JPH10506274A - Gene constructs encoding crop protection agents, and transformed plants containing and expressing such constructs, and methods of regulating disease organisms and pathogens in crops - Google Patents

Abstract

  (57) [Summary] The present invention provides a nucleotide sequence encoding an antibody or a portion thereof that specifically binds to the structure of a diseased organism or pathogen, and a genetic construct comprising nucleotides encoding a protein that is toxic to said organism. The toxic protein can be a toxin or an enzyme with a toxic function. If the pathogen is a fungus or a bacterium, the antibody is specific for an antigen on the cell wall or cell membrane of the pathogen, and if the diseased organism is an insect or nematode, the antibody is a gastrointestinal tract. Can be specific for an antigen. Expression systems and disease resistant plants transformed with these systems, and immunotoxins encoded by the genetic constructs, and organisms containing the constructs or immunotoxins for use as external crop protection agents A body is also provided.

Description

DETAILED DESCRIPTION OF THE INVENTION Genetic constructs encoding crop protection agents, and transformed plants containing and expressing such constructs, as well as methods of regulating plague organisms and pathogens in crops. It relates to a genetic construct suitable for expressing an agent that protects plants against disease organisms and pathogens. Attack of plants by pathogens such as fungi, nematodes, insects, bacteria and viruses constitutes a considerable economic problem, especially for large cultivated crops such as corn, rice, beans, potatoes, tomatoes and grapes. I do. Protection of such plants by chemical means is highly undesirable for environmental reasons. A more effective and more tolerable method of protecting plants against attack by diseased organisms is to provide the plant with genetic information that regulates the effects of such organisms, thereby reducing the effects of organisms that attack the plant. To be resistant to As several recent examples of this type of plant protection show, resistance to the virus may be due to the expression of the viral coat protein (Beachy et al., 1990) or to a single gene against the calcium ion binding domain of the viral coat protein. This can be achieved by expression of an antibody variable fragment of the chain (scFcv) (Tavladoraki et al., 1993). Insect resistance can be obtained by the expression of Bacillus thuringiensis insecticidal crystal protein (ICP) (Vaeck et al., 1987). The resistance to fungi is chitinase and / or glucanase (Alternaria longipes: US Patent Application No. 4,940,840, Fusarium solani: European Patent Application No. 440,304, Botryti s cinerea and Rhizoctonia. Rhizoctonia solani: can be achieved by the expression of Broglie et al., 1989, WO 90-07001. Resistance to plant parasitic nematodes can be achieved by using giant cell-specific promoters in combination with barnase or antisense DNA technology (Opperman et al., 1994). The major disadvantages of these methods are: • Their application is limited to a relatively small number of diseased organisms. For example, ICP is so far only effective against Lepidoptera, Diptera and Coleoptera. This is because the binding domain of the ICP toxin only recognizes the receptors in the digestive tract of insects belonging to these eyes. In addition, each ICP toxin (eg, CryIA (b), CryIC and CryIE) has its own specific spectrum with respect to insect species. There is strong indication that resistance by ICP is not permanent due to mutations in its receptor (Van Rie et al., 1990; McGaughey and Whalon, 1992; Tabashnik). , 1994). -These methods often lead to only partial resistance (viral coat protein, scFv, glucanase / chitinase). It is an object of the present invention to provide means and methods for protecting plants, especially crops, against disease in a more effective and ecologically acceptable manner. This object is met according to the invention by fusing the monoclonal antibody or a part thereof to a toxin or an enzyme having toxic activity, or an effective part of these toxins or enzymes. The chimeric protein can be obtained by fusing sequences encoding the respective portions of the chimera, or by chemically or chemically combining the two portions of the chimeric protein together, for example, as described in WO 9,318,162. It can be made either by chemically linking. Thus, the present invention provides a nucleotide sequence encoding an antibody or a portion thereof that is specific for a diseased organism or pathogen, and a nucleotide encoding a protein having a toxic effect on the diseased organism or pathogen described above. Gene constructs comprising the sequences. The present invention further relates to an antibody or a part thereof which is specific to a diseased organism or pathogen, and to a toxic protein which has a toxic effect on the above-mentioned diseased organism or pathogen and converts the antibody (or a part thereof) to a toxic protein thereof. Alternatively, the present invention relates to chimeric proteins comprising a protein produced by chemically or biochemically linking to an enzyme (or a part thereof). The terms "pathogen" and "disease organism" are both used to describe any organism or agent that affects the growth, development or utilization of a plant. Monoclonal antibodies use hybridoma technology (Kohler and Milstein, 1975) to identify most or any epitopes on any pathogen that are susceptible to attack by toxins or toxic enzymes (and parts thereof). Can appear for most of the structures. Single-chain antibodies can be prepared from hybridomas producing monoclonal antibodies by established molecular techniques. In addition, single-chain antibodies that have an affinity for a selected epitope or molecular structure susceptible to attack by a toxin or toxic enzyme (or a portion thereof) can be obtained from a phage display library (Hogenborn). (Hoogenboom) et al., 1992; Winter et al., 1994). The main advantage of this strategy is that the efficacy of the toxicity can be promoted and that its specificity can be adapted to the target organism. With this strategy, it is possible to create fusion products that are active against a single species or a range of species by selecting the appropriate epitope. In addition, a) selecting antibodies against a conserved epitope, and b) fusing the enzyme or toxin to two or more antibodies (or portions thereof) having different receptors to provide resistance to resistance. It is possible to improve the performance. Antibodies Antibodies can be raised against the structure of diseased organisms that would lead, directly or indirectly, to resistance or partial resistance when these antibodies were fused to an appropriate toxin or enzyme. Examples of these structures are cell membranes and cell walls, especially in bacteria and fungi, or gastrointestinal structures, especially in nematodes and insects, e.g. epithelial antigens, or especially in viruses. It is a coat protein. The antibody is preferably a single-chain antibody. Toxic proteins Toxic proteins include all proteins that have a toxic effect on diseased organisms or pathogens, such as toxins or toxic enzymes. Toxins that can be fused with a monoclonal antibody include: Bacillus thuringiensis insecticidal crystal proteins (ICP). ICPs or δ-endotoxins are described in A family of proteins produced during sporulation in the cytoplasm of B. thuringiensis. These proteins crystallize as paraspore inclusions and are solubilized in the digestive tract of insect larvae. The toxin is highly specific and results in the breakdown of gastrointestinal tract cells of susceptible insect larvae. Many B. elegans producing ICPs of different insect host spectra. A B. thuringiensis strain has been isolated. ICPs are classified according to their specificity spectra (Hoefte and Whiteley, 1989). The genes for various ICPs, including the gene encoding CryIA (b) (Wabiko et al., 1986), have been cloned and sequenced. Cry IA (b) is produced as a 131 kDa protoxin and is activated by removal of the N-terminal and C-terminal propeptides by proteases present in insect gut. This mature toxin (65-66 kDa) contains three domains. The N-terminal (first) domain contains some conserved hydrophobic sequences and is presumed to form pores in the apical membrane of epithelial cells of the gastrointestinal tract. The second domain is highly variable and probably binds to cell membrane receptors. The C-terminal (third) domain also contains the conserved sequence. -Colicins (Pattus et al., 1990). Colicins are a family of bacterially-resistant bacterially encoded proteins that kill bacteria that are closely related to the production strain (generally Escherichia coli). They consist of structural domains and perform different functions such as receptor binding, metastasis and killing. Based on their mode of action, colicins can be classified into two groups. The larger group of colicins causes permeation of the plasma membrane, thereby destroying membrane potential. The C-terminal domain of these colicins forms an ion channel in the artificial membrane. The other group of colicins causes enzymatic cleavage of DNA or 16S rRNA. -Thionines (Bohlmann and Apel, 1991). They are toxic to bacteria and fungi. Cecropins (Fink et al., 1989), which are toxic to bacteria by forming pores in the bacterial membrane. AaIT (Androctonus australis scorpion venom, a 70 amino acid single-chain polypeptide that acts on insect sodium channels, Gordon et al., 1984). These are examples of toxins that disrupt cell membranes and are particularly suitable for obtaining resistance to insects and nematodes. Other toxins, such as ribosome-inactivating proteins, may also be suitable for obtaining resistance to diseased organisms. Further examples are: Saponin (Stripe, 1983; Stripe and Bar bieri, 1986) Eiblin A and C (Wei et al., 1974; Lin et al., 1981) Melittin (a 26 amino acid long hemolytic peptide of bee venom) Gelonin (Stripe et al., 1980) Momordin (Barbieri et al., 1980) rSLTAd7 (Shigella-like toxic substance) Lectins, animals, plants Alternatively, highly specific hydrocarbon binding proteins of microbial origin may be toxic as a result of binding to a part of the organism. Toxic enzymes that can be fused to monoclonal antibodies include: Glucanases, especially β-1,3-glucanases. These PR-2 proteins can be classified into alkaline and acidic forms. The alkaline form is produced after several processing steps of the translation product. First, the N-terminal signal peptide is cleaved during transport to the endoplasmic reticulum, and then the C-terminal portion is glycosylated and removed for transport across the vacuolar membrane. There are both intracellular and extracellular forms of glucanase. The intracellular form is extended by the C-terminal 3-25 amino acids. The sequence of the intracellular β-1,3-glucanase gene is disclosed in EP-A-440,304. Glucanase genes from other plants are also known, such as the corn glucanase (and endochitinase) (Nasser et al., 1988). Effective destruction of cell wall glucans by endo-β-1,3-glucanase sometimes appears to require collaboration with extracellular enzymes such as exoglucanase. -Chitinases. These are PR-3 proteins that contain two domains and one hydrophobic signal peptide that is not present in the active enzyme. Chitinases are subdivided into three types. That is, class I, alkaline chitinase, is localized in the vacuole and also contains a cysteine-rich domain and a C-terminal sequence of six amino acids that are removed post-translationally and are involved in vacuolar targeting; Chitinase lacks a cysteine-rich domain and has lower enzymatic activity. These are located in apoplasts; class III, lysozyme-active chitinase contains conserved sequences different from I and II. Plant chitinase was found to have its chitinase activity derived from a 30 kDa monomer. Chitinase, like glucanase, exists in both intracellular and extracellular forms. The intracellular form is extended by the C-terminal 3-10 amino acids. The catalytic center is located at the C-terminal portion which is the same in both forms. The sequence of the intracellular chitinase gene is disclosed in EP-A-440,304. Chitinase genes from other organisms are also known, such as bean endochitinase (DeBroglie et al., 1986). Chitinase and β-1,3-glucanase are produced at an increased rate upon infection with fungi (Verticillium albo-atrum). Tomato chitinase and β-1,3-glucanase are in vitro (Young and Pegg, 1982; Young and Pegg, 1981) and possibly in vivo (Pegg and Vessie). Vessey), 1973) also inhibit fungal growth. Lipase lysozyme These are examples of enzymes that destroy (part of) the cell wall or cell membrane. This strategy is particularly suitable for obtaining resistance to bacteria and fungi. In principle, cells are broken down, damaging cell walls or disrupting metabolic pathways, replication, transcription, cell division, or other essential to the extent that the pathogen or diseased organism will die or severely impede growth. All enzymes that can interfere with function are suitable as part of the constructs. Constructs Gene constructs include complete antibody molecules, Fab portion, F (ab) 2 portion, scFv portion, divalent scFv [divalent antibody (diabody)] (Hollliger, Prospero and Winter). (Winter), 1993), mini-antibodies (Pack et al., 1993), or any other portion that shows binding to a target (such as a complementarity determining region). In the construct according to the invention, the antibody sequence is fused to the complete sequence encoding the enzyme / toxin or a part thereof which is still functionally active. Chimeric proteins comprising antibodies (fragments) and enzymes / toxins can also be obtained by chemical or biochemical conjugation. The antibody (fragment) and the enzyme / toxin (fragment) are fused directly or using a flexible linker that does not interfere with the structure and function of the two proteins. Such flexible linkers are used, for example, to fuse the variable regions of the heavy and light chains of immunoglobulins to create scFvs, and to create bivalent, bispecific scFvs. Or used in immunotoxins (Whitlow and Filpula, 1991; Kihlberg et al., 1993; Houston et al., 1992; Takinen). Et al., 1991). The linker may also be based on the hinge region (Pack and Pluickthum, 1992; Pack et al., 1993) in the antibody molecule or peptide fragments between the structural domains of the protein. If only the functional part of the toxin is to be conjugated to the antibody (fragment), a linker present between the two domains of the complete toxin itself can be used. Fusions can be made between the enzyme / toxin and the heavy (fragment) or light (fragment) chain of the immunoglobulin, both at the C-terminus and the N-terminus. In the case of a scFv fusion, the variable domain _H -Linker-V _L And V _L -Linker-V _H In both orders. The desired cellular location of the protein can be achieved using a suitable target sequence. Proteins synthesized without the target sequence remain in the cytoplasm of the cell, while others are directed into the secretory pathway by signal peptides. In the absence of other target signals, the latter protein is erroneously secreted. Additional target signals may be present, for example, to direct proteins to the vacuolar compartment of the cell or to keep them in the endoplasmic reticulum (Chrispeels, 1991). Target signals have been described that direct proteins to chloroplasts, mitochondria, peroxisomes or the nucleus (Austen and Westwood, 1991). One example of a target pathway is secretion through the endoplasmic reticulum and Golgi apparatus. Examples of signal sequences for secretion are described by Briggs and Gierasch (1986), Firek et al. (1993), Duiring et al. (1990), and Shirasu et al. (1988). ). If the fusion protein must be expressed in a heterologous organism for the production of the protein itself, to improve expression due to the codon preference of the organism, an unstable motif of the mRNA (eg, AT region, The gene construct may need to be modified to remove the spurious splicing site) and the polyadenylation signal. Target organisms Fungi, bacteria, nematodes, insects, viruses and other diseased organisms or pathogens. Promoter The fusion gene is expressed in plants under the control of any type of promoter that is active in the plant. Examples are: a) a constitutive promoter such as CaMV-35S (Kay et al., 1987). b) Nap et al. (1993) (leaves), De Almeida et al. (1989) (leaves, SSU-promoter), Nap et al. (1992) (potato tubers, patatin promoter), Tissue-specific promoters as described in Hendriks et al. (1991) (potato tubers), Guerche et al. (1990) (species): c) 2 'promoter (Langridge et al. Inducible promoters such as wound inducible promoters (Logemann et al., 1989; Suh et al., 1991) or chemically induced promoters (Williams et al., 1992). . Transformation Transformation can be performed using any method that ensures stable integration of the chimeric gene into the plant genome in such a way that it can still be transcribed. Examples of transformations are: a) Agrobacterium tumefaciens-mediated transformation (Horsch et al., 1985): Bacteria contain a portion of plasmid DNA (T-DNA) in the plant genome. Based on natural transformation systems that integrate stably. The T-DNA includes the gene to be expressed. b) Microprojectile bombardment (Vasil et al., 1992): Particles coated with DNA penetrate the plant cell nucleus at a high rate, during which the DNA is integrated into its genome by the process of host recombination. c) Tissue electroporation (D'Halluin et al., 1992): DNA penetrates into plant cells under the influence of a high electric field and is transferred to the nucleus before integration into the plant genome by host recombination processes. It is. Direct applications The gene constructs can also be used for disease control through external applications on the crop to be protected. Such direct application can be achieved in the form of administration of immunotoxins, including antibodies linked to the expression product of the chimeric gene, ie, a toxic protein. Another form can be by the application of an organism that contains the immunotoxin itself or that contains the gene encoding the immunotoxin and is capable of producing it. Suitable carrier organisms include microorganisms such as bacteria (eg, B. thuringiensis, fungi, yeast, and viruses), which organisms may be alive or dead. Thus, the present invention also relates to an immunotoxin comprising an antibody linked to a protein that is toxic to a disease organism or pathogen, and wherein the immunotoxin is obtained from an expression system as described above. The protein may be purified by known methods.The present invention also relates to an organism containing such an immunotoxin, Also included are organisms that are stably transformed with a genetic construct encoding an immunotoxin, which organisms can be used for external protection of plants against diseased organisms. Pesticidal compositions containing the immunotoxin itself or in encoded form, together with a possible carrier, the composition also comprising a solvent, a substance that prevents the composition from being washed away, a stabilizer, , Attractants, UV absorbers, etc. The present invention also relates to a method of protecting plants against the action of diseased organisms or pathogens, wherein the plant comprises an immune system as described above. It is externally treated with a toxin or an organism containing the immunotoxin or a composition containing the same, such as by spraying, using any suitable equipment, including tractors, airplanes, and the like. Example 1 Monoclonal antibodies against an enzyme having toxic activity or a part thereof fused to a fungus (Verticillium dahliae) Fusion of plant chitinases and glucanases to scFvs from local antibodies The following steps are taken: 1) Antibodies to the mycelium of Verticillium dahliae or purified cell wall components are developed and monoclonal antibodies are isolated 2) Clone the cDNA sequence encoding the antibody variable region to create a single-chain Fv construct. 3) Confirm the functionality of scFv expressed in bacteria. 4) performing an N-terminal or C-terminal fusion between the scFv and chitinase or β-1,3-glucanase using a suitable linker, for example, a CBHI linker (Takkinen et al., 1991); The chimeric gene is inserted into an expression vector, for example, pNem5 or pNem6 (FIGS. 1 and 2). These are derivatives of the vector pHen1 (Hoogenboom et al., 1991). 5) Confirm both the binding activity (by ELISA) and the enzymatic activity (bioassay) of the fusion product expressed in bacteria (E. coli). 6) Transfer the fusion gene together with the appropriate target sequence between the promoter-termination cassette of the plant transformation vector. 7) Confirm expression and functionality in plant cells by transient expression assay. 8) Transfer the expression cassette with the fusion gene and the selectable marker into the plant genome by plant transformation. 9) Screen the regenerated plants for expression of the fusion product. 10) Confirm the activity of the fusion protein in the transgenic plant by bioassay. These steps, along with the appropriate adaptation of antibody production and fusion with the enzyme, are combined with Botrytis cinera, Fusarium oxysporum f.sp. Radicis-lycopersisci and Phytophthora infestans can be followed for the production of transgenic plants resistant to Phytophthora infestans. Fusion with other proteins having toxic activity, such as potato lectin, is also possible. Example 2 Fusion of a Monoclonal Antibody with a Toxin or Part thereof Fusion of CryIA (b) _BT (29-607) to scFv derived from a monoclonal antibody against the digestive tract of insect Spodoptera exigua larvae The following steps Is taken: 1) S.M. Antibodies are raised against the gastrointestinal epithelial tissue of S. exigua larvae and the monoclonal antibodies are isolated. 2) Cloning the cDNA sequence encoding the antibody variable region to create a single-chain Fv construct. 3) Confirm the functionality of scFv expressed in bacteria. 4) N-terminal or C-terminal fusion between scFv and scFv and CryIA (b) _BT (29-607) is performed using a suitable linker, for example, CBHI linker (Takkinen et al., 1991). Then, the chimeric gene is inserted into an expression vector, for example, pNem6 (FIG. 2). This vector is a derivative of pSPORT1 (Gibco, BRL). 5) The binding activity of the chimeric protein expressed in bacteria (E. coli) was determined by using cryo-sections in the midgut of insect larvae (Martens et al., 1994) and insects Western blot analysis on primary culture system of midgut epithelial cells (Baines et al., 1993), ligand blot assay in combination with competition experiments (Bosch et al., 1994). The insecticidal activity of the chimeric protein is confirmed by bioassay, followed by the degradation effect of the chimeric protein using a primary midgut cell culture system. 6) Transfer the fusion gene together with the appropriate target sequence between the promoter-termination cassette of the plant transformation vector for stable transformation. For transient expression, the fusion gene is cloned behind a constitutive promoter (ie, CaMV-35S promoter) and a suitable stop cassette. 7) Confirm expression and functionality in plant cells by transient expression assay. 8) Transfer the expression cassette with the fusion gene and the selectable marker into the plant genome by plant transformation. 9) Screen the regenerated plants for expression of the fusion product. 10) The binding activity of the chimeric protein was determined on cold incisions in the midgut of insect larvae (Martens et al., 1994) and in primary cultures of insect midgut epithelial cells (Baines et al., 1993). ) Above, confirmed by Western blot and ligand blot assay combined with competition experiments. The insecticidal activity of the chimeric protein is confirmed by bioassay, followed by the degradation effect of the chimeric protein using a primary midgut cell culture system. In addition, N- and C-terminal fusions were made between scFv and CryIA (b) _Bt (1-607); CryIA (b) _Bt (29-429); CryIA (b) _Bt (1-1155). create. Further, domain II (or a part thereof) of CryIA (b), which is thought to be responsible for receptor binding, is replaced with scFv. These steps, together with the appropriate adaptation of antibody production and fusion with toxins, can be followed for the production of transgenic plants resistant to nematodes by developing monoclonal antibodies to the nematode gut. Example 3 1) Balb / c mice were immunized with brush border membrane vesicles (BBMV) isolated from the midgut of Spodoptera exigua as described by Bosch et al., 1994. Mice were immunized twice (at 4-week intervals) with subcutaneous injections of BBMV using 50 μg protein equivalents with the addition of Freund's incomplete adjuvant. Four weeks after the last immunization, boosts were administered with BBMV (50 μg protein equivalent) injected into the intraperitoneal cavity. After three days, the tiles were removed and fusion was performed as described by Schotts et al., 1992. 2) Antisera and monoclonal antibodies were confirmed by ELISA following standard procedures for their ability to react with epitopes present in BBMV. 3) Western blot analysis was performed to confirm the reaction pattern of the monoclonal antibody. 250 μg protein equivalents of BBMV were separated on SDS-polyacrylamide gel (12%). The protein was transferred to a PVDF membrane by Western blotting. 4) To determine if the monoclonal antibody can react with epitopes present in the midgut of the insect, a cold incision of the midgut of Spodoptera ex igua was first incubated with the monoclonal antibody. , Followed by incubation with a secondary antibody reactive with the mouse antibody and labeled with FITC. 5) To determine whether the monoclonal antibody is capable of reacting with epitopes present outside the insect midgut and possibly on the lumen of the midgut, A primary epithelial cell culture system of S. exigua midgut was prepared and incubated with a monoclonal antibody followed by incubation with a secondary antibody that reacted with the mouse antibody and was labeled with FITC. 6) Single chain antibodies were purified according to standard procedures as described, for example, in (Johnson and Bird, 1991); (Houston et al., 1992). It was isolated from a hybridoma producing a monoclonal antibody that binds to an epitope present on the luminal side of the membrane of S. exigua and outside of the midgut cells. Example 4 Fusion of the colicin N pore-forming domain with a scFv derived from a monoclonal antibody that appears in the gastrointestinal tract of the insect Spodoptera exigua larvae Follow the steps as in Example 2, but in step 4, convert the scFv to colicin N In addition to the application of binding to the N-terminus of the porogen-forming domain (the C-terminal region of the protein), a peptide that normally links the complete N-terminal portion of N to the porogen-forming domain Fragment, the latter peptide fragment serving as a linker between the two domains of the chimeric protein. Example 5 Sprayable Immune Enzyme Formulation Steps 1) to 5) of Example 1 are repeated and antibodies appear in step 1) against the mycelium or cell wall components of Botrytis cinera. The fusion gene is cloned into the vector pNem5 (FIG. 1). Upon induction with IPTG (isopropylthio-β-galactosidase), the fusion protein is produced through overexpression. The fusion protein (immunotargeted toxic enzyme) is then isolated, purified, and analyzed for its activity against the fungus Botrytis cinera by bioassay, eg, containing 0.1% Tween 20 as a wetting agent. Confirmation by incorporation into a suitable buffer and spraying on a test plant already infected with the fungal culture system on the agar medium and subsequently with the conidia of the fungus. The fusion protein may then be formulated, for example, in a wettable or sprayable powder, and then applied to the fungal-cropped crop. Example 6 Sprayable Immunotoxin Formulation Steps 1) to 5) of Example 2 are repeated. The fusion gene is cloned into the vector pNem6. Upon induction with IPTG, the fusion protein is produced by overexpression. The fusion protein (immunotoxin) is subsequently isolated, purified and its activity against the insect Spodoptera exigua is confirmed by adding it to the artificial feed of the insect. The fusion protein may then be formulated, for example, in a wettable or sprayable powder, and then applied to the insect- threatened crop. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the nucleotide and partial amino acid sequence of the vector pNem5, a derivative of pHen1 (Hoogenboom et al., 1991). The sequence shown was cloned between the HindIII and EcoRI cleavage sites of pHen1. This sequence replaces the multiple cloning site and the gene III encoding the minor coat protein of phage Fd in pHen1. In addition, an extra multiple cloning site was introduced 3 'to the HindIII cleavage site. Single chain antibodies can be cloned, for example, between the SfiI and NotI cleavage sites or SalI (between the 5SmaI cleavage sites. RBS is the prokaryotic ribosome binding site and the pelB signal peptide (Erwinia carotovora). (Hoogenboom et al., 1991) and the sequence encoding the c-myc tag are indicated and the amino acid sequence is given.Figure 2 shows the nucleotide sequence of the pNem6 cloning vector. The sequence shown is between the PstI and SphI cleavage sites (5 ′ and 3 ′ ends, respectively) of the polylinker in such a way that both the original PstI and SphI cleavage sites are disrupted in pSPORT1 ( Gibco, Life Technologies). The sequence begins with a nucleotide changed from G (the last nucleotide of the original PstI cleavage site) to C to confuse the PstI cleavage site.The last restriction enzyme cleavage site (AatII) of the polylinker of pSPORT1 is indicated. Single chain antibodies can be cloned, for example, between the SalI and SmaI cleavage sites, where RBS is the prokaryotic ribosome binding site and the PelB signal peptide (the signal peptide of the pectate lyase of Erwinia carotovora). (Hoogenboom et al., 1991) and the sequence encoding the c-myc tag are indicated.

[Procedure of Amendment] Article 184-8 of the Patent Act [Submission date] September 24, 1996 [Correction contents]                               The scope of the claims 1. Antibodies or one of them that is specific for a diseased organism of a plant or a pathogen of a plant Nucleotide sequence coding for the part, and toxic to the aforementioned pest organisms or pathogens A genetic construct comprising a nucleotide sequence encoding a sex protein. 2. The pathogen is a fungus or a bacterium, and the antibody or one of the above 2. The method according to claim 1, wherein the portion is specific for an antigen on the cell wall or cell membrane of the pathogen. Gene construct. 3. The disease organism is an insect or a nematode, and the antibody or the 2. The gene according to claim 1, wherein a part of said gene is specific for a gastrointestinal tract antigen of said diseased organism. Construct. 4. 4. The method according to claim 1, wherein the protein is a toxic enzyme. Gene construct. 5. 5. The method according to claim 4, wherein said toxic enzyme is chitinase or glucanase. Gene construct. 6. 5. The genetic construct of claim 4, wherein said toxic enzyme is lysozyme. 7. The inheritance according to any one of claims 1 to 3, wherein the protein is a toxin. Child construct. 8. The aforementioned toxin kills Bacillus thuringiensis 8. The gene construct according to claim 7, which is a sex crystal protein (ICP). 9. 8. The gene construct of claim 7, wherein said toxin is colicin. 10. 8. The gene construct of claim 7, wherein said toxin is colicin N. 11. The aforementioned antibody or part thereof is a single-chain antibody or part thereof. 11. The gene construct according to any one of claims 1 to 10. 12. A method according to claim 1 together with a sequence regulating the expression of said gene construct. An expression system comprising the gene construct of any one of claims 11 to 11. 13. 13. The expression system of claim 12, wherein said regulatory sequence comprises a tissue specific promoter. . 14． The gene construct according to any one of claims 1 to 11, or the claim. A diseased organism wherein a plant is stably transformed using the expression system of 12 or 13. Or a method of protecting the plant against the action of pathogens. 15. Inserting the gene construct of any one of claims 1 to 11 into its genome A plant containing and capable of expressing the above-described construct. 16. A plant disease organism or a plant obtained from the expression system according to claim 12. An immunotoxin comprising an antibody that is conjugated to a protein that is toxic to the pathogen. 17． An organism comprising the immunotoxin of claim 16. 18. Stable transformation with the gene construct according to any one of claims 1 to 11 Organism that is 19. An insecticide containing the immunotoxin of claim 16 together with an acceptable carrier Composition. 20. The immunotoxin of claim 16 or the organism of claim 17 or 18 The plant is treated against diseased organisms or pathogens that are treated externally. How to protect.

────────────────────────────────────────────────── ─── Continuation of front page    (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, M C, NL, PT, SE), CA, JP, US (72) Inventor Shiottsu, Ardien             Netherlands Nuel-6708 Lpy Wage             Ningen-Leeubergsweide 208 (72) Inventor Staykema, Wilhelms John             S             Netherlands Nuel-6708 Vee Wage             Ningen Gruthbaide 197 (54) [Title of the Invention] Gene construct encoding a crop protection agent, and transformation containing and expressing such a construct                     Transformed plants, as well as diseased organisms in the crop                     And pathogen control methods

Claims (1)

[Claims] 1. Encodes an antibody or a portion thereof that is specific for a disease organism or pathogen Nucleotide sequences that are toxic to the diseased organism or pathogen described above. A genetic construct comprising a nucleotide sequence encoding a protein. 2. The pathogen is a fungus or a bacterium, and the antibody or one of the above 2. The method according to claim 1, wherein the portion is specific to an antigen on the cell wall or cell membrane of the pathogen. Gene construct. 3. The disease organism is an insect or a nematode, and the antibody or the 2. The gene according to claim 1, wherein a part of the gene is specific for the gastrointestinal tract antigen of the diseased organism. Construct. 4. 4. The method according to claim 1, wherein the protein is a toxic enzyme. Gene construct. 5. 5. The method according to claim 4, wherein said toxic enzyme is chitinase or glucanase. Gene construct. 6. 5. The genetic construct of claim 4, wherein said toxic enzyme is lysozyme. 7. The inheritance according to any one of claims 1 to 3, wherein the protein is a toxin. Child construct. 8. The aforementioned toxin kills Bacillus thuringiensis 8. The gene construct according to claim 7, which is a sex crystal protein (ICP). 9. 8. The gene construct of claim 7, wherein said toxin is colicin. 10. 8. The gene construct of claim 7, wherein said toxin is colicin N. 11. The aforementioned antibody or part thereof is a single-chain antibody or part thereof. 11. The gene construct according to any one of claims 1 to 10. 12. A method according to claim 1 together with a sequence regulating the expression of said gene construct. An expression system comprising the gene construct of any one of claims 11 to 11. 13. 13. The expression system of claim 12, wherein said regulatory sequence comprises a tissue specific promoter. . 14． The gene construct according to any one of claims 1 to 11, or the claim. A diseased organism wherein a plant is stably transformed using the expression system of 12 or 13. Or a method of protecting the plant against the action of pathogens. 15. Inserting the gene construct of any one of claims 1 to 11 into its genome A plant containing and capable of expressing the above-described construct. 16. Toxicity to diseased organisms or pathogens obtained from the expression system of claim 12 An immunotoxin comprising an antibody that is conjugated to a protein that is: 17． An organism comprising the immunotoxin of claim 16. 18. Stable transformation with the gene construct according to any one of claims 1 to 11 Organism that is 19. An insecticide containing the immunotoxin of claim 16 together with an acceptable carrier Composition. 20. The immunotoxin of claim 16 or the organism of claim 17 or 18 The plant is treated against diseased organisms or pathogens that are treated externally. How to protect.