WO2006124502A2 - An inducible genetic cascade for triggering protein expression in subsequent generations of plants - Google Patents

Abstract

The present invention provides a method for inducible expression of a desired trait in a plant where the desired trait is expressed at least one generation removed from the generation of plants in which expression is induced. The method comprises expression of at least three chimeric DNA sequences where the expression is linked through expression of site-specific recombinases. Ordered expression of the recombinases is provided by triggering a first inducible promoter linked to the coding sequence for a first recombinase, which in turn unblocks expression of the next coding sequence in a second step of an expression cascade by removing a blocking sequence from a target chimeric DNA sequence. The temporal expression of DNA coding sequences is determined by selecting misordered pairs of promoters and coding sequences which provide an expression cascade that can span different generations of plants. The invention further provides plants containing chimeric DNA sequences which can be expressed in an expression cascade.

Description

AN INDUCIBLE GENETIC CASCADE FOR TRIGGERING PROTEIN EXPRESSION IN SUBSEQUENT GENERATIONS OF PLANTS

RELATED APPLICATIONS

This application claims priority from U.S. Provisional Patent Application No. 60/679,996, filed on May 12, 2005. TECHNICAL FIELD

The present invention relates generally to plant molecular biology. More specifically, it relates to a method for selectively controlling the expression of one or more genes in a plant to express a desired trait in a specific generation of the plant by applying a specific trigger or stimulus during a prior generation of the plant such that a desired trait is expressed only in progeny of the plant to which the specific trigger or stimulus is applied. The invention further relates to plants containing stably integrated foreign chimeric DNA sequences that provide expression of a desired trait in progeny plants.

BACKGROUND OF THE INVENTION

Current commercial applications of the techniques of molecular biology in crop plants focus on constitutive expression of agronomically useful traits, such as insect or herbicide tolerance. These traits complement existing cultivars and farming practices, and do not interfere with normal plant function. Their constitutive, or always-on, expression in professionally sourced seed allows their use in traditional farming systems. The responsibility to insure trait function in the plant resides with the seed company that grows the seed from pure seed stock and tests seeds and plants for both the presence and function of the trait.

Plant scientists have recognized that for some desired genetic traits, constitutive expression may not be optimal (See for example, Oliver et al., U.S. Patent Nos. 5,723,765, 5,925,808 and 5,977,441; Caddick et al., 1998; Zuo et al. 2001, Masclaux et al., 2004; Chen et al., 2003; Zuo et al., 2000; and Guo et al., 2003, the disclosures of which are incorporated by reference in their entirety). Some traits preferably are expressed in a spatially or temporally controlled manner. Examples of such traits include: (1) pathogen or insect control traits where continued exposure of a pest to the control molecule promotes the rapid development of pest resistance; (2) traits that properly function only in the presence of require higher concentrations of molecules, than can be synthesized by the plant without creating a metabolic burden that might limit yield or growth; and (3) traits that are deleterious or interfere with normal plant function and/or plant-associated beneficial organisms (e.g. mychorrizal fungi, saprophytic fungi and predacious insects and mites) if expressed throughout the plant's life and tissues.

Methods used to control the temporal and spatial expression of genes in plants have employed gene switches in which a trigger applied to a plant subsequently turns on expression of a gene in that same plant. Several examples of single generation gene switches that control gene expression have been developed (See Oliver et al., U.S. Patent No. 5,723,765; Caddick et al. 1998). These systems have value where a gene in seed or in plants in vegetative growth, can be triggered in the same plant generation that benefits from the expression of a desired trait, such as expression of an insect control toxin gene, or an herbicide tolerance gene.

In U.S. Patent No. 5,723,765, Oliver et al. proposed a method to delay gene expression following an activation event. This method used a combination of a chemically- inducible, site-specific recombinase, an excisable block of DNA positioned between a late embryogenesis activated (LEA) promoter and a DNA sequence encoding a seed germination inhibitor to control the fate of transgenes within the environment. This mechanism was subsequently demonstrated to be operational in model crops.

The available expression systems are limited by the requirement that the treatment applied to trigger or activate the expression of a desired trait must be applied to the same plant in which the gene and its resulting desired trait are expressed. However, it would be agronomically and commercially advantageous to delay the actual expression of a desired gene to the generation subsequent to the generation in which activation of a genetic event occurs for a variety of reasons. For example, production of commercial transgenic seeds for crop plants requires assessment of the purity of samples of seed for the transgenic trait. The ability to activate expression of a seed marker trait in transgenic seed permits a rapid purity evaluation of such seeds prior to distribution to farmers. Thus, there is a need for a method to control the expression of one or more transgenes inserted into a plant population by applying a trigger or stimulus to plants such that expression is turned on in progeny of the plants to which the trigger or stimulus is applied. SUMMARY OF THE INVENTION

The present invention provides a method for the inducible expression of a desired trait in a plant where the desired trait is expressed at least one generation subsequent to the generation of plants in which expression is induced. The method comprises expression of at least three chimeric DNA sequences whose expression is linked through the use of site- specific recombinases that remove target blocking sequences from chimeric DNA sequences, which are stably inserted into the genome of a plant cell. The method provides the expression of inheritable genetic traits or events across generational barriers. That is, the desired genetic traits are expressed in a generation subsequent to, or in some embodiments, prior to, the primary induction event that induces the cascade of linked expression of the chimeric DNA sequences. The linked chimeric sequences are inserted into the genome of a transgenic plant using techniques of plant molecular biology.

In one aspect, the method comprises an inducible genetic cascade that comprises at least three chimeric DNA sequences that are stably inserted into the genome of a plant cell. The sequences are constructed such that expression of a desired trait in one generation can be selectively turned on and off using a trigger applied during a prior generation. The chimeric DNA sequences comprise a first chimeric DNA sequence having an inducible promoter operably linked to a DNA sequence encoding a first recombinase; a second chimeric DNA sequence having a seed specific promoter; a blocking sequence having at its 5' and 3' ends the recognition site for the first recombinase and a DNA sequence encoding a second recombinase, different from the first recombinase, such that the blocking sequence prevents the expression of the second recombinase. The third chimeric DNA sequence comprises a plant expressible promoter, a blocking sequence having at its 5' and 3' ends the recognition site for the second recombinase and a DNA sequence that provides a desired trait to be expressed in progeny that is not expressed in the parent plant.

In another aspect, the invention comprises plants having at least three chimeric DNA sequences, whose expression can be linked through a trigger or stimulus that induces the promoter of the first sequence, stably inserted in their genomes.

In another aspect, the invention comprises a method for providing an inducible gene expression cascade in a plant where activation of the expression cascade results in the complete excision of the inserted sequences following expression of the desired trait in a prior generation of the plant.

In another aspect, the invention comprises plants having misordered pairs of chimeric DNA sequences that provide the expression of a desired trait in one generatbn, followed by the complete excision of those DNA sequences in a subsequent progeny generation by inserting a group of misordered pairs of chimeric DNA sequences into the genome of a plant. The linked expression of these misordered pairs in a spatial and temporal order allows expression of a desired trait in one generation, followed by its complete excision in a subsequent generation.

In another aspect, the invention provides a method for expressing a desired trait in a specific generation of a plant, but not in subsequent generations where that trait would have long term deleterious effects on the growth and reproduction of subsequent generations of the plant.

Specific embodiments of the present invention will now be described by way of non- limiting examples of the invention, which are discussed in more detail below. BRIEF DESCRIPTION OF THE FIGURES

The following abbreviations are used in Figs. 1-7 of the present application: 34Sp is the Figwort Mosaic Virus promoter; 35Sp is the Cauliflower Mosaic Virus promoter;

AGP is the Alpha Globulin Promoter, which is expressed during seed development; alcR TF is a transcription factor that turns on the palcA promoter in the presence of ethanol; CRE is the CRE recombinase;

DT is a gene encoding a Desired Trait, indicated as DT1 and DT2; EIP is externally inducible promoter; ESP is an embryo-specific promoter; FLO/LFYp is the floral meristem promoter; FLP is the FLP recombinase; FRT is the FLP recombinase excision site; GERp is the Germination-specific promoter;

GSP is a germination-specific promoter;

LEAp is the Late Embryogenesis Abundant promoter that is turned on when seed is physiologically mature, but not desiccated; LOX is the CRE excision site; mutEPSPS is a mutant enol-pyruvylshikimate phosphate synthase which provides the glyphosate tolerance trait; P is a plant promoter indicated as P1, P2 and P3; palcA is an alcohol responsive promoter which is activated by alcR transcription factor in the presence of ethanol or related compounds; r is a site-specific recombinase recognition site as in M₁ r2, and r3 where M is specific for ssR1 ; r2 is specific for ssR2 and r3 is specific for ssR3; R is the site-specific R recombinase; RNAi CA is an interfering RNA which suppresses the expression of mRNA for carbonic anhydrase, an enzyme whose inhibition reduces oil biosynthesis in plants by approximately 50%; RNAi Sus is an interfering RNA which suppresses the expression of mRNA for sucrose synthase, an enzyme whose inhibition reduces fuzz and lint fiber development on cotton seeds.

RS is the R recombinase excision site; SMT is a seed marker trait coding sequence; SSP is a seed-specific promoter, indicated as SSpI and SSP2; ssR is a site-specific recombinase, indicated as ssR1 and ssR2; and Tnos is the transcription terminator sequence from the nopaline synthase gene of

Agrobacterium.

Fig. 1 is a schematic illustration of the different genetic constructs used to provide the inducible genetic cascade of the present invention and indicates whether the specific constructs are actively expressed during different stages of the life cycle of three generations of a specific plant line.

Fig. 2 is a schematic illustration of three plant life cycles starting from a first transgenic seed. The chimeric DNA sequences used to produce the inducible genetic cascade of the present invention are represented as rectangles labeled as a promoter linked into an expressible chimeric gene. In the first generation the EIP promoter is triggered to express a recombinase (ssR1) which in turn unblocks expression from promoter 1 (P1) and from the seed-specific promoter (SSP1). This leads to expression of the gene encoding desired seed marker trait (SMT) and the ssR2 (ssR2) late in the plant's life cycle. In turn, the ssR2 unblocks expression from P2 and SSP2, also late in the plant's life cycle. In the second generation, promoter 2 is active and expresses the gene encoding Desired Trait 1 (DT1). SSP2 also is active, expressing ssR3, which unblocks expression from P3 late in the progeny plant's life cycle. In generation three, P3 is active, expressing the gene encoding protein that provides DT2.

Fig. 3 is a schematic illustration of two plant life cycles starting from the germination of a first transgenic seed that contains the chimeric genes used to produce an inducible genetic cascade that produces plants containing nutritional proteins encoded by the plants genomic DNA.

Fig. 4 is a schematic illustration of gene cassettes to be used in the inducible gene cascade to prevent escape of a transgene. When vegetative meristems transition into floral meristems and the FLO/LFY homolog promoter is activated, which generates the production of the R recombinase only in the floral meristems. The activation of the R recombinase results in the complete excision of the genetic construction that contains all of the inserted transgenes. The seeds and pollen that derive from these flowers are therefore non-transgenic (non-GMO). Since the FLO/LFYgene has to be activated for flowers to form, this embodiment should be 100% effective in transgene removal.

Fig. 5 is a schematic illustration of gene cassettes for use in the inducible gene cascade to produce low oil seeds in a second generation.

Fig. 6 is a schematic illustration of plant life cycle diagrams illustrating an embodiment of the present invention which provides progeny cotton plants that produce high fiber yield and seeds with a low oil content. In Generation one, the plant expresses the alcR TF, which in combination with ethanol triggers the palcA promoter to express CRE recombinase. CRE then excises the chimeric DNA between the recombinase specific cleavage sites LOX, including the DNA sequence that encodes CRE. CRE also unlocks the expression of RNAi targeted to the Sus gene and the LEA promoter-driven expression of the recombinase FLP. In turn, FLP unblocks the AGP promoter. In generation two, RNAi targeted to carbonic anhydrase is expressed under the control of AGP in a developing cotton embryo, interfering with seed oil production resulting in low oil seed.

Fig. 7 is a schematic illustration of an embodiment of the present invention which provides progeny cotton plants that produce high fiber yield and seeds with a low oil content. In this embodiment CRE recombinase unblocks the PsGNS2 promoter expression of RNAi targeted to the Sus gene and the LEA promoter expression of FLP. DETAILED DESCRIPTION OF THE INVENTION

An embodiment of this invention provides a method for creating inducible genetic cascades that allow traits expressed in one generation to be selectively turned on and off using triggers applied during prior generations. More specifically, the invention includes methods to initiate a series of events in one generation that lead to expression of a desired trait in later generations. This is achieved by initiating an ordered series of heritable events, some of which are plant age- or tissue-specific. The second event occurs earlier in the plant life cycle than the first, extending the time required for the series of events to be accomplished over more than one plant generation. The inducible genetic cascade of this invention is particularly useful when one set of traits is desired in the seed production generation(s) and a different set is desired in the crop production generation.

The minimal set of components necessary to achieve an inducible genetic cascade in a preferred embodiment is: (1) an inducible element or promoter that responds to an input external to the plant, operably linked to a DNA sequence encoding a first recombinase and that when induced, results in expression of a recombinase that links the events in the genetic cascade; (2) a paired set of chimeric DNA sequences that are expressed in a (mis)temporal order such that the event that occurs later in the life cycle of the plant is triggered before the expression that normally occurs earlier in the life cycle; and (3) a gene of interest to be expressed in one generation of a plant, but not in the proceeding generation. The method can also be used to remove genetic components that provide a desired trait in a preceding generation, in which case the gene of interest would be expressed in the proceeding generation, but not the prior generation.

For the preferred inducible cascade to function there must be a functional link between the misordered pairs of chimeric DNA sequences inserted into the genome of the plant that occurs in a temporal sequence. That is, expression of the first gene, which occurs later in the plant life cycle, is required before expression of the second gene can occur. The action of the second genetic element in the pair must occur in a stage of the plant life cycle that precedes the action of first genetic element in the life cycle of an individual plant. This requirement forces the inducible cascade to extend from a sporophytic generation, through a gametaphytic generation and into the next gametophytic generation.

Because the misordered pairs are linked in this way, application of an external trigger that activates the inducible promoter induces the misordered pair of chimeric DNA sequences in the proper sequence to provide proper timing of expression of the desired trait or traits of interest over more than one generation of plants. This functional link is provided by blocking sequences flanked by recombinase excision sites that are present in the chimeric DNA constructs. Specific steps of the cascade involve removal of the blocking sequences in a regulated, ordered way by the site specific recombinases. Persistent regulatory agents may include stable transcription factors; small RNAs, including RNAi; and plant hormones and other second messengers. Each of the misordered paired genetic events must be developmentally regulated such that the timing of the misordered events is distinct enough to avoid overlap in event timing or a reversal in event timing.

Expression of the first of each misordered pair affects the ability of the second of the misordered pair to be expressed with sufficient duration to persist in the germ line cells of the plant. This effect can be permanent, such as an alteration in the chimeric DNA sequences inserted into the genome of germ line cells of the plant, or transitory, such as would occur with accumulation of a persistent regulatory agent in germ line cells.

The first chimeric DNA sequence in the minimal set preferably is an inducible element that activates a plant based system in response to an external trigger that launches the inducible cascade. A number of inducible elements or promoters that can be used as gene switches are available. (See, for example, Potenza et al., 2004.) Suitable inducible promoters include chemically inducible promoters, abiotic stimuli inducible promoters (such as light or heat), biotic stimuli, (such as a viral vector), and the stimulus of the combination of gametes during hybridization. Activation of the inducible element results in the actuation of the first component of a misordered pair. Expression of the first gene can be accomplished by modulating expression of the inducible promoter, by transcriptional regulation of the gene, by post transcriptional regulation of the gene or by post translational regulation of the expressed protein.

One preferred inducible promoter that can be used in a gene switch is the ethanol- inducible promoter, which allows an external input, treatment of plant tissues or seeds with ethanol to trigger gene expression. Other suitable chemical gene switches known in the art are triggered by ecdysone insecticides, tetracycline, estrogen analogs, or non-chemical inputs such as light, and temperature. The process of plant hybridization, that is the process of combining the genomes of two distinct genotypes through fertilization, also can be used as a gene switch.

The misordered pair of genetic elements, of the preferred embodiment generally meet two requirements. For the preferred inducible cascade to work properly, the first genetic element in the misordered pair creates an effect that persists until the second element of the misordered pair is expressed. This effect persists at least through the gametophytic stage of the plant life cycle and into the new sporophytic phase, i.e., it persists during fertilization, embryo development and seed germination. Examples of suitable first elements in the misordered pairs in the preferred embodiment include: (1) a recombinase that excises a blocking sequence to operably link a promoter with a DNA sequence that can be expressed, (2) a recombinase that creates an inversion that either operably links or unlinks a promoter with a gene that is then either expressed or not expressed, (3) an RNA sequence that results in the degradation of a target RNA and in the process self-replicates, (4) an RNA sequence that results in gene methylation, (5) a chemical that accumulates from one generation to the next and can alter subsequent gene expression, and (6) a transcription factor that acetylates or deacetylates histones in the vicinity of a gene or persists from one generation to the next.

The desired trait to be expressed at the end of the inducible cascade includes any trait that would be deleterious to the plant itself. Thus, the cascade can induce a first event which allows the plant to grow normally and produce seed, but which later activates a second event. The present method allows the second event to be one that is deleterious to plant growth or reproduction, as expression of the second event is limited to a specific generation of the plant. The proceeding generation, for example the seed increase generation, will not be affected.

If the second genetic event is provided by a chimeric DNA sequence having a tissue specific promoter, the tissue specific promoter directly or indirectly leads to the expression of the gene of interest in preferred embodiments.

Optional components of the inventive method include additional promoter-gene pair chimeric DNA sequences. Examples of such components include: additional misordered pairs of functionally linked promoter gene sequences to extend the number of plant generations or the time necessary to conclude the inducible genetic cascade. These additional chimeric DNA sequences can provide additional desired traits that can be turned on or off at various stages along the genetic cascade. The invention provides a system with components flexible that can achieve a timed cascade of expression of specific traits that is precisely regulated during subsequent generations and stages of plant growth in response to a single triggering event in a specific plant generation. The inducible inter-generational genetic cascade can generate, for example, (1) a breeder seed assurance marker trait, which tracks the efficiency of the initial triggering event in the seed production generation and (2) a seed quality or yield (seed or biomass) specific trait in the subsequent crop production generation or generations.

The present invention may be used to provide any plant for which transformation methods are available or can be developed with an inducible genetic cascade for expression of a desired trait in a plant that is at least one generation subsequent to the generation in which expression is induced. Preferred plants include cotton, corn, wheat, brassica, soybeans, sugar beets alfalfa, rice, tomatoes and sunflowers. A particularly preferred plant is cotton.

Numerous methods for transforming plants are available to those of skill in the art, including biological and physical methods for inserting chimeric DNA sequences into plant cells. In vitro culture methods for plant cell and tissue culture and for regeneration of transformed plants are also available to those of skill in the art of plant molecular biology. One preferred method is Agrobacterium mediated transformation, which is based on the natural transformation system of these bacteria. Several methods for direct transfer of chimeric DNA molecules are also available, including microprojectile bombardment, and electrophoration of protoplasts.

The first chimeric DNA sequence of the inducible genetic cascade preferably comprises an inducible promoter (ElP) operably linked to the coding sequence for a first site- specific recombinase (ssR1). This recombinase removes the blocking sequence flanked by ssR1 recognition sequences from the genome of the target plant. The blocking sequence preferably is situated in a cis configuration between a second set of plant active promoters with linked coding sequences of target genes that can be used to generate a useful trait and optionally also the next step of the inducible intergenerational genetic cascade. In one embodiment, the ssR1 targets a chimeric DNA sequence which is a plant active promoter (P 1) that directs expression of a gene in the testa or seed coat of seed produced in the seed production generation. The plant active promoter is separated by a blocking sequence flanked by recognition sequences specific to ssR1. When transcribed the gene provides a visible testa or seed coat phenotype. After the blocking sequence is removed by the ssR1 , the expressed gene provides a seed marker trait (SMT) that allows one to physically select seeds from plants in which the ssR1 has been successfully induced. This also permits direct assessment of the efficacy of chemical induction methods during the seed production stage of plants that contain the intergenerational genetic cascade mechanism.

A second preferred target chimeric DNA sequence for ssR1 contains a seed-specific promoter (SSP1) active in the plant embryo that is separated from a linked coding sequence that encodes a second different site-specific recombinase (ssR2) by a blocking sequence flanked by recognition sequences specific to ssR1. When ssR1 is activated by the application of the exogenous inducer, seed-specific expression of ssR2 is effected in the treated plant.

The target chimeric DNA sequence for ssR2 preferably is expressed at a time prior in the plant's life cycle to the time SSP1 is expressed and thus is expressed only in next generation embryos and plants, for example, the crop production generation. Preferred target chimeric DNA sequences for ssR2 contain a promoter (P2) that is not active in seed or embryonic tissues or a promoter that is specifically expressed at a stage or time in seed development that is prior to the expression of the seed-specific promoter (SSP1) that controls the expression of the ssR2.

The promoter and the DNA sequence encoding the third site-specific recombinase 3 (ssR3) are linked to a coding sequence that when transcribed encodes a desired trait, for example, a protein or nucleic acid that improves seed quality or yield (seed or biomass). The promoter and coding sequence are separated by a blocking sequence flanked by recognition sequences specific to ssR2. Since SSP1 is expressed later in the plant's life cycle than P2, ssR2 and SSP1 constitute a misordered pair. The target chimeric DNA sequences for ssR2 are active only in plants that originate from seed produced by the plant treated to activate ssR1 ; i.e., in the second generation or progeny plants.

In addition to the gene that encodes a desired trait such as a seed quality or yield (seed or biomass) protein, other suitable recombinase 2 target chimeric DNA sequences include a third set of plant active promoters with linked coding sequences that can be expressed to generate both useful traits and the next step of the inducible inter-generational genetic cascade. These traits can be activated and expressed in the third generation following the one in which the promoter was first induced.

Thus, a second target chimeric DNA sequence for ssR2 contains a second seed- specific promoter (SSP2) active in the plant embryo and a linked coding sequence that encodes a site-specific recombinase different from site-specific recombinases 1 and 2. The promoter and the coding sequence are separated by a blocking sequence that is flanked by recognition sequences specific to ssR2. Thus, once ssR2 is activated by the expression of the seed-specific promoter (SSP1), an activatable ssR3 encoding chimeric DNA sequence is generated. The seed-specific promoter (SSP2) that constitutes the transcriptional control of the ssR3 gene is active only at a stage of seed development prior to the stage at which SSP1, which controls the transcription of ssR2, is activated.

Expression of SSP1 leads to expression of the promoter, SSP2, which is only activatable at an earlier stage in the plant's life cycle than SSP1. Expression of ssR3 occurs only during the development of seeds that represent the third generation of plants from the generation in which an inducible promoter initially activated the cascade. In preferred embodiments the target chimeric DNA sequences for ssR3 contain a promoter (P3) and a linked coding sequence that encodes a desired trait (DT2) such as improved seed quality or yield (seed or biomass). The promoter P3 is either not active in seed or embryonic tissues, or is specifically expressed at a stage or time in seed development that is prior to the expression of the seed-specific promoter SSP2 that controls the expression of the ssR3. The promoter P3 and sequence encoding the sequence encoding DT2 are separated by a blocking sequence flanked by recognition sequences specific to ssR3. The target genes for ssR3 are activated only in third generation plants.

One embodiment of the present invention provides a method for producing plants which inducibly express linked genes in a cascade that spans more that one generation of plants. In this system, all inserted transgenic technology from a transgenic crop in the second generation is removed from the genome, such that progeny arising from the second generation are functionally transgene free. See Fig. 4. In this embodiment, a site-specific recombinase, recombinase 1 (ssR1), is operably linked to an inducible promoter (EIP) that can respond to an external stimulus to direct the production of ssR1, which then acts to remove blocking sequences positioned between recognition sites specific to recombinase 1 in a specific target chimeric DNA sequence. The target sequences for this embodiment comprise (1), a constitutive plant active promoter (P1) linked to, but separated from, a coding sequence for a protein that confers a desired trait (DT1) such as herbicide tolerance or insect tolerance by a blocking sequence of DNA, flanked at its 5' and 3' ends by excision recognition sequences specific for recombinase 1 ; and (2) a germination specific promoter (GSP) linked to, but separated from, a coding sequence for a second site-specific recombinase (ssR2) by a blocking sequence of DNA. This blocking DNA is also flanked at its 5' and 3' ends by excision recognition sequences specific for recombinase 1. When expression of recombinase 1 is activated by the application of an exogenous signal to a mature plant of generation one, the commercially desirable trait is expressed and the recombinase 2 gene is unblocked, but inactive, since a germination-specific promoter controls its activity. This promoter is not active in mature plants.

When seed (generation two) from generation one plants treated with the exogenous inducer, as discussed above, are planted and germinate, the germination-specific promoter becomes active, the recombinase 2 gene is expressed, producing the recombinase 2 enzyme. This enzyme removes the blocking sequence between recognition sites specific to recombinase 2 within specific target chimeric DNA sequence. As shown in Fig. 4, the target chimeric DNA sequence for recombinase 2 comprises a floral meristem specific promoter (FLO/LFYp) linked to, but separated from, a coding sequence for a third site-specific recombinase (ssR3) by a blocking sequence of DNA. This blocking sequence is flanked by excision recognition sequences specific for recombinase 2.

During the development of floral meristems in the generation two plants, the floral meristem-specific promoter become active and induces expression of ssR3. This third recombinase removes the blocking sequences positioned between recognition sites specific to ssR3, including its own coding region. In this embodiment, the entire system, including recombinase 1, recombinase 2, the commercially desirable trait gene and the recombinase 3 coding sequence itself, which are introduced into the plants that produce generation one, is flanked by excision sequences specific for recombinase 3. As a result, ssR3 expression completely removes of all introduced genes from the cells of the floral meristem and resultant flower structures including seed and pollen. Thus, the seed produced by generation three lacks any functional inserted transgenes.

Recombinase/excision sequence systems which are suitable for use with the invention include any system that selectively removes the recognition sequence-flanked DNA from chimeric DNA sequences inserted into a plant genome. The transgenic excision sequences preferably are unique to the plant to prevent unintended cleavage of the plant genome. Such systems are known to those of skill in the art of plant molecular biology. Examples include those systems discussed in Sauer, 1990, Sadowski, 1993, and U.S. Patent No. 5,723,765. Preferred recombinase/excision sequence systems include the bacteriophage CRE/LOX system, wherein the CRE protein performs site-specific recombination of DNA at LOX sites, the FLP/FRT system (Pan et al., 1993), and the R/RS recombinase system (Onouchi et al., 1995). As one of skill in the art will recognize, other systems which can be used include resolvases (Hall et al., 1993), SSV1 encoded integrase (Muskhekishvili et al., 1993), and the maize Ac/Ds transposon system (Shen and Hohn, 1992).

Inducible promoters which may be used in the chimeric DNA sequences of the present invention include the promoter from the ACE1 system, which responds to copper (Mett et al., 1993); the promoter of the maize lntron 2 gene which responds to benzenesulfonamide herbicide safeners (Hershey et al., 1991 and Gatz et al., 1984), the promoter of the Tet repressor from Tn10 (Gatz et al., 1991), the phosphate-deficiency responsive promoter from a phosphate-starvation responsive beta-glucosidase gene from Arabidopsis (Lefebvre et al., 2001) and the synthetic promoter containing a 235 base pair sulfur deficiency response element from a soybean α-conglycinin gene linked to a 35S core promoter sequence (Fujiwara et al., 2002). Any inducible promoter, which readily responds to an agent or other stimulus that can function as an external signal when applied to plant tissues, such as roots, leaves, or seed in a controlled manner, is suitable for use in methods of the invention as the inducible promoter in the first chimeric DNA sequence expressed in the cascade.

Inducible promoters that respond to an inducing agent to which plants do not normally respond are particularly useful. Preferred promoter systems include the inducible promoter from a steroid hormone gene, the transcriptional activity of which is induced by a glucocorticosteroid hormone (Schena et al., 1991) or the chimeric transcription activator, XVE, which functions in an estrogen receptor-based inducible plant expression system activated by estradiol (Zuo et al., 2000). A preferred inducible promoter is the ethanol- inducible ale gene expression system for transgenic plants (Salter et al., 1998, Caddick et al., 1998, Deveaux et al., 2003).

Suitable constitutive promoters (P) which can be used in the second chimeric DNA sequence of the cascade include the 35S and 19S promoters from cauliflower mosaic virus (CaMV) (Comai et al., 1992. Fraley et al., 1996), the 34S promoter from figwort mosaic virus (FMV) (Comai et al., 2000), the maize ubiquitin promoter (Cigan et al., 1998), the peanut chlorotic streak caulimovirus (PCISV) promoter (Maiti et al., 1998), promoters of Chlorella virus methyltransferase genes (Mitra et al., 1996), the full-length transcript promoter from figwort mosaic virus (FMV) (Rogers, 1995), the rice actin promoter (McElroy et al., 1990), the pEMU promoter (Last et al., 1991), MAS (Velten et al., 1984), the maize H3 histone promoter (Lepetit et al., 1992; Atanassova et al., 1992), and the promoters of Agrobacterium genes (Gelvin, 1988; Hall et al., 1992; Slightom et al., 1993; Barker et al., 1995). Germination specific promoters that are useful for the present invention include the oil seed rape cysteine protease gene promoter (Greenland et al., 2001).

Suitable seed-specific promoters include, but are not limited to, the cotton α-globulin promoter (AGP, Sunilkumar et al., 2002), the napin gene promoter (Kridl et al., 1991), soybean α- and β-conglycinin genes (Barker et al., 1988; Chen et al., 1988; Lessard et al., 1993), and soybean lectin promoter (Townsend and Llewellyn, 2002). Seed-specific promoters also can be generated by operable linkage of genetic elements that direct seed- specific expression to core promoter sequences. Such synthetic seed-specific promoters include, but are not limited to, the use of a concatemerized B-Box element from a 2S napin gene promoter to a 35S minimal promoter sequence (Rask et al., 1998; Ezcurra et ai., 1999), the addition of a G-Box element from the strictosidine synthase gene from Catharanthus roseus (Ouwerkerk and Memelink, 1999), and the linkage of a 68bp seed specific enhancer (SSE) element from a β-phaseolin gene to a 35S basal promoter (-64 to +6) (van der Geest and Hall, 1996). Examples of promoters that are active in both the seed and non-seed tissue include the 35S (Comai et al., 1992; Fraley et al., 5,530,196) and the 34S (Comai et al., U.S. Patent No. 6,051 ,753) promoters.

In a second preferred embodiment, the series of functional chimeric DNA sequences are functional transcriptional units linked on a single DNA molecule where the DNA is flanked on either end by the recognition sequence sites (RS) for the site-specific R recombinase. The chimeric DNA sequence comprises an alcA promoter (palcA) or alternatively a modified constitutive promoter containing the regulatory domains of the alcA promoter, linked to the coding sequence for the site-specific recombinase CRE and a suitable transcription termination signal sequence such as the TrbcS, the termination sequence from the small sub- unit of Rubisco gene in a first transcription unit. (See Cashmore, 1983). The chimeric DNA further includes a second transcription unit comprising a constitutive promoter such as the 35S CaMV promoter or the 34S FMV promoter linked to the coding sequence for the ethanol- dependent alcR transcription factor (alcR-TF) and a suitable transcription termination signal sequence.

The chimeric DNA sequence further comprises a germination specific promoter separated from a coding sequence for the site-specific recombinase FLP by a blocking sequence flanked at its 5' and 3' ends by mutated Lox recognition sequences (sites - Left and Right) specific for the site-specific recombinase. The mutated LOX site is one that favors excision of the blocking sequence and does not allow reinsertion of the DNA into the plant genome.

The chimeric DNA sequence of a further embodiment comprises a constitutive 34S FMV promoter separated from a DNA sequence encoding any protein of commercial importance, for example an herbicide resistance gene such as the EPSPS mutant CP4 from Agrobacterium tumefaciens. The 34S FMV promoter and the coding sequence are separated by a blocking sequence of DNA that is flanked on either end by mutated recognition sequences (LOX sites - Left and Right) as described above, and a suitable transcription termination signal. The chimeric DNA sequence further contains a FLO/LFY homolog promoter isolated from a target plant species, such as cotton, which is separated from a coding sequence for a site-specific R recombinase gene and a suitable transcription termination signal by a blocking sequence of DNA that is flanked on either end by recognition sequences (FRT) specific for the FLP site-specific recombinase.

In this embodiment, the inducible cascade of gene expression is activated in the vegetative stage of the seed production stage (generation one) of the transgenic crop by the application of the signal chemical, ethanol. Ethanol activates the binding of alcR-TF to the alcA promoter, which in turn induces expression of the CRE site-specific recombinase protein. CRE production results in the removal of blocking sequences that are flanked by LOX sites and the unblocking of both a germination specific FLP gene and a constitutive herbicide resistance gene. Thus, the seed production plants (generation one) are rendered herbicide tolerant following the application of ethanol. Since this step occurs in mature plants, the chimeric DNA sequence comprising the germination specific promoter and the FLP gene is inactive. The herbicide tolerance, provides a commercially useful trait for production farming and also can be used as a selection method for only those plants in which the CRE site-specific recombinase has successfully removed blocking sequences, thus ensuring that all of the seed produced in this generation contain the desired herbicide tolerance trait.

In the yield enhanced or production stage (generation two) that is established for commercial use, the FLP site-specific recombinase is activated during germination of the seed derived from the generation one herbicide resistant plants. The activation of the FLP site-specific recombinase is developmentally controlled to occur in all second generation plants.

The production of the FLP site-specific recombinase results in the removal of the blocking sequence that separates the FLO/LFY homolog promoter from a coding sequence for the R site-specific recombinase. The removal of this blocking sequence renders the site- specific R recombinase gene ready to be expressed during the formation of a floral meristem, the developmental stage at which the FLO/LFY homolog promoter is activated.

The complete linear genetic construct, containing all of the aforementioned genetic elements, is flanked by recognition sites for the site-specific R recombinase (RS sequences) so that activation of the site-specific R recombinase results in the complete removal of all introduced genetic elements in the floral meristem tissues. Thus, pollen and seed produced as a result of floral meristem development the generation two plants do not transfer any functional genetic elements to the third generation plants, either by selfing or out-crossing.

A further embodiment of the invention uses a gene such as an herbicide tolerance gene as a production cycle genetic marker to allow one to eliminate plants and plant tissues in which the gene induction system has not been activated. For example, the herbicide tolerance gene is linked to a value added gene for dissemination to the producer. The herbicide tolerance gene comprises a constitutive promoter linked, but separated from, a DNA sequence coding for a plant active mutant 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), such as the mutant disclosed by U.S. Patent No. 4,769,061. The blocking sequence separating the promoter and the mutant EPSPS coding sequence is flanked by recognition sequences specific for the ssR1 , as described above for other embodiments of the invention, to remove inserted transgenic sequences. In this embodiment, the activation of the ssR1 operably links the EPSPS gene with its promoter. When expressed, the EPSPS mutant enzyme renders the plant or plant tissue tolerant to glyphosate.

The presence of herbicide tolerance in plants grown to produce seed allows the identification of plants in which expression of the first chimeric DNA sequence in the cascade has failed. The use of the mutant EPSPS gene, in particular, enables the seed producer to select against seeds derived from germline cells where blocking sequences were not properly removed. Such circumstances could occur if the initial activation of the recombinase 1 gene resulted in a chimeric plant in which some cells fail to generate the recombinase 1 enzyme upon induction. If such cells are contained within the germline progenitor cells, seeds that contain an inactive cascade could be produced. This system is able to remove inactivated reproductive structures because inactivated mutant EPSPS synthase cannot destroy glyphosate. Once a plant is treated, with glyphosate, the herbicide travels to all structures within the plant, in particular the reproductive organs, which act as a metabolic sink. If not destroyed by EPSPS synthase, glyphosate will accumulate and kill the plant cells. Therefore, the seed producer can ensure that each plant generates seed that contains an activated and properly constituted intergenerational cascade for the effective removal of active transgenes in the second generation. As one of skill in the art of plant molecular biology will recognize, chimeric DNA sequences which provide tolerance to other systemic herbicides are also suitable for use in this embodiment.

In another preferred embodiment of the invention an inducible cascade generates both (1) a breeder seed assurance marker trait which also tracks the efficiency of the initial triggering event in the seed production generation, and (2) a seed quality or yield specific trait in the subsequent crop production generation or generations. Preferably, the target crop for the introduction of the inducible intergenerational genetic cascade is cotton; the breeder seed assurance marker trait is a naked seed (Ruan et al., 2003; Ruan et. al., 2005) and the seed quality trait is a seed with low oil content with improved lint production (Hake et. al., U.S. Patent Publication No. 2004133944).

In embodiments of this type, a site-specific recombinase (recombinase 1) is operably linked to an inducible promoter that can respond to an external stimulus which directs the expression of recombinase when activated. The recombinase protein removes blocking sequences positioned between recognition sites specific to recombinase 1 within specific target sequences. The target sequences for the recombinase 1 may comprise; (1) the inducible recombinase 1 gene itself, (2) a constitutive plant active promoter linked to a coding sequence that when expressed produces a visible phenolype in seed of the target plant, but separated from it by a blocking sequence of either DNA flanked by excision recognition sequences specific for recombinase 1, (3), a Late Embryogenesis Abundant (LEA) or germination-specific promoter linked to a coding sequence for recombinase 2, but separated from it by a blocking sequence that is flanked by excision recognition sequences specific for recombinase 1. Once recombinase 1 is activated by the application of an exogenous signal to the mature plant, the blocking sequences flanked by the recombinase 1 excision sequences are removed. The recombinase 1 gene is removed and a seed visible marker is produced. The recombinase 2 gene is unblocked, but inactive, since a LEA or germination- specific promoter controls its activity.

This sequence of events occurs in generation one. When seed (generation two) from these plants is produced, those seed with appropriate activation of the recombines can be selected by virtue of the visible seed marker. Late in seed maturation or upon germination of the activated seeds (generation two), recombinase 2 is expressed. Intervening sequences situated between recognition sites specific to recombinase 2 within specific target genes are then removed.

The target genes for recombinase 2, in this embodiment, which are flanked by the recombinase 2 specific excision sequences, include (1) the inducible recombinase 2 gene itself, (2) the gene encoding the visible seed marker and (3) a plant-specific promoter linked but separated from a coding sequence for a seed or yield value added trait. In generation two, the plants express the desired transgene that adds value to the transgenic crop but do not contain functional genetic elements that comprise the intergenerational genetic cascade.

Preferred embodiments of genetic constructs for use in cotton constitute the inducible intergenerational genetic cascade used to generate both (1) a breeder seed assurance marker trait, which also tracks the efficiency of the initial triggering event in the seed production generation and (2) a seed quality or yield specific trait in the subsequent crop production generation or generations. Genetic constructs, either in linear cis-acting form, or as individual chimeric DNA components that interact in trans are preferably used to form the following embodiments: (1) a mutant LOX(left) excision site (see above) adjacent to an alcA promoter (or a modified constitutive promoter containing the regulatory domains of the alcA promoter) operably linked to the coding sequence for the site-specific recombinase CRE and a suitable transcription termination signal sequence (TrbcS - the termination sequence from the small sub-unit of Rubisco gene) followed by a LOX (right) excision site; (2) a FRT excision site adjacent to a constitutive promoter (for example 35Sp) linked to the coding sequence for the ethanol-dependent alcR transcription factor (alcR-TF) and a suitable transcription termination signal sequence, which is flanked by LOX excision sites adjacent to an RNAi coding sequence specific for the inhibition of the Sus gene of cotton (which if suppressed generates cotton seed with minimal fuzz and lint fibers) followed by a FRT excision sequence; (3) a cotton LEA4 promoter (or a germination specific promoter) separated from a coding sequence for the site-specific recombinase FLP and a suitable transcription termination signal by a blocking sequence of DNA that is flanked on either end by mutated LOX sites; and (4) a seed-specific promoter, preferably the cotton α-globulin promoter (AGP), separated from a coding sequence for an oil suppression gene, preferably a coding sequence that when transcribed generates RNAi molecules specific for the inhibition of cotton carbonic anhydrase (CA) and a suitable transcription termination signal by a blocking sequence of DNA that is flanked on either end by FRT excision sites. A seed coat- specific promoter such as the pea PsGNS2 promoter (Buchner et al., 2002) may be utilized to minimize the expression of RNA-Sus in non seed coat tissues.

In this embodiment the inducible genetic cascade is activated in the vegetative stage of seed production plants (generation one) of the transgenic crop (cotton) by the application of the signal chemical, ethanol. The ethanol activates the alcA promoter, which in turn induces expression of CRE. CRE production results in the removal of blocking sequences that are flanked by LOX sites, which removes the inducible CRE gene itself, establishes a constitutive RNAi for the Sus gene and unblocks a late seed development (LEA) or germination specific FLP gene.

Thus, the seed production plants (generation one) that respond to the ethanol treatment produce naked (lintless) seeds that require less acid delinting and can be easily segregated from seeds derived from ethanol non-responsive plants. In generation two (established for commercial use) the FLP site-specific recombinase activated during the production of seed in the seed production generation (or during the germination of the seed) directs the removal the Sus RNAi, the FLP genes and the blocking sequence that separates the cotton α-globulin promoter (AGP) from the coding sequence that generates RNAi molecules specific for the inhibition of cotton carbonic anhydrase (CA). Thus, the generation two plants produce seed with reduced oil content and improved lint quality and yield. The plant also is free of all other genetic elements that constitute the intergenerational genetic cascade mechanism.

While the invention disclosed herein has been described by reference to details of preferred embodiments, the disclosure is intended to be illustrative and not limiting, as it is contemplated that modifications will readily occur to those skilled in the art which are within the spirit of the invention and the scope of the appended claims.

The complete citations of the references cited throughout the specification, are provided in the following reference list. The disclosures of each are expressly incorporated into the present specification in their entirety.

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1760-327 PCT APP

Claims

We claim:

1. A method for expression of a desired trait in a plant comprising a plant having at least three chimeric DNA sequences stably inserted into its genome, a first chimeric DNA sequence which comprises an inducible promoter operably linked to a DNA sequence encoding a first recombinase; a second chimeric DNA sequence which comprises a seed specific promoter, a blocking sequence having at its 5' and 3' ends recognition sites for the recombinase of the first chimeric DNA sequence; and a DNA sequence encoding a second recombinase; and a third chimeric DNA sequence which comprises a plant expressible promoter, a blocking sequence having at its 5' and 3' ends the recognition site for the second recombinase and a DNA sequence whose expression provides a desired trait wherein induced expression of the first chimeric DNA sequence in a first generation of the plant results in expression of the desired trait only in a progeny plant.

2. The method of claim 1 further comprising a fourth chimeric DNA sequence which comprises a plant expressible promoter and a DNA sequence encoding a seed marker trait.

3. The method of claim 1 further comprising: a fourth chimeric DNA sequence comprising a second seed specific promoter, a blocking sequence flanked at its 5' and 3' ends, and a DNA sequence encoding a third recombinase; and a fifth chimeric DNA sequence comprising a plant expressible promoter and a DNA sequence encoding a protein that provides a second desired trait in progeny of a parent plant where the first and second desired traits are not expressed in the parent plant.

4. The method of claim 1 wherein the DNA sequence of the third chimeric DNA sequence that provides a desired trait comprises a sequence encoding a protein.

5. The method of claim 1 wherein the DNA sequence of the third chimeric DNA sequence comprises a sequence encoding an interfering RNA (RNAi).

6. The method of claim 4, wherein the RNAi is specific for carbonic anhydrase.

7. The method of claim 5, wherein the RNAi specific for carbonic anhydrase provides the trait of low oil content seed.

8. The method of claim 1 wherein the DNA sequence of the third chimeric DNA encodes an EPSPS gene that provides a glyphosate-resistant trait.

9. The method of claim 1 wherein the third DNA sequence encoding the protein that provides the desired trait is a mutant EPSPS gene.

10. The method of claim 1 wherein the DNA sequence of the third chimeric DNA encodes a Bt toxin that provides an insect tolerance trait.

11. The method of claim 1 wherein the inducible promoter of the first chimeric DNA sequence is the alcohol A responsive promoter.

12. The method of claim 1 wherein the first recombinase is the Cre recombinase.

13. The method of claim 1 wherein the first recombinase recognition sites are the Lox recognition sites.

14. The method of claim 1 wherein the second recombinase is the FLP recombinase.

15. The method of claim 1 wherein the promoter of the third chimeric DNA sequence is selected from the group consisting of a figwort mosaic virus promoter and a 35S cauliflower mosaic virus promoter.

16. The method of claim 1 wherein the plant is selected from the group consisting of cotton, corn, soybeans, beets, wheat, alfalfa, rice, tomatoes and sunflower.

17. The method of claim 1 wherein the plant is cotton.

18. The method of claim 1 wherein the inducer of the plant expressible promoter of the first chimeric DNA sequence is applied to plant tissues.

19. The method of claim 1 wherein the inducer of the plant expressible promoter of the first chimeric DNA sequence is applied to seed.

20. A stably transformed parent plant comprising at least three chimeric DNA sequences wherein the first chimeric DNA sequence comprising an inducible promoter operably linked to a DNA sequence encoding a first recombinase; the second chimeric DNA sequence comprising a seed specific promoter, a blocking sequence having at its 5' and 3' ends the recognition site for the first recombinase encoded by the first chimeric gene and a DNA sequence encoding a second recombinase, different from the first recombinase such that the blocking sequence prevents the expression of the second recombinase; and the third chimeric DNA sequence comprising a plant expressible promoter, a blocking sequence having at its 5' and 3' ends the recognition site for the recombinase encoded by the second chimeric DNA sequence and a DNA sequence encoding a protein that provides a desired trait to the progeny of the parent plant that is not expressed in the parent plant; wherein the first and the second chimeric gene are expressed in a parent plant such that a desired trait encoded by the DNA sequence of the third chimeric DNA sequence is not expressed in parent plant.

21. A plant comprising at least three chimeric genes wherein the expression of the third chimeric gene in a progeny is induced by treating the parent of said of progeny with an inducer of an inducible promoter.

22. A stably transformed progeny plant comprising at least three chimeric DNA sequences wherein the first chimeric DNA sequence comprising an inducible promoter operably linked to a DNA sequence encoding a first recombinase; the second chimeric DNA sequence comprising a seed specific promoter, a blocking sequence having at its 5' and 3' ends the recognition site for the first recombinase encoded by the first chimeric gene and a DNA sequence encoding a second recombinase, different from the first recombinase such that the blocking sequence prevents the expression of the second recombinase; and the third chimeric DNA sequence comprising a plant expressible promoter, a blocking sequence having at its 5' and 3' ends the recognition site for the recombinase encoded by the second chimeric DNA sequence and a DNA sequence encoding a protein that provides a desired trait to the progeny of the parent plant that is not expressed in the parent plant; wherein the first and the second chimeric gene are expressed in a parent plant such that a desired trait encoded by the DNA sequence of the third chimeric DNA sequence is not expressed in parent plant.

23. The progeny plant of claim 20 wherein the expression of the third chimeric gene in the progeny plant is induced by treating the parent of said of progeny with an inducer of an inducible promoter of the first chimeric DNA sequence.