Functional Analyses Of Pathogenicity-Related Genes In Pectobacterium Carotovorum Subsp.Carotovorum

Pectobacterium carotovorum subsp. Carotovorum (Pcc) is an important pathogens of bacterial soft rot of Zantedeschia, and has a wide range of serious harm.The protein expression profiles of Pcc grown in2YT medium (Pcc-2YT), Pcc grown in2YT+0.4%stalk extract of Zantedeschia elliotiana (Pcc-2YT-Ze) were analyzed by using two-dimensional electrophoresis (2-DE) and mass technology.The results showed that there were70protein spots in the gels indicated expression high differentially, including ClpP protein、HSP33protein aspartate ammonia-lyase and glycerol-3-phosphate transporter periplasmic binding protein, the encoding gene is clpP, hsl, asp A, ugpB.. To investigate functions of clpP, hsl, aspA, ugpB in Pcc strain PccSl.In this study, the clpP, hsl, aspA, ugpB genes were cloned. We constructed the deletion mutants by using double crossover method, characterized the contribution of these genes to motility, virulence and bacterial growth to speculat the function of the genes. In addition, our results contribute to pathogen’s virulence might be.(I) The clpP genes were amplified by PCR. We constructed AclpP, the deletion mutants from PccSl by using double crossover method, PCR and Southern blot analysis demonstrated that the clpP gene was knocked out successfully. And cell morphology, motility, biofilm formation, difference viabilities under oxidative stress, pathogenic factors and pathogenicity in hosts were tested. We found that:(1)△clpP had the same motility as wild-type PccS1.(2) It showed an obvious reduction of biofilm formation compared with PccS1.(3)△clpP significantly reduced production of pathogenic factor, pectate lyase, cellulase and metalloprotease, The results of qRT-PCR demonstrated that the transcription level of pel,cel were down-regulated in△clpP.(4) Comparing with the wild type PccS1,△clpP showed an obvious reduction of abilities in tolerance to high-temperature, innutrition and oxidative stress.(5)△clpP multiplication in Chinese cabbage was significantly lower than the wild type strain.(6) The pathogenicity on host plant was attenuated in△clpP. These findings demonstrate that clpP gene in P. carotovorum subsp. carotovorum is involved in growth, and play an important role in virulence.(II) The hsl, aspA, ugpB genes were amplified by PCR. We constructed△hsl, AaspA,△ugpB, the deletion mutants from PccS1by using double crossover method, PCR analysis demonstrated that the hsl, aspA, ugpB genes were knocked out successfully. And motility, biofilm formation, difference viabilities under oxidative stress, pathogenic factors and pathogenicity in hosts were tested. We found that there no significant difference with each phenotype between mutants and wild type.And after inoculating, lesion area of mutants were similar with PccS1. The experimental results show that:hsl, aspA, ugpB genes were independent of Pcc pathogenic.