CIHI, A BIOTROPHY-RELATED GENE EXPRESSED SPECIFICALLY AT THE INTRACELLULAR INTERFACE FORMED BETWEEN COLLETOTRICHUM LINDEMUTHIANUM AND FRENCH BEAN
S PERFECT1, J GREEN1 and R O'CONNELL2
1 University of Birmingham, UK; 2IACR-Long Ashton, Bristol, UK
Background and objectives
Colletotrichum is a large genus of plant pathogenic fungi causing anthracnose on a wide range of crops. C. lindemuthianum is a hemibiotrophic species which causes anthracnose of bean, Phaseolus vulgaris. During the inital biotrophic stage of infection, the fungus differentiates infection vesicles and primary hyphae within host epidermal cells. These specialised intracellular hyphae invaginate the host plasma membrane, from which they are separated by a matrix layer. Monoclonal antibodies (MAbs) raised to isolated infection structures have been used to identify proteins present at the fungal-plant interface. This project aims to further characterise these novel molecules.
Results and conclusions
One of these MAbs, designated LTB25, recognises a protein epitope in a 4O-kDa N-linked glycoprotein specific to intracellular hyphae. Indirect immunofluoreseence and EM-immunogold labelling show that the glycoprotein is present in the infection peg, and the fungal walls and matrix surrounding the intracellular hyphae. However, it is not present in secondary necrotrophic hyphae, which suggests that it is specific to biotrophic infection structures. The glycoprotein may therefore be involved in the establishment and maintenance of biotrophy.
A cDNA library has been constructed from total RNA isolated from infected bean hypocotyl epidermis. The MAb UB25 has been used to immunoscreen the library and positive clones have been isolated and sequenced. Southern analysis indicates that the CIHI glycoprotein recognised by LTB25 is fungally encoded. Analysis of the deduced amino acid sequence of CIHI revealed the presence of two distinct domains, one of which is proline-rich and contains short repetitive motifs with tyrosinelysine pairs. Tyrosine residues have been implicated in the oxidative cross-linking of proteins such as extensins. Cross-linking studies of CIHI indicate that this glycoprotein has the potential to be oxidatively cross-linked by peroxidase in the presence of hydrogen peroxide. Gene disruption studies are under way to assess the functional importance of CIHI.