Immunomagnetic isolation of viable intracellular hyphae of Colletotrichum lindemuthianum (Sacc. & Magn.) Briosi & Cav. from infected bean leaves using a monoclonal antibody

Abstract

SUMMARY A method is described for isolating intracellular hyphae (IH, i.e. infection vesicles and primary hyphae) of Colletotrichum lindemuthianum (Sacc. & Magn.) Briosi & Cav. from infected leaves‐of bean (Phaseolus vulgaris L.). 1H were recovered from homogenates of infected leaves after filtration through a 45 μm nylon mesh and isopyenic centrifugation on Percoll. 1H were then affinity‐purified by immunomagnetic separation using Dynabeads coated with monoclonal antibody UB25, specific for IH surface glycoproteins. The method yielded 7 × 104 IH g‐1 f. wt leaf tissue, with 27% purity and 62% viability, as judged by staining with fluorescein diacetate. The Viability of isolated IH was confirmed by their ability to grow in nutrient medium and by the normal ultrastructure of their cytoplasm. The host plasma membrane and matrix layer which surround IH in planta were absent from isolated IH. Staining with lectins. Calcofluor and aniline blue showed that the walls of IH contain N‐acetylgalactosamine. α‐linked mannose residues and β‐linked polysaccharides, including‐chitin and β‐1,3‐glucans. Potential uses of the isolated IH are discussed.

Expression cloning of a fungal proline‐rich glycoprotein specific to the biotrophic interface formed in the Colletotrichum–bean interaction

“…La Victoire were inoculated with conidial suspensions as described by Pain et al (1994a). For immunofluorescence (IF) labelling with MAbs, plant tissue was inoculated by brushing conidial suspensions onto leaves excised from 10 day-old seedlings of P. vulgaris (Pain et al 1994a). Inoculated leaves were then incubated for 3 and 5 days at 17 Ϯ 1°C, to produce a range of fungal infection structures which were labelled in situ by IF (Mackie et al, 1993).…”

“…The top 5 cm of hypocotyls were excised from 6 day-old seedlings of P. vulgaris and were inoculated and incubated as described previously (Pain et al, 1994a). Half of the hypocotyls were incubated for 2 days, the remainder for 3 days.…”

Use of Monoclonal Antibodies to Study Differentiation of Colletotrichum Infection Structures

“…IPC preparations were first incubated with UB25, washed by centrifugation and then incubated with magnetic beads coated with goat anti-mouse IgG antibodies. Magnetic separation enriched the intracellular hyphae lO-fold, yielding a sample which contained 30-40% hyphae, of which 60% were viable [50]. More recently, it has been possible to obtain preparations containing up to 95% intracellular .…”

“…(C), appressorium (A), infection vesicle (IV) and primary hypha (PH). Reprinted from Pain et al [50] with permission of the Trustees of The New Phytologisl.…”

“…These approaches have so far been restricted to infection structures that can be obtained in vitro, such as appressoria. Identification of genes expressed by infection structures formed following host penetration is more difficult due to contamination with host mRNAs, although recent advances in the isolation of such structures from infected tissue may alleviate this problem [19,30,50]. .…”