Benedetta Mattei

Binding of 14-3-3 Protein to the Plasma Membrane H+-ATPase AHA2 Involves the Three C-terminal Residues Tyr946-Thr-Val and Requires Phosphorylation of Thr947

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 (Tyr946-Thr-Val). Binding of 14-3-3 protein to this target was induced by phosphorylation of Thr947 (KD = 88 nm) and was in practice irreversible in the presence of fusicoccin (KD = 7 nm). Mass spectrometry analysis demonstrated that AHA2 expressed in yeast was phosphorylated at Thr947. We conclude that the extreme end of AHA2 contains an unusual high-affinity binding site for 14-3-3 protein.

The specificity of polygalacturonase-inhibiting protein (PGIP): a single amino acid substitution in the solvent-exposed β-strand/β-turn region of the leucine-rich repeats (LRRs) confers a new recognition capability

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.

The crystal structure of polygalacturonase-inhibiting protein (PGIP), a leucine-rich repeat protein involved in plant defense.

Polygalacturonase-inhibiting proteins (PGIPs) are plant cell wall proteins that protect plants from fungal invasion. They interact with endopolygalacturonases 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.

Structural Requirements of Endopolygalacturonase for the Interaction with Pgip (Polygalacturonase-Inhibiting Protein)

To invade a plant tissue, phytopathogenic fungi produce several cell wall-degrading enzymes; among them, endopolygalacturonase (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 (FmPG). The structure of FmPG was useful to study the mode of interaction of the enzyme with PGIP-2 from Phaseolus vulgaris. Several amino acids of FmPG 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.

Polygalacturonase-inhibiting proteins (PGIPs) with different specificities are expressed in Phaseolus vulgaris

The pgip-1 gene of Phaseolus vulgaris, encoding a polygalacturonase-inhibiting protein (PGIP), PGIP-1 (P. Toubart, A. Desiderio, G. Salvi, F. Cervone, L. Daroda, G. De Lorenzo, C. Bergmann, A. G. Darvill, and P. Albersheim, Plant J. 2:367-373, 1992), was expressed under control of the cauliflower mosaic virus 35S promoter in tomato plants via Agrobacterium tumefaciens-mediated transformation. Transgenic tomato plants with different expression levels of PGIP-1 were used in infection experiments with the pathogenic fungi Fusarium oxysporum f. sp. lycopersici, Botrytis cinerea, and Alternaria solani. No evident enhanced resistance, compared with the resistance of untransformed plants, was observed. The pgip-1 gene was also transiently expressed in Nicotiana benthamiana with potato virus X (PVX) as a vector. PGIP-1 purified from transgenic tomatoes and PGIP-1 in crude protein extracts of PVX-infected N. benthamiana plants were tested with several fungal polygalacturonases (PGs). PGIP-1 from both plant sources exhibited a specificity different from that of PGIP purified from P. vulgaris (bulk bean PGIP). Notably, PGIP-1 was unable to interact with a homogeneous PG from Fusarium moniliforme, as determined by surface plasmon resonance analysis, while the bulk bean PGIP interacted with and inhibited this enzyme. Moreover, PGIP-1 expressed in tomato and N. benthamiana had only a limited capacity to inhibit crude PG preparations from F. oxysporum f. sp. lycopersici, B. cinerea, and A. solani. Differential affinity chromatography was used to separate PGIP proteins present in P. vulgaris extracts. A PGIP-A with specificity similar to that of PGIP-1 was separated from a PGIP-B able to interact with both Aspergillus niger and F. moniliforme PGs. Our data show that PGIPs with different specificities are expressed in P. vulgaris and that the high-level expression of one member (pgip-1) of the PGIP gene family in transgenic plants is not sufficient to confer general, enhanced resistance to fungi.

Two Arabidopsis thaliana genes encode functional pectin methylesterase inhibitors1

We have identified, expressed and characterized two genes from Arabidopsis thaliana (AtPMEI‐1 and AtPMEI‐2) encoding functional inhibitors of pectin methylesterases. AtPMEI‐1 and AtPMEI‐2 are cell wall proteins sharing many features with the only pectin methylesterase inhibitor (PMEI) characterized so far from kiwi fruit. Both Arabidopsis proteins interact with and inhibit plant‐derived pectin methylesterases (PMEs) but not microbial enzymes. The occurrence of functional PMEIs in Arabidopsis indicates that a mechanism of controlling pectin esterification by inhibition of endogenous PMEs is present in different plant species.

Mutagenesis of endopolygalacturonase from Fusarium moniliforme: histidine residue 234 is critical for enzymatic and macerating activities and not for binding to polygalacturonase-inhibiting protein (PGIP).

The sequence encoding the endopolygalacturonase (PG) of Fusarium moniliforme was cloned into the E. coli/yeast shuttle vector Yepsec1 for secretion in yeast. The recombinant plasmid (pCC6) was used to transform Saccharomyces cerevisiae strain S150-2B; transformed yeast cells were able to secrete PG activity into the culture medium. The enzyme (wtY-PG) was purified, characterized, and shown to possess biochemical properties similar to those of the PG purified from F. moniliforme. The wtY-PG was able to macerate potato medullary tissue disks and was inhibited by the polygalacturonase-inhibiting protein (PGIP) purified from Phaseolus vulgaris. The sequence encoding PG in pCC6 was subjected to site-directed mutagenesis. Three residues in a region highly conserved in all the sequences known to encode PGs were separately mutated: His 234 was mutated into Lys (H 234-->K), and Ser 237 and Ser 240 into Gly (S 237-->G and S 240-->G). Each of the mutated sequences was used to transform S. cerevisiae and the mutated enzymes were purified and characterized. Replacement of His 234 with Lys abolished the enzymatic activity, confirming the biochemical evidence that a His residue is critical for enzyme activity. Replacement of either Ser 237 or Ser 240 with Gly reduced the enzymatic activity to 48% and 6%, respectively, of the wtY-PG. When applied to potato medullary tissue, F. moniliforme PG and wtY-PG caused comparable maceration, while the variant PGs exhibited a limited (S 234-->G and S 240-->G) or null (H 234-->K) macerating activity. The interaction between the variant enzymes and the P. vulgaris PGIP was investigated using a biosensor based on surface plasmon resonance (BIAlite). The three variant enzymes were still able to interact and bind to PGIP with association constants comparable to that of the wild type enzyme.

Polygalacturonase-inhibiting protein interacts with pectin through a binding site formed by four clustered residues of arginine and lysine

Polygalacturonase-inhibiting protein (PGIP) is a cell wall protein that inhibits fungal polygalacturonases (PGs) and retards the invasion of plant tissues by phytopathogenic fungi. Here, we report the interaction of two PGIP isoforms from Phaseolus vulgaris (PvPGIP1 and PvPGIP2) with both polygalacturonic acid and cell wall fractions containing uronic acids. We identify in the three-dimensional structure of PvPGIP2 a motif of four clustered arginine and lysine residues (R183, R206, K230, and R252) responsible for this binding. The four residues were mutated and the protein variants were expressed in Pichia pastoris. The ability of both wild-type and mutated proteins to bind pectins was investigated by affinity chromatography. Single mutations impaired the binding and double mutations abolished the interaction, thus indicating that the four clustered residues form the pectin-binding site. Remarkably, the binding of PGIP to pectin is displaced in vitro by PGs, suggesting that PGIP interacts with pectin and PGs through overlapping although not identical regions. The specific interaction of PGIP with polygalacturonic acid may be strategic to protect pectins from the degrading activity of fungal PGs.

Identification by 2-D DIGE of apoplastic proteins regulated by oligogalacturonides in Arabidopsis thaliana

Oligogalacturonides (OGs) are elicitors of plant defence responses released from the homogalacturonan of the plant cell wall during the attack by pathogenic micro‐organisms. The signalling pathway mediated by OGs remains poorly understood, and no proteins involved in their signal perception and transduction have yet been identified. In order to shed light into the molecular pathways regulated by OGs, a differential proteomic analysis has been carried out in Arabidopsis. Proteins from the apoplastic compartment were isolated and their expression compared between control and OG‐treated seedlings. 2‐D gels and difference in gel electrophoresis (DIGE) techniques were used to compare control and treated proteomes in the same gel. The analysis of subcellular proteomes from seedlings allowed the identification of novel and low abundance proteins that otherwise remain masked when total cellular extracts are investigated. The DIGE technique showed to be a powerful tool to overcome the high interexperiment variation of 2‐D gels. Differentially expressed apoplastic proteins were identified by MS and included proteins putatively involved in recognition as well as proteins whose PTMs are regulated by OGs. Our findings underscore the importance of cell wall as a source of molecules playing a role in the perception of pathogens and provide candidate proteins involved in the response to OGs.

Targeted Modification of Homogalacturonan by Transgenic Expression of a Fungal Polygalacturonase Alters Plant Growth

Pectins are a highly complex family of cell wall polysaccharides comprised of homogalacturonan (HGA), rhamnogalacturonan I and rhamnogalacturonan II. We have specifically modified HGA in both tobacco (Nicotiana tabacum) and Arabidopsis by expressing the endopolygalacturonase II of Aspergillus niger (AnPGII). Cell walls of transgenic tobacco plants showed a 25% reduction in GalUA content as compared with the wild type and a reduced content of deesterified HGA as detected by antibody labeling. Neutral sugars remained unchanged apart from a slight increase of Rha, Ara, and Gal. Both transgenic tobacco and Arabidopsis were dwarfed, indicating that unesterified HGA is a critical factor for plant cell growth. The dwarf phenotypes were associated with AnPGII activity as demonstrated by the observation that the mutant phenotype of tobacco was completely reverted by crossing the dwarfed plants with plants expressing PGIP2, a strong inhibitor of AnPGII. The mutant phenotype in Arabidopsis did not appear when transformation was performed with a gene encoding AnPGII inactivated by site directed mutagenesis.