WO2017165724A1 - Introducing dna into organisms for transient expression - Google Patents

Abstract

A method for introducing single-stranded nucleic acid into an intact plant cell leading to vastly improved speed and efficiency of DNA uptake and expression; wherein, said nucleic acid is based upon flexible, single-strand DNA which rapidly penetrates intact plant cells and is expressed in the plant cells at comparatively higher levels; wherein said single-stranded DNA is attached to a short piece of primer DNA; and, wherein said nucleic acid is an expression vector based on a Geminivirus.

Description

INTRODUCING DNA INTO ORGANISMS FOR TRANSIENT EXPRESSION

CROSS REFERENCE TO RELATED APPLICATIONS:

[0001] This utility patent application claims priority of a US Provisional Patent Application Serial Nr. 62/390,282 filed on March 24, 2016 which is incorporated in its entirety as a reference.

FIELD OF INVENTION

[0002] The present invention relates to the field of molecular transformation of cells and organisms, in particular to means and methods of efficiently introducing exogenous naked Nucleic acids into living cells or organisms, and the expression of said nucleic acids bearing useful traits.

BACKGROUND OF THE INVENTION

[0003] Organisms have evolved efficient methods of excluding alien molecules from penetrating into their bodies, and when such alien molecules are introduced, organisms have mechanisms that rid themselves of the effects of such molecules. With animals this includes various epithelia as the first line of defense, then the immune system, liver and kidneys as parts of secondary defenses.

[0004] Transfection of animal cells typically involves opening transient pores or "holes" in the cell membrane to allow the uptake of material.

[0005] Similarly, plants have cuticles, bark, seed coats, root-endodermal Casparian strips, cell wall as their first lines of defense, as well a vast array of enzymes (many similar or identical to those in the liver) as a second line of defense. This is evident with small molecules as well large ones. For example, most contact fungicides applied to a leaf surface penetrate and are toxic to pathogens, but do not penetrate the plant cuticles. When applied to isolated cells from the same plant species, these same small molecules are toxic. Herbicides typically penetrate plant cuticles; in crops where they are selective (kill only weeds and not the crop), most herbicides are detoxified by enzymes present in the crop and not the weed. Efforts were made to rationalize the design ways to render potentially toxic small molecules (i.e. toxic if they penetrate the cuticle) and scientists found that a particular range of balance between hydrophilic and lipophilic groups on the molecules facilitated penetration. The only problem with this concept is that two widely used herbicides, paraquat and glyphosate, which easily penetrate leaves are almost totally hydrophilic, demonstrating that there is a large modicum of unpredictability when it comes to discovering and designing small molecules that penetrate the cuticle.

[0006] There is also a large modicum of unpredictability when it comes to discovering and designing large molecules such as DNA that penetrate the cuticle. Carpita et al. (1979) determined that the pore size in cells walls of a cell suspension culture of Acer pseudoplatanus is 3.8 to 4nm. Baron Epel et al. (1988), showed a functional range of diameters for putative trans-wall channels of 6.6-8.6 nm. On the other hand, biophysical studies on the naturally supercoiled state of common bacterial plasmid such as pUC19 - 2.7kb - have determined its radius of gyration to be 65.6 nm and its Stokes radius to be 43.6 nm. Therefore the ability to introduce large DNA fragments or plasmids into intact plant cell seems to be almost impossible. . Half a century, ago it was claimed that one could get radiolabeled naked bacterial DNA into plants by applying it to plant surfaces (see Stroun et al., 1967 and references cited therein). The proofs that the exogenous DNA remained intact and replicated once inside the plants were minimal. The purported finding of exogenous bacterial DNA uptake, spread and replication had no utility as they could find no evidence of expression of any bacterial genes in the plants. It took over a decade to find ways to get exogenous DNA to enter, and be expressed in plants. This was first achieved by inserting the gene of choice into the plasmid of Agrobacterium, infecting the plants, and after the T-DNA incorporates the DNA into nuclear chromosomes, curing the plants from the infection (Windels et al 2008). In this way the delivery of the desired exogenous DNA into the nucleus of a plant cell was overcome by harnessing the agrobacteria natural system.

[0007] Gahan and co-workers reported in 2003 that they managed to transform Solanum aviculare by delivering naked DNA carrying either GUS, NPTII and BAR into cut shoots and the regenerated tissue part permit those reporter gene expression demonstrating their integration to the plant genome.

[0008] There are various methods of introducing foreign DNA into eukaryotic cells: some rely on physical treatment (electroporation, cell squeezing, nanoparticles, magnetofection), other on chemical materials (using calcium phosphate, dendrimers, cationic polymers or by mixing a cationic lipid - such as Lipofectamin - with the material to produce liposomes, which fuse with the cell membrane and deposit their cargo inside.) or biological particles (viruses) that are used as carriers.

[0009] A number of transfection methods, initially developed for mammalian cell lines have been adapted for insect cells but the conditions required for optimal transfection efficiency can vary greatly between vertebrate and invertebrate cell lines. Presently there are several transfection methods and techniques in use with lepidopteran cell lines (Trotter and Wood 1995; Keith et al. 2000; Gundersen-Rindal et al. 2001): calcium phosphate precipitation method, liposome-mediated transfection, electroporation, and polyethylenimine-mediated transfection.

[0010] Similarly, DNA bearing useful traits could be introduced into plants using avirulent viruses (including Gemini viruses) as vectors (e.g. Palmer, 1997, Lomonossoff and Montague 2008, Gleba et al. 2008, Rybicki and Martin 2011), still viral infections were more effective when the cuticular surfaces are abraded. Unlike the viral system, Agrobacterium transformation can reach generative cells and be inherited to subsequent generations, which in many instances is desirable. Conversely, viral transformations are "transient" for single generations, as the viruses do not reach the generative cells. This has the commercial advantage of continued resale, and the biological advantage in that there can be no transgene flow to other plants.

[0011] Subsequently, it was found that one could achieve expression of exogenous naked dsDNA (double stranded DNA) in intact plant tissues, but only when physical force is used to penetrate the cuticle (using biolistic transformation - the naked DNA on microbeads is propelled through the cuticle, electroporation used to cavitate cells and facilitate DNA entry, or put DNA on sharp glass fiber or silicon carbide "whiskers" with the same effect). Similarly, dsDNA can be made to penetrate into cell wall-free protoplasts where it is often incorporated into the nucleus, replicated and expressed in parent and daughter cells after cell walls are re-formed, and in regenerated plants originating from these protoplasts. Intact isolated cells are not easy to transform; the DNA can remain in the space between the cell wall and the plasma membrane (Wu and Cahoon 1995). Naked dsDNA could also be made to enter through cut surfaces of leaves, roots and stems as well as isolated embryos, where it was at least transiently expressed. Topfer et al. 1989, Yoo and Jung 1995, and Mahalakshmi et al. 2000, reported transfection and expression of naked dsDNA, into zygotic embryos. They used membrane permeabilization agents such as DMSO, toluene, saponine to allow or improve transfection. These zygotic embryos were extracted from mature seeds and therefore were wounded tissue and not intact. . It was surprising that such large dsDNA macromolecule could pass through cell wall and plasma membranes. It was proposed that was rendered possible because it occurred during imbibitions of dry tissue to imbibed tissue, possibly through the wounds made in embryo preparation and affected by the permeabilization agent. Senaratna et al 1991 showed dsDNA transfection and expression into dried somatic embryo without help of membrane permeabilization agent. However they showed that pre-imbibition of the somatic embryo prevented the dsDNA uptake, suggesting the non-integrity and relative porous nature of cell wall/plasma membrane in dry embryos, allowing entry of dsDNA upon imbibition.

[0012] Recently Cell-penetrating peptides (CPPs), also known as protein transduction domains (PTDs) have been extensively investigated in various mammalian cells. They are short polypeptides (5-30 amino acids) that can transit across the phospholipid bilayer of living cells. Most common CPPs, such as TAT, p VEC, R9 and Penetratin are characterized by a high number of basic residues, specifically arginine and lysine. It is the cationic nature of these residues which allows CPPs to form complexes (often called polyplexes) with anionic cargoes like DNA. This cationic character also seems to contribute to the cell membrane penetration ability of these peptides through electrostatic interaction with anionic phospholipid head groups. [0013] Optimized transfection systems for introduction of expression cassettes into cultured insect cells have become increasingly important for generation of stably transformed cell lines and investigating and improving transient heterologous protein expression using in vitro systems.

[0014] More recently, as reviewed by Ziemienowicz et al. (2015) and patent application No. US 20110035836 Al, cell-penetrating peptides (CPPs), have been used in plant systems. Many CPPs bind and deliver macromolecular cargoes such as DNA, RNA and protein into living cells. While their use is well established in mammalian cell systems, they have also been explored in the last decade as transfection agents in plant cells. Their efficacy has been demonstrated in both monocots and dicots as well as a variety of tissues and cell cultures, from roots and leaves to protoplasts. For instance, Qi et al 2011, the demonstrated penetration of labeled CPP or complex [GFP-fused-CPP] into intact roots of Barley and Arabidopsis. Factors affecting CPP and CPP-cargo uptake have been addressed with specific attention to the plant cell wall and classes of CPPs utilized in plant cell systems. It has been shown that internalization of macromolecular cargo complexes and conjugates were translocated in plant cells via macropinocytosis.

[0015] Ziemienowicz et al. (2012) used CPP to transfect with ssDNA into triticale microspores, for trying to mimic Agrobacterium transfection, by delivering ssT-DNA- derived nano-complex) .. The "ssDNA" was produced by converting dsDNA into the single-stranded form (ssT-DNA) through heat denaturation by incubation for 10 min at 95°C followed by fast chilling on ice.

[0016] In this case, no transfection was observed without CPP (ie naked "ssDNA"). In addition, transfections were performed with Lipofectamine, a well-known cationic lipid transfectant agent. In transient expression, they observed that "CPP-naked ssDNA" complex provided lower GUS-positive plants (compared with treatment" "CPP- ssDNA+proteins" or "CPP+dsDNA" complexes). The authors explained this by "most likely because of the lack or incomplete protection of DNA from nucleases. Among naked DNA molecules, dsDNA was better protected from nucleolytic degradation than ssDNA. This is not surprising because many plant nucleases show higher specificity for ssDNA (Rangarajan and Shankar, 2001) (Ziemenowicz et al. 2012)".

[0017] Sun et al (2005) described uptake and biological effect of oligo ssDNA (ODN) into barley leaves. The DNA fragment was very short (18mer); and as author mentioned that "usually, naked ODNs are poorly taken up by cells".

[0018] Endocytosis of DNA into isolated plants tissues has been also implicated by Paungfoo-Lonhienne et (2010a and b), where they showed that short ssDNA of 25nt or entire bacteria enter cells of cut roots and are degraded.

[0019] Along the years several attempts have been made to get single-stranded DNA (ssDNA) to enter cells (Ohyma 1978, Holmes et al 1999), and not just for plants. Much work has been presented to knockout genes by transfecting cells with very short antisense ssDNA: linear 18-20 oligonucleotides or circular 60 oligonucleotides , usually delivered with transfecting agent (Moon et al. 2000). For example Holmes et al. 1999, showed that ssDNA fragments are more effective than dsDNA fragments for nuclear delivery into human cells. They studied only the uptake and not the expression, while using heat denaturation process which is not necessarily generate a solution favored of single-stranded form. In addition, the transfection was performed with help of the transfecting agent (Lipofectamine, cationic lipid), and no intranuclear/intracellular fragment was observed with naked ssDNA (which again, in this work produced by heat denaturation). Although the ssDNA was a relatively short 500bp fragment; authors proposed that failure for uptake of naked DNA is due to degradation after uptake or to its size, as it has been reported that intracellular uptake of oligo ssDNA is inversely proportional to the length of the oligonucleotide: oligo(dT)3 >oligo(dT)7 > oligo(dT)15 > oligo(dT)20 (Loke et al, 1989).

[0020] Lee et al 2005 reported transfection of human cells with circular ssDNA of several kb, produced by phage. Their ssDNA sequence is in antisense orientation of gene of interest; and the authors showed effective gene knockdown in transfected cells. For transfection, their constructs was not naked ssDNA but previously mixed with Lipofectamine. Production ssDNA-

[0021] There are several ways to produce ssDNA, such as heating, alkaline denaturation, Ml 3 based phage, enzymatic (Exonuclease; Phi29 DNA polymerase) and synthetic.

[0022] Thermal denaturation can produce both circular and linear DNA; and it is commonly used for further studies (including transfection experiments). However, this method does not guaranty pure ssDNA: on one hand, DNA can be only partially ssDNA (ie in some regions, strands can remain annealed as dsDNA) and on the other hand, heating can damage the DNA integrity, as studied by atomic force microscope (AFM) by Yan and Iwasaki. 2002.

[0023] Alkaline denaturation can produce both circular and linear DNA. Visualization by AFM studied by Yu et al 2008, showed intact circular ssDNA, with stable conformation with unregistered, topologically constrained double strands and intra-strand secondary structure.

[0024] Preparation of ssDNA from phagemids is a well-established technique. It can be laborious, costly and inefficient, but some protocols can improve the process, useful for large-scale preparation of high quality ssDNA (Jupin and Gronenborn 1995; Zhou et al 2009). However, only circular ssDNA can be produced this way.

[0025] As described by Zhang et al 2010, circular ssDNA can be produced enzymatically, by treating circular dsDNA with nicking enzyme followed by Exonuclease III (ExoIII) reaction. Linear ssDNA can also be produced by ExoIII, after treatment with a pair of restriction enzymes: one which generates blunt or 5 'overhanging end and another one which produces an ExoIII resistant 4base 3 'overhang

[0026] Phi29DNA polymerase generates ssDNA concatamer on circular DNA template, and is used for rolling circle amplification (RCA) (Dean et al 2001). Gu and Breaker 2013 used this enzyme on DNA template carrying self-cleaving deoxyribozyme sequence, which allows production of linear ssDNA. These linear ssDNA fragments can eventually be circularized in vitro using ssDNA ligase (like CircLigase, Epicentre). Finally, ssDNA can be synthetized to purity, but the fragments cannot be longer than several hundred of nucleotides. As above, if needed, these linear ssDNA fragments can eventually be circularized in vitro using ssDNA ligase.

[0027] RNA transcription is the first step of gene expression, in which RNA (mRNA) is produced form dsDNA template. Therefore, ssDNA reaching nuclei - such as natural agrobacterium T-DNA or ssDNA viruses - has to be converted into dsDNA for transcription (Liang& Tzfira 2013). This complementary strand replication (CSR) is initiated by RNA/DNA short oligonucleotides (primers) provided by the virus (like the plant Mastreviruses) and/or by the nuclei (Gutierrez, 1999).

[0028] A novel viral base expression system was published on 2007 (Peretz et al 2007). The standard Gemini virus transformation system was improved by using only a portion of the virus to insert exogenous dsDNA into plants (WO 2007/141790), modified coat protein and V2 gene and/or dysfunctional replicase gene. With this they were able to obtain expression of the polycistronic four gene operon required to synthesize the anti-microbial compound pyrrilonitrin, as well as marker genes (Mozes-Koch et al. 2012). They used direct injection to achieve transient expression of these traits. They later demonstrated that the same and similar dsDNA constructs could be taken up through cut roots or grafting and expressed (WO 2010/004561). It was surprising that they later claimed that the same dsDNA constructs could be introduced into plants by seed priming (soaking) while adding the dsDNA into the priming/imbibing solution. This super-coiled dsDNA had to pass through the seed coat and be incorporated into the embryos of the imbibing seeds. . We had been using the technologies described in the above two patent applications (as licensees) and were not satisfied with the inconsistency of results, and the very large amounts of dsDNA required to achieve results. We realized that we must vastly improve DNA uptake and expression. The invention below describes that single-strand DNA form (ssDNA) rapidly penetrates intact cells reaching the nucleus and can be introduced into seed priming solutions (with or without DNase inhibitors) and is expressed in the plant tissues at higher levels using far lower amounts than described in the above two patent applications. We discovered that these much lower amounts of ssDNA are massively expressed when a short piece of primer is attached to the ssDNA, as is also demonstrated in the examples.

[0029] Bipartite begomovirus, such as Bean dwarf mosaic virus (BDMV), are spreading from cell to cell inside the plant via the plasmodesmata with the help of the viral proteins: the Movement Proteins (MP, BC or BL) and the Nuclear shuttle proteins (NSP, BV or BR). These 2 types of proteins are carried by the component B of the bipartite begomoviruses. Two models have been proposed for their action: the "relay-race and the "couple-skating" model (Hehn\e,et al 2004). In the relay-race model, the NSP export the viral DNA from the nucleus to the cytoplasm, where it is displaced by the MP which translocate the viral DNA to the plasmodesmata for its passage to the next cell. In the couple- skating model, the NSP exports the viral DNA from the nucleus to the cytoplasm, where the MP associate, and all the complex translocate and move through the plasmodesmata. Depending on the virus, both models could be true.

[0030] More recently, it has been shown that the MP-NSP-DNA complex is also associated with a histone for movement (Zhou et al. 2011).

[0031] Ye et al. (2015) suggested that NSP indirectly suppresses PTGS (Post Transcriptional Gene Silencing). But no such property has been shown for the bean dwarf mosaic virus MP.

SUMMARY OF THE INVENTION

[0032] According to one aspect of the invention, there is provided a method to introduce nucleic acid into intact cell, in the form of single stranded nucleic acid, linear or circular. Particularly, the introduced nucleic acid is a Geminivirus based expression vector.

[0033] Particularly, this method allows very fast and efficient introduction of a nucleic acid vector into the nucleus of an intact cell, , in the form of ssDNA plasmid. For efficient transcription followed by expression the addition of short complementary nucleic acid (primer) to the single strand form is preferable, either DNA or RNA. Furthermore, this primer is modified in order to provide higher stability and transcription. [0034] Nucleic acid can also be single strand RNA.

[0035] Furthermore the nucleic acid can be partially double strand, where the double strand section may serve as primer to complement of the introduced partial single strand to full double strand as required for expression.

[0036] In addition, the transfection can be performed on intact tissues, such as intact seeds, intact roots, and intact leaves. The material can be also delivered by foliar application, grafting and injection.

[0037] Plants, from mosses, ferns, gymnosperms to angiosperms, including monocotyledons and dicotyledons, including commodity crops, can be transfected by this method.

[0038] Eukaryotic cells, from insect to mammalian, including human, can be transfected by this method.

[0039] The disclosed Geminivirus based expression vector, is composed of geminivirus intergenic region (IR), and lacking Replication associated protein (Rep) gene and, is either lacking coat protein (CP) or comprise a non-modified CP. The vector includes, in same vector or in another helper vector construct, also the BDMV MP as expression enhancer. Any other element known to increase and stabilize expression - such has suppressor of silencing, promoter, transcription regulatory elements - are also provided in same vector or in separate helper vector construct.

[0040] This vector does not induce symptoms nor development disorder. It is not transmitted in subsequent generation nor present in pollen. In addition, it is not transmissible by insect.

[0041] This method and vector are used for gene overexpression, as well as downregulation. It can also be used for gene editing (such as, but not limited to ZFN (Zinc Finger Nucleases), TALEN (Transcriptional Activator-Like Effector Nucleases), CRISPR/Cas9). It can also be used to introduce nucleic marker into the crop variety.

[0042] As described further, this method and vector can be used to provide beneficial traits to crops, such as biotic and abiotic resistance and yield improvement. In addition the method and vector are used for functional genomics purpose in plant. [0043] This method and vector can be used also for Molecular farming, for producing molecule of interest.

[0044] This method can also be used to introduce any plant RNA and DNA virus and viroid vector.

[0045] This method, associated with appropriate vector, is also useful for functional genomics and gene therapy in Mammalian including in humans.

[0046] According to one aspect of the invention there is provided a method to introduce an expression vector, especially Geminivirus based expression vector into intact cell, comprising a separated single strand heterologous polynucleotide sequence encoding a gene or genes of choice and a short primer sequence to allow the complementation of the strand by the plant machinery and to produce the desired dsDNA needed to lead to expression in the treated tissue.

[0047] According to further feature in preferred embodiments of the invention described below:

[0048] According to some embodiments of the invention, the heterologous polynucleotide encodes a polypeptide selected from the group consisting of a reporter molecule, an antiviral molecule, a viral moiety, an antifungal molecule, an antibacterial molecule, an insect resistance molecule, a herbicide resistance molecule, a biotic or abiotic stress tolerance molecule, a pharmaceutical molecule, a growth inducing molecule, and a growth inhibiting molecule.

[0049] According to some embodiments of the invention, the Geminivirus is a begomo virus.

[0050] According to some embodiments of the invention, the Geminivirus is a Tomato yellow leaf curl virus (TYLCV).

[0051] According to some embodiments of the invention, the Geminivirus is a Bean dwarf mosaic virus (BDMV).

[0052] According to some embodiments of the invention, the expression construct is adapted for expression in a plant host selected from the group consisting of Solanaceae, Cucurbitaceae, Apiaceae, Liliacae, Gramineae (Poaceae), Rosaceae, Musaceae, Vitacea, and Brassicaceae.

[0053] According to some embodiments of the invention, the molecule of interest is selected from the group consisting of a reporter molecule, an antiviral molecule, a viral moiety, an antifungal molecule, an antibacterial molecule, an insect resistance molecule, a herbicide resistance molecule, a biotic or abiotic stress tolerance molecule, a pharmaceutical molecule, a growth inducing molecule, a product of genes in a metabolic pathway and a growth inhibiting molecule.

[0054] According to some embodiments of the invention, the genes in the metabolic pathway are encoded by an operon.

a. According to some embodiments of the invention, the plant is selected from the group consisting of members of the families Solanaceae Cucurbitaceae, Apiaceae Liliacae, a Gramineae (Poaceae), Rosaceae. Musaceae, Vitacea and a Brassicaceae .

[0055] Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

[0056] 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. [0057] In the drawings:

[0058] Figure 1: 0.8% Agarose gel showing dsDNA vs. ssDNA of the same plasmid vector.

[0059] Gel electrophoresis analysis of plasmid DNA sized 6 kb, in its double strand form (ds) or its single strand form (ss). The double strand form DNA appears mainly as super coiled (sc). The single strand form gives one band on the gel which is lower than the super coiled band of the double strand form.

[0060] As molecular weight standard (M), the GeneRuler 1Kb DNA ladder 0.5ug/ul (Thermo Scientific) was used.

[0061] Figure 2. ssDNA penetrates nuclei within less than one hour.

[0062] Confocal analysis of various samples of chickpea roots treated with DNA plasmid labeled with Cy3 or Cy5. A- untreated (Mock) sample measured under Cy3 and Cy5 filters , B- treated with Cy5-ssDNA of M13 plasmid, C- Cy3-ssDNA of 1470 , D - Cy5- dsDNA of 1470, E- Free Cy3.

[0063] Figure 3. ssDNA of pl470 labeled with Cy3 penetrates plant root cell nuclei and stay stable for 24hr.

[0064] Confocal analysis of chickpea roots treated with Cy3-ssDNA of 1470, 24 hr post treatment.

[0065] Figure 4: ssDNA with addition of primer leads to expression in Arabidopsis

[0066] Confocal analysis of GFP expression in Arabidopsis Thaliana roots treated with (A) ssDNA 1470, (B) ssl470 with non-modified primer, (C) ssl470 with

phosphorothioate modified primer. Mock- untreated sample.

[0067] Figure 5: Description of various constructs carrying BDMV elements [0068] Plasmid vectors for use in the expression experiment. A. The genome

organization of BDMV DNA-B carrying the movement protein (MP) (BC1) and the nuclear shuttle protein (NSP) (BV1) under the control of the bi-directional promoter.

[0069] B. Binary plasmid constructs carrying the BDMV-MP and the NSP elements under strong promoter (35SCaMV or FiMV)

[0070] Figure 6: BDMV-MP and NSP elements increase GFP expression carried by other vector.

[0071] Binocular analysis of Nicotiana benthamiana leaves 3 days post agroinfiltration treatment with various vectors. Each treatment performed on two leaves (L3 and L4) of two different plants.

[0072] Figure 7: BDMV-MP and NSP elements dramatically increase GFP

expression in cell.

[0073] Leaves of Nicotiana benthamiana (4 weeks old) were introduced by agroinfiltration with the binary plasmid pi 470 cassette carrying GFP under strong promoter (#321) (A-c), or together with the binary vector carrying the BDMV MP and NSP ORFs (#550) (D-G). Confocal analysis performed on Leaves 3 days post agroinfiltration treatment.

[0074] Picture were taken from the center or border of the agroinfiltration area. Zoom size is given in brackets.

[0075] Figure 8: BDMV-MP and NSP elements increase GFP expression, measured by RT-qPCR analysis.

[0076] Real-Time qPCR analysis on RNA extracted from treated leaf tissue was performed.

[0077] The presented result has been normalized to Actin expression. [0078] Figure 9: constructs used in experiments [0079] The description of the Binary constructs of pBIN carrying various expression cassette that were used in the agroinfiltration experiments, is given below.

[0080] Figure 10: BDMV-MP and NSP elements increase GFP expression carried by other vector.

[0081] Binocular microscope analysis of Nicotiana benthamiana leaves 3 days post agroinfiltration treatment with various vectors. Each treatment performed on two leaves (L3 and L4) of two different plants.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0082] In the description and tables that follow, a number of terms are used. In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided:

[0083] 3' non-coding sequences. The "3' non-coding sequences" refer to DNA sequences located downstream of a coding sequence and include polyadenylation recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor. The use of different 3' non-coding sequences is exemplified by Ingelbrecht I L. et al. (1989. Plant Cell 1:671680).

[0084] Cell. Cell as used herein includes a living cell, whether isolated, in tissue culture or incorporated in an organism or organism part.

[0085] Plant cell. Plant cell as used herein includes a plant cell, whether isolated, in tissue culture or incorporated in a plant or plant part.

[0086] Animal cell. The term "animal cells" encompasses, but is not limited to invertebrate, non-mammalian vertebrate (e.g., avian, reptile and amphibian) and mammalian cells.

[0087] Commercial plant seed treatments. Include but are not limited to plant seed treatments such as coating, film coating, dressing and pelleting.

[0088] Commodity seeds/Commodity crops. Commodity seeds or crops are any plant seeds or crops that are traded and used commercially. Generally, they are relatively nonperishable, storable, transportable, and undifferentiated. Commodity crops are crops grown, typically in large volume and at high intensity, specifically for the purpose of sale to the commodities market, as opposed to direct consumption or processing. Some examples include, but are not limited to, corn, soybean, wheat, cotton, rice and the like.

[0089] Comprising the heterologous DNA. The term "comprising the heterologous DNA" when used in reference to a plant or seed refers to a plant or seed that contains at least one heterologous DNA in one or more of its cells. As used herein, the term refers to a plant, a plant structure, a plant tissue, a plant seed or a plant cell that contains at least one heterologous DNA in at least one of its cells. This term includes the primary cell to which the DNA was introduced and cultures and plants derived from that cell without regard to the number of transfers. All progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same functionality as screened for in the original cell to which the DNA was introduced are included in the term.

[0090] Construct. The term "construct" as used herein, refers to an artificially assembled or isolated nucleic acid molecule which includes the heterologous DNA interest. In general a construct may include the heterologous DNA, typically a gene of interest, a marker gene which in some cases can also be the gene of interest and appropriate regulatory sequences. It should be appreciated that the inclusion of regulatory sequences in a construct is optional, for example, such sequences may not be required in situations where the regulatory sequences of a host cell are to be used. The term construct includes vectors but should not be seen as being limited thereto.

[0091] Cotyledon. A cotyledon is a type of seed leaf. The cotyledon contains the food storage tissues of the seed.

[0092] Diploid. A cell or organism having two sets of chromosomes.

[0093] Embryo. The embryo is the small plant contained within a mature seed.

[0094] Enhancer. As used herein, the term an "enhancer" refers to a DNA sequence which can stimulate promoter activity, and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue-specificity of a promoter.

[0095] Expression. As used herein, refers to the production of a functional end-product e.g., an mRNA or a protein

[0096] Gene. The term "gene" refers to a nucleic acid (e.g., DNA or RNA) sequence that comprises coding sequences necessary for the production of RNA or a polypeptide. The term comprises natural as well as man tailored (synthetic) genes. A polypeptide can be encoded by a full-length coding sequence or by any part thereof. The term "parts thereof when used in reference to a gene refers to fragments of that gene ranging in size from a few nucleotides to the entire gene sequence minus one nucleotide. Thus, "a nucleic acid sequence comprising at least a part of a gene" may comprise fragments of the gene or the entire gene.

[0097] The term "gene" also encompasses the coding regions of a structural gene and includes sequences located adjacent to the coding region on both the 5' and 3' ends for a distance of about 1 kb on either. The sequences which are located 5' of the coding region and which are present on the mRNA are referred to as 5' non-translated (or untranslated) sequences (5' UTR). The sequences which are located 3' or downstream of the coding region and which are present on the mRNA are referred to as 3' non-translated (or untranslated) sequences (3' UTR).

[0098] Gene Silencing. The interruption or suppression of the expression of a gene at the level of transcription or translation.

[0099] Genotype. Refers to the genetic constitution of a cell or organism.

[00100] Heterologous DNA. The terms "heterologous DNA" or "exogenous

DNA" refer to a polynucleotide that is not present in its natural environment (i.e., has been altered by the hand of man). For example, a heterologous DNA includes a polynucleotide from one species introduced into another species. As used herein, a heterologous DNA also includes a polynucleotide native to an organism, which may or may not have been altered in some way (e. g., mutated, added in multiple copies, linked to a non-native promoter or enhancer sequence, etc.). Heterologous DNA may comprise gene sequences of plant, bacteria and mammal origin. The gene sequences may comprise cDNA forms of a gene; the cDNA sequences may be expressed in either a sense (to produce mRNA) or anti-sense orientation (to produce an anti-sense RNA transcript that is complementary to the mRNA transcript). Heterologous plant genes are distinguished from endogenous plant genes in that the heterologous gene sequences are typically joined to nucleotide sequences comprising regulatory elements such as promoters that are not found naturally associated with the gene for the protein encoded by the heterologous gene or with plant gene sequences in the chromosome, or are associated with portions of the chromosome not found in nature (e.g., genes expressed in loci where the gene is not normally expressed).

[00101] Naked DNA. Refers to DNA that is devoid of protein coat, or included into a microorganism. For the transfer of genes, naked DNA usually consists of a bacterial plasmid vector with or without plant viral elements and containing the sequence or the gene to be transferred. As used herein, "naked DNA" can be synthetic or purified from a microorganism, and can also be associated with some chemicals to help its penetration into tissues.

[00102] Nucleic Acid. The term "nucleic acid" as used herein refers to DNA that is linear or branched or circular, single or double stranded, or a hybrid thereof.

[00103] Operably linked. The term "operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is regulated by the other. For example, a promoter is operably linked with a coding sequence when it is capable of regulating the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in a sense or antisense orientation.

[00104] Pathogen. Any of a number of microorganisms, such bacteria, fungi and viruses that can impart a disease to a plant or seed.

[00105] Plant. As used herein, the term "plant" includes reference to an immature or mature whole plant, including a plant from which seed, grain, or anthers have been removed. Seed or embryo that will produce the plant is also considered to be the plant.

[00106] Plant parts. As used herein, the term "plant parts" (or a plant organ, or a part thereof) includes but is not limited to protoplasts, leaves, stems, roots, root tips, anthers, pistils, seed, grain, embryo, pollen, ovules, cotyledon, hypocotyl, pod, flower, shoot, tissue, petiole, cells, meristematic cells, and the like.

[00107] Plant tissue. The term "plant tissue" includes differentiated and undifferentiated tissues of plants including those present in roots, shoots, leaves, pollen, seeds and tumors, as well as cells in culture (e.g., single cells, protoplasts, embryos, callus, etc.). Plant tissue may be within the plant, in organ culture, tissue culture, or cell culture.

[00108] Promoter. The terms "promoter element," "promoter," or "promoter sequence" as used herein, refer to a DNA sequence that is located at the 5' end (i.e.

precedes) the coding region of a DNA polymer. The location of most promoters known in nature precedes the transcribed region. The promoter functions as a switch, activating the expression of a gene. If the gene is activated, it is said to be transcribed, or participating in transcription. Transcription involves the synthesis of mRNA from the gene. The promoter, therefore, serves as a transcriptional regulatory element and also provides a site for initiation of transcription of the gene into mRNA. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of some variation may have identical promoter activity. Promoters which cause a gene to be expressed in most cell types at most times are commonly referred to as "constitutive promoters". New promoters of various types useful in plant cells are constantly being discovered; numerous examples may be found in Okamuro J K and Goldberg R B (1989) Biochemistry of Plants 15: 1-82.

[00109] Seed viability. Refers to the percent germination potential of a sample of seed.

[00110] Stable transformation. Stable DNA introduction is referred to as "stable transformation" resulting in "stably transformed" cell or tissue and refers to the introduction and integration of one or more exogenous polynucleotides into the genome of a cell. The term "stable transformant" refers to a cell which has stably integrated one or more exogenous polynucleotides into the genomic or organellar DNA (chloroplast and/or mitochondria). Plants or parts thereof comprising cell stably transformed with exogenous DNA are typically referred to as "transgenic plants", "transgenic plant cell" or, in the context of the present invention "transgenic seeds".

[00111] Symptomless. The virus-based DNA construct of a preferred embodiment of the present invention is symptomless in the plant to which it was introduced. As used herein, the term "symptomless" refers to the ability of the plant plasmid vector to transfect plant tissue, without inducing the characteristic pathogenic symptoms of the virus.

[00112] Transformation The introduction of material including but not limited to

Nucleic Acids through a molecular delivery system.

[00113] Transient transformation. Introduction of a heterologous DNA into a cell may be stable or transient. The term "transient" refers to the introduction of one or more exogenous polynucleotides into a cell in the absence of integration of the exogenous polynucleotide into the host cell's genome. This type of DNA introduction may be also referred to as "transient transformation". The term "transient transformant" thus refers to a cell which has transiently incorporated one or more exogenous polynucleotides.

Transiently transformed cells are typically referred to as "non-transgenic" or "non- genetically modified (non-GMO)".

[00114] Treated plants. Refers to plants into which the heterologous DNA has been introduced, regardless if it has been integrated into the plant genome, into its organelles, or remained free in the cytoplasm, or resided as an episome in the nucleus without integration.

[00115] True leaves. Any leaves of a plant other than the cotyledons.

[00116] Virus based vector. Any plasmid vector that carries any wild type or mutated element or part thereof, which originated from plant viruses, such as promoter, coding sequence or non-coding sequences like coat protein (CP) or an intergenic region (IR). Virus based vectors include vectors that have elements that allow some level of replication and/or spreading.

[00117] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having,"

"including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if the range 10-15 is disclosed, then 11, 12, 13, and 14 are also disclosed. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[00118] In the present invention ssDNA is produced by using standard materials and methods known in the art.

[00119] In order to provide efficient RNA transcription and eventually gene expression, oligonucleotide (primer) might be added in the transfection process.

Furthermore, this primer might be modified (such as phosphorothioate primers) in order to further improve transcription/expression. Alternatively, the transfected DNA could be only partially ssDNA, part of it being dsDNA. a. The present invention, in some embodiments thereof, relates ssDNA form of a plant expression vector such as vector carrying plant geminivirus viral elements, in particular the intergenic region (IR) that are capable of being penetrating intact and non intact plants, becoming incorporated into somatic nuclei, resulting in the expression of an heterologous genes incorporated into the constructs in a symptomless manner. As used herein, the term "symptomless manner" refers to expression of the introduced gene(s) without inducing characteristic Geminivirus symptoms.

[00120] 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 or carried out in various ways.

USES OF THE TECHNOLOGY

[00122] The following are provided as examples of use of the technology and are not intended to limit the scope of the invention.

[00123] The expressed polynucleotide sequence can encode a molecule which would protect the plant from abiotic stress factors such as drought, heat or chill. Examples include antifreeze polypeptides from Myoxocephalus scorpius (WO 00/00512), Myoxocephalus octodecemspinosus, the Arabidopsis thaliana transcription activator CBF1, glutamate dehydrogenases (WO 97/12983, WO 98/11240), calcium- dependent protein kinase genes (WO 98/26045), calcineurins (WO 99/05902), casein kinase from yeast (WO 02/052012), farnesyltransferases (WO 99/06580; Pei Z M et al.

(1998) Science 282:287-290), ferritin (Deak M et al. (1999) Nature Biotechnology 17: 192-196), oxalate oxidase (WO 99/04013; Dunwell J M (1998) Biotechn Genet Eng Rev 15: 1-32), DREB1A factor ("dehydration response element B 1A"; Kasuga M et al.

(1999) Nature Biotech 17:276-286), genes of mannitol or trehalose synthesis such as trehalose-phosphate synthase or trehalose-phosphate phosphatase (WO 97/42326) or by inhibiting genes such as trehalase (WO 97/50561).

[00124] The expressed polynucleotide sequence could be a metabolic enzyme for use in the food-and-feed sector. Examples include, phytases (GenBank Acc. No.: A19451) and cellulases.

[00125] The expressed polynucleotide sequence can confer resistance to viruses, fungi, insects, nematodes and diseases, by directly attacking the pathogen, turning on the host defenses or by leading to an accumulation of certain metabolites or proteins. Examples of include glucosinolates (defense against herbivores), chitinases or glucanases and other enzymes which destroy the cell wall of parasites, ribosome- inactivating proteins (RIPS) and other proteins of the plant resistance and stress reaction as are induced when plants are wounded or attacked by microbes, or chemically, by, for example, salicylic acid, jasmonic acid or ethylene, or lysozymes from nonplant sources such as, for example, T4-lysozyme or lysozyme from a variety of mammals, insecticidal proteins such as Bacillus thuringiensis endotoxin, a-amylase inhibitor or protease inhibitors (cowpea trypsin inhibitor), lectins such as wheatgerm agglutinin, siRNA, antisense RNA, RNAses or ribozymes. Further examples are nucleic acids which encode the Trichoderma harzianum chit42 endochitinase (GenBank Acc. No.: S78423) or the N-hydroxylating, multi-functional cytochrome P- 450 (CYP79) protein from Sorghum bicolor (GenBank Acc. No.: U32624), or functional equivalents thereof.

[00126] Accumulation of glucosinolates as protection from pests (Rask L et al.

(2000) Plant Mol Biol 42:93-113; Menard R et al. (1999) Phytochemistry 52:29-35), the expression of Bacillus thuringiensis endotoxins (Vaeck et al. (1987) Nature 328:33- 37) or the protection against attack by fungi, by expression of chitinases, for example from beans (Broglie et al. (1991) Science 254: 1 194-1 197), is advantageous. Resistance to pests such as, for example, the rice pest Nilaparvata lugens in rice plants can be achieved by expressing the snowdrop (Galanthus nivalis) lectin agglutinin (Rao et al. (1998) Plant J 15(4):469-77).

[00127] The expression of synthetic crylA(b) and crylA(c) genes, which encode

Lepidoptera-specific Bacillus thuringiensis delta-endotoxins can bring about a resistance to insect pests in various plants (Goyal R K et al. (2000) Crop Protection 19(5):307-312).

[00128] Additional genes which are suitable for pathogen defense comprise

"polygalacturonase-inhibiting protein" (PGIP), thaumatin, invertase and antimicrobial peptides such as lactoferrin (Lee T J et al. (2002) J Amer Soc Horticult Sci 127(2): 158- 164).

[00129] The expressed polynucleotide sequence can bring about an accumulation of chemicals such as of tocopherols, tocotrienols or carotenoids. One example of such a polynucleotide is phytoene desaturase. Preferred are nucleic acids which encode the Narcissus pseudonarcissus phytoene desaturase (GenBank Acc. No.: X78815) or functional equivalents thereof. [00130] The expressed polynucleotide sequence can be used for production of nutraceuticals such as, for example, polyunsaturated fatty acids (arachidonic acid, eicosapentaenoic acid or docosahexaenoic acid) examples include, fatty acid elongases and/or desaturases, or for production of proteins with improved nutritional value such as, for example, with a high content of essential amino acids (for example the high- methionine 2S albumin gene of the brazil nut). Preferred are polynucleotide sequence which encode the Bertholletia excelsa high-methionine 2S albumin (GenBank Acc. No.: AB044391), the Physcomitrella patens delta6-acyl-lipid desaturase (GenBank Acc. No.: AJ222980; Girke et al. (1998) Plant J 15:39-48), the Mortierella alpina delta6-desaturase (Sakuradani et al. 1999 Gene 238:445-453), the Caenorhabditis elegans delta5-desaturase (Michaelson et al. 1998, FEBS Letters 439:215-218), the Caenorhabditis elegans A5-fatty acid desaturase (des-5) (GenBank Acc. No.: AF078796), the Mortierella alpina delta5-desaturase (Michaelson et al. JBC 273: 19055-19059), the Caenorhabditis elegans delta6-elongase (Beaudoin et al. 2000, PNAS 97:6421-6426), the Physcomitrella patens delta6-elongase (Zank et al. 2000, Biochemical Society Transactions 28:654-657), or functional equivalents of these.

[00131] The expressed polynucleotide sequence can be used for production of high- quality proteins and enzymes for industrial purposes (for example enzymes, such as lipases) or as pharmaceuticals (such as, for example, antibodies, blood clotting factors, interferons, lymphokins, colony stimulation factor, plasminogen activators, hormones or vaccines, as described by Hood E E, Jilka J M (1999) Curr Opin Biotechnol 10(4):382-6; Ma J K, Vine N D (1999) Curr Top Microbiol Immunol 236:275-92). For example, it has been possible to produce recombinant avidin from chicken albumen and bacterial P-glucuronidase (GUS) on a large scale in transgenic maize plants (Hood et al. (1999) Adv Exp Med Biol 464: 127-47. Review).

[00132] The expressed polynucleotide sequence can be used for obtaining an increased storability in cells which normally comprise fewer storage proteins or storage lipids, with the purpose of increasing the yield of these substances. Examples include, acetyl-CoA carboxylase. Preferred polynucleotide sequence are those which encode the Medicago sativa acetyl-CoA carboxylase (ACCase) (GenBank Acc. No.: L25042), or functional equivalents thereof.

[00133] Additional examples of expressible polynucleotides include Hepatitis B surface antigen [Kumar GBS et al., Planta 222 (3): 484-493, 2005], herbicide resistance [Duke, SO, Pest Management Science 61 :211-218, 2005], interferon [Edelbaum, O. et al., J. Interferon Res. 12: 449-453, 1992], T7-RNA polymerase [Zeitoune et al., Plant Science 141 :59-65., 1997].

[00134] Further examples of polynucleotide sequence which can be expressed by the expression vector of the present invention are mentioned for example in Dunwell J M, Transgenic approaches to crop improvement, J Exp Bot. 2000; 51 pages 487-96.

[00135] The expression vector of the present invention can also be employed for the reduction (suppression) of transcription and/or translation of target genes. Thus, the expression vector of the present invention can express nucleic acids which bring about PTGS (post transcriptional gene silencing) or TGS (transcriptional gene silencing) effects and thus a reduction of the expression of endogenous genes. Such reduction can be achieved for example by expression of an antisense RNA (EP-A1 0 458 367; EP- Al 0 140 308; van der Krol A R et al. (1988) BioTechniques 6(10):658-676; de Lange P et al. (1995) Curr Top Microbiol Immunol 197:57-75, inter alia) or of a double- stranded RNA, each of which has homology with the endogenous target gene to be suppressed. Also, the expression of a suitable sense RNA can bring about a reduction of the expression of endogenous genes, by means of what is known as co-suppression (EP-A1 0 465 572). Especially preferred is the expression of a double-stranded small interfering RNA (siRNA) for reducing the gene expression of a target gene via RNA interference (RNAi). WO 99/32619 and WO 99/53050 describe methods for inhibiting individual target genes using an RNA with double- stranded structure, where the target gene and the region of the RNA duplex have at least partial identity (see also: Montgomery M K et al. (1998) Proc Natl Acad Sci USA 95: 15502-15507; Sharp P A (1999) Genes & Development 13(2): 139-141 ; Fire A et al. (1998) Nature 391:806-11). [00136] The following exemplifies applications where reduction of gene expression can be employed using the expression vector of the present invention.

[00137] Delayed fruit maturation or a modified maturation phenotype (prolonged maturation, later senescence) can be achieved for example by reducing the gene expression of genes selected from the group consisting of polygalacturonases, pectin esterases, beta.- (l ,4)glucanases (cellulases), beta.-galactanases (.beta.-galactosidases), or genes of ethylene biosynthesis, such as 1 -aminocyclopropane- 1 -carboxylate synthase, adenosylmethionine hydrolase (SAMase), aminocyclopropane- 1-carb- oxylate deaminase, aminocyclopropane- 1 -carboxylate oxidase, genes of carotenoid biosynthesis such as, for example, genes of pre-phytoene biosynthesis or phytoene biosynthesis, for example phytoene desaturases, and O-methyltransferases, acyl carrier protein (ACP), elongation factor, auxin-induced gene, cysteine(thiol) proteinases, starch phosphorylases, pyruvate decarboxylases, chalcone reductases, protein kinases, auxin-related gene, sucrose transporters, meristem pattern gene. Further advantageous genes are described for example in WO 91/16440, WO 91/05865, WO 91/16426, WO 92/17596, WO 93/07275 or WO 92/04456. Especially preferred is the reduction of the expression of polygalacturonase for the prevention of cell degradation and mushiness of plants and fruits, for example tomatoes. Nucleic acid sequences such as that of the tomato polygalacturonase gene (GenBank Acc. No.: x 14074) or its homologs are preferably used for this purpose.

[00138] Improved protection against abiotic stress factors (heat, chill, drought, elevated moisture, pollutants, UV radiation). It is preferred to reduce the expression of genes which are implicated in stress reactions.

[00139] The reduction of the gene expression of genes encoding storage proteins

(hereinbelow SPs) has numerous advantages, such as, for example, the reduction of the allergenic potential or modification regarding composition or quantity of other metabolites, such as, for example, oil or starch content.

[00140] Resistance to plant pathogens such as arachnids, fungi, insects, nematodes, protozoans, viruses, bacteria and diseases can be achieved by reducing the gene expression of genes which are essential for the growth, survival, certain developmental stages (for example pupation) or the multiplication of a specific pathogen. Such a reduction can bring about a complete inhibition of the abovementioned steps, or else a delay of same. They can take the form of plant genes which for example make possible the penetration of the pathogen, but may also be homologous pathogen genes. The transgenically expressed nucleic acid sequence (for example the double-stranded RNA) is preferably directed against genes of the pathogen. The antipathogenic agent which acts may be, in this context, the transgenically expressed nucleic acid sequence itself (for example the double-stranded RNA), but also the transgenic expression cassettes or transgenic organisms. The plants themselves, in the form of a transgenic organism, may contain the agents and pass them on to the pathogens, for example in the form of a stomach poison. Various essential genes of a variety of pathogens are known to the skilled artisan (for example for nematode resistance WO 93/10251 , WO 94/17194).

[00141] Virus resistance can be achieved for example by reducing the expression of a viral coat protein, a viral replicase, a viral protease and the like. A large number of plant viruses and suitable target genes are known to the skilled artisan.

[00142] Reduction of undesired, allergenic or toxic plant constituents such as, for example, glucosinolates or patatin. Suitable target genes are described (in WO 97/16559, inter alia). The target genes which are preferred for reduction of allergenic proteins are described for example by Tada Y et al. (1996) FEBS Lett 391(3):341-345 or Nakamura R (1996) Biosci Biotechnol Biochem 60(8): 1215-1221.

[00143] Delayed signs of senescence. Suitable target genes are, inter alia, cinnamoyl-CoA:NADPH reductases or cinnamoyl-alcohol dehydrogenases. Further target genes are described (in WO 95/07993, inter alia).

[00144] Reduction of the susceptibility to bruising of, for example, potatoes by reducing for example polyphenol oxidase (WO 94/03607) and the like.

[00145] Increase of the methionine content by reducing threonine biosynthesis, for example by reducing the expression of threonine synthase (Zeh M et al. (2001) Plant Physiol 127(3):792-802). [00146] It will be appreciated that the nucleic acid construct of the present invention can also express homologues of the above described molecules that exhibit the desired activity (i.e., the biological activity). Such homologues can be, for example, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100 %, identical to any of the expressed sequences described above as determined using the BestFit software of the Wisconsin sequence analysis package, utilizing the Smith and Waterman algorithm, where gap weight equals 50, length weight equals 3, average match equals 10, and average mismatch equals -9.

[00147] Thus, the present invention provides a geminivirus based single strand DNA construct that traverses intact plant tissues, any genes the construct carries are expressed in the host plant, and yet does not induce symptoms of viral disease therein.

[00148] In summary, the nucleic acid construct of the present invention can be utilized for any purpose. Examples of uses include the following:

(i) plant expression of proteins (specific examples provided hereinabove) for various purposes including plant improvement, plant protection, biopharming etc;

(ii) plant expression of nucleic acid molecules (e.g. siRNA, specific examples provided hereinabove);

(iii) produce indicator plants which detect viral infection - a plant carrying a construct including a reporter molecule (e.g. fluorophore) attached to the IR region would express the reporter in when infected by a geminivirus; and

(iv) produce infection-resistant plants - a plant carrying a construct including an anti-viral or anti-plant molecule attached, for example, to the IR region would express such a molecule when infected by a geminivirus; such "immunity" or suicide scheme would only be active when the plant is infected; since the nucleic acid constructs of the present invention are preferably transient and not stably integrated into a genome of the host plant, such a trait would not be inherited by the progeny of the plant nor would it persist in commercial products of the plant.

[00149] The nucleic acid construct of the present invention can be utilized to stably or preferably transiently transform plant cells. In stable transformation, the nucleic acid molecule of the present 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 not integrated into the genome, and as such represents a transient trait.

[00150] There are various methods of introducing foreign genes into both monocotyledonous and dicotyledonous plants (Potrykus, I. (1991). Annu Rev Plant Physiol Plant Mol Biol 42, 205-225; Shimamoto, K. et al. (1989). Fertile transgenic rice plants regenerated from transformed protoplasts. Nature (1989) 338, 274-276).

[00151] The principal methods of the stable integration of exogenous DNA into plant genomic DNA includes two main approaches:

(i) Agrobacterium-mediated gene transfer. See: Klee, H. J. et al. (1987). Annu Rev Plant Physiol 38, 467-486; Klee, H. J. and Rogers, S. G. (1989). Cell Culture and Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of Plant Nuclear Genes, pp. 2-25, J. Schell and L. K. Vasil, eds., Academic Publishers, San Diego, Cal.; and Gatenby, A. A. (1989). Regulation and Expression of Plant Genes in Microorganisms, pp. 93-112, Plant Biotechnology, S. Kung and C. J. Arntzen, eds., Butterworth Publishers, Boston, Mass.

(ii) Direct DNA uptake. See, e.g.: Paszkowski, J. et al. (1989). Cell Culture and Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of Plant Nuclear Genes, pp. 52-68, J. Schell and L. K. Vasil, eds., Academic Publishers, San Diego, Cal.; and Toriyama, K. et al. (1988). Bio/Technol 6, 1072-1074 (methods for direct uptake of DNA into protoplasts). See also: Zhang et al. (1988). Plant Cell Rep 7, 379-384; and Fromm, M. E. et al. (1986). Stable transformation of maize after gene transfer by electroporation. Nature 319, 791-793 (DNA uptake induced by brief electric shock of plant cells). See also: Klein et al. (1988). Bio/Technology 6, 559- 563; McCabe, D. E. et al. (1988). Stable transformation of soybean (Glycine max) by particle acceleration. Bio/Technology 6, 923-926; and Sanford, J. C. (1990). Biolistic plant transformation. Physiol Plant 79, 206-209 (DNA injection into plant cells or tissues by particle bombardment). See also: Neuhaus, J. M. et al. (1987). Theor Appl Genet 75, 30-36; and Neuhaus, J. M. and Spangenberg, G. C. (1990). Physiol Plant 79, 213-217 (use of micropipette systems). See U.S. Pat. No. 5,464,765 (glass fibers or silicon carbide whisker transformation of cell cultures, embryos or callus tissue). See also: DeWet, J. M. J. et al. (1985). "Exogenous gene transfer in maize (Zea mays) using DNA-treated pollen," Experimental Manipulation of Ovule Tissue, G. P. Chapman et al., eds., Longman, New York-London, pp. 197-209; and Ohta, Y. (1986). High-Efficiency Genetic Transformation of Maize by a Mixture of Pollen and Exogenous DNA. Proc Natl Acad Sci USA 83, 715-719 (direct incubation of DNA with germinating pollen).

[00152] The Agrobacterium-mediated system includes the use of plasmid vectors that contain defined DNA segments which 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, R. B. et al. (1988). "Leaf disc transformation." Plant Molecular Biology Manual A5, 1-9, Kluwer Academic Publishers, Dordrecht). A supplementary approach employs the Agrobacterium delivery system in combination with vacuum infiltration. The Agrobacterium system is especially useful for in the creation of transgenic dicotyledenous plants.

[00153] There are various methods of direct DNA transfer into plant cells. In electroporation, the protoplasts are briefly exposed to a strong electric field, opening up mini-pores to allow DNA to enter. In microinjection, the DNA is mechanically injected directly into the cells using micropipettes. In microparticle bombardment, the DNA is adsorbed on microprojectiles such as magnesium sulfate crystals or tungsten particles, and the microprojectiles are physically accelerated into cells or plant tissues. Additional direct DNA transfer techniques include glass or silicone carbide whiskers (see, for example, Dunwell, Methods Mol Biol. 1999;111 :375-82).

[00154] Following stable transformation, plant propagation then occurs. The most common method of plant propagation is by seed. The disadvantage of regeneration by seed propagation, however, is the lack of uniformity in the crop due to heterozygosity, since seeds are produced by plants according to the genetic variances governed by Mendelian rules. In other words, each seed is genetically different and each will grow with its own specific traits. Therefore, it is preferred that the regeneration be effected such that the regenerated plant has identical traits and characteristics to those of the parent transgenic plant. The preferred method of regenerating a transformed plant is by micropropagation, which provides a rapid, consistent reproduction of the transformed plants.

[00155] Micropropagation is a process of growing second-generation plants from a single tissue sample excised from a selected parent plant or cultivar. This process permits the mass reproduction of plants having the preferred tissue and expressing a fusion protein. The newly generated plants are genetically identical to, and have all of the characteristics of, the original plant. Micropropagation allows for mass production of quality plant material in a short period of time and offers a rapid multiplication of selected cultivars with preservation of the characteristics of the original transgenic or transformed plant. The advantages of this method of plant cloning include the speed of plant multiplication and the quality and uniformity of the plants produced.

[00156] 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. During stage one, 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 newly grown tissue samples are divided and grown into individual plantlets. At stage four, the transformed plantlets are transferred to a greenhouse for hardening where the plants' tolerance to light is gradually increased so that they can continue to grow in the natural environment.

[00157] Transient transformation of, for example, leaf cells, meristematic cells, or the whole plant is also envisaged by the present invention.

[00158] Transient transformation can be effected by any of the direct DNA transfer methods described above or by mechanical or vector mediated viral infection using the plant viruses derived plasmid of the present invention.

[00159] According to yet another aspect of the present invention there is provided a method of expressing a molecule of interest in a plant, the method comprising grafting a section of a first plant infected with at least one Geminivirus based expression construct onto a section of a second plant, the expression construct comprising a polynucleotide sequence which encodes the molecule of interest, and further the Geminivirus based expression construct being capable of systemic symptomless spread in a plant host, thereby expressing a molecule of interest in a plant.

[00160] As used herein, the term "grafting" refers to the joining together of the parts of plants so that they bind together and the sap can flow, thus forming a new plant that can grow and develop. A graft therefore consists of two parts: (i) the lower part is the rootstock as referred to herein and essentially consists of the root system and a portion of the stem, and (ii) the upper part, the scion or graft, which gives rise to the aerial parts of the plant. It will be appreciated that a single graft can comprise two parts of the same plant of alternatively a single graft can comprise parts of two different plants.

[00161] Preferably, the first plant and the second plant are compatible for grafting.

[00162] As used herein, the term "compatible" refers to the ability of the grafted rootstock and plant tissue to grow together and survive. It is well known that compatible rootstock and plant tissue grafts do not have to be from the same plant species. For example, tomato scions can be grafted onto eggplant rootstock. Transgenic rootstock can be prepared using standard methods.

[00163] Grafting can be accomplished using standard materials and methods known in the art. (See, for example, Black et al. (2003); Fernandez-Garcia et al. (2004); Edelstein et al. (2004); see Worldwide Website: wwwdotparamount- seedsdotcom/Paramountonline/graftingdothtm; see Worldwide Website: wwwdotagnetdotorg/library/article/eb480dothtml).

[00164] Exemplary plants which can be grafted according to this aspect of the present invention include dicotyledonous plants, such as, for example, peas, alfalfa, tomato, tomatillo, melon, chickpea, chicory, clover, kale, lentil, soybean, tobacco, potato, sweet potato, radish, cabbage, rape, apple trees, grape, cotton, sunflower, citrus (including orange, mandarin, kumquat, lemon, lime, grapefruit, tangerine, tangelo, citron, and pomelo), pepper, bean, and lettuce. Plants within the scope of the present invention also include conifers.

[00165] Examples of tomato rootstock that can be used to prepare pathogen resistant transgenic rootstock includes, but is not limited to, "PG3" and "Beaufort." Examples of tomato cultivars that can be used to provide scions for the present invention include, but are not limited to, "Monroe," "Belle," Summer Set," "Match," Trust," "Better Boy," "Celebrity," "Grace," "Heinz 1439," "Roma," "Rugers," "Ultra Girl," "2710," "BHN 665," "STM 0227," "STM 5206," "Boy oh Boy," "Jubilation," "Sunchief," and "Fabulous."

[00166] Contemplated geminivirus based expression constructs that may be grafted from one plant to another or soaked up by plant roots are those described herein above and further those described in International Patent Application WO2007/141790.

[00167] The terms "comprises", "comprising", "includes", "including", "having" and their conjugates mean "including but not limited to".

[00168] The term "consisting of means "including and limited to". [00169] 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.

[00170] 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.

[00171] 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 sub-combination 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.

[00172] According to yet another aspect of the present invention there is provided a method of transfecting animal cell, including insects, mammals and human, useful for functional genomics and gene therapy in Mammalian including human.

[00173] Insect cell-based systems for the large-scale production of recombinant proteins have become indispensable for biotechnological, pharmaceutical and industrial applications. Insect expression systems (Becker-Pauly and Stocker, 2011) represent an adequate compromise between bacterial and mammalian systems. In insect cells, signal peptides are cleaved as in mammalian cells, disulfide bonds are formed in the endoplasmic reticulum and proprotein-converting enzymes are available for proteolytic processing. Additional continuous cell lines have been established from a large number of insect species, mainly from Lepidoptera and Diptera (Lynn et al. 1988_.) but those derived from Spodoptera frugiperda (IPLB-SF- 21-AE; Vaughn et al. 1_.977 and Sf9 (Summers and Smith ] 7 ) ) and Trichoplusia ra^'(High Five™) have been the most widely used both commercially and in the research laboratory for baculovirus transient gene expression studies and transgenic cell lines. However, the large number of other insect cell lines represents a largely unexplored resource.

[00174] Also, there is an interest of producing genetically modified (G ) insect for various reasons such as agricultural production, oil production and pest control, for instance by engineering insect pests which are either sterile parents or which descendants will not be viable (Fraser 2012). Transgenic insects can be produced by using transposon DNA vector systems

[00175] Transfecting animal cells allow study the function of genes or gene products, by enhancing or inhibiting specific gene expression in cells, and to produce recombinant proteins in mammalian cells. Cultivated mammalian cells have become the dominant system for the production of recombinant proteins for clinical applications - today about 60-70% of all recombinant protein pharmaceuticals are produced in mammalian cells - because of their capacity for proper protein folding, assembly and post-translational modification. Examples are: gene therapy delivering a gene of interest into cells to cure a disease or improve symptoms; induced pluripotent stem cell (iPS cell) generation by transfecting three or four transcription factors; small interference RNA (siRNA) knock-down procedures; and production of human tissue plasminogen activator in immortalized Chinese hamster ovary (CHO) cells for therapeutic purpose.

[00176] Gene therapy is the therapeutic delivery of nucleic acid polymers into a patient's cells as a drug to treat disease. Generally, efforts focused on administering a gene that causes a needed protein to be expressed. More recently, increased understanding of nuclease function has led to more direct DNA editing, using techniques such as zinc finger nucleases and CRISPR/Cas9. The vector incorporates genes into chromosomes. The expressed nucleases then "edit" the chromosome. As of 2014 these approaches involve removing cells from patients, editing a chromosome and returning the transformed cells to patients. The delivery of DNA into cells can be accomplished by multiple methods. The two major classes are recombinant viruses (sometimes called biological nanoparticles or viral vectors) and naked DNA or DNA complexes (non-viral methods). 77] Various embodiments and aspects of the present invention as delineated herein above and as claimed in the claims section below find experimental support in the following examples.

EXAMPLES

METHODS

Preparation and labeling of ssDNA with fluorescent dye

[00179] ssDNA used in the following experiments was either a commercial Ml 3 ssDNA or prepared by the following method:

[00180] Enzymatic preparation from dsDNA by two steps: below is insufficient protocol - one "expert in the art" could not follow it as no sources, amounts, times, buffers, etc.

1] dsDNA plasmid of interest was digested with Nickase bottom enzyme (Nb] to for nicking in the non-sense strand.

2] Digestion of the nicked strand with exonuclease (ExoIII].as above? that digests only linear single strand DNA

The quality of the purified ssDNA by this procedure was verified on agarose gels (Figure D-

[00181] ssDNA was then labeled with Cy3 or Cy5 as described in the Mirus protocol

(Mirus Label IT® Tracker™ - Intracellular Nucleic Acid Localization Kit www.etc.etc,pdf).

Other general methods

A. Preparation of DNA vector carrying GFP under strong promoter (6kb) in the form of single strand.

B. Same sspl470 of group A annealed to short primer, 23bp long with sequence IDs

C. Same sspl470 of group A annealed to phosphorothioate modified primer having the same sequence of primer of group B. with sequence IDs

D. pl470 carrying GFP under strong promoter (6kb) in the form of single strand, with sequence IDs E. Same sspl470 of group A annealed to short primer, 23bp long with sequence IDs

F. Same sspl470 of group A annealed to phosphorothioate modified primer having the same sequence of primer of group B.

G. #321: An IL-60 based vector (Tomato Yellow Leaf Curled Virus (TYLCV] V2 (precoat] and VI (coat protein] ORFs] with a cassette of GFP gene under a FiMV promoter. All flanked by the TYLCV intergenic Region (IR]. with sequence IDs

H. #550: A vector carrying the DNA fragment of the MP and the Nuclear Shuttle Protein (NSP] of BDMV, each under different promoter-the 35S and the FiMV respectively. All flanked by the TYLCV-IR Region, with sequence IDs

I. #557: A vector carrying only the BDMV-NSP cassette under the FiMV promoter, with sequence IDs

J. #558: A vector carrying only the BDMV-MP cassette under the 35S promoter, with sequence IDs

Example 1. ssDNA penetration into cell nuclei of intact plant tissues within less than one hour

[00182] 24hr Chickpea (Cicer aurietinum) seedlings were treated as follows:

a. 3μ1 of ssDNA solution (0.03μg/μl] was spotted on top of emerged root embryo tissue, 24 hr post germination, followed by 30 min incubation at room temperature.

b. At the end of the incubation time the emerging seedling was water washed to remove labeled DNA residuals, and the emerging root tissue was cut out vertically sliced and subjected to confocal microscopic analysis (Leica 510] (Fig 2].

[00183] The following material was tested:

ssDNA M13 plasmid (7.1kb] - labeled with Cy5 sequence ID

ssDNA pl470 plasmid (6 kb] - labeled with Cy3 sequence ID

dsDNA pl470 labeled (6kbp] - with Cy5 sequence ID

Free Cy3 (at labeling working concentration]

[00184] Results: [00185] The nuclei and cytoplasm of cells that were treated with labeled ssDNA are labeled with fluorescence color of Cy3 or Cy5 (Fig.2). The fluorescence of Cy3 (Figure 2 C) and Cy5 (Fig. 2B) was significantly detected in cell nuclei and cytoplasm when treated with ssDNA form. This indicates that ssDNA plasmid rapidly penetrated into nuclei (in less than 1 hour), while passing the cell walls, the cell membranes and the nuclear membranes.

[00186] Treating the tissue with Cy5-dsDNA revealed sporadic cell nuclear penetration of the double-stranded form as compared to the single-stranded (Figure 2D).

[00187] Testing free Cy3 solution (not attached to DNA), as a control for the fluorescence analysis and to eliminate the possibility that fluorescence staining of nuclei was due to free dye, revealed no nuclear staining (Figure 2E). These results clearly indicate that the fluorescence staining analysis received with the Cy labeled DNA was indicative of the DNA penetration and that ssDNA is far more efficient at reaching the nucleus than dsDNA.

[00188] This experiment clearly demonstrated that ss plasmid DNA is rapidly and efficiently incorporated into the nuclei of intact plant tissue when used as a single stranded form.

Example 2.The presence of the introduced ssDNA is detected 24hr post treatment.

[00189] 24 hr old chickpea seedlings were treated with 3μ1 of DNA solution

(0.03μ^μ1) as described in Example 1 and incubated for additional 24hr. Seedlings were grown on germinating paper under controlled condition of 22°C -24°C and long day (16 h day light). The 48hr old germinated root tissue was analyzed as described in example 1.

Results:

[00190] Fluorescence staining was observed in cells of Cy3-single stranded DNA treated tissue 24 hr post treatment. The luminescence was mainly observed in the nucleus and cytoplasm (Figure 3). This demonstrated the presence of the introduced DNA plasmid in nuclei even after 24 hr, which may indicate on the stability of the DNA once introduced to the cell.

Example 3: ssDNA annealed to a primer leads to GFP expression in Arabidopsis

[00191] 5-10 day old Arabidopsis thaliana seedlings, grown on 0.5x Murashige and

Skoog medium (MS), 0.8% agar plate, under controlled condition of 22°C -24°C and long day (16 h day light), were treated with various solutions as follows:

[00192] 3μ1 of DNA solution (0.05μg/μl) was pipeted on intact seedling roots and incubated under the above growth conditions for an additional 24hr or 48hr.

[00193] To monitor GFP expression the whole tested seedlings were placed on glass slides and analyzed under confocal microscope using GFP filter (BP 505-550 and excitation laser of 488 nm), :

[00194] The following material treated groups were tested:

[00195] pl470 carrying GFP under strong promoter (6kb) in the form of single strand, sequence ID

[00196] Same sspl470 of group A annealed to short primer, 23bp long sequence ID

[00197] Same sspl470 of group A annealed to phosphorothioate modified primer having the same sequence of primer of group B. sequence ID

[00198] Each primer was annealed to the ssDNA plasmid prior the treatment, by subjecting the mixture to 5 min incubation at 70°C followed by slow cooling to 25 °C.

[00199] Results:

[00200] GFP fluorescence was observed in all treated groups both at 24hr and 48hr incubation time (Fig. 4). However, the expression in group A and B was inconclusive, where in group C the GFP expression was clearly observed in nucleus and in the cytoplasm of the root cells (Figure 4 C). This suggests that the low expression in group B can be attributed to the high sensitivity of DNA oligonucleotides to endo- and exonucleases within the treated cells. Where the addition of primer modified with phosphorothioate, which confers higher resistance to nuclease attack, may over come degradation and lead to higher expression. Example 4 . Increase expression by adding MP-BDMV element in trans

[00201] The effects of the BDMV MP sequence ID or its Nuclear Shuttle Protein

(NSP) sequence ID, or both, were studied on the expression of a reporter gene located on a different vector,.

[00202] Several Agrobacterium tumefaciens binary vectors were constructed

(Figure 5):

[00203] #321 : An IL-60 based vector (Tomato Yellow Leaf Curled Virus (TYLCV)

V2 (precoat) and VI (coat protein) ORFs) with a cassette of GFP gene under a FiMV promoter. All flanked by the TYLCV intergenic Region (IR).

[00204] #550: A vector carrying the DNA fragment of the MP and the Nuclear

Shuttle Protein (NSP) of BDMV, each under different promoter-the 35S and the FiMV respectively. All flanked by the TYLCV-IR Region.

[00205] #557: A vector carrying only the BDMV-NSP cassette under the FiMV promoter, sequence ID

[00206] #558: A vector carrying only the BDMV-MP cassette under the 35S

Promoter, sequence ID

[00207] Nicotiana benthamiana leaves (aged 3-4 weeks) were agroinfected with vector #321 with and without the addition of vector #550 ("MP+NSP") or #557 ("NSP") or #558 ("MP"), and the GFP fluorescence was monitored by binocular and by confocal microscope 3 days post treatment, as described in Example 3.

Results:

[00208] BDMV-MP and NSP elements (#550) had a dramatic effect of on GFP fluorescence (carried by #321) in infiltrated area (Fig, 6). Cells are more fluorescent: i.e. there is more GFP expression in each cell (Fig. 7) as a result of more RNA per DNA being produced (Fig. 8), as compare to without the addition of the BDMV-MP and NSP elements. [00209] The dramatic effect is related mainly to the MP contribution, where the NSP add minor increase when use without the MP element (Fig. 6).

Example 5:

[00210] Nicotiana benthamiana leaves (aged 4 weeks) were agroinfected with vector #327 sequence ID, which does not carry CP and V2, with and without the addition of vector #550 ("MP+NSP") sequence ID or #557 ("NSP") sequence ID or #558 ("MP") sequence ID (Fig. 9), and the GFP fluorescence was monitored by binocular and by confocal microscope 3 days post treatment.

[00211] As with above example - including construct with CP and V2 - this experiment revealed dramatic effect of BDMV-MP and NSP elements (#550) on GFP fluorescence (carried by #321) in infiltrated area (Figure 5), and is not related to CP and V2.

[00212] The dramatic effect is related mainly to the MP contribution (figure 10).

Claims

What is claimed is:

1. A method for introducing single-stranded nucleic acid into an intact plant cell leading to vastly improved speed and efficiency of DNA uptake and

expression; wherein, said nucleic acid is based upon flexible, single-strand DNA which rapidly penetrates intact plant cells and is expressed in the plant cells at comparatively higher levels; wherein said single-stranded DNA is attached to a short piece of primer DNA; and, wherein said nucleic acid is an expression vector based on a Geminivirus .

2. The method of claim 1, wherein said Geminivirus expression vector comprises a Geminivirus intergenic region (IR) and lacks Replication associated protein (Rep) gene and is either lacking coat protein (CP) or comprises a non- modified (CP) .

3. The method of claim 1 , wherein said expression vector is further comprised of an expression enhancer DMV MP.

4. The method of claim 1, wherein said nucleic acid is a single-strand RNA.

5. The method of claim 1, wherein said intact plant cell is found within intact plant tissue said plant tissue found among intact plant seeds, intact plant roots or intact plant leaves.

6. The method of claim 5 wherein said intact plant cell is found among all plants.

7. The method of claim 1 wherein said Gemini virus is a begomo virus.

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8. The method of claim 1 wherein said Gemini virus is a tomato yellow leaf curl virus.

9. The method of claim 6, wherein said plants are selected from the group consisting of a Solanaceae, a Cucurbitaceae, an Umbelliferae, a Liliacae, a Gramineae, a Rosaceae Musaceae, Vitocea and a Cruciferae.

10. The method of claim 1, wherein said Geminivirus expression vector comprises a separated single-strand heterologos polynucleotide sequence; wherein, said sequence encodes a selected gene or genes.

11. The method of claim 10, wherein said heterologous polynucleotide encodes a polypeptide selected from the group comprising a reporter molecule, an antiviral molecule, a viral moiety, an antifungal molecule, an antibacterial molecule, an insect resistance molecule, a herbicide resistance molecule, a biotic or abiotic stress tolerance molecule, a pharmaceutical molecule, a growth inducing molecule and a growth inhibiting molecule.

12. A method for modifying fruit maturation comprising the step of reducing gene expression with the expression vector according to claim 1; wherein, said genes are selected from the group consisting of poly galacturonases, pectin esterases, beta-(l, 4) gluconases (cellulases), beta-galactanases (beta- galactosidases), or genes of ethylene biosynthesis, genes of carotenoid biosynthesis ,phytoene biosynthesis, O-methyltransferases, acyl carrier protein, elongation factor, auxin-induced gene, cysteine proteinases, starch

phosphorylases, pyruvate decarboxylases, chalcone reductases, protein kinases, auxin-related gene, sucrose transporter and meristem pattern gene.

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13 . A method for improving cell protection against abiotic stress factors, said method employing the expression vector according to claim 1; wherein, said method comprises the step of reducing gene expression of genes which are implicated in stress reactions

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