Polygalacturonase-inhibiting protein accumulates in Phaseolus vulgaris L. in response to wounding, elicitors and fungal infection.

Polygalacturonase-inhibiting protein (PGIP) is a cell wall-associated protein that specifically binds to and inhibits the activity of fungal endopolygalacturonases. The Phaseolus vulgaris gene encoding PGIP has been cloned and characterized. Using a fragment of the cloned pgip gene as a probe in Northern blot experiments, it is demonstrated that the pgip mRNA accumulates in suspension-cultured bean cells following addition of elicitor-active oligogalacturonides or fungal glucan to the medium. Rabbit polyclonal antibodies specific for PGIP were generated against a synthetic peptide designed from the N-terminal region of PGIP; the antigenicity of the peptide was enhanced by coupling to KLH. Using the antibodies and the cloned pgip gene fragment as probes in Western and Northern blot experiments, respectively, it is shown that the levels of PGIP and its mRNA are increased in P. vulgaris hypocotyls in response to wounding or treatment with salicylic acid. Using gold-labeled goat-anti-rabbit secondary antibodies in EM studies, it has also been demonstrated that, in bean hypocotyls infected with Colletotrichum lindemuthianum, the level of PGIP preferentially increases in those cells immediately surrounding the infection site. The data support the hypothesis that synthesis of PGIP constitutes an active defense mechanism of plants that is elicited by signal molecules known to induce plant defense genes.

Purification, characterization, and cell wall localization of an alpha-fucosidase that inactivates a xyloglucan oligosaccharin.

An alpha-fucosidase that releases fucosyl residues from oligosaccharide fragments of xyloglucan, a plant cell wall hemicellulosic polysaccharide, was purified to homogeneity from pea (Pisum sativum) epicotyls using a combination of cation exchange chromatography and isoelectric focusing. The alpha-fucosidase has a molecular mass of 20 kDa according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The alpha-fucosidase has an isoelectric point of 5.5. The substrate specificity of the alpha-fucosidase was determined by high performance anion exchange chromatographic analysis of oligosaccharide substrates and products. The enzyme hydrolyzes the terminal alpha-1,2-fucosidic linkage of oligosaccharides and does not cleave p-nitrophenyl-alpha-L-fucoside. The enzyme does not release measurable amounts of fucosyl residues from large polysaccharides. The subcellular localization of alpha-fucosidase in pea stems and leaves has been studied by immunogold cytochemistry. The alpha-fucosidase accumulates in primary cell walls and is not detectable in the middle lamella or in the cytoplasm of 8-day-old stem tissue and 14-day-old leaf tissue. alpha-Fucosidase activity was readily detected in extracts of 8-day-old stem tissue. No significant alpha-fucosidase activity or immunogold labeling of the alpha-fucosidase was detected in 2- and 4-day-old stem tissue indicating that production of alpha-fucosidase is developmentally regulated.

Antifungal effect of bean endochitinase on Rhizoctonia solani: ultrastructural changes and cytochemical aspects of chitin breakdown.

A chitinase, purified to homogeneity from ethylene-treated bean leaves, was applied to actively growing mycelial cells of Rhizoctonia solani to evaluate a potential antifungal activity. Light microscopic investigations at 30-min intervals following enzyme exposure revealed the induction of morphological changes such as swelling of hyphal tips and hyphal distortions. More precise information concerning fungal cell alteration was obtained by ultrastructural observation and cytochemical detection of chitin distribution in fungal cell walls. Chitin breakdown was found to be an early event preceding wall disruption and cytoplasm leakage. The large amounts of chitin present in the walls of control R. solani cells and the rapid chitin hydrolysis upon chitinase treatment lead us to suggest that this polysaccharide is one of the main components of this fungal cell wall and is readily accessible to chitinase, especially in the apical zone. By 60 min after enzyme treatment, labeled molecules were observed in the vicinity of some fungal cells, suggesting the release of chitin oligosaccharides from fungal cell walls. The antifungal activity of the bean chitinase on cells of R. solani grown in culture is discussed in relation to the potential of genetically modified transgenic plants to resist attack by R. solani through an antimicrobial activity in planta.

Use of pectinases complexed to colloidal gold for the ultrastructural localization of polygalacturonic acids in the cell walls of the fungus Ascocalyx abietina.

Three pectinase--gold complexes were used to localize polygalacturonic acids in the fungus Ascocalyx abietina (Lagerberg) Schlaepfer-Bernhard. With the pectinesterase and pectin lyase--gold complexes, the labelling was uniformly distributed over the fungus walls and did not seem to be significantly influenced by the tissue preparation. With the polygalacturonase--gold complex, differences in the labelling distribution were noted according to the fixation procedure indicating, therefore, that osmication of the tissues could greatly interfere with the localization of the specific enzyme binding sites. These results demonstrate, for the first time, the possibility of detecting polygalacturonic acids by means of different gold-complexed pectinases.