Germ Tube Wall Research Articles

Strawberry Anthracnose: Histopathology ofColletotrichum acutatumandC. fragariae

ABSTRACT Ontogeny of the invasion process by Colletotrichum acutatum and C. fragariae was studied on petioles and stolons of the strawberry cultivar Chandler using light and electron microscopy. The invasion of host tissue by each fungal species was similar; however, each invasion event occurred more rapidly with C. fragariae than with C. acutatum. Following cuticular penetration via an appressorium, subsequent steps of invasion involved hyphal growth within the cuticle and within the cell walls of epidermal, subepidermal, and subtending cells. Both species of fungi began invasion with a brief biotrophic phase before entering an extended necrotrophic phase. Acervuli formed once the cortical tissue had been moderately disrupted and began with the development of a stroma just beneath the outer periclinal epidermal walls. Acervuli erupted through the cuticle and released conidia. Invasion of the vascular tissue typically occurred after acervulus maturation and remained minimal. Chitin distribution in walls of C. fragariae was visualized with gold-labeled wheat germ agglutinin. The outer layer of bilayered walls of conidia, germ tubes, and appressoria contained less chitin than unilayered hyphae in planta.

Development of primary germ tubes by conidia of Blumeria graminis f.sp. hordei on leaf epidermal cells of Hordeum vulgare

Development of primary germ tubes from conidia of Blumeria graminis f.sp. hordei on primary leaf segments of Hordeum vulgare was investigated from 3 to 13 h postinoculation (hpi) using transmission electron microscopy. By 3 hpi, the primary germ tube wall that makes contact with the host cuticle develops a small protrusion that breaches the host cuticle and touches the host cell wall but does not penetrate any further. This protrusion is the cuticular peg. From 3 to 13 hpi, the cuticular peg swells, becomes quite electron dense, and finally develops a loose fibrillar texture. The structure of the primary germ tube with the terminal cuticular peg is consistent with the hypothesis that it allows the conidium to absorb water and solutes present in the host cell wall.Key words: powdery mildew, barley, ultrastructure.

Molecular evidence for induction of phenylalanine ammonia-lyase during Puccinia graminis infection and elicitation in wheat

Degenerate primers corresponding to the conserved sequences in phenylalanine ammonia-lyase (PAL) genes cloned from rice and parsley were used to amplify segments of the PAL genes from wheat (Triticum aestivum). More than 76% base sequence identity with the homologous genes confirmed that a 437 base pairs (bp) fragment was derived from the coding region of PAL. Using this fragment as probe, two PAL genes linked in tandem, Wpalt1 and Wpalt2, were isolated from a wheat genomic library. Northern blot and RNA dot blot analyses using a probe for Wpalt1 demonstrated that Wpalt1 expression was organ specific. Wpalt1 expression was high in roots and moderate in stems, but no detectable expression was found in leaves. Infection of leaves with Puccinia graminis f. sp. tritici (Pgt) induced, within 4 to 8 d, a high level of Wpalt1 transcripts in the Pgt resistant near-isogenic Prelude Sr6 line, while a delayed induction was observed in the Pgt susceptible isogenic Prelude sr6 line. In cell suspension cultures, treatment with chitin oligomers or an elicitor derived from P. graminis germ tube walls activated Wpalt1 gene expression. The Pgt-derived elicitor activated PAL expression earlier than did chitin oligomers, whereas chitin oligomers appeared to be more potent inducers of PAL expression than was the Pgt-derived elicitor.

Purification, characterization, and antifungal activity of chitinase from Fusarium chlamydosporum, a mycoparasiteto groundnut rust, Puccinia arachidis

Chitinase (EC 3.2.1.14) was isolated from the culture filtrate of Fusarium chlamydosporum and purified by ion-exchange chromatography and gel filtration. The molecular mass of purified chitinase was 40 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Chitinase was optimally active at a pH of 5 and stable from pH 4 to 6 and up to 40 degrees C. Among the metals and inhibitors tested, mercuric chloride completely inhibited the enzyme activity. The activity of chitinase was high on colloidal and pure chitin. The purified chitinase inhibited the germination of uredospores of Puccinia arachidis and also lysed the walls of uredospores and germ tubes. The results from these experiments indicated that chitinase of F. chlamydosporum plays an important role in the biocontrol of groundnut rust.

DewA encodes a fungal hydrophobin component of the Aspergillus spore wall

An anonymous cDNA clone, pCAN4, was shown previously to correspond to an mRNA that accumulates preferentially during asexual sporulation of the filamentous fungus Aspergillus nidulans. The peptide encoded by pCAN4 is a fungal hydrophobin, a group of small, hydrophobic cell wall proteins. When the CAN4 gene was disrupted, conidia and conidiophores appeared to be normal, but sporulating colonies wetted more rapidly with detergent solutions than did the wild type. We renamed CAN4 dewA for the detergent wettable phenotype and mapped it to chromosome V, 24 map units from cysC. The A. nidulans rodA gene also encodes a sporulation-specific fungal hydrophobin. Spores of a dewA- rodA- double mutant were less hydrophobic than those of either mutant alone, showing that dewA and rodA contribute independently to spore-wall hydrophobicity. Immunolocalization of DewA by epitope tagging demonstrated that DewA is present in the spore wall, but not in the walls of germ tubes, hyphae or cells of the spore-producing conidiophore. We conclude that dewA encodes a new fungal hydrophobin component of the conidial wall.

Elicitor-active glycoproteins in apoplastic fluids of stemrust-infected wheat leaves

Cell walls of germ tubes from wheat stem rust ( Puccinia graminis f. sp. tritici) contain a glycoprotein with a molecular mass of about 67000 referred to as the Pgt elicitor. This glycoprotein induces a hypersensitive-like response in wheat leaves. In elicitor-active intercellular washing fluid (IWF) from compatible wheat-stem rust interactions we detected several elicitor-active glycoproteins. One of these glycoproteins had an electrophoretic mobility identical to the Pgt elicitor. This IWF-glycoprotein exhibited elicitor activity upon elution from SDS gels. It was recognized by anti-Pgt elicitor antiserum suggesting partial structural identity between the Pgt and IWF elicitors. As with the Pgt elicitor, the elicitor-activity of the IWF glycoprotein resides in the carbohydrate moiety because periodate, but not trypsin or pronase, destroyed activity. These results suggest that the Pgt elicitor is released from hyphal cell walls into the wheat apoplast during stem rust infection.

A monoclonal antibody specific for conidia and mycelium wall layer of Penicillium and Aspergillus.

A monoclonal antibody was obtained from BALB/c mice immunized with Penicillium frequentans mycelium. The specificity of the antibody was evaluated by enzyme-linked immunosorbent and indirect immunofluorescence assays against the same mycelium. This IgM antibody cross-reacted with various strains of the Penicillium and Aspergillus genera. By indirect immunofluorescence assays, the antibody was able to stain about 10% of Penicillium and Aspergillus conidia, but major part of conidia did not absorb the fluorescence-labeled antibody before swelling. During germination of P. frequentans conidia, the germ tube wall which constitutes a continuation of an inner wall layer was also stained. During germination of P. griseofulvum, the protrusion of the germ tube wall was not always recognized by the antibody because the germ tube wall was constituted by a continuation of an outer spore wall layer. The study of the staining patterns of the spores and the protrusions suggests that the antibody specifically recognizes an antigen of the inner spore wall layer. The monoclonal antibody reacts with extracellular galactomannans produced by genera Aspergillus and Penicillium but is not directed against beta-(1,5)-linked galactofuranose units.

Localization of carbohydrate antigens in the walls of Phytophthora megasperma f.sp. glycinea by monoclonal antibodies

A set of five carbohydrate-specific monoclonal antibodies (mAbs) were used to probe the ultrastructure of the walls of the soybean pathogen Phytophthora megasperma f.sp. glycinea, using a combination of immunofluorescence and immuno-gold labelling techniques. Results with β-1,3 glucan-specific antibodies suggest that β-1,3 glucans are present throughout the walls of both germ tubes and cysts, but are more prevalent in the outer portion. In addition, β-1,3 glucans on the surface of hyphal walls, but not cysts, are closely associated with other material, most likely protein, that sterically hinders antibody binding except to non-reducing terminal residues. An antibody whose epitope involved both β-1,4 and β-1,3 glucosyl linkages bound predominantly to the inner portion of the hyphal wall. However, fluorescent labelling with this antibody suggested that β-1,4 linkages are present on the exterior of P. megasperma f.sp. glycinea walls as well. Staining with another antibody indicates that changes in wall composition occur over 50–100 μm from the hyphal tip, a greater distance than previously supposed. The role of the antigens recognized by these mAbs in the plant-pathogen interaction is not known, but potential uses of these and other mAbs are discussed.

Physicochemical Properties of Cell Surfaces from the Different Developmental Stages of the Entomopathogenic Hyphomycete Nomuraea rileyi

Physicochemical properties of cell wall surfaces of conidia, conidial germ tubes, hyphal bodies and mycelia of the entomogenous hyphomycete Nomuraea rileyi were studied. Hydrophobicity was determined using a polystyrene microsphere adhesion assay. Microspheres bound to conidial germ tube and mycelial walls indicating that these surfaces are hydrophobic. Microspheres did not bind to hyphal body walls except at polar areas. Results from these experiments were confirmed using phase exclusion methods.Electrostatic charge characteristics of fungal surfaces were examined using DEAE (positively charged) and CM (negatively charged) beads. Cells from all growth phases adhered to DEAE but not CM beads indicating the presence of negatively charged sites on the surfaces. Degree of electronegativity as shown by FITC-poly-L-lysine binding is greatest on hyphal bodies and at the proximal end of germ tubes. Fluorescence was faint on conidia and mycelia. Cationized ferritin formed a thick capsule around hyphal body cells and a thinner but continuous layer on sheath material surrounding germ tubes; labeling of mycelial sheath was more random possibly due to the dispersed native of this material. Areas of germ tubes or mycelium not covered by sheath were sparsely labeled as were conidial surfaces. Neutralization of hydrophobicity or electronegativity did not affect passive binding of conidial or mycelial cells to monolayers of insect hemocytes.

Physicochemical Properties of Cell Surfaces from the Different Developmental Stages of the Entomopathogenic HyphomyceteNomuraea Rileyi

Physicochemical properties of cell wall surfaces of conidia, conidial germ tubes, hyphal bodies and mycelia ofthe entomogenous hyphomycete Nomuraea rileyi were studied. Hydrophobicity was deter? mined using a polystyrene microsphere adhesion assay. Microspheres bound to conidial germ tube and mycelial walls indicating that these surfaces are hydrophobic. Microspheres did not bind to hyphal body walls except at polar areas. Results from these experiments were confirmed using phase exclusion methods. Electrostatic charge characteristics of fungal surfaces were examined using DEAE (positively charged) and CM (negatively charged) beads. Cells from all growth phases adhered to DEAE but not CM beads indicating the presence of negatively charged sites on the surfaces. Degree of electronegativity as shown by FITC-poly-L-lysine binding is greatest on hyphal bodies and at the proximal end of germ tubes. Fluorescence was faint on conidia and mycelia. Cationized ferritin formed a thick capsule around hyphal body cells and a thinner but continuous layer on sheath material surrounding germ tubes; labeling of mycelial sheath was more random possibly due to the dispersed nature of this material. Areas of germ tubes or mycelium not covered by sheath were sparsely labeled as were conidial surfaces. Neutralization of hydrophobicity or electronegativity did not affect passive binding of conidial or mycelial cells to monolayers of insect hemocytes.

Evidence for expression of the C3d receptor of Candida albicans in vitro and in vivo obtained by immunofluorescence and immunoelectron microscopy

The complement conversion product C3d binds to a receptor on the cell surface of Candida albicans. While the function of this receptor is still uncertain, we investigated whether it is expressed during a murine infection. Rabbit antiserum raised against purified receptor was used in conjunction with immunofluorescence microscopy and immunocolloidal gold electron microscopy to examine kidney tissue and peritoneal lavages from infected mice for receptor expression by C. albicans in vivo. Specificity of the antiserum was indicated by reactivity with purified receptor (55 to 60 kDa) and with a protein of similar molecular mass from whole hyphal extracts in Western blots (immunoblots). In vitro analysis by immunofluorescence microscopy showed that the antiserum reacted with both yeast and pseudohyphal forms of the organism, but reactivity was strongest with pseudohyphae. Immunocolloidal gold electron microscopy of fungal cells from peritoneal lavages revealed intense staining of mother cells of germinative forms, germ tubes, and pseudohyphae. Staining of the mother cells was heaviest at the innermost layers of the cell wall but only scant on the cell surface. In contrast, staining was observed throughout the cell walls of germ tubes and pseudohyphae. In kidney, expression of the C3d receptor was found primarily on the cell walls of hyphae and pseudohyphae, although some staining was observed in the cytoplasm. These data support that the C3d receptor of C. albicans is expressed in vivo.

Specific binding of a hypersensitive lignification elicitor from Puccinia graminis f. sp. tritici to the plasma membrane from wheat (Triticum aestivum L.)

We have recently reported the isolation and characterization of a glycoprotein (Mr 67 000) from germ-tube walls of Puccinia graminis f. sp. tritici which elicits the cellular hypersensitive lignification response in wheat (G. Kogel et al., 1988, Physiol. Mol. Plant Pathol. 33, 173-185). The present study uses this glycoprotein, referred to as Pgt elicitor, to identify putative elicitor targets in wheat cell membranes. In enzyme-linked immunosorbent assays using anti-Pgt elicitor antibodies, specific binding sites for Pgt elicitor were detected in highly purified plasma-membrane vesicles of wheat (Triticum aestivum L.) primary leaf cells. Binding proved to be independent of the presence or absence in wheat of the Sr5 gene for rust resistance, and also occurred on barley (Hordeum vulgare L.) plasma membrane. The binding sites have an Mr of 30 000 and 33 000, respectively, and binding activity was not lost in the presence of sodium dodecyl sulfate. [(14)C]imido-Pgt elicitor was used to determine the apparent K d value for specific binding, found to be 2.0 μM, and the maximum content of binding sites, found to be 250 pmol per mg of plasma-membrane protein. The relevance of the elicitor binding for the outcome of the interaction of P. graminis and wheat is discussed.

In vitro formation of haustoria of the cowpea rust fungus, Uromyces vignae, in the absence of a living plant cell I. Light microscopy

Haustoria of the cowpea rust fungus were induced to form on oil-containing collodion membranes, in the absence of any living plant material, by the application of an inactivated commercial enzyme preparation. No neckband was detected along haustorial necks and mithramycin staining showed that nuclei did not complete their migration into haustoria from the haustorial mother cells (HMCs). Haustorial walls fluoresced only slightly with calcofluor and at an intensity much less than walls of the infection structures or haustorial mother cells. Walls of germ tubes, appressoria, infection pegs, substomatal vesicles, infection hyphae, HMCs and haustoria all fluoresced uniformly but weakly when treated with (FITC)-Concanavalin A, indicating the presence of mannose, glucose or acetyl glucosamine residues of the fungal surface; fluorescence was virtually eliminated by prior treatment of the fungus with unlabelled lectin or by incubating the lectin with its hapten. With FITC-WGA, walls of germ tubes, appressoria, the thickened region of the HMC wall surrounding the infection peg and the haustorial neck wall all fluoresced more brightly than other parts of the fungus. Although fluorescence could be eliminated by mixing the FITC-WGA with chitin hydrolysate, the ability of unlabelled WGA to reduce subsequent binding of FITC-WGA to appressoria and HMCs depended on the concentration of the labelled lectin and in no instance was haustorial wall fluorescence significantly reduced. These and other data suggest that FITC-WGA binding to the haustorium may not indicate the presence of chitin.

Ultrastructural studies of primary spores of Conidiobolus, Erynia, and related Entomophthorales

Wall development during primary spore formation, discharge, and germination of Entomophthorales is emphasized in ultrastructural studies of Conidiobolus, Entomophaga, Neozygites, and Erynia. In the fungi examined, spore and sporophore walls consist of a thick, electron-translucent inner layer and a thin, electron-dense outer layer. During spore formation, cytoplasm of the supporting sporophore cell migrates into the spore initial. As the former cell empties, a septum develops. Discharge is caused by inversion of the papillum, which lacks the electron-dense layer. Only in Erynia did the two spore wall layers separate upon impact. Intracytoplasmic organization of the primary spore is typical of the Zygomycotina; the morphology of organelles was characteristic of species, whereas nuclear ultrastructure was consistent within genera. Conidiobolus nuclei have a prominent nucleolus that lacks heterochromatin, in contrast with the other genera where large patches of heterochromatin were observed. Upon germination, no rupture of the spore outer layer was observed other than at points of germ tube emergence. The germ tube wall was continuous with the inner spore wall layer. The results are discussed in reference to Entomophthorales taxonomy and definition of the terms conidium and monosporous sporangiolum.

Ultrastructure of conidial germ-tube development in vitro by the insect pathogen Entomophaga aulicae

In an effort to understand the factors influencing the formation of infection structures by the insect pathogen Entomophaga aulicae, we examined the ultrastructural development of conidial germ tubes formed in vitro under conditions that resemble those producing appressoria and protoplasts during infection. Conidia germinated on formvar in a nonaqueous environment produced a single viable germ tube, which in turn produced appressorium-like structures or secondary conidia, structures similar to those formed on the host. The formation of appressorium-like structures indicates that stimuli for appressorium formation are relatively nonspecific, whereas development of the infection hypha requires different triggers. Conidia germinated in a liquid culture medium, which supports the growth of the protoplast stage of the fungus, produced a single viable germ tube that continued to grow apically. This apical growth was accompanied by the loss of the outer layer of the germ tube wall and the presence of electron-opaque granules in an extensive system of cytoplasmic membranes. Protoplasts did not form directly from these germ tubes and ultrastructural details of tip growth in this medium did not resemble those previously described in infection hyphae prior to protoplast formation in the host.

A single glycoprotein from Puccinia graminis f. sp. tritici cell walls elicits the hypersensitive lignification response in wheat

Con A-binding glycoproteins were detected in germ tube walls of Puccinia graminis f. sp. tritici and appeared to be potent inducers of the hypersensitive lignification response. This response, which is typical of the large resistance reaction in wheat leaves, was preceded by a increase in extractable phenylalanine ammonia-lyase (PAL) activity, a key enzyme of the phenylpropanoid pathway. Germ tube glycoproteins were isolated from homogenized fungal cell walls by differential centrifugation and affinity chromatography on a Con A-Sepharose column. The most active glycoprotein was purified on a Mono Q anion-exchange column. The relative molecular mass of the molecule was determined in SDS-polyacrylamide gels to be 67 kD. The carbohydrate portion consists mainly of mannose (50%) and galactose (47%) and contains the active part of the glycoprotein as demonstrated by the inability of pronase and trypsin digestion to influence activity. An elicitor-active glycoprotein with identical molecular mass and Con A-binding properties was isolated from intercellular fluids of rust-infected plants of the susceptible wheat cultivar Little Club indicating that the elicitor is released from fungal cell walls during infection. We discuss the role of Con A-binding proteins as inducers of non-host resistance in cereals.

Localization of glycopeptides and race-variable polypeptides in urediosporelings and urediosporeling walls of Puccinia graminis tritici; affinity to concanavalin A, soybean agglutinin, and Lotus lectin

Detergent-soluble polypeptides from urediosporelings and purified germ-tube walls of urediosporelings of wheat stem rust, Puccinia graminis f.sp. tritici, races C1(17), C17(56), and C36(48) were separated by two-dimensional isoelectric focusing – polyacrylamide gel electrophoresis. More than 280 polypeptides were distinguished by Coomassie brilliant blue staining in each race, and there were 18 polypeptides that varied among the three races, 7 of which were coincident with urediosporeling wall polypeptides. Polypeptides transferred from two-dimensional gels onto nitrocellulose membrane were either stained with India ink or probed with concanavalin A – peroxidase for the location of concanavalin A binding glycoproteins. Of 97 concanavalin A binding glycoproteins in urediosporelings, 41 were coincident with polypeptides that stained with Indian ink and 3 were coincident with race-variable polypeptides. The walls of urediosporelings contained few polypeptides that stained with Coomassie brilliant blue or India ink, but most were concanavalin A binding. None of the polypeptides had affinity to soybean agglutinin or Lotus lectin, suggesting that galactose or fucose is not a terminal sugar moiety in these polypeptides. We conclude that race-variable polypeptides are located both within the cytoplasm and in the walls of urediosporelings, but most are not glycosylated.

Identification of two germ-tube-specific cell wall antigens of Candida albicans

Outer cell wall layers of intact yeast- and mycelial-phase Candida albicans B311 were extracted with dithiothreitol. Antisera against mycelial-phase organisms were absorbed with yeast-phase organisms or yeast-phase extract and used to stain Western blots of sodium dodecyl sulfate-polyacrylamide gels loaded with yeast- and mycelial-phase extracts. Autoradiography of gels loaded with extracts from organisms surface labeled with 125I was used to detect surface antigens containing proteins. Antigen bands of interest identified in Western blots were cut from the blots and used to immunize rabbits. Two antigens were identified in the mycelial-phase extract which were not present in the yeast-phase extract. The first was a 19-kilodalton protein that was present in the cell walls of germ tubes but was not expressed on their surfaces. The second was a polysaccharide-rich high-molecular-weight antigen which was expressed on the surface of the germ tube. Treatment of mycelial-phase extract with protease and endo-beta-N-acetylglucosaminidase H demonstrated that this antigen was composed of polysaccharides linked through di-N-acetylchitobiose groups to proteins.

An Elicitor of the Hypersensitive Lignification Response in Wheat Leaves Isolated from the Rust Fungus Puccinia graminis f. sp. tritici I. Partial Purification and Characterization

Several biotic and abiotic clicitors of the hypersensitive reaction in wheat leaves are described. The elicitors induce enhanced activity of phenylalanine ammonia-lyase with subsequent lignifica­tion, visible as a yellow autofluorescence. The deposited material stains positively with phloroglucinol. DEAE-dextran, epoxystearic acid and chitosan are strong elicitors, while the glucans tested have no activity. A biotic elicitor (genuine Pgt-elicitor) was isolated from the germ tube walls of uredospores of Puccinia graminis f. sp. tritici. The high molecular weight, water soluble elicitor is heat stable and unaffected by mild acid and mild alkaline treatments. It seems to be a glycoprotein with the carbohydrate moiety bearing the active residues, as indicated by periodate sensitivity and pro­tease stability. The carbohydrate moiety consists mainly of glucose with some galactose and mannose. Upon ultrafiltration and gel chromatography elicitor activity was associated with fractions of molecular weight of more than 100 kD. When injected into the intercellular space of primary wheat leaves, the elicitor induces lignifica­tion, preceded by an increase in phenylalanine ammonia-lyase activity.

An Elicitor of the Hypersensitive Lignification Response in Wheat Leaves Isolated from the Rust Fungus Puccinia graminis f. sp. tritici II. Induction of Enzymes Correlated with the Biosynthesis of Lignin

The genuine biotic elicitor from germ tube walls of Puccinia graminis f. sp. tritici (Pgt-elicitor) induces lignification, preceded by an increase in phenylalanine ammonia-lyase (PAL) activity, when injected into the intercellular space of primary wheat leaves. This increase in PAL activity is accompanied by an increase in other enzyme activities of the general phenylpropanoid pathway and the specific pathway of lignin biosynthesis: 4-coumarate :CoA ligase (4CL), cinnamyl alcohol dehydrogenase (CAD) and peroxidase (PO). Total extractable activities of 4CL, CAD and PO do not differ significantly in resistant and susceptible near isogenic wheat lines, whereas the dose response curve of induced PAL activity shows significantly higher values in the resistant isoline. This difference is not observed after injection of other biotic and abiotic elicitors of the lignification response. Although total induced PO activities 24 h after elicitor treatment are equal in both isolines, the PO isoenzyme pattern of resistant plants differs markedly from that of susceptible plants. The patterns of PO isoenzymes in the compatible and incompatible interaction 48 h after inoculation with Puccinia graminis f. sp. tritici also show differences, which closely resemble those seen after injection of the Pgt-elicitor. The patterns observed after injection of an artificial biotic elicitor clearly differ from those of the natural interaction.