DE19621572A1 - Localized cell death in plants - Google Patents

Abstract

The invention relates to nucleic acid molecules for production of localised cell death in plants, and vectors containing said nucleic acid molecules. It also relates to plant cells and plants which are genetically modified by said nucleic acid molecules.

Description

The present invention relates to new DNA sequences, one Method of creating new plants that have a new DNA sequence included and when expressing the contained in the DNA sequence These new plants show a localized cell death gene and the use of the DNA sequences to generate a localized cell death in plants.

Plants are consistently of various pathogens attacked. A particular problem related to the attack from Plants caused by pathogens arise in today's form of  Agriculture, which is characterized in that due to the large-scale cultivation of monocultures by means of breeding Muta introduced resistance relatively quickly on the pest side.

There are basically two types of resistance in plants differentiate, the vertical and the horizontal resistance. In the vertical resistance is usually one Gene-by-gene relationship in the sense that a certain avirulence a certain resistance gene of the plant against the pathogen faces. This means that the vertical resistance in is usually monogenic and therefore the easiest will break. The horizontal resistance does not become monogenic inherited, but usually be through a series of loci Right. The horizontal resistance does not lead to complete Resistance, however, is how vertical resistance is usually, since many loci are involved, more permanent.

In the case of vertical resistance, i.e. H. in the case of the existence a resistance gene on the side of the plant and an aviru resistance to the pathogen, resistance expresses itself in the appearance of local necrosis with hypersen plant reaction.

Although the hypersensitive response, hereinafter referred to as "HR" is not fully ver in its details stood, it is clear that the plant host cells suffer rapid cell death in the area of HR. On the one hand this rapid cell death leads to inclusion and thus to localization the pathogen, on the other hand it can be assumed that the pathogen caused by radicals that arise during HR, is damaged. In addition, HR leads in many cases on the phenomenon of so-called systemically acquired resistance. This systemically acquired resistance is generally based on the English term "systemic acquired resistance" as SAR  designated.

The SAR represents an interesting special case of herbal Resistance to pathogens. Characteristic is that a plant that has a SAR due to infection by shows a certain pathogen, the acquired resistance does not only against the pathogen that produces the SAR, but against a much broader spectrum of pathogens.

In this respect, the SAR is a very desirable response from the Plant, as in many cases it is of broad resistance flows which comes close to horizontal resistance (cf. e.g. B. Ryals et al (1992) SEB Symposium 49, 205-229).

The mechanisms that lead to the formation of SAR are partial Roger that. In many cases it has been shown that to SAR formation is a local necrosis of cells associated with an HR is necessary. A necrosis that alone results represents a wound, or due to other abio table stressors is usually not sufficient to generate a SAR. It is also shown that in many cases the SAR has an elevated level correlated to salicylic acid (see e.g. Metraux et al (1990) Science 250, 1004-1006; Malamy et al (1990) Science 250, 1002-1004). This has also been shown by the fact that exogenous Salicylic acid when added to non-infected plants can induce a SAR (see e.g. White (1979) Virology 99, 410-412; Palva et al (1994) Molecular Plant-Microb. Interactions 7, 356-363). Furthermore, it has been observed that in transgenic plants in which the expression of a bacterial salicylate hydroxylase the amount of salicylic acid was reduced, no SAR can be trained (Gaffney et al (1993) Science 261, 754-756). The SAR can also go through a number of other chemicals, such as B. 2,6-dichloro- Isonicotinic acid can be induced (Uknes et al (1992), The Plant Cell 4, 645-656).  

Various systems have been described in the literature to date that lead to localized cell death. Examples for this are the expression of a bacterial ribonuclease in transgenic plants (Strittmatter et al (1995) Biotechnology 13, 1085-1089). Another system is the simultaneous Expression of resistance genes from a host plant and corresponding avirulence genes from pathogens (WO 91/15585). A specific example is the expression of the Avr9 gene the fungus Cladosporium fulvum and the Cf-9 gene from tomato (WO 95/31564; for an overview see also de Witt (1992) Annual Review in Plant Pathology 30, 301-418, for designation further one for such a problem solving Avirulence and resistance genes). In WO 95/31564 in all generally described that the nucleotide sequence or sequences, which are associated with localized cell death and the generation of SAR are described, one or more Genes may include the specific embodiment describes the resistance gene Cf-9 and the avirulence gene Avr9.

As further examples of the use of a nucleotide sequence or the combination of nucleotide sequences that lead to a Plant cell necrosis and a SAR result in WO 95/31564 called the following possible approaches: an ubiquitin conjugating enzyme, the gene VI protein from Cauliflower Mosaic Virus, a simultaneous presence of a viral coat protein a corresponding plant resistance gene, a bacterial les Hairpin protein, the N'-gene from tobacco, the potato virus Coat protein and the avirulent dominant, as well as various Resistance and avirulence gene combinations (e.g. Cf-2 from tomato and Avr2 from C. fulvum, Cf-4 from tomato and Avr4 from C. fulvum). Furthermore, an RNAse, the diphtheria toxin as well as proton pumps such as bacterial proton pumps.

As already described above, the SAR is a very attractive one Resistance phenomenon. The problem, however, is that the  SAR could not yet be treated in breeding because probably too many loci involved and / or the gene products are not known. It is therefore one of the tasks of biotech biological research, alternative ways for both localized cell death as well as the often associated To show SAR.

It is an important object of the invention to create new nucleic acid to provide sequences or proteins with the help of Plants or plant cells a localized cell death and possibly systemically acquired resistance (SAR) was also triggered can be. Another object of the invention is in delivering new nucleic acid molecules that serve can produce male sterility in plants. Further Objects of the invention result from the following description exercise.

These tasks are covered by the subjects of the independent Claims, in particular based on the provision of the DNA sequences according to the invention, their expression in plants localized death of plant cells causes resolved.

We have surprisingly found that certain nucleic acids sequences targeted to trigger localized cell death can be set by producing a protein or trigger protein fragments, its enzymatic activity causes cell death. In particular, we have experimentally gene that expression of a nucleic acid sequence Erwinia amylovora, which is responsible for a secreted form of a Levan encoded, leads to the formation of HR in plants, which can also be coupled with the occurrence of a SAR.

The occurrence of necrosis when expressing a Invertase gene from Saccharomyces cerevisiae in transgenic Plants are observed. In this case, the secretion of the gene product from the plant cell by fusion of the for the  Invertase coding region with N-terminal signal sequences of the proteinase inhibitor II protein from Solanum tuberosum enables.

The present invention thus relates to nucleic acid molecules which code for proteins or fragments thereof, which in Expression lead to cell death. In a preferred one Embodiment is the invention Nucleic acid molecules around nucleic acid molecules that are used for proteins with the biological activity of a fructosyltransferase or encode a biologically active fragment thereof. In a particularly preferred embodiment is the Nucleic acid sequence around a nucleic acid sequence which is for Proteins with the biological activity of a secreted Levansucrase or a biologically active fragment thereof encoded.

In a further particularly preferred embodiment are nucleic acid molecules that are used for proteins with the biological activity of a secreted invertase or one encode biologically active fragments thereof.

Biologically active fragment means in connection with this Invention that the mediated biological activity for Generation of cell death is sufficient.

Nucleic acid sequences necessary for proteins with the enzymatic Encoding activity of a Levansucrase are different Organisms have been described, e.g. B. from, Bacillus amylo liauefaciens (Tang et al. (1990) Gene 96, 89-93), Bacillus subtilis (Fonet et al. (1984) Biochem. Biophys. Res. Commun. 119, 795-800), Erwinia amylovora (Geier and Geider (1993) Physiol. Mol Plant Pathol. 42, 387-404).

A nucleic acid according to the invention particularly preferably contains acid molecule which encodes in Example 1  Region from Erwinia amylovora.

The present invention further relates to nucleic acid molecules whose sequences differ due to the degeneration of the genetic codes of sequences of the above Nucleic acid molecules differ and that for a protein or fragment thereof encode the biological activity of a secreted Levansucrase exhibits.

The nucleic acid molecules according to the invention can be any Be nucleic acid molecules, in particular DNA or RNA molecules, for example cDNA, genomic DNA, mRNA etc. You can naturally occurring or by genetic engineering or chemical Molecules produced synthetic processes.

According to the invention, the nucleic acid molecules of the present Invention linked to regulatory elements that the Ensure transcription and translation in the plant cell Afford. By providing the invention Nucleic acid molecules now have the option of being vegetable Cells using genetic engineering methods change that they cause a new, local cell death have enzymatic activity.

A particular advantage of the invention is that the local cell death in plants using the Invention appropriate nucleic acid sequences are optionally triggered can. In a preferred embodiment, the nucleus is acid sequence that is responsible for local cell death encoded enzymatic activity, pathogenic under control induced promoters. In a particularly preferred The nucleic acid sequence is in combination with the prp1-1 promoter from potato.

Other preferred embodiments include the combination the nucleic acid sequence responsible for cell death with in  Plant active tissue or development specific promo goals. A nucleic acid molecule in which the coding nucleic acid sequence under the control of a anther or wallpaper specific promoter.

Another object of the invention is to use vectors and To deliver microorganisms, the use of which is the manufacture new plants in which localized cell death produces can be made possible. This task is done by the ready position of the vectors and microorganisms according to the invention resolved the new ones causing local cell death Contain nucleic acid sequences.

The present invention thus also relates to vectors, in particular special plasmids, cosmids, viruses, bacteriophages and others in Genetic engineering common vectors that described above Contain and given nucleic acid molecules according to the invention if for the transfer of the nucleic acid according to the invention molecules are used on plants or plant cells can.

The invention also relates to transformed microorganisms, such as bacteria, viruses, fungi, the nucleic acids according to the invention contain acid sequences.

In a preferred embodiment, those are in the vectors contained nucleic acid molecules linked to regulatory Elements related to transcription and translation ensure prokaryotic and eukaryotic cells.

Optionally, the nucleic acid sequences of the invention through enhancer sequences or other regulatory sequences be supplemented. These regulatory sequences include e.g. B. also signal sequences necessary for the transport of the gene product a certain compartment.  

It is also an object of the invention to prepare new plants deliver that are characterized by local cell death, which may be linked to the acquisition of a SAR. Another The task is to deliver plants that are male sterile are.

These tasks are accomplished by transferring the invention appropriate nucleic acid molecules and their expression in plants, which can be controlled in a targeted manner if necessary.

By providing the nucleic acid according to the invention Molecules now have the ability to plant cells to change by means of genetic engineering methods that they have a new enzymatic compared to wild-type cells Exhibit activity, and as a result, local cell death is coming.

In a preferred embodiment, therefore the host cells according to the invention are plant cells which due to the presence and expression of an additional one compared to the inventive nucleic acid molecule non-transformed cells have a new enzymatic activity which is responsible for local cell death. In a particularly preferred embodiment is transgenic plant cells that have a new levan sucrase or Have invertase.

Alternatively, the new enzymatic activity that the Cell death results in a self-replicating system in the cell be introduced.

The invention also relates to transgenic plants which the transgenic plant cells described above, in which the new nucleic acid molecules integrated into the plant genome are present. The invention also relates to Plants in whose cells the nucleic acid according to the invention  sequence is in self-replicating form, d. H. the Plant cell contains the foreign DNA on an independent Nucleic acid molecule.

In the plants with the nucleic acid according to the invention molecules are transformed and in which due to the Introduction of such a molecule one for a localized one Protein responsible for cell death is synthesized, it can are basically any plant. Preferably it is a monocot or dicot crop. examples for monocotyledonous plants are the plants belonging to the genera Avena (oats), Triticum (wheat), Secale (rye), Hordeum (Barley), oryza (rice), panicum, pennisetum, setaria, sorghum (Millet), Zea (corn) belong. With dicotyledonous crops are u. a. to name cotton, legumes, like legumes and in particular alfalfa, soybean, rapeseed, tomato, sugar beet, Potato, ornamental plants, trees. More crops can Fruit (especially apples, pears, cherries, grapes, Citrus, pineapple and bananas), oil palms, tea, cocoa and Coffee bushes, tobacco, sisal and medicinal plants Rauwolfia and be digital. Cereals are particularly preferred, Wheat, rye, oats, barley, rice, corn and millet, sugar turnip, rapeseed, soy, tomato and potato.

The invention also relates to propagation material from plants according to the invention, for example seeds, fruits, Cuttings, tubers, rhizomes etc., this ver propagation material optionally described above transgenes Contains plant cells, as well as parts of these plants such as Protoplasts, plant cells and calli.

The invention also relates to plants which, owing to the Expression of the nucleic acid molecules according to the invention Compared to plants that don't have the nucleic acid molecules contain, have increased salicylic acid concentration. The invention further relates to plants that are compared  to plants that the new nucleic acid molecules don't included, characterized by increased disease resistance.

In a particularly preferred embodiment, it is for plants that are due to the expression of a levan sucrase Gene from E. amylovora or an invertase gene from yeast, one show local cell death, possibly with the form of a SAR goes along.

The present invention also relates to plants which by the expression of one of the nucleic acids according to the invention molecules are male sterile.

In a further embodiment, the invention relates to hosts cells, especially prokaryotic and eukaryotic cells, those with a nucleic acid molecule described above or a Vector were transformed or infected, and cells by such host cells and the described Contain nucleic acid molecules or vectors. The host cells can be bacteria or fungal cells as well as vegetable or be animal cells.

The present invention is also based on the object Process for the production of plants characterized by a distinguish local cell death.

This problem is solved by methods with the help of which Generation of new plants that cause localized cell death show and, if necessary, additionally by acquiring a SAR are marked, as well as plants that are due to the localized cell deaths are male sterile, is possible.

There are various ways to create such new plants Methods. On the one hand, plants or plant cells can With the help of conventional genetic engineering transformation methods  be changed so that the new nucleic acid molecules in the plant genome is integrated, d. H. that stable Transformants are generated. Second, an invention appropriate nucleic acid molecule, its expression in the plants cell causes cell death in the plant cell or the plant as a self-replicating system be. Thus, the nucleic acid molecules according to the invention can be used e.g. B. contained in a virus with which the plant or Plant cell comes into contact.

According to the invention, plant cells, which are due to the Expression of a nucleic acid sequence according to the invention have new activity causing cell death by a Process prepared comprising the following steps:

• a) Production of an expression cassette which contains the following DNA Sequences include:
• - a promoter that translates into ensures plant cells;
• - At least one nucleic acid according to the invention sequence for a protein or fragment encoded thereof, whose enzymatic activity in Planting causes localized cell death, whereby the nucleic acid sequence in sense orientation the 3'-end of the promoter is coupled; and
• - If necessary, a termination signal for the Termination of transcription and addition of a poly A tail to the corresponding one Transcript encoding at the 3′-end Region is coupled.
• b) Transformation of plant cells with that in step a) produced expression cassette.
• c) regeneration of transgenic plants and possibly the Propagation of plants.

Alternatively, one or more nuclei according to the invention acid sequences as a self-replicating system in the plants cell or plant are introduced.

Another object of the invention is to use the nucleic acid sequences according to the invention and that by them to show coded proteins.

This object is achieved by the uses of the invention new DNA molecules to create plants that stand out localized cell death and possibly by comparison Characterize increased disease resistance to wild type, solved.

Furthermore, the invention relates to the use of Nucleic acid sequences according to the invention for generating Plants that are male sterile and therefore particularly suitable for are hybrid breeding.

The invention also relates to the use of the prp1-1 promoter for the production of plants in which a localized cell death occurs and in particular induced can be.

The invention also relates to the use of a Levansucrase gene or an invertase gene for generating Plants that are characterized by localized cell death award.

The invention further relates to the use of a protein the enzymatic activity of a levan sucrase or a Invertase to localize cell death in plants to evoke.

The present invention thus encompasses every possible form of Use of the nucleic acid molecules according to the invention, their  Expression in plants causes cell death, as well the use of the proteins or fragments according to the invention of which the enzymatic activity causes cell death.

In principle, everyone comes into the for the promoter mentioned the transformation chosen plants in functional promoter Consideration that meets the condition that the one he regulates Expression leads to localized necrosis and the plant does not die off too extensively. Particularly useful For this purpose promoters appear that are specific when infested by Pathogens can be induced locally. The term closes Pathogen in the context of the present invention fungi, bacteria, Viruses, insects and nematodes. Such promoters are known and described in the literature, cf. e.g. B. Martini et al (1993) Molecular and General Genetics 236, 179-186, or WO 94/17194. If such promoters are not known, it is Concept for isolating such promoters is known to the person skilled in the art. In a first step, a tissue, which is infected by a certain pathogen, the poly (A) ⁺ RNA isolated and a cDNA library was created. In a second step are generated with the help of cDNA clones based on poly (A) ⁺ RNA molecules len based on a non-infected tissue, from the first Bank uses hybridization to identify those clones their corresponding poly (A) ⁺ RNA molecules only at Infestation can be induced by the pathogen. Then be isolated with the help of these cDNA's promoters identified in this way, which then code for the expression of those described here the nucleic acid sequences can be used.

An alternative to achieving the goal that the plant in many areas due to the expression of the invention Nucleic acid molecules has localized cell death, is to use a constitutive promoter this promoter but from the coding sequence of the inventions DNA molecules according to the invention by inserting a trans separable positive elements (transposon). From transposons is  known to excite in somatic tissues. Such Excisions lead to promoter and coding sequence in be brought into direct contact and thus lead to education those responsible for localized cell death enzymatic activity. Transposons are in the literature described and cloned and are available in rich selection Available, such as B. Ac / Ds (activator / dissociation- Transposon family), En / Spm from maize (enhancer / suppressor mutator transposon family; see. During and Sarlinger (1986) Ann. Rev. Genet. 20, 175 for an overview).

The use of the nucleic acid molecules according to the invention for Local cell death can be generated in addition to that described Use to create systemically acquired resistance (SAR) against pathogens and thus for the generation of Plants that are characterized by increased disease resistance award, also for the production of male sterile plants be used. Male sterile plants play in the Plant breeding, especially in hybrid breeding, a significant role. For this purpose the cell death Nucleic acid encoding causative enzymatic activity sequence with an anther or wallpaper specific promoter coupled. Examples of such promoters are e.g. B. in WO92 / 13956 and WO92 / 13957. This leads to the death of the Anthers and thus to prevent pollen formation. On Restorer gene with the nucleic acid sequence according to the invention can be used together, which would result in cell death suppress enzymatic activity. So z. B. when using the region coding for a levan sucrase the formation of the product, that is, the Levan, by a simultaneous expression of a levanase in the cell space is affected will. Alternatively, the expression of a corresponding Antisense construct that cause localized cell death de enzymatic activity due to antisense suppression influence. Likewise, the expression of the Invention appropriate nucleic acid molecules and thus the resulting  cell killing activity by the phenomenon of cosuppression through the additional introduction of sense constructs into the Plant cell are affected.

Furthermore, the nucleic acid sequences according to the invention used together with promoters used in plants abiotic stimuli such as B. chemicals, ozone, UV radiation, extreme temperatures, dryness, salt. Examples of such promoters can be found in the literature, so z. B. Williams et al. (1992) Biotechnology 10, 540.

There is basically the possibility that the for the local cell death responsible enzymatic activity in each any compartment of the plant cell can. In a preferred embodiment, the shape of the Protein or a fragment thereof chosen in the extra cellular space, d. H. in the apoplast, is localized. In a particularly preferred embodiment is the coding Sequence of levansucrase used as derived from Erwinia amylovora has been isolated.

In a further preferred embodiment, the Invertase gene from S. cerevisiae as a fusion with signal sequences, which secrete the gene product from the cell interior guaranteed, used.

The invention also relates to nucleic acid molecules, those for proteins with the biological activity of a Levan encode sucrase or invertase or biologically active Fragments thereof and those with one of those described above Hybridize nucleic acid molecules. The term biological active fragments refers to fragments that are dying the plant cell can cause. The term "hybridization" means hybridization in the context of this invention conventional hybridization conditions, preferably under stringent conditions, as for example in Sambrook et  al (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

Nucleic acid molecules with the molecules of the invention hybridize, z. B. from genomic or from cDNA libraries be isolated.

The identification and isolation of such nucleic acid Molecules can use the invention Nucleic acid molecules or parts of these molecules or the Reverse complements of these molecules occur, e.g. B. means Hybridization according to standard methods (see e.g. Sambrook et al, supra).

Thus, the invention also relates to the use of a DNA sequence according to the invention or parts thereof Identification and isolation of homologous sequences from plants or other organisms.

As a hybridization probe z. B. nucleic acid molecules used exactly or essentially the above listed nucleotide sequences or parts of these sequences exhibit. In the used as a hybridization probe Fragments can also be synthetic fragments, which have been produced with the help of common synthetic techniques and their sequence essentially with that of an invention according nucleic acid molecule. You have genes identified and isolated that with the invention Hybridizing nucleic acid sequences is a determination of Sequence and an analysis of the properties of this Sequence encoded proteins required. For this stand the Specialist in a range of molecular biological, biochemical and standard biotechnological processes are available.  

The hybridi with the nucleic acid molecules according to the invention Molecules also include fragments, derivatives and allelic variants of the DNA molecules described above, the encode a Levansucrase or Invertase or a biological, d. H. enzymatically active fragment thereof. Among fragments parts of the nucleic acid molecules are understood which are long enough to be a polypeptide with the enzymatic Levansucrase or invertase activity or comparable enzymatic activity that localized Cell death requires coding. The term derivative means in this connection that the sequences of these molecules differ from the sequences of the nucleic acid molecules described above distinguish one or more positions and a high one Show degree of homology to these sequences. Homology means a sequence identity of at least 40%, in particular an identity of at least 60%, preferably over 80% and particularly preferably over 90%. The deviations too the nucleic acid molecules described above can by Deletion, addition, substitution, insertion or recombination have arisen.

Homology also means that functional and / or struc tural equivalence between the nucleic acid in question molecules or the proteins encoded by them. At the nucleic acid molecules homologous to those described above are molecules and represent derivatives of these molecules, are usually variations of these molecules, the modifications represent the same biological radio exercise. It can be both natural occurring variations are, for example, sequences from other organisms, or to mutations, these modifi cations may have occurred naturally or through targeted mutagenesis. Furthermore, it can the variations on synthetically produced sequences act. The allelic variants can be both naturally occurring as well as synthetically manufactured or  act variants generated by recombinant DNA techniques.

The of the different variants of the invention Proteins encoded by nucleic acid molecules have certain common characteristics. For this, e.g. B. Enzyme activity, molecular weight, immunological reactivity, Conformation etc. Other common characteristics can physical properties such. B. the running behavior in gel electrophoresis, chromatographic behavior, sediments tion coefficients, solubility, spectroscopic properties stability, pH optimum, temperature optimum, etc. len. Furthermore, the products of those of the Proteins catalyzed reactions common or similar Features.

Evidence of the enzymatic activity of levansucrase can for example by demonstrating the formation of Levan take place (cf. Ebskamp et al (1994) Bio / Technology 12, 272-275). This detection is based on the fact that a protein with a Levansucrase activity if protein extracts are incubated with sucrose and the resulting Levan as a polyfructan with fructose-specific agents is pointed. It is also possible through shot gun expression in z. B. E. coli when cultivated on sucrose containing medium Levan as polymer in situ when using Evidence of replica plates.

Evidence of the enzymatic activity of the invertase can according to Schaewen et al. (1990) EMBO J.9, 3033-3044 will.

To prepare the introduction of foreign genes into higher plants a large number of cloning vectors are available, which have a replication signal for E. coli and a marker gene Selection of transformed bacterial cells included. Examples of such vectors are pBR322, pUC series,  M13mp series, pAcYC184 etc. The desired sequence can be on a suitable restriction interface in the vector be introduced. The plasmid obtained is used for the Transfor mation of E. coli cells used. Transformed E. coli Cells are grown in a suitable medium and then harvested and lysed. The plasmid is back won. As an analysis method to characterize the plasmid DNA obtained are generally restriction analyzes, gel electrophoresis and other biochemical molecular biological methods used. After every manipulation lation, the plasmid DNA can be cleaved and DNA Fragments can be linked to other DNA sequences. Each Plasmid DNA sequence can be in the same or different plasmids to be cloned.

For the introduction of DNA into a plant host cell There are a variety of known techniques available the person skilled in the art will find the appropriate method without difficulty can determine. These techniques include transformation plant cells with T-DNA using agro bacterium tumefaciens or Agrobacterium rhizogenes as Transformation means, the fusion of protoplasts, the direct gene transfer of isolated DNA in protoplasts that Electroporation of DNA, the introduction of DNA by means of the biolistic method and other possibilities. Alternatively the nucleic acid molecules according to the invention, e.g. B. about a viral infection, as a self-replicating system without subsequent integration into the plant genome in the cells be introduced.

In the injection and electroporation of DNA into plant cells are no special requirements per se Plasmids. The same applies to direct gene transfer. Simple plasmids such as e.g. B. pUC derivatives used will. But should whole cells be transformed from this Plants are regenerated, the presence of a selek  animal marker gene necessary. The experts are familiar Known selection marker and it poses no problem for him represents selecting a suitable marker.

Depending on the method of introducing desired genes into the plants cell may require additional DNA sequences. Will e.g. B. for the transformation of the plant cell the Ti or Ri Plasmid used, at least the right boundary, but often the right and left boundaries of the Ti and Ri plasmid contained T-DNA as the flank region with the one supplying genes.

If Agrobacteria are used for the transformation, the DNA to be inserted is cloned into special plasmids, and either in an intermediate or in a binary Vector. The intermediate vectors can be due to sequences zen, which are homologous to sequences in the T-DNA, by homologous Recombination in the Ti or Ri plasmid of the agrobacteria to get integrated. This also contains those for the transfer the vir region necessary for T-DNA. Intermediate vectors can do not replicate in agrobacteria. Using a helper plasmids can be the intermediate vector on Agrobacterium tumefaciens are transmitted (conjugation). Binary vectors can replicate in both E. coli and Agrobacteria. They contain a selection marker gene and a linker or Polylinker, which is from the right and left T-DNA border region be framed. You can go straight into the agrobacteria be transformed (Holsters et al (1978) Molecular and General Genetics 163, 181-187). That serving as the host cell Agrobacterium is said to be a plasmid that carries a vir region contain. The vir region is for the transfer of the T-DNA into the Plant cell necessary. Additional T-DNA may be present.

The agrobacterium transformed in this way becomes a transfor mation of plant cells used.  

The use of T-DNA for the transformation of plants cells has been intensively examined and is sufficient in EP 120 515; Hoekema in: The Binary Plant Vector System, Offsetdrokkerÿ Kanters B.V., Alblasserdam (1985) Chapter V; Fraley et al (1993) Crit. Rev. Plant. Sci., 4, 1-46 and An et al (1985) EMBO J. 4, 277-287.

For the transfer of the DNA into the plant cell, plant Explants expediently with Agrobacterium tumefaciens or Aarobacterium rhizogenes can be cultivated. From the infected Plant material (e.g. leaf pieces, stem 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 cells whole plants can be regenerated again. The The plants are regenerated according to the usual regeneration methods methods using known culture media. The so obtained plants can then on the presence of imported DNA are examined. Other ways of Introduction of foreign DNA using the biolistic Process or by protoplast transformation are known (see e.g. Wilmitzer L. (1993) Transgenic Plants, in: Biotechnology, A Multi-Volume Comprehensive Treatise (H.J. Rehm, G. Reed, A. Pühler, P. Stadler, eds.) Vol. 2, 627-659, V.C.H. Weinheim - New York - Basel - Cambridge).

While the transformation of dicotyledonous plants via Ti plasmid Vector systems with the help of Agrobacterium tumefaciens probably is established, recent works indicate that too monocot plants of transformation using Agrobacterium based vectors are very accessible (Chan et al (1993) Plant Mol. Biol. 22, 491-506; Hiei et al (1994) Plant J. 6, 271-282; Deng et al (1990) Science in China 33, 28-34; Wilmink et al (1992) Plant Cell Reports 11, 76-80; May et al (1995) Bio / Technology 13, 486-492; Conner and Domiss (1992) Int. J. Plant Sci. 153, 550-555; Ritchie et al (1993) Trans  genic Res. 2, 252-265).

Alternative systems for the transformation of monocotyledonous plants are the transformations using the biolistic approach (Wan and Lemaux (1994) Plant Physiol. 104, 37-48; Vasil et al (1993) Bio / Technology 11, 1553-1558; Ritala et al (1994) Plant Mol. Biol. 24, 317-325; Spencer et al (1990) Theor. Appl. Genet. 79, 625-631), the protoplast transformation, the Electroporation of partially permeabilized cells that Introduction of DNA using glass fibers.

The transformation of maize is specific in the literature variously described (see e.g. WO 95/06128, EP 0 513 849; EP 0 465 875; Fromm et al (1990) Biotechnology 8, 833-844; Gordon-Kamm et al (1990) Plant Cell 2, 603-618; Koziel et al (1993) Biotechnology 11, 194-200). In EP 292 435 a Process described, with the aid of which, starting from a slimy, soft (friable) granular corn callus, fertile Plants can be preserved. Shillito et al ((1989) Bio / Technology 7, 581) have observed in this context that it is also for the regenerability to fertile plants is necessary to start from callus suspension cultures which is a dividing protoplast culture with the ability to regenerate plants, can be produced. After one in in vitro cultivation time of seven to eight months Shillito et al plants with viable offspring that however, abnormalities in morphology and reproduction exhibit.

Prioli and Sondahl ((1989) Bio / Technology 7, 589) regeneration and extraction of fertile plants from maize Protoplasts, the Cateto maize inbred line Cat 100-1. The Authors suspect that protoplast regeneration is too fertile Planting a number of different factors, such as B. from Genotype, the physiological state of the donor cells and the cultivation conditions.  

Also the successful transformation of other types of grain has already been described, e.g. B. for barley (Wan and Lemaux, supra; Ritala et al, supra) and for wheat (Nehra et al (1994) Plant J. 5, 285-297).

Once the inserted DNA is in the genome of the plant cell integrated, so it is usually stable and stays there in the descendants of the originally transformed cell receive. It usually contains a selection marker, the the transformed plant cells resistance to one Biocide or an antibiotic such as kanamycin, G418, bleomycin, Hygromycin, methotrexate, glyphosate, streptomycin, sulfonyl Urea, gentamycin or phosphinotricin u. a. mediated. Of the individually selected markers should therefore select trans formed cells versus cells to which the inserted DNA missing, allow.

The transformed cells grow within the plant in the usual way (see also McCormick et al (1986) Plant Cell Reports 5, 81-84). The resulting plants can be normal to be grown and with plants that transfor the same possessed inheritance or other inheritance, crossed will. The resulting hybrid individuals have that corresponding phenotypic properties. From the plants cells can be harvested.

Two or more generations should be attracted to ensure that the phenotypic trait is stable is retained and inherited. Seeds should also be harvested to ensure that the appropriate phenotype or other peculiarities have been preserved.

Transgenic lines can also be determined by conventional methods that are homozygous for the new nucleic acid molecules and their phenotypic behavior towards one  localized cell death and / or an SAR and / or increased Disease resistance and / or male sterility were examined and compared to that of hemizygotic lines.

Another object of the present invention is Use of the nucleic acid molecules according to the invention or Parts of these molecules or their reverse complement Molecules to identify and isolate homologous molecules the proteins with the enzymatic activity of a levan sucrase or encode invertase or fragments of such proteins Plants or other organisms. For the definition of For the term "homology" see above.

The following examples serve to explain the Invention.

Examples

The following methods are used in the examples:

• 1. Cloning procedure:
  The vector pBluescript II SK (Stratagene) was used for cloning in E. coli.
• 2. Bacterial strains:
  E. coli strain DH5alpha (Bethesda Research Laboratories, Gaithersburgh, USA) was used for the Bluescript vector and the Lss8 constructs.
• 3. Transformation of potatoes:
  Ten small leaves of a potato sterile culture (Solanum tuberosum L. cv.) Desiree) wounded with the scalpel were placed in 10 ml of MS medium (Murashige and Skoog (1962) Physiol. Plant. 15, 473) with 2% sucrose, which was 50 µl of an Agrobacterium tumefaciens culture grown under selection overnight. After gently shaking for 3-5 minutes, incubation was continued for two days in the dark. The leaves were then used for callus induction on MS medium with 1.6% glucose, 5 mg / l naphthylacetic acid, 0.2 mg / l benzylamino purine, 250 mg / l claforan, 50 mg / l kanamycin and 0.8% Bacto agar placed. After a week's incubation at 25 ° C. and 3000 lux, the leaves were induction on MS medium with 1.6% glucose, 1.4 mg / l zeatin ribose, 20 μg / l naphthylacetic acid, 20 μg / l giberellic acid, 250 mg / l Claforan, 50 mg / l kanamycin and 0.8% Bacto agar.
• 4. Transformation of tobacco plants and regeneration of intact plants:
  An overnight culture of the corresponding Agrobacterium tumefaciens clone was centrifuged for three minutes at 6500 rpm and the bacteria were resuspended in YEB medium. Tobacco leaves from a tobacco sterile culture (Nicotiana tabacum cv. Samsun NN) were cut into small pieces of approx. 1 cm² and bathed in the bacteria suspension. The leaf pieces were then placed on MS medium (0.7% agar) and incubated for two days in the dark. The leaf pieces were then sprouted on MS medium (0.7% agar) with 1.6% glucose, 1 mg / l 6-benzylaminopurine, 0.2 mg / l naphthylacetic acid, 500 mg / l claforan and 50 mg / l Kanamycin placed. The medium was changed every seven to ten days. When shoots had developed, the leaf pieces were transferred to glass jars containing the same medium. Resulting shoots were cut off and placed on MS medium with 2% sucrose and 250 mg / l Claforan and whole plants were regenerated from them.
• 5. RNA extraction and Northern blot experiments:
  RNA was isolated from frozen plant material as described in Logemann et al (1987) Anal. Biochem. 163, 21-26. The RNA was denatured in 40% formamide. The RNA was then separated by gel electrophoresis on formaldehyde / agarose gels and, after the gel run, blotted on nylon membranes (Hybond N; Amersham, UK). Hybridization against a radiolabelled DNA probe was carried out according to standard methods (e.g. Sambrook et al, supra).
• 6. Plant husbandry:
  Potato plants (Solanum tuberosum) were kept in the greenhouse at 60% humidity and 22 ° C for 16 hours in the light and 15 ° C for 8 hours in the dark. Tobacco plants (Nicotiana tabacum) were kept in the greenhouse at 60% humidity and 25 ° C for 14 hours in the light and 20 ° C for 10 hours in the dark.
• 7. Determination of salicylic acid contents:
  The methods described in Yalpani et al (1991) The Plant Cell 3, 809-818 and Gaffney et al (1993) Science 261, 754-756 were used to determine the content of salicylic acid.

Description of the pictures

Fig. 1: shows the appearance of necrotic areas in transgenic tobacco plants that have been transformed with the construct Lss8.

Fig. 2: shows the amount of salicylic acid in transgenic tobacco plants ( 1-5 ) compared to wild-type plants ( 6-7 ).

Fig. 3: shows the result of a Northern blot experiment. 30 µg poly (A) (mRNA from different transgenic tobacco plants (lane 1-5 ) and two non-transformed tobacco plants (lane 6-7 ) were used for the analysis. A cDNA of the PR-1-1 gene was used as a probe.

Embodiments Embodiment 1

Construction of a chimeric gene consisting of the coding Region of the levansucrase gene from Erwinia amylovora and one truncated promoter region of the prp1-1 gene from Solanum tuberosum.

First, the restriction enzymes EcoRI and XbaI a fragment of the prp1-1 promoter, which the nucleotides +31 to -402, from plasmid pBS42-1 (Martini et al (1993) Mol. Gene. Genetics 236, 179-186) cut out. This EcoRI XbaI promoter fragment was then inserted into the BINAR vector (Höfgen and Willmitzer (1992) Plant Science 87, 45-54), taking from the vector BINAR using the same enzymes the CaMV 35S promoter was cut out. The resulting one binary vector thus contains the prp1-1 promoter region and that Termination signal of the octopine synthase gene (OCS), separated by restriction sites of the pVC18 polylinker that the Insert coding regions and thus the construction allow appropriate expression cassettes. Subsequently the coding region of Levansucrase was created with the help of the Restriction enzymes XbaI and SalI from the vector pEa-LSK5 (Geier and Geider (1993) Physiol. Mol. Plant Pathology 42, 387-404) cut out and into the analog cut, the prp1-1- Vector containing promoter fragment used. This as Vector designated Lss8 was then in Agrobacterium tumefaciens transformed and to transform tobacco  plants and potato plants.

Embodiment 2 Analysis of transgenic tobacco and potato plants using the vector Lss8 included

Transgenic tobacco and potato plants were transformed, selected and regenerated as described above. After rooting in sterile culture, approx. 50 independent transformants were transferred to soil in the greenhouse. 6 to 8 lines with a phenotype clearly identified by the appearance of necrotic spots could be identified (see Fig. 1). Analysis of these necrotic areas for the presence of Levan using the method described above clearly demonstrated Levan. These plants, which had localized cell death due to the expression of the chimeric gene construct pLSS8, were then subjected to further analyzes in the greenhouse.

Embodiment 3 Determination of the salicylic acid content

The salicylic acid content of various transformants was determined using the methods given above. As can be seen from Fig. 2, the transgenic tobacco plants, which are characterized by localized cell death, have a 2-8 times higher amount of salicylic acid compared to wild-type plants.

Embodiment 4 Analysis of the expression of typical PR proteins

The systemically acquired resistance (SAR) is characterized by the coordinated expression of several genes (Ward et al (1991) The Plant Cell 3, 1085-1094). The gene products of these so-called SAR genes include the PR proteins (pathogenesis-related proteins) (cf. Van Loon (1985) Plant Mol. Biol. 4, 111-116). Since a connection between the expression of the PR proteins and the formation of the SAR is considered to be established, the expression of typical PR proteins can be used as an indication of the formation or the presence of a SAR. The expression of PR proteins in plants which express the chimeric Lss8 gene was examined by means of Northern blot analysis. As can be seen in Fig. 3, the expression of the homologous PR1-1 gene can be detected both in green and in the areas of the tobacco plants which already have necrotic spots, while this is not the case in non-transformed control plants.

Claims (56)

1. DNA sequence I, consisting of the following components, which are strung together in the 5'-3 'orientation:

• a) a promoter that is functional, locally inducible or locally active in plants,
• b) at least one nucleic acid sequence which codes for a protein or a fragment thereof, the enzymatic activity of which causes localized cell death in plants and
• c) optionally a termination signal for the termination of the transcription and the addition of a poly-A tail to the corresponding transcript

and any DNA sequences derived therefrom. 2. DNA sequence I according to claim 1, wherein the promoter is pathogen-induced promoter. 3. DNA sequence I according to claim 2, wherein the promoter of prp1-1 promoter from Solanum tuberosum. 4. DNA sequence I according to claim 1, wherein the promoter is tissue-specific promoter. 5. DNA sequence I according to claim 1, wherein the promoter development-specific promoter or a promoter promoted by an abiotic stimulus can be induced. 6. DNA sequence I according to claim 4 or 5, wherein the Promoter is an anther or wallpaper specific promoter.   7. DNA sequence I according to claim 1, wherein a in plants constitutive promoter with a transposable element like this is combined that the promoter and the at least one Nucleic acid sequence separated by the transposon are. 8. DNA sequence I according to claim 1, wherein the Nucleic acid sequence for a protein or a fragment thereof encodes the enzymatic activity of a Has fructosyl transferase. 9. DNA sequence I according to claim 8, wherein the Fructosyltransferase is a levan sucrase. 10. DNA sequence I according to claim 8 or 9, wherein the Nucleic acid sequence for a protein which is in a Enterobacterium occurs in secreted form, or a Fragment of it encoded. 11. DNA sequence I according to claim 10, wherein the Enterobacterium Erwinia amylovora is. 12. DNA sequence I according to claim 1, wherein the promoter the prp1-1 promoter from Solanum tuberosum and the Nucleic acid sequence a nucleic acid sequence from Erwinia amylovora is responsible for a protein with the enzymatic Activity of a levan sucrase or a fragment thereof. 13. DNA sequence I according to claim 1, wherein the Nucleic acid sequence for a protein or a fragment thereof encodes that has the enzymatic activity of an invertase and whereby this nucleic acid sequence by Signal sequences is modified that in plants the secretion of the gene product is guaranteed.   14. DNA sequence I according to claim 13, wherein the Nucleic acid sequence necessary for an invertase or an active one Coded fragment thereof comes from Saccharomyces cerevisiae. 15. DNA sequence I according to claim 14, wherein as Nucleic acid sequence the invertase gene suc2 or parts thereof be used. 16. DNA sequence I according to claim 13, wherein the signal sequences from N-terminal regions of the proteinase inhibitor II gene originate from Solanum tuberosum. 17. DNA sequence I according to claim 1, wherein the Termination signal that of the octopine synthase gene Agrobacterium tumefaciens is. 18. DNA sequence I according to claim 1, the additional Enhancer sequences or other regulatory sequences contains. 19. DNA sequence I according to claim 1, wherein the coding nucleic acid sequence in the antisense orientation is present. 20. Vectors which the DNA sequence I according to one of the preceding claims included. 21. Vector plasmid Lss8. 22. Transformed microorganisms that Sequence I according to one of the preceding claims. 23. Plants that have a DNA sequence I according to one of the Claims 1 to 21 or a DNA sequence derived therefrom contain as well as parts of these plants or their propagation material such as protoplasts, plant cells, calli, seeds,  Tubers or cuttings, etc., as well as the offspring of these Plants. 24. Plants according to claim 23, the at least one for a systemically acquired resistance typical phenomenon express. 25. Plants according to claim 23 or 24, the one in Compared to plants that do not contain DNA sequence I, have increased salicylic acid concentration. 26. Plants according to any one of claims 23 to 25, the compared to plants that have the DNA sequence I not included, distinguish increased disease resistance. 27. Plants according to claim 23 which are male sterile. 28. Dicotyledonous plants according to one of claims 23 to 27. 29. Plants according to claim 28, which are Useful plants, especially cotton, soybean, rapeseed, tomato, Sugar beet, potato, ornamental plants or trees. 30. Monocot plants according to one of claims 23 to 27. 31. Plants according to claim 30, which are Cereals, in particular oats, wheat, rye, barley, rice, Millet or corn. 32. Plants according to one of claims 23 to 31, in which the DNA sequence I integrated into the genome of the plant is present, as well as the descendants of these plants.   33. Plants according to any one of claims 23 to 31, the DNA sequence I in the form of a self-replicating system, e.g. B. a virus or viral nucleic acid molecules. 34. A method for producing the plants according to one of claims 23 to 33, comprising the following steps:

• a) Preparation of a DNA sequence I, consisting of the following components, which are strung together in the 5'-3 'orientation:
• a promoter which is functional, locally inducible or locally active in plants,
• - at least one nucleic acid sequence coding for a protein or a fragment thereof, the enzymatic activity of which causes localized cell death in plants and
• - if necessary, a termination signal for the termination of the transcription and the addition of a poly-A tail to the corresponding transcript, and, if appropriate, DNA sequences derived therefrom;
• b) transfer of the DNA sequence I to plant cells and optionally integration of the DNA sequence I into the plant genome,
• c) Regeneration of completely transformed plants and, if appropriate, multiplication of these plants.

35. The method of claim 34, wherein the plants or Plant parts of the DNA sequence I in the form of a self-replicating system included. 36. Use of DNA sequence I and / or the vectors according to claim 20 or 21 and / or the transformed Microorganisms according to claim 22 for the transformation of  Plant cells (including protoplasts) and plants (including plant cells, plant parts and seeds). 37. Use of DNA sequence I to generate Plants with localized cell death and in particular can be induced. 38. Use of DNA sequence I to generate Plants that have at least one for a systemically acquired one Resistance typical characteristic. 39. Use of DNA sequence I to generate Plants that one versus plants that the DNA Sequence I not included, increased salicylic acid concentration exhibit. 40. Use of DNA sequence I to generate Plants that one versus plants that the DNA Sequence I not included, increased disease resistance exhibit. 41. Use of DNA sequence I to generate Plants that are male sterile. 42. Use of the prp1-1 promoter to generate Plants with localized cell death and in particular can be induced, and / or the at least one characteristic of a systemically acquired resistance and / or have increased disease resistance. 43. Use of a coding for Levansucrase Nucleic acid sequence from Erwinia amylovora or from parts of causing localized cell death in plants to evoke.   44. Use of a coding for Levansucrase Nucleic acid sequence from Erwinia amylovora or parts thereof to achieve increased disease resistance in plants. 45. Use of a Levansucrase coding Nucleic acid sequence from Erwinia amylovora or parts thereof to produce male sterility in plants. 46. Use of a protein with the enzymatic Activity of a levan sucrase or a fragment thereof in order to Plants cause localized cell death. 47. Use of a protein with the enzymatic Activity of a levan sucrase or a fragment thereof in order to Plants to achieve increased disease resistance. 48. Use of a protein with the enzymatic Activity of a levan sucrase or a fragment thereof in order to Plants to produce male sterility. 49. Use of a coding for an invertase Nucleic acid sequence or parts thereof to be used in plants localized cell death. 50. Use of a coding for an invertase Nucleic acid sequence or parts thereof to be used in plants to achieve increased disease resistance. 51. Use of a coding for an invertase Nucleic acid sequence or parts thereof to be male in plants To produce sterility. 52. Use of a protein with the enzymatic Activity of an invertase or a fragment thereof to convert into Plants cause localized cell death.   53. Use of a protein with the enzymatic Activity of an invertase or a fragment thereof to convert into Plants to achieve increased disease resistance. 54. Use of a protein with the enzymatic Activity of an invertase or a fragment thereof to convert into Plants to produce male sterility. 55. Use of a coding for a Levansucrase Nucleic acid sequence from Erwinia amylovora or parts thereof or their reverse complements for identification and isolation homologous sequences for proteins with the enzymatic Encode activity of a Levansucrase or fragments of such Proteins from plants or other organisms.