The Function Of SigX And FlgM Of Pseudomonasplecoglossicida When Infected Epinephelus Coioides

Pseudomonas plecoglossicida is a kind of Gram-negative aerobic rob-shaped bacteria.The bacteria can lead to ‘visceral white spot disease’ of large yellow croaker,Epihephelus coioides and other economic marine fish,resulting in a large number of fish deaths in a short period of time,causing serious economic losses.Even though P.plecoglossicida causes great harm to mariculture,the research on its pathogenic mechanism is still in the exploratory stage.In previous study,transcriptome data of P.plecoglossicida were obtained about pathogenic temperature and process.In the present study,the NZBD9 strain of highly pathogenic P.plecoglossicida,which was isolated and identified in the previous study,was used as the bacteria object,Epinephelus coioides were used as animal model.Integrating RNAi and RNA-seq to reveal the function of sigX and flgM of P.plecoglossicida when infected E.coioides.The main research contents are as follows:(1)Five sigX-RNAi strains or flgM-RNAi strains of P.plecoglossicida were constructed by RNAi.qRT-PCR was used to determine gene expression,the result showed that sigX-shRNA-216 strain and flgM-shRNA-18 strain exhibited the best efficiency with 99.27% or 99.72% respectively,and named them as sigX-RNAi strain or flgM-RNAi strain of P.plecoglossicida for following study.(2)The infection assay was performed between E.coioides and sigX-RNAi strain or flgM-RNAi strain of P.plecoglossicida.The results indicated that compared with infection with wild-type strain,the infection of sigX-RNAi strain or flgM-RNAi strain resulted in 20% or 75% reduction in mortality on E.coioides respectively,as well as alleviates in the symptoms of E.coioides spleen.The diameter and number of white spots on the surface of spleens infected by sigX-RNAi strain decreased,while there were no obvious white spots on the surface of spleens infected by flgM-RNAi strain.These results indicated that sigX and flgM were virulence-related genes to P.plecoglossicida,and played important roles when infected E.coioides.(3)Taking advantage of the growth assay and spatial and temporal distribution assay,it was found that,compared sigX-RNAi strain or flgM-RNAi strain to wild-type stain of P.plecoglossicida,the growth rate had no significant difference in vitro,however the abundance was reduced significantly post infection.(4)1,2,3,4 days post infection,compared to wild-type strain of P.plecoglossicida,the expression level of sigX of sigX-RNAi strain or flgM of flgM-RNAi strain were both down-regulated significantly.The result indicated that high stability of sigX or flgM silence throughout the infection process.(5)Compared with wild-type strain,the gene silence of sigX in P.plecoglossicida resulted in a signifcant change in transcriptome of infected E.coioides.The result of GO and KEGG analysis on E.coioides showed that genes of serine-type endopeptidase and chemokine signaling pathway,coagulation and complement system,and intestinal immune network for IgA production pathway were mostly affected by sigX of P.plecoglossicida.Meanwhile,the immune genes were associated with different number of miRNA and lncRNA.(6)Compared with infection with the wild-type strain,infection with the flgM-RNAi strain of P.plecoglossicida resulted in a significant change in the transcriptome of the spleens of infected E.coioides and P.plecoglossicida.KEGG analysis for E.coioides showed that genes of 16 immune pathways were most affected by flgM-RNAi of P.plecoglossicida.WGCNA analysis revealed that the immune pathways were coordinated with cellular community,signal transduction,signaling molecules and interaction,circulatory system,amino acid metabolism and cell motility.