RGS4 impacts carbohydrate and siderophore metabolism in Trichoderma reesei.

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.

CLR1 and CLR2 are light dependent regulators of xylanase and pectinase genes in Trichoderma reesei.

Regulation of plant cell wall degradation is of utmost importance for understanding the carbon cycle in nature, but also to improve industrial processes aimed at enzyme production for next generation biofuels. Thereby, the transcription factor networks in different fungi show conservation as well as striking differences, particularly between Trichoderma reesei and Neurospora crassa. Here, we aimed to gain insight into the function of the transcription factors CLR1 and CLR2 in T. reesei, which are crucial for cellulase gene expression in N. crassa. We studied impacts on gene regulation with cellulose, xylan, pectin and chitin, growth on 95 different carbon sources as well as an involvement in regulation of secondary metabolism or development. We found that CLR1 is present in the genome of T. reesei and other Trichoderma spp., albeit with considerably lower homology compared to other ascomycetes. CLR1 and CLR2 regulate pectinase transcript levels upon growth on pectin, no major function was detected on chitin. CLR1 and CLR2 form a positive feedback cycle on xylan and were found to be responsible for balancing co-regulation of xylanase genes in light and darkness with distinct and in part opposite regulatory effects of up to 8fold difference. Our data suggest that CLR1 and CLR2 have evolved differently in T. reesei compared to other fungi. We propose a model in which their main function is in adjustment of regulation of xylanase gene expression to different light conditions and to balance transcript levels of genes involved in plant cell wall degradation according to their individual relevance for this process.

YPR2 is a regulator of light modulated carbon and secondary metabolism in Trichoderma reesei.

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.

SUB1 has photoreceptor dependent and independent functions in sexual development and secondary metabolism in Trichoderma reesei.

Light dependent processes are involved in the regulation of growth, development and enzyme production in Trichoderma reesei. The photoreceptors BLR1, BLR2 and ENV1 exert crucial functions in these processes. We analyzed the involvement of the transcription factor SUB1 in sexual development as well as secondary metabolism and its position in the light signaling cascade. SUB1 influences growth and in contrast to its homologue in N. crassa, SUB1 is not essential for fruiting body formation and male fertility in T. reesei, but required for female fertility. Accordingly, SUB1 is involved in the regulation of the pheromone system of T. reesei. Female sterility of mutants lacking env1 is rescued in triple mutants of blr1, blr2 and env1, but not in double mutants of these genes. Confrontation of strains lacking sub1 results in growth arrest prior to contact of the potential mating partners. This effect is at least in part due to altered secondary metabolite production. Additionally, together with BLR1 and BLR2, SUB1 is essential for spore pigmentation and transcription of pks4, and secondary metabolism is regulated by SUB1 in a light- and nutrient dependent manner. Our results hence indicate rewiring of several pathways targeted by SUB1 in T. reesei.

Plant cell wall deconstruction by ascomycete fungi.

Plant biomass degradation by fungi requires a diverse set of secreted enzymes and significantly contributes to the global carbon cycle. Recent advances in genomic and systems-level studies have begun to reveal how filamentous ascomycete species exploit carbon sources in different habitats. These studies have laid the groundwork for unraveling new enzymatic strategies for deconstructing the plant cell wall, including the discovery of polysaccharide monooxygenases that enhance the activity of cellulases. The identification of genes encoding proteins lacking functional annotation, but that are coregulated with cellulolytic genes, suggests functions associated with plant biomass degradation remain to be elucidated. Recent research shows that signaling cascades mediating cellulolytic responses often act in a light-dependent manner and show crosstalk with other metabolic pathways. In this review, we cover plant biomass degradation, from sensing, to transmission and modulation of signals, to activation of transcription factors and gene induction, to enzyme complement and function.

A Native Threonine Coordinates Ordered Water to Tune Light-Oxygen-Voltage (LOV) Domain Photocycle Kinetics and Osmotic Stress Signaling in Trichoderma reesei ENVOY.

Light-oxygen-voltage (LOV) domain-containing proteins function as small light-activated modules capable of imparting blue light control of biological processes. Their small modular nature has made them model proteins for allosteric signal transduction and optogenetic devices. Despite intense research, key aspects of their signal transduction mechanisms and photochemistry remain poorly understood. In particular, ordered water has been identified as a possible key mediator of photocycle kinetics, despite the lack of ordered water in the LOV active site. Herein, we use recent crystal structures of a fungal LOV protein ENVOY to interrogate the role of Thr(101) in recruiting water to the flavin active site where it can function as an intrinsic base to accelerate photocycle kinetics. Kinetic and molecular dynamic simulations confirm a role in solvent recruitment to the active site and identify structural changes that correlate with solvent recruitment. In vivo analysis of T101I indicates a direct role of the Thr(101) position in mediating adaptation to osmotic stress, thereby verifying biological relevance of ordered water in LOV signaling. The combined studies identify position 101 as a mediator of both allostery and photocycle catalysis that can impact organism physiology.

Omics Analyses of Trichoderma reesei CBS999.97 and QM6a Indicate the Relevance of Female Fertility to Carbohydrate-Active Enzyme and Transporter Levels.

The filamentous fungus Trichoderma reesei is found predominantly in the tropics but also in more temperate regions, such as Europe, and is widely known as a producer of large amounts of plant cell wall-degrading enzymes. We sequenced the genome of the sexually competent isolate CBS999.97, which is phenotypically different from the female sterile strain QM6a but can cross sexually with QM6a. Transcriptome data for growth on cellulose showed that entire carbohydrate-active enzyme (CAZyme) families are consistently differentially regulated between these strains. We evaluated backcrossed strains of both mating types, which acquired female fertility from CBS999.97 but maintained a mostly QM6a genetic background, and we could thereby distinguish between the effects of strain background and female fertility or mating type. We found clear regulatory differences associated with female fertility and female sterility, including regulation of CAZyme and transporter genes. Analysis of carbon source utilization, transcriptomes, and secondary metabolites in these strains revealed that only a few changes in gene regulation are consistently correlated with different mating types. Different strain backgrounds (QM6a versus CBS999.97) resulted in the most significant alterations in the transcriptomes and in carbon source utilization, with decreased growth of CBS999.97 on several amino acids (for example proline or alanine), which further correlated with the downregulation of genes involved in the respective pathways. In combination, our findings support a role of fertility-associated processes in physiology and gene regulation and are of high relevance for the use of sexual crossing in combining the characteristics of two compatible strains or quantitative trait locus (QTL) analysis.IMPORTANCETrichoderma reesei is a filamentous fungus with a high potential for secretion of plant cell wall-degrading enzymes. We sequenced the genome of the fully fertile field isolate CBS999.97 and analyzed its gene regulation characteristics in comparison with the commonly used laboratory wild-type strain QM6a, which is not female fertile. Additionally, we also evaluated fully fertile strains with genotypes very close to that of QM6a in order to distinguish between strain-specific and fertility-specific characteristics. We found that QM6a and CBS999.97 clearly differ in their growth patterns on different carbon sources, CAZyme gene regulation, and secondary metabolism. Importantly, we found altered regulation of 90 genes associated with female fertility, including CAZyme genes and transporter genes, but only minor mating type-dependent differences. Hence, when using sexual crossing in research and for strain improvement, it is important to consider female fertile and female sterile strains for comparison with QM6a and to achieve optimal performance.

Analysis of the Phlebiopsis gigantea genome, transcriptome and secretome provides insight into its pioneer colonization strategies of wood.

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.

Mating type-dependent partner sensing as mediated by VEL1 in Trichoderma reesei.

Sexual development in the filamentous model ascomycete Trichoderma reesei (syn. Hypocrea jecorina) was described only a few years ago. In this study, we show a novel role for VELVET in fungi, which links light response, development and secondary metabolism. Vel1 is required for mating in darkness, normal growth and conidiation. In light, vel1 was dispensable for male fertility but essential for female fertility in both mating types. VEL1 impacted regulation of the pheromone system (hpr1, hpr2, hpp1, ppg1) in a mating type-dependent manner and depending on the mating partner of a given strain. These partner effects only occurred for hpp1 and hpr2, the pheromone precursor and receptor genes associated with the MAT1-2 mating type and for the mating type gene mat1-2-1. Analysis of secondary metabolite patterns secreted by wild type and mutants under asexual and sexual conditions revealed that even in the wild type, the patterns change upon encounter of a mating partner, with again distinct differences for wild type and vel1 mutants. Hence, T. reesei applies a language of pheromones and secondary metabolites to communicate with mating partners and that this communication is at least in part mediated by VEL1.

Comparative genomics of Ceriporiopsis subvermispora and Phanerochaete chrysosporium provide insight into selective ligninolysis.

Efficient lignin depolymerization is unique to the wood decay basidiomycetes, collectively referred to as white rot fungi. Phanerochaete chrysosporium simultaneously degrades lignin and cellulose, whereas the closely related species, Ceriporiopsis subvermispora, also depolymerizes lignin but may do so with relatively little cellulose degradation. To investigate the basis for selective ligninolysis, we conducted comparative genome analysis of C. subvermispora and P. chrysosporium. Genes encoding manganese peroxidase numbered 13 and five in C. subvermispora and P. chrysosporium, respectively. In addition, the C. subvermispora genome contains at least seven genes predicted to encode laccases, whereas the P. chrysosporium genome contains none. We also observed expansion of the number of C. subvermispora desaturase-encoding genes putatively involved in lipid metabolism. Microarray-based transcriptome analysis showed substantial up-regulation of several desaturase and MnP genes in wood-containing medium. MS identified MnP proteins in C. subvermispora culture filtrates, but none in P. chrysosporium cultures. These results support the importance of MnP and a lignin degradation mechanism whereby cleavage of the dominant nonphenolic structures is mediated by lipid peroxidation products. Two C. subvermispora genes were predicted to encode peroxidases structurally similar to P. chrysosporium lignin peroxidase and, following heterologous expression in Escherichia coli, the enzymes were shown to oxidize high redox potential substrates, but not Mn(2+). Apart from oxidative lignin degradation, we also examined cellulolytic and hemicellulolytic systems in both fungi. In summary, the C. subvermispora genetic inventory and expression patterns exhibit increased oxidoreductase potential and diminished cellulolytic capability relative to P. chrysosporium.

Crossroads between light response and nutrient signalling: ENV1 and PhLP1 act as mutual regulatory pair in Trichoderma reesei.

BACKGROUND: Crosstalk between the signalling pathways responding to light-dark cycles and those triggering the adaptation of metabolism to the environment is known to occur in various organisms. This interrelationship of light response and nutrient sigalling is crucial for health and fitness. The tropical ascomycete Trichoderma reesei (syn. Hypocrea jecorina) represents one of the most efficient plant cell wall degraders. Regulation of the enzymes required for this process is affected by nutritional signals as well as other environmental signals including light. Therefore we aimed to elucidate the interrelationship between nutrient and light signaling and how the light signal is transmitted to downstream pathways. RESULTS: We found that the targets of the light regulatory protein ENV1 in light show considerable overlap with those of the heterotrimeric G-protein components PhLP1, GNB1 and GNG1. Detailed investigation of a regulatory interrelationship of these components with ENV1 under conditions of early and late light response indicated a transcriptional mutual regulation between PhLP1 and ENV1, which appears to dampen nutrient signalling during early light response, presumably to free resources for protective measures prior to adaptation of metabolism to light. Investigating the downstream part of the cascade we found support for the hypothesis that ENV1 is necessary for cAMP mediated regulation of a considerable part of the core functions of the output pathway of this cascade, including regulation of glycoside hydrolase genes and those involved in nitrogen, sulphur and amino acid metabolism. CONCLUSIONS: ENV1 and PhLP1 are mutual regulators connecting light signaling with nutrient signaling, with ENV1 triggering the output pathway by influencing cAMP levels.

The transcription factor STE12 influences growth on several carbon sources and production of dehydroacetic acid (DHAA) in Trichoderma reesei.

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.

Mechanisms for plant growth promotion activated by Trichoderma in natural and managed terrestrial ecosystems.

Trichoderma spp. are free-living fungi present in virtually all terrestrial ecosystems. These soil fungi can stimulate plant growth and increase plant nutrient acquisition of macro- and micronutrients and water uptake. Generally, plant growth promotion by Trichoderma is a consequence of the activity of potent fungal signaling metabolites diffused in soil with hormone-like activity, including indolic compounds as indole-3-acetic acid (IAA) produced at concentrations ranging from 14 to 234 µg l-1, and volatile organic compounds such as sesquiterpene isoprenoids (C15), 6-pentyl-2H-pyran-2-one (6-PP) and ethylene (ET) produced at levels from 10 to 120 ng over a period of six days, which in turn, might impact plant endogenous signaling mechanisms orchestrated by plant hormones. Plant growth stimulation occurs without the need of physical contact between both organisms and/or during root colonization. When associated with plants Trichoderma may cause significant biochemical changes in plant content of carbohydrates, amino acids, organic acids and lipids, as detected in Arabidopsis thaliana, maize (Zea mays), tomato (Lycopersicon esculentum) and barley (Hordeum vulgare), which may improve the plant health status during the complete life cycle. Trichoderma-induced plant beneficial effects such as mechanisms of defense and growth are likely to be inherited to the next generations. Depending on the environmental conditions perceived by the fungus during its interaction with plants, Trichoderma can reprogram and/or activate molecular mechanisms commonly modulated by IAA, ET and abscisic acid (ABA) to induce an adaptative physiological response to abiotic stress, including drought, salinity, or environmental pollution. This review, provides a state of the art overview focused on the canonical mechanisms of these beneficial fungi involved in plant growth promotion traits under different environmental scenarios and shows new insights on Trichoderma metabolites from different chemical classes that can modulate specific plant growth aspects. Also, we suggest new research directions on Trichoderma spp. and their secondary metabolites with biological activity on plant growth.

Targets of light signalling in Trichoderma reesei.

BACKGROUND: The tropical ascomycete Trichoderma reesei (Hypocrea jecorina) represents one of the most efficient plant cell wall degraders. Regulation of the enzymes required for this process is affected by nutritional signals as well as other environmental signals including light. RESULTS: Our transcriptome analysis of strains lacking the photoreceptors BLR1 and BLR2 as well as ENV1 revealed a considerable increase in the number of genes showing significantly different transcript levels in light and darkness compared to wild-type. We show that members of all glycoside hydrolase families can be subject to light dependent regulation, hence confirming nutrient utilization including plant cell wall degradation as a major output pathway of light signalling. In contrast to N. crassa, photoreceptor mediated regulation of carbon metabolism in T. reesei occurs primarily by BLR1 and BLR2 via their positive effect on induction of env1 transcription, rather than by a presumed negative effect of ENV1 on the function of the BLR complex. Nevertheless, genes consistently regulated by photoreceptors in N. crassa and T. reesei are significantly enriched in carbon metabolic functions. Hence, different regulatory mechanisms are operative in these two fungi, while the light dependent regulation of plant cell wall degradation appears to be conserved.Analysis of growth on different carbon sources revealed that the oxidoreductive D-galactose and pentose catabolism is influenced by light and ENV1. Transcriptional regulation of the target enzymes in these pathways is enhanced by light and influenced by ENV1, BLR1 and/or BLR2. Additionally we detected an ENV1-regulated genomic cluster of 9 genes including the D-mannitol dehydrogenase gene lxr1, with two genes of this cluster showing consistent regulation in N. crassa. CONCLUSIONS: We show that one major output pathway of light signalling in Trichoderma reesei is regulation of glycoside hydrolase genes and the degradation of hemicellulose building blocks. Targets of ENV1 and BLR1/BLR2 are for the most part distinct and indicate individual functions for ENV1 and the BLR complex besides their postulated regulatory interrelationship.

ENVOY is a major determinant in regulation of sexual development in Hypocrea jecorina (Trichoderma reesei).

Light is one crucial environmental signal which can determine whether a fungus reproduces asexually or initiates sexual development. Mating in the ascomycete Hypocrea jecorina (anamorph Trichoderma reesei) occurs preferentially in light. We therefore investigated the relevance of the light response machinery for sexual development in H. jecorina. We found that the photoreceptors BLR1 and BLR2 and the light-regulatory protein ENV1 have no effect on male fertility, while ENV1 is essential for female fertility. BLR1 and BLR2 were found to impact fruiting body formation although they are not essential for mating. Quantitative reverse transcription-PCR (qRT-PCR) analyses revealed that BLR1, BLR2, and ENV1 negatively regulate transcript levels of both pheromone receptors as well as peptide pheromone precursors in light but not in darkness and in a mating type-dependent manner. The effect of BLR1 and BLR2 on regulation of pheromone precursor and receptor genes is less severe than that of ENV1 as strains lacking env1 show 100-fold (for ppg1) to more than 100,000-fold (for hpp1) increased transcript levels of pheromone precursor genes as well as more than 20-fold increased levels of hpr1, the pheromone receptor receiving the HPP1 signal in a MAT1-1 strain. ENV1 likely integrates additional signals besides light, and our results indicate that its function is partially mediated via regulation of mat1-2-1. We conclude that ENV1 is essential for balancing the levels of genes regulated in a mating-type-dependent manner, which contributes to determination of sexual identity and fruiting body formation.

New cytochalasans from an endophytic Xylaria species associated with Costa Rican Palicourea elata (Rubiaceae).

Four new leucine-derived cytochalasans, possessing a 5,6,5,8-ring (1) and a 5,6,11-ring core (2-4), were isolated from a cultivated endophytic fungus Xylaria sp. strain WH2D4 (Xylariaceae). This fungus was isolated from leaves of the neotropical tree species Palicourea elata (Sw.) Borhidi (Rubiaceae) collected in Costa Rica. The chemical structures were determined by employing IR, MS as well as 1D- and 2D-NMR experiments. The stereochemistry at C-15 of compound 4 was determined by quantum calculations. The isolated compounds did not affect germination and growth of Trichoderma reesei and the opportunistic human fungal pathogen T. longibrachiatum.

MAPkinases regulate secondary metabolism, sexual development and light dependent cellulase regulation in Trichoderma reesei.

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.

Unravelling the molecular basis for light modulated cellulase gene expression - the role of photoreceptors in Neurospora crassa.

BACKGROUND: Light represents an important environmental cue, which exerts considerable influence on the metabolism of fungi. Studies with the biotechnological fungal workhorse Trichoderma reesei (Hypocrea jecorina) have revealed an interconnection between transcriptional regulation of cellulolytic enzymes and the light response. Neurospora crassa has been used as a model organism to study light and circadian rhythm biology. We therefore investigated whether light also regulates transcriptional regulation of cellulolytic enzymes in N. crassa. RESULTS: We show that the N. crassa photoreceptor genes wc-1, wc-2 and vvd are involved in regulation of cellulase gene expression, indicating that this phenomenon is conserved among filamentous fungi. The negative effect of VVD on production of cellulolytic enzymes is thereby accomplished by its role in photoadaptation and hence its function in White collar complex (WCC) formation. In contrast, the induction of vvd expression by the WCC does not seem to be crucial in this process. Additionally, we found that WC-1 and WC-2 not only act as a complex, but also have individual functions upon growth on cellulose. CONCLUSIONS: Genome wide transcriptome analysis of photoreceptor mutants and evaluation of results by analysis of mutant strains identified several candidate genes likely to play a role in light modulated cellulase gene expression. Genes with functions in amino acid metabolism, glycogen metabolism, energy supply and protein folding are enriched among genes with decreased expression levels in the wc-1 and wc-2 mutants. The ability to properly respond to amino acid starvation, i. e. up-regulation of the cross pathway control protein cpc-1, was found to be beneficial for cellulase gene expression. Our results further suggest a contribution of oxidative depolymerization of cellulose to plant cell wall degradation in N. crassa.

The role of pheromone receptors for communication and mating in Hypocrea jecorina (Trichoderma reesei).

Discovery of sexual development in the ascomycete Trichoderma reesei (Hypocrea jecorina) as well as detection of a novel class of peptide pheromone precursors in this fungus indicates promising insights into its physiology and lifestyle. Here we investigated the role of the two pheromone receptors HPR1 and HPR2 in the H. jecorina pheromone-system. We found that these pheromone receptors show an unexpectedly high genetic variability among H. jecorina strains. HPR1 and HPR2 confer female fertility in their cognate mating types (MAT1-1 or MAT1-2, respectively) and mediate induction of fruiting body development. One compatible pheromone precursor-pheromone receptor pair (hpr1-hpp1 or hpr2-ppg1) in mating partners was sufficient for sexual development. Additionally, pheromone receptors were essential for ascospore development, hence indicating their involvement in post-fertilisation events. Neither pheromone precursor genes nor pheromone receptor genes of H. jecorina were transcribed in a strictly mating type dependent manner, but showed enhanced expression levels in the cognate mating type. In the presence of a mating partner under conditions favoring sexual development, transcript levels of pheromone precursors were significantly increased, while those of pheromone receptor genes do not show this trend. In the female sterile T. reesei strain QM6a, transcriptional responses of pheromone precursor and pheromone receptor genes to a mating partner were clearly altered compared to the female fertile wild-type strain CBS999.97. Consequently, a delayed and inappropriate response to the mating partner may be one aspect causing female sterility in QM6a.

Roles of protein kinase A and adenylate cyclase in light-modulated cellulase regulation in Trichoderma reesei.

The cyclic AMP (cAMP) pathway represents a central signaling cascade with crucial functions in all organisms. Previous studies of Trichoderma reesei (anamorph of Hypocrea jecorina) suggested a function of cAMP signaling in regulation of cellulase gene expression. We were therefore interested in how the crucial components of this pathway, adenylate cyclase (ACY1) and cAMP-dependent protein kinase A (PKA), would affect cellulase gene expression. We found that both ACY1 and PKA catalytic subunit 1 (PKAC1) are involved in regulation of vegetative growth but are not essential for sexual development. Interestingly, our results showed considerably increased transcript abundance of cellulase genes in darkness compared to light (light responsiveness) upon growth on lactose. This effect is strongly enhanced in mutant strains lacking PKAC1 or ACY1. Comparison to the wild type showed that ACY1 has a consistently positive effect on cellulase gene expression in light and darkness, while PKAC1 influences transcript levels of cellulase genes positively in light but negatively in darkness. A function of PKAC1 in light-modulated cellulase gene regulation is also reflected by altered complex formation within the cel6a/cbh2 promoter in light and darkness and in the absence of pkac1. Analysis of transcript levels of cellulase regulator genes indicates that the regulatory output of the cAMP pathway may be established via adjustment of XYR1 abundance. Consequently, both adenylate cyclase and protein kinase A are involved in light-modulated cellulase gene expression in T. reesei and have a dampening effect on the light responsiveness of this process.