# Patent

## Title
Plant viral expression vectors and use of same for generating genotypic variations in plant genomes

## Abstract

A method of generating genotypic variation in a genome of a plant is disclosed. The method comprising introducing into the plant at least one viral expression vector encoding at least one chimeric nuclease which comprises a DNA binding domain, a nuclease and a localization signal to a DNA-containing organelle, wherein the DNA binding domain mediates specific targeting of the nuclease to the genome of the plant, thereby generating genotypic variation in the genome of the plant.

## Background
RELATED APPLICATIONS
This Application is a National Phase of PCT Patent Application No. PCT/IL2009/000432 having International filing date of Apr. 21, 2009, which claims the benefit of U.S. Provisional Patent Application No. 61/071,288 filed on Apr. 21, 2008. The contents of the above Applications are all incorporated herein by reference.
FIELD AND BACKGROUND OF THE INVENTION
The present invention, in some embodiments thereof, relates to plant viral expression vectors and, more particularly, but not exclusively, to the use of same for generating genotypic variations in plant genomes.
Genetic modification and improvement of crop plants as well as protection of new varieties is fundamental for modern agriculture. During the past several years an enormous amount of data was obtained from the various large genome-sequencing projects allowing significant progress in agriculture transgenic technologies. Such technologies, including gene expression, gene modification, site-specific gene mutagenesis and gene targeting of plant genome sequences, allow development of basic plant research models and can be directly used for genetic improvement and protection of agronomically important plant species.
Foreign DNA molecules (e.g. T-DNA) delivered by Agrobacterium are integrated in the plant's genome into natural double strand breaks (DSBs) which may be generated by rare-cutting restriction enzymes. These DSBs are recognized and repaired by plant non-homologous end joining (NHEJ) proteins and results in the frequent integration of the foreign DNA into these random sites [Salomon et al. EMBO J (1998) 17: 6086-6095; Tzfira et al. Plant Physiol (2003) 133: 1011-1023, Tzfira et al. Trends Genet (2004) 20: 375-383]. The DSBs may also result in enhanced homologous recombination (HR)-based gene targeting in plant cells [Puchta et al. Proc Natl Acad Sci USA (1996) 93: 5055-5060].
Recent developments in the field of zinc finger nucleases (ZFNs) as novel tools for genome modifications offer new prospects for site-specific induction of DSBs in plant genomes and for the development of NHEJ-based methods for gene targeting in plant species and plant protection. ZFNs are synthetic restriction enzymes which can be specifically designed to bind and cleave virtually any long stretch of dsDNA sequences (see FIG. 1). ZFNs were shown suitable for site-specific genomic DSB induction in plant species using non-viral vectors [Lloyd et al. Proc. Natl. Acad. Sci. U.S. A. (2005) 102: 2232-2237; Tovkach et al. The Plant Journal (2009) 57, 747-757]. Similar effects were shown on human [Moehle et al. Proc Natl Acad Sci USA (2007) 104: 3055-3060] and insect genomes [Beumer et al. Genetics (2006) 172: 2391-2403].
The use of plant viruses as vehicles to introduce and express nonviral genes in plants is well documented [e.g. Donson et al., Proc Natl Acad Sci USA. (1991) 88: 7204-8; Chapman et al. Plant J. (1992) 2: 549-57; Dolja et al., Virology (1998) 252: 269-74]. Infection of plants with modified viruses is simpler and quicker than the regeneration of stably transformed plants (as discussed above) since plant viruses are often small in size (between 3000 and 10,000 nucleotides), are easy to manipulate, have the inherent ability to enter the plant cell, lead to the immediate expression of the heterologous gene and will multiply to produce a high copy number of the gene of interest. Viral vectors have been engineered for delivery of genetic material and expression of recombinant proteins in plants [e.g., Pogue, Annu. Rev. Phytopathol. (2002) 40: 45-74; Gleba, et al., Curr. Opin. Plant Biol. (2004) 7: 182-188; Dolja et al., Proc. Natl. Acad. Sci. USA (1992) 89: 10208-10212; U.S. Pat. No. 5,316,931 and U.S. Pat. No. 5,811,653 for RNA virus vectors]. Viral expression systems are considered transient expression systems as the viral vectors are not integrated into the genome of the host, however, depending on which virus is used, virus multiplication and gene expression can persist for long periods (up to several weeks or months).
To date the use of viral vectors for introducing DSBs in plant genomes was not demonstrated or suggested.
RELATED ART
U.S. Pat. No. 7,229,829 discloses TRV vectors (TRV-RNA1 and TRV-RNA2) carrying heterologous nucleic acid sequences for delivery into plants for transforming plants and plant cells. Specifically, U.S. Pat. No. 7,229,829 teaches vectors for virus induced gene silencing (VIGS) including vectors designed for suppression of host plant gene expression (e.g. antisense transcripts for knocking out expression of genes without the need to genetically transform the plant) or vectors designed for expression of heterologous nucleic acids (e.g. nucleic acids mediating gene silencing or gene suppression).
U.S. Publication Nos. 20050026157 and 20070134796 discloses compositions and methods for targeted cleavage of cellular chromatin and for targeted alterations (e.g. insertions) of cellular nucleotide sequences. To target specific genomic sites, fusion proteins are constructed which comprise a zinc finger domain and a cleavage domain [i.e. zinc finger proteins (ZFPs)]. Moreover, U.S. Publication No. 20070134796 teaches vectors (e.g. bacterial vectors such as plasmid vectors and viral vectors such as adenoviral and retroviral vectors) comprising the ZFPs.
PCT Publication No. WO07139982 discloses methods and compositions for inactivating human genes (e.g. CCR5 gene) using zinc finger nucleases (ZFNs). The ZFNs comprise a zinc finger protein (may include 1, 2, 3, 4, 5, 6 or more zinc fingers) and a cleavage domain or cleavage half-domain (i.e., a nuclease domain). Furthermore, PCT Publication No. WO07139982 teaches vectors comprising ZFNs and/or a donor sequence for targeted integration into a target gene.
U.S. Pat. Nos. 7,309,605 and 6,610,545 disclose nucleotide sequences encoding the enzyme I-SceI (a double-stranded endonuclease that cleaves DNA within its recognition site). These sequences can be incorporated in cloning and expression vectors (such as plasmid, bacteriophage or cosmid vectors) and may be used to transform cell lines and transgenic organisms (e.g. mammals, plants). The vectors disclosed are useful in gene mapping, in site directed genetic recombination and in in-vivo site-directed insertion of genes.
SUMMARY OF THE INVENTION
According to an aspect of some embodiments of the present invention there is provided a method of generating genotypic variation in a genome of a plant, the method comprising introducing into the plant at least one viral expression vector encoding at least one chimeric nuclease which comprises a DNA binding domain, a nuclease and a localization signal to a DNA-containing organelle, wherein the DNA binding domain mediates specific targeting of the nuclease to the genome of the plant, thereby generating genotypic variation in the genome of the plant.
According to an aspect of some embodiments of the present invention there is provided a method of treating a plant infection by a pathogen, the method comprising introducing into the plant at least one viral expression vector encoding at least one chimeric nuclease which comprises a DNA binding domain and a nuclease, wherein the DNA binding domain mediates targeting of the nuclease to the genome of the pathogen, thereby preventing or treating a plant infection by a pathogen.
According to an aspect of some embodiments of the present invention there is provided a method of tagging a genome of a plant, the method comprising introducing into the plant at least one viral expression vector encoding at least one chimeric nuclease which comprises a DNA binding domain, a nuclease and a localization signal to a DNA-containing organelle, wherein the DNA binding domain mediates specific targeting of the nuclease to the genome of the plant, thereby tagging the genome of the plant.
According to an aspect of some embodiments of the present invention there is provided a method of generating male sterility in a plant, the method comprising upregulating in the plant a structural or functional gene of a mitochondria or chloroplast associated with male sterility by introducing into the plant at least one viral expression vector encoding at least one chimeric nuclease which comprises a DNA binding domain, a nuclease and a mitochondria or chloroplast localization signal and a nucleic acid expression construct which comprises at least one heterologous nucleic acid sequence which can upregulate the structural or functional gene of a mitochondria or chloroplast when targeted into the genome of the mitochondria or chloroplast, wherein the DNA binding domain mediates targeting of the heterologous nucleic acid sequence to the genome of the mitochondria or chloroplast, thereby generating male sterility in the plant.
According to an aspect of some embodiments of the present invention there is provided a method of generating a herbicide resistant plant, the method comprising introducing into the plant at least one viral expression vector encoding at least one chimeric nuclease which comprises a DNA binding domain, a nuclease and a chloroplast localization signal, wherein the DNA binding domain mediates targeting of the nuclease to a gene conferring sensitivity to herbicides, thereby generating the herbicide resistant plant.
According to an aspect of some embodiments of the present invention there is provided a plant viral expression vector comprising a nucleic acid sequence encoding at least one chimeric nuclease which comprises a DNA binding domain, a nuclease and a localization signal to a DNA-containing organelle.
According to an aspect of some embodiments of the present invention there is provided a pTRV based expression vector comprising a nucleic acid sequence encoding at least two heterologous polypeptide sequences.
According to an aspect of some embodiments of the present invention there is provided a plant cell comprising at least one chimeric nuclease, wherein the chimeric nuclease comprises a DNA binding domain, a nuclease and a localization signal to a DNA-containing organelle, and wherein the chimeric nuclease induces cleavage of a target sequence.
According to an aspect of some embodiments of the present invention there is provided a transgenic plant comprising the plant viral expression vector of claim 6 or 7.
According to an aspect of some embodiments of the present invention there is provided a method of generating a transgenic plant, the method comprising: introducing into one or more cells of the plant at least one viral expression vector encoding at least one chimeric nuclease which comprises a DNA binding domain, a nuclease and a localization signal to a DNA-containing organelle.
According to an aspect of some embodiments of the present invention there is provided an isolated polynucleotide comprising a nucleic acid sequence as set forth in SEQ ID NOs: 31, 32, 33, 34, 70, 72, 74, 76, 84, 86 or 88.
According to an aspect of some embodiments of the present invention there is provided an isolated polypeptide comprising an amino acid sequence as set forth in SEQ ID NOs: 35, 36, 37, 38, 71, 73, 75, 77, 85, 87 or 89.
According to some embodiments of the invention, generating genotypic variation is transient.
According to some embodiments of the invention, the genotypic variation comprises a nucleotide insertion, a nucleotide deletion or a combination of same.
According to some embodiments of the invention, the tagging comprises a nucleotide insertion, a nucleotide deletion or a combination of same.
According to some embodiments of the invention, the viral expression vector comprises a Tobacco Rattle Virus (TRV) expression vector.
According to some embodiments of the invention, the TRV expression vector comprises a pTRV2 based expression vector.
According to some embodiments of the invention, at least one viral expression vector encodes for two chimeric nucleases.
According to some embodiments of the invention, the at least one viral expression vector comprises two viral expression vectors.
According to some embodiments of the invention, the two viral expression vectors are introduced into the plant concomitantly.
According to some embodiments of the invention, introducing into the plant is effected by an Agrobacterium. 
According to some embodiments of the invention, the Agrobacterium is effected by injection.
According to some embodiments of the invention, introducing the Agrobacterium is effected by leaf infiltration.
According to some embodiments of the invention, introducing into the plant is effected by virion infection.
According to some embodiments of the invention, the at least one chimeric nuclease comprises two chimeric nucleases.
According to some embodiments of the invention, the plant viral expression vector or transgenic plant further comprises a second nucleic acid sequence encoding a heterologous polypeptide.
According to some embodiments of the invention, the plant viral expression vector or transgenic plant comprises a pTRV backbone.
According to some embodiments of the invention, the pTRV is a pTRV1 (GeneBank Accession No: AF406990).
According to some embodiments of the invention, the pTRV is a pTRV2 (GeneBank Accession No: AF406991).
According to some embodiments of the invention, the nucleic acid sequence is devoid of a 2b sequence (SEQ ID NO: 43).
According to some embodiments of the invention, the nucleic acid sequence comprises a Ω enhancer (SEQ ID NO: 44).
According to some embodiments of the invention, the nucleic acid sequence comprises two separate sub genomic promoters (sgPs) for regulating transcription of the at least two heterologous polypeptides.
According to some embodiments of the invention, the at least two heterologous polypeptide sequences are separated by nucleic acid sequence encoding a cleavage domain.
According to some embodiments of the invention, the cleavage domain comprises a T2A-like protein sequence (SEQ ID NO: 40).
According to some embodiments of the invention, the nucleic acid sequence of the at least two heterologous polypeptide sequences is as set forth in SEQ ID NOs: 84, 86 or 88.
According to some embodiments of the invention, the amino acid sequence of at least two heterologous polypeptide sequences are as set forth in SEQ ID NOs: 85, 87 or 89.
According to some embodiments of the invention, the at least two heterologous polypeptide sequences encode for a plant gene.
According to some embodiments of the invention, the at least two heterologous polypeptide sequences comprise chimeric proteins, wherein each of the chimeric proteins comprise a DNA binding domain, a nuclease and a localization signal to a DNA-containing organelle.
According to some embodiments of the invention, the localization signal comprises a ribulose-1,5-bisphospate carboxylase small subunit (RSSU) sequence (SEQ ID NO: 138).
According to some embodiments of the invention, the localization signal comprises an ATPase beta subunit (ATP-β) sequence (SEQ ID NO: 139).
According to some embodiments of the invention, the DNA binding domain binds a 9 nucleotide sequence.
According to some embodiments of the invention, the DNA binding domain comprises at least one zinc finger domain.
According to some embodiments of the invention, the zinc finger domain comprises three zinc finger domains.
According to some embodiments of the invention, the nuclease comprises a cleavage domain of a type II restriction endonuclease.
According to some embodiments of the invention, the type II restriction endonuclease is a FokI restriction endonuclease.
According to some embodiments of the invention, the plant comprises a Petunia hybrida. 
According to some embodiments of the invention, the plant comprises a Nicotiana tabacum. 
According to some embodiments of the invention, the plant in selected from the group consisting of an Arabidopsis thaliana, an Artemisia sp., a Artemisia annua, a Beta vulgaris, a Solanum tuberosum, a Solanum pimpinellifolium, a Solanum lycopersicum, a Solanum melongena, a Spinacia oleracea, a Pisum sativum, a Capsicum annuum, a Cucumis sativus, a Nicotiana benthamiana, a Nicotiana tabacum, a Zea mays, a Brassica napus, a Gossypium hirsutum cv. Siv'on, a Oryza sativa and a Oryza glaberrima. 
According to some embodiments of the invention, the cell is a meristem cell.
According to some embodiments of the invention, the DNA-containing organelle is selected from the group consisting of a nucleus, a chloroplast and a mitochondria.
According to some embodiments of the invention, the specific targeting of the nuclease to the genome of the Petunia hybrida is to a phytoene desaturase (PDS) or a flavanone 3 beta-hydroxylase (FHT) of the Petunia hybrida. 
According to some embodiments of the invention, the mitochondria localization signal comprises an ATPase beta subunit (ATP-β) (SEQ ID NO: 139).
According to some embodiments of the invention, the chloroplast localization signal comprises a ribulose-1,5-bisphospate carboxylase small subunit (Rssu) (SEQ ID NO: 138).
Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.



BRIEF DESCRIPTION OF THE DRAWINGS
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.
In the drawings:
FIG. 1 is a schematic illustration of Zinc-finger nucleases (ZFNs) as a tool for the induction of genomic double-strand breaks (DSBs). FIG. 1A depicts the structure of ZFNs chimeric genes composed of a synthetic DNA-recognition domain consisting of three C2H2 zinc fingers fused to a non-specific DNA restriction enzyme (usually the FokI endonuclease); FIG. 1B depicts custom-made ZFN genes, in which each finger recognizes a three-nucleotide sequence and can potentially be designed to recognize any combination of nine nucleotides (exemplified here by a GGGGAAGAA target sequence, SEQ ID NO: 42). Since FokI functions as a dimer, two sets of ZFNs are used to bind the target DNA, which results in a unique combination of 18 nucleotides; FIG. 1C depicts binding of the ZFNs to the target DNA; FIG. 1D depicts digestion of the DNA by FokI endonuclease domain and creation of a double-strand break (DSB). FIG. 1E depicts repair of the DSBs by non-homologous end-joining (NHEJ) proteins that lead to deletion or insertion mutations at the repair site.
FIG. 2A shows sequence alignment between the sequence of Thosea asigna virus for the self cleaving peptide (Tav-T2A, SEQ ID NO: 51) and the modified sequence according to the codon usage of Petunia (pTRV-T2A, SEQ ID NO: 52).
FIG. 2B shows P1-25 Petunia RB random DNA fragment (SEQ ID NO: 8).
FIG. 3 shows P1-36 Petunia RB random DNA fragment (SEQ ID NO: 9).
FIG. 4 is a schematic illustration of a ZFN structure constructed according to the present teachings. The ZFN construct comprises a nuclear localization signal (NLS), a Zink Finger DNA binding domain and a DNA nuclease domain from FokI.
FIGS. 5A-D shows the sequences of NLS-P1-25-ZFN1, NLS-P1-25-ZFN2, NLS-P1-36-ZFN1 and NLS-P1-36-ZFN2. The sequence is of the full chimera: NLS, ZFN and FokI (d-domain). Sequences of nuclear localization signal (NLS) are depicted in lower case; nuclease (FokI-domain d) first codon and the termination codon are depicted in bold. FIG. 5A shows the sequence of NLS-P1-25-ZFN1 (SEQ ID NO: 31); FIG. 5B shows the sequence of NLS-P1-25-ZFN2 (SEQ ID NO: 32); FIG. 5C shows the sequence of NLS-P1-36-ZFN1 (SEQ ID NO: 33); and FIG. 5D shows the sequence of NLS-P1-36-ZFN2 (SEQ ID NO: 34).
FIG. 5E is a scheme showing the original pET28 vector (SEQ ID NO: 39, commercial available from Novagen) and the modified pET28 vector (SEQ ID NO: 49), pET28c.SX, comprising a modification of MCS. To construct pET28c-SX, nucleotides 179 to 158 (indicated by bold) were deleted from MSC of pET28c by digestion with SalI and XhoI.
FIGS. 6A-D are schematic maps of pTRV2 (GenBank accession No. AF406991) and its modifications. FIG. 6A is a schematic illustration of the complete pTRV2 expression vector; FIG. 6B depicts the removal of the 2b 5′ CDS fragment; FIG. 6C depicts the addition of 5′UTR of TMV (a); and FIG. 6D depicts the addition of sgP-CP from PEBV.
FIG. 6E is schematic maps of part of pTRV1 showing RNA1 (GenBank accession No. AF406990).
FIGS. 7A-E are pictures illustrating the expression of GUS in meristems of petunia plants inoculated with pTRV2-GUS and pTRV2-Δ2b-GUS. FIGS. 7A-B depict meristems from petunia plants 7 days after stem inoculation with the vectors; and FIGS. 7C-D depict meristems from petunia plants 37 days after stem inoculation with the vectors. Plants inoculated with pTRV2-GUS are shown in FIGS. 7A and 7C. Plants inoculated with pTRV2-Δ2b-GUS are shown in FIGS. 7B and 7D. FIG. 7E shows GUS staining in petunia plants propagated in vitro 6 month following inoculation with pTRV2-Δ2b-GUS.
FIGS. 8A-G are pictures illustrating the expression of marker genes in various plants following inoculation with pTRV2 based vectors. FIG. 8A depicts GUS staining (24 days post inoculation) of pepper plants (Capsicum annuum, Endra-1750) inoculated with pTRV2-Δ2b-GUS; FIG. 8B depicts GFP staining (41 days post inoculation) of pepper plants (Capsicum annuum, Endra-1750) inoculated with pTRV2-Δ2b-GFP; FIG. 8C depicts GUS staining (13 and 30 days post inoculation) of Arabidopsis plants inoculated with pTRV2-GUS; FIG. 8D depicts GUS staining (14 days post inoculation) of tomato plants (Solanum pimpinellifolium La121) inoculated with pTRV2-Δ2b-ΩGus; FIG. 8E depicts GFP staining (31 days post inoculation) of Nicotiana benthamiana plants inoculated with pTRV2-Δ2b-GFP, FIG. 8F depicts GUS staining (44 days post inoculation) of Nicotiana benthamiana plants inoculated with pTRV2-35SΩGUS; and FIG. 8G depicts pigmentation of Nicotiana benthamiana inoculated with pTRV2-Δ2b-PAP (76 days post inoculation).
FIGS. 9A-B are pictures illustrating GUS staining 51 days post inoculation of petunia plants with pTRV2-Δ2b-ΩGUS (FIG. 9A) as compared to pTRV2-Δ2b-GUS (FIG. 9B). Of note, arrows point to meristematic regions.
FIGS. 10A-C are pictures illustrating co-expression of two genes in meristems of N. benthamiana plants. FIG. 10A depicts plants inoculated with pTRV2-Δ2b-sgP-GFP. GFP staining was evaluated 17 days post inoculation with pTRV2-Δ2b-sgP-GFP; FIGS. 10B-C depict plants co-inoculated with pTRV2-Δ2b-GUS and pTRV2-Δ2b-GFP. GFP staining was evaluated 17 days post inoculation (FIG. 10B) followed by GUS staining on the same tissue sample (FIG. 10C).
FIGS. 11A-F are pictures illustrating virus-mediated gene expression (DsRed) in cells of different plants. Plants were inoculated with pTRV1 and pTRV2-Δ2b-sgP-DsRed and fluorescence was evaluated using confocal laser scanning microscopy. Upper panel (FIGS. 11A, C, E) shows cells' chlorophyll autofluorescence and lower panel (FIGS. 11B, D, F) shows DsRed fluorescence in the same cells. Autofluorescence was evaluated at excitation (Ex) 488 nm and emission (Em) at more than 650 nm, DsRed was evaluated at ex 545 nm and em between 585-615 nm.
FIGS. 12A-H are pictures illustrating long term gene expression (DsRed) in different plants following inoculation with pTRV1 and pTRV2-Δ2b-sgP-DsRed. Images were obtained using fluorescent stereomicroscope. DsRed fluorescence (FIGS. 12A, C, D, G) was visible in different parts of plants, including roots (FIGS. 12D, F). Lower panel (FIGS. 12B, E, F, H) shows (the same) images taken under bright field. Of note, N. Benthamiana was innoculated with TRV virions and N. tabaccum and Petunia hybrida were innoculated via agro-infiltration.
FIGS. 13A-K are pictures illustrating the applicability of the TRV2 vector for expression of foreign genes in various plants. Expression of the marker genes GUS, GFP and DsRed was demonstrated in various plants belonging to different families following inoculation with TRV vectors: Beta vulgaris (FIG. 13E) were inoculated with pTRV-Δ2b-GUS (sample GUS stained 20 days post inoculation); Solanum melongena (FIGS. 13F-G) was inoculated with pTRV-Δ2b-sgP-Rssu-EGFP (evaluated 5 days post inoculation); Cucumis sativus (FIGS. 13A-B), Gossypium hirsutum cv. Siv'on (FIGS. 13H-I) and Brassica napus (FIGS. 13J-K) were inoculated with pTRV-Δ2b-sgP-DsRed (evaluated 5, 7 and 16 days post inoculation, respectively). Bright field images were also demonstrated. Spinacia oleracea (FIGS. 13C-D) was inoculated with pTRV-Δ2b-GUS (FIG. 13C, sample GUS stained 20 days post inoculation) and pTRV-Δ2b-sgP-DsRed (FIG. 13D, evaluated 12 days post inoculation). All images were taken using fluorescent stereomicroscope.
FIGS. 14A-C are pictures illustrating expression of DsRed2 in Zea mays Var. Royalty coleoptile by TRV viral vectors. Seeds were inoculated with sap containing diluted virions, extracted from Petunia agroinfiltrated with pTRV1 and pTRV2-Δ2b-sgP-DsRed. DsRed was evaluated at 16 days post inoculation (dpi). Visible (FIG. 14A), DsRed (FIG. 14B) and Merged (FIG. 14C).
FIGS. 15A-G are pictures illustrating chloroplast-targeted expression of EGFP in petunia and tobacco following infection with pTRV2-sgP-Rssu-EGFP. N. Tabacuum cv Samsung and Petunia hybrida CV. RB were inoculated with pTRV2-sgP-Rssu-EGFP and the expression of EGFP in chloroplasts was assayed approximately 9 days post inoculation. Autofluorescence (FIGS. 15A, E) of chlorophyll: was evaluated at excitation (ex) 488 nm and emission (em) was evaluated at more than 650 nm. The EGFP (FIGS. 15B, F) was detected by ex at 488 and em between 505-530 nm. Merged signal (FIGS. 15D and G, overlay of pink and green yielding yellow). Inset (FIG. 15C) shows tissue expressing EGFP visualized by fluorescent stereomicroscope.
FIGS. 16A-K are pictures illustrating mitochondrial-targeted expression of EGFP in petunia following infection with pTRV2-sgP-ATPβ-EGFP. Petunia hybrida CV. RB were inoculated with pTRV2-sgP-ATPβ-EGFP and expression of EGFP in mitochondria was assayed approximately 5 days post inoculation. Autofluorescence (FIG. 16A) was evaluated at ex 488 and em at more than 650 nm. EGFP (FIG. 16B) was evaluated at ex. 488 and em between 505-530 nm. Protoplast (FIGS. 16E-K) were prepared from Petunia RB expressing mitochondrial targeted EGFP, stained with MitoTracker and evaluated at ex 545 nm and em between 585-615 nm. Inset (FIG. 16G) shows tissue expressing EGFP visualized by fluorescent stereomicroscope.
FIGS. 17A-D are pictures illustrating co-expression of marker genes in different cellular compartments. DsRed was expressed in the cytosol and GFP in the chloroplasts of N. tabacum cv. Xanthi leaf cells. Plants were co-infected with pTRV1 and pTRV2-Δ2b-sgP-DsRed and pTRV2-Δ2b-sgP-Rssu-EGFP. Autofluorescence (FIG. 17A) was evaluated using ex 488 nm and em of more than 650 nm, GFP (FIG. 17B) was evaluated using ex 488 nm and em between 505-530 nm, DsRed (FIG. 17C) was evaluated using ex 545 nm and em between 585-615 nm, Merge (FIG. 17D) depicts a merge of all three filters (merged signals).
FIGS. 18A-L are pictures illustrating co-expression of DsRed and EGFP in different plants using pTRV2 constructed with the two reporter genes in tandem separated by T2A. Plants (Petunia hybrida, N. tobaccum and N. benthamiana) were inoculated with pTRV1 and pTRV2-Δ2b-sgP-DsRed-T2A-NLS-EGFP. Fluorescence was evaluated using confocal laser scanning microscopy. Cells' chlorophyll autofluorescence (FIGS. 18A, E, I), EGFP (FIGS. 18B, F, J) and DsRed (FIGS. 18C, G, K) and merged signal (FIGS. 18D, H, L) are shown. Autofluorescence was evaluated at ex 488 nm and em at more than 650 nm, EGFP was evaluated at ex 488 nm and em between 505-530 nm, DsRed2 was evaluated at ex 545 nm and em between 585-615 nm.
FIGS. 19A-J are pictures illustrating co-expression of DsRed and GFP in different plants using pTRV2 constructed with the two reporter genes in tandem driven by separate double subgenomic promoters. Plants (N. tobaccum and N. benthamiana) were inoculated with pTRV1 and pTRV2-Δ2b-sgP-GFP-sgP-DsRed. Fluorescence was evaluated using confocal laser scanning microscopy. The cells' chlorophyll autofluorescence (FIGS. 19A, E), GFP (FIGS. 19B, F) and DsRed (FIGS. 19C, G) are shown. FIG. 19D depicts an image in bright field (for N. tobaccum) and FIG. 19H depicts an image of merged signal (for N. benthamiana). FIGS. 19I-J are images, taken by stereomicroscope, of inoculated N. benthamiana tissues (3 dpi). Autofluorescence ex was evaluated at 488 nm and em at more than 650 nm, GFP was evaluated at ex 488 nm and em between 505-530 nm, DsRed2 was evaluated at ex 545 nm and em between 585-615 nm.
FIG. 20 is a picture illustrating digestion of PCR fragments carrying artificial target sites P1-25-1, P1-25-2, P1-36-1 and P1-36-2 (P25-TS1, P25-TS2, P36-TS1, P36-TS2 respectively) by specific ZFNs. PCR fragments (ca. 900 bp) carrying palindrome-like target sequences were incubated with 25-ZFN-1,25-ZFN-2 or 36-ZFN-1,36-ZFN-2 and the digestion products were separated by agarose gel. Of note, the TS (target site) is palindrome-like.
FIG. 21 is a picture illustrating digestion of a NcoI/BamHI (740 bp) fragment from pBS-PI-36, carrying a target P1-36 sequence, with 36-ZFN1 and 36-ZFN2.
FIG. 22 is a picture illustrating digestion of plasmid pBS carrying PI-36 (pBS-PI-36) by a mixture of ZFNs (36-ZFN 1 and 2). Fragment of expected size (515 bp) is indicated by arrow.
FIG. 23 is a picture illustrating digestion of a plasmid carrying target PDS1 or PDS2 palindromic sites. The tested palindromic sites were cut with specific ZFNs (PDS-ZFN1 and PDS-ZFN2) and AgeI which yielded a fragment of approximately 950 bp, as expected. Volumes listed above the columns refer to the amount of enzyme containing crude extract used. Of note, the TS (target site) is palindrome-like.
FIGS. 24A-B are pictures illustrating the expression of DsRFP (FIG. 24B) and GFP (FIG. 24A) in petunia plants inoculated with pTRV2-Δ2b-sgP-CP-PEBV carrying DsRFP and GFP separated by T2A. Figures depict leaf tissues from petunia plants 10 days after stem inoculation with the vector.
FIGS. 25A-B show the N termini of the mutated uidA gene sequence. FIG. 25A depicts the uidA gene sequence containing insert of target sites of QEQ-ZFN (bold) and spacer with stop codon (red). FIG. 25B depicts how miss-repair of the double strand breaks formed by the QEQ-ZFN may lead to elimination of the stop codon and reconstruction of the uidA gene.
FIGS. 26A-J are pictures illustrating TRV-based repair of uidA in planta. Transgenic petunia and tobacco plants carrying mutated uidA were inoculated in vitro or in vivo with pTRV1 and pTRV2-Δ2b-sgP-QEQ-ZFN. At different times after agroinfiltration or inoculation with virions, GUS activity was evaluated in various parts of the plant including in tissues that developed after inoculation. FIG. 26A depicts a mutated uidA transgenic Petunia hybrida line 65 evaluated for GUS expression 12 days post in-vitro agroinoculation. FIG. 26B depicts a mutated uidA transgenic Nicotiana tobaccum line 3 evaluated for GUS expression 22 days post in-vitro agroinoculation. FIG. 26C depicts a mutated uidA transgenic Petunia hybrida line I evaluated for GUS expression 11 days post in-vitro agroinoculation. FIG. 26D depicts a mutated uidA transgenic Nicotiana tobaccum line 3 evaluated for GUS expression 50 days post inoculation. FIG. 26E depicts a mutated uidA transgenic Petunia hybrida line N evaluated for GUS expression 17 days post in-vitro agroinoculation. FIG. 26F depicts a mutated uidA transgenic Petunia hybrida line I in-vitro inoculation with virions evaluated for GUS expression was carried out 15 days post inoculation. FIG. 26G depicts a mutated uidA transgenic Petunia hybrida line I in-vitro agroinoculation with 0.08 OD, evaluated 29 days post inoculation. FIG. 26H depicts a mutated uidA transgenic Petunia hybrida line I in-vitro agro inoculation with 0.8 OD, evaluated 29 days post inoculation. FIG. 26I depicts a mutated uidA transgenic Nicotiana tobaccum line 11, not treated with TRV, GUS tested, and FIG. 26J depicts a mutated uidA transgenic Petunia hybrida line I primordia regeneration evaluated for GUS expression, following in-vitro agroinoculation.
FIG. 27 shows alignment of 20 mutant sequences (SEQ ID NOs: 96-116) in mutated uidA (GUS) identified by DdeI site disruption in the GUS stop codon of transgenic N. tabacum CV Samsung (N_t) as well as Petunia hybrida (Pet) plants. Of note, the results depicted insertions (1 or 2 nucleotides) and deletions (less or equal to 49 nucleotides). Restoration of GUS activity can be ascribed to mutation in Pet30.
FIG. 28A shows the sequence of phytoene desaturase (PDS) exon from Petunia hybrida RB (GenBank accession no AY593974.1, SEQ ID NO: 131). This sequence was confirmed by resequencing (indicated by upper case letters). The highlighted sequences are the target sites (PDS-ZFN1 target site—SEQ ID NO 140 and PDS-ZFN2 target site—SEQ ID NO: 141) of the PDS-ZFNs (SEQ ID NOs: 71 and 73) generated by the present invention (SEQ ID NOs: 70 and 72). SEQ ID NO: 132 depicts a short fragment of a sequence of the complementary strand to which PDS-ZFN2 binds. Recognition site for the MfeI is underlined.
FIG. 28B shows the changes in the PDS nucleic acid sequences (SEQ ID NOs: 119-128) in Petunia hybrida plants inoculated with pTRV1 and pTRV2-Δ2b-sgP-PDS-ZFN1-T2A-PDS-ZFN2 vectors. The mutants were compared to the native PDS sequences in the tested Petunia hybrida plants (PDS-WT, Y10 and G35). TS1 (GGAGATGCA, SEQ ID NO: 135) and TS2 (CACTTCAAT, SEQ ID NO: 136) indicate the binding sites for ZFNs in the PDS gene. The MfeI site (CAATTG, SEQ ID NO: 137) served as a selection tool to isolate ZFNs mediated PDS mutants.
FIG. 29 shows the sequence of flavanone 3 beta-hydroxylase (FHT) exon from Petunia hybrida cv. RB (GenBank accession no AF022142.1, SEQ ID NO: 133). The sequence was confirmed by resequencing. The highlighted sequences (FHT-ZFN1 target site—SEQ ID NO 142 and FHT-ZFN2 target site—SEQ ID NO: 143) were used as the target sites for the FHT-ZFNs generated by the present invention (SEQ ID NOs: 74 and 76). Recognition site for the EcoNI is underlined. SEQ ID NO: 134 depicts a short fragment of a sequence of the complementary strand to which FHT-ZFN2 binds.
FIG. 30 shows the sequence of pTRV2 containing 2b and PEBV-CP subgenomic promoters (sgP) region (SEQ ID NO: 79). For comparison, the sequence lacking 40 nucleotides from 2b-sgP (pTRV2-Δ2b-Δ2bsgP-sgP) is shown in the upper line (SEQ ID NO: 78). Nucleotides) to 72-3′ of CP gene, nucleotides 73 to 237—sgP of 2b, nucleotides 238 to 282—MCS, nucleotides 283 to 466-sgP of CP from PEBV. Nucleotides 206 to 237—deletion from the 3′ of the 2b sgP.
FIG. 31A shows the sequence of NLS-PDS-ZFN1 (SEQ ID NO 70). The sequence is of the full chimera: NLS, ZFN and FokI (d-domain). Sequences of nuclear localization signal (NLS) are depicted in lower case; nuclease (FokI-domain d) first codon and the termination codon are depicted in bold.
FIG. 31B shows the sequence of NLS-PDS-ZFN2 (SEQ ID NO 72). The sequence is of the full chimera: NLS, ZFN and FokI (d-domain). Sequences of nuclear localization signal (NLS) are depicted in lower case; nuclease (FokI-domain d) first codon and the termination codon are depicted in bold.
FIG. 32A shows the sequence of NLS-FHT-ZFN1 (SEQ ID NO 74). The sequence is of the full chimera: NLS, ZFN and FokI (d-domain). Sequences of nuclear localization signal (NLS) are depicted in lower case; nuclease (FokI-domain d) first codon and the termination codon are depicted in bold.
FIG. 32B shows the sequence of NLS-FHT-ZFN2 (SEQ ID NO 76). The sequence is of the full chimera: NLS, ZFN and FokI (d-domain). Sequences of nuclear localization signal (NLS) are depicted in lower case; nuclease (FokI-domain d) first codon and the termination codon are depicted in bold.
FIG. 33 shows a schematic representation of pTRV2-Δ2b-sgP-QEQ-ZFN (SEQ ID NO: 82).
FIG. 34 shows a schematic representation of part of the pTRV2 vector carrying a gene-specific ZFNs (PDS is shown for illustration) fused through T2A sequence, downstream to two subgenomic (2b-sgP and PEBV-CP-sgP) promoters.



DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION
The present invention, in some embodiments thereof, relates to plant viral expression vectors and, more particularly, but not exclusively, to the use of same for generating genotypic variations in plant genomes.
The principles and operation of the present invention may be better understood with reference to the drawings and accompanying descriptions.
Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.
While reducing some embodiments of the present invention to practice, the present inventors have devised an effective tool for generating genotypic variation in plants using viral vectors encoding chimeric polypeptides designed for generating sequence specific double-strand breaks in the plant's genome. As noted in the background section, the formation of DSBs can be used for passively (by plants' repair system) or actively (i.e., directed insertion of heterologous nucleic acid sequences) generating genotypic variation. The aforementioned substantiates beyond any doubt the value of the present tools in generating genomic variations.
As is illustrated in the Examples section which follows, the present inventors have constructed modified tobacco rattle virus (TRV) expression vectors. These vectors were successfully used for introducing and expressing foreign genes of sizes equivalent to the chimeric genes of the present invention (e.g. GUS) in meristematic tissues of different plants (e.g. Petunia, N. benthamiana and N. Tobaccum, e.g. FIGS. 8A-G, FIGS. 9A-B and FIGS. 11A-F). The present inventors were successful in expressing heterologous genes in chloroplast and mitochondrial plastids (FIGS. 15A-G and FIGS. 16A-K, respectively). Moreover, the present inventors were successful in in-planta co-expression of two heterologous genes (e.g. DsRed and GFP) and specifically in different plant compartments (e.g. cytosol, chloroplasts or nucleus) using viral vectors of some embodiments of the invention (see e.g. FIGS. 17A-D and 18A-L). Importantly, the present inventors have generated zinc finger nucleases (ZFNs) which specifically bind and cleave petunia non-coding target sequences (FIGS. 20-22), petunia phytoene desaturase (PDS) genomic sequences (FIGS. 23 and 28B) or petunia flavanone 3 beta-hydroxylase (FHT) genomic sequences. Accordingly, these chimeric nucleases and viral vectors may serve as powerful tools in the field of agriculture transgenic technologies.
Thus, according to one aspect of the present invention there is provided a method of generating genotypic variation in a genome of a plant. The method comprising introducing into the plant at least one viral expression vector encoding at least one chimeric nuclease which comprises a DNA binding domain, a nuclease and a nuclear localization signal, wherein the DNA binding domain mediates specific targeting of the nuclease to the genome of the plant, thereby generating genotypic variation in the genome of the plant.
As used herein the term “plant” refers to whole plants, portions thereof (e.g., leaf, root, fruit, seed) or cells isolated therefrom (homogeneous or heterogeneous populations of cells).
As used herein the phrase “isolated plant cells” refers to plant cells which are derived from disintegrated plant cell tissue or plant cell cultures.
As used herein the phrase “plant cell culture” refers to any type of native (naturally occurring) plant cells, plant cell lines and genetically modified plant cells, which are not assembled to form a complete plant, such that at least one biological structure of a plant is not present. Optionally, the plant cell culture of this aspect of the present invention may comprise a particular type of a plant cell or a plurality of different types of plant cells. It should be noted that optionally plant cultures featuring a particular type of plant cell may be originally derived from a plurality of different types of such plant cells.
Any commercially or scientifically valuable plant is envisaged in accordance with these embodiments of the invention. A suitable plant for use with the method of the invention can be any monocotyledonous or dicotyledonous plant including, but not limited to, maize, wheat, barely, rye, oat, rice, soybean, peanut, pea, lentil and alfalfa, cotton, rapeseed, canola, pepper, sunflower, potato, tobacco, tomato, lettuce, mums, arabidopsis, broccoli, cabbage, beet, quinoa, spinach, cucumber, squash, watermelon, beans, hibiscus, okra, apple, rose, strawberry, chile, garlic, onions, sorghum, eggplant, eucalyptus, pine, a tree, an ornamental plant, a perennial grass and a forage crop, coniferous plants, moss, algae, as well as other plants listed in World Wide Web (dot) nationmaster (dot) com/encyclopedia/Plantae.
Accordingly, plant families may comprise Alliaceae, Amaranthaceae, Amaryllidaceae, Apocynaceae, Asteraceae, Boraginaceae, Brassicaceae, Campanulaceae, Caryophyllaceae, Chenopodiaceae, Compositae, Cruciferae, Cucurbitaceae, Euphorbiaceae, Fabaceae, Gramineae, Hyacinthaceae, Labiatae, Leguminosae-Papilionoideae, Liliaceae, Linaceae, Malvaceae, Phytolaccaceae, Poaceae, Pinaceae, Rosaceae, Scrophulariaceae, Solanaceae, Tropaeolaceae, Umbelliferae and Violaceae.
Such plants include, but are not limited to, Allium cepa, Amaranthus caudatus, Amaranthus retroflexus, Antirrhinum majus, Arabidopsis thaliana, Arachis hypogaea, Artemisia sp., Avena sativa, Bellis perennis, Beta vulgaris, Brassica campestris, Brassica campestris ssp. Napus, Brassica campestris ssp. Pekinensis, Brassica juncea, Calendula officinalis, Capsella bursa-pastoris, Capsicum annuum, Catharanthus roseus, Chemanthus cheiri, Chenopodium album, Chenopodium amaranticolor, Chenopodium foetidum, Chenopodium quinoa, Coriandrum sativum, Cucumis melo, Cucumis sativus, Glycine max, Gomphrena globosa, Gossypium hirsutum cv. Siv'on, Gypsophila elegans, Helianthus annuus, Hyacinthus, Hyoscyamus niger, Lactuca sativa, Lathyrus odoratus, Linum usitatissimum, Lobelia erinus, Lupinus mutabilis, Lycopersicon esculentum, Lycopersicon pimpinellifolium, Melilotus albus, Momordica balsamina, Myosotis sylvatica, Narcissus pseudonarcissus, Nicandra physalodes, Nicotiana benthamiana, Nicotiana clevelandii, Nicotiana glutinosa, Nicotiana rustica, Nicotiana sylvestris, Nicotiana tabacum, Nicotiana edwardsonii, Ocimum basilicum, Petunia hybrida, Phaseolus vulgaris, Phytolacca Americana, Pisum sativum, Raphanus sativus, Ricinus communis, Rosa sericea, Salvia splendens, Senecio vulgaris, Solanum lycopersicum, Solanum melongena, Solanum nigrum, Solanum tuberosum, Solanum pimpinellifolium, Spinacia oleracea, Stellaria media, Sweet Wormwood, Trifolium pratense, Trifolium repens, Tropaeolum majus, Tulipa, Vicia faba, Vicia villosa and Viola arvensis. Other plants that may be infected include Zea maize, Hordeum vulgare, Triticum aestivum, Oryza sativa and Oryza glaberrima. 
According to a specific embodiment of the present invention, the plant comprises a Petunia hybrida. 
According to another specific embodiment of the present invention, the plant comprises a Nicotiana tabacum. 
As used herein the phrase “genotypic variation” refers to a process in which a nucleotide or a nucleotide sequence (at least 2 nucleotides) is selectively altered or mutated at a predetermined genomic site, also termed as mutagenesis. The genomic site may be coding or non-coding (e.g., promoter, terminator, splice site, polyA) genomic site. This alteration can be a result of a deletion of nucleic acid(s), a randomized insertion of nucleic acid(s), introduction of a heterologous nucleic acid carrying a desired sequence, or homologous recombination following formation of a DNA double-stranded break (DSB) in the target gene. Genotypic variation according to the present teachings may be transient as explained in further detail hereinbelow. Genotypic variation in accordance with the present teachings is typically effected by the formation of DSBs, though the present invention also contemplates variation of a single strand. Genotypic variation may be associated with phenotypic variation. The sequence specific or site directed nature of the present teachings thus may be used to specifically design phenotypic variation.
As mentioned hereinabove, the method according to this aspect of the present invention is effected by introducing into the plant at least one viral expression vector encoding at least one chimeric nuclease which comprises a DNA binding domain, a nuclease and a nuclear localization signal.
As used herein the phrase “chimeric nuclease” refers to a synthetic chimeric polypeptide which forms a single open reading frame and mediates DNA cleavage in a sequence specific manner.
As used herein the phrase “DNA binding domain” refers to a native or synthetic amino acid sequence such as of a protein motif that binds to double- or single-stranded DNA with affinity to a specific sequence or set thereof (i.e. target site).
In generating chimeric nucleases any DNA binding domain that recognizes the desired DNA binding sequence with sufficient specificity may be employed.
Examples of DNA binding domains include, but are not limited to, helix-turn-helix (pfam 01381), leucine zipper (ZIP) domain, winged helix (WH) domain, winged helix turn helix domain (wHTH), helix-loop-helix and zinc finger domain.
Thus, a variety of such DNA binding domains are known in the art. In an exemplary embodiment of the present invention, the DNA binding domain is a zinc finger binding domain (e.g., pfam00096).
The zinc finger domain is 30 amino acids long and consists of a recognition helix and a 2-strand beta-sheet. The domain also contains four regularly spaced ligands for Zinc (either histidines or cysteines). The Zn ion stabilizes the 3D structure of the domain. Each finger contains one Zn ion and recognizes a specific triplet of DNA basepairs.
Zinc finger domains can be engineered to bind to a predetermined nucleotide sequence. Each individual zinc finger (e.g. Cys2/His2) contacts primarily three consecutive base pairs of DNA in a modular fashion [Pavletich et al., Science (1991) 252:809-817; Berg et al., Science (1996) 271:1081-1085]. By manipulating the number of zinc fingers and the nature of critical amino acid residues that contact DNA directly, DNA binding domains with novel specificities can be evolved and selected [see, e.g., Desjarlais et al., Proc. Natl. Acad. Sci. USA (1992) 89:7345-7349; Rebar et al., Science (1994) 263:671-673; Greisman et al., Science (1997) 275:657-661; Segal et al., Proc. Natl. Acad. Sci. USA (1999) 96:2758-2763]. Hence, a very wide range of DNA sequences can serve as specific recognition targets for zinc finger proteins. Chimeric nucleases with several different specificities based on zinc finger recognition have been previously disclosed [see for example, Huang et al., J. Protein Chem. (1996) 15:481-489; Kim et al., Biol. Chem. (1998) 379:489-495].
Various methods for designing chimeric nucleases with varied DNA binding domains are known in the art. In one embodiment the DNA binding domain comprises at least one, at least two, at least 3, at least 4, at least 5 at least 6 zinc finger domains, binding a 3, 6, 9, 12, 15, or 18 nucleotide sequence, respectively. It will be appreciated by the skilled artisan that the longer the recognition sequence is, the higher the specificity that will be obtained.
Specific DNA binding zinc fingers can be selected by using polypeptide display libraries. The target site is used with the polypeptide display library in an affinity selection step to select variant zinc fingers that bind to the target site. Typically, constant zinc fingers and zinc fingers to be randomized are made from any suitable C2H2 zinc fingers protein, such as SP-1, SP-1C, TFIIIA, GLI, Tramtrack, YY1, or ZIF268 [see, e.g., Jacobs, EMBO J. 11:4507 (1992); Desjarlais & Berg, Proc. Natl. Acad. Sci. U.S.A. 90:2256-2260 (1993)]. The polypeptide display library encoding variants of a zinc finger protein comprising the randomized zinc finger, one or more variants of which will be selected, and, depending on the selection step, one or two constant zinc fingers, is constructed according to the methods known to those in the art. Optionally, the library contains restriction sites designed for ease of removing constant zinc fingers, and for adding in randomized zinc fingers. Zinc fingers are randomized, e.g., by using degenerate oligonucleotides, mutagenic cassettes, or error prone PCR. See, for example, U.S. Pat. Nos. 6,326,166, 6,410,248, and 6479626.
Zinc fingers can also be selected by design. A designed zinc finger protein is a protein not occurring in nature whose design/composition results principally from rational criteria. Rational criteria for design include application of substitution rules and computerized algorithms for processing information in a database storing information of existing ZFP designs and binding data. See, for example, U.S. Pat. Nos. 6,140,081; 6,453,242; and 6,534,261; see also WO 98/53058; WO 98/53059; WO 98/53060; WO 02/016536 and WO 03/016496.
As illustrated in Example 1 hereinbelow, two sets of chimeric nucleases were designed according to the present teachings each set capable of forming DSBs in specific target sequences of Petunia DNA. Initially, DNA binding sequences were identified in Petunia plants which were suitable for recognition and cleavage by chimeric nucleases. These DNA binding sequences were non-coding, non-repetitive sequences: P25-TS1 (SEQ ID NO: 10), P25-TS2 (SEQ ID NO: 11), P36-TS1 (SEQ ID NO: 12) and P36-TS2 (SEQ ID NO: 13). Next, chimeric nucleases were designed each comprising 3 zinc fingers. As illustrated in FIGS. 21-22, these zinc fingers designated P1-25-ZFN1, P1-25-ZFN2, P1-36-ZFN1 and P1-36-ZFN2 (SEQ ID NOs: 35, 36, 37, or 38, respectively) specifically bound and cleaved the above mentioned Petunia target sites.
Furthermore, as illustrated in Example 1 hereinbelow, according to the present teachings, chimeric nucleases were designed capable of forming specific DSBs in Petunia phytoene desaturase (PDS) genomic sequences or in Petunia flavanone 3 beta-hydroxylase (FHT) genomic sequences. Initially, DNA binding sequences for PDS and FHT were identified in Petunia plants which were suitable for recognition and cleavage by such chimeric nucleases (see FIGS. 28A and 29, respectively). The DNA binding sequences for PDS specific zinc fingers were identified: PDS-ZFN1 (SEQ ID NO: 140) and PDS-ZFN2 (SEQ ID NO: 141) and the chimeric nucleases were designed (SEQ ID NOs: 71 and 73, respectively) which specifically bound and cleaved the PDS Petunia target sites. Likewise, the DNA binding sequences for FHT specific zinc fingers were identified: FHT-ZFN1 (SEQ ID NO: 142) and FHT-ZFN2 (SEQ ID NO: 143) and the chimeric nucleases were designed (SEQ ID NOs: 75 and 77, respectively) which specifically bound and cleaved the FHT Petunia target sites.
According to an embodiment of the present invention the zinc finger binding domain comprises a nucleic acid sequence as set forth in SEQ ID NOs. 17, 18, 19, 22, 23, 24, 25, 26, 27, 28, 29 or 30.
Preferably, the chimeric nucleases of this aspect of the present invention comprise separate domains for DNA binding and for DNA cleavage, such that DNA cleavage is sequence specific.
As used herein the phrase “sequence specific” refers to a distinct chromosomal location at which a double stranded break (cleavage) is introduced. Without being bound by theory, it is believed that the formation of DSB induces a cellular repair mechanism which typically leads to highly efficient recombinational events at that locus.
As used herein the term “nuclease” refers to any polypeptide, or complex comprising a polypeptide, that can generate a strand break in genomic DNA (i.e. comprises DNA cleavage activity). Examples of nucleases which may be used in accordance with the present teachings include restriction enzymes, topoisomerases, recombinases, integrases and DNAses.
It will be appreciated that the nuclease utilized by the present invention may comprise any non-specific DNA cleavage domain, for example, a type II restriction endonuclease such as the cleavage domain of the FokI restriction enzyme (GenBank accession number J04623). FokI restriction enzymes which generally have separate DNA cleavage and DNA binding domains are suitable for construction of the chimeric nucleases. Thus, according to an embodiment of this aspect, the chimeric nucleases are chimeric proteins comprising specific zinc finger binding domains and the DNA cleavage domain of the FokI restriction enzyme (also referred to herein as the FokI cleavage domain).
In accordance with embodiments of the present invention the chimeric nuclease is an isolated polynucleotide comprising a nucleic acid sequence as set forth in SEQ ID NO. 31, 32, 33, 34, 70, 72, 74, 76, 84, 86 or 88.
In accordance with embodiments of the present invention the chimeric nuclease is an isolated polypeptide comprising an amino acid sequence as set forth in SEQ ID NO. 35, 36, 37, 38, 71, 73, 75, 77, 85, 87 or 89.
Since certain nucleases (e.g. FokI) function as dimers, in order to create double stranded breaks in the target gene at least two chimeric nuclease must be employed. Thus, according to an exemplary embodiment, the chimeric nucleases of the present invention form dimers (e.g., via binding to both strands of a target sequence). For example, chimeric nucleases can form a homodimer between two identical chimeric nucleases (e.g., via binding to two identical DNA binding sequences within a target sequence). Alternatively, chimeric nucleases can form a heterodimer between two different chimeric nucleases (e.g., via binding to two different DNA binding sequences within a target sequence, see e.g., FIG. 1). Accordingly, two chimeric nucleases may be employed to create a double-stranded break in a target sequence. Consequently, the DNA binding domain of the chimeric nuclease, or two or more conjointly acting chimeric nucleases may bind a DNA sequence.
Examples of nucleases which can be used according to the present teachings include, but are not limited to, restriction enzymes including Fokl, Scel, I-CeuI, artificial meganucleases, modified meganucleases, homing nucleases; topoisomerases including DNA gyrase, eukaryotic topoisomerase II, bacterial topoisomerase IV and topoisomerase VI; recombinases including Cre recombinase, Hin recombinase, Rad51/RecA; DNAses including deoxyribonuclease I, deoxyribonuclease II and micrococcal nuclease; and integrases.
The phrase “DNA-containing organelle” refers to a subcellular, membrane-encapsulated structure, present in all plant cells.
DNA containing organelles include, the mitochondrion, the nucleus, the chloroplast, the proplastid, the etioplast, the chromoplast and the leukoplast, and any subcellular structure which includes DNA molecules. Typically, the DNA is endogenous but in some cases may refer to exogenous DNA such as of a plant pathogen such as a virus. In the latter case, for example, the DNA-containing organelle is the cytoplasm, in which case the chimeric nuclease may not comprise any localization signal.
It will be appreciated that generating genotypic variation in plant organelles other than the nucleus is of particular interest according to some embodiments of the present invention, as will be detailed infra. Plant organelles (e.g. chloroplast and mitochondria) contain DNA which is a vital participant in plant biochemical pathways. These organelles have a wide structural and functional diversity. As such, they are able to transcribe and translate the information present in their own genome but are strongly dependent on imported proteins that are encoded in the nuclear genome and translated in the cytoplasm.
For example, the chloroplast performs essential metabolic and biosynthetic functions of global significance, including photosynthesis, carotenoids and amino acid biosynthesis. Carotenoids are integral constituents of plants, they are isoprenoids pigments which are involved in a variety of processes including protection against photooxidative stress (through energy-dissipation of excess light absorbed by the antenna pigments); coloring agents in flowers and fruits to attract pollinators, and precursors for the plant growth hormone abscisic acid and vitamin A [Cunningham and Gantt (1998) Annu Rev Plant Physiol Plant Mol Biol 49:557-583]. The carotenoid pigments are synthesized in the plastids of plants where it is derived from the pathways of isoprenoid biosynthesis (Cunningham and Gantt, supra). Two biosynthetic pathways for isoprenoid biosynthesis are present in plants, the mevalonate pathway found in the cytoplasm and the methylerythritol 4-phosphate (MEP) pathway found only in the plastids. The latter biosynthetic route being strongly linked to photosynthesis [Seemann et al. (2006) FEBS Lett. 580: 1547-1552].
Moreover, the aromatic amino acid phenylalanine may be synthesized in chloroplasts from the intermediate prephenate: via arogenate by the activity of prephenate aminotransferase or via phenylpyruvate by the activity of prephenate dehydratase [Jung et al. (1986). Proc. Natl. Acad. Sci. 83: 7231-7235; Rippert et al. (2009) Plant Physiol. 149(3): 1251-1260].
Furthermore, the nitrite reductase and acetolactate synthetase activity of the cell is also located in the plastids. The plastids were found to contain only part of the total glutamine synthetase, aspartate aminotransferase, and triosephosphate dehydrogenase activity in the cell [Miflin B (1974) Plant Physiol. 54(4): 550-555]. The chloroplast is also involved in methionine metabolism in plants, chloroplasts are autonomous for de novo methionine synthesis and can import S-adenosylmethionine from the cytosol [Ravanel et al. (2004). J. Biol. Chem. 279 (21): 22548-22557].
Similarly, the mitochondria comprise key roles in cellular metabolic pathways, catalyzing one or several steps in these pathways (e.g. the synthesis of the vitamins folate and biotin, of the non-vitamin coenzyme lipoate, of the cardiolipin diphosphatidylglycerol. Although the mitochondria lack acetyl-CoA carboxylase, it contains the enzymatic equipment necessary to transform malonate into the two main building units for fatty acid synthesis: malonyl- and acetyl-acyl carrier protein (ACP).
Cytoplasmic male sterility (CMS) in plants, characterized by the suppression of the production of viable pollen and by the non-Mendelian inheritance of this trait, is associated with mitochondrial dysfunction. The genetic determinants for cytoplasmic male sterility reside in the mitochondrial genome. CMS phenotype essentially affects the pollen producing organs due to the high requirement of energy by this tissue. Thus, a mitochondrial dysfunction will dramatically affect pollen production while other plant organs may overcome the consequences of mitochondrial dysfunction.
As used herein the phrase “localization domain” refers to a localization signal which facilitates the transport of the chimeric nucleases to the DNA-containing organelle.
The localization signal can be for example, a nuclear localization signal (NLS), such as a short predominantly basic amino acid sequence, which is recognized by specific receptors at the nuclear pores. In other exemplary embodiments, the localization signal for a DNA containing organelle can be a mitochondrial localization signal (MLS) or a chloroplast localization signal (CLS).
Essentially any NLS may be employed, whether synthetic or a naturally occurring NLS, as long as the NLS is one that is compatible with the target cell (i.e. plant cell).
Although nuclear localization signals are discussed herewith, the present teachings are not meant to be restricted to these localization signals, as any signal directed to a DNA-containing organelle is envisaged by the present teachings. Such signals are well known in the art and can be easily retrieved by the skilled artisan.
Nuclear localization signals which may be used according to the present teachings include, but are not limited to, SV40 large T antigen NLS, acidic M9 domain of hnRNP A1, the sequence KIPIK in yeast transcription repressor Matα2 and the complex signals of U snRNPs, tobacco NLS and rice NLS.
Mitochondrion localization signals which may be used according to the present teachings include, but are not limited to the transition signals of, Beta ATPase subunit [cDNAs encoding the mitochondrial pre-sequences from Nicotiana plumbaginifolia β-ATPase (nucleotides 387-666)], Mitochondrial chaperonin CPN-60 [cDNAs encoding the mitochondrial pre-sequences from Arabidopsis thaliana CPN-60 (nucleotides 74-186] and COX4 [the first 25 codons of Saccharomyces cerevisiae COX4 which encodes the mitochondrial targeting sequence].
According to a specific embodiment of the present invention, the localization signal may comprise a mitochondria localization signal, such as the signal peptide of the ATPase beta subunit (ATP-β) (SEQ ID NO: 139).
Chloroplast localization signals which may be used according to the present teachings include, but are not limited to the transition signals of the ribulose-1,5-bisphosphate carboxylase (Rubisco) small subunit (ats1A) associated transit peptide, the transition signal of LHC II, as well as the N-terminal regions of A. thaliana SIG2 and SIG3 ORFs. See also http://wwwdotspringerlinkdotcom/content/p65013h263617795/.
Alternatively, the chloroplast localization sequence (CLS) may be derived from a viroid [Evans and Pradhan (2004) US 2004/0142476 A1]. The viroid may be an Avsunviroiae viroid, for example, an Avocado Sunblotch Viroid (ASBVd), a Peach Latent Mosaic Virus (PLMVd), a Chrysanthemum Chlorotic Mottle Viroid (CChMVd) or an Eggplant Latent Viroid (ELVd).
According to a specific embodiment of the present invention, the localization signal may comprise a chloroplast localization signal, such as the transit peptide ribulose-1,5-bisphospate carboxylase small subunit (Rssu) (SEQ ID NO: 138).
For efficient gene targeting, the DNA binding domain of the present invention needs to be coupled to the nuclease as to permit DNA cleavage within a workable proximity of the target sequence. A workable proximity is any distance that still facilitates the sequence targeting. Optionally, the DNA binding domain overlaps the target sequence or may bind within the target sequence.
Recombinant DNA technology is typically used to generate the chimeric nucleases of the present invention [see Example 1 of the Examples section which follows and Sambrook et al., Eds., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor University Press, New York (1989); Ausubel et al., Eds., Current Protocols in Molecular Biology, John Wiley & Sons, New York (1998); and Maeder, et al. (2008) Mol Cell 31:294-301, as well as other references which are provided hereinbelow].
Qualifying chimeric nucleases thus generated for specific target recognition can be effected using methods which are well known in the art.
A method for designing a chimeric nuclease for use in gene targeting may include a process for testing the toxicity of the chimeric nuclease on a cell. Such a process may comprise expressing in the cell, or otherwise introducing into a cell, the chimeric nuclease and assessing cell growth or death rates by comparison against a control. The tendency of a chimeric nuclease to cleave at more than one position in the genome may be evaluated by in vitro cleavage assays, followed by electrophoresis (e.g. pulsed field electrophoresis may be used to resolve very large fragments) and, optionally, probing or Southern blotting (see Example 5 in the Examples section which follows). In view of the present disclosure, one of ordinary skill in the art may devise other tests for cleavage specificity.
In one specific embodiment, the present invention provides two sets of chimeric nucleases: P1-25-ZFN1 and P1-25-ZFN2 (shown in SEQ ID NO: 35 and 36, respectively) for gene targeting at the P1-25 site 1 (SEQ ID NO: 10) and P1-25 site 2 (SEQ ID NO: 11) of Petunia, respectively; and P1-36-ZFN1 and P1-36-ZFN2 (shown in SEQ ID NO: 37 and 38, respectively) for gene targeting at the P1-36 site 1 (SEQ ID NO: 12) and P1-36 site 2 (SEQ ID NO: 13) of Petunia. In particular, P1-25-ZFN1 and P1-25-ZFN2 can form a dimer and P1-36-ZFN1 and P1-36-ZFN2 can form a dimer for generating specific double stranded breaks in Petunia target genes.
In another embodiment of the present invention there is provided a set of PDS chimeric nucleases: PDS-ZFN1 and PDS-ZFN2 (shown in SEQ ID NO: 71 and 73, respectively) for gene targeting at the PDS site 1 (SEQ ID NO: 140) and PDS site 2 (SEQ ID NO: 141) of Petunia, respectively. These chimeric nucleases can form a dimer for generating specific double stranded breaks in Petunia PDS gene.
In another embodiment of the present invention there is provided a set of FHT chimeric nucleases: FHT-ZFN1 and FHT-ZFN2 (shown in SEQ ID NO: 75 and 77, respectively) for gene targeting at the FHT site 1 (SEQ ID NO: 142) and FHT site 2 (SEQ ID NO: 143) of Petunia, respectively. These chimeric nucleases can form a dimer for generating specific double stranded breaks in Petunia FHT gene.
As mentioned hereinabove, the chimeric nuclease is introduced into the plant target using a viral expression vector, which is typically used for mediating transient transformation, systemically spreading within the plant such as through the meristem infection.
Thus, according to another aspect of the present invention there is provided a plant viral expression vector comprising a nucleic acid sequence encoding at least one chimeric nuclease which comprises a DNA binding domain, a nuclease and optionally a localization signal.
As used herein a plant viral expression vector refers to a nucleic acid vector including a DNA vector (e.g., a plasmid), a RNA vector, virus or other suitable replicon (e.g., viral vector) encoding for viral genes or parts of viral genes.
Viruses that have been shown to be useful for the transformation of plant hosts include CaMV, TMV and BV. Transformation of plants using plant viruses is described in U.S. Pat. No. 4,855,237 (BGV), EP-A 67,553 (TMV), Japanese Published Application No. 63-14693 (TMV), EPA 194,809 (BV), EPA 278,667 (BV); and Gluzman, Y. et al., Communications in Molecular Biology: Viral Vectors, Cold Spring Harbor Laboratory, New York, pp. 172-189 (1988). Pseudovirus particles for use in expressing foreign DNA in many hosts, including plants, is described in WO 87/06261
Other viruses which may be useful in transformation of plant hosts include tobacco rattle virus (TRV) and its related viruses. TRV is known for its ability to infect meristematic tissues, it comprises a broad host range and different strain isolates. For example strain N5, obtained from narcissus, causes severe necrosis in Nicotiana clevelandii [Harrison et al. (1983) Ann. appl. Biol., 102:331-338]. The hypochoeris mosaic virus (HMV), which is serologically related to TRV [Uhde et al. (1998) Archives of Virology 143:1041-1053], infects the Asteraceae family of plants [Brunt and Stace-Smith (1978) Ann. appl. Biol. 90:205-214]. The tobacco rattle virus strain TCM, originally obtained from tulip, is serologically closely related to the Dutch serotype of Pea early-browning virus [Robinson et al., J. Gen. Virol. (1987) 68:2551-2561). Furthermore, there are also monocotyledons species susceptible to TRV, as for example Avena sativa (family Poaceae) [Cadman and Harrison, Ann. appl. Biol. (1959) 47:542-556].
When the virus is a DNA virus, suitable modifications can be made to the virus itself. Alternatively, the virus can first be cloned into a bacterial plasmid for ease of constructing the desired viral vector with the foreign DNA. The virus can then be excised from the plasmid. If the virus is a DNA virus, a bacterial origin of replication can be attached to the viral DNA, which is then replicated by the bacteria. Transcription and translation of this DNA will produce the coat protein which will encapsidate the viral DNA. If the virus is an RNA virus, the virus is generally cloned as a cDNA and inserted into a plasmid. The plasmid is then used to make all of the constructions. The RNA virus is then produced by replicating the viral sequence of the plasmid and translation of the viral genes to produce the coat protein(s) which encapsidate the viral RNA.
Construction of plant RNA viruses for the introduction and expression in plants of non-viral exogenous nucleic acid sequences such as those included in the construct of the present invention is demonstrated by the above references as well as in U.S. Pat. No. 5,316,931, Dawson, W. O. et al. (1989). A tobacco mosaic virus-hybrid expresses and loses an added gene. Virology 172, 285-292; French, R. et al. (1986) Science 231, 1294-1297; and Takamatsu, N. et al. (1990). Production of enkephalin in tobacco protoplasts using tobacco mosaic virus RNA vector. FEBS Lett 269, 73-76.
In one embodiment, a plant viral nucleic acid is provided in which the native coat protein coding sequence has been deleted from a viral nucleic acid, a non-native plant viral coat protein coding sequence and a non-native promoter, preferably the subgenomic promoter of the non-native coat protein coding sequence, capable of expression in the plant host, packaging of the recombinant plant viral nucleic acid, and ensuring a systemic infection of the host by the recombinant plant viral nucleic acid, has been inserted. Alternatively, the coat protein gene may be inactivated by insertion of the non-native nucleic acid sequence within it, such that a protein is produced. The recombinant plant viral nucleic acid may contain one or more additional non-native subgenomic promoters. Each non-native subgenomic promoter is capable of replicating or expressing adjacent genes or nucleic acid sequences in the plant host and incapable of recombination with each other and with native subgenomic promoters. Non-native (foreign, heterologous) nucleic acid sequences may be inserted adjacent the native plant viral subgenomic promoter or the native and a non-native plant viral subgenomic promoters if more than one nucleic acid sequence is included. The non-native nucleic acid sequences are replicated or expressed in the host plant under control of the subgenomic promoter to produce the desired products.
In a second embodiment, a recombinant plant viral nucleic acid is provided as in the first embodiment except that the native coat protein coding sequence is placed adjacent one of the non-native coat protein subgenomic promoters instead of a non-native coat protein coding sequence.
In a third embodiment, a recombinant plant viral nucleic acid is provided in which the native coat protein gene is adjacent its subgenomic promoter and one or more non-native subgenomic promoters have been inserted into the viral nucleic acid. The inserted non-native subgenomic promoters are capable of replicating or expressing adjacent genes in a plant host and are incapable of recombination with each other and with native subgenomic promoters. Non-native nucleic acid sequences may be inserted adjacent the non-native subgenomic plant viral promoters such that said sequences are replicated or expressed in the host plant under control of the subgenomic promoters to produce the desired product.
In a fourth embodiment, a recombinant plant viral nucleic acid is provided as in the third embodiment except that the native coat protein coding sequence is replaced by a non-native coat protein coding sequence.
The viral vectors are encapsidated by the coat proteins encoded by the recombinant plant viral nucleic acid to produce a recombinant plant virus. The recombinant plant viral nucleic acid or recombinant plant virus is used to infect appropriate host plants. The recombinant plant viral nucleic acid is capable of replication in the host, systemic spread in the host, and transcription or expression of foreign gene(s) (isolated nucleic acid) in the host to produce the desired protein i.e., the chimeric nuclease and optionally other heterologous coding or non-coding nucleic acid sequences.
A viral expression vector comprising a nucleic acid encoding a chimeric nuclease is operably linked to one or more transcriptional regulatory sequences whereby the coding sequence is under the control of transcription signals to permit production or synthesis of the chimeric nuclease. Such transcriptional regulatory sequences include promoter sequences, enhancers, and transcription binding sites.
Promoters which are known or found to cause transcription of a foreign gene in plant cells can be used in the present invention. Such promoters may be obtained from plants or viruses and include, but are not limited to, the 35S promoter of cauliflower mosaic virus (CaMV) (includes variations of CaMV 35S promoter, e.g. promoters derived by means of ligations with operator regions, random or controlled mutagenesis, etc.), promoters of seed storage protein genes such as Zma10 Kz or Zmag12 (maize zein and glutelin genes, respectively), light-inducible genes such as ribulose bisphosphate carboxylase small subunit (rbcS), stress induced genes such as alcohol dehydrogenase (Adh1), or “housekeeping genes” that express in all cells (such as Zmaact, a maize actin gene). For added control, the chimeric nuclease may be under the control of an inducible promoter.
In one embodiment the plant viral expression vector is a tobacco rattle virus (TRV) expression vector.
TRV-based expression vectors have been described in for example U.S. Pat. No. 7,229,829.
TRV is a positive strand RNA virus with a bipartite genome, hence the genome is divided into two positive-sense, single-stranded RNAs, that may be separately encapsidated into viral particles. The two TRV genomic RNA vectors used by the present invention are referred to herein as pTRV1 (GeneBank Accession No: AF406990) and pTRV2 (GeneBank Accession No: AF406991), wherein pTRV1 encodes polypeptides that mediate replication and movement in the host plant while pTRV2 encodes coat proteins.
In certain embodiments, the nucleic acid sequence of pTRV2 is devoid of 2b sequence (SEQ ID NO: 43). Generating a pTRV2 vector devoid of the 300 by of the RNA2 2b gene was carried out by removal from the original vector by digestion with PvuII and EcoRI (see FIGS. 6A-B). The resultant plasmid (pTRV2Δ2b) was identical to the original pTRV2 but lacking the 2b sequence (see Example 1, hereinbelow). According to the present teachings, pTRV2 vectors without the 2b region are much more efficient in gene expression in meristematic tissues (see Example 2, hereinbelow).
In certain embodiments, modification to pTRV2 vector comprises addition of an enhancer. Any enhancer can be inserted into the viral expression vector to enhance transcription levels of genes. For example, a Ω enhancer (SEQ ID NOs: 44 or 47) can be cloned into the pTRV2 vectors of the present invention.
Alternatively, the viral vector of the present invention may be based on TRV related viruses (e.g. tobacco rattle virus strain N5, HMV, or tobacco rattle virus strain TCM).
The selection of the vector may be dependent on the target plant such as monocots. The modified wheat streak mosaic virus (WSMV) has been previously shown to express NPT II and β-glucuronidase (GUS) in monocots (e.g. wheat, barley, oat and maize) [Choi et al., Plant J. (2000) 23:547-555; Choi et al., J Gen Virol (2002) 83:443-450; Choi et al., J. Gen. Virol. (2005) 86:2605-2614]. The work of Choi et al. demonstrated the placement of the foreign genes between the nuclear inclusion b (NIb) and coat protein (CP). For better expression and activity in infected wheat, GUS was inserted immediately downstream of the P1 cleavage site and up stream to HC-Pro of the wheat streak mosaic virus (WSMV) polyprotein ORF. Systemic infection and GUS expression was demonstrated upon inoculation of plants with WSMV in vitro.
The present invention contemplates a viral expression vector comprising at least two heterologous polypeptide sequences.
As used herein the term “heterologous sequence” refers to a sequence that is not normally part of an RNA2 of a naturally occurring TRV. In certain embodiments, a heterologous sequence is a chimeric nuclease as described in detail hereinabove. In certain embodiments, a heterologous sequence is a sequence of interest, such as a plant gene for expression in a plant cell of a heterologous polypeptide. Such plant genes may include, but are not limited to, genes encoding a reporter polypeptide, an antiviral polypeptide, a viral moiety, an antifungal polypeptide, an antibacterial polypeptide, an insect resistance polypeptide, a herbicide resistance polypeptide, a biotic or abiotic stress tolerance polypeptide, a pharmaceutical polypeptide, a growth inducing polypeptide, and a growth inhibiting polypeptide. In certain embodiments, the viral vector comprises both chimeric nucleases and a sequence of interest.
As part of the pTRV vector, the heterologous sequences may comprise separate sub genomic promoters (sgPs), thus may comprise two separate sgPs (e.g. SEQ ID NO: 45 and SEQ ID NO: 48) for replication of the heterologous sequences.
In certain embodiments, the at least two heterologous polypeptide sequences within the viral vector are separated by nucleic acid sequence encoding a cleavage domain. Such a cleavage domain may comprise any cleavage domain known in the art, as for example a T2A-like protein sequence (SEQ ID NOs: 40 and 52).
It will be appreciated that the nucleic acid sequence of the two heterologous polypeptide sequences separated by a cleavage domain may be as set forth in SEQ ID NOs: 84, 86 or 88.
It will be appreciated that the amino acid sequence of the two heterologous polypeptide sequences separated by a cleavage domain may be as set forth in SEQ ID NOs: 85, 87 or 89.
Generally, when introduced into a host plant cell, a pTRV vector provides expression of the heterologous sequence(s) and may also provide expression of other TRV sequences, such as a viral coat protein.
pTRV vectors of the present invention may express a reporter gene so that transformed cells can be identified. Exemplary reporter genes that may be expressed include, but are not limited to, GUS and GFP.
It will be appreciated that two viral expression vectors may be introduced into the same plant cell. These viral vectors may be introduced in the plant cell concomitantly or at separate times. Such viral expression vectors may comprise the same type of vector encoding different heterologous sequences, or alternatively may comprise two different types of vectors (e.g. BV vector and TRV vector, mitovirus vector and TRV vector, TRV1 and TRV2 vectors). For example, pTRV1 and pTRV2 vectors can be introduced concomitantly, as for example at a 1:1 ratio, to enable expression of viral genes in plant cells. Likewise, one vector may comprise the chimeric nuclease/s and another vector may comprise a heterologous gene of interest (as described in detail hereinabove).
It will be appreciated that in order to introduce the heterologous gene of interest (i.e. foreign DNA) into different DNA containing organelles (e.g. nucleus, chloroplast and mitochondria), different types of vectors may be implemented.
Thus, vectors for delivery of foreign DNA may be based on the Geminivirus Abutilon mosaic virus (AbMV), a member of the Begomovirus genus. The AbMV viral DNA has been detected in plastids [Groning et al., (1987) PNAS USA 84: 8996; Groning et al. (1990) Mol. Gen. Gene. 220: 485; Horns & Jeske, (1991) Virol. 181: 580].
The viral vector of the present invention may also be based on the genus Mitovirus, family Narnaviridae such as H. mompa mitovirus 1-18 (HmMV1-18) or O. novo-ulmi mitovirus 6 (OnuMV6). The HmMV1-18 viral dsRNA has been detected in mitochondria [Osaki et al (2005) Virus res. 107, 39-46; Cole et al (2000) Virol. 268, 239-243].
Other DNA virus based vectors that are envisioned by the present invention include, for example, Geminiviridae, Caulimoviridae and Badnaviridae.
For example, Geminiviridae contain circular covalently closed single-stranded (ss) DNA (−2.8 Kbp) genomes, packaged within twinned (so-called geminate) particles.
The sequences regulating DNA replication and transcriptional activity are located in the intergenic regions (IR). The invariant TAATATT_AC sequence is located in the LIR (in mastreviruses), IR (in curtoviruses) and CR (in begomoviruses) and contains the initiation site of rolling-circle DNA replication. The geminivirus replication cycle can be subdivided in several functionally distinct stages. Early during the infection process, viral particles are injected by the insect vector, presumably uncoated, and the viral genome is transported into the host cell nucleus where all later stages occur: conversion of circular ssDNA into covalently closed circular dsDNA intermediates, rolling-circle replication (RCR), production of circular ssDNA genomes for encapsidation [Gutierrez (1999) Cell. Mol. Life Sci. 56 313-329].
Geminiviruses are divided into four genera on the basis of their genome organizations and biological properties [Fauquet et al (2003) Arch Virol 148: 405-421]. Mastreviruses (e.g. Maize streak virus, Panicum streak virus, Sugarcane streak virus, Sugarcane streak Egypt virus, Sugarcane streak Reunion virus, Digitaria streak virus, etc) have monopartite genomes and are transmitted by leafhopper to monocotyledonous plants. Curtoviruses (e.g. Beet curly top virus) have monopartite genomes distinct from those of the mastreviruses and are transmitted by leafhopper vectors to dicotyledonous plants. Topocuviruses (e.g. Tomato pseudo-curly top virus) have monopartite genomes which are transmitted by a treehopper vector to dicotyledonous plants. Begomoviruses (e.g. Bean golden yellow mosaic virus, Tomato yellow leaf curl virus, Abutilon mosaic virus, Tobacco leaf curl virus, African cassava mosaic virus, Mung bean yellow mosaic virus) have bipartite genomes (although numerous begomoviruses with a monopartite genome also occur) and are transmitted by the whitefly Bemisia tabaci to dicotyledonous plants.
Caulimovirus particles contain a single molecule of dsDNA ( ̃8 kbp). Caulimoviruses usually infect hosts systemically; they are found in most mesophyll, parenchyma and epidermal cells and sometimes in phloem sieve tubes and tracheids. Members of the genus include e.g. Cauliflower mosaic virus (CaMV), Soybean chlorotic mottle (SoyCMV), Cassava vein mosaic (CVMV), Petunia vein clearing (PVCV), Rice tungro bacilliform virus (RTBV).
It will be appreciated that the universal vector IL-60 and auxiliary constructs, which has been recently described [WO 2007/141790] may also be used by the present invention. This vector which is, in fact, a disarmed form of Tomato yellow leaf curl virus (begomovirus), is applied as a double-stranded DNA [Peretz et al (2007) Plant Physiology 145:1251-1263]. With IL-60 as the disarmed helper “virus”, transactivation occurs, resulting in an inducible expression/silencing system.
In order to direct the vectors containing the foreign DNA into specific DNA containing organelles, a nuclear localization signal (NLS), chloroplast localization signal (CLS) or mitochondria localization signal (MLS) may be introduced inframe to the heterologous sequence (as is described in further detail hereinabove).
To achieve transformation of plant cells or the whole plant, the viral expression vectors of the present invention can be introduced into the host cell by any method known in the art. For example, transient transformation can be achieved by Agrobacterium-mediated gene transfer, by direct DNA transfer methods, by viral infection (i.e. using the modified plant viruses) or by nematodes, by infiltration, by vacuum, by electroporation or by bombardment.
Agrobacterium-mediated gene transfer as disclosed herein (see for example, Example 1 hereinbelow) includes the use of plasmid vectors that contain defined DNA segments. For example, the present invention teaches the use of Agrobacterium tumefaciens (strain AGLO and EHA-105) transformed with pTRV1, pTRV2 and pTRV2 derivatives containing plasmids as was previously described [see e.g. Liu et al., Plant J (2002) 30: 415-429]. Methods of inoculation of the plant tissue vary depending upon the plant species and the Agrobacterium delivery system. A widely used approach is the leaf-disc procedure, which can be performed with any tissue explant that provides a good source for initiation of whole-plant differentiation [Horsch, R. B. et al. (1988). “Leaf disc transformation.” Plant Molecular Biology Manual AS, 1-9, Kluwer Academic Publishers, Dordrecht]. A supplementary approach employs the Agrobacterium delivery system in combination with vacuum infiltration. The Agrobacterium system is especially useful for in the creation of transgenic dicotyledenous plants. See: Klee, H. J. et al. (1987). Annu Rev Plant Physiol 38, 467-486; Klee, H. J. and Rogers, S. G. (1989). Cell Culture and Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of Plant Nuclear Genes, pp. 2-25, J. Schell and L. K. Vasil, eds., Academic Publishers, San Diego, Cal.; and Gatenby, A. A. (1989). Regulation and Expression of Plant Genes in Microorganisms, pp. 93-112, Plant Biotechnology, S. Kung and C. J. Arntzen, eds., Butterworth Publishers, Boston, Mass. The present teachings also disclose Agrobacterium-mediated gene transfer by injection of Agrobacteria into the plant (e.g. into the exposed shoot surface following removal of the apical meristems) and by leaf infiltration as for example using a syringe without a needle (e.g. Agrobacteria content of the syringe is discharged into the scratched surface of the leaf, see Example 1 of the example section which follows).
Direct DNA transfer methods include for example electroporation, microinjection and microparticle bombardment. See, e.g.: Paszkowski, J. et al. (1989). Cell Culture and Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of Plant Nuclear Genes, pp. 52-68, J. Schell and L. K. Vasil, eds., Academic Publishers, San Diego, Cal.; and Toriyama, K. et al. (1988). Bio/Technol 6, 1072-1074 (methods for direct uptake of DNA into protoplasts). These methods may further be used to direct the foreign DNA containing vectors (as depicted in detail hereinabove) into specific DNA containing organelles. For example, tobacco protoplasts were electroporated co-transformed with both DNA encoding the nuclease and donor DNA [Wright et al. (2005) Plant J 44:693-705].
Infection of viral vectors (e.g. pTRV) into plants can also be carried out by the use of nematodes, including without limitation, N. benthamiana or N. clevelandii (the natural host for TRV). Accordingly, N. benthamiana or N. clevelandii are inoculated with pTRV1, pTRV2 or their derivatives prior to subjection to the plants.
Infection of viral vectors into plants may also be effected by virion infection (as depicted in detail in Example 1, hereinbelow). Virion infection may be carried out, for example, by first inoculating the usual hosts of the virus (e.g. TRV infection of Petunia) with the viral vector (pTRV1, pTRV2, or its derivatives). About 5 to 21 days post infection (dpi) plant leaves are collected and the sap is extracted in 20 mM phosphate buffer pH=6.8 and a surfactant (e.g. 0-0.03% Silwet L-77) by mortar and pestle. The TRV containing sap is then dripped onto cheesecloth or centrifuged to remove cells debris and following addition of carborundum fine powder (to improve infection) stems and leaves of young (approximately 1 month old) plants are gently scratched. Sap infection of in-vitro grown plants may also be carried by first passing the sap through 0.22 μm filter and then stems of tissue culture propagated plants are injured and infected using syringe and needle or by vacuum. For seeds infection (e.g. monocotyledon), seeds may be incubated with the sap during swelling and sprouting (for approximately 1 to 2 weeks).
A transgenic whole plant, callus, tissue or plant cell may be identified and isolated by selecting or screening the engineered plant material for traits encoded by the marker genes present on the viral expression vectors. For instance, selection may be performed by growing the engineered plant material on media containing an inhibitory amount of the antibiotic or herbicide to which the transforming gene construct confers resistance. Further, transgenic plants and plant cells may also be identified by screening for the activities of any visible marker genes (e.g., GFP or GUS) that may be present on the viral expression vectors. Such selection and screening methodologies are well known to those skilled in the art.
Physical and biochemical methods may also be employed to identify transgenic plants or plant cells containing inserted gene constructs. These methods include, but are not limited to, Southern analysis or PCR amplification, Northern blot, enzymatic assays, protein gel electrophoresis, Western blot techniques, immunoprecipitation, or enzyme-linked immunoassays. Additional techniques, such as in situ hybridization, enzyme staining, and immunostaining, also may be used to detect the presence or expression of the heterologous genes in specific plant organs and tissues. The methods for doing all these assays are well known to those skilled in the art.
Other references which may be used to implement the teachings of the present invention are provided infra: Agrobacterium delivery of a Ti plasmid harboring both the ZFNs and a donor DNA construct [Cai et al. (2009) Plant Mol Biol. Accepted: 14 Dec. 2008].
The above mentioned methods, chimeric nucleases and vectors may be used for generating genotypic variation in plants.
The following section provides non-limiting applications for generating such a variation.
Thus, chimeric nucleases of the present invention may be used to generate a signature of randomly inserted nucleic acids in a sequence-specific manner, also referred to herein as tagging. This signature may be used as a “genetic mark”. This term is used herein distinctively from the common term “genetic marker”. While the latter term refers to naturally occurring genetic variations among individuals in a population, the term genetic mark as used herein specifically refers to artificial (man generated), detectable genetic variability, which may be inherited.
The DSB is typically directed into non-coding regions (non open reading frame sequence) so as not to affect the plant's phenotype (e.g. for tagging). However, tagging can also be directed to a coding region. A high quality genetic mark is selected unique to the genome of the plant and endures sequence variation which may be introduced along the generations.
For some, e.g., regulatory, purposes it may be desired to mark commercially distributed plants with publicly known marks, so as to enable regulatory authorities to readily identify the mark, so as to identify the manufacturer, distributor, owner or user of the marked organism. For other purposes secrecy may be advantageous. The latter is true, for example, for preventing an attempt to genetically modify the genetic mark of a supreme event protected by intellectual property laws.
An intellectual property protected organism which is also subject to regulation will therefore be, according to a useful embodiment of the present invention, genetically marked by (a) at least one unique DNA sequence which is known in public; and (b) at least one unique DNA sequence that is unknown, at least not as a genetic mark, in public.
To introduce a heterologous sequence (e.g., coding or non-coding), DSBs will first be generated in plant DNA as described herein. It is well known those of skill in the art that integration of foreign DNA occurs with high frequency in these DNA brake sites [Salomon et al., EMBO J (1998) 17: 6086-6095; Tzfira et al., Plant Physiol (2003) 133: 1011-1023; Tzfira et al., Trends Genet (2004) 20: 375-383, Cai et al. (2009) Plant Mol Biol. Accepted: 14 Dec. 2008]. Once present in the target cell, for example on episomal plasmids, foreign DNA may be cut out from the plasmid using the same ZFN used to generate DSBs in the plant DNA. The foreign DNA released from the episomal plasmid will then be incorporated into the cell DNA by plant non-homologous end joining (NHEJ) proteins. The DSBs may also lead to enhanced homologous recombination (HR)-based gene targeting in plant cells (Puchta et al. Proc Natl Acad Sci USA (1996) 93: 5055-5060).
As mentioned, the present teachings can be used to generate genotypic variation. Thus, the chimeric nucleases of the present teachings can be designed to generate DSBs in coding or non-coding regions of a locus of interest so as to introduce the heterologous gene of interest. Such alterations in the plant genome may consequently lead to additions or alterations in plant gene expression (described in detail hereinabove) and in plant phenotypic characteristics (e.g. color, scent etc.).
Additionally chimeric nucleases can be used to generate genotypic variation by knocking out gene expression. Thus chimeric nucleases can be designed to generate DSBs in coding or non-coding regions of a locus of interest so as to generate a non-sense or mis-sense mutation. Alternatively, two pairs of chimeric nucleases can be used to cleave out an entire sequence of the genome, thereby knocking out gene expression.
Chimeric nucleases of the present invention may also be used to generate variability by introducing non-specific mutations into the plant's genome. This may be achieved by the use of non-specific DNA restrictases or Non-stringent Fok1.
As an alternative, the chimeric nucleases of the present invention may be used to combat infections by plant pathogens.
Thus the present invention envisages a method of treating a plant infection by a pathogen. The method comprising introducing into the plant at least one expression vector encoding at least one chimeric nuclease which comprises a DNA binding domain and a nuclease, wherein the DNA binding domain mediates targeting of the nuclease to the genome of the pathogen, thereby of preventing or treating a plant infection by a pathogen.
As used herein a “plant pathogen” refers to an organism, which causes a disease in the infected plant. Organisms that cause infectious disease include fungi, oomycetes, bacteria, viruses, viroids, virus-like organisms, phytoplasmas, protozoa, nematodes and parasitic plants.
Since complete destruction of the DNA of the pathogen is desired, the chimeric nuclease is designed so as to cleave as much sequence sites on the pathogen's DNA as possible. Thus, repeating sequences may be targeted. Additionally or alternatively a number of distinct sequences are targeted sufficient to induce degradation of the pathogen's DNA.
According to some embodiments of this aspect of the present invention, the chimeric nuclease is designed to cleave the DNA of the pathogen but not that of the plant. To this end, the chimeric nuclease is designed devoid of a localization signal, such that the chimeric nuclease is active in the cytoplasm which comprises the pathogen's (e.g., virus) DNA but not that of the plant.
Alternatively, the nuclease may be designed so as to cleave sequences which are specific for the pathogen but are absent from the plant's genome. This may be achieved using routine bioinformatics analysis such as by the use of alignment software e.g., Blast (http://wwwdotncbidotnlmdotnihdotgov/blast/Blastdotcgi).
A non-limiting list of plant viral pathogens which may be targeted using the teachings of the present invention include, but are not limited to Species: Pea early-browning virus (PEBV), Genus: Tobravirus. Species: Pepper ringspot virus (PepRSV), Genus: Tobravirus. Species: Watermelon mosaic virus (WMV), Genus: Potyvirus and other viruses from the Potyvirus Genus. Species: Tobacco mosaic virus Genus (TMV), Tobamovirus and other viruses from the Tobamovirus Genus. Species: Potato virus X Genus (PVX), Potexvirus and other viruses from the Potexvirus Genus. Thus the present teachings envisage targeting of RNA as well as DNA viruses (e.g. Gemini virus or Bigeminivirus). Geminiviridae viruses which may be targeted include, but are not limited to, Abutilon mosaic bigeminivirus, Ageratum yellow vein bigeminivirus, Bean calico mosaic bigeminivirus, Bean golden mosaic bigeminivirus, Bhendi yellow vein mosaic bigeminivirus, Cassava African mosaic bigeminivirus, Cassava Indian mosaic bigeminivirus, Chino del tomaté bigeminivirus, Cotton leaf crumple bigeminivirus, Cotton leaf curl bigeminivirus, Croton yellow vein mosaic bigeminivirus, Dolichos yellow mosaic bigeminivirus, Euphorbia mosaic bigeminivirus, Horsegram yellow mosaic bigeminivirus, Jatropha mosaic bigeminivirus, Lima bean golden mosaic bigeminivirus, Melon leaf curl bigeminivirus, Mung bean yellow mosaic bigeminivirus, Okra leaf-curl bigeminivirus, Pepper hausteco bigeminivirus, Pepper Texas bigeminivirus, Potato yellow mosaic bigeminivirus, Rhynchosia mosaic bigeminivirus, Serrano golden mosaic bigeminivirus, Squash leaf curl bigeminivirus, Tobacco leaf curl bigeminivirus, Tomato Australian leafcurl bigeminivirus, Tomato golden mosaic bigeminivirus, Tomato Indian leafcurl bigeminivirus, Tomato leaf crumple bigeminivirus, Tomato mottle bigeminivirus, Tomato yellow leaf curl bigeminivirus, Tomato yellow mosaic bigeminivirus, Watermelon chlorotic stunt bigeminivirus and Watermelon curly mottle bigeminivirus.
The present invention also envisages a method of generate male sterility in a plant. The method comprising upregulating in the plant a structural or functional gene of a mitochondria or chloroplast associated with male sterility by introducing into the plant at least one viral expression vector encoding at least one chimeric nuclease which comprises a DNA binding domain, a nuclease and a mitochondria or chloroplast localization signal and a nucleic acid expression construct which comprises at least one heterologous nucleic acid sequence which can upregulate the structural or functional gene of a mitochondria or chloroplast when targeted into the genome of the mitochondria or chloroplast, wherein the DNA binding domain mediates targeting of the heterologous nucleic acid sequence to the genome of the mitochondria or chloroplast, thereby generating male sterility in the plant.
Thus for example, the nucleic acid construct comprises a coding (e.g., for a CMS associated gene) or non-coding (e.g., powerful promoter for enhancing expression of a CMS associated gene) heterologous nucleic acid sequence as well as a binding site for the chimeric nuclease (identical to that on the mitochondria or chloroplast genome). Upon cleavage by the chimeric nuclease, the heterologous nucleic acid sequence is inserted into the predetermined site in the genome of the chloroplast or mitochondria.
As mentioned hereinabove, cytoplasmic male sterility (CMS) is associated with mitochondrial dysfunction. To this effect, the chimeric nucleases are designed to comprise a mitochondria localization signal (as described in detail hereinabove) and cleavage sites which are specific for the mitochondrial genome. Specific genes which may be upregulated include, but are not limited to, the Petunia pcf chimera that is located with close proximity to nad3 and rps12, the Rice (Oryza sativa) sequence which is downstream of B-atp6 gene (i.e. orf79), the Maize T-urf13 and orf221, the Helianthus sp. orf239 downstream to atpA, the Brassica sp. orfs which are upstream to atp6 (e.g. orf139 orf224 or orf138 and orf158). It will be appreciated that in order to induce CMS, these genomic sequences are typically transcribed in the plant, thus the teachings of the present invention envision targeting these sequences (e.g. by adding coding sequences) or overexpression thereof using the above described methods as to achieve CMS.
It will be appreciated that CMS phenotype, generated by the incompatibility between the nuclear and the mitochondrial genomes, is used as an important agronomical trait which prevents inbreeding and favors hybrid production.
As mentioned hereinabove, induction of CMS can also be achieved by overexpression of a chloroplast gene such as β-ketothiolase. Overexpression of β-ketothiolase via the chloroplast genome has been previously shown to induce CMS [Ruiz at al (2005) Plant Physiol. 138 1232-1246]. Thus, the present teachings also envision targeting chloroplast genes or overexpression thereof (e.g. β-ketothiolase) using the above described methods in order to achieve CMS.
The present invention further envisages a method of generating a herbicide resistant plant. The method comprising introducing into the plant at least one viral expression vector encoding at least one chimeric nuclease which comprises a DNA binding domain, a nuclease and a chloroplast localization signal, wherein the DNA binding domain mediates targeting of the nuclease to a gene conferring sensitivity to herbicides, thereby generating the herbicide resistant plant.
It will be appreciated that in the field of genetically modified plants, it is well desired to engineer plants which are resistant to herbicides. Furthermore, most of the herbicides target pathways that reside within plastids (e.g. within the chloroplast). Thus to generate herbicide resistant plants, the chimeric nucleases are designed to comprise a chloroplast localization signal (as described in detail hereinabove) and cleavage sites which are specific for the chloroplast genome. Specific genes which may be targeted in the chloroplast genome include, but are not limited to, the chloroplast gene psbA (which codes for the photosynthetic quinone-binding membrane protein QB, the target of the herbicide atrazine) and the gene for EPSP synthase (a nuclear gene, however, its overexpression or accumulation in the chloroplast enables plant resistance to the herbicide glyphosate as it increases the rate of transcription of EPSPs as well as by a reduced turnover of the enzyme).
Chimeric nucleases and expression constructs of the present teachings may, if desired, be presented in a pack or dispenser device or kit. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for use.
It is expected that during the life of a patent maturing from this application many relevant viral vectors and chimeric nucleases will be developed and the scope of these terms is intended to include all such new technologies a priori.
As used herein the term “about” refers to ±10%
The terms “comprises”, “comprising”, “includes”, “including”, “having” and their conjugates mean “including but not limited to”. This term encompasses the terms “consisting of” and “consisting essentially of”.
The phrase “consisting essentially of” means that the composition or method may include additional ingredients and/or steps, but only if the additional ingredients and/or steps do not materially alter the basic and novel characteristics of the claimed composition or method.
As used herein, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a compound” or “at least one compound” may include a plurality of compounds, including mixtures thereof.
Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.
As used herein the term “method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
As used herein, the term “treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.
It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.
Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.
EXAMPLES
Reference is now made to the following examples, which together with the above descriptions, illustrate the invention in a non limiting fashion.
Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, “Molecular Cloning: A laboratory Manual” Sambrook et al., (1989); “Current Protocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., “Current Protocols in Molecular Biology”, John Wiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide to Molecular Cloning”, John Wiley & Sons, New York (1988); Watson et al., “Recombinant DNA”, Scientific American Books, New York; Birren et al. (eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis, J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J., eds. (1985); “Transcription and Translation” Hames, B. D., and Higgins S. J., Eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide to Molecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317, Academic Press; “PCR Protocols: A Guide To Methods And Applications”, Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategies for Protein Purification and Characterization—A Laboratory Course Manual” CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.
Example 1
Generation of Viral Vectors and Zinc Finger Nucleases
Plant Material
Rooted plantlets of Petunia hybrida lines B1, P720, Burgundy and Royal Blue (Danziger-“Dan” Flower Farm, Mishmar Hashiva, Israel) were routinely used for Agrobacterium tumefaciens infection for transient expression of foreign genes.
Other plants used for inoculation experiments included Solanum pimpinellifolium La121 (Davis University Gene bank), Capsicum annuum indra1750 (S&G Syngeta global LTD.), Arabidopsis thaliana [Greenboim-Wainberg et al. (2005) Plant Cell, 17: 92-102], Artemisia sp. (Danziger-“Dan” Flower Farm, Mishmar Hashiva, Israel), Nicotiana benthamiana [Radian-Sade et al., Phytoparasitica (2000) 28:79-86], Spinacia oleracea and Beta vulgaris (Eden Seeds, Reut, Israel), Nicotiana tabacum CV SAMSUNG [Levy et al. (2004) Plant Physiol 135:1-6], Nicotiana tabacum CV XANTHI [Ovadis et al. (1999) In “Plant Biotechnology and In Vitro Biology”, Kluwer Academic Press, the Netherlands, pp. 189-192], Cucumis sativus (Eden Seeds, Reut, Israel), Solanum melongena (M. Ben-Sachar LTD, Tel-Aviv, Israel), Gossypium hirsutum cv. Siv'on [Saranga et al (2004) Plant, Cell and Environment 27, 263-277) Brassica napus [Nesi et al., C. R. Biologies 331 (2008) 763-771], Zea mays Var. Royalty F1 (M. Ben-Sachar LTD, Tel-Aviv, Israel). All plants were grown in a greenhouse under 25° C./20° C. day/night temperatures and under a natural photoperiod.
Plasmid Construction
pTRV1 a pYL44 binary T-DNA vector carrying the entire sequence of cDNA corresponding to TRV Ppk20 strain RNA1 (GenBank accession No. AF406990) and pTRV2 (GenBank accession No. AF406991) vectors were used [Liu et al. Plant J (2002) 30: 415-429]. pTRV2 containing GUS (pTRV2-Gus) were generated by cloning GUS from pBI101 (Clontech Laboratories) into MCS (XbaI-SacI sites) of pTRV2. pTRV2 containing PAP1 (pTRV2-Δ2b-Pap) were generated by cloning PAP1 from pCHFS-PAP1 [Borevitz et al., The Plant Cell (2000) 12:2383-2393] into MSC (EcoRI-BamHI sites) of pTRV2-Δ2b.
Generating pTRV2 for transient expression of target genes was carried out by removal of the 300 by of the RNA2 2b gene from the original vector. For this, pTRV2 DNA was digested with PvuII and EcoRI and the deleted fragment containing part of 2b was replaced with a PCR fragment. This fragment was generated by PCR using pTRV2 DNA as a template and primers A and B (see Table 1, below). It was digested with PvuII and EcoRI prior to recloning. The resultant plasmid (pTRV2Δ2b) was identical to the original pTRV2 but lacking the 2b sequence. The plasmid pTRV2 without 2b but with GUS (pTRV2Δ2b-Gus) was generated in the same way except that pTRV2-Gus was used as a recipient plasmid instead of pTRV2.
For generation of pTRV2 containing full length of TRV 2b (pTRV2-2b) a PCR fragment of 1.5 Kbp was generated with primers A and C (see Table 1, below) using as a template pK202b-GFP [Vellios et al., Virology (2002) 300:118-124]. The amplified fragment included the 2b gene (from Ppk20 strain GenBank accession No. Z36974) with 5′ and 3′ UTRs plus sub-genomic-promoter (sg-P) of the coat protein (CP) from Pea Early Browning Virus (PEBV) (GenBank accession no X78455). This sg-P was sited downstream to 2b. For generation of pTRV2-2b containing GFP (GenBank accession No. U62637) downstream to sg-P (pTRV2-2b-Gfp), the PvuII-EcoRI fragment was transferred from pK202b-GFP to pTRV2 digested with the same enzymes.
For generation of pTRV2Δ2b containing sg-P with downstream GFP (pTRV2Δ2b-Gfp), a PCR fragment was prepared using primers F and G* (see Table 1, below) and pK202b-GFP as a template. This fragment was then digested with Sad and SmaI and cloned into the pTRV2Δ2b digested with the same enzymes. * Of note, Primer G adds a silence mutation to Gfp in order to eliminate a Sad site upstream to the termination codon.
A Gus gene with and without Ω [Broido et al., Physiologia Plantarum (1993) 88: 259-266] was cloned into the MCS of pTRV2Δ2b-Gfp, upstream to sg-P of PEBV to generate pTRV2Δ2b-ΩGus-Gfp and pTRV2Δ2b-Gus-Gfp, respectively. The GUS fragment was generated following digestion of pTRV2-Gus by EcoRI and Sad. ΩGus fragment was generated by cloning GUS into SalI-BamHI sites downstream to the Ω sequence of TMV (Broido et al., supra) into pDrive (Qiagen) and then digesting the resultant plasmid with XbaI-KpnI to release ΩGus and reconstruct it into pTRV2Δ2b-Gfp.
pTRV2Δ2b containing ΩGus (pTRV2Δ2b-ΩGus) was generated by cloning GUS into SalI-BamHI sites downstream to the Ω sequence of TMV (Broido et al., supra) into a pBluescript SK+ (Stratagen) and then digesting the resultant plasmid with KpnI-SacI to release ΩGus and reconstruct it into pTRV2Δ2b.
A pTRV2Δ2b carrying the Tomato bushy stunt virus silencing suppressor p19 (pTRV2Δ2b-p19) was constructed by transferring a 519-bp PCR fragment encoding p19 (using primers D and E, see Table 1, below) from pCB301-p19 [Voinnet et al., Plant J. (2003) 33: 949-956] into pTRV2Δ2b (Obermeier et al., Phytopathology (2001) 91:797-806].
All newly formed pTRV2 constructs were first transformed into E. coli and into Agrobacterium tumefaciens AGLO [Zuker et al Mol. Breeding (1999) 5:367-375]. Gus activity in Agrobacterium was evaluated qualitatively with X-gluc solution as previously described [Zuker et al., supra]. GFP expression in Agrobacterium was analyzed qualitatively using fluorescence stereomicroscope (Leica Microsystems, Wetzlar, Germany).
pTRV2Δ2b containing sg-P with downstream DsRed2 (DsRed, GenBank accession no AY818373 nucleotides 1395-2074 of DsRed, SEQ ID NO: 129, pTRV-Δ2b-sgP-DsRed) was generated by preparing a PCR fragment using as a template pSAT6-DsRed2-N1 and primers H and I (see Table 1, below). The PCR product was then digested with HpaI and SacI, blunt ended with T4 DNA polymerase and cloned (instead of GFP) into the pTRV-2A2b-GFP digested with HpaI and SmaI.
Two different combinations of two fluorescence genes in pTRV2 were constructed:
A 2A-like 54 nucleotide sequence (GenBank accession no AF062037 nucleotides 502-555, SEQ ID NO: 81), a Thosea asigna virus (TaV-T2A) [Donnelly et al (2001) J. Gen. Virol, 82, 1027-1041; Osborn et al (2005) Mol. Therapy, 12, 569-574], was utilized to create a bicistronic plasmid vector encoding a single, long, ORF consisting of the DsRed2 gene and the NLS-EGFP genes. The sequence (coding for an 18 amino acid peptide), when inserted into single RNA molecule containing two ORFs, allowed separate translation for the two ORFs [Donnelly et al (2001) supra; Osborn et al (2005), supra].
The T2A sequence was modified at the nucleotide level, based on Petunia codon usage (httpllworldwidewebdotkazusadotordotjp/codon/cgi-bin/showcodon.cgi?species=4102) and the modified sequence was termed pTRV-T2A (see FIG. 2A).
A plasmid containing the pTRV-T2A sequence inserted between DsRed2 and NLS-EGFP was generated by first generating a PCR fragment using pSAT6-NLS-P1-36-ZFN1 as a template following triple PCR reaction with two foreword primers J & K (Table 1, below) and reverse primer S (Table 1, below) for FoKI. The resultant product was cloned into BamHI and Sad sites of pBluescript SK (pBS) lacking XhoI site, to generate pBS-T2A-P36-ZFN2. EGFP (GenBank accession no AY818363, SEQ ID NO: 130) was PCR amplified using primers N & O (Table 1, below) and cloned downstream to NLS into XhoI and Sad digested pBS-T2A-P36-ZFN1, instead of ZFN. The resultant plasmid was termed pBS-T2A-NLS-EGFP. The DsRed2 was amplified using primers H & M (Table 1, below). The resultant stop codon-lacking DsRed2 was ligated SalI-BamHI fragment into the pBS T2A-NLS-EGFP upstream to T2A, yielding pBS-DsRed-T2A-NLS-EGFP with the bicistronic ORF. The DsRed-T2A-NLS-EGFP fragment from the plasmid was then ligated into HpaI-SacI sites of pTRV2-Δ2b-sgP to generate pTRV2-Δ2b-sgP-DsRed-T2A-NLS-EGFP.
A pTRV2-Δ2b-sgP containing two fluorescent marker genes was generated under separate subgenomic promoters in which pTRV2-Δ2b-sgP-GFP was first digested with SmaI. The PCR fragment generated using pTRV2-Δ2b-sgP-DsRed as a template and primers P & Q (see Table 1, below) was cloned into this SmaI, to produce pTRV2-Δ2b-sgP-GFP-sgP-DsRed.







TABLE 1







Primers








Primer
Sequence





A
TGGAGTTGAAGAGTTATTACCGAACG



(SEQ ID NO: 1)


 


B
AAGAATT CGAAACTCAAATGCTACCAA



(SEQ ID NO: 2)


 


C (PEBV-sgP R)
TAGAATTCTCGTTAACTCGGGTAAGTGA



(SEQ ID NO: 3)


 


D (P19-F)
AAACTCGAGATGGAACGAGCTATACAAGG



AA



(SEQ ID NO: 4)


 


E (P19-R)
AAACCCGGGAGAGTCTGTCTTACTCGCCTT



CT



(SEQ ID NO: 5)


 


F (5′-PEBV-sgP-F)
AAGAGCTCGAGCATCTTGTTCTGGGGTT



(SEQ ID NO: 6)


 


G (GFPuv-3′-SmaI)
ACCCGGGTTATTTGTAGAGTTCATCCATGC



CA



(SEQ ID NO: 7)


 


H (DsRFP-F-HpaI)
AGTTAACGAGATGGCCTCCTCCGAGA



(SEQ ID NO: 53)


 


I (DsRed2-R-SacI)
TAGAGCTCTCACAGGAACAGGTGGTGGC



(SEQ ID NO: 54)


 


J (T2A-F-BamHI)
TTTGGATCCGAAGGAAGAGGATCTCTTCTT



ACTTGTGGTGATGTTGAAGAG



(SEQ ID NO: 55)


 


K (T2A-NLS-F-
TTACTTGTGGTGATGTTGAAGAGAATCCTG


first)
GACCAAAAAAGAAGAGAAAG



(SEQ ID NO: 56)


 


L (R-FokI SacI)
AAGAGCTCTTAGGATCCAAAGTTTATCTC



(SEQ ID NO: 57)


 


M (DsRFP-R-
AGGATCCCAGGAACAGGTGGTGGC


BamHI)
(SEQ ID NO: 58)


 


N (EGFP-F-XhoI)
A TCT CGA GTG AGC AAG GGC GA



(SEQ ID NO: 59)


 


O (EGFP-R-SacI)
AGAGCTCTACTTGTACAGCTCGTCCATG



(SEQ ID NO: 60)


 


NLS (uppercase)
atggtgCCAAAAAAGAAGAGAAAGGTAGAA



GACCCCtctcgag



(SEQ ID NO: 61)


 


P (2sg F1 Sma)
CCCGGGATTTAAGGACGTGAACTCTGT



(SEQ ID NO: 62)


 


Q (dsRed R678 Sma)
CCCGGGTCACAGGAACAGGTGGT



(SEQ ID NO: 63)


 


R (NLS linking
AGTTAACGAGATGCCAAAAAAGAAGAGAAA


gene)
GGT



(SEQ ID NO: 64)


 


S (FokI-m-R-SacI)
AAGAGCTCTTAaGATCCAAAGTTTATCTC



(SEQ ID NO: 65)


 


T (FokI-m-R-SmaI)
ACCCGGGTTATCCAAAGTTTATCTCGCCGT



(SEQ ID NO: 66)


 


U (F-QEQ-ZFN)
AACTCGAGAAAAACTGCGGAACGGA



(SEQ ID NO: 67)


 


V (Rssu tp-F-HpaI)
AGTTAACGAGATGGCTTCTATGATATCCT



CT



(SEQ ID NO: 68)


 


W (ATP-β-tp-F-HpaI)
AGTTAACGAGATGGCTTCTCGGAGG



(SEQ ID NO: 69)









Cloning of pTRV2 Viral Vectors Allowing Targeting of Gene Products to Plastids
To generate EGFP targeted to chloroplast, inventors amplified a PCR fragment containing transit peptide of Pea ribulose-1,5-bisphosphate carboxylase small subunit (Rssu) (GenBank accession no X00806, nucleotides 1086-1259, SEQ ID NO: 138) fused to EGFP with primers V & O (see Table 1, above) using the plasmid pTEX-Rssu-GFP previously described by Bezawork [Bezawork (2007) M.Sc Thesis, submitted to Agricultural Research Organization, Volcani center and the Faculty of Agriculture] as a template. The PCR product, following blunting with HpaI, was cloned downstream to sgP into pTRV2-Δ2b-sgP to produce pTRV2-Δ2b-sgP-Rssu-EGFP.
To generate EGFP targeted to mitochondria, inventors amplified a PCR fragment containing a signal peptide of Nicotiana sylvestris ATPase beta subunit (ATP-(3) (GenBank accession no U96496, nsatp2.1.1, nucleotides 12 to 167, SEQ ID NO: 139) fused to EGFP with primers W & O (see Table 1, above) using the plasmid pTEX-ATP13-GFP previously described by Bezawork [Bezawork (2007), supra] as a template. The PCR product, following blunting with HpaI, was cloned downstream to sgP into pTRV2-Δ2b-sgP to produce pTRV2-Δ2b-sgP-ATPβ-EGFP.
Inoculation of Plants with TRV Vectors
Agrobacterium tumefaciens (strain AGLO) transformed with pTRV1, pTRV2 and pTRV2 derivatives were prepared as previously described [Liu et al., Plant J (2002) 30: 415-429]. The Agrobacterium culture was grown overnight at 28° C. in LB medium complemented with 50 mg/L kanamycin and 200 μM acetosyringone (A.S.). The cells were harvested and resuspended in inoculation buffer containing 10 mM MES, 200 μM A.S. and 10 mM MgCl2 to an OD600 of 10. Following an additional 3 hours of incubation at 28° C., the bacteria with the pTRV1 was mixed with the bacteria containing the pTRV2 derivates at a 1:1 ratio. When co-infection of more then one pTRV2 was involved, the Agrobacteria with pTRV1 were always 50% in the mixture. 200-400 μl of the Agrobacteria mixture was used for injection into the stem. Agrobacteria were also injected into the exposed shoot surface following removal of the apical meristems.
Another option for infection was leaf infiltration using a syringe without a needle: Agrobacteria content of the syringe was discharged into the scratched surface of the leaf. For infection of plants with TRV, without the use of Agrobacteria, first N. benthamiana or N. clevelandii (the usual host for TRV) was inoculated with pTRV1 and pTRV2 or its derivatives. About 15 to 21 days post infection (dpi) plant leaves (as a source for freshly prepared sap infection) were collected and the sap was extracted in 20 mM phosphate buffer (pH-6.8) by mortar and pestle.
For virion infection of plants (TRV infection without use of agrobacteria), inventors first inoculated Petunia hybrida, Nicotiana tabacum cv Samsung or N. benthamiana, (the usual hosts for TRV) with pTRV1 and pTRV2 (or its derivatives). About 5 to 21 days post infection (dpi) inventors collected plant leaves and extracted the sap in 20 mM phosphate buffer pH=6.8 and a surfactant (e.g. 0-0.03% Silwet L-77) by mortar and pestle. The TRV containing sap was dripped onto cheesecloth or centrifuged and following addition of carborundum fine powder (to improve infection) stems and leaves of young (approximately 1 month old) plants were gently scratched.
Sap infection of in-vitro grown plants: sap was first passed through 0.22 μm filter and then stems of tissue culture propagated plants were injured and infected using syringe and needle.
For Zea mays (monocotyledons) infection, the seeds were incubated with the sap during swelling and sprouting (for approximately 1-2 weeks).
In addition to AGLO strain of Agrobacterium, inventors also successfully used the EHA-105 [Tovkach et al., Plant J. (2009) 57, 747-757] strain for the delivery of various pTRV constructs.
For inoculation of in vitro grown plants using A. tumefaciens AGLO or EHA-105 bacteria, a MS solution [Murashige and Skoog, Physiol Plant (1962) 15:473-497] without glucose but with 10 mM MgSO4 and 50 μg/ml acetosryngone (A.S.) was used and the concentration of the bacteria was reduced to 0.08-0.8 OD 600 nm for each TRV. Infection was performed essentially as above with sap or via vacuum infiltration.
Expression Analysis
Each expression experiment was repeated three times and each experiment included at least four plants per treatment. Tested plant meristems (at least 2 per plant) were collected several times during the experimental course. GFP imaging was completed using UV illumination and photographs were taken using fluorescence stereomicroscope (Leica Microsystems, Wetzlar, Germany) equipped with a digital camera and a filter set for excitation at 455-490 nm and emission at more than 515. Gus activity was evaluated using the substrate 1 mM 5-bromo-4 chloro-3-indolyl-β-D-glucoronic acid (X-gluc., Duchefa Biochemie B.V. Haarlem, Netherlands) in an appropriate buffer (Zuker et al., supra). Prior to an overnight incubation with the substrate mixture at 37° C., plant tissue was vacuum infiltrated with the substrate for 30 minutes. The substrate solution was then exchanges with 75-95% ethanol for a few days for chlorophyll bleaching and the tissue was observed using a stereomicroscope.
DsRed2 imaging was completed using UV illumination and photographs were taken using fluorescence stereomicroscope (Leica Microsystems, Wetzlar, Germany) equipped with a digital camera and a filter set for excitation at 530-560 nm and emission at 590-650.
EGFP and DsRed2 imaging was also generated using a confocal laser-scanning microscope (CLSM510, Zeiss Jena Germany). For EGFP, excitation was set at 488 nm and emission at 505-530 nm, for DsRED2, excitation was set at 545 nm and emission at 585-615 nm. Autofluorescence of chlorophyll, excitation was set at 488 nm and emission at more than 650 nm.
Preparation of Protoplasts
Petunia leaves were used to generate protoplasts as previously described by Locatelli [Locatelli et al, Plant Cell Reports (2003) 21: 865-871].
Transgenic Plants
The binary vector pRCS2[QQR-TS*::GUS] previously described by Tovkach [Tovkach et. al. (2009), supra] was transferred to Agrobacterium tumefaciens which was then used to transform Petunia hybrida cv Burgundy and Nicotiana tobaccum cv. Samsung using the standard leaf disc transformation method [Guterman et al., Plant Mol. Biol. (2006) 60:555-563].
Identification of Non-Coding Genomic Sequences of Petunia 
A genomic DNA of Petunia cv. Royal Blue was prepared using a standard protocol. Initial digestion of the genomic DNA with EcoRI and HindIII was carried out followed by agarose (1%) gel electrophoresis. Next, 1-1.5 Kbp fragments were extracted from the gel by a gel extraction kit (iNtRON Biotechnology, INC. LTD, KOREA). These fragments were ligated to pBS-SK (IRA Company) to form a semi-genomic library in E. coli. Sequences of 110 genomic fragments were generated by Macrogen Inc. (Seoul, Korea). Two BLAST analyses (nucleotide blast and tblastx) were performed with the generated genomic petunia sequences against nucleotide collection and non-human, non-mouse ESTs libraries, to allow elimination of all the putatively transcripted/translated DNAs. All sequences with a BLAST E value higher then 5 were further evaluated to identify those with the shortest ORF, for all six reading frames, and with minimum repetitive AAAA and TTTT regions. Finally two Petunia genomic DNA fragments were selected as non-coding, non repetitive sequences, P1-25 (1.2 Kbp, FIG. 2B) and P1-36 (1.175 Kbp, FIG. 3).
Within these sequences a target site for zinc finger nuclease (ZFN) was designed. Zinc finger proteins are capable of recognizing virtually any 18 by long target sequence, enough to specify a unique address within plant genome. The target sites (artificial-palindrome-like sequence targets, marked in blue in FIGS. 2-3) used were:










P1-25 site 1: 




TCC-TCC-TGC 
(SEQ ID NO: 10)



 



site 2:




GAG-GGG-GAA
(SEQ ID NO: 11)



 



P1-36 site 1:




ACC-ACC-ATC
(SEQ ID NO: 12)



 



site 2:




GGT-TGA-GAG
(SEQ ID NO: 13)






Identification of PDS and FHT Sequences as ZFN Target Sites in Petunia 
The sequence of phytoene desaturase (PDS) exon from Petunia hybrida RB was confirmed by resequencing (based on GenBank accession no AY593974.1, SEQ ID NO: 131) and utilized as target sites for ZFNs. The highlighted sequences (FIG. 28A) were utilized as the target sites of PDS-ZFN proteins (SEQ ID NOs: 71 and 73).
The sequence of flavanone 3 beta-hydroxylase (FHT) exon from Petunia hybrida RB (GenBank accession no AF022142.1, SEQ ID NO: 133) was identified and utilized as target sites for ZFNs. The sequence was confirmed by resequencing. The highlighted sequences (FIG. 29) were utilized as the target sites of FHT-ZFN proteins (SEQ ID NOs: 75 and 77).
Design of Zinc Finger Nucleases (ZFNs)
The zinc finger proteins coding regions were designed based on a zinc-finger-framework consensus sequence formerly developed by Desjarlais and Berg [Desjarlais and Berg, Proc. Natl. Acad. Sci. USA (1993) 90: 2256-2260]. For example, expression of zinc finger endonuclease with the expected affinity to the gagggggaa sequence on P1-25 Petunia random DNA fragment (site 2, SEQ ID NO: 11) the zinc finger 262 by domain was assembled by PFU polymerase (Invitrogen) in a PCR reaction from the set of the following overlapping oligos: BBO1 (5′GAAAAACCTTACAAGTGTCCTGAATGTGGAAAGTCTTTTTCT, SEQ ID NO: 14), BBO2M (5′CAGCGAACACACACAGGTGAGAAGCCATATAAATGCCCAGAATGTGGTA AATCATTCAG, SEQ ID NO: 15), BBO3M (5′CAACGGACCCACACCGGGGAGAAGCCATTTAAATGCCCTGAGTGCGGGA AGAGTTTTT, SEQ ID NO: 16), FtsH2-Z1.1-GAA (SEQ ID NO: 17), P1-25-ZFN2.2 GGG (SEQ ID NO: 18) and P1-25-ZFN2.3 GAG (SEQ ID NO: 19) followed by PCR amplification using the BBO1-XhoI-F (SEQ ID NO: 20) and SDO3-Spe1-R (SEQ ID NO: 21) primers producing the DNA binding domain P1-25-ZFN2bd.
In each PCR reaction, the BBOs and SDOs were mixed at 0.005 μM concentration and amplified for 35 cycles with PFU polymerase (Invitrogen). Similar strategies, only using different oligos (see Table 2, below) have been employed for the assembly of the P1-25-ZFN1bd, P1-36-ZFN1bd and P1-36-ZFN2bd DNA binding domains. An outline of the PCR procedure for assembly of ZF binding domains used in this work is illustrated in FIG. 4.







TABLE 2





Sequences of overlapping oligos used for genera-


tion of DNA binding domains P1-25-ZFN1bd, 


P1-36-ZFN1bd and P1-36-ZFN2bd







P1-25-ZFN1bd









41
P1-25-ZFN1.1
ACCTGTGTGTGTTCGCTGGTGACGTTCAAGATG



GGA
AGCACGCTGAGAAAAAGACTTTCCACA




(SEQ ID NO: 22)


42
P1-25-ZFN1.2
CCCGGTGTGGGTCCGTTGGTGACGTTCAAGAT



GGA
GAGCACGCTGACTGAATGATTTACCACA




(SEQ ID NO: 23)


24
ZFN-IV-Mod
TCCAGTATGAGTACGTTGATGACGACGCAAA



SDO3 GCA
TCTCCAGACTGTGAAAAACTCTTCCCGCAC




(SEQ ID NO: 24)


 







P1-25-ZFN2bd









25
FtsH2-Z1.1-
ACCTGTGTGTGTTCGCTGGTGCTTCTGA



GAA
AGGTTGCTAGACTGAGAAAAAGACTTTCCACA




(SEQ ID NO: 17)


43
P1-25-ZFN2.2
CCCGGTGTGGGTCCGTTGGTGACGA



GGG
ACCAACTTATCAGAACGACTGAATGATTTACCA




CA




(SEQ ID NO: 18)


44
P1-25-ZFN2.3
TCCAGTATGAGTACGTTGATGACGAACCAA



GAG
ATTATCAGAACGTGAAAAACTCTTCCCGCAC




(SEQ ID NO: 19)


 







P1-36-ZFN1bd









45
P1-36-ZFN1.1
ACCTGTGTGTGTTCGCTGGTGACGAA



GGT
CAAGATGTCCAGAAGTAGAAAAAGACTTTCCA




CA




(SEQ ID NO: 25)


29
FtsH2-Z3.2-
CCCGGTGTGGGTCCGTTGGTGACGAACAAG



GGT
ATGTCCAGAAGTACTGAATGATTTACCACA




(SEQ ID NO: 26)


35
FtsH2-Z1a.3
TCCAGTATGAGTACGTTGATGACGAACCAAA



GAT
TTTCCAGAAGTTGAAAAACTCTTCCCGCAC




(SEQ ID NO: 27)


 







P1-36-ZFN2bd









46
P1-36-ZFN2.1 
ACCTGTGTGTGTTCGCTGGTGACGAACCAAA



GAG
TTATCAGAACGAGAAAAAGACTTTCCACA




(SEQ ID NO: 28)


47
P1-36-ZFN2.2
CCCGGTGTGGGTCCGTTGGTGAGAAGCCAAAT



TGA
GTCCAGCCTGACTGAATGATTTACCACA




(SEQ ID NO: 29)


48
P1-36-ZFN2.3
TCCAGTATGAGTACGTTGATGACGAACAAGAT



GGT
GTCCAGAAGTTGAAAAACTCTTCCCGCAC




(SEQ ID NO: 30)









Amplified DNA binding domains were cloned as an XhoI-SpeI fragment into the same sites of pSAT6-NLS-FokI, producing the pSAT6-NLS-P1-25-ZFN1 (SEQ ID NO: 31), pSAT6-NLS-P1-25-ZFN2 (SEQ ID NO: 32), pSAT6-NLS-P1-36-ZFN1 (SEQ ID NO: 33) and pSAT6-NLS-P1-36-ZFN2 (SEQ ID NO: 34) expression vectors (FIGS. 5A-D). pSAT6-NLS-FokI consists of pSAT vector [GeneBank AY818383, Tzfira et al., Plant Mol Biol (2005) 57: 503-516], 30 by of the NLS (nuclear localization signal, SEQ ID NO: 46) cloned into NcoI-XhoI sites and a 584 by fragment of FokI endonuclease (nucleotides 1164 to 1748; GeneBank J04623) cloned into SpeI-BamHI sites of pSAT6.
For expression of His tagged zinc finger endonucleases in E. coli cells, the NLS-P1-25-ZFN1, NLS-P1-25-ZFN2, NLS-P1-36-ZFN1 and NLS-P1-36-ZFN2 fragments were cloned as NcoI-BamHI inserts from their corresponding plasmids into the same sites of a modified pET28 (pET28.5X, FIG. 5E), producing pET28.5X-NLS-P1-36-ZFN1, pET28.5X-NLS-P1-36-ZFN2, pET28.5X-NLS-P1-25-ZFN1 and pET28.5X-NLS-P1-25-ZFN2. In pET28.5X, assembled ZFNs are cloned downstream of T7 promoter of pET28 vector (Novagen). Complete ZFN constructs also contained a sequence coding for 6×His-tag at the C terminus of the protein.
Expression of pET28.5X-NLS-P1-36-ZFN1, pET28.5X-NLS-P1-36-ZFN2, pET28.5X-NLS-P1-25-ZFN1 and pET28.5X-NLS-P1-25-ZFN2 was performed in BL21 GOLD (DE3) PlyS cells (Stratagene). Cell cultures were grown in 100 ml LB medium complemented with Kan (50 ug/ml) and 100 μM ZnCl2 at 22° C. until OD600 was 0.6 and then cells were induced with 0.7 mM IPTG for 3 hours. Cells were harvested by centrifugation, resuspended in 35 ml mixture containing 25 mM Tris-HCl (pH 7.5), 300 mM NaCl, 5% glycerol and 100 μM ZnCl2, and lysed twice through a French Press. The protein was loaded on 0.5 ml Ni-NTA agarose beads (Qiagen) and eluted with 1 ml buffer containing 500 mM imidazole. Eluted protein was stored at −20° C. in 50% glycerol.
Various quantities of E. coli and Ni-NTA purified ZFNs were mixed with 0.5 μg of target DNA for in vitro digestion using NEB4 buffer and digested DNA substrates were separated by agarose gel electrophoresis.
Cloning of pTRV2 Viral Vectors Allowing Expression of ZFNs
ZFN inserts in pSAT constructs (pSAT6-P1-36-ZFN1, pSAT6-P1-36-ZFN2, pSAT6-PDS-ZFN1, pSAT6-PDS-ZFN2, pSAT6-FHT-ZFN1, pSAT6-FHT-ZFN2) start and end with nonspecific domains, NLS and FokI respectively. To generate pTRV2 suitable for delivery and expression of different ZFNs in plant cells, inventors amplified ZFNs using the forward primer R and the reverse primers L (for pSAT6-P1-36-ZFN2, pSAT6-PDS-ZFN1, pSAT6-FHT-ZFN1) or T (for pSAT6-P1-36-ZFN1, pSAT6-PDS-ZFN2, pSAT6-FHT-ZFN2) (see Table 1, above). The resultant amplification products were digested with HpaI and Sad (for primer L) or SmaI (for primer T), blunt ended (in case of Sad) and inserted into HpaI-SmaI sites of pTRV2-Δ2b-sgP. This lead to the generation of pTRV2-Δ2b-sgP-PDS-ZFN1 (FIG. 31A, SEQ ID NO: 70), pTRV2-Δ2b-sgP-PDS-ZFN2 (FIG. 31B, SEQ ID NO: 72); pTRV2-Δ2b-sgP-FHT-ZFN1 (FIG. 32A, SEQ ID NO: 74), pTRV2-Δ2b-sgP-FHT-ZFN2 (FIG. 32B, SEQ ID NO: 76); pTRV2-Δ2b-sgP-P36-ZFN1 (SEQ ID NO: 33) and pTRV2-Δ2b-sgP-P36-ZFN2 (SEQ ID NO: 34).
To generate pTRV2-Δ2b-sgP-QEQ-ZFN (SEQ ID NO: 82), inventors first generated a PCR product using primers U and S (see Table 1, above) and pSAT4.hspP.QQR [Tovkach et al (2009), supra] as a template. The product was digested with XhoI and SpeI and inserted into XhoI and SpeI digested, ZF lacking, plasmid pTRV2-Δ2b-sgP-P36-ZFN2 (FIG. 33).
To generate a construct containing two ZFNs linked with T2A (pTRV2-Δ2b-sgP-P36-ZFN2-T2A-P36-ZFN1, SEQ ID NO: 84), the BamHI fragment from pTRV2-Δ2b-sgP-P36-ZFN2 containing the sgP-NLS-P1-36-ZFN2 was cloned into the unique BamHI site in pBS-T2A-NLS-36-ZFN1. This generated pBS-sgP-P36-ZFN2-T2A-P36-ZFN1. This plasmid was digested with Sad and the released fragment containing sgP-P36-ZFN2-T2A-P36-ZFN1 was cloned into pTRV2-Δ2b linearized with Sad to generate pTRV2-Δ2b-sgP-P36-ZFN2-T2A-P36-ZFN1.
To generate pTRV2-Δ2b-sgP-PDS-ZFN1-T2A-PDS-ZFN2 (SEQ ID NO: 86) and pTRV2-Δ2b-sgP-FHT-ZFN1-T2A-FHT-ZFN2 (SEQ ID NO: 88), inventors first digested pBS-T2A-NLS-36-ZFN1, pSAT6-FHT-ZFN2 and pSAT6-PDS-ZFN2 with XhoI & SpeI (flanking ZF sequences). Inventors then ligated fragments released from the latter two plasmids into digested pBS-T2A-NLS-36-ZFN1 to create pBS-T2A-NLS-PDS-ZFN2 and pBS-T2A-NLS-FHT-ZFN2. Plasmids pTRV2-Δ2b-sgP-PDS-ZFN1 and pTRV2-Δ2b-sgP-FHT-ZFN1 were digested with BamHI to release sgP-PDS-ZFN1 and sgP-FHT-ZFN1, which were then ligated into BamHI digested pBS-T2A-NLS-PDS-ZFN2 and pBS-T2A-NLS-FHT-ZFN2, respectively. These plasmids were digested with Sad and the released fragments were cloned into unique Sad site of the MCS in pTRV2-Δ2b yielding pTRV2-Δ2b-sgP-PDS-ZFN1-T2A-PDS-ZFN2 and pTRV2-Δ2b-sgP-FHT-ZFN1-T2A-FHT-ZFN2, respectively.
Example 2
Expression of Foreign Genes by pTRV2-Δ2b Vectors
The RNA2-2b fragment (300 by of SEQ ID NO: 43 depicted in SEQ ID NO: 50) was entirely removed from pTRV2 (GenBank accession No. AF406991) to generate the pTRV2-Δ2b (see Example 1 hereinabove and FIGS. 6A-B). Inventors were interested in the expression of the reporter genes in the meristematic tissues and hence reporter gene expression was evaluated in these tissues. Petunia plants were inoculated with pTRV2-Δ2b-GUS and pTRV2-GUS and the efficiency of foreign gene expression (i.e. GUS) by these vectors was compared. GUS expression was evaluated a week to two month following infection and percent of meristems expressing GUS out of total number of analyzed meristems was presented (see Table 3, below). As clear from the results, pTRV2 without the 2b region was much more efficient in GUS expression in meristimatic tissues. Furthermore, no GUS staining was noticeable in petunia plants inoculated with TRV lacking GUS. Typical GUS expression in the meristems of inoculated plants is illustrated in FIGS. 7A-E. As clear from FIG. 7E, GUS staining in petunia plants following inoculation with pTRV2-Δ2b-GUS was noticeable even after numerous rounds of propagation (via axillary meristems) in tissue culture. Additional marker genes used to assay applicability of pTRV2 based vector for expression of foreign genes were GFP and PAP1. Inoculation of petunia and other plants (Capsicum annuum, Solanum pimpinellifolium, Nicotiana benthamiana, Arabidopsis thaliana, Artemisia annua, Spinacia olerace and Beta vulgaris) with these vectors led to expression of the reporter genes in meristems of all analyzed plants (FIGS. 8A-G).







TABLE 3







Expression of GUS in Petunia plants inoculated with pTRV2-Δ2b-


GUS and pTRV2-GUS









Days post inoculation













7
13
21
37
48


















pTRV2-GUS
50%
40%
11%
7%
0%



TRV2-Δ2b-GUS
45%
60%
54%
40%
60%










Example 3
Enhancement of Foreign Gene Expression by Ω Translational Enhancer
The 70 by at the 5′UTR of TMV (Ω) is a non-coding sequence shown to be a translational enhancer (SEQ ID NO: 44) [Gallie et al., Nucl. Acid. Res. (1987) 15:8693-8710]. Ω was cloned upstream to the reporter gene in the pTRV2 viral vectors in order to evaluate whether it can promote expression levels of foreign genes (see FIGS. 6A, C). As illustrated in FIGS. 9A-B, inoculation of petunia plants with pTRV2-Δ2b-ΩGUS vectors resulted in higher GUS activity levels as compared to that obtained using pTRV2-Δ2b-GUS vectors (lacking the Ω enhancer). It should be noted that the Ω fragment did not affect the percent of meristems expressing GUS out of total number of analyzed meristems.
Example 4
Co-Expression of Two Foreign Genes by pTRV2 Vectors
Two approaches were developed to allow co-expression of two coding sequences. First, a pTRV2 vector was generated that carries an additional subgenomic promoter (sgP) sequence, hence allowing co-expression of two coding sequences (see FIGS. 6A, D). Coat protein subgenomic promoter of PEBV was used to this end. To test vector activity, GFP reporter gene was cloned downstream to this subgenomic promoter to create pTRV2-Δ2b-sgP-GFP. Inoculation of N. benthamiana plants with this vector, led to expression of GFP in meristematic tissues (FIG. 10A).
In an alternative approach aiming to co-express two foreign genes, N. benthamiana plants were co-inoculated with pTRV2-Δ2b-GUS and pTRV2-Δ2b-GFP (FIGS. 10B-C). Co-expression of both reporter genes was revealed based on the analyses of GFP expression in the tissue followed by GUS staining of the same tissues.
Example 5
Expression of Foreign Genes by pTRV Vectors in a Wide Variety of Plants
Inoculation of different plants (e.g. N. benthamiana, N. tobaccum and Petunia hybrida) with pTRV1 and pTRV2-Δ2b-sgP-DsRed lead to a high expression level of the marker gene in cells of these plants (FIGS. 11A-F). Continuous (several months) strong expression, due to systemic infection, was easily detected in different parts of these plants (FIGS. 12A-H). Inoculation of various plants (e.g. Cucumis sativus, Solanum melongena, Gossypium hirsutum cv. Siv'on (cotton), Brassica napus (canola), Beta vulgaris (beet), Spinacia oleracea) with this vector lead to expression of the reporter genes in all analyzed plants (FIGS. 13A-K).
Example 6
Expression of Foreign Genes by TRV Vectors in Monocots
To assay the applicability of pTRV2 based vectors for expression of foreign genes in monocots (e.g. maize), seeds were incubated with sap generated from petunia plants infected with pTRV1 and pTRV2-Δ2b-sgP-DsRed (as depicted in detail in Example 1, hereinabove). FIGS. 14A-C show clear expression of DsRed in coleoptile.
Example 7
Mitochondrial & Chloroplast Plastids
TRV Vector-Mediated Plastid-Targeted Expression
As described in Example 1, hereinabove, inventors have constructed two vectors pTRV2-Δ2b-sgP-Rssu-EGFP and pTRV2-Δ2b-sgP-ATPβ-EGFP containing a chloroplast transit peptide and a mitochondrial signal peptide, respectively. Inventors agroinfiltrated both pTRV2-Δ2b-2sgP-tp-EGFP (tp here is stand for transit peptide and signal peptide) into Petunia hybrida cv RB and N. benthamiana. The EGFP expression was first analyzed by fluorescent stereomicroscope, then the fluorescent leaf zones were analyzed by confocal laser scanning microscope. The chloroplast size and auto fluorescence (excitation at 488 nm, emission more than 650 nm) enabled to localize the expression of EGFP (excitation at 488 nm, emission 505-530 nm) to the chloroplast (FIGS. 15A-G).
For mitochondrial identification, protoplasts were prepared and red fluorescent mitochondrial specific reagent (MitoTracker® Invitogen inc. USA) was employed. The use of excitation 545 nm and emission 585-615 nm allowed distinguishing the fluorescence of chloroplasts from that of MitoTracker. According to the size and location and mitotracker signal, inventors revealed that the expression of EGFP was localized to mitochondria (FIGS. 16A-K).
Example 8
Co-Expression of Two Reporter Genes in Various Plants
Several approaches were used to simultaneously express two genes in plant (Petunia hybrida, N. benthamiana or N. tabacum) cells. In one approach, plants were inoculated simultaneously with two TRV vectors, one carrying one marker gene and another carrying another marker gene. Specifically, plants were co-infection with pTRV1 and two pTRV2 vectors, pTRV2-Δ2b-sgP-DsRed and pTRV2-Δ2b-sgP-Rssu-EGFP. Results of confocal fluorescent scanning microscopy of in vitro Agroinfiltrated Nicotiana tabacum cv Xanthi plants showed co-expression of both EGFP and DsRed (FIGS. 17A-D).
The second and third approaches for co-expression of two genes were demonstrated using pTRV2 constructed with two reporter genes in tandem. The genes were either separated by T2A (FIGS. 18A-L) or were driven by separate double subgenomic promoters (FIGS. 19A-J). As depicted in FIGS. 18A-L, the co-expression of GFP and DsRed was clear following inoculation of plants with pTRV2-Δ2b-sgP-DsRed-T2A-NLS-EGFP. Similarly, as depicted in FIGS. 19A-J, the co-expression of GFP and DsRed was clear following inoculation of plants with pTRV2-Δ2b-sgP-GFP-sgP-DsRed.
Example 9
Generation of Specific Zinc Finger Nucleases (ZFNs)
Petunia non repetitive putatively non-coding genomic sequences were identified following sequencing of 110 genomic fragments (see Example 1, hereinabove). Two sets of ZFN, 25-ZFN-1, 25-ZFN-2 and 36-ZFN-1, 36-ZFN-2, were synthesized in order to form a double cut in the Petunia's specific DNA sequences, P1-25 and P1-36, respectively. To test nuclease activity of the generated ZFNs, PCR fragments were generated containing target sequences in a palindrome-like form and these fragments were incubated with the specific ZFNs. As illustrated in FIG. 20, PCR fragments were digested by each ZFN to the expected sizes.
To further verify ZFNs activities, pBS vectors carrying P1-36 sequences were generated. Incubation of 740 by fragment of P1-36 carrying target sequences (generated by digestion of pBS-P1-36 with NcoI/BamHI*) with purified ZFNs, 36-ZFN1 and 36-ZFN2, yielded fragments of expected sizes (FIG. 21, depicted by arrows). Of note, BamHI as well as SmaI are part of the multiple cloning sites (MCS) of pBS, right upstream to the cloning site EcoRI. The NcoI site is part of the P1-36 sequence and 200 by downstream to P1-36 site2. Furthermore, as expected, 36-ZFN1 and 36-ZFN2 individually did not yield digestion products. Moreover, as illustrated in FIG. 22, the combination of 36-ZFN-1 and 36-ZFN-2 successfully digests the target sequence P1-36 carried by pBS-P1-36.
Furthermore, Petunia phytoene desaturase (PDS) genomic sequences were identified following sequencing of genomic fragments (see FIG. 28A). Sets of ZFN, PDS-ZFN1 and PDS-ZFN2, were synthesized (as depicted in detail in Example 1, hereinabove) in order to form a double cut in the Petunia's specific PDS DNA sequences. To test nuclease activity of the generated ZFNs, plasmids were constructed to carry semi-palindromic target sequences and these plasmids were incubated with the specific ZFNs. As illustrated in FIG. 23, digestion of plasmids carrying artificial target sites PDS1 and PDS2 (PDS-TS1 and PDS-TS2, respectively) by specific ZFNs was carried out. Plasmids were digested by AgeI and PDS-ZFN1 or PDS-ZFN2 to the expected sizes.
In conclusion, the results conclusively show that foreign genes can be expressed in plants meristems, including petunia meristems. Additionally, these results show the specific digestion in vitro of petunia DNA by ZFNs.
Example 10
Generation of Viral Expression Vectors Comprising ZFNs
The expression of ZFNs by pTRV expression vectors is underway to determine the best approach to co-express ZFNs in petunia. Three approaches are being tested each of which is first tested with two fluorescent reporter genes (GFP and DsRFP) as depicted in detail above. These fluorescent reporter genes are delivered to Petunia plants and their co-expression within meristematic cells are examined using fluorescent microscopy. Based on these results, ZFNs expression vectors will be generated.
In the first approach, each gene is cloned separately into the pTRV2-Δ2b-Ω into the SalI-SacI site. Plants are then co-infected with the two plasmids of pTRV2 (one carrying GFP and the other carrying DsRFP) simultaneously.
In the second approach genes are both introduced into the same pTRV2 (pTRV2-Δ2b-Ω), each gene with a different sub-genomic promoter (sg-P). Plants are then infected with the pTRV2 plasmid.
The third approach is based on a ‘2A like’ protein that is able to cleave itself at the C termini [(Donnelly et al., J. Gen. Virol. (2001) 82: 1027-1041; Osborn et al., Molecu. Therapy (2005) 12: 569-574]. The ‘2A-like’ protein sequence, EGRGSLLTCGDVEENPGP (SEQ ID NO: 41, T2A) of insect virus Thosea asigna mediates an efficient co-translational cleavage event resulting in the release of each individual protein product. The C-termini Pro is the only amino acid that remains with the downstream protein following the self-cleavage. An oligomer was synthesized that encodes these 18 amino acids of the ‘2A-like’ protein (T2A). The nucleotide sequence was designed based on the Petunia codon usage (Codon Usage Database http://wwwdotkazusadotordotjp/codon). To co-translationally express GFP and DsRFP, they are cloned in frame into pTRV2-Δ2b-Ω separated by the T2A 18 amino acids. To deliver ZFNs proteins to the nucleus via this approach, nuclear localization signal (NLS) sequences that start with Pro, are fused to their 5′ ends. Hence, Pro are shared by NLS and T2A, i.e. the last 3′ Pro of the T2A represents the first amino acid of the NLS. This eliminated the need to introduce additional foreign sequences 5′ to NLS. The final insert is KpnI-Ω-NLS-ZFN2-T2A-NLS-ZFN1-SacI. For example, using pTRV2-Δ2b-sgP-CP-PEBV (FIGS. 24A-B) carrying DsRFP and GFP, separated by T2A, petunia tissues co-expressing both reporter genes were generated.
An optional modification to the third approach is to clone one of the ZFNs with the T2A at the N termini (T2A-NLS-ZFN1 fragment) downstream and in frame with TRV1 16 K gene (FIG. 6E). In this case the Agro infection of Petunia plants is performed with modified pTRV1 and pTRV2, each carrying only one foreign ZFN gene.
Example 11
pTRV-Vector-Mediated Activation of GUS Expression in Plants
Inventors have utilized the zinc finger based transgene repair tool that was previously described by Tovkach et al. [Tovkach et al. (2009), supra] in order to generate petunia and tobacco transgenic plants carrying a mutated uidA (GUS) gene. The mutated uidA gene was engineered to carry the TGA (stop) codon within the 6-bp spacer of the QEQ-ZFN target site (see FIG. 25A), leading to premature termination of uidA translation in plant cells. Thus, no GUS expression was detectable in the transgenic plants (FIG. 26I). Digestion of the DNA at the spacer between the ZFNs target site (by the use of specific ZFNs) and its successive repair typically lead to deletion and/or mutation of the stop codon and to the consequent activation of the uidA reporter gene (FIG. 25B). To this end, QEQ-ZFN specific for the mutated uidA gene was cloned into pTRV2-Δ2b-2sgP viral vector (as depicted in detail in Example 1, hereinabove) and the resultant pTRV2-Δ2b-sgP-QEQ-ZFN was used for inoculation of plants transgenic for mutated uidA gene. As clear from the results (FIGS. 26A-J), TRV-driven expression of the QEQ-ZFN in somatic and meristematic tissues lead to activation of GUS expression.
Example 12
Molecular Analysis of Transgenic Tobacco and Petunia Plants with Activated GUS Expression Following Inoculation with pTRV2-Δ2b-sgP-QEQ-ZFN
Total plant DNA was extracted from leaves of GUS transgenic petunia and tobacco plants (carrying a mutated uidA (GUS) gene) before or 7-30 days after inoculation with pTRV1 and pTRV2-42b-sgP-QEQ-ZFN using the phenol-chloroform method. Total DNA was digested with DdeI for 3 hours and the region surrounding the ZFN target site was PCR-amplified using primers 5′-CTATCCTTCGCAAGACCCTTCC-3′ (35S-F, SEQ ID NO: 90) and 5′-GTCTGCCAGTTCAGTTCGTTGTTC-3′ (GUS-R-401, SEQ ID NO: 91). The resulting PCR fragment was redigested with DdeI, and its undigested fraction was reamplified and TA cloned into pGEM-T-easy (Promega inc., WI, USA). Randomly selected colonies were then selected and the DNA fragments sequenced.
FIG. 27 shows the changes in the GUS sequence following activation by QEQ-ZFN, as compared to the original GUS sequence in the transgenic N. tabacum or Petunia plants.
Example 13
Molecular Analysis of Modified PDS in Petunia Plants Following Inoculation with Specific ZFNs
As depicted in detail in Example 1, hereinabove, inventors of the present invention have generated ZFNs which specifically cleave the PDS gene of petunia plants. To analyze the molecular modifications made to the PDS gene following inoculation with pTRV2-42b-sgP-PDS-ZFN1-T2A-PDS-ZFN2, total plant DNA was extracted from leaves of wild type (WT) or pTRV1 and pTRV2-42b-sgP-PDS-ZFN1-T2A-PDS-ZFN2 treated Petunia hybrida plants using the phenol-chloroform method. The region surrounding the ZFN target site (TS) was PCR-amplified using primers 5′-TATTGAGTCAAAAGGTGGCCAAGTC-3′ (phPDS-F 208, SEQ ID NO: 117) and 5′-GCAGATGATCATATGTGTTCTTCAG-3′ (phPDS-R-487, SEQ ID NO: 118). The PCR product was digested with MfeI overnight and the resulting undigested fraction was reamplified and TA cloned into pGEM-T-easy (Promega inc., WI, USA). Inserts from randomly selected colonies were then sequenced. FIG. 28B depicts the changes in the PDS sequence following modification by the specific PDS-ZFNs, as compared to the original PDS sequence in Petunia plants.
Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.
All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.

## Claims

1. A method of generating genotypic variation in a genome of a plant, the method comprising introducing into the plant at least one Tobacco Rattle Virus (TRV) expression vector encoding at least one chimeric nuclease which comprises a zinc finger binding domain, a nuclease, and a localization signal to a DNA-containing organelle, wherein said zinc finger binding domain mediates specific targeting of said nuclease to the genome of the plant, and wherein a nucleic acid sequence of said TRV expression vector is devoid of the 2b sequence as set forth in SEQ ID NO: 43, thereby generating genotypic variation in the genome of the plant.

2. A plant viral expression vector comprising a pTRV backbone and comprising a nucleic acid sequence encoding at least one chimeric nuclease which comprises a zinc finger binding domain, a nuclease, and a localization signal to a DNA-containing organelle, said nucleic acid sequence is devoid of the 2b sequence as set forth in SEQ ID NO: 43.

3. An expression vector comprising a pTRV backbone and comprising a nucleic acid sequence encoding at least two heterologous polypeptides, said nucleic acid sequence is devoid of the 2b sequence as set forth in SEQ ID NO: 43.

4. A transgenic plant comprising the plant viral expression vector of claim 2.

5. The method of claim 1, wherein said generating genotypic variation is transient.

6. The method of claim 1, wherein said at least one expression vector encodes for two chimeric nucleases.

7. The method of claim 1, wherein said at least one chimeric nuclease comprises two chimeric nucleases.

8. The plant viral expression vector of claim 2, further comprising a second nucleic acid sequence encoding a heterologous polypeptide.

9. The plant viral expression vector of claim 2, wherein said nucleic acid sequence comprises a Ω enhancer as set forth in SEQ ID NO: 44.

10. The expression vector of claim 3, wherein said nucleic acid sequence comprises two separate sub genomic promoters (sgPs) for regulating transcription of said at least two heterologous polypeptides.

11. The expression vector of claim 2, wherein the sequences of said at least two heterologous polypeptides are separated by a nucleic acid sequence encoding a cleavage domain.

12. The expression vector of claim 11, wherein said cleavage domain comprises a T2A-like protein sequence as set forth in SEQ ID NO: 52.

13. The expression vector of claim 3, wherein said amino acid sequence of at least two heterologous polypeptide sequences comprise SEQ ID NOs: 85, 87 or 89.

14. The expression vector of claim 3, wherein said at least two heterologous polypeptides comprise chimeric proteins, wherein each of said chimeric proteins comprise a zinc finger binding domain, a nuclease, and a localization signal to a DNA-containing organelle.

15. The method of claim 1, wherein said zinc finger binding domain binds a 9 nucleotide sequence.

16. The method of claim 1, wherein said zinc finger binding domain comprises three zinc finger domains.

17. The method of claim 1, wherein the plant comprises a Petunia hybrida or a Nicotiana tabacum.

18. The method of claim 17, wherein said specific targeting of said nuclease to the genome of said Petunia hybrida is to a phytoene desaturase (PDS) or a flavanone 3 beta-hydroxylase (FHT) of said Petunia hybrida.

19. The method of claim 1, wherein said nuclease comprises a type II restriction endonuclease.

20. The method of claim 1, wherein said nuclease is selected from the group consisting of FokI, I-SceI, I-CeuI, artificial meganuclease, modified meganuclease, homing nuclease, DNA gyrase, eukaryotic topoisomerase II, bacterial topoisomerase W, topoisomerase VI, Cre recombinase, Hin recombinase, Rad51/RecA, deoxyribonuclease I, deoxyribonuclease II and micrococcal nuclease.

21. The method of claim 1, further comprising introducing into the plant a DNA vector.

22. The method of claim 21, wherein said DNA vector is based on an Abutilon mosaic virus (AbMV) or a Tomato yellow leaf curl virus.

23. The method of claim 1, wherein a nucleic acid sequence of said TRV expression vector further comprises two separate sub genomic promoters (sgPs) for regulating transcription of at least two heterologous polypeptides.

24. The method of claim 23, wherein said sub genomic promoters (sgPs) are as set forth in SEQ ID NOs: 45.

25. The method of claim 23, wherein said at least two heterologous polypeptide sequences are separated by nucleic acid sequence encoding a cleavage domain.

26. The method of claim 25, wherein said cleavage domain comprises a T2A-like protein sequence as set forth in SEQ ID NOs: 52.

27. The method of claim 10, wherein said sub genomic promoters (sgPs) are as set forth in SEQ ID NOs: 45.