EP0441884A1

EP0441884A1 - Wound-induced dna sequence from solanum tuberosum and its use

Info

Publication number: EP0441884A1
Application number: EP89912967A
Authority: EP
Inventors: Jürgen Logemann; Lothar Willmitzer; Josef Schell
Original assignee: Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
Current assignee: Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
Priority date: 1988-11-07
Filing date: 1989-11-03
Publication date: 1991-08-21
Also published as: DK82591D0; DE3837752A1; DK82591A; HUT60326A; DE58908980D1; ES2067514T3; CA2002403C; AU4628289A; ZA898479B; ATE118246T1; AU641373B2; JPH04501507A; EP0368167B1; DE3837752C2; WO1990005187A1; IL92219A; HU896870D0; IL92219A0; EP0368167A1; CA2002403A1

Abstract

Wound-induced DNA and part thereof, from Solanum tuberosum, use therof for the expression of gene products in higher plants in the case of wounding or pathogenic diseases, DNA-transmitting vectors containing them and plants or parts of plants containing them. The promotor part of this DNA sequence can be used in higher plants for the expression of gene products in the case of wounding and/or pathogenic diseases.

Description

Wound-stimulated DNA sequence from Solanum tuberosum

and their use

The invention relates to wound-stimulated DNA from Solanum tuberosum and parts thereof, their use for the expression of gene products in higher plants when wounded or

pathogenic infestation, DNA transmission vectors containing them and plants or parts of plants containing them.

It is known that mechanical wounding of plant tissues can cause morphological and physiological changes. The activity of various enzymes increases after the wound, for example phenylalanine ammonium lyase and peroxidases in potato tubers, extensin in the storage tissue of the carrot, fatty acid synthetase in potato tubers and proteinase inhibitors in tomato and potato leaves. For some of these enzymes, the increase in activity is associated with increased mRNA levels. One of the best understood wound-induced genes is the proteinase inhibitor in tomatoes and potatoes; the mRNA for this gene is significantly increased in injured leaves.

The wound-induced expression of genes in plants is of fundamental interest since the promoters which become effective here are able to specifically activate the associated structural gene after a wound. Such promoters, in conjunction with other genes which confer resistance, for example, encode antibiotic substances or which Promoting healing of injuries is of course of great economic interest if it can be infiltrated into the genetic material of other plants.

It has now been found that, in wounded and / or germ-infested potato plants, the formation of relatively high concentrations of induced mRNA occurs specifically. This mRNA could be isolated and characterized; it does not occur in healthy, unwound plants.

The invention accordingly relates to DNA sequences from Solanum tuberosum which are stimulated by injury and / or infection with pathogenic germs, and to their parts, in particular the promoter part and the structural part. The invention further relates to the use of these DNA sequences for the expression of gene products in higher plants in the case of injury and / or pathogenic attack.

The invention further relates to DNA transmission vectors with DNA sequences inserted therein, as defined above. Finally, the invention relates to plant or plant materials which contain such a DNA sequence.

The term "active region" of a gene, its promoter or structural part is understood to mean those nucleotide sequences which are absolutely necessary in the promoter part of the gene for the activation of the structural part and in the structural part for the expression of an effective or active gene product.

“Structural genes” is understood not only to mean that in the homologous system of the DNA sequence described here from Solanum tuberosum, but also structural genes from other origins, that is to say from heterologous systems. As examples, reference is made to the CAT, NPT and GUS structural genes, those with the one described here wound-stimulable promoter part can be fused and used to express the respective structural gene in the potato or foreign plants.

Any suitable DNA molecules that can be introduced into the ultimately receiving bacterium can be used as transmission vectors. These are usually plasmids as described in more detail in the materials section. The insertion techniques are known to the person skilled in the art.

Bacteria of the genus Agrobacterium, in particular Agrobacterium tumefaciens and Agrobacterium rhizogenes, can be used as bacteria for the absorption and transmission of the transmission vector.

EP-A 122 791 describes a DNA transfer vector which comprises T-DNA with a plant gene inserted therein. This plant gene consists of a plant promoter and a plant structural gene, the plant promoter being adjacent to the 5 'end of the plant structural gene and the plant structural gene being located behind the plant promoter in the direction of transcription. EP-A 122 791 describes in detail methods and measures for inserting genetic material into DNA transmission vectors, to which express reference is made here.

Investigations have shown that Erwinia bacteria are latent on potato plants (Solanum tuberosum) and only mainly affect the stem and tuber of the potato in parallel with a tissue wound. This was induced on potato tubers of the Datura variety by slicing the tuber and simultaneously with Erwinia carotovora ssp. atroseptica was incubated. As expected, these tubers had a strongly macerated surface after 18 hours of incubation. It was not possible to isolate mRNA from the underlying tissue using conventional methods. High contamination the causes of RNA due to polysaccharides, poor precipitation and dissolution behavior of the RNA, a high degree of RNA degradation, low RNA yield and high work and material expenditure. By varying and recombining existing methods, a technique could be developed that was also able to isolate high amounts of undegraded RNA from polysaccharide-containing tissue with little expenditure of material and time (Logemann et al., Anal. Bioch. 163, 16, 1987).

A cDNA library was set up on the basis of purified polyA RNA from wounded tubers of the potato variety Granola, which were also incubated with Erwinia. Differential colony hybridization of 4000 cDNA clones against radioactively labeled RNA from unwound and Erwinia-wounded tubers identified 2 clones, here called wun1 and wun2, whose complementary mRNA in tubers from various tetraploid potato varieties and the haploid variety AM 80/5793 were wounded be induced. Wunl mRNA accumulates 30 minutes after the mechanical wounding of a tuber and is therefore involved in wound-induced primary processes, whereas wun2 is not induced until 3.5 hours later. Both clones still show high expression values 24 hours after the wound. In addition to mechanical wounding, if the tuber is grown with Erwinia carotovora ssp. incubated atroseptica, there are no changes to the expression scheme. Overall, the expression studies on the tuber have shown that wun1 and wun2 mRNAs represent genes which are not induced specifically by Erwinia but can be stimulated by all processes which result in the destruction of the tuber tissue.

Wun1 mRNA accumulates in high amounts in wounded potato tubers (quantitatively comparable to proteinase inhibitor llmRNA in unwound potato tubers, see Sanchez-Serrano et al., Mol. Gen. Genet. 203, 15 (1986)), but is in unwound tubers undetectable. It already appears 30 Minutes after wounding with a maximum from the 4th to 24th hour after wounding. In smaller quantities, it was still detectable after 48 hours.

The accumulation of wun1 and wun2 mRNA after wounding is not limited to the tuber, but also takes place in leaves, stems and roots of various tetraploid potato plants with similar kinetics and intensity.

In contrast to the wun2 mRNA, Wunl mRNA is also induced in leaves without wounding if these are sprayed with compatible Phytophtora infestans spores. This result shows that wun1 can be induced not only by mechanical wounding, but also by the presence of (fungal) pathogens.

There were differences in the expression of the wun1 gene depending on whether the tubers were wounded under aerobic conditions (tuber discs were wetted with P buffer and incubated for 18 hours, so they were still exposed to atmospheric oxygen) or wounded under anaerobic conditions (Tuber disks were immersed in P-buffer and incubated in this low-oxygen state for 18 hours).

Wunl mRNA was expressed higher in aerobically wounded tubers than in the corresponding anaerobic approach.

It is important for the use of the promoters according to the invention for the expression of genes whether the detectable wun1 or wun2 mRNA after wounding arises from new synthesis (transcriptional regulation), or is constitutively present, and is only stable in the event of a wound (post-transcriptional regulation ).

Run-off transcription tests were able to determine transcriptional regulation in both wun1 and wun2, which means that the promoters of these genes are responsible for the resynthesis in the event of a wound. A number of Wunl-homologous cDNA clones, some of which were sequenced, were obtained from the tetraploid potato "Granola" by known methods. By cloning wun1-25A2 in M13mp19-P gnawing in both orientations and partial exonuclease digestion, the nucleotide sequence could be determined bidirectionally according to the dideoxy method.

Fig. 1 shows the relative size of the clones used for sequencing. The clone wun1-25A2 exists in two orientations in the Pstl interface, which are referred to as 25A2 ^* MP19 and 25A2MP19 due to the location of the asymmetrical Xhol interface. By successive exonuclease digestions different sized deletion clones could be obtained, which as clones 25A2 ^* MP19delta 1-3 or 25A2MP19delta 1-4

Sequencing were available.

2 shows the nucleotide sequence of the cDNA clone wun1-25A2. Alternative open reading styles are underlined.

The size of the cDNA clone wun1-25A2 is 711 bp. A 14 bp poly A tail can be found at the 3 end (position 697-711). The largest open reading frame spans 105 amino acids (positions 121-438). Also located 5'-further are two short open reading frames which code for position 3 for 3 amino acids and code for position 8 for 8 amino acids (underlined). However, the last two open reading frames listed differ from the reading frame, which encodes the 105 amino acids. Taking into account the genomic sequence data (FIG. 6), the reading frame coding for 105 amino acids is to be regarded as that of the wun1 protein. The translation starting point of the wun1 protein TTTTTGATGCAA is only slightly correct with that of Joshi, Nuc. Acids Res. 15, 6643, (1987) determined consensus sequence for plant translation starts TAAACAATGGCT.

The investigation of the genomic organization of the gene coding for wun1 mRNA in the haploid cardboard cell line AM80 / 5793 shows that this gene is only present in low copy number. The hybridization of radioactively labeled wun1 cDNA against HindllI, PstI and EcoRI-cut leaf DNA led to the expression of one strong and 1 to 2 weak bands in the "Southern blot" analysis. The result of the "Southern blot" analysis of wun1 and wun2 cDNA is shown in FIG. 3.

The "Southern blot" analysis was able to detect only a few genes responsible for the expression of wun1 and wun2 mRNA in the haploid potato line AM80 / 5793. The chance of obtaining an active gene and not an inactive pseudogen was particularly high here.

The presence of only one pronounced band in the "Southern blot" of the haploid potato line suggests a small number of genets per genome. Considering this in connection with a wound-specific and high expression of wun1 mRNA in the haploid line and the transcriptional expression regulation, highly active genes are likely.

To set up a genomic bank, 10 μg EcoRI-cut DNA from AM80 / 5793 were ligated with EcoRI-cut EMBL4 arms and plated out on a C600 bacterial lawn. About 500,000 plagues were obtained, which statistically should represent the potato genome. After transferring these plagues to nitrocellulose, the genomic clone wun1-22 and wun1-85 could be identified and purified using plaque hybridization techniques against radioactively labeled wun1 cDNA.

The isolation of recombinant EMBL4 DNA from wun1-22 and wun1-85 as well as their restriction mapping and hybridization against radioactive wun1 cDNA resulted in the following organization.

The wun1 cDNA-equivalent fragment in wun1-85 has a size of 4 Kb. It thus corresponds exactly to that in wun1 cDNA hybri fragmenting fragment size of EcoRI-digested DNA from the haploid potato. Due to the asymmetric Xhol cleavage in the 5 'region of the cDNA clone, the orientation of the gene in the 4 Kb fragment could be determined. Accordingly, there is an approximately 1 Kb promoter region in front of the wun1 gene, while an approximately 2 Kb non-homologous part is located 3 'of the gene. The additional 8 Kb EcoRI fragment contained in wun1-85 could not be used for a closer analysis of the wun1 gene. Due to the total digestion of the potato DNA with EcoRI, it is likely that during the subsequent ligation two fragments which do not belong together were brought into the EMBL4 vector and therefore cannot be functionally related to one another.

Figure 4 shows the arrangement of wun1 in the genomic clone wun1-85. The 4 kb EcoRI fragment of the genomic clone wun1-85 contains 1.0 kb of the wun1 promoter, 0.8 kb of the wun1 gene and 2.0 kb of the 3 'end.

Since no differences were found in the restriction behavior between wun1-85 and wun1-22, the 4 Kb fragment of wun1-85 was ligated in EcoRI-cut pUC8 for further analysis (see FIG. 4). For sequencing the wun1 promoter and the wun1 gene, the 4 Kb fragment was further cloned into the EcoRI site of M13mp18. Its orientation was determined by means of control digestions at the asymmetrically located Xhol interface. The clones 85 ^* mp18 and 85mp18 represent both orientations of the

Fragments. Cutting these plasmids with SphI and Xbal made it possible to successively digest the 5 'end of the wun1 gene in clone 85mp18 of the 3' end of the wun1 gene and in clone 85 ^* mp18 using the exonucleasells.

5 shows the result of the deletion analysis of the clone wun1-85 in schematic form. The resulting deletion clones with different fragment sizes were used for sequencing. Overall, it was possible to sequence the entire wun1 gene bidirectionally, as well as to analyze approximately 400 bp of the 3 'end unidirectionally.

6 shows the nucleotide sequence of the wun1 promoter and gene from wun1-85. The CAAT box, TATA box and poly-A signal are highlighted, transcription start and stop are marked by arrows. The Sl nuclease mapping method was used to identify the exact start of transcription of the wun1 gene.

7 explains the determination of the size of the S1-protected fragment of the wun1 gene. Hybridization of the 5 'by the

Xhol cleavage region from pLS000 with wun1 mRNA leads to a 162-179 bp long DNA-RNA hybrid (TU) which is protected from the single-stranded Sl nuclease. The actual transcription start is therefore 162-197 bp 5 'from the Xhol interface (A, C, G, T = sequence plot for determining the size of the DNA fragment).

Assuming the longest fragment preserved, the transcription start begins 179 bp upstream from the Xhol site, i.e. with the sequence ACCATAC. This sequence agrees in the central area (CAT) with that of Joshi et al. (1987) determined the consensus sequence for the start of transcription CTCATCA. Further information results from the position of the transcription start (see FIG. 6):

(1) In position-33, seen from the start of transcription, a TATA box CTATATATT can be found, which matches the one described by Joshi et al. (1987) determined consensus consensus TCACTATATATAG.

(2) The method described by Benoist et al., Nuc. Acids Res. 8, 127-142 (1980) CAAT box in the range between -60 and -80 can be found in the wun1 promoter in position -58 (CAAACT). (3) The 5'-untranslated region of the wun1 gene extends over 217 bp and is therefore relatively large.

(4) The gene encoding wun1 mRNA comprises 794 bp, which corresponds very precisely to the size determination of wun1 mRNA based on the "Northern blot" analyzes.

(5) The cDNA clone wun1-25A2 lacks 97 bp of the 5'-untranslated region in the wun1 gene.

(6) In addition to the open reading frames already found in the cDNA clone, no further ones are found in the 5 '-' translated region of the genomic clone.

(7) The wun1 gene does not have introns.

8 shows the arrangement and position of important regions in the wun1 gene. The wun1 promoter is marked in black, the wun1 gene is hatched. Important recognition sequences are framed; The mRNA is represented by a serpentine line, the protein coding region by crosses. The size of the individual areas is given in base pairs (bp).

Figure 9 shows a sequence comparison between the cDNA clone wun1-25A2 and the genomic clone wun1-85. 2368 bp of the genomic clone wun1-85 were compared with 711 bp of the cDNA clone wun1-25A2. Homologous base pairings are identified by a vertical line. The absence of nucleotides is indicated by a dot. The two arrows document the sequence of two 10 bp direct repeats in the cDNA clone.

Comparative analysis shows that the sequence of the genomic clone wun1-85 of the haploid potato AM80 / 5793 is 97% homologous to the sequence of the cDNA clone wun1-25A2 of the tetraploid potato "Granola". A total of 11 base changes could be made be certified, 5 of them in the translated area. All 5 base changes in the translated area did not change the amino acid sequence. The additionally sequenced 400 nucleotides downstream of the transcription stop had no remarkable areas.

The gene codes for a 12,000 dalton protein, the size of which could also be approximately confirmed in hybrid-released translation experiments. Computer evaluations regarding the amino acid composition of the wun1 protein showed that it is a very hydrophilic protein. The amino acid sequence results from FIGS. 2 and 6.

The size of the wun1 protein, its hydrophilic properties, the inducibility of wounding in various tissues and the inducibility by pathogens suggest that it belongs to the PR proteins. The name PR or "pathogenesis-related" protein is used to summarize proteins from different plants which can be induced, inter alia, by pathogenic attack and in some cases have chitinase or glucanase activity, ie the activity of enzymes which can destroy the cell walls of fungi .

The complete genomic wun1 clone including the wun1 promoter and wun1 gene was tested in transgenic tobacco plants using the Agrobacterium transformation system. An expression study was possible at the mRNA level, since tobacco mRNA cross-hybridizes only very weakly with wun1 and additionally has no wun1-homologous 5 'ends, as was evident from S1 nuclease maps. In fact, high amounts of approximately 800 bp wun1 mRNA were detected in wounded leaves in transgenic tobacco plants, which on the one hand documents an active wun1 promoter and on the other hand shows the use of the correct transcription start point.

A 4 Kb fragment from pLS000 which contains the wun1 promoter, the complete wun1 gene and 2 Kb of the 3 'region of the gene holds (wun1-wun-1) were cloned via the EcoRI sites into the mobilization vector pMPK110 required for the Agrobacterium tumefaciens (At) transformation (pLS001). After the transfer into the At3850 _km , the tobacco line Wisconsin 38 (W38) was transformed with this agrobacterium via pieces of leaf. The small calli that appeared after Kanamycin selection after about 3 weeks were regenerated to full plant (LSI) using a shoot induction medium.

In addition to the ability to grow on kanamycin, plants transformed with 3850 _{K m have} the ability to synthesize nopaline, a property that distinguishes them from untransformed plants (Zambryski et al, EMBO J. 1 147-152

(1985)). Wunl-wun1 transformed plants, here called LS1 plants (4 = LS1-4; 6 = LS1-6), were characterized by high amounts of nopaline. In contrast, there was none in untransformed plants

Detectable nopaline. Kanamycin-resistant and nopalin-positive LS1 plants were transferred to the greenhouse and analyzed at the DNA and RNA levels.

Figure 10 shows the construction of wun1-wun1 and wun1-CAT fusions that were used for expression studies based on transient expressions and stable transformations.

(1) Preparation of plasmid pLS001:

Starting from plasmid pLS000 (see FIG. 4), the 4 Kb fragment, which contains the wun1 promoter, the wun1 gene and 2 Kb of the 3 'end, was cloned into the vector pMPK110 via the EcoRI sections (pLS001 ). This pMPK110 derivative allows the transfer of the wun1-wun1 construction to the Agrobacterium 3850 _km .

(2) Preparation of plasmid pLS011:

The wun1 promoter and 179 BP of the 5'-untranslated wun1 gene region were fused to the CAT gene including the 3 'end via the Xhol interface (pLS010). A cloning over PstI in the vector pMPK110 (pLS011) has the advantage that this plasmid can be used on the one hand for transient studies in protoplasts and on the other hand also acts as a mobilization plasmid for the transfer of the wun1-CAT construction into the Agrobacterium 3850 _km .

Southern blot analysis of EcoRI digested DNA from transgenic

LS1 tobacco plants and untransformed W38 tobacco plants resulted in the use of a ³² P-radioactively labeled wun1 cDNA

Sample positive hybridization in both untransformed and transformed plant DNAs. In both cases there was a strong and a weak band. In the transgenic LS1 plants, however, an additional 4 Kb band appeared, whose size corresponded exactly to the size of the EcoRI insert from pLS000. By comparing different amounts of DNA, a 2 to 5-fold copy number of wun1-wun1 could be determined in all LS1 plants tested. Additional bands in the high molecular weight range were found in some LS1 plants, which are probably due to rearranged DNA or undigested DNA.

A functional analysis of the wun1-wun1 DNA of positive LS1 plants was possible at the RNA level. There was only a very weak cross hybridization against a wun1 cDNA sample in both wounded and unwound untransformed tobacco plants. In addition, isolated RNA from untransformed tobacco plants was raised against the 5 'region of the wun1 gene, which is the

Contains the 5 'untranslated region and the wun1 promoter (analogous to the fragment for determining the transcription start of the wun1 gene) via S1 analysis for homologous regions. It was found that there were no wun1-homologous sequences in neither wounded nor wounded untransformed tobacco leaves or stems.

Of 30 nopalin-positive LS1 plants that were examined at the RNA level, 5 plants showed "Northern blot" experiments - high amounts of 800 bp large mRNAs hybridizing with wun1. Their S1 analysis led to the detection of two DNA fragments of the same size, which corresponded in size to the fragments from wounded potato leaves. This was the confirmation that this is a wun1 homologous mRNA. There are several indications that wun1-homologous mRNA is also formed in response to high levels of TMV.

An object of the invention is to construct and isolate genes capable of expressing fused proteins as required. For this it is necessary that the wun1 promoter is solely responsible for the expression of the downstream structural gene, which need not always be the case (eg proteinase inhibitor II).

Transcriptional fusions consisting of the wun1 promoter and 178 bp of the S'-untranslated gene were fused with various structural genes (wun1-CAT, wun1-NPT, wun1-GUS), the detection of which was possible using radioactive or fluorometric methods. (GUS stands for β-glucuronidase (Jefferson et al, Proc. Nat. Acad. Sci. 83, 8447-8451 (1987)).

The construction wun1-CAT was tested for its transient functionality in potato protoplasts. This method makes it possible to analyze promoter activities within a few days by transferring highly purified plasmid DNA, which contains the constructions, to protoplasts using known methods, where they are stable and can also be read. A prerequisite for expression is that the promoter is functional and that the inducing factors are present in protoplasts treated in this way.

In these tests, the wun1 promoter including 179 bp of the 5'-untranslated region was transcriptionally fused with the chloramphenicol acetyl transferase gene (CAT). The construction of the wun1-CAT fusion is shown in FIG. 10. The 35S promoter contained in the pRT101-CAT was via the

Interfaces HincII / XhoI removed and instead the 1.0 Kb wun1 promoter together with 179 bp of the 5'-untranslated region was used via a filled EcoRI interface and Xhol. This chimeric gene wun1-CAT could be brought into the vector pMPK110 via the PstI cleavage sites (pLS011).

High amounts of endogenous wun1 mRNA were found in the potato cell suspensions used for transient tests and the protoplasts obtained from them. It is assumed that the liquid cultivation of cells and above all the protoplasting exert a high stress on the cell, which is expressed in a high expression of wun1-mRNA analogously to the behavior in the potato tuber.

For the transient analysis of the wun1-CAT construction, 1 million protoplasts were isolated from a suspension culture from potato stalks of the Datura variety (D12) and transformed with 20 μg pLS011-DNA according to the CaCl ₂ / PEG method.

CAT analysis of such protoplasts transformed with pLS011 DNA revealed high CAT activities. A parallel test approach with a CAMV-35S-CAT construction (35S promoter from the Cauliflower mosaic virus (CAMV)) (pRT101-CAT) brought about comparable CAT activity, whereas in a control approach without addition of DNA no CAT activity was recognizable. This means that the activity of the wun1 promoter in a transient potato protoplast system is comparable to that of the 35S promoter and can therefore be classified as very high. The CAMV promoter is considered to be the strongest known promoter in plants.

The plasma pLS011 already used in the transient system

(wun1-CAT) could still be used to transform At 3850 _km . 3850 _km :: pLS011 Agrobacteria were used to transform the tobacco variety Samsum NN (SNN) using the method of leaf disc infection and the resulting calli were regenerated to the plant (LS2). The use of this tobacco mosaic virus (TMV) resistant tobacco variety was intended to circumvent the (suspected) inducibility of wun1 by TMV. In the Nopalin test positive and kanamycin-resistant LS2 plants were tested at the DNA level and for the presence of CAT activity. Pstl-cut DNA from Wunl-CAT transformed plants had a 2.2 Kb fragment hybridizing against CAT-CDNA. This corresponded exactly to the expected fragment size of wun1-CAT. No hybridization was seen in untransformed plants. By comparing DNA plots, 2 to 4 copies per haploid genome could be determined in all positive plants.

The functional analysis of LS2 plants extended to the measurement of CAT activities in greenhouse plants and to plants of the F1 generation kept in sterile culture. LS2 plants grown in the greenhouse showed slightly increased CAT activity in their unwounded leaves compared to the background activity of untransformed tobacco leaves. In wounded leaves, an approximately 4-fold higher CAT activity was found compared to unwounded leaves. If, however, an excess of boiled potato tuber extract was added to the wound, the increase was 6 times higher than in the unsealed condition. This effect was independent of the condition of the tubers before extraction (unwound or wounded tubers or tubers freshly removed from the soil (unstored) or stored). Each extract had the same inducing effect, which indicates an inductor present in the extract.

The reason for the relatively low inducibility of wun1-CAT in transgenic tobacco plants can be seen in the activity in unwounded leaves. In order to withstand possible greenhouse-related causes, the seeds of self-grown LS2 plants were harvested and germinated again under sterile conditions. In young leaves of kanamycin-resistant plants, a very weak CAT activity was measurable in the unwound state, which was: little difference from the CAT activity of untransformed leaves. In wounded leaves, CAT activity increased by at least a factor of 40. The addition of boiled supernatants from homogenized potato tubers to the wound base even led to a 60-fold higher expression.

In addition, it was observed that with increasing age of the plant, the activity of the wun1 promoter in the leaves became increasingly constitutive, the level of expression being similar to that of a transgenic CAMV-CAT plant. Processes of senescence may also have an inductive effect here.

The transient system was also used to test which plant species the wun1 promoter is active in and

can be used accordingly. It was found that this promoter is not only active in potato, tobacco and parsley protoplasts, but also in rice protoplasts shows an expression level which corresponds to the activity of a homologous system.

11 shows the production of wun1-NPTII constructions.

Starting from plasmid pLS001, the wun1 promoter including 179 bp of the 5'-untranslated region was cloned via EcoRI-Xhol-blunt in front of an NPT-II gene (neomycin phosphotransferase-II). For better handling for transient experiments, the wun1-NPT-II fusion was cloned into a highly replicating vector (pLS 020).

Using the wun1-NPTII construction, NPT-II activity was found in tobacco and in parsley protoplasts. Interestingly, wun1-dependent NPT-II activity could also be detected in rice protoplasts (Oryza sativa japonica cv Taipai), the intensity of which was comparable to that of wun1 -NPTII activity in potato protoplasts. Comparative transient expression of wun1-NPTII and NOS-NPTII (pGV1103) in rice protoplasts also showed similarly high values. In this together In the meantime, reference is also made to the CAMV-35S-CAT-like transient expression in potato protoplasts.

Overall, these studies prove that the wun1 promoter has a very high activity in both homologous and heterologous systems. Neither in parsley nor in tobacco bones nor in rice protoplasts could be found with wun1-hybridizing mRNA. Factors that stimulate the wun1 promoter must be assumed, which occur equally in all plant species tested.

All in all, all of the important DNA regions are present in the 5 'region of wun1, in order to be able to express a gene fused behind it in large quantities and in a manner which can be induced by the wound.

In addition, the functionality of wun1-CAT and wun1-NPTII in various transient systems (potato, parsley, tobacco, rice) as well as in stably transformed tobacco suggests similar molecular backgrounds.

Experiments carried out in the context of the present invention further showed that it may be advantageous to fuse the wun1 promoter according to the invention with other promoters or enhancer elements.

The fusion of the wun1 promoter with the known TR promoter from the Agrobacterium tumefaciens TR-DNA may be mentioned as an example.

The isolation of this TR promoter from the Agrobacterium tumefaciens TR-DNA is from a work by J. Veiten and J. Schell "Selection-expression vectors for use in genetic transformation of higher plants", published in Nucl. Acids Res., 13: 6981-6997. From this reference it is also known that this promoter is responsible for the expression of mannopine synthase and has the following characteristics: 1. It is bidirectionally active, ie one fuses

both sides of structural genes, so both become more similar

Regulations and intensity read. In this context, reference is also made to the literature reference:

Langridge, W.H.R., Fitzgerald, K.J., Koncz, C, Schell, J.

and Szalay, A.A. (1989). Dual promoter of Agrobacterium

tumefaciens mannopine synthase genes is regulated by plant growth hormones. Proc. Natl. Acad. Be. USA, 86: 3219-3223.

The orientation of the TR promoter can be found with l 'and 2'

fixed.

2. In transgenic plants, the TR promoter can be induced by wounds. A fusion of the TR2 'with the reference gene

beta-galactosidase (beta-gal) leads to a higher beta-gal activity in wounded transgenic tobacco plants than in unwounded ones.

3. The activity of the TR2 'promoter is mainly found in the leaf veins, that is, in the vascular bundle system.

For 2nd and 3rd, reference is made to: Teeri, TH, Lehväslaiho, H., Franck, M., Uotila, J., Heino, P., Palva, ET, Van Montagu, M. and Herrera-Estrella, L. (1989). "Gene fusions to lacZ reveal new expression patterns of chimeric genes in gransgenic plants". EMBO, 8: 343-350.

4. The properties of the TR1 'promoter have so far not been investigated so intensively. However, it is known that the TR1 'promoter is about 4-7 times weaker than the TR2' promoter, but otherwise has similar properties.

In the course of experiments with the aim of a qualitative and quantitative optimization of the wun1 promoter, the TR promoter was fused to the wun1 promoter, namely in the

Orientation TR1'-wun1 (Fig. 12). The marker gene GUS (beta-glucoronidase) (TR1'-wun1-GUS) was used to demonstrate the promoter activity. Using the Agrobacterium tumefaciens transformation system, this construction was used in tobacco

transformed and analyzed. These tobacco plants have the following characteristics in comparison to wun1-GUS transgenic tobacco plants: 1. The tissue specificity has changed.

In wun1-GUS transgenic tobacco, GUS activity is mainly found in the epidermis of leaves and stems. In

TR1'-wun1-GUS transgenic tobacco, the GUS activity is mainly localized in the lead bundle system. So there is the possibility, depending on the scientific question, of being able to direct gene products to different parts of the tissue. In contrast, the wound induction behavior of TR1'-wun1-GUS transgenic tobacco plants is comparable to the behavior of wun1-GUS transgenic tobacco plants.

2. Overall, the activity of the TR1'-wun1 promoter in transgenic tobacco leaves and stems is about 10 times higher than the activity of the wun1 promoter. So that counts

TR1'-wun1 promoter among the strongest promoters available for expression in plants.

3. While promoter activity is hardly a problem in transgenic dicotyledon plants today, there are so far few promoters that are sufficiently active in monocotyledonous plants (to which the economically important cereal plants belong). In transient expression studies in rice protoplasts (they serve as a substitute for studies in transgenic rice plants that are very difficult to obtain), the TR1 'wun1 promoter is characterized by an activity which is about 30 times higher than that of the wun1 promoter. This is all the more remarkable because even the 35S promoter, which is so strong in dicotyledonous plants, is 30 times weaker here than the TR1'-wun1 promoter.

Thus, for example, the wun1 promoter according to the invention can be fusioned with the TR1 'promoter in the following

Change or optimize properties:

(a) The specific activity of the wun1 promoter in the

Leaf or Stenge1 epidermis of transgenic tobacco is shifted into the lead bundle system by the fusion with the TR1 'promoter;

(b) The wound inducibility of the wun1 promoter is not changed by the fusion;

(c) The TR1'-wun1 promoter in transgenic tobacco leaves and stems is about 10 times stronger than the wun1 promoter alone; (d) The TR1'-wun1 promoter is about 30 times stronger than the wun1 promoter in transient experiments with a representative of the monocotyledonous plants (rice).

According to the invention, e.g. increase the expression of the wun1 promoter by fusing it with an enhancer element of the CamV-35S promoter. For example, the fusion of the enhancer element of the CamV-35S promoter to the 5 'end of the wun1 promoter leads to an increased wun1 promoter activity in transgenic tobacco and potato.

Figure 12 shows the organization of the clones wun1-GUS, TR1'-GUS and TR1'-wun1GUS.

Figure 13 shows Northern blot analysis of wounded and wounded transgenic tobacco leaves. Unwound (NW) and wounded (W) leaves of the transgenic tobacco plants wun1-GUS, TR1'-wun1-GUS No. 5 and No. 11 and 35S-GUS were used to isolate RNA. 50 μg of total RNA each was on a

1.2% formaldehyde gel separated by gel electrophoresis, transferred to a nylon membrane, hybridized against a radioactively labeled GUS-DNA sample, and specific bonds visualized on an autoradiogram. (A) shows a 2 day exposure, (B) shows a 6 hour exposure.

The following examples serve to explain the invention in more detail:

Examples 1-8

Materials

media

Culture media for bacteria:

Media used to grow bacteria were prepared according to Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cola Spring, Harbor, New York (1982):

Plant media: The media used are derived from that of Murashige and

Skoog, Physiol. Plan. 15, 473-497 (1962), specified media (MS).

3MS: MS + 3% sucrose

3MSC: MS + 3% sucrose, 500 μg / ml claforan

MSC10: MS + 2% sucrose, 500 μg / ml claforan,

0.2 µg / ml NAA, 1 µg / ml BAP

100 µg / ml kanamycin sulfate

MSC15: MS + 2% sucrose, 500 μg / ml claforan,

100 µg / ml kanamycin sulfate

MS15: MS + 2% sucrose, 100 µg / ml kanamycin sulfate

For solid medium an additional 8 g / l Bacto agar was added.

Tribes and vectors

E. coli strains:

BMH 71-18: (lac-proAB), thi, supE;

F '(laci ^q , ZdeltaM15, proA ⁺ B ⁺ ) (Messing et al., Proc. Nat. Acad. Sci. 74, 3642-3646 (1977))

C 600: = CR34 (Maniatis et al. 1982) GJ23: AB1157 (R64drd11) (pGJ28)

(Van Haute et al., EMBO J. 2, 411-418, (1983))

Agrobacteria

Strains: C58CIpGV3850 _KM (Zambryski et al.,

EMBO J. 1, 147-152, (1983))

Plasmi: de: pUC8 (Vieira and Messing, Gene

19, 259-268 (1982))

pMPK110 (Eckes et al., Mol. Gen. Genet. 205, 14-22 (1986))

pRT101-cat (Pröls et al., Plant Cell Reports, im Druck (1988)) pGVH03 (Hain et al., Mol. Gen. Genet. 199, 166-168 (1985))

Phages: EMBL4 (Frischauf et al., J. Mol.

Biol. 170, 827-842 (1983))

Ml3mpl8 (Yanisch-Perron et al., Gene 33, 103-119, (1985))

M13mp19 (Yanisch-Perron et al., 1985)

Plants:

Solanum tuberosum AM 80/5793 (haploid)

Berolina (tetraploid)

Datura (D12)

granola

Nicotinia tabacum Wisconsin 38 (W38)

Samsum NN (SNN)

Oryza sativa japonica c.v. Taipai

All standard molecular biological methods, e.g. Restriction analysis, plasmid isolation, mini preparation of

Unless otherwise stated, plasmid DNA, transformation of bacteria, etc. are as described in Maniatis et al. (1982).

Wounded plant tissue

Potato bulb material was cut into 3mm thick slices

cut and in a phosphate solution (20 mM phosphate buffer, pH 7.0, incubated with chloramphenicol (50 μg / ml) for 18 h at 28 ° C. with exclusion of light. Leaf, stem and root material was cut into small pieces and incubated under the same conditions. After incubation was complete, the material was either worked up directly or stored at -70 ° C.

Unwound plant tissue

Leaf, stem, root and tuber material was processed immediately after its removal from the living plant or frozen in liquid nitrogen and stored at -70 ° C.

Aerobic wounding of potato tubers

Treatment as described in "wounding", with the tuber discs being only slightly wetted with the phosphate buffer during the incubation. After the incubation, the tuber surface was browned.

Anaerobic wounding of potato tubers

Treatment as described in "wounding", with the tuber disks completely immersed in phosphate buffer during the incubation. After the incubation, the tuber surface was white-yellowish light.

Expression of wun1 mRNA in pathogen infested potato leaves

"Datura" leaves were cut off on their petiole and placed in water with the stem. These leaves were then sprayed with a phytophore spore suspension, in order to isolate the total RNA from one leaf at different times and to examine it using a "Northern slot blot". Three different approaches were used: - water containing the spores of a Phytophtora infestans strain PJ1 compatible with Datura;

- water, with spores of Phytophtora infestans-Starames PJ4, which is incompatible with Datura;

- water, without the addition of spores (control)

The densitometric evaluations of the wun1 hybridization strength showed a 4-fold increase in the amount of wun1 mRNA just two hours after treatment of the leaves with water and spores of the incompatible phytophore species. From the 4th to the 8th hour, the amount of expression remained approximately the same before it gradually decreased to 1.5 to 2 times by the 30th hour. Wun1-mRNA showed the same expression behavior when using water without spores (control).

In the analogous test approach with water and compatible phytophore pores, a 4-fold increase in wun1 mRNA was observed 2 h after infection, which only decreased slightly until the 10 th h. From the 16th h at the latest, however, the expression increased by 6 times the control value in order to reach the factor 9 after a constant increase after 30 h.

Injury with the addition of potato extract

Potato tubers were homogenized without adding buffers at 4 ° C. and centrifuged for 10 minutes at 10,000 rpm in the SS34 rotor. The supernatant was boiled for 10 minutes and centrifuged again. The clear supernatant was added in a ratio of 1:10 to 20 mM phosphate buffer, pH 7.0 and the tissue to be tested was incubated therein under the normal conditions.

B e i s p i e l l

RNA isolation The isolation of RNA from various organs of the potato and tobacco was carried out as in Logemann et al. in anal. Biochem., 163: 16-20 (1987).

The selective enrichment of polyA ⁺ RNA was carried out using mAP paper (Werner et al., Analytical Biochem. 141, 329-336

(1984).

Northern blot analysis

The RNA was electrophoresed on a 1.5% formaldehyde agarose gel (Lehrach et al., Biochemistry 16, 4743, (1977)). As with Willmitzer et al., EMBO J. 1, 139-146,

(1982), the RNA was then transferred to nitrocellulose, fixed and hybridized against ³² P * radiolabeled cDNA, washed and exposed. DNA isolation

Nuclear DNA was obtained from potato leaves by the method of Bedbrook, PMB Newsletter II, 24 (1981) and used for cloning in LambdaPhagen EMBL4. DNA from transformed tissue was purified after the combined disruption with Triton X 100, SDS, and Proteinase K (Wassenegger, dissertation, Cologne (1988)).

Southern blot analysis

DNA was electrophoretically separated on 0.8 to 1.2% agarose gels, transferred to nitrocellulose and fixed

(Southern, J. Mol. Biol. 98, 503-517 (1975)), and hybridized and washed as described by Willmitzer et al. (1982).

Run-off experiments

One method that makes a relatively precise distinction between these two types of regulation is the "run-off" method. Cell nuclei are isolated from wounded and unwound tubers, newly synthesized mRNA is radio-labeled by them and hybridized against single-stranded DNA from the individual clones. No wun1 mRNA was produced in cell nuclei from unwounded clones, but in large numbers in cell nuclei from wounded tubers.

The isolation of cell nuclei from potato tubers and the subsequent in vitro transcription was carried out according to Willmitzer et al., Nucl. Acids Res. 9, 19 (1981). The radioactively labeled RNA transcripts obtained in this way were hybridized against cDNA clones cloned bidirectionally in M13.

Protein isolation and separation

The isolation of proteins from potato tubers and their one- and two-dimensional gel electrophoretic separation is described in Mayer et al., Plant Cell Reports 6, 77-81 (1987).

Example 2 "Hybrid-releasec-translation" experiments The 800 bp fragment of the cDNA clone wun1-25A2 and the 700 bp fragment of the cDNA clone wun2-29C12 were obtained via Pstl

Interface cloned in M13mpl8. The orientation of the inserts in the M13 clones was determined by complementation analyzes (Messing, in: Genetic Engineering of Plants, (1983) Plenum Press, New York, 1983). The M13 clones with the codogenic strand were identified by positive hybridization behavior against radioactively labeled polyA ⁺ RNA from wounded potato tubers.

The isolation of single-stranded DNA from M13 phages (it contains the codogenic strand), the fixation of this DNA to nitrocellulose and hybridization against potato tuber RNA, and the washing and release of the selected RNA is described in Maniatis et al. (1982). The selected RNA and polyA ⁺ RNA isolated from potato tubers were harvested in a rabbit

Reticulocyte lysate (Pelham and Jackson, Eur. J. Biochem. 67, 247 (1976)) translated in the presence of ³⁵ S-methionine. The one- or two-dimensional electrophoretic separation on a polyacrylamide gel was carried out according to Mayer et al. (1987).

Example 3

Create and examine a genomic bank

Isolation of genomic DNA:

Leaf DNA of the haploid line AM isolated according to the Bedbrook (1981) method was used as the genomic potato DNA

80/5793.

This DNA was digested completely with EcoRI.

Isolation of phage DNA:

EMBL4-DNA was cut into three fragments with EcoRI, using gel electrophoretic separation followed by Frag ment isolation the two vector arms could be separated from the middle fragment. In addition, commercially available and cleaned EMBL4 arms were used (Amersham).

Ligation and packaging:

After ligation of the EMBL4 arms with the EcoRI digested genomic DNA, the ligated, high-molecular DNA was packaged in vitro in phage heads (Hohn and Murray, Proc. Nat. Acad. Sci. USA, 74, 3259-63, (1977); Hohn , Meth. Enzyme. 68, 299-309

(1979)). The packaging material comes from a "Lambda in vitro packaging kit" from Amersham. The genomic library was created with a concentration of 25,000 plagues per plate

(25x25cm) plated.

Plaque hybridization:

The cDNA homologous lambda clones were found by

Plaque hybridization according to Benton and Davies, Science 196, 180 (1977) using radioactively labeled cDNA. Plagues that hybridized positively were isolated and tested again.

DNA preparation of recombinant phages:

Chloroform was added to 20 ml of a C600-lysed bacterial culture in order to obtain a bacteria-free supernatant after subsequent centrifugation. The phages were sedimented from the supernatant by centrifugation at 10,000 rpm for 4 hours. The phage sediment was taken up in 500 μl phage buffer (10 mM Tris-HCl, pH 8.0, 10 mM MgCl ₂ ) and treated with DNase and RNase. After extraction of the DNA by repeated phenolization, the phage DNA was EtOH-precipitated, washed with 70% EtOH and taken up in TE.

Example 4

Generation of deletion mutants after exonuclease III treatment. The fragment to be examined was, according to Henikoff et al., Gene 28, 351-359, (1984) in both orientations in M13mp19 klo kidney. Since exonuclease III digested 5 'protruding ends, but spared 3' protruding ends, 20 μg of the DNA to be analyzed were digested with two restriction enzymes, which produced a 5 'and a 3' end on the side of the cloned fragment . Since the 5 'end was facing the fragment, exonuclease digestion could take place into the fragment. By gradually stopping this reaction, 200 bp smaller fragments could be isolated, the protruding ends of which were converted into ligatable blunt ends by a subsequent S1 treatment. Finally, this DNA was transformed into BMH 7118 cells with subsequent single-strand DNA isolation.

Sequencing

Sequencing of single-stranded DNA was carried out according to the chain termination method of Sanger et al., Proc. Natl. Acad. Be. USA 74, 5463, (1977). The reaction batches were separated on 6 and 8% seguence gels (Ansorge and de Mayer, J. Chromatogr. 202, 45-53 (1980); Ansorge and Barker, J. Biochem. Biophys. Meth. 9, 33 (1984) ).

Cloning and sequencing of wun1-25A2

The approximately 711 bp insert of the clone wun1-25A2 was cloned into the M13mp19 vector via the PstI sites, the orientation of the insert being determined via the asymmetric Xhol site. The clones 25A2-4-mp19 and 25A-5-mp19, which differed in the orientation of the insert, were cut with Kpnl / Xbal in order to enable bidirectional exonuclease digestion into the fragment. Deletion clones of different sizes could be obtained by successively stopping the exonuclease reaction, which was prepared as a single-stranded DNA for sequencing according to the method of Sanger et al. (1977) served. Example 5

S1 analysis

The principle of S1 nuclease mapping to determine the

Starting point of the transcription is based on the fact that by hybridizing single-stranded DNA from the 5 'region of the wun1 gene with wun1-mRNA, only those regions are protected against degradation of the single-stranded nuclease S1 which, due to their homology, can form a double strand. The size of the protected DNA fragment can be determined on a sequence gel and the start of transcription can thus be traced back.

The transcription start point was determined according to Berk and Sharp, Proc. Natl. Acad. Be. USA 75, -1274 (1987). For this purpose, the 1.2 kb fragment was isolated from the plasmid pLS000 with EcoRI and Xhol and dephosphorylated by treatment with phosphatase. The 5'OH ends were then radiolabeled by the combination of polynucleotide kinase and y- ³² -P-ATP. After denaturing the DNA fragments, they were hybridized with 50 μg total RNA (hybridization buffer: 80%. Formamide, 0.4 mM NaCl, 40 mM PIPES pH 6.4 and 1 mM EDTA). This condition favors RNA-DNA hybrids compared to DNA-DNA hybrids (Casey and Davidson, Nucl. Acids Res. 4, 1539-1552 (1977)). The hybridization temperature starts at 80 ° C and is reduced to 40 ° C overnight. A subsequent S1 nuclease digestion (120 U / ml) removes unpaired strands. It was expected that the radioactive labeled Xhol site located in the 5'-untranslated region of the wun1 gene would be protected by its homology to the mRNA. Finally, the S1-protected DNA strand is separated electrophoretically on a sequence gel, the sequence of a known DNA fragment serving as a length comparison. Example 6

Transient expression in potato protoplasts

Each 20 μg of highly purified (CsCl gradient) plasmid DNA is transformed into PEG / CaCl ₂ -treated potato protoplasts

(Lipphardt, dissertation, Cologne, 1988). The protoplasts come from a potato stem suspension culture of the Datura variety.

Transient expression in rice protoplasts

20 μg of highly purified (CsCl gradient) plasmid DNA are obtained according to a modified method by Lörz et al., Mol. Gen. Genet. 199: 178-182 (1985). The have been changed

Polyethylene glycol and osmotic concentrations.

Example 7

DNA transfer in agrobacteria

Conjugation:

The DNA cloned in E. coli was prepared according to the method described by Van Haute et al. (1983) transferred the method using the helper strain GJ23 into the agrobacterial strain 3850.

DNA analysis of agrobacteria:

The DNA transfer into the Agrobacterium was checked by isolating Agrobacteria DNA according to that of Murray and Thompson, Nucl. Acid Res. 8, 4321-4325 (1980). Restriction cleavage of the DNA, transfer to nitrocellulose and hybridization against the corresponding radioactive probe gave information about a successful one

DNA transfer to Agrobacterium. Example 8

Transformation of tobacco

Cultivation of agrobacteria:

The 3850 _km agrobacteria required for infection were grown in selective antibiotic medium (Zambrisky et al., 1983), sedimented by centrifugation and washed in YEB medium without antibiotics. After renewed sedimentation and absorption in YEB medium, the bacteria could be used for infection.

Leaf disk infection:

Sterile leaves of the tobacco lines SNN and W38 were used for the leaf disc infection. Pieces of sheet about 1 cm in size were sterilized by incubation in 0.1% HgCl ₂ , 0.1% SDS for 10 minutes and washed three times in sterile water. This was followed by immersion in an agrobacterial suspension as described above, followed by transfer to 3MS medium. After 2 days of incubation at 16 h under light and at 25 to 27 ° C., the leaf pieces were washed several times in liquid 3MS medium and transferred to 3MSC medium. Shoots appearing after 4-6 weeks were cut off and incubated on 2MSC medium. The Nopalin test was carried out as further proof of a successful transformation.

Analysis of the transformed plants

Detection of nopaline synthase activity:

The detection of nopaline synthase activity in transformed plant leaves was carried out according to Otten and Schilperoort, Biochem. Biophys. Acta 527, 497 (1978).

Detection of NPT-II activity:

NPT-II activity in transformed plants was detected according to Reiss et al., Gene 30, 217-223 (1984) and Schreier et al., EMBO J. 4, 25-32 (1985). Evidence of CAT activity:

Chloramphenicol acetyl transferase (CAT) activity was determined by the methods of Veiten and Schell, Nuc. Acids, Res. 13, 6981-6997 (1985) and Herrera-Estrella et al., EMBO J. 2, 987 (1983).

Example 9

Optimization of the wun1 promoter by fusion with the TR promoter (TR1'wun1).

With regard to the media used, reference is made to Examples 1-8.

2.1.4. Tribes and vectors

Agrobacterial strains GV3101pmp90RK (Koncz, C, and Schell, J. "The promotor of TL-DNA gene 5 controls the tissue-specific expression of chimaeric genes carried by a novel type of Agrobacterium binary vetor", (1986), Mol. Gen. Gent 204: 383-396).

Plasmids: pPCV720 (previously unpublished plasmid of

Dr. Koncz, MPI f. Breeding Research, Cologne)

35S-GUS = pRT99-GUS (Töpfer, R., Schell, J. and

Steinbiß, H.H. "Versatile cloning vectors for transient gene expression and direct gene transfer in plant cells." (1988) wun1-CUS (Siebertz, B., Logemann, J., Willmitzer,

L., Schell, J. "Cis-analysis of the wound-inducible promoter wun1 in transgenic tobacco plants and histochemical localization of its expression". The Plant Cell, in press (1989) TR1'-wun1-GUS (Logemann et al., In press)

TR1'-GUS (Logemann et al., In press)

Plants:

Nicotiana tabacum Wisconsin 38 (W38)

Methods

All molecular biological standard methods, such as B.

Restriction analysis, plasmid isolation, mini-preparation of plasmid DNA, transformation of bacteria, etc. unless otherwise stated, are as described in Maniatis et al., (1982)

described.

Wounded and unwounded plant tissue was prepared as described in Examples 1-8. With regard to RNA isolation, "Northern blot" analysis, DNA isolation, transient expression in rice protoplasts, "Southern blot" analysis and DNA analysis of agrobacteria, reference is also made to Examples 1-8.

DNA transfer in agrobacteria

transformation

The DNA cloned in E. coli was prepared according to that of Van Houte, E., Joos, H., Maes, M., Warren G., Van Montagu, M., Schell, J.

"Intergenic transfer and exchange recombination of restriction fragments cloned in pBR322: a novel strategy for reversed genetics of Ti-plasmids of Agrobacterium tumefaciens", EMBO J., 2: 411-418 transferred method using helper strain S17-1 into the Agrobacterium strain GV3101pmp90RK .

Transformation of tobacco

Cultivation of agrobacteria

The GV3101pmp90RK agrobacteria, which contain the desired plasmid, were grown in selective antibiotic medium (hygromacin), sedimented by centrifugation and washed in YEB medium (Maniatis et al, 1982) without antibiotics. After renewed sedimentation and absorption in YEB medium, the bacteria could be used for infection.

Leaf disk infection

The procedure was as described in Example 8, but without using the Nopalin test. Analysis of the transformed plants

Evidence of GUS activity

A: The fluorometric detection of beta-glucoronidase

Activity (GUS activity) takes place after de Mehode of

Jefferson, A.R., Kavanagh, T.A., Bevan, M.W. "Gus mergers:

β-Glucuronidase as a sensitive and versatile gene fusion marker in higher plants ", EMBO J. 6: 3901-3907 (1987).

B: The detection of GUS activity in tissues by means of an X-Gluc staining solution was carried out. X-Gluc solution:

1-2 mM X-Gluc (5-bromo-4-chloro-3-indolyl-glucuronide), and

Dirnethylformamide are pH 50 in 50 mM phosphate buffer

solved.

- Wounded tissue:

Thin tissue sections are incubated as described under "wounded plant tissue" and then stained in X-Gluc solution overnight at 37 ° C. Finally, the chlorophyll is removed by adding 100% methanol at 60 ° C.

-Unwounded tissue:

Thin tissue sections are incubated in X-Gluc solution, which also contains the translational inhibitor cycloheximide (1.8 mM). Incubation period and methanol treatment is identical to the wounded tissue.

-X-Gluc staining of pollen:

Pollen was grown overnight at 28 ° C in 30% sucrose

incubated, which leads to the formation of pollen tubes.

Then double-concentrated 30% X-Gluc solution is added and the pollen is incubated at 28 ° C. overnight.

Alternatively, pollen grains are incubated directly in X-Gluc. without the formation of pollen tubes.

Results

Organization of TR1'-wun1-Gus, TR1'-GUS and wun1-GUS (Fig. 12). wun1-CUS:

1022 bp of the wun1 promoter and 178 bp of the wun1 5 'untranslated region are transcriptionally fused to the beta-glucuronidase gene (GUS). The poly-adenylation signal (pA) of the nopaline synthase gene (NOS) serves as the termination sequence. TR1'-GUS:

The TR1 'promoter becomes transcriptional with the GUS gene

fused and terminated by the pA signal of the NOS gene. TR1'-wun1-GUS:

The pA signal of the octopine synthase gene (OCS) is cloned behind the 1 'promoter of the TR promoter in order to prevent transcription through the wun1 promoter. Behind the OCS pA signal lies the wun1 promoter (1022 bp), which (in homology to the wun1-GUS construction) over 179 bp des

untranslated 5 'region of the wun1 gene is fused to the GUS gene transcriptionally. The pA signal of the 35-S gene serves as the termination sequence for the GUS gene.

This construction (TR1'-wun1-GUS) lies in the binary

Vector pPCV720, which can replicate itself both in E. coli and in agrobacteria (with the help of a helper plasmid).

RNA analysis of TR1 '-wun1-GUS transgenic tobacco

RNA from wounded and from unwounded leaves of various TR1'-wun1-GUS transgenic tobacco plants as well as from wun1-GUS and 35S-GUS leaves was isolated and radioactively labeled GUS-DNA hybridizes.

As the Northers blot in FIG. 13 shows, 1. the GUS mRNA in transgenic TR1'wun1-GUS leaves is as large as in wun1-GUS transgenic leaves. Obviously, the same transcription start is used in both constructs.

2. The amount of RNA is higher both in TR1'-wun1-GUS plants and in wun1-GUS plants in wounded leaves than in unwounded leaves. The wound inducibility already known for wun1-GUS is thus also confirmed for TR1'-wun1-GUS.

3. In a quantitative RNA comparison it can be seen that in wounded TR1'-wun1-GUS transgenic leaves there is sometimes more RNA than in 35S-GUS transgenic leaves. Since the 35S promoter is one of the strongest promoters active in plants, the TR1'-wun1 promoter should also be assessed in this way.

Analysis of GUS activity in transgenic tobacco

A quantitative analysis of the GUS activity of transgenic TR1'-wun1-GUS plants shows that the TR2'1'-wun1 promoter in transgenic leaves can be induced by a factor of 2 to 13 times by wounding (about 6 times on average). This roughly corresponds to the wound inducibility of the wun1 promoter in transgenic leaves (Logemann J., Lipphardt S., Lörz H., Häuser I., Willmitzer L. and Schell J. "5'upstream segments from the wun1-gene are responsible for gene activation by wounding in transgenic plants "The Plant Cell 1: 151-158 (1989A)). Analogous to transgenic wun1-GUS tobacco plants, wound inducibility is highest in the upper, young leaves and lowest in lower, old leaves. The reason for these differences in the inducibility is the increasing activity of the TR1'1-wun1 promoter in unwounded tissue from top to bottom. In old leaves of old plants, the TR1'-wun1 promoter activity in the unwound state is already so high that it can no longer be induced by a wound (analogous to wun1-GUS plants). A relative comparison of promoter activities in transgenic tobacco leaves and stems shows that the wun1 promoter is about 10 times stronger than the TR1 'promoter. The TR1'-wun1GUS

The promoter is 10 times stronger than the wun1 promoter.

Thus, the TR1'-wun1 promoter is about 100 times stronger than the TR1 'promoter.

Localization of GUS activity in transgenic tobacco

- Leaf, stem:

GUS activity in the tissue sections from wounded and unwounded leaves and stems of wun1-GUS and TR1'-wun1-GUS transgenic tobacco was localized using the substrate X-Gluc.

wun1-CUS:

As can be seen from the blue-colored areas, the activity of the wun1 promoter is mainly limited to the epidermis (including trichomes) and to a lesser extent to the

Guide bundle system of wounded leaves (W). On the other hand, in unwounded (NW) leaves, there is little wun1 promoter activity in the epidermis and no activity in the lead bundle system. The same results are achieved with stem cross sections.

The wun1 promoter activity is thus to be regarded as epidermis-specific in leaves and stems.

In TR1'wun1-GUS plants, an intense blue coloration can be seen in the guide bundle system of wounded leaf sections. In other areas (epidermis, parenchyma) the color is only weak. Unwound leaf cuts show a slight blue color in the lead bundle system and no color in the epidermis. The same results are achieved with stem cross sections.

Obviously, TR1 'wun1-GUS is a promoter which is specific in the guide bundle system and expresses in leaves and stems. A relative comparison of promoter activities in transgenic tobacco leaves and stems shows that the wun1 promoter is about 10 times stronger than the TR1 'promoter. The TR1'-wun1GUS

The promoter is 10 times stronger than the wun1 promoter.

Localization of GUS activity in transgenic tobacco

- Leaf, stem:

wun1-CUS:

Obviously, TR1 'wun1-GUS is an in

Leaves and stems of a promoter expressing the guide bundle system. Leaf sections of TR1 'GUS plants served as a control. Because of the weak promoter activity of the TR1 'promoter (10 times weaker than the wun1 promoter), no blue coloration is discernible either in wounded or in unwounded cross sections of the leaf.

- Other:

An X-Gluc staining of anther cross sections and pollen shows the following characteristics that apply to both wun1-GUS and TR1'-wun1-GUS:

A blue color (and thus GUS activity) appears in the stomium

(natural perforation point of the anther to release the pollen) and detected in the pollen. In separated non-germinated and germinated pollen, a blue color can be seen in the pollen grain and in the pollen tube.

Transient expression with rice protoplasts

The constructions TR1'-wun1-GUS, wun1-GUS, TR1'-GUS and

35S-GUS were used for transient expression studies with rice protoplasts of the variety (Oryza sativa japonica cv Taipai). As Table 1 shows, the activity of TR1'-GUS does not differ significantly from the control (GUS activity of rice protoplasts that were transformed without DNA). wun1-GUS and 35S-GUS show 2-3 times higher activity than the control. TR1'-wun1-GUS DNA, on the other hand, shows on average 57 times higher activity than the control and is therefore a highly active promoter in rice protoplasts.

Legend for Tab. 1:

The indicated GUS activity was determined from 5 independent experiments.

The statement in Table 1 can be substantiated by staining the protoplasts with X-Gluc. Only protoplasts transformed with TR1'-wun1-GUS show an intense blue color.

Claims

Patent claims

1. DNA sequence from Solanum tuberosum caused by injury

and / or infection with pathogenic germs is stimulated.

2. promoter part of the DNA sequence according to claim 1 or its

active areas.

3. promoter part of the DNA sequence according to claim 1 or its

active areas fused to the TR promoter.

4. Structural part of the DNA sequence according to claim 1 or its active regions.

5. Use of the DNA sequence according to claim 1 or

Promoter parts according to claims 2 and 3 for expression

of gene products in higher plants when injured

and / or pathogenic infestation.

6. Use according to claim 5,

characterized,

that the promoter part of the DNA sequence according to claim 2 or 3 or its active regions is / are fused with a structural gene of another origin.

7. DNA transmission vector with inserted DNA sequence according to one of claims 1, 2, 3 and / or 4.

8. DNA transfer vector according to claim 7,

characterized,

that it contains the promoter part or its active regions in connection with a structural gene of another origin.

9. Plants or plant material containing a DNA sequence according to claim 1, 2, 3 or 4.