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A Novel Method for In Situ Hybridization in Fungal Cells based on Pricking Micro–injection of Photobiotin Labelled Probes
Author(s) -
Matsuda Y.,
Toyoda H.,
Morita M.,
Ikeda S.,
Tamai T.,
Nishiguchi T.,
Ouchi S.
Publication year - 1994
Publication title -
journal of phytopathology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.53
H-Index - 60
eISSN - 1439-0434
pISSN - 0931-1785
DOI - 10.1111/j.1439-0434.1994.tb01454.x
Subject(s) - biology , in situ hybridization , microbiology and biotechnology , rna , cytochemistry , suppression subtractive hybridization , cytoplasm , complementary dna , biochemistry , gene expression , cdna library , gene , enzyme
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 complementry RNA strand was synthesized in vitro for a probe. Since neither intact nor fixed conidial cells took up FITC–labelled albumin, the photo, biotin–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 phosphatase) which was first bound to the hybridized photobiotinlabelled 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.