CZ382199A3 - Production of plants being tolerant to fungicides due to expression of polypeptides binding fungicides in plant - Google Patents

Abstract

The procedure for the production of fungicide tolerant plants is based on expressing the antibody with the ability to bind a fungicide in plants.

Description

Production of fungicide tolerant plants by expression of fungicide-binding polypeptides in the plant.

Technical field

The present invention relates to a process for producing fungicide tolerant plants by expressing exogenous polypeptides with the ability to bind fungicides to plants or organs thereof. The invention further relates to the use of corresponding nucleic acids encoding antibody polypeptides or antibody portions having fungicide binding properties to transgenic plants, thereby transforming the plant itself.

BACKGROUND OF THE INVENTION

It is known that genetic engineering methods allow the specific transfer of foreign genes into the genome of a plant. This procedure is called transformation and the resulting plant transgenic plant. Transgenic plants are commonly used in various fields of biotechnology. Examples are insect resistant plants (Vaek, et al., Plant Cell, 5, 159-169, 1987), virus resistant plants (Powell, et al., Science, 232, 738-743, 1986) and ozone resistant plants (Van). Camp, et al., BioTech., 12, 165-168, 1994). Examples of improved qualitative characteristics achieved by genetic engineering are: improved fruit storage (Oeller, et al., Science, 254, 437-439, 1991), increased starch production in potatoes (Stark, et al., Science, 242, 419, 1992) , changes in starch (Visser, et al., Mol. Gen. Genet., 225, 289-296, 1991) and lipid composition (Voelker, et al., Science, 257, 72-74, 1992) and formation of foreign polymers (Poirer, et al., Science 256: 520-523 (1992)).

An important goal of work in the field of plant molecular genetics is to create tolerance to herbicides. Herbicide tolerance is characterized by improved tolerability (in terms of type or level) of the plant or plant organs for herbicide application. This can be influenced in several ways. Known methods are the use of metabolic genes, such as ring genes in conjunction with glufosinate resistance (WO 8705629) or the achievement of an herbicide-resistant enzyme such as the synthesis of enolpyruvyl shikimat-3-phosphate (WO 9204449), which is glyphosate resistant herbicide in cell and tissue cultures to select resistant plant cells and resulting resistant plants as described for acetyl CoA carboxylase inhibitors (US Pat. 5,162,602, US Pat. 5,290,696).

Antibodies are proteins as a component of the immune system. The backbone of all antibodies is their spatial spherical structure, the formation of light and heavy chains and

- 2 their basic ability to bind with a high specificity of a molecule or part of a molecular structure (Alberts, et al., In: Molecular biology der Zelle, 2nd ed., 1990, VCH Verlag, ISBN 3-527-27983-0 1198-1237). Based on these properties, antibodies have been used for many tasks. Applications can be divided into applications of antibodies to the animal and human organisms in which they originate, called in-situ and ex-situ applications, ie, the use of antibodies after they have been isolated from the cells or organisms that produce them (Whitelam and Cockburn, TIPSVol 1, (8), 268-272, 1996).

The use of somatic hybrid cell lines (hybridomas) as a source of antibodies against very specific antigens is based on the work of Kohler and Milstein (Nature, 495-97, 1975). This procedure allows so-called monoclonal antibodies having a uniform structure to be produced and which are produced by cell fusion. Spleen cells of immunized mice are allowed to fuse with mouse myeloma cells. This provides hybridoma cells that multiply to infinity. At the same time, the cells secrete specific antibodies against the antigen to which the mice were immunized. Spleen cells allow antibody production, while myeloid cells contribute to unrestricted growth and continuous secretion of antibodies. While each hybridoma cell that is a clone is derived from a single B cell, all of the antibody molecules formed have the same structure including the antigen binding position. This method has brought about the use of antibodies since antibodies having a uniform and known specificity and homogeneous structure are now available in unlimited quantities. Monoclonal antibodies are widely used in immunodiagnostics and as therapeutics.

In recent years, the production of antibodies has been made possible by the so-called phage display method, avoiding the immunosystem and various immunizations of animals. The affinity and specificity of the antibodies are measured in vitro (Winter, et al., Ann. Rev. Immunol., 12, 433-455, 1994; Hoogenboom TIBTech Vol. 15, 62-70, 1997). Gene segments that contain a sequence encoding different antibody sites, i.e., the antigen-binding position, fuse with bacteriophage protein coat genes. The bacteria are then infected with a phage containing such fusion genes. The resulting phage portions are now provided with an envelope comprising a fusion protein similar to that of the antibody, wherein the antibody binding domain is directed outward. Phage Display Libraries Now Available • · • ·

- 3 (phage display library) to isolate a phage containing a fragment of the antibody of interest and that specifically binds to a particular antigen. Each phage isolated in this way produces a monoclonal antigen binding polypeptide corresponding to the monoclonal antibody.

Genes with a particular antigen binding position that are specific for each phage can be isolated from the phage DNA and used to generate complete antibody genes.

In the field of crop protection, antibodies have been used in particular as analytical tools of the ex-site for qualitative and quantitative determination of antibodies. This concerns detection in plant components, herbicides or fungicides in drinking water (Sharp, et al., ACS Symp Ser., 446, 1991; Pestic. Residues Food Saf., 87-95), soil samples (WO 9423018) or plants or their organs and the use of antibodies as adjuvants in the purification of bound molecules.

The generation of immunoglobulins in plants was first described by Hiatt, et al., Nature, 342, 76-78, 1989. This spectrum includes secreted single chain antibodies to multiple antibodies (J. Ma and Mich Hein, Annuals New York Academy of Sciences, 72-81 (1996).

Previous attempts to use in situ antibodies have been directed against viral envelope proteins to protect plants against pathogens, particularly viral expression diseases, to plant cells, specific antibodies or portions thereof (Tavladoraki, et al., Nature, 366, 469-472, 1993; Voss, et al., Mol. Breeding, 1, 39-50 (1995).

An analogous procedure has also been used to protect plants against helminth infections (Rosso, et al., Biochem. Biophys. Res. Corn., 220, 255-263, 1996). In pharmacology, there are examples of applications where expression of antibodies to plants in situ is used for oral immunization (Ma, et al., Science, 268, 716-719, 1995; Mason and Arntzen, Tibtech Vol. 13, 388-392, 1996) . The substance is provided with an antibody produced by a plant and having originated in plants or plant organs suitable for consumption by mouth, throat or digestive tract, and the antibody causes effective immune protection. It has been observed that a single chain anti-abscisic acid antibody of low molecular weight plant hormone that has been expressed to the plant and restricted the availability of plant hormones due to the binding of plant abscisic acid (Artsaenko, et al., The Plant Journal, 8, (5), 745-750, 1995).

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Chemical control of fungi in agronomically important crops requires the use of highly selective fungicides without phytotoxic effects. The phytotoxic effect of fungicides can be based, for example, on inhibiting plant growth, reducing photosynthesis and thereby reducing yield. In certain cases, however, it is difficult to develop sufficiently selective fungicides that can be used for all major crops that do not cause damage to the plant and reduce harvest yields. The introduction of fungicide-resistant or tolerant crops can contribute to addressing this problem and may open new ways of using fungicides for crops that have not yet been able to use fungicides, or that have been possible, but only if harvest losses were still acceptable.

The development of fungicide resistant crops by tissue culture or seed mutagenesis and natural selection is limited. On the one hand, the phytotoxic effect must always be detectable at the tissue culture level, and on the other hand, only those plants that can be successfully regenerated undamaged from the cell cultures can be handled by the technology of the cultured cultures. In addition, subsequent mutagenesis and selection of useful plants may exhibit undesirable characteristics that must be eliminated again, and in some cases the procedure may be repeated by backcrossing. The introduction of resistance using hybrids may be limited to some plants of the same species.

For the above reasons, the contribution of genetic engineering to the isolation of a gene encoding resistance and its transfer to a useful plant is superior to the traditional method of plant propagation in a targeted manner.

The development of useful herbicide tolerant or herbicide resistant plants by molecular biology methods requires knowledge of the mechanism of action of herbicides in the plant as well as of the genes that confer herbicide resistance, and this has been possible only now. A large number of herbicides currently used commercially are in the phase of blocking the biosynthesis of essential amino acid, lipid or pigment enzymes. Herbicide tolerance can be created by altering the genes of these enzymes in such a way that the herbicides are no longer bound and the alternative genes are introduced into the useful plants. An alternative example is to find enzymes analogous to natural ones, for example in microorganisms that exhibit natural resistance to herbicides. These resistance-conferring genes are isolated from such wet organisms, recloned for appropriate 4 4

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- 5 vectors and subsequently, after successful transformation, expression into the useful plants is realized (WO 96/38567).

SUMMARY OF THE INVENTION

It is an object of the present invention to develop a novel, generally applicable genetic engineering method for generating herbicide tolerant transgenic plants. Surprisingly, we have found that this goal can be achieved by expressing exogenous polypeptides, antibodies or antibody portions with the ability to bind fungicides in plants.

The present invention relates first to the production of fungicide binding antibodies and the cloning of the corresponding genes or gene fragments.

The first step is to produce an antibody that binds the fungicide. This can be done, inter alia, by immunizing vertebrates, in most cases mice, rats, dogs, horses, donkeys, or goats with antigen. The antigen in this case is a fungicidally active compound that is associated with or bound to a higher molecular weight carrier, such as bovine serum albumin (BSA), chicken ovoalbumin, stick hemocyanin (KLH), or other functional group carriers. After repeated administration of the antigen, the immune response is monitored by conventional means to isolate the appropriate antiserum. Initially, this method results in polyclonal sera containing antibodies of different specificity. For targeted in situ use, it is necessary to isolate the gene sequence that encodes a single, specific monoclonal antibody. There are different ways to do this. In a first approach, a fusion of antibody producing cells and tumor cells is used to obtain hybridoma cell cultures that continuously produce antibodies and which ultimately lead to clone union and a homogeneous cell line that generates a defined monoclonal antibody.

From such monoclonal cell lines the cDNK for the antibody or a portion thereof is isolated - see the so-called single chain antibody (scFv). These cDNK sequences can then be cloned into expression cassettes and used for functional expression in prokaryotic and eukaryotic organisms, including plants.

Alternatively, antibodies can be selected through phage display libraries; they bind fungicidal molecules and catalytically convert them to a product with non-fungicidal properties. Methods for producing catalytic antibodies are

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6 are described in Janda, et al., Science, 275, 945-948, 1997, Chemical selection for catalysis in combinatorial Antibody libraries; Catalytic Antibodies, 1991, Ciba Foundation Symposium, 159, Wiley-Interscience Publication. Cloning the gene of these catalytic antibodies and expressing them into plants can in principle also lead to a plant resistant fungicide.

In particular, the invention relates to the expression of cassettes whose coding sequence encodes fungicide-binding polypeptides, or a functional equivalent thereof, and the use of these expression cassettes to produce a fungicide tolerant plant. For example, the nucleic acid sequence may be a DNA or cDNK sequence. Encoded sequences suitable for insertion into the expression cassette of the invention are, for example, those comprising a DNA sequence from a hybridoma cell that encodes a polypeptide having fungicide binding properties and thereby imparts resistance to specific fungicides to the host.

In addition, the expression cassettes of the invention contain regulatory nucleic acid sequences that control expression of the encoded sequence into a host cell. The expression cassette which is preferred according to the invention comprises an uplink sequence, i.e. a promoter of the coded sequence terminated at 5 'and a downlink, i.e. polyadenylation signal terminated at 3' and, if desired, additional regulatory elements that are operably linked by the inserted coding sequence. sequences to polypeptides with fungicidal binding properties and / or to a transit peptide. The operative attachment must be understood as meaning the sequence arrangement of the promoter, coding sequence, terminator and, if appropriate, other regulatory elements in such a way that each of the regulatory elements can function as intended by the expression of the coding sequence. Preferred, but not limited to, operative attachment sequences are target sequences to guarantee subcellular localization in apoplast, cell membrane, vacuole, plastids, mitochondria, endoplasmic reticulum (ER), nucleus, liposomes or other compartments and translation enhancers, such as the 5'-control sequence from tobacco mosaic virus (Gallie, et al., Nucl. Acids Res., 15, 8693-8711, 1987).

A suitable promoter of an expression cassette according to the invention is in principle any promoter capable of directing the expression of foreign genes. The promoters which are preferably used are, in particular, plant-derived promoters or promoters.

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- 7 originating from plant viruses. The CaMV 35S promoter from cauliflower mosaic virus is particularly preferred (Franek, et al., Cell, 21, 285-294, 1980). This promoter contains various recognition factors for transcription factors which, in their entirety, lead to the persistent and constitutive expression of the introduced gene (Benfey, et al., EMBO J 8, 2195-2202, 1989).

The expression cassettes of the invention may also contain a chemically initiated promoter such that expression of the exogenous polypeptide into the plant can be controlled within a desired period of time. Such promoters described in the literature that can be used, inter alia, are the PRP1 promoter (Ward, et al., Plant Mol. Biol., 22, 361-366, 1993), a promoter which is initiable by salicylic acid (WO 95/61). 1919443), a promoter which is initiable by benzenesulfonamide (EP 388186), a promoter which is initiable by abscisic acid (EP 335528) or a promoter which is buildable by ethanol or cyclohexanone (WO 9321334).

Other preferred promoters are those that guarantee expression into plant tissues or organs in which a phytotoxic fungicidal effect is exerted. Promoters that guarantee specific expression on a leaf deserve special attention. Attention should be paid to the cytosolic FBPase potato promoter or the ST-LSI potato promoter (Stockhaus, et al., EMBO J., 8, 2245-245, 1989).

Stable expression of single chain antibodies containing up to 0.67% of the total soluble transgenic tobacco plant seed proteins was possible with the seed-specific promoter (Fiedler and Conrad, Bio / Technology, 10, 1090-1094, 1995). Since expression is also possible in seeded seeds or seeds that are in the germination phase and may be suitable for the purposes of the invention, these specific germination and seed promoters are also regulatory elements of the invention. Thus, the expression cassettes of the invention may contain, for example, a seed-specific promoter (preferably a USP or LEB4 promoter), an LEB4 signal peptide, a gene to express and an ER retention signal. The cassette structure is illustrated by way of example in the form of a diagram in Figure 1 with reference to a single chain antibody (scFv gene).

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The expression cassettes of the invention are generated by fusing a suitable promoter with a suitable DNA polypeptide and preferably a DNA that encodes a chloroplast specific transit peptide and that is inserted between the promoter and the DNA polypeptide and a polyadenylation signal using conventional recombinant and cloning technology as described, for example, in T. Maniatis, EFFritsch and J. Samroro, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989, and also in TJShilhavý, MLBerman and LW-Enquist, Experiments with Gene Fusions Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1984 and in Ausubel, FM, et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and WileyInterscience, 1987.

Particularly preferred are sequences that allow targeting to apoplast, plastid, vacuole, plasma membrane, mitochondria, endoplasmic reticulum (ER) or, in the absence of appropriate operative sequences, settle in the production region, namely the cytosol (Kermode, Crit Rev. Plant Sci. 15, 285-423 (1996). Localization in the ER and cell wall has been shown to be particularly useful for quantitative protein accumulation in transgenic plants (Schouten, et al., Plant Mol. Biol., 30, 781-792, 1996; Artsaenko, et al., Plant J, 8, 745-750, 1995).

The invention also relates to expression cassettes whose encoded sequence encodes a fungicide-binding fusion protein, wherein a portion of the fusion protein is a transit peptide that controls the translocation of polypeptides. Particularly preferred are chloroplast-specific transit peptides that are enzymatically cleaved from the fungicidal binding of the polypeptide moiety after the fungicidal binding of the polypeptides has been translocated to plant chloroplasts. Particularly preferred are transit peptides derived from plastid transketolase (TK) or a functional equivalent of such transit peptides (for example, the Rubinsco small subunit transit peptide or NADP oxidreductase ferredoxin transit peptide).

According to the present invention, the polypeptide DNA or polypeptide cDNK required for expression cassettes is preferably extended by polymerase chain reaction (PCR). A method of PCR amplification of DNAs is known, for example, from Innis, et al., PCR Protocols, A Guide to Methods and Applications, Academic Press, 1990. The DNA fragments produced by PCR can advantageously be checked by sequence analysis to provide fc. · Fcfc ·· ti • · · fc ·· fc · fcfc · • fcfcfc * ····· • fcfc fcfc · · fc fcfcfc ··· fcfcfcfc fcfc · · • fc fcfc fcfcfc fcfcfcfc fcfcfcfc

- 9 there was no error in the construction of the polymerase to be expressed.

The inserted nucleotide sequence, which encodes a fungicide-binding polypeptide, may be synthetically produced or obtained naturally, or may consist of a mixture of synthetic and natural DNA components. Generally, a synthetic nucleotide sequence is prepared with codons preferred by the plants. These preferred codons can be determined by the codons whose proteins are most common and which penetrate most species of the desired plant. In preparing the expression cassette, various DNA fragments can be manipulated to obtain a nucleotide sequence that reads in the correct sense and is equipped with the correct reading frame. Adapters and binding components may be added to the DNA fragments for proper interconnection.

Suitably, the promoter and terminator regions of the invention must be provided, in the sense of transcription, with a binding or polybinding unit consisting of one or more restriction positions for insertion of the sequence. As a rule, the linker has 1 to 10, usually 1 to 8, but preferably 2 to 6 restriction sites. In the regulatory regions, the binding unit generally has a dimension of less than 100 bp, more typically less than 60 bp, but at least 5 bp. The promoter of the invention may be both natural or homologous to the host plant and of other foreign or heterologous origin. The expression cassette according to the invention has any sequence and region for transcriptional termination of a promoter comprising transcription 5'-3'-. Depending on the requirements, the different termination areas are interchangeable.

Furthermore, manipulations that create suitable restriction positions or that remove excess DNA or restriction positions can be used. Where insertion, deletion or substitution such as transitions and transversions are possible, in vitro mutagenesis, "primer pairing" can be used. [sic] pair ”), restriction or binding. For possible appropriate manipulations such as restriction, exonuclease digestion (chewing-back), or blunt ends, complementary termination of the fragments can be provided with binding means.

Particularly important for the success of the present invention is the attachment of the specific ER retention signal SEKDEL (Schuoten, A., et al., Plant Mol. Biol., 30, 781-792, 1996), by which the average level of expression is tripled to quadrupled. When creating

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- 10 cassettes can also use other retention signals that naturally occur in plant or animal proteins and are located in the ER.

Preferred polyadenylation signals are plant polyadenylation signals, especially those that essentially correspond to the polyadenylation signals of Agrobacterium tumefaciens T-DNA, in particular gene 3 of the T-DNA (octopine synthase) Ti plasmid pTiACH5 (Gielen, et al., EMBO J., 3, 835 et al., 1984), or functional equivalents. The expression cassettes of the invention may consist, for example, of a constitutional promoter (preferably the CaMV 35 S promoter), a LeB4 signal peptide, a gene to express and an ER retention signal. The cassette structure is shown in the diagram in Figure 2 with reference to a single chain antibody (scFv gene). Preferably, the amino acid sequence KDEL (lysine, aspartic acid, glutamic acid, leucine) is used as the ER retention signal.

Preferably, the fused expression cassette encoding a polypeptide having fungicidal binding properties is cloned into a vector, for example pBin19, which is suitable for Agrobacterium tumefaciens transformation. Agrobacteria which are transformed with such a vector can then be used in a known manner to transform plants, particularly useful plants, e.g. tobacco plants, for example by macerating the damaged leaves or leaf portions in an Agrobacterium solution and subsequently growing them in a suitable medium. Transformation of plants by Agrobacteria is known, inter alia, from F.F.White, Vectors for Gene Transfer in Higher Plants; in Transgenic Plants, Vol. 1, Engineering and Utilization, edited by SDKung and R.Wu, Academic Press, pp. 15-38, 1993 and by SBGelvin, Molecular Genetics of T.DNK Transfer from Agrobacterium to Plants, also in Transgenic Plants, p. 49 -78. Transgenic plants may be regenerated from transformed cells of damaged leaves or leaf parts in a known manner, and these transgenic plants comprise a gene for expressing polypeptides having fungicide binding properties integrated into the cassette of the invention.

In addition, to transform a host plant with a polypeptide encoded by the fungicidal binding of the DNA of the present invention, an expression cassette is inserted as an insert into a recombination vector, wherein the DNA vector contains additional control signals such as replication or integration sequences. A suitable vector is described, between: · mezi · * * * mezi mezi mezi mezi mezi

- 11 others in "Methods in Plant Molecular Biology and Biotechnology" (CRC Press), Chapter 6/7, pp. 71-119, 1993.

Using the above recombination and cloning technologies, the expression cassettes of the invention can be cloned into suitable vectors that allow their multiplication, for example in E. coli. Suitable cloning vectors include, but are not limited to, pBR332, puC series, M13mp series, and pACY184. Particularly suitable are binary vectors that can replicate both E. coli and agrobacteria, for example pBin19 (Bevan, et al., Nucl. Acids Res., 12, 8711, 1980).

The invention further relates to the use of the expression cassettes according to the invention for the transformation of plants, plant cells or plant organs. A preferred use is to mediate resistance to phytotoxic active fungicides.

Depending on the promoter choice, expression can be effected on leaves, seeds or other plant organs. Such transgenic plants, their propagation material and their plant cells, plant tissues or plant organs are further objects of the present invention. The conversion of foreign genes into the genome of a plant is called transformation. This method utilizes the above methods of transforming and regenerating plants from plant tissues or plant cells for transient or permanent transformation. Suitable methods are protoplast transformation by uptake of DNA induced by polyethylene glycol, a biolistic [sic] gene gun approach, electroporation, incubation of dry embryos in DNA containing solution, microinjection and Agrobacterium mediated gene transfer. Such procedures are described, for example, in B. Jenes, et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, Editors: S.D.Kung and R.Wu, Academic Press, 128-143, 1993, and in Potrykus, Annu. Roar. Plant Physiol. plant Molec. Biol., 42, 205-225, 1991. The complex to be expressed is preferably cloned into a vector suitable for the transformation of Agrobacterium tumefaciens, for example pBin19 (Bevan, et al., Nucl. Acids Res., 12, 8711). 1984). The agrobacteria that have been transformed with the expression cassette of the present invention can then be used in a known manner to transform plants, especially useful plants such as cereals, corn, soybeans, rice, cotton, sugar beet, canola, · · · · · · · · · · · · · · · · ·

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- 12 sunflowers, flax, potatoes, tobacco, tomatoes, mustard, alfalfa, lettuce and various shrubs, trees, and various kinds of nuts, drupes, for example coffee, fruit trees such as apple, pear or cherry, walnuts such as walnuts nut or pecan and particularly important vines, for example by macerating damaged leaves or leaf parts in an agrobacterial solution and then growing in a suitable medium.

Functional equivalent sequences encoding fungicidally-binding polypeptides are, according to the invention, those sequences which still have the desired functions despite their nucleotide sequence being different. Thus, functional equivalents include naturally occurring variants of the sequences described herein as well as artificial nucleotide sequences, for example, artificial nucleotide sequences that have been obtained by chemical synthesis and are adapted for codon use in plants.

In particular, a functional or equivalent mutation must be understood to be that natural or artificial mutation of the originally isolated sequence that encodes a polypeptide with the ability to bind a fungicide by the binding that the mutation continues to exhibit the desired function. Mutations include substitutions, additions, deletions, exchanges, or insertions of one or more nucleotide residues. The present invention also encompasses those nucleotide sequences that are obtained by modifying the nucleotide sequence. The purpose of such a modification may be, for example, to further define the coding sequence contained therein, or otherwise, for example, to insert more precise positions for restriction enzymes.

Other functional equivalents are those variants whose function is more or less definite as compared to the parent gene or gene fragment.

Furthermore, artificial DNA sequences are useful when the fungicide imparts the desired tolerance to prevent phytotoxic effect on the useful plants as described above. Such artificial DNA sequences can be identified, for example, by back-translation of proteins having fungicidal binding activity and which have been generated by molecular modeling or in vitro selection. Particularly suitable are DNA coding sequences which have been obtained by back-translation of polypeptide sequences in connection with the use of a plant-specific codon. The specific use of the codon can be immediately determined by a computer scientist by a plant genetics expert

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Processing of another known plant gene to be transformed.

Other suitable equivalent nucleic acid sequences of the invention that must be mentioned are those that encode a fusion of proteins wherein a portion of the non-plant polypeptide fusion protein binds a fungicide or a functional equivalent thereof. For example, the second portion of the fusion protein may be another polypeptide with enzymatic activity, or an antigenic polypeptide sequence to detect the expression of scFvs (e.g., myc-tag or his-tag). However, a conventional protein sequence, such as a signal or transit peptide, that controls the properties of the polypeptides to bind the fungicide to the desired site of action is preferred.

However, the invention also relates to expression products made according to the invention and to the fusion of proteins of transit peptides and polypeptides with fungicide binding ability.

Resistance and tolerance for the purposes of the present invention represents the artificially assured ability of a plant to withstand fungicides with phytotoxic effects. It uses partial and particularly complete insensitivity to inhibitors for at least one generation of the plant. The site of phytotoxic action of the fungicide is generally leaf tissue, so that specific expression of the fungicide-binding polypeptides is capable of providing adequate leaf protection. However, it should be understood at the same time that the phytotoxic effect of fungicides need not be limited to leaf tissue only, but can affect all remaining organs of the plant in a specific manner for the tissue in question.

In addition, constitutive expression of exogenous fungicidal binding polypeptides is preferred. On the other hand, stimulatory expression is also desirable.

The efficacy of transgenically engineered fungicide-binding polypeptides can be determined, for example, by in vitro propagation of inoculated tissue in a medium containing staggered concentrations of fungicide or by germination assays. The tolerance of the test plant to the fungicide, which has been altered with respect to the type and degree of change, can be tested in greenhouse experiments.

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The invention further relates to transgenic plants transformed with the expression cassettes of the invention and to transgenic cells, tissues, organs and propagation material of such plants. Particularly preferred are transgenic crop plants, such as cereals, corn, soybeans, rice, cotton, sugar beet, canola, sunflower, inu, potatoes, tobacco, tomatoes, mustard oil, alfalfa, lettuce and various shrubs, trees, and various nut and drupe species, such as coffee, fruit trees such as apple, pear or cherry, walnuts such as walnut or pecan, and particularly important vines. Transgenic plants, plant cells, plant tissues or plant organs can be treated with fungicides with a phytotoxic effect inhibiting plant enzymes; plants, plant cells, plant tissues or plant organs that have not been successfully transformed die or are damaged. Examples of suitable active ingredients are strobilurins, especially methylmethoxyimino-α- (o-tolyloxy) -o-tolylacetate (BASF 490F), and metabolites and functional derivatives of these compounds. A DNA that encodes polypeptides with fungicidal binding properties and which has been inserted into the expression cassettes of the invention can be used as a selectable marker.

The present invention has particular advantages for those useful plants in which, once they have acquired resistance to fungicides with a phytotoxic effect, these fungicides can be used to control harmful fungi even in larger amounts applied; otherwise, this larger amount would destroy the plants. The compounds of the group below may be considered as examples of such fungicides with a phytotoxic effect, but are not intended to limit the invention in any way:

sulfur, dithiocarbamates and their derivatives, such as ferric dimethyldithiokarbaminan, dimethyldithiokarbaminan zinc ethylenbisdithiokarbaminan zinc ethylenbisdithiokarbaminan manganese ethylendiaminbisdithiokarbaminan manganese, zinc, tetramethylthiuram [sic], ammonia complex (Ν, Nethylenbisdithiokarbaminanu), zinc ammonium complex (Ν, N'propylenbisdithiokarbaminanu) zinc Zinc Ν, Ν'- propylenebisdithiocarbaminate, Ν, Ν'-propylenebis (thiocarbamoyl) disulfide, nitro derivatives such as dinitro (1-methylheptyl) phenyl crotonate, 2-sec-butyl-4,6-dinitrophenyl-3,3-dimethyl acrylate, 2-sec-dimethyl acrylate butyl-4,6-dinitrophenyl isopropyl carbonate, diisopropyl-5-nitroisophthalate, this to this this to this this to this this to this • · ·· ··

- 15 heterocyclic compounds such as 2-heptadecyl-2-imidazoline acetate, 2,4-dichloro-6- (chloroaniline) -s-triazine, 0,0-diethylphthalimidophosphonium thioate, 5-amino-1 [bis (dimethylamino) phosphinyl 3-phenyl-1,2,4-triazole, 2,3-dicyano-1,4-dithioanthraquinone, 2-thio-1,3-dithiol [4,5-b] quinoxaline, methyl-1- (butylcarbamoyl) -2-benzimidazolecarbamate, 2-methoxycarbonylaminobenzimidazole,

2- (2-furyl) benzimidazole, 2- (4-thiazolyl) benzimidazole, N- (1,1,2,2-tetrachloroethylthio) tetrahydrophthalimide, N-trichloromethylthiotetrahydrophthalimide, N-trichloromethylthiophthalimide,

N-dichlorofluoromethylthio-N, N'-dimethyl-N-phenylsulfodiamide,

5-ethoxy-3-trichloromethyl-1,2,3-thiadiazole, 2-thiocyanatomethylthiobenzothiazole,

1,4-dichloro-2,5-dimethoxybenzene,

4- (2-chlorophenylhydrazone) -3-methyl-5-isoxazolone, pyridine-2-thio [sic] 1-oxide, 8-hydroxyquinoline or its copper salt, 2,3-dihydro-5-carboxanilido-6-methyl1. 4-oxathiine, 2,3-dihydro-5-carboxanilido-6-methyl-1,4-oxathiine-4,4-dioxyd, 2-methyl-5,6-dihydro-4H-pyran-3-carboxanilide,

2-methylfuran-3-carboxanilide; 2,5-dimethylfuran-3-carboxanilide; 2,4,5-trimethylfuran-3-carboxanilide, N-cyclohexyl-2,5-dimethylfuran-3-carboxamide, N-cyclohexyl-N-methoxy-2,5-dimethylfuran-3-carboxamide,

2-methylbenzanilide, 2-iodobenzanilide, N-formyl-N-morpholine-2,2,2-trichlorethylacetal, piperazine-1,4-diylbis-1- (2,2,2-trichloroethyl) formamide,

- (3,4-Dichloroanil) -1-formylamino-2,2,2-trichloroethane;

amines such as 2,6-dimethyl-N-tridecylmorpholine or its salts,

2,6-dimethyl-N-cyclododecylmorpholine or its salts,

N- [3- (p-tert-butylphenyl) -2-methylpropyl] -cis-2,6-dimethylmorpholine,

N- [3- (p-tert-butylphenyl) -2-methylpropyl] -piperidine, (8- (1,1-dimethylethyl) -N-ethyl-N-propyl-1,4-dioxaspiro [4,5] decane -2-methanamine, azoles such as 1- [2- (2,4-dichlorophenyl) -4-ethyl-1,3-dioxolan-2-ylethyl] -1H-1,2,4-triazole,

- [2- (2,4-Dichlorophenyl) -4-n-propyl-1,3-dioxolan-2-ylethyl] -1H-1,2,4-triazole, N- (n-propyl) ) -N- (2,4,6-trichlorophenoxyethyl) -N'-imidazolylurea,

- (4-chlorophenoxy) -3,3-dimethyl-1- (1H-1,2,4-triazo-1-yl) -2-butanone,

- (4-chlorophenoxy) -3,3-dimethyl-1- (1H-1,2,4-triazol-1-yl) -2-butanol, (2RS, 3RS) -1- [3- (2- chlorophenyl) -2- (4-fluorophenyl) -oxiran-2-ylmethyl] -1H-1,2,4-triazole, φα · · · • · • · · φ · »·» · φ · φ · · · · · · · · · · · · · · · · · · · · · · · ·

- 16 1- [2- (2,4-Dichlorophenyl) -pentyl] -1H-1,2,4-triazole, 2,4'-difluoro-α- (1H-1,2) , 4-triazolyl-1-methyl) -benzhydryl alcohol,

- ((bis (4-fluorophenyl) methylsilyl) methyl) -1H-1,2,4-triazole, 1 - [(2RS, 4RS; 2RS, 4SR) -4-bromo-2- (2,4-dichlorophenyl) tetrahydrofuryl -1H-1,2,4-triazole, 2- (4-chlorophenyl) -3cyclopropyl-1- (1H-1,2,4-triazol-1-yl) butan-2-ol, (+ ) -4-chloro-4- [4-methyl-2- (1H-1,2,4-triazol-1-ylmethyl) -1,3-dioxolan-2-yl] phenyl-4-chlorophenyl ether, (E) - (R, S) -1- (2,4-dichlorophenyl) -4,4-dimethyl-2- (1H-1,2,4-triazol-1-yl) -pent-1-en-3-ol, 4- (4-chlorophenyl) 2-phenyl-2- (1H-1,2,4-triazolylmethyl) butyronitrile, 3- (2,4-dichlorophenyl) -6-fluoro-2- (1H1,2, 4-triazo-1-yl) quinazolin-4 (3H) -one, (R, S) -2- (dichlorophenyl) -1H-1,2,4-triazo-1-yl) hexane-2- ol, (1RS, 5RS; 1RS, 5SR) -5- (4-Chloro-benzyl) -2,2-dimethyl-1- (1H-1,2,4-triazol-1-ylmethyl) -cyclopentanol, (R, S) - 1- (4-chlorophenyl) -4,4-dimethyl-3- (1H-1,2,4-triazol-1-ylmethyl) pentan-3-ol, (+) - 2- (2,4-dichlorophenyl) ) -3- (1H-1,2,4-triazolyl) -propyl-1,1,2,2-tetrafluoroethyl ether, (E) -1- [1- (4-chloro-2-trifluoromethyl) phenyl] imino] -2-propoxyethyl] -1H-imidazole, 2- (4-chlorophenyl) -2- (1H-1,2,4-triazol-1-ylmethyl) ) -hexane nitrile, α- (2-chlorophenyl) -a- (4-chlorophenyl) -5-pyrimidinemethanol, 5-butyl-2-dimethylamino-4-hydroxy-6-methylpyrimidine, bis (p-chlorophenyl) 3-pyridinmethanol, 1,2 -bis (3-ethoxycarbonyl-2-thioureido) -benzene, 1,2-bis (3-methoxycarbonyl-2-thioureido) benzene, strobilurines such as methyl-E-methoxyimino- [α- (o-tolyloxy) -o-tolyl] acetate, methyl E2- {2- [6- (2-cyanophenoxy) pyrimidin-4-yloxy] phenyl} -3-methoxyacrylate, methyl Emethoxyimino- [α- (2-phenoxyphenyl)] acetamide, methyl E-methoxyimino - [α- (2,5-dimethylphenoxy) -o-tolyl] acetamide, anilinpyrimidines such as N- (4,6-dimethylpyrimidin-2-yl) aniline,

N- [4-Methyl-6- (1-propynyl) -pyrimidin-2-yl] aniline

N- [4-methyl-6-cyclopropylpyrimidin-2-yl-] aniline, penylpyrroles such as 4- (2,2-difluoro-1,3-benzodioxol-4-yl) -pyrrole-3-carbonitrile, cinnamamides such as 3- (4-chlorophenyl) -3- (3,4-dimethoxyphenyl) -acryloylmorpholine, and various fungicides such as dodecylguanidine acetate,

3- [3- (3,5-dimethyl-2-oxycyclohexyl) -2-hydroxyethyl] -glutarimide, Ν-methyl-, N-ethyl (4-trifluoromethyl-2- [3 ', 4'-dimethoxyphenyl] -benzamide [sic], hexachlorobenzene, methyl N- (2,6-dimethylphenyl) -N- (2-furoyl) -DL-alaninate, DL-N- (2,6-dimethylphenyl) N- (2'-methoxyacetyl) alanine methyl ester, ··············

- ¹⁷ N- (2,6-dimethylphenyl) -N-chloroacetyl-D, L-2-aminobutyrolactone, DL-N (2,6-dimethylphenyl) -N- (phenylacetyl) -alanine methyl ester, 5-methyl-5- vinyl-3- (3,5-dichlorophenyl) 2,4-dioxo-1,3-oxazolidine, 3- [3,5-dichlorophenyl- (5-methyl-5-methoxymethyl) -1,3-oxazolidine-2,4- dione [sic], 3- (3,5-dichlorophenyl) -1-isopropylcarbamoylhydantoin, N (3,5-dichlorophenyl) -1,2-dimethylcyclopropane-1,2-dicarboximide, 2-cyano- [N (ethylaminocarbonyl) - 2-methoxyimino] acetamide, N- (chloro-2,6-dinitro-4-trifluoromethylphenyl) -5-trifluoromethyl-3-chloro-2-aminopyridine.

Functionally equivalent derivatives of these fungicides in combination with less, equally or more pronounced phytotoxic activity have a comparable spectrum of action against phytopathogenic fungi as those specifically enumerated.

The invention will now be illustrated by the following non-limiting examples:

EXAMPLES General cloning methods

Cloning steps performed within the scope of the present invention, for example, cleavage restriction, agar gel electrophoresis, DNA fragment purification, transfer of nucleic acids to nitrocellulose and nylon membranes, DNA fragment binding, E. colli transformation, bacterial culture, phage multiplication, and DNA recombination sequence analysis were performed as described in Sambrook, et al., (1989) Cold Spring Harbor Laboratory Press; ISBN 0-87969-309-6.

The bacterial strains used herein (E. colli, XL-I Blue) were obtained from Stratagene. Agrobacterial strains used to transform plants (Agrobacterium tumefaciens, C58C1 with plasmid pGV2260 or pGV3850kan) were described in Deblaere, et al., Nucl. Acids Res., 13, 4777, 1985. Alternatively, agrobacterial strains of LBA4404 (Clontech) or other suitable strains may be used. For cloning purposes, vectors pUC19 (Yanish-Perron, Gene, 33, 103-119, 1985) were used, pBluescript SK- (Stratagene), pGEM-T (Promega), pZerO (Invitrogen), pBin19 (Bevan, et al., Nucl. Acids Res., 12, 8711-8720, 1984) and pBinAR (Hofgen and Willmitzer, Plant Science, 66, 221-230, 1990).

- 18 Sequence analysis of DNA recombinants

Sorting of DNA recombinant molecules was monitored using a Laser fluorescence DNA sequencing device from Pharmacia using the

Sangera (Sanger, et al., Proc. Natl. Acad. Sci. USA, 74, 5463-5467, 1977).

Creation of plant expression cassettes

The 35S CaMV promoter was inserted into plasmid pBin19 (Bevan, et al., Nuci. Acids Res., 12, 8711, 1984) as an EcoRI-Kpn1 fragment (corresponding to nucleotides 6909-7437 of cauliflower mosaic virus - Franck, et al., Cell 21, 285 (1980). The polyadenylation signal of T-DNA DNA gene 3 of plasmid pTiACH5 (Gielen, et al., EMBO J., 3, 835, 1984) of nucleotide 11749-11939 was isolated as a Pvull-HindIII fragment and cloned at the PvuII cleavage site after addition of SphI binding. between the cleavage position of the SphI-HindIII vector. This gave plasmid pBinAR (pBinAR (Hofgen and Willmitzer, Plant Science, 66, 221-230, 1990)).

Practical examples

Example 1

Since fungicides are not immunogenic, they must be bound to a carrier, for example KLH. If the molecule contains a reactive group, the bond may be performed directly; if not, a functional group must be inserted during the synthesis of the fungicide or selection of the reactive precursor during the synthesis to bind these molecules to the carrier molecule by a simple reaction mechanism. Examples of binding reactions can be found in Miroslavic Ferencik in "Handbook of Immunochemistry", Chapman & Halí., 1993, in the chapter Antigens, pp. 20-49.

Repeated injection of this modified carrier molecule (antigen) is used to immunize, for example, Balb / c mice. Once the enzyme-linked immunosorbent assay (ELISA) is detectably sufficient for antigen-binding antibodies, the spleen cells of these animals are removed and fused with myelomatic cells in order to cultivate the hybrids. In addition, 'fungicide-modified BSA' is used as an antigen in the ELISA to differentiate the hapten immune response from the KLH response.

• · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

Monoclonal agents are prepared by similar known methods, for example as described in "Practical Immunology", Leslie Hudson and Frank Hay, Blackwell Scientific Publications, 1989 or as described in "Monoclonal Antibodies: Pronciples and Practice", James Goding, Academic Press, Inc. 1983, or A practical guide to monoclonal antibodies, J. Liddell and A. Cryer, John Willey & Sons, 1991; or in Achim Moller and Franz Emling, "Monoclonal Anticancer Genetics TNF and Derivatives" [Monoclonal Antibodies Against TNF and Their Uses]. European patent EP-A260610.

Example 2

The initial point of investigation was a monoclonal antibody with a specific focus on the fungicide BAS 490F, which additionally had a high binding affinity. The selected hybridoma cell line is characterized in that secreted monoclonal antibodies against the fungicidal antigens of BAS 490F have high affinity and accessible specific immunoglobulin sequences (Berek, C., et al., Nature, 316, 412-418, 1985). These monoclonal antibodies against BAS 490F were the starting point for the construction of a single chain antibody fragment (scFv-antiBAS 490F).

First, hybridoma cells were isolated from mRNK and transcribed to cDNK. This cDNK acted as a template for the amplification of VH and VK variable immunoglobulin genes with specific VH1 BACK and VH FOR-2 primers for high molecular weight chains and VK2 BACK and MJK5 FON X for low molecular weight chains (Clakson, et al., Nature, 352, 624-628 (1991). The starting point for the construction of the single chain antibody fragment (scFv-antiBAS 490F) was various isolated immunoglobulins. Following the fusion of the PCR, the three components HV, VK, and the binding fragment, these components were combined by PCR reaction and scFv-antiBAS 490F amplified (Figure 3).

Functional characterization (antigen binding activity) of the scFv-antiBas 490F gene was performed after expression into the bacterial system. To date, scFv-antiBAS 490F was synthesized in E. colli as a soluble antibody fragment using the method of Hoogenboom, HR, et al., Nucleic Acids Research, 19, 4133-4137, 1991. The activity and specificity of the generated antibody fragment was checked by ELISA. (Figure 4).

To allow the expression of a tobacco seed-specific antibody fragment, it was

The 20 scFv-antiBAS 490F gene was down-cloned from the LeB4 promoter. The LeB4 promoter isolated from Vicia faba shows strictly specific tobacco expression of various foreign genes on seeds (Báumlein, H., et al., Mol. Gen. Genet., 225, 121-128, 1991). Transport of the scFv-antiBAS 490F polypeptide into the endoplasmic reticulum results in stable accumulation of large amounts of antibody fragments. Thus far, the scFvantiBAS 490F gene was fused to a signal peptide sequence that guaranteed entry into the endoplasmic retillary and with a retention ER signal, SEKDEL, that guaranteed the polypeptide to remain in the ER (Wandelt, et al., 1992) (Fig. 5).

The generated expression cassettes were cloned into the binary vector pGSGLUC 1 (Saito, et al., 1990) and transferred to the agrobacterium EHA strain 101 by electroporation. Recombined agrobacterial clones were used for subsequent transformation of Nicotiana tabacum. Upon conversion, 70-140 tobacco plants were regenerated. Seeds were harvested from regenerated tobacco plants at different stages of development after pollination. After extraction by dissolution in an aqueous buffered solution, soluble proteins were obtained from these seeds. Analysis of the transgenic plants shows that fusion of the scFv-antiBAS 490F gene to the ER retention signal of the SEKDEL DNA sequence allowed the accumulation of a maximum of 1.9% of the scFv-antiBAS 490F protein in the mature seed.

The size of the generated scFv-antiBAS 490F gene was about 735 bp. The variable domains were fused to the VH-L-VL sequence.

Specific selectivity was determined from mature tobacco seed extracts by direct ELISA. The values obtained clearly show that protein extracts contain functionally active antibody fragments.

Example 3

Seed-specific expression and single-chain antibody fragment concentration in the endoplasmic reticulum of transgenic tobacco seed cells under the control of the USP promoter.

The starting point of the research was a single chain fragment of anti-fungicide BAS 490F (scFv-antiBAS 490F). Functional characterization (antigen binding activity) of this generated scFv-antiBAS 490F gene was performed after expression on the bacterial system and subsequent expression on tobacco leaves. Activity and specificity • 4 • · • 4 # 4 4 4 4 4 · 4 4 4 • * · * <

^4,444,221 generated antibody fragments were checked by ELISA.

To allow the expression of seed-specific antibody fragments, the scFvantiBAS 490F gene was cloned descending from the USP promoter. The USP promoter, which was isolated from Vicia faba, exhibits strictly specific expression of various foreign genes on tobacco seeds (Fiedler, H., et al., Plant Mol. Biol., 22,669-679, 1993). Transport of the scFv-antiBAS 490F polypeptide to the endoplasmic reticulum results in stable accumulation of a large number of antibody fragments. To date, the scFv-antiBAS 490F genes have been fused to the signal sequence of the polypeptide, which guaranteed entry into the endoplasmic retillary, and with the SEKDEL retention signal, which ensured that the polypeptide would remain in the ER (Wandelt, et al., 1992) (Fig. 1).

The generated expression cassette was cloned into the binary vector pGSLUC 1 (Saito, et al., 1990) and transferred to Agrobacterium EHA 101 strain by electroporation. Recombinant agrobacterial strains were used for subsequent transformation of Nicotiana tabacum. After self pollination, the seeds of transgenic tobacco plants were harvested at various stages of maturity. Soluble proteins were obtained from these seeds after extraction from the aqueous buffer system. Analysis of the transgenic plants shows that the fusion of the scFv-antiBAS 490F genes into the DNA sequence of the SEQ ID NO retention signal SEQ by the USP promoter generated single chain antibody fragments with a binding affinity for BAS 490F as early as 10 days after seed development began.

Example 4

The scFv-antiBAS 490F promoter of the CaMV 35 S promoter was descending cloned in order to achieve the everywhere occurring expression of the antibody fragment in the plant, especially in the leaves. This strong constitutive promoter communicates foreign gene expression to all virtual plant tissues (Benfey and Chua, Science, 250, 956-966 (1990). Transport of scFvantiBAS 490F protein to the endoplasmic reticulum will allow stable accumulation of large numbers of antibody fragments into leaf mass. The scFv-antiBAS 490F gene was first fused to a signal peptide sequence that provides entry into the endoplasmic reticulum and the KDEL ER retention signal, allowing the product to remain in the ER (Wandelt, et al., Plant J., 2, 181-192 , 1992). The generated expression cassette was cloned into the binary vector pGSGLUC 1 (Saito, et al., Plant Cell Rep., 8, 718-).

22221 (1990) and transferred to Agrobacteria EHA 101 strain electroporation. Agrobacterial recombinant clones were used for subsequent transformation of Nicotiana tabacum. About 100 tobacco plants were regenerated. Leaf mass at various stages of development was removed from the regenerated transgenic plants. Soluble proteins were obtained from this leaf mass by extraction from aqueous buffered solution. Subsequent analyzes (Western blot analysis and ELISA) showed that the leaves had a maximum accumulation of more than 2% of the biologically active scFv-antiBAS 490F antigen binding polypeptide. High expression values were determined in fully developed green leaves, but antibody fragments were also detected in the aging leaf mass.

Example 5

Amplification of PCR fragment of cDNK encoding single-chain antibodies against BAS 490F with contribution of synthetic oligonucleotides.

Amplification of the PCR fragment of cDNK encoding single chain antibodies was performed in a DNA cycling thermal cycler from Perkin Elmer.

The reaction mixture contained 8 ng.pl ^{-1 of a} single-stranded cDNK template, 0.5 μΜ of the corresponding oligonucleotides, 200 μΜ of nuclotides (Pharmacia), 50 mM KCl, 10 mM Tris-HCl (pH 8.3 at 25 ° C, 1.5 mM MgCl ₂ ) and 0.02 U. μΓ ¹ Taq polymerase (Perkin Elmer). The amplification conditions were set as follows:

Anelation temperature: 45 ° C

Denaturation temperature: 94 ° C

Elongation temperature: 72 ° C

Number of cycles: 40

As a result, a fragment of approximately 735 base pairs was ligated into the pBluescript vector. The ligation mixture was used to transform E. colli XL-I Blue and the plasmid was amplified. For use and optimization of polymerase chain reaction, see Innis, et al., PCR Protocols, A Guide to Methods and Applications, Academic Press, 1990.

Example 6

Production of transgenic tobacco plants that transmit cDNA coding information of a single chain antibody with fungicide binding ability.

44 »4 4

4 4 4

444 444

4

4 4 4 • 4 4 • 4 4 • 4 4 4 • 4 4 4

4 4 4

4 4 4

Plasmid pGV2260 1 was transformed into Agrobacterium tumefaciens

C58C1: pGV2260. For the transformation of tobacco plants (Nicotiana tabacum cv. Samsun NN), cultivation of positively transformed agrobacterial colonies in Murashige-Skoog medium (Physiol. Plant, 15, 473 an., 1962) containing 2% sucrose (medium 2MS) at 1:50 was used. overnight. The petri dish was incubated with leaf slices of sterile plant (approx. 1 cm ^{2 each} ) for 5-10 minutes in 1:50 agrobacterial medium. This was followed by a two-day incubation in the dark at 25 ° C on a medium containing 0.8% Bacto-Agar. After 2 days, the cultures were continued for 16 hours light, 8 hours dark, and weekly cycles on medium containing 500 mg.l ^-1 Claforan (cefotaximate sodium), 50 mg.l ^-1 kanamycin, 1 mg.l ^-1 benzylaminopurine (BAP), 0.2 mg.l ^-1 naphthylacetic acid and 1.6 g · l ^-1 glucose. The grown buds were transferred to MS medium containing 2% sucrose, 250 mg.l ^-1 Claforan and 0.8% Bacto-Agar.

Example 7

Stable accumulation of a single chain antibody fragment against BAS 490F in the endoplasmic reticulum.

The starting point of the investigation was a fragment of a single chain anti-fungicide BAS 490F (scFv-antiBAS 490F) whose information was generated on tobacco plants. The amount and quality of the synthesized scFv-antiBAS 490F polypeptides was determined by Western blot analysis and ELI SA assay.

To allow expression of the scFv-antiBAS 490F gene into the endoplasmic reticulum, foreign gene expression was performed under the control of the CaMV 53S promoter as a translational fusion with the signal peptide LeB4 (N-terminal) and ER retention signal KDEL (Cterminal). Transport of scFv-antiBAS 490F polypeptides into the endoplasmic reticulum allowed stable accumulation of large amounts of active antibody fragments. After removal of the leaf mass, portions thereof were frozen at -20 ° C (1), lyophilized (2) or dried at room temperature (3). The soluble proteins concerned were obtained from the leaf mass by extraction from aqueous buffer and the scFv-antiBAS 490F polypeptide purified by affinity chromatography. Equal amounts of purified scFv-antiBAS 490F polypeptides (frozen, lyophilized and dried) were used to determine the activity of the antibody fragment (Fig. 6). Giant. 6A depicts the antigen binding activity of the polypeptides. Ftft ft *; * &lt; tb &gt;

- 24 scFv-antiBAS 490F purified from fresh (1), lyophilized (2) and dried (3) leaves. Figure 6B shows the corresponding amounts of scFv-antiBAS 490F protein (approximately 100 ng) as used for the ELISA assay, as determined by Western blot analysis. Standard molar masses of proteins are indicated on the left. Approximately identical antigen binding activities were found.

Example 8

To demonstrate the tolerance of tobacco plants to fungicides caused by fungicide-binding polypeptides, these tobacco plants were treated with varying amounts of BAS 490F. In all cases, it could be demonstrated in the greenhouse that plants with realized scFv-antiBAS 490F gene information showed higher tolerance to the fungicide BAS 490F and less phytotoxic effect.

Claims (20)

Modified-Claims A process for producing methylmethoxyimino-α- (o-tolyloxy) -olyl acetate (BAS 490F) tolerant plants by expressing an exogenous peptide capable of binding methylmethoxyimino-α- (o-tolyloxy) -o-tolyl acetate (BAS 490F) in a plant The method of claim 1, wherein the exogenous polypeptide capable of binding methylmethoxyimino-α- (o-tolyloxy) -o-tolylacetate (BAS 490F) in the plant is a single chain antibody fragment. The method of claim 1, wherein the exogenous polypeptide having the ability to bind methylmethoxyimino-α- (o-tolyloxy) -o-tolylacetate (BAS 490F) is a complete antibody or a fragment derived therefrom. An expression cassette for plants consisting of a promoter, a signal peptide, an expression coding gene, an exogenous polypeptide capable of binding methylmethoxyimino-α- (o-tolyloxy) -o-tolylacetate (BAS 490F), an ER retention signal, and a terminator. Expression cassette according to claim 4, characterized in that the constitutive promoter used is the CaMV 35S promoter. 6. The expression cassette of claim 4, wherein the gene to which the expression relates is a single chain antibody fragment gene. Expression cassette according to claim 4, characterized in that the gene or fragment of the gene to be used for the expression of the gene is a polypeptide capable of binding methylmethoxyimino-α- (o-tolyloxy) -o-tolylacetate (BAS 490F) in the form of translational fusion. with other functional proteins such as enzymes, toxins, chromophores and binding proteins. 8. The expression cassette according to claim 4, wherein the polypeptide gene to be expressed is obtained from hybridoma cells or by other recombination methods, such as an antibody phage display method. frfr frfr frfr frfrfr frfrfr frfrfr frfr frfr frfr I fr • Fr Fr - 26 Use of an expression cassette according to claim 4 for the transformation of monocotyledonous or dicotyledonous plants which constitutively effect expression of an exogenous polypeptide having the ability to bind methylmethoxyimino-α- (o-tolyloxy) -o-tolyl acetate (BAS 490F) specifically by seeds or leaves. Use according to claim 9, characterized in that the expression cassette is transferred to a bacterial strain and the resulting recombinant clones are used to transform monocotyledonous or dicotyledonous plants that constitutively effect expression of exogenous polypeptides with the ability to bind methylmethoxyimino-α- (o-tolyloxy) o - Tolylacetate (BAS 490F) specifically seeds or leaves. Use of an expression cassette according to claim 4 as a selection marker. Use of the transformed plant obtained according to claim 10 for the production of polypeptides with the ability to bind methylmethoxyimino-α- (o-tolyloxy) -o-tolylacetate (BAS 490F). A method for transforming a plant by introducing a gene sequence that encodes a polypeptide having the ability to bind methylmethoxyimino-α- (o-tolyloxy) -olytol acetate (BAS 490F) into a plant cell, into an ocular tissue, into a whole plant, and protoplasts of plant cells. Process according to claim 13, characterized in that the transformation is carried out by means of an agrobacterium, in particular of the species Agrobacterium tumefaciens. Process according to claim 13, characterized in that the transformation takes place by means of electroporation. A method according to claim 13, characterized in that the transformation is carried out using the particle bombardment method. 17. Production of polypeptides with the ability to bind methylmethoxyimino-α- (o-tolyloxy) -olyl acetate (BAS 490F) by expressing a gene that encodes such a polypeptide in a plant or plant cells and subsequently isolating the polypeptides. • te ·· · te · te · te · te · te - 27 The plant comprising the expression cassette of claim 4, wherein the expression cassette confers tolerance to methylmethoxyimino-α- (o-tolyloxy) -olyl acetate (BAS 490F). A method for controlling phytopathogenic fungi in transgenic methylmethoxyimino-α- (o-tolyloxy) -o-tolylacetate tolerant crops (BAS 490F), which comprises using methylmethoxyimino-α- (o-tolyloxy) -o-tolylacetate (BAS 490F) ) against which the useful plant produces polypeptides or antibodies with the ability to bind methylmethoxyimino-α- (o-tolyloxy) -o-tolylacetate (BAS 490F). A polypeptide or antibody having the ability to bind methylmethoxyimino-α- (otolyloxy) -o-tolylacetate (BAS 490F) with a high binding affinity to methylmethoxyimino-α- (o-tolyloxy) -o-tolylacetate (BAS 490F) produced as described in claim 17.