Comparative Analysis Of Proteome And Phosphoproteome Of Neurospora Crassa In Response To Cellulose And Cellobiose

Improving cellulolytic enzyme production by plant biomass degrading fungi holds great potential in reducing costs associated with production of next-generation biofuels generated from lignocellulose.But it is still not clear how fungi sense cellulosic materials and respond by producing and secreting cell wall-degrading enzymes.It is proposed that cellulose hydrolysis products,such as cellobiose,serve as the inducer of cellulase gene expression.However,cellobiose can be hydrolyzed to glucose by ?-glucosidases produced by plant biomass degrading fungi.Here,we applied a mutant of the plant biomass degrading filamentous fungus Neurospora crassa,carrying deletions of three ?-glucosidase enzymes and thus lacking ?–glucosidase activity,to obtain global quantitative proteomic and phosphoproteomic profiles of the N.crassa in response to cellobiose as well as cellulose,sucrose and no carbon.Our results suggest that extensive post-transcriptional regulation occurs in N.crassa in response to exposure to cellobiose or cellulose.Compared with sucrose or no carbon conditions,cellobiose and cellulose significantly affect the expression levels of over 100 proteins.Several hundred serine,threonine or tyrosine residues were identified to have differential phosphorylation in response to cellobiose and crystalline cellulose(Avicel),compared with sucrose or no carbon conditions.Functional category analysis suggests that cellobiose and cellulose particularly affect the expression of proteins for sugar uptake and carbohydrate metabolism.In addition,cellobiose and cellulose modulate the phosphorylation state of transcription factors as well as kinases and receptors for cellular signaling and interactions with the environment,including the Cellodextrin Transporter-Like Protein 1(CLP1).CLP1 is known to affect the expression of the cellodextrin trasnporter CDT-1 and thus cellulases though it has no cellodextrin uptake activities.Our data provides rich information at both the protein and phosphorylation levels of the early cellular responses to cellobiose and cellulose and to carbon starvation and aids in a greater understanding of the underlying post-transcriptional regulatory mechanisms in filamentous fungi.Cellobiose is taken up by N.crassa mainly through cellobiose transporters CDT-1 and CDT-2.The two transporters play important roles in cellulase production and cellulose metabolism.We constructed a random mutagenesis library of CDT-1 and CDT-2 in order to screen for the mutants that lose the cellobiose transport capacity but not affect the induction of cellulase gene expression.These approaches help explore the molecular mechanisms of cellulase production and the dual function of CDT-1 and CDT-2 in transporting cellobiose and mediating downstream activation of cellulase genes expression.With the multiple genes of unknown functions by Proteomics data analysis,we provide the basis and direction to further explore cellobiose in cellulose enzyme system in the process of induction,expression,secretion regulation mechanism.