1.1.22
DISTRIBUTION AND HETEROLOGOUS EXPRESSION OF THE HEPTA-ß-GLUCOSIDE RECEPTOR INVOLVED IN PATHOGEN RECOGNITION IN SOYBEAN
J FLIEGMANN1, A MITHÖFER1, G NEUHAUS-URL2 and J EBEL1

1Botanisches Institut der Ludwig-Maximilians-Universität München, Menzinger Straße 67, D-80638 München, Germany; 2Friedrich Miescher-Institut, PO Box 2543, CH-4002 Basel, Switzerland

Background
High-affinity and specific ß-glucan-binding proteins appear to be responsible for signal perception leading to elicitor-induced defence responses in legumes. In soybean (Glycine max L.), the ß-glucan-binding proteins perceive the extracellular fungal cell wall-derived signal molecules at the plasma membrane and translate the signals into the cell, thereby activating specific cellular defence responses [1].

A low-abundance 75-kDa protein of soybean root membranes which inherits a high-affinity binding site for a hepta-1,3-1,6-ß-glucoside was purified to homogeneity by affinity chromatography [2] and the corresponding full-length cDNA was cloned. Functional expression of this cDNA in different heterologous systems was attempted in order to demonstrate high-affinity binding activity of the encoded protein.

Results and conclusions
The expression of the soybean ß-glucan-binding protein in insect cells (Spodoptera frugiperda Sf9) produced high amounts of the 75-kDa protein. The protein was not membrane-localized, showed no binding activity, but was useful for the generation of a very sensitive polyclonal antiserum. This enabled the detection of a soluble but inactive form of the 75-kDa protein in soybean apart from the expected active membrane-bound species. Transgenic plant cell cultures showed an identical distribution between soluble and membrane-localized forms of the binding protein as seen in soybean. The polyclonal antiserum and the cDNA probe served for the detection of binding proteins and their corresponding transcripts in different tissues of soybean as well as in other legumes. Sequence analysis of one of these homologues displayed high similarity with the ß-glucan-binding protein of soybean. Binding assays with microsomes, but not with the soluble fraction of transgenic plant cells, displayed high-affinity binding of the hepta-ß-glucoside ligand. Membrane targeting of the 75-kDa protein in insect cells was not sufficient to restore binding activity, indicating that a plant-specific component is necessary for membrane association and function.

This work was supported by the Deutsche Forschungsgemeinschaft (SFB 369).

References
1. Ebel J, Scheel D, 1997. The Mycota V Part A, pp. 85-105.
2. Mithöfer A, Lottspeich F, Ebel J, 1996. FEBS Letters 381, 203-207.