Eriocheir sinensis PI3K-mediated 14-3-3ζ phosphorylation at a conserved tyrosine residue enhances ROS production to defend against Spiroplasma eriocheiris infection

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. First, we employed proteomic analysis combined with dsRNA-mediated interference to preliminarily investigate the role of E. sinensis 14-3-3ζ (Es14-3-3ζ) 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 Es14-3-3ζ. 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 Es14-3-3ζ, and co-immunoprecipitation (Co-IP) as well as cellular co-localization experiments were further employed to confirm these protein-protein interactions. Finally, the intracellular reactive oxygen species (ROS) levels following phosphorylation modification and miRNA-mediated silencing were measured using an ROS detection kit. Previous proteomic analysis revealed Es14-3-3ζ was significantly reduced during intracellular pathogen S. eriocheiris infection. RNAi of Es14-3-3ζ enhanced the S. eriocheiris infection and death of E. sinensis. The phosphorylation level of Es14-3-3ζ at the conserved tyrosine residue (Tyr129) was significantly reduced during the early stage of S. eriocheiris infection. The Es14-3-3ζ phosphorylation at Tyr129 significantly enhanced cell viability, reduced apoptosis, and decreased S. eriocheiris load in Drosophila S2 cells. PI3K, Thioredoxin (TRX1), and catalase (CAT) were identified as interacting partners of Es14-3-3ζ. Es14-3-3ζ phosphorylation promoted protein-protein interactions. Overexpression of EsPI3K led to a significant increase in Es14-3-3ζ phosphorylation, whereas RNAi of EsPI3K resulted in reduced phosphorylation. Furthermore, Es14-3-3ζ phosphorylation significantly decreased TRX1 and CAT activity to elevate the ROS levels. Finally, miR-2309 was identified as targeting Es14-3-3ζ. EsPI3K could phosphorylate Es14-3-3ζ, and enhance the interaction between Es14-3-3ζ and antioxidant proteins, leading to the inactivation of antioxidant proteins and positively regulating ROS levels, thereby resisting S. eriocheiris infection. This study is the first to identify a conserved 14-3-3ζ phosphorylation site and define its role in immunity against intracellular infection.