PROTEIN PRODUCTION IN PLANT CELLS

Abstract

Improved methods of producing nucleic acid molecules, proteins and peptides in host cells and genetically engineered plants, vectors and constructs therefor.

Claims

1. An isolated polynucleotide sequence comprising at least one of i) an organellar transgene cassette comprising two origins of replication, one being located adjacent to and at the 5′ end of a left flanking sequence and the second being located adjacent to and at the 3′ end of a right flanking sequence, at least one DNA sequence of interest under operative control of an organellar promoter, and an organellar terminator; and ii) an organellar transgene cassette comprising two origins of replication located at the 5′ and 3′ ends of the cassette, respectively, at least one DNA sequence of interest under operative control of an organellar promoter, wherein the organellar promoter is positioned downstream of the origin of replication at the 5′ end of the transgene cassette, and an organellar terminator and the organellar cassette does not contain left and right flanking sequences; and wherein the said origins of replication are all derived from a geminivirus.

2. An isolated polynucleotide sequence as defined in claim 1 comprising genomic DNA and/or cDNA.

3. Use of a polynucleotide sequence according to claim 1 in the production of a transgenic plant.

4. Use of a polynucleotide sequence according to claim 1 in the production of a polypeptide or protein in a plant.

5. A plant cell transformed with a vector, a transgene cassette, transgene or isolated DNA sequence as defined in claim 1.

6. A plant cell according to claim 5, including transformed organelles selected from plant plastids and mitochondria transformed with a vector, a transgene cassette, transgene or isolated DNA sequence as defined in claim 1.

7. A transformed plant organelle as defined in claim 6.

8. A population of transformed plant organelles according to claim 7 comprised in a plant cell.

9. A population of transformed plant organelles according to claim 8, wherein the organelles are located in plant cells selected from tobacco (Nicotiana tabacum) and other Nicotiana species, arabidopsis, potato, corn(maize), canola (rape), rice, wheat, barley, brassica sp. such as cauliflower, broccoli (e.g. green and purple sprouting), cabbage (e.g. red, green and white cabbages), curly kale, Brussels sprouts, cotton, algae (e.g. blue green species), lemnospora, or moss (e.g. Physcomitrella patens), tomato, capsicum, squashes, sunflower, soyabean, carrot, melons, grape vines, lettuce, strawberry, sugar beet, peas, and sorghum.

10. A population of transformed plant organelles according to claim 8, wherein the organelles are located in plant cells selected from cotton, rice, oilseed Brassica species such as canola, corn(maize) and soyabean.

11. A method of producing a transgenic plant that comprises: 1) introducing into a regenerable plant cell a vector, transgene cassette, transgene or isolated DNA sequence as defined in claim 1; 2) growing said regenerable plant cell of step (1); 3) selecting a plant cell of (2), wherein the transgene or isolated DNA sequence is integrated into the organellar genome or the transgene or isolated DNA sequence is comprised in an independent replicon (mini-chromosome) in the organelle; 4) regenerating a plant from the plant cell of (3); and 5) growing the plant of (4).

12. A method according to claim 11, wherein the plant organellar genome is independently selected from that of plant mitochondria and plant plastids.

13. A method according to claim 11, wherein step (1) additionally comprises introducing a second nucleic acid sequence into the regenerable plant cell comprising a viral Rep gene co-presented on a nuclear cassette comprising a Rep gene fused to an organellar transit peptide, wherein the fused peptide is under operational control of a nuclear promoter and a nuclear terminator.

14. A method according to claim 11, wherein step (1) additionally comprises introducing a second nucleic acid sequence into the regenerable plant cell comprising a viral Rep gene cassette integrated into the organellar genome and is under operational control of a plastid/mitochondria promoter and a organellar terminator.

15. A method according to claim 11, wherein the vector further comprises a Rep gene and is under operational control of a plastid or mitochondrial promoter and an organellar terminator.

16. A method according to claim 11, wherein step (1) is carried out by Agrobacterium transformation, micro projectile bombardment, electroporation, and/or direct DNA uptake.

17. A host cell containing a heterologous polynucleotide or nucleic acid vector as defined in claim 1.

18. A host cell according to claim 17 which is a plant cell or a bacterial cell.

19. A host cell according to claim 17 comprised in a plant, a plant part or a plant propagule, or an extract or derivative of a plant or in a plant cell culture.

20. A plant comprising a plant cell according to claim 5.

21. A plant comprising a plant cell according to claim 20 that is selected from the group consisting of tobacco (Nicotiana tabacum) and other Nicotiana species, such as Nicotiana benthamiana, carrot, vegetable and oilseed Brassicas, melons, Capsicums, grape vines, lettuce, strawberry, sugar beet, wheat, barley, (corn)maize, rice, soybean, peas, sorghum, sunflower, tomato, cotton, and potato.

22. A plant comprising a plant cell according to claim 20 that is selected from the group consisting of cotton, rice, oilseed Brassica species such as canola, corn(maize) and soybean.

Description

FIGURES

[0098] FIG. 1. Schematic presentation of wild type Ec86 retron (A), and reshuffled version of the retron for reverse transcription of TNA (B). Constructs with fusion between Ec86 reverse transcriptase and groupII intron-encoded protein (IEP) such as LtrA, RmInt IEP and a12 IEP were used to reverse transcribe TNA-RNA translocated into the organelles (C). A rigid linker and transit peptide (TP) were added to optimise expression and targeting of the fused peptide to corresponding organelles.

[0099] FIG. 2. GroupII intron-based vectors for TNA-RNA delivery into the plant organelles. TNA was inserted either in domainIV of the intron (A) or flanked by the intron on 5′ or 3′-end of the TNA (B). Each construct contains reshuffled retron at 3′-end for reverse transcription of the TNA-RNA into ssDNA. The Ec86 RT-IEP fusion can both translocate TNA into the organelles and perform reverse transcription of the TNA.

[0100] FIG. 3. Potato Virus Y (PVY)-base vector for TNA-RNA delivery into the plant organelles. PVY polymerase and coat protein were replaced by TNA with the reshuffled retron at 3′-end (A). Thus the vector contains all viral genes at its 5′-end, and the TNA at the 3′-end. Viral VPg protein was functionally fused with chloroplast or mitochondrial transit peptide (TP) for translocation of viral-TNA RNA covalently linked with VPg to specific organelles. A fusion of 35S promoter and viral sequence provides precise transcription start position. Viral polymerase was delivered in trans under constitutive nuclear promoter (B).

[0101] FIG. 4. Schematic presentation of modified PVY-based vector where SpyTag sequence was functionally fused either at 5′-(ST5) or 3′-ends (ST3) of the gene encoding VPg protein.

[0102] FIG. 5. Vectors for overexpression of the SpyCatcher peptide. The SpyCatcher could be expressed either from constitutive nuclear promoter or from inducible promoter, such as DEX-inducible promoter. The SpyCatcher peptide is also fused with chloroplast or mitochondrial transit peptide for translocation of TNA into organelles.

[0103] FIG. 6. Binary vectors containing modified virD2 gene. A cassette containing Agrobacterium virD1 promoter, virD1 gene, cTP- or mTP-virD2 fusion and rrnB terminator was inserted into the pBIN19 binary vector outside of the T-DNA boarders. When the vector delivered into Agrobacterium, modified virD2 protein will be produced in bacteria upon induction with acetosyringon.

[0104] FIG. 7. Binary vectors containing virD2 gene modified by fusion of SpyTag sequence to 5′-(ST5) or 3′-ends (ST3).

[0105] FIG. 8. Vectors for TNA amplification in the organelles using Geminivirus replication system. Two viral origins of replication are provided on flanks of the TNA from Maize Streak Virus (MOR), Beet Curly Top Virus (BOR), and Tomato Golden Mosaic Virus (TOR). As TNA contains LFS and RFS, amplification of the TNA facilitates quick achievement of homoplasmic state of transformants.

[0106] FIG. 9. Vectors for generation of autonomous mini-chromosome in the organelles, based on Geminivirus replication system. As the cassettes do not contain LFS and RFS, they will not be inserted in the genome of organelles, but the cassette will be amplified as long as a source of replicase is provided either from the mini-chromosome, or from plant nucleus. MOR—viral origin of replication from maize streak virus, BOR—viral origin of replication from beet top curly virus, TOR—viral origin of replication from tomato golden mosaic virus.

[0107] FIG. 10. Vectors containing cassette for overexpression of replication initiation protein (Rep) from geminivirus. The Rep gene can be fused to either chloroplast or mitochondrial transit peptides to generate amplification of TNA in organelles.

[0108] FIG. 11. PCR analysis of spectinomycin resistant plants for insertion of transgene into the chloroplast genome of tobacco (A) and rice (B).

[0109] (A): lane 1-3—OTV1; lanes 4-5—OTV2; lanes 6-7—OTV3: lanes 8-9—OTV4, lanes 10-12—OTV5; lanes 13-14—OTV6.

[0110] (B): lane 1—WT DNA of rice; lanes 2-5—OTV7; lanes 6-9—OTV8; lanes 10-13—OTV9; lanes 14-15—OTV10, lane 16—negative control.

[0111] FIG. 12. PCR analysis of spectinomycin resistant plants generated using Potato Virus Y translocation sequence. Lanes 1-4—OTV21; lanes 5-8—OTV22+OTV27; lanes 9-12—OTV23+OTV27.

[0112] FIG. 13. PCR analysis of transgene flanking sequence using virD2 approach for chloroplast transformation in tobacco. Lanes 1-5—OTV21; lanes 6-7—OTV22+OTV27; lanes 8-9—OTV23+OTV27.

[0113] FIG. 14. Southern analysis for amplification of the TNA in tobacco chloroplasts. (A) lanes 1-4—BCTV-based replicon (OTV33+OTV39); lanes 5-6—TGMV-based replicon (OTV35+OTV41). (B) lanes 1-8—MSV-based replicon (OTV34+OTV40).

[0114] FIG. 15. Southern analysis for replication of mini-chromosome in tobacco without insertion into the chloroplast genome. Lanes 1-5—BCTV-based replicon (OTV45+OTV39); lanes 6-10—TGMV-based replicon (OTV46+OTV41).

[0115] FIG. 16. PCR analysis of flanking sequences for mitochondrial transgene insertion in tobacco (A) and rice (B) using groupII intron and PVY-based translocation sequences.

[0116] (A) Lane 1—DNA of WT tobacco; lanes 2-3—OTV11; lanes 4-6 OTV12; lane7—OTV13, lane 8—OTV14, lanes 9-10—OTV15; lane 11—OTV16; lanes 12-13—OTV24; lanes 14-15—OTV25+OTV28; lane 16—OTV26+OTV28, lane 17—negative control.

[0117] (B) lanes 1-3—OTV17; lanes 4-6—OTV18; lane 7-8—OTV19; lane 9—OTV20, lane 10—negative control.

[0118] FIG. 17. PCR analysis of transgene flanking sequence using virD2 approach for mitochondria transformation in tobacco. Lane 1—DNA of WT tobacco; lanes 2-5—OTV30; lanes 6-9—OTV31+OTV28; lanes 10-13—OTV32+OTV28, lane 14—negative control. The expected size of band indicated by arrow.

[0119] FIG. 18. Southern analysis of the TNA mini-chromosome amplification in the mitochondria using Geminivirus replication system. Lanes 1-4—BCTV-based replicon (OTV47+OTV42); lanes 5-8—TGMV-based replicon (OTV48+OTV44).

[0120] FIG. 19. Table of Constructs used in performing the invention and variants 1 and 2

[0121] FIG. 20. Constructs used for chloroplast transformation in tobacco, potato and maize. The AIBW construct (OTV50) contains a replicon cassette located between two viral origins of replication from BCTV (BOR1 and BOR2). The transgene cassette contain 16S promoter from tobacco driving aadA and GFP, while repA gene from BCTV is driven by maize clpP promoter. The construct AJWP (OTV49) was used to generate transiently repA protein expression from the nucleus, to give a boost for replication of the replicon from the AIBW construct (OTV50) in the chloroplasts. It contains 35S promoter, chloroplast transit peptide (cTP) translationally fused to repA gene.

[0122] FIG. 21. Southern analysis confirming replication of transgene cassette from AIBW construct (OTV50) in tobacco (lane 1-5), in potato (lane 6-11) and in maize (lane 12-14). Expected size of replicon is around 2 kb. DNA of homoplasmic chloroplast transgenic line of tobacco was used as a positive control (line 15). WT-DNA of non-transgenic tobacco as a negative control.

EXPERIMENTAL SECTION

[0123] Nucleic Acid Amplification for Plant Organelle Transformation and Gene Expression in Plant Organelles.

Summary

[0124] Sequences employed in the invention are included hereinbelow. Table 1 shows a list of constructs employed in the three variants of the invention.

[0125] We have employed a combination of transgene nucleic acid (TNA) delivery and its amplification in the organelle to improve the efficiency of organelle transformation and transgene expression in plant organelles.

[0126] The RNA approach for transgene nucleic acid delivery utilised complex and conserved structure of group II introns and reverse transcription of the RNA in the organelles using modified retron-specific reverse transcriptase. Utilisation of the covalent link between VPg protein from Potato Virus A (PVA) or Potato Virus Y and viral RNA with transgene nucleic acid or transgene nucleic acid in combination with the SpyTag-SpyCatcher system also gave rise to efficient delivery of transgene nucleic acid into the plant organelles.

[0127] The DNA approach utilised a covalent link between specific protein and transgene nucleic acid to target it to the organelles. Utilisation of virD2 protein directly from Agrobacterium for T-DNA delivery into the organelles is described herein. Improvement of DNA delivery into organelles using a SpyTag-SpyCatcher system is also described herein.

[0128] Amplification of transgene nucleic acid in the plant organelle is achieved by utilising the replication system of plant-specific gemini viruses. Placing of the transgene nucleic acid between two viral origins of replication with simultaneous delivery of viral replication initiation protein into the plant organelles was sufficient to amplify transgene nucleic acid located between two viral origins in linear and circular forms of dsDNA, as well as in the circular form of ssDNA. Amplification of transgene nucleic acid allows efficient saturation of the organelle genome with transgene insertion, or efficient transgene expression in the plant organelle from mini-chromosomes generated from the amplification vector.

Introduction

[0129] Organelle transformation in plants has a great potential for the production of pharmaceuticals in plants, in improving the quality of food, as well as improving environmental stress resistance in plants. However, until the present invention there have been no truly efficient technologies available for organelle transformation in a broad range of crops. To date, only the bombardment method has routinely yielded transformation events in chloroplasts of tobacco, in which, however a few rounds of selection are required to achieve an homoplasmic state of transformation. The bombardment method cannot be used for the transformation of plant mitochondria, because the size of mitochondria is considerably smaller than that of chloroplasts. Thus two problems for organelle transformation needed to be addressed:

[0130] (i) delivery of transgenic nucleic acid (TNA) into organelles; and

[0131] (ii) amplification of the TNA to facilitate rapid achievement of homoplasmic state of transformants.

[0132] We have developed efficient ways for both TNA delivery and amplification to facilitate rapid generation of organelle transformation in a wide range of crops.

[0133] RNA Approach for Delivery of Transgene Nucleic Acids (TNA) into the Organelles.

[0134] The RNA approach of the present invention for delivery and insertion of transgene nucleic acid (TNA) into the plant organelle is based on (i) expression of a TNA cassette from the nucleus, (ii) recruiting TNA RNA from the cytoplasm into the organelles, (iii) reverse transcription of the recruited TNA RNA into single stranded DNA (ssDNA) in the organelles, and (iv) insertion of the TNA into the organelle genome using homologous recombination. A traditional vector is used which contains a constitutive nuclear promoter driving a TNA cassette fused with sequences for RNA translocation into the organelle and reverse transcription. Transformation could be achieved by both transient overexpression and stable transformation of the nuclear cassette.

[0135] Reverse Transcription of RNA-TNA in the Organelles.

[0136] In order to generate insertion of the TNA into the organelle genome, RNA containing the TNA is first reverse transcribed into ssDNA. For this purpose we have utilised a retron-based reverse transcription system.

[0137] A retron is a distinct DNA sequence found in the genome of many bacteria species that codes for reverse transcriptase and a unique single-stranded DNA/RNA hybrid called multicopy single-stranded DNA (msDNA). Retron msr RNA is the non-coding RNA produced by retron elements and is the immediate precursor to the synthesis of msDNA. The retron msr RNA folds into a characteristic secondary structure that contains a conserved guanosine residue at the end of a stem loop. Synthesis of DNA by the retron-encoded reverse transcriptase (RT) results in the DNA/RNA chimera which is composed of a short single-stranded DNA linked to a short single-stranded RNA. The RNA strand is joined to the 5′ end of the DNA chain via a 2′-5′ phosphodiester linkage that occurs from the 2′ position of the conserved internal guanosine residue (Lampson et al., 2005).

[0138] Retron-encoded reverse transcriptase has high efficiency for reverse transcription of fragments of up to 1000 bp, but amplification of longer fragments appears to be difficult due to the processivity—that is to say, fragment size limited processing power—of retron-encoded reverse transcriptase. Attempts at improving reverse transcription using reshuffled retrons have been made (Shimamoto et al., 1998, Rozwadowski and Lydiate, 2003), but no successful amplification of fragments longer than 1000 bp has been reported. Since chloroplast cassette for delivery of TNA exceeds significantly the length of 1000 bp, a more processive or powerful reverse transcriptase had to be engineered. We have optimized a retron-based reverse transcription system by the introduction of a reshuffled retron sequence (FIG. 1B) and fusion of this retron reverse transcriptase to a more processive reverse transcriptase encoded by a group II intron, such as LtrA from Lactococus lactis, RmInt ORF from Sinorhizobium meliloti, and the a12 intron encoded protein from Saccharomyces cerevisiae (FIG. 1C). The combination of the reshuffled retron with an engineered reverse transcriptase significantly improved reverse transcription of longer fragments. Thus, the combination of RNA delivery to plant organelles with an improved reverse transcription system considerably increased the efficiency of organelle transformation.

TABLE-US-00001 Reshuffled Ec86 retron SEQ ID 1 Ctgatgctctccgagccaaccaggaaacccgttttttctgacgtaagggtgcgcaactttcgagctcg cctgctgtgccagccggcgagcgtcgacatgcgcacccttagcgagaggtttatcattaaggtcaacc tctggatgttgtttcggcatcctgcattgaatctgagttactgtctgttttccttgttggaacggaga gcatcgctctagagtctc Eb86 RT-LtrA fusion (the linker is in bold italics) SEQ ID 2 atgaaatccgctgaatatttgaacacttttagattgagaaatctcggcctacctgtcatgaacaattt gcatgacatgtctaaggcgactcgcatatctgttgaaacacttcggttgttaatctatacagctgatt ttcgctataggatctacactgtagaaaagaaaggcccagagaagagaatgagaaccatttaccaacct tctcgagaacttaaagccttacaaggatgggttctacgtaacattttagataaactgtcgtcatctcc tttttctattggatttgaaaagcaccaatctattttaaataatgctaccccgcatattggggcaaact ttatactgaatattgatttggaggattttttcccaagtttaactgctaacaaagtttttggagtgttc cattctcttggttataatcgactaatatcttcagttttgacaaaaatatgttgttataaaaatctgct accacaaggtgctccatcatcacctaaattagctaatctaatatgttctaaacttgattatcgtattc agggttatgcaggtagtcggggcttgatatatacgagatatgccgatgatctcaccttatctgcacag tctatgaaaaaggttgttaaagcacgtgattttttattttctataatcccaagtgaaggattggttat taactcaaaaaaaacttgtattagtgggcctcgtagtcagaggaaagttacaggtttagttatttcac aagagaaagttgggataggtagagaaaaatataaagaaattagagcaaagatacatcatatattttgc ggtaagtcttctgagatagaacacgttaggggatggttgtcatttattttaagtgtggattcaaaaag ccataggagattaataacttatattagcaaattagaaaaaaaatatggaaagaaccctttaaataaag cgaagacc atgaagccaacaatggcaatcctcgaacgaatctctaagaactcacaggagaacatcgacgaggt cttcacaagactttaccgttaccttctccgtcctgacatctactacgtggcatatcagaacctctact ctaacaagggagcttctacaaagggaatcctcgatgatacagctgatggattctctgaggagaagatc aagaagatcatccaatctttgaaggacggaacttactaccctcagcctgtccgaagaatgtacatcgc aaagaagaactctaagaagatgagacctcttggaatcccaactttcacagacaagttgatccaggagg ctgtgagaatcatccttgaatctatctatgagcctgtcttcgaggatgtgtctcacggtttccgacct cagcgaagctgtcacacagctttgaagacaatcaagagagagttcggaggtgcaagatggttcgtgga gggagatatcaagggatgcttcgataacatcgaccacgtcacactcatcggactcatcaaccttaaga tcaaggatatgaagatgagccagttgatctacaagttcctcaaggcaggttacctcgaaaactggcag taccacaagacttacagcggaacacctcagggcggaatcctctctcctctcctcgctaacatctatct tcatgaattggacaagttcgttctccaactcaagatgaagttcgaccgagagagtccagagagaatca cacctgaataccgggagcttcacaacgagatcaaaagaatctctcaccgtctcaagaagttggagggc gaggagaaggctaaggttctcttggaataccaggagaagaggaagaggttgcctacactcccttgtac atcacaaacaaacaaggtcttgaagtacgtccgatacgctgacgacttcatcatctctgttaagggaa gcaaggaggactgtcaatggatcaaggagcaattgaagctcttcatccataacaagctcaagatggaa ttgagtgaggagaagacactcatcacacatagcagtcagcctgctcgtttcctcggatacgacatccg agtcaggagaagtggaactatcaagcgatctggaaaggtcaagaagagaacactcaacgggagtgtgg agcttctcatccctctccaagacaagatccgtcaattcatcttcgacaagaagatcgctatccagaag aaggatagctcatggttcccagttcacaggaagtaccttatccgttcaacagacttggagatcatcac aatctacaactctgaattgagaggtatctgcaactactacggtctcgcaagtaacttcaaccagctca actacttcgcttaccttatggaatactcttgcttgaagactatcgcatctaagcataagggaacactc tcaaagaccatctctatgttcaaggatggaagtggttcttggggaatcccttacgagatcaagcaggg gaagcagaggagatacttcgccaacttcagtgaatgcaaatctccttaccaattcactgatgagatca gtcaagctcctgtgctttacggatacgctcggaacactcttgagaacagacttaaggctaagtgttgt gagctttgtggaacatctgatgagaacacatcttacgagatccaccacgtcaacaaggtcaagaacct taagggaaaggagaagtgggagatggcaatgatcgctaagcagcggaagactcttgttgtttgcttcc attgtcatcgtcacgtgatccataagcacaagtga Ec86 RT-RmInt IEP fusion SEQ ID 3 atgaaatccgctgaatatttgaacacttttagattgagaaatctcggcctacctgtcatgaacaattt gcatgacatgtctaaggcgactcgcatatctgttgaaacacttcggttgttaatctatacagctgatt ttcgctataggatctacactgtagaaaagaaaggcccagagaagagaatgagaaccatttaccaacct tctcgagaacttaaagccttacaaggatgggttctacgtaacattttagataaactgtcgtcatctcc tttttctattggatttgaaaagcaccaatctattttaaataatgctaccccgcatattggggcaaact ttatactgaatattgatttggaggattttttcccaagtttaactgctaacaaagtttttggagtgttc cattctcttggttataatcgactaatatcttcagttttgacaaaaatatgttgttataaaaatctgct accacaaggtgctccatcatcacctaaattagctaatctaatatgttctaaacttgattatcgtattc agggttatgcaggtagtcggggcttgatatatacgagatatgccgatgatctcaccttatctgcacag tctatgaaaaaggttgttaaagcacgtgattttttattttctataatcccaagtgaaggattggttat taactcaaaaaaaacttgtattagtgggcctcgtagtcagaggaaagttacaggtttagttatttcac aagagaaagttgggataggtagagaaaaatataaagaaattagagcaaagatacatcatatattttgc ggtaagtcttctgagatagaacacgttaggggatggttgtcatttattttaagtgtggattcaaaaag ccataggagattaataacttatattagcaaattagaaaaaaaatatggaaagaaccctttaaataaag cgaagacc atgacttcggaaagtacgacagacaagccgtttcgaattgagaaacgtcgagtgtacgaagctta caaagcggtcaaagccaaccgtggcgcggccggggtggacgggcagacgctggagatatttgagaaag accttgcagcaaacctctacaagatctggaatcggatgtcctcgggaacctactttccgccgccggtg cgcgccgtctccattccgaagaaggctggaggcgaaagggttttgggtgtgcccacggtcagcgatcg gatcgcgcagatggtggtcaagcagatgatcgagccggatttggactccctctttcttccggactcct acggttacaggccgggaaaatcggccctggatgctgtcggagtgacgcgtcagcggtgctggaagtat gattgggttttggaattcgacatcaaagggctgtttgacaatcttccgcatgatctcttgctgaaggc ggtcagaaaagacgtcaaatgcaactgggctctgctctacatcgaaagatggctgactgcgcctatgg aaaagaacggagaagtcatcgagcggtcacgcggtaccccacagggaggcgtggttagcccgatcttg gcgaatctctttctgcactatgcatttgatctctggatgacgcggacgcatcccgaccttccatggtg tcgatatgccgacgatggtcttgttcactgccagagcgagcaacaagccgaagccctcagggtggagc tgagttctcggctggcagcgtgcggacttcagatgcatccgacaaagaccaagattgtctactgcaag gatcaacggcgcagggaggcgtatccgaatgtcacgttcgactttctcgggtatcagttccggccgcg acgggtggcgaacacacagcgggacgagttcttctgtggctacacgcctgcggtcagtccgacggcgc tcaagtcgatgcgggcaacgatcaaaagtttgaacatcccgcggcagacgccggggacgctggccgaa atagccaaacagctcaatccactccttcggggatggattgcctactatggacggtacagtcgttcggc cctgtccactctggctgattacgttaatcagaaactcagggcttggatcaggcgaaagttcaaacgct ttcagtcccataagacacgcgccagcctcttcttgcgaaagctggcgcgggaaaatccggggctgttc gtgcattggaaggcgttcggaacgaacacgtttacctga Ec86 RT-a12 IEP fusion SEQ ID 4 atgaaatccgctgaatatttgaacacttttagattgagaaatctcggcctacctgtcatgaacaattt gcatgacatgtctaaggcgactcgcatatctgttgaaacacttcggttgttaatctatacagctgatt ttcgctataggatctacactgtagaaaagaaaggcccagagaagagaatgagaaccatttaccaacct tctcgagaacttaaagccttacaaggatgggttctacgtaacattttagataaactgtcgtcatctcc tttttctattggatttgaaaagcaccaatctattttaaataatgctaccccgcatattggggcaaact ttatactgaatattgatttggaggattttttcccaagtttaactgctaacaaagtttttggagtgttc cattctcttggttataatcgactaatatcttcagttttgacaaaaatatgttgttataaaaatctgct accacaaggtgctccatcatcacctaaattagctaatctaatatgttctaaacttgattatcgtattc agggttatgcaggtagtcggggcttgatatatacgagatatgccgatgatctcaccttatctgcacag tctatgaaaaaggttgttaaagcacgtgattttttattttctataatcccaagtgaaggattggttat taactcaaaaaaaacttgtattagtgggcctcgtagtcagaggaaagttacaggtttagttatttcac aagagaaagttgggataggtagagaaaaatataaagaaattagagcaaagatacatcatatattttgc ggtaagtcttctgagatagaacacgttaggggatggttgtcatttattttaagtgtggattcaaaaag ccataggagattaataacttatattagcaaattagaaaaaaaatatggaaagaaccctttaaataaag cgaagacc atgccgtttcgcttaatttatcactgtattgaagtgttaattgataaacatatctctgtttattc aattaatgaaaactttaccgtatcattttggttctggttattagtagtaacatacatagtatttagat acgtaaaccatatggcttacccagttggggccaactcaacggggacaatagcatgccataaaagcgct ggagtaaaacagccagcgcaaggtaagaactgtccgatggctaggttaacgaattcctgtaaagaatg tttagggttctcattaactccttcccacttggggattgtgattcatgcttatgtattggaagaagagg tacacgagttaaccaaaaatgaatcattagctttaagtaaaagttggcatttggagggctgtacgagt tcaaatggaaaattaagaaatacgggattgtccgaaaggggaaaccctggggataacggagtcttcat agtacccaaatttaatttaaataaagcgagatactttagtactttatctaaattaaatgcaaggaagg aagacagtttagcgtatttaacaaagattaatactacggatttttccgagttaaataaattaatagaa aataatcataataaacttgaaaccattaatactagaattttaaaattaatgtcagatattagaatgtt attaattgcttataataaaattaaaagtaagaaaggtaatatatctaaaggttctaataatattacct tagatgggattaatatttcatatttaaataaattatctaaagatattaacactaatatgtttaaattt tctccggttagaagagttgaaattcctaaaacatctggaggatttagacctttaagtgttggaaatcc tagagaaaaaattgtacaagaaagtatgagaataatattagaaattatctataataatagtttctctt attattctcatggatttagacctaacttatcttgtttaacagctattattcaatgtaaaaattatatg caatactgtaattggtttattaaagtagatttaaataaatgctttgatacaattccacataatatgtt aattaatgtattaaatgagagaatcaaagataaaggtttcatagacttattatataaattattaagag ctggatatgttgataaaaataataattatcataatacaactttaggaattcctcaaggtagtgttgtc agtcctattttatgtaatatttttttagataaattagataaatatttagaaaataaatttgagaatga attcaatactggaaatatgtctaatagaggtagaaatccaatttataatagtttatcatctaaaattt atagatgtaaattattatctgaaaaattaaaattgattagattaagagaccattaccaaagaaatatg ggatccgataaaagttttaaaagagcttattttgttagatatgctgatgatattatcattggtgtaat gggttctcataatgattgtaaaaatattttaaacgatattaataacttcttaaaagaaaatttaggta tgtcaattaatatagataaatccgttattaaacattctaaagaaggagttagttttttagggtatgat gtaaaagttacaccttgggaaaaaagaccttatagaatgattaaaaaaggtgataattttattagggt tagacatcatactagtttagttgttaatgcccctattagaagtattgtaataaaattaaataaacatg gctattgttctcatggtattttaggaaaacccagaggggttggaagattaattcatgaagaaatgaaa accattttaatgcattacttagctgttggtagaggtattataaactattatagattagctaccaattt taccacattaagaggtagaattacatacattttattttattcatgttgtttaacattagcaagtaaat ttaaattaaatactgttaagaaagttattttaaaattcggtaaagtattagttgatcctcattcaaaa gttagttttagtattgatgattttaaaattagacataaaataaatataactgattctaattatacacc tgatgaaattttagatagatataaatatatgttacctagatctttatcattatttagtggtatttgtc aaatttgtggttctaaacatgatttagaagtacatcacgtaagaacattaaataatgctgccaataaa attaaagatgattatttattaggtagaatgattaagataaatagaaaacaaattactatctgtaaaac atgtcattttaaagttcatcaaggtaaatataatggtccaggtttatag

[0139] Delivery of Transgene Nucleic Acid to Organelle Using groupII Intron.

[0140] We utilise groupII introns to deliver RNA of transgene into the organelles. The cassette containing transgene nucleic acid was inserted into domainIV of LtrB intron from Lactococus lactis, RmInt1 intron from Sinorhizobium meliloti, a12 intron from Saccharomyces cerevisiae, tobacco groupII intron from nad1 gene containing matK intron-encoded gene (FIG. 1A). The transgenic nucleic acid can be fused at the 5′ or 3′-prime ends of the groupII intron (FIG. 1B), and is translocated to organelle with the same efficiency as in case when TNA was inserted in domain IV of the groupII intron. We did not observed splicing of the groupII intron in the cytoplasm of the plants and only in environment of the plant organelle intron could be spliced. Thus TNA located at any end of intron can still be translocated to organelles.

TABLE-US-00002 Lactococcus lactis LtrB intron (the cloning site for TNA in domain IV is in bold) SEQ ID 5 Gtgcgcccagatagggtgttaagtcaagtagtttaaggtactactctgtaagataacacagaaaacag ccaacctaaccgaaaagcgaaagctgatacgggaacagagcacggttggaaagcgatgagttacctaa agacaatcgggtacgactgagtcgcaatgttaatcagatataaggtataagttgtgtttactgaacgc aagtttctaatttcggttatgtgtcgatagaggaaagtgtctgaaacctctagtacaaagaaaggtaa gttatggttgtggacttatctgttatcaccacatttgtacaatctgtaggagaacctatgggaacgaa acgaaagcgatgccgagaatctgaatttaccaagacttaacactaactggggataccctaaacaagaa tgcctaatagaaaggaggaaaaaggctatagcactagagcttgaaaatcttgcaagggtacggagtac tcgtagtagtctgagaagggtaacgccctttacatggcaaaggggtacagttattgtgtactaaaatt aaaaattgattagggaggaaaacctcaaaatgaaaccaacaatggcaattttagaaagaatcagtaaa aattcacaagaaaatatagacgaagtttttacaagactttatcgttatcttttacgtccagatattta ttacgtggcgggcgcgccacgcgtgcggccgctgggaaatggcaatgatagcgaaacaacgtaaaact cttgttgtatgctttcattgtcatcgtcacgtgattcataaacacaagtgaatttttacgaacgaaca ataacagagccgtatactccgagaggggtacgtacggttcccgaagagggtggtgcaaaccagtcaca gtaatgtgaacaaggcggtacctccctacttcac Sinorhizobium meliloti RmIntl intron SEQ ID 6 gtgtgctgcagaggcacggaaggagttcaacatgaactaagaccgtggcgtaaagctgcgtgaatgat gggggacggccctccgggatcggctttcaggagcgggtctcaaaccagtccgagctgctgcggtaaag agccgtggtggtgagcgtcggatgaaacgttcggacgagatccgagcaggtgcatgtccaaaagacga acgaaagtgaaccctccgaggacgcgtcgttatgaacgtaagtgtcgtcgaaaccaggaccgtttcgt catcctgggacaagtccgccagatgcctgatgaccgggcgggcggcgaccggcgtagagggggcgtga gttggacataggctttcacgcggaactgcaggaaccaggctcctgatgtcaagggagaagctcaagcg gcgcaaaccgcaaggcgagagtaccgatgcaggagactggggcggatcgccccgtatgagcgtcgagg accctgtaatggggtcggagcaaagggggcggatcaggccgtcgtattgtttgaaacaactggaaaca ggatgacttcggaaagtacgacagacaagccgtttcgaattgagaaacgtcgagtgtacgaagcttac aaagcggtcaaagccaaccgtggcgcggccggggtggacgggcagacgctggagatatttgagaaagg gcgcgccacgcgtgcggccgcgccagcctcttcttgcgaaagctggcgcgggaaaatccggggctgtt cgtgcattggaaggcgttcggaacgaacacgtttacctgatgggagcggtgtgaatcgagaggttcac gcaccgttctgcgagaggccggctggtgaaactcctccggcctactcacc Saccharomyces cerevisiae a12 intron SEQ ID 7 Gcgccgtttcgcttaatttatcactgtattgaagtgttaattgataaacatatctctgtttattcaat taatgaaaactttaccgtatcattttggttctgattattagtagtaacatacatagtatttagatacg taaaccatatggcttacccagttggggccaactcaacggggacaatagcatgccataaaagcgctgga gtaaaacagccagcgcaaggtaagaactgtccgatggctaggttaacgaattcctgtaaagaatgttt agggttctcattaactccttcccacttggggattgtgattcatgcttatgtattggaagaagaggtac acgagttaaccaaaaatgaatcattagctttaagtaaaagttgacatttggagggctgtacgagttca aatggaaaattaagaaatacgggattgtccgaaaggggaaaccctggggataacggagtcttcatagt acccaaatttaatttaaataaagcgagatactttagtactttatctaaattaaatgcaaggaaggaag acagtttagcgtatttaacaaagattaatactacggatttttccgagttaaataaattaatagaaggc gcgccacgcgtgcggccgcatgattaagataaatagaaaacaaattactatctgtaaaacatgtcatt ttaaagttcatcaaggtaaatataatggtccaggtttataataattattatactccttcggggtcgcc gcgggggcgggccggactattaaatatgcgttaaatggagagccgtatgatatgaaagtatcacgtac ggttcggagagggctcttttatatgaatgttattacattcagataggtttgctactctaaa Tobacco nadl intron SEQ ID 8 gtgcggggctttgcatctgacattcgttgggcttctctcttcgggagcctgcgccccggcgtttttgt gcaataaacccctccggccgaagactagtggtaggtggtcctgcggagctttcggaaaagggtagcct tgtgtgtaagcacagcaatgaaccgcggcgaaccctcagacgacctatctaagattagggggggatcc tcagtagtggtgaccctttcactcttccacggactgatacatgtaccgaatgctcatacgggaaagtt tactcctgggtctggaacctggggggttgctccgagaaatcctttctttctcgtccactcaggggggt gcggacacacctgcgcggattacaggtgacagttacaagaatggcggggaagttaacagtacccgacg acattcagggatggatgtagacccatcgggcagggataatcattccggtcctgggagaagtggcgacc attctcaagaaccaaaaagactgagctgagggaagccctatgagtcactgaaacgacggcaggagtgc cctttttctatcaatagagggagcaaaaaacgggctttgctcccctttacaatatgaagaaagaaata agggtcgaagtttagaccgctcacagtagttctacctatagaaaggatcatgaaagaggcgatcagaa tggtactcgaatccatttacgatctcgagtttccagacacatcgcacttccgctcgggtcgaggcttc cactccgtcctaagacggggcgcgccacgcgtgcggccgctagagcttgggaagctcggatccggtca agatccgaacaacaatgagcactcaactactagtaaaaagggagaaagttgactttgagaaagaaggt gcttcttgccgctttattagtaagtaagcttgttttatatctcctcaataaaggcgaaagatcactcc taaaagcaagctttctcttatatacgataccataccacataatttcatttgccttcctgcttaaggca ctagttcggatgga Tobacco matR gene from nadl intron SEQ ID 9 atgaaagaggcgatcagaatggtactcgaatccatttacgatctcgagtttccagacacatcgcactt ccgctcgggtcgaggcttccactccgtcctaagacggatcaaagaagagtggggaacctctcgctggt ttttggaattcgacatcaggaagtgttttcacaccatcgaccgacatcgactcatcccaatctttaag gaagagatcgacgatcccaagttcttttaccccattcagaaagtcttttccgccggacgactcgtagg aggtgagaagggcccttactccgtcccacacagtgtattactatcggccctaccaggcaacatctacc tacacaagctcgatcaggagatagggaggatccgacagaagtacgaaattccgattgttcagagaata agatcggttctattaagaacaggtcgtattgatgaccaagaaaagtcttccgaagaagcaagcttcaa cgctccccaagacaacagagccatcattgtggggaggttaaagagcatccaacgcaaagcggcctttc attcccttgtttcgtcgtggcacaccccccccacaagcaccccccggctcaggggggaccagaaaacg cctttcgttttccacccttcgtcggcccttgccgccttccttaacaagccctcgagcctcctttgcgc cgccttcttcatagaagccgccgggtttacccggaagtccgaattctatggtagagaacgctgtaata ataattgggccatgagagactcttttaagtattgcaaaagaaagggcccgctgatagagctgggcggg gaggcgatacttgttatcaggtcagagagaggcctggcccgtaagctggcccccttaaaaacctatta cttaataaggatttgttacgcgcgatatgccgacgacttactactgggaatcgtgggttccgtcgagc ttctcatagaaatacaaaaacgtatcgcccacttcctacaatctggcttgaacctttgggtagactct gcaggatcaacaaccatagctgcacggagtacggtagaattcctcggtacggtcattcgggaagtccc tccgagggcgactcccatacaattcttgcgagagctggagaagcgtctacgggtaaagcaccgtatcc atataactgcttgccacctacgctccgccatccattcaaagtttaggaacctaggtaatagtatcccg atcaaagagctgacgaaggggatgagcggaacagggagtctactggacgcggttcaactagcggagac tcttggaacagctggagtaagaagtccccaagtgagcgtcttatggggggccgtcaagcacatacggc aaggatcaagggagatctcgttgttgcatagctcaggtcggagcaaggtgccatcggacgttcaacag gtagtctcacgatcgggcactcatgccccgacattgtcattgtatactcccgcgggtcggaaggcggc gggggaaggagggggacactgggcgagatctatcagcagcgaattccccatacaaatagaggcaccta tcaaaaagatacttcgaaggcttcgggatcgaggtctcattagccgaagaagaccctggccaatccac gtggcctgcttgacgaacgtcagcgacggagacatcgtaaattggtccgcgggcatcgcgataagtcc tctgtcctactacaggtgctgcgacaacctttaccaagtccgaacgattgtcgaccaccagatccgct ggtctgcaatattcaccccggcccacaagcacaaatcctcggcgcggaatataatcctaaagtactcc aaagactcaaatatagtcaatcaagaaggtggtaagacccttgcagagttccccaacagcatagagct tgggaagctcggatccggtcaagatccgaacaacaatgagcactcaactactagtaaaaagggagaaa gttga Chloroplast Transit Peptide SEQ ID 10 Atggcttcttctgctcaaatacacggtctcggaaccgcttctttctcttccctcaaaaaaccctcttc catatccggcaactccaaaacccttttcttcggtcagcgactcaattccaaccactctcccttcaccc gcgccgcattccctaaattaagtagcaaaacctttaagaagggtttcactttgagagtt Mitochondria Transit Peptide SEQ ID 11 Atggcttctcggaggcttctcgcctctctcctccgtcaatcggctcaacgtggcggcggtctaatttc ccgatcgttaggaaactccatccctaaatccgcttcacgcgcctcttcacgcgcatcccctaagggat tcctcttaaaccgcgccgtacagtacgctacctccgcagcggcaccggcatctcagccatca Tobacco chloroplast LFS SEQ ID 12 Gcgttcgaactccttcttaaacaacatcgaattaaaccaccatctttccatagagttttcttgccccc tatttgcatgaaaatacaatagatgaatagtcattcgctataaaattatttatttgaatatcttattt cctatcagactaagcatagaaatccaatcactaggattattaactaataaggattgtgagtattgaaa aaaagttctgaatctgggggaacacttcactatatattaatatgttggaaccccctttatattattta aaataatataatttttaataaagggcggcttctcctatgtcgtgtcaaattcgcatcgaaaaaagaga tttgtcctctcctataaagaaataaaaaaataattgtttcgtaaaatctcgtctaatactaatatcta atcactaacaaatctaaaatttaataaaaaaataagtaataaattaaggttctatttcaacacggaac aaaggggacaatatacaggatgggtagaaagaggtgtgatacttggcttgattcagggaaactacaaa ctacaggatagaaaagaatataccaatcctaaggatccgtaggattaattgtggatccaagacaacaa tagaaagatttgag Tobacco chloroplast RFS SEQ ID 13 Ctagattttgtatttcaaatcttgtatatctaggtaagtatatacttagtcaaaatatatgcaataga atctttgttgtattcggctcaatccttttagtaaaagattgggccgagtttaattgcaattcaattaa gagaacgaaggataattacttgagttctttctccttatccttctttatttcctgctaatttatctgct aatgtctactgtttttacttatccaaaacgtccactgctgcaaaattaaatacgatctctttccatac ttcacaagcagcagctagttccgggctccatttgcaagcctcgcgaataatttcattaccttcctgag caagatcacgtccttcattacgagcttttacacatgcttctagagctactcgattagctacggcacct ggcgcattaccccaaggatgtcctaaagttcctccaccgaactgtagtacggaatcatccccaaagat ctcggtcagagcaggcatatgccaaacgtgaatacctcctgaagccacgggtagaacacctggtaaag agacccaatcttgagtgaaataaataccgcgacttcgatcttgttcaacaaaatcatcacgcagtaaa tcaacaaagcccaaagttatgtct Rice chloroplast LFS SEQ ID 14 Ccgtgtcaatcacttccattcctctcatcaacccatctgtagcactcatagctacagctctaactcga ttatttcctaataattgttgtacctcacaagttacattaatttgcttaccgtcagtgtctcgactctt gactaccaaagcattataaatataaggtaacttgcccgggggaaaagtgacatccagcacgggtccaa taatttgatcgatacgccctgtacttttttcttcaattgtagaaaccccgggacgagaagtagtagga ttggttctcataattatcacataattttcaaaaaaaaggaatttatcgaaattttgatttttttcttg ttgaataatgccaaatcaacaccaaaaaaatatccaaaaatccaaaagtcaaaaggaaatgaattagt taattcaataagagagaaaaggggaccagcacttgatttcgttgcccaaacgaatcccattcaatcgt ttactcatggaatgagcccgtcggaaagttcaatcaatctttttttcatatacattttgccttttgta aacgatttgtgcctactctactttcttatctaggacttcgatatacaaaatatatactactgtgaagc atagattgctgtcaacagagaattttcgtagtatttaggtatttccactcaaaataagaaaagggggt ctattaagaacttaataaggattagaagttgatttggggttgcgctatatctattaaagagtatacaa taaagatggatttggtgaatcaaatccatggtttaataacgaagcatgttaacttaccataacaacaa C Rice chloroplast RFS SEQ ID 15 Tcaattcttatcgaattcctatagtagaattcctatagcatagaatgtacacagggtgtacccattat atatgaatgaaacatattatatgaatgaaacatattcattaacttaagcatgccccccattttcttta atgagttgatattaattgaatatcttttttttaagatttttgcaaaggtttcatttacgcctaatcca tatcgagtagaccctgtcgttgtgagaattcttaattcatgagttgtagggagggacgtatgtcacca caaacagaaactaaagcaagtgttggatttaaagctggtgttaaggattataaattgacttactacac cccggagtacgaaaccaaggacactgatatcttggcagcattccgagtaactcctcagccgggggttc cgcccgaagaagcaggggctgcagtagctgccgaatcttctactggtacatggacaactgtttggact gatggacttaccagtcttgatcgttacaaaggccgatgctatcacatcgagcccgttgttggggagga taatcaatatatcgcttatgtagcttatccattagacctatttgaagagggttctgttactaacatgt ttacttccattgtgggtaacgtatttggtttcaaagccctacgcgctctacgtctggaggatctgcga attccccctacttattcaaaaactttccaaggtccgcctcatggtatccaagttgaaagggataagtt gaacaaatacggtcgtcctttattgggatgtactattaaaccaaaattgggattatctgcaaaaaatt atggtagagcatgttatgagtgtctacgcggtgg rrnB terminator SEQ ID 16 aggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggt gaacgctctcctgagtaggacaaatccgccc aadA gene SEQ ID 17 atgagggaagcggtgatcgccgaagtatcgactcaactatcagaggtagttggcgtcatcgagcgcca tctcgaaccgacgttgctggccgtacatttgtacggctccgcagtggatggcggcctgaagccacaca gtgatattgatttgctggttacggtgaccgtaaggcttgatgaaacaacgcggcgagctttgatcaac gaccttttggaaacttcggcttcccctggagagagcgagattctccgcgctgtagaagtcaccattgt tgtgcacgacgacatcattccgtggcgttatccagctaagcgcgaactgcaatttggagaatggcagc gcaatgacattcttgcaggtatcttcgagccagccacgatcgacattgatctggctatcttgctgaca aaagcaagagaacatagcgttgccttggtaggtccagcggcggaggaactctttgatccggttcctga acaggatctatttgaggcgctaaatgaaaccttaacgctatggaactcgccgcccgactgggctggcg atgagcgaaatgtagtgcttacgttgtcccgcatttggtacagcgcagtaaccggcaaaatcgcgccg aaggatgtcgctgccgactgggcaatggagcgcctgccggcccagtatcagcccgtcatacttgaagc tagacaggcttatcttggacaagaagaagatcgcttggcctcgcgcgcagatcagttggaagaatttg tccactacgtgaaaggcgagatcaccaaggtagtcggcaaataa mGFP4 gene SEQ ID 18 atgagtaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggtgatgttaa tgggcacaaattttctgtcagtggagagggtgaaggtgatgcaacatacggaaaacttacccttaaat ttatttgcactactggaaaactacctgttccatggccaacacttgtcactactttctcttatggtgtt caatgcttttcaagatacccagatcatatgaagcggcacgacttcttcaagagcgccatgcctgaggg atacgtgcaggagaggaccatcttcttcaaggacgacgggaactacaagacacgtgctgaagtcaagt ttgagggagacaccctcgtcaacaggatcgagcttaagggaatcgatttcaaggaggacggaaacatc ctcggccacaagttggaatacaactacaactcccacaacgtatacatcatggcagacaaacaaaagaa tggaatcaaagttaacttcaaaattagacacaacattgaagatggaagcgttcaactagcagaccatt atcaacaaaatactccaattggcgatggccctgtccttttaccagacaaccattacctgtccacacaa tctgccctttcgaaagatcccaacgaaaagagagaccacatggtccttcttgagtttgtaacagctgc tgggattacacatggcatggatgaactatacaaataa Tobacco Prrn chloroplast promoter SEQ ID 19 Caatgtgagtttttgtagttggatttgctcccccgccgtcgttcaatgagaatggataagaggctcgt gggattgacgtgagggggcagggatggctatatttctgggagcgaactccgggcgaatatgaagcgca tcgatacaagt Wheat Prrn chloroplast promoter SEQ ID 20 Caatgtgagttttttctattttgacttactcccccgccacgagcgaacgggaatggataagaggcttg tgggattgacgtgatagggtagggttggctatactgctggtggcgaactccaggctaataatctgaag cgcatggatacaagttatccttggaaggaaagacaattccgaatctgctttgtctacgaataaggaag ctataagtaatgcaactatgaatctcatg Tobacco atp9 mitochondrial promoter SEQ ID 21 Gggataagtgaaatcgtatgtatccatccatggtgtatctggtgctctcgtatataagagaagggcag catttatgagtaatcgatctcacaaactatcaatttcataagagaagacgaagacggatcaaattgaa taatcgaagagagatgggaccctagctacgagtcattccctctgacgtcgaatgatctacttgcttgt acttctctttgtcgagattcagttggtcttcagtctaccactccgtgggtataagatcgcaaagaatg cattccaagtgagatgtccaagatcaaaggaacgagggtaagaatcgacgaggaatcaataagatata agataagtga Rice atp6 mitochondrial promoter SEQ ID 22 Acataagccatccgaaaccagtattggaaagtgttcagtttcgttttccattctgaaatgttcatagt agtatagtatgttttccgttgggtcgacgccatgtgatcgctactaaagatagagtttccttggaaaa accgaggccagttgagatcagtctccctttctaggagcagagcttaaaaagatgggaaattcc Tobacco mitochondrial LFS SEQ ID 23 Tatgtgtggaacctggtctttttcggttccagcctctccctcgaatacatagggtaggtagggctggg tgagaaatggttccctcttgccaataaactttccccggccttcgattaaccttactcataaagggtct tacggtcgggagaactacctaactaaagaaaaatagtgttctttctaagagtaggcgtggagagcttt ttgcggggaaacttgcaagtacagtttggggggaggcgggcgtcgaccctaccttatgagtattcgga ctataacagttccgatgaacagtcactcacttttgacagttatacgattccagaagatgatccagaat tgggtcaatcacgtttattagaagtcgacaatagagtggttgtaccagcaaaaagttatatacgtttt attgtaacatctgctgatgtacctcatagttgggctgtaccttccttaggtgtcaaatgtgatgctgt acctggtcgtttaaatcagacctctatttcggtacaacgagaaggagtttactatggtcagtgcagtg agatttgtggaactaatcatgcctttatgcctatcgtcgtagaagctgttcctaggaaagattatggg tctcgggtatccaatcaattaatcccacaaaccggggaagcttaagcggaaatgaaagaggagggtga gggaagccactaaattgagggcttcgctcgctcgctctaacgctcgtttagtagacagcgagtggagt gcataagcccctttagagataggggtgagtactacacgagctcgtaagtaaagtacggaacgagcctt gtctacgaagcagagcgacctcatcttgcttgcttctggcgaagcttctagctctaaataattggaat tctggtatggcaggaatactgtcgaccattacgagcgatagcgaagccaagccgtataaaggcgagca gcccttatagcaatagcaaacggcctacttatagcctat Tobacco mitochondrial RFS SEQ ID 24 Caacaggtcagtcaatatcagtaggggtcctcttgcctaacggagtcagcccaacatggacaatgata ggcagaccaaagatttacgcagtcgttgcgtgcttgctttgcgcaccggcatagcagaattcgaatcc gctggctcagatgagtggctcttggcttcgtaaacatatctatgttgttgctttttcactaccaatga gtaggcagctttggatgcttatggagatatggctttggtaaagatctgcttagcgtgtgctttctcgg gtgctacttagaatagagatagtcagactctaacttgagaatgttatagcgctgtgaaataaggacat tctgatcgacccgattggctctcgttctggtttggcggaaaggtgaaaagcactaaatctttcttcct ggttggtgtactagggcgaggcgaatcccaaccccttcgttagctagcttagctttccctcttttcaa tctatatcagatcctccattacttcttcgccaataccttttagctttcctttagctgctactttttcc cagtccacgcccaatcagagtagtcagtgtgcctgctccgtccttctttgacgaaatggatgctgtag gagaggttgggaaggagggacttcgctaaagatggtctgtctgtgcgcgaggaaggtctttttccttt ctccttccattgcttgactaggttcgctttgcaaggaagggaaggcatccgtgcaggtagaaaaaggc ggaggtcaagctatgggcacaaggaggtaaggtatagtaagttacttcttcgtcttttgcttgtcatt ggattggaagccgcaggcgatgccttcttgcttgtgtagttggccttgcctgcttagtgcggaagtgc gtaaagtaggctcattctttggtttataaagatcttgtagtagccgaaggtagtccgcttgttagatt gaattgaatcttatataacaaccggggccttattaattaagagactttatcaatagtataagtggacc tctcaaaggtataagtagacattagtcttgctggttcgggcggtaaggccctgggtaag Rice mitochondrial LFS SEQ ID 25 Ggtcgatacgatatgactaataataccaaatccaggcagaatgagaatatacacctctggatgaccga agaaccaaaagagatgctggtataatattgggtctccccctcctgcaggatcaaaaaaggttgtatta aagtttcgatcggttaataacattgtaattgcccccgccagtaccggaagtgataataaaagtaggaa tgctgtcactagaacggaccacacaaaaagtggtaatctatgcatagtcattccaggtccacgcatgt tgaagatagttgttataaaattgatagaacctaaaattgatgaaatacctgatagatgaagactaaaa attgctaaatcaactgctcctccagaatggctggtaataccacttaagggcggatagactgtccaccc agtgccgctgcccacttctactaaggctgagcttaataggagcaagagacttggtggcaacaaccaga atgatatattatttaatcgtggaaatgccatgtcaggtgcacctatcagaatcggaacaaaccaatta ccaaatccacctatcatcgccggcataaccataaaaaagatcattaaaaaagcatgagccgttattaa aacattataaagttgatgattcccaccaagaatttgatcgccgggtcgggctaattccatacgaatca gtacggagaagcatgtgcccatcactcctgcaatggcaccgaagatgaaatagagagtcccaatatcc ttgtggttagtagagaagagccatcgaaccatatttgtcattttttatttgagaaatgcaaactttcc ttatcaaagaggggccggggggctggaagagaagaacttgaatactaaacgctggaagagaagaacct taatactaaaccaagtttcgggaacttcttggtgacttgattggttcccttcccccaatttgcaaagg atgattcccgtgaaggtgatctcgatcaccattctatgatatttctggatgcttttgag Rice mitochondrial RFS SEQ ID 26 Ttccttttacctaatgccggctaccgacaacttacttcatgctattactaacacttatgactgagccg cacttgctttccaaaagaaatggaaactatcatgcctgagactagccaatagaagaaagagccacaag caagccatagcagcatcctttttcttcgctttcttcaacaatgcgaatctacctcactcctcatcata actcaaatacaaattcgagttccaaattgatatttcctcacgtaagcaataaaatgtgaaaccaatat tcatcatgaaacttcagacactgatgattgtgaggttctggaagagagacgacgtaggctgaaaaaaa gtaaacagaaaaccaccccttaaactcatttgctcaacattctttccacagcaactagaaaagtggag aaaatccaataaggggaggtcccggtgaatacaaatcaattggaaaccgaaccccgcattcatgtctc taacaaggctgtctaagctaagcggccatggacccatggacccggggaatctgaaccattaggtagag tttcagctgaaagaaaaccaggtcaatcttccgatcgcgagtctttacaagcttgaaacaacttaagc acaggcgggagtcgccccttttaagtcagtatttatgcggcgctgaactaacgagcggatacctaacc ttcgaaggagaagaaaagacggatgtatctttcattcatatcgatcagatgtgctttgctcaggactc ccattttaccattgcttaagccatattacataaagcatagtgagtgatacgcaatgctggtacaccat gtttttttcctcactctgtgtagccacactcgtttgtccatttctacttattatttatgttaaatagt atccgttggttgtagaagcactggcgttcagggattgcaaaatccataatatcaagaagcggtaggaa cctggctaacttcgatgcggataacgcgctgtagaagaaagtggatcaaccaaagtagac Ubiq3At Arabidopsis Promoter SEQ ID 27 taccggatttggagccaagtctcataaacgccattgtggaagaaagtcttgagttggtggtaatgtaa cagagtagtaagaacagagaagagagagagtgtgagatacatgaattgtcgggcaacaaaaatcctga acatcttattttagcaaagagaaagagttccgagtctgtagcagaagagtgaggagaaatttaagctc ttggacttgtgaattgttccgcctcttgaatacttcttcaatcctcatatattcttcttctatgttac ctgaaaaccggcatttaatctcgcgggtttattccggttcaacattttttttgttttgagttattatc tgggcttaataacgcaggcctgaaataaattcaaggcccaactgtttttttttttaagaagttgctgt taaaaaaaaaaaaagggaattaacaacaacaacaaaaaaagataaagaaaataataacaattacttta attgtagactaaaaaaacatagattttatcatgaaaaaaagagaaaagaaataaaaacttggatcaaa aaaaaaacatacagatcttctaattattaacttttcttaaaaattaggtcctttttcccaacaattag gtttagagttttggaattaaaccaaaaagattgttctaaaaaatactcaaatttggtagataagtttc cttattttaattagtcaatggtagatacttttttttcttttctttattagagtagattagaatctttt atgccaagtattgataaattaaatcaagaagataaactatcataatcaacatgaaattaaaagaaaaa tctcatatatagtattagtattctctatatatattatgattgcttattcttaatgggttgggttaacc aagacatagtcttaatggaaagaatcttttttgaactttttccttattgattaaattcttctatagaa aagaaagaaattatttgaggaaaagtatatacaaaaagaaaaatagaaaaatgtcagtgaagcagatg taatggatgacctaatccaaccaccaccataggatgtttctacttgagtcggtcttttaaaaacgcac ggtggaaaatatgacacgtatcatatgattccttcctttagtttcgtgataataatcctcaactgata tcttcctttttttgttttggctaaagatattttattctcattaatagaaaagacggttttgggctttt ggtttgcgatataaagaagaccttcgtgtggaagataataattcatcctttcgtctttttctgactct tcaatctctcccaaagcctaaagcgatctctgcaaatctctcgcgactctctctttcaaggtatattt tctgattctttttgtttttgattcgtatctgatctccaatttttgttatgtggattattgaatctttt gtataaattgcttttgacaatattgttcgtttcgtcaatccagcttctaaattttgtcctgattacta agatatcgattcgtagtgtttacatctgtgtaatttcttgcttgattgtgaaattaggattttcaagg acgatctattcaatttttgtgttttctttgttcgattctctctgttttaggtttcttatgtttagatc cgtttctctttggtgttgttttgatttctcttacggcttttgatttggtatatgttcgctgattggtt tctacttgttctattgttttatttcaggt 35S Promoter SEQ ID 28 Gatctctctgccgacagtggtcccaaagatggacccccacccacgaggagcatcgtggaaaaagaaga cgttccaaccacgtcttcaaagcaagtggattgatgtgacatctccactgacgtaagggatgacgcac aatcccactatccttcgcaagacccttcctctatataaggaagttcatttcatttggagagga UbiqM maize Promoter SEQ ID 29 tgcagcgtgacccggtcgtgcccctctctagagataatgagcattgcatgtctaagttataaaaaatt accacatattttttttgtcacacttgtttgaagtgcagtttatctatctttatacatatatttaaact ttactctacgaataatataatctatagtactacaataatatcagtgttttagagaatcatataaatga acagttagacatggtctaaaggacaattgagtattttgacaacaggactctacagttttatcttttta gtgtgcatgtgttctcctttttttttgcaaatagcttcacctatataatacttcatccattttattag tacatccatttagggtttagggttaatggtttttatagactaatttttttagtacatctattttattc tattttagcctctaaattaagaaaactaaaactctattttagtttttttatttaataatttagatata aaatagaataaaataaagtgactaaaaattaaacaaataccctttaagaaattaaaaaaactaaggaa acatttttcttgtttcgagtagataatgccagcctgttaaacgccgacgacgagtctaacggacacca accagcgaaccagcagcgtcgcgtcgggccaagcgaagcagacggcacggcatctctgtcgctgcctc tggacccctgtcgagagttccgctccaccgttggacttgctccgctgtcggcatccagaaattgcgtg gcggagcggcagacgtgagccggcacggcaggcggcctcctcctcctctcacggcaccggcagctacg ggggattcctttcccaccgctccttcgctttcccttcctcgcccgccgtaataaatagacaccccctc cacaccctctttccccaacctcgtgttgttcggagcgcacacacacacaaccagatctcccccaaatc cacccgtcggcacctccgcttcaaggtacgccgctcgtcctccccccccccccctctctaccttctct agatcggcgttccggtccatggttagggcccggtagttctacttctgttcatgtttgtgttagatccg tgtttgtgttagatccgtgctgctagcgttcgtacacggatgcgacctgtacgtcagacacgttctga ttgctaacttgccagtgtttctctttggggaatcctgggatggctctagccgttccgcagacgggatc gatttcatgattttttttgtttcgttgcatagggtttggtttgcccttttcctttatttcaatatatg ccgtgcacttgtttgtcgggtcatcttttcatgcttttttttgtcttggttgtgatgatgtggtctgg ttgggcggtcgttctagatcggagtagaattaattctgtttcaaactacctggtggatttattaattt tggatctgtatgtgtgtgccatacatattcatagttacgaattgaagatgatggatggaaatatcgat ctaggataggtatacatgttgatgcgggttttactgatgcatatacagagatgctttttgttcgcttg gttgtgatgatgtggtgtggttgggcggtcgttcattcgttctagatcggagtagaatactgtttcaa actacctggtgtatttattaattttggaactgtatgtgtgtgtcatacatcttcatagttacgagttt aagatggatggaaatatcgatctaggataggtatacatgttgatgtgggttttactgatgcatataca tgatggcatatgcagcatctattcatatgctctaaccttgagtacctatctattataataaacaagta tgttttataattattttgatcttgatatacttggatgatggcatatgcagcagctatatgtggatttt tttagccctgccttcatacgctatttatttgcttggtactgtttcttttgtcgatgctcaccctgttg tttggtgttacttctgcag Nos terminator SEQ ID 30 Gtcaagcagatcgttcaaacatttggcaataaagtttcttaagattgaatcctgttgccggtcttgcg atgattatcatataatttctgttgaattacgtgaagcatgtaataattaacatgtaatgcatgacgtt atttatgagatgggtttttatgattagagtcccgcaattatacatttaatacgcgatagaaaacaaaa tatagcgcgcaaactaggataaattatcgcgcgcggtgtcatctatgttactagatcgac Ags terminator SEQ ID 31 gaattaacagaggtggatggacagacccgttcttacaccggactgggcgcgggataggatattcagat tgggatgggattgagcttaaagccggcgctgagaccatgctcaaggtaggcaatgtcctcagcgtcga gcccggcatctatgtcgagggcattggtggagcgcgcttcggggataccgtgcttgtaactgagaccg gatatgaggccctcactccgcttgatcttggcaaagatatttgacgcatttattagtatgtgttaatt ttcatttgcagtgcagtattttctattcgatctttatgtaattcgttacaattaataaatattcaaat cagattattgactgtcatttgtatcaaatcgtgtttaatggatatttttattataatattgatgat

[0141] Delivery on Transgene Nucleic Acid to Organelle Using Covalent Link Between Viral VPg Protein and Viral RNA Containing Transgene Nucleic Acid.

[0142] In order to translocate TNA to the plant organelles, a covalent link between a specific protein and the nucleic acid cassette containing TNA was utilised. It has been shown that some RNA viruses from the genus Potyvirus such as Potato Virus A, Potato virus Y and Sobemovirus such as Rice Yellow Mottle Virus (RYMV) utilise protein primed replication of their genome. A specific VPg protein is covalently linked to 5′-end of viral RNA and serves as a priming mechanism for replication of the viral genome (Ivanov et al., 2014; Rantalainen et al., 2008; Grzela et al., 2008; Olspert et al., 2011). Formation of this covalent bond also facilitates stabilisation and protection of viral RNA from host endonucleases.

[0143] In order to deliver RNA of the TNA into organelles using VPg protein, we used two approaches:

[0144] i) Fusion of VPg Protein with Organelle Transit Peptide

[0145] In this approach we fused VPg protein with an organelle transit peptide. In this case viral polymerase and coat protein of the complete viral genome were replaced with TNA, while polymerase was delivered in trans (FIGS. 2A and B). VPg protein within the viral genome was modified by fusion to a chloroplast or mitochondrial transit peptide. In this approach, although TNA was efficiently delivered to the plant organelle, the replication of viral genome was dramatically reduced, as the majority of the VPg protein was translocated to the organelle.

[0146] ii) Use of a SpyTag-SpyCatcher System

[0147] To avoid the potential problem of reduced viral replication caused by fusion of transit peptide to VPg protein, we have developed a second approach, where we have utilised the SpyTag-SpyCatcher system (see review by Veggiani et al., 2014). The SpyTag-SpyCatcher system was described by Li et al., 2014, and is based on spontaneous isopeptide bond formation. An isopeptide bond is an amide bond in a protein connecting a side chain to a side chain or a side chain to the protein's main chain. Spontaneous intermolecular isopeptide bond formation between adjacent subunits then locks the rings together, forming ‘protein chainmail’ (Wikoff et al., 2000). In summary a small peptide of SpyTag (13 aa) is functionally fused to the viral VPg protein at the N- or C-terminus of the protein. Such a short peptide either does not interfere with, or substantially does not appear to interfere with the function of the VPg protein and does not appear to materially affect the efficiency of viral replication. A SpyCatcher peptide is fused to an organelle transit peptide and expressed under a nuclear inducible or nuclear constitutive promoter. The Spycatcher peptide recognises the shorter SpyTag peptide and forms a strong covalent bond between these two proteins. As SpyCatcher is fused to an organellar transit peptide of choice, all complexes between SpyTag-VPg-TNA and SpyCatcher are subsequently translocated to the organelles.

[0148] Vectors with both N- and C-terminus fusion of the SpyTag to VPg were prepared (FIG. 3). The SpyCatcher sequence was fused to chloroplast or mitochondrial transit peptide under constitutive 35S or inducible DEX promoter (FIG. 4).

TABLE-US-00003 Potato Virus Y base vector with chloroplast transit peptide fused to VPg gene (chloroplast transit peptide is underlined, VPg is presented in bold, cloning site for the TNA is underlined and in bold) SEQ ID 32 aaattaaaacaactcaatacaacataagaaaatcaacgcaaaaacactcacaaaagctttcaactcta attcaaacaatttgttaagtttcaatttcgatcttcatcaaacaaactctttcaatttcagtgtaagc tatcgtaattcagtaagttatttcaaactctcgtaaattgcagaagatcatccatggcaatttacaca tcaacaatccagtttggttccattgaatgcaaacttccatactcacccgctccttttgggctagttgc ggggaaacgagaagtttcaaccaccactgaccccttcgcaagtttggagatgcagctcagtgcgcgat tacgaaggcaggagtttgcaactattcgaacatccaagaatggtacttgcatgtatcgatacaagact gatgtccagattgcgcgcattcaaaagaagcgcgaggaaagagaaagagaggaatataatttccaaat ggctgcgtcaagtgttgtgtcgaagatcactattgctggtggagagccaccttcaaaacttgaatcac aagtgcggaggggtgtcatccacacaactccaaggatgcgcacagcaaaaacatatcacacgccaaag ttgacagagggacaaatgaaccaccttatcaagcaggtgaagcaaattatgtcaaccaaaggagggtc tgtccaactgattagcaagaaaagtacccatgttcactataaagaagttttgggatcacatcgcgcag ttgtttgcactgcacatatgagaggtttacgaaagagagtggactttcggtgtgataaatggaccgtt gtgcgtctacagcatctcgccaggacggacaagtggactaaccaagttcgtgctactgatctacgcaa gggcgatagtggagttatattgagtaatactaatctcaaaggaaactttgggagaagctcggagggcc tattcatagtgcgtgggtcgcacgaaggaaaaatctatgatgcacgttccaaggttactcaaggggtt atggattcaatggttcagttctcaagcgctgaaagcttttggaagggattggacggcaattgggcaca aatgagatatcctacagatcatacatgtgtggcaggcttaccagttgaagactgtggcagagttgcag cgataatgacacacagtattttaccgtgctataagattacctgccctacctgtgcccaacaatatgcc aacttgccagccagtgacttacttaagatattacacaagcacgcaagtgatggtctaaatcgattggg ggcagacaaagatcgctttgtgcatgtcaaaaagttcttgacaatcttagagcacttaactgaaccgg ttgatctgagtctagaaattttcaatgaagtattcaagtctataggggagaagcaacaatcacctttc aaaaacctgaatattctgaataatttctttttgaaaggaaaggaaaatacagctcgtgaatggcaggt ggctcaattaagcttacttgaattggcaagattccaaaagaacagaacggataatatcaagaaaggag acatctcgttctttaggaataaactatctgccaaagcaaattggaacttgtatctgtcatgtgataac cagctggataagaatgcaagcttcctgtggggacagagggaatatcatgctaagcgatttttctcgaa ctatttcgaggaaattgatccagcgaagggctattcagcatacgaaaatcgtttgcatccgaatggga caagaaaacttgcaattggaaacctaattgtaccacttgatctggctgagtttaggcggaagatgaaa ggtgattataaaagacagccaggggtgagtaagaagtgcacgagctcgaaggatggaaactacgtgta tccctgttgttgcactacacttgatgatggctcagctgttgaatcaacattttacccgccaactaaga agcacctcgtaataggtaatagtggcgaccaaaagtatgttgacttaccaaaagggaattctgagatg ttatatattgccaggcaaggcttctgttacattaacattttcctcgcgatgttgattaacattagtga ggaagatgcaaaggatttcactaagaaggttcgtgacatgtgtgtgccaaagcttggaacctggccaa ccatgatggatctggctacaacttgtgctcaaatgaaaatattctaccctgatgttcatgatgcagaa ctgcctagaatactagtcgatcacgaaacgcagacatgccatgtagttgactcgtttggctcacaaac aactgggtatcatattttgaaagcatctagcgtgtcccaacttattttgtttgctaatgatgagttgg agtctgacattaagcactatagagttggtggtattcctggagcatgccctgagcttgggtccacaata tcaccttttagagaaggaggaatcataatgtctgagtcagcagcgctaaaactgctcctaaagggaat ttttaggcccaaagtgatgaagcaattgctactggatgaaccatatttgctcattttatcgatattat ctcctggtatacttatggctatgtacaacaatgggatatttgagttagcggtgaagttgtggatcaat gagaaacaatctatagccatgatagcatcgttattgtccgccttggctttacgagtgtcagcagcaga aacactcgttgcacagaggattataattgacacggcagcaacagatcttctcgatgctacgtgtgatg gattcaatttaaatctgacatatcccactgcactcatggtgttgcaagttgttaagaacagaaatgaa tgtgatgatacgttgtttaaagcaggtttttcacattacaacatgagtgtcgtgcagattatggaaaa aaattatctaagcctcttgggcgatgcctggaaagatttaacctggcgagaaaaattatccgcaacat ggcactcatacaaagcaaagcgctctatcactcagttcataaaacccataggcaaagcagatttaaaa gggttgtacaacatatcaccgcaagcattcttgggtcagggcgtacagagagtcaaaggcaccgcctc agggttgaatgagcgactcaataattatatcaatactaagtgtgtaaatatttcatcctttttcattc gtagaattttccggcgcttgccaacttttgtaactttcattaattcattattagttattagtatgcta actagtgtagtagcagtgtgtcaagcaataattctagatcaaaggaagtatagaaaagaaattgagtt gatgcagattgagaagaatgaaattgtttgtatggagttgtatgcgagtctgcaggtaagtttctgct tctacctttgatatatatataataattatcattaattagtagtaatataatatttcaaatattttttt caaaataaaagaatgtagtatatagcaattgcttttctgtagtttataagtgtgtatattttaattta taacttttctaatatatgaccaaaatttgttgatatgcagcgcaaacttgagcgtgaattcacatggg atgaatatatggaatatttgaaatctgtgaatccccagatagttcaattcgcgcaagctcaaatggaa gaatataatgtgcgacatcagcgctccacaccaggtgttaagaatttagagcaggtggtagcatttat aactctaattatcatgatgtttgatgctgaaaggagcgactgtgtattcaagactctcaacaaattca aaggcatcgtttcttcaatggatcatgaagttaaacaccagtccttggatgatgtaatcaagaatttc gatgaaaggaacgaagttattgattttgagctaaatgaggatacaattaaaacatcatcagtgttgga cacgaagtttagcgactggtgggatcggcaaatccaaatgggacacacacttccccattatagaactg agggacacttcatggaattcacaagggcaactgctgtacaagtggccaacgacatcgcgcatagtgag cacctagactttctagtgaggggagctgttgggtctggaaaatctactggactgcctgtccatctcag tgcagctggatccgtgcttttgatagaaccaactcgaccacttgcagaaaacgtgttcaagcaattat ccagtgaaccgtttttcaagaagccaacactgcgcatgcgaggaaatagtgtgtttggttcctctcca atctccatcatgactagcggctttgcgttgcactactatgctaataatcgctctcagctaactcagtt taatttcataatttttgatgaatgtcatgttttagatccttctgcaatggcatttcgtagcttgttaa gtgtgtatcaccaaacatgcaaagtgttaaaggtgtcagccactccagtgggaagggaggtcgagttc acaacacaacaaccagttaaattggtggttgaggatacactttcattccaatcttttgttgatgcgca aggctcaaaaaccaatgccgacgttgttcagcatggttcgaacatactcgtgtatgtgtcgagttaca atgaagtggatacattagccaagcttctaacagataggaatatggtagtctcaaaagttgatggcaga acaatgaagcacggatgcttagaaattgtaacgaaagggactagtgcaaagccacattttgtcgtagc aaccaacattattgaaaatggagtaactttagatatagatgtagttgtagattttggacttaaagtct caccgtttttagatattgacaataggagcattgcatacaataagattagtgttagctatggagaaaga attcagaggttgggccgtgttgggcgctttaagaagggagtggcattgcgtattggacacaccgaaaa gggaattattgagattccaagtatgattgctagtgaagctgcgcttgcgtgctttgcatacaatttgc cagtaatgacagggggtgtttcaactagcctcattggcaattgtactgttcgtcaagttaaaactatg caacaatttgagctgagtccattctttatacaaaattttgttgcccatgatggatcaatgcatcctgt catacatgacattcttaagaagtataaactgcgagattgtatgacgcccttgtgtgatcaatccatac cttacagagcctcaagcacttggttgtctgttagtgagtacgaacgactcggagtggttttggacatt ccaaaacagatcaagattgcattccacatcaaggacatccctcctaagttgcatgaaatgctttggga aacagttatcaaatataaggatgtttgtttgtttccaagtattcgggcttcatccattagcaaaattg catacacactgcgcactgatctttttgcaattcccagaaccctaattctagttgaaagattgatcgag gaggaacgagtgaaacagagtcaattcagaagtctcattgatgaaggatgctcaagcatgttttcaat tgttaatttaacaaacactcttagagctagatatgcaaaggattacactgcaggtaagtttctgcttc tacctttgatatatatataataattatcattaattagtagtaatataatatttcaaatatttttttca aaataaaagaatgtagtatatagcaattgcttttctgtagtttataagtgtgtatattttaatttata acttttctaatatatgaccaaaatttgttgatatgcagaaaacatacagaagctcgagaaagtgagaa gtcagttaaaggagttctcaaatttaaatggctctgcatgtgaggagaacttaatgaagaggtatgaa tctctacagtttgtgcatcatcaagcaacaacttcactcgcaaaggatttgaagttgaaaggagtttg gaagaagtcattagttgtgcaggacttactcatagcgggtgccgttgctattggtggaatagggctca tctatagttggtttactcaatcagttgaaactgtgtctcaccagatggcttcttctgctcaaatacac ggtctcggaaccgcttctttctcttccctcaaaaaaccctcttccatatccggcaactccaaaaccct tttcttcggtcagcgactcaattccaaccactctcccttcacccgcgccgcattccctaaattaagta gcaaaacctttaagaagggtttcactttgagagttggcaagaacaaatccaaaagaattcaagcattg aagtttcgacacgcccgcgataagagggctggctttgaaattgataacaatgatgatacaatagagga attctttggatctgcatacaggaagaagggaaaaggtaaaggcaccactgttggtatgggcaagtcaa gcaggaggtttgttaatatgtatggatttgacccaacagaatattcattcatccagttcgttgatccg ctcactggagctcaaattgaagagaacgtctatgctgatattagagacatccaagagcgctttagtga tgtccgcaagaaaatggtagaggatgatgaaatcgaattgcaagcattgggcagcaacacaaccattc atgcttacttcaggaaagattggtctgacaaggctctaaaaattgatttgatgccacacaacccactc aaaatctgtgataaatcgaatggcattgctaagtttcctgaaagagaacttgagttgaggcaaactgg gccagcaatagaggttgatgtgaaagacattccaaaacaggaagtggagcatgaagccaaatcactca tgagaggtttaagggatttcaatccaattgctcaaacagtttgcagagtaaaagtgtctgttgaatat ggaacgtctgaaatgtatgggttcggttttggtgcgtatattatagtaaaccaccatctattcaagag cttcaatggatccatggaagtgcgatcaatgcatggaacattcagagtgaagaatttgcatagcttga gcgttttaccgatcaaaggcagagacattatcatcataaagatgccaaaggatttccctgttttccca caaaaactgcacttccgagctccagtgcagaatgagaggatttgtttggttggaactaattttcaaga aaaacatgcatcatcaatcatcacagaaacgagtactacatacaatgtaccgggcagcactttttgga agcattggattgaaacaaatgatgggcattgtggattaccagtagtgagtacagctgatggatgtcta gttggaatacacagcttggcgaataatgtgcaaaccacgaattattattcagcctttgatgaggattt tgaaagtaagtatctccgaactaatgagcataatgagtggaccaaatcgtgggtatataacccagata ctgtgttgtggggtccattgaagctcaaggagagtacccctaaaggcctgtttaagacaacaaaactt gtacaggatttaattgatcatgatgttgttgtagagcaatagggcgcgccacgcgtgcggccgcttgt agtgtctttccggacgatatatagatatttatgtttgcagtaagtattttggcttttcctgtactact tttatcgcaattaataatcgtttgaatattactggcagataggggtggtatagcgattccgtcgttgt agtgaccttagctgtcgtttctgtattattatgtttgtataaaagtgccgggttgttgttgttgtggc tgatctatcgattaggtgatgttgcgatttgtcgtagcagtgactatgtctggatttagttacttggg tgatgctgtgattctgtcatagcagtgactgtaaacttcaatcaggagaccccgggg Potato Virus Y base vector with mitochondrial transit peptide fused to VPg gene (mitochondrial transit peptide is underlined, VPg is presented in bold, cloning site for the TNA is underlined and in bold) SEQ ID 33 aaattaaaacaactcaatacaacataagaaaatcaacgcaaaaacactcacaaaagctttcaactcta attcaaacaatttgttaagtttcaatttcgatcttcatcaaacaaactctttcaatttcagtgtaagc tatcgtaattcagtaagttatttcaaactctcgtaaattgcagaagatcatccatggcaatttacaca tcaacaatccagtttggttccattgaatgcaaacttccatactcacccgctccttttgggctagttgc ggggaaacgagaagtttcaaccaccactgaccccttcgcaagtttggagatgcagctcagtgcgcgat tacgaaggcaggagtttgcaactattcgaacatccaagaatggtacttgcatgtatcgatacaagact gatgtccagattgcgcgcattcaaaagaagcgcgaggaaagagaaagagaggaatataatttccaaat ggctgcgtcaagtgttgtgtcgaagatcactattgctggtggagagccaccttcaaaacttgaatcac aagtgcggaggggtgtcatccacacaactccaaggatgcgcacagcaaaaacatatcacacgccaaag ttgacagagggacaaatgaaccaccttatcaagcaggtgaagcaaattatgtcaaccaaaggagggtc tgtccaactgattagcaagaaaagtacccatgttcactataaagaagttttgggatcacatcgcgcag ttgtttgcactgcacatatgagaggtttacgaaagagagtggactttcggtgtgataaatggaccgtt gtgcgtctacagcatctcgccaggacggacaagtggactaaccaagttcgtgctactgatctacgcaa gggcgatagtggagttatattgagtaatactaatctcaaaggaaactttgggagaagctcggagggcc tattcatagtgcgtgggtcgcacgaaggaaaaatctatgatgcacgttccaaggttactcaaggggtt atggattcaatggttcagttctcaagcgctgaaagcttttggaagggattggacggcaattgggcaca aatgagatatcctacagatcatacatgtgtggcaggcttaccagttgaagactgtggcagagttgcag cgataatgacacacagtattttaccgtgctataagattacctgccctacctgtgcccaacaatatgcc aacttgccagccagtgacttacttaagatattacacaagcacgcaagtgatggtctaaatcgattggg ggcagacaaagatcgctttgtgcatgtcaaaaagttcttgacaatcttagagcacttaactgaaccgg ttgatctgagtctagaaattttcaatgaagtattcaagtctataggggagaagcaacaatcacctttc aaaaacctgaatattctgaataatttctttttgaaaggaaaggaaaatacagctcgtgaatggcaggt ggctcaattaagcttacttgaattggcaagattccaaaagaacagaacggataatatcaagaaaggag acatctcgttctttaggaataaactatctgccaaagcaaattggaacttgtatctgtcatgtgataac cagctggataagaatgcaagcttcctgtggggacagagggaatatcatgctaagcgatttttctcgaa ctatttcgaggaaattgatccagcgaagggctattcagcatacgaaaatcgtttgcatccgaatggga caagaaaacttgcaattggaaacctaattgtaccacttgatctggctgagtttaggcggaagatgaaa ggtgattataaaagacagccaggggtgagtaagaagtgcacgagctcgaaggatggaaactacgtgta tccctgttgttgcactacacttgatgatggctcagctgttgaatcaacattttacccgccaactaaga agcacctcgtaataggtaatagtggcgaccaaaagtatgttgacttaccaaaagggaattctgagatg ttatatattgccaggcaaggcttctgttacattaacattttcctcgcgatgttgattaacattagtga ggaagatgcaaaggatttcactaagaaggttcgtgacatgtgtgtgccaaagcttggaacctggccaa ccatgatggatctggctacaacttgtgctcaaatgaaaatattctaccctgatgttcatgatgcagaa ctgcctagaatactagtcgatcacgaaacgcagacatgccatgtagttgactcgtttggctcacaaac aactgggtatcatattttgaaagcatctagcgtgtcccaacttattttgtttgctaatgatgagttgg agtctgacattaagcactatagagttggtggtattcctggagcatgccctgagcttgggtccacaata tcaccttttagagaaggaggaatcataatgtctgagtcagcagcgctaaaactgctcctaaagggaat ttttaggcccaaagtgatgaagcaattgctactggatgaaccatatttgctcattttatcgatattat ctcctggtatacttatggctatgtacaacaatgggatatttgagttagcggtgaagttgtggatcaat gagaaacaatctatagccatgatagcatcgttattgtccgccttggctttacgagtgtcagcagcaga aacactcgttgcacagaggattataattgacacggcagcaacagatcttctcgatgctacgtgtgatg gattcaatttaaatctgacatatcccactgcactcatggtgttgcaagttgttaagaacagaaatgaa tgtgatgatacgttgtttaaagcaggtttttcacattacaacatgagtgtcgtgcagattatggaaaa aaattatctaagcctcttgggcgatgcctggaaagatttaacctggcgagaaaaattatccgcaacat ggcactcatacaaagcaaagcgctctatcactcagttcataaaacccataggcaaagcagatttaaaa gggttgtacaacatatcaccgcaagcattcttgggtcagggcgtacagagagtcaaaggcaccgcctc agggttgaatgagcgactcaataattatatcaatactaagtgtgtaaatatttcatcctttttcattc gtagaattttccggcgcttgccaacttttgtaactttcattaattcattattagttattagtatgcta actagtgtagtagcagtgtgtcaagcaataattctagatcaaaggaagtatagaaaagaaattgagtt gatgcagattgagaagaatgaaattgtttgtatggagttgtatgcgagtctgcaggtaagtttctgct tctacctttgatatatatataataattatcattaattagtagtaatataatatttcaaatattttttt caaaataaaagaatgtagtatatagcaattgcttttctgtagtttataagtgtgtatattttaattta taacttttctaatatatgaccaaaatttgttgatatgcagcgcaaacttgagcgtgaattcacatggg atgaatatatggaatatttgaaatctgtgaatccccagatagttcaattcgcgcaagctcaaatggaa gaatataatgtgcgacatcagcgctccacaccaggtgttaagaatttagagcaggtggtagcatttat aactctaattatcatgatgtttgatgctgaaaggagcgactgtgtattcaagactctcaacaaattca aaggcatcgtttcttcaatggatcatgaagttaaacaccagtccttggatgatgtaatcaagaatttc gatgaaaggaacgaagttattgattttgagctaaatgaggatacaattaaaacatcatcagtgttgga cacgaagtttagcgactggtgggatcggcaaatccaaatgggacacacacttccccattatagaactg agggacacttcatggaattcacaagggcaactgctgtacaagtggccaacgacatcgcgcatagtgag cacctagactttctagtgaggggagctgttgggtctggaaaatctactggactgcctgtccatctcag tgcagctggatccgtgcttttgatagaaccaactcgaccacttgcagaaaacgtgttcaagcaattat ccagtgaaccgtttttcaagaagccaacactgcgcatgcgaggaaatagtgtgtttggttcctctcca atctccatcatgactagcggctttgcgttgcactactatgctaataatcgctctcagctaactcagtt taatttcataatttttgatgaatgtcatgttttagatccttctgcaatggcatttcgtagcttgttaa gtgtgtatcaccaaacatgcaaagtgttaaaggtgtcagccactccagtgggaagggaggtcgagttc acaacacaacaaccagttaaattggtggttgaggatacactttcattccaatcttttgttgatgcgca aggctcaaaaaccaatgccgacgttgttcagcatggttcgaacatactcgtgtatgtgtcgagttaca atgaagtggatacattagccaagcttctaacagataggaatatggtagtctcaaaagttgatggcaga acaatgaagcacggatgcttagaaattgtaacgaaagggactagtgcaaagccacattttgtcgtagc aaccaacattattgaaaatggagtaactttagatatagatgtagttgtagattttggacttaaagtct caccgtttttagatattgacaataggagcattgcatacaataagattagtgttagctatggagaaaga attcagaggttgggccgtgttgggcgctttaagaagggagtggcattgcgtattggacacaccgaaaa gggaattattgagattccaagtatgattgctagtgaagctgcgcttgcgtgctttgcatacaatttgc cagtaatgacagggggtgtttcaactagcctcattggcaattgtactgttcgtcaagttaaaactatg caacaatttgagctgagtccattctttatacaaaattttgttgcccatgatggatcaatgcatcctgt catacatgacattcttaagaagtataaactgcgagattgtatgacgcccttgtgtgatcaatccatac cttacagagcctcaagcacttggttgtctgttagtgagtacgaacgactcggagtggttttggacatt ccaaaacagatcaagattgcattccacatcaaggacatccctcctaagttgcatgaaatgctttggga aacagttatcaaatataaggatgtttgtttgtttccaagtattcgggcttcatccattagcaaaattg catacacactgcgcactgatctttttgcaattcccagaaccctaattctagttgaaagattgatcgag gaggaacgagtgaaacagagtcaattcagaagtctcattgatgaaggatgctcaagcatgttttcaat tgttaatttaacaaacactcttagagctagatatgcaaaggattacactgcaggtaagtttctgcttc tacctttgatatatatataataattatcattaattagtagtaatataatatttcaaatatttttttca aaataaaagaatgtagtatatagcaattgcttttctgtagtttataagtgtgtatattttaatttata acttttctaatatatgaccaaaatttgttgatatgcagaaaacatacagaagctcgagaaagtgagaa gtcagttaaaggagttctcaaatttaaatggctctgcatgtgaggagaacttaatgaagaggtatgaa tctctacagtttgtgcatcatcaagcaacaacttcactcgcaaaggatttgaagttgaaaggagtttg gaagaagtcattagttgtgcaggacttactcatagcgggtgccgttgctattggtggaatagggctca tctatagttggtttactcaatcagttgaaactgtgtctcaccagatgtatcgtttcgcttctaacctc gcctccaaggcaaggattgctcaaaacgctcgccaggtttccagcagaatgagctggagcaggaacta tggcaagaacaaatccaaaagaattcaagcattgaagtttcgacacgcccgcgataagagggctggct ttgaaattgataacaatgatgatacaatagaggaattctttggatctgcatacaggaagaagggaaaa ggtaaaggcaccactgttggtatgggcaagtcaagcaggaggtttgttaatatgtatggatttgaccc aacagaatattcattcatccagttcgttgatccgctcactggagctcaaattgaagagaacgtctatg ctgatattagagacatccaagagcgctttagtgatgtccgcaagaaaatggtagaggatgatgaaatc gaattgcaagcattgggcagcaacacaaccattcatgcttacttcaggaaagattggtctgacaaggc tctaaaaattgatttgatgccacacaacccactcaaaatctgtgataaatcgaatggcattgctaagt ttcctgaaagagaacttgagttgaggcaaactgggccagcaatagaggttgatgtgaaagacattcca aaacaggaagtggagcatgaagccaaatcactcatgagaggtttaagggatttcaatccaattgctca aacagtttgcagagtaaaagtgtctgttgaatatggaacgtctgaaatgtatgggttcggttttggtg cgtatattatagtaaaccaccatctattcaagagcttcaatggatccatggaagtgcgatcaatgcat ggaacattcagagtgaagaatttgcatagcttgagcgttttaccgatcaaaggcagagacattatcat cataaagatgccaaaggatttccctgttttcccacaaaaactgcacttccgagctccagtgcagaatg agaggatttgtttggttggaactaattttcaagaaaaacatgcatcatcaatcatcacagaaacgagt actacatacaatgtaccgggcagcactttttggaagcattggattgaaacaaatgatgggcattgtgg attaccagtagtgagtacagctgatggatgtctagttggaatacacagcttggcgaataatgtgcaaa ccacgaattattattcagcctttgatgaggattttgaaagtaagtatctccgaactaatgagcataat gagtggaccaaatcgtgggtatataacccagatactgtgttgtggggtccattgaagctcaaggagag tacccctaaaggcctgtttaagacaacaaaacttgtacaggatttaattgatcatgatgttgttgtag agcaatagggcgcgccacgcgtgcggccgcttgtagtgtctttccggacgatatatagatatttatgt ttgcagtaagtattttggcttttcctgtactacttttatcgcaattaataatcgtttgaatattactg gcagataggggtggtatagcgattccgtcgttgtagtgaccttagctgtcgtttctgtattattatgt ttgtataaaagtgccgggttgttgttgttgtggctgatctatcgattaggtgatgttgcgatttgtcg tagcagtgactatgtctggatttagttacttgggtgatgctgtgattctgtcatagcagtgactgtaa acttcaatcaggagaccccgggg Potato Virus Y base vector with SpyTag fused to 5′-end of VPg gene (SpyTag is underlined, VPg is presented in bold, cloning site for the TNA is underlined and in bold SEQ ID 34 aaattaaaacaactcaatacaacataagaaaatcaacgcaaaaacactcacaaaagctttcaactcta attcaaacaatttgttaagtttcaatttcgatcttcatcaaacaaactctttcaatttcagtgtaagc tatcgtaattcagtaagttatttcaaactctcgtaaattgcagaagatcatccatggcaatttacaca tcaacaatccagtttggttccattgaatgcaaacttccatactcacccgctccttttgggctagttgc ggggaaacgagaagtttcaaccaccactgaccccttcgcaagtttggagatgcagctcagtgcgcgat tacgaaggcaggagtttgcaactattcgaacatccaagaatggtacttgcatgtatcgatacaagact gatgtccagattgcgcgcattcaaaagaagcgcgaggaaagagaaagagaggaatataatttccaaat ggctgcgtcaagtgttgtgtcgaagatcactattgctggtggagagccaccttcaaaacttgaatcac aagtgcggaggggtgtcatccacacaactccaaggatgcgcacagcaaaaacatatcacacgccaaag ttgacagagggacaaatgaaccaccttatcaagcaggtgaagcaaattatgtcaaccaaaggagggtc tgtccaactgattagcaagaaaagtacccatgttcactataaagaagttttgggatcacatcgcgcag ttgtttgcactgcacatatgagaggtttacgaaagagagtggactttcggtgtgataaatggaccgtt gtgcgtctacagcatctcgccaggacggacaagtggactaaccaagttcgtgctactgatctacgcaa gggcgatagtggagttatattgagtaatactaatctcaaaggaaactttgggagaagctcggagggcc tattcatagtgcgtgggtcgcacgaaggaaaaatctatgatgcacgttccaaggttactcaaggggtt atggattcaatggttcagttctcaagcgctgaaagcttttggaagggattggacggcaattgggcaca aatgagatatcctacagatcatacatgtgtggcaggcttaccagttgaagactgtggcagagttgcag cgataatgacacacagtattttaccgtgctataagattacctgccctacctgtgcccaacaatatgcc aacttgccagccagtgacttacttaagatattacacaagcacgcaagtgatggtctaaatcgattggg ggcagacaaagatcgctttgtgcatgtcaaaaagttcttgacaatcttagagcacttaactgaaccgg ttgatctgagtctagaaattttcaatgaagtattcaagtctataggggagaagcaacaatcacctttc aaaaacctgaatattctgaataatttctttttgaaaggaaaggaaaatacagctcgtgaatggcaggt ggctcaattaagcttacttgaattggcaagattccaaaagaacagaacggataatatcaagaaaggag acatctcgttctttaggaataaactatctgccaaagcaaattggaacttgtatctgtcatgtgataac cagctggataagaatgcaagcttcctgtggggacagagggaatatcatgctaagcgatttttctcgaa ctatttcgaggaaattgatccagcgaagggctattcagcatacgaaaatcgtttgcatccgaatggga caagaaaacttgcaattggaaacctaattgtaccacttgatctggctgagtttaggcggaagatgaaa ggtgattataaaagacagccaggggtgagtaagaagtgcacgagctcgaaggatggaaactacgtgta tccctgttgttgcactacacttgatgatggctcagctgttgaatcaacattttacccgccaactaaga agcacctcgtaataggtaatagtggcgaccaaaagtatgttgacttaccaaaagggaattctgagatg ttatatattgccaggcaaggcttctgttacattaacattttcctcgcgatgttgattaacattagtga ggaagatgcaaaggatttcactaagaaggttcgtgacatgtgtgtgccaaagcttggaacctggccaa ccatgatggatctggctacaacttgtgctcaaatgaaaatattctaccctgatgttcatgatgcagaa ctgcctagaatactagtcgatcacgaaacgcagacatgccatgtagttgactcgtttggctcacaaac aactgggtatcatattttgaaagcatctagcgtgtcccaacttattttgtttgctaatgatgagttgg agtctgacattaagcactatagagttggtggtattcctggagcatgccctgagcttgggtccacaata tcaccttttagagaaggaggaatcataatgtctgagtcagcagcgctaaaactgctcctaaagggaat ttttaggcccaaagtgatgaagcaattgctactggatgaaccatatttgctcattttatcgatattat ctcctggtatacttatggctatgtacaacaatgggatatttgagttagcggtgaagttgtggatcaat gagaaacaatctatagccatgatagcatcgttattgtccgccttggctttacgagtgtcagcagcaga aacactcgttgcacagaggattataattgacacggcagcaacagatcttctcgatgctacgtgtgatg gattcaatttaaatctgacatatcccactgcactcatggtgttgcaagttgttaagaacagaaatgaa tgtgatgatacgttgtttaaagcaggtttttcacattacaacatgagtgtcgtgcagattatggaaaa aaattatctaagcctcttgggcgatgcctggaaagatttaacctggcgagaaaaattatccgcaacat ggcactcatacaaagcaaagcgctctatcactcagttcataaaacccataggcaaagcagatttaaaa gggttgtacaacatatcaccgcaagcattcttgggtcagggcgtacagagagtcaaaggcaccgcctc agggttgaatgagcgactcaataattatatcaatactaagtgtgtaaatatttcatcctttttcattc gtagaattttccggcgcttgccaacttttgtaactttcattaattcattattagttattagtatgcta actagtgtagtagcagtgtgtcaagcaataattctagatcaaaggaagtatagaaaagaaattgagtt gatgcagattgagaagaatgaaattgtttgtatggagttgtatgcgagtctgcaggtaagtttctgct tctacctttgatatatatataataattatcattaattagtagtaatataatatttcaaatattttttt caaaataaaagaatgtagtatatagcaattgcttttctgtagtttataagtgtgtatattttaattta taacttttctaatatatgaccaaaatttgttgatatgcagcgcaaacttgagcgtgaattcacatggg atgaatatatggaatatttgaaatctgtgaatccccagatagttcaattcgcgcaagctcaaatggaa gaatataatgtgcgacatcagcgctccacaccaggtgttaagaatttagagcaggtggtagcatttat aactctaattatcatgatgtttgatgctgaaaggagcgactgtgtattcaagactctcaacaaattca aaggcatcgtttcttcaatggatcatgaagttaaacaccagtccttggatgatgtaatcaagaatttc gatgaaaggaacgaagttattgattttgagctaaatgaggatacaattaaaacatcatcagtgttgga cacgaagtttagcgactggtgggatcggcaaatccaaatgggacacacacttccccattatagaactg agggacacttcatggaattcacaagggcaactgctgtacaagtggccaacgacatcgcgcatagtgag cacctagactttctagtgaggggagctgttgggtctggaaaatctactggactgcctgtccatctcag tgcagctggatccgtgcttttgatagaaccaactcgaccacttgcagaaaacgtgttcaagcaattat ccagtgaaccgtttttcaagaagccaacactgcgcatgcgaggaaatagtgtgtttggttcctctcca atctccatcatgactagcggctttgcgttgcactactatgctaataatcgctctcagctaactcagtt taatttcataatttttgatgaatgtcatgttttagatccttctgcaatggcatttcgtagcttgttaa gtgtgtatcaccaaacatgcaaagtgttaaaggtgtcagccactccagtgggaagggaggtcgagttc acaacacaacaaccagttaaattggtggttgaggatacactttcattccaatcttttgttgatgcgca aggctcaaaaaccaatgccgacgttgttcagcatggttcgaacatactcgtgtatgtgtcgagttaca atgaagtggatacattagccaagcttctaacagataggaatatggtagtctcaaaagttgatggcaga acaatgaagcacggatgcttagaaattgtaacgaaagggactagtgcaaagccacattttgtcgtagc aaccaacattattgaaaatggagtaactttagatatagatgtagttgtagattttggacttaaagtct caccgtttttagatattgacaataggagcattgcatacaataagattagtgttagctatggagaaaga attcagaggttgggccgtgttgggcgctttaagaagggagtggcattgcgtattggacacaccgaaaa gggaattattgagattccaagtatgattgctagtgaagctgcgcttgcgtgctttgcatacaatttgc cagtaatgacagggggtgtttcaactagcctcattggcaattgtactgttcgtcaagttaaaactatg caacaatttgagctgagtccattctttatacaaaattttgttgcccatgatggatcaatgcatcctgt catacatgacattcttaagaagtataaactgcgagattgtatgacgcccttgtgtgatcaatccatac cttacagagcctcaagcacttggttgtctgttagtgagtacgaacgactcggagtggttttggacatt ccaaaacagatcaagattgcattccacatcaaggacatccctcctaagttgcatgaaatgctttggga aacagttatcaaatataaggatgtttgtttgtttccaagtattcgggcttcatccattagcaaaattg catacacactgcgcactgatctttttgcaattcccagaaccctaattctagttgaaagattgatcgag gaggaacgagtgaaacagagtcaattcagaagtctcattgatgaaggatgctcaagcatgttttcaat tgttaatttaacaaacactcttagagctagatatgcaaaggattacactgcaggtaagtttctgcttc tacctttgatatatatataataattatcattaattagtagtaatataatatttcaaatatttttttca aaataaaagaatgtagtatatagcaattgcttttctgtagtttataagtgtgtatattttaatttata acttttctaatatatgaccaaaatttgttgatatgcagaaaacatacagaagctcgagaaagtgagaa gtcagttaaaggagttctcaaatttaaatggctctgcatgtgaggagaacttaatgaagaggtatgaa tctctacagtttgtgcatcatcaagcaacaacttcactcgcaaaggatttgaagttgaaaggagtttg gaagaagtcattagttgtgcaggacttactcatagcgggtgccgttgctattggtggaatagggctca tctatagttggtttactcaatcagttgaaactgtgtctcaccagggcaagaacaaagcgcatattgtg atggtggatgcgtataaaccgaccaaaggcaagaacaaatccaaaagaattcaagcattgaagtttcg acacgcccgcgataagagggctggctttgaaattgataacaatgatgatacaatagaggaattctttg gatctgcatacaggaagaagggaaaaggtaaaggcaccactgttggtatgggcaagtcaagcaggagg tttgttaatatgtatggatttgacccaacagaatattcattcatccagttcgttgatccgctcactgg agctcaaattgaagagaacgtctatgctgatattagagacatccaagagcgctttagtgatgtccgca agaaaatggtagaggatgatgaaatcgaattgcaagcattgggcagcaacacaaccattcatgcttac ttcaggaaagattggtctgacaaggctctaaaaattgatttgatgccacacaacccactcaaaatctg tgataaatcgaatggcattgctaagtttcctgaaagagaacttgagttgaggcaaactgggccagcaa tagaggttgatgtgaaagacattccaaaacaggaagtggagcatgaagccaaatcactcatgagaggt ttaagggatttcaatccaattgctcaaacagtttgcagagtaaaagtgtctgttgaatatggaacgtc tgaaatgtatgggttcggttttggtgcgtatattatagtaaaccaccatctattcaagagcttcaatg gatccatggaagtgcgatcaatgcatggaacattcagagtgaagaatttgcatagcttgagcgtttta ccgatcaaaggcagagacattatcatcataaagatgccaaaggatttccctgttttcccacaaaaact gcacttccgagctccagtgcagaatgagaggatttgtttggttggaactaattttcaagaaaaacatg catcatcaatcatcacagaaacgagtactacatacaatgtaccgggcagcactttttggaagcattgg attgaaacaaatgatgggcattgtggattaccagtagtgagtacagctgatggatgtctagttggaat acacagcttggcgaataatgtgcaaaccacgaattattattcagcctttgatgaggattttgaaagta agtatctccgaactaatgagcataatgagtggaccaaatcgtgggtatataacccagatactgtgttg tggggtccattgaagctcaaggagagtacccctaaaggcctgtttaagacaacaaaacttgtacagga tttaattgatcatgatgttgttgtagagcaatagggcgcgccacgcgtgcggccgcttgtagtgtctt tccggacgatatatagatatttatgtttgcagtaagtattttggcttttcctgtactacttttatcgc aattaataatcgtttgaatattactggcagataggggtggtatagcgattccgtcgttgtagtgacct tagctgtcgtttctgtattattatgtttgtataaaagtgccgggttgttgttgttgtggctgatctat cgattaggtgatgttgcgatttgtcgtagcagtgactatgtctggatttagttacttgggtgatgctg tgattctgtcatagcagtgactgtaaacttcaatcaggagac Potato Virus Y base vector with SpyTag fused to 3′-end of fused to VPg gene (SpyTag is underlined, VPg is presented in bold, cloning site for the TNA is underlined and in bold) SEQ ID 35 aaattaaaacaactcaatacaacataagaaaatcaacgcaaaaacactcacaaaagctttcaactcta attcaaacaatttgttaagtttcaatttcgatcttcatcaaacaaactctttcaatttcagtgtaagc tatcgtaattcagtaagttatttcaaactctcgtaaattgcagaagatcatccatggcaatttacaca tcaacaatccagtttggttccattgaatgcaaacttccatactcacccgctccttttgggctagttgc ggggaaacgagaagtttcaaccaccactgaccccttcgcaagtttggagatgcagctcagtgcgcgat tacgaaggcaggagtttgcaactattcgaacatccaagaatggtacttgcatgtatcgatacaagact gatgtccagattgcgcgcattcaaaagaagcgcgaggaaagagaaagagaggaatataatttccaaat ggctgcgtcaagtgttgtgtcgaagatcactattgctggtggagagccaccttcaaaacttgaatcac aagtgcggaggggtgtcatccacacaactccaaggatgcgcacagcaaaaacatatcacacgccaaag ttgacagagggacaaatgaaccaccttatcaagcaggtgaagcaaattatgtcaaccaaaggagggtc tgtccaactgattagcaagaaaagtacccatgttcactataaagaagttttgggatcacatcgcgcag ttgtttgcactgcacatatgagaggtttacgaaagagagtggactttcggtgtgataaatggaccgtt gtgcgtctacagcatctcgccaggacggacaagtggactaaccaagttcgtgctactgatctacgcaa gggcgatagtggagttatattgagtaatactaatctcaaaggaaactttgggagaagctcggagggcc tattcatagtgcgtgggtcgcacgaaggaaaaatctatgatgcacgttccaaggttactcaaggggtt atggattcaatggttcagttctcaagcgctgaaagcttttggaagggattggacggcaattgggcaca aatgagatatcctacagatcatacatgtgtggcaggcttaccagttgaagactgtggcagagttgcag cgataatgacacacagtattttaccgtgctataagattacctgccctacctgtgcccaacaatatgcc aacttgccagccagtgacttacttaagatattacacaagcacgcaagtgatggtctaaatcgattggg ggcagacaaagatcgctttgtgcatgtcaaaaagttcttgacaatcttagagcacttaactgaaccgg ttgatctgagtctagaaattttcaatgaagtattcaagtctataggggagaagcaacaatcacctttc aaaaacctgaatattctgaataatttctttttgaaaggaaaggaaaatacagctcgtgaatggcaggt ggctcaattaagcttacttgaattggcaagattccaaaagaacagaacggataatatcaagaaaggag acatctcgttctttaggaataaactatctgccaaagcaaattggaacttgtatctgtcatgtgataac cagctggataagaatgcaagcttcctgtggggacagagggaatatcatgctaagcgatttttctcgaa ctatttcgaggaaattgatccagcgaagggctattcagcatacgaaaatcgtttgcatccgaatggga caagaaaacttgcaattggaaacctaattgtaccacttgatctggctgagtttaggcggaagatgaaa ggtgattataaaagacagccaggggtgagtaagaagtgcacgagctcgaaggatggaaactacgtgta tccctgttgttgcactacacttgatgatggctcagctgttgaatcaacattttacccgccaactaaga agcacctcgtaataggtaatagtggcgaccaaaagtatgttgacttaccaaaagggaattctgagatg ttatatattgccaggcaaggcttctgttacattaacattttcctcgcgatgttgattaacattagtga ggaagatgcaaaggatttcactaagaaggttcgtgacatgtgtgtgccaaagcttggaacctggccaa ccatgatggatctggctacaacttgtgctcaaatgaaaatattctaccctgatgttcatgatgcagaa ctgcctagaatactagtcgatcacgaaacgcagacatgccatgtagttgactcgtttggctcacaaac aactgggtatcatattttgaaagcatctagcgtgtcccaacttattttgtttgctaatgatgagttgg agtctgacattaagcactatagagttggtggtattcctggagcatgccctgagcttgggtccacaata tcaccttttagagaaggaggaatcataatgtctgagtcagcagcgctaaaactgctcctaaagggaat ttttaggcccaaagtgatgaagcaattgctactggatgaaccatatttgctcattttatcgatattat ctcctggtatacttatggctatgtacaacaatgggatatttgagttagcggtgaagttgtggatcaat gagaaacaatctatagccatgatagcatcgttattgtccgccttggctttacgagtgtcagcagcaga aacactcgttgcacagaggattataattgacacggcagcaacagatcttctcgatgctacgtgtgatg gattcaatttaaatctgacatatcccactgcactcatggtgttgcaagttgttaagaacagaaatgaa tgtgatgatacgttgtttaaagcaggtttttcacattacaacatgagtgtcgtgcagattatggaaaa aaattatctaagcctcttgggcgatgcctggaaagatttaacctggcgagaaaaattatccgcaacat ggcactcatacaaagcaaagcgctctatcactcagttcataaaacccataggcaaagcagatttaaaa gggttgtacaacatatcaccgcaagcattcttgggtcagggcgtacagagagtcaaaggcaccgcctc agggttgaatgagcgactcaataattatatcaatactaagtgtgtaaatatttcatcctttttcattc gtagaattttccggcgcttgccaacttttgtaactttcattaattcattattagttattagtatgcta actagtgtagtagcagtgtgtcaagcaataattctagatcaaaggaagtatagaaaagaaattgagtt gatgcagattgagaagaatgaaattgtttgtatggagttgtatgcgagtctgcaggtaagtttctgct tctacctttgatatatatataataattatcattaattagtagtaatataatatttcaaatattttttt caaaataaaagaatgtagtatatagcaattgcttttctgtagtttataagtgtgtatattttaattta taacttttctaatatatgaccaaaatttgttgatatgcagcgcaaacttgagcgtgaattcacatggg atgaatatatggaatatttgaaatctgtgaatccccagatagttcaattcgcgcaagctcaaatggaa gaatataatgtgcgacatcagcgctccacaccaggtgttaagaatttagagcaggtggtagcatttat aactctaattatcatgatgtttgatgctgaaaggagcgactgtgtattcaagactctcaacaaattca aaggcatcgtttcttcaatggatcatgaagttaaacaccagtccttggatgatgtaatcaagaatttc gatgaaaggaacgaagttattgattttgagctaaatgaggatacaattaaaacatcatcagtgttgga cacgaagtttagcgactggtgggatcggcaaatccaaatgggacacacacttccccattatagaactg agggacacttcatggaattcacaagggcaactgctgtacaagtggccaacgacatcgcgcatagtgag cacctagactttctagtgaggggagctgttgggtctggaaaatctactggactgcctgtccatctcag tgcagctggatccgtgcttttgatagaaccaactcgaccacttgcagaaaacgtgttcaagcaattat ccagtgaaccgtttttcaagaagccaacactgcgcatgcgaggaaatagtgtgtttggttcctctcca atctccatcatgactagcggctttgcgttgcactactatgctaataatcgctctcagctaactcagtt taatttcataatttttgatgaatgtcatgttttagatccttctgcaatggcatttcgtagcttgttaa gtgtgtatcaccaaacatgcaaagtgttaaaggtgtcagccactccagtgggaagggaggtcgagttc acaacacaacaaccagttaaattggtggttgaggatacactttcattccaatcttttgttgatgcgca aggctcaaaaaccaatgccgacgttgttcagcatggttcgaacatactcgtgtatgtgtcgagttaca atgaagtggatacattagccaagcttctaacagataggaatatggtagtctcaaaagttgatggcaga acaatgaagcacggatgcttagaaattgtaacgaaagggactagtgcaaagccacattttgtcgtagc aaccaacattattgaaaatggagtaactttagatatagatgtagttgtagattttggacttaaagtct caccgtttttagatattgacaataggagcattgcatacaataagattagtgttagctatggagaaaga attcagaggttgggccgtgttgggcgctttaagaagggagtggcattgcgtattggacacaccgaaaa gggaattattgagattccaagtatgattgctagtgaagctgcgcttgcgtgctttgcatacaatttgc cagtaatgacagggggtgtttcaactagcctcattggcaattgtactgttcgtcaagttaaaactatg caacaatttgagctgagtccattctttatacaaaattttgttgcccatgatggatcaatgcatcctgt catacatgacattcttaagaagtataaactgcgagattgtatgacgcccttgtgtgatcaatccatac cttacagagcctcaagcacttggttgtctgttagtgagtacgaacgactcggagtggttttggacatt ccaaaacagatcaagattgcattccacatcaaggacatccctcctaagttgcatgaaatgctttggga aacagttatcaaatataaggatgtttgtttgtttccaagtattcgggcttcatccattagcaaaattg catacacactgcgcactgatctttttgcaattcccagaaccctaattctagttgaaagattgatcgag gaggaacgagtgaaacagagtcaattcagaagtctcattgatgaaggatgctcaagcatgttttcaat tgttaatttaacaaacactcttagagctagatatgcaaaggattacactgcaggtaagtttctgcttc tacctttgatatatatataataattatcattaattagtagtaatataatatttcaaatatttttttca aaataaaagaatgtagtatatagcaattgcttttctgtagtttataagtgtgtatattttaatttata acttttctaatatatgaccaaaatttgttgatatgcagaaaacatacagaagctcgagaaagtgagaa gtcagttaaaggagttctcaaatttaaatggctctgcatgtgaggagaacttaatgaagaggtatgaa tctctacagtttgtgcatcatcaagcaacaacttcactcgcaaaggatttgaagttgaaaggagtttg gaagaagtcattagttgtgcaggacttactcatagcgggtgccgttgctattggtggaatagggctca tctatagttggtttactcaatcagttgaaactgtgtctcaccagggcaagaacaaatccaaaagaatt caagcattgaagtttcgacacgcccgcgataagagggctggctttgaaattgataacaatgatgatac aatagaggaattctttggatctgcatacaggaagaagggaaaaggtaaaggcaccactgttggtatgg gcaagtcaagcaggaggtttgttaatatgtatggatttgacccaacagaatattcattcatccagttc gttgatccgctcactggagctcaaattgaagagaacgtctatgctgatattagagacatccaagagcg ctttagtgatgtccgcaagaaaatggtagaggatgatgaaatcgaattgcaagcattgggcagcaaca caaccattcatgcttacttcaggaaagattggtctgacaaggctctaaaaattgatttgatgccacac aacccactcaaaatctgtgataaatcgaatggcattgctaagtttcctgaaagagaacttgagttgag gcaaactgggccagcaatagaggttgatgtgaaagacattccaaaacaggaagcgcatattgtgatgg tggatgcgtataaaccgaccaaagtggagcatgaagccaaatcactcatgagaggtttaagggatttc aatccaattgctcaaacagtttgcagagtaaaagtgtctgttgaatatggaacgtctgaaatgtatgg gttcggttttggtgcgtatattatagtaaaccaccatctattcaagagcttcaatggatccatggaag tgcgatcaatgcatggaacattcagagtgaagaatttgcatagcttgagcgttttaccgatcaaaggc agagacattatcatcataaagatgccaaaggatttccctgttttcccacaaaaactgcacttccgagc tccagtgcagaatgagaggatttgtttggttggaactaattttcaagaaaaacatgcatcatcaatca tcacagaaacgagtactacatacaatgtaccgggcagcactttttggaagcattggattgaaacaaat gatgggcattgtggattaccagtagtgagtacagctgatggatgtctagttggaatacacagcttggc gaataatgtgcaaaccacgaattattattcagcctttgatgaggattttgaaagtaagtatctccgaa ctaatgagcataatgagtggaccaaatcgtgggtatataacccagatactgtgttgtggggtccattg aagctcaaggagagtacccctaaaggcctgtttaagacaacaaaacttgtacaggatttaattgatca tgatgttgttgtagagcaatagggcgcgccacgcgtgcggccgcttgtagtgtctttccggacgatat atagatatttatgtttgcagtaagtattttggcttttcctgtactacttttatcgcaattaataatcg tttgaatattactggcagataggggtggtatagcgattccgtcgttgtagtgaccttagctgtcgttt ctgtattattatgtttgtataaaagtgccgggttgttgttgttgtggctgatctatcgattaggtgat gttgcgatttgtcgtagcagtgactatgtctggatttagttacttgggtgatgctgtgattctgtcat agcagtgactgtaaacttcaatcaggagac Potato Virus Y polymerase gene SEQ ID 36 atggctaaacattctgcgtggatgtatgaggctctaacagggaatttgcaagctgtggcgacaatgaa gagtcagctagtgacaaagcacgtggtcaaaggggagtgtcggcacttcaaagagttcttaactgtgg attcggaagcagaagctttcttcaggcctttgatggatgcttatgggaagagcttgttaaatagagaa gcatatataaaggacataatgaaatactcaaagcctattgatgttggaatagtagactgtgatgcttt tgaagaggctatcaatagggttatcatttatctgcaagtgcatggcttccagaaatgcaattacatca ccgatgagcaggaaattttcaaagctctcaatatgaaagctgctgtcggagctatgtatggaggcaag aagaaagactacttcgagcattttactgaggcggataaagaggaaattgttatgcaaagttgctttcg attgtacaagggctcgcttggcatatggaatggatcattgaaagcagaacttcggtgcaaagagaaga tacttgcaaataagacaaggacattcactgctgcacctttagatactctactgggtggaaaggtgtgc gttgatgattttaataatcaattctactcaaagaacattgaatgctgctggactgttggaatgactaa gttttatggaggttgggacaaattgcttcggcgtctacctgaaaattgggtgtactgcgatgccgatg gttcacaattcgatagttcactcaccccatacctaattaatgctgttctcatcatcagaagcacatac atggaagattgggacttggggttgcaaatgttgcgcaatttgtacacagaaataatttacacaccaat ctcaactccagatggaacaattgtcaagaagtttagaggtaataatagcggtcaaccttctaccgttg tggataattctctcatggttgtccttgctatgcattacgctctcattaaggagtgcgttgagtttgaa gaaatcgacagcacgtgtgtattctttgttaatggtgatgacttattgattgctgtgaatccggagaa agagagcattctcgatagaatgtcacaacatttctcagatcttggtttgaactatgatttttcgtcga gaacaagaaggaaggaggaattgtggttcatgtcccatagaggcctgctaatcgaggatatgtacgtg ccaaagcttgaagaagagagaattgtatccattctgcaatgggatagagctgatctgccagagcacag attagaagcgatttgtgcagcaatgatagaatcctggggttattttgagttaacgcaccaaatcagga gattctactcatggttgttgcaacagcaacctttttcaacgatagcacaggaaggaaaagctccatac atagcgagcatggcattgaagaagctgtacatgaataggacagtagatgaggaggaactgaaggcttt cactgaaatgatggttgccttggatgatgaatttgagtgcgatacttatgaagtgcaccatcaatag SpyTag SEQ ID 37 gcgcatattgtgatggtggatgcgtataaaccgaccaaa SpyCatcher SEQ ID 38 atggttgataccttatcaggtttatcaagtgagcaaggtcagtccggtgatatgacaattgaagaaga tagtgctacccatattaaattctcaaaacgtgatgaggacggcaaagagttagctggtgcaactatgg agttgcgtgattcatctggtaaaactattagtacatggatttcagatggacaagtgaaagatttctac ctgtatccaggaaaatatacatttgtcgaaaccgcagcaccagacggttatgaggtagcaactgctat tacctttacagttaatgagcaaggtcaggttactgtaaatggcaaagcaactaaaggtgacgctcata tttaa

[0149] DNA Approach for Delivery of Transgene Nucleic Acid into the Organelles.

[0150] We have developed a simple and reliable system for DNA delivery into plant organelles using Agrobacterium mediated transformation. It has been shown in the past that the virD2 protein is covalently linked with T-DNA in bacterial cells, forming a complex which is then injected into the cytoplasm of the plant cell. At the same time, Agrobacterium injects virE2 protein into the cytoplasm which binds to the T-DNA protecting it from degradation by plant endonucleases, as well as facilitating delivery of the T-DNA into the cell nucleus. We have utilised an Agrobacterium strain where both the virD2 and virE2 gene native functionality was compromised or substantially reduced and/or substantially knocked out so as to inhibit or diminish nuclear transport of the T-DNA to the plant cell nucleus. To replace the functions of bacterial virD2 protein, we modified the virD2 protein by fusing it with organellar transit peptides, such as chloroplast and mitochondrial transit peptides, or by fusing it with a SpyTag peptide, and have introduced such modified virD2 cassettes on a binary vector under the control of a native bacterial promoter (FIGS. 6 and 7) . As a result, the virD2 modified proteins form a covalent complex with T-DNA in the bacterial cell which is then injected into the cytoplasm of the plant cell. The virD2 protein fused with either chloroplast or mitochondrial transit peptide directs delivery of the T-DNA to the organelles instead of the nucleus. The absence of significant virE2 protein functionality also facilitates more efficient translocation of the T-DNA complex to the plant organelles. The SpyTag-SpyCatcher system can also be utilised for translocating T-DNA into the organelles by overexpression of the Transit Peptide-SpyCatcher peptide in plant cells before challenging of the plant cells with Agrobacterium containing virD2-SpyTag gene on the binary vector.

TABLE-US-00004 cTP virD2 cassette (chloroplast transit peptide is underlined, virD2 is in bold) SEQ ID 39 ctgtcgattttgtgaagcggaagtgtgtctgtacttttatttgtgtgtatgattttgcgataattcat aagtaatgtagtaattacctgattttatatttcaattttattgtaatataatttcaattgtaataata taaaaataaatatcccttatgtgttcttgatttcgttttgtatatggctagattcccatctgccacga cgaggaaatgctacggcggggcaagttcagatctttccgtcttctatggaggaagctatgtcgcaagg cagtaggcccacctcaagtgacattgccgtcaaccagcgcgaatgcgtgaaggttgaaggcttcaagg tcgtcagtacccgattaagatcggccgaatatgagagtttttctcatcaggcacgcttgctgggcctc tccgacagcatggccatacgggttgcggtgcgccgcattggtggctttcttgaaatcgacgcagagac tcgtcataggatggaggccatactacaatccataggaacactctcaagcaacattgccgcgctgctat ctgcctatgccgaaaatccgacaatggatttggaggctttgcgagctgaacgtatcgccttcggtaaa tctttcgctgacctcgacggcttgctccgttccattttgtccgtatcacggcggcggatcgacggttg ctcgctgctgaaagacgccttgtagcactgacgtagcacttggcggggaacatattcgatggcttctt ctgctcaaatacacggtctcggaaccgcttctttctcttccctcaaaaaaccctcttccatatccggc aactccaaaacccttttcttcggtcagcgactcaattccaaccactctcccttcacccgcgccgcatt ccctaaattaagtagcaaaacctttaagaagggtttcactttgagagttatgcccgatcgtgctcaag ttatcattcgcattgtgccgggaggtggcaccaagacccttcaacaaattatcaatcagttggagtat ctatcccggaagggcaggctggagctgcagcgttcagcccgacatctcgatattcccctgccaccgga tcaaatccacgaacttgcccgaagctgggttcaagagactggaacttatgacgaaagtcagccagacg aggaaaggcaacaggagttgaccacccatattattgttagcttccccgccggtacaagccaggtagcg gcttatgcggcgagccgggagtgggcagccgagatgtttgggtcaggcgcaggggggggccgatacaa ctatcttacggccttccacatcgatcgcgaccacccacatctgcatgtcgtcgtcaatcggcgcgaac ttttaggacacggctggctgaagatatctcggcgccatccccaactgaattacgacgccctgcgcata aagatggccgagatttcacttcgtcatggcattgccctcgatgcgagccgacgagcagaacgtggcat caccgagcggccgatcacttatgcccaatatcggcgccttgagcgggagcaggctcgccaaatccgtt tcgaagacgcggatttggaacagtcgtcgccgcaaggagatcatccagagttcagccaacctttcgat acatccccatttgaagcatccgcaggcggaccggaggacatgcctcggcccaacaatcggcagaatga gtcgcaagttcatctccaggagccagctggtgtcagcaacgaagccggtgtccttgtgcgggttgcat tggagacggagcgccttgctcaaccattcgtttccgaaaccattctcgcggacgacatagggagcggc tcttcgcgtgttgccgagggccgtgtggagagcgcaaaccgcactcccgatattcctcgcgcagcaac tgaagctgccacgcacacgacacacgaccggcagcggcgtgcaaagcgtcctcatgatgacgacggag ggccgagtggagcaaaacgtgtgacattggaaggcatcgcggttggcccccaggcgaacgccggcgaa caggctggcagtagtggccccttagtacggcaagctggaacgtctcggccatctccaccgacggccac gacgcgggccagcaccgcaaccgcttcattgtctgctacagcccacctccagcaacggagaggtgtcc tttcaaagcgtccgcgtgaagatgatgatggagaaccgagtgaacgcaaacgcgagagagatgagcgc agcaaggacgggcgtgggggaaataggagataggagcttcgacaggcatcaaataaaacgaaaggctc agtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctcctgagtaggacaaat ccgccc mTP-virD2 casette (mitochondrial transit peptide is underlined, virD2 is in bold) SEQ ID 40 ctgtcgattttgtgaagcggaagtgtgtctgtacttttatttgtgtgtatgattttgcgataattcat aagtaatgtagtaattacctgattttatatttcaattttattgtaatataatttcaattgtaataata taaaaataaatatcccttatgtgttcttgatttcgttttgtatatggctagattcccatctgccacga cgaggaaatgctacggcggggcaagttcagatctttccgtcttctatggaggaagctatgtcgcaagg cagtaggcccacctcaagtgacattgccgtcaaccagcgcgaatgcgtgaaggttgaaggcttcaagg tcgtcagtacccgattaagatcggccgaatatgagagtttttctcatcaggcacgcttgctgggcctc tccgacagcatggccatacgggttgcggtgcgccgcattggtggctttcttgaaatcgacgcagagac tcgtcataggatggaggccatactacaatccataggaacactctcaagcaacattgccgcgctgctat ctgcctatgccgaaaatccgacaatggatttggaggctttgcgagctgaacgtatcgccttcggtaaa tctttcgctgacctcgacggcttgctccgttccattttgtccgtatcacggcggcggatcgacggttg ctcgctgctgaaagacgccttgtagcactgacgtagcacttggcggggaacatattcgatgtatcgtt tcgcttctaacctcgcctccaaggcaaggattgctcaaaacgctcgccaggtttccagcagaatgagc tggagcaggaactatatgcccgatcgtgctcaagttatcattcgcattgtgccgggaggtggcaccaa gacccttcaacaaattatcaatcagttggagtatctatcccggaagggcaggctggagctgcagcgtt cagcccgacatctcgatattcccctgccaccggatcaaatccacgaacttgcccgaagctgggttcaa gagactggaacttatgacgaaagtcagccagacgaggaaaggcaacaggagttgaccacccatattat tgttagcttccccgccggtacaagccaggtagcggcttatgcggcgagccgggagtgggcagccgaga tgtttgggtcaggcgcaggggggggccgatacaactatcttacggccttccacatcgatcgcgaccac ccacatctgcatgtcgtcgtcaatcggcgcgaacttttaggacacggctggctgaagatatctcggcg ccatccccaactgaattacgacgccctgcgcataaagatggccgagatttcacttcgtcatggcattg ccctcgatgcgagccgacgagcagaacgtggcatcaccgagcggccgatcacttatgcccaatatcgg cgccttgagcgggagcaggctcgccaaatccgtttcgaagacgcggatttggaacagtcgtcgccgca aggagatcatccagagttcagccaacctttcgatacatccccatttgaagcatccgcaggcggaccgg aggacatgcctcggcccaacaatcggcagaatgagtcgcaagttcatctccaggagccagctggtgtc agcaacgaagccggtgtccttgtgcgggttgcattggagacggagcgccttgctcaaccattcgtttc cgaaaccattctcgcggacgacatagggagcggctcttcgcgtgttgccgagggccgtgtggagagcg caaaccgcactcccgatattcctcgcgcagcaactgaagctgccacgcacacgacacacgaccggcag cggcgtgcaaagcgtcctcatgatgacgacggagggccgagtggagcaaaacgtgtgacattggaagg catcgcggttggcccccaggcgaacgccggcgaacaggctggcagtagtggccccttagtacggcaag ctggaacgtctcggccatctccaccgacggccacgacgcgggccagcaccgcaaccgcttcattgtct gctacagcccacctccagcaacggagaggtgtcctttcaaagcgtccgcgtgaagatgatgatggaga accgagtgaacgcaaacgcgagagagatgagcgcagcaaggacgggcgtgggggaaataggagatagg agcttcgacaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttg tttgtcggtgaacgctctcctgagtaggacaaatccgccc SpyTag-virD2 cassette (SpyTag is underlined, virD2 is in bold) SEQ ID 41 ctgtcgattttgtgaagcggaagtgtgtctgtacttttatttgtgtgtatgattttgcgataattcat aagtaatgtagtaattacctgattttatatttcaattttattgtaatataatttcaattgtaataata taaaaataaatatcccttatgtgttcttgatttcgttttgtatatggctagattcccatctgccacga cgaggaaatgctacggcggggcaagttcagatctttccgtcttctatggaggaagctatgtcgcaagg cagtaggcccacctcaagtgacattgccgtcaaccagcgcgaatgcgtgaaggttgaaggcttcaagg tcgtcagtacccgattaagatcggccgaatatgagagtttttctcatcaggcacgcttgctgggcctc tccgacagcatggccatacgggttgcggtgcgccgcattggtggctttcttgaaatcgacgcagagac tcgtcataggatggaggccatactacaatccataggaacactctcaagcaacattgccgcgctgctat ctgcctatgccgaaaatccgacaatggatttggaggctttgcgagctgaacgtatcgccttcggtaaa tctttcgctgacctcgacggcttgctccgttccattttgtccgtatcacggcggcggatcgacggttg ctcgctgctgaaagacgccttgtagcactgacgtagcacttggcggggaacatattcgatggcgcata ttgtgatggtggatgcgtataaaccgaccaaaatgcccgatcgtgctcaagttatcattcgcattgtg ccgggaggtggcaccaagacccttcaacaaattatcaatcagttggagtatctatcccggaagggcag gctggagctgcagcgttcagcccgacatctcgatattcccctgccaccggatcaaatccacgaacttg cccgaagctgggttcaagagactggaacttatgacgaaagtcagccagacgaggaaaggcaacaggag ttgaccacccatattattgttagcttccccgccggtacaagccaggtagcggcttatgcggcgagccg ggagtgggcagccgagatgtttgggtcaggcgcaggggggggccgatacaactatcttacggccttcc acatcgatcgcgaccacccacatctgcatgtcgtcgtcaatcggcgcgaacttttaggacacggctgg ctgaagatatctcggcgccatccccaactgaattacgacgccctgcgcataaagatggccgagatttc acttcgtcatggcattgccctcgatgcgagccgacgagcagaacgtggcatcaccgagcggccgatca cttatgcccaatatcggcgccttgagcgggagcaggctcgccaaatccgtttcgaagacgcggatttg gaacagtcgtcgccgcaaggagatcatccagagttcagccaacctttcgatacatccccatttgaagc atccgcaggcggaccggaggacatgcctcggcccaacaatcggcagaatgagtcgcaagttcatctcc aggagccagctggtgtcagcaacgaagccggtgtccttgtgcgggttgcattggagacggagcgcctt gctcaaccattcgtttccgaaaccattctcgcggacgacatagggagcggctcttcgcgtgttgccga gggccgtgtggagagcgcaaaccgcactcccgatattcctcgcgcagcaactgaagctgccacgcaca cgacacacgaccggcagcggcgtgcaaagcgtcctcatgatgacgacggagggccgagtggagcaaaa cgtgtgacattggaaggcatcgcggttggcccccaggcgaacgccggcgaacaggctggcagtagtgg ccccttagtacggcaagctggaacgtctcggccatctccaccgacggccacgacgcgggccagcaccg caaccgcttcattgtctgctacagcccacctccagcaacggagaggtgtcctttcaaagcgtccgcgt gaagatgatgatggagaaccgagtgaacgcaaacgcgagagagatgagcgcagcaaggacgggcgtgg gggaaataggagataggagcttcgacaggcatcaaataaaacgaaaggctcagtcgaaagactgggcc tttcgttttatctgttgtttgtcggtgaacgctctcctgagtaggacaaatccgccc virD2-SpyTag cassette (SpyTag is underlined, virD2 is in bold) SEQ ID 42 ctgtcgattttgtgaagcggaagtgtgtctgtacttttatttgtgtgtatgattttgcgataattcat aagtaatgtagtaattacctgattttatatttcaattttattgtaatataatttcaattgtaataata taaaaataaatatcccttatgtgttcttgatttcgttttgtatatggctagattcccatctgccacga cgaggaaatgctacggcggggcaagttcagatctttccgtcttctatggaggaagctatgtcgcaagg cagtaggcccacctcaagtgacattgccgtcaaccagcgcgaatgcgtgaaggttgaaggcttcaagg tcgtcagtacccgattaagatcggccgaatatgagagtttttctcatcaggcacgcttgctgggcctc tccgacagcatggccatacgggttgcggtgcgccgcattggtggctttcttgaaatcgacgcagagac tcgtcataggatggaggccatactacaatccataggaacactctcaagcaacattgccgcgctgctat ctgcctatgccgaaaatccgacaatggatttggaggctttgcgagctgaacgtatcgccttcggtaaa tctttcgctgacctcgacggcttgctccgttccattttgtccgtatcacggcggcggatcgacggttg ctcgctgctgaaagacgccttgtagcactgacgtagcacttggcggggaacatattcgatgcccgatc gtgctcaagttatcattcgcattgtgccgggaggtggcaccaagacccttcaacaaattatcaatcag ttggagtatctatcccggaagggcaggctggagctgcagcgttcagcccgacatctcgatattcccct gccaccggatcaaatccacgaacttgcccgaagctgggttcaagagactggaacttatgacgaaagtc agccagacgaggaaaggcaacaggagttgaccacccatattattgttagcttccccgccggtacaagc caggtagcggcttatgcggcgagccgggagtgggcagccgagatgtttgggtcaggcgcagggggggg ccgatacaactatcttacggccttccacatcgatcgcgaccacccacatctgcatgtcgtcgtcaatc ggcgcgaacttttaggacacggctggctgaagatatctcggcgccatccccaactgaattacgacgcc ctgcgcataaagatggccgagatttcacttcgtcatggcattgccctcgatgcgagccgacgagcaga acgtggcatcaccgagcggccgatcacttatgcccaatatcggcgccttgagcgggagcaggctcgcc aaatccgtttcgaagacgcggatttggaacagtcgtcgccgcaaggagatcatccagagttcagccaa cctttcgatacatccccatttgaagcatccgcaggcggaccggaggacatgcctcggcccaacaatcg gcagaatgagtcgcaagttcatctccaggagccagctggtgtcagcaacgaagccggtgtccttgtgc gggttgcattggagacggagcgccttgctcaaccattcgtttccgaaaccattctcgcggacgacata gggagcggctcttcgcgtgttgccgagggccgtgtggagagcgcaaaccgcactcccgatattcctcg cgcagcaactgaagctgccacgcacacgacacacgaccggcagcggcgtgcaaagcgtcctcatgatg acgacggagggccgagtggagcaaaacgtgtgacattggaaggcatcgcggttggcccccaggcgaac gccggcgaacaggctggcagtagtggccccttagtacggcaagctggaacgtctcggccatctccacc gacggccacgacgcgggccagcaccgcaaccgcttcattgtctgctacagcccacctccagcaacgga gaggtgtcctttcaaagcgtccgcgtgaagatgatgatggagaaccgagtgaacgcaaacgcgagaga gatgagcgcagcaaggacgggcgtgggggaaataggagagcgcatattgtgatggtggatgcgtataa accgaccaaataggagcttcgacaggcatcaaataaaacgaaaggctcagtcgaaagactgggccttt cgttttatctgttgtttgtcggtgaacgctctcctgagtaggacaaatccgccc

[0151] Amplification of the Transgene Nucleic Acid in the Organelles and Mini-Chromosome for Gene Expression in the Organelles.

[0152] Although efficient systems for delivery of transgene nucleic acid (TNA) into organelles were established, a selectable marker and multiple rounds of selection are required to achieve an homoplasmic state of the transformants.

[0153] To address this issue we developed a DNA amplification system of TNA, allowing rapid achievement of an homoplasmic state of the transformants and/or by the introduction of autonomous mini-chromosomes without the need to insert TNA into the organelle genome.

[0154] For this purpose we have employed the replication system of plant ssDNA geminiviruses. It has been shown that some geminiviruses can replicate in non-host organisms such as bacteria and yeast (Selth et al., 2002; Raghavan et al., 2004). Replication of the geminiviruses depends on host cell DNA polymerase, and requires a viral origin of replication and viral Replication Initiation Protein (RIP) encoded by the viral Rep gene. We have designed vectors for both fast achievement of homoplasmic state of the transformants and expression of the TNA in organelles from autonomous mini-chromosome (FIGS. 8 and 9).

[0155] In the first case two viral origins of replication (MOR, BOR or TOR) from Maize Streak Virus (MSV, subgroup I) (MOR), Beet Curly Top Virus (BCTV, subgroup II) (BOR) and Tomato Golden Mosaic Virus (TGMV, subgroup III) (TOR) were introduced on both sides of TNA (FIG. 8). The expression of the viral Rep gene was performed from TNA or from a nuclear cassette where the Rep gene was fused to chloroplast or mitochondrial transit peptides (FIG. 10). We have observed efficient amplification of TNA in the organelles, resulting in fast achievement of the homoplasmic state of the transformants.

[0156] In order to express TNA from the autonomous mini-chromosome, the TNA was modified by removing LFS and RFS, so that only the cassette with genes for expression in organelles was placed between two viral origins of replication (FIG. 9). The expression of viral Rep gene was provided either from the TNA or from nuclear cassette where Rep gene was fused to the chloroplast or mitochondrial transit peptide.

TABLE-US-00005 BCTV viral origin of replication (BOR) SEQ ID 43 gatcctgtactccgatgacgtggcttagcatattaacatatctattggagtattggagtattatatat attagtacaactttcataagggccatccgttataatattaccggatggcccgaaaaaaatgggcaccc aatcaaaacgtgacacgtggaaggggactgttgaatgatgtgacgtttttgagcgggaaacttcctga ag MSV viral origin of replication (MOR) SEQ ID 44 Ccgacgacggaggttgaggctgagggatggcagactggcagctccaaactctatagtatacccgtgcg ccttcgaaatccgccgctcccttgtcttatagtggttgcaaatgggccggaccgggccggcccagcag gaaaagaaggcgcgcactaatattaccgcgccttcttttcctgcgagggcccggtagggcccgagcga tttgatgtaaagtttggtcctgctttgtatgatttatctaaagcagcccat TGMV viral origin of replication (TOR) SEQ ID 45 Gtaattaagaggcttactaccaattgaggaggggctccaaaagttatatgaattggtagtaaggtagc tcttatatattagaagttcctaaggggcacgtggcggccatccgtttaatattaccggatggccgcgc gatcgtcacccgacccgcttccgcaaattacgccgcattgtcgtctaagtggtcccgcatatgtgaag ggccaatcatatttggccctgaaatctaagata BCTV Rep gene (B-rep) SEQ ID 46 Atgcctcctactaaaagatttcgtattcaagcaaaaaacatatttcttacatatcctcagtgttctct ttcaaaagaagaagctcttgagcaaattcaaagaatacaactttcatctaataaaaaatatattaaaa ttgccagagagctacacgaagatgggcaacctcatctccacgtcctgcttcaactcgaaggaaaagtt cagatcacaaatatcagattattcgacctggtatccccaaccaggtcagcacatttccatccaaacat tcagagagctaaatccagctccgacgtcaagtcctacgtagacaaggacggagacacaattgaatggg gagaattccagatcgacggtagaagtgctagaggaggtcaacagacagctaacgactcatatgccaag gcgttaaacgcaacttctcttgaccaagcacttcaaatattgaaggaagaacaaccaaaggattactt ccttcaacatcacaatcttttgaacaatgctcaaaagatatttcagaggccacctgatccatggactc cactatttcctctgtcctcattcacaaacgttcctgaggaaatgcaagaatgggctgatgcatatttc ggggttgatgccgctgcgcggcctttaagatataatagtatcatagtagagggtgattcaagaacagg gaagactatgtgggctagatctttaggggcccacaattacatcacagggcacttagattttagcccta gaacgtattatgatgaagtggaatacaacgtcattgatgacgtagatcccacttacttaaagatgaaa cactggaaacaccttattggagcacaaaaggagtggcagacaaacttaaagtatggaaaaccacgtgt cattaaaggtggtatcccctgcattatattatgcaatccaggacctgagagctcataccaacaatttc ttgaaaaaccagaaaatgaagcccttaagtcctggacattacataattcaaccttctgcaaactccaa ggtccgctctttaataaccaagcagcagcatcctcgcaaggtgactctaccctgtaa MSV Rep gene (M-rep) SEQ ID 47 atggcctcctcctcatccaaccgtcagttctcacaccggaacgctaacacgttcctaacctatccaaa gtgtccagaaaatcctgaaatcgcctgtcagatgatctgggagctcgttgttcgttggattcccaaat acattctatgtgcccgagaggcacataaagatggaagtttgcatttacatgcattgcttcagacagag aagccggtaaggatatctgactcaaggttctttgatataaatgggtttcacccaaatattcagagtgc caagtcagtaaacagggtgagggattacattctcaaggaacctctggctgtgtttgagagaggtactt tcattcctaggaagtcccccttcctaggaaaatctgattcagaggtaaaggaaaaaaagccttctaaa gatgaaataatgcgagacattatttcacacgctacttccaaagaagagtacctctccatgatccagaa agagcttccctttgattggtccacaaaattgcagtattttgaatactctgcaaataagctttttcctg agattcaggaagagttcaccaatcctcatccaccctcatcacctgatttactttgtaatgagtcaatc aatgattggctccagcctaacatcttccagtcatcagatgaaagatcaagaaagcagagcctctacat cgtcggcccaacaagaaccggaaaatctacttgggccagaagcctaggggttcataattactggcaaa ataatgttgattggtcttcatacaacgaagacgcaatctataacatcgtagatgatattccgtttaaa ttctgtccttgttggaaacagttagttggctgtcagagggatttcattgtaaaccccaagtatggtaa aaagaaaaaggtgcagaagaagtctaagcctacaataatcctcgccaactcggatgaagattggatga aggaaatgactccagggcagctggagtatttcgaggcaaactgcatcatttacattatgtcgccgggg gagaaatggtattctccccctgagctgcctcctacggaggcagtacattcagatagatcttga TGMV Rep gene (T-rep) SEQ ID 48 atgccatcgcatccaaaacggtttcaaataaatgccaaaaattattttcttacatatcctcagtgctc cttgtccaaagaagaatcactttctcaattacaagccctaaacactccgattaacaaaaaattcataa aaatctgcagagagcttcatgaagatgggcaacctcacctccacgtgcttattcagttcgagggaaaa tactgctgccaaaatcaacgattcttcgacctggtatccccaacaaggtcagcacatttccatccaaa cattcagagagctaaatcgtcttccgacgtcaagacgtacatcgacaaagacggagatactcttgtat ggggagaattccaggtcgacggtcgaagtgctagaggaggttgccaaacatctaacgacgctgcagca gaggcgttaaatgcttcttccaaagaagaagccctgcagataattagagagaaaatcccagaaaaata tttatttcagttccacaatctaaatagcaatttagataggatatttgataagactcctgaaccatggc ttcctccgttccacgtctcatcatttactaacgtgccagacgagatgagacaatgggctgaaaattat tttggaaagagttccgctgcgcggccggagagacctattagtattatcatcgagggcgatagtcggac gggaaagactatgtgggctcgttcactaggcccacataattatttgagcgggcatttggatctcaatt ctagggtttactcaaacaaggttgagtataacgtcatcgatgatgtcacaccgcaatatctaaagttg aaacattggaaagaactcattggggcccaaagagattggcagactaactgtaaatacggaaagccagt tcaaattaaaggaggtatcccgtcaatcgtgctgtgcaatcctggagagggtgctagctataaagttt tcctcgacaaagaggaaaacactccactaaagaactggactttccataatgcgaaattcgtcttcctc aactcccccctctatcaaagctcaacacagagcagctaa

REFERENCES

[0157] Selth L A, Randles J W, Rezaian M A. Agrobacterium tumefaciens supports DNA replication of diverse geminivirus types. FEBS Lett. 2002, 10; 516(1-3):179-82. [0158] Vineetha Raghavan, Punjab S. Malik, Nirupam Roy Choudhury, and Sunil K. Mukherjee. The DNA-A Component of a Plant Geminivirus (Indian Mung Bean Yellow Mosaic Virus) Replicates in Budding Yeast Cells. J Virol. 2004, 78(5): 2405-2413. [0159] Gianluca Veggiani, Bijan Zakeri, and Mark Howarth. Superglue from bacteria: unbreakable bridges for protein nanotechnology. Trends in Biotechnology. 2014, 32(10):506-12. [0160] Long Li, Jacob O. Fierer, Tom A. Rapoport, and Mark Howarth. Structural Analysis and Optimization of the Covalent Association between SpyCatcher and a Peptide Tag. J Mol Biol. 2014, 23; 426(2): 309-317. [0161] Wikoff, W. R. et al. Topologically linked protein rings in the bacteriophage HK97 capsid. Science. 2000, 289, 2129-2133 [0162] K. I. Ivanov, K. Eskelin, A. Lohmus, K. Makinen. Molecular and cellular mechanisms underlying potyvirus infection. J. Gen. Virol. 2014, 95: 1415-1429. [0163] Rantalainen K I, Uversky V N, Permi P, Kalkkinen N, Dunker A K, Makinen K. Potato virus A genome-linked protein VPg is an intrinsically disordered molten globule-like protein with a hydrophobic core. Virology. 2008, 1; 377(2):280-8. [0164] Grzela R, Szolajska E, Ebel C, Madern D, Favier A, Wojtal I, Zagorski W, Chroboczek J. Virulence factor of potato virus Y, genome-attached terminal protein VPg, is a highly disordered protein. J Biol Chem. 2008, 283(1):213-21. [0165] Allan Olspert, Lauri Peil, Eugenie Hebrard, Denis Fargette and Erkki Truve. Protein-RNA linkage and post-translational modifications of two sobemovirus VPgs. Journal of General Virology. 2011, 92, 445-452. [0166] Lampson B C, Inouye M, Inouye S. Retrons, msDNA, and the bacterial genome”. Cytogenet Genome Res. 2005, 110 (1-4): 491-9 [0167] Rozwadowski K and Lydiate D. 2003. patentscope.wipo.int/search/en/detail.jsf?docId=WO2003104470&recNum=1&maxRec=&office=&prevFilter=&sortOption=&queryString=&tab=PCT+Biblio [0168] Sahoo et al. An improved protocol for efficient transformation and regeneration of diverse indica rice cultivars. Plant MAtheods. 2011, 7:49 [0169] Tadashi Shimamoto, Hideki Kawanishi, Tomofusa Tsuchiya, Sumiko Inouye, and Masayori Inouye. In Vitro Synthesis of Multicopy Single-Stranded DNA, Using Separate Primer and Template RNAs, by Escherichia coli Reverse Transcriptase. J Bacteriol. 1998, 180(11): 2999-3002.

Experimental Examples

[0170] Chloroplast Transformation Using groupII Intron Constructs.

[0171] Reference is made to constructs detailed in Table 1 throughout.

[0172] Note to LS: Table 1 Goes in Here

[0173] We have utilised Agrobacterium-mediated transformation of tobacco (plantsci.missouri.edu/muptcf/protocols/tobacco.html) and rice Sahoo et al., 2011). In order to transform chloroplasts in tobacco, we have used the constructs OTV1-OTV4 (Table 1). The constructs contain TNA in domain IV of the corresponding groupII intron, while the reshuffled retron is flanking 3′-end of the groupII intron. The reverse transcriptase of the retron is fused with corresponding intron encoded protein (IEP), and fulfils three functions, namely translocate TNA-RNA to organelle, initiates reverse transcription from retron to generate priming for reverse transcription of the TNA by the IEP. We expect that reverse transcription is more efficient in this case as it is a natural configuration for reverse transcription by the IEP. The 3′ and 5′-ends of the intron are also reverse transcribed in this case, but they are eliminated by homologous recombination machinery during TNA integration into the organelles genome.

[0174] The tobacco constructs OTV5 and OTV6 contain TNA at the 3′-end of the intron, and utilise direct priming of the TNA without reverse transcription of intron sequence. The reverse transcription in this case generated by combination of RT activities from both retron and the IEP.

[0175] Similar approach was utilised for rice transformation with constructs OTV7-OTV10 (Table 1).

[0176] Successful transformation of tobacco and rice chloroplasts using groupII constructs was confirmed on spectinomycin resistant plants by PCR of flanking sequences and by sequencing of the corresponding PCR products (FIGS. 11A and B).

[0177] The following primers have been used for tobacco to generate a fragment of 720 bp for tobacco:

TABLE-US-00006 TC1 SEQ ID 49 ctgagtaggacaaatccgccc TC2 SEQ ID 50 ggtggagatcatattcactctggtaccgtagt and a fragment of 1100 bp for rice: Rd SEQ ID 51 accccgggacgagaagtagtagga RC2 SEQ ID 52 atcgatcatgagattcatagttgcattact

[0178] Chloroplast Transformation Using PVY-Based Vectors.

[0179] To transform chloroplast in tobacco using Potato Virus Y as a chloroplast translocation sequence, the OTV21, OTV22 and OTV23 constructs has been used. Co-transformation of the construct OTV27 containing SpyCatcher fused to chloroplast transit peptide was performed in combination with OTV22 (N-terminal SpyTag) or OTV23 (C-terminal SpyTag).

[0180] PCR analysis of flanking sequences using T1 and T2 primers on spectinomycin resistant transformants, and sequencing analysis of amplified fragments have confirmed insertion of transgene using this approach (FIG. 12).

[0181] Chloroplast Transformation Using Modified Agrobacterium virD2 Protein.

[0182] Agrobacterium-mediated transformation of the tobacco chloroplasts using modified strain GV3101 with knocked out virD2 and virE2 genes was performed. Complementary virD2 protein modified by fusion of chloroplast transit peptide (OTV29), or N-terminal SpyTag (OTV31) and C-terminal SpyTag (OTV32) was expressed from Agrobacterium virD operon promoter. The cassette carrying virD promoter, modified virD2 gene and bacterial rrnB terminator was integrated on binary vector outside of the T-DNA boarders. The OTV31 and OTV32 constructs carrying SpyTag were transformed in two steps, as SpyCatcher peptide (construct OTV27) should be already expresses in the cytoplast of plant cell before challenging plant cell with these constructs. The tobacco leaves were first infiltrated with Agrobacterium containing OTV27 construct, following second round of transformation of leaf explant from infiltrated plants with OTV31 or OTV32 two days later.

[0183] PCR analysis of flanking sequences using the T1 and T2 primers on the spectinomycin resistant transformants, and sequencing analysis of amplified fragments have confirmed insertion of transgene using this approach (FIG. 13).

[0184] TNA Amplification in the Chloroplast Using Geminivirus Replication System.

[0185] DNA approach for chloroplast transformation using modified virD2 gene has proved to be feasible but not efficient from point of view of copy number of transgene delivered to the chloroplasts. To address this issue, we have developed transgene amplification system in chloroplasts using Geminivirus replication system. It has been shown that Geminivirus could be replicated in Agrobacterium and yeast. Introduction of viral origin of replication and expression of viral Rep gene encoding replication initiation protein (RIR), was sufficient to replicate plasmid in these organisms.

[0186] To evaluate whether Geminivirus can be replicated in the chloroplasts, we have selected Maize Streak Virus-MSV (subclass I), Beet Top Curly Virus-BCTV (subclass II) and Tomato Golden Mosaic Virus-TGMV (subclass III). The constructs were prepared containing two viral origins of replication with chloroplast transformation cassette located between them. Resulted constructs OTV33, OTV34 and OTV35 containing correspondingly BCTV viral origins of replication (BOR), MSV viral origins (MOR), and TGMV viral origins (TOR), were delivered to the tobacco chloroplasts using modified virD2 Agrobacterium approach. The Rep gene for corresponding viral origin of replication was fused to chloroplast transit peptide and was co-expressed from nuclear promoter (OTV39, OTV40 and OTV41).

[0187] We have observed dramatic amplification of transgene nucleic acid with BCTV and TGMV origins (FIG. 14A), while MSV origins were able to amplify transgene with modest efficiency (FIG. 14B).

[0188] Next we wanted to see whether we could maintain transgene in the chloroplasts as mini-chromosome without integration in the chloroplast genome. For this purpose the constructs OTV45 and OTV46 which do not contain LFS and RFS were prepared and co-delivered with the construct OTV39 and OTV41 into the tobacco chloroplasts using combination of Agrobactrium with functional virD2 gene for constructs OTV39 and OTV41, and Agrobacterium with modified virD2 gene fused to chloroplast transit peptide. We have observed efficient delivery amplification of transgene cassette without insertion into the chloroplast genome (FIG. 15).

[0189] Mitochondria Transformation Using groupII Intron Constructs and PVY-Based Vectors.

[0190] Transformation of mitochondria in tobacco and rice was performed in similar way as transformation of chloroplast using constructs OTV11-OTV16 for tobacco and OTV17-OTV20 for rice. Selection was performed for insertion of T-DNA into the nuclear genome, as there is no selectable marker for mitochondria transformation. The OTV24-OTV26 were utilised for PVY-based approach in combination with OTV28 vector. The plants recovered on kanamycin for nuclear insertion were than analysed for insertion of the transgene into the mitochondrial genome using PCR of flanking sequences and by sequencing of the PCR generated fragments. The following primers have been used for amplification of flanking sequences in tobacco to generate fragment of 1050 bp:

TABLE-US-00007 TM1 SEQ ID 53 cgtcccataccttctgcctgtctca TM2 SEQ ID 54 gatggatacatacgatttcacttat

[0191] and a fragment of 1170 bp for rice:

TABLE-US-00008 RM1 SEQ ID 55 gggtaacttttatttatcattcaca RM2 SEQ ID 56 acttcggcgatcaccgcttctgccat

[0192] We observed successful integration events with all approaches (FIG. 16).

[0193] Mitochondria Transformation Using Modified Agrobacterium virD2 Protein.

[0194] Agrobacterium-mediated transformation of the tobacco mitochondria using modified strain GV3101 with knocked out virD2 and virE2 genes was performed. Complementary virD2 protein modified by fusion of mitochondria transit peptide (OTV30), or N-terminal SpyTag (OTV31) and C-terminal SpyTag (OTV32) was expressed from Agrobacterium virD operon promoter. The cassette carrying virD promoter, modified virD2 gene and bacterial rrnB terminator was integrated on binary vector outside of the T-DNA boarders. The OTV31 and OTV32 constructs carrying SpyTag were transformed in two steps, as SpyCatcher peptide (construct OTV28) should be already expresses in the cytoplast of plant cell before challenging plant cell with these constructs. The tobacco leaves were first infiltrated with Agrobacterium containing OTV28 construct, following second round of transformation of leaf explant from infiltrated plants with OTV31 or OTV32 two days later. PCR analysis of flanking sequences has confirmed integration of transgene into the mitochondrial genome of tobacco (FIG. 17).

[0195] TNA Amplification in the Mitochondria Using Geminivirus Replication System.

[0196] Similar to chloroplast approach, to amplify transgene in the mitochondria using Geminivirus replication system we have prepared OTV47 (BOR) and OTV48 (TOR) constructs. These constructs were co-expressed with OTV42 and OTV44 to generate autonomous mini-chromosome of transgene in the mitochondria without its insertion into the mitochondrial genome. Southern analysis of transgenic plants has confirmed that at least BCTV and TGMV-based system could replicate in the mitochondria (FIG. 18).

[0197] Examples of Chloroplast Transformation Using a Replicon Construct.

[0198] To evaluate efficiency of the chloroplast transformation using replicon we utilised particle bombardment procedure described in manual for Bio-Rad particle gun (www.bio-rad.com/webroot/web/pdf/lsr/literature/M1652249.pdf).

[0199] Two constructs were used for transformation of tobacco, potato and maize:

[0200] AIBW construct (OTV 50) contains two genes of interest (aadA and GFP) and a cassette for expression of repA gene flanked by two viral origins of replication (BOR1 and BOR2) from beet curly top virus (BCTV) (FIG. 20). Replication initiation protein repA recruits host DNA polymerase to viral origins of replication and amplify DNA located between BOR1 and BOR2.

[0201] AJWP construct (OTV 49) contains BCTV replication initiation protein repA gene fused to chloroplast transit peptide under constitutive nuclear 35S promoter (FIG. 20).

[0202] Two constructs were co-bombarded into leaf explants of tobacco, potato and maize. The AJWP construct (OTV 49) served as a helper plasmid for establishing replication of the AIBW plasmid (OTV50) in the chloroplasts due to transient production of repA protein from nucleus to boost efficiency of initial replication.

[0203] Tissue culture and regeneration of transgenic plants for potato was performed according Valkov et al., (Transgenic Res (2011) 20:137-151), and for maize according Ahmadabadi et al., (Transgenic Res (2007) 16: 437-448).

[0204] Selection of bombarded explants was performed on medium supplemented with 500 μg/l of spectinomycin.

[0205] We were able to recover plants with the chloroplast transgene replicon in all three plant species (FIG. 21). Chloroplast origin of replicon was confirmed by strong expression of GFP in the chloroplasts. No cytoplasmic or nuclear patterns of GFP expression were detected. The transgene replicon was transferred through the seeds to the subsequent transgene generations.

TABLE-US-00009 SEQ id 57 clpP promoter from maize tctatgtattaatagaatctatagtattcttata gaataagaaaaaaaaaatgaagataataaactgc ggattctttctttctcttccattcttacgtttcc atattaaagtgtagtttttttacttaaatttaat aatattaatctaat

[0206] Variant 1 of the Invention

[0207] Statements on Variant 1

[0208] 1. A method of transforming at least one species of plant cell organelle comprising:

[0209] i) transforming the nucleus of a plant cell with a DNA cassette carrying at least one transgene nucleic acid (TNA) sequence of interest;

[0210] ii) recruiting the transgene nucleic acid RNA generated by the transcription of the transgene nucleic acid sequence of step i) from the cytoplasm and directing it into the at least one species of plant organelle;

[0211] iii) reverse transcribing the transgenic nucleic acid RNA of ii) into single stranded DNA (ssDNA) in the at least one organelle; and

[0212] iv) inserting the single stranded DNA of iii) into the organelle genome via homologous recombination; and

[0213] wherein the reverse transcribing event of step iii) within the organelle is performed by a retron specific reverse transcriptase sequence fused to at least one reverse transcriptase sequence different to the first.

[0214] 2. A method of transforming a plant cell according to statement 1 comprising:

[0215] 1) introducing into the said plant cell a first nucleic acid sequence that comprises a nuclear promoter operably linked to a first nucleic acid sequence comprising a plant organellar transit peptide (TP) located adjacent to a retron specific reverse transcriptase sequence fused to at least one reverse transcriptase sequence different to the first, such as an IEP sequence, and a nuclear terminator;

[0216] 2) introducing into the said plant cell a second nucleic acid sequence that encodes for a group II intron operably linked to a plant nuclear promoter; and

[0217] 3) introducing into the said plant cell a third nucleic acid sequence that encodes for an organellar transgene cassette comprising a left flanking sequence, an organellar promoter, at least one transgene nucleic acid sequence of interest, an organellar terminator and a right flanking sequence;

[0218] 4) introducing a fourth nucleic acid sequence that codes for a retron sequence for reverse transcription of the TNA.

[0219] 3. A method according to statement 1 or statement 2, wherein the transgene nucleic acid sequence is a recombinant DNA sequence or an introduced native, isolated genomic DNA sequence.

[0220] 4. A method according to any one of statements 1 to 3, wherein the third nucleic acid sequence of claim 2 step 3) is inserted into Domain IV of the group II intron of step 2).

[0221] 5. A method according to any one of statements 1 to 3, wherein the third nucleic acid sequence of statement 1 step 3) is located at the 5′ and/or 3′ end of the group II intron of step 2).

[0222] 6. A method according to any one of statements 1 to 3 and 5, wherein the third nucleic acid sequence of 3) is located at the 3′ end of the group II intron of step 2).

[0223] 7. A method according to any one of the preceding statements wherein the plant organelle is selected from a plant mitochondrion, and a plant plastid.

[0224] 8. A method according to any one of the preceding statements, wherein the plant organelle is a mitochondrion.

[0225] 9. A method according to any one of statements 1 to 7, wherein the plant organelle is selected from chloroplasts, proplastids, etioplasts, chromoplasts, amyloplasts, leucoplasts and elaioplasts, and is preferably a chloroplast.

[0226] 10. A method according to any one of the preceding statements, wherein the transgene nucleic acid sequence is selected from a recombinant mammalian nucleic acid sequence, an isolated genomic mammalian nucleic acid sequence, a recombinant plant nucleic acid sequence and an isolated genomic plant nucleic acid sequence and two or more thereof.

[0227] 11. A method according to any one of the preceding statements, wherein the DNA cassette comprises an organellar promoter selected from a mitochondrion specific promoter and a plastid specific promoter.

[0228] 12. A method according to any one of the preceding statements, wherein the mitochondrion specific promoter is selected from mitochondrial promoter nucleotide sequences, such as ATP6, ATP9, Cob, rrn18, Rps13, Rps19, Cox3, Nad6, Nad9 5′ untranslated sequences (promoter region) of tobacco mitochondria, and Arabidopsis mitochondria; and the plastid specific promoter sequence is selected from the group consisting of the RNA polymerase promoter, rpo B promoter element, atpB promoter element, the clpP promoter element, the 16S rDNA promoter element, PrbcL, Prps16, the Prrn16, Prrn-62, Pycf2-1577, PatpB-289, Prps2-152, Prps16-107, Pycf1-41, PatpI-207, PclpP-511, PclpP-173, PaccD-129, PaccD-129 promoter of the tobacco accD gene, the PclpP-53 promoter of the clpP gene, the Prrn-62 promoter of the rrn gene, the Prps16-107 promoter of the rps16 gene, the PatpB/E-290 promoter of the tobacco atpB/E gene, and the PrpoB-345 promoter of the rpoB gene.

[0229] 13. A method according to claim any one of statements 1 to 12, wherein the transgene or isolated nucleic acid sequence is selected from insulin, preproinsulin, proinsulin, glucagon, interferons such as α-interferon, β-interferon, γ-interferon, blood-clotting factors selected from Factor VII, VIII, IX, X, XI, and XII, fertility hormones including luteinising hormone, follicle stimulating hormone growth factors including epidermal growth factor, platelet-derived growth factor, granulocyte colony stimulating factor and the like, prolactin, oxytocin, thyroid stimulating hormone, adrenocorticotropic hormone, calcitonin, parathyroid hormone, somatostatin, erythropoietin (EPO), enzymes such as β-glucocerebrosidase, haemoglobin, serum albumin, collagen, biotic and abiotic stress proteins, such as insecticidal and insect toxic proteins, for example from, or derived from Bacillus thuringiensis, nematicidal proteins, herbicide resistance proteins, (e.g. to glyphosate), salt-tolerance proteins, drought tolerant proteins, proteins capable of conferring cytoplasmic male sterility to plant breeding lines; nutritional enhancement proteins involved in the biosynthesis of phenolics, starches, sugars, alkaloids, vitamins, and edible vaccines, monoclonal antibodies and active fragments thereof, industrial enzymes and active fragments thereof.

[0230] 14. A method according to any one of statements 1 to 13, wherein the transgene or isolated nucleic acid sequence is selected from proteins that confer cytoplasmic male sterility to a plant.

[0231] 15. A method according to any one of the preceding statements, wherein the transgene or isolated nucleic acid sequence that is capable of conferring cytoplasmic male sterility to the plant is selected from the petunia mitochondrion pcf sequence, orf107 sequence of sorghum and orf 79 of rice.

[0232] 16. A method according to any one of the preceding statements wherein the retron is a DNA sequence comprising a msr element encoding an RNA sequence comprising a binding domain for retron-specific reverse transcriptase, and a msd element encoding a DNA component fused to the 3′ end of a nucleic acid sequence or a fragment thereof and/or the 3′ end of TNA, wherein the msr and msd elements comprise pairs of inverted repeat sequences forming double-stranded RNA regions driving reverse transcription of the msd element and/or reverse transcription of the TNA:msd element fusion product.

[0233] 17. A method according to claim any one of the preceding statements, wherein the msr and msd elements comprise pairs of inverted repeat sequences selected from a1 and a2, and b1 and b2 sequences.

[0234] 18. A method according to any one of the preceding statements, wherein the retron msDNA is a bacterial retron msDNA sequence, such as a sequence selected from Ec86, Mx162, Sal63, Ec67, Ec73, and Ec107.

[0235] 19. A method according to any one of the preceding statements, wherein the at least one reverse transcriptase sequence different to the first is a groupII intron or an IEP fragment thereof that encodes reverse transcriptase functionality is selected from the LtrB intron, the RmIntORF, the a12 intron, the tobacco group II intron and the nad1 gene containing matK.

[0236] 20. A method according to any one of the preceding statements wherein the plant organellar transit peptide is independently selected from the mitochondrial signal peptide from tobacco F1-ATPase-1 β subunit, and the Arabidopsis CPN60 protein; and the plastidial transit peptide independently from selected from the tobacco rbcS-cTP, and the Arabidopsis HSP70-cTP protein.

[0237] 21. A plant cell obtained according to any one of statments 1 to 20.

[0238] 22. A plant cell comprising transformed plant organelles as defined in any one of statements 1 to 20, wherein the transformed plant organelles comprise:

[0239] i) an exogenous or heterologous left flanking sequence (LFS) and an exogenous or heterologous right flanking sequence (RFS);

[0240] ii) at least one exogenous or heterologous organelle-specific promoter and at least one exogenous or heterologous organelle-specific terminator sequence; and

[0241] iii) at least one exogenous or heterologous isolated transgene nucleic acid sequence of interest.

[0242] 23. A plant cell according to statement 21, wherein the transformed organelles are selected from plant plastids and mitochondria transformed as defined in any one of statements 1 to 20.

[0243] 24. A transformed plant organelle comprising:

[0244] i) an exogenous or heterologous left flanking sequence (LFS) and an exogenous or heterologous right flanking sequence (RFS);

[0245] ii) at least one exogenous or heterologous organelle-specific promoter and at least one exogenous or heterologous organelle-specific terminator sequence; and

[0246] iii) at least one exogenous or heterologous isolated transgene nucleic acid sequence of interest.

[0247] 25. A transformed plant organelle according to statement 24, wherein the transformed organelle is selected from a plant plastid and a mitochondrion transformed as defined in any one of statements 1 to 20.

[0248] 26. A population of transformed plant organelles as defined in statement 23 or statement 25 comprised in a plant cell.

[0249] 27. A population of transformed plant organelles according to statement 25, wherein the organelles are located in plant cells selected from tobacco (Nicotiana tabacum) and other Nicotiana species, arabidopsis, potato, corn(maize), canola (rape), rice, wheat, barley, brassica sp. such as cauliflower, broccoli (e.g. green and purple sprouting), cabbage (e.g. red, green and white cabbages), curly kale, Brussels sprouts, cotton, algae (e.g. blue green species), lemnospora, or moss (e.g. Physcomitrella patens), tomato, capsicum, squashes, sunflower, soyabean, carrot, melons, grape vines, lettuce, strawberry, sugar beet, peas, and sorghum.

[0250] 28. A population of transformed plant organelles according to statement 26 or statement 27 wherein the organelles are located in plant cells selected from cotton, rice, oilseed Brassica species such as canola, corn(maize) and soyabean.

[0251] 29. A method of producing at least a heterologous or exogenous RNA species in a plant that comprises:

[0252] 1) introducing into a regenerable plant cell a first nucleic acid sequence that comprises a nuclear promoter operably linked to a first nucleic acid sequence comprising a plant organellar transit peptide (TP) located adjacent to a retron specific reverse transcriptase sequence fused to at least one reverse transcriptase sequence different to the first, such as a group II intron sequence or a fragment thereof possessing reverse transcriptase functionality, such as an IEP sequence, and a nuclear terminator;

[0253] 2) introducing into the said plant cell a second nucleic acid sequence that encodes for a group II intron operably linked to a plant nuclear promoter; and

[0254] 3) introducing into the said plant cell a third nucleic acid sequence that encodes for an organellar transgene cassette comprising a left flanking sequence, an organellar promoter, at least one transgene nucleic acid sequence of interest, an organellar terminator and a right flanking sequence; and

[0255] 4) introducing a fourth nucleic acid sequence that codes for a retron sequence for reverse transcription of the TNA.

[0256] 5) growing said regenerable plant cell of steps 1) to 4);

[0257] 6) selecting a plant cell of (5), wherein the transgene comprised within the plant organellar transgene cassette is integrated into the organellar genome;

[0258] 7) regenerating a plant from the plant cell of (6); and

[0259] 8) growing the plant of (7).

[0260] 30. A method according to statement 29, wherein the heterologous or exogenous RNA species encoded by the transgene that is integrated into the organellar genome is expressed as a heterologous or exogenous protein.

[0261] 31. A method according to statement 29 or statement 30, wherein the plant organellar genome is independently selected from that of plant mitochondria and plant plastids.

[0262] 32. An isolated polynucleotide sequence that comprises a plant nuclear promoter operably linked to a first nucleic acid sequence comprising a plant organellar transit peptide (TP) located adjacent to a retron specific reverse transcriptase first nucleic acid sequence that comprises a nuclear promoter operably linked to a first nucleic acid sequence comprising a plant organellar transit peptide (TP) located adjacent to a retron specific reverse transcriptase sequence fused to at least one reverse transcriptase sequence different to the first, such as a group II intron sequence or a fragment thereof possessing reverse transcriptase functionality, such as an IEP sequence and a nuclear terminator; a second nucleic acid sequence that encodes for a group II intron operably linked to a plant nuclear promoter; a third nucleic acid sequence that encodes for an organellar transgene cassette comprising a left flanking sequence, an organellar promoter, at least one transgene nucleic acid sequence of interest, an organellar terminator and a right flanking sequence; and a fourth nucleic acid sequence that codes for a retron sequence for reverse transcription of the TNA for use in a method according to any one of statements 1 to 19 and statements 28 to 30.

[0263] 33. An isolated polynucleotide sequence as defined in any one of statements 1 to 20 and statements 29 to 31, comprising genomic DNA.

[0264] 34. An isolated polynucleotide sequence as defined in any one of statements 1 to 20 and statements 29 to 31, comprising a cDNA component.

[0265] 35. A nucleic acid vector suitable for transformation of a plant cell or a bacterial cell, wherein the cell includes a polynucleotide sequence according to any one of statements 32 to 34.

[0266] 36. A nucleic acid vector according to statement 35 for transformation of a bacterial cell.

[0267] 37. A nucleic acid vector according to statement 36 for transforming an Agrobacterium cell.

[0268] 38. A host cell containing a heterologous polynucleotide or nucleic acid vector according to any one of statements 32 to 37.

[0269] 39. A host cell according to statement 38 which is a plant cell or a bacterial cell.

[0270] 40. A host cell according to statement 38 or statement 39 comprised in a plant, a plant part or a plant propagule, or an extract or derivative of a plant or in a plant cell culture.

[0271] 41. A method of producing a cell according to any one of statements 38 to 40, the method including incorporating said polynucleotide or nucleic acid vector into the cell by means of transformation.

[0272] 42. A method according to statement 41 which includes regenerating a plant from a cell according to any one of statements 38 to 40 from one or more transformed cells.

[0273] 43. A plant comprising a plant cell according to any one of statements 38 to 40.

[0274] 44. A plant comprising a plant cell according to statement 43 that is selected from the group consisting of tobacco (Nicotiana tabacum) and other Nicotiana species, such as Nicotiana benthamiana, carrot, vegetable and oilseed Brassica's, melons, Capsicums, grape vines, lettuce, strawberry, sugar beet, wheat, barley, (corn)maize, rice, soybean, peas, sorghum, sunflower, tomato, cotton, and potato.

[0275] 45. A plant comprising a plant cell according to statement 43 or statement 44 that is selected from the group consisting of cotton, rice, oilseed Brassica species such as canola, corn(maize) and soybean.

[0276] 46. A method of producing a plant, the method including incorporating a polynucleotide sequence or nucleic acid vector according to any one of statements 31 to 36 into a plant cell and regenerating a plant from said cell.

[0277] 47. Use of a polynucleotide sequence according to any one of statements 32 to 37 in the production of a transgenic plant.

[0278] 48. Use of a polynucleotide sequence according to any one of statements 32 to 37 in the production of a polypeptide or protein in a plant.

[0279] All definitions for component parts of statements 1 to 48 of Variant 1 are found either in the accompanying description or in statements 1 to 48. The Experimental section provides technical descriptions of work performed relating to Variant 1.

[0280] Variant 2 of the Invention

[0281] Statements on Variant 2

[0282] 1. A method for use in transforming a transgene nucleic acid of interest into a plant organelle in a plant cell comprising:

[0283] 1(a) deleting viral polymerase and coat protein sequences from the complete viral genome of a potyvirus and replacing them with transgenic nucleic acid in cis, wherein the said transgenic nucleic acid comprises a nuclear promoter operably linked to a viral 5′ UTR sequence linked to the 5′ end of a complete RNA translocation sequence of the potyvirus, wherein

[0284] i) the 5′ end of the potyviral RNA translocation sequence is covalently linked to the VPg protein therein and to an organellar transit peptide; or

[0285] ii) the potyviral RNA translocation sequence is modified by fusing a spytag short peptide sequence to the viral VPg protein at either the N- or C-terminus thereof; and introducing the product of i) or ii) into a plant cell;

[0286] 1(b) introducing into the viral translocation sequence a second component nucleic acid sequence that encodes for an organellar transgene cassette comprising a left flanking sequence, an organellar promoter, at least one transgene nucleic acid sequence of interest, an organellar terminator and a right flanking sequence; and

[0287] 1(c) introducing into the said plant cell a third component nucleic acid sequence that codes for a retron sequence for reverse transcription of the TNA; and

[0288] 1(d)(i) introducing into the said plant cell a fourth component nucleic acid sequence comprising a viral 3′UTR sequence;

[0289] 1(d)(ii) introducing into the plant cell a nucleic acid sequence comprising a nuclear promoter operably linked to a nucleic acid sequence comprising a plant organellar transit peptide (TP) located adjacent to a retron-based reverse transcriptase fused to an intron encoding protein (IEP), and a nuclear terminator; and

[0290] 1(e)(i) introducing into the plant cell either a potyviral polymerase in trans under the control of a plant nuclear promoter sequence and a terminator; or

[0291] 1(e)(ii) a spycatcher peptide fused to an organellar transit peptide, the said fused peptide being expressed under the control of a nuclear promoter.

[0292] 2. A method according to statement 1, wherein the transgene nucleic acid sequence is a recombinant DNA sequence or an introduced native, isolated genomic DNA sequence.

[0293] 3. A method according to statement 1 or statement 2, wherein the plant organelle is selected from a plant mitochondrion, and a plant plastid.

[0294] 4. A method according to any one of the preceding statements, wherein the plant organelle is a mitochondrion.

[0295] 5. A method according to any one of statementss 1 to 14, wherein the plant organelle is selected from chloroplasts, proplastids, etioplasts, chromoplasts, amyloplasts, leucoplasts and elaioplasts, and is preferably a chloroplast.

[0296] 6. A method according to any one of the preceding statementss, wherein the transgene nucleic acid sequence is selected from a recombinant mammalian nucleic acid sequence, an isolated genomic mammalian nucleic acid sequence, a recombinant plant nucleic acid sequence and an isolated genomic plant nucleic acid sequence and two or more thereof.

[0297] 7. A method according to any one of the preceding statements, wherein the DNA cassette comprises an organellar promoter selected from a mitochondrion specific promoter and a plastid specific promoter.

[0298] 8. A method according to any one of the preceding statements, wherein the mitochondrion specific promoter is selected from mitochondrial promoter nucleotide sequences, such as ATP6, ATP9, Cob, rrn18, Rps13, Rps19, Cox3, Nad6, Nad9 5′ untranslated sequences (promoter region) of tobacco mitochondria, and Arabidopsis mitochondria; and the plastid specific promoter sequence is selected from the group consisting of the RNA polymerase promoter, rpo B promoter element, atpB promoter element, the clpP promoter element, the 16S rDNA promoter element, PrbcL, Prps16, the Prrn16, Prrn-62, Pycf2-1577, PatpB-289, Prps2-152, Prps16-107, Pycf1-41, PatpI-207, PclpP-511, PclpP-173, PaccD-129, PaccD-129 promoter of the tobacco accD gene, the PclpP-53 promoter of the clpP gene, the Prrn-62 promoter of the rrn gene, the Prps16-107 promoter of the rps16 gene, the PatpB/E-290 promoter of the tobacco atpB/E gene, and the PrpoB-345 promoter of the rpoB gene.

[0299] 9. A method according to any one of statements 1 to 8, wherein the transgene or isolated nucleic acid sequence is selected from insulin, preproinsulin, proinsulin, glucagon, interferons such as α-interferon, β-interferon, γ-interferon, blood-clotting factors selected from Factor VII, VIII, IX, X, XI, and XII, fertility hormones including luteinising hormone, follicle stimulating hormone growth factors including epidermal growth factor, platelet-derived growth factor, granulocyte colony stimulating factor and the like, prolactin, oxytocin, thyroid stimulating hormone, adrenocorticotropic hormone, calcitonin, parathyroid hormone, somatostatin, erythropoietin (EPO), enzymes such as β-glucocerebrosidase, haemoglobin, serum albumin, collagen, biotic and abiotic stress proteins, such as insecticidal and insect toxic proteins, for example from, or derived from Bacillus thuringiensis, nematicidal proteins, herbicide resistance proteins, (e.g. to glyphosate), salt-tolerance proteins, drought tolerant proteins, proteins capable of conferring cytoplasmic male sterility to plant breeding lines; nutritional enhancement proteins involved in the biosynthesis of phenolics, starches, sugars, alkaloids, vitamins, and edible vaccines, monoclonal antibodies and active fragments thereof, industrial enzymes and active fragments thereof.

[0300] 10. A method according to any one of statementss 1 to 9, wherein the transgene or isolated nucleic acid sequence is selected from proteins that confer cytoplasmic male sterility to a plant.

[0301] 11. A method according to any one of the preceding statements, wherein the transgene or isolated nucleic acid sequence that is capable of conferring cytoplasmic male sterility is the plant is selected from the petunia mitochondrion pcf sequence, orf107 sequence of sorghum and orf 79 of rice.

[0302] 12. A method according to any one of the preceding statements, wherein the plant organellar transit peptide is independently selected from the mitochondrial signal peptide from tobacco F1-ATPase-1 β subunit, and the Arabidopsis CPN60 protein; and the plastidial transit peptide independently from selected from the tobacco rbcS-cTP, and the Arabidopsis HSP70-cTP protein.

[0303] 13. A plant cell obtained according to any one of statements 1 to 12.

[0304] 14. A plant cell comprising transformed plant organelles as defined in statements 1 to 13, wherein the transformed plant organelles comprise:

[0305] i) an exogenous or heterologous left flanking sequence (LFS) and an exogenous or heterologous right flanking sequence (RFS);

[0306] ii) at least one exogenous or heterologous organelle-specific promoter and at least one exogenous or heterologous organelle-specific terminator sequence; and

[0307] iii) at least one exogenous or heterologous isolated transgene nucleic acid sequence of interest.

[0308] 15. A plant cell according to statement 14, wherein the transformed organelles are selected from plant plastids and mitochondria transformed as defined in any one of statements 1 to 13.

[0309] 16. A transformed plant organelle comprising:

[0310] i) an exogenous or heterologous left flanking sequence (LFS) and an exogenous or heterologous right flanking sequence (RFS);

[0311] ii) at least one exogenous or heterologous organelle-specific promoter and at least one exogenous or heterologous organelle-specific terminator sequence; and

[0312] iii) at least one exogenous or heterologous isolated transgene nucleic acid sequence of interest.

[0313] 17. A transformed plant organelle according to statement 16, wherein the plant organelle is selected from a plant plastid and a mitochondrion transformed as defined in any one of statements 1 to 13.

[0314] 18. A population of transformed plant organelles made up of transformed organelles according to statement 16 or statement 17 comprised in a plant cell.

[0315] 19. A population of transformed plant organelles according to statement 18, wherein the organelles are located in plant cells selected from tobacco (Nicotiana tabacum) and other Nicotiana species, arabidopsis, potato, corn(maize), canola (rape), rice, wheat, barley, brassica sp. such as cauliflower, broccoli (e.g. green and purple sprouting), cabbage (e.g. red, green and white cabbages), curly kale, Brussels sprouts, cotton, algae (e.g. blue green species), lemnospora, or moss (e.g. Physcomitrella patens), tomato, capsicum, squashes, sunflower, soyabean, carrot, melons, grape vines, lettuce, strawberry, sugar beet, peas, and sorghum.

[0316] 20. A population of transformed plant organelles according to statement 18 or statement 19, wherein the organelles are located in plant cells selected from cotton, rice, oilseed Brassica species such as canola, corn(maize) and soyabean.

[0317] 21. A method of producing at least a heterologous or exogenous RNA species in a plant that comprises:

[0318] 1(a) deleting viral polymerase and coat protein sequences from the complete viral genome of a potyvirus and replacing them with transgenic nucleic acid in cis, wherein the said transgenic nucleic acid comprises a nuclear promoter operably linked to a 5′ UTR sequence linked to the 5′ end of a complete RNA translocation sequence of the potyvirus forming a potyviral vector, wherein

[0319] i) the potyviral RNA translocation sequence is modified by covalently linking the 5′ end of the VPg protein therein to an organellar transit peptide; or

[0320] ii) the potyviral RNA translocation sequence is modified by fusing a spytag short peptide sequence to the viral VPg protein at either the N- or C-terminus thereof; and introducing the product of i) or ii) into a plant cell;

[0321] 1(b) introducing into the viral translocation sequence a second component nucleic acid sequence that encodes for an organellar transgene cassette comprising a left flanking sequence, an organellar promoter, at least one transgene nucleic acid sequence of interest, an organellar terminator and a right flanking sequence; and

[0322] 1(c) introducing into the said plant cell a third component nucleic acid sequence that codes for a retron sequence for reverse transcription of the TNA; and

[0323] 1(d)(i) introducing into the said plant cell a fourth component nucleic acid acid sequence comprising a viral 3′UTR sequence; and

[0324] 1(d)(ii) introducing into the plant cell a nucleic acid sequence comprising a nuclear promoter operably linked to a nucleic acid sequence comprising a plant organellar transit peptide (TP) located adjacent to a retron-based reverse transcriptase fused to a intron encoding protein (IEP), and a nuclear terminator; and

[0325] 1(e)(i) introducing into the plant cell a further vector that comprises either a potyviral polymerase in trans under the control of a plant nuclear promoter sequence and a terminator; or

[0326] 1(e)(ii) introducing into the plant cell a further vector that does not include a potyviral polymerase-containing vector of 1(e)(i), the vector comprising a spycatcher peptide fused to an organellar transit peptide, the said fused peptide being expressed under the control of a nuclear promoter.

[0327] 2) growing said regenerable plant cell of steps 1a) to 1e);

[0328] 3) selecting a plant cell of (2), wherein the transgene comprised within the plant organellar transgene cassette is integrated into the organellar genome;

[0329] 4) regenerating a plant from the plant cell of (6); and

[0330] 5) growing the plant of (4).

[0331] 22. A method according to statement 21, wherein the heterologous or exogenous RNA species encoded by the transgene that is integrated into the organellar genome is expressed as a heterologous or exogenous protein.

[0332] 23. A method according to statement 21 or statement 22, wherein the plant organellar genome is independently selected from that of plant mitochondria and plant plastids.

[0333] 24. An isolated polynucleotide sequence that comprises

[0334] 1(a) a first component nucleic acid sequence comprising a nuclear promoter operably linked to a 5′ UTR sequence linked to the 5′ end of a complete RNA translocation sequence of a potyvirus forming a potyviral vector, wherein

[0335] i) the potyviral RNA translocation sequence is modified by covalently linking the 5′ end of the VPg protein therein to an organellar transit peptide; or

[0336] ii) the potyviral RNA translocation sequence is modified by fusing a spytag short peptide sequence to the viral VPg protein at either the N- or C-terminus thereof; and introducing the product of i) or ii) into a plant cell;

[0337] 1(b) a second component nucleic acid sequence that encodes for an organellar transgene cassette comprising a left flanking sequence, an organellar promoter, at least one transgene nucleic acid sequence of interest, an organellar terminator and a right flanking sequence; and

[0338] 1(c) a third component nucleic acid sequence that codes for a retron-based reverse transcriptase fused to a reverse transcriptase of a group II intron;

[0339] 1(d) a fourth component nucleic acid acid sequence that is a 3′UTR sequence; and

[0340] 1(e)(i) a fifth component nucleic acid sequence that comprises either a potyviral polymerase in trans under the control of a plant nuclear promoter sequence and a bacterial terminator; or

[0341] 1(e)(ii) a fifth component nucleic acid sequence that does not include a potyviral polymerase-containing vector of 1(e)(i), the vector comprising a spycatcher peptide fused to an organellar transit peptide, the said fused peptide being expressed under the control of a nuclear promoter,

[0342] for use in a method according to any one of statementss 1 to 13 and statements 21 to 23.

[0343] 25. An isolated polynucleotide sequence as defined in any one of statements 1 to 13 and statements 21 to 24, comprising genomic DNA.

[0344] 26. An isolated polynucleotide sequence as defined in any one of statements 1 to 13 and statements 21 to 24, comprising a cDNA component.

[0345] 27. A nucleic acid vector suitable for transformation of a plant cell or a bacterial cell, wherein the cell includes a polynucleotide sequence according to any one of statements 24 to 26.

[0346] 28. A nucleic acid vector according to statement 27 for transformation of a bacterial cell.

[0347] 29. A nucleic acid vector according to statement 28 for transforming an Agrobacterium cell.

[0348] 30. A host cell containing a heterologous polynucleotide or nucleic acid vector according to any one of statements 24 to 29.

[0349] 31. A host cell according to statement 30 which is a plant cell or a bacterial cell.

[0350] 32. A host cell according to statment 30 or statement 31 comprised in a plant, a plant part or a plant propagule, or an extract or derivative of a plant or in a plant cell culture.

[0351] 33. A method of producing a cell according to any one of statements 30 to 32, the method including incorporating said polynucleotide or nucleic acid vector into the cell by means of transformation.

[0352] 34. A method according to statement 33 which includes regenerating a plant from a cell according to any one of statements 30 to 32 from one or more transformed cells.

[0353] 35. A plant comprising a plant cell according to any one of statements 30 to 32.

[0354] 36. A plant comprising a plant cell according to statement 35 that is selected from the group consisting of tobacco (Nicotiana tabacum) and other Nicotiana species, such as Nicotiana benthamiana, carrot, vegetable and oilseed Brassica's, melons, Capsicums, grape vines, lettuce, strawberry, sugar beet, wheat, barley, (corn)maize, rice, soybean, peas, sorghum, sunflower, tomato, cotton, and potato.

[0355] 37. A plant comprising a plant cell according to statement 35 or statement 36 that is selected from the group consisting of cotton, rice, oilseed Brassica species such as canola, corn(maize) and soybean.

[0356] 38. A method of producing a plant, the method including incorporating a polynucleotide sequence or nucleic acid vector according to any one of statements 24 to 29 into a plant cell and regenerating a plant from said cell.

[0357] 39. Use of a polynucleotide sequence according to any one of statements 24 to 26 in the production of a transgenic plant.

[0358] 40. Use of a polynucleotide sequence according to any one of statements 24 to 26 in the production of a polypeptide or protein in a plant.

[0359] All definitions for component parts of statements 1 to 40 of Variant 2 are found either in the accompanying description or in statements 1 to 40. The Experimental section provides technical descriptions of work performed relating to Variant 2.