NZ502951A - Gene coding for androctonine and Protein 1A from tobacco (Nicotiana tabaccum) - Google Patents

Abstract

A nucleic acid fragment encoding for androctonin, a chimeric gene comprising a coding sequence linked to heterologous 5' and 3' regulatory elements, where the coding sequence comprises at least one DNA fragment coding for an androctonin; a cloning or expression vector containing the chimeric gene, a transformed host organism, especially a plant cell, containing the chimeric gene and plants comprising transformed plant cells and seeds are also described.

Description

1 Gene coding for androctonine, vector containing it and disease-resistant transformed plants obtained The present invention relates to a DNA 5 sequence coding for androctonine, to a vector containing it for the transformation of a host organism and to the process for transforming the said organism.

The invention relates more particularly to the transformation of plant cells and plants and to the 10 androctonine produced by the transformed plants, giving them resistance to diseases, in particular diseases of fungal origin.

There is today an increasing need to make plants resistant to diseases, in particular fungal 15 diseases, in order to reduce, or even avoid altogether, the need for treatments with antifungal protection products, in order to protect the environment. One means of increasing this disease-resistance consists in transforming the plants so that they produce substances 2 0 capable of defending them against these diseases.

Various substances of natural origin are known, in particular peptides, which have bactericidal or fungicidal properties, especially against the fungi responsible for plant diseases. However, the problem 25 consists in finding such substances which not only can be produced by transformed plants, but also can conserve their bactericidal or fungicidal properties and confer these properties to the said plants. For the INTEL' ECTUAL P°0?IRtY OrFICE OF N Z 2 a OCT 2001 RECEIVED I — -> purposes of the present invention, the terms bactericidal and fungicidal are understood to refer both to the actual bactericidal or fungicidal properties and to the bacteriostat or fungistat 5 properties.

Androctonine is a peptide produced by-scorpions from the species Androctonus australis. Its preparation by chemical synthesis are described by Ehret-Sabatier et al., along with its in vitro 10 antifungal and antibacterial properties.

Having first identified the androctonine gene, it has also been found that it can be inserted into a host organism, in particular a plant, in order to express androctonine and to give the said host 15 organism properties of resistance to fungal diseases and to diseases of bacterial origin, thereby providing a particularly advantageous solution to the problem outlined above.

The subject of the invention is thus, 20 firstly, an isolated nucleic acid fragment coding for androctonine, a chimeric gene comprising the said fragment coding for androctonine and heterologous regulation elements in the 5' and 3' positions, which can function in a host organism, in particular in 2 5 plants, and a vector for transforming host organisms containing this chimeric gene, and the host organism transformed. The invention also relates to a transformed plant cell containing at least one nucleic acid fragment coding for androctonine, and to a disease-resistant plant containing the said cell, in particular a plant regenerated from this cell. Lastly, the invention relates to a process for transforming 5 plants in order to make them disease-resistant, in which a gene coding for androctonine is inserted using a suitable vector.

According to the invention, the term androctonine is understood to refer to any peptide 10 essentially comprising the peptide sequence of 25 amino acids described by Ehret-Sabatier et al., as well as the equivalent homologous sequences in which certain amino acids are replaced with different but equivalent amino acids on sites which do not induce any 15 substantial change in the antifungal or antibacterial activity of the said homologous sequence. The expression peptide sequence essentially comprising the peptide sequence described by Ehret-Sabatier et al., is understood to refer not only to the mature androctonine 2 0 described in this application and defined by the sequence identifier No. 1 (SEQ ID NO. 1), but also such a sequence comprising, at one or other of its ends, or at both of them, peptide residues required for its expression and targeting in a host organism, in 25 particular a plant cell or a plant.

The present invention thus relates, firstly, to an isolated nucleic acid fragment, in particular a DNA fragment, coding for androctonine. According to the 4 invention, this can be a fragment isolated from Androctonus australis, or alternatively a derived fragment, adapted for the expression of androctonine in the host organism in which the peptide will be 5 expressed. The nucleic acid fragment can be obtained according to the standard methods for isolation and purification, or alternatively by synthesis according to the usual techniques of successive hybridizations of synthetic oligonucleotides. These techniques are 10 described in particular by Ausubel et al.

According to the present invention, the expression "nucleic acid fragment" is understood to refer to a nucleotide sequence which can be of DNA or RNA type, preferably of DNA type, in particular cDNA, 15 especially of double-stranded type.

According to one embodiment of the invention, the nucleic acid fragment coding for androctonine is the DNA sequence described by the sequence identifier No. 1 (SEQ ID NO. 1), a homologous sequence or a 2 0 sequence complementary to the said sequence, more particularly the coding portion of this SEQ ID NO. 1, corresponding to bases 1 to 75.

According to the invention, the term "homologous" is understood to refer to a nucleic acid 25 fragment having one or more sequence modifications when compared with the nucleotide sequence described by the sequence identifier No. 1 coding for androctonine.

These modifications can be obtained according to the usual mutation techniques, or alternatively by selecting the synthetic oligonucleotides used in the preparation of the said sequence by hybridization. With regard to multiple combinations of nucleic acids which 5 can lead to the expression of the same amino acid, the differences between the reference sequence described by the sequence identifier No. 1 and the homologous sequence can be considerable, and all the more so when it concerns a DNA fragment less than 100 nucleic acids 10 in size, which can be produced by synthesis.

Advantageously, the degree of homology will be at least 70% relative to the reference sequence, preferably at least 80% and more preferably at least S0%. These modifications are generally neutral, i.e. they do not 15 affect the primary sequence of the resulting androctonine.

The present invention also relates to a chimeric gene (or expression cassette) comprising a coding sequence and heterologous regulation elements in 20 positions 5' and 3' which can function in a host organism, in particular plant cells or plants, the said coding sequence comprising at least one DNA fragment coding for androctonine as defined above.

The term host organism is understood to refer 25 to any lower-order or higher-order monocellular or multicellular organism into which the chimeric gene according to the invention can be introduced, for the production of androctonine. Such organisms are, in 6 particular, bacteria, for example E. coli, yeasts, in particular yeasts of the genera Saccharomyces or Kluyveromyces, or, preferably, plant cells and plants.

According to the invention, the term "plant 5 cell" is understood to refer to any plant-derived cell which can constitute undifferentiated tissues such as calli, differentiated tissues such as embryos, plant portions, plants or seeds.

According to the invention, the term "plant" 10 is understood to refer to any differentiated multicellular organism capable of photosynthesis, in particular monocotyledons or dicotyledons, more particularly crop plants which may or may not be intended for human or animal consumption, such as corn, 15 wheat, rapeseed, soybean, rice, sugar cane, beetroot, tobacco, cotton, etc.

The regulation elements required for the expression of the DNA fragment coding for androctonine are well known to those skilled in the art as a 20 function of the host organism. They comprise in particular promoter sequences, transcription activators, transit peptides and termination sequences, including start and stop codons. The means and methods for identifying and selecting the regulation elements 25 are well known to those skilled in the art.

The nucleic acid fragment according to the invention can also comprise a nucleic acid sequence fused at the 5' and/or at the 3' end to the sequence 7 coding for androctonine, so as to obtain a "protein-androctonine" fusion protein, whose cleavage by the enzymatic systems of the host organism allows the release of androctonine. This protein fused to 5 androctonine can be a signal peptide or a transit peptide which allows the production of androctonine to be controlled and oriented specifically in one part of the host organism, such as, for example, the cytoplasm or the cell membrane, or, in the case of plants, in 10 tissues of a particular type or in the extracellular matrix.

According to one embodiment, the transit peptide can be a chloroplast-addressing signal or a mitochondrion-addressing signal, which is then cleaved 15 off in the chloroplasts or the mitochondria.

According to another embodiment of the invention, the signal peptide can be an N-terminal signal or "prepeptide", optionally combined with a signal responsible for retaining the protein in the 2 0 endoplasmic reticulum, or a vacuole-addressing peptide or "propeptide". The endoplasmic reticulum is the site at which maturation operations on the protein produced, such as, for example, cleavage of the signal peptide, are undertaken by the "cell machinery". 25 The invention relates more particularly to the transformation of plants. As promoter regulation sequence in plants, it is possible to use any promoter sequence of a gene which is naturally expressed in 8 plants, in particular a promoter of bacterial, viral or plant origin such as, for example, that of a gene for the small subunit of ribulose biscarboxylase (RuBisCO) or of a plant virus gene such as, for example, that of 5 cauliflower mosaic virus (CAMV 19S or 35S), or a promoter which can be induced by pathogens such as tobacco PR-la or asparagus AoPRT-L, it being possible to use any suitable known promoter. Preferably, use is made of a promoter regulation sequence which favours 10 the overexpression of the coding sequence in a constitutive manner or induced by the attack of a pathogen, such as, for example, that comprising at least one histone promoter as described in patent application EP 0,507,698.

According to the invention, it is also possible to use, in combination with the promoter regulation sequence, other regulation sequences which are located between the promoter and the coding sequence, such as transcription activators 2 0 ("enhancers"), such as, for example, the tobacco etch virus (TEV) translation activator described in patent application WO 87/07 644, or transit peptides, which are either single or double, and, in this case, optionally separated by an intermediate sequence, i.e. one 25 comprising, in the direction of transcription, a sequence coding for a transit peptide of a plant gene coding for a plastid localization enzyme, a portion of sequence of the mature N-terminal portion of a plant 9 gene coding for a plastid localization enzyme, and then a sequence coding for a second transit peptide for a plant gene coding for a plastid localization enzyme consisting of a portion of sequence of the mature 5 N-terminal portion of a plant gene coding for a plastid localization enzyme, as described in patent application EP 0,508,909. As transit peptide, mention may be made of the signal peptide for the tobacco PR-la gene described by Cornelissen et al., represented with its 10 coding sequence by the sequence identifier No. 2.

The sequence coding for the PR-la-androctonine signal peptide fusion protein and this fusion protein also form part of the present invention. This sequence is described in particular by 15 the sequence identifier No. 3, more particularly the coding part of this sequence, corresponding to bases 12 to 176.

As polyadenylation or terminator regulation sequence, it is possible to use any corresponding 20 sequence of bacterial origin, such as, for example, the nos terminator of Agrobacterium tumefaciens, or alternatively of plant origin, such as, for example, a histone terminator as described in patent application EP 0,633,317.

According to the present invention, the chimeric gene can also comprise a selection marker adapted to the transformed host organism. Such selection markers are well known to those skilled in the art. Such a marker may be an antibiotic-resistance gene, such as penicillin, or alternatively a herbicide-tolerance gene for plants.

The present invention also relates to a 5 cloning or expression vector for the transformation of a host organism containing at least one chimeric gene as defined above. Besides the above chimeric gene, this vector comprises at least one origin of replication. This vector can consist of a plasmid, a cosmid, a 10 bacteriophage or a virus, which are transformed by introducing the chimeric gene according to the invention. Depending on the host organism to be transformed, such transformation vectors are well known to those skilled in the art and are widely described in 15 the literature.

For the transformation of plant cells or plants, such a vector is, in particular, a virus which can be used for the transformation of the plants developed and also containing its own replication and 20 expression elements. Preferably, the vector for transforming the plant cells or plants according to the invention is a plasmid.

The subject of the invention is also a process for transforming host organisms, in particular 25 plant cells, by incorporating at least one nucleic acid fragment or one chimeric gene as defined above, it being possible for this transformation to be obtained by any suitable known means, which is amply described 11 in the specialized literature, and in particular the references cited in the present application, more particularly by means of the vector according to the invention.

One series of methods consists in bombarding cells or protoplasts with particles to which the DNA sequences are attached. Another series of methods consists in using, as a means of transfer into the plant, a chimeric gene inserted into a Ti plasmid of 10 Agrobacterium tumefaciens or an Ri plasmid of Agrobacterium rhizogenes.

Other methods can be used, such as microinjection or electroporation, or alternatively direct precipitation using PEG.

A person skilled in the art will select the appropriate method as a function of the nature of the host organism, in particular the plant cell or plant.

The subject of the present invention is also transformed host organisms, in particular plant cells 20 or plants, containing an effective amount of a chimeric gene comprising a sequence coding for the androctonine defined above.

The subject of the present invention is also plants containing transformed cells, in particular 25 plants regenerated from the transformed cells. The regeneration is obtained by any suitable process which depends on the nature of the species, as described, for example, in the above references. 12 The plants thus transformed are resistant to certain diseases, in particular to certain fungal or bacterial diseases. Consequently, the DNA sequence coding for androctonine can be inserted with the main 5 aim of producing plants that are resistant to the said diseases, since androctonine is effective against fungal diseases such as those caused by Botrytis, in particular Botrytis cinerea, Cercospora, in particular Cercospora beticola, Cladosporium, in particular 10 Cladosporium herbarum, Fusarium, in particular Fusarium culmorum or Fusarium graminearum, or by Phytophthora, in particular Phytophthora cinnamomi.

In this case, the chimeric gene may also advantageously comprise at least one selection marker, 15 such as one or more herbicide-tolerance genes.

The DNA sequence coding for androctonine can also be inserted as a selection marker during the transformation of plants with other sequences coding for other peptides or proteins of interest, such as, 2 0 for example, herbicide-tolerance genes.

Such herbicide-tolerance genes are well known to those skilled in the art and are described in particular in patent applications EP 115,673, WO 87/04181, EP 337,899, WO 96/38567 or WO 97/04103. 25 Needless to say, the transformed cells and plants according to the invention can also comprise the sequence coding for androctonine, other heterologous sequences coding for other complementary peptides 13 capable of giving the plant resistance to other diseases of bacterial or fungal origin.

The other sequences can be inserted using the same vector comprising a chimeric gene, which comprises 5 a first sequence coding for androctonine, and at least one other sequence coding for another peptide or protein of interest.

They can also be inserted using another vector comprising at least the said other sequence, 10 according to the usual techniques defined above.

The plants according to the invention can also be obtained by crossing parents, one carrying the gene according to the invention coding for androctonine, the other carrying a gene coding for at 15 least one other peptide or protein of interest.

Among the sequences coding for other antifungal peptides, mention may be made of the one coding for drosomycin, described in patent application Fr 2,725,992 and by Fehlbaum et al., (1994), and in the 20 unpublished patent application FR 97/09115 filed on 24 July 1997.

The examples below make it possible to illustrate the invention, the preparation of the sequence coding for androctonine, the chimeric gene, 25 the integration vector and the transformed plants. The attached Figures 1 to 5 describe schematic structures of certain plasmids prepared for the construction of the chimeric genes. In these figures, the various 14 restriction sites are marked in italics.

Example 1; Construction of the chimeric genes All the techniques used below are standard 5 laboratory techniques. The detailed procedures for these techniques are described in particular in Ausubel et al. pRPA-MD-P: Creation of a plasmid containing the signal 10 peptide for the tobacco PR-la gene.

The two complementary synthetic oligonucleotides Oligo 1 and Oligo 2 below are nybridized at 65°C for 5 minutes and then by slowly decreasing the temperature to 30°C over 30 min.

Oligo 1: 5' GCGTCGACGC GATGGGTTTC GTGCTTTTCT CTCAGCTTCC ATCTTTCCTT CTTGTGTCTA CTCTTCTTCT TTTCC 3' Oligo 2: 5' TCGCCGGCAC GGCAAGAGTA AGAGATCACA AGGAAAAGAA GAAGAGTAGA CACAAGAAGG AAAGATGGAA GC 3' After hybridization between Oligo 1 and Oligo 2, the remaining single-stranded DNA serves as a matrix for the klenow fragment of E. coli polymerase 1 (under the standard conditions recommended by the 25 manufacturer (New England Biolabs)) for the creation of the double-stranded oligonucleotide starting from the 3' end of each oligo. The double-stranded oligonucleotide obtained is then digested with the restriction enzymes SacII and Nael and cloned in the plasmid pBS II SK(-) (Stratagene) digested with the same restriction enzymes. A clone comprising the region 5 coding for the signal peptide of the tobacco PR-la gene (SEQ ID NO. 2) is thus obtained. pRPA-PS-PRla-andro: Creation of a sequence coding for androctonine fused to the PR-la signal peptide without 10 an untranscribed 3' region.

The two complementary synthetic oligonucleotide sequences Oligo 3 and Oligo 4 [lacuna] according to the operating conditions described for pRPA-MD-P.

Oligo 3: 5' AGGTCCGTGT GCAGGCAGAT CAAGATCTGC AGGAGGAGGG GTGG 3' Oligo 4: 5' CCGGATCCGT CGACACGTTC GCCTCGCCGA GCTCAGTATG GCCTGTTAGT GCACTTGTAG TAGCAACCAC CCCTCCTCCT 2 0 GCAGATCTTG ATCTGCC 3' After hybridization between Oligo 3 and Oligo 4, the remaining single-stranded DNA serves as a matrix for the klenow fragment of E. coli polymerase 1 2 5 (under the standard conditions recommended by the manufacturer (New England Biolabs)) for the creation of the double-stranded oligonucleotide starting from the 3' end of each oligo. This double-stranded 16 oligonucleotide containing the portion coding for androctonine (SEQ ID NO. 1) is then cloned directly in the plasmid pRPA-MD-P, which was digested with the restriction enzyme Nael. The correct orientation of the 5 clone obtained is verified by sequencing. A clone comprising the region coding for the PR-la-androctonine fusion protein, located between the Ncol restriction site at the N-terminal end and the Seal, SacII and BamHI restriction sites at the C-terminal end (SEQ ID 10 NO. 3), is thus obtained. pRPA-RD-238; Creation of an expression vector in plants comprising the sequence coding for the PR-la androctonine fusion protein.

The plasmid pRTL-2 GUS, derived from the plasmid pUC-19, was obtained from Dr. Jim Carrington (Texas A&M University, not described). This plasmid, whose schematic structure is represented in Figure 1, contains the duplicated CaMV 35S promoter isolated from 2 0 cauliflower mosaic virus (CaMV 2x35S promoter; Odell et al. , 1985) which directs the expression of an RNA containing a 5' untranslated sequence of tobacco etch virus (TEV 5' UTR; Carrington and Freed, 1990), the E. coli a-glucuronidase gene (GUS; Jefferson et al. , 1987) 25 followed by the CaMV RNA 35S polyadenylation site (CaMV polyA; Odell et al., 1985).

The plasmid pRTL-2 GUS is digested with the restriction enzymes Ncol and BamHI and the main DNA 17 fragment is purified. The plasmid pRPA-PS-PRla-andro is digested with the restriction enzymes Ncol and BamHI and the small DNA fragment containing the region coding for the PR-la-androctonine fusion protein is purified.

The two purified DNA fragments are then linked together in an expression cassette in the plants which synthesizes a PR-la-androctonine fusion protein. The schematic structure of this expression cassette is represented in Figure 2. "PR-la-androctonine" 10 represents the region coding for the PR-la-androctonine fusion protein of pRPA-RD-230. The androctonine is transported to the plant's extracelluar matrix by the action of the PR-la peptide signal. pRPA-RD-195: Creation of a plasmid containing a modified multiple cloning site.

The plasmid pRPA-RD-195 is a plasmid derived from pUC-19 which contains a modified multiple cloning site. The complementary synthetic oligonucleotides 2 0 Oligo 5 and Oligo 6 below are hybridized and made double-stranded according to the procedure described for pRPA-MD-P.

Oligo 5: 5' AGGGCCCCCT AGGGTTTAAA CGGCCAGTCA GGCCGAATTC 2 5 GAGCTCGGTA CCCGGGGATC CTCTAGAGTC GACCTGCAGG CATGC 3' Oligo 6: 5' CCCTGAACCA GGCTCGAGGG CGCGCCTTAA TTAAAAGCTT 18 GCATGCCTGC AGGTCGACTC TAGAGG 31 The double-stranded oligonucleotide obtained is then inserted into pUC-19, which was predigested 5 with the restriction enzymes EcoRI and HindiII and made blunt at the ends using the klenow fragment of E. coli DNA polymerase 1. A vector containing multiple cloning sites to facilitate the introduction of the expression cassettes into an Agrobacterium tumefaciens vector 10 plasmid is obtained. The schematic structure of this multiple cloning site is represented in Figure 3. pRPA-RD-233: Introduction of the PR-la-androctonine expression cassette from pRPA-RD-230 into pRPA-RD-195.

The plasmid pRPA-RD-23 0 is digested with the restriction enzyme Hindlll. The DNA fragment containing the PR-la-androctonine expression cassette is purified. The purified fragment is then inserted into pRPA-RP-195, which was predigested with the restriction enzyme 20 HinduI and dephosphorylated with calf intestinal phosphatase. pRPA-RD-174: Plasmid derived from pRPA-BL-150A (EP 0,508,909) containing the bromoxynil-tolerance gene 25 from pRPA-BL-237 (EP 0,508,909).

The bromoxynil-tolerance gene is isolated from pRPA-BL-237 by means of a PCR gene amplification. The fragment obtained has blunt ends, and is cloned in 19 the pRPA-BL-150A EcoRI site, the ends of which were made blunt by the action of klenow polymerase under standard conditions. An Agrobacterium tumefaciens vector which contains the bromoxynil-tolerance gene 5 close to its right-hand end, a kanamycin-tolerance gene close to its left-hand end and a multiple cloning site between these two genes is obtained.

The schematic structure of pRPA-RD-174 is represented in Figure 4. In this figure, "nos" 10 represents the polyadenylation site of Agrobacterium tumefaciens nopaline synthase (Bevan et al., 1983), "NOS pro" represents the Agrobacterium tumefaciens nopaline synthase promoter (Bevan et al., 1983), "NPT II" represents the neomycin phosphotransferase gene of 15 the Tn5 transposon of E. coli (Rothstein et al., 1981), "3 5S pro" represents the 35S promoter isolated from cauliflower mosaic virus (Odell et al., 1985), "BRX" represents the nitrilase gene isolated from K. ozaenae (Stalker et al., 1988), "RB" and "LB" represent, 20 respectively, the right-hand and left-hand ends of the sequence of an Agrobacterium tumefaciens Ti plasmid. pRPA-RD-184: Addition of a new, unique restriction site into pRPA-RD-174.

The complementary synthetic oligonucleotides Oligo 7 and Oligo 8 below are hybridized and made double-stranded according to the procedure described for pRPA-MD-P.

' CCGGCCAGTC AGGCCACACT TAATTAAGTT TAAACGCGGC CCCGGCGCGC CTAGGTGTGT GCTCGAGGGC CCAACCTCAG TACCTGGTTC AGG 31 5' CCGGCCTGAA CCAGGTACTG AGGTTGGGCC CTCGAGCACA CACCTAGGCG CGCCGGGGCC GCGTTTAAAC TTAATTAAGT GTGGCCTGAC TGG 3 ' The hybridized double-stranded 10 oligonucleotide (96 base pairs) is purified after separation on agarose gel (3% Nusieve, FMC). The plasmid pRPA-RD-174 is digested with the restriction enzyme Xmal and the main DNA fragment is purified. The two DNA fragments obtained are then linked together. 15 A plasmid derived from pRPA-RD-174 is obtained, comprising other restriction sites between the bromoxynil-tolerance gene and the selection marker kanamycin gene.

The schematic structure of the plasmid pRPA-20 RD-184 is represented in Figure 5, in which the terms "nos", "NPT II", "NOS pro", "35S pro", "BRX gene", "RB" and "LB" have the same meanings as in Figure 4. pRPA-RD-236: Creation of an Agrobacterium tumefaciens 2 5 vector containing the gene construct coding for androctonine directed towards the extracellular matrix.

The plasmid pRPA-RD-233 is digested with the restriction enzymes Pmel and AscI and the DNA fragment Oligo 7: Oligo 8: 21 containing the PR-la-androctonine gene is purified. The plasmid pRPA-RD-184 is digested with the same restriction enzymes. The DNA fragment containing the PR-la-androctonine expression cassette is then inserted 5 into pRPA-RD-184. An Agrobacterium tumefaciens vector containing the sequence coding for the PR-la-androctonine fusion protein is thus obtained, which leads to the expression of androctonine in the plant's extracellular matrix.

Example 2; Tolerance to herbicides of transformed tobacco plants. 2.1- Transformation The vector pRPA-RD-236 is introduced into the 15 Agrobacterium tumefaciens strain EHA101 (Hood et al., 1987) carrying the cosmid pTVK291 (Komari et al., 1986). The transformation technique is based on the procedure by Horsh et al. (1985). 2.2- Regenerat ion 2 0 Regeneration of the tobacco plant PBD6 (obtained from SEITA France) from foliar explants is carried out on Murashige-Skoog (MS) base medium comprising 30 g/1 of sucrose and 200 ig/ml of kanamycin. The foliar explants are taken from plants 2 5 cultivated in a greenhouse or in vitro and regenerated according to the foliar disc technique (Horsh et al., 1985) in three successive steps: the first step comprises induction of the shoots on a medium 22 supplemented with 30 g/1 of sucrose containing 0.05 mg/1 of naphthylacetic acid (NAA) and 2 mg/1 of benzylaminopurine (BAP) for 2 weeks. The shoots formed during this step are then grown for 10 days by 5 cultivating on MS medium supplemented with 3 0 g/1 of sucrose but containing no hormone. Next, the shoots which have grown are taken and cultivated on an MS rooting medium with half the content of salts, vitamins and sugar and containing no hormone. After about 2 10 weeks, the rooted shoots are placed in earth. 2.3- Tolerance to bromoxynil Twenty transformed plants were regenerated and placed in a greenhouse for the construction of pRPA-RD-236. These plants were treated in the 15 greenhouse, at the 5-leaf stage, with aqueous Pardner suspension corresponding to 0.2 kg of bromoxynil active material per hectare.

All the plants showing complete tolerance to bromoxynil are then used in various experiments which 20 show that the expression of androctonine by the transformed plants makes them resistant to fungal attack. 23 REFERENCES Ausubel, F.A. et al., (eds. Greene). Current Protocols in Molecular Biology. Publ. Wiley & Sons.

Bevan, M. et al., (1983). Nuc. Acids Res. 11:369-385. Carrington and Freed (1990). J. Virol. 64:1590-1597. Ehret-Sabatier et al., (1996). The Journal of Biological Chemistry, 271, 47, 29537-29544.

Horsch et al., (1985). Science 227:1229-1231. 10 Jefferson et al., (1987). EMBO J. 6:3901-3907.

Komari et al., (1986). J. Bacteriol. 166:88-94. Rothstein et al., (1981). Cold Spring Harb. Symp. Quant. Biol. 45:99-105.

Stalker et al., (1988). J. Biol. Chem. 263:6310-6314. 15 Odell, J.T. et al., (1985). Nature 313:810-812. 24 SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES: 14 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 110 base pairs 10 (B) TYPE: nucleotide (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..75 2 0 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: AGG TCC GTG TGC AGG CAG ATC AAG ATC TGC AGG AGG AGG GGT GGT TGC 4 8 Arg Ser Val Cys Arg Gin lie Lys lie Cys Arg Arg Arg Gly Gly Cys 15 10 15 2 5 TAC TAC AAG TGC ACT AAC AGG CCA TAC TGAGCTCGGC GAGGCGAACG 95 Tyr Tyr Lys Cys Thr Asn Arg Pro Tyr 20 25 TGTCGACGGA TCCGG 110 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 106 base pairs 5 (B) TYPE: nucleotide (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 12.. 101 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: GCGTCGACGC C ATG GGT TTC GTG CTT TTC TCT CAG CTT CCA TCT TTC CTT 50 Met Gly Phe Val Leu Phe Ser Gin Leu Pro Ser Phe Leu 15 10 CTT GTG TCT ACT CTT CTT CTT TTC CTT GTG ATC TCT CAC TCT TGC CGT 98 Leu Val Ser Thr Leu Leu Leu Phe Leu Val lie Ser His Ser Cys Arg 20 25 GCC GGCGA 106 2 5 Ala 30 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: 26 (A) LENGTH: 211 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: (A) NAME/KEY: CDS 10 (B) LOCATION: 12..176 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: GCGTCGACGC C ATG GGT TTC GTG CTT TTC TCT CAG CTT CCA TCT TTC CTT 50 15 Met Gly Phe Val Leu Phe Ser Gin Leu Pro Ser Phe Leu 10 CTT GTG TCT ACT CTT CTT CTT TTC CTT GTG ATC TCT CAC TCT TGC CGT 98 Leu Val Ser Thr Leu Leu Leu Phe Leu Val lie Ser His Ser Cys Arg 20 15 20 25 GCC AGG TCC GTG TGC AGG CAG ATC AAG ATC TGC AGG AGG AGG GGT GGT 14 6 Ala Arg Ser Val Cys Arg Gin lie Lys lie Cys Arg Arg Arg Gly Gly 30 35 40 45 TGC TAC TAC AAG TGC ACT AAC AGG CCA TAC TGAGCTCGGC GAGGCGAACG 196 Cys Tyr Tyr Lys Cys Thr Asn Arg Pro Tyr 50 55 TGTCGACGGA TCCGG 211 (2) INFORMATION FOR SEQ ID NO: 4: INTELLECTUAL PROP1RTY OFFICE OF N - 2 3 OCT 2001 RECEIVED 27 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 75 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "synthetic oligonucleotide 1" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: x- GCGTCGACGC GATGGGTTTC GTGCTTTTCT CTCAGCTTCC ATCTTTCCTT CTTGTGTCTA 60 CTCTTCTTCT TTTCC 75 (2) INFORMATION FOR SEQ ID NO: 5: (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 72 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "synthetic oligonucleotide 2" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: TCGCCGGCAC GGCAAGAGTA AGAGATCACA AGGAAAAGAA GAAGAGTAGA CACAAGAAGG 60 28 AAAGATGGAA GC 72 (2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 44 base pairs 5 (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid 10 (A) DESCRIPTION: /desc = "synthetic oligonucleotide 3" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: AGGTCCGTGT GCAGGCAGAT CAAGATCTGC AGGAGGAGGG GTGG 4 4 (2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 97 base pairs 20 (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (n) MOLECULE TYPE: other nucleic acid 25 (A) DESCRIPTION: /desc = "synthetic oligonucleotide 4" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: 29 CCGGATCCGT CGACACGTTC GCCTCGCCGA GCTCAGTATG GCCTGTTAGT GCACTTGTAG 60 TAGCAACCAC CCCTCCTCCT GCAGATCTTG ATCTGCC 97 (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 85 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "synthetic oligonucleotide 5" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: AGGGCCCCCT AGGGTTTAAA CGGCCAGTCA GGCCGAATTC GAGCTCGGTA CCCGGGGATC 60 C7CTAGAGTC GACCTGCAGG CATGC 85 (2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 66 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: single 25 (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "synthetic oligonucleotide 6" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: CCCTGAACCA GGCTCGAGGG CGCGCCCTTAA TTAAAAGCTT GCATGCCTGC AGGTCGACTC 60 TAGAGG 66 (2) INFORMATION FOR SEQ ID NO: 10: (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 93 base pairs 10 (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (n) MOLECULE TYPE: other nucleic acid 15 (A) DESCRIPTION: /desc = "synthetic oligonucleotide 7" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: 2 0 CCGGCCAGTC AGGCCACACT TAATTAAGTT TAAACGCGGC CCCGGCGCGC CTAGGTGTGT 60 GCTCGAGGGC CCAACCTCAG TACCTGGTTC AGG 93 (2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS: 25 (A) LENGTH: 93 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear 31 (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "synthetic oligonucleotide 8" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: CCGGCCTGAA CCAGGTACTG AGGTTGGGCC CTCGAGCACA CACCTAGGCG CGCCGGGGCC 60 GCGTTTAAAC TTAATTAAGT GTGGCCTGAC TGG 93 INTEL' ECTUAL PROPERTY OFFICE OF N.Z. 2 3 OCT 2001 RECEIVED

Claims (28)

1. An isolated nucleic acid fragment, characterized in that is comprises a nucleic acid sequence coding for androctonine. 5

2. Nucleic acid fragment according to claim 1, characterized in that it is a nucleotide sequence of DNA type, in particular cDNA.

3. Nucleic acid fragment according to claim 2, characterized in that the nucleotide sequence 10 of DNA type is the DNA sequence described by the sequence identifier No. 1 (SEQ ID NO. 1), a homologous sequence or a sequence complementary to the said sequence. 15 claim 3, characterized in that the nucleotide sequence of DNA type is the coding portion of SEQ ID NO. 1, corresponding to bases 1 to 75. of claims 1 to 4, characterized m that it comprises a 20 nucleic acid sequence fused at the 5' and/or at the 3' end to the sequence coding for androctonine, so as to obtain a "protem-androctonine" fusion protein. claim 5, characterized in that the protein is a signal 25 peptide or a transit peptide. claim 6, characterized in that the signal peptide is the signal peptide for the tobacco PR-la gene.

4. Nucleic acid fragment according to

5. Nucleic acid fragment according to one

6. Nucleic acid fragment according to

7. Nucleic acid fragment according to 33

8. Nucleic acid fragment according to claim 7, characterized in that it comprises the DNA sequence described by the sequence identifier No. 3 (SEQ ID NO. 3), a homologous sequence or a sequence 5 complementary to the said sequence.

9. Nucleic acid fragment according to claim 8, characterized in that it comprises the coding portion of SEQ ID NO. 3, corresponding to bases 12 to 176.

10 10. "Protem-androctonine" fusion protein, characterized in that the protein is a signal peptide or a transit peptide.

11. Fusion protein, according to claim 10, characterized in that the signal peptide is the signal 15 peptide for the tobacco PR-la gene.

12. Fusion protein, according to claim 11, characterized in that it is described by the sequence identifier No. 3 (SEQ ID NO. 3) .

13. Chimeric gene comprising a coding 20 sequence and heterologous regulation elements in the 5' and 3' positions, which can function in a host organism, in particular plants, characterized in that the coding sequence comprises at least one DNA fragment coding for androctonine as defined m claims 1 to 9. 25

14. Chimeric gene according to claim 13, characterized in that the host organism is chosen from plant cells and plants. 34

15. Chimeric gene according to either of claims 13 and 14, characterized in that it also comprises a selection marker.

16. Cloning or expression vector for 5 transforming a host organism, characterized in that it comprises at least one origin of replication and at least one chimeric gene as defined m claims 13 to 15.

17. Vector according to claim 16, characterized in that it is a virus used for the 10 transformation of the plants developed and also containing its own replication and expression elements.

18. Vector according to claim 16, characterized in uhat it is a plasmid.

19. Transformed host organisms, 15 characterized in that they contain an effective amount of a chimeric gene according to claims 13 to 15.

20. Transformed host organism according to claim 19, characterized m that they are plant cells or plants. 20

21. Transformed host organism according to claim 20, characterized in that it is a plant containing transformed cells.

22. Host organism according to claim 21, characterized m that the plant is regenerated from the 25 transformed cells.

23. Transformed plant cell, characterized in that it contains a nucleic acid fragment according to claims 1 to 9 or a chimeric gene according to claims 13 35 to 15 .

24. Disease-resistant transformed plant, characterized in that it comprises at least one transformed plant cell according to claim 23. 5

25. Transformed plant according to claim 24, characterized in that it is resistant to the diseases caused by Botrytis, m particular Botrytis cinerea, Cercospora, m particular Cercospora beticola, Cladosporium, m particular Cladosporium herbarum, 10 Fusarium, in particular Fusarium culmorum or Fusarium graminearum, or by Phytophthora, m particular Phytophthora cinnamomi.

26. Process for transforming host organisms, m particular plant cells or plants, characterized m 15 that at least one nucleic acid fragment according to claims 1 to 9 or a chimeric gene according to one of claims 13 to 15 is inserted into the said host organism.

27. Process for transforming plants in order 20 to make them resistant to fungal or bacterial diseases, characterized in that at least one nucleic acid fragment according to claims 1 to 9 or a chimeric gene according to claims 13 to 15 is inserted into the plant. 25

28. Process according to either of claims 26 and 27, characterized in that the chimeric gene is inserted using a vector according to one of claims 16 to 18 . END OF CLAIMS