Transfection of plant mitochondria and in organello gene integration

Abstract

Investigation and manipulation of mitochondrial genetics in animal and plant cells remains restricted by the lack of an efficient in vivo transformation methodology. Mitochondrial transfection in whole cells and maintenance of the transfected DNA are main issues on this track. We showed earlier that isolated mitochondria from different organisms can import DNA. Exploiting this mechanism, we assessed the possibility to maintain exogenous DNA in plant organelles. Whereas homologous recombination is scarce in the higher plant nuclear compartment, recombination between large repeats generates the multipartite structure of the plant mitochondrial genome. These processes are under strict surveillance to avoid extensive genomic rearrangements. Nevertheless, following transfection of isolated organelles with constructs composed of a partial gfp gene flanked by fragments of mitochondrial DNA, we demonstrated in organello homologous recombination of the imported DNA with the resident DNA and integration of the reporter gene. Recombination yielded insertion of a continuous exogenous DNA fragment including the gfp sequence and at least 0.5 kb of flanking sequence on each side. According to our observations, transfection constructs carrying multiple sequences homologous to the mitochondrial DNA should be suitable and targeting of most regions in the organelle genome should be feasible, making the approach of general interest.

Figure 1.

Organization of the gene constructs used as substrates for mitochondrial import and in organello recombination assays. Details of cloning and assembly are given in ‘Materials and Methods’ section. (a) Construct DR-Zm/gfp was composed of nucleotides 3881–6927 of the maize mtDNA (accession AY506529) with nucleotides 98–535 of the gfp coding sequence inserted in antisense orientation into the central HindIII site. (b) Construct nad2-St/gfp was composed of the end of intron 3, exon 4, intron 4 and part of exon 5 of the potato mitochondrial nad2 gene with nucleotides 98–535 of the gfp coding sequence replacing in antisense orientation the central SphI fragment. The number of nucleotides in the different components is indicated.

Figure 2.

Specific association of the imported DNA with the main mitochondrial DNA. (a–c) Different [³²P]-labeled DNA substrates (nad2-St/gfp, DR-Zm/gfp, pBluescript and gfp) were imported into isolated maize (a), potato (b) or tobacco (c) mitochondria. The organelles were subsequently incubated in DNA synthesis buffer for 2 h. Nucleic acids were extracted and directly separated on native agarose gels that were transferred onto nylon membranes for autoradiography (Im + Pi). Migration of the mtDNA was confirmed through Southern blot hybridization (Hyb) with a probe for the cob gene (lanes 5, 10 and 15). ‘na’ stands for ‘not applicable’. (d) Following import of radiolabeled nad2-St/gfp construct into isolated tobacco mitochondria and incubation in DNA synthesis buffer, nucleic acids were extracted from the organelle sample and fractionated on native agarose gel. The high molecular weight mtDNA was recovered from the gel and digested with BamHI and ScaI. The digested DNA was in turn fractionated on native agarose gel and transferred onto a nylon membrane for autoradiography (Digest). Migration of import substrates and of reference fragments is indicated in the different panels.

Figure 3.

Stable association of the imported DNA with the main mitochondrial DNA. Following import of [³²P]-labeled nad2-St/gfp into isolated potato mitochondria and incubation in DNA synthesis buffer for 1 or 2 h, nucleic acids were extracted from the organelle samples and directly fractionated on native (a) or denaturing (b) agarose gel. Radioactivity was detected by autoradiography after transfer onto nylon membranes. Migration of the mtDNA, of import substrates and of reference fragments is indicated.

Figure 4.

Integration of the imported nad2-St/gfp construct into the potato mitochondrial genome. Radiolabeled nad2-St/gfp construct was imported into isolated potato mitochondria. Half of the suspension was kept at that stage, whereas the other half was incubated in DNA synthesis buffer for 1 h. Nucleic acids were extracted from both samples, digested with MseI and religated. The religation mixes were used for inverse PCR. Both samples yielded inverse PCR products (4), implying that recombination can start during the import step. The PCR products generated from the import/post-incubation sample (Im + Pi) were cloned and sequenced. Sequencing revealed the fragment expected from integration by homologous recombination [(5); full sequence in Supplementary Data and Supplementary Figure S4a]. Religation by-products were also detected in which mtDNA-derived small MseI fragments were inserted between the MseI sites considered for the inverse PCR strategy (6). (1) Organization of the target region in the potato mtDNA with the original MseI sites; (2) expected organization and MseI site distribution following recombination of the imported nad2-St/gfp construct with the target region in the mtDNA and integration of the gfp sequence; (3) MseI fragment expected from the recombined target region (1415 nt); (4) agarose gel analysis of the inverse PCR products obtained with nucleic acids from organelle samples collected after the import step (Im) or after import and incubation in DNA synthesis buffer (Im + Pi), migration of reference fragments is indicated; (5) content of the inverse PCR products (467 nt) representative for homologous recombination; and (6) content of religation by-products; d1, primer gfp_int3; r1, primer nad2St_inv.

Figure 5.

Integration of the imported nad2-St/gfp construct into the tobacco mitochondrial genome. (a) Following import of radiolabeled DR-Zm/gfp construct or nad2-St/gfp construct into isolated tobacco mitochondria and incubation in DNA synthesis buffer for 2 h, nucleic acids were extracted, digested with MseI and religated. The religation mixes were used for inverse PCR and the reaction products were analyzed on agarose gel; (DR) assay run with the DR-Zm/gfp construct, (nad2) assay run with the nad2-St/gfp construct; migration of reference fragments is indicated. (b) A similar assay as in (a) was run with the nad2-St/gfp construct and the inverse PCR products were cloned and sequenced. Sequencing revealed the fragment expected from integration by homologous recombination [(5); Supplementary Data and Supplementary Figure S4b]. (1) Organization of the target region in the tobacco mtDNA with the original MseI site; (2) expected organization and MseI site distribution following recombination of the imported nad2-St/gfp construct with the target region in the mtDNA and integration of the gfp sequence; (3) MseI fragment expected from the recombined target region (1384 nt); (4) agarose gel analysis of the inverse PCR products obtained after religation of the MseI digest ( + ); as a control, no inverse PCR products were obtained when omitting religation (–); migration of reference fragments is indicated; and (5) content of the inverse PCR products (284 nt) representative for homologous recombination; d1, primer gfp_int3; r2, primer nad2Nt_inv.

Figure 6.

Sequence analysis of the recombined region resulting from the integration of the imported nad2-St/gfp construct into the potato mitochondrial genome. Following import of radiolabeled nad2-St/gfp construct into isolated mitochondria and incubation in DNA synthesis buffer for 2 h, nucleic acids were extracted and used for direct PCR amplification of the 5′ and 3′ recombined regions flanking the integrated gfp sequence. The amplified products were cloned and sequenced. (a) analysis of the direct PCR products on agarose gel; position of the primers is indicated in (b), scheme (2); migration of reference fragments is indicated. (b) Integration of the nad2-St/gfp construct into the potato mtDNA; (1) organization of the nad2-St/gfp construct with the distribution of the sequence markers versus the potato mtDNA (C1–C5); (2) organization of the recombined region in the potato mtDNA showing the origin of the sequence markers, as determined experimentally; the 5′ part of the recombined region was amplified with primers d2 (ex2St) and r3 (gfp_int5); the 3′ part of the recombined region was amplified with primers d1 (gfp_int3) and r4 (ex3St); the two products were sequenced (full sequences in Supplementary Data and Supplementary Figure S5) and the sequences were assembled to yield the recombination product; (3) organization of the target region in the potato mtDNA with the distribution of the sequence markers versus the nad2-St/gfp construct (M1–M5). Distances from the borders of the gfp sequence or of the central mtDNA SphI fragment are indicated in number of nucleotides. (c) Details of the sequence marker motifs as positioned in (b); nucleotides differing between the nad2-St/gfp construct and the potato mtDNA are in underlined grey.

Figure 7.

Sequence analysis of the recombined region resulting from the integration of the imported nad2-St/gfp construct into the tobacco mitochondrial genome. Following import of radiolabeled nad2-St/gfp construct into isolated mitochondria and incubation in DNA synthesis buffer for 2 h, nucleic acids were extracted and used for direct PCR amplification of the entire recombined region. The obtained product served as a template to amplify independently by nested PCR the 5′ and 3′ recombined regions flanking the integrated gfp sequence. Nested PCR products were cloned and sequenced. (a) and (b) analysis of the direct PCR products on agarose gel; original amplification of the entire recombined region (a) and nested PCR for the 5′ and 3′ flanks (b); position of the primers is indicated in (c), scheme (2); migration of reference fragments is indicated. (c) Integration of the nad2-St/gfp construct into the tobacco mtDNA; (1) organization of the nad2-St/gfp construct with the distribution of the sequence markers versus the tobacco mtDNA (C6–C16); (2) organization of the recombined region in the tobacco mtDNA showing the origin of the sequence markers, as determined experimentally; marker 7 was of mixed origin; a fragment spanning the recombined region was first amplified with primers d3 (ex1Nt) and r5 (ex4Nt); the PCR product was subsequently used as a template for nested PCR, so as to amplify the 5′ part of the recombined region with primers d4 (ex2Nt) and r3 (gfp_int5) and the 3′ part of the recombined region with primers d1 (gfp_int3) and r6 (ex3Nt); the two nested PCR products were sequenced (full sequences in Supplementary Data and Supplementary Figure S6) and the sequences were assembled to yield the recombination product; (3) organization of the target region in the tobacco mtDNA with the distribution of the sequence markers versus the nad2-St/gfp construct (M6–M16). Distances from the borders of the gfp sequence or of the central mtDNA SphI fragment are indicated in number of nucleotides. (d) Details of the sequence marker motifs as positioned in (c); nucleotides differing between the nad2-St/gfp construct and the tobacco mtDNA are in underlined grey.