Elucidating the molecular mechanism of how the executor protein Bs3 from pepper triggers plant cell death

阐明辣椒执行蛋白 Bs3 触发植物细胞死亡的分子机制

• 批准号: 388775801
• 负责人: Professor Dr. Thomas Lahaye
• 金额: --
• 依托单位: Abteilung für Allgemeine Genetik
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2017
• 资助国家: 德国
• 起止时间: 2016-12-31 至 2017-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/388775801?language=en
• 关键词: Elucidating; molecular; mechanism; how; executor

The pepper resistance (R) gene Bs3 is transcriptionally activated by the Xanthomonas transcription activator-like effector protein AvrBs3. Bs3 is a structurally unique plant R protein that triggers cell death by unknown means. Bs3 is most related to YUCCAs, a plant-specific family of flavin-dependent monooxygenases (FMOs) that induce cell proliferation but not cell death. YUCCAs bind NADPH2 and O2 to convert indole-3-pyruvate (IPA) into auxin (Indole-3-acetic acid; IAA). Alternatively YUCCAs transfer reduction equivalents directly to O2 resulting in H2O2 production. In planta expression of Bs3 causes increased H2O2- but not increased IAA-levels, suggesting that Bs3 triggers cell death via H2O2 production. Indeed, recombinant Bs3 protein produces 5-fold more H2O2 than YUCCA6, a representative of the Arabidopsis YUCCA family. Photometric analysis suggests that O2-charged Bs3 is less stable than the O2-charged YUCCA6, and rationalizes why Bs3 produces more H2O2 than YUCCA6. Domain-swaps indicate that polymorphisms adjacent to a conserved cysteine determine functional differences between Bs3 and YUCCAs. Yeast two-hybrid (Y2H) studies identified the Arabidopsis transcription factor TCP9 as a putative Bs3 interactor. DNA binding of TCP proteins is redox-regulated and TCP9 activates expression of isochorismate synthase, a key enzyme of salicylic acid (SA) synthesis. Notably, Bs3-triggered cell death correlates with an increase in SA, which is in agreement with a model where Bs3 activates TCP9 via production of H2O2.Two distinct aspects of Bs3-triggered cell death will be studied within this proposal: Firstly, we want to elucidate functional differences between Bs3 and YUCCA proteins and to define the causal polymorphic residues. Secondly, we aim to identify and study the signal components that Bs3 employs to trigger cell death. To study Bs3- and YUCCA6-dependent H2O2 production in vivo both proteins will be fused to visual and functional reporters. In a complementary approach high-resolution microscopy will spatially correlate the presence of Bs3/YUCCA6 and H2O2. Thiol-specific, epitope-tagged probes will be used in conjunction with mass spectrometry (MS) to identify redox-sensitive cysteine residues in Bs3/YUCCA6. Subsequently we will elucidate the exact nature of given thiol modifications and their functional relevance. Coimmunoprecipitation (CoIP) linked to MS will be used to identify putative Bs3/YUCCA6 interactors. H2O2 produced by Bs3 is likely to induce sulfenylation of redox-regulated signal components. We will use sulfenic acid-specific, epitope-tagged probes in CoIP-MS to identify redox-regulated proteins. Subsequently functional relevance of putative Bs3 signalling components identified via CoIP-MS will be clarified by analysis of corresponding Arabidopsis and/or pepper mutant plants. In sum these studies will provide the first insights into how Bs3 triggers a plant defense reaction.

胡椒耐药性（R）基因BS3被Xanthomonas转录激活剂样效应蛋白AVRBS 3转录激活。 BS3是一种结构独特的植物R蛋白，可通过未知均值触发细胞死亡。 BS3与Yuccas是尤卡（Yuccas），这是一种植物特异性的黄素依赖性单加氧酶（FMO），可诱导细胞增殖但不引起细胞死亡。 YUCCAS结合NADPH2和O2，将吲哚-3-丙酮酸（IPA）转化为生长素（吲哚-3-乙酸； IAA）。或者，YUCCAS转移还原等效于直接导致H2O2产生。在BS3的植物表达中，导致H2O2--但没有增加IAA级别，这表明BS3通过H2O2产生触发细胞死亡。实际上，重组BS3蛋白的H2O2产生的H2O2比Yucca6（拟南芥丝兰家族的代表）高5倍。光度法分析表明，O2荷兰BS3的稳定性不如O2电荷YUCCA6，并且合理化为什么BS3产生的H2O2比YUCCA6更多。结构域 - 归结表明，与保守半胱氨酸相邻的多态性决定了BS3和YUCCAS之间的功能差异。酵母双杂交（Y2H）研究将拟南芥转录因子TCP9鉴定为推定的BS3相互作用者。 TCP蛋白的DNA结合是氧化还原调节的，TCP9激活了同义合成酶的表达，这是水杨酸（SA）合成的关键酶。值得注意的是，BS3触发的细胞死亡与SA的增加相关，这与一个模型一致，该模型在此提案中将研究BS3触发的细胞死亡的两个不同方面激活TCP9。首先，我们希望阐明BS3和Yucca Prote蛋白之间的功能差异，并将其固定在Proment中，并将其固定为defim。其次，我们旨在识别和研究BS3用于触发细胞死亡的信号成分。为了研究BS3-和YUCCA6依赖性H2O2在体内的产生，这两种蛋白质都将融合到视觉和功能记者中。在互补方法中，高分辨率显微镜将在空间上与BS3/YUCCA6和H2O2的存在相关。硫醇特异性，表位标记的探针将与质谱法（MS）结合使用，以鉴定BS3/YUCCA6中对氧化还原敏感的半胱氨酸残基。随后，我们将阐明给定硫醇修饰的确切性质及其功能相关性。与MS相关的共免疫沉淀（COIP）将用于识别推定的BS3/YUCCA6相互作用。 BS3产生的H2O2可能诱导氧化还原调节的信号成分的磺苯基化。我们将在COIP-MS中使用硫酸特异性，表位标记的探针来鉴定氧化还原调节的蛋白质。随后通过COIP-MS确定的推定BS3信号传导组件的功能相关性将通过分析相应的拟南芥和/或胡椒突变植物来阐明。总而言之，这些研究将提供有关BS3如何触发植物防御反应的第一个见解。