CA2154576A1 - Aprotinin and synergistic combinations thereof with lectins as larvicides against insect pests of agronomic crops, harvested material thereof, and products obtained from the harvested material - Google Patents
Aprotinin and synergistic combinations thereof with lectins as larvicides against insect pests of agronomic crops, harvested material thereof, and products obtained from the harvested materialInfo
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- CA2154576A1 CA2154576A1 CA002154576A CA2154576A CA2154576A1 CA 2154576 A1 CA2154576 A1 CA 2154576A1 CA 002154576 A CA002154576 A CA 002154576A CA 2154576 A CA2154576 A CA 2154576A CA 2154576 A1 CA2154576 A1 CA 2154576A1
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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)
- 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
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- 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
- A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
- A01N37/44—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a nitrogen atom attached to the same carbon skeleton by a single or double bond, this nitrogen atom not being a member of a derivative or of a thio analogue of a carboxylic group, e.g. amino-carboxylic acids
- A01N37/46—N-acyl derivatives
-
- 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
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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
- C07K14/42—Lectins, e.g. concanavalin, phytohaemagglutinin
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/81—Protease inhibitors
- C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
- C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
- C07K14/8114—Kunitz type inhibitors
- C07K14/8117—Bovine/basic pancreatic trypsin inhibitor (BPTI, aprotinin)
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- 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
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- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Plant Pathology (AREA)
- Pest Control & Pesticides (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Biomedical Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Environmental Sciences (AREA)
- Dentistry (AREA)
- Agronomy & Crop Science (AREA)
- Insects & Arthropods (AREA)
- Virology (AREA)
- Cell Biology (AREA)
- Physics & Mathematics (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Botany (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
Aprotinin has been found to be larvicidal against a number of common insect pests of agricultural crops and stored grains, particularly in combination with an insecticidal or larvicidal lectin. In a preferred embodiment, plant resistance to these insects is produced by inserting into the cells of a plant a gene whose expression causes production of one or more of these proteinase inhibitors in combination with such a lectin, in larvicidal amounts.
Description
~ -215~gS76 WO94/16565 ~ PCT~S94100630 ~ , ~ .
Aprotinin and Synergistic Combinations Thereof with Lectins as Larvicides Against Insect ests of Agronomic Crops, Harvested Material Thereof, and Products Obtained from the ~arvested Material Technical Field This invention relates to materials and methods for killing insect larvae which are harmful to plants, and materials and methods for imparting insect resistance to plants, material harvested from the plants, and products derived from the harvested material.
Background of the Invention Numerous insects are serious pests of common agricul-tural crops. One method of controlling insects has been to apply insecticidal organic or semiorganic chemicals to crops. This method has numerous, art-recognized problems.
A more recent method of control of insect pests has been the use of biological control organisms which are typically natural predators of the troublesome insects. These include other insects, fungi (milky-spore) and bacteria (Bacillus thuringiensis cv., commonly referred to as "Bt"). However, it is difficult to apply biological control organisms to large areas, and even more difficult to have those living organisms remain and survive in the treated area for an extended period. Still more recently, techniques in recombinant DNA have provided the opportunity to insert into plant cells cloned genes which express insecticidal toxins derived from biological control organisms such as Bt. This technology has given rise to additional concerns about eventual insect resistance to well-known, naturally occurring insect toxins, particularly in the face of heavy selection pressure, which may occur in some areas. Thus, a continuing need exists to identify naturally occurring WO94/16565 PCT~S94/00~0 ;i 16 2 -insecticidal toxins which can be formed by plant cells directly by translation of a single strudtural gene.
More recently, certain specific plant lectins have come to be recognized as larvicidal and insecticidal agents of some merit, particularly since they are naturally produced by plant gene expression systems. However, some lectins are active at relatively high levels which limit flexibility in that they require maximal expression systems for most effective larval/insect control. Thus, a continuing need also exists for substances or combinations of substances which are effective in controlling larvae at lower concentrations. It would be particularly desirable to identify compounds or compositions which could be used to potentiate the larvicidal action of existing agents such as lectins. These and other objectives of this invention will be evident from the following disclosure.
Disclosure of the Invention It has now been determined that the serine-specific proteinase inhibitor aprotinin has potent larvicidal activity when administered enterally to the larvae of insects such as European corn borer and corn rootworm.
Thus, this invention provides a method for killing susceptible insect larvae, including larvae of European corn borer and corn rootworm comprising administering enterally to the larvae a larvicidal amount of aprotinin or a serine proteinase inhibitor which is at least 90% homologous to aprotinin by amino acid sequence.
The terms "protease inhibitor" and "proteinase inhibitor" are considered equivalent. The terms "insect"
and "larva", although not equivalent when used specifically, should be understood to include both adult and larval forms of a species when used generically. Thus, the term "insect resistance" should be understood to include resistance to larval forms as well as adults, and "larvicidal" materials should be considered insecticidal, particularly since killing larvae produces a corresponding absence of adults.
WO94/16565 ~ PCT~JS94/00~0 The proteinase inhibitor can be effectively applied to plants, harvested materials or products consumed by the insects by spray; dust or other formulation common to the insecticidal arts. By "harvested plant material" herein is meant any material harvested from an agricultural or horticultural crop, including without limitation grain, fruit, leaves, fibers, seeds, or other plant parts.
Products derived or obtained from such harvested material include flour, meal, and flakes derived from grain; and products in which such materials are admixed, such as, for example, cake, cookie, muffin, pancake and biscuit mixes.
Alternatively, the larvicidal proteinase inhibitor can be incorporated into the tissues of a susceptible plant so that in the course of infesting and consuming the plant, its harvested material, or a product derived from harvested plant material, the larvae consume larvicidal amounts of the proteinase inhibitor. One method of doing this is to incorporate the proteinase inhibitor in a non-phytotoxic vehicle which is adapted for systemic administration to the susceptible plants. This method is commonly employed with insecticidal materials which are designed to attack chewing insects and is well within the purview of one of ordinary skill in the art of insecticide and larvicide formulation, but is a method which may not be as suitable for active enzyme blockers such as proteinase inhibitors.
Alternatively, a dietary bait containing one or more of the selected proteinase inhibitors can be employed, with, optionally, an added pheromonal or other larval attractant material. However, the genes which code for these peptides can be isolated and cloned. Alternatively, they can be synthesized directly using a DNA sequence obtained by working backwards from the known amino acid sequence for aprotinin or a related proteinase inhibitor, preferably using plant-preferred codons. The resulting sequence can be 35 inserted into an appropriate expression cassette and introduced into cells of a susceptible plant species, so that an especially preferred embodiment of this method involves inserting into the genome of the plant a DNA
~ s ~ j WO94/1656~ PCT~S94/00630
Aprotinin and Synergistic Combinations Thereof with Lectins as Larvicides Against Insect ests of Agronomic Crops, Harvested Material Thereof, and Products Obtained from the ~arvested Material Technical Field This invention relates to materials and methods for killing insect larvae which are harmful to plants, and materials and methods for imparting insect resistance to plants, material harvested from the plants, and products derived from the harvested material.
Background of the Invention Numerous insects are serious pests of common agricul-tural crops. One method of controlling insects has been to apply insecticidal organic or semiorganic chemicals to crops. This method has numerous, art-recognized problems.
A more recent method of control of insect pests has been the use of biological control organisms which are typically natural predators of the troublesome insects. These include other insects, fungi (milky-spore) and bacteria (Bacillus thuringiensis cv., commonly referred to as "Bt"). However, it is difficult to apply biological control organisms to large areas, and even more difficult to have those living organisms remain and survive in the treated area for an extended period. Still more recently, techniques in recombinant DNA have provided the opportunity to insert into plant cells cloned genes which express insecticidal toxins derived from biological control organisms such as Bt. This technology has given rise to additional concerns about eventual insect resistance to well-known, naturally occurring insect toxins, particularly in the face of heavy selection pressure, which may occur in some areas. Thus, a continuing need exists to identify naturally occurring WO94/16565 PCT~S94/00~0 ;i 16 2 -insecticidal toxins which can be formed by plant cells directly by translation of a single strudtural gene.
More recently, certain specific plant lectins have come to be recognized as larvicidal and insecticidal agents of some merit, particularly since they are naturally produced by plant gene expression systems. However, some lectins are active at relatively high levels which limit flexibility in that they require maximal expression systems for most effective larval/insect control. Thus, a continuing need also exists for substances or combinations of substances which are effective in controlling larvae at lower concentrations. It would be particularly desirable to identify compounds or compositions which could be used to potentiate the larvicidal action of existing agents such as lectins. These and other objectives of this invention will be evident from the following disclosure.
Disclosure of the Invention It has now been determined that the serine-specific proteinase inhibitor aprotinin has potent larvicidal activity when administered enterally to the larvae of insects such as European corn borer and corn rootworm.
Thus, this invention provides a method for killing susceptible insect larvae, including larvae of European corn borer and corn rootworm comprising administering enterally to the larvae a larvicidal amount of aprotinin or a serine proteinase inhibitor which is at least 90% homologous to aprotinin by amino acid sequence.
The terms "protease inhibitor" and "proteinase inhibitor" are considered equivalent. The terms "insect"
and "larva", although not equivalent when used specifically, should be understood to include both adult and larval forms of a species when used generically. Thus, the term "insect resistance" should be understood to include resistance to larval forms as well as adults, and "larvicidal" materials should be considered insecticidal, particularly since killing larvae produces a corresponding absence of adults.
WO94/16565 ~ PCT~JS94/00~0 The proteinase inhibitor can be effectively applied to plants, harvested materials or products consumed by the insects by spray; dust or other formulation common to the insecticidal arts. By "harvested plant material" herein is meant any material harvested from an agricultural or horticultural crop, including without limitation grain, fruit, leaves, fibers, seeds, or other plant parts.
Products derived or obtained from such harvested material include flour, meal, and flakes derived from grain; and products in which such materials are admixed, such as, for example, cake, cookie, muffin, pancake and biscuit mixes.
Alternatively, the larvicidal proteinase inhibitor can be incorporated into the tissues of a susceptible plant so that in the course of infesting and consuming the plant, its harvested material, or a product derived from harvested plant material, the larvae consume larvicidal amounts of the proteinase inhibitor. One method of doing this is to incorporate the proteinase inhibitor in a non-phytotoxic vehicle which is adapted for systemic administration to the susceptible plants. This method is commonly employed with insecticidal materials which are designed to attack chewing insects and is well within the purview of one of ordinary skill in the art of insecticide and larvicide formulation, but is a method which may not be as suitable for active enzyme blockers such as proteinase inhibitors.
Alternatively, a dietary bait containing one or more of the selected proteinase inhibitors can be employed, with, optionally, an added pheromonal or other larval attractant material. However, the genes which code for these peptides can be isolated and cloned. Alternatively, they can be synthesized directly using a DNA sequence obtained by working backwards from the known amino acid sequence for aprotinin or a related proteinase inhibitor, preferably using plant-preferred codons. The resulting sequence can be 35 inserted into an appropriate expression cassette and introduced into cells of a susceptible plant species, so that an especially preferred embodiment of this method involves inserting into the genome of the plant a DNA
~ s ~ j WO94/1656~ PCT~S94/00630
2 ~5 45~ 6 - 4 -sequence coding for one or more insecticidal proteinase inhibitors selected from aprotinin and serine proteinase inhibitors having at least 90% homology to aprotinin by amino acid sequence, in proper reading frame relative to transcription initiator and promoter sequences active in the plant. Transcription and translation of the DNA sequence under control of the plant-active regulatory sequences causes expression of the larvicidal gene product at levels which provide an insecticidal amount of the proteinase inhibitor in the tissues of the plant which are normally infested by the larvae.
This method offers particular advantages when the potential for insects becoming resistant to these materials is considered. Insecticide-resistant insects become a problem as a result of application of strong selection pressure which highly favors naturally resistant individuals and any resistant mutants which occur. As a result, over the course of a few generations the resistant insects become the predominant type.
Heavy application of insecticidal materials generally to a field or a geographical area by dust or spray or by soil incorporation tends to impose strong selection pressures of the kind described, since insects have no "safe havens" where non-resistant individuals can survive.
However, many insect pests of crop plants also attack non-crop species. Limiting the insecticidal materials to the crop plants in the region by expressing the insecticidal materials only in those plants permits continued survival of non-resistant insects in associated weed plants which provide not only "safe havens" from the toxic compound but food for the insects. This reduces selection pressure significantly and thus slows development and spread of resistant insects.
This method also offers advantages from the standpoint of soil and groundwater contamination, since no application vehicle is required. The insecticidal components themselves are of natural origin and break down naturally when the plant is digested or decomposes. The method offers further WO94/16565 ^ . PCT~S94/00630 ._ .;, - 5 - ~
advantages from the standpoint of cost, since no applicàt~ion expense is involved and the cost of the insecticidal materials is factored into the price of the seed or other reproductive material which the grower purchases.
The plant should be a plant which is susceptible to infestation and damage, or whose harvested material or products are susceptible to infestation and damage by the larvae of European corn borer and corn rootworm. These include corn (Zea mays), wheat (Triticum aestivum) and sorghum (Sorghum bicolor). However, this short list is not to be construed as limiting, inasmuch as these species are among the most difficult commercial crops to reliably transform and regenerate, and these insects (under other common names) also infest other crops. Thus the methods of this invention are readily applicable via conventional techniques to numerous plant species, if they are found to be susceptible to the plant pests listed hereinabove, including, without limitation, species from the genera Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manicot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Cichorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Hemerocallis, Nemesia, Pelargonium, Panicum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browallia, Glycine, Lolium, Triticum, and Datura.
Preferred plants that are to be transformed according to the methods of this invention are cereal crops, including maize, rye, barley, wheat, sorghum, oats, millet, rice, triticale, sunflower, alfalfa, rapeseed and soybean, fiber crops, such as cotton, fruit crops, such as melons, and vegetable crops, including onion, pepper, tomato, cucumber, squash, carrot, crucifer (cabbage, broccoli, cauliflower), eggplant, spinach, potato and lettuce.
The DNA sequence which when expressed imparts insecti-cidal activity is a structural gene which codes for aprotinin, or a proteinase inhibitor having at least 90%
W094/l6565 ~16 ~ PCT~594/00~0 homology to aprotinin. It has been found that these proteinase inhibitors have sufficient insecticidal (larvicidal) activity to be operative in a plant cell expression system. That is, while certain other proteinase inhibitors such as cowpea trypsin inhibitors have some larvicidal activity at high concentrations in pure form, plant cell expression at such high concentrations is either not possible in a living plant cell system, or is not feasible if the commercially useful characteristics of the plant are to be preserved in terms of production of oils, starches, fibers, or other materials. A tissue-specific promoter can be used in any instance where it may be desirable to localize production of the proteinase inhibitor to an infested tissue or to a tissue which is efficient in production of the proteinase inhibitor.
In carrying out this invention, it will be appreciated that numerous plant expression cassettes and vectors are well known in the art. By the term "expression cassette" is meant a complete set of control sequences including initiation, promoter and termination sequences which function in a plant cell when they flank a structural gene in the proper reading frame. Expression cassettes frequently and preferably contain an assortment of restric-tion sites suitable for cleavage and insertion of any desired structural gene. It is important that the cloned gene have a start codon in the correct reading frame for the structural sequence. In addition, the plant expression cassette preferably includes a strong constitutive promoter sequence at one end to cause the gene to be transcribed at ~
high frequency, and a poly-A recognition sequence at the other end for proper processing and transport of the messenger RNA. An example of such a preferred (empty) expression cassette into which the DNA sequence of the present invention can be inserted is the pPHI414 plasmid developed by Beach et al. of Pioneer Hi-Bred International, Inc., Johnston, IA and disclosed in U.S. Patent application No. 07/785,648, filed October 31, 1991. Highly preferred plant expression cassettes will be designed to include one 21;54576 ~ t~ ~YJS '3, WO 94/16565 ~ 4/00630 _, . .
or more selectable marker genes, such as kanamycin resistance or herbicide tolerance genes.
By the term "vector" herein is meant a DNA sequence which is able to replicate and express a foreign gene in a host cell. Typically, the vector has one or more endo-nuclease recognition sites which may be cut in a predictable fashion by use of the appropriate enzyme. Such vectors are preferably constructed to include additional structural gene sequences imparting antibiotic or herbicide resistance, which then serve as selectable markers to identify and separate transformed cells. Preferred selection agents include kanamycin, chlorosulfuron, phosphonothricin, glyphosate, hygromycin and methotrexate, and preferred markers are genes conferring resistance to these agents. A
cell in which the foreign genetic material in a vector is functionally expressed has been "transformed" by the vector and is referred to as a "transformant".
A particularly preferred vector is a plasmid, by which is meant a circular double-stranded DNA molecule that is not a part of the chromosomes of the cell.
As mentioned above, genomic, synthetic and cDNA
encoding the gene of interest may be used in this invention.
The vector of interest may also be constructed partially from a cDNA clone, partially from a synthetic sequence and partially from a genomic clone. When the gene sequence of interest is in hand, genetic constructs are made which contain the necessary regulatory sequences to provide for efficient expression of the gene -in the host cell.
According to this invention, the genetic construct will contain (a) a first genetic sequence coding for the proteinase inhibitor of interest and (b) one or more regulatory sequences operably linked on either side of the structural gene of interest. Typically, the regulatory sequences will be selected from the group comprising of promoters and terminators. The regulatory sequences may be from autologous or heterologous sources.
WO94/16565 PCT~S94/00630 5~6 8 -Promoters that may be used in the genetic sequence include nos, ocs, phaseolin, CaMV, FMV and other promoters isolated from plants or plant pests.
An efficient plant promoter that may be used is an overproducing plant promoter. Overproducing plant promoters that may be used in this invention include the promoter of the small sub-unit (ss) of the ribulose-1,5-biphosphate carboxylase from soybean (Berry-Lowe et al, J. Molecular and App. Gen., 1:483-498 (1982)), and the promoter of the cholorophyll a-b binding protein. These two promoters are known to be light-induced, in eukaryotic plant cells (see, for example, Genetic Engineering of Plants, An Agricultural Perspective, A. Cashmore, Pelham, New York, 1983, pp. 29-38, G. Coruzzi et al., J. Biol. Chem., 258:1399 (1983), and P.
Dunsmuir, et al., J. Molecular and App. Gen., 2:285 (1983)).
The expression cassette comprising the structural gene for the proteinase inhibitor of interest operably linked to the desired control sequences can be ligated into a suitable cloning vector. In general, plasmid or viral (bacterio-phage) vectors containing replication and control sequencesderived from species compatible with the host cell are used.
The cloning vector will typically carry a replication origin, as well as specific genes that are capable of providing phenotypic selection markers in transformed host cells. Typically, genes conferring resistance to anti-biotics or selected herbicides are used. After the genetic material is introduced into the target cells, successfully transformed cells and/or colonies of cells can be isolated by selection on the basis of these markers.
Typically, an intermediate host cell will be used in the practice of this invention to increase the copy number of the cloning vector. With an increased copy number, the vector containing the gene of interest can be isolated in significant quantities for introduction into the desired plant cells. Host cells that can be used in the practice of this invention include prokaryotes, including bacterial hosts such as E. coli, S. typhimurium, and S. marcescens.
2159576 ~
WO94/16~65 -- PCT~S94/00630 _ g _ ~ ~
Eukaryotic hosts such as yeast or filamentous fungi may also be used in this invention.
The isolated cloning vector will then be introduced into the plant cell using any convenient technique, includ-ing electroporation (in protoplasts), retroviruses,microparticle bombardment, and microinjection, into cells from monocotyledonous or dicotyledonous plants, in cell or tissue culture, to provide transformed plant cells containing as foreign DNA at least one copy of the DNA
sequence of the plant expression cassette. Preferably, the monocotyledonous species will be selected from maize, sorghum, wheat and rice, and the dicotyledonous species will be selected from soybean, sunflower, cotton, rapeseed (either edible or industrial), alfalfa, tobacco, and Solanaceae such as potato and tomato. Using known techniques, protoplasts can be regenerated and cell or tissue culture can be regenerated to form whole fertile plants which carry and express the desired gene for the selected protein. Accordingly, a highly preferred embodiment of the present invention is a transformed maize plant, the cells of which contain as foreign DNA at least one copy of the DNA sequence of an expression cassette of this invention.
This invention also provides methods of imparting resistance to European corn borer and corn rootworm to plants of a susceptible taxon, comprising the steps of:
a) culturing cells or tissues from at least one plant from the taxon, b) introducing into the cells of the cell or tiss~le culture at least one copy of an expression cassette compris-ing a structural gene coding for a proteinase inhibitor selected from aprotinin and serine proteinase inhibitors having at least 90% homology thereto by amino acid sequence, or a combination of such proteinase inhibitors, operably linked to plant regulatory sequences which cause the expression of the protein structural gene in the cells, and c) regenerating insect-resistant whole plants from the cell or tissue culture. Once whole plants have been ~ Y f 21S45'~ 5 obtained in this manner, they can be sexually or clonally reproduced in any manner such that at least one copy of the sequence provided by the expression cassette is present in the cells of progeny of the reproduction.
Alternatively, once a single transformed plant has been obtained by the foregoing recombinant DNA method, conven-tional plant breeding methods can be used to transfer the protein structural gene and associated regulatory sequences via crossing and backcrossing. Such intermediate methods will comprise the further steps of a) sexually crossing the insect-resistant plant with a plant from the insect-susceptible taxon;
b) recovering reproductive material from insect-resistant progeny of the cross; and c) growing insect-resistant plants from the reproductive material. Where desirable or necessary, the agronomic characteristics of the susceptible taxon can be substantially preserved by expanding this method to include the further steps of repetitively:
a) backcrossing the insect-resistant progeny with insect-susceptible plants from the susceptible taxon; and b) selecting for expression of insect resistance (or an associated marker gene) among the progeny of the back-cross, until the desired percentage of the characteristics of the susceptible taxon are present in the progeny along with the gene imparting insect resistance. This will be important, for example, where the taxon is a substantially homozygous plant variety, such as an inbred line of maize or a variety of a self-pollinated crop such as soybeans. sy "substantially homozygous" is meant homozygous within the limits commonly accepted in the commercial production of certified seed of the species. For example, an inbred line of maize used in commercial seed production is typically 95%
to 100% homozygous, and preferably 98% to 100% homozygous, as measured by RFLP analysis using 50 to 200 probes well distributed across the genome. If necessary, an RFLP-guided process of self-pollination and selection can be used to achieve this degree of genetic uniformity.
~lS4576`
WO94/16565 PCT~S94/00630 r By the term "taxon" herein is meant a unit of botanical classification of genus or lower. It thus includes genus, species, cultivars, varieties, variants, and other minor taxonomic groups which lack a consistent nomenclature.
It will also be appreciated by those of ordinary skill that the plant vectors provided herein can be incorporated into Agrobacterium tumefaciens or Agrobacterium rhizogenes, which can then be used to transfer the vector into susceptible plant cells, primarily from dicotyledonous species. Thus, this invention provides a method for imparting insect resistance in Agrobacterium-susceptible dicotyledonous plants in which the expression cassette is introduced into the cells by infecting the cells with an Agrobacterium species, a plasmid of which has been modified to include a plant expression cassette of this invention.
Finally, it has now been determined that aprotinin and highly homologous serine proteinase inhibitors strongly potentiate the insecticidal activity of lectins such as wheat germ agglutinin. This effect is surprising and unexpected, since many natural insecticides target the same structures or molecules within the insect and as a result many combinations of such natural insecticides are at best additive and more often competitive, with little or no increase in insecticidal activity, as shown in the experimental results below. In addition to the increased level of activity which this result provides, there are also the practical benefits of increased technical flexibility and feasibility, since the synergy or potentiation between these two groups of plant-expressible materials permits the attainment of effective insect or larval control with lower levels of expression of each component. Since plant cell systems are more amenable to such lower levels of expression, this offers the biotechnological entomologist greater flexibility in formulation and greater likelihood of achieving complete insect suppression in host plants with less-than-maximal levels of gene expression.
The combination of two different, synergistic insecticidal materials in a single system to provide WO94/16565 PCT~S94100630 ~ ~S ~S~ 6 _ 12 -effective insect control also reduces the potential for development of resistant insects. Since such resistances typically arise by spontaneous mutation, developing resistance to a binary system such as aprotinin plus wheat germ agglutinin would require twn mutations, one in each target structure or molecule. `The probability of such a double mutation is potentially (if there is no association between the mutations) as low as the product of the probabilities of the individual mutations, which would be quite low indeed -- perhaps l.0 x l0-1, or less than fifty potentially survivable individuals per year in the entire United States. Also, the low or reduced selection pressure for each individual mutation further reduces its spread within the population.
Accordingly, this invention also provides a method for killing European corn borer and corn rootworm, comprising administering enterally to the larvae of those species a larvicidal combination of (a) aprotinin, a serine proteinase inhibitor having at least 90% homology to aprotinin by amino acid sequence, or a combination thereof; and (b) an insecticidal lectin.
The following description further exemplifies the compositions of this invention and the methods of making and using them. However, it will be understood that other methods, known by those of ordinary skill in the art to be equivalent, can also be employed.
In the following examples the wide variety of insects screened has resulted in several different bioassays being used to determine the effect of aprotinin and combinations of aprotinin plus lectin on larval growth and survivorship.
However, all of the bioassays allow the test materials to be enterally administered to the insect. In vitro bioassays for the European corn borer (Ostrinia nubalis), and southern corn rootworm (Diabrotica undecimpunctata howardii) were done by incorporating the test protein into the artificial diet. This is referred to herein as an "Incorporated Bioassay". This was accomplished by making up a standard artificial diet at 90% of the original water and adding a WO94/16565 PCT~S94/00630 solution of the test protein to this mixture.
Concentrations of the protein in this diet are recorded as mg or ~g of protein per ml of diet. Weight and mortality are recorded after seven days. Specific assays and variations are described in the individual examples.
Éxample l EUROPEAN CORN BORER
Aprotinin was the most effective protease inhibitor against European corn borer with high mortality occurring during a replicated 7-day incorporated bioassay. The results are shown in Table l.
Table l.5 Effect of aprotinin and other protease inhibitors (PI) on European corn borer neonate larvae in incorporated bioassays Treatment Weight Reduction (fold) ~Mortality 20 Control 5.2 -- 0*
Aprotinin -- -- lO0 Soybean PI (Bowman-Birk) 4.7 _ 0 Soybean PI (Kunitz) 3.9 1.3 9 Chicken PI (Type IV) 2.9 l.8 05 *Corrected mortality All materials were tested at 20 mg PI/ml of diet SOU.~KN CORN ROOTWORM
Aprotinin also showed effectiveness against Southern corn rootworm neonate larvae in incorporated bioassays, as seen in Table 2.
W094/16565 215 457 6 PCT~S94/00630 .., Table 2 Effect of aprotinin and other protease inhibitors (PI) on Southern corn rootworm neRnate larvae in incorporated bioa~`6says Treatment Weight Reduction (fold) %Mortality Control 3.3 -- o*
Aprotinin 0.4 8 60 Soybean PI (Bowman-Birk) 2.4 1.5 50 Cystatin 2.5 1.4 30 *Corrected mortality All materials were tested at 20 mg PI/ml of diet WO94/1656~ ~' PCT~S94/00630 Example 2 Combination of Aprotinin plus Wheat Lectin Tests were-,performed employing Wheat Germ Agglutinin (WGA), aprotinin, ~ap,d combinations of the two in 7-day incorporated bioassays~., The results are shown in Table 3.
Table 3 Effect of WGA and aprotinin on European corn borer neonate larvae in incorporated bioassays Treatment Expected mortality from %Mortality aprotinin addition Control 0 1. WGA 0.10 mg/ml 10 2. WGA 0.15 mg/ml 15
This method offers particular advantages when the potential for insects becoming resistant to these materials is considered. Insecticide-resistant insects become a problem as a result of application of strong selection pressure which highly favors naturally resistant individuals and any resistant mutants which occur. As a result, over the course of a few generations the resistant insects become the predominant type.
Heavy application of insecticidal materials generally to a field or a geographical area by dust or spray or by soil incorporation tends to impose strong selection pressures of the kind described, since insects have no "safe havens" where non-resistant individuals can survive.
However, many insect pests of crop plants also attack non-crop species. Limiting the insecticidal materials to the crop plants in the region by expressing the insecticidal materials only in those plants permits continued survival of non-resistant insects in associated weed plants which provide not only "safe havens" from the toxic compound but food for the insects. This reduces selection pressure significantly and thus slows development and spread of resistant insects.
This method also offers advantages from the standpoint of soil and groundwater contamination, since no application vehicle is required. The insecticidal components themselves are of natural origin and break down naturally when the plant is digested or decomposes. The method offers further WO94/16565 ^ . PCT~S94/00630 ._ .;, - 5 - ~
advantages from the standpoint of cost, since no applicàt~ion expense is involved and the cost of the insecticidal materials is factored into the price of the seed or other reproductive material which the grower purchases.
The plant should be a plant which is susceptible to infestation and damage, or whose harvested material or products are susceptible to infestation and damage by the larvae of European corn borer and corn rootworm. These include corn (Zea mays), wheat (Triticum aestivum) and sorghum (Sorghum bicolor). However, this short list is not to be construed as limiting, inasmuch as these species are among the most difficult commercial crops to reliably transform and regenerate, and these insects (under other common names) also infest other crops. Thus the methods of this invention are readily applicable via conventional techniques to numerous plant species, if they are found to be susceptible to the plant pests listed hereinabove, including, without limitation, species from the genera Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manicot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Cichorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Hemerocallis, Nemesia, Pelargonium, Panicum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browallia, Glycine, Lolium, Triticum, and Datura.
Preferred plants that are to be transformed according to the methods of this invention are cereal crops, including maize, rye, barley, wheat, sorghum, oats, millet, rice, triticale, sunflower, alfalfa, rapeseed and soybean, fiber crops, such as cotton, fruit crops, such as melons, and vegetable crops, including onion, pepper, tomato, cucumber, squash, carrot, crucifer (cabbage, broccoli, cauliflower), eggplant, spinach, potato and lettuce.
The DNA sequence which when expressed imparts insecti-cidal activity is a structural gene which codes for aprotinin, or a proteinase inhibitor having at least 90%
W094/l6565 ~16 ~ PCT~594/00~0 homology to aprotinin. It has been found that these proteinase inhibitors have sufficient insecticidal (larvicidal) activity to be operative in a plant cell expression system. That is, while certain other proteinase inhibitors such as cowpea trypsin inhibitors have some larvicidal activity at high concentrations in pure form, plant cell expression at such high concentrations is either not possible in a living plant cell system, or is not feasible if the commercially useful characteristics of the plant are to be preserved in terms of production of oils, starches, fibers, or other materials. A tissue-specific promoter can be used in any instance where it may be desirable to localize production of the proteinase inhibitor to an infested tissue or to a tissue which is efficient in production of the proteinase inhibitor.
In carrying out this invention, it will be appreciated that numerous plant expression cassettes and vectors are well known in the art. By the term "expression cassette" is meant a complete set of control sequences including initiation, promoter and termination sequences which function in a plant cell when they flank a structural gene in the proper reading frame. Expression cassettes frequently and preferably contain an assortment of restric-tion sites suitable for cleavage and insertion of any desired structural gene. It is important that the cloned gene have a start codon in the correct reading frame for the structural sequence. In addition, the plant expression cassette preferably includes a strong constitutive promoter sequence at one end to cause the gene to be transcribed at ~
high frequency, and a poly-A recognition sequence at the other end for proper processing and transport of the messenger RNA. An example of such a preferred (empty) expression cassette into which the DNA sequence of the present invention can be inserted is the pPHI414 plasmid developed by Beach et al. of Pioneer Hi-Bred International, Inc., Johnston, IA and disclosed in U.S. Patent application No. 07/785,648, filed October 31, 1991. Highly preferred plant expression cassettes will be designed to include one 21;54576 ~ t~ ~YJS '3, WO 94/16565 ~ 4/00630 _, . .
or more selectable marker genes, such as kanamycin resistance or herbicide tolerance genes.
By the term "vector" herein is meant a DNA sequence which is able to replicate and express a foreign gene in a host cell. Typically, the vector has one or more endo-nuclease recognition sites which may be cut in a predictable fashion by use of the appropriate enzyme. Such vectors are preferably constructed to include additional structural gene sequences imparting antibiotic or herbicide resistance, which then serve as selectable markers to identify and separate transformed cells. Preferred selection agents include kanamycin, chlorosulfuron, phosphonothricin, glyphosate, hygromycin and methotrexate, and preferred markers are genes conferring resistance to these agents. A
cell in which the foreign genetic material in a vector is functionally expressed has been "transformed" by the vector and is referred to as a "transformant".
A particularly preferred vector is a plasmid, by which is meant a circular double-stranded DNA molecule that is not a part of the chromosomes of the cell.
As mentioned above, genomic, synthetic and cDNA
encoding the gene of interest may be used in this invention.
The vector of interest may also be constructed partially from a cDNA clone, partially from a synthetic sequence and partially from a genomic clone. When the gene sequence of interest is in hand, genetic constructs are made which contain the necessary regulatory sequences to provide for efficient expression of the gene -in the host cell.
According to this invention, the genetic construct will contain (a) a first genetic sequence coding for the proteinase inhibitor of interest and (b) one or more regulatory sequences operably linked on either side of the structural gene of interest. Typically, the regulatory sequences will be selected from the group comprising of promoters and terminators. The regulatory sequences may be from autologous or heterologous sources.
WO94/16565 PCT~S94/00630 5~6 8 -Promoters that may be used in the genetic sequence include nos, ocs, phaseolin, CaMV, FMV and other promoters isolated from plants or plant pests.
An efficient plant promoter that may be used is an overproducing plant promoter. Overproducing plant promoters that may be used in this invention include the promoter of the small sub-unit (ss) of the ribulose-1,5-biphosphate carboxylase from soybean (Berry-Lowe et al, J. Molecular and App. Gen., 1:483-498 (1982)), and the promoter of the cholorophyll a-b binding protein. These two promoters are known to be light-induced, in eukaryotic plant cells (see, for example, Genetic Engineering of Plants, An Agricultural Perspective, A. Cashmore, Pelham, New York, 1983, pp. 29-38, G. Coruzzi et al., J. Biol. Chem., 258:1399 (1983), and P.
Dunsmuir, et al., J. Molecular and App. Gen., 2:285 (1983)).
The expression cassette comprising the structural gene for the proteinase inhibitor of interest operably linked to the desired control sequences can be ligated into a suitable cloning vector. In general, plasmid or viral (bacterio-phage) vectors containing replication and control sequencesderived from species compatible with the host cell are used.
The cloning vector will typically carry a replication origin, as well as specific genes that are capable of providing phenotypic selection markers in transformed host cells. Typically, genes conferring resistance to anti-biotics or selected herbicides are used. After the genetic material is introduced into the target cells, successfully transformed cells and/or colonies of cells can be isolated by selection on the basis of these markers.
Typically, an intermediate host cell will be used in the practice of this invention to increase the copy number of the cloning vector. With an increased copy number, the vector containing the gene of interest can be isolated in significant quantities for introduction into the desired plant cells. Host cells that can be used in the practice of this invention include prokaryotes, including bacterial hosts such as E. coli, S. typhimurium, and S. marcescens.
2159576 ~
WO94/16~65 -- PCT~S94/00630 _ g _ ~ ~
Eukaryotic hosts such as yeast or filamentous fungi may also be used in this invention.
The isolated cloning vector will then be introduced into the plant cell using any convenient technique, includ-ing electroporation (in protoplasts), retroviruses,microparticle bombardment, and microinjection, into cells from monocotyledonous or dicotyledonous plants, in cell or tissue culture, to provide transformed plant cells containing as foreign DNA at least one copy of the DNA
sequence of the plant expression cassette. Preferably, the monocotyledonous species will be selected from maize, sorghum, wheat and rice, and the dicotyledonous species will be selected from soybean, sunflower, cotton, rapeseed (either edible or industrial), alfalfa, tobacco, and Solanaceae such as potato and tomato. Using known techniques, protoplasts can be regenerated and cell or tissue culture can be regenerated to form whole fertile plants which carry and express the desired gene for the selected protein. Accordingly, a highly preferred embodiment of the present invention is a transformed maize plant, the cells of which contain as foreign DNA at least one copy of the DNA sequence of an expression cassette of this invention.
This invention also provides methods of imparting resistance to European corn borer and corn rootworm to plants of a susceptible taxon, comprising the steps of:
a) culturing cells or tissues from at least one plant from the taxon, b) introducing into the cells of the cell or tiss~le culture at least one copy of an expression cassette compris-ing a structural gene coding for a proteinase inhibitor selected from aprotinin and serine proteinase inhibitors having at least 90% homology thereto by amino acid sequence, or a combination of such proteinase inhibitors, operably linked to plant regulatory sequences which cause the expression of the protein structural gene in the cells, and c) regenerating insect-resistant whole plants from the cell or tissue culture. Once whole plants have been ~ Y f 21S45'~ 5 obtained in this manner, they can be sexually or clonally reproduced in any manner such that at least one copy of the sequence provided by the expression cassette is present in the cells of progeny of the reproduction.
Alternatively, once a single transformed plant has been obtained by the foregoing recombinant DNA method, conven-tional plant breeding methods can be used to transfer the protein structural gene and associated regulatory sequences via crossing and backcrossing. Such intermediate methods will comprise the further steps of a) sexually crossing the insect-resistant plant with a plant from the insect-susceptible taxon;
b) recovering reproductive material from insect-resistant progeny of the cross; and c) growing insect-resistant plants from the reproductive material. Where desirable or necessary, the agronomic characteristics of the susceptible taxon can be substantially preserved by expanding this method to include the further steps of repetitively:
a) backcrossing the insect-resistant progeny with insect-susceptible plants from the susceptible taxon; and b) selecting for expression of insect resistance (or an associated marker gene) among the progeny of the back-cross, until the desired percentage of the characteristics of the susceptible taxon are present in the progeny along with the gene imparting insect resistance. This will be important, for example, where the taxon is a substantially homozygous plant variety, such as an inbred line of maize or a variety of a self-pollinated crop such as soybeans. sy "substantially homozygous" is meant homozygous within the limits commonly accepted in the commercial production of certified seed of the species. For example, an inbred line of maize used in commercial seed production is typically 95%
to 100% homozygous, and preferably 98% to 100% homozygous, as measured by RFLP analysis using 50 to 200 probes well distributed across the genome. If necessary, an RFLP-guided process of self-pollination and selection can be used to achieve this degree of genetic uniformity.
~lS4576`
WO94/16565 PCT~S94/00630 r By the term "taxon" herein is meant a unit of botanical classification of genus or lower. It thus includes genus, species, cultivars, varieties, variants, and other minor taxonomic groups which lack a consistent nomenclature.
It will also be appreciated by those of ordinary skill that the plant vectors provided herein can be incorporated into Agrobacterium tumefaciens or Agrobacterium rhizogenes, which can then be used to transfer the vector into susceptible plant cells, primarily from dicotyledonous species. Thus, this invention provides a method for imparting insect resistance in Agrobacterium-susceptible dicotyledonous plants in which the expression cassette is introduced into the cells by infecting the cells with an Agrobacterium species, a plasmid of which has been modified to include a plant expression cassette of this invention.
Finally, it has now been determined that aprotinin and highly homologous serine proteinase inhibitors strongly potentiate the insecticidal activity of lectins such as wheat germ agglutinin. This effect is surprising and unexpected, since many natural insecticides target the same structures or molecules within the insect and as a result many combinations of such natural insecticides are at best additive and more often competitive, with little or no increase in insecticidal activity, as shown in the experimental results below. In addition to the increased level of activity which this result provides, there are also the practical benefits of increased technical flexibility and feasibility, since the synergy or potentiation between these two groups of plant-expressible materials permits the attainment of effective insect or larval control with lower levels of expression of each component. Since plant cell systems are more amenable to such lower levels of expression, this offers the biotechnological entomologist greater flexibility in formulation and greater likelihood of achieving complete insect suppression in host plants with less-than-maximal levels of gene expression.
The combination of two different, synergistic insecticidal materials in a single system to provide WO94/16565 PCT~S94100630 ~ ~S ~S~ 6 _ 12 -effective insect control also reduces the potential for development of resistant insects. Since such resistances typically arise by spontaneous mutation, developing resistance to a binary system such as aprotinin plus wheat germ agglutinin would require twn mutations, one in each target structure or molecule. `The probability of such a double mutation is potentially (if there is no association between the mutations) as low as the product of the probabilities of the individual mutations, which would be quite low indeed -- perhaps l.0 x l0-1, or less than fifty potentially survivable individuals per year in the entire United States. Also, the low or reduced selection pressure for each individual mutation further reduces its spread within the population.
Accordingly, this invention also provides a method for killing European corn borer and corn rootworm, comprising administering enterally to the larvae of those species a larvicidal combination of (a) aprotinin, a serine proteinase inhibitor having at least 90% homology to aprotinin by amino acid sequence, or a combination thereof; and (b) an insecticidal lectin.
The following description further exemplifies the compositions of this invention and the methods of making and using them. However, it will be understood that other methods, known by those of ordinary skill in the art to be equivalent, can also be employed.
In the following examples the wide variety of insects screened has resulted in several different bioassays being used to determine the effect of aprotinin and combinations of aprotinin plus lectin on larval growth and survivorship.
However, all of the bioassays allow the test materials to be enterally administered to the insect. In vitro bioassays for the European corn borer (Ostrinia nubalis), and southern corn rootworm (Diabrotica undecimpunctata howardii) were done by incorporating the test protein into the artificial diet. This is referred to herein as an "Incorporated Bioassay". This was accomplished by making up a standard artificial diet at 90% of the original water and adding a WO94/16565 PCT~S94/00630 solution of the test protein to this mixture.
Concentrations of the protein in this diet are recorded as mg or ~g of protein per ml of diet. Weight and mortality are recorded after seven days. Specific assays and variations are described in the individual examples.
Éxample l EUROPEAN CORN BORER
Aprotinin was the most effective protease inhibitor against European corn borer with high mortality occurring during a replicated 7-day incorporated bioassay. The results are shown in Table l.
Table l.5 Effect of aprotinin and other protease inhibitors (PI) on European corn borer neonate larvae in incorporated bioassays Treatment Weight Reduction (fold) ~Mortality 20 Control 5.2 -- 0*
Aprotinin -- -- lO0 Soybean PI (Bowman-Birk) 4.7 _ 0 Soybean PI (Kunitz) 3.9 1.3 9 Chicken PI (Type IV) 2.9 l.8 05 *Corrected mortality All materials were tested at 20 mg PI/ml of diet SOU.~KN CORN ROOTWORM
Aprotinin also showed effectiveness against Southern corn rootworm neonate larvae in incorporated bioassays, as seen in Table 2.
W094/16565 215 457 6 PCT~S94/00630 .., Table 2 Effect of aprotinin and other protease inhibitors (PI) on Southern corn rootworm neRnate larvae in incorporated bioa~`6says Treatment Weight Reduction (fold) %Mortality Control 3.3 -- o*
Aprotinin 0.4 8 60 Soybean PI (Bowman-Birk) 2.4 1.5 50 Cystatin 2.5 1.4 30 *Corrected mortality All materials were tested at 20 mg PI/ml of diet WO94/1656~ ~' PCT~S94/00630 Example 2 Combination of Aprotinin plus Wheat Lectin Tests were-,performed employing Wheat Germ Agglutinin (WGA), aprotinin, ~ap,d combinations of the two in 7-day incorporated bioassays~., The results are shown in Table 3.
Table 3 Effect of WGA and aprotinin on European corn borer neonate larvae in incorporated bioassays Treatment Expected mortality from %Mortality aprotinin addition Control 0 1. WGA 0.10 mg/ml 10 2. WGA 0.15 mg/ml 15
3. WGA 0.20 mg/ml 20
4. WGA 0.25 mg/ml 25
5. Aprotinin 0.25 mg/ml15
6. Aprotinin 0.5 mg/ml 10
7. Aprotinin 1.0 mg/ml 25
8. Aprotinin 2.0 mg/ml 30 Combinations 4 + 8 90 55 3 + 7 80 45 2 + 7 75 40 3 + 5 70 35 3 + 6 70 30 2 + 6 55 25 When the wheat lectin was replaced with Bauhinea purpùrea lectin, similar results were also obtained.
Replicated 7-day bioassays were also performed to measure effects on growth. Results are shown in Table 4.
WO94/16565g5 ~ 6 16 - PCT~S94/00~0 Table 4 Effect of Aprotinin and WGA Combinations on ECB growth TreatmentWeight Reduction Weight (fold) Control l0.5 --l. Aprotinin 0.l mg/ml 7.8 l.3 2. WGA 0.l mg/ml l0.3 --l + 2 2.5 4.2 The weight and weight reduction is significantly different from all other weights and reductions at p< 0.05.
The foregoing results indicate a synergy between aprotinin and insecticidal lectins in combination, both in terms of mortality and growth inhibition. Based on results with other combinations of insecticidal compounds, an additive or neutral effect would have been expected.
Industrial Applicability I. Isolation of the protein gene and insertion into bacteria In order to isolate the coding sequence for the protein, it is necessary to have nucleotide sequence data which establishes an open reading frame (i.e., the correct triplet code for translation which should have only one "stop" signal at the very end of the gene.) It is also necessary to have an indication of where to look for the protease cleavage junction between the protein and the replicase which precedes it in the sequence. This can be determined from the peptide sequence of the N-terminal portion of the protein or by comparing the protein sequence with that of other homologous proteins. This can generally be accomplished and the necessary information obtained without sequencing the entire gene. Once the sequence at both ends of the gene has been determined, the remainder of the gene can be cloned using restriction enzymes that flank the protein coding region or, more preferably, by cloning the precise protein coding region by oligonucleotide-W094/16565 _ ;7 _ PCT~S94tOO~O
directed amplification of DNA (polymerase chain reaction orPCR).
Once the gene has been isolated, it can be cloned into a bacterial expression vector with linkers added to create all three reading frames (using 8mer, lOmer, and 12mers each of which contain an ATG translational start site). The resulting vectors, containing the fragments of interest, can be inserted into, for example, BRL's Maximum Efficiency DH5 F' IQ transformation competent E. coli cells. All three transformations, one for each linker, are then screened via minipreps for the presence and orientation of insert.
Appropriate clones are then chosen to test for expression of the protein gene.
Clones containing the properly oriented inserts are grown in culture medium conducive to the induction of the gene (LB medium with added IPTG). The cells are lysed and bacterial proteins are subjected to electrophoresis in SDS
polyacrylamide gels and then transferred to nitrocellulose.
The resulting protein blots are easily screened for presence of protein using rabbit polyclonal and mouse monoclonal anti-protein antibody.
Having determined the proper reading frame, it is then necessary to remove the gene from the bacterial expression vector. The linker at the start of the gene region supplies the necessary start codon.
II. Expression of the Protein Gene in Plants A plant expression cassette, employing the regulatory sequences developed by Beach, et al., and containing the protein gene, is constructed. The restriction map of the preferred plasmid, designated pPHI414, is illustrated in Figure 1. This plasmid contains an enhanced 35S promoter spanning nucleotides - 421 to +2 of Cauliflower Mosaic virus with the region from - 421 to - 90 duplicated in tandem, a 79 bp HindIII Sall fragment from pJII101 spanning the 5~
leader sequence of Tobacco Mosaic Virus, a 579 bp fragment spanning the first intron from maize AdH1-S, and a 281 bp W094/16565 215 4 5 7 6 PCT~S94/00~0 fragment spanning the polyadenylation site from the nopaline synthase gene in pTiT37.
Another construct which can be used as an expression cassette is the pPHI412 plasmid shown in Eigure 2. It differs from pPHI414 in that it lacks the AdH intron segment. However, like pPHI414, it is constructed to have numerous restriction sites between the O' segment and the NOS segment, which sites can be conveniently used for splicing any desired protein structural gene into position.
This vector can be cotransformed with a similar plasmid containing a selectable marker for antibiotic resistance into Black Mexican Sweet corn protoplasts by electroporation. These protoplasts can then be induced to regenerate cell walls and develop into callus by conventional techniques. Likewise, this callus can then be subjected to antibiotic selection to select for transformed colonies, and these colonies can be tested for expression of protein with antisera for the appropriate protein using known methods. The efficiency of protection can be measured by infesting callus (or suspension cultures derived from callus) with the target insect and measuring survival percentages.
The protein gene can be introduced into embryogenic maize callus by methods similar to those used for slack Mexican Sweet. Embryogenic callus can be regenerated to whole fertile plants. The insect resistance imparted by the endogenous production of the protein is a simply inherited, dominant trait and can, if desired, be introduced into other plant varieties of the species by simple crossing or backcrossing.
Using the foregoing techniques, aprotinin has been expressed in maize suspension cells as determined by transient assays.
Replicated 7-day bioassays were also performed to measure effects on growth. Results are shown in Table 4.
WO94/16565g5 ~ 6 16 - PCT~S94/00~0 Table 4 Effect of Aprotinin and WGA Combinations on ECB growth TreatmentWeight Reduction Weight (fold) Control l0.5 --l. Aprotinin 0.l mg/ml 7.8 l.3 2. WGA 0.l mg/ml l0.3 --l + 2 2.5 4.2 The weight and weight reduction is significantly different from all other weights and reductions at p< 0.05.
The foregoing results indicate a synergy between aprotinin and insecticidal lectins in combination, both in terms of mortality and growth inhibition. Based on results with other combinations of insecticidal compounds, an additive or neutral effect would have been expected.
Industrial Applicability I. Isolation of the protein gene and insertion into bacteria In order to isolate the coding sequence for the protein, it is necessary to have nucleotide sequence data which establishes an open reading frame (i.e., the correct triplet code for translation which should have only one "stop" signal at the very end of the gene.) It is also necessary to have an indication of where to look for the protease cleavage junction between the protein and the replicase which precedes it in the sequence. This can be determined from the peptide sequence of the N-terminal portion of the protein or by comparing the protein sequence with that of other homologous proteins. This can generally be accomplished and the necessary information obtained without sequencing the entire gene. Once the sequence at both ends of the gene has been determined, the remainder of the gene can be cloned using restriction enzymes that flank the protein coding region or, more preferably, by cloning the precise protein coding region by oligonucleotide-W094/16565 _ ;7 _ PCT~S94tOO~O
directed amplification of DNA (polymerase chain reaction orPCR).
Once the gene has been isolated, it can be cloned into a bacterial expression vector with linkers added to create all three reading frames (using 8mer, lOmer, and 12mers each of which contain an ATG translational start site). The resulting vectors, containing the fragments of interest, can be inserted into, for example, BRL's Maximum Efficiency DH5 F' IQ transformation competent E. coli cells. All three transformations, one for each linker, are then screened via minipreps for the presence and orientation of insert.
Appropriate clones are then chosen to test for expression of the protein gene.
Clones containing the properly oriented inserts are grown in culture medium conducive to the induction of the gene (LB medium with added IPTG). The cells are lysed and bacterial proteins are subjected to electrophoresis in SDS
polyacrylamide gels and then transferred to nitrocellulose.
The resulting protein blots are easily screened for presence of protein using rabbit polyclonal and mouse monoclonal anti-protein antibody.
Having determined the proper reading frame, it is then necessary to remove the gene from the bacterial expression vector. The linker at the start of the gene region supplies the necessary start codon.
II. Expression of the Protein Gene in Plants A plant expression cassette, employing the regulatory sequences developed by Beach, et al., and containing the protein gene, is constructed. The restriction map of the preferred plasmid, designated pPHI414, is illustrated in Figure 1. This plasmid contains an enhanced 35S promoter spanning nucleotides - 421 to +2 of Cauliflower Mosaic virus with the region from - 421 to - 90 duplicated in tandem, a 79 bp HindIII Sall fragment from pJII101 spanning the 5~
leader sequence of Tobacco Mosaic Virus, a 579 bp fragment spanning the first intron from maize AdH1-S, and a 281 bp W094/16565 215 4 5 7 6 PCT~S94/00~0 fragment spanning the polyadenylation site from the nopaline synthase gene in pTiT37.
Another construct which can be used as an expression cassette is the pPHI412 plasmid shown in Eigure 2. It differs from pPHI414 in that it lacks the AdH intron segment. However, like pPHI414, it is constructed to have numerous restriction sites between the O' segment and the NOS segment, which sites can be conveniently used for splicing any desired protein structural gene into position.
This vector can be cotransformed with a similar plasmid containing a selectable marker for antibiotic resistance into Black Mexican Sweet corn protoplasts by electroporation. These protoplasts can then be induced to regenerate cell walls and develop into callus by conventional techniques. Likewise, this callus can then be subjected to antibiotic selection to select for transformed colonies, and these colonies can be tested for expression of protein with antisera for the appropriate protein using known methods. The efficiency of protection can be measured by infesting callus (or suspension cultures derived from callus) with the target insect and measuring survival percentages.
The protein gene can be introduced into embryogenic maize callus by methods similar to those used for slack Mexican Sweet. Embryogenic callus can be regenerated to whole fertile plants. The insect resistance imparted by the endogenous production of the protein is a simply inherited, dominant trait and can, if desired, be introduced into other plant varieties of the species by simple crossing or backcrossing.
Using the foregoing techniques, aprotinin has been expressed in maize suspension cells as determined by transient assays.
Claims (53)
1. A method for killing insects or larvae of European corn borer and Southern corn rootworm, comprising administering enterally to the larvae a larvicidal amount of aprotinin, a serine proteinase inhibitor having at least 90% homology to aprotinin by amino acid sequence, or a combination thereof.
2. A method according to claim 1 wherein the proteinase inhibitor is administered enterally by incorporating it in the diet of the larvae.
3. A method according to claim 2 wherein the diet of the larvae comprises the tissues of a living plant.
4. A method according to claim 3 wherein the proteinase inhibitor is not native to the plant.
5. A method according to any one of claims 1 to 3 wherein the diet of the larvae comprises the tissues of a living plant, harvested material of a plant, or products derived from said harvested material liable to infestation by larvae ofEuropean corn borer and Southern corn rootworm, said plant having inserted into its genome at least one sequence coding for aprotinin, a serine proteinase inhibitorhaving at least 90% homology thereto, or a combination of such proteinase inhibitors, in proper reading frame relative to transcription initiator and promoter sequences active in the plant to cause expression of said coding sequence or sequences at levels which provide a larvicidal amount of the encoded product in the tissues of the plant or harvested material of the plant.
6. A method according to claim 5 wherein said plant is a monocotyledonous species selected from corn, wheat, rice and sorghum.
7. A method according to claim 5 wherein said plant is a dicotyledonous species selected from soybean, sunflower, rapeseed, alfalfa, cotton and tomato.
8. A method of producing a plant as defined in claim 5 comprising the steps of:
a) culturing cells or tissues from a plant liable to infestation by European corn borer or Southern corn rootworm larvae;
b) introducing into the cells of the cell or tissue culture at least one copy of an expression cassette comprising a sequence coding for the proteinase inhibitor or combination of proteinase inhibitors, and c) regenerating resistant whole plants from the cell or tissue culture.
a) culturing cells or tissues from a plant liable to infestation by European corn borer or Southern corn rootworm larvae;
b) introducing into the cells of the cell or tissue culture at least one copy of an expression cassette comprising a sequence coding for the proteinase inhibitor or combination of proteinase inhibitors, and c) regenerating resistant whole plants from the cell or tissue culture.
9. A method according to claim 8 which comprises the further step of sexually or clonally reproducing plants obtained in step (c) in such a manner that at least one copy of the sequence provided by the expression cassette is present in the cells of progeny of the reproduction.
10. A method according to claim 8 in which the expression cassette is introduced into the cells by electroporation.
11. A method according to claim 8 in which the expression cassette is introduced into the cells by microparticle bombardment.
12. A method according to claim 8 in which the expression cassette is introduced into the cells by microinjection.
13. A method according to claim 8 wherein the cells or tissues employed in step (a) are derived from an Agrobacterium tumefaciens-susceptible dicotyledonous plant and the expression cassette is introduced into the cells by infecting the cells with Agrobacterium tumefaciens, a plasmid of which has been modified to include the expression cassette.
14. A method of imparting resistance to European corn borer and Southern corn rootworm to plants of a taxon susceptible to those insects, and thereby to harvested material from the plants and products obtained from the harvested material, comprising the steps of:
(a) selecting a fertile, insect resistant plant prepared by the method of claim 8 from a sexually compatible species;
(b) sexually crossing the insect resistant plant with a plant from the insect susceptible taxon;
(c) recovering reproductive material from insect resistant progeny of the cross; and (d) growing resistant plants from the reproductive material.
(a) selecting a fertile, insect resistant plant prepared by the method of claim 8 from a sexually compatible species;
(b) sexually crossing the insect resistant plant with a plant from the insect susceptible taxon;
(c) recovering reproductive material from insect resistant progeny of the cross; and (d) growing resistant plants from the reproductive material.
15. A method according to claim 14 for imparting insect resistance in a taxon consisting of substantially homozygous plants, and thereby to harvested materialfrom the plants and products obtained from the harvested material, which comprises the further steps of repetitively:
(a) backcrossing the insect resistant progeny with substantially homozygous, insect susceptible plants from the taxon; and (b) selecting for expression of both insect resistance and the other characteristics of the susceptible taxon among the progeny of the backcross, until the desired percentage of the characteristics of the susceptible taxon are present in the progeny along with insect resistance.
(a) backcrossing the insect resistant progeny with substantially homozygous, insect susceptible plants from the taxon; and (b) selecting for expression of both insect resistance and the other characteristics of the susceptible taxon among the progeny of the backcross, until the desired percentage of the characteristics of the susceptible taxon are present in the progeny along with insect resistance.
16. An expression cassette comprising a DNA coding sequence for aprotinin, a serine proteinase inhibitor having at least 90% homology thereto or a combination of such proteinase inhibitors operably linked to plant regulatory sequences which cause the expression of said DNA coding sequence in plant cells.
17. Transformed plant cells containing as foreign DNA at least one copy of the DNA sequence of an expression cassette according to claim 16.
18. Transformed plant cells according to claim 17, which are cells of a monocotyledonous species.
19. Transformed plant cells according to claim 18, which are maize, sorghum, wheat or rice cells.
20. Transformed plant cells according to claim 17, which are cells of a dicotyledonous species.
21. Transformed plant cells according to claim 20, which are soybean, alfalfa, sunflower, rapeseed, cotton or tomato cells.
22. A maize cell- or tissue-culture comprising maize cells according to claim 19.
23. A transformed maize plant, the cells of which contain as foreign DNA at least one copy of the DNA sequence of an expression cassette according to claim 16.
24. A method of killing or controlling European corn borer or Southern corn rootworm larvae comprising applying to harvested plant material liable to infestation with said larvae a composition comprising aprotinin, a proteinase inhibitor having at least 90% homology thereto, or a combination thereof.
25. A method for killing insects or larvae of European corn borer and Southern corn rootworm comprising administering enterally to the larvae a larvicidal combination of (a) aprotinin, a serine proteinase inhibitor having at least 90%
homology to aprotinin by amino acid sequence, or a combination thereof; and (b) an insecticidal lectin.
homology to aprotinin by amino acid sequence, or a combination thereof; and (b) an insecticidal lectin.
26. A method according to claim 25 wherein the proteinase inhibitor and insecticidal lectin are administered enterally by incorporating them in the diet of the larvae.
27. A method according to claim 26 wherein the diet of the larvae comprises the tissues of a living plant.
28. A method according to claim 27 wherein the proteinase inhibitor and lectin are not native to the plant.
29. A method according to any one of claims 25 to 27 wherein the diet of the larvae comprises the tissues of a living plant, harvested material from a plant, or products derived from said harvested material liable to infestation by larvae ofEuropean corn borer and Southern corn rootworm, said plant having inserted in its genome sequences coding for (a) aprotinin, a serine proteinase inhibitor having at least 90%
homology thereto, or a combination of such proteinase inhibitors, and (b) an insecticidal lectin, in proper reading frame relative to transcription initiator and promoter sequences active in the plant to cause expression of said coding sequences at levels which provide a larvicidal amount of the encoded products in the tissues of the plant or harvested material of the plant.
homology thereto, or a combination of such proteinase inhibitors, and (b) an insecticidal lectin, in proper reading frame relative to transcription initiator and promoter sequences active in the plant to cause expression of said coding sequences at levels which provide a larvicidal amount of the encoded products in the tissues of the plant or harvested material of the plant.
30. A method according to claim 29 wherein said plant is a monocotyledonous species selected from corn, wheat, rice and sorghum.
31. A method according to claim 29 wherein said plant is a dicotyledonous species selected from soybean, sunflower, rapeseed, alfalfa, cotton and tomato.
32. A method of producing a plant as defined in claim 29 comprising the steps of:
(a) culturing cells or tissues from a plant liable to infestation by European corn borer or Southern corn rootworm larvae;
(b) introducing into the cells of the cell or tissue culture at least one copy of an expression cassette comprising sequences coding for (i) the proteinase inhibitor or combination of proteinase inhibitors and (ii) the lectin, and c) regenerating resistant whole plants from the cell or tissue culture.
(a) culturing cells or tissues from a plant liable to infestation by European corn borer or Southern corn rootworm larvae;
(b) introducing into the cells of the cell or tissue culture at least one copy of an expression cassette comprising sequences coding for (i) the proteinase inhibitor or combination of proteinase inhibitors and (ii) the lectin, and c) regenerating resistant whole plants from the cell or tissue culture.
33. A method according to claim 32 which comprises the further step of secually or clonally reproducing plants obtained in step (c) in such manner that at least one copy of the sequence provided by the expression cassette is present in the cells of progeny of the reproduction.
34. A method according to claim 32 in which the expression cassette is introduced into the cells by electroporation.
35. A method according to claim 32 in which the expression cassette is introduced into the cells by microparticle bombardment.
36. A method according to claim 32 in which the expression cassette is introduced into the cells by microinjection.
37. A method according to claim 32 wherein the cells or tissues employed in step (a) are derived from an Agrobacterium tumefaciens-susceptible dicotyledonous plant and the expression cssette is introduced into the cells by infecting the cells with an Agrobacterium tumefaciens, a plasmid of which has been modified to include the expression cassette.
38. A method of imparting resistance to European corn borer and Southern corn rootworm to plants of a taxon susceptible to those insects, and thereby to harvested material from the plants and products obtained from the harvested material, comprising the steps of:
(a) selecting a fertile, insect resistant plant prepared by the method of claim 32 from a sexually compatible species;
(b) sexually crossing the insect resistant plant with a plant from the insect susceptible taxon;
(c) recovering reproductive material from insect resistant progeny of the cross; and (d) growing resistant plants from the reproductive material.
(a) selecting a fertile, insect resistant plant prepared by the method of claim 32 from a sexually compatible species;
(b) sexually crossing the insect resistant plant with a plant from the insect susceptible taxon;
(c) recovering reproductive material from insect resistant progeny of the cross; and (d) growing resistant plants from the reproductive material.
39. A method according to claim 38 for imparting insect resistance in a taxon consisting of substantially homozygous plants, and thereby to harvested materialfrom the plants and products obtained from the harvested material, which comprises the further steps of repetitively:
(a) backcrossing the insect resistant progeny with substantially homozygous, insect susceptible plants from the taxon; and (b) selecting for expression of both insect resistance and the other characteristics of the susceptible taxon among the progeny of the backcross, until the desired percentage of the characteristics of the susceptible taxon are present in the progeny along with insect resistance.
(a) backcrossing the insect resistant progeny with substantially homozygous, insect susceptible plants from the taxon; and (b) selecting for expression of both insect resistance and the other characteristics of the susceptible taxon among the progeny of the backcross, until the desired percentage of the characteristics of the susceptible taxon are present in the progeny along with insect resistance.
40. An isolated DNA sequence which codes substantially solely for (a) aprotinin or a serine proteinase inhibitor having at least 90%
homology to aprotinin by amino acid sequence, or a combination of such proteinase inhibitors, and (b) an insecticidal lectin.
homology to aprotinin by amino acid sequence, or a combination of such proteinase inhibitors, and (b) an insecticidal lectin.
41. An expression cassette comprising a DNA sequence according to claim 40 wherein the coding sequences for said serine proteinase inhibitor(s) and said lectin are operably linked to plant regulatory sequences which cause the expression of said DNA coding sequences in plant cells.
42. An expression cassette comprising a DNA sequence according to claim 40 wherein the coding sequences for said serine proteinase inhibitor(s) and said lectin are operably linked to bacterial expression regulatory sequences which cause theexpression of said DNA coding sequences in bacterial cells.
43. Bacterial cells containing as a foreign plasmid at least one copy of an expression cassette according to claim 42.
44. Transformed plant cells containing as foreign DNA at least one copy of the DNA sequence of an expression cassette according to claim 41.
45. Transformed plant cells according to claim 44 which are cells of a monocotyledonous species.
46. Transformed plant cells accoridng to claim 45 which are maize, sorghum, wheat or rice cells.
47. Transformed plant cells according to claim 44 which are cells of a dicotyledonous species.
48. Transformed plant cells according to claim 47 which are soybean, alfalfa, sunflower, rapeseed, cotton or tomato cells.
49. A maize cell- or tissue-culture comprising maize cells according to claim 46.
50. A transformed maize plant, the cells of which contain as foreign DNA at least one copy of the DNA sequence of an expression cassette according to claim 41.
51. A larvicidal composition, comprising a larvicidal amount of a composition consisting of (a) aprotinin, a serine proteinase inhibitor having at least 90%
homology to aprotinin by amino acid sequence, or a combination of such proteinases inhibitors and (b) a larvicidal lectin; in a non-phytotoxic vehicle.
homology to aprotinin by amino acid sequence, or a combination of such proteinases inhibitors and (b) a larvicidal lectin; in a non-phytotoxic vehicle.
52. A composition according to claim 51 wherein the vehicle includes a larval dietary bait for susceptible insects.
53. A composition according to claim 52 wherein the bait comprises a pheromonal larval attractant for susceptible insects.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US792493A | 1993-01-25 | 1993-01-25 | |
| US08/007,924 | 1993-01-25 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2154576A1 true CA2154576A1 (en) | 1994-08-04 |
Family
ID=21728841
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002154576A Abandoned CA2154576A1 (en) | 1993-01-25 | 1994-01-14 | Aprotinin and synergistic combinations thereof with lectins as larvicides against insect pests of agronomic crops, harvested material thereof, and products obtained from the harvested material |
Country Status (6)
| Country | Link |
|---|---|
| EP (1) | EP0680258A1 (en) |
| AU (1) | AU6230194A (en) |
| BR (1) | BR9405668A (en) |
| CA (1) | CA2154576A1 (en) |
| MX (1) | MX9400634A (en) |
| WO (1) | WO1994016565A1 (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| MX9606538A (en) * | 1994-06-17 | 1997-12-31 | Univ Trobe | Biological control of insects. |
| WO1997007681A1 (en) * | 1995-08-24 | 1997-03-06 | Nzym, Inc. | Inhibitors of pepsin or pepsin-like proteinases as acaricides |
| AU7014596A (en) * | 1995-08-24 | 1997-03-19 | Nzym, Inc. | Inhibitors of trypsin or trypsin-like proteinases as acaricides |
| US5824870A (en) * | 1995-11-06 | 1998-10-20 | Baszczynski; Chris | Commercial production of aprotinin in plants |
| GR980100482A (en) | 1998-01-16 | 1999-09-30 | Novartis Ag | Use of insecticides in pest control |
| US6844339B2 (en) | 1998-01-16 | 2005-01-18 | Syngenta Crop Protection, Inc. | Use of neonicotinoids in pest control |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ES2150410T3 (en) * | 1988-06-20 | 2000-12-01 | Novartis Ag | PROCEDURE FOR THE COMBAT OF PARASITES OF PLANTS. |
| ES2074547T3 (en) * | 1989-11-07 | 1995-09-16 | Pioneer Hi Bred Int | LARVICID LECTINES, AND INDUCED RESISTANCE OF PLANTS TO INSECTS. |
| US5258302A (en) * | 1990-07-03 | 1993-11-02 | The Salk Institute Biotechnology/Industrial Associates, Inc. | DNA for expression of aprotinin in methylotrophic yeast cells |
| ATE183884T1 (en) * | 1990-07-30 | 1999-09-15 | Novartis Ag | INSECTICIDAL PROTEINS |
-
1994
- 1994-01-14 EP EP94909462A patent/EP0680258A1/en not_active Withdrawn
- 1994-01-14 AU AU62301/94A patent/AU6230194A/en not_active Abandoned
- 1994-01-14 CA CA002154576A patent/CA2154576A1/en not_active Abandoned
- 1994-01-14 BR BR9405668A patent/BR9405668A/en not_active Application Discontinuation
- 1994-01-14 WO PCT/US1994/000630 patent/WO1994016565A1/en not_active Application Discontinuation
- 1994-01-24 MX MX9400634A patent/MX9400634A/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| WO1994016565A1 (en) | 1994-08-04 |
| MX9400634A (en) | 1994-08-31 |
| EP0680258A1 (en) | 1995-11-08 |
| BR9405668A (en) | 1995-11-21 |
| AU6230194A (en) | 1994-08-15 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| FZDE | Discontinued |