The involvement of Argonaute molecule PIWI in the antiviral immunity of oyster Crassostrea gigas

• Cg PIWI responded to poly (I:C) stimulation in granulocytes of haemocytes. • Cg PIWI and its PAZ domain demonstrated binding activity to ssRNA in vitro. • Cg PIWI and its PIWI domain efficiently cleaved the ssRNA in vitro . The p-element induced wimpy testis (PIWI), as a member of Argonaute superfamily, functions in piRNA pathway of RNAi to achieve target gene silencing. The piRNA pathway plays crucial roles in germline development and regulation of mobile element as well as the antiviral immunity. In the present study, a PIWI homologue ( Cg PIWI) was identified in Crassostrea gigas . The predicted Cg PIWI protein consists of a N domain, a PAZ domain, a MID domain and a PIWI domain, and shared identity ranging from 27.04% to 70.37% with other PIWIs. Transcriptome analysis showed that Cg PIWI was highly expressed in embryonic period (from egg to blastula stage) and then gradually decreased with the development process (from blastula to early D-larva stage). The mRNA transcripts of Cg PIWI were detectable in all the examined tissues of adult oysters with the highest expression in haemocytes. After poly (I:C) stimulation, the mRNA expression level of Cg PIWI in haemocytes were significantly increased, particularly in granulocytes of haemocytes. Immunocytochemistry assay revealed that Cg PIWI proteins were mainly distributed in the cytoplasm of haemocytes. The recombinant proteins of full-length Cg PIWI (r Cg PIWI), and its PAZ domain (r Cg PIWI-PAZ) and PIWI domain (r Cg PIWI-PIWI), were obtained by prokaryotic expression, respectively. Both r Cg PIWI and r Cg PIWI-PAZ demonstrated significant binding activity to ssRNA in vitro , while no binding activity to dsRNA. Additionally, r Cg PIWI and r Cg PIWI-PIWI exhibited substantial RNase activity, efficiently cleaving ssRNA in vitro . These results indicated that Cg PIWI functions as an effector of RNAi-induced silencing and plays a role in the antiviral immune response of oyster.