Functional Analyses Of Two-Component Systemic Response Regulatory Genes Of Pectobacterium PccS1 In Its Pathogenesis

Pectobacterium carotovorum subsp.carotovorum is one of the pathogens with a most widely host range among the strains from the genera of Pactobacterium,causing large areas of soft rot of various crops,resulting in huge economic losses.Therefore,Pcc plays a very important role in the economic significance of agricultural production.The pathogenic bacteria are activated to respond to environmental changes by sensing environmental signals,they can survival and reproduction in the hosts,and result in successful infection or symbiotic interaction with the plants.Two-component systems are ubiquitous in prokaryotic and eukaryotic organisms,it plays a very important role in the signal sensing process of pathogenic bacteria.Previous researches have shown that two-component system also has a variety of regulatory mechanisms based on typical compositional structures in the pathogens,such as metabolic adaptation internal the host and external environment,physiological response to osmotic pressure changes.And It has been revealed that two-component systems influence on the pathogenicity of pathogens.In the present work,three response regulatory genes in PccS1 genome were found based on the completed genome sequencing in Pectobacterium PccS1.In order to understand the roles of these three response regulatory genes to the virulence in PccSl,three strains with a mutant in one of the three reaction regulator genes in PccS1(?PccS1₃334,?PccS1₁979 and ?PccS1₃961)were constructed using homologous recombination,as well as the strains complemented plasmids carrying the fragments of these genes or the empty vector,the gene overexpressing strains,and a strain of all the three genes deleted.The results of biological phenotypic analysis indicated that the growth of the single gene-knockout mutants and the three gene-knockout mutant was the nearly the same with that of the wild type,which revealed that the response regulators of the two-component system in PccS1 did not regulated through the processes of growth to influence the behavior of bacteria.The results of host virulence assay showed that the mutants of the response regulator gene deletion were extremely significant attenuated in pathogenicity,and the activities of the extracellular protease secretion of ?PccS1₃334 were remarkable decreased.The abilities of carbapenem antibiotic synthesis,biofilm formation,sedimentation and swimming in PccS1₃334 mutant strain were also significantly depressed.The results showed that callose deposition was highly induced after infiltrated with the mutant of PccS1₃334 in the leaf cells of both N.benthamiana and potato plants compared with the wild type.Meanwhile,?PccS1₁979 and ?PccS1₃961 showed no differences in other phenotypes compared with that of the wild type except the differences in pathogenicity.Our results indicate that the two regulatory regulators are different from the regulator encoded by PccS1₃334 in the regulatory mechanisms of pathogenicity,and which are wanted for further studies.Additionally,the secretion of Hcp and HexA analyzed using western blotting illustrated only the protein encoding by PccS1₃334 among the three regulatory response proteins was involved in regulatory on the secretion of T6SS other than that of HexA,a central regulator of carbon metabolism.In conclusion,the roles of the three regulatory response proteins in two-component system are different to the virulence in Pectobacterium PccS1.