Fig 1.
Subcellular localization of RNase3 in epidermal cells of Nicotiana benthamiana following expression by agroinfiltration.
(a) RNase3-dsRED (red signals) was detected in cytoplasmic punctate bodies by confocal microscopy at 2 dpi. A few of the many bodies are pointed out with arrowheads. It was also present in the nucleus and subnuclear bodies (arrow). (b) Fibrillarin of A. thaliana (Fib2) was expressed as a fusion with GFP (green) and used as a marker for nucleolus (N) and Cajal bodies (C). (c) Merged image revealed localization of RNase3 in subnuclear bodies (arrow) other than nucleolus or Cajal bodies. Scale bars, 10 μm.
Fig 2.
Co-localization of RNase3 and RDR6 in epidermal cells of N. benthamiana following co-expression by agroinfiltration.
The red signals of RNase3-dsRED and green signals of (a) AtRDR6-GFP (A. thaliana) and (b) NtRDR6-GFP (N. tabacum) co-localized in cytoplasmic, punctate bodies detected by confocal microscopy at 2 dpi. Images in (a) and (b) illustrate optical planes in which many RDR6-containing bodies were observed. Scale bars, 10 μm.
Fig 3.
Interactions of SGS3 with RDR6 and RNase3 in planta.
Protein interactions were tested by BiFC and monitored in epidermal cells of N. benthamiana using epifluorescence microscopy. The name of the protein indicates whether the YFP half was fused to the N- or C-terminus of the test protein (e.g., YN-RNase3 and IbSGS3-YC, respectively). Co-expression of the test proteins with eIF(iso)4E of potato [27] was included as a negative control for interaction (panels b, d, f, h, j and k). (a) Interaction of AtRDR6 and AtSGS3 resulted in SGS3/RDR6 bodies (positive control); (b) negative control. (c) RNase3 did not interact with AtRDR6, but (e) interacted with AtSGS3 (A. thaliana) and (i) IbSGS3 (I. batatas). (g) Catalytically inactive RNase3-Ala was also able to interact with SGS3. Data were collected at 2 dpi. All images were acquired at the same magnification. Scale bar, 20 μm.
Fig 4.
Interactions of RNase3 and AtSGS3, as assessed with BiFC and confocal microscopy, co-localize with cytoplasmic punctate bodies containing AtRDR6 in epidermal cells of N. benthamiana.
The three proteins were co-expressed by agroinfiltration. Images were acquired at an optical plane in which many RDR6-containing bodies were observed at 2 dpi. The dashed circles point out some of the cytoplasmic punctate bodies that show signals of the RNase3-SGS3 interaction and co-localize with RDR6. Scale bar, 10 μm.
Fig 5.
Co-expression of RNase3 and IbSGS3 enhances suppression of sense-mediated gene silencing (gene co-suppression).
(a) and (b) Four sectors (1 to 4) of a leaf of N. benthamiana 16c constitutively expressing gfp were agroinfiltrated to co-express GFP and (1) GUS (negative control), (2) IbSGS3 and RNase3, (3) RNase3, or (4) IbSGS3. If RNase3 was not used, the corresponding Agrobacterium strain was replaced with a strain expressing β-glucuronidase (GUS, negative control). IbSGS3 was expressed with the N-proximal part of YFP fused to the C-terminus. If it was not used, it was replaced with an Agrobacterium strain expressing YN (N-proximal half of yfp) to maintain similar sense-mediated silencing pressure. The treatments are positioned differently in the two leaves in terms of the younger (basal) and older (tip) part of the leaf. Silencing of gfp was observed by the disappearance of GFP fluorescence (sectors 1 and 4), whereas GFP fluorescence above the background level indicated suppression of gfp silencing (sectors 2 and 3). The leaf was photographed under UV light at 6 dpi. Similar results were obtained in five independent experiments. (c) Northern analysis of gfp mRNA and gfp mRNA-derived siRNA in the agroinfiltrated leaf tissues. Co-expression of GUS or IbSGS3 with GFP by agroinfiltration in gfp-transgenic leaves resulted in gfp silencing, as shown by the readily detectable accumulation of gfp-derived siRNA (Fig 5C). In contrast, co-expression of GFP and RNase3 resulted only in low accumulation of gfp siRNA, and no gfp siRNA could be detected following co-expression of GFP, RNase3 and SGS3; however, accumulation of gfp mRNA was enhanced (Fig 5C). Co-expression of the RNase3-Ala mutant (disabled from catalytic activity on dsRNA) with GFP resulted in readily detectable accumulation of gfp siRNA, whereas co-expression of RNase3-Ala, SGS3 and GFP resulted in low accumulation of gfp siRNA (Fig 5C). 25S and 5S ribosomal RNA is shown as a loading control, respectively. (d) Western analysis of RNase3 and IbSGS3-YN in the agroinfiltrated leaf sectors illustrated in (a) by immunoblotting using anti-RNase3 and anti-GFP antibodies, respectively. Molecular masses of the detected proteins (kDa) were estimated by comparison with the protein marker run in the gel.