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, 125 (4), 1585-90

Expression of Bar in the Plastid Genome Confers Herbicide Resistance

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Expression of Bar in the Plastid Genome Confers Herbicide Resistance

K A Lutz et al. Plant Physiol.

Abstract

Phosphinothricin (PPT) is the active component of a family of environmentally safe, nonselective herbicides. Resistance to PPT in transgenic crops has been reported by nuclear expression of a bar transgene encoding phosphinothricin acetyltransferase, a detoxifying enzyme. We report here expression of a bacterial bar gene (b-bar1) in tobacco (Nicotiana tabacum cv Petit Havana) plastids that confers field-level tolerance to Liberty, an herbicide containing PPT. We also describe a second bacterial bar gene (b-bar2) and a codon-optimized synthetic bar (s-bar) gene with significantly elevated levels of expression in plastids (>7% of total soluble cellular protein). Although these genes are expressed at a high level, direct selection thus far did not yield transplastomic clones, indicating that subcellular localization rather than the absolute amount of the enzyme is critical for direct selection of transgenic clones. The codon-modified s-bar gene is poorly expressed in Escherichia coli, a common enteric bacterium, due to differences in codon use. We propose to use codon usage differences as a precautionary measure to prevent expression of marker genes in the unlikely event of horizontal gene transfer from plastids to bacteria. Localization of the bar gene in the plastid genome is an attractive alternative to incorporation in the nuclear genome since there is no transmission of plastid-encoded genes via pollen.

Figures

Figure 1
Figure 1
Introduction of the b-bar1 gene into the tobacco plastid genome. A, Map of the plastid targeting region in plasmid pJEK6 and of the cognate regions in the wild-type (wt) and transplastomic (T) plants. Map positions are shown for: the b-bar1 gene; aadA, the selectable spectinomycin resistance gene; and 16SrDNA and rps12/7, plastid genes (Shinozaki et al., 1986). Arrows indicate direction of transcription. Map position of the probe (2.5 kb) is marked by a heavy line; the wild-type (2.9-kb) and transgenic (3.3-kb, 1.9-kb) fragments generated by SmaI and BglII digestion are marked by thin lines. B, DNA gel blot confirms integration of b-bar1 into the tobacco plastid genome. Data are shown for transplastomic lines Nt-pJEK6-2A through E, Nt-pJEK6-5A through E and Nt-pJEK6-13A and B, and the wild-type parental line. SmaI-BglII digested total cellular DNA was probed with the 2.5-kb ApaI-BamHI plastid targeting sequence (A).
Figure 2
Figure 2
PAT assay confirms b-bar1 expression in tobacco plastids. PAT activity was determined by conversion of PPT into acetyl-PPT using radiolabeled 14C-acetyl-CoA. Data are shown for transplastomic lines Nt-pJEK6-2D, Nt-pJEK6-5A and Nt-pJEK6-13B, nuclear transformant Nt-pDM307-10 and wild type (wt). The unmarked spot is an acetyl-CoA degradation product.
Figure 3
Figure 3
Transplastomic tobacco plants are herbicide resistant. A, Wild-type and pJEK6-transformed plants 2 weeks after Liberty treatment (5 mL, 2% [v/v] solution). B, Seeds from reciprocal crosses with Nt-pJEK6-5A plants germinated on 0, 10, and 50 mg L−1 PPT. Wt × pJEK6-5A, progeny from cross with transplastomic plant used as pollen parent; pJEK6-5A × Wt, progeny from cross with transplastomic plant used as maternal parent.
Figure 4
Figure 4
PAT accumulation in tobacco leaves from plastid b-bar1 (pJEK6-5A), b-bar2 (pKO18-3), and s-bar (pKO3-24) genes. Proteins were separated by SDS-PAGE and stained with Coomassie Blue R250. As control, a sample from non-transformed (Wt) plants was run. Position of PAT, NPTII, and the large (LSU) and small (SSU) Rubisco subunits is marked by arrows. Larger size of PAT in the Nt-pKO3-24 plant is due to a C-terminal extension. Reference for protein levels is NPTII (7% [w/v] of total soluble cellular protein) in the Nt-pHK30 extract (Kuroda and Maliga, 2001).
Figure 5
Figure 5
PAT activity in E. coli carrying s-bar (pKO3), b-bar2 (pKO18), and in non-transformed DH5 α bacteria. Note dilution series. Unincorporated 14C-acetyl-CoA and labeled acetyl-PPT are indicated with arrows.

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