Cloning And Functional Analysis Of Xanthomonas Campestris Pv. Campestris TopoIB Gene From Black Rot Of Cabbage

Xanthomonas campestris pv.campestris?Xcc?is the phytopathogen that causes black rot disease in cruciferous plants,and leads to severe yield reductions worldwide.At the same time,Xcc is also a model organism for studying the interaction between plant and plant pathogenic bacteria,and has high theoretical research and utilization value.DNA topoisomerase is a general term for catalyzing the transformation of DNA topoisomers.It is an important enzyme in cells that regulates the topology of DNA and plays a key role in DNA replication,recombination and transcription.However,there are no reports on the functions of the topoisomerase in plant pathogenic bacteria.This study focused on the function of TopoIB protein encoded by Xc₀034 gene in Xcc 8004.The physicochemical properties of TopoIB protein were analyzed by bioinformatics.The results showed that TopoIB protein contained nuclear localization signal and signal peptide,and no transmembrane domain.The topoIB gene displayed lower expression level based on RT-PCR analysis.The subcellular localization vector p1305-GFP-TopoIB was constructed and Agrobacterium-mediated transient expression was performed in tobacco leaves.The p1305-GFP-TopoIB fusion protein was localized to the nucleus of tobacco leaves.The mutant ATopoIB was constructed by overlapping extension PCR?SOE-PCR?method.The results showed that there is no significant difference in growth status between wild-type Xcc 8004 and ?TopoIB strain,when they grew in plants Arabidopsis thaliana and cabbage?Jinghua No.1?or NYGB and MMX media.Moreover,the yield of extracellular polysaccharide produced by ATopoIB strain was similar to that of 8004.Some virulence factors such as extracellular protease,extracellular cellulase and extracellular amylase were not affected when the topoIB gene was disrupted.When Arabidopsis thaliana and cabbage?Jinghua No.1?were inoculated with Xcc 8004 and ?TopoIB mutants,there is no significant difference in pathogenicity of ?TopoIB compared with Xcc 8004.The prokaryotic expression vector containing topoIB gene was constructed and performed effective expression of topoIB gene.After induction by IPTG,TopoIB fused with His-tag protein was successfully expressed in E.coli BL21 strain with a molecular weight of about 43 kDa.After a nickel column eluted with 200 mM imidazole,the purified protein was obtained at a concentration of 788 ng/?L,indicating that the effective expression of topoIB in vitro.The enzyme activity assay in vitro exhibited that 394 ng of TopoIB recombinant protein could untwist the supercoiled DNA in 1 min of reaction,and the supercoiled DNA could be relaxed in 15 minutes,indicating that TopoIB protein has topoisomerase activity.In summary,this experiment clarified that the topoIB gene in Xcc 8004 can be expressed in a lower level,and the TopoIB was localized in the nucleus of plant cells.The deletion of the topoIB gene had no significant effect on the production of extracellular polysaccharide,extracellular enzyme and its pathogenicity of the strain itself.Significantly,the purified recombinant protein obtained by heterologous expression has higher unwinding activity,which provides an important theoretical basis for the study of topoIB gene in pathogenic bacteria in the future.