Targeted inactivation of the two plastid <i>trnT</i> genes.

<p>(A) Physical map of the <i>trnT-UGU</i> containing region in the tobacco plastid genome (ptDNA; <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003076#pgen.1003076-Shinozaki1" target="_blank">[44]</a>). Genes above the line are transcribed from the left to the right, genes below the line are transcribed in the opposite direction. Selected restriction sites used for cloning or RFLP analysis are indicated. The hybridization probe and the expected sizes of detected DNA fragments are also shown. Introns are represented by open boxes. (B) Map of the transformed plastid genome in <i>ΔtrnT-UGU</i> transplastomic plants. The selectable marker gene <i>aadA</i> (grey box) is inserted into the <i>trnT-UGU</i> gene in the same transcriptional orientation. (C) Physical map of the <i>trnT-GGU</i> containing region in the tobacco ptDNA. (D) Map of the transformed plastid genome in Δ<i>trnT-GGU</i> plants. (E) RFLP analysis of Δ<i>trnT-UGU</i> transplastomic lines. Independently generated transplastomic lines are designated by Arabic numerals following the tRNA gene name. All transplastomic lines remain heteroplasmic and show both the 1.9 kb wild type-specific hybridization band and the 3.1 kb band diagnostic of the transformed plastid genome. Wt: wild type. (F) RFLP analysis of Δ<i>trnT-GGU</i> transplastomic plants. All lines are homoplasmic and show exclusively the 3.7 kb band diagnostic of the transgenic ptDNA. (G) Seed assays to confirm heteroplasmy of Δ<i>trnT-UGU</i> plants and homoplasmy of Δ<i>trnT-GGU</i> plants. Seeds were germinated in the absence or in the presence of spectinomycin. Δ<i>trnT-UGU</i> plants produce mostly antibiotic-sensitive seedlings and a few antibiotic-resistant seedlings, as expected for a heteroplasmic situation. Moreover, many of the resistant seedlings are variegated indicating their composition of tissues possessing and tissues lacking the transgenic plastid genome. In contrast, the Δ<i>trnT-GGU</i> lines produce homogeneous antibiotic-resistant progeny, confirming their homoplasmic status. (H) Analysis of tRNA-Thr(GGU) accumulation in the wild type, a heteroplasmic Δ<i>trnT-UGU</i> line and a homoplasmic Δ<i>trnT-GGU</i> line by northern blotting. Hybridization of electrophoretically separated RNA isolated from purified chloroplasts to a plastid <i>trnT-GGU</i> probe confirms complete lack of mature tRNA-Thr(GGU) in the Δ<i>trnT-GGU</i> homoplasmic knock-out line, whereas its accumulation is unaltered in the heteroplasmic Δ<i>trnT-UGU</i> line. Note accumulation of a ∼1.5 kb hybridizing RNA species in the Δ<i>trnT-GGU</i> line, which corresponds to the tRNA-Thr(GGU) disrupted with the <i>aadA</i> cassette. To control for RNA loading, part of the ethidium bromide-stained gel (containing the two largest 23S rRNA hidden break products) prior to blotting is also shown.