Global Transcriptome and Gene Regulatory Network of MAPK Signaling Pathway in Trichoderma reesei During Sugarcane Bagasse Degradation. Trichoderma reesei

The filamentous fungus Trichoderma reesei is a saprophyte involved with polysaccharides cell wall depolymerization, being the most important industrial source of cellulases, which have been used for bioethanol production. The regulation of cellulase expression is controlled at the transcriptional level and in a carbon source-dependent manner. However, the signaling pathways and mechanisms involved in regulating the expression of these enzymes in T. reesei are still poorly understood. In this study, we demonstrate through transcriptional analysis by RNA-Seq the main changes in gene expression that occur in functional mutants for two genes of the MAPK, tmk1 and tmk2. The results obtained allowed us to identify that these proteins in T. reesei regulate independent processes, but sometimes might regulate the same process through different mechanisms of action, being responsible for modulating gene expression in this organism. Regarding to Δtmk2 strain, it was demonstrated that growth in sugarcane bagasse and glucose modulates the expression of genes involved with chromatin remodeling, metabolism of carbohydrates and cell signaling genes such as GPCR, calcium signaling and phospholipases. On the other hand, deletion of MAPK TMK1 promotes more discrete changes in the transcriptional profile and the main changes are related to the decrease in the expression of the major genes for cellulases and xylanases, repressing the expression of transporters belonging to the MFS family, transporters of amino acids and ions such as Ca2+ and Mg2+. Our results revealed that the MAPK signaling pathway in T. reesei regulates many important processes that allow the fungus to promote the recognition, transport and metabolism of different carbon sources during the process of cell wall degradation. Therefore, the clustering of these data will contribute to the construction of a global model for the events that occur during the degradation of lignocellulose in T. reesei, which will lead to the development of more efficient strains in the plant biomass degradation. Overall design: T. reesei (QM6a and mutant strains ) were grown in Mandels-Andreotti medium, supplemented with 2% of glucose for 48 hours. The cultures were incubated on an orbital shaker (200 rpm) at 30°C for 48 hours using glucose as carbon source. For the sugarcane bagasse induction, the strains were grown in Mandels-Andreotti medium, supplemented with 1% of glycerol for 24 hours. Then, the mycelia was collected and transfered to Mandels-Andreotti medium supplemented with 1% of sugarcane bagasse. The cultures were incubated on an orbital shaker (200 rpm) at 30°C for 48 hours using sugarcane bagasse as carbon source. All experiments were performed in three biological replicates. The resultant mycelia were collected by filtration, frozen and stored at -80°C until RNA extraction. After growth, total RNA was isolated from the mycelia using TRIzol® reagent. RNA-seq experiments were performed by Laboratório Nacional de Ciência e Tecnologia do Bioetanol (CTBE) using the platform Illumina/HiSeq2500.