Differentially expressed transposons between wild-type and <i>fpa-7</i>.

(A) Differential expression of the transposable element gene (At5g10670) in fpa-7. (B) Read-through contiguous RNAs were validated by RT-PCR (red dashed line). Three biological replicates (1, 2 and 3) were used for each genotype: wild-type (WT) and fpa-7. UBIQUITIN LIGASE 21 (UBC21) was used as a control. RT-PCR products were separated on agarose gels and stained with ethidium bromide. (C) Transcripts are either cleaved and polyadenylated in the annotated 3′UTR or at the intergenic sites, as determined by sequencing the cloned RT-PCR products. Red rectangles represent the 3′UTR specific to the read-through transcript and red lines represent 3′UTR introns. (D) Differential expression of the transposable element gene (At5g35935) in fpa-7. Recent re-annotation of At5g35935 [12], [13], [28], [29] defines two transcription units within it: the recently arisen pseudogene psORF and the transposon At5TE50260. DRS data reveal that silencing of psORF is lost in fpa-7. (E) RT-qPCR analysis of psORF in fpa-7 and fpa-8 mutant alleles. Silencing of psORF (p2 and p2b) is lost in fpa-7 but not in fpa-8. Data are the means ± SEM obtained for three independent PCR amplifications of three biological replicates. The y-axis shows the fold change relative to WT (WT set to 1) after normalisation to UBC21 gene expression. Location of the RT-qPCR amplicon is displayed on the left panel. *, P<0.05; Student's t-test. Normalised reads mapping to the different loci are presented for WT and fpa. Genes are orientated 5′–3′; exons are denoted by rectangles, UTRs by adjoining narrower rectangles and introns by lines. Images of normalised read alignments were made using the Integrated Genome Browser [55] and correspond to combined reads from the three sequenced biological replicates for each genotype.