Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
  • C12N15/8237—Externally regulated expression systems
  • C12N15/8238—Externally regulated expression systems chemically inducible, e.g. tetracycline
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
  • A01N37/02—Saturated carboxylic acids or thio analogues thereof; Derivatives thereof
  • A01N37/04—Saturated carboxylic acids or thio analogues thereof; Derivatives thereof polybasic
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
  • A01N37/36—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a singly bound oxygen or sulfur atom attached to the same carbon skeleton, this oxygen or sulfur atom not being a member of a carboxylic group or of a thio analogue, or of a derivative thereof, e.g. hydroxy-carboxylic acids
  • A01N37/38—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a singly bound oxygen or sulfur atom attached to the same carbon skeleton, this oxygen or sulfur atom not being a member of a carboxylic group or of a thio analogue, or of a derivative thereof, e.g. hydroxy-carboxylic acids having at least one oxygen or sulfur atom attached to an aromatic ring system
  • A01N37/40—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a singly bound oxygen or sulfur atom attached to the same carbon skeleton, this oxygen or sulfur atom not being a member of a carboxylic group or of a thio analogue, or of a derivative thereof, e.g. hydroxy-carboxylic acids having at least one oxygen or sulfur atom attached to an aromatic ring system having at least one carboxylic group or a thio analogue, or a derivative thereof, and one oxygen or sulfur atom attached to the same aromatic ring system
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
  • A01N37/42—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing within the same carbon skeleton a carboxylic group or a thio analogue, or a derivative thereof, and a carbon atom having only two bonds to hetero atoms with at the most one bond to halogen, e.g. keto-carboxylic acids
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N61/00—Biocides, pest repellants or attractants, or plant growth regulators containing substances of unknown or undetermined composition, e.g. substances characterised only by the mode of action

Definitions

• the present invention relates to an expression system for use in plants, in particular to an expression system which utilises an exogenous chemical agent as a control mechanism and to the use of certain chemicals as said control agent.
• promoter Gene expression is controlled by regions upstream (5') of the protein encoding region, commonly referred to as the "promoter".
• a promoter may be constitutive, tissue-specific, developmentally-programmed or inducible.
• Manipulation of crop plants to improve characteristics requires the expression of foreign or endogenous genes in plant tissues.
• Such genetic manipulation therefore relies on the availability of means to control gene expression as required; for example, on the availability and use of suitable promoters which are effective in plants. It is advantageous to have the choice of a variety of different promoters so that the most suitable promoter may be selected for a particular gene, construct, cell, tissue, plant or environment. A range of promoters are known to be operative in plants.
• promoters which are inducible by the application of an exogenous chemical inducer This allows particular gene expression to be controlled at particular stages of plant growth. or development, by the presence or absence of a chemical which can applied to the plants or seeds, for example by spraying or using known seed coating techniques. This is sometimes known as a gene "switch".
• the gene which is under the control of the inducible promoter may be the gene which gives rise to the desired characteristic or phenotype itself, or the inducible promoter may control expression of a repressor protein which inhibits expression of a target gene, for example by interacting with an operator sequence upstream of the target gene so as to prevent expression of the gene (for example as known in the bacterial tet and lac operator/repressor systems).
• the gene under the control of the inducible promoter may express a protein which interacts with another protein to inhibit the activity thereof, as for example in the barnase/T-iarstar system which barnase will inhibit or kill cells in the absence of barstar. Gene switches of this type are known in a wide variety of applications.
• promoters include the production of reversible male sterility, a feature which is highly desirable in hybrid plant production as described for instance in WO 90/08830.
• Other applications of such promoters include in germplasm protection, where containment of particular crop plants, in particular transgenic plants, and the control of volunteers is necessary and also in the prevention of pre-harvesting sprouting as described in WO 94/03619.
• Many organisms have mechanisms which allow them to metabolise chemicals such as alcohols or ketones, for example by the production of alcohol dehydrogenase enzymes.
• the promoters of these systems may be useful in gene switches as the promoters may be inducible by the presence of the target alcohol or ketone.
• ADHl alcohol dehydrogenase I
• ale A the enzyme alcohol dehydrogenase I
• ale A the enzyme alcohol dehydrogenase I
• the induction is relayed through a regulator protein encoded by the alcR gene and constitutively expressed.
• inducer alcohol or ketone
• the regulator protein activates the expression of the alcA gene.
• the regulator protein also stimulates expression of itself in the presence of inducer. This means that high levels of the ADHl enzyme are produced under inducing conditions (ie when alcohol or ketone are present).
• the ale A gene and its product, ADHl are not expressed in the absence of inducer. Expression of alcA and production of the enzyme is also repressed in the presence of glucose.
• the alcA gene promoter is an inducible promoter, activated by the alcR regulator protein in the presence of inducer (ie by the protein/alcohol or protein/ketone combination).
• inducer ie by the protein/alcohol or protein/ketone combination.
• the alcR and alcA genes have been cloned and sequenced (Lockington RA et al, 1985, Gene, 33:137-149; Felenbok B et al, 1988, Gene, 73:385-396; Gwynne et al, 1987, Gene, 51 :205-216).
• Alcohol dehydrogenase (adh) genes have been investigated in certain plant species. In maize and other cereals they are switched on by anaerobic conditions. The promoter region of adh genes from maize contains a 300 bp regulatory element necessary for expression under anaerobic conditions. However, no equivalent to the alcR regulator protein has been found in any plant. Hence the alcR/alcA type of gene regulator system is not known in plants. Constitutive expression of alcR in plant cells does not result in the activation of endogenous adh activity.
• WO 93/21334 describes the production of transgenic plants which include such as system as a gene switch.
• This document specifically describes a chemically-inducible plant gene expression cassette comprising a first promoter operatively linked to a regulator sequence which encodes a regulator protein, and an inducible promoter operatively linked to a target gene, the inducible promoter being activated by the regulator protein in the presence of an effective exogenous inducer whereby application of the inducer causes expression of the target gene.
• the alcR/alcA system is utilised in the constructs.
• Exogenous chemical inducers which are applied in this case include those described by Creaser et al., J. Biochem.
• alcohols are generally used as the exogenous chemical inducer.
• such chemicals are often volatile and therefore difficult to handle in an agricultural context, as large volumes of chemical may be lost during spraying.
• the present invention provides the use of an agriculturally acceptable hydrolysable ester in the control of expression of a plant gene, said control being effected by an inducible promoter which requires for activation, the presence of an exogenous chemical which may comprise an alcohol, wherein hydrolysis of said agriculturally acceptable ester results in the production of said alcohol.
• the agriculturally acceptable ester comprises a compound of formula (I)
• R O (I) in which R 1 is a lower alkyl, lower alkenyl or lower alkynyl group, and R 2 is a organic group such that R 2 COOH is an agriculturally acceptable acid. Hydrolysis of a compound of formula (I) yields an alcohol of formula (II)
• lower alkyl as used herein includes C,. 6 alkyl groups, preferably from C alkyl groups which may be straight or branched chain.
• lower alkenyl and lower alkynyl as used herein includes C 2 . 6 alkenyl and C 2 . 6 alkynyl groups respectively, preferably from C 2 ⁇ alkenyl or C 2 . 4 alkynyl groups which may be straight or branched chain.
• Agriculturally acceptable esters for use in the invention such as those of formula (I) are suitably translocated into the target plant in which the gene control system is in place and/or hydrolysed either under enviromental conditions or in the presence of a suitable catalytic moiety such as an enzyme or catalytic antibody, at rates which are appropriate to provide sufficient quantities of the activating alcohol at the required time in the necessary parts of the plant. These may vary depending upon the nature of the plant species being treated, the gene being expressed and the timing of the application of the ester.
• Esters such as the compounds of formula (I) are advantageous in that they are easier to handle than the corresponding alcohols. It has been found that these compounds can produce the desired effect in terms of gene activation.
• the compound should be applied at a sufficient period of time prior to the required gene activation to allow hydrolysis to occur and this should be reasonable depending upon factors such as the growth stage of the plant at which activation is required. If the rate of hydrolysis is relatively slow, the time of application may be earlier in order to ensure that sufficient hydrolysis has occurred by the time the plant is at the growth stage at which gene activation is required. Where this is difficult, more rapidly hydrolysing esters may be selected.
• more than one ester, with differing rates of hydrolysis may be applied in a single treatment.
• an effective "slow release" of activating alcohol can be achieved so that gene expression may be prolonged over the desired period. This means that repeated applications of chemical may be avoided and "one-shot” treatments are possible.
• Particular examples of alcohols of formula (II) include methanol, ethanol, propan-1- ol, propan-2-ol, butan-2-ol or but-3-en-2-ol.
• the alcohol of formula (II) is a lower alkyl alcohol wherein the alkyl group has from 1 to 4 carbon atoms and may be either branched, or linear.
• Preferred groups for R 1 include ethyl, n-propyl and n-butyl.
• a particularly preferred example of a compound of formula (II) is ethanol.
• the precise nature of the R 2 group is immaterial provided that it gives rise to an agriculturally acceptable acid at an appropriate rate in the particular target plant to which it is applied. Rates of hydrolysis can be determined using routine methods for example as described by G. Mitchell et al., Pestic. Sci (1995) 44:49-58. and preferably by testing against whole plant systems. What is appropriate in any particular instance will depend upon a variety of factors including the nature of the gene expression of which is being controlled, the particular plant in which the gene is expressed and other external conditions. The rate of hydrolysis should be sufficient to allow the desired effect, for example, reversible male sterility, to be seen at an appropriate period of time after application of the chemical inducer. R 2 however may be selected such that the resultant acid of formula (III)
• K ⁇ (III) has some useful agrochemical effect.
• it may itself able to act as an inducer of the inducible promoter.
• acids including 3- hydroxybutyric acid, 2-hydroxybutyric acid, pyruvic acid and 3-oxobutyric acid can act as an inducer of the alcR alcA system (Creaser et al., supra.).
• R 2 include optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl or optionally substituted heterocyclyl.
• alkyl includes straight or branched alkyl chains, suitably containing up to 10 carbon atoms, preferably from 1 to 6 carbon atoms.
• alkenyl and alkynyl includes unsaturated straight or branched chains containing up to 10 carbon atoms, preferably from 2 to 6 carbon atoms.
• aryl includes phenyl and naphthyl.
• heterocyclic includes rings containing up to 10, preferably up to 7 atoms, up to three of which are selected from oxygen, sulphur or nitrogen. These rings may be single rings or may be in the form of fused ring systems and these may be aromatic or non-aromatic in nature.
• halo or halogen includes chlorine, fluorine, bromine and iodine.
• alkoxy relates to an alkyl group as defined above, linked with an oxygen atom.
• R 2 is an optionally substituted C 0 alkyl group which may be linear or branched. Preferred alkyl groups R 2 are linear and contain 3 to 8 carbon atoms, in particular 5 carbon atoms.
• Suitable optional substituents for hydrocarbyl groups R", R 12 or R 13 include halo, perhaloalkyl such as trifluoromethyl, mercapto, hydroxy, alkoxy, oxo, heteroaryloxy, alkenyloxy, alkynyloxy, alkoxyalkoxy, aryloxy (where the aryl group may be substituted by halo, nitro, or hydroxy), cyano, nitro, amino, mono- or di-alkyl amino, alkylamido or S(O) p R 14 where m and R 14 are as defined above.
• R 2 are one or more groups selected from oxo; alkoxycarbonyl in particular lower alkoxycarbonyl; cyano; halo such as chloro, fluoro or bromo; phenyl optionally subsituted with amino or mono-or dialkyl amino or alkyl such as methyl; OR 3 where R 3 is alkyl or heterocyclyl optionally substituted by halo or alkyl; S(O) m R" where m is 0 or 2 and R 11 is alkyl or phenyl optionally substituted by alkyl; NR 5 R 6 or C(O)NR 5 R 6 where R 5 is hydrogen, methyl or methoxyethyl and R 6 is alkyl such as methyl, phenyl or benzyl optionally substituted with halo such as fluoro or chloro, alkyl such as methyl or trifiuromethyl or alkoxycarbonyl
• Preferred substituents include, but are not limited to lower alkoxycarbonyl groups and dialkyl acetals. Alkoxycarbonyl groups and dialkyl acetals are of particular interest when the alkyl group of the substituent is the same as R 1 in the compound of formula (I) since on hydrolysis these give rise to more inducer chemical of formula (II) .
• a particular aryl group for R 2 is phenyl.
• Suitable optional substituents for cycloalkyl, aryl and heterocyclyl groups R ⁇ and for aryl or heterocyclyl substituents on the above-mentioned alkyl, alkenyl or alkynyl groups R 2 include halo; haloalkyl; cyano; nitro; amino or mono- or di-alkyl amino; hydroxy; alkoxy, thioalkyl, alkyl or alkoxycarbonyl wherein the alkyl moiety of any of these may be optionally substituted with for example one or more groups selected from halo, alkoxy, cyano, alkoxycarbonyl, amino, mono- or di-alkyl amino, aryl or carboxylate or salts or esters thereof; cycloalkyl; or heterocyclyl.
• Particularly suitable substituents for aryl or heterocyclyl groups R 2 include alkoxy in particular lower alkoxy such as methoxy, alkyl in particular lower alkyl, alkoxycarbonyl in particular lower alkoxycarbonyl and halogen.
• a particular sub-group of compounds of formula (I) are compounds of formula (1A)
• R 1 is as defined above in relation to formula (I)
• n is an integer of from 2 to 4 and R 10 is an alkyl, alkenyl or alkynyl group any of which may be optionally interposed with a heteroatom, a cycloalkyl, heterocyclic group or aryl group, or R 10 is a cycloalkyl or aryl group of valency n.
• R 10 is an alkyl or aryl group of valency n.
• Particularly preferred compounds of formula (I) include :-
• Ethyl 2-n-pentyl-3-oxobutanoate (Compound No. 49); Triethyl 2-carboxyheptan-l,7-dioate (Compound No. 53); and Ethyl 2,4-dimethoxybenzoate ( Compound No. 60).
• Examples of compounds of formula (I) are ethyl esters as shown in Table 1. Table 1
• ompoun s o ormua I are et er nown compoun s o t ey can e prepare rom known compounds using conventional methods.
• Compounds of formula (I) may be hydro lysed in the target plant either chemically, or enzymatically by a naturally occurring enzyme in the target plant or by an enzyme introduced by genetic engineering into the plant and expressed within the plant, or by an appropriate catalytic antibody, or catalytically active portion of a catalytic antibody introduced by genetic engineering into the plant and expressed within the plant.
• Suitable enzymes include, but are not limited to, esterases and Upases.
• Suitable catalytic antibodies may be generated by standard techniques from analogues of a tetrahedral ester hydrolysis transition state, e.g. as for the hydrolysis of the pro-drug ester of chloramphenicol, when appropriate phosphonates were used, Ole K et al., 1998, J. Mol. Biol., 281 :501-511, and for the detoxification of cocaine by methyl ester hydrolysis, Mets B et al., 1998, Proc. Nat. Acad. Sci. USA, 95: 10176-10181.
• a product of this metabolism is ethanol which can act as a chemical inducer as described above.
• the invention provides a method for controlling expression of a target gene in a plant, wherein said plant is transformed with a chemically-inducible plant gene expression cassette comprising a first promoter operatively linked to a regulator sequence which encodes a regulator protein, and an inducible promoter operatively linked to a target gene, the inducible promoter being activated by the regulator protein in the presence of an alcohol such as a compound of formula (II) as defined above, said method comprising applying to said plant an ester which hydrolyses to form said alcohol such as a compound of formula (I) as defined above, so as to cause expression of the target gene.
• the regulator sequence encodes the alcR protein as described above and the inducible promoter is the alcA promoter sequence.
• the plant may also be transformed so that it expresses or overexpresses an enzyme or catalytic antibody or catalytically active fragment thereof, which hydrolyses the compound of formula (I) to form a compound of formula (II).
• Enzymes which are inactive in the absence of enzymes or other moieties which must be engineered into the plant may be preferable in some circumstances since they will be effective only in the target transformed seed.
• nucleic acid sequences which encode the hydrolytic enzyme, antibody or antibody fragment may be included in the construct containing the regulator protein and/or the target gene operatively linked to the inducible promoter or they may be present on a separate construct which is used to co-transform the plant. Such systems however are novel.
• an inducible promoter operatively linked to a target gene, the inducible promoter being activated by the regulator protein in the presence of an effective exogenous inducer of formula (I) as defined above, whereby application of the inducer causes expression of the target gene; and (iii) a sequence which encodes a protein which effects hydrolysis of an ester such as a compound of formula (I) to the corresponding alcohol under the control of a further promoter which allows its expression in pl.ant tissue.
• the target gene may comprise any gene which is required to be introduced into a plant in order to modify the characteristics thereof as outlined above.
• the target gene may be an endogenous plant gene or a foreign gene, and may be a single gene or a series of genes.
• the target gene sequence encodes at least part of a functional protein or an antisense sequence.
• Any transformation method suitable for the target plant or plant cells may be employed, including infection by Agrobacterium tumefaciens containing recombinant Ti plasmids, electroporation, microinjection of cells and protoplasts, microprojectile transformation and pollen tube transformation.
• the transformed cells may then in suitable cases be regenerated into whole plants in which the new nuclear material is stably incorporated into the genome. Both transformed monocot and dicot plants may be obtained in this way.
• Examples of genetically modified plants which may be produced include field crops, cereals, fruit and vegetables such as: canola, sunflower, tobacco, sugarbeet, cotton, soya, maize, wheat, barley, rice, sorghum, tomatoes, mangoes, peaches, apples, pears, strawberries, bananas, melons, potatoes, carrot, lettuce, cabbage, onion.
• the invention further provides a plant cell containing a gene expression system according to the invention.
• the gene expression system may be stably incorporated in the plant's genome by transformation.
• the invention also provides a plant tissue or a plant comprising such cells, and plants or seeds derived therefrom.
• Preferred examples of compounds of formula (I) used in this method are those described above.
• Agriculturally acceptable esters of the invention such as compounds of formula (I) are suitably applied in the form of an agriculturally acceptable composition, in combination with a diluent or carrier.
• Such compositions form a further aspect of the invention.
• the concentration of the agriculturally acceptable ester in the formulation is preferably at a concentration of about 5% wt/wt or less. It is preferably at a concentration between about 2% and 5% wt/wt.
• Suitable carriers or diluents will be apparent to the skilled person and will vary depending upon the particular nature of the compounds of formula (I) employed.
• the compound of formula (I) may require the presence of an emulsifier in order to allow it to be sprayed in aqueous solution.
• Emulsifiers are well known in the art, and a particular example is partially hydrolysed polyvinyl acetate (PVA) or TweenTM.
• a preferred composition comprises an agriculturally acceptable ester such as a compound of formula (I), an emulsifier such as PVA and a diluent such as water.
• an agriculturally acceptable ester such as a compound of formula (I)
• an emulsifier such as PVA
• a diluent such as water.
• the relative amounts of the components will be determined to a large extent by the mutual miscibility of the various components.
• the emulsifier will be present in the composition in amounts of from l-5%w/w, preferably at about 2.5%w/w.
• formulations of the invention include the following: Formulation 1 1.5% Compound of the invention (e.g. Compound 53) 2.5% PVA Balance water
• Formulation 2 1.5%) Compound of the invention (e.g. Compound 53) 5%) acetone 0.05% Tween-20TM Balance water
• compositions which may be employed are similar to those described in our copending British Patent application No. 9902236.0.
• the compositions will comprise the components:
• the diluent (c) may be, for example, water.
• Component (b) of the formulation described above is preferably, a polyethoxylated oleyl, lauryl, stearyl or cetyl alcohol. It is more preferably a polyoxyethylene-oleyl alcohol having a mean molar ethylene oxide content in the range of 0 to 35 and more preferably in the range of 2 to 20. It is most preferably a polyoxyethylene-(2)-oleyl alcohol, a polyoxyethylene-(10)-oleyl alcohol or a polyoxyethylene-(20)-oleyl alcohol.
• Component (b) is, however, preferably a polyoxyethylene-(20)-oleyl alcohol (the number in brackets indicates the mean ethylene oxide content per molecule).
• Such products are commercially available as BRIJ 92TM, BRIJ 97TM and BRIJ 98TM.
• component (b) of the formulation is at a concentration of about 0.5% wt/wt or less. It is preferably at a concentration between about 0.2% wt/'wt and 0.5% wt/wt.
• the formulation includes as component (b), a hydrogen or methyl end-capped trisiloxane polyethoxylate
• component (b) is a methyl end-capped trisiloxane polyethoxylate.
• the methyl end-capped trisiloxane polyethoxylate preferably has a mean molar ethylene oxide content of between 4 and 12 per molecule and is most preferably 8 per molecule.
• Such products are commercially available as SILWET 77TM (SILWET is a trademark of Witco).
• the methyl end-capped trisiloxane polyethoxylate is at a concentration of about 0.5%) wt wt or less. It is preferably at a concentration between about 0.2%> and 0.5%) wt/wt.
• Component (c) of the formulation is preferably at a concentration between about 90%) and 98%o wt/wt.
• additives which may be included in the formulations include dispersants, antibacterial compounds, wetter compounds and anti-evaporants may also be added.
• Leaf samples were removed at various time points and frozen in dry ice/ethanol and stored at -70°C. When all the samples were harvested and stored they were extracted in 200ul of 250mM Tris pH8.0, centrifuged and the supernatant recovered and stored at 4°C overnight. The samples were analysed for cat protein quantification by using a Boehringer Mannheim CAT Elisa kit and the total protein level determined by a Bradford determination. The compounds tested and results are shown on Tables 4, 5, 6, 7 8, 9 and 10. The CAT units are as set out above in Example 1 with each successive number representing an incremental 5,000ng/g range. Results shown in shaded form were obtained in separate but similar trials. Table 4

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