Figure 8.

Localization and activity of the calcineurin complex at the hyphal septum is required for proper hyphal growth. (A) Clustal alignment of the linker region between the catalytic domain and the CnaB-binding helix of CnaA. Residues in red represent the conserved active site residues. (B-C) Expression of the catalytic null mutated CnaAs (CnaA-T359P and CnaA-L365S) along with mcherry-CnaB in the ΔcnaA ΔcnaB strain showed the localization of the calcineurin complex at the hyphal septum (indicated by arrows). Scale bar, 10 μm. (D) Catalytic null mutations in CnaA do not complement hyphal growth. Radial growth of the indicated strains was monitored over a period of 3 days at 37°C on GMM agar medium or GMM supplemented with 1.2 M sorbitol. A total of 1×10⁴ conidia were spotted for each strain. Note that both the T359P and L365S mutations did not restore hyphal growth (upper panel) and were only partially remediated in presence of sorbitol (lower panel). (E) Calcineurin phosphatase activity in the various strains was determined after a 48 h growth period using p-nitrophenyl phosphate as a substrate. The experiment was repeated two times and the results shown are mean ± standard error of six technical replicates. Note the drastic reduction in the catalytic null mutants and the calcineurin deletions strains.