We isolated a Neurospora crassa mutant of the calmodulin (cmd) gene using repeat-induced point mutation and studied its phenotypes. The cmd RIP mutant showed a defect in growth, reduced aerial hyphae, decreased carotenoid accumulation, a severe reduction in viability upon ultraviolet (UV) irradiation, and a fertility defect. Moreover, meiotic silencing of the cmd gene resulted in a barren phenotype. In addition, we also performed site-directed mutational analysis of the calcium/calmodulin-dependent kinase-2 (Ca2+/CaMK-2), a target of the CaM protein encoded by the cmd gene. The camk-2 S247A and the camk-2 T267A mutants in a homozygous cross, or in a cross with a Δcamk-2 mutant, displayed an intermediate phenotype, suggesting that serine 247 and threonine 267 phosphorylation sites of the Ca2+/CaMK-2 are essential for full fertility in N. crassa. Therefore, CaM in N. crassa is required for normal vegetative growth, UV survival, and sexual development. Additionally, serine 247 and threonine 267 phosphorylation sites are important for the Ca2+/CaMK-2 function.
Keywords: Calcium signaling; Calcium/calmodulin-dependent protein kinases; Calmodulin; Meiotic silencing; Neurospora crassa; Repeat-induced point mutation.