Over-expression of Topoisomerase II Enhances Salt Stress Tolerance in Tobacco

Topoisomerases are unique enzymes having an ability to remove or add DNA supercoils and untangle the snarled DNA. They can cut, shuffle and religate DNA strands and remove the torsional stress during DNA replication, transcription or recombination events. In the present study, we over-expressed topoisomerase II (TopoII) in tobacco (Nicotiana tabaccum) and examined its role in growth and development as well as salt (NaCl) stress tolerance. Several putative transgenic plants were generated and the transgene integration and expression was confirmed by PCR and Southern blot analyses, and RT-PCR analysis respectively. Percent seed germination, shoot growth and chlorophyll content revealed that transgenic lines over-expressing the NtTopoIIα-1 gene exhibited enhanced tolerance to salt (150 and 200 mM NaCl) stress. Moreover, over-expression of TopoII lead to the elevation in proline and glycine betaine levels in response to both concentrations of NaCl as compared to wild-type. In response to NaCl stress, TopoII over-expressing lines showed reduced lipid peroxidation derived malondialdehyde (MDA) generation. These results suggest that TopoII plays a pivotal role in salt stress tolerance in plants

RNA-interference in rice against Rice tungro bacilliform virus results in its decreased accumulation in inoculated rice plants

Rice tungro is a viral disease seriously affecting rice production in South and Southeast Asia. Tungro is caused by the simultaneous infection in rice of Rice tungro bacilliform virus (RTBV), a double-stranded DNA virus and Rice tungro spherical virus (RTSV), a single-stranded RNA virus. To apply the concept of RNA-interference (RNAi) for the control of RTBV infection, transgenic rice plants expressing DNA encoding ORF IV of RTBV, both in sense as well as in anti-sense orientation, resulting in the formation of double-stranded (ds) RNA, were raised. RNA blot analysis of two representative lines indicated specific degradation of the transgene transcripts and the accumulation of small molecular weight RNA, a hallmark for RNA-interference. In the two transgenic lines expressing ds-RNA, different resistance responses were observed against RTBV. In one of the above lines (RTBV-O-Ds1), there was an initial rapid buildup of RTBV levels following inoculation, comparable to that of untransformed controls, followed by a sharp reduction, resulting in approximately 50-fold lower viral titers, whereas the untransformed controls maintained high levels of the virus till 40 days post-inoculation (dpi). In RTBV-O-Ds2, RTBV DNA levels gradually rose from an initial low to almost 60% levels of the control by 40 dpi. Line RTBV-O-Ds1 showed symptoms of tungro similar to the untransformed control lines, whereas line RTBV-O-Ds2 showed extremely mild symptoms

RNA interference in Lepidoptera: an overview of successful and unsuccessful studies and implications for experimental design

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Targeting fungal genes by diced siRNAs: a rapid tool to decipher gene function in Aspergillus nidulans.

BACKGROUND: Gene silencing triggered by chemically synthesized small interfering RNAs (siRNAs) has become a powerful tool for deciphering gene function in many eukaryotes. However, prediction and validation of a single siRNA duplex specific to a target gene is often ineffective. RNA interference (RNAi) with synthetic siRNA suffers from lower silencing efficacy, off-target effects and is cost-intensive, especially for functional genomic studies. With the explosion of fungal genomic information, there is an increasing need to analyze gene function in a rapid manner. Therefore, studies were performed in order to investigate the efficacy of gene silencing induced by RNase III-diced-siRNAs (d-siRNA) in model filamentous fungus, Aspergillus nidulans. METHODOLOGY/PRINCIPAL FINDINGS: Stable expression of heterologous reporter gene in A. nidulans eases the examination of a new RNAi-induction route. Hence, we have optimized Agrobacterium tumefaciens-mediated transformation (AMT) of A. nidulans for stable expression of sGFP gene. This study demonstrates that the reporter GFP gene stably introduced into A. nidulans can be effectively silenced by treatment of GFP-d-siRNAs. We have shown the down-regulation of two endogenous genes, AnrasA and AnrasB of A. nidulans by d-siRNAs. We have also elucidated the function of an uncharacterized Ras homolog, rasB gene, which was found to be involved in hyphal growth and development. Further, silencing potency of d-siRNA was higher as compared to synthetic siRNA duplex, targeting AnrasA. Silencing was shown to be sequence-specific, since expression profiles of other closely related Ras family genes in d-siRNA treated AnrasA and AnrasB silenced lines exhibited no change in gene expression. CONCLUSIONS/SIGNIFICANCE: We have developed and applied a fast, specific and efficient gene silencing approach for elucidating gene function in A. nidulans using d-siRNAs. We have also optimized an efficient AMT in A. nidulans, which is useful for stable integration of transgenes

Evaluation of An<i>rasB</i> down-regulation by An<i>rasB</i>-d-siRNA in <i>A. nidulans</i>.

<p><b>(A)</b> Assessment of phenotypic changes in <i>A. nidulans</i>. In upper panel germinating conidia were treated with An<i>rasB</i>-d-siRNA for 12 h, and then light microscopic analysis was performed. The An<i>rasB</i> silenced lines possess the irregular apical branching when compared to control. In the lower panel, fungal tissue was collected from ACM solid agar media containing An<i>rasB</i> silenced lines and control lines, then viewed using OLYMPUS BX-51 microscope under the white light conditions. <b>(B)</b> Radial mycelial growth assay. Germinating conidia were treated with An<i>rasB</i>-d-siRNA and spotted onto center of the ACM agar plate and radial outgrowth of mycelia was monitored 48 h post-inoculation. Radial growth was significantly reduced in An<i>rasB</i> silenced lines compared to controls. <b>(C)</b> Determination of the total biomass. Germinating conidia were treated with An<i>rasB</i>-d-siRNA in ACM broth for 48 h and total fresh biomass formation was determined. The data represent the means of three replicates. Values were compared using <i>t</i> test. * Significant difference at <i>P<0.05</i> as compared to untreated control. <b>(D)</b> Quantification of An<i>rasB</i> silencing by qRT-PCR. Relative An<i>rasB</i> expression was measured in An<i>rasB</i>-d-siRNA treated mycelia and controls using comparative D cycle threshold (CT) method. An<i>rasB</i> values were normalized to An<i>Actin</i> values. The data represent the means of three replicates. Values were compared using <i>t</i> test. * Significant difference at <i>P<0.05</i> as compared to untreated control.</p

T-DNA map of s<i>GFP</i> expression vector.

<p>s<i>GFP</i> gene was cloned under the control of <i>Aspergillus nidulans</i> TrpC promoter and marker gene (<i>HPT</i>) was fused to CaMV35S promoter in the T-DNA back bone of pCAMBIA1300 binary vector.</p

Molecular characterization of <i>A. nidulans</i> s<i>GFP</i> transformants.

<p><b>(A)</b> DNA from several independent hygromycin resistant <i>A. nidulans</i> mycelia was extracted and the presence of s<i>GFP</i> gene (200 bp amplicon) was evaluated by PCR amplification. M. Ladder; PC. pCAMBIA 1300-s<i>GFP</i> plasmid control; 1–4. <i>A. nidulans</i> transformants represented as An<i>GFP</i> 101, An<i>GFP</i> 102, An<i>GFP</i> 103, An<i>GFP</i> 104; UT. Untransformed control. <b>(B)</b> DNA from several independent hygromycin resistant <i>A. nidulans</i> mycelia was extracted and the presence of <i>HPT</i> gene was evaluated by PCR amplification. M. Ladder; UT. Untransformed control; 1–4. An<i>GFP</i> 101, An<i>GFP</i> 102, An<i>GFP</i> 103, An<i>GFP</i> 104 fungal transformants. <b>(C)</b> Southern hybridization was performed using genomic DNA extracted from fungal transformants, DNA was (10 µg) digested with <i>Xho</i>I and probed with radio labelled <i>HPT</i> probe. UT. Untransformed control; 1–4. An<i>GFP</i> 101, An<i>GFP</i> 102, An<i>GFP</i> 103, An<i>GFP</i> 104 fungal transformants. <b>(D)</b> Semi-quantitative RT-PCR analysis for evaluation of expression of s<i>GFP</i> in <i>A. nidulans</i> transformants. Upper panel indicates s<i>GFP</i> expression, 1–4. An<i>GFP</i> 101, An<i>GFP</i> 102, An<i>GFP</i> 103, An<i>GFP</i> 104 fungal transformants; UT. Untransformed control. Lower panel: normalization of gene expression by An<i>Actin</i> as internal control.</p

Expression profiles of <i>Ras</i> family genes in d-siRNA treated lines.

<p><b>(A)</b> Estimation of relative expression levels of <i>Ras</i> family genes in untreated control and An<i>rasA</i>-d-siRNA treated <i>A. nidulans.</i> Relative quantification was performed by qRT-PCR using comparative D cycle threshold (CT) method. Expression levels of An<i>rasB</i>, An<i>rhB</i>, An<i>4873</i>, An<i>medA</i> and An<i>7661</i> in untreated control was showed in the left panel, right panel represents the expression levels of same genes in An<i>rasA</i>-d-siRNA treated <i>A. nidulans</i>. Expression levels of all the genes were normalized to An<i>Actin</i> levels. The data represent the means of three replicates. Values were compared using <i>t</i> test. * Significant difference at <i>P<0.05</i> as compared to untreated control. <b>(B)</b> Estimation of relative expression levels of <i>Ras</i> family genes in untreated control and An<i>rasB</i>-d-siRNA treated <i>A. nidulans</i>. Relative quantification was performed by qRT-PCR using comparative D cycle threshold (CT) method. Expression levels of An<i>rasA</i>, An<i>rhB</i>, An<i>4873</i>, An<i>medA</i> and An<i>7661</i> in untreated control was showed in the left panel, right panel represents the expression levels of same genes in An<i>rasB</i>-d-siRNA treated <i>A. nidulans</i>. Expression levels of all the genes were normalized to An<i>Actin</i> levels. The data represent the means of three replicates.</p