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1.
Nat Commun ; 12(1): 3487, 2021 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-34108468

RESUMO

Fusicoccadiene synthase from Phomopsis amygdali (PaFS) is a unique bifunctional terpenoid synthase that catalyzes the first two steps in the biosynthesis of the diterpene glycoside Fusicoccin A, a mediator of 14-3-3 protein interactions. The prenyltransferase domain of PaFS generates geranylgeranyl diphosphate, which the cyclase domain then utilizes to generate fusicoccadiene, the tricyclic hydrocarbon skeleton of Fusicoccin A. Here, we use cryo-electron microscopy to show that the structure of full-length PaFS consists of a central octameric core of prenyltransferase domains, with the eight cyclase domains radiating outward via flexible linker segments in variable splayed-out positions. Cryo-electron microscopy and chemical crosslinking experiments additionally show that compact conformations can be achieved in which cyclase domains are more closely associated with the prenyltransferase core. This structural analysis provides a framework for understanding substrate channeling, since most of the geranylgeranyl diphosphate generated by the prenyltransferase domains remains on the enzyme for cyclization to form fusicoccadiene.


Assuntos
Alquil e Aril Transferases/química , Diterpenos/metabolismo , Proteínas Fúngicas/química , Alquil e Aril Transferases/metabolismo , Ascomicetos/química , Ascomicetos/enzimologia , Catálise , Domínio Catalítico , Microscopia Crioeletrônica , Ciclização , Dimetilaliltranstransferase/química , Dimetilaliltranstransferase/metabolismo , Proteínas Fúngicas/metabolismo , Glicosídeos/biossíntese , Liases/química , Liases/metabolismo , Enzimas Multifuncionais , Fosfatos de Poli-Isoprenil/metabolismo , Conformação Proteica
2.
Biochim Biophys Acta Proteins Proteom ; 1869(8): 140662, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33887466

RESUMO

Scytalidium catalase is a homotetramer including heme d in each subunit. Its primary function is the dismutation of H2O2 to water and oxygen, but it is also able to oxidase various small organic compounds including catechol and phenol. The crystal structure of Scytalidium catalase reveals the presence of three linked channels providing access to the exterior like other catalases reported so far. The function of these channels has been extensively studied, revealing the possible routes for substrate flow and product release. In this report, we have focussed on the semi-conserved residue Val228, located near to the vinyl groups of the heme at the opening of the lateral channel. Its replacement with Ala, Ser, Gly, Cys, Phe and Ile were tested. We observed a significant decrease in catalytic efficiency in all mutants with the exception of a remarkable increase in oxidase activity when Val228 was mutated to either Ala, Gly or Ser. The reduced catalytic efficiencies are characterized in terms of the restriction of hydrogen peroxide as electron acceptor in the active centre resulting from the opening of lateral channel inlet by introducing the smaller side chain residues. On the other hand, the increased oxidase activity is explained by allowing the suitable electron donor to approach more closely to the heme. The crystal structures of V228C and V228I were determined at 1.41 and 1.47 Å resolution, respectively. The lateral channels of the V228C and V228I presented a broadly identical chain of arranged waters to that observed for wild-type enzyme.


Assuntos
Catalase/genética , Heme/química , Sordariales/enzimologia , Sordariales/genética , Ascomicetos/enzimologia , Ascomicetos/genética , Catalase/química , Catalase/metabolismo , Catálise , Domínio Catalítico , Heme/análogos & derivados , Peróxido de Hidrogênio/química , Modelos Moleculares , Sordariales/metabolismo
3.
Mol Plant Pathol ; 22(5): 580-601, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33742545

RESUMO

Podosphaera xanthii is the main causal agent of cucurbit powdery mildew and a limiting factor of crop productivity. The lifestyle of this fungus is determined by the development of specialized parasitic structures inside epidermal cells, termed haustoria, that are responsible for the acquisition of nutrients and the release of effectors. A typical function of fungal effectors is the manipulation of host immunity, for example the suppression of pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI). Chitin is a major component of fungal cell walls, and chitin oligosaccharides are well-known PAMP elicitors. In this work, we examined the role of PHEC27213, the most highly expressed, haustorium-specific effector candidate of P. xanthii. According to different computational predictions, the protein folding of PHEC27213 was similar to that of lytic polysaccharide monooxygenases (LPMOs) and included a conserved histidine brace; however, PHEC27213 had low sequence similarity with LPMO proteins and displayed a putative chitin-binding domain that was different from the canonical carbohydrate-binding module. Binding and enzymatic assays demonstrated that PHEC27213 was able to bind and catalyse colloidal chitin, as well as chitooligosaccharides, acting as an LPMO. Furthermore, RNAi silencing experiments showed the potential of this protein to prevent the activation of chitin-triggered immunity. Moreover, proteins with similar features were found in other haustorium-forming fungal pathogens. Our results suggest that this protein is a new fungal LPMO that catalyses chitooligosaccharides, thus contributing to the suppression of plant immunity during haustorium development. To our knowledge, this is the first mechanism identified in the haustorium to suppress chitin signalling.


Assuntos
Ascomicetos/enzimologia , Quitina/análogos & derivados , Quitina/imunologia , Cucurbita/microbiologia , Oxigenases de Função Mista/metabolismo , Doenças das Plantas/microbiologia , Ascomicetos/genética , Ascomicetos/fisiologia , Cucurbita/imunologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Oxigenases de Função Mista/genética , Modelos Moleculares , Simulação de Acoplamento Molecular , Padrões Moleculares Associados a Patógenos/imunologia , Doenças das Plantas/imunologia , Imunidade Vegetal , Transdução de Sinais
4.
J Nat Med ; 75(3): 434-447, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33683566

RESUMO

It has been proposed that biosyntheses of many natural products involve pericyclic reactions, including Diels-Alder (DA) reaction. However, only a small set of enzymes have been proposed to catalyze pericyclic reactions. Most surprisingly, there has been no formal identification of natural enzymes that can be defined to catalyze DA reactions (DAases), despite the wide application of the reaction in chemical syntheses of complex organic compounds. However, recent studies began to accumulate a growing body of evidence that supports the notion that enzymes that formally catalyze DA reactions, in fact exist. In this review, I will begin by describing a short history behind the discovery and characterization of macrophomate synthase, one of the earliest enzymes that was proposed to catalyze an intermolecular DA reaction during the biosynthesis of a substituted benzoic acid in a phytopathogenic fungus Macrophoma commelinae. Then, I will discuss representative enzymes that have been chemically authenticated to catalyze DA reactions, with emphasis on more recent discoveries of DAases involved mainly in fungal secondary metabolite biosynthesis except for one example from a marine streptomycete. The current success in identification of a series of DAases and enzymes that catalyze other pericyclic reactions owes to the combined efforts from both the experimental and theoretical approaches in discovering natural products. Such efforts typically involve identifying the chemical features derived from cycloaddition reactions, isolating the biosynthetic genes that encode enzymes that generate such chemical features and deciphering the reaction mechanisms for the enzyme-catalyzed pericyclic reactions.


Assuntos
Ascomicetos/enzimologia , Produtos Biológicos/química , Reação de Cicloadição , Complexos Multienzimáticos/química , Metabolismo Secundário
5.
PLoS Genet ; 17(3): e1009448, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33750960

RESUMO

DNA methylation is found throughout all domains of life, yet the extent and function of DNA methylation differ among eukaryotes. Strains of the plant pathogenic fungus Zymoseptoria tritici appeared to lack cytosine DNA methylation (5mC) because gene amplification followed by Repeat-Induced Point mutation (RIP) resulted in the inactivation of the dim2 DNA methyltransferase gene. 5mC is, however, present in closely related sister species. We demonstrate that inactivation of dim2 occurred recently as some Z. tritici isolates carry a functional dim2 gene. Moreover, we show that dim2 inactivation occurred by a different path than previously hypothesized. We mapped the genome-wide distribution of 5mC in strains with or without functional dim2 alleles. Presence of functional dim2 correlates with high levels of 5mC in transposable elements (TEs), suggesting a role in genome defense. We identified low levels of 5mC in strains carrying non-functional dim2 alleles, suggesting that 5mC is maintained over time, presumably by an active Dnmt5 DNA methyltransferase. Integration of a functional dim2 allele in strains with mutated dim2 restored normal 5mC levels, demonstrating de novo cytosine methylation activity of Dim2. To assess the importance of 5mC for genome evolution, we performed an evolution experiment, comparing genomes of strains with high levels of 5mC to genomes of strains lacking functional dim2. We found that presence of a functional dim2 allele alters nucleotide composition by promoting C to T transitions (C→T) specifically at CpA (CA) sites during mitosis, likely contributing to TE inactivation. Our results show that 5mC density at TEs is a polymorphic trait in Z. tritici populations that can impact genome evolution.


Assuntos
Ascomicetos/enzimologia , Ascomicetos/genética , DNA (Citosina-5-)-Metiltransferase 1/deficiência , Evolução Molecular , Taxa de Mutação , Mutação , 5-Metilcitosina/metabolismo , Alelos , Ascomicetos/classificação , Ascomicetos/isolamento & purificação , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Geografia , Mitose , Filogeografia , Locos de Características Quantitativas
6.
Food Chem ; 351: 129294, 2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-33640774

RESUMO

Cell wall polysaccharides in fruits act a pivotal role in their resistance to fungal invasion. Lasiodiplodia theobromae (Pat.) Griff. & Maubl. is a primary pathogenic fungus causing the spoilage of fresh longan fruit. In this study, the influences of L. theobromae inoculation on the disassembly of cell wall polysaccharides in pericarp of fresh longans and its association with L. theobromae-induced disease and softening development were investigated. In contrast to the control, samples with L. theobromae infection showed more severe disease development, lower firmness, lower amounts of cell wall materials, covalent-soluble pectin, ionic-soluble pectin, cellulose and hemicellulose, whereas higher value of water-soluble pectin, higher activities of cell wall polysaccharide-disassembling enzymes (cellulase, ß-galactosidase, polygalacturonase and pectinesterase). These findings revealed that cell wall polysaccharides disassembly induced by enzymatic manipulation was an essential pathway for L. theobromae to infect harvested longans, and thus led to the disease occurrence and fruit softening.


Assuntos
Ascomicetos/fisiologia , Parede Celular/metabolismo , Polissacarídeos/metabolismo , Sapindaceae/microbiologia , Ascomicetos/enzimologia , Celulase/metabolismo , Celulose/análise , Celulose/metabolismo , Armazenamento de Alimentos , Frutas/química , Frutas/metabolismo , Frutas/microbiologia , Pectinas/metabolismo , Doenças das Plantas/microbiologia , Poligalacturonase/metabolismo , Polissacarídeos/análise , Sapindaceae/metabolismo
7.
Appl Environ Microbiol ; 87(10)2021 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-33637576

RESUMO

More than 30,000 tons of menthol are produced every year as a flavor and fragrance compound or as a medical component. So far, only extraction from plant material and chemical synthesis are possible. An alternative approach for menthol production could be a biotechnological-chemical process with ideally only two conversion steps, starting from (+)-limonene, which is a side product of the citrus processing industry. The first step requires a limonene-3-hydroxylase (L3H) activity that specifically catalyzes hydroxylation of limonene at carbon atom 3. Several protein engineering strategies have already attempted to create limonene-3-hydroxylases from bacterial cytochrome P450 monooxygenases (CYPs, or P450s), which can be efficiently expressed in bacterial hosts. However, their regiospecificity is rather low compared to that of the highly selective L3H enzymes from the biosynthetic pathway for menthol in Mentha species. The only naturally occurring limonene-3-hydroxylase activity identified in microorganisms so far was reported for a strain of the black yeast-like fungus Hormonema sp. in South Africa. We have discovered additional fungi that can catalyze the intended reaction and identified potential CYP-encoding genes within the genome sequence of one of the strains. Using heterologous gene expression and biotransformation experiments in yeasts, we were able to identify limonene-3-hydroxylases from Aureobasidium pullulans and Hormonema carpetanum Further characterization of the A. pullulans enzyme demonstrated its high stereospecificity and regioselectivity, its potential for limonene-based menthol production, and its additional ability to convert α- and ß-pinene to verbenol and pinocarveol, respectively.IMPORTANCE (-)-Menthol is an important flavor and fragrance compound and furthermore has medicinal uses. To realize a two-step synthesis starting from renewable (+)-limonene, a regioselective limonene-3-hydroxylase enzyme is necessary. We identified enzymes from two different fungi which catalyze this hydroxylation reaction and represent an important module for the development of a biotechnological process for (-)-menthol production from renewable (+)-limonene.


Assuntos
Ascomicetos/enzimologia , Aureobasidium/enzimologia , Sistema Enzimático do Citocromo P-450/metabolismo , Limoneno/metabolismo , Mentol/metabolismo , Ascomicetos/genética , Aureobasidium/genética , Biotransformação , Catálise , Sistema Enzimático do Citocromo P-450/genética , Proteínas Fúngicas/genética , Hidroxilação , Microbiologia Industrial
8.
J Vis Exp ; (168)2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33616091

RESUMO

Here, a protocol is presented to facilitate the creation of large volumes (> 100 µL) of micro-crystalline slurries suitable for serial crystallography experiments at both synchrotrons and XFELs. The method is based upon an understanding of the protein crystal phase diagram, and how that knowledge can be utilized. The method is divided into three stages: (1) optimizing crystal morphology, (2) transitioning to batch, and (3) scaling. Stage 1 involves finding well diffracting, single crystals, hopefully but not necessarily, presenting in a cube-like morphology. In Stage 2, the Stage 1 condition is optimized by crystal growth time. This strategy can transform crystals grown by vapor diffusion to batch. Once crystal growth can occur within approximately 24 h, a morphogram of the protein and precipitant mixture can be plotted and used as the basis for a scaling strategy (Stage 3). When crystals can be grown in batch, scaling can be attempted, and the crystal size and concentration optimized as the volume is increased. Endothiapepsin has been used as a demonstration protein for this protocol. Some of the decisions presented are specific to endothiapepsin. However, it is hoped that the way they have been applied will inspire a way of thinking about this procedure that others can adapt to their own projects.


Assuntos
Ascomicetos/enzimologia , Ácido Aspártico Endopeptidases/química , Cristalografia por Raios X/métodos , Cristalografia por Raios X/normas , Proteínas Fúngicas/química , Cristalização
9.
mBio ; 12(1)2021 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-33563819

RESUMO

Two DNA methyltransferase (DNMTase) genes from Cryphonectria parasitica have been previously identified as CpDmt1 and CpDmt2, which are orthologous to rid and dim-2 of Neurospora crassa, respectively. While global changes in DNA methylation have been associated with fungal sectorization and CpDmt1 but not CpDmt2 has been implicated in the sporadic sectorization, the present study continues to investigate the biological functions of both DNMTase genes. Transcription of both DNMTases is regulated in response to infection with the Cryphonectria hypovirus 1 (CHV1-EP713). CpDmt1 is upregulated and CpDmt2 is downregulated by CHV1 infection. Conidium production and response to heat stress are affected only by mutation of CpDmt1, not by CpDmt2 mutation. Significant changes in virulence are observed in opposite directions; i.e., the CpDmt1-null mutant is hypervirulent, while the CpDmt2-null mutant is hypovirulent. Compared to the CHV1-infected wild type, CHV1-transferred single and double mutants show severe growth retardation: the colony size is less than 10% that of the parental virus-free null mutants, and their titers of transferred CHV1 are higher than that of the wild type, implying that no defect in viral replication occurs. However, as cultivation proceeds, spontaneous viral clearance is observed in hypovirus-infected colonies of the null mutants, which has never been reported in this fungus-virus interaction. This study demonstrates that both DNMTases are significant factors in fungal development and virulence. Each fungal DNMTase affects fungal biology in both common and separate ways. In addition, both genes are essential to the antiviral responses, including viral clearance which depends on their mutations.IMPORTANCE Although relatively few in number, studies of DNA methylation have shown that fungal DNA methylation is implicated in development, genome integrity, and genome defense. While fungal DNMTase has been suggested as playing a role in genome defense, studies of the biological function of fungal DNMTase have been very limited. In this study, we have shown distinct biological functions of two DNA methyltransferases from the chestnut blight fungus C. parasitica We have demonstrated that DNMTases are important to fungal development and virulence. In addition, these genes are shown to play an important role in the fungal response to hypoviral CHV1 infection, including severely retarded colonial growth, and in viral clearance, which has never been previously observed in mycovirus infection. These findings provide a better understanding of the biological functions of fungal DNA methyltransferase and a basis for clarifying the epigenetic regulation of fungal virulence, responses to hypovirus infection, and viral clearance.


Assuntos
Ascomicetos/enzimologia , Ascomicetos/patogenicidade , Metilação de DNA/genética , Micovírus/fisiologia , Metiltransferases/genética , Metiltransferases/metabolismo , Ascomicetos/genética , Ascomicetos/virologia , DNA Fúngico , Epigênese Genética , Micovírus/genética , Regulação Fúngica da Expressão Gênica , Metiltransferases/classificação , Doenças das Plantas/microbiologia , Virulência
10.
mBio ; 12(1)2021 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-33563831

RESUMO

Rice blast disease caused by Magnaporthe oryzae is a devastating disease of cultivated rice worldwide. Infections by this fungus lead to a significant reduction in rice yields and threats to food security. To gain better insight into growth and cell death in M. oryzae during infection, we characterized two predicted M. oryzae metacaspase proteins, MoMca1 and MoMca2. These proteins appear to be functionally redundant and can complement the yeast Yca1 homologue. Biochemical analysis revealed that M. oryzae metacaspases exhibited Ca2+-dependent caspase activity in vitro Deletion of both MoMca1 and MoMca2 in M. oryzae resulted in reduced sporulation, delay in conidial germination, and attenuation of disease severity. In addition, the double ΔMomca1mca2 mutant strain showed increased radial growth in the presence of oxidative stress. Interestingly, the ΔMomca1mca2 strain showed an increased accumulation of insoluble aggregates compared to the wild-type strain during vegetative growth. Our findings suggest that MoMca1 and MoMca2 promote the clearance of insoluble aggregates in M. oryzae, demonstrating the important role these metacaspases have in fungal protein homeostasis. Furthermore, these metacaspase proteins may play additional roles, like in regulating stress responses, that would help maintain the fitness of fungal cells required for host infection.IMPORTANCE Magnaporthe oryzae causes rice blast disease that threatens global food security by resulting in the severe loss of rice production every year. A tightly regulated life cycle allows M. oryzae to disarm the host plant immune system during its biotrophic stage before triggering plant cell death in its necrotrophic stage. The ways M. oryzae navigates its complex life cycle remain unclear. This work characterizes two metacaspase proteins with peptidase activity in M. oryzae that are shown to be involved in the regulation of fungal growth and development prior to infection by potentially helping maintain fungal fitness. This study provides new insights into the role of metacaspase proteins in filamentous fungi by illustrating the delays in M. oryzae morphogenesis in the absence of these proteins. Understanding the mechanisms by which M. oryzae morphology and development promote its devastating pathogenicity may lead to the emergence of proper methods for disease control.


Assuntos
Ascomicetos/enzimologia , Ascomicetos/patogenicidade , Caspases/genética , Caspases/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Oryza/microbiologia , Ascomicetos/genética , Ascomicetos/crescimento & desenvolvimento , Caspases/classificação , Biologia Computacional , Regulação Fúngica da Expressão Gênica , Genoma Fúngico , Estresse Oxidativo , Doenças das Plantas/microbiologia , Proteínas de Saccharomyces cerevisiae/genética , Virulência
11.
Int J Biol Macromol ; 176: 165-176, 2021 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-33561463

RESUMO

Pectin, the major non-cellulosic component of primary cell wall can be degraded by polygalacturonases (PGs) and pectin methylesterases (PMEs) during pathogen attack on plants. We characterized two novel enzymes, VdPG2 and VdPME1, from the fungal plant pathogen Verticillium dahliae. VdPME1 was most active on citrus methylesterified pectin (55-70%) at pH 6 and a temperature of 40 °C, while VdPG2 was most active on polygalacturonic acid at pH 5 and a temperature of 50 °C. Using LC-MS/MS oligoprofiling, and various pectins, the mode of action of VdPME1 and VdPG2 were determined. VdPME1 was shown to be processive, in accordance with the electrostatic potential of the enzyme. VdPG2 was identified as endo-PG releasing both methylesterified and non-methylesterified oligogalacturonides (OGs). Additionally, when flax roots were used as substrate, acetylated OGs were detected. The comparisons of OGs released from Verticillium-susceptible and partially resistant flax cultivars identified new possible elicitor of plant defence responses.


Assuntos
Ascomicetos/enzimologia , Hidrolases de Éster Carboxílico/metabolismo , Proteínas Fúngicas/metabolismo , Poligalacturonase/metabolismo , Ascomicetos/genética , Ascomicetos/patogenicidade , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/genética , Linho/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Cinética , Modelos Moleculares , Pectinas/metabolismo , Filogenia , Doenças das Plantas/microbiologia , Raízes de Plantas/metabolismo , Poligalacturonase/química , Poligalacturonase/genética , Eletricidade Estática , Especificidade por Substrato
12.
Planta ; 253(2): 34, 2021 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-33459878

RESUMO

MAIN CONCLUSION: BnPGIPs interacted with Sclerotinia sclerotiorum PGs to improve rapeseed SSR resistance at different levels; the BnPGIP-overexpression lines did not affect plant morphology or seed quality traits. Plant polygalacturonase-inhibiting proteins (PGIPs) play a crucial role in plant defence against phytopathogenic fungi by inhibiting fungal polygalacturonase (PG) activity. We overexpressed BnPGIP2, BnPGIP5, and BnPGIP10 genes in an inbred line 7492 of rapeseed (Brassica napus). Compared with 7492WT, the overexpression of BnPGIP2 lines significantly increased Sclerotinia sclerotiorum resistance in both seedlings and adult plants. BnPGIP5 overexpression lines exhibited decreased S. sclerotiorum disease symptoms in seedlings only, whereas BnPGIP10 overexpression lines did not improve Sclerotinia resistance for seedlings or adult plants. Quantitative real-time PCR analysis of S. sclerotiorum PG1, SsPG3, SsPG5, and SsPG6 genes in overexpressing BnPGIP lines showed that these pathogenic genes in the Sclerotinia resistance transgenic lines exhibited low expression in stem tissues. Split-luciferase complementation experiments confirmed the following: BnPGIP2 interacts with SsPG1 and SsPG6 but not with SsPG3 or SsPG5; BnPGIP5 interacts with SsPG3 and SsPG6 but not with SsPG1 or SsPG5; and BnPGIP10 interacts with SsPG1 but not SsPG3, SsPG5, or SsPG6. Leaf crude protein extracts from BnPGIP2 and BnPGIP5 transgenic lines displayed high inhibitory activity against the SsPG crude protein. BnPGIP-overexpression lines with Sclerotinia resistance displayed a lower accumulation of H2O2 and higher expression of the H2O2-removing gene BnAPX (ascorbate peroxidase) than 7492WT, as well as elevated expression of defence response genes including jasmonic acid/ethylene and salicylic acid pathways after S. sclerotiorum infection. The plants overexpressing BnPGIP exhibited no difference in either agronomic traits or grain yield from 7492WT. This study provides potential target genes for developing S. sclerotiorum resistance in rapeseed.


Assuntos
Ascomicetos , Brassica napus , Resistência à Doença , Proteínas de Plantas , Poligalacturonase , Ascomicetos/enzimologia , Brassica napus/enzimologia , Brassica napus/genética , Brassica napus/microbiologia , Resistência à Doença/genética , Expressão Gênica , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Peróxido de Hidrogênio/metabolismo , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Proteínas de Plantas/metabolismo , Poligalacturonase/metabolismo
13.
J Biosci Bioeng ; 131(5): 475-482, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33495046

RESUMO

Using soil samples, we screened for microbes that produce biogenic manganese oxides (BMOs) and isolated Mn(II)-oxidizing fungus, namely Pleosporales sp. Mn1 (Mn1). We purified the Mn(II)-oxidizing enzyme from intracellular extracts of Mn1. The enzyme oxidized Mn(II) most effectively at pH 7.0 and 45 °C. The N-terminal amino acid sequence of the purified enzyme possessed homology with multicopper oxidases in fungi. The properties of the enzyme and the effects of the pH and inhibitors on the Mn(II)-oxidization activity suggested that the enzyme is a member of the multicopper oxidase family. The X-ray diffraction pattern of the BMOs produced by Mn1 showed a strong correlation with that of a typical poorly crystalized vernadite (δ-MnO2). Since BMOs are some of the most reactive materials in the environment, we investigated a potential new application of BMOs as oxidation catalysts. We confirmed that BMOs oxidized aromatic methyl groups when combined with the purified enzyme and a mediator, 1-hydroxybenzotriazole (HBT). BMO oxidation of 3,4-dimethoxytoluene achieved a better yield than that of abiotic MnO2 and white-rot fungus laccase under acidic and neutral pH conditions. Under neutral pH, the BMOs oxidized 3,4-dimethoxytoluene to yield 200-fold more 3,4-dimethoxybenzaldehyde than that of abiotic MnO2. This is the first report to reveal that BMOs combined with a Mn(II)-oxidizing enzyme and mediator can oxidize aromatic hydrocarbons to yield corresponding aldehydes.


Assuntos
Ascomicetos/enzimologia , Compostos de Manganês/metabolismo , Manganês/metabolismo , Óxidos/metabolismo , Oxirredutases/metabolismo , Tolueno/análogos & derivados , Triazóis/metabolismo , Oxirredução , Tolueno/metabolismo
14.
Mol Biol Evol ; 38(5): 1837-1846, 2021 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-33313834

RESUMO

The RNase II family of 3'-5' exoribonucleases is present in all domains of life, and eukaryotic family members Dis3 and Dis3L2 play essential roles in RNA degradation. Ascomycete yeasts contain both Dis3 and inactive RNase II-like "pseudonucleases." The latter function as RNA-binding proteins that affect cell growth, cytokinesis, and fungal pathogenicity. However, the evolutionary origins of these pseudonucleases are unknown: What sequence of events led to their novel function, and when did these events occur? Here, we show how RNase II pseudonuclease homologs, including Saccharomyces cerevisiae Ssd1, are descended from active Dis3L2 enzymes. During fungal evolution, active site mutations in Dis3L2 homologs have arisen at least four times, in some cases following gene duplication. In contrast, N-terminal cold-shock domains and regulatory features are conserved across diverse dikarya and mucoromycota, suggesting that the nonnuclease function requires these regions. In the basidiomycete pathogenic yeast Cryptococcus neoformans, the single Ssd1/Dis3L2 homolog is required for cytokinesis from polyploid "titan" growth stages. This phenotype of C. neoformans Ssd1/Dis3L2 deletion is consistent with those of inactive fungal pseudonucleases, yet the protein retains an active site sequence signature. We propose that a nuclease-independent function for Dis3L2 arose in an ancestral hyphae-forming fungus. This second function has been conserved across hundreds of millions of years, whereas the RNase activity was lost repeatedly in independent lineages.


Assuntos
Ascomicetos/genética , Evolução Molecular , Exorribonucleases/genética , Família Multigênica , Ascomicetos/enzimologia , Domínio Catalítico/genética , Cryptococcus neoformans/fisiologia , Citocinese , Filogenia , Proteínas de Saccharomyces cerevisiae/genética
15.
Elife ; 92020 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-33275098

RESUMO

The production of reactive oxygen species (ROS) is a ubiquitous defense response in plants. Adapted pathogens evolved mechanisms to counteract the deleterious effects of host-derived ROS and promote infection. How plant pathogens regulate this elaborate response against ROS burst remains unclear. Using the rice blast fungus Magnaporthe oryzae, we uncovered a self-balancing circuit controlling response to ROS in planta and virulence. During infection, ROS induces phosphorylation of the high osmolarity glycerol pathway kinase MoOsm1 and its nuclear translocation. There, MoOsm1 phosphorylates transcription factor MoAtf1 and dissociates MoAtf1-MoTup1 complex. This releases MoTup1-mediated transcriptional repression on oxidoreduction-pathway genes and activates the transcription of MoPtp1/2 protein phosphatases. In turn, MoPtp1/2 dephosphorylate MoOsm1, restoring the circuit to its initial state. Balanced interactions among proteins centered on MoOsm1 provide a means to counter host-derived ROS. Our findings thereby reveal new insights into how M. oryzae utilizes a phosphor-regulatory circuitry to face plant immunity during infection.


Assuntos
Ascomicetos/enzimologia , Proteínas Fúngicas/metabolismo , Regulação Enzimológica da Expressão Gênica/fisiologia , Regulação Fúngica da Expressão Gênica/fisiologia , Oryza/microbiologia , Espécies Reativas de Oxigênio , Adaptação Fisiológica , Ascomicetos/genética , Proteínas Fúngicas/genética , Oryza/metabolismo , Oxirredução , Estresse Oxidativo/fisiologia , Fosforilação , Doenças das Plantas/microbiologia
16.
Sci Rep ; 10(1): 20267, 2020 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-33219291

RESUMO

The efficiency of microorganisms to degrade lignified plants is of great importance in the Earth's carbon cycle, but also in industrial biorefinery processes, such as for biofuel production. Here, we present a large-scale proteomics approach to investigate and compare the enzymatic response of five filamentous fungi when grown on five very different substrates: grass (sugarcane bagasse), hardwood (birch), softwood (spruce), cellulose and glucose. The five fungi included the ascomycetes Aspergillus terreus, Trichoderma reesei, Myceliophthora thermophila, Neurospora crassa and the white-rot basidiomycete Phanerochaete chrysosporium, all expressing a diverse repertoire of enzymes. In this study, we present comparable quantitative protein abundance values across five species and five diverse substrates. The results allow for direct comparison of fungal adaptation to the different substrates, give indications as to the substrate specificity of individual carbohydrate-active enzymes (CAZymes), and reveal proteins of unknown function that are co-expressed with CAZymes. Based on the results, we present a quantitative comparison of 34 lytic polysaccharide monooxygenases (LPMOs), which are crucial enzymes in biomass deconstruction.


Assuntos
Ascomicetos/enzimologia , Proteínas Fúngicas/metabolismo , Oxigenases de Função Mista/metabolismo , Phanerochaete/enzimologia , Polissacarídeos/metabolismo , Biocombustíveis , Proteômica , Especificidade por Substrato
17.
Sci Rep ; 10(1): 16530, 2020 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-33020524

RESUMO

The genus Pseudogymnoascus encompasses soil psychrophilic fungi living also in caves. Some are opportunistic pathogens; nevertheless, they do not cause outbreaks. Pseudogymnoascus destructans is the causative agent of the white-nose syndrome, which is decimating cave-hibernating bats. We used comparative eco-physiology to contrast the enzymatic potential and conidial resilience of P. destructans with that of phylogenetically diverse cave fungi, including Pseudogymnoascus spp., dermatophytes and outdoor saprotrophs. Enzymatic potential was assessed by Biolog MicroArray and by growth on labelled substrates and conidial viability was detected by flow cytometry. Pseudogymnoascus destructans was specific by extensive losses of metabolic variability and by ability of lipid degradation. We suppose that lipases are important enzymes allowing fungal hyphae to digest and invade the skin. Pseudogymnoascus destructans prefers nitrogenous substrates occurring in bat skin and lipids. Additionally, P. destructans alkalizes growth medium, which points to another possible virulence mechanism. Temperature above 30 °C substantially decreases conidial viability of cave fungi including P. destructans. Nevertheless, survival of P. destructans conidia prolongs by the temperature regime simulating beginning of the flight season, what suggests that conidia could persist on the body surface of bats and contribute to disease spreading during bats active season.


Assuntos
Ascomicetos/enzimologia , Ascomicetos/metabolismo , Quirópteros/microbiologia , Animais , Ascomicetos/fisiologia , Cavernas , Quirópteros/fisiologia , República Tcheca , Lipase , Micoses/fisiopatologia , Nariz/microbiologia , Filogenia
18.
J Agric Food Chem ; 68(41): 11449-11458, 2020 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-32924475

RESUMO

Here, we reported that detailed investigation on trace targeted metabolites from nematode-trapping fungus Arthrobotrys oligospora mutant with deletion of P450 gene AOL_s00215g278 led to isolation of 9 new polyketide-terpenoid hybrid derivatives, including four new glycosides of the key precursor farnesyl hydrotoluquinol (1) and, surprisingly, four new sesquiterpenyl epoxy-cyclohexenoids (SECs) analogues. Among them, two major target metabolites 1 and 14 displayed moderate nematode inhibitory ability. Moreover, the mutant lacking AOL_s00215g278 could form far more nematode-capturing traps within 6 h in contact with nematodes and show rapid potent nematicidal activity with killing 93.7% preys, though deletion of the P450 gene resulted in dramatic decrease in fungal colony growth and failure to produce fungal conidia. The results unequivocally revealed that gene AOL_s00215g278 should be involved in not only the SEC biosynthetic pathway in the nematode-trapping fungus A. oligospora but also fungal conidiation and nematicidal activity.


Assuntos
Antinematódeos/farmacologia , Ascomicetos/química , Ascomicetos/metabolismo , Proteínas Fúngicas/genética , Policetídeos/farmacologia , Terpenos/farmacologia , Animais , Antinematódeos/química , Antinematódeos/metabolismo , Ascomicetos/enzimologia , Ascomicetos/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Proteínas Fúngicas/metabolismo , Estrutura Molecular , Mutação , Nematoides/efeitos dos fármacos , Nematoides/crescimento & desenvolvimento , Policetídeos/química , Policetídeos/metabolismo , Terpenos/química , Terpenos/metabolismo
19.
Biochim Biophys Acta Proteins Proteom ; 1868(12): 140533, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32866628

RESUMO

The fungus Thermothielavioides terrestris plays an important role in the global carbon cycle with enzymes capable of degrading polysaccharides from biomass, therefore an attractive source of proteins to be investigated and understood. From cloning to a three-dimensional structure, we foster a deeper characterization of an α-ʟ-arabinofuranosidase, a glycoside hydrolase from the family 62 (TtAbf62), responsible to release arabinofuranose from non-reducing ends of polysaccharides. TtAbf62 was tested with synthetic (pNP-Araf) and polymeric substrates (arabinan and arabinoxylan), showing optimal temperature and pH (for pNP-Araf) of 30 °C and 4.5-5.0, respectively. Kinetic parameters revealed different specific activity for the three substrates, with a higher affinity for pNP-Araf (KM: 4 ± 1 mM). The hydrolyzing activity of TtAbf62 on sugarcane bagasse suggests high efficiency in the decomposition of arabinoxylan, abundant hemicellulose presented in the sugarcane cell wall. The crystal packing of TtAbf62 reveals an exquisite domain swapping, located at the supramolecular arrangement through a disulfide bond. All crystallographic behaviors go against its monomeric state in solution, indicating a crystal-induced artifact. Structural information will form the basis for further studies aiming the development of optimized enzymatic properties to be used in biotechnological applications.


Assuntos
Ascomicetos/enzimologia , Glicosídeo Hidrolases/química , Modelos Moleculares , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Fenômenos Bioquímicos , Catálise , Glicosídeo Hidrolases/metabolismo , Cinética , Ligação Proteica , Proteínas Recombinantes , Análise Espectral , Relação Estrutura-Atividade
20.
Nat Microbiol ; 5(12): 1565-1575, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32958858

RESUMO

Many pathogenic fungi depend on the development of specialized infection structures called appressoria to invade their hosts and cause disease. Impairing the function of fungal infection structures therefore provides a potential means by which diseases could be prevented. In spite of this extraordinary potential, however, relatively few anti-penetrant drugs have been developed to control fungal diseases, of either plants or animals. In the present study, we report the identification of compounds that act specifically to prevent fungal infection. We found that the organization of septin GTPases, which are essential for appressorium-mediated infection in the rice blast fungus Magnaporthe oryzae, requires very-long-chain fatty acids (VLCFAs), which act as mediators of septin organization at membrane interfaces. VLCFAs promote septin recruitment to curved plasma membranes and depletion of VLCFAs prevents septin assembly and host penetration by M. oryzae. We observed that VLCFA biosynthesis inhibitors not only prevent rice blast disease, but also show effective, broad-spectrum fungicidal activity against a wide range of fungal pathogens of maize, wheat and locusts, without affecting their respective hosts. Our findings reveal a mechanism underlying septin-mediated infection structure formation in fungi and provide a class of fungicides to control diverse diseases of plants and animals.


Assuntos
Inibidores Enzimáticos/farmacologia , Proteínas Fúngicas/antagonistas & inibidores , Fungos/efeitos dos fármacos , Fungicidas Industriais/farmacologia , Doenças das Plantas/microbiologia , Septinas/antagonistas & inibidores , Ascomicetos/efeitos dos fármacos , Ascomicetos/enzimologia , Ascomicetos/genética , Ácidos Graxos/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fungos/enzimologia , Fungos/genética , Oryza/microbiologia , Septinas/genética , Septinas/metabolismo
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