Immunomagnetic Purification of Colletotrichum lindemuthianum Appressoria

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.For many fungal plant pathogens, the appressorium is developmentally the first and most important infection structure formed in preparation for invasion of the host. Appressoria increase the area of contact and attachment between the fungus and host surface, provide the mechanical force and enzymes required for penetration, and can promote survival in adverse conditions (10). Species of Colletotrichum, the anthracnose fungi, produce melanized appressoria, and several mRNAs and proteins specific to these structures have been identified. For example, the cap20 gene in Colletotrichum gloeosporioides is induced by the surface wax of its host, avocado, and is expressed during appressorium formation (8). In Colletotrichum lindemuthianum, have been analyzed the cell surface of appressoria and other infection structures using lectins and monoclonal antibodies (MAbs) (12, 13). A plasma membrane-associated glycoprotein specific to appressoria has also been identified using an MAb (16).Our objective in this study was to purify C. lindemuthianum appressoria for further biochemical analysis of the cell surface and to compare appressoria with other fungal structures. To date, there have been no reports of the enrichment of appressoria from infected plant tissue, although appressoria produced on artificial substrata by species of Colletotrichum and Uromyces have been isolated by mechanical scraping (8,9,19). However, appressoria harvested in this way are contaminated with other fungal cell types, e.g., conidia and germ tubes, and there is evidence that appressoria formed in vitro do not have the same composition as appressoria formed on host surfaces (7).A variety of methods have been used to isolate the inter-and intracellular infection structures formed by fungal pathogens inside infected plant tissues. These include enzymatic maceration (2) and mechanical disruption followed by either density gradient centrifugation (1,3,20) or lectin affinity chromatography (4). Immunomagnetic separation (IMS) has been widely used in animal cell biology and microbiology for the purification of cells, bacteria, and viruses from mixed cell populations (18). Magnetic Dynabeads (Dynal) coated ...

Niemann-Pick Type C Proteins Are Required for Sterol Transport and Appressorium-Mediated Plant Penetration of Colletotrichum orbiculare

“…These results indicate that sterol transport by NPC proteins is required for appressorium-mediated plant infection of C. orbiculare . Many but not all Colletotrichum species produce an appressorial cone around the pore ( 45 – 47 ). Although cones are absent from the appressoria of many direct-penetration fungi, including C. truncatum , C. graminicola , and M. oryzae , some direct penetrators form a similar structure in the appressorium ( 45 , 47 ).…”

“…Many but not all Colletotrichum species produce an appressorial cone around the pore ( 45 – 47 ). Although cones are absent from the appressoria of many direct-penetration fungi, including C. truncatum , C. graminicola , and M. oryzae , some direct penetrators form a similar structure in the appressorium ( 45 , 47 ). Thus, although the appressorial cone is not necessary for direct penetration, these structures might play an important role in plant penetration by C. orbiculare .…”

Reusable immunomagnetic beads in an enzyme immunoassay

“…However, the magnetic beads used here posses a relatively larger surface and may covalently bind bioactive molecules. Moreover, the magnetic beads can be collected rapidly in response to a magnetic field (Grant et al 1998;Hutchison et al 2000), making them excellent candidates for application in immunoassays.…”