Vertical box-and-whisker plots summarising real-time PCR data on all constructs bombarded at a 5∶1 weight ratio with infectious clones of diverse MSV strains and another mastrevirus species.

<p>A) MSV-Kom. The plots were constructed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105932#pone-0105932-g006" target="_blank">Figure 6</a>. The number of replicates (i.e. the number of bombarded samples) were as follows: pSK, 34; pSPLITGusUbi, 18; pSPLIT<i>rep</i>^1-219Rb-UbiΔI, 18; pSPLIT<i>rep</i>^1-219Rb-Ubi, 14; pSPLIT<i>rep</i>^III-Rb-Ubi, 8; p<i>rep</i>^1-219Rb-, 9; pMSV-<i>Pst</i>I, 11. Plots in B-C) were constructed as described for A), but this time either pSPLIT<i>rep</i>^III-Rb-Ubi or pSK were co-bombarded with infectious clones of: B) the MSV-B strain isolate VW; and C) the PanSV strain A isolate Kar. The number of replicates for B) were: pSK, 11; pSPLIT<i>rep</i>^III-Rb-Ubi, 14. The number of replicates for C) were: pSK, 16; pSPLIT<i>rep</i>^III-Rb-Ubi, 25. All real-time PCRs were performed on total DNA extracted from BMS cells four days post-bombardment.</p

Primer sequences.

1<p>Underlined letters highlight engineered restriction enzyme (RE) sites (names of the introduced RE sites are incorporated in the primer names).</p><p>Primer sequences.</p

Schematic diagram of synthesised constructs, with restriction enzyme sites incorporated for subsequent cloning.

<p>A) pSPLIT<i>rep</i>^1-219Rb-35S containing “modules” that could be removed and replaced with other sequences by restriction digest. B) Illustration showing how the <i>rep</i>^1-219Rb- transgene was split at the first AGGC (nucleotides 155, 156, 157 and 158 with respect to the start codon). The exon 2, cloned at the 5′ terminus of the split gene cassette in A) therefore began with GC, and the exon 1, cloned at the 3′ terminus, ended in AG. C) The synthesised <i>rep</i>^III-Rb- (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105932#pone-0105932-g001" target="_blank">Fig. 1A</a> for the full-length gene product) exon 2, preceded by the 3′-terminal half of the syntron, flanked by <i>Swa</i>I and <i>Spe</i>I RE sites. The 3′-terminal syntron/<i>rep</i>^1-219Rb- exon 2 in pSPLIT<i>rep</i>^1-219Rb-35S was replaced by the 3′-terminal syntron/<i>rep</i>^III-Rb- exon 2 to create pSPLIT<i>rep</i>^III-Rb-35S. Exon 1 remained the same for both constructs since they share the same 5′-terminal 156 bp. Similarly, other modules were exchanged to create further constructs, such as the CaMV 35S promoter for the maize ubiquitin promoter etc (see text for details).</p

Gus assays to test for cryptic splice sites in the MSV long intergenic region.

<p>A) Gus expression cassettes used in the assays. B) Expression of Gus from p35S-GSLIR²⁴¹ (test construct) as a ratio to p35S-GS (positive control construct), four days after bombardment. Each bar is an average of three replicates; error bars represent 95% confidence intervals. Negative = negative control (protein extract from a non-bombarded Black Mexican sweet sample).</p

Replicative-form specific end-point PCR assay to test the effectiveness of the synthesised split gene cassette, pSPLIT<i>rep</i>^1-219Rb-35S, in interfering with MSV replication.

<p>Black Mexican sweet (BMS) cells were bombarded with an infectious clone of MSV-Kom (pKom602) alone (lanes 1–3); pKom602 and pSPLIT<i>rep</i>^1-219Rb-35S (lanes 7–9), as well as pKom602 and p<i>rep</i>^1-219Rb- (constitutively expressed from the maize ubiquitin promoter <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105932#pone.0105932-Shepherd2" target="_blank">[16]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105932#pone.0105932-Shepherd3" target="_blank">[17]</a>) for comparative purposes (lanes 4–6). W = water control, − = non-bombarded BMS control, + =  positive control (pKom602 plasmid DNA). B = blank. The PCR was performed on total DNA extracted from BMS cells four days post-bombardment.</p

Products of mutated and truncated MSV <i>rep</i> genes used in the split gene cassettes, compared with the wild type.

<p>A) Known sequence motifs and functional domains present in each gene product are highlighted. Amino acid numbering is relative to the N-terminal methionine. Adapted from Shepherd et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105932#pone.0105932-Shepherd3" target="_blank">[17]</a>. B) Three representative <i>Digitaria sanguinalis</i> lines constitutively expressing p<i>rep</i>^1–219Rb- (left), p<i>rep</i>^III-Rb- (middle) or Gus and Bar (from pAHC25 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105932#pone.0105932-Christensen2" target="_blank">[39]</a>; right), illustrate the phenotypic effects of the transgenes. Photo from Shepherd et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105932#pone.0105932-Shepherd3" target="_blank">[17]</a>.</p

Schematic representation of the INPACT system.

<p>MSV-inducible expression from a “split gene cassette” using pSPLIT<i>rep</i>^III-Rb-Ubi is used as an example. NosT = nopaline synthase terminator; UbiP = maize ubiquitin promoter. MSVLIR²⁴¹– =  truncated MSV long intergenic region.</p