Optimization of a fluorescence in situ hybridization method for the

Optimization of a fluorescence in situ hybridization method
for the examination of plant-fungus interactions
Áron Horváth1, Diána Seres1, Dániel G. Knapp1, Pál Vági1, Gábor M. Kovács1
of Plant Anatomy, Eötvös Loránd University, Budapest
Fluorescence in situ hybridization (FISH) methods were optimized to identify and
analyze the symbiotic partners in in vitro inoculated and field root samples. Therefore two
common root colonizing fungi’s ribosomal RNA was fluorescently labeled with singlestranded DNA oligonucleotide probes. Thus, the signal was detected in the cytoplasm of the
target organism instead of its scattered nuclei. The method's applicability was tested to
indicate fungal structures inside root sections of up to 120 micrometer thickness.
Epifluorescence microscopy was used to detect the presence of specific fluorescent
signals in fungal structures. Confocal laser scanning microscope was utilized to reconstruct
the 3D structure of the relatively thick root sections. Since autofluorescence of the plant cell
walls hampered signal detection, we selected plant species that produced lower
autofluorescence background. Fungal cell walls produced their most intense emission at 488
nm excitation maximum. The different laser lines has different intensities, thus, in order to
make fluorescence signals comparable we used neutral density filters. To analyze the effect of
the different modifications we measured the total brightness of a given sample area.
As a result of my work I optimized an RNA FISH protocol, which allows
simultaneous, specific identification and examination of the fungal participants of plantfungus interactions.
Funding was provided by the Hungarian Scientific Research Fund (OTKA, K72776).
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