, Volume 188, Issue 1-2, pp 1-11

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

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The 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.