A novel venom protein from the parasitoid Pteromalus puparum suppresses host melanization by targeting multiple...

Parasitoid venom proteins are known to manipulate host immunity and metabolism. In the present study, a novel venom protein from the parasitoid Pteromalus puparum (Linnaeus) (Hymenoptera: Pteromalidae) (PpVPG) was identified and functional analyses of PpVPG were conducted. PpVPG is abundantly expressed in the venom apparatus, has a low molecular weight, and is a lineage‑specific venom protein with no functional annotation available. Functional analysis via RNA interference revealed that PpVPG is essential for parasitism, as its silencing severely impaired successful parasitism of Pieris rapae (Linnaeus) (Lepidoptera: Pieridae), a major Brassicaceae pest worldwide. Biochemical characterization showed that recombinant PpVPG inhibited PO activation in host hemolymph in a dose-dependent manner but had no effect on pre-activated PO, indicating that it acts upstream of PO activation. Both the N-terminal and C-terminal fragments retained partial inhibitory activity. Target identification by affinity purification coupled with mass spectrometry identified three host proteins as direct interaction partners of PpVPG: two prophenoloxidases (PrPPO1, PrPPO2) and a prophenoloxidase-activating factor (PrPAF2). Surface plasmon resonance confirmed high-affinity binding of PpVPG to these three proteins, as well as to a prophenoloxidase-activating protease (PrPAP1). This multi-target binding strategy, engaging both the zymogen substrates and their activation machinery within a single immune cascade, represents an effective molecular strategy for a small, non-enzymatic venom protein. These findings establish PpVPG as a venom effector that suppresses host melanization through multi-target binding.