Nontransgenic genome modification in plant cells

Abstract

Zinc finger nucleases (ZFNs) are a powerful tool for genome editing in eukaryotic cells. ZFNs have been used for targeted mutagenesis in model and crop species. In animal and human cells, transient ZFN expression is often achieved by direct gene transfer into the target cells. Stable transformation, however, is the preferred method for gene expression in plant species, and ZFN-expressing transgenic plants have been used for recovery of mutants that are likely to be classified as transgenic due to the use of direct gene-transfer methods into the target cells. Here we present an alternative, nontransgenic approach for ZFN delivery and production of mutant plants using a novel Tobacco rattle virus (TRV)-based expression system for indirect transient delivery of ZFNs into a variety of tissues and cells of intact plants. TRV systemically infected its hosts and virus ZFN-mediated targeted mutagenesis could be clearly observed in newly developed infected tissues as measured by activation of a mutated reporter transgene in tobacco (Nicotiana tabacum) and petunia (Petunia hybrida) plants. The ability of TRV to move to developing buds and regenerating tissues enabled recovery of mutated tobacco and petunia plants. Sequence analysis and transmission of the mutations to the next generation confirmed the stability of the ZFN-induced genetic changes. Because TRV is an RNA virus that can infect a wide range of plant species, it provides a viable alternative to the production of ZFN-mediated mutants while avoiding the use of direct plant-transformation methods.

Figure 1.

Structure and key features of pTRV-based expression vectors. A, pTRV-Δ2b-sgP::GOI designed to drive the expression a single gene of interest (GOI) under the control of the sgP constitutive promoter. B, pTRV-Δ2b-sgP::GOI1-T2A-GOI2 designed to drive the coexpression of two genes as a single transcript in which the coding sequences of the two genes (GOI1 and GOI2) are separated by a T2A sequence. C, pTRV-Δ2b-[sgP::GOI1][sgP::GOI2] designed to drive the coexpression of two genes from two independent sg-Ps. The T-DNA region of each vector is presented. Also shown are (1) the constitutive 35S promoter (35sP) and the nopaline synthase terminator (nosT), needed for the production of primary viral transcript following agroinfiltration, and (2) the 5′ and 3′ untranslated regions of the TRV2 needed for viral replication and transcription. The multiple cloning site (MCS) includes EcoRI, XbaI, KpnI, SacI, and XhoI. CP, Coat protein.

Figure 2.

pTRV-mediated expression of a single reporter gene (DsRed2) in newly developed tissues and organs. Plants were infected by pTRV-Δ2b-sgP::DsRed2. Images were taken by fluorescence stereoscope. DsRed2 expression is shown in red.

Figure 3.

pTRV-mediated coexpression of two reporter genes in newly developed plant cells. A, Coexpression of fluorescent reporter genes DsRed2 and EGFP in newly developed leaves of pTRV-Δ2b-sgP::DsRed2-T2A-EGFP-infected plants. DsRed2 and EGFP fluorescence are in orange and green, respectively, and plastid autofluorescence is in dark red. B, Coexpression of DsRed2 and EGFP in newly developed leaves of pTRV-Δ2b-[sgP::DsRed2][sgP::EGFP]-infected tobacco plant. DsRed2 and EGFP fluorescence are in red and green, respectively, and plastid autofluorescence is in dark red. Images in A and B are single confocal sections.

Figure 4.

pTRV-mediated ZFN expression leads to site-specific mutagenesis in newly developed tissues of infected plants. A, Outline of the mGUS reporter gene-repair assay designed to monitor ZFN-mediated mutagenesis in transgenic plants. The mGUS-encoding gene is disrupted by a stop codon (in red) within the 6-bp spacer of the ZFN target site. Reconstruction of active GUS gene occurs by putative deletion of a CTG sequence. The ZFN binding sequences are shown in green and the GUS initiation codon in blue. B to H, Detection of site-specific mutagenesis events in newly developed tissues of pTRV-Δ2b-sgP::QQR infected plants by 5-bromo-4-chloro-3-indolyl-β-glucuronic acid staining. GUS expression was detected in newly developed tobacco (B) and petunia (C) leaves, 13 and 22 d post inoculation, respectively, and in newly developed petunia (D) and tobacco (E) buds, 11 and 50 d post inoculation, respectively. GUS expression was also detected in petunia developing primordia (F) and petunia flower and reproductive tissues (G and H) of the GUS-positive mature plant.

Figure 5.

Molecular analysis of ZFN-mediated mutagenesis events in petunia (P) and tobacco (T) plants. The initiation codon and the ZFN-binding sites on the top strand of the mGUS sequences are in blue and purple, respectively. The stop codon sequence is in red. The predicted outcome of positive (+) or negative (−) GUS expression is indicated on the right.

Figure 6.

Mutant plants can develop directly, without a regeneration step, from virus-infected plants and can stably pass the mutation on to their offspring. Uniformly GUS-stained plantlets, exemplified here with infected petunia (A), which were regenerated from infected plants, and allowed to root, mature (B), and set seed. Also shown are GUS-stained petunia (C) and tobacco (D) seedlings obtained from mature, virus-infected plants.

Figure 7.

Molecular analysis of randomly selected GUS-positive petunia and tobacco seedlings. A, Mutagenesis events in petunia (P) and tobacco (T) offspring. The initiation codon and the ZFN-binding sites on the top strand of the mGUS sequences are in blue and purple, respectively. Stop codon is in red. B, Top section, RT-PCR analysis of pTRV genomes in infected, healthy, and mutated seedlings of petunia and tobacco. Bottom section, RT-PCR analysis of housekeeping gene (the plastid 23S RNA gene). pTRV genomes (identified by RT-PCR amplification of TRV2 sequence coat protein) were detected in infected, but not healthy or mutated seedlings (M, DNA marker ladder).