Schematic showing the alignment of Sanger sequences to scaffolds in benthgenome.com and solgenomics.net.

<p>Schematic showing the alignment of Sanger sequences to scaffolds in benthgenome.com and solgenomics.net.</p

List of primers used in this study.

<p>List of primers used in this study.</p

Schematic of pBIN19-mGFP-ER and Tn5393 and their integration site in 16c.

<p><b>(A–B)</b> Schematic of genes within the T-DNA and transposon integrated in 16c genome. The sequence junctions between <i>N</i>. <i>benthamiana</i> genome-T-DNA-transposon-<i>N</i>. <i>benthamiana</i> genome are highlighted, GenBank accession number KY464890. <b>(C)</b> Log scale density plot of sequence reads aligning to T-DNA, transposon and flanking <i>N</i>. <i>benthamiana</i> genomic regions. <b>(D)</b> Location of primers used for amplification of various regions to confirm site of integration. <b>(E)</b> Log scale density plot of RNAseq reads aligning to T-DNA and transposon. <b>(F)</b> Gel image showing the endpoint (35 cycles) PCR amplicons of the T-DNA insert in 16c with LAB <i>N</i>. <i>benthamiana</i> used as a control. Expected sizes with primer pair B+E is 4174 nt and D+F is 4167 nt. NTC, no template control, 1 kb DNA Ladder (GeneRuler^™). <b>(G)</b> Leaves of <i>N</i>. <i>benthamiana</i> 16c photographed under UV light, showing mobile silencing, 14 days after local induction of RNAi at a lower leaf. Abbreviations are: RB: Right Border, pNOS: nopaline synthase promoter, <i>NPTII</i>: neomycin phosphotransferase II encoding gene, tNOS: nopaline synthase terminator, p35S: Cauliflower mosaic virus 35S promoter. IR: inverted terminal repeat, <i>tnpA</i> transposase gene, res: recombination region, <i>tnpR</i> resolvase gene, IS1133: an insertion element, <i>strA</i> and <i>strB</i>: streptomycin resistance genes, Nb Genome: Flanking DNA of <i>N</i>. <i>benthamiana</i>.</p

Silencing endogene in transient leaf assays to improve the flux of metabolites into transgenic pathways.

<p><b>A.</b> Diagram outlining the flux of lipid metabolites from 18∶1 to 18∶2, via NbFAD2 and limiting the availability of 18∶1 to enter into transgenic pathways such as that catalysed by AtFAE1 and AtDGAT1 to produce 20∶1. <b>B.</b> Diagram outlining the efficient RNAi (hpNbFAD2) silencing of endogenous NbFAD2 activities, to increase the flux of 18∶1 into the engineered pathway to produce 20∶1, whereby both silencing and transgene overexpression are supported with the addition of V2. <b>C.</b> Combinations of hpNbFAD2, V2 or p19, and a two step metabolic pathway, <i>AtFAE1</i> and <i>AtDGAT1</i>, for the production of oil (lower panel), and the weight percentage of 18∶1 and 20∶1 in the total FAME in the oil (upper panel). Data shown are the mean and 5% LSD bars calculated from at least 4–6 infiltrations. When the LSD bars for two metabolites fail to overlap, treatments are significantly different at the 5% level of significance. The complete fatty acid profiles of leaf oils from this experiment are found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052717#pone.0052717.s003" target="_blank">Table S1</a>.</p

Summary of draft assembly of <i>N. benthamiana.</i>

<p>Summary of draft assembly of <i>N. benthamiana.</i></p

V2 supports the overexpression of multistep transgenic pathways in transient leaf assays. A.

<p>Schematic of T-DNA binary constructs for leaf expression of the viral silencing-suppressor proteins (VSP), V2 and p19, and the transgenes GFP, AtFAE1 and AtDGAT1. All ORF constructs were driven by the <i>Cauliflower mosaic virus</i> 35S promoter. <b>B.</b> Time course of GFP expression in transient leaf assays with either no VSP or co-infiltration of V2 or p19. Images show one representative leaf photographed daily from 2 to 7 days post infiltration (dpi), and the last image is used to indicate the placement of infiltrations with a stippled circle showing where complete co-suppression of GFP has occurred 7 dpi. <b>C.</b> Different combinations of VSPs and transgenes, <i>AtFAE1</i> and <i>AtDGAT1</i>, showing the increase in level of the elongated fatty acid 20∶1 in total leaf lipids. Data shown are the means and 5% LSD bars calculated from 10 samples. When the LSD bars for two metabolites fail to overlap, the treatments are significantly different at the 5% level of significance.</p

Size and distribution of sRNA populations generated by a hairpin targeting the endogene <i>NbFAD2.1</i>.

<p><b>A.</b> The ORF of <i>NbFAD2.1</i> is portrayed indicating the region used to generate a 660 bp hairpin, hpNbFAD2. Total RNA was extracted from leaves infiltrated with hpNbFAD2 4 days post infiltration. The size and distribution of the dominate classes of small RNAs on the forward (F) and reverse (R) strand of the <i>NbFAD2.1</i> is illustrated. Absolute read coverage is normalised for each track. <b>B.</b> Absolute numbers and relative percentages of the dominant sRNA size classes generated by hpNbFAD2.</p

Effective silencing of the FAD2 gene family in <i>N. benthamiana</i> leaves. A.

<p>Relative position and length of the 660 bp hairpin, hpNbFAD2, and the 300 bp hairpin, hpNbFAD2-300, against <i>NbFAD2.1</i>. Higher resolution alignments are found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052717#pone.0052717.s002" target="_blank">Figure S2</a>. <b>B.</b> Reduction in expression levels of <i>NbFAD2.1</i> and <i>NbFAD2.2</i> when silenced using hpNbFAD2 and hpNbFAD2-300 hairpin RNA construct. Total RNA extracted from leaves infiltrated with the different hairpin constructs 4 days post infiltration and expression levels of <i>NbFAD2.1</i> and <i>NbFAD2.2</i> were analysed by quantitative real-time, qRT-PCR. Mock represents RNA extracted from leaf tissue infiltrated with agrobacterium only. Data shown are means of 3 biological replicates with 3 technical repeats for each and error bars are the standard errors of the mean. <b>C.</b> Total fatty acid analysis of leaves infiltrated with either the hpNbFAD2 and hpNbFAD2-300 hairpin. Fatty acid methyl esters (FAME) were analysed 4 days post infiltration and the data shown are the mean and 5% LSD bars calculated from 10 independent leaf infiltrations per treatment. When the LSD bars for two metabolites fail to overlap, the metabolite means may be regarded as significantly different at the 5% level of significance. Mock refers to leaves infiltrated with <i>Agrobacterium</i> only. <b>D.</b> Fatty acid profile of phosphatidylcholine (PC) fraction extracted from leaves infiltrated with combinations of V2, p19 and hpNbFAD2. Data shown are the mean and 5% LSD bars calculated from at least 5 infiltrations. When the LSD bars for two metabolites fail to overlap, the treatments may be regarded as significantly different at the 5% level of significance.</p

V2, not p19, allows efficient hairpin-based silencing of a co-infiltrated transgene, GFP. A.

<p>Schematic of the T-DNA binary construct containing a GFP expression cassette and a 380 bp hairpin directed against GFP, hpGFP. <b>B.</b> Time course of GFP expression in transient leaf assays infiltrated with combinations of GFP and hpGFP with V2 or p19. Images show one representative leaf photographed daily from 2 to 7 days post infiltration (dpi), and the last image is used to indicate the placement of infiltrations with stippled circles showing infiltration zones that failed to express GFP. <b>C.</b> Western blot analysis of GFP expression in <i>N. benthamiana</i> leaves infiltrated with combinations of GFP, hpGFP, V2 and p19 as indicated. Samples were collected 4 dpi. The upper panel shows the total protein loaded for the western blot and the lower panel shows the signal generated from an antibody recognising GFP, anti-GFP.</p