Functional Analysis Of The Flagellar Genes And Asparagine Synthetase B Gene Regulated By DSF In Xanthomonas Oryzae PV.Oryzicola

Xanthomonas oryzae pv. oryzae (Xoo) and X. oryzae pv. oryzicola(Xoc) cause leaf blight and leaf streak on rice respectively. To investigate functions of flagellar genes flgD and flgE and asparagine synthetase B gene (asnB) regulated by diffusible signal factor (DSF) in Xoc strain Rs105.In this study, the flgD, flgE and asnB genes were cloned and sequence analyzed from Xoc. We constructed the deletion mutants from Rs105 by using double crossover method, characterized the contribution of these genes to motility, virulence and bacterial growth to speculat the function of the genes. In addition, our results provided molecular evidences that the contribution of DSF-type quorum sensing to pathogen's virulence might be.(Ⅰ) The flgD and flgE genes were amplified by PCR. We constructedΔflgD andΔflgE, the deletion mutants from Rs105 by using double crossover method, and determined cell morphology, motility, pathogenicity in host rice and hypersensitive response (HR) in nonhost tobacco. We tested the differential expression of flgD and flgE gene by reverse transcriptional polymerase chain reaction (RT-PCR) between the wide type andΔrpfF (the deletion mutant of rpfF gene, which could not produce DSF). PCR and Southern blot analysis demonstrated that the flgD and flgE genes were knocked out successfully. Both mutants were non-flagellated and significantly attenuated motility on the 0.3% semi-solid medium. The pathogenicity on rice were obviously attenuated inΔflgD andΔflgE compared to the wild type. All the changes in mutant could be restored through the complementation. However, there was no significant difference in bacterial growth in MMX medium and induction of HR between mutant (ΔflgD orΔflgE) and the wild type. In addition, the results of RT-PCR demonstrated that the transcription level of flgD and flgE were down-regulated inΔrpfF. This study showed that expressions of flgD and flgE were positively regulated by DSF, and necessary for flagellar hook assembly and flagellar structure in Xoc. Meanwhile, FlgD and FlgE contributed to pathogen's virulence, motility and chemotaxis, but no deferences at growth rate in MMX medium and HR in nonhost. (Ⅱ) The asnB gene was amplified by PCR. We constructedΔasnB, the deletion mutants by using double crossover method, and determined cell morphology, motility, pathogenicity in host rice and hypersensitive response (HR) in nonhost tobacco, bacterial growth in rice and MMX medium with different nitrogen sources (Peptone, Yeast extract, Asparagine, Aspartic acid, Glutamine, Glutamate, Ammonium sulfate, Urea, Potassium nitrate or Alanine). We tested the differential expression of asnB gene by reverse transcriptional polymerase chain reaction (RT-PCR) between the wide type andΔrpfF. PCR and Southern blot analysis demonstrated that the asnB gene were knocked out successfully. Compaired with the wide type strain,ΔasnB was no pathogenicity on rice, the change in mutant could be restored through the complementation. The deletion of asnB gene had seriously affected the bacrerial growth, the change could be restored by adding exogenous asparagine. However, there was no significant difference in flagella production, motility and induction of HR between mutant and the wild type. In addition, the results of RT-PCR demonstrated that the transcription level of asnB was down-regulated in ArpfF. This study showed that expressions of asnB were positively regulated by DSF, and necessary for bacterial growth in Xoc. Meanwhile, AsnB contributed to pathogen's virulence in nonhost rice.