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The smut fungus Ustilago esculenta infects Zizania latifolia and induces stem expansion to form a unique vegetable named Jiaobai. Although previous studies have demonstrated that hormonal control is essential for triggering stem swelling, the role of hormones synthesized by Z. latifolia and U. esculenta and the underlying molecular mechanism are not yet clear. To study the mechanism that triggers swollen stem formation, we analyzed the gene expression pattern of both interacting organisms during the initial trigger of culm gall formation, at which time the infective hyphae also propagated extensively and penetrated host stem cells. Transcriptional analysis indicated that abundant genes involving fungal pathogenicity and plant resistance were reprogrammed to maintain the subtle balance between the parasite and host. In addition, the expression of genes involved in auxin biosynthesis of U. esculenta obviously decreased during stem swelling, while a large number of genes related to the synthesis, metabolism and signal transduction of hormones of the host plant were stimulated and showed specific expression patterns, particularly, the expression of ZlYUCCA9 (a flavin monooxygenase, the key enzyme in indole-3-acetic acid (IAA) biosynthesis pathway) increased significantly. Simultaneously, the content of IAA increased significantly, while the contents of cytokinin and gibberellin showed the opposite trend. We speculated that auxin produced by the host plant, rather than the fungus, triggers stem swelling. Furthermore, from the differently expressed genes, two candidate Cys2-His2 (C2H2) zinc finger proteins, GME3058_g and GME5963_g, were identified from U. esculenta , which may conduct fungus growth and infection at the initial stage of stem-gall formation.

In this study, we examined aqueous extracts of the edible mushrooms Pleurotus ostreatus (oyster mushroom) and Lentinula edodes (shiitake mushroom). Proteome analysis was conducted using LC-Triple TOF-MS and showed the expression of 753 proteins by Pleurotus ostreatus , and 432 proteins by Lentinula edodes . Bioactive peptides: Rab GDP dissociation inhibitor, superoxide dismutase, thioredoxin reductase, serine proteinase and lectin, were identified in both mushrooms. The extracts also included promising bioactive compounds including phenolics, flavonoids, vitamins and amino acids. The extracts showed promising antiviral activities, with a selectivity index (SI) of 4.5 for Pleurotus ostreatus against adenovirus (Ad7), and a slight activity for Lentinula edodes against herpes simplex-II (HSV-2). The extracts were not cytotoxic to normal human peripheral blood mononuclear cells (PBMCs). On the contrary, they showed moderate cytotoxicity against various cancer cell lines. Additionally, antioxidant activity was assessed using DPPH radical scavenging, ABTS radical cation scavenging and ORAC assays. The two extracts showed potential antioxidant activities, with the maximum activity seen for Pleurotus ostreatus (IC50 µg/mL) = 39.46 ± 1.27 for DPPH; 11.22 ± 1.81 for ABTS; and 21.40 ± 2.20 for ORAC assays. This study encourages the use of these mushrooms in medicine in the light of their low cytotoxicity on normal PBMCs vis à vis their antiviral, antitumor and antioxidant capabilities.

The ascomycete fungus Fusarium oxysporum f.sp. cucumerinum causes vascular wilt diseases in cucumber. However, few genes related to morphogenesis and pathogenicity of this fungal pathogen have been functionally characterized. BLASTp searches of the Aspergillus fumigatus UgmA and galatofuranosyltransferases (Galf-transferases) sequences in the F. oxysporum genome identified two genes encoding putative UDP-galactopyranose mutase (UGM), ugmA and ugmB, and six genes encoding putative Galf-transferase homologs. In this study, the single and double mutants of the ugmA, ugmB and gfsB were obtained. The roles of UGMs and GfsB were investigated by analyzing the phenotypes of the mutants. Our results showed that deletion of the ugmA gene led to a reduced production of galactofuranose-containing sugar chains, reduced growth and impaired conidiation of F. oxysporum f.sp. cucumerinum. Most importantly, the ugmA deletion mutant lost the pathogenicity in cucumber plantlets. Although deletion of the ugmB gene did not cause any visible phenotype, deletion of both ugmA and ugmB genes caused more severe phenotypes as compared with the ΔugmA, suggesting that UgmA and UgmB are redundant and they can both contribute to synthesis of UDP-Galf. Furthermore, the ΔgfsB exhibited an attenuated virulence although no other phenotype was observed. Our results demonstrate that the galactofuranose (Galf) synthesis contributes to the cell wall integrity, germination, hyphal growth, conidiation and virulence in Fusarium oxysporum f.sp. cucumerinum and an ideal target for the development of new anti-Fusarium agents., Competing Interests: The authors have declared that no competing interests exist.

Trichoderma genus fungi present great potential for the production of carbohydrate-active enzymes (CAZYmes), including glycoside hydrolase (GH) family members. From a renewability perspective, CAZYmes can be biotechnologically exploited to convert plant biomass into free sugars for the production of advanced biofuels and other high-value chemicals. GH54 is an attractive enzyme family for biotechnological applications because many GH54 enzymes are bifunctional. Thus, GH54 enzymes are interesting targets in the search for new enzymes for use in industrial processes such as plant biomass conversion. Herein, a novel metal-dependent GH54 arabinofuranosidase (ThABF) from the cellulolytic fungus Trichoderma harzianum was identified and biochemically characterized. Initial in silico searches were performed to identify the GH54 sequence. Next, the gene was cloned and heterologously overexpressed in Escherichia coli. The recombinant protein was purified, and the enzyme's biochemical and biophysical properties were assessed. GH54 members show wide functional diversity and specifically remove plant cell substitutions including arabinose and galactose in the presence of a metallic cofactor. Plant cell wall substitution has a major impact on lignocellulosic substrate conversion into high-value chemicals. These results expand the known functional diversity of the GH54 family, showing the potential of a novel arabinofuranosidase for plant biomass degradation.

Cobalamin is a cofactor present in essential metabolic pathways in animals and one of the water-soluble vitamins. It is a complex compound synthesized solely by prokaryotes. Cobalamin dependence is scattered across the tree of life. In particular, fungi and plants were deemed devoid of cobalamin. We demonstrate that cobalamin is utilized by all non-Dikarya fungi lineages. This observation is supported by the genomic presence of both B12-dependent enzymes and cobalamin modifying enzymes. Fungal cobalamin-dependent enzymes are highly similar to their animal homologs. Phylogenetic analyses support a scenario of vertical inheritance of the cobalamin usage with several losses. Cobalamin usage was probably lost in Mucorinae and at the base of Dikarya which groups most of the model organisms and which hindered B12-dependent metabolism discovery in fungi. Our results indicate that cobalamin dependence was a widely distributed trait at least in Opisthokonta, across diverse microbial eukaryotes and was likely present in the LECA., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

The p53-like transcription factor (TF) NDT80 plays a vital role in the regulation of pathogenic mechanisms and meiosis in certain fungi. However, the effects of NDT80 on entomopathogenic fungi are still unknown. In this paper, the NDT80 orthologue BbTFO1 was examined in Beauveria bassiana, a filamentous entomopathogenic fungus, to explore the role of an NDT80-like protein for fungal pest control potential. Disruption of BbTFO1 resulted in impaired resistance to oxidative stress (OS) in a growth assay under OS and a 50% minimum inhibitory concentration experiment. Intriguingly, the oxidation resistance changes were accompanied by transcriptional repression of the two key antioxidant enzyme genes cat2 and cat5. ΔBbTFO1 also displayed defective conidial germination, virulence and heat resistance. The specific supplementation of BbTFO1 reversed these phenotypic changes. As revealed by this work, BbTFO1 can affect the transcription of catalase genes and play vital roles in the maintenance of phenotypes associated with the biological control ability of B. bassiana., Competing Interests: The authors have declared that no competing interests exist.

Cellulase production in filamentous fungus Trichoderma reesei is highly responsive to various environmental cues involving multiple positive and negative regulators. XYR1 (Xylanase regulator 1) has been identified as the key transcriptional activator of cellulase gene expression in T. reesei. However, the precise mechanism by which XYR1 achieves transcriptional activation of cellulase genes is still not fully understood. Here, we identified the TrCYC8/TUP1 complex as a novel coactivator for XYR1 in T. reesei. CYC8/TUP1 is the first identified transcriptional corepressor complex mediating repression of diverse genes in Saccharomyces cerevisiae. Knockdown of Trcyc8 or Trtup1 resulted in markedly impaired cellulase gene expression in T. reesei. We found that TrCYC8/TUP1 was recruited to cellulase gene promoters upon cellulose induction and this recruitment is dependent on XYR1. We further observed that repressed Trtup1 or Trcyc8 expression caused a strong defect in XYR1 occupancy and loss of histone H4 at cellulase gene promoters. The defects in XYR1 binding and transcriptional activation of target genes in Trtup1 or Trcyc8 repressed cells could not be overcome by XYR1 overexpression. Our results reveal a novel coactivator function for TrCYC8/TUP1 at the level of activator binding, and suggest a mechanism in which interdependent recruitment of XYR1 and TrCYC8/TUP1 to cellulase gene promoters represents an important regulatory circuit in ensuring the induced cellulase gene expression. These findings thus contribute to unveiling the intricate regulatory mechanism underlying XYR1-mediated cellulase gene activation and also provide an important clue that will help further improve cellulase production by T. reesei., Competing Interests: The authors have declared that no competing interests exist.

Botryosphaeria dothidea is a pathogen with worldwide distribution, infecting hundreds of species of economically important woody plants. It infects and causes various symptoms on apple plants, including wart and canker on branches, twigs, and stems. However, the mechanism of warts formation is unclear. In this study, we investigated the mechanism of wart formation by observing the transection ultrastructure of the inoculated cortical tissues at various time points of the infection process and detecting the expression of genes related to the pathogen pathogenicity and plant defense response. Results revealed that wart induced by B. dothidea consisted of proliferous of phelloderm cells, the newly formed secondary phellem, and the suberized phelloderm cells surrounding the invading mycelia. The qRT-PCR analysis revealed the significant upregulation of apple pathogenesis-related and suberification-related genes and a pathogen cutinase gene Bdo_10846 . The Bdo_10846 knockout transformants showed reduced cutinase activity and decreased virulence. Transient expression of Bdo_10846 in Nicotiana benthamiana induced ROS burst, callose formation, the resistance of N. benthamiana to Botrytis cinerea , and significant upregulation of the plant pathogenesis-related and suberification-related genes. Additionally, the enzyme activity is essential for the induction. Virus-induced gene silencing demonstrated that the NbBAK1 and NbSOBIR1 expression were required for the Bdo_10846 induced defense response in N. benthamiana . These results revealed the mechanism of wart formation induced by B. dothidea invasion and the important roles of the cutinase Bdo_10846 in pathogen virulence and in inducing plant immunity.

Aspergillus niger has the ability to produce a large variety of proteases, which are of particular importance for protein digestion, intracellular protein turnover, cell signaling, flavour development, extracellular matrix remodeling and microbial defense. However, the A. niger degradome (the full repertoire of peptidases encoded by the A. niger genome) available is not accurate and comprehensive. Herein, we have utilized annotations of A. niger proteases in AspGD, JGI, and version 12.2 MEROPS database to compile an index of at least 232 putative proteases that are distributed into the 71 families/subfamilies and 26 clans of the 6 known catalytic classes, which represents ~ 1.64% of the 14,165 putative A. niger protein content. The composition of the A. niger degradome comprises ~ 7.3% aspartic, ~ 2.2% glutamic, ~ 6.0% threonine, ~ 17.7% cysteine, ~ 31.0% serine, and ~ 35.8% metallopeptidases. One hundred and two proteases have been reassigned into the above six classes, while the active sites and/or metal-binding residues of 110 proteases were recharacterized. The probable physiological functions and active site architectures of these peptidases were also investigated. This work provides a more precise overview of the complete degradome of A. niger, which will no doubt constitute a valuable resource and starting point for further experimental studies on the biochemical characterization and physiological roles of these proteases.

Background: AMP-activated protein kinase (AMPK) is an important regulator for lipid accumulation, potentially known to have an inhibitory role in lipid synthesis. It inactivates acetyl-CoA carboxylase (ACC), an important regulatory enzyme required for lipid synthesis. However, in Mucor circinelloides, AMPK and its association with lipid accumulation has not been studied yet., Objectives: To identify AMPK genes in M. circinelloides and to compare their expression levels in high and low lipid-producing strains of M. circinelloides to predict the possible roles of AMPK in lipid metabolism and to select candidate genes for further studies to enhance lipid accumulation., Results: Two genes for α-subunit, one for β-subunit and six for γ-subunit were identified and annotated. Bioinformatic analysis confirmed the presence of typical conserved domains in these genes. Furthermore, transcriptional profiling displayed marked differences in expression kinetics of subunits among the selected strains. The expression of AMPK genes decreased rapidly in WJ11, high lipid producer strain during the lipid accumulation phase while contrasting profile of expression was observed in CBS 277.49, low lipid producer strain., Conclusion: The present study has shown the association of AMPK genes with lipid metabolism at the transcriptional level. The involvement of Snf-α1, Snf-α2, Snf-β, Snf-γ1, Snf-γ4, Snf-γ5 subunits were shown to be more pronounced and could potentially be further explored in future studies.

Introduction: In recent years, the number of diseases caused by fungal pathogens has increased significantly. Many species of fungi are pathogenic for plants, causing a threat to food production and to humans, and are among the causes of chronic diseases., Objective: The aim of the study is to determine the enzyme profiles of fungi, depending on the different types of fruit with which they have contact, and to determine the differences in these profiles in relation to the substrate on which they are grown., Material and Methods: Six strains of fungi identified as Cladosporium sphaerospermum, Fusarium poae, Alternaria alternata, Penicillium expansum, Penicillium verucosum and Acremonium strictum , isolated from fruits, were selected and analyzed for enzymatic profiles. The enzymatic activity was assessed using the API ZYM test (bioMerieux, France)., Results: In the majority of the 6 fungal strains isolated from fruits, enzymes belonging to glycol-hydrolases were the most active. The exception was Acremonium strictum, where phosphatases dominated. Among most fungal isolates, the enzymes β- glucosidase and N-acetyl-β-glucosaminidase showed the highest activity. The highest β-glucosidase activities were found in Cladosporium sphaerospermum and Penicillium expansum . On the other hand, lipase, α-fucosidase and α-chymotrypsin showed the least activity. The least activity of these enzymes or their complete absence was observed in Fusarium poae, Alternaria alternata, Penicillium expansum and Acremonium strictum ., Conclusions: The activity of hydrolytic enzymes in the isolated fungi depended on the addition of fruit and the type of medium. Individual fruits can increase or decrease the activity of the enzymes. Fungi present in fruit have pathogenic properties and can be possible risk factors for fungal infections.

Aspergillus terreus is an allergenic fungus, in addition to causing infections in both humans and plants. However, the allergens in this fungus are still unknown, limiting the development of diagnostic and therapeutic strategies. We used a proteomic approach to search for allergens, identifying 16 allergens based on two-dimensional immunoblotting with A. terreus susceptible patient sera. We further characterized triose-phosphate isomerase (Asp t 36), one of the dominant IgE (IgE)-reactive proteins. The gene was cloned and expressed in Escherichia coli. Phylogenetic analysis showed Asp t 36 to be highly conserved with close similarity to the triose-phosphate isomerase protein sequence from Dermatophagoides farinae, an allergenic dust mite. We identified four immunodominant epitopes using synthetic peptides, and mapped them on a homology-based model of the tertiary structure of Asp t 36. Among these, two were found to create a continuous surface patch on the 3D structure, rendering it an IgE-binding hotspot. Biophysical analysis indicated that Asp t 36 shows similar secondary structure content and temperature sensitivity with other reported triose-phosphate isomerase allergens. In vivo studies using a murine model displayed that the recombinant Asp t 36 was able to stimulate airway inflammation, as demonstrated by an influx of eosinophils, goblet cell hyperplasia, elevated serum Igs, and induction of Th2 cytokines. Collectively, our results reveal the immunogenic property of Asp t 36, a major allergen from A. terreus, and define a new fungal allergen more broadly. This allergen could serve as a potent candidate for investigating component resolved diagnosis and immunotherapy., (Copyright © 2020 © 2020 Karmakar et al. Published by Elsevier Inc. All rights reserved.)

Recent transcriptomic and biochemical studies have revealed that light influences the global gene expression profile and metabolism of the white-rot fungus Cerrena unicolor . Here, we aimed to reveal the involvement of proteases and ubiquitin-mediated proteolysis by the 26S proteasome in the response of this fungus to white, red, blue and green lighting conditions and darkness. The changes in the expression profile of C. unicolor genes putatively engaged in proteolysis were found to be unique and specific to the applied wavelength of light. It was also demonstrated that the activity of proteases in the culture fluid and mycelium measured using natural and synthetic substrates was regulated by light and was substrate-dependent. A clear influence of light on protein turnover and the qualitative and quantitative changes in the hydrolytic degradation of proteins catalyzed by various types of proteases was shown. The analysis of activity associated with the 26S proteasome showed a key role of ATP-dependent proteolysis in the initial stages of adaptation of fungal cells to the stress factors. It was suggested that the light-sensing pathways in C. unicolor are cross-linked with stress signaling and secretion of proteases presumably serving as regulatory molecules.

Fungal chemodiversity is well known in part due to the production of diverse analogous compounds by a single biosynthetic gene cluster (BGC). Usually, similar or the same metabolites are produced by closely related fungal species under a given condition, the foundation of fungal chemotaxonomy. Here, we report a rare case of the production of the cyclodepsipeptide beauveriolides (BVDs) in three insect-pathogenic fungi. We found that the more closely related fungi Beauveria bassiana and Beauveria brongniartii produced structurally distinct analogs of BVDs, whereas the less-close relatives B. brongniartii and Cordyceps militaris biosynthesized structurally similar congeners under the same growth condition. It was verified that a conserved BGC containing four genes is responsible for BVD biosynthesis in three fungi, including a polyketide synthase (PKS) for the production of 3-hydroxy fatty acids (FAs) with chain length variations. In contrast to BVD production patterns, phylogenetic analysis of the BGC enzymes or enzyme domains largely resulted in the congruence relationship with fungal speciation. Feeding assays demonstrated that an FA with a chain length of eight carbon atoms was preferentially utilized, whereas an FA with a chain longer than 10 carbon atoms could not be used as a substrate for BVD biosynthesis. Insect survival assays suggested that the contribution of BVDs to fungal virulence might be associated with the susceptibility of insect species. The results of this study enrich the knowledge of fungal secondary metabolic diversity that can question the reliability of fungal chemotaxonomy. IMPORTANCE Fungal chemotaxonomy is an approach to classify fungi based on the fungal production profile of metabolites, especially the secondary metabolites. We found an atypical example that could question the reliability of fungal chemical classifications in this study, i.e., the more closely related entomopathogenic species Beauveria bassiana and Beauveria brongniartii produced structurally different congeners of the cyclodepsipeptide beauveriolides, whereas the rather divergent species B. brongniartii and Cordyceps militaris biosynthesized similar analogs under the same growth condition. The conserved biosynthetic gene cluster (BGC) containing four genes present in each species is responsible for beauveriolide production. In contrast to the compound formation profiles, the phylogenies of biosynthetic enzymes or enzymatic domains show associations with fungal speciation. Dependent on the insect species, production of beauveriolides may contribute to fungal virulence against the susceptible insect hosts. The findings in this study augment the diversity of fungal secondary metabolisms., (Copyright © 2020 Yin et al.)

Phosphatases, together with kinases and transcription factors, are key components in cellular signalling networks. Here, we present a systematic functional analysis of the phosphatases in Cryptococcus neoformans, a fungal pathogen that causes life-threatening fungal meningoencephalitis. We analyse 230 signature-tagged mutant strains for 114 putative phosphatases under 30 distinct in vitro growth conditions, revealing at least one function for 60 of these proteins. Large-scale virulence and infectivity assays using insect and mouse models indicate roles in pathogenicity for 31 phosphatases involved in various processes such as thermotolerance, melanin and capsule production, stress responses, O-mannosylation, or retromer function. Notably, phosphatases Xpp1, Ssu72, Siw14, and Sit4 promote blood-brain barrier adhesion and crossing by C. neoformans. Together with our previous systematic studies of transcription factors and kinases, our results provide comprehensive insight into the pathobiological signalling circuitry of C. neoformans.

Aspergillus tamarii grows abundantly in naturally composting waste fibers of the textile industry and has a great potential in biomass decomposition. Amongst the key (hemi)cellulose-active enzymes in the secretomes of biomass-degrading fungi are the lytic polysaccharide monooxygenases (LPMOs). By catalyzing oxidative cleavage of glycoside bonds, LPMOs promote the activity of other lignocellulose-degrading enzymes. Here, we analyzed the catalytic potential of two of the seven AA9-type LPMOs that were detected in recently published transcriptome data for A. tamarii, namely AtAA9A and AtAA9B. Analysis of products generated from cellulose revealed that AtAA9A is a C4-oxidizing enzyme, whereas AtAA9B yielded a mixture of C1- and C4-oxidized products. AtAA9A was also active on cellopentaose and cellohexaose. Both enzymes also cleaved the β-(1→4)-glucan backbone of tamarind xyloglucan, but with different cleavage patterns. AtAA9A cleaved the xyloglucan backbone only next to unsubstituted glucosyl units, whereas AtAA9B yielded product profiles indicating that it can cleave the xyloglucan backbone irrespective of substitutions. Building on these new results and on the expanding catalog of xyloglucan- and oligosaccharide-active AA9 LPMOs, we discuss possible structural properties that could underlie the observed functional differences. The results corroborate evidence that filamentous fungi have evolved AA9 LPMOs with distinct substrate specificities and regioselectivities, which likely have complementary functions during biomass degradation., Competing Interests: The authors declare that no competing interests exist. Embrapa, the employer of MMCC, is a not-for-profit, state-funded research organization. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Opportunistic pathogens such as Candida species can use carboxylic acids, like acetate and lactate, to survive and successfully thrive in different environmental niches. These nonfermentable substrates are frequently the major carbon sources present in certain human body sites, and their efficient uptake by regulated plasma membrane transporters plays a critical role in such nutrient-limited conditions. Here, we cover the physiology and regulation of these proteins and their potential role in Candida virulence. This review also presents an evolutionary analysis of orthologues of the Saccharomyces cerevisiae Jen1 lactate and Ady2 acetate transporters, including a phylogenetic analysis of 101 putative carboxylate transporters in twelve medically relevant Candida species. These proteins are assigned to distinct clades according to their amino acid sequence homology and represent the major carboxylic acid uptake systems in yeast. While Jen transporters belong to the sialate:H + symporter (SHS) family, the Ady2 homologue members are assigned to the acetate uptake transporter (AceTr) family. Here, we reclassify the later members as ATO (acetate transporter ortholog). The new nomenclature will facilitate the study of these transporters, as well as the analysis of their relevance for Candida pathogenesis., (Copyright © 2020 Alves et al.)

Carboxylic acid reductases (CARs) are attracting burgeoning attention as biocatalysts for organic synthesis of aldehydes and their follow-up products from economic carboxylic acid precursors. The CAR enzyme class as a whole, however, is still poorly understood. To date, relatively few CAR sequences have been reported, especially from fungal sources. Here, we sought to increase the diversity of the CAR enzyme class. Six new CAR sequences from the white-rot fungus Pycnoporus cinnabarinus were identified from genome-wide mining. Genome and gene clustering analysis suggests that these PcCAR enzymes play different natural roles in Basidiomycete systems, compared to their type II Ascomycete counterparts. The cDNA sequences of all six Pccar genes were deduced and analysis of their corresponding amino acid sequence showed that they encode for proteins of similar properties that possess a conserved modular functional tri-domain arrangement. Phylogenetic analyses showed that all PcCAR enzymes cluster together with the other type IV CARs. One candidate, PcCAR4, was cloned and over-expressed recombinantly in Escherichia coli. Subsequent biotransformation-based screening with a panel of structurally-diverse carboxylic acid substrates suggest that PcCAR4 possessed a more pronounced substrate specificity compared to previously reported CARs, preferring to reduce sterically-rigid carboxylic acids such as benzoic acid. These findings thus present a new functionally-distinct member of the CAR enzyme class., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Laccases belong to ligninolytic enzymes and play important roles in various biological processes of filamentous fungi, including fruiting-body formation and lignin degradation. The process of fruiting-body development in Lentinula edodes is complex and is greatly affected by environmental conditions. In this paper, 14 multicopper oxidase-encoding (laccase) genes were analyzed in the draft genome sequence of L. edodes strain W1-26, followed by a search of multiple stress-related Cis -elements in the promoter region of these laccase genes, and then a transcription profile analysis of 14 laccase genes ( Lelcc ) under the conditions of different carbon sources, temperatures, and photoperiods. All laccase genes were significantly regulated by varying carbon source materials. The expression of only two laccase genes ( Lelcc5 and Lelcc6 ) was induced by sodium-lignosulphonate and the expression of most laccase genes was specifically upregulated in glucose medium. Under different temperature conditions, the expression levels of most laccase genes decreased at 39 °C and transcription was significantly increased for Lelcc1 , Lelcc4 , Lelcc5 , Lelcc9 , Lelcc12 , Lelcc13 , and Lelcc14 after induction for 24 h at 10 °C, indicating their involvement in primordium differentiation. Tyrosinase, which is involved in melanin synthesis, was clustered with the same group as Lelcc4 and Lelcc7 in all the different photoperiod treatments. Meanwhile, five laccase genes ( Lelcc8 , Lelcc9 , Lelcc12 , Lelcc13 , and Lelcc14 ) showed similar expression profiles to that of two blue light receptor genes ( LephrA and LephrB ) in the 12 h light/12 h dark treatment, suggesting the involvement of laccase genes in the adaptation process of L. edodes to the changing environment and fruiting-body formation. This study contributes to our understanding of the function of the different Lelcc genes and facilitates the screening of key genes from the laccase gene family for further functional research.

Most of the edible mushrooms cannot be cultivated or have low yield under industrial conditions, partially due to the lack of knowledge on how basidioma (fruiting body) development is regulated. From winter mushroom ( Flammulina velutipes ), one of the most popular industrially cultivated mushrooms, a transcription factor, PDD1, with a high-mobility group (HMG)-box domain was identified based on its increased transcription during basidioma development. pdd1 knockdown by RNA interference affected vegetative growth and dramatically impaired basidioma development. A strain with an 89.9% reduction in the level of pdd1 transcription failed to produce primordia, while overexpression of pdd1 promoted basidioma development. When the transcriptional level of pdd1 was increased to 5 times the base level, the mushroom cultivation time was shortened by 9.8% and the yield was increased by at least 33%. RNA sequencing (RNA-seq) analysis revealed that pdd1 knockdown downregulated 331 genes and upregulated 463 genes. PDD1 positively regulated several genes related to fruiting, including 6 pheromone receptor-encoding genes, 3 jacalin-related lectin-encoding genes, FVFD16 , and 2 FVFD16 homolog-encoding genes. PDD1 is a novel transcription factor with regulatory function in basidioma development found in industrially cultivated mushrooms. Since its orthologs are widely present in fungal species of the Basidiomycota phylum, PDD1 might have important application prospects in mushroom breeding. IMPORTANCE Mushrooms are sources of food and medicine and provide abundant nutrients and bioactive compounds. However, most of the edible mushrooms cannot be cultivated commercially due to the limited understanding of basidioma development. From winter mushroom ( Flammulina velutipes ; also known as Enokitake), one of the most commonly cultivated mushrooms, we identified a novel transcription factor, PDD1, positively regulating basidioma development. PDD1 increases expression during basidioma development. Artificially increasing its expression promoted basidioma formation and dramatically increased mushroom yield, while reducing its expression dramatically impaired its development. In its PDD1 overexpression mutants, mushroom number, height, yield, and biological efficiency were significantly increased. PDD1 regulates the expression of some genes that are important in or related to basidioma development. PDD1 is the first identified transcription factor with defined functions in mushroom development among commercially cultivated mushroom species, and it might be useful in mushroom breeding., (Copyright © 2019 American Society for Microbiology.)

Glycoside hydrolase family (GH) 16 comprises a large and taxonomically diverse family of glycosidases and transglycosidases that adopt a common β-jelly-roll fold and are active on a range of terrestrial and marine polysaccharides. Presently, broadly insightful sequence-function correlations in GH16 are hindered by a lack of a systematic subfamily structure. To fill this gap, we have used a highly scalable protein sequence similarity network analysis to delineate nearly 23,000 GH16 sequences into 23 robust subfamilies, which are strongly supported by hidden Markov model and maximum likelihood molecular phylogenetic analyses. Subsequent evaluation of over 40 experimental three-dimensional structures has highlighted key tertiary structural differences, predominantly manifested in active-site loops, that dictate substrate specificity across the GH16 evolutionary landscape. As for other large GH families ( i.e. GH5, GH13, and GH43), this new subfamily classification provides a roadmap for functional glycogenomics that will guide future bioinformatics and experimental structure-function analyses. The GH16 subfamily classification is publicly available in the CAZy database. The sequence similarity network workflow used here, SSNpipe, is freely available from GitHub., (© 2019 Viborg et al.)

Objectives: The necrotrophic fungal pathogen Pyrenophora tritici-repentis (Ptr) is the causal agent of tan spot a major disease of wheat. We have generated a new genome resource for an Australian Ptr race 1 isolate V1 to support comparative 'omics analyses. In particular, the V1 PacBio Biosciences long-read sequence assembly was generated to confirm the stability of large-scale genome rearrangements of the Australian race 1 isolate M4 when compared to the North American race 1 isolate Pt-1C-BFP., Results: Over 1.3 million reads were sequenced by PacBio Sequel small-molecule real-time sequencing (SRMT) cell to yield 11.4 Gb for the genome assembly of V1 (285X coverage), with median and maximum read lengths of 8959 bp and 72,292 bp respectively. The V1 genome was assembled into 33 contiguous sequences with a of total length 40.4 Mb and GC content of 50.44%. A total of 14,050 protein coding genes were predicted and annotated for V1. Of these 11,519 genes were orthologous to both Pt-1C-BFP and M4. Whole genome alignment of the Australian long-read assemblies (V1 to M4) confirmed previously identified large-scale genome rearrangements between M4 and Pt-1C-BFP and presented small scale variations, which included a sequence break within a race-specific region for ToxA, a well-known necrotrophic effector gene.

Objective: Label-free quantitative proteomics has emerged as a powerful strategy to obtain high quality quantitative measures of the proteome with only a very small quantity of total protein extract. Because our research projects were requiring the application of bottom-up shotgun mass spectrometry proteomics in the pathogenic yeasts Candida glabrata and Candida albicans, we performed preliminary experiments to (i) obtain a precise list of all the proteins for which measures of abundance could be obtained and (ii) assess the reproducibility of the results arising respectively from biological and technical replicates., Data Description: Three time-courses were performed in each Candida species, and an alkaline pH stress was induced for two of them. Cells were collected 10 and 60 min after stress induction and proteins were extracted. Samples were analysed two times by mass spectrometry. Our final dataset thus comprises label-free quantitative proteomics results for 24 samples (two species, three time-courses, two time points and two runs of mass spectrometry). Statistical procedures were applied to identify proteins with differential abundances between stressed and unstressed situations. Considering that C. glabrata and C. albicans are human pathogens, which face important pH fluctuations during a human host infection, this dataset has a potential value to other researchers in the field.

Pyricularia oryzae is the pathogen for rice blast disease, which is a devastating threat to rice production worldwide. Lysine succinylation, a newly identified post-translational modification, is associated with various cellular processes. Here, liquid chromatography tandem-mass spectrometry combined with a high-efficiency succinyl-lysine antibody was used to identify the succinylated peptides in P. oryzae. In total, 2109 lysine succinylation sites in 714 proteins were identified. Ten conserved succinylation sequence patterns were identified, among which, K*******K suc , and K**K suc , were two most preferred ones. The frequency of lysine succinylation sites, however, greatly varied among organisms, including plants, animals, and microbes. Interestingly, the numbers of succinylation site in each protein of P. oryzae were significantly greater than that of most previous published organisms. Gene ontology and KEGG analysis showed that these succinylated peptides are associated with a wide range of cellular functions, from metabolic processes to stimuli responses. Further analyses determined that lysine succinylation occurs on several key enzymes of the tricarboxylic acid cycle and glycolysis pathway, indicating that succinylation may play important roles in the regulation of basal metabolism in P. oryzae. Furthermore, more than 40 pathogenicity-related proteins were identified as succinylated proteins, suggesting an involvement of succinylation in pathogenicity. Our results provide the first comprehensive view of the P. oryzae succinylome and may aid to find potential pathogenicity-related proteins to control the rice blast disease. Significance Plant pathogens represent a great threat to world food security, and enormous reduction in the global yield of rice was caused by P. oryzae infection. Here, the succinylated proteins in P. oryzae were identified. Furthermore, comparison of succinylation sites among various species, indicating that different degrees of succinylation may be involved in the regulation of basal metabolism. This data facilitates our understanding of the metabolic pathways and proteins that are associated with pathogenicity.

Non-ribosomal peptide synthetase (NRPS)-like enzymes catalyze the non-oxidative homodimerization of aromatic α-keto acids, but the exact reaction mechanism is unknown. The furanone-forming thioesterase domain of the Aspergillus terreus aspulvinone E synthetase MelA displays a predicted quinone-forming motif, whereby its catalytic triad contains an essential cysteine indicating an unusual thioester intermediate. To convert MelA into a quinone-forming atromentin synthetase its thioesterase domain was replaced with that from a Paxillus involutus or A. terreus atromentin synthetase. Phylogenetic proximity of donor and acceptor seems important, as only replacement with the A. terreus thioesterase was functional. Heterologous expression of atromentin synthetases in Aspergillus niger and Aspergillus oryzae revealed host-dependent product formation whereby cross-chemistry directed atromentin biosynthesis in A. niger toward atrofuranic acid. Screening of aspergilli from section Nigri identified an atromentin synthetase in Aspergillus brasiliensis that produced atrofuranic acid in the homologous host. Therefore, cross-chemistry on quinone cores appears common to section Nigri., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The Sterol Regulatory Element Binding Proteins (SREBPs) are basic-helix-loop-helix transcription regulators that control the expression of sterol biosynthesis genes in higher eukaryotes and some fungi. Surprisingly, SREBPs do not regulate sterol biosynthesis in the ascomycete yeasts (Saccharomycotina) as this role was handed off to an unrelated transcription regulator in this clade. The SREBPs, nonetheless, expanded in fungi such as the ascomycete yeasts Candida spp., raising questions about their role and evolution in these organisms. Here we report that the fungal SREBPs diversified their DNA binding preferences concomitantly with an expansion in function. We establish that several branches of fungal SREBPs preferentially bind non-palindromic DNA sequences, in contrast to the palindromic DNA motifs recognized by most basic-helix-loop-helix proteins (including SREBPs) in higher eukaryotes. Reconstruction and biochemical characterization of the likely ancestor protein suggest that an intrinsic DNA binding promiscuity in the family was resolved by alternative mechanisms in different branches of fungal SREBPs. Furthermore, we show that two SREBPs in the human commensal yeast Candida albicans drive a transcriptional cascade that inhibits a morphological switch under anaerobic conditions. Preventing this morphological transition enhances C. albicans colonization of the mammalian intestine, the fungus' natural niche. Thus, our results illustrate how diversification in DNA binding preferences enabled the functional expansion of a family of eukaryotic transcription regulators., Competing Interests: The authors have declared that no competing interests exist.

Background: Trombidid mites have a unique life cycle in which only the larval stage is ectoparasitic. In the superfamily Trombiculoidea ("chiggers"), the larvae feed preferentially on vertebrates, including humans. Species in the genus Leptotrombidium are vectors of a potentially fatal bacterial infection, scrub typhus, that affects 1 million people annually. Moreover, chiggers can cause pruritic dermatitis (trombiculiasis) in humans and domesticated animals. In the Trombidioidea (velvet mites), the larvae feed on other arthropods and are potential biological control agents for agricultural pests. Here, we present the first trombidid mites genomes, obtained both for a chigger, Leptotrombidium deliense, and for a velvet mite, Dinothrombium tinctorium., Results: Sequencing was performed using Illumina technology. A 180 Mb draft assembly for D. tinctorium was generated from two paired-end and one mate-pair library using a single adult specimen. For L. deliense, a lower-coverage draft assembly (117 Mb) was obtained using pooled, engorged larvae with a single paired-end library. Remarkably, both genomes exhibited evidence of ancient lateral gene transfer from soil-derived bacteria or fungi. The transferred genes confer functions that are rare in animals, including terpene and carotenoid synthesis. Thirty-seven allergenic protein families were predicted in the L. deliense genome, of which nine were unique. Preliminary proteomic analyses identified several of these putative allergens in larvae., Conclusions: Trombidid mite genomes appear to be more dynamic than those of other acariform mites. A priority for future research is to determine the biological function of terpene synthesis in this taxon and its potential for exploitation in disease control.

Next-generation sequencing (NGS) of the Flammulina elastica (wood-rotting basidiomycete) genome was performed to identify carbohydrate-active enzymes (CAZymes). The resulting assembly (31 kmer) revealed a total length of 35,045,521 bp (49.7% GC content). Using the AUGUSTUS tool, 12,536 total gene structures were predicted by ab initio gene prediction. An analysis of orthologs revealed that 6806 groups contained at least one F. elastica protein. Among the 12,536 predicted genes, F. elastica contained 24 species-specific genes, of which 17 genes were paralogous. CAZymes are divided into five classes: glycoside hydrolases (GHs), carbohydrate esterases (CEs), polysaccharide lyases (PLs), glycosyltransferases (GTs), and auxiliary activities (AA). In the present study, annotation of the predicted amino acid sequences from F. elastica genes using the dbCAN CAZyme database revealed 508 CAZymes, including 82 AAs, 218 GHs, 89 GTs, 18 PLs, 59 CEs, and 42 carbohydrate binding modules in the F. elastica genome. Although the CAZyme repertoire of F. elastica was similar to those of other fungal species, the total number of GTs in F. elastica was larger than those of other basidiomycetes. This genome information elucidates newly identified wood-degrading machinery in F. elastica , offers opportunities to better understand this fungus, and presents possibilities for more detailed studies on lignocellulosic biomass degradation that may lead to future biotechnological and industrial applications.

Pleurotus ostreatus is a commercially grown mushroom species in China. However, studies on the mechanisms of the fruiting body development and stress response of P. ostreatus are still at a primary stage. In this study, we report the entire genome sequence of P. ostreatus CCMSSC03989. Then, we performed comprehensive genome-wide characterization and expression analysis of the MYB transcription factor family during a series of developmental stages and under the condition of heat stress. A 34.76 Mb genome was obtained through next-generation sequencing (NGS) and Bionano optical mapping approaches. The genome has a scaffold N50 of 1.1 Mb and contains 10.11% repeats, and 10,936 gene models were predicted. A total of 20 MYB genes ( PoMYB ) were identified across the genome, and the full-length open reading frames were isolated. The PoMYBs were classified into 1 repeat (1R), 2R, and 3R-MYB groups according to their MYB domain repeat numbers, and 3R-MYBs possessed relatively more introns than 1R and 2R-MYBs. Based on phylogenetic analysis, the PoMYBs were divided into four groups and showed close relationships with the MYB genes of plants and fungi. RNA-sequencing (RNA-Seq) and quantitative PCR (qPCR) analyses revealed that PoMYB expression showed stage-specific patterns in reproductive stages and could be induced by heat stress. The P. ostreatus draft genome will promote genome-wide analysis, and our study of PoMYBs will promote further functional analysis of MYB genes in mushrooms.

Aspergillus fumigatus is a ubiquitous opportunistic fungus. In this study, systematic analyses were carried out to study the temperature adaptability of A. fumigatus. A total of 241 glycoside hydrolases and 69 proteases in the secretome revealed the strong capability of A. fumigatus to degrade plant biomass and protein substrates. In total, 129 pathogenesis-related proteins detected in the secretome were strongly correlated with glycoside hydrolases and proteases. The variety and abundance of proteins remained at temperatures of 34°C-45°C. The percentage of endo-1,4-xylanase increased when the temperature was lowered to 20°C, while the percentage of cellobiohydrolase increased as temperature was increased, suggesting that the strain obtains carbon mainly by degrading xylan and cellulose, and the main types of proteases in the secretome were aminopeptidases and carboxypeptidases. Only half of the proteins were retained and their abundance declined to 9.7% at 55°C. The activities of the remaining β-glycosidases and proteases were merely 35% and 24%, respectively, when the secretome was treated at 60°C for 2 h. Therefore, temperatures >60°C restrict the growth of A. fumigatus.

Expansins are cell wall proteins that mediate cell wall loosening and promote specific tissue and organ morphogenesis in plants and in some microorganisms. Unlike plant expansins, the biological functions of fungal expansin-like proteins have rarely been discussed. In the present study, an expansin-like protein-encoding fvexpl1 gene, was identified from Flammulina velutipes by using local BLAST. It consisted of five exons with a total length of 822 bp. The deduced protein FVEXPL1 contained 274 amino acids with a predicted molecular mass and isoelectric point of 28,589 Da and pH 4.93, respectively. The first 19 amino acids from the N terminal are the signal peptide. Phylogenetic analysis and multiple protein alignment indicated FVEXPL1 was an expansin-like protein. The expression level of fvexpl1 gene in the stipe was significantly higher than that in the mycelia, primordia, and cap. However, the expression level of fvexpl1 gene was significantly higher in the fast elongation region of the stipe as compared with the slow elongation region. Expression analysis indicated that fvexpl1 gene might have an auxiliary role in the stipe morphogenesis of F. velutipes .

Here, we present evidence that ca. 20 years of experimental N deposition altered the composition of lignin-decaying class II peroxidases expressed by forest floor fungi, a response which has occurred concurrently with reductions in plant litter decomposition and a rapid accumulation of soil organic matter. This finding suggests that anthropogenic N deposition has induced changes in the biological mediation of lignin decay, the rate limiting step in plant litter decomposition. Thus, an altered composition of transcripts for a critical gene that is associated with terrestrial C cycling may explain the increased soil C storage under long-term increases in anthropogenic N deposition. IMPORTANCE Fungal class II peroxidases are enzymes that mediate the rate-limiting step in the decomposition of plant material, which involves the oxidation of lignin and other polyphenols. In field experiments, anthropogenic N deposition has increased soil C storage in forests, a result which could potentially arise from anthropogenic N-induced changes in the composition of class II peroxidases expressed by the fungal community. In this study, we have gained unique insight into how anthropogenic N deposition, a widespread agent of global change, affects the expression of a functional gene encoding an enzyme that plays a critical role in a biologically mediated ecosystem process., (Copyright © 2018 American Society for Microbiology.)

Gene duplication facilitates functional diversification and provides greater phenotypic flexibility to an organism. Expanded gene families arise through repeated gene duplication but the extent of functional divergence that accompanies each paralogous gene is generally unexplored because of the difficulty in isolating the effects of single family members. The telomere-associated (TLO) gene family is a remarkable example of gene family expansion, with 14 members in the more pathogenic Candida albicans relative to two TLO genes in the closely-related species C. dubliniensis. TLO genes encode interchangeable Med2 subunits of the major transcriptional regulatory complex Mediator. To identify biological functions associated with each C. albicans TLO, expression of individual family members was regulated using a Tet-ON system and the strains were assessed across a range of phenotypes involved in growth and virulence traits. All TLOs affected multiple phenotypes and a single phenotype was often affected by multiple TLOs, including simple phenotypes such as cell aggregation and complex phenotypes such as virulence in a Galleria mellonella model of infection. No phenotype was regulated by all TLOs, suggesting neofunctionalization or subfunctionalization of ancestral properties among different family members. Importantly, regulation of three phenotypes could be mapped to individual polymorphic sites among the TLO genes, including an indel correlated with two phenotypes, growth in sucrose and macrophage killing. Different selective pressures have operated on the TLO sequence, with the 5' conserved Med2 domain experiencing purifying selection and the gene/clade-specific 3' end undergoing extensive positive selection that may contribute to the impact of individual TLOs on phenotypic variability. Therefore, expansion of the TLO gene family has conferred unique regulatory properties to each paralog such that it influences a range of phenotypes. We posit that the genetic diversity associated with this expansion contributed to C. albicans success as a commensal and opportunistic pathogen.

The copper-containing superoxide dismutases (SODs) represent a large family of enzymes that participate in the metabolism of reactive oxygen species by disproportionating superoxide anion radical to oxygen and hydrogen peroxide. Catalysis is driven by the redox-active copper ion, and in most cases, SODs also harbor a zinc at the active site that enhances copper catalysis and stabilizes the protein. Such bimetallic Cu,Zn-SODs are widespread, from the periplasm of bacteria to virtually every organelle in the human cell. However, a new class of copper-containing SODs has recently emerged that function without zinc. These copper-only enzymes serve as extracellular SODs in specific bacteria ( i.e. Mycobacteria), throughout the fungal kingdom, and in the fungus-like oomycetes. The eukaryotic copper-only SODs are particularly unique in that they lack an electrostatic loop for substrate guidance and have an unusual open-access copper site, yet they can still react with superoxide at rates limited only by diffusion. Copper-only SOD sequences similar to those seen in fungi and oomycetes are also found in the animal kingdom, but rather than single-domain enzymes, they appear as tandem repeats in large polypeptides we refer to as CSRPs (copper-only SOD-repeat proteins). Here, we compare and contrast the Cu,Zn versus copper-only SODs and discuss the evolution of copper-only SOD protein domains in animals and fungi., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Peptidases are enzymes that cleave peptide bonds, yielding proteins and peptides. Enzymes in this class also perform several other functions, regulating the activation or inactivation of target substrates via proteolysis. Owing to these functions, peptidases have been extensively used in industrial and biotechnological applications. Given their potential functions, it is important to optimize the use of these enzymes, which requires determination of the specificity of each peptidase. The peptidase specificity must be taken into account in choosing a peptidase to catalyze the available protein source within the desired application. The specificity of a peptidase defines the profile of enzyme-substrate interactions, and for this the catalytic site and the arrangement of the amino acid residues involved in peptide bond cleavage need to be known. The catalytic sites of peptidases may be composed of several subsites that interact with amino acid residues for proteolysis. Filamentous fungi produce peptidases with varying specificity, and here we provide a review of those reported to date and their potential applications.

Much can be gained from revealing the mechanisms fungal entomopathogens employ. Especially intriguing are fungal parasites that manipulate insect behavior because, presumably, they secrete a wealth of bioactive compounds. To gain more insight into their strategies, we compared the genomes of five ant-infecting Ophiocordyceps species from three species complexes. These species were collected across three continents, from five different ant species in which they induce different levels of manipulation. A considerable number of (small) secreted and pathogenicity-related proteins were only found in these ant-manipulating Ophiocordyceps species, and not in other ascomycetes. However, few of those proteins were conserved among them, suggesting that several different methods of behavior modification have evolved. This is further supported by a relatively fast evolution of previously reported candidate manipulation genes associated with biting behavior. Moreover, secondary metabolite clusters, activated during biting behavior, appeared conserved within a species complex, but not beyond. The independent co-evolution between these manipulating parasites and their respective hosts might thus have led to rather diverse strategies to alter behavior. Our data indicate that specialized, secreted enterotoxins may play a major role in one of these strategies.

Close to 80 years of research have brought MDR alcohol dehydrogenases (ADHs) from unknown molecular concepts to molecules known in exact detail regarding structural, functional and evolutionary properties. They can be traced backwards in at least six stages of development, to essentially the origin of cellular life, and have been monitored in a long series of biannual meetings on "Carbonyl Metabolism". In between each of these latest meetings, a roughly three-fold increase in known totals of MDR databank entries has been apparent, bringing the total now of known MDR-ADH entries to approaching half a million forms., (Copyright © 2016. Published by Elsevier B.V.)

The reduction of C=C double bond, a key reaction in organic synthesis, is mostly achieved by traditional chemical methods. Therefore, the search for enzymes capable of performing this reaction is rapidly increasing. Old Yellow Enzymes (OYEs) are flavin-dependent oxidoreductases, initially isolated from Saccharomyces pastorianus. In this study, the presence and activation of putative OYE enzymes was investigated in the filamentous fungus Mucor circinelloides, which was previously found to mediate C=C reduction. Following an in silico approach, using S. pastorianus OYE1 amminoacidic sequence as template, ten putative genes were identified in the genome of M. circinelloides. A phylogenetic analysis revealed a high homology of McOYE1-9 with OYE1-like proteins while McOYE10 showed similarity with thermophilic-like OYEs. The activation of mcoyes was evaluated during the transformation of three different model substrates. Cyclohexenone, α-methylcinnamaldehyde and methyl cinnamate were completely reduced in few hours and the induction of gene expression, assessed by qRT-PCR, was generally fast, suggesting a substrate-dependent activation. Eight genes were activated in the tested conditions suggesting that they may encode for active OYEs. Their expression over time correlated with C=C double bond reduction.

Fungi are able to switch between different lifestyles in order to adapt to environmental changes. Their ecological strategy is connected to their secretome as fungi obtain nutrients by secreting hydrolytic enzymes to their surrounding and acquiring the digested molecules. We focus on fungal serine proteases (SPs), the phylogenetic distribution of which is barely described so far. In order to collect a complete set of fungal proteases, we searched over 600 fungal proteomes. Obtained results suggest that serine proteases are more ubiquitous than expected. From 54 SP families described in MEROPS Peptidase Database, 21 are present in fungi. Interestingly, 14 of them are also present in Metazoa and Viridiplantae - this suggests that, except one (S64), all fungal SP families evolved before plants and fungi diverged. Most representatives of sequenced eukaryotic lineages encode a set of 13-16 SP families. The number of SPs from each family varies among the analysed taxa. The most abundant are S8 proteases. In order to verify hypotheses linking lifestyle and expansions of particular SP, we performed statistical analyses and revealed previously undescribed associations. Here, we present a comprehensive evolutionary history of fungal SP families in the context of fungal ecology and fungal tree of life.

Optimum utilization of fermentable sugars from lignocellulosic biomass to deliver multiple products under biorefinery concept has been reported in this work. Alcohol fermentation has been carried out with multiple cell recycling of Kluyveromyces marxianus IIPE453. The yeast utilized xylose-rich fraction from acid and steam treated biomass for cell generation and xylitol production with an average yield of 0.315±0.01g/g while the entire glucose rich saccharified fraction had been fermented to ethanol with high productivity of 0.9±0.08g/L/h. A detailed insight into its genome illustrated the strain's complete set of genes associated with sugar transport and metabolism for high-temperature fermentation. A set flocculation proteins were identified that aided in high cell recovery in successive fermentation cycles to achieve alcohols with high productivity. We have brought biomass derived sugars, yeast cell biomass generation, and ethanol and xylitol fermentation in one platform and validated the overall material balance. 2kg sugarcane bagasse yielded 193.4g yeast cell, and with multiple times cell recycling generated 125.56g xylitol and 289.2g ethanol (366mL)., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

RNA interference (RNAi) is conserved in eukaryotic organisms, and it has been well studied in many animal and plant species and some fungal species, yet it is not well studied in fungal plant pathogens. In the rice blast fungus Magnaporthe oryzae, we examined small RNA (sRNA) and their biogenesis in the context of growth and pathogenicity. Through genetic and genomic analyses, we demonstrate that loss of a single gene encoding Dicer, RNA-dependent RNA polymerase, or Argonaute reduces sRNA levels. These three proteins are required for the biogenesis of sRNA-matching genome-wide regions (coding regions, repeats, and intergenic regions). The loss of one Argonaute reduced both sRNA and fungal virulence on barley leaves. Transcriptome analysis of multiple mutants revealed that sRNA play an important role in transcriptional regulation of repeats and intergenic regions in M. oryzae. Together, these data support that M. oryzae sRNA regulate developmental processes including, fungal growth and virulence.

In this study, the transcriptomic-based response of the white rot fungus Abortiporus biennis to oxalic acid induction was reported. The whole transcriptome of A. biennis was analysed using the RNA-based sequencing technology and Solid 5500 platform. De novo assembly of reads generated 37,719 contigs. A molecular function for 26,280 unique transcripts was assigned. The analysis of the A. biennis transcriptome predicted 635 hypothetical open reading frames encoding carbohydrate active enzymes distributed in 122 families. 82 genes were identified, whose expression level was significantly changed after oxalic acid addition. Among them, 18 genes were up-regulated and 64 genes were down-regulated. Genes coding for putative cellulose and hemicellulose degrading enzymes were predominantly up-regulated in the mycelium induced with oxalic acid; it was in the case of cellulases and xylanases (hemicellulases), in particular, β-glucosidase and endo-1,4-β-xylanases. On the contrary, several genes coding for lignolytic enzymes were down-regulated, with the significant repression level in the case of versatile peroxidase. Finally, we identified putative genes involved in oxalate metabolism. Among the transcripts detected in the A. biennis transcriptome, one was annotated as coding for putative oxalate decarboxylase (ODC) and nine transcripts were annotated as formate dehydrogenases (FDH). The addition of oxalic acid to the culture caused upregulation of the gene coding for ODC and three genes for FDH. Amongst the transcripts of putative FDH genes, one designated as NODE_36057, demonstrated the highest induction level recorded in this study after the oxalic acid addition., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Protein-protein interactions (PPIs) are essential for most living organisms' process. Thus, detecting PPIs is extremely important to understand the molecular mechanisms of biological systems. Although many PPIs data have been generated by high-throughput technologies for a variety of organisms, the whole interatom is still far from complete. In addition, the high-throughput technologies for detecting PPIs has some unavoidable defects, including time consumption, high cost, and high error rate. In recent years, with the development of machine learning, computational methods have been broadly used to predict PPIs, and can achieve good prediction rate. In this paper, we present here PCVMZM, a computational method based on a Probabilistic Classification Vector Machines (PCVM) model and Zernike moments (ZM) descriptor for predicting the PPIs from protein amino acids sequences. Specifically, a Zernike moments (ZM) descriptor is used to extract protein evolutionary information from Position-Specific Scoring Matrix (PSSM) generated by Position-Specific Iterated Basic Local Alignment Search Tool (PSI-BLAST). Then, PCVM classifier is used to infer the interactions among protein. When performed on PPIs datasets of Yeast and H. Pylori , the proposed method can achieve the average prediction accuracy of 94.48% and 91.25%, respectively. In order to further evaluate the performance of the proposed method, the state-of-the-art support vector machines (SVM) classifier is used and compares with the PCVM model. Experimental results on the Yeast dataset show that the performance of PCVM classifier is better than that of SVM classifier. The experimental results indicate that our proposed method is robust, powerful and feasible, which can be used as a helpful tool for proteomics research.

High-throughput microscopy of many single cells generates high-dimensional data that are far from straightforward to analyze. One important problem is automatically detecting the cellular compartment where a fluorescently-tagged protein resides, a task relatively simple for an experienced human, but difficult to automate on a computer. Here, we train an 11-layer neural network on data from mapping thousands of yeast proteins, achieving per cell localization classification accuracy of 91%, and per protein accuracy of 99% on held-out images. We confirm that low-level network features correspond to basic image characteristics, while deeper layers separate localization classes. Using this network as a feature calculator, we train standard classifiers that assign proteins to previously unseen compartments after observing only a small number of training examples. Our results are the most accurate subcellular localization classifications to date, and demonstrate the usefulness of deep learning for high-throughput microscopy., (Copyright © 2017 Parnamaa and Parts.)

Subtilases belong to a superfamily of serine proteases which are ubiquitous in fungi and are suspected to have developed distinct functional properties to help fungi adapt to different ecological niches. In this study, we conducted a large-scale phylogenomic survey of subtilase protease genes in 83 whole genome sequenced fungal species in order to identify the evolutionary patterns and subsequent functional divergences of different subtilase families among the main lineages of the fungal kingdom. Our comparative genomic analyses of the subtilase superfamily indicated that extensive gene duplications, losses and functional diversifications have occurred in fungi, and that the four families of subtilase enzymes in fungi, including proteinase K-like, Pyrolisin, kexin and S53, have distinct evolutionary histories which may have facilitated the adaptation of fungi to a broad array of life strategies. Our study provides new insights into the evolution of the subtilase superfamily in fungi and expands our understanding of the evolution of fungi with different lifestyles.

Karnal bunt disease in wheat is caused by hemibiotrophic fungus, Tilletia indica that has been placed as quarantine pest in more than 70 countries. Despite its economic importance, little knowledge about the molecular components of fungal pathogenesis is known. In this study, first time the genome sequence of T. indica has been deciphered for unraveling the effectors' functions of molecular pathogenesis of Karnal bunt disease. The T. indica genome was sequenced employing hybrid approach of PacBio Single Molecule Real Time (SMRT) and Illumina HiSEQ 2000 sequencing platforms. The genome was assembled into 10,957 contigs (N50 contig length 3 kb) with total size of 26.7 Mb and GC content of 53.99%. The number of predicted putative genes were 11,535, which were annotated with Gene Ontology databases. Functional annotation of Karnal bunt pathogen genome and classification of identified effectors into protein families revealed interesting functions related to pathogenesis. Search for effectors' genes using pathogen host interaction database identified 135 genes. The T. indica genome sequence and putative genes involved in molecular pathogenesis would further help in devising novel and effective disease management strategies including development of resistant wheat genotypes, novel biomarkers for pathogen detection and new targets for fungicide development., Competing Interests: The authors have declared that no competing interests exist.

Self-interacting Proteins (SIPs) play an essential role in a wide range of biological processes, such as gene expression regulation, signal transduction, enzyme activation and immune response. Because of the limitations for experimental self-interaction proteins identification, developing an effective computational method based on protein sequence to detect SIPs is much important. In the study, we proposed a novel computational approach called RVMBIGP that combines the Relevance Vector Machine (RVM) model and Bi-gram probability (BIGP) to predict SIPs based on protein sequence. The proposed prediction model includes as following steps: (1) an effective feature extraction method named BIGP is used to represent protein sequences on Position Specific Scoring Matrix (PSSM); (2) Principal Component Analysis (PCA) method is employed for integrating the useful information and reducing the influence of noise; (3) the robust classifier Relevance Vector Machine (RVM) is used to carry out classification. When performed on yeast and human datasets, the proposed RVMBIGP model can achieve very high accuracies of 95.48% and 98.80%, respectively. The experimental results show that our proposed method is very promising and may provide a cost-effective alternative for SIPs identification. In addition, to facilitate extensive studies for future proteomics research, the RVMBIGP server is freely available for academic use at http://219.219.62.123:8888/RVMBIGP.

Fusarium head blight (FHB) is one of the most destructive diseases of wheat and other cereals worldwide. During infection, the Fusarium fungi produce mycotoxins that represent a high risk to human and animal health. Developing small-molecule inhibitors to specifically reduce mycotoxin levels would be highly beneficial since current treatments unspecifically target the Fusarium pathogen. Culmorin possesses a well-known important synergistically virulence role among mycotoxins, and longiborneol synthase appears to be a key enzyme for its synthesis, thus making longiborneol synthase a particularly interesting target. This study aims to discover potent and less toxic agrochemicals against FHB. These compounds would hamper culmorin synthesis by inhibiting longiborneol synthase. In order to select starting molecules for further investigation, we have conducted a structure-based virtual screening investigation. A longiborneol synthase structural model is first built using homology modeling, followed by molecular dynamics simulations that provided the required input for a protein-ligand ensemble docking procedure. From this strategy, the three most interesting compounds (hits) were selected among the 25 top-ranked docked compounds from a library of 15,000 drug-like compounds. These putative inhibitors of longiborneol synthase provide a sound starting point for further studies involving molecular modeling coupled to biochemical experiments. This process could eventually lead to the development of novel approaches to reduce mycotoxin contamination in harvested grain.

The mitogen-activated protein kinase (MAPK) is characterized by the presence of the T-E-Y, T-D-Y, and T-G-Y motifs in its activation loop region and plays a significant role in regulating diverse cellular responses in eukaryotic organisms. Availability of large-scale genome data in the fungal kingdom encouraged us to identify and analyse the fungal MAPK gene family consisting of 173 fungal species. The analysis of the MAPK gene family resulted in the discovery of several novel activation loop motifs (T-T-Y, T-I-Y, T-N-Y, T-H-Y, T-S-Y, K-G-Y, T-Q-Y, S-E-Y and S-D-Y) in fungal MAPKs. The phylogenetic analysis suggests that fungal MAPKs are non-polymorphic, had evolved from their common ancestors around 1500 million years ago, and are distantly related to plant MAPKs. We are the first to report the presence of nine novel activation loop motifs in fungal MAPKs. The specificity of the activation loop motif plays a significant role in controlling different growth and stress related pathways in fungi. Hence, the presences of these nine novel activation loop motifs in fungi are of special interest.

Background: Black shank is a severe plant disease caused by the soil-borne pathogen Phytophthora nicotianae. Two physiological races of P. nicotianae, races 0 and 1, are predominantly observed in cultivated tobacco fields around the world. Race 0 has been reported to be more aggressive, having a shorter incubation period, and causing worse root rot symptoms, while race 1 causes more severe necrosis. The molecular mechanisms underlying the difference in virulence between race 0 and 1 remain elusive., Findings: We assembled and annotated the genomes of P. nicotianae races 0 and 1, which were obtained by a combination of PacBio single-molecular real-time sequencing and second-generation sequencing (both HiSeq and MiSeq platforms). Gene family analysis revealed a highly expanded ATP-binding cassette transporter gene family in P. nicotianae. Specifically, more RxLR effector genes were found in the genome of race 0 than in that of race 1. In addition, RxLR effector genes were found to be mainly distributed in gene-sparse, repeat-rich regions of the P. nicotianae genome., Conclusions: These results provide not only high quality reference genomes of P. nicotianae, but also insights into the infection mechanisms of P. nicotianae and its co-evolution with the host plant. They also reveal insights into the difference in virulence between the two physiological races.