## 3 Results and Discussion
### Hog Phosphorylation Response to Osmotic Shock
We have established that the conditions without NaCl or with 2.0 M NaCl induce stress responses in _A. sydowii_[29]. Therefore, changing the mycelium from media with 0.5 M NaCl to another condition induces stress responses, which might be different from hyperosmotic or hypoosmotic shock responses. A schematic representation of those experimental variations can be observed in Figure 6D. The differential expression of _Hog1_ and _Hog2_ under these scenarios showed that both genes were downregulated after hyper- or hypoosmotic shocks (Figure 6E), reducing the transcript abundance after long time periods (8 to 24 h post-stimulus). Again, this effect is more pronounced for _Hog2_ gene variant than for _Hog1_. In contrast, _Hog2_ gene expression is mostly upregulated after stress-inducing osmotic shocks, while _Hog1_ expression is not significantly perturbed. The phosphorylation of Hog MAPK variants was analyzed by Western Blot from 5 min to 3 h after an osmotic stimulus and compared to the phosphorylation dynamic of Hog1p in _S. cerevisiae_ (Figure 6F). The independent patterns of Hog1/Hog2 phosphorylation could not be discerned with this experiment as the bands corresponding to both protein products overlapped. Conversely, a higher molecular weight band, below of _S. cerevisiae_ Hog1p could correspond to the MpkC variant, according to its molecular weight prediction. Both or at least one of the Hog1/Hog2 proteins were phosphorylated constitutively in the conditions without NaCl or in 2.0 M NaCl, and to a lesser extent in the 0.5 M NaCl optimal condition. Such phosphorylation status did not change upon hyper- or hypoosmotic shocks, but increased when cells were exposed to stress-inducing osmotic shocks considering the growth optimal condition. These changes were not transient, though, indicating that a sustained phosphorylation of Hog is the normal status of this signaling system in _A. sydowii_. Interestingly, MpkC protein was produced in detectable amounts only in the condition without NaCl where it was constitutively phosphorylated. It remained phosphorylated even after a hyperosmotic shock. In contrast, this protein was transiently phosphorylated upon hypoosmotic shock, especially from 2.0 M NaCl to the medium without NaCl. The phosphorylation of Hog1p in _S. cerevisiae_ and its homolog in _D. hansenii_ occurs transiently and immediately after a mild stress stimulus, and persists up to half an hour in both models [54; 114]. In contrast, in _D. hansenii_ a prolonged Hog1 phosphorylation state was observed in cells subjected to a severe osmotic stress [54; 114; 115]. Interestingly, the phosphorylation of Hog1 remained after cells adapted to severe osmotic stress were subjected to a hypoosmotic shock [114; 115]. Taken together, these results might indicate that the biological activity of Hog1/Hog2 is regulated transcriptionally, while regulation of their activity by phosphorylation is not evident, as they could not be discriminated by Western Blot. The HOG system in _A. sydowii_ is responding to osmotic conditions that induce cellular stress, since the presence of salt in optimal concentrations did not seem to regulate transcript abundance or phosphorylation status of either of the Hog proteins. On the other hand, the Hog homolog MpkC was responsive to hypoosmotic shock. The interplay of these MAPKs on the regulation of saliinity and stress responses should be studied further, as it does not follow the dynamics of other HOG systems in halotolerant or halophile model microorganisms.