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.

辣椒抗性（R）基因Bs3被黄单胞菌属转录激活因子样效应蛋白AvrBs3转录激活。Bs3是一种结构独特的植物R蛋白，通过未知的方式触发细胞死亡。Bs3与YUCCAs最相关，YUCCAs是植物特异性的黄素依赖性单加氧酶（FMOs）家族，其诱导细胞增殖但不诱导细胞死亡。YUCCAs结合NADPH 2和O2，将吲哚-3-丙酮酸（IPA）转化为生长素（吲哚-3-乙酸; IAA）。另外，YUCCAs将还原当量直接转移到O2，导致H2O2的产生。Bs3在植物中的表达导致H2O2水平增加，但不增加IAA水平，表明Bs3通过H2O2产生触发细胞死亡。事实上，重组Bs3蛋白产生的H2O2比拟南芥YUCCA家族的代表YUCCA 6多5倍。光度分析表明，O2充电的Bs3是不稳定的比O2充电的YUCCA 6，并合理化为什么Bs3产生更多的H2O2比YUCCA 6。结构域交换表明邻近保守半胱氨酸的多态性决定了Bs3和YUCCAs之间的功能差异。酵母双杂交（Y2H）的研究确定了拟南芥转录因子TCP9作为一个假定的Bs3相互作用。TCP蛋白的DNA结合受氧化还原调节，TCP9激活水杨酸（SA）合成的关键酶异分支酸合酶的表达。值得注意的是，Bs3触发的细胞死亡与SA的增加相关，这与Bs3通过产生H2O2激活TCP9的模型一致。本研究将从两个方面对Bs3触发的细胞死亡进行研究：首先，我们希望阐明Bs3和YUCCA蛋白的功能差异，并确定致病的多态性残基。其次，我们的目标是识别和研究Bs3用于触发细胞死亡的信号成分。为了研究Bs3-和YUCCA 6-依赖的H2O2在体内的产生，两种蛋白质将融合到视觉和功能报告。在一种互补的方法中，高分辨率显微镜将在空间上关联Bs3/YUCCA 6和H2O2的存在。巯基特异性、表位标记的探针将与质谱法（MS）结合使用，以识别Bs3/YUCCA 6中氧化还原敏感的半胱氨酸残基。随后，我们将阐明巯基修饰的确切性质及其功能相关性。将使用与MS连接的免疫共沉淀（CoIP）来鉴定推定的Bs3/YUCCA 6相互作用物。由Bs3产生的H2O2可能诱导氧化还原调节信号组分的亚磺酰化。我们将在CoIP-MS中使用次磺酸特异性、表位标记的探针来鉴定氧化还原调节蛋白。随后，通过CoIP-MS鉴定的推定的Bs3信号传导组分的功能相关性将通过分析相应的拟南芥和/或辣椒突变体植物来澄清。总之，这些研究将为Bs3如何触发植物防御反应提供第一个见解。