Molecular cloning and functional study of calreticulin from a lepidopteran pest, Pieris rapae

Insects have an effective innate immune system to protect themselves from exogenous invaders. Calreticulin is a multifunctional protein mainly involved in directing proper conformation of proteins, controlling calcium level, and participating in immune responses. Previous suppression subtractive hybridization assay showed that the expression of Pieris rapae calreticulin (PrCRT) was suppressed after injection of Pteromalus puparum venom. In this study, we obtained a full length cDNA of PrCRT and expressed recombinant wild type and the N-domain deleted mutant PrCRT in bacteria. Real time quantitative PCR and western blot analyses showed that PrCRT mRNA and protein were expressed in hemocytes, Malpighian tubule, midgut, epidermis and fat body, with a higher level in hemocytes. PrCRT was probably located in endoplasmic reticulum distributing in the cytoplasm of hemocytes. Recombinant PrCRT was first able to attach and then enter the hemocytes by endocytosis. PrCRT mRNA in hemocytes was significantly induced after injection of yeast or beads, but did not change noticeably after injection of Escherichia coli or Micrococcus lysodeikticus. Recombinant PrCRT enhanced cellular encapsulation by P. rapae hemocytes in vitro, and the N-domain of PrCRT was required for encapsulation. RNAi of PrCRT by dsRNA injection impaired the ability of hemocytes to encapsulate beads. After parasitization by P. puparum, PrCRT mRNA and protein levels in P. rapae pupal hemocytes were significantly suppressed compared to non-parasitized control. Our results suggest that PrCRT is involved in cellular encapsulation and the pupal parasitoid P. puparum can decrease PrCRT expression to impair host cellular immune response.