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.

Polydnavirus are well known to interfere with the host endocrine system, causing immune suppression and other physiological disorders. The Cotesia rubculla polydnavirus gene products, CrV1, are known to be a potent immunosuppressive agent. CrV1 protein express within 12 h after viral infection at oviposition during deposition of parasitoid eggs and are mainly secreted in to host hemocyte, where it functions like phagocytosis and cell spreading. This study identified its homolog in CpBV and analyzed its molecular characteristics motif called “coiled-coil. A point mutation of Alanine to Proline of CpBV-CrV1 could lose the coiled-coil motif from in silico assay. The coiled type CpBV-CrV1 could inhibit host cellular immunity, however, interestingly the mutant CpBV-CrV1 lacking in coiled-coil motif completely lost the immunosuppressive activity. This study suggests that the coiled-coil motif is functional to inhibit host cellular immune responses. RNA interference against CrV1 significantly loses the inhibitory activity and thus further supporting the immunosuppressive activity of CrV1. In this study we also have analyzed the localization of CrV1 by transient transfection in HiFive Cell lines by in situ hybridization.