CN1254381A - Expression of fungicide-binding polypeptides in plants for producing fungicide tolerance - Google Patents

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 for fungicide tolerance

The present invention relates to methods for preparing fungicide-tolerant plants by expressing exogenous fungicide-binding polypeptides in plants or in plant organs. The invention also relates to the use of corresponding nucleic acids encoding polypeptides, antibodies or antibody parts having fungicide-binding properties in transgenic plants and the transformed plants themselves.

Genetic engineering methods are known that allow the specific transfer of foreign genes into the plant genome. This method is called transformation and the resulting plants are transgenic plants. Currently, transgenic plants are used in many fields of biotechnology. Such as 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 (Van Camp et al., Biotechnology 12 ( 1994), 165-168). Examples of improved traits obtained through genetic engineering are: improved fruit shelf life (Oeller et al., Science 254 (1991), 437-439), increased starch production in potato tubers (Stark et al., Science 242 (1992), 419) , starch composition change (Visser et al., Mol.Gen.Genet.225(1991), 289-296) and lipid composition change (Voelker et al., Science 257(1992), 72-74) and exogenous polymer Synthesis (Poirer et al., Science 256 (1992), 520-523).

An important goal of the work carried out in the field of plant molecular genetics is the generation of herbicide tolerance. Herbicide tolerance is characterized by an improved compatibility (either in type or level) of the plant or plant organ with the herbicide used. This can be accomplished in a number of ways. Known methods combine metabolic genes such as the pat gene with glufosinate resistance (WO8705629) or herbicide-resistant target enzymes such as glyphosate-resistant enolpyruvylshikimate-3-phosphate synthase (WO9204449), and Herbicides are used in cell and tissue culture to select for tolerant plant cells and resulting resistant plants as described for acetyl-CoA carboxylase inhibitors (US5162602, US5290696).

Antibodies are proteins that are components of the immune system. Common features of all antibodies are their three-dimensional spherical structure, the organization of the light and heavy chains and their fundamental ability to bind to the structure of molecules or molecular parts with high specificity (Alberts et al., in: Molekularbiologie, der Zelle [Cell Molecular Biology] , 2nd Edition, 1990, VCH Verlag, ISBN 3-527-27983-0, 1198-1237). Based on these characteristics, antibodies are used for a variety of tasks. The application can be divided into the application of the antibody in the animal and human body that produces the antibody, the so-called in situ application, and the ex-situ application, that is, the application of the antibody after the antibody is isolated from the antibody-producing cells and organisms (Whitelam und Cockburn, TIPS No. 1 Vol. 8 (1996), 268-272).

The use of somatic hybrid cell lines (hybridomas) as a source of antibodies to very specific antigens is based on the work performed by Kohler and Milstein (Nature 256 (1975) 495-97). This method allows the production of so-called monoclonal antibodies that have a uniform structure and are produced by cell fusion methods. Splenocytes from immunized mice were fused with mouse myeloma cells. This allows the hybridoma cells to proliferate indefinitely. At the same time, the cells secrete antibodies specific to the antigen used to immunize the mouse. Spleen cells provide the ability to produce antibodies while myeloma cells allow the cells to grow indefinitely and secrete antibodies continuously. As clones, because each hybridoma cell is derived from a single B cell, all antibody molecules produced have the same structure, including the antigen binding site. This approach greatly facilitates the use of antibodies because of the unlimited availability of antibodies with a single, known specificity and common structure. Monoclonal antibodies are widely used in immunodiagnostics and therapeutics.

In recent years, the so-called phage display method has been used to produce antibodies, avoiding the immune system and immunizing animals multiple times. The affinity and specificity of antibodies are measured in vitro (Winter et al., Annal Rev. Immunol. 12 (1994), 433-455; Hoogenboom, TIBTech, Vol. 15 (1997), 62-70). The gene fragment containing the sequence encoding the variable region of the antibody, that is, the antigen-combining site, is fused with the phage coat protein gene. Bacteria are then infected with phages containing such fusion genes. The resulting phage particles have an outer shell containing the antibody-like fusion protein, with the antibody-binding region pointing outward. Such phage display libraries can be used to isolate phage containing desired antibody fragments that specifically bind to an antigen. Each phage isolated in this way produces a monoclonal antigen-binding polypeptide identical to a monoclonal antibody. Each phage's unique antigen-binding site genes can be isolated from phage DNA and used to construct whole antibody genes.

In the field of crop protection, antibodies are used in particular as ex-sita analytical tools for the qualitative and quantitative detection of antigens. This includes plant constituents, herbicides or Detection of fungicides and use of antibodies as an aid in purification of binding molecules.

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

More recent attempts to use antibodies to protect plants in situ against pathogens, especially viral diseases, have been achieved by expressing in plant cells antibodies specific for viral coat proteins or parts thereof (Tavlacloraki et al., Nature 366 (1993), 469 -472; Voss et al., Molecular Breeding 1 (1995), 39-50).

A similar approach has also been used to protect plants from nematode infection (Rosso et al., Biochemical and Biophysical Research Communications, 220 (1996) 255-263). There are also examples of applications in pharmaceuticals, where in situ expression of antibodies in plants is used for oral immunization (Ma et al., Science 268 (1995), 716-719; Mason and Arntzen, Tibtech Vol. 13 (1996), 388- 392). Providing the body with plant-formed or derived plant or plant organ antibodies suitable for consumption through the mouth, throat or alimentary canal confers effective immune protection. Furthermore, single-chain antibodies against the low molecular weight phytohormone abscisic acid have been expressed in plants, and a reduction in the availability of the phytohormone was observed due to the incorporation of abscisic acid in plants (Artsaenko et al., Plant Journal 8(5) (1995), 754-750).

The chemical control of fungi in agronomically important crops requires highly selective fungicides without phytotoxic effects. The phytotoxic effect of fungicides can be based on inhibition of plant growth, reduced photosynthesis and thus reduced yield. However, in some cases it has been difficult to develop fungicides which are sufficiently selective to be usable in all important crops and which do not cause yield-providing plant damage in any crops. The introduction of fungicide-resistant or tolerant crop plants helps to solve this problem and allows the development of new uses of antifungal agents in crops where treatment has hitherto not been possible or only possible when yield loss is acceptable.

Development of fungicide-resistant crop plants by tissue culture or seed mutagenesis and natural selection is limited. On the one hand, the phytotoxic effect must already be detectable at the tissue culture level and, on the other hand, only such plants can be manipulated by tissue culture techniques, successfully regenerating whole plants from cell culture. Furthermore, following mutagenesis and selection, plants may exhibit undesired characteristics that must be removed by, and in some cases repeated, backcrossing. Furthermore, resistance introduced by crossing will be restricted to plants of the same species.

For the above reasons, genetic engineering methods of isolating a resistance-encoding gene and introducing it into crop plants in a targeted manner are superior to traditional plant breeding methods.

Until now, the development of herbicide-tolerant or herbicide-resistant crop plants by molecular biology methods required understanding the mechanism of action of herbicides in plants and the discovery of genes that confer resistance to herbicides. Many herbicides currently in commercial use work by blocking enzymes that are steps in the biosynthesis of essential amino acids, lipids or pigments. Herbicide tolerance is produced by altering the genes for these enzymes in such a way that the herbicide is no longer subject to binding and introducing these altered genes into crop plants. Alternative examples are similar enzymes found in nature, such as in microorganisms, which exhibit natural resistance to herbicides. The resistance-conferring gene is cloned from such microorganisms, subcloned into a suitable vector, and then successfully transformed for expression in herbicide-sensitive crop plants (WO96/38567).

One of the objects of the present invention is to develop novel, general genetic engineering methods for producing fungicide-tolerant transgenic plants.

We have surprisingly found that this object is achieved by expressing in plants an exogenous polypeptide, antibody or antibody part having fungicide binding properties.

The present invention first relates to the preparation of fungicide-binding antibodies and the cloning of related genes or gene fragments.

The first step is to prepare an appropriate fungicide-conjugated antibody. This is achieved in particular by immunizing vertebrates, in most cases mice, rats, dogs, horses, donkeys or goats, with the antigen. In this case, the antigen is associated or conjugated via a functional group to a high molecular weight carrier such as bovine serum albumin (BSA), chicken ovalbumin, keyhole limpet hemocyanin (KLA) or other carriers with fungicidal activity compound of. Following repeated administration of the antigen, suitable antisera are isolated by monitoring the immune response by conventional methods. Initially, this method produced polyclonal sera containing antibodies of different specificities. For targeted in situ application, it is necessary to isolate the gene sequence encoding a single, specific monoclonal antibody. Various approaches are available for this purpose. The first method utilizes the fusion of antibody-producing cells with tumor cells to generate hybridoma cell cultures that continuously produce antibodies, and finally, selection of the resulting clones leads to a homogeneous cell line producing a specific monoclonal antibody.

The cDNAs of antibodies or antibody parts, so-called single-chain antibodies (SCFV), are isolated from such monoclonal cell lines. These cDNA sequences are cloned into expression cassettes and functionally expressed in prokaryotes and eukaryotes, including plants.

Alternatively, it is possible to select antibodies by phage display libraries, and these antibodies bind to the fungicide molecule and subsequently convert it catalytically into a product without fungicidal properties. Methods for making catalytic antibodies are described in Janda et al., Science 275 (1997) 945-948, Chemoselective Catalysis in Conjoint Antibody Libraries; Catalytic Antibodies, 1991, Ciba Foundation Symposium 159, Wiley-Interscience Publication. Theoretically, cloning and expressing this catalytic antibody gene in plants could lead to fungicide resistant plants.

The invention relates in particular to expression cassettes whose coding sequences encode fungicide-binding polypeptides or functional analogues thereof and to the use of these expression cassettes to produce fungicide-tolerant plants. The nucleic acid sequence may be a DNA sequence or a cDNA sequence. Coding sequences suitable for insertion into the expression cassettes according to the invention are, for example, those DNA sequences from hybridoma cells encoding polypeptides with fungicide-binding properties thereby conferring resistance to plant enzyme inhibitors in the host.

Furthermore, the expression cassette according to the invention contains regulatory nucleic acid sequences which control the expression of the coding sequence in the host cell. In a preferred embodiment, the expression cassette according to the invention comprises upstream, i.e. at the 5'-end of the coding sequence; a promoter and downstream, i.e. at the 3'-end, a polyadenylation signal and, if appropriate, further associated with a fungicide. Regulatory elements are operably linked to the coding sequences of the specified polypeptides and/or intermediates of the transfer peptides. Operably linked is understood to mean that the promoter, coding sequence, terminator and, if appropriate, other regulatory elements are arranged in such a sequence that when the coding sequence is expressed, the regulatory elements can function as intended. A preferred sequence for operably linked is, but not limited to, target sequences that ensure subcellular localization in the apoplast, plasma membrane, vacuole, plastid, mitochondria, endoplasmic reticulum (ER), nucleus, liposome, or other compartment and Translational enhancers, such as the 5' leader sequence from tobacco mosaic virus (Gallie et al., Nucleic Acids Res. 15 (1987), 8693-8711).

In principle, a suitable promoter for the expression cassette according to the invention is any promoter which ensures the expression of the foreign gene. Promoters used with preference are in particular promoters from plants or promoters from plant viruses. Particularly preferred is the CaMV35S promoter from cauliflower mosaic virus (Franck et al., Cell 21 (1980) 285-294). This promoter contains multiple recognition sequences for transcriptional effectors, which together lead to permanent constitutive expression of the introduced gene (Benfey et al., EMBO J., 8 (1989) 2195-2202).

The expression cassette according to the invention may also comprise a chemically inducible promoter, by means of which the expression of the foreign polypeptide in plants is controlled to a specific point in time. Such promoters are for example the PRP1 promoter (Ward et al., Plant Mol. Promoters inducible by acetic acid or cyclohexanone (WO9321334) have been described in reference and are applicable in others.

Other particularly preferred promoters are those which ensure expression in the tissues or plant organs in which the phytotoxic fungicidal activity takes effect. Promoters guaranteeing leaf-specific expression deserve special mention. Mention must be made of the potato cytoplasmic FBPase promoter or the potato ST-LST promoter (Stockhans et al., EMBO J. 8 (1989) 2445-245).

With the help of a seed-specific promoter, stable expression of single-chain antibodies can be achieved up to 0.67% of the total soluble seed protein in transgenic tobacco seeds (Fiedler and conrad Bio/Technology 10 (1995), 1090-1094). Such germination and seed-specific promoters are also preferred regulatory elements according to the invention, since sowing or germination seeds can also express and are also desirable for the purposes of the present invention. Thus, the expression cassette according to the invention contains, for example, a seed-specific promoter (preferably the USP or LEB4 promoter), the LEB4 signal peptide, the gene to be expressed and the ER retention signal. The construction of the expression cassette is illustrated schematically in Figure 1 with reference to a single chain antibody (ScFV gene).

According to e.g. T. Maniatis E.F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor (1989), T.J. Silhavy, M.L, Berman and L.W. Enquist, Gene Fusion Experiments, Cold Spring Harbor Laboratory, Cold Spring Hong Kong, NY (1984) and Ausubel, F.M. et al., Contemporary Molecular Biology Methods, Greene Publishing Assoc and Wiley-Interscience (1987) described conventional recombination and cloning techniques, according to the expression cassette of the present invention is the combination of a suitable promoter and a suitable Polypeptide DNA and preferably, inserted between the promoter and the polypeptide DNA is produced by fusing DNA encoding a chloroplast-specific transit peptide and a polyadenylation signal.

Particularly preferred sequences allow localization to apoplasts, plastids, follicles, plasma membranes, mitochondria, endoplasmic reticulum (FR) or, in the absence of suitable operator sequences, stay in the formed compartment, i.e. the cytosol (Kermode , Crit. Rev. Plant Sci. 15, 4 (1996), 285-423). Localization in the ER and cell wall has proven particularly advantageous for quantifying protein accumulation in transgenic plants (Schouten et al., Plant Mol. Biol. 30 (1996), 781-792; Artsaenko et al., The Plant Journal 8 (1995) 745-750) .

The invention also relates to an expression cassette whose coding sequence encodes a fungicide-binding fusion protein, part of which is a transit peptide that controls the transport of the polypeptide. Particularly preferred are chloroplast-specific transit peptides, which are partially cleaved from the fungicide-binding polypeptide following transport of the fungicide-binding polypeptide to the chloroplast of the plant. Particularly preferred transit peptides from plastidic transglycolase (TK) or functional analogs thereof (e.g., ribulose bisphosphate carboxylase-oxygenase small subunit or ferredoxin NADP oxidoreductase) transit peptide).

The polypeptide DNA or polypeptide cDNA required for the preparation of the expression cassette according to the invention is preferably amplified by means of the polymerase chain reaction (PCR). DNA amplification methods using PCR are known, for example, Innis et al., PCR Methods, Guidelines for Methods and Applications, Academic Press (1990). Sequence analysis is conveniently used to detect PCR-generated DNA fragments to avoid polymerase errors in constructs to be expressed.

The inserted nucleotide sequence encoding the fungicide-binding polypeptide may be prepared synthetically or obtained naturally or contain a mixture of synthetic and natural DNA components. In general, synthetic nucleotide sequences containing plant-favored codons are prepared. Identify plant-favored codons by codons for proteins with high frequency and proteins expressed in most plant species of interest. When preparing expression cassettes, multiple DNA fragments can be manipulated to obtain nucleotide sequences in the proper sense reading and equipped with the correct reading frame. To join DNA fragments to each other, adapters or linkers are added to the fragments.

The promoter and terminator regions according to the invention should be provided in the transcriptional sense with a linker or polylinker containing one or more restriction sites for the insertion of this sequence. In principle, linkers have 1-10, usually 1-8, preferably 2-6 restriction sites. Within regulatory regions, the size of the linker is usually less than 100 bp, usually less than 60 bp, but even 5 bp. The promoter according to the invention is native or homologous to the host plant, or foreign or different thereto. The expression cassette according to the invention comprises the promoter according to the invention in the sense of transcription 5'-3', any desired sequence and a transcription termination region. The various termination regions are interchangeable.

Furthermore, manipulations to provide appropriate restriction sites or to remove excess DNA or restriction sites may be employed. If insertions, deletions, substitutions such as transitions and transversions are possible, in vitro mutagenesis, "primer [sic] repair", restriction or ligation may be employed. For suitable manipulations such as restriction, "chewing-back" or filling in to obtain "blunt ends", it is necessary to provide complementary ends of the fragments for ligation.

According to the invention, especially important for success was the addition of the specific ER retention signal SEKDEL which increased the average expression level three-fold to four-fold (Schuoten, A. et al., Plant Mol. Biol. 30 (1996), 781-792). Other ER-localized retention signals that occur naturally in plant and animal proteins can also be used to construct this expression cassette.

A preferred polyadenylation signal is a plant polyadenylation signal, preferably a polyadenylation signal consistent with the T-DNA border sequence from Agrobacterium tumefaciens, especially gene 3 of the T-DNA border sequence of the Ti plasmid pTiACH5 (octopine synthase) (Gielen et al., EMBO J. 3 (1984) 835, et seq.) or a functional analogue thereof.

An expression cassette according to the invention may comprise, for example, a constitutive promoter (preferably the CaMV35S promoter), a LeB4 signal peptide, the gene to be expressed and an ER retention signal. The construction of the expression cassette is shown schematically in Figure 2 with reference to a single chain antibody (scFv gene). The amino acid sequence KDEL (lysine, aspartic acid, glutamic acid, leucine) is preferably used as ER retention signal.

The fusion expression cassette encoding a polypeptide with fungicide binding properties is preferably cloned into a vector, such as pBin19 suitable for transformation of Agrobacterium tumefaciens. Plants, especially agricultural crops such as tobacco, are transformed with Agrobacterium transformed with such a vector by a known method, for example, by immersing calli or leaf parts in an Agrobacterium solution, followed by culturing in a suitable medium. Transformation of plants with Agrobacterium is known, especially by F.F. White, Vectors for Gene Transfer in Higher Plants, Transgenic Plants, Vol. 1, Engineering and Applications, edited by S.D. Kung and R. Wu, Academic Press, 1993, No. 15 - pp. 38 and S.B. Gelvin, Molecular Genetics of T-DNA Border Sequence Transfer from Agrobacterium to Plants, Transgenic Plants, pp. 49-78. Transgenic plants containing a gene expressing a polypeptide with fungicide-binding properties integrated into the expression cassette according to the invention can be regenerated in a known manner from the transformed cells of callus leaves or leaf parts.

For transformation of host plants with the DNA encoding the fungicide-binding polypeptide, the expression cassette according to the invention is integrated by insertion into a recombinant vector whose DNA contains additional functional regulation signals, eg sequences for replication or integration. Suitable vectors are described inter alia in "Methods in Plant Molecular Biology and Biotechnology" (CRC Press), (1993) Chapter 6/7 pp. 71-119.

Using the recombination and cloning techniques described above, the expression cassettes according to the invention can be cloned into suitable vectors allowing their amplification eg in E. coli. Suitable cloning vectors are in particular pBR332, the pUC series, the M13mp series and pACYC184. Especially suitable are binary vectors capable of replicating in E. coli and Agrobacterium, eg pBin19 (Bevan et al. (1980) Nucleic Acids Res. 12, 8711).

The invention furthermore relates to the transformation of plants, plant cells, plant tissues or plant organs with the expression cassette according to the invention. A preferred purpose of use is the modulation of resistance to fungicides with phytotoxic activity.

Depending on the choice of promoter, expression can occur specifically in leaves, seeds or other plant organs. Such transgenic plants, their propagation material and their plant cells, plant tissues or plant organs are further subjects of the present invention.

The transfer of foreign genes into the plant genome is called transformation. In this process, the methods described above for transformation and regeneration of plants from plant tissues or plant cells are used for transient or stable transformation. Suitable methods are protoplast transformation with polyethylene glycol-mediated DNA uptake, the biolistic [sic] method with a gene gun, electroporation, incubation of dried embryos in a DNA-containing solution, microinjection, and Agrobacterium-mediated gene transfer. The mentioned methods are described, e.g., B. Jenes et al., Gene Transfer Technology in: Transgenic Plants, Vol. 1 Engineering and Applications, eds.: S.D.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 suitable for transformation of Agrobacterium tumefaciens, eg pBin19 (Bevan et al., Nucleic Acids Res., 12 (1984) 8711).

The Agrobacterium transformed with the expression cassette according to the present invention can be used to transform plants, especially crops such as corn, corn, soybean, rice, cotton, sugar beet, Canola, sunflower, flax, potato, tobacco, tomato, rapeseed, alfalfa , lettuce and various shrubs, trees, nuts and grape species such as coffee, fruit trees such as apple, pear or cherry, nut trees such as walnut or hickory and especially important vines, e.g. Submerged in an Agrobacterium solution, followed by cultivation in an appropriate medium.

According to the present invention, functionally equivalent sequences encoding fungicide-binding polypeptides still function as expected despite differing nucleotide sequences. Thus, functional analogs include naturally occurring variants of the sequences described herein as well as artificial nucleotide sequences, such as chemically synthesized artificial nucleotide sequences adapted to plant codon usage.

In particular, functional analogues are understood to be natural or artificial mutations of the originally isolated polypeptides encoding fungicide-binding properties, which mutations still exhibit the desired function. Mutations include substitutions, additions, deletions, exchanges or insertions of one or more nucleotide residues. Therefore, the present invention also includes nucleotide sequences obtained by modifying this nucleotide sequence. The purpose of such modification may be, for example, to further restrict multiple cleavage sites in the coding sequence or otherwise, for example, to insert restriction enzymes.

Other functional analogs are those variants which have a reduced or enhanced function compared to the starting gene or gene fragment.

Furthermore, artificial DNA sequences are suitable as long as they induce the desired fungicide resistance as described above to avoid phytotoxic effects on crop plants. Such artificial DNA sequences can be identified, for example, by back-translation of proteins with fungicide binding activity and constructed by molecular modeling or in vitro selection. Particularly suitable are DNA coding sequences which have been back-translated from the polypeptide sequence according to the host plant-specific codon usage. Specific codon usage can be readily determined by an expert familiar with plant genetic methods by computer-aided analysis of other known genes of the transformed plant.

Also necessary to mention suitable analogous nucleic acid sequences according to the invention are sequences encoding fusion proteins, wherein the fusion protein part is a fungicide-binding polypeptide of non-plant origin or a functionally analogous part thereof. For example, the second part of the fusion protein may be an additional enzymatically active polypeptide, or an antigenic polypeptide sequence (eg myc-tag or his-tag) expressed by scFvs that can be assisted by it. However, it is preferably a regulatory protein sequence, eg a signal or transit peptide that directs a polypeptide having fungicide binding properties to the desired site of action.

However, the invention also relates to fusion proteins of expression products and transit peptides prepared according to the invention with polypeptides having fungicide-binding properties.

For the purposes of the present invention, resistance/tolerance refers to the ability of an artificially obtained plant to withstand a fungicide having phytotoxic activity. It includes partial and especially complete insensitivity to inhibitors during at least one plant generation.

The site of phytotoxic action of fungicides is usually leaf tissue, so leaf-specific expression of exogenous fungicide-binding polypeptides can provide adequate protection. However, one can readily understand that the phytotoxic effects of fungicides need not be restricted to leaf tissue, but can also be achieved in all other organs of the plant in a tissue-specific manner.

In addition, constitutive expression of exogenous fungicide-binding polypeptides is advantageous. Alternatively, inducible expression is also desirable.

For example, the efficiency of transgenically expressed polypeptides expressing fungicide binding properties can be determined in vitro by root meristem propagation in media containing a series of fungicide concentration gradients or by seed germination experiments. In addition, fungicide tolerance in test plants of altered type and level can be tested in greenhouse experiments.

The invention additionally relates to transgenic plants transformed with an expression cassette according to the invention, transgenic cells, tissues, organs and propagation material of these plants. Particularly preferred are transgenic crops such as cereals, corn, soybean, rice, cotton, sugar beet, Canola, sunflower, flax, potato, tobacco, tomato, canola, alfalfa, lettuce and various shrub trees, nuts and grape species such as coffee , fruit trees such as apples, pears or cherries, nut trees such as walnuts or hickories and especially important grape vines.

Transgenic plants, plant cells, plant tissues or plant organs can be treated with phytotoxic fungicides which inhibit plant enzymes, whereby the unsuccessfully transformed plants, plant cells, plant tissues or plant organs die or are destroyed. Examples of suitable active ingredients are strobilurins, specifically methylmethoxyimino-α-(O-tolyloxy)-O-tolyl acetate (BAS 490F) and metabolites and functional derivatives of these compounds. A DNA encoding a polypeptide having fungicide-binding properties and inserted into an expression cassette according to the invention can also be used as a selectable marker.

The present invention has the advantage, in particular with regard to crops, that once a selective resistance of the crop plants to fungicides with phytotoxic effects has been induced, such fungicides can be used on these crops at even higher application rates to Controls harmful fungi that can otherwise cause plant destruction. Compounds in the following groups may be mentioned as examples of such phytotoxically active fungicides, without limitation:

Sulfur; dithiocarbamate and its derivatives such as iron(III) dimethyldithiocarbamate, zinc dimethyldithiocarbamate, zinc vinylbisdithiocarbamate Ester, magnesium vinyl bis dithiocarbamate, manganese zinc vinyl diamine bis dithiocarbamate, tetramethyl dihydrocarbyl aminothiocarbonyl disulfide [sic], zinc (N, N '-vinylbisdithiocarbamate) ammonia complex, zinc (N,N'-propylenebisdithiocarbamate) ammonia complex, zinc (N,N'-propylenebisdithiocarbamate) Thiocarbamate), N,N'-polypropylene bis(thiocarbamoyl) disulfide; nitro derivatives such as dinitro(1-methylheptyl)phenyl crotonate, 2-sec-butyl-4,6-dinitrophenyl-3,3-dimethacrylate, 2-sec-butyl-4,6-dinitrophenyl isopropyl carbonate, Diisopropyl 5-nitroisophthalate; heterocyclic substances such as 2-heptadecyl-2-imidazoline acetate, 2,4-dichloro-6-(O-chloroanilino)- Secondary-triazine, O, O-diethylphthalimidophosphonothioate, 5-amino-1-[bis(dimethylamino)phosphoryl]-3-phenyl-1,2, 4-triazole, 2,3-dicyano-1,4-dithioanthraquinone, 2-thio-1,3-dithioalkene[4,5-b]quinoline, methyl-1- (Butylcarbamoyl)-2-benzimidazole carbamate, 2-methoxycarbonylaminobenzimidazole, 2-(2-furyl)benzimidazole, 2-(4-triazolyl)benzene And imidazole, 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-thiodiazole, 2-thiocyanooxymethylthiobenzothiazole, 1,4-dichloro-2,5-dimethoxybenzene, 4-(2-Chlorophenylhydrazino)-3-methyl-5-isoxazolone, pyrimidine-2-thio[sic]1-oxide, 8-alkylquinoline or its copper salt, 2 , 3-dihydro-5-carboxanilide-6-methyl-1,4-oxathione, 2,3-dihydro-5-carboxanilide-6-methyl-1,4 -Oxathione-4,4-dioxide, 2-methyl-5,6-dihydro-4H-pyran-3-carboxanilide, 2-methylfuran-3-carboxyl Aniline, 2,5-dimethylfuran-3-carboxanilide, 2,4,5-dimethylfuran-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-trichloroethyl acetal, piperazine-1,4-diylbis-1-(2,2,2-trichloroethyl)formamide, 1- (3,4) Dichloroanilino-1-formylamino-2,2,2-dichloroethane; Amines such as 2,6-dimethyl-N-tridecylmorpholine or its salts, 2,6-Dimethyl-N-cyclodecanylmorpholine 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 hexamethylenetetramine; azoles such as 1-[2-(2,4-dichlorophenyl)-4- Ethyl-1,3-dioxolan-2-ylethyl]-1H-1,2,4-triazole, 1-[2-(2,4-dichlorophenyl)-4-n- Propyl-1,3-dioxolan-2-ylethyl]-1H-1,2,4-triazole, N-(n-propyl)-N-(2,4,6-trichloro Phenoxyethyl)-N'-imidazolyl urea, 1-(4-chlorophenoxy)-3,3-dimethyl-1-(1H-1,2,4-azido-1- Base)-2-butanone, 1-(4-chlorophenoxy)-3,3-dimethyl-1-(1H-1,2,4-azido-1-yl)-2-butane Alcohol, (2RS, 3RS)-1-[3-(2-chlorophenyl)-2-(4-fluorophenyl)-oxirane-2-ylmethyl]-1H-1,2,4 -triazole, 1-[2-(2,4-dichlorophenyl)-pentyl]-1H-1,2,4-triazole, 2,4'-difluoro-α-(1H-1, 2,4-triazolyl-1-methyl)benzhydryl alcohol, 1-((bis(4-fluorophenyl)methylsilyl)methyl)-1H-1,2,4-tri Azole, 1-[2RS, 4RS; 2RS, 4SR]-4-bromo-2-(2,4-dichlorophenyl)tetrahydrofuryl]-1H-1,2,4-triazole, 2-(4- Chlorophenyl)-3-cyclopropyl-1-(1H-1,2,4-azido-1-yl)-butane-2-ol, (+)-4-chloro-4-[4 -Methyl-2-(1H-1,2,4-azido-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-azido-1-yl)pentyl -1-en-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-azido-1-yl)quinazolin-4(3H)-one, (R,S)-2-( 2,4-dichlorophenyl)-1-H-1,2,4-azido-1-yl)hexan-2-ol, (1RS, 5RS; 1RS, 5SR)-5-(4- Chlorobenzyl)-2,2-dimethyl-1-(1H-1,2,4-azido-1-ylmethyl)cyclopentanol, (R,S)-1-(4- Chlorophenyl)-4,4-dimethyl-3-(1H-1,2,4-azido-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- Azido-1-ylmethyl)capronitrile, α-(2-chlorophenyl)-α-(4-chloropropyl)-5-pyrimidinemethanol, 5-butyl-2-dimethylamino- 4-Hydroxy-6-methylpyrimidine, bis(p-chlorophenyl)-3-pyridinemethanol, 1,2-bis(3-ethoxycarbonyl-2-thioureido)benzene, 1,2-bis (3-Methoxycarbonyl-2-thioureido)benzene; strobilurines such as methyl-E-methoxyimino-[α-(O-methylphenoxy)-O-tolyl]acetate, methyl -[E]-2-{2-[6-(2-cyanophenoxy)pyrimidin-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-cyclopropylpyrimidin-2-yl]aniline; phenylpyrroles such as 4-(2,2-difluoro-1,3-benzodioxole-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-oxocyclohexyl)-2-hydrocarbyl ethyl] glutarimide, N-methyl- or N- Ethyl-(4-trifluoromethyl),-2-[3',4'-dimethoxyphenyl]benzamide [sic], hexafluorobenzene, methyl-N-(2,6- Dimethylphenyl)-N-(2-furoyl)-DL-alanine salt, DL-N-(2,6-dimethylphenyl)-N-(2'-methoxyacetyl ) alanine methyl ester, N-(2,6-dimethylphenyl)-N-chloroacetyl-D, L-2-aminobutyrolactone, DL-N-(2,6-dimethyl Phenyl)-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-(3-chloro-2,6-dinitro-4-tri Fluoromethylphenyl)-5-trifluoromethyl-3-chloro-2-aminopyridine.

Functionally identical derivatives of these fungicides have the same range of action against phytopathogenic fungi as the substances already specifically mentioned, and a lesser, equal or more pronounced phytotoxic activity.

The invention is illustrated but not limited by the following examples: General cloning method:

Steps performed within the scope of the present invention such as restriction cleavage, agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, ligation of DNA fragments, transformation of E. coli cells, bacterial culture, phage manipulation and recombinant DNA sequence analysis was performed as described by Sambrook et al. (1989) Cold Spring Harbor Laboratory Press, ISBN 0-87969-309-6.

The bacterial strain (E. coli XL-I Blue) used below was from Stratagene. The Agrobacterium strain used for plant transformation (Agrobacterium tumefaciens. C58C1 containing plasmid pGV2260 or pGV3850Kan) is described by Deblaere et al. (Nucleic Acids Res. 13 (1985) 4777). Alternatively, Agrobacterium strain LBA4404 (clontech) or other suitable strains may be used. Vector pUC19 (Yanish-Perron, Gene 33 (1985) 103-119), pBluscript.SK-(Stratagene), pGEM-T (Promega), pZero (Invitrogen), pBin19 (Bevan et al., Nucleic Acid Research 12 (1984) 8711- 8720) and pBinAR (Hofgen and Willmitzer, Plant Science 66 (1990) 221-230) were used for cloning purposes. Sequence analysis of recombinant DNA

The sequence of the recombinant DNA molecule was determined by the Sanger method (Sanger et al., Proceedings of the National Academy of Sciences of the United States of America 74 (1977), 5463-5467), and the laser fluorescence DNA sequencing device of Pharmacia Company. Preparation of plant expression cassettes

The 35S CaMV promoter was inserted into plasmid pBin19 (Bevan et al., Nucleic Acids Res. 12, 8711) as an EcoRI-KpnI fragment (corresponding to nucleotides 6909-7437 of Cauliflower Mosaic Virus) (Franck et al., Cell 21 (1980) 285) (1984)). Polyadenylation signal of gene 3 of Ti plasmid pTiACH5 (Gielen et al., EMBO J. 3 (1984) 835) T-DNA border sequence; nucleotides 11749-11939 were isolated as a PvuII-HindIII fragment, plus After the SphI linker; cloned into the PvuII site between the SphI-HindIII cut sites of the vector. The plasmid pBinAR was generated (Hofgen and Willmitzer, Plant Science 66 (1990) 221-230). Application Example Example 1

Because fungicides are not immunogenic, they must be coupled to a carrier material, such as KLH. If the molecules have reactive groups, they can be directly coupled; if not, functional groups are introduced during fungicide synthesis or reactive precursors are selected during synthesis to couple these molecules to carrier molecules in simple reaction steps. Examples of conjugation reactions are found in Miroslavic Ferencik, "Handbook of Immunochemistry", 1993, Chapman & Hall, Chapter Antigens, pp. 20-49.

Repeated injections of this modified carrier molecule (antigen) are used to immunize, eg, Balb/c mice. Once a sufficient amount of antibody bound to the antigen was detected by ELISA (enzyme-linked immunosorbent assay), splenocytes from these animals were removed and fused with myeloma cells to grow hybrids. Additionally "fungicide-modified BSA" was used as the antigen in the ELISA to differentiate immune responses against haptens from those against KLH.

Preparation of monoclonal antibodies by methods similar to known methods, for example in "Practical Immunology" Leslie Hudson and Frank Hay, Blackwell Scientific Publications, 1989 or in "Monoclonal Antibodies: Theory 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 Moller and Franz Emling "Monoklonale Antikorper gegen TNF und deven Verwendung" [Monoclonal Antibodies against TNF and Their Applications ] the method described. European patent specification EP-A260610. Example 2

The starting point of this study was a high-affinity monoclonal antibody that specifically recognizes the fungicide BAS 490F. The selected hybridoma cell line is characterized by the high affinity secreted monoclonal antibody directed against the fungicide antigen BAS 490F and the specific sequence of this immunoglobulin is available (Berek, C. et al., Nature 316, (1985 ) 412-418). This monoclonal antibody against BAS 490F is the starting point for the construction of a single chain antibody fragment (scFv-anti-BAS 490F).

First, mRNA is isolated from hybridoma cells and transcribed into cDNA. This cDNA is used as a template for amplifying variable immunoglobulin genes VH and VK, wherein the specific primers for the heavy chain are VH1BACK and VH FOR-2, and the primers for the light chain are VK2BACK and MJK5FONX (Clackson et al., Nature 352, (1991) 624-628. Isolated variable immunoglobulins are the starting point for the construction of single-chain antibody fragments (scFv-anti-BAS 490F). In subsequent fusion PCR, the three components VH, VK and linker fragments are combined in a PCR reaction , so that ScFv-anti-BAS 490F was amplified (Figure 3).

Functional identification (antigen binding activity) of the constructed scFv-anti-BAS 490F gene was carried out after expression in the bacterial system. In order to achieve this purpose, scFv-anti-BAS 490F was synthesized in Escherichia coli with soluble antibody fragments using the method of Hoogenboom, H.R. et al. Nucleic Acids Research 19 (1991), 4133-4137. The activity and specificity of the constructed antibody fragments were detected by ELISA assay ( FIG. 4 ).

To allow seed-specific expression of antibody fragments in tobacco, the scFv-anti-BAS490F gene was cloned downstream of the LeB4 promoter. The LeB4 promoter isolated from Vicia fqba exhibits strict seed-specific expression of various foreign genes in tobacco (Baumlein, H. et al., Mol. Gen. Genet. 225, (1991) 121-128). Transport of the scFv-anti-BAS 490F polypeptide to the endoplasmic reticulum results in the stable accumulation of large numbers of antibody fragments. To this end, the scFv anti-BAS 490F gene was fused to a signal peptide sequence to ensure its entry into the endoplasmic reticulum and to the ER retention signal SEKDEL to ensure retention of the polypeptide in the ER (Wandelt et al., 1992) (Figure 5).

The constructed expression cassette was cloned into the binary vector pGSGLUC1 (Saito et al., 1990) and transferred into Agrobacterium strain EHA101 by electroporation. Recombinant Agrobacterium clones were used for subsequent tobacco transformation. 70-140 tobacco plants were regenerated per construct. After self-pollination, seeds at different developmental stages were harvested from the regenerated transgenic tobacco plants. Soluble protein in an aqueous buffer system was obtained from these seeds after extraction. Analysis of transgenic plants showed that fusion of the scFv-anti-BAS 490F gene with the ER retention signal SEKDEL allowed a maximum accumulation of 1.9% of the resulting scFv-anti-BAS 490F protein in mature seeds.

The constructed scFv-anti-BAS 490F gene is about 735bp in size. The variable domains are fused to each other in the VH-L-VL sequence.

Identification of specific selectivities in extracts of mature tobacco seeds by direct ELISA. The values obtained clearly indicate that the protein extract contains functionally active antibody fragments. Example 3

Under the control of the USP promoter, scFv fragments were seed-specifically expressed and accumulated in the endoplasmic reticulum of transgenic tobacco seed cells.

The starting point for this study was a single chain antibody fragment (scFv-anti-BAS 490F) directed against the fungicide BAS 490F. Functional identification (antigen binding activity) of the constructed scFv-anti-BAS 490F gene was carried out after expression in the bacterial system and after expression in tobacco leaves. The activity and specificity of the constructed antibody fragments were detected by ELISA assay.

To allow seed-specific expression of antibody fragments in tobacco, the scFv-anti-BAS490F gene was cloned downstream of the USP promoter. The USP promoter isolated from Vicia faba exhibits strict seed-specific expression of various foreign genes in tobacco (Fiedler, U. et al., Plant Molecular Biology 22, (1993) 669-679). Transport of the scFv-anti-BAS 490F polypeptide to the endoplasmic reticulum results in the stable accumulation of large numbers of antibody fragments. For this purpose, the scFv-anti-BAS 490F gene was fused to a signal peptide sequence to ensure its entry into the endoplasmic reticulum and to the ER retention signal SEKDEL to ensure retention of the polypeptide in the ER (Wandelt et al., 1992) (Figure 1).

The constructed expression cassette was cloned into the binary vector pGSGLUC1 (Saito et al., 1990) and transferred into Agrobacterium strain EHA101 by electroporation. Recombinant Agrobacterium clones were used for subsequent tobacco transformation. After self-pollination, seeds at different developmental stages were harvested from the regenerated transgenic tobacco plants. Soluble protein in an aqueous buffer system was obtained from these seeds after extraction. Analysis of transgenic plants showed that fusion of the scFv-anti-BAS 490F gene with the DNA sequence of the ER retention signal SEKDEL under the control of the USP promoter resulted in scFv fragments with binding affinity to BAS 490F as early as day 10 of seed development L ', hvbnm; N. Example 4

To obtain broad expression of the antibody fragment in plants, especially in leaves, the scFv-anti-BAS 490F gene was cloned downstream of the CaMV35S promoter. This strong constitutive promoter mediates the expression of foreign genes in all plant tissues (Benfey and Chaua, Science 250 (1990), 956-966. The transport of the scFv-anti-BAS 490F protein to the endoplasmic reticulum allows the large amount of DNA to be obtained Antibody fragments accumulate stably in leaves. First, the scFv-anti-BAS 490F gene is fused to a signal peptide sequence that ensures entry into the endoplasmic flesh and an ER retention signal KDEL that ensures product retention in the ER (Wandelt et al., Plant Journal 2 (1992) , 181-192). The constructed expression cassette was cloned into the binary vector pGSGLUC1 (Saito et al., Plant Cell Propagation 8 (1990), 718-721) and transferred into Agrobacterium strain EHA101 by electroporation. Recombinant Agrobacterium clone was used for Subsequent tobacco transformation. About 100 tobacco plants are regenerated. The leaves that are in different developmental stages are removed from the transgenic tobacco plants of regeneration. The soluble protein that is in the water buffer system is obtained from these seeds after extraction. Subsequent analysis ( Western blot analysis and ELISA assay) showed that the maximum accumulation of biologically active antigen-binding scFv-anti-BAS 490F polypeptide obtained from leaves was more than 2%. High expression values were identified in fully grown green leaves, but not in senescent leaves. Antibody fragments were also detected in the material. Example 5

A cDNA fragment encoding a single-chain antibody against BAS 490F was PCR amplified with the aid of synthetic oligonucleotides.

PCR amplification of single-chain antibody cDNA was performed on a DNA thermal cycler from Perkin Elmer. The reaction mixture contained 8 ng/μl single-stranded template cDNA, 0.5 μM related oligonucleotides, 200 μM nucleotides (pharmacia), 50 mM KCl, 10 mM Tris-Hcl (pH 8.3 at 25°C, 1.5 mM Mgcl ₂ ) and 0.02 μM /nl Taq polymerase (Perkin Elmer). The reaction conditions were set as follows:

Annealing temperature: 45℃

Denaturation temperature: 94°C

Extension temperature: 72°C

Number of cycles: 40

As a result, a fragment of about 735 base pairs was obtained, which was ligated into the vector pBluescript. The ligation mixture was used to transform E. coli XL-I Blue and the plasmid was amplified. For applications and optimization of the polymerase chain reaction, see: Innis et al., 1990, PCR Methods, Guidelines for Methods and Applications, Academic Press. Example 6 Preparation of transgenic tobacco expressing a single-chain antibody cDNA encoding fungicide-binding properties

The plasmid pGSGLUC1 was transformed into Agrobacterium tumefaciens C58C1:pGV2260. Tobacco plants (Nicotiana cv. Samsun NN) were transformed with a 1:50 dilution of an overnight culture of a transformation-positive Agrobacterium clone grown in Murashige-Skoog medium (2MS medium) containing 2% sucrose. In petri dishes, leaves of sterile plants (approximately 1 cm ² each) were incubated in a 1:50 dilution of Agrobacterium for 5-10 minutes. Then cultured on 2MS medium containing 0.8% Bacto-Agar in the dark for 2 days. After continuing to culture for 2 days under 16 hours of light/8 hours of darkness, in the presence of 500 mg/l cefotaxim (cefotaxim-Sodium), 500 mg/l kanamycin, 1 mg/l benzylaminopurine (BAP), 0.2 mg/l Cultures were continued on a weekly basis in MS medium with NAA and 1.6 g/l glucose. The grown shoots were transferred to MS medium containing 2% sucrose, 250 mg/l cefotaxime and 0.8% Bacto-Agar. Example 7 Stable accumulation of single-chain antibody fragments against the fungicide BAS 490F in the endoplasmic reticulum

The starting point for this study was a single chain antibody (scFv-anti-BAS 490F) expressed in tobacco plants against the fungicide BAS 490F. Quantity and activity of the synthesized scFv-anti-BAS 490F polypeptide were identified by Western blot analysis and ELISA assay.

The exogenous gene to be expressed is expressed as a translational fusion with the LeB4 signal peptide (N-terminus) and the ER retention signal KDEL (C-terminus) under the control of the CaMV35S promoter making it possible for the scFv-anti-BAS 490F gene to be endosomal expressed in the network. The transport of the scFv-anti-BAS 490F polypeptide into the endoplasmic reticulum allows the stable accumulation of large amounts of active antibody fragments. After harvesting the leaf material was frozen at -20°C (1), lyophilized (2) or dried in the greenhouse (3). The soluble protein in water buffer was obtained by extraction from the leaves to be tested and the scFv-anti-BAS 490F polypeptide was purified by affinity chromatography. Equal amounts of purified scFv-anti-BAS 490F polypeptides (frozen, lyophilized and dried) were used to identify the activity of antibody fragments (Figure 6). Figure 6A shows the antigen-binding activity of scFv-anti-BAS 490F polypeptides purified from fresh (1), freeze-dried (2) and dried (3) leaves. Figure 6B shows representative amounts of scFv-anti-BAS 490F protein (approximately 100 ng) used in the ELISA assay as determined by Western blot analysis. The size of the protein molecular weight marker is indicated on the left. The antigen-binding activities were found to be substantially the same. Example 8

To verify the fungicide tolerance of transgenic tobacco plants producing polypeptides with fungicide-binding properties, these tobacco plants were treated with various amounts of BAS 490F. In the greenhouse, all experiments demonstrated that plants expressing the scFv-anti-BAS 490F gene exhibited higher tolerance to the fungicide BAS 490F and less pronounced phytotoxic effects compared to controls.

Claims (28)

CLAIMS 1. A method for preparing fungicide-tolerant plants by expressing exogenous fungicide-binding polypeptides in plants. 2. The method of claim 1, wherein the exogenous fungicide-binding polypeptide is a single chain antibody fragment. 3. The method of claim 1, wherein the exogenous fungicide-binding polypeptide is a whole antibody or a fragment thereof. 4. The method according to claim 1, wherein the fungicide is methylmethoxyimino-alpha-(O-methylphenoxy)-O-tolyl acetate (BAS 490F). 5. The method according to any one of claims 1-3, wherein the plant is a monocot or a dicot. 6. The method of claim 5, wherein the plant is tobacco. 7. The method according to any one of claims 1-6, wherein the exogenous polypeptide is expressed constitutively in the plant. 8. The method according to any one of claims 1-6, wherein expression of the exogenous polypeptide in the plant is induced. 9. The method according to any one of claims 1-6, wherein the exogenous polypeptide is expressed in the leaves of the plant. 10. The method according to any one of claims 1-6, wherein the exogenous polypeptide is expressed in plant seeds. 11. A plant expression cassette consisting of a promoter, a signal peptide, a gene encoding an exogenous fungicide-binding polypeptide, an ER retention signal and a terminator. 12. The expression cassette according to claim 11, wherein the constitutive promoter used is the CaMV35S promoter. 13. The expression cassette according to claim 11, wherein the gene to be expressed is a gene of a single chain antibody fragment. 14. The expression cassette according to claim 11, wherein the gene or gene fragment of the fungicide-binding polypeptide used in translational fusion with other functional proteins such as enzymes, toxins, chromophores and binding proteins is used as the gene to be expressed . 15. The expression cassette according to claim 11, wherein the polypeptide gene to be expressed is obtained from hybridoma cells or other recombinant methods such as antibody phage display method. 16. Use of the expression cassette according to claim 11 for transformation of dicotyledonous or monocotyledonous plants to specifically constitutively express exogenous fungicide-binding polypeptides in seeds or leaves of the plants. 17. The use according to claim 16, wherein the expression cassette is transferred to a bacterial strain and the resulting recombinant clone is used to transform dicotyledonous or monocotyledonous plants to make plant seeds or leaves specifically constitutively express the exogenous fungicide Binding peptides. 18. Use of the expression cassette according to claim 11 as a selectable marker. 19. Use of a transformed plant obtained according to claim 17 or 18 for the preparation of a fungicide-binding polypeptide. 20. A method for transforming plants by introducing a gene sequence encoding a fungicide-binding polypeptide into plant cells, callus tissue, whole plants and plant cell protoplasts. 21. The method according to claim 20, wherein the transformation is performed by means of Agrobacterium, in particular Agrobacterium tumefaciens. 22. The method of claim 20, wherein the transformation is performed by electroporation. 23. The method according to claim 20, wherein the transformation is carried out by means of particle bombardment. 24. A method for the production of a fungicide-binding polypeptide by expressing a gene encoding such a polypeptide in a plant or plant cell and then isolating the polypeptide. 25. A plant containing the expression cassette of claim 11, wherein the expression cassette confers fungicide tolerance. 26. The plant of claim 25, tolerant to methylmethoxyimino-α-(O-methylphenoxy)-O-tolyl acetate (BAS 490F). 27. A method of controlling a phytopathogenic fungus in a transgenic fungicide-resistant crop plant comprising applying a fungicide to the crop plant forming a fungicide-binding polypeptide or antibody. 28. A fungicide-binding polypeptide or antibody with high binding affinity to methylmethoxyimino-α-(O-methylphenoxy)-O-methylphenyl acetate (BAS 490F), according to claim 24 Prepared as described.

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