Missing links in the nutrient signaling cascade of T. reesei

Surviving in a natural environment necessitates identification and efficient utilization of nutrients in the surroundings of living beings. The energy necessary for degradation of the natural substrates and liberation of the chemical building blocks sustaining life must be used wisely to enable successful competition in a complex habitat, where not only growth, but also reproduction and rivalry against other organisms is crucial. The filamentous fungus Trichoderma reesei (syn. Hypocrea jecorina) is optimally adapted to its natural habitat a tropical rain forest and very efficient in degradation of cellulosic plant biomass, which made it one of the most frequently used biotechnological workhorses in industry. Our research showed that regulation of transcription of the important cellulase genes as well as production of cellulolytic enzymes is crucially influenced by environmental factors via the light response pathway and the heterotrimeric G-protein pathway. Our recent findings include a G-protein coupled receptor that is essential for high level production of enzymes under inducing conditions by influencing posttranscriptional regulation. This receptor senses a precisely defined concentration of glucose and signals this nutrient to the cell. Additionally, we found that in the downstream cAMP pathway, deletion of a phosphodiesterase causes a more than 100fold increase in cellulase levels in light. In the course of the proposed project, we consequently intend to understand the message leading T. reesei to focus all resources on enzyme production and how it is transmitted through the signaling pathway all the way to gene expression. Therefore we will evaluate the following hypotheses: The heterotrimeric G-protein pathway is the major pathway for transmission of the signal influencing posttranscriptional regulation of cellulase gene expression. The cAMP pathway and especially phosphodiesterases and/or their targets crucially contribute to cellulase regulation downstream of the G-protein pathway and trigger (de-) phosphorylation of important cellulase regulators and/or transcription factors in response to light. With this work program we aim to gain insight into the reaction of filamentous fungi to their environment, how they sense appropriate substrates and how they distribute their energy for nutrient consumption. The involved genes we investigate will show us how a simple signal like glucose in the correct concentration is integrated into the complex regulation of life sustaining processes of a fungus. From an applied point of view, we want to identify yet unknown missing links that can be manipulated to combine and exploit the effects seen for stable and high level enzyme production in darkness like in an industrial fermentor.

Surviving in a natural environment necessitates identification and efficient utilization of nutrients in the surroundings of living beings. The energy necessary for degradation of the natural substrates and liberation of the chemical building blocks sustaining life must be used wisely to enable successful competition in a complex habitat. The filamentous fungus Trichoderma reesei is very efficient in degradation of cellulosic plant biomass, which made it one of the most frequently used biotechnological workhorses in industry. We could not gain fascinating insight into the interaction of fungi with their environment. T. reesei can sense the presence of a plant and due to the interconnection with cellulase production, it can also distinguish between degradation of dead substrate, for which enzymes are required, and utilization of plant exudates not requiring enzyme production. Therefore the G-protein signaling pathway is of crucial relevance and almost all its components play a role in signal transmission of the glucose signal from outside the fungal cell. The responsible receptor, CSG1, has a light dependent influence on gene regulation and impacts not only regulation of cellulolytic enzymes, but also production of secondary metabolites such as sorbicillins. Thereby, the influence of GPR16, a further G-protein coupled receptor (GPCR), is important, whose gene is not expressed anymore if csg1 is lacking in the genome. GPR16 participates in a feedback mechanism regulating sorbicillin production: Lack of gpr16 in the genome of T. reesei causes overproduction of sorbicillins, negatively influencing growth of the fungus. This effect is dependent on a functional sorbicillin-gene cluster. Considering cellulase regulation, GPR16 is important as well, because the signal it transmits is required for cellulase production. Consequently, with CSG1 and GPR16 we described a pair of sensors, which balances cellulase production with biosynthesis of secondary metabolites and which is crucial for the reaction to plants. Further research towards signaling in T. reesei dealt with the G-protein subunits, which are targets of the G-protein coupled receptors CSG1 and GPR16. Especially GNA1 showed a significant effect on gene regulation during growth on cellulose and influenced regulation of metabolic genes as well as secondary metabolism. For the further subunits GNA2, GNA3, GNB1 and GNG1 we found specific regulation patterns, that were distinct in light and darkness and are subject to further investigation. Finally, we assessed the relevance of the GPCR GPR1 for growth in the presence of increased levels of methionine, found an impact on iron homeostasis for the Regulator of G-protein activity RGS4 and considerable regulation of diverse gene groups for 6 members of the MAPkinase signaling pathway. In summary, our project revealed fascinating new insights into light dependent nutrient signaling, which also highlighted the interdependent regulation of enzyme production and secondary metabolism.