SK137299A3 - Expression of fungicide-binding polypeptides in plants for producing fungicide tolerance - Google Patents

Abstract

Verfahren zur Herstellung von Fungizid-toleranten Pflanzen durch Expression eines Fungizid-bindenden Antikörpers in den Pflanzen.

Description

Expression of fungicide binding polypeptides in plants to achieve fungicide tolerance

Technical field

The present invention relates to a process for the production of fungicide tolerant plants by expressing an exogenous fungicide-binding polypeptide in plants or plant organs. The invention further relates to the use of appropriate nucleic acids which encode a polypeptide, antibody or antibody portion having fungicide binding properties in transgenic plants and the transformed plant itself.

BACKGROUND OF THE INVENTION

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

An important goal of the work done in the field of plant molecular genetics is to generate tolerance to herbicides. Herbicide tolerance is characterized by improved compatibility (in terms of type or level) of the plant or plant organs with the herbicide applied. This can be achieved in various ways. Known methods are the use of a metabolic gene, for example, a pathogen, in conjunction with glufosinate resistance (WO 8705629) or a herbicide-resistant target enzyme, such as enolpyruvyl shikimate-3-phosphate synthesis (WO 9204449), which is resistant to glyphosate, and the use of the herbicide in cell and tissue culture to select tolerant plant cells and resulting resistant plants, as described for acetyl-CoA-carboxylase inhibitors (US 5162602, US 5290696).

Antibodies are proteins, components of the immune system. A common feature of all antibodies is their spatial spherical structure, light and heavy chain construction, and their basic ability to bind molecules or parts of a molecular structure of high specificity (Alberts et al., In: Molecular biology derZelle [Cell Molecular Biology], 2nd edition 1990, VCH Verlag, ISBN 3-52727983-0, 1198-1237). Because of these properties, antibodies are used for several purposes. The use can be divided into the use of antibodies in the animal and human organisms in which they are produced, i.e. so-called in situ applications and ex situ applications, i. utilization of antibodies after isolation from producer cells or organisms (Whitelam und Cockburn, TIPS vol. 1, 8 (1996), 268-272).

The use of somatic hybrid cell lines (hybridomas) as a source of antibodies against very specific antigens is based on the work of Kohler and Milstein (Nature 256 (1975) 495-97). This procedure allows the production of so-called monoclonal antibodies having a uniform structure and which are produced by cell fusion. The spleen cells of the immunized mouse are fused with mouse myeloma cells. Hybridoma cells are thus obtained, which reproduce without limitation. The cells simultaneously secrete specific antibodies against the antigen with which the mouse was immunized. Spleen cells provide the ability to produce antibody, while myeloma cells contribute to the ability of unrestricted growth and sustained secretion of antibodies. Since each hybridoma cell, being 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 has greatly promoted the use of antibodies, since antibodies having a single and known specificity and homogeneous structure are now available in unlimited amounts. Monoclonal antibodies have extensive use in immunodiagnostics and as therapeutics.

In recent years, a so-called bacteriophage display method for producing antibodies has been obtained, which avoids the immune system and various immunizations in the animal. The affinity and specificity of the antibody can be measured in vitro (Winter et al., Ann. Rev. Immunol. 12 (1994), 433-455; Hoogenboom TIBTech vol. 15 (1997), 62-70). Gene segments that contain a sequence that encodes the variable region of antibodies, i. instead of antigen binding, they are fused to bacteriophage coat protein genes. Then, the bacteria are infected with phages that contain such fusion genes. The obtained phage particles are now provided with envelopes containing the antibody-like fusion protein, with the antibody binding domain facing outward. Such a phage display library can now be used to isolate a phage that contains the desired antibody fragment and that binds specifically to a particular antigen. Each phage isolated in this way produces a monoclonal antigen binding polypeptide that corresponds to the monoclonal antibody. Antigen binding site genes that are unique to 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 mainly as ex situ analytical tools for the qualitative and quantitative detection of antigens. This includes the detection of plant components, herbicides or fungicides in drinking water (Sharp et al. (1991) ACS Symp Ser., 446 (Pestic. Residues Food Saf.) 87-95), in soil samples (WO 9423018) or in plants or plant organs, and the use of antibodies as adjuvants for the purification of bound molecules.

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

More recent experiments utilize antibodies in situ to defend plants against pathogens, particularly viral diseases, by expressing specific antibodies or portions thereof in plant cells that target viral envelope proteins (Tavladoraki et al., Nature 366 (1993), 469-472; Voss et al., Mol Breeding 1 (1995), 39-50).

An analogous approach has also been used to protect plants against nematode attack (Rosso et al., Biochem Biophys Res Com, 220 (1996) 255-263). There are examples of applications 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) . Provided to the body are antibodies produced by the plant and derived from plants or plant organs that are suitable for consumption through the oral cavity, throat or digestive tract, which antibodies cause effective immune protection. In addition, a single chain antibody against low molecular weight plant hormone abscisic acid was already expressed in plants and observed reduced availability of plant hormones due to plant abscisic acid binding (Artsaenko et al., The Plant Journal (1995) 8 (5) 745-750).

Chemical control of fungi in agronomically important crops requires the use of highly selective fungicides without phytotoxic effects. The phytotoxic effect of fungicides may be based, for example, on inhibiting plant growth, reduced photosynthesis and, consequently, reduced crop yield. In some cases, however, it is difficult to develop sufficiently selective fungicides that can be used on all important large-scale plant crops and which do not cause damage to the plant that yields the crop of any crop. The introduction of fungicide-resistant or tolerant farm plants can contribute to solving this problem and can open up new uses of fungicides in crops where treatment has so far not been possible or only possible with acceptable crop losses.

The development of fungicide resistant plants by tissue culture or seed mutagenesis and natural selection is limited. On the other hand, the phytotoxic effect must already be detectable at the tissue culture level, and on the other hand only those plants can be manipulated by tissue culture techniques where whole plants can be successfully regenerated from cell cultures. Moreover, after mutagenesis and selection, the crop plants may exhibit undesirable characteristics which need to be removed again in some cases by repeated backcrossing. The introduction of resistance by cross-breeding would also be limited to plants of the same species.

For the above reasons, the genetic engineering approach by isolating the gene encoding resistance and transferring it to farm plants in a targeted manner is better than the traditional method of plant breeding.

Currently, the development of herbicide tolerant or herbicide resistant crop plants by molecular biology methods requires knowledge of the mechanism of action of the herbicide in the plant and that genes conferring herbicide resistance can be found. A large number of herbicides currently used commercially act by blocking the essential amino acid, lipid or pigment enzyme biosynthesis step. Herbicide tolerance can be generated by altering the genes of these enzymes in such a way that the herbicide will no longer be able to bind, and by introducing these altered genes into farm plants. An alternative method is to find analogous enzymes in nature, for example in microorganisms that exhibit natural resistance to a given herbicide. This resistance gene is isolated from such a microorganism, cloned into suitable vectors and then, after successful transformation, expressed in herbicide sensitive farm plants (WO 96/38567).

SUMMARY OF THE INVENTION

It was an object of the present invention to develop a new, generally applicable genetic engineering method for the production of fungicide tolerant transgenic plants.

We have found that this goal can be surprisingly achieved by a process for expressing an exogenous polypeptide, antibody, or antibody portions having fungicide binding properties in plants.

In particular, the present invention relates to the production of a fungicide-binding antibody and the cloning of a particular gene or gene fragment.

The first step is to produce a suitable antibody that binds the fungicide. This can be achieved, inter alia, by immunizing vertebrates, in most cases mice, rats, dogs, horses, donkeys or goats with an antigen. The antigen in this case is a fungicidally active compound that is associated with or associated with a higher molecular weight carrier, such as bovine serum albumin (BSA), chicken ovalbumin, keyhole limpet hemocyanine (KLH) or other carriers via a functional group. Upon repeated administration of the antigen, the immune response is monitored by conventional methods to isolate the appropriate antiserum. This procedure first results in polyclonal serum containing antibodies with different specificities. For targeted in situ use, it is necessary to isolate a gene sequence that encodes a single specific monoclonal antibody. A number of different approaches are available for this purpose. The first approach utilizes the fusion of antibody producing cells and cancer cells to obtain a hybridoma cell culture that continually produces antibodies and which ultimately leads to a homogeneous cell line that produces a defined monoclonal antibody by uniting the clones obtained.

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

Alternatively, antibodies can be selected through phage display libraries and these antibodies bind the fungicide molecules and convert them catalytically into a product that does not have fungicidal properties. Methods for obtaining 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 a plant can also, in principle, also lead to a fungicide-resistant plant.

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

In addition, the expression cassettes of the invention comprise regulatory nucleic acid sequences that direct the expression of a coding sequence in a host cell. In a preferred embodiment, the expression cassette of the invention comprises a promoter at the 5 'end of the coding sequence and a polyadenylation signal at the end of the 3' and, if appropriate, other regulatory elements that are operably linked to an intermediate coding sequence for a polypeptide having fungicide and / or transit peptide binding properties. . The operative linkage is to be understood as meaning the sequence arrangement of the promoter, coding sequence, terminator and, if appropriate, other regulatory elements in such a way that each of the regulatory elements can function as intended when expressing the coding sequence. Sequences preferred inter alia for operative linkage are target sequences to ensure subcellular localization in apoplasts, plasma membrane, vacuole, plastids, mitochondria, endoplasmic reticulum (ER), nucleus, liposomes or other parts, and translation accelerators, such as the tobacco mosaic virus 5 'end leader sequence (Gallie et al., Nucl. Acids Res. 15 (1987) 8693-8711).

Suitable promoters of the expression cassette of the invention are essentially any promoters capable of directing the expression of foreign genes. Preferred promoters are in particular plant promoters or promoters derived from plant viruses. The CaMV 35S promoter from cauliflower mosaic virus is particularly preferred (Franck et al., Cell 21 (1980) 285-294). This promoter contains various recognition sequences for transcriptional effectors that together result in permanent and constitutive expression of the introduced gene (Benfey et al., EMBO J. 8 (1989) 21952202).

The expression cassette of the invention may also contain a chemically inducible promoter by which the expression of an exogenous polypeptide in a plant at a particular time can be controlled. Among others, promoters described in the literature may be used, for example the PRP1 promoter (Ward et al., Plant. Mol. Biol. 22 (1993), 361-366), a salicylic acid-inducible promoter (WO 95/1919443), which is inducible by benzenesulfonamide (EP 388186), a promoter which is inducible by abscisic acid (EP335528), or a promoter which is inducible by ethanol or cyclohexanone (WO9321334).

Other promoters which are particularly preferred are those which guarantee expression in tissues or plant organs in which phytotoxic fungicidal activity takes place. Promoters that guarantee leaf specific expression deserve special mention. Mention may be made of the potato cytosolic FBPase promoter or the potato ST-LSI promoter (Stockhaus et al., EMBO J. 8 (1989) 2445-245).

Stable expression of single chain antibodies, which represented up to 0.67% of total soluble seed protein in the seeds of transgenic tobacco plants, was made possible by the seed-specific promoter (Fiedler and Conrad, Bio / Technology 10 (1995), 1090-1094). Since expression may also be possible in seeds that have been sown or that are germinating and may be desirable for the purposes of the present invention, such germination-specific and seed-specific promoters are also regulatory elements that are preferred according to the invention. The expression cassette of the invention may therefore comprise, for example, a seed-specific promoter (preferably a USP or LEB4 promoter), an LEB4 signal peptide, a gene to be expressed, and an ER retention signal. The cassette construction is illustrated by way of example in the form of a diagram in Figure 1 relating to a single chain antibody (scFv gene).

The expression cassette of the invention is made by fusing a suitable promoter with a suitable polypeptide DNA, and preferably DNA, that encodes a chloroplast-specific transit peptide and which is inserted between the promoter and the polypeptide DNA and a polyadenylation signal using conventional recombination and cloning techniques as described, for example T. Maniatis, EF Fritsch and J. Sambrook, Molecular Cloning: Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) and also in TJ Silhavy, ML Berman and LW Enquist, Experimente with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) and Ausubel, FM et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience (1987).

Particularly preferred are sequences that allow targeting to apoplast, plastid, vacuole, plasma membrane, mitochondria, endoplasmic reticulum (ER) or, in the absence of appropriate operative sequences, residence at the site of origin, particularly cytosol (Kermode, Crit. Rev. Plant Sci. 15, 4 (1996)

The

285-423). Localization in the ER and cell wall has been shown to be particularly beneficial 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 expression cassettes whose coding sequence encodes a fungicide-binding fusion protein, wherein part of the fusion protein is a transit protein that directs the translocation of the polypeptide. Particularly preferred are chloroplast-specific transit peptides that are cleaved enzymatically from the fungicide-binding polypeptide moiety upon translocation of the fungicide-binding polypeptide to the plant chloroplasts. Especially preferred is a transit peptide derived from an plastid transketolase (TK) or a functional equivalent of the transit peptide (for example, a small peptide subunit of Rubisc or ferredoxin NADP oxidoreductase).

Preferably, the polypeptide DNA or polypeptide cDNA required to produce the expression cassettes of the invention is amplified by polymerase chain reaction (PCR). Methods for amplifying DNA by PCR are known, for example, from Innis et al., PCR Protocols, A Guide to Methods and Applications, Academic Press (1990). DNA fragments produced by PCR can be suitably checked by sequence analysis to avoid polymerase errors in the constructs to be expressed.

The inserted nucleotide sequence, which encodes a fungicide-binding polypeptide, can be prepared synthetically or naturally, or it can contain a mixture of synthetic and natural DNA components. In general, synthetic nucleotide sequences with codons that are preferred for plants are prepared. These codons, which are preferred for plants, can be determined from codons whose proteins are most frequent and which are expressed in most of the plant species of interest. In preparing the expression cassette, various DNA fragments can be manipulated to obtain a nucleotide sequence that is read appropriately in the correct sense and which is provided with the correct reading frame. Adapters or linkers can be added to the DNA fragments to join together.

The promoter and terminator regions of the invention should preferably be provided with a linker or polylinker containing one or more restriction sites for insertion of the sequence for transcription. The linker typically has 1 to 10, usually 1 to 8, preferably 2 to 6, restriction sites. In regulatory areas, the linker is generally up to 100 bp, often less than 60 bp, but at least 5 bp. The promoter of the invention may be either native or homologous or even foreign or heterologous to the host plant. The expression cassette according to the invention comprises - in terms of transcription from the end 5 'to the end 3' of the promoter of the invention any desired sequence and region for transcription termination. The different termination regions are interchangeable as needed.

In addition, manipulations that provide suitable restriction sites or that remove excess DNA or restriction sites can be used. Where insertions, deletions or substitutions are possible, for example, transitions or transversions, in vitro mutagenesis, primer repair, restriction, or ligation may be used. In the case of appropriate manipulations such as restriction, chewing-back, or filling of blunt-end projections, complementary ends of the fragments can be provided for ligation purposes.

Of particular importance for the success of the invention is the attachment of a specific ER retention signal by SEKDEL (Schuoten, A. et al. Plant Mol. Biol. 30 (1996), 781 792), by which the average expression level is tripled to quadrupled. Other retention signals that occur naturally in plant and animal proteins found in the ER can also be used to construct the cassette.

Preferred polyadenylation signals are plant polyadenylation signals, preferably those which essentially correspond to T-DNA polyadenylation signals from Agrobacterium tumefaciens, in particular T-DNA gene 3 (octopine synthase) from Ti plasmid pTiACH5 (Gielen et al., EMBO J. 3 (1984) 835 et seq.) Or functional equivalents.

For example, the expression cassette of the invention may comprise a constitutive promoter (preferably the CaMV 35 S promoter), a LeB4 signal peptide, a gene to be expressed, and an ER retention signal. The cassette structure is shown as a diagram in Figure 2 relating to a single chain antibody (scFv gene). Preferably, the KDEL amino acid sequence (lysine, aspartic acid, glutamic acid, leucine) is used as the ER retention signal.

The fused expression cassette that encodes a polypeptide having fungicide binding properties is preferably cloned into a vector, for example pBin19, which is suitable for transforming Agrobacterium tumefaciens. Agrobacteria that are transformed with such a vector can then be used in a known manner to transform plants, especially livestock plants, e.g. tobacco plants, for example by dipping wounded leaves or leaf parts in Agrobacteria solution and then culturing them in a suitable medium. Transformation of plants by Agrobacteria is known among others from 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, p. 15-38, and S. B. Gelvin, Molecular Genetics of T-DNA Transfer from Agrobacterium to Plants, also in Transgenic Plants, p. 49-78. Transgenic plants can be regenerated from transformed cells of injured leaves or leaf portions in a known manner, and these transgenic plants comprise a gene for expressing a fungicide-binding polypeptide integrated into an expression cassette of the invention.

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

Using the above recombinant and cloning techniques, the expression cassettes of the invention can be cloned into suitable vectors that allow them to multiply, for example in E. coli. Suitable cloning vectors include, but are not limited to, pBR332, the pUC series, the M13mp series, and pACYC184. Particularly suitable are binary vectors that can replicate in both E. coli and agrobacteria, for example pBin19 (Bevan et al. (1980) Nucl. Acids Res. 12, 8711).

The invention further relates to the use of an expression cassette according to the invention for the transformation of plants, plant cells, plant tissues or plant organs. A preferred target in use is to mediate resistance to phytotoxically active fungicides.

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

The transfer of foreign genes into the genome of a plant is called transformation. In this process, the above-described methods of transforming and regenerating plants from plant tissues or plant cells to transient or stable transformation are used. Suitable methods are protoplast transformation of poylethylene glycol induced DNA uptake, a biolistic approach using a gene rifle, electroporation, incubation of dry embryos in a DNA-containing solution, microinjection and agrobacterium-mediated gene transfer. Said methods 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, for example pBin19 (Bevan et al., Nucl. Acids Res. 12 (1984) 8711).

The agrobacteria which have been transformed with the expression cassette according to the invention can then be used in a known manner for transforming plants, in particular livestock plants such as cereals, corn, soybeans, rice, cotton, sugar beet, rape, sunflower, rope, potato, tobacco, tomato, rape oilseed, clover, lettuce and various tree, walnut and Vitis species, for example coffee, fruit trees such as apple trees, pears or cherries, walnut trees such as walnut or pecan, and especially vines, for example by dipping injured leaves or leaf parts into an agrobacterial solution and then by cultivation in a suitable medium.

Functionally equivalent sequences that encode a fungicide-binding polypeptide are, according to the invention, those sequences that still have the desired functions despite different nucleotide sequences. Thus, functional equivalents include naturally occurring variants of the sequences described herein as well as artificial nucleotide sequences, for example, artificial nucleotide sequences that have been obtained by chemical synthesis and are adapted to the codon usage of the plant.

In particular, a functional equivalent is to be understood as a natural or artificial mutation of the originally isolated sequence that encodes a fungicide-binding polypeptide that continues to exhibit the desired function. Mutations include substitutions, additions, deletions, exchanges, or insertions of one or more nucleotide residues. Accordingly, the present invention also encompasses those nucleotide sequences that are obtained by modifying the nucleotide sequence. The purpose of such a modification may be, for example, to further limit the coding sequence contained therein, or, for example, to introduce additional restriction enzyme cleavage sites.

Other functional equivalents are those variants whose function is less or more pronounced in comparison to the starting gene or gene fragment.

In addition, artificial DNA sequences are useful as long as they mediate the desired tolerance to fungicides in order to prevent phytotoxic effects on crop plants as described above. Such artificial DNA sequences can be identified, for example, by back-translation of proteins having fungicide binding activity and which have been constructed by molecular modeling or by in vitro selection. Particularly suitable are DNA coding sequences which are obtained by back-translation of a polypeptide sequence in accordance with the use of a host plant-specific codon. The specific use of the codon can be readily determined by one skilled in the art of plant genetics by computer evaluation of other known genes of the plant to be transformed.

Other suitable equivalent nucleic acid sequences of the invention to be mentioned are those that encode fusion proteins, wherein the portion of the fusion protein is a non-plant fungicide-binding polypeptide or a functionally equivalent portion thereof. For example, the second portion of the fusion protein may be another polypeptide with enzymatic activity, or an antigenic polypeptide sequence by which the expression of scFvs (e.g., myc tag or his tag) can be detected. Preferably, however, the polypeptide having fungicide binding properties at the desired site of action is directed by a regulatory protein sequence, for example, a signal or transit peptide.

However, the invention also relates to the expression products obtained according to the invention and to the fusion proteins of the transit peptide and polypeptide having fungicide-binding properties.

Resistance or tolerance for the purposes of the present invention means the artificially acquired ability of plants to resist the action of fungicides with phytotoxic activity. It involves partial and especially complete insensitivity to these inhibitors during at least one generation of the plant.

The phytotoxic site of action of fungicides is generally leaf tissue, so that leaf-specific expression of an exogenous fungicide-binding polypeptide can provide sufficient protection. However, it is understood that the phytotoxic action of the fungicide need not be limited to the leaf tissue, but can be carried out in all other organs of the plant in a tissue-specific manner.

In addition, constitutional expression of an exogenous fungicide-binding polypeptide is preferred. On the other hand, inducible expression may also be desirable.

The efficacy of a transgenically expressed polypeptide with fungicide binding properties can be determined, for example, by in vitro propagation of shoot meristem in a fungicide-containing medium in alternating concentration series or by seed germination assays. In addition, the fungicide tolerance of the test plant, which has been altered with respect to type and level, can be tested in greenhouse experiments.

The invention further relates to transgenic plants transformed with the expression cassette of the invention and to transgenic cells, tissues, organs and propagation material of such plants. Particularly preferred are transgenic farm plants, for example cereals, corn, soybeans, rice, cotton, sugar beet, canola, sunflower, flax, potato, tobacco, tomato, oilseed rape, clover, lettuce and various scrub, tree, nut and Vitis species, for example coffee, fruit trees such as apple trees, pears or cherries, walnut trees such as walnut or pecan, and especially vine.

Transgenic plants, plant cells, plant tissues or plant organs can be treated with a fungicide with a phytotoxic action that inhibits plant enzymes, wherein plants, plant cells, plant tissues or plant organs that have not been transformed, successfully die or are damaged. Examples of suitable active ingredients are strobilurins, in particular methyl methoxyimino-α- (15 15 tolyloxy) -o-tolylacetate (BAS 490F), and metabolites and functional derivatives of these compounds. DNA which encodes a polypeptide having fungicide binding properties and which has been inserted into expression cassettes according to the invention can thus be used as a selectable marker.

The present invention has the advantage, especially in the case of livestock plants, that after inducing selected resistance of the livestock plant to fungicides with phytotoxic activity, such fungicides can be used in these crops to control harmful fungi, even at higher rates of application that otherwise would lead to plant damage. Examples of such fungicides with phytotoxic activity include, but are not limited to, herbicidal compounds of the following groups:

• sulfur, dithiocarbamates and their derivatives, such as ferric dimethyldithiocarbamate, zinc dimethyldithiocarbamate, zinc ethylenebisdithiocarbamate, etylénbisitiokarbamát manganese ethylenebisdithiocarbamate, manganese, zinc, tetramethylthiuram disulfides, ammonia complex (Ν, Ν-ethylenebisdithiocarbamate), zinc ammonia complex (Ν, Ν'-propylenebisdithiocarbamate), zinc Zinc (Ν, Ν'-propylene bis-dithiocarbamate), N, N'-polypropylene bis (thiocarbamoyl disulfide;

Nitro derivatives, for example dinitro (1-methylheptyl) phenyl crotonate, 2-sec-butyl-4,6-dinitrophenyl 3,3-dimethyl acrylate, 2-sec-butyl-4,6-dinitrophenyl isopropyl carbonate, diisopropyl 5-nitroisophthalate;

Heterocyclic substances, for example 2-heptadecyl-2-imidazoline acetate, 2,4-dichloro-6 (o-chloroanilino) -s-triazine, Ο, Ο-diethyl phthalimidophosphonothioate, 5-amino-1- [bis (dimethylamino) phosphinyl 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-methoxycarbonylaminobenzimidazole, 2- (2-furyl) benzimidazole, 2- (4-thiazolyl) benzimidazole, N- (1,1'-tetrachloroethylthiothetrahydrophthalimide, N-trichloromethylthiothetrahydrophthalimide, N-trichloromethylthiomethylthio) N-dimethyl-N-phenylsulfodiamide, 5-ethoxy-3-trichloromethyl-1,2,3-thiadiazole, 2-thiocyanatomethylthiobenzothiazole, 1,4-dichloro-2,516 dimethoxybenzene, 4- (2-chlorophenylhydrazono) -3- methyl-5-isoxazolone, pyridine-2-thio-1-oxide, 8-hydroxyquinoline or its copper salt, 2,3-dihydro-5-carboxanilido-6-methyl-1,4-oxathien, 2,3-dihydro-5-carboxanilido 6-methyl-1,4-oxathiin 4,4-dioxide, 2-methyl-5,6-dihydro-4H-pyran-3-carboxanilide, 2-methylfu ran3-carboxanilide, 2,5-dimethylfuran-3-carboxanilide, 2,4,5-trimethylfuran-3-carboxanilide, N-cyclohexyl-2,5-dimethylfuran-3-carboxamide, N-cyclohexyl-Nmethoxy-2,5-dimethylfuran- 3-carboxamide, 2-methylbenzanilide, 2-iodobenzanilide, Nformyl-N-morpholine-2,2,2-trichloroethyl acetal, piperazine-1,4-diylbis-1- (2,2,2-trichloroethyl) formamide, 1- (3 , 4-dichloroanilino) -1-formylamino-2,2,2-trichloroethane;

Amines, for example 2,6-dimethyl-N-tridecylmorpholine or salts thereof, 2,6-dimethyl-N-cyclododecylmorpholine or salts thereof, N- [3- (p-tert-butylphenyl) -2-methylpropyl] cis-2,6-dimethylmorpholine N- [3- (p-tert-butylphenyl) -2-methylpropyl] piperidine, (8 (1,1-dimethylethyl) -N-ethyl-N-propyl-1,4-dioxaspiro [4.5] decane-2-methanamine), Azoles, 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] -1H-1,2,4-triazole, 1- [2- (2,4-dichlorophenyl) -pentyl] -1H1,2,4-triazole, 2,4'-difluoro-a - (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-dich chlorophenyl) tetrahydrofuryl] -1H-1,2,4-triazole, 2- (4-chlorophenyl) -3cyclopropyl-1- (1H-1,2,4-triazol-1-yl) butan-2-ol (+) - 4-chloro-4- [4-methyl-2- (1H-1,2,4-triazol-1-ylmethyl) -1,3-dioxolan-2-yl] phenyl 4-chlorophenyl ether, (E) - (R, S) -1 (2,4-Dichlorophenyl) -4,4-dimethyl-2- (1H-1,2,4-triazol-1-yl) pent-1-en-3-ol, 4- (4ch, chlorophenyl) -2-phenyl-2- (1H-1,2,4, -triazolylmethyl) butyronitrile, 3- (2,4-dichlorophenyl) 6-fluoro-2- (1H-1, 4-chlorophenyl) 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-triazol-1-ylmethyl) pentan-3-ol, (+) - 2- (2,4 -d their chlorophenyl) -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- (1H17)

1,2,4-triazol-1-ylmethyl) hexanenitrile, α- (2-chlorophenyl) -α- (4-chlorophenyl) -5-pyrimidinethanol, 5-butyl-2-dimethylamino-4-hydroxy-6-methylpyrimidine, bis (chlorophenyl) -3-pyridinemethanol, 1,2-bis (3-ethoxycarbonyl-2-thioureido) benzene, 1,2bis (3-methoxycarbonyl-2-thioureido) benzene, strobilurines, for example methyl E-methoxyimino- [a] - (o-tolyloxy) -o-tolyl] acetate, methyl E-2- {2- [6- (2-cyanophenoxy) pyrimidin-4-yloxy] phenyl} -3-methoxyacrylate, methyl-emethoxyimino- [α- ( 2-phenoxyphenyl) acetamide, methyl E-methoxyimino- [α- (2,5-dimethylphenoxy) -olyl acetamide, • anilinopyrimidines, for example N- (4,6-dimethylpyrimidin-2-yl) aniline, N- [ 4-methyl-6- (1-propynyl) pyrimidin-2-yl] aniline; N- [4-methyl-6-cyclopropylpyrimidin-2-yl] aniline; phenylpyrrols, for example 4- (2,2-difluoro-1; 3-benzodioxol-4-yl) pyrrole-3-carbonitrile, cinamamides, for example 3- (4-chlorophenyl) -3- (3,4-dimethoxyphenyl) acryloylmorpholine, and a series of fungicides, for example dodecylguanidine acetate, 3- (3 - (3,5-dimethyl-2oxycyklohexyl) -2-hydroxyethyl] glutarate imide, Ν-methyl-, N-ethyl- (4-trifluoromethyl, -2 (3 ', 4'-dimethoxyphenyl) benzamide, hexachlorobenzene, methyl N- (2,6-dimethylphenyl) -N (2-furoyl) -DL alaninate, DL-N- (2,6-dimethylphenyl) -N- (2'-methoxyacetyl) alanine methyl ester, N- (2 ₁ 6-dimethylphenyl) -N-chloroacetyl-D, L-2-amino butyrolactone, methyl ₁ DL-N- (2,6-dimethylphenyl) -N- (phenylacetyl) alanine _I 5-methyl-5-vinyl-3- (3,5-dichlorophenyl) -2,4-dioxo-1,3-oxazolidine, 3- [3,5-dichlorophenyl (-5-methyl-5-methoxymethyl) -1,3-oxazolidine-2,4-dione, 3- (3,5-dichlorophenyl) -1-isopropylcarbamoylhydantoin, N- (3,5-dichlorophenyl) ) -1,2-dimethylcyclopropane-1,2-dicarboximide, 2-cyano- [N- (ethylaminocarbonyl) -2-methoxyimino] acetamide, N- (3-chloro-2,6-dinitro-4-trifluoromethylphenyl) -5-trifluoromethyl-3 chloro-2-aminopyridine.

Functionally equivalent derivatives of these fungicides have a comparable spectrum of activity against phytopathogenic fungi than those specifically mentioned in conjunction with less, equally or more pronounced phytotoxic activity.

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

General methods of cloning

Cloning steps performed within the scope of the present invention, for example, restriction digestions, agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, DNA fragment interconnection, transformation of E. coli cells, bacterial culture, phage multiplication and recombinant DNA sequence analysis were performed according to Sambrook et al. (1989) Cold Spring Harbor Laboratory Press; ISBN 0-87969-309-6).

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

Sequence analysis of recombinant DNA

Recombinant DNA molecules were sequenced using a laser fluorescent DNA sequencing instrument from Pharmacia, using the Sanger method (Sanger et al., Proc. Natl. Acad. Sci. USA 74 (1977), 54635467).

Generation of plant expression cassettes

The 35S CaMV promoter was inserted into plasmid pBin19 (Bevan et al., Nucl. Acids Res. 12, 8711 (1984)) as an EcoRI-Kpn1 fragment (corresponding to nucleotides 6909-7437 of cauliflower mosaic virus (Franck et al. Cell 21 (1980)). The polyadenylation signal of the T-DNA gene 3 from the Ti plasmid pTiACH5 (Gielen et al., EMBO J. 3 (1984) 835), nucleotides 11749-11939, was isolated as a Pull-HindIII fragment and cloned after addition of SphI linkers. to the cleavage site

Pvuil between SphI-Hindill cleavage site vector. This gave plasmid pBinAR (Hofgen and Willmitzer, Plant Science 66 (1990) 221-230).

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 depicts a cassette diagram for seed-specific scFV gene expression

Fig. 2 shows a diagram of a cassette for expression of the scFv gene in leaves of transgenic tobacco plants.

Fig. 3 shows a diagram of the construction of the scFv-antiBAS 490F gene (V-variable region, L-linker).

Fig. 4 shows functional characterization of scFv-antiBAS 490F in a direct ELISA.

Fig. 5 shows a cassette diagram for seed-specific expression of the scFvantiBas 490F gene.

Fig. 6 depicts the detection of existing antigen binding activity in the scFv-anti-BAS 490F antibody fragment in sheets after drying or lyophilization by ELISA.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

Since the fungicides are not immunogenic, they must be combined with a carrier material, for example KLH. If the molecule contains a reactive group, the linkage can be made directly; if not, the functional group is introduced when the fungicide is synthesized, or a reactive precursor is selected during synthesis to attach these molecules to the carrier molecule in one reaction step. Examples of coupling reactions can be found in Miroslavic Ferencik in "Handbook of Immunochemistry", 1993, Chapman & Halli, in the chapter Antigens, pages 20-49.

Repeated injection of this modified carrier molecule (antigen) is used to immunize, for example, Balb / c mice. When a sufficient number of antigen-binding antibodies are detectable by an enzyme-linked immunosorbent assay (ELISA), the spleen cells of these animals are obtained and fused with myeloma cells to cultivate the hybrids. In addition, an "fungicide-modified BSA" is used in the ELISA to differentiate the anti-hapten immune response from the KLH response.

Monoclonal antibodies are prepared by methods similar to known methods 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 " Monoclonal Antikorpergegen TNF und deren Verwendung " [monoclonal antibodies against TNF and their use]. European Patent Specification EP-A260610.

Example 2

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

First, mRNA was isolated from hybridoma cells and transcribed into cDNA. This cDNA acted as a template for amplifying the variable immunoglobulin genes VH and VK with 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 immunoglobulins were the starting point for the construction of the single chain antibody fragment (scFv-antiBAS 490F). In the following fusion PCR, the three components VH, VK and linker fragment were combined in a PCR reaction and scFv-antiBAS 490F was amplified (Fig. 3).

Functional characterization (antigen binding activity) of the engineered scFv-antiBAS 490F gene was performed after expression in the bacterial system. To this end, scFv-antiBAS 490F was synthesized in E. coli as a soluble antibody fragment using the method of Hoogenboom, H. R. et al., Nucleic Acids Research 19 (1991), 4133-4137. The activity and specificity of the engineered antibody fragment was checked by ELISA (Fig. 4).

To allow 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, exhibits strictly seed-specific expression of various foreign genes in tobacco (Bäumlein, H. et al., Mol. Gen. Genet. 225 (1991), 121-128). Transport of the scFv-antiBAS 490F polypeptide into the endopasmic reticulum resulted in stable accumulation of large amounts of antibody fragment. To this end, the scFv-antiBAS 490F gene was fused with a signal peptide sequence that guarantees entry into the endopasmic reticulum, and with an ER retention signal SEKDEL, which ensures that the polypeptide remains in the ER (Wandelt et al., 1992) (Fig. 5).

The engineered expression cassette was cloned into the pGSGLUC 1 binary vector (Saito et al., 1990) and transferred to Agrobacterium EHA 101 strain by electroporation. Recombinant agrobacterial clones were used for subsequent transformation of Nicotiana tabacum. Per ΟΙ 40 tobacco plants were regenerated per construct. Seeds at various stages of development were harvested from regenerated transgenic tobacco plants after self-pollination. Soluble proteins were obtained from these seeds in an aqueous buffer system after extraction. Analysis of the transgenic plants demonstrates that fusion of the scFv-antiBAS 490F gene into the DNA sequence of the ER retention signal SEKDEL allowed a maximum accumulation of 1.9% of the scFv-antiBAS 490F protein in mature seeds to be obtained.

The engineered scFv-antiBAS 490F gene was approximately 735 bp in size. The variable domains were fused to one another in the VH-L-VL sequence.

Specific selectivity was determined in mature tobacco seed extracts by direct ELISA. The values obtained clearly demonstrate that protein extracts contain 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 of the study was a single chain antibody fragment against the fungicide BAS 490F (scFv-antiBAS 490F). Functional characterization (antigen binding activity) of the engineered scFv-antiBAS 490F gene was performed after expression in the bacterial system and after expression in the tobacco leaves. The activity and specificity of the engineered antibody fragment was checked by ELISA.

To allow seed-specific expression of the antibody fragment in tobacco, the scFv-antiBAS 490F gene was cloned downstream of the USP promoter. The USP promoter, which was isolated from Vicia faba, exhibits strictly seed-specific expression of various foreign genes in tobacco (Fiedler, H. et al., Plant Mol. Biol. 22 (1993), 669-679). Transport of the scFv-antiBAS 490F polypeptide into the endoplasmic reticulum resulted in stable accumulation of large amounts of antibody fragment. To this end, the scFv-antiBAS 490F gene was fused with a signal peptide sequence that guarantees entry into the endoplasmic reticulum, and with an ER retention signal SEKDEL, which ensures that the polypeptide remains in the ER (Wandelt et al., 1992) (Fig. 1).

The engineered expression cassette was cloned into the pGSGLUC 1 binary vector (Saito et al., 1990) and transferred to Agrobacterium EHA 101 strain by electroporation. Recombinant agrobacterial clones were used for subsequent transformation of Nicotiana tabacum. Seeds at various stages of development were harvested from regenerated transgenic tobacco plants after self-pollination. Soluble proteins were obtained from these seeds in an aqueous buffer system after extraction. Transgenic plant analysis demonstrates that fusion of the scFv-antiBAS 490F gene into the DNA sequence of the ER retention signal SEKDEL under the control of the USP promoter caused synthesis of single chain antibody fragments with binding affinity for BAS 490F as early as day 10 of seed development.

Example 4

The scFv-antiBAS 490F gene was cloned downstream of the CaMV 35 S promoter to achieve expression of the antibody fragment throughout the plant, especially in leaves. This strong constitutional promoter mediates expression of foreign genes in virtually all plant tissues (Benfey and Chua, Science 250 (1990) , 956-966). Transport of the scFv-antiBAS 490F protein into the endoplasmic reticulum allowed stable accumulation of large amounts of antibody fragment in leaf material. First, the scFv-antiBAS 490F gene was fused to a signal peptide sequence that provides entry into the endoplasmic reticulum, and with an ER retention signal KDEL that ensures that the product remains in the ER (Wandelt et al., Plant J. 2 (1992), 181- 192). The engineered expression cassette was cloned into the binary vector pGSGLUC 1 (Saito et al., Plant Cell Rep. 8 (1990), 718-721) and transferred to Agrobacterium EHA 101 strain by electroporation. Recombinant agrobacterial clones were used for subsequent transformation of Nicotiana tabacum. Approximately 100 tobacco plants were regenerated. Leaf material was harvested from the regenerated transgenic tobacco plants at various developmental stages. Soluble proteins were obtained from this sheet material in an aqueous buffer system after extraction. Subsequent analyzes (western blot analysis and ELISA) demonstrated that a maximum accumulation of more than 2% of the biologically active antigen-binding scFv-antiBAS 490F polypeptide was obtained in the leaves. These high expression values were determined in fully grown green leaves, but the antibody fragment was also detected in the aged leaf material.

Example 5

PCR amplification of a cDNA fragment encoding a single chain anti-BAS 490F antibody using synthetic oligonucleotides.

PCR amplification of single chain antibody cDNA was performed in a Perkin Elmer DNA Thermal Cycler. The reaction mixtures contained 8 ng / μΙ single stranded template cDNA, 0.5 μΜ relevant oligonucleotides, 200 μΜ nucleotides (Pharmacia), 50 mM KCl, 10 mM Tris-HCl (pH 8.3 at 25 ° C, 1.5 mM MgCl _2). ) and 0.02 U / μΙ of Taq polymerase (Perkin Elmer). The amplification conditions were as follows:

Annealing temperature: 45 ° C

Denaturation temperature: 94 ° C,

Elongation temperature: 72 ° C,

Number of cycles: 40

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

Example 6

Production of transgenic tobacco plants that express a cDNA encoding a single chain antibody with fungicide binding properties.

Plasmid pGSGLUC 1 was transformed into Agrobacterium tumefaciens C58G1: pGV2260. For the transformation of tobacco plants (Nicotiana tabacum cv. Samsun NN), a 1:50 dilution of a day to day cultivation of a positively transformed agrobacterial colony in Murashige-Skoog medium (Physiol. Plant. 15 (1962) 473 et seq.) Containing 2% sucrose was used. (2MS medium). In a Petri dish, leaf discs of sterile plants (approximately 1 cm ^{2 each} ) were incubated for 5-10 minutes at an agrobacterial dilution of 1:50. This was followed by a two-day incubation in the dark at 25 ° C on 2MS medium containing 0.8% Bacto-Agar. Cultivation continued after 2 days at 16 hours light and 8 hours dark and continued to weekly rhythm on MS medium containing 500 mg / l Claforan (cefotaxime sodium), 50 mg / l kanamycin, 1 mg / l benzylaminopurine (BAP), 0, 2 mg / l naphthylacetic acid and 1.6 g / l glucose. Growing shoots were transferred to MS medium containing 2% sucrose, 250 mg / l Claforan and 0.8% Bacto-Agar.

Example 7

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

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

To allow expression of the scFv-antiBAS 490F gene in the endoplasmic reticulum, the foreign gene was expressed under the control of the CaMV 53S promoter as a translation fusion with the signal peptide LeB4 (N-terminus) and the ER retention signal KDEL (C-terminus). Transport of the scFv-antiBAS 490F polypeptide into the endoplasmic reticulum allowed stable accumulation of large amounts of active antibody fragment. After the leaf material was collected, the sections were frozen at -20 ° C (1), lyophilized (2) or dried at room temperature (3). Soluble proteins were obtained from leaf material by extraction in aqueous buffer and the scFv-antiBAS 490F polypeptide was purified by affinity chromatography. Equal amounts of purified scFv-antiBAS 490F polypeptide (frozen, lyophilized and dried) were used to determine antibody fragment activity (Figure 6). Fig. 6A depicts antigen binding activity for scFv-antiBAS 490F polypeptide purified from fresh (1), lyophilized (2) and dried leaves (3). In FIG. 6B, the respective amounts of scFv-antiBAS 490F protein (about 100 ng) used for ELISA were determined by Western blot analysis. The sizes of the protein molecular weight standards are shown on the left. Approximately identical antigen binding activities were found.

Example 8

To demonstrate fungicide tolerance for transgenic tobacco plants that produce a polypeptide with fungicide-binding properties, these tobacco plants were treated with different amounts of BAS 490F. In all cases, it was possible to demonstrate in the greenhouse that plants expressing the scFv-antiBAS 490F gene showed higher tolerance to the fungicide BAS 490F and less pronounced phytotoxic effects compared to the control.

Claims (20)

Process for the preparation of methyl methoxyimino-α- (o-tolyloxy) o-tolylacetate (BAS 490F) tolerant plants, characterized in that it comprises the expression of an exogenous polypeptide in methyl methoxyimino-α- (o-tolyloxy) -olytolylacetate binding (BAS 490F) in the plant. The method of claim 1, wherein the exogenous methyl methoxyimino-α- (o-tolyloxy) -o-tolylacetate (BAS 490F) binding polypeptide is a fragment of a single chain antibody. The method of claim 1, wherein the exogenous methyl methoxyimino-α- (o-tolyloxy) -o-tolylacetate (BAS 490F) binding polypeptide is a whole antibody or fragment derived therefrom. 4. A plant expression cassette comprising a promoter, a signal peptide, a gene encoding the expression of an exogenous methyl methoxyimino-α- (o-tolyloxy) -o-tolylacetate (BAS 490F) binding polypeptide, an ER retention signal, and a terminator. Expression cassette according to claim 4, characterized in that the constitutive promoter used is the CaMV 35S promoter. 6. The expression cassette of claim 4, wherein the gene to be expressed is a single chain antibody fragment gene. Expression cassette according to claim 4, characterized in that a gene or a gene fragment of a methyl methoxyimino-α- (o-tolyloxy) -o-tolylacetate (BAS 490F) binding polypeptide is used as a translational gene or gene fragment. fusions with other functional proteins, such as enzymes, toxins, chromophores, and binding proteins. Expression cassette according to claim 4, characterized in that the gene to be expressed is obtained from a hybridoma cell or by other recombinant methods, for example an antibody phage display method. Use of an expression cassette according to claim 4 for transforming dicotyledonous or monocotyledonous plants that constitutively express an exogenous methyl methoxyimino-α- (o-tolyloxy) -o-tolylacetate (BAS 490F) binding polypeptide by seed or leaf specificity. The use of claim 9, wherein the expression cassette is transferred to a bacterial strain and the recombinant clones obtained are used to transform dicotyledonous or monocotyledonous plants that constitutively express an exogenous methyl methoxyimino-α- (o-tolyloxy) -olyl acetate (BAS 490F) binding polypeptide. seed or leaf specific. Use of an expression cassette according to claim 4 as a selectable marker. Use of the transformed plant obtained in accordance with claim 10 for the preparation of methyl methoxyimino-α- (o-tolyloxy) -o-tolylacetate binding polypeptide (BAS 490F). 13. A process for transforming a plant by introducing a gene sequence which encodes a methyl methoxyimino-.alpha .- (o-tolyloxy) -olytolylacetate (BAS 490F) binding polypeptide into a plant cell, callus tissue, whole plant and plant cell protoplasts. Process according to claim 13, characterized in that the transformation is carried out by means of an agrobacterium, in particular of the species Agrobacterium tumefaciens. Process according to claim 13, characterized in that the transformation is carried out by electroporation. Method according to claim 13, characterized in that the transformation is carried out by means of a particle bombardment method. 17. Preparation of a methyl methoxyimino-α- (o-tolyloxy) -o-tolylacetate (BAS 490F) binding polypeptide, characterized in that it consists of expressing a gene which encodes the polypeptide in a plant or plant cells and then isolating the polypeptide. The plant comprising the expression cassette of claim 4, wherein the expression cassette provides tolerance to methyl methoxyimino-α- (o-tolyloxy) -olyl acetate (BAS 490F). A method for controlling phytopathogenic fungi in commercial transgenic plants tolerant to methyl methoxyimino-α- (o-tolyloxy) -o-tolylacetate (BAS 490F), characterized in that it comprises the use of methyl methoxyimino-α- (o-tolyloxy) -otolylacetate (BAS 490F) against which this crop plant produces methyl methoxyimino-α- (o-tolyloxy) -olytyl acetate (BAS 490F) polypeptides or antibodies. 20. Methoxyimino-α- (o-tolyloxy) -olytolylacetate (BAS 490F) polypeptide or antibody with high affinity binding to methyl methoxyimino-α- (o-tolyloxy) -o-tolyl acetate (BAS 490F), which is produced according to claim 17. 1/6 Fig. 1 Diagram of a cassette for seed-specific expression of the scFV gene Prom. SP scFv Tag Term r -f " -r with \ SEKDEL 0U3P prorótar temdnator CäW 35 O signal. The peptide was a F4 E3 c-nyc-Tag 2/6 Fig. 2 is a diagram of a scFv gene expression cassette in leaves of transgenic tobacco plants. KDEL _ 5 ' 3 ' Prom. SP scFv Tag Term Dpcarótcr CäW 35S {$ 3 Thematic CäM / 35 ^ signal, pqptid Jj = B4 jeäxostäaaaý IV 3/6 Fig. 3 ScFv-antiBAS 490F gene construction diagram (V - variable region, L - linker) 4/6 Fig. 4 Functional characterization of scFv-antiBAS 490F in direct ELISA 5/6 PV fWl-W Fig. 5 Diagram of the cassette for seed-specific expression of the scFv-anti-BAS 490F gene Rnštzukt ^p XOtn. SP scFv Tag Term 11 ^... “í .......- T ^r SEfCDEL. □ pranótnrLä34 00 tamiratcr C & M7 35 E3 onyo-tag 6/6 PV «Η-Ή Fig. 6 Detection of existing antigen binding activity. in scFv-anti-BAS 490F antibody fragment in sheets after drying or lyophilization by ELISA. Fig. 6A shows antigen binding activity for scFv-anti-BAS 490F polypeptide from fresh (1), lyophilized (2) and dried (3) leaves. Fig. 6B shows appropriate amounts of scFv-anti-BAS 490F protein (approximately 100 ng) used for ELISA assays determined by Western blot analysis. The sizes of the protein molecular weight standards are shown on the left.