Plant mitochondria transformation method

Abstract

Method for heterologous RNA species and protein production in plant cell mitochondria comprising introducing into plant cells nucleic acid components that encode heterologous proteins/RNAS under the control of promoters operative in mitochondria, vectors, host cells, plants and uses thereof.

Claims

1. A method of transforming a plant cell, the method comprising: 1) introducing into the plant cell a first nucleic acid sequence that comprises a plant nuclear promoter operably linked to a first nucleic acid sequence that comprises a plant mitochondrion transgene cassette, a plant mitochondrion translocation sequence from a group II intron RNA, wherein the group II intron is Ll.LtrB, and a primer binding domain; and 2) introducing into the plant cell a second nucleic acid sequence that encodes for a translocation sequence binding protein from an intron encoded protein (IEP) of a group II intron, wherein the IEP is LtrA fused to a plant mitochondrion transit peptide, wherein the second nucleic acid sequence is operably linked to a plant nuclear promoter, and wherein the mitochondrion translocation sequence of the product of 1) binds with the translocation sequence binding protein of the product of 2).

2. The method according to claim 1, wherein the plant mitochondrion transgene cassette comprises: i) a left flanking sequence (LFS) having least one mitochondrion specific promoter (mPRO) and a right flanking sequence (RFS) having at least one mitochondrion specific terminator (mTER) sequence; and ii) at least one isolated nucleic acid of interest.

3. The method according to claim 1, wherein the said isolated nucleic acid sequence is a recombinant DNA sequence or an introduced native, isolated genomic DNA sequence selected from isolated mammalian or plant nucleic acid sequences.

4. The method according to claim 3, wherein the isolated nucleic acid sequence is selected from nucleic acid sequences encoding proteins that confer cytoplasmic male sterility to a plant.

5. The method according to claim 1, wherein the primer binding domain is selected from that of a retrotransposon, or of a retrovirus.

6. A method of producing at least a heterologous or exogenous RNA species in a plant that comprises: 1) introducing into a regenerable plant cell a nucleic acid sequence that comprises a plant nuclear promoter operably linked to a first nucleic acid sequence that comprises a plant mitochondrion transgene cassette, a plant mitochondrion translocation sequence from a group II intron RNA, wherein the group II intron is Ll.LtrB, and a primer binding domain; 2) introducing into the regenerable plant cell a second nucleic acid sequence that encodes for a translocation sequence binding protein from an intron encoded protein (IEP) of a group II intron, wherein the IEP is LtrA fused to a plant mitochondrion transit peptide, and wherein said second nucleic acid sequence is operably linked to a plant nuclear promoter; 3) growing the regenerable plant cell of steps 1) to 2); 4) selecting a plant cell of 3), wherein the transgene comprised within the plant mitochondrion transgene cassette is integrated into the mitochondrial genome; 5) regenerating a plant from the plant cell of 4); and 6) growing the plant of 5).

7. The method according to claim 6, wherein the heterologous or exogenous RNA species encoded by the transgene that is integrated into the mitochondrion is expressed as a heterologous or exogenous protein.

8. An isolated polynucleotide sequence that comprises a plant nuclear promoter operably linked to a first nucleic acid sequence that comprises a plant mitochondrion transgene cassette, a plant mitochondrion translocation sequence from a group II intron RNA, wherein the group II intron is Ll.LtrB, and a primer binding domain for use in a method according to claim 1.

9. An isolated polynucleotide sequence that encodes for a mitochondrion translocation sequence-binding protein fused to a plant mitochondrion transit peptide wherein the polynucleotide sequence is operably linked to a plant nuclear promoter for use in a method according to claim 1.

10. An isolated polynucleotide sequence that encodes for a mitochondrion translocation sequence-binding protein from an intron encoded protein (IEP) of a group II intron, wherein the IEP is LtrA fused to a plant mitochondrion transit peptide, and wherein the polynucleotide sequence is operably linked to a plant nuclear promoter for use in a method according to claim 6.

11. An isolated polynucleotide sequence that encodes for a reverse transcriptase protein fused to a plant mitochondrion transit peptide, wherein the polynucleotide sequence is operably linked to a plant nuclear promoter for use in a method according to claim 1.

12. An isolated polynucleotide sequence that encodes for a reverse transcriptase protein fused to a plant mitochondrion transit peptide wherein the polynucleotide sequence is operably linked to a plant nuclear promoter for use in a method according to claim 6.

13. The isolated polynucleotide sequence according to claim 8 comprising genomic DNA or cDNA.

14. A cell containing a heterologous polynucleotide according to claim 8 comprised in a plant, a plant part, or a plant propagule, or in plant cell culture wherein the plant is selected from the group consisting of tobacco (Nicotiana tabacum) and other Nicotiana species, carrot, vegetable and oilseed Brassicas, melons, capsicums, grape vines, lettuce, strawberry, sugar beet, wheat, barley, corn (maize), rice, peas, sorghum, sunflower, tomato, cotton, and potato.

15. The method according to claim 3, wherein the isolated nucleic acid sequence is selected from nucleic acid sequences encoding proteins selected from the petunia mitochondrion pcf sequence, the orf107 sequence of sorghum and the orf79 of rice.

16. The method according to claim 1, wherein the primer binding domain is selected from the yeast Ty1 retrotransposon and the TnT1 tobacco retrotransposon.

17. The method according to claim 1, wherein the MTS sequence is selected from the group II intron-derived MTS from the Lactococcus lactis Ll.LtrB intron.

Description

(1) There now follow non-limiting examples and figures illustrating the invention.

(2) FIG. 1: Major components of the plant mitochondria transformation system.

(3) (1) Mitochondria transformation unit (MTU) (i) a plant mitochondrion translocation sequence (MTS); (ii) a mitochondrion transgene cassette comprising: a left flanking sequence (LFS).sup.1 and right flanking sequence (RFS).sup.1 to facilitate insertion of the cassette into the mitochondria genome using homologous recombination, a promoter region from tobacco mitochondria (mPro).sup.1, a sequence (transgene) of interest (TG), a transcription terminator from the tobacco mitochondrial genome (mTER).sup.1; and (iii) a primer binding domain (PBD). (2) Reverse Transcriptase-RNase H gene translationally fused to the mitochondria transit peptide from tobacco F1-ATPAse beta subunit (mTP). (3) MTS-Binding peptide translationally fused to the mitochondria transit peptide from Tobacco F1-ATPAse beta subunit (mTP)..sup.1 mPRO and mTER can be part of LFS and RFS respectively, if for example the transgene is fused to a native mitochondrial gene.

(4) FIG. 2: Schematic representation of the mitochondria transformation system

(5) (1) Targeting of RNA-protein complexes to the plant mitochondria.

(6) (a) After transformation of the mitochondria transformation unit (MTU) construct into the plant genome a strong expression of the MTU RNA which contains the mitochondria targeting cassette and mitochondria translocation sequence (MTS) is achieved from a nuclear specific promoter. The MTS-binding protein (MTS-BP) is expressed from a separate cassette. (b) Both MTS-BP and MTU RNAs are transferred from the nucleus into the cytoplasm where MTS-BP is translated. and (c) binds to the MTU RNA via recognition of MTS to form the MTU/MTS-BP nucleoprotein complex. (d) As MTS-BP carries a mitochondria transit peptide it preferentially transfers the MTU/MTS-BP complex into the mitochondria. Once MTU is presented in the plant cell via nuclear transformation, the mitochondria will then be permanently bombarded by the expressed MTU/MTS-BP complex. Such stable and continuous pumping of the complex into the targeted organelle is a prerequisite for achieving a high efficiency of organelle transformation.

(7) (2) Reverse transcription and insertion into the mitochondrial genome.

(8) The third component of the mitochondria transformation system is the mitochondria-targeted reverse transcriptase-RNaseH protein (MRT-RH). (e) MRT-RH is expressed from a nuclear expression cassette and (f) is driven to the mitochondria by its mitochondria transit peptide. (g) Once inside the organelle, MRT-RH catalyses reverse transcription of the MTU RNA primed with tRNA-Met. (h) Insertion of the reverse transcribed cassette into the mitochondrial genome is induced due to homologous recombination between sequences flanking the mitochondria transgene cassette and the homologous sequences in the mitochondrial genome.

(9) FIG. 3: M21 and M28 constructs

(10) M21 Construct

(11) The binary vector pGreen0029 was used as a backbone and enables selection of transgenic plants with kanamycin. It is used for co-expression of the mitochondria transformation unit with the MTS-binding protein mLTRASi in the plant cell.

(12) The mitochondria transformation unit is made up the following components: NtmLFS1 and NtmRFS1 are homologous to two adjacent sequences in the tobacco mitochondrial mitochondria non-coding sequences (position 292641-293235 and 293262-293835, respectively), the GFP gene is placed under the control of Arabidopsis mitochondria ATP6 promoter AtmATP6 PRO and the tobacco ATP6 terminator NtmATP6 TER. PBD (primer-binding domain) is designed to capture the plant mitochondria tRNA-fMet to initiate reverse transcription, LTRBM is based on the LtrB intron sequence from lactococcus lactis and serves as the mitochondria translocation sequence (MTS) into which the first six components were cloned, between the AscI and NotI restriction sites. The whole mitochondria transformation unit was placed under the control of the 35S promoter and the nos terminator.

(13) The second cassette is for expression of the MTS-binding protein MLTRASi placed under the control of the AtUBI3 promoter and ags terminator.

(14) M28 Construct

(15) The pSOUP vector (EU048870) carrying T-DNA from the pGreen0179 vector (EU048866) was used as a backbone and enables selection of transgenic plants with Hygromycin. It is used for expression of the mitochondria-targeted reverse transcriptase-RNaseH protein (mRTRHi-Ty) under the control of the TAF2 promoter and ags terminator.

(16) FIG. 4: M22 construct

(17) The binary vector pGreen0029 was used as a backbone and enables selection of transgenic plants with kanamycin. It is used for co-expression of the mitochondria transformation unit with the MTS-binding protein mLTRASi in the plant cell.

(18) The mitochondria transformation unit is made up the following components: NtmLFS4 and NtmRFS4 correspond to Nicotiana tabacum mitochondria sequences position 24888-24579 and 24578-24269 respectively (on the complementary strand). NtmLFS4 corresponds to the 5 end of the gene coding for ATP6 and can be used for translational fusion with any gene of interest, promoter activity is provided by the ATP6 promoter upstream of NtmLFS4 on the tobacco mitochondrial genome and termination of transcription is achieved with the ATP6 terminator sequence within NtmRFS4. The GFP gene was fused to NtmLFS4 and cloned upstream of NtmRFS4 and PBD. LTRBM is based on the LtrB intron sequence from lactococcus lactis and serves as the mitochondria translocation sequence (MTS) into which the first four components were cloned, between the AscI and NotI restriction sites. The whole mitochondria transformation unit was placed under the control of the 35S promoter and the nos terminator.

(19) The second cassette is for expression of the MTS-binding protein MLTRASi placed under the control of the AtUBI3 promoter and ags terminator.

(20) FIG. 5: M24 construct

(21) The binary vector pGreen0029 was used as a backbone and enables selection of transgenic plants with kanamycin. It is used for co-expression of the mitochondria transformation unit with the MTS-binding protein mLTRASi in the plant cell.

(22) The mitochondria transformation unit is made up the following components: NtmLFS3 and NtmRFS3 correspond to Nicotiana tabacum mitochondria sequences position 85896-86331 and 86332-86860, respectively. the PCFM gene is based on the CMS-inducing PCF gene from petunia mitochondria, PBD (primer-binding domain) is designed to capture the plant mitochondria tRNA-fMet to initiate reverse transcription. LTRBM is based on the LtrB intron sequence from lactococcus lactis and serves as the mitochondria translocation sequence (MTS) into which the first four components were cloned, between the AscI and NotI restriction sites. The whole mitochondria transformation unit was placed under the control of the 35S promoter and the nos terminator.

(23) The second cassette is for expression of the MTS-binding protein MLTRASi placed under the control of the AtUBI3 promoter and ags terminator

(24) FIG. 6: M27 construct

(25) The binary vector pGreen0029 was used as a backbone and enables selection of transgenic plants with kanamycin. It is used for co-expression of the mitochondria transformation unit with the MTS-binding protein MLTRASi in the plant cell.

(26) The mitochondria transformation unit is made up five components: AtmLFS5 and AtmRFS5 are homologous to two adjacent sequences in the Arabidopsis mitochondrial (position 344225-344571 and 344572-344926, respectively), the PCFM gene is based on the CMS-inducing PCF gene from petunia mitochondria, PBD (primer-binding domain) is designed to capture the plant mitochondria tRNA-fMet to initiate reverse transcription, LTRBM is based on the LtrB intron sequence from lactococcus lactis and serves as the mitochondria translocation sequence (MTS) into which the first four components were cloned, between the AscI and NotI restriction sites. The whole mitochondria transformation unit was placed under the control of the 35S promoter and the nos terminator.

(27) The second cassette is for expression of the MTS-binding protein MLTRASi placed under the control of the AtUBI3 promoter and ags terminator.

(28) M28 Construct

(29) The pSOUP vector (EU048870) carrying T-DNA from the pGreen0179 vector (EU048866) was used as a backbone and enables selection of transgenic plants with Hygromycin. It is used for expression of the mitochondria-targeted reverse transcriptase-RNaseH protein (mRTRHi-Ty) under the control of the TAF2 promoter and ags terminator

(30) FIG. 7: Aborted pollen phenotype in tobacco plants transformed with the CMS-inducing pcf gene from petunia mitochondria

(31) (A,B) Pollen from wild type (WT) plants.

(32) (C,D) Pollen from transgenic tobacco line PCFM1, transformed with the M24 vector carrying the CMS-inducing PCF orf from petunia, showing 90% of aborted pollen.

(33) FIG. 8: Modifications of the mitochondria transformation cassette were made by designing primer binding domain and positioning of building blocks on the transgene cassette.

(34) MTUmitochondria transformation unit; MTSmitochondria translocation sequence; PDB-MITprimer binding domain designed for reverse transcription in the mitochondria using tRNA-Met from mitochondria; PBD-CYTprimer binding domain designed for reverse transcription, in the cytoplasm using cytoplasmic tRNA-Met.

(35) The modifications detailed in Example section 1B hereinafter and corresponding figures include modifications of the use of PBD for the binding of cytoplasmic tRNA-Met as primer. As one modification MTS can be located at both the 5- and 3-ends of the transformation cassette, such as in the case with the LtrB intron. The transgene cassette is inserted inside of the LtrB intron (domain IV). The PDB-MIT is located downstream of the LtrB 3-end of the cassette (MTS-3), so that the LtrA protein is able to function as both a translocation protein and reverse transcriptase. The LtrA protein has three major functions: (1) as a maturase (it binds to LtrB RNA and stabilises the secondary structure of the RNA, and assists splicing); (2) as an endonuclease (it induces single-stranded DNA breaks on target site); and (3) as a reverse transcriptase (it performs reverse transcription of the intron RNA after insertion of the LtrB intron RNA into the donor site).

(36) The LtrA protein is unable to perform the reverse transcription reaction efficiently if the PBD-CYT is located adjacent to and in front of a mitochondrion translocation sequence at the 3-end of the MTU (MTS-3) as in FIG. 8(B), but can efficiently reverse transcribe RNA if the PBD is located downstream of a chloroplast translocation sequence (MTS-3) as shown in FIG. 8A. Such a positioning or the combination of components of the transformation cassette as shown in FIG. 8(A) allows both the translocation of the MTU into the mitochondrion and reverse transcription of the MTU by the LtrA protein. Thus, by positioning of the MTS components and of the PBD-MIT as shown in FIG. 8(A) the procedure of transformation is simplified since there is no requirement to co-deliver another gene to provide a reverse transcriptase function.

(37) A similar simplification of the procedure is achieved if a PBD-CYT is used, since there is a significant amount of native endogenous reverse transcriptase in the cytoplasm, and reverse transcription is initiated by endogenous reverse transcriptase using cytoplasmic tRNA-Met. This also eliminates the necessity for the co-delivery of another gene for reverse transcription in the mitochondria.

(38) The case in FIG. 8A and FIG. 8B is attributed to the LtrB intron.

(39) FIG. 9: Tobacco mitochondria transformation constructs (A) M43 construct: PBD-MIT was fused to the 3 end of the LtrB intron (B) M44 construct: PBD-CYT was fused to MTU

(40) PBD-MIT: Primer binding domain designed to anneal with t-RNAmet from mitochondria to initiate reverse transcription. LTRB5, LTRB3: 5 and 3 sequences of the LTRB intron, respectively. NtmLFS3: tobacco mitochondria left flanking sequence. NtmRFS3: tobacco mitochondria left flanking sequence. PCFM: CMS-inducing open reading frame from petunia. 35S pro: promoter from CaMV (Cauliflower mosaic virus). TAF2 Pro: Arabidopsis promoter. 1-beta MTP: mitochondria transit peptide from ATPase 1 beta subunit. LTRASii: sequence coding for the LTRA protein. Ags ter and nos ter: transcription terminator sequences from agrobactrium. KanR: NPTII gene for kanamycin resistance.

(41) FIG. 10: Rice mitochondria transformation constructs (A) M45 construct: PBD-MIT was fused to the 3 end of the LtrB intron (B) M46 construct: PBD-CYT was fused to MTU

(42) PBD-MIT: Primer binding domain designed to anneal with t-RNA Met from mitochondria to initiate reverse transcription. LTRB5, LTRB3: 5 and 3 sequences of the LTRB intron, respectively. osLFS3: rice mitochondria left flanking sequence. osRFS3: rice mitochondria left flanking sequence. PCFM: CMS-inducing open reading frame from petunia. 35S pro: promoter from CaMV (Cauliflower mosaic virus). Act1 Pro: rice promoter from the actin gene. 1-beta MTP: mitochondria transit peptide from ATPase 1 beta subunit. LTRASii: sequence coding for the LTRA protein. Ags ter and nos ter: transcription terminator sequences from agrobactrium. KanR: NPTII gene for kanamycin resistance.

EXPERIMENTAL SECTION 1A

A Novel Approach for Plant Mitochondria Transformation

(43) A new method for transformation of the plant mitochondrial genome comprises

(44) (1) a plant mitochondria transformation unit (MTU) consisting of 3 major domains:

(45) (i) a plant mitochondria translocation sequence (MTS), (ii) a plant mitochondria transgene cassette (iii) a primer binding domain (PBD) which uses plant mitochondria tRNA-fMet or any other mitochondrial RNAs as a primer for reverse transcription;
(2) a reverse Transcriptase-RNase H(RT-RH) from retrotransposons, retroviruses, intron maturases or any protein with reverse transcription activity is fused to a plant mitochondria transit peptide for targeting into the plant mitochondria;
(3) an RNA binding protein that binds to the plant mitochondria translocation sequence (MTS), fused to a plant mitochondria transit peptide (FIG. 1).
Technology Rationale

(46) The process of plant mitochondria transformation comprises two steps (see FIG. 2).

(47) (1) Targeting of an RNA-Protein Complex to the Plant Mitochondria

(48) The mitochondria transformation construct is expressed from the nucleus using a constitutive promoter. After delivery of the mitochondria transformation construct into the plant cell a strong expression of the RNA which contains the mitochondria translocation sequence (MTS), transgene cassette and primer binding domain (PBD) is achieved. The MTS binding protein (MTS-BP) fused to a plant mitochondria transit peptide, is also expressed on co-delivery from the same or a different nuclear transformation vector. It is used to bind to MTS and facilitate the translocation of the MTU RNA into the mitochondria.

(49) Once the plant mitochondria transformation vector is presented in the plant cell via nuclear transformation, the mitochondria will then be permanently bombarded by the expressed MTS-BP/MTU RNA complex. Such stable and continuous pumping of the complex into the targeted organelle is a prerequisite for achieving a high efficiency of organelle transformation. The technology exploits the finding that the plant mitochondria transit sequence is sufficient to permit the whole MTS-BP/MTU RNA complex to be then taken up by the mitochondria.

(50) The plant mitochondrial translocation sequences (MTS) can be selected from a number of RNA sequences such as mitoviruses, viral RNAs (including viral coat protein binding domains), group I and group II intron RNAs, retrotransposon primer binding sites, or any RNA that harbors a domain recognised by RNA binding proteins.

(51) The MTS-binding protein can be any RNA binding protein that recognises and binds to specific RNA domains.

(52) The plant mitochondrial transit peptide can be derived from any mitochondria-targeted proteins.

(53) The fusion of MTS-BP to a mitochondrial transit peptide enables this protein to act as a carrier of RNA molecules into the plant mitochondria provided that these RNA molecules carry the corresponding MTS domain.

(54) (2) Reverse Transcription of the Transgene Cassette and Insertion into the Plant Mitochondria Genome.

(55) Once the MTU RNA is inside the mitochondria, its' primer binding domain (PBD) captures tRNA-fMet as a primer to form a template ready for reverse transcription. Simultaneously, a reverse transcriptase (RT-RH) fused to a plant mitochondria transit peptide is expressed from the nucleus using a constitutive promoter. It is targeted into the mitochondria where it facilitates reverse transcription of the MTU-RNA into single stranded DNA.

(56) This is followed by insertion of the reverse transcribed cassette into the plant mitochondrial genome using homologous recombination between sequences flanking the transgene cassette (LFS, RFS) and the homologous regions in the plant mitochondria genome.

(57) The Primer binding domain (PBD) is designed to capture the RT-RH protein and plant mitochondria tRNA-fMet (or any other plant mitochondrial tRNAs) as a primer, to initiate reverse transcription of the MTU RNA, carrying the plant mitochondria transgene cassette, into single-stranded DNA.

(58) Once the population of organelle genomes has been transformed in the initial plant line, the nuclear encoded transgenes are no longer required and can then be removed through segregation in subsequent plant generations, leaving a clean organelle transformed plant line (FIG. 2).

(59) Materials and Methods.

(60) Part 1Nucleic Acid Sequence Information

(61) 1. Preparation of a Group II Intron-Based Plant Mitochondria Translocation Sequence (MTS).

(62) The LtrB intron from Lactococcus lactis lacking the gene coding for LTRA (intron-encoded maturase) was synthesised by a commercial DNA synthesis provider (Bio S&T Inc., Montreal (Quebec), Canada). Potential splicing donor and acceptor sites were mutagenised to prevent splicing for optimum accumulation of the groupII intron RNA in plant cytoplasm, the resulting group II intron sequence was named LtrBM.

(63) The domain for insertion of the plant mitochondria transgene cassette (AscI-MluI-NotI sites) is underlined and shown in bold letters.

(64) LtrBM Intron Sequence

(65) TABLE-US-00001 SEQIDNO.1 GGATCCCTCGAGGTGCGCCCAGATAGGGTGTTAAGTCAAGTAGTTTA AGGTACTACTCAGTAAGATAACACTGAAAACAGCCAACCTAACCGAA AAGCGAAAGCTGATACGGGAACAGAGCACGGTTGGAAAGCGATGAGT TAGCTAAAGACAATCGGCTACGACTGAGTCGCAATGTTAATCAGATA TAAGCTATAAGTTGTGTTTACTGAACGCAAGTTTCTAATTTCGGTTA TGTGTCGATAGAGGAAAGTGTCTGAAACCTCTAGTACAAAGAAAGCT AAGTTATGGTTGTGGACTTAGCTGTTATCACCACATTTGTACAATCT GTTGGAGAACCAATGGGAACGAAACGAAAGCGATGGCGAGAATCTGA ATTTACCAAGACTTAACACTAACTGGGGATAGCCTAAACAAGAATGC CTAATAGAAAGGAGGAAAAAGGCTATAGCACTAGAGCTTGAAAATCT TGCAAGGCTACGGAGTAGTCGTAGTAGTCTGAGAAGGCTAACGGCCT TTACATGGCAAAGGGCTACAGTTATTGTGTACTAAAATTAAAAATTG ATTAGGGAGGAAAACCTCAAAATGAAACCAACAATGGCAATTTTAGA AAGAATCAGTAAAAATTCACAAGAAAATATAGACGAAGTTTTTACAA GACTTTATCGTTATCTTTTACGTCCTGATATTTATTACGTGGCGGGC GCGCCACGCGTGCGGCCGCTGGGAAATGGCAATGATAGCGAAAGAAC CTAAAACTCTGGTTCTATGCTTTCATTGTCATCGTCACGTGATTCAT AAACACAAGTGAATTTTTACGAACGAACAATAACAGAGCCGTATACT CCGAGAGGGGTACGTACGGTTCCCGAAGAGGGTGGTGCAAACCAGTC ACAGTAATGTGAACAAGGCGGTACCTCCCTACTTCACCATATCATTT TTAATTCTACGAATCTTTATACTGGCAAACAATTTGACTG
2. Mitochondria Transgene Cassettes

(66) Positions of the various mitochondria sequences described below are derived from GenBank sequence accession numbers NC_006581 (Nicotiana tabacum mitochondrion) and NC_001284 (Arabidopsis thaliana mitochondrion).

(67) 2.1 Left and Right Flanking Sequences Used for Homologous Recombination

(68) The mitochondria transgene cassette contains left and right flanking sequences (LFS and RFS) for insertion of the whole cassette into the mitochondrial genome using homologous recombination.

(69) LFS and RFS sequences were amplified from coding and non-coding sequences of the mitochondrial genome of Nicotiana tabacum (NtmLFS, NtmRFS) and Arabidopsis Thaliana (AtmLFS, AtmRFS) using the primers described below.

(70) 2.1.1 NtmLFS1 and NtmRFS1, corresponding to corresponding to Nicotiana tabacum mitochondria non-coding sequences (position 292641-293235 and 293262-293835, respectively) were amplified from tobacco total cellular DNA using the following primers:

(71) NtmLFS1:

(72) TABLE-US-00002 IM101 SEQIDNO.2 GCGGGCGCGCCTATTACTCTCGGTCCTTGTTC IM102 SEQIDNO.3 GCGGAGCTCTACCCTTTAAGACTCAATTACATCGAG
NTmRFS1:

(73) TABLE-US-00003 IM103 SEQIDNO.4 GCATGCATTGCATAAGTAATCTCTTTTCTTATGAG IM104 SEQIDNO.5 ACTAGTAAGGGGATTTGCCACATCGTTG
NtmLFS1 sequence:

(74) TABLE-US-00004 SEQIDNO.6 TATTACTCTCGGTCCTTGTTCTTGGTCTCTGTGAAAGATCCAGTCGA TGGGAATGAATCCATGTTCAAATCTTATTACCGGGTTCGATTACGGG AAGGAAATAGAGAAGGTAAGGGACCGCTTTCCTTGTTCAAGCCGGTA TTGTTTGAGTAAGTAGTAAGTAAGTGAGAAGTGGTGAATTGGCCAGG AGGAATAAAGCTTATTTCAAGTACTAATAAAAGCATTCATTACAAAC TCTTGTGCTCACTTATCCCAAGTATAGGATGTTTTCCCTGAGCCTGT CTGTGTTGAATACGCTTTTTCCGTGTAGAATAGAGATTCTCTCTAAG GTTGATAGAATATACGTTTTCTTTCTCTGATTAAAGGTTGTCCAAAG AGGACTAAGAGACAGATGCTGTGCTTGCAAGTAAGCTTCAGCCAAGC ATCAGATAAACCAAGTTCGGGTTGGGAAAAGGGCTATTTACCCCAGC AATATAGAATAATTATTACCCCCAGCACATCCCCAAATGAGAGCATC GTCTTTACCCCTAGAAAAGGTGCGATGTAATTTCCTGGTTCGATTAC ATTGCTCGATGTAATTGAGTCTTAAAGGGTAGAGCTC
NtmRFS1 sequence:

(75) TABLE-US-00005 SEQIDNO.7 ATTGCATAAGTAATCTCCTTTCTTATGAGAACTACGAATCATCCTCA TGAATAAGCTCTACTCTACCTTAAGGAGATGTGGAGGCAATAGGTCC CGTGCAGCTTTAACTAACTCTACTCCTCCATACGCCTATCCTTTAGT TTAGTGGGCCAGGTCCTCCAGCCTTCCATTAGCTTTCGATTTAGTTT GCATTCAAAGTCTTGGAATGCGAGCTTATGTGCTTTCAGGTATAGGC ACCATTCGCCTGACTTTCTTGAAGTCCTAGGATTCTCCCCTAGTATT CCATTCTCTCCCCCTCTCGGCCTTGCTTTCATTCCTGTCTCATTTGA AATTGCTCCTAAGGCAGGGAGTCTTCTCGAAGCTGTCTAAGTCTTGT AAGGCTCCTATATCTATATATAGAGAGGTCATGGTATGGAGGGAGGA TTTCTACGCGCAACATCGTGGTTGGGGCATTCCTCCTTCTTTTAAAA GAAGACTAGAGGACGAAAGAAGAAGCTCTTACATCGGATAAAGCCTA ATTCCACTGTCCTTTGAAGATTGGAAGATAGTGAAGGCCGACTTCCT TTTTAAAGATCACTCAACGATGTGGCAAATCCCCTTACTAGT

(76) 2.1.2 NtmLFS3 and NtmRFS3, corresponding to Nicotiana tabacum mitochondria sequences (position 85896-86331 and 86332-86860, respectively) were amplified from tobacco total cellular DNA using the following primers:

(77) NtmLFS3

(78) TABLE-US-00006 IM263 GGCGCGCCAGCAGATTTCCTCCCTCTATC SEQIDNO.8 IM264 GCATGCAGATCGACGACGGAACGAAGAAC SEQIDNO.9
NtmRFS3

(79) TABLE-US-00007 IM265 TCTAGATCCAATTTCTTCCGGTATGC SEQIDNO.10 IM375 CCGCGGTACGGTCCGTGCGCCGTT SEQIDNO.11
NtmLFS3 Sequence:

(80) TABLE-US-00008 SEQIDNO.12 AGCAGATTTCCTCCCTCTATCAACTCCTTTTTTATGGTCGGGAGGAT CCACAATTCTTCATTGATCCACAAGACCTGGATTCCATACTGAGGGT GCACCTTGAACCCTTAGAATTCAATCACCCTGCTCTATGCCAGGTCT TAGAAAGTCTATGTGTCGAGAAGCATGATTCCCCTTTTTATCAAGAT GTAAAAATGGCTCAAGCGCATCATTTTCGTGGCTTTATAAACTTAAA GCACCAAGCGAAATTGGAAATGCAACATCGCCTAGAGTTAGGAGAGG TATGGAAATCTCTTGAGAGAAGGAACGCTTTTCTAAGCCAGGAAAAC GCCTCTCTAAGAGAAAAACTTTTAATTCTCGACAGGGAAGCCCCATA GAAATTCTTCTTTGTTGTGTTGCTATCCTAAAATTGCGTTCTTCGTT CCGTCGTCGATCT
NtmRFS3 Sequence:

(81) TABLE-US-00009 SEQIDNO.13 TCTAGATCCAATTTCTTCCGGTATGCCGCTCCGCCAGCAAGGAGCGA AAGAACCAAGTTTTCTGTGGTGATGTCAGAATTTGCACCTATTTGTA TCTATTTAGTGATCAGTCCGCTAGTTTCTTTGCTCCCACTCGGTCTT CCTTTTCTATTTTCTTCCAATTCTTCGACCTATCCAGAAAAATTGTC GGCCTACGAATGTGGTTTCGATCCTTCCGGTGATGCCAGAAGTCGTT TTGATATAAGATTTTATCTTGTTTCCATTTTATTTATTATTCCTGAT CCGGAAGTAACCTTTTCCTTTCCTTGGGCAGTACCTCCCAACAAGAT TGATCCGTTTGGATCTTGGTCCATGATGGCCTTTTTATTGATTTTGA CGATTGGATCTCTCTATGAATGGAAAAGGGGTGCTTCGGATCGGGAG TAACCACTAGTGAGAGGGCAAAAATTGGGGGGAAGGACAAAGGAAAG AGCGATGCCTACATTAAATCAATTGATTCGTCATGGTAGAGAAGAAA AACGGCGCACGGACCGTA
2.1.3 NtmLFS4 and NtmRFS4 corresponding to Nicotiana tabacum mitochondria sequences position 24888-24579 and 24578-24269 respectively (on the complementary strand).

(82) NtmLFS4 corresponds to the 5 end of the gene coding for ATP6 and can be used for translational fusion with any gene of interest, promoter activity is provided by the ATP6 promoter upstream of NtmLFS4 on the tobacco mitochondrial genome and termination of transcription is achieved with the ATP6 terminator sequence within NtmRFS4. Any plant mitochondrial coding sequence can be used instead of ATP6 to achieve expression of any gene of interest.

(83) NtmLFS4 and NtmRFS4 were amplified from tobacco total cellular DNA using the following primers:

(84) NtmLFS4

(85) TABLE-US-00010 IM376 GGCGCGCCAGGGTATGATACCTTATAGCT SEQIDNO.14 IM287 CTCGAGTGAGACTCGCTTTTGTTC SEQIDNO.15
NtmRFS4

(86) TABLE-US-00011 IM289 GAGCTCATGGGTATACTTAGTCGTGG SEQIDNO.16 IM377 CCGCGGCTGAGATAGCTCCGTAAACTAAT SEQIDNO.17
NtmLFS4 Sequence:

(87) TABLE-US-00012 SEQIDNO.18 CCAGGGTATGATACCTTATAGCTTCACAGTTACAAGTCATTTTCTCA TTACTTTGGGTCTCTCATTTTCTATTTTTATTGGCATTACTATAGTG GGATTTCAAAAAAATGGGCTTCATTTTTTAAGCTTCTTATTACCTGC AGGAGTCCCACTGCCATTAGCACCTTTTTTAGTACTCCTTGAGCTAA TCCCTTATTGTTTTCGAGCATTAAGCTCAGGAATACGTTTATTTGCT AATATGATGGCCGGTCATAGTTCAGTAAAGATTTTAAGTGGGTTCGC TTGGACTATGCTATGTATGAATGATCTTTTATATTTCATAGGGGATC TTGGTCCTTTATTTATAGTTCTTGCATTAACCGGTCTGGAATTAGGT GTAGCTATATCACAAGCTCATGTTTCTACGATCTTAATCTGTATTTA CTTGAATGATGCTATAAATCTTCATCAAAGTGCTTCTTTTTTTATAA TTGAACAAAAGCGAGTCTCA
NtmRFS4 Sequence

(88) TABLE-US-00013 SEQIDNO.19 ATGGGTATACTTAGTCGTGGAGCATTCCGAGTATTTGCTTTAGGGAT CGTTCCTGCGCATCTCCTTACTTTATAGCAGTTATTGCTCCGGTTCC AGAAGGTATAGCTCTCGCCTCAGCTTTTTCTTTGAAATCGGAGACTG TTCCAATTTCCTACTGAGATAGGCAAGCGGAGGGAGAACTAGACGTA TCTTGCTAGGCAAAGACAGGTTAGAATGGATAGCTCGCGGGTGGGAT TGACGGGATAGATCACTATTGCAGAAGGAGGTAGAACCGGGGGAAGA ATTATGGCTATAAAGGTCCTCGCCCTCTTAGGCACATGGTTCTAAAG ATTAAATCTCAAAGCGGTACTAAAGATTAGGCAGAAGAAGAACTAGA ACTAGAATTCTTCGCCCCTCCCCTTGTACCAAGAAGCAAGTTCAGAA CATAAGGATAATGGGCTCGTCTATTATAAGTTATTAGTTTACGGAGC TATCTCAG
2.1.4 AtmLFS5 and AtmRFS5 corresponding to Arabidopsis thaliana mitochondria sequences (position 344225-344571 and 344572-344926, respectively) were amplified from Arabidopsis total cellular DNA using the following primers:
AtmLFS5

(89) TABLE-US-00014 SEQIDNO.20 IM398 GGCGCGCCGGGAGGAAGCTGGGCCAGTAGT SEQIDNO.21 IM399 GCATGCGAAAAATAAAGAAAGAAGCAAAAGCCCAT
AtmRFS5

(90) TABLE-US-00015 IM400 ATCGATATGCCGCTTCTTCGCCA SEQIDNO.22 IM401 CCGCGGATTTTGTGCCCTATCACTTTAC SEQIDNO.23
AtmLFS5 Sequence

(91) TABLE-US-00016 SEQIDNO.24 GGGAGGAAGCTGGGCCAGTAGTCCCCTATCCATACAGGAGGGATGAA ATGATTGGGGGGGATAGCGTAGAGGCGATAGAACGCCGCCTTCTGGC GAAATACCCCGAAGGCTCTCCCTCTGCGGAGATCATAGAGATGGCCC GAATAGAGGCCGAAGATCTATTCGAGATCAAAGCCCAAATCATCCAA CGGATGGCTCTATATGACCCAACCGGCGATTGGATGGCGCGTGGGGC TCGGGCCCTCGATAATCCGAGGACCACTAGTGGGGAAGAGTCCTTGG AGCGTCTTTATGATATATGGAAGGACCTCCAAGAAACCGGGCCCCTC TCGGACGAGTTTTCTCGTTTACAAGAGAAAGTATTCCTCAAGAAAGG CGGCCCTGGGGGGGACCCTATCGCATAAGGTCTGCAAGCCTTTCGGG ATGGGCTTTTGCTTCTTTCTTTATTTTTCG

(92) AtmRFS5 Sequence

(93) TABLE-US-00017 SEQIDNO.25 ATATGCCGCTTCTTCGCCAGCAAGGAGCGAGAAAACAAAGTGGGCTG TAATGATGTCAGAATTTGCACCAATTTCTATCTATTTAGTGATTAGT CTGCTAGTTTCTTTGATCCTACTCGGTGTTCCTTTTCCATTTGCTTC CAATAGTTCTACCTACCCAGAAAAATTGTCGGCCTACGAATGTGGTT TCGATCCTTCCGGTGATGCCAGAAGTCGTTTCGATATACGATTTTAT CTTGTTTCAATTTTATTTTTAATCCCTGATCTGGAAGTAACCTTTTT CTTTCCTTGGGCAGTACCTCCCAACAAGATTGATCTGTTTGGATTTT GGTCCATGATGGCCTTTTTATTTATTTTGACGATTGGATTTCTATAT GAATGGAAAAGGGGTGCTTCGGATCGGGAGTAAAGTGATAGGGCACA AAAT
2.2 Plant Mitochondria Promoter and Terminator Sequences
2.2.1 ATP6 promoter sequences were amplified from total cellular DNA of Nicotiana tabacum (NtATP6-PRO) and Arabidopsis thaliana (AtATP6-PRO) using the following primers:
NtATP6-PRO

(94) TABLE-US-00018 IM364 GGCGCGCCTCTAGTCGAATAGAGTATTAG SEQIDNO.26 IM365 ATCTCGAGTGTGATTGAGATAAAAAGATTCC SEQIDNO.27
AtATP6-PRO

(95) TABLE-US-00019 SEQIDNO.28 IM346 CTGCATGCTCCTCTACTGAGTCAGTGACAG SEQIDNO.29 IM347 ATTCTAGAATTGGATTAATTGATTTCAACAAAATG
NtATP6-PRO Sequence

(96) TABLE-US-00020 SEQIDNO.30 CCTCTAGTCGAATAGAGTATTAGTCCGCTCCATTATATTCCCCATTATTT CACTTTCTCGCTATTCGAAATATCATAAGAGAAGAAAGCTGGCAGGTTGG ATCCTAGGGTAGATTCCTGCTGTTGAATGATCGACTAGCTTCCTCTTTAG TTCTTTGATATTGGGTTCGTGTTCAGTGTACCGCTCTTTTTATATATGAA ATTACTTCGTCCTTTTTTTTAGCCCTTTTTCGTTTGTCCATCTTTTTTTC TCCCATGCTTTCCGTTGGTCAACAACCAACCAAAGTGCTCTATACTTCTT CACTACTCGTACAGGCTTGACGGAGTTAAGCTGTATTGAGGGAATCTTTT TATCTCAATCA
AtATP6-PRO Sequence

(97) TABLE-US-00021 SEQIDNO.31 TCCTCTACTGAGTCAGTGACAGAAGTGCAGCAGCCAATAATACGTATATA AGAAGAGGACTGCTTACGGGATCAAACTATCAATCTCATAAGAGAAGAAA TCTCTATGCCCCCTTTTTCTTGGTTTTCTCCCATGCTTTTGTTGGTCAAC AACCAACCACAACTTTCTATAGTTCTTCACTACTCCTAGAGGCTTGACGG AGTGAAGCTGTCTGGAGGGAATCATTTTGTTGAAATCAATTAATCCAAT
2.2.2 ATP6 terminator sequence was amplified from total cellular DNA of Nicotiana tabacum (NtATP6-TER) using the following primers:

(98) TABLE-US-00022 IM289 GAGCTCATGGGTATACTTAGTCGTGG SEQIDNO.32 IM366 CCGCGGCGAGGACCTTTATAGCCATAATTC SEQIDNO.33
NtATP6-TER Sequence

(99) TABLE-US-00023 SEQIDNO.34 ATGGGTATACTTAGTCGTGGAGCATTCCGAGTATTTGCTTTAGGGATCGT TCCTGCGCATCTCCTTACTTTATAGCAGTTATTGCTCCGGTTCCAGAAGG TATAGCTCTCGCCTCAGCTTTTTCTTTGAAATCGGAGACTGTTCCAATTT CCTACTGAGATAGGCAAGCGGAGGGAGAACTAGACGTATCTTGCTAGGCA AAGACAGGTTAGAATGGATAGCTCGCGGGTGGGATTGACGGGATAGATCA CTATTGCAGAAGGAGGTAGAACCGGGGGAAGAATTATGGCTATAAAGGTC CTCG
2.3 Transgene Sequences
2.3.1 Green Fluorescent Protein (GFP) Sequence

(100) The GFP gene was synthesised according to GenBank accession number XXU70496

(101) GFP Sequence

(102) TABLE-US-00024 SEQIDNO.35 ATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGA ATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTG AAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACT GGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCACTTATGG TGTTCAATGCTTTTCAAGATACCCAGATCATATGAAGCGGCACGACTTCT TCAAGAGCGCCATGCCTGAGGGATACGTGCAGGAGAGGACCATCTCTTTC AAGGACGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAGGGAGA CACCCTCGTCAACAGGATCGAGCTTAAGGGAATCGATTTCAAGGAGGACG GAAACATCCTCGGCCACAAGTTGGAATACAACTACAACTCCCACAACGTA TACATCACGGCAGACAAACAAAAGAATGGAATCAAAGCTAACTTCAAAAT TAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAAC AAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTAC CTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCA CATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGG ATGAACTATACAAATAA
2.3.2 Gene for Induction of Cytoplasmic Male Sterility

(103) The sequence coding for the PCFM gene was based on the petunia mitochondria PCF gene responsible for induction of CMS. It comprises a promoter, open reading frame and 3 untranslated sequence. It was modified to remove putative splicing sites for optimum expression of the PCFM RNA in the cytoplasm on transit to the mitochondria.

(104) PCFM was synthesised using a commercial DNA synthesis provider (Bio S&T Inc., Montreal (Quebec), Canada).

(105) The location of mutagenised donor sites are underlined

(106) PCFM Sequence

(107) TABLE-US-00025 SEQIDNO.36 GAACTCAATGGGGCCAGTTATAGCATCCTGCTTCTTCTTACAAAAGAAAT TTCATAAGATAAGAGAGATGAGGCAAAGAAGGAATTGATAGAGGTGCGGC GAGAAGTTCAATACCTTCTTGATCGAGAAAATGTCCTTGCTTGTACTTCT CTTTCTTTATCGAGATTGGGTTGGTGTTCAGTGTACCGCTTGTCTAGCCT ATGCTTTGCATGAACATCTCAATGTCCAAGATAAAAAGAACGAGGGGAAG AATCGACGAGGCCAGTGTTCTCGAAGAGAAAATCGTGATGGAAAAAGCGT GAGGAGAATTCGAAACTCGAGATGTTAGAAGGTGCAAAATCAATGGGTGC AGGAGCTGCTACAATTGCTTCAGCGGGAGCTGCTATCGGTATTGGAAACG TCCTTAGTTCCTCGATTCATTCCGTTTTAGGGATACAAATAACAGACTTA CATCACGATGTCTTTTTCTTCGTTATTCTGATTTTGGTTTTCGTATCATG GATCTTGGGTCGCGCTTTATGGCATTTCCACTATAAAAAAAATCCAATCC CGCAAAGGATTGTTCATGGAACTACTATCGAGATTCTTCGGACCTTATTT CCTAGTATCATCCCTATGTTCATTGCTATACCATCATTTGCCCTGTATGG GTATTCGGACTATAACAGTTCCGATGAACAGTCACTCACTTTTGACAGTT ATACGATTCCAGAAGATGATCCAGAATTGGGTCAATCACGTTTATTAGAA GTCGACAATAGAGTGGTTGTACCAGCAAACAGTTCTCTCCGTTTTATTGT AACATCTGCGGCTGTACCTTCCTTAGGTGTCAAAGGTGATGCTGTGCCTT CCTTAGGTGTCAAAGGTGATGCTGTGCCTTCCTTAGGTGTCAAAGGTGAT GCTGTGCCTGGGCCTGGGCGGGTTTTTCAGACTTGGACCCGAGCTTTTGA GCGTTTGGGCCTGTTGACGGTTGCCCATTGCGCCGGCACCGGAACATCAA GCTCGGGCTCGGTAGTCAGTCTTCCACAGGACGAAATATGGGCCGCCCTT GAGGGCGATCCCCAGGCCCTTCCGGAAGACGGGCAATTTCACGCCGTCGC CCCTGAGGGGAATCCCCAGGCCCTTCCGGAAGACGGGCAATTTCACGCCG TCGCCCCTGAGGGGAATCCCCAGGCCCTTCCGGAAGACGGGCAATTTCAC GCCGTCGCCCCTGAGGGGAATCCCCAGGCCCTTCCGGAAGACGGGCAATT TCACGCCATCGCCTTTGACCCTCTTATAGCAACACGGCAAGACGCGTGGA ATACGCTACTTGTCTTGTTGCGGCGCAGCACCAAAATTGAGCCTAAGGCC AATTTTGTTACTAAAGCAGGGGAAGATCTTGGTATAGATACCGCAGACCC TGTTCGCCTTGACAAGTTAGTACGGGTACTGAACACGTATATCCAACTCG CCCCATTAGAAAGCGGGAGAAAGGTCCTCCAAAACCTGAAAGCCACGATG GCTGAATGGGAAAAGAACGGAAGGCCCTAAGTGGTGTCGTGTACTTTTTT CAATTATAATTAAATAAAAGGAGGTTACCGAATTTACGCGGTGGCCCTTT TATGTATGTTGCTGTCGTAAAGTTTCGTTCT
2.4 Primer Binding Domain for Reverse Transcription

(108) A Primer Binding Domain (PBD) was designed to capture the plant mitochondria tRNA-fMet as described by Friant et al., (1998) to be used as a template for reverse transcription

(109) PBD was constructed by overlapping PCR using the set of following primers:

(110) TABLE-US-00026 IM374 SEQIDNO.37 TTCCGCGGCCTATCTCACATTCACCCAATTGTCA IM368 SEQIDNO.38 TTAGAAGTATCCAATGCACAGTAGGTACCATGACAATTGGGTGAA IM369 SEQIDNO.39 ATTGGATACTTCTAAGGAAGTCCACACAAATCAAGAACCATTAGA IM370 SEQIDNO.40 CTCACATTCTTCTGTTTGGTTAGATGAAACGTCTAATGGTTCTTGA
PBD MIT

(111) TABLE-US-00027 SEQIDNO.41 TATCTCACATTCACCCAATTGTCATGGTACCTACTGTGCATTGGATACTT CTAAGGAAGTCCACACAAATCAAGAACCATTAGACGTTTCATCTAACCAA ACAGAAGAATGTGAGAAGGCTTCCACTAAGGCTAACTCTCAACAGACAAC
3 Mitochondria Transit Peptide (mTP)

(112) The sequence coding for the Mitochondrial ATPase 1 subunit transit peptide (1-mTP) was amplified from total tobacco cellular DNA with the following primers:

(113) Proteins translationally fused to 1-mTP can be driven inside the plant mitochondria.

(114) TABLE-US-00028 IM267 ATACTCGAGTCTCTCTCTACTCCTTTCAC SEQIDNO.42 IM268 ATAGCATGCTGATGGCTGAGATGCCGGTG SEQIDNO.43

(115) 1-mTP Sequence

(116) TABLE-US-00029 SEQIDNO.44 TCTCTCTCTACTCCTTTCACTCTCTCTCTAGCCAAACCCTCCACCATGGC TTCTCGGAGGCTTCTCGCCTCTCTCCTCCGTCAATCGGCTCAACGTGGCG GCGGTCTAATTTCCCGATCGTTAGGAAACTCCATCCCTAAATCCGCTTCA CGCGCCTCTTCACGCGCATCCCCTAAGGGATTCCTCTTAAACCGCGCCGT ACAGTACGCTACCTCCGCAGCGGCACCGGCATCTCAGCCATCA
4 Mitochondria-Targeted Reverse Transcriptase-RNaseH Protein

(117) The reverse transcriptase-RNase H gene from the yeast Ty1-H3 retrotransposon (Boeke et al., Mol. Cellul. Biology (1988), 8: 1432-1442; bank accession No. M18706) was optimised for codon usage in plants and by insertion of 5 introns from the Arabidopsis genome (intron 1from At1g04820, intron 2from At2g29550, intron 3from At1g31810, intron 4 and 5from At1g09170). The gene was synthesised by a commercial DNA synthesis provider and fused to the sequence coding for the mitochondria transit peptide 1-mTP. The resulting gene was named mRTRHi-Ty1. The introns are underlined and shown in bold letters. The sequence coding for 1-mTP is in italics lower case.

(118) mRTRHi-Ty1 Sequence

(119) TABLE-US-00030 SEQIDNO.45 ctcgagtctctctctactcctttcactctctctctagccaaaccctccac catggcttctcggaggcttctcgcctctctcctccgtcaatcggctcaac gtggcggcggtctaatttcccgatcgttaggaaactccatccctaaatcc gcttcacgcgcctcttcacgcgcatcccctaagggattcctcttaaaccg cgccgtacagtacgctacctccgcagcggcaccggcatctcagccatcag catgcATGAACAATTCATCCCACAACATCGTTCCTATCAAGACTCCAACT ACTGTTTCTGAGCAGAACACTGAAGAATCTATCATCGCTGATCTTCCACT TCCTGATCTTCCTCCAGAATCTCCTACTGAATTTCCTGATCCATTCAAAG AACTTCCACCTATCAACTCAAGACAAACTAACTCTTCATTGGGCGGAATT GGCGATTCTAATGCTTACACTACTATCAACTCTAAGAAGAGGTATTGTAG CCAGCCTCAACCAGTCTTTTTGCTGTTACATTTTCTTGGGCTCATCTAAT GTTATTTTCCTATTTTGTTTTCAGGTCACTTGAAGATAATGAAACTGAAA TCAAAGTTTCTAGGGATACATGGAATACTAAGAATATGAGATCACTTGAA CCTCCAAGATCTAAGAAGAGAATCCATCTTATTGCAGCTGTTAAAGCTGT GAAATCAATCAAACCAATTAGAACAACTCTTAGATACGATGAAGCAATTA CATACAACAAAGACATCAAGGAGAAGGAGAAATACATCGAGGCTTACCAC AAAGAAGTTAACCAACTTCTTAAGATGAAAACTTGGGATACTGATGAATA CTACGATAGAAAAGAGATTGACCCTAAGAGAGTTATCAACTCAATGTTCA TCTTCAACAAGAAGAGAGACGGAACTCACAAAGCTAGATTCGTTGCAAGA GGAGATATTCAGCATCCTGACACTTACGATTCAGGTAAGTATTCCAATGT TCTTCGATTATGAGTCAATGTTGTTACTGTATCTGTCTCTGTGGTTTATT GTTTCAGGCTTAGTTATTGATTAGTATTGAAACTTCACTCACATATTTTT TTGTTTGTTTTCTGAATTGTGCAGGTATGCAATCTAATACTGTTCATCAC TACGCATTGATGACATCTCTTTCACTTGCATTGGACAATAACTACTACAT TACACAACTTGACATATCTTCTGCATACCTTTACGCTGATATCAAGGAGG AGCTTTACATTAGACCTCCACCACATTTGGGAATGAATGATAAGTTGATC CGTTTGAAGAAATCACTTTACGGATTGAAACAATCTGGAGCTAATTGGTA CGAAACTATCAAATCATACCTTATTCAGCAATGCGGTATGGAGGAAGTTA GGGGATGGTCATGCGTATTCAAGAACTCTCAAGTTACAATCTGCCTCTTC GTTGATGATATGGTGCTCTTCTCTAAGAATCTTAACTCAAACAAGAGAAT CATTGAGAAGTTGAAGATGCAATACGACACTAAGATCATCAACCTTGGAG AATCTGATGAGGAAATTCAATACGACATTCTTGGATTGGAAATCAAATAC CAAAGAGGTGAGTTATATTTAACAGCTCATCAGTTACTTAAACACTTTTT GGGACAAGCAGTTCAAACTCATGTTCCAATCCTAAAATTAATTGCAATTC ACAGGTAAGTACATGAAGTTGGGAATGGAAAACTCATTGACTGAGAAGAT TCCTAAACTTAACGTTCCTTTGAATCCAAAGGGAAGAAAGCTCTCTGCTC CAGGACAACCAGGACTTTACATTGACCAGGATGAACTTGAGATTGATGAG GATGAATACAAGGAGAAAGTACACGAGATGCAGAAGTTGATTGGACTTGC TTCATACGTTGGATACAAATTCAGATTCGACCTTCTTTACTACATCAACA CACTTGCTCAGCATATACTTTTCCCATCTAGGCAAGTTCTTGACATGACA TACGAGCTTATCCAATTCATGTGGGACACTAGAGACAAGCAACTCATATG GCACAAGAACAAGCCTACAGAGCCAGATAACAAGCTCGTTGCAATCTCTG ATGCTTCTTACGGAAACCAACCATACTACAAATCACAAATTGGAAACATC TACTTGCTTAACGGAAAGGTACTTTTCTCAAAGACTTTACCTTATTGTGG AATATTGAATTTTCTGAAAGACTTCACCTTATCTACATTTGTAATTTTAC TATGGTAATCAGGTGATTGGAGGAAAGAGCACTAAGGCTTCACTTACATG CACTTCAACTACTGAGGCAGAGATCCACGCTATATCAGAATCTGTACCAC TTCTTAACAACCTTTCTTACCTTATCCAAGAGCTTAACAAGAAGCCAATC ATCAAGGGACTTCTTACTGACTCAAGATCAACAATCTCTATCATTAAGTC TACAAATGAAGAGAAATTCAGAAACAGATTCTTCGGAACAAAGGCAATGA GACTTAGAGATGAAGTTTCAGGTAAGTATTAACTTACCAAATGATCAATA TTATTTTGAAATGCAGGTTTTAGAATAATACTCTCTGCCGTTCTTGTTTA TTTCCAGGTAACAACCTTTACGTTTACTACATCGAGACTAAGAAGAACAT TGCTGACGTTATGACAAAGCCTCTTCCTATCAAGACCTTCAAGTTGCTTA CTAACAAATGGATTCATTA
5 MTS-Binding Protein (MTS-BP)

(120) The LtrA protein from Lactococcus lactis encoded by the LtrB intron is able to bind to the LtrB intron-based plant mitochondria translocation sequence (MTS) described in part 1 and therefore serves as a MTS-binding protein. The sequence of the LtrA protein was first optimised for codon usage in plants and 5 plant introns were inserted into the coding sequence to improve LtrA expression in plants. The introns 1, 2 4 are from Arabidopsis gene At5g01290, intron 3 and 5 were selected from Arabidopsis gene At5g43940. The gene was synthesised by commercial DNA synthesis provider and fused to the sequence coding for the mitochondria transit peptide 1-mTP. The resulting gene was named mLTRASi. The mLTRASi protein is able to bind to RNA molecules carrying the LtrB intron MTS and transfer these RNAs into the plant mitochondria.

(121) Plant introns inserted in the coding sequence of the LtrA gene are underlined and shown in bold letters. The sequence coding for 1-mTP is in italics lower case.

(122) mLTRASi Sequence:

(123) TABLE-US-00031 SEQIDNO.46 ctcgagtctctctctactcctttcactctctctctagccaaaccctccac catggcttctcggaggcttctcgcctctctcctccgtcaatcggctcaac gtggcggcggtctaatttcccgatcgttaggaaactccatccctaaatcc gcttcacgcgcctcttcacgcgcatcccctaagggattcctcttaaaccg cgccgtacagtacgctacctccgcagcggcaccggcatctcagccatcag catgcATGAAGCCAACAATGGCAATCCTCGAACGAATCTCTAAGAACTCA CAGGAGAACATCGACGAGGTACAATAACCCATATATATGAATTGATTCAT GTGTTACTCGTACTTGTTTGAATATGTTTGGAGCAAGTTTGATACTTTTG GATGATGATATCGCAAATTCGTTATCTTTTTGGCGTTATAGGTCTTCACA AGACTTTACCGTTACCTTCTCCGTCCTGACATCTACTACGTGGCATATCA GAACCTCTACTCTAACAAGGGAGCTTCTACAAAGGGAATCCTCGATGATA CAGCTGATGGATTCTCTGAGGAGAAGATCAAGAAGATCATCCAATCTTTG AAGGACGGAACTTACTACCCTCAGCCTGTCCGAAGAATGTACATCGCAAA GAAGAACTCTAAGAAGATGAGACCTCTTGGAATCCCAACTTTCACAGACA AGTTGATCCAGGAGGCTGTGAGAATCATCCTTGAATCTATCTATGAGCCT GTCTTCGAGGATGTGTCTCACGGTTTCCGACCTCAGCGAAGCTGTCACAC AGCTTTGAAGACAATCAAGAGAGAGTTCGGAGGTAAATTATATGCTTTGC CACTTCCTCAAAAGATCATTTTAGGTTCATTGGTATGTGGTTTTTTTCTT AACAGGTGCAAGATGGTTCGTGGAGGGAGATATCAAGGGATGCTTCGATA ACATCGACCACGTCACACTCATCGGACTCATCAACCTTAAGATCAAGGAT ATGAAGATGAGCCAGTTGATCTACAAGTTCCTCAAGGCAGGTTACCTCGA AAACTGGCAGTACCACAAGACTTACAGCGGAACACCTCAGGGCGGAATCC TCTCTCCTCTCCTCGCTAACATCTATCTTCATGAATTGGACAAGTTCGTT CTCCAACTCAAGATGAAGTTCGACCGAGAGAGTCCAGAGAGAATCACACC TGAATACCGGGAGCTTCACAACGAGATCAAAAGAATCTCTCACCGTCTCA AGAAGTTGGAGGGCGAGGAGAAGGCTAAGGTTCTCTTGGAATACCAGGAG AAGAGGAAGAGGTTGCCTACACTCCCTTGTACATCACAAACAAACAAGGT TCGTTCTCTCCATTTTCATTCGTTTGAGTCTGATTTAGTGTTTTGTGGTT GATCTGAATCGATTTATTGTTGATTAGTGAATCAATTTGAGGCTGTGTCC TAATGTTTTGACTTTTGATTACAGGTCTTGAAGTACGTCCGATACGCTGA CGACTTCATCATCTCTGTTAAGGGAAGCAAGGAGGACTGTCAATGGATCA AGGAGCAATTGAAGCTCTTCATCCATAACAAGCTCAAGATGGAATTGAGT GAGGAGAAGACACTCATCACACATAGCAGTCAGCCTGCTCGTTTCCTCGG ATACGACATCCGAGTCAGGAGAAGTGGAACTATCAAGCGATCTGGAAAGG TTCAATTCTTTCTTTCACATTTGTACTTGTTCACTCGTTTTATTAATCCT CTTTAGAATGGAGATTCTTACCTCTGTGTGGCCTTTGGCAGGTCAAGAAG AGAACACTCAACGGGAGTGTGGAGCTTCTCATCCCTCTCCAAGACAAGAT CCGTCAATTCATCTTCGACAAGAAGATCGCTATCCAGAAGAAGGATAGCT CATGGTTCCCAGTTCACAGGAAGTACCTTATCCGTTCAACAGACTTGGAG ATCATCACAATCTACAACTCTGAATTGAGAGGTAAGCTGCTACCTCAAAC TTTCTAGTGCTTCCATATTTCCTTTCTTCTGCAAGGCAGAGAACCATTGT GGTTAAGTGTTTTAAATTGTGAATGTATAGGTATCTGCAACTACTACGGT CTCGCAAGTAACTTCAACCAGCTCAACTACTTCGCTTACCTTATGGAATA CTCTTGCTTGAAGACTATCGCATCTAAGCATAAGGGAACACTCTCAAAGA CCATCTCTATGTTCAAGGATGGAAGTGGTTCTTGGGGAATCCCTTACGAG ATCAAGCAGGGGAAGCAGAGGAGATACTTCGCCAACTTCAGTGAATGCAA ATCTCCTTACCAATTCACTGATGAGATCAGTCAAGCTCCTGTGCTTTACG GAACGCTCGGAACACTCTTGAGAACAGACTTAAGGCTAAGTGTTGTGAGT CTTTGTGGAACATCTGATGAGAACACATCTTACGAGATCCACCACGTCAA CAAGGTCAAGAACCTTAAGGGAAAGGAGAAGTGGGAGATGGCAATGATCG CTAAGCAGCGGAAGACTCTTGTTGTTTGCTTCCATTGTCATCGTCACGTG ATCCATAAGCACAAGTGAACTAGTAA
6. Promoter and Terminator Sequences used for Expression of Nuclear Cassettes
6.1 Promoter Sequences
6.1.1 Arabidopsis Ubiquitin Promoter AtUbi3-PRO

(124) The 5 promoter region from Arabidopsis ubiquitin 3 gene was amplified with the following primers:

(125) TABLE-US-00032 SEQ ID NO. 47 IM326 CGAAGCTTGAATTCTACCGGATTTGGAGCCAAGTC SEQIDNO.48 IM327 AAGGATCCTCTAGATGTTTGGTGACCTGAAATAAAACAATAG
AtUbi3-PRO Sequence

(126) TABLE-US-00033 SEQIDNO.49 TACCGGATTTGGAGCCAAGTCTCATAAACGCCATTGTGGAAGAAAGTCTT GAGTTGGTGGTAATGTAACAGAGTAGTAAGAACAGAGAAGAGAGAGAGTG TGAGATACATGAATTGTCGGGCAACAAAAATCCTGAACATCTTATTTTAG CAAAGAGAAAGAGTTCCGAGTCTGTAGCAGAAGAGTGAGGAGAAATTTAA GCTCTTGGACTTGTGAATTGTTCCGCCTCTTGAATACTTCTTCAATCCTC ATATATTCTTCTTCTATGTTACCTGAAAACCGGCATTTAATCTCGCGGGT TTATTCCGGTTCAACATTTTTTTTGTTTTGAGTTATTATCTGGGCTTAAT AACGCAGGCCTGAAATAAATTCAAGGCCCAACTGTTTTTTTTTTTAAGAA GTTGCTGTTAAAAAAAAAAAAAGGGAATTAACAACAACAACAAAAAAAGA TAAAGAAAATAATAACAATTACTTTAATTGTAGACTAAAAAAACATAGAT TTTATCATGAAAAAAAGAGAAAAGAAATAAAAACTTGGATCAAAAAAAAA ACATACAGATCTTCTAATTATTAACTTTTCTTAAAAATTAGGTCCTTTTT CCCAACAATTAGGTTTAGAGTTTTGGAATTAAACCAAAAAGATTGTTCTA AAAAATACTCAAATTTGGTAGATAAGTTTCCTTATTTTAATTAGTCAATG GTAGATACTTTTTTTTCTTTTCTTTATTAGAGTAGATTAGAATCTTTTAT GCCAAGTATTGATAAATTAAATCAAGAAGATAAACTATCATAATCAACAT GAAATTAAAAGAAAAATCTCATATATAGTATTAGTATTCTCTATATATAT TATGATTGCTTATTCTTAATGGGTTGGGTTAACCAAGACATAGTCTTAAT GGAAAGAATCTTTTTTGAACTTTTTCCTTATTGATTAAATTCTTCTATAG AAAAGAAAGAAATTATTTGAGGAAAAGTATATACAAAAAGAAAAATAGAA AAATGTCAGTGAAGCAGATGTAATGGATGACCTAATCCAACCACCACCAT AGGATGTTTCTACTTGAGTCGGTCTTTTAAAAACGCACGGTGGAAAATAT GACACGTATCATATGATTCCTTCCTTTAGTTTCGTGATAATAATCCTCAA CTGATATCTTCCTTTTTTTGTTTTGGCTAAAGATATTTTATTCTCATTAA TAGAAAAGACGGTTTTGGGCTTTTGGTTTGCGATATAAAGAAGACCTTCG TGTGGAAGATAATAATTCATCCTTTCGTCTTTTTCTGACTCTTCAATCTC TCCCAAAGCCTAAAGCGATCTCTGCAAATCTCTCGCGACTCTCTCTTTCA AGGTATATTTTCTGATTCTTTTTGTTTTTGATTCGTATCTGATCTCCAAT TTTTGTTATGTGGATTATTGAATCTTTTGTATAAATTGCTTTTGACAATA TTGTTCGTTTCGTCAATCCAGCTTCTAAATTTTGTCCTGATTACTAAGAT ATCGATTCGTAGTGTTTACATCTGTGTAATTTCTTGCTTGATTGTGAAAT TAGGATTTTCAAGGACGATCTATTCAATTTTTGTGTTTTCTTTGTTCGAT TCTCTCTGTTTTAGGTTTCTTATGTTTAGATCCGTTTCTCTTTGGTGTTG TTTTGATTTCTCTTACGGCTTTTGATTTGGTATATGTTCGCTGATTGGTT TCTACTTGTTCTATTGTTTTATTTCAGGTCACCAAACA
6.1.2 CaMV 35S Promoter

(127) The 35S promoter from Cauliflower mosaic virus (35S-PRO) was synthesised based on GenBank accession number AF502128

(128) 35S-PRO Sequence

(129) TABLE-US-00034 SEQIDNO.50 TTAGCCTTTTCAATTTCAGAAAGAATGCTAACCCACAGATGGTTAGAG AGGCTTACGCAGCAGGTCTCATCAAGACGATCTACCCGAGCAATAATC TCCAGGAAATCAAATACCTTCCCAAGAAGGTTAAAGATGCAGTCAAAA GATTCAGGACTAACTGCATCAAGAACACAGAGAAAGATATATTTCTCA AGATCAGAAGTACTATTCCAGTATGGACGATTCAAGGCTTGCTTCACA AACCAAGGCAAGTAATAGAGATTGGAGTCTCTAAAAAGGTAGTTCCCA CTGAATCAAAGGCCATGGAGTCAAAGATTCAAATAGAGGACCTAACAG AACTCGCCGTAAAGACTGGCGAACAGTTCATACAGAGTCTCTTACGAC TCAATGACAAGAAGAAAATCTTCGTCAACATGGTGGAGCACGACACAC TTGTCTACTCCAAAAATATCAAAGATACAGTCTCAGAAGACCAAAGGG CAATTGAGACTTTTCAACAAAGGGTAATATCCGGAAACCTCCTCGGAT TCCATTGCCCAGCTATCTGTCACTTTATTGTGAAGATAGTGGAAAAGG AAGGTGGCTCCTACAAATGCCATCATTGCGATAAAGGAAAGGCCATCG TTGAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCA CGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGC AAGTGGATTGATGTGATATCTCCACTGACGTAAGGGATGACGCACAAT CCCACTATCCTTCGCAAGACCCTTCCTCTATATAAGGAAGTTCATTTC ATTTGGAGAGAACACGGGGGAC

(130) 6.1.3 TAF2 Promoter Sequence

(131) The 5 untranslated region upstream of the TAF2 gene from Arabidopsis thaliana was synthesised based on sequence At1g73965 from the Arabidopsis genome, (www.arabidopsis.org).

(132) TABLE-US-00035 SEQIDNO.51 GGTACCATGATCGCTTCATGTTTTTATCTAATTTGTTAGCATATTGAA TGATTGATTTTCTTTTAATTTGGATATGTTGATTGTCTTGTTGCATCA TCAATGTATGTTTTATTTAACACCGGAAGATCTTATGATGGGTTCATT ACTTCATAATAATCTCCGAGTTCTACAAGACTACAACTTTCACGTGAC TTTTACAGCGACAAAAAATGCATCTAGCGAAAATTAATCCACAACCTA TGCATTTTTGTCACTCTTCACACGCGTATGTGCATAAATATATAGTAT ATACTCGACAATCGATGCGTATGTGTACACAATTACCAAAACAATTAT TTGAATATTCAGACATGGGTTGACATCACCAAGTAATATTCACAGTAT CTGAAAACTATGTTTTGACATCCCTAAATAGTTTGACTAACCAGTTTA ATATGAGAGCATTTGTAAGAGGCAAGAGCCATGGTTTTGTTGGCTCGT TTAATATGCTCATTTAACCCCCCCAAAAAATACTATTAGATTTAAACG TAAAAGAATTAACGAACACAAGAACTGCTAAAACAAAAAAAAATCAAT GGCCGACATTTCATAGTTCATACATCACTAATACTAAAAGATGCATCA TTTCACTAGGGTCTCATGAAATAGGAGTTGACATTTTTTTTTGTAACG ACAGAAGTTGACATGTTAAGCATCAATTTTTTTAAGAGTGGATTATAC TAGTTTTTTTTTTTTTTTTTAATGTATGGTATGATACAACAACAAAAA CTATAAAATAGAAAAAGTCAGTGAAACCTCAAATTGAAGGAAAAACTT TTGCACAAAAAGAGAGAGAGAGAGAAAGAATGTAAATCCAAATAAATG GGCCTAATTGAGAATGCTTTAACTTTTTTTTTTTGGCTAAAAGAGAAT GCTTTAACTAAGCCCATAAAATGAACATCAAACTCAAAGGGTAAGATT AATACATTTAGAAAACAATAGCCGAATATTTAATAAGTTTAAGACATA GAGGAGTTTTATGTAATTTAGGAACCGATCCATCGTTGGCTGTATAAA AAGGTTACATCTCCGGCTAACATATCGGCAAAAAAGGAACCTCGAG
6.3 Terminator Sequences
6.3.1 Nos Terminator

(133) The nos terminator fragment was synthesised based on GenBank sequence accession EU048864.

(134) nos Terminator Sequence

(135) TABLE-US-00036 SEQIDNO.52 TCTAGAGTCAAGCAGATCGTTCAAACATTTGGCAATAAAGTTTCTTAA GATTGAATCCTGTTGCCGGTCTTGCGATGATTATCATATAATTTCTGT TGAATTACGTGAAGCATGTAATAATTAACATGTAATGCATGACGTTAT TTATGAGATGGGTTTTTATGATTAGAGTCCCGCAATTATACATTTAAT ACGCGATAGAAAACAAAATATAGCGCGCAAACTAGGATAAATTATCGC GCGCGGTGTCATCTATGTTACTAGATCGACCTGCAG
6.3.2 Ags Terminator

(136) The agropine synthase polyA signal (ags terminator) was synthesized based on the GenBank sequence EU181145.

(137) ags Terminator Sequence

(138) TABLE-US-00037 SEQIDNO.53 GAATTAACAGAGGTGGATGGACAGACCCGTTCTTACACCGGACTGGGC GCGGGATAGGATATTCAGATTGGGATGGGATTGAGCTTAAAGCCGGCG CTGAGACCATGCTCAAGGTAGGCAATGTCCTCAGCGTCGAGCCCGGCA TCTATGTCGAGGGCATTGGTGGAGCGCGCTTCGGGGATACCGTGCTTG TAACTGAGACCGGATATGAGGCCCTCACTCCGCTTGATCTTGGCAAAG ATATTTGACGCATTTATTAGTATGTGTTAATTTTCATTTGCAGTGCAG TATTTTCTATTCGATCTTTATGTAATTCGTTACAATTAATAAATATTC AAATCAGATTATTGACTGTCATTTGTATCAAATCGTGTTTAATGGATA TTTTTATTATAATATTGATGAT
Part 2Plant Transformation Methods and Vector Maps

(139) Transformation of plants was carried out using constructs based on the pGreen0029 and pSOUP binary vectors (GenBank accession numbers EU0490266, EU048864 and EU048870) (Hellens et al, 2000, Plant Mol. Biol. 42: 819-832). The pSOUP-0179 vector is carrying the T-DNA from the pGreen0179 (GenBank accession number EU048866) vector into the pSOUP vector (GenBank accession number EU048870)

(140) 1. Transformation of Tobacco Plants

(141) 1.1. Transformation of tobacco plants was performed as described by Horsch et al (1985) (Science 227: 1229-1231) using Agrobacterium strain AGL1.

(142) Plant transformants were selected on regeneration medium supplemented with Kanamycin 300 mg/l and/or Hygromycin 30 mg/l.

(143) 1.2 Vectors for Transformation of Tobacco Mitochondria

(144) 1.2.1 General Vectors for Transgene Expression into Mitochondria

(145) Expression using a Heterologous Promoter

(146) The ATP6 promoter region from Arabidopsis thaliana mitochondria (AtmATP6-PRO) can be used to direct expression of transgenes in tobacco mitochondria.

(147) The gene coding for GFP was cloned between AtmATP6-PRO and the ATP6 terminator sequence from tobacco (NtmATP6-TER). This transgene expression construct was cloned between NtmLFS1 and NtmRFS1 upstream of PCD to form the mitochondria transformation unit (MTU). The mLTRASi sequence was placed under the control of the AtUbi3 promoter and ags terminator and cloned into the pGreen0029 together with the mitochondria transformation unit to generate the M21 vector (FIG. 3).

(148) The mitochondria targeted reverse transcriptase-RNase H mRTRHi-Ty1 was placed under the control of the TAF2 promoter and ags terminator and cloned into the pSOUP-0179 vector to generate the M28 vector (FIG. 3).

(149) The M21 and M28 constructs were co-transformed in Agrobacterium strain AGL1 and used for Nicotiana tabacum transformation.

(150) Transgenic lines were recovered on selection medium supplemented with 300 mg/l of Kanamycin and/or 30 mg/l of Hygromycin:

(151) Expression by Translational Fusion with a Native Mitochondrial Gene

(152) NtmLFS4 corresponds to the 5 end of the gene coding for ATP6 and can be used for translational fusion with any gene of interest, promoter activity is provided by the ATP6 promoter upstream of NtmLFS4 upon insertion in the tobacco mitochondrial genome and termination of transcription is achieved with the ATP6 terminator sequence within NtmRFS4. The gene coding for GFP was fused to NtmLFS4 and cloned together with NtmRFS4 and PBD into domain IV of the LtrBM intron to form the mitochondria transformation unit (MTU). The mLTRASi sequence was placed under the control of the AtUbi3 promoter and ags terminator and cloned into the pGreen0029 together with the mitochondria transformation unit to generate the M22 vector (FIG. 4).

(153) The M22 and M28 constructs were co-transformed in Agrobacterium strain AGL1 and used for Nicotiana tabacum transformation.

(154) Transgenic lines were recovered on selection medium supplemented with 300 mg/l of Kanamycin and/or 30 mg/l of Hygromycin.

(155) 1.2.2 Cytoplasmic-Male Sterility (CMS) Inducing Construct

(156) The CMS-inducing tobacco mitochondria transformation unit containing NtmLFS3, PCFM, NtmLFS3 and primer binding domain (PBD) was inserted into domain IV of the LtrBM intron. The resulting DNA fragment was fused to the 35S promoter and nos terminator and introduced into the pGreen0029 binary vector. The mLTRASi sequence was placed under the control of the AtUbi3 promoter and ags terminator and cloned into the pGreen0029 together with the mitochondria transformation unit to generate the M24 vector (FIG. 5).

(157) The M24 and M28 constructs were co-transformed in Agrobacterium strain AGL1 and used for Nicotiana tabacum transformation.

(158) Transgenic lines were recovered on selection medium supplemented with 300 mg/l of Kanamycin and/or 30 mg/l of Hygromycin.

(159) 2. Transformation of Arabidopsis Thaliana

(160) 2.1. Transformation of Arabidopsis plants was performed as described by Clough & Bent (Clough & Bent (1998) Plant Journal 16:735-743). The Agrobacterium tumefaciens strain GV3101 (Koncz & Schell (1986)Mol Gen Genet. 204:383-396) was used for transformation. The resulting DNA fragment was fused to the 35S promoter and nos terminator and introduced into the pGreen0029 binary vector.
2.2 Vectors for Transformation of Arabidopsis thaliana Mitochondria
Cytoplasmic-Male Sterility (CMS) Inducing Construct

(161) The CMS-inducing arabidopsis mitochondria transformation unit containing AtmLFS5, PCFM, AtmLFS5 and primer binding domain (PBD) was inserted into domain IV of the LtrBM intron. The resulting DNA fragment was fused to the 35S promoter and nos terminator and introduced into the pGreen0029 binary vector. The mLTRASi sequence was placed under the control of the AtUbi3 promoter and ags terminator and cloned into the pGreen0029 together with the mitochondria transformation unit to generate the M27 vector (FIG. 6).

(162) The M27 and M28 constructs (FIG. 3) were co-transformed in Agrobacterium strain GV3101 and used for Arabidopsis (Col-0) transformation.

(163) Transgenic lines were recovered on selection medium supplemented with 300 mg/l of Kanamycin and/or 30 mg/l of Hygromycin.

(164) Part 3Results

(165) The transformation of Nicotiana tabacum and arabidopsis with our vectors containing transgene cassettes generated transgenic plants. In all cases we were able to detect insertion of the transgene cassette into the mitochondria genome using PCR amplification of junction regions.

(166) Five independent transgenic lines were analysed for each construct. Molecular analyses including sequencing of insert junctions showed that there was correct insertion in the mitochondrial genome in 80% of transformed plants.

(167) Furthermore, male sterile tobacco plants transformed with the CMS-inducing open reading frame PCF from petunia mitochondria were generated (FIG. 7).

EXPERIMENTAL SECTION 1B

(168) Modifications of the mitochondria transformation method described in Experimental section 1A can be improved using PBD designed for reverse transcription in the cytoplasm or in mitochondria, and by re-positioning of the building blocks on the transformation cassette (FIG. 8).

(169) A set of constructs was prepared for tobacco and rice transformation with LtrB intron (LtrB-MTS) as the MTS (FIG. 9-10). The positioning of the transgene cassette building blocks was designed as described in FIG. 8, A-B.

(170) PBD-MIT was designed as described previously.

(171) PBD-MIT

(172) TABLE-US-00038 SEQIDNO.41 TATCTCACATTCACCCAATTGTCATGGTACCTACTGTGCATTGGATAC TTCTAAGGAAGTCCACACAAATCAAGAACCATTAGACGTTTCATCTAA CCAAACAGAAGAATGTGAGAAGGCTTCCACTAAGGCTAACTCTCAACA GACAAC

(173) The primer binding domain of the tobacco tnt1 retrotransposon was used as the PBD-CYT, and was amplified from genomic DNA of tobacco cv Petit Gerard using the following primers:

(174) TABLE-US-00039 AS912 gccgcggctttattaccgtgaatatta SEQIDNO.54 AS913 cgcggccgctctgataagtgcaacctgatt SEQIDNO.55
PDB-CYT

(175) TABLE-US-00040 SEQIDNO.56 CTTTATTACCGTGAATATTATTTTGGTAAGGGGTTTATTCCCAACAAC TGGTATCAGAGCACAGGTTCTGCTCGTTCACTGAAATACTATTCACTG TCGGTAGTACTATACTTGGTGAAAAATAAAAATGTCTGGAGTAAAGTA CGAGGTAGCAAAATTCAATGGAGATAACGGTTTCTCAACATGGCAAAG AAGGATGAGAGATCTGCTCATCCAACAAGGATTACACAAGGTTCTAGA TGTTGATTCCAAAAAGCCTGATACCATGAAAGCTGAGGATTGGGCTGA CTTGGATGAAAGAGCTGCTAGTGCAATCAGGTTGCACTTATCAGA

(176) In the first case, PBD-MIT was fused to the 3 end of the LtrB intron (FIG. 8A, FIG. 9A for tobacco and FIG. 10A for rice). As the LtrA protein possesses both LtrB-MTS-binding feature and reverse transcription activity it can fulfil both functions of (i) transgene RNA translocation into mitochondria and (ii) reverse transcription of the RNA cassette using mitochondrial tRNA-Met as a primer.

(177) In the second case, PBD-CYT was fused to MTU (FIG. 8B, FIG. 9B for tobacco and FIG. 10B for rice), so that reverse transcription of the transgene cassette is initiated and performed by endogenous reverse transcriptases in the cytoplasm using cytoplasmic tRNA-Met. The LtrA protein serves as the MTS-binding protein for translocation of RNA:DNA complexes initiated by cytoplasmic reverse transcriptases, into the mitochondria.

(178) Rice PCF Construct M45

(179) Primers used to amplify the rice mitochondria left flanking sequence (osLFS) for insertion of the PCF open reading frame:

(180) TABLE-US-00041 IM416ggatccatatcgagccattgaagcag SEQIDNO.57 IM417_gcatgctcaatcttgtcctttgg SEQIDNO.58
osLFS

(181) TABLE-US-00042 SEQIDNO.59 ggattcatatcgagccattgaagcagcgcgtcgggctacaatcgggca attccatcgtgctatgagcggacaattccgaagaaattgtaagatatg ggtaagagttctcgcagatcttcctattacggggaaacccgcagaagt tcgaatgggaagaggaaaaggaaatcctacgggttggattgctcgtgt gtccacgggacaaatcccatttgaaatggatggtgtgagtttgtcaaa tgctcgacaagccgctagattagcggcgcataaaccatgttcgtcaac caagtttgttcagtggtcgtaacgtaattggttagtggggaaaaaccg ggccgggactcaaaagaatttggcgaagtgtttgttcctgaacgaggg aagtggaaagacaaagagggatagggagctcgcctccttctttttttg aatcgccgaaattgtacgacgacccttcttgttccaggcatacgactc tgagacgtgacggtgtcacttttccggccoggtaaagtgacagttata taaataagaataagaaagagaagcgtgatgttgtcagcaatcaaatta tcgtaaatagatagtacggttgcgttgtttcaatttctgttcgtcggt ccttgggttacgaaggtgtgggcttactaatacggagagggttccgaa tgataaagtgtcatgaaagttcgtgaaagaatgttcttgtttttcgtt ggaaaacccaacgccacggccacaaaacgaaaaagtctcccgtttgtt ttgggagcagagctttaaaaggatatagttaccctatgatgagattta gttcaacggataagaaggatagaagaaatatgctatttgctgctattc catctatttgtgcattcagtgctgccgttcccccggccccaaaggaca agattgagcatgc
Primers used to Amplify the Rice Mitochondria Left Flanking Sequence (osRFS) for Insertion of the PCF Open Reading Frame:

(182) TABLE-US-00043 IM418ttctagagtcgccgctatcacttt SEQIDNO.60 IM419ccgcggctaagactatagaatgttcc SEQIDNO.61
osRFS

(183) TABLE-US-00044 SEQIDNO.62 ttctagagtcgccgctatcactttttttggggggccaatcccgcgaag agttatggaaagattttatagctcaattgaatgaagaaagtgaattca tggacaacattttttttggtgtttacaacgcgagaaacggctatgaaa gcgccacagttcttcagggaatacggatagatttagcgataaacggct atgaaagtgcatttttgtcggaatttgcacctatttgtatctatttag tgatcagtccgctagtttctttgattccactcggtgttccttttccat ttgcttccaatagttcgacctatccagaaaaattgtcggcctacgaat gtggtttcgatccctccggtgatgccagaagtcgtttcgatatacgat tttatccggttcctattttatttattatccctgatctggaagtcacct ttttttttccttgggcagtacctcctaacaagattgatctgtttggat cttggtccatgatggcctttttattgattttgacgattggatttctct atgaatggaaaaggggtgcttcggatcgggagtaaccactagtgaaag ggcaaaggggggaaggacataggaaagagggatgcctacaaaaaatca attgattcgtcatggtagagaagaaaaacagcgcacggaccgtactcg agcttcggatcaatgtccccaaaagcaaggagtatgcctgcgtgtttc gacgagaacaccgaaaaaacctaattcagctctacgtaagatagcaaa agtacggttgagcaatcgacatgatatatttgctcacattccaggcga aggtcataattcgcaggaacattctatagtcttagccgcggcc

(184) The LtrA gene was driven by the actin1 rice promoter amplified using the following primers:

(185) TABLE-US-00045 ARP1gtcattcatatgcttgagaaga SEQIDNO.63 ARP2gcctacaaaaaagctccgcacg SEQIDNO.64
Rice act1 Promoter Sequence

(186) TABLE-US-00046 SEQIDNO.65 gtcattcatatgcttgagaagagagtcgggatagtccaaaataaaaca aaggtaagattacctggtcaaaagtgaaaacatcagttaaaaggtggt ataagtaaaatatcggtaataaaaggtggcccaaagtgaaatttactc ttttctactattataaaaattgaggatgttttgtcggtactttgatac gtcatttttgtatgaattggtttttaagtttattcgcgatttggaaat gcatatctgtatttgagtcggtttttaagttcgttgcttttgtaaata cagagggatttgtataagaaatatctttaaaaaacccatatgctaatt tgacataatttttgagaaaaatatatattcaggcgaattccacaatga acaataataagattaaaatagcttgcccccgttgcagcgatgggtatt ttttctagtaaaataaaagataaacttagactcaaaacatttacaaaa acaacccctaaagtcctaaagcccaaagtgctatgcacgatccatagc aagcccagcccaacccaacccaacccaacccaccccagtgcagccaac tggcaaatagtctccacccccggcactatcaccgtgagttgtccgcac caccgcacgtctcgcagccaaaaaaaaaaaaagaaagaaaaaaaagaa aaagaaaaacagcaggtgggtccgggtcgtgggggccggaaaagcgag gaggatcgcgagcagcgacgaggcccggccctccctccgcttccaaag aaacgccccccatcgccactatatacatacccccccctctcctcccat ccccccaaccctaccaccaccaccaccaccacctcctcccccctcgct gccggacgacgagctcctcccccctccccctccgccgccgccggtaac caccccgcccctctcctctttctttctccgttttttttttcgtctogg tctcgatctttggccttggtagtttgggtgggcgagagcggcttcgtc gcccagatcggtgcgcgggaggggcgggatctcgcggctggcgtctcc gggcgtgagtcggccoggatcctcgcggggaatggggctctoggatgt agatctgcgatccgccgttgttgggggagatgatggggggtttaaaat ttccgccatgctaaacaagatcaggaagaggggaaaagggcactatgg tttatatttttatatatttctgctgcttcgtcaggcttagatgtgcta gatcttctttctttcttctttttgtggtagaatttgaatccctcagca ttgttcatcggtagtttttcttttcatgatttgtgacaaatgcagcct cgtgcggagcttttttgtaggc
Transformation of Rice Immature Embryos.
Immature Embryo Excision
Day 1:

(187) Remove milky/post-milky stage immature seeds from panicles (immature embryos 1-2 mm in size are desired).

(188) Sterilize immature seeds: 50% sodium hypochlorite (12%)+1 drop of tween 20. Shake 10 min.

(189) Rinse 3-5 in sterile deionised water. Drain off surplus water. Aliquot seeds (around 40) in sterile Petri dishes.

(190) Set up a 6015 mm Petri dish containing a 50% sodium hypochlorite solution and next to this a sterile beaker on its side with a sterile filter paper in it. Use sterile forceps to aseptically remove glumes from the first seed. Immerse this seed in the 50% sodium hypochlorite. Remove glumes from a second seed and immerse the second seed into the sodium hypochlorite solution whilst removing the first seed and storing this dehusked/sterilized seed on the filter paper in the beaker. Continue.

(191) After all the glumes are removed:

(192) Sterilize dehusked seeds: 50% sodium hypochlorite: 5 min. with agitation.

(193) Rinse: 5-7 in sterile deionized water, drain.

(194) Place all seeds in a large sterile Petri dish. Aliquot for embryo excision (to keep seeds from drying out, work with only 50-100 in the plate at a time leaving the rest in the master plate).

(195) Remove the embryo from each seed and place embryo, scutellum up, in a 9015 mm Petri dish containing proliferation medium (40-50 embryos/plate). Culture at 28 C. in the dark for 2 days prior to bombardment

(196) Day 3:

(197) Check each Embryo for Contamination before Blasting

(198) Remove the embryos from the proliferation medium. Distribute 35-40 embryos scutellum upwards in an area 1 cm.sup.2 in the centre of a 6015 mm target plate containing 10 ml of proliferation medium+osmoticum (0.6 M). Check each target plate so that the scutellum is straight. Allow enough room so the scutella do not shade each other out.

(199) Bombardment:

(200) TABLE-US-00047 Gun 14 kV Vacuum: 25 inches of Hg 1.sup.st bombardment 4 hours after osmoticum treatment 2.sup.nd bombardment 4 hours after 1.sup.st bombardment
Day 4:

(201) 4-16 hours after the 2nd blast transfer immature embryos to proliferation medium without osmoticum. Culture in the dark at 28 C. for 2 days.

(202) Selection:

(203) Day 5:

(204) Aseptically cut out with scissors the germinating shoot. Transfer 16-20 immature embryos to fresh proliferation medium containing 30-50 mg/l Hygromycin (depending on the genotype); culture in the dark at 28 C.; record total number of embryos.

(205) After 10 days carefully remove the callus from the scutellum by breaking it up into 2-10 small pieces; subculture onto fresh proliferation medium+hygromycin. Do not subculture brown tissue and remaining immature embryo which could inhibit further growth of healthy callus.

(206) Subculture every 10 days by selecting healthy tissue: (embryogenic if present) and transfer it to fresh proliferation medium+hygromycin. Remove brown callus as it could be inhibiting to Embryogenic callus.

(207) 30 to 40 days after bombardment change selection procedure. Instead of eliminating bad-looking tissue keep embryogenic tissue only (eliminate healthy non-embryogenic tissue)

(208) Regeneration:

(209) After 40 to 60 days, transfer established embryogenic callus showing differential growth on proliferation medium+hygromycin to regeneration medium+hygromycin. Culture at 28 C. under low light for 10 days then under high light for 10 additional days. Check plates periodically in the light for the development of embryos and green shoots. As shoots develop it is sometimes beneficial to gently move the developing shoot away from the callus it originated from and remove any dead tissue from the shoot itself to prevent inhibition of growth.

(210) Germination:

(211) Transfer white compact embryos and green shoots initiating roots to the germination medium under high light at 28 C. for 1 to 2 weeks. Check plates periodically. Remove necrotic tissue and divide germinating embryos if necessary.

(212) Results

(213) The transformation of Nicotiana tabacum and rice was performed with group II intron-based vectors containing transgene cassettes for transformation of the mitochondrial genome.

(214) Seven to ten independent transgenic lines were analysed for each construct. Molecular analyses including sequencing of insert junctions showed that there was correct insertion in the mitochondrial genome in 80% of transformed plants. Insertion of the PCF open reading frame in the plant mitochondria was correlated with a sterility phenotype.

(215) The analysis of transgenic plants was performed using PCR for insertion flanking sequences using the following pairs, of primers:

(216) For Tobacco:

(217) TABLE-US-00048 ntLFS3F cccaagttacagcgggctct SEQIDNO.66 PCFMR tatggggcttccctgtcgag SEQIDNO.67 PCFMF gcagcaccaaaattgagcct SEQIDNO.68 ntRFS3R cgagttccagaggcatcttc SEQIDNO.69
For Rice:

(218) TABLE-US-00049 osLFSF actgaatgcggaaagtatgg SEQIDNO.70 PCFMR tatggggcttccctgtcgag SEQIDNO.71 PCFMF gcagcaccaaaattgagcct SEQIDNO.72 osRFSR tagggctactagaaagagga SEQIDNO.73