The small RNA-mediated gene silencing machinery is required in Arabidopsis for stimulation of growth, systemic disease resistance, and suppression of the nitrile-specifier gene NSP4 by Trichoderma atroviride.

Trichoderma atroviride is a root-colonizing fungus that confers multiple benefits to plants. In plants, small RNA (sRNA)-mediated gene silencing (sRNA-MGS) plays pivotal roles in growth, development, and pathogen attack. Here, we explored the role of core components of Arabidopsis thaliana sRNA-MGS pathways during its interaction with Trichoderma. Upon interaction with Trichoderma, sRNA-MGS-related genes paralleled the expression of Arabidopsis defense-related genes, linked to salicylic acid (SA) and jasmonic acid (JA) pathways. SA- and JA-related genes were primed by Trichoderma in leaves after the application of the well-known pathogen-associated molecular patterns flg22 and chitin, respectively. Defense-related genes were primed in roots as well, but to different extents and behaviors. Phenotypical characterization of mutants in AGO genes and components of the RNA-dependent DNA methylation (RdDM) pathway revealed that different sets of sRNA-MGS-related genes are essential for (i) the induction of systemic acquired resistance against Botrytis cinerea, (ii) the activation of the expression of plant defense-related genes, and (iii) root colonization by Trichoderma. Additionally, plant growth induced by Trichoderma depends on functional RdDM. Profiling of DNA methylation and histone N-tail modification patterns at the Arabidopsis Nitrile-Specifier Protein-4 (NSP4) locus, which is responsive to Trichoderma, showed altered epigenetic modifications in RdDM mutants. Furthermore, NSP4 is required for the induction of systemic acquired resistance against Botrytis and avoidance of enhanced root colonization by Trichoderma. Together, our results indicate that RdDM is essential in Arabidopsis to establish a beneficial relationship with Trichoderma. We propose that DNA methylation and histone modifications are required for plant priming by the beneficial fungus against B. cinerea.

Secretome Analysis of Arabidopsis-Trichoderma atroviride Interaction Unveils New Roles for the Plant Glutamate:Glyoxylate Aminotransferase GGAT1 in Plant Growth Induced by the Fungus and Resistance against Botrytis cinerea.

The establishment of plant-fungus mutualistic interaction requires bidirectional molecular crosstalk. Therefore, the analysis of the interacting organisms secretomes would help to understand how such relationships are established. Here, a gel-free shotgun proteomics approach was used to identify the secreted proteins of the plant Arabidopsis thaliana and the mutualistic fungus Trichoderma atroviride during their interaction. A total of 126 proteins of Arabidopsis and 1027 of T. atroviride were identified. Among them, 118 and 780 were differentially modulated, respectively. Bioinformatic analysis unveiled that both organisms' secretomes were enriched with enzymes. In T. atroviride, glycosidases, aspartic endopeptidases, and dehydrogenases increased in response to Arabidopsis. Additionally, amidases, protein-serine/threonine kinases, and hydro-lyases showed decreased levels. Furthermore, peroxidases, cysteine endopeptidases, and enzymes related to the catabolism of secondary metabolites increased in the plant secretome. In contrast, pathogenesis-related proteins and protease inhibitors decreased in response to the fungus. Notably, the glutamate:glyoxylate aminotransferase GGAT1 was secreted by Arabidopsis during its interaction with T. atroviride. Our study showed that GGAT1 is partially required for plant growth stimulation and on the induction of the plant systemic resistance by T. atroviride. Additionally, GGAT1 seems to participate in the negative regulation of the plant systemic resistance against B. cinerea through a mechanism involving H2O2 production.

IPA-1 a Putative Chromatin Remodeler/Helicase-Related Protein of Trichoderma virens Plays Important Roles in Antibiosis Against Rhizoctonia solani and Induction of Arabidopsis Systemic Disease Resistance.

Trichoderma spp. are filamentous fungi that colonize plant roots conferring beneficial effects to plants, either indirectly through the induction of their defense systems or directly through the suppression of phytopathogens in the rhizosphere. Transcriptomic analyses of Trichoderma spp. emerged as a powerful method for identifying the molecular events underlying the establishment of this beneficial relationship. Here, we focus on the transcriptomic response of Trichoderma virens during its interaction with Arabidopsis seedlings. The main response of T. virens to cocultivation with Arabidopsis was the repression of gene expression. The biological processes of transport and metabolism of carbohydrates were downregulated, including a set of cell wall-degrading enzymes putatively relevant for root colonization. Repression of such genes reached their basal levels at later times in the interaction, when genes belonging to the biological process of copper ion transport were induced, a necessary process providing copper as a cofactor for cell wall-degrading enzymes with the auxiliary activities class. RNA-Seq analyses showed the induction of a member of the SNF2 family of chromatin remodelers/helicase-related proteins, which was named IPA-1 (increased protection of Arabidopsis-1). Sequence analyses of IPA-1 showed its closest relatives to be members of the Rad5/Rad16 and SNF2 subfamilies; however, it grouped into a different clade. Although deletion of IPA-1 in T. virens did not affect its growth, the antibiotic activity of Δipa-1 culture filtrates against Rhizoctonia solani diminished but it remained unaltered against Botrytis cinerea. Triggering of the plant defense genes in plants treated with Δipa-1 was higher, showing enhanced resistance against Pseudomonas syringae but not against B. cinerea as compared with the wild type.

TBRG-1 a Ras-like protein in Trichoderma virens involved in conidiation, development, secondary metabolism, mycoparasitism, and biocontrol unveils a new family of Ras-GTPases.

Ras-GTPases are nucleotide hydrolases involved in key cellular processes. In fungi, Ras-GTPases regulate conidiation, development, virulence, and interactions with other fungi or plants. Trichoderma spp. are filamentous saprophytic fungi, widely distributed along all latitudes, characterized by their rapid growth and metabolic diversity. Many species of this genus interact with other fungi, animals or plants. Furthermore, these fungi are used as biocontrol agents due to their ability to antagonize phytopathogenic fungi and oomycetes, through competence, antibiosis, and parasitism. However, the genetic and molecular regulation of these processes is scarcely described in these fungi. In this work, we investigated the role of the gene tbrg-1 product (GenBank accession number XP_013956100; JGI ID: Tv_70852) of T. virens during its interaction with other fungi and plants. Sequence analyses predicted that TBRG-1 bears the characteristic domains of Ras-GTPases; however, its size (1011 aa) is 3- to 4-times bigger compared with classical GTPases. Interestingly, phylogenetic analyses grouped the TBRG-1 protein with hypothetical proteins of similar sizes, sharing conserved regions; whereas other known Ras-GTPases were perfectly grouped with their respective families. These facts led us to classify TBRG-1 into a new family of Ras-GTPases, the Big Ras-GTPases (BRG). Therefore, the gene was named tbrg-1 (TrichodermaBigRas-GTPase-1). Quantification of conidia and scanning electron microscopy showed that the mutants-lacking tbrg-1 produced less conidia, as well as a delayed conidiophore development compared to the wild-type (wt). Moreover, a deregulation of conidiation-related genes (con-10, con-13, and stuA) was observed in tbrg-1-lacking strains, which indicates that TBRG-1 is necessary for proper conidiophore and conidia development. Furthermore, the lack of tbrg-1 affected positively the antagonistic capability of T. virens against the phytopathogens Rhizoctonia solani, Sclerotium rolfsii, and Fusarium oxysporum, which was consistent with the expression patterns of mycoparasitism-related genes, sp1 and cht1, that code for a protease and for a chitinase, respectively. Furthermore, the antibiosis effect of mycelium-free culture filtrates of Δtbrg-1 against R. solani was considerably enhanced. The expression of secondary metabolism-related genes, particularly gliP, showed an upregulation in Δtbrg-1, which paralleled an increase in gliotoxin production as compared to the wt. These results indicate that TBRG-1 plays a negative role in secondary metabolism and antagonism. Unexpectedly, the biocontrol activity of Δtbrg-1 was ineffective to protect the tomato seeds and seedlings against R. solani. On the contrary, Δtbrg-1 behaved like a plant pathogen, indicating that TBRG-1 is probably implicated in the recognition process for establishing a beneficial relationship with plants.

Trichoderma Histone Deacetylase HDA-2 Modulates Multiple Responses in Arabidopsis.

Trichoderma spp. are a rich source of secondary metabolites and volatile organic compounds (VOCs), which may induce plant defenses and modulate plant growth. In filamentous fungi, chromatin modifications regulate secondary metabolism. In this study we investigated how the absence of histone deacetylase HDA-2 in the Trichoderma atroviride strain Δhda-2 impacts its effect on a host, Arabidopsis (Arabidopsis thaliana). The production of VOCs and their impact on plant growth and development were assessed as well. The Δhda-2 strain was impaired in its ability to colonize Arabidopsis roots, thus affecting the promotion of plant growth and modulation of plant defenses against foliar pathogens Botrytis cinerea and Pseudomonas syringae, which normally result from interaction with T. atroviride Furthermore, Δhda-2 VOCs were incapable of triggering plant defenses to counterattack foliar pathogens. The Δhda-2 overproduced the VOC 6-pentyl-2H-pyran-2-one (6-PP), which resulted in enhanced root branching and differentially regulated phytohormone-related genes. Analysis of ten VOCs (including 6-PP) revealed that three of them positively regulated plant growth, whereas six had the opposite effect. Assessment of secondary metabolites, detoxification, and communication with plant-related genes showed a dual role for HDA-2 in T. atroviride gene expression regulation during its interaction with plants. Chromatin immunoprecipitation of acetylated histone H3 on the promoters of plant-responsive genes in Δhda-2 showed, in the presence of Arabidopsis, low levels of epl-1 and abc-2 compared with that in the wild type; whereas ctf-1 presented high constitutive levels, supporting a dual role of HDA-2 in gene regulation. This work highlights the importance of HDA-2 as a global regulator in Trichoderma to modulate multiple responses in Arabidopsis.

Automated, continuous video microscopy tracking of hyphal growth.

The growth of filamentous fungi is a complex process that involves hyphal elongation and branching. Microscopic observations provide a wealth of information on fungal growth, although often requiring laborious manual intervention to record and analyze images. Here, we introduce a novel tool for automated tracking of growth in fungal hyphae that affords quantitative analysis of growth rate and morphology. We supplied a student-grade bright field microscope with stepper motors to enable computer-control of the microscope stage. In addition, we developed an image-processing routine that detects in real-time the tip of a hypha and tracks it as the hypha elongates. To achieve continuous observation of hyphal growth, our system automatically maintains the observed sample within field-of-view and performs periodic autofocus correction in the microscope. We demonstrate automated, continuous tracking of hyphal growth in Trichoderma atroviride with sampling rates of seconds and observation times of up to 14 h. Tracking records allowed us to determine that T. atroviride hyphae grow with characteristic elongation rates of ∼70 nm/s. Surprisingly, we found that prior to the occurrence of an apical branching event the parental hypha stopped growing during a few minutes. These arrest events presented occasionally for subapical branching as well. Finally, from tracking data we found that the persistence length (a measure of filament extension before presenting a change in direction) associated to T. atroviride hyphae is 362 µm. Altogether, these results show how integration of image analysis and computer control enable quantitative microscopic observations of fungal hyphae dynamics.

Histone Deacetylase HDA-2 Regulates Trichoderma atroviride Growth, Conidiation, Blue Light Perception, and Oxidative Stress Responses.

Fungal blue-light photoreceptors have been proposed as integrators of light and oxidative stress. However, additional elements participating in the integrative pathway remain to be identified. In Trichoderma atroviride, the blue-light regulator (BLR) proteins BLR-1 and -2 are known to regulate gene transcription, mycelial growth, and asexual development upon illumination, and recent global transcriptional analysis revealed that the histone deacetylase-encoding gene hda-2 is induced by light. Here, by assessing responses to stimuli in wild-type and Δhda-2 backgrounds, we evaluate the role of HDA-2 in the regulation of genes responsive to light and oxidative stress. Δhda-2 strains present reduced growth, misregulation of the con-1 gene, and absence of conidia in response to light and mechanical injury. We found that the expression of hda-2 is BLR-1 dependent and HDA-2 in turn is essential for the transcription of early and late light-responsive genes that include blr-1, indicating a regulatory feedback loop. When subjected to reactive oxygen species (ROS), Δhda-2 mutants display high sensitivity whereas Δblr strains exhibit the opposite phenotype. Consistently, in the presence of ROS, ROS-related genes show high transcription levels in wild-type and Δblr strains but misregulation in Δhda-2 mutants. Finally, chromatin immunoprecipitations of histone H3 acetylated at Lys9/Lys14 on cat-3 and gst-1 promoters display low accumulation of H3K9K14ac in Δblr and Δhda-2 strains, suggesting indirect regulation of ROS-related genes by HDA-2. Our results point to a mutual dependence between HDA-2 and BLR proteins and reveal the role of these proteins in an intricate gene regulation landscape in response to blue light and ROS. IMPORTANCE: Trichoderma atroviride is a free-living fungus commonly found in soil or colonizing plant roots and is widely used as an agent in biocontrol as it parasitizes other fungi, stimulates plant growth, and induces the plant defense system. To survive in various environments, fungi constantly sense and respond to potentially threatening external factors, such as light. In particular, UV light can damage biomolecules by producing free-radical reactions, in most cases involving reactive oxygen species (ROS). In T. atroviride, conidiation is essential for its survival, which is induced by light and mechanical injury. Notably, conidia are typically used as the inoculum in the field during biocontrol. Therefore, understanding the linkages between responses to light and exposure to ROS in T. atroviride is of major basic and practical relevance. Here, the histone deacetylase-encoding gene hda-2 is induced by light and ROS, and its product regulates growth, conidiation, blue light perception, and oxidative stress responses.

The separation between the 5'-3' ends in long RNA molecules is short and nearly constant.

RNA molecules play different roles in coding, decoding and gene expression regulation. Such roles are often associated to the RNA secondary or tertiary structures. The folding dynamics lead to multiple secondary structures of long RNA molecules, since an RNA molecule might fold into multiple distinct native states. Despite an ensemble of different structures, it has been theoretically proposed that the separation between the 5' and 3' ends of long single-stranded RNA molecules (ssRNA) remains constant, independent of their base content and length. Here, we present the first experimental measurements of the end-to-end separation in long ssRNA molecules. To determine this separation, we use single molecule Fluorescence Resonance Energy Transfer of fluorescently end-labeled ssRNA molecules ranging from 500 to 5500 nucleotides in length, obtained from two viruses and a fungus. We found that the end-to-end separation is indeed short, within 5-9 nm. It is remarkable that the separation of the ends of all RNA molecules studied remains small and similar, despite the origin, length and differences in their secondary structure. This implies that the ssRNA molecules are 'effectively circularized' something that might be a general feature of RNAs, and could result in fine-tuning for translation and gene expression regulation.

Inferring co-expression networks of Arabidopsis thaliana genes during their interaction with Trichoderma spp.

Fungi of the Trichoderma genus are called "biostimulants" because they promote plant growth and development and induce disease resistance. We used conventional transcriptome and gene co-expression analyses to understand the molecular response of the plant Arabidopsis thaliana to inoculation with Trichoderma atroviride or Trichoderma virens. The transcriptional landscape of the plant during the interaction with these fungi showed a reduction in functions such as reactive oxygen species production, defense mechanisms against pathogens, and hormone signaling. T. virens, as opposed to T. atroviride, was more effective at downregulating genes related to terpenoid metabolism, root development, and chemical homeostasis. Through gene co-expression analysis, we found functional gene modules that closely link plant defense with hypoxia. Notably, we found a transcription factor (locus AT2G47520) with two functional domains of interest: a DNA-binding domain and an N-terminal cysteine needed for protein stability under hypoxia. We hypothesize that the transcription factor can bind to the promoter sequence of the GCC-box that is connected to pathogenesis by positioned weight matrix analysis.

The Small GTPases in Fungal Signaling Conservation and Function.

Monomeric GTPases, which belong to the Ras superfamily, are small proteins involved in many biological processes. They are fine-tuned regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Several families have been identified in organisms from different kingdoms. Overall, the most studied families are Ras, Rho, Rab, Ran, Arf, and Miro. Recently, a new family named Big Ras GTPases was reported. As a general rule, the proteins of all families have five characteristic motifs (G1-G5), and some specific features for each family have been described. Here, we present an exhaustive analysis of these small GTPase families in fungi, using 56 different genomes belonging to different phyla. For this purpose, we used distinct approaches such as phylogenetics and sequences analysis. The main functions described for monomeric GTPases in fungi include morphogenesis, secondary metabolism, vesicle trafficking, and virulence, which are discussed here. Their participation during fungus-plant interactions is reviewed as well.

Humic Substances Mediate Anaerobic Methane Oxidation Linked to Nitrous Oxide Reduction in Wetland Sediments.

Humic substances are redox-active organic molecules, which play pivotal roles in several biogeochemical cycles due to their electron-transferring capacity involving multiple abiotic and microbial transformations. Based on the redox properties of humic substances, and the metabolic capabilities of microorganisms to reduce and oxidize them, we hypothesized that they could mediate the anaerobic oxidation of methane (AOM) coupled to the reduction of nitrous oxide (N2O) in wetland sediments. This study provides several lines of evidence indicating the coupling between AOM and the reduction of N2O through an extracellular electron transfer mechanism mediated by the redox active functional groups in humic substances (e.g., quinones). We found that the microbiota of a sediment collected from the Sisal wetland (Yucatán Peninsula, southeastern Mexico) was able to reduce N2O (4.6 ± 0.5 µmol N2O g sed. -1 day-1) when reduced humic substances were provided as electron donor in a close stoichiometric relationship. Furthermore, a microbial enrichment derived from the wetland sediment achieved simultaneous 13CH4 oxidation (1.3 ± 0.1 µmol 13CO2 g sed. -1 day-1) and N2O reduction (25.2 ± 0.5 µmol N2O g sed. -1 day-1), which was significantly dependent on the presence of humic substances as an extracellular electron shuttle. Taxonomic characterization based on 16S rRNA gene sequencing revealed Acinetobacter (a É£-proteobacterium), the Rice Cluster I from the Methanocellaceae and an uncultured archaeon from the Methanomicrobiaceae family as the microbes potentially involved in AOM linked to N2O reduction mediated by humic substances. The findings reported here suggest that humic substances might play an important role to prevent the emission of greenhouse gases (CH4 and N2O) from wetland sediments. Further efforts to evaluate the feasibility of this novel mechanism under the natural conditions prevailing in ecosystems must be considered in future studies.

Characterization of the trypsin-III from Monterey sardine (Sardinops caeruleus): Insights on the cold-adaptation from the A236N mutant.

Trypsins (E.C. 3.4.21.4) are digestive enzymes that catalyze the hydrolysis of peptide bonds containing arginine and lysine residues. Some trypsins from fish species are active at temperatures just above freezing, and for that are called cold-adapted enzymes, having many biotechnological applications. In this work, we characterized a recombinant trypsin-III from Monterey sardine (Sardinops caeruleus) and studied the role of a single residue on its cold-adapted features. The A236N mutant from sardine trypsin-III showed higher activation energy for the enzyme-catalyzed reaction, it was more active at higher temperatures, and exhibited a higher thermal stability than the wild-type enzyme, suggesting a key role of this residue. The thermodynamic activation parameters revealed an increase in the activation enthalpy for the A236N mutant, suggesting the existence of more intramolecular contacts during the activation step. Molecular models for both enzymes suggest that a hydrogen-bond involving N236 may contact the C-terminal α-helix to the vicinity of the active site, thus affecting the biochemical and thermodynamic properties of the enzyme.

Trichoderma as a Model to Study Effector-Like Molecules.

Plants are capable of perceiving microorganisms by coordinating processes to establish different forms of plant-microbe relationships. Plant colonization is governed in fungal and bacterial systems by secreted effector molecules, suppressing plant defense responses and modulating plant physiology to promote either virulence or compatibility. Proteins, secondary metabolites, and small RNAs have been described as effector molecules that use different mechanisms to establish the interaction. Effector molecules have been studied in more detail due to their involvement in harmful interactions, leading to a negative impact on agriculture. Recently, research groups have started to study the effectors in symbiotic interactions. Interestingly, most symbiotic effectors are members of the same families present in phytopathogens. Nevertheless, the quantity and ratio of secreted effectors depends on the microorganism and the host, suggesting a complex mechanism of recognition between the plant and their associated microorganisms. Fungi belonging to Trichoderma genus interact with plants by inducing their defense system and promoting plant growth. Research suggests that some of these effects are associated with effector molecules that Trichoderma delivers during the association with the plant. In this review, we will focus on the main findings concerning the effector molecules reported in Trichoderma spp. and their role during the interaction with plants, mainly in the molecular dialogue that takes place between them.

Unraveling the photoactive annihilation mechanism of nanostructures as effective green tools for inhibiting the proliferation of the phytopathogenic bacterium Pseudomonas syringae.

The infectious proliferation of phytopathogenic microorganisms depends on a complex sequence of biological events involving host defense, environmental conditions, and chemical and physical interactions between the surface of a plant and microorganisms, which in numerous cases display resistance to conventional microbicides. Among these microorganisms, Pseudomonas syringae (P. syringae) is a Gram-negative bacterium that attacks wounded parts of plants before invading healthy tissues. In order to control P. syringae, considering it to be a phytopathogenic model, an effective method featuring silver nanoparticles (AgNPs) functionalized on titanate nanotubes (Nts) used as photoactive antibacterial agents was investigated to understand the effective photoactive annihilation mechanism. The high dispersion of AgNPs over the Nts boosted charge carrier separation by generating reactive oxygen species (ROS) under visible-light, which stressed the bacteria and enhanced the biocidal effect by quickly preventing the rod-shaped P. syringae bacteria from proliferating. Biological transmission and scanning electron microscopy revealed damaged P. syringae cells that underwent the formation of outer membrane vesicles, caused by photo-assisted annihilation, which is considered to be an indication of a critical defense mechanism. The unusual synergistic properties of the Nts, and their low cost and practical synthesis, made these nanocomposites promising green tools that can positively and swiftly photokill P. syringae within 30 min.

Microbial contamination in methanol biofilters inoculated with a pure strain of Pichia pastoris: A potential limitation for waste revalorization.

Novel biotechnologies to valorize waste emissions are based on the use of specialized microbial groups that produce different compounds of industrial interest. On this scenario, the retention of such specific microorganisms in the system is of critical interest; however, the potential limitations of working with simplified cultures in a competitive open environment are neither fully explored nor well understood. In this work, a series of biofilters treating methanol vapors coupled with heterologous endochitinase production were used to evaluate the performance of a specialized microbial population during a typical open-to-environment operation. The biofilters were inoculated with a transformed strain of Pichia pastoris and were operated identically for about 90 days. The results showed that the biofiltration performance became diverse with time in terms of the elimination capacity (EC) shifting from a variation coefficient of 1.5% (EC = 274 ± 24, 279 ± 5, and 281.9 ± 25 g/[m3 h]) at the beginning of the operation to 33% (EC = 297 ± 9, 338 ± 7, and 341 ± 2 g/[m3 h]) at the end of operation. Epifluorescence analysis and cloning-sequencing suggested that P. pastoris remained as the dominant microorganism of methanol degradation, whereas diverse airborne bacteria, including Ochrobactrum spp. and Klebsiella oxytoca, played a secondary role possibly associated with the consumption of intermediates. Overall, this study found that low diversity systems operated under non-sterile conditions could be susceptible to contamination with external microorganisms causing a diversifying behavior at the performance and microbial community levels. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2715, 2019.

Silencing of OCH1 unveils the role of Sporothrix schenckii N-linked glycans during the host-fungus interaction.

BACKGROUND: Sporothrix schenckii is a neglected fungal pathogen for the human being and other mammals. In several fungal systems, Och1 is a Golgi α1,6-mannosyltransferase with a key function in the synthesis of N-linked glycans; which are important elements during the host-fungus interplay. The role of OCH1 in fungal virulence seems to be species-specific, being an essential component for Candida albicans virulence and dispensable during the interaction of Aspergillus fumigatus with the host. METHODS: Here, we silenced S. schenckii OCH1 and characterized the phenotype of the mutant strains. RESULTS: The mutant strains did not show defects in the cell or colony morphology, the growth rate or the ability to undergo dimorphism; but the cell wall changed in both composition and exposure of inner components at the surface. When interacting with human monocytes, the silenced strains had a reduced ability to stimulate TNFα and IL-6 but stimulated higher levels of IL-10. The interaction with human macrophages was also altered, with reduced numbers of silenced cells phagocytosed. These strains showed virulence attenuation in both Galleria mellonella and in the mouse model of sporotrichosis. Nonetheless, the cytokine levels in infected organs did not vary significantly when compared with the wild-type strain. CONCLUSION: Our data demonstrate that OCH1 silencing affects different aspects of the S. schenckii-host interaction.

Light induces oxidative damage and protein stability in the fungal photoreceptor Vivid.

Flavin-binding photoreceptor proteins sense blue-light (BL) in diverse organisms and have become core elements in recent optogenetic applications. The light-oxygen-voltage (LOV) protein Vivid (VVD) from the filamentous fungus Neurospora crassa is a classic BL photoreceptor, characterized by effecting a photocycle based on light-driven formation and subsequent spontaneous decay of a flavin-cysteinyl adduct. Here we report that VVD presents alternative outcomes to light exposure that result in protein self-oxidation and, unexpectedly, rise of stability through kinetic control. Using optical absorbance and mass spectrometry we show that purified VVD develops amorphous aggregates with the presence of oxidized residues located at the cofactor binding pocket. Light exposure increases oxidative levels in VVD and specific probe analysis identifies singlet oxygen production by the flavin. These results indicate that VVD acts alternatively as a photosensitizer, inducing self-oxidative damage and subsequent aggregation. Surprisingly, BL illumination has an additional, opposite effect in VVD. We show that light-induced adduct formation establishes a stable state, delaying protein aggregation until photoadduct decay occurs. In accordance, repeated BL illumination suppresses VVD aggregation altogether. Furthermore, photoadduct formation confers VVD stability against chemical denaturation. Analysis of the aggregation kinetics and testing of stabilizers against aggregation reveal that aggregation in VVD proceeds through light-dependent kinetic control and dimer formation. These results uncover the aggregation pathway of a photosensor, where light induces a remarkable interplay between protein damage and stability.

Generation of Sporothrix schenckii mutants expressing the green fluorescent protein suitable for the study of host-fungus interactions.

Sporotrichosis is an infection caused by members of the Sporothrix genus, and among them, Sporothrix schenckii is one of the etiological agents. Both, the disease and the causative agent have gained interest in the recent years, because of the report of epidemic outbreaks, and the description of the disease transmission from animals to human beings. Despite the relevance of S. schenckii in the clinical field, there are basic aspects of its biology poorly explored. So far, Agrobacterium tumefaciens-mediated transformation has been reported as an alternative for genetic manipulation of this fungal pathogen. Here, we report the optimization of the transformation method and used this to generate insertional mutants that express the green fluorescent protein in S. schenckii. We obtained five mutant strains that showed mitotic stability and expression of the reporter gene. The strains displayed normal cell wall composition, and a similar ability to interact ex vivo with human monocytes and monocyte-derived macrophages. Moreover, the virulence in larvae of Galleria mellonella was similar to that obtained with the wild-type control strains. These data indicate that these fluorescent mutants with normal ability to interact with the host could be used in bioimaging to track the host-Sporothrix interaction in vivo.

Histone acetyltransferase TGF-1 regulates Trichoderma atroviride secondary metabolism and mycoparasitism.

Some filamentous fungi of the Trichoderma genus are used as biocontrol agents against airborne and soilborne phytopathogens. The proposed mechanism by which Trichoderma spp. antagonizes phytopathogens is through the release of lytic enzymes, antimicrobial compounds, mycoparasitism, and the induction of systemic disease-resistance in plants. Here we analyzed the role of TGF-1 (Trichoderma Gcn Five-1), a histone acetyltransferase of Trichoderma atroviride, in mycoparasitism and antibiosis against the phytopathogen Rhizoctonia solani. Trichostatin A (TSA), a histone deacetylase inhibitor that promotes histone acetylation, slightly affected T. atroviride and R. solani growth, but not the growth of the mycoparasite over R. solani. Application of TSA to the liquid medium induced synthesis of antimicrobial compounds. Expression analysis of the mycoparasitism-related genes ech-42 and prb-1, which encode an endochitinase and a proteinase, as well as the secondary metabolism-related genes pbs-1 and tps-1, which encode a peptaibol synthetase and a terpene synthase, respectively, showed that they were regulated by TSA. A T. atroviride strain harboring a deletion of tgf-1 gene showed slow growth, thinner and less branched hyphae than the wild-type strain, whereas its ability to coil around the R. solani hyphae was not affected. Δtgf-1 presented a diminished capacity to grow over R. solani, but the ability of its mycelium -free culture filtrates (MFCF) to inhibit the phytopathogen growth was enhanced. Intriguingly, addition of TSA to the culture medium reverted the enhanced inhibition growth of Δtgf-1 MFCF on R. solani at levels compared to the wild-type MFCF grown in medium amended with TSA. The presence of R. solani mycelium in the culture medium induced similar proteinase activity in a Δtgf-1 compared to the wild-type, whereas the chitinolytic activity was higher in a Δtgf-1 mutant in the absence of R. solani, compared to the parental strain. Expression of mycoparasitism- and secondary metabolism-related genes in Δtgf-1 was differentially regulated in the presence or absence of R. solani. These results indicate that histone acetylation may play important roles in the biocontrol mechanisms of T. atroviride.

The Genomes of Three Uneven Siblings: Footprints of the Lifestyles of Three Trichoderma Species.

The genus Trichoderma contains fungi with high relevance for humans, with applications in enzyme production for plant cell wall degradation and use in biocontrol. Here, we provide a broad, comprehensive overview of the genomic content of these species for "hot topic" research aspects, including CAZymes, transport, transcription factors, and development, along with a detailed analysis and annotation of less-studied topics, such as signal transduction, genome integrity, chromatin, photobiology, or lipid, sulfur, and nitrogen metabolism in T. reesei, T. atroviride, and T. virens, and we open up new perspectives to those topics discussed previously. In total, we covered more than 2,000 of the predicted 9,000 to 11,000 genes of each Trichoderma species discussed, which is >20% of the respective gene content. Additionally, we considered available transcriptome data for the annotated genes. Highlights of our analyses include overall carbohydrate cleavage preferences due to the different genomic contents and regulation of the respective genes. We found light regulation of many sulfur metabolic genes. Additionally, a new Golgi 1,2-mannosidase likely involved in N-linked glycosylation was detected, as were indications for the ability of Trichoderma spp. to generate hybrid galactose-containing N-linked glycans. The genomic inventory of effector proteins revealed numerous compounds unique to Trichoderma, and these warrant further investigation. We found interesting expansions in the Trichoderma genus in several signaling pathways, such as G-protein-coupled receptors, RAS GTPases, and casein kinases. A particularly interesting feature absolutely unique to T. atroviride is the duplication of the alternative sulfur amino acid synthesis pathway.