WO1994018335A2 - Procede d'elimination de champignons pathogenes des plantes - Google Patents

Procede d'elimination de champignons pathogenes des plantes Download PDF

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WO1994018335A2
WO1994018335A2 PCT/US1994/000844 US9400844W WO9418335A2 WO 1994018335 A2 WO1994018335 A2 WO 1994018335A2 US 9400844 W US9400844 W US 9400844W WO 9418335 A2 WO9418335 A2 WO 9418335A2
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plant
ribonuclease
sequence
promoter
protein
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PCT/US1994/000844
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WO1994018335A3 (fr
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Catherine Michiko Hironaka
Quang Khai Huynh
Dilipkumar Maganlal Shah
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Monsanto Company
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/50—Isolated enzymes; Isolated proteins
    • 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
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • 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

  • This invention relates to a method of controlling plant pathogenic fungi by a protein which may be applied directly to the plant or produced thereon by microorganisms or by genetically modifying the plant to produce the protein, and to genes, microorganisms, and plants useful in that method.
  • BACKGROUND OF THE INVENTION Ribonucleases are enzymes which cleave RNA by a variety of mechanisms.
  • the enzyme may attack the terminal diester bond of the nucleic acid; it may hydrolyze the 3'-phosphate, or it may split the nucleotide chain without hydrolysis. There are various mechanisms, but the end result is a degradation of RNA. [Wilson]
  • Plant ribonucleases have been evaluated for their function in self- incompatibility, which is a mechanism to prevent inbreeding in plants.
  • the enzymes are typically glycoproteins which operate as gametophytic or sporophytic incompatibility systems. [Haring, et al.] Until now no other practical function was known.
  • ribonucleases have antifungal properties. It is an object of the present invention to provide a ribonuclease from a plant or microorganism that is capable of reducing or eliminating the damage caused by plant fungal pathogens and genes useful in producing such proteins. It is a further object of the present invention to provide genetic constructs for and methods of inserting such genetic material into plant- colonizing microorganisms and plant cells. It is another object of the present invention to provide transformed microorganisms and plants containing such genetic material.
  • the plants may also be transformed to co-express other antifungal proteins or insecticidal proteins, for example, using Bacillus thurengiensis (B.t.) genes.
  • Bacillus thurengiensis (B.t.) genes are disclosed in European Patent Publication No. 0 385 962, which corresponds to U.S. Serial Number 07/476,661, filed February 12, 1990 [Fischhoff et al.], which is incorporated herein by reference.
  • Another example of genes which may be coexpressed with the proteins of the present invention is the group of PR proteins having antifungal activity, as discussed above. Examples are found in EP 0 460 753 [Woloshuk] and copending U.S. Serial No.
  • a method of controlling fungal damage to plants by providing a ribonuclease to the plant locus comprising in operative sequence: a) a promoter which functions in plant cells to cause the production of an RNA sequence; and b) a structural coding sequence that codes for production of a ribonuclease; c) a 3' non-translated region which functions in plant cells to cause the addition of polyadenylate nucleotides to the 3' end of the RNA sequence, said promoter being heterologous with respect to the structural coding sequence.
  • a method of producing genetically transformed plants which express an antifungal amount of a ribonuclease comprising the steps of: a) inserting into the genome of a plant cell a recombinant, double- stranded DNA molecule comprising
  • a promoter which functions in plant cells to cause the production of an RNA sequence;
  • a structural coding sequence that codes for production of a ribonuclease;
  • a 3' non-translated region which functions in said plant cells to cause the addition of polyadenylate nucleotides to the 3' end of the RNA sequence, said promoter being heterologous with respect to the structural coding sequence;
  • ribonuclease is used to indicate an enzyme from plants or microorganisms which uses RNA as a substrate.
  • controlling fungal damage is used to indicate causing a reduction in damage to a crop due to infection by a fungal pathogen.
  • structural coding sequence means a DNA sequence which encodes for a polypeptide, which may be made by a cell following transcription of the DNA to mRNA, followed by translation to the desired polypeptide.
  • plant locus means the area immediately surrounding a plant and including the plant and its root zone.
  • the method of the present invention may be carried out in a variety of ways.
  • the antifungal protein prepared by various techniques, may be directly applied to plants in a mixture with carriers or other additives, including other antifungal agents.
  • the protein may be expressed by bacterial or yeast cells which have been applied to the plant.
  • plant cells are transformed by one or more means to contain the gene encoding a ribonuclease which is expressed constitutively or in certain plant parts or upon exposure of the plant to the fungal infection.
  • One embodiment of the present invention comprises a protein isolated from the leaves of Engelmannia pinnatifida, commonly known as Engelman's daisy.
  • This protein has been purified to homogeneity by ammonium sulfate precipitation, Mono-Q ion exchange and C ⁇ 8 reverse phase column chromatography.
  • the purified protein has a molecular weight of almost 30.0 kD. It inhibits the growth of the agronomically important pathogens, Phytophthora infestans (Pi), a causal fungal pathogen of late blight disease in potato and tomato, and Gaeumannomyces graminis var tritici (Ggt), a causal fungal pathogen of Take-all disease in cereals, with an amount as little as 50 ng under the assay conditions.
  • Phytophthora infestans Pieris
  • Gaeumannomyces graminis var tritici Gaeumannomyces graminis var tritici
  • N-terminal amino acid sequence analysis of the purified P-8 protein indicates that it has some degree of homology to a known phosphate starvation-induced ribonuclease from tomato [Loeffler], but P-8 was isolated from a perennial plant not under any known extraordinary stress.
  • Another embodiment of the present invention comprises a ribonuclease derived from a microorganism.
  • ribonucleases from Bacillus cereus, E. coli, and Physarum polycephalum. If plant expression of such proteins is desired, the genes may be extracted by known methods and inserted into the genome of plants, optionally after synthesizing new genes having improved expression levels in planta.
  • the plants that may be protected by the methods of the present invention will depend on the level of protection needed for the fungal pathogens of that plant type. For example, many vegetables such as potatoes and tomatoes may be protected from Pi by the present methods. However, other Phytophthora species are pathogenic to many other plants, such as fruit trees or turf, and thus these plants may also be protected by the methods of the present invention. Furthermore, wheat and barley plants may be protected from Ggt by the present method.
  • the antifungal proteins of the present invention may be used in combination with other antifungal proteins so as to provide a broad spectrum of activity, i.e., control additional pathogens, and/or provide multiple modes of action for the inhibition of same fungal pathogen. Sources of such antifungal proteins might be plants, such as the proteins of the present invention, or may be microbial or other nonplant organisms. BIOEFFICACY ASSAYS Antifungal assays with P8
  • Assays for activity against Pi and Ggt were conducted with P8 protein.
  • the growth medium for the Pi and Ggt assays was made from 100 mL V8® vegetable juice, 2 g calcium carbonate, 15 g bacto agar, and 900 mL water. The calcium carbonate was added to the V8® juice; then the mixture was decanted and combined with the rest of the ingredients. The medium was then autoclaved for 30 minutes. All reagents used were of the highest grade commercially available.
  • Antifungal activity of the protein was determined using a hyphal extension-inhibition assay as described by Roberts and Selitrennikoff.
  • fungal mycelium was harvested from actively growing fungus and placed in the center of a sterile Petri dish containing nutrient agar. After incubation of the dish at 20 °C for 48-72 hr to allow for mycelial growth in a symmetrically circular shape, sterile paper discs (Difco concentration disc, 174) were positioned on the agar approximately 1.5 cm from the mycelium. 35 ⁇ L of a Tris buffer solution (25 mM, pH 8.0) containing ⁇ 1 ⁇ g of the protein was applied to each disc. The plate was incubated at 20 °C overnight.
  • the antifungal activity was determined based on the zone of hyphal extension inhibition exhibited in the vicinity of the discs.
  • P8 protein demonstrated inhibition of Pi and Ggt with as little as 50 ng of protein per disc. Activity is noted by the formation of a crescent shaped zone of inhibition at the edge of the mycelial growth that was approaching the disc.
  • Assays for activity against Pi and Septoria nodorum (Sn) were conducted with two microbial ribonucleases. Preparations of a ribonuclease from Bacillus cereus and one from Physarum polycephalum were obtained from United States Biochemical Corporation (Cleveland, Ohio) and tested at a concentration equal to 100 units of activity in assays designed for a 96-well plate. One "unit" is sufficient enzyme activity to give a uniform partial digestion of 3 ⁇ g of RNA when incubated at 55 °C for 12 min in 6 ⁇ l of assay buffer.
  • Tests against Pi were conducted in Medium #303, prepared as follows: One liter contains 1 g MgSO 4 7H 2 0; 2 g KH 2 P0 4 ; 0.5 g NaCl; 1 g CaC0 3 ; 1 ml ZnS0 4 -7H 2 O stock- 1 mg ml; 1 ml FeS0 4 -7H 2 0 stock - 1 mg ml; 0.5 ml FeEDTA stock - 100 mM; 20 g Maltrin M-100; 20 g Casein; 5 g Yeast Extract; 5 g Glucose; 3.02 g Pipes 10 mM; pH adjusted to 6.5 and filter sterilized.
  • Pi is seeded at 5 x 103 sporangia per well and allowed to incubate at 18 °C for 24-48 hours. Assessment of growth is made by measuring the OD at 595 nm.
  • the ribonuclease from Bacillus cereus provided 59% inhibition; the ribonuclease from Physarum polycephalum provided 55% inhibition.
  • Tests against Sn were conducted in CDAA .1% media prepared as follows: 35 g 1 Difco Czapek Dox Broth, 1 g 1 Proline, 500 mg/1 Asparagine, 500 mg/1 Cysteine, and 1 g/1 Agar are autoclaved for 23 minutes, and filter- sterilized vitamins (1 ppm Thiamine and 1 ppm Biotin) are added. A seven day old sporulating culture of Septoria nodorum on YMA agar is used to make the spore suspension. A small amount ( ⁇ 1 ml) of CDAA media is dropped onto an area of the culture with pink spore masses oozing from the pycnidia.
  • the spores are mixed with the CDAA media by repeatedly drawing up and expelling them from the pipetter.
  • the concentrated suspension is added to the total volume of CDAA required for the test, adjusting spore concentration to 50,000 spores/ml.
  • Assay incubation is at 24° C in darkness.
  • the spore suspension is dispensed at 50 ⁇ l/well in a 96 well microtiter plate. These plates are then placed in an incubator (lOhr/day fight at 12 °C) for 24 hours prior to sample application. 50 ⁇ l of sample is added to the 50 ⁇ l of inoculum (prepared 24 hours earlier) resulting in a total well volume of 100 ⁇ J/treated well/replicate treatment.
  • Assay plates are incubated for 48 hours and the results are determined by reading optical density (OD) with a BioRad microtiter plate reader model 3550 at a single wavelength of 595 nm. An OD reading is made at time zero (to) which is made immediately after sample application, and an OD reading is made at 48 hours after sample application (t 4 s).
  • Fungal growth estimate is determined by the difference in OD readings between to and t ⁇ multiplied by a calculation value for fungal biomass. (The calculation value for fungal biomass is the relationship between fungal growth and optical density and was determined in separate experiments.
  • the relationship between fungal growth and optical density was determined by growing fungi in 96 well microtiter plates, and harvesting the mycelium over time, at absorbance intervals of approximately 0.1 OD.
  • the calculation value comes from the linear relationship between fungal biomass and OD for the specific fungus. It is the slope value obtained from the linear realtionship.
  • the calculation value for Sn is 0.508. Then % inhibition is determined from the difference between the biomass of the treatments and the biomass of the controls.
  • the ribonuclease from Bacillus cereus provided no inhibition at the concentration tested; the ribonuclease from Physarum polycephalum provided 53% inhibition.
  • the active protein P8 from Engelmannia pinnatif ⁇ da was isolated, purified, partially sequenced, and identified as having homology to a plant ribonuclease.
  • Other plant derived ribonucleases may be isolated by similar techniques.
  • Bacteria and other microorganisms which are known or found to produce ribonucleases may be cultured by known methods and the ribonuclease extracted from the culture medium.
  • the dialyzed solution was then concentrated to about 25 mL using an Amicon centripep concentrator with 10 kDa molecular weight cutoff.
  • the concentrated solution was passed over a PD-10 column equilibrated with Buffer B to remove any remaining small molecular weight molecules.
  • the high molecular weight fraction was then applied on a Mono-Q column which was equilibrated with Buffer B. Separated components were eluted from the column with a gradient of NaCl (0 to 1.0 M) in Buffer B at a flow rate of 1 mL per min using a Waters Associate HPLC system (model 510).
  • Active fractions were pooled and further purified on a Vydac C s reversed phase column equilibrated with 0.1% TFA in H 2 O (Buffer C). After washing with Buffer C for 5 min, the column was eluted at a flow rate of 1 mL per min at 25°C with a linear gradient of 0 to 80% acetonitrile in Buffer C. All fractions were collected, concentrated to dryness using a Savant concentrator system, redissolved in 0.5 mL of Buffer A and assayed for antifungal activity. A fraction which eluted at about 60% acetonitrile showed strong antifungal activity. SDS-PAGE showed that this fraction contained a homogeneous protein with a molecular weight of almost 30 kDa.
  • Protein samples were hydrolyzed for 24 hrs in 6 N HCl at 110 °C in vacuo and analyzed on a Beckman 630 High Performance Amino Acid Analyzer. Automated Edman degradation was carried out on an Applied Biosystems model 470A Protein Sequenator. The respective PTH-amino acid derivatives were identified by reversed phase analysis in an on-line fashion employing an Applied Biosystems model 120 PTH Analyzer. N- terminal sequencing of the P8 protein identified 16 amino acids with undetermined amino acids at positions 1, 2, and 14 of the mature protein.
  • the full length P8 cDNA was isolated in two separate stages.
  • a partial cDNA was obtained using Polymerase Chain Reaction (PCR) based protocols of mixed oligonucleotide primed amplification of cDNA (MOPAC) [Lee] and rapid amplification of cDNA ends (R.A.C.E.) [Frohman].
  • First strand cDNA was generated from Engelmannia pinnatifida leaf poly A+ RNA and served as the template for MOPAC reactions.
  • Primers for P8 gene specific amplification were mixed oligonucleotide 17-mers (32 fold degenerate) made to amino acids at positions 4 through 9 of the mature protein (SEQ ID NO:l).
  • a second nested mixed oligonucleotide 18-mer (512 fold degenerate) was made to amino acids at positions 6 through 11 of the mature protein to verify the MOPAC PCR product as P8 specific (SEQ ID NO:2).
  • a 934 bp fragment was identified and subcloned into pUCll ⁇ vector [Vieira] at the Smal site (blunt ligation) resulting in pMON8969.
  • the partial cDNA was sequenced and the deduced 5' sequence matched the N-terminal protein sequence obtained, specifically amino acid positions 9 through 16 (region independent of PCR primer).
  • the P8 gene was isolated from a genomic library, which was made using DNA isolated from leaf tissue of Engelmannia pinnatifida.
  • the genomic library was constructed from genomic DNA partially digested with Mbol ligated into BamHI site of the lambda EMBL3 vector. [Frischauf et al.]
  • the library was screened using pMON8969 cDNA insert and a 5'- specific, 34-mer oligonucleotide probe (SEQ ID NO:3).
  • SEQ ID NO:3 5'- specific, 34-mer oligonucleotide probe
  • the P8 gene was localized to two AccI fragments, a 4.7 kb fragment encoding the 5' region and a 1.5 kb fragment encoding the 3' region.
  • the 4.7 kb AccI fragment was subcloned and the 5' region of the P8 gene was sequenced.
  • the 5' DNA sequence encoded a 33 amino acid N-terminal signal sequence that is removed during protein processing and is not present in the mature protein.
  • the first two N-terminal amino acids of the mature protein were identified as glutamic acid and histidine.
  • the full length P8 cDNA was generated by PCR using a 5' gene specific primer from the start codon (ATG) of the signal peptide with nested BamHI and Bglll restriction sites (SEQ ID NO:4) and a 3' gene specific primer after the stop codon (TGA) with nested EcoRI and Hindlll restriction sites (SEQ ID NO:5).
  • the full length P8 cDNA PCR product was subcloned as a 822 bp BglH/EcoRI fragment into a previously constructed E.coli cassette vector containing an enhanced CaMV 35S promoter.
  • the 3' nontranslated polyadenylation sequence of the ssRUBISCO E9 gene was also provided as the terminator.
  • the vector also contained a multilinker site between the leader and the terminator sequences, NotI sites before and after the promoter and the terminator sequences, and an ampicillin resistance gene.
  • the full length P8 cDNA was sequenced and thus determined to be that shown in SEQ ID NO: 10.
  • the deduced translated protein sequence is shown as SEQ ID NO:ll.
  • RNA For expression of a ribonuclease in heterologous systems, baculovirus or plant host, it was necessary to engineer a signal peptide to the N-terminus of the near full length mature protein (missing the first two amino acids), to target the protein to the vacuole or to the extracellular space, away from the cytoplasm where RNA is present.
  • Two signal peptides were selected, one from an Arabidopsis thaumatin-like protein (ATLP) and one from aNicotiana alata self incompatibility protein (NA2- 2), a known ribonuclease. [McClure]
  • nested BamHI/Bglll restriction sites were designed at the 5' end upstream of the ATG start of the ATLP synthetic signal peptide and a BsaAI restriction site at the 3' end of the synthetic signal peptide (SEQ ID NO:6).
  • a Dral restriction site was engineered at the 5' end of the near full length P8 cDNA (SEQ ID NO:7). Blunt ligation of the BsaAI and Dral restriction sites inserted codons for Ala and Thr as the first two amino acids of the modified mature P8 protein.
  • Ala and Thr are the first two amino acids found in the native ATLP mature protein and should conserve the context around the signal peptide proteolytic cleavage site.
  • Nested EcoRI/Hindlll restriction sites were engineered after the stop codon TGA of the near full length P8 cDNA (SEQ ID NO:5). These modifications allow the intact chimeric protein gene to be mobilized as a cassette (a nested BamHI Bglll (5') - EcoRI/Hindlll (3') restriction fragment of 795 bp) with minimal noncoding flanking sequences, into pUCH9 [Vieira] to create pMON8989.
  • nested BamHI/Bglll restriction sites were designed at the 5' end upstream of the ATG start codon of the NA2-2 synthetic signal peptide and a BstBI restriction site at the 3' end of the synthetic signal peptide (SEQ ID NO:8).
  • a Narl restriction site was engineered at the 5' end of the near full length P8 cDNA (SEQ ID NO:9).
  • Ligation of BstBI and Narl restriction sites inserts codons for Ala, Phe, Ala, and Thr as the first four amino acids of the modified mature P8 protein. Ala and Phe are the first two amino acids found in native NA2-2 mature protein and should ⁇ conserve the context around the signal peptide proteolytic cleavage site.
  • EcoRI/Hindlll restriction sites were engineered after the stop codon TGA of the near full length P8 cDNA (SEQ ID NO:5). These modifications allow the intact chimeric protein to be mobilized as a cassette, (nested BamHI/Bglll (5') - EcoRI/Hindlll (3') restriction fragment of 800 bp) with minimal noncoding flanking sequences, into pUCH9 [Vieira] to create pMON8990.
  • a Bglll-EcoRI fragment containing the chimeric P8 gene was inserted into pVL1392, a vector for transfection of baculovirus with a heterologous gene. [Luckow] This cloning placed the chimeric P8 under the control of the polyhedrin promoter. The gene was detected in the transfected virus using dot blot analysis. The expressed protein was inhibitory to Pi in a hyphal extension plate assay.
  • RNA polymerase enzyme messenger RNA
  • 3' non-translated region which adds polyadenylate nucleotides to the 3' end of the RNA.
  • Transcription of DNA into mRNA is regulated by a region of DNA usually referred to as the "promoter.”
  • the promoter region contains a sequence of bases that signals RNA polymerase to associate with the DNA and to initiate the transcription of mRNA using one of the DNA strands as a template to make a corresponding strand of RNA.
  • promoters which are active in plant cells have been described in the literature. These include the nopaline synthase (NOS) and octopine synthase (OCS) promoters (which are carried on tumor-inducing plasmids of Agrobacterium tumefaciens), the cauliflower mosaic virus (CaMV) 19S and 35S promoters, the Figwort Mosaic Virus (FMV) 35S promoter, and the light-inducible promoter from the small subunit of ribulose 1,5-bis-phos-phate carboxylase (ssRUBISCO, a very abundant plant polypeptide). All of these promoters have been used to create various types of DNA constructs which have been expressed in plants.
  • U.S. Patent Number 5,034322 (Fraley et al., 1991), herein incorporated by reference, discloses such uses.
  • the promoters utilized in the double-stranded DNA molecules may be selected to confer specific expression of a ribonuclease protein in response to fungal infection.
  • the infection of plants by fungal pathogens triggers the induction of a wide array of proteins, termed defense-related or pathogenesis-related (PR) proteins [Bowles; Bol et al.; Linthorst].
  • PR pathogenesis-related
  • Such defense-related or PR genes may encode enzymes (such as phenylalanine ammonia lyase, chalcone synthase, 4-coumarate coA ligase, coumaric acid 4-hydroxylase) of phenylpropanoid metabolism, proteins that modify plant cell wall (such as hydroxyproline-rich glycoproteins, glycine-rich proteins, peroxidases), enzymes (such as chitinases and glucanases) that degrade the fungal cell wall, thaumatin-like proteins, or proteins of as yet unknown function.
  • the defense-related or PR genes have been isolated and characterized from a number of plant species.
  • the promoters of these genes may be used to attain expression of ribonuclease in transgenic potato plants when challenged with Pi. Such promoters may derive from defense-related or PR genes isolated from potato itself [Fritzemeier et al.; Cuypers et al.; Logemann et al.; Matton and Brisson; Taylor et al.; Matton et al.; Schroder et al.]. In order to place the ribonuclease under the control of a promoter induced by infection with P. infestans the promoter reported by Taylor et al. may be preferred.
  • the particular promoter selected should be capable of causing sufficient expression of the enzyme coding sequence to result in the production of an effective amount of ribonuclease.
  • a preferred promoter is a constitutive promoter such as FMV 35S or CaMV 35S.
  • the promoters used in the DNA constructs (i.e. chimeric plant genes) of the present invention may be modified, if desired, to affect their control characteristics.
  • the CaMV 35S promoter may be ligated to the portion of the ssRUBISCO gene that represses the expression of ssRUBISCO in the absence of light, to create a promoter which is active in leaves but not in roots.
  • the resulting chimeric promoter may be used as described herein.
  • the phrase "CaMV 35S" promoter thus includes variations of CaMV 35S promoter, e.g., promoters derived by means of ligation with operator regions, random or controlled mutagenesis, etc.
  • the promoters may be altered to contain multiple "enhancer sequences" to assist in elevating gene expression. Examples of such enhancer sequences have been reported by Kay et al.
  • An enhanced CaMV 35S promoter has been constructed as follows. A fragment of the CaMV 35S promoter extending between position -343 and +9 was previously constructed in pUC13. [Odell et al.] This segment contains a region identified as being necessary for maximal expression of the CaMV 35S promoter. It was excised as a Clal-Hindlll fragment, made blunt ended with DNA polymerase I (Clal fragment) and inserted into the Hindi site of pUC18.
  • This upstream region of the 35S promoter was excised from this plasmid as a Hindlll-EcoRV fragment (extending from - 343 to -90) and inserted into the same plasmid between the Hindlll and PstI sites.
  • the enhanced CaMV 35S promoter hereafter "CaMV E35S" thus contains a duplication of sequences between -343 and -90.
  • the RNA produced by a DNA construct of the present invention also contains a 5' non-translated leader sequence.
  • This sequence can be derived from the promoter selected to express the gene, and can be specif- ically modified so as to increase translation of the mRNA.
  • the 5' non- translated regions can also be obtained from viral RNA's, from suitable eukaryotic genes, or from a synthetic gene sequence.
  • the present invention is not limited to constructs wherein the non-translated region is derived from the 5' non-translated sequence that accompanies the promoter sequence. Rather, the non-translated leader sequence can be derived from an unrelated promoter or coding sequence.
  • the petunia heat shock protein 70 (Hsp70) contains such a leader.
  • the 3' non-translated region of the chimeric plant genes of the present invention contains a polyadenylation signal which functions in plants to cause the addition of adenylate nucleotides to the 3' end of the RNA.
  • preferred 3' regions are (1) the 3' transcribed, non-translated regions containing the polyadenylate signal of Agrobacterium tumor-inducing (Ti) plasmid genes, such as the nopaline syn ⁇ thase (NOS) gene and (2) plant genes like the soybean 7s storage 'protein genes and the pea ssRUBISCO E9 gene.
  • An E. coli plasmid cassette vector designated pMON8999, was preliminarily constructed for transformation of dicotyledonous plants.
  • the Bglll-EcoRI fragment containing the ATLP signal peptide-P8 protein coding sequence from pMON8989 was inserted into a multilinker cloning site of a previously constructed vector containing a CaMV E35S promoter.
  • the 3' ⁇ ontranslated polyadenylation sequence from pea ssRUBISCO E9 gene was provided as the terminator downstream of the multilinker cloning site.
  • the vector also contains NotI sites before the promoter and after the terminator sequences, and an ampicillin resistance gene.
  • This vector contains the glyphosate resistance gene described by Barry for future selection of transformed plants.
  • An additional E.coli plasmid cassette vector designated pMON8992, was constructed by inserting the Bglll-EcoRI fragment containing the NA2-2 signal peptide-P8 protein coding sequence into a previously constructed vector containing the FMV 35S promoter with a petunia Hsp70 leader sequence.
  • the 3' nontranslated polyadenylation sequence of the NOS gene was also provided as the terminator.
  • the vector also contained a multilinker site between the leader and the terminator sequences, NotI restriction sites before and after the promoter and the terminator sequences, and an ampicillin resistance gene.
  • the Notl-NotI 2.17 kb fragment from pMON22518 containing the CaMV E35S promoter, the native full length P8 cDNA (SEQ ID NO: 10), and the E9 3' terminator, was inserted in both orientations into the NotI site of pMON17227, a Ti plasmid vector described above, to produce pMON22519 and pMON22520.
  • Vectors pMON 22508, 22509, 22512, 22513, 22519 and 22520 were introduced into disarmed Agrobacterium ABI and used to transform potato explants in tissue culture. After selection for glyphosate resistance and plant regeneration, whole potato plants containing the ATLP-P8, the NA2-2-P8, and the native P8 coding sequences have been recovered and disease resistance assays have been performed. At least one transformation using pMON22509 and one using pMON22512 have resulted in potato plants which are more disease resistant than nontransformed or hollow vector control plants. The test was performed as follows:
  • Plantlets were inoculated with a suspension of 5 x 104 sporangia / ml to uniform wetness, and incubated at 19 °C. Plantlets were scored for late blight development seven, eight, and nine days post inoculation, or until disease levels in the non transformed controls exceeded 80%.
  • P8 protein is in the creation of nematode resistant plants.
  • proteins which are toxic intracellularly such as ribonucleases may be placed under the control of a nematode-induced promoter which is specific to the root cells on which nematodes feed.
  • ribonucleases may be placed under the control of a nematode-induced promoter which is specific to the root cells on which nematodes feed.
  • production of the ribonuclease is induced and the cell dies or is otherwise rendered unsuitable for nematodes to feed upon. Wild-type or mutated P8 would be useful in such a method of producing nematode resistant plants, particularly potatoes.
  • a method of producing genetically transformed plants which are resistant to nematode damage would comprise the steps of: a) inserting into the genome of a plant cell a recombinant, double- stranded DNA molecule comprising (i) a promoter which functions in plant cells to cause the production of an RNA sequence in response to nematode attack of the root tissue;
  • RNA sequence (ii) a structural coding sequence that causes the production of a ribonuclease; (iii) a 3' non-translated region which functions in said plant cells to cause the addition of polyadenylate nucleotides to the 3' end of the RNA sequence; b) obtaining transformed plant cells; and c) regenerating from the transformed plant cells genetically transformed plants which express an amount of a ribonuclease effective to prevent the formation of nematode feeding sites.
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • AAG TTA CCA AAG TTT GCA TTC TTG GAT GGC
  • ACG TCA
  • Tyr Met Pro lie Leu Ser Cys Asn Gly lie Glu Ser Cys Asp Asn Lys 115 120 125

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Abstract

Les ribonucléases tirées des plantes ou micro-organismes permettent de lutter contre les champignons et les nématodes provoquant des dégâts sur les plantes. Des genès codant ces protéines peuvent être clonés en vecteurs de transformation de micro-organismes ou plantes infestant des plantes; on obtient ainsi un procédé empêchant les champignons ou les nématodes de provoquer des dégâts sur les plantes.
PCT/US1994/000844 1993-01-29 1994-01-24 Procede d'elimination de champignons pathogenes des plantes WO1994018335A2 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996015251A1 (fr) * 1994-11-10 1996-05-23 Bayer Aktiengesellschaft Sequence d'adn et son utilisation

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0344029A1 (fr) * 1988-04-28 1989-11-29 Plant Genetic Systems, N.V. Plantes avec des cellules d'étamine modifiées
EP0502718A1 (fr) * 1991-03-04 1992-09-09 Pioneer Hi-Bred International, Inc. Protéines naturelles et synthétiques avec activité inhibitrice contre des microorganismes pathogènes

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0344029A1 (fr) * 1988-04-28 1989-11-29 Plant Genetic Systems, N.V. Plantes avec des cellules d'étamine modifiées
EP0502718A1 (fr) * 1991-03-04 1992-09-09 Pioneer Hi-Bred International, Inc. Protéines naturelles et synthétiques avec activité inhibitrice contre des microorganismes pathogènes

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ACTA BOTAN. IND., vol.17, 1989 pages 248 - 251 J.P. SHARMA AND H.S. CHAWLA; 'Ribonucleases and soluble proteins during leaf rust pathogenesis in wheat' *
EUR. J. BIOCHEM., vol.198, 1991 W. JOST ET AL.; 'Amino acid sequence of an extracellular, phosphate-starvation- induced ribonuclease from cultured tomato (Lycopersicon esculentum) cells' *
PHYTOPATHOLOGY, vol.78, 1988 pages 270 - 272 R.C.GERGERICH AND H.A. SCOTT; 'The enzymatic function of ribonuclease determines plant virus transmission by leaf-feeding beetles' *
SCIENCE, vol.254, 1991 pages 1194 - 1197 K. BROGLIE ET AL.; 'Transgenic plants with enhanced resistance to the fungal pathogen Rhizoctonia solani' cited in the application *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996015251A1 (fr) * 1994-11-10 1996-05-23 Bayer Aktiengesellschaft Sequence d'adn et son utilisation
US6063988A (en) * 1994-11-10 2000-05-16 Bayer Aktiengesellschaft DNA sequences encoding stilbene synthases and their use

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WO1994018335A3 (fr) 1995-01-12

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