The results are the means ± standard deviation of four sets of experiments. Figure 3 Overepression of A. fumigatus AfCrzA increased the mRNA accumulation of several genes. Fold increase in mRNA levels after the growth of the wild type and alcA::crzA
mutant strain in MM+2% glycerol+2% ethanol for 6 hours at 37°C of (A) AfpmcB (Afu3g10690), (B) AfzfpA (Afu8g05010), (C) A. fumigatus Phospholipase D (Afu2g16520), (D) AfctfA (Afu4g03960), (E) Af BAR adaptor protein (Afu3g14230), (F) AfrcnA (Afu2g13060), (G) AfrfeF (Afu4g10200), (H) Af AAA ATPase (Afu4g04800), and (I) Afscf1 (Afu1g17370). The see more relative quantitation of all the genes and tubulin gene expression was determined by a standard curve (i.e., CT -values plotted against logarithm of the DNA copy number). The results are the means 3-MA mouse ± standard deviation c-Kit inhibitor of four sets of experiments. The values represent the number of times the genes are expressed compared to the corresponding
wild type control strain (represented absolutely as 1.00). A. fumigatus AfRcnA belongs to a class of endogenous calcineurin regulators, calcipressins, a family of calcineurin-binding proteins, conserved from yeast to mammals [34, 35]. A phylogenetic analysis was performed to determine the relationship of AfRcnA to calcipressin homologues in several different organisms (Additional file 3, Figure S1). The mechanism how this protein family functions still remains controversial. There are reports showing that calcipressins can both stimulate and inhibit the calcineurin pathway 34 35 36. Induction of S. cerevisiae RCN1-lacZ in response to calcium was completely blocked
by addition of FK506 or by deletion of the genes encoding Tcn1p or calcineurin . The S. cerevisiae RCN1 is also induced by calcium, repressed by calcium+FK506 and in the crz1 background . Another member of this family, Cbp1, was identified in Cryptococcus neoformans, and is required for mating but not for growth at 37°C . We have observed that AfrcnA mRNA accumulation upon calcium stress is dependent on both calcineurin and AfCrzA (Figure 1A). These results suggest that both S. cerevisiae and A. fumigatus RCAN homologues may Ketotifen be downstream targets of the calcineurin-dependent transcription factor. This fits a model where increased A. fumigatus AfRcnA regulation in response to calcineurin signaling is possibly a negative-feedback mechanism modulating calcineurin acitivity. We constructed an A. nidulans alcA::AncrzA also by replacing the endogenous AncrzA promoter region homologously with the A. nidulans alcA promoter. We investigate the genetic interactions between ΔAncnaA and ΔAncrzA mutations and a double mutant ΔAncnaA ΔAncrzA displays the same growth behavior than the ΔAncnaA mutant indicating as expected that AncnaA is epistatic to AncrzA (data not shown).