Physiological Function Of HOG Pathway Kinase SakA During Stipe Elongation Of P. Cinerea

Coprinopsis cinerea is a model fungus used to study the developmental mechanism of basidiomycete fruiting bodies.Under the stimulus of adverse external environmental conditions such as light,low temperature,lack of nutrients and other external environmental conditions,the growth stage of basidiomycetes will be transferred from the growth stage of vegetative hyphal to the growth and development stage of fruiting bodies.The HOG pathway is important for eukaryotes to respond to adverse environmental signal stimuli from the outside world.In fungi of ascomycetes,HOG pathway responds to environmental signals such as hyperosmotic pressure and oxidative stress,and participates in the physiological process of asexual spore formation and stress resistance.But at present,the function and mechanism of HOG pathway in the development of macrofungi fruit bodies are still unclear.In this study,the protein in different regions of the fruiting body stipes in Coprinopsis cinerea AmutBmut strain was extracted,and the phosphorylation level of the key kinase SakA in the different regions of the stipes was detected by Western Blot.The results showed that the phosphorylation level of the kinase SakA in the faster elongation stipe regions in the apical was higher than the slower elongation stipe regions in the median and the non-elongation stipe regions in the basal.It shows that the key kinase SakA in the HOG pathway is involved in the elongation and growth of the stipe.Further detection and analysis showed that the tissue osmotic pressure in the fast elongation region of the apical was lower and the endogenous reactive oxygen species(ROS)level was higher,it indicates that the key kinase SakA in the HOG pathway in the apical of the stipes may be activated by responding to osmotic stress or oxidative stress signals.In this study,a SakA interference(pCC-SakAi)vector was constructed.Using a PEG-Ca Cl₂ mediated genetic transformation system,the SakAi strain was obtained.The real-time quantitative PCR detecting system results showed that the expression of SakA in SakAi strains decreased by 76.8%.After analyzing the growth of the fruiting bodies in Mock strains and SakAi strains,we found that the radial growth rate of the hyphae in SakAi strains were significantly slowed down,and the length of the fruiting bodies were also significantly lower than that in Mock strains;After detecting the expression of genes related to stipe elongation,we found that the expression of the cell wall expansion protein chitinase ChiE1 in the apical of the fruiting bodies in SakAi strains were significantly reduced.The results showed that SakA gene silencing may lead to a decrease in the expression of the cell wall expansion protein ChiE1,which resulted in a slowdown in the stipe elongation rate of Coprinopsis cinerea fruiting bodies.In addition,our study also constructed a recombinant expression plasmid of SakA constitutively phosphorylated mutant protein(T170E+Y172D).Analyzing the growth of Mock strains and recombinant expression strains of SakA constitutively phosphorylated mutant protein(SakA^T170E+Y172D),we found that the radial growth rate of the hyphae in SakA^T170E+Y172D strains were significantly faster,and the length of stipes were also significantly higher than that in Mock strains,and the expression of ChiE1 in SakA^T170E+Y172D strains also increased significantly.The above results indicate that the key kinase SakA in the HOG pathway may be involved in the stipe elongation process of Coprinopsis cinerea fruiting bodies by regulating the expression of the cell wall expansion protein chitinase ChiE1.This study proves that during the development of fruiting bodies in Coprinopsis cinerea,the key kinase SakA of the HOG pathway may be involved in regulating the elongation and growth of the fruiting body stipes by regulating the expression of the cell wall expansion protein ChiE1.