DataSheet_1_Host Range Determinants of Pseudomonas savastanoi Pathovars of Woody Hosts Revealed by Comparative Genomics and Cross-Pathogenicity Tests.docx

The study of host range determinants within the Pseudomonas syringae complex is gaining renewed attention due to its widespread distribution in non-agricultural environments, evidence of large variability in intra-pathovar host range, and the emergence of new epidemic diseases. This requires the establishment of appropriate model pathosystems facilitating integration of phenotypic, genomic and evolutionary data. Pseudomonas savastanoi pv. savastanoi is a model pathogen of the olive tree, and here we report a closed genome of strain NCPPB 3335, plus draft genome sequences of three strains isolated from oleander (pv. nerii), ash (pv. fraxini) and broom plants (pv. retacarpa). We then conducted a comparative genomic analysis of these four new genomes plus 16 publicly available genomes, representing 20 strains of these four P. savastanoi pathovars of woody hosts. Despite overlapping host ranges, cross-pathogenicity tests using four plant hosts clearly separated these pathovars and lead to pathovar reassignment of two strains. Critically, these functional assays were pivotal to reconcile phylogeny with host range and to define pathovar-specific genes repertoires. We report a pan-genome of 7,953 ortholog gene families and a total of 45 type III secretion system effector genes, including 24 core genes, four genes exclusive of pv. retacarpa and several genes encoding pathovar-specific truncations. Noticeably, the four pathovars corresponded with well-defined genetic lineages, with core genome phylogeny and hierarchical clustering of effector genes closely correlating with pathogenic specialization. Knot-inducing pathovars encode genes absent in the canker-inducing pv. fraxini, such as those related to indole acetic acid, cytokinins, rhizobitoxine, and a bacteriophytochrome. Other pathovar-exclusive genes encode type I, type II, type IV, and type VI secretion system proteins, the phytotoxine phevamine A, a siderophore, c-di-GMP-related proteins, methyl chemotaxis proteins, and a broad collection of transcriptional regulators and transporters of eight different superfamilies. Our combination of pathogenicity analyses and genomics tools allowed us to correctly assign strains to pathovars and to propose a repertoire of host range-related genes in the P. syringae complex.

针对丁香假单胞菌复合群（Pseudomonas syringae complex）内寄主范围决定因子的研究再度受到关注，这是因为该类群广泛分布于非农业环境中，存在致病变种（pathovar，pv.）内寄主范围高度变异的证据，且新的流行性病害不断出现。这一研究方向亟需构建适宜的模型致病系统，以整合表型组、基因组与进化组相关数据。沙斯塔诺伊假单胞菌沙斯塔诺伊致病变种（Pseudomonas savastanoi pv. savastanoi）是橄榄树的模式病原菌，本研究报道了菌株NCPPB 3335的封闭组装基因组，以及分别从夹竹桃（pv. nerii）、白蜡树（pv. fraxini）和金雀花（pv. retacarpa）中分离的3株菌株的草图基因组序列。随后，我们对这4株新测序基因组与另外16株已公开的基因组进行了比较基因组学分析，涵盖了这4种木本寄主来源的沙斯塔诺伊假单胞菌致病变种的共20株菌株。尽管各致病变种的寄主范围存在重叠，但基于4种植物寄主的交叉致病性试验可清晰区分这些致病变种，并完成了2株菌株的致病变种重分类。至关重要的是，这些功能验证试验为协调系统发育与寄主范围的关联、明确致病变种特异性基因库提供了关键支撑。本研究构建了包含7953个直系同源基因家族的泛基因组，共鉴定出45个III型分泌系统（type III secretion system, T3SS）效应基因，其中包括24个核心基因、仅存在于pv. retacarpa中的4个基因，以及若干编码致病变种特异性截短蛋白的基因。值得注意的是，这4种致病变种与明确界定的遗传谱系一一对应，核心基因组系统发育与效应基因层级聚类结果均与病原菌致病特化程度高度相关。结瘤型致病变种携带有溃疡诱导型pv. fraxini所缺失的基因，例如与吲哚乙酸、细胞分裂素、根瘤毒素及细菌光敏色素相关的基因。其余致病变种特异性基因则编码I型、II型、IV型及VI型分泌系统蛋白、植物毒素phevamine A、铁载体、c-di-GMP相关蛋白、甲基趋化蛋白，以及八大不同超家族的转录调控因子与转运蛋白。本研究通过整合致病性分析与基因组学手段，实现了菌株的准确致病变种归类，并为丁香假单胞菌复合群中寄主范围相关基因库的挖掘提供了候选集合。