WO2016125143A1 - Mdmv based vector for gene expression and silencing - Google Patents

Mdmv based vector for gene expression and silencing Download PDF

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Publication number
WO2016125143A1
WO2016125143A1 PCT/IL2016/050095 IL2016050095W WO2016125143A1 WO 2016125143 A1 WO2016125143 A1 WO 2016125143A1 IL 2016050095 W IL2016050095 W IL 2016050095W WO 2016125143 A1 WO2016125143 A1 WO 2016125143A1
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Prior art keywords
plant
expression vector
vector
mdmv
polypeptide
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PCT/IL2016/050095
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French (fr)
Inventor
Ayelet OMID
Ira Marton
Barry A. COHEN
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Danziger Innovations Ltd.
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Publication of WO2016125143A1 publication Critical patent/WO2016125143A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C12N15/8203—Virus mediated transformation

Definitions

  • the present invention in some embodiments thereof, relates to a Maize dwarf mosaic virus (MDMV) expression vector and, more particularly, but not exclusively, to the use of same for generating genotypic variations in plants.
  • MDMV Maize dwarf mosaic virus
  • MDMV Maize dwarf mosaic virus
  • VPg viral-encoded protein
  • MDMV strains are known to infect maize, sorghum and Johnson grass. Generally, infected plants develop distinct chlorotic mosaics, mottles or streaks on green tissues (typically observed on young leaves). Infected plants are characterized by stunting and shortening of the upper internodes. Ear development may be arrested, leading to an incomplete grain filling and direct yield loss. Plants infected early may produce nubbins or can be totally barren.
  • Israeli MDMV (MDMV-IL) infected maize plants typically show non-severe symptoms, corn plants reach adulthood, ear formation and development may slow and may cause grain yield loss.
  • MDMV is transmitted by several species of aphids in a non-persistent manner.
  • the coat protein (CP) and the helper component-protease (HC-Pro) are required for aphid transmission, through the CP DAG and the HC-Pro KITC motifs.
  • Aphids transmit MDMV disease effectively fifteen to thirty minutes after feeding on infected plants. Virus is also transmitted efficiently by mechanical inoculation and is not transmitted by contact between plants. Symptoms appear 5-8 days after mechanical inoculation.
  • MDMV complete genome sequences have been reported from different regions of the world including Bulgaria, Spain, Hungary and United States. The strains identity is approximately 85 %.
  • the two main applications of viral vectors known in the literature are gene expression and gene silencing (virus-induced gene silencing (VIGS)).
  • VIGS virus-induced gene silencing
  • BSMV Barley stripe mosaic virus
  • BMV Brome mosaic virus
  • a clone of BSMV was made into a vector for use in barley [Holzberg et al., Plant J (2002) 30: 315- 327] and wheat [Triticum aestivum; Scofield et al., Plant physiol.
  • the second VIGS system based on BMV was developed for rice (Oryza sativa), maize (Zea mays), and barley (Hordeum vulgare) [Ding et al., Mol Plant Microbe In (2006), 19: 1229-1239].
  • U.S. Patent No. 5,428,144 provides methods and materials for isolation of the coat protein gene (MDMVA-CP) from Maize Dwarf Mosaic Virus Strain A (MDMVA) used as a vaccine against MDMV infection.
  • MDMVA-CP is incorporated into a plant expression cassette in which the cDNA clone is operably linked to plant regulatory sequence which causes the expression of the cDNA clone in living plant cells.
  • the plant expression cassette preferably includes a strong constitutive promoter sequence at one end to cause the gene to be transcribed at a high frequency, and a poly-A recognition sequence at the other end for proper processing and transport of the messenger RNA.
  • the resulting transformation vector is then introduced into maize callus to provide cross -protection to MDMV or related viral infections.
  • U.S. Patent No. 5,530,193 provides the nucleic acid and amino acid sequences of Maize Dwarf Mosaic Virus (MDMV) strains A, B, and KS1 as well as constructs and vectors used to produce transformed plants comprising MDMV resistance (plants are transformed with the coat protein gene of one of the various strains of MDMV).
  • MDMV Maize Dwarf Mosaic Virus
  • U.S. Patent Application No. 20050132440 provides a gene silencing vector that suppresses the expression of specific target gene in a host.
  • U.S. 20050132440 provides a gene silencing vector comprising a promoter, an enhancer sequence (downstream of the promoter), and a gene encoding a potyvirus -origin coat protein e.g. of potato virus Y (downstream of the enhancer sequence).
  • the vector is used with a specific target gene or a gene that is homologous to the target gene inserted in the vicinity of the gene encoding the coat protein.
  • U.S. Patent Application No. 20030031648 provides a recombinant vector for expression of a heterologous peptide at the amino-terminus of a potyvirus coat protein.
  • the vector includes sufficient potyvirus nucleic acid sequence (e.g. ZYMV, MDMV, etc.) to permit viral replication and spread within a plant infected by the vector.
  • the vector further includes a heterologous nucleic acid sequence inserted at the amino- terminus of the potyvirus coat protein.
  • potyvirus nucleic acid sequence e.g. ZYMV, MDMV, etc.
  • ZYMV Zucchini Yellow Mosaic Potyvirus
  • U.S. Patent Application No. 20050257287 provides a nucleic acid vector for concurrently imparting herbicide resistance to a plant and cross protecting the plant.
  • the vector includes sufficient potyvirus nucleic acid sequence (ZYMV) to permit viral replication and spread.
  • ZYMV potyvirus nucleic acid sequence
  • the vector further includes mutations which attenuate symptoms of viral infection in the plant and which abolish transmission of the virus by an insect vector (e.g. aphid).
  • the vector further includes an additional nucleic acid sequence encoding a protein (e.g. phosphinothricin acetyltransferase) which imparts resistance to an herbicide when expressed in the infected plant.
  • a protein e.g. phosphinothricin acetyltransferase
  • WO 1999/051749 provides a potyvirus infectious nucleic acid construct for providing protection against viral infection in plants.
  • the construct of WO 1999051749 comprises a full length clone characterized in that its HC-Pro gene conserved FRNK box sequence contains a substitution (e.g. a substitution of Arg). This substitution renders the construct infectious, however, when introduced to plants, induces little or no symptom development.
  • the construct of WO 1999/051749 further contains a substitution which effectively abolishes aphid transmissibility.
  • WO 1999/051749 further relates to transient expression of foreign nucleic acid genes in plants using the construct. Specifically, WO 1999051749 relates to a method for cross protection of cucurbits against ZYMV infection.
  • a plant expression vector comprising a nucleic acid sequence encoding a polyprotein product, the polyprotein product comprising Maize Dwarf Mosaic Virus (MDMV) polypeptides and a heterologous polypeptide of interest, the heterologous polypeptide of interest being cleaved of the MDMV polypeptides upon expression in a plant cell, and wherein the plant expression vector is capable of spreading in a plant.
  • MDMV Maize Dwarf Mosaic Virus
  • a plant expression vector comprising a nucleic acid sequence encoding a polyprotein product, the polyprotein product comprising Maize Dwarf Mosaic Virus (MDMV) polypeptides, wherein the MDMV polypeptides comprise a PI polypeptide and a coat protein polypeptide, and a heterologous polypeptide of interest, the heterologous polypeptide of interest being cleaved of the MDMV polypeptides upon expression in a plant cell infected with a helper virus or an MDMV virus, and wherein the plant expression vector is capable of spreading in a plant.
  • a plant expression vector system comprising: (i) the plant expression vector of some embodiments of the invention; and (ii) an MDMV helper virus.
  • a plant cell comprising the plant expression vector or vector system of some embodiments of the invention.
  • a plant comprising the plant expression vector or vector system of some embodiments of the invention.
  • a method of generating a plant comprising introducing into one or more cells of the plant the plant expression vector or vector system of some embodiments of the invention.
  • a method of transiently expressing a heterologous polypeptide of interest in a plant comprising introducing into at least one cell of the plant the plant expression vector or vector system of some embodiments of the invention, thereby transiently expressing the heterologous polypeptide of interest in the plant.
  • a method of generating genotypic variation in a genome of a plant comprising introducing into the plant the plant expression vector or vector system of some embodiments of the invention, wherein the nuclease mediates cleavage in a genome of the plant and enables an alteration in the cleavage site, thereby generating genotypic variation in the genome of the plant.
  • a method of tagging a genome of a plant comprising introducing into the plant the plant expression vector or vector system of some embodiments of the invention, wherein the nuclease mediates cleavage in a genome of the plant and enables an alteration in the cleavage site, thereby tagging the genome of the plant.
  • a method of generating a herbicide resistant plant comprising introducing into the plant the plant expression vector or vector system of some embodiments of the invention, wherein the nuclease mediates cleavage in a gene conferring sensitivity to herbicides and enables an alteration in the cleavage site, thereby generating the herbicide resistant plant.
  • a method of generating a pathogen resistant plant comprising introducing into the plant the plant expression vector or vector system of some embodiments of the invention, wherein the nuclease mediates cleavage in a gene conferring sensitivity to a pathogen or in a gene inhibiting the resistance pathway and enables an alteration in the cleavage site, thereby generating the pathogen resistant plant.
  • a method of generating a plant with increased abiotic stress tolerance comprising introducing into the plant the plant expression vector or vector system of some embodiments of the invention, wherein the nuclease mediates cleavage in a gene of the plant conferring sensitivity to abiotic stress and enables an alteration in the cleavage site, thereby generating the plant with increased abiotic stress tolerance.
  • a method of generating male sterility in a plant comprising upregulating in the plant a structural or functional gene of a mitochondria or plastid associated with male sterility by introducing into the plant the plant expression vector or vector system of some embodiments of the invention 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 plastid when targeted into a genome of the mitochondria or plastid, wherein the nuclease mediates cleavage in a genome of the mitochondria or plastid and enables insertion of the heterologous nucleic acid sequence into the cleavage site, thereby generating male sterility in the plant.
  • the plant expression vector of some embodiments of the invention being a satellite vector.
  • the plant expression vector is non-transmittable by aphids.
  • the vector comprises an amino acid alteration which renders the vector non-transmittable by the aphids.
  • the vector comprises an amino acid alteration comprising a DAE to DTE substitution in a N terminal region of a Coat Protein (CP).
  • CP Coat Protein
  • the vector comprises an amino acid alteration comprising a DAG to DTG substitution in a N terminal region of a coat protein (CP).
  • CP coat protein
  • the vector comprises an amino acid alteration comprising a KITC to EITC substitution in a helper component proteinase (HCPro).
  • HCP helper component proteinase
  • the nucleic acid sequence encoding the polyprotein product encodes the full set of proteins of the MDMV.
  • the heterologous polypeptide of interest is translationally fused N terminally to the MDMV polypeptides in the polyprotein product.
  • the heterologous polypeptide of interest is translationally fused C terminally to the MDMV polypeptides in the polyprotein product.
  • the heterologous polypeptide of interest is flanked by the MDMV polypeptides.
  • the heterologous polypeptide of interest is flanked by a PI polypeptide and a HCPro polypeptide of the MDMV, wherein the PI polypeptide is N-terminally positioned to the heterologous polypeptide of interest and the HCPro polypeptide is C-terminally to the heterologous polypeptide of interest.
  • the heterologous polypeptide of interest is flanked by a NIb-RNA replicase polypeptide and a CP polypeptide of the MDMV, wherein the NIb-RNA replicase polypeptide is N-terminally positioned to the heterologous polypeptide of interest and the CP polypeptide is C-terminally to the heterologous polypeptide of interest.
  • the heterologous polypeptide of interest is flanked by the PI polypeptide and the coat protein polypeptide of the MDMV.
  • the PI polypeptide is N- terminally positioned to the heterologous polypeptide of interest and the coat protein polypeptide is C-terminally to the heterologous polypeptide of interest.
  • the heterologous polypeptide of interest is directly translationally fused to at least one protease cleavage site.
  • the protease cleavage site comprises a NIa protease cleavage site as set forth in SEQ ID NO: 42.
  • cleavage of the heterologous polypeptide of interest of the MDMV polypeptides upon expression in a plant cell is effected by a viral protease.
  • the viral protease comprises a NIa protease.
  • the vector further comprises at least one heterologous promoter sequence for directing expression of the polyprotein in the plant cell.
  • the heterologous promoter sequence comprises a 35S promoter.
  • the at least one heterologous promoter sequence comprises two heterologous promoter sequences.
  • the plant expression vector comprises a pGreen backbone.
  • the heterologous polypeptide of interest is selected from the group consisting of 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, a growth inhibiting polypeptide, an enzyme, a transcription factor and a transposase.
  • the nucleic acid sequence encoding the heterologous polypeptide of interest encodes for two heterologous polypeptides of interest.
  • the nucleic acid sequence encoding the polyprotein product comprises a full genome sequence of the MDMV.
  • the heterologous polypeptide of interest comprises a nuclease.
  • the nuclease is selected from the group consisting of a meganuclease, a Cas and a RISC.
  • the Cas comprises Cas9.
  • the nuclease comprises a chimeric nuclease.
  • the chimeric nuclease comprises a nucleic acid binding domain and a nuclease.
  • the chimeric nuclease is selected from the group consisting of a zinc finger nuclease (ZFN), a TALENs and a compact- TALENs.
  • ZFN zinc finger nuclease
  • TALENs TALENs
  • compact- TALENs compact- TALENs
  • the nuclease is selected from the group consisting of a restriction enzyme, a topoisomerase, a recombinase, an integrase, a homing endonucleases and a DNAse.
  • the nucleic acid binding domain of the chimeric nuclease is selected from the group consisting of a meganuclease binding domain, a helix-turn-helix binding domain, a leucine zipper (ZIP) binding domain, a winged helix (WH) binding domain, a winged helix turn helix domain (wHTH) binding domain, a helix-loop-helix binding domain, a transcription activatorlike (TAL) binding domain, a recombinase, and a zinc finger binding domain.
  • ZIP leucine zipper
  • WH winged helix
  • wHTH winged helix turn helix domain
  • TAL transcription activatorlike
  • the nuclease is attached to a localization signal to a DNA-containing organelle.
  • the DNA-containing organelle is selected from the group consisting of a nucleus, a plastid and a mitochondria.
  • the gene conferring sensitivity to a pathogen or the gene inhibiting the resistance pathway is knocked-out to thereby increase resistance to the pathogen.
  • the gene conferring sensitivity to a pathogen comprises an elF4E (translation initiation factor 4E) gene or a Mlo gene.
  • the gene inhibiting the resistance pathway comprises a transcription factor.
  • the gene inhibiting the resistance pathway comprises a rice OsSSI2 gene or a rice NRR gene.
  • the alteration in the cleavage site comprises an amino acid mutation, insertion or deletion.
  • the plastid comprises a chloroplast.
  • the plant expression vector further comprises a chloroplast localization signal.
  • the chloroplast localization signal comprises a ribulose-l,5-bisphospate carboxylase small subunit (Rssu) (SEQ ID NOs: 76 or 77).
  • the plant expression vector further comprises a mitochondria localization signal.
  • the mitochondria localization signal comprises an ATPase beta subunit ( ⁇ - ⁇ ) (SEQ ID NO: 78).
  • the plant is a monocot.
  • the monocot plant is selected from the group consisting of maize, rice, wheat, barley, sugar cane, sorghum, Johnson grass, grasses, bamboo, palm, agave, pineapple, banana, ginger, garlic, onion, oat, rye, turf grass, millet, spelt, triticale, fonio, aloe, asparagus, yam or ubi, orchid, iris, lily, amaryllis, canna-lily arum or gabi, lemon grass, pandan or screwpine, arrow root, rush, pipewort and sedge.
  • the cell is selected from the group consisting of a meristem cell, a leaf cell, a male inflorescence cell, a pollen cell, a female inflorescence cell, an ovule cell and a cell of first node derived calli.
  • the introducing is effected by particle bombardment, agroinfection or sap mechanical infection.
  • FIG. 1 is a 5' MDMV characterization using 5' Race procedure.
  • Four ATPs characterize the beginning of the MDMV genome.
  • the end of the poly G nucleotides indicates the beginning of the viral genome (indicated by the red box, SEQ ID NO: 69).
  • FIGs. 2A-E illustrate the nucleic acid sequence of MDMV isolated in Israel (MDMV-IL) - clone #3076 (9563 bp).
  • 5' UTR dark red, SEQ ID NO: 44
  • PI protease red, SEQ ID NO: 45
  • HcPro yellow, SEQ ID NO: 46
  • P3 light green, SEQ ID NO: 47
  • 6K1 dark green, SEQ ID NO: 48
  • CI light blue, SEQ ID NO: 49
  • 6K2 dark blue, SEQ ID NO: 50
  • NIa-VPg grey, SEQ ID NO: 51
  • NIa-Pro dark grey, SEQ ID NO: 52
  • NIb-RNA replicase purple, SEQ ID NO: 53
  • CP light purple, SEQ ID NO: 54
  • 3' UTR black, SEQ ID NO: 55.
  • the underlined sequence in Figure 2A indicates the start codon of the first viral protein.
  • the underlined green sequence in Figure 2B indicates P3N-PIPO protein expressed by a -1 ribosomal frameshifting from the P3 ORF (SEQ ID NO: 56)
  • the underlined sequence in Figure 2E indicates the stop codon.
  • FIG. 2F is a schematic illustration of clone #3076 vector map.
  • FIG. 3 is a schematic illustration of the addition of multiple cloning sites (MCS) (Agel, Apal) and NIa protease cleavage site (IDVKHQA - SEQ ID NO: 42) in between the PI and HcPro genes of the MDMV sequence (appears in blue). Black arrows point on protease recognition sites.
  • MCS multiple cloning sites
  • IDVKHQA - SEQ ID NO: 42 NIa protease cleavage site
  • FIG. 4 is a schematic illustrations of the addition of MCS (Nhel, Avrll) and NIa protease cleavage site (IDVKHQA - SEQ ID NO: 42) in between the Nib and CP genes of the MDMV sequence (from Biomatik). Black arrows point on protease recognition site.
  • FIG. 5 is a schematic illustration of generation of the MDMV-IL DsRed infective clones.
  • FIGs. 6A-C are photographs illustrating the maize seedlings bombardment procedure.
  • Figure 6A illustrates the 1000/He Biolistic® Particle Delivery System (BioRad);
  • Figure 6B illustrates the maize seedlings before gene shot; and
  • Figure 6C illustrates the particle bombardment chamber containing maize seedlings.
  • FIG. 7 is a photograph illustrating the MDMV-IL phenotype appearance one week post bombardment of pGreen-35s-MDMV (#3076) as compared to a healthy maize leaf.
  • FIGs. 8A-B are photographs illustrating several sorghum cultivars SB 102, SB273 and Israeli cultivar SB 153 which are sensitive to MDMV. Maize infective leaf sap brushed against sorghum young seedling leaves caused, one week later, harsh MDMV leaf symptoms.
  • FIGs. 9A-S are photographs illustrating expression of DsRed by #3101 MDMV- IL clone.
  • Figures 9A-D illustrate the expression of MDMV carrying DsRed in maize leaves;
  • Figures 9E-I illustrate male inflorescence spikelets showing expression of DsRed;
  • Figures 9J-N illustrate male inflorescence anthers showing expression of DsRed;
  • Figures 90-S illustrate that MDMV enters maize pollen.
  • FIGs. 10A-B illustrate the results of an aphid transmission test.
  • Myzus persicae aphids were introduced to feed on #3302 and WT virus infected plants.
  • aphids were removed to 2 healthy maize plant groups.
  • Eight days post aphid inoculation 5 out of 10 plants were infected with WT virus, while none were infected from the #3302 mutated MDMV clone. The results were confirmed by an ELISA test (A) and RT-PCR (B).
  • FIGs. 11A-E illustrate GUS expression from MDMV aphid non-transmissible viral vector.
  • Figure 11A illustrates insertion of GUS, al800 bp reporter gene, in the 5' MCS of the MDMV vector to create clone #3304;
  • Figures 11B-E demonstrate GUS expression by viral vector in several maize leaves.
  • expression of GUS in young (non bombarded) leaves illustrates that the virus can replicate and move within the plant regardless of its foreign insertion (GUS).
  • FIGs. 12A-D are photographs illustrating an increase in Dsred expression in maize B73 explants from 18 to 25 days following bombardment (FB). Maize tissue culture was infected with the viral vector #3101.
  • Figure 12A shows a bright field of explants (18FB);
  • Figure 12B shows Dsred expression in explants (18FB);
  • Figure 12C shows a bright field of explants (25FB);
  • Figure 12D shows Dsred expression in explants (25FB).
  • FIG. 13 is a schematic illustration of an environmental safe viral clone - replacement of DAE and DAG into DTE and DTG motifs, respectively, in the coat protein (SEQ ID NO: 67).
  • FIG. 14 is a schematic illustration of an environmental safe viral clone - replacement of KITC into EITC motif in the HCPro amino acid sequence (SEQ ID NO: 59).
  • the present invention in some embodiments thereof, relates to a Maize dwarf mosaic virus (MDMV) expression vector and, more particularly, but not exclusively, to the use of same for generating genotypic variations in plants.
  • MDMV Maize dwarf mosaic virus
  • MDMV expression vector comprising the whole genome of MDMV under the expression of a double heterologous promoter (e.g., 35S). Since MDMV genome size is large compared to other viruses, cloning of its approximately 10,000 bp was done in several parts while achieving one long open reading frame (ORF) from the beginning of the PI gene until the end of the CP gene (see Figure 5).
  • ORF long open reading frame
  • the expression vector was constructed such that the viral 3' terminal sequence contained a PolyA region.
  • MCS multiple cloning sites
  • Each of the MCS was built from the insertion of 2 unique restriction enzymes, so as to enable cleavage and separation of the foreign gene from the viral proteins upon translation in plants.
  • Cloning of the two reporter genes DsRed (0.7 Kbp in the MDMV 5' or 3' site) and GUS (1.8 Kbp in the MDMV 5' site) allowed stable systemic infection and expression of these genes by the MDMV viral vector (see FIGs. 9A-S and FIGs. 12A-D for DsRed and FIGs. 11A-E for GUS).
  • expression of DsRed was evident in maize leaves ( Figures 9A-D) as well as in male inflorescence ( Figures 9E-N) and its pollen ( Figures 90-S).
  • the MDMV viral vector of the invention was shown to infect young maize seedling ( Figures 6A-C) as well as embryogenic/organogenic callus ( Figures 12A-D) by bombardment procedure.
  • two central motifs that have an effect on the virus transmission by aphids were changed. Specifically, DAG motif located in the coat protein gene (CP), and KITC motif found in the HcPro gene were changed to non-functional motifs, DTG and EITC respectively (see Figures 13-14). Aphid tests proved that once the vector infected maize plants, it could not be transmitted by aphids to new healthy plants ( Figures 10A-B). Accordingly, these novel MDMV viral vectors may serve as powerful tools in the field of agriculture transgenic technologies.
  • a plant expression vector comprising a nucleic acid sequence encoding a polyprotein product, the polyprotein product comprising Maize Dwarf Mosaic Virus (MDMV) polypeptides and a heterologous polypeptide of interest, the heterologous polypeptide of interest being cleaved of the MDMV polypeptides upon expression in a plant cell, and wherein the plant expression vector is capable of spreading in a plant.
  • MDMV Maize Dwarf Mosaic Virus
  • a plant expression vector refers to a nucleic acid vector including a DNA vector, a RNA vector, virus or other suitable replicon (e.g., viral vector) encoding for viral genes or parts of viral genes, as well as for heterologous expression products such as RNA and proteins (e.g. polypeptides).
  • the expression vector of some embodiments of the invention synonymously refers to a plasmid, a vector, an expression vector, a construct and an expression construct.
  • polyprotein or "polyprotein product” as used herein refers to a protein product that is cleaved into separate smaller proteins each with a distinctive biological function.
  • the polyprotein of the invention comprises maize dwarf mosaic virus (MDMV) polypeptides as well as a heterologous polypeptide of interest.
  • MDMV dwarf mosaic virus
  • MDMV Maize Dwarf Mosaic Virus
  • RNA virus which is a member of the genus Potyvirus and of the family Potyviridae.
  • MDMV genome is expressed as a polyprotein which is cleaved by viral proteases and processed into mature proteins.
  • the MDMV genome is expressed as a recombinant polyprotein.
  • nucleic acid sequence encoding a MDMV polypeptide may be used according to the present teachings, such as the nucleic acid sequences encoding MDMV- A, MDMV-C, MDMV-D, MDMV-E and MDMV-F.
  • the MDMV of the invention is Israeli type MDMV (MDMV-IL) or a fragment thereof.
  • the full length MDMV-IL gene (set forth in SEQ ID NO: 43) comprises a 5' UTR sequence (SEQ ID NO: 44), a PI protease sequence (SEQ ID NO: 45), a helper component proteinase (HcPro) sequence (SEQ ID NO: 46), a P3 protein sequence (SEQ ID NO: 47), a P3N-PIPO protein sequence (SEQ ID NO: 56), a 6K1 protein sequence (SEQ ID NO: 48), a CI protein sequence (SEQ ID NO: 49), a 6K2 protein sequence (SEQ ID NO: 50), a N-terminal viral protein genome- linked (NIa-VPg) sequence (SEQ ID NO: 51), a nuclear inclusion protein-a protease (NIa-Pro) sequence (SEQ ID NO: 52), a NIb-RNA replicase sequence (SEQ ID NO: 53), a coat protein
  • the nucleic acid sequence encoding the polyprotein product comprises the full genome sequence of the MDMV (SEQ ID NO:
  • the nucleic acid sequence encoding the polyprotein product comprises a partial sequence of the MDMV genome.
  • the nucleic acid sequence encoding the polyprotein product comprises the entire sequence of the MDMV genome without the nucleic acid sequence encoding the coat protein polypeptide.
  • the nucleic acid sequence encoding the polyprotein product only comprises the MDMV sequences for PI polypeptide.
  • the nucleic acid sequence encoding the polyprotein product only comprises the MDMV sequences for the coat protein polypeptide.
  • the nucleic acid sequence encoding the polyprotein product comprises the MDMV sequences for PI polypeptide and for the coat protein polypeptide.
  • the partial sequence comprising the nucleic acid sequence encoding any of the MDMV proteins, e.g. 5' UTR sequence (SEQ ID NO:
  • PI protease SEQ ID NO: 45
  • HcPro SEQ ID NO: 46
  • P3 SEQ ID NO: 47
  • P3N-PIPO SEQ ID NO: 56
  • 6K1 SEQ ID NO: 48
  • CI SEQ ID NO: 49
  • 6K2 SEQ ID NO: 50
  • NIa-VPg SEQ ID NO: 51
  • NIa-Pro SEQ ID NO: 52
  • NIb-RNA replicase SEQ ID NO: 53
  • CP SEQ ID NO: 54
  • 3' UTR SEQ ID NO: 55
  • the plant expression vector of the invention comprises a nucleic acid sequence encoding a polyprotein product which enables spreading in a plant.
  • spreading in a plant refers to the expression of the polyprotein product in a cell or organ other than the cell or organ initially infected by the vector.
  • spreading in a plant includes expression of the polyprotein product in meristematic tissues which allows efficient expression throughout the plant. Spreading in plants is discussed in further detail hereinbelow.
  • the nucleic acid sequence encodes the full set of proteins of the MDMV (SEQ ID NO: 57).
  • the nucleic acid sequence encodes for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of the MDMV proteins.
  • the nucleic acid sequence may encode for any of a PI protease (SEQ ID NO: 58), a HcPro (SEQ ID NO: 59), a P3 protein (SEQ ID NO: 60), a P3N-PIPO protein (SEQ ID NO: 68), a 6K1 protein (SEQ ID NO: 61), a CI protein (SEQ ID NO: 62), a 6K2 protein (SEQ ID NO: 63), a NIa-VPg (SEQ ID NO: 64), a NIa-Pro (SEQ ID NO: 65), a NIb-RNA replicase (SEQ ID NO: 66) and/or a coat protein (CP) (SEQ ID NO: 67).
  • SEQ ID NO: 58 PI protease
  • HcPro SEQ ID NO: 59
  • P3 protein SEQ ID NO: 60
  • the nucleic acid sequence encodes for a PI protease, a heterologous polypeptide of interest, a HcPro, a P3 protein, a 6K1 protein, a CI protein, a 6K2 protein, a NIa-VPg, a NIa-Pro, a NIb-RNA replicase (e.g. NIb-RdRp), and a coat protein (CP).
  • the nucleic acid sequence encodes for a PI protease, a HcPro, a P3 protein, a 6K1 protein, a CI protein, a 6K2 protein, a NIa-VPg, a NIa-Pro, a NIb-RNA replicase (e.g. NIb-RdRp), a heterologous polypeptide of interest, and a coat protein (CP).
  • a PI protease a HcPro, a P3 protein, a 6K1 protein, a CI protein, a 6K2 protein, a NIa-VPg, a NIa-Pro, a NIb-RNA replicase (e.g. NIb-RdRp), a heterologous polypeptide of interest, and a coat protein (CP).
  • the plant expression vector of the invention comprises an amino acid alteration which renders the vector non-transmittable by an aphid [e.g. any aphid capable of transmitting MDMV including but not limited to the corn leaf aphid (Rhopalosiphum maidis), the greenbug (Schiz phis graminum), and the green peach aphid (Myzus persicae)] .
  • the amino acid alteration may be at any site or multiple sites of MDMV known to one of skill in the art to contribute to aphid transmission, as for example, in the coat protein or the helper component-protease (HC-Pro) typically required for aphid transmission.
  • the amino acid alteration comprises a DAE to DTE substitution at the N terminal region of a coat protein. This alteration results in an inability of the virus to attach to the transmission aphid.
  • the amino acid alteration comprises a DAG to DTG substitution in a N terminal region of a coat protein. Similarly, this alteration results in an inability of the virus to attach to the transmission aphid.
  • the amino acid alteration comprises a KITC to EITC substitution in a helper component proteinase (HCPro). Similarly, this alteration results in an inability of the virus to attach to the transmission aphid.
  • the amino acid alteration comprises a DAE to DTE substitution and/or a DAG to DTG substitution at the N terminal region of a coat protein. Additionally or alternatively, the amino acid alteration may comprise a KITC to EITC substitution in a helper component proteinase (HCPro).
  • HCP helper component proteinase
  • the plant expression vector of the invention may comprise an amino acid alteration which renders the MDMV symptoms nonsignificant or inhibits MDMV symptoms altogether.
  • the plant expression vector of the invention further comprises at least one heterologous polypeptide of interest.
  • the heterologous polypeptide of interest being cleaved of the MDMV polypeptides upon expression in a plant cell.
  • the polypeptide of the invention is expressed in a plant cell, the polypeptide is cleaved into individual proteins and the heterologous polypeptide is cleaved of the polypeptide, i.e. is not attached to any of the MDMV peptides (as discussed in further detail hereinbelow).
  • heterologous refers to exogenous, not-naturally occurring in MDMV and/or not occurring within a native cell of a plant.
  • polypeptide is used interchangeably with the terms “peptides”, “oligopeptides” and “proteins” and refers to a biomolecule composed of amino acids of any length, linked together by peptide bonds, unless mentioned herein otherwise.
  • heterologous polypeptide or “heterologous polypeptide of interest”, as used herein, refers to a biomolecule composed of two or more amino acids (including truncation products and full length proteins) that is expressed from a "heterologous gene” or “heterologous coding sequence” as defined below. Accordingly, the heterologous polypeptide produced in a plant is exogenous to, or not naturally occurring in MDMV.
  • the heterologous polypeptide may be also heterologous to the plant, indicating that it is not expressed in a plant, not expressed in a particular plant species, or is expressed at a different expression level or localization in the plant.
  • the heterologous polypeptide can be, for example, a plant polypeptide, a bacterial polypeptide, a viral polypeptide a mammalian polypeptide or a synthetic polypeptide (e.g., chimeric nuclease, nuclease e.g. cas9).
  • the heterologous polypeptide of interest may be a plant polypeptide or protein that is a variant or mutated form of a plant polypeptide or protein or a polypeptide or protein not naturally found in the producing plant species, line or variety.
  • heterologous gene or “heterologous coding sequence” refers to polynucleotide (nucleic acid sequence) that is exogenous to, or not naturally found in, the MDMV genome and that encodes an expression product e.g., the heterologous polypeptide of interest.
  • polynucleotide refers to a single or double stranded nucleic acid sequence which is isolated and provided in the form of an RNA sequence, a complementary polynucleotide sequence (cDNA), a genomic polynucleotide sequence and/or a composite polynucleotide sequences (e.g., a combination of the above).
  • isolated refers to at least partially separated from the natural environment.
  • the heterologous polypeptide of interest may include, but is not limited to, a reporter polypeptide, an antiviral polypeptide, a viral moiety, an antiviral polypeptide, 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, a growth inhibiting polypeptide, an enzyme, a transcription factor and a transposase.
  • Exemplary proteins which may be produced include, but are not limited to: nucleases, kinases, proteases, enzymes, hormones, tumor suppressors, blood clotting proteins, cell cycle proteins, metabolic proteins, neuronal proteins, cardiac proteins, proteins deficient in specific disease states, structural proteins, antibodies, antigens, proteins that provide resistance to diseases, antimicrobial proteins, antiviral proteins, interferons, cytokines, growth factors, receptors, ligands, and signaling molecules.
  • the heterologous polypeptide of interest comprises two or more (e.g., 2, 3, 4) heterologous polypeptides.
  • the heterologous polypeptide of interest enables modifying the plant genome, e.g., nuclease.
  • nuclease refers to any polypeptide, or complex comprising a polypeptide, that can generate a strand break in the genome, e.g. in genomic DNA.
  • the cleavage is site specific usually conferred by an auxiliary subunit, alternatively the nuclease is inherently specific to a target sequence of interest.
  • cleavage refers to the breakage of the covalent backbone of a DNA molecule. Both single- stranded cleavage and double- stranded cleavage are possible, and double-stranded cleavage can occur as a result of two distinct single-stranded cleavage events. DNA cleavage can result in the production of either blunt ends or staggered ends.
  • nucleases which may be used in accordance with the present teachings include restriction enzymes (e.g. type II restriction endonuclease), topoisomerases [e.g. DNA gyrase, eukaryotic topoisomerase II (topo II), and bacterial topoisomerase IV (topo IV)], recombinases (e.g. Cre recombinase, Hin recombinase), integrases, DNAses, endo-exonucleases (e.g. micrococcal nuclease) and homing endonucleases.
  • restriction enzymes e.g. type II restriction endonuclease
  • topoisomerases e.g. DNA gyrase, eukaryotic topoisomerase II (topo II), and bacterial topoisomerase IV (topo IV)
  • recombinases e.g. Cre recombinase, Hin recombinas
  • the nuclease utilized may comprise a non-specific DNA cleavage domain, for example, a type II restriction endonuclease such as the cleavage domain of the Fokl restriction enzyme (GenBank accession number J04623).
  • the nuclease is a meganuclease.
  • the term "meganuclease” refers to a double- stranded endonuclease having a large polynucleotide recognition site, e.g. DNA sequences of at least 12 base pairs (bp) or from 12 bp to 40 bp.
  • the meganuclease may also be referred to as rare-cutting or very rare-cutting endonuclease.
  • the meganuclease of the invention may be monomeric or dimeric.
  • the meganuclease may include any natural meganuclease such as a homing endonuclease, but may also include any artificial or man-made meganuclease endowed with high specificity, either derived from homing endonucleases of group I introns and inteins, or other proteins such as zinc finger proteins or group II intron proteins, or compounds such as nucleic acid fused with chemical compounds.
  • Artificial meganucleases of the invention include, but are not limited to, custom- made meganucleases which are meganucleases derived from any initial meganuclease, either natural or not, presenting a recognition and cleavage site different from the site of the initial meganuclease, i.e. the custom-made meganuclease cleaves a novel site with an efficacy at least 10 fold, at least 50 fold or at least 100 fold more than the natural meganuclease.
  • Custom-made meganucleases may be produced by any method known in the art, for example, by preparing a library of meganuclease variants and isolating, by selection and/or screening, the variants able to cleave the targeted DNA sequence.
  • the diversity could be introduced in the meganuclease by any method known to one skilled in the art, for example, the diversity may be introduced by targeted mutagenesis (i.e. cassette mutagenesis, oligonucleotide directed codon mutagenesis, targeted random mutagenesis), by random mutagenesis (i.e. mutator strains, Neurospora crassa system (U.S. Pat. No.
  • the diversity may be introduced at positions of the residues contacting the DNA target or interacting (directly or indirectly) with the DNA target, or may be introduced specifically at the positions of the interacting amino acids.
  • the 20 amino acids can be introduced at the chosen variable positions.
  • the amino acids present at the variable positions are the amino acids well-known to be generally involved in protein-DNA interaction. More particularly, these amino acids are generally the hydrophilic amino acids, e.g. comprise D, E, H, K, N, Q, R, S, T, Y. Synthetic or modified amino acids may also be used.
  • the custom-made meganuclease may be derived from any initial meganuclease.
  • the initial meganuclease is selected so as its natural recognition and cleavage site is the closest to the targeted DNA site.
  • the initial meganuclease is a homing endonuclease. Homing endonucleases fall into 4 separated families on the basis of well conserved amino acids motifs, namely the LAGLIDADG family, the GIY-YIG family, the His-Cys box family, and the HNH family (Chevalier et al., 2001, N.A.R, 29, 3757-3774).
  • the homing endonuclease is a I-Dmo I, PI-Sce I, I-Scel, PI-Pfu I, I-Cre I, I- Ppo I, or a hybrid homing endonuclease I-Dmo I/I-Cre I called E-Dre I (as taught in Chevalier et al., 2001, Nat Struct Biol, 8, 312-316).
  • the nuclease comprises an oligonucleotide-dependant nuclease such as Cas or a RISC.
  • RISC enzymes are taught in Martinez J, Tuschl T. RISC is a 5' phosphomonoester-producing RNA endonuclease. Genes Dev. 2004;18:975-980. Also contemplated are sequence modifications to improve plant expression i.e., homologs that are at least 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 %. Homology and identity are also contemplated herein (e.g., using Blast(N)/(P) with default parameters).
  • the Cas9 or RISC is attached to a single guide RNA (sgRNA) to cleave genomic DNA in a sequence specific manner.
  • sgRNA single guide RNA
  • a single guide RNA or “sgRNA” refers to a chimeric RNA molecule which is composed of a clustered regularly interspersed short p_alindromic repeats (CRISPR) RNA (crRNA) and trans-encoded CRISPR RNA (tracrRNA).
  • CRISPR CRISPR
  • crRNA clustered regularly interspersed short p_alindromic repeats
  • tracrRNA trans-encoded CRISPR RNA
  • the crRNA defines a site-specific targeting of the Cas9 protein.
  • the sequence is 19-22 nucleotides long e.g., 20 consecutive nucleotides complementary to the target and is typically located at the 5' end of the sgRNA molecule.
  • the crRNA may have 100 % complementation with the target sequence although at least 80 %, 85 %, 90 %, and 95 % global homology to the target sequence are also contemplated according to the present teachings.
  • the tracrRNA is 100-300 nucleotides long and provides a binding site for the nuclease e.g., Cas9 protein forming the CRISPR/Cas9 complex.
  • a plurality of sgRNAs are provided to the plant cell that are complementary to different target nucleic acid sequences and the nuclease e.g., Cas9 enzyme cleaves the different target nucleic acid sequences in a site specific manner.
  • the nuclease e.g., Cas9 enzyme
  • the sgRNA may be encoded from the same expression vector as the nuclease, e.g. Cas9. Additionally or alternatively, the sgRNA may be encoded from another nucleic acid construct and thus the CRISPR-Cas9 complex is encoded from a nucleic acid construct system.
  • sgRNA is encoded from the plant expression vector of the invention.
  • the nuclease e.g. Cas9
  • the nucleic acid construct e.g., which may be MDMV-based as described herein, or not
  • the CRISPR-Cas9 complex is encoded from a nucleic acid construct system.
  • the plurality of sgRNAs may be encoded from a single vector or from a plurality of vectors as described herein.
  • the use of a plurality of sgRNAs allows multiplexing.
  • RNA-guided endonuclease of the invention comprises at least one nuclease (e.g. Cas9 or RISC) and at least one RNA binding domain (e.g. CRISPR).
  • CRISPR/Cas proteins of the invention may comprise a nuclease domain, DNA binding domain, helicase domain, RNAse domain, protein-protein interaction domain and/or a dimerization domain.
  • the CRISPR/Cas protein can be a wild type
  • CRISPR/Cas protein a modified CRISPR/Cas protein, or a fragment of a wild type or modified CRISPR/Cas protein.
  • the CRISPR/Cas protein can be modified to increase nucleic acid binding affinity and/or specificity, or to alter an enzymatic activity of the protein.
  • nuclease i.e., Cas9 domains of the CRISPR/Cas protein can be modified.
  • Non-limiting examples of suitable Cas proteins which may be used in accordance with the present teachings include Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8al, Cas8a2, Cas8b, Cas8c, Cas9, CaslO, Casl Od, CasF, CasG, CasH, Csyl, Csy2, Csy3, Csel (or CasA), Cse2 (or CasB), Cse3 (or CasE), Cse4 (or CasC), Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3,Csxl7, Csxl4, CsxlO, Csxl6, CsaX
  • the cas nuclease is Cas9.
  • Cas9 is a monomeric DNA nuclease guided to a DNA target sequence adjacent to the protospacer adjacent motif (PAM).
  • the Cas9 protein comprises two nuclease domains homologous to RuvC and HNH nucleases.
  • the HNH nuclease domain cleaves the complementary DNA strand whereas the RuvC-like domain cleaves the non-complementary strand and, as a result, a blunt cut is introduced in the target DNA.
  • the CRISPR/Cas system comprises a wild type Cas9 protein or fragment thereof.
  • the CRISPR/Cas system comprises a modified Cas9 protein.
  • the amino acid sequence of the Cas9 protein may be modified to alter one or more properties (e.g., nuclease activity, affinity, stability, etc.) of the protein.
  • domains of the Cas9 protein not involved in RNA-guided cleavage can be eliminated from the protein such that the modified Cas9 protein is smaller than the wild type Cas9 protein.
  • the Cas9 protein can be modified to lack at least one functional nuclease domain. According to one embodiment, the Cas9 protein can be modified to lack all nuclease activity. According to another embodiment, the CRISPR/Cas system is fused with various effector domains, such as DNA cleavage domains.
  • the DNA cleavage domain can be obtained from any endonuclease or exonuclease. Non-limiting examples of endonucleases from which a DNA cleavage domain can be derived include, but are not limited to, restriction endonucleases and homing endonucleases (see, for example, New England Biolabs Catalog or Belfort et al. (1997) Nucleic Acids Res.).
  • the cleavage domain of the CRISPR/Cas system is a Fokl endonuclease domain or a modified Fokl endonuclease domain.
  • the nuclease comprises a chimeric nuclease.
  • chimeric nuclease refers to a synthetic chimeric polypeptide which forms a single open reading frame (ORF) and mediates DNA cleavage in a sequence specific manner.
  • the chimeric nucleases of this aspect of the present invention comprise separate domains for nucleic acid binding (e.g. DNA binding) and for nucleic acid cleavage (e.g. DNA cleavage), such that cleavage is sequence specific.
  • sequence specific refers to a distinct chromosomal location at which nucleic acid cleavage (e.g. DNA cleavage) is introduced.
  • nucleic acid 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 or RNA in a sequence- specific manner (i.e. target site).
  • the nucleic acid (e.g. DNA) binding domain of the present invention needs to be coupled to a DNA cleavage domain (e.g. 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.
  • the DNA binding domain overlaps the target sequence or may bind within the target sequence.
  • the chimeric nuclease induces a single stranded or a double stranded cleavage in the target site.
  • any DNA or RNA binding domain that recognizes the desired target sequence e.g. DNA binding sequence
  • a variety of such DNA and RNA binding domains are known in the art.
  • DNA binding domains include, but are not limited to, a meganuclease binding domain, a helix-turn-helix (pfam 01381) binding domain, a leucine zipper (ZIP) binding domain, a winged helix (WH) binding domain, a winged helix turn helix domain (wHTH) binding domain, a helix-loop-helix binding domain, a transcription activator-like (TAL) binding domain, a recombinase, and a zinc finger binding domain.
  • a meganuclease binding domain a helix-turn-helix (pfam 01381) binding domain, a leucine zipper (ZIP) binding domain, a winged helix (WH) binding domain, a winged helix turn helix domain (wHTH) binding domain, a helix-loop-helix binding domain, a transcription activator-like (TAL) binding domain, a recombinase, and a
  • the DNA binding domain is a zinc finger binding domain.
  • the chimeric nuclease is a chimeric protein comprising a specific zinc finger binding domain (e.g., pfam00096) and the DNA cleavage domain, such as that of the Fokl restriction enzyme (also referred to herein as the Fokl cleavage domain), termed herein zinc finger nuclease (ZFN).
  • 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].
  • DNA binding domains with novel specificities can be evolved and selected [see, e.g., Desjarlais et al., Proc. Natl. Acad. Sci.
  • 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.
  • 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.
  • 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.
  • the chimeric nuclease is a TALENs or a compact-TALENs (cTALENs).
  • TALENs or "Transcription Activator-Like Effector Nucleases” refers to the artificial restriction enzymes generated by fusing the TAL effector DNA binding domain to a DNA cleavage domain. TALENs of the invention enable efficient, programmable, and specific DNA cleavage.
  • TALEs Transcription activator-like effectors
  • TALEN is broad and includes a monomeric TALEN that can cleave double stranded DNA without assistance from another TALEN.
  • the term TALEN is also used to refer to one or both members of a pair of TALENs that are engineered to work together to cleave DNA at the same site. TALENs that work together may be referred to as a left-TALEN and a right-TALEN. Further details relating to TALENS can be found in U.S. Pat. No. 8,450,471; U.S. Pat. No. 8,440,431; U.S. Pat. No. 8,440,432; and U.S. Pat. Applic. No. 20140256798 all of which are incorporated herein by reference in their entirety.
  • TALEs are proteins secreted by Xanthomonas bacteria.
  • the DNA binding domain of TALEs contains a highly conserved 33-34 amino acid sequence with the exception of the 12th and 13th amino acids. These two locations are highly variable [Repeat Variable Diresidue (RVD)] and show a strong correlation with specific nucleotide recognition. This simple relationship between amino acid sequence and DNA recognition has allowed for the engineering of specific DNA binding domains by selecting a combination of repeat segments containing the appropriate RVDs.
  • RVD Repeat Variable Diresidue
  • TALENs of the invention are typically constructed using a non-specific DNA cleavage domain, such as the non-specific DNA cleavage domain of Fokl endonuclease.
  • a non-specific DNA cleavage domain such as the non-specific DNA cleavage domain of Fokl endonuclease.
  • wild-type Fokl cleavage domain may be used as well as Fokl cleavage domain variants with mutations designed to improve cleavage specificity and cleavage activity.
  • the Fokl domain functions as a dimer, requiring two constructs with unique DNA binding domains for sites in the target genome with proper orientation and spacing. Both the number of amino acid residues between the TALEN DNA binding domain and the DNA cleavage domain (e.g. Fokl cleavage domain) and the number of bases between the two individual TALEN binding sites are parameters for achieving high levels of activity.
  • the number of amino acid residues between the TALEN DNA binding domain and the DNA cleavage domain may be modified by introduction of a spacer between the plurality of TAL effector repeat sequences and the nuclease (e.g. Fokl endonuclease domain).
  • the spacer sequence may be 12 to 30 nucleotides.
  • compact TALENs may be used according to the present teachings.
  • These cTALENs are typically designed with the partially specific I- Tevl catalytic domain and are monomeric DNA-cleaving enzymes, i.e. TALENs which are half- size, single-polypeptide compact transcription activator- like effector nucleases (see Beurdeley M. et al., Nature Communications (2013) 4: 1762, which is incorporated herein by reference in its entirety).
  • TALEN binding domain The relationship between amino acid sequence and DNA recognition of the TALEN binding domain allows for designable proteins.
  • software programs e.g. DNA Works
  • DNA Works may be used which calculate oligonucleotides suitable for assembly in a two step PCR; oligonucleotide assembly followed by whole gene amplification.
  • Modular assembly schemes for generating engineered TALE constructs may also be used. Both methods offer a systematic approach to engineering DNA binding domains that are conceptually similar to the modular assembly method for generating zinc finger DNA recognition domains (described hereinabove).
  • Qualifying the nucleases e.g. ZFN, TALENs and CRISPR/Cas
  • meganucleases thus generated for specific target recognition can be effected using methods which are well known in the art.
  • a method for designing the nucleases for use in gene targeting may include a process for testing the toxicity of the nuclease on a cell. Such a process may comprise expressing in the cell, or otherwise introducing into a cell, the nuclease and assessing cell growth or death rates by comparison against a control.
  • the tendency of a 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.
  • electrophoresis e.g. pulsed field electrophoresis may be used to resolve very large fragments
  • the heterologous polypeptide of interest may further comprise at least one nuclear localization signal (NLS) which facilitates the transport of the nuclease to the DNA-containing organelle.
  • NLS nuclear localization signal
  • an NLS comprises a stretch of basic amino acids which is recognized by specific receptors at the nuclear pores.
  • the NLS can be located at the N-terminus, the C-terminal, or in an internal location of the nuclease.
  • 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).
  • 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 Al, the sequence KIPIK in yeast transcription repressor Mata2 and the complex signals of U snRNPs, tobacco NLS and rice NLS.
  • the localization signal for a DNA containing organelle can be a mitochondrial localization signal (MLS) or a chloroplast localization signal (CLS).
  • Mitochondrion localization signals (MLS) 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 ⁇ -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].
  • the localization signal may comprise a mitochondria localization signal, such as the signal peptide of the ATPase beta subunit ( ⁇ - ⁇ ) (SEQ ID NO: 78).
  • 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 (atslA) 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 www(dot)springerlink(dot)com/content/p65013h263617795/.
  • the chloroplast localization sequence may be derived from a viroid [Evans and Pradhan (2004) US 2004/0142476 Al].
  • 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).
  • the localization signal may comprise a chloroplast localization signal, such as the transit peptide ribulose-l,5-bisphospate carboxylase small subunit (Rssu) (SEQ ID NOs: 76 or 77).
  • a chloroplast localization signal such as the transit peptide ribulose-l,5-bisphospate carboxylase small subunit (Rssu) (SEQ ID NOs: 76 or 77).
  • the heterologous polypeptide of interest (e.g. nuclease) further comprises at least one cell-penetrating domain.
  • the cell- penetrating domain can be a cell-penetrating peptide (CPP) sequence derived from Tat, Tat2, arginine-rich intracellular delivery peptides (AID), pVEC, transportan and penetratin.
  • CPP cell-penetrating peptide
  • the CPP sequence comprises a dimmer of the Tat molecule (Tat2, RKKRRQRRRRKKRRQRRR, SEQ ID NO: 79) which has an increased ability to translocate across plant cell membranes because of the presence of high number of arginine residues.
  • the CPP sequence comprises an 18 amino acid peptide of vascular endothelial-cadherin (pVEC) (LLIILRRRIRKQ AH AS K SEQ ID NO: 80).
  • the heterologous polypeptide of interest is introduced into the plant target using a plant expression vector which is typically used for mediating transient transformation through systemic spreading within the plant.
  • a plant expression vector which is typically used for mediating transient transformation through systemic spreading within the plant.
  • Constructs useful in the methods according to some embodiments of the invention may be constructed using recombinant DNA technology well known to persons skilled in the art.
  • the gene constructs may be inserted into vectors, which may be commercially available, suitable for transforming into plants and suitable for expression of the gene of interest in the transformed cells.
  • vectors which may be commercially available, suitable for transforming into plants and suitable for expression of the gene of interest in the transformed cells.
  • a nucleic acid sequence encoding the polyprotein is cloned into an expression vector, for example in to a binary vector, the expression vector is introduced into a plant cell, as described in the examples see below, and the polyprotein is expressed in the host cell.
  • binary vectors examples include pBIN19, pBHOl, pBinAR, pGPTV, pCAMBIA, pBIB-HYG, pBecks, pGreen or pPZP (Hajukiewicz, P. et al., Plant Mol. Biol. 25, 989 (1994), and Hellens et al., Trends in Plant Science 5, 446 (2000)).
  • the vector is a pGreen vector.
  • cloning may be carried out in several parts (e.g. gene sequences may be cloned into several vectors, such as pJET or pGEM (Amp ), as described in detail in the Examples section which follows) and may then be combined into one vector (e.g. pGreen) to achieve one long open reading frame (ORF) (e.g. from the beginning of the PI gene of MDMV until the end of the CP gene of MDMV, see Figure 2F).
  • the expression vector may further be constructed to include a PolyA region (e.g. at the viral 3' terminal sequence).
  • the expression vector of the invention may be constructed such that the nucleic acid sequence encoding the heterologous polypeptide of interest is cloned at the N- terminal end or at the C-terminal end of the nucleic acid sequence encoding the MDMV polypeptides, i.e. such that the heterologous polypeptide of interest is directly translationally fused N-terminally or C-terminally, respectively, to the MDMV polypeptides in the polyprotein product.
  • the expression vector of the invention may be constructed such that the heterologous polypeptide of interest is flanked by the MDMV polypeptides.
  • the vector of the invention may be constructed to include multiple cloning sites (MCS) at different locations within the MDMV genome and without interfering with the readthrough of the ORF or the polyprotein.
  • MCS may be inserted, for example, between the PI gene and a HCPro gene of MDMV, or between the NIb-RdRp gene and a CP gene of the MDMV, by insertion of restriction enzymes (e.g. on the 5' MCS Agel and Apal and on the 3' MCS Nhel and Avrll).
  • the heterologous polypeptide of interest is flanked by a PI polypeptide and a HCPro polypeptide of the MDMV, wherein the PI polypeptide is N-terminally positioned to the heterologous polypeptide of interest and the HCPro polypeptide is C-terminally to the heterologous polypeptide of interest.
  • the heterologous polypeptide of interest is flanked by a NIb-RNA replicase (e.g. NIb-RdRp) polypeptide and a CP polypeptide of the MDMV, wherein the NIb-RNA replicase (e.g. NIb-RdRp) polypeptide is N-terminally positioned to the heterologous polypeptide of interest and the CP polypeptide is C-terminally to the heterologous polypeptide of interest.
  • a NIb-RNA replicase e.g. NIb-RdRp
  • CP polypeptide is C-terminally to the heterologous polypeptide of interest.
  • the expression vector is a satellite vector.
  • Satellites either encapsidated or in the form of naked nucleic acid, depend on a helper virus for their replication.
  • Geminivirus-associated, 682-base-long DNA satellite was first reported by Dry et al. in 1997, Proc. Natl. Acad. Sci. USA 94, 7088- 7093.
  • Other gemini-associated satellites have been discovered since then (Briddon et al., 2001, Virology 285, 234-243; Mansoor et al., 1999, Virology 259, 190-199; Zhou et al., 2003, J. Gen. Virol. 84, 237-247).
  • the satellite determines symptom severity.
  • these are encapsidated and replicated via factors provided by helper viruses.
  • a satellite associated with a particular virus may be supported for replication by other viruses as well.
  • the helper virus may be a wild type MDMV virus or may be an MDMV virus deficient of one or more peptides (e.g. lacking a coat protein gene). Satellite vectors of the invention enable expression of a large heterologous polypeptide (of about 4000 bases) in a target cell.
  • the plant expression vector comprises a nucleic acid sequence encoding a polyprotein product, the polyprotein product comprising Maize Dwarf Mosaic Virus (MDMV) polypeptides, wherein the MDMV polypeptides comprise a PI polypeptide and a coat protein polypeptide, and a heterologous polypeptide of interest, the heterologous polypeptide of interest being cleaved of the MDMV polypeptides upon expression in a plant cell infected with a helper virus or an MDMV virus, and wherein the plant expression vector is capable of spreading in a plant.
  • MDMV Maize Dwarf Mosaic Virus
  • the heterologous polypeptide of interest is flanked by a PI polypeptide and a coat protein polypeptide of the MDMV.
  • the PI polypeptide is N-terminally positioned to the heterologous polypeptide of interest and the coat protein polypeptide is C-terminally to the heterologous polypeptide of interest.
  • the expression vector is included in a system.
  • the plant expression vector system comprises one plant expression vector of some embodiments of the invention (e.g satellite vector) and an MDMV helper virus (as discussed above).
  • the vector of the invention is designed such that the heterologous polypeptide or polypeptides are cleaved and separated of the MDMV polypeptides upon expression in a plant cells.
  • the nucleic acid sequence encoding the heterologous polypeptide of interest is directly translationally fused to the protease cleavage site.
  • the heterologous polypeptide is cleaved of the viral polypeptides with no functional remnants of viral proteins remaining in the heterologous polypeptide.
  • the protease cleavage site is a viral protease cleavage site.
  • the protease cleavage site is a NIa protease cleave site (e.g. SEQ ID NO: 42).
  • the nucleic acid sequence encoding the heterologous polypeptide of interest encodes for two heterologous polypeptides of interest.
  • the nucleic acid sequence encoding the heterologous polypeptide of interest encodes for 2, 3, 4, 5 or more heterologous polypeptides of interest.
  • heterologous polypeptide In cases in which more than one heterologous polypeptide is expressed in a cell, additional protease cleavage sites may be added between the heterologous polypeptides as to enable cleavage thereof.
  • At least two heterologous polypeptide sequences within the vector are separated by a nucleic acid sequence encoding a cleavage domain.
  • a cleavage domain may comprise any cleavage domain known in the art, as for example a NIa protease cleave site (e.g. SEQ ID NO: 42).
  • the expression vector of the invention is may be constructed such that the nucleic acid sequence encoding the polyprotein product is operably linked to one or more regulatory sequences allowing expression in the plant cells.
  • the regulatory sequence is a plant-expressible promoter.
  • plant-expressible refers to a promoter sequence, including any additional regulatory elements added thereto or contained therein, is at least capable of inducing, conferring, activating or enhancing expression in a plant cell, tissue or organ, preferably a monocotyledonous or dicotyledonous plant cell, tissue, or organ. Examples of preferred promoters useful for the methods of some embodiments of the invention are presented in Table I, II, III and IV. Table I
  • PR00122 chlorophyll a/lo-binding protein very weak in shoot
  • PR00171 reversibly glycosylated protein weak constitutive
  • PRO0200 OSH1 very weak in meristem of young plants
  • the promoter is a heterologous promoter i.e. a promoter not naturally found in the plant (i.e. not-native to the plant).
  • the heterologous promoter comprises a 35S promoter.
  • the vector comprises a nucleic acid sequence operably linked to two or more promoter sequences.
  • the vector comprises a nucleic acid sequence operably linked to a double 35s promoter (i.e. two 35S promoters one after the other).
  • Nucleic acid sequences of the polypeptides of some embodiments of the invention may be optimized for plant expression. Examples of such sequence modifications include, but are not limited to, an altered G/C content to more closely approach that typically found in the plant species of interest, and the removal of codons atypically found in the plant species commonly referred to as codon optimization.
  • an optimized gene or nucleic acid sequence refers to a gene in which the nucleotide sequence of a native or naturally occurring gene has been modified in order to utilize statistically-preferred or statistically-favored codons within the plant.
  • the nucleotide sequence typically is examined at the DNA level and the coding region optimized for expression in the plant species determined using any suitable procedure, for example as described in Sardana et al. (1996, Plant Cell Reports 15:677-681).
  • the standard deviation of codon usage may be calculated by first finding the squared proportional deviation of usage of each codon of the native gene relative to that of highly expressed plant genes, followed by a calculation of the average squared deviation.
  • a table of codon usage from highly expressed genes of dicotyledonous plants is compiled using the data of Murray et al. (1989, Nuc Acids Res. 17:477-498).
  • Codon Usage Database contains codon usage tables for a number of different species, with each codon usage table having been statistically determined based on the data present in Genbank.
  • a naturally- occurring nucleotide sequence encoding a protein of interest can be codon optimized for that particular plant species. This is effected by replacing codons that may have a low statistical incidence in the particular species genome with corresponding codons, in regard to an amino acid, that are statistically more favored.
  • one or more less- favored codons may be selected to delete existing restriction sites, to create new ones at potentially useful junctions (5' and 3' ends to add signal peptide or termination cassettes, internal sites that might be used to cut and splice segments together to produce a correct full-length sequence), or to eliminate nucleotide sequences that may negatively effect mRNA stability or expression.
  • codon optimization of the native nucleotide sequence may comprise determining which codons, within the native nucleotide sequence, are not statistically-favored with regards to a particular plant, and modifying these codons in accordance with a codon usage table of the particular plant to produce a codon optimized derivative.
  • a modified nucleotide sequence may be fully or partially optimized for plant codon usage provided that the protein encoded by the modified nucleotide sequence is produced at a level higher than the protein encoded by the corresponding naturally occurring or native gene. Construction of synthetic genes by altering the codon usage is described in for example PCT Patent Application 93/07278.
  • some embodiments of the invention encompasses nucleic acid sequences described hereinabove; fragments thereof, sequences hybridizable therewith, sequences homologous thereto, sequences orthologous thereto, sequences encoding similar polypeptides with different codon usage, altered sequences characterized by mutations, such as deletion, insertion or substitution of one or more nucleotides, either naturally occurring or man induced, either randomly or in a targeted fashion.
  • 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, DsRed, GUS and GFP.
  • an enhancer sequence can be inserted into the plant expression vector to enhance transcription levels of genes.
  • an ⁇ enhancer can be cloned into the vectors of the present invention.
  • Plant cells may be transformed stabley or transiently with the nucleic acid constructs of some embodiments of the invention.
  • stable transformation the nucleic acid molecule of some embodiments of the invention is integrated into the plant genome and as such it represents a stable and inherited trait.
  • transient transformation the nucleic acid molecule is expressed by the cell transformed but it is not integrated into the genome and as such it represents a transient trait.
  • transient transformation is presently preferred, stable transformation of leaf cells, meristematic cells or the whole plant is also envisaged by some embodiments of the invention.
  • acetosyringone is included with Agrobacterium for the infection of monocotyledonous plants.
  • the Agrobacterium system includes the use of plasmid vectors that contain defined DNA segments that integrate into the plant genomic DNA. 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 et al. in Plant Molecular Biology Manual A5, Kluwer Academic Publishers, Dordrecht (1988) p. 1-9. A supplementary approach employs the Agrobacterium delivery system in combination with vacuum infiltration. The Agrobacterium system is especially viable in the creation of transgenic dicotyledonous plants.
  • DNA transfer into plant cells There are various methods of direct DNA transfer into plant cells.
  • electroporation the protoplasts are briefly exposed to a strong electric field.
  • microinjection the DNA is mechanically injected directly into the cells using very small micropipettes.
  • microparticle bombardment the DNA is adsorbed on microprojectiles such as magnesium sulfate crystals or on gold, tungsten or platinum particles, and the microprojectiles are physically accelerated into cells or plant tissues.
  • direct DNA uptake by protoplasts can be stimulated by chemicals like polyethylene glycol (PEG), i.e. PEG-mediated DNA transfer.
  • PEG polyethylene glycol
  • PEG polyethylene glycol
  • Micropropagation is a process of growing new generation plants from a single piece of tissue that has been excised from a selected parent plant or cultivar. This process permits the mass reproduction of plants having the preferred tissue expressing the fusion protein.
  • the new generation plants which are produced are genetically identical to, and have all of the characteristics of, the original plant.
  • Micropropagation allows mass production of quality plant material in a short period of time and offers a rapid multiplication of selected cultivars in the preservation of the characteristics of the original transgenic or transformed plant.
  • the advantages of cloning plants are the speed of plant multiplication and the quality and uniformity of plants produced.
  • Micropropagation is a multi-stage procedure that requires alteration of culture medium or growth conditions between stages.
  • the micropropagation process involves four basic stages: Stage one, initial tissue culturing; stage two, tissue culture multiplication; stage three, differentiation and plant formation; and stage four, greenhouse culturing and hardening.
  • stage one initial tissue culturing
  • stage two tissue culture multiplication
  • stage three differentiation and plant formation
  • stage four greenhouse culturing and hardening.
  • stage one initial tissue culturing
  • the tissue culture is established and certified contaminant-free.
  • stage two the initial tissue culture is multiplied until a sufficient number of tissue samples are produced to meet production goals.
  • stage three the tissue samples grown in stage two are divided and grown into individual plantlets.
  • the transformed plantlets are transferred to a greenhouse for hardening off, i.e.
  • Transient transformation i.e. transient gene expression
  • Agrobacterium-mediated gene transfer by viral infection using modified plant viruses, by aphids, by nematodes, by infiltration, by vacuum, by electroporation or by bombardment.
  • 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.
  • the virus 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 transcribing the viral sequence of the plasmid and translation of the viral genes to produce the coat protein(s) which encapsidate the viral RNA.
  • a plant viral nucleic acid 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.
  • 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 transcribing 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) 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 transcribed or expressed in the host plant under control of the subgenomic promoter to produce the desired products.
  • 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.
  • a recombinant plant viral nucleic acid 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 transcribing 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 the sequences are transcribed or expressed in the host plant under control of the subgenomic promoters to produce the desired product.
  • 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.
  • nucleic acid molecule of some embodiments of the invention can also be introduced into a plastid genome (e.g. chloroplast genome) thereby enabling plastid (e.g. chloroplast) expression.
  • a plastid genome e.g. chloroplast genome
  • a technique for introducing exogenous nucleic acid sequences to the genome of the plastids is known. This technique involves the following procedures. First, plant cells are chemically treated so as to reduce the number of chloroplasts per cell to about one. Then, the exogenous nucleic acid is introduced via particle bombardment into the cells with the aim of introducing at least one exogenous nucleic acid molecule into the chloroplasts. The exogenous nucleic acid is selected such that it is integratable into the chloroplast's genome via homologous recombination which is readily effected by enzymes inherent to the chloroplast.
  • the exogenous nucleic acid includes, in addition to a gene of interest, at least one nucleic acid stretch which is derived from the chloroplast's genome.
  • the exogenous nucleic acid includes a selectable marker, which serves by sequential selection procedures to ascertain that all or substantially all of the copies of the chloroplast genomes following such selection will include the exogenous nucleic acid. Further details relating to this technique are found in U.S. Pat. Nos. 4,945,050; and 5,693,507 which are incorporated herein by reference.
  • a polypeptide can thus be produced by the protein expression system of the chloroplast and become integrated into the chloroplast's inner membrane.
  • Infection of viral vectors into plants can also be carried out by the use of aphids, including without limitation, the corn leaf aphid (Rhopalosiphum maidis), the greenbug (Schizaphis graminum), and the green peach aphid (Myzus persicae) (the natural hosts for MDMV).
  • aphids including without limitation, the corn leaf aphid (Rhopalosiphum maidis), the greenbug (Schizaphis graminum), and the green peach aphid (Myzus persicae) (the natural hosts for MDMV).
  • Rhopalosiphum maidis, Schizaphis graminum, and Myzus persicae are inoculated with the plant expression vectors of the invention or their derivatives prior to subjection to the plants.
  • 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 expression vectors. Such selection and screening methodologies are well known to those skilled in the art.
  • any visible marker genes e.g., GFP or GUS
  • 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 immuno staining, 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.
  • the vectors of some embodiments of the invention may be used for generating a plant.
  • the plant may be generated using a single expression vector or using several expression vectors (e.g. designed for expression of different heterologous polypeptides of interest).
  • plant refers to whole plants, portions thereof (e.g., leaf, root, stem, fruit, seed) or cells isolated therefrom (homogeneous or heterogeneous populations of cells, including meristem cells, leaf cells, pollen cells, ovule cells, male inflorescence cell, female inflorescence cells, microspores, megaspores, embryogenic calli cells or cells of first node derived calli).
  • the plant is a seedling.
  • the plant may be an adult plant such as one which comprises a gamete i.e. male and female reproductive plant organs including the anther and ovary (i.e. organs producing pollen and ovules, respectively) or a gamete producing tissue, i.e. any tissue which may give rise to gametes, such as but not limited to, a floral meristem tissue and flowers.
  • a gamete i.e. male and female reproductive plant organs including the anther and ovary (i.e. organs producing pollen and ovules, respectively) or a gamete producing tissue, i.e. any tissue which may give rise to gametes, such as but not limited to, a floral meristem tissue and flowers.
  • isolated plant cells refers to plant cells which are derived from dissociated plant cell tissues or plant cell cultures.
  • plant cell culture refers to any type of native
  • 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.
  • 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, barley, 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, sugar cane, watermelon, beans, hibiscus, okra, apple, rose, strawberry, chile, garlic, onions, sorghum, Johnson grass, turf grass, bamboo, palm, banana, ginger, 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
  • plant families may comprise Acanthaceae, Alismataceae, Amaranthaceae, Amaryllidaceae, Annonaceae, Apiaceae, Apocynaceae, Araceae, Araucariaceae, Arecaceae, Asteraceae, Asclepiadaceae, Bignoniaceae, Boraginaceae, Brassicaceae, Bromeliaceae, Cactaceae, Campanulaceae, Caryophyllaceae, Casuarinaceae, Celastraceae, Clusiaceae, Combretaceae, Commelinaceae, Convolvulaceae, Crassulaceae, Cucurbitaceae, Cyperaceae, Ericaceae, Euphorbiaceae, Fabaceae, Fagaceae, Iridaceae, Lamiaceae, Lauraceae, Liliaceae, Lythraceae, Magnoliaceae, Malpighiacea
  • 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, Cheiranthus cheiri, Chenopodium album, Chenopodium amaranticolor, Chenopodium foetidum, Chenopodium quinoa, Coriandrum sativum, Cucumis melo, Cucumis sativus, Glycine max, Gomphrena globosa, Gossypium hirsutum cv.
  • the plant is a monocot.
  • the plant comprises maize, rice, wheat, barley, sugar cane, sorghum, Johnson grass, grasses, bamboo, palm, agave, pineapple, banana, ginger, garlic, onion, oat, rye, turf grass, millet, spelt, triticale, fonio, aloe, asparagus, yam or ubi, orchid, iris, lily, amaryllis, canna-lily arum or gabi, lemon grass, pandan or screwpine, arrow root, rush, pipewort and sedge.
  • a method of generating genotypic variation in a genome of a plant comprising introducing into the plant the plant expression vector of some embodiments of the invention, wherein the nuclease mediates cleavage in the genome of the plant and enables an alteration in the cleavage site.
  • genomic 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.
  • DSB DNA double- stranded break
  • Genotypic variation according to the present teachings may be transient as explained in further detail hereinabove. 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.
  • two plant expression vectors may be introduced into the same plant cell. These plant expression vectors may be introduced in the plant cell concomitantly or at separate times. Such expression vectors may comprise nucleic acid sequences encoding different heterologous sequences. For example, an expression vector comprising a nucleic acid sequence encoding a nuclease and an expression vector comprising a nucleic acid sequence encoding, for example, a herbicide resistance polypeptide. The two expression vectors can be introduced concomitantly, as for example at a 1: 1 ratio, to enable expression of heterologous genes in plant cells.
  • two plant expression vectors are introduced into a plant, the first expression vector comprising a satellite vector comprising the heterologous polypeptide of interest (e.g. a nucleic acid sequence comprising the MDMV coat protein, the MDMV PI polypeptide and a heterologous polypeptide of interest).
  • the second expression vector comprising a MDMV virus or a helper virus (e.g. a nucleic acid sequence of the MDMV genome without the gene for the coat protein).
  • a helper virus e.g. a nucleic acid sequence of the MDMV genome without the gene for the coat protein.
  • nucleases of some embodiments 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.
  • 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.
  • 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].
  • foreign DNA may be cut out from the plasmid using the same nuclease 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.
  • NHEJ plant non-homologous end joining
  • 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).
  • the present teachings can be used to generate genotypic variation.
  • the 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 a 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.).
  • nucleases can be used to generate genotypic variation by knocking out gene expression.
  • 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.
  • two pairs of nucleases e.g. or combinations of same can be used to cleave out an entire sequence of the genome, thereby knocking out gene expression.
  • Nucleases of the present invention may also be used to generate genotypic variations in gametes and seeds of the plant.
  • the nucleases of the present invention may be used to generate specific or non-specific mutations in gametes which, following fertilization, will generate genotypically modified seeds and consequently modified plants.
  • Nucleases of the present invention may also be used to generate genotypic variations in calli of the plant.
  • the nucleases of the present invention may be used to generate specific or non-specific mutations in embryogenic calli cells, including in immature embryo scutella and mature embryo scutella cells, in cells of a first node derived calli, in split seedling nodes, in split seeds, in inner leaf sheathes of seedlings, in zygotes of fertilized embryo sacs and in immature male and female inflorescences.
  • plant calli of the invention can differentiate into a whole plant (e.g. regenerate) thereby generating plants comprising the genotypic variation.
  • the 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 nucleases such as the DNA restrictases or Non-stringent Fokl.
  • nucleases of the present invention may be used to combat infections by plant pathogens.
  • the present invention envisages a method of treating a plant infection by a pathogen.
  • the method comprising generating a pathogen resistant plant, the method comprising introducing into the plant the expression vector of some embodiments of the invention, wherein the nuclease mediates cleavage of a gene conferring sensitivity to a pathogen or in a gene inhibiting the resistance pathway, and enables an alteration in the cleavage site, thereby generating the pathogen resistant plant.
  • a "plant pathogen” refers to an organism, which causes a disease in a plant.
  • Organisms that cause infectious disease include fungi, oomycetes, bacteria, viruses, viroids, virus-like organisms, phytoplasmas, protozoa, nematodes and parasitic plants.
  • the gene conferring sensitivity to a pathogen is knocked-out to thereby increase resistance to the pathogen.
  • the gene comprises an elF4E (translation initiation factor 4E) gene or a Mlo gene.
  • elF4E translation initiation factor 4E
  • the gene inhibiting the resistance pathway comprises a transcription factor.
  • An exemplary gene includes the rice fatty-acid desaturase gene OsSSI2 (which was previously shown to act upstream of WRKY45 to negatively regulate WRKY45- dependent resistance, see Jiang et al. Molecular Plant-Microbe Interactions (2009) 22(7): 820-829).
  • Another exemplary gene includes the rice NRR, a negative regulator of disease resistance (which was previously shown to interact with Arabidopsis NPR1 and rice NH1, see Chern et al. Plant J. 2005 Sep;43(5):623-35).
  • generating a pathogen resistant plant may be carried out by introducing into the plant the expression vector of some embodiments of the invention, wherein the nuclease mediates cleavage in a gene of a pathogen and enables an alteration in the cleavage site, thereby generating the pathogen resistant plant.
  • the nuclease e.g. chimeric nuclease
  • the nuclease is designed so as to cleave as much sequence sites on the pathogen's nucleic acid (DNA or RNA) as possible.
  • repeating sequences may be targeted.
  • a number of distinct sequences are targeted sufficient to induce degradation of the pathogen's genome.
  • the nuclease (e.g chimeric nuclease) is designed to cleave the genome (DNA or RNA) of the pathogen but not that of the plant.
  • the nuclease is designed devoid of a localization signal, such that the nuclease is active in the cytoplasm which comprises the pathogen's (e.g., virus) DNA but not that of the plant.
  • 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 (www(dot)ncbi(dot)nlm(dot)nih(dot)gov/blast/Blast(dot)cgi).
  • Blast www(dot)ncbi(dot)nlm(dot)nih(dot)gov/blast/Blast(dot)cgi).
  • 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.
  • PEBV Pea early - browning virus
  • Genus Tobravirus.
  • Pepper ringspot virus PepRSV
  • Genus Tobravirus.
  • WMV Watermelon mosaic virus
  • TMV Tobacco mosaic virus Genus
  • TMVX Tobamovirus and other viruses from the Tobamovirus Genus.
  • PVX Potato
  • 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 tomate 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 big
  • the present invention also envisages 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 plastid (e.g. chloroplast) associated with male sterility by introducing into the plant the plant expression vector of some embodiments of the invention 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 plastid (e.g. chloroplast) when targeted into the genome of the mitochondria or plastid (e.g. chloroplast), wherein the nuclease, mediates cleavage in the genome of the mitochondria or plastid (e.g. chloroplast) and enables insertion of the heterologous nucleic acid sequence into the cleavage site, thereby generating male sterility in the plant.
  • a mitochondria or plastid e.g. chloroplast
  • the nucleic acid expression 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 nuclease (identical to that on the mitochondria or plastid e.g. chloroplast genome).
  • a coding e.g., for a CMS associated gene
  • non-coding e.g., powerful promoter for enhancing expression of a CMS associated gene
  • the heterologous nucleic acid sequence is inserted into the predetermined site in the genome of the plastid (e.g. chloroplast) or mitochondria.
  • cytoplasmic male sterility is associated with mitochondrial dysfunction.
  • the 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 rpsl2, the Rice (Oryz sativa) sequence which is downstream of B-atp6 gene (i.e. orf79), the Maize T-urfl3 and orf221, the Helianthus sp. orf239 downstream to atpA, the Brassica sp.
  • orfs which are upstream to atp6 (e.g. orfl39 orf224 or orfl38 and orfl58).
  • atp6 e.g. orfl39 orf224 or orfl38 and orfl58.
  • 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.
  • 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.
  • induction of CMS can also be achieved by overexpression of a chloroplast gene such as ⁇ -ketothiolase.
  • a chloroplast gene such as ⁇ -ketothiolase.
  • Overexpression of ⁇ - ketothiolase via the chloroplast genome has been previously shown to induce CMS [Ruiz at al. (2005) Plant Physiol. 138 1232-1246].
  • the present teachings also envision targeting chloroplast genes or overexpression thereof (e.g. ⁇ -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 the plant expression vector of some embodiments of the invention, wherein the nuclease mediates cleavage in a gene conferring sensitivity to herbicides and enables an alteration in the cleavage site, thereby generating the herbicide resistant plant.
  • nucleases are designed to comprise a chloroplast localization signal (as described in detail hereinabove) and cleavage sites which are specific for the chloroplast genome.
  • chloroplast 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 Q B , 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).
  • psbA which codes for the photosynthetic quinone-binding membrane protein Q B , the target of the herbicide atrazine
  • 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).
  • herbicide resistance may be introduced into a plant by upregulating an expression of a protein (e.g. phosphinothricin acetyltransferase) which imparts resistance to an herbicide when expressed in the plant.
  • a nucleic acid expression construct comprising a heterologous nucleic acid sequence (e.g. phosphinothricin acetyltransferase) is introduced into the plant for expression of the protein conferring herbicide resistance.
  • the present invention further envisages a method of generating a plant with increased abiotic stress tolerance.
  • the method comprising introducing into the plant the plant expression vector of some embodiments of the invention, wherein the nuclease, mediates cleavage in a gene of the plant conferring sensitivity to abiotic stress and enables an alteration in the cleavage site, thereby generating the plant with increased abiotic stress tolerance.
  • abiotic stress refers to any adverse effect on metabolism, growth, reproduction and/or viability of a plant. Accordingly, abiotic stress can be induced by suboptimal environmental growth conditions such as, for example, salinity, osmotic stress, water deprivation, drought, flooding, freezing, low or high temperature, heavy metal toxicity, anaerobiosis, nutrient deficiency (e.g., nitrogen deficiency or limited nitrogen), atmospheric pollution or UV irradiation.
  • suboptimal environmental growth conditions such as, for example, salinity, osmotic stress, water deprivation, drought, flooding, freezing, low or high temperature, heavy metal toxicity, anaerobiosis, nutrient deficiency (e.g., nitrogen deficiency or limited nitrogen), atmospheric pollution or UV irradiation.
  • abiotic stress tolerance refers to the ability of a plant to endure an abiotic stress without suffering a substantial alteration in metabolism, growth, productivity and/or viability.
  • compositions, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
  • a compound or “at least one compound” may include a plurality of compounds, including mixtures thereof.
  • 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.
  • 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.
  • Finnzymes Fusion Hot start II high-fidelity DNA polymerase
  • Amplified cDNA was cloned into pJET or pGEM (Amp ), respectively, and grown in E. coli Xl-lBlue cells.
  • dCTPs tailed first strand cDNA using TdT enzyme (Fermentas), then amplified using a 3' end MDMV primer (primer no. 4, see Table 1) and a 5' poly-dG adaptor (primer no. 41, see Table 1).
  • the Israeli MDMV full-length sequence was constructed and fully sequenced using several MDMV primers (primers 1-25, as listed in Table 1 below, SEQ ID NO: 43).
  • a synthetic DNA fragment was synthesized in order to fuse 35S promoter to 5' MDMV genome (pSAT-Synthetic block).
  • the 1-5429 bp MDMV fragment was inserted downstream to the 35S promoter in the pSAT-Synthetic block plasmid (using Nrul and Banll).
  • Creation of a full-length sequence MDMV under 35S promoter was accomplished by triple ligation of 35S promoter- 1-5429, 3661-polyA and pGreen (digested with Agel, AlwNi and Kpnl) (#3076) (as illustrated in Figures 2A-E).
  • the Israeli MDMV contained all of the expected potyvirus genes.
  • the 5' untranslated region (UTR) extended from 1-138 nt (SEQ ID NO: 44).
  • Protease cleavage sites within the polyprotein were predicted based on comparisons with other potyvirus species. Based on sequence predictions of cleavage sites, the PI protease was encoded at nt 139-837 (SEQ ID NO: 45), HC-Pro at nt 838- 2217 (SEQ ID NO: 46), the P3 protease from nt 2218 to 3258 (SEQ ID NO: 47), 6K1 from nt 3259 to 3459 (SEQ ID NO: 48), CI from nt 3460 to 5373 (SEQ ID NO: 49), 6K2 from nt 5374 to 5532 (SEQ ID NO: 50), NIa-VPg from nt 5533 to 6099 (SEQ ID NO: 51), NIa-Pro from nt 6100 to 6825 (SEQ ID NO: 52), NIb-RNA replicase from nt
  • MCS multiple cloning sites
  • the second MCS was designed between the Nib-CP viral genes containing Nhel, Avrll restriction sites, NIa protease cleavage site and alteration of 2 aphid transmission motifs DAE and DAG to DTE and DTG, respectively (as illustrated in Figure 4). MDMV sequences containing these modifications were synthesized and ordered from Biomatik Company (#3091 and #3093, respectively).
  • DsRed a 700 bp reporter gene
  • the DsRed was inserted into the native MDMV infective clone #3076 by Bbcl and Sphl or by Swal and Bsu36I to create MDMV-DsRed infective clones #3101 and #3096, respectively (as illustrated in Figure 5).
  • MDMV-935R ATCGTAGGTGTGTGCTCTGT 71 Particle bombardment procedure - Infecting plants with MDMV clones
  • Gold microcarriers were prepared following the manufacturer instructions (Bio- Rad Laboratories, Richmond, CA, USA). Specifically, 15 milligrams of 1 ⁇ diameter gold particles were suspended in 0.5 ml of 100 % ethanol, sonicated for 15 seconds and then centrifuged at 3000 rpm for 60 seconds. The recovered particle pellet was washed twice before suspension in 0.5 ml of sterile distilled water. For each bombardment shot, 1-2 ⁇ g of MDMV construct, 8 ⁇ of CaC12 (2.5 M), and 2.5 ⁇ of spermidine (0.1 M) were added one by one into an aliquot of 8 ⁇ of gold particle suspension. The mixture was vortexed for 10 min, and then centrifuged at 5,000 rpm for 12 seconds.
  • Maize B73 explants used for bombardment were derived from the split 1st nodes of 1 week old germinated seedlings which were cultured for approximately 8 weeks to produce both organogenic structures (shoot and root) and embryogenic callus outgrowths. This material was used to successfully demonstrate the spread and intensity of Dsred expression in explants following particle bombardment with plasmid DNA of the MDMV expression #3101 vector clone ( Figures 12A-D).
  • Infectivity tests were performed by rubbing homogenates of virus-infected plants on maize seedlings. Using a sterile scalpel, a small piece of a fresh symptomatic plant leaf was excised and homogenized in a small amount of H 2 0 using a sterile pestle and mortar to yield a finely ground virus suspension. The homogenate was rubbed immediately onto carborundum dusted fully expanded leaves using sterile cotton swaps. After mechanical inoculations the inoculated leaves were sprayed with water to remove inoculum and to reduce excessive evaporation. 3-5 leave seedlings were the most susceptible plant, hence inoculation experiments were done on seedlings at that stage.
  • Plants were scored for symptom development from 5 days to 2 weeks post- infection/bombardment. Infection of symptomatic plants was confirmed by RT-PCR using primers no. 70 and 71 (see Table 1, above). In some cases a serological test was performed using MDMV antibodies (CAB 18000, Agdia inc.). Mechanical inoculation infection rates by rubbing virus suspension were close to 100 %. Particle bombardment infection rates were 40-60 %. EXAMPLE 1
  • a MDMV expression vector (IL type MDMV) was generated as indicated in the materials and experimental section above. After cloning the first native MDMV-IL clone (#3076), plasmid clone was tested using bombardment procedure. One week later typical MDMV symptoms were seen on the bombarded maize seedlings ( Figure 7). Next, several sorghum cultivars were tested for MDMV-IL susceptibility. Young seedlings of SB 102, SB273 and Israeli cultivar SB 153 were brushed with virus suspension. One week later harsh MDMV leaf symptoms were detected on each sorghum cultivar ( Figures 8A-B).
  • the first goal was to demonstrate that the MDMV expression vector can express a foreign gene.
  • DsRed a 700 bp reporter gene
  • Seven days post bombardment of the #3096 clone infected plants were detected by viral symptoms on the first new growing leaf, DsRed was detected too.
  • DsRed expression persisted for up to two weeks from initial expression in the bombarded leaves, (data not shown).
  • Clone #3101 showed viral symptoms and DsRed expression in the new developing leaves, a week after bombardment procedure ( Figures 9A-D).
  • infected plants continued to express the DsRed marker gene until maturity (as compared to the #3096 clone).
  • Another way to determine viral infectivity of an infected plant is by questioning its ability to infect a new healthy plant. A new infection of healthy plants was illustrated by rubbing #3101 infected plant's sap ( Figures 9A-S). Another goal was to develop a safe environment viral clone.
  • aphid transmission motifs were mutated with the insertion of the 3'MCS, namely DAE and DAG were changed to DTE and DTG, respectively (motifs are at 8467 bp and 8622 bp from the beginning of the viral genome, see Figure 13 and SEQ ID NOs: 72- 73).
  • aphid transmissible motif was changed in the MDMV clone. Specifically, a KITC motif which appears in the beginning of the HcPro gene was changed by site directed mutagenesis into EITC (motif present 1030 bp from the beginning of the MDMV genome, see Figure 14 and SEQ ID NOs: 74-75).
  • GUS a different reporter gene, i.e. GUS
  • GUS a relatively big reporter gene (1800 bp)
  • FIG. 11 A GUS expression by the MDMV vector was illustrated in several maize leaves (see Figures 11B-E). Expression of GUS was further illustrated in young, non-bombarded leaves, an indication that the virus is capable of replicating and moving in the plant regardless of its foreign insertion (GUS).
  • the MDMV viral vector of the invention was further shown to infect embryogenic and/ or meristematic callus, i.e. DsRed expression in maize B73 explants was illustrated from 18 to 25 days following bombardment ( Figures 12A-D). It was further shown that the MDMV virus proliferates and spreads throughout the plant/callus and expresses a foreign reporter gene ( Figures 9A-R, 11A-E and Figures 12A-D).
  • the MDMV viral vector of the invention can dually express two reporter genes. Specifically, a MDMV vector was designed in which GUS was inserted in the 5'MCS and DsRed was inserted in the 3'MCS (clone no #3182). The results illustrated that both reporter genes were expressed together in the same infected leaf (bombarded leaf, data not shown).
  • a MDMV expression vector (IL type MDMV) was constructed to include a sequence encoding a meganuclease, the meganuclease comprising I-Scel.
  • the gene encoding the meganuclease was inserted in a MCS site, between the PI gene and the HCPro gene of the MDMV.
  • the MDMV expression vector was constructed such that the meganuclease was directly followed by a NIa protease cleavage site.
  • the meganuclease was selected to target a specific sequence in the plant genome for efficient gene editing. The results illustrated a base deletion in the gene (data not shown).
  • a MDMV expression vector (IL type MDMV) is constructed to include a sequence encoding a meganuclease.
  • the gene encoding the meganuclease is inserted in one of the MCS sites, e.g. between the NIb-RdRp gene and the CP gene of the MDMV or between the PI gene and the HCPro gene of the MDMV.
  • the MDMV expression vector is constructed such that the meganuclease is directly followed by a NIa protease cleavage site.
  • the meganuclease is selected to target a specific sequence in the plant genome for efficient gene editing.
  • a MDMV expression vector (IL type MDMV) is constructed to include a sequence encoding a zinc finger nuclease (ZFN).
  • ZFN zinc finger nuclease
  • the gene encoding the ZFN is inserted in one of the MCS sites, e.g. between the NIb-RdRp gene and the CP gene of the MDMV or between the PI gene and the HCPro gene of the MDMV.
  • the MDMV expression vector is constructed such that the ZFN is directly followed by a NIa protease cleavage site. The ZFN is selected to target a specific sequence in the plant genome for efficient gene editing.
  • a MDMV expression vector (IL type MDMV) is constructed to include a sequence encoding a Cas9.
  • the gene encoding the Cas9 is inserted in one of the MCS sites, e.g. between the NIb-RdRp gene and the CP gene of the MDMV or between the PI gene and the HCPro gene of the MDMV.
  • the MDMV expression vector is constructed such that the Cas9 is directly followed by a NIa protease cleavage site.
  • An additional vector is used for expression of CRISPR for specific gene targeting of the CRISPR/Cas9 system.
  • a MDMV expression vector (IL type MDMV) is constructed to include a sequence encoding transcription activator-like effector nucleases (TALENs) or compact TALENs (c TALENs).
  • the gene encoding the TALENs or cTALENs is inserted in one of the MCS sites, e.g. between the NIb-RdRp gene and the CP gene of the MDMV or between the PI gene and the HCPro gene of the MDMV.
  • the MDMV expression vector is constructed such that the TALENs or the cTALENs is directly followed by a NIa protease cleavage site.
  • the TALENs or cTALENs is selected to target a specific sequence in the plant genome for efficient gene editing.
  • Co-expression of two foreign genes by one MDMV expression vector is carried out by construction of a vector including two foreign sequences successive to each other (e.g. two ZFNs, two TALENS, two meganucleases, etc.), each gene being followed by a NIa protease cleavage site.
  • the foreign genes are placed in each of the MCSs each being followed by a NIa protease cleavage site.
  • All MDMV expression vectors are constructed with reduced aphid transmission capabilities. Specifically, to inhibit aphid transmission of the MDMV vectors, alteration of 2 aphid transmission motifs are carried out, i.e. DAE and DAG to DTE and DTG, respectively, at the N terminal region of the coat protein (CP). To achieve full protection from aphid transmission another aphid transmissible motif is changed in the MDMV vector, namely, an amino acid alteration comprising a KITC to EITC substitution in a helper component proteinase (HCPro).
  • HCPro helper component proteinase

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Abstract

A plant expression vector comprising a nucleic acid sequence encoding a polyprotein product is disclosed. The polyprotein product comprising Maize Dwarf Mosaic Virus (MDMV) polypeptides and a heterologous polypeptide of interest, the heterologous polypeptide of interest being cleaved of the MDMV polypeptides upon expression in a plant cell, and wherein the plant expression vector is capable of spreading in a plant. A plant cell, a plant and methods of producing plants are also provided.

Description

MDMV BASED VECTOR FOR GENE EXPRESSION AND SILENCING
FIELD AND BACKGROUND OF THE INVENTION
The present invention, in some embodiments thereof, relates to a Maize dwarf mosaic virus (MDMV) expression vector and, more particularly, but not exclusively, to the use of same for generating genotypic variations in plants.
Maize dwarf mosaic virus (MDMV), a plant pathogenic virus, is a member of the genus Potyvirus, family Potyviridae. MDMV is a positive- strand RNA virus. The MDMV genome is expressed as a polyprotein cleaved by three viral proteases and processed into 11 putative mature proteins. Virions are flexuous filaments of 750 nm in length and 13 nm in diameter, composed of about 2000 subunits of a single 31-kDa protein. The genome RNA size is 9.5 kb covalently linked to a viral-encoded protein (the VPg) at the 5' end, and with a 3' poly A tail.
Symptoms of maize dwarf mosaic diseases vary widely depending on host genotype, time of infection, and viral strain. MDMV strains are known to infect maize, sorghum and Johnson grass. Generally, infected plants develop distinct chlorotic mosaics, mottles or streaks on green tissues (typically observed on young leaves). Infected plants are characterized by stunting and shortening of the upper internodes. Ear development may be arrested, leading to an incomplete grain filling and direct yield loss. Plants infected early may produce nubbins or can be totally barren. Israeli MDMV (MDMV-IL) infected maize plants typically show non-severe symptoms, corn plants reach adulthood, ear formation and development may slow and may cause grain yield loss.
MDMV is transmitted by several species of aphids in a non-persistent manner. The coat protein (CP) and the helper component-protease (HC-Pro) are required for aphid transmission, through the CP DAG and the HC-Pro KITC motifs. Aphids transmit MDMV disease effectively fifteen to thirty minutes after feeding on infected plants. Virus is also transmitted efficiently by mechanical inoculation and is not transmitted by contact between plants. Symptoms appear 5-8 days after mechanical inoculation.
MDMV complete genome sequences have been reported from different regions of the world including Bulgaria, Spain, Hungary and United States. The strains identity is approximately 85 %. The two main applications of viral vectors known in the literature are gene expression and gene silencing (virus-induced gene silencing (VIGS)). Currently, two viruses are used for VIGS in maize, Barley stripe mosaic virus (BSMV) and Brome mosaic virus (BMV), both of which do not belong to the potyvirus family. A clone of BSMV was made into a vector for use in barley [Holzberg et al., Plant J (2002) 30: 315- 327] and wheat [Triticum aestivum; Scofield et al., Plant physiol. (2005) 138:2165- 2173], and is the most widely employed grass VIGS vector. The second VIGS system based on BMV was developed for rice (Oryza sativa), maize (Zea mays), and barley (Hordeum vulgare) [Ding et al., Mol Plant Microbe In (2006), 19: 1229-1239].
Potyvirus based viral vectors have been previously demonstrated for tobacco etch virus (TEV), Plum pox virus (PPV), Zucchini yellow mosaic virus (ZYMV) and Wheat stripe mosaic virus (WSMV). Of these, only WSMV has been implicated for maize gene expression applications [Tatineni et al., Virology. (2011) 410: 268-281].
Arazi et al. [Journal of Biotechnology 87 (2001) 67-82] engineered zucchini yellow mosaic potyvirus as a non-pathogenic vector for expression of heterologous proteins in cucurbits.
Stewart et al. [Virus Research 165 (2012) 219-224] investigated two MDMV isolates (Ohiol and Ohio2) which are missing 19 aa from the CP 5' terminal region. According to Stewart et al., creation of a MDMV viral vector failed as their full length MDMV exhibited toxicity in E. coli and also was not recovered at high concentrations. In order to accomplish infectivity of their clone, the Ohiol MDMV full genome strain was inserted into pSPORT vector, which served as a template for in vitro transcription, and the transcripts (RNA) were used for inoculating maize plants.
In another work on Hungarian MDMV strain, carried out by Gyongyver Monika Gell from Szent Istvan University in Hungary (see szie(dot)hu//file/tti/archivum/Gell_Gy_thesis(dot)pdf), a full length MDMV clone was created and inserted into a plasmid, but infection was not achieved.
Additional background art include:
U.S. Patent No. 5,428,144 provides methods and materials for isolation of the coat protein gene (MDMVA-CP) from Maize Dwarf Mosaic Virus Strain A (MDMVA) used as a vaccine against MDMV infection. According to the teachings of U.S. 5,428,144, MDMVA-CP is incorporated into a plant expression cassette in which the cDNA clone is operably linked to plant regulatory sequence which causes the expression of the cDNA clone in living plant cells. The plant expression cassette preferably includes a strong constitutive promoter sequence at one end to cause the gene to be transcribed at a high frequency, and a poly-A recognition sequence at the other end for proper processing and transport of the messenger RNA. The resulting transformation vector is then introduced into maize callus to provide cross -protection to MDMV or related viral infections.
U.S. Patent No. 5,530,193 provides the nucleic acid and amino acid sequences of Maize Dwarf Mosaic Virus (MDMV) strains A, B, and KS1 as well as constructs and vectors used to produce transformed plants comprising MDMV resistance (plants are transformed with the coat protein gene of one of the various strains of MDMV).
U.S. Patent Application No. 20050132440 provides a gene silencing vector that suppresses the expression of specific target gene in a host. Specifically, U.S. 20050132440 provides a gene silencing vector comprising a promoter, an enhancer sequence (downstream of the promoter), and a gene encoding a potyvirus -origin coat protein e.g. of potato virus Y (downstream of the enhancer sequence). In order to cause gene silencing of a specific target gene in a host plant, the vector is used with a specific target gene or a gene that is homologous to the target gene inserted in the vicinity of the gene encoding the coat protein.
U.S. Patent Application No. 20030031648 provides a recombinant vector for expression of a heterologous peptide at the amino-terminus of a potyvirus coat protein. The vector includes sufficient potyvirus nucleic acid sequence (e.g. ZYMV, MDMV, etc.) to permit viral replication and spread within a plant infected by the vector. The vector further includes a heterologous nucleic acid sequence inserted at the amino- terminus of the potyvirus coat protein. Particularly, U.S. 20030031648 teaches a Zucchini Yellow Mosaic Potyvirus (ZYMV) vector capable of expressing at least a portion of a heterologous peptide on the surface of virions so that isolated virions or a portion of a plant, for example a cucurbit fruit, infected therewith may be used as a source of material for vaccination, pharmaceutical or diagnostic applications.
U.S. Patent Application No. 20050257287 provides a nucleic acid vector for concurrently imparting herbicide resistance to a plant and cross protecting the plant. The vector includes sufficient potyvirus nucleic acid sequence (ZYMV) to permit viral replication and spread. The vector further includes mutations which attenuate symptoms of viral infection in the plant and which abolish transmission of the virus by an insect vector (e.g. aphid). The vector further includes an additional nucleic acid sequence encoding a protein (e.g. phosphinothricin acetyltransferase) which imparts resistance to an herbicide when expressed in the infected plant.
PCT publication no. WO 1999/051749 provides a potyvirus infectious nucleic acid construct for providing protection against viral infection in plants. The construct of WO 1999051749 comprises a full length clone characterized in that its HC-Pro gene conserved FRNK box sequence contains a substitution (e.g. a substitution of Arg). This substitution renders the construct infectious, however, when introduced to plants, induces little or no symptom development. The construct of WO 1999/051749 further contains a substitution which effectively abolishes aphid transmissibility. WO 1999/051749 further relates to transient expression of foreign nucleic acid genes in plants using the construct. Specifically, WO 1999051749 relates to a method for cross protection of cucurbits against ZYMV infection.
SUMMARY OF THE INVENTION
According to an aspect of some embodiments of the present invention there is provided a plant expression vector comprising a nucleic acid sequence encoding a polyprotein product, the polyprotein product comprising Maize Dwarf Mosaic Virus (MDMV) polypeptides and a heterologous polypeptide of interest, the heterologous polypeptide of interest being cleaved of the MDMV polypeptides upon expression in a plant cell, and wherein the plant expression vector is capable of spreading in a plant.
According to an aspect of some embodiments of the present invention there is provided a plant expression vector comprising a nucleic acid sequence encoding a polyprotein product, the polyprotein product comprising Maize Dwarf Mosaic Virus (MDMV) polypeptides, wherein the MDMV polypeptides comprise a PI polypeptide and a coat protein polypeptide, and a heterologous polypeptide of interest, the heterologous polypeptide of interest being cleaved of the MDMV polypeptides upon expression in a plant cell infected with a helper virus or an MDMV virus, and wherein the plant expression vector is capable of spreading in a plant. According to an aspect of some embodiments of the present invention there is provided a plant expression vector system comprising: (i) the plant expression vector of some embodiments of the invention; and (ii) an MDMV helper virus.
According to an aspect of some embodiments of the present invention there is provided a plant cell comprising the plant expression vector or vector system of some embodiments of the invention.
According to an aspect of some embodiments of the present invention there is provided a plant comprising the plant expression vector or vector system of some embodiments of the invention.
According to an aspect of some embodiments of the present invention there is provided a method of generating a plant, the method comprising introducing into one or more cells of the plant the plant expression vector or vector system of some embodiments of the invention.
According to an aspect of some embodiments of the present invention there is provided a method of transiently expressing a heterologous polypeptide of interest in a plant, the method comprising introducing into at least one cell of the plant the plant expression vector or vector system of some embodiments of the invention, thereby transiently expressing the heterologous polypeptide of interest in the plant.
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 the plant expression vector or vector system of some embodiments of the invention, wherein the nuclease mediates cleavage in a genome of the plant and enables an alteration in the cleavage site, 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 tagging a genome of a plant, the method comprising introducing into the plant the plant expression vector or vector system of some embodiments of the invention, wherein the nuclease mediates cleavage in a genome of the plant and enables an alteration in the cleavage site, 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 a herbicide resistant plant, the method comprising introducing into the plant the plant expression vector or vector system of some embodiments of the invention, wherein the nuclease mediates cleavage in a gene conferring sensitivity to herbicides and enables an alteration in the cleavage site, thereby generating the herbicide resistant plant.
According to an aspect of some embodiments of the present invention there is provided a method of generating a pathogen resistant plant, the method comprising introducing into the plant the plant expression vector or vector system of some embodiments of the invention, wherein the nuclease mediates cleavage in a gene conferring sensitivity to a pathogen or in a gene inhibiting the resistance pathway and enables an alteration in the cleavage site, thereby generating the pathogen resistant plant.
According to an aspect of some embodiments of the present invention there is provided a method of generating a plant with increased abiotic stress tolerance, the method comprising introducing into the plant the plant expression vector or vector system of some embodiments of the invention, wherein the nuclease mediates cleavage in a gene of the plant conferring sensitivity to abiotic stress and enables an alteration in the cleavage site, thereby generating the plant with increased abiotic stress tolerance.
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 plastid associated with male sterility by introducing into the plant the plant expression vector or vector system of some embodiments of the invention 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 plastid when targeted into a genome of the mitochondria or plastid, wherein the nuclease mediates cleavage in a genome of the mitochondria or plastid and enables insertion of the heterologous nucleic acid sequence into the cleavage site, thereby generating male sterility in the plant.
According to some embodiments of the invention, the plant expression vector of some embodiments of the invention being a satellite vector.
According to some embodiments of the invention, the plant expression vector is non-transmittable by aphids.
According to some embodiments of the invention, the vector comprises an amino acid alteration which renders the vector non-transmittable by the aphids. According to some embodiments of the invention, the vector comprises an amino acid alteration comprising a DAE to DTE substitution in a N terminal region of a Coat Protein (CP).
According to some embodiments of the invention, the vector comprises an amino acid alteration comprising a DAG to DTG substitution in a N terminal region of a coat protein (CP).
According to some embodiments of the invention, the vector comprises an amino acid alteration comprising a KITC to EITC substitution in a helper component proteinase (HCPro).
According to some embodiments of the invention, the nucleic acid sequence encoding the polyprotein product encodes the full set of proteins of the MDMV.
According to some embodiments of the invention, the heterologous polypeptide of interest is translationally fused N terminally to the MDMV polypeptides in the polyprotein product.
According to some embodiments of the invention, the heterologous polypeptide of interest is translationally fused C terminally to the MDMV polypeptides in the polyprotein product.
According to some embodiments of the invention, the heterologous polypeptide of interest is flanked by the MDMV polypeptides.
According to some embodiments of the invention, the heterologous polypeptide of interest is flanked by a PI polypeptide and a HCPro polypeptide of the MDMV, wherein the PI polypeptide is N-terminally positioned to the heterologous polypeptide of interest and the HCPro polypeptide is C-terminally to the heterologous polypeptide of interest.
According to some embodiments of the invention, the heterologous polypeptide of interest is flanked by a NIb-RNA replicase polypeptide and a CP polypeptide of the MDMV, wherein the NIb-RNA replicase polypeptide is N-terminally positioned to the heterologous polypeptide of interest and the CP polypeptide is C-terminally to the heterologous polypeptide of interest.
According to some embodiments of the invention, the heterologous polypeptide of interest is flanked by the PI polypeptide and the coat protein polypeptide of the MDMV. According to some embodiments of the invention, the PI polypeptide is N- terminally positioned to the heterologous polypeptide of interest and the coat protein polypeptide is C-terminally to the heterologous polypeptide of interest.
According to some embodiments of the invention, the heterologous polypeptide of interest is directly translationally fused to at least one protease cleavage site.
According to some embodiments of the invention, the protease cleavage site comprises a NIa protease cleavage site as set forth in SEQ ID NO: 42.
According to some embodiments of the invention, cleavage of the heterologous polypeptide of interest of the MDMV polypeptides upon expression in a plant cell is effected by a viral protease.
According to some embodiments of the invention, the viral protease comprises a NIa protease.
According to some embodiments of the invention, the vector further comprises at least one heterologous promoter sequence for directing expression of the polyprotein in the plant cell.
According to some embodiments of the invention, the heterologous promoter sequence comprises a 35S promoter.
According to some embodiments of the invention, the at least one heterologous promoter sequence comprises two heterologous promoter sequences.
According to some embodiments of the invention, the plant expression vector comprises a pGreen backbone.
According to some embodiments of the invention, the heterologous polypeptide of interest is selected from the group consisting of 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, a growth inhibiting polypeptide, an enzyme, a transcription factor and a transposase.
According to some embodiments of the invention, the nucleic acid sequence encoding the heterologous polypeptide of interest encodes for two heterologous polypeptides of interest.
According to some embodiments of the invention, the nucleic acid sequence encoding the polyprotein product comprises a full genome sequence of the MDMV. According to some embodiments of the invention, the heterologous polypeptide of interest comprises a nuclease.
According to some embodiments of the invention, the nuclease is selected from the group consisting of a meganuclease, a Cas and a RISC.
According to some embodiments of the invention, the Cas comprises Cas9.
According to some embodiments of the invention, the nuclease comprises a chimeric nuclease.
According to some embodiments of the invention, the chimeric nuclease comprises a nucleic acid binding domain and a nuclease.
According to some embodiments of the invention, the chimeric nuclease is selected from the group consisting of a zinc finger nuclease (ZFN), a TALENs and a compact- TALENs.
According to some embodiments of the invention, the nuclease is selected from the group consisting of a restriction enzyme, a topoisomerase, a recombinase, an integrase, a homing endonucleases and a DNAse.
According to some embodiments of the invention, the nucleic acid binding domain of the chimeric nuclease is selected from the group consisting of a meganuclease binding domain, a helix-turn-helix binding domain, a leucine zipper (ZIP) binding domain, a winged helix (WH) binding domain, a winged helix turn helix domain (wHTH) binding domain, a helix-loop-helix binding domain, a transcription activatorlike (TAL) binding domain, a recombinase, and a zinc finger binding domain.
According to some embodiments of the invention, the nuclease is attached to a localization signal to a DNA-containing organelle.
According to some embodiments of the invention, the DNA-containing organelle is selected from the group consisting of a nucleus, a plastid and a mitochondria.
According to some embodiments of the invention, the gene conferring sensitivity to a pathogen or the gene inhibiting the resistance pathway is knocked-out to thereby increase resistance to the pathogen.
According to some embodiments of the invention, the gene conferring sensitivity to a pathogen comprises an elF4E (translation initiation factor 4E) gene or a Mlo gene.
According to some embodiments of the invention, the gene inhibiting the resistance pathway comprises a transcription factor. According to some embodiments of the invention, the gene inhibiting the resistance pathway comprises a rice OsSSI2 gene or a rice NRR gene.
According to some embodiments of the invention, the alteration in the cleavage site comprises an amino acid mutation, insertion or deletion.
According to some embodiments of the invention, the plastid comprises a chloroplast.
According to some embodiments of the invention, the plant expression vector further comprises a chloroplast localization signal.
According to some embodiments of the invention, the chloroplast localization signal comprises a ribulose-l,5-bisphospate carboxylase small subunit (Rssu) (SEQ ID NOs: 76 or 77).
According to some embodiments of the invention, the plant expression vector further comprises a mitochondria localization signal.
According to some embodiments of the invention, the mitochondria localization signal comprises an ATPase beta subunit (ΑΤΡ-β) (SEQ ID NO: 78).
According to some embodiments of the invention, the plant is a monocot.
According to some embodiments of the invention, the monocot plant is selected from the group consisting of maize, rice, wheat, barley, sugar cane, sorghum, Johnson grass, grasses, bamboo, palm, agave, pineapple, banana, ginger, garlic, onion, oat, rye, turf grass, millet, spelt, triticale, fonio, aloe, asparagus, yam or ubi, orchid, iris, lily, amaryllis, canna-lily arum or gabi, lemon grass, pandan or screwpine, arrow root, rush, pipewort and sedge.
According to some embodiments of the invention, the cell is selected from the group consisting of a meristem cell, a leaf cell, a male inflorescence cell, a pollen cell, a female inflorescence cell, an ovule cell and a cell of first node derived calli.
According to some embodiments of the invention, the introducing is effected by particle bombardment, agroinfection or sap mechanical infection.
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 SEVERAL VIEWS OF THE DRAWINGS
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 5' MDMV characterization using 5' Race procedure. Four ATPs characterize the beginning of the MDMV genome. In these sequences, the end of the poly G nucleotides indicates the beginning of the viral genome (indicated by the red box, SEQ ID NO: 69).
FIGs. 2A-E illustrate the nucleic acid sequence of MDMV isolated in Israel (MDMV-IL) - clone #3076 (9563 bp). 5' UTR (dark red, SEQ ID NO: 44), PI protease (red, SEQ ID NO: 45), HcPro (yellow, SEQ ID NO: 46), P3 (light green, SEQ ID NO: 47), 6K1 (dark green, SEQ ID NO: 48), CI (light blue, SEQ ID NO: 49), 6K2 (dark blue, SEQ ID NO: 50), NIa-VPg (grey, SEQ ID NO: 51), NIa-Pro (dark grey, SEQ ID NO: 52), NIb-RNA replicase (purple, SEQ ID NO: 53), CP (light purple, SEQ ID NO: 54), 3' UTR (black, SEQ ID NO: 55). The underlined sequence in Figure 2A indicates the start codon of the first viral protein. The underlined green sequence in Figure 2B indicates P3N-PIPO protein expressed by a -1 ribosomal frameshifting from the P3 ORF (SEQ ID NO: 56) The underlined sequence in Figure 2E indicates the stop codon.
FIG. 2F is a schematic illustration of clone #3076 vector map.
FIG. 3 is a schematic illustration of the addition of multiple cloning sites (MCS) (Agel, Apal) and NIa protease cleavage site (IDVKHQA - SEQ ID NO: 42) in between the PI and HcPro genes of the MDMV sequence (appears in blue). Black arrows point on protease recognition sites.
FIG. 4 is a schematic illustrations of the addition of MCS (Nhel, Avrll) and NIa protease cleavage site (IDVKHQA - SEQ ID NO: 42) in between the Nib and CP genes of the MDMV sequence (from Biomatik). Black arrows point on protease recognition site.
FIG. 5 is a schematic illustration of generation of the MDMV-IL DsRed infective clones.
FIGs. 6A-C are photographs illustrating the maize seedlings bombardment procedure. Figure 6A illustrates the 1000/He Biolistic® Particle Delivery System (BioRad); Figure 6B illustrates the maize seedlings before gene shot; and Figure 6C illustrates the particle bombardment chamber containing maize seedlings.
FIG. 7 is a photograph illustrating the MDMV-IL phenotype appearance one week post bombardment of pGreen-35s-MDMV (#3076) as compared to a healthy maize leaf.
FIGs. 8A-B are photographs illustrating several sorghum cultivars SB 102, SB273 and Israeli cultivar SB 153 which are sensitive to MDMV. Maize infective leaf sap brushed against sorghum young seedling leaves caused, one week later, harsh MDMV leaf symptoms.
FIGs. 9A-S are photographs illustrating expression of DsRed by #3101 MDMV- IL clone. Figures 9A-D illustrate the expression of MDMV carrying DsRed in maize leaves; Figures 9E-I illustrate male inflorescence spikelets showing expression of DsRed; Figures 9J-N illustrate male inflorescence anthers showing expression of DsRed; and Figures 90-S illustrate that MDMV enters maize pollen.
FIGs. 10A-B illustrate the results of an aphid transmission test. Myzus persicae aphids were introduced to feed on #3302 and WT virus infected plants. Half an hour later, aphids were removed to 2 healthy maize plant groups. Eight days post aphid inoculation, 5 out of 10 plants were infected with WT virus, while none were infected from the #3302 mutated MDMV clone. The results were confirmed by an ELISA test (A) and RT-PCR (B).
FIGs. 11A-E illustrate GUS expression from MDMV aphid non-transmissible viral vector. Figure 11A illustrates insertion of GUS, al800 bp reporter gene, in the 5' MCS of the MDMV vector to create clone #3304; Figures 11B-E demonstrate GUS expression by viral vector in several maize leaves. Of note, expression of GUS in young (non bombarded) leaves illustrates that the virus can replicate and move within the plant regardless of its foreign insertion (GUS). FIGs. 12A-D are photographs illustrating an increase in Dsred expression in maize B73 explants from 18 to 25 days following bombardment (FB). Maize tissue culture was infected with the viral vector #3101. Figure 12A shows a bright field of explants (18FB); Figure 12B shows Dsred expression in explants (18FB); Figure 12C shows a bright field of explants (25FB); and Figure 12D shows Dsred expression in explants (25FB).
FIG. 13 is a schematic illustration of an environmental safe viral clone - replacement of DAE and DAG into DTE and DTG motifs, respectively, in the coat protein (SEQ ID NO: 67).
FIG. 14 is a schematic illustration of an environmental safe viral clone - replacement of KITC into EITC motif in the HCPro amino acid sequence (SEQ ID NO: 59).
DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION
The present invention, in some embodiments thereof, relates to a Maize dwarf mosaic virus (MDMV) expression vector and, more particularly, but not exclusively, to the use of same for generating genotypic variations in plants.
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.
Production of heterologous proteins in monocot plants is associated with various challenges as monocots possess unique morphological features and seed biology not present in dicots. Yet, maize, sugarcane, bamboo and other monocot species, being essential crops for feeding humans and livestock in many parts of the world, are imperative for the production of proteins, biofuels and other industrial products. Thus, developing tools and expertise to produce recombinant proteins in monocot plants is necessary. While reducing the present invention to practice, the present inventors have constructed a full length Israeli MDMV (MDMV-IL) expression construct which can be used as an efficient tool for gene expression, especially in monocotyledons plants.
As is shown hereinbelow and in the Examples section which follows, the present inventors have constructed through laborious experimentation a MDMV expression vector comprising the whole genome of MDMV under the expression of a double heterologous promoter (e.g., 35S). Since MDMV genome size is large compared to other viruses, cloning of its approximately 10,000 bp was done in several parts while achieving one long open reading frame (ORF) from the beginning of the PI gene until the end of the CP gene (see Figure 5). The expression vector was constructed such that the viral 3' terminal sequence contained a PolyA region. In order to make it an expression vector, two multiple cloning sites (MCS) were inserted at two positions of the virus genome. Minimal MCSs were generated to reduce potential problems associated with virus infectivity. Each of the MCS was built from the insertion of 2 unique restriction enzymes, so as to enable cleavage and separation of the foreign gene from the viral proteins upon translation in plants. Cloning of the two reporter genes DsRed (0.7 Kbp in the MDMV 5' or 3' site) and GUS (1.8 Kbp in the MDMV 5' site) allowed stable systemic infection and expression of these genes by the MDMV viral vector (see FIGs. 9A-S and FIGs. 12A-D for DsRed and FIGs. 11A-E for GUS). Moreover, expression of DsRed was evident in maize leaves (Figures 9A-D) as well as in male inflorescence (Figures 9E-N) and its pollen (Figures 90-S). The MDMV viral vector of the invention was shown to infect young maize seedling (Figures 6A-C) as well as embryogenic/organogenic callus (Figures 12A-D) by bombardment procedure. In order to make the viral vector environmentally safe, two central motifs that have an effect on the virus transmission by aphids were changed. Specifically, DAG motif located in the coat protein gene (CP), and KITC motif found in the HcPro gene were changed to non-functional motifs, DTG and EITC respectively (see Figures 13-14). Aphid tests proved that once the vector infected maize plants, it could not be transmitted by aphids to new healthy plants (Figures 10A-B). Accordingly, these novel MDMV 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 plant expression vector comprising a nucleic acid sequence encoding a polyprotein product, the polyprotein product comprising Maize Dwarf Mosaic Virus (MDMV) polypeptides and a heterologous polypeptide of interest, the heterologous polypeptide of interest being cleaved of the MDMV polypeptides upon expression in a plant cell, and wherein the plant expression vector is capable of spreading in a plant.
As used herein a plant expression vector refers to a nucleic acid vector including a DNA vector, a RNA vector, virus or other suitable replicon (e.g., viral vector) encoding for viral genes or parts of viral genes, as well as for heterologous expression products such as RNA and proteins (e.g. polypeptides). The expression vector of some embodiments of the invention synonymously refers to a plasmid, a vector, an expression vector, a construct and an expression construct.
The term "polyprotein" or "polyprotein product" as used herein refers to a protein product that is cleaved into separate smaller proteins each with a distinctive biological function. The polyprotein of the invention comprises maize dwarf mosaic virus (MDMV) polypeptides as well as a heterologous polypeptide of interest.
The term "Maize Dwarf Mosaic Virus" or "MDMV" as used herein refers to the plant RNA virus which is a member of the genus Potyvirus and of the family Potyviridae. Typically the MDMV genome is expressed as a polyprotein which is cleaved by viral proteases and processed into mature proteins.
According to some embodiments of the invention the MDMV genome is expressed as a recombinant polyprotein.
Any nucleic acid sequence encoding a MDMV polypeptide may be used according to the present teachings, such as the nucleic acid sequences encoding MDMV- A, MDMV-C, MDMV-D, MDMV-E and MDMV-F.
According to one embodiment, the MDMV of the invention is Israeli type MDMV (MDMV-IL) or a fragment thereof. The full length MDMV-IL gene (set forth in SEQ ID NO: 43) comprises a 5' UTR sequence (SEQ ID NO: 44), a PI protease sequence (SEQ ID NO: 45), a helper component proteinase (HcPro) sequence (SEQ ID NO: 46), a P3 protein sequence (SEQ ID NO: 47), a P3N-PIPO protein sequence (SEQ ID NO: 56), a 6K1 protein sequence (SEQ ID NO: 48), a CI protein sequence (SEQ ID NO: 49), a 6K2 protein sequence (SEQ ID NO: 50), a N-terminal viral protein genome- linked (NIa-VPg) sequence (SEQ ID NO: 51), a nuclear inclusion protein-a protease (NIa-Pro) sequence (SEQ ID NO: 52), a NIb-RNA replicase sequence (SEQ ID NO: 53), a coat protein (CP) sequence (SEQ ID NO: 54) and a 3' UTR sequence (SEQ ID NO: 55). In general, the MDMV viral proteases PI protease and HcPro cleave themselves, while NIa-Pro cleaves itself and the rest of the MDMV proteins.
According to one embodiment, the nucleic acid sequence encoding the polyprotein product comprises the full genome sequence of the MDMV (SEQ ID NO:
43) .
According to one embodiment, the nucleic acid sequence encoding the polyprotein product comprises a partial sequence of the MDMV genome.
According to one embodiment, the nucleic acid sequence encoding the polyprotein product comprises the entire sequence of the MDMV genome without the nucleic acid sequence encoding the coat protein polypeptide.
According to one embodiment, the nucleic acid sequence encoding the polyprotein product only comprises the MDMV sequences for PI polypeptide.
According to one embodiment, the nucleic acid sequence encoding the polyprotein product only comprises the MDMV sequences for the coat protein polypeptide.
According to one embodiment, the nucleic acid sequence encoding the polyprotein product comprises the MDMV sequences for PI polypeptide and for the coat protein polypeptide.
According to a specific embodiment, the partial sequence comprising the nucleic acid sequence encoding any of the MDMV proteins, e.g. 5' UTR sequence (SEQ ID NO:
44) , PI protease (SEQ ID NO: 45), HcPro (SEQ ID NO: 46), P3 (SEQ ID NO: 47), P3N-PIPO (SEQ ID NO: 56), 6K1 (SEQ ID NO: 48), CI (SEQ ID NO: 49), 6K2 (SEQ ID NO: 50), NIa-VPg (SEQ ID NO: 51), NIa-Pro (SEQ ID NO: 52), NIb-RNA replicase (SEQ ID NO: 53), CP (SEQ ID NO: 54) and/or 3' UTR (SEQ ID NO: 55).
The plant expression vector of the invention comprises a nucleic acid sequence encoding a polyprotein product which enables spreading in a plant.
As used herein, the phrase "spreading in a plant" refers to the expression of the polyprotein product in a cell or organ other than the cell or organ initially infected by the vector. According to an embodiment, spreading in a plant includes expression of the polyprotein product in meristematic tissues which allows efficient expression throughout the plant. Spreading in plants is discussed in further detail hereinbelow. According to one embodiment, the nucleic acid sequence encodes the full set of proteins of the MDMV (SEQ ID NO: 57).
According to another embodiment, the nucleic acid sequence encodes for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of the MDMV proteins. Thus, the nucleic acid sequence may encode for any of a PI protease (SEQ ID NO: 58), a HcPro (SEQ ID NO: 59), a P3 protein (SEQ ID NO: 60), a P3N-PIPO protein (SEQ ID NO: 68), a 6K1 protein (SEQ ID NO: 61), a CI protein (SEQ ID NO: 62), a 6K2 protein (SEQ ID NO: 63), a NIa-VPg (SEQ ID NO: 64), a NIa-Pro (SEQ ID NO: 65), a NIb-RNA replicase (SEQ ID NO: 66) and/or a coat protein (CP) (SEQ ID NO: 67).
According to a specific embodiment, the nucleic acid sequence encodes for a PI protease, a heterologous polypeptide of interest, a HcPro, a P3 protein, a 6K1 protein, a CI protein, a 6K2 protein, a NIa-VPg, a NIa-Pro, a NIb-RNA replicase (e.g. NIb-RdRp), and a coat protein (CP).
According to a specific embodiment, the nucleic acid sequence encodes for a PI protease, a HcPro, a P3 protein, a 6K1 protein, a CI protein, a 6K2 protein, a NIa-VPg, a NIa-Pro, a NIb-RNA replicase (e.g. NIb-RdRp), a heterologous polypeptide of interest, and a coat protein (CP).
In order to reduce, and preferably inhibit, aphid transmission of the virus, the plant expression vector of the invention comprises an amino acid alteration which renders the vector non-transmittable by an aphid [e.g. any aphid capable of transmitting MDMV including but not limited to the corn leaf aphid (Rhopalosiphum maidis), the greenbug (Schiz phis graminum), and the green peach aphid (Myzus persicae)] . The amino acid alteration may be at any site or multiple sites of MDMV known to one of skill in the art to contribute to aphid transmission, as for example, in the coat protein or the helper component-protease (HC-Pro) typically required for aphid transmission.
According to one embodiment, the amino acid alteration comprises a DAE to DTE substitution at the N terminal region of a coat protein. This alteration results in an inability of the virus to attach to the transmission aphid.
According to one embodiment, the amino acid alteration comprises a DAG to DTG substitution in a N terminal region of a coat protein. Similarly, this alteration results in an inability of the virus to attach to the transmission aphid. According to one embodiment, the amino acid alteration comprises a KITC to EITC substitution in a helper component proteinase (HCPro). Similarly, this alteration results in an inability of the virus to attach to the transmission aphid.
According to one embodiment, the amino acid alteration comprises a DAE to DTE substitution and/or a DAG to DTG substitution at the N terminal region of a coat protein. Additionally or alternatively, the amino acid alteration may comprise a KITC to EITC substitution in a helper component proteinase (HCPro).
In order to reduce symptoms of viral infection (e.g. chlorotic mosaics, mottles or streaks on green tissues; stunting and shortening of the upper internodes; or arrested ear development and incomplete grain filling), the plant expression vector of the invention may comprise an amino acid alteration which renders the MDMV symptoms nonsignificant or inhibits MDMV symptoms altogether.
As mentioned, the plant expression vector of the invention further comprises at least one heterologous polypeptide of interest. The heterologous polypeptide of interest being cleaved of the MDMV polypeptides upon expression in a plant cell. Thus, once the polypeptide of the invention is expressed in a plant cell, the polypeptide is cleaved into individual proteins and the heterologous polypeptide is cleaved of the polypeptide, i.e. is not attached to any of the MDMV peptides (as discussed in further detail hereinbelow).
The term "heterologous" as used herein refers to exogenous, not-naturally occurring in MDMV and/or not occurring within a native cell of a plant.
As used herein, the term "polypeptide" is used interchangeably with the terms "peptides", "oligopeptides" and "proteins" and refers to a biomolecule composed of amino acids of any length, linked together by peptide bonds, unless mentioned herein otherwise.
The term "heterologous polypeptide" or "heterologous polypeptide of interest", as used herein, refers to a biomolecule composed of two or more amino acids (including truncation products and full length proteins) that is expressed from a "heterologous gene" or "heterologous coding sequence" as defined below. Accordingly, the heterologous polypeptide produced in a plant is exogenous to, or not naturally occurring in MDMV. The heterologous polypeptide may be also heterologous to the plant, indicating that it is not expressed in a plant, not expressed in a particular plant species, or is expressed at a different expression level or localization in the plant. The heterologous polypeptide can be, for example, a plant polypeptide, a bacterial polypeptide, a viral polypeptide a mammalian polypeptide or a synthetic polypeptide (e.g., chimeric nuclease, nuclease e.g. cas9). Thus, the heterologous polypeptide of interest may be a plant polypeptide or protein that is a variant or mutated form of a plant polypeptide or protein or a polypeptide or protein not naturally found in the producing plant species, line or variety.
A "heterologous gene" or "heterologous coding sequence" refers to polynucleotide (nucleic acid sequence) that is exogenous to, or not naturally found in, the MDMV genome and that encodes an expression product e.g., the heterologous polypeptide of interest.
As used herein the term "polynucleotide" refers to a single or double stranded nucleic acid sequence which is isolated and provided in the form of an RNA sequence, a complementary polynucleotide sequence (cDNA), a genomic polynucleotide sequence and/or a composite polynucleotide sequences (e.g., a combination of the above).
The term "isolated" refers to at least partially separated from the natural environment.
According to one embodiment, the heterologous polypeptide of interest may include, but is not limited to, a reporter polypeptide, an antiviral polypeptide, a viral moiety, an antiviral polypeptide, 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, a growth inhibiting polypeptide, an enzyme, a transcription factor and a transposase.
Exemplary proteins which may be produced, include, but are not limited to: nucleases, kinases, proteases, enzymes, hormones, tumor suppressors, blood clotting proteins, cell cycle proteins, metabolic proteins, neuronal proteins, cardiac proteins, proteins deficient in specific disease states, structural proteins, antibodies, antigens, proteins that provide resistance to diseases, antimicrobial proteins, antiviral proteins, interferons, cytokines, growth factors, receptors, ligands, and signaling molecules.
According to one embodiment, the heterologous polypeptide of interest comprises two or more (e.g., 2, 3, 4) heterologous polypeptides. According to one embodiment, the heterologous polypeptide of interest enables modifying the plant genome, e.g., nuclease.
As used herein the term "nuclease" refers to any polypeptide, or complex comprising a polypeptide, that can generate a strand break in the genome, e.g. in genomic DNA. According to an embodiment, the cleavage is site specific usually conferred by an auxiliary subunit, alternatively the nuclease is inherently specific to a target sequence of interest.
As used herein, the term "cleavage" or "DNA cleavage" refers to the breakage of the covalent backbone of a DNA molecule. Both single- stranded cleavage and double- stranded cleavage are possible, and double-stranded cleavage can occur as a result of two distinct single-stranded cleavage events. DNA cleavage can result in the production of either blunt ends or staggered ends.
Exemplary nucleases which may be used in accordance with the present teachings include restriction enzymes (e.g. type II restriction endonuclease), topoisomerases [e.g. DNA gyrase, eukaryotic topoisomerase II (topo II), and bacterial topoisomerase IV (topo IV)], recombinases (e.g. Cre recombinase, Hin recombinase), integrases, DNAses, endo-exonucleases (e.g. micrococcal nuclease) and homing endonucleases.
According to one embodiment, the nuclease utilized may comprise a non-specific DNA cleavage domain, for example, a type II restriction endonuclease such as the cleavage domain of the Fokl restriction enzyme (GenBank accession number J04623).
According to one embodiment, the nuclease is a meganuclease.
As used herein, the term "meganuclease" refers to a double- stranded endonuclease having a large polynucleotide recognition site, e.g. DNA sequences of at least 12 base pairs (bp) or from 12 bp to 40 bp. The meganuclease may also be referred to as rare-cutting or very rare-cutting endonuclease. The meganuclease of the invention may be monomeric or dimeric. The meganuclease may include any natural meganuclease such as a homing endonuclease, but may also include any artificial or man-made meganuclease endowed with high specificity, either derived from homing endonucleases of group I introns and inteins, or other proteins such as zinc finger proteins or group II intron proteins, or compounds such as nucleic acid fused with chemical compounds. Artificial meganucleases of the invention include, but are not limited to, custom- made meganucleases which are meganucleases derived from any initial meganuclease, either natural or not, presenting a recognition and cleavage site different from the site of the initial meganuclease, i.e. the custom-made meganuclease cleaves a novel site with an efficacy at least 10 fold, at least 50 fold or at least 100 fold more than the natural meganuclease.
Custom-made meganucleases may be produced by any method known in the art, for example, by preparing a library of meganuclease variants and isolating, by selection and/or screening, the variants able to cleave the targeted DNA sequence. The diversity could be introduced in the meganuclease by any method known to one skilled in the art, for example, the diversity may be introduced by targeted mutagenesis (i.e. cassette mutagenesis, oligonucleotide directed codon mutagenesis, targeted random mutagenesis), by random mutagenesis (i.e. mutator strains, Neurospora crassa system (U.S. Pat. No. 6,232,112; WO 01/70946, error-prone PCR), by DNA shuffling, by directed mutation or a combination of these technologies (See Current Protocols in Molecular Biology, Chapter 8 "Mutagenesis in cloned DNA", Eds Ausubel et al., John Wiley and Sons). The diversity may be introduced at positions of the residues contacting the DNA target or interacting (directly or indirectly) with the DNA target, or may be introduced specifically at the positions of the interacting amino acids. In libraries generated by targeted mutagenesis, the 20 amino acids can be introduced at the chosen variable positions. According to an embodiment, the amino acids present at the variable positions are the amino acids well-known to be generally involved in protein-DNA interaction. More particularly, these amino acids are generally the hydrophilic amino acids, e.g. comprise D, E, H, K, N, Q, R, S, T, Y. Synthetic or modified amino acids may also be used.
The custom-made meganuclease may be derived from any initial meganuclease. According to one embodiment the initial meganuclease is selected so as its natural recognition and cleavage site is the closest to the targeted DNA site. According to an embodiment, the initial meganuclease is a homing endonuclease. Homing endonucleases fall into 4 separated families on the basis of well conserved amino acids motifs, namely the LAGLIDADG family, the GIY-YIG family, the His-Cys box family, and the HNH family (Chevalier et al., 2001, N.A.R, 29, 3757-3774). According to one embodiment, the homing endonuclease is a I-Dmo I, PI-Sce I, I-Scel, PI-Pfu I, I-Cre I, I- Ppo I, or a hybrid homing endonuclease I-Dmo I/I-Cre I called E-Dre I (as taught in Chevalier et al., 2001, Nat Struct Biol, 8, 312-316).
Further details relating to meganucleases are found in U.S. Pat. No. 8,697,395 which is incorporated herein by reference.
According to another embodiment, of the present invention, the nuclease comprises an oligonucleotide-dependant nuclease such as Cas or a RISC.
RISC enzymes are taught in Martinez J, Tuschl T. RISC is a 5' phosphomonoester-producing RNA endonuclease. Genes Dev. 2004;18:975-980. Also contemplated are sequence modifications to improve plant expression i.e., homologs that are at least 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 %. Homology and identity are also contemplated herein (e.g., using Blast(N)/(P) with default parameters).
According to one embodiment, the Cas9 or RISC is attached to a single guide RNA (sgRNA) to cleave genomic DNA in a sequence specific manner.
As used herein "a single guide RNA" or "sgRNA" refers to a chimeric RNA molecule which is composed of a clustered regularly interspersed short p_alindromic repeats (CRISPR) RNA (crRNA) and trans-encoded CRISPR RNA (tracrRNA). The crRNA defines a site-specific targeting of the Cas9 protein. The sequence is 19-22 nucleotides long e.g., 20 consecutive nucleotides complementary to the target and is typically located at the 5' end of the sgRNA molecule. The crRNA may have 100 % complementation with the target sequence although at least 80 %, 85 %, 90 %, and 95 % global homology to the target sequence are also contemplated according to the present teachings.
The tracrRNA is 100-300 nucleotides long and provides a binding site for the nuclease e.g., Cas9 protein forming the CRISPR/Cas9 complex.
According to a specific embodiment a plurality of sgRNAs are provided to the plant cell that are complementary to different target nucleic acid sequences and the nuclease e.g., Cas9 enzyme cleaves the different target nucleic acid sequences in a site specific manner.
It will be appreciated that the sgRNA may be encoded from the same expression vector as the nuclease, e.g. Cas9. Additionally or alternatively, the sgRNA may be encoded from another nucleic acid construct and thus the CRISPR-Cas9 complex is encoded from a nucleic acid construct system.
According to another embodiment, sgRNA is encoded from the plant expression vector of the invention. In such a case the nuclease, e.g. Cas9, may be encoded from another nucleic acid construct (e.g., which may be MDMV-based as described herein, or not) and thus the CRISPR-Cas9 complex is encoded from a nucleic acid construct system.
Likewise, the plurality of sgRNAs may be encoded from a single vector or from a plurality of vectors as described herein. The use of a plurality of sgRNAs allows multiplexing.
Thus, the RNA-guided endonuclease of the invention comprises at least one nuclease (e.g. Cas9 or RISC) and at least one RNA binding domain (e.g. CRISPR). CRISPR/Cas proteins of the invention may comprise a nuclease domain, DNA binding domain, helicase domain, RNAse domain, protein-protein interaction domain and/or a dimerization domain.
According to one embodiment, the CRISPR/Cas protein can be a wild type
CRISPR/Cas protein, a modified CRISPR/Cas protein, or a fragment of a wild type or modified CRISPR/Cas protein. Furthermore, the CRISPR/Cas protein can be modified to increase nucleic acid binding affinity and/or specificity, or to alter an enzymatic activity of the protein. For example, nuclease (i.e., Cas9) domains of the CRISPR/Cas protein can be modified.
Non-limiting examples of suitable Cas proteins which may be used in accordance with the present teachings include Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8al, Cas8a2, Cas8b, Cas8c, Cas9, CaslO, Casl Od, CasF, CasG, CasH, Csyl, Csy2, Csy3, Csel (or CasA), Cse2 (or CasB), Cse3 (or CasE), Cse4 (or CasC), Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3,Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Cszl, Csxl5, Csfl, Csf2, Csf3, Csf4, and Cul966.
According to a specific embodiment, the cas nuclease is Cas9. Cas9 is a monomeric DNA nuclease guided to a DNA target sequence adjacent to the protospacer adjacent motif (PAM). The Cas9 protein comprises two nuclease domains homologous to RuvC and HNH nucleases. The HNH nuclease domain cleaves the complementary DNA strand whereas the RuvC-like domain cleaves the non-complementary strand and, as a result, a blunt cut is introduced in the target DNA.
In some embodiments, the CRISPR/Cas system comprises a wild type Cas9 protein or fragment thereof.
In other embodiments, the CRISPR/Cas system comprises a modified Cas9 protein. For example, the amino acid sequence of the Cas9 protein may be modified to alter one or more properties (e.g., nuclease activity, affinity, stability, etc.) of the protein. Alternatively, domains of the Cas9 protein not involved in RNA-guided cleavage can be eliminated from the protein such that the modified Cas9 protein is smaller than the wild type Cas9 protein.
According to one embodiment, the Cas9 protein can be modified to lack at least one functional nuclease domain. According to one embodiment, the Cas9 protein can be modified to lack all nuclease activity. According to another embodiment, the CRISPR/Cas system is fused with various effector domains, such as DNA cleavage domains. The DNA cleavage domain can be obtained from any endonuclease or exonuclease. Non-limiting examples of endonucleases from which a DNA cleavage domain can be derived include, but are not limited to, restriction endonucleases and homing endonucleases (see, for example, New England Biolabs Catalog or Belfort et al. (1997) Nucleic Acids Res.). In exemplary embodiments, the cleavage domain of the CRISPR/Cas system is a Fokl endonuclease domain or a modified Fokl endonuclease domain.
Various methods for designing CRISPR/Cas are known in the art and may be implemented in accordance with the present teachings. Further details relating to CRISPR/Cas can be found in PCT publication no. WO 2014089290 which is incorporated herein by reference in its entirety. According to another embodiment of the present invention, the nuclease comprises a chimeric nuclease.
As used herein the phrase "chimeric nuclease" refers to a synthetic chimeric polypeptide which forms a single open reading frame (ORF) and mediates DNA cleavage in a sequence specific manner.
According to a specific embodiment, the chimeric nucleases of this aspect of the present invention comprise separate domains for nucleic acid binding (e.g. DNA binding) and for nucleic acid cleavage (e.g. DNA cleavage), such that cleavage is sequence specific.
As used herein the phrase "sequence specific" refers to a distinct chromosomal location at which nucleic acid cleavage (e.g. DNA cleavage) is introduced.
As used herein the phrase "nucleic acid 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 or RNA in a sequence- specific manner (i.e. target site).
In order to induce efficient gene targeting, the nucleic acid (e.g. DNA) binding domain of the present invention needs to be coupled to a DNA cleavage domain (e.g. 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.
According to one embodiment, the chimeric nuclease induces a single stranded or a double stranded cleavage in the target site.
In generating chimeric nucleases any DNA or RNA binding domain that recognizes the desired target sequence (e.g. DNA binding sequence) with sufficient specificity may be employed. A variety of such DNA and RNA binding domains are known in the art.
Examples of DNA binding domains include, but are not limited to, a meganuclease binding domain, a helix-turn-helix (pfam 01381) binding domain, a leucine zipper (ZIP) binding domain, a winged helix (WH) binding domain, a winged helix turn helix domain (wHTH) binding domain, a helix-loop-helix binding domain, a transcription activator-like (TAL) binding domain, a recombinase, and a zinc finger binding domain.
In an exemplary embodiment of the present invention, the DNA binding domain is a zinc finger binding domain.
Thus, according to an embodiment of this aspect, the chimeric nuclease is a chimeric protein comprising a specific zinc finger binding domain (e.g., pfam00096) and the DNA cleavage domain, such as that of the Fokl restriction enzyme (also referred to herein as the Fokl cleavage domain), termed herein zinc finger nuclease (ZFN). 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 zinc finger 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.
According to another embodiment, the chimeric nuclease is a TALENs or a compact-TALENs (cTALENs).
As used herein, the term "TALENs" or "Transcription Activator-Like Effector Nucleases" refers to the artificial restriction enzymes generated by fusing the TAL effector DNA binding domain to a DNA cleavage domain. TALENs of the invention enable efficient, programmable, and specific DNA cleavage.
It will be appreciated that Transcription activator-like effectors (TALEs) can be quickly engineered to bind practically any DNA sequence. The term TALEN, as used herein, is broad and includes a monomeric TALEN that can cleave double stranded DNA without assistance from another TALEN. The term TALEN is also used to refer to one or both members of a pair of TALENs that are engineered to work together to cleave DNA at the same site. TALENs that work together may be referred to as a left-TALEN and a right-TALEN. Further details relating to TALENS can be found in U.S. Pat. No. 8,450,471; U.S. Pat. No. 8,440,431; U.S. Pat. No. 8,440,432; and U.S. Pat. Applic. No. 20140256798 all of which are incorporated herein by reference in their entirety.
TALEs are proteins secreted by Xanthomonas bacteria. The DNA binding domain of TALEs contains a highly conserved 33-34 amino acid sequence with the exception of the 12th and 13th amino acids. These two locations are highly variable [Repeat Variable Diresidue (RVD)] and show a strong correlation with specific nucleotide recognition. This simple relationship between amino acid sequence and DNA recognition has allowed for the engineering of specific DNA binding domains by selecting a combination of repeat segments containing the appropriate RVDs.
TALENs of the invention are typically constructed using a non-specific DNA cleavage domain, such as the non-specific DNA cleavage domain of Fokl endonuclease. Thus, wild-type Fokl cleavage domain may be used as well as Fokl cleavage domain variants with mutations designed to improve cleavage specificity and cleavage activity. The Fokl domain functions as a dimer, requiring two constructs with unique DNA binding domains for sites in the target genome with proper orientation and spacing. Both the number of amino acid residues between the TALEN DNA binding domain and the DNA cleavage domain (e.g. Fokl cleavage domain) and the number of bases between the two individual TALEN binding sites are parameters for achieving high levels of activity. The number of amino acid residues between the TALEN DNA binding domain and the DNA cleavage domain (e.g. Fokl cleavage domain) may be modified by introduction of a spacer between the plurality of TAL effector repeat sequences and the nuclease (e.g. Fokl endonuclease domain). The spacer sequence may be 12 to 30 nucleotides.
Furthermore, compact TALENs (c TALENs) may be used according to the present teachings. These cTALENs are typically designed with the partially specific I- Tevl catalytic domain and are monomeric DNA-cleaving enzymes, i.e. TALENs which are half- size, single-polypeptide compact transcription activator- like effector nucleases (see Beurdeley M. et al., Nature Communications (2013) 4: 1762, which is incorporated herein by reference in its entirety).
The relationship between amino acid sequence and DNA recognition of the TALEN binding domain allows for designable proteins. In this case software programs (e.g. DNA Works) may be used which calculate oligonucleotides suitable for assembly in a two step PCR; oligonucleotide assembly followed by whole gene amplification. Modular assembly schemes for generating engineered TALE constructs may also be used. Both methods offer a systematic approach to engineering DNA binding domains that are conceptually similar to the modular assembly method for generating zinc finger DNA recognition domains (described hereinabove). Qualifying the nucleases (e.g. ZFN, TALENs and CRISPR/Cas) and meganucleases thus generated for specific target recognition can be effected using methods which are well known in the art.
A method for designing the nucleases (e.g. chimeric nucleases, ZFN, TALENs, Cas9, RISC, meganucleases) for use in gene targeting may include a process for testing the toxicity of the nuclease on a cell. Such a process may comprise expressing in the cell, or otherwise introducing into a cell, the nuclease and assessing cell growth or death rates by comparison against a control. The tendency of a 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. In view of the present disclosure, one of ordinary skill in the art may devise other tests for cleavage specificity.
The heterologous polypeptide of interest (e.g. nuclease) disclosed herein may further comprise at least one nuclear localization signal (NLS) which facilitates the transport of the nuclease to the DNA-containing organelle. In general, an NLS comprises a stretch of basic amino acids which is recognized by specific receptors at the nuclear pores. The NLS can be located at the N-terminus, the C-terminal, or in an internal location of the nuclease.
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 Al, the sequence KIPIK in yeast transcription repressor Mata2 and the complex signals of U snRNPs, tobacco NLS and rice NLS.
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). Mitochondrion localization signals (MLS) 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 (ΑΤΡ-β) (SEQ ID NO: 78).
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 (atslA) 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 www(dot)springerlink(dot)com/content/p65013h263617795/.
Alternatively, the chloroplast localization sequence (CLS) may be derived from a viroid [Evans and Pradhan (2004) US 2004/0142476 Al]. 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-l,5-bisphospate carboxylase small subunit (Rssu) (SEQ ID NOs: 76 or 77).
In some embodiments, the heterologous polypeptide of interest (e.g. nuclease) further comprises at least one cell-penetrating domain. In one embodiment, the cell- penetrating domain can be a cell-penetrating peptide (CPP) sequence derived from Tat, Tat2, arginine-rich intracellular delivery peptides (AID), pVEC, transportan and penetratin.
According to a specific embodiment of the present invention, the CPP sequence comprises a dimmer of the Tat molecule (Tat2, RKKRRQRRRRKKRRQRRR, SEQ ID NO: 79) which has an increased ability to translocate across plant cell membranes because of the presence of high number of arginine residues. According to a specific embodiment of the present invention, the CPP sequence comprises an 18 amino acid peptide of vascular endothelial-cadherin (pVEC) (LLIILRRRIRKQ AH AS K SEQ ID NO: 80).
As mentioned hereinabove, the heterologous polypeptide of interest is introduced into the plant target using a plant expression vector which is typically used for mediating transient transformation through systemic spreading within the plant. Thus, once the genes encoding the polyprotein product (i.e. MDMV polyprotein and heterologous polypeptide of interest) have been assembled they are inserted into a vector. The vector is then introduced into the target cell where the gene products are expressed and optionally enter the nucleus to access the genome.
Constructs useful in the methods according to some embodiments of the invention may be constructed using recombinant DNA technology well known to persons skilled in the art. The gene constructs may be inserted into vectors, which may be commercially available, suitable for transforming into plants and suitable for expression of the gene of interest in the transformed cells. For example, a nucleic acid sequence encoding the polyprotein is cloned into an expression vector, for example in to a binary vector, the expression vector is introduced into a plant cell, as described in the examples see below, and the polyprotein is expressed in the host cell. Examples for binary vectors are pBIN19, pBHOl, pBinAR, pGPTV, pCAMBIA, pBIB-HYG, pBecks, pGreen or pPZP (Hajukiewicz, P. et al., Plant Mol. Biol. 25, 989 (1994), and Hellens et al., Trends in Plant Science 5, 446 (2000)). According to a specific embodiment, the vector is a pGreen vector.
In cases where the polyprotein product is large (e.g. the MDMV genome size is approximately 10,000 bp), cloning may be carried out in several parts (e.g. gene sequences may be cloned into several vectors, such as pJET or pGEM (Amp ), as described in detail in the Examples section which follows) and may then be combined into one vector (e.g. pGreen) to achieve one long open reading frame (ORF) (e.g. from the beginning of the PI gene of MDMV until the end of the CP gene of MDMV, see Figure 2F). The expression vector may further be constructed to include a PolyA region (e.g. at the viral 3' terminal sequence).
The expression vector of the invention may be constructed such that the nucleic acid sequence encoding the heterologous polypeptide of interest is cloned at the N- terminal end or at the C-terminal end of the nucleic acid sequence encoding the MDMV polypeptides, i.e. such that the heterologous polypeptide of interest is directly translationally fused N-terminally or C-terminally, respectively, to the MDMV polypeptides in the polyprotein product.
The term "directly translationally fused" as used herein relates to the immediate position of the two sequences, i.e. with no intervening nucleic acid sequences which may stop the translation.
Alternatively, the expression vector of the invention may be constructed such that the heterologous polypeptide of interest is flanked by the MDMV polypeptides. Accordingly, the vector of the invention may be constructed to include multiple cloning sites (MCS) at different locations within the MDMV genome and without interfering with the readthrough of the ORF or the polyprotein. Without being bound to theory and as explained in further detail in the examples section which follows, MCS may be inserted, for example, between the PI gene and a HCPro gene of MDMV, or between the NIb-RdRp gene and a CP gene of the MDMV, by insertion of restriction enzymes (e.g. on the 5' MCS Agel and Apal and on the 3' MCS Nhel and Avrll).
According to a specific embodiment, the heterologous polypeptide of interest is flanked by a PI polypeptide and a HCPro polypeptide of the MDMV, wherein the PI polypeptide is N-terminally positioned to the heterologous polypeptide of interest and the HCPro polypeptide is C-terminally to the heterologous polypeptide of interest.
According to another specific embodiment, the heterologous polypeptide of interest is flanked by a NIb-RNA replicase (e.g. NIb-RdRp) polypeptide and a CP polypeptide of the MDMV, wherein the NIb-RNA replicase (e.g. NIb-RdRp) polypeptide is N-terminally positioned to the heterologous polypeptide of interest and the CP polypeptide is C-terminally to the heterologous polypeptide of interest.
According to one embodiment of the present invention, the expression vector is a satellite vector.
The association of viral satellites with RNA viruses is a well-documented phenomenon. Satellites, either encapsidated or in the form of naked nucleic acid, depend on a helper virus for their replication. Geminivirus-associated, 682-base-long DNA satellite was first reported by Dry et al. in 1997, Proc. Natl. Acad. Sci. USA 94, 7088- 7093. Other gemini-associated satellites have been discovered since then (Briddon et al., 2001, Virology 285, 234-243; Mansoor et al., 1999, Virology 259, 190-199; Zhou et al., 2003, J. Gen. Virol. 84, 237-247). In many cases of geminiviral infection, the satellite determines symptom severity. In the case of geminiviral satellites, these are encapsidated and replicated via factors provided by helper viruses. A satellite associated with a particular virus may be supported for replication by other viruses as well. Thus, for example, the helper virus may be a wild type MDMV virus or may be an MDMV virus deficient of one or more peptides (e.g. lacking a coat protein gene). Satellite vectors of the invention enable expression of a large heterologous polypeptide (of about 4000 bases) in a target cell.
According to one embodiment, the plant expression vector comprises a nucleic acid sequence encoding a polyprotein product, the polyprotein product comprising Maize Dwarf Mosaic Virus (MDMV) polypeptides, wherein the MDMV polypeptides comprise a PI polypeptide and a coat protein polypeptide, and a heterologous polypeptide of interest, the heterologous polypeptide of interest being cleaved of the MDMV polypeptides upon expression in a plant cell infected with a helper virus or an MDMV virus, and wherein the plant expression vector is capable of spreading in a plant.
According to another specific embodiment, the heterologous polypeptide of interest is flanked by a PI polypeptide and a coat protein polypeptide of the MDMV.
According to another specific embodiment, the PI polypeptide is N-terminally positioned to the heterologous polypeptide of interest and the coat protein polypeptide is C-terminally to the heterologous polypeptide of interest.
According to an embodiment of the present invention, the expression vector is included in a system. Thus, according to one embodiment, the plant expression vector system comprises one plant expression vector of some embodiments of the invention (e.g satellite vector) and an MDMV helper virus (as discussed above).
Regardless of the cloning site, the vector of the invention is designed such that the heterologous polypeptide or polypeptides are cleaved and separated of the MDMV polypeptides upon expression in a plant cells. Thus, according to one embodiment, the nucleic acid sequence encoding the heterologous polypeptide of interest is directly translationally fused to the protease cleavage site. According to one embodiment the heterologous polypeptide is cleaved of the viral polypeptides with no functional remnants of viral proteins remaining in the heterologous polypeptide.
According to one embodiment, the protease cleavage site is a viral protease cleavage site.
According to a specific embodiment, the protease cleavage site is a NIa protease cleave site (e.g. SEQ ID NO: 42).
According to one embodiment, the nucleic acid sequence encoding the heterologous polypeptide of interest encodes for two heterologous polypeptides of interest.
According to another embodiment, the nucleic acid sequence encoding the heterologous polypeptide of interest encodes for 2, 3, 4, 5 or more heterologous polypeptides of interest.
In cases in which more than one heterologous polypeptide is expressed in a cell, additional protease cleavage sites may be added between the heterologous polypeptides as to enable cleavage thereof.
Thus, in certain embodiments, at least two heterologous polypeptide sequences within the vector are separated by a nucleic acid sequence encoding a cleavage domain. Such a cleavage domain may comprise any cleavage domain known in the art, as for example a NIa protease cleave site (e.g. SEQ ID NO: 42).
The expression vector of the invention is may be constructed such that the nucleic acid sequence encoding the polyprotein product is operably linked to one or more regulatory sequences allowing expression in the plant cells.
In a particular embodiment of some embodiments of the invention the regulatory sequence is a plant-expressible promoter.
As used herein the phrase "plant-expressible" refers to a promoter sequence, including any additional regulatory elements added thereto or contained therein, is at least capable of inducing, conferring, activating or enhancing expression in a plant cell, tissue or organ, preferably a monocotyledonous or dicotyledonous plant cell, tissue, or organ. Examples of preferred promoters useful for the methods of some embodiments of the invention are presented in Table I, II, III and IV. Table I
Exemplary constitutive promoters for use in the performance of embodiments of the invention
Figure imgf000036_0001
Table II
Exemplary seed-preferred promoters for use in the performance of some embodiments of the invention
Gene Source Expression Pattern Reference
Seed specific seed Simon, et al., Plant Mol. Biol. 5.
191, 1985; Scofield, et al., J. Biol. Chem. 262: 12202, 1987.; Baszczynski, et al., Plant Mol. Biol. 14: 633, 1990.
Brazil Nut albumin seed Pearson' et al., Plant Mol. Biol.
18: 235- 245, 1992.
legumin seed Ellis, et al. Plant Mol. Biol. 10:
203-214, 1988
Glutelin (rice) seed Takaiwa, et al., Mol. Gen. Genet. 208: 15-22, 1986;
Takaiwa, et al., FEBS Letts. 221 : 43-47, 1987
zein seed Matzke et al. Plant Mol Biol,
143)323-32 1990
napA seed Stalberg, et al., Planta 199: 515- 519, 1996
Wheat LMW and HMW, endosperm Mol Gen Genet 216:81-90, glutenin-1 1989; NAR 17:461-2,
Wheat SPA seed Albanietal, Plant Cell, 9: 171- 184, 1997
Wheat a, b and g gliadins endosperm EMBO3: 1409-15, 1984
Barley ltrl promoter endosperm
Barley B l, C, D hordein endosperm Theor Appl Gen 98: 1253-62,
1999; Plant J 4:343-55, 1993; Mol Gen Genet 250:750- 60, 1996
Barley DOF endosperm Mena et al., The Plant Journal,
116(1): 53- 62, 1998
Biz2 endosperm EP99106056.7
Synthetic promoter endosperm Vicente-Carbajosa et al., Plant J.
13: 629-640, 1998
Rice prolamin NRP33 endosperm Wu et al., Plant Cell Physiology
39(8) 885- 889, 1998
Rice -globulin Glb-1 endosperm Wu et al., Plant Cell Physiology
398) 885-889, 1998
Rice OSH1 embryo Sato et al., Proc. Nati. Acad.
Sci. USA, 93: 8117-8122
Rice alpha-globulin REB/OHP- endosperm Nakase et al. Plant Mol. Biol. 1 33: 513-S22, 1997 endosperm Trans Res 6: 157-68, 1997
Rice ADP-glucose PP
Maize ESR gene family endosperm Plant J 12:235-46, 1997
Sorgum gamma- kafirin endosperm PMB 32: 1029-35, 1996
KNOX embryo Postma-Haarsma et al., Plant
Mol. Biol. 39:257-71, 1999
Rice oleosin embryo and aleuton Wu et at, J. Biochem., 123:386, 1998
Sunflower oleosin Seed (embryo and dry seed) Cummins, et al., Plant Mol.
Biol. 19: 873- 876, 1992
Table III
Exemplary flower-specific promoters for use in the performance of the invention
Figure imgf000038_0001
Table IV
Alternative rice promoters for use in the performance of the invention
PRO # gene expression
PR00001 Metallothionein Mte transfer layer of embryo + calli
PR00005 Putative beta-amylase transfer layer of embryo
PR00009 Putative cellulose synthase weak in roots
PR00012 Lipase (putative)
PR00014 Transferase (putative)
PR00016 peptidyl prolyl cis-trans isomerase
(putative)
PR00019 unknown
PR00020 prp protein (putative)
PR00029 noduline (putative)
PR00058 Proteinase inhibitor Rgpi9 seed
PR00061 beta expansine EXPB9 weak in young flowers
PR00063 Structural protein young tissues+calli+embryo
PR00069 xylosidase (putative)
PR00075 Prolamine lOKda strong in endosperm
PR00076 allergen RA2 strong in endosperm
PR00077 prolamine RP7 strong in endosperm
PR00078 CBP80
PR00079 starch branching enzyme I PR00080 Metallothioneine-like ML2 transfer layer of embryo + calli
PR00081 putative caffeoyl- CoA 3-0 shoot
methyltransferase
PR00087 prolamine RM9 strong in endosperm
PR00090 prolamine RP6 strong in endosperm
PR00091 prolamine RP5 strong in endosperm
PR00092 allergen RA5
PR00095 putative methionine embryo
aminopeptidase
PR00098 ras-related GTP binding protein
PR00104 beta expansine EXPB 1
PR00105 Glycine rich protein
PR00108 metallothionein like protein
(putative)
PR00110 RCc3 strong root
PROOl l l uclacyanin 3 -like protein weak discrimination center / shoot meristem
PR00116 26S proteasome regulatory very weak meristem specific particle non-ATPase subunit 11
PR00117 putative 40S ribosomal protein weak in endosperm
PR00122 chlorophyll a/lo-binding protein very weak in shoot
precursor (Cab27)
PR00123 putative protochlorophyllide strong in leaves
reductase
PR00126 metallothionein RiCMT strong discrimination center shoot meristem
PR00129 GOS2 strong constitutive
PR00131 GOS9
PR00133 chitinase Cht-3 very weak meristem specific
PR00135 alpha- globulin strong in endosperm
PR00136 alanine aminotransferase weak in endosperm
PR00138 Cyclin A2
PR00139 Cyclin D2
PR00140 Cyclin D3
PR00141 Cyclophyllin 2 shoot and seed
PR00146 sucrose synthase SS I (barley) medium constitutive
PR00147 trypsin inhibitor ITR1 (barley) weak in endosperm PR00149 ubiquitine 2 with intron strong constitutive
PR00151 WSI18 embryo and following stress
PR00156 HVA22 homologue (putative)
PR00157 EL2
PR00169 aquaporine medium constitutive in young plants
PR00170 High mobility group protein strong constitutive
PR00171 reversibly glycosylated protein weak constitutive
RGP1
PR00173 cytosolic MDH shoot
PR00175 RAB21 embryo and following stress
PR00176 CDPK7
PR00177 Cdc2-1 very weak in meristem
PR00197 sucrose synthase 3
PRO0198 OsVPl
PRO0200 OSH1 very weak in meristem of young plants
PRO0208 putative chlorophyllase
PRO0210 OsNRTl
PRO0211 EXP 3
PRO0216 phosphate transporter OjPTl
PRO0218 oleosin 18kd aleurone + embryo
PRO0219 ubiquitine 2 without intron
PRO0220 RFL
PRO0221 maize UBI delta intron not detected
PRO0223 glutelin-1
PRO0224 fragment of prolamin RP6
promoter
PRO0225 4xABRE
PRO0226 glutelin OSGLUA3
PRO0227 BLZ-2_short (barley)
PR00228 BLZ-2_long (barley)
According to one embodiment, the promoter is a heterologous promoter i.e. a promoter not naturally found in the plant (i.e. not-native to the plant).
According to one embodiment, the heterologous promoter comprises a 35S promoter. According to a specific embodiment, the vector comprises a nucleic acid sequence operably linked to two or more promoter sequences.
According to another specific embodiment, the vector comprises a nucleic acid sequence operably linked to a double 35s promoter (i.e. two 35S promoters one after the other).
Nucleic acid sequences of the polypeptides of some embodiments of the invention may be optimized for plant expression. Examples of such sequence modifications include, but are not limited to, an altered G/C content to more closely approach that typically found in the plant species of interest, and the removal of codons atypically found in the plant species commonly referred to as codon optimization.
The phrase "codon optimization" refers to the selection of appropriate DNA nucleotides for use within a structural gene or fragment thereof that approaches codon usage within the plant of interest. Therefore, an optimized gene or nucleic acid sequence refers to a gene in which the nucleotide sequence of a native or naturally occurring gene has been modified in order to utilize statistically-preferred or statistically-favored codons within the plant. The nucleotide sequence typically is examined at the DNA level and the coding region optimized for expression in the plant species determined using any suitable procedure, for example as described in Sardana et al. (1996, Plant Cell Reports 15:677-681). In this method, the standard deviation of codon usage, a measure of codon usage bias, may be calculated by first finding the squared proportional deviation of usage of each codon of the native gene relative to that of highly expressed plant genes, followed by a calculation of the average squared deviation. The formula used is: 1 SDCU = n = 1 N [ ( Xn - Yn ) / Yn ] 2 / N, where Xn refers to the frequency of usage of codon n in highly expressed plant genes, where Yn to the frequency of usage of codon n in the gene of interest and N refers to the total number of codons in the gene of interest. A table of codon usage from highly expressed genes of dicotyledonous plants is compiled using the data of Murray et al. (1989, Nuc Acids Res. 17:477-498).
One method of optimizing the nucleic acid sequence in accordance with the preferred codon usage for a particular plant cell type is based on the direct use, without performing any extra statistical calculations, of codon optimization tables such as those provided on-line at the Codon Usage Database through the NIAS (National Institute of Agrobiological Sciences) DNA bank in Japan (www(dot)kazusa(dot)or(dot)jp/codon/). The Codon Usage Database contains codon usage tables for a number of different species, with each codon usage table having been statistically determined based on the data present in Genbank.
By using the above tables to determine the most preferred or most favored codons for each amino acid in a particular species (for example, rice), a naturally- occurring nucleotide sequence encoding a protein of interest can be codon optimized for that particular plant species. This is effected by replacing codons that may have a low statistical incidence in the particular species genome with corresponding codons, in regard to an amino acid, that are statistically more favored. However, one or more less- favored codons may be selected to delete existing restriction sites, to create new ones at potentially useful junctions (5' and 3' ends to add signal peptide or termination cassettes, internal sites that might be used to cut and splice segments together to produce a correct full-length sequence), or to eliminate nucleotide sequences that may negatively effect mRNA stability or expression.
The naturally-occurring encoding nucleotide sequence may already, in advance of any modification, contain a number of codons that correspond to a statistically- favored codon in a particular plant species. Therefore, codon optimization of the native nucleotide sequence may comprise determining which codons, within the native nucleotide sequence, are not statistically-favored with regards to a particular plant, and modifying these codons in accordance with a codon usage table of the particular plant to produce a codon optimized derivative. A modified nucleotide sequence may be fully or partially optimized for plant codon usage provided that the protein encoded by the modified nucleotide sequence is produced at a level higher than the protein encoded by the corresponding naturally occurring or native gene. Construction of synthetic genes by altering the codon usage is described in for example PCT Patent Application 93/07278.
Thus, some embodiments of the invention encompasses nucleic acid sequences described hereinabove; fragments thereof, sequences hybridizable therewith, sequences homologous thereto, sequences orthologous thereto, sequences encoding similar polypeptides with different codon usage, altered sequences characterized by mutations, such as deletion, insertion or substitution of one or more nucleotides, either naturally occurring or man induced, either randomly or in a targeted fashion. It will be appreciated that 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, DsRed, GUS and GFP.
In certain embodiments, further modifications to vector may be carried out such as the addition of an enhancer sequence. Any enhancer sequence can be inserted into the plant expression vector to enhance transcription levels of genes. For example, an Ω enhancer can be cloned into the vectors of the present invention.
Plant cells may be transformed stabley or transiently with the nucleic acid constructs of some embodiments of the invention. In stable transformation, the nucleic acid molecule of some embodiments of the invention is integrated into the plant genome and as such it represents a stable and inherited trait. In transient transformation, the nucleic acid molecule is expressed by the cell transformed but it is not integrated into the genome and as such it represents a transient trait.
There are various methods of introducing foreign genes into both monocotyledonous and dicotyledonous plants (Potrykus, I., Annu. Rev. Plant. Physiol., Plant. Mol. Biol. (1991) 42:205-225; Shimamoto et al., Nature (1989) 338:274-276).
Although transient transformation is presently preferred, stable transformation of leaf cells, meristematic cells or the whole plant is also envisaged by some embodiments of the invention.
The principle methods of causing stable integration of exogenous DNA into plant genomic DNA include two main approaches:
(i) Agrobacterium-mediated gene transfer: Klee et al. (1987) Annu. Rev. Plant Physiol. 38:467-486; Klee and Rogers in Cell Culture and Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of Plant Nuclear Genes, eds. Schell, J., and Vasil, L. K., Academic Publishers, San Diego, Calif. (1989) p. 2-25; Gatenby, in Plant Biotechnology, eds. Kung, S. and Arntzen, C. J., Butterworth Publishers, Boston, Mass. (1989) p. 93-112. According to one embodiment, acetosyringone is included with Agrobacterium for the infection of monocotyledonous plants.
(ii) direct DNA uptake: Paszkowski et al., in Cell Culture and Somatic Cell
Genetics of Plants, Vol. 6, Molecular Biology of Plant Nuclear Genes eds. Schell, J., and Vasil, L. K., Academic Publishers, San Diego, Calif. (1989) p. 52-68; including methods for direct uptake of DNA into protoplasts, Toriyama, K. et al. (1988) Bio/Technology 6: 1072-1074. DNA uptake induced by brief electric shock of plant cells: Zhang et al. Plant Cell Rep. (1988) 7:379-384. Fromm et al. Nature (1986) 319:791-793. DNA injection into plant cells or tissues by particle bombardment, Klein et al. Bio/Technology (1988) 6:559-563; McCabe et al. Bio/Technology (1988) 6:923- 926; Sanford, Physiol. Plant. (1990) 79:206-209; by the use of micropipette systems: Neuhaus et al., Theor. Appl. Genet. (1987) 75:30-36; Neuhaus and Spangenberg, Physiol. Plant. (1990) 79:213-217; glass fibers or silicon carbide whisker transformation of cell cultures, embryos or callus tissue, U.S. Pat. No. 5,464,765 or by the direct incubation of DNA with germinating pollen, DeWet et al. in Experimental Manipulation of Ovule Tissue, eds. Chapman, G. P. and Mantell, S. H. and Daniels, W. Longman, London, (1985) p. 197-209; and Ohta, Proc. Natl. Acad. Sci. USA (1986) 83:715-719.
The Agrobacterium system includes the use of plasmid vectors that contain defined DNA segments that integrate into the plant genomic DNA. 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 et al. in Plant Molecular Biology Manual A5, Kluwer Academic Publishers, Dordrecht (1988) p. 1-9. A supplementary approach employs the Agrobacterium delivery system in combination with vacuum infiltration. The Agrobacterium system is especially viable in the creation of transgenic dicotyledonous plants.
There are various methods of direct DNA transfer into plant cells. In electroporation, the protoplasts are briefly exposed to a strong electric field. In microinjection, the DNA is mechanically injected directly into the cells using very small micropipettes. In microparticle bombardment, the DNA is adsorbed on microprojectiles such as magnesium sulfate crystals or on gold, tungsten or platinum particles, and the microprojectiles are physically accelerated into cells or plant tissues.
According to another embodiment, direct DNA uptake by protoplasts can be stimulated by chemicals like polyethylene glycol (PEG), i.e. PEG-mediated DNA transfer. Following stable transformation plant propagation is exercised. The most common method of plant propagation is by seed. Regeneration by seed propagation, however, has the deficiency that due to heterozygosity there is a lack of uniformity in the crop, since seeds are produced by plants according to the genetic variances governed by Mendelian rules. Basically, each seed is genetically different and each will grow with its own specific traits. Therefore, it is preferred that the transformed plant be produced such that the regenerated plant has the identical traits and characteristics of the parent transgenic plant. Therefore, it is preferred that the transformed plant be regenerated by micropropagation which provides a rapid, consistent reproduction of the transformed plants.
Micropropagation is a process of growing new generation plants from a single piece of tissue that has been excised from a selected parent plant or cultivar. This process permits the mass reproduction of plants having the preferred tissue expressing the fusion protein. The new generation plants which are produced are genetically identical to, and have all of the characteristics of, the original plant. Micropropagation allows mass production of quality plant material in a short period of time and offers a rapid multiplication of selected cultivars in the preservation of the characteristics of the original transgenic or transformed plant. The advantages of cloning plants are the speed of plant multiplication and the quality and uniformity of plants produced.
Micropropagation is a multi-stage procedure that requires alteration of culture medium or growth conditions between stages. Thus, the micropropagation process involves four basic stages: Stage one, initial tissue culturing; stage two, tissue culture multiplication; stage three, differentiation and plant formation; and stage four, greenhouse culturing and hardening. During stage one, initial tissue culturing, the tissue culture is established and certified contaminant-free. During stage two, the initial tissue culture is multiplied until a sufficient number of tissue samples are produced to meet production goals. During stage three, the tissue samples grown in stage two are divided and grown into individual plantlets. At stage four, the transformed plantlets are transferred to a greenhouse for hardening off, i.e. for building up of the cuticle on the leaves by the gradual exposure of the still fragile plants to air movement, sun and lower humidity. The thicker cuticle prevents less water transpiration and stronger resilience of the plantlets and enables the plants to be grown in a natural environment. Transient transformation (i.e. transient gene expression) can be effected by any of the direct DNA transfer methods described above, by Agrobacterium-mediated gene transfer, by viral infection using modified plant viruses, by aphids, by nematodes, by infiltration, by vacuum, by electroporation or by bombardment.
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.
Construction of plant RNA viruses for the introduction and expression of non- viral exogenous nucleic acid sequences in plants is demonstrated by the above references as well as by Dawson, W. O. et al., Virology (1989) 172:285-292; Takamatsu et al. EMBO J. (1987) 6:307-311; French et al. Science (1986) 231: 1294-1297; and Takamatsu et al. FEBS Letters (1990) 269:73-76.
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 transcribing 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 some embodiments of the invention is demonstrated by the above references as well as in U.S. Pat. No. 5,316,931. 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 transcribing 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) 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 transcribed 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 transcribing 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 the sequences are transcribed 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.
In addition to the above, the nucleic acid molecule of some embodiments of the invention can also be introduced into a plastid genome (e.g. chloroplast genome) thereby enabling plastid (e.g. chloroplast) expression.
A technique for introducing exogenous nucleic acid sequences to the genome of the plastids (e.g. chloroplasts) is known. This technique involves the following procedures. First, plant cells are chemically treated so as to reduce the number of chloroplasts per cell to about one. Then, the exogenous nucleic acid is introduced via particle bombardment into the cells with the aim of introducing at least one exogenous nucleic acid molecule into the chloroplasts. The exogenous nucleic acid is selected such that it is integratable into the chloroplast's genome via homologous recombination which is readily effected by enzymes inherent to the chloroplast. To this end, the exogenous nucleic acid includes, in addition to a gene of interest, at least one nucleic acid stretch which is derived from the chloroplast's genome. In addition, the exogenous nucleic acid includes a selectable marker, which serves by sequential selection procedures to ascertain that all or substantially all of the copies of the chloroplast genomes following such selection will include the exogenous nucleic acid. Further details relating to this technique are found in U.S. Pat. Nos. 4,945,050; and 5,693,507 which are incorporated herein by reference. A polypeptide can thus be produced by the protein expression system of the chloroplast and become integrated into the chloroplast's inner membrane.
Infection of viral vectors (e.g. MDMV) into plants can also be carried out by the use of aphids, including without limitation, the corn leaf aphid (Rhopalosiphum maidis), the greenbug (Schizaphis graminum), and the green peach aphid (Myzus persicae) (the natural hosts for MDMV). Accordingly, Rhopalosiphum maidis, Schizaphis graminum, and Myzus persicae are inoculated with the plant expression vectors of the invention or their derivatives prior to subjection to the plants.
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 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 immuno staining, 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.
The vectors of some embodiments of the invention may be used for generating a plant. The plant may be generated using a single expression vector or using several expression vectors (e.g. designed for expression of different heterologous polypeptides of interest).
As used herein the term "plant" refers to whole plants, portions thereof (e.g., leaf, root, stem, fruit, seed) or cells isolated therefrom (homogeneous or heterogeneous populations of cells, including meristem cells, leaf cells, pollen cells, ovule cells, male inflorescence cell, female inflorescence cells, microspores, megaspores, embryogenic calli cells or cells of first node derived calli).
According to one embodiment the plant is a seedling.
According to an embodiment of the present invention, the plant may be an adult plant such as one which comprises a gamete i.e. male and female reproductive plant organs including the anther and ovary (i.e. organs producing pollen and ovules, respectively) or a gamete producing tissue, i.e. any tissue which may give rise to gametes, such as but not limited to, a floral meristem tissue and flowers.
As used herein the phrase "isolated plant cells" refers to plant cells which are derived from dissociated plant cell tissues 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, barley, 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, sugar cane, watermelon, beans, hibiscus, okra, apple, rose, strawberry, chile, garlic, onions, sorghum, Johnson grass, turf grass, bamboo, palm, banana, ginger, 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 Acanthaceae, Alismataceae, Amaranthaceae, Amaryllidaceae, Annonaceae, Apiaceae, Apocynaceae, Araceae, Araucariaceae, Arecaceae, Asteraceae, Asclepiadaceae, Bignoniaceae, Boraginaceae, Brassicaceae, Bromeliaceae, Cactaceae, Campanulaceae, Caryophyllaceae, Casuarinaceae, Celastraceae, Clusiaceae, Combretaceae, Commelinaceae, Convolvulaceae, Crassulaceae, Cucurbitaceae, Cyperaceae, Ericaceae, Euphorbiaceae, Fabaceae, Fagaceae, Iridaceae, Lamiaceae, Lauraceae, Liliaceae, Lythraceae, Magnoliaceae, Malpighiaceae, Malvaceae, Melastomataceae, Moraceae, Myrsinaceae, Myrtaceae, Nyctaginaceae, Nymphaceae, Oleaceae, Orchidaceae, Oxalidaceae, Papaveraceae, Piperaceae, Plantaginaceae, Poaceae, Polygonaceae, Portulaceae, Proteaceae, Ranunculaceae, Rhamnaceae, Rosaceae, Rubiaceae, Rutaceae, Sapindaceae, Sapotaceae, Solanaceae, Urticaceae, Verbenaceae, Violaceae, Zingiberaceae, Sapindaceae, Sapotaceae, Solanaceae, Urticaceae, Verbenaceae, Violaceae, Zingiberaceae.
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, Cheiranthus 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 is a monocot.
According to a specific embodiment, the plant comprises maize, rice, wheat, barley, sugar cane, sorghum, Johnson grass, grasses, bamboo, palm, agave, pineapple, banana, ginger, garlic, onion, oat, rye, turf grass, millet, spelt, triticale, fonio, aloe, asparagus, yam or ubi, orchid, iris, lily, amaryllis, canna-lily arum or gabi, lemon grass, pandan or screwpine, arrow root, rush, pipewort and sedge.
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 the plant expression vector of some embodiments of the invention, wherein the nuclease mediates cleavage in the genome of the plant and enables an alteration in the cleavage site.
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 hereinabove. 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.
It will be appreciated that two plant expression vectors may be introduced into the same plant cell. These plant expression vectors may be introduced in the plant cell concomitantly or at separate times. Such expression vectors may comprise nucleic acid sequences encoding different heterologous sequences. For example, an expression vector comprising a nucleic acid sequence encoding a nuclease and an expression vector comprising a nucleic acid sequence encoding, for example, a herbicide resistance polypeptide. The two expression vectors can be introduced concomitantly, as for example at a 1: 1 ratio, to enable expression of heterologous genes in plant cells.
According to a specific embodiment, two plant expression vectors are introduced into a plant, the first expression vector comprising a satellite vector comprising the heterologous polypeptide of interest (e.g. a nucleic acid sequence comprising the MDMV coat protein, the MDMV PI polypeptide and a heterologous polypeptide of interest). The second expression vector comprising a MDMV virus or a helper virus (e.g. a nucleic acid sequence of the MDMV genome without the gene for the coat protein). Typically expression of the heterologous polypeptide only occurs when both constructs are co-expressed.
The following section provides non-limiting applications for generating such a variation.
Thus, nucleases of some embodiments 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 nuclease 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 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 a 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 nucleases can be used to generate genotypic variation by knocking out gene expression. Thus 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 nucleases (e.g. or combinations of same) can be used to cleave out an entire sequence of the genome, thereby knocking out gene expression.
Nucleases of the present invention may also be used to generate genotypic variations in gametes and seeds of the plant. Thus, the nucleases of the present invention may be used to generate specific or non-specific mutations in gametes which, following fertilization, will generate genotypically modified seeds and consequently modified plants.
Nucleases of the present invention may also be used to generate genotypic variations in calli of the plant. Thus, the nucleases of the present invention may be used to generate specific or non-specific mutations in embryogenic calli cells, including in immature embryo scutella and mature embryo scutella cells, in cells of a first node derived calli, in split seedling nodes, in split seeds, in inner leaf sheathes of seedlings, in zygotes of fertilized embryo sacs and in immature male and female inflorescences.
It will be appreciated that plant calli of the invention can differentiate into a whole plant (e.g. regenerate) thereby generating plants comprising the genotypic variation.
The 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 nucleases such as the DNA restrictases or Non-stringent Fokl.
Additionally, the 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 generating a pathogen resistant plant, the method comprising introducing into the plant the expression vector of some embodiments of the invention, wherein the nuclease mediates cleavage of a gene conferring sensitivity to a pathogen or in a gene inhibiting the resistance pathway, and enables an alteration in the cleavage site, thereby generating the pathogen resistant plant.
As used herein a "plant pathogen" refers to an organism, which causes a disease in a plant. Organisms that cause infectious disease include fungi, oomycetes, bacteria, viruses, viroids, virus-like organisms, phytoplasmas, protozoa, nematodes and parasitic plants.
It is advisable to generate a plant lacking a gene which is needed for the pathogen' s infection of the plant. Thus, according to one embodiment, the gene conferring sensitivity to a pathogen is knocked-out to thereby increase resistance to the pathogen.
According to a specific embodiment, the gene comprises an elF4E (translation initiation factor 4E) gene or a Mlo gene.
It is further advisable to generate a plant lacking a gene which inhibits the plant resistance pathway.
According to one embodiment, the gene inhibiting the resistance pathway comprises a transcription factor.
An exemplary gene includes the rice fatty-acid desaturase gene OsSSI2 (which was previously shown to act upstream of WRKY45 to negatively regulate WRKY45- dependent resistance, see Jiang et al. Molecular Plant-Microbe Interactions (2009) 22(7): 820-829). Another exemplary gene includes the rice NRR, a negative regulator of disease resistance (which was previously shown to interact with Arabidopsis NPR1 and rice NH1, see Chern et al. Plant J. 2005 Sep;43(5):623-35).
Alternatively, generating a pathogen resistant plant may be carried out by introducing into the plant the expression vector of some embodiments of the invention, wherein the nuclease mediates cleavage in a gene of a pathogen and enables an alteration in the cleavage site, thereby generating the pathogen resistant plant.
Since complete destruction of the DNA of the pathogen is desired, the nuclease (e.g. chimeric nuclease) is designed so as to cleave as much sequence sites on the pathogen's nucleic acid (DNA or RNA) 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 genome.
According to some embodiments of this aspect of the present invention, the nuclease (e.g chimeric nuclease) is designed to cleave the genome (DNA or RNA) of the pathogen but not that of the plant. To this end, the nuclease is designed devoid of a localization signal, such that the 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 (www(dot)ncbi(dot)nlm(dot)nih(dot)gov/blast/Blast(dot)cgi).
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 tomate 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 generating male sterility in a plant. The method comprising upregulating in the plant a structural or functional gene of a mitochondria or plastid (e.g. chloroplast) associated with male sterility by introducing into the plant the plant expression vector of some embodiments of the invention 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 plastid (e.g. chloroplast) when targeted into the genome of the mitochondria or plastid (e.g. chloroplast), wherein the nuclease, mediates cleavage in the genome of the mitochondria or plastid (e.g. chloroplast) and enables insertion of the heterologous nucleic acid sequence into the cleavage site, thereby generating male sterility in the plant.
Thus for example, the nucleic acid expression 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 nuclease (identical to that on the mitochondria or plastid e.g. chloroplast genome). Upon cleavage by the nuclease, the heterologous nucleic acid sequence is inserted into the predetermined site in the genome of the plastid (e.g. chloroplast) or mitochondria.
As mentioned hereinabove, cytoplasmic male sterility (CMS) is associated with mitochondrial dysfunction. To this effect, the 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 rpsl2, the Rice (Oryz sativa) sequence which is downstream of B-atp6 gene (i.e. orf79), the Maize T-urfl3 and orf221, the Helianthus sp. orf239 downstream to atpA, the Brassica sp. orfs which are upstream to atp6 (e.g. orfl39 orf224 or orfl38 and orfl58). 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 the plant expression vector of some embodiments of the invention, wherein the nuclease mediates cleavage in a gene conferring sensitivity to herbicides and enables an alteration in the cleavage site, 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 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).
Alternatively, herbicide resistance may be introduced into a plant by upregulating an expression of a protein (e.g. phosphinothricin acetyltransferase) which imparts resistance to an herbicide when expressed in the plant. Thus, a nucleic acid expression construct comprising a heterologous nucleic acid sequence (e.g. phosphinothricin acetyltransferase) is introduced into the plant for expression of the protein conferring herbicide resistance.
The present invention further envisages a method of generating a plant with increased abiotic stress tolerance. The method comprising introducing into the plant the plant expression vector of some embodiments of the invention, wherein the nuclease, mediates cleavage in a gene of the plant conferring sensitivity to abiotic stress and enables an alteration in the cleavage site, thereby generating the plant with increased abiotic stress tolerance.
The phrase "abiotic stress" as used herein refers to any adverse effect on metabolism, growth, reproduction and/or viability of a plant. Accordingly, abiotic stress can be induced by suboptimal environmental growth conditions such as, for example, salinity, osmotic stress, water deprivation, drought, flooding, freezing, low or high temperature, heavy metal toxicity, anaerobiosis, nutrient deficiency (e.g., nitrogen deficiency or limited nitrogen), atmospheric pollution or UV irradiation.
The phrase "abiotic stress tolerance" as used herein refers to the ability of a plant to endure an abiotic stress without suffering a substantial alteration in metabolism, growth, productivity and/or viability.
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". The term "consisting of means "including and limited to".
The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
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.
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, Maryland (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, CT (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, CA (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.
GENERAL MATERIALS AND EXPERIMENTAL PROCEDURES
Construction of Israeli MDMV infective clones
The MDMV isolate used herein was collected from maize in Israel. Total RNA was purified from MDMV infected maize leaves using Tri-reagent solution (Sigma). cDNA was reverse-transcribed with oligo-dT primer (primer no. 27, see Table 1). Full length cDNA was obtained using superscript® III Reverse (Invitrogene) with Reverse primer no. 28 (see Table 1). Two overlapping fragments of MDMV 1-5429 bp and 3661-polyA were amplified by RT-PCR utilizing Fusion Hot start II high-fidelity DNA polymerase (Finnzymes), amplification was done with primers 1 with 18 and 2 with 26 (see Table 1), respectively. Amplified cDNA was cloned into pJET or pGEM (Amp ), respectively, and grown in E. coli Xl-lBlue cells. To obtain 5' end cDNA sequences, dCTPs tailed first strand cDNA using TdT enzyme (Fermentas), then amplified using a 3' end MDMV primer (primer no. 4, see Table 1) and a 5' poly-dG adaptor (primer no. 41, see Table 1). The Israeli MDMV full-length sequence was constructed and fully sequenced using several MDMV primers (primers 1-25, as listed in Table 1 below, SEQ ID NO: 43).
Further on, to achieve virus transcription via plants, a synthetic DNA fragment was synthesized in order to fuse 35S promoter to 5' MDMV genome (pSAT-Synthetic block). The 1-5429 bp MDMV fragment was inserted downstream to the 35S promoter in the pSAT-Synthetic block plasmid (using Nrul and Banll). Creation of a full-length sequence MDMV under 35S promoter was accomplished by triple ligation of 35S promoter- 1-5429, 3661-polyA and pGreen (digested with Agel, AlwNi and Kpnl) (#3076) (as illustrated in Figures 2A-E). The Israeli MDMV contained all of the expected potyvirus genes. The 5' untranslated region (UTR) extended from 1-138 nt (SEQ ID NO: 44). Protease cleavage sites within the polyprotein were predicted based on comparisons with other potyvirus species. Based on sequence predictions of cleavage sites, the PI protease was encoded at nt 139-837 (SEQ ID NO: 45), HC-Pro at nt 838- 2217 (SEQ ID NO: 46), the P3 protease from nt 2218 to 3258 (SEQ ID NO: 47), 6K1 from nt 3259 to 3459 (SEQ ID NO: 48), CI from nt 3460 to 5373 (SEQ ID NO: 49), 6K2 from nt 5374 to 5532 (SEQ ID NO: 50), NIa-VPg from nt 5533 to 6099 (SEQ ID NO: 51), NIa-Pro from nt 6100 to 6825 (SEQ ID NO: 52), NIb-RNA replicase from nt 6826 to 8388 (SEQ ID NO: 53), and CP from nt 8389 to 9261 (SEQ ID NO: 54). The 3' UTR extended from 9262-9500 nt (SEQ ID NO: 55) and was followed by a poly(A) tail.
Next, two multiple cloning sites (MCS) were designed, each followed by an originally NIa protease cleavage site (IDVKHQA - SEQ ID NO: 42). These MCS sites enable to easily clone the genes of interest and the NIa protease cleavage site enables release of the protein from the viral polyprotein. The first MCS was designed in-between the Pl-HcPro viral genes, containing Agel, Apal and the NIa protease cleavage site (as illustrated in Figure 3). The second MCS was designed between the Nib-CP viral genes containing Nhel, Avrll restriction sites, NIa protease cleavage site and alteration of 2 aphid transmission motifs DAE and DAG to DTE and DTG, respectively (as illustrated in Figure 4). MDMV sequences containing these modifications were synthesized and ordered from Biomatik Company (#3091 and #3093, respectively).
DsRed, a 700 bp reporter gene, was amplified with no start or stop codon using primers listed in Table 1 below (primer nos. 29-32) and cloned into Biomatik plasmids MCS separately (#3092 and #3095). Then the DsRed was inserted into the native MDMV infective clone #3076 by Bbcl and Sphl or by Swal and Bsu36I to create MDMV-DsRed infective clones #3101 and #3096, respectively (as illustrated in Figure 5).
To develop a safe environmental viral clone, two aphid transmission motifs were mutated with the insertion of the 3'MCS (as mentioned above). To achieve full protection from aphid transmission another known aphid transmissible motif was changed in the MDMV clone, namely, KITC motif which appears in the beginning of the HcPro gene was changed by site directed mutagenesis into EITC. An aphid transmission assay was carried out as previously taught [Antignus et al., Phytoparasitica (1989) 17, 289-287].
Table 1: Primers used for MDMV cloning and sequencing
SEQ ID
Primer Name Primer Sequence
NO
MDMV-F1 AAAAACAACAAGACTCAACACAAC 1
MDMV-141F GGCAGGAACTTGGACTCACG 2
MDMV 327F GCTCAACCAAAGAAGTGTGC 3
MDMV-R350 TCTGTGCACACTTCTTTGG 4
MDMV 1095F DEG CAACAGRGTGARTATCTWGC 5
MDMV 1912F DEG TKAAYGARGATTCAGCYAARG 6
MDMV 2032F CCCAGAGATCAAAAACGCTGAGTTGC 7
MDMV 2416F CCAGGGAGTAGCAGCAATGT 8
MDMV 2870F AGTCGAAGTCAAGCCAGCTC 9
MDMV 3248R CTCCTGTCCCTGTCAGTTCG 10
MDMV 3298R DEG CATYGCTTGCTCYAGRTTRA 11
MDMV 3661F AAGCAGCATCTGTGAGCATTAG 12
MDMV 3739R TCTTGATCCGACTGCACCTC 13
MDMV 3960R TTGTCAGGGTTGTTTGCGTA 14
MDMV 4082-R GAACTCACATTCCCTTCCTGGTGGTGT 15
MDMV 4925R DEG CYARTTCAAGATCRCAKCCA 16
MDMV F5087 GCTTTRCCACGAACAATYGC 17
MDMV 5429R AGTCTCCGTTCCACCTTCCT 18
MDMV 5596R AGCGTATTTGTTGTCGCGTGCCTGCCT 19
MDMV 5950F CATGACTCCACACGAACCAT 20
MDMV 6395F DEG TYAARTTTCARGCACCYARTC 21
Potyvirus Degenerate NIB For (774 GGHAAYAAYAGYGGHCARCC 22
MDMV 7865R DEG ACACTTCGTCTGCYTYATCY 23
MDMV 8891-9110 F TGGACMATGATGGAYGGAGA 24
MDMV 9203-9184 R CAAGACCAAACAWCCGTGTG 25
3 RACE-R Kpnl AAAGGTACCGACTCGAGTCGACATCG 26
3'T (RACE) primer GACTCGAGTCGACATCGA (T)17 27
3 prim (RACE) GACTCGAGTCGACATCG 28
DsRed-F-Agel ATAACCGGTGCCTCCTCCGAGAACG 29
DsRed-R-Apal AGGGCCCCAGGAACAGGTGGTGGC 30
DsRed-F-Nhel AAAGCTAGCGCCTCCTCCGAGAACG 31
DsRed-R-Avrll CCTAGGCAGGAACAGGTGGTGGC 32
MDMV_secondDAG_change-R AGCCTGAGGTTCCTGTATCCACGTCTTT 33
NheI-Gus3F ATATGGCTAGCTTACGTCCTGTAGAAACCC 34
AvrII-Gusl806R ATCCTAGGTTTGCCTCCCTGCTG 35
AgeI-GUS-F4 ATATACCGGTTTACGTCCTGTAGAAACCC 36
Apa-GUS-1806R ATATGGGCCCTTTGCCTCCCTGCTG 37
Agel-I-Scel ATATACCGGTCCAAAAAAAAAAAGAAAAGTTG 38
Apa-I-Scel-R ATATGGGCCCTTTCAGGAAAGTTTCGGAG 39
Apa-I-SceI_TCtag-R ATATGGGCCCGCAGCATCCTGGGCAGCATTTCAGGAAAGTT 40
5' RACE GGCCACGCGTCGACTAGTACGGG1IGGGUGGG1IG 41
MDMV-784F CCGAGGAAGACTTAAAGGCG 70
MDMV-935R ATCGTAGGTGTGTGCTCTGT 71 Particle bombardment procedure - Infecting plants with MDMV clones
Gold microcarriers were prepared following the manufacturer instructions (Bio- Rad Laboratories, Richmond, CA, USA). Specifically, 15 milligrams of 1 μιη diameter gold particles were suspended in 0.5 ml of 100 % ethanol, sonicated for 15 seconds and then centrifuged at 3000 rpm for 60 seconds. The recovered particle pellet was washed twice before suspension in 0.5 ml of sterile distilled water. For each bombardment shot, 1-2 μg of MDMV construct, 8 μΐ of CaC12 (2.5 M), and 2.5 μΐ of spermidine (0.1 M) were added one by one into an aliquot of 8 μΐ of gold particle suspension. The mixture was vortexed for 10 min, and then centrifuged at 5,000 rpm for 12 seconds. The pellet was washed with 250 μΐ of absolute cold ethanol and was resuspended in 15 μΐ of absolute ethanol. Biolistic transformation was carried out using the PDS-1000/He Biolistic® Particle Delivery System (Bio-Rad Laboratories) (Figures 6A-B) by bombardment of pGreen-MDMV's plasmid into 2-3 leaf maize seedlings. Each bombardment was performed once at 1,100-1,350 psi helium pressure, 9 cm distance from the stopping plate to the target tissue, and with 28 mmHg vacuum pressure. Bombarded seedlings were transferred on to soil medium just after bombardment procedure (Figure 6C).
MDMV viral vector in maize tissue culture
Maize B73 explants used for bombardment were derived from the split 1st nodes of 1 week old germinated seedlings which were cultured for approximately 8 weeks to produce both organogenic structures (shoot and root) and embryogenic callus outgrowths. This material was used to successfully demonstrate the spread and intensity of Dsred expression in explants following particle bombardment with plasmid DNA of the MDMV expression #3101 vector clone (Figures 12A-D).
Mechanical Plant inoculation
Infectivity tests were performed by rubbing homogenates of virus-infected plants on maize seedlings. Using a sterile scalpel, a small piece of a fresh symptomatic plant leaf was excised and homogenized in a small amount of H20 using a sterile pestle and mortar to yield a finely ground virus suspension. The homogenate was rubbed immediately onto carborundum dusted fully expanded leaves using sterile cotton swaps. After mechanical inoculations the inoculated leaves were sprayed with water to remove inoculum and to reduce excessive evaporation. 3-5 leave seedlings were the most susceptible plant, hence inoculation experiments were done on seedlings at that stage.
Plants were scored for symptom development from 5 days to 2 weeks post- infection/bombardment. Infection of symptomatic plants was confirmed by RT-PCR using primers no. 70 and 71 (see Table 1, above). In some cases a serological test was performed using MDMV antibodies (CAB 18000, Agdia inc.). Mechanical inoculation infection rates by rubbing virus suspension were close to 100 %. Particle bombardment infection rates were 40-60 %. EXAMPLE 1
MDMV expression vector
A MDMV expression vector (IL type MDMV) was generated as indicated in the materials and experimental section above. After cloning the first native MDMV-IL clone (#3076), plasmid clone was tested using bombardment procedure. One week later typical MDMV symptoms were seen on the bombarded maize seedlings (Figure 7). Next, several sorghum cultivars were tested for MDMV-IL susceptibility. Young seedlings of SB 102, SB273 and Israeli cultivar SB 153 were brushed with virus suspension. One week later harsh MDMV leaf symptoms were detected on each sorghum cultivar (Figures 8A-B).
After substantiating the MDMV-IL infective ability, the first goal was to demonstrate that the MDMV expression vector can express a foreign gene. DsRed, a 700 bp reporter gene, was amplified and cloned into the MCS creating MDMV-DsRed infective clones #3101 and #3096 (Figure 5). Seven days post bombardment of the #3096 clone, infected plants were detected by viral symptoms on the first new growing leaf, DsRed was detected too. DsRed expression persisted for up to two weeks from initial expression in the bombarded leaves, (data not shown). Clone #3101 showed viral symptoms and DsRed expression in the new developing leaves, a week after bombardment procedure (Figures 9A-D). As well as month later, infected plants continued to express the DsRed marker gene until maturity (as compared to the #3096 clone). Another way to determine viral infectivity of an infected plant is by questioning its ability to infect a new healthy plant. A new infection of healthy plants was illustrated by rubbing #3101 infected plant's sap (Figures 9A-S). Another goal was to develop a safe environment viral clone. For this purpose, two aphid transmission motifs were mutated with the insertion of the 3'MCS, namely DAE and DAG were changed to DTE and DTG, respectively (motifs are at 8467 bp and 8622 bp from the beginning of the viral genome, see Figure 13 and SEQ ID NOs: 72- 73).
To achieve full protection from aphid transmission another aphid transmissible motif was changed in the MDMV clone. Specifically, a KITC motif which appears in the beginning of the HcPro gene was changed by site directed mutagenesis into EITC (motif present 1030 bp from the beginning of the MDMV genome, see Figure 14 and SEQ ID NOs: 74-75).
An aphid transmission assay demonstrated that the MDMV clone could not be transmitted by aphids (see Figures 10A-B).
Next, it was shown that a different reporter gene, i.e. GUS, can be expressed from the MDMV aphid non-transmissible viral vector. Specifically, GUS, a relatively big reporter gene (1800 bp), was inserted in the 5' MCS of the MDMV vector to create clone #3304 (see Figure 11 A). GUS expression by the MDMV vector was illustrated in several maize leaves (see Figures 11B-E). Expression of GUS was further illustrated in young, non-bombarded leaves, an indication that the virus is capable of replicating and moving in the plant regardless of its foreign insertion (GUS).
The MDMV viral vector of the invention was further shown to infect embryogenic and/ or meristematic callus, i.e. DsRed expression in maize B73 explants was illustrated from 18 to 25 days following bombardment (Figures 12A-D). It was further shown that the MDMV virus proliferates and spreads throughout the plant/callus and expresses a foreign reporter gene (Figures 9A-R, 11A-E and Figures 12A-D).
It was further shown that the MDMV viral vector of the invention can dually express two reporter genes. Specifically, a MDMV vector was designed in which GUS was inserted in the 5'MCS and DsRed was inserted in the 3'MCS (clone no #3182). The results illustrated that both reporter genes were expressed together in the same infected leaf (bombarded leaf, data not shown).
A MDMV expression vector (IL type MDMV) was constructed to include a sequence encoding a meganuclease, the meganuclease comprising I-Scel. The gene encoding the meganuclease was inserted in a MCS site, between the PI gene and the HCPro gene of the MDMV. The MDMV expression vector was constructed such that the meganuclease was directly followed by a NIa protease cleavage site. The meganuclease was selected to target a specific sequence in the plant genome for efficient gene editing. The results illustrated a base deletion in the gene (data not shown). EXAMPLE 2
Expression of foreign genes by MDMV expression vectors A MDMV expression vector (IL type MDMV) is constructed to include a sequence encoding a meganuclease. The gene encoding the meganuclease is inserted in one of the MCS sites, e.g. between the NIb-RdRp gene and the CP gene of the MDMV or between the PI gene and the HCPro gene of the MDMV. The MDMV expression vector is constructed such that the meganuclease is directly followed by a NIa protease cleavage site. The meganuclease is selected to target a specific sequence in the plant genome for efficient gene editing.
A MDMV expression vector (IL type MDMV) is constructed to include a sequence encoding a zinc finger nuclease (ZFN). The gene encoding the ZFN is inserted in one of the MCS sites, e.g. between the NIb-RdRp gene and the CP gene of the MDMV or between the PI gene and the HCPro gene of the MDMV. The MDMV expression vector is constructed such that the ZFN is directly followed by a NIa protease cleavage site. The ZFN is selected to target a specific sequence in the plant genome for efficient gene editing.
A MDMV expression vector (IL type MDMV) is constructed to include a sequence encoding a Cas9. The gene encoding the Cas9 is inserted in one of the MCS sites, e.g. between the NIb-RdRp gene and the CP gene of the MDMV or between the PI gene and the HCPro gene of the MDMV. The MDMV expression vector is constructed such that the Cas9 is directly followed by a NIa protease cleavage site. An additional vector is used for expression of CRISPR for specific gene targeting of the CRISPR/Cas9 system.
A MDMV expression vector (IL type MDMV) is constructed to include a sequence encoding transcription activator-like effector nucleases (TALENs) or compact TALENs (c TALENs). The gene encoding the TALENs or cTALENs is inserted in one of the MCS sites, e.g. between the NIb-RdRp gene and the CP gene of the MDMV or between the PI gene and the HCPro gene of the MDMV. The MDMV expression vector is constructed such that the TALENs or the cTALENs is directly followed by a NIa protease cleavage site. The TALENs or cTALENs is selected to target a specific sequence in the plant genome for efficient gene editing.
Co-expression of two foreign genes by one MDMV expression vector is carried out by construction of a vector including two foreign sequences successive to each other (e.g. two ZFNs, two TALENS, two meganucleases, etc.), each gene being followed by a NIa protease cleavage site. Alternatively, the foreign genes are placed in each of the MCSs each being followed by a NIa protease cleavage site.
All MDMV expression vectors are constructed with reduced aphid transmission capabilities. Specifically, to inhibit aphid transmission of the MDMV vectors, alteration of 2 aphid transmission motifs are carried out, i.e. DAE and DAG to DTE and DTG, respectively, at the N terminal region of the coat protein (CP). To achieve full protection from aphid transmission another aphid transmissible motif is changed in the MDMV vector, namely, an amino acid alteration comprising a KITC to EITC substitution in a helper component proteinase (HCPro).
Inoculation of different seedlings (e.g. maize, sorghum) with the different MDMV expression vectors as described above is expected to lead to a high expression level of the foreign genes in different parts of these plants (e.g. leaves, gametes etc).
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 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

WHAT IS CLAIMED IS:
1. A plant expression vector comprising a nucleic acid sequence encoding a polyprotein product, said polyprotein product comprising Maize Dwarf Mosaic Virus (MDMV) polypeptides and a heterologous polypeptide of interest, said heterologous polypeptide of interest being cleaved of said MDMV polypeptides upon expression in a plant cell, and wherein said plant expression vector is capable of spreading in a plant.
2. The plant expression vector of claim 1, being a satellite vector.
3. A plant expression vector comprising a nucleic acid sequence encoding a polyprotein product, said polyprotein product comprising Maize Dwarf Mosaic Virus (MDMV) polypeptides, wherein said MDMV polypeptides comprise a PI polypeptide and a coat protein polypeptide, and a heterologous polypeptide of interest, said heterologous polypeptide of interest being cleaved of said MDMV polypeptides upon expression in a plant cell infected with a helper virus or an MDMV virus, and wherein said plant expression vector is capable of spreading in a plant.
4. A plant expression vector system comprising:
(i) the plant expression vector of claim 2 or 3; and
(ii) an MDMV helper virus.
5. The plant expression vector of claim 1, 2 or 3, or vector system of claim 4, wherein said plant expression vector is non-transmittable by aphids.
6. The plant expression vector or vector system of claim 5, wherein said vector comprises an amino acid alteration which renders said vector non-transmittable by said aphids.
7. The plant expression vector of claim 1, 2 or 3, or vector system of claim 4, wherein said vector comprises an amino acid alteration comprising a DAE to DTE substitution in a N terminal region of a Coat Protein (CP).
8. The plant expression vector of claim 1, 2 or 3, or vector system of claim 4, wherein said vector comprises an amino acid alteration comprising a DAG to DTG substitution in a N terminal region of a coat protein (CP).
9. The plant expression vector of claim 1 or 2, or vector system of claim 4, wherein said vector comprises an amino acid alteration comprising a KITC to EITC substitution in a helper component proteinase (HCPro).
10. The plant expression vector of claim 1, wherein said nucleic acid sequence encoding said polyprotein product encodes the full set of proteins of said MDMV.
11. The plant expression vector or vector system of any one of claims 1-10, wherein said heterologous polypeptide of interest is translationally fused N terminally to said MDMV polypeptides in said polyprotein product.
12. The plant expression vector or vector system of any one of claims 1-10, wherein said heterologous polypeptide of interest is translationally fused C terminally to said MDMV polypeptides in said polyprotein product.
13. The plant expression vector or vector system of any one of claims 1-10, wherein said heterologous polypeptide of interest is flanked by said MDMV polypeptides.
14. The plant expression vector of any one of claims 1, 5-10, wherein said heterologous polypeptide of interest is flanked by a PI polypeptide and a HCPro polypeptide of said MDMV, wherein said PI polypeptide is N-terminally positioned to said heterologous polypeptide of interest and said HCPro polypeptide is C-terminally to said heterologous polypeptide of interest.
15. The plant expression vector of any one of claims 1, 5-10, wherein said heterologous polypeptide of interest is flanked by a NIb-RNA replicase polypeptide and a CP polypeptide of said MDMV, wherein said NIb-RNA replicase polypeptide is N- terminally positioned to said heterologous polypeptide of interest and said CP polypeptide is C-terminally to said heterologous polypeptide of interest.
16. The plant expression vector or vector system of any one of claims 3-9, wherein said heterologous polypeptide of interest is flanked by said PI polypeptide and said coat protein polypeptide of said MDMV.
17. The plant expression vector or vector system of claim 16, wherein said PI polypeptide is N-terminally positioned to said heterologous polypeptide of interest and said coat protein polypeptide is C-terminally to said heterologous polypeptide of interest.
18. The plant expression vector or vector system of any one of claims 1-17, wherein said heterologous polypeptide of interest is directly translationally fused to at least one protease cleavage site.
19. The plant expression vector or vector system of claim 18, wherein said protease cleavage site comprises a NIa protease cleavage site as set forth in SEQ ID NO: 42.
20. The plant expression vector or vector system of any one of claims 1-19, wherein cleavage of said heterologous polypeptide of interest of said MDMV polypeptides upon expression in a plant cell is effected by a viral protease.
21. The plant expression vector or vector system of claim 20, wherein said viral protease comprises a NIa protease.
22. The plant expression vector or vector system of any one of claims 1-21, wherein the vector further comprises at least one heterologous promoter sequence for directing expression of said polyprotein in said plant cell.
23. The plant expression vector or vector system of claim 22, wherein said heterologous promoter sequence comprises a 35S promoter.
24. The plant expression vector or vector system of claim 22, wherein said at least one heterologous promoter sequence comprises two heterologous promoter sequences.
25. The plant expression vector or vector system of any one of claims 1-24, wherein said plant expression vector comprises a pGreen backbone.
26. The plant expression vector or system of any one of claims 1-25, wherein said heterologous polypeptide of interest is selected from the group consisting of 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, a growth inhibiting polypeptide, an enzyme, a transcription factor and a transposase.
27. The plant expression vector of any one of claims 1-3, or vector system of claim 4, wherein said nucleic acid sequence encoding said heterologous polypeptide of interest encodes for two heterologous polypeptides of interest.
28. The plant expression vector of claim 1, wherein said nucleic acid sequence encoding said polyprotein product comprises a full genome sequence of said MDMV.
29. The plant expression vector or vector system of any one of claims 1-25, wherein said heterologous polypeptide of interest comprises a nuclease.
30. The plant expression vector or vector system of claim 29, wherein said nuclease is selected from the group consisting of a meganuclease, a Cas and a RISC.
31. The plant expression vector or vector system of claim 30, wherein said Cas comprises Cas9.
32. The plant expression vector or vector system of claim 29, wherein said nuclease comprises a chimeric nuclease.
33. The plant expression vector or vector system of claim 32, wherein said chimeric nuclease comprises a nucleic acid binding domain and a nuclease.
34. The plant expression vector or vector system of claim 32, wherein said chimeric nuclease is selected from the group consisting of a zinc finger nuclease (ZFN), a TALENs and a compact-TALENs.
35. The plant expression vector or vector system of claim 29, 32 or 33, wherein said nuclease is selected from the group consisting of a restriction enzyme, a topoisomerase, a recombinase, an integrase, a homing endonucleases and a DNAse.
36. The plant expression vector or vector system of claim 33, wherein said nucleic acid binding domain of said chimeric nuclease is selected from the group consisting of a meganuclease binding domain, a helix-turn-helix binding domain, a leucine zipper (ZIP) binding domain, a winged helix (WH) binding domain, a winged helix turn helix domain (wHTH) binding domain, a helix-loop-helix binding domain, a transcription activator-like (TAL) binding domain, a recombinase, and a zinc finger binding domain.
37. The plant expression vector or vector system of any one of claims 29-33, wherein said nuclease is attached to a localization signal to a DNA-containing organelle.
38. The plant expression vector or vector system of claim 37, wherein said DNA-containing organelle is selected from the group consisting of a nucleus, a plastid and a mitochondria.
39. A plant cell comprising the plant expression vector or vector system of any one of claims 1-38.
40. A plant comprising the plant expression vector or vector system of any one of claims 1-38.
41. A method of generating a plant, the method comprising introducing into one or more cells of the plant the plant expression vector or vector system of any one of claims 1-38.
42. A method of transiently expressing a heterologous polypeptide of interest in a plant, the method comprising introducing into at least one cell of the plant the plant expression vector or vector system of any one of claims 1-38, thereby transiently expressing the heterologous polypeptide of interest in the plant.
43. A method of generating genotypic variation in a genome of a plant, the method comprising introducing into the plant the plant expression vector or vector system of any one of claims 29-38, wherein said nuclease mediates cleavage in a genome of the plant and enables an alteration in the cleavage site, thereby generating genotypic variation in the genome of the plant.
44. A method of tagging a genome of a plant, the method comprising introducing into the plant the plant expression vector or vector system of any one of claims 29-38, wherein said nuclease mediates cleavage in a genome of the plant and enables an alteration in the cleavage site, thereby tagging the genome of the plant.
45. A method of generating a herbicide resistant plant, the method comprising introducing into the plant the plant expression vector or vector system of any one of claims 29-38, wherein said nuclease mediates cleavage in a gene conferring sensitivity to herbicides and enables an alteration in the cleavage site, thereby generating the herbicide resistant plant.
46. A method of generating a pathogen resistant plant, the method comprising introducing into the plant the plant expression vector or vector system of any one of claims 29-38, wherein said nuclease mediates cleavage in a gene conferring sensitivity to a pathogen or in a gene inhibiting the resistance pathway and enables an alteration in the cleavage site, thereby generating the pathogen resistant plant.
47. The method of claim 46, wherein said gene conferring sensitivity to a pathogen or said gene inhibiting the resistance pathway is knocked-out to thereby increase resistance to said pathogen.
48. The method of claim 47, wherein said gene conferring sensitivity to a pathogen comprises an elF4E (translation initiation factor 4E) gene or a Mlo gene.
49. The method of claim 47, wherein said gene inhibiting the resistance pathway comprises a transcription factor.
50. The method of claim 47, wherein said gene inhibiting the resistance pathway comprises a rice OsSSI2 gene or a rice NRR gene.
51. A method of generating a plant with increased abiotic stress tolerance, the method comprising introducing into the plant the plant expression vector or vector system of any one of claims 29-38, wherein said nuclease mediates cleavage in a gene of the plant conferring sensitivity to abiotic stress and enables an alteration in the cleavage site, thereby generating the plant with increased abiotic stress tolerance.
52. The method of any one of claims 43, 44, 45, 46 or 51, wherein said alteration in said cleavage site comprises an amino acid mutation, insertion or deletion.
53. 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 plastid associated with male sterility by introducing into the plant the plant expression vector or vector system of any one of claims 29-38 and a nucleic acid expression construct which comprises at least one heterologous nucleic acid sequence which can upregulate said structural or functional gene of a mitochondria or plastid when targeted into a genome of said mitochondria or plastid, wherein said nuclease mediates cleavage in a genome of the mitochondria or plastid and enables insertion of said heterologous nucleic acid sequence into the cleavage site, thereby generating male sterility in the plant.
54. The plant expression vector or vector system of claim 38, or method of claim 53, wherein said plastid comprises a chloroplast.
55. The method of any one of claims 43, 44, 45, 46, 51 or 54, wherein said plant expression vector further comprises a chloroplast localization signal.
56. The method of claim 55, wherein said chloroplast localization signal comprises a ribulose-l,5-bisphospate carboxylase small subunit (Rssu) (SEQ ID NOs: 76 or 77).
57. The method of any one of claims 43, 44, 45, 46, 51 or 53, wherein said plant expression vector further comprises a mitochondria localization signal.
58. The method of claim 57, wherein said mitochondria localization signal comprises an ATPase beta subunit (ΑΤΡ-β) (SEQ ID NO: 78).
59. The plant expression vector of any one of claims 1-3, plant cell of claim 39, plant of claim 40, or method of any one of claims 43, 44, 45, 46, 51 or 53, wherein the plant is a monocot.
60. The plant expression vector, plant cell, plant, or method of claim 59, wherein the monocot plant is selected from the group consisting of maize, rice, wheat, barley, sugar cane, sorghum, Johnson grass, grasses, bamboo, palm, agave, pineapple, banana, ginger, garlic, onion, oat, rye, turf grass, millet, spelt, triticale, fonio, aloe, asparagus, yam or ubi, orchid, iris, lily, amaryllis, canna-lily arum or gabi, lemon grass, pandan or screwpine, arrow root, rush, pipewort and sedge.
61. The plant expression vector of any one of claims 1-3, 20 or 22, plant cell of claims 39 or 60, or method of claim 41 or 42, wherein the cell is selected from the group consisting of a meristem cell, a leaf cell, a male inflorescence cell, a pollen cell, a female inflorescence cell, an ovule cell and a cell of first node derived calli.
62. The method of any one of claims 41, 42, 43, 44, 45, 46, 51 or 53, wherein said introducing is effected by particle bombardment, agroinfection or sap mechanical infection.
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CN110747200A (en) * 2019-11-12 2020-02-04 成都大学 Nucleotide sequence, primer and method for inhibiting candida albicans protein by quinoa
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CN111206038B (en) * 2020-03-16 2021-12-07 南京林业大学 Bambusa multiplex transcription factor BmbZIP62 gene and application thereof
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