WO1998049329A1

WO1998049329A1 - Expression of fungicide-binding polypeptides in plants for producing fungicide tolerance

Info

Publication number: WO1998049329A1
Application number: PCT/EP1998/002242
Authority: WO
Inventors: Eberhard Ammermann; Earle Butterfield; Jens Lerchl; Gisela Lorenz; Achim Möller; Udo Rabe; Ralf-Michael Schmidt; Udo Conrad
Original assignee: Basf Aktiengesellschaft
Priority date: 1997-04-30
Filing date: 1998-04-16
Publication date: 1998-11-05
Also published as: TR199902681T2; NO995291L; ID22915A; KR20010020387A; JP2001523101A; EP0979295A1; IL132252A0; BG103840A; PL336661A1; DE19718251A1; CA2288432A1; HUP0003594A2; AU7335698A; BR9808698A; HUP0003594A3; AU737242B2; ZA983594B; GEP20032959B; NZ500181A; AR015626A1

Abstract

The invention relates to a method for producing fungicide-tolerant plants by expressing a fungicide-binding antibody therein.

Description

Expression of fungicide-binding polypeptides in plants to produce fungicide tolerance

5

description

The present invention relates to a method for producing fungicide-tolerant plants by expressing an exogenous one

10 fungicide-binding polypeptides in plants or parts of plants. The invention further relates to the use of the corresponding nucleic acids coding for a polypeptide, an antibody or parts of an antibody with fungicide-binding properties in transgenic plants and the transformed in this way

15 plant itself.

It is known that with the help of genetic engineering methods, specific foreign genes can be transferred into the genome of a plant. This process is called the transformation and the resulting

20 plants are called transgenic plants. Transgenic plants are currently used in various biotechnological areas. Examples are insect-resistant plants (Vaek et al. Plant Cell 5 (1987), 159-169), virus-resistant plants (Powell et al. Science 232 (1986), 738-743) and ozone-resistant plants.

25 resistant plants (Van Camp et al. BioTech. 12 (1994), 165-168). Examples of genetically engineered quality improvements are: increasing the shelf life of fruit (Oeller et al. Science 254 (1991), 437-439), increasing the starch production in potato tubers (Stark et al. Science 242 (1992), 419), changing the

30 starch (Visser et al. Mol. Gen. Genet. 225 (1991), 289-296) and ipid composition (Voelker et al. Science 257 (1992), 72-74) and production of non-plant polymers (Poirer et al. Science 256: 520-523 (1992).

^ ⁵ An important goal of plant molecular genetic work is the generation of herbicide tolerance. The herbicide tolerance is characterized by an increased type or level of tolerance of the plant or of parts of plants to the applied herbicide. This can be accomplished in various ways. The known methods are the use of a metabolism gene such as, for example, the pat gene in connection with glufosinate resistance (WO 8705629) or a target enzyme which is resistant to the herbicide, as in the case of enolpyruvylshikimate -3-phosphate synthase (WO 9204449), which is resistant to ⁵ glyphosate, and the use of a herbicide in cell and tissue culture for the selection of tolerant plant cells and resulting resistant plants such as acetyl CoA carboxylase Inhibitors described (US 5162602, US 5290696).

Antibodies are proteins that are part of the immune system. Common to all antibodies is their spatial, globular structure, the structure of light and heavy chains as well as their basic ability to bind molecules or parts of a molecular structure with high specificity (Alberts et al., In: Molecular Biology of the Cell, 2nd edition 1990, VCH Verlag, ISBN 3-527-27983-0, 1198-1237). Because of these properties, antibodies have been used for a variety of tasks. A distinction is made here between the use of the antibodies in animal and human organisms that produce them, the so-called in-situ applications and the ex-situ applications, i.e. the use of the antibodies after isolation from the producing cells or organisms (Whitelam and Cockburn, TIPS Vol.1, 8 (1996), 268-272).

The use of hybrid somatic cell lines (hybridomas) as a source of antibodies against very specific antigens goes back to work by Köhler and Milstein (Nature 256 (1975) 495-97). This process can be used to produce so-called monoclonal antibodies, which have a uniform structure and are generated by cell fusion. Spleen cells from an immunized mouse are fused with cells from a mouse myeloma. This creates hybridoma cells that proliferate indefinitely. At the same time, the cells secrete specific antibodies against the antigen with which the mouse was immunized. The spleen cells provide the ability to produce antibodies, while the myeloma cells contribute the unlimited growth ability and the continuous antibody secretion. Since each hybridoma cell is derived from a single B cell as a clone, all of the antibody molecules produced have the same structure, including the antigen binding site. This method has greatly promoted the use of antibodies, as antibodies with a single, known specificity and a homogeneous structure are now available indefinitely. Monoclonal antibodies are widely used in immunodiagnostics and as therapeutic agents.

The so-called phage display method for producing antibodies has been around for a few years, in which the immune system and the various immunizations in the animal are bypassed. Here, the affinity and specificity of the antibody is tailored in vitro (Winter et al., Ann. Rev. Immunol. 12 (1994), 433-455; Hoogenboom TIBTech Vol 15 (1997), 62-70). Gene segments which contain the coding sequence of the variable region of antibodies, ie the antigen binding site, are fused with genes for the coat protein of a bacteriophage. Then you infect bacterial with phages containing such fusion genes. The resulting phage particles now have envelopes with the antibody-like fusion protein, with the antibody-binding domain pointing outwards. The phage which contains the desired antibody fragment and specifically binds to a specific antigen can now be isolated from such a phage display library. Each phage isolated in this way produces a monoclonal, antigen-binding polypeptide which corresponds to a monoclonal antibody. The genes for the antigen binding site, which are unique for each phage, can be isolated from the phage DNA and used to construct complete antibody genes.

In the field of crop protection, antibodies have been used, in particular, as an analytical means ex situ for the qualitative and quantitative detection of antigens. This includes the detection of plant constituents, herbicides or fungicides in drinking water (Sharp et al. (1991) ACS Symp Ser., 446 (Pestic. Resides Food Saf.) 87-95), soil samples (WO 9423018) or in plants or parts of plants, and the use of antibodies

20 as an aid for the purification of bound molecules.

The production of immunoglobulins in plants was first described by Hiatt et al., Nature, 342 (1989), 76-78. The spectrum ranges from single-chain antibodies to multimeric se¬

25 cretoric antibodies (J. Ma and Mich Hein, 1996, Annuals New York Academy of Sciences, 72-81).

More recent experiments use antibodies in situ to ward off pathogens in 30 plants, in particular viral diseases by expression of specific antibodies or parts thereof directed against virus coat proteins in plant cells (Tavladoraki et al., Nature 366 (1993), 469-472; Voss et al ., Mol. Breeding 1 (1995), 39-50).

35 An analogous approach has also been used to ward off the infection of the plant by nematodes (Rosso et al., Biochem Biophys Res Com, 220 (1996) 255-263). For a pharmaceutical application, examples are known which use antibody expression in-situ in plants for oral immunization (Ma et al., Science

1995, 40: 268: 716-719; Mason and Arntzen, Tibtech Vol 13 (1996), 388-392). Antibodies formed by the plant are supplied to the body from plants or parts of plants suitable for consumption via the mouth, throat or digestive tract and cause effective immune protection. Furthermore, in plants

45 already expressed a single-chain antibody against the low-molecular plant hormone abscisic acid and reduced plant hormone availability due to abse- Acid binding in the plant was observed (Artsaenko et al., The Plant Journal (1995) 8 (5), 745-750).

The chemical control of fungi in agronomically important crops requires the use of highly selective fungicides without phytotoxic effects. The phytotoxic effect of fungicides can, for example, be based on an inhibition of plant growth, reduced photosynthetic performance and the associated reduced yield. In some cases

^■ * ^• "it difficult to develop fungicides with sufficient selectivity that can be used in all major vegetable wholesale cultures and no culture damage to the yield -. Plant cause The introduction of fungicide-resistant or tolerant crops can help to solve this problem in -

¹⁵ wear and develop new uses of fungicides in crops that were previously not treatable or could only be treated with reduced yield.

There are limits to the development of fungicide-resistant crops through tissue culture or seed mutagenesis and natural selection. On the one hand, the phytotoxic effect must be detectable in tissue culture and on the other hand, only those plants can be manipulated using tissue culture techniques whose regeneration to whole plants from cell cultures is successful. 5 In addition, after mutagenesis and selection, crop plants can show undesirable properties which have to be eliminated by repeated cross-breeding in some cases. The introduction of resistance by crossing to plants of the same species would also be restricted. 0

For these reasons, the genetic engineering approach of isolating a gene coding for resistance and transferring it specifically in crop plants is superior to the classic breeding method.

The molecular biological development of herbicide-tolerant or herbicide-resistant crop plants has hitherto presupposed that the mechanism of action of the herbicide in the plant is known and that 0 genes which impart resistance to the herbicide can be found. Many currently used herbicides work by blocking an enzyme of essential amino acid, lipid or pigment biosynthesis. By changing the genes of these enzymes such that the herbicide can no longer be bound and by introducing these changed genes into crop plants, herbicide tolerance can be generated. Alternatively, for example, enzymes analogous in nature can be found, for example, in micro- Organisms are found that show a natural resistance to the herbicide. This resistance-imparting gene is isolated from such a microorganism, cloned into suitable vectors and then, after successful transformation, expressed in herbicide-sensitive crop plants (WO 96/38567).

The object of the present invention was to develop a novel, generally applicable, genetic engineering method for producing fungicide-tolerant transgenic plants.

This object was surprisingly achieved by a method of expressing an exogenous polypeptide, antibody or parts of an antibody with fungicide-binding properties in the plants.

A first object of the present invention relates to the production of a fungicide-binding antibody and the cloning of the associated gene or gene fragment.

A suitable antibody is first generated that binds the fungicide. This can include by immunizing a vertebrate, usually a mouse, rat, dog, horse, donkey or goat with an antigen. The antigen is a fungicidally active compound that is linked or associated via a functional group to a higher molecular carrier such as bovine serum albumin (BSA), chicken egg white (ovalbumin), keyhole limpet hemocyanin (KLH) or other carriers. After repeated application of antigen, the immune response is reproduced using common methods, thus isolating a suitable antiserum. This approach initially provides a polyclonal serum that contains antibodies with different specificities. For targeted in-situ use, it is necessary to isolate the gene sequence coding for a single specific monoclonal antibody. There are various ways of doing this. The first approach uses the fusion of antibody-producing cells with cancer cells to form a hybridoma cell culture that constantly produces antibodies, which, by separating the clones it contains, ultimately leads to a homogeneous cell line that produces a defined monoclonal antibody.

The cDNA for the antibody or parts of the antibody, the so-called single chain antibody (scFv), is isolated from such a monoclonal cell line. These cDNA sequences can then be cloned in expression cassettes and for functional expression in prokaryotic and eukaryotic Organisms, including plants, can be used.

It is also possible to use phage display banks to select antibodies that bind fungicide molecules and catalytically convert them into a product with non-fungicidal properties. Methods for producing catalytic antibodies are described in Janda et al., Science 275 (1997) 945-948, Chemical selection for catalysis in combinatorial Antibody libraries; Catalytic Antibodies, 1991, Ciba Foundation Symposium 159, Wiley-Interscience Publication. In principle, a fungicide-resistant plant can also be produced by cloning the gene of this catalytic antibody and expressing it in a plant.

The invention particularly relates to expression cassettes, the coding sequence of which codes for a fungicide-binding polypeptide or its functional equivalent, and their use for producing a fungicide-tolerant plant. The nucleic acid sequence can e.g. be a DNA or a cDNA sequence. Coding sequences suitable for insertion into an expression cassette according to the invention are, for example, those which contain a DNA sequence from a hybridoma cell which codes for a polypeptide with fungicide-binding properties and which thus give the host resistance to certain fungicides.

The expression cassettes according to the invention also contain regulatory nucleic acid sequences which control the expression of the coding sequence in the host cell. According to a preferred embodiment, an expression cassette according to the invention comprises a promoter upstream, ie at the 5 'end of the coding sequence, and downstream, ie at the 3' end, a polyadenylation signal and, if appropriate, further regulatory elements which are associated with the coding sequence in between are operatively linked to the polypeptide with fungicide-binding properties and / or transit peptide. An operative link is understood to mean the sequential arrangement of promoter, coding sequence, terminator and, if appropriate, further regulatory elements in such a way that each of the regulatory elements can fulfill its function as intended when expressing the coding sequence. The sequences preferred but not limited to the operative linkage are targeting sequences to ensure subcellular localization in the apoplast, in the plasma membrane, in the vacuole, in plastids, in the mitochondrion, in the endoplasmic reticulum (ER), in the cell nucleus, in oil - corpuscles or other compartments and translation enhancers such as the 5 'leadership sequence from the tobacco mosaic virus (Gallie et

shown as an example.

An expression cassette according to the invention is produced by fusing a suitable promoter with a suitable polypeptide DNA and preferably a DNA coding for a chloroplast-specific transit peptide and a polyadenylation signal inserted between the promoter and polypeptide DNA and a polyadenylation signal according to common recombination and cloning techniques, such as, for example in T. Maniatis, EF Fritsch and J. Sambrook, Molecular Cloning: A Laboratory manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) and in T.J. Silhavy, M.L. Berman and L.W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) and in Ausubel, F.M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience (1987).

Particularly preferred are sequences which ensure targeting in the apoplasts, plastids, the vacuole, in the plasma membrane, the mitochondrium, the endoplasmic reticulum (ER) or by a lack of corresponding operative sequences, ensuring that they remain in the compartment of formation, the cytosol (Kermode , Crit. Rev. Plant Sei. 15, 4 (1996), 285-423). Localization in the ER and in the cell wall has proven to be particularly beneficial for the amount of protein accumulation in transgenic plants (Schouten et al., Plant Mol. Biol. 30 (1996), 781-792; Artsaenko et al., Plant J. 8 ( 1995) 745-750).

The invention also relates to expression cassettes, the coding sequence of which codes for a fungicide-binding fusion protein, part of the fusion protein being a transit peptide which controls the translocation of the polypeptide. Chloroplast-specific transit peptides which are split off enzymatically from the fungicide-binding polypeptide part after transposition of the fungicide-binding polypeptide into the plant chloroplasts are particularly preferred. The transit peptide is particularly preferably derived from plastidic transketolase (TK) or a functional equivalent of this transit peptide (e.g. the transit peptide of the small subunit of the Rubisco or the ferredoxin NADP oxidoreductase).

The polypeptide DNA or cDNA required for the production of expression cassettes according to the invention is preferably amplified with the aid of the polymerase chain reaction (PCR). Methods for DNA amplification by means of PCR are known, for example from Innis et al., PCR Protocols, A Guide to Methods and Applications, Academic Press (1990). The PCR-generated DNA fragments can expediently be checked by sequence analysis to avoid polymerase errors in constructs to be expressed.

⁵ The inserted nucleotide sequence coding for a fungicide-binding polypeptide can be produced synthetically or obtained naturally or contain a mixture of synthetic and natural DNA components. In general, synthetic nucleotide sequences with codons are generated which are preferred by plants

10 are added. These plant preferred codons can be determined from the highest protein frequency codons expressed in most interesting plant species. When preparing an expression cassette, various DNA fragments can be manipulated to a nucleotide sequence to - ^'■ -' obtained which expediently reads in the correct direction and is equipped with a correct reading frame. To connect the DNA fragments to one another, adapters or linkers can be attached to the fragments.

20th

The promoter and terminator regions according to the invention should expediently be provided in the transcription direction with a linker or polylinker which contains one or more restriction sites for the insertion of this sequence. As a rule, the linker has 1 to 10, usually 1 to 8, preferably 2 5 to 6 restriction sites. In general, the linker has a size of less than 100 bp, often less than 60 bp, but at least 5 bp within the regulatory ranges. The promoter according to the invention can be both native or homologous and foreign or heterologous to the host plant. The expression cassette according to the invention contains, in the 5 '-3' transcription direction, the promoter according to the invention, any sequence and a region for the transcriptional termination. Different termination areas are interchangeable. 5

Manipulations which provide suitable restriction sites or which remove superfluous DNA or restriction sites can also be used. Where insertions, deletions or substitutions such as transitions and trans versions are possible, in vitro mutagenesis, "primer pair", restriction or ligation can be used. With suitable manipulations, such as restriction, "chewing-back" or filling in overhangs for "blunt ends", complementary ends 5 of the fragments can be provided for the ligation. _^ SH _^ Φ

To transform a host plant with a DNA coding for a fungicide-binding polypeptide, an expression cassette according to the invention is inserted as an insert in a recombinant vector, the vector DNA of which contains additional functional regulatory signals, for example sequences for replication or integration. Suitable vectors are inter alia in "Methods in Plant Molecular Biology and Biotechnology" (CRC Press), Chap. 6/7, p.71-119 (1993).

Using the recombination and

Cloning techniques can be cloned the expression cassettes according to the invention into suitable vectors that allow their multiplication, for example in E. coli. Suitable cloning vectors include pBR332, pUC series, M13mp series and pA-CYC184. Binary vectors which can replicate both in E. coli and in agrobacteria, such as e.g. pBin19 (Bevan et al. (1980) Nucl. Acids Res. 12, 8711).

Another object of the invention relates to the use of an expression cassette according to the invention for the transformation of plants, plant cells, plant tissues or parts of plants. The aim of the use is preferably to impart resistance to fungicides with phytotoxic activity.

Depending on the choice of the promoter, the expression can take place specifically in the leaves, in the seeds or in other parts of the plant. Such transgenic plants, their reproductive material and their plant cells, tissue or parts are a further object of the present invention.

The transfer of foreign genes into the genome of a plant is called transformation. The methods described for the transformation and regeneration of plants from plant tissues or plant cells for transient or stable transformation are used. Suitable methods are protoplast transformation by polyethylene glycol-induced DNA uptake, the biolistic approach with the gene cannon, electroporation, the incubation of dry embryos in DNA-containing solution, microinjection and Agrobacterium-mediated gene transfer. The methods mentioned are described, for example, in B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, edited by SD Kung and R. Wu, Academic Press (1993) 128-143 and in Potrykus Annu.Rev. Plant Physiol. Plant Molec.Biol. 42 (1991) 205-225). The construct to be expressed is preferably cloned into a vector which is suitable for transcribing Agrobacterium tumefaciens form, for example pBin19 (Bevan et al., Nucl. Acids Res. 12 (1984) 8711).

Agrobacteria transformed with an expression cassette according to the invention can then be used in a known manner to transform plants, in particular crop plants, such as cereals, maize, soybeans, rice, cotton, sugar beet, canola, sunflower, flax, potato, tobacco, tomato, rapeseed, alfalfa, lettuce and the various bush, tree, nut and wine species, such as coffee, fruit trees such as apple, pear or cherry, nut trees such as walnut or pecan and particularly importantly used in vines, for example by bathing wounded leaves or leaf pieces in an agrobacterial solution and then cultivating them in suitable media.

Functionally equivalent sequences which encode a fungicide-binding polypeptide are, according to the invention, those sequences which, despite a different nucleotide sequence, still have the desired functions. Functional equivalents thus include naturally occurring variants of the sequences described herein as well as artificial, e.g. Artificial nucleotide sequences obtained by chemical synthesis and adapted to the codon usage of a plant.

A functional equivalent is understood to mean, in particular, natural or artificial mutations of an originally isolated sequence encoding the fungicide-binding polypeptide, which furthermore show the desired function. Mutations include substitutions, additions, deletions, exchanges or insertions of one or more nucleotide residues. Thus, for example, the present invention also encompasses those nucleotide sequences which are obtained by modification of this nucleotide sequence. The aim of such a modification can e.g. further narrowing down the coding sequence contained therein or e.g. also the insertion of further restriction enzyme interfaces.

Functional equivalents are also those variants whose function is weakened or enhanced compared to the original gene or gene fragment.

In addition, artificial DNA sequences are suitable as long as, as described above, they impart the desired tolerance to fungicides to avoid phytotoxic effects on crop plants. Such artificial DNA sequences can be constructed, for example, by back-translation using molecular modeling Proteins that have fungicide-binding activity or are determined by in vitro selection. Coding DNA sequences which are obtained by back-translating a polypeptide sequence in accordance with the codon usage 5 specific for the host plant are particularly suitable. The specific codon usage can easily be determined by a person skilled in plant genetic methods by computer evaluations of other, known genes of the plant to be transformed.

_ As further suitable equivalent nucleic acid

Sequences are to be called sequences which code for fusion proteins, part of the fusion protein being a non-vegetable fungicide-binding polypeptide or a functionally equivalent part thereof. The second part of the fusion protein may be ^5, for example, another polypeptide having enzymatic activity or be an antigenic polypeptide sequence by means of which detection of scFvs expression is possible (for example myc-tag or his-tag). However, this is preferably a regulatory protein sequence, such as a signal or transit peptide, which directs the polypeptide with fungicide-binding properties to the desired site of action.

The invention also relates to the expression products produced according to the invention and to fusion proteins composed of a transit peptide and a polypeptide with fungicide-binding properties.

Resistance or tolerance in the context of the present invention means the artificially acquired resistance of plants to fungicides with phytotoxic activity. It includes the partial and, in particular, the complete insensitivity to these inhibitors for at least one generation of plants. 5

The phytotoxic site of action of fungicides is generally the leaf tissue, so that leaf-specific expression of the exogenous fungicide-binding polypeptide can offer adequate protection. However, it is obvious that the phytotoxic effect of a fungicide need not be restricted to the leaf tissue, but can also be tissue-specific in all other parts of the plant.

In addition, constitutive expression of the exogenous fungicide-binding polypeptide is advantageous. On the other hand, inducible expression may also appear desirable. The efficacy of the transgenically expressed polypeptide with fungicide-binding properties can be determined, for example, in vitro by increasing the number of shoots on fungicide-containing medium using graduated series of concentrations or using seed germination tests. In addition, a fungicide tolerance of a test plant which has been modified in terms of type and amount can be tested in greenhouse experiments.

The invention furthermore relates to transgenic plants, trans- ^υ formed with an inventive expression cassette, as well as transgenic cells, tissue, parts and propagation material of such plants. Transgenic crop plants, such as, for example, cereals, maize, soybeans, rice, cotton, sugar beet, canola, sunflower, flax, potato, tobacco, tomato, rapeseed, alfalfa, Sa ³ lat and the various bush, tree, Nut and wine species such as coffee, fruit trees such as apple, pear or cherry, nut trees such as walnut or pecan and particularly important the vine.

The transgenic plants, plant cells, tissue or parts can be treated with a fungicide with a phytotoxic effect which inhibits the plant enzymes, as a result of which the plants, cells, tissue or plant parts which have not been successfully transformed die or are damaged. Examples of suitable active ingredients are strobilurins, in particular 5 methyl methoxyimino-α- (o-tolyloxy) -o-tolylacetate (BAS 490F), and metabolites and functional derivatives of these compounds. The DNA inserted into the expression cassettes according to the invention and coding for a polypeptide with fungicide-binding properties can thus also be used as a selection marker. 0

In particular in the case of crop plants, the present invention offers the advantage that, after induction of a selective resistance of the crop plant to fungicides having a phytotoxic effect, 5 such fungicides can also be used in these crops at higher application rates to control harmful fungi which would otherwise lead to plant damage. Compounds from the following groups can be mentioned as non-limiting examples of such fungicides with phytotoxic activity:

Sulfur, dithiocarbamates and their derivatives, such as ferridimethyldithiocarbamate, zinc dimethyldithiocarbamate, zinc ethylene bisdithiocarbamate, manganese ethylene bisdithiocarbamate, manganese zinc ethylenediamine bisdithiocarbamate, tetramethylthiammonodiaminodisulfate , Ammonia complex of zinc (N, N '-propylene-bis-dithiocarbamate), Zinc (N, N '-propylenebis-dithiocarbamate), N, N' -polypropylene-bis- (thiocarbamoyl) disulfide;

Nitroderivate, such as dinitro- (1-methylheptyl) phenylcrotonate, 2-sec-butyl-4, 6-dinitrophenyl-3, 3-dimethylacrylate, 2-sec-butyl-4, 6-dinitrophenyl-isopropyl carbonate, 5- Di-isopropyl nitro-isophthalate;

Heterocyclic substances, such as 2-heptadecyl-2-imidazoline acetate, 2,4-dichloro-6- (o-chloroanilino) -s-triazine, 0.0-diethyl-phthalimidophosphonothioate, 5-amino-l- [ bis- (dimethylamino) - phosphinyl] -3-phenyl-l, 2, 4-triazole, 2, 3-dicyano-l, -dithioanthraquinone, 2-thio-l, 3-dithiolo [4, 5-b] quinoxaline, l- (butylcarbamoyl) -2-benzimidazole-carbamic acid methyl ester, 2-methoxycarbonylamino-benzimidazole, 2- (furyl- (2)) -benzimidazole, 2- (thiazolyl- (4)) -benzimidazole, N- (1, 1, 2, 2-tetrachloroethylthio) tetrahydrophthalimide, N-trichloromethylthio-tetrahydrophthalimide,

N-trichloromethylthio-phthalimide,

• N-dichlorofluoromethylthio-N ', N' -dimethyl-N-phenylsulfuric acid diamide, 5-ethoxy-3-trichloromethyl-1,2,3-thiadiazole, 2-rhodanomethylthiobenzothiazole, 1,4-dichloro-2 , 5-dimethoxybenzene,

4- (2-chlorophenylhydrazono) -3-methyl-5-isoxazolone, pyridine-2-thio-1-oxide, 8-hydroxyquinoline or its copper salt, 2,3-di-hydro-5-carboxanilido-6-methyl- l, 4-oxathiin, 2, 3-dihydro-5-car-boxanilido-6-methyl-l, -oxathiin-4, 4-dioxide, 2-methyl-5, 6-dihydro-4H-pyran-3 -carboxylic acid anilide, 2-methyl-furan-3-carboxylic acid anilide, 2, 5-dimethyl-furan-3-carboxylic acid anilide, 2,4,5-trimethyl-furan-3-carboxylic acid anilide, 2, 5-dimethyl -furan-3-carboxylic acid- recyclohexylamide, N-cyclohexyl-N-methoxy-2, 5-dimethyl-furan-3-carboxamide, 2-methyl-benzoic acid anilide, 2-iodo-benzoic acid anilide, N -Formyl-N-morpholine-2,2,2-trichloroethyl-talc, piperazin-1,4-diylbis-l- (2,2,2-trichloroethyl) -formamide, 1- (3,4-dichloroanilino) -1 -formylamino-2,2,2-trichloroethane,

• Amines such as 2, 6-dimethyl-N-tridecyl-morpholine or its salts, 2, 6-dimethyl-N-cyclododecyl-morpholine or its salts, N- [3- (p-tert-butylphenyl) -2 -methylpropyl] -cis-2, 6-dimethyl-morpholine, 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-l, 3-dioxolan-2-yl-ethyl] -IH-l, 2,4-triazole, 1- [2- (2nd , 4-dichlorophenyl) -4-n-propyl-1,3-dioxolan-2-yl-ethyl] -1H-1,2,4-triazole, N- (n-propyl) -N- ( 2, 4, 6-trichlorophenoxyethyl) -N '-imidazol-yl-urea, 1- (4-chlorophenoxy) -3, 3-dimethyl-l- (1H-1, 2, 4-triazol-l-yl) - 2-butanone, 1- (4-chlorophenoxy) -3, 3-dimethyl-l- (1H-1,2, 4-triazol-l-yl) -2-butanol, (2RS, 3RS) - 1- [3- (2-chlorophenyl) -2- (4-fluoro-phenyl) -oxiran-2-ylmethyl] -1H-1,2,4-triazole, 1- [2- (2,4-dichloro- phenyD-pentyl] -1H-1, 2, 4-triazole, 2, 4 '-difluoro- α- (1H-1, 2, 4-triazolyl-l-methyl) -benzhydryl alcohol,, 1- ((bis- (4-fluorophenyl) -methylsilyl) -methyl) -1H-1, 2, 4-triazole, 1 - [2RS, 4RS; 2RS, 4SR) -4-bromo-2- (2, 4-dichlorophenyl) tetrahydrofuryl] -lH-l, 2,4-triazole, 2-- (4-chlorophenyl) -3- cyclopropyl-1- (1H-1,2,4-triazol-l-yl) butan-2-ol,

(+) -4-chloro-4- [4-methyl-2- (1H-1,2,4-triazol-l-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-l-yl) pent-l-ene -3-ol, 4- (4-chlorophenyl) -2-phenyl-2- (1H-1, 2,4, -triazolylmethyl) butyronitrile, 3- (2, 4-dichlorophenyl) -6- fluoro-2- (1H-1,2,4-triazol-l-yl) quinazolin-4 (3H) -one, (R, S) -2- (2,4-dichlorophenyl) -lHl, 2,4- triazol-l-yl) -hex-ano-2-ol, (1RS, 5RS; 1RS, 5SR) -5- (4-chlorobenzyl) -2, 2-dimethyl-1- (1H-1, 2, 4-triazol-l-ylmethyl) cyclopentanol, (R, S) -1- (4-chlorophenyl) -4,4-dimethyl-3- (1H-1, 2, 4-triazol-l-ylmethyl) pentane -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] 1 H -imidazole, 2- (4-chlorophenyl) -2- (1H -1, 2, 4-triazol-l-ylmethyl) hexanitrile, α- (2-chlorophenyl) -α- (4-chlorophenyl) -5-pyrimidine-methanol, 5-butyl-2-dimethylamino-4- hydroxy-6-methyl-pyrimidine, bis- (p-chlorophenyl) -3-pyridine-methanol, 1,2-bis- (3-ethoxycarbonyl-2-thiou-reido) -benzene, 1,2-bis- (3-methoxycarbonyl-2-thioureido) -benzene,

Strobilurins such as methyl-E-methoxyimino- [α- (o-tolyloxy) -o-tolyl] acetate, methyl-E-2- {2- [6- (2-cyanophenoxy) pyrimidine-4-yloxy ] -phenyl} -3-methoxyacrylate, methyl-E-methoxyimino- [α- (2-phenoxyphenyl)] acetamide, methyl-E-methoxyimino- [α- (2, 5-dimethylphenoxy) -o-tolyl] -acetamide,

Anilinopyrimidines such as N- (4, 6-dimethylpyrimidin-2-yl) aniline, N- [4-methyl-6- (1-propynyl) pyrimidin-2-yl] aniline,

N- [4-methyl-6-cyclopropyl-pyrimidin-2-yl] aniline, • phenylpyrroles such as 4- (2, 2-difluoro-1, 3-benzodioxol-4-yl) pyrrole-3-carbonitrile ,

• Cinnamic acid amides such as 3- (4-chlorophenyl) -3- (3, 4-dimethoxyphenyl) acrylic morpholide, as well as various fungicides, such as dodecylguanidine acetate, 3- [3- (3, 5-dimethyl-2-oxycyclohexyl) -2 -hydroxyethyl] -glutarimide, N-methyl-, N-ethyl- (4-trifluoromethyl, -2- [3 ', 4' -dimethoxyphenyl] -benzoic acid amide, hexachlorobenzene, DL-methyl-N- (2, 6- dimethyl-phenyl) -N-furoyl (2) -alaninate, DL-N- (2, 6-dimethyl-phenyl) -N- (2'-methoxyacetyl) -alanine-methyl-ester, N- ( 2,6-dimethylphenyl) -N-chloroacetyl-D, -2-aminobutyrolactone, DL-N- (2,6-dimethylphenyl) -N- (phenylacetyl) -alanine methyl ester, 5-methyl-5-vinyl-3- (3, 5-dichlorophenyl) -2, 4-dioxo-l, 3-oxazoli- din, 3- [3,5-dichlorophenyl (-5-methyl-5-methoxymethyl] -1,3-oxazolidine-2,4-dione, 3- (3,5-dichlorophenyl) -1-isopropylcarbamoylhydantoin , N- (3, 5-dichlorophenyl) -1, 2-dimethylcyclopropane-l, 2-dicarboximide, 2-cyano- [N- (ethylaminocarbonyl) -2-methoxi- 5 mino] -acetamide, N- (3rd -Chlor-2, 6-dinitro-4-trifluoromethyl-phenyl) -5-trifluoromethyl-3-chloro-2-aminopyridine

Functions equivalent derivatives of these fungicides have a ^u comparable spectrum of activity against phytopathogenic fungi as the specifically named substances, with lower, the same or higher phytotoxic activity.

The invention is illustrated by the following examples, but FIG. 5 is not restricted to these:

General cloning procedures

0 The cloning steps carried out in the context of the present invention, such as Restriction cleavages, agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, linking of DNA fragments, transformation of E. coli cells, cultivation of 5 bacteria, multiplication of phages and sequence analysis of recombinant DNA were as in Sambrook et al. (1989) Cold Spring Harbor Laboratory Press; ISBN 0-87969-309-6).

The bacterial strains used below (E. coli, XL-I Blue) ^ were obtained from Stratagene. The Agrobacterium strain used for plant transformation (Agrobacterium tumefaciens, C58C1 with the plasmid pGV2260 or pGV3850kan) was developed by Deblaere et al. (Nucl. Acids Res. 13 (1985) 4777). Alternatively, the agrobacterial strain LBA4404 (Clontech) or other ⁵ suitable strains can be used. For cloning, the vectors pUC19 (Yanish-Perron, Gene 33 (1985), 103-119) pBlues- script SK- (Stratagene), pGEM-T (Promega), pZerO (Invitrogen), pBinl9 (Bevan et al., Nucl Acids Res. 12 (1984) 8711-8720) and pBinAR (Höfgen and Willmitzer, Plant Science 66 (1990) 221-230) ⁰ .

Sequence analysis of recombinant DNA

The sequencing of recombinant DNA molecules was carried out with a laser fluorescence DNA sequencer from Pharmacia according to the method of Sanger (Sanger et al., Proc. Natl. Acad. Sei. USA 74 (1977), 5463-5467).

Production of plant expression cassettes

5

In plasmid pBin19 (Bevan et al., Nucl. Acids Res. 12, 8711

(1984)), a 35S CaMV promoter was inserted as an EcoRI-Kpnl fragment (corresponding to nucleotides 6909-7437 of the Cauliflower mosaic virus (Franck et al. Cell 21 (1980) 285). The polyadenylation signal of gene 3 der T-DNA of the Ti plasmid pTiACH5

10 (Gielen et al., EMBO J. 3 (1984) 835), nucleotides 11749-11939 was isolated as a PvuII-HindIII fragment and cloned between the SpHI-HindIII interface of the vector after addition of Sphl linkers to the PvuII interface . The plasmid pBinAR was formed (Höfgen and Willmitzer, Plant Science 66 (1990) 221-230).

15

Examples of use

example 1

20th

Since fungicides are not immunogenic, they have to be attached to a carrier material such as e.g. KLH can be coupled. If there is a reactive group in the molecule, this coupling can take place directly, otherwise a functional function is carried out during the synthesis of the fungicide.

25 nelle group introduced or a reactive precursor selected during the synthesis in order to couple these molecules to the carrier molecule in a simple reaction step. Examples of couplings are given by Miroslavic Ferencik in "Handbook of Immunochemistry", 1993, Chapman & Hall, in the chapter Antigens, page 20 -

30 49 described.

By repeated injection of this modified carrier molecule (antigen) e.g. Balb / c mice immunized. As soon as there is enough antibody in the serum to bind to the antigen in the ELISA (enzyme

35 linked immunosorbent assay) are detectable, the spleen cells of these animals are removed and fused with myeloma cells in order to cultivate hybrids. In the ELISA "fungicide-modified BSA" is also used as an antigen to distinguish the immune response against the hapten from the KLH response. 0

Monoclonal antibodies are produced based on known methods, as described, for example, in "Practical Immunology", Leslie Hudson and Frank Hay, Blackwell Scientific 5 Publications, 1989 or in "Monoclonal Antibodies: Principles and Practice", James Goding, 1983 , Academic Press, Inc., or in "A practical guide to monoclonal antibodies", J. Liddell and A. Cryer, 1991, John Wiley &Sons; or Achim Möller and Franz Emling "Monoconal antibodies against TNF and their use". European patent EP-A260610.

⁵ Example 2

The starting point for the investigation was a monoclonal antibody that specifically recognizes the BAS 490F fungicide and also has a high binding affinity. The selected hybrid cell line is characterized in that the secreted monoclonal antibodies directed against the fungicide antigen BAS 490F have a high affinity and the specific sequences of the immunoglobulins are available (Berek, C. et al., Nature 316, 412- 418, 1985). This monoclonal antibody against BAS 490F 5 was the starting point for the construction of the single-chain antibody fragment (scFv-antiBAS 490F).

First, mRNA was isolated from the hybridoma cells and rewritten into cDNA o. This cDNA served as a template for the amplification of the variable immunoglobulin genes VH and VK with the specific primers VH1 BACK and VH FOR-2 for the heavy chain and VK2 BACK and MJK5 FON X for the light chain (Clackson et al., Nature 352 , 624-628, 1991). The isolated variable immunoglobulin lines were the starting point for the construction of a single-chain antibody fragment (scFv-antiBAS 490F). In the subsequent fusion PCR, three components VH, VK and a linker fragment were combined in a PCR reaction mixture and the scFv-antiBAS 49OF was amplified (Fig. 3). 0

The functional characterization (antigen binding activity) of the constructed scFv-antiBAS 490F gene was carried out after expression in a bacterial system. The scFv-antiBAS 49OF was made using the method of Hoogenboom, H.R. et al., Nucleic Acids Research 19, 4133-4137, 1991 as a soluble antibody fragment in E. coli. The activity and the specificity of the constructed antibody fragment were checked in ELISA tests (Fig. 4).

To enable seed-specific expression of the antibody fragment in tobacco, the scFv-antiBAS 490F gene was cloned downstream of the LeB4 promoter. The LeB4 promoter isolated from Vicia faba shows a strictly seed-specific expression of various foreign genes in tobacco (Bäumlein, H. et al., Mol. Gen. Genet. 225, 121-128, 1991). By transporting the scFv-antiBAS 490F polypeptide into the endoplasmic reticulum, a stable accumulation of large amounts of antibody fragments was achieved. The scFv For this purpose, the antiBAS 490F gene was merged with a signal peptide sequence that fuses entry into the endoplasmic reticulum and the ER retention signal SEKDEL, which ensures that it remains in the ER (Wandelt et al., 1992) (Fig. 5). 5

The constructed expression cassette was cloned into the binary vector pGSGLUC 1 (Saito et al., 1990) and transferred into the Agrobacterium strain EHA 101 by electroporation. Recombinant agrobacterial clones were used for the subsequent transformation of ^x "Nicotiana tabacum. 70-140 tobacco plants were regenerated per construct. From the regenerated transgenic tobacco plants, seeds of various developmental stages were harvested after self-fertilization. From these seeds, the soluble proteins were obtained after extraction in an aqueous buffer system -

^■ "th. The analysis of the transgenic plants demonstrates that by the fusion of the scFv-antiBAS 490F gene to the DNA sequence of the ER-tion retentate signal SEKDEL mature a maximum accumulation of 1.9% scFv-antiBAS 490F protein in Seeds could be obtained.

20th

The constructed scFv-antiBAS 490F gene had a size of approx.

735 bp. The variable domains were fused together in the order VH-L-VL.

25 The specific selectivity was determined in the extracts of the ripe tobacco seeds using a direct ELISA. The values obtained show clearly that the protein extracts contain functionally active antibody fragments.

0 Example 3

Seed-specific expression and enrichment of single-chain antibody fragments in the endoplasmic reticulum of cells of transgenic tobacco seeds controlled by the USP promoter. 5

The starting point for the investigations was a single chain antibody fragment against the fungicide BAS 490F (scFv-anti BAS 490F). The functional characterization (antigen binding activity) of this constructed scFv-anti-BAS 490F gene was carried out after expression 0 in a bacterial system and after expression in tobacco leaves. The activity and the specificity of the antibody fragment constructed was checked in ELISA tests.

5 To enable seed-specific expression of the antibody fragment in tobacco, the scFv-antiBAS 490F gene was cloned downstream of the USP promoter. The one isolated from Vicia faba ι-Λ

The soluble proteins were obtained after extraction in an aqueous buffer system. Subsequent analyzes (Western blot analyzes and ELISA tests) showed that a maximum accumulation of greater than 2% of biologically active, antigen-binding scFv-antiBAS 490F polypeptide could be achieved in the leaves. The high expression values were determined in adult green leaves, but the antibody fragment could also be detected in senescent leaf material.

10 Example 5

PCR amplification of a fragment of the cDNA coding for the single-chain antibody against BAS 490F using synthetic oligonucleotides.

15

The PCR amplification of the one-chain antibody cDNA was carried out in a DNA thermal cycler from Perkin Elmer. The reaction mixtures contained 8 ng / μl of single-stranded template 20 cDNA, 0.5 μM of the corresponding oligonucleotides,

200 μM nucleotides (Pharmacia), 50 mM KC1, 10 M Tris-HCl (pH 8.3 at 25oC, 1.5mM MgC12) and 0.02 U / μl Taq polymerase (Perkin Elmer). The amplification conditions were set as follows:

25 annealing temperature: 45 ° C

Denaturation temperature: 94 ° C,

Elongation temperature: 72 ° C,

Number of cycles: 40

•on

The result was a fragment of approximately 735 base pairs, the m den

Vector pBluescript was ligated. E. coli XL-I Blue was transformed with the ligation mixture and the plasmid was amplified. For application and optimization of the polymerase chain reaction see: Innis et al., 1990, PCR Protocols, a Guide to Methods and Applications, Academic Press.

Example 6

Production of transgenic tobacco plants which express a cDNA coding for a single-chain antibody with fungicide-binding properties.

The plasmid pGSGLUC 1 was transformed into Agrobacterium tumefaciens 5 C58Cl: pGV2260. For the transformation of tobacco plants (Nicotiana tabacum cv. Samsun NN) a 1:50 dilution of an overnight culture of a positively transformed agrobact colony in Murashige-Skoog medium (Physiol. Plant. 15 (1962) 473 ff.) with 2% sucrose (2MS medium). Leaf disks of sterile plants (each about 1 cm ² ) were incubated in a Petri dish with a 1:50 agrobacterial dilution for 5-10 minutes. This was followed by a 2-day incubation in the dark at 25 ° C. on 2MS medium with 0.8% Bacto agar. The cultivation was continued after 2 days with 16 hours of light / 8 hours of darkness and on a weekly basis on MS medium with 500 mg / 1 claforan (cefotaxime sodium), 50 mg / 1 kanamycin, 1 mg / 1 benzylaaminopurine (BAP ), 0.2 mg / 1 naphthylacetic acid and 1.6 g / 1 glucose continued. Growing shoots were transferred to MS medium with 2% sucrose, 250 mg / 1 Claforan and 0.8% Bacto agar.

Example 7

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

The starting point of the investigations was a single-chain antibody fragment expressed against the fungicide BAS 490F (scFv-anti BAS 490F) expressed in tobacco plants. The amount and activity of the synthesized scFv-antiBAS 490F polypeptide were determined in Western blot analyzes and ELISA tests.

To enable expression of the scFv-antiBAS 490F gene in the endoplasmic reticulum, the foreign gene was controlled under the control of the CaMV 53S promoter as a translation fusion with the LeB4 signal peptide (N-th inal) and the ER retention signal KDEL (C- terminal). By transporting the scFv-antiBAS 490F polypeptide into the endoplasmic reticulum, a stable accumulation of high amounts of active antibody fragment was achieved. After harvesting the leaf material, pieces were frozen at -20 ° C (1), lyophilized (2) or dried at room temperature (3). The soluble proteins were obtained from the respective leaf material by extraction in an aqueous buffer and the scFv-antiBAS 490F polpypeptide was purified by affinity chromatography. Equal amounts of purified scFv-antiBAS 490F polypeptide (frozen, lyophilized and dried) were used to determine the activity of the antibody fragment (Fig. 6). Fig. 6 A shows the antigen binding activity of the fresh (1), lyophilized (2) and dried leaves (3) of purified scFv-antiBAS 490F polypeptides. In Fig. 6 B the respective amounts of scFv-antiBAS 490F protein (about 100 ng), which were used for the ELISA analyzes, are determined by means of Western blot analyzes. The sizes of the protein molecular weight standards are shown on the left. Thereby about the same antigen binding activity was found.

Example 8

To demonstrate the fungicide tolerance of the transgenic tobacco plants producing a polypeptide with fungicide-binding properties, these were treated with different amounts of BAS 490F. In all cases it could be shown in the greenhouse that the plants expressing a scFv-antiBAS 490F gene compared to the

Control show a higher tolerance to the fungicide BAS 490F and lower phytotoxic effects.

Claims

claims

1. Process for the production of fungicide-tolerant plants by expression of an exogenous fungicide-binding polypeptide in the plants.

2. The method according to claim 1, characterized in that the exogenous fungicide-binding polypeptide is an ^x ^ single-chain antibody fragment.

3. The method according to claim 1, characterized in that the exogenous fungicide-binding polypeptide is a complete antibody or a fragment derived therefrom

15 acts.

4. The method according to claim 1, characterized in that the fungicide is methyl methoxyimino-α- (o-toly-loxy) -o-tolylacetate (BAS 490F).

20th

5. The method according to any one of claims 1-3, characterized in that it is mono- or dicotyledonous plants.

²⁵ 6. The method according to claim 5, characterized in that the plant is tobacco.

7. The method according to any one of claims 1-6, characterized in that the expression of the exogenous polypeptide constant

30 tutive in the plant.

8. The method according to any one of claims 1-6, characterized in that the expression of the exogenous polypeptide is induced in the plant.

35

9. The method according to any one of claims 1-6, characterized in that the expression of the exogenous polypeptide takes place in the leaves of the plant.

40 10. The method according to any one of claims 1-6, characterized in that the expression of the exogenous polypeptide takes place in the seeds of the plant.

Sign.

45

11. Expression cassette for plants consisting of a promoter, a signal peptide, a gene coding for the expression of an exogenous fungicide-binding polypeptide, an ER retention signal and a terminator.

5

12. Expression cassette according to claim 11, characterized in that the CaMV 35S promoter is used as the constitutive promoter.

13 13. Expression cassette according to claim 11, characterized in that the gene of a single-chain antibody fragment is used as the gene to be expressed.

14. Expression cassette according to claim 11, characterized in that the gene or gene fragment of a fungicide-binding polypeptide is used as a translation fusion with other functional proteins such as enzymes, toxins, chromophores and binding proteins as the gene to be expressed.

15. Expression cassette according to claim 11, characterized in that the polypeptide gene to be expressed from a hybridoma cell or by means of other recombinant methods - such as e.g. the antibody phage display method - is obtained.

25 16. Use of the expression cassette according to claim 11 for

Transformation of dicotyledonous or monocotyledonous plants which constitutively express an exogenous fungicide-binding polypeptide in a seed- or leaf-specific manner.

17. Use according to claim 16, characterized in that the expression cassette is transferred into a bacterial strain and the resulting recombinant clones for the transformation of dicotyledonous or monocotyledonous plants which are constitutively seed- or leaf-specific, an exogenous fungicide-binding

35 Express polypeptide used.

18. Use of the expression cassette according to claim 11 as a selection marker.

40 19. Use of a transformed plant as claimed in claim 17 or 18 for the preparation of a fungicide-binding polypeptide.

45

20. A method for transforming a plant by introducing a gene sequence coding for a fungicide-binding polypeptide into a plant cell, into callus tissue, a whole plant and protoplasts from plant cells.

5

21. The method according to claim 20, characterized in that the transformation is carried out with the aid of an Agrobacterium, in particular the type Agrobacterium tumefaciens.

10. The method according to claim 20, characterized in that the transformation is carried out with the aid of electroporation.

23. The method according to claim 20, characterized in that the transformation using the particle bombardment method

15 follows.

24. Production of a fungicide-binding polypeptide by expression of a gene coding for such a polypeptide in a plant or cells of a plant and subsequent

20 Isolation of the polypeptide.

25. Plant containing an expression cassette according to claim 11, characterized in that the expression cassette imparts tolerance to a fungicide.

25

26. Plant according to claim 25, characterized in that it is tolerant of methyl methoxyimino-α- (o-tolyloxy) -o-tolylacetate (BAS 490F).

30 27. A method for combating phytopathogenic fungi in transgenic fungicide-tolerant crop plants characterized in that fungicides are used against which the crop plant forms fungicide-binding polypeptides or antibodies.

35

28. Fungicide-binding polypeptides or antibodies with high

Binding affinity to methyl methoxyimino-α- (o-tolyloxy) -o-toly-lacetate (BAS 490F), characterized in that they are prepared according to claim 24. 0

5 Expression of fungicide-binding polypeptides in plants to produce fungicide tolerance

Summary

Process for the preparation of fungicide-tolerant plants by expression of a fungicide-binding antibody in the plants.