Journal: Nature plants

Article Title: Targeted introduction of heritable point mutations into the plant mitochondrial genome

doi: 10.1038/s41477-022-01108-y

Figure Lengend Snippet: ( a ) Binding sites of the three designed TALEN pairs in the nad9 CDS (coding sequence), and schematic view of the binary vectors used for plant transformation. Nucleotide positions refer to the start codon ( A TG=1). Scissors indicate the predicted TALEN cut sites, the BsrGI restriction site is boxed. LB/RB, T-DNA left/right borders; 35S, CaMV 35S promoter; mt PS, mitochondrial presequence (from the Arabidopsis IVD protein); HA, 3xHA-tag; TALE N-term, TALE N-terminus; Repeats, TALE DNA-binding units; FokI, endonuclease domain; OCS, octopine synthase terminator ( Agrobacterium ); FLAG, 3xFLAG-tag; NOS (octagon), nopaline synthase terminator ( Agrobacterium ); nptII (kan R ), neomycin phosphotransferase II encoding kanamycin resistance; NOS (arrow), nopaline synthase promoter ( Agrobacterium ); arrows, TALEN binding sites. ( b ) Detection of TALEN activity by Southern blot analysis. Predicted sizes of hybridizing restriction fragments and the location of the probe are depicted. Scissors mark the cut sites of the restriction enzymes PvuII, EcoRV and BsrGI, and of the pJF1006-encoded TALEN pair. BsrGI cuts 2 bp away from the predicted TALEN cut site. In addition to PvuII/EcoRV, BsrGI was included in a digestion reaction with wild-type DNA (WT + BsrGI; right) to simulate the TALEN cut. The numbers below the gel give the relative intensities of the TALEN cleavage products). Arrowheads mark the positions of the three expected signals (magenta: uncut, cyan: TALEN-cut). ( c ) PCR and RT-PCR assays to test for the presence of TALEN arms in the lines shown in panel (b). The binding sites of the PCR primers are shown in the upper panel (dashed lines: RT-PCR primers), the lower panels display the PCR results. A sequence from the β-TUBULIN gene was amplified as internal control. Arrowheads indicate the expected sizes for β-TUBULIN (black, 412 bp/303 bp for DNA/cDNA), the HA arm (cyan, 311 bp/210 bp) and the FLAG arm (magenta, 251 bp/212 bp), respectively. H 2 O, water control; M, DNA size marker. Only the FLAG-TALEN arm is present in Nt-JF1006-30. Pools of kanamycin-resistant T 1 seedlings obtained by selfing were used for nucleic acid extractions. See also . The experiments in (b) and (c) were performed once.

Article Snippet: The nad9 sequences in the T 0 plants were screened for mutations by PCR amplification with primers oJF271 and oJF272, followed by bulk Sanger sequencing of the amplification products (LGC Genomics).

Techniques: Binding Assay, Sequencing, Transformation Assay, Activity Assay, Southern Blot, Reverse Transcription Polymerase Chain Reaction, Amplification, Control, Marker

Journal: Nature plants

Article Title: Targeted introduction of heritable point mutations into the plant mitochondrial genome

doi: 10.1038/s41477-022-01108-y

Figure Lengend Snippet: ( a ) End-mapping strategy to verify that the TALENs cut at the predicted sites. Arrows indicate oligonucleotides or primer-binding sites. mtDNA cut by the TALENs in vivo is blunted by mung bean exonuclease in vitro , followed by adapter ligation and PCR amplification. Note that PCR 2 is a half-nested PCR using the products of PCR 1 as template. P, 5’ monophosphate group of oligonucleotides. ( b ) PCR results obtained from execution of the strategy depicted in panel (a). Arrows indicate the expected sizes of products derived from TALEN-cut mtDNA. Cyan arrow, expected size (270 bp) of the amplification product of upstream site PCR 1; magenta arrow, expected product size (350 bp) for downstream site PCR 1; green arrow, expected amplicon size (192 bp) for upstream site PCR 2; orange arrow, expected amplicon size (288 bp) for downstream site PCR 2; line-30, line Nt-JF1006-30; WT, wild-type control; H 2 O, water control; M, DNA size marker. One of two similar experiments with similar results is shown. ( c ) Sequencing chromatograms for the products of PCR 2. The chromatograms show the DNA sequence obtained from sequencing the PCR products marked by the green (upstream site) and orange (downstream site) arrows, respectively, in panel (b). For comparison, the sequences of the adapter oligonucleotide duplex and the nad9 TALEN target site are also given (boxed). Black arrows indicate the direction of the sequencing reactions.

Article Snippet: The nad9 sequences in the T 0 plants were screened for mutations by PCR amplification with primers oJF271 and oJF272, followed by bulk Sanger sequencing of the amplification products (LGC Genomics).

Techniques: TALENs, Binding Assay, In Vivo, In Vitro, Adapter Ligation, Amplification, Nested PCR, Derivative Assay, Control, Marker, Sequencing, Comparison

Journal: Nature plants

Article Title: Targeted introduction of heritable point mutations into the plant mitochondrial genome

doi: 10.1038/s41477-022-01108-y

Figure Lengend Snippet: ( a ) Extension of the analyses shown in to a larger set of plants. See legend to for details. ( b ) Analyses of the same plants as shown in (a) by amplification of full-length nad9 cDNA with primers oJF311 and oJF748 (yielding a product of 769 bp, indicated by the green arrowhead; left panel) for subsequent analysis by Sanger sequencing, and partial nad9 cDNA with primers oJF496 and oJF748 (465 bp, indicated by the green arrowhead; right panel) and full-length atp9 cDNA with primers oJF1145 and oJF748 (568 bp, purple arrowhead) for semiquantitative analysis of expression strength. Primers for first strand cDNA synthesis were oJF1113 ( nad9 ) and oJF1144 ( atp9 ). Sequencing analyses showed all full-length nad9 RT-PCR products to be fully edited and free of mutations. The loading scheme is identical for all five gels. For primer sequences, see Extended Data Table 2. The experiments shown in (a) and (b) were performed once.

Article Snippet: The nad9 sequences in the T 0 plants were screened for mutations by PCR amplification with primers oJF271 and oJF272, followed by bulk Sanger sequencing of the amplification products (LGC Genomics).

Techniques: Amplification, Sequencing, Expressing, cDNA Synthesis, Reverse Transcription Polymerase Chain Reaction

Journal: Nature plants

Article Title: Targeted introduction of heritable point mutations into the plant mitochondrial genome

doi: 10.1038/s41477-022-01108-y

Figure Lengend Snippet: ( a ) Schematic overview of the experimental procedures. Transgenic plants with confirmed TALEN activity (cf. ) were raised from seeds, leaves were harvested, cut into pieces and placed onto shoot induction medium. Leaves from regenerated shoots were genotyped for mutations in nad9 (‘Discovery of mutation’). If no mutation was detected, a new regeneration round was initiated. When a point mutation was found, an additional regeneration round was conducted to promote genome segregation and facilitate isolation of homochondriomic lines (‘Purification of mutation’). Homochondriomic mutant shoots were rooted and transferred to the greenhouse for seed production. In some experiments, the mutagens (M) ethidium bromide or N-nitroso-N-ethylurea were added to the shoot induction medium or applied during seed imbibition. See text, Methods and for details. ( b ) Exemplary sequencing chromatograms showing the successful isolation of a homochondriomic nad9 mutant carrying a Ser-to-Gly exchange in amino acid position 91 of the Nad9 protein (corresponding to an A-to-G substitution in nucleotide position 271 of the nad9 reading frame). The mutated position is indicated by arrowheads in the sequencing chromatograms of the S91G-1 mutant line and the wild type. The sequencing primer was oJF271 .

Article Snippet: The nad9 sequences in the T 0 plants were screened for mutations by PCR amplification with primers oJF271 and oJF272, followed by bulk Sanger sequencing of the amplification products (LGC Genomics).

Techniques: Transgenic Assay, Activity Assay, Mutagenesis, Isolation, Purification, Sequencing

Journal: Nature plants

Article Title: Targeted introduction of heritable point mutations into the plant mitochondrial genome

doi: 10.1038/s41477-022-01108-y

Figure Lengend Snippet: ( a ) Genotyping of an S91N-1 mutant plant carrying the G272A mutation. Sequencing of PCR products amplified from total plant DNA (with primers oJF271 and oJF272; ) suggested persistent heteroplasmy (i.e., presence of residual copies of wild-type nad9 alleles), as evidenced by an A+G double peak (arrowhead, left sequence). However, when the same PCR product was generated from purified mtDNA as template, a clean single A peak is seen (right sequence). Sequencing was done with primer oJF271. ( b ) Overview of all TALEN-induced mutations obtained in nad9 . Nucleotide positions in the nad9 coding sequence ( A TG = 1) and amino acid positions in the Nad9 protein are given. Block arrows denote the pJF1006 TALEN binding sites. The ‘left’ (HA-)TALEN (not present in line Nt-JF1006-30) extends from nucleotide position 237 to position 255, the ‘right’ (FLAG-)TALEN from position 268 to 286. Scissors point to the predicted TALEN cut site. ( c ) Analysis of TALEN cleavage activity by Southern blotting using pools of kanamycin-resistant seedlings (from back-crosses of the original mutants with the wild type). The same restriction enzymes and hybridization probe as in were used, but electrophoretic separation was done in a 2% agarose gel. To determine relative cutting efficiencies, the percentage of cut nad9 was calculated for each lane and then divided by the respective value for control line 1 (contr. 1). The smaller of the two nad9 cleavage products was used for quantification, because it is covered by a larger portion of the hybridization probe. Contr. 1 and contr. 2, descendants of Nt-JF1006 plants having gone through the same mutagenesis procedure as the point mutants, but retaining a wild-type nad9 sequence. Line D93E/E94K-1 is represented twice (descendants of two different vegetative clones of the original mutant plant). The full Southern blot with enhanced contrast settings is presented in . In contrast to all other mutants, line R85L-1 may not be the result of a gene drive effect. A technical replicate of the Southern blot yielded similar results.

Article Snippet: The nad9 sequences in the T 0 plants were screened for mutations by PCR amplification with primers oJF271 and oJF272, followed by bulk Sanger sequencing of the amplification products (LGC Genomics).

Techniques: Mutagenesis, Sequencing, Amplification, Generated, Purification, Blocking Assay, Binding Assay, Activity Assay, Southern Blot, Hybridization, Agarose Gel Electrophoresis, Control, Clone Assay

Journal: Nature plants

Article Title: Targeted introduction of heritable point mutations into the plant mitochondrial genome

doi: 10.1038/s41477-022-01108-y

Figure Lengend Snippet: The mitochondrial genomes of mutant lines D93N-1, E94K-1 and S91N-1 were sequenced. Nucleotide positions [bp] on the x-axis are according to the tobacco mitochondrial reference genome sequence (NC_006581). The y-axis shows the number of reads per position. Upper panel: D93N-1 (black), middle panel: E94K-1 (blue), lower panel: S91N-1 (red). The arrow indicates the position of nad9 .

Article Snippet: The nad9 sequences in the T 0 plants were screened for mutations by PCR amplification with primers oJF271 and oJF272, followed by bulk Sanger sequencing of the amplification products (LGC Genomics).

Techniques: Mutagenesis, Sequencing

Journal: Nature plants

Article Title: Targeted introduction of heritable point mutations into the plant mitochondrial genome

doi: 10.1038/s41477-022-01108-y

Figure Lengend Snippet: The highly conserved glutamate residue 94 is highlighted in red. Asterisks (*) indicate perfect conservation, colons (:) denote residues belonging to a group of amino acids exhibiting strong similarity, and dots (.) mark residues belonging to a group of amino acids exhibiting weak similarity. For the tobacco Nad9 protein, the (unedited) genomic sequence was used.

Article Snippet: The nad9 sequences in the T 0 plants were screened for mutations by PCR amplification with primers oJF271 and oJF272, followed by bulk Sanger sequencing of the amplification products (LGC Genomics).

Techniques: Residue, Sequencing