F. Leckie

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.

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.

The promoter of a gene encoding a polygalacturonase-inhibiting protein of Phaseolus vulgaris L. is activated by wounding but not by elicitors or pathogen infection

Abstract. Polygalacturonase-inhibiting proteins (PGIPs), leucine-rich repeat (LRR) proteins evolutionarily related to several plant resistance genes, bind to and regulate the action of fungal endopolygalacturonases. In Phaseolus vulgaris L., PGIPs are encoded by a gene family comprising at least five members. As a start for a systematic analysis of the regulation of the pgip family, we have analysed the ability of the promoter of the bean gene pgip-1 to direct expression of β-glucuronidase (GUS) in transfected tobacco protoplasts, microbombarded bean and tobacco leaves, and transgenic tobacco plants. In protoplasts, the pgip-1 gene region from nucleotide (nt) −2004 to nt +27 directed a level of expression that was as high as that directed by the cauliflower mosaic virus (CaMV) 35S promoter and could not be further induced by elicitor treatment; alteration of the region immediately following the TATAA sequence at nt −29 abolished expression. Upon stable integration into tobacco plants of the pgip-1 promoter-GUS construct, as well as of a −394 deletion, expression was detected for both constructs mainly in the stigma and, to a lesser extent, in the anthers and in the conductive vascular tissue. The promoter responded to wounding but not to oligogalacturonides, fungal glucan, salicylic acid, cryptogein, or pathogen infection. This expression pattern does not mirror that of the whole pgip gene family.

Perception of fungal elicitors and signal transduction

Plants, like animals, are continually exposed to a vast array of potential fungal pathogens; in many cases, they resist attack by blocking fungal development soon after penetration. As plants lack a circulatory system and antibodies, they have evolved defense mechanisms that are distinct from the vertebrate immune system. In contrast to animal cells, each plant cell is capable of defending itself by means of a combination of constitutive mechanisms and induced responses. After the perception of the pathogen (recognition), the plant cell at the site of infection transmits the information inside the cell across the plasma membrane as well as to neighboring cells. As a consequence, a number of defense reactions are induced, which include a rapid and localized cell death (hypersensitive response), a rapid oxidative burst, cross-linking and strengthening of the plant cell wall, the induction of the phenylpropanoid pathway and synthesis of lignin, the accumulation of antimicrobial compounds named phytoalexins, the synthesis of hydroxyproline-rich glycoproteins (HRGPs) and fungal wall degrading enzymes (chitinases, glucanases), and the production of ethylene. The effectiveness of the plant defense responses against a pathogen depends both on the magnitude and on the rapidity of their onset (Dixon and Lamb, 1990).

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.

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.

Molecular Analysis of the Polygalacturonase-Inhibiting Protein (PGIP) Gene Family in Phaseolus Vulgaris L.

In Phaseolus vulgaris (bean), PGIP is encoded by a gene family. We have isolated cDNA clones corresponding to two different pgip genes (pgip-1 and pgip-2). These genes have been separately expressed in Nicotiana benthamiana using the potato virus X (PVX) as a vector. PGIP-1 and PGIP-2 in crude protein extracts of PVX-infected TV. benthamiana, and a PGIP-1 purified from transgenic tomato plants carrying the pgip-1 coding sequence under the control of the CaMV 35S promoter, have been assayed for specificity of interaction with several fungal PGs. PGIP-1 from both plant sources exhibited a similar specificity, which was different from that of PGIP-2 and of the bulk PGIP purified from bean. Notably, PGIP-1 was unable to interact with homogeneous PG from Fusarium moniliforme, as determined by surface plasmon resonance analysis, while PGIP-2 and the bulk bean PGIP interacted with this enzyme.

Accumulation of Pgip, a Leucine-Rich Receptor-Like Protein, Correlates with the Hypersensitive Response in Race-Cultivar Interactions

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. Accumulation of pgip mRNA has been followed in different race-cultivar interactions (either compatible or incompatible) between Colletotrichum lindemuthianum and Phaseolus vulgaris 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, each derived from modifications of a 24-amino acid peptide. A similar modular structure has been observed in several proteins implicated in protein-protein interactions and in the extracellular domain of a cloned Arabidopsis leucine-rich receptor-like protein kinase (RLK5). 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.