Mitochondrial dysfunction in yeast expressing the cytoplasmic male sterility T-urf13 gene from maize: analysis at the population and individual cell level

Summary The urf13TW gene, which is derived from the mitochondrial T-urf13 gene responsible for Texas cytoplasmic male sterility in maize, was expressed in Saccharomyces cerevisiae by targeting its translation product into mitochondria. Analysis by oxygraphy at the population level revealed that in the presence of methomyl the oxygen uptake of intact yeast cells carrying the targeted protein is strongly stimulated only with ethanol as respiratory substrate and not with glycerol, lactate, pyruvate, or acetate. When malate is the substrate oxidized by isolated mitochondria, interaction between the targeted protein and methomyl results in significant inhibition of oxygen uptake. This inhibition is eliminated and oxygen uptake is stimulated by subsequent addition of NAD⁺. Using 3,3-dihexyloxacarbocyanine iodide [DiOC₆(3)] as probe, interactive laser scanning and flow cytometry, which permit analysis at the individual cell level, demonstrated that specific staining of the mitochondrial compartment is obtained and that DiOC₆(3) fluorescence serves as a measure of the membrane potential. Finally, it was shown that, as in T cytoplasm maize mitochondria, HmT toxin and methomyl dissipate the membrane potential of yeast mitochondria that carry the foreign protein. Furthermore, the results suggest that the HmT toxin and methomyl response is related to the plasmid copy number per cell and that the deleterious effect induced by HmT toxin is stronger than that of methomyl.     

Phenotypically normal transgenic T-cyt tobacco plants as a model for the investigation of plant gene expression in response to phytohormonal stress

The tumour-inducing T-DNA gene 4 (T-cyt gene) of the nopaline Ti plasmid pTiC58 was cloned and introduced into tobacco cells by leaf disc transformation using Agrobacterium plasmid vectors. Tobacco shoots exposed to elevated cytokinin levels were unable to develop roots and lacked apical dominance. Using exogenously applied phytohormone manipulations we were able to regenerate morphologically normal transgenic tobacco plants which differed in endogenous cytokinin levels from normal untransformed plants. Although T-cyt gene mRNA levels, as revealed by dot-blot hybridization data, in these rooting plants were only about half those in primary transformed shoots the total amount of cytokinins was much lower than in crown gall tissue or cytokinin-type transformed shoots as reported by others. Nevertheless the cytokinin content in T-cyt plants was about 3 times greater than in control tobacco plants.Elevated cytokinin levels have been shown to change the expression of several plant genes, including some nuclear genes encoding chloroplast proteins. Our results show that the mRNA levels of chloroplast rbcL gene increase in cytokinin-type transgenic tobacco plants as compared with untransformed plants. Data obtained suggest that T-cyt transgenic plants are a good model for studying plant gene activity in different parts of the plant under endogenous cytokinin stress.     

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 geneorf221, 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 oforf221 incms-T and normal cytoplasms is not increased.     

URF13, a ligand-gated,pore-forming receptor for T-toxin in the inner membrane ofcms-T mitochondria

URF13 is the product of a mitochondrial-encoded gene (T-urfl3) found only in maize plants containing the Texas male-sterile cytoplasm (cms-T), and it is thought to be responsible for both cytoplasmic male sterility and the susceptibility ofcms-T maize to the fungal pathogensBipolaris maydis race T andPhyllosticta maydis. Mitochondria isolated fromcms-T maize are uniquely sensitive to pathotoxins (T-toxin) produced by these fungi and to methomyl (a commercial insecticide). URF13 acts as a receptor that specifically binds T-toxin to produce hydrophilic pores in the inner mitochondrial membrane. When expressed inEscherichia coli cells, URF13 also forms hydrophilic pores in the plasma membrane if exposed to T-toxin or methomyl. Topological studies established that URF13 contains three membrane-spanning -helices, two of which are amphipathic and can contribute to pore formation. Chemical crosslinking of URF13 was used to demonstrate the existence of URF13 oligomers incms-T mitochondria andE. coli cells. The ability of the carboxylate-specific reagent,N,N-dicyclohexycarbodiimide, to cross-link URF13 was used in conjunction with site-directed mutagenesis to establish that the URF13 tetramer has a central core consisting of a four--helical bundle which undergoes a conformational change after interaction with T-toxin or methomyl. Overall, the experimental evidence indicates that URF13 functions as a ligand-gated, pore-forming T-toxin receptor incms-T mitochondria.     

Expression of a chimaeric heat-shock-inducible Agrobacterium 6b oncogene in Nicotiana rustica

The T-6b gene of Agrobacterium tumefaciens strain Tm4 induces tumours on Nicotiana rustica by an as yet unknown mechanism. These tumours cannot be regenerated into normal plants. To study the effect of the T-6b gene product on normal plant cells, the T-6b gene was placed under control of the Drosophila melanogaster hsp70 heat-shock promoter and introduced into N. rustica. Progeny of an hsp70-T-6b transformant developed into normal plants. The inducibility of the hsp70-T-6b construct was shown by northern analysis and by heat-shock-dependent growth alterations on the level of whole seedlings. Upon wounding at normal temperature conditions hsp70-T-6b plants formed small tumours on leaves and stems. Grafts between transformed plants and normal plants led to a wound callus which remained limited to transformed tissues, indicating that the T-6b gene product does not diffuse. Protoplasts of hsp70-T-6b plants divided in the same way as control protoplasts under standard culture conditions. However, when protoplast cultures were started in the absence of hormones, normal cells rapidly lost their sensitivity towards hormones, whereas hsp70-T-6b cells remained sensitive for a significantly longer period. Thus, the T-6b gene product alters hormone sensitivity during the initial phases of protoplast culture.     

In planta analysis of the Agrobacterium tumefaciens T-cyt gene promoter: identification of an upstream region essential for promoter activity in leaf,stem and root cells of transgenic tobacco

The promoter region of the Agrobacterium tumefaciens T-cyt gene was fused to a -glucuronidase (gusA) reporter gene and introduced into tobacco plants. Detection of gusA expression in transgenic F1 progeny revealed that the T-cyt promoter is active in many, if not all, cell types in leaves, stems and roots of fully developed plants. Developmental stage-dependent promoter activity was observed in seedlings. Analysis of 5-deleted promoter fragments showed that sequences located between positions–185 and –139 with respect to the T-cyt translational start codon are essential for T-cyt promoter activity in transfected tobacco protoplasts as well as in transformed tobacco plants.     

Changes in the tissue-specific prevalence of translatable mRNAs in transgenic tobacco shoots containing the T-DNA cytokinin gene

Two-dimensional gel electrophoresis of in vitro translation products was used to examine differences between the steady state RNA populations of an untransformed tobacco plant line and a non-rooting tobacco shoot line transformed with a T _l -DNA segment from Agrobacterium tumefaciens carrying the cytokinin gene (T-cyt). The analysis comprised about 240 translation products representing the more abundant mRNAs. Approximately 8% of the translation products were found to have significantly different concentrations, due to both increases and decreases, when the shoot parts of the transformed and untransformed lines were compared. Only a few of these differences were specific for the comparison of transformed and untransformed shoots. Most of the differences were also observed when the shoot and root parts of the untransformed line were compared. This implies that the shoot or root prevalence of several mRNA species in normal plants is altered in transgenic T-cyt shoots. The observed changes in the mRNA population of transgenic T-cyt shoots are discussed in relation to the transformed phenotype and previously cloned mRNAs showing similar changes in tissue-specific prevalence.     

A fungal chitinase gene fromRhizopus oligosporus confers antifungal activity to transgenic tobacco

We have studied whether a chitinase involved in cell autolysis of a filamentous fungus,Rhizopus oligosporus, can operate as an antifungal defense system in tobacco. Thechi1 gene was introduced into tobacco by theAgrobacterium tumefaciens leaf disc system. Among 22 transgenic tobacco plants, 2 were selected and their individual homozygous progeny, Tch1-1 and Tch2-1, were studied. Chitinase activity in the extracts of young leaves from Tch1-1 or Tch2-1, in which thechi1 gene product was detected by Western blot analysis, was three- to four-fold higher than that from the control plants. A fungal infection assay on the leaves infected with the discomycete pathogensSclerotinia sclerotiorum andBotrytis cinerea revealed that the symptoms observed with these two were remarkably suppressed as compared with the control leaves.Abbreviations CaMV Cauliflower mosaic virus - Km ^r kanamycin resistant - Km ^s kanamycin sensitive - MS Murashige and Skoog - PCR polymerase chain reaction - PDA potato dextrose agar - PR pathogenesis-related     

Improved salt tolerance in tobacco plants by co-transformation of a betaine synthesis gene <Emphasis Type="Italic">BADH</Emphasis> and a vacuolar Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter gene <Emphasis Type="Italic">SeNHX1</Emphasis>

Three types of transgenic tobacco plants were acquired by separate transformation or co-transformation of a vacuolar Na⁺/H⁺ antiporter gene, SeNHX1, and a betaine synthesis gene, BADH. When exposed to 200 mM NaCl, the dual gene-transformed plants displayed greater accumulation of betaine and Na⁺ than their wild-type counterparts. Photosynthetic rate and photosystem II activity in the transgenic plants were less affected by salt stress than wild-type plants. Transgenic plants exhibited a greater increase in osmotic pressure than wild-type plants when exposed to NaCl. More importantly, the dual gene transformed plants accumulated higher biomass than either of the single transgenic plants under salt stress. Taken together, these findings indicate that simultaneous transformation of BADH and SeNHX1 genes into tobacco plants can enable plants to accumulate betaine and Na⁺, thus conferring them more tolerance to salinity than either of the single gene transformed plants or wild-type tobacco plants. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Recovery of morphologically normal transgenic tobacco from hairy roots co-transformed with Agrobacterium rhizogenes and a binary vector plasmid

Summary Co-transformation of tobacco (Nicotiana tabacum) leaf explants with Agrobacterium rhizogenes harbouring pRi1855 and the binary vector pBin19 was achieved at a frequency of 67%. The kanamycin resistant hairy roots were cultured via a callusing phase to regenerate plants which were partially fertile when outcrossed with wild-type pollen. Phenotypic and molecular analysis of the F₁ progeny demonstrated the efficient segregation of the hairy root marker from the kanamycin resistance marker, enabling morphologically normal plants to be recovered which retained the binary vector marker gene. This co-transformation strategy provides a means of introducing non-selectable genes into plants in cases where antibiotic resistance markers are undesirable.     

Cloning of Helicobacter pylori urease subunit B gene and its expression in tobacco (Nicotiana tabacum L.)

Vaccines produced by transgenic plants would have the potential to change the traditional means of production and inoculation of vaccines, and to reduce the cost of vaccine production. In the present study, an UreB antigen gene from a new Helicobacter pylori strain ZJC02 was cloned into the binary vector pBI121 which contains a CaMV35S promoter and a kanamycin resistance gene, and then transformed UreB into tobacco leaf-disc by Agrobacterium-mediated method. A total of 50 regenerated plants with kanamycin resistance were obtained in the selection media. The 35 putative transgenic individuals were tested and verified the presence and integration of the UreB into the nuclear genome of tobacco plants by PCR, PCR-southern, and Southern analyses. Expression of UreB gene in the tobacco plants was confirmed by RT-PCR and Western Blot analysis using polyclonal human antiserum. To our knowledge, this is the first report of the expression of Helicobacter pylori UreB antigen gene in a plant system, suggesting a major step in the production of plant-based vaccines for Helicobacter pylori.     

Meiotic stability of transgene expression is unaffected by flanking matrix-associated regions

DNA repeats are associated with gene instability and silencing phenomena in plants. Therefore, the presence of a direct repeat of matrix-associated region (MAR) DNA, that considerably reduced position effects between independent transformants, may increase (epi)genetic instability. To investigate the influence of such a repeat on the stability of the expression of embedded transgenes, the meiotic stability of transgene expression was assessed in eighteen homozygous 1-locus transgenic tobacco lines carrying the kanamycin resistance (NPTII) and the -glucuronidase (GUS) gene. Half of the lines carry a 3 kb direct repeat of MAR DNA flanking the transgenes. Large progeny populations, totalling over a million seedlings, were screened for kanamycin resistance with the help of a newly developed high-density seedling screen. Kanamycin-sensitive seedlings were detected in selfed progeny at a frequency of 0.5-5.9×10^-4. The frequency became as high as 2×10^-2 when embryo development occurred under heat and/or drought stress. In backcrossed progeny only, a joint loss of NPTII and GUS gene expression was observed at an average frequency of 2.9×10^-5. In selfed and backcrossed progeny we observed similar frequencies of reversion to kanamycin sensitivity, indicating that epigenetic silencing mechanisms rather than MAR repeat-related homologous recombination underlie the reversal to kanamycin sensitivity. Different lines, hence different areas of the tobacco genome, differed in their genetic stability. No significant differences in reversal frequencies were apparent between lines with or without the MAR elements. The use of the MAR repeat is, therefore, not compromised by any increased (epi)genetic instability.     

Agrobacterium-mediated transformation of germinating seeds of Arabidopsis thaliana: A non-tissue culture approach

Summary Germinating seeds of Arabidopsis thaliana were cocultivated with an Agrobacterium tumefaciens strain (C58Clrif) carrying the pGV3850:pAK1003 Ti plasmid. This Ti plasmid contains the neomycin phosphotransferase II gene (NPT II) which confers resistance to kanamycin and G418. Seeds (T1 generation) imbibed for 12 h before a 24 h exposure to Agrobacterium gave rise to the highest number of transformed progeny (T2 generation). Over 200 kanamycin-resistant T2 seedlings were isolated. Some of the T2 seedlings and T3 families were characterized for genetic segregation of functional NPT II gene(s), NPT II activity, and the presence of T-DNA inserts (Southern analysis). Ninety percent of the T2 individuals transmitted the resistance factor to the T3 families in a Mendelian fashion. Of the T3 families segregating in a Mendelian fashion (n=111), 62% segregated for one functional insert, 29% for two unlinked or linked functional inserts, 5% for three unlinked inserts, 1% for four unlinked inserts, whereas 3% appeared to be homozygous for the insert(s). The 13 families that did not exhibit Mendelian segregation ratios fell into 2 classes, both of which had a deficiency of kanamycin-resistant seedlings. In the Group I T3 families (n=6) only 0%–2% of the seedlings were resistant to kanamycin (100 mg/l), whereas in the Group II families (n=7) 8%–63% of the seedlings were resistant. All of the kanamycin-resistant plants that were tested were found to possess NPT II activity. Southern analysis revealed that all of the resistant plants contained at least one copy of the T-DNA and that the majority of the plants had multiple inserts. Explants from kanamycin-resistant plants survived and formed callus when cultured on callus-inducing medium containg G418.     

Transgenic antibiotic resistance may be differentially silenced in germinating pollen grains

Transgenic tobacco (Nicotiana tabacum L.) plants, carrying the neomycin phosphotransferase (NPT II) gene from E. coli, are resistant to kanamycin when grown from seeds on kanamycin containing medium. Tissue and cell cultures derived from those transformants also express resistance and regenerate complete plantlets in the presence of the antibiotic. This unspecific response to the selective condition has led to the belief that the foreign gene is continuously active or uniformly inducible in all cells of the transgenic plant. However, our experiments show that this view is not true for pollen grains during in vitro germination. Pollen grains isolated from kanamycin resistant tobacco plants carry and transmit the foreign gene but do not express resistance when germinating in vitro. This data presents evidence for differential silencing of a foreign gene in a mature gamete. On the other hand, immature pollen grains (microspores) appear to express resistance. The point of the downregulation of the neomycin transferase gene during pollen maturation is discussed.Abbreviations kan kanamycin sulfate - NPT II neomycin phosphotransferase II - sr streptomycin sulfate     

Phenotypic changes in T-cyt-transformed potato plants are consistent with enhanced sensitivity of specific cell types to normal regulation by root-derived cytokinin

From over forty independently isolated potato lines transformed with wild-type and promoter-mutated T-cyt genes [12], a number of lines were selected for examination of phenotypic changes in growth and development for plants grown in soil in a controlled environment. The three lines chosen for most detailed examination showed a wide spectrum of phenotypic changes. In comparisons with control potato cv. Désirée, the plants of one line had a two- to three-fold increase in biomass production during early vegetative growth, advanced senescence and a shortened plant life-span. Another line showed abnormal cellulytic senescence. In two lines there were increases in tuber numbers and more skewed tuber size distributions which correlated with reduced shoot apical dominance and shortened dormancy of the stored tubers. None of the lines showed altered timing of onset of tuberization or flowering, although tuberization was consistently delayed when expressed as a function of increasing total plant weight. A hypothesis is proposed to explain the diverse phenotypes which postulates that (1) T-cyt transformation causes enhanced sensitivity to cytokinins in specific types of shoot cells which are already targets for regulation by normal root-derived cytokinins; (2) two distinct types of shoot target cells are present, one in shoot meristems and one in leaves; (3) the two types can acquire enhanced sensitivity, either separately or in combination depending on the particular T-cyt transformation event. The scope for using the transformed plants in subsequent physiological, biochemical and molecular studies, aimed at examining the molecular basis of the model or selected consequences of T-cyt transformation in altering regulation of potato plant growth and development, is discussed. The attention is drawn to the possible involvement at the subcellular level of sucrose phosphate synthase in mediating the phenotypic effects caused by T-cyt transformation.     

Relocation of urf a from the mitochondrion to the nucleus cures the mitochondrial mutator phenotype in the fission yeast Schizosaccharomyces pombe

In previous papers we have reported the characterisation of mitochondrial mutator mutants of Schizosaccharomyces pombe. In contrast to nuclear mutator mutants known from other eucaryotes, this mutator phenotype correlates with mutations in an unassigned open reading frame (urf a) in the mitochondrial genome. Since an efficient biolistic transformation system for fission yeast mitochondria is not yet available, we relocated the mitochondrial urf a gene to the nucleus. As host strain for the ectopic expression, we used the nonsense mutant ana ^r -6, which carries a premature stop codon in the urf a gene. The phenotype of this mutant is characterised by continuous segregation of progeny giving rise to fully respiration competent colonies, colonies that show moderate growth on glycerol and a fraction of colonies that are unable to grow on glycerol. The phenotype of this mutant provides an excellent tool with which to study the effects on the mutator phenotype of ectopic expression of the urf a gene. Since a UGA codon encoding tryptophan is present in the original mitochondrial gene, we constructed two types of expression cassettes containing either the mitochondrial version of the urf a gene (mt-urf a) or a standard genetic code version (nc-urf a; UGA replaced by UGG) fused to the N-terminal import leader sequence of the cox4 gene of Saccharomyces cerevisiae. We show that the expression of the mt-urf a gene in its new location is able to cure, at least in part, the phenotype of mutant ana ^r -6, whereas the expression of the nc-urf a gene completely restores the wild-type (non-mutator) phenotype. The significant similarity of the urf a gene to the mitochondrial var1 gene of S. cerevisiae and homologous genes in other yeasts suggests that the urf a gene product might be a ribosomal protein with a dual function in protein synthesis and maintenance of mitochondrial DNA integrity. Received: 13 May 1997 / Accepted: 14 January 1998     

Optimization of Agrobacterial (<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>) Transformation of Maize Embryogenic Callus

The method for genetic transformation of maize (Zea mays L.) via embryogenic callus infection with Agrobacterium tumefaciens was developed. Calli were co-cultivated with the overnight culture of A. tumefaciens strain LBA4404 harboring the pBI121 plasmid with the nptII and uidA genes. Thereafter, the sensitivity of calli and regenerated plantlets to kanamycin (Km) was determined. It was shown that kanamycin selection was more efficient at the stage of regenerated plantlets than in callus culture. Both vacuum infiltration at the infection step and preliminary activation of Agrobacterium by acetosyringone or by tobacco leaves exudate increased the frequency of Km-resistant plants. The frequency of Km-resistant plants also varied depending on the morphogenic ability of calli. Polymerase chain reaction confirmed the presence of the nptII gene in the genome of regenerated plants and their progeny. β-Glucuronidase gene expression was observed in roots of T1 plants.__________Translated from Fiziologiya Rastenii, Vol. 52, No. 4, 2005, pp. 600–607.Original Russian Text Copyright © 2005 by Danilova, Dolgikh.     

Agrobacterium tumefaciens T-DNA gene 6b stimulates rol-induced root formation, permits growth at high auxin concentrations and increases root size

Summary All Agrobacterium tumefaciens strains studied up to now transfer an active 6b gene to plant cells. However, the role of this gene in natural tumour induction is unknown. Various effects of 6b on plant cell growth have been described, but the precise mechanism by which 6b causes these effects has not been elucidated. Earlier experiments indicated that the 6b gene might increase auxin sensitivity as do the A. rhizogenes rol genes. The 6b gene from Tm4 (T-6b) was therefore compared with the rolB and rolABC genes. Although T-6b was unable to induce root formation, it strongly interfered with root induction and root elongation. In rolABC/T-6b coinfection experiments on carrots, T-6b-transformed cells stimulated root outgrowth of rolABC-transformed cells, indicating that the biologically active T-6b product is diffusible. Carrot rolABC roots containing the T6b gene rapidly developed into unorganized calli. Nicotiana rustica roots with rolABC and T-6b continued their development, but became very large. Fragments of such roots formed callus at -naphthaleneacetic acid concentrations which inhibited growth of rolABC and normal root fragments, suggesting that the role of 6b genes in natural tumour induction may be to reduce the inhibitory effects of high auxin levels and to keep cells in an undifferentiated state.     

Overexpression of tomato <Emphasis Type="Italic">tAPX</Emphasis> gene in tobacco improves tolerance to high or low temperature stress

In order to investigate the function of chloroplast ascorbate peroxidase under temperature stress, the thylakoid-bound ascorbate peroxidase gene from tomato leaf (TtAPX) was introduced into tobacco. Transformants were selected for their ability to grow on medium containing kanamycin. RNA gel blot analysis confirmed that TtAPX in tomato was induced by chilling or heat stress. Over-expression of TtAPX in tobacco improved seed germination under temperature stress. Two transgenic tobacco lines showed higher ascorbate peroxidase activity, accumulated less hydrogen peroxide and malondialdehyde than wild type plants under stress condition. The photochemical efficiency of photosystem 2 in the transgenic lines was distinctly higher than that of wild type plants under chilling and heat stresses. Results indicated that the over-expression of TtAPX enhanced tolerance to temperature stress in transgenic tobacco plants.     

Combination of kanamycin resistance and nitrate reductase deficiency as selectable markers in one nuclear genome provides a universal somatic hybridizer in plants

Summary The combination in the nuclear genome of a dominant resistance marker (to select against unfused wild-type cells) and a recessive deficiency marker (to select against unfused mutant cells) in a cell line should provide a system for selecting fusion hybrids between the mutant line and any wild-type line. To test this idea, we fused protoplasts from a non-morphogenic cell line of Nicotiana tabacum which was kanamycin resistant (by transformation) and deficient in nitrate reductase (NR^-K⁺) with protoplasts from N. tabacum cv. Petit Havana clone SR1, which provided resistance against streptomycin as an additional selectable marker (NR⁺K^-SR⁺). Putative hybrids were selected using a culture medium containing no available reduced nitrogen source and 50 mg/l kanamycin sulphate. After regeneration into plants, the hybrid character was demonstrated from: (i) the morphological variation of the regenerants; (ii) the chromosome number; (iii) the ability to grow on medium without a reduced nitrogen source and containing kanamycin sulphate at 50 mg/l; (iv) the presence of nitrate reductase activity; (v) the presence of the gene coding for neomycin phosphotransferase, which provides resistance to kanamycin sulphate; (vi) callus formation from leaves on medium containing 1 g/l streptomycin or 50 mg/l kanamycin sulphate; (vii) F₁ plants containing nitrate reductase and the gene for neomycin phosphotransferase. Fusions between the mutant cell line (NR^-K⁺) and three wild-type tobacco species and subsequent cultivation on medium containing no available nitrogen source but 50 mg/l kanamycin sulphate resulted in callus formation with all combinations, while hybrid plants were only regenerated when N. sylvestris was the fusion partner.