9 research outputs found
Anti-viral RNAi and its suppression in plants.
As a defense against viral infection, plants are thought to use RNA-induced
silencing complexes (RISCs) to target and cleave viral RNA. To counteract this, some
viruses have evolved proteins to inhibit RISC-mediated activity, thus ensuring
continued virulence. This research focused on the study and analysis of the anti-viral
RNAi response to various viruses in plants to gain an understanding of how the plant
defense operates on the molecular and biochemical levels. Nicotiana benthamiana plants
were infected with Tomato bushy stunt virus (TBSV) and Tobacco rattle virus (TRV).
These plants were subjected to column chromatography methods, and fractions
contained a virus-specific ribonuclease activity, co-eluting with small interfering RNAs
(siRNA), that was shown to be sensitive to inhibition with EDTA and enhanced by the
addition of divalent metal cations. This ribonuclease activity co-purified with proteins
that contained a domain from the hallmark RISC protein Argonaute family. To further
study host responses to viral infection, monocots were infected with Panicum mosaic
virus (PMV) and satellite panicum mosaic virus (SPMV) and also were subjected to
column chromatography following infection. Preliminary studies show that fractions
contained ribonuclease activity as well as siRNAs and proteins containing an Argonaute
domain. Additionally, silencing suppressors have been directly implicated in interfering
with RNAi pathways in plants. Studies involving Agrobacterium- and virus-vectored
cDNA to express green fluorescent protein (GFP) were used to establish that cointroduced
suppressors of RNAi can extend the production of a foreign protein for
enhancement of biotechnological applications. It was found that the hordeivirus protein
?b contributes to enhancement of expression for the foreign protein GFP early in the infection, while the potyvirus protein HcPro and tombusvirus protein P19 enhance and
extend protein production later in the infection
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
Cardiovirus Leader proteins bind exportins: Implications for virus replication and nucleocytoplasmic trafficking inhibition
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
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
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 (1×10<sup>5</sup> 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; 1×10<sup>5</sup> 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
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