Imaging of viroids in nuclei from tomato leaf tissue by in situ hybridization and confocal laser scanning microscopy.
AUTOR(ES)
Harders, J
RESUMO
The intracellular localization of viroids has been investigated by viroid-specific in situ hybridization and analysis by digital microscopy of the distribution of the fluorescent hybridization signals. Isolated nuclei from green leaf tissue of tomato plants infected with potato spindle tuber viroid (PSTVd) were bound to microscope slides, fixed with formaldehyde and hybridized with biotinylated transcripts of cloned PSTVd cDNA. The bound probe was detected with lissamine--rhodamine conjugated streptavidin. Nucleoli were identified by immunofluorescence using the monoclonal antibody Bv96 and a secondary FITC-conjugated antibody. In plants infected with either a lethal or an intermediate PSTVd strain, the highest intensity of fluorescence that arose from hybridization with the probe specific for the viroid (+)strand was found in the nucleoli, confirming results of previous fractionation studies. A similar distribution was found for (-)strand replication intermediates of PSTVd using specific (+)strand transcripts as hybridization probes. In order to determine if viroids are located at the surface or in the interior of the nucleoli, the distribution of the fluorescence hybridization signals was studied with a confocal laser scanning microscope (CLSM). It was shown by three-dimensional reconstruction that viroids are neither restricted to the surface of the nucleoli nor to a peripheral zone, but are instead homogeneously distributed throughout the nucleolus. The functional implications of the intranucleolar location of viroids and their replication intermediates are discussed with respect to proposed mechanisms of viroid replication and pathogenesis.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=401569Documentos Relacionados
- Activity and three-dimensional distribution of toluene-degrading Pseudomonas putida in a multispecies biofilm assessed by quantitative in situ hybridization and scanning confocal laser microscopy.
- Subcellular localization of viroids in highly purified nuclei from tomato leaf tissue.
- Analysis of banded human chromosomes and in situ hybridization patterns by scanning force microscopy.
- Differentiation of Methanosaeta concilii and Methanosarcina barkeri in Anaerobic Mesophilic Granular Sludge by Fluorescent In Situ Hybridization and Confocal Scanning Laser Microscopy†
- Three dimensional analysis of the retinal vasculature using immunofluorescent staining and confocal laser scanning microscopy.