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. 2000 Aug;66(8):3464-7.
doi: 10.1128/AEM.66.8.3464-3467.2000.

Immunomagnetic purification of Colletotrichum lindemuthianum appressoria

K A Hutchison  1 , S E Perfect, R J O'Connell, J R Green
Affiliations

Immunomagnetic purification of Colletotrichum lindemuthianum appressoria

K A Hutchison et al. Appl Environ Microbiol. 2000 Aug.

Abstract

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.

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Figures

FIG. 1
FIG. 1
Appressoria (asterisks) of C. lindemuthianum purified by IMS and viewed with differential interference contrast microscopy. The Dynabeads (arrowheads) are attached to the surfaces of single appressoria (A) and groups of appressoria that have become aggregated during the isolation procedure (B). Bars, 10 μm.
FIG. 2
FIG. 2
Transmission electron micrographs showing appressoria of C. lindemuthianum purified by IMS and prepared by propane jet freezing and freeze-substitution. (A) The appressorium contains no cytoplasm but retains an intact appressorial cone (ac) around the basal penetration pore (asterisk) and remnants of the extracellular matrix (arrowheads). Bar, 1 μm. (B and C) Portions of empty appressoria. Despite the loss of cytoplasm, the plasma membrane (arrowheads) remains closely associated with the cell wall but is undulating in profile. The appressorial wall is composed of a moderately electron-opaque inner layer (iw) and a highly electron-opaque outer layer (ow), surrounded by extracellular matrix (ecm). Bar, 0.2 μm.
FIG. 3
FIG. 3
Western blots of cell wall glycoproteins from purified appressoria (A) and mycelium (B and C) of C. lindemuthianum probed with MAb UB22 (A and B) or UBIM22 (C).
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