<i>TAD3-1</i> is methylated in Nok-1 and Est-1, preventing its expression.

<p>(A) DNA methylation of <i>TAD3-1</i> 5’-UTR analysed by digesting the indicated genomic DNA with <i>McrBC</i> followed by PCR amplification. Regions amplified correspond to <i>PCR#2</i> and <i>PCR#3</i> (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006551#pgen.1006551.g002" target="_blank">Fig 2A</a>) and are specific for <i>TAD3-1</i> (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006551#pgen.1006551.s004" target="_blank">S4 Fig</a>). (B) <i>TAD3-1</i> is silenced in Nok-1 and Est-1. Plants were grown on medium containing 0 (-) or 10 (+) μg/ml of 5-aza-2’deoxycytidine (<i>Aza</i>) for seven days. RNAs of plants were extracted and cDNAs were amplified using primers corresponding to PCR#3 (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006551#pgen.1006551.g002" target="_blank">Fig 2A</a>), specific for chromosome 5. <i>ATEF</i> amplifications served as controls. (C) <i>TAD3-1</i> qRT-PCR analyses using PCR#3 primers that are specific for chromosome 5 and plants described in (B).</p

Sequence length distribution of the small RNAs over the <i>TAD3-1</i> locus.

<p>sRNA reads were counted in three different genomic regions: the first one (Chr5: 8,446,240–8,447,463) comprises two transposons upstream of <i>TAD3-1</i>, the second includes the promoter of <i>TAD3-1</i> (Chr5: 8,447,463–8,447,954) and the last one corresponds to the <i>TAD3-1</i> gene (Chr5: 8,447,954–8,451,218). Transposable elements are depicted in orange and <i>TAD3-1</i> in grey. Counts are given in reads per million of mapped reads. The precise distribution of sRNA reads is described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006551#pgen.1006551.s011" target="_blank">S11 Fig</a>.</p

Expression profiles of <i>TAD3-1</i> alleles in Col-0 x Nok-1 hybrids determined by pyrosequencing.

<p>Expression profiles of <i>TAD3-1</i> alleles in Col-0 x Nok-1 hybrids determined by pyrosequencing.</p

Segregation analysis of the two <i>TAD3</i> loci in different epigenetic mutant backgrounds.

<p>Segregation analysis of the two <i>TAD3</i> loci in different epigenetic mutant backgrounds.</p

Methylation patterns of <i>TAD3-1</i> in incompatible plants carrying a <i>cmt3</i> mutation.

<p>(A) Methylation patterns of <i>TAD3-1</i> in incompatible plants carrying a <i>cmt3</i> mutation. Genomic DNAs (300 ng) were digested with <i>McrBC (+McrBC</i>) and then amplified using primers specific for the PCR fragments indicated (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006551#pgen.1006551.g002" target="_blank">Fig 2A</a> to localise the amplicons within the <i>TAD3-1</i> gene). Plants noted “<i>CN</i>” or “<i>CC</i>” are all in the <i>cmt3-11</i> background. “<i>C</i>” indicates that the Col-0 allele is fixed, “<i>N</i>” indicates that the Nok-1 allele is fixed. For the Nok-1 plants, only PCR#2 and #3 are specific for chromosome 5. (B) Methylation rates within the promoter regions in incompatible plants carrying a <i>cmt3-11</i> mutation. Data (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006551#pgen.1006551.s013" target="_blank">S13 Fig</a>) were obtained by amplifying from leaf genomic DNA the promoter region corresponding to PCR#7 (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006551#pgen.1006551.s007" target="_blank">S7A Fig</a>). The numbers indicate the plants analyzed, as shown in (A). In a <i>cmt3</i> background, the allele inherited from Nok-1 at chromosome 5 becomes specifically hypomethylated in the CHG context compared to the Nok-1 parent (plants #6 and #46). (C) Segregation of the Nok-1 and Col-0 alleles at chromosome 5 in the progeny (n = 128) of a <i>cmt3</i> mutant. The progeny of a plant fixed Col-0 at chromosome 1 and heterozygous Col-0/Nok-1 at chromosome 5 and sibling of the plants presented on (A) and (B) were genotyped.</p

The methylation of the <i>tad3-1 epiallele</i> is reversible.

<p>(A) Genomic DNAs (300 ng) from the F9 plants indicated were digested with <i>McrBC (+McrBC</i>) and then amplified using primers specific for the regions indicated (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006551#pgen.1006551.g002" target="_blank">Fig 2A</a> to localize the amplicons within <i>TAD3</i>). We verified the presence of the polymorphisms between Nok-1 and Col-0 (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006551#pgen.1006551.g002" target="_blank">Fig 2A</a>) by sequencing the PCR fragments obtained without <i>McrBC</i> treatment (-<i>McrBC</i>) for PCR#2, #3, #5 and #6, excluding a genetic recombination between Col-0 and Nok-1 in this region. All plants are from the progeny of an F8 revertant that was fixed Col-0 at chromosome 1 and heterozygous Col-0/Nok-1 at chromosome 5. <i>K1ColK5Nok</i> are F9 plants fixed Col-0 at chromosome 1 and Nok-1 at chromosome 5. <i>K1ColK5Col</i> are F9 plants fixed Col-0 at both chromosomes. For the Nok-1 plants, only PCR#2 and #3 are specific for chromosome 5. (B) The methylation rates within the promoter and the gene body of <i>TAD3</i> were determined in plants described in (A). Data were obtained by amplifying the regions indicated, namely ‘<i>Prom</i>.’ for the promoter and ‘<i>Gene’</i> for the gene body, after bisulfite conversion (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006551#pgen.1006551.s007" target="_blank">S7 Fig</a>). (C) Expression of <i>TAD3</i> analyzed by qRT-PCR in plants described in (A) using primers corresponding to PCR#3 (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006551#pgen.1006551.g002" target="_blank">Fig 2A</a>), specific for chromosome 5. (D) Segregation of the Nok-1 and Col-0 alleles at chromosome 5 in the progeny of the revertant. We genotyped the progeny of two F9 plants (#3 and #2), descending from the revertant, and fixed Col-0 at chromosome 1 and heterozygous Col-0/Nok-1 at chromosome 5. The control is an F9 plant fixed Col-0 at chromosome 1 and heterozygous Col-0/Nok-1 at chromosome 5, coming from a lineage independent of the revertant. The numbers of plants genotyped are indicated in parentheses.</p

Unmethylated <i>TAD3-1</i> alleles are converted in hybrids.

<p>(A) Methylation rates within the promoter of <i>TAD3-1</i> in four hybrids obtained by crossing Col-0 and Nok-1 (Plants #1 to #4). Data (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006551#pgen.1006551.s009" target="_blank">S9 Fig</a>) were obtained by amplifying from leaf genomic DNA the promoter region corresponding to PCR#9 (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006551#pgen.1006551.s008" target="_blank">S8 Fig</a>), differentiating the Col-0 or Nok-1 alleles with the TTT insertion. (B) Expression of <i>TAD3-1</i> determined by qRT-PCR in leaves of seven different hybrids (Plants #1 to #7) including the four hybrids shown in (A).</p

<i>TAD3</i> is the candidate gene for the incompatibility between Nok-1/Est-1 and Col-0.

<p>(A) The mapping interval localized on chromosome 5 in Col-0 contains four predicted genes. <i>TER2</i> encodes a non-coding RNA associated with the telomerase [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006551#pgen.1006551.ref024" target="_blank">24</a>]. <i>AT5G24655</i> and <i>AT5G24660</i> are homologous genes involved in sulfur response [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006551#pgen.1006551.ref053" target="_blank">53</a>]. <i>AT5G24670</i> encodes an enzyme involved in tRNA editing (<i>TAD3</i>) and GABI_141G12 was described [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006551#pgen.1006551.ref023" target="_blank">23</a>]. The T-DNA positions in individual mutants isolated are represented by triangles. We recovered homozygous mutants for lines depicted in blue, and heterozygous mutants for lines depicted in red. T-DNAs in both SALK_121147 and SAIL_363_B11 are inserted in the 5’-UTR of <i>TAD3</i>, disrupting <i>TER2</i>. UTRs of <i>TAD3</i> are represented by black boxes. (B) Siliques of a <i>tad3</i>/<i>TAD3</i> heterozygous T-DNA plant (GABI_141G12) are similar to siliques of a F8 RIL plant (4RV355) carrying Col-0 alleles at chromosome 1 and heterozygous Col-0/Nok-1 for the chromosome 5 locus. Red arrows indicate aborted seeds. (C) Number of defective seeds per silique in three different RILs (F8 generation) carrying Col-0 alleles at chromosome 1 and heterozygous Col-0/Nok-1 for the chromosome 5 locus. One to five siliques per plant were phenotyped, representing a total of 400 embryos for 4RV207, 227 embryos for 4RV355 and 565 embryos for 4RV415.</p