Oligodeoxynucleotides as probes forin situhybridization with transmission electron microscopy to specifically localize phytoplasma in plant cells

• Published in: Molecular and cellular probes Vol. 13; no. 1; pp. 41 - 47
• Main Authors: Lherminier, J., Bonfiglioli, R.G., Daire, X., Symons, R.H., Boudon-Padieu, E.
• Format: Journal Article
• Language: English
• Published: Elsevier Ltd 01.02.1999
• Subjects: in situhybridization, transmission electron microscopy, rDNA, oligoprobes, gold labelling; in situhybridization, transmission electron microscopy, rDNA, oligoprobes, gold labelling
• ISSN: 0890-8508; 1096-1194
• DOI: 10.1006/mcpr.1998.0213

Phytoplasmas are plant-pathogenic mollicutes restricted to phloem. They belong to several groups in a unique phylogenetic clade. Non-related phytoplasmas may infect the same plant species, often with similar symptoms. Hence methods are needed to specifically localize phytoplasmas and to study their multiplication and movement in their hosts. Conditions for post-embeddingin situhybridization (ISH) with transmission electron microscopy using oligodeoxynucleotides as probes for labelling of phytoplasmas in plant tissues have been searched. Sections of acrylic resin-embedded tissues of phytoplasma-infected periwinkle were submitted to ISH using digoxigenin or biotin-labelled oligoprobes (22 mers). These probes were the complementary sequences of primers used in group-specific polymerase chain reaction (PCR) amplification of 16S rDNA of stolbur and elm yellows phytoplasma, respectively. Together with preliminary digestion with pepsin, differentin situdenaturation conditions and formamide concentrations were tested. The grids were incubated in the hybridization mixture at 37°C overnight. Detection of hybridized material was performed with gold immunocytochemistry. Specificity of labelling was checked with appropriate controls. Stringency conditions could be found to ensure specific hybridization with such short probes. A specific labelling was obtained for stolbur phytoplasma on groups of mature as well as senescent phytoplasma cells. The results show that oligonucleotides may be used as probes for phytoplasma identification in post-embedding ISH with electron microscopy.