EP0688363A1 - Method of controlling insects in plants - Google Patents

Method of controlling insects in plants

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Publication number
EP0688363A1
EP0688363A1 EP19940911451 EP94911451A EP0688363A1 EP 0688363 A1 EP0688363 A1 EP 0688363A1 EP 19940911451 EP19940911451 EP 19940911451 EP 94911451 A EP94911451 A EP 94911451A EP 0688363 A1 EP0688363 A1 EP 0688363A1
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EP
European Patent Office
Prior art keywords
plant
patatin
seq
sequence
coding sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP19940911451
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German (de)
English (en)
French (fr)
Inventor
Sherri Marie Brown
John Thomas Greenplate
Barbara Guenther Isaac
Michael Girard Jennings
Elaine Beatrice Levine
John Patrick Purcell
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Monsanto Technology LLC
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Monsanto Co
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Publication of EP0688363A1 publication Critical patent/EP0688363A1/en
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    • 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)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/12—Processes for modifying agronomic input traits, e.g. crop yield
    • A01H1/122—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • A01H1/1245—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, e.g. pathogen, pest or disease resistance
    • A01H1/127—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, e.g. pathogen, pest or disease resistance for insect resistance
    • 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
    • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
    • A01N65/08—Magnoliopsida [dicotyledons]
    • A01N65/38—Solanaceae [Potato family], e.g. nightshade, tomato, tobacco or chilli pepper
    • 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
    • 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
    • C12N15/8286—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 insect 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

  • This invention relates to a method of controlling insect infestation of plants by providing 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 microorganisms and plants useful in that method.
  • insecticidal proteins are found in plants. These include lectins, amylase inhibitors, and protease inhibitors, which can affect insect growth and development when ingested at high doses [Boulter et al.,1989; Broadway and Duffey, 1986; Czapla and Lang, 1990; Gatehouse et al., 1986; Heusing et al., 1991; Ishimoto and K. Kitamura, 1989; Murdock et al., 1990; Shukle and Murdock, 1983], but do not provide the acute mortality afforded by B.t. proteins.
  • WCRW western corn rootworm
  • SCRW southern corn rootworm
  • BWV boll weevil
  • Patatins are lethal to some larvae and will stunt the growth of survivors so that maturation is prevented or severely delayed resulting in no reproduction.
  • These proteins which are known to have esterase (lipid acyl hydrolase) activity, may be applied directly to plants or introduced in other ways such as through the application of plant-colonizing microorganisms, which have been transformed to produce the enzymes, or by the plants themselves after similar transformation.
  • Patatins are a family of proteins found in potato [Gaillaird, 1971; Racusen, 1984; Andrews et al., 1988] and other plants, particularly in solanaceous plants [Ganal et al., 1991; Vancanneyt et al., 1989].
  • the patatins are found predominantly in tubers, but also at much lower levels in other plant organs [Hofgen and Willmitzer, 1990].
  • the esterase substrate specificities of several patatin isozymes have been examined [Hofgen and Willmitzer, 1990; Racusen, 1986] Genes that encode patatins have been previously isolated by
  • patatin genes can be heterologously expressed by plants.
  • Genes for patatins may be similarly isolated and inserted into appropriate transformation vector cassettes which are then (1) used to transform plant-colonizing microorganisms which when applied to plants express the genes producing a patatin, thereby providing control of insects, or (2) incorporated into the genome of a plant, which then protects itself from attack by insects by expressing the gene and producing a patatin.
  • the plant may also be transformed or bred to co-express one or more B.t. genes which code for proteins for the control of insects. This would provide plants that are either (1) protected from a wider range of pests and/or (2) have two modes of action against some pests, which is an important tool in resistance management. Examples of plants transformed to express B.t. genes are disclosed in European Patent Publication No. 0 385 962, which corresponds to U.S. Serial Number 476,661, filed February 12, 1990, [Fischhoff et al.], which is incorporated herein by reference.
  • the plant may also be transformed or bred to co-express proteinase inhibitor genes, such as those encoding potato papain inhibitor [Rodis and Hoff, 1984] or soybean trypsin inhibitor [for review see Ryan, 1990] as proteinase inhibitors have been shown to potentiate the activity of other insecticidal proteins.
  • proteinase inhibitor genes such as those encoding potato papain inhibitor [Rodis and Hoff, 1984] or soybean trypsin inhibitor [for review see Ryan, 1990] as proteinase inhibitors have been shown to potentiate the activity of other insecticidal proteins.
  • a method of controlling insect infes ⁇ tation of plants comprising providing an effective amount of an insecticidal patatin for ingestion by the insect.
  • This method may be effected by providing plant-colonizing microorganisms which have been transformed to express a gene for a patatin and which are introduced to the plant, express such gene, and provide an insecticidally effective amount of a patatin.
  • This method may also be effected by genetically transforming the plant to be protected with a DNA molecule comprising
  • RNA sequence (i) a promoter which functions in plant cells to cause the production of an RNA sequence;
  • 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, wherein said promoter is heterologous with respect to said structural coding sequence and wherein said promoter is operatively linked with said structural coding sequence, which is in turn operably linked with said non- translated region.
  • the plant will express patatin at a level of about 0.1-0.5% of total protein.
  • Also provided by the present invention are genetically transformed, insect-resistant corn, cotton, tomato and potato plants.
  • controlling insect infestation means reducing the number of insects which cause reduced beneficial yield, either through mortality, retardation of larval development (stunting), or reduced reproductive efficiency.
  • insecticidal means capable of reducing the number of insects which cause reduced beneficial yield, either through mortality, retardation of larval development (stunting), or reduced reproductive efficiency.
  • 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.
  • the term "patatin” means a plant protein having 75% or more homology to the protein encoded by SEQ ID NO:31, shown below, or more preferably at least 80% homology, or even more preferably at least 85% homology. This term also includes proteins produced from synthetic DNA sequences which have been designed for improved expression in monocots.
  • Patatins are a family of esterases found in potato [Gaillaird, 1971;
  • patatins are found predominantly in tubers, but also at very low levels in other plant organs [Hofgen and Willmitzer, 1990].
  • the esterase substrate specificities of several patatin isozymes have been examined [Hofgen and Willmitzer, 1990; Racusen, 1986] and found to have broad substrate specificity, showing that these enzymes have limited substrate requirements.
  • the use of all plant-derived patatins and their equivalents, both those disclosed in detail herein and homologous proteins, whether derived from natural DNA sequences or synthetic DNA sequences, for the purpose of controlling insect infestation of plants is within the scope of the present invention.
  • Crude patatin preparations from potato are available commercially.
  • Sigma Chemical Company, St. Louis, MO offers potato protein preparations denominated by Sigma as acid phosphatase (P-1146 and P-3752) or apyrase (A-9149).
  • Potato tubers may also be acquired and protein extracts can be prepared by methods described in the literature (Racusen and Foote, 1980; Park et al., 1983). BIOEFFICACY ASSAYS Artificial Diet Bioassavs
  • Test samples for activity against larvae of SCRW, BWV, Colorado potato beetle (CPB), Leptinotarsa decemlineata, and European corn borer (ECB), Ostrinia nubilalis are carried out by overlaying the test sample on an agar diet similar to that described for SCRW by Marrone et al., 1985.
  • Test samples were prepared by solubilization of the protein in 4-5 mL 10 mM HEPES, pH 7.5, followed by dialysis in this same buffer using 3500 molecular weight cutoff tubing. Neonate larvae are allowed to feed on the treated diet at 26 °C and mortality and growth stunting are evaluated at 5 or 6 days.
  • the results of the assays of P-3752 (Sigma) are given in Table 1. This crude potato preparation showed broad spectrum insecticidal activity.
  • the proteinaceous nature of the insecticidal component of P-3752 which is active against southern corn rootworm (SCRW) and boll weevil (BWV) was determined by heat lability, ammonium sulfate precipitation, molecular size fractionation, and protease susceptibility experiments.
  • SCRW southern corn rootworm
  • BWV boll weevil
  • a long term (25 day) assay of P-3752 against SCRW utilized 2nd instar larvae and several transfers of surviving insects onto freshly-treated diet. At the end of the study, all of the control larvae had pupated. In contrast, 50% of the treatment larvae were dead and the other 50% had increased in body weight by only 16% of their initial weight (2.48 mg vs. 2.14 mg). This demonstrates that the larval development is arrested, not just slowed. This has important ramifications from an insect control standpoint as the larvae will not develop to adulthood. Thus the number of rootworms in future generations will be reduced.
  • WCRW western corn rootworm
  • Diabrotica virgifera can only be used in laboratory experiments in the 2nd instar larval stage.
  • P-3752 against WCRW a side-by-side assay with 2nd instar SCRW larvae was designed.
  • P-3752 treatment resulted in only 13% and 11% weight gain, respectively, of SCRW and WCRW 2nd instar larvae.
  • Control SCRW increased in weight by 474% and WCRW grew 200% in 7 days. This suggests that patatin activity against WCRW is roughly equivalent to its activity against SCRW.
  • P-3752 was slightly active against tobacco budworm (TBW), Heliothis ⁇ irescens, beet armyworm (BAW), Spodoptera exigua, corn earworm, Helico ⁇ erpa zea, pink boll worm, Pectinop-hora gossypiella, and tobacco hornworm, Manduca sexta, with stunting ratings of 1 to 1.5 at the same concentration at which a stunting rating of 3 is demonstrated for SCRW.
  • P-3752 gave a stunting rating of 2.5 for black cutworm, Agrotis ipsilon. (The stunting ratings are defined above in Table 1.) It was inactive against green peach aphid, Myzus persicae, at the concentration tested. Plant Tissue Bioassays
  • Potato One g of crude P-3752 was dissolved in 4 mL 25 mM Tris, pH 7.5 buffer, then dialyzed and filtered through a 0.2 ⁇ m membrane. Triton® X-100 was added to generate a 0.1% solution. Potato leaves were dipped into the enzyme preparation and placed on moistened filter paper in petri dishes. CPB larvae were added and the plates were incubated at 27 °C for 3 days. P-3752 treatment of potato leaves resulted in stunting and reduced feeding of CPB larvae. At the conclusion of the assay, significantly less leaf tissue remained on control leaves compared to P-3752-treated leaves.
  • Excised corn roots and shoots were vacuum-infiltrated (Inflt.) with crude P-3752 or 25 mM Tris, pH 7.5 buffer.
  • the control sample was tissue submersed in Tris buffer. Approximately 10-15 pieces of root or 3 pieces of shoot tissue were placed in wells of a 24- well tissue culture plate and replicated 4 times. Four neonate SCRW larvae were added to each well.
  • the assay was incubated at 26 °C for 4 days, at which time observations were made with respect to mortality and average larval weight. The results of these assays are shown in Table 4.
  • patatin the insecticidally active component of P-3752
  • PROTEIN IDENTIFICATION The insecticidally active component from P-3752 has been purified, partially sequenced, and characterized.
  • the active agent(s) has been identified as patatin, a family of lipid acyl hydrolases from potato. Protein Isolation
  • SCRW activity bv anion exchange chromatographv -
  • the SCRW-active component from P-3752 was purified by Q-Sepharose (Pharmacia) anion exchange chromatography followed by MONO-Q (HR 5/5, Pharmacia) anion exchange chromatography.
  • the protein levels in SCRW-active fractions indicated that the observed ** stunting was achieved with a protein concentration of 31 ppm of diet.
  • SDS-PAGE indicated that three major protein bands (M r 42,000, -26,000 and -16,000) were present in the active fractions.
  • the SDS-PAGE profile of the SCRW-active fractions was very similar to the profile observed in active fractions from the 5-step purification and the anion exchange purification.
  • the IEF gel showed that the proteins fractionate from pH 4.6 to 5.1, consistent with the reported pi range for patatin (Racusen and Foote, 1980).
  • NH 2 -te ⁇ ninal amino acid sequence was obtained on all the protein bands (M r 42,000, -26,000 and -16,000) in the SCRW-active chromatography fraction from the anion exchange purification and the five step purification. Overall, sequence data were generated for all bands in the active fractions. Most of the bands showed >85% homology with a 15- amino acid sequence at either the NH 2 -terminus (SEQ ID NO:l) or an internal sequence (SEQ ID NO:7) of an isozyme of patatin (Stiekema et al., 1988). One of the 17 kD bands showed 75% homology with the initial eight amino acids of the published NH 2 -terminus sequence of patatin.
  • the other 17 kD band showed >85% homology with the initial eight amino acids of the pubKshed internal sequence. These bands represent proteolyzed products of patatin. The presence of isozymes is clearly indicated by variability in amino acids at positions 1 and 3 for both NH 2 -terminus and internal sequences.
  • M r 55,000 band was not observed in the heated sample, which indicates that the heat treatment in SDS eliminates the esterase activity. In the absence of the M r 55,000 band in the heated sample, the originally observed M r 42,000 band was observed with coomassie staining.
  • p-Nitrophenyl substrate specificity studies A series ofp- nitrophenyl esters (C-2, C-4, C-6, C-8, C-10, C-12, C-14, and C-16 esters) was tested to determine the substrate specificity. p-NP C-8 and C-10 esters were consistently the best substrates for the esterase activity of most of the patatins tested, relative to the other esters.
  • Lipid ester substrates A SCRW-active purified fraction (from 5- step purification) was tested for the abihty to hydrolyze several Upids Each Upid was dissolved and incubated with an ahquot of a SCRW-active purified fraction. Samples were analyzed by TLC utiUzing a three solvent development system (Pernes et al., 1980). Four Upids showed marked modifications by TLC. These included oleoyl lysolecithin, dioleoyl L- ⁇ - phosphatidylcholine, 1-monoUnolenoyl-rac-glycerol, and diolein (Sigma).
  • WCRW midguts were removed from third instar larvae feeding on corn roots. Midgut Upids were extracted, dissolved and incubated at the pH of the midgut (pH 6.55) with the SCRW-active purified fraction. Samples were analyzed by TLC utilizing the above method. The purified SCRW- active fraction demonstrated esterase activity on WCRW midgut phosphoUpids at the pH of the midgut. This illustrates a possible mode-of- action for the insecticidal activity of patatin. Alternate Sources of Patatin Because all initial experiments were carried out with P-3752, a commercially available enzyme preparation (Sigma) from Minnesota Russet var.
  • Extracts of S. berthaultii, S. kurtzianum, and S. tarijense were bioassayed against two additional target insects, CPB and ECB.
  • Bioassay data is summarized below in Table 6. Very little activity was noted with these extracts against CPB whereas the ECB larvae were moderately to severely stunted at the IX rate. However, the ECB larvae appear to be slightly less sensitive to these potato extracts than the SCRW larvae, as indicated by a complete absence of activity at 0.1X against ECB.
  • DNA sequences for these homologous proteins can be readily obtained by one of ordinary skill in the art and inserted into plants or other organisms by known means.
  • the insecticidal properties of such proteins can be best tested after heterologous expression, for example, from baculovirus or E. coli.
  • other proteins which can be used in the methods of the present invention may be obtained with a normal amount of experimentation using known methods and thereafter used to provide plants with protection from insect infestation.
  • GM203 Genes for patatins have been cloned by several investigators.
  • SEQ ID NO: 11 was constructed with the signal sequence of PS20 and the cDNA coding portion of GM203, hereinafter referred to as PatA+. It also contains an Ncol restriction site and an EcoRI site immediately following the translation termination codon.
  • Russet Burbank and sequenced The deduced amino acid sequences show that these cDNAs encode eleven different patatin isozymes. These eleven proteins are from about 82% to 100% identical as compared to PatA+, SEQ ID NO:ll, with differences occurring at numerous positions throughout the length of the cDN A.
  • the sequences for eleven different representative cDNAs encoding the eleven different patatin isozymes are denominated as shown in Table 7.
  • the cDNAs were engineered by PCR procedures using primers SEQ ID NO:26 and SEQ ID NO:27, corresponding to the 5' nucleotides encoding the first few codons of the signal sequence and the 3' end of the coding sequence, respectively, for later cloning manipulations.
  • Patatin cDNAs from the diploid potato S. berthaultii were isolated by reverse transcription of tuber mRNA followed by PCR with primers SEQ ID NO:26 and SEQ ID NO:27, described above. Multiple independent PCR reactions were performed to avoid the isolation of duplicate clones due to the amplification process.
  • a total of 14 patatin cDNAs were partially sequenced. All fourteen cDNAs (denominated Patl through Patl4) appear to have a unique nucleotide sequence, suggesting that at least 14 different patatin mRNAs are expressed in S. berthaultii tubers.
  • the sequence for Pat3+ is SEQ ID NO:28.
  • the sequence for Patl0+ is SEQ ID NO:29.
  • the deduced amino acid sequence shows that the 14 cDNAs encode at least 11 different proteins.
  • the cDNA sequences from the S. berthaultii tubers were very similar. Only 12 amino acid positions of the total 367 residues (3%) showed sequence variabiUty. The amino acid residues present in each of those positions is shown in Table 8.
  • Ten cD ⁇ A clones were generated via PCR utilizing mR ⁇ A isolated from Solanum cardiophyllum tubers as described above. Nucleotide sequence was obtained on at least 75% of the length of each clone. The full length sequence of one clone denominated Patl7+ is SEQ ID NO:30. SEQ ID NO:31 is the engineered mature form, Patl7 m . The S. cardiophyllum clones were almost identical, with only random nucleotide sequence changes that could be actual differences or PCR errors. However at positions 54 and 519, several clones were observed to have identical changes, suggesting that they are not due to the ampUfication process. The patterns of nucleotides at these positions indicated that there are at least 4 different mRNAs represented. mRNAs from two of the groups were isolated several times and mRNA from the other two groups were only isolated once in this set of cDNA clones.
  • the deduced amino acid sequences of the S. cardiophyllum clones were also extremely similar. There were 8 unique amino acid sequence groups, each differing from the other sequences by a single residue. cDNA clones encoding an amino acid sequence identical to the Patl7+ sequence were recovered twice and the other seven cDNAs (Pat 18+, 19+, 20+, 21+, 22+, 23+, and 24+) contained a single unique residue.
  • patatin genes can be isolated from various plant sources. One or more of these genes may then be used to transform bacterial cells or plant cells to enable the production of patatin and carry out the methods of this invention. Examples of how this may be done with various sequences for patatin are given below. Engineering of the Patatin cDNAs
  • SEQ ID NO: 12 substituted two amino acids (methionine-alanine) for lysine at the N-terminus of the mature protein and introduced an Ncol site, and SEQ ID NO: 13 added a second termination codon and an EcoRI site.
  • PatA mj SEQ ID NO: 14 The DNA coding sequence for PatA m (SEQ ID NO:14) was inserted into pMON5766, an E. coli expression vector derived from pBR327 (Soberon et al., 1980) with a recA promoter and a G10 leader (Olins et al., 1989).
  • the resulting vector, pMON19714 was mobiUzed into E. coli strain JMlOl, which subsequently produced PatA m as confirmed by Western blot analysis and esterase activity using p-nitrophenyl C-10 ester.
  • the DNA coding sequence for Patl7 m as well as that for PatA m were each inserted into an E. coli expression vector derived from pMON6235 with the AraBAD promoter (inducible when cells are grown in arabinose), a G10 leader, and an ampicilUn resistance marker gene.
  • the resulting vectors, pMON25213, containing Patl7 m , and pMON25216, containing PatA m were introduced into E. coli strain JMlOl.
  • Patatin is expressed by the transformed E. coli; however, it is compartmentalized in refractile bodies (RBs). Intact cells and solubiUzed RBs were used in SCRW assays. The results are shown in Table 9. Table 9
  • patatins in a plant-colonizing bacterium, and then apply this bacterium to the plant. As the insect feeds on the plant, it ingests a toxic dose of patatin produced by the plant colonizers.
  • Plant-colonizers can be either those that inhabit the plant surface, such as Pseudomonas or Agrobacterium species, or endophytes that inhabit the plant vasculature such as Clavibacter species.
  • the patatin gene may be inserted into a broad host range vector capable of replicating in these Gram-negative hosts.
  • patatin gene can be inserted into the chromosome by homologous recombination or by incorporation of the gene onto an appropriate transposon capable of chromosomal insertion in these endophytic bacteria.
  • Patatin genes were cloned into the baculovirus donor vector pMON14327, described in co-pending U.S. Serial Number 07/941,363, filed September 4, 1992, which is hereby incorporated by reference, as Ncol/ EcoRI fragments.
  • Donor vector pMON14327 contains an ampicillin resistance gene, the left and right arms of the Tn7 transposon, and, between these arms, a gentamicin resistance gene, the strong baculovirus polyhedrin promoter and a polyUnker.
  • the baculovirus shuttle vector or bacmid is composed of a mini-attTn7 site in frame within the lacZ gene and a kanamycin resistance gene recombined into the AcNPV viral genome.
  • pMON7124 With the help of a tetracycline-resistant helper plasmid, pMON7124, recombinant AcNPV virus were produced by transposition of the patatin or GUS genes and marker genes into the viral genome (Luckow et al., 1993). The following genes were inserted into pMON14327: the genes listed in Table 7 along with Pat3+, Patl0+, and Patl7+.
  • 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 Uterature.
  • Such promoters may be obtained from plants or plant viruses and include, but are not Umited to, 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 light-inducible promoter from the small subunit of ribulose 1,5-bis-phosphate carboxylase (ssRUBISCO, a very abundant plant polypeptide), and the Figwort Mosaic Virus (FMV) 35S promoter.
  • NOS nopaline synthase
  • OCS octopine synthase
  • CaMV cauliflower mosaic virus
  • ssRUBISCO the light-inducible promoter from the small subunit of ribulose 1,5-bis-phosphate carboxylase
  • FMV Fig
  • telomeres have been used to create various types of DNA constructs which have been expressed in plants (see e.g., PCT pubUcation WO 84/02913).
  • a root-specific promoter may be used to limit expression to the root or a root-enhanced promoter may be used to increase levels of active protein in the roots. This is preferred for plants susceptible to root-eating insects.
  • Certain plant promoters are also more effective in monocots.
  • the rice actin promoter described in WO 91/09948 is efficacious for expression in corn.
  • the maize ubiquitin promoter, described in EP 0 342 926, may also be used in monocots.
  • 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 CaMV35S promoter may be ligated to the portion of the ssRUBISCO gene that represses the expres ⁇ sion 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.
  • CaMV35S thus includes variations of CaMV35S promoter, e.g., promoters derived by means of Ugation with operator regions, random or controlled mutagenesis, etc. Furthermore, 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. (1987). 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 patatin.
  • a preferred promoter is the CaMV E35S promoter (enhanced CaMV35S).
  • 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 specifi ⁇ cally modified so as to increase translation of the mRNA.
  • the 5' non-trans ⁇ lated regions can also be obtained from viral RNA's, from suitable eukary- otic genes, or from a synthetic gene sequence.
  • the present invention is not Umited to constructs wherein the non-translated region is derived from the 5' non-translated sequence that accompanies the promoter sequence.
  • 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 synthase (NOS) gene and (2) plant genes Uke the soybean 7s storage protein genes and the pea ssRUBISCO E9 gene.
  • Ti Agrobacterium tumor-inducing
  • NOS nopaline synthase
  • Vectors containing the patatin cassettes described above express the active protein in the cytoplasm or vacuoles of the plant cell. It may be desirable to direct most or all of the patatin into the plant secretory pathway. To achieve this, it may be advantageous to use a signal sequence derived from a bacterial or plant gene, but a plant gene is expected to be preferred. Examples of such signal sequences are those from the endoproteinase B gene (Koehler and Ho) and the tobacco PR lb gene (Cornelissen et al.).
  • pMON10824 disclosed in EP Publ. 0 385 962, is a plant transformation vector designed for the expression of the lepidopteran active B.t. kurstaki protein.
  • the B.t.k. coding sequence is fused to the PRlb signal sequence plus 10 amino acids of the mature PRlb coding sequence.
  • pMON10824 is cut with Bglll and Ncol and the small Bglll-Ncol fragment that contains the PRlb signal is isolated.
  • the small Bglll-Ncol pMON10824 fragment is mixed with the 1.0 kb NcoI-EcoRI fragment from pMON19714 and Ba HI-EcoRI digested pMON19470 (Brown et al.).
  • This reaction constructs a plasmid in which the patatin coding sequence is fused to the secretory signal from the PRlb gene and driven by the CaMV35S promoter and an intron for monocot expression.
  • a similar reaction may be performed.
  • the Notl-NotI fragment of the dicot expression vector may be inserted into a dicot transformation vector as described below and mobiUzed into a disarmed Agrobacterium host and used to transform dicots.
  • the Notl-NotI fragment of this monocot plasmid may be inserted into a corn transformation vector (such as pMON18181 described above) to produce a corn plant which secretes patatin.
  • a corn transformation vector such as pMON18181 described above
  • CTP chloroplast transit peptide
  • One CTP that has worked to locaUze heterologous proteins to the chloroplast is that derived from the RUBISCO small subunit gene of Arabidopsis, denoted atslA.
  • a variant of this transit peptide that encodes the transit peptide, 23 amino acids of mature RUBISCO sequence, plus a reiteration of the transit peptide cleavage site has been constructed for the successful chloroplast localization of the B.t.k. protein.
  • pMON19643 described in Brown et al., contains the Arabidopsis atslA transit peptide fused to the GOX gene and may be used as the base for constructing vectors for the chloroplast localization of the patatin.
  • a complete EcoRI and partial Ncol digestion of pMON19643 is performed and the large (4.0 kb) fragment is isolated.
  • the NcoI-EcoRI fragment from pMON19714 is mixed with the large fragment of pMON19643. This reaction constructs a plasmid in which the patatin coding sequence is fused to the Arabidopsis transit peptide with 23 amino acids of mature
  • RUBISCO and driven by the CaMV E35S promoter.
  • a similar plasmid may be constructed to replace the promoter with the FMV35S promoter.
  • Such plasmids are mobiUzed into disarmed Agrobacterium hosts and used to transform dicots.
  • the Notl- NotI fragment is cloned into a corn transformation vector, as described above.
  • plants can be generated which produce patatin that is locaUzed to the chloroplast.
  • a chimeric plant gene containing a structural coding sequence of the present invention can be inserted into the genome of a plant by any suitable method.
  • Suitable plant transformation vectors include those derived from a Ti plasmid of Agrobacterium tumefaciens, as well as those disclosed, e.g., by Herrera-Estrella (1983), Bevan (1983), Klee (1985) and EPO pubUcation 0 120 516 (Schilperoort et al.).
  • Ri root-inducing
  • alternative methods can be used to insert the DNA con ⁇ structs of this invention into plant cells. Such methods may involve, for example, the use of liposomes, electroporation, chemicals that increase free DNA uptake, free DNA delivery via microprojectile bombardment, and transformation using viruses or pollen.
  • a particularly useful plasmid cassette vector for transformation of dicotyledonous plants is pMON11794.
  • the expression cassette pMON 11794 consists of the CaMV E35S promoter, the petunia Hsp70 5' untrans- lated leader, and the 3' end including polyadenylation signals from the NOS gene.
  • pMON11794 includes Ncol and EcoRI sites for insertion of coding sequences and Notl-NotI sites flanking the plant gene expression cassette.
  • PatA+ (SEQ ID NO:ll), PatB+ (SEQ ID NO:16), PatC+ (SEQ ID NO:17), and PatG+ (SEQ ID NO:22), were each inserted into pMON11794 to produce pMON19745, pMON19742, pMON19743, and pMON19744 respectively.
  • Each of these vectors was electroporated into tobacco protoplasts. Expression of patatin by the transformed tobacco cells was confirmed by Western blot analysis. Stable Transformation of Dicots Stable transformation of a dicot with a patatin gene has been repor ⁇ ted by Rosahl et al. Tobacco was transformed with a patatin gene under the control of a leaf and stem specific promoter. Patatin was expressed.
  • the Notl-NotI fragment from pMON19745 was inserted into pMON17227, a Ti plasmid vector disclosed and described by Barry et al. in WO 92/04449, incorporated herein by reference, to produce pMON22566.
  • This vector contains the glyphosate resistance gene described by Barry for selection of transformed plants.
  • SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:22 were used to make vectors pMON22563, 22564, and 22565, respectively.
  • These vectors were introduced into disarmed Agrobacterium ABI and used to transform tomato explants in tissue culture. After selection for glyphosate resistance and plant regeneration, whole plants expressing the patatin gene were recovered. Expression of the patatin gene was confirmed by Western blot analysis, and preUminary results indicate expression at levels between 0.1 and 0.5% of total protein. Bioassays with insect larvae are underway.
  • the resulting plasmid, pMON19731 was digested with NotI and the resulting fragment inserted into pMON10081, also described by Brown et al., to give pMON19740.
  • This plasmid was electroporated into corn leaf protoplasts as described by Sheen, 1991. Expression of patatin by the transformed corn protoplasts was confirmed by Western blot analysis.
  • the NcoI-EcoRI fragment of pMON19714 was inserted into pMON19433 to produce pMON19730.
  • the NotI fragment of pMON19730 was inserted into pMON10081 and the resulting plasmid, pMON19739, was electroporated into corn leaf protoplasts, which produced patatin, as confirmed by Western blot analysis.
  • Patl7 with and without targeting signals, was also expressed in corn protoplasts.
  • pMON19761 was constructed by inserting the 1.1 Kb NcoI-EcoRI fragment (SEQ ID NO:30) encoding the protein Patl7+ (the mature Patl7 protein and its own signal sequence for vacuolar targeting) into pMON19648 .
  • pMON19761 contains the CaMV E35S promoter, the Hsp 70 intron, the Patl7+ gene, and the NOS terminator for expression in corn cells.
  • Patl7+ sequence in pMON19761 was replaced by a NcoI-EcoRI fragment encoding the Patl7 m protein (SEQ ID NO:31) from pMON25213 to form the construct pMON25223.
  • pMON25224 was made by inserting two fragments, 0.3 Kb Xbal- Ncol fragment containing the chloroplast transit peptide (CTP) from the Arabidopsis thaliana SSU la gene (Timko et al.) from pMON19643 (Brown, et al.) and the 1Kb NcoI-EcoRI fragment for Patl7 m from pMON25213, inserted into pMON19761 (Xbal-EcoRI).
  • CTP chloroplast transit peptide
  • pMON25224 contains the CaMV E35S promoter, the Hsp 70 intron, CTP/Patl7 m coding sequence, and the NOS terminator.
  • the 5' end of the endoproteinase B cDNA (Koehler and Ho) encoding the extracellular signal peptide of the secreted protein was joined to the gene for Patl7 m from pMON25213.
  • a Bglll- EcoRI fragment containing the chimeric gene was made by a splicing overlap extension technique (Horton et al.) and inserted into pMON19761 (BamHI-EcoRI) to make pMON25225.
  • the corn transformation vector, pMON18181 was constructed from pMON19653 and pMON19643 (Brown et al.). This construct contains a cassette of the CaMV E35S promoter, the Hsp70 intron, the CP4 glyphosate selection marker, and the NOS terminator; a cassette of the CaMV E35S promoter, the Hsp70 intron, the GOX glyphosate selection marker, and the NOS terminator; and a single NotI site for insertion of a gene expression cassette containing a patatin gene. SEQ ID NO: 11 and SEQ ID NO:30 were each inserted as Notl-NotI fragments into pMONl8181 to produce pMON19746 and pMON19764, respectively.
  • Modification of coding sequences has been demonstrated to improve expression of other insecticidal protein genes such as the delta endotoxin sequences from Bacillus thuringiensis (Fischhoff and Perlak; WO 93/07278, Ciba-Geigy).
  • a modified coding sequence was thus designed to improve patatin expression in plants, especiaUy corn.
  • the modified Patl7+ sequence is shown in SEQ ID NO:33.
  • a DNA fragment containing SEQ ID NO:33 will be synthesized and inserted into a corn expression cassette vector such as pMON19470 (Brown et al.).
  • MOLECULE TYPE cDNA
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:28:

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US6329574B1 (en) 1990-01-22 2001-12-11 Dekalb Genetics Corporation High lysine fertile transgenic corn plants
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US6326527B1 (en) 1993-08-25 2001-12-04 Dekalb Genetics Corporation Method for altering the nutritional content of plant seed
US5824864A (en) * 1995-05-25 1998-10-20 Pioneer Hi-Bred International, Inc. Maize gene and protein for insect control
ES2196340T3 (es) 1996-06-18 2003-12-16 Unilever Nv Procedimiento de esterificacion enzimatica.
US6080913A (en) * 1996-09-25 2000-06-27 Pioneer Hi-Bred International, Inc. Binary methods of increasing accumulation of essential amino acids in seeds
US6057491A (en) * 1997-05-29 2000-05-02 Borad Of Regents For University Of Oklahoma Protein having insecticidal activities and method of use
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