Jonathan R. Green

Cloning and characterisation of glutamine synthetase from Colletotrichum gloeosporioides and demonstration of elevated expression during pathogenesis on Stylosanthes guianensis

Abstract Experiments were designed to clone and identify genes of the fungal phytopathogen Colletotrichum gloeosporioides expressed at high levels during growth on the compatible host Stylosanthes guianensis when compared with expression in axenic culture. A cDNA clone (pCgGS) that hybridised preferentially to a cDNA probe prepared from infected leaves was isolated by the differential screening of a cDNA library from a nitrogen-starved axenic culture of C. gloeosporioides. The DNA sequence of pCgGS is highly homologous to genes for glutamine synthetase (GS) in other organisms. pCgGS contained all of the conserved regions assigned as catalytic domains in GS enzymes. Comparison with genomic sequences indicated that in C. gloeosporioides the GS gene is present as a single copy with three introns. To our knowledge this is the first report of the cloning of a GS from a filamentous fungus. A second clone (pCgRL1) was also isolated and represented a partial cDNA of the 25s rRNA of C. gloeosporioides. Because pCgRL1 did not hybridise to plant rRNA under high-stringency hybridisation conditions, it was used as a reference to quantify the expression of fungal GS mRNA during pathogenesis in S. guianensis compared to fungal growth in axenic culture. The results indicated that elevated expression of GS occurred during pathogenesis of C. gloeosporioides on S. guianensis, particularly at early stages of infection where expression was about six-times higher than during growth in rich culture media. This work also demonstrates that fungal-specific 25s rRNA fragments, such as pCgRL1, have considerable utility as a reference for quantifying pathogen gene expression in infected plants.

Monoclonal antibodies to cell surface components of zoospores and cysts of the fungusPythium aphanidermatum reveal species-specific antigens

Abstract A panel of monoclonal antibodies (MAbs) designated PA1 to PA8 has been raised against cell surface components of zoospores and cysts of the pathogenic fungus Pythium aphanidermatum . The antibodies were selected on the basis of binding assays using indirect immunofluorescence. Four binding patterns were observed: PA1 labeled the entire zoospore surface including both flagella, PA2 binding was restricted to the anterior flagellum, PA3–PA6 bound to the adhesive cell coat secreted by zoospores during encystment, and PA7 and PA8 labeled zoospores and the cyst cell wall. Electron microscopic immunogold labeling of zoospores showed that PA2 bound to the mastigonemes on the anterior flagellum. The MAbs were tested for binding to zoospores and cysts of several isolates of P. aphanidermatum , and to zoospores and cysts of several species of Pythium, Phystophthora, Aphanomyces , and Saprolegnia . The results showed that the antigens recognized by MAbs PA1–PA6 were restricted to P. aphanidermatum , whereas those recognized by PA7 and PA8 occurred on all species tested.

Molecular differentiation in pea powdery-mildew haustoria : Identification of a 62-kDa N-linked glycoprotein unique to the haustorial plasma membrane.

Monoclonal antibodies have been raised against haustorial complexes isolated from pea (Pisum sativum L.) leaves infected by the biotrophic powdery mildew fungus Erysiphe pisi D.C. Immuno-localisation studies, using isolated haustorial complexes and infected pea leaf material, have shown that one of the antibodies, designated UB7, binds to fungal wall and plasma membranes present in both haustoria and mycelia. However, a second antibody, UB8, binds specifically to the haustorial plasma membrane, and does not label fungal plasma membranes in mycelia. Western blotting and antigen-modification techniques have shown that UB8 recognises a protein epitope of a 62-kDa antigen. A reduction in molecular weight of this component after endo-F treatment indicates that the antigen is an N-linked glycoprotein. UB7 also recognises a 62-kDa glycoprotein, which is susceptible to endo-F treatment, and the antibody binds to a carbohydrate epitope. Differences in molecular weights of the products after endo-F treatment of antigens show that the 62-kDa glycoproteins recognised by the antibodies are distinct molecules, in accordance with the localisation results. Overall, the results provide evidence for molecular differentiation associated with the development of haustoria in a biotrophic infection.

Monoclonal antibodies which show restricted binding to four Colletotrichum species: C. lindemuthianum, C. malvarum, C. orbiculare and C. trifolii☆

Monoclonal antibodies (MAbs) were raised to Colletotrichum lindemuthianum race γ germlings. When screened against eleven other Colletotrichum species using an ELISA, two of these MAbs, UB20 and UB22, showed restricted cross-reaction with three other species, viz. C. malvarum, C. orbiculare and C. trifolii. It was also observed that UB20 and UB22 bound only to isolates of C. lindemuthianum from bean and not cowpea. These findings suggest that the four Colletotrichum species recognized by the two MAbs are very closely related, confirming previous evidence. The MAbs did not bind to species of Fusarium or Phoma. UB20 and UB22 bound to carbohydrate epitopes on two distinct sets of glycoproteins. The glycoproteins recognized by the two MAbs were present on the surface of different infection structures in varying amounts: UB20 antigens were predominantly located on conidia, whereas UB22 antigens appeared to be most abundant in fibrillar material around germ-tubes. Both MAbs labelled the wall and matrix around intracellular infection hyphae. Potential applications of the MAbs for the identification of the above pathogens are discussed.

Composition and organisation of extracellular matrices around germ tubes and appressoria ofColletotrichum lindemuthianum

SummaryThe ultrastructure and composition of the extracellular matrices (ECMs) associated with germ tubes and appressoria ofColletotrichum lindemuthianum have been examined. Flexuous fibres (fimbriae), up to 6 μm long and 4–30 nm in diameter, protruded from the surface of germ tubes and appressoria. Anionic colloidal gold and lectin cytochemistry showed that ECMs of germ tubes and appressoria contain basic proteins, α-D-mannose and α-D-galactose residues. A monoclonal antibody, UB26, was raised to infection structures isolated from leaves ofPhaseolus vulgaris infected withC. lindemuthianum. UB26 recognised a protein epitope on two glycoproteins (Mr 133,000 and 146,000). Reductions in the Mr of these proteins after treatment with peptide-N-glycosidase and trifluoromethane sulphonic acid suggest that they carry N- and O-linked side-chains. Immunofluorescence and EM-immunogold labelling showed that glycoproteins recognised by UB26 were restricted to the ECMs around germ tubes and appressoria but fimbriae were not labelled. Unlike appressorial germ tubes formed in vitro, intracellular infection hyphae were not labelled, suggesting that the glycoproteins recognised by UB26 are not present on fungal structures formed within host cells. In liquid culture, these glycoproteins were not released into the medium, suggesting they are physically linked to the cell wall. Also, the glycoproteins were not removed from glass surfaces by ultrasonication. These results suggest that glycoproteins recognised by UB26 may be involved in the adhesion of germ tubes and appressoria to substrata. Our results show that the ECMs of germ tubes and appressoria differ markedly in structure and composition from those of conidia and intracellular hyphae, and that extracellular glycoproteins are associated with specific regions of the fungal cell surface.

Ultrastructure and composition of the cell surfaces of infection structures formed by the fungal plant pathogen Colletotrichum lindemuthianum

A variety of microscopical techniques and molecular probes have been used to study the ultrastructure and composition of the cell surfaces of the conidia (i.e. spores) and infection structures produced by the hemibiotrophic fungal plant pathogen Colletotrichum lindemuthianum. The fungal conidium germinates to produce a germ‐tube, the tip of which swells to produce a domed, melanized appressorium which adheres firmly to the plant surface. Penetration of the cuticle and cell wall is followed by the development of a biotrophic intracellular hypha, which is surrounded by an invagination of the host plasma membrane. Freeze‐substitution of C. lindemuthianum germlings showed that conidia are coated with a dense layer of fibrillar material. This ‘spore coat’ contains irregularly shaped pores, giving it a reticular appearance. Negative staining of germlings revealed the presence of numerous long, flexuous fibres or fimbriae, protruding from the surfaces of germ‐tubes and appressoria. Colloidal gold was used to visualize fungal extracellular proteins. The colloidal gold stained a fibrillar sheath around germ‐tubes, whereas appressoria were surrounded by a halo, comprising an inner unstained region and a stained perimeter. The carbohydrate composition of the cell surfaces of the conidia and infection structures was studied by labelling cells with rhodamine‐ and fluorescein‐conjugated lectins. The results showed that the extracellular matrices of germ‐tubes and appressoria are very similar in composition, but differ from those of conidia and intracellular hyphae. Monoclonal antibodies have been prepared to germlings and infection structures of C. lindemuthianum and their use has provided further evidence that the extracellular matrices around germ‐tubes and appressoria have several glycoproteins in common. The results also show that the cell surface of C. lindemuthianum becomes specialized during biotrophic development inside host cells.

Monoclonal antibodies to sperm surface antigens of the brown alga Fucus serratus exhibit region-, gamete-, species- and genus-preferential binding

A panel of twelve monoclonal antibodies (MAbs), designated FS1 to FS12, have been raised against surface antigens of Fucus serratus sperm. The antibodies were selected on the basis that they show region-, gamete-, species- or genus-preferential binding. Indirect immunofluorescence shows that the antigens bound by the MAbs are distributed non-randomly over the cell surface. Seven MAbs (FS1, FS3, FS4, FS6, FS8, FS9, FS10) bind antigens located primarily on the cell body, while the others (FS2, FS5, FS7, FS11, FS12) bind antigens located primarily on the anterior flagellum. Of the MAbs that label the anterior flagellum, FS2, FS5, FS7 and FS12 form a ‘halo’ at the perimeter of the flagellum. Electron microscopic-immunogold studies indicate that the ‘halo’ results from labelling of the mastigonemes, as opposed to the flagellar plasmamembrane. Gamete-preferential binding of antibodies was detected using an enzyme-linked immunosorbent assay with egg membrane vesicles. Eight of the MAbs bind sperm antigens not common to eggs, though FS2, FS4, FS5 and FS9 bind antigens present on both sperm and eggs. In studies of species- and genus-specificity FS2, FS3, FS5, FS6, FS7, FS8, FS10, FS11 and FS12 exhibit genus-preferential binding, labelling sperm of F. serratus and F. vesiculosus more intensely than that of Ascophyllum nodosum. Only FS10 showed marked species-preferential binding, labelling sperm of F. serratus much more intensely than that of F. vesiculosus.

Surface characteristics of necrotrophic secondary hyphae produced by the bean anthracnose fungus, Colletotrichum lindemuthianum

During infection of bean (Phaseolus vulgaris), the hemibiotrophic anthracnose pathogen, Colletotrichum lindemuthianum, initially produces biotrophic primary hyphae that are large-diameter and entirely intracellular, followed by necrotrophic secondary hyphae that are narrower and either intercellular or intracellular. In the present study, transmission electron microscopy of infected tissues prepared by high-pressure freezing and freeze-substitution showed that secondary hyphae have much thinner cell walls (25–40 nm) than primary hyphae (100–130 nm) and are not surrounded by an extracellular matrix. Immunofluorescence labelling with a panel of monoclonal antibodies showed that glycoproteins which are present on conidia, germ-tubes, appressoria, primary hyphae and mycelium grown in vitro are absent from the surface of secondary hyphae. Chitin, detected with the lectin wheat germ agglutinin, was the only surface component shared by secondary hyphae and the other fungal cell types. The results suggest that the fungal cell surface becomes modified during necrotrophic growth, with none of the glycoproteins associated with earlier stages of the infection process being produced.

The molecular nature of Fucus serratus sperm surface antigens recognised by monoclonal antibodies FS1 to FS12.

Sperm of the brown alga Fucus serratus are highly differentiated, biflagellate, naked cells. Immunolocalisation studies, employing monoclonal antibodies (MAbs — designated FS1 to FS12) raised against antigens of these sperm cells, have revealed that some sperm surface components are distributed over the entire cell, whereas others are restricted to, or occur preferentially on, the surface of the anterior flagellum or cell body. This report describes the use of these MAbs in Western-blot procedures and antigen-modification binding assays to determine the nature of these sperm surface components. Monoclonal antibodies which bind to antigens found on the cell body and both flagella (FS3, FS4, FS6, FS8, FS10) recognise carbohydrate epitopes of a high-molecular-weight glycoprotein (Mr=205 kDa). These MAbs were initially chosen at random from a much larger number of antibodies which bound to sperm in a similar fashion, indicating that this glycoprotein is an immunodominant antigen. Though these MAbs compete under conditions of limited antigen availability, differences in the effects of periodate on antibody binding and differences in other binding data indicate that the MAbs recognise epitopes of this glycoprotein which are neighbouring or overlapping, rather than common. The MAb FS9, which has a similar binding pattern to the above antibodies, also seems to bind to carbohydrate epitopes, but the antigen recognised by this antibody could not be identified in Western-blotting procedures. The MAbs FS7 and FS12, which bind to the mastigonemes on the anterior flagellum and to the cell body and posterior flagellum, recognise a set of glycoproteins in the molecular-weight range 40–250 kDa. The evidence indicates that the antibodies are binding to N-linked carbohydrate side chains of these glycoproteins. Three MAbs that bind to the anterior flagellum (FS2, FS5 and FS11) recognise protein antigens in the molecular-weight range 90–250 kDa; it is not known whether these antigens are glycosylated. The MAb FS1, which binds primarily to the sperm cell body, could not be used in enzyme-linked immunosorbent assays or Western-blotting procedures and the antigen recognised by this antibody is so far uncharacterised.

Identification and localisation of glycoproteins in the extracellular matrices around germ-tubes and appressoria of Colletotrichum species

A monoclonal antibody (MAb), UB31, is described that binds to the extracellular matrix (ECM) surrounding germtubes and appressoria, but not conidia, of the bean anthrachose fungus, Colletotrichum lindemuthianum. Comparative localisation studies with MAb UB26, which has the same cell type specificity, suggest that the ECM is heterogeneous in composition. Immunofluorescence showed that UB31 labelled appressoria more intensely than germ-tubes, whereas UB26 labelled these structures to a similar extent. Immunofluorescence and TEM-immunogold labelling showed that UB31 antigens were located close to the appressorial wall, while UB26 antigens extended further away from the wall. MAb UB31 bound to the ECMs of all six Colletotrichum species tested. Western blotting and ELISA indicated that the antibody recognises a high M r glycoprotein (> 200000) that may be linked to melanin. The glycoprotein recognised by UB31 was not removed from substrata by ultrasonication, suggesting it may contribute to germling adhesion.