The Function Of SecY And DksA Genes In The Interaction Between Aquatic Pathogenic Pseudomonas Plecoglossicida And Larimichthys Crocea

Larimichthys crocea is the largest marine fish produced in China,and Pseudomonas plecoglossicida is the causative agent of visceral white spot disease in cultured L.crocea.In the early stage of this laboratory,the pathogen P.plecoglossicida NZBD9 was isolated and identified.The transcriptome data analysis showed that the secY and dksA genes of P.plecoglossicida were significantly expressed during infection.This article discusses the function of secY and dksA genes in the interaction between crocea by combining RNA interference and dual RNA-seq technology.First design and synthesize 5 pairs of shRNA for the sequence of secY and dksA genes respectively for the construction of silenced strains,Then the qRT-PCR method was used to select the mutants with the best silencing effect of the two genes,and the virulence of the pathogen was determined by intraperitoneal injection of healthy L.crocea at a dose of 10~4 cfu/g fish body weight.Finally,the spleen of the L.crocea infected with P.plecoglossicida 48 hours after infection was subjected to dual RNA-seq,and compared with the dual RNA-seq data of the spleens of L.crocea infected with wild strains for 48 hours.The main results are as follows:(1)The shRNA designed and synthesized in this paper can reduce the expression of secY gene and dksA gene in varying degrees,among which secY-shRNA-1165 has the best silencing effect,with the silencing efficiency of 82.4%;dksA-shRNA-87 has the best silencing effect,with the silencing efficiency of 96.23%.(2)The results of artificial infection of large yellow croaker showed that compared with the wild type strain injection group,the death time of L.crocea in the RNAi strain injection group was delayed,the mortality rate of the secY RNAi strain injection group was significantly reduced by 35%,and the death rate of the dksA RNAi strain injection group at 25%,the spleen of the RNAi strain injection group showed only a few slight white nodules,the density was significantly reduced.After the same dose of infection,the bacterial load of the secY RNAi strain in the spleen,liver,kidney and blood of the L.crocea was significantly lower than that of the wild strain in the early stage of infection.It was significantly lower than the wild strain,and the relative bacterial load of the secY and dksA RNAi strains increased rapidly with the progress of infection.These results indicate that secY and dksA can participate in and affect the virulence regulation of P.plecoglossicida and the process of infection.(3)Comparative analysis of the dual RNA-seq data of spleen of L.crocea after 48 hours of infection with different strains of P.plecoglossicida found that:The silence of the secY gene affects the core related gene secD of the translocation device of the“sec secretion system”of P.plecoglossicida,which may affect the bacterial protein output and reduce the strain’s virulence.Silencing of the dksA gene significantly down-regulates the cellular components of the pathogen,flagella and ribosome assembly-related genes,thereby reducing the virulence of P.plecoglossicida;the fliC gene is significantly up-regulated during infection,which may be P.plecoglossicida a way to maintain bacterial virulence.(4)The results of GO and KEGG enrichment analysis showed that multiple immune-related pathways of L.crocea infected with secY and dksA RNAi strains were affected.Among them,the Toll-like receptor signaling pathway,complement and coagulation cascade of the secY RNAi strain infection group were most significantly affected;Toll-like receptor signaling pathway,TNF signaling pathway,hematopoietic cell lineage,and interaction of cytokines and cytokine receptors in the dksA RNAi infection group significantly affected the four KEGG pathways.In summary,secY and dksA are the virulence-related genes of P.plecoglossicida.Silencing of these two genes can significantly reduce the virulence of P.plecoglossicida against L.crocea.The results of dual RNA-seq found that the two genes were different in the affected genes of P.plecoglossicida after silencing,and the response of spleen immune related genes of L.crocea to the infection of these two gene silenced strains is also very different.In this paper,the function of SecY and dksA genes in pathogen host interaction was studied by dual RNA-seq.The results are helpful to improve the understanding of pathogenicity of P.plecoglossicida and provide a reference for the study of pathogenicity mechanism of aquatic pathogenic bacteria.