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The model organism Neurospora crassa undergoes programmed cell death when exposed to staurosporine. Here, we show that staurosporine causes defined changes in cytosolic free Ca²⁺ ([Ca²⁺]_c) dynamics and a distinct Ca²⁺ signature that involves Ca²⁺ influx from the external medium and internal Ca²⁺ stores. We investigated the molecular basis of this Ca²⁺ response by using [Ca²⁺]_c measurements combined with pharmacological and genetic approaches. Phospholipase C was identified as a pivotal player during cell death, because modulation of the phospholipase C signaling pathway and deletion of PLC-2, which we show to be involved in hyphal development, results in an inability to trigger the characteristic staurosporine-induced Ca²⁺ signature. Using Δcch-1, Δfig-1 and Δyvc-1 mutants and a range of inhibitors, we show that extracellular Ca²⁺ entry does not occur through the hitherto …