EP0994957A1 - Plantes transgeniques employant le gene de tdc (tryptophane decarboxylase) en vue d'une amelioration des cultures - Google Patents

Plantes transgeniques employant le gene de tdc (tryptophane decarboxylase) en vue d'une amelioration des cultures

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Publication number
EP0994957A1
EP0994957A1 EP98938251A EP98938251A EP0994957A1 EP 0994957 A1 EP0994957 A1 EP 0994957A1 EP 98938251 A EP98938251 A EP 98938251A EP 98938251 A EP98938251 A EP 98938251A EP 0994957 A1 EP0994957 A1 EP 0994957A1
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Prior art keywords
tryptamine
tdc
plant tissue
gene
nucleic acid
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German (de)
English (en)
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John Sanford
Alan D. Blowers
Franzine Smith
Joyce Van Eck
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Sanford Scientific Inc
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Sanford Scientific Inc
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Publication of EP0994957A1 publication Critical patent/EP0994957A1/fr
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8281—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for bacterial resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8282—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for fungal resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8285—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for nematode resistance
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the present invention relates to transgenic plants having surprisingly improved fungal, bacterial, and/or nematode disease resistance, wherein the enhanced resistance arises from enhanced expression of a tryptophan decarboxylase (TDC) gene.
  • TDC tryptophan decarboxylase
  • defensins Other antimicrobial peptides, termed defensins (for review, see Broekaert et al. (1995) Plant Physiol. 108, 1353-1358) have been isolated from radish (Terras et al. (1992) J. Biol. Chem. 267, 15301-15309) and barley (Mendenez et al. (1990) Eur. J. Biochem. 194, 533-539) and feature a more complex three-dimensional structure that includes cysteine-stabilized triple anti-parallel ⁇ sheets together with an ⁇ -helix. Terras et al.
  • ribosome-inactivating proteins which act by inhibiting protein synthesis in target cells by a modification of the 28S rRNA.
  • RLP's do not affect the ribosomes of the plants in which they are produced, but can be effective against fungal ribosomes.
  • a barley RIP under control of a wound-inducible promoter was reported to show increased resistance to Rhizoctonia solani (Logemann et al. (1992) Biotech 10, 305-308). Finally, Alexander et al. (1993) ProcNatl. Acad.
  • Plants produce a rich diversity of secondary metabolites, which do not seem necessary for their basic metabolism, but appear to contribute to their environmental fitness and adaptability. These secondary compounds are responsible for aroma (monote ⁇ ene indole alkaloids) and color (anthocyanins and carotenoids) and are commercial sources of numerous important pharmaceutical (alkaloids) and industrial chemicals. Their importance has encouraged intensive investigation of the regulation and control of the biosynthetic pathways, and more recently, how these pathways can be manipulated through "metabolic engineering,” first coined by Bailey, J.E. (1991) Science 251, 1668- 1675, as "the improvement of cellular activities by manipulation of enzymatic, transport, and regulatory functions of the cell with the use of recombinant DNA technology.”
  • the enzymology associated with the biosynthesis of alkaloids has been the subject of much study.
  • the tropical plant, Catharanthus roseus (periwinkle) forms a wide range of te ⁇ enoid indole alkaloids (TIA's), some of which have important medicinal applications.
  • TIA's te ⁇ enoid indole alkaloids
  • the leaf-derived alkaloids, vinblastine and vincristine, and the root-derived alkaloids, ajmalicine and se ⁇ entine are valuable drugs for treatment of cardiac/circulatory diseases and tumors, respectively. It is generally accepted that protoalkaloid production is the first committed step in the TIA pathway of Catharanthus.
  • tryptophan decarboxylase the enzyme that catalyzes this bridge reaction between primary (amino acid) and secondary (alkaloid) metabolic pathways, has drawn much attention.
  • TDC catalyzes the decarboxylation and conversion of L-tryptophan into tryptamine. Tryptamine and horrin, another secondary compound, are then condensed to form strictosidine, the precursor for all TIA's in Catharanthus.
  • anthranilate synthase a heterodimeric enzyme composed of an ⁇ and ⁇ subunit, which catalyzes the conversion of chorismate to anthranilate. Tryptophan has been demonstrated to be a negative feedback regulator of the microbial enzyme. In microbes, when tryptophan levels are sufficiently high, tryptophan binds to an allosteric site on the ⁇ subunit, thereby inactivating the enzyme. Although similar observations have not been made for the plant enzyme, other studies suggest that AS is the rate-limiting enzyme for tryptophan biosynthesis. Plant mutants characterized by elevated tryptophan levels ( ⁇ 3-fold) have been found to contain amino acid changes in their anthranilate synthase coding regions.
  • TDC steady-state transcript levels were most abundant in roots, moderately abundant in leaves and barely detectable in the flowers and stems of 3-month-old Catharanthus plants. They further demonstrated that TDC expression in cell suspension cultures was down-regulated by addition of auxin, but strongly induced by treatment with fungal elicitors. This report, along with others (Roewer et al. (1992) Plant Cell Rep. 11, 86-89; Berlin et al. (1993) Transgenic Res. 2, 336-3444; Goddijn et al. (1992) Plant Mol. Biol. 18, 1113-1120; and Goddijn et al. (1995) Transgenic Res. 4, 315-323), helped establish the importance of TDC as the first committed step in TIA biosynthesis.
  • transgenic canola In contrast to tobacco, transgenic canola, which overexpressed the same TDC transgene, exhibited a very striking phenotype. Chavadej et al. (1994) Proc. Natl. Acad. USA 91, 2166-2170, reported that their best transgenic canola line never expressed >9% of the TDC-specific activity and only accumulated 2% of the tryptamine found in the most active transgenic tobacco line.
  • tryptamine accumulation was found to be very tissue specific (Yao et al. (1995) Plant Cell 7, 1787- 1799). While tryptamine accumulated to high levels in the leaves of transgenic potato plants, tryptamine was undetectable in the tubers and was only detected after wounding or fungal elicitor treatment. The re-direction of tryptophan into tryptamine resulted in a dramatic decrease in the levels of soluble tryptophan, phenylalanine, and phenylalanine derived phenolic compounds, including chlorogenic acid, the major soluble phenolic ester in potato tubers.
  • tryptamine can effectively inhibit in vitro and in vivo growth of both phytopathogenic bacteria and fungi. Furthermore, tryptamine is shown herein to inhibit phytopathogenic nematodes.
  • the present invention affords constitutive expression in plants of the gene encoding tryptophan decarboxylase (TDC), the enzyme that converts tryptophan to tryptamine, to confer enhanced resistance to infection to a broad spectrum of phytopathogenic fungi, bacteria, and nematodes.
  • TDC tryptophan decarboxylase
  • AS gene can boost this pathway, making more ty ⁇ tamine.
  • the present invention further provides a gene construct containing a promoter and a DNA sequence encoding a protein with tryptophan decarboxylase activity and/or AS activity.
  • the invention also provides transgenic plants and multicellular plant tissue having enhanced fungal and/or bacterial disease resistance and/or nematode resistance, wherein the enhanced resistance is a result of expression of a TDC and/or AS transgene.
  • FIG. 1 Chimeric (A) E35S::TDC::nos and (B) UBQ3::TDC::nos constructs.
  • a DNA fragment containing the duplicated enhancer region, promoter, transcription initiation site and 5 ' untranslated region (UTR) from the CaMV 35S RNA region was fused to the 5 ' UTR of the TDC cDNA from plasmid pTDC5.
  • a second DNA fragment containing the promoter, transcription initiation site and 5 ' UTR from the Arabidopsis thaliana UBQ3 gene was fused to the 5 'UTR of the
  • TDC cDNA from plasmid pTDC5.
  • a DNA fragment containing the polyA addition signal from the nopaline synthase gene (nos) is responsible for transcript maturation at the 3 ' end in both transgenes.
  • the present inventors have su ⁇ risingly discovered (as is demonstrated in the Examples, infra) that enhanced TDC expression can confer on plants resistance to a broad spectrum of both bacterial, fungal, and nematode phytopathogens.
  • the present invention comprises methods and nucleic acid constructs for enhancing resistance in plants to phytopathogenic bacteria, fungi, and nematodes by transforming plant cells with the gene coding for tryptophan decarboxylase (TDC).
  • TDC tryptophan decarboxylase
  • the invention is not limited by any theory of action, we believe that enhanced TDC expression results in enhanced phytopathogenic resistance through increased levels of tiyptamine and/or alkaloids produced therefrom.
  • the invention further comprises transgenic plant tissue thereby produced.
  • the invention provides nucleic acid constructs comprising the TDC gene. Any plant TDC gene can be used. Upon transformation of plant cells, these constructs are useful for conferring enhanced fungal, bacterial, and/or nematode resistance to a wide variety of plants and against a broad spectrum of phytopathogenic fungi, bacteria, and nematodes.
  • the invention provides nucleic acid constructs comprising an AS (preferably AS ⁇ l) and a TDC transgene. Any plant AS gene can be used.
  • Co-introduction of constitutively- expressed AS (preferably AS ⁇ l) and TDC transgenes should provide increased tryptophan levels for immediate conversion into tryptamine.
  • AS constitutively- expressed transgenes
  • a single plasmid harboring both constitutively-expressed transgenes can be viewed as portable expression cassette for increasing tryptamine levels in virtually any plant.
  • an AS transgene could be used by itself.
  • the nucleic acid constructs according to this aspect of the invention will further comprise targeting regions at the 3' and 5' ends, which regions target the constructs according to the invention to the plant nucleus or plastid.
  • any plant promoter can be operatively linked to the TDC and/or AS ⁇ l genes.
  • the constructs according to this aspect of the invention will preferably be operatively linked to the UBQ3, UBQIO, CaMV 35S RNA, or the enhanced version of the CaMV 35S RNA (E35S) promoter.
  • the constructs further comprise a pUC-based vector containing the 3 ' flanking region of the nopaline synthase gene (nos) from Agrobacterium tumefaciens.
  • one or both of the transgenes can be fused and thereby operationally linked to tissue-specific promoters.
  • tissue-specific promoters for example, in petunia, one or both of the transgenes can be fused to petal-specific promoters to confer resistance to infection by Botrytis cinerea.
  • Constructs according to this aspect of the invention can also comprise a selectable marker gene, expression of which by the transformed cell enables one to identify and isolate the transformed cell or cells from amongst other cells. Any plant selectable marker gene known in the art can be used in the present invention.
  • the selectable marker gene is the nptll (neomycin phosphotransferase II) or hph (hygromycin phosphotransferase) gene.
  • Cells expressing these preferred selectable marker genes are resistant to kanamycin (for nuclear nptll transformation) and hygromycin (for nuclear transformation) or glyphosate (for plastid hph transformation) and can be selected for by exposing cells subject to transformation (by, e.g., biolistic delivery) to media containing the minimum level of kanamycin, hygromycin, or glyphosate that kill untransformed cells.
  • the agent to which transformed cells are subject for selection pu ⁇ oses should correspond to the selectable marker gene employed in the transformation.
  • the sequence and structure of the constituent elements of the nucleic acid constructs according to this aspect of the invention are publicly available, and constructs according to this aspect of the invention can be made by routine, art recognized techniques. Exemplary methods are described, e.g., in Example 1, infra.
  • the invention provides methods for enhancing the resistance of plants to phytopathogenic fungi, bacteria, and nematodes.
  • the method comprises transforming plant tissue with a construct according to the first aspect of the invention.
  • the two genes when co-transformation with both the TDC and AS ⁇ l genes is desired, can be on separate expression vectors and co-transformed simultaneously or sequentially. Transformation can be accomplished in either the nucleus or the plastid, as determined by the targeting regions of the nucleic acid construct. Details of plastid transformation can be found, e.g., in co-pending international application PCT/US98/**** (WO 99/*****) ? entitled, "Improved Plastid Transformation Of Higher Plants And Production Of Transgenic Plants With Herbicide Resistance," filed July 23, 1998, and U.S. Application Serial No. 08/899,061, filed July 23, 1997. Any of the numerous methods for transformation can be used, e.g., Agrobacterium (for nuclear transformation), PEG treatment, electroporation, and biolistic delivery. Preferably, biolistic delivery is employed.
  • the invention provides transgenic plant tissue that expresses the TDC gene and/or the AS ⁇ l gene.
  • plant tissue includes a plant cell or cells, multicellular plant tissue, and whole plants.
  • Transgenic plants according to this aspect of the invention can be made according to the second aspect of the invention using nucleic acid constructs according to the first aspect of the invention.
  • cells e.g., cell suspensions or calli
  • plant tissue samples e.g., leaf or other plant parts
  • TDC- and/or AS-containing constructs To increase tryptamine production throughout the entire plant, the TDC gene was preferably placed under the control of two very active, constitutively expressed promoters.
  • the CaMV 35S RNA promoter from the CaMV genome is a very well-characterized promoter for expression of transgenes in both dicotyledonous and monocotyledonous plants.
  • the enhanced version (Kay et al, (1987) Science 236, 1299-1302,) of the CaMV 35S RNA promoter (E35S) was preferably used.
  • a second promoter, UBQ3, is derived from Arabidopsis thaliana and normally directs expression of a member of the polyubiquitin gene family. The present inventors have determined that this promoter directs high levels of reporter gene expression throughout the entire plant in transgenic petunias.
  • Both promoters were moved into a pUC -based vector already containing the 3 ' flanking region of the nopaline synthase gene (nos) from Agrobacterium tumefaciens.
  • This DNA sequence element possesses the recognition site for polyA addition to the transcript.
  • Also already resident on this plasmid was a multi-cloning site region containing a number of unique restriction enzyme sites for insertion of additional DNA sequence elements.
  • the CaMV 35S and UBQ3 promoters were moved into this plasmid (while maintaining a number of unique restriction sites between the promoter and the nos 3' sequence element) to create plasmids pSAN14 and pSAN151, respectively.
  • the TDC gene was originally cloned from Catharanthus roseus, or vinca (De Luca et al., (1989), supra.
  • a 1.75 kbp fragment containing the full-length cDNA was cloned from a cDNA library by immunodetection methods.
  • the TDC cDNA contains an open reading frame coding for a protein of 500 amino acids, corresponding to a molecular mass of -56 kDa.
  • the cDNA from plasmid pTDCS (De Luca et al., 1989, supra) was removed by digestion with Pst I and Xho 1, the single-strand overhangs at each end removed by treatment with T4 DNA polymerase, and cloned into the Sma I sites of plasmids ⁇ SAN14 and pSAN151 to create plasmids pSAN213 and pSAN247, respectively ( Figure 1).
  • TDC-containing plasmids were to be co-bombarded with a second plasmid containing a plant selectable marker gene like nptll (neomycin phosphotransferase 11) or hph (hygromycin phosphotransferase), no marker gene was added to the TDC-containing plasmid.
  • a plant selectable marker gene like nptll (neomycin phosphotransferase 11) or hph (hygromycin phosphotransferase
  • Construct pSAN368 contains the AS ⁇ l gene driven by the UBQl 0 promoter and, constructs pSAN369 and pSAN310 contained in addition to the AS ⁇ l gene the TDC gene driven by the UBQ3 pormoter.
  • TDC transgenes were introduced into petunia by particle bombardment. Briefly, two plasmids, one containing the TDC transgene and the second containing an nptll transgene, were co- precipitated in equivalent amounts onto - 1 ⁇ m M-10 tungsten particles. The DNA coated particles were then bombarded into petunia leaf explants that had been placed on a nutrient agar media. After two successive bombardments, the leaf explants were allowed to recover on the nutrient media for four to five days. After recovery, the leaf explants were cut into small pieces and placed onto selective agar media containing kanamycin. Kanamycin-resistant transformed shoots were regenerated and shoots were transferred to fresh media and rooted in the absence of kanamycin.
  • MIC Tryptamine minimum inhibitory concentration
  • a MIC minimum tryptamine concentration required to inhibit all fungal growth after 48 hours, not assayed
  • Phytophthora parasitica were inhibited by 0.25 mg/ml tryptamine. Germinated spores from both Phytophthora isolates were sensitive to 0.25-0.50 mg/ml tryptamine. Similar results were observed for Thielaviopsis basicola (germinated and non-germinated spores). Regarding the Fusarium species, non-germinated spores from F. graminearum were somewhat sensitive to tryptamine while those from F. solani were considered insensitive. However, germinated spores from F. solani were sensitive. Also, growth from nongerminated spores of Botrytis cinerea were inhibited by 0.50 mg/ml tryptamine. Rhizoctonia solani mycelial fragments were unaffected by 1 mg/ml tryptamine. In general, nearly all the fungi exhibited some level of sensitivity to treatment with tryptamine.
  • tryptamine's mode of action was fungicidal or fungistatic.
  • Botrytis cinerea spores were exposed to toxic levels of tryptamine for various times, then diluted to sub-lethal concentrations to permit growth. If the spores now germinated and grew, then tryptamine's effect was considered to be fungistatic. If no growth was observed, the effect was concluded to be fungicidal.
  • Botrytis spores maintained in 0.50 mg/ml tryptamine throughout the entire experiment showed essentially no growth while all growth was inhibited at 1 mg/ml.
  • Botrytis spores exposed to 1 mg/ml tryptamine for 22 h showed no growth, but when diluted to 0.25 mg/ml, germinated and grew normally over the next 48 h.
  • spores exposed to 2 mg/ml for 22 h showed no growth, but when diluted to 0.5 mg/ml, germinated and grew, albeit relatively poorly.
  • Tryptamine acts primarily as a fungistatic agent against Botrytis cinerea rather than a fungicidal one
  • MIC Tryptamine minimum inhibitory concentration
  • MIC minimum tryptamine concentration required to inhibit all bacterial growth after 24 hrs.
  • Petunia line PE-261 contains the TDC gene under control of the E35S promoter while lines PE-310 and PE-312 contain the TDC gene under control of the UBQ3 promoter.
  • Tryptamine levels were measured in the leaf tissue of both in vitro and greenhouse-grown plants.
  • Leaf tissue from in vitro and greenhouse plants and petal tissue from greenhouse plants (all -10-20 mgs fresh weight) were excised and homogenized in 0.2 ml cold 50 mM Na 3 PO 4 , pH 7.4 buffer. Cell debris was removed by centrifugation at 10,000 x g for 5 min at 4 °C.
  • the tryptamine levels ranged from 141-310 ⁇ g/gfw.
  • the levels decreased approximately 4-fold for all three lines to 42-80 ⁇ g/gfw. It is especially worth noting that this decrease can most likely be attributed to a reduction in the level of available tryptophan in the cell.
  • the levels of tryptophan in the leaves of the untransformed V26 control and each of the transgenic lines dropped -4-fold when plants were transferred to the greenhouse. In all three transgenic lines, the ratio of tryptamine to tryptophan content in the cells remained unchanged.
  • Transgenic TDC-expressing Petunia resistance to powdery mildew Greenhouse-grown petunia lines PE-261 , PE-310, PE-312 and an untransformed V26 control were inoculated on their leaf surface with a 5 ⁇ l droplet containing 10 3 powdery mildew spores. The third leaf (from the top) on each of five shoots of a single plant was inoculated. Each line was represented by four plants (for a total of 20 inoculation sites). Disease progression was monitored for approximately two weeks by recording the percentage of inoculated sites infected and measuring the diameter of the fungal colony with calipers to calculate their size (area). The results are displayed in Table 6.
  • V26 control and PE-261 lines showed equal susceptibility to infection by Botrytis.
  • lines PE-310 and PE-312 still exhibited greatly reduced rates of infection by Botrytis.
  • PE-310 flowers still showed only a small number of isolated lesions. Disease symptoms were only slightly more advanced in PE312 flowers as the lesions were more numerous and larger.
  • leaf disks of transgenic petunia lines were inoculated with the foliar nematode Aphelenchoides fragariae. Briefly, leaf disks (2.1 cm diameter) were cut from surface sterilized greenhouse grown leaves of the transgenic V26 petunia lines (PE-310 and PE-312) and the non-transgenic control. Disks were placed onto wet filter paper in a multi-well dish (1 disk/well) and then inoculated with a drop containing 50 A.fragariae nematodes that had been raised on a sterile tobacco callus culture.
  • Inoculated disks were incubated for eighteen days at room temperature and then the number of nematodes per well was quantified. Although, there was an increase in nematode population in each well, the two transgenic lines (PE- 310, PE-312) showed only a slight increase (from 50 to 74 or 67 respectively) while the non- transgenic control increased four fold (from 50 to 194).
  • TDC transgenic plants of any species overexpressing the TDC and/or AS gene should be resistant to nematodes, as long as the target tissue has adequate amounts of tryptamine. In the plants we have tested, tryptophan and tryptamine levels are low in the roots.
  • Botrytis cinerea resistance in other transgenic plant species Several other plant species (poinsettia cv. Angelika, geranium Designer Scarlet, lisianthus and another bedding plant) were transformed with the TDC gene. A measurable increase in tryptamine was found in transgenic poinsettia and geranium lines. Transgenic poinsettia lines demonstrated resistance to Botrytis cinerea in a leaf disk assay. Briefly, twelve leaf disks (8 mm in diameter) from each tissue culture-maintained transformed lines (and an untransformed control) were punched out with a cork borer and placed onto moistened Whatman 3M paper inside a sterile plastic bioassay dish.
  • a freshly-prepared suspension of Botrytis spores (10 3 spores in 2.5 ⁇ l) was then pipetted onto the leaf disk surface.
  • the humidity chamber was sealed and the leaf disks left at 20°C to permit disease development.
  • timeline is species-dependent
  • disease progression was monitored and recorded as percentage of leaf disks infected.
  • Transgenic geranium lines demonstrated resistance to Botrytis cinerea infection when flowers were inoculated.
  • transgenic plant species we observed increased tryptophan and tryptamine concentration when the AS/TDC combination was used.

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  • Enzymes And Modification Thereof (AREA)

Abstract

La présente invention concerne des produits de recombinaison d'acides nucléiques et des procédés utiles pour la production de plantes transgéniques présentant une résistance accrue à des champignons, des bactéries et/ou des nématodes, cette résistance accrue provenant de l'expression accrue d'un produit de recombinaison de gène de tryptophane décarboxylase (TDC).
EP98938251A 1997-07-31 1998-07-31 Plantes transgeniques employant le gene de tdc (tryptophane decarboxylase) en vue d'une amelioration des cultures Withdrawn EP0994957A1 (fr)

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US5431697P 1997-07-31 1997-07-31
US54316P 1997-07-31
PCT/US1998/016033 WO1999006581A1 (fr) 1997-07-31 1998-07-31 Plantes transgeniques employant le gene de tdc (tryptophane decarboxylase) en vue d'une amelioration des cultures

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EP0994957A1 true EP0994957A1 (fr) 2000-04-26

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EP98938251A Withdrawn EP0994957A1 (fr) 1997-07-31 1998-07-31 Plantes transgeniques employant le gene de tdc (tryptophane decarboxylase) en vue d'une amelioration des cultures

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EP (1) EP0994957A1 (fr)
JP (1) JP2001512028A (fr)
AU (1) AU8682198A (fr)
CA (1) CA2298882A1 (fr)
WO (1) WO1999006581A1 (fr)

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US6972197B1 (en) 1999-03-18 2005-12-06 The University Of Chicago Plant chromosome compositions and methods
CA2904855A1 (fr) * 1999-03-18 2000-09-21 The University Of Chicago Compositions de chromosomes vegetaux et methodes
EP1542527B1 (fr) * 2001-05-04 2012-02-08 Renessen LLC Plantes transgeniques a contenu eleve en tryptophane
EP2216405A1 (fr) 2002-05-03 2010-08-11 Monsanto Technology LLC Promoteurs USP spécifiques de sémences pour l'expression de gènes dans des plantes
BR0309860A (pt) 2002-05-03 2007-11-06 Monsanto Technology Llc plantas transgênicas com elevado teor de triptofano
US7078234B2 (en) 2002-12-18 2006-07-18 Monsanto Technology Llc Maize embryo-specific promoter compositions and methods for use thereof
US7294760B2 (en) 2003-03-28 2007-11-13 Monsanto Technology Llc Plant promoters for use in early seed development
ES2346224T4 (es) * 2003-11-10 2011-06-01 Icon Genetics Gmbh Sistema de expresión de planta derivado de virus de arn.
CA2598307C (fr) 2005-02-26 2014-12-30 Basf Plant Science Gmbh Cassettes d'expression destinees a une expression preferentielle de semences chez des plantes
US8034994B2 (en) 2005-04-19 2011-10-11 Basf Plant Science Gmbh Starchy-endosperm and/or germinating embryo-specific expression in mono-cotyledonous plants
US7790873B2 (en) 2005-05-10 2010-09-07 Basf Plant Science Gmbh Expression cassettes for seed-preferential expression in plants
EP2074219B1 (fr) 2007-02-16 2013-11-20 BASF Plant Science GmbH Séquences d'acides nucléiques pour la régulation de l'expression spécifique de l'embryon dans des plantes monocotyles
CA2758824A1 (fr) 2009-04-17 2010-10-21 Basf Plant Science Company Gmbh Promoteur vegetal apte a agir dans l'endosperme et ses utilisations
WO2011003901A1 (fr) 2009-07-10 2011-01-13 Basf Plant Science Company Gmbh Cassettes d'expression pour l'expression spécifiquement dans l'endosperme dans des plantes
WO2011011693A1 (fr) 2009-07-23 2011-01-27 Chromatin, Inc. Séquences de centromère et mini-chromosomes de sorgho
BR112012013156A2 (pt) 2009-12-03 2017-06-13 Basf Plant Science Co Gmbh cassete de expressão, vetor, célula horpedeira, tecido de planta transgênica e método para a produção de um tecido de planta transgênica
EP2547195A4 (fr) * 2010-03-18 2014-01-08 Basf Plant Science Co Gmbh Plantes possédant des caractéristiques favorisant le rendement, et leur procédé d'obtention
JP2013036889A (ja) * 2011-08-09 2013-02-21 Hamamatsu Photonics Kk 植物体の病原菌感染診断装置及び植物体の病原菌感染診断方法

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AU8682198A (en) 1999-02-22
JP2001512028A (ja) 2001-08-21
CA2298882A1 (fr) 1999-02-11
WO1999006581A1 (fr) 1999-02-11

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