Naomi A. Pain

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.

Use of Monoclonal Antibodies to Study Differentiation of Colletotrichum Infection Structures

Successful penetration and colonization of plant tissues by most fungal pathogens requires differentiation of specialised cell types or infection structures, e.g. germ tubes, appressoria, penetration hyphae, infection hyphae and haustoria. Each cell type is adapted to a particular role in the infection process, e.g. adhesion, contact-sensing, penetration and nutrient uptake [22,32]. Molecular genetic techniques, such as differential or subtractive hybridization and mutational analysis, are being used to identify genes involved in the morphogenesis and function of these infection structures. For example, many genes that are specifically expressed or up-regulated during the formation of appressoria by Colletotrichum, Magnaporthe and rust fungi have now been cloned [7,8,23,24,27,29,55,69,76]. Some of these genes have been sequenced and disrupted to determine their role in the infection process [24,69]. These approaches have so far been restricted to infection structures that can be obtained in vitro, such as appressoria. Identification of genes expressed by infection structures formed following host penetration is more difficult due to contamination with host mRNAs, although recent advances in the isolation of such structures from infected tissue may alleviate this problem [19,30,50].

A plasma membrane-associated protein is a marker for differentiation and polarisation ofColletotrichum lindemuthianum appressoria

SummaryThe appressorium formed by the facultative biotrophic fungusColletotrichum lindemuthianum on bean tissues is a specialised cell involved in penetration of the host cuticle and epidermal cell wall, leading to the formation of intracellular hyphae. A monoclonal antibody designated UB27, raised against infection structures isolated from infected leaves ofPhaseolus vulgaris, bound specifically to appressoria, as shown by immunofluorescence, EM-immunogold and Western blotting. It did not bind to other fungal or plant structures. Immunogold labelling of appressoria formed on bean hypocotyls showed that UB27 bound to the appressorial plasma membrane and a layer of cytoplasm just beneath this membrane. Labelling stopped abruptly at the point at which the appressorial wall contacted the plant cuticle, leaving a region that included the penetration pore and appressorial cone unlabelled. Labelling in appressoria formed on polycarbonate membranes was similar, except that the diameter of the unlabelled region was smaller. UB27 recognised a 48–50 kDa protein. The mobility of this protein was unaffected by peptide-N-glycosidase treatment, but trifluoromethane sulphonic acid treatment resulted in a reduction of Mr of approx. 16000. This suggests that the protein is glycosylated, possibly withO-linked carbohydrate side chains. After solubilisation and phase-separation of appressorial proteins in Triton X-114, the protein recognised by UB27 partitioned primarily into the detergent phase, suggesting that it is an integral membrane protein. A proportion of the protein remained unsolubilised, suggesting that there are interactions between the protein and cytoskeletal and/or cell wall components. Overall, the results show that the plasma membrane of appressoria ofC. lindemuthianum is differentiated into two distinct domains and the distribution of the protein identified by UB27 provides evidence for polarisation of appressoria.

A monoclonal antibody that recognizes a carbohydrate epitope on N -linked glycoproteins restricted to a subset of chitin-rich fungi

Previous studies with a monoclonal antibody (designated UB7) raised against isolated haustorial complexes formed by the pea powdery mildew fungus Erysiphe pisi showed that it recognized an abundant glycoprotein found in cell walls and plasma membranes of haustoria and the surface mycelium. In this paper, Western blotting and phase-partitioning in the detergent Triton X-114 have been used to show that the plasma membrane antigens recognized by UB7 comprise a 62 kDa integral glycoprotein and a set of integral membrane glycoproteins of lower molecular weight. A 59 kDa glycoprotein recognized by UB7 has been extracted from the fungus using aqueous buffer, and the evidence suggests that this represents the cell wall form of the antigen. Binding of UB7 is abolished by pre-treatment of glycoproteins with peptide-N-glycosidase, but is retained after endo-F treatment. This suggests that the epitope to which UB7 binds is the innermost N-acetylglucosamine residue of N-linked carbohydrate side chains of glycoproteins, possibly substituted with one or more sugars. When tested for cross-reactivity with other fungi, UB7 bound to some, but not all, of those examined, and recognized different sets of glycoproteins compared with those detected in E. pisi. However, UB7 did not bind to any higher plants nor to any animal glycoproteins tested. This antibody thus identifies a carbohydrate epitope restricted to glycoproteins in a subset of the fungi.

DIFFERENTIATION AND DEVELOPMENT OF THE SPECIALISED INFECTION STRUCTURES FORMED BY BIOTROPHIC FUNGAL· PLANT PATHOGENS

Biotrophic fungal plant pathogens, including the powdery mildew, downy mildew and rust fungi, allow the infected plant cells and tissues to remain alive and active for extensive periods in order to obtain nutrients. They all develop specialised infection structures called haustoria, within plant cells, that form an interface between the fungal parasite and host plant [1]. The key interface is the extrahaustorial membrane (ehm), which is an invagination of the host plasma membrane, and this is likely to be involved in the transport of nutrients to the fungus and recognition/signalling between the plant and the fungus [1]. Organisms such as Colletotrichum lindemuthianum also have a biotrophic phase of infection involving a specialised infection structure, the intracellular hypha (IH). However, after several days, biotrophy breaks down, leading to a necrotrophic phase of development in the plant, and such organisms are termed hemibiotrophs or facultative biotrophs [2].