An RNA-mediated interference screen for small GTPases reveals novel GTPases functionally linked to <i>exoc-7</i> and <i>exoc-8</i>.

<p>(A) The design of the screen. (B) The synthetic lethality rates of the candidates identified in the screen. Underlining/bold indicates genes where the enhanced phenotypes were also observed in the wild type N2 background.</p

<i>exoc-7</i>, <i>exoc-8</i> single and <i>exoc-7;exoc-8</i> double mutations affect the <i>rab-10</i> RNAi-induced endocytic phenotype.

<p>(A) <i>rab-10</i> RNAi causes enhanced accumulation of the trans-membrane protein hTAC in <i>exoc-8</i> and <i>exoc-7;exoc-8</i> double mutants (WT n = 37, <i>exoc-7</i> n = 29, <i>exoc-8</i> n = 30, <i>exoc-7;exoc-8</i> n = 31, WT treated <i>rab-10</i> RNAi n = 27, <i>exoc-7</i> treated <i>rab-10</i> RNAi n = 32, <i>exoc-8</i> treated <i>rab-10</i> RNAi n = 38, <i>exoc-7;exoc-8</i> treated <i>rab-10</i> RNAi n = 36). (B) Early endosome marker GFP::RAB-5 is accumulated in <i>exoc-8</i> and <i>exoc-7;exoc-8</i> double mutants subjected to <i>rab-10</i> RNAi (WT n = 27, <i>exoc-7</i> n = 29, <i>exoc-8</i> n = 30, <i>exoc-7;exoc-8</i> n = 29, WT treated <i>rab-10</i> RNAi n = 27, <i>exoc-7</i> treated <i>rab-10</i> RNAi n = 25, <i>exoc-8</i> treated <i>rab-10</i> RNAi n = 33, <i>exoc-7;exoc-8</i> treated <i>rab-10</i> RNAi n = 28). (C) The accumulation of GFP::RME-1 in <i>rab-10</i> RNAi-treated <i>exoc-7</i>, <i>exoc-8</i> and <i>exoc-7;exoc-8</i> double mutant animals (WT n = 27, <i>exoc-7</i> n = 22, <i>exoc-8</i> n = 30, <i>exoc-7;exoc-8</i> n = 27, WT treated <i>rab-10</i> RNAi n = 23, <i>exoc-7</i> treated <i>rab-10</i> RNAi n = 26, <i>exoc-8</i> treated <i>rab-10</i> RNAi n = 31, <i>exoc-7;exoc-8</i> treated <i>rab-10</i> RNAi n = 29). In each sub-figure the upper panels show representative images of the GFP tagged protein in intestinal cells. The lower panels show the quantification of the normalized average fluorescence intensity. Asterisks denote statistical significance as compared to controls, with a P value less than 0.05 (*), 0.01 (**) and 0.001 (***).</p

Characterization of the behavioral phenotypes in <i>exoc-7</i>, <i>exoc-8</i> single and <i>exoc-7;exoc-8</i> double mutant worms.

<p>(A) Quantification of the growth rate to adulthood in wild type (n = 15), <i>exoc-7</i> (n = 18), <i>exoc-8</i> (n = 23) and <i>exoc-7;exoc-8</i> (n = 16) worms. (B) Quantification of wild type (n = 35), <i>exoc-7</i> (n = 42), <i>exoc-8</i> (n = 33) and <i>exoc-7;exoc-8</i> (n = 39) animal locomotion defect. (C) A schematic presentation of the experimental setup used in the Cu²⁺ avoidance assay. NaN₃ was spotted on one side of the plate divided by a 100 mM Cu²⁺ (or Cd²⁺) barrier spread on the midline of the plate. For each experiment 200-400 washed adult worms were placed on the opposite side [B] to the NaN₃ and their ability to traverse the Cu²⁺ to the other side [A] was scored. The index was calculated as A/(A+B). (D) Quantification of the Cu²⁺-sensitivity for wild type, <i>exoc-7, exoc-8</i> and <i>exoc-7;exoc-8</i> worms. (E) Quantification of the sensitivity to gentle nose touch for wild type (n = 25), <i>exoc-7</i> (n = 21), <i>exoc-8</i> (n = 27) and <i>exoc-7;exoc-8</i> (n = 29) worms. (F) The effect of thermal shock (35°C) on viability of wild type, <i>exoc-7</i>, <i>exoc-8</i> and <i>exoc-7;exoc-8</i> worms. (G) and (H) Analysis the resistance to aldicarb and levamisole in wild type, <i>exoc-7</i>, <i>exoc-8</i> and <i>exoc-7;exoc-8</i> worms. Asterisks denote statistical significance as compared to controls, with a P value less than 0.05 (*), 0.01 (**) and 0.001 (***).</p

<i>exoc-8</i> and <i>exoc-7;exoc-8</i> mutations result in up-regulation of RAB-10 protein expression.

<p>(A) GFP::RAB-10 fluorescence is increased in <i>exoc-8</i> and <i>exoc-7;exoc-8</i> mutants. (B) Normalized average intensity of GFP fluorescence in transgenic strains Is[<i>rab-10</i>::GFP::RAB-10] of wild type (n = 36), <i>exoc-7</i> (n = 43), <i>exoc-8</i> (n = 47) and <i>exoc-7;exoc-8</i> (n = 40) mutant background. (C) A representative Western blot for GFP::RAB-10 in WT, <i>exoc-7</i>, <i>exoc-8</i> and <i>exoc-7;exoc-8</i> mutants. (D) Quantification of the GFP::RAB-10 Western blot result from four independent experiments. α-tubulin was used for normalization. Asterisks denote statistical significance as compared to controls, with a P value less than 0.05 (*), 0.01 (**) and 0.001 (***).</p

Expression patterns of exocyst subunit <i>exoc-7</i> in <i>C. elegans</i>.

<p>Confocal images of adult hermaphrodites expressing P<i>exoc-7</i>::GFP under the 2954 bp promoter (Ex[P<i>exoc-7</i>::GFP; <i>pRF4</i>]).</p

<i>ral-1</i> and <i>rab-8</i> display differential genetic interactions with <i>exoc-7</i>, <i>exoc-8</i> and <i>exoc-7;exoc-8</i> in the Cu²⁺-sensing assay.

<p>The analysis for Cu²⁺ avoidance in <i>ral-1</i>, <i>rab-8</i> RNAi, <i>exoc-7</i>, <i>exoc-8</i> and <i>exoc-7;exoc-8</i> mutants and their different combinations. The experimental setup was identical to that described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032077#pone-0032077-g002" target="_blank">Figure 2C</a>. Asterisks denote statistical significance as compared to controls, with a P value less than 0.05 (*), 0.01 (**) and 0.001 (***).</p

Immunoblot analysis of proteins in <i>Drosophila melanogaster</i> S2 cells transfected to co-express LacZ and viral RNase III proteins.

<p>S2 cells were transfected to co-express sense and antisense transcripts of LacZ to silence LacZ expression, and they were additionally co-transfected to express (<b>A</b>) CSR3 or CSR3-Ala (Crinivirus) or (<b>B</b>) PPR3 or PPR3-Ala (Iridovirus) and tested for their ability to suppress <i>LacZ</i> silencing. In some experiments, GFP was co-expressed as an internal control to verify similar levels of protein expression, as shown in A. All genes were expressed under a metallothionein promoter, which allowed simultaneous induction of expression of all genes by treatment of cultures with CuSO₄. Cells were harvested for analysis 72 h post-induction. All proteins were expressed with the V5 tag fused to the C-terminus and detected by immunoblotting using monoclonal anti-V5. Co-expression of LacZ from two plasmids in an attempt to induce sense-mediated silencing (lane 4, both panels) or co-expression of LacZ and antisense luciferase (<i>luc</i>) transcripts (lane 5 in both panels) had no significant effect on LacZ expression level. In contrast, LacZ expression was reduced significantly in the cells co-transfected with LacZ and the anti-LacZ construct (lanes 3, 6 and 7), irrespective of whether CSR3 or CSR3-Ala (lanes 6 and 7 in panel A) or PPR3 or PPR3-Ala (lanes 6 and 7 in panel B) were co-expressed in the same cells. These results indicated that the tested viral proteins could not suppress antisense-mediated gene silencing in <i>D</i>. <i>melanogaster</i>. S2 cells were transfected or co-transfected for expression of the following constructs. “RNase III” refers to CSR3 (panel A) and PPR3 (panel B): lane 1, an empty plasmid; lane 2, LacZ; lane 3, LacZ and antisense-LacZ; lane 4, LacZ and LacZ; lane 5, LacZ and antisense-luc; lane 6, LacZ and antisense-LacZ and RNase III; lane 7, LacZ and antisense-LacZ and RNase III-Ala; lane 8, LacZ and LacZ and RNase III; lane 9, LacZ and LacZ and RNase III-Ala; lane 10, LacZ and RNase III; lane 11, LacZ and RNase III-Ala; lane 12, RNase III. Size markers (kDa) are shown to the left of each blot.</p

Ability of CSR3 and CSR3-Ala to interfere with <i>gfp</i> silencing in <i>gfp</i>-transgenic <i>C</i>. <i>elegans</i> (strain RT476) expressing GFP under an intestine-specific promoter.

<p>(<b>A, B</b>) Upper panel: representative images of <i>C</i>. <i>elegans</i> intestine using bright field (BF) illumination or UV light to observe fluorescence of GFP or the red fluorescent protein dTomato. Lower panel: normalized average intensity of GFP fluorescence in four independent experiments. Error bars indicated S.E. (n = 35–50). In (<b>A</b>), the <i>gfp</i>-transgenic strain RT476 was transformed to express CSR3 or CSR3-Ala under a heat shock—inducible promoter, and two transgenic lines expressing each protein were included in experiments. Sense-mediated silencing of <i>gfp</i> expression was induced in the nematodes by feeding them <i>E</i>. <i>coli</i> expressing <i>gfp</i> mRNA (RNAi), which reduced GFP fluorescence significantly (<i>gfp</i>-transgenic nematodes fed bacteria harboring an insert-less plasmid were used as a control) (Student’s t-test, p < 0.001). At 72 h, expression of CSR3 and CSR3-Ala was induced in the <i>gfp</i>-silenced nematodes by heat shock. At 24 h post-induction, however, no significant difference (Student’s t-test) in GFP fluorescence was detected in nematodes expressing CSR3 or CSR3-Ala as compared with the <i>gfp</i>-silenced strain (RNAi). In (<b>B</b>), similar experiments as above were carried out with the <i>gfp</i>-transgenic strain RT476 transformed to express CSR3 or CSR3-Ala fused with dTomato. No recovery of GFP fluorescence was observed. (<b>C</b>) Immunoblot analysis using a rabbit polyclonal antibody specific to CSR3 detected the dTomato-CSR3 fusion protein in nematodes (B) in which dTomato-CSR3 expression was induced. (<b>D</b>) Simultaneous induction of CSR3 expression and GFP-silencing. CSR3 was not able to delay silencing.</p

Suppression of antiviral RNAi by PPR3 in <i>C</i>. <i>elegans</i>.

<p>(<b>A</b>) GFP fluorescence and stunted growth of the heat shock—treated <i>C</i>. <i>elegans</i> strain 123/PPR3 expressing PPR3 and a mutated flock house virus (FHV) genome (FR1gfp), in which the <i>B2</i> gene for an RNAi suppressor had been replaced with <i>GFP</i>, and in <i>C</i>. <i>elegans</i> strain 123/FHVB2 expressing FHV <i>B2</i> in the FR1gfp background under control of a heat shock—inducible promoter. Strain 123/rde-4 is defective in RdRp function, hence deficient in RNAi and allows GFP expression in pharyngeal cells. In contrast, the lines expressing PPR3-Ala, CSR3, or CSR3-Ala show only faint signals of the pharyngeal-specifically expressed cyan fluorescent protein used as a co-injection marker. Images taken under a microscope in bright field (BF) or with a GFP fluorescence filter (GFP) were merged (M). Scale bars, 100 μm. (<b>B</b>) Transgene expression was detected by RT-PCR on DNase-treated total RNA extracted from worms. PCR template (100 ng) was cDNA (indicated by a +) or extracted RNA that was DNase-treated but not reverse transcribed (—). Positive controls (DNA): C1, FHV RNA1 plasmid (includes <i>B2</i>); C2, PPR3 plasmid; C3, CSR3 plasmid. Detection of actin mRNA was used as an additional control. PCR products were verified by sequencing. L, Gene Ruler DNA ladder mix.</p

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.</p