BACKGROUND Reactive oxygen species (ROS) production is an early event in the immune response of plants. ROS production affects the redox-based modification of cysteine residues in redox proteins, which contribute to protein functions such as enzymatic activity, protein-protein interactions, oligomerization, and intracellular localization. Thus, the sensitivity of cysteine residues to changes in the cellular redox status is critical to the immune response of plants. METHODS We used disulfide proteomics to identify immune response-related redox proteins. Total protein was extracted from rice cultured cells expressing constitutively active or dominant-negative OsRacl, which is a key regulator of the immune response in rice, and from rice cultured cells that were treated with probenazole, which is an activator of the plant immune response, in the presence of the thiol group-specific fluorescent probe monobromobimane (mBBr), which was a tag for reduced proteins in a differential display two-dimensional gel electrophoresis. The mBBr fluorescence was detected by using a charge-coupled device system, and total protein spots were detected using Coomassie brilliant blue staining. Both of the protein spots were analyzed by gel image software and identified using MS spectrometry. The possible disulfide bonds were identified using the disulfide bond prediction software. Subcellular localization and bimolecular fluorescence complementation analysis were performed in one of the identified proteins: Oryza sativa cold shock protein 2 (OsCSP2). RESULTS We identified seven proteins carrying potential redox-sensitive cysteine residues. Two proteins of them were oxidized in cultured cells expressing DN-OsRac1, which indicates that these two proteins would be inactivated through the inhibition of OsRac1 signaling pathway. One of the two oxidized proteins, OsCSP2, contains 197 amino acid residues and six cysteine residues. Site-directed mutagenesis of these cysteine residues revealed that a Cys140 mutation causes mislocalization of a green fluorescent protein fusion protein in the root cells of rice. Bimolecular fluorescence complementation analysis revealed that OsCSP2 is localized in the nucleus as a homo dimer in rice root cells. CONCLUSIONS The findings of the study indicate that redox-sensitive cysteine modification would contribute to the immune response in rice.

中文翻译：

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水稻培养细胞中的二硫键蛋白质组学对水稻中的OsRacl和丙二唑相关的免疫信号通路的响应。

背景技术活性氧物质（ROS）的产生是植物免疫应答中的早期事件。ROS的产生会影响氧化还原蛋白中基于半胱氨​​酸残基的氧化还原修饰，从而有助于蛋白质功能，例如酶活性，蛋白质-蛋白质相互作用，寡聚和细胞内定位。因此，半胱氨酸残基对细胞氧化还原状态变化的敏感性对植物的免疫应答至关重要。方法我们使用二硫键蛋白质组学来鉴定与免疫反应相关的氧化还原蛋白。从表达组成性活性或显性负性OsRacl的水稻培养细胞中提取总蛋白，OsRacl是水稻免疫应答的关键调节剂，并从经过丙二唑处理的水稻培养细胞中提取总蛋白，Pronazole是植物免疫应答的激活剂，硫醇基团特异性荧光探针单溴二苯醚（mBBr）的存在，它是差异显示二维凝胶电泳中还原蛋白的标签。使用电荷耦合器件系统检测mBBr荧光，并使用考马斯亮蓝染色检测总蛋白斑点。通过凝胶图像软件分析两个蛋白质斑点，并使用MS光谱法鉴定。使用二硫键预测软件可以识别可能的二硫键。亚细胞定位和双分子荧光互补分析是在一种鉴定的蛋白质：水稻冷激蛋白2（OsCSP2）中进行的。结果我们确定了七个带有潜在的氧化还原敏感的半胱氨酸残基的蛋白质。其中两种蛋白质在表达DN-OsRac1的培养细胞中被氧化，这表明这两种蛋白质将通过抑制OsRac1信号通路而失活。两种氧化蛋白之一OsCSP2包含197个氨基酸残基和6个半胱氨酸残基。这些半胱氨酸残基的定点诱变表明，Cys140突变会导致水稻根细胞中绿色荧光蛋白融合蛋白的错误定位。双分子荧光互补分析表明，OsCSP2在水稻根细胞中以同源二聚体的形式存在于细胞核中。结论研究的结果表明，氧化还原敏感的半胱氨酸修饰将有助于水稻的免疫反应。这表明这两种蛋白将通过抑制OsRac1信号通路而失活。两种氧化蛋白之一OsCSP2包含197个氨基酸残基和6个半胱氨酸残基。这些半胱氨酸残基的定点诱变表明，Cys140突变会导致水稻根细胞中绿色荧光蛋白融合蛋白的错误定位。双分子荧光互补分析表明，OsCSP2在水稻根细胞中以同源二聚体的形式存在于细胞核中。结论研究的结果表明，氧化还原敏感的半胱氨酸修饰将有助于水稻的免疫反应。这表明这两种蛋白将通过抑制OsRac1信号通路而失活。两种氧化蛋白之一OsCSP2包含197个氨基酸残基和6个半胱氨酸残基。这些半胱氨酸残基的定点诱变表明，Cys140突变会导致水稻根细胞中绿色荧光蛋白融合蛋白的错误定位。双分子荧光互补分析表明，OsCSP2在水稻根细胞中以同源二聚体的形式存在于细胞核中。结论研究的结果表明，氧化还原敏感的半胱氨酸修饰将有助于水稻的免疫反应。这些半胱氨酸残基的定点诱变表明，Cys140突变会导致水稻根细胞中绿色荧光蛋白融合蛋白的错误定位。双分子荧光互补分析表明，OsCSP2在水稻根细胞中以同源二聚体的形式存在于细胞核中。结论研究的结果表明，氧化还原敏感的半胱氨酸修饰将有助于水稻的免疫反应。这些半胱氨酸残基的定点诱变表明，Cys140突变会导致水稻根细胞中绿色荧光蛋白融合蛋白的错误定位。双分子荧光互补分析表明，OsCSP2在水稻根细胞中以同源二聚体的形式存在于细胞核中。结论研究的结果表明，氧化还原敏感的半胱氨酸修饰将有助于水稻的免疫反应。