The Function Of RK21_RS10315 Virulence Gene In The Process Of Pseudomonas Plecoglossicida Infection In Epinephelus Coioides

Pseudomonas plecoglossicida is a widely distributed Gram-negative bacterium and can cause serious economic losses to the aquaculture industry.However,the study on the pathogenesis of Pseudomonas plecoglossicida is still in its infancy,and the proven virulence genes are also few.This study based on RNA interference and dual RNA-seq technology revealed the function of a transcriptional regulator gene(RK21_RS10315 gene)in the process of pathogen-host interaction,using Pseudomonas plecoglossicida as a pathogen and Epinephelus coioides as the host.Firstly,the RK21_RS10315 gene which was highly expressed was selected by comparing the difference in transcriptome data of Pseudomonas plecoglossicida(Pseudomonas plecoglossicida cultivated in vivo vs in the spleen of Epinephelus coioides 24 h,48h,72 h,and 96 h after infection by Pseudomonas plecoglossicida).Five shRNAs were designed to silence RK21_RS10315 gene,and the silencing strains were constructed using pCM130/tac plasmids,respectively.The silencing efficiency was detected by qRT-PCR(real-time fluorescent quantitative PCR).The results showed that the silencing effect of the shRNA-574 was the best,and the silencing efficiency reached 96.1%.When Epinephelus coioides was infected by the silent strain and the wild-type strain of Pseudomonas plecoglossicida respectively,it was found that the fish infected by wild-type strain had no obvious characteristics on the surface and the surface of the spleen had with different sizes dense white nodules,while the fish infected by RK21_RS10315-RNAi strain resulted in the death time delay and 90% reduction in mortality of Epinephelus coioides.The alleviation in the symptoms of Epinephelus coioides spleen,which confirmed the pathogenicity of the RK21_RS10315 virulence candidate gene.Dual RNA-seq were conducted with Epinephelus coioides' s spleen,which the samples were collected 48 h post infected by the wild type strain and RK21_RS10315-RNAi strain,respectively.The function of RK21_RS10315 gene in pathogen-host interaction between Pseudomonas plecoglossicida and Epinephelus coioides was analyzed by comparison of two sets of data.The infected spleen contains two parts of transcriptome data,including pathogen data of Pseudomonas plecoglossicida and host data of Epinephelus coioides.22010 differential genes were found in the transcriptome data of Epinephelus coioides.Among them,there were 4321 differentially up-regulated,17689 differentially down-regulated.Meanwhile,65 differentially expressed genes were found in the transcriptome data of Pseudomonas plecoglossicida.Among them,there were 50 differentially up-regulated,15 differentially down-regulated.The differentially expressed genes in the transcriptome data of Epinephelus coioides were observed by KEGG enrichment,and 16 immunologically significant pathways were found.The two enrichment pathways of ko04672 and ko04670 accounted for a certain proportion.Then,19 differential genes(euk-DEMs)in the two enrichment pathways of ko04672 and ko04670 were co-expressed with 19 differential genes(pro-DEMs)in the transcriptome data of Pseudomonas plecoglossicida.There were varying degrees of correlation between them,and it has also been found that differential genes such as ipxA,grpE,yhbJ,truD and suhB from the Pseudomonas plecoglossicida transcriptome data occupy an important position in the co-expression network.These expression may be likely to be associated with silencing of the RK21_RS10315 gene in Pseudomonas plecoglossicida.In summary,the RK21_RS10315 gene affects the pathogenicity of Pseudomonas plecoglossicida,and the dual RNA-seq simultaneously detects the spleen of Epinephelus coioides,which the samples are collected 48 h post infected by the wild type strain and RK21_RS10315-RNAi strain,respectively.Further bioinformatics analysis of the transcriptome data revealed a correlation between the differential genes of the pathogen-host.The pathogen-host interaction after gene silencing confirmed the close association with the RK21_RS10315 gene in Pseudomonas plecoglossicida.