The Role Of Ceramide Synthases In Regulating Sphingolipid Homeostasis,Mycelial Growth,Stress Responses And Ganderic Acids Biosynthesis In Ganoderma Lucidum

The medicinal fungus Ganoderma lucidum contains many pharmacological active ingredients,and there is widespread interest in investigating this organism.The reliable gene silencing system and genome-wide sequence make G lucidum become a widely used research materials for studying the secondary metabolism and mycelial growth patterns in filamentous fungi.Ceramide synthases belong to the longevity-assurance gene(LAG)family.Ceramide synthases are ubiquitous in eukaryotes,and have attracted widespread attention because it is at the center of sphingolipid metabolism and participates in physiological processes such as growth,stress response and signal transduction.However,the functions of ceramide synthases in basidiomycetes are still unclear.Firstly,in this article,three homologues of ceramide synthase,encoded by the three distinct genes LAG1,LAG2 and LAG3,were distinguished in G.lucidum.To inquire into the function of ceramide synthase,we constructed LAG1 single-silenced strains,LAG2 single-silenced strains and LAG3 single-silenced strains.When LAG1,LAG2 and LAG3 respectively were inhibited by RNA interference,LAG1 single-silencing strains showed obvious changes in sphingolipid homeostasis,mainly due to decreased content of ceramides and complex sphingolipids containing long-chain fatty acid chains(C16/C18),unaffected mycelial growth,and reduced levels of ganoderic acids(GA).However,the sphingolipid content of LAG2 single-silencing strains and LAG3 single-silencing strains did not change significantly and the growth rate was not affected.Therefore,LAG2 and LAG3 may have functional redundancy.Therefore,we constructed LAG2/3 double-silencing strains.After LAG2 and LAG3 were silenced by RNA interference,the content of ceramides and complex sphingolipids containing very long chain fatty acid chains(C22/C24/C26)decreased,the mycelial growth rate decreased,and the content of GA increased.However,in the face of various abiotic stresses(such as oxidative stress,cell wall stress and osmotic stress),LAG1 single-silencing strains and LAG2/3 double-silencing strains showed different degrees of sensitivity.Next,we initially explored the mechanism that LAG1 and LAG2/3 affect the biosynthesis of GA.When LAG2 and LAG3 were simultaneously inhibited,ROS levels increased,resulting in increased GA content.Adding ROS scavengers could reduce the GA accumulation due to silencing of LAG2 and LAG3 genes.These results indicated ROS signaling is involved in the regulation of GA synthesis by LAG2 and LAG3.Consequently,we explored the source of intracellular ROS.When LAG2 and LAGS were simultaneously inhibited,NADPH oxidase activity and transcriptional level did not change.Mitochondria are also one of the main sources of ROS.When and were simultaneously inhibited,resulting in mitochondrial ROS outbreak,respiratory rate reduction,and reduced the enzymatic activities of mitochondrial complexes Ⅱ and Ⅳ.Ceramides are not only a precursor to the synthesis of complex sphingolipids,but also an effective signal molecule.When LAG2 and LAG3 were simultaneously inhibited,resulted in a significant increase in most ceramide containing long-chain fatty acid chains(C16/C18),and inhibited the enzymatic activities of mitochondrial complexes Ⅱ and Ⅳ.The regulation of sphingolipid homeostasis on mitochondrial enzyme activity remains to be further studied.In conclusion,these results demonstrate the potential role of LAG 1,LAG2 and LAG3 in G.lucidum in influencing sphingolipid homeostasis,growth,stress response and GA biosynthesis,laying the foundation for subsequent research.