Isolation of sealed plasma membrane vesicles from Phytophthora megasperma f. sp. glycinea: II. Partial characterization of Ca2+ transport and glyceollin effects

Calcium uptake was examined in sealed plasma membrane vesicles isolated from the plant pathogenic fungus, Phytophthora megasperma f. sp. glycinea. Calcium uptake was ATP-dependent and by the addition of various ionophores in the presence of ATP, it was shown that Ca2+ transport was mediated by a nH+/Ca2+ antiport. Further evidence for this antiport mechanism included Ca2+ uptake driven by an imposed pH gradient and the observation that calcium could dissipate a steady-state pH gradient across the vesicle membrane. Transport mediated by the nH+/Ca2+ antiport was optimal at pH 7.0, and demonstrated saturation kinetics for Ca2+ with a Km of about 7 microM. Glyceollin, a soybean phytoalexin, was found to inhibit Ca2+ transport consistent with its ability to increase H+ conductance. In the presence of glyceollin, calcium leakage from Phytophthora membrane vesicles also increased. This study provides basic information about calcium transport in a plant pathogenic fungus as well as demonstrating a possible mode of action of a phytoalexin.     

Isolation of plasma membrane ofPhytophthora megasperma f. sp.glycinea and some properties of the associated ATPase

Plasma membrane vesicles were isolated fromPhytophthora megasperma f. sp.glycinea using conventional methods of mechanical disruption followed by differential and density gradient centrifugation. The validity of presumed biochemical markers was confirmed using electron microscopy and the phosphotungstic acid-chromic acid staining procedure, which was judged to be specific for plasma membrane when performed under suitable conditions. The plasma membrane fraction showed a peak equilibrium density of 1.14 g/ml and was identified by its vanadate-sensitive Mg²⁺-dependent ATPase with an optimum temperature of 42°C and a pH optimum of 6.0 to 6.5. The activity was weakly stimulated by K⁺ and strongly inhibited by Ca²⁺. The enzyme showed a marked specificity for ATP as a substrate compared to other nucleoside mono-, di-, and triphosphate substrates or other general phosphatase substrates. The divalent cation requirement could be met equally well by Mg²⁺ and Co²⁺ and, to a lesser extent, by Mn²⁺, but not by Ni²⁺, Ba²⁺, Zn²⁺, Sr²⁺, Ca²⁺, Hg²⁺, Cu²⁺, or Fe²⁺ (in decreasing order of preference). Contamination by intact mitochondria (density 1.21 g/ml) or mitochondrial fragments (density 1.16 g/ml) was minimal and could be monitored by measuring cytochromec oxidase or oligomycin-sensitive pH 8.5 ATPase.     

Enzyme induction in soybean infected by Phytophthora megasperma f.sp. glycinea

Laminin, the glycoprotein of basement membranes, consists of two subunits of 200,000 (α) and 400,000 (β) M_r on gel electrophoresis after reduction. We evaluated the relative proteolytic susceptibility of the two subunits using a variety of serine proteases. Human α-thrombin degraded the β subunit without altering the density or size of the α subunit. Chymotrypsin, plasmin, and cathepsin G all degraded both the β and α subunits producing limited digestion products. Chymotrypsin and cathepsin G both produced two major fragments of 160,000 and 130,000 M_r whereas plasmin produced two fragments of 180,000 and 140,000 M_r. Time course digestion studies demonstrated that the 400-kd β subunit was digested much more rapidly than the α subunit, and suggested that the major fragments (greater than 100,000 M_r) produced by chymotrypsin, plasmin, and cathepsin G were derived from the α subunit. The latter supposition was confirmed by first digesting laminin with thrombin to completely remove the β subunit, followed by digestion with chymotrypsin, cathepsin G, or plasmin. We conclude that the β subunit of laminin is highly protease labile. In contrast, the α subunit contains a large region resistant to serine proteases. Electron microscopic studies of the purified fragment of laminin derived from digestion with cathepsin G demonstrated that the protease resistant region of the α subunit contained three arms of similar appearance (32 nm) and included the intersection of the three short arms of the laminin molecule.     

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.     

Races of Phytophthora megasperma f.sp. glycinea (Phytophthora Rot) Affecting Soybeans in North Alabama

Phytophthora megasperma Drechs. f. sp. glycinea Kuan and Erwin (Pmg) was isolated for the first time from the northern counties of Alabama (MORGAN and MADISON). TWO predominant Pmg races, R₂ and R₁₁, were identified by the reactions of standard soybean differential cultivars. Other soil-borne pathogens isolated from the soil and suspected Pmg-affected root samples in Morgan, Adadison, and Limestone counties were Pytbium spp., Fusarium ssp., Sclerotium rolfsii, and Rbizoctonia solani. Further study is in progress to obtain a proper assessment of Pmg races in Alabama.     

Different cell-wall components from Phytophthora megasperma f. sp. glycinea elicit phytoalexin production in soybean and parsley

Different components of a crude cell-wall preparation from the phytopathogenic fungus, Phytophthora megasperma f. sp. glycinea, act as elicitors of phytoalexin accumulation in parsley (Petroselinum crispum) and soybean (Glycine max). Treatments of cultured parsley cells and protoplasts or soybean cells and cotyledons with proteinase-digested or deglycosylated elicitor preparations identify proteinaceous constituents as active eliciting compounds in parsley, which are inactive in soybean. The proteinase-treated elicitor as well as a defined heptaglucan are active in soybean but do not stimulate phytoalexin synthesis in parsley. Soybean and parsley cells therefore not only perceive different signals from cell walls of Phytophthora megasperma f. sp. glycinea, but are unable to respond to the fungal compounds primarily recognized by the other plant.Abbreviations Pmg Phytophthora megasperma f. sp. glycinea     

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).     

Effect of antimicrobial peptides on ATPase activity and proton pumping in plasma membrane vesicles obtained from mycobacteria

The potential usefulness of antimicrobial peptides (AMPs) as antimycobacterial compounds has not been extensively explored. Although a myriad of studies on AMPs from different sources have been done, some of its mechanisms of action are still unknown. Maganins are of particular interest since they do not lyse non-dividing mammalian cells. In this work, AMPs with well-recognized activity against bacteria were synthesized, characterized, purified and their antimycobacterial activity and influence on ATPase activity in mycobacterial plasma membrane vesicles were assessed. Using bioinformatics tools, a magainin-I analog peptide (MIAP) with improved antimicrobial activity was designed. The influence of MIAP on proton (H(+)) pumping mediated by F(1)F(0)-ATPase in plasma membrane vesicles obtained from Mycobacterium tuberculosis was evaluated. We observed that the antimycobacterial activity of AMPs was low and variable. However, the activity of the designed peptide MIAP against M. tuberculosis was 2-fold higher in comparison to magainin-I. The basal ATPase activity of mycobacterial plasma membrane vesicles decreased approximately 24-30% in the presence of AMPs. On the other hand, the MIAP peptide completely abolished the F(1)F(0)-ATPase activity involved in H(+) pumping across M. tuberculosis plasma membranes vesicles at levels similar to the specific inhibitor N,N' dicyclohexylcarbodiimide. These finding suggest that AMPs can inhibit the H(+) pumping F(1)F(0)-ATPase of mycobacterial plasma membrane that potentially interferes the internal pH and viability of mycobacteria.     

Investigation of the mechanism of glyceollin accumulation in soybean infected by Phytophthora megasperma f. sp. glycinea

Soybean seedlings (Glycine max, cv. Harosoy 63) which had been inoculated in the hypocotyls with mycelium from either race 1 (incompatible) or race 3 (compatible) of Phytophthora megasperma f. sp. glycinea were pulse labeled with ¹⁴CO₂. The time course of accumulation of glyceollin and daidzein and of ¹⁴C incorporation into these compounds was determined. Metabolic rates of glyceollin were measured by pulse-chase experiments. Differences in glyceollin accumulation between the incompatible and compatible interaction were not apparent before about 14 h after inoculation. Subsequently glyceollin accumulated to a higher level in the incompatible interaction. This difference is also reflected in the rate of ¹⁴C incorporation, which declines more rapidly in the compatible interaction. The apparent half-life of glyceollin metabolism was 28 ± 7 h for inoculation with race 1, while no metabolism was observed with race 3. In contrast to a previous report (M. Yoshikawa, K. Yamauchi, and H. Masago (1979)Physiol. Plant Pathol.14, 157–169), our data prove that the higher accumulation of glyceollin in the incompatible interaction is due to a longer duration of synthetic activity and that the level of glyceollin in both the incompatible and compatible interaction is determined predominantly by its rate of synthesis.     

Monoclonal Antibodies to Glycoprotein Antigens of a Fungal Plant Pathogen, Phytophthora megasperma f. sp. glycinea

Genetic studies of plants and their pathogens indicate that dominant alleles for resistance in hosts are complemented by corresponding dominant alleles for avirulence in pathogens. Products of these genes have not yet been identified. We have produced murine monoclonal antibodies (mAbs) to extracellular antigens of the fungal soybean pathogen Phytophthora megasperma f. sp. glycinea (Pmg, race 1) as part of a larger effort to identify antigenic determinants associated with particular avirulence genes. Thirty-six independent mAbs have been characterized by binding to Western blots of Pmg extracellular glycoproteins and by enzyme-linked immunosorbent assay with glycoproteins modified by treatment with periodate, α-mannosidase, and endo-β-N-acetylglucosaminidase H. The mAbs are predominantly carbohydrate-specific and can be placed in six groups based on interactions with Pmg glycoproteins. Binding patterns of various mAbs to Western blots indicate that Pmg proteins may have single or multiple types of attached carbohydrate antigens. Races of Pmg with differing avirulence genes exhibit more characteristic differences by Western blot analysis than by protein staining of glycoprotein profiles. Several of the mAbs show much higher reaction levels to glycoproteins from race 1 than from two other races. All of the glycoprotein-specific mAbs cross-react with purified mycelial walls.     

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)     

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.     

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     

Quantitative Localization of the Phytoalexin Glyceollin I in Relation to Fungal Hyphae in Soybean Roots Infected with Phytophthora megasperma f. sp. glycinea

A radioimmunoassay specific for glyceollin I was used to quantitate this phytoalexin in roots of soybean (Glycine max [L.] Merr. cv Harosoy 63) after infection with zoospores of either race 1 (incompatible) or race 3 (compatible) of Phytophthora megasperma Drechs. f. sp. glycinea Kuan and Erwin. The sensitivity of the radioimmunoassay and an inmmunofluorescent stain for hyphae permitted quantitation of phytoalexin and localization of the fungus in alternate serial cryotome sections from the same root. The incompatible interaction was characterized by extensive fungal colonization of the root cortex which was limited to the immediate vicinity of the inoculation site. Glyceollin I was first detected in extracts of whole roots 2 hours after infection, and phytoalexin content rose rapidly thereafter. Significant concentrations of glyceollin I were present at the infection site in cross-sections (42 micrometers thick) of such roots by 5 hours, and exceeded 0.6 micromoles per milliliter (EC₉₀in vitro for glyceollin I) by 8 hours after infection. Longitudinal sectioning (14 micrometers thick) showed that glyceollin I accumulated particularly in the epidermal cell layers, but also was present in the root cortex at inhibitory concentrations. No hyphae were observed in advance of detectable levels of the phytoalexin and, in most roots, glyceollin I concentrations dropped sharply at the leading edge of the infection. In contrast, the compatible interaction was characterized by extensive unchecked fungal colonization of the root stele, with lesser growth in the rest of the root. Only small amounts of glyceollin I were detected in whole root extracts during the first 14 hours after infection. Measurable amounts of glyceollin I were detected only in occasional cross-sections of such roots 11 and 14 hours after infection. The phytoalexin was present at inhibitory concentrations in the epidermal cell layers, but the inhibitory zone did not extend appreciably into the cortex. Altogether, these data support the hypothesis that the accumulation of glyceollin I is an important early response of soybean roots to infection by P. megasperma, but may not be solely responsible for inhibition of fungal growth in the resistant response.     

Race:cultivar-specific induction of enzymes related to phytoalexin biosynthesis in soybean roots following infection with Phytophthora megasperma f. sp. glycinea

Primary roots of soybean [Glycine max (L.), 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 (Pmg) and the activities of phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), isoflavone synthase, and dihydroxypterocarpan 6a-hydroxylase related to phytoalexin (glyceollin) biosynthesis, and of glucose-6-phosphate dehydrogenase (Glc-6-PDH) and glutamate dehydrogenase (Glu-DH) were determined at various times after inoculation. About 2-4 h after inoculation with race 1, the activities of PAL, CHS, and pterocarpan 6a-hydroxylase were higher than after inoculation with race 3 and increased considerably thereafter. In contrast, activities of these enzymes in the compatible interaction were equal to or only slightly higher than in the controls over the entire infection period investigated (2-8 h). Isoflavone synthase did not increase until 7 h after inoculation with race 1. There were no significant differences in activities for Glc-6-PDH and Glu-DH between inoculated roots and controls. The results show that infection of soybean roots with zoospores of Pmg race 1 causes a race:cultivar-specific early induction of enzymes involved in glyceollin synthesis, whereas such an induction does not occur with zoospores of race 3. These findings are in agreement with the race:cultivar-specific accumulation of glyceollin in soybean roots reported previously [M. G. Hahn, A. Bonhoff, and H. Grisebach (1985) Plant Physiol. 77, 591-601].     

ATPase activity and ATP-dependent proton translocation in plasma membrane vesicles of turtle bladder epithelial cells

ATP-induced quenching of fluorescence of acridine orange (a pH probe) or Oxonol V (a potential difference probe) is evoked in turtle bladder membrane vesicles in suspending media of appropriate ionic composition and is insensitive to oligomycin, valinomycin, and ouabain. These effects are ascribed to a membrane-bound, ouabain-resistant ATPase which mediates an active electrogenic proton transport.     

Proton pumping by vesicles reconstituted from two fractions of solubilized rose-cell plasma membrane ATPase

Two forms of K⁺ -stimulated ATPase, which can be solubilized from purified plasma membrane preparations of suspension-cultured rose cells and separated by molecular sieve chromatography, both catalyze the ATP-dependent accumulation of protons into artificial phospholipid/cholesterol vesicles. The higher-molecular weight form of ATPase is highly sensitive to ultraviolet light, and the proton pumping ability of this form is similarly sensitive.