Enhanced disease resistance conferred by expression of an antimicrobial magainin analog in transgenic tobacco.

Magainins are a group of short peptides originally isolated from frog skin and thought to function as a natural defense mechanism against infection due to their antimicrobial properties. The engineered magainin analog peptide Myp30 was found to inhibit spore germination of the oomycete, Peronospora tabacina (Adam) in vitro, and the growth of a bacterial pathogen Erwinia carotovora subsp. carotovora (Jones). Transgenic tobacco (Nicotiana tabacum L.) plants expressing Myp30 were evaluated for resistance to these pathogens. The expression of the peptide only to an extracellular location resulted in significant reduction in sporulation and lesion size due to P. tabacina infection. A significant increase in resistance to the bacterial pathogen was also observed regardless of the targeting location of the peptide.

Structure and promoter/leader deletion analysis of mirabilis mosaic virus (MMV) full-length transcript promoter in transgenic plants.

A full-length transcript (FLt) promoter fragment was isolated from a genomic clone of mirabilis mosaic virus (MMV), a double-stranded DNA plant pararetrovirus belonging to the caulimovirus family. The boundaries required for maximal promoter expression were defined by 5' and 3' deletion analysis of the MMV promoter fragments coupled to a GUS reporter gene. The expression patterns of these chimeric gene constructs were evaluated both in transgenic Nicotiana tabacum cv. Samsun NN plants and in protoplast transient expression experiments. A 360 bp FLt promoter fragment (sequence -297 to +63 from the transcription start site) was found sufficient for strong promoter activity. The transcription start site (TSS) of the MMV FLt promoter was determined by primer extension analysis using total RNA isolated from transgenic plants containing a MMV promoter:uidA fusion gene. Analysis of the 5' and 3' deletion constructs showed that an upstream region (sequence -248 to -193 from the transcription start site) is required for the MMV FLt promoter activity along with the as-1, TATA box regions. In addition, a 31 bp sequence (+33 to +63 from the transcription start site) located downstream of a TATA box is also essential for the maximum expression of the MMV FLt promoter. Analysis of transcripts (mRNA) from these chimeric constructs also indicated that the MMV FLt promoter fragment (-297 to +63 from the transcription start site) has the highest promoter activity. In a comparative analysis the MMV FLt promoter showed much greater activity than the CaMV 35S promoter.

Gene expression regulated by gene VI of caulimovirus: transactivation of downstream genes of transcripts by gene VI of peanut chlorotic streak virus in transgenic tobacco.

Here we document that the gene VI product of peanut chlorotic streak virus (PClSV), a newly characterized member of the group, transactivates the translation of dicistronic transcripts. Dicistronic expression units have been analyzed both in protoplast transient expression experiments and in transgenic tobacco plants. Transgenic plants containing a dicistronic transcription unit (PClSV-gene VII-GUS) under the control of PClSV full-length transcript promoter with its long leader sequence show a relatively high abundance of the expected transcript but very little, or no, GUS activity. However, high GUS activity is found when gene VI protein is then provided by subsequent infection with PClSV. The efficient translation of polycistronic mRNAs mediated by gene VI of caulimovirus has potential value in product engineering of plants.

Isolation and expression analysis of peanut chlorotic streak caulimovirus (PClSV) full-length transcript (FLt) promoter in transgenic plants.

A promoter fragment from peanut chlorotic streak caulimovirus (PClSV) full-length transcript (FLt) was identified and later modified to have duplicated enhancer domain. The FLt promoter with its single or double enhancer domains, fused with the GUS reporter gene to form chimeric gene constructs, showed a high level of expression of these genes in cells and transgenic plants. The FLt promoter with its double enhancer domain gives an average threefold greater expression of genes compared to the FLt promoter with its single enhancer domain in transgenic plants. In young seedlings the expression was in the order root > leaf > stem. The histochemical GUS assay in young seedlings showed more activity in root tips and leaf midribs, veins, and other vascular tissues. The expression from the PClSV FLt promoter was compared with that from the figwort mosaic virus promoter in transgenic plants. These constitutive promoters were comparable in respect to GUS expression level.

Promoter/leader deletion analysis and plant expression vectors with the figwort mosaic virus (FMV) full length transcript (FLt) promoter containing single or double enhancer domains.

The boundaries required for maximal expression from the promoter/leader region of the full length transcript of figwort mosaic virus (FLt promoter) coupled to reporter genes were defined by 5' and 3' deletion analyses. In transient expression assays using protoplasts of Nicotiana edwardsonii, a 314 bp FLt promoter fragment sequence (-249 to +65 from the transcription start site) was sufficient for strong expression activity. Plant expression vectors developed with modified FLt promoters were tested with GUS or CAT as reporter genes in transgenic plants. The FLt promoter is a strong constitutive promoter, with strength comparable to or greater than that of the CaMV 35S promoter. The FLt promoter with its double enhancer domain linked to GUS or CAT reporter genes provides an average 4-fold greater activity than the FLt promoter with a single enhancer domain (-55 to -249 bp upstream fragment) in tests with transgenic plants and in protoplast transient expression assays.

Plants that express a potyvirus proteinase gene are resistant to virus infection.

Transgenic tobacco plants that express the genome-linked protein/proteinase-coding region of the potyvirus tobacco vein mottling virus (TVMV) were produced and tested for their reaction to inoculation with TVMV and two other potyviruses. These plants did not develop disease symptoms after being inoculated with large doses of TVMV but were as susceptible to infection by the other potyviruses as were control plants. Lines of tobacco that express the coat protein- or the nonstructural cylindrical inclusion protein-coding regions were also produced. The coat protein transgenic plants were protected against all three potyviruses, and the cylindrical inclusion transgenic plants were susceptible to all three potyviruses. These results indicate that some, but not all, TVMV genes can be used to confer protection against potyviruses in plants. The results also suggest that combinations of viral genes in transgenic plants might improve protection against potyviruses.

Tissue partitioning of cadmium in transgenic tobacco seedlings and field grown plants expressing the mouse metallothionein I gene.

Since agricultural crops contribute > 70% of human cadmium (Cd) intake, modification of crops to reduce accumulation of this pollutant metal during plant growth is desirable. Here we describe Cd accumulation characteristics of seedlings and field grown tobacco plants expressing the Cd-chelating protein, mouse metallothionein I. The objective of the transformation is to entrap Cd in roots as Cd-metallothionein and thereby reduce its accumulation in the shoot. Transformed and control seedlings were exposed for 15 days in liquid culture at a field soil-solution-like Cd concentration of 0.02 microM. Transformed seedlings of Nicotiana tabacum cultivar KY 14 contained about 24% lower Cd concentration in shoots and about 5% higher Cd concentration in roots than control seedlings. Dry weights of transformed and control tissues did not differ significantly. In the field in 1990, mature transformed N. tabacum cv. KY 14 plants exposed only to endogenous soil Cd contained about 14% lower leaf lamina Cd concentration than did controls. Differences were significant at the p < or = 0.1 level in 13 of 16 leaf positions. Leaf dry weight did not differ significantly but transformed field plants had 12% fewer leaves and were 9% shorter than the controls. Copper (Cu) concentration was significantly higher (ca10%) in the bottom nine leaf positions of transformed plants suggesting that reduced leaf number and plant height may be due to Cu deficiency or toxicity. Alternatively, somaclonal variation or gene position effects may be involved. No differences were found in zinc levels. With N. tabacum cv. Petit Havana, transformed seedlings contained no less Cd in shoots but 48% higher Cd concentration in roots.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction and turnover of catfish (Heteropneustes fossilis) metallothionein.

Induction of metallothionein by cadmium in catfish was a dose-dependent, transcriptionally-controlled process. Metallothionein mRNA was detected after Cd-exposure. Chronic doses produced more metallothionein than a single acute dose. Zn and Cu induced metallothionein to a lower extent compared to Cd. A few other low molecular weight proteins were induced in cadmium-exposed catfish liver, besides metallothionein. Isoelectric point of catfish metallothionein was 3.9. The rate of depletion of Cd and metallothionein was very slow from liver and almost unchanged from kidney following its induction by cadmium.

Comparison of the immunological properties of mammalian (rodent), bird, fish, amphibian (toad), and invertebrate (crab) metallothioneins.

Immunoassays such as rocket immunoelectrophoresis, western dot blot hybridization, and competitive ELISA analysis were used to estimate and compare homology among fishes (several species), crab, toad, chicken and rodent metallothioneins. The relative abundance of tissue-specific metallothionein in catfish was highest in liver followed by kidney and pancreas like mammalian systems. Immunologically, considerable homology exists among fish metallothioneins, although the extent of homology differs to some extent. No homology was found between fish or chicken metallothioneins. The amphibian (toad) and invertebrate (crab) metallothioneins showed only partial homology with fish metallothioneins in antigenic determinants.

Inheritance and expression of the mouse metallothionein gene in tobacco: impact on cd tolerance and tissue cd distribution in seedlings.

Genetically engineered seedlings obtained from self-fertilized transgenic tobacco (Nicotiana tabacum) contained and expressed the mouse metallothionein and kanamycin resistance marker genes and were more tolerant to cadmium stress than untransformed controls. Cadmium accumulation in leaves of transgenic seedlings exposed to a low, field-like Cd concentration (0.02 micromolar) was about 20% lower than that in untransformed controls. Genetic analysis of R1 and R2 progeny showed inheritance of the marker gene to be as a dominant Mendelian trait. These results suggest the possibility of developing transgenic plants with modified tolerance to heavy metal stress and food crops having lower Cd content.

Seed-transmissible expression of mammalian metallothionein in transgenic tobacco.

A binary plasmid was constructed to contain the mouse metallothionein c-DNA, the constitutive 35S promoter from cauliflower mosaic virus, the polyadenylation signal from the pea rbcS-E9 gene and several selectable markers. The plasmid was transferred to Agrobacterium tumefaciens and the leaf disc method was used to transform tobacco. Callus and shoots were regenerated in the presence of kanamycin and transformed plants were obtained. Southern, Northern and Western blot analysis demonstrated integration and expression of the metallothionein gene in transformed callus and transgenic plants. The gene is transmitted to and expressed in seed derived progeny as a dominant Mendelian trait.

Purification and immunological characterization of catfish (Heteropneustes fossilis) metallothionein.

Catfish hepatic metallothionein was purified to homogeneity by Sephadex G-75 gel filtration, DEAE-Sephadex A-25 column chromatography and preparative polyacrylamide gel electrophoresis. Induction by cadmium and zinc, characteristic UV spectrum, cadmium binding property and its low MW established that it was a metallothionein. Antibody was raised in rabbit against catfish metallothionein. Catfish antimetallothionein cross-reacted with other fish metallothioneins but not with chicken or rodent metallothionein. Catfish metallothionein is more electronegative as compared to mouse, rat, chicken or hamster metallothionein. Catfish MT appeared to aggregate readily on storage and to be less electronegative.

Immunological and biochemical properties of metallothioneins of golden hamster, mouse and rat.

Golden hamster, mouse and rat hepatic cadmium metallothioneins (MT) were purified by Sephadex G-75 gel filtration, DEAE-Sephadex A-25 chromatography and activated Thiol-Sepharose 4B affinity chromatography. Metallothioneins were separated by DEAE-Sephadex A-25 chromatography into two forms: MT-1 and MT-2. In mouse and golden hamster liver, MT-1 was the major form. The purified proteins were homogeneous as judged by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. In non-denaturing polyacrylamide gel electrophoresis, migration of mouse, rat and golden hamster hepatic metallothioneins were found to be different. Antibodies to mouse hepatic MT-1 was raised in rabbits. The antiserum cross reacted with mouse and hamster MT-1 and MT-2 giving a single precipitin band. Mouse, rat and hamster hepatic MTs are immunologically identical but electrophoretically different. The kidney and pancreatic MTs of rat and golden hamster were purified by Sephadex G-75 gel filtration. They were immunologically distinct. Pancreas MT formed a line of partial identity with hepatic MTs. Kidney MTs form two precipitin band one identical with the pancreatic form and another of complete identity with the hepatic MTs. This indicates the presence of tissue specific MTs.

Biochemical and genetic characterization of respiration-deficient mutants of Chinese hamster cells with a Gal- phenotype.

Four Chinese hamster somatic cell mutants A13G9, 34A13G32, 2A13G14, and V6IG15 with a Gal- phenotype have the following characteristics: (1) a low respiration rate; (2) a reduced Krebs cycle activity; (3) a low level of stimulation of oxygen consumption of mutant mitochondria by malate; (4) an absolute dependence on an ample supply of glucose to sustain a high rate of glycolysis; (5) a defect in the electron transport chain from NADH to coenzyme Q; and (6) no appreciable activity of rotenone-sensitive NADH oxidase in mutant mitochondria. These four mutants and another mutant, P12GX1, were analyzed by complementation analysis using seven other respiratory mutants of Dr. Scheffler which define seven complementation groups (I-VII). P12GX1 fails to complement mutant CCL16-B9 (group IV). A13G9 and 34A13G32 do not complement each other. Mutants V6IG15, A13G9, and 34A13G32 define two new groups of complementation (VIII and IX), while 2A13G14 does not complement mutants of groups II and VI.

Prevention of fungal infection of plants by specific inhibition of cutinase.

Specific antibodies prepared against cutinase from Fusarium solani pisi and diisopropylfluorophosphate, a potent inhibitor of this enzyme, prevented infection of the host (pea epicotyl) by this organism, without affecting the viability of the spores. This finding shows that enzymatic penetration of cuticle is involved in pathogenesis.

Evidence for a Functional myo-Inositol Oxidation Pathway in Lilium longiflorum Pollen.

Addition of myo-inositol to pentaerythritol-based germination media repressed the conversion of d-[1-(14)C]glucose to labeled uronosyl and pentosyl units of tube wall pectic substance in lily pollen (Lilium longiflorum Thunb.). Conversion of d-[1-(14)C]glucose to labeled glucosyl, galactosyl, and rhamnosyl units was unaffected. The reverse experiment, addition of d-glucose to pentaerythritol-based media, failed to affect the conversion of myo-[2-(3)H]inositol to uronosyl and pentosyl units although the flow of label into products of myo-inositol-linked glucogenesis was blocked. Results of these experiments are discussed in terms of a functional myo-inositol oxidation pathway.d-[1-(14)C]Glucose-labeled pollen tubes contain a labeled, 70% ethyl alcohol-soluble, acidic compound whose formation is blocked by the myo-inositol antagonist, 2-O,C-methylene-myo-inositol (Chen et al. 1977 Plant Physiol. 59: 658). This compound has been identified as l-malic acid.

myo-Inositol metabolism in germinating wheat.

During imbibition, exogenous myo-inositol (MI) was readily introduced into the free MI pool of germinating wheat (Triticum aestivum L.). Maximum uptake, 70 µg per caryopsis or 1.5 mg g(-1) of caryopsis, was reached at 0.05 M MI. Movement of free MI within the germinating caryopsis was traced with [2-(3)H]MI by two procedures, uptake by imbibition and injection into softened endosperm. The former procedure was useful during initial stages of germination; the latter provided a means of tracing the metabolic fate of MI generated by hydrolysis of phytate during mobilization of reserves within the caryopsis. In both procedures, the bulk of the added label was transferred to the seedling where it appeared in uronosyl and pentosyl units of 80% ethanol-insoluble polysaccharides, 2-O, C-Methylene-MI, an inhibitor of the MI oxidation pathway, blocked the utilization of [2-(3)H]MI as well as D-[1(14)C]glucose for biogenesis of pentose-and uronic-acid-containing polysaccharides.