Cooperation of R and Rab5 regulates crayfish anti-disease immune response by triggering apoptosis.

Toll-like receptors (TLRs) are involved in innate immunity in aquatic animals. The comprehensive regulation characteristic of TLRs in the immune of crayfish (Procambarus clarkii) has been less elucidated. In this study, we investigated the regulatory pathways of TLRs in the crayfish by identifying the proteins interacting with TLRs encoded by the resistance (R)-gene identified in our previous study. In vivo pull-down analysis indicated an interaction between the R protein and myeloid differentiation factor 88 (MyD88) and the Ras-like small GTPase Rab5. In vitro pull-down assays verified that R directly interacted with MyD88, but not with Rab5. Many differentially expressed genes involved in the Toll signaling pathway were identified using transcriptomes analysis of RNAi-Rab5 and RNAi-GFP crayfish hemocytes. Tumor susceptibility gene 101 and CD9 (encoding a tetraspanin protein) related to exosomes were identified, and their protein expression was validated using western blotting. We hypothesize that the R protein receives a signal upon pathogen challenge and triggers apoptosis during immune responses by interacting with MyD88, with the cooperation of Rab5-secreting exosomes. We anticipate this study to provide preliminary evidence for the involvement of exosomes in the TLR-mediated immune regulatory pathway and advance the understanding of this pathway in crayfish immune resistance.