WO2003044200A1 - Nucleotide sequence coding for an enzyme having acetolactase synthase activity, plant cell and plant containing same, and crop weeding method - Google Patents

Abstract

The invention concerns a nucleotide sequence coding for an enzyme having acetolactase synthase (ALS) activity, functional in the genus Cichorium, characterized in that said enzyme has reduced affinity for sulphonylurea or imidazolinone compounds. The invention also concerns an enzyme encoded by said sequence, a plant cell or a plant containing same. The invention further concerns a method for detecting a herbicide-tolerant plant, as well as a crop weeding method.

Description

(54) Title: NUCLEOTIDE SEQUENCE ENCODING AN ENZYME HAVING ACETOLACTATE SYNTHASE ACTIVITY OF CICHORIUM

NUCLEOTIDE SEQUENCE ENCODING AN ENZYME HAVING ACETOLACTATE CICHORIUM SYNTHASE ACTIVITY

The present invention relates to nucleotide sequences associated with herbicide tolerance in plants, in particular of the genus Cichorium.

Chicory (Cichorium intybus) is a plant whose early stages of development are slow. Indeed, it does not take less than two months before covering the ground. Culture then finds itself in direct competition with a large number of weeds, which during this period show very active development. In the absence of an effective and selective chicory herbicide, it is frequent to have to proceed to the elimination of the remaining weeds by mechanical hoeing supplemented by manual weeding which generate a very significant additional cost. 15 If the selectivity of a herbicide in relation to a plant results in its non-toxicity (at least for the dose used) towards the latter, specificity designates the categories of plants liable to be destroyed by this product, in a word, its spectrum of activity.

A herbicide is therefore all the more effective as it displays a limited selectivity (at best the cultivated plant), a broad specificity and a low persistence, not penalizing for subsequent crops.

The alternative is therefore either to develop a new herbicide having the specificity and selectivity characteristics required for a given plant, or to have a plant modified to be tolerant to a known herbicide.

The second solution has a certain advantage because it makes it possible to avoid carrying out all of the toxicity tests necessary for the placing on the market of these products.

Acetolactate synthase (ALS) is the first enzyme in the pathway

30 biosynthesis of the branched chain amino acids Val, Leu and Ile. Four classes of herbicides inhibit this enzyme, including imidazolinones, triazolopyrimidine sulfoanilides, pyrimidinylthiobenzoates and sulfonylureas. These herbicides are widely used because they have a high efficiency (a few grams per hectare is enough), a broad spectrum of activity and low toxicity towards mammals.

Chicory is not naturally resistant to ALS inhibitors. An objective of the present invention is to obtain mutants of Cichorium intybus resistant to chlorsulfuron (herbicide belonging to the class of sulfonylureas), in particular by in vitro selection of cell suspension in prolonged contact with the herbicide. It is generally accepted that if resistance is linked to the mutation of a "wild" gene, this occurs naturally with a probability of the order of 10 ^" to 10 ^" under in vitro culture conditions. The enzymatic analyzes indicate that the acquisition of tolerance is due to the alteration of the sensitivity of the ALS vis-à-vis these inhibitors. The 50% inhibition of ALS activity requires, in the homozygous tolerant, a dose of chlorsulfuron 570 times greater than that producing the same effect in the sensitive (20-25nM). The sensitivity of ALS from the tolerant line to Pimazamethabenz is 125 times lower (Northern Blot, Figure 1). Furthermore, no overproduction of this target was noted. This alteration of ALS is transmitted from generation to generation, like a Mendelian character encoded by a single nuclear gene. Analysis of the genome of homozygous tolerant chicories confirms this monogenic character. The tolerant allele "T", indifferently designated resistant in this text, is semi-dominant, the sensitive allele "s" is recessive; a homozygote s / s is therefore sensitive to these herbicides. The specific activity of ALS tolerant to sulfonylureas is reduced compared to that of sensitive plants.

The activity of ALS is known to be negatively regulated by the end products, valine and leucine, of the biosynthetic pathway that it initiates. The ALS extracted from tolerant chicories has a reduced sensitivity (10 times for leucine and 3 times for valine) to this feedback. Thus a nucleotide sequence according to the invention codes for a functional ALS in the genus

Cichorium and with a reduced affinity for the amino acids leucine and valine.

Previous work has shown that acetolactate synthase is an enzyme of 670 amino acids. Two mutation regions confer tolerance to sulfonylureas in different species, a first ALS region A between positions 121 and 209 and a second ALS region B between positions 560 and 665 (Wright et al., 1998, and Figure 6). Document FR 2 765 240 describes a mutation in ALS conferring tolerance in Cichorium between amino acids 377 and 670 Resistance results in Xanthium from the mutation in position 574 of a Trp in Leu (Banasconi et al., 1995, and figure 6). In addition, in view of the work already carried out by Dewaele on the ALS of

Cichorium (doctoral thesis, 1997), the need remains to know the nucleotide sequence coding for amino acids 1 to 376 of ALS, and the possible mutation or mutations which confer tolerance to herbicides. The inventors therefore sought to identify the complete sequence of natural functional ALS, and the complete sequence of mutated ALS with respect to natural ALS, this or these mutations conferring resistance to herbicides of the sulfonylurea and / or imidazolinone type. .

To this end, the subject of the invention is, according to a first aspect, an isolated nucleotide sequence corresponding to SEQ ID No. 1 and coding for an acetolactate synthase (ALS) functional in plants of the genus Cichorium. The amino acid sequence deduced from the nucleotide sequence SEQ ID No. 1 of the ALS of this species corresponds to SEQ ID No. 2.

The invention also relates to a nucleotide sequence chosen from: a) a nucleotide sequence comprising at least 70, in particular at least 80% identity with SEQ ID No. 1; b) a nucleotide sequence complementary to SEQ ID No. 1 or a sequence defined in a) or b) or the RNA sequence; c) a nucleotide sequence comprising a sequence as defined in a), or b); , said sequence defined in a), or b) coding for an ALS of sequence SEQ

ID No. 2 or a functionally equivalent polypeptide sequence.

By functionally equivalent ALS protein is meant that the biological properties with respect to herbicides, more particularly to sulfonylureas and imidazolinones, are identical or very close to natural non-mutated ALS, either due to an absence of mutation, either in the possible presence of a mutation but without consequence on the sensitivity to these herbicides. The inventors have succeeded in obtaining the sequence of the non mutated ALS between amino acids 103 and 670. The list of the annexed sequences therefore corresponds to sequences starting from amino acid 103. For example SEQ ID N ° 2 is numbered from 1 to 568, which corresponds to amino acids 103 to 670 respectively. The offset is therefore 102 amino acids, thus the mutation in position 197 of the mutated ALS mentioned below is found in position (197-102 = 95) on SEQ ID N ° 2 (i.e. Pro for l 'Non mutated ALS and Ser on the mutated ALS referenced SEQ ID N ° 4).

The term “nucleic acid, nucleic or nucleic acid sequence, polynucleotide, oligonucleotide, polynucleotide sequence, nucleotide sequence, terms which are used interchangeably in the present description, is intended to denote a precise sequence of nucleotides, modified or not, making it possible to define a fragment or region of a nucleic acid, which may or may not contain unnatural nucleotides, and which may correspond to both double-stranded DNA, single-stranded DNA and transcripts of said DNAs. Thus, the nucleic acid sequences according to the invention also include PNA (Peptid Nucleic Acid), or the like.

It should be understood that the present invention does not relate to nucleotide sequences in their natural chromosomal environment, that is to say in the natural state. These are sequences which have been isolated and / or purified, that is to say that they have been taken directly or indirectly, for example by copying, their environment having been at least partially modified. This also means the nucleic acids obtained by chemical synthesis.

By “percentage of identity” between two nucleic acid or amino acid sequences within the meaning of the present invention is meant a percentage of identical nucleotides or amino acid residues between the two sequences to be compared, obtained after the best alignment, this percentage being purely statistical and the differences between the two sequences being distributed randomly and over their entire length. The term “best alignment” or “optimal alignment” is intended to denote the alignment for which the percentage of identity determined as below is the highest. Sequence comparisons between two nucleic acid or amino acid sequences are traditionally carried out by comparing these sequences after having optimally aligned them, said comparison being performed by segment or by “comparison window” to identify and compare local regions of sequence similarity. The optimal alignment of the sequences for the comparison can be carried out, besides manually, by means of the algorithm of local homology of Smith and Waterman (1981, Ad. App. Math. 2: 482), by means of the algorithm of local homology by Neddleman and Wunsch (1970, J. Mol. Biol. 48: 443), using the similarity search method of Pearson and Lipman (1988, Proc. Natl. Acad. Sci. USA 85: 2444 ), using computer software using these algorithms (GAP, BESTFIT, BLAST P, BLAST N, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI). In order to obtain optimal alignment, the BLAST program is preferably used with the BLOSUM 62 matrix. The PAM or PAM250 matrices can also be used.

The percentage of identity between two nucleic acid or amino acid sequences is determined by comparing these two optimally aligned sequences in which the nucleic acid or amino acid sequence to be compared may include additions or deletions compared to the reference sequence for optimal alignment between these two sequences. The percentage of identity is calculated by determining the number of identical positions for which the nucleotide or the amino acid residue is identical between the two sequences, by dividing this number of identical positions by the total number of positions compared and by multiplying the result obtained by 100 to obtain the percentage of identity between these two sequences.

By nucleic acid sequences having a percentage identity of at least 80%, preferably 85% or 90%, more preferably 95% or even 98%, after optimal alignment with a reference sequence, is meant the nucleic acid sequences having , with respect to the reference nucleic acid sequence, certain modifications such as in particular a deletion, a truncation, an elongation, a chimeric fusion and / or a substitution, in particular punctual, and whose nucleic sequence has at least 80%, preferably 85 %, 90%, 95% or 98% of identity after optimal alignment with the reference nucleic sequence. They are preferably sequences whose complementary sequences are capable of hybridizing specifically with the reference sequences. Of preferably, the specific hybridization or high stringency conditions will be such that they ensure at least 80%, preferably 85%, 90%, 95% or 98% of identity after optimal alignment between one of the two sequences and the complementary sequence of the other. Hybridization under conditions of high stringency means that the conditions of temperature and ionic strength are chosen in such a way that they allow hybridization to be maintained between two complementary DNA fragments. By way of illustration, high stringency conditions of the hybridization step for the purpose of defining the polynucleotide fragments described above are advantageously as follows.

DNA-DNA or DNA-RNA hybridization is carried out in two stages: (1) prehybridization at 42 ° C for 3 hours in phosphate buffer (20 mM, pH 7.5) containing 5 x SSC (1 x SSC corresponds to a 0.15 M NaCl + 0.015 M sodium citrate solution), 50% formamide, 7% sodium dodecyl sulfate (SDS), 10 x Denhardt's, 5% dextran sulfate and 1% salmon sperm DNA; (2) actual hybridization for 20 hours at a temperature depending on the size of the probe (ie: 42 ° C, for a probe of size> 100 nucleotides) followed by 2 washes of 20 minutes at 20 ° C in 2 x SSC + 2% SDS, 1 wash for 20 minutes at 20 ° C in 0.1 x SSC + 0.1% SDS. The last washing is carried out in 0.1 × SSC + 0.1% SDS for 30 minutes at 60 ° C. for a probe of size> 100 nucleotides. The conditions of high stringency hybridization described above for a polynucleotide of defined size, can be adapted by the skilled person for oligonucleotides of larger or smaller size, according to the teaching of Sambrook et al., ( 1989, Molecular cloning: a laboratory manual. 2 ^nd Ed. Cold Spring Harbor).

Thanks to the techniques described later in the application which make it possible to measure the ALS activity, a person skilled in the art has the appropriate data to directly identify without excessive effort the nucleic acids corresponding to the homologies mentioned above. Furthermore, the inventors carrying out the cloning of 85% of the ALS gene from amino acid 103 identified a point mutation conferring resistance to the sulfonylureas of the line studied. Until now, the sequence of the ALS gene in chicory between amino acids 103 and 377 has not been known. Thus, according to a second aspect the invention relates to an isolated nucleotide sequence characterized in that it corresponds to SEQ ID No. 3 and codes for a mutated acetolactate synthase (ALS) having at least one mutation in the region of amino acids 103 to 377 of ALS, the said mutation or mutations conferring on the plant resistance to a herbicide. No mutation conferring said resistance in chicory had yet been identified and this very useful result was obtained by the inventors thanks to the work not obvious to the skilled person described below. The invention also relates to a nucleotide sequence isolated from a plant of the genus Cichorium chosen from: a) a nucleotide sequence corresponding to SEQ ID No. 3 b) a nucleotide sequence comprising at least 80% identity with SEQ ID No. 3 c) a nucleotide sequence complementary to SEQ ID No. 3 or of a sequence defined in b) or of the RNA sequence d) a nucleotide sequence comprising a sequence as defined in a), b) or c); and in that it codes for a mutated acetolactate synthase having at least one mutation in the amino acid region 103 to 377 of the ALS, the said mutation or mutations conferring on the plant resistance to a herbicide.

In the following description, the term mutated nucleotide sequence will denote a nucleotide sequence coding for a mutated ALS, that is to say conferring tolerance (indifferently designated resistance) to these herbicides. Such a mutated sequence is typically at least 80% homologous to the non-mutated sequence.

By sequencing the ALS of several sensitive and tolerant individuals, the inventors have in fact demonstrated that the same point mutation was found in the same position on the polypeptide chain, in position 197, giving the plant resistance or very strongly increasing resistance herbicides tested. According to a preferred embodiment, the mutation in position 197 is a Proline to Serine mutation. The herbicide is chosen from sulfonylureas, imidazolinones.

However, any functionally equivalent mutation (in particular due to the structural modification of the ALS due to the substitution of proline) also forms part of the invention. Those skilled in the art knowing such an amino acid modification at position 197 deduces the corresponding modification on the nucleotide sequence.

In addition, the invention also relates to a nucleotide sequence isolated from a plant of the genus Cichorium comprising at least 80% and typically at least 90.95.98.99% of identity with SEQ ID No. 3, said sequence coding for a mutated ALS exhibiting at least one mutation in the region of amino acids 103 to 670, the said mutation or mutations conferring on the plant resistance to herbicides, more particularly to sulfonylureas and / or imidazolinones. According to another aspect, the invention relates to a nucleotide sequence which is a representative fragment of a nucleotide sequence, of the natural ALS described above.

According to another aspect, the invention relates to a nucleotide sequence which is a representative fragment of a nucleotide sequence of the mutated ALS described above. By fragment representative of a mutated ALS nucleotide sequence is meant according to a first embodiment a nucleotide sequence usable as a primer for the identification and / or selection of plants of the genus Cichorium resistant to a sulfonylurea or imidazolinone herbicide, of a length of at least 10 bases, typically at least 20 bases, and capable of amplifying a mutated nucleotide sequence described above.

By fragment representative of a mutated ALS nucleotide sequence is meant according to a second embodiment a nucleotide sequence usable as a probe for the identification and / or selection of plants of the genus Cichorium tolerant to a sulfonylurea or imidazolinone herbicide, and capable of hybridize with a mutated nucleotide sequence described above.

A probe or primer is defined, within the meaning of the invention, as being a single-stranded nucleic acid fragment or a denatured double-stranded fragment, and having a specificity of hybridization under conditions determined to form a hybridization complex with a target nucleic acid.

According to one embodiment, the probes and primers according to the invention are marked directly or indirectly with a radioactive or non-radioactive compound, in order to obtain a detectable and / or quantifiable signal. The labeling of primers or probes according to the invention is typically carried out by radioactive elements or by non-radioactive molecules. Among the radioactive isotopes used, mention may be made of P, P, S, H or I. Non-radioactive entities are selected from ligands such as biotin, avidin, streptavidin, digoxygenin, haptens, dyes, luminescent agents such as radioluminescent, chemoluminescent, bioluminescent, fluorescent, phosphorescent agents.

The invention also relates to the nucleic acids capable of being obtained by amplification using primers according to the invention.

The previously mentioned polynucleotides of the invention can thus be used as a primer and / or probe in methods using in particular the PCR technique (polymerase chain reaction) (Rolfs et al., 1991, Berlin: Springer-Nerlag) . This technique requires the choice of pairs of oligonucleotide primers framing the fragment which must be amplified. One can, for example, refer to the technique described in American patent US Pat. No. 4,683,202. The amplified fragments can be identified, for example after agarose or polyacrylamide gel electrophoresis, or after a chromatographic technique such as gel filtration or ion exchange chromatography, and then sequenced. The specificity of the amplification can be controlled by using, as primer, the nucleotide sequences of polynucleotides of the invention as template, plasmids containing these sequences or else the derived amplification products. The amplified nucleotide fragments can be used as reagents in hybridization reactions in order to demonstrate the presence, in a biological sample, of a target nucleic acid of sequence complementary to that of said amplified nucleotide fragments. Other techniques for amplifying the target nucleic acid can advantageously be used as an alternative to PCR, so-called PCR-like methods using pairs of primers of nucleotide sequences according to the invention. By PCR- like is intended to denote all the methods implementing direct or indirect reproductions of the nucleic acid sequences, or else in which the labeling systems have been amplified. These techniques are of course known, in general it is the amplification of DNA by a polymerase. There are currently many methods for this amplification, such as the SDA (Strand Displacement Amplification) technique or strand displacement amplification technique (Walker et al., 1992, Nucleic Acids Res. 20: 1691), the technique TAS (Transcription-based Amplification System) described by Kwoh et al. (1989, Proc. Natl. Acad. Sci. USA, 86, 1173), the 3SR technique (Self-Sustained Sequence Replication) described by Guatelli et al. (1990, Proc. Natl. Acad. Sci. USA 87: 1874), the NASBA (Nucleic Acid Sequence Based Amplification) technique described by Kievitis et al. (1991, J. Virol. Methods, 35, 273), the TMA technique (Transcription Mediated Amplification), the LCR technique (Ligase Chain Reaction) described by Landegren et al. (1988, Science 241, 1077), the RCR (Repair Chain Reaction) technique described by Segev (1992, Kessler C. Springer Nerlag, Berlin, New York, 197-205), the CPR (Cycling Probe Reaction) technique described by Duck et al. (1990, Biotechniques, 9, 142), the Q-beta-replicase amplification technique described by Miele et al. (1983, J. Mol. Biol., 171, 281). Some of these techniques have since been perfected. In the case where the target polynucleotide to be detected is an mRNA, it is advantageous to use, prior to the implementation of an amplification reaction using the primers according to the invention or to the implementation of a method detection using the probes of the invention, an enzyme of reverse transcriptase type in order to obtain a cDNA from the mRNA contained in the biological sample. This is the ALS mRNA of the plant sample analyzed. The cDNA obtained will then serve as a target for the primers or probes used in the amplification or detection method according to the invention. It is thus possible to use the techniques of RT-PCR, of quantitative PCR.

Regarding the use of probes, the probe hybridization technique can be carried out in various ways (Matthews et al, 1988, Anal. Biochem., 169, 1-25). The most general method consists in immobilizing the nucleic acid extracted from cells of different tissues or from cells in culture on a support designated support immobilization (such as nitrocellulose, nylon, polystyrene) and to incubate, under well-defined conditions, the target nucleic acid immobilized with the probe. After hybridization, the excess probe is eliminated and the hybrid molecules formed are detected by the appropriate method (measurement of radioactivity, fluorescence or enzymatic activity linked to the probe).

According to a particular embodiment, the nucleic acid probes according to the invention can be used as capture probes. In this case, a probe, called a “capture probe”, is immobilized on a support and is used to capture by specific hybridization the target nucleic acid obtained from the biological sample to be tested and the target nucleic acid is then detected. thanks to a second probe, called a “detection probe”, marked by an easily detectable element.

According to another aspect the invention relates to a support on which the probes or primers mentioned above are immobilized, covalently or non-covalently. In particular, the support can be a DNA chip or a high density filter.

The term “DNA chip or high density filter” is intended to denote a support on which DNA sequences are fixed, each of which can be identified by its geographic location. These chips or filters differ mainly in their size, the material of the support, and possibly the number of DNA sequences attached to them.

The probes or primers can be fixed on solid supports, in particular DNA chips, by various manufacturing methods. In particular, a synthesis can be carried out in situ by photochemical addressing or by ink jet. Other techniques consist in carrying out an ex situ synthesis and in fixing the probes on the support of the DNA chip by mechanical, electronic or inkjet addressing. These different methods are well known to those skilled in the art.

A nucleotide sequence (probe or primer) according to the invention therefore allows the detection and / or amplification of specific nucleic sequences. In particular, the detection of these sequences is facilitated when the probe is fixed to a DNA chip, or to a high density filter.

According to another aspect, the invention relates to a polypeptide with acetolactate synthase activity of sequence SEQ ID No. 4 comprising the mutation in position 197, or a fragment of said polypeptide, and coded by a mutated nucleotide sequence as described above. Such a polypeptide or fragment is a polypeptide consisting of the sequence SEQ ID No. 4 or of a sequence having at least 80% identity, preferably at least 85%, 90%, 95%, 98% and 99% d with SEQ ID N ° 4 after optimal alignment, and having the mutation in position 197. By polypeptide whose amino acid sequence having a percentage identity of at least 80%, preferably at least 85 %, 90%, 95%, 98% and 99% after optimal alignment with a reference sequence, it is intended to denote the polypeptides having certain modifications compared to the reference polypeptide, such as in particular one or more deletions, truncations, an elongation, a chimeric fusion, and / or one or more substitutions. Among the polypeptides whose amino acid sequence has a percentage identity of at least 80%, preferably at least 85%, 90%, 95%, 98% and 99% after optimal alignment with the sequences SEQ ID No. 4 or with one of their fragments according to the invention, the variant polypeptides encoded by the variant nucleotide sequences as defined above are preferred, in particular the polypeptides whose amino acid sequence has at least one corresponding mutation in particular to a truncation, deletion, substitution and / or addition of at least one amino acid residue with respect to the sequences SEQ ID No. 4 or with one of their fragments.

According to yet another aspect, the invention relates to a process for obtaining a Cichorium plant resistant to a sulfonylurea or imidazolinone herbicide comprising the selection of plants comprising at least one mutation in the region 103 to 670, in particular 103 to 377 of l acetolactate synthase, more specifically a mutation in position 197.

According to another aspect, the invention relates to a method for the qualitative detection of a plant of the genus Cichorium tolerant to a sulfonylurea or imidazolinone herbicide comprising a step of demonstration in a tested plant of a mutated nucleotide sequence (mutation in position 197 of ALS) as described above.

According to one embodiment, this method comprises the following steps: a) optionally, isolation of the DNA from the biological sample to be analyzed, or obtaining a cDNA from the RNA of the biological sample; b) specific amplification of the isolated DNA using at least one primer as described above; c) highlighting of the amplification products.

According to one embodiment, this detection can be carried out using primers, corresponding to fragments located respectively on either side of the identified mutation characteristic of tolerance or resistance.

According to another aspect, the invention relates to a process for obtaining, within the framework of a conventional selection program, a plant of the genus Cichorium resistant to a sulfonylurea or imidazolinone herbicide comprising the selection of plants comprising at least one mutation in the region. 103 to 670, in particular 103 to 377, of acetolactate synthase, more especially a mutation in position 197.

According to another aspect, the invention relates to a method for distinguishing a sensitive plant homozygous (s / s), from a plant homozygous (T / T) or heterozygous (T / s) tolerant to herbicides of the sulfonylureas group and imidazolinones. According to one embodiment, this method of distinction comprises a step in which it is demonstrated, from DNA extracted from a plant which it is desired to test, the presence, on one or two alleles, of a mutated nucleotide sequence ( mutation conferring tolerance / resistance) according to the invention. The distinction between these individuals allows breeders to very advantageously improve downstream breeding patterns.

According to a variant, this method of distinction comprises the analysis of the alleles of the ALS gene, making it possible to distinguish the sensitive allele (not mutated in position 197) from the resistant allele (mutated in position 197). According to one embodiment, an SSCP or similar method is used. Tools of the kit type or the like capable of implementing these methods, in particular with reagents necessary for PCR amplification, also form part of the invention.

According to a variant, this method of distinction comprises the determination of the enzymatic activity of ALS in the presence of herbicide and in particular more particularly of sulfonylurea, the level of activity of ALS being directly correlated to the genotype of the individual. According to an embodiment in the presence of 26.6 nM of chlorsulfuron, the residual enzymatic activity is around 30% for sensitive homozygous individuals, around 60 to 70%> for tolerant heterozygotes, and around 100% for tolerant homozygotes.

An enzyme encoded by the mutated sequences according to the invention, having a reduced affinity for the attachment of herbicides from the group of sulfonylureas and imidazolinones, called in the following ALS-, is also included in the invention.

A plant cell comprising a nucleotide sequence as defined above, with the elements necessary for the expression of said sequence and / or comprising an ALS- enzyme, constitutes another object of the invention.

Such a cell has an increased concentration of leucine, isoleucine and valine relative to the concentration of these amino acids in a wild cell; this results from the decrease in sensitivity of ALS- to feedback by these amino acids. This plant cell preferably contains at least part of the genome of the genus Cichorium, in particular of Cichorium intybus.

A strain of chicory Witloof (Cichorium intybus L. var Witloof) tolerant to sulfonylureas and / or imidazolinones was obtained.

In the absence of any mutagen, calluses tolerant to chlorsulfuron, initiated from cell suspensions, were selected on culture medium containing 28 nM of chlorsulfuron (lethal dose for unselected chicories). The latter have enabled the regeneration of fertile plants capable of withstanding doses of herbicides 1,500 to 2,000 times higher for tolerant individuals homozygous, and 300 times higher for individuals heterozygous. In the absence of herbicide, no cost consecutive to the acquisition of tolerance is measurable, as much on vegetative development, as on the reproductive potential of the tolerant line.

The subject of the invention is therefore also a plant which contains a mutated nucleotide sequence as defined above, an ALS- enzyme or a plant cell containing the elements explained above, said plant being tolerant to herbicides from the group of sulfonylureas and imidazolinones . The invention also relates to a process for weeding plants using a herbicide inhibiting the ALS enzyme, in which the plant comprises a mutated nucleotide sequence as defined above coding for an ALS- enzyme, an ALS enzyme - or a cell as defined above.

The following examples are intended to illustrate the invention. In these examples, reference is made to the following figures: FIG. 1: A: Comparison of the ALS protein contents in wild (S) and tolerant (T) strains. The amount of total protein deposited in each condition is indicated in μg.

B: Comparison of transcriptional activity in wild (S) and tolerant (R) strains.

Northern blot analysis of the expression of the messenger RNA corresponding to the ALS of sensitive and tolerant chicory line. Total RNA was extracted and 10 μg of each sample were subjected to electrophoresis, transferred to a nylon membrane and revealed by a fragment of chicory ALS labeled with

32P.

Figure 2: Inhibition of the ALS enzyme activity extracted from sensitive, tolerant and heterozygous plants (sensitive female x tolerant male), by chlorsulfuron. The values are expressed as a percentage of the untreated controls, and represent the average of the results obtained in three independent experiments.

Figure 3: Effect of increasing concentration of imadazolinone-imazamethabenz on the ALS activity extracted from wild-type chicory and tolerant to chlorsulfuron. The values, expressed as a percentage of the untreated controls, on the average of the results obtained with two different enzymatic preparations.

Figure 4: Consequence of the mutation on the feedback of ALS by the branched chain amino acids. The sensitivity of ALS extracted from chicory tolerant to chlorsulfuron and wild type to inhibition by feedback by each of the three branched amino acids was evaluated by measuring the enzymatic activity in the presence of increasing concentrations of isoleucine, leucine and valine. The results, expressed as a percentage of untreated controls, are the means of data obtained in three independent experiments.

Figure 5: Intraspecific polymorphism: comparison of sequences between fragments of genes coding for ALS, coming from sensitive and tolerant lines of Cichorium intybus (nt 309 to nt 2015, ie 85% "of the gene coding for ALS).

Nucleotide sequence and prediction of the amino acid sequence of Cichorium intybus acetolactate synthase from genotypes sensitive (S) and tolerant (T) to sulfonylureas. Identical nucleotides are indicated by a dash.

The positioning of the primers ALS-L1 and ALS-R1 corresponds to the nucleotide sequences on a gray background. These primers were used to obtain the 5 'part of the ALS gene.

The nucleotide sequences of the primers ALS-L2 and ALS-R2 are boxed. These primers were used to obtain the 3 'part of the ALS gene.

The nucleotide sequences of the primers ALS-L3 and ALS-R3 used to differentiate sensitive individuals from tolerant individuals homozygous and heterozygous by the SSCP technique are underlined

Figure 6: Interspecific polymorphism.

Alignment on 85% of the amino acid sequence deduced from the nucleotide sequence of the ALS of Cichorium intybus with the 11 closest ALS protein sequences from different plant species. Sequence alignment is performed using the CLUSTALW Network Protein Séquence @nalysis sequence alignment software (IBCP, France), available on the Internet.

Region A and Region B: Mutating regions conferring tolerance to sulfonylureas in different plant species (Wright et al., Weed Sci. 46, 13-23, 1998)

Figure 7: Comparison of two methods (assay of the enzymatic activity of ALS and SSCP analysis) which make it possible to distinguish in Cichorium intybus individuals who are sensitive homozygotes (s / s), tolerant homozygotes (T / T) and tolerant heterozygotes ( T / s) to sulfonylureas on 44 F2 plants treated with sulfonylureas and the sensitive parent MS8 of the F2 progeny. 1) Main results obtained from Cichorium intybus L. var Witloof We first successively describe: example 1: obtaining a natural line resistant to chlorsulfuron example 2: a method for characterizing resistance to chlorsulfuron example 3: l of the mutated ALS sequence example 4: a method of distinguishing individuals sensitive and tolerant to sulfonylureas

EXAMPLE 1 Obtaining a Line Resistant to Chlorsulfuron

Young seedlings from aseptic germination of achenes were used to obtain this line. The cotyledons are lacerated and deposited on a static liquid medium until calogenic explants are obtained. Then these explants are placed in a liquid medium with stirring. These explants are removed as the subcultures continue until only a heterogeneous suspension remains (cells and minicals). It is only when the cultures are stabilized, with a delay of 10-15 days between transplanting, that the selection can begin. The calluses and the cells are spread on an agar medium containing the herbicide, as well as on a non-selective medium.

After 30 to 40 days of culture, the non-necrotic cell clusters in the presence of the herbicides are subcultured on a selection medium of the same composition. This operation is repeated until it is possible to select cells capable of multiplying normally in the presence of herbicide. These cultures are then deposited on a selective medium with control cells which have never undergone selection pressure so as to compare the behavior of sensitive strains or those supporting the herbicide.

Vitroplants are then produced by holding the calluses on the induction media until they form meristematic nodules and then transplanting onto a bud development medium. The young vitroplants once rooted are removed and transplanted in mini greenhouses. We thus isolate an RI strain OK which normally develops in the presence of chlorsulfuron and sulfometuron (herbicides of the sulfonylurea family).

Example 2: Characterization of resistance to chlorsulfuron 1. Materials and Methods

- Plant material RI OK

Seeds obtained from chicory of the wild type, homozygous resistant to chlorsurfuron (RI OK) and heterozygote (obtained by pollination of wild type plants with pollen RI OK) were surface sterilized for 15 min in an aqueous chloride solution 0.1% mercuric), rinsed three times with sterile distilled water and germinated on agarized Heller salts (Heller, 1953) containing 20 g / l of sucrose. The seedlings are cultivated at 22 ± 1 ° C, at a rate of 16 hours of day (250 μE m-2 s-1 density of the photosynthetic fluid) and 8 hours of night. After one month, the plants are harvested and used for RNA extraction and enzyme analysis.

- Extraction of acetolactate synthase and dosage

The ALS activity was extracted and assayed as described by Forlani et al (Plant Growth Regul. 14, 203-209, 1994). Briefly, 5 g of leaves are reduced to powder in liquid nitrogen and resuspended in 5 ml.g-1 of 50 mM phosphate buffer (pH 7.5) containing 0.5 mM dithiothreitol, 1 mM MgC12, 0.1 mM thiamine pyrophosphate (TPP), 0.01 mM flavin adenine dinucleotide (FAD), 20%) of glycerol and 2% (w / v) of polyvinyl polypyrrolidone. After centrifugation for 20 minutes at 18,000 g, the supernatant is added with solid ammonium sulphate (70% of saturation) and centrifuged. The pellets are dissolved in half-strength extraction buffer and desalted by passage through a column of Sephadex G25 (Pharmacia). The enzymatic activity is measured as the amount of acetolactate produced. The aliquots corresponding to approximately 5 pkat of ALS activity are incubated for up to 90 min at 35 ° C. in the presence of 20 mM potassium phosphate buffer (pH 7.5) 40 mM sodium pyruvate, 1 mM MgCl 2, TPP 0 , 1 mM and 0.01 mM FAD. After acid catalyzed decarboxylation of acetolactate to acetoin, the latter is determined by colorimetry. Cookies are made to quantify the background noise. The averages of specific ESL activities are calculated on at least five independent determinations on seedlings having uniform growth, harvested at the same stage of development. Enzyme inhibition was estimated by adding increasing concentrations of chlorsulfuron, imazamethabenz, isoleucine, leucine and valine to the reaction medium; three measurements were made for each assay. Each experiment was repeated twice on different enzyme preparations; the means of the two repetitions are presented, expressed as a percentage of the untreated controls. - Northern blot analysis

Total RNA was extracted and 10 μg aliquots were denatured and subjected to electrophoresis on 1.5% agarose gels, containing 3% formaldehyde. The gels are transferred to Hybond-N membranes in the 20 X SSC buffer. The chicory probe used for the detection of the ALS transcript was generated by PCR, with primers homologous to the gene sequence on tobacco A, comprising positions 1274 to 1295 and 1883 to 1902 (Mazur et al, Plant Physiol. 85, 1110-1117, 1987). The 630 bp fragment obtained was cloned into the PCRTM II vector (Invitrogene) using standard cloning procedures and sequenced by the dideoxy termination procedure (Sanger et al, 1977, Proc Natl. Acad. Sci. USA 74, 5463- 5467, 1977). The probe was labeled with 32P using the T7-Quick-Prime kit (promega) according to the manufacturer's instructions. Hybridization was performed at 60 ° C. The autoradiograms were analyzed on a Microtek Color / GaryTM densitometer (Biorad) and the data processed with the Cricket GraphTM program.

Example 3: Isolation of the sequence of 1 "mutated ALS, and identification of a mutation in position 197 conferring the tolerance - Plant material used

A cross was made within Cichorium intybus between an industrial sterile male chicory gene sensitive to sulfonylureas (MS8, Ets FLORIMOND DESPREZ) and an endive chicory of the RI OK line homozygous resistant to sulfonylureas (Ets HOQUET Seeds), the aim being to transfer the trait for resistance to sulfonylureas from the endive cultigroup to that of industrial chicory. An F2 progeny was obtained and treated with sulfonylureas at the rate of 30g / ha of Glean (70% chlorsulfuron, Du Pont). At this dose, theoretically, only plants resistant to sulfonylureas survive, whether homozygous or heterozygous. The material used for the study corresponds to the F2 plants which survived the treatment. As a control, the sensitive genotype MS8, parent of the F2 progeny, is used. - Isolation of the gene coding for the ALS of Cichorium intybus a) Extraction of genomic DNA.

The extraction of chicory genomic DNA is carried out using the “Dneasy plant mini kit” sold by Qiagen. Fifty mg of leaves are crushed and the elution after passage through a Qiagen column is done in 2 x 100 μl of buffer. b) Obtaining the 5 ′ part of the gene by PCR.

The comparison of sequences of acetolactate synthase genes from different plant species made it possible to define the degenerate sense primer (ALS-L1) in a conserved region of the gene.

Sense primer (ALS-Ll): 5'-CTCGTCGAAGCYCTKGARCG-3 'where Y = C / T, K = G / T and R = G / A

The antisense sequence (ALS-Rl) was determined from the partial chicory ALS sequence obtained by Eric Dewaele, clone DCIALSEB32 (Acquisition of resistance to sulfonylureas in a chicory strain (Cichorium intybus L. var. Witloof) and consequences on the metabolism of branched chain amino acids, Doctoral thesis, University of Sciences and Technologies of Lille, 1997).

Antisense primer (ALS-Rl):

5'-AAGCTTCAGCAAACTTCA-3 'The PCR was carried out in a final volume of 20 μl containing 0.2 μM of each of the 2 primers ALS-Ll and ALS-Rl, 1 mM of MgCl ₂ , 0.1 mM of dNTP, 1 ng chicory genomic DNA, 0.6 U of Taq polymerase Appligene (15 U / μl) and PCR buffer x 1. The reaction mixture was incubated in a thermocycler (PE 9600, Perkin Elmer) for 40 cycles including for each cycle 1 min of denaturation at 94 ° C, 1 min of hybridization at 52 ° C and 1 min of elongation at 72 ° C. Two independent PCRs were performed, one with DNA from a sensitive chicory (MS8) and the other with DNA from a chicory tolerant to sulfonylureas of the F2 progeny. Agarose gel electrophoresis of the PCR products revealed a 1.5 kb band, corresponding to the expected size according to the ALS sequence alignments of different plant species. The PCR products were purified using Qiaquick columns and cloned into the vector pGEM-T Easy (Promega). The E. coli JM109 bacteria (Promega) were transformed with this construct. Three clones containing susceptible plant DNA and 8 clones containing tolerant plant DNA were sequenced with an automatic Licor DNA sequencer (Lincoln, USA). The sequence reactions were carried out according to the Sanger method using the “ABI PRISM Dye Primer Cycle Sequencing Core Kit” kit (Perkin Elmer). The primers were labeled with 1TRD-800, a fluorescent compound. c) Obtaining the 3 ′ part of the gene by PCR.

The sequences of the sense (ALS-L2) and antisense (ALS-R2) primers were determined from the partial sequence of chicory ALS obtained by Eric Dewaele, clone DCIALSEB32.

Sense primer (ALS-L2):

5 '-CAGAGCCAAAATCGTTCACA-3'

Antisense primer (ALS-R2):

5'-TTCATTCTGCCATCACCATC-3 'Independent PCR reactions were carried out with the two primers

ALS-L2 and ALS-R2 on DNA from a sensitive chicory (MS8) and 6 F2 tolerant chicories, under the same conditions as above but for 30 cycles and at a hybridization temperature of 55 ° C. The PCR fragments obtained of 850 bp, corresponding to the expected size, were cloned into the vector pGEM-T Easy. Two clones containing MS8 susceptible plant DNA and 4 clones per F2 tolerant plant were sequenced.

EXAMPLE 4 Distinction Methods in Cichorium intybus of Sensitive Individuals, Tolerant Homozygotes and Heterozygotes Tolerant to Sulphonylureas a) Colorimetric determination of the residual enzymatic activity of ALS in the presence of chlorsulfuron. The extraction and the assay of the activity of the ALS were carried out according to the method described in Example 2 on fresh leaves one month old, b) Analysis of the polymorphism of the ALS by SSCP. Principle: The SSCP (Single Strand Conformation Polymorphism) method makes it possible to distinguish the alleles of a gene when these present point differences at the nucleotide level. It involves PCR amplification of DNA fragments and analysis of denatured DNA fragments on a non-denaturing polyacrylamide gel. Each primary sequence has a unique three-dimensional structure and will migrate at its own speed. The sensitivity of the mutation detection is high because a change of a base is enough to alter the mobility of the DNA strand.

The sequences of the sense (ALS-L3) and antisense (ALS-R3) primers were determined from the nucleotide sequence presented in FIG. 5. They allow the theoretical amplification of a 310 bp fragment comprising the mutated site in individuals. tolerant. Sense primer (ALS-L3): 5'-CTAACCCGGTCAAGCATCAT-3 'Antisense primer (ALS-R3): 5' -CGGAAGAGGCGAGATGAAA-3 'PCR reactions were performed with the two primers ALS-L3 and ALS-

R3 and the DNA of the plants on which the biochemical test was carried out, under the same conditions as in Example 3 b) "Obtaining the 5 'part of the gene by PCR" but with a hybridization temperature of 55 ° C. The amplified DNA fragments were then denatured for 5 min at 94 ° C. in the presence of formamide. About 1/10 ^th of the PCR reaction was deposited on non-denaturing polyacrylamide gel - gel 0.75 mm thick and 20 cm long, MDE x0.5 (TEBU) -. The electrophoresis was carried out at a temperature of 10 ° C. in Protean Ilxi tanks (Biorad) with TBE x0.6 buffer (Tris-borate-EDTA). The separation time is 3000 Vh for a fragment of 300 bp. After migration, the bands were revealed with silver nitrate according to the method of Bassam et al. (Anal. Biochem. 196, 80-83, 1991). 2. Results and discussion

- Herbicide tolerance:

The first elements taken into consideration are the quantity of messenger RNA-ALS and the level of specific activity ALS. Northern blot experiments with both susceptible and resistant plants did not show a substantial difference between the two lines (Figure 1). On the contrary, the enzymatic analysis revealed a significant difference as regards the specific activity ALS, which is 12.2 +/- 1.1 pkat.mg-1 of total protein in the wild type and 7.9 + / - 0.8 pkat.mg-1 protein in resistant plants. These results exclude the possibility that resistance is derived partially or completely from an increase in ALS levels. It may be noted that despite a 35% decrease in specific enzymatic activity, resistant plants do not show a reduction or alteration in vegetative or reproductive characteristics. Since the level of mRNA is not changed, a possible explanation for this reduction could be the occurrence of a mutation affecting the stability of the enzyme.

- Comparison of in vitro sensitivity to chlorsulfuron between ALS from chicory resistant to herbicides and sensitive:

The homozygous ALS activity of resistant and sensitive plants was assayed in the presence of increasing concentrations of chlorsulfuron. The results showed that ALS from resistant plants is not inhibited up to herbicide concentrations on the order of micromolar, whereas that from wild plants was already affected by nanomolar levels (Figure 2). The concentration required to inhibit 50% of the ALS activity of resistant plants is 12 μM, while the ID50 for the enzyme of wild type plants is 0.021 μM, corresponding to a difference of 570 times. Such markedly different behaviors strongly suggest a decreased sensitivity of ALS to the inhibitory effect of chlorsulfuron as a determinant of the selected tolerance.

ALS was also extracted from heterozygous chicory obtained by pollination of sensitive plants with RI OK pollen, and the enzymatic activity was measured in the presence of the herbicide. Results show a biphasic profile intermediate between wild type and homozygous profiles resistant (Figure 2), indicating the concurrent presence of a resistant form of ALS with a sensitive form, and that the character of resistance to chlorsulfuron is inherited semi-dominantly.

The enzyme resistant to chlorsulfuron shows cross tolerance towards the herbicide imidazolinone: imazamethabenz.

The ALS activity of wild type and resistant type respectively was measured in the presence of increasing concentrations of imazamethabenz. As can be seen in FIG. 3, the ALS activity of resistant plants has not been affected at concentrations of up to 10 μM, the ID50 being approximately 200 μM, while that for ALS from plants wild type, activity was inhibited by 50%) at 1.8 μM. Initially selected for its resistance to chlorsulfuron, the RI OK line thus appears to have an ALS activity which is also 125 times more resistant to a herbicide belonging to another class of ALS inhibitors. In the chicory mutant, the difference in the level of resistance to chlorsulfuron and imazamethabenz is apparently consistent with the existence of a single mutation site with specific binding domains overlapping.

- Sensitivity to ALS feedback by leucine and valine.

Branched chain amino acids modulate their own synthesis by cooperatively inhibiting the first enzyme in their biosynthetic pathway. ALS activity was thus measured in extracts from wild type plants and RI OK in the presence of increasing concentrations of valine, leucine, or isoleucine. As expected, valine and leucine markedly decrease the activity of wild type ALS with similar inhibition rates (ID50 about 1 mM) while isoleucine, which controls the flow of carbon through its pathway at threonine deaminase is substantially ineffective (ID50 greater than 100 mM; Figure 4). When the activity resistant to chlorsulfuron extracted from chicory RI OK was examined, it was found significantly less sensitive to the inhibitory effect of valine (ID50 = 3 mM), or even more of leucine (ID50 around 10 mM; Figure 4). It is therefore probable that the resistant form of ALS of the RI OK line results from one or several mutational events which concern both the site of attachment to the herbicides and the field involved in feedback control with valine and leucine.

This decrease in sensitivity to feedback (10 times for leucine, and 3 times for valine) results in an increase in the content of free valine (+ 140%>) and leucine (+ 80%>) in these plants, like this appears in the following table:

Content expressed in nmol.g-1 of fresh matter

Amino acid Sensitive Tolerant

Valine 10.1 ± 1.4 24.3 ± 1.8

Leucine 9.0 ± 1.9 16.0 ± 1.6

Isoleucine 6.9 ± 1.1 8.2 ± 1.2

Total (20 aa) 1502 1600

- Isolation of the gene coding for ALS

The global nucleotide sequences obtained (5 ′ and 3 ′ part of the gene) and the deduced amino acid sequences, of chicory-sensitive and sulfonylurea-tolerant ALS, are presented in Figure 5. Only 85% of the gene is represented, of the nucleotide 309 at nucleotide 2015. The first 308 nucleotides are missing in 5 ', the part of the ALS gene in 3' is complete and goes to the STOP codon. The chicory ALS sequence, however, contains the entire two regions A and B (Figure 6) where mutations conferring tolerance to sulfonylureas in other plant species have been identified (Wright et al., Weed Sci. 46, 13 -23, 1998). At the nucleotide level, the only difference between sensitive and tolerant alleles is located at position 591 where a Cytosine (C) for the sensitive allele is mutated into Thymine (T) for the tolerant allele, which causes a change in amino acid level of a proline (P) into serine (S) at position 197. This mutation obtained in chicory is located in domain A containing known mutation sites conferring tolerance to sulfonylureas in other plant species.

At the protein level, the chicory ALS sequence has been aligned with those of other plants (Figure 6). The closest ALS sequence to chicory is that of lettuce. This substitution is also found in the same place (position 197) in Lactuca sativa (Eberlein et al., Weed Sci. 47, 383-392, 1999) and L. seriola (Guttieri et al., Weed Sci. 40, 670 -678, 1992) but the proline (P) in the sensitive individual is mutated to histidine (H) in the tolerant homozygous individual. On the other hand, we find exactly the same mutation of a (P) in (S) conferring tolerance to sulfonylureas in the same place (position 197) in tobacco (Harms et al., 1992) and in A. thaliana (Haughn and al., 1988) which are however more botanically distant from chicory than lettuce.

In addition, it can be noted that the chicory ALS gene does not contain an intron like the ALS genes of Arabidopsis thaliana, Bassia scoparia, beetroot, Brassica napus, cotton, lettuce and tobacco. This would appear to be the case for all ALS genes.

- Colorimetric determination of the residual enzymatic activity of ALS in the presence of chlorsulfuron.

The assay of the residual enzymatic activity of ALS in the presence of 26.6 nM of chlorsulfuron makes it possible to determine the genotype of the individuals. Indeed, out of 44 F2 plants treated with sulfonylureas and the sensitive MS8 parent whose residual enzymatic activity of ALS has been measured, a sensitive homozygous individual (s / s) has an average of 29.5% ± 0.71 d residual enzyme activity of ALS, a heterozygous tolerant individual (T / s) 63% ± 4 and a homozygous tolerant individual (T / T) 103%) ± 3.6 (Figure 7). It should be noted that the plant F2 No. 11, although having survived the sulfonylurea treatment, appears according to this sensitive homozygous test (s / s) with a residual ALS activity of 30%.

- Analysis of the ALS polymorphism by SSCP Analysis on SSCP gel of the fragments originating from 44 F2 plants treated with sulfonylureas and from the sensitive parent MS8 (same plants analyzed as in Example 4 a) “Colorimetric determination of the activity residual enzyme of ALS in the presence of chlorsulfuron ”) shows that a sensitive individual (s / s) always has a single band (corresponding to wild alleles bb), the tolerant homozygous individual (T / T) also shows a single band (corresponding to the mutated alleles aa) but at a level of migration different from the first and the heterozygous tolerant individual (T / s) shows two bands corresponding to that of the sensitive plant and the homozygous tolerant plant (corresponding to the ab alleles) (Figure 7). It should be noted that the plant F2 No. 11, although having survived the sulfonylurea treatment, appears from this sensitive homozygous test (s / s) because it has the two alleles bb. Note: plant F2 n ° l 1 has a sensitive homozygous genotype (s / s) whatever the detection method used. She "escaped" Glean treatment, possibly due to a poor distribution of the herbicide.

The biochemical (colorimetric assay of the residual ALS activity) and molecular (SSCP analysis) experiments are perfectly correlated and allow the genotype at the ALS locus to be unambiguously determined. The nucleotide substitution of a C at T at position 591 of the sequence presented in Figure V is therefore responsible for the sulfonylurea resistance of the RI OK line.

The molecular method has a number of advantages over the enzymatic method: it is safe, faster, can be performed on elderly individuals and DNA can be extracted from dried leaves. This molecular test can be performed routinely on a large number of individuals.

BIBLIOGRAPHY

Bassam, B.J. et al.:"Fast and sensitive silver staining of DNA in polyacrylamide gels "Anal. Biochem., Vol. 196, 1991, Pages 80-83.

Bernasconi, P. et al .: "A naturally occurring point mutation confers broad range tolerance to herbicides that target acetolactate synthase" J. Biol. Biochem., Vol. 270, 1995, Pages 17381-17385.

Dewaele, E .: "Acquisition of resistance to sulfonylureas in a chicory strain (Cichorium intybus L. var. Witloof) and consequences on the metabolism of branched chain amino acids" Doctoral thesis, University of Sciences and Technologies of Lille, 1997.

Eberlein, C.V. et al .: "Physiological consequences of mutation for ALS-inhibitor resistance" Weed Sci., Vol. 47, 1999, Pages 383-392.

Guttieri, M.J. et al.:"DNA sequence variation in Domain A of the acetolactate synthase genes of herbicide-resistant and -usceptible weed biotypes "Weed Sci., Vol. 40, 1992, Pages 670-678.

Harms, C.T. et al .: "Herbicide resistance due to amplification of a mutant acetohydroxyacid synthase gene" Mol. Gen. Broom, Vol. 233, 1992, Pages 427-435.

Haughn, G.W. et al.:"Transformation with a mutant Arabidopsis acetolactate synthase gene renders tobacco resistant to sulfonylurea herbicides "Mol. Gen. Broom, Vol. 211, 1988, Pages 266-271.

Wright, T.R. et al. : "Biochemical mechanism and molecular basis for ALS- inhibiting herbicide resistance in sugarbeet (Beta vulgaris) somatic cell selections" Weed Sci., Vol. 46, 1998, Pages 13-23.

Claims

1) Nucleotide sequence isolated from a plant of the genus Cichorium, characterized in that it is chosen from: a) a nucleotide sequence corresponding to SEQ ID No. 3 b) a nucleotide sequence comprising at least 80%> of identity with SEQ ID No. 3 c) a nucleotide sequence complementary to SEQ ID No. 3 or of a sequence defined in b) or of the RNA sequence d) a nucleotide sequence comprising a sequence as defined in a), b) or c ); and in that it codes for a mutated acetolactate synthase having at least one mutation in the amino acid region 103 to 377 of the ALS, the said mutation or mutations conferring on the plant resistance to a herbicide.

2) Isolated nucleotide sequence characterized in that it comprises at least 80% and typically at least 90.95.98% identity with SEQ ID No. 3, said sequence coding for a mutated ALS having at least one mutation in the region of amino acids 103 to 670, the said mutation or mutations giving the plant resistance to a herbicide.

3) Nucleotide sequence according to claim 2 characterized in that it codes for an acetolactate synthase mutated in position 197.

4) Nucleotide sequence according to claim 3 characterized in that the mutation is a mutation from a Proline to a Serine, or any functionally equivalent mutation.

5) Nucleotide sequence according to any one of claims 1 to 4 characterized in that the herbicide is chosen from sulfonylureas, imidazolinones. 6) Nucleotide sequence characterized in that it is a fragment representative of a nucleotide sequence according to any one of claims 1 to 5.

7) Nucleotide sequence according to claim 6, usable as a primer for the identification and / or selection of plants of the genus Cichorium tolerant to a sulfonylurea or imidazolinone herbicide characterized in that it comprises at least 10 bases and is capable of amplifying a nucleotide sequence according to any one of claims 1 to 4.

8) Nucleotide sequence usable as a probe for the identification and / or selection of plants of the genus Cichorium resistant to a sulfonylurea or imidazolinone herbicide characterized in that it is capable of hybridizing with a nucleotide sequence according to any one of claims 1 to 4.

9) Sequence according to claim 7 or 8 characterized in that it is marked by radioactivity, fluorescence or colorimetric reagent.

10) Polypeptide with acetolactate synthase activity modified with respect to the non-mutated polypeptide, or fragment of said polypeptide, characterized in that it is of sequence SEQ ID No. 4 or comprises at least 80%> of identity with SEQ ID No. 4 .

11) Process for obtaining a plant of the genus Cichorium resistant to a sulfonylurea or imidazolinone herbicide, characterized in that it comprises the selection of plants comprising at least one mutation in the region 103 to 670 of acetolactate synthase, more particularly a transfer to position 197.

12) Method for the qualitative detection of a plant of the genus Cichorium resistant to a sulfonylurea or imidazolinone herbicide, characterized in that it comprises a step of demonstration in a tested plant of a mutated nucleotide sequence according to any one of claims 1 to 4. 13) Method for distinguishing a plant of the genus Cichorium homozygous (s / s) sensitive, from a plant homozygous (T / T) or heterozygous (T / s) tolerant to herbicides from the group of sulfonylureas and imidazolinones comprising a step in which it is demonstrated, from DNA extracted from a plant which it is desired to test, the presence, on one or two alleles, of a mutated nucleotide sequence according to any one of claims 1 to 4.

14) A method of distinction according to claim 13 of a plant of the genus Cichorium sensitive homozygous (s / s), of a plant homozygous (T / T) or heterozygous (T / s) tolerant to herbicides from the group of sulfonylureas and imidazolinones, comprising a step of measuring the enzymatic activity of ALS and the deduction of its genotype.

15) Method according to claim 13 or 14 characterized in that it comprises the following steps: a) optionally, isolation of the DNA from the biological sample to be analyzed, or obtaining a cDNA from the Biological sample RNA; b) specific amplification of the DNA isolated using at least one primer according to claim 7; c) highlighting of the amplification products.

16) Kit or kit for the detection of plants of the genus Cichorium resistant to sulfonylurea herbicides, characterized in that it comprises a primer nucleotide sequence according to claim 7 and of reagents necessary for the amplification of this sequence, and / or a nucleotide sequence probe according to claim 10, and reagents necessary for the hybridization of this sequence.

17) Plant cell characterized in that it is modified so as to comprise a nucleotide sequence according to any one of claims 1 to 4 or a polypeptide according to claim 10. 18) Use of a nucleic acid according to any one of claims 1 to 4 for obtaining a plant resistant to sulfonylurea or imidazolinone herbicides.

19) Method for weeding plants using a herbicide inhibiting the ALS enzyme, in which the plant comprises a mutated nucleotide sequence according to any one of claims 1 to 4.