Pectin is synthesized in a highly methylesterified form in the Golgi cisternae and partially de‐methylesterified in muro by pectin methylesterases (PMEs). Arabidopsis thaliana produces a local and strong induction of PME activity during the infection of the necrotrophic fungus Botrytis cinerea. AtPME17 is a putative A. thaliana PME highly induced in response to B. cinerea. Here, a fine tuning of AtPME17 expression by different defence hormones was identified. Our genetic evidence demonstrates that AtPME17 strongly contributes to the pathogen‐induced PME activity and resistance against B. cinerea by triggering jasmonic acid–ethylene‐dependent PDF1.2 expression. AtPME17 belongs to group 2 isoforms of PMEs characterized by a PME domain preceded by an N‐terminal PRO region. However, the biochemical evidence for AtPME17 as a functional PME is still lacking and the role played by its PRO region is not known. Using the Pichia pastoris expression system, we demonstrate that AtPME17 is a functional PME with activity favoured by an increase in pH. AtPME17 performs a blockwise pattern of pectin de‐methylesterification that favours the formation of egg‐box structures between homogalacturonans. Recombinant AtPME17 expression in Escherichia coli reveals that the PRO region acts as an intramolecular inhibitor of AtPME17 activity.

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 32P-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 Mg2+ 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 C-terminal 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.

We have recently reported on the purification of the fusicoccin (FC) receptor from corn (Zea mays L.) and its identification by photoaffinity labeling (P. Aducci, A. Ballio, V. Fogliano, M.R. Fullone, M. Marra, N. Proietti [1993] Eur J Biochem 214: 339–345). 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 14–3–3like protein from corn, named GF14. 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.

Fusicoccin receptors are proteins that are widespread in plasma membrane of higher plants. They act as a perception system for the fungal metabolite fusicoccin, a toxin affecting plasma membrane transport by the stimulation of proton ATPase. FC represents a unique tool to elucidate the regulatory mechanisms underlying transport processes at the plasma membrane for its ability to elicit several hormone-like responses rapidly. This review will summarize the studies so far reported on localization, biochemical properties and purification of fusicoccin receptors and will also deal with more recent data on the role played by 14-3-3-like proteins in fusicoccin signalling.

Crude fusicoccin binding proteins and a partially purified plasma membrane H+-transporting ATPase (EC 3.6.1.34), both solubilized from maize tissues, were simultaneously inserted into liposomes by the freeze-thaw method. ATP-driven intravesicular acidification in the proteoliposomes, measured by the fluorescence quenching of the dye 9-amino-6-chloro-2-methoxyacridine, markedly increased upon addition of fusicoccin to the reconstituted system. This effect could not be observed when binding sites and ATPase preparations were separately reconstituted into the proteoliposomes, thus demonstrating that fusicoccin binding to its receptor is a prerequisite for ATPase stimulation.

Fusicoccin was shown to stimulate the ATP-driven, intravesicular acidification of liposomes reconstituted with crude fusicoccin receptors and the H⁺-translocating ATPase, both solubilized from maize (Zea mays L.) plasma membrane. The present paper reports optimal conditions for dual reconstitution and fusicoccin activation as well as the biochemical characterization of the effect of fusicoccin on this system. Fusicoccin stimulation of proton pumping was dependent on pH and fusicoccin concentration. Its specificity was demonstrated by the positive effect of two cotylenins that have a high affinity for fusicoccin receptors and by the negative response to 7,9-epideacetylfusicoccin, an inactive fusicoccin derivative. Kinetic measurements at different ATP concentrations showed that fusicoccin increases the V_max of the enzyme. Fusicoccin stimulation of maize H⁺-ATPase was also maintained when receptors from maize were substituted by those from spinach (Spinacia oleracea L.).