Enhanced Transgene Expression in Sugarcane by Co-Expression of Virus-Encoded RNA Silencing Suppressors

Post-transcriptional gene silencing is commonly observed in polyploid species and often poses a major limitation to plant improvement via biotechnology. Five plant viral suppressors of RNA silencing were evaluated for their ability to counteract gene silencing and enhance the expression of the Enhanced Yellow Fluorescent Protein (EYFP) or the β-glucuronidase (GUS) reporter gene in sugarcane, a major sugar and biomass producing polyploid. Functionality of these suppressors was first verified in Nicotiana benthamiana and onion epidermal cells, and later tested by transient expression in sugarcane young leaf segments and protoplasts. In young leaf segments co-expressing a suppressor, EYFP reached its maximum expression at 48-96 h post-DNA introduction and maintained its peak expression for a longer time compared with that in the absence of a suppressor. Among the five suppressors, Tomato bushy stunt virus-encoded P19 and Barley stripe mosaic virus-encoded γb were the most efficient. Co-expression with P19 and γb enhanced EYFP expression 4.6-fold and 3.6-fold in young leaf segments, and GUS activity 2.3-fold and 2.4-fold in protoplasts compared with those in the absence of a suppressor, respectively. In transgenic sugarcane, co-expression of GUS and P19 suppressor showed the highest accumulation of GUS levels with an average of 2.7-fold more than when GUS was expressed alone, with no detrimental phenotypic effects. The two established transient expression assays, based on young leaf segments and protoplasts, and confirmed by stable transgene expression, offer a rapid versatile system to verify the efficiency of RNA silencing suppressors that proved to be valuable in enhancing and stabilizing transgene expression in sugarcane

High frequency direct shoot regeneration from Kazakh commercial potato cultivars

Potato (Solanum tuberosum L.) is the third most economically important crop in the world and has a high nutritional value. In this study, the in vitro culture response of four widely grown in Kazakhstan potato cultivars, Astanalyk, Monument Kunaev, Tokhtar, and Aksor, was investigated using stem and leaf explants. Published protocols were evaluated and optimized to develop a more efficient protocol for the regeneration of plants from local potato cultivars in tissue culture, which is a prerequisite to facilitate potato genome modification. The explants were cultured on solid Murashige and Skoog medium supplemented with different concentrations and combinations of zeatin, 6-benzylaminopurine (BAP), gibberellic acid (GA3), 1-naphthaleneacetic acid (NAA) and indole-3-acetic acid (IAA). The maximum regeneration was induced from the stem internodal explants. A significant effect of the explant source on direct regeneration was confirmed with statistical analysis. The number of shoots obtained from the internode was 10.0 from cv. Aksor followed by cvs. Tokhtar and Astanalyk. The medium DRM-VIII with 1 mg/l zeatin, 0.1 mg/l IAA and 7.0 mg/l GA3 was considered the best for direct shoot regeneration and multiple shoot formation from all cultivars. To conclude, we outline a protocol for direct plant regeneration from four potato cultivars. Our findings suggest commercial cultivars Astanalyk and Aksor are good candidates for developing the genome-edited plants through direct shoot regeneration

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>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 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

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

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