WO1994016087A1 - Plant virus-resistant transgenic plants and method for producing same - Google Patents

Abstract

A potyvirus-resistant plant having in its genome one or more DNA fragments expressing transcripts corresponding to a protein or protein fraction of a donor virus, e.g. a protein involved in the formation of a potyvirus replication complex, a protein involved in the transport of a potyvirus between cells, or a protein having a similarity with the cleavage sites of potyvirus viral proteins. A method for producing plant virus-resistant plants comprises inserting vectors from a donor virus complementary cDNA libray into cells or tissue fragments from said plants, and selecting plants displaying plant virus resistance.

Description

"Transgenic plants resistant to plant viruses and process for obtaining them"

The subject of the present invention is transgenic plants resistant to plant viruses and a process for obtaining plants.

It also relates to fragments. DNA, RNA and proteins that provide protection to plants against these viruses.

Viruses are among the major pathogens of cultivated plants. Due to the dependence of viruses on their host cells, it is difficult to destroy them without running the risk of damaging the organisms they parasitize. The means of control ^s' therefore essentially preventive: health seed selection, use of resistant varieties, sanitation plants infected with thermotherapy or meristem culture.

Certain genotypes have resistance genes for certain viruses, but these characters take an extremely long time to be introduced into other lines by the conventional pathways of genetics.

The number of plant species accessible to genetic transformation is increasing steadily. Strategies for introducing resistance genes by this route are therefore highly desirable. No natural resistance gene has yet been isolated, so current methods involve the construction of artificial genes.

The potyviruses form by their number and their agronomic importance the most important group of plant viruses. The filamentous viral particle 60 _0-900 nm in length contains a single RNA molecule of positive polarity of approximately 10 kb. Each RNA molecule carries a protein covalently linked at its 5 'end (VPg) and is polyadenylated at its 3 'end. In the plant cell, these viruses induce the formation of cylindrical and sometimes nuclear cytoplasmic inclusions of characteristic shape. They are transmitted naturally by aphids in a non-persistent fashion. Most have a relatively narrow host spectrum.

Potato virus Y (PVY) is the typical member of this group. There are three main strains (O, N and C) characterized by the differences in symptoms they induce. PVY is pathogenic for many Solanaceae of agronomic interest, such as tobacco, tomatoes, peppers and various varieties of potatoes. In this ^last case head yields can be reduced by 10 to 80% depending on the virus strain, cultivar and the time of infection. Early tobacco infection can cause yield loss of around 30%; necrosis of the ribs disease, caused by the N strain on certain varieties of tobacco, can cause a total loss of the crop of this plant.

The RNA of potyviruses, like that of other associated plant-type picorna viruses (nepovirus and comovirus) is translated into a polypeptide, which is then mature by proteolytic cleavage to give the viral proteins. The review by Riechmann et al. (J. Gen. Virol. (1992), 73, 1-16) summarizes the results obtained recently.

It has been shown that there is a great similarity in the genomic organization of the proteins of the replicative complex of potyviruses, comoviruses and nepoviruses.

Analysis of the nucleotide sequences of the potyviruses confirmed the existence of a single long open reading phase, between an AUG codon initiator and a translation stop codon; viral RNA codes for a polyprotein, which is later cleaved by enzymatic proteolysis. A partial genetic map of these viruses has been established and is represented for the PVY virus in FIG. 1. It has been shown that one of the nuclear inclusions (Nia) is a protease (Carrington and Dougherty 1987, J. Virol. 61 , 2540-2547), and sequence homologies demonstrated with the proteins encoded by picornaviruses suggest that the other nuclear inclusion (Nlb) could be involved in replication (Domier et al. 1987 Virology 158, 20-27) . The other known proteins are those of the helper factor necessary for the transmission by aphids, of cylindrical inclusion, of the VPg bound in 5 'of the viral RNA, and finally of the capsid. To this are added two proteins framing the assistant factor.

One with a mass of 31 kD, called PI, could be involved in cell-to-cell transport (Domier, (1987), Virology, 158, 20-27) while the other with a mass of 38 kD called P3 is implicated in replication (Gamble - Klein et al, Abstracts of the third international congress of plant molecular biology, Tuckson, Arizona, October 6-11, 1991).

In 1985 Sanford and Johnston (J. Theor. Biol. 113, 395-405) introduced the concept of pathogen-derived resistance, whereby nucleotide sequences from a pathogen can be used to genetically modify the host to that it becomes resistant to this pathogen.

According to this principle, resistance to plant viruses has already been obtained by transformation of plants using DNA fragments carrying genes coding for the capsid proteins.

A bibliographic review recorded the results obtained on various plant viruses (Beachy et al. Annual Review of Phytopathol, 1990, 28: 451-74).

This technique was more precisely developed in the case of potyviruses and resulted in the filing of PCT application FR-89 00 260 (published under the number O-89 121 00). In this application, the construction of a gene coding for the capsid protein of potyvirus is described in conditions such that its function is not altered.

Transgenic plants for this gene ^" and resistant to potyviruses can be obtained. Another publication mentions obtaining plants resistant to tobacco mosaic viruses by the introduction of a gene supposed to code for a non-structural protein which could be a component of the replication complex of this virus (Golemboski et al., Proc. National Acad. Sci. USA, Vol.87, pp.6311-6315, 1990). The authors show that plants transformed using a vector carrying the sequence of this gene exhibit resistance to the virus. Although the techniques described above make it possible in certain cases to obtain plants exhibiting resistance to plant viruses, they are nevertheless not applicable to all species of plants and for all plant viruses: in the case of certain potyviruses, the results could only lead to low resistance.

In addition, mutations could intervene in viruses, rendering ineffective the resistance induced in plants transformed by capsid proteins.

It would therefore be desirable to generate other types of resistance genes in order to be able to combine several resistances in transgenic plants, and to combine the effects of weak resistances.

Such an approach has already been tested, without success by AUDY et al. (1992, Canadian Journal of Plant Pathology, 14, 240-249) who processed tomatoes and potatoes with DNA encoding the PVB virus PVY protein from potatoes. However, the authors do not mention obtaining resistant transgenic plants.

This short report could therefore not encourage a person skilled in the art to use the Nlb gene to confer on plants resistance to potyviruses.

Another report of a Symposium held in April 1992 (Maiti and Hunt, J. Cell Biochem. Suppl. Volume 16F, 1992, page 217) describes an attempt to obtain resistant plants, in particular PVY by expression of the Nia protein of the entire Tobacco Vein Mottling Virus (TVMV). The authors state that the studies performed are preliminary and that they simply indicate that expression of the protease gene may affect the response of plants to a homologous virus.

It will be noted that in this document, the transgenic plants obtained express the entire and active protease and not a part of this protein.

It therefore appears from the analysis of the state of the art that the very preliminary results obtained on transgenic plants for Nia and Nlb genes could in no way encourage the skilled person to test the potyvirus resistance of transgenic plants expressing fragments of these proteins or proteins other than proteins of capsid.

The applicant set out to develop a process for obtaining transgenic plants resistant to potyviruses transformed by non-capsid gene sequences, which do not have the abovementioned drawbacks.

According to the invention, it has been surprisingly shown that the introduction into plants of a DNA fragment expressing transcripts corresponding to at least part of a protein of a potyvirus other than a capsid protein, gave these plants effective resistance against these viruses.

The inventors have also shown, just as surprisingly, that plants resistant to plant viruses can be selected in sufficient quantities by direct introduction of a DNA library into the cells or tissues of these plants.

The subject of the present invention is a plant resistant to potyviruses, characterized in that it carries in its genome one or more DNA fragments expressing transcripts corresponding to a protein or to a part of a protein of a donor virus, with the exception of a capsid protein and the protease Nia of the whole TVMV, said plant being capable of being obtained by introduction using techniques known to those skilled in the art, such as electroporation, vectors from a DNA library of the donor virus, and selection according to criteria of resistance to potyviruses.

By donor virus is meant a virus whose genome is going to be at the origin of the DNA fragments to be inserted into the genome of the plant.

According to a first embodiment of the invention, said plant is characterized in that that it carries a DNA fragment expressing transcripts corresponding to a protein or part of protein involved in the formation of the replicative complex of a potyvirus. Such a DNA fragment may contain part of the sequence coding for the protein P3, in particular a part of the sequence between nucleotides 2853 and 3492 of the complementary DNA (cDNA) of the PVY potyvirus.

Such a DNA fragment may also contain part of the sequence coding for the central part of the protease Nia, in particular a part of the sequence between nucleotides 5999 and 6660 of the DNA sequence complementary to the PVY potyvirus. A third type of DNA fragment may contain part of the sequence coding for the C-terminal end of the Nia protease and the N-terminal end of the Nlb polymerase, in which case the DNA fragment is preferably between nucleotides 6652 and 7080 of the DNA sequence complementary to the PVY potyvirus.

The complementary DNA sequences corresponding to the sequence of potyvirus Y as well as the amino acids composing the corresponding polyprotein were described in the above-mentioned application WO 89-1100. Reference is therefore made to this document with regard to the numbering of the nucleotides and amino acids of this polyprotein.

However, this application does not mention the particular sequences which are the subject of the present invention.

According to a second embodiment, said plant can carry a DNA fragment expressing transcripts corresponding to a protein involved in the transport of a potyvirus from a cell to a other. In this case, the DNA fragment can code for part of the PI protein, and in particular be between nucleotides 428 and 1044 of the DNA sequence complementary to the PVY potyvirus. According to a third embodiment of the invention, said plant can also be defined in that it carries a DNA fragment expressing transcripts corresponding to a protein or a part of protein having similarity with the cleavage sites. viral proteins from a potyvirus.

Preferably, the potyvirus against which the plants are resistant is the potato virus Y.

Said plant can be any plant susceptible to plant viruses and in particular to potyviruses.

The present invention can also be applied to the resistance of plants to other plant viruses such as nepoviruses and comoviruses. The DNA fragments carried by the plants are then preferably fragments expressing transcripts corresponding to proteins or parts of protein having similarities with the central part of the Nia protease or the part of the polyprotein corresponding to the C-terminal end. of the Nia protease and the N-terminus of the Nlb polymerase.

This plant in the case of PVY advantageously belongs to the Solanaceae family including in particular tobacco, tomato, pepper and potato.

The present invention further relates to a DNA fragment as defined above and providing protection to plants against plant viruses, and in particular potyviruses. The present invention further relates to RNA capable of being transcribed from one of these DNA fragments.

Another object of the present invention is a protein fragment or a protein as defined above and providing plants with resistance to potyviruses.

More generally, the present invention further relates to a process for obtaining plants resistant to a plant virus in which: - vectors are introduced into cells or tissue fragments of said plants. DNA complementary to a donor virus, - plants are regenerated from said cells or said tissue fragments, and - plants selected having resistance against the plant virus are selected.

Advantageously, such a method can be applied to the selection of plants resistant to a potyvirus, a nepovirus or a comovirus, but the method can be applied to obtaining plants resistant to any plant virus. .

The vectors originating from the complementary DNA library can be introduced by electroporation or by any method known to a person skilled in the art making it possible to introduce DNA into a plant cell.

Preferably, the donor virus as defined above is a virus belonging to the same family as that for which it is sought to obtain resistant plants. Thus, a DNA bank of a potyvirus will preferably be used to obtain plants resistant to a potyvirus.

The present invention is illustrated without however being limited by the examples which follow. FIG. 1 schematically represents the known structure of the polyprotein of the potato virus Y (1A) and the peptides translated from this virus (1B). The curved arrows indicate the proteolytic cleavages.

FIG. 2 represents the general scheme for obtaining plants resistant to viruses from DNA complementary to PVY virus.

FIG. 3 shows more precisely the method for cloning the viral complementary DNA fragments which resulted in the manufacture of a complementary DNA library.

FIGS. 4 to 7 illustrate the resistances respectively of the descendants of the clones FCD3E1, FCD3B6, FCD4A2 and FCD3I1.

The percentage of infected plants is expressed on the ordinate while the number of days following inoculation of the virus with the plant is indicated on the abscissa. FIGS. 8 and 9 respectively illustrate the resistances of first and second generation plants homozygous for the introduced gene resulting from crosses from the clone T3A5.

FIGS. 10 to 13 represent the nucleotide sequence of the DNA expressed in the T3A5 clones,

FCD3I1, FCD4A2 and FCD3B6 and their corresponding peptide sequences in the three possible reading frames denoted a, b, and c.

Figure 14 is a schematic genetic map of the PVY virus, in which the black arrows indicate the positions of the virus fragments corresponding to the DNA fragments integrated into the lines T3A5, FCD3I1, FCD4A2 and FCD3B6. EXAMPLE 1; Construction of the expression vector of the complementary DNA bank

The process for constructing this vector is shown diagrammatically in FIGS. 2 and 3.

In FIG. 2, the complementary DNA (cDNA) of the viral genome (PVY RNA) is synthesized by primer at random in step (1).

The cDNA obtained in (2) is cloned into a Sma I site of the plasmid pRT103NPT II (-) shown schematically in (3) and gives rise to a cDNA library shown schematically in (4). The cDNA library (4) is then introduced by electroporation into a preparation of tobacco protoplasts (5) then the clones are selected and regenerated (6) ^' and subjected to PVY resistance tests (7). Figure 3 summarizes more precisely the manufacturing of the bank.

An 800 base pair fragment from the Hind III digestion of the plasmid pABDl (Paskowski et al., 1984, EMBO, 3,2717-2722) was purified and filled in with Klenow polymerase. This fragment corresponds to the coding part of the gene conferring resistance to kanamycin.

It is then cloned at the BamHI site of the plasmid pRt103 filled with the Klenow polymerase (Topfer et al., 1987, N.A.R., Vol.15, n ° 14).

The plasmid thus obtained, called pRtl03NPTII (-), is composed by the promoter of the RNA 35S of the cauliflower mosaic virus (CaMV) followed by a translation initiation codon which is not in phase with the coding region of the gene conferring resistance to kanamycin derived from the bacterial transposon Tn5, coding for the protein neomycin phosphotransferase II (NPTII). This plasmid further comprises the transcription terminator of the cauliflower mosaic virus. DNA complementary to the viral RNA of a strain N of the potato virus Y obtained by random priming (Sambrook et al., 1989, Cold

Spring Harbor Laboratory press) was cloned into the plasmid pRtl03NPTII (-) at the Smal site.

Since the NPTII gene is not placed in phase with the translation initiation codon, the insertion of a complementary DNA fragment coding for a fraction of the viral polyprotein can restore the continuity of the reading phase between the initiation codon and the coding region of the NPTII gene. Such an event leads to the assembly of a transcriptional unit which can result in the synthesis of a fusion protein comprising an N-terminal end derived from the viral polyprotein and a C-terminal end corresponding to the neomycin phosphotransferase II (NPTII) .

It should be noted that the NPTII protein is known to support N-terminal extensions which do not affect its activity. EXAMPLE 2:

Obtaining transgenic plants.

The complementary DNA library obtained in Example 1 was introduced by electroporation (Guerche et al., 1987, Biochemistry, 69: 621) in protoplasts of Nicotiana tabacu (Xanthi variety, diploid clone D8).

Calluses resistant to kanamycin were selected, and plants were regenerated from these calluses by techniques known to those skilled in the art.

The offspring from self-fertilization of the plants thus obtained were collected and sown in vitro on a selective medium. Segregation for resistance to Kanamycin is of the dominant monogenic Mendelian type in the descendants of the clones obtained.

EXAMPLE 3:

Resistance of transgenic plants to the virus.

The descendants of transgenic plants were obtained by self-fertilization from these primary transformants. The behavior of these plants towards a viral infection was then evaluated.

Twenty plants of the progeny were mechanically inoculated by a 1/2000 dilution of an extract of plants infected with the Yn virus, as well as twenty non-transgenic control plants.

Five clones whose progeny have a phenotype of resistance to the PVYn virus were obtained (out of 53 clones tested).

The results obtained are presented on the curves of FIGS. 4 to 7 (respectively descendants of the clones FCD3E1, FCD3B6, FCD4A2 and FCD3I1) and 8 (descendants of the clone T3A5).

For these five clones, the results obtained clearly demonstrated an increased resistance to infection by potyviruses compared to a control plant.

The resistance of the plants has been confirmed by an ELISA test (SANOFI, Phyto Diagnostic) which allows the capsid protein of the virus to be determined, thus representing the level of infection of the plants. EXAMPLE 4:

Resistance of first generation and second generation plants from the T3A5 clone.

A self-fertilization of the plant from the T3A5 clone was carried out. The resistance of the plants obtained, known as the first generation, was tested. The results are shown on the curve of Figure 8, as indicated above. Among these plants, those which were homozygous for the kanamycin resistance marker associated with the viral cDNA fragment were sought.

The progeny of self-fertilization of a homozygous plant have been studied. The results are shown in Figure 9.

These results clearly show that resistance to virus Y is sexually transmitted and is maintained at an equivalent level from one generation to the next. EXAMPLE 5:

Determination of the sequences expressed in transgenic plants.

Complementary DNAs corresponding to the transcripts of the chimeric genes expressing a piece of viral protein, resistant plants, were obtained by the PCR retrotranscription technique (Sambrook et al, 1989, Cold Spring Harbor Laboratory Press).

The two oligonucleotides synthesized which made it possible to amplify the complementary DNAs have the following sequences:

** primer 1 covers the sequence of the expression vector pRtl03NPTII (-) ranging from nucleotide + 1 of the transcription to the ATG codon (Odell et al, 1985 Nature 313, 810)

5 'ACCTCGAGTGGCCACCATG 3'

** primer 2 covers the complementary sequence of the NPTII gene of pABDl going from nucleotide 1489 to nucleotide 1508: 5 'GGCCGGAGAACCTGCGTG 3' Only one amplification product is obtained for each plant studied.

The amplified DNA fragments were purified and sequenced (Applied Biosystem, Taq Dye Deoxy Terminator Cycle Sequencing Kit) using these oligonucleotides in order to determine the part of the viral genome which is expressed in these plants.

The sequence expressed by the clone T3A5 is as follows: from base 428 to base 1044 of the viral genome. It is represented in FIG. 10 with the corresponding peptide sequences.

This gene codes for the terminal C part of the first protein (PI) of the viral polyprotein.

The descendants of the T3A5 clone are therefore resistant to the Y virus and have a fragment of the gene coding for the PI protein integrated into their genome.

The sequences expressed by the clones FCD3I1, FCD4A2 and FCD3B6 range respectively from 2853 to 3492, from 5999 to 6660 and from 6552 to 7080. (Figures 11, 12 and 13).

The sequence expressed by the clone FCD3I1 can code for part of the P3 protein involved in replication. The sequence expressed by the clone FCD4A2 can code for a part of the Nia protein comprising the proteolytic cleavage site between the VPg part and the protease part of this protein (Dougherty and Parks (1991) - Virology. 182 449-456). The sequence expressed by the clone FCDB6 can code for a protein comprising the C-terminal end of the Nia protease and the N-terminal end of the Nlb polymerase and comprises the proteolytic cleavage site separating these two proteins. A diagram representing the different sequences expressed in the clones T3A5, FCD3I1, FCD4A2 and FCD3B6 is given in FIG. 14.

Claims

1. Plant resistant to potyviruses, characterized in that it carries in its genome one or more DNA fragments expressing transcripts corresponding to a protein or part of a protein of a donor virus, with the exception of 'A capsid protein and protease Nia of the entire TVMV, said plant being capable of being obtained by introduction of vectors from a DNA library of the donor virus, and selection according to criteria of resistance to potyvirus. 2. Plant according to claim 1, characterized in that it carries a fragment of ΑDN expressing transcripts corresponding to a protein or part of protein involved in the formation of the replicative complex of a potyvirus. 3. Plant according to claim 2, characterized in that the DNA fragment contains part of the sequence coding for the protein P3. 4. Plant according to claim 3, characterized in that the DNA fragment is between nucleotides 2853 and 3492 of the DNA complementary to the potyvirus Y. 5. Plant according to claim 2, characterized in that the DNA fragment contains part of the sequence coding for the central part of the protease Nia 6. Plant according to claim 5, characterized in that the DNA fragment is between nucleotides 5999 and 6660 of the DNA sequence complementary to potyvirus Y. 7. Plant according to claim 2, characterized in that the DNA fragment contains part of the sequence coding for the C-terminal end of the Nia protease and the N- end Nlb polymerase terminal. 8. Plant according to claim 7, characterized in that the DNA fragment is between nucleotides 6652 and 7080 of the DNA sequence complementary to potyvirus Y. 9. Plant according to claim 1, characterized in that it carries a DNA fragment expressing transcripts corresponding to a protein or a fragment of a protein involved in the transport of a potyvirus between cells. 10. Plant according to claim 9, characterized in that the DNA fragment codes at least for part of the PI protein. 11. Plant according to claim 10, characterized in that the DNA fragment is between nucleotides 428 and 1044 of the DNA sequence complementary to the potyvirus Y. 12. Plant according to claim 1, characterized in that it carries a DNA fragment expressing transcripts corresponding to a protein or a part of protein having a similarity with the cleavage sites of the viral proteins of a potyvirus. 13. Plant according to one of claims 1 to 12, characterized in that it is resistant to the potato virus. 14. Plant according to one of claims 1 to 13, characterized in that it belongs to the Solanaceae family. 15. DNA or DNA fragment as defined in one of claims 1 to 12 and providing plants with resistance to a potyvirus. 16. RNA capable of being transcribed from a DNA fragment according to claim 15. 17. Protein fragment or protein such as defined in one of claims 1 to 12 and providing plants with resistance to a potyvirus. 18. A method of obtaining plants according to any one of claims 1 to 14 wherein: - is introduced into cells or tissue fragments of said plants vectors from a DNA library complementary to a donor virus , - plants are regenerated from said cells or said tissue fragments and - plants selected having resistance against potyviruses are selected. 19. The method of claim 18, characterized in that the vectors from the complementary DNA library are introduced by electroporation.