G. De Lorenzo

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.

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.

A leucine-rich repeat receptor-like protein kinase (LRPKm1) gene is induced in Malus × domestica by Venturia inaequalis infection and salicylic acid treatment

A cDNA clone encoding a leucine-rich repeat (LRR) receptor-like protein kinase (LRPKm1) of Malus × domestica cv. Florina has been isolated using as a heterologous probe a cloned gene encoding a polygalacturonase-inhibiting protein (PGIP) of Phaseolus vulgaris L. A genomic clone containing the 5′-regulatory region and a 5′ portion of the open reading frame of the LRPKm1 gene has also been isolated. An open reading frame of 2997 nt (999 amino acids) was present in the cDNA clone, encoding a receptor-like protein comprising a 21 amino acid signal peptide for secretion, a leucine zipper, 23 LRRs, a putative membrane-spanning region and a serine/threonine protein kinase domain. LRPKm1 shows homology to the A. thaliana receptor-like protein kinase RLK5 and, to a minor extent, to PGIP. The LRPKm1 region from +5 to +600 exhibits an alternative reading frame that encodes a product corresponding to a proline-rich protein fragment homologous to several hydroxyproline-rich proteins. Southern blot analysis showed that LRPKm1 belongs to a multigene family and that there is length polymorphism of the hybridizing restriction fragments among different M. × domestica cultivars. Northern blot analysis was carried out on mRNA extracted from infected leaves of either cv. Florina (resistant to Venturia inaequalis) or cv. Golden Delicious (susceptible to V. inaequalis), and from tissues treated with salicylic acid. A 3500 bp transcript hybridizing at high stringency with the LRPKm1 cDNA accumulated in response to infection or salicylic acid treatment. Transcript accumulation was more intense in the incompatible interaction than in the compatible one. The possible involvement of this receptor-like protein kinase in resistance of apple to phytopathogenic fungi is discussed.

Fungal Invasion Enzymes and Their Inhibition

The first lines of defense of a plant against phytopathogenic fungi are the external cuticle and the polysaccharide-rich cell wall (Fig. 1). The vast majority of fungi need to breach these barriers to gain access to the plant tissue, and, once inside the tissue, to degrade the cell wall components in order to sustain their growth and to complete the invasion process. It is generally accepted that the enzymatic arsenal of the fungus contributes, together with mechanical forces (Howard et al. 1991; Chap. 3, this Vol.), to the degradation of both cuticle and cell walls.

A Polygalacturonase-Inhibiting Protein in the Flowers of Phaseolus vulgaris L.

Summary The localization of the endopolygalacturonase inhibiting protein (PGIP) has been studied in Phaseolus vulgaris L. by a simple infiltration-extraction procedure. Virtually all of the PGIP activity was recovered from the bean stem apoplast without significantly disturbing the symplastic component of the tissue. Activity of PGIP was determined in different parts and organs of developing bean seedlings. PGIP was present in all the tissues and organs tested (root, leaf, cotyledon, flower, stem, seed, embryo). Very low levels of PGIP were found in the roots. Higher specific activities were detected in all the other parts of the plant, the highest PGIP levels being detected in the vegetative apex and in the flower. PGIP levels in the stems increased during plant growth. At different stages of development a differential expression of PGIP along the stem was observed and an acropetal shift of the zone of maximum PGIP expression occurred. PGIP from Phaseolus vulgaris flowers was purified 32-fold by DEAE-cellulose chromatography followed by affinity chromatography through a Sepharose-endopolygalacturonase column. The purified PGIP, subjected to SDS-PAGE, showed a mobility similar but not identical to that of PGIP purified from hypocotyls. A molecular mass of 42 kDa was calculated for the flower PGIP, 1kDa larger than the molecular mass of the PGIP purified from stems.

Cytological localization of thePGIP genes in the embryo suspensor cells ofPhaseolus vulgavis L

Polygalacturonase-inhibiting protein (PGIP) is a cell wall protein which inhibits fungalendopolygalacturonases. A small gene family encodesPGIP in the genome of common bean, as indicated by Southernblot experiments performed at high-stringency conditions. Southern-blot analysis of DNA extracted from different cultivars ofPhaseolus vulgaris and fromPhaseolus coccineus showed length polymorphism of the hybridizing restriction fragments. The cytological localization of thePGIP genes was determined in polytene chromosomes of theP. vulgaris embryo suspensor cells. In-situ hybridization experiments using the clonedPGIP gene revealed labelling over a single region of the pericentromeric heterochromatin of chromosome pair X, next to the euchromatin, suggesting thatPGIP gene family may be clustered in one chromosomal region.

Transgenic peas (Pisum sativum) expressing polygalacturonase inhibiting protein from raspberry (Rubus idaeus) and stilbene synthase from grape (Vitis vinifera)

The pea (Pisum sativum L.) varieties Baroness (United Kingdome) and Baccara (France) were transformed via Agrobacterium tumefaciens-mediated gene transfer with pGPTV binary vectors containing the bar gene in combination with two different antifungal genes coding for polygalacturonase-inhibiting protein (PGIP) from raspberry (Rubus idaeus L.) driven by a double 35S promoter, or the stilbene synthase (Vst1) from grape (Vitis vinifera L.) driven by its own elicitor-inducible promoter. Transgenic lines were established and transgenes combined via conventional crossing. Resveratrol, produced by Vst1 transgenic plants, was detected using HPLC and the PGIP expression was determined in functional inhibition assays against fungal polygalacturonases. Stable inheritance of the antifungal genes in the transgenic plants was demonstrated.

Expression and localization of polygalacturonase during the outgrowth of lateral roots in Allium porrum L.

The presence of polygalacturonase and its correlation with the formation of lateral roots in leek (Allium porrum L.) seedlings have been investigated. During root growth, a steady increase in polygalacturonase activity was associated with that of the lateral root primordia. Fractionation of root extract by fast protein liquid chromatography resolved at least two polygalacturonase isoforms. One of the isoforms, a 75-kdalton protein, strongly reacted on Western blots probed with a polyclonal antibody raised against tomato polygalacturonase. It also reacted with both polyclonal and monoclonal antisera raised against Fusarium moniliforme polygalacturonase. In situ localization with these three antibodies showed that polygalacturonase was present over the meristems of lateral root primordia. Antibodies against pectins (Knox et al. 1990, Planta 181, 512–521) detected large amounts of pectic material filling the area between the apex of the primordium and the mother root tissues. We suggest that a polygalacturonase plays an important role in leek root morphogenesis, particularly during lateral root outgrowth.