Quantitative assessment of the effect of viral RNA silencing suppressors on transient expression the <i>GUS</i> reporter gene in sugarcane protoplasts.

<p>The GUS activity of protoplasts was monitored for 24 h after co-transfection of protoplasts (1×10⁵ protoplasts; 100 µL) with 5 µg of <i>GUS</i> (pUbi:<i>GUS</i>:Tnos; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066046#pone.0066046.s003" target="_blank">Figure S3</a>) and 10 µg each of RNA silencing suppressors (under the control of the Ubi promoter and nos terminator; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066046#pone.0066046.s003" target="_blank">Figure S3</a>), <i>P1/HC-Pro</i>, <i>γb</i>, <i>P19</i>, <i>P19/R43W</i> and <i>SCBV OrfI</i>. Vector with no suppressor (pUbi:Tnos; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066046#pone.0066046.s003" target="_blank">Figure S3</a>) was used as a negative control. Values represent means with standard error from three independent experiments and six replicates per experiment. Means with the same letter are not significantly different (<i>p</i>>0.05).</p

Dosage effect of the TBSV-encoded P19 RNA silencing suppressor on transient expression of the <i>GUS</i> reporter gene in sugarcane protoplasts.

<p>DNA (5 µg) from pUbi:<i>GUS</i>:Tnos (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066046#pone.0066046.s003" target="_blank">Figure S3</a>) was co-transfected into a protoplast suspension (100 µL; 1×10⁵ protoplasts) with three concentrations of <i>P19</i> DNA (pUbi:<i>P19</i>:Tnos) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066046#pone.0066046.s003" target="_blank">Figure S3</a>), respectively, and GUS activity of protoplasts was measured at 24 h post-transfection. Vector with no <i>P19</i> suppressor (pUbi:Tnos; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066046#pone.0066046.s003" target="_blank">Figure S3</a>) and sterile water were used as controls. Values represent three biological samples and six technical repeats, and are reported with the standard error. Means with the same letter are not significantly different (<i>p</i>>0.05).</p

Quantitative assessment of the effect of viral RNA silencing suppressors on transient expression of the <i>EYFP</i> reporter gene in sugarcane young leaf segments.

<p><i>EYFP</i> expression as measured by foci count (a) and expression level (b) was monitored in 3 day-pre-cultured young leaf segments for 240 h after co-bombardment with 0.25 µg (per shot) of <i>EYFP</i> (pUbi:<i>EYFP</i>:Tnos; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066046#pone.0066046.s003" target="_blank">Figure S3</a>) and 0.5 µg (per shot) each of RNA silencing suppressors (under the control of the Ubi promoter and nos terminator; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066046#pone.0066046.s003" target="_blank">Figure S3</a>), <i>P1/HC-Pro</i>, <i>γb</i>, <i>P19</i>, <i>P19/R43W</i> and <i>SCBV OrfI</i>. Vector with no suppressor (pUbi:Tnos; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066046#pone.0066046.s003" target="_blank">Figure S3</a>) was used as a negative control. Values represent means with standard error from three independent experiments and 8–10 replicates per experiment. Quantitation of <i>EYFP</i> foci counts and expression levels is provided in Materials and Methods. gamma-b: γb.</p

Enhanced expression of the <i>GUS</i> reporter gene by stable co-expression of the TBSV-encoded <i>P19</i> suppressor in transgenic sugarcane.

<p>(a) Relative abundance of <i>P19</i> and <i>GUS</i> transcripts was determined by northern blot and quantitative RT-PCR (qRT-PCR) analyses in two representative <i>P19-GUS</i> transgenic sugarcane lines co-expressing <i>GUS</i> and <i>P19</i> (two plants per line). Lines expressing <i>GUS</i> with no suppressor were used as a control. Blots of RNA (15 µg per sample) were probed with radioactively labeled <i>P19</i> DNA, stripped and then reprobed with <i>GUS</i> DNA. Normalized qRT-PCR <i>P19</i> expression levels of the <i>P19-GUS</i> lines are reported as a percentage, relative to that of the highest expressing plant. GUS activity (pmoles of 4-methylumbelliferone/min/µg protein) of the <i>P19-GUS</i> lines is also indicated. Values represent three biological samples and three technical repeats, and are reported with the standard error. (b) Methylation status of the coding region and promoter of the <i>GUS</i> reporter gene in the <i>P19-GUS</i> transgenic sugarcane lines. Southern blot of genomic DNA (10 µg per sample) of two representative <i>P19-GUS</i> lines, one non-silenced (Line 1, plants 4 and 5) and one silenced (Line 3, plants 12 and 16), digested with methylation-sensitive <i>Hpa</i>II (H), and methylation-insensitive <i>Msp</i>I (M), restriction endonucleases, were probed with the <i>GUS</i> gene or the Ubi promoter. Shifts in DNA hybridization fragments indicate methylation.</p

Dosage effect of the TBSV-encoded P19 RNA silencing suppressor on transient expression of the <i>EYFP</i> reporter gene in sugarcane young leaf segments.

<p><i>EYFP</i> (pUbi:<i>EYFP</i>:Tnos; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066046#pone.0066046.s003" target="_blank">Figure S3</a>) (0.25 µg per shot) was co-bombarded with each of five concentrations of <i>P19</i> DNA (pUbi:<i>P19</i>:Tnos; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066046#pone.0066046.s003" target="_blank">Figure S3</a>), and <i>EYFP</i> expression as measured by foci count (a) and expression level (b) was monitored for 120 h post-bombardment of sugarcane young leaf segments (3 day-pre-cultured). Vector with no suppressor (pUbi:Tnos; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066046#pone.0066046.s003" target="_blank">Figure S3</a>) was used as a negative control. Values represent two independent experiments and ten technical repeats, and are reported with the standard error. Quantitation of <i>EYFP</i> foci counts and expression levels is provided in Materials and Methods.</p

Effect of the TBSV-encoded P19 RNA silencing suppressors on the expression of the <i>GUS</i> reporter transgene in transgenic sugarcane.

<p>Average GUS activity was measured in leaves of 4-month-old sugarcane transgenic lines co-expressing pUbi:<i>GUS</i> and pUbi:<i>P19</i> (7 lines; 41 plants analyzed). pUbi:<i>GUS</i> transgenic (3 lines; 5 plants analyzed) and non-transgenic sugarcane (3 plants) were included as controls. For each set of experiments, the range of GUS activity values is indicated in parenthesis. Values represent three biological samples and three technical repeats, and are reported with the standard error. Values with the same letter are not significantly different (<i>p</i>>0.05). 20% and 39% represent the percentage of plants that are transgenic for the <i>P19</i> and <i>GUS</i> genes.</p