Components signalling the presence of cellulose to cellulase gene expression in Trichoderma reesei

Hypocrea Jecorina (Trichoderma reesei) is a filamentous fungus which is employed for commercial cellulose production since more than 30 years. Formation of cellulases is inducible by cellulose, and this induction occurs at the transcriptional level. We have previously been investigating the mechanisms by which an insoluble molecule like cellulose can trigger gene transcription, using the major conidial bound isoenzyme - cellobiohydrolase II (gene cbh2) as a model. Our data, hitherto obtained, suggest the synergistic and constitutive binding of a protein complex, consisting of HAP2/3/5 homologues and of a unique zinc finger protein to a 5`-ATTGGGTAATA-3` target sequence, which is essential for cbh2 gene transcription. Data from EMSA suggest that induction is mediated by the binding of a further protein to this complex, however without contacting the DNA. We also have preliminary evidence that phosphorylation of one of the compounds of this complex may play a role in induction. To further continue with our studies on cbh2 gene transcription, we now plan to clone the gene encoding this further binding protein by the aid of the yeast two-hybrid system, making use of the availability of the genes encoding the components binding to 5`-ATTGGGTAAT-3`. In addition, we will use REMI mutagenesis of a recombinant strain bearing the pyr4 gene under the cbh2 promoter, in order to obtain mutants both affected in cellulose gene transcription (5-FOA resistant) and constitutively transcribing from the cbh2 promoter (uridine prototroph). The REMI mutants will provide us with a set of genes encoding components of the cellulose signalling pathway, which will aid in the elucidation of the mechanism of cellulose induction in Trichoderma reesei.

The aim of this project was to identify components signalling the presence of cellulose to the fungus Trichoderma reesei (Hypocrea jecorina), thereby eliciting the induction of cellulase gene expression. Two different molecular approaches were employed: using the One Hybrid System we intended to isolate the protein(s) that bind to GTAATA sequence within the CAE motif in the cbh2-promoter of Hypocrea jecorina, which has been previously shown to be involved in cellulase induction. After two rounds of the One Hybrid screening procedure and analysis of the resulting clones 10 of them met the expectations of the experiment and were analysed further. Several of the clones showed homologies to transcription factors of sometimes closely related organisms but also to genes that are not directly correlated with transcriptional activation. However, many of those act together in the complex machinery of gene regulation in other organisms. One clone showed differential expression in the wild-type strain QM9414 and a cellulase-negative mutant. This cellulase-negative mutant fails to induce cellulase gene expression because it is defective in the functional formation of the protein complexes binding to the responsible CAE motif but not in the binding of the respective proteins to the target sequence itself. Our second strategy therefore involved Rapid Subtraction Hybridization (RaSH) to identify genes with elevated expression in the wildtype strain Hypocrea jecorina QM9414 under inducing conditions (sophorose) but not in the mutant strain QM9978. Thereby, 23 genes, some of them present in more than one clone, were identified: most of them encoded proteins without homologies to other proteins from databases, but some clones encoding proteins with significant similarity to conserved domains in transcription factors or signal transducing proteins were also obtained. Northern blots confirmed differential expression and the chromosomal sequence of the respective clones was isolated and analysed. Knock-out mutants of these genes were constructed to obtain evidence for their functional involvement in cellulase gene regulation in Hypocrea jecorina. One gene, encoding envoy - a PAS/PAC domain-containing protein showed the expected phenoytpe and is thus the first component detected to be involved in fungal cellulose signalling.