KR20010020387A - Expression of Fungicide-Binding Polypeptides in Plants for Producing Fungicide Tolerance - Google Patents

Abstract

The present invention relates to a method for producing fungicide resistant plants by expressing fungicide binding antibodies.

Description

Expression of Fungicide-Binding Polypeptides in Plants for Producing Fungicide Tolerance}

The present invention relates to a method for producing fungicide resistant plants by expression of exogenous fungicide binding polypeptides in plants or plant organs. The invention also relates to the use of the corresponding nucleic acid encoding a polypeptide, antibody or part of an antibody having fungicidal binding properties in a transgenic plant and the transformed plant itself.

Genetic engineering methods are known to enable specific transfer of heterologous genes into plants' genomes. This method is called transformation, and the resulting plant is called a transgenic plant. Transgenic plants are currently being used for biotechnology in various fields. Examples include pest 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. BioTech. 12 (1994) 165-168. Examples of quality characteristics improved by genetic engineering include improved shelf life of fruits [Oeller et al. Science 254 (1991) 437-439], and increased starch production in potato tubers [Stark et al. Science 242 (1992) 419], changes in starch composition [Visser et al. Mol. Gen. Genet. 225 (1991) 289-296] and changes in lipid composition [Voelker et al. Science 257 (1992) 72-74 and the production of foreign polymers [Poirer et al. Science 256 (1992) 520-523.

An important research goal in the field of plant molecular genetics is to make them resistant to fungicides. Fungicide resistance is characterized by improved suitability (in terms of type and concentration) of the fungicides applied to the plants or plant organs. This can be done in several ways. Known methods are for metabolism genes associated with glufosinate resistance (International Publication No. 8705629), for example pat genes, or for enolpyruville shchimate-3-phosphate synthase resistant to glyphosate (international Using a target enzyme that is resistant to fungicides, such as in Publication No. 9204449, and also in the case of acetyl-CoA-carboxylase inhibitors (US Pat. No. 5,060,602, 5290696). The use of fungicides in cell and tissue cultures for the selection of resistant plant cells and the resulting resistant plants.

Antibodies are proteins as components of the immune system. A common feature of all antibodies is their basic ability to bind their spatial, small, spherical structures, the construction of light and heavy chains, and the parts of molecules and molecular structures with high specificity (Alberts et al., In: Molekularbiologie der Zelle [ Molecular Biology of the Cell, 2nd Edition 1990, VCH Verlag, ISBN 3-527-27983-0, 1198-1237. Based on these properties, antibodies have been used for many tasks. Such use may be divided into the use of the antibody in the animal and human organisms in which the antibody is formed, ie the so-called in situ use, and the out-of-situ use, ie the use of the antibody after the antibody is isolated from the producing cell or organism. Whitelam und Cockburn, TIPS Vol. 1, 8 (1996) 268-272.

The use of somatic hybrid cell lines (hybridomas) as a source of antibodies to highly specific antigens is based on studies performed by Kohler and Milstein (Nature 256 (1975) 495-497). This method allows for the formation of so-called monoclonal antibodies having a homogeneous structure and produced by cell fusion. Splenocytes of immunized mice are fused with mouse myeloma cells. This gives hybridoma cells that proliferate indefinitely. At the same time, the cells secrete specific antibodies to the antigens that have immunized mice. Splenocytes offer the possibility of antibody production, while myeloma cells contribute to the non-limiting growth and continuous secretion ability of the antibody. Since each hybridoma cell that is a clone is derived from a single B cell, all the antibody molecules produced have the same structure, including the antigen binding site. This method greatly facilitated the use of antibodies because antibodies with a single known specificity and homogeneous structure are currently available in non-limiting amounts. Monoclonal antibodies are widely used in the diagnosis of immune responses and also as therapeutics.

Recently, so-called phage display methods have become available for the production of antibodies, where the immune system and various immunizations in animals are avoided. The affinity and specificity of the antibodies were measured in vitro [Winter et al., Ann. Rev. Immunol. 12 (1994) 433-455; Hoogenboom TIBTech Vol 15 (1997) 62-70]. The gene segment containing the variable region of the antibody, ie the sequence encoding the antigen binding site, is fused with the gene for the envelope protein of the bacteriophage. Thereafter, the bacteria are infected by phages containing such fusion genes. The resulting phage particles have an outer shell containing the antibody-like fusion protein, with the antibody binding domain facing outward. Such phage display libraries can be used to isolate phages containing the antibody fragment of interest and specifically binding to any antigen. Each phage isolated in this manner produces a monoclonal antigen binding polypeptide corresponding to a monoclonal antibody. Genes for the antigen binding site, unique for each phage, can be isolated from phage DNA and used to construct complete antibody genes.

In the field of crop protection, antibodies have been used, in particular, as in situ analytical tools for qualitative and quantitative detection of antigens. This is because drinking water [Sharp et al. (1991) ACS Symp Ser., 446 (Pestic. Residues Food Saf.) 87-95], detection of plant components, fungicides or fungicides in soil samples (International Publication No. 9423018) or plants or plant organs, And the use of antibodies as adjuvants for the purification of binding molecules.

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

More recent attempts use in situ antibodies to defend plants against pathogens, particularly viral diseases, by expressing in a plant cell specific antibodies against viral envelope proteins [Tavladoraki et al., Nature 366 (1993). ), 469-472; Voss et al., Mol. Breeding 1 (1995) 39-50].

Similar methods have also been used to defend plants against infection by nematodes [Rosso et al., Biochem Biophys Res Com. 220 (1996) 255-263. Examples of use can be found in pharmacology 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. Antibodies from the plant or plant organs formed by the plant and suitable for ingestion through the mouth, throat or digestive system are provided to the body, and the antibodies produce efficient immunoprotection. In addition, single-chain antibodies to low molecular weight plant hormone abstic acid have already been expressed in plants, and reduced availability of plant hormones has been observed due to the binding of absic acid in plants [Artsaenko et al., The Plant Journal (1995). 8 (5) 754-750].

Chemical control of weeds in crops important for agricultural operations requires the use of highly selective fungicides without adversely affecting the plants. The deleterious effects of fungicides on plants can be based, for example, on the inhibition of plant growth, on photosynthesis and thus on the yield. In some cases, however, it is difficult to develop sufficiently selective fungicides that can be used in any significant large-scale harvest of the plant in any crop and do not damage the profitable plant. The introduction of fungicide-resistant or resistant crops can contribute to solving this problem, and it is possible to begin the use of new fungicides on crops that are not treatable or possible to date and allow for reduced yields. It became.

The development of fungicide resistant crops by tissue culture or seed mutagenesis and natural selection is limited. While deleterious effects on plants must have been detected at the tissue culture level in advance, only these plants can be manipulated through tissue culture techniques capable of successfully regenerating the entire plant from cell culture. In addition, following mutagenesis and selection, crops may exhibit undesirable properties that in some cases should be re-excluded by repeated backcrossing. In addition, the introduction of resistance by performing crosses will be limited to plants of the same species.

For this reason, genetic engineering methods of isolating resistance coding genes as target methods and transferring them to crops are superior to traditional plant variety improvement methods.

To date, the development of herbicide-tolerant or herbicide-tolerant crops by molecular biology methods requires knowledge of the mechanism of action of herbicides in plants and the discovery of genes that confer resistance to herbicides. A large number of currently used herbicides work by blocking enzymes of essential amino acid, lipid or pigment biosynthesis steps. Herbicide resistance can be formed by modifying the gene of this enzyme so that the herbicide can no longer be bound and introducing the modified gene into the crop. Another example is to find similar enzymes in nature, for example in microorganisms that exhibit natural resistance to herbicides. This resistance conferring gene is isolated from such microorganisms, recloned into a suitable vector and then expressed in herbicide sensitive crops after successful transformation (International Publication No. 96/38567).

It is an object of the present invention to develop a new, generally available genetically engineered method for producing fungicide resistant transgenic plants.

The inventors have surprisingly found that this object is achieved by a method of expressing in a plant an exogenous polypeptide, antibody or portion of an antibody having fungicidal binding properties.

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

The first step is to produce a suitable antibody that binds to the fungicide. This can be done in particular by immunizing vertebrates, usually mice, rats, dogs, horses, donkeys or goats with antigens. The antigen in this case is a fungicidally active compound which is bound or coupled via a functional group to a high molecular weight carrier such as bovine serum albumin (BSA), chicken ovalbumin, keyhole limpet hemocyanin (KLH) or other carrier. After the antigen is repeatedly applied, the immune response is monitored in a conventional manner, and the appropriate antiserum is thus isolated. This method initially produces polyclonal serum containing antibodies with different specificities. For targeted in situ use, it is necessary to isolate the gene sequence encoding a single, specific monoclonal antibody. Several methods can be used for this purpose. The first method is to provide hybridoma cell cultures that induce homologous cell lines that produce specific monoclonal antibodies by continuously generating antibodies and unicating the resulting clones using fusion of antibody producing cells and cancer cells. .

The cDNA, or so-called single chain antibody (scFv), for an antibody, or portion of an antibody, is isolated from such monoclonal cell lines. These cDNA sequences are then cloned into expression cassettes and used for functional expression in eukaryotes, including prokaryotes and plants.

It is also possible to select antibodies through phage display libraries, which bind catalytically and convert catalytically into products with fungicidal properties. Methods of forming 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. Cloning the gene of this catalytic antibody and expressing it in plants in principle leads to the production of fungicide resistant plants.

The invention particularly relates to expression cassettes whose coding sequences encode fungicide binding polypeptides or functional equivalents thereof, and the use of these expression cassettes for the production of fungicide resistant plants. The nucleic acid sequence can be, for example, a DNA sequence or a cDNA sequence. Suitable coding sequences for insertion into expression cassettes according to the invention contain DNA sequences from hybridoma cells, for example, encoding polypeptides having fungicidal binding properties and thus conferring resistance to plant enzyme inhibitors to the host. will be.

In addition, the expression cassette according to the present invention contains regulatory nucleic acid sequences that regulate the expression of the coding sequence in the host cell. In a preferred embodiment, the expression cassette according to the present invention comprises an upstream, ie on the 5 'end, a promoter and a down, ie on the 3'-end, a polyadenylation signal and, if appropriate, other regulatory sequences of the coding sequence, between And operably linked with a coding sequence of a polypeptide having a fungicide binding property and / or a transient peptide located at. Operable linkage should be understood to mean that the promoter, coding sequence, terminator and, where appropriate, other regulatory elements that each function as intended when the coding sequence is expressed are arranged sequentially. Preferred sequences for operative linkage include, but are not limited to, targeting sequences and translations that ensure subcellular localization in apoplasts, plasma membranes, vacuoles, pigments, mitochondria, vesicles (ER), nuclei, liposomes, or other septa. Enhancers such as the 5'-leader sequence from tobacco mosaic virus [Gallie et al., Nucl. Acids Res. 15 (1987) 8693-8711.

Suitable promoters of the expression cassettes according to the invention are in principle any promoter capable of controlling the expression of heterologous genes. Promoters used preferably are especially plant-derived promoters or promoters derived from plant viruses. Especially preferred is the CaMV 35S promoter from Cauliflower mosaic virus (Franck et al., Cell 21 (1980) 285-294). This promoter contains several recognition sequences for transcriptional effectors that induce permanent and constitutive expression of the introduced gene as a whole (Benfey et al., EMBO J. 8 (1989) 2195-2202).

Expression cassettes according to the invention may also comprise chemically induced promoters which allow the expression of exogenous polypeptides in plants to be regulated at particular times. Such promoters, for example the PRP1 promoter [Ward et al., Plant. Mol. Biol. 22 (1993) 361-366], promoters which can be induced by salicylic acid (International Publication No. 95/1919443), promoters which can be induced by benzenesulfonamide (European Patent No. 388186), induced by absic acid Promoter (European Patent No. 335528) or promoter which can be induced by ethanol or cyclohexanone (International Publication No. 9321334) is described in the literature and can be preferably used among many.

Another particularly preferred promoter is to ensure expression in tissues or plant organs that exhibit fungicidal activity that is harmful to plants. Of particular note are promoters that ensure leaf specific expression. Potato cytosol FBPase promoters or potato ST-LSI promoters are also mentioned (Stockhaus et al., EMBO J. 8 (1989) 2445-245).

Stable expression of single chain antibodies of up to 0.67% of the total soluble seed protein in the seed of transgenic tobacco plants was made possible by species specific promoters (Fiedler and Conrad, Bio / Technology 10 (1995) 1090-1094). Since expression may be possible in seeds that are in the process of sowing or germination and may be necessary for the purposes of the present invention, such germination and species specific promoters are also preferred regulatory elements in accordance with the present invention. Thus, expression cassettes according to the invention may contain, for example, species specific promoters (preferably USP or LEB4 promoters), LEB4 signal peptides, genes to be expressed and ER bearing signals. The structure of the cassette is illustrated in the diagrammatic form of FIG. 1 with reference to single chain antibodies (scFv gene).

Expression cassettes according to the invention are described, for example, in T. Maniatis, E.F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989), and also T.J. Silhavy, M.L. Berman and L.W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) and Ausubel, F.M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience (1987), using conventional recombination and cloning techniques, a suitable promoter can be used to encode a suitable polypeptide DNA, and preferably a chloroplast specific transient peptide and be inserted between the promoter and the polypeptide DNA, And by fusion with a polyadenylation signal.

Especially preferred are those present in the forming diaphragm with apoplasts, pigments, vacuoles, plasma membranes, mitochondria, endoplasmic reticulum (ER), or by the absence of suitable operating sequences, ie, targeting to cytosol [Kermode, Crit. Rev. Plant Sci. 15, 4 (1996) 285-423. ER and accumulation in cell walls have proved particularly advantageous for quantitative 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 an expression cassette encoding a portion of the fungicide binding fusion protein, a fusion protein, which is a transient peptide whose coding sequence regulates the translocation of the polypeptide. Particularly preferred are chloroplast specific transient peptides which are enzymatically degraded from fungicide binding polypeptide residues after the fungicide binding polypeptide is transferred to the chloroplasts of the plant. Particularly preferred are the temporal peptides derived from the pigmentary transketolase (TK) or functional equivalents of these temporal peptides (e.g., temporal peptides of small subunits of Rubisco or peredoxin NADP redox enzymes).

The polypeptide DNA or polypeptide cDNA required for the production of the expression cassette according to the invention is preferably amplified by polymerase chain reaction (PCR). DNA amplification methods using PCR are known, for example, from Innis et al., PCR Protocols, A Guide to Methods and Applications, Academic Press (1990). PCR generated DNA fragments can be conveniently checked by sequencing to avoid polymerase errors in the construct to be expressed.

An inserted nucleotide sequence, which encodes a fungicide binding polypeptide, may be produced synthetically or obtained naturally, or may comprise a mixture of synthetic and natural DNA components. In general, synthetic nucleotide sequences are made with the desired codons in plants. Preferred codons for plants are the most common in their protein and can be determined from codons expressed in most of the plant species. When preparing an expression cassette, several DNA fragments can be manipulated to obtain nucleotide sequences that are read with the correct sense for convenience and with the correct reading frame. To connect the DNA fragments to each other, an adapter or linker may be added to the fragments.

The promoter and terminator regions according to the invention should be equipped with a linker or polylinker comprising one or more restriction sites for insertion of this sequence in the sense of transcription. In general, the linker has 1 to 10 restriction sites, generally 1 to 8, preferably 2 to 6 sites. Within the regulatory region, linkers are generally less than 100 bp in size, sometimes less than 60 bp and more than 5 bp. Promoters according to the invention may be natural or homologous or heterologous to the host plant or heterologous. The expression cassette according to the invention comprises within the 5'-3'-sense of transcription the promoter according to the invention, certain sequences and regions for transcription termination. The various terminating areas are interchangeable as desired.

In addition, manipulations to provide suitable restriction sites or to remove excess DNA or restriction sites can be used. In in vitro mutations, “primer repair”, restriction or ligation may be employed where insertion, deletion or substitution, eg metastasis and conversion, is possible. In the case of suitable manipulations such as cleavage by restriction enzymes for the "blunt ends", "chewing-back" or filling-up, the complementary ends of the fragments may be provided for ligation purposes. have.

Of particular importance in achieving the object of the present invention is the attachment of the specific ER retention signal SEKDEL, which results in a three to four fold average expression level [Schuoten, A. et al. Plant Mol. Biol. 30 (1996) 781-792. Other retention signals naturally occurring in plant and animal proteins located in the ER can also be used to construct cassettes.

Preferred polyadenylation signals correspond essentially to plant polyadenylation signals, preferably T-DNA polyadenylation signals from Agrobacterium tumefaciens, in particular gene 3 (Octo) of T-DNA of Ti plasmid pTiACH5. Pin synthase) (see Gilen et al., EMBO J. 3 (1984) 835 hereinafter) or a functional equivalent thereof.

Expression cassettes according to the invention may comprise, for example, a constitutive promoter (preferably a CaMV 35S promoter), a LeB4 signal peptide, a gene to be expressed and an ER bearing signal. The construct of the cassette is shown in the diagram of FIG. 2 for the single chain antibody (scFv gene). The amino acid sequence KDEL (lysine, aspartic acid, glutamic acid, leucine) is preferably used as the ER retention signal.

The fused expression cassette encoding the polypeptide having fungicidal binding properties is preferably cloned into a vector suitable for transforming Agrobacterium tumefaciens, for example pBin19. Agrobacteria transformed with such vectors are known methods for transforming plants, in particular crops, for example tobacco plants, for example soaking a wounded leaf or leaf fragment in an Agrobacteria solution and then propagating it in a suitable medium. Can be used. Transformation of plants by Agrobacteria is described in F.F. White, Vectors for Gene Transfer in Higher Plants; in Transgenic Plants, Vol. 1, Engineering and Utilization, edited by S.D. Kung and R. Wu, Academic Press, 1993, pp. 15-38, and S.B. Gelvin, Molecular Genetics of T-DNA Transfer from Agrobacterium to Plants, also in Transgenic Plants, pp. 49-78. Transgenic plants can be regenerated from transformed cells of wound leaves or leaf fragments by known methods, and these transgenic plants are capable of expressing the polypeptide having fungicidal binding properties, integrated in the expression cassette according to the invention. Contains the gene for

In order to transform a host plant with DNA encoding a fungicide binding polypeptide, the expression cassette according to the invention can be applied to a recombinant vector whose vector DNA contains further functional regulatory signals, eg sequences for replication or integration. It is incorporated as an insert. Suitable vectors are described in "Methods in Plant Molecular Biology and Biotechnology" (CRC Press), (1993) chapter 6/7, pp 71-119.

Using the recombination and cloning techniques cited above, the expression cassettes according to the invention are for example E. coli. It can be cloned into a suitable vector to propagate it in Collie. Suitable cloning vectors are in particular pBR332, pUC series, M13mp series and pACYC184. Especially suitable is this. Binary vectors capable of replicating both coli and Agrobacteria, for example pBin19. [Bevan et al. (1980) Nucl. Acids Res. 12, 8711].

The invention also relates to the use of the expression cassette according to the invention for transforming a plant, plant cell, plant tissue or plant organ. A preferred goal in use is to mediate resistance to fungicides with harmful activity to plants.

Depending on the choice of promoter, expression can occur specifically in leaves, in seeds or in other plant organs. Such transgenic plants, their proliferating materials and their plant cells, plant tissues or plant organs are another subject of the invention.

The transfer of heterologous genes to the genome of a plant is called transformation. In this process, the method of transforming and regenerating a plant from plant tissue or plant cells is used for transient or stable transformation. Suitable methods include protoplast transformation by polyethylene glycol induced DNA uptake, infrared radiation using gene guns, electroporation, incubation of dry embryos in DNA-containing solutions, microinjection and Agrobacterium mediated gene transfer It is a law. These methods described above are described, for example, in B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, editors: S.D. Kung and R. Wu, Academic Press (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 the transformation of Agrobacterium tumefaciens, eg pBin19 [Bevan et al., Nucl. Acids Res. 12 (1984) 8711].

Agrobacteria transformed with the expression cassettes according to the invention are plants, in particular crops, for example cereals, corn, soybeans, rice, cotton, sugar beets, canola, sunflower, flax, potatoes, tobacco, tomatoes, oilseed rape, often Canis, lettuce and various shrubs, trees, nuts and Vitis species, for example fruit trees such as coffee, apples, pears or cherries, nut trees such as hodo or pecans, and more importantly, to transform vines In a known manner, it can be used, for example, by soaking a wounded leaf or leaf fragment in an Agrobacteria solution and then propagating it in a suitable medium.

Functionally equivalent sequences encoding fungicide binding polypeptides according to the present invention are sequences that still have the desired function despite other nucleotide sequences. Thus, functional equivalents include naturally occurring variants of the sequences described herein, and also artificial nucleotide sequences, such as artificial nucleotide sequences obtained by chemical synthesis and suitable for plant codon use.

In particular, functional equivalents should be understood as natural or artificial mutations of the original isolated sequence encoding the fungicide binding polypeptide, which continues to exhibit the desired function. Mutations include substitution, addition, deletion, exchange or insertion of one or more nucleotide residues. Thus, the present invention also encompasses nucleotide sequences obtained by modifying this nucleotide sequence. The purpose of such modifications can be, for example, further restriction of the coding sequence contained therein, or the insertion of more cleavage sites, for example for restriction enzymes.

Another functional equivalent is a variant whose function is less or more pronounced than the starting gene or gene fragment.

In addition, artificial DNA sequences are suitable as long as they induce the desired resistance to fungicides as described above. Such artificial DNA sequences can be identified, for example, by reverse translation of proteins having fungicidal binding activity and constructed by molecular modeling or by in vitro selection. Particularly suitable are coding DNA sequences obtained by reverse translation of polypeptide sequences in accordance with codon usage specific to the host plant. Specific codon usage can be readily determined by one skilled in the method of plant genetics by computer-assisted assessment of other known genes of the plant to be transformed.

Another suitable equivalent nucleic acid sequence according to the invention which should be mentioned is a sequence encoding a fusion protein, some of which are non-plant derived fungicide binding polypeptides or functionally equivalent parts thereof. For example, the second portion of the fusion protein may be another polypeptide having enzymatic activity, or an antigen polypeptide sequence that allows detection of scFvs expression (eg myc-tag or his-tag). However, it is preferably a regulatory protein sequence, such as a signal or a transient peptide, that directs a polypeptide having fungicidal binding properties to the desired site of action.

However, the present invention also relates to fusion proteins of expression products produced according to the invention and polypeptides having transient peptide and fungicide binding properties.

Resistance / resistant for the purposes of the present invention means the artificially obtained ability of plants to withstand the action of plant enzyme inhibitors. It includes partial and especially complete insensitivity to these inhibitors for one or more periods of plant manufacture.

Since the primary site of fungicide action is generally leaf tissue, leaf specific expression of exogenous fungicide binding polypeptides may provide sufficient protection. However, those skilled in the art will readily understand that the action of fungicides is not limited to leaf tissue and may be exerted in all remaining organs of the plant in a tissue specific manner.

In addition, constitutive expression of exogenous fungicide binding polypeptides is advantageous. On the other hand, induced expression may be preferred.

The efficacy of polypeptides expressed by transduction with fungicidal binding properties can be confirmed in vitro, for example, by sprouting meristem growth on a series of fungicide containing media with different concentrations, or via seed germination tests. . Fungicide resistance of test plants, which varied with the type and concentration of test plants, can be tested in greenhouse experiments.

The invention also relates to transgenic plants transformed with the expression cassettes according to the invention and transgenic cells, tissues, organs and proliferating materials of such plants. Particularly preferred are transgenic crops such as cereals, corn, soybeans, rice, cotton, sugar beets, canola, sunflower, flax, potatoes, tobacco, tomatoes, oilseed rape, alfalfa, lettuce and various shrubs, trees, nuts and Vitis species, for example, fruit trees such as coffee, apples, pears or cherries, nut trees such as hodo or pecans, and more importantly vines.

Transgenic plants, plant cells, plant tissues or plant organs can be treated with fungicides with harmful activity that inhibits plant enzymes, thereby killing plants, plant cells, plant tissues or plant organs that have not been successfully transformed or Damage. Examples of suitable active ingredients are especially methyl methoxyimino-α- (o-tolyloxy) -o-tolylacetate (BAS 490F) and metabolites and functional derivatives of these compounds. DNA encoding a polypeptide having fungicidal binding properties and inserted into an expression cassette according to the invention may also be used as a selection marker.

The present invention provides that such inhibitors can be used to inhibit harmful fungi without harming the plant even at high application rates, as long as the selected resistance of the crop to fungicides with harmful activity to the plant is induced, especially in the case of crops. Has an advantage. Fungicide compounds from the following groups may be mentioned as examples of such inhibitors, but are not limited to:

Sulfur, dithiocarbamates and derivatives thereof such as dimethyldithiocarbamate iron (III), dimethyldithiocarbamate zinc, ethylenebisdithiocarbamate zinc, ethylenebisdithiocarbamate manganese, ethylenediamine Manganese zinc bisdithiocarbamate, tetramethylthiuram disulfide, ammonia complex of (N, N-ethylenebisdithiocarbamic acid) zinc, ammonia complex of (N, N'-propylenebisdithiocarbamic acid) zinc, (N, N'-propylenebisdithiocarbamic acid) zinc, N, N'-polypropylenebis (thiocarbamoyl) disulfide;

Nitro derivatives such as dinitro (1-methylheptyl) phenyl crotonate, 2-sec-butyl-4,6-dinitrophenyl-3,3-dimethylacrylate, 2-sec-butyl-4 , 6-dinitrophenyl isopropyl carbonate, diisopropyl 5-nitroisophthalate;

Heterocyclic materials such as 2-heptadecyl-2-imidazoline acetate, 2,4-dichloro-6- (o-chloroaniline) -s-triazine, O, O-diethyl phthalimidofos Phonothioate, 5-amino-1- [bis (dimethylamino) phosphonyl] -3-phenyl-1,2,4-triazole, 2,3-dicyano-1,4, -dithioanthraquinone, 2-thio-1,3-dithiolo [4,5-b] quinoxaline, methyl-1- (butylcarbamoyl) -2-benzimidazolecarbamate, 2-methoxycarbonylaminobenzimi Dazole, 2- (2-furyl) benzimidazole, 2- (4-triazolyl) benzimidazole, N- (1,1,2,2-tetrachloroethylthio) tetrahydrophthalimide, N-trichloro Romethylthiotetrahydrophthalimide, N-trichloromethyltyrophthalimide,

N-dichlorofluoromethylthio-N ', N'-dimethyl-N-phenylsulfodiamide, 5-ethoxy-3-trichloromethyl-1,2,3-thiadiazole, 2-thiocyane Itomethylthiobenzothiazole, 1,4-dichloro-2,5-dimethoxybenzene, 4- (2-chlorophenylhydrazono) -3-methyl-5-isoxazolone, pyridine-2-thio-1- Oxide, 8-hydroxyquinoline or a copper salt thereof, 2,3-dihydro-5-carboxanilideo-6-methyl-1,4-oxathiin, 2,3-dihydro-5-carbox Anilido-6-methyl-1,4-oxatiin, 4,4-dioxide, 2-methyl-5,6-dihydro-4H-pyran-3-carboxanilide, 2-methylfuran-3- Carboxanilide, 2,5-dimethylfuran-3-carboxanilide, 2,4,5-trimethylfuran-3-carboxanilide, N-cyclohexyl-2,5-dimethylfuran-3-carboxamide, N-cyclohexyl-N-methoxy-2,5-dimethylfuran-3-carboxamide, 2-methylbenzanilide, 2-iodobenzanilide, N-formyl-N-morpholine-2,2, 2-trichloro Tyl Acetal, Piperazine-1,4-diylbis-1- (2,2,2-trichloroethyl) formamide, 1- (3,4-dichloroanilino) -1-formylamino-2,2 , 2-trichloroethane;

Amines such as 2,6-dimethyl-N-tridecylmorpholine or salts thereof, 2,6-dimethyl-N-cyclododecylmorpholine or salts thereof, N- [3- (pt-butylphenyl ) -2-methylpropyl] -cis-2,6-dimethylmorpholine, N- [3- (pt-butylphenyl) -2-methylpropyl] piperidine, (8- (1,1-dimethylethyl) -N-ethyl-N-propyl-1,4-dioxaspiro [4.5] decene-2-methanamine,

Azoles, for example 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-trichlorophenoxyethyl) -N'-imidazolylurea, 1- (4-chlorophenoxy) -3,3-dimethyl-1- (1H-1,2, 4-triazol-1-yl) -2-butanone, 1- (4-chlorophenoxy) -3,3-dimethyl-1- (1H-1,2,4-triazol-1-yl)- 2-butanol, (2RS, 3RS) -1- [3- (2-chlorophenyl) -2- (4-fluorophenyl) -oxiran-2-ylmethyl] -1 H-1,2,4-tria Sol, 1- [2- (2,4-dichlorophenyl) -pentyl] -1 H-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-triazole, 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-triazol-1-yl) -butan-2-ol, (+)-4- Chloro-4- [4-methyl-2- (1H-1,2,4-triazol-1-ylmethyl) -1,3-dioxolan-2-yl] phenyl 4-chlorophenyl ether, (E) -(R, S) -1- (2,4-dichlorophenyl) -4,4-dimethyl-2- (1H-1,2,4-triazol-1-yl) -1-penten-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-1-yl) quinazolin-4 (3H) -one, (R, S) -2- (2,4-dichlorophenyl) -1H-1,2, 4-triazol-1-yl) hexan-2-ol, (1RS, 5RS; 1RS, 5SR) -5- (4-chlorobenzyl) -2,2-dimethyl-1- (1H-1,2,4 -Triazol-1-ylmethyl) cyclopentanol), (R, S) -1- (4-chlorophenyl) -4,4-dimethyl-3- (1H-1,2,4-triazole-1 -Ylmethyl) pentan-3-ol, (+)-2- (2,4-dichlorophenyl) -3- (1H-1,2,4-triazolyl) propyl-1,1,2,2-tetra Fluoroethyl ether, (E) -1- [1- [4-chloro-2-trifluoromethyl) phenyl] imino) -2-propoxyethyl] -1H-imidazole, 2- (4-chloro Phenyl) -2- (1H-1,2,4-triazol-1-ylmethyl) hexanonitrile, α- (2-chlorofe ) -α- (4-chlorophenyl) -5-pyrimidinmethanol, 5-butyl-2-dimethylamino-4-hydroxy-6-methylpyrimidine, bis (p-chlorophenyl) -3-pyrimidinemethanol , 1,2-bis (3-ethoxycarbonyl-2-thioureido) benzene, 1,2-bis (3-methoxycarbonyl-2-thioureido) benzene,

Strobilurines, for example methyl E-methoxyimino- [α- (o-tolyloxy) -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, for example N- (4,6-ditylpyrimidin-2-yl) aniline, N- [4-methyl-6- (1-propynyl) pyrimidin-2-yl] aniline , N- [4-methyl-6-cyclopropylpyrimidin-2-yl] aniline,

Phenylpyrrole, for example 4- (2,2-difluoro-1,3-benzodioxol-4-yl) pyrrole-3-carbonitrile,

Cinnamic amides such as 3- (4-chlorophenyl) -3- (3,4-dimethoxyphenyl) acryloyl morpholine, and

Various fungicides, for example dodecylguanidine acetate, 3- [3- (3,5-dimethyl-2-oxycyclohexyl) -2-hydroxyethyl] glutarimidide, N-methyl-, N- Ethyl- (4-trifluoromethyl-2- [3 ', 4'-dimethoxyphenyl] benzamide, hexachlorobenzene, methyl N- (2,6-dimethylphenyl) -N- (2-furoyl) -DL-alanine, 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-dimethylphenyl) -N- (phenylacetyl) alanine methyl ester, N- (2,6-dimethylphenyl) -N-chloroacetyl- D, L-2-aminobutyrolactone, DL-N- (2,6-dimethylphenyl) -N- (phenylacetyl) alanine methyl ester, 5-methyl-5-vinyl-3- (3,5-dichloro Phenyl) -2,4-dioxo-1,3-oxazolidine, 3- [3,5-dichlorophenyl (5-methyl-5-methoxymethyl] -1,3-oxazolidine-2,4 -Dione, 3- (3,5-dichlorophenyl) -1-isopropylcarbamoylhydantoin, N- (3,5-dichloro Phenyl) -1,2-dimethylcyclopropane-1,2-dicarboxyimide, 2-cyano- [N- (ethylaminocarbonyl) -2-methoxyimino] acetamide, N- (3-chloro- 2,6-dinitro-4-trifluoromethylphenyl) -5-trifluoromethyl-3-chloro-2-aminopyridine.

The range of action of functionally similar derivatives of fungicides is essentially similar to that of named substances, while the detrimental activity on plants is lower, the same or higher.

The invention is now illustrated by, but not limited to, the following examples.

<General Cloning Method>

Cloning steps performed within the scope of the present invention, eg, cleavage by restriction enzymes, agarose gel electrophoresis, purification of DNA fragments, delivery of nucleic acids to nitrocellulose and nylon membranes, ligation of DNA fragments, e. Transformation of coli cells, bacterial cultures, phage proliferation and sequencing of recombinant DNA are described in 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 obtained from Stratagene. Agrobacterial strains (C58C1 with Agrobacterium tumefaciens, plasmid pGV2260 or pGV3850kan) used for plant transformation are described in Deblaere et al. (Nucl. Acids Res. 13 (1985) 4777). In addition, Agrobacteria strain LBA4404 (Clontech) or other suitable strain may be used. Vectors pUC19 (Yanish-Perron, Gene 33 (1985), 103-119), pBluescript SK- (Stratagene), pGEM-T (Promega), pZerO (Invitrogen), pBin19 (Bevan et al., Nucl. Acids Res. 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

Recombinant DNA molecules were sequenced using Pharmacia's laser fluorescence DNA sequencing device using Sanger's method [Sanger et al., Proc. Natl. Acad. Sci. USA 74 (1977) 5463-5467.

<Formation of Plant Expression Cassette>

The 35S CaMV promoter was plasmid pBin19 in the form of EcoRI-KpnI fragment (corresponding to nucleotides 6909-7437 of Cauliflower mosaic virus [Franck et al. Cell 21 (1980) 285)] [Bevan et al., Nucl. Acids Res. 12, 8711 (1984). Polyadenylation signal of gene 3 of T-DNA of Ti plasmid pTiACH5 (Gielen et al., EMBO J. 3 (1984) 835), nucleotides 11749-11939 in the form of PvuII-HindIII fragments and after addition of SphI linker It was cloned into the PvuII cleavage site between the SphI-HindIII cleavage sites of the vector. This gave plasmid pBinAR (Hofgen and Willmitzer, Plant Science 66 (1990) 221-230).

<Use example>

<Example 1>

Because fungicides are not immunogenic, they must be coupled to a carrier material, for example KLH. If the molecule contains a reactive group, the coupling can be carried out directly, otherwise the functional group is introduced when a fungicide is synthesized or a reactive precursor is selected during synthesis to couple the molecule to the carrier molecule in a simple reaction step. do. Examples of coupling reactions can be found in Miroslavic Ferencik in "Handbook of Immunochemistry", 1993, Chapman & Hall, in the chapter Antigens, pages 20-49.

Repeated infusion of this modified carrier molecule (antigen) is used to immunize Balb / c mice, for example. As long as a sufficient number of antibodies bound to the antigen can be detected by ELISA (enzyme labeled immunoassay), spleen cells of the animal are removed and fused with myeloma cells for culturing the hybrid. "Fungicide modified BSA" is also used as an antigen in ELISA to distinguish the immune response against hapten from the KLH response.

Monoclonal antibodies are described, for example, in "Practical Immunology", Leslie Hudson and Frank Hay, Blackwell Scientific 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 Moller and Franz Emling "Monoklonale Antikorper gegen TNF und deren Verwendung" [Monoclonal antibodies against TNF, and their use]. European Patent Publication No. 260610.

<Example 2>

The starting point for this study was a monoclonal antibody that specifically recognized the fungicide BAS 490F and also had a high binding affinity. Selected hybridoma cell lines are characterized by the high affinity of the secreted monoclonal antibodies against the fungicide antigen BAS 490F and the availability of specific sequences of immunoglobulins [Berek, C. et al., Nature 316, (1985). 412-418]. This monoclonal antibody against BAS 490F was the starting point for the construction of single chain antibody fragments (scFv-antiBAS 490F).

First, mRNA was isolated from hybridoma cells and transcribed into cDNA. This cDNA served as a template for the amplification of the variable immunoglobulin genes VH and VK by 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 (1991) 624-628. Isolated variable immunoglobulin was the starting point for the construction of single chain antibody fragments (scFv-antiBAS 490F). In the following fusion PCR, three component VH, VK and linker fragments were combined in a PCR reaction and amplified scFv-antiBAS 490F (FIG. 3).

Functional characterization (antigen binding activity) of the constructed scFv-antiBAS 490F gene was performed after expression in the bacterial system. For this purpose, scFv-antiBAS 490F is described by Hoogenboom, H.R. et al., Nucleic Acids Research, 19 (1991) 4133-4137 using E. coli as a soluble antibody fragment. Synthesized in Collie. The activity and specificity of the constructed antibody fragments were checked by ELISA assay (FIG. 4).

To enable seed specific expression of antibody fragments in tobacco, the scFv-antiBAS 490F gene was cloned down the LeB4 promoter. The LeB4 promoter, isolated from Vicia faba, accurately shows seed specific expression of several heterologous genes in tobacco [Baumlein, H. et al., Mol. Gen. Genet. 225 (1991) 121-128]. Transfer of the scFv-antiBAS 490F polypeptide to the endoplasmic reticulum resulted in stable accumulation of large amounts of antibody fragments. To this end, the scFv-antiBAS 490F gene was fused with a signal peptide sequence to ensure influx into the endoplasmic reticulum and also with the ER bearing signal SEKDEL allowing the polypeptide to remain in the ER [Wandelt et al., 1992] (FIG. 5). .

The constructed expression cassette was cloned into binary vector pGSGLUC 1 (Saito et al., 1990) and transferred to Agrobacterium strain EHA 101 by electroporation. Recombinant Agrobacterium clones were used for the following transformation of Nicotiana tabacum. 70-140 tobacco plants were regenerated per building. Seeds of different developmental stages were harvested from transgenic tobacco plants that were regenerated and then regenerated. After extraction, soluble proteins were obtained from these seeds in an aqueous buffer system. Analysis of transgenic plants demonstrates that fusion of the scFv-antiBAS 490F gene to the DNA sequence of the ER bearing signal SEKDEL allows for the maximum accumulation of 1.9% scFv-antiBAS 490F protein to be obtained in mature seeds.

The constructed scFv-antiBAS 490F gene was about 735 bp in size. The variable domains were fused to each other in the sequences VH-L-VL.

Direct ELISA was used to confirm specific selectivity in extracts of mature tobacco seeds. The values obtained clearly demonstrate that the protein extract contains functionally active antibody fragments.

<Example 3>

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

The starting point for this study was the single chain antibody fragment (scFv-antiBAS 490F) against the fungicide BAS 490F. Functional characterization (antigen binding activity) of this scFv-antiBAS 490F gene was performed following expression in the bacterial system and also in tobacco leaves. The activity and specificity of the constructed antibody fragments were checked by ELISA assay.

To enable seed specific expression of antibody fragments in tobacco, the scFv-antiBAS 490F gene was cloned down the USP promoter. The USP promoter, isolated from Vicia faba, accurately represents seed specific expression of several heterologous genes in tobacco [Fiedler, U. et al., Plant Mol. Biol. 22 (1993) 669-679. Transfer of the scFv-antiBAS 490F polypeptide to the endoplasmic reticulum resulted in stable accumulation of large amounts of antibody fragments. To this end, the scFv-antiBAS 490F gene is fused with a signal peptide sequence that ensures entry into the endoplasmic reticulum and also with the ER bearing signal SEKDE, which allows the polypeptide to remain in the ER [Wandelt et al., 1992] (FIG. 1). ).

The constructed expression cassette was cloned into binary vector pGSGLUC 1 [Saito et al., 1990] and transferred to Agrobacterium strain EHA 101 by electroporation. Recombinant Agrobacterium clones were used for subsequent transformation of Nicotiana tabacum. Seeds at different stages of growth were harvested from transgenic tobacco plants following self-pollination. After extraction, soluble proteins were obtained from these seeds in an aqueous buffer system. Analysis of the transgenic plants revealed that the fusion of the scFv-antiBAS 490F gene to the DNA sequence of the ER-bearing signal SEKDEL under the control of the USP promoter ensured that the single-chain antibody fragment with binding affinity for BAS 490F was already synthesized on day 10 of seed development. Prove it.

<Example 4>

To achieve ubiquitous expression of antibody fragments in plants, especially in leaves, the scFv-antiBAS 490F gene was cloned down the CaMV 35S promoter. This strong constitutive promoter mediates the expression of heterologous genes in practically all plant tissues (Benfey and Chua, Science 250 (1990) 956-966). Transfer of the scFv-antiBAS 490F protein to the endoplasmic reticulum ensures that stable accumulation of large amounts of antibody fragments is obtained from the leaf material. First, the scFv-antiBAS 490F gene is fused to a signal peptide sequence that ensures influx into the endoplasmic reticulum, and to an ER-bearing signal KDEL that allows the product to remain in the ER [Wandelt et al., Plant J. 2 (1992). 181-192]. The constructed expression cassette was cloned into binary vector pGSGLUC 1 [Saito et al., Plant Cell Rep. 8 (1990) 718-721], and transferred to Agrobacterium strain EHA 101 by electroporation. Recombinant Agrobacteria clones were used for subsequent transformation of Nicotiana tabacum. About 100 tobacco plants were regenerated. Leaf material at various stages of development was removed from the regenerated transgenic tobacco plants. After extraction, soluble protein was obtained from the leaf material in an aqueous buffer system. Subsequent analysis (Western blot analysis and ELISA analysis) demonstrated that a maximum accumulation of more than 2% of the biologically active antigen binding scFv-antiBAS 490F polypeptide was obtained in the leaves. Although high expression values were identified in fully grown green leaves, antibody fragments were also detected in aging leaf material.

<Example 5>

PCR amplification of fragments of cDNA encoding single chain antibodies against BAS 490F by synthetic oligonucleotides

PCR amplification of the single chain antibody cDNA was performed in a DNA heat cycler from Perkin Elmer. The reaction mixture consists of 8 ng / μl single strand template cDNA, 0.5 μM of related oligonucleotide, 200 μM nucleotide (Pharmacia), 50 mM KCl, 10 mM Tris-HCl (pH 8.3 at 25 ° C., 1.5 mM MgCl ₂ ) and 0.02 U / Μl Taq polymerase (Perkin Elmer). The amplification conditions were set as follows:

Annealing Temperature: 45 ℃

Denaturation temperature: 94 ℃

Elongation Temperature: 72 ℃

Number of cycles: 40

The resultant is a fragment of about 735 base pairs, which was ligated into the vector pBluescript. Ligation mixture. Coli XL-I Blue was used to transform and plasmids were amplified. See Innis et al., 1990, PCR Protocols, A Guide to Methods and Applications, Academic Press, for the use and optimization of polymerase chain reactions.

<Example 6>

Preparation of Transgenic Tobacco Plants Expressing cDNA Encoding Single Chain Antibodies Having Fungicide Binding Properties

Plasmid pGSGLUC 1 was transformed with Agrobacterium tumefaciens C58C1: pGV2260. To transform tobacco plants (Nicotiana tabacum cv.Samsun NN), the Agrobacterium colonies transformed with the amount in Murashige-Skoog medium (2MS medium) containing 2% sucrose. A 1:50 dilution of overnight culture was used. In Petri dishes, discoidal leaves of sterile plants (about 1 cm 2 each) were incubated for 5-10 minutes in a 1:50 Agrobacterial dilution. Thereafter, the cells were incubated for 2 days in a dark place at 25 ° C. on 2MS medium containing 0.8% bacto-agar. Incubation was continued after 2 days in 16 h light / 8 h dark and 500 mg / l claporan (Chemtaxam-sodium), 50 mg / l kanamycin, 1 mg / l benzylaminopurine (BAP), 0.2 mg / l The cycle was repeated periodically weekly on MS medium containing naphthylacetic acid and 1.6 g / L glucose. Growing shoots were transferred to MS medium containing 2% sucrose, 250 mg / L claphoran and 0.8% bacto-agar.

<Example 7>

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

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

To enable the expression of the scFv-antiBAS 490F gene in the endoplasmic reticulum, the heterologous gene is controlled under the control of the CaMV 53S promoter as a translational fusion with the LeB4 signal peptide (N-terminus) and the ER bearing signal KDEL (C-terminus). Expression. Transfer of the scFv-antiBAS 490F polypeptide to the endoplasmic reticulum allowed stable accumulation of large amounts of active antibody fragments. After harvesting the leaf material, the sections were frozen (1), lyophilized (2) or dried at room temperature (3). Soluble protein was obtained from the leaf material in question by extraction in aqueous buffer and the scFv-antiBAS 490F polypeptide was purified by affinity chromatography. The same amount of purified scFv-antiBAS 490F polypeptide (freeze, lyophilized and dried) was used to measure the activity of antibody fragments (FIG. 6). 6A shows the antigen binding activity of scFv-antiBAS 490F polypeptides obtained from fresh (1), lyophilized (2) and dried (3) leaves. FIG. 6B shows each amount (about 100 ng) of scFv-antiBAS 490F protein, used in ELISA assay and confirmed by Western blot analysis. The size of the protein molecular weight reference is shown on the left. The antigen binding activity was found to be nearly identical.

<Example 8>

To demonstrate the fungicide resistance of transgenic tobacco plants that produce polypeptides with fungicide binding properties, the tobacco plants were treated with varying amounts of BAS 490F. In all cases, it could be demonstrated that plants expressing scFv-antiBAS 490F in greenhouses each exhibited resistance to the fungicides according to the invention comparable to controls.

Claims (28)

A method for producing fungicide resistant plants by expression of exogenous fungicide binding polypeptides in plants. The method of claim 1, wherein the exogenous fungicide binding polypeptide is a single chain antibody fragment. The method of claim 1, wherein the exogenous fungicide binding polypeptide is a complete antibody or fragment derived therefrom. The method of claim 1 wherein the fungicide is methyl methoxyimino-α- (o-tolyloxy) -o-tolylacetate (BAS 490F). The method according to any one of claims 1 to 3, wherein the plant is a monocotyledonous or dicotyledonous plant. The method of claim 5 wherein the plant is tobacco. 7. The method of claim 1, wherein the exogenous polypeptide is constitutively expressed in a plant. The method of claim 1, wherein the expression of exogenous polypeptides in a plant is induced. The method according to any one of claims 1 to 6, wherein the exogenous polypeptide is expressed in the leaves of the plant. The method according to any one of claims 1 to 6, wherein the exogenous polypeptide is expressed in seeds of the plant. An expression cassette for use in a plant, comprising a promoter, a signal peptide, a gene encoding the expression of an exogenous fungicide binding polypeptide, an ER retention signal and a terminator. The expression cassette of claim 11, wherein the constitutive promoter used is a CaMV 35S promoter. The expression cassette of claim 11, wherein the gene to be expressed is a gene of a single chain antibody fragment. The expression cassette of claim 11, wherein a gene or gene fragment of a fungicide binding polypeptide in the form of a translational fusion with other functional proteins, such as enzymes, toxins, chromophores and binding proteins, is used as the gene to be expressed. The expression cassette of claim 11, wherein the polypeptide gene to be expressed is obtained from a hybridoma cell or by other recombinant methods, such as antibody phage display methods. Use of an expression cassette according to claim 11 for the transformation of a dicotyledonous or monocotyledonous plant which specifically expresses an exogenous fungicide binding polypeptide in a seed or leaf. 17. The use according to claim 16, wherein the expression cassette is transferred to a bacterial strain and the recombinant clones formed are used for transformation of a dicotyledonous or monocotyledonous plant that specifically expresses an exogenous fungicide binding polypeptide to the seed or leaf. . Use of the expression cassette according to claim 11 as a selection marker. Use of the transformed plant obtained according to claim 17 or 18 for the preparation of fungicide binding polypeptides. A method of transforming a plant, characterized by introducing a gene sequence encoding a fungicide binding polypeptide into plant cells, callus tissue, intact plants and protoplasts of the plant cells. The method of claim 20, wherein the transformation is carried out by the genus Agrobacterium, in particular Agrobacterium tumefaciens. The method of claim 20, wherein the transformation is performed by electroporation. The method of claim 20, wherein the transformation is performed by particle bombardment. A method for producing a fungicide-binding polypeptide, comprising expressing a gene encoding a fungicide-binding polypeptide in a plant or plant cell, and then isolating the polypeptide. A plant comprising the expression cassette according to claim 11 conferring resistance to fungicides. The plant of claim 25, which is resistant to methyl methoxyimino-α- (o-tolyloxy) -o-tolylacetate (BAS 490F). A method of inhibiting phytopathogenic fungi in a transgenic fungicide resistant crop comprising using a fungicide against a transgenic fungicide resistant crop that forms a fungicide binding polypeptide or an antibody. A fungicide binding polypeptide or antibody having a high binding affinity for methyl methoxyimino-α- (o-tolyloxy) -o-tolylacetate (BAS 490F), prepared as described in claim 24.