Mixed Transcriptome Analysis Revealed the Possible Interaction Mechanisms between Zizania latifolia and Ustilago esculenta Inducing Jiaobai Stem-Gall Formation.

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.

Proteome Analysis and In Vitro Antiviral, Anticancer and Antioxidant Capacities of the Aqueous Extracts of Lentinula edodes and Pleurotus ostreatus Edible Mushrooms.

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 Cutinase Bdo_10846 Play an Important Role in the Virulence of Botryosphaeria dothidea and in Inducing the Wart Symptom on Apple Plant.

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.

Bioinformatic mapping of a more precise Aspergillus niger degradome.

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.

Diversity of Omega Glutathione Transferases in mushroom-forming fungi revealed by phylogenetic, transcriptomic, biochemical and structural approaches.

The Omega class of glutathione transferases (GSTs) forms a distinct class within the cytosolic GST superfamily because most of them possess a catalytic cysteine residue. The human GST Omega 1 isoform was first characterized twenty years ago, but it took years of work to clarify the roles of the human isoforms. Concerning the kingdom of fungi, little is known about the cellular functions of Omega glutathione transferases (GSTOs), although they are widely represented in some of these organisms. In this study, we re-assess the phylogeny and the classification of GSTOs based on 240 genomes of mushroom-forming fungi (Agaricomycetes). We observe that the number of GSTOs is not only extended in the order of Polyporales but also in other orders such as Boletales. Our analysis leads to a new classification in which the fungal GSTOs are divided into two Types A and B. The catalytic residue of Type-A is either cysteine or serine, while that of Type-B is cysteine. The present study focuses on Trametes versicolor GSTO isoforms that possess a catalytic cysteine residue. Transcriptomic data show that Type-A GSTOs are constitutive enzymes while Type-B are inducible ones. The crystallographic analysis reveals substantial structural differences between the two types while they have similar biochemical profiles in the tested conditions. Additionally, these enzymes have the ability to bind antioxidant molecules such as wood polyphenols in two possible binding sites as observed from X-ray structures. The multiplication of GSTOs could allow fungal organisms to adapt more easily to new environments.

Lighting Conditions Influence the Dynamics of Protease Synthesis and Proteasomal Activity in the White Rot Fungus Cerrena unicolor.

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.

Succinyl-proteome profiling of Pyricularia oryzae, a devastating phytopathogenic fungus that causes rice blast disease.

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*******Ksuc, and K**Ksuc, 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.

Involvement of the cysteine protease BcAtg4 in development and virulence of Botrytis cinerea.

Autophagy serves as a survival mechanism against starvation and has been reported to be important for cell growth and differentiation in eukaryotes. Here, we investigated the function of a cysteine protease BcAtg4 in the gray mold fungus Botrytis cinerea. Yeast complementation experiments revealed that Bcatg4 can functionally replace the counterpart of yeast. Subcellular localization exhibited that BcAtg4 diffused in cytoplasm at different developmental stages. Targeted gene deletion of Bcatg4 (ΔBcatg4) led to autophagy blocking and a significant retardation in growth and conidiation. In addition, ΔBcatg4 failed to form sclerotia. Infection tests demonstrated that ΔBcatg4 was severely attenuated in virulence on different host plant tissues. All of the phenotypic defects were restored by reintroducing an intact copy of Bcatg4 into ΔBcatg4. These results indicate that Bcatg4 plays multiple roles in the developmental processes and pathogenesis of B. cinerea.

Cross-Chemistry Leads to Product Diversity from Atromentin Synthetases in Aspergilli from Section Nigri.

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.

Functional diversification accompanies gene family expansion of MED2 homologs in Candida albicans.

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.

Anthropogenic N Deposition Alters the Composition of Expressed Class II Fungal Peroxidases.

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.

Specificity of peptidases secreted by filamentous fungi.

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.

Eukaryotic copper-only superoxide dismutases (SODs): A new class of SOD enzymes and SOD-like protein domains.

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.

Identification of self-interacting proteins by exploring evolutionary information embedded in PSI-BLAST-constructed position specific scoring matrix.

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.

Involvement of the Fusarium graminearum cerato-platanin proteins in fungal growth and plant infection.

The genome of Fusarium graminearum, a necrotrophic fungal pathogen causing Fusarium head blight (FHB) disease of wheat, barley and other cereal grains, contains five genes putatively encoding for proteins with a cerato-platanin domain. Cerato-platanins are small secreted cysteine-rich proteins possibly localized in the fungal cell walls and also contributing to the virulence. Two of these F. graminearum proteins (FgCPP1 and FgCPP2) belong to the class of SnodProt proteins which exhibit phytotoxic activity in the fungal pathogens Botrytis cinerea and Magnaporthe grisea. In order to verify their contribution during plant infection and fungal growth, single and double gene knock-out mutants were produced and no reduction in symptoms severity was observed compared to the wild type strain on both soybean and wheat spikes. Histological analysis performed by fluorescence microscopy on wheat spikelets infected with mutants constitutively expressing the dsRed confirmed that FgCPPs do not contribute to fungal virulence. In particular, the formation of compound appressoria on wheat paleas was unchanged. Looking for other functions of these proteins, the double mutant was characterized by in vitro experiments. The mutant was inhibited by salt and H2O2 stress similarly to wild type. Though no growth difference was observed on glucose, the mutant grew better than wild type on carboxymethyl cellulose. Additionally, the mutant's mycelium was more affected by treatments with chitinase and ß-1,3-glucanase, thus indicating that FgCPPs could protect fungal cell wall polysaccharides from enzymatic degradation.

Sequences of stilboflavin C: towards the peptaibiome of the filamentous fungus Stilbella (= Trichoderma) flavipes.

Filamentous fungi of the genus Stilbella are recognized as an abundant source of naturally occurring α-aminoisobutyric acid-containing peptides. The culture broth of Stilbella (Trichoderma) flavipes CBS 146.81 yielded a mixture of peptides named stilboflavins (SF), and these were isolated and separated by preparative TLC into groups named SF-A, SF-B, and SF-C. Although all three of these groups resolved as single spots on thin-layer chromatograms, HPLC analysis revealed that each of the groups represents very microheterogeneous mixtures of closely related peptides. Here, we report on the sequence analysis of SF-C peptides, formerly isolated by preparative TLC. HPLC coupled to QqTOF-ESI-HRMS provided the sequences of 10 16-residue peptides and five 19-residue peptides, all of which were N-terminally acetylated. In contrast to the previously described SF-A and SF-B peptaibols, SF-C peptaibols contain Ser-Alaol or Ser-Leuol, which are rarely found as C-termini, and repetitive Leu-Aib-Gly sequences, which have not been detected in peptaibols before. Taking the previously determined sequences of SF-A and SF-B into account, the entirety of peptides produced by S. flavipes (the 'peptaibiome') approaches or exceeds 100 non-ribosomally biosynthesized peptaibiotics. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Genomes and virulence difference between two physiological races of Phytophthora nicotianae.

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.

Genome Sequence and Architecture of the Tobacco Downy Mildew Pathogen Peronospora tabacina.

Peronospora tabacina is an obligate biotrophic oomycete that causes blue mold or downy mildew on tobacco (Nicotiana tabacum). It is an economically important disease occurring frequently in tobacco-growing regions worldwide. We sequenced and characterized the genomes of two P. tabacina isolates and mined them for pathogenicity-related proteins and effector-encoding genes. De novo assembly of the genomes using Illumina reads resulted in 4,016 (63.1 Mb, N50 = 79 kb) and 3,245 (55.3 Mb, N50 = 61 kb) scaffolds for isolates 968-J2 and 968-S26, respectively, with an estimated genome size of 68 Mb. The mitochondrial genome has a similar size (approximately 43 kb) and structure to those of other oomycetes, plus several minor unique features. Repetitive elements, primarily retrotransposons, make up approximately 24% of the nuclear genome. Approximately 18,000 protein-coding gene models were predicted. Mining the secretome revealed approximately 120 candidate RxLR, six CRN (candidate effectors that elicit crinkling and necrosis), and 61 WY domain-containing proteins. Candidate RxLR effectors were shown to be predominantly undergoing diversifying selection, with approximately 57% located in variable gene-sparse regions of the genome. Aligning the P. tabacina genome to Hyaloperonospora arabidopsidis and Phytophthora spp. revealed a high level of synteny. Blocks of synteny show gene inversions and instances of expansion in intergenic regions. Extensive rearrangements of the gene-rich genomic regions do not appear to have occurred during the evolution of these highly variable pathogens. These assemblies provide the basis for studies of virulence in this and other downy mildew pathogens.

Characterization and application of a novel class II thermophilic peroxidase from Myceliophthora thermophila in biosynthesis of polycatechol.

A peroxidase from the thermophilic fungus Myceliophthora thermophila that belongs to ascomycete Class II based on PeroxiBase classification was functionally expressed in methylotrophic yeast Pichia pastoris. The putative peroxidase from the genomic DNA was successfully cloned in P. pastoris X-33 under the transcriptional control of the alcohol oxidase (AOX1) promoter. The heterologous production was greatly enhanced by the addition of hemin with a titer of 0.41 U mL(-1) peroxidase activity at the second day of incubation. The recombinant enzyme was purified to homogeneity (50 kDa) and characterized using a series of phenolic substrates that indicated similar characteristics with those of generic peroxidases. In addition, the enzyme was found thermostable, retaining its activity for temperatures up to 60 °C after eight hours incubation. Moreover, the enzyme displayed remarkable H2O2 stability, retaining more than 80% of its initial activity after 24h incubation in 5000-fold molar excess of H2O2. The ability of the peroxidase to polymerize catechol at high superoxide concentrations, together with its high thermostability and substrate specificity, indicate a potential commercial significance of the enzyme.

A ribosomal protein AgRPS3aE from halophilic Aspergillus glaucus confers salt tolerance in heterologous organisms.

High salt in soils is one of the abiotic stresses that significantly reduces crop yield, although saline lands are considered potential resources arable for agriculture. Currently, genetic engineering for enhancing salt tolerance is being tested as an efficient and viable strategy for crop improvement. We previously characterized a large subunit of the ribosomal protein RPL44, which is involved in osmotic stress in the extremely halophilic fungus Aspergillus glaucus. Here, we screened another ribosomal protein (AgRPS3aE) that also produced high-salt tolerance in yeast. Bioinformatics analysis indicated that AgRPS3aE encodes a 29.2 kDa small subunit of a ribosomal protein belonging to the RPS3Ae family in eukaryotes. To further confirm its protective function against salinity, we expressed AgRPS3aE in three heterologous systems, the filamentous fungus Magnaporthe oryzae and two model plants Arabidopsis and tobacco. Overexpression of AgRPS3aE in all tested transformants significantly alleviated stress symptoms compared with controls, suggesting that AgRPS3aE functions not only in fungi but also in plants. Considering that ribosomal proteins are housekeeping components in organisms from prokaryotes to eukaryotes, we propose that AgRPS3aE is one of the optimal genes for improving high-salt tolerance in crops.