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

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Competing interests

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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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.).

Author information

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.

Competing interests

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’.

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

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Supplementary Figs. 1–10 and Supplementary Tables 1–3.

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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.