CA1340769C - Inducible virus resistance in plants - Google Patents

Abstract

The present invention relates to a novel process which, based on genetic engineering techniques, makes it possible to produce an inducible virus resistance in plants. The present invention further relates to the use of this process for the production of virus-resistant plants and to the plants themselves that have been obtained by this process and provided with an inducible virus resistance, and to the progeny thereof.

The present invention also includes chimeric genetic constructs, cloning vehicles and host organisms and methods or transferring an inducible virus resistance to plants. A further aspect of the present invention concerns the use of said recombinant DNA
molecules for the "immunisation" of plants against undesired virus attack.

Description

Inducible virus resistance in plants The present invention relates to a process which, based on genetic engineering techniques, makes it possible to produce an in<iucible virus resistance in plants. The present inveni~ion further relates to the use of this process for the production of virus-resistant plants and to the plants provided with an inducible virus resist-ance that are obtainable by this process, and to the progeny thereof.
A large number of the diseases known today that affect our most important cultivated plants and that result time and again in considerable losses in yield and in the harvested product in fruit, vegetable and cereal cultivation are caused by plant viruses. These phyto-pathogenic viruses are transmitted especially by sucking or biting phyt:ophagaus insects and nematodes, which act as vectors.
Efforts have therefore been made for a very long time now in plant cultivation to develop suitable processes for the protecaion of cultivated plants from virus attack.
One of these measures, which, however, is of only limited effect: and moreover involves considerable risks, has become known by the name "cross protection". This involves the a.rtific:ial infection of healthy crops with weakened strains of pathogenic viruses, which is intended to prevent a second infection with the actual pathogen. This method has been used, for example, to reduce the losses in yield in tomatoes, potatoes and citrus fruits that are caused by pathogenic viruses ~3~i~rl ~9~

such as tomato mosaic virus, potato spindle tuber viroid and citrus t:risteza virus. (Broadbent L, Ann.
Rev. Phytopathol., 14: 75, 1976; Fernow KH, Phytopath-ology, 57: 1347, 1967; Costa AS and Muller GW, Plant Dis., 64: 538, 1980).
The use of this process for the protection of healthy crops against. a virus infection involves a great many disadvantages and dangers, however.
For example, the weakened virus strain used for the artificial infection may undergo mutation into a highly virulent strain and thus itself cause an outbreak of a disease.
It is also pe~ssible: for a particular virus strain to be perfectly suitable for the protection of one particular plant species but t:o act as a pathogen and cause severe damage in another :species.
In addition, synergistic actions of two or more types of virus have been described which give rise to new, previously unknown disease symptoms (Garces-Orejuela C
and Pound GS, Phytapathology, 57: 232, 1957).
With the introduction of genetic engineering techniques also into plant genetics and with the possibilities this has provided for inserting foreign genetic material into plants, new processes have been developed which are based on the expression in the corresponding plants of individual virus gE:nes only instead of infection with the entire virus genome which has been practised hitherto.
In the new processes mentioned, for example, coat protein genes of certain viruses (tobacco mosaic virus, - 3 - 134Ur1~~9 - alfalfa mosa_LC virus) are incorporated into the plant genome with i:he aid of recombinant DNA techniques (Bevan MW et al., EPZBO J., 4(8): 1921-1926, 1985; Abel PP et al., Science, 232: 738-743, 1986).
Upon expression of the integrated coat protein gene in the plant, the coat protein produced is able to achieve a certain protective action which protects the plant from infection with the corresponding virus or weakens any symptoms of the disease that occur. The precise-mechanism underlying this protective action by the coat protein are :Largely unknown at present.
The expression of other virus genes also can result in a protective a~~tion against infection with the correspon-ding virus. For example, in transgenic plants that synthesise a:ntisense RNA of parts of the corresponding viruses, inhibition of the virus expression occurs.
The expressi~~n of satellite RNA in transgenic plants also can res,alt in a protective action, presumably because of c~~mpetition with the infecting virus for replicase (Baulcombe DC et al., Nature, 321: 446-449, 1 986 ) .
Although these processes afford a certain protection against a virus infection and/or its sequelae, they too have crucial disadvantages:
a) at high inoculation concentrations of the virus the protection factor can be titrated out, so that a protective function is no longer ensured.
b) the protection factor itself may cause disease-like states to be produced in the plant synthesising the protection factor.

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For example, the virus coat protein is capable of penetrating into biological membranes (de Zoeten GA and Gaard G, Virus Research, 1:
713-725, 1984) and damaging them. This can consequently lead to functional disturbances within the cell, especially in chloroplasts and mitochondria.
c) The protection factor genes are continuously (constitutively) expressed even when there is no virus infection, which results in marked impairment of the energy balance of the plants in question..
In order to overcome the disadvantages mentioned above, the object of the present invention is, therefore, to develop a system in which the protection factor is no longer produced constitutively by the transformed plant but in which the synthesis of said protection factor can be switched on or off in the producing plant as required.
Surprisingly, it has been possible to achieve this object within the scope of the present invention by simple means, by providing a system that renders possible the induction of genes based on virus-intrinsic control mechanisms.
The invention accordingly relates to a process for the protection of plants from a virus infection and/or its sequelae, which process is characterised in that there is introduced into said plants an inducible protection factor gene that is expressed as required, that is to say at the beginning of an infection with a virus, and controls the production of the protection factor.
The invention also relates to a process for the protection of plants against a virus infection and/or its sequelae, characterised in that an inducible virus resistance due 1340'~G~
4a to a gene encoding a virus resistance factor under the control of a promoter and/or a virus specific control region which regulates expression of the control factor is introduced into said plants using genetic eng:Lneering techniques.
The invention further relates to a recombinant DNA
molecule, characterised in that it confers an inducible virus resistance upon p:Lants due to a gene encoding a virus resistance factor under the ~:ontrol of a promoter and/or a virus specific control region wh:Lch regulates expression of the control factor.
The invention also relates to a process for the preparation of a :recombinant DNA molecule as described above, which process is characterised by the following stepsz a) isolation and cloning of a DNA sequence coding for a protection factor, b) functional linking of said DNA sequence coding for a protection factor with a virus-specific control region in such a manner that the induction of protection factor formation is ensured when a virus infection begins, c) splicing in of the hybrid gene construct described in b) in operable manner between expression signals that are active in plant cells.
The invention also relates to a use of a DNA module of the present invention to immunize a plant against undesired virus attack.
The present invention further relates to a process for the protection of plants from infection by phytopatho-13407&9 -S-genic viruses, which process is characterised in that there is introduced into said plants an inducible protection factor gene that is operably linked with virus-specific control elements and with expression signals that are active in plant cells, so that the expression of said protection factor gene and hence the production of the protection factor is controlled by the infecting virus itself .
In addition, 'the present invention relates to recom-binant DNA molecules that confer an inducible virus resistance upon plants, the infecting viruses being neutralised b:y a protection factor synthesised by the plant.
The present invention relates especially to transgenic virus-resistant plants and to viable parts of virus-resistant transgenic plants transformed with a recom-binant DNA mo:Lecule that codes for an inducible protec-tion factor that is responsible for the neutralisation of the infecting virus, and to mutants and variants of said transgen:LC plaints.
Within the scope of the present invention the term "virus-resistant" plant is defined as a plant that survives at a normally effective virus concentration and preferabl~T exhibits normal growth.
The present invention further relates to plants regene-rated from transformed plant cells containing said recombinant DrdA molecule, and to the seeds thereof, and also to the pwogeny of plants regenerated from said transgenic plant ce:Lls, and to mutants and variants thereof .
According to t:he invention, the expression "mutants and 1340~~~~-- variants of transgenic plants" shall be understood as meaning those plants that still possess the character-istic properties of the original plant that that plant obtained as a result of transformation with an r-DNA
molecule acct>rding to vhe._invention.
The present invention also comprises alL hybridization and fusion products with the transformed plant material mentioned above.
The invention further relates to a process for the production of virus-resistant transgenic plants, which process is characterised in that a plant or viable parts thereof is(are) transformed with a recombinant DNA
molecule.
Without constituting any limitation of the subject of the invention, viable parts of plants are defined as being, for example, plant protoplasts, cells, cell aggregates, callus, seeds, pollen, ova, zygotes or embryos.
The present invention also includes chimeric genetic constructs containing a protection factor gene, and processes for the production thereof, cloning vehicles and host organisms and methods of conferring an induc-ible virus resistance upon plants.
A further aspE:ct of the present invention concerns the use of said rE:combinant DNA molecules for the "immuni-sation" of plants against undesired virus attack.
In the following description, a number of expressions that are customary :in recombinant DNA technology and in plant genetic=c is used.

_,_ In order to ensure a clear and consistent understanding of the description and the claims and of the scope allotted to said expressions, the following definitions are given:
Heterologous ~~ene(s) or DNA: a DNA sequence that codes for a specific product or products or for a biological function and that originates from a species other than th;~t into which said gene is inserted; said DNA sequence :is also referred to as "a foreign gene" or "foreign DNA".
Homologous gene s or DNA: a DNA sequence that codes for a specific product or products or for a biological function and i~hat originates from the same species as that into whi~~h said gene is inserted.
Synthetic gene; s) o:r DNA: a DNA sequence that codes for a specifi~~ product or products or for a biological function and i:hat has been produced synthetically.
Plant promoter: a control sequence of DNA expression that ensures i~he transcription of any homologous or heterologous I)NA gene sequence in a plant provided that said gene sequence .is operably linked with such a promoter.
Over-producinc; plant promoter (OPP): plant promoter that is capabT_e of causing the expression in a trans-genic plant cE:ll of any operably linked functional gene sequences) to a degree (measured in the form of the amount of RNA or of polypeptide) that is distinctly higher than treat observed naturally in host cells that have not been transformed with said OPP.
Plant: any photosynthetically active member of the 13~0~~~
_8-kingdom Planta that: is characterised by a membrane-enclosed nucleus, genetic material organised in the form of chromosomes, a membrane-enclosed cytoplasmic organelle and the ability to undergo meiosis.
Plant cell: structural and physiological unit of the plant, consisting of a protoplast and a cell wall.
Plant tissue: a group of plant cells organised in the form of a structural and functional unit. -Plant organ: a defined and visibly clearly differ-entiated part of a plant, such as, for example, a root, stem, l~saf or embryo.
Protection factor: a one-component or multi-component system that directly or indirectly interacts with infec-ting viruses within the plant in such a manner as to ensure a protective action against a virus infection and/or its sec~uelae.
Protection facaor gene(s): nucleotide sequences) coding for a protection factor as defined above.
Virus-specific: control elements: regions on the virus genome that interact with a virus product in such a manner as to influence replication, transcription, translation or other post-transcriptional processes (in "cis").
DNA expression vector: cloning vehicle, such as, e.g., a plasmid or a bacteriophage, containing all the signal sequences necessary for the expression of an inserted DNA in a suitable host cell.
DNA transfer ~~ector: transfer vehicle, such as, e.g., 1340~1~9-- g _ - a Ti-plasmid or a virus, that enables the insertion of genetic material into a suitable host cell.
A brief descriptiora of the Figures is given below:
Figure 1 shows the construction of plasmid pHS1 which is described in detail in Example 1.1.
Figure 2A-C show the various possible methods of trans-activat:ion in CaMV.
Figure 3 shows the restriction map of plasmid pCIB200.
Figure 4 shows the restriction map of plasmid V374.
The present invention relates especially to recombinant DNA molecules that are capable of conferring an induc-ible virus resistance upon plants.
The virus resistance of the plants is mediated by so-called protection factors which are produced by the transgenic plant itself and which, by specific inter-action with the infecting plant virus, impair the latter's ability to reproduce to such an extent that the outbreak of an infection is prevented and/or the seque-lae thereof a:re diminished.
Two categories of protection factor are basically distinguished. On the one hand, there are antiviral substances which attack the virus directly and thereby adversely affect its ability to reproduce, for example by inhibiting enzymes that are essential for the virus's cycle of development.
A second cate~~ory is concerned with cell-toxic subs-- 10 - 1340~1~9 tances which poison and/or kill the cells attacked by the virus and thus isolate the focus of infection from the remaining, hea:Lthy tissue. In this case, therefore, the virus's ability to multiply is restricted indirectly by artificia7_ly triggering a "hypersensitive reaction".
Within the scope of the present invention, therefore, protection factors are to be understood as being one-component: or multi-component systems that directly or indirectl~r interact with the infecting viruses within the plant so as to ensure a protective action against a virus infection and/or the sequelae thereof.
Examples of :;uch protection factors, which do not, however, imply any limitation of the subject of the invention, are:
1) Aritiviral substances, such as, e.g., antivirus-antibodies, F~R-prot:eins (Ahl P et al., Plant Mol.
Biol., 4: 37, 1985), virus-specific protease inhib-itors (Jamet E and Fritig B, Plant Mol. Biol., 6:
69-80, 1986), protE~ases, virus-specific polymerase inhibitors, a.nd interferon-like proteins.
2) Cell-toxic. substances such as, e.g., toxins, active subunits of toxins, PR-proteins, and other proteins that naturally cause hypersensitivity, such as S-gluca-nase (Mohnen D, et al., EMBO J., 4: 1631-1635, 1985).
In connection. with the present invention, particular importance is. attached to the ribosome-inactivating proteins [RIP'S] (Lard et al., Oxford Sur. of Plant Mol. Cell. Biol., 1: 85-101, 1984) which cause an irreversible inhibition of ribosomes and lead to an immediate cessation of protein synthesis.

Examples of ouch ribosome-inactivating proteins are abrin (from ~~brus precatorius seeds) (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 Modecca diqitata roots) (Stirpe et al., FEBS Lett., 85: 65-67, 1978), viscumin (from Viscum album leaves) (Stirpe _et _al., Biochem. J., 190: 843-845, 1980; Olsnes et _al., J. Biol.
Chem., 257: 13263-'13270, 1982) and ricin (from Ricinus communis) (Ol.snes and Pihl, in: Cuatrecasas P. (Ed.) The Specificity of Action of Animal, Bacterial and Plant Toxins, Chapman and Hall, pp. 129-173, 1976). The pokeweed toxn (Owens et al., Virol-og~, 56: 390, 1973) also belongs to this group. All of the compounds ment-ioned have a great structural and functional similarity.
It has been found especially advantageous if the induc-tion of the protection factor gene and hence of protec-tion factor p~roduct:ion is effected by the infecting virus itself. In this manner, the synthesis of the protection factor remains limited to those cases in which a virus. infection occurs and the protection factor is actually needed in order to give the plant the appropriate protection.
If the plant is attacked by a corresponding virus, the virus-specific control region is switched on and hence the protection factor gene is expressed. This results in the synthesis of the protection factor, which neut-ralises the infecting virus. The more infecting virus present, the more protection factor will be produced and the more virus will be neutralised.
Thus, it has become possible for the first time within the scope of the present invention to create a closed control circuit that is self-regulating and affords the 1340 rG9 plant optimum protection from a virus infection.
Consequently, energy that is urgently needed for growth and fruit formation does not have to be expended by the plant unnecessarily in order to maintain lasting pro-tection against a virus infection. This can be achieved according to the invention, for example, by linking the protection factor gene to virus-specific control elem-ents and thus placing the expression thereof under the control of said control elements.
A further aspect of the present invention therefore relates to a process for the protection of plants from a virus infection and/or the sequelae thereof, which process is characterised in that there is introduced into said plants an inducible protection factor gene that is operably linked with virus-specific control elements and with expression signals that are active in plant cells, so that the expression of the protection factor gene is regulated by the infecting virus itself.
A large number of viruses are known to have developed specific coni::rol mechanisms that are essential to the coordination of individual virus functions.
The mentioned virus-specific control functions include, for example:
a) transactivation of virus genes at the transcription and the post--transcription levels 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 (CalE;nder, Biotechnology, 4: 1074, 1986).
There are fundamentally different levels at which a transactivat~ng mechanism can have effect. The most simple and most obvious route is the.transactivation of transcription. '.this type of transactivation has been described, for example, as one of the principal mech-anisms for the human HTLV system. In this case, 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, for example, the tat gene, code for transactivators that enable the expression of the virus genes gag-pol-env. The tat gene product is a polypeptide of molecular weight 15,000 that in its structure and prop-erties is reminiscent of nucleic acid binding proteins, such as, for example, the transcription factor VIII A of Xenopus laevis and others.
Accordingly, a possible mechanism considered for trans-activation is a cooperative binding of a regulator to a recognition site. Such a recognition site exists, for example, in the region of the RNA start region.
At the post-transcriptional level, on the other hand, a large number of different transactivation mechanisms is available which can have effect at very different sites.
The possibilities range from the control of mRNA elong-ation (Grayha~ck et al., Cell, _42: 259, 1985; Yi-Kao et al., Nature, 330: 489 1987), of mRNA transport from the nucleus into the cytoplasm, of mRNA splicing, of mRNA polyaden_~rlation (Derse, J. Virol., 62, 1115, 1988), of translation initiation (Jay et al., Proc. Natl. Acad.

Sci, USA, 78.: 2927, 1981) to the differentiated use of different ORFs ('Open Reading Frame') on the RNA.
A third level of the possible transactivation mechanisms is concerned with the post-translational modification of the protein product in the form of phosphorylation or of protein processing.
In the case ~of cauliflower mosaic virus (CaMV), all three of the above-mentioned transactivation levels are relevant.
CaMV possess.=_s a double-stranded (ds) circular DNA
genome the r~:plication of which is presumably carried out by means of a reverse transcription mechanism. In that process, two RNA intermediates are formed, a 35S
RNA and a 19:> RNA, which correspond to two operons on the CaMV genome. 'The 19S promoter transcribes a subgenomic RtJA (19S RNA transcript) which encodes the CaMV protein VI which forms the main portion of the virus-specific inclusion body. The 35S RNA consists of a leader region comprising 600 nucleotides followed (in series) by the reading frames VII, I, II, III, IV, V and VI. It is assumed that all these reading frames, with the exception of reading frame VI, are translated by the 35S RNA. (Pf:eiffer P and Hohn T, Cell, _33: 781-789, 1983; Kridl ,:rC and Goodman RM, Bio Essays, _4: 4 - 8, 1 986 ) .
Within the scope of the present invention it has been possible to verify the following control mechanisms for the CaMV system:
1) The 19S promoter is activated by the protein product of the 19S operon in the form of an autocatalysis (see Fig. 2A).

2) In addition, the use of polycistronic RNA is regul-ated by said protein product of the 19S operon (see Fig.
2B).
3) The release of a specific protein product occurs from a polyprotein as a result of the activity of viral proteases (Fig. 2C).
In certain strand-positive (+) R~1'A viruses, a variation of this viral t:ranscriptional transactivation is found. In these cases, production of a subgenomic virus (messen-ger) RNA occurs starting from the minus strand RNA, catalysed by virus-specific polymerases.
This special form of transcriptional transactivation occurs, for example, in brome mosaic virus (BMV) which attacks plani:s of the monocotyledon group, especially numerous species of gramineae, such as, e.g., maize, barley, bromE:-grass and other wild grasses, and, in addition, several dicotyledonous plants.
The genome of: BMV :is composed of a total of three RNA
components. The transcripts of two of these components (RNA 1 and RD1A 2) are essential for the replication of the viral genome in the plant. For example, the repli-cation of the third component (RNA 3), a dicistronic RNA coding for a protein of molecular weight 32,000 and for the coat protein, and the production of subgenomic coat protein mRNA is dependent on the presence of the RNA 1 and RNP, 2 transcripts (French _et _al., Science, 231 , 1 294-1 2f7, 1 9E36 ) .
The mechanism serving for the production of subgenomic mRNA for the expression of the genes present on RNA 3 is presumably an internal initiation of a virus-specific replicase that is formed very early on in the course of the infection cycle (possibly encoded by yen-omic RNA 1 and/or RNA 2) on the minus (-) strand of the genomic RNA 3 (Miller et al., Nature, 313: 68-70, 1985).
Furthermore, it is ;possible to proceed from the assump-tion that the copy number of genomic nucleic acids (DNA
or RNA) can bE: increased very substantially if they are provided with viral replication signals, this being due to the activity of apecific viral polymerases. ' All of these :>ystems mentioned can be used in the process according to the invention for the construction of vectors that are capable of conferring an inducible virus resistance upon plant material.
Within the scope of this invention, the use of virus-specific control elE:ments of cauliflower mosaic virus (CaMV) is especially preferred; however, this does not limit the scoF>e of the present invention in any way but serves merely to demonstrate that the process according to the invention functions.
The present invention further relates therefore to the production of hybrid gene constructs composed of one or more virus-specific control regions and of homologous or heterologou.s DNA sequences that code for one or more protection factors ~, using methods and processes that are to a large extent known and customary in the field of recombinant DNA technology and that are described, inter alia, in the following publications:
"Molecular Cloning", Maniatis T, Fritsch EF and 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, Tokyo, 1983.

- _ The region coding for a protection factor gene, which is used according to the present invention may be of homologous or heterologous origin in relation to the plant cell or the plant that is to be transformed. At all events, however, it is necessary for the gene sequence coding for the protection factor to be expressed and lead to the production of an enzyme or polypeptide capable of functioning in the resulting plant cell.
DNA sequences of heterologous origin are to be under-stood according to 'the invention as those which origi-nate from othE:r plant species or from organisms belong-ing to another. taxonomic unit, for example from microbes or from mammals, or those of synthetic origin, which DNA
sequences are capab:Le of achieving the desired protec-tive function against a virus attack in the particular desired plant species.
The coding region of the hybrid gene construct can also encode a prote<aion factor that differs from a naturally occurring protection factor but that still substantially performs the protective function of the natural protection factor. Such a coding sequence will usually be a variant: of a natural coding region. A
"variant" of a natural DNA sequence is defined within the scope of this invention as being a modified form of a natural sequence, which, however, stillperforms the same function. The variant can be a mutant or a synthetic DNA sequence and is substantially homologous to the corresponding' natural sequence. Within the scope of this invention, a DNA sequence is substantially homologous to a second DNA sequence if at least 70 ~, preferably at least 80 ~s, but especially at least 90 of the active portions of the DNA sequence are homolo-gous. According to this definition of the expression _ _ I34U ~s~
"substantiall:y homologous", two different nucleotides in a DNA sequence of a coding region can be considered to be homologous if the exchange of one nucleotide for the other represents a silent mutation.
The invention therefore includes any hybrid gene encod-ing an amino acid sequence and mediating the desired protective function that~meets the disclosed and claimed requirements. Special preference is given to a nucleo-tide sequence that :is substantially homologous with at least that part or those parts of the natural sequence that is (are) responsible for the protective function against a virus attack.
The DNA sequence coding for the protection factor may be constructed e~:elusively from genomic DNA, from cDNA or from synthetic' DNA. Another possibility is the constru-ction of a hybrid DNA sequence consisting both of cDNA
and of genomic DNA and/or synthetic DNA. In that case, the cDNA may originate from the same gene as does the genomic DNA or, alternatively, both the cDNA and the genomic DNA may originate from different genes. In either case, however, both the genomic DNA and/or the cDNA can each be produced from the same gene or from different genes.
If the DNA sequence contains portions from more than one gene, these genes may originate either from one and the same organism, from several organisms belonging to more than one strain, variety or species of the same genus, or from organisms belonging to more than one genus of the same or another taxonomic unit (kingdom).
The various regions of DNA sequence can be linked to one another to form a complete DNA sequence coding for the protection factor using methods that are known per se.

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Suitable methods include, for example, the _in vivo recombination of DNA sequences having homologous regions and the in vitro linking of restriction fragments.
Thus, the broad concept of this invention covers a great number of embodiments. _ One of these embodiments according to the invention is characterised by a c:himeric gene sequence containing:
(a) one or more gene sequences coding_for one or more protection factor polypeptides that, when the gene has been expressed. in a given plant cell, produce a protec-tive function against a virus infection and/or the sequelae thereof, and (b) one or more gene: sequences coding for one or more virus-specific control regions that are operably linked with the region coding for the protection factor, and (c) one or more additional gene sequences that are operably linked with both ends of the region coding for the protection factor and also, where appropriate, for the associated virus-specific control elements. These additional gene sequences contain promoter and termi-nator regions and, optionally in addition, regulatory sequences of the 3'- and 5'-non-translated regions. The plant regulatory seguences may be heterologous or homo-logous in relation to the host cell.
The functional linking of the region coding for a protection factor with a virus-specific control region can be achieved according to the invention, for example, by isolating virus-intrinsic coding DNA sequences that are known to be controlled naturally in the course of the infection cycle by specific functions of the intact 1340~~~

virus, and replacing the coding region by a protection factor gene, thus bringing the protection factor gene under viral control.
As examples of such coding viral regions, which are not to be regarded as being in any way limiting, there may be mentioned the reading frames I and VI of the CaMV
genome, and also the coat protein-coding DNA sequence on the dicistron.ic RNA 3 of BMV.
Any promoter and any terminator capable of causing the expression of a DNA sequence coding for a protection factor can be used as a component of the chimeric gene sequence. Examples of suitable promoters and termin-ators are those of the nopaline synthase genes (nos), the octopine synthase genes (ocs) and of the cauliflower mosaic virus genes (CaMV).
An effective representative of a plant promoter that can be used is an over-producing plant promoter. Provided that this kind. of promoter is operably linked with the gene sequence for the protection factor gene, it should be capable of mediating the expression of said protec-tion factors in such a manner that the transformed plant is protected against: a virus attack.
Over-producing plant promoters that can be used within the scope of the present invention include the promoter of the small subunit (ss) of ribulose-1,5-bisphosphate carboxylase fr~~m soybeans [Berry-Lowe _et _al., J. Molec-ular and App. ~:~en., 1: 483-498 (1982)) and the promoter of the chlorophyll-a/b-binding protein. These two promoters are known to be induced by light in eukaryotic plant cells [sEae, for example, Genetic En ineerin of Plants, an Aqr:icultural Per~ective, A. Cashmore, Plenum, New York 1983, pages 29-38; Coruzzi G et al., The Journal of Biological Chemistry, 258: 1399 (1983) and Dunsmuir P
et al., Journal of Molecular and A lied Genetics, 2: 285 (1983)]. _ The use of strong promoters ensures that, even in the absence of the virus, small amounts of protection factor protein are s=~nthesised continuously, so that in the event of an acute virus infection a very rapid first reaction to the occurrence of infection is possible.
A gene construct that is preferred within the scope of the present invention is composed of the CaMV 35S
promoter, a lE:ader Sequence, the CaMV reading frame VII, a CaPdV readincf fram~a I / protection factor gene fusion and a transcr9:ption terminator.
Also preferred within the scope of this invention is a gene construct. composed of the CaMV 19S promoter, a protection facaor-coding DNA sequence in the ORF VI of the CaMV genome and a CaMV termination sequence.
The chimeric gene sequence, which consists of one or more virus-spE:cific control sequences and one or more protection facaor genes and is operably linked with expression signals that are active in plant cells, can be spliced into a suitable cloning vector. Plasmid or virus (bacteri.ophage) vectors having replication and control sequences originating from species that are compatible with the host cell are generally used.
The cloning vector normally carries an origin of repli-cation, and, in addition, specific genes that result in phenotypical selectable markers in the transformed host cell, especially in resistance to antibiotics or to certain herbicides. The transformed vectors can be selected by means of these phenotypical markers after - zz - 1340'~~0 transformation in a. host cell.
Suitable host cells within the scope of this invention are prokaryotes, including bacterial hosts, such as, e.g. A. tumef~~ciens, A. rhizoc~enes, E, coli, S. typhi-murium and Se:rratia marcescens, and cyanobacteria.
Eukaryotic ho;sts-such as yeasts, mycelium-forming fungi and plant cel:Ls can also-be used within the scope of this invention.
The cloning vE:ctor and the host cell transformed with that vector are used according to the invention to increase the copy number of the vector. 6Vith an increased cop;r number it is possible to isolate the vector carrying the protection factor gene and use it, for example, t:o insert the chimeric gene sequence into the plant cell..
The insertion of DNA into host cells can be carried out using processes than are known per se. For example, bacterial host: cells can be transformed after treatment with calcium chloride. In plant cells, DNA can be inserted by direct contact with protoplasts produced from the cells. Another possible method of introducing DNA into plant. cells comprises bringing the cells into contact with viruses~ or with Agrobacterium. This can be achieved by infection of sensitive plant cells or by co-cultivation of protoplasts derived from plant cells.
These processes will. be discussed again in detail below.
There is a number of processes available for directly inserting DNA into plant cells. For example, the genetic material contained in a vector can be micro-injected directly into the plant cells with the aid of micropipettes for the mechanical transfer of recombinant 1340r16~

DNA. The genetic material can alternatively be intro-duced into protopla;sts after the latter have been treated with polyethylene glycol. [Paszkowski J _et _al., EMBO J., 3: 2i'17-2722 (1984)].
A further aspE:ct of the present invention relates to the insertion of a protection factor gene into a plant cell by means of el.ectroporation [Shillito R _et _al., Bio-technoloc~y, 3: 1099-1103 (1985); Fromm M _et _al., Proc.
Natl. Acad. Sci. USA, 82: 5824 (1985)].
In this technique, plant protoplasts are subjected to electropora.tion in the presence of plasmids containing the protection factor gene.
High-intensity electrical pulses produce a reversible increase in the permeability of biomembranes and thus enable the insertion of the plasmids. Electroporated plant protoplasts renew their cell wall, divide and form callus tissue. Selection of the transformed plant cells containing the expressed protection factor can be carried out with the aid of the phenotypical markers described above.
Cauliflower mosaic virus (CaMV) also can be used within the scope of this invention as a vector for the inser-tion of a protection factor gene into a plant (Hohn _et al., in "Molecular Biology of Plant Tumors", Academic Press, New York, 1982, pages 549-560; Howell, US Patent No. 4,407,956).
For this, the ~=ntire viral DNA genome of CaMV is integrated into a bacterial parent plasmid to give a recombinant DNA molecule that can be multiplied in bacteria. Aft~sr cloning, the recombinant plasmid is cleaved with the aid of restriction enzymes either 13407~~

randomly or ai: quite specific non-essential sites within the viral pari~ of t:he recombinant plasmid, for example within the gene coding for the ability of the virus to be transmitted by aphids, so that the protection factor gene sequence can be incorporated.
A small oligonucleotide, a so-called linker, which possesses a single, specific restriction recognition site, can also be integrated. The recombinant plasmid modified in this manner is cloned again and further modified by s~~licing the protection factor gene sequence into a restricaion site that occurs only once.
The modified viral part of the recombinant plasmid is then excised from the bacterial parent plasmid and used for inoculation of plant cells or of entire plants.
Another method of inserting a hybrid gene construct of the invention into a cell relies upon the infection of a plant cell with Agrobacterium tumefaciens or Agrobacterium rhizogenes which is transformed by the protection factor gE:ne in such a manner that said protection factor gene replaces the tumour-producing genes between the T-DNA border sequences. Another possibility within the scope of this invention consists of a mixed infection using both A. rhizo enes and transformed A. tumefaciens, as has been described by Petit et al. (Petit et al., Mol. Gen. Genet.
202: 388, 1986) for the transformation of carrots.
The ratio of the constituents of the mixture to each other must be so selected that the rootlet colonies formed as a result of the A, rhizo enes transformation also contain a high proportion of A. tumefaciens Ti-plasmid. This can be achieved according to the invention by applying A, rhizo eves and A. tumefaciens together to the plant material, in known manner, in a - 13 4 0'~~9 ratio of from 1 :1 t:o 1 :100, but preferably of 1 :10. The transgenic plant cells are then cultivated under suit-able cultivation conditions known to the skilled person so that they form shoots and roots and, finally, entire plants result. The two Agrobacteria species are advantageously mixed only shortly before the actual inoculation.
Gene sequences encoding the protection factor can be transferred into suitable plant cells using, for example, the 'ri-plasmid of Agrobacterium tumef_aciens [DeCleene et <~1. , Bot. Rev. . , _47: 1 47-1 94 ( 1 981 ) ;
Bot. Rev., 42: 389-466 (1976)] and the-Ri-plasmid of A. rhizo enes (M Tepfer and F Casse-Delbart, Micro-biol. Sci., 4~(1~: 24-28, 1987). The Ti-plasmid is transferred to a plant during the course of infection by Agrobacterium tumefaciens and integrated into the plant genome p!n a stable manner. [Horsch _et _al., Science, 233: 496-498 1;1984); Fraley et al., Proc. Natl. Acad. Sci.
USA, 80: 4803 (1983)].
For plants whose cells are not susceptible to infection with Agrobacte:rium recourse can be had 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 these, the transfer DNA region,. is transferred to the plant and leads to the induction of tumours. The other, the virulence-conferring (vir) region, is essential only for forming and not, however, for maintaining the tumours.
The dimensions of the transfer DNA region can be enlarged by incorporating a protection factor gene sequence without the transferability being thereby impaired. By removing the tumour-causing genes, as a result of which the transgenic plant cells remain non-tumorous, and by incorporating a selectable marker the modified Ti-plasmid can be used as a vector for the transfer of the gene construct of the invention into a suitable plant cell.
The vir region effects the transfer of the T-DNA region from Agrobacterium to th-e genome of the plant cell irrespective of whether the T-DNA region and the vir region are on the same vector or on different vectors within the same A~robacterium cell. A vir region on a chromosome also induces the transfer of the T-DNA from a vector into a plant cell.
A preferred s:~stem for transferring a T-DNA region from an Agrobacter:ium into plant cells is characterised in that the vir :region and the T-DNA region are located on different vect=ors. Such a system is known by the name "binary vector system" and the vector containing the T-DNA is referred to as a "binary vector".
Any vector that contains T-DNA and that is capable of being transferred into plant cells and that permits selection of t:he transformed cells is suitable for use within the scope of this invention.
Any vector that has a vir region and that effects the transfer of a T-DNA region from Agrobacterium to plant cells can be used within the scope of this invention.
Another embodiment within the scope of the present invention concerns a process that combines the advan-tages of a virus-mediated transformation and transfor-mation by means of Agrobacterium. This process is described in European Patent Application No. 201 904.
In that process, viral DNA or parts of viral DNA which 1340'~~9 may or may not contain passenger DNA incorporated therein is (are) integrated into a T-replicon in such a manner that an incorporation of the viral DNA in the plant genome takes place.
Incorporation of viral DNA ~.nto the T-replicon is advan-tageously effected in the region of the transfer DNA
region of the T-replicon so that the viral DNA of at least one of the T-DNA. border sequences that are essential for the integration of the transfer DNA into the plant genome is flanked.
In this case also, transfer of the T-DNA region from Agrobacterium into a plant cell can be effected using a binary vector system.
Especially preferred within the scope of the present invention is a hybrid genetic construct that contains the complete CaM'il 35S enhancer/promoter region, the leader sequence, the reading frame ORF VII, the small intergenomic region, 16 codons of the reading frame ORF I, 17 codons of linker and spacer sequences, the region coding fo:r a protection factor gene, spacer sequences, a CaM'il polyadenylation site and sequences of the plasmid pUC18 (Norrander J et al., Gene 26: 101-106, 1983).
Said hybrid genetic construct is formed by linking an Eco RV/Eco RI fragment of the CaMV strain CM4.184 (Dixon 1~40'~~9 L et al., Virologv, 150: 463-468, 1986) comprising the 35S promoter region, the leader sequence, the reading frame ORF VII, the small intergenomic region and the first 16 ~:odons of the reading frame ORF I, with the large Sma I/Eco RV fragment of the plasmid pDH51 (Pietrzak M ei= al., Nucl. Acids Res., _14: 5857-5868, 1 986 ) .
A DNA fragment: coding for a protection factor gene can then be splicE:d into the polylinker region of the resulting pla:~mid using methods that are known per, se, producing the hybrid genetic construct characterised in detail above.
Another genetic construct that is preferred within the scope of this invention uses a DNA fragment that contains the 19S promoter of cauliflower mosaic virus.
This DNA fragment is cloned together with the CaMV
transcription terminator. The reading frame of a structural gene, preferably of a structural gene coding for a protection factor, is inserted into the resulting plasmid, between those regulatory CaMV sequences, in such a manner that t:he translation of said structural gene begins directly at the start codon of the gene VI
protein.
In order to achieve this, a deletion mutagenesis is advantageously carried out using an oligonucleotide.
This is done starting with a single-stranded DNA that comprises the corresponding part of the CaMV genome and can be produced with the aid of M13 helper phages.
The resulting circular single-stranded DNA can then be used as a substrate for the in vitro deletion using oligonucleotides.
In a manner analogous to the construction of expression - 29 - 13~0'~f~9 vectors that are active in plants using CaMV control systems which. has been described above, the control system characaeristic of strand-positive (+) RNA viruses and described. above using the BMV virus as an example can be used for the vector construction.
As already mentioned before, in strand-positive (+) RNA
viruses, the production of subgenomic mRNA is effected starting from minus (-) strand RNA. In the BMV system, the production of a subgenomic RNA 4 which codes for a coat protein is presumably initiated by a partial trans-cription of the minus (-) strand RNA 3 with the partici-pation of virus-intrinsic replicases.
By producing and cloning complementary cDNA it is possible to use the recombinant DNA techniques discussed in detail hereinbefore also for a genetic manipulation of RNA viruses.
In a manner analogous to the CaMV system discussed above, therefore, i.t is possible also in the case of strand-positive (+) RNA viruses to carry out replacement of the regions coding for a viral protein (in the case of BMV the region on the RNA 3 coding for the coat protein) by a protection factor gene. This hybrid gene construct can then be inserted between expression signals active in plant cells in such a manner that, in addition to the protection factor gene, also the regions controlling the viral gene that was originally present are retained in functioning form. In BMV these are the DNA regions located upstream of the ORF of the coat protein that 'was originally present (French _et _al., Science, 231: 1294-1297, 1986).
Plant cells or plants that have been transformed in accordance with the present invention can be selected - 1340'~~9 with the aid of a suitable phenotypical marker that, in addition to t:he protection factor gene and the virus-specific control sequences, is a component part of the DNA. Examples of such phenotypical markers, which are not be regarded as limiting, however, comprise antibiotic-resistant markers, such as, for example, kanamycin-resistance and hygromycin-resistance genes, or herbicide-resistance markers. Other phenotypical markers are known to the skilled person and can also be used within the scope of this invention.
All plants whose cells are susceptible to transforma-tion by the direct insertion of DNA, by viral or bacterial vectors, such as, e.g., CaMV or Agrobact-erium, or by ether suitable vectors and can subsequently be regenerated into complete plants can be subjected to the process of the invention for the production of transgenic entire plants containing the transferred protection fa~~tor gene. There is a steadily growing number of indications suggesting that, in principle, all plants can be regenerated from cultivated cells or tissues, including, but not limited to, all important cereal species, sugar cane, sugar beet, cotton, fruit trees and othE:r types of tree, leguminous plants and vegetables.
The process oi= the :invention is suitable for the transformation of all plants, especially those belonging to the systematic groups Angios ermae and Gymnospermae.
Of particular interest among the Gymnos ermae are plants of the Coniferae class.
Of particular interest among the Angiospermae are, in addition to deciduous trees and shrubs, plants of the Solanaceae, Cruciferae, Compositae, Liliaceae, Vita-- 1340~r69 ceae, Chenopodiaceae, Rutaceae, Alliaceae, Amaryllida-ceae, Asparaq~iceae, Orchidaceae, Palmae, Bromeliaceae, Rubiaceae, Thf:aceae, Musaceae or Gramineae families and of the order Leguminosae and, of these, especially the Papiliona<:eae family. Representatives of the Solanaceae, Crucife:rae, Leguminosae and Gramineae are preferred,.
Target crops within the scope of the present invention also include, for example, those selected from the group consisting of:; Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Tri onel:la, Vi na, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lyco-persicon, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Cichorium, Helianthus, Lactuca, Bromus, Asparagus, Ant:irrhinum, Hemerocallis, Nemesia, Pelar-onium, Panicum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browallia, Glycine, Gossypium, Lolium, Zea, Triticum and Sorghum, including those selected from the group consisting of: Ipomoea, Passi-flora, CyclamE:n, Ma:Lus, Prunus, Rosa, Rubus, Populus, Santalum, Alli.um, Lilium, Narcissus, Ananas, Arachis, Phaseolus and Pisum.
This list of examples serves merely to illustrate the present invention in more detail and should not be regarded as a limitation of the subject of the invention.
By using newly developed transformation techniques it has meanwhile been possible to transform _in vitro also those plant species which are not natural host plants for Agrobacterium. For example, monocotyledonous plants, especially species of cereal and grasses, are not natural hosts for Agrobacterium.

~3~407~g In the meantime, there have been increasing indications that monocotyledons too can be transformed with Agro=
bacterium, so that, using new experimental strategies that are now becoming available, cereals and species of grasses also are svusceptible to a transformation [Grimsley N, et al., Nature, 32.x: 177-179 (1987)].
The regeneration of protoplasts kept in culture into entire plants has been described in Evans, et _al., "Protoplast Isolation and Culture", in Handbook of Plant Cell Culture, 1: 124-17Ei (MacMillan Publishing Co. New York 1983); MR Davey, "Recent Developments in the Cul-ture and Regeneration of Plant Protoplasts", Protoplasts, 1983 - Lecture Proceedings, pages 19-29, (Birkh~user, Basle 1983); PJ Dale, "Protoplast Culture and Plant Regeneration of Cereals and Other Recalcitrant Crops", in Protoplasts 1983 - Lecture Proceedings, pages 31-41, (Birkhduser, Basle 1983); and H Binding "Regenera-tion of Plants", in Plant Protoplasts, pages 21-37 (CRC Press, Boca Raton 1985).
Regeneration differs from plant species to plant species. In general, however, first of all a suspension of transformed protoplasts, cells or tissue containing numerous copies of the protection factor genes is prep-ared. Starting with these suspensions, the induction of the embryo formation can then be carried out. The embryos are allowed to develop as far as the stage of maturity and germination, as is also the case with naturally occurring embryos. The culture media normally contain various amino acids and hormones, such as, for example, auxins and cytokinins. It has also proved advantageous to add glutamic acid and proline to the medium, especially in species such as maize and alfalfa.
Shoot and root formatiora generally take place simultan-eously. Efficient regeneration depends especially on the medium, t:he genotype and on the previous history of the culture. If these three variables are sufficiently controlled, z-egeneration is completely reproducible and repeatable.
In view of ne:w developments in the field of in vitro cultivation of plants, primarily in the area of plant regeneration, it has meanwhile become possible to regen-erate whole plants, starting from plant protoplasts, also in the case of representatives of the Gramineae family. Examples of successful regeneration experiments with Gramineae are described, inter alia, 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 maize protoplasts (Biotech-nology, 6: 56-59, 1988).
Accordingly, there are especially preferred within the scope of this invention also representatives of the Gramineae family, such as, for example, plants that are planted over a large area and give high yields.
Examples that may be mentioned are: maize, rice, wheat, barley, rye, oats, millet or sorghum and also meadow grasses.
Examples of other target crops that are especially preferred for use in the process of the invention are, therefore, plants of the genera Allium, Avena, Hordeum, Ory_z,a, Panicum, Saccharum, Secale, Setaria, Sorghum, Triticum, Zea, Musa, Cocos, Phoenix and Elaeis.
Mature plants that have been raised from transformed plant cells a:re crossed with themselves for the purpose of seed production. Some of the seeds contain the genes -- ~ I34D ~~~

responsible i:or an increased protective action in a ratio that a}:actly obeys the established laws of heredity. These seeds can be used for the production of virus-resistant plants. The degree of virus-resistance achieved by t:he transformed plants can be determined by artificial infection with pathogenic virus strains.
Homozygous lines can be obtained by repeated self-fertilisation and production of inbred lines. These inbred lines can then be used in turn for the devel-opment of virus-resistant hybrids. In this process, a virus-resistant inbred line is crossed with another inbred line in order to produce virus-resistant hybrids.
The present invention relates also to parts that can be obtained from regenerated plants, such as, for example, blossom, seeds, leaves, branches, fruits, etc., provided that these parts contain virus-resistant cells. The invention relates also to progeny (including hybrid progeny), varieties and mutants of the regenerated plants.
In order to illustrate the rather general description and for a better understanding of the present invention, reference will now be made to a few specific working examples which are not of a limiting nature unless specified as such.

Non-limiting Exemplary Embodiments General recorlbinant DNA technigues Since many oi= the recombinant DNA techniques used in this invention are routine for the skilled person, a brief description of these generally used-techniques is given here rather 'than at the points in the text where they appear. Unless additionally indicated, all of these methods are described in the reference work by Maniatis et al., ('1982).
A. Cleaving with restriction endonucleases The reaction mixture will typically contain about 50 to 500 ~g/ml DNA in the buffer solution recommended by the manufacturer, New England Biolabs, Beverly, MA.. From 2 to 5 units of restriction endonuclease are added for every ~g of DNA and the reaction mixture is incubated at the temperature recommended by the manufacturer for from one to three hours. The reaction is stopped 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 to 106 of the Maniatis et a.l. reference.
B. Treatment of the DNA with polymerase to produce blunt ends 50 to 500 ~g/ml DNA fragments are added to a reaction mixture in th.e buffer recommended by the manufacturer, New England H.iolabs. The reaction mixture contains all four deoxynucleotide triphosphates in concentrations of 0.2 mM. The reaction is carried out for 30 minutes at 15°C and is then stopped by heating for 10 minutes at 65°C. For fragments obtained by cleaving with restric-tion endonuclE;ases that produce 5'-cohesive ends, such as EcoRI and BamHI, the large fragment, or Klenow fragment, of I)NA po:lymerase is used. For fragments obtained by endonucleases that produce 3'-cohesive ends, such as PstI and SacI, T4 DNA polymerase is used. The use of these t:wo enzymes is described on pages 113 to 121 of the Maniatis et al, reference work.
C. Agarose ge7_ electrophoresis and isolating DNA
fragments from gels Agarose gel e7_ectrophoresis is carried out in a horizontal apparatus as described on pages 150 to 163 of the Maniatis et al. reference work. The buffer used is the Tris-borate buffer described therein. The DNA
fragments are stained with 0.5 ~g/ml ethidium bromide which either i.s present in the gel or tank buffer during electrophoresis or is added after electrophoresis. The DNA is made visible by illumination with long-wave ultra-violet light. If the fragments are not to be separated from the gel, an agarose that gels at low temperature, obtainable from Sigma Chemical, St. Louis, Missouri, is used. After electrophoresis, the desired fragment is excised, placed in a small plastics tube, heated at 65°C', for about 15 minutes, extracted three times with phenol and precipitated twice with ethanol.
This method ha.s been changed slightly with respect to the method described by Maniatis et _al. on page 170.
Alternatively, the DNA can be isolated from the agarose with the aid o~f the Geneclean Kit (Bio 101 Inc., La Jolla, CA, USA.) .
D. Addition of synthetic linker fragments to DNA ends If it is desired to add a new endonuclease cleavage site onto the end of a DNA molecule, the molecule is, if 1340r1~~

necessary, first treated with DNA polymerase in order to produce blunt ends as described in the section above.
About 0.1 to 'i .0 fag of this fragment is added to about ng of phosphorylated linker DNA, obtained from New England Biolabs, in a volume of from 20 to 30 )Z1 with 2 ~1 of T4 DNA ligase from New England Biolabs, and 1mM
ATP in the bu:Efer recommended by the manufacturer.
After incubating overnight at 15°C, the reaction is stopped by heating for 10 minutes at 65°C. The reaction mixture is di:Luted to about 100 )Z1 in a buffer that is correct for the restriction endonuclease that cleaves the synthetic linker sequence. Approximately from 50 to 200 units of this endonuclease are added thereto. The mixture is incubated at the appropriate temperature for from 2 to 6 hours and then the fragment is subjected to agarose gel a:Lectrophoresis and isolated as described above. The resulting fragment will now have ends whose endings have been produced by cleaving with the restric-tion endonuclease. These ends are usually cohesive, so that the resu7lting .fragment can then readily be linked to other fragments having the same cohesive ends.
E. Removal of 5'-terminal phosphates from DNA fragments During the plasmid cloning steps, treatment of the vector plasmi~i with phosphatase reduces the recircul-arisation of t:he ver_tor (discussed on page 13 of the Maniatis et al., reference work). After cleaving the DNA with the correct restriction endonuclease, one unit of calf intestinal alkaline phosphatase, obtained from Boehringer-Mannheim, Mannheim, is added. The DNA is incubated for one hour at 37°C and then extracted twice with phenol arid precipitated with ethanol.

38 - 134o~s9 F. Linking of the DNA fragments If fragments having complementary cohesive ends are to be linked to one another, about 100 ng of each fragment are incubated with about 0.2 unit of T4 DNA ligase from New England B:iolabs in the buffer recommended by the manufacturer :in a reaction mixture of from 20 to 40 ~Z1.
The incubation is carried out for from 1 to 20 hours at 15°C. If DNA fragments having blunt ends are to be linked, they <~re incubated as described above except that the amount of T4 DNA ligase is increased to from 2 to 4 units.
G. Transformation of DNA in E. coli E. coli strain HB101 is used for most experiments.
DNA is introduced into E. coli using the calcium chloride method described by Maniatis et al., pages 250 to 251.
H. Screening of E. coli for plasmids After transformation, the resulting colonies of E.
coli are examined for the presence of the desired plasmid by a rapid plasmid isolation process. Two commonly used processes are described on pages 366 to 369 of the Maniatis et al. reference work.
I. Large-scale isolation of plasmid DNA
Processes for the large-scale isolation of plasmids from E. coli are described on pages 88 to 94 of the Maniatis et al. reference work.

134~~~69 J. Cloning in M13 phage vectors In the following description, the double-stranded replicative form of the phage M13 derivatives is used for routine methods, such as cleavage with restriction endonuclease, linking etc..
Example 1: Transactivation of a CAT gene in tobacco ~~otoplasts 1.1 Construction of the plasmid pHS1 The EcoRV/Eco~RI fragment of the CaMV strain CM4.184 (Dixon L et al., Virology, 150: 463-468, 1986), which comprises the 35S promoter region, the leader sequence, the reading frame ORF VII, the small inter-genomic regie~n and the first 16 codons of the reading frame ORF I, is isolated from a polyacrylamide gel and then treated with the Klenow fragment of DNA polymerase (hereinafter abbreviated to "Klenow") in order to fill-_in the F;coRI cohesive ends.
This EcoRV/EcoRI fragment isligated (Fig. 1A) with the large SmaI/EcoRV fragment of plasmid pDH51 described in Pietrzak et al., 1986 (Pietrzak M et al., Nucl. Acids Res., 14: 5857 - 5868, 1986). This construct is cloned in E. coli strain DH1. Subsequently, restriction analysis is used to determine those transformants in which the EcoRV restriction site has been re-established whilst the SmaI site is missing and which have restric-tion fragments of a corresponding size (Fig. 1B).
The resulting plasmid is then cleaved with XbaI in the region of the polylinker region, and the cohesive ends are ~ f i lled-in. us inch "Klenow" . A Hind I I I fragment that carries the coding region of bacterial chloramph-enicol transac:etylase (CAT) (Alton NK et Vapnek D, Nature, 282: 864-869, 1979) is isolated and the cohesive ends are filled-in with "Klenow".
The supplemented Hind III and XbaI fragments are then ligated with each other and used for the transformation of the E, coli strain DH1. The resulting transformants that have a rE:-established XbaI restriction site and also the correct orientation of the insert are selected.
Accordingly, t:he suitable genetic construct possesses the complete C:aMV 3aS enhancer/promoter region, the leader sequence, the reading frame ORF VII, the small intergenomic region, 16 codons of the reading frame ORF
I, 17 codons of linker and spacer sequences, the coding region of the bacterial CAT gene, spacer sequences, a CaMV polyadenylation site and also sequences of the plasmid pUC18 (Norrander J et al., Gene 26: 101 - 106, 1983).
The ORF I, spacer codons and CAT codons together form a fused reading frame. The resulting plasmid confers on transformed plant protoplasts a CAT activity that is ~ - 20 ~ of that of the control plasmid pDW2 (Pietrzak et al., 1986, supra).
1.2 Transformation of tobacco ~rotoplasts Tobacco protoplasts of Nicotiana tabacum c.v. Petite Havana are produced from a tobacco suspension culture by conventional methods (Potrykus I and Shillito RD, Methods in Enzymoloqy, Vol. 118, Plant Molecular Biology, eds. A. and H. Weissbach, Academic Press, Orlando, 1986). Under sterile conditions, completely developed leaves of six-week-old shoot cultures are removed and thoroughly wetted with an enzyme solution of the following composition:

1340'~~~~

Enzyme solution: H20 ~ 70 ml sucrose ~ 13 g Macerozyme R 10 1 g cellula~ 2 g "Onozuka" R 10 (Yakult Co. Ltd., Japan) Drisellase (Chemische Fabrik Schweizerhalle, Switzerland) 0.13 g 2(n-morpholine)ethane-sulphonic acid (MES) 0.5 ml pH 6.0 Leaves are then cut into squares of 1 - 2 cm2 and floated on the above-mentioned enzyme solution. Incubation is carried out overnight in the dark at a temperature of 26°C. This mixture is then stirred gently and incubated for a further 30 minutes until digestion is complete.
This suspension is then filtered through a steel sieve of mesh width 100 Vim, rinsed thoroughly with 0.6M
sucrose (MES, pH 5.6) and subsequently centrifuged for minutes at 4000 - 5000 rev/min. The protoplasts collect on the surface of the medium which is then drawn off from under the protoplasts, for example using a sterilised injection syringe.
The protoplasts are resuspended in a K3A medium [sucrose (102.96 g/1); xylose (0.25 g/1); 2,4-D (0.10 mg/1); NAA
(1.00 mg/1); BAP (0.20 mg/1); pH 5.8] that contains 0.4M
sucrose.
The protoplasts are then washed (3 times) by flotation of the protoplasts and removal of the medium in the manner described above.
Tra c~e Ma r ,~-Transformation of the protoplasts with plasmid DNA is carried out by means of direct gene transfer (Paszkowski J et al . , EMFiO J. , 3 : 271 7 - 2722, 1 984 ) using the electroporati.on technique (Fromm M et al., Proc. Natl.
Acad. Sci. U~>A, 82: 5824 - 5829, 1985).
The electroporation of the tobacco protoplasts is carried out i.n a disposable semimicrocuvette lined with aluminium foil (Greiner, Nuremberg).
In said cuvetae, the tobacco protoplasts are incubated under sterilE: conditions, in a population density of 2 x 106 protoplasts/ml, with 15 ~g of plasmid DNA in 0.7 ml of electroporation buffer (Fromm et al., 1985, supra) of the: following composition:
f final concentration 10mM Hepes pH 7.2 -150mM NaCL
-5mM CaCl2 -0.2M mannitc>1.
The electroporation of the protoplasts is carried out by discharging a capacitance of 820 )ZF which had been charged beforehand at 220 V (Fromm et al., 1985, supra).
After an incubation period of 10 minutes at 4°C and subsequent incubation for a further 10 minutes at room temperature, the treated protoplasts are diluted with ml of AA medium of the following composition:

- 43 _ AA medium for 1 litre -37 g/1 MgSO~~ 10 ml -17 g/1 KH2P04 20 ml -44 g/1 CaCl~, 10 ml -KC1 2 .95 g -(1MS) EDTA 5 ml -(MS) FeCl3 5 ml -(MS or NT) microelements 1 ml -1000 x vitamins (;gee below) 1 ml -100 x amino acids (see below) 10 ~ml -100 x micro~;ugar (see below) 10 ml -sorbitol 63 g -22,4-D, 20 mg/100 ml 5 ml -kinetin, 20 mg/100 ml 1 ml -3GA3, 20 mg/100 ml 0.5 ml -pH 5.8 to 6 (NaOH), osmotic pressure 530-570 mOs 1000 x vitamins (100 ml):
-inositol 2 g -nicotinic acid 50 mg -thiamine 40 mg -pyridoxine 10 mg -MES 1 bi pH 7 1 ml made up with H20 to 100 ml 1 MS = Murashige and Skoog Medium (Murashige T and Skoog F, Physiol. Plant, _15: 473, 1962) 2 2,4-D = 2,4-dichlorophenoxyacetic acid 3 GA3 = gibberellic acid - 4 4 - ~. 3 4 C1'~ ~ 9 100 x amino acids mix:
-glutamic acid 43.8 g -aspartic acid 13.3 g -arginine-HCI. 8,7 g -glycine 0.375 g -pH approx . Ei , made up with H20 to 500 m1 100 x microsugar mix:
-xylose 1.5 g -arabinose 1~.5 g -glucose 1,8 g -inositol 0.8 g made up with H20 to 100 ml The various s;olutians are sterilised by autoclaving (amino acids mix) or by sterile-filtration.
The protoplasts are then incubated in the dark for 2 days at 28°C. The cells are then lysed using ultrasound anal extracted in accordance with the method described by Fromm et al., 1985, supra.
1.3. CAT assay The resulting extract is subsequently examined, using 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 make it clear that radioactively labelled acetylchloramphenicol is detectable in the chromatogram of the protoplasts 45 _ 134~7~
treated with plasmid pDW2, i.e. that the protoplasts have a CAT activity.
In the cells treated with plasmid pHS1, on the other hand, no corresponding activity could be detected.
If, in addition, 15 ~g of CaMV DNA are added to the electroporation mixture containing tobacco protoplasts and plasmid pHS1, then the pHS1-treated cells too exhibit CAT activity.
Control experiments carried out in the same manner using calf thymus I>NA gave negative results (no CAT activity), however.
These result:. clearly demonstrate, therefore, that an expression in the position of the CAT reading frame in plasmid pHS1 takes place only in the presence of virus DNA and, accordingly, that induction of CAT activity is possible by using virus DNA.
In place of t:he CAT gene in plasmid pHS1 it is possible to use any gene that is suitable within the scope of the present invention, such as, for example, a protection factor gene.
Example 2: Transactivation of a CAT gene in Brassica na~pus plants 2.1. Construction of the plasmid ~CIB 200 TJS75kan is produced by digesting the plasmid pTJS75 (Schmidhauser and Helinski, J. Bacteriol., 164:
446-455, 1985) with the restriction enzyme NarI to obtain the tEaracycline-resistance gene and subsequently inserting an AccI fragment of pUC4K (Vierra and Messing, Gene, 19: 259--268, 1982) that carries the NptI gene.
pCIB200 is then produced by linking XhoI linkers with the EcoRV fragment of pCIB7 [this fragment contains the left and righi~ 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 XhoI-digested fragment into the SalI cleavage site of TJS75kan.
2.2. Production of the cointegrated vector pCIB200/HS1 Plasmid pCIB200, whose construction is described in Example 2.1, and plasmid pHS1 from Example 1.1 are used as starting material for the production of the cointe-grated vector pCIB200/HS1.
Both parent p:Lasmids are first of all cleaved with the restriction enzyme KpnI, mixed with each other in one of the incubation buffers customarily used and then'ligated with each other by adding suitable enzymes (ligases).
E. coli HB101 cells are then transformed with this mixture by thE: method described by Maniatis et al., 1982 (see Maniatis et al., pages 250-251) and ampicillin/kanamycin-resistant E. coli colonies are selected.
Resistant colonies are examined with the aid of restriction analysis for the presence of the desired cointegrated plasmid pCIB200/HS1 by resolving the latter into the linear forms of the parent plasmids again by KpnI.

I34~~~~9 2.3. Transferring the cointegrated vector pCIB200/HS1 into Agrobacterium tumefaciens by "triparental mating"
The starting materials (parents) used for the "triparental mating" are E. coli HB101 (pRK2013) [described by Tepfer and Casse-Delbart, Microbio7_. Sci., 4:24, 1987], E, coli HB101 (pCIB200/HS1) that contains the above-described cointegration vector pCIB200/HS1, and Agrobacterium tumefaciens C58 (pCIB542).
Plasmid pCIB542 is an apathogenic helper plasmid whose construction has been dE:scribed in detail in European Patent Application EP 256 223.
The crossing of the 3 parent strains and, therewith, the transfer of the cointegrated plasmid pCIB200/HS1 into Agrobacterium is carried out in accordance with the method described by Tepfer and Casse-Delbart, 1987. The Agrobacteria resulting from this "tri.parental mating" are designated C58 (pCIB542:pCIB200/HSl). They thus possess, in addition to the helper plasmid pCIB542, also the cointegrated plasmid pCIB200/HS1.
2.4. Production of transgenic Brassica napus plants Petioles of Brassi~~a napus are transformed by the method Described in Guerc:ze et al., Mol. Gen. Genet., 206: 382-386, 1987. Instead of the A~crobacterium rhizogenes strains used therein, the tranfo rmation of Brassica napus is in this case carried out using ~~ mixture of Agrobacterium rhizogenes and Agrobacterium tume:Eaciens C58 (pCIB542:pCIB200/HS1) the production of which has been described in Example 2.3. The ratio of A. rhizogenes to A. tumefaciens C58 (pCIB542:pCIB200/HS1) is preferably 9:1.
X

1340~~~

2.4.1. Cultiv<~tion of the Agrobacterium strains and preparation o:E the inoculation solution Before inocul<~tion, the Agrobacteria strains are plated out on YEB medium [Bacto beef extract 5 g/1, Bacto yeast extract 1 g/1, peptone 5 g/1, sucrose 5 g/1, MgS04 2mM, pH 7.2] that Jzas previously been enriched with 100 ~g/ml ampicillin and 25 ~Zg/ml kanamycin and solidified with 1.5 ~ agar. After a cultivation period of 48 hours at a temperature o:E 28°C, single colonies are used to inocul-ate liquid cu:Ltures. This is carried out in 100 ml Erlenmeyer flasks in a liquid YEB medium that has been enriched with antibiotics in the concentration given above. Cultivation is carried out at a temperature of 28°C on a gyx<<tory shaker at a speed of 200 rev~min.
The cultivation period is 24 hours.
Subsequently, a second subcultivation is carried out in liquid medium with a dilution ratio of 1:20 under otherwise identical conditions. The incubation period is in this case 20 hours.
These measures resu:Lt in a population density of living Agrobacteria of about 109/ml.
The bacterial cells are harvested by centrifugation and are then resuspended in an equivalent volume of a 10mM
MgS04 solution without antibiotics.
2.4.2. Inoculation of Brassica napus etioles Petioles of Brassica napus are first surface-sterilised foi: 10 minutes in a calcium hypochlorite solution (70 g/1) and rinsed with distilled water. They are then cut into small pieces 6-10 mm thick and transferred to Petri dishes. The latter contain i34opr~~

sterilised tap water or a Monnier salt solution (Monnier, Revue de Cytologie et de Biolo ie Ve etales, Vol. 31: 78, '1976) r~uhich is solidified by the addition of 0.7 ~ agar,.
For the inocu7lation, well grown Agrobacteria cultures having an OD6~;0 of about 1.00 (this corresponds to a number of about 109 cells/ml) are used.
For the mixed inoculation of A~robacterium rhizogenes and Agrobactex-ium tumefaciens the bacteria are first of all cultivated separately until the wecessary cell density has bE:en achieved and are mixed, in a ratio of 9:1 (A. rhizoce! nes,:A. tumefaciens) only immediately before inoculation :Ls envisaged.
When inoculation has been carried out, the Petri dishes are sealed well and incubated for about 2 to 3 weeks at room temperature. Successful transformation with _A.
rhizogenes can then be recognised very easily by the appearance of hairy rootlets.
Transformation: by A. tumefaciens, on the other hand, can be detected only by means of the kanamycin resis-ta nce conf erred .
In the ratio o~f 9:1 used here, up to 95 $ of the rootlet colonies have kanamycin resistance caused by A, tumefac-iens transformation.
2.5. Regeneration of entire lants The regeneration of entire plants from root colonies is carried out in. accordance with known processes, as are described, for example, in David _et _al., Biotechnolo y, 1: 73, 1982, Tepfer and Casse-Delbart, 1987 or in I340~~9 Guerche et al., Mol. Gen. Genet., 206: 382-386, 1 987 .
Root tissue is. transferred onto a Monnier medium that contains 0.36 ~M 2,4-D and 0.72 ~M kinetin as additive and is solidified with 0.8 ~ agar. After a cultivation period of 4 weeks; the resulting callus tissue is transferred to a liquid, hormone-free Monnier medium and incubated on a~ gyratory shaker at 150 rev~min and at a temperature of from 22°C to 25°C.
The callus disintegrate and forms a suspension culture from which embryos differentiate after about 1 month. These embryos are further cultivated in Petri dishes on hormone-free N(onnier medium where they further differ-entiate into small plants.
2.6. Transacti.vation of transgenic plants by CaMV
Transgenic Bra.ssica nagus plants are infected with cauliflower mosaic virus according to Lebeurier et al., 1980.
The CAT activity before and after CaMV infection has been carried out is determined in leaf extracts of transgenic Bra~ssica napus plants in accordance with the method de~:cribed by Fromm et al., 1982.
In that method, first of all leaf material (50 mg to 100 mg) is conuninuted in an extraction buffer (10 ~ v/v glycerol, 0.01 ~ w/v sodium dodecylsulphate, 5 ~ v/v meraptoethanol., 0.005 $ bromophenol blue, 0.0625 M
Tris pH 6.8). The particulate part of the extract so otained is removed by centrifuging. The supernatant is heated for 10 minutes at a temperature of 65°C (inact-ivating plant substances that impair the CAT activity), then cooled to room temperature and used in the CAT
assay described under section 1.3. The plants trans-formed with the HS1 construct exhibit an increased ability of th~~ CAT gene to be activated which may be as much as 50 times that of the controls.
Example 3: Tr<insactivation of CaMV 19S transcripts 3.1. Construct=ion of p19SCAT
The vector usE;d is plasmid pDH19u which is composed of the CaMV 35S ~~romoter-terminator cassette from plasmid pDH51 (Pietrz<~k M et al., Nucl. Acids Res., 14, 5857-5868, 1986) and plasmid pTZ19u (Mead DA, et al., B-, Prot. Enginee~_-ing, 1: 67-74, 1986). For this, pDH51 is digested with EcoRI, the resulting fragments are treated with ~~4 DNA polymerase and the CaMV promoter-terminator fragment is cloned into the large PvuII
fragment of p'.~Z19u. After digesting pDH19u with NcoI
and SmaI the projecting NcoI end is filled-in using "Klenow" DNA polymerase and the large fragment is isolated. As the C~aMV 19S promoter fragment, the genome region from position 4875 to position 5355 of CaMV
(strain CM4.184, Dixon L et al., Virology, 150: 463-468, 1986) is isolated as MnlI fragment and ligated with the large NcoI/SmaI fragment of pDH19u. As a result, plasmid p19S .Ls obtained which contains the CaMV 19S
promoter, pol~rlinker sequences and the CaMV transcrip-tion terminator. Plasmid pl9S is opened with BamHI and SalI and ligated with the small BamHI/SalI CAT fragment from plasmid pZL811 (along, DT et al., J. Virol., _55: 223-231, 1985). In thE: last step the ATG start codon of the CAT
reading frame is fused with the ATG start codon of the CaMV protein VI by deletion mutagenesis using an oligo-nucleotide. Plasmid V374 (see Figure 4) is used as the 13~O~~g transactivator plasrnid. It contains the EcoRV fragment from CaMV (strain CM4.184), the gene VI, cloned into the SmaI restriction site of plasmid pDH51.
3.2 Transformaaion of Brassica raga protoplasts Turgescent leaves of: Brassica raps are first surface-sterilised by placing them in a 0.5 ~ calcium hypo-chlorite solution for 30 minutes. The leaves are then washed 5 times with sterile water for 1 to 2 minutes each time.
The leaves so treated are cut roughly into pieces and introduced into an Enzyme solution and are there cut into strips about 1 mm in width. Incubation is carried out overnight in the dark at a temperature of 26°C.
Enzyme solution: mannitol 6.15 g (100 ml) CaCl2 0.63 g cellulose 1.0 g "nnozuka" R10 (Yakult Co . Ltd . , Japan ) Macerozyme R10 0.1 g pH 5.4 After incubat9_on overnight, the enzyme solution, containing protoplasts and leaf remnants, is poured over a 50 ~m steel sieve and filtered. The protoplasts separated in i:his manner are transferred into centrifuge tubes and ceni:rifuged for 10 minutes at 4000-5000 rev/min.
The supernatant (enzyme solution) is discarded and the pelleted protoplast;s are taken up again in 10 ml of electroporation buffer. The whole procedure is repeated and the protoplast suspension is adjusted to 3 x 106/ml.

X34~~~~

The electroporation. of Brassica raps is carried out in a manner analogous to that described for tobacco proto-plasts in Example 1.2., but in this case the capacitors are charged with 180 V.
Incubation of the electroporated Brassica protoplasts is carried out in A. medium (see below) instead of the AA
medium used for the tobacco protoplasts.
A medium 1 litre:
A macro 100 ml A micro ~ B5 micro 1 ml MS iron each 5 ml A vita ~ B5 vita 10 ml A OS 25 ml glucose 80 g/1 xylose 250 mg/1 casein hydrol:ysates500 mg/1 (= N-Z-amines) 42,4-D 1 mg/1 SNAA 0.1 mg/1 66BAP 0.5 mg/1 pH 5.7~-5.8 Stock solutions (A medium):
A macro (per '100 ml~:
NaH2P04~1 75 mg KH2P04 170 mg KN03 2200 mg NH4N03 600 mg 4 2,4-D 2,4-dichlorophenoxyacetic acid NAA naphthyl-1-acetic acid 6 6BAP 6-benzylaminopurine i3407~~

(NH4)2S04 75 mg MgS04 ~ 7 31 0 mg CaCl2~2 295 mg A micro (per 100 ml):
MnS04 1 1 000 mg Na2Mo042 25 mg H3B03 300 mg ZnS047 200 mg CuS045 2.5 mg CoCl26 25 mg KI 75 mg A vita (per 100 ml):
inositol 1000 mg nicotinic acid 10 mg pyridoxineHC1 10 mg thiamineHC1 100 mg A OS ( per 1 00- ml ) sodium pyruvate 20 mg citric acid 40 mg malic acid 40 mg fumaric acid 40 mg (pH adjusted to 5.5 with NH40H) 3.3 Results The induced C.AT activity is measured 20 hours after electroporation has taken place, using the CAT assays described in section 1~3. Analysis shows that the electroporation with p19SCAT results in only very low CAT expression. If, however, a plasmid from which the CaMV protein VI can. be expressed is electroporated into the protoplasts together with p19SCAT, then an approxi-1340~G0 mately 20-fold stimulation of the CAT activity is to be observed.

Claims (42)

1. A process for the protection of plants against a virus infection and/or its sequelae, characterised in that an inducible virus resistance due to a gene encoding a virus resistance factor under the control of a promoter and/or a virus specific control region which regulates expression of the control factor is introduced into said plants using genetic engineering techniques.

2. A process according to claim 1, characterised in that an inducible protection factor gene is introduced into the plant, which gene is caused to express said protection factor when a virus infection begins by the addition of suitable inducers and/or by the infesting virus itself.

3. A process according to claim 2, characterised in that the protection factor gene is operably linked with virus-specific control sequences and with expression signals that are active in plant cells, so that the induction of the protection factor gene and hence the production of the protection factor is controlled by the infecting virus itself.

4. A process according to claim 3, characterised in that the virus-specific induction of the protection factor gene is effected by means of a transactivation of transcription.

5. A process according to claim 4, characterised in that the virus-specific induction of the protection factor gene occurs at the post-transcriptional level.

6. A process according to claim 5, characterised in that the virus-specific induction of the protection factor gene is effected by means of -56a-a) a control of the mRNA, elongation b) a control of the mRNA, transport from the nucleus into the cytoplasm, c) a control of mRNA splicing, d) a control of mRNA polyadenylation, e) a control of the initiation of translation, or f) a differentiated use of different ORFs on a polycistronic RNA.

7. A process according to claim 3, characterised in that the virus-specific induction of the protection factor gene is effected by post-translational modification of the protein product.

8. A process according to claim 2, characterised in that the inducible protection factor gene is operably linked with expression signals that are active in plant cells and that ensure the expression of said protection factor gene in the plant.

9. A process according to claim 2, characterised in that said protection factor gene is a naturally occurring DNA sequence coding for a protection factor.

10. A process according to claim 9, characterised in that said DNA sequence codes for a protection factor that differs from a naturally occurring protection factor but still performs substantially the same protective function.

11. A process according to claim 9, characterised in that said protection factor is an antiviral substance.

12. A process according to claim 9, characterised in that said protection factor is a cell-toxic substance.

13. A process according to claim 11, characterised in that said antiviral substance is an antivirus-antibody, a PR-protein, a virus-specific protease inhibitor, a protease, a virus-specific polymerase inhibitor or an interfernon-like protein.

14. A process according to claim 12, characterised in that said cell-toxic substance is a toxin, an active subunit of a toxin, a PR-protein or a protein that naturally triggers a hypersensitive reaction.

15. A process according to claim 14, characterised in that said cell-toxic substance is a ribosome-inactivating protein.

16. A recombinant DNA molecule, characterised in that it confers an inducible virus resistance upon plants due to a gene encoding a virus resistance factor under the control of a promoter andlor a virus specific control region which regulates expression of the control factor.

17. A recombinant DNA molecule according to claim 16, characterised in that it has a DNA sequence that codes for an inducibe protection factor.

18. A recombinant DNA molecule according to claim 17, characterised in that said DNA
sequence coding for a protection factor consists exclusively of genomic DNA, of cDNA or of synthetic DNA, or alternatively of a mixture of said DNAs.

19. A recombinant DNA molecule according to claim 17, characterised in that said DNA-sequence coding for a protection factor gene is operably linked with a control region that ensures induction of the protection factor gene.

20. A recombinant DNA molecule according to claim 19, characterised in that said control region is a virus-specific control region.

21. A recombinant DNA molecule according to claim 19, characterised in that said DNA-sequence coding for an inducible protection factor gene is operably linked with expression signals that are active in plant cells.

22. A recombinant DNA molecule according to claim 20, characterised in that said virus-specific control region comprises ORF VII and the intergenomic region between ORF VII and ORF I of cauliflower mosaic virus.

23. A recombinant DNA molecule according to claim 21, characterised 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 codons of linker and spacer sequences, the region coding for a protection factor gene, spacer sequences, a CaMV polyadenylation site and sequences of the plasmid pUCl8.

24. A recombinant DNA molecule according to claim 21, characterised in that the DNA-sequence coding for an inducible protection factor gene is operably linked with a 19S promoter and a CaMV transcription terminator.

25. A process for the preparation of a recombinant DNA molecule according to claim 16, which process is characterised by the following steps:
a) isolation and cloning of a DNA sequence coding for a protection factor, b) functional linking of said DNA sequence coding for a protection factor with a virus-specific control region in such a manner that the induction of protection factor formation is ensured when a virus infection begins, c) splicing in of the hybrid gene construct described in b) in operable manner between expression signals that are active in plant cells.

26. A process according to claim 25, characterised in that the functional linking between the DNA-sequence coding for a protection factor and the virus-specific control region is effected by replacement with viral coding DNA sequences that are themselves controlled by said virus-specific control regions.

27. A recombinant DNA molecule according to claim 21, characterised in that it is a cDNA that is complementary to RN,A3 of BMV and that contains, incorporated therein, a protection factor gene in place of the DNA region coding for a coat protein.

28. A process according to claim 26, characterised in that said virus-specific control sequences are CaMV control sequences.

29. A process according to claim 26, characterised in that said virus-specific control sequences are BMV control sequences.

30. A process according to claim 26, characterised in that said functional linking is effected by replacing a coding DNA region of ORF I on the CaMV genome by a protection factor gene.

31. A process according to claim 26, characterised in that said functional linking is effected by replacing a coding DNA region of ORF VI on the CaMV genome by a protection factor gene.

32. A process according to claim 26, characterised in that said functional linking is effected by replacing a cDNA region coding for a coat protein that is complementary to the corresponding region on the RNA3 of BMV, by a protection factor gene.

33. A process according to claim 25, characterised in that said expression signals are promoter and termination sequences, and other regulatory sequences of the 3' and 5'-non-translated region that: are active in plant cells.

34. A DNA transfer vector, characterised in that it contains a recombinant DNA
molecule according to claim 16.

35. A DNA expression vector, characterised in that it contains a recombinant DNA
molecule according to claim 16.

36. A host cell, characterised in that it contains a) a DNA transfer vector which contains a recombinant DNA molecule according to claim 16 or b) a DNA expression vector which contains a recombinant DNA molecule according to claim 16.

37. A host cell according to claim 36, characterised in that it is a microorganism.

38. A host cell according to claim 36, characterised in that it is a plant cell.

39. A plant cell, characterised in that it has been transformed with a recombinant DNA
molecule according to claim 16.

40. A plant cell according to claim 39, characterised in that it is a component part of an entire plant

41. A process for the production of virus-resistant transgenic plants, characterised in that a plant or viable parts thereof is (are) transformed with a recombinant DNA
molecule according to claim 16.

42. A use of a DNA molecule according to claim 16 to immunize a plant against undesired virus attack.