Genômica estrutural e funcional de fungos do gênero Trichoderma

Tese (doutorado)—Universidade de Brasília, Instituto de Ciências Biológicas, Programa de Pós-Graduação em Biologia Molecular, 2016.O controle biológico é um processo complexo que inclui diferentes mecanismos e uma diversidade de vias metabólicas. Espécies de Trichoderma harzianum são conhecidas por sua atividade de biocontrole contra patógenos de plantas. Para melhor entender os mecanismos utilizados por T. harzianum no controle biológico, no presente trabalho foi sequenciado o genoma do isolado TR274 usando sequenciamento Illumina, assim como seu respectivo transcritoma na interação direta com Scletotinia sclerotiorum ou na presença de sua parede celular. A montagem do genoma feita utilizando o programa AllPaths-LG cobertura máxima de 100x, resultou em 2282 contigs, tamanho do genoma de 40,8 Mb e um conteúdo GC de 47.7%, similar aos outros genomas de Trichoderma. Um total de 13932 genes foram anotados. Análise do Core Eukariotic Genes Dataset (CEGMA) sugere que o genoma está 100% completo e 97,9% das sequencias de RNA-seq alinharam corretamente no genoma. A análise filogenética usando proteínas ortólogas com todas as espécies de Trichoderma sequenciadas no JGI, confirmam a divisão nas seções Tricoderma (T. asperellum e T. atroviride), Longibrachiatum (T. reesei, T. citrinoviride e T. longibrachiatum) e Pachibasium (T. harzianum e T. virens). Das proteínas ortólogas anotadas, 8242 compõem proteínas compartilhadas por todas as espécies, as proteínas espécie específicas variam de 262 (T. reesei) a 1803 (T. longibrachiatum). Os dois genomas de T. harzianum analisados sugerem uma alta similaridade entre eles, mesmo tendo sido isolados de locais e continentes distintos, um de solo de cerrado no Brasil e outro de solo de jardim na Inglaterra. Análises de genes envolvidos com o metabolismo secundário, CAZymes, transportadores, proteases e fatores de transcrição foram feitas. A seção Pachibasium expandiu virtualmente todas as categorias analisadas quando comparada com as outras seções. Análise CAFE mostrou uma correlação positiva entre estas expansões e o tamanho dos genomas. O subgrupo C1 das quitinases foi completamente perdido pela seção Longibrachiatum. Estes resultados sugerem que estas famílias proteicas tem um importante papel nos seus respectivos fenótipos. As abordagens transcritômicas mostraram que a interação entre T. harzianum e S. sclerotiorum é bem complexa e envolve a produção de metabólitos secundários e síntese de transportadores antes e durante o contato, com uma modulação principalmente de enzimas hidrolíticas após o contato. Dos genes encontrados diferencialmente expressos na condição de crescimento em parede celular, 86,8% foram também encontrados diferencialmente expressos na interação direta. Cerca de 25% de todo o genoma de T. harzianum foi modulado durante a interação com S. sclerotiorum.Biological control is a complex process, which requires many mechanisms and a high diversity of biochemical pathways. Trichoderma harzianum species complex are well known for their biocontrol activity against many plant pathogens. To gain new insights into the biocontrol mechanism employed by T. harzianum, we sequenced genome of the isolate TR274 with its transcriptome during direct interaction with the fungal pathogen Sclerotinia sclerotiorum and its cell wall, using Illumina sequencing. Whole genome assembly was performed using AllPaths-LG, with a maximum coverage of 100x. The assembly resulted in 2282 contigs, with an estimated genome size of 40.8 Mb and GC content of 47.7%, similar to other Trichoderma genomes. Using the JGI Annotation Pipeline we predicted 13,932 genes, with high transcriptome support. Core Eukariotic Genes Dataset (CEGMA) tests suggested 100% genome completeness and 97.9% of RNA-SEQ reads mapped to the genome. The phylogenetic comparison using orthologous proteins with all Trichoderma genomes sequenced at JGI, corroborates the Trichoderma section division described previously (T. asperellum and T. atroviride), Longibrachiatum (T. reesei, T. citrinoviride and T. longibrachiatum) and Pachibasium (T. harzianum and T. virens). A Venn diagram was built with orthologs proteins, with 8242 composing the core protein group and species specific varying from 262 proteins (T. reesei) to 1803 (T. longibrachiatum). The comparison between two Trichoderma harzianum CBS 226.95 and TR274 isolates, suggests a high genome similarity. Analyses of the secondary metabolites, CAZymes, transporters, proteases and transcription factors were performed. The Pachybasium section expanded virtually all categories analyzed compared with the other sections. CAFE analysis showed positive correlation between these families and genome size. The chitinase subgroup C1 was completely absent in Longibrachiatum section members. These results suggest that these proteins families play an important role on their respective phenotypes. Transcriptome analysis suggests that the interaction between T. harzianum and S. sclerotiorum is complex, involving production of secondary metabolites and transporters before and during the contact, with a modulation of CAZymes after contact. A total of 86.8% of differentially expressed genes during growth on Sclerotinia sclerotiorum cell wall were found during direct interaction. Approximately the 25% of whole T. harzianum genome was seen to be modulated during the interaction with S. sclerotiorum

Biochemical characterization of a 27kDa 1,3-β-d-glucanase from Trichoderma asperellum induced by cell wall of Rhizoctonia solani

AbstractTrichoderma asperellum produces two extracellular 1,3-β-d-glucanase upon induction with cell walls from Rhizoctonia solani. A minor 1,3-β-d-glucanase was purified to homogeneity by ion exchange chromatography on Q-Sepharose and gel filtration on Sephacryl S-100. A typical procedure provided 13.8-fold purification with 70% yield. SDS–PAGE of the purified enzyme showed a single protein band of molecular weight 27kDa. The enzyme exhibited optimum catalytic activity at pH 3.6 and 45°C. It was thermostable at 40°C, and retained 75% activity after 60min at 45°C. The Km and Vmax values for 1,3-β-d-glucanase, using laminarin as substrate, were 0.323mgml−1 and 0.315Umin−1, respectively. The enzyme was strongly inhibited by Hg2+ and SDS. The enzyme was only active toward glucans containing β-1,3-linkages. Peptide sequences showed similarity with two endo-1,3(4)-β-d-glucanases from Aspergillus fumigatus Af293when compared against GenBank non-redundant database

Identification of differentially expressed genes from Trichoderma harzianum during growth on cell wall of Fusarium solani as a tool for biotechnological application

Background: The species of T. harzianum are well known for their biocontrol activity against many plant pathogens. However, there is a lack of studies concerning its use as a biological control agent against F. solani, a pathogen involved in several crop diseases. In this study, we have used subtractive library hybridization (SSH) and quantitative real-time PCR (RT-qPCR) techniques in order to explore changes in T. harzianum genes expression during growth on cell wall of F. solani (FSCW) or glucose. RT-qPCR was also used to examine the regulation of 18 genes, potentially involved in biocontrol, during confrontation between T. harzianum and F. solani. Results: Data obtained from two subtractive libraries were compared after annotation using the Blast2GO suite. A total of 417 and 78 readable EST sequence were annotated in the FSCW and glucose libraries, respectively. Functional annotation of these genes identified diverse biological processes and molecular functions required during T. harzianum growth on FSCW or glucose. We identified various genes of biotechnological value encoding to proteins which function such as transporters, hydrolytic activity, adherence, appressorium development and pathogenesis. Fifteen genes were up-regulated and sixteen were down-regulated at least at one-time point during growth of T. harzianum in FSCW. During the confrontation assay most of the genes were up-regulated, mainly after contact, when the interaction has been established. Conclusions: This study demonstrates that T. harzianum expressed different genes when grown on FSCW compared to glucose. It provides insights into the mechanisms of gene expression involved in mycoparasitism of T. harzianum against F. solani. The identification and evaluation of these genes may contribute to the development of an efficient biological control agent.This work was funded by Research and Projects Financing (FINEP) and the National Council for Scientific and Technological Development (CNPq) and Fundação de Amparo à Pesquisa do Estado de Goiás (FAPEG). C.J.U is supported by a biotechnology research grant (FAPEGO and CNPq). PMV has benefited awarded with a scholarship from Capes

Il Cristo in forma Pietatis del Rosso Fiorentino fra devozione e bellezza

<p>Differential distribution pattern of root- identified proteins in the four conditions described: TT, 42T, 42FS and 42RS.</p

Construction and analisys of a cDNA library of Trichoderma harzianum grown in presence of Fusarium solani cell wall

Made available in DSpace on 2014-07-29T15:16:27Z (GMT). No. of bitstreams: 1 dissertacao andrei.pdf: 2548450 bytes, checksum: dedf19335cba3686a528075fb5da8300 (MD5) Previous issue date: 2010-02-26Species of Trichoderma are commercially applied as biological control agents against plant fungal pathogens. Their action is based on a set of events, such as the production of antifungal metabolites, competition for space and nutrients and mycoparasitism. However the biocontrol mechanism of the interaction between Trichoderma harzianum and Fusarium solani is not yet understanded at the transcriptome level. This study aimed to initiate the preliminary development of an expressed sequence tag (EST) database for Trichoderma harzianum and thereby gain potentially useful information on Trichoderma gene sequences in order to elucidate the integrated biocontrol mechanism. Partial sequencing of anonymous cDNA clones is a widely used technique for gene identification. A directional cDNA library has been constructed from mycelium of Trichoderma harzianum grown on cell wall isolated from Fusarium solani. 3984 clones have been randomly selected, subjected to single-pass sequencing from the 5 end of the vector, and identified by sequence similarity searches against gene sequences in GenBank fungal database. Of the 3984 mycelium clones, 40.8% exhibit similarity to known genes. Analysis of the identified clones indicated sequence similarity to a broad diversity of genes encoding proteins such as enzymes, structural proteins, and regulatory factors. A significant proportion of genes identified were involved in processes related to mycoparasitism (3,81%), as would be expected in biocontrol fungus. These results present the successful application of EST analysis in the interaction of Trichoderma harzianum and Fusarium solani to provide a preliminary indication of gene expression.Espécies do gênero Trichoderma são comercialmente aplicadas como agentes de controle biológico contra fungos patógenos de plantas. Sua ação é baseada em diferentes mecanismos como a produção de antibióticos voláteis e não-voláteis, competição por espaço e nutrientes e micoparasitismo. O mecanismo de biocontrole da interação entre Trichoderma harzianum e Fusarium solani ainda não foi explorado ao nível de transcriptoma. Este estudo buscou iniciar o desenvolvimento de uma base de dados de EST (Expressed Sequence Tags) para assim obter informações úteis nas sequências de Trichoderma harzianum e entender melhor as vias integradas do mecanismo de biocontrole. Uma biblioteca direcional de cDNA foi construída a partir do micélio de Trichoderma harzianum crescido em parede celular isolada de Fusarium solani. 3984 clones foram selecionados aleatoriamente, submetidos ao seqüenciamento da região 5 do vetor e identificados por similaridade contra a base de dados de seqüências de fungos GenBank. Dentre os 3984 clones, 40,8% mostraram similaridade com genes conhecidos e descritos na literatura. Análises dos genes identificados indicam similaridade com uma grande diversidade de genes que codificam para enzimas, proteínas estruturais e fatores de regulação. Uma proporção significante dos genes identificados está envolvida com processos relacionados ao icoparasitismo (3,81%), como esperado para um fungo envolvido no controle biológico. Estes resultados mostram uma aplicação bem sucedida da análise de ESTs na interação entre Trichoderma harzianum e Fusarium solani e dar uma sugetão da expressão gênica

Physiological characterization and transcriptome analysis of Pichia pastoris reveals its response to lignocellulose-derived inhibitors.

The negative effects of lignocellulose-derived inhibitors such as acetic acid and furaldehydes on microbial metabolism constitute a significant drawback to the usage of biomass feedstocks for the production of fuels and chemicals. The yeast Pichia pastoris has shown a great biotechnological potential for producing heterologous proteins and renewable chemicals. Despite its relevance, the performance of P. pastoris in presence of lignocellulose-derived inhibitors remains unclear. In this work, our results show for the first time the dose-dependent response of P. pastoris to acetic acid, furaldehydes (HMF and furfural), and sugarcane biomass hydrolysate, both at physiological and transcriptional levels. The yeast was able to grow in synthetic media with up to 6&nbsp;g.L-1 acetic acid, 1.75&nbsp;g.L-1 furaldehydes or hydrolysate diluted to 10% (v/v). However, its metabolism was completely hindered in presence of hydrolysate diluted to 30% (v/v). Additionally, the yeast was capable to co-consume acetic acid and glucose. At the transcriptional level, P. pastoris response to lignocellulose-derived inhibitors relays on the up-regulation of genes related to transmembrane transport, oxidoreductase activities, RNA processing, and the repression of pathways related to biosynthetic processes and central carbon metabolism. These results demonstrate a polygenetic response that involves detoxification activities, and maintenance of energy and cellular homeostasis. In this context, ALD4, OYE3, QOR2, NTL100, YCT1, and PPR1 were identified as target genes to improve P. pastoris tolerance. Altogether, this work provides valuable insights into the P. pastoris stress tolerance, which can be useful to expand its use in different bioprocesses

Trichoderma harzianum expressed sequence tags for identification of genes with putative roles in mycoparasitism against Fusarium solani

The plant pathogen Fusarium solani causes a disease root rot of common bean (Phaseolus vulgaris) resulting in great losses of yield in irrigated areas of the Southeast and Midwest regions of Brazil. Species of the genus Trichoderma have been used in the biological control of this pathogen as an alternative to chemical control. To gain new insights into the biocontrol mechanism used by Trichoderma harzianum against the phytopathogenic fungus, Fusarium solani, we performed a transcriptome analysis using expressed sequence tags (ESTs) and quantitative real-time PCR (RT-qPCR) approaches. A cDNA library from T. harzianum mycelium (isolate ALL42) grown on cell walls of F. solani (CWFS) was constructed and analyzed. A total of 2927 high quality sequences were selected from 3845 and 37.7% were identified as unique genes. The Gene Ontology analysis revealed that the majority of the annotated genes are involved in metabolic processes (80.9%), followed by cellular process (73.7%). We tested twenty genes that encode proteins with potential role in biological control. RT-qPCR analysis showed that none of these genes were expressed when T. harzianum was challenged with itself. These genes showed different patterns of expression during in vitro interaction between T. harzianum and F. solani. (C) 2012 Elsevier Inc. All rights reserved.FINEPFINEPNational Council for Scientific and Technological Development (CNPq) (FINEP/CT-AGRO/Acao) [01.07.0551.00, 0845/07]National Council for Scientific and Technological Development (CNPq) (FINEP/CTAGRO/Acao)FAPEGOFAPEGOCNPqCNP

Draft Genome Sequence of Stenotrophomonas maltophilia Strain PE591, a Polyethylene-Degrading Bacterium Isolated from Savanna Soil.

We report the genome sequence of a polyethylene-degrading bacterial strain identified as Stenotrophomonas maltophilia strain PE591, which was isolated from plastic debris found in savanna soil. The genome was assembled in 16 scaffolds with a length of 4,751,236 bp, a GC content of 66.5%, and 4,432 predicted genes

Draft Genome Sequence of Stenotrophomonas maltophilia Strain PE591, a Polyethylene-Degrading Bacterium Isolated from Savanna Soil.

We report the genome sequence of a polyethylene-degrading bacterial strain identified as Stenotrophomonas maltophilia strain PE591, which was isolated from plastic debris found in savanna soil. The genome was assembled in 16 scaffolds with a length of 4,751,236 bp, a GC content of 66.5%, and 4,432 predicted genes