WO2002077209A1

WO2002077209A1 - Transgenic plants with resistance against tobacco rattle virus and corresponding nucleotide sequence

Info

Publication number: WO2002077209A1
Application number: PCT/SE2002/000533
Authority: WO
Inventors: Margareta Melander
Original assignee: Plant Science Sweden Ab
Priority date: 2001-03-26
Filing date: 2002-03-20
Publication date: 2002-10-03
Also published as: SE0101048D0

Abstract

The invention relates to a nucleotide sequence molecule comprising at least part of a structural gene encoding a tobacco rattle virus replicase polypeptide, the nucleotide sequence molecule of a plant transformation vector suitable for transformation of transformation of a plant cell and the plant transformation vector in a procaryotic cell. The invention also relates to a transformation method to transfer the nucleotide sequence molecule in the plant transformation vector into a plant cell, identification of the transgenic plant cell and regeneration of a plant from the transgenic plant cell. The invention also relates to a molecular marker for the detection of the transgenic cell, plant or product obtained from or containing parts of the plant.

Description

Transgen i c p l ants wi th res i stance aga i nst tobacco ratt l e v i rus and correspond i ng nuc l eot i de sequence .

FIELD OF INVENTION

The invention relates to means and methods to obtain and detect transgenic plants or part of transgenic plants with resistance against tobacco rattle virus. The resistance is achieved by the introduction of at least part of a structural gene encoding part of a Tobacco rattle virus replicase into the plant to obtain resistance in plants such as potato.

BACKGROUND OF INVENTION

Many agricultural crops are susceptible to plant virus infection. These viruses can severely damage the crop and thereby dramatically decrease the economic value for the grower. Furthermore this results in higher prices for the consumer of the products manufactured from the crop. There have been efforts to prevent and control these viral diseases by several methods, such as a) chemicals which eliminates the virus host harbouring the virus, b) breeding for new varieties resistant or tolerant against the disease, c) genetic engineering by introducing new genetic material into the crop to obtain plants resistant or tolerant against the viral disease. One example is the potato crop {Solanum tuberosum), susceptible to inter alia tobacco rattle virus (TRV), belonging to the genus Tobravirus. The disease is called corky ringspot (CRS) and the main symptoms of the disease are necrotic local lesions, systemic chlorotic or necrotic spots, which in the tubers occurs as rings of dark brown corky layers. The disease significantly affects acres of the cool areas where the potato is cultivated for the potato processing industry in the Eurasian region, the North, South and central America, China, Japan and the former USSR. It has also been found in New Zeeland and Australia (ICTV database, 1998). The disease may results in considerable economic losses. Infection with TRV influences the yield but of more importance is the qualitative damage of dark brown corky layers which are considered unacceptable for fresh market or for potatoes that are processed into French fries or other pre-cooked products.

TRV has a very wide host range and may furthermore infect and cause disease in other plants such as ringspot in asters, notched leaf of gladiolus and yellow mottle in spinach. Many different weed species can also be infected with the virus. TRV is transmitted by the stubby root nematodes Trichodorus and Para- trichodorus spp. These nematodes are persistent carriers of TRV and can obtain the virus from various hosts such as nightshades, pigweed, shepherd's-purse, purslade, cocklebur and sunflower. Young potato roots and tubers are infected with TRV when virus-infected nematodes feed on them. The Trichodorus and Paratrichodorus vectors transmitting the infection can be combatted by nematicide treatment of the soil. However, this treatment is very difficult to apply and the chemicals used have environmental drawbacks. In many countries the use of nematicides is not allowed and the potato industry have had to rely on resistant or tolerant varieties. However, there are only few varieties resistant to the TRV and the mechanism underlying the resistance is unknown and difficult to use in conventional breeding.

The use of varieties tolerant or resistant to TRV will enable the growers to cultivate potato plants in areas infected with TRV where potato is one of the essen- tial crops for crop rotation and thereby significantly influence the growers income. Scientists have developed means of using genetic engineering to obtain resistance against viruses in plants, which are advantageous to the use of chemicals since the introduced new genetic material will be inherited from one generation of plants to another. Furthermore, the use of genetic engineering in general, is based on the use of a single gene to obtain the resistance as compared to conventional breeding, which often is based on several genes to achieve the same goal. The genetic engineered material is thereby easier to maintain throughout the breeding programme.

The first reports on genetic engineering for resistance to viruses were those concerning the use of the coat protein (Abel et al., 1986. Science 232:738-743, Turner et al., 1987. EMBO J 6:1181-1188). They showed that expression of an introduced coat protein gene into a susceptible plant showed resistance at least to the homologous virus. This type of resistance has proven to be useful at least for certain viruses, but not in all cases. In unsuccessful cases, other approaches have been ex- plored, such as antisense coat protein (Cuozzo et al., 1988. Bio/Technology 6:549- 557), satellite RNA (Harrisson et al., 1987. Nature 328:799-802), ribozymes (Walbot and Bruening, 1988. Nature 334:196-197), defective interfering molecules (Morch et al., 1987. Nucleic Acids Research 15(10):4123-4130), antiviral proteins (Irvin et al., 1980. Archives of Biochemistry and Biophysics 200(2):418-425), re- plicases or portions thereof as well as different proteins involved in virus cell-to-cell movement (Abel et al., 1986. Science 232:738-743; Anderson et al., 1992. Proc Natl Acad Sci 89:8759-8763, Lapidot et al., 1993. The Plant J 4(6):59-970). The approach or approaches to be chosen depends on several things such as, the virus and it's corresponding genus, the transmitting vector, the part of the crop in which the disease occurs as well as the possibility to transform/introduce the new quality into the crop. For most crops often the logistical problems force the inventors to concentrate on a limited number of resistance approaches dependent on which crop they are focused on. TRV is a single-stranded RNA virus possessing a bipartite genome. The two genome RNA species (RNA-1 and RNA-2) are of positive polarity. The RNA-1 is 6790 bases and RNA-2 is 3251 bases (Sudarshana & Berger, 1998, Arch Virol, 143:1535-44). The 5'- part of RNA-1 encodes the replicase gene and the complete replicase gene occupies nearly 75% of RNAl . In the C-terminal regoin of the replicase gene there is a readthrough portion encoding a 54 kDa protein. RNA-2 encodes the coat protein of 9 kDa and two other protein of 29 kDa and 18 kDa (MacFarlane et al., 1999, J Gen Virol, 80:273-276).

Other viruses within the Tobravirus genus are pepper ringspot virus (PepRSV) and pea-early-browning virus (PEBV). All of them are transmitted by nematodes and there is a highly specific relationship between virus and nematode, so that particular virus isolates are transmitted only by certain nematode species (MacFarlane, 1999, J. of Gen. Virol. 80:2799-2807).

A part of the replicase gene (the C-terminal region encoding the 54 kDa protein) from PEBV has been introduced into Nicotiana Bentamiana and conferred resistance to infection of the plants by PEBV (MacFarlane & Davies, 1992, Proc. Natl. Acad. Sci. 89:5829-5833). However, the PEBV virus was cultivated on Nicotiana Bentamiana plants, purified and mechanically inoculated onto the plants in a buffer, i.e. no natural infection using PEBV infected nematodes was used. Furthermore, it is not solely the presence of a resistance gene within a plant that confers resistance against the homologous virus. The promoter present upstream of the gene is also a critical parameter since promoters often are specific for expression in a certain type of tissue and the expression of the promoter may also vary in between species. Dependent on which type of tissue that is affected by the virus, for example in the tuber of a potato different types of promoters might be chosen for the expression of the resistance against the viral disease.

There is a continuing need for transgenic expression of genes derived from Tobacco rattle virus, which confer resistance to infection by this virus. The object of the invention is thereby to provide virus resistance against these viruses by genetic engineering.

SUMMARY OF THE INVENTION

The object of the invention is to provide means and methods to be able to produce and detect genetically engineered plants with at least part of a structural gene encoding the tobacco rattle virus replicase polypeptide.

Accordingly, in a first aspect the invention relates to a nucleotide sequence molecule which comprises; at least one infection site specific promoter region which functions in plant cells, the promoter region is operably linked at least part of a structural gene encoding a tobacco rattle virus replicase polypeptide, which is operably linked to at least one non-translated region which functions in plants cells.

In another aspect the invention relates to plant transformation vectors and procaryotic cells used to enable the transfer of the nucleotide sequence molecule from the plant transformation vector into the plant genome to obtain resistance against a tobacco rattle virus.

In a further aspect, the invention relates to a method for producing a transgenic plant with resistance to infection by tobacco rattle virus, the method comprises: transforming a plant cell from the plant with a nucleotide sequence molecule or a plant transformation vector or a procaryotic cell; regeneration of the plant cell and production of transgenic plants and selecting a transgenic plant which transcribes the introduced nucleotide sequence molecule and which provides resistance to the TRV.

In still further aspect the invention relates to a transgenic plant comprising a nucleotide sequence molecule according to the invention and propagation parts thereof including seeds, tubers, pollen, cuttings and microtubers obtained from the transgenic plant.

In another aspect the invention relates to a method for the detection of a transgenic plant, the method comprises a step in which a molecular marker is used, the molecular marker comprises at least a part of the nucleotide sequence molecule according to the invention.

In still another aspect the invention relates to food products based on a transgenic plant or parts of a transgenic plant.

The invention provides completely new genetically engineered plants resistant to the tobacco rattle virus. The method and means according to the invention enables the possibility to render susceptible potato plants resistant by using solely a single gene which is easy to follow and maintain during a breeding programme. By providing such plants the grower is able to grow potatoes in fields infected by the tobacco rattle vims and thereby increase the economic value of the yield obtained from such an infected field.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG 1 illustrates the physical map of the vector (plasmid) pTRM:l . 54 kD = 54kD part of the TRVreplicase Rl, P35S = CaMV 35s promoter, pAnos = nopaline syn- thase polyA sequence, NPTII = neomycin phosphotransferase II for kanamycin resistance, Pnos = nopaline synthase promoter, RB = right border, LB = left border and Kan = bacterial kanamycin resistance

FIG 2 illustrates the physical map of the vector (plasmid) pTRV-TS 54 kD = 54kD part of the TRVreplicase Rl, pGBSS = granule-bound starch synthase promoter, pAnos = nopaline synthase polyA sequence, NPTII = neomycin phosphotransferase II for kanamycin resistance, Pnos = nopaline synthase promoter, RB = right border, LB = left border and Kan = bacterial kanamycin resistance FIG 3 illustrates the incidence of TRV infection determined by scoring of symp- toms in the first greenhouse trial. Line M6-M567 are transgenic lines. FIG 4 shows the results from the second greenhouse trial.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

In the context of the present application and invention the following definitions apply:

The term "nucleotide sequence" is intended to mean a sequence of two or more nucleotides. The term "nucleotide sequence molecule" is intended to indicate a consecutive stretch of three or more regions of nucleotide sequences. The nucleotide sequence molecule comprises a promoter region, structural gene and a non-translated region. The nucleotide sequence or nucleotide sequence molecule may be of genomic, cDNA, RNA, semi-synthetic or synthetic origin, or a combination thereof. The nucleotide sequence molecule is designed to express a structural gene located within the nucleotide sequence molecule when the nucleotide sequence molecule is integrated in the genome of a plant.

The term "promoter region" is intended to mean one or more nucleotide sequences involved in the expression of a structural gene, e.g. promoter nucleotide sequences, as well as nucleotide sequences involved in regulation and/or enhancement of the expression of the structural gene. An example of the latter regulation is a signal peptide functional in a plant tissue, which directs the translated polypeptide to a specific tissue. A promoter region comprises a promoter nucleotide sequence involved in the expression of a structural gene, and normally other functions such as enhancer elements and/or signal peptides. In the present context the promoter regions are regions, which function in a plant cell at the site of the infection (infection site specific), such as in a potato plant. The promoter region may be selected from a plant, virus and bacteria or it may be of semi-synthetic or synthetic origin or a mixture thereof. Examples of promoter regions are constitutive promoter nucleotide sequences, which are expressed where the infection occurs, such as in the tubers of a potato plant. Other useful promoter regions include those, which are capable of expressing the tobacco rattle virus replicase gene in an inducible manner or in a tissue-specific manner in certain plant cell types in which the tobacco rattle infection occurs. One example of a suitable promoter region is the granule-bound starch synthase (GBSS) promoter mentioned in WO92/11376, which express the GBSS in the tubers. Another example is the patatin I promoter (Mignery et al., 1988. Gene 62:27-44). These two types of promoter regions are promising in obtaining resistance against the TRV since the virus enters into the plant through the tubers and the main problem of the TRV disease is the symptoms of rings of dark brown corky layers in the tubers of the potato. However, other promoter regions, which successfully express a gene solely or to a level sufficient to achieve resistance against the TRV, such as in the tubers are also suitable promoter region candidates to be used. The GBSS promoter mentioned above is highly expressed in the tubers, up to 25 times the expression of the conventional 35S promoter (Visser et al., 1991. Plant Mol Biol 17: 691 -699). Thereby that specific promoter either alone or in combination with other control sequences is a suitable candidate to be used in combination with a gene in which the expression is desired to be a high level within the tubers. The term "structural gene" is intended to mean a nucleotide sequence encoding part of a tobacco rattle virus replicase polypeptide, such as the C-terminal part of the replicase gene encoding a protein of 54 kDa. The nucleotide sequence of the structural gene preferably have an identity of at least 75 %, at least 80 %, at least 85 % at least 90 %, at least 95 % or substantially the same as the nucleotide sequence shown in SEQ ID NO 1. The structural gene, being involved in the inhibition of replication of the corresponding virus, i.e. TRV. In other words to obtain a structural gene having at least 75 % identity to the nucleotide sequence shown in SEQ ID NO 1, up to 25 % of the nucleotide sequence residues may be inserted, deleted or substituted with another nucleotide sequence residue. The same discussion applies for 80 %, 85 %, 90 % and 95 %. These alterations of nucleotide sequence shown in SEQ ID NO 1 may occur at the 5' or the 3' terminal position, as well as interspersed either individually among the nucleotide sequence residues or in one or more contiguous groups within the nucleotide sequence shown in SEQ ID NO 1. The structural gene may be native or synthetic or a mixture thereof.

The term "a non-translated region " also called termination region is intended to mean a region of nucleotide sequences, which typically cause the termination of transcription and the polyadenylation of the 3' region of the RNA sequence. The non-translated region may be of native or synthetic origin as long as it functions in a plant cell according to the definition above. Suitable non-translated regions are 3' transcribed, non-translated regions containing a polyadenylation signal downstream of a plant gene, such as the nopaline synthase (nos) gene, octopine synthase gene and the CaMV 35 S gene.

The term "operably linked" is intended to mean the covalent joining of two or more nucleotide sequences by means of enzymatic ligation, in a configuration which enables the normal functions of the sequences ligated to each other. For example a promoter region is operably linked to a signal peptide region and/or a coding sequence of a structural gene to direct and/or enable transcription of the structural gene. Another example is a structural gene operably linked to a 3' non- translated region for termination of transcription of the structural gene. Generally, "operably linked" means that the nucleotide sequences being linked are continuously and in reading frame. Linking is normally accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic adaptors or the like are used in conjunction with standard recombinant DNA techniques well-known for a person skilled in the art. The term "propagation part" is intended to mean any part of the plant which may be used for propagation and multiplication of the plant including, e.g., seeds, cuttings, pollen, tubers and microtubers or parts thereof.

The term "molecular marker " is intended to indicate a sequence of two or more nucleotide sequences of a nucleotide sequence molecule as defined above. The molecular marker may be of genomic, DNA, cDNA, RNA, mRNA, semi- synthetic or synthetic origin, or any combination thereof. The molecular marker may be a probe used in hybridisation assays or a primer used in all kind of polymer- ase chain reactions (PCR's).

The term "hybridisation assay" generally refers to a method used for the detection of a complementary stretch of a nucleotide sequence using a hybridisation probe. In general the hybridisation probe is labelled either radioactively, or with some other detectable molecule, such as biotin, digoxigenin or fluoroscein. Examples of hybridisation techniques are Southern blot analysis, Northern blot analysis and dot blot analysis. The term "polymerase chain reaction" or "PCR" generally refers to a method for amplification of a desired nucleotide sequence in vitro as described, for example, in US 4,683,195. In general, the PCR method involves repeated cycles of primer extension synthesis, using oligonucleotide primers capable of hybridising preferentially to a template nucleic acid, wherein the primers are complementary to the template nucleic acid.

The term "resistance to tobacco rattle virus" is intended to mean, that the plant minimises or avoids the replication of the virus and thereby inhibit or block the spread of the infection within the plant and only a minor local infection may occur. The plant has the ability to suppress or retard the multiplication of a virus, which gives a decrease in virus accumulation as compared to a non-resistant plant. Resistance can also be manifested as suppression or retardation of the development of pathogenic symptoms.

In the present context, amino acid names and atom names are used as defined by the Protein DataBank (PNB) (www.pdb.org), which is based on the IUPAC nomenclature (IUPAC Nomenclature and Symbolism for Amino Acids and Pep- tides (residue names, atom names etc.), Eur J Biochem., 138, 9-37 (1984) together with their corrections in Eur J Biochem., 152, 1 (1985). The term "amino acid" is intended to indicate an amino acid from the group consisting of alanine (Ala or A), cysteine (Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), phenyl- alanine (Phe or F), glycine (Gly or G), histidine (His or H), isoleucine (He or I), lysine (Lys or K), leucine (Leu or L), methionine (Met or M), asparagine (Asn or N), proline (Pro or P), glutamine (Gin or Q), arginine (Arg or R), serine (Ser or S), threonine (Thr or T), valine (Val or V), tryptophan (Trp or W) and tyrosine (Tyr or Y), or derivatives thereof.

Description

The invention will now be described in detail using the potato plant as an example. The invention should not be limited thereto The structural gene comprises at least a nucleotide sequence encoding part of a tobacco rattle virus replicase, such as the nucleotide sequence shown in SEQ ID NO 1. Another example is the nucleotide sequences shown in SEQ ID NO 2.

Several methods exist to isolate the structural gene from TRV (Hamilton et al., 1987, J. Gen. Virol, 68:2563-2575). To do so, a person having ordinary skill in the art can use information about the genomic organisation of TRV to locate and isolate the replicase gene. TRV may be cultivated in a suitable host such as Nicotiana tabacum or Nicotiana clevelandi. The virus is multiplied in the host and harvested using well-known techniques. Viral RNA is purified from the harvested material and double stranded cDNA prepared using a conventional method such as the c-DNA Synthesis System Plus kit from Amersham, UK (see example 1 and 2). The nucleotide sequence encoding part of the replicase gene, such as the C-terminal part of the replicase gene encoding a protein of 54kDa located between nucleotide 3811-5418 within the nucleotide sequence published by Hamilton et al., 1987. J Gen Virol 68:2563-2575 may be amplified from the cDNA. Sequence specific homologous primers are used, specific to the 5' and 3' part of the nucleotide sequence corresponding to the C-terminal part of the replicase gene encoding a protein of 54kDa, such as in example 2. The primers may include restriction enzyme sites to enable further cloning into a suitable vector. However, it is understood that the particular nucleotide and/or amino acid sequences disclosed herein are representative in the sense that equivalent genes or portions thereof may be obtained and/or generated according to this disclosure. By the term "equivalent" means that the gene would function in substantially the same way as the part of the TRV replicase gene encoding a polypeptide of 54 kDa, disclosed herein and provide resistance to TRV in plants. The amplified PCR product of 1608 nucleotides cor- responding to the nucleotide sequence 3811-5418 mentioned above is purified from the PCR mixture and cloned into a suitable vector by the aid of DNA-ligase (see example 2). The vector is propagated in a suitable host in which the vector is able to replicate. Numerous vectors exist that have been described in the literature, such as pBluescript SK- (Stratagene, CA, USA). A suitable host is E.coli, such as DH5α E.coli. The nucleotide sequence of the 1608 nucleotide fragment may furthermore comprise minor modifications such as one or more substituted nucleotides without influencing the resistance mechanism within the plant. An amplified nucleotide sequence of the above, mentioned structural gene of at least 100 nucleotides is also possible to achieve the same results.

The nucleotide sequence of the amplified PCR products may be analysed by any method, known for a person skilled in the art, such as restriction enzyme analysis and/or nucleotide sequence analysis.

The structural gene encoding part of the replicase gene, such as the C- terminal part of the replicase gene encoding a protein of 54 kDa may be inserted into a plant transformation vector (see example 3). In general, the structural gene is integrated into a nucleotide sequence comprising (from the 5' end to the 3' end); at least one promoter region which functions in plant cells, which is operably linked to the structural gene encoding part of a tobacco rattle virus replicase polypeptide and/or fragments thereof, which is operably linked to at least one non-translated region which functions in a plant cell.

The nucleotide sequence is produced by conventional cloning means known for a person skilled in the art and examples are found under Examples.

According to the invention, a plant transformation vector preferably includes all elements necessary for transforming a plant cell. In general a plant transformation vector comprise beyond the nucleotide sequence mentioned above, T-DNA borders, selectable marker genes, genes to easily detect the transgenic cell(s), such as screenable markers. Examples of transformation vectors are pBHOl and pBI121 (Clonetech Laboratories, CA, USA), in which the structural gene encoding the β- glucuronidase may be replaced by another structural gene while maintaining the Cauliflower Mosaic Virus (CaMV) 35S promoter region and the nos non-translated region. (The CaMV 35S promoter-structural gene-non-translated region comprising the nucleotide sequence). Other examples of transformation vectors are those based on pGPTV-kan (Becker et al, 1992, , Plant Mol. Biol 20:1195-1197) and the pPZP- family (Hajdukiewicz et al, 1994, Plant Mol. Biol. 25, 989-994). A number of promoters functional in plant cells are known in the art and may be employed in the practice of the present invention. Useful promoters include promoters, which are capable of expressing the tobacco rattle virus replicase gene or part thereof in an inducible manner or in a tissue-specific manner in certain cell types in which the infection occurs, such as in the tubers where the TRV infection occurs. One example of a useful promoter is the granule-bound starch synthase (GBSS) promoter disclosed in W092/11376, which expresses the GBSS in the tubers. Another example is a patatin promoter (Mignery et al, 1988. Gene 62:27-44). These two types of promoters may be preferred in obtaining resistance against the TRV since the virus enters into the plant through the tubers and the main problem of the disease is the symptoms of rings of dark brown corky layers within the tuber of the potato.

The non-translated region can be native with the promoter region, native with the structural gene, or can be derived from another source or be of synthetic origin as long as it may function in a plant. Suitable non-translated regions are 3' transcribed, non-translated regions containing a polyadenylation signal downstream of a plant gene, such as the nopaline synthase (nos) gene and the CaMV 35S gene.

In developing the nucleotide sequence and the plant transformation vector, the elements thereof will normally be inserted into a convenient cloning vector, which is capable of replicating in a bacterial host such as E.coli. Examples of suitable vectors and furthermore descriptions of cloning techniques are well known for a person skilled in the art and can for instance be found in Sambrook et al, 1989. Mol Cloning, a laboratory manual, second edition, Cold Spring Harbour Laboratory Press.

A number of techniques are available for the introduction of genetic material into plants (transformation techniques). However, the technique used to introduce the genetic material into the host is not critical to the invention and any technique available for the crop can be used as long as sufficient independently transformed cells are obtained. One technique useful for the transformation of plants is a technique in which a bacterial cell is used to transfer the nucleotide sequence as defined above into the plant cell. A bacterial technique in which the bacteria harbours the vector and during transformation the nucleotide sequence is transferred from the host into the plant and becomes integrated into the plant genome such as the bacte- ria Agrobacte ium tumefaciens and Ag obacterium rhizogenes might be used. Examples of useful Agrobacterium tumefaciens strains are LBA4404, EHA101, C58C1, GV3101, C68C1 and A281. Alternative methods that can be used involve the nucleotide sequence or DNA constructs according to the invention for direct transfer into the host by the use of electroporation, microinjection, microprojectile bombardment or chemically mediated transformation.

A selectable marker may be used in the plant transformation vector for the selection of a transgenic cell among other cells. There are several selectable marker genes available including those using a herbicide, antibiotic or a carbohydrate source as the selectable agent. Examples of antibiotic selectable agents are kanamy- cin, gentamycin, G418, hygromycin, streptomycin, spectinomycin, tetracycline, chloramphenicol and the like. Herbicides, which might act as selectable markers are glyphosate, sufonylurea, phosphinotricine, imidazolinones or bromoxynil and the like. As a carbohydrate source mannose or desoxyglucose-phosphate may be used. Additional means of selection, might be those in which selection is performed against the protein encoded by the structural gene within the nucleotide sequence molecule.

Furthermore a screening marker may be used to facilitate the selection of a transgenic cell among non-transgenic cells. Examples of such screening markers are the β-glucuronidase gene or the green fluorescence gene.

The choice of plant tissue source or cultured plant cells for transformation will depend on the nature of the host plant and the transformation protocol. Useful tissue sources include callus, suspension culture cells, stomata guard cells, protoplasts, leaf segments, pollen, embryos, hypocotyls, tuber segments, meristematic regions and the like. Preferably the plant tissue source or plants cells are from a potato plant such as Solanum tuber osum.

Following treatment of the plant cells with the vector containing the nucleotide sequence molecule, the plant cells may be selected on a suitable selection medium containing a selectable agent, such as those described herein above. The selection medium may be formulated to maintain the plant cells in an undifferen- tiated stage. If the vector is harboured in Agrobacterium antibiotics against the bacteria may be included. Cells growing on the selectable medium presumed to be transgenic can be subjected to protocols aiming in regeneration of the transgenic plant. Such protocols are well known for a person skilled in the art, for example Visser, 1991, Plant Tissue Culture Manual (Lindsey K, ed) B5 : 1 -9.

The transgenic plant cells may be identified in several ways including PCR using primers homologous to at least part of the nucleotide sequences aimed to be transferred into the plant genome during transformation, such as primers specific for the selectable marker or the structural gene within the nucleotide sequence mole- cule. Alternative methods are detection of the expression of the integrated gene or genes at the level of niRNA by methods, such as Northern blot analysis, RT-PCR or ribonuclease protection assay. Additional analysis may be identification of the protein encoded by any of the genes which have been transferred into the plant using an enzymatic assay, antibody detection assay or any other suitable method. After identification of the transgenic plant cells the cells are regenerated into a complete plant, such as a potato plant. Any known method for the regeneration of a potato plant may be used according to the invention.

A plant containing the nucleotide sequence molecule mentioned above might be cultivated using conventional means known to those skilled in the art. The transgenic plant or transgenic plant material may at any stage from in vitro culture to a fully developed plant be analysed for the number of integrated copies of the nucleotide sequence in the genome. The number of integrated copies may be determined in DNA extracted from the plant by means of Southern blot analysis, using a probe specific for the nucleotide sequence molecule or any other part of the vector which end up in the genome of the transgenic plant. The transgenic plant contains at least one copy of the nucleotide sequence molecule. However, the plants may contain two or more copies of the integrated nucleotide sequence molecule, which might be difficult to determine. Copies may be located in the same position in the genome or at different locations in the genome. Successful transcription of the introduced gene may be determined by Northern blot analysis or ribonuclease protection assay using total RNA extracted from the transgenic plant and a probe specific to the introduced structural gene. The presence of the transgenic protein, expressed from the introduced structural gene may be detected by ELISA or Western blot analysis using antibodies specific against the expressed protein.

Tobacco rattle virus resistance can be evaluated in greenhouse and/or field trials. Transgenic plants containing the above mentioned structural gene encoding a tobacco rattle virus replicase polypeptide may be evaluated in greenhouse and field trials for their ability to protect the plant from TRV infection. Greenhouse trials for the identification of resistant non-transgenic plants may be used for the identification of genetically engineered plants capable of coping with TRV infection, such as the greenhouse in Example 5. In such a greenhouse trial the plants are cultivated in a greenhouse in soil infected with the Paratrichodorus or Trichodorus spp. s har- bouring the TRV (natural infection and not solely virus inoculation). The plant material in the greenhouse is cultivated in pots to full maturity. The tubers in each pot is counted and each tuber sliced and inspected for dark brown corky layers and the transgenic material is compared with the non-transgenic material.

The transgenic plant material according to the present invention comprising the tobacco rattle virus replicase gene or part thereof integrated into the genome includes all part of the potato plant including plants cells, plants, tubers, microtubers, cuttings, pollen and seeds. Plant parts, includes propagation parts of the plant.

The transgenic plant material may be detected and identified by a molecular marker. Identification may be performed at all stages from the in vitro stage, breeding, cultivation by the farmers in a field, processing of the plants and tubers and the final product commercialised on the market, such as a food product. Examples of food products are potatoes in the form of potatoes or processed potatoes, potato chip or just part of the potato in a certain product. The food product may be a product, which partly consist of potato in any form as described above.

The molecular marker comprising at least part of the nucleotide sequence molecule which has been transferred into the plant during transformation.

Preferably, the molecular marker comprising the structural gene encoding a tobacco rattle virus replicase polypeptide, such as the replicase polypeptides shown in SEQ ID NO 1 or SEQ ID NO 2.

The molecular marker utilised as a probe in a hybridisation assay may be used under such hybridisation conditions that the molecular marker only hybridises to the nucleotide sequence molecule mentioned above or part thereof in a material, such as a food product comprising the transgenic plant material transformed with the nucleotide sequence molecule.

A molecular marker used as a primer (primer 1) in PCR for the amplification of at least part of the nucleotide sequence molecule present in the transgenic plant material transformed with the nucleotide sequence molecule. For the ability to amplify a product by PCR the primer 1 used as a molecular marker needs to be combined with another primer (primer 2) as a person skilled in the art will understand. The primer 1 in this case may be combined with another primer 2 located in the nucleotide sequence molecule or outside the nucleotide sequence molecule within a selectable marker or even within the genome of the plant outside of the nucleotide sequence originating from the non-transgenic plant material used for transformation.

The invention provides an alternative approach to obtain virus resistance in a plant against TRV, such as in potato (Solanum tuberosum). The TRV resistance is obtained by expression of an isolated structural gene in at least part of a plant, such as in the leaf, tubers, stem, seeds, roots and flowers, preferably in the tubers of the plant. The isolated structural gene is integrated into the plant genome as part of a nucleotide sequence molecule.

By providing transgenic plants, such as potato plants (Solanum tuberosum) resistant against TRV, the breeders get the opportunity to produce varieties resistant against TRV and thereby the growers get the possibility to cultivate potato plants in areas infected with TRV, areas where potato is one of the essential crops for crop rotation and thereby significantly influence the growers income.

The invention will furthermore be explained by examples and should not be limited thereto. EXAMPLES

EXAMPLE 1

Virus isolate and RNA-isolation Purified virus particles from a Swedish isolate of TRV, 91-013, was kindly provided by Professor Klas Lindsten, Swedish University of Agricultural Sciences, Uppsala, Sweden.

100 μg virus particles were incubated for 30 minutes at 37°C in 0.02 M sodium phosphate buffer, pH 7.4, 4 mM dithiotreitol, 1.5 u/μl RNAsin ribonuclease inhibitor (Promega) and 0.5 mg/ml proteinase K. SDS was added to a concentration of 1% (w/v), sodium acetate, pH 5.0, to 0.2 M and finally an equal volume of phenolxhloroform (1 :1) was added. The sample was mixed and centrifuged at 7000 x g, 5 min room temp. RNA was precipitated from the aqueous phase by the addition of an equal volume of isopropanol at -20°C. The RNA was collected by cen- trifugation at 7000 x g, 10 min at room temp and the pellet was washed with 70 % ethanol, air dried and resuspended in water. The RNA was reprecipitated once by the addition of sodium acetate to 0.2 M and 2.5 volumes of ethanol at -20 °C. The final pellet was dissolved in water and the RNA-concentration was determined by measuring the absorbance at 260 nm.

EXAMPLE 2 cDNA synthesis and PCR amplification

Double stranded cDNA was prepared with the cDNA Synthesis System Plus (Amersham, UK) using random hexanucleotide primers for first strand synthesis. To isolate the sequence coding for the 54 kDa part of the replicase, primers were selected according to a published sequence of TRV RNA-1 (Hamilton et al, 1987, J. Gen. Virol. 68:2563-2575). Amplification with the primer pair 5 '-TTG CGC CGT TCC AGA TTC AG-3 ' and 5 '-CGT CCA CAA ACA ATT CAA TGG C-3 ' would replicate nucleotides 3811-5418 giving a product of 1608 nucleotides. The PCR reaction was carried out with recombinant Pfu-polymerase (Stratagene, CA, USA) in the provided buffer according to the provided protocol. After an initial denaturing period at 95 °C for 5 minutes the polymerase was added and 35 cycles with denatu- ration at 95 °C for 30 seconds, annealing at 56 °C for 30 seconds and extension at 75 °C for 90 seconds were performed, followed by a final extension for 5 minutes at 75 °C. The PCR-reaction products were purified with Geneclean (Bio 101, CA, USA) and then blunted and ligated into the Smal site of pBluescript SK- (Stratagene, CA, USA) using a DNA blunting kit (Amersham, UK). The ligated plasmids were transformed into competent DH5 E. coli cells which were grown on LB-plates containing ampicillin (50 μg/ml), X-gal (40 μg/ml) and IPTG (0.04 mM). Random white colonies were selected for plasmid mini-preparations by alkaline lysis (Sambrook et al, 1989. Molecular cloning, a laboratory manual, second ed. Cold Spring Harbour Laboratory Press). After restriction enzyme analysis one construct, Rl containing a fragment of expected size, was chosen for further studies. The construct was propagated and purified with a Plasmid Maxi Kit (Qiagen,

Germany). The Rl fragment was cut into three subsequences, which were cloned in pBluescript SK- and sequenced on an Applied Biosystems automatic sequencer with M13-primers.

EXAMPLE 3

Transformation vectors

As binary vector pBI121 (Clonetech, CA, USA) where the GUS-gene was cut out with Smal and Sstl was used. The Rl fragment was excised with EcoRV and Xbal from pBluescript. Both the pBI121 vector and the fragment were blunted and then ligated together using the DNA blunting kit. The ligations were transformed to DH5α cells which were plated on LB-plates containing kanamycin (25 μg/ml). Random colonies were selected for plasmid mini-preparations (as above) and analysed by restriction enzyme analysis. The construct containing the Rl fragment in correct orientation was chosen for further work. The construct containing the Rl fragment was named pTRM: 1 (FIG 1).

Another construct was made with the tuber specific promoter from granule bound starch synthase (GBSS) from potatoes (Hofvander et al, 1991, WO 92/11376). This promoter was derived from a vector based on pUC19 were the GBSS-promoter and the nopaline synthase (nos) terminator from pBI121 had been inserted between Hindlll and EcoRI. The Rl fragment was excised from pTRM: 1 with EcoRI followed by blunting and BamHI. The GBSS-vector was cut with Sail followed by blunting and BamHI. The excised Rl fragment was then ligated into the GBSS-vector and the ligation transformed to E. coli XLl-Blue MRF' cells (Stratagene, CA, USA). After plasmid mini-preparations and restriction enzyme analysis a plasmid named p2Virl was derived. From this plasmid the GBSS-Rl-nos fragment was subsequently excised with EcoRI and Hindlll and ligated into EcoRI and Hindlll of pPTV-pA (a derivative of the binary vector pGPTV-Kan (Becker et al, 1992, Plant Mol Biol 20: 1195-1197) in which the GUS gene has been replaced with the poly linker of pUC19). The ligation was transformed to XLl-Blue cells. After plasmid mini-preparations and restriction enzyme analysis a final binary vector construct named pTRV-TS (FIG 2) was derived.The constructs pTRM: 1 and pTRV-TS were introduced into A. tumefaciens strain LBA4404 (pAL4404, Life Technologies, UK) by direct transformation (Walkerpeach & Velten, 1994, Plant Mol Biol Manual, second edition, (Gelvin % Schilperoort ed) B 1-19). Transformed bacteria were selected on YEB-plates (Walkenpeach &Velten, 1994, Plant Mol Biol Manual, second edition, (Gelvin % Schilperoort ed) BL1-19) containing kanamycin (50 μg/ml). Colonies were chosen for plasmid mini -preparation and subsequent restriction enzyme analysis.

EXAMPLE 4 Transformation of potato

The potato cultivars (cv) Matilda (Svalδf Weibull AB, Sweden) and Desiree were transformed according to Visser (1991) by Agrobacterium mediated transformation using pTRM: 1 and pTRV-TS respectively. Leaf segments were precultured for 1-3 days on plates containing MS-medium with the addition of 2 mg/1 NAA, 1 mg/1 BA and 2.5 g/1 gellan at pH 5.8 overlayered with MS 30 medium containing 0.5 mg/1 thiamine-HCl, 0.5 mg/1 pyridoxine-HCl, 1 mg/1 nicotinic acid, 29.8 mg/1 FeS0₄ x 7H₂0, 1 mg/1 2,4-D, 0.5 mg/1 kinetin and 2 g/1 casein hydrolysate at pH 6.5. The leaf segments were removed from the plates and incubated with the A. tumefaciens containing either pTRM: 1 or pTRV-TS in MS- medium containing 10 g/1 sucrose at pH 5.8. After incubation for 10 min the leaf segments were retransferred to the original plates and incubated for another 1-3 days. The leaf segments were transferred to plates containing MS-medium with the addition of 10 g/1 sucrose, 2 mg/1 zeatin, 0.01 mg/1 NAA, 0.1 mg/1 GA₃, 400 mg/1 claforan, 2.5 g/1 gellan at pH 5.8 and incubated for 3-5 days. Thereafter the leaf segments were transferred to new plates containing the same medium but with the addition of 50 mg/1 kanamycin. The tissue was transferred to new plates with the same medium every two weeks until the formation of callus. Callus were removed and transferred to plates with MS-medium containing 10 g/1 sucrose, 0.25 mg/1 BA, 0.1 mg/1 GA₃, 400 mg/1 claforan, 50 mg/1 kanamycin and 2.5 g/1 gellan at pH 5.8. When shoots were formed these were removed and transferred to MS-medium with 30 mg/1 sucrose, 50 mg/1 kanamycin and 2.5 g/1 gellan at pH 5.8.

Generated shoots were analysed by PCR for presence of the NPTII- and 54 kDa-sequences. For the detection of NPTII, 5 '-TTT TGT CAA GAC CGA CCT GTC C-3 ' was used as forward primer and 5 '-AAC TCG TCA AGA AGG CGA TAG AAG-3 ' as backward primer, generating a fragment of 648 nucleotides corresponding to nucleotide 291-938 of the NPTII-gene (Beck et al, 1982, Gene 19:327- 336). The 54 kDa-gene was detected by the forward primer 5'-TCA ACG CGA GAG TTG GTC AAG-3 ' and the backward primer 5 '-TGC AAC ACG TCT CCG GTT AAA G-3 ' generating a fragment of 794 nucleotides corresponding to base 448-1241 of the Rl sequence. Ten positive shoots of cv. Matilda were transferred to the greenhouse together with one untransformed shoot of the same cultivar as negative control. Tubers from these transformants were planted in the greenhouse the next season for multiplication and to study any eventual side effects of the transformation.

EXAMPLE 5 Greenhouse trial — test of the replicase gene

First greenhouse trial

The greenhouse trial was performed by Dr. David Robinson at the Scottish Crop Research Institute. Soil was obtained from a site near Tayport, Fife, Scotland, where Paratrichodorus pachydermus transmitting a TRV-strain serologically similar to PRN are known to occur. Tobacco seedlings, cv. White Burley, were planted in pots containing the collected soil. After 3-4 weeks the roots of each plant were ground and used to inoculate Chenopodium amaranthicolor to study the production of local lesions. Only pots where the soil was demonstrated to contain viruliferous nematodes were further used. Five tubers from each transformed line (including controls) were individually planted in the pots where the potato plants were grown to full maturity and then allowed to die back. The tubers in each pot were counted and each tuber was sliced and scored for dark brown corky layers. A scale of 0 (no symptoms) to 9 (very severe symptoms) was used for scoring (FIG 3).

Second greenhouse trial

In the first greenhouse trial all ten transgenic lines of cultivar Matilda showed fewer symptoms compared to the control (FIG 3). The infection pressure in the first trial was rather low, and therefore the two most promising lines, M6 and M565, were analysed further in a second greenhouse trial. This time there was a good infection pressure as judged from the non-transgenic controls. Still, there was a clear reduction in symptoms observed in the transgenic lines. The mean symptom scores for lines M6 and M565 are significantly lower than the score for the untransformed Matilda control according to Dunnett's test (FIG 4). For line M565 the symptom score is reduced by 78% compared to the Matilda control.

Claims

1. A nucleotide sequence molecule which comprises; a) at least one infection site specific promoter region which functions in plant cells, the promoter region is operably linked to b) at least part of a structural gene encoding part of a tobacco rattle virus replicase polypeptide, which is operably linked to c) at least one non-translated region which functions in plant cells.

2. The nucleotide sequence molecule according to claim 1, wherein the structural gene having at least 75 % identity to the nucleotide sequence shown in SEQ ID

NO l.

3. The nucleotide sequence molecule according to claim 2, wherein the structural gene having at least 80 % identity to the nucleotide sequence shown in SEQ ID NO 1.

4. The nucleotide sequence molecule according to claim 3, wherein the structural gene having at least 85 % identity to the nucleotide sequence shown in SEQ ID NO l .

5. The nucleotide sequence molecule according to claim 4, wherein the structural gene having at least 90 % identity to the nucleotide sequence shown in SEQ ID NO l.

6. The nucleotide sequence molecule according to claim 5, wherein the structural gene having at least 95 % identity to the nucleotide sequence shown in SEQ ID NO l.

7. The nucleotide sequence molecule according to claim 6, wherein the infection site specific promoter is a promoter capable of being active mainly in the tubers of a potato, such as the GBSS promoter or the patatin I promoter.

8. The nucleotide sequence molecule according to any of claims 1-7 for use in a method for the production of plants having an increased resistance against infection by tobacco rattle virus.

9. A plant transformation vector comprising a nucleotide sequence molecule according to any of claims 1-8.

10. The plant transformation vector according to claim 9, wherein the plant transformation vector is based on pBHOl, pBI121, pGPTV-kan or pPZP.

11. A procaryotic cell comprising a plant transformation vector according to any of claims 9 or 10.

12. The procaryotic cell according to claim 11, wherein the cell is selected from the group consisting of cells from Agrobacterium tumefaciens and Agrobacterium rhizogenes.

13. The procaryotic cell according to claim 12 is selected from the group consisting of LBA4404, EHA 101, C68C1, GV3101 and A281.

14. A method for producing a transgenic plant with resistance to infection by tobacco rattle virus in a plant, the method comprises: a) transforming a plant cell from the plant with a nucleotide sequence molecule according to any of claims 1-7 or a vector according to claim 9 or 10 or a procaryotic cell according to any of claims 11-13; b) regeneration of the plant cell and production of transgenic plants; c) selecting a transgenic plant which transcribes the introduced nucleotide sequence molecule and which provides resistance to the TRV.

15. The method according to claim 14, wherein the plant cell is a potato plant cell.

16. The method according to claim 15, wherein the plant cell is a cell from Solanum tuberosum.

17. A transgenic plant produced by a method according to any of the claims 14-16 and parts thereof including seeds, tubers, cuttings, pollen, and microtubers obtained from the transgenic plant.

18. The transgenic plant according to claim 17, wherein the plant, is a potato plant, such as Solanum tuberosum.

19. The transgenic plant according to any of claims 17 or 18, wherein the transgenic plant comprises at least one copy of the nucleotide sequence molecule.

20. A method for the detection of a transgenic plant according to 17-19 or part of the transgenic plant, the method comprises a step in which a molecular marker is used, the molecular marker comprises at least a part of the nucleotide sequence molecule according to any of the claims 1-7.

21. A food product based on a transgenic plant or a propagation part of a transgenic plant according to the claims 17-19.

22. The food product according to claim 21, in the form of potatoes or processed potatoes.

23. The food product according to claim 21 comprising processed potatoes .

24. The method according to claim 20, for the detection of a food product according to any of the claims 21-23.