Elucidating the Role of CO2 Sensing in Fruiting Body Formation of Schizophyllum commune

Abstract

Abstract: Fruiting body formation of fungi is usually controlled by environmental factors, as is the case for the basidiomycetous fungus Schizophyllum commune. For this fungus blue-light and CO2 concentration seem to be the two major factors influencing fructification. Blue-light is sensed through the WC-1/2 complex that acts as both a receptor and a transcriptional activator. Transcription factors involved in mushroom development have also been described. However, how CO2 is sensed by S. commune and how this signal is transduced is largely unknown. The proposed pathway describes how CO2 is converted into bicarbonate by carbonic anhydrases, bicarbonate subsequently stimulates soluble adenylyl cyclases to form cAMP, the raised cAMP concentration is necessary to activate protein kinases A, which can finally phosphorylate many different targets inhibiting fructification. This pathway is therefore further investigated. The role of bicarbonate was tested by directly adding it to medium. While able to inhibit fruiting body formation it was argued that this effect was caused by a pH change of the medium. The carbonic anhydrase inhibitor acetazolamide and pomegranate peel extract were also tested. With their ability to inhibit certain CAs they could prevent bicarbonate levels of reaching a threshold for activating adenylyl cyclases. While the effect of acetazolamide seemed insignificant the peel extract influenced fruiting body formation both by initial inhibition and apparent subsequent stimulation. Purified compounds present in pomegranates might be investigated in future research. Using the novel compound TDI-10229 and a known fungal quorum sensing associated compound called farnesol, it was tried to inhibit the fungal soluble adenylyl cyclase. Expecting increased fructification this was only the case for TDI-10229, which was able to induce fructification under inhibiting conditions for the control. Farnesol inhibited fructification when added to the medium and caused a fluffy phenotype, which was unexpected. TDI-10229 was able to partly cancel out this inhibiting effect of farnesol. It could not be shown that farnesol can act as a volatile organic compound on S. commune. Lowering glucose concentrations of the medium was expected to lower intracellular levels of ATP, resulting in expedited fructification. While glucose concentration did seem to affect fruiting body size and number, no significant change in fructification rate could be observed. This study also investigated the possibility of using a TET-ON system in S. commune as a method of inducible gene expression, using a previously created transcriptional activator strain that was further transformed to express the fluorescent gene dTomato behind a Tet promotor region. Fluorescent microscopy analysis of dTomato expression showed to be unpromising, so further analysis needs to be done to determine whether there is inducible expression. Finally, an attempt at creating a Pde2 knockout transformant line was unsuccessful possible due to mutations in the antibiotic resistance cassette of the construct. While not all experiments came to full fruition, this study does provide new insights on the CO2 sensing pathway.