Abstract
The 14-3-3 proteins interact with a wide variety of cellular proteins for many diverse functions in biological processes. In this study, a yeast two-hybrid assay revealed that two 14-3-3ε isoforms (14-3-3ES and 14-3-3EL) interacted with Rab11 in the white shrimp Litopenaeus vannamei (LvRab11). The interaction of 14-3-3ε and LvRab11 was confirmed by a GST pull-down assay. The LvRab11 open reading frame was 645 bp long, encoding a protein of 214 amino acids. Possible complexes of 14-3-3ε isoforms and LvRab11 were elucidated by in silico analysis, in which LvRab11 showed a better binding energy score with 14-3-3EL than with 14-3-3ES. In shrimp challenged with the white spot syndrome virus (WSSV), the mRNA expression levels of LvRab11 and 14-3-3ε were significantly upregulated at 48 h after challenge. To determine whether LvRab11 and binding between 14-3-3ε and LvRab11 are active against WSSV infection, an in vivo neutralization assay and RNA interference were performed. The results of in vivo neutralization showed that LvRab11 and complexes of 14-3-3ε/LvRab11 delayed mortality in shrimp challenged with WSSV. Interestingly, in the RNAi experiments, the silencing effect of LvRab11 in WSSV-infected shrimp resulted in decreased ie-1 mRNA expression and WSSV copy number. Whereas suppression of complex 14-3-3ε/LvRab11 increased WSSV replication. This study has suggested two functions of LvRab11 in shrimp innate immunity; (1) at the early stage of WSSV infection, LvRab11 might play an important role in WSSV infection processes and (2) at the late stage of infection, the 14-3-3ε/LvRab11 interaction acquires functions that are involved in immune response against WSSV invasion.
Highlights
The 14-3-3 protein family is highly conserved and expressed in all eukaryotic organisms. 14-3-3 proteins are known for their ability to bind multiple cellular p roteins[1,2]
Rab11-mediated trafficking events were reported to have a role in several aspects of innate immunity[8], in Macrobrachium rosenbergii, MrRab11A was involved in antibacterial and anti-white spot syndrome virus (WSSV) innate immunity in prawns[9]
We confirmed the binding of LvRab[11] to 14-3-3ε with in vitro Glutathione S‐Transferase (GST) pull-down assays, performed using purified GST-14-3-3ES, GST-14-3-3EL and His-LvRab[11] recombinant proteins
Summary
The 14-3-3 protein family is highly conserved and expressed in all eukaryotic organisms. 14-3-3 proteins are known for their ability to bind multiple cellular p roteins[1,2]. In Drosophila melanogaster, 14-3-3ε is required for the Rab11-positive vesicle function, which in turn enables antimicrobial peptide secretion during an innate immune r esponse. Of the large number of Rab proteins, 11 isoforms are known to be involved in the immune response endocytic pathway, namely, Rab3a, Rab4a, Rab4b, Rab5a, Rab5b, Rab5c, Rab7a, Rab9a, Rab9b, Rab11a and Rab11b7. Rab[11] mediates several cellular processes that involve intracellular vesicle trafficking, including the Scientific Reports | (2021) 11:19188. Rab11-mediated trafficking events were reported to have a role in several aspects of innate immunity[8], in Macrobrachium rosenbergii, MrRab11A was involved in antibacterial and anti-WSSV innate immunity in prawns[9]. Molecular biological experiments were used to detail the characteristics and innate immune response function of LvRab[11] in L. vannamei during viral infection
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