Mutated <i>rdm1</i> sequences are transcribed and differentially complement defects in Pol V transcription in an <i>rdm1</i> mutant.

<p>Top: Confirmation of <i>rdm1</i> transgene expression using RT-PCR. RNA from the corresponding genotypes was reverse transcribed into cDNA and amplified by PCR using gene-specific primers (Methods). The PCR product was then digested with <i>Pag</i>I (recognition site created by <i>rdm1-4</i> mutation). After <i>Pag</i>1 digestion, the transcript from the <i>rdm1-4</i> allele yields a shorter fragment around 250 bp (<i>rdm1-4</i>, lanes 4-9). The transcripts from a wild-type <i>RDM1</i> gene (lanes 1–3) or <i>rdm1-4</i> mutants complemented with the wild-type <i>RDM1</i> sequence (lane 5) or various mutated <i>rdm1</i> sequences (lanes 5–9) that lack the <i>rdm1-4</i> mutation remain undigested and produce a product around 500 bp (WT/TG). The larger PCR products were sequenced to confirm the expected mutations in the <i>rdm1-d1d2</i>, <i>rdm1</i>-<i>p1p2</i> and <i>rdm1</i>-<i>M50A</i> transgenes. Middle: Pol V-dependent <i>IGN5</i> transcripts are detectable by RT-PCR in wild-type Col-0 and T+S plants (lanes 1 and 2) and are reduced in <i>nrpe1</i> and <i>rdm1</i> mutants (lanes 3 and 4). <i>IGN5</i> transcripts can be detected after complementing the <i>rdm1</i> mutant with the wild-type <i>RDM1</i> construct (lanes 5 and 6) and with <i>rdm1</i>-<i>M50A</i> (lane 9). Low levels of <i>IGN5</i> transcript were observed with the <i>rdm1</i>-<i>d1d2</i> construct in an <i>rdm1</i> background(lane 7) but the <i>rdm1</i>-<i>p1p2</i> construct did not support transcription of <i>IGN5</i> above the level seen in the <i>rdm1</i> mutant (lane 8). RT-PCR analysis using actin primers is shown as loading control. Contamination by genomic was checked by omitting the RT step (No RT). Genomic DNA from a wild-type non-transgenic plant (Col) was used as positive control for the PCR reaction (No RT, lane 10).