Release of highly elicitor-active glucans by germinating zoospores of Phytophthora megasperma f. sp. glycinea

An in-vitro culture system allowing the simultaneous germination of cysts was used to study the early host-independent release of phytoalexin elicitors by Phytophthora megasperma f. sp. glycinea, a soybean pathogen. Significant elicitor activity could be detected in the culture medium as early as 2 h after germination of P.m. f. sp. glycinea, race 1, cysts. The phytoalexin elicitor was heat-stable and heterogeneous in size. The apparent molecular mass ranged from 3 to 80 kDa. Anion exchange and lectin-affinity chromatography followed by sugar analysis confirmed that the elicitor activity resided primarily in glucans. The time course of elicitor release could then be accurately monitored by means of a competitive radioligand-displacement assay using the -glucan elicitor-binding sites of soybean (Glycine max (L.) Merr.) membranes. Linkage-composition analysis of the glucan elicitors showed that they were primarily (1 3)-linked with (1 6)--branches, a composition similar to that of glucans obtained by heat release from mature mycelium but different from that of elicitors obtained by acid hydrolysis or from spontaneous autohydrolytic release by senescent cultures. The naturally released elicitors displayed a biological activity in soybean cotyledon bioassays higher than purified acid-hydrolysed glucan elicitor or than the hepta-(1 3, 1 6)--glucoside, the smallest known carbohydrate elicitor for soybean. The present results demonstrate that elicitor release from the pathogen and perception by the potential host can take place in this system as early as during germ-tube formation and independent of the presence of host-produced endoglucanases.Abbreviations EC₅₀ effector concentration necessary for halfmaximal response - GC-MS gas chromatography-mass spectrometry - HG-APEA 1-[2-(4-aminophenyl)ethyl]amino-1-[hexagluco-syl]-deoxyglucitol - IC₅₀ inhibitor concentration necessary for half-maximal inhibition - P.m. f. sp. glycinea Phytophthora megasperma f. sp. glycinea - V₀ void volume Deceased on March 19, 1990This work was supported by the Deutsche Forschungsgemeinschaft (SFB 206). We thank Dr. H. Mayer, C. Warth and D. Borowiak (Max-Planck-Institut für Immunbiologie, Freiburg) for helpful discussions and experienced technical assistance (glucosyl-linkage analysis).     

Biochemical reactions of different tissues of potato (Solanum tuberosum) to zoospores or elicitors from Phytophthora infestans

The time courses of sesquiterpenoid phytoalexin accumulation were examined in compatible and incompatible interactions of leaves and tubers from five different R genotypes of potato (Solanum tuberosum) with corresponding pathotypes of Phytophthora infestans, as well as in non-host interactions of all five potato cultivars with Phytophthora megasperma f. sp. glycinea and in elicitor-treated tubers from five, and cell suspension cultures from two, of the cultivars. In tubers, rishitin and several structurally related sesquiterpene derivatives accumulated rapidly in non-host incompatible interactions, less rapidly in host incompatible interactions, and more slowly in compatible interactions. Treatment of tubers or cell cultures with fungal culture filtrate or arachidonic acid elicited in most cases a transient accumulation of the sesquiterpenoid phytoalexins. None of these compounds was detectable under any of the applied conditions either in infected or in elicitortreated leaves. Sesquiterpenoid phytoalexins might therefore be helpful, but appear not to be essential, in disease resistance of potato.Abbreviations CF concentrated culture filtrate of Pi - cv. cultivar - Pi Phytophthora infestans (numbering indicates pathotypes corresponding to R genes in potato) - Pmg Phytophthora megasperma f. sp. glycinea     

Elicitor-induced accumulation of glyceollin and callose in soybean roots and localized resistance against Phytophthora megasperma f.sp. glycinea

Immersion of roots of 2-day-old soybean seedlings (Glycine max cv. Harosoy 63) into solutions of several glucan elicitors caused the accumulation to various degrees of the soybean phytoalexin glyceollin. Laminarin and polytran proved to be more effective elicitors in this system than the glucan elicitor from Phytophthora megasperma f.sp. glycinea (Pmg). Digitonin and tomatin caused, in addition to glyceollin accumulation, the deposition of callose in the rhizodermis. Pretreatment of the soybean roots with laminarin effected an increase in resistance of the seedlings against a compatible race of Pmg.     

Phytoalexin Elicitor Activity of Carbohydrates from Phytophthora megasperma f.sp. glycinea and Other Sources

Three unique classes of carbohydrates were isolated from the hyphal cell walls of Phytophthora megasperma f.sp. glycinea (Pmg) and compared with other substances for their activity as elicitors of the phytoalexin glyceollin in soybean tissues. Glucomannans extracted from cell walls with soybean β-1,3-endoglucanase were purified and proved to be the most active elicitors yet reported. They were approximately 10 times more active in soybean cotyledons than the heterogeneous β-glucan elicitor fraction extracted from Pmg walls. In addition, the glucomannan fraction gave race-specific elicitor activity in soybean hypocotyls. Pronase was found to be a suitable reagent for the mild extraction of glycopeptides from Pmg cell walls. All of the carbohydrates isolated from Pmg cell walls possessed significant elicitor activity, but other glucans, a glucomannan and mannan from other sources, were much less active. Chitin and chitosan, reported to function as elicitors in other plants, had low activity in soybean cotyledons. Arachidonic acid was inactive, despite its previously observed elicitor activity in potato tubers. The results indicated that, for Pmg, the carbohydrate elicitor most probably involved in the initiation of phytoalexinmediated defense during fungus infection of soybean plants is the glucomannan fraction liberated by endoglucanase.     

Rapid induction of phenylalanine ammonia-lyase and chalcone synthase mRNAs during fungus infection of soybean (Glycine max L.) roots or elicitor treatment of soybean cell cultures at the onset of phytoalexin synthesis

The differential regulation of the activities and amounts of mRNAs for two enzymes involved in isoflavonoid phytoalexin biosynthesis in soybean was studied during the early stages after inoculation of primary roots with zoospores from either race 1 (incompatible, host resistant) or race 3 (compatible, host susceptible) of Phytophthora megasperma f.sp. glycinea, the causal fungus of root rot disease. In the incompatible interaction, cloned cDNAs were used to demonstrate that the amounts of phenylalanine ammonia-lyase and chalcone synthase mRNAs increased rapidly at the time of penetration of fungal germ tubes into epidermal cell layers (1–2 h after inoculation) concomitant with the onset of phytoalxxin accumulation; highest levels were reached after about 7 h. In the compatible interaction, only a slight early enhancement of mRNA levels was found and no further increase occurred until about 9 h after inoculation. The time course for changes in the activity of chalcone synthase mRNA also showed major differences between the incompatible and compatible interaction. The observed kinetics for the stimulation of mRNA expression related to phytoalexin synthesis in soybean roots lends further support to the hypothesis that phytoalexin production is an early defense response in the incompatible plant-fungus interaction. The kinetics for the enhancement of mRNA expression after treatment of soybean cell suspension cultures with a glucan elicitor derived from P. megasperma cell walls was similar to that measured during the early stages of the resistant response of soybean roots.Abbreviations cDNA copy DNA - CHS chalcone synthase - PAL phenylalanine ammonia-lyase     

Induction of enzymes of phytoalexin synthesis in cultured soybean cells by an elicitor from Phytophthora megasperma f. sp. glycinea

The glucan elicitor from cell walls of the fungal pathogen, Phytophthora megasperma f. sp. glycinea, induced rapid but transient increases in enzyme activities of general phenylpropanoid metabolism (phenylalanine ammonia-lyase and 4-coumarate: CoA ligase) and of the flavonoid pathway (chalcone synthase) in cell suspension cultures of soybean (Glycine max). After transferring cells into fresh medium, two peaks of inducibility for the enzymes by elicitor were observed, one shortly after transfer (stage I), and one at the end of the linear growth phase (stage II). Only one of the two isoenzymes of 4-coumarate: CoA ligase (isoenzyme 2), for which a specific involvement in flavonoid biosynthesis has been postulated, was affected by the elicitor. For two of the induced enzymes, phenylalanine ammonia-lyase and chalcone synthase, the changes in activity at stage I were shown to be preceded by large changes in their rates of synthesis, as determined by in vivo labelling with [³⁵S] methionine and immunoprecipitation.Abbreviations Pmg Phytophthora megasperma f. sp. glycinea - glyceollin is a term used to designate the 3 isomers which accumulate in challenged soybean tissue (Moesta and Grisebach 1981b)     

Chitosan-elicited synthesis of callose and of coumarin derivatives in parsley cell suspension cultures

In suspension cultured cells of parsley (Petroselinum crispum), chitosan elicited a rapid deposition of the 1,3-ß-glucan callose on the cell wall and a slower formation of coumarins. With cells remaining in conditioned growth medium, fully N-deacetylated chitosans and partially N-acetylated chitosans were about equally active, the potency increased with the degree of polymerization up to several thousand and addition of reduced glutathione increased the sensitivity of the cells. These results indicate common initial events in the induction of callose and coumarin synthesis although two fully independent metabolic pathways are involved. When the cells were suspended in fresh growth medium, less chitosan was required, and fully N-deacetylated chitosan became the best callose elicitor.Abbreviations DP average degree of polymerization - GSH reduced glutathione - PE pachyman equivalents - Pmg Phytophthora megasperma f. sp.glycinea     

Dependence of the level of phytoalexin and enzyme induction by fungal elicitor on the growth stage of Petroselinum crispum cell cultures

The extent of induction of some metabolic activities in cultured parsley cells (Petroselinum crispum) by an elicitor preparation from Phytophthora megasperma f. sp. glycinea varied with the growth stage of the cell culture. On the basis of cell fresh weight, the induction of phytoalexin accumulation was high until cell mass reached a maximum, and then declined to a low level which was indistinguishable from a level caused by an endogenous mechanism operating at this late growth stage. The induction of phenylalanine ammonia-lyase and 4-coumarate:CoA ligase activities by the elicitor showed a high degree of coordination and a sharp maximum preceding the stage of maximal cell mass. 1,3--Glucanase activity was induced to about the same level throughout all growth stages, with a large contribution by an endogenous mechanism at late stages.Abbreviations PAL Phenylalanine ammonia-lyase (EC 4.3.1.5) - 4CL 4-Coumarate:CoA ligase (EC 6.2.1.12)     

Race cultivar-specific differences in callose deposition in soybean roots following infection with Phytophthora megasperma f.sp. glycinea

Primary roots of soybean (Glycine max (L.), Merrill, cv. Harosoy 63) seedlings were inoculated with zoospores from either race 1 (incompatible, host resistant) or race 3 (compatible, host susceptible) of Phytophthora megasperma f.sp. glycinea and total callose was determined at various times after inoculation. From 4 h onward, total callose was significantly higher in roots showing the resistant rather than the susceptible response. Local callose deposition in relation to location of fungal hyphae was determined in microtome sections by its specific fluorescence with sirofluor and was quantified on paper prints with an image-analysis system. Callose deposition, which occurs adjacent to hyphae, was found soon after inoculation (2, 3 and 4 h post inoculation) only in roots displaying the resistant response, and was also higher at 5 and 6 h after inoculation in these resistant roots than in susceptible roots. Early callose deposition in the incompatible root-fungus reaction could be a factor in resistance of soybean against P. megasperma.Abbreviation pi post inoculation     

Effects of R-(1-amino-2-phenylethyl)phosphonic acid on glyceollin accumulation and expression of resistance to Phytophthora megasperma f.sp. glycinea in soybean

(R)-(1-Amino-2-phenylethyl)phosphonic acid (R-APEP), an inhibitor of phenylalanine ammonia-lyase (PAL), was applied to the tap root of 42-h-old soybean (Glycine max. (L.) Merrill cv. Harosoy 63) seedlings during inoculation with zoospores of the incompatible race 1 of Phytophthora megasperma f.sp. glycinea (Pmg1) for 2 h and during a subsequent incubation period. In contrast to L-2-aminooxy-3-phenylpropionic acid, R-APEP was not toxic to the zoospores which remained virulent in presence of the inhibitor. A 50% inhibition of PAL activity in vitro was observed with 4.2 M R-APEP and with 36 M of the S-enantiomer. When R-APEP at 330 M was applied for a total of 36 h to the seedlings, resistance against Pmg 1 was abolished. Such seedlings were indistinguishable in appearance from those seedlings which had been inoculated with the compatible race 3 of Pmg. Roots treated with R-APEP at 330 M showed a reduction of about 47% in glyceollin content when measured 12 h after inoculation, and with 1 mM a 67% reduction. In contrast, treatment with S-APEP (1 mM) caused only a 20% reduction in glyceollin content. As determined by indirect immunofluorescence of fungal hyphae in cryotome cross-sections of roots, the growth pattern of the incompatible race 1 of Pmg changed to that of the compatible race 3 under conditions where R-APEP caused loss of resistance against Pmg 1. The results support the concept of an important role of glyceollin in resistance of soybean against incompatible races of the fungus.Abbreviations R-APEP, S-APEP R.S enantiomers of (1-amino-2-phenylethyl)phosphonic acid - L-AOPP L-2-aminooxy-3-phenylpropionic acid - PAL phenylalanine ammonia-lyase (EC 4.3.1.5) - Pmg 1 Phytophthora megasperma f.sp. glycinea race 1 - Pmg 3 Phytophthora megasperma f.sp. glycinea race 3     

Elicitation of 2,3-Dihydroxybenzoic Acid and Ajmalicine in a Catharanthus roseus Suspension Culture*

Addition of an elicitor preparation from cell walls of Phytophthora megasperma f. sp. glycinea (Pmg elicitor) to a newly established cell suspension culture of Catharanthus roseus induced extracellular free 2,3-dihydroxybenzoic acid, suggesting its role in pathogen defense. The same substance also accumulated intracellularly in a bound form. Treatment of the crude Pmg elicitor preparation with trypsin abolished elicitor activity, suggesting that the active fraction is proteinaceous. The cells became more sensitive to low but not to elevated elicitor concentrations when they were pretreated with 2,6-dichloroisonicotinic (DCIA) or 5-chlorosalicylic (5CSA) acid for about 1 day before addition of the elicitor. This indicates that the elicitor reception/transduction system becomes improved by these compounds known to be related to systemic acquired resistance against plant pathogens. The newly established cell line initially accumulated also the indole alkaloid ajmalicine, a process enhanced by Pmg elicitor. This potency was lost during subculturing for about 1 year and was also not restored by preincubation with DCIA or 5CSA. In contrast, elicitation of 2,3-dihydroxybenzoic acid synthesis was undiminished, suggesting that the Pmg elicitor perception system was still functioning and not the cause for the decline in elicited indole alkaloid production.     

Differential induction of enzyme in soybean cell cultures by elicitor or osmotic stress

Soybean cell cultures were challenged either by glucan elicitor from Phytophthora megasperma f.sp. glycinea or by osmotic stress (0.4 M glucose). Osmotic stress induced production of a microsomal NADPH-dependent flavone synthase (flavone synthase II) which catalyses conversion of (2S)-naringenin to apigenin. In one of our cell-lines this enzyme activity was not detected either in unchallenged cells or in cells treated with glucan elicitor. Inducibility of flavone synthase II by 0.4 M glucose was highest at the end of the linear growth phase. Changes in the activities of a number of other enzymes were determined after treatment of the cells with elicitor or 0.4 M glucose. The activities of phenylalanine ammonialyase, cinnamate 4-hydroxylase, chalcone synthase and dihydroxypterocarpan 6a-hydroxylase all increased with elicitor and with osmoticum, albeit to a different degree. The rise in enzyme activity occurred later with osmoticum than with elicitor. The prenyltransferase involved in glyceollin synthesis was induced strongly by elicitor but only very weakly by osmoticum, whereas isoflavone synthase and NADPH: cytochrome-c reductase were only induced by elicitor. The activity of glucose-6-phosphate dehydrogenase did not change with elicitor or with osmoticum. Different product patterns were also obtained: whereas with elicitor, glyceollin I was the major product, intermediates of the glyceollin pathway (7,4-dihydroxyflavanone, trihydroxypterocarpan) accumulated with osmoticum.     

An endogenous factor from soybean (Glycine max L.) cell cultures activates phosphorylation of a protein which is dephosphorylated in vivo in elicitor-challenged cells

The existence of specific binding sites for a -glucan elicitor of phytoalexin synthesis derived from the fungus Phytophthora megasperma f.sp. glycinea at the plasma membrane of soybean (Glycine max L.) tissues (W.E. Schmidt, J. Ebel (1987) Proc. Natl. Acad. Sci. USA 84, 4117–4121) might imply that stimulation of phytoalexin formation by the elicitor is a membrane-mediated process. Addition of the -glucan elicitor to soybean cellsuspension cultures, which has previously been shown to induce phytoalexin accumulation, also results in rapid changes in the phosphate turnover of several phosphoproteins. The effect of the elicitor on protein phosphorylation was tested after labeling of the cells with [³²P]orthophosphate. As shown by analysis using one-and two-dimensional gel electrophoresis, decreases as well as increases in the labeling of several phosphoroteins occurred rapidly, being detectable within 5 min after elicitor application, and persisted for at least 15 min. As judged by their relative molecular masses (M_r) and isoelectric points (pI), a number of proteins which were radioactively labeled in vivo were also phosphorylated in vitro by endogenous protein-kinase activity in the presence of Ca²⁺. The most pronounced effect was observed with a protein substrate with M_r=69000 and pI=5.7 (pp69) whose phosphate labeling markedly decreased in response to elicitor treatment in vivo. Phosphorylation of pp69 in vitro in the presence of -[³²P]ATP was strongly enhanced by a phosphorylation-stimulating factor (effector) derived from soybean cell cultures and occurred predominantly at serine residues. The effector possessed a low apparent M_r (1000), was negatively charged at pH 7.3, and was relatively heat stable. The effector was inactivated by treatment with alkaline phosphatase from calf intestine. Phosphorylation of pp69 was only slightly stimulated by Ca²⁺, and was insensitive to cAMP, cGMP, calmodulin, a lipid mixture, a ganglioside mixture, or spermine under the assay conditions used. A 10 mM concentration of 3-phosphoglycerate increased pp69 phosphorylation to the extent of about 50% of that induced by the soybean effector. There was no evidence, however, that such concentrations of 3-phosphoglycerate occurred in effector preparations. The results are discussed in relation to hypothetical signal transduction during elicitor action on soybean cells.Abbreviations M_r relative molecular mass - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - TPCK L-1-tosylamide-2-phenylethyl chloromethyl ketone     

Localization of α-Mannan in the Hyphal Wall of Phytophthora megasperma f. sp. glycinea and it's Possible Relevance to the Host-Pathogen Interaction of the Fungus With Soybeans (Glycine max)

At the apices of growing hyphae of Phytophthora megasperma f. sp. glycinea (Pmg) distinctly more fluorescein-isothiocyanate labelled Concanavalin A was bound than else-where in the mycelium. In combination with some other analytical evidence this observation conclusively indicates that the α-mannan in the cell wall of the fungus is predominantly concentrated or exposed at the hyphal tips. The possible relevance of this fact to the host-pathogene interaction between Pmg and soybean is discussed.     

Host-Pathogen Interactions: XII. Response of Suspension-cultured Soybean Cells to the Elicitor Isolated from Phytophthora megasperma var. sojae, a Fungal Pathogen of Soybeans

The glucan elicitor isolated from the mycelial walls of Phytophthora megasperma var. sojae, the fungus which causes stem and root rot in soybeans, stimulates the activity of phenylalanine ammonia-lyase and the accumulation of glyceollin in suspension-cultured soybean cells. Nigeran, a commercially available fungal wall glucan, was the only other compound tested which has any activity in this system. Glyceollin is a phenylpropanoid-derived phytoalexin which is toxic to P. megasperma var. sojae. Evidence is presented to support the hypothesis that the action of elicitors in stimulating phytoalexin synthesis is not species or variety specific but, rather, is part of a general defensive response of plants.     

Rapid Accumulation of Anionic Peroxidases and Phenolic Polymers in Soybean Cotyledon Tissues following Treatment with Phytophthora megasperma f. sp. Glycinea Wall Glucan

Phytophthora megasperma Drechs. f. sp. glycinea Kuan & Erwin (PMG) cell wall glucan has been extensively characterized as an elicitor of the pterocarpan phytoalexins, the glyceollins in soybean (Glycine max L.). Just recently, this glucan was shown to be a potent elicitor of conjugates of the isoflavones, daidzein and genistein as well. Here we report that PMG wall glucan also induces a rapid and massive accumulation of phenolic polymers in soybean cotyledon cells proximal to the point of elicitor application. Deposition of phenolic polymers is over then times that in wounded controls within just 4 hours of elicitor treatment and reaches a maximum by 24 hours. In the same tissues, isoflavone conjugates begin to accumulate at 8 hours and glyceollin at 12 hours. By 24 hours, the total deposition of wall bound phenolics in elicitor-treated tissues is several times greater than the peak glyceollin and isoflavone responses combined. Histochemical stains and quantitation of phenolic residues released after saponification and nitrobenzene or copper oxide oxidation suggest that the covalently linked phenolics include both lignin- and suberin-like polymers as well as simple esterified coumaric and ferulic acid monomers. Accumulations of phenolic polymers are accompanied by equally rapid and massive increases in activity of a specific group of anionic peroxidases. Although increases in peroxidase activity are not strictly limited to cells immediately adjacent to the area of elicitor treatment, the deposition of phenolic polymers is significantly less extensive in distal cells.     

Host-Pathogen Interactions : XVIII. ISOLATION AND BIOLOGICAL ACTIVITY OF GLYCINOL, A PTEROCARPAN PHYTOALEXIN SYNTHESIZED BY SOYBEANS

A previously unrecognized phytoalexin has been isolated from soybean cotyledons that had been infected with bacteria or exposed to ultraviolet light. The phytoalexin has been purified to homogeneity by silica gel flash chromatography and high pressure liquid chromatography. It has been structurally characterized by its ultraviolet, circular dichroism and nuclear magnetic resonance spectra, polarimetry, and its mass spectrometric fragmentation pattern. The phytoalexin, (6aS,11aS)-3,6a,9-trihydroxypterocarpan, is a compound that had previously been detected in CuCl₂-treated soybeans and is structurally related to the previously identified soybean phytoalexins glycerollins I to IV. It is proposed that the trivial name glycinol be used for this phytoalexin. Glycinol is a broad spectrum antibiotic capable of prolonging the lag phase of growth of all six bacteria examined, namely Erwinia carotovora, Pseudomonas glycinea (races 1 and 3), Escherichia coli, Xanthomonas phaseoli, and Bacillus subtilis. Glycinol also inhibits the growth of the fungi Phytophthora megasperma f. sp. glycinea (race 1), Saccharomyces cerevisiae, and Cladosporium cucumerinum. Glycinol is a static agent against the six bacterial species listed above and against S. cerevisiae, and appears to be static against the other fungi examined. As with other phytoalexins, there is no correlation between the pathogenicity of a microorganism and its sensitivity to glycinol.