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. 2023 Sep 15:14:1253640.
doi: 10.3389/fpls.2023.1253640. eCollection 2023.

Cytoplasmic male sterility and abortive seed traits generated through mitochondrial genome editing coupled with allotopic expression of atp1 in tobacco

Ralph E Dewey  1 , Devarshi Selote  1 , H Carol Griffin  1 , Allison N Dickey  2 , Derek Jantz  3 , J Jeff Smith  3 , Anna Matthiadis  4 , Josh Strable  5 , Caitlin Kestell  1 , William A Smith  1
Affiliations

Cytoplasmic male sterility and abortive seed traits generated through mitochondrial genome editing coupled with allotopic expression of atp1 in tobacco

Ralph E Dewey et al. Front Plant Sci. .

Abstract

Allotopic expression is the term given for the deliberate relocation of gene function from an organellar genome to the nuclear genome. We hypothesized that the allotopic expression of an essential mitochondrial gene using a promoter that expressed efficiently in all cell types except those responsible for male reproduction would yield a cytoplasmic male sterility (CMS) phenotype once the endogenous mitochondrial gene was inactivated via genome editing. To test this, we repurposed the mitochondrially encoded atp1 gene of tobacco to function in the nucleus under the transcriptional control of a CaMV 35S promoter (construct 35S:nATP1), a promoter that has been shown to be minimally expressed in early stages of anther development. The endogenous atp1 gene was eliminated (Δatp1) from 35S:nATP1 tobacco plants using custom-designed meganucleases directed to the mitochondria. Vegetative growth of most 35S:nATP1/Δatp1 plants appeared normal, but upon flowering produced malformed anthers that failed to shed pollen. When 35S:nATP1/Δatp1 plants were cross-pollinated, ovary/capsule development appeared normal, but the vast majority of the resultant seeds were small, largely hollow and failed to germinate, a phenotype akin to the seedless trait known as stenospermocarpy. Characterization of the mitochondrial genomes from three independent Δatp1 events suggested that spontaneous recombination over regions of microhomology and substoichiometric shifting were the mechanisms responsible for atp1 elimination and genome rearrangement in response to exposure to the atp1-targeting meganucleases. Should the results reported here in tobacco prove to be translatable to other crop species, then multiple applications of allotopic expression of an essential mitochondrial gene followed by its elimination through genome editing can be envisaged. Depending on the promoter(s) used to drive the allotopic gene, this technology may have potential application in the areas of: (1) CMS trait development for use in hybrid seed production; (2) seedless fruit production; and (3) transgene containment.

Keywords: allotopic expression; atp1; custom-designed meganucleases; hybrid seed systems; mitoarcus; stenospermocarpy; substoichiometric shifting; transgene containment.

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Conflict of interest statement

RD, DJ, JJS, and AM are named on a joint patent application Patent Applicants: North Carolina State University, Elo Life Systems, and Precision BioSciences; application number – PCT/US2022/025965. Specific aspects of manuscript covered in patent application: CMS trait, hybrid seed system description, seedless fruit production technology, ARCUS nuclease sequences, and compositions for targeting ARCUS enzymes to plant mitochondria. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Mitochondrial genome editing of atp1 in 35S:nATP1 transformed plants. (A) 35S:nATP1 construct. LB, left border; RB, right border; 35S pro, CaMV 35S promoter; TP, transit peptide; UTR, untranslated region; Ter, terminator; HPTII, hygromycin phosphotransferase II. (B) atp1-targeting vector SP3379 containing mitoARCUS cassettes ATP-7/8 and ATP-5/6. FSgt-PFlt pro, Figwort Mosaic Virus – Peanut Chlorotic Streak Caulimovirus chimeric promoter; MMV pro, Mirabilis Mosaic Virus promoter; NPTII, neomycin phosphotransferase II. (C) Tobacco atp1 gene. MitoARCUS cleavage sites are indicated with thunderbolts. Nucleotide positions are in accordance to the tobacco mitochondrial reference genome BA000042. (D) PCR analysis of individual T0 plants transformed with SP3379. Each reaction contained primers designed to simultaneously amplify atp1, and atp6 as a control. M, molecular size markers; WT, wild type K326 tobacco; Neg, negative control lacking template DNA. (E) T0 35S:nATP1 plants lacking a functional mitochondrial atp1 gene (Δatp1) six weeks after transplanting to soil. Phenotypically normal plants are shown in the upper panel; individuals with aberrant leaf or growth phenotypes are shown in the lower panel. K326 EV#13, empty vector control plant.
Figure 2
Figure 2
35S:nATP1/Δatp1 plants have abnormal flowers, misshapen anthers, and fail to produce pollen. (A) Flower development in K326 WT and 35S:nATP1/Δatp1#22 (Δatp1) individuals at Stages 1, 5, 10 and 12 (as defined in Koltunow et al., 1990). (B) Anthers and stigmas at Stages 10 and 12 in K326 WT and 35S:nATP1/Δatp1#22 mutant plants. (C) Staining of Stage 10 anthers from K326 WT and 35S:nATP1/Δatp1#22 individuals. For each genotype, the panel on the left shows the contents expelled upon firmly pressing the stained anthers on a microscope slide with a cover slip. Viable pollen grains are stained purple. Panels on the right show a cross section of the anthers after pollen expulsion. (D) Histological analysis of Stage 1, Stage 5 and Stage 10 anthers. C, connective tissue; E, epidermis; En, endothecium; Msp, microspores; PS, pollen sac; St, stomium; Spt, septum; Th, theca; V, vascular bundle. Scale bars are indicated.
Figure 3
Figure 3
Seed development is aborted in cross-fertilized 35S:nATP1/Δatp1 plants. (A) Capsules fail to develop in unfertilized 35S:nATP1/Δatp1#8 (Δatp1#8) and 35S:nATP1/Δatp1#22 (Δatp1#22) flowers, but appear normal when crossed to a 35S:nATP1 pollen parent. (B) Seeds produced from X 35S:nATP1/Δatp1 X 35S:nATP1 crosses are smaller and lighter in color than EV control tobacco seeds. (C) Hundred-seed weights of 35S:nATP1/Δatp1#1, #8 and #22 plants fertilized with 35S:nATP1 or WT pollen. (D) 35S:nATP1/Δatp1 X 35S:nATP1 seeds are translucent and fail to germinate on solid MS media. DAP, days-after-plating. (E) Stained histological sections of developing WT and 35S:nATP1/Δatp1 X 35S:nATP1 seeds at 14 and 21 days-after-fertilization (DAF). Em, embryo; En, endosperm; SC, seed coat. Scale bars are indicated.
Figure 4
Figure 4
Long read sequence coverage of the mitochondrial genomes of 35S:nATP1/Δatp1#8 (A), #16 (B), and #22 (C). x-axis, reference genome BA000042; y-axis, PacBio sequence coverage after removal of 33% clipped reads. (D) Position and extent of sequences missing in Δatp1 mutants. Known functional genes cox3, atp1 and trnM are highlighted in green; uncharacterized ORFs greater than 100 codons in length are highlighted blue.
Figure 5
Figure 5
Predicted recombination and SSS events leading to the elimination of atp1 in 35S:nATP1/Δatp1 plants. (A) Linear representation of the tobacco mitochondrial reference genome BA000042 showing the locations of atp1, atp6 and the three long direct repeats Rep1, Rep2 and Rep3. Junctions 5’ and 3’ of atp1 derived via recombination or SSS are shown for T0 events 35S:nATP1/Δatp1#8 (B), 35S:nATP1/Δatp1#16 (C), and 35S:nATP1/Δatp1#22 (D). Lightning bolts depict mitoARCUS cleavage sites. Blue boxes and nucleotides indicate the homologous regions across which recombination is predicted to have occurred in response to mitoARCUS cleavage of atp1, or where pre-existing species were amplified by SSS. Nucleotide positions are in accordance to BA000042. Predictions of more than one possibility adjacent to one the major repeats shown in (C) and (D) are supported by PacBio sequencing-derived contigs and/or PCR and DNA sequence analysis.
Figure 6
Figure 6
Analysis of 35S:nATP1/Δatp1#8 X 35S:nATP1 F1 progeny. (A) Germination of ~500 mg of 35S:nATP1/Δatp1#8 X 35S:nATP1 seed on potting soil. PCR analysis of six 35S:nATP1/Δatp1#8 X 35S:nATP1 progeny, designated 8-3 through 8-8, using primers designed to amplify: atp1 (B), the atp1-targeting ARCUS transgene (C), the nptII selectable marker gene and a control endogenous tobacco gene (CYP82E10) (D), and the 5’ break junction (E) and the 3’ break junction (F) unique to T0 event 35S:nATP1/Δatp1#8. Flowers and anthers of F1 generation Δatp1#8 plants are shown in (G) and (H), respectively. The specific primers used in (B) through (F) are found in Table S1 . WT, wild type K326; +Con, unrelated transgenic plant used as nptII positive control; Neg, negative control with no DNA template included in the reaction.
Figure 7
Figure 7
Proposed CMS-based hybrid seed production system based on allotopic expression combined with mitochondrial genome editing. The CMS inbred is propagated by crossing to the maintainer line as the pollen parent; hybrid seed is produced by crossing the CMS line with the restorer line. Fertility in the field is restored by the Con-prom:nEMtG construct. Anth-def-prom, promoter expressed in all tissues except anthers; Con-prom, promoter expressed in all plant tissues; nEMtG, essential mitochondrial gene repurposed to function in the nucleus; emtg-mut, essential mitochondrial gene rendered nonfunctional through genome editing. Maize is depicted as the example crop species.
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Grants and funding

This work was funded in part by a grant from Elo Life Systems to RED (NCSU grant 2019-1417). Support for the bioinformatic analyses was provided by the NCSU Plant Breeding Consortium.

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