MXPA00001394A - Tolerance of trichothecene mycotoxins in plants and animals through the modification of the ribosomal protein l3 gene - Google Patents

Abstract

Fusarium graminearum is a plant pathogen, attacking a wide range of plant species including corn (ear and stalk rot), barley, and wheat (head blight). Fusarium epidemics result in millions of dollars of losses in crop revenues. Fusarium graminearum infection in the cereals reduces both grain yield and quality. Mycotoxins are produced by many fungal Fusarium species and thus the grain becomes contamined with these mycotoxins, such as the trichothecenes. The major trichothecene produced by F. graminearum as well as F. culmorum is deoxynivalenol (abbreviated as DON, also known as vomitoxin). Trichothecenes are potent protein synthesis inhibitors and are quite toxic to humans and livestock. A yeast gene has been identified which is resistant to the trichothecene, trichodermin. A corresponding plant gene has been prepared, which has been used to transform plants and would be suitable to transform animals. These transformed plants have an increased tolerance to trichothecenes and could be more resistant to Fusarium infestation. Potentially, transformed animals could have an increased tolerance to the trichothecene mycotoxins. This modified gene could also be used as a selectable marker in transformation experiments.

Description

TOLERANCE OF MYCOTOXINS OF TRICOTECHENES IN PLANTS AND ANIMALS THROUGH THE MODIFICATION OF THE L3 RIBOSOMAL PROTEIN GENE The present invention relates to a modified gene, wherein a host transformed with this gene is resistant to trichothecene mycotoxins, wherein the wild-type form of the gene encodes a ribosomal L3 protein. The present invention also relates to a method for using this gene to transform plants to provide increased resistance against trichothecene mycotoxins. The present invention also relates to a method for using the gene to transform animals to increase the tolerance of the animal to trichothecene mycotoxins. The present invention further relates to a method for using the gene as a selectable marker in the transformation.

BACKGROUND OF THE INVENTION Globally, Fusa ri um gramin ea rum is a major plant pathogen, which attacks a wide range of plant species that include many important crop plants such as corn (spike rot and stem) , barley and wheat (head rust). Favorable environmental conditions (contributing temperatures and high humidity) can result in Fusa ri um epidemics and millions of dollars lost in crop income. Infection by F. gramin ea rum in cereals reduces both the production and the quality of the grain. The reduction of the quality is the result of the mycotoxins produced by this species of fungus; These fungal toxins remain in the contaminated cereal after harvesting and pose serious health risks to animals and humans who may consume the grain. Low levels of pollution in non-epidemic years still account for 5% of grain loss in corn farmers in Ontario, a figure that translates into approximately $ 27 Million for the pork industry that uses this corn for food. In epidemic years, this figure in dollars can double or triple. These direct losses to growers include crop and animal losses associated with reduced feed and lower quality feed. Overall, the United Nations FOA estimates that 25% of the world's food crops are affected by mycotoxins each year (Mannon and Johnson, 1985, Fungi down on the Farm, New Scientist 105: 12-16). The mycotoxins of Fusa ri um are found in the main cereals species that include corn, wheat, barley, oats, rice and others. The disease is more prevalent in temperate climates. Mycotoxins or fungal toxins are produced by many fungal species. The species Fusa ri um graminearum as well as F. sambucin um, F. poa e, F. sporo tri chi oides, F. culmorum and F. crookwel l teach are capable of producing a class of compounds known as trichothecenes. This large family of sesquiterpene epoxides is closely related and varies by the position and number of hydroxylations and substitutions of a basic chemical structure. The main tricothecene produced by F. Gramin ea rum is deoxynivalenol (DON) also known as vomitoxin for its ability to induce vomiting. These chemicals are potent inhibitors of the synthesis of eukaryotic proteins, toxic to both humans and animals, and other organisms such as plants. Due to its toxicity, safety threshold levels have been recommended for contamination with DON mycotoxin in the grain used for human food and animal feed (Van Egmond, 1989, Food Addit., Contam 6: 139-188; Underhill, CFIA Fact Sheet, Mycotoxins, 1996). The most dangerous for livestock producers is that if the livestock animals are fed with contaminated grain they suffer severe health hazards, which include reduced intake of food, reduced growth rate, reduced fertility, immunosuppression, diarrhea, vomiting and possible death. Some of these effects are directly observable and therefore measurable, such as weight loss, while other effects, such as immunosuppression, are more delicate and less quantifiable. In general, a reduction of 10 to 20% of the pig's litter rate combined with a reduction of 10 to 20% in the growth rates of the animal can cause an approximate reduction of 17 to 44% in the profit margin for the animals. pig producers. The effects of mycotoxins on poultry and livestock are less quantified since these species are less sensitive to DOW contamination in their diet, and no detailed, economic threshold assessments have been made. During years of epidemics with Fusa ri um, Canadian grain that is above the safety threshold of 2 ppm DON for human consumption must be degraded for animal feed. If the grain contains more than 10 ppm of DON, it becomes incapable to be animal feed and must be discarded. Since many farmers use their own grains for feeding animals on the farm, and may not be able to assess the level of grain mycotoxin contamination, a considerable amount of food contaminated with DON is used. In this way, it is important to minimize the level of trichothecenes in foodstuffs, which can be accompanied by the control of the epidemics of the Fusa ri um species in the cultivated cereal species. Chemical treatment has been used in the past to control the biosynthesis of trichothecenes. One such inhibitor is ancymidol, which has been described in U.S. Patent No. 4,816,406. However, in the present environment, it is desirable to avoid chemical control, especially in foodstuffs. Thus, there is a need for a method to control the epidemics of Fu sa ri um species, particularly F. gram inea rum by using non-chemical methods. It has been shown that trichothecenes act as virulence factors in the head of the wheat. This was demonstrated by inoculating wheat heads with non-trichothecene-producing mutants of F. grami n ea rum in which the first gene specific to the trichothecene biosynthetic pathway has been broken through genetic management (Desjardins et al., 1996 , Mol. Plant-Micr. Int. 9: 775-781). In two years of trial without field, the non-trichothecene producing strains were less virulent than the trichothene producing or reverting strains, as measured by various disease parameters. Similar results have been obtained from the inoculation of corn grown in the field with these trichothene-producing strains of Fusa ri um. Therefore, the increase in tolerance of wheat or maize to the effects of trichothecenes should lead to a disease reduced.

BRIEF DESCRIPTION OF THE INVENTION Studies with animals have concluded that the biological response to trichothecene mycotoxins is rapid if the route of administration is oral, topical or parenteral. Before its excretion from the body that usually occurs in the space of 24 to 72 hours after injection, the highest concentration of toxins is usually found in the bile, gallbladder, kidneys, liver and intestines. The mode of action of all trichothecenes is related to their ability to bind to the 60S ribosomal subunit and essentially inhibit the activity of the peptidyl transferase. This is achieved either by inhibiting the initiation of protein synthesis, elongation of the peptide chain or growth peptide termination (Freinberg and McLaughlin, 1989, Biochemical mechanism of actinic trichothecene mycotoxins, p27, In: Trichothocene Mycotoxicosis: Pathophysilogic Effects Vol 1 CRC Press, Boca Ratón Fl.). The effect of these toxins on protein synthesis is observed in a diverse array of eukaryotic cells such as the mammalian and yeast cell lines. Each ribosome apparently has a single binding site for the toxin, and research suggested that all trichothecenes compete for the same ribosomal binding site on the L3 ribosomal protein. The mutant of Sa ccharomyces cer evisiae (yeast) that was isolated spontaneously by Schindler et al. (1974, Nature, 248: 548-536) showed to be able to grow in the trichodermin of the tricothecene drug. This yeast line was shown to have altered the function of the 60S ribosomal subunit and when the responsible gene was cloned, it was found to code for the ribosomal L3 protein (Rpl 3) (Schultz and Friesen, 1983, J. Bacteriol. 155: 8-14). In one aspect of the present invention, the information obtained by comparing the wild-type yeast gene and the mutant yeast gene was used to modify the corresponding gene of the Oryza sa t i va rice, a cereal plant species. The transgenic tobacco plants were then created, using the modified rice gene, and these plants demonstrated greater tolerance to trichothecene mycotoxins than wild type tobacco plants, or plants transformed with the wild type rice gene. Transgenic maize embryo cultures containing the modified rice Rpl 3 gene also exhibited a higher tolerance to tricothecene DON, compared to cultures containing the wild type rice Rpl 3 gene. In this way, this modified rice gene can provide protection against trichothecene mycotoxins and thus provide resistance to a Fusa ri um pest in other plant species. Thus, according to the present invention, a modified gene is provided, wherein a host transformed with the gene has an increased resistance to trichothecene mycotoxins, wherein the wild-type form of the gene encodes a ribosomal L3 protein. In one embodiment of this aspect of the invention, the gene encoding the ribosomal L3 protein is from rice. The present invention further provides a suitable cloning vector containing the modified ribosomal L3 protein gene. In a further aspect of the invention, there is provided a transformed plant, transformed with the modified Rpl 3 gene wherein the transformed plant has increased resistance to the Fu sa ri um pest. The present invention also includes the seed from the transformed plant, referred to above. In yet a further aspect of the invention, there is provided a transformed animal, transformed with the modified Rpl 3 gene, wherein the transformed animal has an increased tolerance to the trichothecene mycotoxins. In still an aspect of the present invention, a method is provided for increasing the resistance to the pest of Fusari um by transforming a suitable plant with a modified gene., wherein the plant transformed with the gene has increased resistance to the mycotoxins of tricot eceans, where the wild-type form of the gene codes for the L3 ribosomal protein. The present invention also provides a method for increasing tolerance to mycotoxins of trichothecenes by transforming a suitable animal with a modified gene, wherein the animal transformed with the gene has increased tolerance to trichothecene mycotoxins, and wherein the wild type of the gene codes for a ribosomal L3 protein. In a further aspect in the present invention, there is provided a method for using the modified gene of the invention as a selectable marker in transformation experiments.

BRIEF DESCRIPTION OF THE DRAWINGS These and other features of the invention will become more apparent from the following description in which reference is made to the accompanying drawings, wherein: Figure 1 shows a comparison of the amino acid sequence of Wild type yeast Rpl 3 (RPL13WT; SEQ ID No .: 1), the upper line, and the tricodermin resistant yeast sequence (SCRP 13 PRO; SEQ ID No .: 2), the bottom line. The change of amino acids W-255 to C-255 is shown .. The access number in the GenBank for the mutant yeast gene is J01351. Figure 2 shows the comparison of the rice Rpl 3 sequence (SEQ ID No. 3), the upper line, and the sequence of the tricodermin-resistant yeast (SEQ ID No. 2), the lower line. This comparison led to the predicted change from residue W258 (rice numbering) to C258, to create the Rpl 3: c2 5 8 gene of mycotoxin-tolerant rice. The access number in the GenBank for the rice gene is D12630. Figure 3 shows the plasmid map of the binary vector pBin 19 of Agroba cteri um t ume fa in s for the transformation of the plant (Bevan, M. 1984, Nucleic Acids Research 12: 8711-8721). Figure 4 shows the plasmid pCAMterX, which was used to clone the Rpl 3 genes at the multiple cloning site (MCS). The Rpl 3 genes were expressed under the direction of the cauliflower mosaic virus (CAMV 35S promoter) arranged in tandem (70S promoter). Figure 5 shows the growth rate of the transgenic tobacco cells containing either the Rpl 3 gene of wild-type rice (C3 cells; Figure 5A), or the modified version of Rpl 3 (C4 cells; Figure 5B). Cells were cultured in medium containing either no toxin or 25 ppm DON.

DETAILED DESCRIPTION OF THE INVENTION In accordance with the present invention there is provided a gene of modified L3 ribosomal protein, whose gene product provides resistance to trichothecene mycotoxins. Previous work has shown that trichothecenes bind to a single or individual in the eukaryotic 60S ribosome. A spontaneous mutant from yeast S. cerevi s i a e, which is the drug resistant tricothecene, tricodermin, has been identified. The corresponding wild-type gene was identified and the nature of the mutant gene was found to result from an individual amino acid change at position 255 of the proposed Rpl 3 protein (Figure 1). This mutant represents only one example of a number of possible mutants of the same gene that will result in resistance to the trichodermin drug of trichothecenes. In this manner, the present invention is directed to a gene of the modified ribosomal L3 protein, wherein the modified gene provides resistance to the trichothecenes. Not waiting to be bound by any particular theory, it is believed that the mycotoxin binds to the wild-type protein, but not to the gene product, mutant. In this way, the modified Rpl 3 gene in the present invention will obtain that it allow the function of the peptidyl transferase in the ribosomal complex, but it will be modified to a sufficient degree to reduce the binding capacities of the mycotoxin. If the mycotoxin has a reduced effect, the plant is better able to defend itself against the fungus and thus reduce the incidence of disease. In one embodiment of this aspect of the invention, the gene encoding Rpl 3 is from a plant. In one example of this embodiment, the Rpl 3 gene of the corresponding rice was identified and modified to reflect the modification in the yeast mutant gene. The resulting Rpl 3 gene also showed resistance to trichothecenes. A plant source of the Rpl 3 gene was chosen in place of the yeast gene, since it was anticipated that the plant gene will have an improved expression in a plant host, which will be the yeast gene. Rice was chosen because it is more closely related to wheat and corn, two examples of plant host. Although the Rpl 3 gene of rice was used as an example, other suitable plant genes could also have been used. Suitable examples include: the corresponding Arabidopsi s tha l i ana gene and the monocotyledonous sources, e.g., wheat and maize. For animal transformation, the corresponding bovine gene will be a suitable target for the modification. As noted previously, the invention is not limited to the use of modified plant Rpl 3 genes to confer resistance to trichothecenes. Any suitable modified modified plant or animal gene that confers resistance to trichothecenes can be used in accordance with the present invention to transform plants or animals to provide resistance to trichothecenes. The area of modification in the yeast gene is a highly conserved area. shown below in Table 1 is the amino acid homology that occurs around this critical part of the protein, in plants, rats, mice, human yeast, C. elegans and Escherichia Coli. Any of these can be used as source material for the Rpl 3 gene. In each case, the amino acid sequence will be aligned with the mutant yeast gene and the corresponding mutation made in the corresponding Rpl 3 gene. As the entire area between the amino acid residue 240 and 263, based on amino acid numbering is yeast, is highly conserved, it is considered part of the present invention to modify any of the amino acids within this region to obtain a modified gene sequence . The modification may include substitutions or deletions of short lengths, additions or inversions. As previously noted, the modified gene product must continue to allow the function of peptidyl transferase activity, but has reduced binding capacities to the mycotoxin TABLE 1 COMPARISON OF THE SEQUENCE OF VARIOUS L3 RIBOSOMAL PROTEINS BETWEEN WASTE 240 AND 263 Amino Acid Sequence 240 258 263 Rice R Arabidopsis 1 R Arabidops? S 2 R L I I Bars represent amino acids identical to the wild type yeast RPL3 sequence The present invention further provides a suitable cloning vector containing the modified Rpl 3 gene. Any cloning vector can be used. The chosen cloning vector will of course reflect the host in which the final transformation will be made. The present invention includes both animals and transformed plants. Suitable plant cloning vectors may include: Agrobacterium binary vectors, such as Bin 19 (Bevan, M., 1984 Nucleic Acids Research 12: 8711-8721) and vectors used for microprojectile bombardment of monocotyledons. For the transformation of plants, the cloning vector may additionally comprise a 3 'untranslated region. A 3 'non-translated region refers to that portion of a gene comprising a DNA segment containing a polyadenylation signal and any other regulatory signal capable of effecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by the direction of the addition of the polyadenylic acid tracks to the 3 'end of the mRNA precursor. Polyadenylation signals are commonly recognized by the presence of homology to the canonical form 5'-AATAAA-3 'although variations are not common. Examples of suitable 3 'regions are the 3' transcribed, non-translated regions containing a polyadenylation signal of the Agrobacterium (Ti) tumor induction plasmid genes, such as the nopaline-synthase genes (gene Nos) and plant such as the genes of soy storage protein and the small subunit of the ribulose-1, 5-bisphosphate-carboxylase gene (ssRUBISCO). The region not translated 3 'from the Rpl 3 gene and modified from the present construct can be used for expression in plants, without any additional region. The vectors of the present invention may also contain a suitable promoter. In the plant transformation examples of the present invention, any strong promoter will be suitable. Suitable examples include, but are not limited to, cauliflower mosaic virus (CAMV 35S). It can be used alone or together with other plant promoters. The cloning vector of the present invention may also additionally include enhancers, either translational or transcriptional enhancers, as may be required. These integrating regions are well known to those skilled in the art and can include the ATG initiation codon and adjacent sequences. The initiation codon must be in phase with the reading frame of the coding sequence to ensure translation of the complete sequence. The translation control signals and the initiation codons can be from a variety of origins, both natural and synthetic. The transductional initiation regions can be provided from the source of the transcriptional initiation region, or from the structural gene. The sequence can also be derived from the promoter selected to express the gene, and can be modified specifically to increase the translation of the mRNA. To assist in the identification of the transformed plant cells, the vector of this invention can be further manipulated to include selectable plant markers. Useful selectable markers include enzymes that provide resistance to an antibiotic such as gentamicin, hygromycin, kanamycin, and the like. Similarly, enzymes are useful in the production of a compound identifiable by color change such as GUS (ß-glucuronidase) or luminescence., such as luciferase. Also considered part of this invention are the transgenic plants that contain the modified Rpl 3 gene of the present invention. Methods for regenerating whole plants from plant cells are known in the art, and the method for obtaining transformed and regenerated plants is not critical to this invention. In general, the transformed plant cells are cultured in an appropriate medium, which may contain selective agents such as antibiotics, where selectable markers are used to facilitate the identification of transformed plant cells. Once the callus is formed, the formation of the shoots can be encouraged by using the appropriate plant hormones according to known methods and the sprouts transferred to the regeneration medium for the regeneration of plants. Then, the plants can be used to establish repetitive generations, either from seeds or using vegetative propagation techniques. The vector constructs of the present invention can be introduced into the plant cells using Ti-plasmids, Ri-plasmids, plant virus vectors, direct DNA transformation, microinjection, electroporation, etc. For reviews of these techniques, for example, Weissbach, and Weissbach, Methods for Plant Molecular Biology, Academy Press, New York VIII, pp. 421-463 (1988); and Geierson and Corey, Plant Molecular Biology, 2d Ed. (1988). Suitable plant hosts include but are not limited to corn, barley, wheat, rice, rye, oats and millet. Techniques for generating transgenic animals have been developed and optimized in mice (Hogan et al., 1986, Manipulation of the mouse embody: a laboratory manual, Cold Spring Harbor Laboratory Press: New York), sheep (Wright et al., 1991, Biotechnology NY 9: 831-834), goats (Ebert and Schindler, 1993, Teriogenology, 39: 121-135) and creeds (Rexroad and Purcel, 1988, Proc. Llth Int. Congress of Animal Reproduction and Artificial Insem. 5: 29- 35). In general, these methods are based on the pro-nuclear microinjection of fertilized zygotes taken from superovulated female animals. The zygote pronuclei are microinjected with several thousand copies of the new gene construct and then transferred to the recipient females for the remainder of the gestation period. Confirmation of the integration of the transgene is by Southern hybridization of the somatic tissues taken from the offspring, and the analysis of the gene product of the gene function. Gene replacement experiments will allow the insertion of a modified Rpl 3 instead of an endogenous wild type (susceptible) gene. Of the animal, which can confer the animal with a higher level of resistance to the effect of the mycotoxin (Stacey et al., 1994, Mol Cell Biol .. 14: 1009-1016). Suitable animal hosts include any animal that has, at least as a part of its diet, the food grains referred to above as hosts of susceptible plants. These animals will include, but are not limited to, cows, sheep, goats, pigs, horses, poultry and even man. As noted previously, pigs are very sensitive to mycotoxins. When referring to specific sequences in the present invention, it is understood that these sequences include within their scope, sequences that are "substantially homologous" to these specific sequences. The sequences are "substantially homologous" when at least about 80% and more preferably at least about 90 to 95% of the nucleotides correspond over a defined length of the molecule. Sequences that are "substantially homologous" include any substitution, deletion or addition within the sequence. DNA sequences that are substantially homologous can be identified in Southern hybridization experiments, for example, under severe hybridization conditions (see, Maniatis et al., In Molecular Cloning (A Laboratory Manual), Cold Spring Harbor Laboratory (1982) p 387 to 389). Specific sequences, referred to in the present invention, also include sequences that are "functionally equivalent" to the specific sequences. In the present invention, functionally equivalent sequences refer to sequences which, while not identical to the specific sequences, provide the same or substantially the same function. DNA sequences that are functionally equivalent include any substitution, deletion or addition within the sequence. With reference to the present invention, functionally equivalent sequences will provide resistance to trichothecenes. As described above, the modified gene of the present invention must still allow peptidyl transferase activity but has reduced binding capacities for the mycotoxin. Thus, a further aspect of the invention is a transformed plant, transformed with the modified Rpl 3 gene, wherein the transformed plant has increased resistance to the Fusa ri um pest. In a further aspect of the invention, there is provided a transformed animal, transformed with the modified Rpl 3 gene, wherein the transformed animal is more tolerant to the mycotoxins of the trichothecenes. In yet another aspect of the present invention, there is provided a method for conferring Fusa ri um pest resistance, comprising the steps of: providing a modified gene, wherein the wild-type form of the gene encodes a ribosomal L3 protein; and transform a suitable plant with the modified gene. In yet another aspect of the present invention, there is provided a method for increasing the tolerance in animals to the mycotoxins of the trichothecenes comprising: providing a modified gene, wherein the wild-type form of the gene encodes a Lioresombo protein L3; and transform an appropriate animal with the modified gene. Another aspect of the present invention is the use of the modified gene as a selectable marker in transformation experiments. Selectable marker genes such as neomycin phosphotransferase npt II from bacterial transposons, or hygromycin-phosphotransferase hpt, or the dhfr gene of mammalian hydrofolate reductase, have been successfully used in many plant systems (Sproule et al. , 1991, Theor, Appl. Genet, 82: 450-456, Dijak et al., 1991, Cell Tissue Plan and Organ Culture 25: 189-197). These genes have allowed the use of antibiotics. kanamycin, hygromycin and methotrexate, respectively, in the selection of transgenic plants and at the level of protoplasts for the selection of somatic hybrids. Alternatively, selection strategies are essentially useful for carrying out multiple transformations, which is the repeated transformation of a plant with several different genes. To effect this, new and effective selective agents are desirable. New screening strategies based on genes that detoxify compounds other than antibiotics are also useful in cases where the use of detoxification or antibiotic degradation genes is not allowed or desired in the transgenic organism. Under these cases, it would be desirable to have a gene that confers a useful phenotype (resistance to disease) as a selectable marker. In accordance with the present invention, the plant or animal cells that were exposed to DON are unable to proliferate in the presence of this toxin. Cell lines transformed with the modified gene of the present invention are more resistant to DON and will grow in a medium containing from 0.1 ppm to 50 ppm DON. In an example of the present invention, 0.5 to 10 ppm of DON can be used in a selection medium. In this manner, the modified gene can be used as a selectable marker in transformation experiments, wherein only cell lines that have been transformed with a vector containing the modified gene will grow in a selection medium containing DON. Thus, for example, the modified gene of the present invention could be used as a selectable marker in transforming elements in plants or animals, in the same way as genes that provide resistance to gentamicin, hydromycin, kanamycin, and the like, are used currently. While this invention is described in detail with particular reference to preferred embodiments thereof, these embodiments are offered to illustrate but not limit the invention.

EXAMPLES EXAMPLE 1: MODIFICATION OF THE RICE RPL3 GENE The wild type DNA sequence of the gene Tcml of yeast was obtained from M. Bolotin-Fukuhara from the yeast genome sequencing project. In the comparison of the tcmI DNA sequence with the mutant tcml sequence, an individual base pair change was observed. This change converts a tryptophan (Tcml) to a cysteine (tcml) at residue 255 in the proposed Rpl 3 protein (Figure 1). In this example of the present invention, the corresponding rice Rpl 3 gene was converted to a form resembling that of the tricodermin resistance gene of yeast (tcml). A rice Rpl 3 cDNA, containing a 5 'non-coding region, of 21 bp, a coding region of 1170 bp, and a 3' non-coding region of 177 bp (including a partial polyA extremity ), was kindly provided by Dr. A. Kato (Hokkaido University, Japan). The cDNA (originally named T82, renamed pOSRLP3) was received as a 1368 bp insert at the Smal / EcoRI site of pIBI31. This rice cDNA was randomly cloned from the rice suspension culture cells (Uchimaya et al., 1992, Plant J. 2: 1005-1009). A search in the database revealed the homology of the sequence with numerous genes of the ribosomal L3 protein (Nishi et al., 1993, Biochimica et Biophysica 1216: 110-112). The proposed proteins encoded by the Rpl 3 genes of rice and yeast Tcml shared 65% amino acid identity. The change of tryptophan to cysteine observed between alleles of the wash gene is within a well conserved region in the rice gene; 17 amino acids of 5 ', the tryptophan residue itself, and 3 3' amino acids of tryptophan are completely conserved between rice and yeast (Figure 2 and Table 1). In this manner, sequence specific mutagenesis was employed to modify the rice Rpl 3 cDNA to resemble the yeast tcml gene at the critical site.

POSRpl 3 was digested with Xbal and Nael, yielding a 1722 bp fragment encompassing the Rpl 3 cDNA. This fragment was subcloned into the Xbal / Hpal site of the pALTER-EXl vector (Promega) and named p 1Rpl 3. An 18 bp oligomer (5'-GGCTGGATGGCAGGCACC; SEQ ID No .: 4) was used to produce the desired mutation with the help of the Altered Sites (Promega) team. DNA sequencing confirmed that the mutagenesis was successful and the resulting clone was named pALTRPLC4.

EXAMPLE 2 VECTOR CONSTRUCTION AND TRANSFORMATION The Xbal site in the 5 'direction and an EcoRI site 8 bp beyond the rice Rpl 3 terminator codon were used to subclone either the unmodified or modified form of the gene into pCAMterX. PCAMterX is derived from pBIN19 (Bevan, M., 1984, Nucleic Acids Research, 12: 8711-8721; Figure 3) and has had a 70S CaMV promoter, a multiple cloning site and the 3 'terminator we added. Plasmids having modified and unmodified Rpl 3 genes, subcloned into pCAMterX (Figure 4) were named pCARpl 3 and pCARPLC4, respectively. These two clones were transformed into strain GV3101 / prnp90 from Agrobacterium which was subsequently used to transform Nicotiana tabacum, cultivar Delgold and N. debneyi. The transformed lines of N. tabacum and N. debneyi were selected in the regeneration medium (Sproule et al., 1991, Theor, Appl. Genet, 82: 450-456) containing 150 mg / L of kanamycin.

EXAMPLE 3 TRANSFORMATION OF TOBACCO The vectors containing the Rpl 3 genes not modified and modified. { pCARpl 3 and pCARPLC4, respectively) were used to transform wild-type tobacco (Nicotiana tobacum) and a wild diploid species, N. Debneyi. Both genes were transferred in these tobacco species at equal frequencies suggesting that no rice gene has a negative effect on growth, regeneration or seed production. For example, 70 and 363 independent transgenic lines were recovered from N. Debneyi for the pCARpl 3 and pCARPLC4 genes, respectively. Southern hybridization data and progeny testing of the seeds from these transgenic plants was used to verify that the plants chosen for the detailed analysis had individual copy inserts.

EXAMPLE 4 ISOLATION AND CULTURE OF PROTOPLASTS Seed harvested from Nicotiana tabacum and N. Debneyi transgenic were sterilized on the surface in 70% Javex solution for 2-3 monitos followed by 5 rinses in distilled, sterile water. SE planted (20 seeds per petri dish of 60 x 20 mm) on the surface of solidified medium B5 agar (Gibco) containing 150 mg / L kanamycin and kept at 25 ° C in 16 hours during the day of 100 uE m seconds. Those seedlings that germinated and remained green after two weeks of selection were transferred to fresh petri dishes containing medium concentration MS medium (Gibco) lacking kanamycin. These plants were kept in sterile Magenta containers in a culture room at 25 ° C for a duration of the day in 16 hours of 100 uE seconds. The isolation of the protoplasts from the cells of the leaf mesophiles was as described by Sproule et al., (1991, Theor, Appl. Genet, 82: 450-456). A solution of enzymes of 1% (w / v) cellulase R-10 and macerozyme R-10 in 0.45 M mannitol saline was sterilized by filter and 20 ml aliquot was taken to sterilize 100 x 15 mm petri dishes . Five leaves of each donor plant were excised and floated from the abbot side down on the enzyme solution. The petri dishes for film were sealed, incubated in a moist room and in a dark culture chamber at 28 ° C for 17 hours with gentle agitation. The liberated protoplasts were separated from the tissue debris by filtration through a sterile, 800 μm mesh nylon funnel. The protoplast-enzyme solution was taken in aliquots and put into sterile, round-bottomed glass test tubes, and centrifuged at 900 rpm for 10 minutes. The isolated protoplasts were separated from cell debris by flotation on the surface of 4 ml of the sterile 0.6 M sucrose solution with a 0.5 ml coating of SCM (0.45 M sorbitol, 10 mg / L CaCl2-2H20, 5 mg / L of MES-morpholinoethane-sulfonic acid, pH 5.8). The purified protoplasts were recovered from the SCM interface with sterile pipettes. The protoplasts were adjusted to a density of 5 x 104 cells / ml with a hemacytometer in a liquid NT medium (Nagata and Takebe, 1991, Plan 99: 12-20), which contains 0.4 M glucose in osmotic. A concentrated solution of DON, produced according to the method of Greenhalgh et al. (1986, J. Agri. Food Chem. 34: 98-102) was used to adjust the concentration of the DON toxin in some protoplast cultures to either 0, 0.1, 1.0, 5.0, or 10.0 ppm. All the protoplast cultures were 2 ml of liquid, incubated in petri dishes of 60 x 10 mm, sterile, at 28 ° C in the dark. After one week of culture, the osmotic concentration of the medium was adjusted by the addition of 0.5 ml of the NT medium containing 0.3 M glucose, and the protoplast cultures were moved in low light (10 μm m seconds) at 25 ° C. Wild type plants were shown to be susceptible to DON at 0.5 to 10 ppm in the culture medium. The effect of DON on these protoplasts was to reduce the ability of protoplasts to reform cell pairs, reduce the frequency of division (mitotic index of cells), and reduce the efficiency of plating (number of microcolonies formed) of protoplasts in relation to those cultivated in the absence of DON. The viability of protoplasts of genotype Rpl 3: 258 (line C) was not significantly affected by culture for 20 days in the medium supplemented with 0.5 to 25 ppm of DON. Whereas the viability of protoplasts containing Rpl 3: c258 in the absence of DON was about 654% by 56% when these protoplasts were cultured in the presence of 25 ppm of DON. Protoplasts from wild-type tobacco plants when grown in NT medium supplemented with 25 ppm DON had a viability of 18%, whereas those from transgenic plants with the Rpl 3 gene of rice (lines) had less viability. This effect on the protoplasts of the mesophiles of the leaf was not due to the general effect of each genotype, since in the absence of DON each line had viabilities in the NT medium that vary from 58% to 66%. The pronounced differences between the genotypes became apparent when the protoplasts were cultured in the presence of the DON mycotoxin. Protoplasts were also cultured on 2 ml sub-layers of agarose (0.4% w / v) in sterile 60 x 15 mm petri dishes. The agarose sublayers contained either 0, 0.1, 1.0, 10 or 25 ppm of DON. The protoplasts in these cultures were dispersed in the liquid NT medium at a density of 1 x 105 per ml and cultured as Sroule et al. (191, Theor, Appl. Genet, 82: 450-456). When the protoplasts were cultured in the medium supplemented with DON, remarkable differences were observed in the formation of microcolonies (cell colonies of isolated protoplasts). Colonies from protoplasts containing Rpl 3 frequently did not develop in streets, and therefore were not transferred to the regeneration medium while micro-colonies containing Rpl 3: c258 were able to transfer to the regeneration medium.

EXAMPLE 5 CELLULAR SUSPENSION CROPS Cell suspension cultures from primary wild type or transgenic tobacco plants were initiated from the leaf callus cultures. 2 grams of callus were cultured in a sterile mixer, and the homogenized tissue was used to inoculate 33 ml of the liquid MS medium containing 2 mg / L of 2,4-D in a sterile 125 ml Erlenmeyer flask. The cell suspensions were maintained on an orbital shaker at 150 rpm under a 16-hour daylight at 22 ° C with weekly sub-cultures of 5-10 ml of cells in 33 ml of fresh medium. Growth measurements of cell suspensions were taken after the cultures had reached equilibrium under growth conditions for 12 weeks. The measurement of the weight gain was determined by plating one ml of the cell suspension in a sterile Whatman filter paper placed on the 10 ml surface of the MS medium solidified on agar containing either 0 or 25 ppm DON. At 4-day intervals, the fresh weight of each filter paper was determined under aseptic conditions and then the cells were re-cultured medium. The cells of both genotypes were equally able to grow when they were transferred to the solidified medium on kanamycin supplemented agar, indicating the stability and presence of the transgenes in these cultures. The increase in cell volume was measured by inoculating 5 ml of cells in 35 ml of liquid MS medium supplemented with either 0 or 25 ppm of DON. At 3-day intervals, the entire contents of each flask were transferred to calibrated, conical, sterile tubes, and the packed cell volume was recorded (Table 2). The cells were returned to the culture in the same solution.

TABLE 2 INCREASE IN THE AVERAGE VOLUME OF TOBACCO CELLS TRANSGENIC CULTIVATED IN THE PRESENCE OR ABSENCE OF MICOTOXIN DON FOR 12 DAYS Cell Line Concentration of% Increase DON (ppm) Volumetric, Average C4 0 45 C3 0 40 C4 25 41 C3 25 13 The DON at 25 ppm was sufficient to inhibit the packed cell volume, and the fresh weight gain of the cell suspensions of the pCAPRL3 plants. These levels of don had a less serious impact on the packed cell volume, or on the fresh, cellular weight gain of the cultures of the pCARPLC4 plants (Table 3 and Figure 5a and Figure 5b).

TABLE 3 SPEED OF AVERAGE CREAM OF AS TOBACCO CELLS TRANSGENIC CULTIVATED IN THE RESENCE OR ABSENCE OF THE MICOTOXIN DON FOR 16 DAYS Cell Line Concentration of% Increase DON (ppm) by weight, Average C4 0 22.5 C3 0 24.5 C4 25 21.5 C3 25 8.5 DON was also able to inhibit callus formation in leaf explants cultivated m vitro from leaves of wild type plants and pCAPRL3, while explant from pCARPLC4 plants were capable of regeneration in the presence of DON.

EXAMPLE 6 TRANSFORMATION OF MONOCOTILEDONEAS Unmodified Rpl 3 and modified Rpl 3: c258 genes were cloned into a monocotyledonous expression vector under the control of the rice actin promoter and introns elements (pC0Rl3 provided by Prof. Ray Wu, University of Cornell, NY) to provide pAct.pI.3 and pActRPLC4, respectively, for constitutive expression in monocots. These constructs were introduced by particle bombardment into the cells of embryonic corn tissue cultures derived from the immature Fl embryos of corn A188xB73. To obtain the transgenic lines, each construct was co-bombarded with a pAHC25 gene resistant to herbicide, selectable, containing the Bar gene (provided by Dr Peter Quail, UC Berkeley Ca) and cultures resistant to fos f inotrichin were established. Southern blot analysis of these cultures identified lines with Rpl 3 and RPLC4 integrated into the DNA of high molecular weight maize.

EXAMPLE 7 TRANSGENIC MONOCOTILEDON CROPS Numerous researchers have shown inhibition of growth of several monocot tissues by DON. Bruis et al. (1993), Plant Sci, 94: 195-206) showed that DON reduces callus tissue growth derived from the wheat antenna. DON concentrations of 10 mg / l were sufficient to significantly inhibit the growth of mature maize embryos (McLean, 1996, Mycopathologia 132: 173-183). A dose of 100 mg / l of DON was lethal to most wheat callus (Menke-Milczarek and Jimny, 1991, Mycotox, Res 7: 146-149). A line of each of Rpl 3 and RPLC4 was chosen that exhibited a low copy number of the transgene by Southern analysis. The calluses from these two lines have been subjected to identical selection regimes and were of the same age. These two lines were tested for their ability to grow in medium containing 0 to 25 mg / l of DON. The growth of the callus in the media containing DON showed that the RPLC4 line is substantially more tolerant to mycotoxin. The growth of Rpl 3 was reduced to 15% of the control by 5 mg / l of DON while the RPLC4 line was reduced to only 63% of the control value by the same level of DON (Table 4). To reduce the growth of the RPLC4 line to 15% of the control values required 50 mg / l of DON. This represents a 10-fold increase in tolerance to DON by the call of RPLC4.

TABLE 4 THE EFFECT OF DON ON THE GROWTH OF EBRYONIC CROPS MAIZE (A188xB73) TRANSFORMED WITH PACTRPJ..3 AND PACTRPLC4 Initial dry weight. -Rpl3 = l .4 mg, RPLC4 = 1.5 mg. Initial dry weight after 3 weeks of culture in the dark at 25 ° C. 12 explant / treatment.

DON mg / l 0 5 10 25 50 pActRpl3 31.1 4.9 3.3 3.2 2.2 pActRPLC4 29.8 18.7 13.1 7.0 4.4 These plants were autocrossed to establish homozygous uniform lines and are increasing in the field for Fusarium resistance studies. All scientific publications and patent documents are incorporated herein by reference. The present invention has been described with respect to preferred embodiments. However, it will be obvious to a person skilled in the art that a number of variations and modifications may be made without departing from the scope of the invention as described in the following claims.

LIST OF SEQUENCES i) GENERAL INFORMATION (i) APPLICANT: (A) NAME: His Majesty in Canadian Law As Represented by the Minister of Agriculture and Food of Canada (B STREET: Central Experimental Farm (C CITY: Ottawa (D STATE: Ontario (E COUNTRY: Canada (F ZIP CODE): K1A 0C6 (A NAME: Linda J. Harris (B STREET: 6495 Wheatfield Cres. (C CITY: Greely (D STATE: Ontario (E COUNTRY: Canada 20 (F ZIP CODE): K4P 1E8 (A NAME: Stephen C. Gleeddie (B STREET: 33 Leonard Ave. (C CITY: Ottawa 25 (D STATE: Ontario (E) COUNTRY: Canada (F) ZIP CODE: KIS 4T8 (A) NAME: John A. Simmonds (B) STREET: 130 Amberwood Cres. (C) CITY: Nepean (D) STATE: Ontario (E) COUNTRY: Canada (F) ZIP CODE: K2E 7H8 (ii) TITLE OF THE INVENTION: Tolerance of Trichothecene Mycotoxins in Plants and Animals Through the Modification of the Ribosomal L3 Protein Gene (iii) NUMBER OF SEQUENCES: 4 (iv) COMPUTER LEGIBLE FORM: (A) TYPE OF MEDIA: Flexible disk (B) COMPUTER: compatible with IBM (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) PROGRAM: Patentln Relay # 1.0, Version # 1.30 (EPO) (2) INFORMATION FOR SEQ ID NO: 1: i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 387 amino acids (B) TYPE: amino acid (C) TYPE OF HEBRA: individual (D) TOPOLOGY: linear D TYPE OF MOLECULE: protein (xi) DESCRIPTION FOR SEQ. ID No: 1: Met Ser His Arg Lys Tyr Glu Wing Pro Arg His Gly His Leu Gly Phe 1 5 10 15 Leu Pro Arg Lys Arg Ala Ala Ser lie Arg Ala Arg Val I, and s Ala Phe 20 25 30 Pro Lys Asp Asp Arg Ser Lys Pro Val Wing Leu Thr Ser Phe Leu Gly 3S 40 45 Tyr Lys Wing Gly Met Thr Thr He Val Arg Asp Leu Asp Arg Pro Gly 50 55 60 Ser Lys Phe His Lys Arg Glu Val Val Glu Ala Val Thr Val Val Asp 65 70 75 B0 Thr Pro Pro Val Val Val Val Val Val Val Gly Tyr Val Glu Thr Pro 85 90 95 Arg Gly Leu Arg Ser Leu Thr Thr Val Trp Wing Glu His Leu Ser Asp 100 105 110 Glu Val Lys Arg Arg Phe Tyr Lys Asn Trp Tyr Lys Ser Lys Lys Lys 115 120 125 Wing Phe Thr Lys Tyr Wing Wing Lys Tyr Wing Gln Asp Gly Wing Gly He 130 135 140 Glu Arg Glu Leu Wing Arg He Lys Lys Tyr Wing Ser Val Val Arg Val 145 150 155 ISO Leu Val His Thr Gln He Arg Lys Thr Pro Leu Wing GLn Lys Lys Wing 165 170 175 His Leu Ala Glu He Glp Leu Asn Gly Gly Ser He Ser Glu Lys Val 180 18S 190 Asp Trp Wing Arg Glu His Phe Glu Lys Thr Val Wing Val Asp Ser Val 195 200 205 Phe Glu Gln Asn Glu Met He Asp Wing He Wing Val Thr Lys Gly Eis 210 215 220- Gly Phe Glu Gly Val Thr His Arg Trp Gly Thr Lys Lys Leu Pro Arg 225 230 235 240 Lys Thr His Arg Gly Leu Arg Lys Val Wing Cys He Gly Wing Trp His 245 250 255 Pro Ala His Val Met Trp Ser Val Ala Arg Ala Gly Gln Arg Gly Tyr 260 265 270 His Ser Arg Thr Ser He Asn His Lys He Tyr Arg Val Gly Lys Gly 275 280 285 Asp Asp Glu Wing Aap Gly Wing Thr Ser Phe Asp Arg Thr Lys Lys Thr 290 29S 300 He Thr Pro Met Gly Gly Phe Val His Tyr Gly Glu He Lys Asn Asp 305 310 315 320 Phe He Met Val Lys Gly Cys He Pro Gly Asn Arg Lys Arg He Val 325 330 335 Thr Leu Arg Lys Ser Leu Tyr Thr Asn Thr Ser Arg Lys Ala Leu Glu 340 345 350 Glu Val Ser Leu Lys Trp He Asp Thr Wing Ser Lys Phe Gly Lys Gly 355 360 365 Arg Phe Gln Thr Pro Wing Glu Lys His Wing Phe Met Gly Thr Leu Lys 370 375 380 Lys Asp Leu 365 (2) INFORMATION FOR SEQ ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 387 amino acids (B) TYPE: amino acid (C) TYPE OF HEBRA: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRI PCIÓN FOR SEQ. ID No: 2: Met Ser His Arg Lys Tyr Glu Wing Pro Arg His Gly His Leu Gly Phe 1 5 10 15 Leu Pro A-rg Lys Axg Wing Wing Be He Arg Wing A-rg Val Lys Wing Phe 20 25 30 Pro Lys Asp Asp Arg Ser Lys Pro Val Wing Leu Thr Ser Phe Leu Gly 35 40 45 Tyr Lys Wing Gly Met Thr Thr He Val Arg Asp Leu Asp Arg Pro Gly 50 55 60 Ser Lys Phe His Lys Arg Glu Val Val Glu Ala Val Thr Val Val Asp 65 70 75 80 Thr Pro Pro Val Val Val Val Gly Val Val Gly Tyr Val Glu Thr Pro 85 90 95 Arg Gly Leu Arg Ser Leu Thr Thr Val Trp Wing Glu His Leu Ser Asp 100 105 110 Glu Val Lys Arg Arg Phe Tyr Lys Asn Trp Tyr Lys Ser Lys Lys Lys 115 120 125 Wing Phe Thr Lys Tyr Wing Wing Lys Tyr Wing Gln Asp Gly Wing Gly He 130 135 140 Glu Arg Glu Leu Wing Arg He Lys Lys Tyr Wing Ser -Val Val Arg Val 145 150 155 160 Leu Val His Thr Gln He Arg Lys Thr Pro Leu Wing Gln Lys Lys Wing 165 170 175 His Leu Ala Glu He Gln Leu Asn Gly Gly Ser He Ser Glu Lys Val 180 185 190 Asp Trp Wing Arg Glu His Phe Glu Lys Thr Val Wing Val Asp Ser Val 195 200 205 Phe Glu Gln Asn Glu Met He Asp Wing He Wing Val Thr Lys Gly His 210 215 220 Gly Phe Glu Gly Val Thr His Arg Trp Gly Thr Lys Lys Leu Pro Arg 225 230 235 240 Lys Thr His Arg Gly Leu Arg Lys Val Wing Cys He Gly Wing Cys His 245 250 255 Pro Ala His Val Val Trp Ser Val Ala Arg Ala Gly Gln Arg Gly Tyr 260 265 270 His Ser Arg Thr Ser He Asn His Lys He Tyr Arg Val Gly Lys Gly 275 280 285 Asp Asp Glu Wing Asn Gly Wing Thr Ser Phe Asp Arg Thr Lys Lys Thr 290 295 300 He Thr Pro Met Gly Gly Phe Val His Tyr Gly Glu He Lys Asn Asp 305 310 315 320 Phe He Met Val Lys Gly Cys He Pro Gly Asn Arg Lys Arg He Val 325 330 335 Thr Leu Arg Lys Ser Leu Tyr Thr Asn Thr Ser Arg Lys Ala Leu Glu 340 345 350 Glu Val Ser Leu Lys Trp He Asp Thr Wing Ser Lys Phß Gly Lys Gly 355 360 365 Arg Phe Gln Thr Pro Wing Glu Lys His Wing Phe Met Gly Thr Leu Lys 370 375 380 Lys Asp Leu 335 [2) INFORMATION FOR SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 389 amino acids (B) TYPE: amino acid (C) TYPE OF HEBRA: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein ; xi) DESCRIPTION FOR SEQ. ID No: 3: Met Ser His Arg Lys Phe Glu His Pro Arg His Gly Ser Leu Gly Phe 1 5 10 15 Leu Pro Arg Lys Arg Ser Ser Arg His Arg Gly Lys Val Lys Ser Phe 20 25 30 Pro Lys Asp Asp Val Ser Lys Pro Cys His Leu Thr Ser Phe Val Gly 35 40 45 Tyr Lys Wing Gly Met Thr His He val Arg Glu Val Glu Lys Pro Gly 50 55 60 Ser Lys Leu His Lys Lys Glu Thr Cys Glu Ala Val Thr He He Glu 65 70 75 80 Thr Pro Pro Leu Val He Val Gly Leu Val Wing Tyr Val Lys Thr Pro 85 90 95 Arg Gly Leu Arg Ser Leu Asn Ser Val Trp Wing Glp His Leu Ser Glu 100 105 110 Glu Val Arg Arg Arg Phe Tyr Lys Asn Trp Cys Lys Ser Lys Lys Lys 115 120 125 Wing Phe Thr Lys Tyr Wing Leu Lys Tyr Asp Ser Asp Wing Gly Lys Lys 130 135 140 Glu He Gln Met Gln Leu Glu Lys Met Lys Lys Tyr Ala Ser He Val 145 150 155 160 Arg Val He Wing His Thr Gln He Arg Lys Met Lys Gly Leu Lys Gln 165 170 175 Lys Lys Wing His Leu Met Glu He Gln He Asn Gly Gly Thr He Wing 1SD IBS 190 Asp Lys Val Asp Tyr Gly Tyr Lys Phe Phe Glu Lys Glu He Pro Val 195 200 205 Asp Ala Val Phe Gln Lys Asp Glu Met He Asp He He Gly Val Thr 210 215 220 Lys Gly Lys Gly Tyr Glu Gly Val Val Thr Arg Trp Gly Val Thr Arg 225 230 235 240 Leu Pro Arg Lys Thr His Arg Gly Leu Arg Lys Val Wing Cys He Gly 245 250 255 Ala Trp His Pro Ala Arg Val Ser Tyr Thr Val Ala Arg Ala Gly Gla 260 265 270 Asn Gly Tyr His His Arg Thr Glu Met Asn Lys Lys Val Tyr Lys He 275 2B0 285 Gly Lys Ser Gly Gln Glu Ser His Wing Ala Cys Thr Glu Phe Asp Arg 290 295 300 Thr. Glu Lys Asp He Thr Pro Met Gly Gly Phe Pro His Tyr Gly Val 305 310 315 320 Val Lys Gly Asp Tyr Leu Met lie Lys Gly Cys Cys Val Gly Pro Lys 325 330 335 Lys Arg Val Val Thr Leu Arg Gln Ser Leu Leu Lys Gln Thr Ser Arg 340 345 350 Leu Ala Leu Glu Glu He Lys Leu Lys Phe He Asp Thr Ser Ser Lys 355 360 365 Phe Gly His Gly Arg Phe Gln Thr Thr Asp Glu Lys Gln Arg Phe Phe 370 375 380 Gly Lys Leu Lys Wing 385 (2) INFORMATION FOR SEQ ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / des = "oligomer" (xi) DESCRIPTION FOR SEQ. ID No:: GGCTGGATGG CAGGCACC

Claims (25)

1. A modified gene, wherein the wild-type form of the gene encodes a ribosomal L3 protein; wherein the modification occurs between amino acid 240 and 263, based on the amino acid numbering of the yeast protein; and wherein a host transformed with the modified gene has increased resistance to trichothecene mycotoxins, provided that the gene is not Sa ccha romyces cerevi si a e.

2 . The modified gene according to claim 1, wherein the gene is modified by a substitution, deletion, addition or inversion of the base pairs, wherein the modification is sufficient to reduce the binding capacities of the mycotoxin, but is insufficient to destroy the function of the ribosomal protein gene.

3. The modified gene according to claim 2 wherein the source of the gene encoding the ribosomal L3 protein is selected from the group consisting of: rice, Arabidopsis Thaliana, monocotyledons, rat, mice, human, yeast, C. egan sy Es ch eri ch ia col i.

4. The modified gene according to claim 3, wherein the gene encoding the Rpl 3 gene is a rice gene.

5. The modified gene according to claim 4, wherein the gene has a sequence that will code for the amino acid sequence shown in SEQ ID No. : 3, with the sequence coding for a protein at position 258, or a functional equivalent thereof.

6. A cloning vector containing a modified Rpl 3 gene as defined in claim 1.

7. The cloning vector according to claim 6, wherein the gene is modified by a substitution, deletion, addition or inversion of the pairs of base, where the modification is sufficient to reduce the binding capacities of the mycotoxin, but insufficient to destroy the function of the gene as a ribosomal protein.

The cloning vector according to claim 7, wherein the gene encoding the ribosomal L3 protein is selected from the group consisting of: rice, Arabidopsis Thaliana, monocotyledons, rat, mice, human, yeast, C. elegans and Escherichia coli.

9. The cloning vector according to claim 8, wherein the gene encoding the ribosomal L3 protein, is a rice gene. The cloning vector according to claim 9, wherein the gene has a sequence that will code for the amino acid sequence shown in SEQ ID NO: 3, with the sequence coding for a cysteine at position 258, or an equivalent functional of it. 11. A transformed plant, transformed with a modified Rpl 3 gene of claim 1, wherein the transformed plant has increased resistance to the Fusa ri um pest. The plant according to claim 11, wherein the gene is modified by a substitution, deletion, addition or inversion of base pairs, wherein the modification is sufficient to reduce the binding capacities of the mycotoxin, but insufficient to destroy the function of the gene as a ribosomal L3 protein. The plant according to claim 12, wherein the gene encoding the ribosomal L3 protein is selected from the group consisting of: rice, Arabidopsis Thaliana, monocotyledons, rat, mice, human, yeast, C. elegans and Escherichia coli. The plant according to claim 13, wherein the gene encoding the Rpl 3 gene is a rice gene. The plant according to claim 14, wherein the gene has a sequence that will code for the amino acid sequence shown in SEQ ID No.:3, with the sequence coding for a cysteine at position 258, or an equivalent function of 16. The plant according to claim 11, wherein the plant is selected from the group consisting of: corn, barley, wheat, rice, rye, oats and my o. 17. Seeds from a transformed plant as defined in claims 12 to 17. 18. A transformed animal, transformed with a modified Rpl 3 as defined in claim 1, wherein the transformed animal has an increased tolerance to the mycotoxins of the trichothecenes. 19. The animal according to claim 18, wherein the gene is modified by a substitution, suppression, addition or inversion of base pairs, wherein the modification is sufficient to reduce the binding capacities of the mycotoxin, but insufficient to destroy the function of the gene as a ribosomal L3 protein. The animal according to claim 19, wherein the gene encoding the ribosomal L3 protein is selected from the group consisting of: rice, Arabidopsis Thaliana, monocotyledons, rat, mice, human, yeast, C. elegans and Escherichia coli The animal according to claim 20, wherein the gene encoding the ribosomal L3 protein is a rice gene. The animal according to claim 21, wherein the gene has a sequence that will code for the amino acid sequence shown in SEQ ID No. 3, with the sequence coding for a cysteine at position 258, or a functional equivalent of the same. 23. A method for increasing the resistance to a pest of Fusa ri um by transforming a suitable plant with a modified gene according to claim 1, wherein the plant transformed with the gene has increased resistance to the mycotoxins of the trichothecenes and wherein the The method comprises the steps of: providing a modified gene, and transforming an appropriate plant with the gene. 24. A method for increasing mycotoxin tolerance of trichothecenes by transforming a suitable animal with a modified gene according to claim 1, wherein the animal transformed with the gene has increased resistance to the mycotoxins of the trichothecenes and where the method comprises the steps of: providing a modified gene, and transforming a suitable animal with the gene. 25. A method for using the modified gene according to claim 1, as a selectable marker in animal or plant transformation experiments. SUMMARY OF THE INVENTION Fusa ri um gramin ea rum is a plant pathogen, which attacks a wide range of plant species including corn (spike and stem rot), barley and wheat (head plague). The epidemics of Fusa ri um result in millions of dollars of losses in crop income. Infection by Fusa ri um gramin ea rum in cereals reduces both the production and the quality of the grain. Mycotoxins are produced by many fungal species of Fusa ri um and in this way the grain becomes contaminated with these mycotoxins, such as trichothecenes. The main trichothecene produced by F. gram in ea rum as well as F. culm orum is deoxynivalenol (abbreviated as DON, also known as vomitoxin). Trichothecenes are potent inhibitors of protein synthesis and are completely toxic to humans and livestock. A yeast gene that is resistant to tricothecene, trichodermin, has been identified. A corresponding plant gene has been prepared, which has been used to transform plants and will be suitable for transforming animals. These transformed plants have an increased tolerance to trichothecenes and could be more resistant to the pest by Fusa ri um. Potentially increased animals could have an increased tolerance to trichothecene mycotoxins. This modified gene could also be used as a selectable marker in transformation experiments.