C. Caprari

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.

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.

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.

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.

The polygalacturonase-inhibiting protein PGIP2 of Phaseolus vulgaris has evolved a mixed mode of inhibition of endopolygalacturonase PG1 of Botrytis cinerea

Botrytis cinerea is a phytopathogenic fungus that causes gray mold in >1,000 plant species. During infection, it secretes several endopolygalacturonases (PGs) to degrade cell wall pectin, and among them, BcPG1 is constitutively expressed and is an important virulence factor. To counteract the action of PGs, plants express polygalacturonase-inhibiting proteins (PGIPs) that have been shown to inhibit a variety of PGs with different inhibition kinetics, both competitive and noncompetitive. The PG-PGIP interaction promotes the accumulation of oligogalacturonides, fragments of the plant cell wall that are general elicitors of plant defense responses. Here, we characterize the enzymatic activity of BcPG1 and investigate its interaction with PGIP isoform 2 from Phaseolus vulgaris (PvPGIP2) by means of inhibition assays, homology modeling, and molecular docking simulations. Our results indicate a mixed mode of inhibition. This is compatible with a model for the interaction where PvPGIP2 binds the N-terminal portion of BcPG1, partially covering its active site and decreasing the enzyme affinity for the substrate. The structural framework provided by the docking model is confirmed by site-directed mutagenesis of the residues that distinguish PvPGIP2 from the isoform PvPGIP1. The finding that PvPGIP2 inhibits BcPG1 with a mixed-type kinetics further indicates the versatility of PGIPs to evolve different recognition specificities.

A gene for plant protection: expression of a bean polygalacturonase inhibitor in tobacco confers a strong resistance against Rhizoctonia solani and two oomycetes

We have tested whether a gene encoding a polygalacturonase-inhibiting protein (PGIP) protects tobacco against a fungal pathogen (Rhizoctonia solani) and two oomycetes (Phytophthora parasitica var. nicotianae and Peronospora hyoscyami f. sp. tabacina). The trials were performed in greenhouse conditions for R. solani and P. parasitica and in the field for P. hyoscyami. Our results show that expression of PGIP is a powerful way of engineering a broad-spectrum disease resistance.

The Interaction between Fungal Endopolygalacturonase and Plant Cell Wall Pgip (Polygalacturonase-Inhibiting Protein)

The characterization of the genes encoding the endopolygalacturonase of Fusariwm moniliforme and the PGIP of Phaseolus vulgaris is reported. Studies on the regulation of the genes are also described. A model of the involvement of polygalacturonase and PGIP in resistance of plants to fungi is presented.

Recognition and signalling in the cell wall: The case of endopolygalacturonase, PGIP and oligogalacturonides

The ongoing research carried out in our laboratory to elucidate the roles of polygalacturonase, PGIP and oligogalacturonides is reviewed.

The role of polygalacturonase, PGIP and pectin oligomers in fungal infection

Abstract The interaction between fungal endopolygalacturonases and a plant cell wall PGIP (PolyGalacturonase-Inhibiting Protein) in plant-pathogen recognition is being investigated. This protein-protein interaction has been shown to favour the formation of oligogalacturonides able to elicit plant defense responses. A single mutation in the endo polygalacturonase gene of Fusarium moniliforme abolishes the hydrolytic activity but does not affect the elicitor activity of the enzyme and its ability to interact with PGIP. Accumulation of pgip mRNA in different race-cultivar interactions (either compatible or incompatible) between Colletotrichum lindemuthianum and Phaseolus vulgaris has been followed by Northern blot and in situ hybridisation analyses. Rapid accumulation of pgip mRNA correlates with the appearance of the hypersensitive response in incompatible interactions, while a more delayed increase, coincident with the onset of lesion formation, occurs in compatible interactions. PGIP exhibits a modular structure: its amino acid sequence can be divided into a set of 10.5 leucine-rich tandemly repeated units (LRR=leucinerich repeats), each derived from modifications of a 24-amino acid peptide. A LRR structure has been observed in several proteins implicated in protein-protein interactions and in the extracellular domain of a cloned Arabidopsis receptor-like protein kinase (RLK5); a LRR structure has also been observed in the products of several resistance genes recently cloned. A plasma membrane-associated high molecular weight protein cross-reacting with an antibody prepared agaist PGIP is being purified in our laboratory. We suggest that PGIP may belong to a class of receptor complexes specialized for defense against microbes.

ARTIFICIAL EVOLUTION CORRECTS A REPULSIVE AMINO ACID IN POLYGALACTURONASE INHIBITING PROTEINS (PGIPs)

Polygalacturonase-inhibiting proteins (PGIPs), extracellular proteins that specifically inhibit fungal endopolygalacturonases (PGs), play a critical role in plant protection by favouring the accumulation of oligogalacturonides (OGs), which are elicitors of plant defence responses. The genes encoding PGIP2 of P. vulgaris and the variant PGIP2.Q224K were subjected to error prone PCR (epPCR) to generate mutated inhibitors with novel and improved recognition capabilities. Using a Pichia pastoris expression library and a high-throughput screening method, two mutated PvPGIP2.Q224Kderived inhibitors active against the PG produced by the phytopathogenic fungus F. phyllophilum (FpPG) were isolated. Both variants were better inhibitors than PGIP2.Q224K and were characterized by the replacement of the lysine in position 224, supporting the view that the absence of this positively charged amino acid at position 224 is a primary requirement for gaining the inhibition capability against FpPG.