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。本文研究的系统是植物病原真菌巨藻疫霉（Phytophthora megasperma f. sp. glycinea）菌丝壁上产生的寡糖（激发子）诱导植物抗菌素在大豆（Glycine max）中积累的防御反应。关于从菌丝壁葡聚糖衍生的激发子（支链庚- β -葡萄糖苷）的结构，以及引发子诱导的编码植物抗毒素生物合成所需酶的基因的身份和调控，已经获得了相当多的信息。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。本提案所描述的研究重点是激发子刺激信号转导途径的第一步，即肝- β -葡萄糖苷激发子被质膜定位受体识别。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鉴定、纯化和表征庚- β -葡萄糖苷激发子结合蛋白（ebp）。已有证据表明，起源于质膜的大豆根微粒体中存在一类特异性高亲和力（解离常数为1 nM）的ebp。这些ebp已成功地用洗涤剂从膜上溶解，并且溶解后的ebp对激发剂保持了高亲和力和特异性。具有庚- β -葡萄糖苷激发子结合活性的蛋白的数量和特性将通过光亲和标记来确定。ebp将通过亲和层析纯化，并制备针对结合蛋白的抗体。第二个目标是分离和鉴定编码ebp的cDNA序列。携带EPB序列的克隆将通过放射性标记的hepta- β -葡萄糖苷激发子筛选cDNA表达文库进行鉴定。或者，从纯化的ebp中获得的部分蛋白质序列将用于制备合成核酸探针，用于筛选cDNA文库。这些克隆及其衍生序列将用于鉴定ebp中可能与它们在信号转导中的作用相关的结构和功能域。第三个目标，如果时间允许，将进行，是证明在这些研究中确定的ebp是触发子的生理受体。序列比较和截断的EBP基因的表达将用于识别和描述EBP的功能域。野生型和突变型ebp在不含内源性ebp的植物细胞中的转基因表达，ebp的组织特异性定位与植物防御反应定位的关系，并确定反义EPB序列对hepta- β -葡萄糖苷诱导的植物防御反应表达的影响，以证明ebp在激发剂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。正在研究的系统是大豆植物防御反应的诱导。防御反应是由植物暴露于某些碳水化合物（激发子）引发的，这些碳水化合物来自一种专门感染大豆的致病真菌的丝壁。作为对激发子的反应，大豆植物合成并积累了被称为植物抗毒素的保护性化学物质。本项目更直接的目标是鉴定和表征激发子的植物受体。激发子受体的详细表征将是理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.