一株人参细菌性软腐病致病假单胞菌的基因组特征与毒力基因分析

Genomic characteristics and virulence genes of Pseudomonas glycinae, the causal agent of bacterial soft rot in ginseng

摘要:

【背景】细菌性软腐病是植物根部主要病害之一，可在作物生长期和储藏期造成巨大损失。2023年本团队首次发现并报道了由Pseudomonas glycinae引起的人参细菌性软腐病。【目的】探索Pseudomonas glycinae XJFL-1菌株的基因组特征，并通过与属内和种内代表性菌株的比较基因组学分析，初步探讨P. glycinae的遗传特性以及菌株XJFL-1基因组中可能编码的致病相关的毒力因子和Ⅱ型、Ⅲ型分泌系统效应因子。【方法】通过基因组注释、比较基因组学分析和基因家族分析等探索基因组的特征、遗传特性及毒力基因。【结果】基因组注释发现编码了碳水化合物、氨基酸和蛋白质代谢相关酶类，以及与鞭毛运动、膜形成和群体感应相关的重要基因；平均核苷酸一致性(average nucleotide identity, ANI)、数字DNA-DNA杂交分析(digital DNA-DNA hybridization, dDDH)及共线性分析结果确认该菌株为P. glycinae，并且P. glycinae物种的直系同源基因集具有较高的种内保守性；植物细胞壁降解酶系(plant cell wall degrading enzyme, PCWDE)分析结果显示，菌株XJFL-1可编码37种PCWDE，主要为糖苷水解酶(glycoside hydrolase, GH)、碳水化合物酯酶(carbohydrate esterase, CE)和多糖裂解酶(polysaccharide lyase, PL)；预测到的Ⅱ型分泌系统效应因子基因主要有编码脂肪酶的lapA、lapB和lapC，编码碱性磷酸酶的phoA2，编码磷脂酶的plcA和编码几丁质酶的cbpD；Ⅲ型分泌系统中E3泛素连接酶效应因子是XJFL-1编码的优势基因，包括IpaH家族的ipaH9.8、ipaH7.8、ipaH4.5、ipaH3和ipaH1.4，SLRP家族的slrP，以及sspH2、sspH1和NGR_a03640，但未注释到编码果胶酶的相关基因。果胶酶活力测定结果显示，菌株XJFL-1确实不能分泌果胶酶类。【结论】比较基因组学和基因家族分析结果明确了菌株XJFL-1与假单胞菌属(Pseudomonas)内代表性菌株的亲缘关系，以及P. glycinae的种内遗传特性；毒力基因预测结果发现，菌株编码了丰富的PCWDEs基因和独特的与致病相关的Ⅱ型和Ⅲ型分泌系统效应因子。

Abstract:

[Background] Bacterial soft rot poses a significant threat to plant roots, resulting in substantial agricultural losses. Our team identified Pseudomonas glycinae as the causative agent of bacterial soft rot in ginseng in 2023. [Objective] To delve into the genomic characteristics of P. glycinae XJFL-1, conduct a comparative genomics analysis for XJFL-1 with intra- and intergenus strains, and predict putative virulence factors and effectors of type II and III secretion systems (T2SS and T3SS, respectively). [Methods] We performed genome annotation, comparative genomics analysis, and gene family analysis to predict the genomic characteristics, genetic characteristics, and virulence genes. [Results] Genome annotation revealed the presence of genes encoding the enzymes involved in carbohydrate, amino acid, and protein metabolism, as well as key genes associated with flagellar movement, membrane biogenesis, and quorum sensing. Strain XJFL-1 was identified as P. glycinae based on ANI, dDDH, and collinearity analyses, and a high degree of intraspecific gene conservation was found for P. glycinae. Strain XJFL-1 encoded 37 plant cell wall-degrading enzymes (PCWDEs) including glycoside hydrolases (GHs), carbohydrate esterases (CEs), and polysaccharide lyases (PLs). In addition, the strain carried the genes encoding effectors of T2SS (e.g. lapA, lapB, lapC, phoA2, plcA, and cbpD) and T3SS (e.g. ipaH9.8, ipaH7.8, ipaH4.5, ipaH3, ipaH1.4, slrP, sspH2, sspH1, and NGR_a03640). However, pectinase genes were conspicuously absent. The results of the pectinase activity assay indicated that strain XJFL-1 was unable to secrete pectinase. [Conclusion] The results of comparative genomics and gene family analyses clarified the genetic relationship of strain XJFL-1 with representative strains of Pseudomonas and the intraspecific genetic characterization of P. glycinae. Additionally, the strain carried abundant genes encoding PCWDEs and effectors of T2SS and T3SS associated with pathogenicity.