水稻细菌性条斑病菌RpfCxoc/RpfGxoc双组分系统的功能

Function of a two-component system RpfCxoc/RpfGxoc in Xanthomonas oryzae pv.oryzicola

摘要:

摘要:【目的】旨在阐明双组分系统RpfCxoc/RpfGxoc 在水稻细菌性条斑病菌(Xanthomonas oryzae pv．oryzicola，Xoc)DSF(diffusible signal factor)合成、致病性等相关方面的生物学功能。【方法】以Xoc 野生型菌株Rs105 为母体，利用自杀载体pK18mobsacB 缺失突变rpfCxoc、rpfGxoc 和rpfGCxoc(rpfCxoc 和rpfGxoc 双基 因) ，测定突变体及其互补菌株的DSF 合成水平、对水稻的致病性、胞外多糖(extracellular polysaccharide，EPS) 产量、菌体形态及群体结构。【结果】从Rs105 基因组中克隆了rpfCxoc 和rpfGxoc 基因，并获得了相应的单基因或双基因缺失突变体。与Rs105 相比，ΔrpfCxoc 和ΔrpfGCxoc 过量合成DSF 信号分子，但是ΔrpfGxoc 合成DSF 的能力显著下降;rpfCxoc 和rpfGxoc 单基因或双基因的缺失突变均导致Xoc 的致病性丧失，EPS 合成水平下降34.1%－48.5%，形成菌体高度聚集的生物膜结构。【结论】RpfCxoc/RpfGxoc 双组分系统调控Xoc 的DSF 生物合成、EPS 产生和生物膜的驱散，是Xoc保持致病性所必需的因子。

Abstract:

Abstract:［Objective］ To elucidate the biological functions of a two-component system RpfCxoc/RpfGxoc in Xanthomonas oryzae pv． oryzicola (Xoc)．［Method］ Based on the genome template from Xoc wild-type strain Rs105，the rpfCxoc and rpfGxoc genes were amplified by polymerase chain reaction (PCR)．The in-frame deletion mutations of rpfCxoc，rpfGxoc and rpfGCxoc (rpfCxoc and rpfGxoc double genes) were performed by the suicide vector pK18mobsacB，and determined diffusible signal factor (DSF) biosynthesis，pathogenicity in host rice，biofilm，extracellular polysaccharide (EPS) production and cell morphology．［Result］ rpfCxoc and rpfGxoc were cloned from the genomic DNA of Rs105．PCR analysis demonstrated that the rpfCxoc，rpfGxoc and rpfGCxoc genes were in-frame deleted successfully． Compared to the wild-type strain Rs105，DSF were overproduced in ΔrpfCxoc and ΔrpfGCxoc， but DSF production was remarkably decreased in ΔrpfGxoc． The DSF production of these mutants was restored by introducing the complemented cosmid pUFRrpfCxoc，pUFR-rpfGxoc and pUFR-rpfGCxoc，respectively． Subsequent experimental results indicated that mutation of rpfCxoc，rpfGxoc and rpfGCxoc resulted in pathogenicity loss of Xoc in host rice，and decreased biosynthesis level of EPS at 34.1%－48.5% compared to that of Rs105． In L medium (Tryptoen，10 g/L; yeast extract，5 g/L; sodium chloride，5g/L;D-glucose，1g/L;pH7. 0)，Rs105 was growing at planktonic pattern，but the mutation of rpfCxoc and rpfGxoc led to Xoc cell aggregation at the wall of the flaks at the air-liquid interfaces，and ΔrpfGxoc generated reticulation biofilm at the bottom of the flaks． But ΔrpfGCxoc only generated reticulation biofilm at the bottom of the flaks． ［Conclusion］The twocomponent system RpfCxoc /RpfGxoc modulated DSF biosynthesis，EPS production and biofilm dispersal of Xoc，which was required for the pathogenicity of Xoc in host rice．