Novel insights into host-pathogen interactions of large yellow croakers(Larimichthys crocea)and pathogenic bacterium Pseudomonas plecoglossicida using time-resolved dual RNA-seq of infected spleens

Host-pathogen interactions are highly complex,involving large dynamic changes in gene expression during infection. These interactions are fundamental to understanding anti-infection immunity of hosts, as well as the pathogenesis of pathogens. For bacterial pathogens interacting with animal hosts, timeresolved dual RNA-seq of infected tissue is difficult to perform due to low pathogen load in infected tissue. In this study, an acute infection model of Larimichthys crocea infected by Pseudomonas plecoglossicida was established. The spleens of infected fish exhibited typical symptoms, with a maximum bacterial load at two days post-injection(dpi). Time-resolved dual RNA-seq of infected spleens was successfully applied to study hostpathogen interactions between L. crocea and P.plecoglossicida. The spleens of infected L. crocea were subjected to dual RNA-seq, and transcriptome data were compared with those of noninfected spleens or in vitro cultured bacteria. Results showed that pathogen-host interactions were highly dynamically regulated, with corresponding fluctuations in host and pathogen transcriptomes during infection. The expression levels of many immunogenes involved in cytokine-cytokine receptor,Toll-like receptor signaling, and other immunerelated pathways were significantly up-regulated during the infection period. Furthermore, metabolic processes and the use of oxygen in L. crocea were strongly affected by P. plecoglossicida infection. The WGCNA results showed that the metabolic process was strongly related to the entire immune process.For P. plecoglossicida, the expression levels of motility-related genes and flagellum assemblyrelated genes were significantly up-regulated. The results of this study may help to elucidate the interactions between L. crocea and P.plecoglossicida.

Dual RNA-seq provides novel insight into the roles of dks A from Pseudomonas plecoglossicida in pathogenhost interactions with large yellow croakers(Larimichthys crocea)

Pseudomonas plecoglossicida is a rod-shaped,gram-negative bacterium with flagella.It causes visceral white spot disease and high mortality in Larimichthys crocea during culture,resulting in serious economic loss.Analysis of transcriptome and quantitative real-time polymerase chain reaction(PCR)data showed that dks A gene expression was significantly up-regulated after 48 h of infection with Epinephelus coioides(log2 FC=3.12,P<0.001).RNAi of five sh RNAs significantly reduced the expression of dks A in P.plecoglossicida,and the optimal silencing efficiency was 96.23%.Compared with wild-type strains,the symptoms of visceral white spot disease in L.crocea infected with RNAi strains were reduced,with time of death delayed by 48 h and mortality reduced by 25%.The dks A silencing led to a substantial down-regulation in cellular component-,flagellum-,and ribosome assembly-related genes in P.plecoglossicida,and the significant up-regulation of fli C may be a way in which virulence is maintained in P.plecoglossicida.The GO and KEGG results showed that RNAi strain infection in L.crocea led to the down-regulation of inflammatory factor genes in immune-related pathways,which were associated with multiple immune response processes.Results also showed that dks A was a virulence gene in P.plecoglossicida.Compared with the wild-type strains,RNAi strain infection induced a weaker immune response in L.crocea.