Studies On The Interaction Between Larimichthys Crocea And Pseudomonas Plecoglossicida Based On Dual RNA-seq

Large yellow croaker(Larimichthys crocea)is an economical important farmed fish in China.“Visceral White Spot Disease” caused by Pseudomonas plecoglossicida is a disease with a high mortality rate in cage-cultured L.crocea in recent years and resulted in heavy economy lossess every year.Host-pathogen interactions are highly complex,involving large dynamic changes in gene expression during infection.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.At present,the research on P.plecoglossicida is still in its infancy,and there is no report on the study of the complete infection process of P.plecoglossicida-L.crocea based on time-resolved dual RNA-seq.In view of this,this article used dual RNA-seq technology to research on the interaction between L.crocea and P.plecoglossicida,the main contents were as follows:An acute infection model of L.crocea infected by P.plecoglossicida was established,and used dual RNA-seq technology to simultaneously monitor their transcriptome changes during infection.The spleens of infected fish exhibited typical symptoms,with a maximum bacterial load at 2 days post-injection(dpi).Time-resolved dual RNA-seq of infected spleens was successfully applied to study host-pathogen interactions between L.crocea and P.plecoglossicida.The spleens of infected L.crocea were subjected to dual RNA-seq,and were compared with those of spleens of L.crocea injected with PBS or in vitro cultured bacteria.Results showed that the interaction between the pathogen and the host was highly dynamically regulated,with corresponding fluctuations in host and pathogen transcriptomes during infection.For L.crocea,many immunogenes related pathways such as cytokine-cytokine receptor interaction,Toll-like receptor signaling,were significantly up-regulated during infection.In addition,P.plecoglossicida infection seriously affects the metabolic process and the use of oxygen in fish,which is reflected in the whole infection process.For P.plecoglossicida,the expression levels of genes related to motility and flagella assembly pathway were significantly up-regulated,indicating that motility plays an important role in the infection of P.plecoglossicida.WGCNA analysis showed that the metabolic process of pathogen was strongly related to the host immune process.Based on time-resolved dual RNA-seq,we preliminarily analysed the specific performance of the interaction between L.crocea and P.plecoglossicida at the transcriptome level during the entire infection stage,and unearthed potential interaction function genes.Then we selected the P.plecoglossicida gene clpV whose expression level was significantly up-regulated during the infection process by time-resolved dual RNA-seq data analysis.Five shRNAs were designed and synthesized to silence this gene in P.plecoglossicida,with a maximum silencing efficiency of 96.1%.Compared with the wild-type strain,the onset time of clpV-RNAi strain infected L.crocea was delayed for 2 days,a 25% reduction in mortality and the white spots on the surface of the spleen were also significantly reduced.Dual RNA-seq of infected spleens at 2 dpi which has the highest bacterial load was subjected,compared with the wild-strain group,the results of L.crocea infected by clpV-RNAi strain changed considerably.The result of KEGG enrichment showed that cytokine-cytokine receptor interaction,Toll-like receptor signaling pathway,C-type lectin receptor signaling pathway and MAPK signaling pathway of L.crocea were most affected by the clpV gene in P.plecoglossicida.RNAi of clpV resulted in the downregulation of genes in flagella assembly pathway and a weaker immune response of host.In summary,time-resolved dual RNA-seq of infected spleens was applied to study host-pathogen interactions between L.crocea and P.plecoglossicida.The analysis showed that the anti-infection ability of L.crocea was closely related to metabolism,and the motility is an important part of P.plecoglossicida of infection ability.Further combined with RNA interference technology,analyzed the effect of the P.plecoglossicida gene clpV on pathogen virulence.The results of this paper may help to deepen the knowledge on interactions between L.crocea and P.plecoglossicida and provide a foundation for further study in the related fields.