DE10033750A1 - Pathogen resistance in organisms - Google Patents

Abstract

The invention relates to the use of a polypeptide which binds a cyclical ketone that can be derived from oxazole, for generating pathogen resistance in organisms, especially plants and animals.

Description

The present invention relates to the use of a polypeptide comprising a Cyclic ketone derived from oxazole binds to produce Pathogen resistance in organisms, especially plants and animals.

Plants and animals are a variety of pathogens, i.e. H. Pathogens, exposed, that can trigger diseases. Such pathogens are e.g. B. fungi, bacteria and Viruses. In plants, the pathogens are often via the leaves, tubers and Roots ingested, while this is often via the skin, breathing and in animals Food is done. Many strategies are followed to help with these organisms To create resistance to the pathogens. For example, in plants tries to find resistance genes via classic breeding or genetic engineering Integrate procedures into current plant varieties. For animals, ins to name special active and passive immunization methods by means of which activated pathogens or antibodies directed against them administered to the animals can be. However, these procedures often require large financial and time expenditure. Furthermore, they often do not lead to the hoped for Success.

The present invention is therefore based on the technical problem, a means provide with which in organisms, especially plants and animals Resistance to pathogens can be generated.

This technical problem is solved by the subject matter in the claims solved.  

The present invention is based on the knowledge of the applicant that plants and animals containing a polypeptide that is cyclic from oxazole ketone binds, have resistance to pathogens. In particular he found that transgenic potato plants that contain 2-phenyloxazol-5-one express binding antibody, resistance to pathogens, e.g. B. the phytopathogenic fungus Phytophthora infestans or the bacterial pathogen Erwi nia carotovora. He also found that transgenic mice that express such an antibody, resistance to pathogens e.g. B. that Bacterium Staphylococcus aureus. It will refer to the following Examples referenced.

According to the invention, these findings of the applicant are used a polypeptide for the generation of pathogen resistance in organisms, in particular plants, animals and humans used, the polypeptide cyclic ketone derived from oxazole binds.

The term "polypeptide" includes a (poly) peptide of any kind and origin which can bind a cyclic oxazole-derived ketone, in particular 3-phenyloxazol-5-one, the ketone being present as such or within a compound. In particular, the polypeptide can be an antibody or a fragment thereof, e.g. B. a Fab, an Fv or a single chain (sc) antibody, in particular an scFv antibody and very particularly those with the amino acid sequence of FIG. 1. The polypeptide can be provided by various methods. A method is favorable in which a polypeptide or antibody expression library is screened with the above ketone or a compound containing it (for example HuCAL, Morphosys, Germany).

According to the invention, the polypeptide can be used to generate pathogen resistance in Organisms, especially plants, animals and humans, are used become. Below is first the use of the polypeptide in plants described. For this purpose it can be advantageous if the polypeptide is a signal peptide has, whereby it trans in certain cell compartments of a plant cell can be ported. Suitable signal peptides and methods for linking the  Signal peptides with a desired protein, e.g. B. the present polypeptide are known to the person skilled in the art. For example, the α- Barley amylase for apoplast (Düring et al., Plant Journal 3 (1993), 587-598), on a mouse signal peptide, on the combination of one Mouse signal peptide and the KDEL-ER retention signal with respect to the ER (Artsa enko et al., Molecular Breeding 4 (1998), 313-319), on the targeting signal of a Mammalian α-2,6-sialyltransferase with respect to the Golgi apparatus (Wee et al., Plant Cell IV (1998), 1759-1768, on the vacuole localization signal of a vacuole ren cucumber chitinase with regard to vacuoles (Neuhaus et al., Proc. Natl. Acad. Sci. USA 88: 10362-10366 (1991), to the ferredoxin transit peptide with regard to chloroplasts and plastids, and to the trypto transit peptide yeast phanyl-t RNA snetetase with regard to mitochondria (Schmitz and Lonsdale, Plant Cell 1 (1989), 783-791). The polypeptide can be by means of various processes such. B. via media such as culture media, a plant or Parts of these, in particular plant cells, are administered.

Furthermore, the polypeptide in the form of a nucleic acid encoding it, e.g. B. DNA or RNA, administered to plants or parts thereof. This is it necessary that the nucleic acid is present in an expression vector or with Sequences of such a ligated, it may be advantageous if this or these enable expression of the nucleic acid in cell compartments. Such Expression vectors or sequences of these are known to the person skilled in the art. at for example, Svab et al., Proc. Natl. Acad. Sci. USA 87 (1990) 8526- 8530; Khan and Maliga, Nature Biotechnology 17 (1999), 910-915; Sidorov et al., Plant Journal 19 (1999), 209-216.

Methods for constructing the expression vectors that contain the desired gene, e.g. B. for the present polypeptide, contained in expressible form, are known in the art known and described for example in common standard works (see e.g. Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). The expressions vectors can be on a plasmid, cosmid, virus, bacteriophage or a other vector common in genetic engineering. These vectors can  have other functional units that stabilize the vector in the Effect plant. They can also use "left border" and "right border" sequences contain agrobacterial T-DNA, which ensures stable integration into the genome of plants. A termination sequence can also be used the correct completion of the transcription as well as the addition serves a poly-A sequence to the transcript. Such elements are in the Literature described (see. Gielen et al., EMBO J. 8 (1989), 23-29) and any interchangeable.

For the expression of the for the desired protein, e.g. B. the present polyp gene encoding tids, suitable promoters are known to the person skilled in the art and these include, for example, the Cauliflower Mosaic Virus 35S promoter (Odell et al., Nature 313 (1995), 810-812), Agrobacterium tumefaciens Nopalin Syn thase promoter and the mannopine synthase promoter (Harpster et al., Molecular and General Genetics 212 (1988), 182-190). The one for the desired protein, z. B. the gene encoding the present polypeptide can also be induced with an cash promoter be linked to what z. B. controlling the synthesis of ge wanted protein e.g. B. in a transgenic plant, to a desired one Time allowed. Suitable promoters are known to the person skilled in the art and for this purpose include, for example, the anaerobically inducible Gap C4 promoter from maize (Bülow et al., Molecular Plant-Microbe Interactions 12 (1999), 182-188), PR promoters, such as L-phenylalanine ammonium, lyase, chalcon synthase and "hydroxyproline rich glycoprotein "promoters inducible by ethylene (Ecker and Davies, Proc. Natl. Acad. Sci. USA 84: 5202-5210 (1987) and inducible by dexamethasone chimeric transcription induction system (Kunkel et al., Nature Biotechnology 17 (1990), 916-918), IncW promoter from maize, inducible by sucrose or D- Glucose (Chen et al., Proc. Natl. Acad. Sci. USA 96: 10512-10517 (1999). In addition, Datla et al., Biotechnology Annual Review 3 (1997), 269-290 and Gatz and Denk, Trends in Plant Science 3 (1998), 352-358.

One is available for producing the expression vectors for introduction into plants large number of cloning vectors are available that provide a replication signal for E.coli and a marker gene for the selection of transformed bacterial cells.  

Examples of such vectors are pBR322, pUC series, M13mp series, pA- CYC184, etc. The desired sequence can be placed on a suitable restriction interface into the vector. The vector obtained is used for the Transformation of E. coli cells used. Transformed E.coli cells are in grown in a suitable medium, then harvested and lysed. The vector is then recovered. As analysis methods to characterize the obtained vector DNA are generally restriction analyzes, Gelelek trophoresis and other biochemical-molecular biological methods. After each manipulation, the vector DNA can be cleaved and DNA Fragments can be linked to other DNA sequences. Any vector DNA sequence can be cloned in the same or different vectors.

For the introduction of the above expression vectors into a plant cell a variety of techniques are available. These techniques include the Transformation of plant cells with T-DNA using Agrobacteri um tumefaciens or Agrobacterium rhizogenes as transformation agents which Fusion of protoplasts, injection, electroporation of DNA, the one Bringing DNA using the biolistic method and other possibilities.

When injecting and electroporation of DNA into plant cells are in themselves no special requirements were placed on the vectors used. It can simple plasmids, such as B. pUC derivatives can be used. But should out whole plants should be regenerated in such a transformed cells selectable markers are available. Suitable selectable markers are the Known to those skilled in the art and include, for example, neomycin phosphotrans ferase II gene from E. coli (Beck et al., Gene 19 (1982), 327-336), the sulfonamide Resistance gene (EP-369637) and the hygromycin resistance gene (EP-186425). ever according to the method of introducing the desired genes into the plant cell additional DNA sequences may be required. Is z. B. for the transformation of Plant cell that uses the Ti or Ri plasmid, at least the right one Limitation, but often the right and left limitation of the Ti and Ri Plasmid T-DNA linked to the genes to be introduced as a flank region become.  

If Agrobacteria are used for the transformation, the one to be introduced DNA are cloned into special vectors, either in an intermediate or a binary vector (see also the following examples play 1 to 3). The intermediate vectors can be due to sequences that are homologous to sequences in the T-DNA, by homologous recombination in the Ti or Ri plasmid of the agrobacteria can be integrated. This also contains the vir region necessary for the transfer of the T-DNA. Intermediate vectors cannot replicate in agrobacteria. Using a helper plasmid, the intermediate vector can be transferred to Agrobacterium tumefaciens. binary Vectors can replicate in both E. coli and agrobacteria. It contains a selection marker gene and a linker or polylinker, which of the right and left T-DNA border region are framed. You can jump in the agrobacteria are transformed. The Agrobacterium serving as the host cell should contain a plasmid bearing a vir region. The vir region is for the Transfer of the T-DNA into the plant cell necessary. Additional T-DNA can to be available. The agrobacterium transformed in this way becomes a transformation used by plant cells.

Plant explants can be used to transfer the DNA into the plant cell moderately with Agrobacterium tumefaciens or Agrobacterium rhizogenes be co-cultivated. From the infected plant material (e.g. leaf pieces, stems segments, roots, but also protoplasts or suspension-cultivated plants cells) can then in a suitable medium, which antibiotics or biocides for the selection of transformed tents can contain whole plants again be cured. The plants thus obtained can then be switched on in the presence of the led DNA to be examined. Alternative systems for transforming monocotyledonous plants are the transformation using the biolistic approach, the electrically or chemically induced DNA uptake in protoplasts, the elec troporation of partially permeabilized cells, macro injection of DNA into Inflorescences, the microinjection of DNA into microspores and pro-embryos DNA uptake by germinating pollen and DNA uptake in embryos by swelling (for an overview: Potrykus, Physiol. Plant (1990), 269-273). currency rend the transformation of dicotyledonous plants with the help of Ti plasmid vector systems  of Agrobacterium tumefaciens is well established, recent work points to it hints that monocotyledonous plants of the transformation means on Agrobacterium based vectors are very accessible.

In a preferred embodiment, the used according to the invention Expression vectors Localization signals for localization in cell compartments elements, especially endoplasmic reticulum (ER), apoplasts, Golgi Apparatus, plastids, peroxisomes, mitochondria and / or vacuoles. It's going on referenced above regarding signal peptides. Especially before The localization signals are the KDEL-ER targeting peptide, the Golgi Localization signal of β-1,2-N-acetylglucosamine transferase (GnTI), the transit peptide from the small subunit of ribulose bisphosphate carboxylase and / or the vacuolar targeting signal SKNPIN.

According to the invention, the above polypeptide is administered to plants. there can in principle be plants of any plant species, d. H. both monocot and dicot plants. The term "Pflan ze "also includes Gramineae, Chenopodia, Leguminosae, Brasicaceen, Solana ceen, mushrooms, mosses and algae. It is preferably useful plants, for. B. around plants such as wheat, barley, rice, corn, sugar beet, sugar cane, rapeseed, mustard, Turnips, flax, peas, bean lupine, tobacco and potato. The one for expression Plant parts of the polypeptide desired in principle relate to any part Plant part, at least propagation material of these plants, for example Seeds, tubers, rhizomes, seedlings, cuttings etc.

Furthermore, the present invention also relates to plant seeds, a plant cell or a plant that expresses a polypeptide above.

With the present invention it is also possible to develop pathogen resistance in To produce animals and humans. The above polypeptide can be used for this as such or in combination with a signal peptide on animals, humans or cells can be administered. Such a signal peptide can e.g. B. a Mouse signal peptide, a combination of a mouse signal peptide and the  KDEL-ER retention signal or the targeting signal of a mammalian alpha-2,6 Sialyltransferase with respect to the Golgi apparatus. Furthermore, the polypeptide in the form of a nucleic acid encoding it, e.g. B. DNA or RNA, on animals, the People or cells of this can be administered. For administration in the form a nucleic acid must be present in an expression vector, or is ligated with sequences of such. It is based on the above all general explanations regarding expression vectors and their production directed. In addition, reference is made to vectors that are suitable for gene therapy suitable for animals. In these the nucleic acid can be under the control of a inducible or tissue-specific promoters, such as metallothionein I or Polyhedrin promoter. Preferred vectors are e.g. B. viruses, such as retroviruses, Adenoviruses, adeno-associated viruses or vaccinia viruses. Examples of retroviruses are MoMuLV, HaMuSV, MUMTV, RSV or GaLV. Furthermore, the for Nucleic acid encoding polypeptide in the form of colloidal dispersions to the Target cells are transported. These include e.g. B. liposomes and lipoplexes (Mannino et al., 1988 Biotechniques 6: 682).

According to the invention, the above polypeptide is used on animals, humans and Cells administered this. In principle, it can be any animal Trade animal species. It is preferably useful and pets, e.g. B. Cattle, horses, sheep, pigs, goats, dogs, cats, etc.

Furthermore, the present invention also relates to animals and cells thereof, which the express the above polypeptide.

The present invention is characterized in that in plants and animals or human resistance to pathogens can be generated. The The term "pathogens" used encompasses any pathogens mentioned in the organisms can cause disease. Examples of such pathogens are Bacteria, fungi and viruses. Regarding plants, phytopathogenic in particular Fungi such as Phytophthora infestans, Botrytis cinerea, Alternaria alternata, Fusarium oxysporum, Ustilago maydis, and bacterial pathogens such as Erwinia carotovora, Pseudomonas syringae, Ralstonia solanacearum, Xanthomonas campestris, Clavibacter  michiganense to call. Regarding animals and humans are ins special animal pathogenic fungi, the diseases, such as Candidamycosen, Crypto coccoses, aspergilloses, dermatomycoses, hystopolasmoses, coccidiomycoses and blastomycoses, and bacterial pathogens such as Micrococcaceae (e.g. Staphy lococci), Lactobacteriaceae (e.g. Streptococci), Neisseriaceae (e.g. Neisseriae), Corynebacteriaceae, Spirillaceae, Listeria bacteriae, Mycobacteriaceae, Enter obacteriaceae (e.g. Escherichia bacteriae), Salmonellae, Brucellaceae (e.g. Pasteu rella bacteriae), anaerobic and aerobic spore-forming agents (e.g. Bacillaceae, Clostridia) and Rickettsiales to name. Overall, the present one is suitable Invention best, in plant and animal breeding as well as in human medicine to be used.

The present invention also provides a pharmaceutical for this use Composition ready containing a protruding polypeptide as such, in Kombina tion with a signal peptide or in the form of a nucleic acid encoding it and usual auxiliaries, such as diluents, solvents, preservatives, Contains extenders, etc. Also the composition can be in different Shapes, e.g. B. as an ointment, paste, cream, gel or aqueous solution. Depending on its shape, the composition can be injected or externally be applied.

Brief description of the figures

Fig. 1 shows the DNA and amino acid sequence of a scFv antibody according to the invention which binds 2-phenyloxazol-5-one.

Fig. 2 shows the proportion of the leaf surface with disease symptoms after infection of potato leaves with Phytophthora infestans (mixture of breeds 1-11, about 200 spores per 20 ul) and incubation for 3 days. The transgenic lines contain the gene coding for the antibody which binds 2-phenyloxazol-5-one (fused with the KDEL localization peptide for the endoplasmic reticulum); Line Dk1 contains only the nptll marker gene; Désirée: non-transgenic parent variety as a control. Columns with * differ significantly from Désirée (Wilcoxon test, p = 0.05).

Fig. 3 shows remaining intact potato tuber tissue after inoculation of tuber slices with 2000 Erwinia carotovora ssp. atroseptica bacteria in 2 µl and incubation for 3 days according to Düring et al. (1993), supra. Lines 9/7, 9/49, 9/51, 9/52 contain the gene encoding the antibody that binds 2-phenyloxazol-5-one (fused to the KDEL localization peptide for the endoplasmic reticulum); Line DK1 contains only the nptll marker gene; Désirée: non-transgenic parent variety as a control.

The invention is illustrated by the examples below.

example 1 Generation of resistance in transgenic potato plants gene Phytophthora infestans

Transgenic potato plants that have an scFv antibody with a KDEL signal pep express tid, whereby the antibody binds 2-phenyloxazolon-5-one (Artsaenko et al., supra, were grown in the greenhouse and a phytopathological Subjected to testing.

For this purpose, round leaf disks with a diameter of 20 mm were produced from potato leaves. These leaf disks were laid out on a moist filter paper, which was spread in a translucent plastic can on a stainless steel grid, and inoculated with a 20 µl drop of spore suspension (approx. 200 sporangia) from Phytophthora infestans race 1-11. The sporangia suspension was prepared from leaf disks already infected and cooled to 4 ° C. for about 15 minutes before inoculation to stimulate the zoospores hatch. Incubation takes place in illuminated incubators with a day period of 14 hours and a day / night temperature of 17 ° / 10 ° C. After five and six days, evaluations were carried out, the percentage of the infested area of the total leaf disk area being determined. The results of 8 transgenic lines are shown in Fig. 2.

It was found that by using the above according to the invention Polypeptide the symptoms of the disease could be significantly reduced, which highlights the generation of pathogen resistance in plants.

Example 2 Generation of resistance to transgenic potato tubers Erwinia carotovora

The potato plants described in Example 1 were grown in greenhouses grown. After the potato plants had ripened, the tubers were harvested and made for the phytopathological test is stored.

The resistance properties of the transgenic potato tubers to the bacterial pathogen Erwinia carotovora were tested in a tuber disc test. For this, tubers were peeled and 1 cm thick cylinders were cut out. These were again cut into 3 mm thick slices. The basic experimental procedure is described in Düring et al. (Plant Journal (1993) 3, 587-598). The bulbous disks laid out on a wet filter paper were freshly pierced in the middle and a suspension of 2000 Erwinia carotovora ssp. atroseptica bacteria was applied in a volume of 2 µl. After three days, the macerated tissue was rinsed off and the remaining solid potato tissue was weighed after drying. The results of 4 transgenic lines are shown in Fig. 3.

It was found that by using the above according to the invention Polypeptide the symptoms of the disease could be significantly reduced, which highlights the generation of pathogen resistance in plants.

Example 3 Generation of resistance in transgenic mice to Staphy lococcus aureus

About 20 female CB6F1 mice aged 4-5 weeks were superovulated by intra peritoneal injection of 5 U PMS (pregnant mare's serum) on day 1 and another intra peritoneal injection of 5 U hCG (human chorionic gonadotropin) on day 3 and in the evening mated the same day with male CB6F1 animals. On the morning of the 4th day, the animals were examined for the presence of a vaginal plug, positive animals were killed by cervical dislocation and the oviducts were removed. The egg cells were emptied from the oviduct in M2 medium, the cumulus cells were detached by brief incubation with hyaluronidase, the egg cells were washed thoroughly and incubated in an incubator (5% CO ₂ , 85% humidity, 37 ° C) with paraffin oil over M16 medium ) kept.

On day 4, a DNA from FIG. 1 was injected into the male pre-nucleus of fertilized egg cells, which is under the control of the WAP promoter (whey acidic protein), ie this promoter is produced by lactotropic hormones derived from mammals in pregnancy or Lactation period are induced. The injected eggs were then incubated in the incubator until retransmission the following day. To provide pseudopregnant Foster mice, about 25 female B6CBAF1 mice aged 8 to 12 weeks were mated with vasectomized male mice on the evening before the microinjection. On the morning of the 5th day, the animals with vaginal plugs were selected for retransfer of the injected eggs. The cells, which were cultured overnight and proliferated and microinjected to the two-cell stage, were re-implanted on the 5th day. 10-15 embryos were washed into the infundibulum of an oviduct of an anesthetized Foster mouse. The implanted embryos were born 19-20 days after retransfer. 9 transgenic mice were obtained which express the scFv antibody shown in FIG. 1. A cut of approx. 1 cm in length was added to the back of these mice. A suspension of Staphylococcus aureus was placed in the wound and the mice were observed for the development of disease symptoms, ie pus formation.

It was found that by using the above according to the invention Polypeptide significantly reduced disease symptoms compared to controls  could be what a generation of pathogen resistance in animals underlines.

Claims (16)

1. Use of a polypeptide that is a cyclic derived from oxazole of ketone binds to create pathogen resistance in organisms. 2. Use according to claim 1, wherein the organisms are plants. 3. Use according to claim 1, wherein the organisms are animals and humans are. 4. Use according to any one of claims 1 to 3, wherein the ketone within a connection exists. 5. Use according to one of claims 1 to 4, wherein the polypeptide is a 2- Phenyloxazol-5-one binds. 6. Use according to any one of claims 1-5, wherein the polypeptide is a for a localization signal suitable for localization in cell compartments includes. 7. Use according to claim 6, wherein the cell compartment the endoplas is matical reticulum. 8. Use according to claim 6 or 7, wherein the localization signal KDEL is. 9. Use according to any one of claims 1 to 8, wherein the polypeptide Is antibody. 10. Use according to claim 9, wherein the antibody is an scFv antibody.   11. Use according to any one of claims 8-10, wherein the antibody comprises the amino acid sequence shown in Fig. 1. 12. Use according to any one of claims 1-11, wherein the polypeptide in Form of a nucleic acid encoding it is present. 13. Use according to claim 12, wherein the nucleic acid in an express ion vector is present. 14. Use according to any one of claims 2 and 4 to 13, wherein the plants Gramineae, Chenopodia, legumes, Brasicaceae, Solanaceae, mushrooms, Include mosses and algae. 15. Use according to any one of claims 2 and 4 to 13, wherein the plants Wheat, barley, rice, corn, sugar beet, sugar cane, rapeseed, mustard, turnips, Include flax, pea, bean, lupine, tobacco and potato. 16. Use according to any one of claims 3-13, wherein the animals are domestic and Farm animals are.