A novel method for in situ hybridization in fungal cells based on pricking micro-injection of photobiotin labelled probes
1994
Matsuda, Y. (Kinki Univ. (Japan). Faculty of Agriculture. Lab. of Plant Pathology) | Toyoda, H. | Morita, M. | Ikeda, S. | Tamai, T. | Nishiguchi, T. | Ouchi, S.
A novel method, a combination of a micro-injection and in situ hybridization cytochemistry, was developed for the examination of gene expression in Erysiphe graminis f. sp. hordei. In view of its high cellular content, hence high probability of hybridization with corresponding probes, cytoplasmic rRNA was chosen as the target and hybridized with a micro-injected photobiotin-labelled nucleic acid probe. The rDNA sequence was isolated from a genomic library of the fungus by the use of cDNA derived from 28S RNA of Fusarium oxysporum f. sp. lycopersici, and the complementary RNA strand was synthesized in vitro for a probe. Since neither intact nor fixed conidial cells took up FITC-labelled albumin, the photobiotin-conjugated RNA probe was introduced into cytoplasm of conidiospores by a pricking method. Positive hybridization was visualized by the colour-generating reaction catalyzed by biotinylated enzyme (alkaline phosphastase) which was first bound to the hybridized photobiotin-labelled probe. Specific hybridization was detected in cytoplasm of more than 80% of pricked conidiospores. A similar result was obtained when a probe was introduced into appressoria and haustoria formed on/in barley coleoptile epidermal cells. Hybridization was also observed in these structures when a double-stranded rDNA probe was introduced by pricking
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