HK1017381A - Juvenile hormone or one of its agonists as a chemical ligand to control gene expression in plants by receptor mediated transactivation - Google Patents

Description

juvenile hormone or one of its agonists as chemical ligand for controlling plant gene expression by receptor-mediated transactivation

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The present invention relates to chemical control of plant gene expression. In particular, it relates to methods of using juvenile hormone or one of its agonists as a chemical ligand to modulate the expression of a target polypeptide receptor mediated in plant cells, and transgenic plant cells, plant material or plants and their progeny containing a suitable expression cassette.

In some instances, it is desirable to control the timing or extent of expression of a phenotypic trait in a plant, plant cell, or plant tissue. It is desirable that the modulation of the expression of the trait is triggered by chemicals that are easily applied to field crops, decorative shrubs, and the like. One such system (not known whether naturally occurring in plants) that can be used to achieve this ideal state of regulated gene expression is the steroid and thyroid hormone superfamily of nuclear receptors. The steroid and thyroid hormone superfamily of nuclear receptors is found in mammals and insects and consists of over 100 known proteins. Some of the receptors found in this superfamily in mammals are Retinoic Acid Receptors (RARs), Vitamin D Receptors (VDRs), thyroid hormone receptors (Ts)₃R) and retinoic acid X receptor (RXR). These and other receptors of this superfamily bind to the 5' regulatory region of target genes and, when a chemical ligand binds to the receptor, transactivates target gene expression.

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 kafetos, biochemical and biophysical research communications, 193: 1318 (1993); henrich et al, nucleic acid research, 18: 4143(1990). Ecdysone receptor (EcR) binds the insect steroid hormone 20-hydroxyecdysone and, when heterodimeric with the Ultraspiracle gene (USP) product, transactivates gene expression. Other chemical ligands (e.g., hormone agonists) besides 20-hydroxyecdysone also bind to EcR under similar conditions and cause transactivation of the target gene.

USP has been shown to be a member of the nuclear receptor steroid and thyroid superfamily, although it is considered to be an "orphan" receptor because its ligand has not been identified (Seagraves, cells, 67: 225-. USP is related in sequence to RXR α (Oro et al, Nature, 347: 298-. Methoxyproline and its derivatives methoprene acid, which are agonists of juvenile hormone, have been shown to transcriptionally activate recombinant reporter genes in insect and mammalian cells by RXR α action (Harmon et al, Proc. Natl. Acad. Sci. USA: 92: 6157-6160 (1995)). However, juvenile hormone cannot induce RXR α mediated transactivation (Harmon et al). To date there has been no clear evidence for the nuclear juvenile hormone receptor (Harmon et al; Henrich and Brown, insect biochemistry and molecular biology, 25: 881-897(1995)), although it has been shown that the orphan receptor USP may be a candidate (Harmon et al; see also Seagraves; Oro et al, Current genetics and development: 2: 269-274 (1992)).

Juvenile hormones and agonists thereof provide previously unrecognized opportunities for chemically controlling plant gene expression because the use of these chemicals in agriculture is known. What is lacking to date is a method by which these chemicals can be used to induce transactivation of a target gene in a transgenic plant. Juvenile hormone and agonists thereof are shown herein to be ligands of the orphan receptor USP. This finding allows for a protocol for plant gene control using a nuclear receptor that is not naturally present in plants. This means that the only effect of the application of juvenile hormone or one of its agonists is to induce the expression of the genetically engineered target gene. As demonstrated by the present invention, USP receptor polypeptides and plant-expressed genes encoding them have been formed that function in plant cells to control the expression of a target polypeptide, wherein the USP receptor polypeptide activates the 5' regulatory region of the target expression cassette in the presence of juvenile hormone or one of its agonists. Such methods of controlling plant gene expression are useful for controlling various agronomic traits of interest, such as plant fertility.

Methods of controlling gene expression in plants are described. In particular, the method comprises transforming a plant with a USP receptor expression cassette encoding a USP receptor polypeptide, at least one target expression cassette encoding a target polypeptide, and optionally a second receptor expression cassette encoding a second receptor polypeptide different from the USP receptor polypeptide. Contacting said transformed plant with juvenile hormone or one of its agonists activates or inhibits expression of the target polypeptide in the presence of said USP receptor polypeptide. Alternatively, another "second" receptor expression cassette may be used, wherein the second receptor expression cassette encodes a receptor polypeptide other than USP. The method is useful for controlling various important agronomic traits, such as plant fertility.

The invention also describes transgenic plants comprising a USP receptor expression cassette and a target expression cassette capable of being activated by juvenile hormone or one of its agonists. The invention also encompasses methods of identifying previously unknown ligands for USP that are effective in a plant cell environment. The substances to be tested are identified by contacting them with plant cells transformed with a USP receptor expression cassette and a target expression cassette. The target expression cassette encodes a reporter polypeptide, the expression of which can be determined quantitatively or qualitatively, from which the test substance is identified as a ligand for USP.

FIG. 1 shows a schematic representation of a plant cell containing the VP16-USP receptor expression cassette and a target expression cassette with direct repeat response elements in the 5' regulatory region. The VP16-USP receptor activates expression of the target polypeptide in the presence of juvenile hormone or one of its agonists.

FIG. 2 shows a schematic representation of plant cells containing the expression cassettes for USP-VP16 and GAL4-EcR receptor. The activation of target polypeptide expression by the combination of GAL4-EcR and USP-VP16 receptor polypeptides is reversed when contacted with juvenile hormone or one of its agonists.

Fig. 3 corresponds to fig. 2 except that in the presence of a chemical ligand, tebufenozide (also known as RH 5992). In the presence of juvenile hormone or one of its agonists, is also activated in reverse in these cases.

"juvenile hormones" refers to a class of chemical compounds produced by insects. Juvenile hormones control larval metamorphosis by causing the maintenance of the juvenile characteristics of insects and thereby preventing maturation. The action of juvenile hormones is biologically demonstrated as behavioral, biochemical or molecular effects. Some naturally occurring juvenile hormones have been isolated and identified. Agonists of juvenile hormone refer to a class of compounds that exhibit one or more of the biological activities of juvenile hormone. The juvenile hormone agonist may or may not be a structural analogue of juvenile hormone. Also included within this specification are compounds that are metabolic precursors of those compounds that directly produce the aforementioned juvenile hormone-like biological effects. For example, methoprene is a metabolic precursor of methoprene acid, which directly produces the juvenile hormone-like biological effects observed with the application of methoprene.

As used herein, "receptor polypeptide" refers to a polypeptide that activates or inhibits expression of a target polypeptide in response to an administered chemical ligand. The receptor polypeptide consists of a ligand binding region, a DNA binding region and a transactivation region. The ligand binding region comprises an amino acid sequence whose structure non-covalently binds a complementary chemical ligand. Thus, the ligand binding region and its chemical ligand form a complementary binding pair. The DNA binding region comprises an amino acid sequence that non-covalently binds a specific nucleotide sequence called a Response Element (RE). One or more response elements are located in the 5' regulatory region of the target expression cassette. Each RE comprises a pair of half-sites, each half-site having a 5-6 base pair core, wherein a single DNA binding region recognizes a single half-site. The half-sites may be arranged in a direct repeat, palindromic repeat or inverted repeat of each other in a relatively linear orientation. A nucleotide sequence of a half site; the spacing and linear orientation determine which one or more DNA binding regions form complementary binding pairs with the reaction element. The transactivation domain comprises one or more amino acid sequences that serve as subdomains that influence the operation of transcription factors during pre-initiation and assembly in the TATA box. The role of the transactivation domain is to allow repeated transcription initiation events to occur, resulting in higher levels of gene expression.

A "receptor expression cassette" includes a 5 'regulatory region nucleotide sequence operably linked to a nucleotide sequence encoding a receptor polypeptide and an untranslated 3' termination region (stop codon and polyadenylation sequence). The 5' regulatory region promotes expression in plants.

"USP" refers to the receptor Ultraspiracle found in Drosophila. It is also known as "XR 2C" and has been isolated and cloned, and its ligand binding domain has been identified by sequence homology to known ligand binding domains (Henrich et al, nucleic acids Res. 18: 4143-4148(1990)), although no chemical ligand has been known to bind to it so far. The name "USP" as used herein refers to the native form of the receptor and mutant or chimeric forms thereof. This includes, but is not limited to, those mutant or chimeric forms disclosed herein, as well as chimeric forms of USP and mutants thereof that minimally contain the ligand binding region of the native USP. More than one form of USP may be used simultaneously in the present invention.

A "second receptor expression cassette" comprises a nucleotide sequence of a 5 'regulatory region operably linked to a nucleotide sequence encoding a receptor polypeptide other than USP operably linked to a 3' termination region. Such second receptor expression cassettes include, but are not limited to, EcR, RXR, DHR38(Kafatos et al, Proc. Natl. Acad. Sci. USA 92: 7966-7970(1995) and mutant and chimeric forms thereof.

"partial molecule" refers to a portion of the receptor polypeptide from the indicated source. For example, "USP-moiety" refers to a partial receptor polypeptide from a native Ultraspiracle receptor. As used herein, a moiety includes one or more regions, at a minimum, the ligand binding region of the receptor to which the moiety is named.

The term "chimera" is used to refer to a receptor polypeptide consisting of multiple regions, at least one of which has a heterologous origin with respect to the other regions present. "heterologous" means that one or more regions present in the receptor polypeptide differ in their natural origin from the other regions present. For example, if the transactivation domain from the herpes simplex VP16 protein is operably linked to the USP receptor from Drosophila, then the VP16 transactivation domain is heterologous to the USP-part molecule. Furthermore, if a region of USP is operably linked to a region of RXR to produce a functional receptor, the chimeric fusion has regions that are heterologous to each other. These chimeric receptor polypeptides are encoded by a nucleotide sequence that has been operably linked to produce a coding sequence that is not found in nature. The chimeric receptor polypeptides of the invention are referred to by the linear nomenclature from the N-terminus to the C-terminus portion of the polypeptide. Using this nomenclature, a chimeric receptor polypeptide having a transactivation region of VP16 added to the N-terminal region of the USP receptor was designated VP 16-USP. Conversely, if VP16 is added to the C-terminal region of the USP receptor, the chimeric receptor polypeptide can be designated USP-VP 16.

When naming a genetic construct that involves a 5 'regulatory region and its operably linked coding sequence, the 5' regulatory region is named before the slash (/), and the coding sequence is named after the slash. For example, the genetic construct 35S/USP-VP16 names the 35S promoter of cauliflower mosaic virus operably linked to a DNA sequence encoding the chimeric receptor USP-VP16, wherein the VP16 transactivation region has been added to the C-terminal region of USP. When directed to a receptor polypeptide, the promoter is not named. For example, the above gene construct encodes a USP-VP16 polypeptide.

A "target expression cassette" comprises a nucleotide sequence of a 5' regulatory region operably linked to a nucleotide sequence encoding a target polypeptide, the expression of which is activated or inhibited by a receptor polypeptide in the presence of a chemical ligand. The 5' regulatory region of the target gene includes the core promoter sequence, the start of the transcription sequence and the response element or response elements necessary for complementary binding of the receptor polypeptide. The 5' regulatory region promotes expression in plants. The target expression cassette also has a 3' termination region (stop codon and polyadenylation sequence).

Juvenile hormones I, II, III and O are known, and are substituted forms of I, e.g., the basis of insect physiology, M.S. Blum edition, John Wiley&Sons, new york, 1985. The structural formula of the juvenile hormone I is methyl 10, 11-epoxy-7-ethyl-3, 11-dimethyl-trans-2, 6-tridecadienoic acid. The juvenile hormone has the following structure:

juvenile hormone agonists are compounds that exhibit one or more of the biological activities of juvenile hormone. Compounds with this characteristic and bearing structures associated with juvenile hormones include, but are not limited to, juvenin, methoprene, hydroprene and methoprene acid. The structure of juvenin, which is an example of these juvenile hormone agonists, is shown below:

juvenile hormone agonists that are not structurally related to juvenile hormone are also known. Such compounds include, but are not limited to, the polycyclic non-isoprenoid compounds non-nocarbon oxides, which are well known juvenile hormone agonists. The structural formula of the fenoxanil is [2- (4-phenoxyphenoxy) ethyl]And (3) urethane. The structure of the non-noroxycarbides is shown below:

another juvenile hormone agonist useful in the present invention is the compound diofenolan, a diphenyl ether compound. The structural formula of the diofenolan is 4- (2-ethyl-1, 3-dioxolan-4-yl methoxy) phenyl ether. The compound has known juvenile hormone activity and is expected to function in a manner similar to the compound non-oxycarbide or methoprene.

The use of juvenile hormone agonists in the present invention provides several advantages. First, the compounds are synthetic and readily available. Second, many of these compounds have benefits that have been tested for use in agricultural production, making such compounds "ready-to-use" for field application to crops.

The present invention utilizes the findings that: juvenile hormone or one of its agonists acts as a chemical ligand for the USP receptor. Previously unknown chemical ligands for USP have been used in the present invention in combination with plant expressible USP receptor expression cassettes and appropriate target expression cassettes to create a novel method for controlling gene expression in plants.

Many insect growth regulators have been found to inhibit molting in insects and are likely to act directly on receptors involved in initiating molting. Such insect growth regulators include, but are not limited to, triflu-muron ((1- (2-chlorobenzoyl) -3- (4-trifluoromethoxyphenyl) urea), hexam-flurouron (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-. alpha.,. alpha. -trifluoro-p-tolyloxy) -2-fluorophenyl ] -3- (2, 6-difluorobenzoyl) urea), flucycloxuron (1- [ alpha- (4-chloro-. alpha. -cyclopropylbenzylideneamino-oxy) -p-tolyl ] -3- (2, 6-difluorobenzoyl) urea), and lufenuron (1- [2, 5-dichloro-4- (1, 1, 2, 3, 3, 3-hexafluoropropoxy) phenyl ] -3- (2, 6-difluorobenzoyl) urea). Other benzoylphenylurea insecticides, including but not limited to diflubenzuron and chlorfluzuron, may also be used in the present invention.

Retinoic acid or its derivatives may also be useful ligands for controlling gene expression in transgenic plants. Recently, retinoic acid has been shown to activate heterologously expressed USP gene products in cultured mammalian cell lines (Harmon et al). Thus, insect receptors can be modulated by applying retinoic acid derivatives to transgenic plants carrying an appropriate combination of receptor and target gene constructs, as described below.

Natural sources can also serve as ligands for insect receptors expressed in transgenic plants such as transgenic maize or wheat. Many plants have been found to synthesize compounds that accelerate or inhibit insect molting. For example, one of the most active ecdysteroids, muris-terone, is isolated from plant sources. Plants of many families are known to produce ecdysteroid or juvenile hormone activity.

Juvenile hormone agonists may also be used as ligands for modulating gene expression in transgenic plants. Examples of such ligands are 4- [ 2-tert-butylcarbonyloxy ] -benzoic acid ethyl ester; bis (thiocarbamate), 5-methoxy-6- [1- (4-methoxyphenyl) ethyl ] -1, 3-benzodioxole or ethyl E-3-methyl-2-dodecenoate. The agonist ligands can be used to activate low level expression of transgenes or to inhibit high level gene expression in heterologous plant systems expressing modified insect receptors.

The methods of the invention comprise transforming a plant cell or plant with a USP receptor expression cassette and a target expression cassette. Expression of the USP receptor polypeptide in the resulting plant cells, plants or progeny thereof in the presence of juvenile hormone or one of its agonists activates the 5' regulatory region of the target expression cassette in the transgenic cells or plants (figure 1). Juvenile hormone or one of its agonists is essential to the present invention as it is believed to bind to the USP ligand binding region.

Controlling the expression of the target expression cassette may also be achieved by optionally expressing in the plant another second receptor polypeptide or a polypeptide other than USP (figures 2 and 3). Examples of additional second receptor polypeptides encompassed by the present invention include, but are not limited to, EcR, RXR, DHR38(Kafatos et al, Proc. Natl. Acad. Sci. USA 92: 7966-7970(1995)) and mutant or chimeric forms thereof. The use of these receptors to mediate ligand-induced transactivation is described in International application No. PCT/EP96/00686 filed on 2/19 1996, which is incorporated herein by reference.

The ligand binding region of the USP receptor polypeptide provides a means for chemically controlling the activation of the 5' regulatory region of the target expression cassette with juvenile hormone or one of its agonists. USP is similar to the steroid receptor RXR α, which has 9-cis retinoic acid as a chemical ligand. USP has been shown to form heterodimers with EcR receptor polypeptides and to modulate the expression of target polypeptides in response to administration of ecdysone, an insect hormone that binds EcR, in transformed mouse kidney cells, independently of juvenile hormone and agonists thereof (WO 94/01558). In the present invention it has been found that the receptor USP and its ligand binding region are particularly useful for controlling plant target polypeptide expression in response to the application of juvenile hormone or one of its agonists, as described in the examples below.

Chimeric forms of USP receptor polypeptides may also be used in the present invention to activate expression of a target polypeptide in the presence of juvenile hormone or one of its agonists. The DNA-binding or transactivation domain of the chimeric USP receptor polypeptide may be selected from heterologous sources based on its effectiveness in transactivation or DNA binding. The said region of the chimeric receptor polypeptide can be obtained from any organism such as plants, insects and mammals having similar transcription regulatory functions. In one embodiment of the invention, these regions are selected from the steroid and other members of the thyroid hormone superfamily of nuclear receptors. The use of chimeric receptor polypeptides has the benefit of combining regions of different origin. The chimeric USP receptor polypeptides provided herein provide the advantage of combining optimal transactivation activity or altered RE binding or recognition with a specific response element of juvenile hormone or one of its agonists as ligand. Thus, chimeric polypeptides can be constructed that are tailored for a particular purpose. These chimeric receptor polypeptides also provide improved function in a plant cell heterologous environment.

It is also contemplated that a portion of the present invention is that the transactivation, ligand binding and DNA binding regions may be assembled in any functional arrangement within the chimeric receptor polypeptide. For example, if a sub-region of a transactivation domain is found in the N-terminal portion of a naturally occurring receptor, a chimeric receptor polypeptide of the invention may include a transactivation sub-region at the C-terminus in place of or in addition to the N-terminal sub-region. Chimeric receptor polypeptides disclosed herein can also have multiple regions of the same type, e.g., more than one transactivation region (or two subregions) per receptor polypeptide.

Accordingly, one embodiment of the present invention provides a USP receptor polypeptide which activates expression of a target polypeptide in the presence of juvenile hormone or one of its agonists and which also has the superior property of transactivation. The transactivation region may be defined as an amino acid sequence that enhances the productive transcription initiation of RNA polymerase. (see generally Ptashne, Nature 335: 683-689 (1988)). Different transactivation regions are known to have varying degrees of potency in their ability to enhance transcription initiation. It is desirable in the present invention to use a transactivation domain that has excellent transactivation efficacy in plant cells in order to produce high levels of target polypeptide expression in response to the presence of juvenile hormone or one of its agonists. Transactivation domains that have been shown to be particularly effective in the methods of the invention include, but are not limited to, VP16 (isolated from herpes simplex virus). In a preferred embodiment of the invention, the transactivation domain of VP16 is operably linked to a USP-moiety to produce a chimeric USP receptor polypeptide for controlling expression of a target polypeptide in a plant. Other transactivation domains are also effective.

The DNA binding region is an amino acid sequence with certain functional characteristics that is responsible for the binding of the USP receptor polypeptide to a specific nucleotide sequence, called a response element, present in the 5' regulatory region of the target expression cassette. The structure of the steroid and thyroid superfamily DNA binding domains of nuclear receptors are highly conserved across species and therefore have limited variation in response elements for forming complementary binding pairs (Evans, science 240: 889-895 (1988)). However, considerable variability can be introduced in other ways in methods of controlling gene expression using response elements. In a preferred embodiment of the invention, multiple copies, and preferably 1 to 11 copies, of a suitable response element are placed in the 5' regulatory region, resulting in multiple sites for binding of the USP or optional second receptor polypeptide, resulting in a greater degree of activation.

Additional variability in the gene control methodology can be achieved by altering the linear orientation or position of the response element in the 5' regulatory region. The response element recognized by the type II receptor protein has a "two-fold" symmetry consisting of two "half-sites". (Evans, science 240: 889-895 (1988)). Each receptor polypeptide binds to a "half-site". These "half-sites" may be oriented in direct repeats, inverted repeats or palindromic fashion. In one embodiment of the invention, more than one USP receptor polypeptide molecule recognizes a Direct Repeat (DR) response element, thereby effecting activation of the target expression cassette in the presence of juvenile hormone or one of its agonists.

Other changes in the control of gene expression using the present invention may be obtained through the use of DNA binding domains and response elements from other transcriptional activators including, but not limited to, LexA or GAL4 proteins. The DNA binding region of the LexA protein encoded by the LexA gene from E.coli and its complementary binding site can be used (Brent and Ptashne, cell 43: 729-736, (1985), which describes the LexA/GAL4 transcriptional activator). Another useful source is the GAL4 protein from yeast (Sadowski et al, Nature 335: 563-564(1988), which describes a GAL4-VP16 transcriptional activator). In a preferred embodiment of the invention, the optional chimeric form of the second receptor polypeptide is constructed by fusing the GAL4DNA binding domain to a portion of the molecule containing the EcR ligand binding domain.

The 5' regulatory region of the USP and optional second receptor expression cassette further comprises a promoter which allows expression in plant tissues and cells. Suitable promoters for the receptor expression cassettes are selected so that expression of the receptor polypeptide can be constitutive, developmentally regulated, tissue specific, cell specific or cell compartment specific. The promoter may also be selected so that expression of the receptor polypeptide itself is chemically induced in the plant, thereby increasing the level of promoter induction by the ligand. The combination of a promoter element that provides specific expression and a promoter element that provides chemically inducible expression allows the receptor polypeptide to be expressed or activated in specific cells or tissues of the plant in response to the application of chemicals.

The nucleotide sequence encoding the receptor polypeptide may be modified to improve expression in a plant, to improve function, or both. Such modifications include, but are not limited to, altering codon usage, inserting introns, or generating mutations. In one embodiment of the invention, expression of the target polypeptide is activated in the presence of juvenile hormone or one of its agonists using an expression cassette comprising an anther-specific or pistil-specific promoter operably linked to a nucleotide sequence encoding a USP receptor polypeptide.

Also disclosed are target polypeptides whose expression is activated by a receptor polypeptide in the presence of juvenile hormone or one of its agonists. The present invention can be used to control the expression of any coding sequence, provided that a promoter operably linked to the coding sequence is engineered to contain one or more response elements complementary to the DNA binding region of the USP receptor, and optionally one or more response elements required for a second receptor. For example, a USP receptor polypeptide may be used to activate a target polypeptide useful for controlling plant fertility in the presence of juvenile hormone or one of its agonists.

The invention also encompasses mutants of USP receptor polypeptides. Mutants with reduced background activation levels of the target expression cassette can be made that induce greater expression relative to the uninduced background. Furthermore, mutants may be formed in which the binding to juvenile hormone or one of its agonists is altered. Mutants with altered binding properties respond to different agonists in a manner specific for these agonists. For example, mutant USP receptors can be formed that respond only to the agonist non-nocarbon oxide and not to hydroprene, thereby distinguishing isoprenoid from non-isoprenoid juvenile hormone agonists. Useful mutagenesis methods such as chemical mutagenesis or site-directed mutagenesis are known in the art.

In another method, a mutant receptor polypeptide is prepared by PCR mutagenesis of a nucleotide sequence encoding the ligand binding region of USP. These mutant receptor polypeptides are expressed in host organisms such as yeast suitable for conventional screening and isolation techniques. However, screening for mutant receptor polypeptides exhibiting reduced basal activity and greater fold induction in this host organism only provided candidates for further testing in plant cells, since it is clear from work with the Glucocorticoid Receptor (GR) that receptors of the superfamily of retinoids and thyroid hormones could function in yeast but could not be expected to function in transgenic plants (Lloyd et al, science, 226: 436 (1994)). Further limiting the use observed with yeast cells expressing GR was that yeast cells were not responsive to the commonly used chemical ligand dexamethasone, which was functional in other heterologous systems (Schena et al, proceedings of the American academy of sciences 88: 10421-10425 (1991)).

Further testing in plant cells can be achieved by preparing a receptor expression cassette encoding a mutant receptor polypeptide and transforming it into a plant cell in conjunction with the target expression cassette. The transformed plant cells are tested for activation of the 5' regulatory region of the target expression cassette by the mutant receptor polypeptide in the presence of juvenile hormone or one of its agonists. Mutant receptor polypeptides that induce low basal expression of a target polypeptide in the absence of juvenile hormone or one of its agonists and high level expression of the target polypeptide in the presence of juvenile hormone or one of its agonists are useful for controlling gene expression in plants.

As described above, the methods of the invention can be used to statistically increase gene expression relative to a minimum basal level. However, the present invention can also be used to statistically reduce or inhibit the activation of gene expression mediated by a complex formed by receptors such as USP and EcR. The control of gene expression in plants mediated by this receptor complex is the subject of PCT/EP96/00686, filed 3/1995, which is incorporated herein by reference. Reversal of activation mediated by these receptor complexes is caused in the presence of juvenile hormone or one of its agonists, which are chemical ligands for the USP receptor polypeptide (see figures 2 and 3). In the presence of juvenile hormone or one of its agonists, USP is disrupted: EcR complex, thereby reversing the activation. For example, in transgenic plants expressing USP and GAL4-EcR-C1 receptor polypeptides and comprising a target expression cassette with a GAL4 binding site element, activation of gene expression of the target polypeptide by the complex is reversed. This reversal may occur in the presence of tebufenozide (also known as RH5992) or other chemical ligand that binds to the EcR ligand binding region, or in the absence of such chemical ligand.

For expression in plants, a suitable promoter must be selected that can be used for both the receptor expression cassette and the target expression cassette. Unless otherwise indicated, promoters discussed below may be used to direct expression of the receptor polypeptide or target polypeptide in a plant. These promoters include, but are not limited to, constitutive, inducible, temporally regulated, developmentally regulated, chemically regulated, tissue-preferred and tissue-specific promoters. Preferred constitutive promoters include, but are not limited to, the CaMV35S and 193 promoters (U.S. Pat. No. 5,352,605). Other preferred promoters include, but are not limited to, one of several actin genes known to be expressed in most cell types. General molecular genetics 231, by McElroy et al: the promoter described in 150-160(1991) can be incorporated into the receptor expression cassettes of the invention relatively easily and is particularly suitable for use in monocotyledonous plant hosts. Yet another preferred constitutive promoter is from ubiquitin, another gene product known to accumulate in many cell types. Ubiquitin promoters have been cloned from several species for transgenic plants (e.g., sunflower-Binet et al, plant science 79: 87-94 (1991); maize-Christensen et al, plant molecular biology 12: 619 (1989)). The maize ubiquitin promoter has been developed in transgenic monocot systems and its sequences and vectors constructed for transformation of monocots are disclosed in EP-A-342926. Ubiquitin promoters are suitable for use in transgenic plants of the invention, particularly monocots. Other useful promoters are the U2 and U5 snRNA promoters from maize (Brown et al, nucleic acids Res. 17: 8991(1989)) and the promoter from alcohol dehydrogenase (Dennis et al, nucleic acids Res. 12: 3983 (1984)).

Tissue-specific or tissue-preferred promoters useful in the plants of the invention, particularly maize, are those that direct expression in roots, medulla, leaves or pollen. Such promoters are disclosed in WO93/07278, which is hereby incorporated by reference in its entirety. Other useful promoters that provide seed-specific expression are, for example, Schernthaner et al, EMBO J.7: 1249(1988), the anther-specific promoters ant32 and ant43D disclosed in EP-A-578611 (incorporated herein by reference in their entirety), the anther (tapetum) -specific promoter B6(Huffman et al, J. Cell Biochemical 17B: Abstract # D209 (1993)); medullary-specific promoters, such as the modified S13 promoter (Dzelkals et al, plant cell 5: 855 (1993)).

Also useful in the present invention are chemically inducible promoters. Specific promoters useful in this class for directing the expression of receptor or target polypeptides in plants are disclosed, for example, in EP-A-332104, which is 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. Many sequences have been found to enhance gene expression in transgenic plants. For example, many non-translated leader sequences from viruses are known to enhance expression. In particular, leader sequences from tobacco mosaic virus (TMV, "omega sequences"), Maize Chlorotic Mottle Virus (MCMV) and Alfalfa Mosaic Virus (AMV) have been shown to be effective in enhancing expression (e.g., Gallie et al, nucleic acids Res. 15: 8693-. Other leader sequences known in the art include, but are not limited to:

picornavirus leaders, e.g., the EMCV leader (the 5' non-coding region of encephalomyocarditis virus) (Elroy-Stein, O., Fuerst, T.R., and Moss, B.PNAS USA 86: 6126-6130 (1989));

potyvirus leaders, e.g., the TEV leader (tobacco etch Virus) (Allison et al, (1986); MDMV leader (maize dwarf mosaic Virus); virology 154: 9-20);

human immunoglobulin heavy chain binding protein (BiP) leader sequence, (Macejak, D.G., and Sarnow, P., Nature 353: 90-94 (1991);

untranslated leader sequence (AMVRNA 4) from E.melissa mosaic virus capsid protein mRNA, (Jobling, S.A., and Gehrke, L., Nature, 325: 622-;

tobacco mosaic Virus leader sequence (TMV), (Gallie, D.R., et al, molecular biology of RNA, pp 237-

Maize chlorotic mottle virus leader sequence (MCMV) (Lommel, S.A. et al, virology, 81: 382-385(1991) see also Della-Cioppa et al, plant physiology, 84: 965-968 (1987).

Various intron sequences have been shown to enhance expression when added to the 5' regulatory region, particularly in monocotyledonous plant cells. For example, it has been found that the intron of the maize Adh1 gene significantly enhances expression of the wild-type gene under its homologous promoter when introduced into maize cells (Callis et al, Gene development 1: 1183-1200 (1987)).

In addition to incorporating one or more of the above elements into the 5' regulatory region of the target expression cassette, other elements specific to the target expression cassette may be incorporated. Such elements include, but are not limited to, minimal promoters. The minimal promoter renders the basal promoter element inactive or nearly inactive due to the lack of binding sites for upstream activators. Such promoters have low background activity in plants in the absence of transactivators or in the absence of enhancer or response element binding sites. One minimal promoter that is particularly useful for plant target genes is the Bz1 minimal promoter obtained from the maize bronze1 gene. The Bz1 core promoter was obtained from the "myc" mutant Bz 1-luciferase construct pBz1LucR98 by cleavage at the Nhe I site located-53 to-58 (Roth et al, plant cell 3: 317 (1991)). The Bz1 core promoter fragment thus generated extends from-53 to +227 and includes the Bz1 intron-1 in the 5' untranslated region when used in transgenic maize.

In addition to promoters, various 3' transcription terminators may be used in the present invention. The transcription terminator is responsible for the termination of transcription and proper mRNA polyadenylation. Suitable transcription terminators and terminators known to function in plants include the CaMV35S terminator, the tml terminator, the nopaline synthase terminator, the pea rbcS E9 terminator and other terminators known in the art. These are useful in both monocotyledons and dicotyledons.

The expression cassettes of the invention can be introduced into plant cells in a number of ways known in the art. The person skilled in the art will appreciate that the choice of method depends on the type of plant targeted for transformation, i.e.monocotyledonous or dicotyledonous plants. Suitable methods for transforming plant cells include, but are not limited to, microinjection (Crossway et al, Biotechnology 4: 320-334(1986)), electroporation (Riggs et al, Proc. Natl. Acad. Sci. USA 83: 5602-. See also Weissinger et al, yearbook of genetics 22: 421-477 (1988); san-ford et al, particle science and technology 5: 27-37(1987) (onions); christou et al, plant physiology 87: 671-674(1988) (Glycine max); McCabe et al, bio/technique 6: 923-; datta et al, Bio/technology 8: 736 (1990) (Oryza sativa); klein et al, proceedings of the American academy of sciences, 85: 4305-; klein et al, bio/technology 6: 559-563(1988) (maize); klein et al, plant physiology 91: 440-444(1988) (maize); fromm et al, bio/technology 8: 833-839(1990) (maize); and Gordon-Kamm et al, plant cell 2: 603-618(1990) (maize); svab et al, proceedings of the American academy of sciences 87: 8526-8530(1990) (tobacco chloroplasts); koziel et al, Biotechnology 11: 194-200(1993) (maize); shimamoto et al, Nature 338: 274-277(1989) (rice); christou et al, Biotechnology 9: 957-962(1991) (Rice); EP-A-332581(Orchardgrass and other Pooideae); vasil et al, Biotechnology 11: 1553 1558(1993) (wheat); weeks et al, plant physiology 102: 1077, 1084(1993) (wheat).

One particularly preferred set of embodiments for introducing the expression cassettes of the invention into maize in microprojectile bombardment is described in Koziel et al, Bio/technology 11: 194, 200, 1993, the entire contents of which are incorporated herein by reference. Another preferred embodiment is the protoplast transformation method for maize disclosed in EP-A-292435, which is incorporated herein by reference in its entirety. A particularly preferred set of embodiments for introducing the expression cassette of the invention into wheat by microprojectile bombardment can be found in WO94/13822, which is incorporated herein by reference in its entirety.

Transformation of plants can be carried out using a single DNA molecule or multiple DNA molecules (i.e., co-transformation), and these techniques are applicable to the expression cassettes of the invention. Many transformation vectors are available for plant transformation, and the expression cassettes of the invention can be used in combination with any such vector. The choice of vector will depend on the preferred transformation technique and the type of target being transformed.

There are many vectors for transformation using Agrobacterium tumefaciens. They typically carry at least one T-DNA border sequence and include vectors such as pBIN19 (Bevan, nucleic acids research (1984)). In a preferred embodiment, the expression cassette of the invention can be inserted into the binary vectors pCIB200 and pCIB2001 for use with Agrobacterium. Expression cassettes for agrobacterium-mediated transformation were constructed in the following manner. pTJS75(Schmidhauser and Helinski, J.Bacteriol 164: 446-455(1985)) was subjected to Narl digestion to excise the tetracycline resistance gene, followed by insertion of the Acc I fragment from pUC4K harboring NPT II (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)) to produce pTJS75 kan. The Xho I linker was ligated to the pCIB7EcoRV fragment containing the left and right T-DNA borders, the plant selectable nos/npt II chimeric gene and the pUC polylinker (Rothstein et al, Gene 53: 153-161(1987)), and the Xho I digested fragment was cloned into SｃA II digested pTJS75kan to generate pCIB200 (see also EP-A-332104, example 19). pCIB200 contains the following single polylinker restriction sites: EcoRI, Sst I, Kpn I, Bg III, Xba I and Sa II. Plasmid pCIB2001 is a derivative of pCIB200, which is generated by the insertion of additional restriction sites into the polylinker. The unique restriction sites in the polylinker of pCIB2001 are EcoRI, Sst I, Kpn I, Bg III, Xba I, Sa II, Mlu I, Bc II, Avr II, Apa I, Hpa I and Stu I. In addition to containing these unique restriction sites, pCIB2001 also has plant and bacterial kanamycin selection genes, left and right T-DNA borders for Agrobacterium-mediated transformation, trfA function from RK2 for translocation between E.coli and other hosts, and OriT and OriV functions from PK 2. The pCIB2001 polylinker is suitable for cloning plant expression cassettes containing their own regulatory signals.

Another vector useful for Agrobacterium-mediated transformation is the binary vector pCIB10, which contains the gene encoding kanamycin resistance for selection in plants, the T-DNA right and left border sequences and the insertion of sequences from the broad-spectrum host plasmid pRK252, making it replicable in both E.coli and Agrobacterium. Rothstein et al, gene 53: 153-161(1987) describe its construction. Various derivatives of pCIB10 have been constructed in which Gritz et al, Gene 25: 179-188(1983) on the gene for hygromycin B phosphotransferase. These derivatives enable the selection of transgenic plant cells on hygromycin only (pCIB743) or on both hygromycin and kanamycin (pCIB715, pCIB 717).

Methods using direct gene transfer or Agrobacterium-mediated transfer formats are typically, but not necessarily, performed with selectable markers that provide resistance to antibiotics (e.g., kanamycin, hygromycin or methotrexate) or herbicides (e.g., phosphinothricin). However, the selection of a selectable marker for plant transformation is not critical to the present invention.

For certain plant species, different antibiotic or herbicide selection markers may be preferred. Selection markers routinely used in transformation include the npt II gene which 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 which confers resistance to the herbicide phosphinothricin (White et al, nucleic acids Res. 18: 1062 (1990)), Spencer et al, genetics and applications 79: 625-631(1990)), the hph gene which confers resistance to the antibiotic hygromycin (Blochinger and Diggelmann, molecular cytobiology 4: 2929-2931), the dhfr gene which confers resistance to methotrexate (Bourouis et al, EMBO J. 2: 1099-1104 (1983)).

One such vector that is useful for direct gene transfer technology in combination with the herbicide Basta (or phosphinothricin) selection is pCIB 3064. The vector is based on the plasmid pCIB246, comprising the CaMV35S promoter operably fused to the GUS gene of E.coli and the transcription terminator CaMV35S, and is described in WO93/07278, which is incorporated herein by reference. One gene that is useful for providing resistance to phosphinothricin is the bar gene from S.viridochromogenes (Thompson et al, EMBO J.6: 2519-2523 (1987)). The vector is suitable for cloning plant expression cassettes containing their own regulatory signals.

Another transformation vector is pSOG35 which utilizes the E.coli gene dihydrofolate reductase (DHFR) as a selectable marker for resistance to methotrexate. PCR was used to amplify the 35S promoter (. about.800 bp), maize Adh1 gene intron 6 (. about.550 bp) and the GUS untranslated leader sequence of pSOG10 at 18 bp. A250 bp fragment encoding the E.coli dihydrofolate reductase type II gene was also amplified using PCR, and these two PCR fragments were assembled with the Sac I-Pst I fragment from pBI221(Clon-tech) containing the pUC19 vector backbone and the nopaline synthase terminator. The assembly of these fragments yielded pSOG19, which contained the 35S promoter fused to the intron 6 sequence, the GUS leader sequence, the DHFR gene and the nopaline synthase terminator. Vector pSOG35 was generated by replacing the GUS leader sequence of pSOG19 with the leader sequence from maize chlorotic mottle virus check (MCMV). pSOG19 and pSOG35 carry the ampicillin resistance gene from pUC and have Hind III, Sph I, Pst I and EcoR I sites into which foreign sequences can be cloned.

An advantageous aspect of the present invention is its use in controlling plant fertility in field conditions. Effective fertilization results from the formation of a viable zygote and can be measured as the percentage of seeds forming a viable zygote. According to the present invention, fertilization can be controlled by incorporating a nucleotide sequence encoding a suitable target into a target expression cassette, wherein expression of said target polypeptide interferes with plant fertilization, i.e., it statistically reduces or increases plant fertilization. In a preferred embodiment of the invention, said target polypeptide causes the fertilization process to be ineffective, i.e. hampers or prevents the formation of a viable zygote. This inefficient fertilization can be measured as the percentage of seeds that do not form viable zygotes and can be caused by various means. These include, but are not limited to, 1) disruption or alteration of those processes critical to the formation of viable gametes, 2) nonfunctional pollen or ovule (if formed), or3) failure of the normal development of the embryo sac, pistil, stigma or duct. In the present invention, juvenile hormone or one of its agonists is administered to or contacted with a transgenic plant under field conditions, wherein expression of the target polypeptide is activated, thereby resulting in failure of fertilization. In another embodiment of the invention, expression of said target polypeptide enhances or restores fertility in a plant.

It will be appreciated that different degrees of effective or ineffective fertilization may be achieved using the present invention. In a preferred embodiment, over 80% and more preferably over 95% of ineffective fertilization can be achieved. The ability to provide variability in fertility levels allows the present invention to be adapted to a variety of agronomic purposes.

Useful coding sequences for target polypeptides include, but are not limited to, any sequence that encodes a product that can lead to ineffective fertilization. These coding sequences may be of homologous or heterologous origin. Gene products of these coding sequences include, but are not limited to:

diphtheria toxin a-chain (DTA), which inhibits protein synthesis, Greenfield et al, proceedings of the american academy of sciences, 80: 6853 (1983); palmiter et al, cell, 50: 435 (1987);

pectate lyase pelE from erwinia chrysanthemi EC16, which degrades pectin causing cell lysis. Keen et al, journal of bacteriology, 168: 595 (1986);

t-urf13(TURF-13) from the cms-T maize mitochondrial genome; this gene encodes a polypeptide designated URF13, which disrupts the mitochondrial or cytoplasmic membrane. Braun et al, plant cells, 2: 153 (1990); dewey et al, proceedings of the American college of sciences, 84: 5374 (1987); dewey et al, cell, 44: 439 (1986);

beta-1, 3-glucanase, which causes premature lysis of the microspore callose wall, Worral et al, plant cell 4: 759-;

gin recombinase from bacteriophage Mu, which gene encodes a site-specific DNA recombinase, which causes genomic rearrangement and loses cellular activity when expressed in plant cells. Maeser et al, molecular general genetics, 230: 170-176 (1991);

indoleacetic acid lysine synthetase (iaaL) from pseudomonas syringae, which encodes an enzyme that links lysine to indoleacetic acid (IAA). When expressed in plant cells, IAA causes developmental alterations due to removal from the cell via ligation. Romano et al, genes and development, 5: 438-446 (1991); spena et al, molecular general genetics, 227: 205-212 (1991); roberto et al, proceedings of the american academy of sciences, 87: 5795-;

ribonucleases from bacillus amyloliquefaciens, also known as bacillus rnases, digest mRNA in cells in which they are expressed, leading to cell death. Mariani et al, nature 347: 737-741 (1990); mariani et al, nature 357: 384-; and

CytA toxin gene from Bacillus thuringiensis israeliensis, which encodes a mosquito-killing and haemolytic protein. When expressed in plant cells, cell death occurs due to disruption of the cell membrane. McLean et al, J bacteriology 169: 1017-1023 (1987); ellar et al, U.S. Pat. No. 4,918,006 (1990).

Such polypeptides also include Adenine Phosphoribosyltransferase (APRT), Moffatt and Somerville, plant physiology, 86: 1150-1154 (1988); dnase, rnase, protease, salicylic acid hydroxylase, and the like.

It will also be appreciated that the target expression cassette may include a 5' regulatory region operably linked to a nucleotide sequence which, when transcribed, produces an antisense form of the coding sequence (e.g., APRT) critical to the formation of viable gametes. Alternatively, ribozymes can be utilized which target the mRNA of genes critical to gametogenesis or function. The ribozyme comprises a hybridizing region of about 9 nucleotides which is complementary in nucleotide sequence to at least a portion of the target RNA and a catalytic region suitable for cleaving the target RNA. Ribozymes are described in EP-A-321201 and WO88/04300, which are incorporated herein by reference. See also Haseloff and Gerlach, nature, 334: 585-591 (1988); fedor and Uhlenbeck, proceedings of the american academy of sciences, 87: 1668-1672 (1990); cech and Bass, yearbook of biochemistry, 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 will be appreciated that the above-described nucleotide sequence encoding the target polypeptide may also be operably linked to a 5' regulatory sequence which directs its expression in a tissue or cell specific manner. The manner of providing this tissue-or cell-specific expression has been described above. This specificity in expression ensures that the potency of the target polypeptide acts only on those tissues or cells that are essential for the formation of viable zygotes and is not detrimental to the plant except for affecting fertility.

It is recognized within the scope of the invention that the male fertility of the transgenic plant, the female fertility of the transgenic plant, or both can be controlled. Male sterility is the failure or inability to produce functional or viable pollen. Male sterility can be caused by a defect that results in the absence of pollen formation or the lack of function of pollen when formed. Thus, either pollen cannot form or, if it does, it is not viable or cannot be fertilized effectively under normal conditions.

Female sterility is the failure or inability to produce functional or viable megaspores or embryo sacs or other tissues required for pollen germination, growth or fertilization. Female sterility can be caused by a defect that results in the failure to form a megaspore or embryo sac or ovary, ovule, pistil, stigma or duct to develop normally. Thus, either a viable blastocyst cannot develop or, if formed, cannot be effectively fertilized under normal conditions.

For example, transgenic plants can be obtained that express a USP receptor polypeptide or polypeptides in anthers using an anther-specific promoter operably linked to a suitable nucleotide sequence. In addition, the transgenic plants also contain a target expression cassette with 5' regulatory sequences comprising a suitable response element sequence with a core promoter element from Bz1 operably linked to a Bacillus ribonuclease RNAse coding sequence. When transgenic plants expressing USP receptor polypeptides are administered juvenile hormone or one of its agonists, activation of the 5' regulatory sequence of the target expression cassette results in the subsequent production of the target polypeptide barnase. The specific expression of the barnase in the anther causes cell death and thus male sterility. The same combination of receptor polypeptide and target expression cassette can produce female sterility using a pistil-specific promoter operably linked to a nucleotide sequence encoding the receptor polypeptide.

Alternatively, plants can be engineered in which expression of the target polypeptide restores fertility to a male-sterile or female-sterile plant. For example, a plant may be obtained which expresses a barstar gene under the control of the Ant43D, Ant32 or B6 promoter, or alternatively, as in Mariani et al, Nature 347: 737-741(1990) and Mariani et al, Nature 357: 384-387(1992) under the control of the TA29 promoter. These plants also contain a receptor expression cassette for the USP receptor polypeptide and any optional second receptor polypeptide from the same anther-specific promoter or a constitutive promoter such as maize ubiquitin, 35S or rice actin promoter. These plants further comprise a target expression cassette having 5' regulatory sequences comprising a suitable response element sequence having a Bz1 core promoter element operably linked to the coding sequence for the barstar. The plant is male sterile, but when juvenile hormone or one of its agonists is administered, activation of the 5' regulatory sequence of the target expression cassette results in the subsequent production of the target polypeptide barnase inhibitor. The barnase inhibitor inhibits the ribonuclease activity of barnase polypeptide, and anthers and pollen develop normally. Thereby restoring fertility.

A similar approach can be used to control female sterility. By using a promoter specifically expressed in female reproductive tissues to replace an anther-specific promoter to start expression of the barnase, female sterile plants can be obtained. Induction of a target expression cassette comprising a barstar coding sequence with juvenile hormone or one of its agonists results in restoration of female fertility.

The above methods may utilize any female or male sterile gene for which a restorer gene can be designed. Potential restorer genes for non-barstar RNase inhibitors are described in EP-A-412911.

The genetic traits engineered into the above transgenic plants and their seeds are transmitted by sexual reproduction or asexual growth and are thus maintained and propagated in progeny plants. The so-called maintenance and propagation make use of known agricultural methods, such as cultivation, sowing or harvesting, which are developed to suit specific purposes. Special methods such as hydroponics or greenhouse techniques may also be applied. Since growing crops are susceptible to attack and damage or infection by insects, and to competition by weed plants, methods have been employed to control weeds, plant diseases, insects, nematodes and other harmful conditions to increase yield. These methods include mechanical methods such as soil cultivation or weed removal and plant infection, and the application of pesticides, such as herbicides, fungicides, gametocides, nematocides, growth regulators, ripeners and insecticides.

Plant breeding can also be carried out using the beneficial genetic properties of the transgenic plants and seeds according to the invention to form plants with improved properties, such as tolerance to pests, herbicides, or stress, improved nutritional value, increased yield, or improved architecture to cause reduced lodging or defoliation. Various breeding steps are identified in a mature human intervention approach, such as selecting lines to be crossed, directing pollination of parental lines, or selecting appropriate progeny plants. Different breeding measures are taken according to the required characteristics. Related techniques are well known in the art and include, but are not limited to, hybridization, inbreeding, backcrossing, mixed line hybridization; variety fusion, interspecific hybridization, aneuploidy techniques, and the like. Thus, the transgenic plants according to the invention and their seeds can be used to develop improved plant lines, for example, to enhance the efficacy of conventional methods (such as herbicide or pesticide treatment) or to allow the omission of said methods due to their modified genetic properties. Alternatively, new crops with enhanced stress tolerance can be obtained, which, due to their optimized genetic "equipment", can result in better quality harvested products than products that cannot tolerate rather harmful developmental conditions.

In seed production, the germination quality and uniformity of seeds is an essential product characteristic, while the germination quality and uniformity of seeds harvested and sold by farmers is not important. Due to the difficulty of keeping crops free of other crops and weed seeds, seed producers have developed fairly extensive and well-defined seed production practices, which are experienced in the field of pure seed growth, adaptation and distribution, in order to control seed congenital diseases and produce seeds with better germination. Thus, farmers often purchase certified seeds that meet certain quality standards without using seeds harvested from their own crops. Propagation material used as seeds is usually treated with a protective coating comprising herbicides, insecticides, fungicides, bactericides, nematicides, molluscicides or mixtures thereof. Commonly used protective coatings include, for example, cycloheximide, carboxin, tetramethylthiuram disulfide (TMTD)^R)，methalaxyl(Apron^R) And pirimiphos-methyl (Actellic)^R) The compound of (1). These compounds may be formulated, if desired, with other carriers, surfactants or application-promoting adjuvants conventionally used in the art of compositions to provide protection against damage caused by bacterial, fungal or animal pests. The protectant coatings may be applied by impregnating the propagation material with a liquid formulation or coating with a combined wet or dry formulation. Other methods, such as treatment directly on the shoots or fruits, may also be applied.

Another aspect of the present invention is to provide novel agronomic methods, such as the methods exemplified above, which are characterized in that transgenic plants, transgenic plant material or transgenic seeds according to the invention are used.

The present invention can be used with any plant that can be transformed and regenerated into a transgenic plant. Male sterility, female sterility, or both may be controlled by administration of a suitable chemical ligand. Control of plant fertility is particularly useful for producing hybrid seed. In order to produce hybrid seed uncontaminated by selfed seed, pollination control must be performed to ensure that the hybrid is pollinated without self-pollination. This is usually done with mechanical, genetic or Chemical Hybridising Agents (CHA). For example, in corn, current practice is to mechanically remove tassels from the female (or seed) parents, which is a time consuming and laborious process. In wheat, mechanical fertility control on a seed production scale is not achievable and no genetic source of fertility control is established. The use of the present invention in producing hybrid seed provides the advantages of reliability, ease of use and control of male or female fertility.

Transgenic plants containing the appropriate receptor expression cassette and target expression cassette can be made homozygous and maintained indefinitely. To obtain hybrid seed, homozygous lines of parent 1 and parent 2 are crossed. In one example of the use of the present invention to produce hybrid seed, parent 1 is engineered to be male sterile in the presence of juvenile hormone or one of its agonists, while parent 2 is engineered to be female sterile in the presence of juvenile hormone or one of its agonists. Both parent 1 and parent 2 are contacted with juvenile hormone or one of its agonists and the only successful seed production results from the pollen of parent 2 fertilizing the ovule of parent 1. In a second embodiment using the present invention; parent 1 is engineered to be male sterile in the absence of juvenile hormone or one of its agonists, while parent 2 is engineered to be female sterile in the absence of juvenile hormone or one of its agonists. Parent 1 and parent 2 are contacted with juvenile hormone or one of its agonists to maintain each line by self-fertilization. To produce hybrid seed, the two parental lines are interplanted and only hybrid seed is obtained. The introduced restoring gene can restore the fertility of the hybrid plant. Any desired hybrid seed can be produced in these methods.

Chemical control of plant transgene expression is useful for modulating significant developmental alterations in target crops, altering crop plants to be more adapted to harsh environments, altering the flow of specific metabolic pathways, or simply inducing high levels of expression of individual desired protein products. Chemical control of plant development programs allows farmers to dictate when to initiate specific events such as flower development, picking of leaves or fruits or other important developmental stages. Chemical control of specific developmental events allows farmers greater flexibility in planting and harvesting time and their ability to respond to specific environmental conditions, such as early winter or the prediction of rainy spring. This change can be achieved by controlling genes critical to development or conditional response pathways, similar to the homeogene of Drosophila.

It is also useful to control gene expression of metabolic pathways. It is believed that plants can only consume a fixed amount of energy and any enhancement of a particular pathway such as storage of protein biosynthesis will result in a simultaneous reduction in the amount of biosynthesis via energy-competitive pathways, such as starch or lipid synthesis. In cases where such a change in the biosynthetic pathway is acceptable only after the plant has reached a certain stage of development (i.e., maturity and growth), chemical regulation of gene expression may be useful or even necessary to effect a particular biosynthetic change.

Chemical regulation of gene expression is useful for high-level overexpression of a particular protein. Certain proteins are known to be toxic to cells when expressed in heterologous hosts, exogenous subcellular compartments, or even expressed at abnormally high levels. Chemical control of the expression of this protein is advantageous for normal plant growth or even necessary to obtain sufficient plant quantities to confirm the use of plants as biosynthetic protein factories. Proteins useful for large scale biosynthesis in plants include industrial enzymes, pharmaceutical proteins, antigens and other proteins.

Bioassays for identifying ligands for steroid hormone receptors are known (Evans et al, U.S. patent No. 5,298,429). The disclosed receptors are limited to glucocorticoid, mineralocorticoid, estrogen-related hormone and thyroid hormone receptors. These receptors were transformed into mouse kidney cell cultures (CV-1 or COS cell lines) and tested for their ability to transactivate chimeric CAT gene expression in the presence of appropriate mammalian hormones. There is no disclosure of transforming plant cells or constructing plant expressed genes with these receptors, or those receptors other than those having a mammalian hormone as a ligand.

USP is considered to be an "insect retinoid receptor" in Oro and Evans, WO 91/14695. In a prophetic example, XR2C encoding USP was transformed into insect cell cultures (S2 cell line) to transactivate the chimeric CAT gene in response to the addition of retinoic acid. This document suggests that insect or animal cells transformed with the "insect retinoid receptor" can be used to screen for compounds that lead to receptor activation. However, Oro et al later reported that USP was not activated by retinoic acid in Drosophila cell culture assays and that USP did not respond to any of the retinoids or juvenile hormones tested, including methoprene, under conditions in which RXR responds (Harmon et al and references therein).

Since it is disclosed herein that juvenile hormone and its agonists are ligands of USP, and that USP can be used to activate target gene expression in plant cells in the presence of juvenile hormone or one of its agonists, it is now possible to find new ligands for the USP receptor that are effective in a plant cell environment. The screening is based on the expression of a target expression cassette encoding a receptor-regulated reporter gene in a transgenic plant or plant cell, wherein the transgenic plant or plant cell also expresses a transgenic USP receptor polypeptide and optionally a second receptor polypeptide different from the USP receptor polypeptide. A chemical substance to be tested for its ability to induce USP receptor mediated activation of target polypeptide expression is contacted with the transgenic plant or plant cell at various concentrations and then assayed for reporter gene expression to determine target polypeptide expression. Test agents that exhibit activation or a statistically significant increase in expression of the target polypeptide and test agents that exhibit inhibition or a statistically significant decrease in expression of the target polypeptide are identified as ligands for the USP receptor polypeptide. The method allows for identifying as its ligand a test substance not previously known to be a ligand for USP in a plant cell environment or validating as its ligand a test substance suspected of being a ligand for USP in a plant cell environment. It is therefore now possible to generate ligands for USP receptor polypeptides by performing the following method steps.

-synthesis of new test substances according to conventional methods known in the chemical art;

-transforming a plant cell with a USP receptor expression cassette encoding a USP receptor polypeptide and a target expression cassette encoding a target polypeptide;

-culturing progeny cells of said transformed plant cells;

-expressing USP receptor polypeptide in progeny cells;

-contacting the progeny cells with the new test substance synthesized as above;

-determining the expression of the target polypeptide;

-repeating the above two method steps with other new test substances;

-selecting a test agent that significantly activates or inhibits expression of the target polypeptide; and

-repeating the chemical synthesis of the selected substance.

Ligands for USP receptor polypeptides obtainable by the above-described process steps form a further subject of the present invention.

In addition, the method can be used to identify antagonists or inhibitors of USP receptor mediated activation of target polypeptide expression. The antagonist is identified by its ability to reduce ligand-induced activity of target polypeptide expression.

Different reporter genes can be used as target expression cassettes in the screening method. One useful reporter gene is firefly luciferase. Its use in target expression cassettes is described in examples 7 and 9 below. Another useful reporter gene is GUS or glucuronidase, which catalyzes the cleavage of a chromogenic substrate such as 5-bromo-4-chloro-3-indolyl- β -D-glucuronide or o-nitrophenyl- β -D-glucuronide. The GUS reporter gene has the advantage of producing a chromogenic reaction product that can be detected quantitatively, e.g., by spectrophotometry or qualitatively by visual inspection. Receptor expression cassettes useful in the screening method are USP, or chimeric forms of USP, such as USP-VP16 or VP 16-USP.

Since USP is related in sequence to the mammalian RXR α receptor and RXR is able to form a heterodimer with EcR (Thomas et al, Nature 362: 471-475(1993)), receptor expression cassettes encoding RXR can also be used in this screening method. In this way, the USP ligand may be found to be useful as a ligand for RXR in plant cells, or a chemical substance other than juvenile hormone or one of its agonists may be identified as a suitable chemical ligand for RXR in plant cells.

Examples

The following examples further describe the materials and methods used in the practice of the present invention and the results obtained. They are provided by way of illustration and the description thereof should not be construed as limiting the invention. Example 1: construction of plant-expressible receptor expression cassettes encoding ecdysone receptors

The DNA coding region for Drosophila ecdysone receptor (EcR) was isolated from a cDNA library prepared in lambda gt11(Clontech, Cat. No. IL1005b) from Canton S pupae (6 days) and from a genomic PCR-generated fragment using oligonucleotides designed with the published sequence for the EcR B1 isoform (Koelle et al, cell 67: 59, 1991). The B1 isoform EcR sequences were confirmed by automated sequence analysis and sequence comparison to published sequences using standard methods (Talbot et al, cell, 73: 1323, 1993). The full length EcR coding region expressed was modified in a PCR reaction to contain a BamH I site just upstream of the start codon using oligonucleotide SF43 (5'-CGC GGA TCC TAA ACA ATG AAG CGG CGC TGG TCG AACAAC GGC-3'; SEQ ID NO: 1). Plant expression vectors pMF6 and pMF7 contain a cauliflower mosaic virus 35S promoter (CaMV35S), maize Adh1 intron 1, and nopaline synthase polyadenylation and termination signals (see Goff et al, Gene and development 5: 298, 1991). The vectors pMF6 and pMF7 differ only in the orientation of the polylinker used to insert the desired coding sequence. The full-length EcR coding sequence was ligated into the plant CaMV35S expression vector pMF6 by using flanking BamH I restriction sites. This receptor expression cassette is designated 35S/EcR. Example 2: construction of a plant-expressible receptor expression cassette encoding an Ultraspiracle receptor

Henrich et al, nucleic acids research 18: 4143(1990) describes a cDNA encoding the Drosophila natural Ul-traspiracle receptor (USP). The full-length USP coding sequence with flanking 5 'and 3' untranslated regions was ligated to the plant expression vector pMF7 (described in example 1) using flanking EcoR1 restriction sites. This receptor expression cassette is designated 35S/USP. Example 3: construction of receptors having DNA binding region of GAL4 and ligand binding region of EcR

Expression cassette

A receptor expression cassette was constructed in which the DNA binding domain of EcR was replaced with the DNA binding domain of GAL4 fused to the N-terminal position. The DNA coding region of Drosophila EcR was obtained as described in example 1. The coding sequence for the DNA binding region of GAL4, Ma and Ptashne, cell, 48: 847(1987).

The receptor expression cassette encoding the GAL4-EcR chimeric receptor polypeptide is constructed by fusing the DNA binding domain of GAL4 to the ligand binding domain and the carboxy terminus of EcR. To prepare the fusions, oligonucleotide SF23, (5'-CGC GGG ATC CAT GCG GCCGGA ATG CGT CGT CCC G-3'; SEQ ID NO: 2) was used to introduce a BamH I site by PCR into the cDNA sequence of EcR at the nucleotide position corresponding to amino acid residue 330 (just after the EcR DNA binding region). The resulting truncated EcR coding sequence (EcR)^330-878) Subcloning into plasmid pKS + (Stratagene).

The GAL4 subclone was obtained from plasmid pMA210 containing the coding sequence for the DNA binding region (amino acids 1-147) by subcloning the amino-terminal DNA sequence of GAL4 into the Cla I site into pSK + (Strata-gene) as described previously (Goff et al, Gene and development 5: 298, 1991). This plasmid was designated pSKGAL2 and was cleaved with Cla I and Kpn I and the following double-stranded oligonucleotides were inserted:

5′-CGGGGGATCCTAAGTAAGTAAGGTAC-3′(SEQ ID NO：3)

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3 '-CCCCTAGGATTCATTCATTC-5' (SEQ ID NO: 4) the resulting plasmid was designated pSKGAL2.3. Use of pSK⁺DNA binding domain and pKS of middle GAL4⁺Middle EcR^330-878The BamH I site in the polylinker flanking part of the molecule produced a complete fusion 35S/GAL4-EcR^330-78. These coding sequences were ligated into the monocot expression vector pMF6 (described in example 1) by using flanking EcoR I restriction sites. This receptor expression cassette is designated 35S/GAL4-EcR^330-878. Example 4: construction of ligand binding region with Ultraspiracle and transactivation of VP16

Plant-expressible receptor expression cassettes for regions

A receptor expression cassette was constructed containing the ligand binding domain of USP and the transactivation domain of VP16 fused to the N-or C-terminus of the USP polypeptide.

To construct a receptor expression cassette encoding a chimeric polypeptide having the transactivation domain of VP16 at the C-terminal position, the Xho I site at coding sequence USP nucleotide number 1471 was used for subcloning into pKS⁺(Stratagene) the carboxy terminus and stop codon of the receptor USP cDNA (described in example 2) were removed. The resulting USP subclones encoding amino acids 1 to 490 were fused to the transactivation region of VP16 using the flanking KpnI restriction site of the USP subclone and the KpnI site of pSJT1193CRF3 (Triezenberg et al, Gene and development 2: 718-729(1988)) encoding the 80 carboxy-terminal amino acids of VP 16. The resulting USP-VP16 fusion was cloned into the CaMV35S plant expression vector pMF7 (described in example 1) using EcoRI and BamH I restriction sites flanking the coding sequence of USP-VP 16. These receptor expression cassettes are designated 35S/USP-VP 16.

USP derivatives with transcriptional activation regions fused to the amino terminus were constructed by first engineering the BamH I site adjacent to the USP start codon in a PCR reaction using oligonucleotide SF42 (5'-CGC GGA TCC ATG GAC AACTGC GAC CAG GAC-3'; SEQ ID NO: 5). The stop codon of VP16 was removed, and the flanking BamH I site introduced using oligonucleotide SF37 (5'-GCG GGA TCCCCC ACC GTA CTC GTC AAT TC-3'; SEQ ID NO: 6) and the start codon with the plant consensus sequence just upstream of the start codon and the BamH I site were introduced at the amino terminus in a PCR reaction using oligonucleotide SA115 (5'-GTC GAG CTC TCG GAT CCT AAA ACA ATGGCC CCC CCG ACC GAT GTC-3'; SEQ ID NO: 7) as a primer. The resulting VP16 activation region and USP coding sequence (encoding amino acids 1 to 507) were ligated into the framework with adjacent BamH I sites, and VP16-USP coding sequence was inserted into CaMV35S plant expression vector pMF7 with 5 'BamH I and 3' EcoR I sites. This receptor expression cassette was designated 35S/VP 16-USP. Example 5: construction of DNA binding and ligand binding regions with EcR and maize C1 Regulation

Trans-activation region receptor expression cassette for segment gene

EcR was generated by placing the start codon on the full-length EcR coding sequence immediately before the EcR DNA binding region using the oligonucleotide SF30(5 '-CGC-GGA-TCC-ATG-GGT-CGC-GAT-GAT-CTC-TCG-CCF-TC-3'; SEQ ID NO: 8) used in the PCR reaction^227-825-C1 fusion. The coding sequence for the transcriptional activation region of the maize C1 protein (amino acids 219-273) (Goff et al, Gene and development 5: 298-309(1991)) is fused in-frame to the coding sequence for EcR amino acids 51-825 (at the EcR Kpn I restriction enzyme site). The C1 transactivation domain was linked to EcR with a polylinker encoding VPGPPSRSRV-SISLHA (SEQ ID NO: 9). 35S/EcR is constructed by inserting a BamH I fragment carrying the coding sequence into the pMF7 vector^227-825-C1 plant expression vector fusion. The receptor expression cassette is designated 35S/EcR^227-825-C1. Example 6: construction of DNA-binding domain having GAL4, ligand-binding domain of EcR and Jade

Receptor expression cassette for transactivation region of rice C1 regulatory gene

GAL4-EcR as described in example 3 was used^330-878Constructs and EcR of example 5^227-825Construction of the-C1 construct GAL4-EcR^330-825-C1 fusion. The sequence of the EcR coding region (beginning at amino acid 456) is exchanged at the Aat II site. The receptor expression cassette is designated 35S/GAL4-EcR^330-825-C1. Example 7: construction of a fluorescent light encoding firefly with GAL4DNA binding domain response elements

Plant-expressible target expression cassette for cellulase

Plant-expressible target expression cassettes encoding firefly luciferase with the response element for the GAL4DNA binding region were constructed as follows. The core promoter of maize Bronze-1(Bz1) which initiates firefly luciferase synthesis was removed from the Bz1 reporter gene pBz1LucR98(Roth et al, plant cells 3: 317, 1991) via Nhe I and Sph I sites and placed in a pUC 6S-derived plasmid carrying the luciferase gene. The modified Bz1 core promoter contained the Nhe I site (GCTAGC) and the Bz1 promoter sequence up to nucleotide position-53 (Roth et al, plant cell 3: 317, 1991). The reporter gene pGALLue2(Goff et al, Gene and development 5: 298, 1991), which was regulated by GAL4 by digestion with EcoR I and Pst I, was stripped of 10GAL4 binding sites and inserted into pBluescript (Stratagene) using the same restriction enzyme sites. The Hind III site 5' to the GAL4 binding site was changed to a BamH I site by inserting Hind III/BamH I/Hind III adaptors, and the resulting BamH I fragment containing the GAL4 binding site was removed and placed in the Bgl II site upstream of the luciferase-promoting Bz1 core promoter. This target expression cassette is designated (GAL 4)_b.s.)10-Bz1_TATAand/Luc. Example 8: construction of a plant encoding firefly luciferase with direct repeat response elements

Target expression cassette for physical expression

Plant-expressible target expression cassettes encoding firefly luciferase with Direct Repeat (DR) response elements containing an EcRE half-site and an RXR-preferred half-site were constructed as follows: the maize Bz1 core promoter luciferase construct in a plasmid from pUC6S as described in example 7 was used as the origin. Combination of Chinese herbsA3 base pair double stranded synthetic oligonucleotide (SF 77: 5'-GAT CCG TAG GGG TCA CGA AGT TCA CTCGCA-3'; SEQ ID NO: 10) (SF 78: 5'-GAT CTG CGA GTG AACTTC GTG ACC CCT ACG-3'; SEQ ID NO: 11) with DR RE and spacer half sites with BamH I and Bgl II cohesive ends was phosphorylated, annealed and ligated upstream of the Bz1 core promoter via insertion of a single Bgl II site. 3 copies of RE were obtained by sequential Bgl II digestions and insertion of another double stranded oligonucleotide. This target expression cassette is called (OR3)₃-Bz1_TATAand/Luc. Example 9: transformation of plant cells and the use of receptor polypeptides in the presence of chemical ligands

Controlling expression of target polypeptides

Simultaneous transformation of plant cells with the necessary gene constructs by using high-speed microprojectile bombardment followed by biochemical assays for the presence of the target polypeptide can indicate that expression of the target polypeptide is controlled by a variety of receptor polypeptides, including chimeric receptor polypeptides of the invention. The essential gene construct comprises a USP receptor expression cassette encoding a USP receptor polypeptide (figure 1). Optionally, the USP receptor expression cassette may be transformed with a second receptor expression cassette encoding a receptor polypeptide different from USP (figures 2 and 3). In addition, a target expression cassette encoding the target polypeptide is also necessary.

The expression cassettes were simultaneously delivered to maize suspension cells cultured in liquid N6 medium by high-speed microprojectile bombardment using standard techniques of DNA precipitation on microprojectiles and high-speed bombardment driven by compressed helium (PDS-100/He, BioRad, Hercules, Calif.) (Chu et al, Sci-entia Sinica XV III, 659-668, 1975). Transfected cells were cultured in liquid suspension in N6 medium in the presence of a suitable chemical ligand for about 48 hours. After culturing, the transformed cells were harvested and then homogenized at 0 ℃. The extract was centrifuged at 10,000g at 4 ℃ for 5 minutes to remove debris.

Expression of the target polypeptide is detected by assaying the extract for the presence of a product encoded by the target expression cassette. A commonly used coding sequence for a target polypeptide for testing the control of expression of a receptor polypeptide in the presence of a chemical ligand is fireflyA luciferase. Firefly luciferase activity was determined by quantifying chemiluminescence generated by luciferase-catalyzed phosphorylation using an analytical luminometer type 2001 using ATP as a substrate (Promega luciferase kit, cat # E1500). Example 10: receptor polypeptide GAL4-EcR^330-825-C1 and USP-VP16 activated transplantation

Expression and activation of target polypeptide in biological cells inhibited by juvenile hormone agonists

Using the transformation procedure of example 9, the receptor expression cassette 35S/GAL4-EcR^330-825-C1 (example 6), receptor cassette 35S/USP-VP16 (example 4) and target cassette (GAL 4)_b.s.)₁₀-Bz/_TATAthe/Luc (example 7) was co-transformed into maize cells. Transformed cells were cultured in the presence of 10 μ M of fenoxycarbon or methoprene as a chemical ligand for about 48 hours. Luciferase assays were performed as described in example 9. The results are provided in table 1.

TABLE 1

Receptors	Chemical ligands	Luciferase Activity (light Unit)
			Experiment #1	Methoprene free of fenoxanide	2,503277,86277,41814,786
35S-free/GAL 4-EcR330-825-C1+35S/USP-VP1635S/GAL4-EcR330-825-C1+35S/USP-VP1635S/GAL4-EcR330-825-C1+35S/USP-VP16
	Experiment #2	Non-methoprene	1,302178,0925,730
35S-free/GAL 4-EcR330-825-C1+35S/USP-VP1635S/GAL4-EcR330-825-C1+35S/USP-VP16

The above results indicate that the 5' regulatory region of the target expression cassette containing the GAL4 response element can be activated in plant cells by the receptor polypeptides GAL4-EcR-C1 and USP-VP16, and that this activation is reversed in the presence of a juvenile hormone agonist. The expression level of the target polypeptide luciferase is 3.5 to 31-fold lower in the presence of the juvenile hormone agonist compared to in its absence. Example 11: receptor polypeptide GAL4-EcR^330-825-C1 and USP-VP16 activated transplantation

Expression of the target polypeptide in the biological cell and inhibition of this activation by juvenile hormone agonists

Using the transformation procedure of example 9, the receptor expression cassette 35S/GAL4-EcR^330-825-C1 (example 6), receptor cassette 35S/USP-VP16 (example 4) and target cassette (GAL 4)_b.s.)₁₀-Bz1_TATAthe/Luc (example 7) was co-transformed into maize cells. Transformed cells were cultured in the presence of 10. mu.M of tebufenozide and without 10. mu.M of either non-oxycarbide or methoprene as the chemical ligand for approximately 48 hours. Luciferase assays were performed as described in example 9.

TABLE 2

Receptors	Chemical ligands	Luciferase Activity (light Unit)
			35S-free/GAL 4-EcR330-825-C1+35S/USP-VP1635S/GAL4-EcR330-825-C1+35S/USP-VP1635S/GAL4-EcR330-825-C1+35S/USP-VP16	Without Tebufenozides Tebufenozide + methoprene	1,302178,092908,912159,873

The above results indicate that the 5' regulatory region of the target expression cassette containing the GAL4 response element is activated by the receptor polypeptides GAL4-EcR-C1 and USP-VP16 in response to the chemical ligand tebufenozide in plant cells and that this activation is inhibited in the presence of the juvenile hormone agonist methoprene. The level of tebufenozide-induced activation of luciferase gene expression increased approximately 6-fold when used alone, but was completely inhibited in the presence of the juvenile hormone agonist methoprene. Example 12: receptor polypeptide VP16-USP activates expression of target polypeptide in plant cells

Using the transformation procedure of example 9, the receptor-containing cassette 35S/VP16-USP (example 4) and the target cassette (DR3)₃-Bz1_TATAThe plasmid of/Luc (example 8) was co-transformed into maize cells. Transformed cells were cultured for 48 hours in the presence of 10. mu.M methoprene as a chemical ligand. Luciferase assays were performed as described in example 9. The results are provided in table 3.

TABLE 3

Receptors	Chemical ligands	Luciferase Activity (light Unit)
			No 35S/VP16-USP35S/VP16-USP	Non-methoprene	5,69029,967485,458

The above results indicate that the 5' regulatory region of the target expression cassette containing the direct repeat response element can be activated by the receptor polypeptide VP16-USP in plant cells in the presence of a juvenile hormone agonist. The expression level of the target polypeptide luciferase is approximately 16-fold higher than that observed in the absence of the juvenile hormone agonist. Example 13: construction of reporter expressing EcR, USP or RXR derivatives and carrying receptor modulation

Vectors of genes of interest for transformation of Arabidopsis plants

Agrobacterium T-DNA vector plasmids were constructed from the previously described plasmids pGPTV-Kan and pGPTV-Hyg (Becker et al, plant molecular biology 20: 1195-1197, (1992)). The Sac I/Hind IIIuid A (GUS) reporter genes of pGPTV-Kan and pGPTV-Hyp plasmids were replaced by Sac I/Hind III polylinkers from pGEM4Zf (+), pSPORT1, pBluescriptKS (+), pIC20H or pUC18 to yield plasmids pSGCFW, pSGCFX, pSGCFY, pSGCFZ, pSGCGA, pSGCGC, pSGCGD, pSGCGE, pSGCGF and pSGCGG, respectively. The GAL 4-regulated luciferase reporter gene as the target expression cassette was constructed as a T-DNA Agrobacterium plasmid by first subcloning the 328bpKpn I/Hind III fragment with 10GAL4 binding sites and maize Bronze-1 TATA as described in example 7 into the Kpn I/Hind III sites of the modified luciferase reporter plasmid pSPLuc + (Promega) to generate plasmid pSGCFO 1. A1.991 Kb Kpn I/Xba I fragment of pSGCFO1 containing the GAL4 binding site-Bz 1 TATA-luciferase reporter gene was subcloned into the T-DNA vector by ligation to the 7.194 Nde I/Spe I fragment of pSGCFX1 and the 4.111 Nde I/Kpn I fragment of pSGCFZ1 described above. The resulting plasmid was designated pSGCGL1 and carried the nos promoter-driven NPT II selectable marker conferring kanamycin resistance in transgenic plants and a GAL 4-regulated luciferase reporter gene. In a similar manner, a GAL 4-regulated GUS reporter gene was constructed containing a 10GAL4 binding site, a 35S TATA region and a GUS coding region and was designated pAT 86. A Direct Repeat (DR) response element reporter having 3 copies of DR RE, Bz1 TATA, the luciferase coding region and a nos terminator similar to that described in example 8 was also constructed in a similar manner as described for pSGCGL1 and was designated pSGCHU 1. The receptor expression cassettes described in examples 3-6 above were used to construct an analogous Agrobacterium T-DNA construct carrying the CaMV35S promoter and the nos polyadenylation signal. Single and dual receptor constructs were generated by subcloning the appropriate expression cassette into the GAL 4-luciferase reporter gene pSGCGL 1. Example 14: generation of a transgene expressing VP16-USP and carrying the DR-luciferase receptor

Genus Arabidopsis

Ara-bidopsis thaliana (Columbia) was transformed with an Agrobacterium vector containing the CaMV35S promoter and DR-luciferase reporter gene (as described in example 13 above) by the following vacuum infiltration procedure. By culturing Agrobacterium GV3101 in 2 XYT medium at 28 deg.C for 24-30 hr under aeration to OD₆₀₀Electrocompetent GV3101 Agrobacterium cells were prepared at 0.5-0.7 units, frozen on ice for 10-30 minutes, and centrifuged at 5,000RPM for 5 minutes at 4 ℃. The supernatant was discarded and the cell pellet was resuspended in1 volume of ice-cold 10% glycerol. The cells were again centrifuged at 5,000RPM for 5 minutes at 4 ℃. The supernatant was discarded and the cell pellet was resuspended in 0.05 volume of ice-cold 10% glycerol. The cells were again centrifuged at 5,000RPM for 5 minutes at 4 ℃, the supernatant was discarded, and the cell pellet was resuspended in 0.02 volume of ice-cold 10% glycerol. The cells were centrifuged again at 5,000RPM for 5 minutes at 4 ℃. The supernatant was discarded and the cell pellet was resuspended in 0.02 volume of ice-cold 10% glycerol. The cells were centrifuged again at 5,000RPM for 5 minutes at 4 ℃. The supernatant was discarded and the cell pellet was resuspended in 0.01 volume of ice-cold 10% glycerol. The cells were aliquoted in 200. mu.l/1.5 ml microfuge tube in liquid N₂Rapidly freezing at medium temperature, and storing at-80 deg.C. Electrocompetent cells were used before 6 weeks of storage at-80 ℃. Frozen electrocompetent cells were thawed on ice and 40. mu.l were transferred to a pre-cooled 1.5ml microcentrifuge tube. To the resuscitated cells 1ml of the appropriate Agrobacterium plasmid DNA (2-10ng) was added and mixed on ice. The cell/plasmid mixture was transferred into a pre-cooled 0.2cm Bio-Rad electroporation cuvette and electroporated with a time constant of 2.0 kilovolts, 600 ohms, 25 μ farads and 6msecAnd (4) a hole. Add 1+ ml 2 XYT medium to the electroporation cuvette, mix the cell/plasmid solution with the pipette tip, transfer the mixture to a new 1.5ml microcentrifuge tube, and then culture the cells on a 200RFM shaker at 37 ℃ for 1 hour. The cells were centrifuged in an Eppendorph speed adjustable microcentrifuge for 2 minutes at 6 steps, the supernatant was discarded, and the cell pellet was resuspended in the remaining liquid. The resuspended cells were plated on LB medium plates containing the appropriate antibiotic. The plates were incubated at 28-30 ℃ for 2-3 days. A single transformed colony was used to inoculate 50ml of LB medium in a 250ml flask containing 100. mu.g/ml rifampicin and 25. mu.g/ml gentamicin and 100. mu.g/ml kanamycin. Cultures were incubated at 28 ℃ at 250RPM for 24-36 hours and 10ml of culture was used to inoculate 500ml LB + antibiotics in 2 liter bottles. The culture was incubated overnight at 28 ℃ with shaking at 250 RPM. Plasmid DNA was isolated from this Agrobacterium culture and confirmed by restriction enzyme analysis.

Arabidopsis plants were grown for 4-5 weeks on net-covered soil in 3-inch square plastic pots in an artificial climate chamber set at 16 hours light, 8 hours dark, and 20 ℃. Plants were grown until the flower meristem was approximately 2 inches high. The floral meristem of the Arabidopsis plant to be transformed was removed 2 days before exposure to Agrobacterium. The Agrobacterium culture was centrifuged at 5000RPM for 5 minutes and the resulting pellet resuspended in 500ml of infiltration medium (4.3g MS salts/L, 5% sucrose, 0.01mg/ml benzylaminopurine, 100 ml/L Silwet L77, pH 5.8). Arabidopsis plants were soaked in water to saturate the soil. 500ml of bacterial cell suspension was transferred to the bottom of a sterile vacuum desiccator. Arabidopsis plants in their pots were placed into the agrobacterium solution with the top down. The desiccator was evacuated for 5 minutes and then slowly released. This vacuum treatment was repeated 3 times to wash the plants of excess Agrobacterium and returned to the growth chamber. The vacuum infiltrated plant is matured, flowers and produces seeds. The resulting seeds were dried in a low humidity drying chamber at 95 ℃ for about 5-10 days. The seeds were removed from the dried flowers by pressing and then filtered through a 425 micron mesh screen. It takes about 5 weeks to obtain seeds after vacuum infiltration. Once completely dried, approximately 240mg of seeds were sterilized by adding 1ml of 70% E-tOH, vortexing thoroughly and incubating at room temperature for 2 minutes. The seeds were briefly centrifuged at high speed in Eppendorf centrifuge tubes and the supernatant removed. The precipitated seeds were resuspended in 1ml of sterilization buffer (1 part 10% Triton X-100, 10 parts bleach, 20 parts double distilled water), vortexed and incubated at room temperature for 30 minutes. The seeds were briefly centrifuged at high speed in an Eppendorph microcentrifuge and the supernatant removed. The seeds were resuspended in 1ml sterile double distilled water, vortexed, centrifuged at high speed in a microfuge and the supernatant removed. This washing step was repeated 3 times, then the seeds were transferred to 50ml centrifuge tubes and washed once in 5ml double distilled water. Seeds were briefly centrifuged at high speed in a Beckman bench top centrifuge. The supernatant was discarded, the seeds were resuspended in 24ml of sterile 0.8 w/v% low-melting agarose at 50 ℃, mixed and divided into 8ml aliquots on 3 plates of 150mm Germination Medium (GM) containing the antibiotic used for selection (50. mu.g/ml kanamycin or 50. mu.g/ml hygromycin) and 500. mu.g/ml carbenicillin (Murashige and Skoog, plant physiology 15: 473-497, 1962). The plated seeds were incubated in the dark at 4 ℃ for 24 hours and then moved into a growth chamber at 20 ℃ with 16 hour light daily and 8 hour dark cycles. Selecting germinated seedlings on the plate for 5-10 days, transplanting the plantlets to a fresh selection plate, and transplanting to soil for re-selection after 5-10 days. The newly transplanted plantlets were covered with plastic film for 2-3 days and then grown until the beginning of the floral meristem. Example 15: chemical induction of isolated transgenic plant tissue

Transgenic plants were tested for inducible gene expression using the following techniques. Two leaves of approximately the same size were removed from the transgenic plants and cultured in water containing 50. mu.g/ml kanamycin (or 25. mu.g/ml hygromycin if the transgene carries the marker), 0.1% ethanol or 0.1% ethanol and 10. mu.M methoprene or a non-noroxy oxide. The leaves were cultured for approximately 24 hours under standard growth conditions as described in example 14. After incubation with the inducing compound, the cells were incubated at 500. mu.l of 100mM KPO₄Leaf extracts were prepared by homogenizing leaves at 0 ℃ in 1mM DTT, pH7.8 buffer. The extract was centrifuged in an Eppendorf microcentrifuge at 4 ℃ for 5 minutes and stored at 0 ℃ until assayed. Using an analytical luminescence 2010 luminometer and a Promega luciferase assay SystemLuciferase activity was measured in each extract according to the manufacturer's recommendations. The protein concentration of the extract was determined using the Pierce BCA protein assay (Smith et al, analytical biochemistry 150: 76-85). Luciferase values are expressed as light units per 10 seconds per 100. mu.g of extracted protein at room temperature. Nonooxycarbide treatment resulted in a 6.2-fold increase in luciferase activity, and methoprene resulted in a 25-fold increase in luciferase activity, as shown in Table 4 below.

TABLE 4

Receptors	Chemical ligands	Luciferase Activity (light Unit)
			Is free of	Is free of	638
35S/VP16-USP	Nonoxides of carbon	3,991
			35S/VP16-USP	Methoprene (MxPrin)	15,790

Example 16: use of reporter groups expressing USP or RXR derivatives and carrying receptor modulation

Screening of transgenic plants or plant cells for novel ligands that bind USP

Novel ligands for USP or RXR that are effective in plant cell environments can be discovered through screening methods based on the expression of receptor-regulated reporter genes as target expression cassettes in transgenic plants or plant cells that also express the appropriate receptor polypeptides. In this way, the chemical substance to be tested for its ability to mediate the expression of the USP or RXR activated target polypeptide is contacted with the transgenic plant or plant cell at various concentrations and then assayed for reporter gene expression. For example, 1) a transgenic plant or plant cell carrying a GAL 4-regulated luciferase reporter gene and GAL4-EcR and USP-VP16 receptor expression cassettes as target expression cassettes can be contacted with the substance to be tested for testing using a light amplification instrument such as a Hamamatsu light detection device and compared to an uncontacted plant, 2) a transgenic plant or plant cell carrying a GAL 4-regulated GUS reporter gene and GAL4-EcR-C1 and VP16-RXR receptor expression cassettes as target expression cassettes can be contacted with the substance to be tested for testing its ability to catalyze the cleavage of a chromogenic substrate such as 5-bromo-4-chloro-3-indolyl- β -D-glucuronide or o-nitrophenyl- β -D-glucuronide and compared to an uncontacted plant, 3) a transgenic plant or plant cell carrying a DR RE-regulated luciferase reporter gene and 16-USP expression cassette Plants or plant cells are contacted with the substance to be tested for detection using a light amplification instrument such as a Hamamatsu light detection device and compared to the non-contacted plants. Positive controls that may be found useful in the screening methods described above include, but are not limited to, tebufenozide and methoprene. In each of the above cases, detecting a higher level of expression of the target polypeptide in the presence of the test agent than in the absence of the test agent indicates that the test agent is a ligand for USP or RXR depending on the receptor expression cassette used in the method. In this manner, a test substance not previously known to be a ligand for USP in a plant cell environment can be identified as being, or a test substance suspected of being a ligand for USP in a plant cell environment can be identified as being, a positive. Example 17: isolation of receptor polypeptide mutants with reduced basal Activity

Using Leung et al, technique 1: 11-15(1989) the PCR mutagenesis described in vitro generated mutations in the ligand binding region of the Ultraspiracle receptor (USP). The PCR fragment of the mutated USP ligand binding region was cloned into a yeast expression vector operably linked to the VP16 transcriptional activation region. Transformation of the mutant constructs into yeast GAL4 reporter strain GGY 1: : 171. Yeast transformants were plated into medium containing the indicator X-Gal. Mutants with a basal level of USP receptor polypeptide activity on heterodimers produced white to light blue colonies on X-Gal indicator plates, while transformants expressing non-mutagenized USP receptor polypeptide produced dark blue colonies. Basal and chemical ligand-induced levels of receptor polypeptide activity were examined for white to bright blue colonies by culturing yeast cells representing the selected colonies in S medium containing glycerol, ethanol and galactose as carbon sources. The resulting culture was divided into two portions, one treated with juvenile hormone or one of its agonists, the other served as a control in the absence of chemical ligands. After contact with juvenile hormone or one of its agonists, the cultures of the treated and control fractions were assayed for beta-galactosidase activity according to the method of Miller (experiments in molecular genetics, p.352-355, J.H.Miller, eds., Cold spring harbor laboratory, Cold spring harbor, New York, 1972). Nucleotide sequences encoding mutant receptor polypeptides isolated and identified by this technique are useful as candidates for further testing, as the encoded receptor polypeptides may exhibit reduced basal activity in plant cells, induce expression of a target gene more fold in the presence of juvenile hormone or one of its agonists or produce a different response to a different agonist of juvenile hormone. Example 18: identification of mutant receptor polypeptides with enhanced function in plant cells

Receptor expression cassettes encoding the mutant USP receptor polypeptides of example 15 were prepared according to examples 2 and 4 above. These receptor expression cassettes are transformed into plant cells according to the method of example 9 in combination with the target expression cassette of example 8. The transformed plant cells are tested for activation of the 5' regulatory region of the target expression cassette by the mutant receptor polypeptide in the presence of juvenile hormone or one of its agonists. Mutant USP receptor polypeptides that produce a low basal expression of the target polypeptide in the absence of a chemical ligand and a high expression of the target polypeptide in the presence of juvenile hormone or one of its agonists are useful for controlling gene expression in plants. Example 19: breeding progeny of transgenic plants

Arabidopsis thaliana (Columbia) transformed plants prepared in example 14 were grown for 4-5 weeks on net-covered soil in 3-inch square plastic pots in a phytotron set at 16 hours of light, 8 hours of darkness and 20 ℃. The plant contains exogenous DNA in the form of a receptor and target expression cassette according to the invention integrated into its genome. Due to the life cycle of the transformed plant, the integrated DNA is transferred from one generation of plant to the next through the fertilization process.

Fertilization is the process by which male gametophytes interact with sporophytes or the female tissues of the gametophytes to successfully produce zygotes. Mature pollen grains are produced in the anther and stored on the stigma surface (pollinated), where they hydrate and germinate to grow pollen tubes. In the pollen tube the sperm cells are transferred to the embryo sac in the ovary (gynoecium) where the actual fertilization (gamete fusion) event occurs to produce a zygote. The zygotes are used in the form of seeds to obtain the next generation of plant lines. This next generation is called the "progeny" of the transformed plant.

Self-fertilization can result in the formation of progeny in which male gametophyte and female gametophyte tissues are produced from the same plant. This means that the individual plants are the source of the next generation of genomic DNA. Alternatively, progeny may be produced by crossing two different plants to fertilize by contacting the male gametophyte from one plant with the female sporophyte tissue of the other plant to produce the next generation plant. In this case, the progeny genomic DNA is from two different plants. Furthermore, when a transformed plant is crossed with an untransformed plant for fertilization, the progeny genomic DNA consists of genomic DNA from a transgene from one plant and genomic DNA from an untransformed plant from another plant. Regardless of whether progeny of a transformed plant are produced by self-fertilization or by cross fertilization, some progeny will receive unequal genetic assignments due to the presence of exogenous DNA integrated into the genome. This unequal genetic allocation can be confirmed using classical genetic and molecular biological techniques.

To produce the next generation of plants containing the receptor and target expression cassettes according to the invention, the original transformed plants are matured, flowered and seed-produced under controlled environmental conditions. The resulting seeds were further dried in a low humidity drying chamber at 95F for about 5-10 days. Seeds were removed from the dried flowers by squeezing the siliques and then separating the seeds from the other plant material by filtration through a 425 μm mesh screen. The seeds can be used to propagate progeny plants.

Although described with respect to arabidopsis plants, this method of producing next generation transformed plants is generally applicable to all angiosperms having the receptor and target expression cassettes according to the invention integrated into their genome.

All publications and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims.

Description of sequences

(1) General information:

(i) the applicant:

(A) name: CIBA-GEIGY AG

(B) Street: klybeckstr.141

(C) City: basel

(E) The state is as follows: switzerland

(F) And E, postcode: 4002

(G) Telephone: +4161691111

(H) Faxing: +41616967976

(I) Electric transmission: 962991

(ii) The invention provides a subject: juvenile hormone or one of its agonists as chemical ligand for controlling plant gene expression by receptor-mediated transactivation

(iii) Sequence number: 11

(iv) Computer readable form

(A) Media type: floppy disk

(B) A computer: IBM PC compatible machine

(C) Operating the system: PC-DOS/MS-DOS

(D) Software: PatentIn Release #1.0, Version #1.30B

(2) SEQ ID NO: 1, information:

(i) sequence characteristics:

(A) length: 42 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topology: wire type

(ii) Molecular type: other nucleic acids

(A) The following steps are described: "oligonucleotide SF 43" (/ desc) "

(iii) Suppose that: is free of

(xi) Description of the sequence: SEQ ID NO: 1:

CGCGGATCCT AAACAATGAA GCGGCGCTGG TCGAA-

CAACG GC 42

(2) SEQ ID NO: 2, information:

(i) sequence characteristics:

(A) length: 34 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topology: wire type

(ii) Molecular type: other nucleic acids

(A) The following steps are described: "oligonucleotide SF 23" (/ desc) "

(iii) Suppose that: is free of

(xi) Description of the sequence: SEQ ID NO: 2:

CGCGGGATCC ATGCGGCCGG AATGCGTCGT CCCG 34

(2) SEQ ID NO: 3, information:

(i) sequence characteristics:

(A) length: 26 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topology: wire type

(ii) Molecular type: other nucleic acids

(A) The following steps are described: "Positive strand oligonucleotide for the construction of pSKGAL2.3"

(iii) Suppose that: is free of

(xi) Description of the sequence: SEQ ID NO: 3:

CGGGGGATCC TAAGTAAGTA AGGTAC 26

(2) SEQ ID NO: 4:

(i) sequence characteristics:

(A) length: 20 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topology: wire type

(ii) Molecular type: other nucleic acids

(A) The following steps are described: "complementary strand oligonucleotide for the construction of pSKGAL2.3"

(iii) Suppose that: is free of

(xi) Description of the sequence: SEQ ID NO: 4:

CTTACTTACT TAGGATCCCC 20

(2) SEQ ID NO: 5, information:

(i) sequence characteristics:

(A) length: 30 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topology: wire type

(ii) Molecular type: other nucleic acids

(A) The following steps are described: "oligonucleotide SF 42" (/ desc) "

(iii) Suppose that: is free of

(xi) Description of the sequence: SEQ ID NO: 5:

CGCGGATCCA TGGACAACTG CGACCAGGAC 30

(2) SEQ ID NO: 6:

(i) sequence characteristics:

(A) length: 29 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topology: wire type

(ii) Molecular type: other nucleic acids

(A) The following steps are described: "oligonucleotide SF 37" (/ desc) "

(iii) Suppose that: is free of

(xi) Description of the sequence: SEQ ID NO: 6:

GCGGGATCCC CCACCGTACT CGTCAATTC 29

(2) SEQ ID NO: 7, information:

(i) sequence characteristics:

(A) length: 45 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topology: wire type

(ii) Molecular type: other nucleic acids

(A) The following steps are described: "oligonucleotide SA 115"

(iii) Suppose that: is free of

(xi) Description of the sequence: SEQ ID NO: 7:

GTCGAGCTCT CGGATCCTAA AACAATGGCC CCCCCGAC

CG ATGTC 45

(2) SEQ ID NO: information of 8:

(i) sequence characteristics:

(A) length: 35 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topology: wire type

(ii) Molecular type: other nucleic acids

(A) The following steps are described: "oligonucleotide SF 30" (/ desc) "

(iii) Suppose that: is free of

(xi) Description of the sequence: SEQ ID NO: 8:

CGCGGATCCA TGGGTCGCGA TGATCTCTCG CCTTC 35

(2) SEQ ID NO: 9, information:

(i) sequence characteristics:

(A) length: 16 amino acids

(B) Type (2): amino acids

(C) Chain type: single strand

(D) Topology: wire type

(ii) Molecular type: peptides

(iii) Suppose that: is free of

(v) Fragment type: inner part

(xi) Description of the sequence: SEQ ID NO: 9:

Val Pro Gly Pro Pro Ser Arg Ser Arg Val Ser Ile Ser leu His Ala

1 5 10 15

(2) SEQ ID NO: 10, information:

(i) sequence characteristics:

(A) length: 30 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topology: wire type

(ii) Molecular type: other nucleic acids

(A) The following steps are described: primer SF77 "

(iii) Suppose that: is free of

(xi) Description of the sequence: SEQ ID NO: 10:

GATCCGTAGG GGTCACGAAG TTCACTCGCA 30

(2) SEQ ID NO: 11, information:

(i) sequence characteristics:

(A) length: 30 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topology: wire type

(ii) Molecular type: other nucleic acids

(A) The following steps are described: primer SF78 "

(iii) Suppose that: is free of

(xi) Description of the sequence: SEQ ID NO: 11:

GATCTGCGAG TGAACTTCGT GACCCCTACG 30

Claims (31)

1. A transgenic plant cell, plant material or plant and the progeny thereof comprising:

a) a USP receptor expression cassette encoding a USP receptor polypeptide, and

b) a target expression cassette encoding a target polypeptide.

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

3. A method for producing a plant cell or plant according to claim 1, comprising treating a plant cell or plant with:

a) a USP receptor expression cassette encoding a USP receptor polypeptide, and

b) a target expression cassette encoding a target polypeptide,

transforming a plant cell or plant.

4. A method according to claim 3, comprising obtaining plant cells or progeny of plants transformed with said expression cassette.

5. The plant according to claim 1, wherein said plant is maize.

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

7. A method of controlling gene expression in a plant according to claim 1 or such plant additionally comprising a second receptor expression cassette encoding a second receptor polypeptide different from the USP receptor polypeptide comprising:

a) expressing one or more of the receptor polypeptides in said plant, and

b) contacting said plant with juvenile hormone or one of its agonists.

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

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

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

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

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

13. The method according to claim 7, wherein said agonist is selected from the group consisting of fenoxate, diofenolan, meprobrine, hydroprene, diofenolan, triflumuron of methoprene acid, hemamflumuron, teflubenzuron, flufenoxuron, flucycloxuron, and lufenuron, diflubenzuron and chlorfluzuron.

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

15. A method of controlling the fertility of a plant according to claim 2, or a plant further comprising a second receptor expression cassette encoding a second receptor polypeptide, comprising:

a) expressing one or more receptor polypeptides in said plant; and

b) contacting said plant with juvenile hormone or one of its agonists.

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

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

18. The method according to claim 15, wherein the USP or second receptor expression cassette comprises an anther-specific promoter operably linked to a coding sequence for a receptor polypeptide.

19. The method according to claim 15, wherein the USP or second receptor expression cassette comprises a pistil-specific promoter operably linked to a coding sequence for a receptor polypeptide.

20. The method according to claim 15, wherein the target polypeptide is a barnase of the ribonuclease bacillus.

21. The method according to claim 15, wherein the target expression cassette encodes an antisense sequence that causes fertilization invalidity.

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 the ribonuclease inhibitor barnase inhibitor.

24. A method of identifying a ligand for a USP receptor polypeptide that activates or inhibits expression of a target expression cassette in a plant cell environment, comprising the steps of:

a) transforming a plant cell with a USP receptor expression cassette encoding a USP receptor polypeptide and a target expression cassette encoding a target polypeptide;

b) expressing a USP receptor polypeptide in said plant cell;

c) contacting a plant cell with a test substance;

d) detecting expression of the target polypeptide; and

e) identifying a test agent that significantly activates or inhibits expression of the target polypeptide.

25. The method according to claim 24 further comprising transforming said plant cells with a second receptor expression cassette encoding a second receptor polypeptide different from the USP receptor polypeptide and expressing said second 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 quantitatively.

28. A method of producing a ligand for a USP receptor polypeptide comprising the steps of:

a) synthesis of new test substances according to methods known in the art;

b) transforming a plant cell with a USP receptor expression cassette encoding a USP receptor polypeptide and a target expression cassette encoding a target polypeptide;

c) culturing progeny cells of said transformed plant cell;

d) expressing a USP receptor polypeptide in progeny cells;

e) contacting the progeny cells with the test substance of step a);

f) determining expression of the target polypeptide;

g) repeating the process of steps e) and f) with different test substances according to step a);

h) selecting a test agent that significantly activates or inhibits expression of the target polypeptide; and

i) repeating the process of step a) for the substance selected in step h).

29. A ligand for the USP receptor obtainable by the method according to claim 28.

30. An agronomic method in which transgenic plant material or plants comprising:

a) a USP receptor expression cassette encoding a USP receptor polypeptide; and

b) a target expression cassette encoding a target polypeptide.

31. Use of a juvenile hormone or a juvenile hormone agonist for controlling the expression of a target polypeptide in a plant according to claim 1.