Figure 1. Sense Transgene-induced Silencing is Impaired in dcl2 Mutant Plants, but Enhanced in dcl4 Mutants.

(A) The diagram shows the coding region of the silenced GUS sense transgene in line L1, plus the coordinates and positions of the probes used throughout this work for detecting GUS mRNA and siRNA. Antisense polarity probes will be indicated by an asterisk. The position and length in base pairs (bp) of the stem, deletion, and loop regions of the ΔGUS-SUG hairpin construct (Figures 2–3) are also shown. (B) Accumulation of GUS mRNA, GUS siRNAs, and siR255 in wild type (wt) and mutant plants carrying the L1 GUS locus was determined using RNA gel blot analysis. ³²P-labelled RNA probes were used for hybridization except that a DNA oligonucleotide probe was used for the siR255 blots and a full length GUS cDNA probe was used for the high molecular weight (HMW) RNA blot of lanes 18–21. Otherwise, probe 3*, which has antisense polarity, was used to detect GUS mRNA. Probes for GUS siRNA all had sense polarity and, therefore, detected the antisense strand. The positions of 21- and 22-nt RNA size markers (Ambion Decade™ Marker system) are indicated on the right of the low molecular weight (LMW) RNA blots. Grouped lanes are all from the same gel, blot, and exposure. A longer exposure of the GUS mRNA band in lanes 3–6 and 9–12 is shown directly below the rRNA band. LMW RNA blots were successively stripped and hybridized with the indicated probes. Genotypes and the zygosity of the L1 locus are indicated at the top of the lanes. The designation “mix” indicates that a segregating F2 population that was a mix (theoretically about 2:1) of L1 GUS hemizygotes and homozygotes was used for RNA isolation; +/− and +/+ indicate hemizygous and homozygous for L1 GUS, respectively. Ethidium bromide (EtBr) stained rRNA and the major RNA species in LMW RNA are shown as loading controls. (C) Relative levels of transcription of the GUS transgene in plants wild type for DCL2 (lane 1) and in dcl2-1 mutant plants homozygous (lane 2) or hemizygous (lane 3) for L1 GUS were determined in isolated nuclei. Nuclear transcripts from these plants were labeled with ³²P by run-off transcription and then hybridized to slot blots loaded with plasmid DNA containing GUS, actin, and pUC19 empty vector sequence.