Table_9_Complete Genome Sequence of Sequevar 14M Ralstonia solanacearum Strain HA4-1 Reveals Novel Type III Effectors Acquired Through Horizontal Gene Transfer.XLSX

Ralstonia solanacearum, which causes bacterial wilt in a broad range of plants, is considered a “species complex” due to its significant genetic diversity. Recently, we have isolated a new R. solanacearum strain HA4-1 from Hong’an county in Hubei province of China and identified it being phylotype I, sequevar 14M (phylotype I-14M). Interestingly, we found that it can cause various disease symptoms among different potato genotypes and display different pathogenic behavior compared to a phylogenetically related strain, GMI1000. To dissect the pathogenic mechanisms of HA4-1, we sequenced its whole genome by combined sequencing technologies including Illumina HiSeq2000, PacBio RS II, and BAC-end sequencing. Genome assembly results revealed the presence of a conventional chromosome, a megaplasmid as well as a 143 kb plasmid in HA4-1. Comparative genome analysis between HA4-1 and GMI1000 shows high conservation of the general virulence factors such as secretion systems, motility, exopolysaccharides (EPS), and key regulatory factors, but significant variation in the repertoire and structure of type III effectors, which could be the determinants of their differential pathogenesis in certain potato species or genotypes. We have identified two novel type III effectors that were probably acquired through horizontal gene transfer (HGT). These novel R. solanacearum effectors display homology to several YopJ and XopAC family members. We named them as RipBR and RipBS. Notably, the copy of RipBR on the plasmid is a pseudogene, while the other on the megaplasmid is normal. For RipBS, there are three copies located in the megaplasmid and plasmid, respectively. Our results have not only enriched the genome information on R. solanacearum species complex by sequencing the first sequevar 14M strain and the largest plasmid reported in R. solanacearum to date but also revealed the variation in the repertoire of type III effectors. This will greatly contribute to the future studies on the pathogenic evolution, host adaptation, and interaction between R. solanacearum and potato.

青枯菌（Ralstonia solanacearum）可引发多种植物的细菌性萎蔫病，因其显著的遗传多样性，被归类为“物种复合群（species complex）”。近期，我们从中国湖北省红安县分离得到一株全新的青枯菌菌株HA4-1，经鉴定其属于系统型I（phylotype I）、序列变种14M（sequevar 14M，即phylotype I-14M）。有趣的是，我们发现该菌株可在不同基因型马铃薯中引发多种病害症状，且与亲缘关系相近的菌株GMI1000相比，其致病行为存在显著差异。为解析HA4-1的致病机制，我们结合Illumina HiSeq2000、PacBio RS II以及BAC末端测序（BAC-end sequencing）三种测序技术对其全基因组进行了测序。基因组组装结果显示，HA4-1的基因组包含一条常规染色体、一个巨型质粒以及一个143 kb的质粒。对HA4-1与GMI1000开展比较基因组分析发现，二者在分泌系统、运动性、胞外多糖（exopolysaccharides, EPS）以及关键调控因子等通用毒力因子方面保守性较高，但在III型效应因子（type III effectors）的组成与结构上存在显著差异——这类效应因子可能是二者在特定马铃薯物种或基因型中致病表型分化的决定因素。我们鉴定出两个全新的III型效应因子，它们大概率通过水平基因转移（horizontal gene transfer, HGT）获得。这两个全新的青枯菌效应因子与多个YopJ家族与XopAC家族成员具有同源性，我们将其命名为RipBR和RipBS。值得注意的是，质粒上的RipBR拷贝为假基因（pseudogene），而巨型质粒上的拷贝则为功能性基因。对于RipBS而言，其共有三个拷贝，分别位于巨型质粒与质粒上。本研究不仅通过测序首株序列变种14M菌株以及目前已报道的青枯菌中最大质粒，丰富了青枯菌物种复合群的基因组信息，同时揭示了其III型效应因子组成的变异特征。上述研究成果将为未来青枯菌致病演化、宿主适应性以及青枯菌与马铃薯互作机制的相关研究提供重要支撑。