FR2700235A1 - Transgenic plants resistant to plant viruses and method of obtaining same. - Google Patents

Abstract

Plant resistant to potyviruses carrying 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 such as a protein involved in the formation of the replicative complex of a potyvirus, a protein involved in the transport of a potyvirus between cells or a protein showing similarity to the cleavage sites of viral proteins of a potyvirus. Process for obtaining plants resistant to a plant virus in which vectors obtained from a DNA bank complementary to a donor virus are introduced into cells or fragments of tissue of said plants and in that the plants are selected showing resistance to the plant virus.

Description

 The present invention relates to transgenic plants resistant to plant viruses and a process for obtaining plants.

 It also relates to fragments of DNA, RNA and proteins providing 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 the risk of damaging the organisms they parasitize. The means of control are thus essentially preventive: sanitary selection of the seeds, use of resistant varieties, purification of the plants infected by thermotherapy or culture of meristems.

 Some genotypes have resistance genes for some viruses, but these characters are extremely long to be introduced into other lineages by the classical pathways of genetics.

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

 Potyviruses form by their number and agronomic importance the largest group of plant viruses. The filamentous viral particle 600 to 900 nm long contains a single molecule of RNA of positive polarity of about 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 form.

They are naturally transmitted by the aphids in the non-persistent mode. 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, tomato, pepper and various varieties of potato. In the latter species the yields can be decreased from 10 to 80% depending on the strain of the virus, the cultivar and the time of infection. Early infection of tobacco can induce a yield loss of about 30%; the necrosis of the veins, caused by the strain N on some varieties of tobacco, may result in a total loss of the crop of this plant.

The potyvirus RNA, like that of other associated picorna-like plant viruses (nepoviruses and comoviruses) is translated into a polypeptide, which is then processed by proteolytic cleavage to yield the viral proteins. The review of
Riechmann et al. (J. Gen. Virol (1992), 73, 1-16) summarizes recent results.

 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.

 Potyvirus nucleotide sequence analysis confirmed the existence of a single long open reading phase between an initiator AUG codon and a translation stop codon; the viral RNA encodes a polyprotein, which is subsequently cleaved by enzymatic proteolysis. A partial genetic map of these viruses has been established and is shown for the PVY virus in Figure 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 picornavirus-encoded proteins suggest that the other nuclear inclusion (NIb) might be involved in replication (Domier et al., 1987 Virology 158, 20-27). ). The other known proteins are those of the helper component required for aphid transmission, cylindrical inclusion, 5 'bound VPg of the viral RNA, and finally the capsid. In addition, there are two proteins that surround the assistant factor.

One of a 31 kD mass, called P1, could be involved in cell-to-cell transport (Domier, (1987), Virology, 158, 20-27) while the other of a mass of 38 kD called P3 would be involved in replication (Gamble - Klein et al, Abstracts of the Third International Congress of Plant Molecular Biology, Tuckson, Arizona, 6-11).
October 1991).

 In 1985, Sanford and Johnston (J. Theor Biol 113, 395-405) issued the concept of pathogen-derived resistance, under which nucleotide sequences derived from a pathogen can be used to genetically modify the host to to become resistant to this pathogen.

According to this principle, resistance against plant viruses has already been obtained by transformation of the plants using DNA fragments carrying genes coding for capsid proteins.
A literature review has identified the results obtained on different plant viruses (Beachy et al.
Review of Phytopathol, l990, 28: 451-74)
This technique was more specifically developed in the case of potyviruses and resulted in the filing of the PCT application
FR-89 00 260 (published as WO-89 121 00) in which application the construction of a gene coding for the capsid protein of potyvirus under conditions such that its function is not impaired is described. .

 Transgenic plants for this gene and resistant to potyvirus can be obtained.

Another publication mentions obtaining plants resistant to tobacco mosaic virus by introducing a gene that is supposed to encode a non-structural protein that could be a component of the replication complex of this virus (Golemboski et al., National Proc.
Acad. Sci. USA, Vol. 87, pp.6311-6315, 1990). The authors show that plants transformed with a vector carrying the sequence of this gene exhibit resistance to the virus.

 Although the previously described techniques allow in some cases to obtain plants with resistance to plant viruses, they are nevertheless not applicable to all plant species and all plant viruses.

 Indeed, in the case of some potyviruses, the results could only lead to weak resistance.

 On the other hand, mutations could occur in viruses, rendering the resistance induced in plants transformed by capsid proteins ineffective.

 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.

 Accordingly, the Applicant has endeavored to develop a method for obtaining transgenic plants resistant to potyvirus transformed by non-capsidic gene sequences, not having the aforementioned drawbacks.

 According to the invention, it has surprisingly been found that the introduction into plants of a DNA fragment expressing transcripts corresponding to at least a part of a protein of a potyvirus other than a capsid protein, conferred on these plants an 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 cells or tissues of these plants.

The subject of the present invention is a plant resistant to potyvirus, characterized in that it carries in its genome one or more DNA fragments expressing transcripts corresponding to a protein or a part of a protein of a donor virus, with the exception of a capsid protein, said plant being capable of being obtained by introducing, using techniques known to those skilled in the art, such as electroporation, vectors derived from a library of DNA of the donor virus, and selection according to potyvirus resistance criteria
The term "donor virus" is intended to mean a virus whose genome will 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 it carries a DNA fragment expressing transcripts corresponding to a protein or part of a protein involved in the formation of the replicative complex of a potyvirus. . Such a DNA fragment may contain a part of the sequence coding for the P3 protein, in particular a part of the sequence between the nucleotides 2853 and 3492 of the complementary DNA (cDNA) of the PVY potyvirus.
Such a DNA fragment may also contain a 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 PVY potyvirus complementary DNA sequence.
A third type of DNA fragment may contain a portion of the coding sequence for the C-terminus of the NIa protease and the N-terminus of the polymerase
NIb, 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 the potyvirus Y as well as the amino acids composing the corresponding polyprotein have been described in the application
WO-89-12100 mentioned above. This document is therefore referred to with regard to the numbering of the nucleotides and amino acids of this polyprotein.

 Nevertheless, this application does not mention the particular sequences that are the subject of the present invention.

 According to a second embodiment, said plant may carry a DNA fragment expressing transcripts corresponding to a protein involved in the transport of a potyvirus from one cell to another. In this case, the DNA fragment can encode part of the P1 protein, and in particular be between the nucleotides 428 and 1044 of the sequence of the PVY potyvirus complementary DNA.

According to a third embodiment of the invention, said plant may also be defined by carrying a DNA fragment expressing transcripts corresponding to a protein or a portion of protein having a similarity with the cleavage sites. viral proteins of a potyvirus
Preferably, the potyvirus against which the plants are resistant is virus Y of the potato
Said plant may 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 portions of protein having similarities to the central portion of the NIa protease or that portion of the polyprotein corresponding to the C-terminus of the NIa protease and the N-terminus of the NIb polymerase.

This plant in the case of PVY belongs advantageously to the family Solanaceae including including 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 subject of the present invention is moreover RNAs that can be 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
Cells derived from a DNA library complementary to a donor virus are introduced into cells or tissue fragments of said plants.
plants are regenerated from said cells or said tissue fragments, and
plants which have a resistance against the plant virus are selected.

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

The vectors from the complementary DNA library may be introduced by electroporation or by any method known to those skilled in the art to introduce a
DNA in a plant cell.

Preferentially, 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, it will preferentially use a DNA library of a potyvirus to obtain plants resistant to a potyvirus.

The present invention is illustrated without being limited by the following examples
Figure 1 schematically shows the known structure of the polyprotein of potato virus Y (1A) and the peptides translated from this virus (1B).

Curved arrows indicate proteolytic cleavages.

 Figure 2 shows the general scheme for obtaining virus-resistant plants from PVY virus-complementary DNA.

FIG. 3 more precisely represents the process for cloning the viral complementary DNA fragments that resulted in the production of a complementary DNA library.
FIGS. 4 to 7 illustrate the resistances of the descendants of clones FCD3E1, FCD3B6, respectively,
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 the first and second generation plants homozygous for the introduced gene resulting from crosses from the T3A5 clone.

FIGS. 10 to 13 represent the nucleotide sequence of the DNA expressed in the clones T3A5, FCD3I1
FCD4A2 and FCD3B6 and their corresponding peptide sequences in the three possible reading frames noted 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 in the lines T3A5, FCD3I1, FCD4A2 and FCD3B6.

EXAMPLE 1
Construction of the Expression Vector of Complementary DNA Banaue
The method for constructing this vector is shown diagrammatically in FIGS.
In Figure 2 of the complementary DNA (cDNA) of the viral genome (PVY RNA) is synthesized by random primer 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 tobacco protoplast preparation (5) and then the clones are selected and regenerated (6) and subjected to PVY resistance tests (7).

 Figure 3 summarizes more precisely the manufacture of the bank.

An 800 base pair fragment resulting from Hind III digestion of plasmid pABD1 (Paskowski et al., 1984,
EMB0,3,2717-2722) was purified and filled with the polymerase of
Klenow. This fragment corresponds to the coding part of the gene conferring resistance to kanamycin.

It is then cloned at the BamHI site of plasmid pRt103 filled with Klenow polymerase (Topfer et al., 1987,
NAR, Vol.15, n014).

 The plasmid thus obtained, called pRt103NPTI1 (-), is composed of the 35S RNA promoter of cauliflower mosaic virus (CaMV) followed by a translation initiation codon which is not in phase with the coding region of the kanamycin resistance conferring gene derived from the Tn5 bacterial transposon, encoding the neomycin phosphotransferase II protein (NPTII). This plasmid further comprises the transcription terminator of cauliflower mosaic virus.

 DNA complementary to the viral RNA of a strain N of the virus virus Y obtained by random priming (Sambrook et al., 1989, Cold Spring Harbor Laboratory Press) was cloned into the plasmid pRt103NPTII ( -) to the SmaI 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 gene
NPTII. Such an event leads to the assembly of a transcriptional unit that 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 neomycin phosphotransferase II (NPTII). .

It should be noted that the NPTII protein is known to support N-terminal extensions that do not affect its activity.
EXAMPLE 2
Obtaining transsenis plants
The complementary DNA library obtained in Example 1 was introduced by electroporation (Guerche et al., 1987
Biochemistry, 69: 621) in protoplasts of Nicotiana tabacum (Xanthi variety, diploid clone D8).

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

 The progeny resulting from self-fertilization of the plants thus obtained was harvested and sown in vitro on a selective medium.

Segregation for kanamycin resistance is of the dominant monogenic Mendelian type in the offspring of the clones obtained
EXAMPLE 3
Resistance of transgenic plants to the virus.

 The descendants of transgenic plants were obtained by inbreeding from these primary transformants. The behavior of these plants with respect to a viral infection was then evaluated.

Twenty offspring plants were mechanically inoculated with a 1/2000 dilution of Yn virus-infected plant extract and twenty non-transgenic control plants.
Five clones whose offspring exhibited a PVYn virus resistance phenotype 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 clearly showed increased resistance to infection by potyvirus compared to a control plant.

Plant resistance was confirmed by a test
ELISA (SANOFI, Phyto Diagnostic) which allows to dose the capsidial protein of the virus thus representing the level of infection of the plants
EXAMPLE 4
Resistance of first generation and second veneration plants from clone T3A5.

A self-fertilization of the plant resulting from clone T3A5 was carried out
The resistance of the plants obtained, said first generation was tested. The results appear on the curve of Figure 8, as previously indicated
Among these plants, those that were homozygous for the kanamycin resistance marker associated with the viral cDNA fragment were searched for.

 The offspring of a selfing of a homozygous plant has been studied.

The results are shown in Figure 9
These results clearly show that resistance to virus Y is transmitted sexutively and is maintained at an equivalent level from one generation to the next.

EXAMPLE 5
Determination of the sequences expressed in transsniau plants.

Complementary DNAs corresponding to the transcripts of 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 synthesized oligonucleotides which made it possible to amplify the complementary DNAs have the following sequences:
** Primer 1 covers the sequence of the expression vector pRt103NPTI1 (-) from the 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 gene
NPTII of pABD1 ranging from nucleotide 1489 to nucleotide 1508 ::
5 'GGCCGGAGAACCTGCGTG 3'
A single 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 to determine which part of the viral genome is expressed in these plants
The sequence expressed by the T3A5 clone 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 encodes the C-terminal portion of the first protein (P1) of the viral polyprotein.

 The descendant plants of the T3A5 clone are therefore resistant to the virus Y and possess a fragment of the gene coding for the P1 protein integrated into their genome.

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

 The sequence expressed by clone FCD3I1 can encode a portion of the P3 protein involved in replication.

The sequence expressed by clone FCD4A2 can encode a portion of the NIa protein comprising the proteolytic cleavage site between the VPg portion and the protease portion of this protein (Dougherty and Parks (1991)
Virology 183,449-456).

The sequence expressed by clone FCDB6 can encode a protein comprising the C-terminal end of the protease
Nia and the N-terminus of the NIb polymerase and comprises the proteolytic cleavage site separating these two proteins.

 A diagram representing the different sequences expressed in clones T3A5, FCD3I1, FCD4A2 and FCD3B6 is given in FIG. 14.

Claims (19)

 1. plant resistant to potyvirus, characterized in that it carries in its genome one or more DNA fragments expressing transcripts corresponding to a protein or a part of a protein of a donor virus, except for a capsid protein, said plant being obtainable by introduction of vectors from a DNA library of the donor virus, and selection according to potyvirus resistance criteria.  2. Plant according to claim 1, characterized in that it carries a DNA fragment expressing transcripts corresponding to a protein or a portion 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 a part of the sequence coding for the P3 protein.  4. Plant according to claim 3, characterized in that the DNA fragment is between the nucleotides 2853 and 3492 of the complementary DNA of the potyvirus Y  5. Plant according to claim 2, characterized in that the DNA fragment contains a 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 sequence of the complementary DNA of potyvirus Y.  Plant according to claim 2, characterized in that the DNA fragment contains a part of the coding sequence for the C-terminus of the protease NIa and the N-terminus of the polymerase NIb.  8. Plant according to claim 7, characterized in that the DNA fragment is between nucleotides 6652 and 7080 of the complementary DNA sequence of 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 the cells.  10. Plant according to claim 9, characterized in that the DNA fragment encodes at least a portion of the P1 protein.  11. Plant according to claim 10, characterized in that the DNA fragment is between nucleotides 428 and 1044 of the complementary DNA sequence of potyvirus Y.  12. Plant according to claim 1, characterized in that it carries a DNA fragment expressing transcripts corresponding to a protein or a portion 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  Plant according to one of claims 1 to 13, characterized in that it belongs to the family of Solanaceae.  15. DNA or DNA fragment as defined in one of claims 1 to 12 and providing the plants resistance to a potyvirus  16. RNA capable of being transcribed from a DNA fragment according to claim 15.  17. Fragment of protein or protein as defined in one of claims 1 to 12 and providing the plants resistance to a potyvirus  18. Process for obtaining plants according to any one of claims 1 to 14 in which  Cells derived from a DNA library complementary to a donor virus are introduced into cells or tissue fragments of said plants.  plants are regenerated from said cells or said tissue fragments and  - plants with 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.