MXPA98002807A - Youth hormone or one of its agonists like a chemical ligand to control the genetic expression in plants by means of transactivation mediated by the recep - Google Patents

Abstract

The present invention relates to a method for controlling gene expression in plants. In a specific manner, the method comprises transforming a plant with an expression cassette of the USP receptor encoding a USP receptor, and at least one target expression cassette encoding a target polypeptide. The contact of this transformed plant with juvenile hormone or one of its agonists activates the expression of the target polypeptide in the presence of this USP receptor polypeptide. Optionally, additional "secondary" receptor expression cassettes may be used, wherein the secondary receptor expression cassette encodes a receptor polypeptide other than USP. The method is useful to control different traits of agronomic importance, such as plant fertility. A method for identifying ligands previously unknown to USP is also disclosed. The substances to be tested are identified by placing them in contact with cells of transformed plants with an expression cassette of the USP receptor and an objective expression cassette. The target expression cassette encodes a reporter polypeptide whose expression can be determined in a quantitative or qualitative manner, whereby the test substance is identified as a ligand for U.

Description

YOUTH HORMONE OR ONE OF ITS AGONISTS AS A LIGANDO CHEMISTRY TO CONTROL GENETIC EXPRESSION IN PLANTS THROUGH MEDIATED TRANSACTION BY THE RECEIVER The present invention relates to the chemical control of gene expression in plants. In particular, it relates to a method by which the juvenile hormone or one of its agonists is used, as a chemical ligand to regulate receptor-mediated expression of a target polypeptide in a plant cell, as well as to plant cells transgenic, to the plant material, or to the plants, and to the progeny thereof containing appropriate expression cassettes. In some cases, it is desirable to control the timing or degree of expression of a phenotypic trait in plants, plant cells, or plant tissues. An ideal situation would be the regulation of the expression of that trait to taste, triggered by a chemical that can be easily applied to field crops, ornamental shrubs, and so on. A system of regulation of the genetic expression that could be used to achieve this ideal situation, as it is still unknown that it is naturally present in plants, is the superfamily of nuclear receptors of thyroid hormone and steroids. The superfamily of thyroid hormone and steroid hormone receptors is found in mammals and insects, and is composed of more than 100 known proteins. Some of the receptors within this superfamily found in mammals are the Retinoic Acid Receptor (RAR), the Vitamin D Receptor (VDR), the Thyroid Hormone Receptor (T3R), and the Retinoic X Receptor (RXR). These and other receptors of the superfamily are bound to the 5 'regulatory region of the target gene, and by attaching a chemical ligand to the receptor, they transactivate the expression of the target gene. In addition to the receptors found in mammals, as described above, receptors of similar structure and activity have been identified in the insect Drosophila. Koelle et al., Cell 67: 59 (1991); Christianson and Kafatos, Biochem. Biophys. Res. Comm. 193: 1318 (1993); Henrich et al., Nucleic Acids Res. 18: 4143 (1990). The Ecdysone Receptor (EcR) fixes the insect steroid hormone 20-hydroxyecdysone, and when it is heterodimerized with the Ultraspiracle gene product (USP), it transactivates gene expression. Additional chemical ligands in addition to 20-hydroxyecdysone, such as hormone agonists, will also bind to EcR under similar conditions, and will cause the transactivation of a target gene. It has also been shown that USP is a member of the nuclear superfamily of thyroid and steroid receptors, although it is considered an "orphan" receptor, since its ligand has not been identified (Seagraves, Cell 67: 225-228 (1991 )). USP is related in sequence to RXRa (Gold et al., Nature, 347: 298-301 (1990)), and RXR is capable of forming heterodimers with EcR (Thomas et al., Nature 362: 471-475 ( 1993)). It has been shown that methoprene and its methoprene acid derivative, which are juvenile hormone agonists, transcriptionally activate a recombinant reporter gene in both insect and mammalian cells, acting through the RXRa (Harmon et al., Proc. Nati. Acad. Sci. 92: 6157-6160 (1995)). However, the juvenile hormone does not induce transactivation mediated by RXRa (Harmon et al.). To date, there has been no definitive evidence of a juvenile nuclear hormone receptor (Harmon et al., Henrich and Brown, Insect Biochem.Molec. Biol. 25: 881-897 (1995)), although it has been suggested that the USP of the orphan receptor can be a candidate (Harmon et al., see also Seagraves; Gold et al., Current Opinion in Genetics and Development 2: 269-274 (1992)). Juvenile hormone and its agonists offer previously unrecognized opportunities for chemical control of gene expression in plants, since these chemicals are already known for their agricultural use. What has been missing to date, is a means by which these chemical products can be used to induce the transactivation of a target gene in a transgenic plant. In the present, it has been shown that the juvenile hormone and its agonists are ligands for the USP of the orphan receptor. This discovery allows the implementation of gene control strategies for plants, which uses a nuclear receptor that does not occur naturally in plants. This means that the only effect of the application of the juvenile hormone or one of its agonists will be to induce the expression of a genetically designed target gene. As demonstrated in the present invention, USP receptor polypeptides have been developed, and the genes expressible in plants encoding them, which function in plant cells to control the expression of a target polypeptide, wherein the USP receptor polypeptide activates the 5 'regulatory region of a target expression cassette in the presence of the juvenile hormone or one of its agonists. This method to control the genetic expression in plants is useful to control different traits of agronomic importance, such as the fertility of the plant. The present invention relates to a method for controlling gene expression in plants. Specifically, the method comprises transforming a plant with a cassette of expression of the USP receptor encoding a USP receptor polypeptide, with at least one target expression cassette encoding a target polypeptide, and optionally with a secondary receptor expression cassette encoding a secondary receptor polypeptide other than the USP receptor polypeptide. The contact of the transformed plant with the juvenile hormone or one of its agonists, activates or inhibits the expression of the target polypeptide in the presence of the USP receptor polypeptide. Optionally, additional "secondary" receptor expression cassettes may be used, wherein the secondary receptor expression cassette encodes a receptor polypeptide other than USP. The method is useful to control different traits of agronomic importance, such as the fertility of the plant. The invention further relates to transgenic plants comprising an expression cassette of the USP receptor and an objective expression cassette capable of being activated by the juvenile hormone or one of its agonists. The invention also encompasses a method for identifying ligands previously unknown to USP, which are effective in an environment of plant cells. The substances to be tested are identified by placing them in contact with transformed plant cells with a cassette of USP receptor expression and an objective expression cassette. The target expression cassette encodes a reporter polypeptide whose expression can be determined in a quantitative or qualitative manner, wherein the test substance is identified as a ligand for USP.

Figure 1 gives a pictorial representation of a plant cell comprising an expression cassette of the VP16-USP receptor, and a cassette of i or target expression, with a direct repeat response element present in the 5 'regulatory region. In the presence of the juvenile hormone or one of its agonists, the VP16-USP receptor activates the expression of the target polypeptide. Figure 2 gives a pictorial representation of a plant cell comprising both the expression cassette of the USP-VP16 receptor and GAL4-EcR. To the When exposed to the juvenile hormone or one of its agonists, the activation of the expression of the target polypeptide caused by the combination of the GAL4-EcR and USP-VP16 receptor polypeptides is reversed. Figure 3 corresponds to Figure 2, with the exception that the chemical ligand tebufenozide (also known as RH 5992) is present. In the presence of the juvenile hormone or one of its agonists, activation is also reversed under these circumstances.

"Juvenile hormone" refers to a class of chemical compounds that are produced by insects. The juvenile hormone controls the larval metamorphosis causing the retention of the juvenile characteristics of the insect, and the consequent prevention of maturation. The action of the juvenile hormone is biologically manifested as behavioral, biochemical, or molecular effects. Several naturally occurring juvenile hormones have been isolated and characterized. Juvenile hormone agonists refer to a class of compounds that exhibit one or more of the biological activities of the juvenile hormone. The juvenile hormone agonists may or may not be structural analogues of the juvenile hormone. Also included in this disclosure are compounds that may be metabolic precursors of the compound that directly produces the aforementioned biological effects of the juvenile hormone type. For example, methoprene is a metabolic precursor of methoprene acid, which in turn directly produces the biological effects of the juvenile hormone type observed upon methoprene application. "Receptor polypeptide", as used herein, refers to polypeptides that can activate or inhibit the expression of an objective polypeptide in response to an applied chemical ligand. The receptor polypeptide is composed of a ligand binding domain, a DNA binding domain and a transactivation domain. The binding domain of the ligand comprises a sequence of amino acids whose structure binds in a non-covalent manner, a complementary chemical ligand. Accordingly, a ligand binding domain and its chemical ligand form a complementary binding pair. The DNA binding domain comprises an amino acid sequence that binds in a non-covalent manner, a specific nucleotide sequence known as a response element (RE). One or more response elements are located in the regulatory region 5 'of the target expression cassette. Each response element comprises a pair of media-sites, each half-site having a core of 5 to 6 base pairs, wherein a single DNA binding domain recognizes a single half-site. The media sites can be configured in a relatively linear orientation with respect to each other, such as direct repeats, palindromic repeats, or inverted repeats. The nucleotide sequence, the separation, and the linear orientation of the mediated sites, determine which domain or which DNA binding domains will form a binding pair complementary to the response element. The transactivation domain comprises one or more amino acid sequences that act as subdomains that affect the operation of the transcription factors during the preinitiation and assembly of the TATA box. The effect of the transactivation domain is to allow repeated transcription initiation events, which lead to higher levels of gene expression. A "receptor expression cassette" comprises a nucleotide sequence for a 5 'regulatory region operably linked to a nucleotide sequence encoding a receptor polypeptide and a 3' terminating region (stop codon and polyadenylation sequence). The 5 'regulatory region is capable of promoting expression in plants.

"USP" refers to the Ultraspiracle receptor that is found in Drosophila. It is also known as "XR2C", it has been isolated and cloned, and its ligand binding domain has been identified by sequence homology with known ligand binding domains (Henrich et al., Nucleic Acids 5 Research 18: 4143-4148 ( 1990)). Although the ligand or the chemical ligands that do not bind to it are unknown until now. The designation of "USP", as used herein, refers to the native forms of the receptor, as well as mutant or chimeric forms thereof. This includes, but is not limited to, those mutant or chimeric forms disclosed herein, as well as the forms I or USP chimerics comprising at least the ligand binding domain of the native USP and the mutants thereof. More than one form of USP may be used simultaneously in the present invention. A "secondary receptor expression cassette" comprises a nucleotide sequence for a 5 'regulatory region operably linked to a nucleotide sequence encoding a receptor polypeptide other than USP operably linked to a 3 'termination region. The expression cassette of the secondary receptor includes, but is not limited to, EcR, RXR, DHR38 (Kafatos et al, Proc.Nat.Acid.Sci.92: 7966-7970 (1995)), as well as mutant and chimeric forms thereof. 0 A "fraction" refers to the portion of a receptor polypeptide that is derived from the indicated source. For example, a "USP fraction" refers to the portion of the receptor polypeptide that was derived from the native Ultraspiracle receptor. The moiety, as used herein, may comprise one or more domains, and at a minimum comprises the ligand binding domain of the receptor for which the moiety is named. The term "chimeric" is used to indicate that the receptor polypeptide is composed of domains, at least one of which has an origin that is heterologous with respect to the other domains present. "Heterologist" means that one or more of the domains present in a receptor polypeptide differ in their natural origin with respect to the other domains present. For example, if the transactivation domain of the herpes simplex VP16 protein is operably linked to the Drosophila USP receptor, then the transactivation domain VP16 is heterologous with respect to the USP fraction. In addition, if a USP domain is operably linked to an RXR domain to make a functional receiver, then the chimeric merger would have domains that are heterologous to each other. These chimeric receptor polypeptides are encoded by nucleotide sequences that have been operably linked, resulting in a coding sequence that does not occur naturally. The chimeric receptor polypeptides of the present invention are referenced by a linear nomenclature from the N-terminal portion to the C-terminal portion of the polypeptide. Using this nomenclature, a chimeric receptor polypeptide having the transactivation domain of VP16 added to the N-terminal region of the USP receptor, would be designated VP16-USP. In contrast, if VP16 was added to the C-region region of the USP receptor, the chimeric receptor polypeptide would be designated USP-VP16.

Genetic constructs are termed in terms of a 5 'regulatory region and its operably linked coding sequence, wherein the 5' regulatory region is designated before a diagonal (/) and the coding sequence is designated after the diagonal. For example, the genetic construct 35S / USP-VP16 designates the 35S promoter of Cauliflower Mosaic Virus operably linked to the DNA sequence encoding the chimeric receptor USP-VP16, where the transactivation domain of VP16 has been added to the C-terminal region of USP. When reference is made to the receptor polypeptide, no promoter is designated. For example, the construction of the above gene encodes the USP-VP16 polypeptide. An "objective expression cassette" comprises a nucleotide sequence for a 5 'regulatory region operably linked to a nucleotide sequence encoding a target polypeptide whose expression is activated or inhibited by the receptor polypeptides in the presence of a chemical ligand. The 5 'regulatory region of the target gene comprises a core promoter sequence, a transcription initiation sequence, and a response element or response elements necessary for the complementary binding of the receptor polypeptides. The 5 'regulatory region is capable of promoting expression in plants. The target expression cassette also possesses a 3 'termination region (the stop codon and the polyadenylation sequence). Juvenile hormones I, II, III, and O are known, as well as the substituted version of I. For example, Fundamentáis of Insect Physiology, M.S. Blum, Ed., John Wiley & amp;; Sons, New York, 1985. Juvenile hormone I has the formula: 10,11-epoxy-7-ethyl-3,11-dimethyl-ura / 7s-2,6-tridecadienoate methyl. The structure of juvenile hormone I is shown below: Juvenile hormone agonists are compounds that exhibit one or more of the biological activities of the juvenile hormone. Compounds having this property, which have a structural relationship with the juvenile hormone, include, but are not limited to, quinoprene, methoprene, hydroprene, and methoprene acid. The structure of the quinoprene as an example of these juvenile hormone agonists is shown below: The juvenile hormone agonists that are not structurally related to juvenile hormone are also known. These compounds include, but are not limited to, the polycyclic compound that is not soprenoid, phenoxycarb, which is a well-known juvenile hormone agonist. The formula for phenoxycarb is ethyl [2- (4-phenoxyphenoxy) ethyl] carbamate. The structure of fenoxicarb is shown below: Another juvenile hormone agonist useful in the invention is the di-phenolan compound, a diphenyl ether compound. The formula for di-phenolan is phenyl ether of 4- (2-ethyl-1,3-dioxolan-4-ylmethoxy) phenyl. This compound has known juvenile hormone activity, and is expected to function in a manner analogous to that of the phenoxycarb or methoprene compounds. The use of juvenile hormone agonists in the present invention offers several advantages. First, the compounds are synthetic and readily available. Second, many of these compounds have the benefit of having already been examined in agricultural production, making these chemicals "ready to use" for field application to crops. The present invention makes use of the discovery that the juvenile hormone or one of its agonists acts as a chemical ligand for the USP receptor. The chemical ligand for USP, previously unknown, has been used in the present invention in conjunction with expressible USP receptor expression cassettes in plants, and appropriate target expression cassettes to create a novel method for controlling gene expression in plants. It has been found that many of the growth regulators of insects inhibit moult in insects, and possibly work directly on the receptors involved in the initiation of moult. These insect growth regulators include, but are not limited to, triflumuron ((1- (2-chlorobenzoyl) -3- (4-trifluoromethoxyphenyl) urea), hexamflumuron (1- [3,5-dichloro-4- ( 1,1-2,2-tetrafluoroethoxy) phenyl] -3- (2,6-difluorobenzoyl) urea), teflubenzuron (1- (3,5-dichloro-2,4-difluorophenyl) -3- (2,6- difluorobenzoyl) urea), flufenoxuron (1- [4- (2-chloro-a, a, α-trifluoro-p-tolyloxy) -2-fluorophenyl] -3- (2,6-difluorobenzoyl) urea), flucycloxuron (1 - [α- (4-chloro-α-cyclopropylbenzylideneamino-oxy) -p-tolyl] -3- (2,6-difluoro-benzoyl) urea), and lufenuron (1- [2,5-dichloro-4- ( 1, 1, 2,3,3,3-hexafluoropropoxy) phenyl] -3- (2,6-difluorobenzoyl) urea.) Additional benzoylphenylurea insecticides, including, but not limited to, diflubenzuron and chlorfluzuron, can also be used with The present invention Retinoic acid or its derivatives can also be a useful ligand for controlling gene expression in transgenic plants. to a USP gene product heterologously expressed in cultured mammalian cell lines (Harmon et al.). Accordingly, insect receptors can be regulated by the application of retinoic acid derivatives to transgenic plants bearing the appropriate combination of receptors and target gene constructs, as discussed below. Natural sources can also act as ligands for insect receptors, expressed in transgenic plants, such as transgenic corn or wheat. It has been discovered that a number of plants synthesize compounds that accelerate or inhibit the moult of insects. For example, one of the most active ecdysteroids, muristerone, is isolated from plant sources. Many families of plants that produce ecdysteroid or juvenile hormone activities are known. Juvenile hormone antagonists can also serve as ligands to regulate gene expression in transgenic plants. Examples of these ligands are 4- [2-tertiary butyl-carboxyloxy-ethylbenzoate]; bistiocarbamate, 5-methoxy-6- [1- (4-methoxyphenyl) ethyl] -1,3-benzodioxole, or ethyl E-3-methyl-2-dodecenoate. These antagonist ligands could serve to activate the low basal expression of transgenes, or to inhibit high basal gene expression in a heterologous plant system that expresses modified insect receptors. The method of the present invention comprises transforming a plant cell or a plant with an expression cassette of the USP receptor and an objective expression cassette. Expression, in the presence of juvenile hormone or one of its agonists, of the USP receptor polypeptide within the obtained plant cells, of the plants, or of the progeny thereof, activates the 5 'regulatory region of the expression cassette target inside the transgenic cells or plants (Figure 1). The juvenile hormone or one of its agonists, having been recognized herein as being bound to the ligand binding domain of USP, are essential for the present invention. Control of the expression of an objective expression cassette can also be achieved by optional expression within a plant, an additional secondary receptor polypeptide, or polypeptides other than USP (Figures 2 and 3). Examples of additional secondary receptor polypeptides encompassed by the invention include, but are not limited to, EcR, RXR, DHR38 (Kafatos et al., Proc.Nat.Acid.Sci.92: 7966-7970 (1995)), as well as the mutant or chimeric forms thereof. The use of these receptors to mediate ligand-induced transactivation is described in International Application Number PCT / EP 96/00686, filed on February 19, 1996, and incorporated herein by reference. The ligand binding domain of the USP receptor polypeptide provides the means to chemically control the activation of the 5 'regulatory region of the target expression cassette by the juvenile hormone or one of its agonists. USP is similar to the steroid receptor RXRa, which has a chemical ligand i or 9-cis-retinoic acid. It has also been shown that USP forms heterodimers with the EcR receptor polypeptide, and regulates the expression of an objective polypeptide in transformed mouse kidney cells, in response to the application of ecdysone, an insect hormone that binds to EcR, and that is not related to the juvenile hormone and its agonists (International Publication WO 94/01558). In the present invention, it has been discovered that the receptor USP and its ligand binding domain are particularly useful for controlling the expression of the target polypeptide in plants, in response to the application of the juvenile hormone or one of its agonists, as described in the examples below. In the present invention, the chimeric forms of the USP receptor polypeptides can also be used to activate the expression of a target polypeptide in the presence of juvenile hormone or one of its agonists. The DNA binding domain or the transactivation domain of a chimeric USP receptor polypeptide can be selected from a heterologous source, based on its effectiveness for transactivation or DNA binding. These domains of the chimeric receptor polypeptide can be obtained from any organism, such as from plants, insects, and mammals, which have similar transcriptional regulatory functions. In one embodiment of the invention, these domains are selected from other members of the steroid and thyroid hormone superfamily of nuclear receptors. The use of chimeric receptor polypeptides has the benefit of combining domains from different sources. The chimeric USP receptor polypeptides, as provided herein, offer the advantage of combining an optimal transactivation activity, or a binding or recognition of the altered response element of a specific response element with the juvenile hormone or one of its agonists. as a ligand. Accordingly, a chimeric polypeptide that is tailor-made for a specific purpose can be constructed. These chimeric receptor polypeptides also provide improved functionality in the heterologous environment of a plant cell. It is also considered part of the present invention that transactivation, ligand binding, and DNA binding domains can be assembled into the chimeric receptor polypeptide in any functional configuration. For example, where a subdomain of a transactivation domain is found in the N-terminal portion of a naturally occurring receptor, the chimeric receptor polypeptide of the present invention may include a subdomain of transactivation in the C term instead of, or in addition to, a subdomain in the term N. Chimeric receptor polypeptides, as disclosed herein, may also have multiple domains of the same type, for example, more than one transactivation domain (or two subdomains) per receptor polypeptide. Accordingly, one embodiment of the invention provides a USP receptor polypeptide that activates the expression of a target polypeptide in the presence of juvenile hormone or one of its agonists, and which also possesses superior characteristics for transactivation. The transactivation domains can be defined as amino acid sequences that increase the initiation of productive transcription by RNA polymerases. (See generally Ptashne, Nature 335: 683-689 (1988)). Different transactivation domains are known that have different degrees of effectiveness in their ability to increase the initiation of transcription. In the present invention, it is desirable to use transactivation domains that have superior transactivation effectiveness in plant cells, in order to create a high level of expression of the target polypeptide, in response to the presence of a juvenile hormone or one of its agonists. The transactivation domains that have been shown to be particularly effective in the method of the present invention include, but are not limited to, VP16 (isolated from herpes simplex virus). In a preferred embodiment of the present invention, the transactivation domain from VP16 is operably linked to a USP fraction to create a chimeric USP receptor polypeptide, in order to control the expression of the target polypeptide in plants. Other transactivation domains will also be effective.

The DNA binding domain is an amino acid sequence having certain functional characteristics that are responsible for binding the USP receptor polypeptide to a specific nucleotide sequence, termed the response element, present in the 5 'regulatory region of the expression cassette. objective. The structure of the DNA binding domains for the nuclear superfamily of steroid and thyroid receptors is highly conserved from one species to another, and consequently, there is limited variation in the response elements used to form a complementary fixation pair ( Evans, Science 240: 889-895 (1988)). However, considerable flexibility in the method for controlling gene expression can be introduced by using the response elements in other ways. In a preferred embodiment of the invention, multiple copies, and preferably between 1 and 11 copies of the appropriate response element, are placed in the 5 'regulatory region, which allows having multiple sites for the attachment of USP or secondary receptor polypeptides. optional, resulting in a greater degree of activation. Greater flexibility in the method of genetic control can be achieved by changing the linear orientation or the position of the response elements in the 5 'regulatory region. The response elements that are recogn by the Class II receptor proteins have a "diad" symmetry composed of two "half sites" (Evans, Science 240: 889-895 (1988)). Each receptor polypeptide is fixed to a "half site". These "means sites" can be oriented either in a direct repetition, an inverted repetition, or in a palindromic form. In one embodiment of the present invention, more than one USP receptor polypeptide molecule recogn a direct repeat response (DR) element, where activation of the target expression cassette is achieved in the presence of a juvenile hormone or one of its agonists. Additional flexibility in the control of gene expression can be obtained by the present invention, using DNA binding domains and response elements from other transcription activators, including, but not limited to, the LexA or GAL4 proteins. The DNA binding domain from the LexA protein encoded by the lexA gene of E. coli, and its complementary binding site (Brent and Ptashne, Cell 43: 729-736 (1985), which describes a LexA transcription activator / GAL4) can be used. Another useful source is from the yeast GAL4 protein (Sadowzki et al., Nature 563-564 (1988), which describes a transcription activator GAL4-VP16). In a preferred embodiment of the invention, a chimeric version of the optional secondary receptor polypeptide is constructed by fusing the GAL4 DNA binding domain to a fraction containing the ligand binding domain from EcR. The 5 'regulatory region of the USP, and the optional secondary receptor expression cassettes, further comprise a promoter that allows expression in tissues and cells of plants. Appropriate promoters are chosen for the cassettes of expression of the receptor, such that the expression of the receptor polypeptides can be constitutive, tissue-specific, cell-specific, cell-specific, or developmentally regulated. The promoters can also be selected such that the expression of the receptor polypeptides themselves can be chemically induced in the plant, thereby increasing the level of promoter induction by the ligand. By combining the promoter elements that confer specific expression, with those that confer chemically-induced expression, the receptor polypeptides can be expressed or activated within specific cells or tissues of the plant, in response to chemical application. The nucleotide sequence encoding the receptor polypeptide, it can be modified for better expression in plants, better functionality, or both. These modifications include, but are not limited to, altering codon usage, inserting introns, or creating mutations. In one embodiment of the invention, expression cassettes comprising an anther-specific or pistil-specific promoter, operably linked to a nucleotide sequence encoding a USP receptor polypeptide, are used to activate the expression of a target polypeptide in the presence of juvenile hormone or one of its agonists. Target polypeptides whose expression is activated by the receptor polypeptides in the presence of a juvenile hormone or one of its agonists are also disclosed. The expression of any coding sequence can be controlled by the present invention, provided that the promoter operably linked to this coding sequence has been designed to contain the response element or response elements that are complementary to the domain of DNA binding of the USP receptor, and optionally, the response element or the response elements necessary for the secondary receptor. For example, target polypeptides that are useful for controlling plant fertility are activated by the USP receptor polypeptide in the presence of a juvenile hormone or one of its agonists. Mutants of the USP receptor polypeptide are also encompassed by the invention. Mutants having the property of a reduced level of background activation of the target expression cassette can be prepared, such that the induction is large relative to the non-induced background expression. In addition, mutants may develop that are altered in their attachment to the juvenile hormone or one of its agonists. Mutants that have altered binding properties will respond to different agonists in unique ways for those agonists. For example, mutant USP receptors can be developed that respond only to the phenoxycarb agonist, and not to the hydroprene, thus distinguishing between juvenile isoprenoid and non-isoprenoid hormone agonists. Useful methods of mutagenesis, such as chemical mutagenesis or site-directed mutagenesis, are known in the art. In another method, mutant receptor polypeptides are prepared by polymerase chain reaction mutagenesis of the nucleotide sequence encoding the USP ligand binding domain. These mutant receptor polypeptides are expressed in a host organism that lends itself to convenient selection and isolation techniques, such as yeast. The selection for mutant receptor polypeptides that exhibit a decrease in basal activity, and a greater fold induction in this host organism, however, will only provide candidates for additional testing in plant cells, since it is clear, from the work with the glucocorticoid receptor (GR), which although the receptors of the thyroid hormone and steroid superfamily, can function in the yeast, this does not predict the functionality in transgenic plants (Lloyd et al., Science 226: 436 (1994)). Further limiting the application of yeast results is the observation that yeast cells that express the glucocorticoid receptor do not respond to the commonly used chemical ligand dexamethasone, although this ligand is functional in other heterologous systems (Schena et al. Proc. Nati, Acad. Sci. USA 88: 10421-10425 (1991)). Another test is performed on plant cells, by preparing i or receptor expression cassettes that encode the mutated receptor polypeptides, and transforming them into plant cells in combination with an objective expression cassette. Transformed plant cells are tested for activation of the 5 'regulatory region of the target expression cassette, by mutant receptor polypeptides in the presence of juvenile hormone or of one of its agonists. Mutant receptor polypeptides that cause a low basal expression of a target polypeptide, in the absence of juvenile hormone or one of its agonists, and a high expression of the target polypeptide in the presence of juvenile hormone or one of its agonists, are useful for control gene expression in plants. As described above, the method of the present invention can be used to statistically increase gene expression at a minimum basal level. However, the present invention can be used to statistically decrease or inhibit the activation of gene expression that has been mediated by a complex consisting of receptors, such as USP and EcR. The control of gene expression in plants, mediated by these receptor complexes, is the subject of Publication Number PCT / EP 96/00686, filed on March 3, 1995, incorporated herein by reference. The inversion of the activation mediated by these receptor complexes is caused by the presence of a juvenile hormone or one of its agonists, which are the chemical ligands for the USP receptor polypeptide (see Figures 2 and 3). In the presence of the juvenile hormone or one of its agonists, the USP: EcR complex is altered, thus inverting the activation. For example, in a transgenic plant expressing the USP and GAL4-EcR-C1 receptor polypeptides, and comprising an objective expression cassette having a GAL4 binding site element, the activation of the gene expression of the obtained polypeptide will be reversed , caused by the complex. This inversion would occur in the presence of tebufenozide (also known as RH 5992), or of another chemical ligand that binds to the ligand binding domain of EcR, or in the absence of that chemical ligand. For expression in plants, suitable promoters must be selected for both the receptor expression cassettes and the target expression cassette. Unless specifically noted, the promoters discussed below can be used for direct expression in plants either of receptor polypeptides or of the target polypeptide. These promoters include, but are not limited to, constitutive, inducible, temporarily regulated, developmentally regulated, chemically regulated, tissue-preferred, and tissue-specific promoters. Preferred constitutive promoters include, but are not limited to, CaMV 35S and 19S promoters (U.S. Patent Number 5,352,605). The additionally preferred promoters include, but are not limited to, one of several of the actin genes, which are known to be expressed in most cell types. The promoter described by McEIroy et al., Mol. Gen. Genet. 231: 150-160 (1991), can be easily incorporated into the expression cassettes of the receptor of the present invention, and are particularly suitable for use in monocotyledonous hosts. Yet another preferred constitutive promoter is derived from ubiquitin, which is another genetic product that is known to accumulate in many cell types. The ubiquitin promoter has been cloned from several species for use in transgenic plants (eg, sunflower - Binet et al., Plant Science 79: 87-94 (1991); maize - Christensen et al., Plant Molec. Biol. : 619-632 (1989)). The corn ubiquitin promoter has been developed in transgenic monocotyledonous systems, and its sequence and vectors constructed for the transformation of monocotyledonous plants are disclosed in European Patent Number EP-A-342,926. The ubiquitin promoter is suitable for use in the present invention in transgenic plants, especially monocotyledons. Other useful promoters are the promoters of U2 and U5 snRNAs of corn (Brown et al., Nuc Acids Res.17: 8991 (1989)), and the alcohol dehydrogenase promoter (Denis et al., Nucleic Acids Res. 12: 3983 (1984)). The tissue-specific or tissue-preferential promoters useful in the present invention in plants, particularly in corn, are those that direct expression in the root, in the marrow, in the leaves, or in the pollen. These promoters are disclosed in International Publication Number WO 93/07278, incorporated herein by reference in its entirety. Also useful are promoters that confer specific expression of the seed, such as those disclosed by Schernthaner et al., EMBO J. 7: 1249 (1988).; the specific promoters of the ant32 and ant43D anthers disclosed in European Patent Number EP-A578,611, incorporated herein by reference in its entirety; the anther-specific promoter (tapetal) B6 (Huffman et al., J. Cell. Biochem. 17B: Abstract # D209 (1993)); the specific promoters of the pistil, such as a modified S13 promoter (Dzelkains et al., Plant Cell 5: 855 (1993)). In the present invention, chemically induced promoters are also useful. Particular promoters of this category useful for directing the expression of the receptor polypeptides or the target polypeptide in plants are disclosed, for example, in European Patent Number EP-A-332,104, incorporated herein by reference in its entirety. The 5 'regulatory region of the receptor expression cassette or target expression cassette may also include other enhancer sequences. It has been found that numerous sequences improve gene expression in transgenic plants. For example, it is known that a number of untranslated leader sequences derived from viruses improve expression. Specifically, it has been demonstrated that the leading sequences of Tobacco Mosaic Virus (TMV, the "O sequence"), Corn Chlorotic Speck Virus (MCMV), and Alfalfa Mosaic Virus (AMV), are effective in improving expression (eg, Gallie et al., Nucí Acids Res. 15: 8693-8711 (1987); Skuzeski et al., Plant Molec. Biol. 65-79 (1990)). Other leaders known in the art include, but are not limited to: • leaders of picornaviruses, for example the leader of EMCV (5 'non-coding region of encephalomyocarditis) (Elroy-Stein, O., Fuerst, TR, and Moss, B PNAS USA 86: 6126-6130 (1989)); • Potivirus leaders, for example the leader of VTE (Tobacco Scab Virus) (Allison et al., (1986); MDMV leader (Maize Dwarf Mosaic Virus); Virology, 154: 9-20); I leader of human immunoglobulin heavy chain binding protein (BiP) (Macejak, DG, and Sarnow, P., Nature, 353: 90-94 (1991); V leader untranslated from virus coat protein mRNA alfalfa mosaic (AMV RNA 4), (Jobling, SA, and Gehrke, L., Nature, 325: 622-625 (1987)) • Tobacco mosaic virus leader (TMV), (Gallie, DR et al. , Molecular Biology of RNA, pp. 237-256 (1989); and • Leader of Corn Chlorotic Mottled Virus (MCMV) (Lommel, SA et al., Virology, 81: 382-385 (1991) See also, Della-Cioppa and collaborators, Plant Physiology, 84: 965-968 (1987)).

It has been shown that different introns sequences improve expression when added to the 5 'regulatory region, particularly in monocotyledonous cells. For example, it has been discovered that the introns of the Adhl gene of corn significantly improve the expression of the wild type gene under its known promoter, when introduced into corn cells (Callis et al., Genes Develop 1: 1183-1200 (1987)). In addition to incorporating one or more of the aforementioned elements in the 5 'regulatory region of an objective expression cassette, other peculiar elements can also be incorporated into the objective expression cassette. These elements include, but are not limited to, a minimum promoter. Minimum promoter means that the elements of the basal promoter are inactive or almost i or inactive, with no binding sites for the upstream activators. This promoter has a low background activity in the plants when there is no transactivator present, or when the enhancer or the binding sites of the response element are absent. A minimal promoter that is particularly useful for the target genes in plants, is the minimal Bz1 promoter that is obtained from the bronzel gene of corn The Bz1 core promoter was obtained from the construction of mutant βz7-luciferase mutant "myc" pBz1 LucR98 by means of dissociation at the Nhel site located at -53 to -58 (Roth et al., Plant Cell 3: 317 (1991)) . The promoter fragment of the Bz1 core derived in this way extends from -53 to +227, and includes, when used for transgenic corn, Bz1 intron-1 in the 5 'non-translated region. In addition to the promoters, there is also a variety of 3 'transcription terminators available for use in the present invention. Transcription terminators are responsible for the termination of transcription and polyadenylation of the correct mRNA. Suitable transcription terminators and those known to work in plants include the CaMV 35S terminator, the tml terminator, the nopaline synthase terminator, the rbcS pea E9 terminator, and others known in the art. These can be used in both monocots and dicots. The expression cassettes of the present invention can be introduced into the plant cell in a number of ways recognized in the art. The experts in this field will appreciate that the choice of the method could depend on the type of plant, that is, monocotyledon or dicotyledon, directed for the transformation. Suitable methods for transforming plant cells include, but are not limited to, microinjection (Crossway et al., Bio Techniques 4: 320-334 (1986)), electroincorporation (Riggs et al., Proc. Nati. Acad. Sci. USA 83 : 5602-5606 (1986)), Agrobacterium-mediated transformation (Hinchee et al., Biotechnology 6: 915-921 (1988)), direct gene transfer (Paszkowski et al., EMBO J. 3: 2717-2722 (1984)), and acceleration of ballistic particles using devices available from Agracetus, Inc., Madison, Wisconsin, and BioRad, Hercules, California (see, for example, Sanford et al., U.S. Patent No. 4,945,050; and McCabe et al., Biotechnology 6: 923-926 (19888)). Also see Weissinger et al., Annual Rev. Genet. 22: 421-477 (1988); Sanford et al., Particulate Science and Technology 5: 27-37 (1987) (onion); Christou et al., Plant Physiol. 87: 671-674 (1988) (soybean); McCabe et al., Bio / Technology 6: 923-926 (1988) (soybeans); Datta et al., Bio / Technology 8: 736-740 (1990) (rice); Klein et al., Proc. Nati Acad. Sci. USA, 85: 4305-4309 (1988) (corn); Klein et al., Bio / Technology 6: 559-563 (1988) (corn); Klein et al., Plant Physiol. 91: 440-444 (1988) (corn); Fromm et al., Bio / Technology 8: 833-839 (1990) (corn); and Gordon-Kamm et al., Plant Cell 2: 603-618 (1990) (corn); Svab et al., Proc. Nati Acad. Sci. USA 87: 8526-8530 (1990) (tobacco chloroplast); Koziel et al., Biotechnology 11: 194-200 (1993) (corn); Shimamoto et al., Nature 338: 274-277 (1989) (rice); Christou et al., Biotechnology 9: 957-962 (1991) (rice); European Patent Number i or EP-A-332,581 (orchard grass and other Pooideae); Vasil et al., Biotechnology 11: 1553-1558 (1993) (wheat); Weeks and collaborators, Plant Physiol. 102: 1077-1084 (1993) (wheat). A particularly preferred set of embodiments for the introduction of the expression cassettes of the present invention into corn by microprojectile bombardment, is described in Koziel et al., Bio / Technology 11: 194-200, 1993, incorporated herein by reference in its entirety. A further preferred embodiment is the protoplast transformation method for corn as disclosed in European Patent Number EP-A-292,435, incorporated herein by reference in its entirety. A particularly preferred set of embodiments for the introduction of the expression cassettes of the present invention into wheat by bombardment of microprojectiles, can be found in International Publication Number WO 94/13822, incorporated herein by reference in its entirety.

The transformation of the plants can be undertaken with a single DNA molecule or with multiple DNA molecules (i.e., co-transformation), and both techniques are suitable for use with the expression cassettes of the present invention. There are numerous transformation vectors available for the transformation of plants, and the expression cassettes of this invention can be used in conjunction with any of these vectors. The selection of the vector will depend on the preferred transformation technique and on the target species for the transformation. There are many vectors available for transformation using Agrobacterium tumefaciens. These normally carry at least one T-DNA limit sequence, and include vectors such as pBIN19 (Bevan, Nucí Acids Res. (1984)). In a preferred embodiment, the expression cassettes of the present invention can be inserted into any of the binary vectors pCIB200 and pCIB2001 for use with Agrobacterium. These vector cassettes for the Agrobacterium-mediated transformation were constructed in the following manner. PTJS75an was created by Narl digestion of pTJS75 (Schmidhauser and Helinski, J Bacteriol 164: 446-455 (1985)), allowing the separation of the tetracycline resistance gene, followed by the insertion of an Accl fragment of pUC4K carrying an NPTII (Messing and Vierra, Gene 19: 259-268 (1982); Bevan et al., Nature 304: 184-187 (1983); McBride et al., Plant Molecular Biology 14: 266-276 (1990)). Xhol linkers were ligated to the EcoRV fragment of pCIB7 containing the boundaries of left and right T-DNA, a chimeric us / nptll gene selectable in plants, and the pUC polylinker (Rothstein et al., Gene 53: 153-161 (1987)) , and the fragment digested by XhoI was cloned in pTJS75kan digested with SalI to create pCIB200 (see also European Patent Number EP-A-332,104, Example 19). PCIB200 contains the following restriction sites of the single polylinker: EcoRI, Sstl, Kpnl, BglII, Xbal, and SalI. Plasmid 5 pCIB2001 is a derivative of pCIB200 that was created by inserting additional restriction sites into the polylinker. The unique restriction sites in the polylinker of pCIB2001 are EcoRI, Sstl, Kpnl, BglII, XbaI, SalI, Mul, Bell, Avrll, Apal, Hpal and Stul. PCIB2001, in addition to containing these unique restriction sites, also has selection of kanamycin in plants and bacterial, the limits of left and right T-DNA for Agrobacterium-mediated transformation, the function of trAA derived from RK2 for the mobilization between .coli and other guests, and the functions of OriT and OriV also from RK2. The polylinker pCIB2001 is suitable for the cloning of expression cassettes in plants containing their own regulatory signals. 15 An additional vector useful for the transformation mediated by Agrobacterium is the binary vector pCIBIO, which contains a gene that codes for resistance to kanamycin for plant selection, right and left T-DNA border sequences, and incorporates sequences from the host wide-range plasmid pRK252 that they allow replication in both E. coli and 0 in Agrobacterium. Its construction is described by Rothstein et al., Gene 53: 153-161 (1987). Several pCIBIO derivatives have been constructed that incorporate the gene for hygromycin B phosphotransferase described by Gritz et al., Gene 25: 179-188 (1983). These derivatives make it possible to select cells from transgenic plants on hygromycin alone (pCIB743), or on hygromycin and kanamycin (pCIB715, pCIB717). Methods that use either a form of direct gene transfer, or alternatively Agrobacterium-mediated transfer, usually, but not necessarily, are undertaken with a selectable marker that can provide resistance to an antibiotic (eg, kanamycin, hygromycin, or methotrexate) or to a herbicide (for example, phosphinothricin). The choice of the selectable marker for transformation into plants, however, is not critical to the invention. For certain plant species, different antibiotic or herbicide selection markers may be preferred. The selection markers routinely used in the transformation include the nptll gene that confers resistance to kanamycin and related antibiotics (Messing and Vierra, Gene 19: 259-268 (1982)).; Bevan et al., Nature 304: 184-187 (1983)), the bar gene that confers resistance to the herbicide phosphinothricin (White et al., Nucí Acids Res 18: 1062 (1990), Spencer et al., Theor Appl Genet 79: 625- 631 (1990)), the hph gene that confers resistance to the antibiotic hygromycin (Blochinger and Diggelmann, Mol Cell Biol 4: 2929-2931), and the dhfr gene, which confers resistance to methotrexate (Bourouis et al., EMBO J. 2: 1099-1 104 (1983)). A useful vector for direct gene transfer techniques in combination with selection by the herbicide Basta (or phosphinothricin) is pCIB3064. This vector is based on the plasmid pCIB246, which comprises the CaMV 35S promoter in fusion operative with the GUS gene of E. coli, and the transcription terminator of CaMV 35S, and is described in International Publication Number WO 93/07278, incorporated to the present as a reference. A gene useful for conferring resistance to phosphinothricin is the bar gene of Streptomyces viridochromogenes (Thompson et al., EMBO J. 6: 2519-2523 (1987)). This vector is suitable for cloning of expression cassettes in plants containing their own regulatory signals. A further transformation vector is pSOG35 which uses the dihydrofolate reductase (DHFR) of the E. coli gene as a selectable marker that confers resistance to methotrexate. The io polymerase chain reaction was used to amplify the 35S promoter (approximately 800 base pairs), intron 6 of the maize Adhl gene (approximately 550 base pairs), and 18 base pairs of the leader sequence not translated from GUS from pSOGIO. A fragment of 250 base pairs encoding the type II dihydrofolate reductase gene of E. coli was also amplified by chain reaction of polymerase, and these two fragments of the polymerase chain reaction were assembled with a Sacl-Pstl fragment from pBI221 (Clontech) which would comprise the base structure of the pUC19 vector and the nopaline synthase terminator. The assembly of these fragments generated pSOG19 containing the 35S promoter in fusion with the intron 6 sequence, the GUS leader, the DHFR gene, and the nopaline synthase 0 terminator. The replacement of the GUS leader in pSOG19 with the leading Corn Chlorotic Mottled Check (MCMV) sequence generated the vector pSOG35. PSOG19 and pSOG35 carry the gene derived from pUC for ampicillin resistance, and have the Hindlll, Sphl, Pstl, and EcoRI sites available for the cloning of foreign sequences. One of the convenient aspects of the present invention is its use in controlling the fertility of plants under field conditions. Effective fertilization results from the formation of viable zygotes, and can be measured as the percentage of seeds that form viable zygotes. According to the present invention, fertility can be controlled by incorporating a nucleotide sequence encoding an appropriate target into the target expression cassette, wherein the expression of this target polypeptide interferes with the fertility of the plant, meaning that statistically reduces or increases the fertility of the plant. In a preferred embodiment of the invention, this target polypeptide makes the fertilization process ineffective, meaning that the formation of viable zygotes will be prevented. This ineffective fertilization can be measured as the percentage of seeds that do not form viable zygotes, and can be caused by a variety of means. These include, but are not limited to: 1) interruption or alteration of processes that are critical for the formation of viable gametes, 2) pollen or ovules that, if formed, are not functional, or 3) failure of the embryo sac, of the pistil, of the stigma or of the transmitting tract, to develop properly. In the present invention, the juvenile hormone or one of its agonists is applied to, or contacted with, transgenic plants under field conditions, where the expression of a target polypeptide is activated, thereby rendering the fertilization ineffective. . In another embodiment of the present invention, the expression of the target polypeptide is increased, or the fertility of a plant is restored. It is recognized that different degrees of effective or ineffective fertilization can be achieved with the present invention. In a preferred embodiment, more than 80 percent, and more preferably more than 95 percent ineffective fertilization can be achieved. The ability to provide variability in the level of fertility allows the invention to be tailored for a variety of agricultural purposes. Useful coding sequences for the target polypeptide include, but are not limited to, any sequence that encodes a product capable of rendering the fertilization ineffective. These coding sequences may be of homologous or heterologous origin. The gene products of these coding sequences include, but are not limited to: • Diphtheria Toxin Chain A (DTA), which inhibits protein synthesis, Greenfield et al., Proc. Nati Acad. Sci. USA, 80: 6853 (1983);.

Palmiter et al., Cell, 50: 435 (1987); • pectate lyase pelt from Erwinia chrysanthemi EC16, which degrades pectin, causing cell lysis. Keen et al., J. Bacteriology, 168: 595 (1986); • T-urf13 (TURF-13) from mitochondrial genomes of cms-T corn; this gene encodes a polypeptide designated as URF-13 that alters the mitochondrial or plasma membranes. Braun et al., Plant Cell, 2: 153 (1990); Dewey et al., Proc. Nati Acad. Sci .: USA, 84: 5374 (1987); Dewey et al., Cell, 44: 439 (1986); F Beta-1, 3-glucanase, which causes the premature dissolution of the callosum wall of microspores. Worral et al., Plant Cell 4: 759-771 (1992); • Juniper recombinase from the Mu a phage gene, which encodes a site-specific DNA recombinase, which causes reconfigurations of the genome, and loss of cell viability when expressed in plant cells. Maeser and collaborators, Mol. Gen. Genet., 230: 170-176 (1991); • Indoleacetic acid-lysine synthetase (iaaL) from Pseudomonas syringae, which encodes an enzyme that combines lysine with indoleacetic acid (IAA). When expressed in the cells of plants, it causes altered developments due to the removal of indoleacetic acid from the cell by means of conjugation. Romano et al., Genes and Development, 5: 438-446 (1991); Spena et al., Mol. Gen. Genet., 227: 205-212 (1991); Roberto and collaborators, Proc. Nati Acad. Sciz USA, 87: 5795-5801; • Bacillus amyloliquefaciens ribonuclease, also known as barnase, which digests mRNA in the cells in which it is expressed, leading to cell death. Mariani et al., Nature 347: 737-741 (1990); Mariani et al., Nature 357: 384-387 (1992); and • CytA toxin gene from Bacillus thuringiensis israeliensis that encodes a protein that is mosquitocidal and hemolytic. When expressed in plant cells, it causes the death of the cell due to the alteration of the cell membrane. McLean et al., J. Bacteriology, 169: 1017-1023 (1987); Ellar et al., United States Patent Number 4,918,006 (1990). • These polypeptides also include the Moffatt and Somerville of Adenine Fosforribosyltransferase (APRT), Plant Physiol., 86: 1150-1154 (1988); DNase, RNAse; protease; salicylate hydroxylase; etc. It is further recognized that the target expression cassette may comprise a regulatory region 5"operably linked to a nucleotide sequence which, upon transcription, produces an anti-sense version of a critical coding sequence for the formation of viable gametes, such as APRT Alternatively, ribozymes directing the mRNA can be used from the gene that is critical for gamete formation or function.These ribozymes will comprise a hybridization region of about 9 nucleotides that is complementary to the nucleotide sequence with at least part of the target RNA, and a catalytic region that is adapted to dissociate the target RNA The ribozymes are described in European Patent Number EP-A-321, 201 and in International Publication Number WO 88/04300, incorporated herein by reference. See also Haseloff and Gerlach, Nature, 334: 585-591 (1988), Fedor and Uhlenbeck, Proc. Nati, Acad. Sci .: USA, 87: 1668-1672 (1990); Cech and Bass, Ann. Rev. Biochem., 55: 599-629 (1986); Cech, T.R. 236: 1532-1539 (1987); Cech, T.R. Gene, 73: 259-271 (1988); and, Zang and Cech, Science, 231: 470-475 (1986). It is recognized that the above nucleotide sequences, which encode a target polypeptide, can also be operably linked to a 5 'regulatory sequence that directs its expression in a specific manner to the tissue or cell. The means for providing this specific expression of the tissue or cell have been described above. This specificity in the expression ensures that the effect of the target polypeptide is exerted only on those tissues or cells that are necessary for the formation of viable zygotes, and that are not harmful to the plant beyond its effect on fertility. It is recognized within the scope of the invention, that male fertility of transgenic plants, female fertility of transgenic plants, or both can be controlled. Male sterility is the failure or inability to produce functional or viable pollen. Male sterility can result from defects leading to lack of pollen formation, or lack of functional capacity in pollen when formed. Therefore, either the pollen is not formed, or if it is formed, it is not viable or otherwise incapable of effective fertilization under normal conditions. Female infertility is the failure or inability to produce functional or viable embryonic or functional megaespores or sacs, or other tissues required for germination, growth, or pollen fertilization. Female infertility can result from defects that lead to the lack of formation of the mega-spores or the embryo sac, or failure of the ovary, ovule, pistil, stigma, or the transmitting tract to develop properly. Therefore, either a viable embryo sac is not developed, or if it is formed, it is unable to have an effective fertilization under normal conditions.

For example, a transgenic plant expressing the USP polypeptide or polypeptides in the anthers can be obtained by using an anther-specific promoter operably linked to the appropriate nucleotide sequences. In addition, the transgenic plant will further comprise an objective expression cassette having a 5 'regulatory sequence comprising the sequence of the appropriate response element with the core promoter elements from Bz1, operably linked to the coding sequence for the ribonasease barnase. Upon application of the juvenile hormone or one of its agonists to the transgenic plant expressing the USP receptor polypeptides, activation of the 5 'regulatory sequence of the target expression cassette occurs, with the subsequent production of the polypeptide barnase. objective. The resulting expression of barnase specifically in the anthers, causes cell death and consequent male sterility. A similar combination of receptor polypeptides and target expression cassette, utilizing a specific pistil promoter operably linked to the nucleotide sequences encoding the receptor polypeptides, can produce female sterility. Alternatively, a plant can be designed wherein the expression of the target polypeptide restores fertility to a male-sterile or female-sterile plant. For example, a plant expressing the barnase gene under the control of the promoter Ant43D, Ant32, or B6, or as described in Mariani et al., Nature 347: 737-741 (1990), and Mariani et al., Can be obtained. Nature 357: 384-387 (1992), under the control of the TA29 promoter. These plants additionally comprise the receptor expression cassettes for the USP receptor polypeptide, and any optional secondary receptor polypeptide either from the same anther-specific promoter, or from a constitutive promoter, such as corn ubiquitin, 35S, or the rice actin. These plants further comprise an objective expression cassette having a 5 'regulatory sequence comprising the sequence of the appropriate response element, with the core promoter elements from Bz1, operably linked to the coding sequence for the barstar of the inhibitor. of barnase. The plants are male-sterile, but, after the application of the juvenile hormone or one of its agonists, activation of the 5 'regulatory sequence of the target expression cassette occurs, with the subsequent barstar production of the target polypeptide. Barstar inhibits the ribonuclease activity of the barnase polypeptide, and the development of anthers and pollen proceeds normally. In this way, fertility is restored. A similar approach can be employed to control female sterility. By using specific promoters for expression in female reproductive tissues, female-sterile plants can be obtained in place of the anther-specific promoters to boost barnase expression. Induction of the target expression cassette comprising the barstar coding sequence by the juvenile hormone or one of its agonists, results in the restoration of female fertility. The above approaches can use any female or male sterility gene for which a resetting gene can be devised. Other potential restorative genes different from barstar are described in European Patent Number EP-A-412,911. The genetic properties designed in the transgenic plants described above and their seeds, are surpassed by sexual reproduction or vegetative growth, and therefore, can be maintained and propagated in the plants of the progeny. In general, this maintenance and propagation makes use of known agricultural methods developed to suit specific purposes, such as tillage, sowing, or harvesting. Specialized processes, such as hydroponics or greenhouse technologies, can also be applied. Since the growing crop is vulnerable to attack and damage caused by insects or infections, as well as competition for wild plants, measures are taken to control weeds, plant diseases, insects, nematodes, and others. adverse conditions, to improve performance. These include mechanical measures, such as tillage of land or removal of the herbs or the infected plants, as well as the application of agrochemicals such as herbicides, fungicides, gametocides, nematicides, growth regulators, ripening agents, and insecticides. The use of the suitable genetic properties of the transgenic plants and seeds according to the invention, can be constituted 0 additionally in the breeding of plants that has as objective the development of plants with better properties, such as tolerance to pests, to herbicides , or to the effort better nutritional value, greater yield, or better structure that causes less loss of accommodation. The different breeding steps are characterized by a well defined human intervention, such as the selection of the lines to be crossed, the direction of the pollination of the parent lines, or the selection of the appropriate progeny plants. Depending on the desired properties, different breeding measures are taken. Relevant techniques are well known in the art, and include, but are not limited to, hybridization, inbreeding, backcross breeding, multi-line breeding, variety blending, interspecific hybridization, aneuploid techniques, and the like. Accordingly, the transgenic plants according to the invention, and their seeds, can be used for the breeding of better plant lines which, for example, increase the effectiveness of conventional methods, such as treatment with herbicides or pesticides, or to eliminate these methods due to their modified genetic properties. In an alternative way, new crops with better tolerance to effort can be obtained which, due to their optimized genetic "equipment", produce a harvested product of better quality than the products that could not tolerate comparable adverse development conditions. In the production of seeds, the quality and uniformity of seed germination are essential characteristics of the product, while the quality and uniformity of germination of the seeds harvested and sold by the farmer is not important. Since it is difficult to keep a crop free of other crops and herbal seeds, control seed diseases, and produce seeds with good germination, seed producers have developed extensive well-defined seed production practices, which have experimented in the technique of cultivation, conditioning, and marketing of pure seeds.

It is therefore a common practice of the farmer to buy certified seeds that meet specific quality standards, instead of using seeds harvested from his own crop. The propagation material to be used as seed is customarily treated with a protective coating comprising herbicides, insecticides, fungicides, bactericides, nematicides, molluscicides, or mixtures thereof. The customary protective coatings comprise compounds such as captan, carboxin, thiram (TMTDMR), metalaxyl (ApronR), and pirimiphos-methyl (Actellic). If desired, these compounds are formulated together with other vehicles, surfactants, or application promoting aids customarily employed in the art of formulation, to provide protection against damage caused by bacterial, fungal, or animal pests. The protective coatings can be applied by impregnating the propagation material with a liquid formulation, or by coating with a combined wet or dry formulation. Other methods of application are also possible, such as treatment directed to shoots or fruit. It is a further aspect of the present invention to provide new agricultural methods, such as the methods exemplified above, which are characterized by the use of transgenic plants, transgenic plant material, or transgenic seeds in accordance with the present invention. The present invention can be used in any plant that can be transformed and regenerated to a transgenic plant. The male sterility, female sterility, or both, can be controlled by applying the appropriate chemical ligand. The control of the fertility of the plant is particularly useful for the production of hybrid seeds. In order to produce hybrid seeds not contaminated with pure seeds, pollination control methods should be implemented to ensure cross-pollination and non-self-pollination. This is usually done by mechanical, genetic, or chemical hybridization agents (CHAs). For example, in corn, the current practice is to mechanically overflow the female mother (or seed), which is a time-consuming and labor-intensive process. In wheat, it is impractical to control fertility through mechanical elements on a scale of seed production, and no genetic sources of fertility control are established. The use of the present invention in the production of hybrid seeds offers the advantages of reliability, ease of use, and control of male or female fertility. Transgenic plants containing the appropriate receptor expression cassettes, and the target expression cassette, can be made homozygous, and can be maintained indefinitely. To obtain hybrid seeds, the homozygous lines of Progenitor 1 and Progenitor 2 are crossed. In one example of use of the present invention to produce hybrid seeds, Progenitor 1 is designed to be sterile male in the presence of juvenile hormone or one of its agonists, while Progenitor 2 is designed to be sterile female in the presence of juvenile hormone or one of its agonists. By contacting Progenitor 1 and Progenitor 2 with the juvenile hormone or one of its agonists, the only successful seed production will be a result of the Progenitor 2 pollen fertilizing the Progenitor 1's ovules. In a second example of Use of the present invention, Progenitor 1 is designed to be sterile male in the absence of juvenile hormone or one of its agonists, and Progenitor 2 is designed to be sterile female in the absence of the juvenile hormone or one of its agonists. By contacting Progenitor 1 and Progenitor 2 with the juvenile hormone or one of its agonists, maintenance of each line is allowed through self-fertilization. To produce hybrid seeds, the two parental lines are interplanted, and only hybrid seeds are obtained. Fertility is restored for the hybrid plants of the progeny, by means of an introduced restoration gene. By this means, any desired hybrid seeds can be produced. Chemical control of the transgenic expression of the plant may be useful to regulate its large, large developmental changes in the target crop, to change the crop plant to be more compatible with a hostile environment, to alter the flow of metabolic pathways specific, or simply to induce the high-level expression of a single desired protein product. Chemical control of plant development programs can allow the farmer to decide when specific events begin, such as the development of the flower, the separation of the leaf or fruit, or other important stages of development. This chemical control over specific development events would allow the farmer to have greater flexibility in the time of planting and harvesting, as well as allowing him to respond to specific environmental conditions, such as the forecast of an early winter or a wet spring. These changes could be made by controlling genes critical for development or for the trajectory of conditional response, in analogy to the homeotic genes of Drosophila.

It may also be useful to control the genetic expression of metabolic trajectories. It is believed that only a fixed amount of energy can be expended by the plant, and that any improvement of a specific path, such as the biosynthesis of storage proteins, will result in a simultaneous loss of biosynthetic flux through the pathways that they compete for energy, such as the synthesis of starch or lipids. In a situation where these changes in biosynthetic trajectories were only acceptable after the plant had reached a certain stage of development (ie, maturation versus growth), chemical regulation of gene expression could be useful, or perhaps even would require, for specific biosynthetic changes to be achieved. The chemical regulation of gene expression may be useful to overexpress a specific protein at high levels. It is known that certain proteins are toxic to the cell when expressed in a heterologous host, a foreign subcellular compartment, or even when they are expressed at an extraordinarily high level. The chemical control of the expression of these proteins can be convenient for the normal growth of the plant, or even it can be required to obtain sufficient mass of the plant to justify the use of the plant for a factory of biosynthetic proteins. Proteins that may be useful for large scale biosynthesis in plants include industrial enzymes, pharmaceutical proteins, antigens, as well as other proteins. Bioassays are known to identify ligands for steroid hormone receptors (Evans et al., US Pat. No. 5,298,429). The receptors given are limited to the glucocorticoid, mineralocorticoid, estrogen-related, and thyroid hormone receptors. These receptors were transformed into mouse kidney cell cultures (CV-1 or COS cell lines), and were tested for their ability to transactivate the expression of a chimeric CAT gene in the presence of an appropriate mammalian hormone. The transformation of plant cells wthese receptors, the construction of genes that can be expressed in plants, or other receptors different from those that have a mammalian hormone as a ligand are not disclosed. i o It was suggested that the USP is a "insect retinoid receptor" in Gold and Evans, International Publication Number WO 91/14695. In a prophetic example, the USP encoding XR2C is transformed into insect cell cultures (S2 cell line) to transact a chimeric CAT gene in response to the addition of retinoic acid. The disclosure suggests that the insect or animal cells 1 transformed wthese "insect retinoid receptors" can be used to select compounds that are capable of driving the activation of the receptors. However, Oro and colleagues later reported that USP is not activated wretinoic acid in Drosophila cell culture assays, and under conditions in which RXR responds, USP does not respond to any of the 0 tested retinoids or juvenoids , including methoprene acid (Harmon et al. and references therein). Wthe discovery of the present, that the juvenile hormone and its agonists are the ligand for USP, and that USP can be used to activate the expression of the target gene in the presence of the juvenile hormone or one of its agonists in cells of plant, it is now possible to discover new legands for the USP receptor that are effective in an environment of plant cells. The selection is based on the expression of an objective expression cassette encoding a reporter gene regulated by the receptor in plants or in cells of transgenic plants, which also express a transgenic USP receptor polypeptide., and optionally a secondary receptor polypeptide other than the USP receptor polypeptide. The chemicals to be tested for their ability to induce USP receptor mediated activation of the expression of the target polypeptide are contacted with the transgenic plant or with the plant cells in different concentrations, after which conducts an assay for the expression of the reporter gene, in order to determine the expression of the target polypeptide. Test substances showing activation or a statistically significant increase in expression of the target polypeptide, as well as test substances showing inhibition or a statistically significant decrease in expression of the target polypeptide, are identified as ligands of the USP receptor polypeptide. The method allows previously unknown test substances to be ligands for USP in an environment of plant cells, to be identified as such, or to confirm a test substance that is suspected to be a USP ligand in a cell environment of plant. Accordingly, it is now possible to produce ligands of the USP receptor polypeptide, by performing the following steps of the process: synthesizing novel test substances according to routine methods known in the chemical art; transforming a plant cell with a cassette of USP receptor expression encoding a USP receptor polypeptide, and an objective expression cassette encoding a target polypeptide; cultivate progeny cells of these transformed plant cells; expressing the USP receptor polypeptide in the cells of the progeny; - contacting a cell of the progeny with a novel test substance as synthesized above; and determining the expression of the target polypeptide; repeat the previous two steps of the process with other novel test substances; - selecting a test substance that activates or significantly inhibits the expression of the target polypeptide; and repeat the chemical synthesis of the selected substance.

Ligands of the USP receptor polypeptide that can be obtained by following the above process steps, constitute an additional subject matter of the present invention. Additionally, this method can be used to identify antagonists, or inhibitors, of the USP receptor-mediated activation of the expression of the target polypeptide. These antagonists are identified by their ability to reduce the activity induced by the ligand of the expression of the target polypeptide. Different reporter genes can be used as the target expression cassette in the screening method. A useful reporter is firefly luciferase. Its use in a target expression cassette is described in Examples 7 and 9 below. Another useful reporter gene is GUS, or glucuronidase, which catalyzes the dissociation of a chromogenic substrate, such as 5-bromo-4-chloro-3-indolyl-β-D-glucuronide or o-nitrophenyl-β-D-glucuronide. The GUS reporter has the advantage of producing a chromogenic reaction product that can be detected quantitatively, for example, by spectrophotometry, or qualitatively by visual inspection. The receptor expression cassettes useful in the screening method are USP, or the chimeric versions of USP, such as USP-VP16 or VP16-USP. Since the USP is related in sequence to the mammalian RXRa receptor, and the RXR is capable of forming heterodimers with EcR (Thomas et al., Nature 362: 471-475 (1993)), expression cassettes of the receiver that encode RXR in the tracking method. In this way, it can be discovered to what extent the ligands for USP are useful as ligands for RXR in plant cells, or chemical substances other than juvenile hormone or one of its agonists can be identified, as chemical ligands suitable for RXR in cells of plant.

EXAMPLES The following examples further describe the materials and methods employed in carrying out the invention, and the subsequent results. They are offered by way of illustration, and their text should not be considered as a limitation of the claimed invention. Example 1: Construction of an Expression Cassette of the Expressable Receptor in the Plant that Codifies the Ecdysone Receptor The coding region of DNA for the Ecdysone Receptor (EcR) I O of Drosophila, was isolated from a cDNA library derived from Chrysalides of Canton S (day 6) prepared in? Gtl 1 (Clontech, catalog number IL 1005b), and from fragments generated by genomic polymerase chain reaction with oligonucleotides designed from the published sequence of EcR isoform B1 (Koelle et al., Cell 67:59, 1991). The The sequence of the B1 isoform of EcR was confirmed by automated sequence analysis, using conventional methods, and aligning with the published sequence (Talbot et al., Cell 73: 1323, 1993). The coding region of Full length EcR expressed was modified to contain a BamHl site immediately upstream from the start codon, using the 0 oligonucleotide SF43 (5'-CGC GGA TCC TAA ACA ATG AAG CGG CGC TGG TCG AAC AAC GGC-3 '; SEQ ID NO: 1) in a polymerase chain reaction. The plant expression vectors pMF6 and pMF7 contain a 35S promoter from Cauliflower Mosaic Virus (CaMV 35S), an Adhl 1 intron from maize, and a termination and polyadenylation signal from nopaline synthetase (see Goff et al., Genes and Development 5: 298, 1991). Vectors pMF6 and pMF7 differ only in the orientation of the polylinker used for the insertion of the desired coding sequence. The full-length EcR coding sequence was ligated into the expression vector of CaMV 35S in plant pMF6, by using the flanking BamHI restriction sites. This cassette of receptor expression is referred to as 35S / EcR.

Example 2: Construction of a Expressable Receptor Expression Catheter in the Plant that Encodes the Ultraspiracle Receptor The cDNA encoding the native Ultraspiracle Receptor (USP) of Drosophila, is described by Henrich et al., Nucleic Acids Research 18: 4143 (1990). The full-length USP coding sequence, with the 5 'and 3"flanking non-translated regions, was ligated into the plant expression vector pMF7 (described in Example 1), using the EcoRI flanking restriction sites. This receiver expression cassette is referred to as 35S / USP.

Example 3: Construction of a Receptor Expression Cassette having the DNA Fixation Domain from GAL4, and the Ligand Fixation Domain from EcR A cassette for expression of the receptor was constructed, wherein the binding domain of the EcR DNA is replaced by the DNA binding domain of GAL4 fused at the N-terminal position. The coding region of the DNA for the Drosophila EcR was obtained as described in Example 1. The coding sequence for the domain of GAL4 DNA binding was subcloned from plasmid pMA210. Ma and Ptashne, Cell, 48: 847 (1987). An expression cassette of the receptor encoding a GAL4-EcR chimeric receptor polypeptide was constructed by fusion of the GAL4 DNA binding domain with the ligand binding domain and the carboxy terminus of EcR. To make the fusion, the oligonucleotide SF23 (5'-CGC GGGATC CAT GCG GCC GGA ATG CGT CGT CCC G-3 ', SEQ ID NO: 2) was used to introduce, by polymerase chain reaction, a BamHl site in the cDNA sequence for EcR at the position of the nucleotide equivalent to amino acid residue 330 (immediately following the DNA binding domain of EcR). The resulting truncated EcR coding sequence (EcR330"878) was subcloned into the plasmid pKS + (Stratagene) .A subclone of GAL4 was obtained from plasmid pMA210, which contained the coding sequence of the DNA binding domain (amino acids 1). -147), by subcloning the DNA sequence encoding the amino terminus of GAL4 into the ClaS site of pSK + (Stratagene) as described above (Goff et al., Genes and Development 5: 298, 1991). designated as pSKGAL2, and cut with Clal and Kpnl, and the following double-stranded oligonucleotide was inserted: The resulting plasmid was designated PSKGAL23. The complete fusion of 35S / GAL4-EcR330 878 was generated using the BamH1 sites in the polylinkers flanking the GAL4 DNA binding domain in pKS +, and the EcR330 fraction "878 in pKS +." These coding sequences were ligated into the vector 5 of monocotyledonous expression pMF6 (described in Example 1) by using the EcoRI flanking restriction sites This cassette of expression of the receptor is referred to as 35S / GAL4-EcR330"878.

Example 4: Construction of an Ise expressible receptor cassette in plants, which has a ligand binding domain from ultraspiracle, and the transactivation domain from VP16. A receiver expression cassette was constructed, which comprises the binding domain of the USP ligand with the transactivation domain of VP16 fused with the term N or with the C-terminus of the USP polypeptide. To construct the expression cassette of the receptor encoding a chimeric polypeptide having the transactivation domain of VP16 in the C-terminal position, the carboxy terminus and the stop codon of the cDNA for the receiving USP (described in Example 2) were removed. by subcloning into pKS + 0 (Stratagene) using the Xhol site at the nucleotide number 1471 of USP of the coding sequence. The resulting USP subclone, encoding amino acids 1 to 490, was fused to the VP16 domain, using the Kpnl restriction site flanking the USP subclone, and the Kpnl site of pSJT1 193CRF3 encoding the carboxy-terminal 80 amino acids from VP16 (Triezenberg et al., Genes Develop, 2: 718-729 (1988)). The resulting USP-VP16 fusion was cloned into the pMF7 plant expression vector of Camv 35S (described in Example 1), using the restriction enzyme sites EcoRI and BamH1 flanking the USP-VP16 coding sequence. This cassette of receptor expression is referred to as 35S / USP-VP16. The USP derivative, with the transcription activation domain fused to the amino terminus, was constructed by first designing a BamHl site adjacent to the USP start codon, using the oligonucleotide SF42 (5'-CGC GGA TCC ATG GAC AAC TGC GAC CAG GAC-3 '; SEQ ID NO: 5) in a polymerase chain reaction. The stop codon was removed in VP16, and a flanking BamH1 site was introduced, using oligonucleotide SF37 (5'-GCG GGA TCC CCC ACC GTA CTC GTC AAT TC-3 '; SEQ ID NO: 6), and introduced a start codon with a plant consensus sequence immediately upstream of the start codon, as well as a BamH1 site, at the end of the amino terminus, using oligonucleotide SA115 (5'-GTC GAG CTC TCG GAT CCT AAA ACA ATG GCC CCC CCG ACC GAT GTC-3 '; SEQ ID NO: 7) as primers in a polymerase chain reaction. The resulting VP16 activation domain, and USP coding sequence (encoding amino acids 1 to 507) were joined in-frame by the adjacent BamH1 sites, and the VP16-USP coding sequence was inserted into the vector of expression in pMF7 plant of CaMV 35S, through the 5 'BamHl and 3' EcoRl sites. This cassette of receptor expression is referred to as 35S? / P16-USP.

Example 5: Construction of a Capsule of Expression of the Receptor, which has the Domain of DNA Fixation and the Domain of Fixation of Ligand from EcR, and the Domain of Transactivation to 5 from the Regulator Gene C1 of Maize The fusion was generated EcR227825-C1, by placing a start codon immediately before the binding domain of the EcR DNA, with the oligonucleotide SF30 (d'CGC-GGA-TCC-ATG-GGT-CGC-GAT-GAT-CTC-TCG -CCT-TC-3 '; SEQ ID NO: 8) used in a polymerase chain reaction, on the full-length EcR coding sequence. The coding sequence for the transcriptional activation domain (amino acids 219-273) of the corn protein C1 (Goff et al., Genes and Develop. 5: 298-309 (1991)) was fused into the frame, with the sequence of coding for amino acids 51 to 825 of EcR (at the site of the restriction enzyme Kpnl of EcR). The domain of transactivation C1 was linked to EcR by a polylinker encoding VPGPPSRSRVSISLHA (SEQ ID NO: 9). The fusion of the plant expression vector 35S / EcR227 825-C1 was constructed by inserting a BamHI fragment carrying the coding sequence into the pMF7 vector. This cassette of receptor expression is referred to as 35S / EcR227 825-C1. 0 Example 6: Construction of a Receptor Expression Cassette having the DNA Fixation Domain from GAL4, the Ligand Fixation Domain from EcR, and the Transactivation Domain from the C1 Regulating Gene of Maize Se built a GAL4-EcR330 825-C1 fusion using the construction GAL4-EcR330-878 described in Example 3, and construction EcR227 825-C1 of Example 5. The sequence of the coding region of EcR (starting at the amino acid 456) was exchanged at the Aatll site. This cassette of receptor expression is referred to as 35S / GAL4-EcR330"825-C1.

Example 7: Construction of an expressible objective expression cassette in i or Plants, which encodes Lucifernaga Luciferase, which has the Response Element for the GAL4 DNA Fixation Domain The expressible target expression cassette was constructed, which encodes the firefly luciferase, which has the response element for the DNA binding domain of GAL4, in the following manner. The promoter of the core Bronze-1 (Bz1) of corn, which drives the synthesis of firefly luciferase, of the reporter pBz1 LucR98 of Bz1 (Roth et al., Plant Cell 3: 317, 1991), was removed by means of the Nhel sites. and Sphl, and placed on a plasmid 0 derived from pUC6S carrying the luciferase gene. The modified ßz7 core promoter contains an Nhel site (GCTAGC), and the Bz1 promoter is sequenced to the position of nucleotide -53 (Roth et al., Plant Cell 3: 317, 1991). Ten binding sites of GAL4 were removed from the reporter pGALLuc2 regulated by GAL4 (Goff et al., Genes and Development 5: 298, 1991), by digestion with EcoRI and Pstl, and inserted into pBlueScript (Stratagene) using the same sites of restriction enzyme. The Hindlll site at the 5 'end of the GAL4 binding sites was changed to a BamHI site by the insertion of a HindIII / BamHI / HindIII adapter, and the resulting BamHl fragment containing the GAL4 binding sites was removed. , and was placed in a Bgll site upstream of the Bz1 core promoter that drives the luciferase. This target expression cassette is referred to as (GAL4b s) 10-Bz1TATA / Luc. io EXAMPLE 8: Construction of a Target Expression Target Capsule, Which Encodes Lucifernaga Luciferase, Which Has a Direct Repetition Response Element The expression cassette expressible target in plants, which encodes firefly luciferase, which has a direct repeat response element (DR), with a half-site of EcRE, and a half-site preferred by RXR, was constructed in the following manner: the core promoter Bz1 construct of corn-luciferase was used in the plasmid derived from pUC6S, as described in Example 7, as the starting point. Synthetic double-layered oligonucleotides containing a direct repeat response element and a separation of three base pairs between the half sites were synthesized, with cohesive ends BamHl and Bgll (SF77: 5'-GAT CCG TAG GGG TCA CGA AGT TCA CTC GCA-3 '; SEQ ID NO: 10) (SF78: 5'-GAT CTG CGA GTG AAC TTC GTG ACC CCT ACG-3 '; SEQ ID NO: 11), were phosphorylated, annealed, and ligated upstream of the Bz1 core promoter by its insertion in a unique Bglll site. Three copies of the response element were obtained by digestion with BglII in sequence, and insertion of additional double-stranded oligonucleotides. This target expression cassette is referred to as (DR3) 3-Bz1TATA / Luc. Example 9: Transformation of Plant Cells, and Control of Expression of the Target Polypeptide by the Receptive Polypeptides in the Presence of a Chemical Ligand Control of the Expression of the Target Polypeptide by Different I or receptor polypeptides, including the chimeric receptor polypeptides of the present invention, can be demonstrated by the simultaneous transformation of plant cells with the necessary genetic constructs, using high-speed microprojectile bombardment, followed by biochemical assay for the presence of the target polypeptide. The necessary genetic constructions comprise a cassette expression of the USP receptor encoding a USP receptor polypeptide (Figure 1 ). Optionally, the expression cassette of the USP receptor can be transformed together with a secondary receptor expression cassette encoding a receptor polypeptide other than USP (Figures 2 and 3). In addition, an objective expression cassette encoding a target polypeptide is also necessary. 0 The expression cassettes were delivered simultaneously to cells in suspension of corn grown in a liquid N6 medium (Chu et al.

Scientia Sinica XVII 1: 659-668, 1975) by bombarding microprojectiles at high speed, using conventional techniques of DNA precipitation on microprojectiles, and high speed bombardment driven by compressed helium (PDS-1000 / He, BioRad, Hercules, CA ). The transfected cells were incubated in the liquid suspension in the presence of the appropriate chemical ligand for about 48 hours in the N6 medium. After incubation, the transformed cells were harvested, and then homogenized to ODC. Extract waste was removed by centrifugation at 10,000 g at 4DC for 5 minutes. Expression of the target polypeptide was detected by the assay of the extract in the presence of the product encoded by the target expression cassette. A coding sequence commonly used for the target polypeptide, when testing the expression control by the receptor polypeptides in the presence of a chemical ligand, is firefly luciferase. Firefly luciferase activity is determined by quantification of the chemiluminescence produced by luciferin phosphorylation catalyzed by luciferin luciferase, using ATP as the substrate (Promega Luciferase Case, catalog number E1500), using an Analytical Luminescence Luminometer 2001 model.

Example 10: The GAL4-EcR33 (, 25-C1 and USP-VP16 Receptor Polypeptides Activate Expression of a Target Polypeptide in Plant Cells, and Activation is Blocked by Juvenile Hormone Agonists Using the transformation method of Example 9, the expression cassette of the 35S / GAL4-Ecr330825-C1 receptor (Example 6), the expression cassette of the 35S / USP-VP16 receptor (Example 4), and the target expression cassette (GAL4b s) 10- were co-transformed. Bz1TATA / Luc (Example 7) in maize cells Transformed cells were incubated in the presence of 10 μM of fenoxycarb or methoprene as chemical ligands for about 48 hours Luciferase assays were performed as described in Example 9. The results are presented in Table 1.

Table 1 Chemical Ligand Receptor Luciferase activity (light units) Experiment # None None 2,503 35S / GAL4-EcR330"8 5-C1 + None 277,862 35S / USP-VP16 Fenoxicarb 77,418 35S / GAL4-EcR330-825-C1 + Metoprene 14,786 35S / USP-VP16 35S / GAL4-EcR330-825-C1 + 35S / USP-VP16 Experiment # 2 None None 1, 302 35S / GAL4-EcR330-825-C1 + None 178,092 35S / USP-VP16 Metoprene 5,730 35S / GAL4-EcR330 825-C1 + 35S / USP-VP16 The above results show that the 5 'regulatory region of the cassette of target expression comprising the response elements of GAL4, can be activated in plant cells by the GAL4-EcR-C1 and USP-VP16 receptor polypeptides, and that this activation is reversed in the presence of a juvenile hormone agonist. The level of expression of the luciferase of the target polypeptide was 3.5 to 31 times lower in the presence of juvenile hormone agonists, compared to its absence.

Example 11: GAL4-EcR33M 5-C1 and USP-VP16 Receptor polypeptides Activate Expression of a Target Polypeptide in Plant Cells, and Activation is Blocked by Juvenile Hormone Agonists Using the transformation method of Example 9, they were cotransformed the expression cassette of the 35S / GAL4-EcR330825-C1 receptor (Example 6), the expression cassette of the 35S / USP-VP16 receptor (Example 4), and the target expression cassette (GA4b s) 10Bz1TATA / Luc (Example 7) ), in corn cells. Transformed cells were incubated in the presence of 10 μM tebufenozide with and without 10 μM phenoxycarb or methoprene as chemical ligands for approximately 48 hours. Luciferase assays were performed as described in Example 9. The results are presented in Table 2.

Table 2 Chemical Ligand Receptor Luciferase activity (light units) None None 1, 302 35S / GAL4-EcR330-825- None 178.092 C1 + 35S / USP-VP16 Tebufenozide 908.912 35S / GAL4-ECR330"825- Tebufenozide + Metop 159.873 C1 + 35S / USP-VP16 35S / GAL4-EcR330" 825- C1 + 35S / USP-VP16 The above results show that the 5 'regulatory region of the target expression cassette, which comprises the GAL4 response elements, can be activated in plant cells by the GAL4-EcR-C1 and USP-VP16 receptor polypeptides in response to the ligand. chemical of tebufenozide, and that this activation is blocked in the presence of the juvenile hormone agonist: methoprene. The level of activation induced by tebufenozide from the expression of I or luciferase gene was increased by approximately 6 times when used alone, and completely blocked by the presence of the juvenile hormone agonist: methoprene.

Example 12: The VP16-USP Receptor Polypeptide Activates the Expression of a Target Polypeptide in Plant Cells Using the transformation method of Example 9, plasmids containing the 35S / VP16-USP receptor expression cassette (Example 4), and the target expression cassette (DR3) 3-Bz1TATA / Luc (Example 8) were co-transformed. in corn cells. Transformed cells were incubated in the presence of 10 μM methoprene as a chemical ligand for 48 hours. Luciferase assays were performed as described in Example 9. The results are presented in Table 3.

Table 3 Chemical Ligand Receptor Luciferase activity (light units) None None 5,690 35S / VP16-USP None 29,967 35S / VP16-USP Metoprene 485,458 The above results show that the 5 'regulatory region of the target expression cassette, comprising the direct repeat response elements, can be activated in plant by the VP16-USP receptor polypeptide in the presence of a juvenile hormone agonist. The level of expression of the luciferase of the target polypeptide was approximately 16 times higher than that observed in the absence of the juvenile hormone agonist.

Example 13: Construction of Vectors to Transform Arabidopsis Plants Expressing Derivatives of EcR, USP, or RXR, and i or carrying a Reporter Regulated by the Recipient Agrobacterium T-DNA vector plasmids were constructed from the plasmids described above pGPTV-Kan and pGPTV-Hyg (Becker et al., Plant Mol. Biol. 20: 1195-1 197, (1992)). The uidA reporter gene (GUS) Sacl / Hindlll from both plasmids pGPTV-Kan and pGPTV-Hyg was replaced by the polylinker Sacl / Hindlll from pGEM4Zf (+), pSPORTI, pBluescriptKS (+), plC20H, or pUC18, to give the plasmids pSGCFW, pSGCFX , pSGCFY, pSGCFZ, pSGCGA, pSGCGC, pSGCGD, pSGCGE, pSGCGF, and pSGCGG, respectively. A luciferase reporter regulated by GAL4 was constructed as the target expression cassette, as a T-DNA Agrobacterium plasmid, first subcloning a 328 base pair Kpnl / Hindlll fragment with ten GAL4 binding sites and one Bronze- 1 TATA of corn, as described in Example 7, at the Kpnl / Hindlll sites of the modified luciferase reporter plasmid pSPLuc + (Promega), to create plasmid pSGCFOL A 1,991 Kb Kpnl / Xbal fragment was subcloned from pSGCFOl, which contained the GAL4-Bz1 TATA-reporter Luciferase reporter binding sites, into a T-DNA vector by means of ligand to a Ndel / Spel fragment of 7,194 kb of pSGCFXI, and a Ndel / Kpnl fragment of 4,111 kb of pSGCFZI described above. The resulting plasmid was designated pSGCGLI, and carries a selectable NPTII marker driven by a promoter that confers resistance to kanamycin in the transgenic plant, a luciferase reporter regulated by GAL4. A GUS reporter regulated by GAL4 with 10 GAL4 fixation sites, a 35S TATA region, and a GUS coding region, was constructed in a similar manner, and designated as pAT86. A direct repeat response element (DR) reporter was also constructed with 3 copies of the direct repeat response element, a Bz1 TATA, a luciferase coding region, and a terminator analogous to that described in Example 8, of a manner similar to that described for pSGCGLI, and designated pSGCHUL. The receptor expression cassettes described in Examples 3 to 6 above were used to construct analogue Agrobacterium T-DNA constructs carrying the CaMV 35S promoter, and the signals of polyadenylation of nos. Single and double receptor constructs were generated by subcloning the appropriate expression cassette into the pSGCGLI reporter of GAL4 luciferase: Example 14: Generation of Transgenic Arabidopsis expressing VP16-USP, and carrying a Direct Repetition Luciferase Reporter Arabidopsis thaliana (Columbia) was transformed with an Agrobacterium vector harboring a CaMV 35S promoter, and a luciferase reporter from direct repetition (as described in Example 13 above), following the vacuum infiltration procedure. Electrocompetent Agrobacterium 5 GV3101 cells were prepared by incubating Agrobacterium GV3101 in 2 times the YT medium at 28DC with aeration for 24 to 30 hours, to an OD600 of 0.5 to 0.7 units. The cells were frozen on ice for 10 to 30 minutes, and centrifuged at 5,000 RPM for 5 minutes at 4DC. The supernatant was discarded, and the cell pellet was resuspended in a volume of 10 percent glycerol or ice cream. The cells were centrifuged again at 5,000 RPM for 5 minutes at 4DC. The supernatant was discarded, and the cell granule was resuspended in 0.05 volumes of ice-cold 10% glycerol. The cells were centrifuged again at 5,000 RPM for 5 minutes at 4DC. The supernatant was discarded, and the cell granule was resuspended in 0.02 volumes of 10 glycerol. cent ice cream. The cells were centrifuged again at 5,000 RPM for 5 minutes at 4pC. The supernatant was discarded, and the cell granule was resuspended in 0.02 volumes of ice-cold 10% glycerol. The cells were again centrifuged at 5,000 RPM for 5 minutes at 4 C. The supernatant was discarded, and the cell pellet was resuspended in 0.01 volumes of ice cold glycerol or 10 percent. The cells were aliquoted in amounts of 200 milliliters by 1.5 milliliter microfuge tubes, quickly frozen in liquid N2, and stored at -80 ° C. The electrocompetent cells were used before 6 weeks of storage at -80pC. Frozen electrocompetent cells were thawed on ice, and 40 milliliters were transferred to a previously frozen 1.5 milliliter microfuge tube. 1 milliliter of the appropriate Agrobacterium plasmid DNA (2 to 10 nanograms) was added to the thawed cells, and mixed on ice. The cell / plasmid mixture was transferred to a 0.2 cm BioRad electroincorporation cuvette previously frozen, and electroencupied at 2.0 KVolts, 600 Ohms, 25 microfarads, and with a time constant of 6 milliseconds. Media 1 ml of 2x YT was added to the electrocorporation cuvette, the cell / plasmid solution was mixed with a pipette tip, and the contents transferred to a fresh 1.5 milliliter microfuge tube. The cells were then incubated at 37 C! C for 1 hour on a shaker at 200 RPM. The cells were centrifuged for 2 minutes at position 6 in an Eppendorph adjustable speed microcentrifuge, the supernatant was decanted, and the cell granule was resuspended in the remaining liquid. The resuspended cells were spread on a medium plate LB with the appropriate antibiotic. Plates were incubated at 28-30 CC for 2 to 3 days. A culture of 50 milliliters of LB was inoculated with a single colony transformed into a 250 milliliter flask with Rifampin at 100 micrograms / milliliter, and Gentomycin at 25 micrograms / milliliter, and kanamycin at 100 micrograms / milliliter. The culture was incubated for 24 to 36 hours at 28 LJC at 250 RPM, and 10 milliliters of the culture was used to inoculate 500 milliliters of LB + antibiotics in a 2 liter flask. This culture was incubated overnight at 28 C with shaking at 250 RPM. Plasmid DNA was isolated from this Agrobacterium culture, and verified by restriction analysis. Arabidopsis plants were cultivated in a mesh covered with earth, in square plastic pots of 7.62 centimeters in a phytotron adjusted for 16 hours of light, 8 hours of darkness, at 20ºC, for 4 to 5 weeks. The plants were grown until the floral meristem was approximately 5.08 centimeters tall. The floral meristems of Arabidopsis plants to be transformed were removed 2 days before exposure to Agrobacterium. The Agrobacterium culture was centrifuged at 5, 000 RPM for 5 minutes, and the resulting granule was resuspended in 500 milliliters of Infiltration Media (4.3 grams of MS / Liter salts, 5 percent sucrose, 0.01 milligrams / milliliter of benzylaminopurine, 100 milliliters / liter of Silwet L77, pH of 5.8). The Arabidopsis plants were soaked in water until the soil was saturated. 500 milliliters of the bacterial cell suspension were transferred to the bottom of a sterile vacuum desiccator, and the Arabidopsis plants in their pots were placed turned down in the Agrobacterium solution. A vacuum was applied to the desiccator for 5 minutes, and then released slowly. This vacuum treatment was repeated three times, the plants were rinsed of excess Agrobacterium, and returned to the culture chamber. Plants infiltrated under vacuum were allowed to mature, bloom, and produce seeds. The resulting seeds were further dried in a drying chamber with low humidity at 95 ° C for about 5 to 10 days. The seeds were removed from the dried flowers by milling, then filtered through a 425 micron sieve. Approximately 5 weeks are needed to obtain seeds after vacuum infiltration. Once completely dry, approximately 240 milligrams of seeds were sterilized by adding 1 milliliter of 70 percent EtOH, centrifuged completely, and incubated for 2 minutes at room temperature. The seeds were centrifuged briefly at high speed in an Eppendorf microcentrifuge, and the supernatant was removed. The granulated seeds were resuspended in 1 milliliter of sterilization buffer (one part of 10 percent Triton X-100, 10 parts of bleach, 20 parts of dd H2O), centrifuged, and incubated at room temperature during 30 minutes. The seeds were centrifuged briefly at high speed in an Eppendorf microcentrifuge, and the supernatant was removed. The seeds were resuspended in 1 milliliter of sterile H2Odd, swirled, centrifuged at high speed in a microcentrifuge, and the solvent was removed. This wash step was repeated three times, and then the seeds were transferred to a 50 milliliter centrifuge tube. for a final wash in 5 milliliters of H2Odd. The seeds were centrifuged briefly at a higher speed in a Beckman table centrifuge. The supernatant was decanted, and the seeds were resuspended in 24 milliliters of sterile low melting point agarose at 0.8 weight percent / volume, at 50 ° C, mixed, and formed into 8 milliliter aliquots in each of three plates of germination medium (GM) 150 millimeters (Murashige and Skoog, Physiol Plant 51: 473-497, 1962), containing antibiotic for selection (either 50 micrograms / milliliter of kanamycin, or 50 micrograms / milliliter of hygromycin), and 500 micrograms / milliliter of carbenicillin. The coated seeds were incubated at 4HC in the dark for 24 hours, then taken to a growth chamber set at 20 ° C with a cycle of 16 hours of light and 8 hours of darkness per day. The germinated seedlings were selected on plates for 5 to 10 days, the seedlings were transplanted to fresh selection plates, and transplanted to the soil immediately after 5 to 10 days of further selection. The recently transplanted seedlings were covered with a plastic wrap for 2 to 3 days, and then grown until the initiation of the floral meristems.

Example 15: Chemical Induction of Isolated Transgenic Plant Tissues The transgenic plants were tested for inducible gene expression by the following technique. Two leaves of approximately the same size as the transgenic plant were removed, and were incubated in water containing 50 micrograms / milliliter of kanamycin (or 25 micrograms / milliliter of hygromycin if the transgene carried this marker), with 0.1 percent ethanol or 0.1% ethanol and 10 μM methoprene or phenoxycarb. The leaves were incubated for approximately 24 hours under standard growth conditions described in Example 14. Following incubation with the inducing compound, a leaf extract was prepared by homogenizing the sheet in 500 milliliters of 100 mM KPO4, DTT 1 mM, regulator of a pH of 7.8, to OpC. The extracts were centrifuged for 5 minutes in an Eppendorf microcentrigo at 4] C, and stored at 0 ° C until assayed. The luciferase activity in each extract was determined using a Model 2010 Analytical Luminescence Luminometer, and the Promega Luciferase Assay System, according to the manufacturer's recommendations. The protein concentration of the extract was determined using the Pierce BCA Protein Assay (Smith et al., Anal. Biochem. 150: 76-85). Luciferase values are expressed as light units for 10 seconds at room temperature per 100 micrograms of extract protein. Treatment with phenoxycarb resulted in a 6.2 fold increase in luciferase activity, and methoprene resulted in a 25-fold increase in luciferase activity, as shown in the following Table 4.

Table 4 Chemical Ligand Receptor Luciferase activity (light units) None None 638 35S / VP16-USP Fenoxicarb 3,991 35S / VP16-USP Metoprene 15,790 Example 16: Tracking of New Ligands that are Fixed to USP, Using Plants or Cells of Transgenic Plants Expressing USP or RXR Derivatives, and Carrying a Reporter Regulated by the Recipient New ligands for USP or RXR that are effective in an environment of plant cells, by utilizing a selection method based on the expression of a reporter regulated by the receptor as the target expression cassette in transgenic plant or plant cells that also express the appropriate receptor polypeptide. In this way, the chemical substances to be tested for their ability to mediate the activation of USP or RXR of the expression of the target polypeptide are contacted with a plant or with transgenic plant cells in varying concentrations, after which is conducted an assay for the expression of the reporter gene. For example, 1) the plants or cells of transgenic plants carrying a GAL4-regulated luciferase reporter as the target expression cassette, and the receptor expression cassettes for GAL4-EcR and USP-VP16, can be exposed to the substances to be tested, and compared with non-exposed plants using a light amplification instrument, such as the Hamamatsu Light Detection Device, 2) the plants or cells of transgenic plants carrying a GUS reporter regulated by GAL as the target expression cassette, and the receptor expression cassettes for GAL4-EcR-C1 and VP16RXR, can be exposed to the substances to be tested, and compared with non-exposed plants for their ability to catalyze the dissociation of a chromogenic substrate, such as 5-bromo-4-chloro-3-indolyl-β-D-glucuronide or o-nitrophenyl-β-D-glucuronide, 3) plants or transgenic plant cells carrying a reporter of lucifera sa regulated by the direct repeat response element, and an expression cassette for VP16-USP, can be exposed to the substances to be tested, and compared with non-exposed plants using a light amplification instrument, such as the Hamamatsu light detection device. Positive controls that may find utility in the above screening methods include, but are not limited to, tebufenozide and methoprene. In each of the above cases, a higher level of detection of the expression of the target polypeptide in the presence of a test substance, compared to the level of expression in the absence of the test substance, indicates that the tested substance is a ligand either for USP or for RXR, depending on the expression cassette of the receiver used in the method. In this way, test substances that were previously not known to be ligands for USP in a plant cell environment can be identified as such, or a test substance suspected of being a ligand can be confirmed. for USP in an environment of plant cells as such.

Example 17: Isolation of Receptor Polypeptide Mutants Having Lower Basal Activity Mutations were generated in the binding domain of the Ultraspiracle receptor ligand (USP) in vitro, using polymerase chain reaction mutagenesis, as described by Leung et al. , Technique 1: 11-15 (1989). Fragments of the polymerase chain reaction of the binding domain of the mutant USP ligand were cloned into a yeast expression vector operably linked to the transcription activation domain of VP16. The mutant constructs were transformed into the GGY1 :: 171 strain of the yeast GAL4 reporter. The yeast transformants were coated on the medium containing the X-Gal indicator. Mutants that had a decreased baseline level of USP receptor polypeptide activity for the heterodimer generated white to light blue colonies on the X-Gal indicator plates, whereas transformants expressing the non-mutagenized USP receptor polypeptide generated dark blue colonies. . The white to light blue colonies were tested to determine the basal level and induced by the chemical ligand of the receptor polypeptide activity, by culturing yeast cells representing those colonies selected in S medium containing glycerol, ethanol, and galactose as sources of carbon. The resulting culture was divided into two portions, one of which was treated with juvenile hormone or one of its agonists, and the other was used as a control in the absence of chemical ligand. After being exposed to the juvenile hormone or one of its agonists, both treated and control portions of the culture were assayed for β-galactosidase activity according to the Miller method (Experiments in Molecular Genetics, pages 352-355 , JH Miller, Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1972). The nucleotide sequences encoding the mutant receptor polypeptides isolated and identified by this technique are candidates for another test, since the receptor polypeptides they encode may exhibit, in plant cells, a lower basal activity, a greater fold induction of the expression of the target gene in the presence of juvenile hormone or one of its agonists, or a different response to different agonists of juvenile hormone.

Example 18: Identification of Mutant Receptor Polypeptides with Better Function in Plant Cells [0121] Receptor expression cassettes encoding the mutated USP receptor polypeptides of Example 15 were prepared according to Examples 2 and 4 above. These receptor expression cassettes, in combination with the target expression cassettes of Example 8, were transformed into plant cells according to the procedure of Example 9. The transformed plant cells were tested to determine the activation of the regulatory region. "of the target expression cassette by the mutant receptor polypeptides in the presence of juvenile hormone or one of its agonists. The mutant USP receptor polypeptides that produce, in the plant cells, a low basal expression of a target polypeptide in the absence of chemical ligand, and a high expression of the target polypeptide in the presence of juvenile hormone or one of its agonists, They are useful for controlling gene expression in plants. Example 19: Rearing the Progeny of the Transgenic Plants Transformed plants of Arabidopsis thaliana (Columbia) prepared in Example 14, in mesh covered with soil, in ½ square plastic pots of 7.62 centimeters, are cultivated in a phytotron apparatus to have 16 hours of light and 8 hours of darkness, at 20ºC, for 4 to 5 weeks. The plants contain, in their genome, foreign DNA in the form of the expression cassettes of the receptor and target according to the invention. The integrated DNA is transferred from one generation of plants to the next, through the process of fertilization, as a consequence of the life cycle of the transformed plant. Fertilization is a process by which the male gametophyte and sporophytic or gametophytic female tissues interact to achieve the successful production of a zygote. Mature pollen grains are produced in the anthers of the flower, and are deposited on the surface of stigma (pollination), where it hydrates and germinates to grow a pollen tube. The sperm cells of the pollen tube are delivered to the embryo sac present in the ovary (gynoecium) where the actual events of fertilization (gamete fusion) take place to produce the zygote. The zygote, in the form of a seed, is the realization of the next generation of a plant line. This next generation is called the 'progeny' of the transformed plant. Progeny can be formed by self-fertilization, where the male gametophyte and female gametophytic tissue leave the same individual plant. This means that a single plant is the source of genomic DNA for the next generation. Alternatively, the progeny can be produced by cross-fertilization of two separate plants, placing the male gametophyte of a plant in contact with the female sporophytic tissues of a separate plant, in order to produce the next generation of plants. In this case, the genomic DNA of the progeny is derived from two separate plants. In addition, when a transformed plant is cross-fertilized with an untransformed plant, the genomic DNA of the progeny is composed of transgenic genomic DNA from a plant, and non-transgenic genomic DNA from a separate plant. Regardless of whether the progeny of the transformed plant is produced by self-fertilization or cross-fertilization, some of the progeny will receive an unequal genetic contribution, due to the presence of foreign DNA integrated into the genome. This unequal genetic contribution can be determined using the techniques of classical genetics and molecular biology. In order to produce the next generation of plants containing the expression cassettes of the recipient and target according to the inven, the original transformed plants are allowed to ripen, flower, and produce seeds under controlled environmental conditions. The resulting seeds are further dried in a drying chamber with a low humidity at 35 ° C for about 5 to 10 days. The seeds are removed from the dried flowers by grinding the siliques, and then filtered through a 425 micron sieve, to separate the seeds from the other plant material. Then you can use the seeds to grow other generations of plants. This process for producing a next generation of transformed plants, although described for Arabidopsis, can be applied in general to all angiosperm plants having integrated in their genome, the cassettes of expression of the receptor and target according to the inven.

All publications and patent applications mened in this specification indicate the level of ability of the experts in the field to which this inven pertains. All publications and patent applications are hereby incorporated by reference to the same extent as if each individual patent application or publication was specifically and individually indicated as incorporated by reference. Although the above inven has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

LIST OF SEQUENCES (1. GENERAL INFORMATION: (i) APPLICANT: (A) NAME: CIBA-GEIGY AG (B) STREET: Klybeckstr. 141 (C) CITY: Basel (E) COUNTRY: Switzerland (F) ZIP CODE (ZIP): 4002 (G) TELEPHONE: +41 61 69 11 11 (H) TELEFAX: + 41 61 696 79 76 (I) TELEX: 962 991 (ii) TITLE OF THE INVENTION: YOUTH HORMONE OR ONE OF ITS AGONISTS AS A CHEMICAL LIGAND TO CONTROL THE GENETIC EXPRESSION IN PLANTS BY MEANS OF TRANSACTION MEDIATED BY THE RECEIVER. (iii) NUMBER OF SEQUENCES: 11 (iv) COMPUTER LEGIBLE FORM: (A) TYPE OF MEDIA: Flexible disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE : Patentln Relay # 1.0, Version # 1 30B (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 42 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear 10 (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "oligonucleotide SF43" (iii) HYPOTHETIC: NO I 5 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: CGCGGATCCT AAACAATGAA GCGGCGCTGG TCGAACAACG GC 42 (2) INFORMATION FOR SEQ ID NO: 2: 0 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "oligonucleotide SF23" (iii) HYPOTHETIC: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: CGCGGGATCC ATGCGGCCGG AATGCGTCGT CCCG 34 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "positive chain oligonucleotide used to create pSKGAL2.3" (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: CGGGGGATCC TAAGTAAGTA AGGTAC 26 (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (I) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "complementary chain oligonucleotide used to create pSKGAL2.3" (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: CTTACTTACT TAGGATCCCC 20 (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (¡I) UPO DE MOLECULA: other nucleic acid (A) DESCRIPTION: / desc = "oligonucleotide SF42" (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: CGCGGATCCA TGGACAACTG CGACCAGGAC 30 (2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "oligonucleotide SF37" (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: GCGGGATCCC CCACCGTACT CGTCAATTC 29 (2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "oligonucleotide SA1 15" (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: GTCGAGCTCT CGGATCCTAA AACAATGGCC CCCCCGACCG ATGTC 45 (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "oligonucleotide SF30" (iii) HYPOTHETIC: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: CGCGGATCCA TGGGTCGCGA TGATCTCTCG CCTTC 35 (2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS: 5 (A) LENGTH: 16 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear I O (ii) TYPE OF MOLECULE: peptide (iii) HYPOTHETIC: NO (v) TYPE OF FRAGMENT: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: 15 Val Pro Gly Pro Pro Ser Arg Ser Arg Val Ser lie Ser Leu His Wing 1 5 10 15 (2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS: 20 (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "SF77 primer" (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: GATCCGTAGG GGTCACGAAG TTCACTCGCA 30 (2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear ( ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "SF78 primer" (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: GATCTGCGAG TGAACTTCGT GACCCCTACG 30

Claims (31)

1. A transgenic plant cell, the material of the plant, or the plant and the progeny thereof, comprising: a) a cassette for expression of the USP receptor encoding a USP receptor polypeptide; and b) an objective expression cassette encoding a target polypeptide. I OR

2. The plant cell, the plant material, or the plant according to claim 1, wherein the expression of the target expression cassette interferes with the fertility of the plant.

3. A method for producing a plant cell or a plant according to claim 1, which comprises transforming a plant cell 15 or a plant, with: a) an expression cassette of the USP receptor encoding a USP receptor polypeptide; and b) an objective expression cassette encoding a target polypeptide.

4. The method according to claim 3, which comprises obtaining the progeny of the cell of the plant or of the transformed plant with these expression cassettes.

5. The plant according to claim 1, wherein the plant is corn.

6. The plant according to claim 1, wherein the plant is wheat.

7. A method for controlling gene expression in a plant according to claim 1, or this plant further comprising an expression cassette of a secondary receptor encoding a secondary receptor polypeptide other than the USP receptor polypeptide, comprising: a) expressing the polypeptide or receptor polypeptides in this plant; and b) contacting the plant with juvenile hormone or one of its agonists.

The method according to claim 7, wherein the expression of the target polypeptide is increased or activated in the presence of 15 juvenile hormone or one of its agonists.

The method according to claim 7, wherein the expression of the target polypeptide is decreased or inhibited in the presence of juvenile hormone or one of its agonists.

10. The method according to claim 7, wherein the 0 USP receptor polypeptide comprises a heterologous transactivation domain.

The method according to claim 10, wherein the heterologous transactivation domain is the transactivation domain from the VP16 protein of herpes simplex.

12. The method according to claim 7, wherein the secondary receptor polypeptide is selected from the group consisting of EcR, DHR38, and RXR.

The method according to claim 7, wherein the agonist is selected from the group consisting of phenoxycarb, di-phenolamine, quinoprene, methoprene, hydroprene, di-phenolamine, methoprene acid, triflumuron, hexamflumuron, teflubenzuron, flufenoxuron, flucycloxuron, and lufenuron, diflubenzuron, and chlorfluzuron.

The method according to claim 7, wherein the target expression cassette comprises a 5 'regulatory region comprising between 1 and 11 copies of a response element.

A method for controlling the fertility of a plant according to claim 2, or this plant further comprising a cassette for expression of the secondary receptor encoding a secondary receptor polypeptide, which comprises: a) expressing the polypeptide or polypeptides receptors in this plant. b) contact the plant with juvenile hormone or one of its agonists.

16. The method according to claim 15, wherein the expression of the target polypeptide is increased or activated in the presence of juvenile hormone or one of its agonists.

17. The method according to claim 15, wherein the expression of the target polypeptide is decreased or inhibited in the presence of juvenile hormone or one of its agonists.

18. The method according to claim 15, wherein the USP 5 or the secondary receptor expression cassette comprises an anther-specific promoter operably linked to the coding sequence for the receptor polypeptide. The method according to claim 15, wherein the USP or the expression cassette of the secondary receptor comprises a specific promoter. IO of the pistil operably linked to the coding sequence for the receptor polypeptide. 20. The method according to claim 15, wherein the target polypeptide is barnase ribonuclease. 21. The method according to claim 15, wherein the target expression cassette encodes an anti-sense sequence that renders fertilization ineffective. 22. The method according to claim 15, wherein the target polypeptide restores effective fertilization. 23. The method according to claim 22, wherein the target polypeptide is barstar ribonuclease inhibitor. 24. A method for identifying a ligand of a USP receptor polypeptide that, in a plant cell environment, activates or inhibits the expression of an objective expression cassette, comprising the steps of: a) transforming a plant cell with a cassette expression of the USP receptor encoding a USP receptor polypeptide and an objective expression cassette encoding a target polypeptide; b) expressing the USP receptor polypeptide in this plant cell; c) contacting the plant cell with a test substance; d) determining the expression of the target polypeptide; and e) identifying test substances that activate or inhibit in a significant manner the expression of the target polypeptide. 25. The method according to claim 24, further comprising transforming the plant cell with a secondary receptor expression cassette encoding a secondary receptor polypeptide other than the USP receptor polypeptide, and expressing the secondary receptor polypeptide. 26. The method according to claim 24, wherein the expression of the target polypeptide is determined qualitatively. 27. The method according to claim 24, wherein the expression of the target polypeptide is determined in a quantitative manner. 28. A method for producing a ligand of a USP receptor polypeptide, comprising the steps of: a) synthesizing novel test substances according to the processes known in the art; b) transforming a plant cell with a cassette of USP receptor expression by coding a USP receptor polypeptide and an objective expression cassette encoding a target polypeptide; c) culturing the progeny cells of these transformed plant cells; d) expressing the USP receptor polypeptide in the cells of the progeny; e) contacting a cell of the progeny with a test substance from step a); and f) determining the expression of the target polypeptide; g) repeating the steps of process e) and f) with a different test substance according to step a); h) selecting a test substance that activates or significantly inhibits the expression of the target polypeptide; and i) repeating step a) for the substance selected in process step h). 29. A ligand of a USP receptor that can be obtained by a process according to claim 28. 30. An agricultural method wherein use is made of plant material, or transgenic plants, which comprises: a) a cassette of expression of the USP receptor encoding a USP receptor polypeptide; and b) an objective expression cassette encoding a target polypeptide. 31. The use of juvenile hormone or a juvenile hormone agonist to control the expression of a target polypeptide in a plant according to claim 1.