Infection structures of biotrophic and hemibiotrophic fungal plant pathogens.

Summary Biotrophic plant pathogenic fungi are one of the major causes of crop losses. The infection processes they exhibit are typified by infected host plant cells remaining alive for several days. This requires the development of specialized infection structures such as haustoria which are produced by obligate biotrophs, and intracellular hyphae which are produced by many hemibiotrophs. These infection hyphae are surrounded by the host plant plasma membrane, and in the case of haustoria the extrahaustorial membrane differs biochemically and structurally from the normal membrane. An interfacial matrix separates haustoria and intracellular hyphae from the invaginated membrane and this seems to be characteristic of biotrophic interactions. There is clear evidence for molecular differentiation of the haustorial plasma membrane in powdery mildews and rusts in comparison with the other fungal membranes. Relatively few pathogenicity genes related to biotrophy, and the switch from biotrophy to necrotrophy in hemibiotrophs, have been identified.

Immunomagnetic purification of Colletotrichum lindemuthianum appressoria.

We developed a method to purify appressoria of the bean anthracnose fungus Colletotrichum lindemuthianum for biochemical analysis of the cell surface and to compare appressoria with other fungal structures. We used immunomagnetic separation after incubation of infected bean leaf homogenates with a monoclonal antibody that binds strongly to the appressoria. Preparations with a purity of >90% could be obtained. Examination of the purified appressoria by transmission electron microscopy showed that most had lost their cytoplasm. However, the plasma membrane was retained, suggesting that there is some form of attachment of this membrane to the cell wall. The purified appressoria can be used for studies of their cell surface, and we have shown that there are clear differences in the glycoprotein constituents of cell walls of appressoria compared with mycelium.

The distribution and expression of a biotrophy-related gene, CIH1, within the genus Colletotrichum.

Abstract During the biotrophic phase of the infection process of the hemibiotrophic anthracnose fungus Colletotrichum lindemuthianum, an intracellular hypha develops within epidermal cells of its host, Phaseolus vulgaris. This is followed by the formation of secondary hyphae during the necrotrophic phase. Previous work using a monoclonal antibody, UB25, has identified a glycoprotein that is specific to the interfacial matrix that forms between the wall of the intracellular hypha and the invaginated host plasma membrane. The gene encoding the protein identified by UB25 was cloned by immunoscreening and designated CIH1. The predicted amino acid sequence revealed a proline-rich glycoprotein, and biochemical evidence suggested that it formed a cross-linked structure at the biotrophic interface. Although CIH1 is a fungal gene, its product has several similarities to plant cell wall proteins. In this paper, we have surveyed the distribution and expression of CIH1 within the genus Colletotrichum, encompassing both necrotrophic and hemibiotrophic species. The results show that homologues of the CIH1 gene are present in all the Colletotrichum species tested. Northern blot studies of the time course of the infection process in planta have shown that CIH1 is expressed by both C. lindemuthianum in bean and C. trifolii in alfalfa during the biotrophic phase of fungal development. Immunofluorescence labelling of infected epidermal strips with UB25 revealed that the intracellular hyphae formed by C. destructivum as it infects alfalfa were specifically labelled in a similar way to those formed by C. lindemuthianum in bean. Northern and Western analysis showed that CIH1 was also expressed by C. lindemuthianum in vitro, though not constitutively. Overall, the evidence supports a role for CIH1 in biotrophy within the genus Colletotrichum.

Colletotrichum: A model genus for studies on pathology and fungal-plant interactions.

Species of Colletotrichum use diverse strategies for invading host tissue, ranging from intracellular hemibiotrophy to subcuticular intramural necrotrophy. In addition, these pathogens develop a series of specialized infection structures, including germ tubes, appressoria, intracellular hyphae, and secondary necrotrophic hyphae. Colletotrichum species provide excellent models for studying the molecular basis of infection structure differentiation and fungal-plant interactions. In this review we cover the various stages of the infection processes of Colletotrichum species, including spore adhesion and germination, germ tube and appressorium differentiation and functions, and biotrophic and necrotrophic development. The contribution of molecular, biochemical, and immunological approaches to the identification of genes and proteins relevant to each stage of fungal development will be considered. As well as reviewing results from several groups, we also describe our own work on the hemibiotrophic pathogen, C. lindemuthianum.

Expression cloning of a fungal proline-rich glycoprotein specific to the biotrophic interface formed in the Colletotrichum-bean interaction.

The monoclonal antibody, UB25, recognises a glycoprotein specifically located at the biotrophic interface formed in the Colletotrichum lindemuthianum-bean interaction. The antibody labels the walls of intracellular hyphae and the interfacial matrix which separates them from the invaginated host plasma membrane. In Western blots, UB25 recognises a ladder of bands which are multiples of M(r) 40.5 kDa. A full length cDNA encoding the glycoprotein recognised by UB25 has been isolated by expression cloning and designated CIH1 (Colletotrichum Intracellular Hypha 1). In vitro transcription/translation of CIH1, and transfection of mammalian COS cells, showed that UB25 recognized the expressed product in both procedures confirming that the clones isolated were true positives. Southern analysis of bean and C. lindemuthianum genomic DNA indicated that the CIH1 glycoprotein is fungally encoded and Northern analysis showed that it is only expressed in planta. Analysis of the deduced amino acid sequence of CIH1 indicates the presence of an N-terminal signal sequence and two possible sites for N-glycosylation. The N-terminal domain of the mature protein is rich in proline and contains several short repetitive motifs. CIH1 is thus a fungal proline-rich glycoprotein which appears to form a cross-linked structure in planta and, as such, resembles plant cell wall proline- and hydroxyproline-rich proteins. Possible functions for the CIH1 protein in the establishment and maintenance of biotrophy are discussed.