DE4442179C2 - Pathogen resistant plants and processes for their production - Google Patents

Description

The invention relates to pathogen-resistant plants and processes for their production according to claims 1 and 4.

Plants are unable to change their location on their own. As a result they become frequently changing physical, chemical and biological stressful situations exposed. In the course of evolution, they have developed protective mechanisms that one Enable adaptation to changing environmental conditions.

One of these reactions is the so-called "hypersensitive reaction" after pathogen attack. After infestation, rapid cell death takes place in the affected tissues, which leads to inclusion of the pathogen. This causes the multiplication and spread of the pathogen prevented (summarized in Dixon et al. (1994) Annu. Rev. Phytopathol. 32, 479). As As a result of the local reaction, messenger substances are transmitted to other tissues, causing a systemic resistance is achieved in the case of renewed infestation in unaffected tissues (Ross (1961) Virology 14, 340). Salicylic acid is discussed as a possible messenger (Raskin (1992) Annu. Rev. Plant Physiol. Plant Mol. Biol. 43, 439). Parallel to the local and systemic resistance, the appearance of new soluble proteins is observed (van Loon & van Kammen (1970) Virology 40, 199). These proteins are made by the plant and are not pathogen specific. Since the proteins are induced by pathogens, they have been "Pathogen related proteins" (PR proteins) called (Van Loon (1985) Plant Molec. Biol. 4, 11). Through their occurrence with local and systemic resistance, they become functions in the Inhibition of pathogen multiplication and / or distribution awarded. The currently Known PR proteins have been serologically related due to their properties in five Groups summarized. Due to their isoelectric point, the PR proteins in divided basic and acidic isoforms. The acidic isoforms are secreted and accumulate in the intercellular fluid, whereas the basic forms in the Accumulate vacuole.

Establishing a systemic virus infection requires the virus particles to be removed the infection can be transported to other areas of the plant. This spreading process is divided into two sections: (i) short-distance transport from cell to cell and (ii) Long-distance transport in vascular tissue (McLean et al. (1993) Trends in Microbiol. 1, 105). The short-distance transport runs via plasmodesmata, which are caused by viral "movement" Proteins are changed in their transport properties (Wolf & Lucas (1994) Plant Cell Environ. 17, 573; Lucas & Gilbertson (1994) Annu. Rev. Phytopathol. 32, 387). The Interaction between plant and viral proteins is essential for the spread of  Virus particles necessary (Meshi et al. (1989) Plant Cell 1, 515). About the molecular Mechanisms of long-distance transport are not known.

Methods for suppressing viral infections described so far

• (1) Immunization of crops by inoculating necrotizing agents. This Process is called "cross protection". Inoculation with an attenuated viral Pathogen suppresses a second infection, which minimizes crop losses can be. Potential dangers with this procedure are (a) mutations that lead to Pathogenicity cannot be excluded, (b) the viruses used could act as pathogens on other plant species and (c) synergistic interactions Several types of virus can lead to new symptoms of the disease.
• (2) Expression of viral proteins in transgenic plants. In this procedure, the Example virus coat proteins overexpressed (EP 0480310 A2), whereby the virus spread in infected plants is suppressed (summarized in Beachy et al. (1990) Annu. Rev. Phytopathol. 28, 451). Another example is the combination of viral expression Proteins and herbicide resistance (DE 40 03 045 A1). The expression of viral proteins includes the dangers of inactive viruses present in the plant genome due to complementation activated and the properties of other viruses can be changed. Furthermore offers this procedure provides limited protection against the selected pathogens. In addition to the method for suppressing viral coat proteins, methods for Suppression of viral replicases (WO 93106710; Baulcomb (1994) Curr. Opinion Biotech. 5, 117) and for the expression of defective envelope proteins (Smith et al. (1994) Plant Cell 6, 1441) described. Although recombination events are minimized with these methods, they only offer limited protection against the selected viruses.
• (3) Expression of cell destructive enzymes in transgenic plants. Two variants for expression cell destructive enzymes have been described so far. In one of these procedures, the Expression of the toxic protein controlled by viral replicases after infection (EP 0479 180 A2). In the second method, expression (e.g. an RNase) is controlled by a inducible promoter (WO 93/06710). The first procedure is directed like that The procedure listed under point 2 only against certain viruses and is therefore only limited use. The second method runs the risk of being affected by environmental influences unwanted expression of the chimeric gene cannot be ruled out Harvest losses can be caused.
• (4) Inhibition of protein biosynthesis in transgenic plants. DE 40 40 954 A1 describes a Process for the inducible expression of a protein synthesis inhibitor gene from barley described. This method is said to be essential for the multiplication of pathogens Protein biosynthesis can be inhibited after infestation, thereby protecting the transgenic plant  is achieved. The so-called RIP proteins (ribosome inhibiting proteins) influence plant-specific ribosomes not, while non-plant ribosomes are inhibited. There Viruses that use the plant's own ribosomes to reproduce are offered by this The method does not protect against virus attacks and is only used to combat bacterial and suitable for fungal pathogens
• (5) Overexpression of plant PR proteins in transgenic plants. The reports on this Procedures in the literature are contradictory. Overexpression of the individual PR proteins in Transgenic tobacco plants led to none (Linthorst et al. (1989) Plant Cell 1, 285) or one limited resistance to certain pathogens (Uknes et al. (1993) Plant Response to environments. 2, 1; CRC Press). One conclusion from these experiments is that further factors are required for complete resistance.

The aim of the invention is to isolate new PR proteins (VIP proteins) and with the help of suitable ones Transfer gene constructs into plants.

Part of the present invention is the provision of DNA sequences which the VIP Encode proteins. Plants are also provided which have an altered VIP protein expression.

The present invention relates to plants which are derived from transformed plant cells contain said recombinant DNA molecule, their seeds, as well as Descendants of the plants that have arisen from said transformed plant cells as well as mutants and variants thereof. The present invention also encompasses all of them Crossover and fusion products with the aforementioned transformed plant material.

As regulatory sequences for the expression of VIP proteins in transgenic plants Promoters and termination sequences used that are active in plants. As promoters sequences can be used which are constitutive (e.g. the 35S promoter of the Cauliflower mosaic virus), a light-regulated (e.g. the ST-LS1 promoter from potatoes), a leaf-specific (cy-FBPase promoter from potato) or an inducible one (e.g. the PR- 1b promoter from tobacco) mediate expression.

For the construction of the chimeric gene constructs intended for plant transformation a large number of cloning vectors are available in E. coli. About suitable Restriction sites allow the chimeric genes to be introduced into the vectors. The Plasmids obtained are transformed into E. coli and the resulting cells in suitable Media attracted. The plasmids can be isolated from the E. coli cells and one Sequence or restriction analysis are subjected. Depending on the implementation method desired DNA genes in plants may require additional DNA sequences. Become a Example for transformation Ti plasmids used, at least the right one Limitation of the T-DNA to be present as a flanking region. To select the transformed plant cells, the DNA to be integrated normally contains one  Selection marker. The selection marker often conveys resistance to one Biocide or an antibiotic.

Various methods are available for transforming plant cells. she involve transformation with T-DNA using Agrobacteria (tumefaciens or rhizogenes), fusion, injection or electroporation, and other options. Binary vectors are preferably used for transformation with agrobacteria. These Vectors can replicate in both E. coli and Agrobacteria. After transfer of the desired DNA from E. coli in agrobacteria, the cells thus obtained can Transformation of plants can be used. After the transformation, you can the choice of suitable media containing antibiotics or biocides for selection, whole Plants are regenerated. The plants thus obtained can then be checked for the presence of imported DNA can be tested. You have increased pathogen resistance.

Embodiments Genetic engineering processes on which the exemplary embodiments are based 1. General cloning procedures

Cloning procedures such as For example: restriction cleavages, agarose gel electophoresis, purification of DNA fragments, transfer of nucleic acids to nitocellulose and nylon membranes, Linking DNA fragments, transforming E. coli cells, growing bacteria, Multiplication of phages and sequence analysis of recombinant DNA were carried out as in Sambrook et al. (1989) Cold Spring Harbor Laboratory Press; ISBN 0-87969-309-6) carried out. The transformation of Agrobacterium tumefaciens was carried out according to the Höfgen and Willmitzer method (Nucl. Acid Res. (1988) 16, 9877). The The agrobacteria were grown in YEB medium (Vervliet et al. J. Gen. Virol. (1975) 26, 33).

2. Generation of cDNA libraries

Total RNA from leaves was used to produce leaf-specific cDNA libraries of untransformed control plants (wild type) and PPa1 plants (Sonnewald (1992) Plant J. 2, 571) according to one of Logemann et al. (Anal. Biochem. (1987) 163, 21) Method isolated. The poly (A) RNA was then analyzed via oligo (dT) cellulose type 7 (Pharmacia Biotech Europe, Munzgerstr. 9, 79111 Freiburg) according to the manufacturer cleaned. After determination of the photometric concentration, 5 μg of the thus obtained were RNA used for cDNA synthesis. All necessary for the production of the cDNA  Chemicals and enzymes were obtained from Stratagene (11099 North Torrey Pines Road, La Jolla, CA 92037, USA). The methods used have been specified carried out by the manufacturer. The synthesis of the first and second strand of the cDNA was performed with the ZAP-cDNA synthesis kit. The double-stranded cDNAs obtained were then provided with EcoRI-NotI adapters and digested in an EcoRI Lambda ZAPII vector cloned. After in vitro packaging (Gigapack II packaging extract) the recombinant lambda DNA was transformed into XL-1 E. coli cells (Stratagene). By The titer of the cDNA libraries was determined by counting the plaques formed.

3. Generation of a subtractive cDNA library

The production of a subtractive cDNA library is shown schematically in Fig. 1. An advantage of the Lambda ZAPII vectors is that the addition of a helper phage results in plasmid vectors which carry the cDNA fragments. This process is called in vivo excision and can be carried out according to the manufacturer's (Stratagene) instructions. To generate a subtractive bank, after in vivo excision and propagation of the recombinant plasmids, the wild-type cDNA was cleaved with the restriction enzyme NotI and the PPa1 cDNA with the restriction enzyme EcoRI. After the restriction had been carried out, the cDNA fragments were separated in an agarose gel and fragments between 1.5 and 0.5 kb in length were isolated from the gel. The wild-type cDNA fragments were photobiotinylated for later separation. Photobiotinylation was carried out according to a protocol by Strauss and Ausubel (Proc. Natl. Acad. Sci. USA 87, 1889 (1990)). Following photobiotinylation, the wild-type and PPa1 cDNA fragments were mixed in a ratio of 10: 1, denatured at 95 ° C and then renatured by lowering the temperature to 25 ° C. Wild-type and hybrid cDNA molecules were removed from the cDNA mixture by adding avidin (Vectrex avidin, sales: Camon Labor Service, Bahnstrasse 9a, 65183 Wiesbaden), followed by centrifugation. The remaining PPa1-specific cDNA molecules were then cloned into EcoRI digested Lambda ZAPII vectors and used to transform E. coli cells as described above. The cDNA library obtained in this way contained 10-fold enriched PPa1-specific clones.

4. Differential screening of a subtractive cDNA library

For the isolation of PPa1-specific cDNA clones, 2 × 10⁵ phages were placed on agar plates attracted. After transferring the phage DNA to nylon filters (Hybond N, Amersham Buchler) the filters were first hybridized with the radioactively labeled wild-type cDNA. Hybridizing phage DNAs were visualized by autoradiography. The filters were then hybridized with the radioactively labeled PPa1 cDNA and also subjected to autoradiography. By comparing the two autoradiograms  phage DNAs were identified which hybridize specifically with the PPa1 cDNA. To Purification of the phages obtained and subsequent in vivo excision became the identity of the individual clones determined by sequencing.

5. Bacterial strains

E. coli (XL-1 Blue) bacteria were purchased from Stratagene. The for Plant transformation used Agrobacterium tumefaciens strain (C58C1 with the Plasmid pGV 3850kan) was developed by Debleare et al. (1985, Nucl. Acid Res. 13, 4777) described.

6. Tobacco transformation

To transform tobacco plants (Nicotiana tabacum L. cv. Samsun NN), 10 ml an overnight culture of Agrobacterium tumefaciens grown under selection centrifuged, the supernatant discarded, and the bacteria in the same volume of antibiotics free medium resuspended. Leaf disks became more sterile in a sterile petri dish Plants (diameter approx. 1 cm) bathed in this bacterial solution. Then the Leaf disks in petri dishes on MS medium (Murashige and Skoog, Physiol. Plant. (1962) 15, 473) with 2% sucrose and 0.8% Bacto agar. After 2 days of incubation in In the dark at 25 ° C they were on MS medium with 100 mg / l kanamycin, 500 mg / l claforan, 1 mg / l benzylaminopurine (BAP), 0.2 mg / l naphthylacetic acid (NAA), 1.6% glucose and 0.8% Bacto agar transfer and cultivation (16 hours light / 8 hours dark) continued. Growing sprouts were placed on hormone-free MS medium with 2% sucrose, 250 mg / l Claforan and 0.8% Bacto agar transferred.

7. Analysis of total RNA from plant tissues

Total RNA from plant tissues was determined as in Logemann et al. (Anal. Biochem. (1987) 163, 21). For the analysis, 20-40 µg RNA were in one Formaldehyde-containing 1.5% agarose gel separated. After electrophoretic separation the RNA molecules were transferred to a nylon membrane by capillary transfer transfer. The detection of specific transcripts was carried out as for Amasino (Anal. Biochem. (1986) 152, 304). The cDNA fragments used as a probe were radioactive with a Random Primed DNA Labeling Kit (Boehringer, Mannheim) marked.

8. Immunological detection of virus particles

Plant material was mixed with PBS buffer (per liter: 8 g NaCl, 0.24 g KH₂PO₄, 1.44 g Na₂HPO₄ × 12 H₂0, 0.2 g KCl, pH 7.4) mixed with 0.5 ml / l Tween-20, 2%  Polyvinylpyrollidone 25000, 9.2% bovine serum albumin) homogenized (to 1 g Plant material 20 ml buffer) and by means of monoclonal antibodies against KVY (BIOREBA AG, 4153 Reinach, Switzerland) in the "Double Antibody Sandwich Test". The Elisa Test procedure was carried out according to the manufacturer (BIOREBA AG).

9. Infection of tobacco plants with KVY by mechanical rubbing

The infectious material for rubbing the leaves was made from infected leaf material, by homogenizing it in 50 mM phosphate buffer, pH 7.2 (1:20). The too infecting leaves were dusted with carborundum and then with the homogenate rubbed off with a pestle. After a few minutes, the leaves were washed with water hosed.

Non-limiting embodiments 1. Screening of the subtractive cDNA library

By differential screening of the subtractive cDNA library with the radioactive labeled wild-type and PPa1 cDNA fragments could be identified in nine clones are specifically enriched in the cDNA library. Sequence analysis of the clones obtained showed that four of the isolated clones correspond to clones already described (Acc oxidase, SAR-8.2, PR-1b and PR-3). The remaining five clones are so far unpublished cDNA clones. The inducibility of the isolated cDNA clones by Pathogens were tested in Northern blot experiments. One clone showed a clear one Induction in pathogen infestation and was therefore subjected to a more detailed analysis.

2. Cloning of the VIP cDNAs

The EcoRI cDNA insert of the clone isolated from the subtractive cDNA library was used to screen the starting PPa1 cDNA library. From several hundred hybridizing plaque areas, 25 independent to single plaques were enriched. After in vivo excisions, the cDNA clones were analyzed by restriction digestion and sequencing. It turned out that, due to their different sequences, the cDNAs could be classified into three classes, which were designated VIP-20a, -20b or -20c. In FIGS. 2a, b and c, the cDNA sequence is in each case a clone of the three classes listed.

3. Sequence analysis of the VIP cDNAs

Homology comparisons of the three VIP cDNA classes showed that the VIP-20a cDNAs has a significantly higher similarity to the VIP-20b cDNAs (90.6% identity) than to the VIP-20c cDNAs (75.2% identity). A 70.5% identity was found between the VIP-20b and -20c cDNAs. The search for protein coding regions led to the identification of a reading frame in all VIP cDNA classes. The information on the positions of the start and stop codons is shown in Fig. 3. The homologies between the supposedly coding regions were significantly higher than those of the total cDNAs; VIP-20a / VIP-20b: 94.8%, VIP-20a / VIP-20c: 78.1% and VIP-20b / VIP-20c: 79%. In the supposedly 3 ′ untranslated regions, there were no homologies between the cDNA classes except for shorter areas. In the 5 ′ untranslated sequences, an 82.4% identity was found between the VIP-20a and VIP-20b cDNAs; the DNA sequences between the other classes showed little similarity. These results suggest that the reading frames identified are indeed protein-coding regions. The reading frames would code for proteins with a theoretical molecular weight of 20 kDa. Homology comparisons at the amino acid level ( Fig. 4) essentially reflected the similarities at the DNA level. The VIP-20a and -20b proteins were significantly more similar (90.3% identity) than the VIP-20a and -20c proteins (78.1% identity) and the VIP-20b and -20c proteins (79% identity). The three VIP protein classes showed the same profile in hydropathy diagrams. All VIP proteins have a hydrophobic region of 28 amino acids at the N-terminus, which probably acts as a signal peptide.

4. Evidence of induction of WP mRNAs in the case of virus infection

To demonstrate that VIP mRNAs were induced by viruses, wild-type tobacco plants were infected with potato virus Y by mechanical rubbing of a lower leaf. On several days after infection, samples for RNA were taken from the infected as well as systemic leaves (the 1st and 3rd leaves above the infected) and in Northern blots with the VIP-20a cDNA under less stringent conditions, which hybridized all three Allow VIP mRNA classes, analyzed. Samples taken in parallel were examined for the presence of virus particles using the ELISA technique. It was found that VIP mRNAs were already induced in the locally infected leaves after 4 days and that there was a further accumulation up to the 9th day ( FIG. 5A). The VIP mRNAs were slightly induced in the systemic leaves on the 8th day, transcript quantities increased up to the 12th day (the last examined) ( Fig. 5B). Virus particles could be detected on the 3rd (in the local) or on the 6th day (in the 3rd above the infected leaf). Amazingly, there were no virus particles in the first systemic leaf. This proves that VIP mRNAs can be induced both locally and systemically.

5. Reduced virus spread in transgenic plants

Northern blot analysis showed that the VIP mRNA amounts in the PPa1 plants were clearly induced compared to wild-type plants ( FIG. 6). As a result, it was reasonable to assume that these plants could have preconditioned protection against viruses due to the constitutive presence of the VIP proteins. To test this assumption, PPa-1 (8 plants) and control wild-type plants (9 plants) (each 12-14 leaf stage) were infected with potato virus Y. At different times after infection, the same leaf areas of the local and systemically infected leaves were punched out, homogenized and different dilutions of the homogenate were examined for the presence of virus by means of the Elisa test. It was shown that the local virus spread and / or multiplication of infected leaves in ppa-1 is greatly reduced compared to wild-type plants (Table 1). In addition, the systemic spread in the ppa1 plants was extremely delayed, which indicates an inhibition of viral long-distance transport through the vascular system. While wild-type plants were systemically infected as early as the 12th day after infection, this was the case with ppa-1 plants only after 32 days (Table 2).

6. Production of the plasmids BinAR-VIP-20a and -20c sense and transformation of Tobacco plants

Studies on the virus-induced expression of the VIP proteins have shown that the expression of the VIP proteins is stimulated after a virus attack. By amino acid comparison it was found that the VIP proteins 20a and 20b have a very high degree of homology to one another. The amino acid sequence of the VIP-20c proteins differs significantly from the other two. In order to generate plants which can activate their defense mechanisms more quickly by constitutive expression of the VIP proteins, the cDNA clones VIP-20a and VIP-20c were therefore provided with a promoter which causes constitutive expression and a plant termination signal. The plasmids BinAR-VIP-20a sense and BinAR-VIP-20c sense consist of the three fragments A, the respective cDNA and C (see Fig. 7 B and C) and were inserted into the expression vector pBinAR by inserting the corresponding cDNA sequences ( Fig. 7 A).

Fragment A contains the 35S CaMV promoter. It contains a fragment which comprises nucleotides 6909 to 7437 of the Cauliflower Mosaic Virus (CaMV) (Franck et al. (1980) Cell 21, 285). It was isolated as an EcoRI-KpnI fragment from the plasmid pDH51 (Pietrzak et al. (1986) Nucleic. Acid Res. 14, 5857). The VIP-20a cDNA was cloned from the pBluescript SK- ( Fig. 3) as SalI-BamlII fragment and the VIP-20c cDNA as Asp718-BamHI fragment directed into the pBinAR vector ( Fig. 7). Fragment C contains the polyadenylation signal of gene 3 of the T-DNA of the Ti plasmid pTiACH5 (Gielen et al. (1984); EMBO J. 3, 835), nucleotides 11749-11939, which is a PvuII-HindIII fragment from the plasmid pAOV 40 (Herrera-Estrella et al. (1983); Nature 303, 209) and was cloned after addition of SphI linkers to the PvuII site between the SphI-HindIII site of the vector.

The resulting plasmids BinAR-VIP-20a and c sense were analyzed using the Agrobacterium system transformed into tobacco.

Description of the pictures Fig. 1 Schematic representation for the creation of a subtractive cDNA library

Wild type corresponds to a leaf-specific cDN A library from untransformed Tobacco plants. PPaI corresponds to a leaf-specific cDNA library from transgenic Tobacco plants with reduced virus spread. The samples for creating the cDNA Libraries were grown after 8 hours of exposure to adult tobacco plants taken.

Fig. 2a-c Representation of the VIP-20a, -20b and -20c nucleic acid sequences

The cDNA clones are given in the 5'-3 'direction based on the open reading frame. The Start and stop codons are underlined. The clone VIP-20a consists of 727 bp, clone VIP-20b from 804 bp and clone VIP-20c from 817 bp (bp = base pairs).

Fig. 3 Schematic representation of the VIP-20 cDNA clones in the plasmid pBlue SK-

After in vivo excision, the cDNA fragments isolated as lambda phages lie in the vector pBlueScript SK- (Stratagene) as EcoRI / NotI fragments. Those designated as A and B. Areas represent the left (5 ′) or right (3 ′) polylinker sequence. Start corresponds to that Initiation Codon of Translation (ATG). Stop corresponds to the termination signal for the Translation (TAA). The numbers in brackets correspond to the positions of the start or stop codons within the cDNA fragments. The arrows give the orientation of the cDNA clones. The fragment lengths of the individual clones are within the arrows in Base pairs (bp) indicated.

Fig. 4

Comparison of the amino acid sequences derived from the VIP-20a, -20b and -20c DNAs  Dots correspond to identical amino acids; Dashes symbolize missing amino acids. The amino acid comparison was based on the MacMolly program (Compare subroutine) a Macintosh computer.

Fig. 5

Local and systemic induction of the VIP-20 transcripts in KVY infected tobacco leaves RNA was isolated from the infected (A) and systemic leaves (B) and in Northern blot Analyzes with radioactively labeled VIP-20a cDNA fragment under less stringent Conditions hybridized (hybridization buffer: 25% formamide, hybridization at 42 ° C). The figures refer to the day of sampling after infection.

Fig. 6 Evidence of accumulation of VIP-20 specific transcripts in sheets of PPa1 Tobacco plants

RNA was derived from adult leaves of wild-type plants (con) and ppa1- Tobacco plants isolated and in Northern blot analyzes with radio-labeled VIP-20a cDNA fragment hybridized under less stringent conditions.

Fig. 7 Construction of plasmids for overexpression of VIP proteins in transgenic plants

A: Herbal expression cassette consisting of the following fragments:

A = Fragment A (529 bp): Contains the 35S promoter of the Cauliflower mosaic virus (CaMV). It contains a fragment which contains the nucleotides 6909 to 7437 of the CaMV includes.

B = fragment B contains the multiple cloning site with the restriction sites: KpnI, SmaI, BamHI, XbaI, SalI and SphI from the polylinker of the plasmid puc18 Yanish-Perron et al. (1985) Gene 33, 103) for inserting gene sequences.

C = fragment C (192 bp): contains the polyadenylation signal of gene 3 of the T-DNA of the Ti plasmids pTiACH5.

The fragments A to C were in EcoRI / HindIII in the binary vector Bin 19 (Bevan (1984) Nucl. Acid Res. 12, 8711).

B: Plant expression cassette for overexpression of the VIP-20a protein in transgenic Plants. Fragments A and C correspond to the fragments described in Figure 6A. The black bars indicate the polylinker sequences from the plasmid pBlue SK-, which in the Cloning of the VIP-20a cDNA sequence were transferred into the expression cassette. The  The cDNA fragment was inserted into the plant expression cassette as a BamHI / SalI fragment cloned.

C: Herbal expression cassette for overexpression of the VIP-20c protein in transgenic Plants. Fragments A and C correspond to the fragments described in Figure 6A. The black bars indicate the polylinker sequences from the plasmid pBlue SK-, which in the Cloning of the VIP-20c cDNA sequence into the expression cassette were transferred. The The cDNA fragment was inserted as a KpnI / BamHI fragment in the plant expression cassette cloned.  

Tables Table 1: Accumulation of the KVY coat proteins in infected leaves of transgenic tobacco plants

The numbers are relative OD values measured at 405 nm after performing the Elisa test Procedure. They are mean values with derived standard deviations 9 (wild type) or 8 (ppa-1) different plants

Table 2: Systemic spread of KVY in leaves of transgenic tobacco plants

Claims (5)

1. A process for the production of pathogen-resistant plants, characterized in that a chimeric gene construct consisting of in the genetic material of the plants

• - an active promoter,
• a region coding for virus-induced proteins (VIP proteins)
• - and a polyadenylation signal,

introduces. 2. The method according to claim 1, characterized in that one uses as active promoters sequences that a mediate constitutive, organ-specific or inducible expression. 3. The method according to claim 1, characterized in that DNA sequences which are isolated from transgenic tobacco plants and which have the following DNA sequences are used as the region encoding VIP proteins. 4. Plants and parts of plants, which is modified by the method according to claims 1-3 Have genetic makeup.