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1.
Fungal co-culture increases ligninolytic enzyme activities: statistical optimization using response surface methodology.
Jiménez-Barrera, D, Chan-Cupul, W, Fan, Z, Osuna-Castro, JA
Preparative biochemistry & biotechnology. 2018;(9):787-798
Abstract
The optimization of ligninolytic enzyme (LE) activities in a novel fungal co-culture between Pycnoporus sanguineus and Beauveria brongniartii were studied using a Plackett-Burman experimental design (PBED) and a central composite design (CCD). In addition, H2O2 role was analyzed. Laccase (EC. 1.10.3.2) and MnP (EC 1.11.1.14) activities of P. sanguineus increased 6.0- and 2.3-fold, respectively, in the co-culture with B. brongniartii. The H2O2 content was higher in the co-culture (0.33-7.12-fold) than in the P. sanguineus monoculture. The PBED revealed that yeast extract (YE), FeSO4, and inoculum amount were significant factors for laccase and MnP activities and H2O2 production in the co-culture, which increased by 8.2-, 5.2- and 1.03-fold, respectively. The YE and FeSO4 were studied using a CCD to optimize the studied response variables. Laccase activity was enhanced 1.5-fold by CCD, the optimal amount of YE was 0.366 g L-1. Quadratic term of FeSO4 modulated MnP activity and was associated with a 4.28-fold increase compared to the PBED. Both YE and its quadratic term significantly affected H2O2 production; however, the CCD did not enable an increase in H2O2 production. Pearson correlation indicated an increase in laccase (r2=0.4411, p = 0.0436) and MnP (r2=0.5186, p = 0.0198) activities following increases in H2O2 in the co-culture system.
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2.
Selection of Reference Genes for qRT-PCR Analysis in Lentinula edodes after Hot-Air Drying.
Gao, S, Wang, G, Huang, Z, Lei, X, Bian, Y, Liu, Y, Huang, W
Molecules (Basel, Switzerland). 2018;(1)
Abstract
Volatile sulfur compounds gradually develop in Lentinula edodes after hot-air drying, and many genes are involved in the generation of these sulfur compounds. The expression stability of reference genes may vary in a particular experimental treatment when analyzing their expressions by quantitative real-time polymerase chain reaction (qRT-PCR). In this study, the expression profile of 17 candidate genes was assessed in L. edodes under treatment at 50 °C for 0, 1, 2, and 3 h, and the expression stability of each reference gene was analyzed by three statistical algorithms, including geNorm, NormFinder, and BestKeeper. Results indicated that the two optimal reference genes for mycelium and fruiting body were CAC and DAHP as well as CAC and NUP, respectively. Additionally, CAC and DAHP were found to be the two most stable reference genes across the mycelium and fruiting body set. Our results will provide a genetic foundation for further research on the metabolism genes of sulfur compounds in L. edodes.
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3.
Proteomics as a Tool to Identify New Targets Against Aspergillus and Scedosporium in the Context of Cystic Fibrosis.
Ramirez-Garcia, A, Pellon, A, Buldain, I, Antoran, A, Arbizu-Delgado, A, Guruceaga, X, Rementeria, A, Hernando, FL
Mycopathologia. 2018;(1):273-289
Abstract
Cystic fibrosis (CF) is a genetic disorder that increases the risk of suffering microbial, including fungal, infections. In this paper, proteomics-based information was collated relating to secreted and cell wall proteins with potential medical applications from the most common filamentous fungi in CF, i.e., Aspergillus and Scedosporium/Lomentospora species. Among the Aspergillus fumigatus secreted allergens, β-1,3-endoglucanase, the alkaline protease 1 (Alp1/oryzin), Asp f 2, Asp f 13/15, chitinase, chitosanase, dipeptidyl-peptidase V (DppV), the metalloprotease Asp f 5, mitogillin/Asp f 1, and thioredoxin reductase receive a special mention. In addition, the antigens β-glucosidase 1, catalase, glucan endo-1,3-β-glucosidase EglC, β-1,3-glucanosyltransferases Gel1 and Gel2, and glutaminase A were also identified in secretomes of other Aspergillus species associated with CF: Aspergillus flavus, Aspergillus niger, Aspergillus nidulans, and Aspergillus terreus. Regarding cell wall proteins, cytochrome P450 and eEF-3 were proposed as diagnostic targets, and alkaline protease 2 (Alp2), Asp f 3 (putative peroxiredoxin pmp20), probable glycosidases Asp f 9/Crf1 and Crf2, GPI-anchored protein Ecm33, β-1,3-glucanosyltransferase Gel4, conidial hydrophobin Hyp1/RodA, and secreted aspartyl protease Pep2 as protective vaccines in A. fumigatus. On the other hand, for Scedosporium/Lomentospora species, the heat shock protein Hsp70 stands out as a relevant secreted and cell wall antigen. Additionally, the secreted aspartyl proteinase and an ortholog of Asp f 13, as well as the cell wall endo-1,3-β-D-glucosidase and 1,3-β-glucanosyl transferase, were also found to be significant proteins. In conclusion, proteins mentioned in this review may be promising candidates for developing innovative diagnostic and therapeutic tools for fungal infections in CF patients.
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4.
The Elicitor Protein AsES Induces a Systemic Acquired Resistance Response Accompanied by Systemic Microbursts and Micro-Hypersensitive Responses in Fragaria ananassa.
Hael-Conrad, V, Perato, SM, Arias, ME, Martínez-Zamora, MG, Di Peto, PLÁ, Martos, GG, Castagnaro, AP, Díaz-Ricci, JC, Chalfoun, NR
Molecular plant-microbe interactions : MPMI. 2018;(1):46-60
Abstract
The elicitor AsES (Acremonium strictum elicitor subtilisin) is a 34-kDa subtilisin-like protein secreted by the opportunistic fungus Acremonium strictum. AsES activates innate immunity and confers resistance against anthracnose and gray mold diseases in strawberry plants (Fragaria × ananassa Duch.) and the last disease also in Arabidopsis. In the present work, we show that, upon AsES recognition, a cascade of defense responses is activated, including: calcium influx, biphasic oxidative burst (O2⋅- and H2O2), hypersensitive cell-death response (HR), accumulation of autofluorescent compounds, cell-wall reinforcement with callose and lignin deposition, salicylic acid accumulation, and expression of defense-related genes, such as FaPR1, FaPG1, FaMYB30, FaRBOH-D, FaRBOH-F, FaCHI23, and FaFLS. All these responses occurred following a spatial and temporal program, first induced in infiltrated leaflets (local acquired resistance), spreading out to untreated lateral leaflets, and later, to distal leaves (systemic acquired resistance). After AsES treatment, macro-HR and macro-oxidative bursts were localized in infiltrated leaflets, while micro-HRs and microbursts occurred later in untreated leaves, being confined to a single cell or a cluster of a few epidermal cells that differentiated from the surrounding ones. The differentiated cells initiated a time-dependent series of physiological and anatomical changes, evolving to idioblasts accumulating H2O2 and autofluorescent compounds that blast, delivering its content into surrounding cells. This kind of systemic cell-death process in plants is described for the first time in response to a single elicitor. All data presented in this study suggest that AsES has the potential to activate a wide spectrum of biochemical and molecular defense responses in F. ananassa that may explain the induced protection toward pathogens of opposite lifestyle, like hemibiotrophic and necrotrophic fungi.
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5.
Amyloid-Like β-Aggregates as Force-Sensitive Switches in Fungal Biofilms and Infections.
Lipke, PN, Klotz, SA, Dufrene, YF, Jackson, DN, Garcia-Sherman, MC
Microbiology and molecular biology reviews : MMBR. 2018;(1)
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Abstract
Cellular aggregation is an essential step in the formation of biofilms, which promote fungal survival and persistence in hosts. In many of the known yeast cell adhesion proteins, there are amino acid sequences predicted to form amyloid-like β-aggregates. These sequences mediate amyloid formation in vitro. In vivo, these sequences mediate a phase transition from a disordered state to a partially ordered state to create patches of adhesins on the cell surface. These β-aggregated protein patches are called adhesin nanodomains, and their presence greatly increases and strengthens cell-cell interactions in fungal cell aggregation. Nanodomain formation is slow (with molecular response in minutes and the consequences being evident for hours), and strong interactions lead to enhanced biofilm formation. Unique among functional amyloids, fungal adhesin β-aggregation can be triggered by the application of physical shear force, leading to cellular responses to flow-induced stress and the formation of robust biofilms that persist under flow. Bioinformatics analysis suggests that this phenomenon may be widespread. Analysis of fungal abscesses shows the presence of surface amyloids in situ, a finding which supports the idea that phase changes to an amyloid-like state occur in vivo. The amyloid-coated fungi bind the damage-associated molecular pattern receptor serum amyloid P component, and there may be a consequential modulation of innate immune responses to the fungi. Structural data now suggest mechanisms for the force-mediated induction of the phase change. We summarize and discuss evidence that the sequences function as triggers for protein aggregation and subsequent cellular aggregation, both in vitro and in vivo.
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6.
Influence of light on lignin-degrading activities of fungal genus Polyporus s. str.
Grassi, E, Robledo, G, Levin, L
Journal of basic microbiology. 2018;(11):947-956
Abstract
Six strains belonging to five species of Polyporus (P. arcularius, P. arcularioides, P. tricholoma, P. cfr. tricholoma, and P. varius), collected from an Atlantic Forest area in Misiones (Argentina), where species usually grow exposed to high temperatures and humidity, were identified by morphological and molecular analyses. P. tricholoma (BAFC 4536) and P. arcularioides (BAFC 4534) were selected by their lignin-degrading enzyme production, their ability to produce primordial of basidiomes under submerged fermentation, and the decrease in lignin content caused in Poplar wood (up to 29% after 45 days). Among several variables evaluated with a Plackett-Burman design (glucose, copper, vanillic acid and manganese concentration, incubation period, and light incidence), the most important factor affecting laccase and Mn-peroxidase (MnP) production by both strains, was light incidence. Light induced fruit body development but diminished laccase and MnP production. Moreover, a modified isoenzymatic laccase pattern was observed, showing additional isoenzymes when fungi were cultivated under darkness and differences in optimal temperature. Although the studied strains did not produce high laccase and MnP titers (uppermost detected 4230 and 90 U L-1 , respectively), their laccases showed thermal stability and optimal temperature above 70 °C, representing an interesting source in the search of thermo-tolerant enzymes for biotechnological applications.
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The binding mechanism between azoles and FgCYP51B, sterol 14α-demethylase of Fusarium graminearum.
Qian, H, Duan, M, Sun, X, Chi, M, Zhao, Y, Liang, W, Du, J, Huang, J, Li, B
Pest management science. 2018;(1):126-134
Abstract
BACKGROUND Fusarium graminearum is the main pathogen of Fusarium head blight (FHB), a worldwide plant disease and a major disease of wheat in China. Control of FHB is mainly dependent on the application of demethylase inhibitor (DMI) fungicides. Fungal sterol 14α-demethylase enzymes (CYP51) are the main target for DMI fungicides. A molecular modeling study and biological evaluation were performed to investigate the binding mechanism between azoles and CYP51B in F. graminearum. RESULTS A homology model based on the crystal structure of Aspergillus fumigatus was built. Molecular docking and molecular dynamics (MD) simulations were then used to identify the optimum binding mode of propiconazole (PRP), diniconazole (DIN), triadimenol (TRL), tebuconazole (TEC) and triadimefon (TRN) with FgCYP51B. Furthermore, the binding free energy of the five protein-inhibitor complexes was calculated using molecular mechanics generalized Born surface area and Poisson-Boltzmann surface area (MM-GB/PBSA) methods. Key residues in the selective binding of azoles to FgCYP51B were recognized by per-residue free energy decomposition analysis. The five ligands have a similar binding mode in the active pocket. The binding free energy to the enzyme for inhibitors PRP and TEC is more favorable than that of TRN, TRL and DIN. Furthermore, the amino acid residues Phe511, Val136, Ile374, Ala308, Ser312 and Try137 of FgCYP51B are key residues interacting with azoles fungicides. From the experimental evaluation, the 50% effective concentration (EC50 ) values for PRP, TEC, DIN, TRL and TRN are 0.024, 0.047, 0.148, 0.154 and 0.474 mg L-1 , respectively. These five molecules exhibit potential inhibitory activity against CYP51B protein from F. graminearum. CONCLUSION Azole fungicides for FgCYP51B should possess more hydrophobic groups interacting with residues Phe511, Val136, Ile374, Ala308, Ser312 and Tyr137. PRP and TEC are preferable for the control of FHB than DIN, TRL and TRN. © 2017 Society of Chemical Industry.
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Influence of rye flour enzymatic biotransformation on the antioxidant capacity and transepithelial transport of phenolic acids.
de Lima, FA, Martins, IM, Faria, A, Calhau, C, Azevedo, J, Fernandes, I, Mateus, N, Macedo, GA
Food & function. 2018;(3):1889-1898
Abstract
Phenolic acids have been reported to play a role on the antioxidant activity and other important biological activities. However, as most polyphenolics in food products are either bound to cellular matrices or present as free polymeric forms, the way they are absorbed has not been totally clear until now. Hydrolytic enzymes may act to increase functionalities in polyphenolic-rich foods, enhancing the bioaccessibility of phenolic compounds and minerals from whole grains. The aim of this study was to evaluate the action of tannin acyl hydrolase (tannase) on the total phenols, phenolic acid profile, antioxidant capacity and in vitro bioaccessibility of phenolic acids found in whole rye flour (RF). Besides increasing total phenols and the antioxidant capacity, tannase treatment increased the amounts of ferulic, sinapic and vanillic acids identified in RF, evidencing a new type of feruloyl esterase catalytic action of tannase. Vanillic and sinapic acids in tannase-treated whole rye flour (RFT) were higher than RF after in vitro gastrointestinal digestion, and higher amounts of transported vanillic acid through the Caco-2 monolayer were detected in RFT. However, the bioaccessibility and the transport efficiency of RF phenolic acids were higher than RFT. Underutilized crops like rye and rye-derived products may be an important source of phenolic acids. The tannase biotransformation, even influencing the total phenolics and antioxidant capacity of RF, did not increase the bioaccessibility of phenolic acids under the experimental conditions of this study.
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9.
Suppression or Activation of Immune Responses by Predicted Secreted Proteins of the Soybean Rust Pathogen Phakopsora pachyrhizi.
Qi, M, Grayczyk, JP, Seitz, JM, Lee, Y, Link, TI, Choi, D, Pedley, KF, Voegele, RT, Baum, TJ, Whitham, SA
Molecular plant-microbe interactions : MPMI. 2018;(1):163-174
Abstract
Rust fungi, such as the soybean rust pathogen Phakopsora pachyrhizi, are major threats to crop production. They form specialized haustoria that are hyphal structures intimately associated with host-plant cell membranes. These haustoria have roles in acquiring nutrients and secreting effector proteins that manipulate host immune systems. Functional characterization of effector proteins of rust fungi is important for understanding mechanisms that underlie their virulence and pathogenicity. Hundreds of candidate effector proteins have been predicted for rust pathogens, but it is not clear how to prioritize these effector candidates for further characterization. There is a need for high-throughput approaches for screening effector candidates to obtain experimental evidence for effector-like functions, such as the manipulation of host immune systems. We have focused on identifying effector candidates with immune-related functions in the soybean rust fungus P. pachyrhizi. To facilitate the screening of many P. pachyrhizi effector candidates (named PpECs), we used heterologous expression systems, including the bacterial type III secretion system, Agrobacterium infiltration, a plant virus, and a yeast strain, to establish an experimental pipeline for identifying PpECs with immune-related functions and establishing their subcellular localizations. Several PpECs were identified that could suppress or activate immune responses in nonhost Nicotiana benthamiana, N. tabacum, Arabidopsis, tomato, or pepper plants.
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10.
Reconstitution of Enzymatic Carbon-Sulfur Bond Formation Reveals Detoxification-Like Strategy in Fungal Toxin Biosynthesis.
Scharf, DH, Dworschak, JD, Chankhamjon, P, Scherlach, K, Heinekamp, T, Brakhage, AA, Hertweck, C
ACS chemical biology. 2018;(9):2508-2512
Abstract
Gliotoxin is a virulence factor of the human pathogen Aspergillus fumigatus, the leading cause of invasive aspergillosis. The activity of this metabolite is mediated by a transannular disulfide bond, a hallmark of the epipolythiodiketopiperazine (ETP) family. Through the creation of fungal gene deletion mutants and heterologous protein expression, we unveiled the critical role of the cytochrome P450 monooxygenase (CYP450) GliC for the stepwise bishydroxylation of the diketopiperazine (DKP) core. We show for the first time the formation of the C-S bond from the DKP in a combined assay of GliC and the glutathione- S-transferase (GST) GliG in vitro. Furthermore, we present experimental evidence for an intermediary imine species. The flexible substrate scope of GliC and GliG in combination parallels P450/GST pairs used in eukaryotic phase I/II detoxification pathways.