Erwinia amylovora Novel Plasmid pEI70: Complete Sequence, Biogeography, and Role in Aggressiveness in the Fire Blight Phytopathogen

Comparative genomics of several strains of Erwinia amylovora, a plant pathogenic bacterium causal agent of fire blight disease, revealed that its diversity is primarily attributable to the flexible genome comprised of plasmids. We recently identified and sequenced in full a novel 65.8 kb plasmid, called pEI70. Annotation revealed a lack of known virulence-related genes, but found evidence for a unique integrative conjugative element related to that of other plant and human pathogens. Comparative analyses using BLASTN showed that pEI70 is almost entirely included in plasmid pEB102 from E. billingiae, an epiphytic Erwinia of pome fruits, with sequence identities superior to 98%. A duplex PCR assay was developed to survey the prevalence of plasmid pEI70 and also that of pEA29, which had previously been described in several E. amylovora strains. Plasmid pEI70 was found widely dispersed across Europe with frequencies of 5–92%, but it was absent in E. amylovora analyzed populations from outside of Europe. Restriction analysis and hybridization demonstrated that this plasmid was identical in at least 13 strains. Curing E. amylovora strains of pEI70 reduced their aggressiveness on pear, and introducing pEI70 into low-aggressiveness strains lacking this plasmid increased symptoms development in this host. Discovery of this novel plasmid offers new insights into the biogeography, evolution and virulence determinants in E. amylovora.对若干株引发火疫病的植物病原菌解淀粉欧文氏菌（Erwinia amylovora）进行比较基因组学分析，结果显示其遗传多样性主要由质粒构成的柔性基因组所决定。本研究团队近期完成了一种新型65.8 kb质粒pEI70的全序列测定与鉴定。基因组注释结果显示，该质粒未携带已知的毒力相关基因，但存在一段独特的整合接合元件，其与其他植物及人类病原菌的同源整合接合元件具有相关性。通过BLASTN比对分析发现，pEI70的全序列几乎完全覆盖了来自梨果附生比尔欧文氏菌（Erwinia billingiae）的质粒pEB102，二者序列一致性高达98%以上。本研究开发了一种双重聚合酶链式反应（duplex PCR）检测方法，用于调查质粒pEI70以及此前在多株解淀粉欧文氏菌中报道过的pEA29质粒的流行率。检测结果显示，pEI70质粒在欧洲范围内广泛分布，检出率为5%至92%，但在欧洲以外地区的解淀粉欧文氏菌检测种群中均未检出该质粒。限制性酶切分析与核酸杂交实验证实，至少13株菌株所携带的pEI70质粒序列完全一致。将解淀粉欧文氏菌菌株中的pEI70质粒消除后，其对梨的致病力显著下降；而将pEI70质粒转入不携带该质粒的低致病力菌株后，该菌株对宿主梨的致病症状显著加重。该新型质粒的发现，为解淀粉欧文氏菌的生物地理学、进化机制以及毒力决定因子研究提供了全新视角。