Molecular Mechanism Of Nucleoplasmic Transport Of Yki Protein Driven By Bacterial Infection In The Chinese Mitten Crab Eriocheir Sinensis

The Chinese mitten crab(Eriocheir sinensis)is an important freshwater crab for economic breeding in China.With the flourishing of the crab farming industry,the crab has also faced a great risk of disease occurrence in recent years.Therefore,an in-depth study on the immune mechanism of this crab will not only provide an important theoretical basis for disease prevention and control in the artificial culture of this crab,but also effectively promote the development of a green,healthy and sustainable aquaculture industry in China.Invertebrates lack an acquired immune system,so their defense against pathogenic microorganisms relies mainly on their strong innate immune system.The Hippo signaling pathway and its core effector,Yap/TAZ/Yki,have played an important role in the regulation of innate immune signaling pathways.In this study,the nucleic acid sequence of EsYki was obtained by gene cloning in E.sinensis,and after correlation bioinformatics analysis,EsYki was found to contain three major structural domains,namely,the NES region at the N-terminal end,the NLS region at the C-terminal end,and two WW structural domains located in the middle of the two.Subsequent expression pattern analysis revealed that EsYki had the highest expression in the hepatopancreas and the lowest expression in the hemocytes.Under bacterial stimulation,EsYki did not change significantly at either the m RNA or protein level in the hemocytes of the crab,while immunofluorescence results showed that EsYki protein was transferred from the nucleus to the cytoplasm upon bacterial stimulation,and this occurred with the assistance of a lepromycin B-sensitive transporter protein.Subsequently,in order to investigate the molecular mechanism of nucleoplasmic transfer of EsYki,we focused on the transporter protein Es CRM1 and Es Sd,which is homologous to TEAD protein.Bioinformatics analysis revealed that Es CRM1 contains three structural domains,namely,the IBN-N structural domain located at the N terminus,the CRM1-C structural domain located at the C terminus,and the Xpo I structural domain located in the middle;while Es Sd contains two structural domains,namely,the TEA/ATTS structural domain located at the N terminus and the YBD structural domain located at the YBD.Analysis of the expression patterns of both genes after bacterial stimulation showed that they were both up-regulated in response to bacterial stimulation,while knockdown of Es CRM1 combined with nucleoplasmic isolation and immunofluorescence analysis confirmed that Es CRM1 mediated the nucleoplasmic translocation of EsYki and regulated the expression of the organism’s antimicrobial peptide,and that knockdown of Es CRM1 significantly reduced the organism’s ability to clear bacteria and increased mortality.In contrast,Es Sd interference only slightly increased EsYki protein translocation from the nucleus to the cytoplasm,but was not significantly different from the control.It was further confirmed by immunoprecipitation that EsYki protein can interact with Es CRM1 or Es Sd,and a conserved phosphorylation site S26 was found in the amino acid sequence of the NES region of EsYki protein,which can be phosphorylated by a relatively conserved phosphokinase PRP4 k.Immunoprecipitation experiments were performed by constructing phosphorylated mutants of the EsYki S26 site with Es CRM1 or Es Sd,respectively,and showed that phosphorylation of the S26 site directly affected the interaction between EsYki protein and Es CRM1,but had no significant effect on the interaction between EsYki protein and Es Sd.For the analysis of EsPRP4 k expression pattern under bacterial stimulation,the results showed that EsPRP4 k could respond to bacterial stimulation by up-regulating expression,but the nucleoplasmic translocation of EsYki protein was significantly diminished or even disappeared when EsPRP4 k was disturbed by bacterial stimulation of crab hemocytes.These results suggest that when E.sinensis is stimulated by bacteria,EsPRP4 k induces nucleoplasmic translocation by phosphorylating the S26 site of EsYki protein and binding it to Es CRM1 transporter protein,which in turn deregulates the inhibition of dorsal by Cact and allows it to enter the nucleus to regulate the expression of antimicrobial peptide-related genes.In summary,this paper reveals the nucleoplasmic translocation of EsYki,a core component of the Hippo signaling pathway in E.sinensis,in response to bacterial stimulation and its molecular mechanism.This not only deepens the understanding of the regulation of innate immunity by Yap/TAZ/Yki,but also enriches the theory of innate immunity in crustaceans such as shrimp and crab.