Studies On Functions Of Two Venom Serpins In Endoparasitiod Wasp,Micropilits Mediator

Parasitic wasps are important natural enemy insects whose parasitic process can lead to host mortality and are therefore widely used for biological control of pests.Microplitis mediator is an endoparasitic wasp that lays eggs in hosts such as Helicoverpa armigera and Pseudaletia separate,which are agricultural pests of the family Lepidoptera.The successful survival of endoparasitic wasps in their hosts is due to a variety of parasitic factors such as venom,ovarian fluids,polydnavirus(PDV)and virus-like particles,which help the parasitic wasps to escape or resist the host’s immune response and eventually survive the mutualistic competition.Serpin(serine protease inhibitor,serpin)acts specifically on the target serine protease to inhibit the activation of immune signaling pathways such as prophenoloxidase(PPO)and Toll pathways,preventing damage caused by excessive immune responses.The proteomic and transcriptomic data of the M.mediator indicate the presence of serpins in its venom,but their role in the parasitization process is not clear.M.mediator and its host H.armigera and P.separate were used as the research objects.Bioinformatics analysis,RNA interference,prokaryotic expression,western blot and pull-down experimental techniques were performed to investigate the role of MmvSPN-1 and MmvSPN-2 in the interaction between M.mediator and its hosts.The main findings are as follows:1.Functional structure analysis and tissue expression pattern of MmvSPN-1and MmvSPN-2.Bioinformatics analysis showed that both MmvSPN-1 and MmvSPN-2have signal peptides,which are secretory proteins.Amino acid residues at their cleavage sites are arginine(R),implying that they can be recognized and cleaved by trypsin and inactivate trypsin.q RT-PCR results showed that MmvSPN-1 and MmvSPN-2 were mainly highly expressed in the ovaries and venom apparatus.2.Prokaryotic expression and purification of recombinant proteins MmvSPN-1and MmvSPN-2 with inhibitory activity,and preparation of polyclonal antibodies.The recombinant plasmids p ET28a-MmvSPN-1 and p ET28a-MmvSPN-2 were constructed in vitro,and the recombinant proteins were expressed and purified in Escherichia coli cells.The recombinant proteins rMmvSPN-1 and rMmvSPN-2 showed significant inhibitory effects on trypsin.Specific polyclonal rabbit antibodies were prepared using the recombinant proteins,and the two proteins were detected in parasitic wasp venom using these antibodies.3.MmvSPN-1 and MmvSPN-2 inhibite the PPO signaling pathway of the host P.separate and H.armigera.Knockdown of MmvSPN-1 and MmvSPN-2 expression caused higher PO activity and enhanced PPO activation in hosts P.separate and H.armigera.After incubation of recombinant MmvSPN-1 and MmvSPN-2 proteins with H.armigera plasma,a significant decrease in PO activity and PPO activation were detected.These results suggest that MmvSPN-1 and MmvSPN-2 negatively regulate the PPO signaling pathway of hosts.4.MmvSPN-1 and MmvSPN-2 inhibite the expression levels of antimicrobial peptides(AMP)in the hosts P.separate and H.armigera.Knockdown of MmvSPN-1 and MmvSPN-2 resulted in higher expression of AMP genes in the parasitized P.separate and H.armigera.The larvae pre-injected with recombinant MmvSPN-1 orMmvSPN-2 protein showed reduced AMPs expression after challenge with bacteria.Our results suggest that MmvSPN-1 and MmvSPN-2 play roles to down-regulate the expression of AMPs in in H.armigera.5.Identification of H.armigera plasma proteins that interact with MmvSPN-1and MmvSPN-2 respectively.The target protease HacSP29 of MmvSPN-1 and MmvSPN-2 was captured using pull-down and LC-MS/MS identification analysis techniques.After expression and purification of r Haproc SP29,the hemolymph was added to activate the zymogen.The MmvSPN-1-HacSP29 and MmvSPN-2-HacSP29 complexes were detected when activated HacSP29 incubated with recombinant proteins rMmvSPN-1and rMmvSPN-2 in vitro.These results suggest that MmvSPN-1 and MmvSPN-2negatively regulate the host PPO activation and antimicrobial peptides expression by directly inhibiting the activity of hemolyph protease HacSP29.6.MmvSPN-1 and MmvSPN-2 inhibit the activation of the downstream serine proteases in H.armigera plasma.HacSP29 is a protease that blongs to the initiation module of serine protease cascade.We detected the activation of serine protease Haproc SP4,Haproc SP7,and Haproc SP8,which are in the downstream of HacSP29,were inhibited in the presence of recombinant MmvSPN-1 and MmvSPN-2 proteins.These results suggest that the activity of downstream proteases Haproc SP4,Haproc SP7 and Haproc SP8 are suppressed,mostly because MmvSPN-1 and MmvSPN-2 lead to the inactivation of HacSP29.Combined with the phylogenetic analysis,these two venom serpins were deduced to inhibit the host H.armigera immunity through HacSP29-HacSP4-HacSP8 cascade and HacSP29-HacSP4-HacSP7 cascade.In conclusion,this thesis showed that the venom proteins MmvSPN-1 and MmvSPN-2from M.mediator venom inhibit the activation of PPO pathway and the expression level of antimicrobial peptide genes in the host P.separate.Based on this phenomenon,the inhibition mechanism is resolved in another host,H.armigera,revealing that MmvSPN-1and MmvSPN-2 respectively form a covalent complex with the serine protease HacSP29 of the host.It may inhibit the activation of PPO pathway in H.armigera through HacSP29-HacSP4-HacSP8 cascade and the expression level of antimicrobial peptide gene through HacSP29-HacSP4-HacSP7 cascade.It provides guidance for the exploitation of parasitic wasp venom resources and the development of highly effective and specific biopestcides.