Isolation And Identification Of Pectobacterium Carotovorum Subsp. Carotovorum And Functional Analysis Of Hemolysin Co-regulated Protein(Hcp)

Pectobacterium carotovorum subsp.carotovorum(Pcc)is a plant pathogenic bacterium causing soft rot in many different hosts,such as vegetables,ornamental and medicinal plants.In the genera of Pectobacterium,the genetic diversity is very rich,and host ranges of the most strains are very wide.Researches have revealed that pathogenic factors of these pathogens are complex.At present,there is no effective prevention and treatment measure for this disease.Pcc can produce a series of virulence factors(effector proteins,pectinases,proteases,cellulases,etc.)to infect the host.Previous studies have shown that there are at least nine types of secretory systems in Gram-negative bacteria,namely the type ?,?,?,?,?,?,?,? and ? secretion systems,we have known much about the type ? to ? secretion systems,but fewer about the type ?,? and ?secretion system.The type ? secretion system(T6SS)was firstly reported at 1996,but it has not been named until 2006.T6SS has been Known as a potential virulence determinant in many pathogenic bacteria,it exists in most Gram-negative pathogens and participates in interaction of bacterial cells and of bacterial-pant.T6SS is a complex composed of a series of proteins such as structural proteins,effectors,regulatory proteins and chaperones.Among them,haemolysin co-regulated proteins(Hcps)are the main structural proteins and effector proteins of T6SS,which can be secreted by all bacteria with T6SS function,and are often used as markers for judging whether the bacteria have T6SS function.In our previous works,based on the completed genome sequence of the strain Pectobacterium PccS1,it was found that a complete gene cluster of T6SS exist in it,,one hcp gene is included in the cluster,and 8 hcp homologous genes dispersed in the genome of PccS1 the 9 strains(Km resistant)with mutant in one of the 9 hcp genes constructed by homologous recombination showed a similar maceration level as the wild type in the tissue of yellow calla and Chinese cabbage plants,and unchanged ability of Hcp protein secretion.Therefore,it is speculated that there is functional complementation between the hcp genes.The main purpose of this study was to understand the effects of hcp genes completely deletion on the function of T6SS and the pathogenicity of PccSl and how the hcp genes function complementarily,and further to demonstrate the role of hcp genes in T6SS.Using a double-crossover homologous recombination and Cre-loxP binding method for multi-gene knockout,9 strains with deletion mutant in one of the 9 hcp genes without any markers,8 multigene deletion mutants including a strain of 9 hcp genes whole deleted,10 strains overexpressed with the fragment of hcp from the cluster and 10 complementary strains were constructed.The results of phenotypic determination showed that the pathogenicity and growth characteristics of all mutants showed no significant difference compared with wild type PccS1.and the results of western blotting showed that both VgrG and Hcp could be secreted normally though the hcps completely deleted.Meanwhile,qRT-PCR results showed that the expression of the remaining 16 genes in T6SS cluster was not affected.In conclusion,deletion or overexpression of the hcp gene does not affect pathogenicity in PccS1,and an objective of whether the components of the T6SS except Hcp assemble in a T6SS-like for secretion in PccS1 is waiting for further study.Additionally,in this work,a strain(NJAU170)caused soft rot in red radish(Raphanus sativus cv.Manshenhong)in Nanjing was collected,and identified using molecular biological methods as P.carotovorum subsp.brasiliense(Pcb).