Faculty of Health and Life Sciences, University of the West of England, Bristol, United Kingdom.
PLoS Pathog. 2011 Mar;7(3):e1002010. doi: 10.1371/journal.ppat.1002010. Epub 2011 Mar 31.
Pseudomonas syringae pv. phaseolicola is the causative agent of halo blight in the common bean, Phaseolus vulgaris. P. syringae pv. phaseolicola race 4 strain 1302A contains the avirulence gene avrPphB (syn. hopAR1), which resides on PPHGI-1, a 106 kb genomic island. Loss of PPHGI-1 from P. syringae pv. phaseolicola 1302A following exposure to the hypersensitive resistance response (HR) leads to the evolution of strains with altered virulence. Here we have used fluorescent protein reporter systems to gain insight into the mobility of PPHGI-1. Confocal imaging of dual-labelled P. syringae pv. phaseolicola 1302A strain, F532 (dsRFP in chromosome and eGFP in PPHGI-1), revealed loss of PPHGI-1::eGFP encoded fluorescence during plant infection and when grown in vitro on extracted leaf apoplastic fluids. Fluorescence-activated cell sorting (FACS) of fluorescent and non-fluorescent PPHGI-1::eGFP F532 populations showed that cells lost fluorescence not only when the GI was deleted, but also when it had excised and was present as a circular episome. In addition to reduced expression of eGFP, quantitative PCR on sub-populations separated by FACS showed that transcription of other genes on PPHGI-1 (avrPphB and xerC) was also greatly reduced in F532 cells harbouring the excised PPHGI-1::eGFP episome. Our results show how virulence determinants located on mobile pathogenicity islands may be hidden from detection by host surveillance systems through the suppression of gene expression in the episomal state.
丁香假单胞菌 pv. phaseolicola 是普通菜豆（Phaseolus vulgaris）晕斑病的病原体。丁香假单胞菌 pv. phaseolicola 4 型菌株 1302A 含有无毒基因 avrPphB（syn. hopAR1），该基因位于 PPHGI-1 上，这是一个 106kb 的基因组岛。在暴露于超敏反应（HR）后，丁香假单胞菌 pv. phaseolicola 1302A 失去 PPHGI-1，导致毒性发生变化的菌株进化。在这里，我们使用荧光蛋白报告系统深入了解 PPHGI-1 的移动性。对双标记的丁香假单胞菌 pv. phaseolicola 1302A 菌株 F532（染色体上的 dsRFP 和 PPHGI-1 中的 eGFP）进行共焦成像，发现在植物感染期间和在提取的叶片质外体液中体外生长时，PPHGI-1::eGFP 编码的荧光丢失。荧光激活细胞分选（FACS）对荧光和非荧光 PPHGI-1::eGFP F532 群体进行分选，结果表明，当 GI 缺失时，不仅当 GI 缺失时，而且当 GI 切除并作为环状染色体外体存在时，细胞也会失去荧光。除了 eGFP 的表达减少外，通过 FACS 分离的亚群进行定量 PCR 显示，携带切除的 PPHGI-1::eGFP 染色体外体的 F532 细胞中，PPHGI-1 上的其他基因（avrPphB 和 xerC）的转录也大大减少。我们的结果表明，位于可移动致病性岛上的毒力决定因素如何通过在染色体外体状态下抑制基因表达而逃避宿主监测系统的检测。
