Eliane Ferreira Noronha

Identification of mycoparasitism-related genes against the phytopathogen Sclerotinia sclerotiorum through transcriptome and expression profile analysis in Trichoderma harzianum

BackgroundThe species of T. harzianum are well known for their biocontrol activity against plant pathogens. However, few studies have been conducted to further our understanding of its role as a biological control agent against S. sclerotiorum, a pathogen involved in several crop diseases around the world. In this study, we have used RNA-seq and quantitative real-time PCR (RT-qPCR) techniques in order to explore changes in T. harzianum gene expression during growth on cell wall of S. sclerotiorum (SSCW) or glucose. RT-qPCR was also used to examine genes potentially involved in biocontrol, during confrontation between T. harzianum and S. sclerotiorum.ResultsData obtained from six RNA-seq libraries were aligned onto the T. harzianum CBS 226.95 reference genome and compared after annotation using the Blast2GO suite. A total of 297 differentially expressed genes were found in mycelia grown for 12, 24 and 36 h under the two different conditions: supplemented with glucose or SSCW. Functional annotation of these genes identified diverse biological processes and molecular functions required during T. harzianum growth on SSCW or glucose. We identified various genes of biotechnological value encoding proteins with functions such as transporters, hydrolytic activity, adherence, appressorium development and pathogenesis. To validate the expression profile, RT-qPCR was performed using 20 randomly chosen genes. RT-qPCR expression profiles were in complete agreement with the RNA-Seq data for 17 of the genes evaluated. The other three showed differences at one or two growth times. During the confrontation assay, some genes were up-regulated during and after contact, as shown in the presence of SSCW which is commonly used as a model to mimic this interaction.ConclusionsThe present study is the first initiative to use RNA-seq for identification of differentially expressed genes in T. harzianum strain TR274, in response to the phytopathogenic fungus S. sclerotiorum. It provides insights into the mechanisms of gene expression involved in mycoparasitism of T. harzianum against S.sclerotiorum. The RNA-seq data presented will facilitate improvement of the annotation of gene models in the draft T. harzianum genome and provide important information regarding the transcriptome during this interaction.

Insecticidal activity of two proteases against Spodoptera frugiperda larvae infected with recombinant baculoviruses

BackgroundBaculovirus comprise the largest group of insect viruses most studied worldwide, mainly because they efficiently kill agricutural insect pests. In this study, two recombinant baculoviruses containing the ScathL gene from Sarcophaga peregrina (vSynScathL), and the Keratinase gene from the fungus Aspergillus fumigatus (vSynKerat), were constructed. and their insecticidal properties analysed against Spodoptera frugiperda larvae.ResultsBioassays of third-instar and neonate S. frugiperda larvae with vSynScathL and vSynKerat showed a decrease in the time needed to kill the infected insects when compared to the wild type virus. We have also shown that both recombinants were able to increase phenoloxidase activity in the hemolymph of S. frugiperda larvae. The expression of proteases in infected larvae resulted in destruction of internal tissues late in infection, which could be the reason for the increased viral speed of kill.ConclusionsBaculoviruses and their recombinant forms constitute viable alternatives to chemical insecticides. Recombinant baculoviruses containing protease genes can be added to the list of engineered baculoviruses with great potential to be used in integrated pest management programs.

Heterologous production of Aspergillus fumigatus keratinase in Pichia pastoris

Aspergillus fumigatus Fresenius was previously shown to grow in mineral medium containing chicken feather flour as carbon and nitrogen source. Substantial proteolytic keratin-degrading activity was present in the culture supernatant after 24–72 h of growth at 42 °C. The keratinase was successfully purified by a single ion exchange chromatographic procedure and had a molecular mass of 31 kDa as determined by SDS–PAGE. The keratinase cDNA was expressed in Pichia pastoris cells and the recombinant clones were shown to be able to produce substantial caseinolytic, azo-keratinolytic and keratinolytic activities. SDS–PAGE and Western-blotting analysis using antibody against keratinase of A. fumigatus showed the presence of a single protein in the culture supernatants of several recombinant P. pastoris cells. This protein had a molecular mass corresponding to that of the A. fumigatus keratinase. The enzyme production profile showed that theP. pastoris recombinant cells produced an increasing amount of proteolytic and azo-keratinolytic activities over a 72 h growth period. Dry weight determination analysis indicated that 10% of the keratin flour was hydrolysed over a 24 h incubation period with 510 U (caseinolytic activity) of the recombinant keratinase.

Analysis of Phaseolus vulgaris Response to Its Association with Trichoderma harzianum (ALL-42) in the Presence or Absence of the Phytopathogenic Fungi Rhizoctonia solani and Fusarium solani

The present study was carried out to evaluate the ability of Trichoderma harzianum (ALL 42-isolated from Brazilian Cerrado soil) to promote common bean growth and to modulate its metabolism and defense response in the presence or absence of the phytopathogenic fungi Rhizoctonia solani and Fusarium solani using a proteomic approach. T. harzianum was able to promote common bean plants growth as shown by the increase in root/foliar areas and by size in comparison to plants grown in its absence. The interaction was shown to modulate the expression of defense-related genes (Glu1, pod3 and lox1) in roots of P. vulgaris. Proteomic maps constructed using roots and leaves of plants challenged or unchallenged by T. harzianum and phytopathogenic fungi showed differences. Reference gels presented differences in spot distribution (absence/presence) and relative volumes of common spots (up or down-regulation). Differential spots were identified by peptide fingerprinting MALDI-TOF mass spectrometry. A total of 48 identified spots (19 for leaves and 29 for roots) were grouped into protein functional classes. For leaves, 33%, 22% and 11% of the identified proteins were categorized as pertaining to the groups: metabolism, defense response and oxidative stress response, respectively. For roots, 17.2%, 24.1% and 10.3% of the identified proteins were categorized as pertaining to the groups: metabolism, defense response and oxidative stress response, respectively.

New dioxygenase from metagenomic library from Brazilian soil: insights into antibiotic resistance and bioremediation

AbstractObjectives Putative new dioxygenases were identified in a metagenomic β-lactam-resistance screening and, given their key role on aromatic metabolism, we raise the hypothesis that these enzymes maybe concomitantly related to antibiotic resistance and aromatic degradation.ResultsORFs of three putative dioxygenases were isolated from resistant metagenomic clones. One of them, CRB2(1), was subcloned into pET24a expression vector and subjected to downstream phenotypic and bioinformatics analyses that demonstrated the “dual effect” of our metagenomic dioxygenase, on antibiotic and aromatic resistance. Furthermore, initial characterization assays strongly suggests that CRB2(1) open-reading frame is an extradiol-dioxygenase, most probably a bicupin domain gentisate 1,2-dioxygenase. This observation is, to our knowledge, the first description of a metagenomic dioxygenase and its action on β-lactam resistance.ConclusionUnraveling the diversity of antibiotic resistance elements on the environment could not only identify new genes and mechanisms in which bacteria can resist to antibiotics, but also contribute to biotechnology processes, such as in bioremediation.

Production of Brazilian human norovirus VLPs and comparison of purification methods.

Abstract Noroviruses (NVs) are responsible for most cases of human nonbacterial gastroenteritis worldwide. Some parameters for the purification of NV virus-like particles (VLPs) such as ease of production and yield were studied for future development of vaccines and diagnostic tools. In this study, VLPs were produced by the expression of the VP1 and VP2 gene cassette of the Brazilian NV isolate, and two purification methods were compared: cesium chloride (CsCl) gradient centrifugation and ion-exchange chromatography (IEC). IEC produced more and purer VLPs of NV compared to CsCl gradient centrifugation.

Biochemical and structural characterization of a protein complex containing a hyaluronidase and a CRISP-like protein isolated from the venom of the spider Acanthoscurria natalensis

Spider venoms are composed of a complex mixture of bioactive molecules. The structural and functional characterization of these molecules in the venom of the Brazilian spider Acanthoscurria natalensis, has been little explored. The venom was fractionated using reversed-phase liquid chromatography. The fraction with hyaluronidase activity was named AnHyal. The partial sequencing of AnHyal revealed the presence of a CRISP-like protein, in addition to hyaluronidase, comprising 67% coverage for hyaluronidase from Brachypelma vagans and 82% for CRISP-like protein from Grammostola rosea. 1D BN-PAGE zymogram assays of AnHyal confirmed the presence of enzymatically active 53 kDa monomer and 124 and 178 kDa oligomers. The decomposition of the complexes by 2D BN/SDS-PAGE zymogram assays showed two subunits, 53 (AnHyalH) and 44 kDa (AnHyalC), with sequence similarity to hyaluronidase and CRISP proteins, respectively. The secondary structure of AnHyal is composed by 36% of α-helix. AnHyal presented maximum activity at pH between 4.0 and 6.0 and 30 and 60 °C, showed specificity to hyaluronic acid substrate and presented a KM of 617.9 μg/mL. Our results showed that hyaluronidase and CRISP proteins can form a complex and the CRISP protein may contribute to the enzymatic activity of AnHyalH.

Heterologous expression and characterization of a putative glycoside hydrolase family 43 arabinofuranosidase from Clostridium thermocellum B8

An extensive list of putative cellulosomal enzymes from C. thermocellum is now available in the public databanks, however, most of these remain unvalidated with regard to their activity and expression control mechanisms. This is particularly true of those enzymes putatively involved in hemicellulose deconstruction. Our research group has been working on mapping and characterization of glycoside hydrolases produced by C. thermocellum B8, that are critical for lignocellulosic biomass deconstruction. One of the identified genes expressed during growth on sugar cane bagasse and straw is axb8, which encodes a putative cellulosomal GH43_29 α-arabinofuranosidase (EC 3.2.1.55) that has not previously been characterized at the molecular or kinetic levels. The AxB8 predicted amino acid sequence presented GH43 and dockerin domains, as well as a family 6 carbohydrate-binding module (CBM6). Also, it is a close homologue of Firmicutes putatives α-arabinofuranosidases, including cellulosomal proteins. Multiple alignment analysis grouped AxB8 in a cluster with four uncharacterized putative GH43_29 subfamily enzymes, all containing dockerin type I domain and CBM6 modules. Purified heterologously expressed AxB8 showed activity against the synthetic substrates pNPX (p-nytrophenyl-β-d-xylopyranoside) and pNPA (p-nytrophenyl-α-l-arabinofuranoside), as well as against the natural substrate wheat arabinoxylan (WAX), with maximal activity at 50°C and pH between 5.0 and 6.0. The WAX degradation profile by AxB8 is different from those typically seen for α-arabinofuranosidases, presenting mainly xylose as a hydrolysis product, instead of arabinose. In addition, unlike other GH43_29 enzymes already characterized, AxB8 did not present activity against arabinan. Kinetic parameters using pNPA as a substrate were Km of 23±3mM and kcat of 104±7s-1. Despite its activity against pNPX, we did not observe AxB8 saturation with this substrate. AxB8 is the first member in its clade to be characterized regarding kinetic parameters, and together with its closest homologues could represent a large group of glycoside hydrolases with particular properties within the GH43_29 subfamily.

Trichoderma : A Dual Function Fungi and Their Use in the Wine and Beer Industries

The genus Trichoderma was first described in 1794 and presents a link to the sexual state Hypocrea . Whilst classification in the genus Trichoderma or Hypocrea using morphological characters is limited, an online identification tool was developed for accurate identification of all species within the genus Trichoderma. A group of Trichoderma species are described which share interesting features, including adaptation to different environmental conditions, metabolism of different biomolecules or compounds, and fast growth rate. These metabolic features place these Trichoderma species as the principal decomposers of the ecosphere and also raise attention of researchers for exploitation of their metabolism for industrial purposes. Indeed, Trichoderma species have been applied in different biotechnological applications such as a biological control, biofertilizers, as a source of industrial enzymes and as protein producers. The present chapter will present an introduction to industrial Trichoderma species and their application in wine and beer industries.