DE19718251A1 - Expression of fungicide-binding polypeptides in plants to produce fungicide tolerance - Google Patents

Abstract

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

Description

The present invention relates to a method for manufacturing of fungicide-tolerant plants by expression of an exogenous Fungicide-binding polypeptides in plants or parts of plants. The invention further relates to the use of the the nucleic acids coding for a polypeptide, an antibody or parts of an antibody with a fungicide-binding property in transgenic plants and the one transformed in this way Plant yourself.

It is known that ge targets foreign genes in the genome of a plant nen. This process is called the transformation and the resulting Plants are called transgenic plants. Transgenes Plants are currently used in different biotechnological areas Areas used. Examples are insect-resistant plants (Vaek et al. Plant Cell 5 (1987), 159-169), virus resistant Plants (Powell et al. Science 232 (1986), 738-743) and ozonre resistant plants (Van Camp et al. BioTech. 12 (1994), 165-168). Examples of genetically engineered quality improvements are: Increasing the shelf life of fruit (Oeller et al. Science 254 (1991), 437-439), increase in starch production in potato knol len (Stark et al. Science 242 (1992), 419), change in Starch- (Visser et al. Mol. Gen. Genet. 225 (1991), 289-296) and Lipid composition (Voelker et al. Science 257 (1992), 72-74) and production of non-plant polymers (Poirer et al. Science 256: 520-523 (1992).

An important goal of plant molecular genetic work is the generation of herbicide tolerance. The herbicide tolerance is ge characterized by an increased in type or amount of contract of the plant or parts of plants compared to the applicator herbicide. This can be done in several ways will. The known methods are the use of a metabolis musgens such as B. the pat gene in connection with the glufosinate Resistance (WO 8705629) or one against the herbicide resi constant target enzyme as in the case of enolpyruvylshiki mat-3-phosphate synthase (WO 9204449), which is resistant to Glyphosate, as well as the use of a herbicide in cell and Ge weaving culture for the selection of tolerant plant cells and re cultivating resistant plants like Acetyl CoA carboxylase  Inhibitors described (US 5162602, US 5290696).

Antibodies are proteins that are part of the immune system. Everyone Antibodies have in common their spatial, globular structure, the Structure from light and heavy chain as well as their principal Ability to make molecules or parts of a molecular structure with high To bind specificity (Alberts et al., In: Molecular Biolo the cell, 2nd edition 1990, VCH Verlag, ISBN 3-527-27983-0, 1198-1237). Because of these properties, antibodies for diverse tasks used. A distinction is made between the users formation of antibodies in animal and human organisms, that they produce, the so-called in-situ applications and the ex-situ applications, d. H. the use of antibodies after isolation from the producing cells or organisms (Whitelam and Cockburn, TIPS Vol. 1, 8 (1996), 268-272).

The use of hybrid somatic cell lines (hybridomas) as Source for antibodies against certain antigens goes to Ar from Köhler and Milstein (Nature 256 (1975) 495-97). So-called monoclonal anti Manufacture bodies that have a uniform structure and generated by cell fusion. Spleen cells become one immunized mouse fused with cells of a mouse myeloma. So hybridoma cells are created that proliferate indefinitely. Soon the cells secrete specific antibodies against the Antigen with which the mouse had been immunized. The spleen cells deliver the ability to produce antibodies while the mye lom cells the unlimited viability and the continuous Contribute antibody secretion. Because every hybridoma cell is different as a clone derived from a single B cell, they all have it antibody molecules produced the same structure including the Antigen binding site. This method has the use of anti strongly promoted because now antibodies with a single, known specificity and a homogeneous structure unlimited To be available. Monoclonal antibodies are widely used in immunodiagnostics and as therapeutic agents.

The so-called phage display method has been around for a few years for the production of antibodies in which the immune system and the various immunizations in the animal can be avoided. Here the affinity and specificity of the antibody is measured in vitro Schneider (Winter et al., Ann. Rev. Immunol. 12 (1994), 433-455; Hoogenboom TIBTech Vol 15 (1997), 62-70). Gene segments that the contains coding sequence of the variable region of antibodies, d. H. the antigen binding site, with genes for the envelope pro tein of a bacteriophage fused. Then you infect bacteria  with phages containing such fusion genes. That arise the phage particles now have shells with the antibody-like Chen fusion protein, the antibody-binding domain after eating shows. Such a phage display library can be used Now isolate the phage that contains the desired antibody fragment contains and binds specifically to a specific antigen. Everyone like that Isolated phage creates a monoclonal, antigen-binding poly peptide that corresponds to a monoclonal antibody. The genes for the antigen binding site that is unique to each phage are isolated from the phage DNA and used for construction use complete antibody genes.

Antibodies have been used in the field of crop protection especially as an analytical tool ex-situ for qualitative and quantitative detection of antigens. This excludes the Detection of plant ingredients, herbicides or fungicides in Drinking water (Sharp et al. (1991) ACS Symp Ser., 446 (Pestic. Re sidues Food Saf.) 87-95), soil samples (WO 9423018) or in Plants or parts of plants as well as the use of antibodies as an aid for the purification of bound molecules.

The production of immunoglobulins in plants was first started by Hiatt et al., Nature, 342 (1989), 76-78. The The spectrum ranges from one-chain antibodies to multimeric se cretoric antibodies (J. Ma and Mich Hein, 1996, Annuals New York Academy of Sciences, 72-81).

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

An analogous approach is also used to ward off the infection of the plant have been used by nematodes (Rosso et al., Biochem Biophys Res Com, 220 (1996) 255-263). For a pharmaceutical application Examples are known that the antibody expression in situ in Use plants for oral immunization (Ma et al., Science 1995, 268: 716-719; Mason and Arntzen, Tibtech Vol 13 (1996), 388-392). Antibodies produced by the plant are thereby released Plants or parts of plants suitable for consumption via the Mouth, throat or digestive tract fed to the body and doomed effective immune protection. Would continue in plants already a single chain antibody against expresses the low molecular plant hormone abscisic acid and decreased plant hormone availability due to Absci  Acid binding observed in the plant (Artsaenko et al., Plant Journal 8 (1995) 754-750).

Chemical control of fungi in agronomically important Cultures continues the use of highly selective fungicides without phytotoxic effect ahead. The phytotoxic effects of fun For example, gicides can inhibit plants growth, reduced photosynthesis and associated with it based on reduced earnings. In some cases however, it is difficult to add fungicides with sufficient selectivity develop that in all major plant crops are settable and in no culture damage to the yield cause plant. The introduction of fungicide-resistant or - Tolerant crops can help solve this problem wear and new uses of fungicides in up cultures that cannot be treated or can only be treated with reduced yield open up.

The development of fungicide-resistant crops by Ge weaving or seed mutagenesis and natural selection are great zen set. On the one hand, the phytotoxic effect must already be be detectable in tissue culture and on the other hand only those can some plants are manipulated using tissue culture techniques, their regeneration to whole plants from cell cultures succeed. In addition, after mutagenesis and selection, crop plants can un show desired properties by partially repeated Backcrosses have to be eliminated again. Also would be the one bringing resistance to plants by crossing them Kind of limited.

For these reasons, the genetic engineering approach is one for the Resistance coding gene to isolate and ge in crops aims to transcend the classic breeding process gene.

The molecular biological development of herbicide-tolerant or Herbicide-resistant crops previously required that Mechanism of action of the herbicide in the plant is known and that Genes that confer resistance to the herbicide are found can. Many herbicides currently in commercial use by using an enzyme of an essential amino acid, lipid or Block pigment biosynthesis. By changing the genes of this Enzymes such that the herbicide is no longer bound can and by introducing these modified genes into culture planting can produce herbicide tolerance. Alternatively, you can for example enzymes analogous in nature, for example in micro  organisms are found that have a natural resistance to show over the herbicide. This resistance mediating gene will isolated from such a microorganism, in suitable Vek cloned and then after a successful transforma tion in herbicide-sensitive crops for expression ge brings (WO 96/38567).

The object of the present invention was to develop a new one like generally applicable, genetic engineering process for Generation of fungicide-tolerant transgenic plants.

This task was surprisingly solved by a process expression of an exogenous polypeptide, antibody or Share an antibody with fungicide binding properties in the plants.

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

A suitable antibody is first generated, which has the fun binds gicide. This can a. by immunizing a vertebra animals, mostly mouse, rat, dog, horse, donkey or goat with one Antigen. The antigen is a fungicidally active Connection that goes through a functional group to a higher one molecular carriers such as bovine serum albumin (BSA), chicken egg white (Ovalbumin), keyhole limpet hemocyanin (KLH) or other carriers coupled or associated. The immune response becomes after repeated application of antigen using common methods gene and so isolated a suitable antiserum. This approach was first produces a polyclonal serum, the antibody with under contains different specificities. For the targeted in-situ Ge need it is necessary for a single specific isolate gene sequence encoding monoclonal antibodies. To There are various ways of doing this. The first approach uses the fusion of antibody-producing cells with cancer cells to a hybridoma cell that constantly produces antibodies culture that ultimately by separating the contained clones a homogeneous, a defined monoclonal antibody produ ornamental cell line leads.

Such a monoclonal cell line becomes the cDNA for the Antibodies or parts of the antibody, the so-called one-chain anti body (single chain antibody - scFv) isolated. This cDNA Se sequences can then be cloned into expression cassettes and used for functional expression in prokaryotic and eukaryotic  Organisms, including plants, can be used.

It is also possible to use antibodies for phage display banks read, bind the fungicide molecules and catalytically into one Implement product with non-fungicidal properties. Methods of Production of 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 described. By cloning the gene of this catalytic anti body and its expression in a plant can in principle a fungicide-resistant plant can also be produced.

The invention particularly relates to expression cassettes, their coding sequence for a fungicide-binding polypeptide or encoded its functional equivalent, as well as their Use to make a fungicide-tolerant plant. The Nucleic acid sequence can z. B. a DNA or a cDNA Se be a quenz. For insertion into an expression according to the invention Cassette-suitable coding sequences are, for example, sol che which contain a DNA sequence from a hybridoma cell which encoded for a polypeptide with fungicide-binding properties and thus ver the host resistance to certain fungicides lend.

The expression cassettes according to the invention also include regulatory nucleic acid sequences which express the expression of the co control the sequence in the host cell. According to one before preferred embodiment comprises an expression according to the invention cassette upstream, d. H. at the 5 'end of the coding sequence a promoter and downstream, d. H. a polyadeny at the 3 'end lation signal and, if necessary, further regulatory ele elements with the intervening coding sequence for the polypeptide with fungicide-binding properties and / or Transit peptide are operatively linked. Under an operative Linkage means the sequential arrangement of promoter, coding sequence, terminator and possibly other regulatory Elements such that each of the regulatory elements has its radio tion in the expression of the coding sequence as intended can meet. Those preferred for the operative linkage sequences that are not restricted to this are targeting sequences Ensuring subcellular localization in the apoplast, in the vacuole, in plastids, into the mitochondrion, in the endo plasma reticulum (ER), in the cell nucleus, in oil cells or other compartments and translation enhancers such as the 5 'guide sequence from the tobacco mosaic virus (Gallie et al., Nucl.  Acids Res. 15 (1987) 8693-8711).

As promoters of the expression cassette according to the invention basically any promoter suitable for the expression of Can control foreign genes. It is preferably used in particular a plant promoter or a promoter that is a plant zen virus originated. The CaMV 35S-Pro is particularly preferred motor from the cauliflower mosaic virus (Franck et al., Cell 21 (1980) 285-294). This promoter contains different Er identifier sequences for transcriptional effectors, which in their Whole to a permanent and constitutive expression of the introduced gene (Benfey et al., EMBO J. 8 (1989) 2195-2202).

The expression cassette according to the invention can also be a che contain mix-inducible promoter by which the expression of the exogenous polypeptide in the plant at a given time point can be controlled. Such promoters such. B. the PRPI promoter (Ward et al., Plant. Mol. Biol. 22 (1993), 361-366) promoter induced by salicylic acid (WO 95/1919443) by benzene sulfonamide inducible (EP 388186), a by Ab scisic acid-inducible (EP 335528) or one by ethanol or Cyclohexanone-inducible (WO 9321334) promoters are the Literature described and u. a. be used.

Furthermore, promoters are preferred which are Ex Ensure pressure in tissues or parts of plants in which the phytotoxic fungicidal effect unfolds. In particular, too are promoters that have a leaf-specific expression guarantee. The promoter of the cytosolic FBPase should be mentioned from potato or the ST-LSI promoter from potato (Stockhaus et al., EMBO J. 8 (1989) 2445-245).

With the help of a seed-specific promoter, single-chain antibodies were able to stably express up to 0.67% of the total soluble seed protein in the seeds of transgenic tobacco plants (Fiedler and Conrad, Bio / Technology 10 (1995), 1090-1094). Since an expression in sown or germinating seeds may also be possible and may be desirable in the sense of the present invention, corresponding regulating elements according to the invention are likewise preferred regulating and seed-specific promoters. The expression cassette according to the invention can therefore contain, for example, a seed-specific promoter (preferably the USP or LEB4 promoter, the LEB4 signal peptide, the gene to be expressed and an ER retention signal. The structure of the cassette is shown in FIG. 1 using the example of a single chain antibody (scFv gene) is shown schematically as an example.

The production of an expression cassette according to the invention follows by fusing a suitable promoter with a suitable one th polypeptide DNA and preferably one between promoter and Polypeptide DNA inserted for a chloroplast specific DNA encoding transit peptide and a polyadenylation signal according to common recombination and cloning techniques as they are for example in T. Maniatis, E.F. Fritsch and J. Sambrook, Mo lecular cloning: A Laboratory manual, Cold Spring Harbor Labora tory, Cold Spring Harbor, NY (1989) and in T.J. Silhavy, M.L. Berman and L.W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) and in Ausubel, F.M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience (1987) ben are.

Sequences which target in the Apoplasts, plastids, the vacuole, the mitochondrion, the endo plasmatic reticulum (ER) or due to a lack of it operative sequences remain in the compartment of the first hens, the cytosol (Kermode, Crit. Rev. Plant Sci. 15: 4 (1996), 285-423). For the amount of protein accumulation in A transgenic plant has proven particularly beneficial Localization in the ER and the cell wall (Schouten et al., Plant Mol. Biol. 30: 781-792 (1996); Artsaenko et al., Plant J. 8 (1995) 745-750).

The invention also relates to expression cassettes, the coding sequence for a fungicide-binding fusion protein ko diert, wherein part of the fusion protein is a transit peptide that controls the translocation of the polypeptide. Particularly preferred are chloroplast-specific transit peptides, which according to Trans location of the fungicide-binding polypeptide in the plant chloro plastically from the fungicide-binding polypeptide part enzymatically be split off. The transit peptide is particularly preferred derived from plastid transketolase (TK) or a functio equivalent of this transit peptide (e.g. the transit peptide the small subunit of the Rubisco or the ferredoxin NADP Oxidoreductase).

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

The inserted nucleotide sequence coding for a fungicide bin The polypeptide can be produced synthetically or naturally be delightful or a mixture of synthetic and natural Contain DNA components. Generally synthetic Nucleotide sequences with codons generated by plants before be moved. These codons preferred by plants can be obtained from Co dons with the highest protein frequency, which are determined in most interesting plant species can be expressed. At The preparation of an expression cassette can be different DNA fragments are manipulated to create a nucleotide sequence receive, which reads appropriately in the correct direction and which is equipped with a correct reading frame. For the The DNA fragments can be connected to one another to form the fragments Adapters or linkers can be used.

The promoter and the terminator regions in the direction of transcription with a Lin ker or polylinker, the one or more restriction sites contains for the insertion of this sequence. In the The linker usually has 1 to 10, mostly 1 to 8, preferably 2 up to 6 restriction sites. In general, the left has inner half of the regulatory areas a size of less than 100 bp, often less than 60 bp, but at least 5 bp. Of the The promoter according to the invention can be native or homologous as well strange or heterologous to the host plant. The fiction appropriate expression cassette contains in the 5'-3 'transcript onsrichtung the promoter according to the invention, any Se quenz and a region for transcriptional termination. Ver Different termination areas are arbitrary against each other exchangeable.

Manipulations can also find the appropriate restriction cut provide or dispose of the superfluous DNA or restriction Remove interfaces, be used. Where insertions, Deletions or substitutions such. B. Transitions and Trans versions may be considered in vitro mutagenesis, "primerre pair ", restriction or ligation can be used. With suitable Manipulations, such as B. restriction, "chewing-back" or replenishment len of overhangs for "bluntends" can have complementary ends of the fragments are made available for ligation.  

This is of particular importance for the success according to the invention Append the specific ER retention signal SEKDEL (Schuoten, A. et al. Plant Mol. Biol. 30 (1996), 781-792) by Average expression level is tripled to four fold. There may also be other retention signals that are more natural wise in plant and animal protei localized in the ER be used for the assembly of the cassette.

Preferred polyadenylation signals are vegetable polyadenylation lation signals, preferably those which are essentially T-DNA Polyadenylation signals from Agrobacterium tumefaciens, ins particular of gene 3 of T-DNA (octopine synthase) of the Ti plasmid correspond to pTiACH5 (Gielen et al., EMBO J. 3 (1984) 835 ff) or functional equivalents.

An expression cassette according to the invention can contain, for example, a constitutive promoter (preferably the CaMV 35 S promoter), the LeB4 signal peptide, the gene to be expressed and the ER retention signal. The structure of the cassette is shown schematically in Fig. 2 using the example of a single-chain antibody (scFv gene). The amino acid sequence KDEL (lysine, aspartic acid, glutamic acid, leucine) is preferably used as the ER retention signal.

Preferably, the fused expression cassette used for encodes a polypeptide with fungicide-binding properties, in cloned a vector, for example pBin19, which is suitable Transform Agrobacterium tumefaciens. With one Vector-transformed agrobacteria can then be known Way to transform plants, especially culture plants, such as B. tobacco plants can be used by for example wounded leaves or leaf pieces in an agro bacterial solution bathed and then in suitable media be cultivated. The transformation of plants through Agrobak terien is known from F.F. White, Vectors for Gene Transfer to higher plants; in Transgenic Plants, Vol. 1, Enginee ring and utilization, edited by S.D. Kung and R. Wu, Academic Press, 1993, pp. 15-38 and from S.B. Gelvin, Molecular Ge netics of T-DNA Transfer from Agrobacterium to Plants, the same if in Transgenic Plants, pp. 49-78. From the transformed Cells of the wounded leaves or leaf pieces can be known ter transgenic plants are regenerated, the one in the Expression cassette integrated gene according to the invention for the Ex pression of a polypeptide with fungicide-binding properties contain.  

To transform a host plant with one for a fungicide DNA encoding binding polypeptide becomes an inventive Expression cassette as an insertion into a recombinant vector built in, whose vector DNA additional functional regulati on signals, for example sequences for replication or inte gration contains. Suitable vectors are inter alia in "Methods in Plant Molecular Biology and Biotechnology" (CRC Press), chap. 6/7, pp. 71-119.

Using the recombination and The expression cases according to the invention can be cloned set to be cloned into appropriate vectors that match their tion, for example in E. coli. Suitable clony tion vectors are u. a. pBR332, pUC series, M13mp series and pA CYC184. Binary vectors which are particularly suitable in E. can replicate coli as well as in agrobacteria, e.g. B. pBin19 (Bevan et al. (1980) Nucl. Acids Res. 12, 8711).

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

Depending on the choice of the promoter, the expression can be specific in the leaves, seeds or other parts of the plant consequences. Such transgenic plants, their reproductive material as well their plant cells, tissues or parts are another Ge subject of the present invention.

The transfer of foreign genes into the genome of a plant is made referred to as transformation. There are the described Methods for transforming and regenerating plants Plant tissues or plant cells for transient or stable Transformation used. Protoplasts are suitable methods transformation by polyethylene glycol-induced DNA uptake, the biolistic approach with the gene gun, electroporation, the incubation of dry embryos in DNA-containing solution, the Microinjection and Agrobacterium -mediated gene transfer. The The methods mentioned are described, for example, in B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, En gineering and Utilization, edited by 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). Before preferably the construct to be expressed is converted into a vector cloned, which is suitable for trans Agrobacterium tumefaciens  form, for example pBin19 (Bevan et al., Nucl. Acids Res. 12 (1984) 8711).

Transformed with an expression cassette according to the invention Agrobacteria can then be transformed in a known manner of plants, in particular of crop plants, such as cereals, Corn, soybeans, rice, cotton, sugar beet, canola, sunflower, Flax, potato, tobacco, tomato, rapeseed, alfalfa, lettuce and various bush, tree, nut and wine species, such as wise coffee, fruit trees like apple, pear or cherry, nut trees like walnut or pecan and especially important in vines be, e.g. B. by wounded leaves or leaf pieces in one Agrobacteria solution bathed and then in suitable media be cultivated.

Functionally equivalent sequences that contain a fungicide-binding Coding polypeptide are, according to the invention, such sequences, wel che despite the differing nucleotide sequence the desired Have functions. Functional equivalents thus include na Naturally occurring variants of the sequences described here as well as artificial, e.g. B. obtained by chemical synthesis to the Artificial nucleotide sequences adapted to the codon usage of a plant Zen.

A functional equivalent is understood to mean in particular also natural or artificial mutations of one originally isolated sequence encoding the fungicide-binding polypeptide, which still show the desired function. Mutations around include substitutions, additions, deletions, exchanges or insertions of one or more nucleotide residues. So be for example, such nucleotide sequences by the vorlie The present invention includes which can be modified by this Receives nucleotide sequence. Such a modification can be the target e.g. B. the further limitation of the encoding contained therein Sequence or z. B. also the insertion of further restriction enzyme Interfaces.

Functional equivalents are also those variants whose radio tion compared to the parent gene or gene fragment is weakening or strengthening.

In addition, artificial DNA sequences are suitable as long as they as described above, the desired tolerance to fungizi ver to avoid phytotoxic effects on crops average. Such artificial DNA sequences can, for example constructed by back translation using molecular modeling  Proteins that have fungicide-binding activity or by in in vitro selection can be determined. Codes are particularly suitable DNA sequences generated by back-translating a polypeptide sequence according to the codon usage specific for the host plant were obtained. The specific codon usage can be with Plant genetic methods familiar expert through Compute evaluations of other known genes to be transformed Easily identify the plant.

As further suitable equivalent nucleic acid Sequences are to be called sequences which are for fusion proteins encode, a component of the fusion protein being a non-plant fungicide-binding polypeptide or a functionally equivalent lent part of it. The second part of the fusion protein can e.g. B. be another polypeptide with enzymatic activity or an antigenic polypeptide sequence with the help of which a detection on scFvs expression is possible (e.g. myc-tag or his-tag). However, this is preferably a regulatory pro tine sequence, such as B. a signal or transit peptide that Polypeptide with fungicide binding properties to the desired directs the place of action.

The invention also relates to the invention produced expression products, as well as fusion proteins from one Transit peptide and a polypeptide with fungicide-binding properties create.

Resistance or tolerance means in the context of the present Er the artificially acquired resistance of plants against fungicides with phytotoxic effects. It includes the partial and, in particular, complete insensitivity versus these inhibitors for at least one for the duration Plant generation.

The phytotoxic location of fungicides is generally that Leaf tissue, so that a leaf-specific expression of the exogenous Fungicide-binding polypeptides can provide adequate protection. However, it is obvious that the phytotoxic effect of a Fungicide need not be limited to the leaf tissue, but also tissue-specific success in all other parts of the plant can.

In addition, there is a constitutive expression of exogenous fun Gicide-binding polypeptides are an advantage. On the other hand, however inducible expression also appears desirable.  

The effectiveness of the transgenically expressed polypeptide with fungi zid-binding properties can, for example, in vitro Shoot meristem propagation on medium containing fungicide via abge graduated series of concentrations or via seed germination tests be communicated. In addition, a fungicide that has changed in type and level tolerance of a test plant in greenhouse experiments be steady.

The invention also relates to transgenic plants, trans formed with an expression cassette according to the invention, and transgenic cells, tissues, parts and propagation material of such plants Zen. Transgenic crop plants, such as e.g. B. cereals, corn, soybeans, rice, cotton, sugar beet, canola, Sunflower, flax, potato, tobacco, tomato, rapeseed, alfalfa, Sa lat and the various bush, tree, nut and wine species such as for example coffee, fruit trees such as apple, pear or cherry, Nut trees like walnut or pecan and especially important the vine.

The transgenic plants, plant cells, tissue or parts can with a fungicide with a phytotoxic effect that inhibited herbal enzymes, treated, causing the plants, cells, tissues or Plant parts die or are damaged. Examples of ge suitable active ingredients are strobilurins in particular Methyl methoxyimino-α- (o-tolyloxy) -o-tolylacetate (BAS 490F), as well as metabolites and functional derivatives of these compounds. The inserted into the expression cassettes according to the invention for a polypeptide with fungicide-binding properties DNA, can also be used as a selection marker.

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

• - Sulfur, dithiocarbamates and their derivatives, such as ferridimes ethyldithiocarbamate, zinc dimethyldithiocarbamate, zinc ethylene bis dithiocarbamate, manganese ethylene bisdithiocarbamate, manganese zinc ethylenediamine bis-dithiocarbamate, tetramethylthiuram disulfide, Ammonia complex of zinc (N, N-ethylene-bis-dithiocarbamate), Am monia complex of zinc (N, N'-propylene-bis-dithiocarbamate),  Zinc (N, N'-propylene bis-dithiocarbamate), N, N'-polypropylene bis- (thiocarbamoyl) disulfide;
• Nitroderivatives, such as dinitro- (1-methylheptyl) phenylcrotonate, 2-sec-butyl-4,6-dinitrophenyl-3,3-dimethylacrylate, 2-sec-Bu tyl-4,6-dinitrophenyl-isopropyl carbonate, 5-nitro-isoph di-isopropyl thalate;
• - Heterocyclic substances, such as 2-heptadecyl-2-imidazolin-ace tat, 2,4-dichloro-6- (o-chloroanilino) -s-triazine, O, O-diethyl phthalimidophosphonothioate, 5-amino-1- [bis- (dimethylamino) - phosphinyl] -3-phenyl-1,2,4-triazole, 2,3-dicyano-1,4-dithioane thrachinone, 2-thio-1,3-dithiolo [4,5-b] quinoxaline, 1- (butylcar bamoyl) -2-benzimidazole-carbamic acid methyl ester, 2-methoxycar bonylamino-benzimidazole, 2- (furyl- (2)) -benzimidazole, 2- (thiazo lyl- (4)) - benzimidazole, N- (1,1,2,2-tetrachloroethylthio) tetrahy drophthalimide, N-trichloromethylthio-tetrahydrophthalimide, N-trichloromethylthio-phthalimide,
• - N-dichlorofluoromethylthio-N ', N'-dimethyl-N-phenylsulfuric acid diamide, 5-ethoxy-3-trichloromethyl-1,2,3-thiadiazole, 2-rhodanme thylthiobenzthiazole, 1,4-dichloro-2,5-dimethoxybenzene, 4- (2-chlorophenylhydrazono) -3-methyl-5-isoxazolone, pyridine-2- thio-1-oxide, 8-hydroxyquinoline or its copper salt, 2,3-di hydro-5-carboxanilido-6-methyl-1,4-oxathiine, 2,3-dihydro-5-car boxanilido-6-methyl-1,4-oxathiin-4,4-dioxide, 2-methyl-5,6-dihy dro-4H-pyran-3-carboxylic acid anilide, 2-methyl-furan-3-carboxylic acid reanilide, 2,5-dimethyl-furan-3-carboxylic acid anilide, 2,4,5-trime thyl-furan-3-carboxylic acid anilide, 2,5-dimethyl-furan-3-carboxylic acid recyclohexylamide, N-cyclohexyl-N-methoxy-2,5-dimethyl-fu ran-3-carboxamide, 2-methyl-benzoic acid anilide, 2-iodine-ben zoic acid anilide, N-formyl-N-morpholine-2,2,2-trichloroethylace tal, piperazin-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 its salts, 2,6-dimethyl-N-cyclododecyl-morpholine or its salts, N- [3- (p-tert-Butylphenyl) -2-methylpropyl] cis-2,6-dimethyl morpholine, N- [3- (p-tert-butylphenyl) -2-methylpropyl] piperi din, (8- (1,1-dimethylethyl) -N-ethyl-N-propyl-1,4-dioxas piro [4.5] decane-2-methanamine,
• Azoles such as 1- [2- (2,4-dichlorophenyl) -4-ethyl-1,3-dioxolan-2-yl-ethyl] -1H-1,2,4-triazole, 1- [2- (2nd , 4-dichlorophenyl) -4-n-propyl-1,3-dioxolan-2-yl-ethyl] -1H-1,2,4-triazole, N- (n-propyl) -N- (2, 4,6-trichlorophenoxyethyl) -N'-imidazol-yl urea, 1- (4-chlorophenoxy) -3,3-dimethyl-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] -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-triazole, 1- [2RS, 4RS; 2RS, 4SR) -4-bromo-2- (2,4-dichlorophenyl) tetrahydrofuran] -1H-1,2,4 -triazol, 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) pent-1-en-3-ol, 4- (4- Chl orphenyl) -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) -1-H-1,2,4-triazol-1-yl) -hex ano -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-triazol-1-yl methyl) pentan-3-ol, (+) - 2- ( 2,4, -Dichlorophenyl) -3- (1H-1,2,4-triazolyl) propyl-1,1,2,2-tetrafluoroethyl ether, (E) -1- [1- [4-chloro 2-trifluoromethyl) -phenyl] -imino) -2-propoxyethyl] -1H-imidazole, 2- (4-chlorophenyl) -2- (1H-1,2,4-triazol-1-ylmethyl) -hexanenitrile,
  α- (2-Chlorophenyl) -α- (4-chlorophenyl) -5-pyrimidine-methanol, 5-butyl-2-dimethylamino-4-hydroxy-6-methyl-pyrimidine, bis- (p-chlorophenyl) - 3-pyridine methanol, 1,2-bis (3-ethoxycarbonyl-2-thiou reido) benzene, 1,2-bis (3-methoxycarbonyl-2-thioureido) benzene,
• - Strobilurins such as methyl-E-methoxyimino- [α- (o-tolyloxy) -o-to lyl] acetate, methyl E-2- {2- [6- (2-cyanophenoxy) pyrimi din-4-yloxy] phenyl} -3-methoxyacrylate, methyl-E-methoxyi mino- [α- (2- phenoxyphenyl)] - acetamide, methyl-E-methoxyimino- [α- (2,5-dime thylphenoxy) -o-tolyl] -acetamide,
• Anilinopyrimidines such as N- (4,6-dimethylpyrimidin-2-yl) aniline, N- [4-methyl-6- (1-propynyl) pyrimidin-2-yl] aniline, N- [4-methyl-6-cyclopropyl-pyrimidin-2-yl] aniline,
• Phenylpyrroles such as 4- (2,2-difluoro-1,3-benzodioxol-4-yl) pyr rol-3-carbonitrile,
• - Cinnamic acid amides such as 3- (4-chlorophenyl) -3- (3,4-dimethoxyphe nyl) acrylic acid morpholide,
• - and various fungicides, such as dodecylguanidine acetate, 3- [3- (3,5-dimethyl-2-oxycyclohexyl) -2-hydroxyethyl] glutarimide, N- Methyl, N-ethyl- (4-trifluoromethyl-2- [3 ', 4'-dimethoxyphe nyl] -benzoic acid amide, hexachlorobenzene, DL-methyl-N- (2,6-dime thyl-phenyl) -N-furoyl (2) -alaninate, DL-N- (2,6-dimethyl-phe nyl) -N- (2'-methoxyacetyl) alanine methyl ester, N- (2,6-dime thylphenyl) -N-chloroacetyl-D, L-2-aminobutyrolactone, DL- N- (2,6-dimethylphenyl) -N- (phenylacetyl) alanine methyl ester, 5-methyl-5-vinyl-3- (3,5-dichlorophenyl) -2,4-dioxo-1,3-oxazoli  din, 3- [3,5-dichlorophenyl (-5-methyl-5-methoxymethyl] -1,3-oxazo lidin-2,4-dione, 3- (3,5-dichlorophenyl) -1-isopropylcarbamoylhy dantoin, N- (3,5-dichlorophenyl) -1,2-dimethylcyclopropane-1,2-di carboximide, 2-cyano- [N- (ethylaminocarbonyl) -2-methoxy mino] acetamide, N- (3-chloro-2,6-dinitro-4-trifluoromethyl-phe nyl) -5-trifluoromethyl-3-chloro-2-aminopyridine.

Functionally equivalent derivatives of these fungicides have one comparable spectrum of activity against phytopathogenic fungi such as the specifically named substances, with lower, equal or higher phytotoxic activity.

The invention is illustrated by the following examples, but is not limited to these:

General cloning procedures

The clone carried out in the context of the present invention steps such as B. restriction cleavages, agarose gel electrophoresis, purification of DNA fragments, transfer of nu small acids on nitrocellulose and nylon membranes, linking of DNA fragments, transformation of E. coli cells, cultivation of Bacteria, phage propagation and recombinant sequence analysis DNA was as in Sambrook et al. (1989) Cold Spring Harbor La boratory press; ISBN 0-87969-309-6).

The bacterial strains used below (E. coli, XL-I Blue) were sourced from Stratagene. The one for plant transformation Agrobacterium strain used (Agrobacterium tumefaciens, C58C1 with the plasmid pGV2260 or pGV3850kan) was developed by Deblaere et al. (Nucl. Acids Res. 13 (1985) 4777). Alternatively can also be the agrobacterial strain LBA4404 (Clontech) or others suitable strains are used. For cloning, the Vectors pUC19 (Yanish-Perron, Gene 33 (1985), 103-119) pBlues cript SK- (Stratagene), pGEM-T (Promega), pZerO (Invitrogen), pBin19 (Bevan et al., Nucl. Acids Res. 12 (1984) 8711-8720) and pBinAR (Höfgen and Willmitzer, Plant Science 66 (1990) 221-230) used.

Sequence analysis of recombinant DNA

Recombinant DNA molecules were sequenced using a Laser fluorescence DNA sequencer from Pharmacia after the Sanger's method (Sanger et al., Proc. Natl. Acad. Sci. USA  74 (1977), 5463-5467).

Production of plant expression cassettes

In the plasmid pBin19 (Bevan et al., Nucl. Acids Res. 12, 8711 (1984)) became a 35S CaMV promoter as an EcoRI-KpnI fragment (corresponding to nucleotides 6909-7437 of the Cauliflower mosaic- Virus (Franck et al. Cell 21 (1980) 285). The polyad nylation signal of gene 3 of the T-DNA of the Ti plasmid pTiACH5 (Gielen et al., EMBO J. 3 (1984) 835), nucleotides 11749-11939 was isolated as a PvuII-HindIII fragment and after addition of SphI linkers to the PvuII interface between the SpHI-HindIII Vector cloned interface. The plasmid pBi was formed nAR (Höfgen and Willmitzer, Plant Science 66 (1990) 221-230).

Examples of use example 1

Since fungicides are not immunogenic, they must be carried on a carrier material such as B. KLH can be coupled. There is a reak tive group in the molecule, this coupling can take place directly otherwise, a function will occur during the synthesis of the fungicide nelle group or a reactive precursor during the Synthesis selected to make these molecules in a simple reaction coupling step to the carrier molecule. Examples of cop Miroslavic Ferencik's "Handbook of Immunoche mistry ", 1993, Chapman & Hall, in the chapter Antigens, pages 20-49 described.

By repeated injection of this modified carrier molecule (Antigen) are e.g. B. Balb / c mice immunized. Once in the serum sufficient antibodies with binding to the antigen in the ELISA (enzyme linked immuno sorbent assay) are detectable, the spleen cells from these animals were taken and fused with myeloma cells Cultivate hybrids. In the ELISA is also used as an antigen "Fungicide-modified BSA" used to combat the hapten distinguish directed immune response from the KLH response.

The production of monoclonal antibodies is based on to known methods such. B. described in "Practical Immuno logy ", 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 Möller and Franz Emling "Monoconal antibodies to TNF and their use". European cal patent EP-A260610.

Example 2

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

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

The functional characterization (antigen binding activity) of the constructed scFv-antiBAS 490F gene was carried out after expression in a bacterial system. The scFv-antiBAS 490F was synthesized according to the method of Hoogenboom et al., 1991 as a soluble antibody fragment in E. coli. The activity and the specificity of the constructed antibody fragment were checked in ELISA tests ( Fig. 4).

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

The expression cassette was constructed into the binary vector pGSGLUC 1 (Saito et al., 1990) cloned and by electroporation transferred to the Agrobacterium strain EHA 101. Recombinant Agrobacterial clones were used for the subsequent transformation of Nicotiana tabacum used. 70-140 tobacco were used per construct plants regenerated. From the regenerated transgenic tobacco after self-fertilization, seeds of various species were planted development stages harvested. Of these seeds, the soluble ones Obtain proteins after extraction in an aqueous buffer system The analysis of the transgenic plants shows that the Fu sion of the scFv-antiBAS 490F gene with the DNA sequence of ER-Reten tion signal SEKDEL a maximum accumulation of 1.9% scFv-an tiBAS 490F protein could be obtained in mature seeds.

The constructed scFv-antiBAS 490F gene had a size of approx. 735 bp. The variable domains were in the order VH-L-VL merged with each other.

The specific selectivity was in the extracts of the mature Tobacco seeds determined with a direct ELISA. The one there values obtained clearly show that the protein extracts are radio contain tionally active antibody fragments.

Example 3

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

The starting point for the investigations was a single chain antibody fragment against the fungicide BAS 490F (scFv-anti BAS 490F). The functional characterization (antigen binding activity) of this constructed scFv-anti-BAS 490F genes took place after expression in a bacterial system and after expression in tobacco leaf tern. The activity and specificity of the antibody constructed perfragmentes was checked in ELISA tests.

To enable seed-specific expression of the antibody fragment in tobacco, the scFv-anti-BAS 490F gene was cloned downstream from the USP promoter. The USP promoter isolated from Vicia faba shows strictly seed-specific expression of various foreign genes in tobacco (Fiedler et al., 1993). By transporting the scFv-antiBAS 490F polypeptide into the endoplasmic reticulum, a stable accumulation of high antibody fragments was achieved. For this purpose, the scFv-antiBAS 490F gene was fused with a signal peptide sequence that fuses entry into the endoplasmic reticulum and the ER retention signal SEKDEL, which ensures that it remains in the ER (Wandelt et al., 1992) ( Fig. 1) .

The expression cassette was constructed into the binary vector pGSGLuC1 (Saito et al., 1990) cloned and by electroporation transferred to the Agrobacterium strain EHA 101. Recombinant Agrobacterial clones were used for the subsequent transformation of Nicotiana tabacum used. From the regenerated transgenic Ta Bacon plants were seeded differently after self-fertilization Stages of development harvested. From these seeds the soluble proteins after extraction in an aqueous buffer system hold. The analysis of the transgenic plants shows that by the Fusion of the scFv-antiBAS 490F gene with the DNA sequence of the ER-Re tention signal SEKDEL under the control of the USP promoter already from day 10 of seed development with single chain antibody fragments Binding affinity for BAS 490F were synthesized.

Example 4

For ubiquitous expression of the antibody fragment in the Plant, especially in leaves, the scFv-anti- BAS 490F gene cloned downstream of the CaMV 35S promoter. The This strong constitutive promoter mediates expression of Foreign genes in almost all plant tissues (Benfey and Chua, Science 250 (1990), 956-966. By transporting the scFv-antiBAS 490F protein in the endoplasmic reticulum was a sta bile accumulation of large amounts of antibody fragments in the leaf material reached. The scFv-antiBAS 490F gene was first treated with an Si Signal peptide sequence that enters the endoplasmic reti kulum and the ER retention signal KDEL, which remains in the ER guaranteed (Wandelt et al., Plant J. 2 (1992), 181-192) fu based. The constructed expression cassette was in the binary ren vector pGSGLUC 1 (Saito et al., Plant Cell Rep. 8 (1990), 718 - 721) cloned and by electroporation into the agrobacterium - Strain EHA 101 transferred. Recombinant agrobacterial clones were that for the subsequent transformation of Nicotiana tabacum used. About 100 tobacco plants were regenerated. From The regenerated transgenic tobacco plants became leaf material taken from different stages of development. From this sheet size After extraction, the soluble proteins were mixed in one  receive aqueous buffer system. Subsequent analyzes (Western Blot analyzes and ELISA tests) showed that a maximum accumulation of more than 2% of biologically active, anti gene-binding scFv-antiBAS 490F polypeptide could be achieved. The high expression values were in full-grown green leaves determined, but also in senescent leaf material Antibody fragment can be detected.

Example 5

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

The PCR amplification of the one-chain antibody cDNA was carried out in a DNA thermal cycler from Perkin Elmer.

The reaction mixtures contained 8 ng / µl single-stranded templates cDNA, 0.5 µM of the corresponding oligonucleotides, 200 µM nucleotides (Pharmacia), 50 mM KCl, 10 mM Tris-HCl (pH 8.3 at 25 ° C, 1.5 mM MgCl2 ) and 0.02 U / El Taq polymerase (Perkin El mer). The amplification conditions were set as follows:
Annealing temperature: 45 ° C
Denaturation temperature: 94 ° C,
Elongation temperature: 72 ° C,
Number of cycles: 40.

The result was a fragment of approximately 735 base pairs, which in the Vector pBluescript was ligated. With the ligation approach E. coli XL-I Blue transformed and the plasmid amplified. For Application and optimization of the polymerase chain reaction see: Innis et al., 1990, PCR Protocols, a Guide to Methods and Ap plications, Academic Press.

Example 6

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

The plasmid pGSGLUC 1 was transformed into Agrobacterium tumefaciens C58C1: pGV2260. For the transformation of tobacco plants (Nicotiana tabacum cv. Samsun NN) a 1:50 dilution of an overnight culture of a positively transformed agrobacteria colony in Murashige-Skoog medium (Physiol. Plant. 15 (1962) 473 ff.) With 2% sucrose (2MS- Medium) is used. Leaf disks of sterile plants (each about 1 cm ² ) were incubated in a Petri dish with a 1:50 agrobacterial dilution for 5-10 minutes. This was followed by a 2-day incubation in the dark at 25 ° C. on 2MS medium with 0.8% Bacto agar. The cultivation was continued after 2 days with 16 hours of light / 8 hours of darkness and in a weekly rhythm on MS medium with 500 mg / l claforan (cefotaxime sodium), 50 mg / l kanamycin, 1 mg / l benzyla minopurin (BAP ), 0.2 mg / l naphthylacetic acid and 1.6 g / 1 glucose continued. Growing shoots were transferred to MS medium with 2% sucrose, 250 mg / l Claforan and 0.8% Bacto agar.

Example 7

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

The starting point of the investigations was an expri in tobacco plants mated single-chain antibody fragment against the fungicide BAS 490F (scFv-anti BAS 490F). Amount and activity of the synthesized scFv-antiBAS 490F polypeptides were analyzed in Western blot and ELISA tests.

To enable expression of the scFv-antiBAS 490F gene in the endoplasmic reticulum, the foreign gene was controlled under the control of the CaMV 53S promoter as a translation fusion with the LeB4 signal peptide (N-terminal) and the ER retention signal KDEL (C -terminal). By transporting the scFv-anti-BAS 490F polypeptide into the endoplasmic reticulum, a stable accumulation of large amounts of active antibody fragment was achieved. After harvesting the leaf material, pieces were frozen at -200 ° C, lyophilized or dried at room temperature. The soluble proteins were obtained from the respective leaf material by extraction in an aqueous buffer and the scFv antiBAS 490F polpypeptide was purified by affinity chromatography. Equal amounts of purified scFv-antiBAS 490F polypeptide (one frozen, lyophilized and dried) were used to determine the activity of the antibody fragment ( Fig. 6). The same antigen binding activities were found.

Example 8

To detect the fungicide tolerance of a polypeptide with fungi Transgenic tobacco plant producing zid-binding properties These were treated with different amounts of BAS 490F.  

In all cases it could be shown in the greenhouse that the one plants expressing scFv-antiBAS 490F gene compared to Control a higher tolerance to the BAS 490F fungicide and show less phytotoxic effects.

Claims (28)

1. Process for the preparation of fungicide-tolerant plants by expression of an exogenous fungicide-binding poly peptides in plants. 2. The method according to claim 1, characterized in that it the exogenous fungicide-binding polypeptide Single chain antibody fragment. 3. The method according to claim 1, characterized in that it the exogenous fungicide-binding polypeptide complete antibody or a fragment derived therefrom acts. 4. The method according to claim 1, characterized in that it the fungicide is methyl methoxyimino-α- (o-toly loxy) -o-tolylacetate (BAS 490F). 5. The method according to any one of claims 1-3, characterized records that it is mono- or dicotyledonous plants delt. 6. The method according to claim 5, characterized in that it the plant is tobacco. 7. The method according to any one of claims 1-6, characterized net that the expression of the exogenous polypeptide is constitutive takes place in the plant. 8. The method according to any one of claims 1-6, characterized records that the expression of the exogenous polypeptide in the Plant is induced. 9. The method according to any one of claims 1-6, characterized records that the expression of the exogenous polypeptide in the Leaves the plant. 10. The method according to any one of claims 1-6, characterized records that the expression of the exogenous polypeptide in the Plant seeds are made.   11. Expression cassette for plants consisting of one Promoter, a signal peptide, a gene coding for the Ex pression of an exogenous fungicide-binding polypeptide, a ER retention signal and a terminator. 12. Expression cassette according to claim 11, characterized in that that as a constitutive promoter the CaMV 35S promoter is used. 13. Expression cassette according to claim 11, characterized in that that the gene to be expressed is a single chain anti gene body fragment is used. 14. Expression cassette according to claim 11, characterized in that that the gene or gene fragment of a gene to be expressed Fungicide-binding polypeptide as a translation fusion other functional proteins such as enzymes, To xinen, chromophores and binding proteins is used. 15. Expression cassette according to claim 11, characterized in that that the polypeptide gene to be expressed is from a hybridoma cell or using other recombinant methods - such as e.g. B. the antibody phage display method - is obtained. 16. Use of the expression cassette according to claim 11 for Transformation of dicotyledonous or monocotyledonous plants that constitutively seed- or leaf-specific an exogenous fungi Express cid-binding polypeptide. 17. Use according to claim 16, characterized in that one the expression cassette is transferred into a bacterial strain and the resulting recombinant clones for transformation of dicotyledonous or monocotyledonous plants which are constitutive sa an exogenous fungicide binding specific to the individual or leaf Express polypeptide used. 18. Use of the expression cassette according to claim 11 as Se lesson markers. 19. Use of a transformed plant as claimed in claim 17 or 18 obtained to produce a fungicide-binding Polypeptide.   20. Process for transforming a plant by insertion a gene sequence for a fungicide-binding polypeptide encoded, in a plant cell, in callus tissue, a whole Plant and protoplasts of plant cells. 21. The method according to claim 20, characterized in that the Transformation with the help of an Agrobacterium in particular of the species Agrobacterium tumefaciens. 22. The method according to claim 20, characterized in that the Transformation is done with the help of electroporation. 23. The method according to claim 20, characterized in that the Transformation using the particle bombardment method follows. 24. Preparation of a fungicide-binding polypeptide by Ex pression of a gene coding for such a polypeptide in a plant or cells of a plant and subsequent Isolation of the polypeptide. 25. Plant containing an expression cassette according to claim 11, characterized in that the expression cassette Tole mediated against a fungicide. 26. Plant according to claim 25, characterized in that it to lerant to methyl-methoxyimino-α- (o-tolyloxy) -o-tolyla cetate (BAS 490F). 27. Process for combating phytopathogenic fungi in trans characterized by fungicide-tolerant crops net that fungicides are used against which the culture plant fungicide-binding polypeptides or antibodies forms. 28. Fungicide-binding polypeptides or antibodies with high Binding affinity for methyl-methoxyimino-α- (o-tolyloxy) -o-toly lacetate (BAS 490F), characterized in that it according to Claim 24 are made.