Hendrickson E L, Guevera P, Peñaloza-Vàzquez A, Shao J, Bender C, Ausubel F M
Department of Genetics, Harvard Medical School, Bosston, Massachusetts 02114, USA.
J Bacteriol. 2000 Jun;182(12):3498-507. doi: 10.1128/JB.182.12.3498-3507.2000.
We cloned the rpoN (ntrA and glnF) gene encoding sigma(54) from the phytopathogen Pseudomonas syringae pv. maculicola strain ES4326. The P. syringae ES4326 rpoN gene complemented Pseudomonas aeruginosa, Escherichia coli, and Klebsiella aerogenes rpoN mutants for a variety of rpoN mutant phenotypes, including the inability to utilize nitrate as sole nitrogen source. DNA sequence analysis of the P. syringae ES4326 rpoN gene revealed that the deduced amino acid sequence was most similar (86% identity; 95% similarity) to the sigma(54) protein encoded by the Pseudomonas putida rpoN gene. A marker exchange protocol was used to construct an ES4326 rpoN insertional mutation, rpoN::Km(r). In contrast to wild-type ES4326, ES4326 rpoN::Km(r) was nonmotile and could not utilize nitrate, urea, C(4)-dicarboxylic acids, several amino acids, or concentrations of ammonia below 2 mM as nitrogen sources. rpoN was essential for production of the phytotoxin coronatine and for expression of the structural genes encoding coronamic acid. In addition, ES4326 rpoN::Km(r) did not multiply or elicit disease symptoms when infiltrated into Arabidopsis thaliana leaves, did not elicit the accumulation of several Arabidopsis defense-related mRNAs, and did not elicit a hypersensitive response (HR) when infiltrated into tobacco (Nicotiana tabacum) leaves. Furthermore, whereas P. syringae ES4326 carrying the avirulence gene avrRpt2 elicited an HR when infiltrated into Arabidopsis ecotype Columbia leaves, ES4326 rpoN::Km(r) carrying avrRpt2 elicited no response. Constitutive expression of ES4326 hrpL in ES4326 rpoN::Km(r) partially restored defense-related mRNA accumulation, showing a direct role for the hrp cluster in host defense gene induction in a compatible host-pathogen interaction. However, constitutive expression of hrpL in ES4326 rpoN::Km(r) did not restore coronatine production, showing that coronatine biosynthesis requires factors other than hrpL.
我们从植物病原菌丁香假单胞菌致病变种黄斑假单胞菌菌株ES4326中克隆了编码σ⁵⁴的rpoN（ntrA和glnF）基因。丁香假单胞菌ES4326的rpoN基因弥补了铜绿假单胞菌、大肠杆菌和气杆菌克雷伯氏菌rpoN突变体的多种rpoN突变表型，包括无法利用硝酸盐作为唯一氮源。对丁香假单胞菌ES4326的rpoN基因进行DNA序列分析表明，推导的氨基酸序列与恶臭假单胞菌rpoN基因编码的σ⁵⁴蛋白最为相似（同一性为86%；相似性为95%）。采用标记交换方案构建了ES4326的rpoN插入突变体rpoN::Km(r)。与野生型ES4326相比，ES4326 rpoN::Km(r)不具有运动性，且无法利用硝酸盐、尿素、C₄-二羧酸、几种氨基酸或浓度低于2 mM的氨作为氮源。rpoN对于植物毒素冠菌素的产生以及编码冠氨酸的结构基因的表达至关重要。此外，将ES4326 rpoN::Km(r)浸润到拟南芥叶片中时，它不会繁殖或引发疾病症状，不会引发几种拟南芥防御相关mRNA的积累，浸润到烟草叶片中时也不会引发超敏反应（HR）。此外，携带无毒基因avrRpt2的丁香假单胞菌ES4326浸润到拟南芥生态型哥伦比亚叶片中时会引发HR，而携带avrRpt2的ES4326 rpoN::Km(r)则无反应。在ES4326 rpoN::Km(r)中组成型表达ES4326的hrpL可部分恢复防御相关mRNA的积累，表明hrp簇在亲和性宿主-病原体相互作用中对宿主防御基因诱导具有直接作用。然而，在ES4326 rpoN::Km(r)中组成型表达hrpL并不能恢复冠菌素的产生，这表明冠菌素生物合成需要hrpL以外的因子。
