WO1996009398A1 - Produits d'assemblage geniques codant des agents de protection des cultures, plantes transformees renfermant et exprimant de tels produits d'assemblage, et procedes de lutte contre les agents pathogenes et organismes pesteux des cultures - Google Patents

Produits d'assemblage geniques codant des agents de protection des cultures, plantes transformees renfermant et exprimant de tels produits d'assemblage, et procedes de lutte contre les agents pathogenes et organismes pesteux des cultures Download PDF

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WO1996009398A1
WO1996009398A1 PCT/NL1995/000310 NL9500310W WO9609398A1 WO 1996009398 A1 WO1996009398 A1 WO 1996009398A1 NL 9500310 W NL9500310 W NL 9500310W WO 9609398 A1 WO9609398 A1 WO 9609398A1
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gene construct
organism
antibody
pathogen
plague
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PCT/NL1995/000310
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Jacob Bakker
Arjen Schots
Wilhelmus Johannes Stiekema
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Rijkslandbouwuniversiteit Wageningen
Stichting Voor De Technische Wetenschappen
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Priority to EP95932974A priority Critical patent/EP0782626A1/fr
Priority to JP8510769A priority patent/JPH10506274A/ja
Publication of WO1996009398A1 publication Critical patent/WO1996009398A1/fr

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    • 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
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/50—Isolated enzymes; Isolated proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/245—Escherichia (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
    • C07K14/325—Bacillus thuringiensis crystal protein (delta-endotoxin)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/14—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from fungi, algea or lichens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8282—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for fungal resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/244—Endo-1,3(4)-beta-glucanase (3.2.1.6)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2442—Chitinase (3.2.1.14)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01006—Endo-1,3(4)-beta-glucanase (3.2.1.6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01014—Chitinase (3.2.1.14)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • 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
    • 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
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to gene constructs suitable for expressing agents which protect a plant against plague organisms and pathogens.
  • attack of plants by pathogens such as fungi, nematodes, insects, bacteria and viruses, constitutes a considerable economic problem, in particular for large-scale culture crops such as corn, rice, beans, potatoes, tomatoes and grapes. Protection of such plants by chemical means is highly undesired for environmental reasons.
  • a more effective and more acceptable way of protecting plants against attack by plague organisms consists in making the plant resistant to the action of attacking organisms by providing it with genetic information controlling the effect of such organisms.
  • resistance to viruses can be achieved by expression of viral coat protein (Beachy et al, 1990), or by expression of a single chain variable antibody fragment (scFcv) against the Ca 2+ binding domain of a viral coat protein (Tavladoraki et al, 1993). Resistance to insects can be obtained by expression of Bacillus thuringiensis insecticidal crystal proteins (ICP's) (Vaeck et al, 1987).
  • ICP's Bacillus thuringiensis insecticidal crystal proteins
  • Resistance to fungi can be achieved by expression of chitinase and/or glucanase (Alternaria longipes: US-A-4,940,840, Fusarium solani: EP-A-440304, Botrytis cinerea and Rhizoctonia solani : Broglie et al, 1989, WO- A-90- 07001).
  • Resistance to plant-parasitic nematodes can be achieved by using giant cell specific promoters in combination with barnase or anti-sense DNA techniques (Opperman et al, 1994).
  • ICP-toxin e.g. Cry ⁇ A(b), CrylC and CrylE
  • the objective of the present invention is to provide means and methods for protecting plants, in particular agricultural crops, against plagues in a more effective and ecologically acceptable manner.
  • This objective is met according to the invention, by fusing monoclonal antibodies or parts thereof to toxins or enzymes having toxic activities or to effective parts of these toxins or enzymes.
  • the chimeric protein can be made either by fusing the sequences encoding the respective parts of the chimera or by chemically or biochemically linking the two parts of the chimeric protein together as for instance described in WO-A-9318162.
  • the invention relates to gene constructs comprising a nucleotide sequence encoding an antibody or part thereof which is specific for a plague organism or pathogen, and a nucleotide sequence encoding a protein which has a toxic effect on said plague organism or pathogen.
  • the invention furthermore relates to chimeric proteins consisting of an antibody or parts thereof, which is specific for a plague organism or pathogen and a protein which has a toxic effect on a said plague organism or pathogen and which has been constructed by chemically or biochemically linking the antibody (or parts thereof) to the toxic protein or enzyme (or parts thereof).
  • Monoclonal antibodies can be raised to almost any epitope or to almost any molecular structure of a pathogen which is vulnerable to toxins or toxic enzymes (and parts thereof), using the hybridoma technique (K ⁇ hler and Milstein, 1975).
  • Single chain antibodies can be prepared from monoclonal antibody producing hybridoma's by established molecular techniques.
  • single chain antibodies with affinities for selected epitopes or molecular structures which are vulnerable to toxins or toxic enzymes (or parts thereof) can be obtained from phage display libraries (Hoogenboom et al. 1992; Winter et al., 1994).
  • a major advantage of this strategy is that the effectiveness of the toxicity can be enhanced and furthermore that the specificity can be tuned to the target organisms.
  • Antibodies can be raised to structures of the plague organism which will directly or indirectly lead to resistance or partial resistance when these antibodies are fused to the appropriate toxin or enzyme.
  • these structures are cell membranes and cell walls, especially in the case of bacteria and fungi; or alimentary tract structures, e.g. epithelial antigens, especially in the case of nematodes and insects; or coat proteins, especially in the case of viruses.
  • the antibodies are preferably single chain antibodies.
  • Toxic proteins include all proteins that have a toxic effect on plague organisms or pathogens, such as toxins and toxic enzymes.
  • Toxins that can be fused with monoclonal antibodies include the following:
  • ICP Bacillus thuringiensis insecticidal crystal proteins
  • ICP's or ⁇ -endo-toxins are a family of proteins produced during sporulation in the cytoplasm of B. thuringien- sis. These proteins crystallize as parasporal inclusion, which are solubilized in the insect larvae gut.
  • the toxins are highly specific and effect lysis of gut cells of susceptible insect larvae.
  • Many B. thuringiensis strains producing ICP's with different insect host spectra have been isolated. ICP's are classified according to their specificity spectrum (Hofte and Whiteley, 1989).
  • CryIA(b) is produced as a 131 kDa protoxin which is activated by removal of an N- and a C-terminal propeptide by proteases present in the insect gut.
  • the mature toxin (65-66 kDa) comprises three domains.
  • the N-terminal (first) domain which contains several conserved hydrophobic sequences, is assumed to form a pore in the apical membrane of gut epidermis cells.
  • the second domain is highly variable and presumably binds to a receptor in the cell membrane.
  • the C-terminal (third) domain also contains conserved sequences.
  • Colicins are a family of plasmid encoded antibiotical proteins, which kill bacteria closely related to the producing strain (generally
  • Escherichia coli are composed of structural domains, which exert different functions, such as receptor binding, translocation and killing. Based on their mode of action colicins can be classified into two groups. The major group of colicins causes permeabilisation of the cytoplasmic membrane, thereby destroying the membrane potential. The C-terminal domain of these colicins form ion channels in artificial membranes. The other group of colicins causes enzymatic cleavage of DNA or 16S rRNA. - thionins (Bohlmann and Apel, 1991); these are toxic for bacteria and fungi.
  • toxins like ribosome-inactivating proteins may be suitable as well to obtain resistance against plague organisms. Further examples are: - Saporin (Stirpe 1983; Stirpe and Barbieri, 1986)
  • Toxic enzymes that can be fused to monoclonal antibodies include:
  • PR-2 proteins can be classified in an alkaline form and acidic form.
  • the alkaline form is produced after several processing steps of the translation product. First the N-terminal signal peptide is cleaved off during transport to the endoplasmatic reticulum, and then the C-terminal part is glycosylated and removed for transport over the vacuole membrane.
  • glucanases both an intracellular and an extracellular form exist. The intracellular form is extended by 3-25 aminoacids at the C-terminus.
  • the sequence of intra ⁇ cellular ⁇ -l,3-glucanase gene is disclosed in EP-A-440304.
  • Glucanase genes from other plants are also known, e.g. glucanase (and endochitinase) from maize (Nasser et al. 1988). Effective destruction of cell wall glucans by endo- ⁇ -l,3-glucanases sometimes appears to require cooperation by exoenzymes such as exoglucanases. - chitinases. These are PR-3 proteins comprising two domains and a hydrophobic signal peptide which is absent in the active enzyme.
  • Chitinases are subdivided in three classes: class I, alkaline chitinases, are localised in the vacuole and contain a cysteine-rich domain, and a C-terminal sequence of 6 aminoacids that is removed after translation and are involved in vacuolar targeting; class II, acid chitinases, lack a cysteine-rich domain and have a lower enzyme activity; they are localised in the apoplast; class III, lysozyme-active chitinases, contain other conserved sequences than I and II. Plant chitinase was found to derive its chitinase activity from a 30 kDa monomer.
  • chitinases as for glucanases, both an intracellular and an extracellular form exist.
  • the intracellular form is extended by 3-10 aminoacids at the C-terminus.
  • the catalytic centre is localised in the C-terminal part which is the same in both forms.
  • the sequence of intracellular chitinase gene is disclosed in EP- A-440304.
  • Chitinase genes from other organisms are also known, e.g. bean endo ⁇ chitinase, (DeBroglie et al. 1986).
  • Chitinase and ⁇ -l,3-glucanase are produced at an increased rate upon infection with fungi (Verticillium albo-atrum); Chitinase and ⁇ -l,3-glucanase from tomato inhibit growth of fungi in vitro (Young and Pegg, 1982, Young and Pegg 1981) and probably also in vivo (Pegg and Vessey, 1973).
  • Gene constructs may comprise nucleotide sequences encoding the complete antibody molecule, the Fab part, the F(ab)2part, scFv part, bivalent scFv (diabody) (Holliger, Prospero & Winter, 1993), minibody (Pack et al., 1993), or any other part (like complementarity determining regions) which shows binding to the targets.
  • the antibody sequence is fused to a complete sequence encoding an enzyme/toxin or to a part thereof which is still functionally active.
  • the chimeric protein consisting of an antibody (fragment) and an enzyme/toxin can also be obtained by chemical or biochemical linkage.
  • the antibody (fragment) and the enzyme/toxin (fragment) is fused directly or using a flexible linker which does not interfere with the structure and function of the two proteins.
  • a flexible linker which does not interfere with the structure and function of the two proteins.
  • Such flexible linkers are for instance those which have been used to fuse the variable domains of the heavy and light chain of immunoglobulms to construct a scFv, those used to create bivalent bispecific scFvs or those used in immunotoxins (see Whitlow and Filpula, 1991; Kihlberg et al., 1993; Huston et al., 1992; Takkinen et al., 1991).
  • Linkers can also be based on hinge regions in antibody molecules (Pack and Pl ⁇ ckthun, 1992; Pack et al., 1993) or on peptide fragments between structural domains of proteins. When only a functional part of the toxin is to be conjugated to the antibody (fragment) the linker present between two domains of the complete toxin itself could be used. Fusions can be made between the enzyme/toxin and the heavy chain (fragment) or the light chain (fragment) of the immunoglobulin at both the C and N terminus. In the case of a scFv fusion the variable domains can be in both the order V H -linker-V L and V L -linker-V H .
  • the desired cellular location of the proteins can be achieved using the appropriate targeting sequences. Proteins synthesized without targeting sequences stay in the cytoplasm of the cell, whereas others are directed into the secretory pathway by a signal peptide. When no other targeting signal is present, the latter proteins are secreted by default. Additional targeting signals can be present to direct the proteins for example to the vacuolar compartment of the cell or to retain them in the endoplasmic reticulum (Chrispeels, 1991). Targeting signals to direct proteins to the chloroplast, mitochondria, peroxisomes or nucleus have been described (Austen and Westwood, 1991). An example of a targeting route is the secretion via endoplasmic reticulum and golgi apparatus. Examples of signal sequences for secretion are described in Briggs and
  • the fusion protein has to be expressed in a heterologous organism for production of the protein as such, it may be necessary to modify the gene construct in order to improve expression because of the codon preference of this organism, to remove mRNA instability motifs (e.g. AT regions, false splice sites) and polyadenylation signals.
  • mRNA instability motifs e.g. AT regions, false splice sites
  • polyadenylation signals e.g. AT regions, false splice sites
  • the fusions genes are expressed in plants under control of any type of promoter which is active in plants.
  • Examples are: a) constitutive promoters such as the CaMV-35S (Kay et al., 1987) b) tissue specific promoters such as described in Nap et al. (1993) (leave), De Almeida et al. (1989) (leave, SSU-promoter), Nap et al. (1992) (potato tuber, patatin promoter), Hendriks et al. (1991) (potato tuber), Guerche et al.
  • inducible promoters such as the 2' promoters (Langridge et al., 1994), wound inducible promoters (Logemann et al., 1989; Suh et al., 1991) or chemically induced promoters (Williams et al., 1992).
  • Transformation can be done using any method which ensures a stable integration of the chimeric gene in the plant genome in such a way that it can still be transcribed.
  • Examples of transformation are: a) Agrobacterium tumefaciens mediated transformation (Horsch et al., 1985): based on a natural transformation system in which the bacterium stably incorporates part of a plasmid DNA (T-DNA) into the plant genome.
  • T-DNA includes the gene to be expressed.
  • Microprojectile bombardment Vasil et al., 1992): particles coated with DNA penetrate the plant cell nuclei at high velocity where the DNA is integrated into the genome by host recombination processes.
  • the gene constructs can also be used for plague control through external application on crops which are to be protected. Such direct application can be achieved in the form of administering the expression product of the chimeric gene, i.e. the immunotoxin comprising an antibody linked to a toxic protein.
  • Another form can be by applying an organism containing the immunotoxin as such or containing a gene encoding the immunotoxin and capable of producing it.
  • Suitable carrier organisms include microorganisms such as bacteria (e.g. B. thuringiensis), fungi, yeasts and viruses. The organisms may be alive or dead.
  • the invention also relates to an immunotoxin comprising an antibody linked to a protein which is toxic for a plague organism or pathogen, and which immunotoxin is obtainable from an expression system as described above and can be used for external protection of plants against plague organisms.
  • the protein may be purified by known methods.
  • the invention also comprises organisms which contain such an immunotoxin and organisms which are stably transformed with the gene construct encoding the immunotoxin. These organisms can be used for external protection of plants against plague organisms.
  • the invention further relates to pesticidal compositions containing an immunotoxin as such or in encoded form, together with an acceptable carrier.
  • the compositions may also contain solvents, agents preventing the composition from washing away, stabilisers, attractants, UV-absorbers, and the like.
  • the invention also relates to a process for protecting a plant against the action of a plague organism or a pathogen, wherein the plant is externally treated with an immunotoxin as described or an organism containing the immunotoxin, or with a composition containing it. Treatment may be done by spraying and the like, by hand using any suitable equipment including tractors, aircraft etc.
  • Fusion of monoclonal antibodies or parts thereof with enzymes having toxic activities Fusion of a plant chitinase and glucanase to a scFv derived from a monoclonal antibody against a fungus (Verticillium dahliae) The following steps are taken: 1) Antibodies against mycelium or purified cell wall components of Verticillium dahliae are raised, and monoclonals are isolated. 2) cDNA sequences encoding antibody variable region are cloned to create a single- chain Fv construct.
  • N- or C-terminal fusion between scFv and chitinase or ⁇ -l,3-glucanase is performed using a suitable linker, e.g. CBHI linker (Takkinen et al. 1991), and the chimeric gene is inserted in an expression vector, e.g. pNem5 or pNem ⁇ ( Figures
  • Regenerated plants are screened for expression of fusion product.
  • the steps can be followed, with appropriate adaptations of antibody production and fusion with enzyme, for producing transgenic plants with resistance to Botrytis cinerea, Fusarium oxysporum f.sp. radicis-ly coper sisci and Phytophthora infestans.
  • Fusions with other proteins having toxic activities are also possible, e.g. with potato lectine.
  • N- or C-terminal fusion between scFv and CryIA(b)_BT(29-607) is performed using a suitable linker, e.g. CBHI linker (Takkinen et al. 1991), and the chimeric gene is inserted in an expression vector, e.g. pNem6 ( Figure 2), which is a derivative of pSPORTl (Gibco, BRL).
  • a suitable linker e.g. CBHI linker (Takkinen et al. 1991)
  • pNem6 Figure 2
  • Figure 2 is a derivative of pSPORTl (Gibco, BRL).
  • the binding activity of the bacterially (E. col ⁇ ) expressed chimeric protein will be analyzed by Western blot analysis, ligand blot assays in combination with competition experiments (Bosch et al., 1994), on cryo-sections of midguts of insect larvae (Martens et al., 1994) and on primary cultures of epithelial cells of insect midguts (Baines et al., 1993). Insecticidal activity of the chimeric protein will be checked by bioassays and lysing effect of the chimeric proteins will be followed using primary midgut cell cultures.
  • the fusion gene is transferred, together with suitable targeting sequences, to the plant transformation vector in between a promoter-termination cassette for stable transformation.
  • a promoter-termination cassette for stable transformation.
  • the fusion gene will be cloned behind a constitutive promoter (i.e. CaMV-35S promoter) and a suitable termination cassette.
  • the expression cassette with fusion gene and selection marker is transferred into the plant genome by plant transformation. 9) Regenerated plants are screened for expression of fusion product.
  • the binding activity of the chimeric protein will be checked by western analysis and ligand blot assays in combination with competition experiments, on cryo-sections of midguts of insect larvae (Martens et al., 1994) and on primary cultures of epithelial cells of insect midguts (Baines et al., 1993). Insecticidal activity of the chimeric protein will be checked by bioassays and lysing effect of the chimeric proteins will be followed using primary midgut cell cultures.
  • N- and C-terminal fusions will be made between a scFv and CryIA(b)_Bt(l-607); CryIA(b)_Bt(29-429); CryIA(b)_Bt(l-1155).
  • domain II (or part of it) of Cry ⁇ A(b) which is thought to be responsible for receptor binding, will be replaced by a scFv.
  • the steps can be followed, with appropriate adaptations of antibody production and fusion with toxins, for producing transgenic plants with resistance to nematodes by raising monoclonal antibodies against the intestine of the nematode.
  • Example 3 Balb/c mice were immunized with brush border membrane vesicles (BBMV's), isolated from the midgut of Spodoptera exigua as described by Bosch et al., 1994. The mice were immunized twice (with a four week interval) by subcutaneous injection of BBMV's using the equivalent of 50 ⁇ g protein with the addition of Freund's incomplete adjuvans. Four weeks after the last immunization a boost was given with BBMV's (50 ⁇ g protein equivalent) injected intraperitoneally. Three days later the spleen was removed and the fusion was carried out as described by Schots et al, 1992. 2) Antisera and monoclonal antibodies were checked for their ability to react with epitopes present in BBMV's with ELISA according to standard procedures.
  • BBMV's brush border membrane vesicles
  • Single chain antibodies were isolated from hybridoma's producing monoclonals which bound to epitopes present at the luminal side of the membrane and at the outside of the midgut cells of S. exigua, according to standard procedures as, for instance, described in (Johnson & Bird, 1991); (Huston et al., 1992).
  • step 4 the scFv is coupled to the N-terminus of the pore-forming domain of colicin N (C-terminal region of the protein) (Pugsley, 1987), with in front of this domain the peptide fragment which normally links the N-terminal part of the complete colicin N to its pore-forming domain, the latter peptide fragment serving as a linker between the two domains of the chimeric protein.
  • Steps 1) to 5) of Example 1 are repeated, the antibodies being raised in step 1) against the mycelium or cell wall components of Botrytis cinerea.
  • the fusion gene is cloned into vector pNem5 (Fig. 1).
  • IPTG isopropylthio- ⁇ - galactosidase
  • the fusion protein is produced through overexpression.
  • the fusion protein is then isolated, purified, and its activity against the fungus Botrytis cinerea is checked through a bioassay, e.g.
  • fusion protein can then be formulated e.g. into a wettable powder or spraying powder and then be applied on crops threatened with the fungus.
  • Example 2 The steps 1) to 5) of Example 2 are repeated.
  • the fusion gene is cloned into vector pNem ⁇ .
  • IPTG IPTG
  • the fusion protein is produced by over- expression.
  • the fusion protein (immunotoxin) is then isolated, purified, and its activity against insect Spodoptera exigua checked by adding it to the artificial diet for this insect.
  • the fusion protein can then be formulated e.g. into a wettable powder or spraying powder and then be applied on crops threatened with the insect.
  • Fig. 1 shows the nucleotide and partial aminoacid sequences of the vector pNem5, a derivative of pHenl (Hoogenboom et al. 1991). The sequence shown was cloned between the Hindlll and EcoRI sites of pHenl. This sequence replaces the multiple cloning site and gene III encoding a minor coat protein of phage Fd in pHenl. In addition an extra multiple cloning site was introduced 3' of the Hindlll site. Single chain antibodies can, for instance be cloned between the Sfil and NotI sites or the Sail and Smal sites.
  • RBS is a prokaryotic ribosomal binding site; the sequence encoding the pelB signal peptide (signal peptide of pectate lyase of Erwinia carotovora) (Hoogen ⁇ boom et al., 1991) and a c-myc tag are indicated and their amino acid sequences given.
  • Fig. 2 shows the nucleotide sequence of the pNem ⁇ cloning vector. The sequence shown was cloned in pSPORTl (Gibco, Life Technologies) between PstI and SphI sites of the poly linker (5' and 3' ends respectively), in such a way that both the original PstI and the SphI sites were destroyed.
  • the sequence begins with the nucleotide that was changed from G (last nucleotide of the original PstI site) to C in order to disrupt PstI site.
  • the last restriction site (Aatll) of the polylinker of pSPORTl is indicated.
  • Single chain antibodies can for instance cloned between the Sail and the Smal sites.
  • RBS is a prokaryotic ribosomal binding site; the sequences encoding the PelB signal peptide (signal peptide of pectate lyase of Erwinia carotovora) (Hoogenboom et al., 1991) and a c-myc tag are indicated.

Abstract

Produits d'assemblage géniques comportant une séquence nucléotidique codant un anticorps ou une partie de celui-ci qui se lie de manière spécifique aux structures d'un agent pathogène ou organisme pesteux, et un nucléotide codant une protéine toxique vis-à-vis de cet organisme. La protéine toxique peut être une toxine ou une enzyme à fonction toxique. Si l'agent pathogène est un champignon ou une bactérie, ledit anticorps peut être spécifique des antigènes de membrane ou paroi cellulaire de l'agent pathogène. Si l'organisme pesteux est un insecte ou un nématode, ledit anticorps peut être spécifique des antigènes du tube digestif. On a également prévu des systèmes d'expression et des plantes résistantes à la peste transformées à l'aide de ces systèmes, ainsi que des immunotoxines codées par les produits d'assemblage géniques, et des organismes renfermant les produits d'assemblage ou les immunotoxines et servant d'agents externes de protection des cultures.
PCT/NL1995/000310 1994-09-19 1995-09-19 Produits d'assemblage geniques codant des agents de protection des cultures, plantes transformees renfermant et exprimant de tels produits d'assemblage, et procedes de lutte contre les agents pathogenes et organismes pesteux des cultures WO1996009398A1 (fr)

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JP8510769A JPH10506274A (ja) 1994-09-19 1995-09-19 農作物保護剤をコードする遺伝子構築物、ならびにそうした構築物を含有しかつ発現する形質転換された植物、ならびに農作物における病害生物体（ｐｌａｇｕｅ ｏｒｇａｎｉｓｍｓ）および病原体の調節方法

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WO2000023593A2 (fr) * 1998-10-16 2000-04-27 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Pathogenicide moleculaire induisant une resistance a la maladie chez des vegetaux
WO2002016625A2 (fr) 2000-08-25 2002-02-28 Basf Plant Science Gmbh Polynucleotides vegetaux codant de nouvelles proteases prenyle
WO2003089475A2 (fr) * 2002-04-22 2003-10-30 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Resistance des plantes aux moisissures exercee par des anticorps, des anticorps recombines, des fragments et des fusions d'anticorps recombines
WO2009027313A2 (fr) * 2007-08-31 2009-03-05 Basf Plant Science Gmbh Gènes de lutte contre les agents pathogènes et procédés d'utilisation de ces gènes dans des plantes
WO2011023522A1 (fr) * 2009-08-24 2011-03-03 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Résistance d'une plante vis-à-vis d'oomycètes médiée par la fusion à un anticorps
US8367066B2 (en) * 2004-12-10 2013-02-05 Pheromonicin Biotech, Ltd Antiviral bifunctional molecules, methods of construction and methods of treating virus-induced cancer therewith
US8394379B2 (en) 2003-05-15 2013-03-12 Iogenetics, Llc Targeted cryptosporidium biocides
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WO2014177595A1 (fr) * 2013-04-29 2014-11-06 Agrosavfe N.V. Compositions agrochimiques comprenant des anticorps se liant à des sphingolipides
WO2016071438A3 (fr) * 2014-11-05 2016-06-23 Agrosavfe Nv Plante transgénique comprenant un polynucléotide codant un domaine variable d'anticorps à chaîne lourde

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WO1998044137A3 (fr) * 1997-04-03 1998-12-17 Novartis Ag Lutte contre des ennemis des plantes
US6291156B1 (en) 1997-04-03 2001-09-18 Syngenta Participations Ag Plant pest control
WO2000023593A2 (fr) * 1998-10-16 2000-04-27 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Pathogenicide moleculaire induisant une resistance a la maladie chez des vegetaux
WO2000023593A3 (fr) * 1998-10-16 2000-07-27 Fraunhofer Ges Forschung Pathogenicide moleculaire induisant une resistance a la maladie chez des vegetaux
US6825325B1 (en) * 1998-10-16 2004-11-30 Fraunhofer Gesellschaft Zur Forderung Der Angewandten Forschung E.V. Molecular pathogenicide mediated plant disease resistance
WO2002016625A2 (fr) 2000-08-25 2002-02-28 Basf Plant Science Gmbh Polynucleotides vegetaux codant de nouvelles proteases prenyle
WO2003089475A3 (fr) * 2002-04-22 2004-06-03 Fraunhofer Ges Forschung Resistance des plantes aux moisissures exercee par des anticorps, des anticorps recombines, des fragments et des fusions d'anticorps recombines
WO2003089475A2 (fr) * 2002-04-22 2003-10-30 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Resistance des plantes aux moisissures exercee par des anticorps, des anticorps recombines, des fragments et des fusions d'anticorps recombines
US7531522B2 (en) 2002-04-22 2009-05-12 Fraunhofer-Gesellschaft Zur Forderung Der Angewandten Forschung E.V. Antibodies, recombinant antibodies, recombinant antibody fragments and fusions mediated plant disease resistance against fungi
AU2003224073B2 (en) * 2002-04-22 2009-09-24 AgroProtect GmbH Antibodies, recombinant antibodies, recombinant antibody fragments and fusions mediated plant disease resistance against fungi
EP2090591A3 (fr) * 2002-04-22 2009-10-28 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Anticorps, anticorps recombinants, fragments anticorps recombinants et fusions liées à la maladie des plantes résistantes contre les champignons
AU2003224073C1 (en) * 2002-04-22 2010-03-11 AgroProtect GmbH Antibodies, recombinant antibodies, recombinant antibody fragments and fusions mediated plant disease resistance against fungi
US8703134B2 (en) 2003-05-15 2014-04-22 Iogenetics, Llc Targeted cryptosporidium biocides
US8394379B2 (en) 2003-05-15 2013-03-12 Iogenetics, Llc Targeted cryptosporidium biocides
US8367066B2 (en) * 2004-12-10 2013-02-05 Pheromonicin Biotech, Ltd Antiviral bifunctional molecules, methods of construction and methods of treating virus-induced cancer therewith
US8722050B2 (en) 2004-12-10 2014-05-13 Pheromonicin Biotech, Ltd. Method of treating virus-induced cancer
US8802397B2 (en) 2004-12-10 2014-08-12 Pheromonicin Biotech, Ltd. Method of producing the polypeptide for treating virus-induced cancer
US8802837B2 (en) 2004-12-10 2014-08-12 Pheromonicin Biotech, Ltd. Nucleic acids molecule encoding the polypeptide for treating virus-induced cancer
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