Osmotic-adaptation Response Of SakA/hogA Gene To Aflatoxin Biosynthesis And Pathogenicity In Aspergillus Flavus

Aspergillus flavus is a saprophyte fungus,and it belongs to the family of Aspergillus genus.This fungus is considered to be an opportunistic pathogen which is capable of contaminating most of seed crops and living organisms including animals and humans.In severe cases,Aspergillus flavus is capable of causing death or chronic diseases due to its highly toxic secondary metabolites.As a result,all the factors influencing the growth and distribution of Aspergillus flavus as well as its aflatoxin biosynthetic pathways,clustered genes and their regulatory mechanisms have been the subject of several studies for the past few decades.The above include but are not limited to the mitogen-activated protein kinase signal of the high osmolarity glycerol?MAPK/HOG?pathway which responds to osmotic stress.Although this pathway has been extensively studied in some of the fungi for many years,however,its mechanism in Aspergillus flavus is not well known yet.SakA?hog1/hogA?is believed to be an essential part of the MAPK/HOG pathway,so this study focused on determining the properties and the biological function of SakA in Aspergillus flavus.In this study,AfsakA gene was knocked-out??AfsakA?and complemented??AfsakA::AfsakA?using homologous recombination method,and osmotic stress was subsequently induced by 1.2moL/L of Sodium Chloride?NaCl?,1.2 moL/L of D-sorbital,as well as 0.93 and 0.99 water activity.It is found that?AfsakA mutant caused a significant influence on conidial production in contrast to both wild type?WT?and?AfsakA::AfsakA strains.The qRT-PCR assay showed that the expression level of abaA and brlA genes were down-regulated in?AfsakA than that in WT and?AfsakA::AfsakA strains.It was also found that AfsakA responds to both the cell wall stress and the osmotic stress.The expression level of HPS,GPD,GRE and STL osmolarity-related genes revealed that the transcription levels of these four genes were significantly lower in?AfsakA than that in WT and?AfsakA::AfsakA strains.In the absence of osmotic stress induced by the above compounds,?AfsakA mutant produced more sclerotia compared to other strains,whereas all strains failed to produce any sclerotia in the presence of osmotic stress.The transcription levels of nsdC and nsdD genes related to sclerotia production showed that,with no stress the transcription levels of these two genes were higher in?AfsakA than that in WT and?AfsakA::AfsakA strains.Regarding the aflatoxin production,this study revealed that?AfsakA mutant inhibited aflatoxin B₁?AFB₁?production under 0.93a_w osmotic stress conditions.However,under 1.2 moL/L D-sorbital,0.93a_w and 0.99a_w osmotic stress conditions,the deletion of AfsakA resulted in the significance increase of AFB₁ production which was different from WT and?AfsakA::AfsakA strains.The expression levels of aflatoxin biosynthesis catalyzing genes?aflO and aflQ?and aflatoxin transcriptional regulator genes?aflR and aflS?were further examined by qRT-PCR in the absence of stress.The results showed that all these aflatoxin biosynthesis and regulating genes were up-regulated in?AfsakA strain compared to WT and?AfsakA::AfsakA stains.The pathogenicity test performed on peanut seeds and maize kernels revealed that,?AfsakA strains produced more AFB₁ and conidia than WT and?AfsakA::AfsakA strains.Results on subcellular localization demonstrated that,AfSakA-mCherry was located to the cytoplasm without osmotic stimuli,whereas it translocated to the nucleus upon exposure to the osmotic stress.In conclusion,this study includes but not limited to the provision of new insights on the development of A.flavus and evaluation of aflatoxin biosynthesis.It is believed that this work will mark the first step towards the prevention of the seeds damages orchestrated by Aspergillus flavus,which causes significant financial losses to famers as well as numerous diseases and cancers around the world.