Production of a subunit vaccine candidate against porcine post-weaning diarrhea in high-biomass transplastomic tobacco

Post-weaning diarrhea (PWD) in piglets is a major problem in piggeries worldwide and results in severe economic losses. Infection with Enterotoxigenic Escherichia coli (ETEC) is the key culprit for the PWD disease. F4 fimbriae of ETEC are highly stable proteinaceous polymers, mainly composed of the major structural subunit FaeG, with a capacity to evoke mucosal immune responses, thus demonstrating a potential to act as an oral vaccine against ETEC-induced porcine PWD. In this study we used a transplastomic approach in tobacco to produce a recombinant variant of the FaeG protein, rFaeG(ntd/dsc), engineered for expression as a stable monomer by N-terminal deletion and donor strand-complementation (ntd/dsc). The generated transplastomic tobacco plants accumulated up to 2.0 g rFaeG(ntd/dsc) per 1 kg fresh leaf tissue (more than 1% of dry leaf tissue) and showed normal phenotype indistinguishable from wild type untransformed plants. We determined that chloroplast-produced rFaeG(ntd/dsc) protein retained the key properties of an oral vaccine, i.e. binding to porcine intestinal F4 receptors (F4R), and inhibition of the F4-possessing (F4+) ETEC attachment to F4R. Additionally, the plant biomass matrix was shown to delay degradation of the chloroplast-produced rFaeG(ntd/dsc) in gastrointestinal conditions, demonstrating a potential to function as a shelter-vehicle for vaccine delivery. These results suggest that transplastomic plants expressing the rFaeG(ntd/dsc) protein could be used for production and, possibly, delivery of an oral vaccine against porcine F4+ ETEC infections. Our findings therefore present a feasible approach for developing an oral vaccination strategy against porcine PWD

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

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

Rapid generation of hypomorphic mutations

Hypomorphic mutations are a valuable tool for both genetic analysis of gene function and for synthetic biology applications. However, current methods to generate hypomorphic mutations are limited to a specific organism, change gene expression unpredictably, or depend on changes in spatial-temporal expression of the targeted gene. Here we present a simple and predictable method to generate hypomorphic mutations in model organisms by targeting translation elongation. Adding consecutive adenosine nucleotides, so-called polyA tracks, to the gene coding sequence of interest will decrease translation elongation efficiency, and in all tested cell cultures and model organisms, this decreases mRNA stability and protein expression. We show that protein expression is adjustable independent of promoter strength and can be further modulated by changing sequence features of the polyA tracks. These characteristics make this method highly predictable and tractable for generation of programmable allelic series with a range of expression levels

Plastid Transformation of Micro-Tom Tomato with a Hemipteran Double-Stranded RNA Results in RNA Interference in Multiple Insect Species

Plant-mediated RNA interference (RNAi) holds great promise for insect pest control, as plants can be transformed to produce double-stranded RNA (dsRNA) to selectively down-regulate insect genes essential for survival. For optimum potency, dsRNA can be produced in plant plastids, enabling the accumulation of unprocessed dsRNAs. However, the relative effectiveness of this strategy in inducing an RNAi response in insects using different feeding mechanisms is understudied. To investigate this, we first tested an in vitro-synthesized 189 bp dsRNA matching a highly conserved region of the v-ATPaseA gene from cotton mealybug (Phenacoccus solenopsis) on three insect species from two different orders that use leaf-chewing, lacerate-and-flush, or sap-sucking mechanisms to feed, and showed that the dsRNA significantly down-regulated the target gene. We then developed transplastomic Micro-tom tomato plants to produce the dsRNA in plant plastids and showed that the dsRNA is produced in leaf, flower, green fruit, red fruit, and roots, with the highest dsRNA levels found in the leaf. The plastid-produced dsRNA induced a significant gene down-regulation in insects using leaf-chewing and lacerate-and-flush feeding mechanisms, while sap-sucking insects were unaffected. Our results suggest that plastid-produced dsRNA can be used to control leaf-chewing and lacerate-and-flush feeding insects, but may not be useful for sap-sucking insects

Plastid Transformation of Micro-Tom Tomato with a Hemipteran Double-Stranded RNA Results in RNA Interference in Multiple Insect Species

Plant-mediated RNA interference (RNAi) holds great promise for insect pest control, as plants can be transformed to produce double-stranded RNA (dsRNA) to selectively down-regulate insect genes essential for survival. For optimum potency, dsRNA can be produced in plant plastids, enabling the accumulation of unprocessed dsRNAs. However, the relative effectiveness of this strategy in inducing an RNAi response in insects using different feeding mechanisms is understudied. To investigate this, we first tested an in vitro-synthesized 189 bp dsRNA matching a highly conserved region of the v-ATPaseA gene from cotton mealybug (Phenacoccus solenopsis) on three insect species from two different orders that use leaf-chewing, lacerate-and-flush, or sap-sucking mechanisms to feed, and showed that the dsRNA significantly down-regulated the target gene. We then developed transplastomic Micro-tom tomato plants to produce the dsRNA in plant plastids and showed that the dsRNA is produced in leaf, flower, green fruit, red fruit, and roots, with the highest dsRNA levels found in the leaf. The plastid-produced dsRNA induced a significant gene down-regulation in insects using leaf-chewing and lacerate-and-flush feeding mechanisms, while sap-sucking insects were unaffected. Our results suggest that plastid-produced dsRNA can be used to control leaf-chewing and lacerate-and-flush feeding insects, but may not be useful for sap-sucking insects

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

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

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

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