Functional Analysis Of CAMP/PKA Pathway Associated Genes In Aspergillus Flavus

The ubiquitious fungus Aspergillus flavus is notorious for its infection of many important crops and animals with the carcinogenic mycotoxin,aflatoxin(AF).AF biosynthesis is extremely sophisticated,which is co-regulated at multiple levels.This dual ability to cause disease on both animals and plants exacerbates the importance of A.flavus as a pathogen.Thus this fungus causes enormous agricultural economic losses and health problems at several levels worldwide.Recent studies demonstrated that Cyclic AMP signaling is involved in regulation of Aspergillus asexual development and sterigmatocystin biosynthesis.Here,we identified and characterized genes involeved in cAMP/PKA signaling pathway,and investigated their functions during different development stage and fungal virulence of this fungus.We identified and characterized six RGS(Regulator of G protein signaling)proteins,named as RgsA,RgsB,RgsC,RgsD,RgsE and FlbA.All of these protiens contain the specific conserve RGS domain,while olny half of them have the transmembrane domain.The results of northern blot indicated that these RGS genes had different roles in fungal development and secondary metabolism of this fungus.To investigate the functions of rgs genes in A.flavus,we generated the deletion mutants of these six genes.The results showed that rgsA,rgsC and rgsE were important for A.flavus vegetative growth,sclerotia formation and aflatoxins biosynthesis.Severe defects in sporulation and sclerotia formation were observed in the ?flbA mutant,which is consistent with those in A.nidulans.However,deletion of rgsB or rgsD didn't demonstrate any phynotype defects in A.flavus.An increasing level of cAMP was found both in ?rgsA and ?rgsC mutants,leading to the activation of PKA activity.We further showed the interaction activities of RgsA?RgsB?RgsD and FlbA with Ga submit FadA.And rgs genes negtivatively regulated the function of Ga submit in asexual development and AF biosynthesis.Together,these data indicated that RGS proteins play negative roles in regulation of cAMP/PKA pathway.Since cyclic AMP signaling controls a range of physiological processes,like fungal development and infection when responding to extracellular stimuli in fungal pathogens,in this study,we investigated the function of adenylate cyclase,a core component of cAMP signaling,in aflatoxins biosynthesis and virulence on plant seeds in A.flavus.A gene replacement strategy was used to generate the deletion mutant of acyA that encodes the adenylate cyclase.Severe defects in fungal growth,sporulation and sclerotia formation were observed in the acyA deletion mutant.The defect in radical growth could be partially rescued by exogenous cAMP analog.The acyA mutant was also significantly reduced in aflatoxins production and virulence.Similar to the former studies in other fungi,The acyA mutant showed enhancing tolerance to oxidative stress,but more sensitive to heat stress.Overall,the pleiotropic defects of the acyA deletion mutant indicates that the cAMP-PKA pathway is involved in fungal development,aflatoxins biosynthesis and plant seed invasion in A.flavus.Cap(for cyclase-associated protein)was found to be important for the activation of AcyA.Although the cAMP signaling pathway has been shown to play important roles in various plant pathogenic fungi,to date no Cap genes have been functionally studied in Aspergillus.In this study,we identified and characterized the Cap gene in A.flavus.The Acap mutant was defective in fungal development,conidiation,and virulence.In addition,deletion of cap affected spore gemination and the toleration of osmotic stress and genomic toxicity stress in A.flavus.Domain deletion analysis revealed that the CARP domain of Cap,located in the C terminal,was important for its function in the proper regulation of vegetative growth,conidiation,AF produciton and full virulence.In addition,discruption of cap in A.flavus decreased the cAMP level and PKA activity,and the mutation of each Cap domain caused the dysfuntion of Cap in regulating cAMP synthesis and PKA activation.Overall,our results indicate that Cap is important for the activation of adenylate cyclase,fungal development,and virulence in A.flavus.In this study,we also investigated the function of the high-affinity(PdeH)and low-affinity(PdeL)cAMP phosphodiesterase from the carcinogenic aflatoxin producing fungus A.flavus,and found that instead of PdeL,inactivation of PdeH exhibited a reduction in conidiation and sclerotia formation.However,the ?pdeL/?pdeH mutant exhibited an enhanced phenotype defects,a similar phenotype defects to wild-type strain treated with exogenous cAMP.The activation of PKA activity was inhibited in the ?pdeH or ?pdeL/?pdeH mutant,both of whom exhibited increasing AF production.Further analysis by qRT-PCR revealed that pdeH had a high transcriptional level compared to pdeL in wild-type strain,and affected pdeL transcription.Green fluorescent protein tagging at the C-terminus of PDEs showed that PdeH-GFP is broadly compartmentalized in the cytosol,while PdeL-GFP localized mainly to the nucleus.Overall,our results indicated that PdeH plays a major role,but has overlapping function with PdeL,in vegetative growth,development and AF biosynthesis in A.flavus.Taken together,this study deomosntrated that regulation of cAMP level and activation of PKA acitvity by cAMP/PKA is important for A.flavus development,Aflatoxins biosynthesis and fungal virulence.This work provides valuable information for new control strategies of this fungal pathogen,and advance our understanding in regulation of secondary metabolites in filamentous fungi.