Curing cytoplasmic male sterility via TALEN-mediated mitochondrial genome editing

Abstract

Sequence-specific nucleases are commonly used to modify the nuclear genome of plants. However, targeted modification of the mitochondrial genome of land plants has not yet been achieved. In plants, a type of male sterility called cytoplasmic male sterility (CMS) has been attributed to certain mitochondrial genes, but none of these genes has been validated by direct mitochondrial gene-targeted modification. Here, we knocked out CMS-associated genes (orf79 and orf125) of CMS varieties of rice and rapeseed, respectively, using transcription activator-like effector nucleases (TALENs) with mitochondria localization signals (mitoTALENs). We demonstrate that knocking out these genes cures male sterility, strongly suggesting that these genes are causes of CMS. Sequencing revealed that double-strand breaks induced by mitoTALENs were repaired by homologous recombination, and that during this process, the target genes and surrounding sequences were deleted. Our results show that mitoTALENs can be used to stably and heritably modify the mitochondrial genome in plants.

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Fig. 1: MitoTALEN transformation to knock out orf79 in rice and recover male fertility.
Fig. 2: Modification of the genomic region around orf79.
Fig. 3: MitoTALEN transformation to knock out orf125 in rapeseed and recover male fertility.
Fig. 4: Illumina sequencing analysis of mitoTALEN-induced disappearances and recombination in the rapeseed mitochondrial genome.
Fig. 5: PCR analyses of the T1 and F1 populations of rapeseed TAL2-2.
Fig. 6: Changes in the mitochondrial genome of mito-cTAL7-3.
Fig. 7: Changes in the mitochondrial genome of TAL2-5.

Data availability

All data are available in the main text or the Supplementary Information.

References

  1. 1.

    Altpeter, F. et al. Advancing crop transformation in the era of genome editing. Plant Cell 28, 1510–1520 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. 2.

    Bock, R. Engineering plastid genomes: methods, tools, and applications in basic research and biotechnology. Annu. Rev. Plant Biol. 66, 211–241 (2015).

    CAS  Article  Google Scholar 

  3. 3.

    Gualberto, J. M. & Newton, K. J. Plant mitochondrial genomes: dynamics and mechanisms of mutation. Annu. Rev. Plant Biol. 68, 225–252 (2017).

    CAS  Article  Google Scholar 

  4. 4.

    Knoop, V. Plant mitochondrial genome peculiarities evolving in the earliest vascular plant lineages. J. Syst. Evol. 51, 1–12 (2013).

    Article  Google Scholar 

  5. 5.

    Kubo, T. & Newton, K. J. Angiosperm mitochondrial genomes and mutations. Mitochondrion 8, 5–14 (2008).

    CAS  Article  Google Scholar 

  6. 6.

    Beurdeley, M. et al. Compact designer TALENs for efficient genome engineering. Nat. Commun. 4, 1762 (2013).

    Article  Google Scholar 

  7. 7.

    Bacman, S. R., Williams, S. L., Pinto, M., Peralta, S. & Moraes, C. T. Specific elimination of mutant mitochondrial genomes in patient-derived cells by mitoTALENs. Nat. Med. 19, 1111–1113 (2013).

    CAS  Article  Google Scholar 

  8. 8.

    Reddy, P. et al. Selective elimination of mitochondrial mutations in the germline by genome editing. Cell 161, 459–469 (2015).

    CAS  Article  Google Scholar 

  9. 9.

    Hanson, M. R. & Bentolila, S. Interactions of mitochondrial and nuclear genes that affect male gametophyte development. Plant Cell 16, S154–S169 (2004).

    CAS  Article  Google Scholar 

  10. 10.

    Akagi, H., Sakamoto, M., Shinjyo, C., Shimada, H. & Fujimura, T. A unique sequence located downstream from the rice mitochondrial atp6 may cause male-sterility. Curr. Genet. 25, 52–58 (1994).

    CAS  Article  Google Scholar 

  11. 11.

    Kazama, T., Nakamura, T., Watanabe, M., Sugita, M. & Toriyama, K. Suppression mechanism of mitochondrial ORF79 accumulation by Rf1 protein in BT-type cytoplasmic male sterile rice. Plant J. 55, 619–628 (2008).

    CAS  Article  Google Scholar 

  12. 12.

    Grelon, M., Budar, F., Bonhomme, S. & Pelletier, G. Ogura cytoplasmic male-sterility (CMS)-associated orf138 is translated into a mitochondrial membrane polypeptide in male-sterile Brassica cybrids. Mol. Gen. Genet. 243, 540–547 (1994).

    CAS  Article  Google Scholar 

  13. 13.

    Iwabuchi, M., Koizuka, N., Fujimoto, H., Sakai, T. & Imamura, J. Identification and expression of the kosena radish (Raphanus sativus cv. Kosena) homologue of the ogura radish CMS-associated gene, orf138. Plant Mol. Biol. 39, 183–188 (1999).

    CAS  Article  Google Scholar 

  14. 14.

    Kazama, T. & Toriyama, K. Whole mitochondrial genome sequencing and re-examination of a cytoplasmic male sterility-associated gene in Boro-Taichung-type cytoplasmic male sterile rice. PloS ONE 11, e0159379 (2016).

    Article  Google Scholar 

  15. 15.

    Iwabuchi, M., Kyozuka, J. & Shimamoto, K. Processing followed by complete editing of an altered mitochondrial atp6 RNA restores fertility of cytoplasmic male sterile rice. EMBO J. 12, 1437–1446 (1993).

    CAS  Article  Google Scholar 

  16. 16.

    Wang, Z. et al. Fusion primer and nested integrated PCR (FPNI-PCR): a new high-efficiency strategy for rapid chromosome walking or flanking sequence cloning. BMC Biotechnol. 11, 109 (2011).

    Article  Google Scholar 

  17. 17.

    Sakai, T. & Imamura, J. Alteration of mitochondrial genomes containing atpA genes in the sexual progeny of cybrids between Raphanus sativus CMS line and Brassica napus cv. Westar. Theor. Appl. Genet. 84, 923–929 (1992).

    CAS  Article  Google Scholar 

  18. 18.

    Arimura, S. I., Yanase, S., Tsutsumi, N. & Koizuka, N. The mitochondrial genome of an asymmetrically cell-fused rapeseed, Brassica napus, containing a radish-derived cytoplasmic male sterility-associated gene. Genes Genet. Syst. 93, 143–148 (2018).

    Article  Google Scholar 

  19. 19.

    Koizuka, N. et al. Genetic characterization of a pentatricopeptide repeat protein gene, orf687, that restores fertility in the cytoplasmic male-sterile Kosena radish. Plant J. 34, 407–415 (2003).

    CAS  Article  Google Scholar 

  20. 20.

    Sakai, T. & Imamura, J. Intergeneric transfer of cytoplasmic male sterility between Raphanus sativus (CMS line) and Brassica napus through cytoplast–protoplast fusion. Theor. Appl. Genet. 80, 421–427 (1990).

    CAS  Article  Google Scholar 

  21. 21.

    Ogura, H. Studies on the new male sterility in Japanese radish, with special references to the utilization of this sterility towards the practical raising of hybrid seeds. Mem. Fac. Agric. Kagoshima Univ. 6, 39–78 (1968).

    Google Scholar 

  22. 22.

    He, S., Abad, A. R., Gelvin, S. B. & Mackenzie, S. A. A cytoplasmic male sterility-associated mitochondrial protein causes pollen disruption in transgenic tobacco. Proc. Natl Acad. Sci. USA 93, 11763–11768 (1996).

    CAS  Article  Google Scholar 

  23. 23.

    Wang, Z. et al. Cytoplasmic male sterility of rice with boro II cytoplasm is caused by a cytotoxic peptide and is restored by two related PPR motif genes via distinct modes of mRNA silencing. Plant Cell 18, 676–687 (2006).

    CAS  Article  Google Scholar 

  24. 24.

    Luo, D. et al. A detrimental mitochondrial-nuclear interaction causes cytoplasmic male sterility in rice. Nat. Genet. 45, 573–577 (2013).

    CAS  Article  Google Scholar 

  25. 25.

    Bonhomme, S. et al. Sequence and transcript analysis of the Nco2.5 Ogura-specific fragment correlated with cytoplasmic male sterility in Brassica cybrids. Mol. Gen. Genet. 235, 340–348 (1992).

    CAS  Article  Google Scholar 

  26. 26.

    Voytas, D. F. Plant genome engineering with sequence-specific nucleases. Annu. Rev. Plant Biol. 64, 327–350 (2013).

    CAS  Article  Google Scholar 

  27. 27.

    Gammage, P. A. et al. Genome editing in mitochondria corrects a pathogenic mtDNA mutation in vivo. Nat. Med. 24, 1691–1695 (2018).

    CAS  Article  Google Scholar 

  28. 28.

    Kohl, S. & Bock, R. Transposition of a bacterial insertion sequence in chloroplasts. Plant J. 58, 423–436 (2009).

    CAS  Article  Google Scholar 

  29. 29.

    Kanazawa, A., Tsutsumi, N. & Hirai, A. Reversible changes in the composition of the population of mtDNAs during dedifferentiation and regeneration in tobacco. Genetics 138, 865–870 (1994).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Janska, H., Sarria, R., Woloszynska, M., Arrieta-Montiel, M. & Mackenzie, S. A. Stoichiometric shifts in the common bean mitochondrial genome leading to male sterility and spontaneous reversion to fertility. Plant Cell 10, 1163–1180 (1998).

    CAS  Article  Google Scholar 

  31. 31.

    Small, I., Suffolk, R. & Leaver, C. J. Evolution of plant mitochondrial genomes via substoichiometric intermediates. Cell 58, 69–76 (1989).

    CAS  Article  Google Scholar 

  32. 32.

    Val, R. et al. Organelle trafficking of chimeric ribozymes and genetic manipulation of mitochondria. Nucleic Acids Res. 39, 9262–9274 (2011).

    CAS  Article  Google Scholar 

  33. 33.

    Sultan, L. D. et al. The reverse transcriptase/RNA maturase protein MatR is required for the splicing of various group II introns in Brassicaceae mitochondria. Plant Cell 28, 2805–2829 (2016).

    CAS  Article  Google Scholar 

  34. 34.

    Colas des Francs-Small, C., Vincis Pereira Sanglard, L. & Small, I. Targeted cleavage of nad6 mRNA induced by a modified pentatricopeptide repeat protein in plant mitochondria. Commun. Biol. 1, 166 (2018).

    Article  Google Scholar 

  35. 35.

    Kazama, T., Itabashi, E., Fujii, S., Nakamura, T. & Toriyama, K. Mitochondrial ORF79 levels determine pollen abortion in cytoplasmic male sterile rice. Plant J. 85, 707–716 (2016).

    CAS  Article  Google Scholar 

  36. 36.

    Kleinstiver, B. P. et al. The I-TevI nuclease and linker domains contribute to the specificity of monomeric TALENs. Genes Genom. Genet. 4, 1155–1165 (2014).

    CAS  Google Scholar 

  37. 37.

    Doyle, E. L. et al. TAL effector-nucleotide targeter (TALE-NT) 2.0: tools for TAL effector design and target prediction. Nucleic Acids Res. 40, W117–W122 (2012).

    CAS  Article  Google Scholar 

  38. 38.

    Sakuma, T. et al. Repeating pattern of non-RVD variations in DNA-binding modules enhances TALEN activity. Sci. Rep. 3, 3379 (2013).

    Article  Google Scholar 

  39. 39.

    Arimura, S. & Tsutsumi, N. A dynamin-like protein (ADL2b), rather than FtsZ, is involved in Arabidopsis mitochondrial division. Proc. Natl Acad. Sci. USA 99, 5727–5731 (2002).

    CAS  Article  Google Scholar 

  40. 40.

    Karimi, M., Inze, D. & Depicker, A. GATEWAYTM vectors for Agrobacterium-mediated plant transformation. Trends Plant Sci. 7, 193–195 (2002).

    CAS  Article  Google Scholar 

  41. 41.

    Nagaya, S., Kawamura, K., Shinmyo, A. & Kato, K. The HSP terminator of Arabidopsis thaliana increases gene expression in plant cells. Plant Cell Physiol. 51, 328–332 (2010).

    CAS  Article  Google Scholar 

  42. 42.

    Kohno-Murase, J., Murase, M., Ichikawa, H. & Imamura, J. Effects of an antisense napin gene on seed storage compounds in transgenic Brassica napus seeds. Plant Mol. Biol. 26, 1115–1124 (1994).

    CAS  Article  Google Scholar 

  43. 43.

    Kent, W. J. BLAT–the BLAST-like alignment tool. Genome Res. 12, 656–664 (2002).

    CAS  Article  Google Scholar 

  44. 44.

    Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics 25, 1754–1760 (2009).

    CAS  Article  Google Scholar 

  45. 45.

    Krzywinski, M. et al. Circos: an information aesthetic for comparative genomics. Genome Res. 19, 1639–1645 (2009).

    CAS  Article  Google Scholar 

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Acknowledgements

We thank M. Ito (Tohoku University) for technical assistance in the mito-cTAL transformation and I. Small (University of Western Australia) for his careful reading of the manuscript and points raised. This research was partly supported by grants from the Japanese Science and Technology Agency (PRESTO to S.-i.A.) and the Japan Society for the Promotion of Science (grant number 24248001 to N.T., and 16H06182, 17K19256 and 18H02172 to T.K., and 16H06279, 18H0431 and 18K19202 to S.-i.A.).

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S.-i.A., T.K., K.T., N.K. and N.T. initiated and designed the project. Y.W. and S.Y. constructed the vectors. T.K. performed rice experiments. N.K. and C.K. performed rapeseed transformations, crossing and DNA isolations. S.Y., H.S. and Y.T. implemented rapeseed PCR analyses. M.O., A.T., T.I. and S.-i.A. analysed NGS data. T.K., N.K., K.T. and S.-i.A. wrote this manuscript.

Corresponding authors

Correspondence to Tomohiko Kazama or Nobuya Koizuka or Shin-ichi Arimura.

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A patent for the method described in this paper is pending in Japan (application no. 2017-024923) and the USA (application no. 15895118), entitled ‘Method for editing plant mitochondrial genome’.

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Kazama, T., Okuno, M., Watari, Y. et al. Curing cytoplasmic male sterility via TALEN-mediated mitochondrial genome editing. Nat. Plants 5, 722–730 (2019). https://doi.org/10.1038/s41477-019-0459-z

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