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Supporting figure legends
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Figure S1: Outline of the mutagenesis approach
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The parental lines (P) homozygous for the TARGET (Kchr1-1) or the SILENCER (H)
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transgene were crossed with each other (Fischer et al., 2008). Obtained F1 hemizygous
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for both transgenes (K/-;H/-) were grown on soil and allowed to set seeds by self-
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pollination. To identify lines homozygous for both transgenes, the resulting F2 progeny
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was grown on soil again and again allowed to set seeds by self-pollination. Resulting F3
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seeds were harvested from individual F2 plants and were germinated on medium
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containing 20 mg/l hygromycin. If the parental F2 plant had been homozygous for the
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SILENCER transgene, 100% of the F3 derived from this F2 were HygR. The F3 seedlings
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from such batches were then subjected to a GUS activity test, as, if the parental F2 plant
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had been homozygous for the TARGET transgene, 100% of the F3 should be GUS+. F3
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plants from such batches showing 100% HygR GUS+, and thus containing only
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individuals homozygous for both transgenes (K/K;H/H), were allowed to set seeds by
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self-pollination and resulting F4 seeds were harvested in bulk. Bulks of F4 seeds were
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treated with mutagen ethyl methanesulfonate (EMS) and then allowed to germinate on
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soil to give rise to mutagenized M1 plants. M1 plants were allowed to set seeds by self-
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pollination and resulting M2 seeds were harvested in bulk. M2 (equal to F5) are the first
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generation in which EMS-induced mutations can become homozygous and thus, if
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recessive, result in selectable phenotypes. Thus, M2 progeny was tested for resistance on
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medium containing 200 mg/l kanamycin or 200 mg/l kanamycin and 20 mg/l
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hygromycin and obtained KanR or KanR HygR mutant candidates were counterchecked
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again in the M3 (F6) next generation obtained by self-pollination.
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Figure S2: Rough mapping of the position of mutation nrd3-1 using a Col-0 x Ler M3F2
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population
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The frequencies of homozygosity for Col-0 alleles at 46 Col-0 / Ler polymorphic SNP
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markers in the A. thaliana genome are given as tested by Illumina GoldenGate assays.
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Results for 34 HygR KanR nrd3-1 M3F2 plants (later found to include 2 “false positive”
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ones; green columns) are compared to results for 66 HygR GUS+ control C3F2 plants
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from a cross of non-mutagenized Col-0 (K/K;H/H) to Ler (grey columns) that were
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tested for the presence of SILENCER and TARGET transgenes. Elevated frequencies of
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homozygosity for the Col-0-version of markers in HygR KanR nrd3-1 M3F2 identify the
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putative position of the mutation causative for the release of RdTGS of the TARGET
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ProNOS-NPTII reporter. Black diamonds: positions of centromeres, red dot: position of
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the DRM2 gene locus;
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Figure S3: TARGET ProNOS methylation patterns in K/K;H/H wild-type control, M3
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nrd3-1 and M3 nrd3-2 plants
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Methylation levels at individual cytosines in CG context (black columns), CHG context
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(blue columns) and CHH context (red columns). The positions of recognition sites for
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restriction enzymes Psp1406I, NheI, Alw26I and NcoI are indicated, a black arrowhead
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marks the ProNOS transcription start site. Numbers of clones sequenced per target and
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genotype were 19 for K/K;H/H wild-type control, 17 for M3 nrd3-1 and 19 for M3 nrd3-
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2 plants.
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Figure S4: TARGET ProNOS methylation patterns in F1, F2, F3 and F4 plants in presence
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of the SILENCER
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Methylation levels at individual cytosines in CG context (black columns), CHG context
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(blue columns) and CHH context (red columns). The positions of recognition sites for
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restriction enzymes Psp1406I, NheI, Alw26I and NcoI are indicated, a black arrowhead
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marks the ProNOS transcription start site. Numbers of clones sequenced per target and
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genotype were seven for F1 K/-;H/-, ten for F2 K/K;H/-, twelve for F3 K/K;H/- and ten
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for F4 K/K;H/- plants.
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Figure S5: TARGET ProNOS methylation patterns in F2, F3 and F4 plants after
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segregation of the SILENCER
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Methylation levels at individual cytosines in CG context (black columns), CHG context
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(blue columns) and CHH context (red columns). The positions of recognition sites for
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restriction enzymes Psp1406I, NheI, Alw26I and NcoI are indicated, a black arrowhead
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marks the ProNOS transcription start site. Numbers of clones sequenced per target and
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genotype were seven for F1 K/-, ten for F2 K/K*, 21 for F3 K/K* and 22 for F4 K/K*
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plants.
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Figure S6: Mutation suvh4 does not affect TARGET ProNOS RNA-directed
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transcriptional gene silencing and DNA methylation maintenance.
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(a) Plants homozygous for the mutant suvh4R302 allele were separately crossed to K/K
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and H/H plants homozygous for TARGET or SILENCER, respectively. The resulting F1
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plants were crossed with each other to obtain progeny segregating for all three loci.
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Among resulting F2 progeny, plants homozygous for either the SUVH4 wild-type or the
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suvh4R302 mutant allele and containing the TARGET in absence of the SILENCER or
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TARGET and SILENCER simultaneously (in either hemi- or homozygous state) were
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identified by PCR and used for NPTII determination. A F2 plant from this set
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homozygous for the suvh4R302 mutant allele, hemizygous for the SILENCER and
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containing the TARGET was allowed to self-pollinate and the resulting F3 were
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genotyped by PCR to identify individual plants hemizygous for the SILENCER and
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containing the TARGET. These were again allowed to set seeds by self-pollination, and
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among the resulting F4 progeny plants containing the TARGET in absence of the
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SILENCER or TARGET and SILENCER simultaneously were identified by PCR and
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used for TARGET ProNOS DNA methylation tests. (b) Quantification of NPTII protein
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by ELISA in F2 plants: NPTII protein amounts in relation to total soluble protein were
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measured by ELISA in extracts from leaves of 8-week-old plants. Results are displayed
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relative to the mean value for un-silenced expression in K/K plants (set to 1). (c)
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TARGET ProNOS DNA methylation in F4 segregating for the SILENCER. Cytosine
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methylation was determined by quantitative PCR after cleavage of genomic DNA from
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mature plants with methylation-sensitive restriction enzymes Psp1406I (grey –
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symmetric CG context), NheI (blue – CHG and CHH context), Alw26I (orange –
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asymmetric CHH context) and NcoI (yellow – asymmetric CHH context, control
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outside of the methylated region, see legend of Figure 1). N in (b) and (c) indicates the
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number of individual plants tested per genotype.
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Table S1: Candidate genes for nrd3-2
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Table S2: Primers for methylation-sensitive restriction cleavage-quantitative PCR and
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miRNA hybridisation
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Table S3: Primers for bisulfite sequencing
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Table S4: Illumina GoldenGate® markers for Col-0 / Ler polymorphisms
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Table S5: Primers for PCR-based detection of Col-0 / Ler polymorphisms
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Table S6: Primers for chromatin immunoprecipitation-quantitative PCR
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Methods S1: Supporting experimental procedures
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