Shen H, Keen N T
Department of Plant Pathology, University of California, Riverside 92521.
J Bacteriol. 1993 Sep;175(18):5916-24. doi: 10.1128/jb.175.18.5916-5924.1993.
The avirulence gene D (avrD) from Pseudomonas syringae pv. tomato comprises the first open reading frame (ORF) of a putative operon consisting of at least five tandem ORFs. The promoter of the avrD operon was localized to a 150-bp DNA fragment occurring 5' to the avrD gene by using the Tn7-lux and gus reporter systems. The avrD promoter in P. syringae pv. tomato and P. syringae pv. glycinea was poorly expressed when bacteria were grown in complex culture media but was activated during bacterial growth in plants. The timing and level of induction were similar in compatible and incompatible plant-pathogen interactions. When bacteria were grown in minimal culture medium, promoter activity was repressed by certain carbon sources, high concentrations of nitrogen compounds, and pH values above 6.5. Primer extension experiments on RNA from bacteria grown in minimal medium identified two transcription initiation sites 87 and 41 nucleotides upstream from the translational start site. Only the -41 transcriptional start site was identified in bacteria grown in soybean leaves. A sigma 54 promoter consensus sequence (GG-10 bp-GC) occurred 14 bp upstream of the -41 transcriptional start, and 3' deletions into this region completely abolished promoter activity. Little expression was observed when a gus fusion with the avrD promoter was introduced into an ntrA mutant strain of P. syringae pv. phaseolicola deficient in the sigma 54 cofactor. Expression from the avrD promoter also required the hrp regulatory genes, hrpS and hrpL. Deletions from the 5' end of the promoter region and base substitution analyses also identified two upstream elements important for expression. Sequence comparison of these elements with other cloned avirulence genes revealed the presence of a conserved consensus sequence elements with other cloned avirulence genes revealed the presence of a conserved consensus sequence (GGAACC-N15/16-CCAC) in the promoters of nine different avirulence genes from P. syringae pathovers.
丁香假单胞菌番茄致病变种的无毒基因D（avrD）包含一个推测操纵子的首个开放阅读框（ORF），该操纵子至少由五个串联ORF组成。通过使用Tn7 - lux和gus报告系统，avrD操纵子的启动子定位于avrD基因5'端的一个150 bp DNA片段。丁香假单胞菌番茄致病变种和丁香假单胞菌大豆致病变种中的avrD启动子在细菌于复杂培养基中生长时表达较差，但在植物中细菌生长期间被激活。在亲和与非亲和植物 - 病原体相互作用中，诱导的时间和水平相似。当细菌在基本培养基中生长时，启动子活性受到某些碳源、高浓度氮化合物以及pH值高于6.5的抑制。对在基本培养基中生长的细菌的RNA进行引物延伸实验，确定了两个转录起始位点，分别位于翻译起始位点上游87和41个核苷酸处。在大豆叶片中生长的细菌中仅鉴定出 - 41转录起始位点。一个σ54启动子共有序列（GG - 10 bp - GC）出现在 - 41转录起始位点上游14 bp处，向该区域的3'端缺失完全消除了启动子活性。当将与avrD启动子融合的gus导入缺乏σ54辅因子的丁香假单胞菌菜豆致病变种的ntrA突变菌株时，几乎没有观察到表达。来自avrD启动子的表达也需要hrp调控基因hrpS和hrpL。从启动子区域5'端的缺失和碱基置换分析还确定了两个对表达重要的上游元件。将这些元件与其他克隆的无毒基因进行序列比较，发现在来自丁香假单胞菌致病型的九个不同无毒基因的启动子中存在一个保守共有序列（GGAACC - N15/16 - CCAC）。
