C-terminal conserved motifs of Neprilysin1 in Cotesia plutellae are not required for immune suppression of the Diamondback moth, Plutella xylostella (L.)

Abstract

Abstract Cotesia plutellae, an endoparasitoid wasp, parasitizes Plutella xylostella larvae and disrupts host immune responses through parasitic factors. These immune disruptive factors are known as maternal factors (venom proteins, polydnavirus, and ovary proteins) and an embryonic factor (teratocytes). In this study, we identified neprilysin-1 (Cp-NEP1) known as a potential immunosuppressive gene from the transcriptome of venom glands in C. plutellae. Cp-NEP1 encoding 451 amino acids belonged to the hymenopteran NEP1 family through phylogenetic analysis. Based on the structural comparison, Cp-NEP1 has lacked in conserved motifs such as a substrate binding (NAYY/F), zinc-binding (HExxH and ExxxD), and protein folding and maturation (CxxW). In order to investigate the function of Cp-NEP1 in host immune response, we constructed a recombinant Cp-NEP1 (rCp-NEP1) harboring N-terminally fused 6X His-tag. rCp-NEP1 was successfully expressed in Escherichia coli and purified. Pre-heated E. coli induced the spike of nodule formation whereas co-injection of rCp-NEP1 with pre-heated E. coli exhibited suppression of nodule formation. Quantitative real-time PCR showed that expression of phenoloxidase related to nodule formation was also suppressed when rCp-NEP1 was co-injected with preheated E. coli. These results suggest that Cp-NEP1 contributes to immunosuppression and that conserved motifs lacking in Cp-NEP1 are not necessary for immune suppression in the host.