Trichoderma is a genus of mycotrophic filamentous fungi (teleomorph Hypocrea) which possess a bright variety of biotrophic and saprotrophic lifestyles. The ability to parasitize and/or kill other fungi (mycoparasitism) is used in plant protection against soil-borne fungal diseases (biological control, or biocontrol). To investigate mechanisms of mycoparasitism, we compared the transcriptional responses of cosmopolitan opportunistic species and powerful biocontrol agents Trichoderma atroviride and T. virens with tropical ecologically restricted species T. reesei during confrontations with a plant pathogenic fungus Rhizoctonia solani.

Mycoparasites, e.g. fungi feeding on other fungi, are prominent within the genus Trichoderma and represent a promising alternative to chemical fungicides for plant disease control. We previously showed that the seven-transmembrane receptor Gpr1 regulates mycelial growth and asexual development and governs mycoparasitism-related processes in Trichoderma atroviride. We now describe the identification of genes being targeted by Gpr1 under mycoparasitic conditions. The identified gene set includes a candidate, sfp2, encoding a protein of the fungal-specific Sur7 superfamily, whose upregulation in T. atroviride upon interaction with a fungal prey is dependent on Gpr1. Sur7 family proteins are typical residents of membrane microdomains such as the membrane compartment of Can1 (MCC)/eisosome in yeast. We found that GFP-labeled Gpr1 and Sfp2 proteins show partly overlapping localization patterns in T. atroviride hyphae, which may point to shared functions and potential interaction during signal perception and endocytosis. Deletion of sfp2 caused heavily altered colony morphology, defects in polarized growth, cell wall integrity and endocytosis, and significantly reduced mycoparasitic activity, whereas sfp2 overexpression enhanced full overgrowth and killing of the prey. Transcriptional activation of a chitinase specific for hyphal growth and network formation and strong downregulation of chitin synthase-encoding genes were observed in Δsfp2. Taken together, these findings imply crucial functions of Sfp2 in hyphal morphogenesis of T. atroviride and its interaction with prey fungi.

Mycoparasitism, a lifestyle where one fungus is parasitic on another fungus, has special relevance when the prey is a plant pathogen, providing a strategy for biological control of pests for plant protection. Probably, the most studied biocontrol agents are species of the genus Hypocrea/Trichoderma.

Trichoderma reesei, a mitosporic green mould, was recognized during the WW II based on a single isolate from the Solomon Islands and since then used in industry for production of cellulases. It is believed to be an anamorph (asexual stage) of the common pantropical ascomycete Hypocrea jecorina.

Single-molecule localization microscopy has boosted our understanding of biological samples by offering access to subdiffraction resolution using fluorescence microscopy methods. While in standard mammalian cells this approach has found wide-spread use, its application to filamentous fungi has been scarce. This is mainly due to experimental challenges that lead to high amounts of background signal because of ample autofluorescence. Here, we report the optimization of labeling, imaging and data analysis protocols to yield the first single-molecule localization microscopy images of the filamentous fungus Trichoderma atroviride. As an example, we show the spatial distribution of the Sur7 tetraspanin-family protein Sfp2 required for hyphal growth and cell wall stability in this mycoparasitic fungus.

Trichoderma atroviride (Ascomycota, Sordariomycetes) is a well-known mycoparasite applied for protecting plants against fungal pathogens. Its mycoparasitic activity involves processes shared with plant and human pathogenic fungi such as the production of cell wall degrading enzymes and secondary metabolites and is tightly regulated by environmental cues. In eukaryotes, the conserved Target of Rapamycin (TOR) kinase serves as a central regulator of cellular growth in response to nutrient availability. Here we describe how alteration of the activity of TOR1, the single and essential TOR kinase of T. atroviride, by treatment with chemical TOR inhibitors or by genetic manipulation of selected TOR pathway components affected various cellular functions. Loss of TSC1 and TSC2, that are negative regulators of TOR complex 1 (TORC1) in mammalian cells, resulted in altered nitrogen source-dependent growth of T. atroviride, reduced mycoparasitic overgrowth and, in the case of Δtsc1, a diminished production of numerous secondary metabolites. Deletion of the gene encoding the GTPase RHE2, whose mammalian orthologue activates mTORC1, led to rapamycin hypersensitivity and altered secondary metabolism, but had an only minor effect on vegetative growth and mycoparasitic overgrowth. The latter also applied to mutants missing the npr1-1 gene that encodes a fungus-specific kinase known as TOR target in yeast. Genome-wide transcriptome analysis confirmed TOR1 as a regulatory hub that governs T. atroviride metabolism and processes associated to ribosome biogenesis, gene expression and translation. In addition, mycoparasitism-relevant genes encoding terpenoid and polyketide synthases, peptidases, glycoside hydrolases, small secreted cysteine-rich proteins, and G protein coupled receptors emerged as TOR1 targets. Our results provide the first in-depth insights into TOR signaling in a fungal mycoparasite and emphasize its importance in the regulation of processes that critically contribute to the antagonistic activity of T. atroviride.

Mycoparasitism is a key feature of Trichoderma (Hypocreales, Ascomycota) biocontrol agents. Recent studies of intracellular signal transduction pathways of the potent mycoparasite Trichoderma atroviride revealed the involvement of Tmk1, a mitogen-activated protein kinase (MAPK), in triggering the mycoparasitic response. We previously showed that mutants missing Tmk1 exhibit reduced mycoparasitic activity against several plant pathogenic fungi. In this study, we identified the most robustly regulated targets that were governed by Tmk1 during mycoparasitism using transcriptome and proteome profiling. Tmk1 mainly exerts a stimulating function for T. atroviride during its mycoparasitic interaction with the fungal plant pathogen Rhizoctonia solani, as reflected by 89% of strongly differently responding genes in the ∆tmk1 mutant compared to the wild type. Specifically, 54% of these genes showed strong downregulation in the response with a deletion of the tmk1 gene, whereas in the wild type the same genes were strongly upregulated during the interaction with the fungal host. These included the gene encoding the mycoparasitism-related proteinase Prb1; genes involved in signal transduction pathways such as a candidate coding for a conserved 14-3-3 protein, and a gene coding for Tmk2, the T. atroviride cell-wall integrity MAP kinase; genes encoding a specific siderophore synthetase, and multiple FAD-dependent oxidoreductases and aminotransferases. Due to the phosphorylating activity of Tmk1, different (phospho-)proteomics approaches were applied and identified proteins associated with cellular metabolism, energy production, protein synthesis and fate, and cell organization. Members of FAD- and NAD/NADP-binding-domain proteins, vesicular trafficking of molecules between cellular organelles, fungal translational, as well as protein folding apparatus were among others found to be phosphorylated by Tmk1 during mycoparasitism. Outstanding downregulation in the response of the ∆tmk1 mutant to the fungal host compared to the wild type at both the transcriptome and the proteome levels was observed for nitrilase, indicating that its defense and detoxification functions might be greatly dependent on Tmk1 during T. atroviride mycoparasitism. An intersection network analysis between the identified transcripts and proteins revealed a strong involvement of Tmk1 in molecular functions with GTPase and oxidoreductase activity. These data suggest that during T. atroviride mycoparasitism this MAPK mainly governs processes regulating cell responses to extracellular signals and those involved in reactive oxygen stress.

Single molecule localization microscopy is currently revolutionizing the life sciences as it offers, for the first time, insights into the organization of biological samples below the classical diffraction limit of light microscopy. While there have been numerous examples of new biological findings reported in the last decade, the technique could not reach its full potential due to a set of limitations immanent to the samples themselves. Particularly, high background signals impede the proper performance of most single-molecule identification and localization algorithms. One option is to exploit the characteristic blinking of single molecule signals, which differs substantially from the residual brightness fluctuations of the fluorescence background. To pronounce single molecule signals, we used a temporal high-pass filtering in Fourier space on a pixel-by-pixel basis. We evaluated the performance of temporal filtering by assessing statistical parameters such as true positive rate and false discovery rate. For this, ground truth signals were generated by simulations and overlaid onto experimentally derived movies of samples with high background signals. Compared to the nonfiltered case, we found an improvement of the sensitivity by up to a factor 3.5 while no significant change in the localization accuracy was observable.

The filamentous fungus Trichoderma parareesei is the asexually reproducing ancestor of Trichoderma reesei, the holomorphic industrial producer of cellulase and hemicellulase. Here, we present the genome sequence of the T. parareesei type strain CBS 125925, which contains genes for 9,318 proteins.

Pectin is one of the major and most complex plant cell wall components that needs to be overcome by microorganisms as part of their strategies for plant invasion or nutrition. Microbial pectinolytic enzymes therefore play a significant role for plant-associated microorganisms and for the decomposition and recycling of plant organic matter. Recently, comparative studies revealed significant gene copy number expansion of the polysaccharide lyase 1 (PL1) pectin/pectate lyase gene family in the Clonostachys rosea genome, while only low numbers were found in Trichoderma species. Both of these fungal genera are widely known for their ability to parasitize and kill other fungi (mycoparasitism) and certain species are thus used for biocontrol of plant pathogenic fungi.

The identification and characterization of the transcriptional regulatory networks governing the physiology and adaptation of microbial cells is a key step in understanding their behaviour. One such wide-domain regulatory circuit, essential to all cells, is carbon catabolite repression (CCR): it allows the cell to prefer some carbon sources, whose assimilation is of high nutritional value, over less profitable ones. In lower multicellular fungi, the C2H2 zinc finger CreA/CRE1 protein has been shown to act as the transcriptional repressor in this process. However, the complete list of its gene targets is not known.

The diversity of Trichoderma (Hypocreales, Ascomycota) colonizing leaf litter as well as the rhizosphere of Garcinia macrophylla (Clusiaceae) was investigated in primary and secondary rain forests in Colombian Amazonia. DNA barcoding of 107 strains based on the internal transcribed spacers 1 and 2 (ITS1 and 2) of the ribosomal RNA gene cluster and the partial sequence of the translation elongation factor 1 alpha (tef1) gene revealed that the diversity of Trichoderma was dominated (71 %) by three common cosmopolitan species, namely Trichoderma harzianum sensu lato (41 %), Trichoderma spirale (17 %) and Trichoderma koningiopsis (13 %). Four ITS 1 and 2 phylotypes (13 strains) could not be identified with certainty. Multigene phylogenetic analysis and phenotype profiling of four strains with an ITS1 and 2 phylotype similar to Trichoderma strigosum revealed a new sister species of the latter that is described here as Trichoderma strigosellum sp. nov. Sequence similarity searches revealed that this species also occurs in soils of Malaysia and Cameroon, suggesting a pantropical distribution.

Unlike most other fungi, molds of the genus Trichoderma (Hypocreales, Ascomycota) are aggressive parasites of other fungi and efficient decomposers of plant biomass. Although nutritional shifts are common among hypocrealean fungi, there are no examples of such broad substrate versatility as that observed in Trichoderma. A phylogenomic analysis of 23 hypocrealean fungi (including nine Trichoderma spp. and the related Escovopsis weberi) revealed that the genus Trichoderma has evolved from an ancestor with limited cellulolytic capability that fed on either fungi or arthropods. The evolutionary analysis of Trichoderma genes encoding plant cell wall-degrading carbohydrate-active enzymes and auxiliary proteins (pcwdCAZome, 122 gene families) based on a gene tree / species tree reconciliation demonstrated that the formation of the genus was accompanied by an unprecedented extent of lateral gene transfer (LGT). Nearly one-half of the genes in Trichoderma pcwdCAZome (41%) were obtained via LGT from plant-associated filamentous fungi belonging to different classes of Ascomycota, while no LGT was observed from other potential donors. In addition to the ability to feed on unrelated fungi (such as Basidiomycota), we also showed that Trichoderma is capable of endoparasitism on a broad range of Ascomycota, including extant LGT donors. This phenomenon was not observed in E. weberi and rarely in other mycoparasitic hypocrealean fungi. Thus, our study suggests that LGT is linked to the ability of Trichoderma to parasitize taxonomically related fungi (up to adelphoparasitism in strict sense). This may have allowed primarily mycotrophic Trichoderma fungi to evolve into decomposers of plant biomass.

The fungal cell wall is composed of a cross-linked matrix of chitin, glucans, mannans, galactomannans, and cell wall proteins with mannan chains. Cell wall mannans are directly attached to the cell wall core, while the majority of mannoproteins is produced with a glycosylphosphatidylinositol (GPI) anchor and then transferred to β-1,6-glucan in the cell wall. In this study, we functionally characterized the transmembrane protein Dfg5 of the glycoside hydrolase family 76 (GH76) in the fungal mycoparasite Trichoderma atroviride, whose ortholog has recently been proposed to cross-link glycoproteins into the cell wall of yeast and fungi. We show that the T. atroviride Dfg5 candidate is a GPI-anchored, transmembrane, 6-hairpin member of the GH76 Dfg5 subfamily that plays an important role in hyphal morphology in this mycoparasite. Alterations in the release of proteins associated with cell wall remodeling as well as a higher amount of non-covalently bonded cell surface proteins were detected in the mutants compared to the wild-type. Gene expression analysis suggests that transcript levels of genes involved in glucan synthesis, of proteases involved in mycoparasitism, and of the Tmk1 mitogen-activated protein kinase (MAPK)-encoding gene are influenced by Dfg5, whereas Tmk3 governs Dfg5 transcription. We show that Dfg5 controls important physiological properties of T. atroviride, such as osmotic stress resistance, hyphal morphology, and cell wall stability.