High-efficiency biolistic transformation of Chlamydomonas mitochondria can be used to insert mutations in complex I genes

Abstract

Mitochondrial transformation of Chlamydomonas reinhardtii has been optimized by using a particle-gun device and cloned mitochondrial DNA or PCR fragments. A respiratory-deficient strain lacking a 1.2-kb mitochondrial DNA region including the left telomere and part of the cob gene could be rescued as well as a double-frameshift mutant in the mitochondrial cox1 and nd1 genes. High transformation efficiency has been achieved (100-250 transformants per microgram of DNA), the best results being obtained with linearized plasmid DNA. Molecular analysis of the transformants suggests that the right telomere sequence can be copied to reconstruct the left telomere by recombination. In addition, both nondeleterious and deleterious mutations could be introduced. Myxothiazol-resistant transformants have been created by introducing a nucleotide substitution into the cob gene. Similarly, an in-frame deletion of 23 codons has been created in the nd4 mitochondrial gene of both the deleted and frameshift recipient strains. These 23 codons are believed to encode the first transmembrane segment of the ND4 protein. This Deltand4 mutation causes a misassembly of complex I, with the accumulation of a subcomplex that is 250-kDa smaller than the wild-type complex I. The availability of efficient mitochondrial transformation in Chlamydomonas provides an invaluable tool for the study of mitochondrial biogenesis and, more specifically, for site-directed mutagenesis of mitochondrially encoded subunits of complex I, of special interest because the yeast Saccharomyces cerevisiae, whose mitochondrial genome can be manipulated virtually at will, is lacking complex I.

Fig. 1.

Partial physical map of the 15.8-kb mitochondrial genome of C. reinhardtii. The rectangles represent protein-coding genes: cob, gene encoding apocytochrome b of complex III; nd1, 2, 4, 5, and 6, genes encoding the corresponding subunits of complex I; cox1, gene encoding the subunit 1 of complex IV, rtl: reverse transcriptase-like protein. The inverted telomeric ends are represented by short arrows, the bidirectional origin of transcription between nd5 and cox1 by longer arrows. Only restriction sites used in this work are presented: Nc, NcoI; C, ClaI; N, NdeI; S, SstI. Mu, resistance to myxothiazol. Positions of the dum11 and dum22 deletions and of the dum19 and dum25 point mutations are indicated. Fragments of mitochondrial genome contained in pShort, pShortΔ, pLong, and pLongΔ are shown as well as the primers used for PCR amplifications. Using the GenBank u03843 numbering for the Chlamydomonas mitochondrial DNA, primers positions are Tel, 1–21; cob-F, 431–450; cob-R, 564–545; nd4-F, 2765–2780; nd4-R, 3301–3282; cox1, 6653–6634; nd2-F, 6636–6655; and nd2-R, 7343–7323.

Fig. 2.

Southern blot analysis of mitochondrial transformants using dum11 as recipient strain. (A) TTC⁺ (T1 to T6) and TTC⁻ (T7 to T12) transformants isolated after transformation by using pShort (T1, T2, T3, T7, T8, and T9) or a Tel/cox1 PCR fragment (T4, T5, T6, T10, T11, and T12). Total undigested DNA was probed with a nd2 PCR fragment amplified with primers nd2-F/R (Fig. 1). Wild type (WT) and dum11 mutant are shown as controls. Equal amounts (10 μg) of each total DNA preparation were loaded per lane. As shown, dum11 and the TTC⁻ transformants repeatedly gave lower signal intensities, reflecting the fact that deletion mutant strains contain less mitochondrial DNA (17). (B) TTC⁺ transformants (T13 to T16) isolated after transformation by using a cob-F/cox1 PCR fragment (T13, T14) or a Tel/cox1 PCR fragment (T15, T16). Total DNA digested with NcoI was hybridized with a probe covering the telomeric region (primers Tel/cob-R). The WT and the dum11 mutant are shown as controls. In this case, the dum11 lane has been overloaded to ensure the correct detection of the fragments.

Fig. 3.

Detection of the Δnd4 mutation by PCR amplification. PCR on 12 transformants (D1 to D12) was performed by using primers nd4-F and nd4-R. D2 and D6 present the 69-bp NdeI deletion in nd4 at the heteroplasmic (D2) and homoplasmic (D6) state. The 100-bp ladder (Eurogentec, Liège, Belgium) is shown at the right.

Fig. 4.

Analysis of mitochondrial NADH dehydrogenases from Δnd4 transformants after BN/PAGE. (A) Membrane extracts (120 μg of protein) of wild type, dum19 dum25, dum22, and two transformants. D6 carries the Δnd4 mutation, and D34 harbors both the Δnd4 and dum25 mutations. The NADH/NBT staining revealed purple bands corresponding to complexes at 950 and 700 kDa. The green 600-kDa complex, which is not detected by the staining, is shown as a loading control and probably corresponds to Photosystem I associated to light-harvesting complex (20). (B) The gel was blotted and probed with an antiserum against the N. crassa whole complex I.