L. Nuss

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.

Developmental and pathogen-induced accumulation of transcripts of polygalacturonase-inhibiting protein in Phaseolus vulgaris L.

Abstract. Expression of the polygalacturonase-inhibiting-protein gene ( pgip) during development of French bean (Phaseolus vulgaris L. cv. Red Kidney) was analysed in various organs, including leaves, stems and roots of etiolated and light-grown seedlings, light-grown adult plants, as well as flowers, pods, and seeds. In light-grown seedlings and plants, pgip transcripts were detected in all tissues examined, with higher levels found in the transition zone between the elongating and mature region of young hypocotyls, and in the basal region of the stem of adult plants. In etiolated hypocotyls, levels of pgip transcripts in the elongating region were five- to six fold higher than in light-grown hypocotyls. High levels of transcripts were also observed in pods. Accumulation of pgip mRNA was also followed in both compatible and incompatible race-cultivar interactions between Colletotrichum lindemuthianum races α and γ, respectively, and P. vulgaris by Northern blot analysis and in situ hybridisation. In situ experiments showed, in incompatible interactions, a rapid, intense and transient accumulation of pgip mRNA in epidermal cells proximal to the site of infection, and, less intense, within the cortical parenchyma underneath. In compatible interactions, no substantial accumulation of pgip mRNA was observed in hypocotyls; a very weak accumulation was observed in leaves during lesion formation.

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.