METHOD FOR INDUCING VIRAL RESISTANCE INTO A PLANT

Abstract: The present invention concerns a method for inducing resistance to a virus comprising a TGB2 sequence into a cell plant or a plant, comprising the following steps: preparing a nucleotide construct comprising a nucleotide sequence corresponding to at least 70 % of the nucleotide sequence of TGB2 of said virus or its complementary cDNA, being operably linked to one or more regulatory sequence(s) active in a plant, transforming a plant cell with the nucleotide construct, and possibly regenerating a transgenic plant from the transformed plant cell. The present invention is also related to the plant obtained.

Description: METHOD FOR INDUCING VIRAL RESISTANCE INTO A PLANT Field of the invention The present invention is related to a method for inducing viral resistance into a cell and a plant, especially BNYW-resistance into a sugar beet cell and plant . Background of the invention and state of the art The widespread viral disease of the sugar beet plant (Beta vulgari s) called Rhizomania is caused by a benyvirus, the beet necrotic yellow vein virus (BNYW) (23, 24) which is transmitted to the root of the beet by the soilborne fungus Polymyxa betae (25) . The disease significantly affects acreages where the sugar beet plant is grown for industrial use in Europe, USA and Japan and is still in extension in several places in Western Europe (26, 27) . As there exists no practical method to effectively control the spread of the virus at a large scale by chemical or physical means (28) , neither in the plants nor in the soil, the main focus has been to identify natural sources of resistance within the sugar beet germplasm and to develop by breeding, varieties of sugar beet plants expressing the resistance genes. A variety of such tolerance genes to the virus have been identified and, some have been successfully used in the breeding of commercial sugar beet varieties (29, 30, 31) . Only the use of BNYW-resistant or tolerant varieties will enable farmers to grow sugar beet plants m BNYW- infected areas where the sugar beet plant is an essential component of the crop rotation and contributes significantly to the grower's income. A number of detailed studies have shown that a difference m susceptibility to the BNYW- infection among sugar beet genotypes or varieties, generally reflect difference m the diffusion or translocation of the virus m the root tissues (32) . However, there are still few reports which indicate clearly that the tolerance genes, even from differing sources of sugar beet germplasm or wild relatives germplasm (33), would provide distinct mechanisms of resistance. Such a situation would represent a more manageable situation to design long lasting BNYW- resistance strategies. Since 1986, a number of reports and publications have described the use of isolated viral gene sequences expressed m plants to confer a high level of tolerance against the virus or even to confer a broad spectrum type of resistance against a number of related viruses (34, 35, 36) . One of the most documented viral resistance strategy based on genetic engineering, m many cultivated species such as potato, squash, cucumber or tomato, is the use of the viral gene sequence which under the control of plant regulatory elements, encodes the coat- protem of the target virus (37) . However, for coat-protein mediated resistance, the expression of a certain level of resistance m the transgenic plant might be attributed to different mechanisms such as RNA co-suppression and not necessarily to the production of tne protein sequence. In general, the virus sequence will be transferred m an appropriate cell or tissue culture of the plant species using an Agroba cterium mediated transformation system or a direct gene transfer method according to the constraints of the tissue culture or cell culture method which can be successfully applied m a given species A whole plant will be regenerated and the expression of the transgene will be characterized. Though sugar beet is known as a recalcitrant species m cell culture, limiting the extent of practical genetic engineering applications m that species, there are number of isolated reports of successful transformation and regeneration of whole plants (38) . A few examples of engineering tolerance to the BNYW by transforming and expressing the BNYW coat -protein sequence m the sugar beet genome have also been published (39, W091/13159) though they rarely report data on whole functional transgenic sugar beet plants (40) . In particular, reports show limited data on the level of resistance observed m infected conditions with transgenic sugar beet plants transformed with a gene encoding a BNYW coat-protein sequence (41, 42) . A complete technology package including a sugar beet transformation method and the use of the expression of the BNYλ/N coat-protem sequence as resistance source m the transgenic sugar beet plant obtained by said transformation method has been described m the Patent Application W091/13159. Based on the information published, it can not be concluded that the coat-protem mediated resistance mechanism provides any potential for conferring to the sugar beet plant a total immunity to the BΝYW- infection by inhibiting completely the virus multiplication and diffusion mechanisms. To identify a resistance mechanism which significantly blocks the spread of the virus at the early stage of the infection process would be a major step toward successfully developing such a transgenic resistance. In addition, such resistance would diversify the mechanisms of resistance available. Because the disease is shown to expand m many countries or areas, at a speed depending upon the combination of numerous local environmental and agricultural factors, there is a strong interest diversifying genetic resistance mechanisms which may, alone or m combination, confer a stable and long lasting resistance strategy m the current and future varieties of sugar beet plants which are grown for industrial use. The genome of beet necrotic yellow vein benyvirus (BNYλ N) consists of five plus-sense RΝAs , two of which (RΝAs 1 and 2) encode functions essential for infection of all plants while the other three (RΝAs 3, 4 and 5) are implicated m vector-mediated infections of host plants (Beta macrocarpa , Beta vulgar is , Spinacear oleracea, Chenopodium quinoa, etc.) roots (1). Cell-to-cell movement of BΝYλ/N is governed by a set of three successive, slightly overlapping viral genes on RΝA 2 known as the triple gene block (TGB) (2), which encode the viral proteins P42, P13 and P15 (gene products are designated by their calculated M r m kilodalton (3) . In the following description, the TGB genes and the corresponding proteins will be identified by the following terms: TGBl, TGB2 , TGB3 or by their encoded viral protein number P42, P13 and P15. TGB counterparts are present m other plant viruses and the characteristics of their TGB have allowed the classification of said viruses m two groups: the viruses of group I which include hordeiviruses, benyviruses, pecluviruses and pomoviruses and the viruses of group II represented by potexviruses and carlaviruses (4, 5, 6, 44) . For the viruses of group II, capsid protein is also involved m the cell-to-cell movement of viruses. The development of a resistance to viral infections into a plant by blocking the cell-to-cell movement has been described for the potato viruses X (PVX) (45) and for the white clover mosaic virus (WC1MV) (46) m Nicotiana benthamiana . These two viruses belong to the above-described group II. In both cases, various ammo acids were replaced by Alanme the hydrophilic part of the TGB sequence downstream of the N-terminal hydrophobic domain of said ammo acid sequence. However, it was not possible with said mutants to obtain total resistance, especially when a virus challenger concentration is increasing into the plant . Aims of the invention The present invention aims to provide a new method for introducing various viral resistances into a cell and a plant and the viral resistant cell and plant obtained. A ma aim of the invention is to provide a new method for introducing BNY resistance into a cell and a plant and the BNYW-resistant cell and plant, particular a sugar beet cell and plant (Beta vulgar is ssp . ) , obtained. Summary of the invention The present invention provides the use of an alternative sequence of plant virus, especially the BNYλ/N, to obtain a high degree of tolerance to the viral infection, particular to ensure a rapid and total blocking of virus multiplication and diffusion mechanisms a plant, especially m the sugar beet plant (Beta vulgar i s) , including fodder beet, Swiss chard and table beet, which may also be subject to this viral infection. Expression of the resistance will be obtained m transgenic cell and plant, especially sugar beet cells and plants produced by the transformation method subject to the Patent Application WO95/10178 or by other transformation methods based on Agrobacteriu tumefaci ens or direct gene transfer. Because of its high efficiency, the transformation method as described WO95/10178 enables the production of large numbers of transformed plants, especially sugar beet plants, and will be preferred to develop transgenic plants which may be analysed and characterized for their level of viral resistance, especially BNYW Resistance, including their field evaluation. In the table 1 are represented viruses having a TGB2 sequence, the molecular weight of TGB2 of said viruses, their host and references. Table 1 The Inventors propose herewith a new method for providing resistance to plant viruses into a plant by blocking virus multiplication and diffusion mechanisms into said plant, especially into its root tissue. In order to demonstrate said resistance, the Inventors describe hereafter the effect of the overexpression of TGB2 sequence alone or combination upon BNYW multiplication and diffusion mechanism plants of C . qumoa which are also the hosts of the BNYW virus and which could be more easily manipulated by the man skilled the art. It is known that BNYλ/N does not require synthesis of viral coat protein for production of local lesions on leaves of hosts such as Chenopodium qumoa (7) , indicating that virion formation is not required for cell- to-cell movement. However, the manner which the TGB components assist m the movement process is not understood although computer-assisted sequence comparisons have detected characteristic conserved sequences which may provide clues to their function. Thus, the 5 ' -proximal TGB protein (TGBl) invariably contains a series of sequence motifs characteristic of an ATP/GTP-b dmg helicase while the second protein (TGB2) always has two potentially membrane-spanning hydrophobic domains separated by a hydrophilic sequence which contains a highly conserved peptide motif of unknown significance (6) . So far, no example has been reported of a virus of group I which the three TGB members are arranged differently on the same RΝA or are parcelled out to different genome RΝAs, suggesting that their association m a particular order might be important regulating their function. The present invention concerns a method for inducing viral resistance to a virus of group I comprising the triple gene block (TGB2) . Said viruses of group I comprise hordeiviruses , benyviruses, pecluviruses and pomoviruses, preferably viruses selected from the group consisting of the beet necrotic yellow vein virus, the barley stripe mosaic virus, the potato mop top virus, the peanut clump virus and tne beet soil -borne virus; said method comprises the following steps: - preparing a nucleotide construct comprising a nucleotide sequence corresponding to at least 70% of the wild-type nucleotide sequence of TGB2 of said group I virus or its corresponding cDNA, being operably linked to one or more regulatory sequence (s) active a plant, - transforming a plant cell with the nucleotide construct, and possibly - regenerating the transgenic plant from the transformed plant cell . Advantageously, the nucleotide sequence corresponding to at least 70% of the wild-type nucleotide sequence of TGB2 or its corresponding cDNA comprise the substitution of at least one ammo acid into another different ammo acid m the TGB2 wild-type sequence SEQ ID NO. 1 (Fig. 1) . Preferably, the substitution of at least one ammo acid into another different ammo acid is made m regions rich m hydrophilic ammo acids usually present at the surface of the corresponding protein m its native configuration. Preferably, a modification is made m the hydrophilic region of the wild-type sequence downstream the N-terminal hydrophobic domain and ust upstream the conserved central domain. According to a preferred embodiment of the present invention, said ammo acids are each substituted by the ammo acid Alanme. Preferably, the plant or plant cell is a plant or plant cell which may be infected by the above- described virus and is preferably selected from the group consisting of potato, barley, peanut and sugar beet. The present invention concerns also the obtained plant cell and transgenic (or transformed) plant (made of said plant cells) resistant to said viruses and comprising said nucleotide construct. The Inventors have also discovered unexpectedly that it is possible to induce BNYW-resistance into a plant by a method wnich comprises the following steps : - preparing a nucleotide construct comprising a nucleotide sequence corresponding to at least 70%, preferably at least 80%, more preferably at least 90%, of the wild-type nucleotide sequence comprised between the nucleotides 3287 and 3643 of the 5' strand of the genomic or subgenomic wild-type RNA 2 of the BNYW or its corresponding cDNA, being operably linked to one or more regulatory sequence (s) active m a plant, - transforming a plant cell with said construct, and possibly - regenerating a transgenic plant from the transformed plant cell . The nucleotide sequence comprised between the nucleotides 3287 and 3643 of the 5' strand of the genomic or subgenomic RNA 2 encoding the P13 protein is described the Fig. 1 (SEQ ID NO. 1) . A preferred mutated nucleotide sequence and its corresponding mutated ammo acid sequence are described the following specification as SEQ ID NO. 3 (Fig. 2) . Another aspect of the present invention concerns a plant cell and a transgenic plant (made of said plant cells) resistant to BNYW and comprising a nucleotide construct having a nucleotide sequence corresponding to at least 70%, preferably at least 80%, more preferably at least 90%, of the nucleotide sequence comprised between the nucleotides 3287 and 3643 of the 5' strand of the genomic or subgenomic wild-type RNA 2 of BNYλ/N or its corresponding cDΝA, being operably linked to one or more regulatory sequence (s) active m the plant. Preferably, said plant cell or transgenic plant (made of said plant cells) resistant to BΝYVN is obtained by the method according to the invention. The variants of the wild-type nucleotide sequence (SEQ ID NO. 1) comprise insertion, substitution or deletion of nucleotides encoding the same or different ammo acιd(s) (see Fig 2) . Therefore, the present invention concerns also said variants of the nucleotide sequence of SEQ ID NO. 1, for example SEQ ID NO. 3, which present at least 70%, preferably at least 80%, more preferably at least 90%, homology with said nucleotide sequence and which are preferably able to hybridise to said nucleotide sequence m stringent or non-stringent conditions as described by Sambrook et al . , §§ 9.47-9.51 m Molecular Cloning : A Labora tory Manual , Cold Spring Harbor, Laboratory Press, Cold Spring Harbor, New York (1989) . A nucleotide sequence corresponding to at least 70%, preferably at least 80%, more preferably at least 90%, of the nucleotide sequence comprised between the nucleotides 3287 and 3643 of the 5' strand of the genomic or subgenomic wild-type RNA 2 of BNYW or its corresponding cDNA, is preferably a sequence comprising a substitution of at least one ammo acid into another different ammo acid m the wild-type RNA2 sequence of the BNYW or its corresponding cDNA. Preferably said substitution is made m regions m which hydrophilic ammo acids are usually present at the surface of the protein its native configuration (47) as described m Fig. 2 (A = substitution by Alanme) . Preferably, said substitution of one or more ammo acids is a mutation which allows the substitution of one or more ammo acids into one or more Alanme ammo acids . According to a preferred embodiment of the present invention, said nucleotide sequence is SEQ ID NO . 3. Preferably, said sequences are also able to induce BNYW resistance into a plant. The terms "induce a viral resistance into a plant" mean inducing a possible reduction or a significant delay into the appearance of infection symptoms, virus multiplication or its diffusion mechanisms into the plant, especially m the root tissues. In Fig. 3 are represented results snowing the capacity of a plant comoculated with virus containing a replicon construct with the nucleotide sequence according to the invention, especially the sequence SEQ ID NO. 3, to inhibit the movement by BNYλ N m C. Qumoa . The infectious factor of BΝYλ/N is shown by the appearance of local lesions of leaves of said plant after co- oculation of wild-type virus S12. Fig. 3 presents the number of local lesions upon leaves of a plant by a BΝYW S12 isolate (comprising RΝA1 and RΝA2) when co- inoculated with various replicons incorporating either mutated sequences including SEQ ID NO. 3 identified Fig. 2 or a wild-type nucleotide sequence (T) . Eight days after said inoculation, the local lesions are identified. The results of three experiments show that the decreasing of said effect is mostly observed with the co-moculation of the mutated sequence SEQ ID NO. 3 (up to 100% inhibition) . This effect is not due to a possible blocking effect upon RNA1 and RNA2 replication, but the replicons according to the invention allow a blocking of the biochemical mechanisms involved m cell-to- cell movements by the infectious virus. The regulatory sequence (s) of the nucleotide sequence according to the invention are promoter sequence (s) and terminator sequence (s) active into a plant. The nucleotide construct may also include a selectable marker gene, which could be used to identify the transformed cell or plant and express the nucleotide construct according to the invention Preferably, the cell is a stomatal cell and the plant is a sugar beet (Beva vulgaπs ssp . ) made of said cells . According to the invention, the promoter sequence is a constitutive or foreigner promoter sequence. Examples are 35S Cauliflower Mosaic Virus promoter sequence, polyubiquitin Arabidopsi s thai i ana promoter (43), a promoter which is mainly active in root tissues such as the par promoter of the haemoglobin gene from Perosponia andersonii (Landsman et al . , Mol. Gen . Genet. 214 : 68-73 (1988)) or a mixture thereof. A last aspect of the present invention is related to a transgenic plant tissue such as fruit, stem, root, tuber, seed of the transgenic plant according to the invention or a reproducible structure (preferably selected from the group consisting of calluses, buds or embryos) obtained from the transgenic plant or the cell according to the invention. The techniques of plant transformation, tissue culture and regeneration used in the method according to the invention are the ones well known by the person skilled in the art. Such techniques are preferably the ones described in the International Patent Applications WO95/10178 or W091/13159 corresponding to the European Patent Application EP-B-0517833 , which are incorporated herein by reference. These techniques are preferably used for the preparation of transgenic sugar beets according to the invention. REFERENCES 1. Richards K.E. & Tamada T., Annu . Rev. Phytopathol . 30, pp. 291-313 (1992) 2. Gilmer D. et al . , Virology 189, pp. 40-47 (1992) 3. Bouzoubaa S. et al . , J " . Gen. Virol. 68, pp. 615-626 (1987) 4. Herzog E. et al . , J. Gen. Virol. 18, pp. 3147-3155 (1994) 5. Scott K. P. et al., J " . Gen. Virol. 75, pp. 3561-3568 (1994) 6. Koonin E.V. & Dolja V.V., Crit. Rev. Biochem. and Mol. Biol. 28, pp. 375-430 (1993) 7. Schmitt C. et al . , Proc . Natl . Acad. Sci . USA. 89, pp. 5715-5719 (1992) 8. Morozov S.Y. et al . , J. Gen. Virol. 72, pp. 2039-2042 (1991) 22. Pelletier J. & Sonenberg N, Nature 334, pp. 320-325 (1988) 23. Tamada T. & Baba T., Annals of the Phytopathological Society of Japan 39, pp. 325-332 (1973) 24. Kuszala M.& Putz C, Annals of Phytopathology 9, pp. 435-446 (1977) 25. Tamada T., CMI/AAB Description of Plants Viruses 44, (1975) 26. Asher M.J.C., Rhizomania In The sugar beet crop, ed. D.A. Cooke and R.K. Scott, Chapman & Hall, London, pp. 312-338 (1993) 27. Richard-Molard M., Rhizomanie In Institut franςais de la betterave industrielle . 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Kallerhof J. et al . , Plant Cell Reports 9 , pp. 224-228 (1990) 40. Ehlers U. et al . , Theoreti cal and Appli ed Geneti c 81, pp. 777-782 (1991) 41. Kraus J. et al . , Field performance of transgenic sugar beet plants expresing BNYW coat protein plants, Fourth International Congress of Plant Molecular Biology, Int . Soc . for Plant Molecular Biology, Amsterdam (1994) 42. Maiss E. et al . , Proceedings of the Third Interna ti onal Symposium on the Biosafety Resul ts of Field Tests of Geneti cally Modifi ed Plants and Microorgani sms, Monterey, pp. 129-139 (1994) 43. Norris et al . , Plan t Mol ecular Bi ology 21, pp. 895-906 (1993) 44. Solovyev et al . , Virology 219, pp. 9-18 (1996) 45. Seppanen P. et al . , J. of General Virology 78, pp. 1241-1246 (1997) 46. Beck et al . , Proc . Natl . Acad . Sci . USA 91, pp. 10310- 10314 (1994) 47. Cunningham et Wells, Sci ences 244, pp. 1081-1085 (1989)

Claims: CLAIMS 1. Method for inducing resistance to a group I virus comprising a TGB2 sequence into a plant cell or a plant, comprising the following steps: - preparing a nucleotide construct comprising a nucleotide sequence corresponding to at least 70% of the nucleotide sequence of TGB2 of said virus or its complementary cDNA, being operably linked to one or more regulatory sequence (s) active m a plant, - transforming a plant cell with the nucleotide construct, and possibly - regenerating a transgenic plant from the transformed plant cell . 2. Method according to the claim 1, characterized m that the nucleotide sequence of the nucleotide construct corresponds to at least 80%, preferably at least 90%, of the nucleotide sequence of TGB2 of said virus or its complementary cDNA. 3. Method according to the claim 1 or 2 , characterized m that the group I virus is selected from the group consisting of hordeiviruses , benyviruses, pecluviruses and pomoviruses, preferably selected from the group consisting of the beet necrotic yellow vein virus, the barley stripe mosaic virus, the potato mop top virus, the peanut clump virus and the beet soil -borne virus. 4. Method according to any of the preceding claims, characterized m that the plant cell is a stomatal cell. 5. Method according to any of the preceding claims, characterized m that the plant is selected from the group consisting of sugar beet, potato, barley or peanut . 6. Method according to claim 1 or 2, characterized m that the virus is BNYW, the nucleotide sequence of TGB2 of said virus is comprised between the nucleotide 3287 and 3643 of the 5' strand of genomic or subgenomic RNA 2 of the BNYW and the plant is a beet, preferably a sugar beet (Beta vulgaris) . 7. Method according to any of the preceding claims, characterized m that the regulatory sequence comprises a promoter sequence or a terminator sequence active m a plant. 8. Method according to claim 7 characterized m that the promoter sequence is a constitutive or a foreigner promoter sequence. 9. Method according to the preceding claim 7, characterized m that the promoter sequence is selected from the group consisting of 35S Cauliflower Mosaic Virus promoter, and/or the polyubiquitm Arabidopsis thaliana promoter. 10. Method according to any of the claim 7 to 9, characterized m that the promoter sequence is a promoter which is capable of being active mainly into the root tissues of plants, such as the par promoter of the haemoglobin gene from Perosponia andersonn. 11. Transgenic plant resistant to a group I virus comprising a nucleotide construct having a nucleotide sequence corresponding to at least 70% of the nucleotide sequence of TGB2 of said virus or its corresponding cDNA, being operably linked to one or more regulatory sequence (s) active m a plant. 12. Transgenic plant according to the claim 11, characterized m that the nucleotide construct has a nucleotide sequence corresponding to at least 80%, preferably at least 90%, of the nucleotide sequence of TGB2 of said virus or its complementary cDNA. 13. Transgenic plant according to the claim 11 or 12, characterized m that the virus is selected from the group consisting of hordeiviruses, benyviruses, pecluviruses and pomoviruses, preferably selected from the group consisting of the beet necrotic yellow vein virus, the barley stripe mosaic virus, the potato mop top virus, the peanut clump virus and the beet soil -borne virus. 14. Transgenic plant according to the claims 11 to 13 being a plant selected from the group consisting of sugar beet, potato, barley or peanut. 15. Transgenic plant according to the claims 11 or 12, characterized m that the transgenic plant being a beet, preferably a sugar beet (Beta vulgar i s) the virus is BNYW and the nucleotide sequence of TGB2 of said virus is comprised between the nucleotides 3287 and 3643 of the 5 ' strand of genomic or subgenomic RNA 2 of BNYW or its corresponding cDNA. 16. Transgenic plant according to any of the preceding claims 11 to 15, characterized m that the regulatory sequence comprises a promoter sequence and a terminator sequence active a plant . 17. Transgenic plant according to any of the preceding claims 11 to 16, characterized m that the regulatory sequence (s) comprise a promoter sequence which is a constitutive or a foreigner promoter sequence. 18. Transgenic plant according to the claim 17, characterized m that promoter sequence is selected from the group consisting of 35S Cauliflower Mosaic Virus promoter, and/or the polyubiquitm Arabidopsis thaliana promoter. 19. Transgenic plant according to claim 17 or 18 characterized m that the promoter sequence is a promoter which is capable of being active mainly into root tissues, such as the par promoter of the haemoglobin gene from Perospoma andersom i . 20. Transgenic plant tissue selected from the group consisting of fruit, stem, root, tuber, seed of a plant according to any of the preceding claims 11 to 19. 21. Transgenic plant according to any one of the claims 11 to 19, characterised m that it further carries natural tolerance to Group I viruses . 22. Transgenic plant according to any one of the claims 11 to 19 and 21, characterised m that it further comprises a pesticide, herbicide or fungicide resistance, preferably a resistance selected from the group consisting of nematode resistance, glyphosate resistance, glufosomate resistance and/or acetochloride resistance.

Inventors: GUILLEY HUBERT, JONARD GERARD, LAUBER EMMANUELLE, RICHARDS KENNETH, GUILLEY, HUBERT, JONARD, GERARD, LAUBER, EMMANUELLE, RICHARDS, KENNETH
Applicants: CENTRE NAT RECH SCIENT, GUILLEY HUBERT, JONARD GERARD, LAUBER EMMANUELLE, RICHARDS KENNETH, CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE (CNRS), GUILLEY, HUBERT, JONARD, GERARD, LAUBER, EMMANUELLE, RICHARDS, KENNETH
Application year: 2000
Main classification: C12N
IPC codes: C07K14, C12N15