RESUMEN
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.
Asunto(s)
Carbono , Proteínas Fúngicas , Regulación Fúngica de la Expresión Génica , Factores de Transcripción , Carbono/metabolismo , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Hypocreales/metabolismo , Hypocreales/genética , Hypocreales/crecimiento & desarrollo , Celulosa/metabolismo , Celulosa/biosíntesis , Metabolismo SecundarioRESUMEN
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.
Asunto(s)
Arabidopsis , Etilenos , Trichoderma , Ecosistema , Trichoderma/metabolismo , Desarrollo de la Planta , Reguladores del Crecimiento de las Plantas/metabolismo , Plantas/metabolismo , Suelo , Raíces de Plantas/microbiologíaRESUMEN
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.
Asunto(s)
Celulasa , Trichoderma , Sideróforos/metabolismo , Trichoderma/metabolismo , Metabolismo Secundario , Carbohidratos , Regulación Fúngica de la Expresión Génica , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Celulasa/genética , Celulasa/metabolismoRESUMEN
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.
Asunto(s)
Celulasa , Trichoderma , Celulasa/metabolismo , Metabolismo Secundario , Trichoderma/metabolismo , Desarrollo Sexual , Regulación Fúngica de la Expresión Génica , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismoRESUMEN
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.
Asunto(s)
Rubiaceae , Xylariales , Humanos , Costa Rica , Rubiaceae/química , Xylariales/química , Espectroscopía de Resonancia Magnética , Citocalasinas/químicaRESUMEN
Sensing the environment and interpretation of the received signals are crucial competences of living organisms in order to properly adapt to their habitat, succeed in competition and to reproduce. G-protein coupled receptors (GPCRs) are members of a large family of sensors for extracellular signals and represent the starting point of complex signaling cascades regulating a plethora of intracellular physiological processes and output pathways in fungi. In Trichoderma spp. current research involves a wide range of topics from enzyme production, light response and secondary metabolism to sexual and asexual development as well as biocontrol, all of which require delicate balancing of resources in response to the environmental challenges or biotechnological needs at hand, which are crucially impacted by the surroundings of the fungi and their intercellular signaling cascades triggering a precisely tailored response. In this review we summarize recent findings on sensing by GPCRs in Trichoderma, including the function of pheromone receptors, glucose sensing by CSG1 and CSG2, regulation of secondary metabolism by GPR8 and impacts on mycoparasitism by GPR1. Additionally, we provide an overview on structural determinants, posttranslational modifications and interactions for regulation, activation and signal termination of GPCRs in order to inspire future in depth analyses of their function and to understand previous regulatory outcomes of natural and biotechnological processes modulated or enabled by GPCRs.
Asunto(s)
Trichoderma , Humanos , Trichoderma/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , EcosistemaRESUMEN
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.
RESUMEN
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.
RESUMEN
Transformation enables the transfer of DNA into fungal cells for subsequent integration into the genome. Due to its versatility in industrial application, transformation is of utmost importance in Trichoderma reesei and hence continuously optimized. As one of the most crucial obstacles in fungal transformation efforts, removal of the cell wall is required to efficiently target genome modification cassettes to the genome. Here we describe resistance marker-mediated gene gun (biolistic) transformation of fungal spores of T. reesei as an alternative to protoplast transformation.
Asunto(s)
Biolística/métodos , Hypocreales/genética , Transformación Genética , ADN de Hongos/genética , Marcadores GenéticosRESUMEN
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.
RESUMEN
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.
RESUMEN
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.
RESUMEN
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.
RESUMEN
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.
RESUMEN
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.
Asunto(s)
Endo-1,4-beta Xilanasas/genética , Hypocreales/genética , Luz , Poligalacturonasa/genética , Factores de Transcripción/genética , Pared Celular/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Endo-1,4-beta Xilanasas/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulación Enzimológica de la Expresión Génica/efectos de la radiación , Regulación Fúngica de la Expresión Génica/efectos de la radiación , Genes Fúngicos , Hypocreales/metabolismo , Hypocreales/efectos de la radiación , Mutación , Filogenia , Poligalacturonasa/metabolismo , Metabolismo Secundario/genética , Factores de Transcripción/metabolismo , Xilanos/metabolismoRESUMEN
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.
RESUMEN
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.
RESUMEN
Trichoderma reesei represents one of the most prolific producers of plant cell wall degrading enzymes. Recent research showed broad regulation by phosphorylation in T. reesei, including important transcription factors involved in cellulase regulation. To evaluate factors crucial for changes in these phosphorylation events, we studied non-essential protein phosphatases (PPs) of T. reesei. Viable deletion strains were tested for growth on different carbon sources, osmotic and oxidative stress response, asexual and sexual development, cellulase and protease production as well as secondary metabolism. Six PPs were found to be positive or negative regulators for cellulase production. A correlation of the effects of PPs on protease activities and cellulase activities was not detected. Hierarchical clustering of regulation patterns and phenotypes of deletion indicated functional specialization within PP classes and common as well as variable effects. Our results confirmed the central role of catalytic and regulatory subunits of PP2A which regulates several aspects of cell growth and metabolism. Moreover we show that the additional homologue of PPH5 in Trichoderma spp., PPH5-2 assumes distinct functions in metabolism, development and stress response, different from PPH5. The influence of PPs on both cellulase gene expression and secondary metabolite production support an interrelationship in the underlying regulation mechanisms.