Integration of RNAi and RNA-seq uncovers the immune responses of Epinephelus coioides to L321_RS19110 gene of Pseudomonas plecoglossicida.

Pseudomonas plecoglossicida is a threatening and important pathogen in aquaculture and causes heavy losses. Expression of L321_RS19110 was found significant up-regulated at 18 °C than at 28 °C, which was confirmed by quantitative real-time PCR. RNAi significantly reduced the content of L321_RS19110 mRNA of P. plecoglossicida, and exhibited the best efficiency of gene silencing with a reduction of 84.9%. Compared with the wild type strain, the infection of L321_RS19110-RNAi-1 strain resulted in the onset time delay, and 30% reduction in mortality of Epinephelus coioides, as well as alleviates in the symptoms of E. coioides spleen. Moreover, compared with wild type strain, the gene silence of L321_RS19110 in P. plecoglossicida resulted in a significant change in transcriptome of infected E. coioides. The results of KEGG analysis showed that genes of chemokine signaling pathway and cytokine-cytokine receptor interaction, digestion and absorption of proteins (non-specific immune) and antigen processing and presentation pathways were most affected by L321_RS19110-RNAi of P. plecoglossicida. In these immune pathways, the most affected immune genes were associated with different number of non-coding RNAs. Among them, most lncRNAs and miRNAs exerted negative regulation on immune genes. The results indicated that L321_RS19110 was an important virulent gene of P. plecoglossicida, the up-regulation of the immune pathways made E. coioides more likely to remove L321_RS19110-RNAi strain than the wild type strain of P. plecoglossicida, the expression of immune genes were regulated by miRNA and lncRNA in a complex way.