Functional Analyses Of Pota And Mreb In Pectobacterium Carotovorum Subsp.Carotovorum

Pectobacterium carotovorum subsp. carotovorum (Pcc) is an important pathogen causing bacterial soft rot disease. It can infect a wide variety of ornamental plants and economic crops and cause economic loss, and there is still lack of effective prevention and control measures. Therefore, in recent years, research on pathogenicity of Pcc gradually deepening, to provide basis for the prevention and control method is proposed. For previous research in our lab indicated that over70proteins were differential expressed in PccS1interacted with colored calla lily (Zantedeschia elliotiana) in vivo, out of many proteins expressed up-regulated, PotA (spermidine/putrescine ABC transporter ATPase) and MreB (rod shape-determining protein) were selected.To investigate functions of potA and mreB in pathogenicity of Pcc, six gene deletion mutants of PccS1were constructed, and the phenotypes related with pathogenicity were compared between the mutants and wild type strains. The genes coding4subunits of the spermidine/putrescine transport system, potA, potB, potC, potD and potCD deletion mutants were constructed by using homologous recombination method. The pathogenicity test results showed that only that of ApotA decreased significantly compared with PccSl, and the results of RT-PCR indicated that in5mutant the transcription levels of speE, the key gene of spermidine synthesis, were all up-regulated. The phenotypes, including settlement, swimming ability, the capacities of carbapenem producing, biofilm formation and antioxidation and protease activity, proved the results of pathogenicity tests of ApotA and PccS1, despite of the unchanged activities of cellulase and pectinase. Meanwhile, the growth rates in different media and hypersensitive response to the non-host were the same between the mutants and wild type of PccS1.The results of transcriptomic analysis by RNA-sequencing showed that332genes were expressed differentially in ApotA comparing with PccSl.86out of these genes coding proteins for tranportion and nitrate reduction were up-regulated expressed, and the others for chemotaxis, secretion and transcription were down-regulated experssed. Moreever, the orientation of alterated transcriptional levels of some pathogenic factors, such as secD, clpV, and impC were vertified by RT-PCR analysis.Additionally, MreB, rod shape-determining protein, was studied in the same way. The results showed that the pathogenicity of mreB gene deletion mutant strains were decreased significantly compared with PccS1, the cell appearance changed from rod to ball,△mreB could not growth in MMX, a culture medium with basic elements and grew in LB with a lower ability, lost motility in water arar, and producing ability of extracellular enzymes, such as pectase, protease and carbapenem, although the settlement rate was improved, and the ability to produce cellulase and hypersensitive response of no-host plant had not been altered.In conclusoin, either potA or mreB in P. carotovorum subsp. carotovorum play an important role in virulence through a comprehensive regulating pathway which remains to be further studied.