Isolation and biochemical characterization of fusicoccin-insensitive variant lines of Arabidopsis thaliana (L.) Heynh

Abstract

Arabidopsis thaliana lines have been isolated that are insensitive to the fungal toxin fusicoccin (FC). Initial screening was done by selecting for plants that either grew well on high concentrations of FC or did not respond to FC by increases in H⁺-extrusion. All selected plants were tested, in several additional rounds of screening, for binding to microsomal proteins of a ³H-labeled radioligand of fusicoccin. A novel assay allowing for the direct selection of individual plants exhibiting reduced binding of FC was developed and used as screening procedure. Independent variant lines (43) with stably expressed, reduced binding of FC were isolated and subjected to a detailed characterization of their binding sites. The lines could be subdivided into several distinct classes with respect to these characteristics. In class-I lines, the data indicate a partial conversion of high-affinity binding sites to a low-affinity state. In class-II lines, the affinity of the binding site to FC is strongly reduced while the number of sites, as well as several other biochemical parameters, is completely unchanged, suggesting a specific alteration in the properties of the fusicoccin-binding protein. In class-III lines, the ligand-binding protein complex, while retaining its high affinity, is destabilized at supraoptimal concentrations of FC (such as those used for screening). In wild-type plants, only the high-affinity binding site was detected. Combined, these data prove that the high-affinity sites represent the plant's FC receptor.