水稻细菌性条斑病菌中受DSF 调控的鞭毛基因flgD、flgE 的功能分析

Analysis of the flgD and flgE genes regulated by diffusible signal factor in Xanthomonas oryzae pv. oryzicola

摘要:

摘要:【目的】为了阐明可扩散性信号分子( diffusible signal factor，DSF) 调控的鞭毛基因对水稻细菌性条斑病菌(Xanthomonas oryzae pv． oryzicola，Xoc)Rs105 的致病性等方面的影响。【方法】采用PCR 的方法克隆靶标基因flgDxoc 和flgExoc，用同源重组法构建缺失突变体，测定突变体及其互补菌株的菌体形态、运动性、致病性及过敏性反应等表型，用反转录PCR(RT-PCR) 的方法验证Rs105 和ΔrpfFxoc( rpfFxoc 基因

Abstract:

Abstract:［Objective］ To investigate functions of flgDxoc and flgExoc genes regulated by diffusible signal factor (DSF) in Xanthomonas oryzae pv． oryzicola(Xoc)Rs105．Methods］The flgDxoc and flgExoc genes were amplified by PCR．We constructed ΔflgDxoc and ΔflgExoc，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 flgDxoc and flgExoc gene by reverse transcriptional polymerase chain reaction(RT-PCR) between the wide type and ΔrpfFxoc ( the deletion mutant of rpfFxoc gene，which could not produce DSF)．Results］We cloned flgDxoc and flgExoc from genomic DNA of Rs105． PCR and Southern blot analysis demonstrated that the flgDxoc and flgExoc 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 in ΔflgDxoc and ΔflgExoc 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 (ΔflgDxoc or ΔflgExoc ) and the wild type． In addition，the results of RT-PCR demonstrated that the transcription level of flgDxoc and flgExoc were downregulated in ΔrpfFxoc．Conclusion］ This study showed that expressions of flgDxoc and flgExoc 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 differences at growth rate in MMX medium and HR in nonhost．In addition，our results provided molecular evidences that the contribution of DSF-type quorum sensing to pathogen's virulence might be，at least partially，dependent on acterial flagellar in Xoc．