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

辣椒抗性(R)基因Bs3被Xanthomonas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，它以未知的方式触发细胞死亡。BS3与丝兰关系最密切，丝兰是一种植物特有的黄素依赖单加氧酶(FMOS)家族，可以诱导细胞增殖，但不会导致细胞死亡。丝兰与NADPH2和O2结合，将吲哚-3-丙酮酸(IPA)转化为生长素(吲哚-3-乙酸；IAA)。或者，丝兰将还原当量直接转移到氧气中，从而产生过氧化氢。在植物中，BS3的表达导致过氧化氢水平增加，而不是IAA水平增加，这表明BS3通过产生过氧化氢来触发细胞死亡。事实上，重组BS3蛋白产生的过氧化氢比拟南芥丝兰家族的代表YUCCA6多5倍。光度分析表明，带O2的BS3比带O2的YUCCA6更不稳定，这解释了为什么BS3比YUCCA6产生更多的过氧化氢。结构域交换表明，BS3和丝兰之间的功能差异是由保守的半胱氨酸附近的多态决定的。酵母双杂交(Y2H)研究证实拟南芥转录因子TCP9可能是BS3相互作用因子。TCP9可激活水杨酸(SA)合成关键酶--异支酸合成酶的表达，使其与DNA结合受氧化还原调节。值得注意的是，BS3触发的细胞死亡与SA的增加相关，这与BS3通过产生过氧化氢激活TCP9的模型是一致的。在这一建议中，我们将研究BS3触发的细胞死亡的两个不同方面：首先，我们想要阐明BS3和丝兰蛋白之间的功能差异，并定义原因的多态残基。其次，我们的目标是鉴定和研究BS3用来触发细胞死亡的信号成分。为了研究依赖于BS3和YUCCA6的体内过氧化氢的产生，这两种蛋白质将被融合到视觉和功能报道中。在一种互补的方法中，高分辨率显微镜将在空间上关联BS3/YUCCA6和过氧化氢的存在。硫醇特异性、表位标记的探针将与质谱仪(MS)结合使用，以识别BS3/YUCCA6中对氧化还原敏感的半胱氨酸残基。随后，我们将阐明给定的硫醇修饰的确切性质及其功能相关性。与MS相连的免疫共沉淀(CoIP)将用于鉴定BS3/YUCCA6可能的相互作用。BS3产生的H_2O_2可能诱导氧化还原调节的信号成分的亚硫基化。我们将在CoIP-MS中使用磺酸特定的、表位标记的探针来识别氧化还原调节的蛋白质。随后，通过CoIP-MS鉴定的可能的BS3信号成分的功能相关性将通过对相应的拟南芥和/或辣椒突变植株的分析来阐明。总之，这些研究将为BS3如何触发植物防御反应提供第一个洞察力。

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.