## 3 Results and Discussion
### Hog Phosphorylation Response to Osmotic Shock  
Using several model organisms, it has been established that the HO pathway coordinates responses to cellular osmotic stress. In _S. cerevisiae_ and _D. hansenii_, the activation of this pathway leads to the transient phosphorylation of Hog upon a hyperosmotic shock [111]. In _H. werneckii_, the _hog_ gene is duplicated while the protein phosphorylation dynamics is similar to Hog1p from _S. cerevisiae_[18]. However, in _W. ichthyophaga_, the HOG system responds in the opposite direction, since the kinase is phosphorylated in the lowest salinities and is transiently dephosphorylated when the cells were exposed to a hypersaline medium [11; 18; 112]. These diverse signaling pathway configurations suggest that the HOG system is a key component of the mechanisms of adaptation to salinity and other stress-inducing conditions. Therefore, we studied the responses of the _A. sydowii_ Hog orthologues to salinity stress. Few fungi, like several Aspergillus, have more than one copy of the Hog MAPK [113]. For example, _A. nidulans_ has two orthologues of the _S. cerevisiae_ Hog1p, named SakA/HogA and MpkC. The former is responsible for most of the osmo-protective stress responses in _A. nidulans_, while the latter is involved in conidial and oxidative stress response [113]. Interestingly, _A. sydowii_ has a third copy of the Hog MAPK that cannot be found in its close relatives _A. versicolor_ and _A. mulundensis_. Here we named these MAPKs as Hog1, Hog2, and MpkC, which respectively have protein identifiers 141488, 372814, and 47278 in the published _A. sydowii_ CBS 593.65 genome (Figure 6A). All three orthologues have complete protein kinase (PK) domains and the conserved TGY phosphorylation site (Figure 6B), which implicates that they could be phosphorylated and biologically active. This allowed us to detect the phosphorylation of the _A. sydowii_ MAPK orthologues using antibodies with cross-reactivity to the _S. cerevisiae_ Hog1p. Figure 6: Hog MAPK system in _A. ydovvii_. (**A**) Reconstruction of MAPK phylogeny in selected Aspergillus, including Hog1, Hog2, and MpkC genes of _A. ydovvii_. A more extensive phylogenetic tree can be observed in Supplementary Figure S3. (**B**) Hog gene homologs on _A. ydovvii_ showing protein size, molecular weight, conserved phosphorylation motifs, protein kinase (PK) domain, and the region corresponding to Y-215 and D3F9 recognized by the antibodies used to detect phosphorylated and total Hog, respectively, by Western blot. (**C**) Relative expression of _Hog1_ and _Hog2_ transcripts in salt-adapted _A. ydovvii_ growing without NaCl, 0.5 M or 2.0 M NaCl, representing the average and standard deviations of  
three biological replicates (\(n=3\)) and two technical qPCR replicates. (**D**) Diagram of the shock conditions used to test the phosphorylation dynamics of Hog1 and Hog2 (**E**) Relative expression of _Hog1_ and _Hog2_ transcripts after osmotic shock in _A. sydowii_, representing the average and standard deviations of three biological replicates (\(n=3\)) and two technical qPCR replicates. The statistical significance (*) was assessed by a randomization test performed with the software Rest [46]. The dashed horizontal lines correspond to a cutoff logFC = \(\pm 2\). (**F**) Phosphorylation of Hog MAPK homologs after different osmotic shocks. Extracts from _S. cerevisiae_ cultures shifted from a medium without NaCl to a medium with 1.0 M NaCl were used as positive controls for Hog1 phosphorylation. The transcript levels of the _Hog1_ variant might be higher than those of _Hog2_, according to previous RNA-seq experiments (Supplementary Figure S4) [29]. More important, the expression of both genes was similar under stress conditions (No NaCl or 2.0 M NaCl) but was lower under optimal salinity conditions (Figure 6C). This effect was more pronounced for _Hog2_, suggesting that this variant might play a more significant role in the salt stress response.