A PLASMA MEMBRANE-ASSOCIATED PROTEIN IS A MARKER FOR DIFFERENTIATION AND POLARIZATION OF COLLETOTRICHUM-LINDEMUTHIANUM APPRESSORIA

The appressorium formed by the faculative biotrophic fungus Colletotri chum lindenmuthianum on bean tissues is a specialised cell involved in penetration of the host cuticle and epidermal cell wall, leading to t he formation of intracellular hyphae. A monoclonal antibody designated UB27, raised against infection structures isolated from infected leav es of Phaseolus, 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 app ressoria formed on bean hypocotyls showed that UB27 bound to the appre ssorial plasma membrane and a layer of cytoplasm just beneath this mem brane. Labelling stopped abruptly at the point at which the appressori al wall contacted the plant cuticle, leaving a region that included th e penetration pore and appressorial cone unlabelled. Labelling in appr essoria formed on porycarbonate membranes was similar, except that the diameter of the unlabelled region was smaller. UB27 recognised a 48-5 0 kDa protein. The mobility of this protein was unaffected by peptide- N-glycosidase treatment, but trifluoromethane sulphonic acid treatment resulted in a reduction of M(r) of approx. 16000. This suggests that the protein is glycosylated, possibly with O-linked carbohydrate side chains. After solubilisation and phase-separation of appressorial prot eins in Triton X-114, the protein recognised by UB27 partitioned prima rily into the detergent phase, suggesting that it is an integral membr ane protein. A proportion of the protein remained unsolubilised, sugge sting that there are interactions between the protein and cytoskeletal and/or cell wall components. Overall, the results show that the plasm a membrane of appressoria of C. lindemuthianum is differentiated into, two distinct domains and the distribution of the protein identified b y UB27 provides evidence for polarisation of appressoria.