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.