Fig. 5

Transmission electron micrographs showing subcellular localization of Citrus exocortis viroid (CEVd) and Hop stunt viroid (HSVd) in two citrus cultivars. The locations of CEVd and HSVd in three tissues of blood oranges and Murcott mandarins were detected by in situ hybridization with digoxigenin (DIG)-labeled and biotinylated anti-sense riboprobes, respectively. The locations of CEVd DIG-labeled probes were detected by anti-DIG monoclonal antibody as a primary antibody and a 10-nm diameter colloidal gold conjugate of Alexa Fluor 488 goat anti-rabbit IgG as a secondary antibody. HSVd biotin-labeled probes were detected with 20-nm colloidal streptavidin-gold from Streptomyces avidinii. Ultrathin sections of viroid-negative roots (a), rootstock bark (d) and twig bark (g) hybridized with the same probes revealed neither CEVd nor HSVd signals in the nucleus nor in any other subcellular structures. b Ultrathin sections of mature blood orange roots infected by the two viroids. Most of the probe signals were associated with the nucleus and present near the plasma membrane and cytoplasm. c Ultrathin sections of mature roots of co-infected Murcott mandarin. The viroid signals were associated with the vacuole and cell wall. e Ultrathin sections of rootstock bark of co-infected blood orange. The probes were associated with the nucleoplasm or cytoplasm. f Ultrathin sections of rootstock bark of co-infected Murcott mandarins. The viroid signals were associated with cell walls, cytoplasm and the nucleoplasm. h Ultrathin sections of twig bark of co-infected blood orange. The probes were associated with the cytoplasm and cell walls. i Ultrathin sections of twig bark of co-infected Murcott mandarins. The viroid signals were associated with the cytoplasm. NP = nucleoplasm; V = vacuole; PM = plasma membrane; CP = cytoplasm; CW = cell wall