Glyceraldehyde-3-phosphate dehydrogenase is a mediator of hemocyte-spreading behavior and molecular target of immunosuppressive factor CrV1

Abstract

Cellular immunity is accompanied by hemocyte-spreading behavior, which undergoes cytoskeletal rearrangement. Polydnaviral factor CpBV-CrV1 can inhibit the hemocyte-spreading behavior and suppress host immune response of Plutella xylostella. However, host target molecule of CpBV-CrV1 that inhibits the hemocyte behavior has not been identified yet. This study used a pull-down approach to identify the target molecule of CpBV-CrV1. A protein bound to CpBV-CrV1 was co-precipitated and identified to be glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by LC-MS/MS analysis. RNA interference (RNAi) specific to GAPDH of P. xylostella was found to be able to inhibit the hemocyte-spreading behavior, while RNAi treatments with other glycolytic genes had no effect on the spreading behavior. An addition of recombinant CpBV-CrV1 on hemocyte monolayer interrupted the association between GAPDH and α-tubulin in the cytoplasm. Overlay of mutant proteins (Y492A or Y501A with tyrosine to alanine at putative GAPDH-binding site) of CpBV-CrV1 on hemocyte monolayer revealed that they could enter hemocytes unlike a mutant in the N-terminal coiled-coil domain. However, they failed to inhibit the hemocyte-spreading behavior without any binding affinity to GAPDH. These results suggest that GAPDH plays a critical role in hemocyte-spreading behavior during immune challenge as a molecular target of viral factor CpBV-CrV1.