Cotesia plutellae, an endoparasitoid wasp, parasitizes larvae of Plutella xylostella, and disrupt immune response of the host through parasitic factors. These immune disruption factors are maternal (venom proteins, polydnavirus, and ovary proteins) and embryonic (teratocytes) factors. In this study, we performed transcriptome analysis of venom glands of C. plutellae and identified neprilysin-1 (NEP1) known to be potential immunosuppression gene. Cp-NEP1 encoded 451 amino acid and belongs to hymeopteran NEP1 via phylogenetic analysis. Based on the structural comparison Cp-NEP1 lacks in conserved motifs such as substrate binding (NAYY/F), zinc-binding site (HExxH), zinc-binding site, protein folding and maturation (CxxW). To investigate function of Cp-NEP1, we constructed a recombinant Cp-NEP1 harboring N-terminally fused 6X His tag. Peptide sequencing revealed successful expression of the recombinant Cp-NEP1 in Escherichia coli. Pre-heated E. coli as antigen induced spike of nodule formation whereas co-injection of the recombinant Cp-NEP1 and pre-heated E. coli exhibited suppression of nodule formation in the host. Quantitative real-time PCR revealed that expression of phenoloxidase related to nodule formation was suppressed under co-injection of the recombinant Cp-NEP1 and E. coli. These results suggest that Cp-NEP1 contributes to immunosuppression of P. xylostella via phenoloxidase suppression and conserved motifs of neprilysin family are not required for host immune suppression.

One of the endoparasitoid wasp, Cotesia plutellae (Braconidae), parasitizes young larvae of the diamondback moth, Plutella xylostella. For the successful parasitization, C. plutellae required suppression of immune response in P. xylostella. Maternal (polydnavirus, venom proteins and ovary proteins) and embryonic (teratocytes) factors have been involved in immune-suppression. In this study, we performed transcriptome analysis of venom of C. plutellae and identify neprilysin-1 (Cp-NEP1) as a potential immunosuppressive protein. Cp-NEP1 encoded 451 amino acids and largely belongs to the hymeopteran neprilysin family via phylogenetic analysis. It is of interest that Cp-NEP1 has no conserved motifs such as zinc-binding domain (HExxH), substate binding domain (NAYY/F) and protein folding and maturation domain (CxxW) generally identified in other neprilysin family. In order to examine the biochemical function of Cp-NEP1, the recombinant Cp-NEP1 tagged with N-terminally 6X His was constructed and expressed in Escherichia coli. Expression of Cp-NEP1 was confirmed with SDS-PAGE and peptide sequencing. Recombinant Cp-NEP1 significantly suppressed nodule formation when the co-injection with E. coli. These results suggest that Cp-NEP1 contributes to suppression of immune response in P. xylostella and that the conserved motifs reported from other neprilysin do not involve immunosupperssion.