Eggplant latent viroid is located in the chloroplasts and nuclei of eggplant infected cells

Abstract

Viroids that belong to genera Avsunviroid and Pelamovirod (family Avsunviroidae) replicate and accumulate in the chloroplasts of infected cells. In this report, we confirmed by RNA in situ hybridization using digoxigenin-UTP-labelled riboprobes that the positive strands of eggplant latent viroid (ELVd), the only member of genus Elaviroid within the family Avsunviroidae, also accumulate in the chloroplasts of infected cells. However, comparison of ELVd in situ hybridization signals with those from bona fide chloroplastic and nuclear non-coding RNAs, such as chloroplast 5S rRNA and U1 small nuclear RNA, supports the notion that this viroid is also present in the nuclei of infected cells. These results suggest that the subcellular localization of viroids within the family Avsunviroidae may be more complex than previously assumed with dynamic presence in several compartments during the infectious cycle.

Keywords: Avsunviroidae; Elaviroid; Non-coding RNA; Subcellular localization; Viroid.

Fig. 1

RT-PCR analysis of ELVd infection in eggplants. Plants were mechanically inoculated with ELVd RNA. The presence of ELVd RNA in upper non-inoculated tissues was analyzed one month after inoculation by RT-PCR followed by PAGE analysis of the products. Lane 0, DNA ladder marker with size of some of the molecules (in bp) on the left; lanes 1 to 12, ELVd-inoculated eggplants; lanes 13 and 14, mock-inoculated eggplants; lane 15, RT-PCR negative control with no template added. Arrow on the right points the 333-bp ELVd cDNA amplified in samples corresponding to infected eggplants

Fig. 2

In situ hybridization of mock-inoculated (left images) and ELVd-infected (right images) eggplant tissues with an ELVd DIG-labelled probe of minus polarity. Representative DIC microscope images are shown. Arrows, in detail insets, indicate hybridization signals in chloroplasts of the palisade and lacunar parenchyma (upper right image), and phloem cells (lower right image). Ep, epidermis; Pp and Pl, palisade and lacunar parenchyma, respectively; Ph, phloem; Xy, xylem. Bars represent 50 μm, as indicated

Fig. 3

In situ hybridization of ELVd-infected eggplant tissues with DIG-labelled RNA probes complementary to ELVd (upper row), and eggplant chloroplast 5S rRNA (central row) and snRNA U1 (lower row). Representative DIC microscope images are shown on the left column. After reveling in situ hybridization, sections were subjected to DAPI staining (images on the central column). Merged images are shown on the right column. Arrows point to some representative nuclei that are stained with DAPI and also show hybridization signal from the ELVd and snRNA U1 probes, but not from that corresponding to chloroplast 5S rRNA. Merged images were obtained with Fiji IJ software. Bars represent 50–100 μm, as indicated