Role of Calcium signalling in cell fusion in the filamentous fungus Neurospora crassa

Self fusion between genetically identical cells in the form of hyphal fusion is a defining feature of colony morphogenesis in filamentous fungi. Cell fusion occurs at numerous stages during the vegetative and sexual phases of the life cycle of filamentous fungi. Fusion between conidia and/or conidial germlings involves formation and interaction of specialized hyphae, called conidial anastomosis tubes (CATs). Recent genetic and cell biological studies on CATs in Neurospora crassa have established it as a model to analyse vegetative hyphal fusion. While proteins involved in the initial two steps of CAT formation - CAT induction and homing - were already isolated and characterized, no protein involved in the third stage - CAT fusion - has been identified yet and additionally the mechanistic basis of self-signalling during CAT formation has not been addressed yet. Prof. Read`s group at the University of Edinburgh annotated the Ca2+ signalling machinery in Neurospora and they identified 48 Ca2+ signalling proteins in N. crassa and showed that its Ca2+ signalling machinery is significantly more complex than that found in the budding yeast. With regard to signalling in CATs, Prof. Read`s group has recently obtained evidence that Ca2+ signalling is involved in CAT induction because this process is impaired in the presence of the calcium chelator BAPTA, and by the Ca2+ channel blocker KP4. The proposed project will test the hypothesis that calcium signalling is involved in CAT induction, homing and/or fusion. The role of the Neurospora MID1 calcium channel in CAT induction, homing and fusion will by studied by analysis of a MID1 knockout mutant and a MID1-GFP fusion protein with live-cell imaging techniques. Cytosolic free Ca2+ ([Ca 2+] c) during CAT formation will be measured by imaging [Ca 2+] c with a recombinant Ca2+-sensitive probe during CAT induction, homing and fusion to determine whether there is (a) elevated [Ca 2+] c associated with CAT induction or (b) a tip-focused [Ca 2+] c gradient associated with the tips of homing and/or non-homing CATs. The methods which Verena Seidl will learn during the project in Edinburgh will be beneficial to her work at the TU Vienna, which will be continued after her return to Austria. This project addresses the roles of chitinases and the mechanism of biocontrol in Trichoderma. The learned microscopic techniques will enable the investigation of the effect of chitinases on cell wall architecture and assembly during germination and hyphal growth.