| Bacterial leaf blight(BLB)is a serious disease of rice in China.The BLB pathogen,Xanthomonas oryzae pv.oryzae(Xoo),is a gram-negative bacterium that can substantially reduces rice yields.Bismerthiazol[N,N-methylene-bis(2-amino-5-mercapto-1,3,4-thiadiazole)]is a kind of bactericide which is produced in China and used to control BLB,rice bacterial leaf streak,citrus canker and other diseases.The field-resistance strains were found to be more sensitive than wild-type strain to bismerthiazol in vitro,and the laboratory-resistance strains were found to have lost their pathogenicity in vivo.To further explain the difference between bismerthiazol-sensitive and bismerthiazol-resistance strains,the field wild-type strain ZJ173 was used to induce several bismerthiazol-resistance strains by rice leaves treated with high concentration bismerthiazol repeatedly.The resistant strains 1-1-1,2-1-1,4-1-1 and the sensitive stain ZJ173 were then stored in our laboratory as the model variables of this research.There were multiple point mutations in the genome sequence of 2-1-1.In 2-1-1,the 959th base of Xoo3738(hypothetical protein gene)was mutated from A to G which causing the 320th amino acid changed from tyrosine to cysteine(Y320C)and the 319th base of Xoo3640(TonB-dependent outer membrane receptor protein gene)was changed from C to T which causing the 107th amino acid changed from proline to arginine(P107R).Then,Xoo3738 and Xoo3640 of 2-1-1 were replaced to ZJ173 to construct point mutant strains,however,the point mutant strains did not show the resistance to bismerthiazol in vivo.Besides gene point mutations,the resistance of strains may also be caused by the changes of gene expression levels.Our laboratory has measured the transcriptome differences between 2-1-1 and ZJ173 and the transcriptome differences between ZJ173-treated and untreated bismerthiazol in vitro,we found that the gene expression levels of ClpP and its subunits ClpA,ClpB,and ClpX,which code the ClpP enzyme and its subunits and regulate bacterial response by the environment,were different in the sensitive strain and resistant strain.RT-qPCR was used in this research to detect the gene expression of ClpP and its subunits gene.Results showed that the expressions of ClpP genes were higher in the bismerthiazol resistance strain(1-1-1,2-1-1,and 4-1-1)than that in their parental susceptible WT strain(ZJ173).Under the treatment of bismerthiazole,the expression of ClpP genes increased in early growth phase and decreased in middle growth phase of ZJ173,but not of the resistant strains.To improve that the resistance of bismerthiazol-resistant strains was related to the higher expression of ClpP and its subunits,the ClpP and its subunits genes were overexpressed in ZJ173 and 2-1-1 respectively.The results showed that all the overexpression mutants of ZJ173 and 2-1-1 were more sensitive to bismerthiazol in vitro and more resistant to bismerthiazol in vivo than ZJ173 and 2-1-1.But the resistance level was lower for the overexpression mutants of ZJ173 than for 2-1-1 in vivo.The results therefore demonstrated that the resistance of Xoo to bismerthiazol did not depend solely on the gene expression of ClpP and its subunits genes.Measuring the influence of ClpP and its subunits gene expression levels on the biological phenotype of Xoo,it was found that overexpression of ClpP and its subunits genes could reduce the inhibition of the EPS production,mobility,and biofilm by bismerthiazol.To study the gene function of ClpP and its subunits in Xoo,we compared various biological phenotypes of ClpA,ClpB,ClpP,ClpX knockout mutants and ZJ173.The results showed that ClpP and its subunits genes positively regulated virulence,growth,biofilm formation,motility,and EPS production of Xoo,and negatively regulated the rice defense response.To further study the difference between Xoo sensitive and resistant strains by bismerthiazol in vivo,we analyzed the transcription of ZJ173 and 2-1-1 which inoculated to rice leaves with and without bismerthiazol.The results of RT-qPCR verification showed that bismerthiazol could inhibit the expression level of type VI secretion system T6SS-1 related genes of sensitive strain in vivo,but not of resistant strains;When treated rice with streptomycin,which has no cross-resistance with bismerthiazol,streptomycin could not inhibit the expression level of T6SS-1 core genes of both sensitive and resistant strains;When treated AMT(2-amino-5mercapto-1,3,4-thiadiazole),which has cross-resistance with bismerthiazol,AMT could inhibit the expression level of T6SS-1 core genes of sensitive strain,but not of resistant strains.To determine whether T6SS-1 related genes also have important functions in Xoo,we randomly knocked out 2 core genes and 6 related genes of T6SS-1 in ZJ173.The results proved that,T6SS-1 related genes positively regulated the pathogenicity,biofilms and defense response of rice,and did not affect growth,motility,and extracellular polysaccharide production of Xoo,and the expression levels of the T6SS-1 ClpV and ClpP and its subunits genes were mutually regulated.The movement of bacteria is related to cluster induction and affects the pathogenicity of bacteria.In this paper,we found that low concentrations of bismerthiazol could promote the swimming of ZJ173 and 2-1-1;medium concentrations of bismerthiazol could only inhibit the swimming of ZJ173,but not of 2-1-1;high concentrations of bismerthiazol could inhibit the swimming of both ZJ173 and 2-1-1.We analyzed the transcriptome data of 2-1-1 and ZJ173 and summarized the gene which were related to motility differences between the sensitive and resistant strains.After verification,the results showed that bismerthiazol could inhibit the methyl-accepting chemotactic receptor protein gene Xoo1370 of ZJ173,but not of 2-1-1.Bismerthiazol could inhibit 12 swimming-related genes such as flagellar genes and chemotaxis genes downstream of Xoo1370 in ZJ173,but not in 2-1-1.Knocking out the Xoo1370 of ZJ173,it was proved that Xoo1370 positively regulated the biological phenotypes of biofilm production,motility,and pathogenicity of Xoo,and positively regulated the downstream flagellar genes and chemotactic proteins related to bacterial swimming.In summary,in Xoo sensitive strain,bismerthiazol could inhibit the type VI secretion system T6SS-1 related genes,swimming related genes,stress protein ClpP and its subunits genes and then inhibited the pathogenicity and chemotaxis ability of Xoo;in Xoo resistant strain,bismerthiazol could not significantly inhibit the above gene pathways,and the expression level of ClpP and its subunits genes of resistant strains were higher than that of sensitive strain which caused that the resistant strains had a more strong ability against the change of environment and enhanced the resistance to bismerthiazol in vivo. |
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