The Role Of Tor1 In Regulating Growth,Cell Wall Integrity And Ganderic Acids Biosynthesis In Ganoderma Lucidum

Ganoderma lucidum is a famous edible and medicinal fungi and has a potential effect,such as anti-cancer,anti-virus,anti-aging effect and so on.In China,G.lucidum has a extensive and long history of application.In recent years,the separation and purification of pharmacologically active compounds from G.lucidum have been received more and more attention.Ganoderic acids are the major active compounds in G.lucidum and the research about ganoderic acids has become a hot spot.With the completion of G.lucidum genome sequencing and establishment of genetic methods,G.lucidum has become a potential model basidiomycetes strain to research growth,development and secondary metabolism.TOR(target of rapamycin)is an evolutionarily conserved serine/threonine protein kinase that plays an important role in regulating growth and metabolism as well as response to environmental cues.Rapamycin,as a specific inhibitor of TOR,binding to the intracellular cofactor FKBP12(FK506 binding protein 12),forming a complex that then inhibits TOR activity.When TOR was inhibited by RNAi or rapamycin treatment,the growth of G.lucidum mycelium was inhibited,the distance between hyphal branching increased,the cell wall components changed,and the content of secondary metabolites ganoderic acids decreased.In this study,The results indicated that Slt2-MAPK was involved in TOR regulating cell wall components in G.lucidum.when TOR signaling was inhibited by RNAi or treatment with rapamycin,G.lucidum was not sensitive to the cell wall damage agents CFW and CR treatment.In addtion,the content of the cell wall components ?-1,3-glucan and chitin increased.Moreover,we used electron microscope to detect cell wall thickness and found that cell wall thickness increased when TOR signaling was inhibited.Slt2-MAPK plays an important role in regulating cell wall integrity,we detected the phosphorylation level of Slt2-MAPK by Western Blot and found that Slt2-MAPK phosphorylation levels increased when TOR signaling was inhibited.To further explore whether TOR signaling affected cell wall components through Slt2-MAPK,we used Rapamycin to treat Slt2-silenced starins and the results showed that when Slt2-silenced starins were treated with rapamycin,the content of ?-1,3-glucan and chitin were significantly lower than WT with rapamycin treatment.These results indicated that Slt2-MAPK is involved in Tor1 regulating cell wall components in G.lucidum.Since TOR plays an important role in the regulation of cellular metabolism,the effect of TOR kinase on the synthesis of ganoderic acids was investigated.The results showed that the content of ganoderic acids was decreased when TOR was inhibited by rapamycin treatment or RNAi.Due to TOR was able to sense intracellular ATP,we detected the content of intracellular ATP and found that when inhibition of TOR kinase,intracellular ATP content was decreased.Interestingly,adding exogenous ATP can compensate the decrease of ganoderic acids content when TOR was inhibited.These results indicated that Tor1 regulates the biosynthesis of ganoderic acids through intracellular ATP.In G.lucidum,the mechanism of Tor1 regulating intracellular ATP remains to be further studied.