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Abstract The filamentous ascomycete Trichoderma reesei, known for its prolific cellulolytic enzyme production, recently also gained attention for its secondary metabolite synthesis. Both processes are intricately influenced by environmental factors like carbon source availability and light exposure. Here, we explore the role of the transcription factor STE12 in regulating metabolic pathways in T. reesei in terms of gene regulation, carbon source utilization and biosynthesis of secondary metabolites. We show that STE12 is involved in regulating cellulase gene expression and growth on carbon sources associated with iron homeostasis. STE12 impacts gene regulation in a light dependent manner on cellulose with modulation of several CAZyme encoding genes as well as genes involved in secondary metabolism. STE12 selectively influences the biosynthesis of the sorbicillinoid trichodimerol, while not affecting the biosynthesis of bisorbibutenolide, which was recently shown to be regulated by the MAPkinase pathway upstream of STE12 in the signaling cascade. We further report on the biosynthesis of dehydroacetic acid (DHAA) in T. reesei, a compound known for its antimicrobial properties, which is subject to regulation by STE12. We conclude, that STE12 exerts functions beyond development and hence contributes to balance the energy distribution between substrate consumption, reproduction and defense.

Abstract Background Adaptation to complex, rapidly changing environments is crucial for evolutionary success of fungi. The heterotrimeric G-protein pathway belongs to the most important signaling cascades applied for this task. In Trichoderma reesei, enzyme production, growth and secondary metabolism are among the physiological traits influenced by the G-protein pathway in a light dependent manner. Results Here, we investigated the function of the SNX/H-type regulator of G-protein signaling (RGS) protein RGS4 of T. reesei. We show that RGS4 is involved in regulation of cellulase production, growth, asexual development and oxidative stress response in darkness as well as in osmotic stress response in the presence of sodium chloride, particularly in light. Transcriptome analysis revealed regulation of several ribosomal genes, six genes mutated in RutC30 as well as several genes encoding transcription factors and transporters. Importantly, RGS4 positively regulates the siderophore cluster responsible for fusarinine C biosynthesis in light. The respective deletion mutant shows altered growth on nutrient sources related to siderophore production such as ornithine or proline in a BIOLOG phenotype microarray assay. Additionally, growth on storage carbohydrates as well as several intermediates of the D-galactose and D-arabinose catabolic pathway is decreased, predominantly in light. Conclusions We conclude that RGS4 mainly operates in light and targets plant cell wall degradation, siderophore production and storage compound metabolism in T. reesei.

Abstract The filamentous fungus Trichoderma reesei is a prolific producer of plant cell wall degrading enzymes, which are regulated in response to diverse environmental signals for optimal adaptation, but also produces a wide array of secondary metabolites. Available carbon source and light are the strongest cues currently known to impact secreted enzyme levels and an interplay with regulation of secondary metabolism became increasingly obvious in recent years. While cellulase regulation is already known to be modulated by different mitogen activated protein kinase (MAPK) pathways, the relevance of the light signal, which is transmitted by this pathway in other fungi as well, is still unknown in T. reesei as are interconnections to secondary metabolism and chemical communication under mating conditions. Here we show that MAPkinases differentially influence cellulase regulation in light and darkness and that the Hog1 homologue TMK3, but not TMK1 or TMK2 are required for the chemotropic response to glucose in T. reesei. Additionally, MAPkinases regulate production of specific secondary metabolites including trichodimerol and bisorbibutenolid, a bioactive compound with cytostatic effect on cancer cells and deterrent effect on larvae, under conditions facilitating mating, which reflects a defect in chemical communication. Strains lacking either of the MAPkinases become female sterile, indicating the conservation of the role of MAPkinases in sexual fertility also in T. reesei. In summary, our findings substantiate the previously detected interconnection of cellulase regulation with regulation of secondary metabolism as well as the involvement of MAPkinases in light dependent gene regulation of cellulase and secondary metabolite genes in fungi.

The filamentous ascomycete Trichoderma reesei is a biotechnological workhorse used in the production of homologous and heterologous proteins for diverse applications including biofuel production, textile finishing and food additives. This fungus uses a complex adaptation machinery to regulate its cellulases in response to environmental conditions. Detailed understanding of this regulation allows for improvement of enzyme production using the strong enzyme gene promotors. Here, we selected six genes with characteristic transcript levels associated with cellulase production to be tested for their enzyme regulatory function. Machine learning for inference of a gene regulatory network (GRN) was applied to support the association of these genes with cellulase induction. Additionally, we screened available transcriptomic data for genes with strong constitutive transcript levels and selected the promoter of the gene cfe1, whose transcript levels were above those of tef1 and cDNA1 and near those of cbh1, for gene overexpression testing. Using this promoter, we explored the relevance to cellulose degradation efficiency of three transporters, two ferric reductases and one gene of unknown function, which were overexpressed in T. reesei grown on cellulose. This promoter enabled up to 400 fold overexpression and yielded transcript levels above those of tef1 or cDNA1. We provide evidence of effects of a ferric reductase, an ammonium permease and a gene of unknown function on the regulatory machinery of cellulase expression in T. reesei. In summary we identified the cfe1 promoter, a novel constitutive promoter with promising efficiency, as well as three genes relevant to cellulase regulation in T. reesei.

The genus Trichoderma is among the best studied groups of filamentous fungi, largely because of its high relevance in applications from agriculture to enzyme biosynthesis to biofuel production. However, the physiological competences of these fungi, that led to these beneficial applications are intriguing also from a scientific and ecological point of view. This review therefore summarizes recent developments in studies of fungal genomes, updates on previously started genome annotation efforts and novel discoveries as well as efforts towards bioprospecting for enzymes and bioactive compounds such as cellulases, enzymes degrading xenobiotics and metabolites with potential pharmaceutical value. Thereby insights are provided into genomes, mitochondrial genomes and genomes of mycoviruses of Trichoderma strains relevant for enzyme production, biocontrol and mycoremediation. In several cases, production of bioactive compounds could be associated with responsible genes or clusters and bioremediation capabilities could be supported or predicted using genome information. Insights into evolution of the genus Trichoderma revealed large scale horizontal gene transfer, predominantly of CAZyme genes, but also secondary metabolite clusters. Investigation of sexual development showed that Trichoderma species are competent of repeat induced point mutation (RIP) and in some cases, segmental aneuploidy was observed. Some random mutants finally gave away their crucial mutations like T. reesei QM9978 and QM9136 and the fertility defect of QM6a was traced back to its gene defect. The Trichoderma core genome was narrowed down to 7000 genes and gene clustering was investigated in the genomes of multiple species. Finally, recent developments in application of CRISPR/Cas9 in Trichoderma, cloning and expression strategies for the workhorse T. reesei as well as the use genome mining tools for bioprospecting Trichoderma are highlighted. The intriguing new findings on evolution, genomics and physiology highlight emerging trends and illustrate worthwhile perspectives in diverse fields of research with Trichoderma.

Abstract Background Trichoderma reesei represents a model system for investigation of plant cell wall degradation and its connection to light response. The cyclic adenosine monophosphate pathway (cAMP pathway) plays an important role in both physiological outputs, being crucial for regulation of photoreceptor function as well as for cellulase regulation on different carbon sources. Phosphorylation of photoreceptors and of the carbon catabolite repressor CRE1 was shown in ascomycetes, indicating a relevance of protein kinase A in regulation of the target genes of these transcription factors as well as an impact on regulation of induction specific genes. Moreover, the cAMP pathway impacts growth and development. Results Here, we investigated gene regulation by the catalytic subunit of protein kinase A (PKAc1) upon growth on cellulose. We found distinct gene sets for regulation upon growth in light and darkness with an overlap of only 13 genes. PKAc1 regulates metabolic genes as well as transport and defense functions. The overlap of gene regulation by PKAc1 with the genes representing the cAMP dependent regulatory output of the photoreceptor ENV1 indicates an involvement of PKA in this pathway, which counteracts its effects by contrasting regulation. Moreover, we found considerable overlap with the gene sets regulated under cellulase inducing conditions and by the carbon catabolite repressor CRE1. Our analysis also showed that PKAc1 regulates the genes of the SOR cluster associated with the biosynthesis of sorbicillinoids. The homologue of gin4, encoding a CAMK type kinase, which is regulated by PKAc1, CRE1 and YPR2 showed a moderate impact on trichodimerol production. We isolated trichodimerol as representative sorbicillin compound and established a method for its quantification in large sample sets using high performance thin layer chromatography (HPTLC), which can be broadly applied for secondary metabolite screening of mutants or different growth conditions. Due to the high expression levels of the SOR cluster under conditions of sexual development we crosschecked the relevance of PKAc1 under these conditions. We could show that PKAc1 impacts biosynthesis of trichodimerol in axenic growth and upon mating. Conclusions We conclude that PKAc1 is involved in light dependent regulation of plant cell wall degradation, including carbon catabolite repression as well as secondary metabolism and development in T. reesei.

Abstract Background Filamentous fungi have evolved to succeed in nature by efficient growth and degradation of substrates, but also due to the production of secondary metabolites including mycotoxins. For Trichoderma reesei, as a biotechnological workhorse for homologous and heterologous protein production, secondary metabolite secretion is of particular importance for industrial application. Recent studies revealed an interconnected regulation of enzyme gene expression and carbon metabolism with secondary metabolism. Results Here, we investigated gene regulation by YPR2, one out of two transcription factors located within the SOR cluster of T. reesei, which is involved in biosynthesis of sorbicillinoids. Transcriptome analysis showed that YPR2 exerts its major function in constant darkness upon growth on cellulose. Targets (direct and indirect) of YPR2 overlap with induction specific genes as well as with targets of the carbon catabolite repressor CRE1 and a considerable proportion is regulated by photoreceptors as well. Functional category analysis revealed both effects on carbon metabolism and secondary metabolism. Further, we found indications for an involvement of YPR2 in regulation of siderophores. In agreement with transcriptome data, mass spectrometric analyses revealed a broad alteration in metabolite patterns in ∆ypr2. Additionally, YPR2 positively influenced alamethicin levels along with transcript levels of the alamethicin synthase tex1 and is essential for production of orsellinic acid in darkness. Conclusions YPR2 is an important regulator balancing secondary metabolism with carbon metabolism in darkness and depending on the carbon source. The function of YPR2 reaches beyond the SOR cluster in which ypr2 is located and happens downstream of carbon catabolite repression mediated by CRE1.

Fungi of the genus Trichoderma are of high importance for biotechnological applications, in biocontrol and for production of homologous and heterologous proteins. However, sexual crossing under laboratory conditions has so far only been achieved with the species Trichoderma reesei, which was so far only isolated from tropical regions. Our isolation efforts aimed at the collection of Trichoderma strains from Austrian soils surprisingly also yielded 12 strains of the species T. reesei, which was previously not known to occur in Europe. Their identity was confirmed with tef1- and rpb2-sequencing and phylogenetic analysis. They could clearly be distinguished from tropical strains including the common laboratory wildtypes by UP-PCR and genetic variations adjacent to the mating type locus. The strains readily mated with reference strains derived from CBS999.97. Secreted cellulase and xylanase levels of these isolates were up to six-fold higher than those of QM6a indicating a high potential for strain improvement. The strains showed different responses to injury in terms of induction of sporulation, but a correlation to alterations in the nox1-gene sequence was not detected. Several synonymous SNPs were found in the sequence of the regulator gene noxR of the soil isolates compared to QM6a. Only in one strain, non-synonymous SNPs were found which impact a PEST sequence of NoxR, suggesting altered protein stability. The availability of sexually fertile strains from middle Europe naturally producing decent amounts of plant cell wall degrading enzymes opens up novel perspectives for non-GMO strain improvement and biological pretreatment of plant biomass for bioethanol production. Moreover, the varied response of these strains to injury in terms of sporulation, which is independent of Nox1 and NoxR suggests that additional regulators impact this phenomenon in T. reesei.

Changing environmental conditions are of utmost importance for regulation of secondary metabolism in fungi. Different environmental cues including the carbon source, light and the presence of a mating partner can lead to altered production of compounds. Thereby, the heterotrimeric G-protein pathway is of major importance for sensing and adjustment of gene regulation. Regulation of secondary metabolism is crucial in the biotechnological workhorse Trichoderma reesei for knowledge-based adjustment in industrial fermentations, but also with respect to the potential use as a host for heterologous compound production. We investigated the function of the class VII G-protein coupled receptor (GPCR) gene gpr8 that is localized in the vicinity of the SOR cluster, which is responsible for biosynthesis of sorbicillinoids. GPR8 positively impacts regulation of the genes in this cluster in darkness. Accordingly, abundance of trichodimerol and dihydrotrichotetronine as well as other secondary metabolites is decreased in the deletion mutant. Transcriptome analysis moreover showed the major role of GPR8 being exerted in darkness with a considerable influence on regulation of secondary metabolism. Genes regulated in Δgpr8 overlap with those regulated directly or indirectly by the transcription factor YPR2, especially concerning genes related to secondary metabolism. The predicted FAD/FMN containing dehydrogenase gene sor7, one of the positive targets of the cascade triggered by GPR8, has a positive effect on secondary metabolite production, but also cellulase gene expression. Hence SOR7 has some overlapping, but also additional functions compared to GPR8. The G-protein coupled receptor GPR8 exerts a light dependent impact on secondary metabolism, which is in part mediated by the transcription factor YPR2 and the function of SOR7. Hence, T. reesei may apply GPR8 to adjust production of secondary metabolites and hence chemical communication to signals from the environment.

Trichoderma strains used in biological control products usually exhibit high efficiency in the control of plant diseases. However, their behavior under field conditions is difficult to predict. In addition, the potential of indigenous strains has been poorly assayed as well as their possible behavior as endophytes. Hence, niche colonization is a key feature for an effective protection. In this study, we aimed to: (i) explore the possibility of using a new Trichoderma strain isolated from vine to control pathogens, (ii) study the in planta interaction with the pathogen Phaeoacremonium minimum W. Gams, Crous, M.J. Wingf. & L. Mugnai (formerly Phaeoacremonium aleophilum), a pioneer fungus involved in Grapevine Trunk Diseases (GTDs) such as esca. For this purpose, fluorescently tagged Trichoderma sp. T154 and a P. minimum strain were used for scanning electron microscopy and confocal scanning laser microscopy analyses. Data showed that the Trichoderma strain is able to colonize plants up to 12 weeks post inoculation and is located in xylem, fibers, as well as in parenchymatic tissues inside the wood. The beneficial fungus reduced colonization of the esca-related pathogen colonizing the same niches. The main observed mechanism involved in biocontrol of Trichoderma against the esca pathogen was spore adhesion, niche exclusion and only few typical hypha coiling was found between Trichoderma and the pathogen. These results suggest that the Trichoderma strain has potential for reducing the colonization of Phaeoacremonium minimum and thus, an inoculation of this biological control agent can protect the plant by limiting the development of GTD, and the strain can behave as an endophyte.

The necrotrophic mycoparasite Trichoderma atroviride is a biological pest control agent frequently applied in agriculture for the protection of plants against fungal phytopathogens. One of the main secondary metabolites produced by this fungus is 6-pentyl-α-pyrone (6-PP). 6-PP is an organic compound with antifungal and plant growth-promoting activities, whose biosynthesis was previously proposed to involve a lipoxygenase (Lox). In this study, we investigated the role of the single lipoxygenase-encoding gene lox1 encoded in the T. atroviride genome by targeted gene deletion. We found that light inhibits 6-PP biosynthesis but lox1 is dispensable for 6-PP production as well as for the ability of T. atroviride to parasitize and antagonize host fungi. However, we found Lox1 to be involved in T. atroviride conidiation in darkness, in injury-response, in the production of several metabolites, including oxylipins and volatile organic compounds, as well as in the induction of systemic resistance against the plant-pathogenic fungus Botrytis cinerea in Arabidopsis thaliana plants. Our findings give novel insights into the roles of a fungal Ile-group lipoxygenase and expand the understanding of a light-dependent role of these enzymes.

The complex environment of fungi requires a delicate balance between the efforts to acquire nutrition, to reproduce, and to fend off competitors. In Trichoderma reesei, an interrelationship between regulation of enzyme gene expression and secondary metabolism was shown. In this study, we investigated the physiological relevance of the unique YPK1-type kinase USK1 of T. reesei. Usk1 is located in the vicinity of the SOR cluster and is involved in regulation of several genes from this secondary metabolite cluster as well as dihydrotrichotetronine and other secondary metabolites. Moreover, USK1 is required for biosynthesis of normal levels of secondary metabolites in liquid culture. USK1 positively influences cellulase gene regulation, secreted cellulase activity, and biomass formation upon growth in constant darkness on cellulose. Positive effects of USK1 on transcript abundance of the regulator of secondary metabolism, vel1, and the carbon catabolite repressor gene cre1 are in agreement with these functions. In summary, we found that with USK1, T. reesei comprises a unique kinase that adds an additional layer of regulation to the connection of secondary metabolism and enzyme production in fungi.

Abstract Background Trichoderma reesei is one of the most frequently used filamentous fungi in industry for production of homologous and heterologous proteins. The ability to use sexual crossing in this fungus was discovered several years ago and opens up new perspectives for industrial strain improvement and investigation of gene regulation. Results Here we investigated the female sterile strain QM6a in comparison to the fertile isolate CBS999.97 and backcrossed derivatives of QM6a, which have regained fertility (FF1 and FF2 strains) in both mating types under conditions of sexual development. We found considerable differences in gene regulation between strains with the CBS999.97 genetic background and the QM6a background. Regulation patterns of QM6a largely clustered with the backcrossed FF1 and FF2 strains. Differential regulation between QM6a and FF1/FF2 as well as clustering of QM6a patterns with those of CBS999.97 strains was also observed. Consistent mating type dependent regulation was limited to mating type genes and those involved in pheromone response, but included also nta1 encoding a putative N-terminal amidase previously not associated with development. Comparison of female sterile QM6a with female fertile strains showed differential expression in genes encoding several transcription factors, metabolic genes and genes involved in secondary metabolism. Conclusions Evaluation of the functions of genes specifically regulated under conditions of sexual development and of genes with highest levels of transcripts under these conditions indicated a relevance of secondary metabolism for sexual development in T. reesei. Among others, the biosynthetic genes of the recently characterized SOR cluster are in this gene group. However, these genes are not essential for sexual development, but rather have a function in protection and defence against competitors during reproduction.

Abstract Trichoderma reesei (syn. Hypocrea jecorina) is the model organism for industrial production of plant cell wall degradating enzymes. The integration of light and nutrient signals for adaptation of enzyme production in T. reesei emerged as an important regulatory mechanism to be tackled for strain improvement. Gene regulation specific for cellulase inducing conditions is different in light and darkness with substantial regulation by photoreceptors. Genes regulated by light are clustered in the genome, with several of the clusters overlapping with CAZyme clusters. Major cellulase transcription factor genes and at least 75% of glycoside hydrolase encoding genes show the potential of light dependent regulation. Accordingly, light dependent protein complex formation occurs within the promoters of cellulases and their regulators. Additionally growth on diverse carbon sources is different between light and darkness and dependent on the presence of photoreceptors in several cases. Thereby, also light intensity plays a regulatory role, with cellulase levels dropping at higher light intensities dependent in the strain background. The heterotrimeric G-protein pathway is the most important nutrient signaling pathway in the connection with light response and triggers posttranscriptional regulation of cellulase expression. All G-protein alpha subunits impact cellulase regulation in a light dependent manner. The downstream cAMP pathway is involved in light dependent regulation as well. Connections between the regulatory pathways are mainly established via the photoreceptor ENV1. The effect of photoreceptors on plant cell wall degradation also occurs in the model filamentous fungus Neurospora crassa. In the currently proposed model, T. reesei senses the presence of plant biomass in its environment by detection of building blocks of cellulose and hemicellulose. Interpretation of the respective signals is subsequently adjusted to the requirements in light and darkness (or on the surface versus within the substrate) by an interconnection of nutrient signaling with light response. This review provides an overview on the importance of light, photoreceptors and related signaling pathways for formation of plant cell wall degrading enzymes in T. reesei. Additionally, the relevance of light dependent gene regulation for industrial fermentations with Trichoderma as well as strategies for exploitation of the observed effects are discussed.

Fungal plant cell wall degradation processes are governed by complex regulatory mechanisms, allowing the organisms to adapt their metabolic program with high specificity to the available substrates. While the uptake of representative plant cell wall mono- and disaccharides is known to induce specific transcriptional and translational responses, the processes related to early signal reception and transduction remain largely unknown. A fast and reversible way of signal transmission are post-translational protein modifications, such as phosphorylations, which could initiate rapid adaptations of the fungal metabolism to a new condition. To elucidate how changes in the initial substrate recognition phase of Neurospora crassa affect the global phosphorylation pattern, phospho-proteomics was performed after a short (2 min) induction period with several plant cell wall-related mono- and disaccharides. The MS/MS-based peptide analysis revealed large-scale substrate-specific protein phosphorylation and de-phosphorylations. Using the proteins identified by MS/MS, a protein-protein-interaction (PPI) network was constructed. The variance in phosphorylation of a large number of kinases, phosphatases and transcription factors indicate the participation of many known signaling pathways, including circadian responses, two-component regulatory systems, MAP kinases as well as the cAMP-dependent and heterotrimeric G-protein pathways. Adenylate cyclase, a key component of the cAMP pathway, was identified as a potential hub for carbon source-specific differential protein interactions. In addition, four phosphorylated F-Box proteins were identified, two of which, Fbx-19 and Fbx-22, were found to be involved in carbon catabolite repression responses. Overall, these results provide unprecedented and detailed insights into a so far less well known stage of the fungal response to environmental cues and allow to better elucidate the molecular mechanisms of sensory perception and signal transduction during plant cell wall degradation.

The soil-borne fungus Dactylonectria torresensis is the most common causal agent of black-foot disease in Europe. However, there is a lack of understanding on how this fungus can provoke plant symptoms. In this study, we sequenced, annotated and analyzed the genomes of three isolates of D. torresensis collected from asymptomatic vine, weed and soil. Sequenced genomes were further compared to those of 27 fungal species including root and aerial pathogens, white rot degraders, indoor biodeterioration agents, saprotrophs, dark septate endophytes and mycorrhiza. Strains of D. torresensis present genomes with between 64 and 65 Mbp and with up to 18,548 predicted genes for each strain. Average Nucleotide Identity (ANI) shows that strains are different according to genome contents. Clusters of orthologous groups were compared, and clusters of genes related to necroses were particularly detected in all strains of D. torresensis (necrosis inducing peptides and proteins, and ethylene inducing peptides) as well as several genes involved in resistance against fungicides frequently used in viticulture such as copper. Interestingly, an expanded high number of genes related to carbohydrate-active enzymes were detected in each Dactylonectria strain, especially those related to glycoside hydrolases that could be involved in penetration of plant tissues or pathogenicity. An increased number of candidate genes for CAZyme classes AA9 and AA3-1 supports the ability of strains to efficiently degrade plant material. High numbers of genes of D. torresensis related to secretome and small secreted proteins were further characterized. Moreover, the presence of several gene clusters such as fujikurin-like genes was detected and were normally found in Fusariumfujikuroi, that have been linked to fungal pathogenicity. The phenotypes of the three strains investigated showed further difference in light response. We found that Dactylonectria strains have an increased number of photoreceptor encoding genes and we showed sequence alterations. Altogether, the results highlight several gene clusters present in D. torresensis strains that could be linked to endophytic lifestyle, pathogenicity, plant maceration and degradation of plant tissues as well as adaptation to soil contaminated with metals and metalloids and light response.

In Trichoderma reesei light is an important factor in the regulation of glycoside hydrolase gene expression. We therefore investigated the influence of different light intensities on cellulase activity and protein secretion. Differentially secreted proteins in light and darkness as identified by mass spectrometry included members of different glycoside hydrolase families, such as CBH1, Cel3A, Cel61B, XYN2, and XYN4. Several of the associated genes showed light-dependent regulation on the transcript level. Deletion of the photoreceptor genes blr1 and blr2 resulted in a diminished difference of protein abundance between light and darkness. The amount of secreted proteins including that of the major exo-acting beta-1,4-glucanases CBH1 and CBH2 was generally lower in light-grown cultures than in darkness. In contrast, cbh1 transcript levels increased with increasing light intensity from 700 to 2,000 lux but dopped at high light intensity (5,000 lux). In the photoreceptor mutants Δblr1 and Δblr2 cellulase activity in light was reduced compared to activity in darkness, showing a discrepancy between transcript levels and secreted cellulase activity. Furthermore, evaluation of different light sensitivities revealed an increased light tolerance with respect to cellulase expression of QM9414 compared to its parental strain QM6a. Investigation of one of the differentially expressed proteins between light and darkness, CLF1, revealed its function as a factor involved in regulation of secreted protease activity. T. reesei secretes a different set of proteins in light compared to darkness, this difference being mainly due to the function of the major known photoreceptors. Moreover, cellulase regulation is adjusted to light intensity and improved light tolerance was correlated with increased cellulase production. Our findings further support the hypothesis of a light intensity dependent post-transcriptional regulation of cellulase gene expression in T. reesei.

ABSTRACT In fungi, most metabolic processes are subject to regulation by light. Trichoderma reesei is adapted to degradation of plant cell walls and regulates production of the required enzymes in a manner dependent on the nutrient source and the light status. Here we investigated the interrelated relevance of two regulation levels of the transcriptome of T. reesei: light regulation and carbon source-dependent control. We show that the carbon source (cellulose, lactose, sophorose, glucose, or glycerol) is the major source of variation, with light having a modulating effect on transcript regulation. A total of 907 genes were regulated under cellulase-inducing conditions in light, and 947 genes were regulated in darkness, with 530 genes overlapping (1,324 in total). Only 218 of the 1,324 induction-specific genes were independent of light and not regulated by the BLR1, BLR2, and ENV1 photoreceptors. Analysis of the genomic distribution of genes regulated by light upon growth on cellulose revealed considerable overlap of light-regulated clusters with induction-specific clusters and carbohydrate-active enzyme (CAZyme) clusters. Further, we found evidence for the operation of a sensing mechanism for solid cellulosic substrates, with regulation of genes such as swo1, cip1, and cip2 or of genes encoding hydrophobins which is related to the cyclic AMP (cAMP)-dependent regulatory output of ENV1. We identified class XIII G-protein-coupled receptors (GPCRs) CSG1 and CSG2 in T. reesei as putative cellulose/glucose-sensing GPCRs. Our data indicate that the cellulase regulation pathway is bipartite, comprising a section corresponding to transcriptional regulation and one corresponding to posttranscriptional regulation, with the two connected by the function of CSG1. IMPORTANCE In fungi, most metabolic processes are subject to regulation by light. For Trichoderma reesei, light-dependent regulation of cellulase gene expression is specifically shown. Therefore, we intended to unravel the relationship between regulation of enzymes by the carbon source and regulation of enzymes by light. Our two-dimensional analysis included inducing and repressing carbon sources which we used to compare light-specific regulation to dark-specific regulation and to rule out effects specific for a single carbon source. We found close connections with respect to gene regulation as well as significant differences in dealing with carbon in the environment in light and darkness. Moreover, our analyses showed an intricate regulation mechanism for substrate degradation potentially involving surface sensing and provide a basis for knowledge-based screening for strain improvement.

Sexual development is regulated by a complex regulatory mechanism in fungi. For Trichoderma reesei, the light response pathway was shown to impact sexual development, particularly through the photoreceptor ENVOY. Moreover, T. reesei communicates chemically with a potential mating partner in its vicinity, a response which is mediated by the velvet family protein VEL1 and its impact on secondary metabolism. We therefore studied the regulatory interactions of ENV1 and VEL1 with a focus on sexual development. Although individual mutants in both genes are female sterile under standard crossing conditions (light-dark cycles), an altered light regime enabled sexual development, which we found to be due to conditional female sterility of Δenv1, but not Δvel1. Phenotypes of growth and asexual sporulation as well as regulation of the peptide pheromone precursors of double mutants suggested that ENV1 and VEL1 balance positive and negative regulators of these functions. Additionally, VEL1 contributed to the strong deregulation of the pheromone system observed in env1 mutants. Female sterility of Δvel1 was rescued by deletion of env1 in darkness in MAT1-1, indicating a block of sexual development by ENV1 in darkness that is balanced by VEL1 in the wild-type. We conclude that ENV1 and VEL1 exert complementing functions in development of T. reesei. Our results further showed that the different developmental phenotypes of vel1/veA mutants in T. reesei and Aspergillus nidulans are not due to the presence or function of ENV1 in the VELVET regulatory pathway in T. reesei.

Changing light conditions, caused by the rotation of earth resulting in day and night or growth on the surface or within a substrate, result in considerably altered physiological processes in fungi. For the biotechnological workhorse Trichoderma reesei, regulation of glycoside hydrolase gene expression, especially cellulase expression was shown to be a target of light dependent gene regulation. Analysis of regulatory targets of the carbon catabolite repressor CRE1 under cellulase inducing conditions revealed a secondary metabolite cluster to be differentially regulated in light and darkness and by photoreceptors. We found that this cluster is involved in production of trichodimerol and that the two polyketide synthases of the cluster are essential for biosynthesis of dihydrotrichotetronine (syn. bislongiquinolide or bisorbibutenolide). Additionally, an indirect influence on production of the peptaibol antibiotic paracelsin was observed. The two polyketide synthetase genes as well as the monooxygenase gene of the cluster were found to be connected at the level of transcription in a positive feedback cycle in darkness, but negative feedback in light, indicating a cellular sensing and response mechanism for the products of these enzymes. The transcription factor TR_102497/YPR2 residing within the cluster regulates the cluster genes in a light dependent manner. Additionally, an interrelationship of this cluster with regulation of cellulase gene expression was detected. Hence the regulatory connection between primary and secondary metabolism appears more widespread than previously assumed, indicating a sophisticated distribution of resources either to degradation of substrate (feed) or to antagonism of competitors (fight), which is influenced by light.

Collectively classified as white-rot fungi, certain basidiomycetes efficiently degrade the major structural polymers of wood cell walls. A small subset of these Agaricomycetes, exemplified by Phlebiopsis gigantea, is capable of colonizing freshly exposed conifer sapwood despite its high content of extractives, which retards the establishment of other fungal species. The mechanism(s) by which P. gigantea tolerates and metabolizes resinous compounds have not been explored. Here, we report the annotated P. gigantea genome and compare profiles of its transcriptome and secretome when cultured on fresh-cut versus solvent-extracted loblolly pine wood. The P. gigantea genome contains a conventional repertoire of hydrolase genes involved in cellulose/hemicellulose degradation, whose patterns of expression were relatively unperturbed by the absence of extractives. The expression of genes typically ascribed to lignin degradation was also largely unaffected. In contrast, genes likely involved in the transformation and detoxification of wood extractives were highly induced in its presence. Their products included an ABC transporter, lipases, cytochrome P450s, glutathione S-transferase and aldehyde dehydrogenase. Other regulated genes of unknown function and several constitutively expressed genes are also likely involved in P. gigantea's extractives metabolism. These results contribute to our fundamental understanding of pioneer colonization of conifer wood and provide insight into the diverse chemistries employed by fungi in carbon cycling processes.

Almost all creatures have invented sophisticated mechanisms to adjust their developmental and metabolic processes to the changing light intensities in day and night. Recent findings suggest that one such mechanism is signaling via heterotrimeric G-proteins.1 The Trichoderma reesei (anamorph of Hypocrea jecorina) G-alpha subunit gene gna3 was found to be responsive to light and influenced by the light regulatory protein ENVOY.2 GNA3 significantly impacts regulation of cellulase gene expression only in light.1 While the exact mechanism of this regulation remains to be determined, first hints point to a regulation at the transcriptional level, since we observed light induced complex formation within the gna3 promotor. At least some of the components of this putatively regulatory protein complex also bind to the env1- promotor. These data indicate that the signal related by GNA3 is of light-dependent significance for H. jecorina and that the pathway of heterotrimeric G-protein signaling may be a target of the light perception machinery in fungi.

The industrially important cellulolytic filamentous fungus Trichoderma reesei is the anamorph of the pantropical ascomycete Hypocrea jecorina. H. jecorina CBS999.97 strain undergoes a heterothallic reproductive cycle, and the mating yields fertilized perithecia imbedded in stromata. Asci in the perithecia contain 16 linearly arranged ascospores. Here, we investigated H. jecorina sexual development under different light regimes, and found that visible light was dispensable for sexual development (stroma formation and ascospore discharge). By contrast, constant illumination inhibited stroma formation, and an interruption of the darkness facilitated timely stroma formation in a 12 h/12 h light-dark photoperiod. The results of genetic analyses further revealed that H. jecorina blue-light photoreceptors (BLR1, BLR2) and the photoadaptation protein ENV1 were not essential for sexual development in general. BLR1, BLR2 and ENV1 are orthologues of the conserved Neurospora crassa WC-1, WC-2 and VVD, respectively. Moreover, BLR1 and BLR2 mediate both positive and negative light-dependent regulation on sexual development, whereas ENV1 is required for dampening the light-dependent inhibitory effect in response to changes in illumination. Comparative genome-wide microarray analysis demonstrated an overview of light-dependent gene expression versus sexual potency in CBS999.97 (MAT1-2) haploid cells. Constant illumination promotes abundant asexual conidiation and high levels of hpp1 transcripts. hpp1 encodes a h (hybrid)-type propheromone that exhibits features of both yeast a and a pheromone precursors. Deletion of hpp1 could rescue stroma formation but not ascospore generation under constant illumination. We inferred that the HPP1-dependent pheromone signaling system might directly prevent stroma formation or simply disallow the haploid cells to acquire sexual potency due to abundant asexual conidiation upon constant illumination.