The Effect Of Acetylation Of Cyclophilin D On The Apoptosis Of Host Hemocytes Induced By MbBV In Spodoptera Litura

Spodoptera litura(S.litura)has high adaptability to different ecological environme nts because of its high mobility and spawning ability,and it has become one of the major agricultural pests to be controlled urgently.S.litura can be parasitized by Microplitis bicoloratus(M.bicoloratus).When M.bicoloratus parasitizes S.litura larvae,MbBV was injected together with one or several eggs into the body of S.litura larvae,MbBV Infects and induces apoptosis of blood cel s to inhibit the S.litura immune responses.Cyclinophil in(CypD)is a member of the immunophilin protein family.The earliest found immunophil in protein is immunophilin A,which is the most abundant in the cytoplasm,and CypD is currently the only type of immunophilin to be localized in mitochondria,encoded by the nuclear gene ppif.Studies have shown that CypD is a component and an important regulator of mitochondrial membrane permeability transition pore(mPTP)and participates in the regulation of mPTP to regulate apoptosis and non-apoptotic death.The transcripto me sequencing results of our previous research revealed that CypD mRNA level was significantly up-regulated in S.litura hemocytes after parasitization,suggesting that CypD may be involved in host immune suppression caused by M.bicoloratus parasitism.To further explore the molecular mechanisms that CypD may be involved in the host immune response,this article first detected the expression level and acetylation level of CypD in hemocytes of S.litura before and after parasitism by Western blot.It was found that the expression level and acetylation level of CypD were significantly increased after parasitization,and hemocyte apoptosis was increased too;Secondly,infected Spli221 cells with purified MbBV,similar results were observed with S.litura after paratisizm.Both CypD expression and acetylatio n levels were elevated.Third,in order to determine that CypD is involved in the regulation of cell apoptosis by acetylation,the acetylation site of Spodoptera litura was found to be K125 by comparison.The pIZT-CypDK125 R plasmid was constructed by site-directed mutagenesis.The pIZT-CypDK125 R expressed protein cannot be acetylated.When the two plasmids(pIZT-CypD and pIZT-CypDK125R)were transfected into MbBV-infected Hi5 cel s,it was found that pIZT-CypDK125 R significantly down-regulated the apoptosis of Hi5 cel s caused by pIZT-CypD,similar to the results of treatment of MbBV-infected Spli221 cel s with an acetylation inhibitor of CypD.Finally,the study also found that compared with the pIZT-CypD-expressed protein,the binding ability of the pIZT-CypDK125R-expressed protein to Adenine nucleotide translocase 1(ANT1)was decreased,and both parasitic and MbBV infection of Spli221 cel s resulted in a decrease in the concentration of Cyt C in the mitochondria.These results suggest that one of the ways MbBV induced host immunosuppression during the process of S.litura was parasitized by M.bicoloratus is probably by enhancing the expression and acetylation of CypD in host hemocytes,and promoting CypD binding to ANT1 on the mitochondrial membrane,to regulate mitochond r ia opening and releases Cyt C,leading to apoptosis of hemocytes.The achievement of these research results has laid a certain foundation for further investigation of the molecular mechanism of insect immunosuppression.