Transgenic induction of mitochondrial rearrangements for cytoplasmic male sterility in crop plants

Abstract

Stability of the mitochondrial genome is controlled by nuclear loci. In plants, nuclear genes suppress mitochondrial DNA rearrangements during development. One nuclear gene involved in this process is Msh1. Msh1 appears to be involved in the suppression of illegitimate recombination in plant mitochondria. To test the hypothesis that Msh1 disruption leads to the type of mitochondrial DNA rearrangements associated with naturally occurring cytoplasmic male sterility in plants, a transgenic approach for RNAi was used to modulate expression of Msh1 in tobacco and tomato. In both species, these experiments resulted in reproducible mitochondrial DNA rearrangements and a condition of male (pollen) sterility. The male sterility was, in each case, heritable, associated with normal female fertility, and apparently maternal in its inheritance. Segregation of the transgene did not reverse the male sterile phenotype, producing stable, nontransgenic male sterility. The reproducible transgenic induction of mitochondrial rearrangements in plants is unprecedented, providing a means to develop novel cytoplasmic male sterile lines for release as non-GMO or transgenic materials.

Fig. 1.

Evidence of transgenically induced CMS in tobacco. (A) Prepared mitochondrial DNA from Xanthi [wild-type (wt)], T₂ male sterile plant 23-5-39, and T₃ male sterile plant 23-32-4-17, digested with PstI and SstII and fractionated by 0.6% agarose gel electrophoresis. Upper polymorphic band, 6.5 kb; lower band, 5.5 kb (designated by arrows). (B) Seed capsules from Xanthi (wt) and the semisterile progeny plant no. 4 from Xanthi × P-23-32 (ss). In this case the transgene was transmitted through pollen to effect the sterility phenotype. The small size of the capsule is associated with dramatically reduced seed set, but plants classified as semisterile with small capsules do produce very small amounts of viable, selfed seed. Fully male sterile plants produce fully collapsed, detached capsules. (C) Fully collapsed, detached seed capsule characteristic of male sterile tobacco plants. (D) Evidence of subtle leaf variegation in a tobacco transformant. Variegation was not evident until the T₂ generation, was infrequent, and was not restricted to fully male sterile plants. (E) Detached anthers from Xanthi (wt), the Xanthi semisterile transgenic T₀ plant no. 23 (ss), and a petaloid anther (pa). Several but not all of the tobacco male sterile and semisterile plants were characterized by the production of extremely small amounts of visible pollen. (F) Detached floral branches from Xanthi (wt) and the semisterile progeny plant no. 4 from Xanthi × P-23-32 (ss), in which the transgene was transmitted through pollen. Semisterility is characterized by both collapsed capsules that immediately detach (arrow) and small capsules that produce minute amounts of seed (see B). Fully male sterile plants produce only collapsed capsules that immediately detach.

Fig. 2.

Assay for reduced Msh1 transcript levels in transgenic tomato (A) and tobacco (B) lines. Shown are quantitative RT-PCR amplifications with Msh1-specific primers, giving a fragment of 629 bp, versus an 18S internal control amplification, giving a fragment of 315 bp. RNA was prepared from young leaves of tomato lines T17-12 (Rutgers transgenic), MM-12 (Moneymaker transgenic), and Rutgers (wild type) and from tobacco lines 23-5, 23-32, and Xanthi (wild type). Results shown are from two independent experiments using different numbers of PCR cycles because of the low abundance of MSH1 transcript. M designates molecular weight marker lanes.

Fig. 3.

The male sterility phenotype observed in transgenic tomato lines. (A) DNA gel blot hybridization analysis of total genomic DNA from Rutgers (wt) and Rutgers male sterile (ms) T₀ transgenic line 17 digested with PstI/SalI. The mitochondrial DNA probe, derived from A. thaliana BAC clone T5E7, encompasses eight mitochondrial genes and/or ORFs. Evidence of DNA polymorphism in the male sterile line is shown by an arrowhead. (B) Some leaves from a transgenic Rutgers T₁ semisterile plant display a green-white variegation pattern that appears to be associated with the mitochondrial DNA rearrangement shown in A. (C) The male sterility phenotype is associated with bud drop, the premature senescence of flowers after their opening. (D) The male sterile Rutgers transgenic plants produce parthenocarpic fruit or fruit containing a small number of seeds upon self-pollination (Self). Pollination of these plants with wild-type Rutgers pollen produces normal seed set (Cross). (E) Anther morphology is modified in the male sterile transgenic plants (ms) relative to those from wild-type Rutgers (wt). (F) DNA gel blot analysis of T₁ transgenic tomato plants. The male sterile selections, T17-12, T17-15, and T20-4, are partially sterile and variegated and contain a single copy of the transgene. The male fertile selections, T17-17 and T20-5, are nonvariegated and lack the transgene. Rutgers-GUS is a control transformant that contains the uidA transgene. Total genomic DNA was double-digested with PstI and SstII and hybridized with a DNA probe that encompasses mitochondrial genes atp9, nad1, nad5, and rps13.