Roberto Nascimento Silva

Comparative metabolism of cellulose, sophorose and glucose in Trichoderma reesei using high-throughput genomic and proteomic analyses

BackgroundThe 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.ResultsWe 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.ConclusionsOur 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.

Biochemical and metabolic profiles of Trichoderma strains isolated from common bean crops in the Brazilian Cerrado, and potential antagonism against Sclerotinia sclerotiorum

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.

Friends or foes? Emerging insights from fungal interactions with plants

Fungi interact with plants in various ways, with each interaction giving rise to different alterations in both partners. While fungal pathogens have detrimental effects on plant physiology, mutualistic fungi augment host defence responses to pathogens and/or improve plant nutrient uptake. Tropic growth towards plant roots or stomata, mediated by chemical and topographical signals, has been described for several fungi, with evidence of species-specific signals and sensing mechanisms. Fungal partners secrete bioactive molecules such as small peptide effectors, enzymes and secondary metabolites which facilitate colonization and contribute to both symbiotic and pathogenic relationships. There has been tremendous advancement in fungal molecular biology, omics sciences and microscopy in recent years, opening up new possibilities for the identification of key molecular mechanisms in plant–fungal interactions, the power of which is often borne out in their combination. Our fragmentary knowledge on the interactions between plants and fungi must be made whole to understand the potential of fungi in preventing plant diseases, improving plant productivity and understanding ecosystem stability. Here, we review innovative methods and the associated new insights into plant–fungal interactions.

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

BackgroundThe 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.ResultsData 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.ConclusionsThis 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.

Expression pattern of cellulolytic and xylanolytic genes regulated by transcriptional factors XYR1 and CRE1 are affected by carbon source in Trichoderma reesei.

Trichoderma reesei is the most important fungus for the industrial production of enzymes to biomass deconstruction. Most of the genes encoding cellulases and hemicellulases are regulated by the transcription factors CRE1 and XYR1. In this work, the regulation of 22 genes of cellulases and xylanases by these transcription factors was investigated under three different carbon sources. Analysis of gene expression and enzymatic profiles of CMCase, β-glucosidase, and xylanases showed different regulation that was depended of the carbon source in both Δxyr1 and Δcre1 mutants. In the presence of glucose, the majority of genes evaluated (82%) showed increased expression levels in the Δcre1 mutant compared to the parental QM9414 strain. In the Δxyr1 mutant, it was observed that expression of cellulase and xylanase genes was reduced compared to the parental QM9414 strain, when cultured in the presence of cellulose or sophorose. Interesting, in the presence of glucose, approximately 60% of the analyzed genes had increased expression in the Δxyr1 mutant compared to parental strain. Furthermore, no correlation between gene expression and the number of putative binding sites of XYR1 and CRE1 to promoter region of cellulolytic and xylanolytic studied genes was observed. Therefore, these results demonstrated that the regulation of cellulase and xylanase by the transcription factors CRE1 and XYR1 is influenced by different carbon sources.

Defining the genome-wide role of CRE1 during carbon catabolite repression in Trichoderma reesei using RNA-Seq analysis.

The ascomycete Trichoderma reesei is one of the most well-studied cellulolytic fungi and is widely used by the biotechnology industry in the production of second generation bioethanol. The carbon catabolite repression (CCR) mechanism adopted by T. reesei is mediated by the transcription factor CRE1. CCR represses genes related to cellulase production when a carbon source is readily available in the medium. Using RNA sequencing, we investigated CCR during the synthesis of cellulases, comparing the T. reesei Δcre1 mutant strain with its parental strain, QM9414. Of 9129 genes in the T. reesei genome, 268 genes were upregulated and 85 were downregulated in the presence of cellulose (Avicel). In addition, 251 genes were upregulated and 230 were downregulated in the presence of a high concentration of glucose. Genes encoding cellulolytic enzymes and transcription factors and genes related to the transport of nutrients and oxidative metabolism were also targets of CCR, mediated by CRE1 in a carbon source-dependent manner. Our results also suggested that CRE1 regulates the expression of genes related to the use of copper and iron as final electron acceptors or as cofactors of enzymes that participate in biomass degradation. As a result, the final effect of CRE1-mediated transcriptional regulation is to modulate the access of cellulolytic enzymes to cellulose polymers or blocks the entry of cellulase inducers into the cell, depending on the glucose content in the medium. These results will contribute to a better understanding of the mechanism of carbon catabolite repression in T. reesei, thereby enhancing its application in several biotechnology fields.

The impact of chromatin remodelling on cellulase expression in Trichoderma reesei.

BackgroundTrichoderma reesei is used for industry-scale production of plant cell wall-degrading enzymes, in particular cellulases, but also xylanases. The expression of the encoding genes was so far primarily investigated on the level of transcriptional regulation by regulatory proteins. Otherwise, the impact of chromatin remodelling on gene expression received hardly any attention. In this study we aimed to learn if the chromatin status changes in context to the applied conditions (repressing/inducing), and if the presence or absence of the essential transactivator, the Xylanase regulator 1 (Xyr1), influences the chromatin packaging.ResultsComparing the results of chromatin accessibility real-time PCR analyses and gene expression studies of the two prominent cellulase-encoding genes, cbh1 and cbh2, we found that the chromatin opens during sophorose-mediated induction compared to D-glucose-conferred repression. In the strain bearing a xyr1 deletion the sophorose mediated induction of gene expression is lost and the chromatin opening is strongly reduced. In all conditions the chromatin got denser when Xyr1 is absent. In the case of the xylanase-encoding genes, xyn1 and xyn2, the result was similar concerning the condition-specific response of the chromatin compaction. However, the difference in chromatin status provoked by the absence of Xyr1 is less pronounced. A more detailed investigation of the DNA accessibility in the cbh1 promoter showed that the deletion of xyr1 changed the in vivo footprinting pattern. In particular, we detected increased hypersensitivity on Xyr1-sites and stronger protection of Cre1-sites. Looking for the players directly causing the observed chromatin remodelling, a whole transcriptome shotgun sequencing revealed that 15 genes encoding putative chromatin remodelers are differentially expressed in response to the applied condition and two amongst them are differentially expressed in the absence of Xyr1.ConclusionsThe regulation of xylanase and cellulase expression in T. reesei is not only restricted to the action of transcription factors but is clearly related to changes in the chromatin packaging. Both the applied condition and the presence of Xyr1 influence chromatin status.

Understanding the Role of the Master Regulator XYR1 in Trichoderma reesei by Global Transcriptional Analysis

We defined the role of the transcriptional factor—XYR1—in the filamentous fungus Trichoderma reesei during cellulosic material degradation. In this regard, we performed a global transcriptome analysis using RNA-Seq of the Δxyr1 mutant strain of T. reesei compared with the parental strain QM9414 grown in the presence of cellulose, sophorose, and glucose as sole carbon sources. We found that 5885 genes were expressed differentially under the three tested carbon sources. Of these, 322 genes were upregulated in the presence of cellulose, while 367 and 188 were upregulated in sophorose and glucose, respectively. With respect to genes under the direct regulation of XYR1, 30 and 33 are exclusive to cellulose and sophorose, respectively. The most modulated genes in the Δxyr1 belong to Carbohydrate-Active Enzymes (CAZymes), transcription factors, and transporters families. Moreover, we highlight the downregulation of transporters belonging to the MFS and ABC transporter families. Of these, MFS members were mostly downregulated in the presence of cellulose. In sophorose and glucose, the expression of these transporters was mainly upregulated. Our results revealed that MFS and ABC transporters could be new players in cellulose degradation and their role was shown to be carbon source-dependent. Our findings contribute to a better understanding of the regulatory mechanisms of XYR1 to control cellulase gene expression in T. reesei in the presence of cellulosic material, thereby potentially enhancing its application in several biotechnology fields.

Deciphering the Cis-Regulatory Elements for XYR1 and CRE1 Regulators in Trichoderma reesei

In this work, we report the in silico identification of the cis-regulatory elements for XYR1 and CRE1 proteins in the filamentous fungus Trichoderma reesei, two regulators that play a central role in the expression of cellulase genes. Using four datasets of condition-dependent genes from RNA-seq and RT-qPCR experiments, we performed unsupervised motif discovery and found two short motifs resembling the proposed binding consensus for XYR1 and CRE1. Using these motifs, we analysed the presence and arrangement of putative cis-regulatory elements recognized by both regulators and found that shortly spaced sites were more associated with XYR1- and CRE1-dependent promoters than single, high-score sites. Furthermore, the approach used here allowed the identification of the previously reported XYR1-binding sites from cel7a and xyn1 promoters, and we also mapped the potential target sequence for this regulator at the cel6a promoter that has been suggested but not identified previously. Additionally, seven other promoters (for cel7b, cel61a, cel61b, cel3c, cel3d, xyn3 and swo genes) presented a putative XYR1-binding site, and strong sites for CRE1 were found at the xyr1 and cel7b promoters. Using the cis-regulatory architectures nearly defined for XYR1 and CRE1, we performed genome-wide identification of potential targets for direct regulation by both proteins and important differences on their functional regulons were elucidated. Finally, we performed binding site mapping on the promoters of differentially expressed genes found in T. reesei mutant strains lacking xyr1 or cre1 and found that indirect regulation plays a key role on their signalling pathways. Taken together, the data provided here sheds new light on the mechanisms for signal integration mediated by XYR1 and CRE1 at cellulase promoters.

Evidence of cAMP involvement in cellobiohydrolase expression and secretion by Trichoderma reesei in presence of the inducer sophorose

BackgroundThe signaling second messenger cyclic AMP (cAMP) regulates many aspects of cellular function in all organisms. Previous studies have suggested a role for cAMP in the regulation of gene expression of cellulolytic enzymes in Trichoderma reesei (anamorph of Hypocrea jecorina).MethodsThe effects of cAMP in T. reesei were analyzed through both activity and expression of cellulase, intracellular cAMP level measurement, western blotting, indirect immunofluorescence and confocal microscopy.ResultsTo elucidate the involvement of cAMP in the cellulase expression, we analyzed the growth of the mutant strain ∆acy1 and its parental strain QM9414 in the presence of the inducers cellulose, cellobiose, lactose, or sophorose, and the repressor glucose. Our results indicated that cAMP regulates the expression of cellulase in a carbon source-dependent manner. The expression cel7a, and cel6a genes was higher in the presence of sophorose than in the presence of cellulose, lactose, cellobiose, or glucose. Moreover, intracellular levels of cAMP were up to four times higher in the presence of sophorose compared to other carbon sources. Concomitantly, our immunofluorescence microscopy and western blot data suggest that in the presence of sophorose, cAMP may regulate secretion of cellulolytic enzymes in T. reesei.ConclusionsThese results allow us to better understand the role of cAMP and expand our knowledge on the signal transduction pathways involved in the regulation of cellulase expression in T. reesei. Finally, our data may help develop new strategies to improve the expression of cel7a and cel6a genes, and therefore, favor their application in several biotechnology fields.