Toll-receptor 9 gene in the black tiger shrimp (Penaeus monodon) induced the activation of the TLR NF-κB signaling pathway

Toll receptors are important pathogen recognition receptors (PRRs) in shrimps, which play a vital role in defending against virus and bacterial challenge. In this paper, the characterization and functional analysis of a Toll9 receptor gene from Penaeus monodon was performed in HEK293T cells. Data showed that PmToll9 can activate the NF-kappa B promoter activities of TLR pathway, while ISRE and IFN-beta promoter cannot be activated obviously in HEK293T cells using dual-luciferase reporter system. The downstream immune factors of IL-8, I kappa Ba, and TRAF6 were activated by PmToll9 and IL-8 showed the most significant up-regulation in expression levels, indicating the activities of NF-KB can be mediated by PmToll9. Six LRRs-deletion mutants were constructed and results showed these mutants had obvious declines in luciferase activities, among which the mutant pCMV-DeLRR4 showed the most significant decline. qPCR data indicated LRRs-deletion mutants efficiently impaired the activities of the downstream immune factors IL-8, I kappa B-alpha, and TRAF6. It demonstrates that LRRs-deletion mutants could result in the weaken abilities of PmToll9 in signaling transduction. Overexpression of PmToll9-GFP fusion protein in Hela cells revealed the primary cellular localization of PmToll9 is in the cytoplasm.