十字花科黑腐病菌一个新的Ⅲ型效应物的鉴定(英文)

Identification of a novel typeⅢsecreted effector in Xanthomonas campestris pv.campestris

【目的】在全基因组范围内鉴定十字花科黑腐病菌Xcc8004中新的Ⅲ型效应物(typeⅢsecreted effector,T3SE)基因。【方法】通过构建与AvrBs159-445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Xcc8004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。

[Objective]To identify a novel typeⅢsecreted effector(T3SE)gene in the genome of Xanthomonas campestris pv.campestris(Xcc)strain 8004.[Methods]A Tn5 transposon system integrated with Avr Bs159-445 was constructed for library screening.The mutant library was generated based on avr Bs1-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 Avr Bs1 and elicit the HR of ECW-10R.The results ofβ-glucuronidase(GUS)activity assay demonstrated that the expression of XC_0438a was induced in nutrition sterile medium and activated by the key regulatory proteins Hrp G and Hrp X.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 typeⅢsecretion system.