Purification and Cloning of Hepta-beta Glucoside Elicitor- binding Protein(s) from Soybean

9723685哈恩：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。这些研究中使用的模型系统是通过源自植物致病的胚乳菌疫霉菌（Phytophthora sojae var. glycines）菌丝壁的寡糖（激发子）诱导植物防御反应，特别是大豆（Glycine max）中植物抗菌素的积累。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，以及激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，已经积累了相当多的信息。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。本提案中描述的研究重点是激发子刺激信号转导途径的第一步，即质膜定位受体对hepa - β -葡萄糖苷激发子的识别。最近的研究有力地表明，先前被认为是hepta- β -葡萄糖苷激发物结合蛋白（EBP）的75kda多肽实际上是一种非特异性葡聚糖结合蛋白。利用改进的提取程序和光亲和标记的初步研究已经确定了一个90 kDa的多肽作为候选EBP。本研究的总体目标是从大豆根膜中鉴定、纯化和克隆ebp，并获得ebp是hepta- β -葡萄糖苷激发子的生理受体的证据。第一个特定的目标是识别和纯化ebp。具有庚- β -葡萄糖苷激发子结合位点的可溶性膜蛋白制剂中多肽的数量和特性将通过光亲和标记来确定。光亲和标记的特异性将在与hepta- β -葡萄糖苷引发子结构相关的低聚葡萄糖苷的l配体竞争分析中严格建立。首先消除存在于膜上的与激活剂无活性β -葡聚糖结合的多肽，然后用配体亲和层析纯化ebp。部分氨基酸序列将被确定的多肽(s)发现有一个特定的hepta- β -葡萄糖苷激发子结合位点。第二个具体目标是获得针对ebp的抗体。这些抗体将用于在植物组织中定位ebp并分离编码ebp的基因。第三个具体目标是克隆编码ebp的cDNA。将合成退化寡核苷酸，并利用聚合酶链反应（PCR）从大豆根mRNA合成的异质单链cdna群体中扩增出双链DNA探针。该探针将用于筛选lambda gt10文库中编码ebp的cdna。或者，针对亲和纯化的EBP产生的抗体将用于筛选表达EBP序列的克隆的cDNA表达文库。获得的任何cDNA克隆都将被纯化和测序。由克隆的cDNA编码的蛋白特异性和高亲和力结合hepta- β -葡萄糖苷激发子的能力将被确定。cDNA克隆及其衍生序列将用于鉴定ebp可能的结构和功能域，这些结构和功能域可能与它们在信号转导中的作用有关。本更新申请中概述的研究将为未来的工作奠定基础，这些工作将测试ebp是否作为生理受体起作用，并识别和表征与ebp相互作用的成分，以及可能在诱导植物细胞中导致植物抗毒素积累的细胞信号通路下游受体中起作用的成分。这些研究的结果应该为植物细胞感知和响应细胞外信号的机制提供更多的见解。这些研究也增加了我们对碳水化合物信号分子（低聚糖）在多种生物过程调节中的作用的理解。植物感知并响应病原体的攻击。对病原体感染的感知是通过检测与攻击相关的特定化合物称为激发子来介导的。细胞表面蛋白检测这些激发子的存在，这些激发子在植物细胞内发出各种过程的信号，以指挥防御反应。哈恩博士发现了一种大豆细胞表面蛋白，它能结合源自疫霉菌病原体的激发子。这种结合似乎对单一蛋白质具有高度特异性，并具有使其成为激发子受体候选物的特征。获得这个奖项后，哈恩博士将纯化并分离受体蛋白。从纯化蛋白中获得的数据将用于获得启动子结合蛋白的cDNA克隆。这项研究的结果将为植物感知病原体感染的机制提供信息。这些信息对于了解植物如何在恶劣环境中保持生长非常重要，并且在改善和修改植物对病原体挑战的反应方面具有潜在的实际应用价值。* * *

9723685 Hahn The long-term goal of this research is to understand how plant cells perceive and respond to extracellular signals. The model system used in these studies is the induction of plant defense responses, specifically phytoalexin accumulation, in soybean (Glycine max) by oligosaccharides (elicitors) originating from the mycelial wall of a phytopathogenic oomycete, Phytophthora sojae var. glycines. Considerable information has accrued 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, namely the recognition of a hepta-beta-glucoside elicitor by a plasma membrane-localized receptor. Recent studies strongly suggest that the 75 kDa polypeptide previously thought to be a hepta-beta-glucoside elicitor-binding protein (EBP) is, in fact, a non-specific glucan- binding protein. Preliminary studies using modified extraction procedures and photo-affinity labeling have now identified a 90 kDa polypeptide as a candidate EBP. The overall goal of the research proposed in this application is to identify, purify, and clone EBPs from soybean root membranes and to obtain evidence that the EBPs are physiological receptors for the hepta-beta-glucoside elicitor. The first specific goal is to identify and purify EBPs. The number and identity of polypeptides in solubilized membrane protein preparations that have hepta-beta-glucoside elicitor-binding sites will be established by photo- affinity labeling. The specificity of the photo-affinity labeling will be rigorously established in l igand competition assays with oligoglucosides structurally related to the hepta-beta-glucoside elicitor. Polypeptides present in the membranes that bind to elicitor-inactive beta-glucans will be eliminated first and then the EBPs will be purified by ligand-affinity chromatography. Partial amino acid sequences will be determined for the polypeptide(s) found to have a specific hepta-beta-glucoside elicitor-binding site. The second specific goal is to obtain antibodies against the EBPs. These antibodies will be used to localize EBPs in plant tissues and to isolate genes encoding EBPs. The third specific goal is to clone cDNA(s) that encode EBPs. Degenerate oligonucleotides will be synthesized and used to amplify, by PCR, a double-stranded DNA probe from a heterogeneous population of single-stranded cDNAs made from soybean root mRNA. This probe will be used to screen a lambda gt10 library for cDNAs encoding EBPs. Alternatively, antibodies generated against affinity-purified EBPs will be used to screen a cDNA expression library for clones expressing EBP sequences. Any cDNA clones that are obtained will be purified and sequenced. The ability of the protein(s) encoded by the cloned cDNA(s) to bind the hepta- beta-glucoside elicitor specifically and with high affinity will be determined. The cDNA 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 research outlined in this renewal application will lay the groundwork for future work that will test whether the EBPs function as physiological receptors and to identify and characterize components that interact with EBPs and might function downstream of the receptor in the cellular signaling pathway that results in the accumulation of phytoalexins in elicited plant cells. The results of these studies should provide additional insight into the mechanisms used by plant cells to perceive and respond to extracellular signals. These studies should also increase our understanding of the roles of carbohydrate signal molecules (oligosaccharins) in the regulation of biological processes in diverse organisms. Plants perceive and respond to pathogen attack. The perception of the infection of a pathogen is mediated by the detection of specific compounds termed elicitors are associated with the attack. Cell surface proteins detect the presence of these elicitors which signal a variety of processes within the plant cell to marshal defense reactions. Dr. Hahn has identified a soybean cell-surface protein that binds an elicitor originating from the pathogen Phytophthora. The binding appears to be highly specific to a single protein and has characteristics that make it a candidate for the elicitor receptor. With this award Dr. Hahn will purify and isolate the receptor protein. Data obtained from the purified protein will be used to obtain a cDNA clone of the elicitor binding protein. The results of this research will yield information on the mechanisms by which plants perceive infection of pathogens. This information is extremely important in understanding how plants maintain growth in a hostile world and it has potential practical applications I improving and modifying a plants response to pathogen challenge. ***