AbstractBackgroundTrichoderma reesei is the main filamentous fungus used in industry to produce cellulases. Over the last decades, there have been a strong increase in the understanding of the regulatory network controlling cellulase-coding gene expression in response to a number of inducers and environmental signals. In this sense, the role of calcium and the Calcineurin-responsive protein (CRZ1) has been investigated in industrially relevant strains of T. reesei RUT-C30, but this system has not been investigated in wild-type reference strain of this fungus.ResultsHere, we investigated the role of CRZ1 and Ca2+ signaling in the fungus T. reesei QM6a. For this, we first searched for potential CRZ1 binding sites in promoter regions of key genes coding holocellulases, as well as transcriptional regulators and sugar and calcium transporters. Using a nearly constructed T. reesei Δcrzl strain, we demonstrated that most of the genes expected to be regulated by CRZ1 were affected in the mutant strain induced with sugarcane bagasse (SCB) and cellulose. In particular, our data demonstrate that Ca2+ acts synergistically with CRZ1 to modulate gene expression, but also exerts CRZ1-independent regulatory role in gene expression in T. reesei, suggesting the existence of additional Ca2+ sensing mechanisms in this fungus.ConclusionsThis work presents new evidence on the regulatory role of CRZ1 and Ca2+ sensing in the regulation of cellulolytic enzymes in T. reesei, evidencing significant and previously unknown function of this Ca2+ sensing system in the control key transcriptional regulators (XYR1 and CRE1) and on the expression of genes related to sugar and Ca2+ transport. Taken together, the data obtained here provide new evidence on the regulatory network of T. reesei related to plant biomass deconstruction.

Transcription factors (TFs) are responsible for regulating the rate of transcription of genes in all organisms. These factors can be represented computationally by position weight matrices (PWM). TFound was developed to allow the detailed visualization of predicted binding sites of transcription factors in multiple sequences based on the PWMs, by using the graphic user interface (GUI). The tool was loaded with the genome of Saccharomyces cerevisiae and PWMs from the YeTFaSCo database (http://yetfasco.ccbr.utoronto.ca/), also allowing the insertion of new sequences and PWMs. Thus, the user is allowed to load custom PWMs and genomes to perform easily mining and visualization of binding site motifs of cis-regulatory elements of interest, permitting an efficient way to inspect DNA assembly projects for complex synthetic circuits. This work describes the functionality of the current version of the tool, which is coded in Python and is freely available at the repository https://github.com/adri4nogomes/TFound. MOTIVATIONThe ability to coordinate the expression of thousands of genes by processing a wide range of environmental inputs is a fundamental feature of any living organism 1 . To decipher the logic underlying such complex regulatory network is crucial to understand the basic signaling principles of life machinery, providing knowledge to reprogram it to execute novel synthetic functions 2,3 . However, despite being essential to engineering, the inference of such complexity remains a major bottleneck, especially to eukaryotes 4-7 .Moreover, Fungi are a powerful resource for biotechnological applications, from biomass conversion aiming biofuels production to the synthesis of industrially relevant compounds 8,9 .