十字花科黑腐病菌一个新的III型效应物的鉴定

Identification of a novel type III secreted effector in Xanthomonas campestris pv. campestris

摘要:

【目的】在全基因组范围内鉴定十字花科黑腐病菌Xcc 8004中新的Ⅲ型效应物(type Ⅲ secreted effector，T3SE)基因。【方法】通过构建与AvrBs1_59-445整合的Tn5转座子系统，进行文库筛选。在avrBs1缺失突变体背景下生成突变文库，在辣椒ECW-10R上进一步筛选。【结果】大规模HR测定筛选出7个具有明显过敏反应(hypersensitive response，HR)的克隆。通过质粒拯救和测序发现除了插入已知T3SE基因的3个突变体外，还鉴定了一个位于XC_0438和XC_0439之间且在Xcc 8004中未注释的新基因。结合生物信息学，我们将其命名为一个新的基因XC_0438a。易位实验证实XC_0438a信号区可引导报告蛋白AvrBs1的分泌和易位，并诱导辣椒ECW-10R产生HR反应。β-葡萄糖醛酸酶(β-glucuronidase，GUS)活性测定，XC_0438a在基本培养基中诱导表达，并由关键调控蛋白HrpG和HrpX激活。然而，在我们的实验条件下，XC_0438a对Xcc的致病力没有显著的贡献。【结论】我们鉴定了一种新的依赖于Ⅲ型分泌系统的效应基因XC_0438a。

Abstract:

[Objective] To identify a novel type III secreted effector (T3SE) gene in the genome of Xanthomonas campestris pv. campestris (Xcc) strain 8004. [Methods] A Tn5 transposon system integrated with AvrBs1_59-445was constructed for library screening. The mutant library was generated based on avrBs1-deleted mutant and then screened on Capsicum annuum cv. ECW-10R. [Results] Seven clones with visible hypersensitive response (HR) were screened out via large-scale HR assay. In addition to 3 mutants inserted into known T3SE genes, a new locus was identified, which was located between XC_0438 and XC_0439 and un-annotated in Xcc 8004 by plasmid rescue and sequencing. We annotated the new gene designated as XC_0438a according to the bioinformatics analysis results. The translocation assay confirmed that the signal region of XC_0438a could guide the secretion and translocation of the reporter protein AvrBs1 and elicit the HR of ECW-10R. The results of β-glucuronidase (GUS) activity assay demonstrated that the expression of XC_0438a was induced in nutrition sterile medium and activated by the key regulatory proteins HrpG and HrpX. However, in our tested conditions, XC_0438a had no significant contribution to the pathogenesis of Xcc. [Conclusion] In summary, we identified a novel effector gene XC_0438a dependent on the type III secretion system.