Host-Pathogen Interactions

Abstract

ABSTRACrHeat-labile elicitors of phytoalexin accumulation in soybeans (Glycine max L. Merr. cv Wayne) were detected in culture filtrates of Erwinia carotovora grown on a defed medium containing citrus pectin as the sole carbon source. The heat-labile elicitors were highly purified by cation-exchange chromatography on a CM-Sephadex (C-50) column, followed by agarose-affinity chromatography on a Bio-Gel A-05m gel filtration column. The heat-labile elicitor activity co-purified with two a- (14) and from citrus pectin (25).The Gram-negative bacterium E. carotovora has been shown to elicit the accumulation of pterocarpan phytoalexins in wounded soybean cotyledons (33). Ninety-five per cent of this elicitor activity was abolished by killing the bacteria with heat treatments or antibiotics (13). These results suggested that viable E. carotovora cells produce an elicitor when in contact with plant tissue. We report in this paper the purification and characterization of heat-labile elicitors produced by E. carotovora grown on a defined medium containing citrus pectin. These elicitors were found to be PGA2 lyases, pectin-degrading enzymes that have been shown to be secreted by many plant pathogens (3). We also present evidence that the release, by PGA lyase, of oligosaccharides from the pectic polymers of plant cell walls triggered the elicitation of phytoalexin accumulation. This evidence suggests that the release of endogenous elicitors (14, 25) from plant cell walls by pectin-degrading enzymes plays a role in general plant disease resistance to microorganisms. A preliminary report of these results has been published (10).MATERIALS AND METHODS Chemicals. Electrophoresis-grade acrylamide and N,N'-methylenebisacrylamide, low-mol-wt protein standards, silver-stain reagents, dye reagent concentrate for protein assay, and Bio-Gel A-0.5m agarose beads were from Bio-Rad. Sephadex G-15, CMSephadex (C-50), BSA (fraction V), streptomycin sulfate, and Tris were from Sigma. Ion-exchange resin PB 118 and Pharmalyte pH 8-10.5 ampholytes were from Pharmacia. Electrophoresis-grade SDS was from BDH Chemicals Ltd., D-galacturonic acid was from Aldrich, and D-glucose was from Fisher. All other chemicals and solvents were of reagent grade or better.Polysaccharides. Sodium polypectate (grade II), polygalacturonic acid (grade III), and citrus pectin were from Sigma. Cell walls from soybean stems were prepared by M. Woodward and M. Hahn, as described (14). Before use, the cell walls (1 g) were washed with 1 L 5 mm Tris-HCl, 1 mm CaCl2 at pH 8.5, and 1 L deionized H20. The walls were then dried in a vacuum oven. A crude glucan elicitor from Phytophthora megasperma var glycinea was a gift from J. K. Sharp of this laboratory.

Host-Pathogen Interactions

“…An apparently homogeneous preparation of the PGA lyase was shown to induce phytoalexin accumulation in soybean cotyledons and also to release uronic-acid rich, heatstable elicitors from soybean cell walls, citrus pectin, and sodium polypectate (7). Similar results have been obtained by West et al.…”

“…Recent work has demonstrated that a heat-labile elicitor of phytoalexins produced by the phytopathogenic bacterium Erwinia carotovora var carotovora is a pectin-degrading enzyme, PGA lyase3 (7). An apparently homogeneous preparation of the PGA lyase was shown to induce phytoalexin accumulation in soybean cotyledons and also to release uronic-acid rich, heatstable elicitors from soybean cell walls, citrus pectin, and sodium polypectate (7).…”

“…495) as described (7). The enzyme activity of the PGA lyase was measured spectrophotometrically (7). One unit of PGA lyase activity was the amount required to release 1 umol unsaturated products/min at 30°C.…”

“…Homogeneous a-1,4-D-endopolygalacturonic acid lyase (EC 4.2.2.2) was prepared from cultures of Erwinia carotovora var carotovora (ATCC No. 495) as described (7). The enzyme activity of the PGA lyase was measured spectrophotometrically (7).…”

“…The modification of the soybean-cotyledon assay previously described was used to determine elicitor activities (7). All samples assayed for elicitor activity were desalted by gel permeation chromatography on Sephadex G-10 columns (1.0 x 18 cm) equilibrated with deionized H20.…”