins, Fba1 aldolase and Met6 methionine synthetase, supported that hypothesis. Thus, we have discovered widespread zinc sparing mechanisms and obtained evidence of a high accumulation of zinc proteins that lack their cofactor during deficiency.
Assuntos
Proteínas de Transporte/metabolismo , Regulação Fúngica da Expressão Gênica , Proteoma/análise , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Zinco/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimentoRESUMO
The genus Fusarium contains some of the most studied and important species of plant pathogens that economically affect world agriculture and horticulture. Fusarium spp. are ubiquitous fungi widely distributed in soil, plants as well as in different organic substrates and are also considered as opportunistic human pathogens. The identification of specific enzymes essential to the metabolism of these fungi is expected to provide molecular targets to control the diseases they induce to their hosts. Through applications of traditional techniques of sequence homology comparison by similarity search and Markov modeling, this report describes the characterization of enzymatic functionalities associated to protein targets that could be considered for the control of root rots induced by Fusarium oxysporum. From the analysis of 318 F. graminearum enzymes, we retrieved 30 enzymes that are specific of F. oxysporum compared to 15 species of host plants. By comparing these 30 specific enzymes of F. oxysporum with the genome of Arabidopsis thaliana, Brassica rapa, Glycine max, Jatropha curcas and Ricinus communis, we found 7 key specific enzymes whose inhibition is expected to affect significantly the development of the fungus and 5 specific enzymes that were considered here to be secondary because they are inserted in pathways with alternative routes.
Assuntos
Proteínas Fúngicas/genética , Fusarium/enzimologia , Proteínas de Plantas/genética , Plantas/enzimologia , Fusarium/genética , Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Cadeias de Markov , Doenças das Plantas/microbiologia , Raízes de Plantas/genética , Plantas/genética , Plantas/microbiologia , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
Organisms regulate gene expression through changes in the activity of transcription factors (TFs). In yeast, the response of genes to changes in TF activity is generally assumed to be encoded in the promoter. To directly test this assumption, we chose 42 genes and, for each, replaced the promoter with a synthetic inducible promoter and measured how protein expression changes as a function of TF activity. Most genes exhibited gene-specific TF dose-response curves not due to differences in mRNA stability, translation, or protein stability. Instead, most genes have an intrinsic ability to buffer the effects of promoter activity. This can be encoded in the open reading frame and the 3' end of genes and can be implemented by both autoregulatory feedback and by titration of limiting trans regulators. We show experimentally and computationally that, when misexpression of a gene is deleterious, this buffering insulates cells from fitness defects due to misregulation.
Assuntos
Regulação Fúngica da Expressão Gênica , Regiões Promotoras Genéticas , Saccharomyces cerevisiae/genética , Sequência de Bases , Mecanismo Genético de Compensação de Dose , Retroalimentação Fisiológica , Genes Fúngicos , Homeostase , Modelos Genéticos , Fases de Leitura Aberta/genética , Ploidias , Fatores de Transcrição/metabolismoRESUMO
Candida albicans is an opportunistic pathogenic fungus which causes superficial and systemic infections in immunocompromised patients. It is important to characterize the roles of genes involved in its pathogenesis, virulence, and drug resistance. Several genetic manipulation toolkits have been developed for gene function research in C. albicans. Here, we describe efficient vector systems that allow economical and rapid C-terminal and N-terminal epitope-tagging, inducible and constitutive promoter replacements, and ectopic gene overexpression in C. albicans. These systems use modularized genetic elements (conventional and non-conventional selection markers, epitope tags and promoters) and universal primers. These advantages should greatly reduce laboratory work and costs of strain construction for C. albicans.
Assuntos
Candida albicans/genética , Epitopos/genética , Regulação Fúngica da Expressão Gênica , Engenharia Genética/métodos , Vetores Genéticos , Doxiciclina/farmacologia , Escherichia coli/genética , Proteínas Fúngicas/genética , Marcação de Genes , Engenharia Genética/economia , Humanos , Regiões Promotoras Genéticas , Transformação GenéticaRESUMO
Fungal immunomodulatory proteins (FIPs) have been identified from a series of fungi, especially in Ganoderma species. However, little is known about the FIPs from G. applanatum. In this study, two novel FIP genes, termed as FIP-gap1 and FIP-gap2, were cloned from G. applanatum, characterized and functionally expressed after codon optimization in Pichia pastoris GS115. Results showed that FIP-gap1 and FIP-gap2 comprised 342-bp encoding peptides of 113 amino acids, which shared a high homology with other Ganoderma FIPs. The yield of recombinant FIP-gap1 and FIP-gap2 increased significantly after codon optimization and reached 247.4 and 197.5 mg/L, respectively. Bioactivity assay in vitro revealed that both rFIP-gap1 and rFIP-gap2 could agglutinate mouse, sheep, and human red blood cells. Besides, rFIP-gap1 and rFIP-gap2 obviously stimulated the proliferation of mouse splenocytes and enhanced IL-2 and IFN-γ release. Cytotoxicity detection indicated that IC50 of rFIP-gap1 towards A549 and HeLa cancer cells were 29.89 and 8.34 µg/mL, respectively, whereas IC50 of rFIP-gap2 to the same cancer cells were 60.92 and 41.05 µg/mL, respectively. Taken together, novel FIP gaps were cloned and functionally expressed in P. pastoris, which can serve as feasible and stable resources of rFIP gaps for further studies and potential applications.
Assuntos
Códon/genética , Ganoderma/genética , Regulação Fúngica da Expressão Gênica/genética , Pichia/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Células A549 , Aglutinação/efeitos dos fármacos , Animais , Sobrevivência Celular/efeitos dos fármacos , Clonagem Molecular , Eritrócitos/efeitos dos fármacos , Proteínas Fúngicas/genética , Proteínas Fúngicas/farmacologia , Proteínas Fúngicas/toxicidade , Células HeLa , Humanos , Fatores Imunológicos/genética , Fatores Imunológicos/farmacologia , Fatores Imunológicos/toxicidade , Camundongos , Proteínas Recombinantes/toxicidadeRESUMO
Cordyceps sinensis has gained increasing attention due to its nutritional and medicinal properties. Herein, we employed label-free quantitative mass spectrometry to explore the proteome differences between naturally- and artificially-cultivated C. sinensis. A total of 22,829 peptides with confidence ≥95%, corresponding to 2541 protein groups were identified from the caterpillar bodies/stromata of 12 naturally- and artificially-cultivated samples of C. sinensis. Among them, 165 proteins showed significant differences between the samples of natural and artificial cultivation. These proteins were mainly involved in energy production/conversion, amino acid transport/metabolism, and transcription regulation. The proteomic results were confirmed by the identification of 4 significantly changed metabolites, thus, lysine, threonine, serine, and arginine via untargeted metabolomics. The change tendencies of these metabolites were partly in accordance with changes in abundance of the proteins, which was upstream of their synthetic pathways. In addition, the nutritional value in terms of the levels of nucleosides, nucleotides, and adenosine between the artificially- and naturally-cultivated samples was virtually same. These proteomic data will be useful for understanding the medicinal value of C. sinensis and serve as reference for its artificial cultivation. SIGNIFICANCE: C. sinensis is a precious and valued medicinal product, the current basic proteome dataset would provide useful information to understand its development/infection processes as well as help to artificially cultivate it. This work would also provide basic proteome profile for further study of C. sinensis.
Assuntos
Cordyceps/crescimento & desenvolvimento , Proteínas Fúngicas/biossíntese , Regulação Fúngica da Expressão Gênica , Proteoma/biossíntese , Proteômica , Transcrição GênicaRESUMO
The fungal genus Heterobasidion includes some of the most devastating conifer pathogens in the boreal forest region. In this study, we showed that the alphapartitivirus Heterobasidion partitivirus 13 from Heterobasidion annosum (HetPV13-an1) is the main causal agent of severe phenotypic debilitation in the host fungus. Based on RNA sequencing using isogenic virus-infected and cured fungal strains, HetPV13-an1 affected the transcription of 683 genes, of which 60% were downregulated and 40% upregulated. Alterations observed in carbohydrate and amino acid metabolism suggest that the virus causes a state of starvation, which is compensated for by alternative synthesis routes. We used dual cultures to transmit HetPV13-an1 into new strains of H. annosum and Heterobasidion parviporum The three strains of H. parviporum that acquired the virus showed noticeable growth reduction on rich culturing medium, while only two of six H. annosum isolates tested showed significant debilitation. Based on reverse transcription-quantitative PCR (RT-qPCR) analysis, the response toward HetPV13-an1 infection was somewhat different in H. annosum and H. parviporum We assessed the effects of HetPV13-an1 on the wood colonization efficacy of H. parviporum in a field experiment where 46 Norway spruce trees were inoculated with isogenic strains with or without the virus. The virus-infected H. parviporum strain showed considerably less growth within living trees than the isolate without HetPV13-an1, indicating that the virus also causes growth debilitation in natural substrates.IMPORTANCE A biocontrol method restricting the spread of Heterobasidion species would be highly beneficial to forestry, as these fungi are difficult to eradicate from diseased forest stands and cause approximate annual losses of 800 million in Europe. We used virus curing and reintroduction experiments and RNA sequencing to show that the alphapartitivirus HetPV13-an1 affects many basic cellular functions of the white rot wood decay fungus Heterobasidion annosum, which results in aberrant hyphal morphology and a low growth rate. Dual fungal cultures were used to introduce HetPV13-an1 into a new host species, Heterobasidion parviporum, and field experiments confirmed the capability of the virus to reduce the growth of H. parviporum in living spruce wood. Taken together, our results suggest that HetPV13-an1 shows potential for the development of a future biocontrol agent against Heterobasidion fungi.
Assuntos
Basidiomycota/crescimento & desenvolvimento , Basidiomycota/genética , Basidiomycota/virologia , Doenças das Plantas/microbiologia , Vírus de RNA/fisiologia , Atropina/metabolismo , Basidiomycota/patogenicidade , Agentes de Controle Biológico , Metabolismo dos Carboidratos , Ciclo Celular , Diazepam/metabolismo , Combinação de Medicamentos , Emodina/análogos & derivados , Emodina/metabolismo , Europa (Continente) , Florestas , Regulação Fúngica da Expressão Gênica , Genótipo , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/fisiologia , Metabolismo , Mitocôndrias/metabolismo , Micélio/genética , Micélio/crescimento & desenvolvimento , Micélio/virologia , Noruega , Fenótipo , Fenilpropanolamina/metabolismo , Picea/microbiologia , Doenças das Plantas/economia , Infecções por Vírus de RNA , Vírus de RNA/genética , RNA Viral/genética , RNA Viral/isolamento & purificação , Análise de Sequência de RNA , Tri-Iodotironina/metabolismoRESUMO
The notion of state for a system is prevalent in the quantitative sciences and refers to the minimal system summary sufficient to describe the time evolution of the system in a self-consistent manner. This is a prerequisite for a principled understanding of the inner workings of a system. Owing to the complexity of intracellular processes, experimental techniques that can retrieve a sufficient summary are beyond our reach. For the case of stochastic biomolecular reaction networks, we show how to convert the partial state information accessible by experimental techniques into a full system state using mathematical analysis together with a computational model. This is intimately related to the notion of conditional Markov processes and we introduce the posterior master equation and derive novel approximations to the corresponding infinite-dimensional posterior moment dynamics. We exemplify this state reconstruction approach using both in silico data and single-cell data from two gene expression systems in Saccharomyces cerevisiae, where we reconstruct the dynamic promoter and mRNA states from noisy protein abundance measurements.
Assuntos
Regulação Fúngica da Expressão Gênica/fisiologia , Modelos Biológicos , Regiões Promotoras Genéticas/fisiologia , RNA Fúngico/biossíntese , RNA Mensageiro/biossíntese , Saccharomyces cerevisiae/metabolismo , Cadeias de Markov , Saccharomyces cerevisiae/citologiaRESUMO
BACKGROUND: Cryptic unstable transcripts (CUTs) are a largely unexplored class of nuclear exosome degraded, non-coding RNAs in budding yeast. It is highly debated whether CUT transcription has a functional role in the cell or whether CUTs represent noise in the yeast transcriptome. We sought to ascertain the extent of conserved CUT expression across a variety of Saccharomyces yeast strains to further understand and characterize the nature of CUT expression. RESULTS: We sequenced the WT and rrp6Δ transcriptomes of three S.cerevisiae strains: S288c, Σ1278b, JAY291 and the S.paradoxus strain N17 and utilized a hidden Markov model to annotate CUTs in these four strains. Utilizing a four-way genomic alignment we identified a large population of CUTs with conserved syntenic expression across all four strains. By identifying configurations of gene-CUT pairs, where CUT expression originates from the gene 5' or 3' nucleosome free region, we observed distinct gene expression trends specific to these configurations which were most prevalent in the presence of conserved CUT expression. Divergent pairs correlate with higher expression of genes, and convergent pairs correlate with reduced gene expression. CONCLUSIONS: Our RNA-seq based method has greatly expanded upon previous CUT annotations in S.cerevisiae underscoring the extensive and pervasive nature of unstable transcription. Furthermore we provide the first assessment of conserved CUT expression in yeast and globally demonstrate possible modes of CUT-based regulation of gene expression.
Assuntos
Regulação Fúngica da Expressão Gênica , Estabilidade de RNA , RNA não Traduzido/genética , Saccharomyces/genética , Exossomos/genética , Genoma Fúngico , Cadeias de Markov , Nucleossomos/genética , RNA Fúngico/genética , Saccharomyces/classificação , Análise de Sequência de RNA , Sintenia , TranscriptomaRESUMO
The economy of protein production is central to cell physiology, being intimately linked with cell division rate and cell size. Attempts to model cellular physiology are limited by the scarcity of experimental data defining the molecular processes limiting protein expression. Here, we distinguish the relative contribution of gene transcription and protein translation to the slower proliferation of budding yeast producing excess levels of unneeded proteins. In contrast to widely held assumptions, rapidly growing cells are not universally limited by ribosome content. Rather, transcription dominates cost under some conditions (e.g., low phosphate), translation in others (e.g., low nitrogen), and both in other conditions (e.g., rich media). Furthermore, cells adapted to enforced protein production by becoming larger and increasing their endogenous protein levels, suggesting limited competition for common resources. We propose that rapidly growing cells do not exhaust their resources to maximize growth but maintain sufficient reserves to accommodate changing requirements.
Assuntos
Regulação Fúngica da Expressão Gênica/fisiologia , Biossíntese de Proteínas/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/fisiologia , Transcrição Gênica/fisiologiaRESUMO
Hidden Markov models (HMMs) have been extensively used to dissect the genome into functionally distinct regions using data such as RNA expression or DNA binding measurements. It is a challenge to disentangle processes occurring on complementary strands of the same genomic region. We present the double-stranded HMM (dsHMM), a model for the strand-specific analysis of genomic processes. We applied dsHMM to yeast using strand specific transcription data, nucleosome data, and protein binding data for a set of 11 factors associated with the regulation of transcription.The resulting annotation recovers the mRNA transcription cycle (initiation, elongation, termination) while correctly predicting strand-specificity and directionality of the transcription process. We find that pre-initiation complex formation is an essentially undirected process, giving rise to a large number of bidirectional promoters and to pervasive antisense transcription. Notably, 12% of all transcriptionally active positions showed simultaneous activity on both strands. Furthermore, dsHMM reveals that antisense transcription is specifically suppressed by Nrd1, a yeast termination factor.
Assuntos
DNA Fúngico/genética , DNA/genética , Regulação Fúngica da Expressão Gênica , Genoma Fúngico , Cadeias de Markov , Saccharomyces cerevisiae/genética , DNA/metabolismo , DNA Fúngico/metabolismo , Genômica , Nucleossomos/química , Nucleossomos/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
In ribosome biogenesis, a large fraction of ribosomes is used for producing ribosomal proteins themselves. Here, we applied simulation and experimentation to determine what fraction of ribosomes should be allocated for the synthesis of ribosomal proteins to optimize cellular economy for growth. We define the "r-fraction" as the fraction of mRNA of the ribosomal protein genes out of the total mRNA, and we simulated the effect of the r-fraction on the number of ribosomes. We then empirically measured the amount of protein and RNA in fission yeast cells cultured with high and low nitrogen sources. In the cells cultured with a low nitrogen source, the r-fraction decreased from 0.46 to 0.42 with a 40% reduction of rRNA, but the reduction of the total protein was smaller at 30%. These results indicate that the r-fraction is internally controlled to optimize the efficiency of protein synthesis at a limited cellular cost.