14-3-3 proteins play a regulatory role in a diverse array of cellular functions such as apoptosis, regulation of the cell cycle, and regulation of gene transcription. The phytotoxin fusicoccin specifically induces association of virtually any 14-3-3 protein to plant plasma membrane H(+)-ATPase. The 14-3-3 binding site in the Arabidopsis plasma membrane H(+)-ATPase AHA2 was localized to the three C-terminal residues of the enzyme (Tyr(946)-Thr-Val). Binding of 14-3-3 protein to this target was induced by phosphorylation of Thr(947) (K(D) = 88 nM) and was in practice irreversible in the presence of fusicoccin (K(D) = 7 nM). Mass spectrometry analysis demonstrated that AHA2 expressed in yeast was phosphorylated at Thr(947). We conclude that the extreme end of AHA2 contains an unusual high-affinity binding site for 14-3-3 protein.

Polygalacturonase-inhibiting proteins (PGIPs) are plant cell wall proteins that protect plants from fungal invasion. They interact with endo polygalacturonases secreted by phytopathogenic fungi, inhibit their enzymatic activity, and favor the accumulation of oligogalacturonides, which activate plant defense responses. PGIPs are members of the leucine-rich repeat (LRR) protein family that in plants play crucial roles in development, defense against pathogens, and recognition of beneficial microbes. Here we report the crystal structure at 1.7-Å resolution of a PGIP from Phaseolus vulgaris . The structure is characterized by the presence of two β-sheets instead of the single one originally predicted by modeling studies. The structure also reveals a negatively charged surface on the LRR concave face, likely involved in binding polygalacturonases. The structural information on PGIP provides a basis for designing more efficient inhibitors for plant protection.

Two members of the pgip gene family ( pgip‐1 and pgip‐2 ) of Phaseolus vulgaris L. were expressed separately in Nicotiana benthamiana and the ligand specificity of their products was analysed by surface plasmon resonance (SPR). Polygalacturonase‐inhibiting protein‐1 (PGIP‐1) was unable to interact with PG from Fusarium moniliforme and interacted with PG from Aspergillus niger ; PGIP‐2 interacted with both PGs. Only eight amino acid variations distinguish the two proteins: five of them are confined within the β‐sheet/β‐turn structure and two of them are contiguous to this region. By site‐directed mutagenesis, each of the variant amino acids of PGIP‐2 was replaced with the corresponding amino acid of PGIP‐1, in a loss‐of‐function approach. The mutated PGIP‐2s were expressed individually in N.benthamiana , purified and subjected to SPR analysis. Each single mutation caused a decrease in affinity for PG from F.moniliforme ; residue Q253 made a major contribution, and its replacement with a lysine led to a dramatic reduction in the binding energy of the complex. Conversely, in a gain‐of‐function approach, amino acid K253 of PGIP‐1 was mutated into the corresponding amino acid of PGIP‐2, a glutamine. With this single mutation, PGIP‐1 acquired the ability to interact with F.moniliforme PG.

Summary: Recognition of endogenous molecules acting as ‘damage‐associated molecular patterns’ (DAMPs) is a key feature of immunity in both animals and plants. Oligogalacturonides (OGs), i.e. fragments derived from the hydrolysis of homogalacturonan, a major component of pectin are a well known class of DAMPs that activate immunity and protect plants against several microbes. However, hyper‐accumulation of OGs severely affects growth, eventually leading to cell death and clearly pointing to OGs as players in the growth‐defence trade‐off. Here we report a mechanism that may control the homeostasis of OGs avoiding their deleterious hyper‐accumulation. By combining affinity chromatography on acrylamide‐trapped OGs and other procedures, an Arabidopsis thaliana enzyme that specifically oxidizes OGs was purified and identified. The enzyme was named OG OXIDASE 1 (OGOX1) and shown to be encoded by the gene At4g20830. As a typical flavo‐protein, OGOX1 is a sulphite‐sensitive H₂O₂‐producing enzyme that displays maximal activity on OGs with a degree of polymerization >4. OGOX1 belongs to a large gene family of mainly apoplastic putative FAD‐binding proteins [Berberine Bridge Enzyme‐like (BBE‐like); 27 members], whose biochemical and biological function is largely unexplored. We have found that at least four BBE‐like enzymes in Arabidopsis are OG oxidases (OGOX1–4). Oxidized OGs display a reduced capability of activating the immune responses and are less hydrolysable by fungal polygalacturonases. Plants overexpressing OGOX1 are more resistant to Botrytis cinerea, pointing to a crucial role of OGOX enzymes in plant immunity.

To invade a plant tissue, phytopathogenic fungi produce several cell wall-degrading enzymes; among them, endo polygalacturonase (PG) catalyzes the fragmentation and solubilization of homogalacturonan. Polygalacturonase-inhibiting proteins (PGIPs), found in the cell wall of many plants, counteract fungal PGs by forming specific complexes with them. We report the crystal structure at 1.73 Å resolution of PG from the phytopathogenic fungus Fusarium moniliforme ( Fm PG). The structure of Fm PG was useful to study the mode of interaction of the enzyme with PGIP-2 from Phaseolus vulgaris. Several amino acids of Fm PG were mutated, and their contribution to the formation of the complex with PGIP-2 was investigated by surface plasmon resonance. The residues Lys-269 and Arg-267, located inside the active site cleft, and His-188, at the edge of the active site cleft, are critical for the formation of the complex, which is consistent with the observed competitive inhibition of the enzyme played by PGIP-2. The replacement of His-188 with a proline or the insertion of a tryptophan after position 270, variations that both occur in plant PGs, interferes with the formation of the complex. We suggest that these variations are important structural requirements of plant PGs to prevent PGIP binding.