The Texas cytoplasm of maize: cytoplasmic male sterility and disease susceptibility.

The Texas cytoplasm of maize carries two cytoplasmically inherited traits, male sterility and disease susceptibility, which have been of great interest both for basic research and plant breeding. The two traits are inseparable and are associated with an unusual mitochondrial gene, T-urf13, which encodes a 13-kilodalton polypeptide (URF13). An interaction between fungal toxins and URF13, which results in permeabilization of the inner mitochondrial membrane, accounts for the specific susceptibility to the fungal pathogens.

Restriction endonuclease analysis of mitochondrial DNA from normal and Texas cytoplasmic male-sterile maize.

Mitochondrial DNA from normal and cytoplasmic male-sterile maize was digested with restriction endonucleases RI from Escherichia coli or dIII from Hemophilus influenzae. Electrophoresis of resulting fragments revealed distinctions between the two cytoplasmic types. These distinctions suggest that factors responsible or cytoplasmic male sterility are located in the mitochondrial DNA, and that the mitochondrial genome is not inherited paternally.

A 13-kilodalton maize mitochondrial protein in E. coli confers sensitivity to Bipolaris maydis toxin.

The Texas male-sterile cytoplasm (cms-T) of maize carries the cytoplasmically inherited trait of male sterility. Mitochondria isolated from cms-T maize are specifically sensitive to a toxin (BmT-toxin) produced by the fungal pathogen Bipolaris maydis, race T, and the carbamate insecticide methomyl. A mitochondrial gene unique to cms-T maize, which produces a 13-kilodalton polypeptide associated with cytoplasmic male sterility, was expressed in Escherichia coli. After addition of BmT-toxin or methomyl, inhibition of whole cell respiration and swelling of spheroplasts were observed in Escherichia coli cultures producing the novel mitochondrial protein; these effects are similar to those observed with isolated cms-T mitochondria. The amino-terminal region of the 13-kilodalton polypeptide appears to be essential for proper interaction with the BmT-toxin and methomyl. These results implicate the 13-kilodalton polypeptide in conferring toxin sensitivity to mitochondria of cms-T maize.

Cytoplasmic Reversion of cms-S in Maize: Association with a Transpositional Event.

Spontaneous reversion to fertility in S male-sterile cytoplasm of maize is correlated with the disappearance of the mitochondrial plasmid-like DNA's, S-1 and S-2, and changes in the mitochondrial chromosomal DNA. Hybridization data indicate that one of the plasmid-like DNA's, S-2, is prominently involved in the mitochondrial DNA rearrangements.

The relationship between the mitochondrial gene T-urf13 and fungal pathotoxin sensitivity in maize.

Mitochondria isolated from maize containing cms-T cytoplasm are specifically sensitive to pathotoxins (T-toxins) produced by the fungi Bipolaris maydis race T and Phyllosticta maydis. T-toxins interact with a 13 kDa membrane-bound toxin receptor protein, URF13, to produce hydrophillic pores in the membrane. Expression of URF13 in Escherichia coli produces bacterial cells that form hydrophillic pores in the plasma membrane when exposed to T-toxin or methomyl. Topological studies have established that URF13 contains three membrane-spanning alpha-helices, two of which are amphipathic and may contribute to pore formation. URF13 specifically binds T-toxin in a cooperative manner. Oligonucleotide-directed mutagenesis of URF13 led to the isolation of methomyl/T-toxin-resistant mutations at 39 separate positions throughout the URF13 primary sequence. Chemical cross-linking of URF13 demonstrated the presence of URF13 oligomers and established that the pore-forming species is oligomeric. The ability of the carboxylate-specific reagent, dicyclohexycarbodiimide to cross-link URF13 has been used in conjunction with site-directed mutagenesis to establish that the URF13 tetramer has a central core consisting of a four-alpha-helical bundle that may undergo a conformational change after T-toxin or methomyl binding. Experimental evidence indicates that URF13 acts as a ligand-gated, pore-forming T-toxin receptor.

Heterogeneity of Maize Cytoplasmic Genomes among Male-Sterile Cytoplasms.

Maize mitochondrial and chloroplast DNA's were prepared from normal (fertile) lines or single crosses and from members of the T, C, and S groups of male-sterile cytoplasms. Restriction endonucleases HindIII, BamI, EcoRI, and SalI were used to restrict the DNA, and the resultant fragments were electrophoresed in agarose gels. The results show that the N (fertile), T, C, and S cytoplasms each contained distinct mitochondrial DNA (mtDNA). These distinctive patterns were unaffected by nuclear genotype. No evidence of paternal inheritance of mtDNA was observed. Chloroplast DNA (ctDNA) from the N, C, and T cytoplasms was indistinguishable by HindIII, SalI, or EcoRI endonuclease digestion. The S cytoplasm ctDNA, however, was slightly different from that of other cytoplasms, as indicated by a slight displacement of one band in HindIII digests. The molecular weight of maize ctDNA was estimated to be as high as 88 x 10(6). Estimates of the minimum molecular weight of maize mtDNA ranged from 116-131 x 10(6), but the patterns were to complex for an unambiguous determination. Based on HindIII data, a comparison of the molecular weight of mtDNA bands common to the N, T. C, and S cytoplasms suggests that C cytoplasm most closely resembles N cytoplasm. The T and S sources are more divergent from the C and N cytoplasms. These results indicate a possible gradation of relatedness among male-sterile cytoplasms. The marked variation in mtDNA, with apparently less variation in ctDNA, represents circumstantial, but compelling, evidence that mtDNA may be involved in the male sterility and disease susceptibility traits in maize.

Patterns of mitochondrial DNA variation in indigenous maize races of latin america.

Mitochondrial DNAs (mtDNAs) were isolated from 93 diverse races of maize from Latin America. DNAs were examined by agarose gel electrophoresis of undigested DNA and by BamHI and EcoRI cleavage fragment analysis. Eighteen races contained plasmid-like mtDNAs. One race contained the S-1 and S-2 molecules associated with the S cytoplasmic male-sterile, and 17 were found to have the R-1 and R-2 plasmid-like DNAs. BamHI digestion of mtDNAs generated ten distinct electrophoretograms, and Eco RI digestion produced eight different fragment patterns. Races were assigned to one of 18 groups according to EcoRI and BamHI fragment patterns and whether or not they contained plasmid-like DNAs. Eight races produced restriction patterns similar to one of the characterized cytoplasmic male-steriles C, T, or S. Races from Meso-America and some from South America with Meso-American affinities were separated from other South American races. South American races were placed in three general classes of related groups. There was considerable agreement among the groupings here and those based on morphological and cytological affinities.

Maize mitochondrial plasmid S-1 sequences share homology with chloroplast gene psbA.

The linear, 6397-base pair (bp), mitochondrial S-1 DNA molecule from maize contains a 420-bp segment that is homologous with the chloroplast gene (psbA) that codes for the quinone binding protein of photosystem II. This is the first report of a chloroplast sequence in a naturally occurring viral-like or plasmid DNA. The complete sequence of the S-1 chloroplast segment has been compared with homologous regions of six different chloroplast genes. The S-1 segment has diverged from the other genes both by length mutation and base substitution. Several of the length mutations are exact adjacent tandem duplications of 4 and 5 bp similar to "footprints" left after excision of transposable elements in maize nuclear DNA.

Functional analysis of a recently originating, atypical presequence: mitochondrial import and processing of GUS fusion proteins in transgenic tobacco and yeast.

A gene family of at least five members encodes the tobacco mitochondrial Rieske Fe-S protein (RISP). To determine whether all five RISPs are translocated to mitochondria, fusion proteins containing the putative presequences of tobacco RISPs and Escherichia coli beta-glucuronidase (GUS) were expressed in transgenic tobacco, and the resultant GUS proteins were localized by cell fractionation. The amino-terminal 75 and 71 residues of RISP2 and RISP3, respectively, directed GUS import into mitochondria, where fusion protein processing occurred. The amino-terminal sequence of RISP4, which contains an atypical mitochondrial presequence, can translocate the GUS protein specifically into tobacco mitochondria with apparently low efficiency. Consistent with the proposal of a conserved mechanism for protein import in plants and fungi, the tobacco RISP3 and RISP4 presequences can direct import and processing of a GUS fusion protein in yeast mitochondria. Plant presequences, however, direct mitochondrial import in yeast less efficiently than the yeast presequence, indicating subtle differences between the plant and yeast mitochondrial import machineries. Our studies show that import of RISP4 may not require positively charged amino acid residues and an amphipathic secondary structure; however, these structural properties may improve the efficiency of mitochondrial import.

The protein-encoding gene T-urf13 is not edited in maize mitochondria.

RNA editing of T-urf13, a gene specific to the mitochondria of cytoplasmic male-sterile, type-T (cms-T) maize, and an adjacent, cotranscribed gene orf221, have been studied by cDNA sequencing. No editing was detected in 22 cDNA clones. This is the only report of a polypeptide-encoding gene in higher-plant mitochondria that is not edited. T-urf13 may not be edited because it is derived largely from the coding and flanking regions, which are rarely edited, of a ribosomal RNA gene. orf221 is edited; however, the similarity between the predicted amino acid sequences of orf221 in cms-T and normal cytoplasms is not increased.

Transcription of the S-2 maize mitochondrial plasmid.

Mitochondria from S (male-sterile) and RU (male-fertile) maize cytoplasms contain both autonomous plasmids and copies of plasmid sequences integrated into the mitochondrial genome. The S-2 plasmid in S, which appears essentially the same as the R-2 plasmid in RU, contains two large open reading frames (ORFs) that may encode gene products required for plasmid maintainance and replication. Transcripts from both ORF1 and ORF2 are readily detected in cytoplasms having free plasmids. The 5' ends of these RNAs, which are transcribed from opposite ends of the plasmid, correspond to nucleotide position 32 within each of the 208 bp terminal inverted repeats (IRs). In Normal (fertile) and Revertant S (now fertile) cytoplasms that have only integrated copies of the S-2 sequence, transcripts from the S-2 ORFs are either present in greatly reduced amounts, or not detectable. The loss of transcriptional activity is correlated with deletions of, or nucleotide changes within, the IRs associated with the integrated S-2 ORFs. Evidence for transcription of non-plasmid DNA adjacent to integrated IRs suggests that both free-plasmid and integrated forms of the plasmid IR have promoter-like activity.

RNA editing of a chimeric maize mitochondrial gene transcript is sequence specific.

RNA editing was analysed in the mitochondrial ATPase complex subunit 6 gene (atp6) transcripts of the C male-sterile cytoplasm (cms-C) of maize. The only copy of atp6 in cms-C, designated C-atp6, is a triple gene fusion product comprised of DNA sequences derived from atp9, atp6, and an unknown origin. Sequences of cDNAs revealed 19 C to U alterations resulting in 16 amino acid residue changes compared to the genomic sequence. The only C to U edit in the 39-nucleotide sequence similar to atp9 was comparable to a change in the complete atp9 mRNAs of Petunia, Oenothera, wheat, and sorghum. The 442 nucleotides of unknown origin were not edited. The 18 editing events within the atp6 homologous region were similar to those in the atp6 transcripts of sorghum. RNA editing in maize C-atp6 transcripts introduces a translational stop codon at the same position where it is created by editing in sorghum and Oenothera atp6 mRNAs and is already present in atp6 open reading frames of most other plant and non-plant organisms. Our results, along with other reports on editing in chimeric transcripts, indicate that RNA editing is not influenced by rearrangements but instead is sequence specific.

Baculovirus expression of the maize mitochondrial protein URF13 confers insecticidal activity in cell cultures and larvae.

The URF13 protein, which is encoded by the mitochondrial gene T-urf13, is responsible for cytoplasmic male sterility and pathotoxin sensitivity in the Texas male-sterile cytoplasm (cms-T) of maize. Mitochondrial sensitivity to two host-specific fungal toxins (T toxins) is mediated by the interaction of URF13 and T toxins to form pores in the inner mitochondrial membrane. A carbamate insecticide, methomyl, mimics the effects of T toxins on isolated cms-T mitochondria. URF13 was expressed in Spodoptera frugiperda (fall army-worm) cells (Sf9) in culture and in Trichoplusia ni (cabbage looper) larvae with a baculovirus vector. In insect cells, URF13 forms oligomeric structures in the membrane and confers T toxin or methomyl sensitivity. Adding T toxin or methomyl to Sf9 cells producing URF13 causes permeabilization of plasma membranes. In addition, URF13 is toxic to insect cells grown in culture without T toxins or methomyl; even a T-toxin-insensitive mutant form of URF13 is lethal to cell cultures. Baculoviruses expressing URF13 are lethal to T. ni larvae, at times postinjection comparable to those obtained by injecting a baculovirus expressing an insect neurotoxin. This result suggests that URF13 could be useful as a biological control agent for insect pests. Our data indicate that URF13 has two independent mechanisms for toxicity, one that is mediated by T toxin and methomyl and one that is independent of these toxins. Similarly, male sterility and toxin sensitivity in cms-T maize may be due to independent mechanisms.

Nucleotide sequence of the S-2 mitochondrial DNA from the S cytoplasm of maize.

Mitochondria from the S male-sterile cytoplasm (cms-S) of maize contain two plasmid-like DNAs, S-1 and S-2, that appear to be prominently involved with the cytoplasmic male sterility trait. The complete nucleotide sequence of the S-2 DNA molecule was determined by the chain termination method. The linear S-2 DNA molecule contains 5,452 base pairs and is terminated by exact 208-base-pair inverted repetitions. Two large open reading frames were identified in the S-2 DNA, suggesting the possibility of protein-encoding genes. The nucleotide sequence of the S-2 termini are discussed with regard to models proposed for the replication of linear DNA molecules.