Targeted manipulation of maize genes in vivo using chimeric RNA/DNA oligonucleotides

Abstract

Site-specific heritable mutations in maize genes were engineered by introducing chimeric RNA/DNA oligonucleotides. Two independent targets within the endogenous maize acetohydroxyacid synthase gene sequence were modified in a site-specific fashion, thereby conferring resistance to either imidazolinone or sulfonylurea herbicides. Similarly, an engineered green fluorescence protein transgene was site-specifically modified in vivo. Expression of the introduced inactive green fluorescence protein was restored, and plants containing the modified transgene were regenerated. Progeny analysis indicated Mendelian transmission of the converted transgene. The efficiency of gene conversion mediated by chimeric oligonucleotides in maize was estimated as 10(-4), which is 1-3 orders of magnitude higher than frequencies reported for gene targeting by homologous recombination in plants. The heritable changes in maize genes engineered by this approach create opportunities for basic studies of plant gene function and agricultural trait manipulation and also provide a system for studying mismatch repair mechanisms in maize.

Figure 1

Nuclear localization of rhodamine-labeled chimeric ONDs in onion epidermal cells (A–C) and maize BMS cells (D–F) 1 hr after bombardment, illustrated by rhodamine signal (Top), cellular organization (Middle), and their superimposed images (Bottom).

Figure 2

Chimeric ONDs and target sequences of (A) AHAS Ser-621–Asn, (B) AHAS Pro-165–Ala, and (C) Ubi∷PAT/GFP fusion Ter-996–Tyr. DNA residues are indicated in uppercase, and the modified RNA residues are shown in lowercase. Nucleotides in bold differ between the target sequence and chimeric OND. ∗ indicates the nucleotide that should be introduced into the target sequence. The overscored sequence highlights the BfaI restriction site and the underlined sequence indicates the site after sequence modification.

Figure 3

AHAS621 conversion examined by restriction fragment length polymorphism and sequence analysis. (A) A map of the amplified AHAS621 target sequence from both wild-type and mutant alleles indicates the positions of PCR primers and the BfaI restriction site. (B) Polymorphism of wild-type and mutant alleles in target PCR fragments from positive control (P), negative control (N), and a representative event (E) before (U) and after BfaI restriction (R). (C) Sequence comparison of cloned AHAS621 alleles from the above samples. (D) Sequence comparison of cloned AHAS165 alleles from a positive control event (P), a negative control event (N), and two events with the predicted nucleotide conversion (E). Sequences were generated directly from PCR-amplified DNA from maize tissues. Because multiple AHAS genes exist in maize, as expected, both unconverted wild-type and converted mutant alleles are present in the events, as represented by the two overlapping peaks and the N nucleotide designation in the chromatograms.

Figure 4

GFP expression in T₀ callus (A), leaves (B), germinating embryo (C), and roots (D) after PAT/GFP conversion. (E) Chromatogram of the junction region of the targeted PAT/GFP showing sequence obtained directly from PCR amplification. (F) T₁ progeny segregation. In seedlings with GFP expression, strong green fluorescence was detected in the coleoptiles that lack chlorophyll. In contrast, coleoptiles in seedlings without GFP expression were semitransparent under the excitation and emission spectra used, and leaf chlorophyll autofluorescence could be seen through coleoptile tissue.