Specific binding of the Xanthomonas campestris pv. vesicatoria AraC-type transcriptional activator HrpX to plant-inducible promoter boxes
Specific binding of the Xanthomonas campestris pv. vesicatoria AraC-type transcriptional activator HrpX to plant-inducible promoter boxes
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DOI：
10.1128/jb.00795-06
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发表时间：
2006-11-01
影响因子：
3.2
通讯作者：
Bonas, Ulla
中科院分区：
文献类型：
--
作者：
Koebnik, Ralf;Krueger, Antje;Bonas, Ulla
植物病原细菌野油菜黄单胞菌的致病性。 vesicatoria 依赖于由 23 kb hrp（过敏反应和致病性）基因簇编码的 III 型分泌系统。 hrp 操纵子的表达在植物中和特殊的基本培养基中被强烈诱导，并且依赖于两种调节蛋白：HrpG 和 HrpX。在这项研究中，DNA 亲和富集被用来证明 AraC 型转录激活剂 HrpX 与保守的顺式调控元件、植物诱导启动子 (PIP) 盒 (TTCGC-N-15-TTCGC) 结合，该元件存在于四个 hrp 操纵子的启动子区域。当使用缺乏 PIP 盒的 DNA 片段时，没有观察到 HrpX 的结合。 HrpX 还与含有不完美 PIP 盒 (TTCGC-N-8-TTCGT) 的 DNA 片段结合。 PIP 盒每个半位点的二核苷酸替换强烈降低了 HrpX 的结合，而两个半位点同时的二核苷酸替换则完全消除了结合。基于 Xanthomonas Campestris pv. 的完整基因组序列。在 vesicatoria 中，几乎所有 HrpX 激活基因的启动子区域都鉴定出了由 PIP 盒和 -10 启动子基序组成的推定植物诱导型启动子。生物信息分析和逆转录-PCR 实验揭示了新的 HrpX 依赖性基因，其中包括 NUDIX 水解酶基因和几个在植物细胞壁降解中具有预测作用的基因。我们得出结论，HrpX 是 hrp 调节级联的最下游组件，建议通过与相应的 PIP 盒结合来直接激活 hrpX 调节子的大多数基因。
The pathogenicity of the plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria depends on a type III secretion system which is encoded by the 23-kb hrp (hypersensitive response and pathogenicity) gene cluster. Expression of the hrp operons is strongly induced in planta and in a special minimal medium and depends on two regulatory proteins, HrpG and HrpX. In this study, DNA affinity enrichment was used to demonstrate that the AraC-type transcriptional activator HrpX binds to a conserved cis-regulatory element, the plant-inducible promoter (PIP) box (TTCGC-N-15-TTCGC), present in the promoter regions of four hrp operons. No binding of HrpX was observed when DNA fragments lacking a PIP box were used. HrpX also bound to a DNA fragment containing an imperfect PIP box (TTCGC-N-8-TTCGT). Dinucleotide replacements in each half-site of the PIP box strongly decreased binding of HrpX, while simultaneous dinucleotide replacements in both half-sites completely abolished binding. Based on the complete genome sequence of Xanthomonas campestris pv. vesicatoria, putative plant-inducible promoters consisting of a PIP box and a -10 promoter motif were identified in the promoter regions of almost all HrpX-activated genes. Bioinformatic analyses and reverse transcription-PCR experiments revealed novel HrpX-dependent genes, among them a NUDIX hydrolase gene and several genes with a predicted role in the degradation of the plant cell wall. We conclude that HrpX is the most downstream component of the hrp regulatory cascade, which is proposed to directly activate most genes of the hrpX regulon via binding to corresponding PIP boxes.