A tool for functional plant genomics: chimeric RNA/DNA oligonucleotides cause in vivo gene-specific mutations

Abstract

Self-complementary chimeric oligonucleotides (COs) composed of DNA and modified RNA residues were evaluated as a means to (i) create stable, site-specific base substitutions in a nuclear gene and (ii) introduce a frameshift in a nuclear transgene in plant cells. To demonstrate the creation of allele-specific mutations in a member of a gene family, COs were designed to target the codon for Pro-196 of SuRA, a tobacco acetolactate synthase (ALS) gene. An amino acid substitution at Pro-196 of ALS confers a herbicide-resistance phenotype that can be used as a selectable marker in plant cells. COs were designed to contain a 25-nt homology domain comprised of a five-deoxyribonucleotide region (harboring a single base mismatch to the native ALS sequence) flanked by regions each composed of 10 ribonucleotides. After recovery of herbicide-resistant tobacco cells on selective medium, DNA sequence analyses identified base conversions in the ALS gene at the codon for Pro-196. To demonstrate a site-specific insertion of a single base into a targeted gene, COs were used to restore expression of an inactive green fluorescent protein transgene that had been designed to contain a single base deletion. Recovery of fluorescent cells confirmed the deletion correction. Our results demonstrate the application of a technology to modify individual genetic loci by catalyzing either a base substitution or a base addition to specific nuclear genes; this approach should have great utility in the area of plant functional genomics.

Figure 1

Target sequence of ALS SuRA and SuRB alleles (a) and COs ALS-1 and ALS-2 (b). The single nucleotide difference between the ALS SuRA and SuRB alleles in the target region is underlined. Lowercase letters represent 2′-O-methyl-RNA residues. Bold letters represent the codon for Pro-196. DNA residues represented by uppercase letters.

Figure 2

Confocal photomicrograph demonstrating nuclear localization of rhodamine-tagged COs in tobacco Nt-1 cell protoplasts.

Figure 3

Generation and selection of Glean-resistant Nt-1 cells grown on selective medium. Three-day-old plate cultures of Nt-1 cell suspension were used in ALS CO biolistic delivery experiments. Bombarded cell cultures were maintained on 15 ppb chlorsulfuron; putatively converted Nt-1 cell masses were transferred to medium containing 50 ppb chlorsulfon.

Figure 4

Automated nucleotide sequence analyses of gene modification events. Electropherograms displaying the sequence of ALS-specific PCR products derived from DNA isolated from untreated Nt-1 cells (wt) and herbicide-resistant Nt-1 cells recovered as a result of an introduced CO (–4) indicate an alteration in the native sequence for codon 196 (CCA). The conversion (C → A; arrow) in codon 196 (CCA) was detected (–4) and is consistent with a targeted mutation induced by the CO. Because there are multiple copies of ALS alleles, modification of a single gene can confer herbicide resistance, but sequence analyses will indicate base composition heterogeneity at the revised position. To characterize individual species from the heterogeneous population of the ALS-specific PCR products derived from the DNA of herbicide resistant cells, shown in electropherograms 1–4, these products were cloned and sequenced. Sequence analysis of 1 of the 24 randomly selected clones, shown (Cloned), demonstrates the C → A conversion.

Figure 5

Target sequence of the mutant GFP(Δ) and the GFP1 CO (a). Fluorescence photomicrograph of a putative conversion to the GFP phenotype in leaf callus from the tobacco GFP-mutant line Δ6 (b).