| Pseudosciaena crocea(P.crocea)is one of the most economically important maricultured fish species in southeast China.In recent years,P.crocea aquaculture industry has been limited by diseases.“White spots disease” is the most common disease outbreaks in P.crocea in intensive cage cultured zones,which causes serious economic losses.Nowdays,Pseudomonas plecoglossicida is found to be the pathogenic bacterium which leads to “white spots disease”.P.plecoglossicida also infected a variety of economic animals besides P.crocea,and caused financial losses.“White spots disease” is water temperature dependent,according to published reports and previous research in our lab.Our lab used bioinformatics and transcriptome high-throughput sequencing technology in previous studies to compare the gene expression pattern of P.plecoglossicida cultured in 12 ℃,18 ℃ and 28 ℃,which showed that htpB’s gene expression level at virulent temperature(18 ℃)was apparently higher than avirulent temperature(12 ℃ and 28 ℃).That means htpB may be related to the pathogenicity of P.plecoglossicida,and change of temperature regulates htpB’s gene expression might be the major inducement.Hence,we used the P.plecoglossicida strain named NZBD9 to explore the functions and regulatory mechanism about htpB in the process of infection.The main contents of this study are listed as follows:(1)Five htpB-RNAi strains were constructed,and qRT-PCR was used to detect htpB’s gene expression level,in order to pick out the best silenced strain for further research.(2)Artificial infection revealed that mortality was remarkably lower in the group treated with htpB-RNAi strains compared to the wild-type strain.Meanwhile,the abundances of htpB-RNAi strain in the spleen,liver,head kidney,trunk kidney,and blood were significantly reduced compared with the wild-type P.plecoglossicida at most of the time points after injection.These indicated that htpB is close related to the virulence of P.plecoglossicida.(3)The spleen is the main lesion in fish of disease caused by P.plecoglossicida.qRT-PCR was used to detect htpB’s gene expression level changes over time,which showed that the highest gene expression level of htpB was in spleen at 2 d post-infection by wild-type strain.So we choose the spleens uninfected and spleens infected by htpB-RNAi strain or wild-type strain at 2 d for dual host-pathogen interaction sequencing.After htpB was silenced,throughRNA-seq we found that various virulence genes of P.plecoglossicida was reduced,such as flgD,flgE,fliG,rpsE and rpsS,which are closely related to the ability to form biofilms and move,besides the formation of ribosomes in bacteria.These indicated that htpB played a key role through regulating other genes in the pathogenic process of P.plecoglossicida.Comepared with wild-type strain infected fish spleen,genes expression in htpB-RNAi strain infected fish spleen was significantly changed.From this,we deduced the htpB-dependent virulent mechanism,htpB-dependent defense mechanism,and htpB-independent defense mechanism.By constructing the pathogen-host integration network,we could found that infection induce the expression of host genes related to immune response,while infection reduce the expression of bacterial virulence genes.Close integration was found between host immune genes and bacterial virulence genes,while IL6,IL1R2,IL1 B and TLR5 played key role in the network.(4)By GeneMANIA analysis,we could found flgC and rpsE might play key role during the htpB-dependent virulence regulation,which was accordance with the reduced biofilm production and motibility in htpB-RNAi strain.Meanwhile,IL1R2 was found to play key role during the fefense of P.plecoglossicida.Further research would be helpful to validate this.(5)With bioinformatics analysis,microbiological and molecular biological techniques,the present study proved that dual RNA-Seq of pathogen and host is illuminating and with high accuracy,which would be a powerful tool for the aquaculture diseases research in the future. |
PDF Full Text Request