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

【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。

[ Objective ] To elucidate the biological functions of a twocomponent system RpfCxoc/RpfGxoc in Xanthomonas oryzae pv. oryzicola （Xoc）. [Method] Based on the genome template from Xoc wildtype strain Rsl05, the rpfCxoc and rpfGxoc genes were amplified by polymerase chain reaction （PCR）. The inframe 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 Rsl05. PCR analysis demonstrated that the rpfCxoc, rpfGxoc and rpfGCxoc genes were inframe deleted successfully. Compared to the wildtype strain Rsl05, DSF were overproduced in ArpfCxoc and ArpfGCxoc, but DSF production was remarkably decreased in ArpfGxoc. The DSF production of these mutants was restored by introducing the complemented cosmid pUFRrpfCxoc, pUFRrpfGxoc and pUFRrpfGCxoc, 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 Rsl05. In L medium （Tryptoen, 10 g/L; yeast extract, 5 g/L; sodium chloride, 5 g/L; Dglucose, 1 g/L;pH7.0） , Rsl05 was growing at planktonic pattern, but the mutation ofrpfCxoc and rpfGxoc led to Xoc cell aggregation at the wall of the flaks at the airliquid interfaces, and ArpfGxoc generated reticulation biofilm at the bottom of the flaks. But ArpfGCxoc 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.