Functional Analysis Of Type ? Secretion System In Pectobacterium Carotovorum Subsp.Carotovorum

Pectobacterium carotovorum subsp.carotovorum(Pcc)is an important phytopathogen causing soft rot disease in many crops and ornamental plants.Effectors and extracellular hydrolases such as pectinase,protease and cellulose,are the main pathogenic factors of Pcc,for destroying cells of host plants quickly.It has been discovered that this pathogen possesses six kinds of secretion system(type ?,?,?,?,? and ?)as many other bacteria,which can transport the pathogenic effetors and extracelluar enzymes to plant cells crossing the membranes both of bacteria and plants.Unlike type I to V secretion system,type ? secretion system(T6SS)was discovered recently and attracted widely attention.Studies have shown that T6SS is involved in pathogenicity and plays important roles in cell-cell interations in some animal pathogenic bacteria,such as Vibrio cholerae and Pseudomonas aeruginosa.but there are few reports about T6SS in plant pathogens.This work focuses on the functions of T6SS genes in Pcc,especially on pathogenicity related of them.Genome sequencing and alignment analysis of nucleic acid sequence of PccS1 indicated that it's T6SS was composed by a T6SS cluster consisting of 17 genes and some homologous genes outside this cluster,including 8 hcp genes and 4 vgrG genes.We constructed 17 gene deletion mutants of each gene in T6SS cluster(?vgrG,?hcp,?vasL,?impL,?impA,?vasI,?vasH,?clpB,?impK,?impJ,?vasD,?impI,?impH,?impG,?impF,?impC and ?impB)by homologous recombination,and the results of pathogenicity assay showed that AimpG was the only one mutant of virulence significantly attenuated,and while the other 16 genes had no difference of pathogenicity on the hosts compairing with that of the wild type.It was observed that the activities of extracellular enzymes and the gene transcription of celC,pell and prtW were all significantly repressed in ?impG.Meanwhile,carbapenem secrated by AimpG was almost lost,and the expression of carC was severely reduced.CarC is important in the synthesis of carbapenem by which the pathogen competes with environmental organisms,one of the T6SS functions in bacteria.The growth rates in different media and colonization in the host were identical between AimpG and the wild type.Additionally,the results showed that AimpG could not induce hypersensitive response in tobacco and the expression of hrpN,hrpA,hrpi.hrpQ and hrcN were all significantly down-regulted because of impG deleted.Furthermor,the protein expression demonstrated that impG is also crucial to Hcp exo-secretion as many other genes in the cluster.The mutants of the orthologous genes of T6SS outside the cluster in PccS1 were also constructed,and the 12 mutants(?hcpl,?hcp2,?hcp3,?hcp4,?hcp5,?hcp6,?hcp,?hcp8,AvgrG1,?vgrG2,AvgrG3 and AvgrG4)showed no difference in pathogenicity comparing with the wild type.In conclusion,among the 29 genes consisting the T6SS in Pectobacterium carotovorum subsp.carotovorum PccS1,impG is the only one gene strong association with virulence and hypersensitive response of PccS1,it encodes the conserved protein ImpG setting the secretion system with many of the components of T6SS for secretion of Hcp,but mechanisms of the regulating pathways in virulence,carbapenem synthsis and Hcp secretion are remaining.