WO2003060134A1

WO2003060134A1 - Herbicide mode of action determination

Info

Publication number: WO2003060134A1
Application number: PCT/EP2003/000512
Authority: WO
Inventors: Willem Broekaert; Dirk Inze
Original assignee: Cropdesign N.V.
Priority date: 2002-01-16
Filing date: 2003-01-14
Publication date: 2003-07-24
Also published as: AU2003235582A1

Abstract

The present invention relates to methods to determine the molecular target of an agrochemical or candidate agrochemical compound. The methods of the present invention can also be used as screening assay to identify new agrochemicals with pesticidal potency or with growth regulatory potency (such as herbicides or growth stimulators). The invention further relates to agrochemical and agrochemical compositions and methods for preparing these. The invention further provides plants that are tolerant to an agrochemcial compound.

Description

HERBICIDE MODE OF ACTION DETERMINATION

Field of the invention

The invention relates to a method to rapidly discover the mode of action of an agrochemical or candidate agrochemical compound such as a herbicide, insecticide, nematicide or fungicide.

Background to the invention

Crop protection needs continuous innovation in order to find better and more efficacious ways to use agrochemicals, such as pesticides. There is a need for agrochemical compounds (e.g. herbicides) that exhibit greater potency, that have a broader action spectrum and that have a short half-life in the environment. However such powerful agrochemicals still need to retain low crop phytotoxicity.

Pesticides generally interact with, and totally or partially inactivate a particular protein or enzyme in the target organism, and in this way they disturb a vital process in the target organism and kill it . So far only a limited n umber of pesticides, i n p articular h erbicides,^" a re known of which the full mode of action is identified, while knowing the precise mode of action of the pesticide or herbicide would allow to optimize the action of the pesticide or herbicide. Knowing the mode of action of the pesticide would thus allow using it in a more efficacious way, which would have beneficial impact on the economical, agricultural and the environmental aspects of pesticide use.

Summary of the invention

The present invention provides a high throughput method to rapidly discover the mode of action of an agrochemical or candidate agrochemical compound such as a pesticide, herbicide, insecticide, nematicide or fungicide. The invention is also useful to optimise the action of an existing agrochemical based on the knowledge of the molecular target of the herbicide. Further the invention is also applicable as a screening assay to identify new agrochemicals or new agrochemical compounds. Such agrochemical compounds can have pesticidal potency (such as herbicides) or can have growth regulatory potency (such as growth inhibitors or growth stimulators). Finally, the invention may also be applied to develop agrochemical resistant plants. In particular, the methods of the present invention are based on the following steps: (i) a set of transgenic lines of a target organism is made, with each transgenic line overexpressing a different "candidate target gene"; (ii) a population of individuals of the target organism, consisting of a sufficient number of transgenic individuals for each overexpressed candidate target gene, is then treated with the agrochemical at various concentrations. I he concentrations of the agrochemical are chosen such as to kill a large majority of the transgenic organisms and/or the large majority of the non-transgenic target organism); (iii) lines of the target organism transformed with and overexpressing the gene that represents the true molecular target of the applied agrochemical are exhibiting enhanced tolerance to the agrochemical compound and therefore better survive the treatment with the agrochemical compound. Treatment of a population composed of the different overexpressor lines and identification of the gene contained in those individuals with enhanced tolerance to the compound, is therefore a rapid method to identify the mode of action (i.e. the molecular target) of candidate agrochemicals.

Knowledge about the molecular target of an agrochemical or candidate agrochemical compound gives an important indication of the features of the compound, such as likely toxicity, selectivity, spectrum, etc. Also, knowledge about the molecular target of an agrochemical or candidate agrochemical compound is an important element to estimate the novelty of such compounds and to guide decisions concerning commercial exploitation of it. Therefore this information is of crucial importance in agrochemical research and development.

Detailed Description of the Invention

Standard molecular biology techniques allow making libraries of large numbers of candidate genes in a full-length form. Once the genes are cloned in a library vector they can be easily cloned in an appropriate expression vector and transformed into the target organism. In this way, u sing a variety of standard techniques, it is possible to o btain a certain type of target organism, e.g. plants, large numbers of different transgenic lines, each line overexpressing a different candidate target gene.

Literature describes several cases in which overexpression of a particular target gene in a host organism leads to enhanced tolerance of the host organism to an agrochemical compound. This method of developing agrochemical tolerant organisms, such as herbicicde resistant crop plants, as a general m ethod is not commercially interesting, since overexpression of target genes will not usually lead to a commercially useful phenotype of the host organism. Therefore, for the development of herbicide tolerant crops, other strategies have been developed. In order to obtain herbicide tolerant crops it is not sufficient t o o verexpress the herbicide target gene, but mutations in the target gene altering the affinity of the target protein for the herbicide need to be introduced. Also the introduction of genes that can break down the herbicide are utilised to achieve commercially useful tolerance levels in crop plants. The present invention now teaches how to utilise the low levels of tolerance of organisms to a certain agrochemical, generally obtained by overexpressing the agrochemical target gene, in a practically useful way, i.e. use of this level of tolerance as a means to identify the true molecular target from a (large) set of candidate target genes.

Therefore, according to a first embodiment the invention relates to a method to rapidly determine the mode of action of an agrochemical or candidate agrochemical by identifying the molecular target of said agrochemical or candidate agrochemical compound, comprising the steps of: (a) providing a population of transgenic individuals, wherein different individuals overexpress different genes selected from a candidate target gene set;

(b) applying to said population of individuals said agrochemical or candidate agrochemical compound in a concentration that affects or kills a large majority of transgenic individuals of said population or a large majority of non-transgenic individuals; and

(c) identifying transgenic individuals exhibiting enhanced tolerance to said agrochemical or candidate agrochemical compound.

The terms "agrochemical" and "candidate agrochemical" comprise pesticides, herbicides, insecticides, nematicides, fungicides a nd the l ike, a nd the respective candidate compounds having similar properties. The terms "pesticide", "agrochemical", "candidate pesticide" and "candidate agrochemical" are used interchangeable with the term "compound" anywhere it is intended that the compounds should have agrochemical or pesiticidal or candidate agrochemical or pesticidal properties.

With the expression "population of transgenic individuals" is meant a group of plants that were transformed with a genetic construct. These plants can originate from the same transformation event and are part of a transformed plant line or alternatively, these plants can originate from different transformation events and can belong to different transformed plant lines. The term "lines" is meant herein as "cell line" or "tissue line" or any line of parts of organisms or even complete organisms that can be grown.

With the expression "determine the mode of action" is meant the identification of the particular protein or enzyme, or a gene encoding such protein or enzyme, with which the agrochemical or candidate agrochemical interferes or interacts, resulting in for example a pesticidal adtion. To determine the mode of action thus means the identification of the molecular target of the agrochemical or candidate agrochemical. Furthermore, according to the invention, the population of individuals in the methods of the invention are chosen from a population of cells, tissues or parts of an organism or an organism. Furthermore, according to the invention, said organism is chosen from a prokaryote, such as a bacteria or a microorganism, or is chosen from an eukaryote, such as a yeast, a fungus, an insect, a plant, a mammal or a human.

The plant cells or plants used in the methods of the present invention include all plants or cells of plants which belong to the superfamily Vi diplantae, including both monocotyledonous and dicotyledonous plants. Particular interesting plants for use in the methods of the invention are Arabidopsis thaliana and Crop plants such as Oryza sativa (rice), maize, wheat, oats, rye, sorghum, barley, millet, turf and forage grasses, cotton, sugar cane, sugar beet, oilseed rape and soybean, or plant cells or tissues derived thereof. Furthermore, the choice of target species depends on the result to be achieved. For instance if the goal is the determination of the mode of action of a broad spectrum candidate herbicide, use of for example Arabidopsis thaliana is recommended. Use of rice or another gramineae is for instance recommended if the focus is for example on mode of action of selective grass herbicides.

In using the invention it is of particular importance to select an appropriate set of candidate target genes to transform into the target organism that will be treated with the agrochemical compound. In general, the ideal set of "candidate target genes" is composed of all genes of a given organism, so that every possible target gene of the agrochemical is represented in the set. However, the workload associated with cloning and creating a library of a full gene set of an organism as well as the upset of the necessary infrastructure to generate a population of transgenic individuals which is extensive enough to cover overexpression of all genes, may sometimes be practically inconceivable or uneconomical.

Therefore according to other embodiments of the invention, the candidate target gene set will be more limited. A preferred way of restricting the candidate target gene set is to restrict to all the genes expressed in a particular tissue of the target organisms (preferably the tissue on which the pesticide is known or suspected to act upon). Alternatively or additionally, the target gene set is restricted to the genes that are expressed in the target organism under particular circumstances (preferably under circumstances under which the pesticide is known or suspected to act). Many other ways of restricting the candidate target gene set can be used, and these restrictions can be based on particular information on the action of the agrochemical or candidate agrochemical compound that is available or predicted. Such information that may be available is for example the plant species where the (candidate) agrochemial works upon; whether it has a contact action (such as locally on the leaves when sprayed upon the leaves) or a systemic action, whether it also acts on isolated leaf disks, or there may be any information available from other mode of action studies such as radiotrace studies, expression array etc.

In certain cases there may be biological information about the mode of action of the tested agrochemical compound, not in terms of its precise molecular target protein, but in terms of the general biological process with which it interferes. This biological information allows to restrict the candidate target gene set to a limited gene set of for example between several tens and several hundreds of genes. These genes can be specifically selected from a functionally annotated gene database, from literature or from other information sources and may take all part in the same biological process(ses) in the target organism. This is a particularly useful approach for target genes from an organism of which complete or quasi-complete genome sequence information is available. In a particular example of the present invention this limited "target gene set" is selected so that it covers a specific biological pathway in its entirety.

Accordingly, in particular embodiments of the present invention, the candidate target gene set is composed by one of the following approaches,

(i) gene selection on the basis of specific biological information (100's range); (ii) selection of all genes in a cDNA library comprising a subset of the genome, such as a leaf specific cDNA library (1 ,000's range); and

(iii) genome wide approach (10,000's range).

The first approach (i) may be recommended when there is sufficient basic knowledge about the action of the compound and when the genes can be selected from a functionally annotated sequence database for example the annotated Arabidopsis and other public databases.

Examples of "biological information" about the action of the agrochemical could be for example that the agrochemical compound is known to interact with a certain biosynthetic pathway and the genes involved in that pathway are known.

Also biological studies, such as differential gene expression profiles in the presence and in the absence of a selected compound, can provide information on which genes or gene products are influenced by application of the compound. Again, this information allows making an intelligent selection of candidate target genes.

The second approach (ii) to make a selection of candidate target genes may be recommended when there is not sufficient information to make a rational gene selection as described here- above, but when it is possible to identify a well defined place or cirumstance in which the agrochemical compound acts. The third approach (iii) could be considered in case a "universal" tool is to be developed for mode of action determination of a (candidate) agrochemical. This approach could also be based on an extension of the second approach using several libraries or could be based on PCR products designed to cover all genes of an organisms, for example a plant.

The method of the invention thus allows to take "mode of action research" to a different level of understanding namely from g eneral i nformation a bout the b iological p rocess w ith which the compound interferes to the precise elucidation of the molecular target of the agrochemical. For example, when it i s generally k nown that the mode of a ction of a candidate h erbicide i s to inhibit photosynthesis, than the method of the present invention allows the identification of the precise molecular target from a pool of photosynthesis related genes.

According to a more specific embodiment the present invention thus relates to any of the methods herein described, wherein said candidate target gene set is selected from at least one of the following:

(a) . a complete cDNA library of a selected organism;

(b) a cDNA library of all genes expressed in a specific tissue or part of said organism;

(c) a cDNA library of all genes involved in a specific biological pathway of said organism,;

(d) a cDNA library of all genes expressed in a cell, tissue or part of said organism under specific environmental condition, for example under stress conditions;

(e) a gene set corresponding to the genes of at least one functionally annotated gene database; and (f) a gene set selected from a functionally annotated gene database, wherein said selection is based on a common functional feature of the encoded polypeptide.

In a preferred embodiment of the invention, the candidate target genes are involved in a specific biological pathway. Preferably the specific biological process is an essential biological process, such the cell cycle, preferably the target gene is an essential gene for the cell, such as a cell cycle gene. Alternatively, the target genes are involved in the biosynthesis of a particular cellular compound, such as a particular amino acid or are involved in certain signalling pathways. Further preferably, the candidate target gene is involved in a plant specific pathway, such as the biosynthesis of a plant specific compound, or photosynthesis. Further preferably, the selected (candidate) target genes are plant specific target genes. Plant specific processes, involving plant specific proteins, occur for example during mitosis of the cell cycle or karyo-and cytokinesis and formation of the cell wall or cytoskeleton. One way of performing the methods of the invention is to prepare a full-length and normalized cDNA library from a cell, tissue or o rganism, sequence a n umber of clones that is likely to cover the large majority of expressed genes (e.g. 20,000) and to then select a full length clone of each gene (e.g. a few 1000's) for transformation of the library. The invention thus further relates to any of the herein described methods wherein said cDNA library is a normalised full-length cDNA library.

With a "normalised cDNA library" is meant a cDNA library in which the high variation in abundance among the clones of the cDNA library that represent individual mRNA species is reduced (Sankhavaram et al., 1991, Proc Natl. Acad. Sci. USA 88: 1943-1947; Soares et al., 1994; Proc. Natl. Acad. Sci. USA, 91 : 9228-9232). The normalization of a cDNA library can be checked by the content of a control gene for example the redundancy of an abundant housekeeping gene such as actin). For example: when a cDNA library is prepared and normalized using standard protocols it may be checked by Southern blot analysis if the prepared batch of cDNA displays the same discrete band when hybridised with a β-actin cDNA probe (Sankhavaram et al., 1991).

The invention further relates to any of the methods of the invention wherein overexpression of the (candidate) target gene is achieved by introducing said gene in an expressible format in the target individual. This can be achieved for example by operably linking the (candidate) target gene to regulatory elements allowing expression of the gene in the target individual and transforming that genetic construct into the target organism. Optionally the organism is stably transformed by stably integrating the target gene with or without said regulatory elements into the genome of said individual.

Overexpression can be achieved by operably linking the transgene to a promoters (e.g. a strong promoter) and transforming into a host cell. Alternatively and/or additionally, the transgene or e ndogenous gen can be o perably linked to a transcription a nd/or translational enhancer.

The gene that is to be overexpressed can be the endogenous gene or transgene.

The term "expressible form" should be understood as containing the control or regulatory sequences or elements needed for expression. The expression "regulatory element" refers to regulatory nucleic acid sequences which are necessary to effect the expression of sequences to which they are l igated. The regulatory elements differ depending upon the intended host organism and upon the nature of the sequence to be expressed. For expression of a protein, in prokaryotes, the regulatory elements generally include a promoter, a ribosomal binding site, and a terminator. In eukaryotes, regulatory elements generally include promoters, terminators and, in some instances, enhancers, introns, and/or 5' and 3' untranslated sequences. The term "regulatory elements" is intended to include, at a minimum, all components necessary for expression, and may also include additional advantageous components.

One way of expression the (candidate) agrochemical target gene according to the invention relates to "overexpression" of a gene or a protein which refers to expression patterns and/or expression levels of the gene or protein normally not occurring under natural conditions. Overexpression can be achieved in a n umber of ways including o perably l inking the target gene to a homologous (from the same originating source) or heterologous (from a different origination source) promoter, preferably a strong promoter, in order to reach high expression levels. Alternatively, it is possible to achieve overexpression by building a chimeric gene and/or operably linking said coding sequence to its own isolated promoter (i.e. the unisolated promoter naturally driving expression of said protein) in order to crease a recombinant gene duplication o r g ene multiplication effect. P referred promoters to use i n the methods of the present invention are strong constitutive promoters and/or strong ubiquitous promoters, for example CaMV35S promoter, GOS2, Actin 2 promoter, ubiquitin promoter etc... Alternatively, overexpression can be achieved by using translational and/ort transcriptional enhancers. In all the methods described above the (candidate) agrochemical target gene to be overexpressed, can be a transgene or can be the endogenous gene of the target organism. In the latter case, the control elements of the endogenous gene can be modulated or replaced to reach higher expression levels, or the endogenous gene can be multiplicated.

According to the present invention, the concentration of the agrochemical or candidate agrochemical as applied in any of the methods herein described, will be defined in relation to its capacity to kill a certain percentage of the non-protected individuals. That concentration is described as the lethal concentration (LC), for example a concentration of the agrochemical ofLC80 and LC99.9 is expected to kill between 80% and 99.9% of the non protected individuals, which are the non-transformed individuals or transformed individuals which do not overexpress the true m olecular target of the agrochemical. Preferably the agrochemical as used in the methods of the present invention is applied at a lethal concentration of LC80, LC85, LC90, LC95, LC99, LC99.5, LC99.9, which is expected to kill 80%, 85%, 90%, 95%, 99%, 99.5%, 99.9% or 99.99%, respectively, of the non-protected individuals.

The LC value is thus a parameter which depends on different factors such as the type of agrochemical applied, but also the type of target individual used. Therefore, the mode of action screen will be developed for each of the selected target species, for example a plant species. Practically, in a first step prior to the screening method of the present invention, for example the LC99.9 is determined for the applied agrochemical in combination with the used target species, as well as the percentage of surviving cells or organisms at 0.1 , 1, 1.5, 2, 5, 10 and 100 times this doses. Than the doses of for example LC 99.9 is applied in the screening method of the present invention, as well as 0.1 , 1, 10 or.100 times this concentration.

The set of overexpressor l ines (for instance around 1 00 plants per l ine, i.e. 100 p lants p er (candidate) target gene) is sown and treated with the various concentrations of the agrochemical or test agrochemical.

With "set of overexpressor lines" as used herein also falls under the meaning of the "population of individuals", since each of the "lines" is overexpressing a single gene which differs between different "lines".

Genes, of whom the overexpression in the target organism resultis in differential tolerance to the agrochemical compound, are identified and are prime candidate target genes for the tested agrochemical compound. Therefore, according to the invention, if in any of the methods of the invention, the growth of an individual or an overexpressor line is positively affected or influenced, the gene or gene product overexpressed is regarded as being the target of said agrochemical compound tested.

A review of the nature of the identified genes in combination with possible additional (biological or biochemical) studies (to be defined on a case by case basis) may be required for final confirmation of the results.

The knowledge of the target gene of the agrochemical and the way it protects the target organism against the applied agrochemical, will allow the person skilled in the art to further design and develop a target organism with the desired phenotypic features, for instance enhanced resistance to the agrochemical. The further development of herbicide tolerant crops may allow the use of powerful herbicides that normally exhibit unfavorable crop phytotoxicity. These powerful herbicides would than also be useful for killing the unwanted weeds without damaging the crop plants.

The methods of the present invention can also be used as a screening assay to identify agrochemical compounds with pesticidal potency. Accordingly, in another embodiment, the present invention relates to the use of a molecular target gene of an agrochemical, identifiable by any of the methods of the invention, as a molecular target for identifying a herbicidal, insecticidal, nematicidal or fungicidal compound.

The use of herbicides to control the growth of unwanted weeds in the fields is common practice by farmers all over the world. However, problems occur with weeds that become resistant against the used herbicide. Therefore the use of herbicides needs to be efficiently managed and t here must be s ufficient innovation to overcome these particular problems of newly developed resistance to the agrochemical. One solution is to find a new agrochemical. Another solution is to o ptimize the efficiency of t he existing h erbicide. The methods of the present invention, providing information of the molecular target of the agrochemical and therefore constitutes and important source of information to optimize the efficiency and to test the efficiency of an existing or an improved version of an existing herbicide. To that purpose the pool of target genes that is chosen for the methods of the present invention, may be variants of an existing target gene and the agrochemical may be a variant of an existing agrochemical. Such variants of an existing target gene may be allelic variants, splice variants, substitution or deletion variants, mutants. Such variants of an existing agrochemical may also be chemical or biochemical modifications, or in case the agrochemical is a protein, also genetic modifications as described for the target gene. Furthermore since a lot of pesticides are known to work suboptimally, the present invention, by providing knowledge of the specific molecular target of a specific agrochemical compound, allows optimisation of the target's activity towards the compound or optimisation of the agrochemical's activity towards its target, for example by increasing the binding capacity to the target molecule. The invention thus relates to any of the methods described above further comprising modulating the activity between said agrochemical and its molecular target identifiable by any of said methods. In a further embodiment of the invention, said modulation results in an enhanced or decreased binding between s aid agrochemical or candidate agrochemical compound and its molecular target.

The expression "modulating the activity between" relates to changing the activity in a positive or negative sense, or altering the affinity between the compound and the target in a positive or negative sense, for instance by enhancing or decreasing the binding or activity or sensitivity of the compound towards its target, or by enhancing or decreasing the binding or activity or sensitivity of the target towards the compound. For instance, this can be done by modulating the compound and/or target molecules themselves, for example by modulating the binding pocket or binding site of the target molecule or the pesticide (for instance by site directed mutagenesis of the target molecule or by chemical modification of the pesticide). The present invention thus also relates to such methods wherein the sensitivity ot a molecular target tor a specific pesticide is modulated.

The expression "modulating the sensitivity of a molecular target for a specific pesticide" when used herein can have multiple functions and/or applications. In one way the former expression may mean, "increase the sensitivity" hereby resulting in a more sensitive target. In another way the expression may mean, "reducing the sensitivity" hereby resulting in more resistant target. According to a further embodiment of the i nvention, i n any of the methods of the invention where enhanced tolerance of the organisms to a certain agrochemical compound needs to be measured, this measurement is achieved by assaying cell viability or organism viability. The viability assayed on a visual basis or alternatively is measured with techniques well known in the art, such as for instance by vital staining, cell counting, light scattering or reporter gene expression (e.g. GFP expression).

In a further embodiment, the invention relates to a m olecular target of an agrochemical or candidate agrochemical identifiable by any of the methods described herein.

The invention further relates to a composition comprising a compound or a compound identifiable as a pesticidal agrochemical by any of the methods described herein. The invention further relates to a method for the production of a herbicide, insecticide, nematicide or fungicide composition comprising formulating a compound or a composition as defined above in a suitable form for the application in agriculture, or in plant cell or tissue culture. According to another embodiment, the invention also relates to a method for identifying a gene or a gene product that is involved in an essential cellular process, comprising identifying a gene or gene product by at least one of the methods described above and determining the polypeptide from the sequence of the gene.

According to another embodiment, the invention also relates to a method for identifying the target gene of an agrochemical which is a growth enhancer or candidate growth enhancer comprising identifying a g ene p roduct by at least one of the methods described e arlier and determining the polypeptide from the sequence of the gene.

According to yet another embodiment, the invention relates to an individual organism identifiable during any of the methods described herein wherein said individual organism exhibits an enhanced tolerance to said agrochemical or candidate agrochemical compound by overexpressing a gene encoding said molecular target. The invention further relates to a method for the production of a transgenic organism comprising overexpressing a gene encoding a molecular target identifiable by any of the methods described earlier. In said method, the organism is chosen from a microorganism, a prokaryote, a eukaryote, a yeast, a fungus, a plant, a mammal or a human.

The invention also relates to the use of an organism obtainable by said methods in agriculture. Those skilled in the art will be aware that the invention described herein is subject to variations and modifications other than those specifically described. It is to be understood that the invention described herein includes all such variations and modifications. The invention also includes all such steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any or more of said steps or features.

The invention, now being generally described, will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present invention and are not intended to limit the invention.

Description of the figures Figure 1: Schematic representation of the principle of "Mode of Action determination" according to the present invention

Examples

Example 1. Elucidation of the mode of action of candidate herbicidal compounds A given candidate herbicide is known to kill different species of plants including dicotyledonous plants such as Arabidopsis thaliana. The candidate herbicide is known to act as a post- emergence herbicide, when a pplied on the leaves. The action of the candidate h erbicide is rapid bleaching and this effect is also observed on isolated leaf discs. In this particular case Arabidopsis thaliana is selected as the target organism of choice for identifying mode of action since it is one of the plants most amenable to large scale transformation.

A normalized full-length c-DNA library is used as the source of the candidate target gene set, since it can be deduced from experimental data that the target must be present in the leaves. A sufficient number of clones from the library (e.g. 30,000) is sequenced and a full length clone of each different gene (eliminating redundancy and arriving at e.g. 2000) is cloned behind a strong constitutive promoter such as pssu in a suitable plant expression cassette. Alternatively p35S or other well known constitutive promoters can be used.

The Arabidopsis plants are then transformed with this cDNA library via flower dip transformation.

For each gene bulked seed are harvested on 20 primary transformants and stored. In a preferred way to practice the invention individual transformants are not further characterized, but a sufficient number of different individuals is taken to achieve a very high probability of making true overexpressors part of the population. Alternatively it is conceivable, particularly when the candidate target gene set is rather small, to characterize the overexpressors for their level of transcript, and to compose a smaller population of characterized individuals. For each of the 2000 genes, 100 seeds are taken from the bulked seed harvested on the primary tranformants, and all seeds are sown in large trays. The 200,000 seedlings are than sprayed with a concentration of the compound that is expected to kill e.g. 99 % of the non- transformed plants (= LC 99).

A likely target gene is a gene for which several plantlets (e.g. m ore than one) survive the treatment.

One can vary the concentration used from e.g. LC 95 to LC 99,9 or even from LC 80 to LC 99,99, or different populations can be sown and treated with different concentrations chosen from the above-mentioned range.

Further d ose response studies o n the surviving plants p rogeny I eads to confirmation of the importance of the gene thus identified as a molecular target for the candidate herbicide

Claims

1. A method to identify the molecular target of an agrochemical or candidate agrochemical compound comprising the steps of:

(a) providing a population of individuals, wherein said individuals each overexpress the same or a different gene to other said individual, selected from a candidate target gene set,

(b) applying to said population of individuals said agrochemical or candidate agrochemical compound in a concentration that affects or kills a large majority of individuals of a non-transgenic population, and (c) identifying individuals exhibiting enhanced tolerance to said agrochemical or candidate agrochemical compound.

2. A m ethod according to claim 1 wherein said population of individuals is chosen from a population of cells, tissues or parts of an organism or an organism.

3. A method according to claim 2 wherein said organism is chosen from a microorganism, a prokaryote, an eukaryote, a yeast, a fungus, an insect, a plant, a mammal or a human.

4. A method according to any of claims 1 to 3 wherein said concentration of -said agrochemical or candidate agrochemical compound ranges between LC 80 and LC 99.9, wherein LC80 and LC99.9 are defined as t he lethal concentrations to k ill 80% (LC80) and 99.9% (LC99.9), respectively of said individuals.

5. A method according to any of claims 1 to 4 wherein said candidate target gene set is selected from at least one of the following:

(a) a complete cDNA library of the genome of a selected organism,

(b) a cDNA library of all genes expressed in a specific tissue or part of said organism, (c) a cDNA library of all genes expressed under certain developmental stages

(d) a cDNA library of all genes expressed in a specific biological pathway of said organism,

(e) a cDNA library of all genes involved in a cell, tissue or part of said organism under specific environmental conditions or stress conditions, (f) a gene set corresponding to the genes of at least one functionally annotated gene database, and

(g) a gene set selected from a functionally annotated gene database, wherein said selection is based on a common functional feature of the encoded polypeptides.

6. A method according to claim 5 wherein said cDNA library is a normalized full length cDNA library.

7. A method according to any of claims 1 to 6 wherein overexpression of said gene is achieved by introducing said gene in an expressible format into said organism.

8. A method according to any of claims 1 to 7 wherein enhanced tolerance ot the organism to the agrochemical compound is measured by assaying cell or organism viability.

9. A method according to claim 8 wherein cell or organism viability is measured by cell or organism growth.

10. A method according to claim 9 wherein, if the growth of an individual of said population is positively affected, the gene or gene product overexpressed in said individual is identified as the target of said agrochemical or candidate agrochemical compound.

11. A molecular target for an agrochemical or candidate agrochemical identifiable by any of the methods of claims 1 to 10.

12. Use of a molecular target as defined in claim 11 for identifying a herbicidal, insecticidal, nematicidal or fungicidal compound.

13. A composition comprising a compound or a compound which acts on a molecular target identifiable by any of the methods of claims 1 to 10.

14. Method according to any of claims 1 to 10 further comprising modulating the activity between said agrochemical or candidate agrochemical compound and its molecular target identifiable by any of the methods of any of claims 1 to 10.

15. Method according to claim 14 wherein said modulation results in an enhanced binding between said agrochemical or candidate agrochemical compound and its molecular target.

16. Method for the production of a herbicide, insecticide, nematicide or fungicide composition comprising formulating a compound or a composition as defined in claim 13 in a suitable form for the application in agriculture, or in plant cell or tissue culture.

17. Method for i dentifying a g ene or a g ene p roduct that is involved i n an essential cellular process, comprising identifying a gene or gene product by at least one of the methods described earlier and determining the polypeptide from the sequence of the gene.

18. Method for identifying the target gene of an agrochemical which is a growth enhancer or candidate growth enhancer comprising identifying a gene product by at least one of the methods described earlier and determining the polypeptide from the sequence of the gene.

19. Individual identifiable by any of the methods of claims 1 to 10 wherein said individual exhibits an enhanced tolerance to said agrochemical or candidate agrochemical compound by overexpressing a gene encoding said molecular target.

20. Method for the production of a transgenic organism comprising overexpressing a gene encoding a molecular target identifiable by any of the methods of claims 1 to 10.

21. Method according to claim 20 wherein said organism is chosen from a microorganism, a prokaryote, an eukaryote, a yeast, a fungus, an insect, a plant, a mammal or a human.

22. Use of an organism obtainable by the method of claim 20 or 21 in agriculture.