J. Lherminier et al., Oligodeoxynucleotides as probes for in situ hybridization with transmission electron microscopy to specifically localize phytoplasma in plant cells, MOL CELL PR, 13(1), 1999, pp. 41-47
Phytoplasmas are plant-pathogenic mollicutes restricted to phloem. They bel
ong to several groups in a unique phylogenetic clade. Non-related phytoplas
mas may infect the same plant species, often with similar symptoms. Hence m
ethods are needed to specifically localize phytoplasmas and to study their
multiplication and movement in their hosts. Conditions for post-embedding i
n situ hybridization (ISH) with transmission electron microscopy using olig
odeoxynucleotides as probes for labelling of phytoplasmas in plant tissues
have been searched. Sections of acrylic resin-embedded tissues of phytoplas
ma-infected periwinkle were submitted to ISH using digoxigenin or biotin-la
belled oligoprobes (22 mers). These probes were the complementary sequences
of primers used in group-specific polymerase chain reaction (PCR) amplific
ation of 16S rDNA of stolbur and elm yellows phytoplasma, respectively. Tog
ether with preliminary digestion with pepsin, different in situ denaturatio
n conditions and formamide concentrations were tested. The grids were incub
ated in the hybridization mixture at 37 degrees C overnight. Detection of h
ybridized 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 s
pecific labelling was obtained for stolbur phytoplasma on groups of mature
as well as senescent phytoplasma cells. The results show that oligonucleoti
des may be used as probes for phytoplasma identification in post-embedding
ISH with electron microscopy. (C) 1999 Academic Press.