Abstract
Genome editing has become more and more popular in animal and plant systems following the emergence of CRISPR/Cas9 technology. However, target sequence modification by CRISPR/Cas9 has not been reported in the plant mitochondrial genome, mtDNA. In plants, a type of male sterility known as cytoplasmic male sterility (CMS) has been associated with certain mitochondrial genes, but few genes have been confirmed by direct mitochondrial gene-targeted modifications. Here, the CMS-associated gene (mtatp9) in tobacco was cleaved using mitoCRISPR/Cas9 with a mitochondrial localization signal. The male-sterile mutant, with aborted stamens, exhibited only 70% of the mtDNA copy number of the wild type and exhibited an altered percentage of heteroplasmic mtatp9 alleles; otherwise, the seed setting rate of the mutant flowers was zero. Transcriptomic analyses showed that glycolysis, tricarboxylic acid cycle metabolism and the oxidative phosphorylation pathway, which are all related to aerobic respiration, were inhibited in stamens of the male-sterile gene-edited mutant. In addition, overexpression of the synonymous mutations dsmtatp9 could restore fertility to the male-sterile mutant. Our results strongly suggest that mutation of mtatp9 causes CMS and that mitoCRISPR/Cas9 can be used to modify the mitochondrial genome of plants.
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Data availability
The transcriptomic data has been successfully uploaded to CNGBdb (https://db.cngb.org/) (Submission ID: sub032099; BioProject ID: CNP0003097). All data generated or analyzed during this study are included within the article and its additional files.
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Acknowledgements
We are very thankful to Professor Liu Yaoguang from South China Agricultural University for providing Cas9 sequence used for mtCas9 synthesis.
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This research was financially supported by the QingHai Science and Technology Department (2021-ZJ-931).
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Conceptualization, Baolong Liu; data curation, Yun LI; investigation, Yanzi Chang and Yanyan Jiang; methodology, Baolong Liu; project administration, Baolong Liu and Yun LI; resources, Yongju Liu; software, Dong Cao; supervision, Baolong Liu and Yun LI; writing—original draft, Yun LI; writing—review and editing, Yun LI.
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Yanzi Chang and Baolong Liu contributed equally to this work.
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ESM 1:
Fig. S1. PCR detection of mtCas9 region in transgenic plants. 1 to 27 represent independent regenerated plants.. Fig. S2. Detection of mtCas9 in mitochondria of leaves by western immunoblotting. Fig. S3. Partial nucleotide sequence alignment of mtatp9 alleles containing 79G->A at PAM regain. Fig. S4. Principal component analysis in stamens between the WT and AST. WT: wild type; AST: abortive stamen type. Fig. S5. Heat map of gene expression in stamens between the WT and AST . Fig. S6. Volcano shows differentially expressed genes (DEGs) in stamens between the WT and AST. Fig. S7. The KEGG pathway analysis of DEGs in stamens between the wild type and abortive stamen type. Fig. S8. Structural genes in oxidative phosphorylation pathway with different expression levels in stamens between the wild type and abortive stamen type. Fig. S9. The plant and capsule of tobacco. Table S1. Primer sequences used in this research. Table S2. DEGs related to glycolysis and tricarboxylic acid cycle metabolism in stamens between the wild type and stamen abortive type. Table S3 BLAST score results for the target sequence of mtatp9 based on transcriptomic data.
ESM 2:
Excel S1. The Hi-tom analysis of mtatp9 in stamens of wild type and mutant tobacco with three biological replicates. Reads number: the reads number of the sequence in the high-throughput sequencing result; Left/Right variation type: the variation type of left/right reads, WT means the typical mtatp9 allele, D means delete, I means indel and SNP means the single base substitution; Left/Right variation: variation description of left/right reads; Left/Right reads seq: the sequences of left/right reads.
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Chang, Y., Liu, B., Jiang, Y. et al. Induce male sterility by CRISPR/Cas9-mediated mitochondrial genome editing in tobacco. Funct Integr Genomics 23, 205 (2023). https://doi.org/10.1007/s10142-023-01136-7
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DOI: https://doi.org/10.1007/s10142-023-01136-7