## 3 Discussion  
It has been demonstrated that MAPK signaling pathway plays an important role in eukaryotic organisms and cascades of MAPK involves in regulating the apoptosis, growth, expression of the genes, hyperosmoregulation, cell division, cell endurance and ascospore development [17; 19]. Similarly, in both _A. fumigatus_ and _A. nidulans_, _sakA_ gene intervenes in MAPK signaling cascades in HOG pathway as an osmotic regulator. _SakA_ gene is also a homologue of _hog1_ within the _S. cerevisiae_[13; 14; 19] and acts as an essential response to numerous types of stress [13]. In the present study, our main interest was to understand how MAPK _AfsakA_ gene responds to osmotic stress signals in _A. flavus_. During the identification process of our target gene in _A. flavus_, we realized that _sakA_ gene was conserved among all _Aspergillus_ species. The sequence alignments of all the analyzed fungal species revealed that _sakA_ has high similarity as they all share a highly conserved protein kinase domain. All these results confirmed that _sakA_ is conserved in MAP kinase/SAPKs family, which is in consistence with earlier studies that _sakA_ gene was found to encode a part of MAPK gene family in both _A. nidulans_[19] and _P. marneffei_[20]. The function of _AfsakA_ in responses to osmotic stress was examined. _\(\Delta\)AfsakA_ proved to be more sensitive to 1.2 mol/L D-Sorbitol than WT and \(\Delta\)AfsakA::AfsakA_ strains and our results also demonstrated that growth rate in \(\Delta\)AfsakA could be related to the expression of the _HPS_, _GPD_, _GRE_ and _STL_ genes which are related with osmolarity. Surprisingly, when using 1.2 mol/L NaCl, no growth rate differences were detected in all the strains. As a result, we suspect that _AfsakA_ involves in multiple mechanisms that contribute to osmotic regulation. The above was corroborated by [PERSON] et al. [23] who reiterated that _A.fumigatus sakA_ deletion affect growth and conidia germination at a high osmolarity (1 mol/L NaCl). The current results were also supported by the findings of [PERSON] et al. [20] and [PERSON] et al. [31] who previously reported that _A. fumigatus sakA_ gene contributes to the regulation of hyperosmotic stress. Conidiophores are known as the multicellular structures which produces conidia during the vegetative growth under favorable conditions and in a particular period of time [32]. In the present study, we evaluated the role of _AfsakA_ gene in asexual development (conidiation) and we found that \(\Delta\)AfsakA mutant produced less conidia than other strains in the absence of environmental stress. In the presence of extracellular stress induced by 1.2 mol/L NaCl, \(\Delta\)AfsakA exhibited a significant  
Figure 9: Phenotype and inhibition of growth rate of WT, \(\Delta\)AfsakA and \(\Delta\)AfsakA::AfsakA strains under cell wall stress only or together with osmotic stress. (**A**) All strains were grown on a control and on a YES agar media with various cell wall stress agents such as 100 \(\upmu\)g/mL SDS (Sodium dodecyl sulfate) and 200 \(\upmu\)g/mL CR (Congo Red) only or together with 1.2 mol/L NaCl. (**B**) Inhibition of mycelial growth rate. The line bar in column of the graph indicates standard errors of all three replicates and the asterisks demonstrate significant difference levels between WT and other strains (\({}^{**}\)\(p\)\(\leq\) 0.01). increase in conidial formation, whereas with 1.2 mol/L D-sorbitol, \(\Delta\)_AfsaK_ illustrated a decrease in conidial formation in contrast to WT and \(\Delta\)_AfsaK_::_AfsaK_ strains. We concurrently found that without stress the expression levels of \(abaA\) and _brIA_ genes which are related with conidia production were significantly down-regulated in \(\Delta\)_AfsaK_ compared to that in both WT and complemented strains. Previous studies indicated that an increased amount of sorbitol increases the conidia production in _A. nidulans_[15; 35]. [PERSON] et al. [36] and [PERSON] et al. [15] also indicated that a decrease in conidia production was observed when _A.