The Role of Pectic Fragments of the Plant Cell Wall in Elicitation by a Fungal

Endopolygalacturonase isolated from culture ifitrates of the fungus Rhizopus stolonifer was shown previously to act as an elicitor ofbiosynthetic capacity for the antifungal agent, casbene, in castor bean (Ricinus commais L.) seedlings (S.-C. Lee, C.A. West 1961 Plant Physiology 67:633-639). Selective amidation of exposed carboxyl groups of the pure fungal endopolygalacturonase using intermediate activation with a watersoluble carbodiimide under mild conditions leads to inactivatin of its enzymic activity. Tests of active and partially inactivated preparations of the enzyme reveal a close correlation between the levels of catalytic and elicitor activities. This suggests that the catalytic activity of the enzyme is necessary for its function as an elicitor. Treatment of the celi-free particulate fraction of homogenates of castor bean seedligs with the active fungal endopolygalacturonase results in the production of a heat-stable, water-soluble component which is highly active as an elicitor of casbene synthetase activity. Several additional lines of evidence, including the susceptibility of the heat-stable elicitor fraction to partial inactivation following prolonged treatment with endopolygalacturonase, indicate that the heat-stable elicitor is most likely a pectic fragment of the plant cell wail and that it is a required intermediate in the process of elicitation of casbene synthetase activity by the fungal endopolygalacturonase. castor bean plant as a phytoalexin (18). A number ofthe biochemical characteristics of casbene biosynthesis have been elucidated (26). Substances which trigger the production of phytoalexins in higher plants are termed elicitors. A wide variety of fungal molecules, including polysaccharide wall components, polypeptides, glycoproteins and lipids, have been implicated as elicitors (25). Two lines of investigation have shown that fungal enzymes can also act as elicitors. Swinburne (22, 23) has implicated a fungal proteinase as an elicitor of benzoic acid production in infected apple fruit. In addition, recent work in our laboratory (11, 12) has shown that homogeneous a1,4-endopolygalacturonase from culture filtrates of the fungus Rhizopus stolonifer elicits casbene synthetase activity in castor bean (R communis L.) seedlings. In both cases, heat-treatment of the enzyme preparations leads to equivalent losses of both enzymic activity and elicitor ability. This suggests that the elicitor abilities of these enzymes may be dependent on their catalytic activities. The work described in this paper demonstrates that pectic cell wall fragments released from particulate fractions of castor bean seedling homogenates through the action ofR stolonifer endopolygalacturonase possess casbene synthetase elicitor activity and supports a model in which these elicitor fragments serve as obligate intermediates in the process of elicitation by the enzyme in vivo. Many species of higher plants have been shown to produce antifungal compounds known as phytoalexins (4, 6, 10). The presence of low or undetectable levels of phytoalexins in healthy plant tissues and the greatly increased accumulation of these substances in the region of fungal infection are distinctive features of phytoalexin production. Because of these characteristics and their general toxicities for a broad range of fungi, phytoalexins are believed to play a defensive role against invasion of the plant by fungal predators. Casbene is one of the five cyclic diterpene hydrocarbons produced from GGPP2 in cell-free extracts of young seedlings of the castor bean (Ricinus communis L.) (14, 15). Extracts of healthy seedlings produce little or no casbene, whereas extracts ofseedlings exposed for several hours to any of several phytopathogenic fungi show a greatly enhanced capacity for casbene synthesis (18). This characteristic coupled with demonstration of the antifungal properties of casbene led to the proposal that casbene may serve the ' This work was supported by National Science Foundation Grant PCM-79-23 142. 2Abbreviations: GGPP: geranylgeranyl pyrophosphate; EPGase: endopolygalacturonase; Rs EPGase: Rhizopus stolonifer endopolygalacturonase; EDC: l-ethyl-3(3-dimethylaminopropyl) carbodiimide; PGA: polygalacturonic acid; PIIF, proteinase inhibitor inducing factor. MATERIALS AND METHODS Elicitor Bioassay for Casbene Synthetase. The elicitor activity of various test solutions was measured using a bioassay for the levels of casbene synthetase activity in treated seedlings as described by Lee and West (11). Castor bean seeds were shelled, surface-sterilized in a dilute solution of NaOCl, and germinated under sterile conditions at room temperature for 53 to 55 h. For each test solution, 10 seedlings with the radicles removed were cut along the plane dividing the cotyledons with a double-edged razor blade and placed, cut surface down, in a sterile Petri dish containing two layers of Whatman GF/A glass fiber filter and a single layer of cheesecloth. The test solution (10 ml) was added to the Petri dish which was covered and incubated for 10 to 12 h at 25°C in the dark. After incubation, the set of pooled seedlings from one treatment was homogenized in cold homogenization buffer. The homogenates were centrifuged and portions of the supernatant were removed and assayed for casbene synthetase activity. In a typical assay, 100 ,u enzyme extract and 300 pil buffer were incubated with 100 ,u 50 AM radiolabeled GGPP at 30°C for 30 min. The reaction was quenched with ethanol and the casbene was extracted with petroleum ether. The casbene was separated from other substances by TLC and quantitated by measuring the radioactivity associated with the appropriate region of each plate. Some casbene synthetase activity was observed in seedlings which had been subjected to the experimental assay procedure, 1181 www.plantphysiol.org on July 15, 2017 Published by Downloaded from Copyright © 1982 American Society of Plant Biologists. All rights reserved. Plant Physiol. Vol. 69, 1982 but which had not been treated with elicitor. These control values generally ranged between 5,000 and 6,000 dpm in casbene when 4,4'[3HIGGPP was used as the substrate in a 30-min assay. A control was run with each set of test samples and the control value was subtracted from the test sample values. Under optimal conditions, elicitor-treated seedlings could accumulate up to five times the control level of casbene synthetase activity. A typical doseresponse curve with Rs EPGase as the elicitor is shown in Figure 1. At the lower concentrations of elicitor, the amount of casbene synthetase activity follows a nearly linear relationship with the amount of elicitor tested. Higher levels of elicitor, however, saturate the response. The amount of EPGase tested in elicitor bioassays was usually between 0.2 and 0.3 units in order to produce responses which were in the approximately linear region of the dose response curve. Synthesis of Radiolabeled Geranylgeranyl Pyropbosphate. 4,4'[3H]Geranylgeraniol (10.0 mCi/mmol) was synthesized from farnesylacetone in a manner similar to that described by Upper and West (24) as later modified by Simcox (17). The tritium label was introduced into farnesylacetone by refluxing a solution of famesylacetone and tritiated water in dioxane for 40 h. A small amount of NaOH was added to the mixture prior to reflux to catalyze the exchange of hydrogens bound to carbons alpha to the carbonyl function with tritium atoms of the tritiated H20. A water-cooled condenser was kept in place above the reaction vessel and the reaction mixture was maintained under dry nitrogen throughout the period of refluxing. Pyrophosphorylation of the alcohol was achieved with the method recently introduced by Dixit et al (5) in which tetra (tetrabutylammonium) pyrophosphate is employed as a pyrophosphorylating agent with geranylgeranyl bromide. The tritiated GGPP synthesized in this manner was used in all reported experiments except where specifically indicated. Purification of Endopolygalacturonase from Rhizopus stolonfer. EPGase from culture filtrates of R stoloni'er was purified to homogeneity following the techniques described by Lee and West (11). The purification sequence involved chromatography of the concentrated culture filtrate on Sephadex G-25, G-75, and CM50 under conditions suitable for the preservation of enzyme activity. All samples ofRs EPGase employed in this work were purified to homogeneity in this manner. In some instances, the homogeneous enzyme was chromatographed on Sephadex G-10 (coarse grade) in 25 mm NaCl when EPGase in a low salt solution was required.