A Cell Wall-degrading Endopolygalacturonase Secreted by Colletotrichum lindemuthianum

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191

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.

Section: Resultssupporting

confidence: 58%

Host-Pathogen Interactions

Davis

Lyon²,

Darvill³

et al. 1984

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191

“…The solubilization of wall components resulting from the action of the endopolygalacturonase on isolated sycamore cell walls is complete in 1 hr when 20 units of endopolygalacturonase are used for each 5 mg of walls (32). If the walls are de-esterified by treatment with base before the endopolygalacturonase treatment, the extent of the reaction is the same as with untreated walls.…”

Section: The Portion Of Sycamore Cell Walls Solubilized By Endopolygamentioning

confidence: 83%

“…The only purified enzyme that we have examined which is capable of appreciable degradation of intact cell walls is the endopolygalacturonase isolated from Colletotrichum lindemuthianum culture medium (32). The reaction of endopolygalacturonase with cell walls and the fractionation and characterization of the portion of the wall solubilized by the action of this enzyme are described below.…”

Section: The Portion Of Sycamore Cell Walls Solubilized By Endopolygamentioning

confidence: 99%

See 1 more Smart Citation

The Structure of Plant Cell Walls

Talmadge¹,

Keegstra²,

Bauer³

et al. 1973

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500

162

This is the first in a series of papers dealing with the structure of cell walls isolated from suspension-cultured sycamore cells (Acer pseudoplatanus) . These studies have been made possible by the availability of purified hydrolytic enzymes and by recent improvements in the techniques of methylation analysis. These techniques have permitted us to identify and quantitate the macromolecular components of sycamore cell walls. These walls are composed of 10% arabinan, 2% 3,6-linked arabinogalactan, 23% cellulose, 9 % oligo-arabinosides (attached to hydroxyproline), 8% 4-linked galactan, 10% hydroxyproline-rich protein, 16 % rhamnogalacturonan, and 21% xyloglucan.The structures of the pectic polymers (the neutral arabinan, the neutral galactan, and the acidic rhamnogalacturonan) were obtained, in part, by methylation analysis of fragments of these polymers which were released from the sycamore walls by the action of a highly purified endopolygalacturonase. The data suggest a branched arabinan and a linear 4-linked galactan occurring as side chains on the rhamnogalacturonan. Small amounts or pieces of a xyloglucan, the wall hemicellulose, appear to be covalently linked to some of the galactan chains. Thus, the galactan appears to serve as a bridge between the xyloglucan and rhamnogalacturonan components of the wall.The rhamnogalacturonan consists of an a-(1 -; 4)-linked galacturonan chain which is interspersed with 2-linked rham. nosyl residues. The rhamnosyl residues are not randomly distributed in the chain but probably occur in units of rhamnosyl-(1 -4 4)-galacturonosyl-(1 -e 2)-rhamnosyl. This sequence appears to alternate with a homogalacturonan sequence containing approximately 8 residues of 4-linked galacturonic acid. About half of the rhamnosyl residues are branched, having a substituent attached to carbon 4. This is likely to be the site of attachment of the 4-linked galactan.The hydroxyprolyl oligo-arabinosides of the hydroxyproline. rich glycoprotein contain 3-linked, 2-linked, and terminal arabinosyl residues. The structure of the hydroxyprolyl oligoarabinosides deduced from our methylation studies agrees with the structure reported for similar oligosaccharides. Cellulose, hemicellulose, pectic polysaccharide, structural protein, and lignin have been identified as the major components of the plant cell wall. These components have been discussed in several recent reviews (4,9,26,35,47,52,63,64,68). Lignin is a characteristic component of secondary walls and is therefore not further discussed.Crystalline cellulose fibers make up an important part of the framework of the cell walls of all higher plants. Electron microscopy and x-ray diffraction have led to a rather detailed description of the structure of this wall component (35,57,69

“…The results demonstrated that the oligogalacturonides with the highest specific elicitor activity were closely related, but not identical, to those obtained by partial acid hydrolysis of soybean cell walls and citrus pectin (28) and by treatment of polygalacturonic acid with endopolygalacturon-ical Co. D-GalUA was from Aldrich Chemical Co. and D-glucose was from Fisher Scientific Co. a-1 ,4-Trigalactosyluronic acid was prepared as described (12). Galactaric acid and a-D-GalUA-( 1 -* 4)-a-D-GalUA41 --4)-a-galactaric acid were prepared from D-GalUA and a-1 ,4-trigalactosyluronic acid, respectively, by bromine oxidation as described (33).…”

mentioning

confidence: 99%

Host-Pathogen Interactions

Davis¹,

Darvill²,

Albersheim³

et al. 1986

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159

Recent studies have demonstrated that an apparently homogeneous preparation of an a-1,4-D-endopolyplacturonic acid lyase (EC 4.2.2.2) isolated from the phytopathogenic bacterium Erwinia carotovora induced phytoalexin accumulation in cotyledons of soybean (Glycine max jL.1Merr. cv Wayne) and that this pectin-degrading enzyme released heatstable elicitors of phytoalexins from soybean cell walls, citrus pectin, and sodium polypectate Plant Physiol 74: 52-60). The present paper reports the purification, by anion-exchange chromatography on QAE-Sephadex columns followed by gel-permeation chromatography on a Bio-Gel P6 column, of the two fractions with highest specific elicitor activity present in a crude elicitor-preparation obtained by lyase treatment of sodium polypectate. Structural analysis of the fraction with highest specific elicitor activity indicated that the major, if not only, component was a decasaccharide of a-1,4-D-galactosyluronic acid that contained the expected product of lyase cleavage, 4-deoxy-,6-L-5-threohexopyranos-4-enyluronic acid (4,5-unsaturated galactosyluronic acid), at the nonreducing terminus. This modified decagalacturonide fraction exhibited half-maximum and maximum elicitor activity at 1 microgram/ cotyledon (6 micromolar) and 5 micrograms/cotyledon (32 micromolar) plactosyluronic acid equivalents, respectively. Reducing 90 to 95% of the carboxyl groups of the galactosyluronic acid residues abolished the elicitor activity of the decagalacturonide fraction. The second most elicitor-active fraction contained mostly undeca-a-1,4-D-galactosyluronic acid that contained 4,5-unsaturated gaactosyluronic acid at the nonreducing termini. This fraction exhibited half-maximum and maximum elicitor activity at approximately 3 micrograms/cotyledon (17 micromolar) and 6 micrograms/cotyledon (34 micromolar) galactosyluronic acid equivalents, respectively. These results confirm and extend previous observations that oligogalacturonides derived from the pectic polysaccharides of plant cell walls can serve as regulatory molecules that induce phytoalexin accumulation in soybean. These results are consistent with the hypothesis that oligogalacturonides play a role in disease resistance in plants. Although (1, 1 1). Phytoalexins are a diverse group of compounds of low mol wt that have broad antibiotic activity against many prokaryotic and eukaryotic organisms (32, 36). In some hostpathogen combinations, phytoalexins accumulate at the site of attempted infection rapidly enough and in sufficient concentration to prevent further growth of the invading microorganism (19,24).Phytoalexin accumulation is also induced by molecules called elicitors. Various compounds isolated from microorganisms have been found to have elicitor activity (reviewed in 1, 2, 6, 37). Molecules derived from plant tissues also induce phytoalexin accumulation (14-16, 23, 28).Recent work has demonstrated that a heat-labile elicitor of phytoalexins produced by the phytopathogenic bacterium Erwinia carotovora var carotovora is a p...