AU620039B2 - Inducible virus resistance in 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

i I 620039 S F Ref: 63187 FORM COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952 COMPLETE SPECIFICATION

(ORIGINAL)

FOR OFFICE USE: Class Int Class 9 94 9O 9 9 94 9*94 9 9,4" 9 9* 44 0 9 9* 0 49 .44 8 4 4*4 S44 4 9* Complete Specification Lodged: Accepted: Published: Priority: Related Art: Name and Address of Applicant: Address for Service: Ciba-Geigy AG Klybeckstrasse 141 4002 Basle

SWITZERLAND

Spruson Ferguson, Patent Attorneys Level 33 St Martins Tower, 31 Market Street Sydney, New South Wales, 2000, Australia Complete Specification for the invention entitled: Inducible Virus Resistance in Plants The following statementis a full description of this invention, including the best method of performing it known to me/us 5845/3 5-16553/+/ZFM Inducible virus resistance in plants 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 virusresistant 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.

a The present invention also includes chimeric genetic constructs, cloning vehicles and host organisms and methods of 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.

a l .t r L

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5-16553/+/ZFM Inducible virus resistance in plants The present invention relates to a 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 i to the plants provided with an inducible virus resistance 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 4time and again in considerable losses in yield and in the harvested product in fruit, vegetable and cereal cultivation are caused by plant viruses. These phytopathogenic viruses are transmitted especially by sucking or biting phytophagous insects and nematodes, which act as vectors.

S, Efforts have therefore been made for a very long time t now in plant rultivation to develop suitable processes i for the protection of cultivated plants from virus attack.

One of these measures, which, however, is of only i limited effect and moreover involves considerable risks, has become known by the name "cross protection". This involves the artificial 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 ~c I 2 such as tomato mosaic virus, potato spindle tuber viroid and citrus tristeza virus. (Broadbent L, Ann.

Rev. Phytopathol., 14: 75, 1976; Fernow KH, Phytopathology, 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 possible for a particular virus strain to be perfectly suitable for the protection of one particular S* plant species but to act as a pathogen and cause severe Sdamage in another species.

it In addition, synergistic actions of two or more types of virus have been described which give rise to new, epreviously unknown disease symptoms (Garces-Orejuela C S\ and Pound GS, Phytopathology, 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 genes 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 alfalfa mosaic virus) are incorporated into the plant genome with the aid of recombinant DNA techniques (Bevan MW et al., EMBO 1921-1926, 1985; Abel PP et al., Science,' 232: 738-743, 1986).

Upon expression of the integrated coat protein gene in the plant, the cbat 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 action against infection with the corresponding virus. For example, in transgenic plants that synthesise antisense RNA of parts of the corresponding e viruses, inhibition of the virus expression occurs.

The expression of satellite RNA in transgenic plants also can result in a protective action, presumably S* because of competition with the infecting virus for replicase (Baulcombe DC et al., Nature, 321: 446-449, i 1986).

I 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.

-4- 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 mitochondra.

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 scooe of the present invention by simple means, by providing a system that renders possible the induction of genes based on virus-intrinsic control mechanisms.

According to a first embodiment of this invention, there is provided a process for the protection of plants against a virus infection S 20 and/or its sequelae, which process comprises the following steps: ,:ct isolating a virus specific control region from the genome of a S suitable DNA or RNA virus; constructing a chimeric gene, wherein one or more protection factor gene(s), which i5(are) under the control of plant active expression signals, is(are) operably linked to a virus-specific control region isolated In step in such manner that the induction of the associated protection factor(s) by a suitable inducer or the infecting virus itself is ensured; transforming the above chimeric gene construct into a recipient plant; expressing the Introduced protein factor gene(s) upon addition of a suitable inducer or infection with the appropriate virus.

The present Invention further describes a process for the protection of plants from infection by phytopathogenic viruses, which process Is characterised in that there Is introduced into said plants an inducible protection factor gene that is operably linked with virusspecific control elements and with expression signals that are active in plant cells, so that the expression of said protection factor gene and I LMM/1197v jqs 5 hence the production of the protection factor is controlled by the infecting virus itself.

According to a second embodiment of this invention, there is ;1 provided a recombinant DNA molecule, which upon transformation into a j 5 plant confers an inducible virus resistance upon plants, characterized in I that it comprises a DNA-sequence coding for an expressible protection factor, said DNA sequence being operably linked with a virus-specific control region that ensures induction of protection factor synthesis in the transformed plant by the infecting virus itself via a transactivation mechanism.

According to a third embodiment of this invention, there is provided a process for the preparation of a recombinant DNA molecule which confers an inducible virus resistance upon plants, which process comprises the following steps: isolating a virus specific control region from the genome of a suitable DNA or RNA virus; constructing a chimeric gene, wherein one or more protection j I factor gene(s), which is(are) under the control of plant active j I dexpression signals, is(are) operably linked to a virus-specific control 20 region isolated in step In such manner that the induction of the associated protection factor(s) by the infecting virus itself is ensured.

According to a fourth embodiment of this invention, there is provided a DNA transfer vector, characterised in that it contains a recombinant DNA molecule according to the second embodiment.

According to a fifth embodiment of this invention, there is j lprovided a DNA expression vector, characterised in that it contains a recombinant DNA molecule according to the second embodiment.

According to a sixth embodiment of this invention, there is provided a host cell, characterised in that it contains I 30 a DNA transfer vector according to the fourth embodiment and a DNA expression vector according to the fifth embodiment.

According to a seventh embodiment of this invention, there is provided a transgenic plant and the seeds thereof, which is protected against a virus infection and/or its sequelae, produced by a process according to the first embodiment.

According to an eighth embodiment of this Invention, there is provided a transgenic plant the cells of which are transformed in whole or in part with a recombinant DNA molecule according to the second embodiment, as well as the seeds of said transgenlc plant.

SLMM/GSA/i 1 97v 9 n^B i 5A According to a ninth embodiment of this invention, there is provided a plant cell, characterised in that it has been transformed with a recombinant DNA molecule according to the second embodiment.

According to a tenth embodiment of this invention, there is provided a transgenic plant and the seeds thereof according to the ninth embodiment, which is a monocotyledonous plant.

According to an eleventh embodiment of this invention, there is I provided transformed viable parts of plants according to the seventh, eighth and tenth embodiments, characterised in that they are protoplasts, cells, cell aggregates, callus, seeds, pollen, ova, zygotes or embryos.

According to a twelfth embodiment of this invention, there is provided hybridization and fusion products with the plant material of the invention that still exhibit characteristic properties of the transformed starting material.

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 preferauly exhibits normal growth.

The present invention further describes plants regenerated from I to transformed plant cells containing said recombinant DNA molecule, and to 20 the seeds thereof, and also to the progeny of plants regenerated from said transgenic plant cells, and to mutants and variants thereof.

According to the invention, the expression "mutants and LMM/GSA/1197V A49, 6 variants of transgenic plants" shall be understood as meaning those plants that still possess the characteristic properties of the original plant that that plant obtained 'as a result of transformation with an r-DNA molecule according to the-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.

pP Without constituting any limitation of the subject of o0 the invention, viable parts of plants are defined as 0 being, for example, plant protoplasts, cells, cell 0 it 0 aggregates, callus, seeds, pollen, ova, zygotes or embryos.

The present invention also includes chimeric genetic constructs containing a protection factor gene, and i processes for the production thereof, cloning vehicles 0, and host organisms and methods of conferring an inducible virus resistance upon plants.

S A further aspect of the present invention concerns the use of said recombinant DNA molecules for the "immunisation" of plants against undesired virus attack.

In the following description, a number of expressions that are customary in recombinant DNA technology and in plant genetics is used.

7 7_ 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 gene(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 that 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 that originates from the same species as that into which said gene is inserted.

Synthetic gene(s) or DNA: a DNA sequence that codes for a specific product or products or for a biological *l 'function and that has been produced synthetically.

Plant promoter: a control sequence of DNA expression 4I that ensures the transcription of any homologous or heterologous DNA gene sequence in a plant provided that '1 said Qgno sequence is operably linked with such a promoter.

Over-producing plant promoter (OPP): plant promoter that is capable of causing the expression in a transgenic plant cell of any operably linked functional gene sequence(s) to a degree (measured in the form of the amount of RNA or of polypeptide) that is distinctly higher than that observed naturally in host cells that have not been transformed with said OPP.

Plant: any photosynthetically active member of the 8 kingdom Planta that is characterised by a membraneenclosed 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 differentiated part of a plant, such as, for example, a root, stem, leaf or embryo.

o Protection factor: a one-component or multi-component system that directly or indirectly interacts with infecting viruses within the plant in such a manner as to Sg ensure a protective action against a virus infection and/or its sequelae.

Protection factor gene(s): nucleotide sequence(s) Scoding for a protection factor as defined above.

Virus-specific control elements: regions on the virus o, 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 vector: transfer vehicle, such as, e.g., 27 "i..w 9 a Ti-plasmid or a virus, that enables genetic material into a suitable host the insertion of cell.

A brief description of the Figures is given below: Figure 1 shows the construction of is described in detail in Figure 2A-C show the various possible activation in CaMV.

plasmid pHS1 which Example 1.1.

methods of transof plasmid a.

*0 **9ft aft a a a ft Ia ft ftaiB ft ft ft Figure 3 Figure 4 shows the restriction map pCIB200.

shows the restriction map of plasmid V374.

ft.

8I fc t The present invention relates especially to recombinant DNA molecules that are capable of conferring an inducible 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 interaction 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 sequelae thereof are 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 category is concerned with cell-toxic subs- 10 10 tances which poison and/or kill the cells attacked by the virus and thus isolate the focus of infection from the remaining, healthy tissue. In this case, therefore, the virus's ability to multiply is restricted indirectly by artificially 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 ifdirectly interact with the infecting viruses within the plant so as to ensure a protective action against a virus infection and/or the sequelae thereof.

si Examples of such protection factors, which do not, 9*> however, imply any limitation of the subject of the invention, are: 1) Antiviral substances, such as, antivirusantibodies, PR-proteins (Ahl P et al., Plant Mol.

Biol., 4: 37, 1985), virus-specific protease inhibitors (Jamet E and Fritig B, Plant Mol. Biol., 6: 69-80, 1986), proteases, virus-specific polymerase inhibitors, and interferon-like proteins.

2) Cell-toxic substances such as, toxins, active subunits of toxins, PR-proteins, and other proteins that naturally cause hypersensitivity, such as -glucanase (Mohnen D. et al., EMBO 4: 1631-1635, 1985).

In connection with the present invention, particular importance is attached to the ribosome-inactivating proteins [RIPS] (Lord 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.

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I. l i- 11 Examples of such ribosome-inactivating proteins are abrin (from Abrus precatorius seeds) (Olsnes et al., in: Cohen P. and Van Heyninger S. Molecular Actions of Toxins and Viruses, Biomedical Press, pp. 51-105, 1982), modeccin (from Modecca digitata roots) (Stirpe et al., FEBS Lett., 85: 65-67, 1978), viscumin (from Viscum album leaves) (Stirpe et al., Biochem. 190: 843-845, 1980; Olsnes et al., J. Biol.

Chem., 257: 13263-13270, 1982) and ricin (from Ricinus communis) (Olsnes and Pihl, in: Cuatrecasas P. (Ed.) The Specificity of Action of Animal, Bacterial and Plant .9 Toxins, Chapman and Hall, pp. 129-173, 1976). The too pokeweed toxin (Owens et al., Virology, 56: 390, 1973) "also belongs to this group. All of the compounds mentioned have a great structural and functional similarity.

It has been found especially advantageous if the induction of the protection factor gene and hence of protection factor production 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 S; in the synthesis of the protection factor, which neutralises 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 "~i 12 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 protection against a virus infection. This can be achieved according to the-invention, for example, by linking the protection factor gene to virus-specific control elements and thus placing the expression thereof under the virus infection and/or the sequelae thereof, which process is characterised in that there is introduced .ccinto said plants an inducible protection factor gene that is operably linked with virus-specific control I elements and with expression signals that are active in S plant cells, so that the expression of the protection *factor gene is regulated by the infecting virus itself.

E a A large number of viruses are known to have developed specific control 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 8' 13 been observed almost exclusively in bacterial and animal systems (Calender, Biotechnology, 4: 1074, 1986).

There are fundamentally different levels at which a transactivating 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 mechanisms 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).

S" 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 jagpol-env. The tat gene product is a polypeptide of molecular weight 15,000 that in its structure and properties 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 transactivation is a cooperative binding of a regulator to a t 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 elongation (Grayhack et al., Cell, 42: 259, 1985; Yi-Kao et aj., Nature, 330: 489 1987), of mRNA transport from the nucleus into the cytoplasm, of mRNA splicing, of mRNA polyadenylation (Derse, J. Virol., 62, 1115, 1988), of translation initiation (Jay et al., Proc. Natl. Acad.

I 1 -14 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 processi-ng.

In the case of cauliflower mosaic virus (CaMV), all three of the above-mentioned transactivation levels are relevant.

CaMV possesses a double-stranded (ds) circular DNA genome the replication of which is presumably carried out by means of a reverse transcription mechanism. In that process, two RNA intermediates are formed, a RNA and a 19S RNA, which correspond to two operons on the CaMV genome. The 19S promoter transcribes a subgenomic RNA (19S RNA transcript) which encodes the CaMV protein VI which forms the main portion of the virusspecific 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 RNA. (Pfeiffer P and Hohn T, Cell, 33: 781-789, 1983; Kridl JC and Goodman RM, Bio Essays, 4: 4 8, 1986).

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).

i 15 2) In addition, the use of polycistronic RNA is regulated 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 RNA viruses, a variation of this viral transcriptional transactivation is found. In these cases, production of a subgenomic virus (messenger) RNA occurs starting from the minus strand RNA, catalysed by virus-specific polymerases.

This special form of transcriptional transactivation 4occurs, for example, in brome mosaic virus (BMV) which attacks plants of the monocotyledon group, especially numerous species of gramineae, such as, maize, barley, brome-grass and other wild grasses, and, in '4 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 RNA 2) are essential for the replication of the viral genome in the plant. For example, the repli- J t cation of the third component (RNA a dicistronic RNA coding for a protein of molecular weight 32,000 and Ifor the coat protein, and the production of subgenomic coat protein mRNA is dependent on the presence of the SRNA 1 and RNA 2 transcripts (French et al., Science, 231, 1294-1297, 1986).

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 virusspecific replicase that is formed very early on in the course of the infection cycle (possibly encoded by gen- 16omic 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 assumption 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 specific viral polymerases.

All of these systems mentioned can be used in the S 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- I specific control elements of cauliflower mosaic virus (CaMV) is especially preferred; however, this does not i limit the scope of the present invention in any way but 1 serves merely to demonstrate that the process according to the invention functions.

SThe 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 heterologous DNA sequences that code for one or more protection factors X, 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.

17 i- 17- 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 understood according to the invention as those which origi- .'~.nate from other plant species or from organisms belong- S. ing to another taxonomic unit, for example from microbes or from mammals, or those of synthetic origin, which DNA L1 sequences are capable of achieving the desired protective function against a virus attack in the particular desired plant species.

SThe coding region of the hybrid gene construct can S. also encode a protection factor that differs from a naturally occurring protection factor but that still substantially performs the protective function of the Snatural 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 but especially at least 90 of the active portions of the DNA sequence are homologous. According to this definition of the expression

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i 18 "substantially 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 encoding an amino acid sequence and mediating the desired protective function that meets the disclosed and claimed requirements. Special preference is given to a nucleotide 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 exclusively from genomic DNA, from cDNA or from synthetic DNA. Another possibility is the construction 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.

r; ~i-~Llc~ t 19 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 chimeric gene sequence containing: t one or more gene sequences coding.for one or more foot protection factor polypeptides that, when the gene has been expressed in a given plant cell, produce a protecof tive function against a virus infection and/or the sequelae thereof, and ftf**b ft 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 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 terminator regions and, optionally in addition, regulatory sequences of the and 5'-non-translated regions. The plant regulatory sequences may be heterologous or homo- A 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 I 20 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 dicistronic 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- 4 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 I be used is an over-producing plant promoter. Provided S 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 protection factors in such a manner that the transformed plant p 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 I the small subunit (ss) of carboxylase from soybeans [Berry-Lowe et al., J. Molecular and App. Gen., 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 [see, for example, Genetic Engineering of Plants, an Agricultural Perspective, A. Cashmore, Plenum, New York 1983, pages 29-38; Coruzzi G et al., The Journal i i 1 21 of Biological Chemistry, 258: 1399 (1983) and Dunsmuir P et al., Journal of Molecular and Applied 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 synthesised 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 promoter, a leader sequence, the CaMV reading frame VII, a CaMV reading frame I protection factor gene fusion I and a transcription terminator.

Also preferred within the scope of this invention is a S#t gene construct composed of the CaMV 19S promoter, a k protection factor-coding DNA sequence in the ORF VI of the CaMV genome and a CaMV termination sequence.

i The chimeric gene sequence, which consists of one or more virus-specific control sequences and one or more protection factor 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 (bacteriophage) 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 replication, 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 11 22 transformation in a host cell.

Suitable host cells within the scope of this invention are prokaryotes, including bacterial hosts, such as, e.g. A. tumefaciens, A. rhizogenes, E. coli, S. yphimurium and Serratia marcescens, and cyanobacteria.

Eukaryotic hosts such as yeasts, mycelium-forming fungi and plant cells can also be used within the scope of this invention.

The cloning vector and the host cell transformed with that 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 protection factor gene and use it, i for example, to insert the chimeric gene sequence into the plant cell.

The insertion of DNA into host cells can be carried out "using processes that are known per se. For example, bacterial host cells can be transformed after treatment S' 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.

SThese 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 microinjected directly into the plant cells with the aid of micropipettes for the mechanical transfer of recombinant i- 23 DNA. The genetic material can alternatively be introduced into protoplasts after the latter have been treated with polyethylene glycol. [Paszkowski J et al., EMBO 3: 2717-2722 (1984)].

A further aspect of the present invention relates to the insertion of a protection factor gene into a plant cell by means of electroporation [Shillito R at al., Biotechnology, 3: 1099-1103 (1985); Fromm, M et al., Proc.

Natl. Acad. Sci. USA, 82: 5824 (1985)].

S In this technique, plant protoplasts are subjected to electroporation in the presence of plasmids containing the protection factor gene.

9 4* 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 insertion 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 entire viral DNA genome of CaMV is integrated into a bacterial parent plasmid to give a recombinant DNA molecule that can be multiplied in bacteria. After cloning, the recombinant plasmid is cleaved with tie aid of restriction enzymes either 24 randomly or at quite specific non-essential sites within the viral part of the 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 splicing the protection factor gene sequence into a restriction site that occurs only once.

4 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 gene 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. rhizogenes and transformed At 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. rhizogenes transformation also contain a high proportion of A. tumefaciens Ti-plasmid. This can be achieved according to the invention by applying A. rhizogenes and A. tumefaciens together to the plant material, in known manner, in a i 25 ratio of from 1:1 to 1:100, but preferably of 1:10. The transgenic plant cells are then cultivated under suitable 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 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 S. A. rhizogenes (M Tepfer and F Casse-Delbart, Microbiol. Sci., 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 in a stable manner. [Horsch et al., Science, 233: 496-498 (1984); Fraley et al., Proc. Natl. Acad. Sci.

S' USA, 80: 4803 (1983)].

For plants whose cells are not susceptible to infection with Agrobacterium recourse can be had to the cocultivation 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 11 r 26 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 the 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 Agrobacterium cell. A vir region on a chromosome also induces the transfer of the T-DNA from a vector into a plant cell.

A preferred system for transferring a T-DNA region from an Agrobacterium into plant cells is characterised in that the vir region and the T-DNA region are located on r different vectors. Such a system is known by the name "binary vector system" and the vector containing the

S

t 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 the 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 advantages of a virus-mediated transformation and transformation 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 -27may 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 into the T-replicon is advantageously 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 Sthe 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.

9* Especially preferred within the scope of the present 94 invention is a hybrid genetic construct that 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 I, 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 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 i 28 L et al., Virology, 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 codons of the reading frame ORF I, with the large Sma I/Eco RV fragment of the plasmid pDH51 (Pietrzak M et al., Nucl. Acids Res.,.14: 5857-5868, 1986).

A DNA fragment coding for a protection factor gene can then be spliced into the polylinker region of the 'resulting plasmid using methods that are known per se, rj| producing the hybrid genetic construct characterised in r detail above.

r Another genetic construct that is preferred within the J 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 Stranscription terminator. The reading frame of a t structural gene, preferably of a structural gene coding for a protection factor, is inserted into the resulting S plasmid, between those regulatory CaMV sequences, in such a manner that the 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

:I-I

29 vectors that are active in plants using CaMV control systems which has been described above, the control system characteristic 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 transcription of the minus strand RNA 3 with the participation of virus-intrinsic replicases.

4 0* 4 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.

4 In a manner analogous to the CaMV system discussed above, therefore, it 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 I i 30 with the aid of a suitable phenotypical marker that, in addition to the protection factor gene and the virusspecific control sequences, is a component part of the DNA. Examples of such phenotypical markers, which are Inot 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 transformation by the direct insertion of DNA, by viral or SLE bacterial vectors, such as, CaMV or Agrobact- 1 erium, or by other suitable vectors and can subsequently tI be regenerated into complete plants can be subjected to Ithe process of the invention for the production of I transgenic entire plants containing the transferred S* protection factor gene. There is a steadily growing 4i 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 other types of tree, leguminous plants and vegetables.

The process of the invention is suitable for the transformation of all plants, especially those belonging to the systematic groups Angiospermae and Gymnospermae.

Of particular interest among the Gymnospermae 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- 31ceae, Chenopodiaceae, Rutaceae, Alliaceae, Amaryllidaceae, Asparagaceae, Orchidaceae, Palmae, Bromeliaceae, Rubiaceae, Theaceae, Musaceae or Gramineae families and of the order Leguminosae and, of these, especially the Papilionaceae family. Representatives of the Solanaceae, Cruciferae, Leguminosae and Gramineae are preferred.

Target crops within the scope of the present invention also include, for example, those selected from the group

O

consisting of: Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, e* Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Cichorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Hemerocallis, Nemesia, Pelargonium, Panicum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browallia, Glycine, Gossypium, Lolium, Zea, Triticum and Sorghum, including those selected from the group consisting of: Ipomoea, Passiflora, Cyclamen, Malus, Prunus, Rosa, Rubus, Populus, Santalum, Allium, 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 Sinvention.

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.

32- In the meantime, there have been increasing indications that monocotyledons-too can- be transformed with Agrobacterium, so that, using new experimental strategies that are now becoming available, cereals and species of grasses also are susceptible to a transformation [Grimsley N, et al., Nature, 325: 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: 124-176 (MacMillan Publishing Co. New York 1983); MR Davey, "Recent Developments in the Cul- Sture and Regeneration of Plant Protoplasts", Protoplasts, 1983 Lecture Proceedings, pages 19-29, (Birkhauser, Basle 1983); PJ Dale, "Protoplast Culture and Plant Regeneration of Cereals and Other Recalcitrant Crops", in Protoplasts 1983 Lecture Proceedings, pages 31-41, (BirkhRuser, Basle 1983); and H Binding "Regeneration of Plants", in Plant Protoplasts, pages 21-37 (CRC Press, Boca Raton 1985).

SRegeneration 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 prepared. 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 formation generally take place simultaneously. Efficient regeneration depends especially on i 33 the medium, the genotype and on the previous history of the culture. -If these three variables are sufficiently controlled, regeneration is completely reproducible and repeatable.

In view of new developments in the field of in vitro cultivation of plants, primarily in the area of plant regeneration, it has meanwhile become possible t6 regenerate whole plants, starting from plant protoplasts, also in the case of representatives of the Gramineae S.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, et al., Theor Appl Genet, 73: 16-19, 1986, Yamada, et al., Plant Cell Rep., 5: 85-88, 1986 (rice) and in Rhodes et al., for maize protoplasts (Biotechnology, 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.

S

l 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 L- preferred for use in the process of the invention are, therefore, plants of the genera Allium, Avena, Hordeum, Oryza, Panicum, Saccharum, Secale, Setaria, Sorghum, Triticum, Zea, Musa, Cocos, Phoenix and Elaeis.

Mature plants that have been raised from transformed plant cells are crossed with themselves for the purpose of seed production. Some of the seeds contain the genes t 34 responsible for an increased protective action in a ratio that exactly 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 the transformed plants can be determined by artificial infection with pathogenic virus strains.

Homozygous lines can be obtained by repeated selffertilisation and production of inbred lines. These inbred lines can then be used in turn for the development of virus-resistant hybrids. In this process, a virus-resistant inbred line is crossed with another *e 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 j 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.

J4s r Non-limiting Exemplary Embodiments General recombinant DNA techniques Since many of 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).

I Cleaving with restriction endonucleases The reaction mixture will typically contain about 50 to 500 ug/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 pg 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 0 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 al. reference.

B. Treatment of the DNA with polymerase to produce blunt ends to 500 pg/ml DNA fragments are added to a reaction mixture in the buffer recommended by the manufacturer, New England Biolabs. The reaction mixture contains all four deoxynucleotide triphosphates in concentrations of 0.2 mM. The reaction is carried out for 30 minutes at and is then stopped by heating for 10 minutes at C. For fragments obtained by cleaving with restric- -36 tion endonucleases that produce 5'-cohesive ends, such as EcoRI and BamHI, the large fragment, or Klenow fragment, of DNA polymerase 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 two enzymes is described on pages 113 to 121 of the Maniatis et al. reference work.

C. Agarose gel electrophoresis and isolating DNA fragments from gels SAgarose gel electrophoresis is carried out in a horizontal apparatus as described on pages 150 to 163 of S" the Maniatis et al. reference work. The buffer used is the Tris-borate buffer described therein. The DNA fragments are stained with 0.5 pg/ml ethidium bromide i, which either is present in the gel or tank buffer during electrophoresis or is added after electrophoresis. The S' DNA is made visible by illumination with long-wave ultra-violet light. If the fragments are not to be t 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 has 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 of 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 37necessary, first treated with DNA polymerase in order to produce blunt ends as described in the section above.

About 0.1 to 1.0 pg 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 Fl with 2 pl of T4 DNA ligase from New England Biolabs, and 1mM ATP in the buffer recommended by the manufacturer.

After incubating overnight at 15 0 C, the reaction is stopped by heating for 10 minutes at 65 0 C. The reaction Smixture is diluted to about 100 pl in a buffer that is Scorrect for the restriction endonuclease that cleaves i' the synthetic linker sequence. Approximt .ely from 50 to 200 units of this endonuclease are added thereto. The S* mi.-:.ire is incubated at the appropriate temperature ;or S from 2 to 6 hours and then the fragment is subjected to agarose gel electrophoresis and isolated as described S above. The resulting fragment will now have ends whose endings have been produced by cleaving with the restric- Stion endonuclease. These ends are usually cohesive, so that the resulting fragment can then readily be linked to other fragments having the same cohesive ends.

SE. Removal of 5'-terminal phosphates from DNA fragments During the plasmid cloning steps, treatment of the vector plasmid with phosphatase reduces the recircularisation of the vector (discussed on page 13 of the Maniatis et al. reference work). After cleaving the DUA 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 0 C and then extracted twice with phenol and precipitated with ethanol.

together to the plant material, in known manner, in a ^4 38 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 Biolabs in the buffer recommended by the manufacturer in a reaction mixture of from 20 to 40 pl.

The incubation is carried out for from 1 to 20 hours at 0 C. If DNA fragments having blunt ends are to be linked, they are incubated as described above except I t *that the amount of T4 DNA ligase is increased to from 2 to 4 units.

*4 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 t *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, fe I. Large-scale isolation of plasmid DNA Processes for the large-scale isolation of plasmids from E. coli are described on ages 88 to 94 of the Maniatis et al. reference work.

39 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 protoplasts

V

1.1 Construction of the plasmid pHS1 S1',The EcoRV/EcoRI fragment of the CaMV strain CM4.184 S\(Dixon L et al., Virology, 150: 463-468, 1986), *i which comprises the 35S promoter region, the leader sequence, the reading frame ORF VII, the small inter- .genomic region 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 EcoRT cohesive ends.

SThis EcoRV/EcoRI fragment is ligated (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 J which the EcoRV restriction site has been re-established whilst the SmaI site is missing and which have restriction fragments of a corresponding size (Fig. 1B).

The resulting plasmid is then cleaved with Xbal in the region of the polylinker region, and the cohesive ends are -filled-in using "Klenow". A Hind III fragment that carries the coding region of bacterial chloramph- 40 enicol transacetylase (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, the suitable genetic construct possesses the complete CaMV 35S enhancer/promoter region, the go* leader sequence, the reading frame ORF VII, the small intergenomic region, 16 codons of the reading frame ORF 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 S' fused reading frame. The resulting plasmid confers on transformed plant protoplasts a CAT activity that is 4 ta 10 20 of that of the control plasmid pDW2 t (Pietrzak et al., 1986, supra).

1.2 Transformation of tobacco protoplasts 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 Enzymology, 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: 41 Enzyme solution: H20 70 ml sucrose 13 g Macerozyme R 10 1 g cellulase 2 g "Onozuka" R 10 (Yakult Co. Ltd., Japan) Drisellase (Chemische Fabrik Schweizerhalle, Switzerland) 0.13 g I 2(n-morpholine)ethane- S*t sulphonic acid (MES) 0.5 ml r pH Leaves are then cut into squares of 1- 2 cm 2 and floated St etc t Son the above-mentioned enzyme solution. Incubation is carried out overnight in the dark at a temperature of i 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 pm, 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 xylose (0.25 2,4-D (0.10 mg/1); NAA (1.00 mg/l); BAP (0.20 mg/l); 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.

a a.

aa** a a *a ae Sa a a *c a a .e S,a a.

aP aQ i a *ri 42 Transformation of the protoplasts with plasmid DNA is carried out by means of direct gene transfer (Paszkowski J et al., EMBO 3: 2717 2722, 1984) using the electroporation technique (Fromm M et al., Proc. Natl.

Acad. Sci. USA, 82: 5824 5829, 1985).

The electroporation of the tobacco protoplasts is carried out in a disposable semimicrocuvette lined with aluminium foil (Greiner, Nuremberg).

In said cuvette, the tobacco protoplasts are incubated under sterile conditions, in a population density of 2 x 106 protoplasts/ml, with 15 pg of plasmid DNA in 0.7 ml of electroporation buffer (Fromm et al., 1985, supra) of the following composition: final concentration 10mM Hepes pH 7.2 -150mM NaCL -5mM CaC12 -0.2M mannitol.

The electroporation of the protoplasts is carried out by discharging a capacitance of 820 pF which had been charged beforehand at 220 V (Fromm et al., 1985, supra).

After an incubation period of 10 minutes at 4 0 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: r -43- AA medium for 1 litre -37 g/l MgSO 4 10 ml -17 g/l KH 2

PO

4 20 ml -44 gil CaC1 2 10 ml -KC1 2.95 g 1 MS) EDTA 5 ml FeCl 3 5 ml 0-(MS or NT) microelements 1 ml -1000 x vitamins (see below) 1 ml -100 x amino acids (see below) 10 ml too. -100 x microsugar (see below) 10 ml -sorbitol 63 g 2,-,20 mg/100 ml 5 ml -kinetin, 20 mg/i 00 ml 1 ml 3 GA3, 20 mg/i 00 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 A-pyridoxine 10 mg -MES 1M pH 7 1 ml made up with H1 2 0 to 100 ml 1 MS Murashige and Skoog Medium (Murashige T and 2 Skoog F, Physiol. Plant, 15: 473, 1962) 22,4-D =2,4-dichlorophenoxyacetic acid 3GA3 gibberellic acid

I--

I _J7 p 44 100 x amino acids mix: -glutamic acid -aspartic acid -arginine-HCl -glycine -pH approx. 6, made up with H 2 0 to 500 ml 100 x microsugar mix: -xylose -arabinose -glucose -inositol made up with H 2 0 to 100 ml 43.8 g 13.3 g 8.7 g 0.375 g 4 41 eQ .r Ur 4 4r t Ur 1.5 g 1.5 g 1.8 g 0.8 g The various solutions are sterilised by autoclaving (amino acids mix) or by sterile-filtration.

The protoplasts are then incubated in the dark for 2 days at 28 0 C. The cells are then lysed using ultrasound and 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

I,

treated with plasmid pDW2, i.e. that the protoplasts have a CAT activity.

In the cells treated with plasmid pHSJ, on the other hand, no corresponding activity could be detected.

If, in addition, 15 pg 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 Scalf thymus DNA gave negative results (no CAT activity), however.

These results 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

S..

DNA and, accordingly, that induction of CAT activity is possible by using virus DNA.

In place or the 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 napus plants 2.1. Construction of the plasmid pCIB 200 is produced by digesting the plasmid (Schmidhauser and Helinski, J. Bacteriol., 164: 446-455, 1985) with the restriction enzyme Narl to obtain the tetracycline-resistance gene and subsequently inserting an AccI fragment of pUC4K (Vierra and Messing, 1 t

I

46 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 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 XhoI-digested fragment into the Sall cleavage site of #ee t. 2.2. Production of the cointegrated vector pCIB200/HS1 i e* 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 cointegrated vector pCIB200/HS1.

9 0e I Both parent plasmids are first of all cleaved with the restriction enzyme KpnI, mixed with each other in one of I the incubation buffers customarily used and then ligated with each other by adding suitable enzymes (ligases).

9 t I 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 Scointegrated plasmid pCIB200/HS1 by resolving the latter into the linear forms of the parent plasmids again by Kpnl.

I- ii F""L 47 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, Microbiol. Sci., 4: 24, 1987], E. coli HB101 (pCIB200/HS1) that contains the above-described cointegration vector pCIB200/HS1, and Agrobacterium tumefaciens C58 (pCIB542).

e4 Plasmid pCIB542 is an apathogenic helper plasmid whose construction has been described in detail in European Patent Application EP 256 223 and which is incorporated herein by reference.

*4 S, 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 "triparental mating" are designated C58 (pCIB542:pCIB200/HS1). They thus possess, in addition to the helper plasmid pCIB542, also the conintegrated plasmid pCIB200/HS1.

2.4. Production of transgenic Brassica napus plants Petioles of Brassica napus are transformed by the method described in Guerche et al., Mol. Gen. Genet., 206: 382-386, 1987. Instead of the Agrobacterium rhizogenes strains used therein, the transformation of Brassica napus is in this case carried out using a mixture of Agrobacterium rhizogenes and Agrobacterium tumefaciens 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.

-48 2.4.1. Cultivation of the Agrobacterium strains and preparation of-the inoculation solution Before inoculation, the Agrobacteria strains are plated out on YEB medium [Bacto beef extract 5 g/1, Bacto yeast extract 1 g/l, peptone 5 g/l, sucrose 5 g/1, MgSO 4 2mM, pH 7.2] that has previously been enriched with 100 ug/ml ampicillin and 25 pg/ml kanamycin and solidified with 1.5 agar. After a cultivation period of 48 hours at a temperature of 28 0 C, single colonies are used to inoculate liquid cultures. This is carried out in 100 ml Erlenmeyer flasks in a liquid YEB medium that has been S' enriched with antibiotics in the concentration given above. Cultivation is carried out at a temperature of S| .280C on a gyratory shaker at a speed of 200 rev/min.

0: The cultivation period is 24 hours.

i Subsequently, a second subcultivation is carried out in liquid medium with a dilution ratio of 1:20 under j otherwise identical conditions. The incubation period t «is in this case 20 hours.

These measures result in a population density of living Agrobacteria of about 10 9 /ml.

The bacterial cells are harvested by centrifugation and are then resuspended in an equivalent volume of a 1 0mM MgSO 4 solution without antibiotics.

2.4.2. Inoculation of Brassica napus petioles Pet;.oles of Brassica napus are first surfacesterilised for 10 minutes in a calcium hypochlorite solution (70 g/l) and rinsed with distilled water. They are then cut into small pieces 6-10 mm thick and transferred to Petri dishes. The latter contain 49 sterilised tap water or a Monnier salt solution (Monnier, Revue de Cytologie et de Biologie Vegetales, Vol. 31: 78, 1976) which is solidified by the addition of 0.7 agar.

For the inoculation, well grown Agrobacteria cultures having an OD 680 of about 1.00 (this corresponds to a -number of about 109 cells/ml) are used.

S For the mixed inoculation of Agrobacterium rhizogenes and Agrobacterium tumefaciens the bacteria are first of all cultivated separately until the necessary cell density has been achieved and are mixed, in a ratio of 9:1 rhizogenes:A. tumefaciens) only immediately before inoculation is 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 resistance conferred.

In the ratio of 9:1 used here, up to 95 of the rootlet colonies have kanamycin resistance caused by A. tumefaciens transformation.

Regeneration of entire plants 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., Biotechnology, 1: 73, 1982, Tepfer and Casse-Delbart, 1987 or in i- -C I~ 11 50 Guerche et al., Mol. Gen. Genet., 206: 382-386, 1987.

Root tissue is transferred onto a Monnier medium that contains 0.36 pM 2,4-D and 0.72 pM kinetin as additive and is solidified with 0.8 agar. After a cultivation period of 4 weeks, the resulting callus tissue is s' transferred to a liquid, hormone-free Monnier medium and incubated on a gyratory shaker at 150 rev/min and S at a temperature of from 22 0 C to 25 0

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 i hormone-free Monnier medium where they further differ- Sentiate into small plants.

2.6. Transactivation of transgenic plants by CaMV 1 infection iS Transgenic Brassica napus plants are infected with ia 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 Brassica napus plants in accordance with the method described by Fromm et al., 1982.

In that method, first of all leaf material (50 mg to 100 mg) is comminuted in an extraction buffer (10 v/v glycerol, 0.01 w/v sodium dodecylaulphate, 5 v/v meraptoethanol, 0.005 bromophenol blue, 0.0625 M Tris pH The particulate part of the extract so otained is removed by centrifuging. The supernatant is heated for 10 minutes at a temperature of 65 0 C (inact- 51 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 transformed with the HS1 construct exhibit an increased ability of the CAT gene to be activated which may be as much as 50 times that of the controls.

Example 3: Transactivation of CaMV 19S transcripts r*r 3.1. Construction of p19SCAT I m* The vector used is plasmid pDH19u which is composed of the CaMV 35S promoter-terminator cassette from plasmid pDH51 (Pietrzak M et al., Nucl. Acids Res., 14, 5857- *5868, 1986) and plasmid pTZ19u (Mead DA, et al., B, SProt. Engineering, 1: 67-74, 1986). For this, pDH51 is digested with EcoRI, the resulting fragments are treated with T4 DNA polymerase and the CaMV promoterterminator fragment is cloned into the large PvuII fragment of pTZ19u. After digesting pED19u with NcoI S and SmaI the projecting NcoI end is fil' i-in using "Klenow" DNA polymerase and the large fragment is isolated. As the CaMV 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 ligatedwith the large NcoI/SmaI fragment of pDH19u. As a result, plasmid p19S is obtained which contains the CaMV 19S i promoter, polylinker sequences and the CaMV transcription terminator. Plasmid p19S is opened with BamHI and Sall and ligated with the small BamHI/SalI CAT fragment from plasmid pZL811 (Wong, 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 oligonucleotide. Plasmid V374 (see Figure 4) is used as tho.

II 52 transactivator plasmid. It contains the EcoRV fragment from CaMV (strain CM4.184), the gene VI, cloned into the SmaI restriction site of plasmid pDH51.

3.2 Transformation of Brassica rapa protoplasts Turgescent leaves of Brassica rapa are first surfacesterilised by placing them in a 0.5 calcium hypochlorite 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 c, out overnight in the dark at a temperature of 26 0

C.

Enzyme solution: mannitol 6.15 g (100 ml) CaCl 2 0.63 g cellulose 1.0 g S, "Onozuka" R10 (Yakult Co. Ltd., Japan) Macerozyme R10 0.1 g pH 5.4 After incubation overnight, the enzyme solution, containing ,rotoplasts and leaf remnants, is poured over a 50 pm steel sieve and filtered. The protoplasts separated in this manner are transferred inti centrifuge tubes and centrifuged for 10 minutes at 40, U-600 rev/min.

The supernatant (enzyme solution) is discatr'd and the pelleted protoplasts are taken up again in 1C ml of electroporation buffer. The whole procedure is repeated and the protoplast suspension is adjusted to 3 x 10 6 /ml.

53 The electroporation of Brassica. rapa is carried out in a manner analogous to 'that described for tobacco protoplasts in Example 1 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.

q~O

S.

#5 S C 5*

S

*5

S

*5

'S

S

9*S 5* 5 5 #5 A medium 1 litre: A macro A micro B5 micro MS iron A vita B5 vita A OS glucose xy lose casein hydrolysates N-Z-.amines) 4 2,4-D 5

NAA

66 BAP pH 5.7-5.8 100 ml 1 ml each 5 ml 10 ml 25 ml 80 g/l 250 mg/l1 500 mg/l 1 mg/l 0.1 mg/l 0.5 mg/l

II

Stock solutions (A medium): A macro (per 100 ml): NaH 2

PO

4 KHqPO 4 j

KNO

3

NH

4

NO

3 75 mg 170 mg 2200 mg 600 mg 42,4-D 2,4-dichiorophenoxyacetic acid naphthyl-1 -acetic acid 66BAP 6-benzylaminopurine -54

(NH

4 2

SO

4 75 mg MgSO 4 -7 310 mg CaC12 2 295 mg A micro (per 100 ml): MnSO 4 *1 1000 mg Na 2 MoO 4 *2 25 mg

SH

3

B

3 300 mg ZnS04-7 200 mg 4 CuSO 4 *5 2.5 mg S. CoC1 2 -6 2.5 mg KI 75 mg A vita (per 100 ml): inositol 1000 mg nicotinic acid 10 mg S' pyridoxine*HCl 10 mg thiamine*HCl 100 mg A OS (per 100 ml): sodium pyruvate 20 mg citric acid 40 mg malic acid 40 mg fumaric acid 40 mg (pH adjusted to 5.5 with 3.3 Results The induced CAT 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- I mately 20-fold stimulation of the CAT activity is to be observed.

Claims (34)

1. A process for the protection of plants against a virus Infection and/or its sequelae, which process comprises the following steps: isolating a virus specific control region from the genome of a suitable DNA or RNA virus; constructing a chimeric gene, wherein one or more protection factor gene(s), which is(are) under the control of plant active expression signals, is(are) operably linked to a virus-specific control region isolated in step in such manner that the induction of the associated protection factor(s) by a suitable inducer or the infecting virus itself is ensured; transforming the above chimeric gene construct into a recipient plant; 15 expressing the introduced protein factor gene(s) upon ad ition of a suitable inducer or infection with the appropriate virus.

2. A process according to claim 1, characterised in that the virus-specific induction of the protection factor gene is effected by j, means of a transactivation of transcription. ,I 20 3. A process according to claim 1 or 2, characterised in that the Svirus-specific induction of the protection factor gene occurs at the post-transcriptional level.

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 control of the mRNA elongation, a control of mRNA transport from the nucleus into the cytoplasm, a control of mRNA splicing, a control of mRNA polyadenylation, a control of the initation of translation, or a differentiated use of different ORFs on a polycistronic RNA. A process according to any one of claims 1 to 4, characterised in that the virus-specific induction of the protection factor gene is effected by post-translational modification of the protein product.

6. A process according to any one of claims 1 to 5, characterised In that said protection factor gene is a naturally occurring DNA sequence coding for a protection factor. SVrLWM/GSA/1197 -I 57

7. A process according to any one of claims 1 to 6, 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.

8. A process according to any one of claims 1 to 7, characterised in that said protection factor is an antiviral substance.

9. A process according to any one of claims 1 to 7, characterised in that said protection factor is a cell-toxic substance. A process according to claim 8, characterised in that said antiviral substance is selected from the group consisting of an antivirus-antibody, a PR-protein, a virus-specific protease inhibitor, a protease, a virus-specific polymerase inhibitor and an interferon-like protein. I 11. A process according to claim 9, characterised in that said cell-toxic substance is selected from the group consisting of a toxin, an active subunit of a toxin, a PR-protein and a protein that naturally triggers a hypersensitive reaction.

12. A process according to claim 9, characterised in that said cell-toxic substance Is a ribosome-inactivating protein. 20 13. A recombinant DNA molecule, which upon transformation Into a plant confers an inducible virus resistance upon plants, characterized in i that it comprises a DNA-sequence coding for an expressible protection factor, said DNA sequence being operably linked with a virus-specific control region that ensures induction of protection factor synthesis in the transformed plant by the infecting virus itself via a transactivation Smechanism. S14. A recombinant DNA molecule according to claim 13, wherein the expressible protection factor is selected from the group consisting of an antivirus-antibody, a PR-protein, a virus-specific protease inhibitor, a protease, a virus-specific polymerase inhibitor, an interferon-like protein, a toxin, an active subunit of a toxin and a protein that naturally triggers a hypersensitive reaction, a ribosome-inactivating protein, and a coat protein or an antisense DNA. A recombinant DNA molecule according to claim 13 or 14, wherein Induction results from a transactivation mechanism which is realized by CaMV, selected from the group consisting of transactlvatio, associated genes at the transcription and the post-transcription levels; 'LMM/1197v A r.i" 58 production of subgenomic RNA by virus-encoded replicases and; activation of proteins by processing, phosphorylation and other modifications.

16. A recombinant DNA molecule according to any one of claims 13 to 15, 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.

17. A recombinant DNA molecule according f:o any one of claims 13 to 16, 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. 18, A recombinant DNA molecule according to any one of claims 13 to 17, 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.

19. A recombinant DNA molecule according to any one of claims 13 to 18, 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 I, 17 *20 codons of linker and spacer sequences, the region coding for a protection factor gene, spacer sequences, a CaMV polyadenylation site and sequences I of the plasmid pUC18.

20. A recombinant DNA molecule according to claim 17, characterised in that the said expression signals active in plant cells are the 19S promoter and the CaMV transcription terminator.

21. A process for the preparation of a recombinant DNA molecule which confers an inducible virus resistance upon plants, which process comprises the following steps: isolating a virus specific control region from the genome of a suitable DNA or RNA virus; J constructing a chimeric gene, wherein one or more protection factor gene(s), which is(are) under the control of plant active expression signals, is(are) operably linked to a virus-specific control region isolated in step in such manner that the induction of the associated protection factor(s) by the infecting virus itself is ensured.

22. A process according to claim 21, characterised in that said virsu-specific control sequences are CaMV control sequences.

23. A process according to claim 21, characterised in that said virus-specific control sequences are BMV control sequences. LMM/GSA/1197v 59 59

24. A process according to any one of claims 21 to 23, characterised in that the operable 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. A process according to claim 24, 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.

26. A process according to claim 24, 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.

27. A process according to claim 24, 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 of the RNA3 of BMV, by a protection factor gene.

28. A process according to any one of claims 21 to 27, characterised In that said expression signals are promoter and termination sequences, and other regulatory sequences of the 3' and region that are active in plant cells. 20 29. A DNA transfer vector, characterised in that it contains a recombinant DNA molecule according to any one of claims 13 to *30. A DNA expression vector, characterised in that it contains a recombinant DNA molecule according to any one of claims 13 to

31. A host cell, characterised in that it contains S 25 a DNA transfer vector according to claim 29 and a DNA expression vector according to claim

32. A host cell according to claim 31, characterised in that It is a microorganism.

33. A host cell according to claim 31, characterised in that it is a plant cell.

34. A transgenic plant and the seeds thereof, which is protected against a virus infection and/or its sequelae, produced by a process according to any one of claims 1 to 12. A transgenlc plant the cells of which are transformed in whole or In part with a recombinant DNA molecule according to any one of claims 13 to 20, as well as the seeds of said transgenic plant.

36. A plant cell, characterised In that It has been transformed with a recombinant DNA molecule according to any one of claims 13 to kX4 3 LMM/1197v i r *6 60

37. A plant cell according to claim 36, characterised in that it is a component part of an entire plant.

38. A transgenic plant and the seeds thereof according to claim 36, which is a monocotyledonous plant.

39. The progeny of a transgenic plant according to claim characterised in that said progeny has been regenerated from plant cells that have been transformed with a recombinant DNA molecule conferring an S inducible virus resistance to plants and that said progeny includes Smutants and variants that still show the inducible virus resistance of 10 the starting material. I,

40. Transformed viable parts of plants according to any one of claims 34 to 35 and 38 to 39, characterised in that they are protoplasts, cells, cell aggregates, callus, seeds, pollen, ova, zygotes or embryos.

41. Hybridization and fusion products with the plant material defined in claims 34 to 40 that still exhibit characteristic properties of the transformed starting material. 'I't 42. A process for the production of virus-resistant transgenic plants, characterised in that a plant or viable parts thereof is (are) S transformed with a recombinant DNA molecule according to any one of claims 13 to

43. The use of a recombinant DNA molecule according to any one of claims 13 to 20 for the "immunisation" of plants against undesired virus attack. 4, t t

44. A process for the protection of plants against a virus infection and/or its sequelae which process is substantially as hereinbefore described with reference to any one of the Examples. A recombinant DNA molecule, which upon transformation into a plant establishes into said plant a system that renders possible the induction of genes based on virus-intrinsic control mechanisms, substantially as hereinbefore described with reference to any one of the Examples.

46. A process for the preparation of a recombinant DNA molecule, which establishes into a plant a system that renders possible the Induction of genes based on virus-intrinsic control mechanisms which process Is substantially as hereinbefore described with reference to any one of the Examples. 47, A DNA transfer vector substantially as hereinbefore described with reference to Figure 4. W,4 W/\4yS r!MM/GSA/TCW/1l97v I 61

48. A DNA expression vector substantially as hereinbefore described with reference to Figure 4.

49. A host cell substantially as hereinbefore described with reference to any one of the Examples but excluding any comparative examples. A transgenic plant and the seeds thereof substantially as hereinbefore described with reference to any one of the Examples. DATED this TWENTIETH day of NOVEMBER 1991 Ciba-Geigy AG Patent Attorneys for the Applicant SPRUSON FERGUSON t te flit I If (I C I11) 9 0 1Ft I I I LMMGS/TC/197 *4fl S S a a a a a. a a a a a a A 44, r- EcoRl NCO] EcoRV RNA KK)nli.SScl- CoRT-IfindIll Comv CaMV pUC18 ,s 6909 7437 7439 7632 Kpnl-Smal-BarWil Xbal-Sall-PstI-SphI B EcoRl 1 'pUCi 8 6909 C Ecom! 7632 PatI .Sphl Figure I A C 1. ~"I A\ 19S 19S CAT p pa r a rsrp 4r r a B 9* a *C a I 3-vi CAT R IVAno II CAT PROTEIN VJRIUS PROTEASE C i: i :i Figure 2A C tp (Eco RY'O/Sal a, a 4 *4 *4aa a a, a a ~4 a aa a. a o aa a a44Aa4 a a a a a a a *a a a ase ti a, a *1 lt I Sma I To9O3 Neo pst I Fkiaate23 4 a 9 a a a S S S a as S P A a a a 4 sat S P P a S P P 4 SOJ P P 44 5 1* a I a Bard-lI xbaI Sail BspmI Ucol Ppu4 FinI Figure 4