TUNEL analysis of genomic fragmentation in WT and RNAi/<i>DUT1</i> lines.

<p>WT (Ws) and RNAi/<i>DUT1</i> leaf tissues were subjected to TUNEL labelling and observed by fluorescence microscopy. Numbers in parenthesis indicate the number of nuclei that were observed. Nuclear DNA was stained with DAPI.</p

Frequency of homologous recombination events in relation to the 5FU sensitivity of RNAi/<i>DUT1</i> lines and control plants.

<p>A – Schema showing the luciferase <i>in planta</i> substrate for recombination that allows to measure HR. B - We compared the 5FU sensitivity (50 µM) of three independent transformants and then we gathered the empty vector control together (58F/RNAi/0-luc), the 5FU sensitive RNAi/<i>DUT1</i> lines together (58F/RNAi/<i>DUT1</i>-luc-5FUsensitive) and the 5FU resistant RNAi/<i>DUT1</i> line alone (58F/RNAi/<i>DUT1</i>-luc9), with the aim to represent the trend lines in the homologous recombination assay. C – Table showing the percentage of seedling we counted with different number of luciferase foci. D – Three independent control transformants were used (58F/0-luc1, -luc2 and -luc3) and four independent RNAi/<i>DUT1</i> transformants, 58F/RNAi/<i>DUT1</i>-luc2, -luc4, -luc5 and -luc9 to assess somatic recombination in line 58F.</p

<i>DUT1</i> expression levels and dUTPase activities in RNAi/<i>DUT1</i> lines compared to control plants.

<p>A - <i>DUT1</i> expression level was analyzed by quantitative real time RT-PCR in young leaves of RNAi/<i>DUT1</i>-4 and -5 compared with WT plants (Ws ecotype). The relative mRNA normalized to <i>ACTIN</i> is reported. Error bars correspond to standard deviations. B - Level of dUTPase activity in RNAi/<i>DUT1-</i>4 and -5 plant lines compared to control plant (empty vector, RNAi/0). Activity was determined by measuring the dUMP produced by UPLC, after supplementation of plant extract with dUTP at 30°C for increasing lengths of time (three points in two independent experiments). Error bars correspond to standard deviations.</p

Expression level of <i>RAD51</i> and <i>PARP2</i> in RNAi/<i>DUT1</i> lines compared to control plants (WT, irradiated or not).

<p><i>RAD51</i> and <i>PARP2</i> expression in young leaves of RNAi/<i>DUT1</i>-4 and -5 was compared to WT plants (Ws), irradiated or not (Gamma rays at a 10 Krad dose). The relative mRNA normalized to <i>ACTIN</i> is reported. Error bars correspond to standard deviations.</p

RNAi/<i>DUT1</i>-4 siliques in a WT versus <i>rad51</i>^−/− background.

<p>Siliques were found to have the same “unfolded” floral phenotype appearance in the RNA/<i>DUT1</i>-4 <i>rad51</i>^−/− mutant plants as in the WT plants, while also being shorter due to the sterility of the <i>rad51</i>^−/− mutation.</p

<i>Arabidopsis DUT1</i> can complement a <i>dut-1</i> mutation in <i>E. coli</i> and a <i>dut1-1</i> mutation in yeast.

<p>A – <i>E. coli</i> WT and <i>dut-1</i> strains transformed with an empty vector or a vector containing At<i>DUT1</i> were tested for their sensitivity to uracil (U) or 5-fluoro-uracil (5FU). B – <i>E. coli</i> WT, <i>recA</i>(Ts) or <i>recBC</i>(Ts) and <i>dut-1</i> or <i>dut-I recA</i>(Ts) and <i>dut-1 reBC</i>(Ts) strains were transformed with an empty vector or a vector containing At<i>DUT1</i> and grown at permissive (20°C) or non-permissive (37°C) temperature. C -Yeast WT and <i>dut1-1</i> strains transformed with an empty vector or a vector containing At<i>DUT1</i> were tested for their sensitivity to 5FU. Rapidly growing cultures were serially diluted 10-fold at each step and 15 µl of each were spotted in rows. The left most column is not diluted; the right most column is a 10⁻⁵ dilution.</p

Effects of ethanol inducible expression on the viability of seedlings transformed by different RNAi constructs.

<p>Plants carrying the p35S:AlcR construct and a Nos terminator (PSRN) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018658#pone.0018658-Deveaux1" target="_blank">[19]</a> were transformed with RNAi constructs under the pAlcA promoter in order to inactivate either <i>DMC1</i> (a meiotic gene), or <i>EMB506</i> (a gene which is embryo-lethal when mutated) or <i>DUT1</i> (two independent lines) expression. All the plants are of the same Ws ecotype as the WT or PSRN control seedlings.</p