Calcineurin and Calcium Channel CchA Coordinate the Salt Stress Response by Regulating Cytoplasmic Ca 2+ Homeostasis in Aspergillus nidulans

Abstract

ABSTRACT The eukaryotic calcium/calmodulin-dependent protein phosphatase calcineurin is crucial for the environmental adaption of fungi. However, the mechanism of coordinate regulation of the response to salt stress by calcineurin and the high-affinity calcium channel CchA in fungi is not well understood. Here we show that the deletion of cchA suppresses the hyphal growth defects caused by the loss of calcineurin under salt stress in Aspergillus nidulans . Additionally, the hypersensitivity of the Δ cnaA strain to extracellular calcium and cell-wall-damaging agents can be suppressed by cchA deletion. Using the calcium-sensitive photoprotein aequorin to monitor the cytoplasmic Ca 2+ concentration ([Ca 2+ ] c ) in living cells, we found that calcineurin negatively regulates CchA on calcium uptake in response to external calcium in normally cultured cells. However, in salt-stress-pretreated cells, loss of either cnaA or cchA significantly decreased the [Ca 2+ ] c , but a deficiency in both cnaA and cchA switches the [Ca 2+ ] c to the reference strain level, indicating that calcineurin and CchA synergistically coordinate calcium influx under salt stress. Moreover, real-time PCR results showed that the dysfunction of cchA in the Δ cnaA strain dramatically restored the expression of enaA (a major determinant for sodium detoxification), which was abolished in the Δ cnaA strain under salt stress. These results suggest that double deficiencies of cnaA and cchA could bypass the requirement of calcineurin to induce enaA expression under salt stress. Finally, YvcA, a member of the transient receptor potential channel (TRPC) protein family of vacuolar Ca 2+ channels, was proven to compensate for calcineurin-CchA in fungal salt stress adaption. IMPORTANCE The feedback inhibition relationship between calcineurin and the calcium channel Cch1/Mid1 has been well recognized from yeast. Interestingly, our previous study (S. Wang et al., PLoS One 7: e46564, 2012, http://dx.doi.org/10.1371/journal.pone.0046564 ) showed that the deletion of cchA could suppress the hyphal growth defects caused by the loss of calcineurin under salt stress in Aspergillus nidulans . In this study, our findings suggest that fungi are able to develop a unique mechanism for adapting to environmental salt stress. Compared to cells cultured normally, the NaCl-pretreated cells had a remarkable increase in transient [Ca 2+ ] c . Furthermore, we show that calcineurin and CchA are required to modulate cellular calcium levels and synergistically coordinate calcium influx under salt stress. Finally, YvcA, a member of of the TRPC family of vacuolar Ca 2+ channels, was proven to compensate for calcineurin-CchA in fungal salt stress adaption. The findings in this study provide insights into the complex regulatory links between calcineurin and CchA to maintain cytoplasmic Ca 2+ homeostasis in response to different environments.