Proteome profiling of enriched membrane-associated proteins unraveled a novel sophorose and cello-oligosaccharide transporter in Trichoderma reesei.

BACKGROUND: Trichoderma reesei is an organism extensively used in the bioethanol industry, owing to its capability to produce enzymes capable of breaking down holocellulose into simple sugars. The uptake of carbohydrates generated from cellulose breakdown is crucial to induce the signaling cascade that triggers cellulase production. However, the sugar transporters involved in this process in T. reesei remain poorly identified and characterized. RESULTS: To address this gap, this study used temporal membrane proteomics analysis to identify five known and nine putative sugar transporters that may be involved in cellulose degradation by T. reesei. Docking analysis pointed out potential ligands for the putative sugar transporter Tr44175. Further functional validation of this transporter was carried out in Saccharomyces cerevisiae. The results showed that Tr44175 transports a variety of sugar molecules, including cellobiose, cellotriose, cellotetraose, and sophorose. CONCLUSION: This study has unveiled a transporter Tr44175 capable of transporting cellobiose, cellotriose, cellotetraose, and sophorose. Our study represents the first inventory of T. reesei sugar transportome once exposed to cellulose, offering promising potential targets for strain engineering in the context of bioethanol production.

The Cerato-Platanin EPL2 from Trichoderma reesei Is Not Directly Involved in Cellulase Formation but in Cell Wall Remodeling.

Trichoderma reesei is a saprophytic fungus that produces large amounts of cellulases and is widely used for biotechnological applications. Cerato-platanins (CPs) are a family of proteins universally distributed among Dikarya fungi and have been implicated in various functions related to fungal physiology and interaction with the environment. In T. reesei, three CPs are encoded in the genome: Trire2_111449, Trire2_123955, and Trire2_82662. However, their function is not fully elucidated. In this study, we deleted the Trire2_123955 gene (named here as epl2) in the wild-type QM6aΔtmus53Δpyr4 (WT) strain and examined the behavior of the Δepl2 strain compared with WT grown for 72 h in 1% cellulose using RNA sequencing. Of the 9143 genes in the T. reesei genome, 760 were differentially expressed, including 260 only in WT, 214 only in Δepl2, and 286 in both. Genes involved in oxidative stress, oxidoreductase activity, antioxidant activity, and transport were upregulated in the Δepl2 mutant. Genes encoding cell wall synthesis were upregulated in the mutant strain during the late growth stage. The Δepl2 mutant accumulated chitin and glucan at higher levels than the parental strain and was more resistant to cell wall stressors. These results suggest a compensatory effect in cell wall remodeling due to the absence of EPL2 in T. reesei. This study is expected to contribute to a better understanding of the role of the EPL2 protein in T. reesei and improve its application in biotechnological fields.

Use of carbohydrate-directed enzymes for the potential exploitation of sugarcane bagasse to obtain value-added biotechnological products.

Microorganisms, such as fungi and bacteria, are crucial players in the production of enzymatic cocktails for biomass hydrolysis or the bioconversion of plant biomass into products with industrial relevance. The biotechnology industry can exploit lignocellulosic biomass for the production of high-value chemicals. The generation of biotechnological products from lignocellulosic feedstock presents several bottlenecks, including low efficiency of enzymatic hydrolysis, high cost of enzymes, and limitations on microbe metabolic performance. Genetic engineering offers a route for developing improved microbial strains for biotechnological applications in high-value product biosynthesis. Sugarcane bagasse, for example, is an agro-industrial waste that is abundantly produced in sugar and first-generation processing plants. Here, we review the potential conversion of its feedstock into relevant industrial products via microbial production and discuss the advances that have been made in improving strains for biotechnological applications.

Blastocladiella emersonii spliceosome is regulated in response to the splicing inhibition caused by the metals cadmium, cobalt and manganese.

Blastocladiella emersonii is an aquatic fungus of the phylum Blastocladiomycota, localized near the base of the fungal tree. Previous studies have shown that B. emersonii responds to heat shock and cadmium exposure inducing the transcription of a high number of genes. EST sequencing from heat shocked and cadmium exposed B. emersonii cells has shown that exposure to cadmium causes strong splicing inhibition. Despite the knowledge about splicing inhibition by cadmium, it is still unclear if other metal contaminants can cause the same response. In the present study, we have demonstrated that the effect of cadmium exposure on splicing inhibition is much stronger than that of other divalent metals such as cobalt and manganese. Data presented here also indicate that intron retention occurs randomly among the fungal transcripts, as verified by analyzing differently affected transcripts. In addition, we identified in the genome of B. emersonii the genes encoding the snRNA splicing components U1, U2, U4, U5 and U6 and observed that spliceosome snRNAs are upregulated in the presence of metals, in particular snRNA U1 in cells under cadmium exposure. This observation suggests that snRNA upregulation might be a defense of the fungal cell against the metal stress condition.

Small heat shock protein genes are developmentally regulated during stress and non-stress conditions in Blastocladiella emersonii.

Small heat shock proteins (sHsps) are molecular chaperones of low molecular weight involved in an early association with misfolded proteins. In response to heat shock, B. emersonii induces the synthesis of a number of proteins. As sHsps are still poorly studied in B. emersonii and in fungi overall, the aim of this work was to carry out a in-depth characterization of sHsps during B. emersonni life cycle, as well as in response to thermal stress. We verified a strong induction of the hsp17 gene in cells exposed to heat shock both in germination and sporulation stages, and that Hsp17 protein levels show the same pattern of variation of its mRNA. Unlike hsp17 and hsp30, hsp16 gene is not significantly induced during heat shock, in germination or sporulation cells. However, at normal temperatures, the hsp16 gene presents high mRNA levels in sporulation cells, whereas the hsp30 gene presents high mRNA levels in germination cells. Interestingly, heat shock mRNA levels for hsp17 and hsp30 genes are 10 times higher in germination cells than in sporulation cells. Thus, our data show that the expression of these sHsp genes is quite distinct, both under normal temperature as during heat shock.

Trichoderma harzianum transcriptome in response to cadmium exposure.

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.

Hsp genes are differentially expressed during Trichoderma asperellum self-recognition, mycoparasitism and thermal stress.

Heat shock proteins (Hsp) are important factors in the response of organisms to oscillations in environmental conditions. Although Hsp have been studied for a long time, little is known about this protein class in Trichoderma species. Here we studied the expression of Hsp genes during T. asperellum growth, and mycoparasitism against two phytopathogens: Sclerotinia sclerotiorum and Fusarium oxysporum, as well as during thermal stress. The expression levels of these genes were observed by real-time PCR and they showed to be differentially expressed under these conditions. We verified that the TaHsp26c, TaHsp70b and TaHsp70c genes were differentially expressed over time, indicating that these genes can be developmentally regulated in T. asperellum. Except for TaHsp26a, all other genes analyzed were induced in the post-contact condition when T. asperellum was cultured in a confrontation plate assay against itself. Additionally, TaHsp26b, TaHsp26c, TaHsp90, TaHsp104a and TaHsp104b were induced during initial contact between T. asperellum hyphae, suggesting that these proteins must play a role in the organism´s self-recognition mechanism. When we examined gene expression during mycoparasitism, we observed that some genes were induced both by S. sclerotiorum and F. oxysporum, while others were not induced during interaction with either of the phytopathogens. Furthermore, we observed some genes induced only during confrontation against S. sclerotiorum, indicating that the expression of Hsp genes during mycoparasitism seems to be modulated by the phytopathogen. To assess whether such genes are expressed during temperature oscillations, we analyzed their transcription levels during thermal and cold shock. We observed that except for the TaHsp70c gene, all others presented high transcript levels when T. asperellum was submitted to high temperature (38 °C), indicating their importance in the response to heat stress. The TaHsp70c gene was significantly induced only in cold shock at 4 °C. Our results show the importance of Hsp proteins during self-recognition, mycoparasitism and thermal stress in T. asperellum.

Plant-associated bacteria mitigate drought stress in soybean.

Agriculture accounts for ~ 70% of all water use and the world population is increasing annually; soon more people will need to be fed, while also using less water. The use of plant-associated bacteria (PAB) is an eco-friendly alternative that can increase crop water use efficiency. This work aimed to study the effect of some PAB on increasing soybean tolerance to drought stress, the mechanisms of the drought tolerance process, and the effect of the PAB on promoting plant growth and on the biocontrol of Sclerotinia sclerotiorum. PAB were isolated from soybean rhizosphere and S. sclerotiorum sclerotia. The strains identified as UFGS1 (Bacillus subtilis), UFGS2 (Bacillus thuringiensis), UFGRB2 and UFGRB3 (Bacillus cereus) were selected on their ability to grow in media with reduced water activity. Soybean plants were inoculated with the PAB and evaluated for growth promotion, physiological and molecular parameters, after drought stress. Under drought stress, UFGS2 and UFGRB2 sustained potential quantum efficiency of PSII (Fv/Fm), while a decrease was found in the control plants. Moreover, UFGS2 and UFGRB3 maintained the photosynthetic rates in non-stressed conditions compared to the control. UFGS2-treated plants showed a higher stomatal conductance and higher transpiration than the control, after drought stress. Some PAB-treated plants also had other beneficial phenotypes, such as increases in fresh and dried biomass relative to the control. Differential gene expression analysis of genes involved in plant stress pathways shows changes in expression in PAB-treated plants. Results from this study suggest that PAB can mitigate drought stress in soybean and may improve water efficiency under certain conditions.