Figure 1.
Graphic map of TDNA containing the 35S::AtMYB90 construct.
The main map shown represents the TDNA construct (containing a 35S::AtMYB90 transgene) used to transform N. tabacum. Flagged vertical lines indicate TDNA/plant DNA junctions cloned from the Myb27 line, with plant segments shown above the main map (NtL, tobacco sequence at the TR border; NtR-B1 and NtR-B2, tobacco sequences at the TL border). Segments of the original plasmid TDNA that are absent at the Myb27 locus are shown as a faded graphic. Labels include: TR (right TDNA border); PClSV-Pro (Peanut Chlorotic Streak Virus promoter); BAR (basta resistance coding region); 35S-Ter (CaMV 35S transcription termination signal); 35S-Pro (CaMV 35S promoter with duplicated enhancer region [Enh-1, Enh-2]); AtMYB90 (AtMYB90 coding region [the Myb R2–R3 repeats are indicated]); g7-Ter (transcription termination signal from the gene-7 of octopine TDNA); TL (left TDNA border). The locations of primers used for qRTPCR are indicated by blue arrowheads and the segment of the AtMYB90 coding region used in a hairpin expression vector (pKOihpMyb) is indicated by a red box. Dashed lines show the boundaries of PCR amplimers used to confirm the structure of the T-DNA insert (“+” indicates predicted PCR product observed, “−” indicates no product of the predicted size; primer sequences are listed in Additional files, Table S1). The PCR templates used were total plant leaf DNA from the Myb27 line and from untransformed tobacco, Nt (SR1). The BAR and Myb probes used for blot hybridization are indicated by green boxes above the map and restriction enzyme cleavage sites used for Southern blot analysis, including resulting fragment sizes, are shown below the main map.
Figure 2.
Plant pigmentation phenotypes.
Phenotypes of 35S::AtMyb90 transgenic lines Myb27 and Myb237 that are hemizygous (27Hmi, 237Hmi) or homozygous (27Hmo, 237Hmo) for the transgene. Also shown are 27Hmi and 237Hmo plants displaying systemic silencing (27Hmi-SysSil, 237Hmo-SysSil) induced by pKOhpMyb-Agrobacterium infiltration (see hairpin stem, Fig. 1) of older leaves (infiltration sites indicated by ‘Inf’ arrows). A similar pattern of silencing is seen in the offspring from a 27Hmo×237Hmo cross (the plant shown is hemizygous for both the Myb27 and Myb237 transgene loci [27Hmi–237Hmi]).
Figure 3.
35S::AtMyb90 transcription in Myb27 and Myb237 lines.
A. Northern blot autoradiogram of total RNA from 35S::AtMyb90 transgenic plants. The AtMYB90 coding region was used as probe (see Fig. 1) against equal RNA loadings (confirmed by 28S rRNA fluorescence, bottom row) from 27Hmi and 237Hmo leaf material (leaves used were similar to the 5th True Leaf pictured in 3C). The 27Hmo blot shows two bands of approximately 1050 nt and 600 nt. B. Comparison of mRNA levels (qRTPCR) using leaf tissue from 27Hmo (partially-green), 27Hmi (purple), 237Hmo (purple) and 237Hmi (purple) plants. The individual samples were normalized to total RNA from a pooled leaf collection of 27Hmi leaves (5th true leaf through the third from the top were combined). Error bars represent standard error for 2–3 biological replicates. C. AtMYB90 mRNA quantification (qRTPCR) from 27Hmo and 27Hmi matched leaves of varying ages (shown at the left). In order to match leaf development between plants sampling was limited to the oldest (the 3rd, 4th, 5th and 6th true leaves) and youngest (top leaf, Top-1, Top-2, Top-3) leaves. Error bars represent standard error for 2–3 biological replicates. The ratio of homozygous/hemizygous values for each pair of matched leaves is indicated at the right. Individual samples were normalized to the same pooled 27Hmi leaf RNA used for part B.
Figure 4.
Southern Blot Analysis of Myb27 TDNA Insert.
27Hmo DNA digested with EcoRI plus NcoI, or EcoRI plus NcoI plus NdeI (see Fig. 1) were electrophoretically separated, blotted and probed with either BAR or AtMyb90 coding regions. The single large EcoRI+NcoI bands (BAR, ∼4.5 kb and AtMyb90, ∼5 kb) that were shortened by NdeI digestion to a smaller band for each probe (BAR, 925 bp and Myb, 2001 bp).
Figure 5.
Graphic representation of GenBank matches to cloned tobacco flanking sequences in Myb27.
The top horizontal bar represents the color scale for NCBI/BLAST alignment scores represented by the alignment bars shown below. The NtL, NtR-B1 and NtR-B2 sequencers were combined into a single string prior to BLAST alignment (the blue triangle indicates the T-DNA insertion site). The combined tobacco junction sequences were BLAST aligned [discontiguous megablast] against tobacco genomic sequences [Genomic Survey Sequence - tobacco, taxid:4097], or non-human, non-mouse expressed sequence tags [est_others]. All of the indicated alignments represent N. tabacum GenBank entries.
Table 1.
Plant mature miRNA reads in small RNA sequence data sets.
Figure 6.
Small RNA sequence alignments mapped to the Myb27 TDNA and flanking tobacco sequence.
Short RNA sequence reads (SOLiD platform) from plant samples displaying a silenced (A) or purple (B) phenotype were BLAST aligned to the Myb27 Nt-TDNA-Nt sequence. A structural map of the alignment target sequence is shown at the top (see Fig. 1 for labels). The TDNA contains two areas of duplicated sequence: Dup-X (X1 = 35S-PolyA: 841–1000; X2 = 3280–3439) and Dup-Y (duplicated 35S promoter enhancer region: Y1 = 1264–1590; Y2 = 1592–1918). It is impossible to determine which repeat represents the actual origin of any of the small number of matching siRNAs and the point of homology has been marked at both copies. For each RNA sample the total number of unfiltered sequence reads, and the number of TDNA alignments, is indicated. Yellow vertical lines indicate the segment of AtMYB90 displayed in Fig. 9 (2367–2532).
Figure 7.
Normalized siRNA profiles summed across the Nt-TDNA-Nt from Myb27.
The number of aligned sequence reads from each sample (Fig. 6) were divided by the total number of matching reads for the corresponding sample (plus [above 0 line] and minus [below 0 line] strands were treated separately) and the resulting fractions summed across the Nt-TDNA-Nt sequence (from 0 at position 1 to 1.0 at position 4329). Profiles from silenced (A) or non-silenced (B) phenotypes are aligned to a structural map of the Nt-TDNA-Nt target sequence (diagrammed at the top, see Fig. 1 for labels). See the legend for Fig. 6 regarding how regions of duplicated sequence were addressed. The boundaries of the AtMYB90 transcript are indicated by yellow vertical lines.
Figure 8.
SmRNA size profiles for siRNAs homologous to the AtMYB90 transcribed region.
Each bar represents the number of sense (top) or antisense (bottom) siRNAs of the indicated size divided by the total number of siRNA sequence reads aligning to the AtMYB90 transcribed region (2010–3009 bp of Fig. 1, sense and antisense data were independently normalized). Sequence from replicate samples of the 27Hmo treatment (O1 and LL6) were used to generate the 27Hmo values shown (standard error indicated, n = 2).
Figure 9.
siRNA sequence alignments to Nt-TDNA-Nt segment 2367–2532.
siRNA sequence reads from 27Hmo-O1 and 27Hmo-LL6 (LL6 text is faded and offset to the right ∼20 nt) siRNA sequence reads directly aligned to the 2367 to 2532 segment of the Myb27 Nt-TDNA-Nt. The number of sequence reads for each alignment is shown in parenthesis next to the corresponding smRNA sequence. This sequence segment was chosen as it contains potential smRNA recognition sites identified in Arabidopsis. Homology to ath-miR828 (blue) and ath-TAS4-siR81 (red) are indicated. ath-TAS4-siRNA81 associated cleavage of AtMYB90 and miRNA828 directed cleavage of the closely related AtMYB113 mRNA (arrows) have been detected in Arabidopsis [43].