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. ***
