DD294501A5 - INDUCIBLE VIRUS RESISTANCE FOR PLANTS - Google Patents

Abstract

A process based on genetic engineering methods makes it possible to develop inducible virus resistance in plants. The invention further relates to the use of the process and to the plants with inducible virus resistance obtainable by this process, and to their offspring. The invention also embraces chimeric genetic constructs as well as cloning vectors and host organisms, and methods for transferring an inducible virus resistance to plants. Said recombinant DNA molecules are used to "immunise" plants against unwanted viral attack. <IMAGE>

Description

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The present invention is a method which based on genetic engineering ·· methods the formation of an inducible virus resistance be ^! Allows plants. Furthermore, the present invention relates to the use of this method for the production of virus-resistant plants and to the obtainable by this method, equipped with an inducible virus resistance plants themselves and their offspring.

Much of today's known diseases of our most important crops, which again and again lead to considerable losses of yield and yield in fruit, vegetable and cereal crops, are caused by plant viruses. The transmission of these plant-pathogenic viruses is primarily by sucking or biting phytophagous insects and nematodes, which act as vectors.

Therefore, it has long been tried in crop cultivation, suitable methods for protecting the crop before a Develop virus attack. However, one of these measures, which is only partially effective and involves considerable risks, has been taken under the Concept of 'cross protection' known. It is the artificial infection of healthy cultures with weakened Strains of pathogenic viruses, whereby a secondary infection with the actual pathogen should be prevented. This Procedure was z. B. for reducing the yield losses in tomato, potato and citrus growing, by

pathogenic viruses, such as the tomato mosaic virus, the potato spindle tuber viroid and the citrus tristeza virus. (Broadbent L, Ann., Rev. Phytopathol ,, 14: 75, 1976; Fernow KH, Phytopathology, 57: 1347, 1967; Costa

AS and Muller GW, Plant DIt., 64: 538, 1980). However, the application of this method to protect healthy cultures against viral infection has numerous disadvantages

and dangers with it.

Thus, for example, the attenuated virus strain used for the artificial infection may change into a

highly virulent strain and thus cause even the onset of disease.

Furthermore, it is possible that a particular virus strain may well be suitable for the protection of a particular plant species

In contrast, in another species it acts as a pathogen and causes severe damage.

In addition, synergistic effects of two or more types of virus are described, leading to new hitherto unknown Disease symptoms (Garces-Orejuela C and Pound GS, Phytopathology, 57: 232, 1957). With the introduction of genetic engineering methods also in plant genetics and the resulting Ways of introducing foreign genetic material into plants, new methods have been developed that are based on the Expression of only single virus genes in the corresponding plants based, instead of the previously practiced infection with the

entire virus genome.

In the new methods mentioned, for example, coat protein genes of certain viruses (tobacco mosaic virus, alfalfa Mosaic virus) into the plant genome using recombinant DNA technology

(Bevan MW et al., EMBO J., 4 (8): 1921-1926, 1985; Abel PP et al., Science, 232: 738-743, 1986).

In the case of aer expression of the integrated envelope protein gene in the plant, a certain amount of protein can be produced by the produced envelope protein Protective effect can be achieved, which protects the plant against infection with the corresponding virus or occurring Kankheitssymptome weakens. The exact mechanistic background for this protective effect by the coat protein

are so far largely unknown.

The expression of other virus genes can also lead to a protective effect against infection with the relevant virus. So

For example, in transgenic plants, the antisense RNA of parts of the corresponding virus syntheses leads to an inhibition of virus expression. Also, the expression of satellite RNA in transgenic plants can lead to a protective effect, presumably due to competition with the infecting virus for the replicase (Baulcombe

DC et al., Nature, 321: 446-449, 1986). While these procedures provide some protection against a viral infection and / or its consequences, they do have

also decisive disadvantages:

a) At high Inokulationskonzentrationen of the virus, the protection factor can be titrated off, so that a protective function is no longer guaranteed.

b) the protection factor itself may be the cause of the triggering of disease-like conditions in the protection factor synthesizing plant.

For example, the virus envelope protein is able to invade and damage biological membranes (de Zoeten GA and Gaard G, Virus Research, 1: 713-725, 1984). This can subsequently lead to dysfunctions within the cell, especially chloroplasts and mitochondrion.

c) The protective factor genes are constantly (constitutively) expressed, even if there is no viral infection, which leads to a significant deterioration in the energy balance of the plants concerned. The aim of the present invention is therefore to develop a system in which the protective factor of the transformed plant is no longer constitutively produced, but rather the synthesis of the said protective factor as required in the producing plant in order to overcome the abovementioned disadvantages can be switched on or off.

This task has now been solved in the context of the present invention, surprisingly with simple measures

By providing a system that detects the induction of genes based on the virus

Control mechanisms. An object of the present invention thus relates to a method for protecting plants from a viral infection

and / or their consequences, characterized by introducing an inducible factor of protection into said plants which, if necessary, d. H. in case of incipient infection with a virus, exprimi jrt becomes and the production of the

Protection factor controls. Another object of the present invention relates to a method for protecting plants from infection with

phytopathogenic viruses characterized by introducing into said plants an inducible protection factor which is inoperably linked to virus-specific control elements as well as to plant cell-active expression signals such that the expression of said protective factor gene and thus the production of the protective factor is controlled by the infecting virus itself becomes.

Moreover, the present invention relates to recombinant DNA molecules that raise an inducible virus resistance

in which the infecting viruses have been neutralized by a protective factor synthesized by the plant.

In particular, the present invention relates to transgenic virus-resistant plants as well as viable parts of virus-resistant

Transgenic plants that have been transformed with a recombinant DNA molecule, which is an inducible

Protective factor is responsible for the neutralization of the infecting virus and mutants and variants of said

transgenic plants.

In the context of the present invention, a "virus-resistant" plant is defined as a plant,

which survives at a normally effective virus concentration and preferably exhibits normal growth.

The present invention further relates to plants which are regenerated from transformed plant cells containing said recombinant DNA molecule and their seeds, moreover the progeny of plants regenerated from said transgenic plant cells and mutants and variants thereof.

According to the invention, mutants and variants of transgenic plants are to be understood as meaning those plants which still possess the characteristic properties of the starting plant which it has obtained as a result of the transformation with an r-DNA molecule according to the invention.

Also included in the present invention are all cross and fusion products with the aforementioned transformed plant material.

Furthermore, the invention relates to methods for producing virus-resistant transgenic plants, which is characterized by transforming a plant or viable parts thereof with a recombinant DNA molecule.

By viable parts of plants are by definition. Without this being a limitation of the subject invention, z. As plant protoplasts, cells, cell aggregates, callus, seeds, pollen, oocytes, zygotes or embryos can be understood.

The present invention also encompasses cellular genetic constructs containing a protective factor gene, as well as

Process for their preparation, furthermore cloning vehicle and host organisms as well as methods for the transmission of an inducible virus resistance to plants.

Another object of the present invention relates to the use of said recombinant DNA molecules for "immunizing" plants against an undesired virus attack.

In the following description, a number of terms are used which are common in recombinant DNA technology as well as in plant genetics.

In order to ensure a clear and consistent understanding of the description and claims as well as the scope of the said terms, the following definitions are set forth:

Heterologous (s) QEN (s) or DNA: A DNA sequence encoding a specific product, products or biological function derived from a species other than that into which said gene is introduced; said DNA sequence is also referred to as foreign gene or foreign DNA.

Homologous gene (s) or DNA: A DNA sequence encoding a specific product, product or biological function derived from the same species into which said gene is introduced.

Synthetic gene (s) or DNA: A DNA sequence encoding a specific product, product or biological function synthesized synthetically.

Plant promoter: A DNA expression control sequence that ensures the transcription of any homologous or heterologous DNA gene sequence in a plant, if said gene sequence is operably linked to such a promoter.

Overproducing Plant Promoter (OPP): A plant promoter capable of effecting, in a transgenic plant cell, the expression of any operably linked functional gene sequence (s) to an extent (as measured in terms of RNA or polypeptide amount) which is significantly higher than is naturally observed in host cells that are not transformed with said OPP.

Plant: Jode's photosynthetic active member from the Planta realm characterized by a membrane-bound nucleus, a chromosome-organized genetic material, membrane-coated cytoplasmic organelles, and the ability to perform meiosis.

Plant cell: Structural and physiological unit of the plant consisting of a protoplast and a cell wall. Plant Waste: A group of plant cells organized in the form of a single structural and functional unit.

Plant organ: A delimited and distinctly differentiated part of a plant, such as root, stem, leaf or embryo.

Protection factor: Single or multi-component system which directly or indirectly interacts with infecting viruses within the plant in a manner that ensures a protective effect against a viral infection and / or its consequences.

Protective factor gene (s): nucleotide sequence (s) that encode a protective factor as defined above.

Virus-specific control elements: Regions on the viral genome that interact with a viral product in a way that affects replication, transcription, translation, or other post-translational processes (in ".").

DNA Expresslone Vector: Cloning vehicle, such as. A plasmid or a bacteriophage containing all the signal sequences necessary for the expulsion of an inserted DNA into a suitable host cell.

DNA transfer vector: transfer vehicle, such as. B. a Ti plasmid or a virus that allows the introduction of genetic material into a suitable host cell. The following is a brief description of the figures will be given

Figure 1: shows the construction of plasmid pHS 1, which is described in detail in Example 1.1.

Figure 2A-C: shows the different possibilities of transactivation in CaMV.

Figure 3: shows the restriction map of plasmid pCIB200.

Figure 4: shows the restriction map of plasmid V374.

The present invention relates primarily to recombinant DNA molecules capable of conferring inducible virus resistance on plants.

The virus resistance of the plants is mediated by so-called protective factors, which are produced by the transgenic plant itself and impair its reproductive capacity through a specific interaction with the infecting plant virus to such an extent that an outbreak of infection is prevented and / or their consequences are reduced ,

There are basically two categories of protection factors. On the one hand, these are antiviral substances that directly attack the virus and thus have a negative effect on its reproductive capacity. By inhibiting enzymes essential to the developmental cycle of the virus.

A second category concerns cytotoxic substances which poison and / or kill the cells affected by the viruses and thus isolate the source of infection from the rest of the healthy tissue. In this case, therefore, the reproductive capacity of the virus is indirectly limited by the artificial triggering of a "hypersensitive reaction".

In the context of the present invention, SchuU factors are therefore to be understood as meaning single-component or multicomponent systems which interact directly or indirectly with the infecting viruses within the plant, so that a protective effect from a viral infection and / or its consequences is ensured.

Examples of such protective factors, which are not a limitation of the subject invention, are:

1) Antiviral substances, such as. Antivirus antibodies, PR proteins (Ah P et al., Plant Mol. BIol., 4: 37, 1985), virus-specific protease inhibitors (Jamet E and Fritig B, Plant Mol. BIol., P: 69-80, 1986) ) Proteases, virus-specific polymerase inhibitors, and interferon-like proteins.

2) Celltoxic substances, such as. Toxins, active subunits of toxins, PR proteins, as well as other proteins that naturally cause hypersensitivity, such as the β-glucanase (Mohnen D. et al., EMBO J., 4: 1631-1635, 1985).

Of particular importance in the context of the present invention are the Ribosome Inactivating Proteins [RIPS] (Lord et al., Oxford Sur. Plant Mol. Cell BIoI., 1: 85-101, 1984) which cause irreversible inhibition of the ribosomes and lead to an immediate cessation of protein synthesis.

Examples of such ribosome-inactivating proteins are the abrin (from Abrus precatorius seed) (Olsnes et al., (In: Cohen P.).

and Van Heyninger S. (Ed.) Molecular Actions of Toxins and Viruses, Biomedical Press, pp. 51-105, 1982) Modeccin (from Medecca digitate roots) (Stirpe et al., FEBS Lett. 85: 65-67, 1978), Viscumin (from Viscum album leaves) (Stirpe et al., Blochem J., 190 :

843-845.1980; Olsnes et al., J. Biol. Chem., 257: 13263-13270, 1982) and ricin (from Ricinus communls) (Olsnes and Pihl, In:

Cuatrecasas P. (Ed.) The Specificity of Animal Action, Bacterial and Plant Toxins. Chapman and Hall, pp. 129-173.1976).

Also included in this group is the pokeweed toxin (Owens et al. Virology, 56: 390, 1973).

All these compounds have a great structural as well as similar similarity.

It proves to be particularly advantageous if the induction of Schutzfaktorgens and thus the protection factor production by the infecting virus itself. In this way, the synthesis of the protection factor is limited to the cases in which a virus infection takes place and the protection factor is actually needed to afford the plant the appropriate protection.

If the plant is attacked by a corresponding virus, then the virus-specific control region is turned on and thus the Schutzufaktorgen expressed. Subsequently, the synthesis of the protective factor neutralizes the infecting virus.

The more infectious virus is present, the more protection factor is produced and the more virus is neutralized.

Thus, in the context of the present invention, it has been possible for the first time to construct a self-contained control circuit which regulates itself and ensures the plant optimal protection against a virus infection.

As a result, the plant does not need to expend unnecessary energy to maintain lasting protection against viral infection, which is desperately needed for growth and fruiting.

This can be achieved according to the invention, for example, by linking the protective factor gene with virus-specific control elements and thus placing their expression under the control of said control elements.

Another object of the present invention relates to hearing a method for the protection of plants against a viral infection and / or their consequences, which is characterized in that one introduces an inducible protective factor genes in said plants, with virus-specific control elements and active in plant cells expression signals is operably linked so that the expression of the protection factor is self-regulated by the infecting virus.

It is known that a variety of viruses have developed specific control mechanisms that are essential for the coordination of the individual virus functions.

The so-called virus-specific control functions include, for example:

a) the transactivation of viral genes at the transcriptional and post-transcriptional level

b) Production of subgenomic RNA by virus-encoded replicases

c) Activation of proteins by processing, phosphorylation and other modifications.

The phenomenon of viral transactivation has hitherto been observed almost exclusively in bacterial and animal systems (Calender, Biotechnology, 4: 1074, 1986).

There are basically different levels on which a transactivation mechanism can attack.

The simplest and most obvious way is to transactivate the transcription.

This type of transactivation has been described, for example, as one of the major mechanisms of action for the human HTLV system.

The transactivator regulates transcription by reaction with the viral LTR (Long Terminal Repeat) (Muesing MA et al., Cell, 48: 691-701, 1987).

Certain reading frames of this virus (HIV-LTR), such as. As the tat gene encode transactivators that allow the expression of the virus genes gag-pol-env. The tat gene product is a polypeptide of MG 15000, which is similar in structure and properties to nucleic acid binding proteins such. B. the transcription factor VIII A of

Xenopus laevlt u.a.

As a possible mechanism for the transactivation, therefore, a cooperative binding of a regulator to a recognition site is considered. Such a recognition site exists, for example, in the region of the RNA start region.

On the posttranscriptional level, on the other hand, a variety of different transactivation mechanisms are available that can attack at very different sites.

The possibilities range from the control of mRNA extension (Grayhack et al., Cell, 42: 259, 1985; Yi-Kao et al., Nature, 330: 4891987), via the control of mRNA transport from the nucleus to the cytoplasm, of mRNA secretion, mRNA polyadenylation (Derse, J. Virol., 62, 115, 1988) of the Tianslations initiation (Jay et al., Proc Natl. Acad. Sei, USA, 78: 2927, 1981) to the differentiated Using different ORF's ('Open Reading Frame') on the RNA. A third level of potential transactivation mechanisms involves post-translational modification of the protein product in the form of phosphorylations or protein processing.

In the case of Cauliflower Mosaic Virus (CaMV), all three of the aforementioned transactivation levels are relevant. CaMV has a double-stranded (ds) circular DNA genome, which is believed to be implicated by a reverse transcriptional mechanism. Two RNA intermediates are formed, a 35S RNA and a 19S RNA corresponding to two operons on the CaMV genome. The 19S promoter transcribes a subgenomic RNA (19S RNA transcript) encoding the CaMV protein Vl, which forms the bulk of the virus-specific inclusion bodies. The 35S RNA consists of a 600 nucleotide leader region followed (in order) by the reader rasters VII, I, II, III, IV, V and Vl. It is assumed that all of these reading frames, except for the reading key V1, are translated by the 35S. (Pfeifer, P., and Hohn, T., Cell, 33: 781-789, 1983; Kridl JC and Goodman RM, Bio Essays, 4: 4-8, 1986).

In the context of the present invention, the following control mechanisms for the CaMV system have been verified:

1) The 19 S promoter is activated by the protein product of the 19 S operon in the form of autocatalysis (see Fig. 2 A).

2) In addition, the use of polycistronic RNA is regulated by the said protein product of the 19S operon (see Fig. 2 B).

3) The release of a specific protein product from a polyprotein by the activity of viral proteases (Figure 2 C).

Certain strand-p positive (+) viruses find a variation in this viral transcriptional transactivation. In these cases, subgenomic virus (messenger) RNA is produced from minus-strand RNA catalyzed by virus-specific polymerases.

This special form of transcriptional transactivation is realized, for example, in the Brome mosaic virus (3MV), the plants of the group of monocotyledons in particular numerous Graminea species, such. As maize, barley, bromeliads and other wild grasses, but also various dicotyledonous plants attacks.

The genome of BMV is composed of a total of three RNA components. The transcripts of two of these components (RNA 1 and RNA2) are essential for the replication of the viral genome in the plant. For example, the replication of the third component (RNA3), a dicistronic RNA encoding a 32,000 MW protein and the coat protein, is dependent on the presence of the RNA1 and RNA2 transcripts for the production of subgonomic coat protein mRNA (French et al., Science , 231: 1294-1297, 1986).

As a mechanism for the production of subgenomic mRNA for the expression of the genes located on the RNA3, an internal initiation of a virus-specific replicase, which is formed very early in the course of the infection cycle (possibly encoded by genomic RNA1 and / or RNA2) at the minus (-. ) Strand of genomic RNAC (Miller et al. Nature, 313:

68-70.1985).

In addition, it can be expected that the copy number of genomic nucleic acids (DNA or RNA) can be greatly increased when they are provided with viral replication signals due to the activity of specific viral polymerases.

All of these systems can be used in the method according to the invention for the construction of vectors which are capable of transmitting an inducible virus resistance to plant material.

Particularly preferred in the context of this invention is the use of the virus-specific control elements of Cauliflower Mosaic Virus (CaMV), which, however, in no way restricts the scope of the present invention, but merely serves to demonstrate the functioning of the method according to the invention.

Another object of the present invention thus relates to the production of hybrid gene constructs which are composed of one or more virus-specific control regions as well as of homologous or heterologous DNA sequences which code for one or more protective factors X, using methods and methods which are widely known and used in the field of recombinant DNA technology, and are described inter alia in the following publications:

"Molecular Cloning", Maniatis T., Fritsch, EF, J. Sambrook, Cold Spring Harbor Laboratory, 1982, and "Recombinant DNA Techniques", Rodriguez RL and RC Tait, Addison-Wesley Publishing Comp., London, Amsterdam, Don Mils , Ontario, Sydney,

The protective factor gene coding region used according to the present invention may be of homolous or heterologous origin relative to the plant cell or plant to be transformed. However, it is in any case necessary that the gene sequence encoded for the protection factor be expressed and result in the production of a functional enzyme or polypeptide in the resulting plant cell.

According to the invention, DNA sequences of heterologous origin are to be understood as meaning those derived from other plant species or from organisms belonging to another taxonomic unit, such as e.g. Microbial or mammalian or of synthetic origin and which are capable of achieving the desired protection against virus challenge in any desired plant species.

The coding region of the hybrid gene design may also encode a protection factor that differs from a naturally occurring one, but still substantially fulfills the protective function of the natural protection factor.

Such a coding sequence will usually be a variant of a natural coding region. For the purposes of the present invention, a "variant" of a natural DNA sequence is defined as a modified form of a natural sequence which, however, still fulfills the same function: The variant can be a mutant or a synthetic DNA sequence and is essentially the corresponding one In the context of this invention, a DNA sequence of a second DNA sequence is then substantially homologous when at least 70%, preferably

at least 80%, but especially at least 90% of the active portions of the DNA sequence are homologous. As defined herein, the term "substantially homologous" will have two distinct nucleotides in one

DNA sequence of a coding region then considered to be homologous when the replacement of the one nucleotide against the

another represents a silent mutation.

The invention thus includes any hybrid gene encoding an amino acid sequence which has the desired protective function

which fulfills the disclosed and claimed requirements. Particularly preferred is a nucleotide sequence which is substantially homologous to at least the portion or portions of the natural sequence responsible for the protective function against virus attack.

The coding for the protective factor DNA sequence can exclusively from genomic, cDNA or synthetic DNA

be constructed. Another possibility is to construct a hybrid DNA sequence consisting of both cDNA and genomic DNA and / or synthetic DNA. In this case, the cDNA may be from the same gene as the genomic DNA, or both the cDNA and the genomic DNA may be from different genes. In either case, however, both the genomic DNA and / or the cDNA, each by itself, can be made from the same or different genes.

If the DNA sequence contains portions of more than one gene, these genes can either be of one and the same Organism, of several organisms, more than one strain, one variety or species of the same genus

or from organisms belonging to more than one genus of the same or another taxonomic entity (Reich).

The various DNA sequence sections can be combined with one another by means of methods known per se

linked to the protective factor coding DNA sequence. Suitable methods include, for example, in vivo

Recombination of DNA sequences that have homologous sections and the in vitro linkage of Restriction fragments with a. Thus, a variety of embodiments are encompassed by the broad concept of this invention! One of these embodiments of the invention is characterized by a chimeric gene sequence containing:

(a) one or more gene sequences coding for one or more protective polypeptides, which after the expression of the gene in a given plant cell has a protective function against a viral infection and / or its consequences, and

(b) one or more gene sequences encoding one or more virus specific control regions operably linked to the protective factor coding region, and

(c) one or more additional gene sequences which are operably linked to both sides of the coding region for the protection factor and optionally for the associated virus-specific control elements. These additional gene sequences contain promoter and terminator regions and, optionally, additional regulatory sequences of the 3 'and 5' untranslated regions. The plant regulatory sequences may be heterologous or homologous relative to the host cell.

Functionally linking the protection factor coding region to a virus specific control region can

according to the invention z. This can be accomplished, for example, by isolating virus-own coding DNA sequences, known to be naturally controlled during the infection cycle by specific functions of the intact virus, and replacing the coding portion with a protective factor gene, and thus subjecting the protective factor to viral control brings.

As examples of such coding viral regions, which are in no way to be regarded as limiting, the Reading frame I and VI of the CaMV genome, as well as the coat protein-encoding DNA sequence on the dicistronic

RNA3 of the BMV.

Any promoter and terminator capable of expressing a protective fiber-coding DNA sequence

can be used as part of the chimeras Genseqeunz. Examples of suitable promoters and

Terminators are z. Such as the nopaline synthase genes (nos), the octopine synthase genes (ocs) and the Cauliflower mosaic Virus genes (CaMV). An effective member of a plant promoter that can be used is an overproducing plant promoter. This type of promoter should, if operably linked to the protective factor gene sequence, be found in the Be able to mediate the expression of said protective factors in such a way that the transformed plant against a Virus infestation is protected. Overproducing plant promoters that can be used in the present invention,

include the promotor of the small subunit (ss) of soybean ribulose-1,5-bisphosphate carboxylase [Berry-Lcwe et al., J. Molecular and App. Gen., 1: 483-498 (1982)) and the promoter of the chlorophyll a / b binding protein. Both of these are known to be induced by light in eukaryotic plant cells [see, e.g. Genotlc Eivwoerlng of Plants, to Agricultural Perspective, A. Cashmore, Plenum, New York 1983, pages 29-38; Coruzzi, G., et al., The Journal of Qiologlcel Chemistry, 258: 1399 (1983) and Dunsmuir P., et al., Journal of Molecular and Applied Genetics, 2: 285 (1983)].

By using strong promoters ensures that even in the absence of the virus constantly small amounts of Protector protein can be synthesized so that in an acute viral infection a very fast first response to the Infection event is possible. A preferred gene construction in the context of the present invention is composed of the CaMV35-S promoter,

from a leader sequence, the CaMV reading frame VIi, a CaMV reading frame / protection factor merger, and a

Transcription terminator. Also preferred in the context of this invention is a gene construction consisting of the CaMV 19S promoter, a protective factor

coding DNA sequence in the ORF Vl of the CaMV genome and a CaMV termination sequence is composed.

The chimeric gene sequence consisting of one or more virus-specific control sequences and one or more Schutzfaktorgenen and which is operably linked to active in plant cells expression signals, can

be spliced into a suitable cloning vector. In general, one uses plasmid or virus (bacteriophage) vectors with replication and control sequences derived from species compatible with the host cell

The cloning vector usually carries a replicatin origin, as well as specific genes that lead to phenotypic selection characteristics in the transformed host cell, in particular resistance to antibiotics or to certain herbicides. The transformed vectors can be selected from these phenotypic markers after transformation in a host cell.

As host cells in the context of this invention, propkaryotes in question, including bacterial hosts such. B. A.tumefaclens, A. rhizogenes, E.coll, S. typhlmurium and Serratia marcetcens, as well as cyanobacteria. Further, eukaryotic hosts such as yeasts, mycelium-forming fungi and plant cells may also be used in the invention. The cloning vector and the host cell transformed with this vector are used according to the invention to increase the copy number of the vector. With an increased copy number, it is possible to isolate the vector carrying the r'as protection factor gene, and e.g. B. for the introduction of the chimeric gene sequence in the plant cell to ve 'ν end. The introduction of DNA into host cells can be performed by methods known per se. For example, bacterial host cells can be transformed after treatment with calcium chloride. In plant cells, DNA can be introduced by direct contact with the protoplasts produced from the cells. Another way to introduce DNA into plant cells is to bring the cells into contact with viruses or with Agrobacterium. This can be accomplished by infection of sensitive plant cells or by co-culturing of protoplasts derived from plant cells

 These procedures will be discussed in detail below.

There are a number of methods available for the direct introduction of DNA into plant cellon. For example, the genetic material contained in a vector can be microinjected directly into the plant cells by means of micropipettes for the mechanical transfer of the recombinant DNA. The genetic material may also be introduced into the protoplast with polyethylene glycol after treatment. (Paszkowski J et al., EMBO J., 3: 2717-22 [1984]).

Another object of the present invention relates to the introduction of the protective factor gene into the plant cell by means of electroporation (Shillito R et al .. Biotechnology, 3: 1099-1103 (1985); Romm M et al., Proc. Natl. Acad. Sci. USA, 82: 5824 (1985)).

In this technique, plant protoplasts are electroporated in the presence of plasmids containing the protection factor gene.

Electrical impulses of high field strength lead to a reversible increase in the permeability of biomembranes and thus facilitate the introduction of the plasmids. Electroporated herbal f'rotoplasts renew their cell wall, divide and form callus tissue. The selection of the transformed plant cells with the expressed protection factor can be carried out with the help of the previously described phenotypic markers.

The cauliflower mosaic virus (CaMV) can also be used as a vector for the introduction of the protective factor gene into the plant in the context of this invention (Hohn et al., In Molecular Biology of Plant Tumors, Academic Press, New York, 1982, p 549-560; Howell, U.S. Patent No. 4,407,956).

The entire viral DNA genome of CaMV is thereby integrated into a bacterial parent plasmid to form a recombinant DNA molecule that can be propagated in bacteria. After cloning, the recombinant plasmid is cleaved by restriction enzymes either randomly or at very specific nonessential sites within the viral portion of the recombinant plasmid, e.g. Within the gene coding for the transmissibility of the virus by aphids in order to be able to incorporate aprotective factor gene sequence.

A small oligonucleotide, a so-called linker, which has a unique, specific restriction recognition site can also be integrated. The thus modified recombinant plasmid is re-mated and further modified by splicing the protection factor gene sequence into a unique restriction site.

The modified viral portion of the recombinant plasmid is then excised from the parent bacterial plasmid and used for inoculation of the plant cells or the whole plants.

Another method for introducing the hybrid gene construction into the cell according to the invention involves infection of the plant cell with Agrobacterium tumefaclens or Agrobacterium rhizogenes, which is transformed with the protective factor gene in such a way that said protection factor gene blocks the tumor-producing genes between the T cells -DNA border sequences replaced. Another possibility in the context of this invention is a mixed infection using both A.rhlzogenes and transformed A.tumefaclens as described by Petit et al. (Petit et al., Mol. Gen. Genet. 202: 388, 1986) for the transformation of carrots. The mixing ratio must be coordinated so that the caused by the A. rhizogenes transformation Würzeichenkolonien also contain a high proportion of A.tumefaclens Ti plasmid. This can be achieved according to the invention by applying A. rhizogenes and A. tumefaciens in a mixing ratio of 1: 1 to 1: 100, but preferably 1:10 in a known manner together to the plant material. The transgenic plant cells are then cultured under suitable culturing conditions known to those skilled in the art, so that they form shoots and roots and ultimately result in whole planton.

The mixture of the two Agrobacteria species advantageously takes place shortly before the actual inoculation. The protection factor-encoding gene sequences can be determined, for example, using the Ti plasmid of Agrobacterium tumefaciens (DeCleene et al., Bot. Rev., 47: 147-194 (1981); Bot. Rev., 42: 389-466 (1976)). and the Ri plasmid of A. rhizogenes (M. Tepfer and FCasse-Delbart, Mlcroblol ScI., 4 (1): 24-28, 1987) into suitable plant cells. The Ti plasmid is transferred to the plant during the infection by Agrobacterium tumefaciens and stably integrated into the plant genome. (Horsch et al., Science, 233: 496-498 (1984); Fraley et al., Proc Natl Acad ScI, USA, 80: 4803 (1983)). For plants whose cells are susceptible to infection with Agrobacterium are not sensitive, one can resort to the co-cultivation of Agrobacterium with the corresponding protoplasts.

Ti plasmids have two regions that are essential for the production of transformed cells. One of them, the transfer DNA region, is transferred to the plant and leads to the induction of tumors. The other, the virulence-conferring (vir) region, is essential only for training, but not for the maintenance of tumors. The transfer DNA region can be increased in size by incorporation of the protection factor gene sequence without transference

is impaired. By removing the tumorigenic genes, leaving the transgenic plant cells non-tumorous and incorporating a selectable marker, the modified Ti plasmid can be used as a vector for the transfer of the gene design of the invention into a suitable plant cell.

The vir region effects the transfer of the T-DNA region of Agrobacterium to the genome of the plant cell, regardless of whether the T-DNA region and the Vir region are present on the same vector or on different vectors within the same Agrobacterium cell. A vir region on a cnomosome also induces the transfer of T-DNA from a vector into a plant cell.

Preferred is a system for transferring a T-DNA region from an Agrobacterium to plant cells, characterized in that the vir region and the T-DNA region are on different vectors. Such a system is known by the term "binary vector system" and the vector containing the T-DNA is referred to as a "binary vector". Any T-DNA containing vector that is transmissible in plant cells and that allows for selection of the transformed cells is suitable for use in this invention.

Any vector having a vir region which effects the transfer of a T-DNA region of Agrobacterium to plant cells may be used in this invention.

A further embodiment within the scope of the present invention relates to a method which combines the advantages of a virus-mediated transformation and the transformation with the aid of Agrobacterium. This process is described in European Patent Application No. 201904. In this case, viral DNA or parts of viral DNA, which may optionally contain incorporated cargo DNA, integrated in such a way in a T-replicon that incorporation of the viral DNA in the plant genome takes place.

The incorporation of the viral DNA into the T-replicon is advantageously carried out in the region of the transfer DNA region of the T-replicon, so that the viral DNA is flanked by at least one of the T-DNA border sequences, which are responsible for the integration of the transfer DNA into Plant genomes are essential.

Also in this case, the transfer of the T-DNA region of Agrobacterium into a plant cell can be done using a binary vector system.

Particularly preferred in the context of the present invention is a hybrid genetic construct comprising the complete CaMV 35S enhancer / promoter region, the leader sequence, the reading frame ORF VII, the small intergenomic region, 16 codons of the reading frame ORFI, 17 Kodo'.is from Linker and spacer sequences containing a Schutifactorgen coding region, spacer sequences, a CaMV polyadenylation site and sequences of the plasmid pUC 18 (Norrander J et al, Gene 26: 101-106, 1983).

Said hybrid genetic construction is accomplished by linking an Eco RV / Eco RI fragment of the CaMV strain CM4.184 (Dixon L et al., Virology, 150: 463-468, 1986) containing the 35S promoter region, the leader sequence, the ORFVII reading frame, the small intergenomic region, and the first 16 codons of the ORF I lysing frame, with the large Sma I / Eco RV fragment of the plasmid pDH 51 (Pietrzak M et al, Nucl. Acids Res., 14: 5857-5868, 1986). A DNA fragment encoding a protective factor can then be spliced into the polylinkore region of the resulting plasmid by methods known per se, to form the hybrid genetic construct characterized in more detail above.

Another genetic design preferred in this invention utilizes a DNA fragment containing the 1S promoter of Cauliflower mosaic virus.

This DNA fragment is cloned together with the CaMV transcriptional terminator. The reading frame of a structural gene, preferably a protective factor-encoding structural gene, is inserted into the resulting plasmid between these regulatory CaM V sequences in such a way that translation of said structural gene begins directly at the start codon of the gene V protein.

To achieve this, it is advantageous to carry out a deletion mutagenesis with the aid of an oligonucleotide. It is based on single-stranded DNA, which includes the corresponding part of the CaMV genome and which can be prepared with H if θ of M13 helper phage.

The resulting single stranded circular DNA can be used as a substrate for in vitro deletion using oligonucleotides.

In an analogous manner to the previously described construction of plant-active expression vectors using the CaMV rolling systems, the non-control system characteristic of strand-positive (+) RNA viruses and previously described using the example of the BMV virus can be used for vector construction.

As already mentioned above, in the case of strand-positive (+) RNA viruses, the production of subgenomic mRNA proceeds from minus (-) strand RNA. Ini BMV system will initiate the production of a subgenomic RNA 4 encoding a coat protein, presumably by a partial transcription of the minus (-) strand RNA3, involving viral replicases. Through the production and cloning of complementary cDNA, it is possible to use the recombinant DNA techniques previously discussed in detail for a genetic manipulation of RNA viruses. Analogously to the previously discussed CaMV system, therefore, in the case of strand-positive (+) RNA viruses, an exchange of the sections coding for a viral protein (in the case of BMV of the coat protein coding section on the RNA 3) with a Schutzfaktorgen be made.

It is possible to splice in this hybrid gene construction between expression signals active in plant cells in such a way that, in addition to the protective factor gene, the sections which control the original viral gene remain in functional form.

In BMV, these are the DNA portions located upstream of the ORF of the original coat protein gene (French et al., Science, 231: 1294-1297, 1986).

Plant cells or plants transformed in accordance with the present invention may be incorporated by means of a suitable phenotypic marker, in addition to the protection factor gene and the virus-specific control sequences

the DNA is to be selected. Examples of such phenotypic markers, but are not intended to be limiting, include antibiotic resistance markers, such as kanamycin resistance and hygromycin resistance genes, or herbicide resistance markers. Other phenotypic markers are known to those skilled in the art and may also be used within the scope of this invention.

All plants whose cells are characterized by direct introduction of DNA by using viral or bacterial vectors, such as

z. B. CaMV or Agrobacterlum or other suitable vectors can be transformed and then regenerated into whole plants can be subjected to the inventive method for producing transgenic whole plants containing the transmitted protective factor gene. There is an ever increasing body of evidence that, in principle, all plants can be regenerated from cultured cells or tissues, including, but not limited to, all major cereals, sugarcane, sugarbeet, cotton, fruit trees and other tree species, legumes, and so on vegetables.

The inventive method is suitable for the transformation of all plants, in particular those of the systematic groups Angiospermae and Gymnospermae.

Among the gymnospermae the plants of the class Coniferae are of special interest.

Among the angiosperms are in addition to deciduous trees and shrubs of particular interest plants of the families Solanaceae, Cruciferae, Compositae, Liliaceae, Vitaceae, Chenopodiaceae, Rutaceae, Alliaceae, Amaryllldaceae, Asparagaceae, Orchidaceae, Palmae, Bromeliaceae, Rubiaceae, Theaceae, Musaceae or Gramineae and the Order Leguminosae and especially the family Papilionaceae. Preferred are representatives of Solanaceae, Cruciferae, Leguminosae and Gramineae.

Among the target cultures in the present invention include, for example, those selected from the series: Fragaria, Lotus, Medicago, Onobrychls, Trlfolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arebldopsls, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Cichorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Hemerocallis, Nemesis, Pelargonium, Panlcum, Pennisetum, Ranunculus, Senecio, Salplglossis, Cucumis, Browallia, Glycine, Gossypium, Lolium, Zea, Triticum and Sorghum, including those selected from the series: Ipomoea, Passiflora, Cyclamen, Malus, Prunus, Rosa, Rubus, Populus, Snntalum, Allium, Lilium, Narcissus, Pineapple, Arachis, Phaseolus and Pisum.

This exemplary list is only intended to illustrate the present invention and must not be construed as limiting the scope of the subject invention.

By using newly developed transformation techniques, it is now also possible to transform in vitro those plant species which are not natural host plants for Agrobacterium. For example, monocotyledonous plants, especially the cereals and grasses, are not natural hosts for Agrobacterium.

In the meantime, there is increasing evidence that monocotyledons can also be transformed with Agrobacterium so that, using new experimental approaches that are now becoming available, even cereals and Giaser species are susceptible to transformation (Grimsley N, et al. Nature, 325: 177-179 (19871).

Regeneration of cultured protoplasts to whole plants is described in Evcis, et al., Protoplast Isolation and Culture, Handbook of Plant Cell Culture, 1: 124-176 (MacMiane Publishing Co., New York, 1983); MR Davey, Recent Developments in the Culture and Regeneration of Plant Protoplasts, Protoplasts, 1983 Lecture Proceedings, pages 19-29, (Birkhäuser, Basel 1983); PJ Dale, "Protoplast Culture and Plant Regeneration of Cereals and Other Recalcitrant Crops," in Protoplast's 1983 Lecture Proceedings, pages 31-41, (Birkhäuser, Basel 1983); and H Binding, "Regeneration of Plants," in Plant Protoplasts, Pages 21-37, (CRC Press, Boca Raton 1985).

The regeneration differs from plant species to plant species. In general, however, a suspension of transformed protoplasts, cells, or tissues containing numerous copies of the protection factor genes is first prepared. On the basis of these suspensions, the induction of embryo formation can subsequently take place. The development of the embryos is allowed to proceed to the stage of roife and germination, as is the case with naturally occurring embryos. The culture media usually contain various amino acids and hormones, such as

z. Auxin and cytokinins. It also proves to be advantageous to add glutamic acid and proline to the medium, especially in species such as maize and alfalfa. Sprouting and rooting generally occur simultaneously. Efficient regeneration depends primarily on the medium, the genotype and the history of the culture. If these three variables are adequately controlled, the regeneration is completely reproducible and repeatable.

Due to recent developments in the field of in vitro cultivation of plants, mainly in the field of plant regeneration, it is now possible to regenerate whole plants, including representatives of the family Gramineae, starting from plant protoplasts. Examples of successful regeneration experiments with gramineae are i.a. in Abdullah, R., et al., Bio / Technology, 4: 1087-1090, 1986, Fujimura T., et al., Plant Tissue Culture Lett, 2:

74-75, 1985, Toriyama, K., et al., Theor Appl Genet, 73: 16-19, 1986, Yamada, Y., et al., Plant Cell Rep, 5: 85-88, 1986 (Rice). and in Rhodes et al. for corn protoplasts (Biotechnology, 6: 56-69, 1988).

Therefore, in the context of this invention, representatives of the family of Gramineae are particularly preferred, such. B. plants that are grown over a large area and provide high yields. Examples include corn, rice, wheat, barley, rye, oats, millet or sorghum and pasture grasses.

Further target crops which are particularly preferred for use in the method according to the invention are therefore, for example, plants of the genera Allium, Avena, Hordeum, Oryza, Panicum, Saccharum, Seeale, Setaria, Sorghum, Triticum, Zea, Musa, Cocos, Phoenix and Elaels.

The mature plants grown from transformed plant cells are crossed with themselves for semen production. Some of the seeds contain the genes that are responsible for increasing the effect of shearing, in a proportion that obeys the established laws of inheritance. These seeds can be used to produce virus-resistant plants. The degree of virus resistance of the transformed plants can be achieved by artificial infection mi!

determine pathogenic virus strains.

Homozygous lines can be obtained by repeated self-fertilization and inbred line production. These inbred lines can in turn be used to develop virus-resistant hybrids. In this method, a virus-resistant inbred line is crossed with another inbred line to produce virus-resistant hybrids.

Parts of dio regenerated plants can be obtained, such. B. flowers, seeds, leaves, branches, fruits u. a., Are also part of the present invention, provided that these parts include virus-resistant cells. Progeny (including hybrid progeny), varieties and mutants of the regenerated plants are also part of this invention.

For purposes of illustrating the more general description and understanding of the present invention, reference will now be made to specific embodiments, which are not limiting, unless specifically noted.

Non-explanatory embodiments:

General recombinant DNA techniques

As many of the recombinant DNA techniques used in this invention are routine to those skilled in the art, a brief description of these commonly used techniques is included rather than where they appear. Unless otherwise noted, all of these methods are in the reference of Maniatis et al. (1982).

A. Cutting with restriction endonucleases

Typically, about 50 to 500 pg / ml of DNA is included in the reaction mixture in the buffer solution recommended by the manufacturer, New England Biolabs, Beverly, MA. 2 to 5 units of restriction endonucleases are added for each pg of DNA and the reaction is incubated at the manufacturer's recommended temperature for one to three hours. The reaction is terminated by heating at 65 ° C for 10 minutes or by extraction with phenol, followed by precipitation of the DNA with ethanol. This technique is also described on pages 104-106 of the Maniatis et al. Referent.

B. Treatment of the DNA with polymerase to produce blunt ends

50 to 500 μg / ml DNA fragments are added to a reaction mixture in the buffer recommended by the manufacturer, New England Bioblabs. The reaction contains all four deoxynucleotide triphosphates in concentrations of 0.2 mM. The reaction takes place for 30 minutes at 15 ° C and is then terminated by heating at 65 ⁰ C lOminütiges. For fragments obtained by cleavage with restriction endonucleases which produce 5 'protruding ends, such as EcoRI and BamHI, the large fragment or Klenow fragment of the DNA polymerase is used. For fragments obtained by endonucleases which produce 3 'protruding ends, such as PstI and SacI, T4 DNA polymerase is used. The use of these two enzymes is described on pages 113 to 121 of the Maniatis et al. Reference.

C. Agarose gel electrophoresis and purification of DNA fragments from gels

Agarose gel electrophoresis is performed in a horizontal apparatus as described on pages 150-163 of the Maniatis et al. Reference. The buffer used is the Tris-borate buffer described therein. The DNA fragments are stained by 0.5 μg / ml ethidium bromide, which is either present in the gel or tank buffer during electrophoresis or added after electrophoresis. The DNA is visualized by illumination with long-wave ultraviolet light. When the fragments are not to be separated from the gel, an agarose which can be gelled at low temperature and purchased from Sigma Chemical, St. Louis, Missouri is used. After the electrophoresis, the desired fragment is excised, placed in a plastic tube, heated to 65 ^° C. for about 15 minutes, extracted three times with phenol and precipitated twice with ethanol. This method is opposite to that of Maniatis et al. on page 170 slightly changed. Alternatively, the DNA can be isolated from the agarose using the Geneclean kit (Bio 101 Inc., La JoIIa, CA).

D. Adding Synthetic Linker Fragments to DNA Ends

If it is desired to add a new endonuclease cleavage site to the end of a DNA molecule, the molecule may be first treated with DNA polymerase to create blunt ends as described in the section above. About 0.1 to 1 g of this fragment is added to approximately 100 μg of phosphorylated linker DNA obtained from New England Biolabs in a volume of 20 to 30 μM with 2 μM T4 DNA IgG from New England Biolabs, and 1 mM ATP in the Manufacturer recommended buffers. After an overnight incubation at 15 ⁰ C, the reaction by lOminütiges heating at 65 ⁰ C is terminated. The reaction is diluted to about 100 μΙ in a buffer appropriate for the restriction endonuclease that cuts the synthetic linker sequence. Approximately 50 to 200 units of this endonuclease are added. The mixture is incubated for 2 to 6 hours at the appropriate temperature, then the fragment is subjected to agarose gel electrophoresis and purified as described above. The resulting fragment will now have ends with endings generated by restriction endonuclease cleavage. These ends are usually cohesive so that the resulting fragment can now be easily attached to other fragments having the same cohesive ends.

E. Removal of o'-teminal phosphates from DNA fragments

During the plasmid cloning steps, treatment of the vector plasmid with phosphatase reduces the recircularization of the vector (discussed on page 13 of the Maniatis et al. Reference). After cleaving the DNA with the correct restriction endonuclease, one unit of calf alkaline phosphatase is added from calves purchased from Boehringer-Mannheim, Mannheim. The DNA is incubated for one hour at 37 ⁰ C and then extracted twice with phenol and precipitated with ethanol.

F. Linking the DNA fragments

When fragments with complementary cohesive ends are to be linked together, approximately 100 ng of each fragment in a 20-40 μM reaction mixture is incubated with approximately 0.2 unit T4 DNA ligase from New England Biolabs in the manufacturer's recommended buffer. The incubation is carried out at 15 ° C for 1 to 20 hours. When DNA fragments are to be blunt-ended, they are incubated as above, except that the amount of T4 DNA ligase is increased to 2 to 4 units.

G. Transformation of DNA In E. coll

E. coll strain HB101 is used for most experiments. DNA is introduced into E. coli by the calcium chloride method as described by Maniatis et al., Pages 250-251.

H. Screening E. coli for plasmids

After transformation, the resulting E. coli colonies are checked for the presence of the desired plasmid by a rapid plasmid isolation procedure. Two common methods are described on pages 366 to 369 of the Maniatis et al. Reference.

I. Isolation of Plasmid DK'A on a Large Scale

Methods for large scale isolation of plasmids from E. coli are described on pages 88-94 of Maniatis et al., Reference.

J. Cloning In M 13 Phage Vectors

The following description is to be understood as using the double-stranded replicative form of phage M 13 derivatives for routine procedures such as restriction endonuclease cutting, linking, etc.

Example 1: Transactivation of a CAT gene in tobacco protoplasts

1.1. Construction of the plasmid pHS1

The EcoRV / EcoRI fragment of the CaMV strain CM4.184 (Dixon, L., et al., Virclogy, 150: 463-468, 1986) containing the 35S promoter region, the leader sequence, ORFVII, the small intergenomic region and the first 16 codons of the ORFI reading frame is isolated from a polyacrylamide gel and subsequently treated to supplement the EcoRI cohesive ends with the Klenow fragment of DNA polymerase (abbreviated "Kleno.v").

This EcoRV / EcoRI fragment is ligated to the large Smal / EcoRV fragment of the plasmid pDH51 described in Pietrzak et al., 1986 (Pietrzak, M., et al., Nucl. Adds Res. 14: 5857-5868, 1986). is linked (Figure 1 A). This construct is described in the E.

coli strain DH1 cloned. Subsequently, the restriction analysis identifies those transformants in which the EcoRV restriction site is restored, while the SmaI site is missing and which restriction fragments have a corresponding size (FIG. 1B).

The resulting plasmid is then cut in the region of the polylinker region with XbaI and the cohesive ends are supplemented using the "Klenow." A Hind III fragment carrying the bacterial chloramphenicol transacetylase (CAT) coding region (Alton, NK et Vapnek , D., Nature, 282: 864-869, 1979), is isolated, and the cohesive ends are filled in with "Klenow".

The supplemented HindIII and XbaI fragments are then linked together and used for the transformation of E. coli strain DH1. The resulting transformants having a restored XbaI restriction site and having the correct orientation of the insert are selected. Thus, the appropriate genetic construct will have the complete CaMVSSS enhancer / promoter region, the leader sequence, the ORFVII reading frame, the small intergenomic region, 16 ORFI reading frame codes, 17 codon of linker and spacer sequences, the coding sequence of the bacterial CAT gene Spacer sequences, a CaMV polyadenylation site and sequences of the plasmid pUC 18 (Norrander, J. et al., Gene 26: 101-106, 1983).

The ORFI, spacer and CAT codons together form a fused reading frame. The resulting plasmid confers a CAT activity to transformed plant protoplasts that is 10% -20% compared to the control plasmid pDWÜ (Pietrzak et al., 1986, supra).

1.2. Transformation of tobacco protoplasts

Tobacco protoplasts of Nicotiana tabacum c. v. Petite Havana are prepared by conventional procedures from a tobacco suspension culture (Potrykus, I. and Shillito, RD, Methods in Enzymology, Vo1118, Plant Molecular Biology, eds. A. and H., Weissbach, Academic Press, Orlando, 1986). Fully unfolded leaves of 6 week old shoot cultures are removed under sterile conditions and thoroughly wetted with an enzyme solution of the following composition.

Enzyme solution: H2O 70 ml sucrose 13g Macerozyme R 10 ig cellulase 2g "Onozuka" R10 (Yakult Co. Ltd., Japan) Drisellase (Chemical Factory Schweizerhalle, Switzerland) 0.13 g 2 (n-morpholine) -ethanesulfonic acid (MES) 0.5 ml pH 6.0

Leaves are then cut into 1-2 cm squares and allowed to float on the above enzyme solution. The incubation takes place overnight at a temperature of 26 ⁰ C in the dark. This mixture is then gently stirred and incubated for a further 30 minutes until complete digestion. .

This suspension is then filtered through a 100 mm mesh steel egg, rinsed with 0.6 M sucrose (MES, pH 5.6) and then centrifuged for 10 minutes at 4000-5000 rpm. The protoplasts collect on the surface of the medium, which is then stripped off under the protoplasts, e.g. B. using a sterilized syringe. The protoplasts are in a K3A medium (sucrose [102.96 g / l], xylose [0.25 g / l], 2.4D [0.10 mg]; NAA (1,00mg / l); BAP (0.20 mg / l, pH 5.8) containing 0.4 M sucrose.

The proioplasts are then washed by flotation of the protoplasts and removal of the medium in the manner previously described (3x).

The transformation of the protoplasts with plasmid DNA takes place by means of direct gene transfer (Paszkowski, J. et al., EMBO J., 3: 2717-2722, 1984) using the electroporation technique (Fromm, M. et al., Proc. Natl. Acad. Sei, USA, 82: 5824-5829,

The electroporation of the tobacco protoplasts is carried out in a disposable semi-microcuvette (Greiner, Nuremberg) designed with aluminum foil.

In said cuvette, the tobacco protoplasts are grown at a population density of 2 × 10 ⁶ protoplasts / ml in 0.7 ml electroporation buffer (Fromm et al., 1985, supra) of the following composition:

final concentration

10mMHepespH7,2

- 15MMNaCI

5mM CaCl ₂

- 0.2M mannitol

incubated with 15pg of plasmid DNA under sterile conditions.

Electroporation of the protoplasts is accomplished by discharging a capacity of 82OpF previously charged at 220V (Fromm et al., 1985, supra).

After an incubation period of 10min. at 4 ° C and subsequent incubation for a further 10 min at room temperature, the treated protoplasts are diluted with 10 ml AA medium of the following composition:

AA MediumfüMI 10ml 37 g / l MgSO ₄ 20 ml - 17 g / l KH ₂ PO ₄ 10ml - 44 g / l CaCl ₂ 2.95 g - KCI 5ml - ( ¹ MS) EDTA 5ml - (MS) FeCI ₃ 1ml - (MSorNT) microelements 1ml - 1 00Ox vitamins (see below) 10ml - 100x amino acids (see below) 10ml - 100x micro sugar (see below) 63 g - sorbitol 5 ml - ² 2,4-D, 20 mg / 100 ml 1ml - Kinetin, 20mg / 100ml 0.5 ml - ³ GA3,20mg / 100ml - pH 5.8-6 (NaOH). 530-57OmOs osmotic pressure 1000X Vitamins (100 ml): 2g - inositol 50 mg - nicotinic acid 40 mg - thiamine 10mg - Pyridoxine 1ml - MESiMpH7 Make up to 100 ml with H ₂ O 100X AmlnosSure mix: 43.8 g - glutamic acid 13.3 g - asparagine 8.7 g - Arginine HCl 0.375 g - Glycine - pH about 6 Make up to 500 ml with H ₂ O 100X micro sugar mix: 1.5g - xylose 1.5g - arabinose 1.8g - glucose 0.8 g - inositol Make up to 100 ml with H ₂ O

1 MS = Murashige and Shook Medium (Murashige, T. and Skoog, F., Physlol. Plant, 15: 473, 1962).

2 2,4-0 2,4-dichlorophenoxyacetic acid.

3 GA3gibberellic acid.

The various solutions are sterilized by autoclaving (amino acid mix) or filter sterilization. This is followed by a 2-day incubation of the protoplasts at 28 ⁰ C in the dark. The cells are then used according to the method described by Fromm et

al., 1985, supra, and ultrasonically lysed and extracted.

1.3. CAT assay

The extract thus obtained is then analyzed by thin layer chromatography for its ability to acetylate radioactive chloramphenicol (CAT activity, Gormann, M. et al., Mol. Cell. Biol., 2: 1044-1051, 1982).

1.4. Results:

The results of the CAT assay indicate that radioactively labeled protoplasts are present in the protoplasts treated with the plasmid pDW2 Acetylchloramphenicol is detectable in the chromatogram, that is, that the protoplasts include a CAT activity. In contrast, no activity was detected in cells treated with the plasmid pHS1. If the electroporation mixture containing tobacco protoplasts and the plasmid pHS 1, in addition to 15pg CaMV DNA

the pHS 1-treated cells also show CAT activity.

However, control experiments using calf thymus DNA performed in the same way yielded negative results Results (no CAT activity). These results thus clearly show that expression in the position of the CAT reading frame in the plasmid pHS 1 is only in Virus DNA is present and thus induction of CAT activity by use of viral DNA is possible Instead of the CAT gene in the plasmid pHS 1, any gene suitable in the context of the present invention, such as

For example, a Schutzfaktorgen be used.

Example 2 Transactivation of a CAT Gene In Brasslca napus Plants

2.1. Construction of the plasmid pCIB200

TJS75kan is prepared by digesting the plasmid pTJS75 (Schmidhauser and Helinski, J. Bacteriol., 164: 446-455, 1985) with the restriction enzyme Narl to obtain the tetracycline resistance gene and then inserting an Accl fragment of pUC4K (Vierra and Messing, Gene, 19: 259-268, 1982) carrying the NptI gene. pCIB200 is then generated by linking Xhol linkers to the EcoRV fragment of pCIB7 (this fragment contains the left and right T-DNA border sequence, a chimeric nos / nptII gene and the pUC polylinker region (Rothstein, SJ et al. Gene, 53: 153-161, 1987]) and cloning the Xhol digested fragment into the Sall site of TJS75kan.

2.2. Preparation of the cointegrated vector pCIB200 / HS1

The plasmid pCIB200, whose construction is described in Example 2.1, and plasmid pHS 1 from Example 1.1 are described as Starting material for the preparation of the cointegrated vector pCIB200 / HS 1 used. Both parent plasmids are first cut with the restriction enzyme Kpnl, together in one of the usual

incubation buffer used mixed and then linked together with the addition of suitable enzymes (ligases).

Subsequently, E. coll HB 101 cells are treated with this mixture according to the method described in Maniatis et al., 1982 (cf. Maniatis et al, pp. 250-251) and selection of ampicillin / kanamycin resistant E. coli colonies. Resistant colonies are screened for the presence of the desired cointegrated plasmid by restriction analysis

pCIB200 / HS 1 by decomposing it back into the linear forms of the parent plasmids by Kpnl.

2.3. Conversion of the cointegrated vector pCIB200 / HS 1 into Agrobacterium tumefaclens by "trlparnntal mating"

The starting material (parent) for triparental mating is E. coli HB101 (pRK2013) (described by Tepfer and Casse-Delbart, Microbiol. Sci., 4: 24,1987), E. coli HB101 (pCIB 200 / HS1), containing the cointegration vector described above

pCIB200 / HS1 and Agrobacterium tumefaciens C58 (pCIB542).

The plasmid pCIB542 is an apathogenic helper plasmid whose construction in the European Patent application EP 256223 is described in detail and in the form of a reference is a part of the present invention The crossing of the 3 parental strains and thus the transfer of the cointegrated plasmid pCIB 200 / HS 1 into Agrobacterium

is carried out according to the method described by Tepfer and Casse-Delbart, 1987.

The agrobacteria resulting from this "triparental mating" are designated C58 (pCIB 542: pCIB 200 / HS 1)

Thus, in addition to the helper plasmid pCIB542, the cointegrated plasmid pCIB 200 / HS 1 is also available.

2.4. Production of transgenic Brasslca napus plants

Petioles of Brasslca napus are isolated according to the method described by Guerche et al., Mol. Genet., 206: 382-386, 1987. Instead of the Agrobacterium strains strains used there, the transformation of Brassica napus in this case takes place with the aid of a mixture of Agrobacterium rhuczogenes and Agrobacterium tumefaclens C58 (pCIB542: pCIB200 / HS1), the preparation of which in Example 2.3. is described. The mixing ratio of A.rhlzngenes and A. tumefaclens C58 (pCIB542: pCIB200 / HS1) is preferably 9: 1.

2.4.1. Culturing the Agrobacterium strains and preparing the inoculation solution Before inoculation, the Agrobacterium strains are seeded on YEB medium (Bacto beef extract: 5 g / L, Bacto yeast extract 1 g / L, Peptone 5 g / I, sucrose 5 g / L, MgSO 2, 2 mM, pH 7.2), which was previously enriched with 100 μg / ml ampicillin and 25 μg / ml kanamycin and solidified with 1.5% agar. After culturing for 48 hours at a temperature of 28 ^° C., a single colony is used to inoculate a liquid culture. This is carried out in 100 ml Erlenmeyer flasks in a liquid YEB medium enriched with antibiotics in the concentration indicated above. The cultivation takes place at a temperature of 28 ^° C. on a shaking machine at a speed of 200 rpm. The cultivation time is 24 hours.

Subsequently, a second subculturing is carried out in liquid medium with a dilution ratio of 1: 2Q under otherwise identical conditions. The incubation period is 20h in this case.

These measures lead to a population density of living Agrobacteria of about 10 ⁹ / ml.

The bacterial cells are harvested by centrifugation and then resuspended in an equivalent volume of 10 mM MgSO ₄ solution containing no antibiotics.

2.4.2. Inoculation of Brasslca napus leaf trunks

Leaf stems of Brasslca napus are first surface sterilized for 10 min in a calcium hypochlorite solution (70 g / l) and rinsed with distilled water. These are then cut into small pieces 6-10 mm thick and transferred to Petri dishes. These contain sterile tap water or a saline solution according to Monnier (Monnier, Revue des Cytologie et de Biologie Vogetales, tome 31: 78,1976), which is solidified by the addition of 0.7% agar.

For inoculation, well grown Agrobacterium cultures are used which have an OD ₆ S ₀ of about 1.00 (this corresponds to a number of about 10 ⁹ cells / ml).

For the mixed inoculation of Agrobacterium rhizogenes and Agrobacterium tumefaclens, the bacteria are first cultured separately until the required cell density is reached and mixed immediately before the intended inoculation in a ratio of 9: 1 (A. rhigogenes: A. tumefaclens).

After inoculation, the Petri dishes are well sealed and incubated for about 2 to 3 weeks at room temperature.

A successful transformation with A. rhizogenes can then be easily recognized by the appearance of hairy radicles.

The transformation by A. tumefaclens, however, can only be detected by the acquired kanamycin resistance.

At the mixing ratio of 9: 1 used here contain up to 95% of the Würzeichenkolonien one

A. tumefaclens transformation caused kanamycin resistance.

2.5. Regeneration of whole plants

The regeneration of whole plants from root colonies is carried out by known methods, as described, for example, in David et al., Biotechnology, 1: 73,1982, Tepfer and Casse-Delbart, 1987 or in Guerche et al., Mol. Genet., 206: 382-386 1987.

Root tissue is transferred to a Monnier medium containing 0.36μΜ 2,4 D and 0.72 μΜ kinetin as additive and solidified with 0.8% agar.

After 4 weeks of culture, the resulting callus tissue is transferred to a liquid, hormone-free medium to Monnier and incubated on a rotary shaker at 150Upm and a temperature of 22 ⁰ C to 25 ⁰ C.

The callus dissolves and forms a suspension culture, from which differentiate embryos after about 1 month. These embryos are further cultured in Petri dishes on hormone-free Monnier medium, where they further differentiate into small plants.

2.6. Transactivation of Transgenic Plants by CaMV Infection

Transgenic Brasslca napus plants are infected according to Lebeurier et al., 1980 with cauliflower mosaic virus.

The CAT activity before and after the CaMV infection is determined in leaf extracts of transgenic Brasslca napus plants according to the method described by Fromm et al., 1982.

Firstly, leaf material (50 mg to 100 mg) is comminuted in an extraction buffer (10% v / v glycerol, 0.01% w / v sodium dodecyl sulfate, 5% v / v mercaptoethanol, 0.005% bromophenol blue, 0.0625M Tris pH6.8) , The particulate part of the extract thus obtained is removed by means of centrifugation.

The supernatant is heated at a temperature of 65 ⁰ C for 10 min (inactivation of plant substances that affect the CAT activity), then cooled to room temperature and in the point 1.3. described CAT assay used. The plants transformed with the HS 1 construct show an increased activatability of the CAT gene, which can be up to 50 times compared to the controls.

Example 3: Transactivation of CaMV 19S transcripts

3.1. Construction of p19SCAT

The vector used is the plasmid pDH 19 ¹ J obtained from the CaMV35 S promoter-terminator cassette from the plasmid pDH 51 (Pietrzak, M. et al., Nucl. Adds Res., 14: 5857-5868, 1986). and the plasmid pTZ19u (Mead, D. Α., et al., B., Prot. Engineering, 1:

67-74, 1986). For this, pDH 51 is digested with EcroRI, the resulting fragments are treated with T4 DNA polymerase and the CaMV35S promoter-terminator fragment is cloned into the large Pvull fragment of pTZ 19u.

After digestion of pDH 19u with Ncol and SmaI, the overhanging NcoI end is filled in using Klenow DNA polymerase and the large fragment isolated The CaMV 19S promoter fragment becomes the genomic region from position 4875 to position 5355 of CaMV (Strain CM 4.184, Dixon, L. et al., Virology, 150: 463-468, 1986) as a MnII fragment and linked to the large NcoI / Smal fragment of pDH 19u to give the plasmid p19S, containing the CaMV19S promoter, polylinker sequences and the CaMV transcriptional terminator.

Plasmid ρ 19S is opened with BamHI and Sail and ligated with the small BamHI / SalI CAT fragment from plasmid pZL811 (Wong,

D.T. et al., J. Virol., 55: 223-231, 1985). In the last step, the ATG Starkodon the CAT reading frame is fused by deletion mutagenesis using an oligonucleotide with the ATG start codon of the CaMV protein Vl. The transactivator plasmid used was plasmid V374 (see Figure 4). It contains the EcoRV fragment from CaMV (strain CM4.184) containing the gene Vl cloned into the Smal restriction site of plasmid pDH51.

3.2. Transformation of Brassica rapa protoplasts

Turgescent leaves of Brassica rapa are first surface sterilized by placing in a 0.5% calcium hypochlorite solution for 30 minutes. Subsequently, the leaves are washed with sterile water for 1 to 2 minutes (5x). The leaves treated in this way are cut into coarse pieces and introduced into an enzyme solution, where they are cut into strips about 1 mm wide. The incubation takes place overnight in the dark at a temperature of 26 ^° C.

Enzyme solution: mannitol 6.15 g (100 ml) CaCl ₂ 0.63 g cellulose 1.0 g "Onozuka" R10 (Yakult Co., Ltd., Japan) MacerozymeFMO 0.01 pH 5.4

After the overnight incubation, the enzyme solution containing protoplasts and leaf residues is placed on a 50% steel strainer and filtered. The protoplasts separated in this way are transferred into centrifuge tubes and centrifuged for 10 min at 4000-5000 rpm. The supernatant (enzyme solution) is discarded and the pelleted protoplasts are resumed in 10 ml electroporation buffer. The whole process is repeated again and the protoplast suspension adjusted to 3 × 10 ⁶ / ml.

The electroporation of the Brassica rapa protoplasts is carried out in a manner analogous to that in Example 1.2. For tobacco protoplasts described method, but in this case, the charging of the capacitors with 180 V.

Incubation of the electroporated Brassica protoplasts is carried out in Α-medium (see below) in place of the AA medium used for tobacco protoplasts.

A Medium1l: 100ml A macro 1 ml A micro = B 5 m icro 5 ml MS iron ever 10ml A vita ύ, B 5 vita 25ml AOS 80 g / l glucose 250mg / l xylose 500mg / l casein hydrolysates (= N-Z-amines) 1 mg / l <2,4 D 0.1 mg / l ⁶ N.AA 0.5 mg / l ⁶ 6BAP pH 5.7-5.8 Stock solutions (A-Medium): A macro (per 100ml): 75 mg NaH ₂ PO ₄ -I 170 mg KH ₂ PO ₄ 2 200 mg KNO ₃ 600 mg NH ₄ NO ₃ 75 mg (NH ₄ J ₂ SO ₄ 310 mg MgSO ₄ -7 295 mg CaCl ₂ -2 A micro (per 100 ml): 1000 mg _MnSO.sub.4 -I 25 mg Na ₂ MoO ₄ -2 300 mg H ₃ BO ₃ 200 mg ZnSO ₄ -7 2.5 mg CuSO ₄ -5 2.5 mg CoCI ₂ -6 75 mg KJ A vita (per 100ml): 1000 mg inositol 10mg nicotinic acid 10mg Pyridoxine HCI 100 mg Thiamine HCI

4 2,4 D 2,4-dichlorophenoxyacetic acid.

5 NAANaphthyl-1-acetic acid.

6 eBAPe-Benzylaminopurine.

A OS (per 100ml): 20 mg sodium pyruvate 40 mg citric acid 40mg Aepelsäure 40 mg fumaric acid

(adjust pH to 5.5 with NH ₄ OH)

3.3. Results

Induced CAT activity is measured 20 hours after electroporation using the methods described in Section 1.3. described CAT assays. The analysis shows that electroporation with ρ19SCAT only leads to a very low CAT expression. However, when a plasmid is electroporated together with ρ 19 SCAT into the protoplasts from which the CaMV protein Vl can be expressed, then an approximately 20-fold stuttering of the CAT activity can be observed.

Claims (50)

1. A method for protecting plants from a viral infection and / or their consequences, characterized in that introducing into said plants by means of genetic engineering methods an inducible virus resistance. 2. The method according to claim 1, characterized in that one introduces an inducible protective factor gene in the plant, which is caused in an incipient virus infection by the addition of suitable inducers and / or by the infecting virus itself for the expression of said protection factor. 3. The method according to claim 2, characterized in that one combines the Schutzfaktorgen with virus-specific control sequences as well as active in plant cells expression signals in an operable manner, so that the induction of Schutzfaktorgens and thus the production of the protection factor is controlled by the infecting virus itself. 4. The method according to claim 3, characterized in that the virus-specific induction of Schutzfaktorgens takes place via transactivation of the transcription. 5. The method according to claim 4, characterized in that the virus-specific induction of Schutzfaktorgens takes place on the posttranscriptional level. 6. The method according to claim 5, characterized in that the virus-specific induction of Schutzfaktorgens via a) control of mRNA extension, b) a control of the mRNA transport from the nucleus to the cytoplasm, c) a control of mRNA splicing, d) a control of mRNA polyadenylation, e) a control of the translation initiation or f) a differentiated use of different ORFs takes place on a polycistronic RNA. 7. The method according to claim 3, characterized in that the virus-specific induction of Schutzfaktorgens done by post-translational modification of the protein product. 8. The method according to any one of claims 2 or 3, characterized in that the inducible Schutzfaktorgen operably linked to expression signals that are active in the plant cell and ensure the expression of said Schutzfaktorgens in the plant. 9. The method according to claim 2, characterized in that said Schutzfaktorgen is a naturally occurring, encoding a protective factor DNA sequence. 10. The method according to claim 9, characterized in that said DNA sequence encodes a protection factor that differs from a naturally occurring protection factor, but still fulfills essentially the same protective function. 11. The method according to claim 9, characterized in that said protection factor is an antiviral substance. 12. The method according to claim 9, characterized in that said protection factor is a cytotoxic substance. 13. The method according to claim 11, characterized in that said antiviral substance is an antiviral antibody, a P _n protein, a virus-specific protease inhibitor, a protease, a virus-specific polymerase inhibitor or an interferon-like protein. 14. The method according to claim 12, characterized in that said cell-toxic substance is a toxin, an active subunit of a toxin, a PR protein or a protein, which naturally causes a hypersensitive reaction. 15. The method according to claim 14, characterized in that said cell-toxic substance is a ribosome-inactivating protein. 16. A recombinant DNA molecule, characterized in that it transmits an inducible virus resistance to plants. 17. A recombinant DNA molecule according to claim 16, characterized in that it has a DNA sequence coding for an inducible protective factor. 18. A recombinant DNA molecule according to claim 17, characterized in that said protective factor encoding DNA sequence consists exclusively of genomic DNA, cDNA or synthetic DNA or a mixture of said DNA. 19. A recombinant DNA molecule according to claim 17, characterized in that said protective factor-encoding DNA sequence is operably linked to a control region which ensures induction of the protective factor gene. 20. A recombinant DNA molecule according to claim 19, characterized in that said control region is a virus-specific control region. 21. A recombinant DNA molecule according to claim 19, characterized in that said inducible protective factor-encoding DNA sequence is operably linked to expression signals active in plant cells. 22. A recombinant DNA molecule according to claim 20, characterized in that said virus-specific control region comprises the ORF VII and the intergenomic region between ORF VII and ORF I of Cauliflower mosaic virus. 23. Recombinant DNA molecule according to claim 21, characterized in that it contains the complete CaMV 35S enhancer / promoter region, the leader sequence, the reading frame ORF VII, the small intergenomic region, 16 codons of the reading frame ORF 1.17 condyls of linker and spacer sequences containing a protective factor gene coding region, spacer sequences, a CaMV polyadenylation site and sequences of the pUC 18 plasmid. 24. Recombinant DNA molecule according to claim 21, characterized in that it contains the reading frame of a protective factor-encoding structural gene between the 19S promoter and the CaMV transcription terminator. 25. A method for producing a recombinant DNA molecule according to claim 16, which is characterized by the following method steps: a) isolation and cloning of a protective factor-encoding DNA sequence, b) functionally linking said DNA sequence encoding a protective factor with a virus specific control region in a manner that ensures the induction of protection factor formation upon incipient virus infection, and c) Splicing the hybrid gene construction described under b) in an operable manner between expression signals active in plant cells. 26. The method according to claim 25, characterized in that the functional linkage between the protective factor-encoding DNA sequence and virus-specific control region by exchanging viral, coding DNA sequences takes place, which are themselves controlled by said virus-specific control region. 27. A recombinant DNA molecule according to claim 21, characterized in that it is a cDNA which is complementary to the RNA3 of BMV and which contains a protective factor incorporated in the place of the coat protein coding for a DNA segment. 28. The method according to claim 26, characterized in that said virus-specific control sequences are CaMV control sequences. 29. The method according to claim 26, characterized in that they are BMV control sequences. 30. The method according to claim 26, characterized in that said functional linkage by replacing a coding DNA portion of the ORF 1 on the CaMV genome against a Schutzfaktorgen. 31. The method according to claim 26, characterized in that said functional linkage by replacement of a coding DNA portion of the ORFVI on the CaMV genome takes place against a Schutzfaktorgen. 32. The method according to claim 26, characterized in that said functional linkage by replacement of a coat protein-encoding cDNA portion which is complementary to the corresponding region on the RNA3 of BMV, takes place against a Schutzfaktorgen. 33. The method according to claim 25, characterized in that said expression signals are promoter and termination sequences as well as other regulatory sequences of the 3 'and 5'-untranslated region, which are active in plant cells. 34. A DNA transfer vector, characterized in that it contains a recombinant DNA molecule according to any one of claims 16 to 24. 35. A DNA expression vector, characterized in that it contains a recombinant DNA molecule according to any one of claims 16 to 24. 36. A host cell, characterized in that a) contains a DNA transfer vector according to claim 34 b) contains a DNA expression vector according to claim 35. 37. A host cell according to claim 16, characterized in that it is a microorganism. 38. A host cell according to claim 36, characterized in that it is a plant cell. 39. Virus-resistant transgenic plant and its seeds, prepared by a method according to any one of claims 1 to 15. 40. Virus-resistant transgenic plant whose cells are transformed in whole or in part with a recombinant DNA molecule according to any one of claims 16 to 24 and the seeds of said transgenic plant. 41. A plant cell, characterized in that it is transformed with a recombinant DNA molecule according to any one of claims 16 to 24. 42. A plant cell according to claim 41, characterized in that it is a component of a whole plant. 43. A virus-resistant transgenic plant and its seeds according to any one of claims 39 or 40, selected from the group consisting μms Solanaceae, Cruclferae, Composltae, Lillaceae, Vitaceae, Chenopodlaceae, Rutaceae, Alliaceae, Amaryllidaceae, Asparagaceae, Orchidaceae, Palmae, Bromelibceae, Rubiaceae, Theaceae, Musaceae or Gramineae and the order Legumlnosae and especially the family Papilionaceae. 44. A virus-resistant transgenic plant and seeds thereof according to claim 43 selected from the group consisting of Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trlgonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapls , Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Cichorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Hemerocallis, Nemesia, Pelargonium, Panicum, Pennisetum, Ranunculus, Seneclo, Salpiglossis, Cucumis , Browallia, Glycine, Gossypium, Lolium, Zea, Trlticum and Sorghum, Ipomoea, Passiflora, Cyclamen, Malus, Prunus, Rosa, Rubus, Populus, Santalum, Allium, Ulium, Narcissus, Pineapple, Arachis, Phaseolus and Pisum. 45. A virus-resistant transgenic plant and its seeds according to claim 43, selected from the group Allium, Avena, Hordeum, Oryza, Panicum, Saccharum, Seeale, Setaria, Sorghum, Triticum, Zea, Musa, Cocos, Phoenix and Elaeis, 46. A progeny of a virus-resistant transgenic plant according to claim 40, characterized in that said progeny have been regenerated from plant cells transformed with a recombinant DNA molecule conferring inducible virus resistance on plants and said progeny mutants and variants include you. 47. Transformed viable parts of plants according to any one of claims 39 or 40, characterized in that they are protoplasts, cells, cell aggregates, callus, seeds, pollen, ova, zygotes or embryos. 48. Cross and fusion products with the plant material defined in claims 39 to 47, which still has the characteristic properties of the transformed starting material. 49. A process for the production of virus-resistant, transgenic plants, characterized in that one transforms a plant or viable parts thereof with a recombinant DNA molecule according to any one of claims 16 to 24. 50. Use of a recombinant DNA molecule according to any one of claims 16 to 24, for the "immunization" of plants against an unwanted virus attack.