利用gusA基因在水稻组织中示踪和定量水稻白叶枯病菌的方法

 水稻白叶枯病菌(Xanthomonas oryzae pv. oryzae, Xoo)从自然孔口水孔或者伤口处侵入水稻叶片,在维管束中定殖、繁殖和扩展,引起典型的叶枯症状。可视化这个动态的病程过程和快速定量水稻组织中细菌的群体是Xoo-水稻互作研究中亟待突破的技术难点。本研究在PXO99^A菌株中表达gusA基因,将其置于lacZ启动子、hrpX启动子和不含有(T₁)₄终止子的hrpX启动子下,构建了3个示踪菌株PXO99^A_GUSRBS、PXO99^A_GUSX和PXO99^A_GUSX-。水稻上毒性测定结果显示,这3个示踪菌株与野生型PXO99^A展现相同的毒性。利用示踪菌株,通过注射接种法,能够观察到Xoo在水稻维管束中定殖和扩展的动态变化;通过喷雾接种法,能够观察到Xoo从叶尖或者叶缘侵入水稻叶片引起发病的特性;发现PXO99^A_GUSX和PXO99^A_GUSX-比PXO99^A_GUSRBS能够更加灵敏地反映这些病程。同时发现,PXO99^A_GUSRBS的细菌数量与受其侵染的水稻组织的GUS活性显著正相关。本研究也评价了利用GUS活性测定法精确和快速地定量水稻组织中细菌群体的可行性。以上结果表明这套GUS系统是非常有效的,能够示踪病原菌的侵染过程和监测细菌在水稻组织中的群体数量,这将为Xoo-水稻互作研究提供有力的技术支持。

An efficient method using gusA as reporter gene for tracking and quantifying Xanthomonas oryzae pv. oryzae in rice

Xanthomonas oryzae pv. oryzae infects rice leaves through natural openings such as hydathodes or wounds and colonizes the vascular tissues, then bacteria multiply and spread into the whole leaf through the xylem leading to a typical leaf blight symptom. However, it is still bothered to visualize symptom progress and to determine bacterial population rapidly in rice tissues in Xoo-rice interaction system. In this study, we constructed three GUS tracking strains PXO99^A_GUSRBS, PXO99^A_GUSX and PXO99^A_GUSX- in PXO99^A expressing gusA as reporter gene under control of the lacZ promoter, hrpX promoter and the hrpX promoter without (T₁)₄ terminators, respectively. Pathogenicity assays showed that three strains displayed same virulence as the wild type strain PXO99^A. By using the tracking strains, the dynamic changes of localization and propagation of Xoo were successfully detected from rice vascular system when injection inoculation was applied. Meanwhile, the invasion through hydathodes at the leaf tip and expansion along leaf margin were observed in the spraying inoculation method. PXO99^A_GUSX and PXO99^A_GUSX- were found to be more sensitive than PXO99^A_GUSRBS in tracing the deve-loping progress of blight symptom. Based on statistical analysis, bacterial population of PXO99^A_GUSRBS showed significantly positive correlation with the GUS activity of infected rice tissues, implying that the GUS activity was truly derived from Xoo cell numbers. As a conclusion, a GUS marker system was developed for accurate and rapid determination of bacterial growth in rice tissues in this study. This system is useful for tracking the bacterial infection and monitoring bacterial population in rice, which will provide an efficient method for the research on Xoo-rice interaction.