1.3.40
ULTRASTRUCTURE AND COMPOSITION OF THE CELL SURFACE OF COLLETOTRICHUM LINDEMUTHIANUM CONIDIA

B HUGHES1, R CARZANIGA2, RJ O'CONNELL2 and JR GREEN1

1School of Biological Sciences, University of Birmingham, Birmingham B15 2TT, UK; 2IACR-Long Ashton Research Station, Department of Agricultural Sciences, University of Bristol, Long Ashton, Bristol, BSI 8 9AF, UK

Background and objectives
The conidia of Colletotrichum lindemuthianum, the causal agent of bean anthracnose, are coated with a fibrillar layer arranged perpendicular to the cell wall [1]. This spore coat is stained by silver proteinate and removed after protease digestion, indicating the presence of glycoproteins. The monoclonal antibody UB20 was raised against germlings growing in vitro and recognizes a carbohydrate epitope carried by glycoproteins on the spore surface [2]. The purpose of our investigation is to characterize these glycoproteins with a view to determining their location and function at the spore surface.

Results and conclusions
Western blotting demonstrates that the glycoproteins recognized by LTB20 are extracted from conidia by hot water and SDS. Electron microscopy shows that the spore coat is also removed by these treatments. The major glycoprotein extracted from the cell is identified at 110 kDa, and biotinylation of washed spores shows that this glycoprotein is located at the cell surface. The same set of glycoproteins are extracted by O-mercaptoethanol at alkaline pH. Further characterization of the 110-kDa glycoprotein demonstrates that it contains both O-linked and N-linked carbohydrate side chains. Monoclonal antibodies have been raised against the 110-kDa glycoprotein, and these are being used to screen a cDNA library for the gene encoding the glycoprotein and to determine the precise location of the glycoprotein within the cell wall.

References
1. O'Connell RJ, Pain NA, Hutchison KA et al., 1996. Journal of Microscopy 181, 204-212.
2. Pain NA, O'Connell RJ, Bailey JA, Green JR, 1992. Physiological and Molecular Plant Pathology 41, 111-126.