A 17-amino acid peptide was selectively cleaved from the highly variant C terminus of the 33-kDa 14-3-3 isoform occurring in fusicoccin receptor preparations from maize and was sequenced. The determined C-terminal sequence was identical to that of the already known maize 14-3-3 homolog GF14-6, thus prompting the use of recombinant GF14-6 in an in vitro protein-protein interaction study. The cDNA of GF14-6 was expressed in Escherichia coli as a 32 P-phosphorylatable glutathione S-transferase fusion protein and was used as a probe in overlay experiments with H ؉ -ATPase partially purified from maize roots. The results demonstrated that the recombinant protein specifically bound to H ؉ -ATPase. The binding was dependent on Mg 2؉ and was strongly increased by fusicoccin. Controlled trypsin digestion of H ؉ -ATPase abolished the association with GF14-6, a finding that was suggestive of an interaction with the C terminus of the enzyme. To confirm this result, the Cterminal domain of H ؉ -ATPase was expressed as a glutathione S-transferase fusion peptide and was used in overlay experiments. GF14-6 was also able to bind to the isolated C terminus, but only in the presence of fusicoccin.

Plant pathogens negatively affect agricultural production by reducing the plant yield and worsening the nutritional and qualitative characteristics of the harvest. To limit the damage caused by pathogen infection, crops are treated with large doses of pesticides, which cause soil and groundwater pollution. The use of crop varieties genetically resistant to necrotrophs represents a more sustainable solution but is limited by the scarcity of resistance genes to be integrated into crops. For this reason, the identification of new

Pure preparations of FC receptors, obtained under nondenaturing conditions, showed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis two doublets of proteins with apparent molecular masses of 30 and 90 kD. In the present paper we describe the isolation and identification of the primary structure of the 30-kD doublet proteins. Sequencing studies of peptides resulting from the digestion of the 30-kD protein showed a full identity with a 14-3-3-like protein from corn, named CF14. The 14-3-3 family is a class of proteins that is widely distributed in eukaryotes and is known to play various regulatory roles. The 30-kD protein has been immunologically identified by specific antibodies prepared against a synthetic peptide based on the determined amino acid sequence. A similar protein is recognized in partially purified FC receptor preparations from bean and spinach leaves.Although in the last few years progress has been made in the elucidation of mode of action of FC (Aducci et al., 1988; Johansson et al., 1993; Rasi-Caldogno et al., 1993), the primary structure of the FC receptor is still unknown. Different groups have reported on the purification of FC-binding proteins from various plants (de Boer et al., 1989; Feyerabend and Weiler, 1989; Oecking and Weiler, 1991), showing that the putative FC receptor is a protein in the range of 30 to 35 kD.Recently we have described the purification of the FC receptor from com (Zea mays L.) shoots using HPLC (Aducci et al., 1993). SDS-PAGE of highly purified samples revealed the occurrence of two doublets corresponding to proteins of * ComesDondine author; fax 39-6-2023500. tritiated azido-FC, resulted in labeling of the 90-kD protein under conditions that did not affect FC binding. In contrast, labeling of the 30-kD protein occurred only under conditions that severely impaired the binding activity. From our data we inferred that the minimal functional FC receptor has an apparent molecular mass of 90 kD. However, the presence of the 30-kD protein in fractions retaining an FC-binding capacity after extensive purification (5000-fold increase of specific FC-binding activity) suggests a tight association of the two proteins during chromatographic separations, including gel filtration, when performed under nondissociating conditions. This suggests a possible regulatory role for the 30-kD protein in the FC signaling pathway. These results prompted us to further purify the FC receptor to obtain pure samples of the 30-and 90-kD proteins suitable for primary structure determination, cloning, and expression.In this paper we report on the separation of the 30-and 90-kD proteins from com by RP HPLC chromatography and partial elucidation of the primary structure of the 30-kD protein and its immunological identification by antibodies directed to a synthetic peptide based on the determined amino acid sequence. MATERIALS AND METHODS ChemicalsFC was prepared as described by Ballio et al. (1968).[3H]FC was obtained according to Ballio et al. (1980). Chemicals used for gel electrophor...

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