Cloning And Funcitional Analysis Of Genes Induced By Calla Lily Tissue Extract In Pectobacterterium Carotovorum Subsp. Carotovorum

Pectobacterterium carotovorum subsp. carotovorum (Pcc) causes soft-rotting disease on a wide variety of crops and ornamental plants. Research about the soft rot disease caused by Pcc has become the hot subjects of plant pathology, especially after the publication of its genome sequence, and research on the pathogenicity has become more thorough. In order to investgate the relationship between the host and virulent genes of Pcc, we used the susceptive species (Zantedeschia. elliotiana) and resistant species (Zantedeschia. aethiopica) to study the interaction with calla lily-Pcc, and to provide the theoretical basis of the pathogenic mechanisms of Pcc.The mutant library of P. carotovorum subsp. carotovorum strain PccS1was successfully constructed by mating mariner transpson into strain PccS1. And the mariner transposon which containing a Km resistant gene and there is no promoter before the Km gene. About500mutants induced by tissue extract were selected from over6000mutants. With the test of pathogenicity, two mutants, PM86and PM5, which could be induced by the tissue extract of Z. elliotiana (the susceptive species) and Z. aethiopica (the resistant species), respectively, were obtained. With the addition of the host tissues extract, mutant PM86could be induced obviously. Pathogenicity test showed that virulence of mutant PM86was reduced on Z. elliotiana and the lesion area was about one-sixth of the wild type. In addition, non-pathogenicity was observed on the Z. aethiopica and chinese cabbage. Virulence of Mutant PM5was reduced on all the three hosts. The lesion area was about half of the wild type on the Z elliotiana and Z. aethiopica, while approximate one quarter of the wild type on the cabbage. With subclone, the transposon insertion site was identified as a rplY gene and a ubiG gene in PM86and PM5, respectively.In order to do further research about the function of both rplY and ubiG genes, deletion mutants were constructed by homologue recombination technology. Compared with the wild type, the pathogenicity of△rplY was reduced on the Z elliotiana, and the lesion area was about one-fifth of the wild type. In addition, non-pathogenicity were observed on the Z. aethiopica and chinese cabbage. Phenotype analysis showed that ArplY had loss of motility, and the sedimentation rate markedly accelerated. More important, production of Protease, Celluase and Pectate lyase were reduced. Part of the phenotypes of the mutant were restored in complemented strain.The pathogenicity of△ubiG was reduced on all the three hosts when compared with the wild type. The lesion area was about one half of the wild type on the Z. elliotiana and Z. aethiopica, while about one-fifth on the cabbage. Analysis of the virulence-related phenotypes revealed that sedimentation rate of△ubiG obviously accelerated, and production of biofilm formation was approximate half of the wild type. In addition, resistance to streptomycin and neomycin were significantly enhanced, about3times and27.5times, respectively, compared to the wild type PccSl. Furthermore, there was no production of extracellular Protease. Part of the phenotypes of the mutant were restored in complemented strain.