Construction Of Egfp Marked-Mutants From Pseudomonas Syringae Pv.tomato And Pv.tabaci

Type III secretory system (TTSS) of plant pathogenic bacteria is an important protein secretion system for Pseudomonas syringae infection. TTSS is regulated by Hrp gene. It is helpful to further explore the the function of hrp gene and reveal the molecular pathogenicity of pathogens that to study the hrp gene expression during the infection. To construct egfp marked-mutants is an important strategy for studying the interaction between plants -bacteriaIn this study, we intended to use enhanced green fluorescent protein (egfp) gene as a reporter, and hrpA gene and hrpZ gene of Pseudomonas syringae pv.tomato and Pseudomonas syringae pv.tabaci as the targets, to construct egfp-marked fusion mutants and egfp-marked deletion mutants, respectively, with recombination in vitro and parental conjugation. The major results are as follows:(1)hrpA,hrpZ gene and its down-stream and up-stream were amplified from the genomic DNA of P.s. pv.tomato DC3000 and P.s.pv.tabaci 4. The egfp full-legth ORF was amplified from plasmid pEGFP-C1 DNA. After recombination in vitro by PCR, 4 expected fragments CDE3,CTE1,CTE3,CTE4 were obtained that laid the foundation for construction of fusion mutants and deletion mutants.(2)After the expected recombined-fragments were cloned to vector pMD19-T, the positive cloning vector pMD19-CDE3, pMD19-CTE1, pMD19-CTE3, pMD19-CTE4 and the suicide plasmid pKSM1 was double digested with BamHI and XbaI, respectively. And the digested fragments were collected and ligated. So the research got plasmids pKSM-CDE3, pKSM-CTE1, pKSM-CTE3, pKSM-CTE4 which were suitable for parental conjugation.(3)The research obtained fusion mutant hrpZPto::egfp through the parental conjugation between Ecoli S17-1 containing the plasmid pKSM-CDE3 and P.s.pv.tomato wild type strain DC3000. Meantime, the research obtained fusion mutants hrpAPsta::egfp,hrpZPsta::egfp and deletion mutant△hrpZPsta::egfp through the parental conjugation between Ecoli S17-1 containing the plasmids pKSM-CTE1, pKSM-CTE3, pKSM-CTE4 separately and P.s.pv.tabaci wild type 4. When the fusion mutants and deletion mutant were cultured on KB plate(30μg/ml Nalr),only the fusion mutant hrpZPsta::egfp had significant green fluorescent colonies, while fusion mutants hrpZPto::egfp,hrpAPsta::egfp and deletion mutant hrpZPsta::egfp not.(4)Compared with the wild type strains, the resultant 4 egfp-marked mutants artificially inoculated to tabaco lealves showed different HRP capacity. The fusion mutant hrpZPto::egfp revealed a significantly reduced hypersensitivity-inducing, while the other 3 egfp marked mutants hrpAPsta::egfp, hrpZPsta::egfp and△hrpZPsta::egfp showed significant reduced pathogenicity to tobacco.