Furutani Ayako, Tsuge Seiji, Ohnishi Kouhei, Hikichi Yasufumi, Oku Takashi, Tsuno Kazunori, Inoue Yasuhiro, Ochiai Hirokazu, Kaku Hisatoshi, Kubo Yasuyuki
Laboratory of Plant Pathology, Graduate School of Agriculture, Kyoto Prefectural University, Kyoto 606-8522, Japan.
J Bacteriol. 2004 Mar;186(5):1374-80. doi: 10.1128/JB.186.5.1374-1380.2004.
Xanthomonas oryzae pv. oryzae is a causal agent of bacterial leaf blight of rice. Recently, an efficient hrp-inducing medium, XOM2, was established for this bacterium. In this medium, more than 10 proteins were secreted from the wild-type strain of X. oryzae pv. oryzae. Many of these proteins disappeared or decreased in amount in culture on XOM2 when incubated with the strain that has a mutation in the hrp regulatory gene. Interestingly, the secretory protein profile of a mutant lacking a type III secretion system (TTSS), components of which are encoded by hrp genes, was similar to that of the wild-type strain except that a few proteins had disappeared. This finding suggests that many HrpXo-dependent secretory proteins are secreted via systems other than the TTSS. By isolating mutant strains lacking a type II secretion system, we examined this hypothesis. As expected, many of the HrpXo-dependent secretory proteins disappeared or decreased when the mutant was cultured in XOM2. By determining the N-terminal amino acid sequence, we identified one of the type II secretory proteins as a cysteine protease homolog, CysP2. Nucleotide sequence analysis revealed that cysP2 has an imperfect plant-inducible-promoter box, a consensus sequence which HrpXo regulons possess in the promoter region, and a deduced signal peptide sequence at the N terminus. By reverse transcription-PCR analysis and examination of the expression of CysP2 by using a plasmid harboring a cysP2::gus fusion gene, HrpXo-dependent expression of CysP2 was confirmed. Here, we reveal that the hrp regulatory gene hrpXo is also involved in the expression of not only hrp genes and type III secretory proteins but also some type II secretory proteins.
水稻白叶枯病菌是水稻细菌性条斑病的病原菌。最近，针对该细菌建立了一种高效的hrp诱导培养基XOM2。在这种培养基中，水稻白叶枯病菌野生型菌株分泌了10多种蛋白质。当与hrp调控基因突变的菌株一起在XOM2上培养时，这些蛋白质中的许多在培养物中消失或数量减少。有趣的是，缺乏III型分泌系统（TTSS）的突变体的分泌蛋白谱与野生型菌株相似，只是有几种蛋白质消失了。这一发现表明，许多依赖HrpXo的分泌蛋白是通过TTSS以外的系统分泌的。通过分离缺乏II型分泌系统的突变菌株，我们检验了这一假设。正如预期的那样，当突变体在XOM2中培养时，许多依赖HrpXo的分泌蛋白消失或减少。通过确定N端氨基酸序列，我们鉴定出一种II型分泌蛋白为半胱氨酸蛋白酶同源物CysP2。核苷酸序列分析表明，cysP2有一个不完美的植物诱导型启动子框，这是HrpXo调控子在启动子区域所具有的共有序列，并且在N端有一个推导的信号肽序列。通过逆转录PCR分析以及使用携带cysP2::gus融合基因的质粒检测CysP2的表达，证实了CysP2依赖HrpXo的表达。在此，我们揭示hrp调控基因hrpXo不仅参与hrp基因和III型分泌蛋白的表达，还参与一些II型分泌蛋白的表达。
