12 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
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 (nβ=β3).</p
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. 2AβF 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. 2GβL 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. (nβ=β3). (<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. (nβ=β3).</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. (nβ=β3). (<b>B</b>) Electrophoretic mobility shift assay (EMSA) with increasing amounts (lanes 1β5) 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. (nβ=β3). (<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. (nβ=β3).</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 (nβ=β55) 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
<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. (nβ=β3). (<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. (nβ=β3).</p
Global methylation level.
<p>Ratio of the number of mapped Cs divided by the number of mapped C plus T for all classified Cs. Black: Cs that are called methylated, White: C that are not called methylated.</p
Comparison of mapping results for BiSS and selected other aligning programs<sup>1</sup>.
<p>Default parameters unless otherwise specified.</p>1<p>A3M β results reported by <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041528#pone.0041528-Lister1" target="_blank">[4]</a>.</p>2<p>BSMAPv1 parameters: -p 8 -s 12 -r 2 -w 100 -n 1 -v 5 -g 5, recommended by the authors, maximal 5 mismatches.</p>3<p>RMAP parameters: -m 5 βv, default parameters.</p
Examples for validation by individual bisulfite sequencing.
<p>The plots show the correlation between calculated and validated methylation levels (C/(C+T)) from regions selected for congruency (A) or disagreement (BβD) between BiSS and A3M. Each point represents one cytosine position. The x-axis corresponds to the methylation levels calculated from either BiSS (filled circles and black regression lines) or A3M (open circles and dotted regression lines); the y-axis shows the result of individual bisulfite sequencing. The legends show the Pearson correlation coefficients. (A) Methylated region according to both methods (M/M). (B) A region called methylated by BiSS but not by A3M (M/U); the rectangles indicate experimentally validated Cs congruent to BiSS (filled) and discrepant to A3M (open). (C) A region called methylated by BiSS but not by A3M due to insufficient sequencing coverage (M/X). (D) A region called methylated by A3M but not by BiSS (U/M), the rectangle symbols are the same as in (B).</p
Congruency between methylation calling by A3M and BiSS.
<p>M: methylated, U: unmethylated, X: not determined due to lack of sufficient sequencing coverage. Percentages refer to the total number of genomic cytosines.</p