Characterization Of Three Novel MAPK-Containing Regulatory Pathways That Control Cuticle Penetration Or Conidiation In Metarhizium Robertsii

Recently,the abuse of chemical insecticide has received more and more attention.Metarhizium robertsii,one of the main representative entomopathogenic fungi,has been developed as fungal insecticides to control agricultural and forest pests.Metarhizium robertsii can switch among parasitic,saprophytic and symbiotic lifestyles.For this reason,it is not merely an emerging fungal model for investigating the pathogenic mechanisms but a great material for investigating the mechanisms of carbon and nitrogen metabolisms.The pathogenic process of Metarhizium robertsii is divided into cuticle penetration and colonization of hemocoel and the former is decisive for pathogenicity.However,the mechanism of cuticle penetration is poorly understood.Futhermore,as the active constituent of fungal insecticide,the conidia of Metarhizium robertsii determines the efficiency of fungal insecticides.Explanation of conidiation mechanism can provide significant insights into the efficiency of fungal insecticides.Our Studies focus on the pathogenicity and conidiation mechanisms of three signal cascades that are regulated by MAPK.The main results are as follows:1.Previous study found that the transcription factor RNS1(regulator of nutrient selection 1)was positively regulated by Fus3.Compared to the WT strain,the fungal virulence was depleted in the deletion mutant of Rns1.In this study,we found that Fus3 phosphorylated the Thr-215 and Ser-226 in RNS1,which facilitated the entry of RNS1 into nuclei.The phosphorylated RNS1 binds to the BM2 motif(CCCAGAC)in its own promoter to activate the expression of Rns1.Furthermore,RNS1 positively regulates the expression of lipase,chitinase and protease for degrading cuticle,which not merely facilitate aquisition of nutrients for Metarhizium robertsii,but open a channel for entering the hemocoel of insects.Besides the lipid,chitin and protein from insect cuticle,we further found that Fus3 phosphorylated RNS1 and induced its expression in non-insect complex organic carbon and nitrogen sources(hydrocarbons,colloid chitin,and casein).Fus3/RNS1 cascade induces the expression of genes for utilizing non-insect complex carbon and nitrogen sources,which is inhibited by favored organic carbon and nitrogen nutrients including glucose and glutamine or less-favored carbon and nitrogen nutrients(argnine,proline,Glc NAc,raffinose,and trehalose).In addition,we failed to identify the relationship between RNS1 and the regulator of carbon and nitrogen metabolism(CRR1,AREA,G6 PD,Tps1,Snf1,TOR kinase).2.Compared to the WT strain,the deletion mutant(35)Rns1 appears more “fluffy” on PDA plates(14 days after inoculation).The conidial yields of(35)Rns1 are significant less than the WT strain.Compared to the WT strain,the structure of phialides develops abnormally in the deletion mutant(35)Rns1.Moreover,Rns1 expresses highly in phialides and conidia.Above studies indicated that RNS1 regulated the conidiation of Metarhizium robertsii.Transcription factors Brl A and Aba A are two components of the central regulation pathway of conidiation and Brl A regulates the expression of Aba A directly.There are two transcripts(Brl Aα and Brl Aβ)in Brl A.The start site of Brl Aβ transcript is 835 bp upstream of the start site of Brl Aα transcript.RNS1 binds to the BM2 motif on the Brl A promoter and promotes its expression.However,the expression of Aba A regulated by RNS1 is mediated by Brl A.We futher found that Slt2 positively regulated the conidiation regulatory pathway mediated by RNS1.3.Previous study found that transcription factor AFTF1 controled cuticle penetration by regulating the formation of appressorium.Although the expression of Aftf1 is regulated by Fus3,the mechanism of which remains incompletely understood.Our study further found that the regulation of Aftf1 by Fus3 was mediated by transcription factor Mr St12.The deletion mutant of Mr St12 fails to form the appressorium structure and is thus nonpathogenic.Fus3 physically contacts and phosphorylates Mr St12.The activated Mr St12 binds to the cis-acting element(ATGAAACA)on the promoter of Aftf1 and promotes its expression during cuticle penetration.RNS1,Mr St12 and AFTF1 are three transcription factors that regulated by Fus3,but we failed to identifiy the relationship between RNS1 and Mr St12/AFTF1.Our study indicated that Fus3/RNS1 and Fus3/Mr St12/AFTF1 were two independent cascades during cuticle penetration.In conclusion,our studies found that MAPK and three transcription factors constituted three regulatory pathways Fus3/RNS1,Slt2/RNS1/Brl A/Aba A and Fus3/Mr St12/AFTF1.Two pathways that contain Fus3 are involved in regulating cuticle penetration,but there is no direct correlation between them.Slt2/RNS1 controls the conidial development by regulating conidial central regulation pathway.