Figure 1.

Construction of the cnaA and cnaB deletion strains in A. fumigatus and radial growth analysis of the calcineurin mutants. (A) Schematic representation of the genomic locus of the WT and the cnaA and cnaB deletion strains. The entire coding sequence of the A. fumigatus cnaA gene was replaced with the A. fumigatus argB gene by homologous recombination. Southern analysis with xhoI-digested genomic DNA and cnaA left flank probe shows the replacement of cnaA by argB as a 5.6 kb fragment in the ΔcnaA strain. (B) Genomic DNA from both the wild type and the ΔcnaA ΔcnaB strains was digested with SalI; The DIG-labeled probe bound to a 1.6 kb and a 6.2 kb band in the wild type and the ΔcnaA ΔcnaB strains, respectively, showing the replacement of cnaB by A. parasiticus pyrG as a 6.2 kb fragment in the ΔcnaA strain. (C). Radial growth of the wild-type, the calcineurin single mutants (ΔcnaA and ΔcnaB) and the double mutant ΔcnaA ΔcnaB strain, and the corresponding complemented strains over a period of 3 days at 37°C on GMM agar medium. A total of 1×10⁴ conidia were spotted for each strain. (D) Different concentrations of inocula as indicated were spotted on GMM agar plates and growth of the strains was monitored over a period of 2 days at 37°C to observe the severe growth defect of the ΔcnaA ΔcnaB strain in comparison to the single mutants. (E). Different concentrations of inocula were spotted on SDA plates and growth of the strains was monitored over a period of 3 days at 37°C to observe the growth defect of the calcineurin mutants. (F). A total of 1×10⁵ conidia from each calcineurin mutant were spotted on GMM agar supplemented with 1.2 M sorbitol and observed for growth remediation over a period of 3 days at 37°C. Sorbitol-dependent partial growth remediation in the absence of CnaA may be noted in the ΔcnaA and ΔcnaA ΔcnaB + cnaB strains.