Mutated rdm1 sequences are transcribed and differentially complement defects in Pol V transcription in an rdm1 mutant.
Top: Confirmation of rdm1 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 PagI (recognition site created by rdm1-4 mutation). After Pag1 digestion, the transcript from the rdm1-4 allele yields a shorter fragment around 250 bp (rdm1-4, lanes 4-9). The transcripts from a wild-type RDM1 gene (lanes 1–3) or rdm1-4 mutants complemented with the wild-type RDM1 sequence (lane 5) or various mutated rdm1 sequences (lanes 5–9) that lack the rdm1-4 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 rdm1-d1d2, rdm1-p1p2 and rdm1-M50A transgenes. Middle: Pol V-dependent IGN5 transcripts are detectable by RT-PCR in wild-type Col-0 and T+S plants (lanes 1 and 2) and are reduced in nrpe1 and rdm1 mutants (lanes 3 and 4). IGN5 transcripts can be detected after complementing the rdm1 mutant with the wild-type RDM1 construct (lanes 5 and 6) and with rdm1-M50A (lane 9). Low levels of IGN5 transcript were observed with the rdm1-d1d2 construct in an rdm1 background(lane 7) but the rdm1-p1p2 construct did not support transcription of IGN5 above the level seen in the rdm1 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).