14 research outputs found
How a Retrotransposon Exploits the Plant's Heat Stress Response for Its Activation
<div><p>Retrotransposons are major components of plant and animal genomes. They amplify by reverse transcription and reintegration into the host genome but their activity is usually epigenetically silenced. In plants, genomic copies of retrotransposons are typically associated with repressive chromatin modifications installed and maintained by RNA-directed DNA methylation. To escape this tight control, retrotransposons employ various strategies to avoid epigenetic silencing. Here we describe the mechanism developed by <i>ONSEN</i>, an LTR-copia type retrotransposon in <i>Arabidopsis thaliana</i>. <i>ONSEN</i> has acquired a heat-responsive element recognized by plant-derived heat stress defense factors, resulting in transcription and production of full length extrachromosomal DNA under elevated temperatures. Further, the <i>ONSEN</i> promoter is free of CG and CHG sites, and the reduction of DNA methylation at the CHH sites is not sufficient to activate the element. Since dividing cells have a more pronounced heat response, the extrachromosomal <i>ONSEN</i> DNA, capable of reintegrating into the genome, accumulates preferentially in the meristematic tissue of the shoot. The recruitment of a major plant heat shock transcription factor in periods of heat stress exploits the plant's heat stress response to achieve the transposon's activation, making it impossible for the host to respond appropriately to stress without losing control over the invader.</p></div
Loss of CHH DNA methylation at the promoter is not sufficient to activate the element but enhances the response to heat stress.
<p>(<b>A</b>) Total CHH methylation at LTRs of <i>ONSEN 1 and 2</i> (contributing the majority of exDNA) and <i>ONSEN 8</i> (not activated) in non-stressed and 30 h HS wild type plants. Only CHH positions shared between all three LTRs were considered. CHH methylation profiles along the LTRs and data for individual genomic copies are shown in Supplementary Fig. 2A–F and Supplementary Dataset S1. (<b>B</b>) Total CHH methylation of the same <i>ONSEN</i> LTRs in non-stressed and 30 h HS drm1/drm2/cmt3 triple mutant plants (ddc). Only CHH positions shared between all three LTRs were considered. CHH methylation profiles along the LTRs and data for individual genomic copies are shown in Supplementary Fig. 2G–L and Supplementary Dataset S1. (<b>C</b>) Relative amount of <i>ONSEN RNA</i> determined by quantitative RT-PCR in three week old wild type and ddc mutant plants, non-stressed and after 30 h HS. Bars represent the <i>ONSEN</i> transcripts in relation to that of <i>AtSAND</i> and normalized to WT at 0 h HS. Error bars correspond to the s.e.m. (n = 3). (<b>D</b>) Quantification of <i>ONSEN</i> extrachromosomal DNA from the same samples as in F. Bars represent the ratio between extrachromosomal and integrated copies determined by densitometry after Southern blot analysis. Error bars correspond to the s.d. (n = 3).</p
<i>ONSEN</i> is activated by heat stress.
<p>(<b>A</b>) Schematic representation of the experiments. White and black boxes represent light or darkness intervals; blue and red boxes represent periods of standard growth temperature or heat stress, arrows indicate sampling time points. (<b>B</b>) Relative amounts of <i>ONSEN</i> RNA determined by quantitative RT-PCR in three week-old wild type seedlings harvested as indicated in A. Bars represent the <i>ONSEN</i> transcripts in relation to that of AtSAND (equal level in all samples) and normalized to WT at 0 h HS. Error bars correspond to the s.e.m. (n = 3). (<b>C</b>) Southern blot analysis of undigested genomic DNA isolated from the same material as in B and hybridized to an <i>ONSEN</i>-specific probe. Upper and lower arrows indicate integrated and extrachromosomal <i>ONSEN</i> copies, respectively. The EtBr image indicates the loading of genomic DNA. (<b>D</b>) Quantification of <i>ONSEN</i> extrachromosomal DNA based on C. Bars represent the ratio between signal intensities of extrachromosomal and integrated copies determined by densitometry. Error bars correspond to the s.d. (n = 3).</p
Heat shock factors are necessary for <i>ONSEN</i> activation by heat stress.
<p>Relative amounts of <i>ONSEN</i> RNA and extrachromosomal DNA prepared from three week-old, non-stressed or 30 h HS treated seedlings of wild type Wassilewskija (WT2, background of the mutants), triple or quadruple mutants lacking three or all functional HSFA1 proteins (A, B, D, E). RNA and exDNA were determined by quantitative RT-PCR and densitometry after Southern blot analysis as described before (n = 3).</p
HSFA2 acts downstream of HSFA1 and is able to bind to the <i>ONSEN</i> LTR.
<p>(<b>A</b>) Relative amount of <i>HSFA2</i> RNA determined by quantitative RT-PCR in three week old WT (Col-0), WT2 (Ws), triple and quadruple <i>HSFA1</i> mutant seedlings. Bars represent the <i>HSFA2</i> transcript in relation to <i>AtSAND</i> and normalized to Col-0 WT at 0 h HS. Error bars correspond to the s.e.m. (n = 3). (<b>B</b>) Electrophoretic mobility shift assay (EMSA) with increasing amounts (lanes 1–5) of recombinant HSFA2 protein and constant amounts of radioactively labeled <i>ONSEN</i> LTR fragment derived from <i>ONSEN 1</i>. Competition by unlabeled DNA of the full length LTR in 50-fold excess (lane 6) or the fragment containing only the heat-responsive element (Supplementary <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004115#pgen.1004115.s007" target="_blank">Table S2</a>) in a 200-fold excess (lane 7) demonstrate the binding specificity. (<b>C</b>) Relative amount of <i>ONSEN</i> RNA determined by quantitative RT-PCR in wild type and <i>hsfa2-1</i> mutant plants after 30 h HS. Bars represent the <i>ONSEN</i> transcripts in relation to that of <i>AtSAND</i> and normalized to WT at 0 h HS. Error bars correspond to the s.e.m. (n = 3). (<b>D</b>) Quantification of <i>ONSEN</i> extrachromosomal DNA from the same samples as in C. Bars represent the ratio between extrachromosomal and integrated copies determined by densitometry after Southern blot analysis. Error bars correspond to the s.d. (n = 3).</p
Several <i>ONSEN</i> genomic copies contribute to the extrachromosomal DNA.
<p>The pie chart indicates the ratio of extrachromosomal <i>ONSEN</i> sequences (n = 55) present after 30 h HS, distinguished by element-specific polymorphisms (color-coded). Polymorphisms are listed in Supplementary <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004115#pgen.1004115.s006" target="_blank">Table S1</a>.</p
Additional file 1: of Arabidopsis thaliana ambient temperature responsive lncRNAs
TableS1. LncRNAs annotation. List of lncRNAs retrieved in this study and corresponding identification in the Plant long non-coding RNA database (PlncDB) and Araport11. (XLSX 422 kb
Additional file 5: of Arabidopsis thaliana ambient temperature responsive lncRNAs
Figure S1. Temperature induced flowering for T-DNA insertion lines. Col-8 plants were used for comparison with AtLnc2 (A), AtLnc120 (B), and AtLnc1524 (D) T-DNA insertion lines, while wild-type and T-DNA carrying plants from segregating population were compared for AtLnc213 (C). The experiment was performed using four biological replicates with 13 plants per replicate for each genotype/condition. The T-DNA insertion did not affect temperature-induced flowering in any of these mutants since no significant difference was observed in the ratio of flowering time at the different temperatures between wild-type and mutant plants. (TIF 642 kb
Additional file 7: of Arabidopsis thaliana ambient temperature responsive lncRNAs
Figure S3. A. Expression of flowering-related genes in WT and flinc plants. Expression was measured by qPCR in rosettes of twenty days-old WT and flinc grown at 16 °C in long day conditions. Material was harvested at ZT6. The graph shows the average of three biological replicates, each composed of a pool of 10 plants. Bars indicate SEM of the replicates. B. Expression of genes with sequence similarity to FLINC in wild-type (WT) and flinc mutant plants (Mut), as measured by qPCR. The graph shows the average of three biological replicates, each composed of a pool of 10 two weeks-old plants growing at 21 °C in long day. Bars indicate SEM of the replicates. C. FLINC sequences are also found in other plant species. (PDF 561 kb
Additional file 4: of Arabidopsis thaliana ambient temperature responsive lncRNAs
Table S4. Pearson correlation between lncRNAs and protein coding genes. For the NATs expression value of the corresponding sense transcript while for the lincRNAs the expression value of their two direct flanking protein coding genes in our data set was used. NA, indicates absence of detectable expression in our RNA-seq. (XLSX 15 kb