WO1997045547A2

WO1997045547A2 - Localised cell death in plants

Info

Publication number: WO1997045547A2
Application number: PCT/EP1997/002749
Authority: WO
Inventors: Lothar Willmitzer; Bernd Müller-Röber
Original assignee: MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.
Priority date: 1996-05-29
Filing date: 1997-05-27
Publication date: 1997-12-04
Also published as: DE19621572A1; WO1997045547A3; AU3168497A

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

Localized cell death in plants

The present invention relates to nucleic acid molecules which, when expressed in plants, lead to the production of localized cell death, and to plant cells and plants which contain such nucleic acid molecules.

Plants are attacked by various pathogens throughout. A particular problem with regard to the attack of plants by pathogens arises in the current form of agriculture, which is characterized in that, due to the large-scale cultivation of monocultures, resistance introduced by means of breeding methods is overcome relatively quickly by mutations on the part of the pests.

In principle, two types of resistance are distinguished in plants, vertical and horizontal resistance. The vertical resistance is generally a gene-for-gene relationship in the sense that a certain avirulence gene of the pathogen is opposed to a certain resistance gene of the plant. This means that vertical resistance is usually monogenic and therefore the easiest to break through. The horizontal resistance is not inherited monogenously, but is usually determined by a series of loci. The horizontal resistance does not lead to complete resistance, as vertical resistance can, but is generally more permanent since many loci are involved.

In the case of vertical resistance, ie in the presence of a resistance gene on the plant side and an avirulence gene on the pathogen side, the resistance manifests itself in the occurrence of local necrosis, which is associated with a hypersensitive reaction on the part of the plant goes hand in hand.

Although the details of the hypersensitive reaction, which is referred to below as "HR", are not completely understood, 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 the inclusion and localization of the pathogen, on the other hand, it can be assumed that the pathogen is damaged by radicals that arise during HR. In addition, HR leads in many cases to the phenomenon of so-called systemically acquired resistance. This systemically acquired resistance is generally referred to as SAR after the English term "systemic acquired resistance".

The SAR represents an interesting special case of plant resistance to pathogens. It is characteristic here that a plant which has a SAR due to an infection by a specific pathogen does not only acquire the acquired resistance to the pathogen producing the SAR owns, but towards a much broader spectrum of pathogens.

In this respect, the SAR is a very desirable reaction of the plant, since in many cases it results in a broad resistance that comes close to horizontal resistance (see, for example, Ryals et al. (1992) SEB Symposium 49, 205-229. The mechanisms that lead to the formation of the SAR are partially understood. In many cases it has been shown that local necrosis of cells associated with HR is necessary for the formation of an SAR. A necrosis which is solely the result of a wound, or induced due to other abiotic stressors is generally not sufficient to produce an SAR. Furthermore, it has been shown that in many cases SAR correlates with an increased level of salicylic acid (cf. eg Metraux et al (1990) Science 250, 1004-1006; Malamy et al. (1990 ⁾ Science 250, 1002-1004. This is also shown by what that exogenous salicylic acid, when it is not added to infizier ^¬ ten plants, can induce the SAR (see, for example, White (1979) Virology 99, 410-412; Palva et al (1994) Molecular Plant-Microb Interactions 7, 356th -363). Ren the wide ^¬ has been observed that m transgenic plants in which the salicylic acid was decreased by the expression of a bacterial Saiicylat- hydroxylase, no SAR can be formed (Gaffney et al. (1993) Science 261, 754-756). The SAR can also be induced by a number of other chemicals, such as, for example, 2,6-dichloroisonicotmic acid (Uknes et al. (1992), The Plant Cell 4, 645-656).

Various systems have so far been described in the literature which lead to localized cell death. Examples include 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 Ayr9 gene from 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, to name other avirulence and resistance genes that come into question for such a solution to the problem). WO 95/31564 describes in a general way that the nucleotide sequence or sequences which are described in connection with localized cell death and the generation of SAR can comprise one or more genes, the specific exemplary embodiment being the resistance gene Cf-9 and the Avirulenzgen Ayr9 describes.

As further examples for the use of a nucleotide sequence or the combination of nucleotide sequences which lead to plant cell necrosis and an SAR, ιr. WO 95/31564 mentions the following possible approaches: an enzyme-conjugating enzyme, the gene VI protein from Cauliflower Mosaic Virus, a viral coat protein in simultaneous presence of a corresponding plant resistance gene, a bacterial 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 Ayr2 from C. fulvum, Cf-4 from tomato and Ayr4 from C. fulvum). Furthermore, an RNAse, the diphtheria toxin and proton pumps such as bacterial proton pumps are mentioned.

As already described above, SAR is a very atractive resistance phenomenon. The problem, however, is that the SAR has not yet been able to be treated in breeding because presumably too many loci are involved and / or the gene products are unknown. It is therefore one of the tasks of bio-technological research to show alternative ways for both localized cell death and the SAR that is often associated with it.

The present invention is therefore based on the object of providing nucleic acid molecules with the aid of which localized cell death and optionally additionally systemically acquired resistance (SAR) can be triggered in plants or plant cells. Another object of the invention is to provide nucleic acid molecules which can serve to produce male sterility in plants. Further objects of the invention will become apparent from the following description.

These tasks are solved by providing the embodiments characterized in the claims.

It has surprisingly been found that certain nucleic acid sequences can be used specifically to trigger localized cell death by triggering the generation of a protein or protein fragment, the enzymatic activity of which causes cell death. In particular, it could be shown experimentally that the expression of a nucleic acid sequence from Erwinia amylovora, which is a secreted form a Levansucrase coded, leads to the formation of HR in plants, which can also be coupled with the occurrence of SAR.

The present invention thus relates to nucleic acid molecules which encode proteins or fragments thereof which, when expressed, lead to the death of the cell. In a preferred embodiment, the nucleic acid molecules according to the invention are nucleic acid molecules which encode proteins with the biological activity of a fructosyl transferase or a biologically active fragment thereof. In a particularly preferred embodiment, the nucleic acid sequence is a nucleic acid sequence which encodes proteins with the biological activity of a levan sucrase or a biologically active fragment thereof, in particular a secreted levan sucrase. In the context of this invention, biologically active fragment means that the mediated biological activity is sufficient to produce cell death.

Nucleic acid sequences which encode proteins with the enzymatic activity of a levan sucrase have been described from various organisms, e.g. from, Bacillus amyloliquefaciens (Tang et al. (1990) Gene 96, 89-93), Bacillus subtilis (Fönet et al. (1984) Biochem. Biophys. Res. Commun. 119, 795-800), Erwinia amylovora ( Geier and Geider (1993) Physiol. Mol. Plant Pathol. 42, 387-404; DE-Al 42 27 061).

In a particularly preferred embodiment, a nucleic acid molecule according to the invention comprises the coding region from Erwinia amylovora mentioned in Example 1.

Furthermore, the present invention relates to nucleic acid molecules whose coding regions differ from sequences of the above-mentioned nucleic acid molecules due to the degeneration of the genetic code and which are a protein or encode a fragment thereof that has the biological activity of a levan sucrase, in particular a secreted levan sucrase.

The nucleic acid sequence which encodes a protein with the biological activity of a levan sucrase or a biologically active fragment thereof can also be sequences which hybridize with one of the nucleic acid molecules described above. The term biologically active fragments refers to fragments that can cause the plant cell to die. In the context of this invention, the term “hybridization” means hybridization under conventional hybridization conditions, preferably under stringent conditions, as described, 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 sequences that hybridize with the above-mentioned molecules can e.g. can be isolated from genomic or from cDNA libraries.

Such nucleic acid sequences can be identified and isolated using the known nucleic acid molecules described above or parts of these molecules or the reverse complements of these molecules, e.g. by means of hybridization according to standard methods (see e.g. Sambrook et al., op. cit.).

For example, nucleic acid molecules can be used as the hybridization probe that have exactly or essentially the nucleotide sequences listed above or parts of these sequences. The used as a hybridization probe fragments may also be synthetic fragments spindles han ^¬, HERGÉ by means of the conventional synthesizing methods and the sequence of which were ^¬ represents coincides with that of a known levansucrase-encoding Nucleinsäuremole- CRWT substantially. Once genes have been identified and isolated that hybridize with the known nucleic acid sequences, a determination of the sequence and an analysis of the intrinsic The proteins encoded by this sequence are required. For this purpose, a number of standard molecular, biological, biochemical and biotechnological methods are available to the person skilled in the art.

The molecules hybridizing with the known nucleic acid molecules also include fragments, derivatives and allelic variants of the DNA molecules described above, which encode a levan sucrase or a biological, i.e. enzymatically active fragment thereof. Fragments are understood to mean parts of the nucleic acid molecules that are long enough to encode a polypeptide with the enzymatic activity of a Levan sucrase or a comparable enzymatic activity that causes localized cell death. The term derivative in this context means that the sequences of these molecules differ from the sequences of the above-described nucleic acid molecules at one or more positions and have a high 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 from the nucleic acid molecules described above may have arisen through deletion, addition, substitution, insertion or recombination.

Homology also means that there is functional and / or structural equivalence between the nucleic acid molecules in question or the proteins encoded by them. The nucleic acid molecules which are homologous to the molecules described above and represent derivatives of these molecules are generally variations of these molecules which represent modifications which have the same biological function. This can involve both naturally occurring variations, for example sequences from other organisms, or mutations, it being possible for these modifications to have occurred naturally or to have been introduced by targeted mutagenesis. Furthermore, the variations can be synthetic. act sequences. The allelic variants can be both naturally occurring and synthetically produced variants or those produced by recombinant DNA techniques.

The proteins encoded by the different variants of the nucleic acid molecules have certain common characteristics. For this, e.g. Enzyme activity, molecular weight, immunological reactivity, conformation etc. belong. Other common characteristics can include physical properties such as e.g. represent the running behavior in gel electrophoresis, chromatographic behavior, sedimentation coefficients, solubility, spectroscopic properties, stability, optimum pH, optimum temperature, etc. Furthermore, the products of the reactions catalyzed by the proteins can of course have common or similar features. In particular, the proteins encoded by the nucleic acid sequences have levan sucrase activity. The detection of the enzymatic activity of levan sucrase can be carried out, for example, by the detection of the formation of levan (cf. Ebskamp et al. (1994) Bio / Technology 12, 272-275). This detection is based on the fact that a protein with a levan sucrase activity can be detected if protein extracts are incubated with sucrose and the levan which forms is detected as a polyfructan with fructose-specific agents. It is also possible to use shot gun expression in e.g. Detect E. coli when cultivated on sucrose-containing medium Levan as a polymer in situ when using replica plates.

The nucleic acid molecules according to the invention can be any nucleic acid molecules, in particular DNA or RNA molecules, for example cDNA, genomic DNA, mRNA etc. They can be naturally occurring molecules or those produced by genetic engineering or chemical synthesis methods.

According to the invention the nucleic acid molecules are present ^¬ the invention to regulatory elements linked to the ensure transcription and translation in the plant cell. By providing the nucleic acid molecules according to the invention, there is now the possibility of changing plant cells by means of genetic engineering methods to the effect that they have an enzymatic activity which causes local cell death.

A particular advantage of the invention is that local cell death in plants can optionally be triggered in a targeted manner using the nucleic acid molecules according to the invention. In principle, any promoter which is functional in the plants selected for the transformation and which fulfills the condition that the expression regulated by it leads to localized necrosis and that the plant does not become too large is suitable for the promoter mentioned Dying brings. Promoters which are specifically induced locally when infected by pathogens appear to be particularly useful for this purpose. The term pathogen in the context of the present invention includes fungi, bacteria, viruses, insects and nematodes. Such promoters are known and described in the literature, cf. eg Martini et al. (1993) Molecular and General Genetics 236, 179-186, or WO 94/17194. If such promoters are not known, the concept of isolating such promoters is known to the person skilled in the art. In a first step, the poly (a) ⁺ RNA is isolated from a tissue infected with a specific pathogen and a cDNA library is created. In a second step, cDNA clones based on poly (A) ⁺ RNA molecules from a non-infected tissue are used to identify those clones from the first bank whose hybrid poly (A) ⁺ RNA is used -Molecules are only induced when the pathogen is infected. Promoters are then isolated with the help of these cDNAs identified in this way, which can then be used for the expression of the coding nucleic acid sequences described here. In a preferred embodiment there is the nucleic acid sequence which the enzymatic activations responsible for the local cell death tat coded, therefore under the control of a pathogen-induced promoter. In a particularly preferred embodiment, the nucleic acid sequence is in combination with the potato prpl-1 promoter.

Further preferred embodiments include the combination of the nucleic acid sequence responsible for cell death with tissue-specific or development-specific promoters active in plants. A nucleic acid molecule in which the coding nucleic acid sequence is under the control of an anther- or tapetum-specific promoter is particularly preferred.

An alternative to achieving the goal that the plant has localized cell death in many areas due to the expression of the nucleic acid molecules according to the invention is to use a constitutive promoter, but this promoter from the coding sequence of the nucleic acid molecules according to the invention by insertion to separate a transposable element (transposon). Transposons are known to excite in somatic tissues. Such excisions result in the promoter and coding sequence being brought into direct contact and thus lead to the formation of the enzymatic activity responsible for the localized cell death. Transposons are described and cloned in the literature and are available in a wide range, e.g. Ac / Ds (activator / dissociation transposon family), En / Spm from maize (enhancer / suppressor mutator transposon family; see Düring and Sarlinger (1986) Ann. Rev. Genet. 20, 175 for an overview) .

The use of the nucleic acid molecules according to the invention for generating a local cell death can in addition to the be¬ signed application to generate a systemic erwor ^¬ surrounded resistance (SAR) to pathogens and thus to He ^¬ generation of plants, the integrated resistance by an increased Krank¬ distinguished, even for the production of male sterile plants can be used. Male sterile plants play an important role in plant breeding, especially in hybrid breeding. For this purpose, the nucleic acid sequence coding for the enzymatic activity causing the cell death is coupled with an anther- or tape-specific promoter. Examples of such motors are mentioned, for example, in W092 / 13956 and W092 / 13957. This leads to the death of the anthers and the sub ^¬ connection pollen formation. A restorer gene, which can be used together with the nucleic acid sequence according to the invention, would suppress the enzymatic activity causing the cell death. For example, if the region encoding a levan sucrase were used, the formation of the product, ie the levan, would be influenced by a simultaneous expression of a levanase in the cell space. Alternatively, the expression of a corresponding antisense construct would influence the enzymatic activity causing the localized cell death due to antisense suppression. Likewise, the expression of the nucleic acid molecules according to the invention and thus the resulting cell-killing activity could be influenced by the phenomenon of cosuppression by the additional introduction of sense constructs into the plant cell.

Furthermore, the nucleic acid sequences according to the invention can be used together with promoters which are induced in plants by abiotic stimuli, e.g. Chemicals, ozone, UV radiation, extreme temperatures, drought, salt can be induced. Examples of such promoters can be found in the literature, e.g. in Williams et al. (1992) Biotechnology 10, 540.

There is basically the possibility that the enzymatic activity responsible for local cell death can be localized in any compartment of the plant cell. In a preferred embodiment, the shape of the protein or a fragment thereof is selected which is localized in the extracellular space, ie in the apoplast. In a particularly preferred embodiment, the coding sequence of levansucrase is used, as has been isolated from Erwinia amylovora.

In a further preferred embodiment, the nucleic acid molecule according to the invention contains a termination signal for the termination of the transcription and the addition of a poly-A tail to the corresponding transcript. Such termination signals are known and can be interchanged with one another as desired. An example of such a sequence is the termination signal of the octopine synthase gene from Agrobacterium tumefaciens.

Optionally, the nucleic acid sequences of the invention can be supplemented by enhancer sequences or other regulatory sequences. These regulatory sequences include e.g. also signal sequences that ensure that the gene product is transported to a specific compartment. A nucleic acid molecule according to the invention preferably comprises a nucleotide sequence which codes an amino acid sequence which ensures the secretion of the protein.

The present invention further relates to vectors, in particular plasmids, cosmids, viruses, bacteriophages and other vectors common in genetic engineering, which contain the above-described nucleic acid molecules according to the invention and, if appropriate, for the transfer of the nucleic acid molecules according to the invention into plants or plant cells can be used.

The invention also relates to host cells which are genetically modified with a nucleic acid molecule or vector according to the invention. These can be prokaryotic as well as eukaryotic cells. In particular, it may be Mikroor ^¬ organisms, such as bacteria, viruses, fungi containing the OF INVENTION ^¬ to the invention nucleic acid sequences, that Mikroor- ganisms that are genetically modified with a nucleic acid molecule according to the invention. In addition, it can also tie ^¬ generic cells.

By providing the nucleic acid molecules according to the invention, there is now the possibility, using genetic engineering methods, to modify plant cells to the extent that they have a new enzymatic activity compared to wild-type cells and, as a result, to localize in plants which contain such cells Cell death can come.

In a preferred embodiment, the host cells according to the invention are plant cells which, owing to the presence and expression of an additionally introduced nucleic acid molecule according to the invention, have an enzymatic activity in comparison to non-transformed cells which is responsible for local cell death is. In a particularly preferred embodiment, these are transgenic plant cells which express a levan sucrase under the control of a pathogen-specific promoter.

The nucleic acid molecule according to the invention can be stably integrated into the genome of the plant cell. Alternatively, the nucleic acid molecule encoding the enzymatic activity that causes cell death can be introduced into the cell as a self-replicating system.

The invention also relates to transgenic plants which contain the transgenic plant cells according to the invention described above, in which the nucleic acid molecules are integrated into the plant genome. Plants are also the subject of the invention, in the cells of which a nucleic acid molecule according to the invention is present in self-replicating form, ie the plant cell contains the foreign DNA on an independent nucleic acid molecule. The plants which are transformed with the nucleic acid molecules according to the invention and in which a protein responsible for localized cell death is synthesized due to the introduction of such a molecule can in principle be any plant. It is preferably a monocotyledon or dicotyledon crop. Examples of monocot plants are the plants belonging to the genera Avena (oats), Triticum (wheat), Seeale (rye), Hordeum (barley), Oryza (rice), Panicum, Pennisetum, Setaria, Sorghum (millet), Zea (corn) belong. In the case of the dicotyledonous crops, cotton, legumes, such as legumes and in particular alfalfa, soybean, rapeseed, tomato, sugar beet, potato, ornamental plants, trees are to be mentioned among others. Other useful plants can be fruit (in particular apples, pears, cherries, grapes, citrus, ananas and bananas), oil palms, tea, cocoa and coffee bushes, tobacco, sisal and, in the case of medicinal plants, rauwolfia and digitalis. Cereals, wheat, rye, oats, barley, rice, corn and millet, sugar beet, rapeseed, soy, tomato and potato are particularly preferred.

The invention furthermore relates to propagation material from plants according to the invention, for example seeds, fruits, cuttings, tubers, rhizomes etc., this propagation material containing transgenic plant cells described above. The present invention also relates to parts of the plants according to the invention, such as e.g. Protoplasts, plant cells and calli that contain nucleic acid molecules according to the invention.

The invention also relates to plants which, owing to the expression of the nucleic acid molecules according to the invention, have an increased salicylic acid concentration in comparison to plants which do not contain the nucleic acid molecules. The invention further relates to plants which, in comparison to plants, contain the nucleic acid molecules according to the invention not included, characterized by increased disease resistance.

In a particularly preferred embodiment, these are plants which, owing to the expression of a levan sucrase gene from E. amylovora, show local cell death which may be associated with the expression of a SAR.

The present invention also relates to plants which are male-sterile due to the expression of a nucleic acid molecule according to the invention.

Furthermore, the present invention relates to a method by means of which it is possible to produce plants which show a localized cell death and which may additionally be characterized by the acquisition of an SAR, and plants which are male-sterile due to the localized cell death .

Various methods are available for producing such plants. On the one hand, plants or plant cells can be modified with the aid of conventional genetic engineering transformation methods such that a nucleic acid molecule according to the invention is integrated into the plant genome, i.e. that stable transformants are generated. On the other hand, a nucleic acid molecule according to the invention, the expression of which in the plant cell causes the cell to die, can be contained in the plant cell or the plant as a self-replicating system. Thus the nucleic acid molecules according to the invention can e.g. be contained in a virus with which the plant or plant cell comes into contact.

According to the invention, plant cells which have an activity causing cell death due to the expression of a nucleic acid molecule according to the invention are produced by a process which comprises the following steps: (a) Production of an expression cassette which comprises the following sequences:

- a promoter that ensures transcription in plant cells;

at least one nucleic acid sequence which encodes a protein or a fragment thereof, the enzymatic activity of which causes localized cell death in plants, the nucleic acid sequence being coupled in sense orientation to the 3 'end of the promoter; and

- if necessary, a termination signal for the termination of the transcription and the addition of a poly-A tail to the corresponding transcript which is coupled to the 3 'end of the coding region.

(b) Transformation of plant cells with that in step

(a) manufactured expression cassette.

(c) Regeneration of transgenic plants and, if appropriate, the multiplication of the plants.

Alternatively, one or more nucleic acid molecules according to the invention can as a self-replicating system in ^¬ These crops zenzelle or plant are introduced.

The invention is also the use of a pathogen-specific promoter, in particular the prpl-1 promoter for the production of plants in which a cell death occurs lokali ^¬ lized and / or the at least one typical for a systemic acquired resistance trait and / or increased Have disease resistance compared to wild ^¬ type plants.

The invention also relates to the use of nucleic acid sequences ^¬ that tivity a protein having the enzymatic Akti ^¬ a levansucrase encode for generating of plant zen, which are characterized by a localized cell death, as well as for the production of a localized cell death in plants. Also, the present invention relates to the Verwen¬ dung such nucleic acid to produce a systemic acquired resistance in plants, to increase the salicylic acid concentration in plants and / or He ^¬ disease resistance heightening in plants.

The invention further relates to the use of a protein with the enzymatic activity of a levan sucrase in order to cause localized cell death in plants.

The present invention also relates to the use of nucleic acid sequences which encode proteins with the biological activity of a levan sucrase, or such proteins, for producing male sterility in plants.

The present invention thus encompasses every possible form of use of the nucleic acid molecules according to the invention, the expression of which in plants causes the death of cells, and of the proteins or fragments thereof according to the invention, the enzymatic activity of which causes cell death.

To prepare the introduction of foreign genes into higher plants, a large number of cloning vectors are available which contain 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, paCYC184 etc. The desired sequence can be introduced into the vector at a suitable restriction interface. The plasmid obtained is used for the transformation of E. coli cells. Transformed E. coli cells are grown in a suitable medium and then harvested and lysed. The plasmid is recovered. As analysis method for characterizing the plasmid DNA obtained, generally restriction analyzes, gel electrophoresis and other biochemical-molecular biological methods are used. After each manipulation, the plasmid DNA can be cleaved and the DNA fragments obtained can be linked to other DNA sequences. Each plasmid DNA sequence can be cloned into the same or other plasmids.

A large number of known techniques are available for introducing DNA into a plant host cell, and the person skilled in the art can determine the appropriate method in each case without difficulty. These techniques include the transformation of plant cells with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as transformation agents, the fusion of protoplasts, the direct gene transfer of isolated DNA into protoplasts, the 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, for example can be introduced into the cells via a viral infection as a self-replicating system without subsequent integration into the plant genome.

In the injection and electroporation of DNA into plant cells, no special requirements are made of the plasmids used per se. The same applies to direct gene transfer. Simple plasmids such as e.g. pUC derivatives are used. However, if whole plants are to be regenerated from such transformed cells, a selectable marker gene should be present. The conventional selection markers are known to the person skilled in the art and it is not a problem for him to select a suitable marker.

Depending on the method of introducing a gene into the plant cell, additional DNA sequences may be required. If, for example, the Ti or Ri plasmid is used for transformation of the plant cell, it should be at least the right border, but often the right and left Be ^¬ limitation of the T-DNA contained in the Ti- and Ri-plasmid as Flanking region to the genes to be introduced linked to ^¬.

If agrobacteria are used for the transformation, the DNA to be introduced should be cloned into special plasmids, either in an intermediate or in a binary vector. The intermediate vectors can on ^¬ Due to sequences that are homologous to sequences in the T-DNA by homologous recombination into the Ti or Ri-Plas¬ the Agrobacteria mid be integrated. This also contains the vir region necessary for the transfer of the T-DNA. Intermediate vectors cannot replicate in agrobacteria. The intermediate vector can be transferred to Agrobacterium tumefaciens by means of a helper plasmid

(Conjugation). Binary vectors can replicate in E. coli as well as in Agrobacteria. They contain a selection marker gene and a linker or polylinker, which are framed by the right and left T-DNA border region. They can be transformed directly into the agrobacteria

(Holsters et al. (1978) Molecular and General Genetics 163, 181-187). The Agrobacterium serving as the host cell should contain a plasmid which carries a vir region. The vir region is usually necessary for the transfer of the T-DNA into the plant cell. Additional T-DNA may be present.

The agrobacterium transformed in this way is used for the transformation of plant cells.

The use of T-DNA for the transformation of plant cells has been intensively investigated and is sufficient in EP 120 515; Hoekema in: The Binary Plant Vector System, Offsetdrokkerij Kanters BV, Alblasserdam (1985) Chapter V; Fraley et al. (1993) Crit. Rev. Plant. Be. 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 can expediently be cultivated with Agrobacterium tumefaciens or Agrobacterium rhizogenes. From the infected plant material (e.g. leaf pieces, stem Segments, roots, but also protoplasts or suspension-cultivated plant cells) can then regenerate whole plants again in a suitable medium, which can contain antibiotics or biocides for the selection of transformed cells. The plants are regenerated by customary regeneration methods using known nutrient media. The plants thus obtained can then be examined for the presence of the introduced DNA. Other possibilities of introducing foreign DNA using the biolostic method or by means of protoplast transformation are known (cf., for example, Willmitzer L. (1993) Transgenic Plants, in: Biotechnology, A Multi-Volume Comprehensive Treatise (HJ Rehm, G. Reed , A. Pühler, P. Stadler, eds., Vol. 2, 627-659, VCH Weinheim - New York - Basel - Cambridge).

While the transformation of dicotyledonous plants via Ti plasmid vector systems with the help of Agrobacterium tumefaciens is well established, recent work indicates that monocotyledonous plants are also very well amenable to Agrobacterium-mediated transformation using Agrobacterium-based vectors (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 Sei. 153, 550-555; Ritchie et al. (1993) Transgenic Res. 2, 252-265).

Zen Alternative systems for Tansformation of monocotyledonous Pflan ^¬ are the transformation by means of the biolistic An¬ set (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, the introduction of DNA using glass fibers. The transformation of maize is described in various ways in the literature (see, for example, 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). EP 292 435 describes a process by means of which fertile plants can be obtained starting from a slimy, soft (friable) granular corn callus. Shillito et al. ((1989) Bio / Technology 7; 581) have observed in this connection that it is also necessary, in order to be able to regenerate into fertile plants, to start from callus suspension cultures from which a dividing protoplast culture with the ability to plants to regenerate, is producible. After an in vitro cultivation period of seven to eight months, Shillito et al. Plants with viable offspring that, however, have abnormalities in morphology and reproduction. Prioli and Sondahl ((1989) Bio / Technology 7, 589) describe the regeneration and the production of fertile plants from maize protoplasts, the Cateto maize inbred line Cat 100-1. The authors suspect that protoplast regeneration to fertile plants depends on a number of different factors, such as, for example, the genotype, the physiological state of the donor cells and the cultivation conditions. The successful transformation of other cereals has also been described, for example for barley (Wan and Lemaux, loc. Cit., Ritala et al., Loc. Cit.) And for wheat (Nehra et al. (1994) Plant J. 5, 285-297).

Once the introduced DNA is integrated in the genome of the plant cell, it is generally stable there and is also retained in the progeny of the originally transformed cell. It normally contains a selection marker which imparts resistance to the transformed plant cells to a biocide or an antibiotic such as kanamycin, G418, bleomycin, hygromycin, methotrexate, glyphosate, streptomycin, sulfonylurea, gentamycin or phosphinotricin and others. The individually selected marker should therefore allow the selection of transformed cells over cells that lack the inserted DNA. 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 grown normally and crossed with plants that have the same transformed genetic makeup or other genetic makeup. The resulting hybrid individuals have the corresponding phenotypic properties. Seeds can be obtained from the plants.

Two or more generations should be grown to ensure that the phenotypic trait is stably maintained and inherited. Seeds should also be harvested to ensure that the appropriate phenotype or other characteristics have been preserved. Likewise, transgenic lines can be determined by conventional methods, which are homozygous for the new nucleic acid molecules and which investigate and investigate their phenotypic behavior with regard to localized cell death and / or SAR and / or increased disease resistance and / or male sterility with that of hemizygote lines. Figure 1: shows the appearance of necrotic areas in transgenic tobacco plants with the construct

Lss8 have been transformed.

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

Figure 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 l gene was used as a probe. 23

The following examples serve to explain the invention.

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 Contained 50 μl of an Agrobacte¬ rium tumefaciens overnight culture grown under selection. After shaking gently for 3-5 minutes, a further incubation was carried out for two days in the dark. The leaves were then used for callus induction on MS medium with 1.6% glucose, 5 mg / 1 naphthylacetic acid, 0.2 mg / 1 benzylaminopurine, 250 mg / 1 claforan, 50 mg / 1 kanamycin and 0.8 % Bacto agar placed. After incubation for one week at 25 ° C. and 3000 lux, the leaves were induction on MS medium with 1.6% glucose, 1.4 mg / 1 zeatin ribose, 20 μg / 1 naphthylacetic acid, 20 μg / 1 giberellic acid, 250 mg / 1 Claforan, 50 mg / 1 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 carried out at 6500 rpm for three minutes centrifuged 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 measuring approx. 1 cm ² and bathed in the bacterial suspension. The leaf pieces were then placed on MS medium (0.7% agar) and incubated for two days in the dark. Then the leaf pieces for shoot induction on MS medium (0.7% agar) with 1.6% glucose, 1 mg / 1 6-benzylaminopurine, 0.2 mg / 1 naphthylacetic acid, 500 mg / 1 claforan and 50 mg / 1 kanamycin. The medium was changed every seven to ten days. When shoots had developed, the leaf pieces were transferred to glass vessels which contained the same medium. Resulting shoots were cut off and placed on MS medium with 2% sucrose and 250 mg / 1 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 blotted on nylon membranes (Hybond N; Amersham, UK) after the gel run. Hybridization against a radioactively labeled DNA probe was carried out according to standard methods (e.g. Sambrook et al, op. Cit.).

6. Plant husbandry:

Potato plants (Solanum tuberosum) were kept in the greenhouse ^¬ house at 60% humidity and 22 ° C for 16 hours in the light and 15 ° C for 8 hours in darkness. 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:

To determine the salicylic acid content, the methods as described by Yalpani et al. (1991) The Plant Cell 3, 809-818 and Gaffney et al. (1993) Science 261, 754-756.

example 1

Construction of a chimeric gene consisting of the coding region of the levansucrase gene from Erwinia amylovora and a shortened promoter region of the prpl-1 gene from Solanum tuberosum

First, using the restriction enzymes EcoRI and Xbal, a fragment of the prpl-1 promoter, which encompasses nucleotides +31 to -402, was made from plasmid pBS42-1 (Martini et al. (1993) Mol. Gen. Genetics 236, 179 -186) cut out. This EcoRI-Xbal promoter fragment was then ligated into the vector BINAR (Höfgen and Willmitzer (1992) Plant Science 87, 45-54), the CaMV 35S promoter being excised from the vector BINAR using the same enzymes. The resulting binary vector thus contains the prpl-1 promoter region and the termination signal of the octopine synthase gene (OCS) separated by restriction sites of the pUC18 polylinker, which allows the insertion of coding regions and thus the construction of appropriate expression cassettes. The coding region of the levan sucrase was then cut out of the vector pEa-LSK5 (Geier and Geider (1993) Physiol. Mol. Plant Pathology 42, 387-404) using the restriction enzymes Xbal and Sall and the prpl-1 Vector containing promoter fragment. This vector, designated Lss8, was then transformed into Agrobacterium tumefaciens and used for the transformation of tobacco plants and potato plants. Example 2

Analysis of transgenic tobacco and potato plants that contain the vector Lss8

Transgenic tobacco and potato plants were transformed, selected and regenerated as described above. After rooting in stone culture, approx. 50 independent transformants were transferred to soil in the greenhouse. 6 to 8 lines which had a phenotype clearly identified by the appearance of necrotic spots could be identified (see FIG. 1). The analysis of these necrotic areas for the presence of Levan by the method described above clearly provided evidence of 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.

Example 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 Figure 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.

Example 4

Analysis of the expression of typical PR proteins

Systemic acquired resistance (SAR) is defined by the coor ^¬ ordinated expression of multiple genes in (Ward et al. (1991) The Plant Cell 3, 1085-1094). To the gene products these so-called SAR genes include the PR proteins (pathogenesis-related proteins) (cf. Van Loon (1985) Plant Mol. Biol. 4, 111-116). Since a relationship between the expression of the PR proteins and the formation of the SAR is considered to be secured, 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 PRI-1 gene can be detected both in green and in areas of the tobacco plants which already have necrotic spots, while in non-transformed areas Control plants are not.

Claims

Patent claims

1. Nucleic acid molecule consisting of the following constituents which are linked to one another in a 5 '-3' orientation:

(a) a promoter which is functional in plants is selected from:

(i) a pathogen-induced promoter, or (ii) a promoter constitutively active in plants in combination with a transposable element, the transposable element separating the promoter from the nucleic acid sequence to be transcribed; and

(b) at least one nucleic acid sequence which encodes a protein with the enzymatic activity of a levan sucrase.

2. The nucleic acid molecule according to claim 1, wherein the promoter is the prpl-1 promoter from Solanum tuberosum.

3. Nucleic acid molecule according to claim 1 or 2, wherein the nucleic acid sequence encodes a levansucrase from Erwinia amylovora.

4. Nucleic acid molecule according to one of claims 1 to 3, wherein the nucleic acid sequence comprises a sequence which ensures the secretion of levan sucrase.

5. Nucleic acid molecule according to one of claims 1 to 4, which further contains a termination signal for the termination of the transcription and the addition of a poly-A tail to the corresponding transcript, the ^¬ ses arranged behind the nucleic acid sequence mentioned under (b) is.

6. Nucleic acid molecule according to claim 5, wherein the termination signal is that of the octopine synthase gene from Agrobacterium tumefaciens.

7. Nucleic acid molecule according to one of claims 1 to 5, which contains additional enhancer sequences.

8. vector containing a nucleic acid molecule according to any one of claims 1 to 7.

9. host cell containing a nucleic acid molecule according to any one of claims 1 to 7 or a vector according to claim 8.

10. Transgenic plant cell that is genetically modified with a nucleic acid molecule according to one of claims 1 to 7 or with a vector according to claim 8.

11. Plant containing plant cells according to claim 10.

12. Plant according to claim 11, which forms at least one phenomenon typical of a systemically acquired resistance.

13. Plant according to claim 11 or 12, which has an increased salicylic acid concentration in comparison to non-transformed plants.

14. Plant according to one of claims 11 to 13, which have an increased disease resistance compared to non-transformed plants.

15. Use of a nucleic acid sequence, which encodes a polypeptide with the enzymatic activity of a levan sucrase, for producing a localized cell death in plants.

16. Use of a nucleic acid sequence which encodes a polypeptide with the enzymatic activity of a levan sucrase to generate a systemically acquired resistance in plants.

17. Use of a nucleic acid sequence, which encodes a polypeptide with the enzymatic activity of a levan sucrase, to increase the salicylic acid concentration in plants.

18. Use of a nucleic acid sequence that encodes a polypeptide with the enzymatic activity of a levan sucrase for the production of plants with increased disease resistance.