Class 1 RNaseIII endoribonucleases of PPIV (PPR3) and SPCSV (CSR3) suppress RNAi in leaves of <i>Nicotiana benthamiana</i>.

<p>(<b>A</b>) The RNaseIII signature motif (blue) and dsRNA-binding domain (red) conserved in Class 1 RNaseIII endoribonucleases, and substitution of two conserved amino acid residues of the catalytic site are depicted (mutated proteins PPR3-Ala and CSR3-Ala). (<b>B</b>) Suppression of sense ssRNA—induced RNAi. The left and right side of leaves of <i>gfp</i>-transgenic <i>N</i>. <i>benthamiana</i> line 16c were infiltrated with <i>Agrobacterium tumefaciens</i> (Agro) strains expressing <i>gfp</i> to induce silencing of the constitutively expressed <i>gfp</i> transgene (note green fluorescence in leaf veins) and co-infiltrated with Agro strains for expression of PPR3, PPR3-Ala, CSR3, CSR3-Ala, or GUS (β-glucuronidase, negative control) or mock-infiltrated with buffer (negative control). Leaves were photographed and analyzed 3 days post-infiltration. Accumulation of <i>gfp</i> mRNA and siRNA was analyzed by northern blotting. Ethidium bromide—stained gels of rRNA were used as loading controls. (<b>C</b>) Immunoblot of the RNaseIII proteins in the infiltrated tissues shown in (B). Control indicates purified recombinant CSR3 (positive control). (<b>D</b>) Suppression of dsRNA (hairpin RNA)-induced RNAi. Co-infiltration was carried out as in (B), except that an Agro strain expressing double-stranded (hairpin) <i>gfp</i> was used. Agro strains expressing GUS or the SPCSV p22 silencing suppressor were used as a negative and a positive control, respectively. Accumulation of <i>gfp</i> mRNA and siRNA was determined by northern blotting. (<b>E</b>) Reversion of RNAi. Suppression of the <i>gfp</i> transgene was achieved by sense-mediated silencing as in (B). After 24 h, the same leaf spots were infiltrated with an Agro strain for expression of PPR3, PPR3-Ala, CSR3, CSR3-Ala, GUS (negative control), or the HCpro silencing suppressor of <i>Potato virus A</i> (positive control) and photographed 3 days later.