Piptocephalis Virginiana Research Articles

Isolation and partial characterization of a complementary protein from the mycoparasite Piptocephalis virginiana that specifically binds to two glycoproteins at the host cell surface

Immunofluorescence microscopy was used to detect in the mycoparasite Piptocephalis virginiana the presence of a complementary glycoprotein that binds specifically to the host cell surface glycoproteins b and c, reported earlier from our laboratory. Germinated spores of P. virginiana treated with cell wall extract of the host Mortierella pusilla, primary antibody prepared against cell wall glycoproteins b and c, and fluorescein isothiocyanate (FITC) – goat anti-rabbit IgG conjugate showed fluorescence. Immunobinding analysis identified from the mycoparasite a protein of 100 kDa that binds with the host glycoproteins b and c, separately as well as collectively. Its purification was achieved by (i) 60% ammonium sulfate precipitation, (ii) heat treatment, (iii) Sephadex G-100 gel filtration, and (iv) preparative polyacrylamide gel electrophoresis (PAGE). The purity was ascertained by sodium dodecyl sulphate (SDS) – PAGE and Western blot analysis. Positive reaction to periodic acid – Schiff s reagent revealed its glycoprotein nature, and mannose was identified as a major sugar component. The specificity of the polyclonal antibody raised against electrophoretically purified complementary protein in rabbit was confirmed by dot immunobinding and Western blot analyses. Immunofluorescence microscopy revealed surface localization of the protein on the germ tubes of P. virginiana. Fluorescence was also observed at the surface of the germinated spores and hyphae of the host M. pusilla, after treatment with complementary protein from P. virginiana, primary antibody prepared against the complementary protein, and FITC – goat anti-rabbit IgG conjugate.Key words: biotrophic mycoparasite, cell surface agglutinin, glycoprotein immunobinding, immunofluorescence, mucoraceous host.

Immunocytochemical and cytochemical localization of chitinase and chitin in the infected hosts of a biotrophic mycoparasite, Piptocephalis virginiana

Localization of chitinase and chitin in the uninfected and infected mucoraceous hosts Choanephora cucurbitarum and Phascolomyces articulosus of the mycoparasite, Piptocephlis virginiana, was investigated immunocytochemically with an antibody raised against purified chitinase and cytochemically with wheat germ lectin for the detection of N-acetyl-D-glucosamine oligomers. In the uninfected susceptible host (C. cucurbitarum), chitinase was found in cytoplasmic vacuoles, whereas in the resistant host (P. articulosus) chitinase was observed in the cell wall, as well as in vacuoles. In both hosts, infection by the mycoparasite destroyed vacuolar integrity and the chitinase was dispersed into the cytoplasm. Only in the resistant host, was accumulation of chitinase observed at the penetration sites. N-acetyl-D-glucosamine oligomers were detected in the cell wall of both host fungi, as well as in the mycoparasite and its haustorium. In the infected resistant host, papillae and the extrahaustorial matrix also contained N-acetyl-D-glucosamine, indicating their cell wall-like composition. Differences in the spatial localization of chitinases and N-acetyl-D-glucosamine oligomers in the two fungal hosts are implicated in their compatible and incompatible interactions with the mycoparasite.

Immunocytochemical and Cytochemical Localization of Chitinase and Chitin in the Infected Hosts of a Biotrophic Mycoparasite, Piptocephalis virginiana

Immunocytochemical and Cytochemical Localization of Chitinase and Chitin in the Infected Hosts of a Biotrophic Mycoparasite, Piptocephalis virginiana

Immuno-cytochemical localization of host glycoproteins involved in the attachment of the mycoparasite Piptocephalis virginiana

Polyclonal antibodies prepared against the two glycoproteins combined (M r 100 and 85 kDa) that are involved in the attachment of the mycoparasite, Piptocephalis virginiana, to its hosts, Mortierella pusilla and Phascolomyces articulosus, susceptible and resistant, respectively, were employed to localize the antigens at their cell surfaces. An indirect immuno-cytochemical technique using secondary antibodies labelled with either fluorescein isothiocyanate or gold particles, was used. FITC-labelled antibodies revealed a discontinuous pattern of fluorescence on the hyphae of Mortierella pusilla and no fluorescence on the hyphae of Phascolomyces articulosus. Intensity of fluorescence was high in the germinating spores of both fungi. Fluorescence could be observed on P. Articulosus hyphae pretreated with a commercial proteinase. Fluorescence was not observed on either hyphae or germinating spores of the non-host Mortierella candelabrum and the mycoparasite P. virginiana. Antibodies labelled with gold particles showed a different pattern of antigen localization on the hyphal walls of the susceptible and resistant hosts. Patches of gold particles were observed all over the whole cell wall of the susceptible host but only on the inner cell wall layer of the resistant host. Attachment of the mycoparasite to cell wall fragments of the susceptible host, but not those of the resistant host, was partially inhibited by the antibody preparation. Implications of different spatial distribution of the antigen in the resistant host and its absence in the non-host are discussed.

Involvement of fimbriae in fungal host-mycoparasite interaction

Germ tubes of the biotrophic haustorial mycoparasite, Piptocephalis virginiana, show directed growth towards hyphae of the mucoraceous fungi Mortierella pusilla, Phascolomyces articulosus and M. candelabrum. The mycoparasite will subsequently attach to cell walls of both the susceptible, M. pusilla, and resistant, P. articulosus, hosts but not to the non-host M. candelabrum. Fimbriae were observed by electron microscopy on the surfaces of the host and non-host species but not the mycoparasite. Polyclonal antiserum prepared against the fimbrial protein of Ustilago violacea crossreacted with 64 kDa proteins from both M. pusilla and P. articulosus and 60 and 57 kDa proteins from M. candelabrum. These proteins were electroeluted from polyacrylamide gels and were shown subsequently to form fibrils with the same diameter as the cell surface fimbriae. To ascertain the role of fimbriae in host-mycoparasite interactions, the antiserum was incubated with P. virginiana and M. pusilla, and with P. virginiana and P. articulosus. Contacts between mycoparasite and host were blocked significantly by the antiserum. This inhibition was not due to any effect of the antiserum on the linear growth rate of mycoparasite germ tubes. Thus, it was proposed that the recognition of fimbriae by the mycoparasite leads to directed growth and contact between mycoparasite germ tubes and the hyphae of a potential host.

Cell Surface Characterization of Five Haustorial Mycoparasites with Lectin Probes

Five zygomycetous, haustorial mycoparasites (Piptocephalis virginiana, P. xenophila, and Syncephalis sphaerica: Piptocephalidaceae, Zoopagales; and Dimargaris cristalligena, Dispira cornuta: Dimargaritaceae, Dimargaritales), which differ in their strategies of parasitism, were investigated. Identification of components of the outer surface of germ tube cell walls, which come in contact with the host hyphae, may be an important step in understanding host-parasite interactions. A variety of FITC-labeled lectins were used as probes. Binding with WGA, PAA and Con A, specific for N-acetyl glucosamine and D-mannose/glucose, was observed on the surface of all five mycoparasites. Dimargaris and Dispira also revealed galactose at their cell surface, but N-acetyl galactosamine was observed at the surface of Dimargaris only. Most notable was the inaccessibility of fucose at the cell surface of all five mycoparasites. The binding pattern varied with different lectins in different mycoparasites, but there was no change in the lectin binding pattern during various growth stages of the same mycoparasite.

Isolation and partial characterization of host cell surface agglutinin and its role in attachment of a biotrophic mycoparasite

Cell surface proteins obtained by alkaline extraction from isolated cell walls of Mortierella pusilla and M. candelabrum, host and nonhost, respectively, of the mycoparasite Piptocephalis virginiana, were tested for their ability to agglutinate mycoparasite spores. The host cell wall protein extract had a high agglutinating activity (788 agglutination units/mg) compared with that of the nonhost extract (21 agglutination units/mg). Sodium dodecyl sulfate – polyacrylamide gel electrophoresis of the crude extract of the host revealed four bands, a, b, c, and d, with respective Mr of 117 000, 100 000, 85 000 and 64 000; these bands except for a faint band c, were absent from the nonhost surface. Deletion of proteins b or c from the crude protein extract of the host significantly reduced its agglutinating activity. Proteins b and c, purified by a series of procedures, were shown to be glycoproteins with glucose and N-acetylglucosamine as major saccharides. The agglutinating activity of a mixture of pure proteins b and c was over 500 times that of either glycoprotein alone, suggesting an involvement of both glycoproteins in the agglutination process. Further characterization showed that the two glycoproteins were heat-resistant with respect to their agglutinin function, which could be totally inhibited by three sugars: arabinose, glucose and N-acetyglucosamine. It is suggested that glycoproteins b and c are the two subunits of a carbohydrate-binding agglutinin present at the host cell surface and involved in agglutination and attachment of the mycoparasite germ tubes. Key words: agglutinin, attachment, cell surface, sugars, glycoproteins, mycoparasitism.

Involvement of cell surface sugars in recognition, attachment, and appressorium formation by a mycoparasite

Fluorescein isothiocyanate labeled lectin binding techniques have revealed differences in the distribution pattern of glycosyl residues at the cell wall level between fungi that are hosts and those that are nonhosts of the mycoparasite Piptocephalis virginiana, and at the protoplast level between compatible and incompatible hosts. The cell wall of the compatible hosts (Choanephora cucurbitarum and Mortierella pusilla) and an incompatible host (Phascolomyces articulosus), as well as that of the mycoparasite itself, contains glucose and N-acetylglucosamine. However, the cell wall of a nonhost (Mortierella candelabrum) tested positive with lectins specific for various sugars, including not only glucose and N-acetylglucosamine, but also fucose, N-acetylgalactosamine, and galactose. These latter sugars could also be exposed at the surfaces of hosts and of the mycoparasite, but only after mild treatment with proteinase or when grown in a liquid culture. Pretreatment of the mycoparasite with glucose and N-acetylglucosamine inhibited its attachment to the host cell surface, but had no obvious effect on appressorium formation. On the other hand, appressorium formation was inhibited by heat treatment of host cell wall fragments which still permitted attachment, thus indicating that the factors responsible for attachment and for appressorium formation are different. The protoplast surfaces of compatible hosts contained all the sugars listed above and these protoplasts could attach to the germ tube of the mycoparasite. Only lectins specific for N-acetylglucosamine and for glucose were bound at the protoplast surface of the incompatible host; these protoplasts did not attach to the mycoparasite germ tube. Key words: mycoparasite, appressorium formation, lectins, host cell surface, attachment, protoplast surface.

In vitro regulation of chitinase and chitin synthase activity of two mucoraceous hosts of a mycoparasite

Effects of partially purified preparations of proteinases extracted from Choanephora cucurbitarum and Phascolomyces articulosus, susceptible and resistant hosts of Piptocephalis virginiana, respectively, were studied in vitro on the activities of chitinase and chitin synthase of the same hosts. Both chitinase and chitin synthase are membrane bound, zymogenic, and activated by partial proteolysis. Host proteinases of acid and neutral type stimulated chitinase activity, but only the acid proteinase stimulated chitin synthase activity of the two hosts. Neutral proteinase of C. cucurbitarum inhibited its chitin synthase, whereas that of Ph. articulosus increased its chitin synthase activity. Partially purified preparations of neutral proteinase from the mycoparasite, Piptocephalis virginiana, however, enhanced chitinase and suppressed chitin synthase activity of both the hosts alike. The role of host proteinases in regulating the activity of chitinase and chitin synthase of the two host species is suggested.

Proteinase, Chitinase, and Chitosanase Activities in Germinating Spores ofPiptocephalis Virginiana

Proteinase, Chitinase, and Chitosanase Activities in Germinating Spores of<i>Piptocephalis Virginiana</i>

Proteinase, Chitinase, and Chitosanase Activities in Germinating Spores of Piptocephalis virginiana

Proteinase, Chitinase, and Chitosanase Activities in Germinating Spores of Piptocephalis virginiana

Suppression of host cell wall synthesis at penetration sites in a compatible interaction with a mycoparasite

Differences in cell wall reactions at the penetration sites on a resistant host, Phascolomyces articulosus Boedijn ex Benny and Benjamin, and on a susceptible host, Choanephora cucurbitarum (Berk. &amp; Rav.) Thaxter, were studied by autoradiography at 18 h after inoculation with a mycoparasite, Piptocephalis virginiana Leadbeater and Mercer. Localized incorporation of a radioactive chitin precursor, N-[3H]acetylglucosamine ([3H]GlcNAc), was observed at the penetration sites on the resistant but not the susceptible host. Autoradiography of mechanically wounded hyphae of the susceptible and resistant hosts showed similar patterns of localized incorporation of [3H]GlcNAc within 5 min after sonication. Label incorporation at the penetration as well as at the wounding sites was inhibited by polyoxin D. Cycloheximide treatment greatly increased the label in subapical regions and reduced that at the hyphal tips in both the hosts, whereas this treatment did not prevent localized incorporation at the wounding or the penetration sites on the resistant host. Suppression of localized incorporation of [3H]GlcNAc at penetration sites in C. cucurbitarum was not due to the lack of mobility of chitin synthase to the penetration site or its activating factor.

Specificity of mycoparasite attachment to the host cell surface

The use of isolated cell wall fragments of Choanephora cucurbitarum (Berk. &amp; Rav.) Thaxter (a host), and of Linderina pennispora Raper and Fennell (a nonhost), has provided not only a convenient method to quantify attachment of the parasite, Piptocephalis virginiana Leadbeater and Mercer, by the artificial inoculation and washing-off procedure, but also an excellent material for investigations on the molecular basis of specificity and host recognition. The parasite germ tubes are attached to the cell wall fragments of the host but not of the nonhost. Attachment was inhibited by the addition of sugars, chitobiose and chitotriose, and by treatment with acid or alkali indicating the involvement of proteins or glycoproteins in recognizing sugar residues at the cell surface. Both host and nonhost showed a positive binding reaction with fluorescent lectins specific for N-acetyl-D-glucosamine oligomer. The cell surface of the nonhost also contains D-galactose and N-acetyl-D-galactosamine residues as lectin binding sites. Sodium dodecyl sulfate – polyacrylamide gel electrophoresis of cell wall extracts of host and nonhost revealed four bands of glycoproteins common to both fungi and two were specific to the host.

Properties of chitin synthase from mucoraceous hosts of a mycoparasite

Crude chitin synthase extracted from young (24 h) hyphae of Choanephora cucurbitarum and Phascolomyces articulosus, susceptible and resistant hosts, respectively, to the mycoparasite, Piptocephalis virginiana, was identified and characterized by measuring the incorporation of the substrate [14C]UDP – N-acetylglucosamine into chitin. The enzyme activity was mainly associated with the mixed membrane fraction. Properties of the enzyme preparation such as activation with proteases, N-acetylglucosamine, magnesium, inhibition with polyoxin D, Vmax, apparent Km value for UDP – N-acetyl-D-glucosamine (UDP-GlcNAc), and response to pH were examined. Enzyme activity from both fungi displayed basically the same features as the corresponding enzymes reported from other mucoraceous fungi. However, the two enzyme preparations (from P. articulosus and C. cucurbitarum) differed from each other in their response to various proteases and storage at 4 °C. Enzyme preparation from P. articulosus was activated by all proteases, whereas the C. cucurbitarum preparation was activated by acid protease, slightly activated by trypsin over a narrow concentration range, and was inhibited by neutral protease. Enzyme preparation from C. cucurbitarum showed a rapid decrease in activity within the first 5 h of storage at 4 °C and also exhibited relatively higher activity of endogenous proteases than that from P. articulosus.

Cell surface characteristics of Mortierella species and their interaction with a mycoparasite

Cell surface characteristics of three Mortierella species differing in their response to a mycoparasite, Piptocephalis virginiana, were examined. Their cell wall composition was typical of mucoraceous fungi with chitin and chitosan as major polysaccharides. Electron microscopy revealed that the mycoparasite penetrated and formed haustoria in the hyphae of susceptible hosts, M. pusilla and M. isabellina. The failure of the parasite to establish contact and penetrate a hypha of the nonhost, M. candelabrum, was not due to cell wall thickness, rigidity, or chitin contents. Markedly different protein patterns obtained from crude alkali extracts of host and nonhost cell walls by sodium dodecyl sulfate – polyacrylamide gel electrophoresis might explain the difference in host and nonhost response to the mycoparasite. Whereas most of the bands differed only in intensity after staining with either Coomassie blue or periodic acid – Schiff reagent, there were two distinct bands of glycoproteins (76 000 and 74 000) observed in the host species which were absent in the nonhost species.

Host-Parasite Relations in a Mycoparasite. VIII. Age-Related Host Resistance inChoanephora Cucurbitarum

Isolated cell walls obtained from young (1 da) and mature (5 da) cultures of Choanephora cucurbitarum, susceptible and resistant, respectively, to the biotrophic mycoparasite, Piptocephalis virginiana, were analyzed by microchemical and electron microscopic techniques. Comparison of the chemical composition of both young and mature cell walls revealed no qualitative difference. However, as the cultures aged, an increase in chitin, protein, neutral sugars (especially glucose and mannose), and a decrease in chitosan content were noted. Glutaraldehyde-osmium-fixed thin sections and metal shadow replicas revealed that the cell walls of hyphae in young cultures had a single layer of randomly distributed microfibrils of chitin, whereas the hyphal walls in mature cultures possessed two layers, randomly- and parallel-oriented microfibrils. The presence of a secondary layer of cell wall in mature hyphae coincided with the ability of the host to form a papilla at the infection site when challenged by the parasite. The role of the host cell wall in age-related resistance to a mycoparasite is discussed.

Spore Swelling, Germination, and Germ Tube Formation in Axenic Culture among Four Species of Piptocephalis

The nutritional requirements for spore swelling, germination, and germ tube formation in axenic culture were compared among four species of Piptocephalis. The spores of P. lepidula did not require exogenous nutrients for swelling, germination, or germ tube formation, although best germ tube elongation occurred on media containing selected nitrogen sources. In contrast, significant swelling, germination, and germ tube elongation of P. digitata and P. xenophila spores only occurred on media containing orange juice or yeast extract. Piptocephalis Virginiana spores swelled on several media that did not support germination or germ tube formation. Best germ tube elongation by P. Virginiana occurred on a medium containing yeast extract.

Host-Parasite Relations in a Mycoparasite. VII. Light and Scanning Electron Microscopy of Interactions of Piptocephalis Virginiana with Host and Nonhost Species

SUMMARY Spores of the mycoparasite Piptocephalis virginiana on germination usually produced two germ tubes. The germ tube nearest to a host hypha continued to grow and made contact with it. The tip of the germ tube in contact with the host formed an appressorium. Further events of parasitic infection depended on the nature of the host. In compatible interaction with a susceptible host, Choanephora cucurbitarum, the parasite penetrated the host cell wall without any apparent reaction of the host and formed a slender haustorium. Interaction with a resistant host, Phascolomyces articulosus, resulted in the development of host cell wall thickening at the site of penetration which probably arrested the further advance of the parasite. A marked difference between the penetration sites on the susceptible and resistant hosts was revealed by scanning electron microscopy. The parasite germinated equally well in the presence of host and nonhost species, but it neither made contact nor attempted penetration of hypha of a nonhost, Linderina pennispora. An earlier investigation described ultrastructural features of the

Host-Parasite Relations in a Mycoparasite. VII. Light and Scanning Electron Microscopy of Interactions of Piptocephalis virginiana with Host and Nonhost Species

SUMMARYSpores of the mycoparasite Piptocephalis virginiana on germination usually produced two germ tubes. The germ tube nearest to a host hypha continued to grow and made contact with it. The tip of the germ tube in contact with the host formed an appressorium. Further events of parasitic infection depended on the nature of the host. In compatible interaction with a susceptible host, Choanephora cucurbitarum, the parasite penetrated the host cell wall without any apparent reaction of the host and formed a slender haustorium. Interaction with a resistant host, Phascolomyces articulosus, resulted in the development of host cell wall thickening at the site of penetration which probably arrested the further advance of the parasite. A marked difference between the penetration sites on the susceptible and resistant hosts was revealed by scanning electron microscopy. The parasite germinated equally well in the presence of host and nonhost species, but it neither made contact nor attempted penetration of hypha of a nonhost, Linderina pennispora.

Host–parasite relations in a mycoparasite. VI. Leakage of electrolytes, amino acids, and sugars after infection with Piptocephalis virginiana

The leakage of electrolytes, amino acids, and sugars from Choanephora cucurbitarum infected by a biotrophic haustorial mycoparasite, Piptocephalis virginiana was investigated and compared with published reports of leakage from plant hosts infected by biotrophic pathogens. During early stages of infection (24 h after inoculation), the mycoparasitic system showed a significant increase in the leakage of electrolytes, amino acids, and sugars contrary to plant host–pathogen systems which exhibit pronounced leakage during later stages of disease development. Among the 26 amino acids and amino compounds, aspartic acid, glutamic acid, and alanine exuded in higher amounts. No new species of amino acid or amino compound was detected in the leachate of the infected host. Sugar analysis of leachate showed the presence of eight different sugars among which glucose was found in significant amounts (50–53%). Comparison between leachate sugars and internal pool sugars of the infected and control hosts suggested that the leakage of sugars was selective. The nutritional implication of this preferential leakage is discussed.