Purification and Cloning of Elicitor Binding Protein(s) from Soybean

这项研究的长期目标是了解植物细胞如何感知和响应细胞外信号。目前正在研究的系统是由植物病原真菌巨型疫霉菌丝壁产生的寡糖(激发子)诱导植物防御反应，诱导大豆中植物防御素的积累。甘西尼娜。关于菌丝壁葡聚糖的激发子(一种分枝的七β-葡萄糖苷)的结构，以及由激发子诱导的编码植物保卫素生物合成所需的酶的基因的识别和调控，已经获得了大量的信息。然而，关于植物细胞感知激发子的机制，或者该信号如何传递到细胞核以启动基因表达的变化，人们知之甚少。这项研究集中在激发子刺激的信号转导途径的第一步，即通过质膜上定位的受体识别七β-葡萄糖苷激发子。这项拟议的研究有三个主要目标。第一个目标是从大豆根微粒体膜中分离、纯化和鉴定七-β-葡萄糖苷激发子结合蛋白(EBPS)。已有证据表明，起源于质膜的大豆根微体中存在一类特异的、高亲和力(解离常数为1 nM)的EBPs。这些EBPs已经被成功地用洗涤剂从膜上增溶，并且溶解的EBPs保持了它们对激发子的高亲和力和特异性。具有七-β-葡萄糖苷激发子结合活性的蛋白质的数量和鉴定将通过光亲和标记来确定。通过亲和层析对EBPs进行纯化，制备抗结合蛋白(S)抗体。第二个目标是分离和鉴定编码EBPs的cdna序列。携带EPB序列的克隆将通过用放射性标记的七β-葡萄糖苷激发子筛选cDNA表达文库来鉴定。或者，从纯化的EBPs中获得的部分蛋白质序列将被用来制备合成的核酸探针，用来筛选cDNA文库。这些克隆及其衍生序列将被用来确定EBPs中可能与其在信号转导中的作用有关的结构和功能结构域。第三个目标将在时间允许的情况下进行，以证明在这些研究中确定的EBPs是激发子的生理受体(S)。序列比较和截短的EBP基因的表达将被用来识别和描绘EBPs的功能结构域。野生型和突变型EBPs在不含内源EBPs的植物细胞中的转基因表达，EBPs的组织特异性定位与植物防御反应的定位，以及反义EPB序列对七-β-葡萄糖苷诱导的植物防御反应表达的影响的测定，以证明EBPs在激发子介导的信号转导中的生理意义。这项研究的长期目标是了解植物细胞如何感知和响应细胞外信号。正在研究的系统是在大豆植株中诱导防御反应。这种防御反应是由植物暴露于某些碳水化合物(激发子)触发的，这些碳水化合物来自一种专门感染大豆的病原真菌的丝状壁。作为对激发子的响应，大豆植株合成并积累了被称为植物抗毒素的保护性化学物质。这个项目更直接的目标是为激发子鉴定和表征植物受体。对激发子受体的详细描述将是了解植物检测和响应病原体的信号转导途径的重要一步。

The long term goal of this research is to understand how plant cells perceive and respond to extracellular signals. The system being studied is the induction of plant defense responses, phytoalexin accumulation in soybean (Glycine max) induced by oligosaccharides (elicitors) originating from the mycelial wall of a phytopathogenic fungus, Phytophthora megasperma f. sp. glycinea. Considerable information has been obtained about the structure of an elicitor (a branched hepta-beta-glucoside) derived from mycelial wall glucans and about the identity and regulation of elicitor- induced genes encoding enzymes required for the biosynthesis of the phytoalexins. However, little is known about the mechanisms by which plant cells perceive the elicitor, or how that signal is transmitted to the cell nucleus to initiate changes in gene expression. The research described in this proposal focuses on the first step in the elicitor-stimulated signal transduction pathway, that is, the recognition of a hepta-beta-glucoside elicitor by a plasma-membrane localized receptor. The proposed research has three major goals. The first goal is the identification, purification, and characterization of hepta-beta-glucoside elicitor binding protein(s) (EBPs) from soybean root microsomal membranes. Evidence has already been obtained that a single class of specific, high-affinity (dissociation constant of 1 nM) EBPs exist in soybean root microsomes originating from the plasma membrane. These EBPs have been successfully solubilized from the membranes using detergents, and the solubilized EBPs retain their high affinity and specificity for the elicitor. The number and identity of the proteins that have hepta-beta-glucoside elicitor binding activity will be established by photo-affinity labeling. The EBPs will be purified by affinity chromatography and antibodies against the binding protein(s) will be prepared. The second goal is the isolation and characterization of cDNA sequences that encode EBPs. Clones carrying EPB sequences will be identified by screening cDNA expression libraries with radiolabeled hepta-beta-glucoside elicitor. Alternatively, partial protein sequences obtained from the purified EBPs will be used to prepare synthetic nucleic acid probes with which to screen cDNA libraries. These clones and their derived sequences will be used to identify possible structural and functional domains in the EBPs that might relate to their role in signal transduction. The third goal, which will be undertaken if time permits, is to demonstrate that the EBPs identified in these studies are physiological receptor(s) for the elicitor. Sequence comparisons and expression of truncated EBP genes will be used to identify and delineate functional domains of the EBPs. Transgenic expression of wild-type and mutant EBPs in plant cells that do not contain endogenous EBPs, tissue-specific localization of EBPs in relation to localization of plant defense responses, and determination of the effect of anti-sense EPB sequences on the expression of hepta-beta-glucoside-induced plant defense responses will be undertaken to demonstrate the physiological significance of the EBPs in elicitor-mediated signal transduction. %%% The long term goal of this research is to understand how plant cells perceive and respond to extracellular signals. The system being studied is the induction of a defense response in soybean plants. The defense response is triggered by exposure of the plant to certain carbohydrates (elicitors) which originate from the filament wall of a pathogenic fungus which specifically infects the soybean. In response to the elicitors the soybean plant synthesizes and accumulates protective chemicals known as phytoalexins. The more immediate goals of this project are to identify and characterize the plant receptors for the elicitors. Detailed characterization of the elicitor receptor will be an important step toward understanding of the signal transduction pathway by which plants detect and respond to pathogens.