The Mechanism Of FgSnt1 And FgBlm10 Genetic Interaction With The CAMP-PKA Signal Pathway In Fusarium Graminearum

Fusarium head blight（FHB）which is caused by Fusarium graminerium is a global disease that endangers food production and food safety.It is prone to outbreaks under suitable climatic conditions.It is difficult to avoid and has a wide range of hazards.Therefore,deep inside to research the mechanism between F.graminerium and wheat is particularly important for preventing FHB.Previous research showed that deleted of PKA regulatory subunit PKR had severe defects in growth,conidiation,and plant infection.When cultured on PDA or any other medium,the pkr mutant was unstable and spontaneous suppressors with faster growth rates were often produced.In this study,we assay the growth rate,conidiation and pathogenicity of 60 suppressor strains from the previously study.The main results are as follows:（1）All 60 supressor strains partly rescued the growth decfects of pkr mutant.Base on the growth rate,the suppressor strains were categorized into three groups.Type I suppressor strains had the fastest growth rate which is over 2 folds of that of the pkr mutant.Even fully restore than the wild type.Type II suppressor strains had the faster growth rate which is1.51-1.93 folds than that of pkr.Type III is only 1.02-1.36 folds than the pkr mutant in growth rate.Although all suppressor stains of the pkr mutant have been partially restored the defective in vegetative growth,most suppressor stains do not restore the conidiation and pathogenicity defective of pkr mutant.Then selected 10 suppressor strains for Whole Genome Sequencing（WGS）.The result show that there are 16 genes are detected.Nine of them have homology in yeast,including SNT1,BLM10,PRE5,PRE6,SGF73,TPK2,MIG1,CYC8,and NHP6A/6B.The others seven gens have no homology in yeast.Total,11suppressor mutations had mutation in Fg Snt1 and 10 suppressor mutations had mutation in Fg Blm10 were indentified.These two genes are likely to be important factors in revealing the spontaneous mutation of the pkr mutant.SNT1 is a core component of the Set3 HDAC complex in yeast.BLM10,PRE5 and PRE6 are components of the 26S proteasome.（2）Removed the 393 to 1959 amino acids of Fg Blm10,and then knock out PKR.The double knockout mutant partly restores the defects of pkr mutant in growth,morphology of conidia and conidiation.PKA activity cannot be detected after knocking out the PKA regulatory submit PKR.Through the proteasome inhibitor MG132 treatment,the PKA activity of the pkr mutant is restored.When culturing the pkr mutant on PDA medium with MG132,the production of the suppressors was inhibited.In addition,the Cpk1 band can not detect in pkr mutant by Western Blotting.While,the degradation of Cpk1 was inhibited with the treatment with MG132,or C-terminal region deletion of Fg BLM10.These results indicate that the regulate subunit Pkr protects the catalytic subunit of PKA,which is degraded by 26S proteasome in the absence of Pkr.The dysfunction of 26S proteasomes such as spontaneous mutations in Fg Blm10 or the treatment of its inhibitor MG132stabilized Cpk1 and partially restores its function.（3）When tested the PKA activity of suppressor strains which mutations in Fg Snt1,and we find that PKA activity can not be detected.This result shows that Fg Snt1 does not suppress the defective of pkr mutant by restoring its PKA activity.To clarify the role of Fg Snt1 in the spontaneous mutation of pkr mutant,we double knock out Fg SNT1 and PKR.It is interesting that Fgsnt1 pkr double mutant cannot restore the defects of pkr mutant.Only deleted the C-terminal 98 amino acids of Fg Snt1,the defects of growth,conidiation,sexual reproduction and infection in pkr mutant could be restored.Through yeast two-hybrid and immunoprecipitation（IP）assays,Fg Snt1^ΔC98 interacts with Hdf1 instead of Fg Set3,while Fg Snt1 perfer to interact with Fg Set3.The differential interaction may resuce the reduced the histone H4 acetylation level in pkr mutant.Furthermore,we also find that Fg Snt1 may has a conserved PKA phosphorylation site S443.The dominate active form Fg Snt1 S443D can also restore the defects of pkr mutant.In addition,the C-terminus binds to the Fg Snt1S443.These results indicated that the C-terminus of Fg Snt1 has an important role in its interaction with the N teriminal of Fg Snt1,which may change the Fg Snt1 conformation and its interaction with the other component of Set3 complex.In summary,this study confirms that the the relationship of the regulatory and catalytic subunits of PKA,the mechanism of Cpk1 degradation in the absence of Pkr,therefore the reason for of the loss of PKA acitivity in pkr mutant.In addition,it is also verified that histone acetylation modification is an important factor in restoring the defects of pkr mutant.