THE PSEUDOMONAS-SYRINGAE PV SYRINGAE-61 HRPH PRODUCT, AN ENVELOPE PROTEIN REQUIRED FOR ELICITATION OF THE HYPERSENSITIVE RESPONSE IN PLANTS
THE PSEUDOMONAS-SYRINGAE PV SYRINGAE-61 HRPH PRODUCT, AN ENVELOPE PROTEIN REQUIRED FOR ELICITATION OF THE HYPERSENSITIVE RESPONSE IN PLANTS
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DOI：
10.1128/jb.174.21.6878-6885.1992
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发表时间：
1992-11-01
影响因子：
3.2
通讯作者：
COLLMER, A
中科院分区：
文献类型：
--
作者：
HUANG, HC;HE, SY;COLLMER, A
丁香假单胞菌 pv. syringae 61 包含一个 25 kb 的 hrp 基因簇，这些基因是在烟草中引发过敏反应 (HR) 所必需的。含有 hrp 簇的粘粒 pHIR11 的 TnphoA 诱变揭示了编码输出蛋白或内膜跨膜蛋白的两个基因（H.-C. Huang、S. W. Hutcheson 和 A. Collmer, Mol. Plant-Microbe Interact. 4:469-476, 1991）。互补组 X 中的基因（指定为 hrpH）在 3.1 kb SalI 片段上亚克隆到 pCPP30（一种广泛宿主范围的可移动载体）中。该亚克隆恢复了 hrpH 突变体丁香假单胞菌 pv. 的能力。丁香菌 61-2089 引发烟草中的 HR。 3.1-kb SalI 片段的 DNA 序列分析揭示了一个编码 81,956-Da 前蛋白的单一开放阅读框，具有典型的氨基末端信号肽，并且没有可能的跨膜内疏水区域。 hrpH 在[S-35]蛋氨酸存在下通过使用 T7 RNA 聚合酶启动子系统和载体 pT7-3 在大肠杆菌中表达，并在十二烷基硫酸钠-聚丙烯酰胺凝胶上显示编码表观分子量为 83,000 的蛋白质。大肠杆菌中的 HrpH 蛋白位于膜部分，周质和细胞质中不存在。 HrpH 蛋白与已知参与蛋白质或噬菌体分泌的几种外膜蛋白具有相似性，包括产酸克雷伯氏菌 PulD 蛋白、小肠结肠炎耶尔森氏菌 YscC 蛋白和丝状大肠杆菌噬菌体的 pIV 蛋白。所有这些蛋白质在羧基末端附近都具有可能的分泌基序 GG(X)12VP(L/F)LXXIPXIGXL(F/L)，并且它们缺乏羧基末端苯丙氨酸，这与其他没有已知分泌功能的外膜蛋白相反。这些结果表明丁香假单胞菌 pv。丁香菌 HrpH 蛋白参与蛋白质 HR 激发子的分泌。
Pseudomonas syringae pv. syringae 61 contains a 25-kb cluster of hrp genes that are required for elicitation of the hypersensitive response (HR) in tobacco. TnphoA mutagenesis of cosmid pHIR11, which contains the hrp cluster, revealed two genes encoding exported or inner-membrane-spanning proteins (H.-C. Huang, S. W. Hutcheson, and A. Collmer, Mol. Plant-Microbe Interact. 4:469-476, 1991). The gene in complementation group X, designated hrpH, was subcloned on a 3.1-kb SalI fragment into pCPP30, a broad-host-range, mobilizable vector. The subclone restored the ability of hrpH mutant P. syringae pv. syringae 61-2089 to elicit the HR in tobacco. DNA sequence analysis of the 3.1-kb SalI fragment revealed a single open reading frame encoding an 81,956-Da preprotein with a typical amino-terminal signal peptide and no likely inner-membrane-spanning hydrophobic regions. hrpH was expressed in the presence of [S-35]methionine by using the T7 RNA polymerase-promoter system and vector pT7-3 in Escherichia coli and was shown to encode a protein with an apparent molecular weight of 83,000 on sodium dodecyl sulfate-polyacrylamide gels. The HrpH protein in E. coli was located in the membrane fraction and was absent from the periplasm and cytoplasm. The HrpH protein possessed similarity with several outer membrane proteins that are known to be involved in protein or phage secretion, including the Klebsiella oxytoca PulD protein, the Yersinia enterocolitica YscC protein, and the pIV protein of filamentous coliphages. All of these proteins possess a possible secretion motif, GG(X)12VP(L/F)LXXIPXIGXL(F/L), near the carboxyl terminus, and they lack a carboxyl-terminal phenylalanine, in contrast to other outer membrane proteins with no known secretion function. These results suggest that the P. syringae pv. syringae HrpH protein is involved in the secretion of a proteinaceous HR elicitor.