Calmodulin, Calcium/Calmodulin-Dependent Kinases-1 and 2 Regulate Expression of the Heat Shock Proteins for Heat Shock Tolerance and Pheromone Signaling Genes for Sexual Development in Neurospora crassa.

Calmodulin (CaM) is a primary Ca2+ sensor that binds and activates numerous target proteins and regulates several cellular processes in eukaryotes. CaM is essential in Neurospora crassa; therefore, we generated a CaM mutant using repeat-induced point (RIP) mutation and investigated the cmdRIP mutant phenotypes. We also studied knockout mutants of four Ca2+/CaM kinases (camk-1, 2, 3, and 4) for their role during stress conditions and sexual development. The cmdRIP, ∆camk-1, and ∆camk-2 mutants showed reduced survival and growth rates under heat stress, oxidative stress, pH, and ER stress conditions. In addition, under the heat stress conditions, expression of the heat shock protein genes hsp70 and hsp80 was reduced in the cmdRIP, ∆camk-1, and ∆camk-2 mutants. The cmdRIP mutant was also defective in cell fusion, its vegetative hyphae could not support the fertilized wild type perithecia graft, and female sterile. Furthermore, the expression of pheromone signaling genes pre-1, pre-2, ccg-4, mfa-1, and fmf-1 was reduced in the cmdRIP, ∆camk-1, and ∆camk-2 mutants. Therefore, CaM, Ca2+/CaMK-1 and 2 are involved in the tolerance to heat stress conditions and sexual development by regulating the heat shock and pheromone response pathways, respectively, in N. crassa. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-023-01091-8.

Multiple calcium signaling genes play a role in the circadian period of Neurospora crassa.

The Ca2+ signaling genes cpe-1, plc-1, ncs-1, splA2, camk-1, camk-2, camk-3, camk-4, cmd, and cnb-1 are necessary for a normal circadian period length in Neurospora crassa. In addition, the Q10 values ranged between 0.8 and 1.2 for the single mutants lacking cpe-1, splA2, camk-1, camk-2, camk-3, camk-4, and cnb-1, suggesting that the circadian clock exhibits standard temperature compensation. However, the Q10 value for the ∆plc-1 mutant was 1.41 at 25 and 30 °C, 1.53 and 1.40 for the ∆ncs-1 mutant at 20 and 25 °C, and at 20 and 30 °C, respectively, suggesting a partial loss of temperature compensation in these two mutants. Moreover, expression of frq, a regulator of the circadian period, and the blue light receptor wc-1, were increased >2-fold in the Δplc-1, ∆plc-1; ∆cpe-1, and the ∆plc-1; ∆splA2 mutants at 20 °C. The frq mRNA level was increased >2-fold in the Δncs-1 mutant compared to the ras-1bd strain at 20 °C. Therefore, multiple Ca2+ signaling genes regulate the circadian period, by influencing expression of the frq and wc-1 genes that are critical for maintaining the normal circadian period length in N. crassa.