Investigation On Regulatory Mechanisms Under AGC Signal Pathway In Sporisorium Scitamineum Sexual Mating/filamentation

Sporisorium scitamineumis the fungal pathogen that causes sugarcane smut.It belongs to the genus Basidiomycetes,and contains two different mating types,MAT-1 and MAT-2.S.scitamineum invades host sugarcane by dikaryotic hyphae,formed after sexual mating of haploidspores(sporidia)of two opposite mating types.Therefore,mating/filamentation is critical to S.scitamineum pathogenicity,while its molecular mechanism has not yet been resolved.The AGC kinase family is a group of serine/threonine(Ser/Th)protein kinases conserved in eukaryotic organisms and has a variety of physiological functions including cell growth,metabolism,differentiation,and cell death.In this study,we identified an AGC kinase named Ss Agc1 and studied its function by reverse genetics and biochemical analysis.The results showed that Ss Agc1 is essential for S.scitamineum tolerance to oxidative stress and mating/filamentation.We further found that exogenous additions of the known fungal quorum-sensing(QS)molecules,as well as the intermediate products of the Ehrlich pathway or Kynurenine pathway,both of which are responsible for trytophane metabolism,could partially recoverssagc1(35)mating/filamenation.Both transcriptome analysis and q RT-PCR validation indicated that Ss Agc1 regulates the expression of genes governing fungal mating/filamentation and/or tryptophanecatabolism.We infer that Agc1 signaling may integrate pheromone response and trytophane catabolism/tryptophol biosynthesis,in S.scitamineummating/filamentation.Tryptophol is one of the established fungal quorum-sensing molecules(QSMs),and is known to be produced by the Ehrlich pathway in Saccharomyces cerevisiae.So far it has not been reported about the physiological function,synthetic pathway,and transcriptional regulation of trytophol in the pathogenic development of S.Scitamineum.The genes encoding the key enzymes(Aro8,Aro9,Aro10,and DC)of the Ehrlich pathway were shown to be differentially regulated in the ssagc1(35)mutant compared to the wild-type strain,indicating that they may be involved in Agc1-mediated regulation on S.Scitamineummating/filamentation.To investigate their function in biosynthesis of trytophol productionand pathogenic development in S.Scitamineum,we deleted ARO8,ARO9,ARO10,and DCgenes individually.The results showed that ARO8/9 was essential for S.scitamineummating/filamentation,while ARO10/PDC1 was not.We further found that exogenous addition oftryptopholor the genetic complementation ARO8 gene couldboth at least partially restore ssaro8Δ mating/filamentation,suggesting that S.Scitamineum Aro8 may actually participate in the Ehrlich pathway to produce tryptophol and promote mating/filamentation.Overall,our work revealed a signaling pathways mediated by AGC protein kinase to regulate fungal mating/filamentation,likely through sensing and responding to pheromone and/or quorum-sensing signal molecule tryptophol.We also found that the key enzyme of aromatic alcohol metabolism pathway,Aro8 and Aro9,may regulate the S.scitamineum mating/filamentationlikely by regulating tryptopholproduction.