Identification Of Key Virulence Gene In Pseudomonas Plecoglossicida And Epinephelus Coioides Interactions Based On Dual RNA-seq

Pseudomonas plecoglossicida is a Gram-negative bacterium that has been found in both seawater and freshwater,which has been frequently associated with some fish of economic importance and leads to high mortality.However,at present,the research on the pathogenic mechanism of P.plecoglossicida is still in the early stage,and the study of the pathogenesis of P.plecoglossicida has not been reported based on the dynamic interaction transcriptome.In the current study,in order to identify the key virulence gene of pathopoiesis of P.plecoglossicida on Epinephelus coioides,thus enhancing the understanding of the pathogenic mechanism of P.plecoglossicida and host-pathogen interactions,P.plecoglossicida and its infected E.coioides(pathological model)were used as the research objects.The main research contents are as follows:After the genomic DNA was extracted from different organs or tissues of wild type P.plecoglossicida-injected E.coioides at different time points,the distribution of pathogenic bacteria in various organs or tissues of the fish was tested using digital PCR.The results indicated that P.plecoglossicida in E.coioides spread primarily by blood and caused a systemic infection in a very short period of time;meanwhile,the load changes of P.plecoglossicida in most of the tissues decreased briefly in the early stage of infection,then increased gradually and reached the maximum at 4 days post-injection(dpi),and then decreased.At most of the time points after the injection,the load of P.plecoglossicida in the spleen was higher than that in other tissues(more than 200 times at 4 dpi),the results showed that the spleen was the main target organ of E.coioides infected by P.plecoglossicida strain NZBD9,this provided a good guidance for organ sampling in the application of dual RNA-seq technology in this study.After RNA was extracted from the spleen of the P.plecoglossicida-infected E.coioides,the cDNA library was sequenced by dual RNA-seq,and all the original sequencing data were filtered.The high quality sequencing data(clean reads)of Q20 greater than 97% were obtained from different samples.After the clean reads of P.plecoglossicida and E.coioides were used separately for analysis of differentially expressed genes(DEGs),the number of DEGs of the pathogenic bacteria and the host was obtained: compared with the P.plecoglossicida in pure culture,the number of DEGs of P.plecoglossicida at 24,48,72 and 96 hours post-injection(hpi)was 258、 10,72 、808 and 894,respectively;compared with the uninfected E.coioides,the number of DEGs of infected E.coioides at 24,48,72 and 96 hpi was 66,852、77,771、61,369 and73,776,respectively.The number of DEGs of pathogen and host was the most at 48 hpi,and the expression level and quantity distribution of DEGs of pathogen and host showed dynamic significant change in different infection periods.After weighted gene co-expression network analysis(WGCNA)was independently carried out on DEGs of the pathogen and the host,KEGG-enrichment analysis of gene coexpression modules was performed,respectively.Then the main virulence related pathways(‘bacterial secretion system’ and ‘flagellar assembly’)and immune response-related pathways(‘complement and coagulation cascades’,‘natural killer cell mediated cytotoxicity’ and ‘hematopoietic cell lineage’)were significantly enriched.In order to identify the key virulence gene of pathogenicity of P.plecoglossicida against E.coioides,first,DEGs in the virulence and immune related pathways were used for construction of gene regulatory network.It was predicted that the ‘Sec pathway’ and ‘flagellar assembly’pathway have a more important role in the pathogenicity against the host.Then the key virulence gene in the ‘Sec pathway’ and ‘flagellar assembly’ pathway was predicted by WGCNA as secY and fli N,respectively.Dynamic distribution experiments showed that the load of secY and fliN-RNAi strains in the different tissues of host was significantly reduced compared with the P.plecoglossicida strain expressing a non-sense shRNA at most of the time points after injection.The infection experiment of secY and fliN-RNAi strains revealed that E.coioides survival rate infected with the secY-RNAi strain was significantly higher than for the fli N-RNAi strain,indicating that the secY gene may be more important to P.plecoglossicida virulence compared to the fli N gene.In summary,the spleen was the main target organ of pathogenicity of P.plecoglossicida against E.coioides.The dynamics of interaction transcriptome changes of both P.plecoglossicida and host spleen were simultaneously monitored by dual RNA-seq.Further bioinformatics analysis and in vivo experiments showed that secY was the key virulence gene for pathogenicity of P.plecoglossicida against E.coioides.