| Ralstonia solanacearum is one of the most important phytopathogenic bacteria worldwide, bothin terms of host range and destructive capability. A new hierarchical classification scheme wasestablished to distinguish the genetic diversity within the R. solanacearum species complex. Underthis classification, the phylotyping scheme was used to determine the genetic diversity of286strainsof R. solanacearum from17plant species in13Chinese provinces in previous study. Po82, areference strain, isolated from potato in Mexico, was resolve into phylotype II/sequevar4, in groupwith phylotype II/sequevar4NPB strain and banana disease-causing strains. Pathogenicity testrevealed that Po82possess pathogenic traits of both NPB and moko strains, thereby proving that itwas a pathogenicity variation strain.In order to elucidate the molecular mechanism of pathogenicity variation, the method ofcomparative genomics was used; Suppressive subtractive hybridization (SSH) approach was used toinvestigate the differential genes of the Po82strain; the complete genome of Po82has been sequenced,annotated, and compared to the genomes of sequenced R. solanacearum strains; In this paper,candidate pathogenicity-related genes were selected for further analysis based on the pan-genomeconcept.1. Screening strain-specific genes of Po82strain by comparative genomicsSuppressive subtractive hybridization (SSH) approach was used to investigate the pathogenicitydeterminant of the Po82strain. By comparison of the genome sequences of the Po82strain and mokostrain RUN92, there was no specific sequences were obtained. When using the Po82strain as thetester and NPB strain RUN292as driver,46differential genes were identified.The R. solanacearum strain Po82chromosome and megaplasmid sequences have been depositedin GenBank with accession numbers CP002819and CP002820. In comparison to the six sequenced R.solanacearum strains,390strain-specific genes were identified within the genome of strain Po82,most of which encoded hypothetical and conserved hypothetical proteins. Fifty-three genomic islandswere predicted by Islandsviewer website. Using bioinformatics to analyze the46differential genesand the390strain-specific genes, five candidate genes were selected for further study.2. Study on role of type III seretion system regulator hrpB gene of Po82strainThe mutant of hrpB gene was constructed by using homologous recombination and namedPo82ΔhrpB. Based on the mutant strain, the complement strain was also constructed. Thepathogenicity and the biological function of wild type, mutant strain and the complement strain weretested. Pathogenicity test showed that disease index of the mutant Po82ΔhrpB was decreasedcompared with the wild type Po82. Growth curve analysis indicated that Po82ΔhrpB mutant grew asfast as the Po82strain in rich medium, whereas, in Boucher's minimal medium, Po82ΔhrpB mutantgrew faster than the Po82. There were no significant differences between the mutants and wild typestrain in the ability of the mobility. 3. The Po82strain-specific hopAF1gene encoding putative T3SS effectorThe hopAF1gene was one of strain-specific genes and had high consistency with the T3SSeffector of Pseudomonas syringae pv. tomato DC3000. The hopAF1gene mutant strain and itscomplement strain were constructed. The pathogenicity test revealed that hopAF1gene mutant strainwas significantly reduced in pathogenicity. The results of semi RT-PCR showed that hopAF1expression was increased in hrp-inducing conditions in a manner similar to hrpB. To determine ifHopAF1is translocated into plant cells by the T3SS, the adenylate cyclase (CyaA) fusion assay wasemployed. These data confirm that HopAF1-CyaA was translocated into plant cells by the T3SS ofPo82. The expression pattern and translocation of HopAF1are all hallmarks of a T3E. There were nosignificant differences among the wild-type, mutant and the complement strain in the ability of thegrowth condition, mobility and the biofilm formation.4. Functional analysis of four strain-specific genes in Po82strainThe bioinformatic analysis found that the c00283and m00778genes were speculated to beputative T3SS effectors and m00553gene belonged to MarR gene family. In the process ofconstructing the mutant strain of c01536gene, there was no colony appeared. So we speculated thatthe lethal mutant occurred. Compared with the wild-type strain, the pathogenicity test results showedthat disease index of the mutant strains of c00283, m00553, m00778genes were decreased by15.8%,28.5%and17.2%, respectively. The results of semi RT-PCR suggested that the c00283and m00778genes expression were increased in hrp-inducing conditions in a manner similar to hrpB. Finally, toanalyze the basic biological function of three genes above, there were no significant differencesamong the wild-type, mutant and the complement strain in the ability of the growth condition,mobility and the biofilm formation.5. Functional analysis of type III effectors AvrPtoB and XopX analogues in strain Po82According to the related studies, two genes encongding type III effectors AvrPtoB and XopXanalogues were founded in the moko-strain Molk2. Both the two genes above were screened from thecomplete genome of Po82strain. The c01857gene had homology with the avrPtoB gene of P.syringae and the m00718genes had homology with the xopx gene of Xanthomonas campestris pv.vesicatoria. The c01857gene product had an E3ubiquitin ligase domain in the C-terminal. Thepathogenicity test suggested that the two genes played important role in pathogenicity of Po82strain.Semi RT-PCR results showed that two genes expression were increased in hrp-inducing conditions ina manner similar to hrpB. Basic biological function experiment demonstrated that c01857andm00718genes had no effects on the growth condition, mobility and the biofilm formation. |
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