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

辣椒抗性基因Bs3是由黄单胞菌转录激活因子样效应蛋白AvrBs3转录激活的。Bs3是一种结构独特的植物R蛋白，通过未知的方式引发细胞死亡。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，可诱导细胞增殖但不诱导细胞死亡。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将还原当量直接传递给O2，从而产生H2O2。在植物中，Bs3的表达导致H2O2-升高，但不增加iaa -水平，表明Bs3通过产生H2O2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YUCCA6多5倍。光度分析表明，带o2电荷的Bs3比带o2电荷的YUCCA6更不稳定，并解释了Bs3比YUCCA6产生更多H2O2的原因。结构域交换表明，Bs3和YUCCAs之间的功能差异取决于邻近保守半胱氨酸的多态性。酵母双杂交（Y2H）研究发现拟南芥转录因子TCP9可能是Bs3相互作用因子。TCP蛋白的DNA结合受氧化还原调控，TCP9激活异choris酸合成酶的表达，异choris酸合成酶是水杨酸合成的关键酶。值得注意的是，Bs3触发的细胞死亡与SA的增加相关，这与Bs3通过产生H2O2激活TCP9的模型一致。本提案将研究Bs3触发细胞死亡的两个不同方面：首先，我们希望阐明Bs3和YUCCA蛋白之间的功能差异，并定义因果多态性残基。其次，我们的目标是识别和研究Bs3触发细胞死亡的信号成分。为了研究Bs3-和yucca6依赖的H2O2在体内的产生，这两种蛋白将融合到视觉和功能报告蛋白中。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H2O2的存在。硫醇特异性、表位标记探针将与质谱（MS）结合使用，以鉴定Bs3/YUCCA6中氧化还原敏感的半胱氨酸残基。随后，我们将阐明给定巯基修饰的确切性质及其功能相关性。结合MS的共免疫沉淀（CoIP）将用于鉴定推定的Bs3/YUCCA6相互作用物。Bs3产生的H2O2可能诱导氧化还原调控信号组分的亚砜化。我们将在CoIP-MS中使用亚磺酸特异性、表位标记探针来鉴定氧化还原调节蛋白。随后，通过CoIP-MS鉴定的Bs3信号成分的功能相关性将通过分析相应的拟南芥和/或辣椒突变植物来澄清。总之，这些研究将首次揭示Bs3是如何引发植物防御反应的。

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.