Data_Sheet_1_RNA Interference in the Tobacco Hornworm, Manduca sexta, Using Plastid-Encoded Long Double-Stranded RNA.pdf

RNA interference (RNAi) is a promising method for controlling pest insects by silencing the expression of vital insect genes to interfere with development and physiology; however, certain insect Orders are resistant to this process. In this study, we set out to test the ability of in planta-expressed dsRNA synthesized within the plastids to silence gene expression in an insect recalcitrant to RNAi, the lepidopteran species, Manduca sexta (tobacco hornworm). Using the Manduca vacuolar-type H+ ATPase subunit A (v-ATPaseA) gene as the target, we first evaluated RNAi efficiency of two dsRNA products of different lengths by directly feeding the in vitro-synthesized dsRNAs to M. sexta larvae. We found that a long dsRNA of 2222 bp was the most effective in inducing lethality and silencing the v-ATPaseA gene, when delivered orally in a water droplet. We further transformed the plastid genome of the M. sexta host plant, Nicotiana tabacum, to produce this long dsRNA in its plastids and performed bioassays with M. sexta larvae on the transplastomic plants. In the tested insects, the plastid-derived dsRNA had no effect on larval survival and no statistically significant effect on expression of the v-ATPaseA gene was observed. Comparison of the absolute quantities of the dsRNA present in transplastomic leaf tissue for v-ATPaseA and a control gene, GFP, of a shorter size, revealed a lower concentration for the long dsRNA product compared to the short control product. We suggest that stability and length of the dsRNA may have influenced the quantities produced in the plastids, resulting in inefficient RNAi in the tested insects. Our results imply that many factors dictate the effectiveness of in planta RNAi, including a likely trade-off effect as increasing the dsRNA product length may be countered by a reduction in the amount of dsRNA produced and accumulated in the plastids.</p

Purification of rFaeG_ntd/dsc from crude plant extract and quantification.

<p>(a) rFaeG_ntd/dsc was extracted from 5 g of mature transplastomic leaf tissue and purified. The initial volume of the extract was 50 ml; 3 µl of the extract from each step of the procedure were resolved by SDS-PAGE and stained. Lane 1 - Initial extract from leaf tissue, pH = 7.5; lane 2 - extract acidified to pH = 2 and centrifuged; lane 3 - clarified extract neutralized to pH = 7.4; Lane 4 - flowthrough from IMAC column; Lane 5 - wash with 20 mM imidazole; Lane 6 - elution of purified rFaeG_ntd/dsc; Lane 7 - 0.5 µg of BSA as loading control; kDa - protein molecular weight marker. (b) Purified rFaeG_ntd/dsc was quantified using densitometry. Dilutions of the purified rFaeG_ntd/dsc protein (lanes 1 through 7) were resolved in SDS-PAGE gel along with known amounts of BSA (lanes 8–14; 1.0, 0.8, 0.6, 0.4, 0.2, 0.1, 0.05 µg BSA, respectively) and stained. BSA bands were used for generation of a standard curve (<i>R²</i> = 0.987; <i>p</i> = 0.01) and extrapolating rFaeG_ntd/dsc concentration. kDa - molecular weight marker.</p

Accumulation levels of rFaeG_ntd/dsc in transplastomic leaf tissue.

<p>(a) Samples of equal volume (4 µl) were prepared from crude extract fractions. Lane 1 - WT extract (negative control); lanes 2, 3 and 4 represent crude extract of 0.4 mg of leaf tissue, re-extracted pellet, and clarified extract, respectively, where clarified extract contains 5 µg TSP. The rFaeG_ntd/dsc yield was estimated using a standard curve (<i>R²</i> = 0.993) of known amounts of purified rFaeG_ntd/dsc (lanes 5 through 8∶2 µg, 1 µg, 0.5 µg and 0.25 µg, respectively). (b) No variation in rFaeG_ntd/dsc accumulation was observed in transplastomic clones (C1, C2) after dark (D) or after light (L) periods. Image is representative of sampling on three different days, 1 µg TSP was used per lane. WT =  untransformed control.</p

Spatial accumulation of rFaeG_ntd/dsc in transplastomic tobacco plants.

<p>(a) Schematic showing the 10 leaves sampled to assess the spatial accumulation of rFaeG_ntd/dsc in transplastomic tobacco plants. (b) Samples examined on SDS-PAGE stained gel (upper panel) and western blot (lower panel). Each lane was loaded with an extract from either ∼2.3 mg of leaf tissue (stained gel), or ∼0.5 mg (immunoblotted gel). WT =  leaf 4 from an untransformed plant. A band of the predicted size (29 kDa, indicated with a black rhomb) corresponding to rFaeG_ntd/dsc was observed in all transplastomic leaf samples, but was absent in the WT. This band was immunoreactive with anti-FaeG serum on the Western blot. kDa - protein molecular weight marker.</p

Chloroplast-produced rFaeG_ntd/dsc protein is recognized in F4 fimbriae-specific ELISA, partially polymerizes and specifically binds to the brush border of F4R+ small intestinal villi.

<p>(a) Both rFaeG_ntd/dsc and F4 fimbriae are recognized by a monoclonal anti-F4_ad fimbriae antibody in ELISA. (b) Purified F4 fimbriae (lane 1) and purified rFaeG_ntd/dsc (lane 2) were resolved under non-reducing conditions to assess polymerization. The F4 fimbriae sample displayed the formation of native FaeG polymers, number of subunits is indicated by stacked black triangles next to each band. Most of the rFaeG_ntd/dsc is present as monomers (denoted by black rhomb); formation of rFaeG_ntd/dsc dimers and trimers was also observed (two and three stacked black rhombs). (c) Adhesion of the rFaeG_ntd/dsc protein to the brush border of F4R+ small intestinal villi. Binding to the F4-specific receptors present on the apical surface of the epithelial cells, which line the brush border of F4R+ small intestinal villi is shown as a bright line on the edge of the sample, the result of excited FITC fluorochrome (indicated with white arrows, lower panel). rFaeG_ntd/dsc fails to bind to brush border of F4R− small intestinal villi. Images are representative of rFaeG_ntd/dsc adhesion to isolated villi of three F4R+ and two F4R− piglets. Bar: 50 µm.</p

Stability of rFaeG_ntd/dsc under simulated gastrointestinal conditions.

<p>Time course analysis of the stability of chloroplast-expressed rFaeG_ntd/dsc in simulated gastric fluid (SGF; a) and simulated intestinal fluid (SIF; b). rFaeG_ntd/dsc was present in similar amounts either as purified protein (“Purified”) or as lyophilized and powdered transplastomic leaf tissue (“Biomass”) and was visualized by western blotting. SGF digestion of leaf biomass was done at two different pH values: pH = 3.5 and pH = 4.5. SGF and SIF fluids with no substrate [SGF (−) and SIF (−), respectively] represent negative controls. The rFaeG_ntd/dsc band is indicated with an arrow.</p

Chloroplast-produced rFaeG_ntd/dsc inhibits the adhesion of F4+ ETEC to porcine small intestinal villi.

<p>Adhesion of F4+ ETEC to F4R− villi (a) and F4R+ villi (b), white arrows indicate bacterial cells. Bar: 50 µm. (c) Competitive inhibition of adhesion of F4+ ETEC to porcine small intestinal villi by the rFaeG_ntd/dsc protein or F4 fimbriae, determined at different protein concentrations. The data represent the mean ±SE (n = 4).</p

Accumulation of chloroplast-targeted, transiently-expressed rFaeG_ntd/dsc.

<p>Transient expression of the rFaeG_ntd/dsc protein via agroinfiltration in <i>Nicotiana benthamiana</i> leaves was examined by SDS-PAGE and staining (a), and immunoblot analysis (b). Lanes 1 and 2−5.0 µg of protein extract of leaves co-infiltrated with <i>Agrobacteria</i> carrying chloroplast-targeted rFaeG_ntd/dsc and the p19 viral suppressor of post-transcriptional gene silencing (1), or p19 alone as negative control (2). rFaeG_ntd/dsc is indicated with a black rhomb, higher bands likely correspond to rFaeG_ntd/dsc with partially cleaved transit peptide; Lane 3−0.5 µg purified F4_ad fimbriae as positive control, the F4 native FaeG is indicated with a black triangle; the ∼2 kDa difference in size of rFaeG_ntd/dsc (29 kDa) and the native FaeG (27 kDa) is due to the additional complementing fused domain.</p

Homoplastomic lines show normal phenotype.

<p>(a) A schematic representation of the chloroplast transformation cassette (pCT-rFaeG_ntd/dsc). The cassette was designed to integrate between the <i>trnI (tRNA-Ile)</i> and <i>trnA (tRNA-Ala)</i> genes of the tobacco plastome. The wild type (WT) plastome <i>trnI - trnA</i> region is shown at the bottom. Expected sizes of <i>Rsr</i> II-digested fragments are indicated. Thick black lines represent hybridization sites for the probe used in Southern blot analyses. IEE  =  intercistronic expression element with the Shine-Dalgarno sequence from the 5′ UTR of bacteriophage T7 gene 10 fused to the 3′ end; aadA  =  gene encoding aminoglycoside 3′ adenylyltransferase for spectinomycin resistance; T<i>psbC</i>  = 3′ UTR of <i>psbC</i> from white poplar plastome; P<i>psbA</i>  = 5′ UTR and promoter of tobacco <i>psbA</i> gene. <i>rfaeG_ntd/dsc</i>  =  gene encoding the rFaeG_ntd/dsc protein variant. T<i>rbcL</i>  = 3′ UTR of <i>rbcL</i> from white poplar plastome. (b) Phenotypes of mature transplastomic tobacco cv. I 64 plants transformed with pCT-rFaeG_ntd/dsc (1 and 2) were indistinguishable from WT plants (3). A one-meter ruler was photographed to the left of each plant as size reference. (c) Confirmation of homoplastomy. Southern blot analysis of total plant DNA from 2 independent transformants and 1 untransformed plant displayed a single band of the expected size.</p