Plant materials
Tobacco (Nicotiana tabacum) ‘Samsun’ plants were grown in a greenhouse under controlled conditions with a 16-h photoperiod and a constant temperature of 25°C. ‘Samsun’ tobacco material and plasmids were provided by the Key Laboratory of Crop Molecular Breeding, Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining, Qinghai Province, China. All primers used in this study and listed in Supplemental Tab. 1) were synthesized by Sangon Bioengineering Co., Ltd (Shanghai, China).
Construction of the CRISPR/mtCas9 vector
The SpCas9 gene from the Streptococcus pyogenes type II CRISPR/Cas system was optimized according to the codon usage bias of the plant and the mitochondrial targeting sequence (cytochrome C oxidase subunit IV presequence from Saccharomyces cerevisiae, COX IV) was added. The designed target sequence (GCTTCAGCGGGAGCTGCTAT) was selected at the 5'-coding region of the tobacco typical mtatp9 gene by online software CRISPR-GE (https://skl.scau.edu.cn/targetdesign/). A complete single guide RNA (sgRNA) expression frame contained the 3' region of the ubiquitin small nuclear RNA (snRNA) promoter AtU3d and the target sgRNA sequence. The total sequence of the mtCas9 gene and the sgRNA expression frame was then synthesized by Genewiz Biotechnology Co., Ltd (Suzhou, China). The mito-Cas9 vector was constructed by ligating the synthesized sequence with linearized pCambia 1300 binary vector digested by HindⅢ and KpnⅠ.
Tobacco transformation
The vector was transferred into Agrobacterium tumefaciens strain LBA4401 competent cells (Biomed Gene Technology Co., Ltd, Beijing, China) by the freeze-thaw method. The positive clone identified by screening with PCR using the specific primers Cas9F and Cas9R (Supplemental Tab. 1) was cultured in liquid MG-medium containing 100 mg/L kanamycin and 100 mg/L rifampicin at 28℃ and 200 rpm for 48 h. When the OD600 value reached 0.8, genetic transformation of small, young tobacco leaves was carried out using the leaf disk transformation/regeneration method (Horsch et al., 1985). Firstly, the leaves were placed in the co-culture medium (Horsch et al., 1985) in the dark for 2 d, transferred to solid differentiation medium (Horsch et al., 1985) for 7 d, and then transferred to solid differentiation medium containing 30 mg/L hygromycin for 14 d. Shoots were transferred to rooting medium until they rooted, after which the plantlets were acclimated and transplanted into soil.
To identify transgenic tobacco plantlets, genomic DNA was extracted from leaves from regenerated tobacco plantlets using Genomic DNA Extraction Kit (Takara, Beijing, China) and PCR was performed using specific primers Cas9F and Cas9R (Supplemental Tab. 1). The PCR reaction volume was 20 μL, in which DNA template was 1 μL, 2×SanTaq PCR Mix 10 μL (Sangon, Shanghai, China), primers were each 1 μL, and ddH2O 7μL. PCR reaction conditions were pre-denaturation at 94℃ for 5 min, denaturation at 94℃ for 30 s, annealing at 58℃ for 30 s, extension at 72℃ for 1 min for 32 cycles, then a final extension at 72℃ for 10 min. An aliquot (3 μL) of the products was separated by electrophoresis on 1.0% agarose gel.
Staining of pollen activity by the TTC method
The viability of different tobacco pollens was determined by the 2, 3, 5-triphenyl tetrazolium chloride (TTC) viability assay. Mature pollens were placed onto glass slides and stained with 10 μl 2% (w/v) TTC solution (Solarbio, Beijing, China) at 35°C for 15min. The phenotypes of the pollen grains (stained = viable, unstained = non-viable) were observed and images were captured using an Olympus BX53 light microscope (Olympus Corp, Tokyo, Japan). The experiment was performed in three biological replicates, with five fields-of-view observed in each replicate.
Relative quantification of the mtDNA copy number in stamens
Total genomic DNA and mitochondrial DNA were extracted from stamens using Genomic DNA Extraction Kit (Takara, Beijing, China) for real-time quantitative PCR (qPCR). The quality of the genomic DNA was determined spectrophotometrically (NanoDrop 2000c; Thermo, Shanghai, China). The total genomic DNA of each sample was diluted to 40 ng/μL. Primers for mtatp9, mtatp1 and mtorfX (Supplemental Tab. 1) were annealed to mtDNA sequences at different distances from cleavage sites to monitor mtDNA copy number. The housekeeping gene α-Tubulin was co-amplified as a control for normalizing DNA templates (Supplemental Tab. 1). qPCR was performed using the 7500 Fast Real-Time PCR System (ABI, Carlsbad, USA). The reaction mixture (20 μl) contained 2 μl of DNA, 0.8 μl each of forward and reverse primers (from 10 μM stock), 10 μl TB green, 0.4 μl ROX (TB GreenTM Premix Ex TaqTM II; Takara, Tokyo, Japan), and 6 μl nuclease-free water. Each sample was analyzed with three biological replicates. The PCR program was set up with an initial denaturation step at 95°C for 3 min, followed by 40 cycles at 95°C for 20 s and 60°C for 1 min. The dissociation curve for checking the specificity of PCR production was acquired by adding a step at 95°C for 15 s. The relative copy number of target genes was calculated by the 2−ΔΔCt method (Livak and Schmittgen 2001). The expression patterns of the transcripts were plotted using Microsoft Excel 2007
High-throughput tracking of mutations
The proportion of mtatp9 alleles in stamens of wild-type and mutant tobacco were explored using the High-throughput Tracking of Mutations (Hi-TOM) method (Liu et al. 2019). Genomic DNA of stamens of wild-type and mutant tobacco was extracted and used as template for the first round of PCR amplification, using mtatp9-specific primers, TOMATP-F and TOMATP-R (carrying the bridging sequence; Supplemental Table 1). Then, the second round of PCR amplification was carried out with Hi-TOM Mix (Novogene, Tianjin, China). The second-round PCR reaction system (20 μL) consisted of Hi-TOM Mix 10 μL, first-round PCR product 1.0 μL, and ddH2O 9.0 μL. The PCR reaction procedure involved pre-denaturation at 94℃ for 5 min, denaturation at 94℃ for 30 s, annealing at 58℃ for 30 s, extension at 72℃ for 1 min for 32 cycles, then a final extension at 72℃ for 10 min. An aliquot (3 μL) of the products was separated by electrophoresis on 1.0% agarose gel. High-throughput sequencing of the second round of PCR amplification products was carried out by Novogene Co., Ltd (Tianjin, China). The results of sequencing were analyzed using the Hi-TOM online tool (https://www.hi-tom.net/hi-tom/).
RNA-Sequencing
The stamens of mutant and wild-type tobacco were selected to construct cDNA libraries. Six libraries, each with three biological replicates, were prepared and sequenced using an Illumina HiSeq 2000 system (Novogene, Tianjin, China). The original sequencing results were preprocessed before data assembly, and the low-quality sequences were removed to obtain high-quality sequences. High-quality sequencing data were assembled using Trinity, a short-read assembly program, to obtain reliable transcripts. Gene function was annotated using the following databases, Gene Ontology (GO) and Kyoto Encyclopedia of Gene and Genome (KEGG). Gene expression levels were calculated using fragments per kb per million reads (FPKM) values. Significantly enriched GO and KEGG terms were obtained using the R platform.
Expression levels of genes encoded by mtDNA
For cDNA synthesis, total RNA of stamens was extracted using an RNAprep Pure Plant Kit (Tiangen, Beijing, China). The quality of the RNA was determined spectrophotometrically (NanoDrop 2000c; Thermo, Shanghai, China), and 800 ng RNA was reverse-transcribed using a PrimeScript™ RT reagent Kit with gDNA Eraser (Takara, Tokyo, Japan). The ten-fold dilution of cDNAs was used as template for real-time quantitative PCR applications. Using the appropriate primers (Supplemental Tab. 1), mitochondrion-encoded genes of interest were amplified. The housekeeping gene α-Tubulin was co-amplified as a control for normalizing cDNA templates and qPCR was performed as described earlier. The expression levels of target genes were calculated by the 2−ΔΔCt method (Livak and Schmittgen 2001).
Effect of overexpression of atp9 on fertility recovery
The target region of mtatp9 gene was codon modified by overlapping extended PCR to generate synonymous mutations (dsatp9) so that the CRISPR/Cas9 ribonucleoprotein complexes could not cleave the dsatp9 gene sequence of the overexpressed vector. Moreover, the COX IV presequence which targeted mitochondria was added to the 5' end of the dsatp9 gene sequence to ensure that the dsATP9 protein could enter mitochondria smoothly. The overexpression vector of dsatp9 was constructed by linking the linearized vector pCambia2301-KY, digested by restriction endonucleases BamHI and SacI, with the fragment containing the COX IV presequence and dsatp9 by homologous recombination, using ClonExpress II One Step Cloning Kit (Vazyme Biotech Co., Ltd. Nanjing, China). The overexpression vector of dsatp9 was introduced into abortive stamen type tobacco leaves via the leaf disk transformation/regeneration method (Horsch et al., 1985). Regenerated tobacco plantlets were obtained using kanamycin as the selection agent.
Statistical analysis
The statistical analysis was performed with Tukey’s post hoc ANOVA for multiple comparisons and Student’s 𝑡-test for comparison of two groups. Statistical comparisons were made with PASW Statistics 18 (IBM SPSS, Chicago, USA). A value of p < 0.05 was considered to be statistically significant, and all values from a sample were expressed as the mean ± standard deviation. Each experiment was replicated three times independently.