Abstract

Background Eriocheir sinensis , also known as the river crab or hairy crab, is an economically important freshwater species in China with significant ecological and economic value. As market demand continues to grow, its aquaculture scale has been expanding. According to statistics, the farming production of E. sinensis reached 888,629 tons in 2023. Then the 14-3-3 proteins are recognized to play an important role in pathogen transmission and infection processes. Meanwhile, protein phosphorylation, as one of the most common post-translational modifications, can alter the physicochemical properties of proteins, thereby affecting their spatial structure and regulating physiological and biochemical processes such as neural activity and cell proliferation. Through phosphorylation-dependent interactions, 14-3-3 proteins occupy a central position in signaling pathways, dynamically regulating host growth, development, and stress responses. However, their role during pathogen infection in crabs remains unclear. Methods First, we employed proteomic analysis combined with dsRNA-mediated interference to preliminarily investigate the role of E. sinensis 14-3-3ζ ( Es 14-3-3ζ) in the host immune process. Subsequently, phosphoproteomic analysis along with a phospho-tyrosine specific antibody was utilized to validate the Phosphoinositide-3 kinase (PI3K)-mediated phosphorylation of Es 14-3-3ζ. To explore the functional impact of this phosphorylation modification, a site-directed mutagenesis assay was conducted in the Drosophila S2 cell line. In vitro experiments primarily included assessments of cell viability, apoptosis, and the copy number of Spiroplasma eriocheiris ( S. eriocheiris ). His-pull down assays were performed to screen for proteins interacting with Es 14-3-3ζ, and co-immunoprecipitation (Co-IP) as well as cellular co-localization experiments were further employ