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Microbial communities in the world ocean are affected strongly by oceanic circulation, creating characteristic marine biomes. The high connectivity of most of the ocean makes it difficult to disentangle selective retention of colonizing genotypes (with traits suited to biome specific conditions) from evolutionary selection, which would act on founder genotypes over time. The Arctic Ocean is exceptional with limited exchange with other oceans and ice covered since the last ice age. To test whether Arctic microalgal lineages evolved apart from algae in the global ocean, we sequenced four lineages of microalgae isolated from Arctic waters and sea ice. Here we show convergent evolution and highlight geographically limited HGT as an ecological adaptive force in the form of PFAM complements and horizontal acquisition of key adaptive genes. Notably, ice-binding proteins were acquired and horizontally transferred among Arctic strains. A comparison withTaraOceans metagenomes and metatranscriptomes confirmed mostly Arctic distributions of these IBPs. The phylogeny of Arctic-specific genes indicated that these events were independent of bacterial-sourced HGTs in Antarctic Southern Ocean microalgae.

Humicola grisea var. thermoidea is a thermophilic ascomycete and important enzyme producer that has an efficient enzymatic system with a broad spectrum of thermostable carbohydrate-active (CAZy) enzymes. These enzymes can be employed in lignocellulose biomass deconstruction and other industrial applications. In this work, the genome of H. grisea var. thermoidea was sequenced. The acquired sequence reads were assembled into a total length of 28.75 Mbp. Genome features correlate with what was expected for thermophilic Sordariomycetes. The transcriptomic data showed that sugarcane bagasse significantly upregulated genes related to primary metabolism and polysaccharide deconstruction, especially hydrolases, at both pH 5 and pH 8. However, a number of exclusive and shared genes between the pH values were found, especially at pH 8. H. grisea expresses an average of 211 CAZy enzymes (CAZymes), which are capable of acting in different substrates. The top upregulated genes at both pH values represent CAZyme-encoding genes from different classes, including acetylxylan esterase, endo-1,4-β-mannosidase, exoglucanase, and endoglucanase genes. For the first time, the arsenal that the thermophilic fungus H. grisea var. thermoidea possesses to degrade the lignocellulosic biomass is shown. Carbon source and pH are of pivotal importance in regulating gene expression in this organism, and alkaline pH is a key regulatory factor for sugarcane bagasse hydrolysis. This work paves the way for the genetic manipulation and robust biotechnological applications of this fungus. IMPORTANCE Most studies regarding the use of fungi as enzyme producers for biomass deconstruction have focused on mesophile species, whereas the potential of thermophiles has been evaluated less. This study revealed, through genome and transcriptome analyses, the genetic repertoire of the biotechnological relevant thermophile fungus Humicola grisea. Comparative genomics helped us to further understand the biology and biotechnological potential of H. grisea. The results demonstrate that this fungus possesses an arsenal of carbohydrate-active (CAZy) enzymes to degrade the lignocellulosic biomass. Indeed, it expresses more than 200 genes encoding CAZy enzymes when cultivated in sugarcane bagasse. Carbon source and pH are key factors for regulating the gene expression in this organism. This work shows, for the first time, the great potential of H. grisea as an enzyme producer and a gene donor for biotechnological applications and provides the base for the genetic manipulation and robust biotechnological applications of this fungus.

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.

Large losses before crop harvesting are caused by plant pathogens, such as viruses, bacteria, oomycetes, fungi, and nematodes. Among these, fungi are the major cause of losses in agriculture worldwide. Plant pathogens are still controlled through application of agrochemicals, causing human disease and impacting environmental and food security. Biological control provides a safe alternative for the control of fungal plant pathogens, because of the ability of biocontrol agents to establish in the ecosystem. Some Trichoderma spp. are considered potential agents in the control of fungal plant diseases. They can interact directly with roots, increasing plant growth, resistance to diseases, and tolerance to abiotic stress. Furthermore, Trichoderma can directly kill fungal plant pathogens by antibiosis, as well as via mycoparasitism strategies. In this review, we will discuss the interactions between Trichoderma/fungal pathogens/plants during the pre-harvest of crops. In addition, we will highlight how these interactions can influence crop production and food security. Finally, we will describe the future of crop production using antimicrobial peptides, plants carrying pathogen-derived resistance, and plantibodies.

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 g.L−1 acetic acid, 1.75 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.

Forest fires generate a large amount of carbon that remains resident on the site as dead and partially 'pyrolysed' (i.e. burnt) material that has long residency times and constitutes a significant pool in fire-prone ecosystems. In addition, fire-induced hydrophobic soil layers, caused by condensation of pyrolysed waxes and lipids, increase post-fire erosion and can lead to long-term productivity losses. A small set of pyrophilous fungi dominate post-fire soils and are likely to be involved with the degradation of all these compounds, yet almost nothing is currently known about what these fungi do or the metabolic processes they employ. In this study, we sequenced and analysed genomes from fungi isolated after Rim fire near Yosemite National Park in 2013 and showed the enrichment/expansion of CAZymes and families known to be involved in fruiting body initiation when compared to other basidiomycete fungi. We found gene families potentially involved in the degradation of the hydrophobic layer and pyrolysed organic matter, such as hydrophobic surface binding proteins, laccases (AA1_1), xylanases (GH10, GH11), fatty acid desaturases and tannases. Thus, pyrophilous fungi are important actors to restate the soil's functional capabilities.

Trichoderma species are known for their ability to produce lytic enzymes, such as exoglucanases, endoglucanases, chitinases, and proteases, which play important roles in cell wall degradation of phytopathogens. &beta, glucanases play crucial roles in the morphogenetic-morphological process during the development and differentiation processes in Trichoderma species, which have &beta, glucans as the primary components of their cell walls. Despite the importance of glucanases in the mycoparasitism of Trichoderma spp., only a few functional analysis studies have been conducted on glucanases. In the present study, we used a functional genomics approach to investigate the functional role of the gluc31 gene, which encodes an endo-&beta, 1,3-glucanase belonging to the GH16 family in Trichoderma harzianum ALL42. We demonstrated that the absence of the gluc31 gene did not affect the in vivo mycoparasitism ability of mutant T. harzianum ALL42, however, gluc31 evidently influenced cell wall organization. Polymer measurements and fluorescence microscopy analyses indicated that the lack of the gluc31 gene induced a compensatory response by increasing the production of chitin and glucan polymers on the cell walls of the mutant hyphae. The mutant strain became more resistant to the fungicide benomyl compared to the parental strain. Furthermore, qRT-PCR analysis showed that the absence of gluc31 in T. harzianum resulted in the differential expression of other glycosyl hydrolases belonging to the GH16 family, because of functional redundancy among the glucanases.

The plant cell wall is a source of fermentable sugars in second-generation bioethanol production. However, cellulosic biomass hydrolysis remains an obstacle to bioethanol production in an efficient and low-cost process. Clostridium thermocellum has been studied as a model organism able to produce enzymatic blends that efficiently degrade lignocellulosic biomass, and also as a fermentative microorganism in a consolidated process for the conversion of lignocellulose to bioethanol. In this study, a C. thermocellum strain (designated B8) isolated from goat rumen was characterized for its ability to grow on sugarcane straw and cotton waste, and to produce cellulosomes. We also evaluated C. thermocellum gene expression control in the presence of complex lignocellulosic biomasses. This isolate is capable of growing in the presence of microcrystalline cellulose, sugarcane straw and cotton waste as carbon sources, producing free enzymes and residual substrate-bound proteins (RSBP). The highest growth rate and cellulase/xylanase production were detected at pH 7.0 and 60 °C, after 48 h. Moreover, this strain showed different expression levels of transcripts encoding cellulosomal proteins and proteins with a role in fermentation and catabolic repression.

Trichoderma species are known for their ability to produce lytic enzymes, such as exoglucanases, endoglucanases, chitinases, and proteases, which play important roles in cell wall degradation of phytopathogens. β-glucanases play crucial roles in the morphogenetic-morphological process during the development and differentiation processes in Trichoderma species, which have β-glucans as the primary components of their cell walls. Despite the importance of glucanases in the mycoparasitism of Trichoderma spp., only a few functional analysis studies have been conducted on glucanases. In the present study, we used a functional genomics approach to investigate the functional role of the gluc31 gene, which encodes an endo-β-1,3-glucanase belonging to the GH16 family in Trichoderma harzianum ALL42. We demonstrated that the absence of the gluc31 gene did not affect the in vivo mycoparasitism ability of mutant T. harzianum ALL42; however, gluc31 evidently influenced cell wall organization. Polymer measurements and fluorescence microscopy analyses indicated that the lack of the gluc31 gene induced a compensatory response by increasing the production of chitin and glucan polymers on the cell walls of the mutant hyphae. The mutant strain became more resistant to the fungicide benomyl compared to the parental strain. Furthermore, qRT-PCR analysis showed that the absence of gluc31 in T. harzianum resulted in the differential expression of other glycosyl hydrolases belonging to the GH16 family, because of functional redundancy among the glucanases.

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.

Xylitol is a five-carbon polyol of economic interest that can be produced by microbial xylose reduction from renewable resources. The current study sought to investigate the potential of two yeast strains, isolated from Brazilian Cerrado biome, in the production of xylitol as well as the genomic characteristics that may impact this process. Xylose conversion capacity by the new isolates Spathaspora sp. JA1 and Meyerozyma caribbica JA9 was evaluated and compared with control strains on xylose and sugarcane biomass hydrolysate. Among the evaluated strains, Spathaspora sp. JA1 was the strongest xylitol producer, reaching product yield and productivity as high as 0.74 g/g and 0.20 g/(L.h) on xylose, and 0.58 g/g and 0.44 g/(L.h) on non-detoxified hydrolysate. Genome sequences of Spathaspora sp. JA1 and M. caribbica JA9 were obtained and annotated. Comparative genomic analysis revealed that the predicted xylose metabolic pathway is conserved among the xylitol-producing yeasts Spathaspora sp. JA1, M. caribbica JA9 and Meyerozyma guilliermondii, but not in Spathaspora passalidarum, an efficient ethanol-producing yeast. Xylitol-producing yeasts showed strictly NADPH-dependent xylose reductase and NAD+-dependent xylitol-dehydrogenase activities. This imbalance of cofactors favors the high xylitol yield shown by Spathaspora sp. JA1, which is similar to the most efficient xylitol producers described so far.

Eighty-one Rhizoctonia-like isolates were identified based on morphology and nuclei-staining methods from natural and agricultural soils of the Cerrado (Brazilian savanna). The nucleotide similarity analysis of ITS1-5.8S-ITS2 regions identified 14 different taxa, with 39.5% of isolates assigned to Waitea circinata (zeae, oryzae, and circinata varieties), while 37.0% belonged to Thanatephorus cucumeris anastomosis groups (AGs) AG1-IB, AG1-ID, AG1-IE, AG4-HGI, and AG4-HGIII. Ceratobasidium spp. AG-A, AG-F, AG-Fa, AG-P, and AG-R comprised 23.5%. Rhizoctonia zeae (19.8%), R. solani AG1-IE (18.6%), and binucleate Rhizoctonia AG-A (8.6%) were the most frequent anamorphic states found. Root rot severity caused by the different taxa varied from low to high on common beans, and tended to be low to average in maize. Twenty-two isolates were pathogenic to both hosts, suggesting difficulties in managing Rhizoctonia root rots with crop rotation. These results suggest that cropping history affects the geographical arrangement of AGs, with a prevalence of AG1 in the tropical zone from central to north Brazil while the AG4 group was most prevalent from central to subtropical south. W. circinata var. zeae was predominant in soils under maize production. To our knowledge, this is the first report on the occurrence of W. circinata var. circinata in Brazil.

Background The growing importance of the ubiquitous fungal genus Trichoderma (Hypocreales, Ascomycota) requires understanding of its biology and evolution. Many Trichoderma species are used as biofertilizers and biofungicides and T. reesei is the model organism for industrial production of cellulolytic enzymes. In addition, some highly opportunistic species devastate mushroom farms and can become pathogens of humans. A comparative analysis of the first three whole genomes revealed mycoparasitism as the innate feature of Trichoderma. However, the evolution of these traits is not yet understood. Results We selected 12 most commonly occurring Trichoderma species and studied the evolution of their genome sequences. Trichoderma evolved in the time of the Cretaceous-Palaeogene extinction event 66 (±15) mya, but the formation of extant sections (Longibrachiatum, Trichoderma) or clades (Harzianum/Virens) happened in Oligocene. The evolution of the Harzianum clade and section Trichoderma was accompanied by significant gene gain, but the ancestor of section Longibrachiatum experienced rapid gene loss. The highest number of genes gained encoded ankyrins, HET domain proteins and transcription factors. We also identified the Trichoderma core genome, completely curated its annotation, investigated several gene families in detail and compared the results to those of other fungi. Eighty percent of those genes for which a function could be predicted were also found in other fungi, but only 67% of those without a predictable function. Conclusions Our study presents a time scaled pattern of genome evolution in 12 Trichoderma species from three phylogenetically distant clades/sections and a comprehensive analysis of their genes. The data offer insights in the evolution of a mycoparasite towards a generalist. Electronic supplementary material The online version of this article (10.1186/s12864-019-5680-7) contains supplementary material, which is available to authorized users.

Cadmium (Cd) is a heavy metal present in the environment mainly as a result of industrial contamination that can cause toxic effects to life. Some microorganisms, as Trichoderma harzianum, a fungus used in biocontrol, are able to survive in polluted environments and act as bioremediators. Aspects about the tolerance to the metal have been widely studied in other fungi although there are a few reports about the response of T. harzianum. In this study, we determined the effects of cadmium over growth of T. harzianum and used RNA-Seq to identify significant genes and processes regulated in the metal presence. Cadmium inhibited the fungus growth proportionally to its concentration although the fungus exhibited tolerance as it continued to grow, even in the highest concentrations used. A total of 3767 (1993 up and 1774 down) and 2986 (1606 up and 1380 down) differentially expressed genes were detected in the mycelium of T. harzianum cultivated in the presence of 1.0 mg mL−1 or 2.0 mg mL−1 of CdCl2, respectively, compared to the absence of the metal. Of these, 2562 were common to both treatments. Biological processes related to cellular homeostasis, transcription initiation, sulfur compound biosynthetic and metabolic processes, RNA processing, protein modification and vesicle-mediated transport were up-regulated. Carbohydrate metabolic processes were down-regulated. Pathway enrichment analysis indicated induction of glutathione and its precursor’s metabolism. Interestingly, it also indicated an intense transcriptional induction, especially by up-regulation of spliceosome components. Carbohydrate metabolism was repressed, especially the mycoparasitism-related genes, suggesting that the mycoparasitic ability of T. harzianum could be affected during cadmium exposure. These results contribute to the advance of the current knowledge about the response of T. harzianum to cadmium exposure and provide significant targets for biotechnological improvement of this fungus as a bioremediator and a biocontrol agent.

We report the whole-genome sequence of Muricauda sp. strain K001 isolated from a marine cyanobacterial culture. This genome sequence will improve our understanding of the influence of heterotrophic bacteria on the physiology of cyanobacteria and may contribute to the development of new natural products.

Expansins refer to a family of closely related non-enzymatic proteins found in the plant cell wall that are involved in the cell wall loosening. In addition, expansins appear to be involved in different physiological and environmental responses in plants such as leaf and stem initiation and growth, stomata opening and closing, reproduction, ripening and stress tolerance. Sugarcane (Saccharum spp.) is one of the main crops grown worldwide. Lignocellulosic biomass from sugarcane is one of the most promising raw materials for the ethanol industry. However, the efficient use of lignocellulosic biomass requires the optimization of several steps, including the access of some enzymes to the hemicellulosic matrix. The addition of expansins in an enzymatic cocktail or their genetic manipulation could drastically improve the saccharification process of feedstock biomass by weakening the hydrogen bonds between polysaccharides present in plant cell walls. In this study, the expansin gene family in sugarcane was identified and characterized by in silico analysis. Ninety two putative expansins in sugarcane (SacEXPs) were categorized in three subfamilies after phylogenetic analysis. The expression profile of some expansin genes in leaves of sugarcane in different developmental stages was also investigated. This study intended to provide suitable expansin targets for genetic manipulation of sugarcane aiming at biomass and yield improvement.

© 2018 Santiago et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Expansins refer to a family of closely related non-enzymatic proteins found in the plant cell wall that are involved in the cell wall loosening. In addition, expansins appear to be involved in different physiological and environmental responses in plants such as leaf and stem initiation and growth, stomata opening and closing, reproduction, ripening and stress tolerance. Sugarcane (Saccharum spp.) is one of the main crops grown worldwide. Lignocellulosic biomass from sugarcane is one of the most promising raw materials for the ethanol industry. However, the efficient use of lignocellulosic biomass requires the optimization of several steps, including the access of some enzymes to the hemicellulosic matrix. The addition of expansins in an enzymatic cocktail or their genetic manipulation could drastically improve the saccharification process of feedstock biomass by weakening the hydrogen bonds between polysaccharides present in plant cell walls. In this study, the expansin gene family in sugarcane was identified and characterized by in silico analysis. Ninety two putative expansins in sugarcane (SacEXPs) were categorized in three subfamilies after phylogenetic analysis. The expression profile of some expansin genes in leaves of sugarcane in different developmental stages was also investigated. This study intended to provide suitable expansin targets for genetic manipulation of sugarcane aiming at biomass and yield improvement.

A small protein, cysteine-rich, designated SM1, produced by Trichoderma virens and Trichoderma atroviride, acts as elicitor for triggering plant defense reactions. We analyzed Sm1 gene expression of eight different strains of Trichoderma spp. grown on glucose, seeds or roots of beans. Regardless of the carbon source, T37 strain had significantly higher Sm1 expression and was chosen for further studies. When grown on different carbon sources, Sm1 expression was highest on galactose, bean seed, glucose and starch. Sm1 gene from T37 strain was cloned; it had a single exon, and encoded a protein of 138 amino acids, showing high sequence identity with some proteins belonging to the cerato-platanin family.

The ascomycete Trichoderma reesei is one of the most well studied cellulolytic microorganisms. This fungus is widely used in the biotechnology industry, mainly in the production of biofuels. Due to its importance, its genome was sequenced in 2008, opening new avenues to study this microorganism. In this 'post-genomic' era, a transcriptomic and proteomic era has emerged. Here, we present an overview of new findings in the gene expression regulation network of T. reesei. We also discuss new rational strategies to obtain mutants that produce hydrolytic enzymes with a higher yield, using metabolic engineering. Finally, we present how synthetic biology strategies can be used to create engineered promoters to efficiently synthesize enzymes for biomass degradation to produce bioethanol.

Some species of Trichoderma have successfully been used in the commercial biological control of fungal pathogens, e.g., Sclerotinia sclerotiorum, an economically important pathogen of common beans (Phaseolus vulgaris L.). The objectives of the present study were (1) to provide molecular characterization of Trichoderma strains isolated from the Brazilian Cerrado; (2) to assess the metabolic profile of each strain by means of Biolog FF Microplates; and (3) to evaluate the ability of each strain to antagonize S. sclerotiorum via the production of cell wall-degrading enzymes (CWDEs), volatile antibiotics, and dual-culture tests. Among 21 isolates, we identified 42.86% as Trichoderma asperellum, 33.33% as Trichoderma harzianum, 14.29% as Trichoderma tomentosum, 4.76% as Trichoderma koningiopsis, and 4.76% as Trichoderma erinaceum. Trichoderma asperellum showed the highest CWDE activity. However, no species secreted a specific group of CWDEs. Trichoderma asperellum 364/01, T. asperellum 483/02, and T. asperellum 356/02 exhibited high and medium specific activities for key enzymes in the mycoparasitic process, but a low capacity for antagonism. We observed no significant correlation between CWDE and antagonism, or between metabolic profile and antagonism. The diversity of Trichoderma species, and in particular of T. harzianum, was clearly reflected in their metabolic profiles. Our findings indicate that the selection of Trichoderma candidates for biological control should be based primarily on the environmental fitness of competitive isolates and the target pathogen.

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.

Trichoderma 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 27 kDa. The enzyme exhibited optimum catalytic activity at pH 3.6 and 45 °C. It was thermostable at 40 °C, and retained 75% activity after 60 min at 45 °C. The Km and Vmax values for 1,3-β- d -glucanase, using laminarin as substrate, were 0.323 mg ml−1 and 0.315 U min−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.

Trichoderma harzianum is a fungus well known for its potential as a biocontrol agent against many fungal phytopathogens. The aim of this study was to characterize the proteins secreted by T. harzianum ALL42 when its spores were inoculated and incubated for 48 h in culture media supplemented with glucose (GLU) or with cell walls from Fusarium solani (FSCW), a phytopathogen that causes severe losses in common bean and soy crops in Brazil, as well as other crop diseases around the world. Trichoderma harzianum was able to grow in Trichoderma Liquid Enzyme Production medium (TLE) and Minimal medium (MM) supplemented with FSCW and in TLE+GLU, but was unable to grow in MM+GLU medium. Protein quantification showed that TLE+FSCW and MM+FSCW had 45- and 30- fold, respectively, higher protein concentration on supernatant when compared to TLE+GLU, and this difference was observable on 2D gel electrophoresis (2DE). A total of 94 out of 105 proteins excised from 2DE maps were identified. The only protein observed in all three conditions was epl1. In the media supplemented with FSCW, different hydrolases such as chitinases, β-1,3-glucanases, glucoamylases, α-1,3-glucanases and proteases were identified, along with other proteins with no known functions in mycoparasitism, such as npp1 and cys. Trichoderma harzianum showed a complex and diverse arsenal of proteins that are secreted in response to the presence of FSCW, with novel proteins not previously described in mycoparasitic-related studies.

Trichoderma harzianum ALL42 were capable of overgrowing and degrading Rhizoctonia solani and Macrophomina phaseolina mycelia, coiling around the hyphae with formation of apressoria and hook-like structures. Hyphae of T. harzianum ALL42 did not show any coiling around Fusarium sp. hyphae suggesting that mycoparasitism may be different among the plant pathogens. In this study, a secretome analysis was used to identify some extracellular proteins secreted by T. harzianum ALL42 after growth on cell wall of M. phaseolina, Fusarium sp., and R. solani. The secreted proteins were analyzed by two-dimensional electrophoresis and MALDI-TOF mass spectrometry. A total of 60 T. harzianum ALL42 secreted proteins excised from the gel were analyzed from the three growth conditions. While seven cell wall-induced proteins were identified, more than 53 proteins spots remain unidentified, indicating that these proteins are either novel proteins or proteins that have not yet been sequenced. Endochitinase, β-glucosidase, α-mannosidase, acid phosphatase, α-1,3-glucanase, and proteases were identified in the gel and also detected in the supernatant of culture.

The regulation of the gene encoding the extracellular exo-beta-1,3-glucanase (tag83) produced by the mycoparasite Trichoderma asperellum was studied. Enzyme activity was detected in all carbon sources, but the highest levels were found when starch and purified cell walls from Rhizoctonia solani were used. These results are supported by the appearance of one strong band with enzyme activity in non-denaturing PAGE. Experiments using RT-PCR showed that exo-beta-1,3-glucanase induction in T. asperellum occurred at the transcriptional level. We used RT-PCR and real-time PCR analysis to examine the expression of tag83 gene during in vivo assay of T. asperellum against R. solani. We showed that the expression of tag83 is significantly increased by the presence of R. solani.

The involvement of the G-alpha protein GNA3 in the production of cell wall-degrading enzymes (CWDEs) by Trichoderma reesei during antagonism against Pythium ultimum was investigated. cAMP content was 2.8-fold higher in the T. reesei mutant gna3QL than in the parental TU-6. The gna3QL, like TU-6, inhibited the growth of P. ultimum in dual culture assays. Scanning electron microscopy showed that the gna3QL promoted more morphological alterations of P. ultimum cell wall than TU-6. In general, gna3QL produced higher activities of CWDEs than TU-6. We therefore suggest that CWDEs production during mycoparasitism by T. reesei against P. ultimum may be associated with the level of GNA3 activity.

Trichoderma reesei (Hypocrea jecorina) is widely explored in industry and its potential for using in agriculture as a biocontrol agent against phytophatogenic fungi has just began to be investigated. We have investigated the involvement of G proteins during mycoparasitism against plant pathogens. Here we described the role of GNA1, a G-alpha protein that belongs to αi group in Cell Wall Degrading Enzymes (CWDEs) production by T. reesei during antagonism against Pythium ultimum. For that, two mutants were used: Δgna1 and gna1QL (=constitutively activated version of GNA1). The gna1QL mutant of T. reesei, like the parental TU-6, inhibited the growth of P. ultimum in plate confrontation assay and grew faster than the parental TU-6 while the Δgna1 did not grow over P. ultimum. Scanning electron microscopy showed that the gna1QL mutant promoted more morphological alterations of P. ultimum cell wall than the parental TU-6 while the Δgna1 caused no effects. The mutant Δgna1 showed less CWDEs activity than gna1QL and TU-6 during in vitro cultivations. The gna1QL mutant showed a better performance in production of CWDEs such as endochitinase, N-Acetyl-β-D-glucosaminidase (NAGase), lipase and acid phosphatase, after 72 hours of incubation. However, the parental TU-6 showed higher cellulase activity than gna1QL and Δgna1. The intracellular content of cAMP in the strains after 72 hours of incubation in the presence of P. ultimum cell wall was: gna1QL (79.85 ± 12), Δgna1 (268.65 ± 8.5) and TU-6 (109.70 ± 9.2) pmol/mg protein. RT-qPCR results showed a low level of transcripts of mycoparasitism-specific genes in Δgna1 strain. We therefore suggest that the production of some CWDEs during mycoparasitism by T. reesei against P. ultimum can be mediated by GNA1 activity or cAMP levels.

Trichoderma spp. are known for their biocontrol activity against several plant pathogens. A specific isolate of Trichoderma harzianum, 303/02, has the potential to inhibit the growth of Sclerotinia sclerotiorum, an important agent involved in several crop diseases. In this study, the interaction between T. harzianum 303/02 and mycelia, sclerotia and apothecia of S. sclerotiorum was studied by scanning electron microscopy. RT-qPCR was used to examine the expression of 11 genes potentially involved in biocontrol. T. harzianum 303/02 parasitizes S. sclerotiorum by forming branches that coil around the hyphae. The fungus multiplied abundantly at the sclerotia and apothecia surface, forming a dense mycelium that penetrated the inner surface of these structures. The levels of gene expression varied according to the type of structure with which T. harzianum was interacting. The data also showed the presence of synergistic action between the cell-wall degrading enzymes.

Members of the genus Trichoderma are among of the most commonly disseminated fungi in nature. They can grow in a great variety of carbon and nitrogen substrates. Trichoderma spp. have a fine metabolic regulation that is able to respond to environmental changes and to nutrient and oxygen limitations. They therefore produce a range of enzymes to degrade homopolysaccharides and heteropolysaccharides, which are important for survival. Fungal strains of the genus Trichoderma are also well-known producers of secondary metabolites with antibiotic activity that are important for the biocontrol of phytopathogens. Due to the commercial importance, the genomes of many Trichoderma species have been sequenced completely, and the organisms have been studied in detail by functional genomics and molecular biochemistry. The accumulated knowledge allows a better understanding of the metabolic diversity among different species of Trichoderma, which is important for metabolic engineering in Trichoderma for commercial purposes.

Trichoderma spp. are used for biocontrol of several plant pathogens. However, their efficient interaction with the host needs to be accompanied by production of secondary metabolites and cell wall-degrading enzymes. Three parameters were evaluated after interaction between four Trichoderma species and plant-pathogenic fungi: Fusarium solani, Rhizoctonia solani and Sclerotinia sclerotiorum. Trichoderma harzianum and T. asperellum were the most effective antagonists against the pathogens. Most of the Trichoderma species produced toxic volatile metabolites, having significant effects on growth and development of the plant pathogens. When these species were grown in liquid cultures with cell walls from these plant pathogens, they produced and secreted β-1,3-glucanase, NAGAse, chitinase, acid phosphatase, acid proteases and alginate lyase.

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.

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.

Background: The filamentous fungus Trichoderma reesei is a major producer of lignocellulolytic enzymes utilized by bioethanol industries. However, to achieve low cost second generation bioethanol production on an industrial scale an efficient mix of hydrolytic enzymes is required for the deconstruction of plant biomass. In this study, we investigated the molecular basis for lignocellulose-degrading enzyme production T. reesei during growth in cellulose, sophorose, and glucose. Results: We examined and compared the transcriptome and differential secretome (2D-DIGE) of T. reesei grown in cellulose, sophorose, or glucose as the sole carbon sources. By applying a stringent cut-off threshold 2,060 genes were identified as being differentially expressed in at least one of the respective carbon source comparisons. Hierarchical clustering of the differentially expressed genes identified three possible regulons, representing 123 genes controlled by cellulose, 154 genes controlled by sophorose and 402 genes controlled by glucose. Gene regulatory network analyses of the 692 genes differentially expressed between cellulose and sophorose, identified only 75 and 107 genes as being specific to growth in sophorose and cellulose, respectively. 2D-DIGE analyses identified 30 proteins exclusive to sophorose and 37 exclusive to cellulose. A correlation of 70.17% was obtained between transcription and secreted protein profiles. Conclusions: Our data revealed new players in cellulose degradation such as accessory proteins with non-catalytic functions secreted in different carbon sources, transporters, transcription factors, and CAZymes, that specifically respond in response to either cellulose or sophorose.

Background The 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. Results Data 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. Conclusions The 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.

Humicola grisea var. thermoidea is a thermophilic ascomycete and important enzyme producer that has an efficient enzymatic system with a broad spectrum of thermostable carbohydrate-active (CAZy) enzymes. These enzymes can be employed in lignocellulose biomass deconstruction and other industrial applications. In this work, the genome of H. grisea var. thermoidea was sequenced. The acquired sequence reads were assembled into a total length of 28.75 Mbp. Genome features correlate with what was expected for thermophilic Sordariomycetes. The transcriptomic data showed that sugar-cane bagasse significantly upregulated genes related to primary metabolism and polysaccharide deconstruction, especially hydrolases, at both pH 5 and pH 8. However, a number of exclusive and shared genes between the pH values were found, especially at pH 8. H. grisea expresses an average of 211 CAZy enzymes (CAZymes), which are capable of acting in different substrates. The top upregulated genes at both pH values represent CAZyme-encoding genes from different classes, including acetylxylan esterase, endo-1,4-b-mannosidase, exoglucanase, and endoglucanase genes. For the first time, the arsenal that the thermophilic fungus H. grisea var. thermoidea possesses to degrade the lignocellulosic biomass is shown. Carbon source and pH are of pivotal importance in regulating gene expression in this organism, and alkaline pH is a key regulatory factor for sugarcane bagasse hydrolysis. This work paves the way for the genetic manipulation and robust biotechnological applications of this fungus. Made available in DSpace on 2021-09-16T02:15:06Z (GMT). No. of bitstreams: 1 Insights-into-the-Lignocellulose-Degrading-Enzyme-System-2021.pdf: 2194226 bytes, checksum: 419ea07eb34b3577d6fdd82e1f710085 (MD5) Previous issue date: 2021