Accumulation of 21–22 nt siRNA854 negatively regulates transgene transcripts with the <i>UBP1b</i> 3′ UTR.

<p>(A) Plants homozygous for a transgene constitutively expressing the GUS reporter protein fused to the <i>UBP1b</i> 3′ UTR (35S:GUS-<i>UBP1b</i> 3′UTR) were transformed with an artificial microRNA (amiRNA) with the siRNA854 sequence (35S:amiRNA-854), or a second control sequence that does not target <i>UBP1b</i> (35S-amiRNA-control). GUS activity was monitored using a quantitative assay (left) and inflorescence staining (right). Plants expressing the siRNA854 sequence as an artificial microRNA show significantly reduced GUS levels. (B) Col and <i>ddm1</i> plants homozygous for a constitutively expressed GUS transgene (35S:GUS) or the same reporter transgene with the <i>UBP1b</i> 3′ UTR from part A. As in A, GUS activity was monitored using a quantitative assay (left) and plant staining (right). Wt Col plants show no differential regulation between the two transgene variations, while in the <i>ddm1</i> mutant background the GUS activity of the <i>UBP1b</i> 3′ UTR transgene is significantly less than the control 35S:GUS transgene. (C) A 35S:GUS-<i>UBP1b</i> 3′UTR transgene in the wt Col background was crossed to a <i>ddm1</i> homozygote, and the GUS activity was measured in the F1 plant. GUS activity of the same hemizygous transgene in the wt Col background is shown as a control. (D) RT-PCR of the same transgenic individuals from part B. The GUS activity differences observed in part B are not reflected in transgene transcript levels, demonstrating that this regulation is not due to post-transcriptional mRNA degradation. In A, B and C, the box plot whiskers represent the minimum and maximum of the dataset, the top and bottom of the box are the 75^th and 25^th percentile (respectively), the middle line is the median, and + is the mean. The number of individuals assayed (n) is shown in or near the box.