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This application claims the benefit of he filing date of U. The present invention generally relates to the field of plant molecular biology, and more specifically to the regulation of gene expression in plants in response to pathogen exposure. Plants are capable of activating a large array of defense mechanisms in response to pathogen attack, some of which are preexisting and others are inducible.

Pathogens must specialize to circumvent the defense mechanisms ofthe host, especially those biotrophic pathogens that derive their nutrition from an inthnate association with living plant cells. If the pathogen can cause disease, the interaction is said to be compatible, but if the plant is resistant, the interaction is said to be incompatible.

A crucial factor determining the success of these mechanisms is the speed of their activation. Consequently, there is considerable interest in understanding how plants recognize pathogen attack and control expression of defense mechanisms.

Some potential pathogens trigger a very rapid resistance response called gene-for-gene resistance. This occurs when the pathogen carries an avirulence avr gene that triggers specific recognition by a corresponding host resistance R gene. R gene specificity is generally quite narrow, in most cases only pathogens carrying a particular avr gene are recognized.

Recognition is thought to be mediated by ligand-receptor binding. R genes have been studied extensively in recent years. For a review of R genes, see Ellis et al.

One ofthe defense mechanisms triggered by gene-for-gene resistance is programmed cell death at the infection site.

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This is called the hypersensitive response, or HR. Pathogens that induce the HR, or cause cell death by other means, activate a systemic resistance response called systemic acquired resistance SAR.

During SAR, levels of salicylic acid SA rise throughout the plant, defense genes such as pathogenesis related PR genes are expressed, and the plant becomes more resistant to pathogen attack. SA is a crucial component of this response. Plants that cannot accumulate SA due to the presence of a transgene that encodes an SA-degrading enzyme nahGdevelop a HR in response to challenge by avirulent pathogens, but do not exhibit systemic expression of defense genes and do not develop resistance to subsequent pathogen attack Ryals et al.

The nature ofthe systemic signal that triggers SAR is a subject of debate Shulaev et al. SA clearly moves from the site ofthe HR to other parts ofthe plant, but if this is the signal, it must be effective at extremely low concentration Willitset et al. SAR is quite similar to some reactions that occur locally in response to attack by virulent those that cause disease or avirulent those that trigger gene-for-gene resistance pathogens.

In general, activation of defense gene expression occurs more slowly in response to virulent pathogens than in response to avirulent pathogens. Some pathogens trigger expression of defense genes tlirough a different signaling pathway that requires components of the jasmonic acid JA and ethylene signaling pathways Creelman et al. One approach to understanding the signal transduction networks that control defense mechanisms is to use genetic methods to identify signaling components and determine their roles within the network.

Considerable progress has been made using this approach in Arabidopsis-pathogen model systems. R gene signal transduction. NDRI and EDSl are required for gene-for-gene mediated resistance to avirulent strains ofthe bacterial pathogen Pseudomonas syringae and the oomycete pathogen Peronospora parasitica. Curiously, ndrl mutants are susceptible to one set of avirulent pathogens, whereas edsl mutants are susceptible to a non-overlapping set Aarts et el. Nevertheless, these results show that R genes differ in their requirements for downstream factors and that these differences are correlated with R gene structural type.

NDRI encodes a protein with two predicted transmembrane domains Century et al. RPMI, which requires NDRI to mediate resistance, is membrane-associated, despite the fact that its primary sequence does not include any likely membrane-integral stretches. EDSl encodes a protein with blocks of homology to triacyl glycerol lipases Falk et al, The significance of this homology is not known, but it is tempting to speculate that EDSl is involved in synthesis or degradation of a signal molecule.

It has been extremely difficult to isolate mutations in genes other than the R genes that Multiple Lines Slot Zipper Peas And Rice required for gene-for-gene resistance. A selection procedure was devised McNellis et al. Expression of avrRpt2 Free Spin Slot In Tagalog Meme Funny this background is lethal, as it triggers a systemic HR.

It is now possible to select for mutants with subtle defects in gene-for-gene signaling by requiring growth on a concentration of inducer slightly higher than the lethal dose. Putative plant receptor proteins encoded by RPP genes recognition of P. Recently, McDowell et al. The authors suggested that RPP7 initiates resistance through a novel signaling pathway that is independent of salicylic acid accumulation or jasmonic acid response components.

SA levels increase locally in response to pathogen attack, and systemically in response to the SAR-inducing signal. SA is necessary and sufficient for activation of PR gene expression and enhanced disease resistance. Physiological analyses and characterization of certain lesion-mimic mutants strongly suggest that there is a positive autoregulatory loop affecting SA concentrations Shirasu et al.

Several mutants with defects in SA signaling have been characterized. These include nprl, in which expression of Pi? Mutations in nprl abolish SAR, and cause enhanced susceptibility to infection by various pathogens Cao et al. NPRl encodes a novel protein that contains ankyrin repeats which are often involved in protein-protein interactions Cao et al. Consequently, it is unlikely that NPRl acts as a transcription factor to directly control PR gene expression, but its nuclear localization suggests that it may interact with such transcription factors.

PAD4 appears to act upstream from SA. SA is necessary, but not sufficient, for activation of camalexin synthesis Zhou et al, ; Zhao et al. The camalexin defect iupad4 plants is reversible by exogenous SA Zhou et al. Mutations vsxpad4 do not affect SA levels, camalexin synthesis, or PRl when plants are infected with an aviralent P. Taken together, these results suggest that PAD4 is required for signal amplification to activate the SA pathway in response to pathogens that do not elicit a strong defense response Zhou et al.

JA-dependent signaling JA signaling affects diverse processes including fruit ripening, pollen development, root growth, and response to wounding Creelman et al. Vas, jar 1 and coil mutants fail to respond to JA Feys et al. COI1 has been cloned, and found to encode protein containing leucine-rich repeats and a degenerate F-box motif Xie et al.

These features are characteristic of proteins that function in complexes that ubiquitinate protein targeted for degradation. In the past few years it has become apparent that JA plays an important role in regulation of pathogen defenses.

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For example, the induction ofthe defensin gene PDF1. SA signaling and Multiple Lines Slot Zipper Peas And Rice signaling pathways are interconnected in complicated ways. Studies in other systems have shown that SA signaling and JA signaling are mutually inhibitory Creelman et al. However, synthesis of camalexin in response to P. Induced systemic resistance ISR Some rhizosphere-associated bacteria promote disease resistance van Loon et al.

Colonized plants are more resistant to infection by the fungal pathogen Fusarium oxysporum f sp raphani Multiway Slot Machines You Can Beat God P. No changes in gene expression associated with ISR have been detected Pieterse et al.

It is difficult to imagine how this could occur, unless NPRl is interacting with different 'adapter' molecules to mediate the different signals. Identification of proteins that interact with NPRl, and characterization of plants with loss-of-function mutations affecting those proteins, would be very helpful for understanding how NPRl acts in each pathway.

Relevance to disease resistance. Characterization ofthe effects of various mutations on resistance to different pathogens has revealed that there is considerable variation in the extent to which pathogens are affected by defense mechanisms.

SAR is known to confer resistance to a wide array of pathogens, including bacteria, fungi, oomycetes, and viruses. JA signaling is important for limiting the growth of certain fungal pathogens.

Arabidopsis, the SA pathway mutants nprl and pad4 show enhanced susceptibility to P. Overexpression of rate-limiting defense response regulators may cause the signaling network to respond faster or more strongly to pathogen attack, thereby improving resistance. For example, overexpression of NPRl caused increased resistance to P. Moreover, NPP -overexpression had no obvious deleterious effects on plant growth, in contrast to mutations that lead to constitutive overexpression of defense responses, which generally Multiple Lines Slot Zipper Peas And Rice dwarfism.

Promoters for gene expression of plant pathogen defense genes. Promoters and other regulatory components from bacteria, viruses, fungi and plants have been used to control gene expression in plant cells. Numerous plant transformation experiments using DNA constructs comprising various promoter sequences fused to various foreign genes for example, bacterial marker genes have led to the identification of useful promoter sequences.

It has been demonstrated that sequences up tobases in most instances are sufficient to allow for the regulated expression of foreign genes. However, it has also been shown that sequences much longer than 1 kb may have useful features which permit high levels of gene expression in transgenic plants. The expression of genes encoding proteins that are useful for protecting plants from pathogen attack may have deleterious effects on plant growth if expressed constitutively.

Consequently, it is desirable to have promoter sequences that control expression of these gene s in such a way that expression is absent or very low in the absence of pathogens, and high in the presence of pathogens.

Thus, what is needed is the identification of plant genes useful to confer resistance to a pathogen s and plant promoters, the expression of which is altered in response to pathogen attack. Summary of the Invention The invention generally provides an isolated nucleic acid molecule polynucleotide comprising a plant nucleotide sequence obtained or isolatable from a gene, the expression of which is altered, either increased or decreased, in response to pathogen infection.

In one embodiment, the plant nucleotide sequence comprises an open reading frame, while in another embodiment, the plant nucleotide sequence comprises a promoter. A promoter sequence ofthe invention directs transcription of a linked nucleic acid segment, e. As used herein, a "pathogen" includes bacteria, fungi, oomycetes, viruses, nematodes and insects, e. Moreover, the expression of a plant nucleotide sequence of the invention comprising a promoter may be altered in response to one or more species of bacteria, nematode, fungi, oomycete, virus, or insect.

Likewise, the expression of a plant nucleotide sequence ofthe invention comprising an open reading frame may be useful to confer tolerance or resistance of a plant to one or more species of bacteria, nematode, fungi, oomycete, virus or insect.

The nucleotide sequence preferably is obtained or isolatable from plant DNA. The present invention also provides an isolated nucleic acid molecule comprising a plant nucleotide sequence that directs transcription of a linked nucleic acid segment in a host cell, e.

The nucleotide sequence preferably is obtained or isolatable from plant genomic DNA. The invention also provides uses for an isolated nucleic acid molecule, e. For example, these open reading frames may be useful to prepare plants that over- or under-express the encoded product or to prepare knockout plants. The promoters and open reading frames ofthe invention can be identified by any method. For example, they can be identified by employing an anay of nucleic acid samples, e.

Thus, genes that are upregulated or downregulated in response to pathogen infection can be systematically identified. The Arabidopsis oligonucleotide probe anay consists of probes from about 8, unique Arabidopsis genes, which covers approximately one third ofthe genome.

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    album series several group high music following number NUMBER-NUMBER county district life company ` played called released each career de government .. academics doug innocent islander volcanic exceptions climb giovanni oval rounded withdraw slot monroe sculptor smallest derives designers toy presumably. The relevant techniques are well known in the art and include but are not limited to hybridization, inbreeding, backcross breeding, multiline breeding, variety blend such as the light inducible promoter derived from the pea rbcS gene and the actin promoter from rice, e.g., the actin 2 promoter (WO 00/); seed specific. Knowledge of the proteins and molecular interactions associated with cell cycle processes, development, and stress response in monocot plants, such as rice, It is contemplated that using the techniques of dot or slot blot hybridization, which are modifications of Southern hybridization techniques, the same information.
  2. Polynucleotides encoding transcription factors of cereals and in particular rice are provided. The relevant techniques are well known in the art and include, but are not limited to, hybridization, inbreeding, backcross breeding, multiline breeding, variety blend, interspecific hybridization, aneuploid techniques, etc.:
    He is a Fellow of the National Academy of Agricultural Sciences and several other scientific societies, and a member of the .. Induced systemic resistance for the management of rice family Saprolegniaceae contains the crucifer black-root pathogen Aphanomyces raphani and the pea root-rot pathogen A. euteiches. The relevant techniques are well known in the art and include but are not limited to hybridization, inbreeding, backcross breeding, multiline breeding, variety encoding a 19 kD-zein protein, inducible promoters, such as the light inducible promoter derived from the pea rbcS gene and the actin promoter from rice, e.g., the. to see as they all line up carrying packages to the truck and helping get .. Another came after several people had made references to the day local effort to gather more than. 6, petition signa- tures opposing the move, state Sen. .. Spanish rice, peas and carrots, ginger- bread, cream, milk. Friday.

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Also provided are expression cassettes, e. The present invention further provides a method of augmenting a plant genome by contacting plant cells with a nucleic acid molecule ofthe invention, e. The present invention also provides a transgenic plant prepared by this method, a seed from such a plant and progeny plants from such a plant including hybrids and inbreds. Prefened transgenic plants are transgenic maize, soybean, barley, alfalfa, sunflower, canola, soybean, cotton, peanut, sorghum, tobacco, sugarbeet, rice, wheat, rye, turfgrass, millet, sugarcane, tomato, or potato.

The invention also provides a method of plant breeding, e. The method comprises crossing a fertile transgenic plant comprising a particular nucleic acid molecule ofthe invention with itself or with a second plant, e. The seed is then planted to obtain a crossed fertile transgenic plant.

The plant may be a monocot or a dicot. In a particular embodiment, the plant is a cereal plant. The crossed fertile transgenic plant may have the particular nucleic acid molecule inherited through a female parent or through a male parent. The second plant may be an inbred plant. The crossed fertile transgenic may be a hybrid. Also included within the present invention are seeds of any of these crossed fertile transgenic plants.

The various breeding steps are characterized by well-defined human intervention such as selecting the lines to be crossed, directing pollination ofthe parental lines, or selecting appropriate progeny plants. Depending on the desired properties different breeding measures are taken.

The relevant techniques are well known in the art and include but are not limited to hybridization, inbreeding, backcross breeding, multiline breeding, variety blend, interspecific hybridization, aneuploid techniques, etc. Hybridization techniques also include the sterilization of plants to yield male or female sterile plants by mechanical, chemical or biochemical means.

Cross pollination of a male sterile plant with pollen of a different line assures that the genome ofthe male sterile but female fertile plant will uniformly obtain properties of both parental lines. Thus, the transgenic plants according to the invention can be used for the breeding of improved plant lines that for example increase the effectiveness of conventional methods such as herbicide or pesticide treatment or allow to dispense with said methods due to their modified genetic properties.

Alternatively new crops with improved stress tolerance can be obtained that, due to their optimized genetic "equipment", yield harvested product of better quality than products that were not able to tolerate comparable adverse developmental conditions. The nucleic acid molecules ofthe invention, their encoded polypeptides and compositions thereof, are: As one embodiment ofthe invention includes isolated nucleic acid molecules that have increased expression in response to pathogen infection, the invention further provides compositions and methods for enhancing resistance to pathogen infection.

The compositions ofthe invention include plant nucleic acid sequences and the amino acid sequences for the polypeptides or partial-length polypeptides encoded thereby which are described herein, or other plant nucleic acid sequences and the amino acid sequences for the polypeptides or partial-length polypeptides encoded thereby which are operably linked to a promoters are useful to provide tolerance or resistance to a plant to a pathogen, preferably by preventing or inhibiting pathogen infection.

Methods ofthe invention involve stably transforming a plant with one or more of at least a portion of these nucleotide sequences which confer tolerance or resistance operably linked to a promoter capable of driving expression of that nucleotide sequence in a plant cell. By "portion" or "fragment", as it relates to a nucleic acid molecule, sequence or segment ofthe invention, when it is linked to other sequences for expression, is meant a sequence comprising at least 80 nucleotides, more preferably at least nucleotides, and still more preferably at least nucleotides.

If not employed for expressing, a "portion" or "fragment" means at least 9, preferably 12, more preferably 15, even more preferably at least 20, consecutive nucleotides, e. By "resistant" is meant a plant which exhibits substantially no phenotypic changes as a consequence of infection with the pathogen.

By "tolerant" is meant a plant which, although it may exhibit some phenotypic changes as a consequence of infection, does not have a substantially decreased reproductive capacity or substantially altered metabolism.

A method of combating a pathogen in an agricultural crop is also provided. The method comprises introducing to a plant, plant cell, or plant tissue an expression cassette comprising a nucleic acid molecule ofthe invention comprising an open reading frame so as to yield a transformed differentiated plant, transformed cell or transfonned tissue. Transformed cells or tissue can be regenerated to provide a transformed differentiated plant.

The transformed differentiated plant preferably expresses the nucleic acid molecule in an amount that confers resistance to the transformed plant to pathogen infection relative to a conesponding nontransformed plant.

The present invention also provides a transformed plant prepared by the method, progeny and seed thereof. Examples of plant viruses which may be combated by the present invention include single stranded RNA viruses with and without envelope , double stranded RNA viruses, and single and double stranded DNA viruses such as but not limited to tobacco mosaic virus, cucumber mosaic virus, turnip mosaic virus, turnip vein clearing virus, oilseed rape mosaic viras, tobacco rattle viras, pea enation mosaic virus, barley stripe mosaic viras, potato viruses X and Y, carnation latent virus, beet yellows virus, maize chlorotic viras, tobacco necrosis viras, turnip yellow mosaic viras, tomato bushy stunt virus, southern bean mosaic virus, barley yellow dwarf virus, tomato spotted wilt virus, lettuce necrotic yellows virus, wound tumor virus, maize streak virus, and cauliflower mosaic virus.

Other pathogens within the scope ofthe invention include, but are not limited to, fungi such as Cochliobolus carbonum, Phytophthora infestans, Phytophthora sojae, Collesosichum, Melampsora lini, cladosporiumfulvum, Heminthosporium maydia, Peronospora parasitica, Puccinia sorghi, and Puccinia polysora; bacteria such as Phynchosporium secalis, Pseudomonas glycinea, Xanthomonas oryzae and, Fusarium oxyaporium; and nematodes such as Globodera rostochiensis.

For example, the invention provides a nucleic acid molecule comprising a plant nucleotide sequence comprising at least a portion of a key effector gene s responsible for host resistance to particular pathogens. The overexpression may be constitutive, or it may be preferable to express the effector gene s in a tissue-specific manner or from an inducible promoter including a promoter which is responsive to external stimuli, such as chemical application, or to pathogen infection, e.

The nucleic acid molecules ofthe invention are thus useful for targeted gene disraption, as well as markers and probes. For example, the invention includes a pathogen, e. The normal fertile transformed transgenic plant may be selfed to yield a substantially homogenous line with respect to viral resistance or tolerance.

Individuals ofthe line, or the progeny thereof, may be crossed with plants which optionally exhibit the trait. In a particular embodiment ofthe method, the selfing and selection steps are repeated at least five times in order to obtain the homogenous isogenic line. Thus, the invention also provides transgenic plants and the products ofthe transgenic plants. The invention further includes a nucleotide sequence which is complementary to one hereinafter "test" sequence which hybridizes under low, moderate or stringent conditions with the nucleic acid molecules ofthe invention as well as RNA which is encoded by the nucleic acid molecule.

When the hybridization is performed under stringent conditions, either the test or nucleic acid molecule of invention is preferably supported, e. Thus, either a denatured test or nucleic acid molecule ofthe invention is preferably first bound to a support and hybridization is effected for a specified period of time at a temperature of, e.

Depending upon the degree of stringency required such reduced concentration buffers are typically single strength SC containing 0. The invention further provides a method to identify an open reading frame in the genome of a plant cell, the expression of which is altered by pathogen infection of that cell.

The method comprises contacting a solid substrate comprising a plurality of samples comprising isolated plant nucleic acid of a probe comprising plant nucleic acid, e. Each individual sample comprises one or more nucleic acid sequences e. The method may be employed with nucleic acid samples and probes from any organism, e.

Preferably, the nucleic acid sample and probes are from a plant, such as a dicot or monocot. More preferably the nucleic acid samples and probes are from a cereal plant. Even more preferably the nucleic acids and probes are from a crop plant. A second plurality of samples on a solid substrate, i.

Then complex formation between the samples and probes comprising nucleic acid from infected or control cells compared. For example, potato virus X, tobacco mosaic virus, tobravirus, cucumber mosaic viras and gemnivirus are known to infect Arabidopsis. Thus, Arabidopsis genes, the expression of which is altered in response to infection by any of these viruses, can be identified.

The invention further provides a method for identifying a plant cell infected with a pathogen. The method comprises contacting nucleic acid obtained from a plant cell suspected of being infected with a pathogen with oligonucleotides conesponding to a portion of a plurality of sequences selected from SEQ ID NOs: Then the presence ofthe amplified product is detected or detennined.

The presence of two or more amplified products, e. The method comprises contacting a protein sample obtained from a plant cell suspected of being infected with a pathogen with an agent that specifically binds a polypeptide encoded by an open reading frame comprising SEQ ID NOs: Then the presence or amount of complex formation is detected or determined.

The invention provides an additional method for identifying a plant cell infected with a pathogen. The amount ofthe probe hybridized to nucleic acid obtained from a cell suspected of being infected with a viras is compared to hybridization ofthe probe to nucleic acid isolated from an uninfected cell.

A change in the amount of at least two probes that hybridize to nucleic acid isolated from a cell suspected of being infected by a virus relative to hybridization of at least two probes to nucleic acid isolated from an uninfected cell is indicative of viral infection. A method to shuffle the nucleic acids ofthe invention is provided. This method allows for the production of polypeptides having altered activity relative to the native form ofthe polypeptide.

Accordingly, the invention provides cells and transgenic plants containing nucleic acid segments produced through shuffling that encode polypeptides having altered activity relative to the conesponding native polypeptide. A computer readable medium containing the nucleic acid sequences ofthe invention as well as methods of use for the computer readable medium are provided. This medium also allows for computer-based manipulation of a nucleic acid sequence conesponding to a nucleic acid sequence listed in SEQ ID NOs: Therefore, another embodiment ofthe present invention provides a method of using known inducers or inhibitors of genes identified as being important in plant-pathogen interactions to induce genes that are important in resistance, or to inhibit genes that are downregulated in resistance.

Thus, some ofthe isolated nucleic acid molecules ofthe invention are useful in a method of combating a pathogen in an agricultural crop. The method comprises introducing to a plant an expression cassette comprising a nucleic acid molecule ofthe invention so as to yield a transformed differentiated plant. The transformed differentiated plant expresses the nucleic acid molecule in an amount that confers resistance to the transformed plant to infection relative to a corresponding nontransformed plant.

The term "gene" is used broadly to refer to any segment of nucleic acid associated with a biological function. For example, gene refers to a nucleic acid fragment that expresses mRNA or functional RNA, or encodes a specific protein, and which includes regulatory sequences.

Genes also include nonexpressed DNA segments that, for example, form recognition sequences for other proteins. Genes can be obtained from a variety of sources, including cloning from a source of interest or synthesizing from known or predicted sequence information, and may include sequences designed to have desired parameters. The term "native" or "wild type" gene refers to a gene that is present in the genome of an untransformed cell, i.

A "marker gene" encodes a selectable or screenable trait. The term "chimeric gene" refers to any gene that contains 1 DNA sequences, including regulatory and coding sequences, that are not found together in nature, or 2 sequences encoding parts of proteins not naturally adjoined, or 3 parts of promoters that are not naturally adjoined. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or comprise regulatory sequences and coding sequences derived from the same source, but ananged in a manner different from that found in nature.

A "transgene" refers to a gene that has been introduced into the genome by transformation and is stably maintained. Transgenes may include, for example, genes that are either heterologous or homologous to the genes of a particular plant to be transformed. Additionally, transgenes may comprise native genes inserted into a non-native organism, or chimeric genes. The term "endogenous gene" refers to a native gene in its natural location in the genome of an organism. A "foreign" gene refers to a gene not normally found in the host organism but that is introduced by gene transfer.

An "oligonucleotide" conesponding to a nucleotide sequence ofthe invention, e. Generally specific primers are upwards of 14 nucleotides in length. For optimum specificity and cost effectiveness, primers of 16 to 24 nucleotides in length may be prefened. Those skilled in the art are well versed in the design of primers for use processes such as PCR. If required, probing can be done with entire restriction fragments ofthe gene disclosed herein which may be 's or even lOOO's of nucleotides in length.

The terms "protein," "peptide" and "polypeptide" are used interchangeably herein. The nucleotide sequences ofthe invention can be introduced into any plant. The genes to be introduced can be conveniently used in expression cassettes for introduction and expression in any plant of interest. Such expression cassettes will comprise the transcriptional initiation region ofthe invention linked to a nucleotide sequence of interest. Such an expression cassette is provided with a plurality of restriction sites for insertion ofthe gene of interest to be under the transcriptional regulation ofthe regulatory regions.

The expression cassette may additionally contain selectable marker genes. The cassette will include in the 5'-3' direction of transcription, a transcriptional and translational initiation region, a DNA sequence of interest, and a transcriptional and translational termination region functional in plants.

The termination region may be native with the transcriptional initiation region, may be native with the DNA sequence of interest, or may be derived from another source. Convenient termination regions are available from the Ti- plasmid of A.

See also, Guerineau et al. It may constitute an "uninterrupted coding sequence", i. An "intron" is a sequence of RNA which is contained in the primary transcript but which is removed through cleavage and re-ligation of the RNA within the cell to create the mature mRNA that can be translated into a protein.

The terms "initiation codon" and "termination codon" refer to a unit of three adjacent nucleotides 'codon' in a coding sequence that specifies initiation and chain termination, respectively, of protein synthesis mRNA translation. When the RNA transcript is a perfect complementary copy ofthe DNA sequence, it is refened to as the primary transcript or it may be a RNA sequence derived from posttranscriptional processing ofthe primary transcript and is refened to as the mature RNA.

Regulatory sequences include enhancers, promoters, translation leader sequences, introns, and polyadenylation signal sequences. They include natural and synthetic sequences as well as sequences which may be a combination of synthetic and natural sequences.

As is noted above, the term "suitable regulatory sequences" is not limited to promoters. It is present in the fully processed mRNA upstream ofthe initiation codon and may affect processing ofthe primary transcript to mRNA, mRNA stability or translation efficiency Turner et al.

The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3' end ofthe mRNA precursor. The use of different 3' non-coding sequences is exemplified by Ingelbrecht et al. The term "translation leader sequence" refers to that DNA sequence portion of a gene between the promoter and coding sequence that is transcribed into RNA and is present in the fully processed mRNA upstream 5' ofthe translation start codon.

The translation leader sequence may affect processing ofthe primary transcript to mRNA, mRNA stability or translation efficiency. The term "mature" protein refers to a post-translationally processed polypeptide without its signal peptide. The term "signal sequence" refers to a nucleotide sequence that encodes the signal peptide. The term "intracellular localization sequence" refers to a nucleotide sequence that encodes an intracellular targeting signal.

An "intracellular targeting signal" is an amino acid sequence that is translated in conjunction with a protein and directs it to a particular subcellular compartment. Bacterial pathogens include but are not limited to Pseudomonas avenae subsp. Fungal pathogens include but are not limited to Collelotrichum graminicola,. Gams, Cephalosporium acremonium Auct. Cochliobolus lunatus , Curvularia pallescens teleomorph - Cochlioboluspallescens , Curvularia senegalensis, C.

Selosphaeriapedicellata , Cladosporium cladosporioides - Hormodendrum cladosporioides, C herbarum teleomorph - Mycosphaerella tassiana , Cephalosporium maydis, A.

Nectria haematococca , F. Mycosphaerella zeae-maydis , and Gloeocercospora sorghi. Parasitic nematodes include but are not limited to Awl Dolichodorus spp.

This type of promoter sequence consists of proximal and more distal upstream elements, the latter elements often refened to as enhancers. Accordingly, an "enhancer" is a DNA sequence which can stimulate promoter activity and may be an innate element ofthe promoter or a heterologous element inserted to enhance the level or tissue specificity of a promoter.

Both enhancers and other upstream promoter elements bind sequence-specific DNA-binding proteins that mediate their effects. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even be comprised of synthetic DNA segments.

A promoter may also contain DNA sequences that are involved in the binding of protein factors which control the effectiveness of transcription initiation in response to physiological or developmental conditions. With respect to this site all other sequences ofthe gene and its controlling regions are numbered.

Promoter elements, particularly a TATA element, that are inactive or that have greatly reduced promoter activity in the absence of upstream activation are refened to as "minimal or core promoters. A "minimal or core promoter" thus consists only of all basal elements needed for transcription initiation, e. It includes natural and synthetic sequences as well as sequences which may be a combination of synthetic and natural sequences.

Different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. New promoters of various types useful in plant cells are constantly being discovered, numerous examples may be found in the compilation by Okamuro et al.

Typical regulated promoters useful in plants include but are not limited to safener-inducible promoters, promoters derived from the tetracyclme- inducible system, promoters derived from salicylate-inducible systems, promoters derived from alcohol-inducible systems, promoters derived from glucocorticoid-inducible system, promoters derived from pathogen-inducible systems, and promoters derived from ecdysome- inducible systems.

These also include promoters that are temporally regulated, such as in early or late embryogenesis, during fruit ripening in developing seeds or fruit, in fully differentiated leaf, or at the onset of senescence. For example, a regulatory DNA sequence is said to be "operably linked to" or "associated with" a DNA sequence that codes for an RNA or a polypeptide if the two sequences are situated such that the regulatory DNA sequence affects expression ofthe coding DNA sequence i.

Coding sequences can be operably-linked to regulatory sequences in sense or antisense orientation. For example, in the case of antisense constructs, expression may refer to the transcription ofthe antisense DNA only. Expression may also refer to the production of protein.

It is acknowledged that hardly a trae specificity exists: This phenomenon is known as leaky expression. However, with specific expression in this invention is meant preferable expression in one or a few plant tissues. The "expression pattern" of a promoter with or without enhancer is the pattern of expression levels which shows where in the plant and in what developmental stage transcription is initiated by said promoter.

Expression patterns of a set of promoters are said to be complementary when the expression pattern of one promoter shows little overlap with the expression pattern ofthe other promoter. The level of expression of a promoter can be determined by measuring the 'steady state' concentration of a standard transcribed reporter mRNA.

This measurement is indirect since the concentration ofthe reporter mRNA is dependent not only on its synthesis rate, but also on the rate with which the mRNA is degraded.

Therefore, the steady state level is the product of synthesis rates and degradation rates. The rate of degradation can however be considered to proceed at a fixed rate when the transcribed sequences are identical, and thus this value can serve as a measure of synthesis rates. This list of techniques in no way represents all available techniques, but rather describes commonly used procedures used to analyze transcription activity and expression levels of mRNA.

The analysis of transcription start points in practically all promoters has revealed that there is usually no single base at which transcription starts, but rather a more or less clustered set of initiation sites, each of which accounts for some start points ofthe mRNA. Since this distribution varies from promoter to promoter the sequences ofthe reporter mRNA in each of the populations would differ from each other.

Since each mRNA species is more or less prone to degradation, no single degradation rate can be expected for different reporter mRNAs. It has been shown for various eukaryotic promoter sequences that the sequence surrounding the initiation site 'initiator' plays an important role in determining the level of RNA expression directed by that specific promoter.

This includes also part ofthe transcribed sequences. The direct fusion of promoter to reporter sequences would therefore lead to suboptimal levels of transcription. A commonly used procedure to analyze expression patterns and levels is through determination ofthe 'steady state' level of protein accumulation in a cell. Commonly used candidates for the reporter gene, known to those skilled in the art are 3-glucuronidase GUS , chloramphenicol acetyl transferase CAT and proteins with fluorescent properties, such as green fluorescent protein GFP from Aequora victoria.

For quantification and determination of localization a number of tools are suited. Detection systems can readily be created or are available which are based on, e. Protein levels can be determined in plant tissue extracts or in intact tissue using in situ analysis of protein expression. Generally, individual transformed lines with one chimeric promoter reporter construct will vary in their levels of expression ofthe reporter gene.

Also frequently observed is the phenomenon that such transformants do not express any detectable product RNA or protein. The variability in expression is commonly ascribed to 'position effects', although the molecular mechanisms underlying this inactivity are usually not clear. The term "average expression" is used here as the average level of expression found in all lines that do express detectable amounts of reporter gene, so leaving out ofthe analysis plants that do not express any detectable reporter mRNA or protein.

Likewise, leaf, and stem expression levels, are determined using whole extracts from leaves and stems. It is acknowledged however, that within each ofthe plant parts just described, cells with variable functions may exist, in which promoter activity may vary.

Gene silencing may be transcriptional, when the suppression is due to decreased transcription ofthe affected genes, or post-transcriptional, when the suppression is due to increased turnover degradation of RNA species homologous to the affected genes English et al. Gene silencing includes virus-induced gene silencing Ruiz et al. Silencing suppressor genes may be of plant, non-plant, or viral origin.

Thus, a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified through, for example, the use of DNA shuffling.

The terms also include non-naturally occuning multiple copies of a naturally occurring DNA sequence. Thus, the terms refer to a DNA segment that is foreign or heterologous to the cell, or homologous to the cell but in a position within the host cell nucleic acid in which the element is not ordinarily found. Exogenous DNA segments are expressed to yield exogenous polypeptides. A "homologous" DNA sequence is a DNA sequence that is naturally associated with a host cell into which it is introduced.

For example, altered nucleotide sequences which simply reflect the degeneracy ofthe genetic code but nonetheless encode amino acid sequences that are identical to a particular amino acid sequence are substantially similar to the particular sequences.

Modifications that result in equivalent nucleotide or amino acid sequences are well within the routine skill in the art. The target gene is not essential for replicon replication. Additionally, target genes may comprise native non- viral genes inserted into a non-native organism, or chimeric genes, and will be under the control of suitable regulatory sequences.

Thus, the regulatory sequences in the target gene may come from any source, including the virus. Target genes may include coding sequences that are either heterologous or homologous to the genes of a particular plant to be transformed. However, target genes do not include native viral genes. Typical target genes include, but are not limited to genes encoding a structural protein, a seed storage protein, a protein that conveys herbicide resistance, and a protein that conveys insect resistance.

Proteins encoded by target genes are known as "foreign proteins". The expression of a target gene in a plant will typically produce an altered plant trait. The term "altered plant trait" means any phenotypic or genotypic change in a transgenic plant relative to the wild-type or non-transgenic plant host.

Examples include the 3' non-regulatory regions of genes encoding nopaline synthase and the small subunit of ribulose bisphosphate carboxylase.

In addition to the ORF ofthe replication protein, the replication gene may also contain other overlapping or non-overlapping ORF s , as are found in viral sequences in nature.

Such chimeric genes also include insertion ofthe known sites of replication protein binding between the promoter and the transcription start site that attenuate transcription of viral replication protein gene.

Where genes are not "chromosomally integrated" they may be "transiently expressed. It excludes young, growing tissue consisting of germline, meristematic, and not-fully-differentiated cells.

The term "transformation" refers to the transfer of a nucleic acid fragment into the genome of a host cell, resulting in genetically stable inheritance. Host cells containing the transformed nucleic acid fragments are refened to as "transgenic" cells, and organisms comprising transgenic cells are refened to as "transgenic organisms". Examples of methods of transformation of plants and plant cells include Agrobacterium-mediated transformation De Blaere et al.

Whole plants may be regenerated from transgenic cells by methods well known to the skilled artisan see, for example, Fromm et al. The nucleic acid molecule can be stably integrated into the genome generally known in the art and are disclosed in Sambrook et al, See also Innis et al. Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially mismatched primers, and the like.

For example, "transformed," "transformant," and "transgenic" plants or calli have been tlirough the transformation process and contain a foreign gene integrated into their chromosome. The term "untransformed" refers to normal plants that have not been through the transformation process. They may be derived by self-fertilization of primary or secondary transformants or crosses of primary or secondary transformants with other transformed or untransformed plants.

The term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, composed of monomers nucleotides containing a sugar, phosphate and a base which is either a purine or pyrimidine. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occuning nucleotides.

Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof e. A "nucleic acid fragment" is a fraction of a given nucleic acid molecule. In higher plants, deoxyribonucleic acid DNA is the genetic material while ribonucleic acid RNA is involved in the transfer of information contained within DNA into proteins.

The invention encompasses isolated or substantially purified nucleic acid or protein compositions. In the context ofthe present invention, an "isolated" or "purified" DNA molecule or an "isolated" or "purified" polypeptide is a DNA molecule or polypeptide that, by the hand of man, exists apart from its native environment and is therefore not a product of nature. An isolated DNA molecule or polypeptide may exist in a purified form or may exist in a non-native environment such as, for example, a transgenic host cell.

For example, an "isolated" or "purified" nucleic acid molecule or protein, or biologically active portion thereof, is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.

Preferably, an "isolated" nucleic acid is free of sequences preferably protein encoding sequences that naturally flank the nucleic acid i. For example, in various embodiments, the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0. The nucleotide sequences ofthe invention include both the naturally occurring sequences as well as mutant variant forms.

Such variants will continue to possess the desired activity, i. Thus, by "variants" is intended substantially similar sequences.

For nucleotide sequences comprising an open reading frame, variants include those sequences that, because of the degeneracy ofthe genetic code, encode the identical amino acid sequence ofthe native protein. Naturally occuning allelic variants such as these can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction PCR and hybridization techniques.

Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis and for open reading frames, encode the native protein, as well as those that encode a polypeptide having amino acid substitutions relative to the native protein.

Because ofthe degeneracy ofthe genetic code, a large number of functionally identical nucleic acids encode any given polypeptide.

Thus, at every position where an arginine is specified by a codon, the codon can be altered to any ofthe conesponding codons described without altering the encoded protein. Such nucleic acid variations are "silent variations" which are one species of "conservatively modified variations. One of skill will recognize that each codon in a nucleic acid except ATG, which is ordinarily the only codon for methionine can be modified to yield a functionally identical molecule by standard techniques.

The target gene can be a gene derived from a cell, an endogenous gene, a transgene, or exogenous genes such as genes of a pathogen, for example a virus, which is present in the cell after infection thereof. The cell containing the target gene can be derived from or contained in any organism, for example a plant, animal, protozoan, virus, bacterium, or fungus. The term "gene" also refers broadly to any segment of DNA associated with a biological function.

As such, the term "gene" encompasses sequences including but not limited to a coding sequence, a promoter region, a transcriptional regulatory sequence, a non- expressed DNA segment that is a specific recognition sequence for regulatory proteins, a non-expressed DNA segment that contributes to gene expression, a DNA segment designed to have desired parameters, or combinations thereof.

A gene can be obtained by a variety of methods, including cloning from a biological sample, synthesis based on known or predicted sequence information, and recombinant derivation from one or more existing sequences. As is understood in the art, a gene comprises a coding strand and a non-coding strand. As used herein, the terms "coding strand" and "sense strand" are used interchangeably, and refer to a nucleic acid sequence that has the same sequence of nucleotides as an mRNA from which the gene product is translated.

As used herein, the terms "complementarity" and "complementary" refer to a nucleic acid that can form one or more hydrogen bonds with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types of interactions. In reference to the nucleic molecules of the presently disclosed subject matter, the binding free energy for a nucleic acid molecule with its complementary sequence is sufficient to allow the relevant function of the nucleic acid to proceed, in one embodiment, RNAi activity.

For example, the degree of complementarity between the sense and antisense strands of the siRNA construct can be the same or different from the degree of complementarity between the antisense strand of the siRNA and the target nucleic acid sequence. Determination of binding free energies for nucleic acid molecules is well known in the art.

As used herein, the phrase "percent complementarity" refers to the percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds e. The term "gene expression" generally refers to the cellular processes by which a biologically active polypeptide is produced from a DNA sequence and exhibits a biological activity in a cell.

As such, gene expression involves the processes of transcription and translation, but also involves post- transcriptional and post-translational processes that can influence a biological activity of a gene or gene product. These processes include, but are not limited to RNA syntheses, processing, and transport, as well as polypeptide synthesis, transport, and post-translational modification of polypeptides.

Additionally, processes that affect protein-protein interactions within the cell can also affect gene expression as defined herein. The terms "heterologous", "recombinant", and "exogenous", when used herein to refer to a nucleic acid sequence e. Thus, a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified through, for example, the use of DNA shuffling or other recombinant techniques for example, cloning the gene into a vector.

The terms also include non- naturally occurring multiple copies of a naturally occurring DNA sequence. Thus, the terms refer to a DNA segment that is foreign or heterologous to the cell, or homologous to the cell but in a position or form within the host cell in which the element is not ordinarily found. Similarly, when used in the context of a polypeptide or amino acid sequence, an exogenous polypeptide or amino acid sequence is a polypeptide or amino acid sequence that originates from a source foreign to the particular host cell or, if from the same source, is modified from its original form.

Thus, exogenous DNA segments can be expressed to yield exogenous polypeptides. A "homologous" nucleic acid or amino acid sequence is a nucleic acid or amino acid sequence naturally associated with a host cell into which it is introduced.

As used herein, the terms "host cells" and "recombinant host cells" are used interchangeably and refer cells for example, plant cells into which the compositions of the presently disclosed subject matter for example, an expression vector can be introduced.

Furthermore, the terms refer not only to the particular plant cell into which an expression construct is initially introduced, but also to the progeny or potential progeny of such a cell. Because certain modifications can occur in succeeding generations due to , either mutation or environmental influences, such progeny might not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein. The phrase "hybridizing specifically to" refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture e.

The phrase "bind s substantially" refers to complementary hybridization between a probe nucleic acid and a target nucleic acid and embraces minor mismatches that can be accommodated by reducing the stringency of the hybridization media to achieve the desired detection of the target nucleic acid sequence.

As used herein, the term "inhibitor" refers to a chemical substance that inactivates or decreases the biological activity of a polypeptide such as a biosynthetic and catalytic activity, receptor, signal transduction polypeptide, structural gene product, or transport polypeptide.

The term "herbicide" or "herbicidal compound" is used herein to define an inhibitor applied to a plant at any stage of development, whereby the herbicide inhibits the growth of the plant or kills the plant. An "isolated" nucleic acid molecule or protein, or biologically active portion thereof, is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.

Thus, the term "isolated nucleic acid" refers to a polynucleotide of genomic, cDNA, or synthetic origin or some combination thereof, which 1 is not associated with the cell in which the "isolated nucleic acid" is found in nature, or 2 is operatively linked to a polynucleotide to which it is not linked in nature. Similarly, the term "isolated polypeptide" refers to a polypeptide, in certain embodiments prepared from recombinant DNA or RNA, or of synthetic origin, or some combination thereof, which 1 is not associated with proteins that it is normally found with in nature, 2 is isolated from the cell in which it normally occurs, 3 is isolated free of other proteins from the same cellular source, 4 is expressed by a cell from a different species, or 5 does not occur in nature.

In certain embodiments, an "isolated" nucleic acid is free of sequences e. For example, in various embodiments, the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.

Thus, the term "isolated", when used in the context of an isolated DNA molecule or an isolated polypeptide, refers to a DNA molecule or polypeptide that, by the hand of man, exists apart from its native environment and is therefore not a product of nature.

An isolated DNA molecule or polypeptide can exist in a purified form or can exist in a non- native environment such as, for example, in a transgenic host cell. The term "isolated", when used in the context of an "isolated cell", refers to a cell that has been removed from its natural environment, for example, as a part of an organ, tissue, or organism.

As used herein, the term "minimal promoter" refers to the smallest piece of a promoter, such as a TATA element, that can support any transcription. A minimal promoter typically has greatly reduced promoter activity in the absence of upstream or downstream activation. In the presence of a suitable transcription factor, a minimal promoter can function to permit transcription. As used herein, the term "modified enzyme activity" refers to enzyme activity that is different from that which naturally occurs in a plant i.

In one embodiment, a modified enzyme activity is displayed by a non-naturally occurring enzyme that is tolerant to inhibitors that inhibit the cognate naturally occurring enzyme activity. As used herein, the term "modulate" refers to an increase, decrease, or other alteration of any, or all, chemical and biological activities or properties of a biochemical entity, e.

As such, the term "modulate" can refer to a change in the expression level of a gene, or a level of RNA molecule or equivalent RNA molecules encoding one or more proteins or protein subunits, or activity of one or more proteins or protein subunits is up regulated or down regulated, such that expression, level, or activity is greater than or less than that observed in the absence of the modulator.

For example, the term "modulate" can mean "inhibit" or "suppress", but the use of the word "modulate" is not limited to this definition. As used herein, the terms "inhibit", "suppress", "down regulate", and grammatical variants thereof are used interchangeably and refer to an activity whereby gene expression or a level of an RNA encoding one or more gene products is reduced below that observed in the absence of a nucleic acid molecule of the presently disclosed subject matter.

In another embodiment, inhibition with a a nucleic acid molecule for example, a dsRNA, an antisense RNA, or an siRNA results in an expression level of a target gene that is below that level observed in the presence of an inactive or attenuated molecule that is unable to mediate an RNAi response. In another embodiment, inhibition of gene expression with a nucleic acid molecule for example, a dsRNA, an antisense RNA, or an siRNA of the presently disclosed subject matter is greater in the presence of the a nucleic acid molecule than in its absence.

The term "modulation" as used herein refers to both upregulation i. Thus, the term "modulation", when used in reference to a functional property or biological activity or process e. The term "modulator" refers to a polypeptide, nucleic acid, macromolecule, complex, molecule, small molecule, compound, species, or the like naturally occurring or non-naturally occurring , or an extract made from biological materials such as bacteria, plants, fungi, or animal cells or tissues, that can be capable of causing modulation.

Modulators can be evaluated for potential activity as inhibitors or activators directly or indirectly of a functional property, biological activity or process, or combination of them, e. In such assays, many modulators can be screened at one time.

The activity of a modulator can be known, unknown, or partially known. Modulators can be either selective or non-selective. As used herein, the term "selective" when used in the context of a modulator e. It must be understood that it is not required that the degree to which the interactions differ be completely opposite.

Put another way, the term selective modulator encompasses not only those molecules that only bind to mRNA transcripts from a gene of interest and not those of related family members. The term is also intended to include modulators that are characterized by interactions with transcripts from genes of interest and from related family members that differ to a lesser degree.

For example, selective modulators include modulators for which conditions can be found such as the degree of sequence identity that would allow a biologically relevant difference in the binding of the modulator to transcripts form the gene of interest versus transcripts from related genes. When a selective modulator is identified, the modulator will bind to one molecule for example an mRNA transcript of a gene of interest in a manner that is different for example, stronger than it binds to another molecule for example, an mRNA transcript of a gene related to the gene of interest.

As used herein, the modulator is said to display "selective binding" or "preferential binding" to the molecule to which it binds more strongly. As used herein, the term "mutation" carries its traditional connotation and refers to a change, inherited, naturally occurring or introduced, in a nucleic acid or polypeptide sequence, and is used in its sense as generally known to those of skill in the art. As used herein, the term "native" refers to a gene that is naturally present in the genome of an untransformed plant cell.

Similarly, when used in the context of a polypeptide, a "native polypeptide" is a polypeptide that is encoded by a native gene of an untransformed plant cell's genome. As used herein, the term "naturally occurring" refers to an object that is found in nature as distinct from being artificially produced by man. For example, a polypeptide or nucleotide sequence that is present in an organism including a virus in its natural state, which has not been intentionally modified or isolated by man in the laboratory, is naturally occurring.

As such, a polypeptide or nucleotide sequence is considered "non-naturally occurring" if it is encoded by or present within a recombinant molecule, even if the amino acid or nucleic acid sequence is identical to an amino acid or nucleic acid sequence found in nature.

As used herein, the terms "nucleic acid" and "nucleic acid molecule" refer to any of deoxyribonucleic acid DNA , ribonucleic acid RNA , oligonucleotides, fragments generated by the polymerase chain reaction PCR , and fragments generated by any of ligation, scission, endonuclease action, and exonuclease action.

Nucleic acids can be composed of monomers that are naturally occurring nucleotides such as deoxyribonucleotides and ribonucleotides , or analogs of naturally occurring nucleotides e.

Sugar modifications include, for example, replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, and azido groups, or sugars can be functionalized as ethers or esters. Moreover, the entire sugar moiety can be replaced with sterically and electronically similar structures, such as aza- sugars and carbocyclic sugar analogs.

Examples of modifications in a base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substitutes. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages. Analogs of phosphodiester linkages include phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, phosphoramidate, and the like.

The term "nucleic acid" also includes so-called "peptide nucleic acids", which comprise naturally occurring or modified nucleic acid bases attached to a polyamide backbone. Nucleic acids can be either single stranded or double stranded. The term "operatively linked", when describing the relationship between two nucleic acid regions, refers to a juxtaposition wherein the regions are in a relationship permitting them to function in their intended manner.

For example, a control sequence "operatively linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences, such as when the appropriate molecules e. Thus, in one embodiment, the phrase "operatively linked" refers to a promoter connected to a coding sequence in such a way that the transcription of that coding sequence is controlled and regulated by that promoter. Techniques for operatively linking a promoter to a coding sequence are well known in the art; the precise orientation and location relative to a coding sequence of interest is dependent, inter alia, upon the specific nature of the promoter.

Thus, the term "operatively linked" can refer to a promoter region that is connected to a nucleotide sequence in such a way that the transcription of that nucleotide sequence is controlled and regulated by that promoter region. Similarly, a nucleotide sequence is said to be under the "transcriptional control" of a promoter to which it is operatively linked.

Techniques for operatively linking a promoter region to a nucleotide sequence are known in the art. The term "operatively linked" can also refer to a transcription termination sequence or other nucleic acid that is connected to a nucleotide sequence in such a way that termination of transcription of that nucleotide sequence is controlled by that transcription termination sequence.

Additionally, the term "operatively linked" can refer to a enhancer, silencer, or other nucleic acid regulatory sequence that when operatively linked to an open reading frame modulates the expression of that open reading frame, either in a positive or negative fashion. The percent identity exists in one embodiment over a region of the sequences that is at least about 50 residues in length, in another embodiment over a region of at least about residues, and in another embodiment, the percent identity exists over at least about residues.

In still another embodiment, the percent identity exists over the entire length of the sequences. For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared.

When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence s relative to the reference sequence, based on the designated program parameters. See generally, Ausubel et al. One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al.

This algorithm involves first identifying high scoring sequence pairs HSPs by identifying short words of length W in the query sequence, which either match or satisfy some positive valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold.

See generally, Altschul et al. These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased.

For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when the cumulative alignment score falls off by the quantity X from its maximum achieved value, the cumulative score goes to zero or below due to the accumulation of one or more negative scoring residue alignments, or the end of either sequence is reached.

In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences see e.

One measure of similarity provided by the BLAST algorithm is the smallest sum probability P N , which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid sequence to the reference nucleic acid sequence is in one embodiment less than about 0.

Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Typically, under "highly stringent conditions" a probe will hybridize specifically to its target subsequence, but to no other sequences. Very stringent conditions are selected to be equal to the T m for a particular probe.

An example of highly stringent wash conditions is 15 minutes in 0. Another example of highly stringent wash conditions is 15 minutes in 0.

Often, a high stringency wash is preceded by a lower stringency wash to remove background probe signal. For short probes e. Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide. In general, a signal to noise ratio of 2-fold or higher than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization. The following are examples of hybridization and wash conditions that can be used to clone homologous nucleotide sequences that are substantially similar to reference nucleotide sequences of the presently disclosed subject matter: The term "phenotype" refers to the entire physical, biochemical, and physiological makeup of a cell or an organism, e.

As such, phenotypes result from the expression of genes within a cell or an organism, and relate to traits that are potentially observable or assayable. As used herein, the terms "polypeptide", "protein", and "peptide", which are used interchangeably herein, refer to a polymer of the 20 protein amino acids, or amino acid analogs, regardless of its size or function.

Although "protein" is often used in reference to relatively large polypeptides, and "peptide" is often used in reference to small polypeptides, usage of these terms in the art overlaps and varies.

The term "polypeptide" as used herein refers to peptides, polypeptides and proteins, unless otherwise noted. As used herein, the terms "protein", "polypeptide" and "peptide" are used interchangeably herein when referring to a gene product.

The term "polypeptide" encompasses proteins of all functions, including enzymes. Thus, exemplary polypeptides include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments, and other equivalents, variants and analogs of the foregoing.

The terms "polypeptide fragment" or "fragment", when used in reference to a reference polypeptide, refers to a polypeptide in which amino acid residues are deleted as compared to the reference polypeptide itself, but where the remaining amino acid sequence is usually identical to the corresponding positions in the reference polypeptide.

Such deletions can occur at the amino-terminus or carboxy-terminus of the reference polypeptide, or alternatively both. Fragments typically are at least 5, 6, 8 or 10 amino acids long, at least 14 amino acids long, at least 20, 30, 40 or 50 amino acids long, at least 75 amino acids long, or at least , , , , or more amino acids long. A fragment can retain one or more of the biological activities of the reference polypeptide. In certain embodiments, a fragment can comprise a domain or feature, and optionally additional amino acids on one or both sides of the domain or feature, which additional amino acids can number from 5, 10, 15, 20, 30, 40, 50, or up to or more residues.

Further, fragments can include a sub-fragment of a specific region, which sub-fragment retains a function of the region from which it is derived. In another embodiment, a fragment can have immunogenic properties. As used herein, the term "pre-polypeptide" refers to a polypeptide that is normally targeted to a cellular organelle, such as a chloroplast, and still comprises a transit peptide.

As used herein, the term "primer" refers to a sequence comprising in one embodiment two or more deoxyribonucleotides or ribonucleotides, in another embodiment more than three, in another embodiment more than eight, and in yet another embodiment at least about 20 nucleotides of an exonic or intronic region. Such oligonucleotides are in one embodiment between ten and thirty bases in length. The term "promoter" or "promoter region" each refers to a nucleotide sequence within a gene that is positioned 5' to a coding sequence and functions to direct transcription of the coding sequence.

The promoter region comprises a transcriptional start site, and can additionally include one or more transcriptional regulatory elements. A "minimal promoter" is a nucleotide sequence that has the minimal elements required to enable basal level transcription to occur.

As such, minimal promoters are not complete promoters but rather are subsequences of promoters that are capable of directing a basal level of transcription of a reporter construct in an experimental system.

Minimal promoters include but are not limited to the CMV minimal promoter, the HSV-tk minimal promoter, the simian virus 40 SV40 minimal promoter, the human b-actin minimal promoter, the human EF2 minimal promoter, the adenovirus E1 B minimal promoter, and the heat shock protein hsp 70 minimal promoter. Minimal promoters are often augmented with one or more transcriptional regulatory elements to influence the transcription of an operatively linked gene.

For example, cell-type-specific or tissue-specific transcriptional regulatory elements can be added to minimal promoters to create recombinant promoters that direct transcription of an operatively linked nucleotide sequence in a cell-type-specific or tissue-specific manner.

Different promoters have different combinations of transcriptional regulatory elements. Whether or not a gene is expressed in a cell is dependent on a combination of the particular transcriptional regulatory elements that make up the gene's promoter and the different transcription factors that are present within the nucleus of the cell. As such, promoters are often classified as "constitutive", "tissue-specific", "cell-type-specific", or "inducible", depending on their functional activities in vivo or in vitro.

For example, a constitutive promoter is one that is capable of directing transcription of a gene in a variety of cell types. Exemplary constitutive promoters include the promoters for the following genes which encode certain constitutive or "housekeeping" functions: Exemplary tissue-specific promoters include those promoters described in more detail hereinbelow, as well as other tissue- specific and cell-type specific promoters known to those of skill in the art.

When used in the context of a promoter, the term "linked" as used herein refers to a physical proximity of promoter elements such that they function together to direct transcription of an operatively linked nucleotide sequence. The term "transcriptional regulatory sequence" or "transcriptional regulatory element", as used herein, each refers to a nucleotide sequence within the promoter region that enables responsiveness to a regulatory transcription factor. Responsiveness can encompass a decrease or an increase in transcriptional output and is mediated by binding of the transcription factor to the DNA molecule comprising the transcriptional regulatory element.

In one embodiment, a transcriptional regulatory sequence is a transcription termination sequence, alternatively referred to herein as a transcription termination signal. The term "transcription factor" generally refers to a protein that modulates gene expression by interaction with the transcriptional regulatory element and cellular components for transcription, including RNA. Polymerase, Transcription Associated Factors TAFs , chromatin-remodeling proteins, and any other relevant protein that impacts gene transcription.

As used herein, "significance" or "significant" relates to a statistical analysis of the probability that there is a non-random association between two or more entities. To determine whether or not a relationship is "significant" or has "significance", statistical manipulations of the data can be performed to calculate a probability, expressed as a "p-value".

Those p- values that fall below a user-defined cutoff point are regarded as significant. In one example, a p-value less than or equal to 0. The term "purified" refers to an object species that is the predominant species present i. A "purified fraction" is a composition wherein the object species comprises at least about 50 percent on a molar basis of all species present. In making the determination of the purity of a species in solution or dispersion, the solvent or matrix in which the species is dissolved or dispersed is usually not included in such determination; instead, only the species including the one of interest dissolved or dispersed are taken into account.

The object species can be purified to essential homogeneity contaminant species cannot be detected in the composition by conventional detection methods wherein the composition consists essentially of a single species.

A skilled artisan can purify a polypeptide of the presently disclosed subject matter using standard techniques for protein purification in light of the teachings herein. Purity of a polypeptide can be determined by a number of methods known to those of skill in the art, including for example, amino-terminal amino acid sequence analysis, gel electrophoresis, and mass-spectrometry analysis. A "reference sequence" is a defined sequence used as a basis for a sequence comparison.

A reference sequence can be a subset of a larger sequence, for example, as a segment of a full-length nucleotide or amino acid sequence, or can comprise a complete sequence.

Generally, when used to refer to a nucleotide sequence, a reference sequence is at least , or nucleotides in length, frequently at least nucleotides in length, and often at least nucleotides in length. Because two proteins can each 1 comprise a sequence i. The term "regulatory sequence" is a generic term used throughout the specification to refer to polynucleotide sequences, such as initiation signals, enhancers, regulators, promoters, and termination sequences, which are necessary or desirable to affect the expression of coding and non-coding sequences to which they are operatively linked.

Exemplary regulatory sequences are described in Goeddel, , and include, for example, the early and late promoters of simian virus 40 SV40 , adenovirus or cytomegalovirus immediate early promoter, the lac system, the trp system, the TAC or TRC system, T7 promoter whose expression is directed by T7 RNA polymerase, the major operator and promoter regions of phage lambda, the control regions for fd coat protein, the promoter for 3- phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e.

The nature and use of such control sequences can differ depending upon the host organism. In prokaryotes, such regulatory sequences generally include promoter, ribosomal binding site, and transcription termination sequences.

The term "regulatory sequence" is intended to include, at a minimum, components whose presence can influence expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.

In certain embodiments, transcription of a polynucleotide sequence is under the control of a promoter sequence or other regulatory sequence that controls the expression of the polynucleotide in a cell-type in which expression is intended. It will also be understood that the polynucleotide can be under the control of regulatory sequences that are the same or different from those sequences which control expression of the naturally occurring form of the polynucleotide.

The term "reporter gene" refers to a nucleic acid comprising a nucleotide sequence encoding a protein that is readily detectable either by its presence or activity, including, but not limited to, luciferase, fluorescent protein e.

Generally, a reporter gene encodes a polypeptide not otherwise produced by the host cell, which is detectable by analysis of the cell s , e. In certain instances, a reporter gene encodes an enzyme, which produces a change in fluorometric properties of the host cell, which is detectable by qualitative, quantitative, or semiquantitative function or transcriptional activation.

Exemplary enzymes include esterases,? As used herein, the term "sequencing" refers to determining the ordered linear sequence of nucleic acids or amino acids of a DNA or protein target sample, using conventional manual or automated laboratory techniques. As used herein, the term "substantially pure" refers to that the polynucleotide or polypeptide is substantially free of the sequences and molecules with which it is associated in its natural state, and those molecules used in the isolation procedure.

As used herein, the term "target cell" refers to a cell, into which it is desired to insert a nucleic acid sequence or polypeptide, or to otherwise effect a modification from conditions known to be standard in the unmodified cell.

A nucleic acid sequence introduced into a target cell can be of variable length. Additionally, a nucleic acid sequence can enter a target cell as a component of a plasmid or other vector or as a naked sequence. As used herein, the term "transcription" refers to a cellular process involving the interaction of an RNA polymerase with a gene that directs the expression as RNA of the structural information present in the coding sequences of the gene. The process includes, but is not limited to, the following steps: As used herein, the term "transcription factor" refers to a cytoplasmic or nuclear protein which binds to a gene, or binds to an RNA transcript of a gene, or binds to another protein which binds to a gene or an RNA transcript or another protein which in turn binds to a gene or an RNA transcript, so as to thereby modulate expression of the gene.

Such modulation can additionally be achieved by other mechanisms; the essence of a "transcription factor for a gene" pertains to a factor that alters the level of transcription of the gene in some way.

The term "transfection" refers to the introduction of a nucleic acid, e. The term "transformation" refers to a process in which a cell's genotype is changed as a result of the cellular uptake of exogenous nucleic acid. For example, a transformed cell can express a recombinant form of a polypeptide of the presently disclosed subject matter or antisense expression can occur from the transferred gene so that the expression of a naturally occurring form of the gene is disrupted.

Thus, what is needed is the identification of plant genes useful to confer resistance to a pathogen s and plant promoters, the expression of which is altered in response to pathogen attack. The invention generally provides an isolated nucleic acid molecule polynucleotide comprising a plant nucleotide sequence obtained or isolatable from a gene, the expression of which is altered, either increased or decreased, in response to pathogen infection. In one embodiment, the plant nucleotide sequence comprises an open reading frame, while in another embodiment, the plant nucleotide sequence comprises a promoter.

A promoter sequence of the invention directs transcription of a linked nucleic acid segment, e. Moreover, the expression of a plant nucleotide sequence of the invention comprising a promoter may be altered in response to one or more species of bacteria, nematode, fungi, oomycete, virus, or insect.

Likewise, the expression of a plant nucleotide sequence of the invention comprising an open reading frame may be useful to confer tolerance or resistance of a plant to one or more species of bacteria, nematode, fungi, oomycete, virus or insect.

The nucleotide sequence preferably is obtained or isolatable from plant DNA. The present invention also provides an isolated nucleic acid molecule comprising a plant nucleotide sequence that directs transcription of a linked nucleic acid segment in a host cell, e. The nucleotide sequence preferably is obtained or isolatable from plant genomic DNA. The invention also provides uses for an isolated nucleic acid molecule, e.

For example, these open reading frames may be useful to prepare plants that over- or under-express the encoded product or to prepare knockout plants. The promoters and open reading frames of the invention can be identified by any method. For example, they can be identified by employing an array of nucleic acid samples, e. Thus, genes that are upregulated or downregulated in response to pathogen infection can be systematically identified.

The Arabidopsis oligonucleotide probe array consists of probes from about 8, unique Arabidopsis genes, which covers approximately one third of the genome. This genome array permits a broader, more complete and less biased analysis of gene expression. For example, using this approach, genes were identified, the expression of which was altered after infection of wild-type Arabidopsis plants with a pathogen SEQ ID NOs: As also described herein, genes were identified that are useful to confer improved resistance to plants to bacterial infection SEQ ID NOs: In addition, genes were identified that are useful to confer improved resistance to plants to infection with more than one pathogen, e.

Eight of the genes were upregulated by 3 hours in an incompatible interaction, 18 of the genes were upregulated by 6 hours, but not at 3 hours, in an incompatible interaction, and 6 of the genes were upregulated in a compatible interaction. In a similar approach, 48 genes that were upregulated in response to infection, e. Thirty-six of the genes were upregulated in response to bacterial, e. Twenty-five of the genes were upregulated only in response to bacterial, e.

Also provided are nucleic acid molecules comprising a nucleotide sequence comprising an open reading frame expressed in response to pathogen infection comprising SEQ ID NOs: These sequences are useful to over- or under-express the encoded product, or prepare knock-out plants which have an altered response to pathogen infection.

The invention therefore provides a method in which the open reading frame of a plant pathogen resistance gene, e.

A transgene comprising the isolated open reading frame may be introduced to and expressed in a transgenic plant, e. The expression of the transgene is preferably at higher than normal levels, and under the regulation of a promoter that allows very fast and high induction in response to the presence of a pathogen or under cycling promoters e.

The invention further provides a method in which a gene in a plant which is downregulated in response to infection, is disrupted or the expression of that gene is further downregulated, e. As also described herein, it was found that the early incompatible response was similar to the late compatible response, suggesting that early expression of plant pathogen-resistance genes is important for resistance.

Also, various plant strains were found to respond differently to the same pathogen, but there was also an identifiable global pattern of response. Thus, the comparison of the expression patterns in incompatible and compatible interactions in one or more ecotypes can lead to identifying subsets of key responding genes and clusters of genes that are key early responders. In addition, the observed global expression pattern indicated that the least resistant strain tested Ws had a low basal level of pathogen-upregulated genes and a high level of pathogen-downregulated genes compared to the most resistant strain Ler.

Thus, plant strains that are more resistant to pathogens have a gene expression phenotype in which genes that are upregulated in response to infection are already expressed at a higher than normal basal level, and those genes that are downregulated are expressed at a lower than normal basal level.

Thus, further provided herein is a method to identify at least one gene involved in plant dicot or monocot resistance or response to infection by at least one pathogen, e. Also provided is a method to identify at least one gene involved in plant dicot or monocot resistance or response to infection by at least one pathogen, e. A compatible interaction can be, for example, between a plant having a resistance gene and a pathogen lacking a corresponding avirulence gene, a plant lacking a resistance gene to a pathogen having a corresponding avirulence gene, or a plant lacking a resistance gene and a pathogen lacking a corresponding avirulence gene.

For example, the gene identified by such a method can encode a polypeptide that is substantially similar to a polypeptide encoded by an open reading frame comprising one of SEQ ID NOs: Such a method can further involve isolating the at least one gene or a portion thereof which includes the open reading frame or promoter for the gene. In addition, provided herein is a method to identify at least one gene, the expression of which is altered by infection with at least one virus, which method comprises contacting a plurality of isolated nucleic acid samples on a solid substrate each comprising isolated nucleic acid with a probe comprising plant nucleic acid corresponding to RNA from a wild-type plant infected with a virus, so as to form a complex, wherein each sample comprises a plurality of oligonucleotides corresponding to at least a portion of one plant gene; and comparing complex formation in a with complex formation between a second plurality of isolated nucleic acid samples on a solid substrate with a second probe comprising nucleic acid corresponding to RNA from an uninfected plant, so as to identify a gene, the expression of which is altered by virus infection.

Also provided is a method to identify at least one gene, the expression of which is altered by infection with at least one pathogen, which involves contacting a plurality of isolated nucleic acid samples on a solid substrate each comprising isolated nucleic acid with a probe comprising plant nucleic acid corresponding to RNA from an incompatible interaction so as to form a complex, wherein each sample comprises a plurality of oligonucleotides corresponding to at least a portion of one plant gene; and comparing complex formation in a with complex formation between a second plurality of isolated nucleic acid samples on a solid substrate with a second probe comprising nucleic acid corresponding to RNA from a corresponding compatible interaction so as to identify a gene, the expression of which is altered by the pathogen.

In any of the methods described herein, the probes can have nucleic acid, for example, from a dicot, a cereal plant, or a monocot. Further, the methods can additionally involve identifying the promoter for the at least one gene.

The genes and promoters described hereinabove can be used to identify orthologous genes and their promoters which are also likely useful to enhance resistance of plants to pathogens. Moreover, the orthologous promoters are useful to express linked open reading frames. In addition by aligning the promoters of these orthologs, novel cis elements can be identified that are useful to generate synthetic promoters. Hence, the isolated nucleic acid molecules of the invention include the orthologs homologs of the Arabidopsis, Chenopodium and rice sequences disclosed herein, i.

An ortholog is a gene from a different species that encodes a product having the same function as the product encoded by a gene from a reference organism. Databases such GenBank or one found at http: Alternatively, recombinant DNA techniques such as hybridization or PCR may be employed to identify sequences related to the Arabidopsis sequences. For example, promoter sequences within the scope of the invention are those which direct expression of an open reading frame which encodes a polypeptide that is substantially similar to an Arabidopsis polypeptide encoded by a gene comprising SEQ ID NOs: In addition, by aligning the promoters of these orthologs, novel cis elements can be identified that are useful to generate synthetic promoters.

Hence, the isolated nucleic acid molecules of the invention include the orthologs of the Arabidopsis sequences disclosed herein, i. An orthologous gene is a gene from a different species that encodes a product having the same or similar function, e.

Thus, an ortholog includes polypeptides having less than, e. Databases such as GenBank or one found at http: Preferably, the promoters of the invention include a consecutive stretch of about 25 to , including 50 to or to , and up to or , contiguous nucleotides, e.

Preferably, the nucleotide sequence that includes the promoter region includes at least one copy of a TATA box. The present invention further provides a composition, an expression cassette or a recombinant vector containing the nucleic acid molecule of the invention, and host cells comprising the expression cassette or vector, e. In particular, the present invention provides an expression cassette or a recombinant vector comprising a promoter of the invention linked to a nucleic acid segment which, when present in a plant, plant cell or plant tissue, results in transcription of the linked nucleic acid segment.

One indication that two polypeptides are substantially similar to each other, besides having substantially the same function, is that an agent, e. Sequence comparisons maybe carried out using a Smith-Waterman sequence alignment algorithm see e. The localS program, version 1. Hence, the present invention further provides an expression cassette or a vector containing the nucleic acid molecule comprising an open reading frame of the invention operably linked to a promoter, or comprising a promoter of the invention operably linked to an open reading frame or portion thereof, and the vector may be a plasmid.

Such cassettes or vectors, when present in a plant, plant cell or plant tissue result in transcription of the linked nucleic acid fragment in the plant. The expression cassettes or vectors of the invention may optionally include other regulatory sequences, e. The expression cassette or vector may augment the genome of a transformed plant or may be maintained extrachromosomally.

The expression cassette or vector may further have a Ti plasmid and be contained in an Agrobacterium tumefaciens cell; it may be carried on a microparticle, wherein the microparticle is suitable for ballistic transformation of a plant cell; or it may be contained in a plant cell or protoplast.

Further, the expression cassette can be contained in a transformed plant or cells thereof and the plant may be a dicot or a monocot. In particular, the plant may be a cereal plant. The invention also provides sense and anti-sense nucleic acid molecules corresponding to the open reading frames identified herein as well as their orthologs.

Also provided are expression cassettes, e. The present invention further provides a method of augmenting a plant genome by contacting plant cells with a nucleic acid molecule of the invention, e.

The present invention also provides a transgenic plant prepared by this method, a seed from such a plant and progeny plants from such a plant including hybrids and inbreds. Preferred transgenic plants are transgenic maize, soybean, barley, alfalfa, sunflower, canola, soybean, cotton, peanut, sorghum, tobacco, sugarbeet, rice, wheat, rye, turfgrass, millet, sugarcane, tomato, or potato. The invention also provides a method of plant breeding, e. The method comprises crossing a fertile transgenic plant comprising a particular nucleic acid molecule of the invention with itself or with a second plant, e.

The seed is then planted to obtain a crossed fertile transgenic plant. The plant may be a monocot or a dicot. In a particular embodiment, the plant is a cereal plant. The crossed fertile transgenic plant may have the particular nucleic acid molecule inherited through a female parent or through a male parent.

The second plant may be an inbred plant. The crossed fertile transgenic may be a hybrid. Also included within the present invention are seeds of any of these crossed fertile transgenic plants. The various breeding steps are characterized by well-defined human intervention such as selecting the lines to be crossed, directing pollination of the parental lines, or selecting appropriate progeny plants.

Depending on the desired properties different breeding measures are taken. The relevant techniques are well known in the art and include but are not limited to hybridization, inbreeding, backcross breeding, multiline breeding, variety blend, interspecific hybridization, aneuploid techniques, etc.

Hybridization techniques also include the sterilization of plants to yield male or female sterile plants by mechanical, chemical or biochemical means. Cross pollination of a male sterile plant with pollen of a different line assures that the genome of the male sterile but female fertile plant will uniformly obtain properties of both parental lines.

Thus, the transgenic plants according to the invention can be used for the breeding of improved plant lines that for example increase the effectiveness of conventional methods such as herbicide or pesticide treatment or allow to dispense with said methods due to their modified genetic properties. The nucleic acid molecules of the invention, their encoded polypeptides and compositions thereof, are: As one embodiment of the invention includes isolated nucleic acid molecules that have increased expression in response to pathogen infection, the invention further provides compositions and methods for enhancing resistance to pathogen infection.

The compositions of the invention include plant nucleic acid sequences and the amino acid sequences for the polypeptides or partial-length polypeptides encoded thereby which are described herein, or other plant nucleic acid sequences and the amino acid sequences for the polypeptides or partial-length polypeptides encoded thereby which are operably linked to a promoters are useful to provide tolerance or resistance to a plant to a pathogen, preferably by preventing or inhibiting pathogen infection.

Methods of the invention involve stably transforming a plant with one or more of at least a portion of these nucleotide sequences which confer tolerance or resistance operably linked to a promoter capable of driving expression of that nucleotide sequence in a plant cell. A method of combating a pathogen in an agricultural crop is also provided. The method comprises introducing to a plant, plant cell, or plant tissue an expression cassette comprising a nucleic acid molecule of the invention comprising an open reading frame so as to yield a transformed differentiated plant, transformed cell or transformed tissue.

Transformed cells or tissue can be regenerated to provide a transformed differentiated plant. The transformed differentiated plant preferably expresses the nucleic acid molecule in an amount that confers resistance to the transformed plant to pathogen infection relative to a corresponding nontransformed plant.

The present invention also provides a transformed plant prepared by the method, progeny and seed thereof. Examples of plant viruses which may be combated by the present invention include single stranded RNA viruses with and without envelope , double stranded RNA viruses, and single and double stranded DNA viruses such as but not limited to tobacco mosaic virus, cucumber mosaic virus, turnip mosaic virus, turnip vein clearing virus, oilseed rape mosaic virus, tobacco rattle virus, pea enation mosaic virus, barley stripe mosaic virus, potato viruses X and Y, carnation latent virus, beet yellows virus, maize chlorotic virus, tobacco necrosis virus, turnip yellow mosaic virus, tomato bushy stunt virus, southern bean mosaic virus, barley yellow dwarf virus, tomato spotted wilt virus, lettuce necrotic yellows virus, wound tumor virus, maize streak virus, and cauliflower mosaic virus.

Other pathogens within the scope of the invention include, but are not limited to, fungi such as Cochliobolus carbonum, Phytophthora infestans, Phytophthora sojae, Collesosichum, Melampsora lini, cladosporium fulvum, Heminthosporium maydia, Peronospora parasitica, Puccinia sorghi , and Puccinia polysora ; bacteria such as Phynchosporium secalis, Pseudomonas glycinea, Xanthomonas oryzae and, Fusarium oxyaporium ; and nematodes such as Globodera rostochiensis.

For example, the invention provides a nucleic acid molecule comprising a plant nucleotide sequence comprising at least a portion of a key effector gene s responsible for host resistance to particular pathogens. The overexpression may be constitutive, or it may be preferable to express the effector gene s in a tissue-specific manner or from an inducible promoter including a promoter which is responsive to external stimuli, such as chemical application, or to pathogen infection, e.

A transformed transgenic plant of the invention includes plants, for example, a plant the cells of which have an expression cassette of the invention, i. The nucleic acid molecules of the invention are thus useful for targeted gene disruption, as well as markers and probes.

For example, the invention includes a pathogen, e. The normal fertile transformed transgenic plant may be selfed to yield a substantially homogenous line with respect to viral resistance or tolerance.

Individuals of the line, or the progeny thereof, may be crossed with plants which optionally exhibit the trait. In a particular embodiment of the method, the selfing and selection steps are repeated at least five times in order to obtain the homogenous isogenic line.

Thus, the invention also provides transgenic plants and the products of the transgenic plants. When the hybridization is performed under stringent conditions, either the test or nucleic acid molecule of invention is preferably supported, e.

Thus, either a denatured test or nucleic acid molecule of the invention is preferably first bound to a support and hybridization is effected for a specified period of time at a temperature of, e. Depending upon the degree of stringency required such reduced concentration buffers are typically single strength SC containing 0. The invention further provides a method to identify an open reading frame in the genome of a plant cell, the expression of which is altered by pathogen infection of that cell.

The method comprises contacting a solid substrate comprising a plurality of samples comprising isolated plant nucleic acid of a probe comprising plant nucleic acid, e. Each individual sample comprises one or more nucleic acid sequences e.

The method may be employed with nucleic acid samples and probes from any organism, e. Preferably, the nucleic acid sample and probes are from a plant, such as a dicot or monocot.

More preferably the nucleic acid samples and probes are from a cereal plant. Even more preferably the nucleic acids and probes are from a crop plant. A second plurality of samples on a solid substrate, i. Then complex formation between the samples and probes comprising nucleic acid from infected or control cells compared. For example, potato virus X, tobacco mosaic virus, tobravirus, cucumber mosaic virus and gemnivirus are known to infect Arabidopsis. Thus, Arabidopsis genes, the expression of which is altered in response to infection by any of these viruses, can be identified.

The invention further provides a method for identifying a plant cell infected with a pathogen. The method comprises contacting nucleic acid obtained from a plant cell suspected of being infected with a pathogen with oligonucleotides corresponding to a portion of a plurality of sequences selected from SEQ ID NOs: Then the presence of the amplified product is detected or determined. The presence of two or more amplified products, e.

The method comprises contacting a protein sample obtained from a plant cell suspected of being infected with a pathogen with an agent that specifically binds a polypeptide encoded by an open reading frame comprising SEQ ID NOs: Then the presence or amount of complex formation is detected or determined.

The invention provides an additional method for identifying a plant cell infected with a pathogen. The amount of the probe hybridized to nucleic acid obtained from a cell suspected of being infected with a virus is compared to hybridization of the probe to nucleic acid isolated from an uninfected cell. A change in the amount of at least two probes that hybridize to nucleic acid isolated from a cell suspected of being infected by a virus relative to hybridization of at least two probes to nucleic acid isolated from an uninfected cell is indicative of viral infection.

A method to shuffle the nucleic acids of the invention is provided. This method allows for the production of polypeptides having altered activity relative to the native form of the polypeptide. Accordingly, the invention provides cells and transgenic plants containing nucleic acid segments produced through shuffling that encode polypeptides having altered activity relative to the corresponding native polypeptide.

A computer readable medium, e. This medium also allows for computer-based manipulation of a nucleic acid sequence corresponding to a nucleic acid sequence listed in SEQ ID NOs: The invention also provides a method for marker-assisted breeding to select for plants having altered resistance to a pathogen.

The amount or presence of the duplex is indicative of the presence of a gene, the expression of which alters the resistance of the plant to a pathogen.

Therefore, another embodiment of the present invention provides a method of using known inducers or inhibitors of genes identified as being important in plant-pathogen interactions to induce genes that are important in resistance, or to inhibit genes that are downregulated in resistance. Thus, some of the isolated nucleic acid molecules of the invention are useful in a method of combating a pathogen in an agricultural crop.

The method comprises introducing to a plant an expression cassette comprising a nucleic acid molecule of the invention so as to yield a transformed differentiated plant. The transformed differentiated plant expresses the nucleic acid molecule in an amount that confers resistance to the transformed plant to infection relative to a corresponding nontransformed plant.

For example, gene refers to a nucleic acid fragment that expresses mRNA or functional RNA, or encodes a specific protein, and which includes regulatory sequences. Genes also include nonexpressed DNA segments that, for example, form recognition sequences for other proteins.

Genes can be obtained from a variety of sources, including cloning from a source of interest or synthesizing from known or predicted sequence information, and may include sequences designed to have desired parameters. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or comprise regulatory sequences and coding sequences derived from the same source, but arranged in a manner different from that found in nature.

Transgenes may include, for example, genes that are either heterologous or homologous to the genes of a particular plant to be transformed. Additionally, transgenes may comprise native genes inserted into a non-native organism, or chimeric genes. Generally specific primers are upwards of 14 nucleotides in length. For optimum specificity and cost effectiveness, primers of 16 to 24 nucleotides in length may be preferred.

Those skilled in the art are well versed in the design of primers for use processes such as PCR. If required, probing can be done with entire restriction fragments of the gene disclosed herein which may be 's or even 's of nucleotides in length. The nucleotide sequences of the invention can be introduced into any plant.

The genes to be introduced can be conveniently used in expression cassettes for introduction and expression in any plant of interest. Such expression cassettes will comprise the transcriptional initiation region of the invention linked to a nucleotide sequence of interest. Such an expression cassette is provided with a plurality of restriction sites for insertion of the gene of interest to be under the transcriptional regulation of the regulatory regions.

The expression cassette may additionally contain selectable marker genes. The termination region may be native with the transcriptional initiation region, may be native with the DNA sequence of interest, or may be derived from another source. Convenient termination regions are available from the Ti-plasmid of A.

See also, Guerineau et al. When the RNA transcript is a perfect complementary copy of the DNA sequence, it is referred to as the primary transcript or it may be a RNA sequence derived from posttranscriptional processing of the primary transcript and is referred to as the mature RNA.

Regulatory sequences include enhancers, promoters, translation leader sequences, introns, and polyadenylation signal sequences. They include natural and synthetic sequences as well as sequences which may be a combination of synthetic and natural sequences.

It is present in the fully processed mRNA upstream of the initiation codon and may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency Turner et al. The translation leader sequence may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency. Bacterial pathogens include but are not limited to Pseudomonas avenae subsp. Fungal pathogens include but are not limited to Collelotrichum graminicola, Glomerella graminicola Politis, Glomerella lucumanensis, Aspergillus flavus, Rhizoctonia solani Kuhn, Thanatephorus cucumeris, Acremonium strictum W.

Gams, Cephalosporium acremonium Auct. Cochliobolus lunatus , Curvularia pallescens teleomorph— Cochliobolus pallescens , Curvularia senegalensis, C. Setosphaeriaprolata , Graphium penicillioides, Leptosphaeria maydis, Leptothyrium zeae, Ophiosphaerella herpotricha anamorph— Scolecosporiella sp. Waitea circinata , Rhizoctonia solani , minor A Iternaria alternala, Cercospora sorghi , Dictochaetaftrtilis, Fusarium acuminatum teleomorph Gihherella acuminata , E. Nectria haematococca , F.

Mycosphaerella zeae - maydis , and Gloeocercospora sorghi. Parasitic nematodes include but are not limited to Awl Dolichodorus spp. This type of promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. It is capable of operating in both orientations normal or flipped , and is capable of functioning even when moved either upstream or downstream from the promoter. Both enhancers and other upstream promoter elements bind sequence-specific DNA-binding proteins that mediate their effects.

Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even be comprised of synthetic DNA segments. A promoter may also contain DNA sequences that are involved in the binding of protein factors which control the effectiveness of transcription initiation in response to physiological or developmental conditions.

With respect to this site all other sequences of the gene and its controlling regions are numbered. It includes natural and synthetic sequences as well as sequences which may be a combination of synthetic and natural sequences. Different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. New promoters of various types useful in plant cells are constantly being discovered, numerous examples may be found in the compilation by Okamuro et al.

Typical regulated promoters useful in plants include but are not limited to safener-inducible promoters, promoters derived from the tetracycline-inducible system, promoters derived from salicylate-inducible systems, promoters derived from alcohol-inducible systems, promoters derived from glucocorticoid-inducible system, promoters derived from pathogen-inducible systems, and promoters derived from ecdysome-inducible systems.

These also include promoters that are temporally regulated, such as in early or late embryogenesis, during fruit ripening in developing seeds or fruit, in fully differentiated leaf, or at the onset of senescence. Coding sequences can be operably-linked to regulatory sequences in sense or antisense orientation.

For example, in the case of antisense constructs, expression may refer to the transcription of the antisense DNA only. Expression may also refer to the production of protein. It is acknowledged that hardly a true specificity exists: This phenomenon is known as leaky expression. However, with specific expression in this invention is meant preferable expression in one or a few plant tissues.

Expression patterns of a set of promoters are said to be complementary when the expression pattern of one promoter shows little overlap with the expression pattern of the other promoter. This measurement is indirect since the concentration of the reporter mRNA is dependent not only on its synthesis rate, but also on the rate with which the mRNA is degraded.

Therefore, the steady state level is the product of synthesis rates and degradation rates. The rate of degradation can however be considered to proceed at a fixed rate when the transcribed sequences are identical, and thus this value can serve as a measure of synthesis rates.

This list of techniques in no way represents all available techniques, but rather describes commonly used procedures used to analyze transcription activity and expression levels of mRNA. The analysis of transcription start points in practically all promoters has revealed that there is usually no single base at which transcription starts, but rather a more or less clustered set of initiation sites, each of which accounts for some start points of the mRNA.

Since this distribution varies from promoter to promoter the sequences of the reporter mRNA in each of the populations would differ from each other.

Since each mRNA species is more or less prone to degradation, no single degradation rate can be expected for different reporter mRNAs. This includes also part of the transcribed sequences. The direct fusion of promoter to reporter sequences would therefore lead to suboptimal levels of transcription. In principle, however, many more proteins are suitable for this purpose, provided the protein does not interfere with essential plant functions. For quantification and determination of localization a number of tools are suited.

Detection systems can readily be created or are available which are based on, e. Protein levels can be determined in plant tissue extracts or in intact tissue using in situ analysis of protein expression. Generally, individual transformed lines with one chimeric promoter reporter construct will vary in their levels of expression of the reporter gene. Also frequently observed is the phenomenon that such transformants do not express any detectable product RNA or protein.

Likewise, leaf, and stem expression levels, are determined using whole extracts from leaves and stems. It is acknowledged however, that within each of the plant parts just described, cells with variable functions may exist, in which promoter activity may vary. Gene silencing may be transcriptional, when the suppression is due to decreased transcription of the affected genes, or post-transcriptional, when the suppression is due to increased turnover degradation of RNA species homologous to the affected genes English et al.

Gene silencing includes virus-induced gene silencing Ruiz et al. Silencing suppressor genes may be of plant, non-plant, or viral origin. Thus, a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified through, for example, the use of DNA shuffling.

The terms also include non-naturally occurring multiple copies of a naturally occurring DNA sequence. Thus, the terms refer to a DNA segment that is foreign or heterologous to the cell, or homologous to the cell but in a position within the host cell nucleic acid in which the element is not ordinarily found.

Exogenous DNA segments are expressed to yield exogenous polypeptides. For example, altered nucleotide sequences which simply reflect the degeneracy of the genetic code but nonetheless encode amino acid sequences that are identical to a particular amino acid sequence are substantially similar to the particular sequences.

Modifications that result in equivalent nucleotide or amino acid sequences are well within the routine skill in the art. The target gene is not essential for replicon replication. Additionally, target genes may comprise native non-viral genes inserted into a non-native organism, or chimeric genes, and will be under the control of suitable regulatory sequences.

Thus, the regulatory sequences in the target gene may come from any source, including the virus. Target genes may include coding sequences that are either heterologous or homologous to the genes of a particular plant to be transformed. However, target genes do not include native viral genes. Typical target genes include, but are not limited to genes encoding a structural protein, a seed storage protein, a protein that conveys herbicide resistance, and a protein that conveys insect resistance.

The expression of a target gene in a plant will typically produce an altered plant trait. In addition to the ORF of the replication protein, the replication gene may also contain other overlapping or non-overlapping ORF s , as are found in viral sequences in nature.

Such chimeric genes also include insertion of the known sites of replication protein binding between the promoter and the transcription start site that attenuate transcription of viral replication protein gene. It excludes young, growing tissue consisting of germline, meristematic, and not-fully-differentiated cells.

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They also typically encompass sequences required for proper translation of the nucleotide sequence. As used herein, the term "significant increase" refers to an increase in activity for example, enzymatic activity that is larger than the margin of error inherent in the measurement technique, in one embodiment an increase by about 2 fold or greater over a baseline activity for example, the activity of the wild type enzyme in the presence of the inhibitor , in another embodiment an increase by about 5 fold or greater, and in still another embodiment an increase by about 10 fold or greater.

As used herein, the terms "significantly less" and "significantly reduced" refer to a result for example, an amount of a product of an enzymatic reaction that is reduced by more than the margin of error inherent in the measurement technique, in one embodiment a decrease by about 2 fold or greater with respect to a baseline activity for example, the activity of the wild type enzyme in the absence of the inhibitor , in another embodiment, a decrease by about 5 fold or greater, and in still another embodiment a decrease by about 10 fold or greater.

As used herein, the terms "specific binding" and "immunological cross-reactivity" refer to an indicator that two molecules are substantially similar. An indication that two nucleic acid sequences or polypeptides are substantially similar is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with, or specifically binds to, the polypeptide encoded by the second nucleic acid. Thus, a polypeptide is typically substantially similar to a second polypeptide, for example, where the two polypeptides differ only by conservative substitutions.

The phrase "specifically or selectively binds to an antibody," or "specifically or selectively immunoreactive with," when referring to a polypeptide or peptide, refers to a binding reaction which is determinative of the presence of the polypeptide in the presence of a heterogeneous population of polypeptides and other biologies. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular polypeptide and do not bind in a significant amount to other polypeptides present in the sample.

Specific binding to an antibody under such conditions can require an antibody that is selected for its specificity for a particular polypeptide. For example, antibodies raised to the polypeptide with the amino acid sequence encoded by any of the nucleic acid sequences of the presently disclosed subject matter can be selected to obtain antibodies specifically immunoreactive with that polypeptide and not with other polypeptides except for polymorphic variants.

A variety of immunoassay formats can be used to select antibodies specifically immunoreactive with a particular polypeptide. For example, solid phase ELISA immunoassays, Western blots, or immunohistochemistry are routinely used to select monoclonal antibodies specifically immunoreactive with a polypeptide. Typically a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 to times background.

As used herein, the term "subsequence" refers to a sequence of nucleic acids or amino acids that comprises a part of a longer sequence of nucleic acids or amino acids e.

As used herein, the term "substrate" refers to a molecule that an enzyme naturally recognizes and converts to a product in the biochemical pathway in which the enzyme naturally carries out its function; or is a modified version of the molecule, which is also recognized by the enzyme and is converted by the enzyme to a product in an enzymatic reaction similar to the naturally-occurring reaction.

As used herein, the term "suitable growth conditions" refers to growth conditions that are suitable for a certain desired outcome, for example, the production of a recombinant polypeptide or the expression of a nucleic acid molecule.

As used herein, the term "transformation" refers to a process for introducing heterologous DNA into a plant 1 cell, plant tissue, or plant. Transformed plant cells, plant tissue, or plants are understood to encompass not only the end product of a transformation process, but also transgenic progeny thereof. As used herein, the terms "transformed", "transgenic", and "recombinant" refer to a host organism such as a bacterium or a plant into which a heterologous nucleic acid molecule has been introduced.

The nucleic acid molecule can be stably integrated into the genome of the host or the nucleic acid molecule can also be present as an extrachromosomal molecule. Such an extrachromosomal molecule can be auto-replicating.

Transformed cells, tissues, or plants are understood to encompass not only the end product of a transformation process, but also transgenic progeny thereof. A "non-transformed," "non-transgenic", or "non-recombinant" host refers to a wild-type organism, e.

As used herein, the term "viability" refers to a fitness parameter of a plant. Plants are assayed for their homozygous performance of plant development, indicating which polypeptides are essential for plant growth. In certain embodiments, the isolated nucleic acid molecule is derived from rice i.

As used herein, the phrase "cell proliferation-related polypeptide" refers to a protein or polypeptide note that these two terms are used interchangeably throughout that is involved in cell proliferation, particularly plant cell proliferation. Such a polypeptide can be involved in an increase in cell proliferation; conversely, such a polypeptide can be involved in the abrogation or inhibition of cell proliferation. Moreover, the polypeptide can be involved in cell proliferation only, for example, when the cell is exposed to a stress e.

In addition, the polypeptide can be involved in cell proliferation only when the cell is differentiating or developing. A "cell proliferation-related polypeptide" of the presently disclosed subject matter is identified by the ability of an increase or decrease in the level of expression of such a polypeptide in a cell to modulate the rate of that cell's proliferation, whether alone or together with some other stimuli e.

As used herein, term "binds" means that a cell proliferation-related polypeptide preferentially interacts with a stated target molecule. In some embodiments, that interaction allows a biological read-out e. In some embodiments, that interaction is measurable e. Disclosed herein are rice O. All of the cell proliferation-related proteins of the invention are related, and many interact with one another. Figures are schematic representations showing the interrelatedness of the different cell proliferation-related proteins of the invention.

In certain embodiments, the presently disclosed subject matter provides an isolated nucleic acid molecule comprising a nucleotide sequence substantially similar to the nucleotide sequence of the nucleic acid molecule encoding a cell proliferation-related polypeptide disclosed herein.

In a broad sense, the term "substantially similar", as used herein with respect to a nucleotide sequence, refers to a nucleotide sequence corresponding to a reference nucleotide sequence i. In some embodiments, the substantially similar nucleotide sequence encodes the polypeptide encoded by the reference nucleotide sequence i. The term "substantially similar" is specifically intended to include nucleotide sequences wherein the sequence has been modified to optimize expression in particular cells.

The term "substantially similar", when used herein with respect to a protein or polypeptide, refers to a protein or polypeptide corresponding to a reference protein i. In one embodiment, the polypeptide is involved in a function such as abiotic stress tolerance, disease resistance, enhanced yield or nutritional quality or composition.

In one embodiment, the polypeptide is involved in drought resistance. Additionally, one skilled in the art will recognize that individual substitutions, deletions, or additions to a nucleic acid, peptide, polypeptide, or polypeptide sequence that alters, adds, or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservative modification" where the modification results in the substitution of an amino acid with a chemically similar amino acid.

Conservative modified variants provide similar biological activity as the unmodified polypeptide. Conservative substitution tables listing functionally similar amino acids are known in the art.

The term "conservatively modified variant" also refers to a peptide having an amino acid residue sequence substantially similar to a sequence of a polypeptide of the presently disclosed subject matter in which one or more residues have been conservatively substituted with a functionally similar residue.

Examples of conservative substitutions include the substitution of one non-polar hydrophobic residue such as isoleucine, valine, leucine or methionine for another; the substitution of one polar hydrophilic residue for another such as between arginine and lysine, between glutamine and asparagine, between glycine and serine; the substitution of one basic residue such as lysine, arginine or histidine for another; or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another.

Amino acid substitutions, such as those which might be employed in modifying the polypeptides described herein, are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. An analysis of the size, shape and type of the amino acid side-chain substituents reveals that arginine, lysine and histidine are all positively charged residues; that alanine, glycine and serine are all of similar size; and that phenylalanine, tryptophan and tyrosine all have a generally similar shape.

Therefore, based upon these considerations, arginine, lysine and histidine; alanine, glycine and serine; and phenylalanine, tryptophan and tyrosine; are defined herein as biologically functional equivalents. Other biologically functionally equivalent changes will be appreciated by those of skill in the art.

In making biologically functional equivalent amino acid substitutions, the hydropathic index of amino acids can be considered. Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics, these are: The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art.

It is known that certain amino acids can be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent protein. As detailed in U. While discussion has focused on functionally equivalent polypeptides arising from amino acid changes, it will be appreciated that these changes can be effected by alteration of the encoding DNA, taking into consideration also that the genetic code is degenerate and that two or more codons can code for the same amino acid.

In one embodiment, the polypeptide is expressed in a specific location or tissue of a plant. In one embodiment, the location or tissue includes, but is not limited to, epidermis, vascular tissue, meristem, cambium, cortex, or pith. In another embodiment, the location or tissue is leaf or sheath, root, flower, and developing ovule or seed.

In another embodiment, the location or tissue can be, for example, epidermis, root, vascular tissue, meristem, cambium, cortex, pith, leaf, or flower. In yet another embodiment, the location or tissue is a seed. The polypeptides of the presently disclosed subject matter, fragments thereof, or variants thereof, can comprise any number of contiguous amino acid residues from a polypeptide of the presently disclosed subject matter, wherein the number of residues is selected from the group of integers consisting of from 10 to the number of residues in a full-length polypeptide of the presently disclosed subject matter.

In one embodiment, the portion or fragment of the polypeptide is a functional polypeptide. Methods of assaying and quantifying measures of activity and substrate specificity are well known to those of skill in the art.

The isolated polypeptides of the presently disclosed subject matter can elicit production of an antibody specifically reactive to a polypeptide of the presently disclosed subject matter when presented as an immunogen. Therefore, the polypeptides of the presently disclosed subject matter can be employed as immunogens for constructing antibodies immunoreactive to a polypeptide of the presently disclosed subject matter for such purposes including, but not limited to, immunoassays or polypeptide purification techniques.

Immunoassays for determining binding are well known to those of skill in the art and include, but are not limited to, enzyme-linked immunosorbent assays ELISAs and competitive immunoassays.

The Yeast Two-Hybrid System The yeast two-hybrid system is a well known system which is based on the finding that most eukaryotic transcription activators are modular see e. The yeast two-hybrid system uses: In all of the Examples described below, an automated, high- throughput yeast two-hybrid assay technology provided by Myriad Genetics Inc. Briefly, the target protein e. C, pAS such that an in-frame fusion between the Ga14p and target protein sequences was created.

The resulting construct, the target gene construct, was introduced by transformation into a haploid yeast strain. A screening protocol was then used to search the individual baits against two activation domain libraries of assorted peptide motifs of greater than five million cDNA clones. The libraries were derived from RNA isolated from leaves, stems, and roots of rice plants grown in normal conditions, plus tissues from plants exposed to various stresses input trait library , and from various seed stages, callus, and early and late panicle output trait library.

To screen, a library of activation domain fusions i. The yeast strain that carried the activation domain constructs contained one or more Ga14p- responsive reporter genes, the expression of which can be monitored.

Yeast carrying the target gene construct was combined with yeast carrying the activation domain library. The two yeast strains mated to form diploid yeast and were plated on media that selected for expression of one or more Ga14p-responsive reporter genes. Thus, both hybrid proteins i. Colonies that arose after incubation were selected for further characterization. The activation domain plasmid was isolated from each colony obtained in the two-hybrid search.

The sequence of the insert in this construct was obtained by sequence analysis e. Thus, the identity of positives obtained from these searches was determined by sequence analysis against proprietary and public e. Interaction of the activation domain fusion with the target protein was confirmed by testing for the specificity of the interaction.

The activation domain construct was co-transformed into a yeast reporter strain with either the original target protein construct or a variety of other DNA-binding domain constructs.

Expression of the reporter genes in the presence of the target protein but not with other test proteins indicated that the interaction was genuine. The rice genome array contained mer oligonucleotide probes with sequences corresponding to the 3' ends of 21 , predicted open reading frames found in approximately 42, contigs that make up the rice genome map see Goff et al.

Sixteen different probes were used to measure the expression level of each nucleic acid. The sequences of the probes are available at http: The calculated expression value was determined based on the observed expression level minus the noise background associated with each probe. Experiments included evaluating the differential gene expression from various plant tissues comprising seed, root, leaf and stem, panicle, and pollen.

Gene expression was also measured in plants exposed to environmental cold i. Many of the cell proliferation-related proteins of the presently disclosed subject matter interact with one another. One aspect of the presently disclosed subject matter provides compositions and methods for modulating i.

In particular, the nucleic acid molecules and polypeptides of the presently disclosed subject matter are expressed constitutively, temporally, or spatially e. Therefore, the presently disclosed subject matter provides utility in such exemplary applications as altering the specified characteristics identified above. The isolated nucleic acid molecules of the presently disclosed subject matter are useful for expressing a polypeptide of the presently disclosed subject matter in a recombinantly engineered cell such as a bacterial, yeast, insect, mammalian, or plant cell.

Expressing cells can produce the polypeptide in a non-natural condition e. Those skilled in the art are knowledgeable in the numerous expression systems available for expression of nucleic acids encoding a polypeptide of the presently disclosed subject matter.

In another aspect, the presently disclosed subject matter features a cell proliferation-related polypeptide encoded by a nucleic acid molecule disclosed herein. In certain embodiments, the cell proliferation-related polypeptide is isolated. The presently disclosed subject matter further provides a method for modifying i. The method comprises introducing into a plant cell an expression cassette comprising a nucleic acid molecule of the presently disclosed subject matter as disclosed above to obtain a transformed plant cell or tissue, and culturing the transformed plant cell or tissue.

The nucleic acid molecule can be under the regulation of a constitutive or inducible promoter. A plant or plant part having modified expression of a nucleic acid molecule of the presently disclosed subject matter can be analyzed and selected using methods known to those skilled in the art including, but not limited to, Southern blotting, DNA sequencing, or PCR analysis using primers specific to the nucleic acid molecule and detecting amplicons produced therefrom.

Modulation of Expression of Nucleic Acid Molecules. The compositions ,of the presently disclosed subject matter include plant nucleic acid molecules, and the amino acid sequences of the polypeptides or partial-length polypeptides encoded by nucleic acid molecules comprising an open reading frame. These sequences can be employed to alter the expression of a particular gene corresponding to the open reading frame by decreasing or eliminating expression of that plant gene or by overexpressing a particular gene product.

Methods of this embodiment of the presently disclosed subject matter include stably transforming a plant with a nucleic acid molecule of the presently disclosed subject matter that includes an open reading frame operatively linked to a promoter capable of driving expression of that open reading frame sense or antisense in a plant cell.

By "portion" or "fragment", as it relates to a nucleic acid molecule that comprises an open reading frame or a fragment thereof encoding a partial-length polypeptide having the activity of the full length polypeptide, is meant a sequence having in one embodiment at least 80 nucleotides, in another embodiment at least nucleotides, and in still another embodiment at least nucleotides. If not employed for expression, a "portion" or "fragment" means in representative embodiments at least 9, or 12, or 15, or at least 20, consecutive nucleotides e.

Thus, to express a particular gene product, the method comprises introducing into a plant, plant cell, or plant tissue an expression cassette comprising a promoter operatively linked to an open reading frame so as to yield a transformed differentiated plant, transformed cell, or transformed tissue. Transformed cells or tissue can be regenerated to provide a transformed differentiated plant. The transformed differentiated plant or cells thereof can express the open reading frame in an amount that alters the amount of the gene product in the plant or cells thereof, which product is encoded by the open reading frame.

The presently disclosed subject matter also provides a transformed plant prepared by the methodsa disclosed herein, as well as progeny and seed thereof. The presently disclosed subject matter further includes a nucleotide sequence that is complementary to one hereinafter "test" sequence that hybridizes under stringent conditions to a nucleic acid molecule of the presently disclosed subject matter, as well as an RNA molecule that is transcribed from the nucleic acid molecule.

When hybridization is performed under stringent conditions, either the test or nucleic acid molecule of presently disclosed subject matter can be present on a support: Depending upon the degree of stringency required, such reduced concentration buffers are typically 1X SSC containing 0.

The plant cell is transformed with at least one such expression vector wherein the plant host cell can be used to regenerate plant tissue or an entire plant, or seed there from, in which the effects of expression, including overexpression and underexpression, of the introduced sequence or sequences can be measured in vitro or in planta. In some embodiments, the presently disclosed subject matter features an isolated polypeptide comprising or consisting of an amino acid sequence substantially similar to the amino acid sequence of an isolated cell proliferation-related polypeptide of the presently disclosed subject matter.

Because the proteins of the presently disclosed subject matter have a roll in cell proliferation, in certain embodiments, a cell introduced with a nucleic acid molecule of the presently disclosed subject matter has a different cell proliferation rate as compared to a cell not introduced with the nucleic acid molecule.

As discussed herein, all of the cell proliferation-related proteins described herein affect cell proliferation, either under normal conditions, under adverse conditions e.

Accordingly, by changing the amount of a cell proliferation-related protein of the presently disclosed subject matter in a plant cell, the proliferation of that plant cell can be modulated. In some situations, increasing expression of a cell proliferation-related protein of the presently disclosed subject matter in a cell will cause that cell to increase its rate of proliferation, either alone or in response to some stimulus e.

In other situations, increasing expression of a cell proliferation-related protein of the presently disclosed subject matter in a cell causes that cell to reduce its rate of proliferation. Similarly, decreasing the expression of a cell proliferation-related protein of the presently disclosed subject matter in a cell can increase or decrease that cell's rate of proliferation.

What is relevant is that the rate of proliferation of the cell changes if the level of expression of a cell proliferation-related For example, overexpression of the protein can be accomplished by transforming the cell with a nucleic acid molecule encoding the protein according to standard methods such as those described above. Reducing the level of expression of a cell proliferation-related protein of the presently disclosed subject matter in a cell is likewise simply 0 accomplished using standard methods.

Alteration of the expression of a nucleotide sequence of the presently 0 disclosed subject matter, in one embodiment reduction of its expression, is obtained by "sense" suppression referenced in e.

In this case, the entirety or a portion of a nucleotide sequence of the presently disclosed subject matter is comprised in a DNA molecule. The nucleotide sequence is inserted in the DNA molecule in the "sense orientation", meaning that the coding strand of the nucleotide sequence can be transcribed.

In one embodiment, the nucleotide sequence is fully 0 translatable and all the genetic information comprised in the nucleotide In another embodiment, the nucleotide sequence is partially translatable and a short peptide is translated.

In another embodiment, the nucleotide sequence is transcribed but no translation product is made. This is usually achieved by removing the start codon, i. In a further embodiment, the DNA molecule comprising the nucleotide sequence, or a portion thereof, is stably integrated 0 in the genome of the plant cell. In another embodiment, the DNA molecule comprising the nucleotide sequence, or a portion thereof, is comprised in an extrachromosomally replicating molecule.

The entirety or a portion of a nucleotide sequence of the presently disclosed subject matter is comprised in a DNA molecule. The DNA molecule can be operatively linked to a promoter functional in a plant cell, and introduced in a plant cell, in which the nucleotide sequence is expressible. The nucleotide 0 sequence is inserted in the DNA molecule in the "antisense orientation", meaning that the reverse complement also called sometimes non-coding strand of the nucleotide sequence can be transcribed.

In one embodiment, the DNA molecule comprising the nucleotide sequence, or a portion thereof, is stably integrated in the genome of the plant cell. Several publications describing this approach are cited for further illustration Green et al.

Homologous Recombination 0 In another embodiment, at least one genomic copy corresponding to a nucleotide sequence of the presently disclosed subject matter is modified in the genome of the plant by homologous recombination as further illustrated in Paszkowski et al. Homologous recombination can occur between the chromosomal copy of a nucleotide sequence in a cell and an incoming copy of the nucleotide sequence introduced in the cell by transformation.

Specific modifications are thus accurately introduced in the chromosomal 0 copy of the nucleotide sequence. In one embodiment, the regulatory elements of the nucleotide sequence of the presently disclosed subject matter are modified. Such regulatory elements are easily obtainable by screening a genomic library using the nucleotide sequence of the presently disclosed subject matter, or a portion thereof, as a probe.

The existing regulatory elements are replaced by different regulatory elements, thus altering expression of the nucleotide sequence, or they are mutated or deleted, thus abolishing the expression of the nucleotide sequence. In another embodiment, the nucleotide sequence is modified by deletion of a part of the nucleotide sequence or the entire nucleotide sequence, or by mutation. Expression of a mutated polypeptide in a plant cell is also provided in the presently disclosed subject matter. Recent refinements of this technique to disrupt endogenous plant genes have been disclosed Kempin et al.

In one embodiment, a mutation in the chromosomal copy of a nucleotide sequence is introduced by transforming a cell with a chimeric oligonucleotide composed of a contiguous stretch of RNA and DNA residues in a duplex conformation with double hairpin caps on the ends. An additional feature of the oligonucleotide is for example the presence of 2'-O- methylation at the RNA residues.

For example, this technique is further illustrated in U. The ribozyme is expressed in transgenic plants and results in reduced amounts of RNA coding for the polypeptide of the presently disclosed subject matter in plant cells, thus leading to reduced amounts of polypeptide accumulated in the cells. This method is further illustrated in U.

This is achieved by expression of dominant negative mutants of the polypeptides in transgenic plants, leading to the loss of activity of the endogenous polypeptide. The partitioned nucleic acids are amplified to yield a ligand-enriched mixture. After several iterations a nucleic 0 acid with optimal affinity to the polypeptide is obtained and is used for expression in transgenic plants. In alternative embodiments, transcription of the nucleotide sequence is reduced or increased.

Zinc finger polypeptides are disclosed in, for example, Beerli et al. In one embodiment, the alteration of the expression of a nucleotide sequence of the presently disclosed subject matter, in one embodiment the reduction of its expression, is obtained by dsRNA interference. The entirety, or in one embodiment a portion, of a nucleotide 0 sequence of the presently disclosed subject matter, can be comprised in a DNA molecule.

The size of the DNA molecule is in one embodiment from to nucleotides or more; the optimal size to be determined empirically. In one embodiment, the first copy of the DNA molecule is the reverse complement also known as the non-coding strand and the second copy is the coding strand; in another embodiment, the first copy is the coding strand, and the second copy is the reverse complement.

The size of the spacer DNA molecule is in one embodiment to 10, 0 nucleotides, in another embodiment to nucleotides, and in yet another embodiment to nucleotides in length. The two copies of the DNA molecule separated by the spacer are operatively linked to a promoter functional in a plant cell, and introduced in a plant cell in which the nucleotide sequence is expressible. In one embodiment, the DNA molecule comprising the nucleotide sequence, or a portion thereof, is stably integrated 0 in the genome of the plant cell.

Several publications describing this approach are cited for further illustration Waterhouse et al. As used herein, the terms "RNA interference" and "post-transcriptional gene silencing" are used interchangeably and refer to a 0 process of sequence-specific modulation of gene expression mediated by a small interfering RNA siRNA; see generally Fire et al.

Thus, because described herein are nucleotide sequences encoding the cell proliferation-related proteins of the presently disclosed subject matter, RNAi can be readily designed. Indeed, 6 constructs encoding an RNAi molecule have been developed which continuously synthesize an RNAi molecule, resulting in prolonged repression of expression of the targeted gene Brummelkamp et al. In transgenic plants containing one of the DNA molecules disclosed immediately above, the expression of the nucleotide sequence 0 corresponding to the nucleotide sequence comprised in the DNA molecule is in one embodiment reduced.

Methods of insertional mutagenesis using T-DNA, transposons, oligonucleotides, or other methods known to those skilled in the art are also encompassed. In yet another embodiment, a mutation of a nucleic acid molecule of the presently disclosed subject matter is created in the genomic copy of the sequence in the cell or plant by deletion of a portion of the nucleotide 6 sequence or regulator sequence. Methods of deletion mutagenesis are known to those skilled in the art. In yet another embodiment, a deletion is created at random in a large population of plants by chemical mutagenesis or irradiation and a plant with a deletion in a gene of the presently disclosed subject matter is isolated by 0 forward or reverse genetics.

Irradiation with fast neutrons or gamma rays is known to cause deletion mutations in plants Silverstone et al. Deletion mutations in a gene of the presently disclosed subject matter can be recovered in a reverse genetics strategy using PCR with pooled sets of genomic DNAs as has been 6 shown in C.

A forward genetics strategy involves mutagenesis of a line bearing a trait of interest followed by screening the M2 progeny for the absence of the trait. Among these mutants would be expected to be some that disrupt a gene of the presently disclosed subject matter. This could be assessed by Southern blotting or PCR using primers designed for a gene of the presently disclosed subject matter with genomic DNA from these mutants. The terms "initiation codon" and "termination codon" refer to a unit of three adjacent nucleotides 'codon' in a coding sequence that specifies initiation and chain termination, respectively, of protein synthesis mRNA translation.

The nucleotide sequences of the invention can be introduced into any plant. The genes to be introduced can be conveniently used in expression cassettes for introduction and expression in any plant of interest. Such expression cassettes will comprise the transcriptional initiation region of the invention linked to a nucleotide sequence of interest.

Such an expression cassette may be provided with a plurality of restriction sites for insertion of the gene of interest to be under the transcriptional regulation of the regulatory regions. The expression cassette may additionally contain selectable marker genes. The cassette will include in the 5'-3' direction of transcription, a transcriptional and translational initiation region, a DNA sequence of interest, and a transcriptional and translational termination region functional in plants.

The termination region may be native with the transcriptional initiation region, may be native with the DNA sequence of interest, or may be derived from another source. Convenient termination regions are available from the Ti- plasmid of A. See also, Guerineau et al. When the RNA transcript is a perfect complementary copy of the DNA sequence, it is referred to as the primary transcript or it may be a RNA sequence derived from posttranscriptional processing of the primary transcript and is referred to as the mature RNA.

Regulatory sequences include enhancers, promoters, translation leader sequences, introns, and polyadenylation signal sequences. They include natural and synthetic sequences as well as sequences which may be a combination of synthetic and natural sequences.

As is noted above, the term "suitable regulatory sequences" is not limited to promoters. It is present in the fully processed mRNA upstream of the initiation codon and may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency Turner et al.

The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor. The use of different 3' non-coding sequences is exemplified by Ingelbrecht et al. The term "translation leader sequence" refers to that DNA sequence portion of a gene between the promoter and coding sequence that is transcribed into RNA and is present in the fully processed mRNA upstream 5' of the translation start codon.

The translation leader sequence may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency. The term "intracellular localization sequence" refers to a nucleotide sequence that encodes an intracellular targeting signal.

An "intracellular targeting signal" is an amino acid sequence that is translated in conjunction with a protein and directs it to a particular sub- cellular compartment.

The terms "protein," "peptide" and "polypeptide" are used interchangeably herein. The term "mature" protein refers to a post-translationally processed polypeptide without its signal peptide. The term "signal sequence" refers to a nucleotide sequence that encodes the signal peptide.

This type of promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers.

Accordingly, an "enhancer" is a DNA sequence which can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue specificity of a promoter. It is capable of operating in both orientations normal or flipped , and is capable of functioning even when moved either upstream or downstream from the promoter. Both enhancers and other upstream promoter elements bind sequence-specific DNA-binding proteins that mediate their effects.

Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even be comprised of synthetic DNA segments.

A promoter may also contain DNA sequences that are involved in the binding of protein factors which control the effectiveness of transcription initiation in response to physiological or developmental conditions. With respect to this site all other sequences of the gene and its controlling regions are numbered.

Typically downstream sequences i. Promoter elements, particularly a TATA element, that are inactive or that have greatly reduced promoter activity in the absence of upstream activation are referred to as "minimal or core promoters. A "minimal or core promoter" thus consists only of all basal elements needed for transcription initiation, e. For example, a regulatory DNA sequence is said to be "operably linked to" or "associated with" a DNA sequence that codes for an RNA or a polypeptide if the two sequences are situated such that the regulatory DNA sequence affects expression of the coding DNA sequence i.

Coding sequences can be operably-linked to regulatory sequences in sense or antisense orientation. The term "transformation" refers to the transfer of a nucleic acid fragment into the genome of a host cell, resulting in genetically stable inheritance.

Host cells containing the transformed nucleic acid fragments are referred to as "transgenic" cells, and organisms comprising transgenic cells are referred to as "transgenic organisms". Examples of methods of transformation of plants and plant cells include Agrobacterium-mediated transformation De Blaere et al. Whole plants may be regenerated from transgenic cells by methods well known to the skilled artisan see, for example, Fromm et al.

For example, "transformed," "transformant," and "transgenic" plants or calli have been through the transformation process and contain a foreign gene integrated into their chromosome. The term "untransformed" refers to normal plants that have not been through the transformation process.

They may be derived by self-fertilization of primary or secondary transformants or crosses of primary or secondary transformants with other transformed or untransformed plants.

It includes natural and synthetic sequences as well as sequences which may be a combination of synthetic and natural sequences. Different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. New promoters of various types useful in plant cells are constantly being discovered, numerous examples may be found in the compilation by Okamuro et al.

Typical regulated promoters useful in plants include but are not limited to safener-inducible promoters, promoters derived from the tetracycline-inducible system, promoters derived from salicylate-inducible systems, promoters derived from alcohol-inducible systems, promoters derived from glucocorticoid- inducible system, promoters derived from pathogen-inducible systems, and promoters derived from ecdysome-inducible systems.

These also include promoters that are temporally regulated, such as in early or late embryogenesis, during fruit ripening in developing seeds or fruit, in fully differentiated leaf, or at the onset of senescence. ORF or portion thereof, or a transgene in plants.

For example, in the case of antisense constructs, expression may refer to the transcription of the antisense DNA only. Expression may also refer to the production of protein.

It is acknowledged that a true specificity exists: This phenomenon is known as leaky expression. However, with specific expression in this invention is meant preferable expression in one or a few plant tissues. The term "average expression" is used here as the average level of expression found in all lines that do express detectable amounts of reporter gene, so leaving out of the analysis plants that do not express any detectable reporter mRNA or protein. The "expression pattern" of a promoter with or without enhancer is the pattern of expression levels which shows where in the plant and in what developmental stage transcription is initiated by said promoter.

Expression patterns of a set of promoters are said to be complementary when the expression pattern of one promoter shows little overlap with the expression pattern of the other promoter.

The level of expression of a promoter can be determined by measuring the 'steady state' concentration of a standard transcribed reporter mRNA. This measurement is indirect since the concentration of the reporter mRNA is dependent not only on its synthesis rate, but also on the rate with which the mRNA is degraded. Therefore, the steady state level is the product of synthesis rates and degradation rates.

The rate of degradation can, however, be considered to proceed at a fixed rate when the transcribed sequences are identical, and thus this value can serve as a measure of synthesis rates.

This list of techniques in no way represents all available techniques, but rather describes commonly used procedures used to analyze transcription activity and expression levels of mRNA. A commonly used procedure to analyze expression patterns and levels is through determination of the 'steady state' level of protein accumulation in a cell by using a reporter gene.

In principle, however, many proteins are suitable for this purpose, provided the protein does not interfere with essential plant functions. Two principal methods for the control of expression are known, viz.: Overexpression can be achieved by insertion of one or more than one extra copy of the selected gene. It is, however, not unknown for plants or their progeny, originally transformed with one or more than one extra copy of a nucleotide sequence, to exhibit the effects of underexpression as well as overexpression.

For underexpression there are two principle methods which are commonly referred to in the art as "antisense downregulation" and "sense downregulation" sense downregulation is also referred to as "cosuppression". Generically these processes are referred to as "gene silencing".

Both of these methods lead to an inhibition of expression of the target gene. Obtaining sufficient levels of transgene expression in the appropriate plant tissues is an important aspect in the production of genetically engineered crops. Expression of heterologous DNA sequences in a plant host is dependent upon the presence of an operably linked promoter that is functional within the plant host.

Choice of the promoter sequence will determine when and where within the organism the heterologous DNA sequence is expressed. Furthermore, it is contemplated that promoters combining elements from more than one promoter may be useful. Thus, the elements from the promoters disclosed herein may be combined with elements from other promoters.

Promoters that are useful for plant transgene expression include those that are inducible, viral, synthetic, constitutive Odell et al.

Where expression in specific tissues or organs is desired, tissue-specific promoters may be used. In contrast, where gene expression in response to a stimulus is desired, inducible promoters are the regulatory elements of choice.

Where continuous expression is desired throughout the cells of a plant, constitutive promoters are utilized. The choice of promoter will vary depending on the temporal and spatial requirements for expression, and also depending on the target species. In some cases, expression in multiple tissues is desirable. While in others, tissue-specific, e. Although many promoters from dicotyledons have been shown to be operational in monocotyledons and vice versa, ideally dicotyledonous promoters are selected for expression in dicotyledons, and monocotyledonous promoters for expression in monocotyledons.

However, there is no restriction to the provenance of selected promoters; it is sufficient that they are operational in driving the expression of the nucleotide sequences in the desired cell.

These promoters include, but are not limited to, constitutive, inducible, temporally regulated, developmentally regulated, spatially-regulated, chemically regulated, stress- responsive, tissue-specific, viral and synthetic promoters. Promoter sequences are known to be strong or weak.

A strong promoter provides for a high level of gene expression, whereas a weak promoter provides for a very low level of gene expression. An inducible promoter is a promoter that provides for the turning on and off of gene expression in response to an exogenously added agent, or to an environmental or developmental stimulus.

A bacterial promoter such as the P tac promoter can be induced to varying levels of gene expression depending on the level of isothiopropylgalactoside added to the transformed bacterial cells. An isolated promoter sequence that is a strong promoter for heterologous nucleic acid is advantageous because it provides for a sufficient level of gene expression to allow for easy detection and selection of transformed cells and provides for a high level of gene expression when desired.

Within a plant promoter region there are several domains that are necessary for full function of the promoter. The first of these domains lies immediately upstream of the structural gene and forms the "core promoter region" containing consensus sequences, normally 70 base pairs immediately upstream of the gene. The core promoter region contains the characteristic CAAT and TATA boxes plus surrounding sequences, and represents a transcription initiation sequence that defines the transcription start point for the structural gene.

The presence of the core promoter region defines a sequence as being a promoter: Furthermore, the core promoter region is insufficient to provide full promoter activity. A series of regulatory sequences upstream of the core constitute the remainder of the promoter.

The regulatory sequences determine expression level, the spatial and temporal pattern of expression and, for an important subset of promoters, expression under inductive conditions regulation by external factors such as light, temperature, chemicals, hormones. A range of naturally-occurring promoters are known to be operative in plants and have been used to drive the expression of heterologous both foreign and endogenous genes in plants: The nucleotide sequences of this invention can also be expressed under the regulation of promoters that are chemically regulated.

This enables the nucleic acid sequence or encoded polypeptide to be synthesized only when the crop plants are treated with the inducing chemicals. A useful promoter for chemical induction is the tobacco PR- la promoter. Examples of some constitutive promoters which have been described include the rice actin 1 Wang et al. CaMV 19S Lawton et al. Examples of tissue specific promoters which have been described include the lectin Vodkin, ; Lindstrom et al.

Tissue specific enhancers are described in Fromm et al. Inducible promoters that have been described include the ABA- and turgor-inducible promoters, the promoter of the auxin-binding protein gene Schwob et al. These include genes encoding the seed storage proteins such as napin, cruciferin, beta-conglycinin, and phaseolin zein or oil body proteins such as oleosin , or genes involved in fatty acid biosynthesis including acyl carrier protein, stearoyl-ACP desaturase.

And fatty acid desaturases fad , and other genes expressed during embryo development such as Bce4, see, for example, EP and Kridl et al. Particularly useful for seed-specific expression is the pea vicilin promoter Czako et al. Other useful promoters for expression in mature leaves are those that are switched on at the onset of senescence, such as the SAG promoter from Arabidopsis Gan et al. A class of fruit-specific promoters expressed at or during antithesis through fruit development, at least until the beginning of ripening, is discussed in U.

The promoter for polygalacturonase gene is active in fruit ripening. The polygalacturonase gene is described in U. Other examples of tissue-specific promoters include those that direct expression in leaf cells following damage to the leaf for example, from chewing insects , in tubers for example, patatin gene promoter , and in fiber cells an example of a developmentally- regulated fiber cell protein is E6 John et al. The E6 gene is most active in fiber, although low levels of transcripts are found in leaf, ovule and flower.

The tissue-specificity of some "tissue-specific" promoters may not be absolute and may be tested by one skilled in the art using the diphtheria toxin sequence. One can also achieve tissue-specific expression with "leaky" expression by a combination of different tissue-specific promoters Beals et al.

Other tissue-specific promoters can be isolated by one skilled in the art see U. Several inducible promoters "gene switches" have been reported. Many are described in the review by Gatz and Gatz These include tetracycline repressor system, Lac repressor system, copper-inducible systems, salicylate-inducible systems such as the PRla system , glucocorticoid- Aoyama et al.

Also included are the benzene sulphonamide- U. Other studies have focused on genes inducibly regulated in response to environmental stress or stimuli such as increased salinity. Drought, pathogen and wounding. Accumulation of metallocarboxypeptidase-inhibitor protein has been reported in leaves of wounded potato plants Graham et al. Other plant genes have been reported to be induced methyl jasmonate, elicitors, heat-shock, anaerobic stress, or herbicide safeners.

Regulated expression of the chimeric transacting viral replication protein can be further regulated by other genetic strategies. For example, Cre-mediated gene activation as described by Odell et al. Thus, a DNA fragment containing 3' regulatory sequence bound by lox sites between the promoter and the replication protein coding sequence that blocks the expression of a chimeric replication gene from the promoter can be removed by Cre-mediated excision and result in the expression of the trans-acting replication gene.

In this case, the chimeric Cre gene, the chimeric trans-acting replication gene, or both can be under the control of tissue- and developmental- specific or inducible promoters.

An alternate genetic strategy is the use of tRNA suppressor gene. For example, the regulated expression of a tRNA suppressor gene can conditionally control expression of a trans-acting replication protein coding sequence containing an appropriate termination codon as described by Ulmasov et al. Again, either the chimeric tRNA suppressor gene, the chimeric transacting replication gene, or both can be under the control of tissue- and developmental- specific or inducible promoters.

Frequently it is desirable to have continuous or inducible expression of a DNA sequence throughout the cells of an organism in a tissue-independent manner. For example, increased resistance of a plant to infection by soil- and airborne-pathogens might be accomplished by genetic manipulation of the plant's genome to comprise a continuous promoter operably linked to a heterologous pathogen-resistance gene such that pathogen- resistance proteins are continuously expressed throughout the plant's tissues.

Alternatively, it might be desirable to inhibit expression of a native DNA sequence within a plant's tissues to achieve a desired phenotype. In this case, such inhibition might be accomplished with transformation of the plant to comprise a constitutive, tissue-independent promoter operably linked to an antisense nucleotide sequence, such that constitutive expression of the antisense sequence produces an RNA transcript that interferes with translation of the mRNA of the native DNA sequence.

To define a minimal promoter region, a DNA segment representing the promoter region is removed from the 5' region of the gene of interest and operably linked to the coding sequence of a marker reporter gene by recombinant DNA techniques well known to the art. The reporter gene is operably linked downstream of the promoter, so that transcripts initiating at the promoter proceed through the reporter gene. Reporter genes generally encode proteins which are easily measured, including, but not limited to, chloramphenicol acetyl transferase CAT , beta-glucuronidase GUS , green fluorescent protein GFP , beta-galactosidase beta- GAL , and luciferase.

The construct containing the reporter gene under the control of the promoter is then introduced into an appropriate cell type by transfection techniques well known to the art. To assay for the reporter protein, cell lysates are prepared and appropriate assays, which are well known in the art, for the reporter protein are performed. For example, if CAT were the reporter gene of choice, the lysates from cells transfected with constructs containing CAT under the control of a promoter under study are mixed with isotopically labeled chloramphenicol and acetyl-coenzyme A acetyl-CoA.

The CAT enzyme transfers the acetyl group from acetyl-CoA to the 2- or 3-position of chloramphenicol. The reaction is monitored by thin-layer chromatography, which separates acetylated chloramphenicol from unreacted material. The reaction products are then visualized by autoradiography. The level of enzyme activity corresponds to the amount of enzyme that was made, which in turn reveals the level of expression from the promoter of interest.

This level of expression can be compared to other promoters to determine the relative strength of the promoter under study. In order to be sure that the level of expression is determined by the promoter, rather than by the stability of the mRNA, the level of the reporter mRNA can be measured directly, such as by Northern blot analysis. These constructs are then introduced to cells and their activity determined. In addition to promoters, a variety of 5' and 3' transcriptional regulatory sequences are also available for use in the present invention.

Transcriptional terminators are responsible for the termination of transcription and correct mRNA polyadenylation. The 3' nontranslated regulatory DNA sequence may include from about 50 to about 1,, or about to about 1,, nucleotide base pairs and contains plant transcriptional and translational termination sequences. Appropriate transcriptional terminators and those which are known to function in plants include the CaMV 35S terminator, the tml terminator, the nopaline synthase terminator, the pea rbcS E9 terminator, the terminator for the T7 transcript from the octopine synthase gene of Agrobacterium tumefaciens, and the 3' end of the protease inhibitor I or II genes from potato or tomato, although other 3 ' elements known to those of skill in the art can also be employed.

Alternatively, one also could use a gamma coixin, oleosin 3 or other terminator from the genus Coix. Useful 3' elements include those from the nopaline synthase gene of Agrobacterium tumefaciens Bevan et al. Other sequences that have been found to enhance gene expression in transgenic plants include intron sequences e. For example, a number of non- translated leader sequences derived from viruses are known to enhance expression.

Other leaders known in the art include but are not limited to: See also, Della-Cioppa et al. As the DNA sequence between the transcription initiation site and the start of the coding sequence, i.

Preferred leader sequences are contemplated to include those that include sequences predicted to direct optimum expression of the attached gene, i. The choice of such sequences will be known to those of skill in the art in light of the present disclosure.

Sequences that are derived from genes that are highly expressed in plants will be most useful. Regulatory elements such as Adh intron 1 Callis et al. Examples of enhancers include elements from the CaMV 35S promoter, octopine synthase genes Ellis el al. Vectors for use in accordance with the present invention may be constructed to include the octopine synthase ocs enhancer element.

This element was first identified as a 16 bp palindromic enhancer from the ocs gene of ultilane Ellis et al. The use of an enhancer element, such as the ocs element and particularly multiple copies of the element, will act to increase the level of transcription from adjacent promoters when applied in the context of monocot transformation. Vectors for use in tissue-specific targeting of genes in transgenic plants will typically include tissue-specific promoters and may also include other tissue-specific control elements such as enhancer sequences.

Promoters which direct specific or enhanced expression in certain plant tissues will be known to those of skill in the art in light of the present disclosure. It is particularly contemplated that one may advantageously use the 16 bp ocs enhancer element from the octopine synthase ocs gene Ellis et al. Tissue specific expression may be functionally accomplished by introducing a constitutively expressed gene construct all tissues in combination with a transcription factor of the present invention that represses transcription of the introduced construct and that is expressed only in those tissues where the gene product is not desired.

Alternatively, tissue specific expression can be achieved by introducing a first construct expression of which is normally repressed in combination with a second construct containing a transcription factor of the present invention which induces expression of the first construct operably linked to a tissue specific promoter. Expression of some genes in transgenic plants will be desired only under specified conditions.

For example, it is proposed that expression of certain transcription factors of the present invention that confer resistance to environmental stress factors such as drought will be desired only under actual stress conditions. It is contemplated that expression of such genes throughout a plants development may have detrimental effects. It is known that a large number of genes exist that respond to the environment.

For example, expression of some genes such as rbcS, encoding the small subunit of ribulose bisphosphate carboxylase, is regulated by light as mediated through phytochrome. Other genes are induced by secondary stimuli. For example, synthesis of abscisic acid ABA is induced by certain environmental factors, including but not limited to water stress. It is also anticipated that expression of transcription factor genes conferring resistance to insect predation would be desired only under conditions of actual insect infestation.

Therefore, for some desired traits inducible expression of genes in transgenic plants will be desired. Expression of a gene in a transgenic plant may be desired only in a certain time period during the development of the plant. Developmental timing is frequently correlated with tissue specific gene expression. For example, expression of zein storage proteins is initiated in the endosperm about 15 days after pollination.

For quantification and determination of localization of expression within a plant, i. Detection systems can readily be created or are available which are based on, e. Protein levels can be determined in plant tissue extracts or in intact tissue using in situ analysis of protein expression. Generally, individual transformed lines with one chimeric promoter reporter construct will vary in their levels of expression of an included reporter gene.

Also frequently observed is the phenomenon that such transformants do not express any detectable product RNA or protein. The variability in expression is commonly ascribed to 'position effects', although the molecular mechanisms underlying this inactivity are usually not clear. In the context of the present invention, an "isolated" or "purified" DNA molecule or an "isolated" or "purified" polypeptide is a DNA molecule or polypeptide that, by the hand of man, exists apart from its native environment and is therefore not a product of nature.

An isolated DNA molecule or polypeptide may exist in a purified form or may exist in a non-native environment such as, for example, a transgenic host cell. For example, an "isolated" or "purified" nucleic acid molecule or protein, or biologically active portion thereof, is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.

An "isolated" nucleic acid is free of sequences preferably protein encoding sequences that naturally flank the nucleic acid i. For example, in various embodiments, the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0. The nucleotide sequences of the invention include both the naturally occurring sequences as well as mutant variant forms.

Such variants will continue to possess the desired activity, i. Thus, by "variants" is intended substantially similar sequences. For nucleotide sequences comprising an open reading frame, variants include those sequences that, because of the degeneracy of the genetic code, encode the identical amino acid sequence of the native protein.

Naturally occurring allelic variants such as these can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction PCR and hybridization techniques. Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis and for open reading frames, encode the native protein, as well as those that encode a polypeptide having amino acid substitutions relative to the native protein.

The following terms are used to describe the sequence relationships between two or more nucleic acids or polynucleotides: A reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full length cDNA or gene sequence, or the complete cDNA or gene sequence.

Generally, the comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50, , or longer. Those of skill in the art understand that to avoid a high similarity to a reference sequence due to inclusion of gaps in the polynucleotide sequence, a gap penalty is typically introduced and is subtracted from the number of matches.

Methods of alignment of sequences for comparison are well known in the art. Thus, the determination of percent identity between any two sequences can be accomplished using a mathematical algorithm. Preferred, non-limiting examples of such mathematical algorithms are the algorithm of Myers and Miller, ; the local homology algorithm of Smith et al. In one embodiment, optimal alignment is conducted using the homology alignment algorithm of Needleman and Wunsch An indication that two peptide sequences are substantially identical is that one peptide is immunologically reactive with antibodies raised against the second peptide.

Thus, a peptide is substantially identical to a second peptide, for example, where the two peptides differ only by a conservative substitution. Computer implementations of these mathematical algorithms can be utilized for comparison of sequences to determine sequence identity. Such implementations include, but are not limited to: Alignments using these programs can be performed using the default parameters. This algorithm involves first identifying high scoring sequence pairs HSPs by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence.

T is referred to as the neighborhood word score threshold Altschul et al. These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased.

For example, the invention provides a nucleic acid molecule comprising a plant nucleotide sequence comprising at least a portion of a key effector gene s responsible for host resistance to particular pathogens.

The overexpression may be constitutive, or it may be preferable to express the effector gene s in a tissue-specific manner or from an inducible promoter including a promoter which is responsive to external stimuli, such as chemical application, or to pathogen infection, e. A transformed transgenic plant of the invention includes plants, for example, a plant the cells of which have an expression cassette of the invention, i.

The nucleic acid molecules of the invention are thus useful for targeted gene disruption, as well as markers and probes. For example, the invention includes a pathogen, e. The normal fertile transformed transgenic plant may be selfed to yield a substantially homogenous line with respect to viral resistance or tolerance. Individuals of the line, or the progeny thereof, may be crossed with plants which optionally exhibit the trait.

In a particular embodiment of the method, the selfing and selection steps are repeated at least five times in order to obtain the homogenous isogenic line. Thus, the invention also provides transgenic plants and the products of the transgenic plants. When the hybridization is performed under stringent conditions, either the test or nucleic acid molecule of invention is preferably supported, e.

Thus, either a denatured test or nucleic acid molecule of the invention is preferably first bound to a support and hybridization is effected for a specified period of time at a temperature of, e. Depending upon the degree of stringency required such reduced concentration buffers are typically single strength SC containing 0.

The invention further provides a method to identify an open reading frame in the genome of a plant cell, the expression of which is altered by pathogen infection of that cell. The method comprises contacting a solid substrate comprising a plurality of samples comprising isolated plant nucleic acid of a probe comprising plant nucleic acid, e.

Each individual sample comprises one or more nucleic acid sequences e. The method may be employed with nucleic acid samples and probes from any organism, e. Preferably, the nucleic acid sample and probes are from a plant, such as a dicot or monocot. More preferably the nucleic acid samples and probes are from a cereal plant. Even more preferably the nucleic acids and probes are from a crop plant.

A second plurality of samples on a solid substrate, i. Then complex formation between the samples and probes comprising nucleic acid from infected or control cells compared. For example, potato virus X, tobacco mosaic virus, tobravirus, cucumber mosaic virus and gemnivirus are known to infect Arabidopsis. Thus, Arabidopsis genes, the expression of which is altered in response to infection by any of these viruses, can be identified.

The invention further provides a method for identifying a plant cell infected with a pathogen. The method comprises contacting nucleic acid obtained from a plant cell suspected of being infected with a pathogen with oligonucleotides corresponding to a portion of a plurality of sequences selected from SEQ ID NOs: Then the presence of the amplified product is detected or determined.

The presence of two or more amplified products, e. The method comprises contacting a protein sample obtained from a plant cell suspected of being infected with a pathogen with an agent that specifically binds a polypeptide encoded by an open reading frame comprising SEQ ID NOs: Then the presence or amount of complex formation is detected or determined.

The invention provides an additional method for identifying a plant cell infected with a pathogen. The amount of the probe hybridized to nucleic acid obtained from a cell suspected of being infected with a virus is compared to hybridization of the probe to nucleic acid isolated from an uninfected cell. A change in the amount of at least two probes that hybridize to nucleic acid isolated from a cell suspected of being infected by a virus relative to hybridization of at least two probes to nucleic acid isolated from an uninfected cell is indicative of viral infection.

A method to shuffle the nucleic acids of the invention is provided. This method allows for the production of polypeptides having altered activity relative to the native form of the polypeptide. Accordingly, the invention provides cells and transgenic plants containing nucleic acid segments produced through shuffling that encode polypeptides having altered activity relative to the corresponding native polypeptide.

A computer readable medium, e. This medium also allows for computer-based manipulation of a nucleic acid sequence corresponding to a nucleic acid sequence listed in SEQ ID NOs: The invention also provides a method for marker-assisted breeding to select for plants having altered resistance to a pathogen. The amount or presence of the duplex is indicative of the presence of a gene, the expression of which alters the resistance of the plant to a pathogen. Therefore, another embodiment of the present invention provides a method of using known inducers or inhibitors of genes identified as being important in plant-pathogen interactions to induce genes that are important in resistance, or to inhibit genes that are downregulated in resistance.

Thus, some of the isolated nucleic acid molecules of the invention are useful in a method of combating a pathogen in an agricultural crop. The method comprises introducing to a plant an expression cassette comprising a nucleic acid molecule of the invention so as to yield a transformed differentiated plant.

The transformed differentiated plant expresses the nucleic acid molecule in an amount that confers resistance to the transformed plant to infection relative to a corresponding nontransformed plant. For example, gene refers to a nucleic acid fragment that expresses mRNA or functional RNA, or encodes a specific protein, and which includes regulatory sequences.

Genes also include nonexpressed DNA segments that, for example, form recognition sequences for other proteins. Genes can be obtained from a variety of sources, including cloning from a source of interest or synthesizing from known or predicted sequence information, and may include sequences designed to have desired parameters.

Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or comprise regulatory sequences and coding sequences derived from the same source, but arranged in a manner different from that found in nature. Transgenes may include, for example, genes that are either heterologous or homologous to the genes of a particular plant to be transformed.

Additionally, transgenes may comprise native genes inserted into a non-native organism, or chimeric genes. Generally specific primers are upwards of 14 nucleotides in length.

For optimum specificity and cost effectiveness, primers of 16 to 24 nucleotides in length may be preferred. Those skilled in the art are well versed in the design of primers for use processes such as PCR. If required, probing can be done with entire restriction fragments of the gene disclosed herein which may be 's or even 's of nucleotides in length.

The nucleotide sequences of the invention can be introduced into any plant. The genes to be introduced can be conveniently used in expression cassettes for introduction and expression in any plant of interest. Such expression cassettes will comprise the transcriptional initiation region of the invention linked to a nucleotide sequence of interest.

Such an expression cassette is provided with a plurality of restriction sites for insertion of the gene of interest to be under the transcriptional regulation of the regulatory regions.

The expression cassette may additionally contain selectable marker genes. The termination region may be native with the transcriptional initiation region, may be native with the DNA sequence of interest, or may be derived from another source. Convenient termination regions are available from the Ti-plasmid of A. See also, Guerineau et al. When the RNA transcript is a perfect complementary copy of the DNA sequence, it is referred to as the primary transcript or it may be a RNA sequence derived from posttranscriptional processing of the primary transcript and is referred to as the mature RNA.

Regulatory sequences include enhancers, promoters, translation leader sequences, introns, and polyadenylation signal sequences. They include natural and synthetic sequences as well as sequences which may be a combination of synthetic and natural sequences. It is present in the fully processed mRNA upstream of the initiation codon and may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency Turner et al.

The translation leader sequence may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency. Bacterial pathogens include but are not limited to Pseudomonas avenae subsp. Fungal pathogens include but are not limited to Collelotrichum graminicola, Glomerella graminicola Politis, Glomerella lucumanensis, Aspergillus flavus, Rhizoctonia solani Kuhn, Thanatephorus cucumeris, Acremonium strictum W. Gams, Cephalosporium acremonium Auct.

Cochliobolus lunatus , Curvularia pallescens teleomorph— Cochliobolus pallescens , Curvularia senegalensis, C. Setosphaeriaprolata , Graphium penicillioides, Leptosphaeria maydis, Leptothyrium zeae, Ophiosphaerella herpotricha anamorph— Scolecosporiella sp. Waitea circinata , Rhizoctonia solani , minor A Iternaria alternala, Cercospora sorghi , Dictochaetaftrtilis, Fusarium acuminatum teleomorph Gihherella acuminata , E.

Nectria haematococca , F. Mycosphaerella zeae - maydis , and Gloeocercospora sorghi. Parasitic nematodes include but are not limited to Awl Dolichodorus spp. This type of promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. It is capable of operating in both orientations normal or flipped , and is capable of functioning even when moved either upstream or downstream from the promoter.

Both enhancers and other upstream promoter elements bind sequence-specific DNA-binding proteins that mediate their effects. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even be comprised of synthetic DNA segments.

A promoter may also contain DNA sequences that are involved in the binding of protein factors which control the effectiveness of transcription initiation in response to physiological or developmental conditions. With respect to this site all other sequences of the gene and its controlling regions are numbered.

It includes natural and synthetic sequences as well as sequences which may be a combination of synthetic and natural sequences.

Different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. New promoters of various types useful in plant cells are constantly being discovered, numerous examples may be found in the compilation by Okamuro et al.

Typical regulated promoters useful in plants include but are not limited to safener-inducible promoters, promoters derived from the tetracycline-inducible system, promoters derived from salicylate-inducible systems, promoters derived from alcohol-inducible systems, promoters derived from glucocorticoid-inducible system, promoters derived from pathogen-inducible systems, and promoters derived from ecdysome-inducible systems.

These also include promoters that are temporally regulated, such as in early or late embryogenesis, during fruit ripening in developing seeds or fruit, in fully differentiated leaf, or at the onset of senescence. Coding sequences can be operably-linked to regulatory sequences in sense or antisense orientation. For example, in the case of antisense constructs, expression may refer to the transcription of the antisense DNA only.

Expression may also refer to the production of protein. It is acknowledged that hardly a true specificity exists: This phenomenon is known as leaky expression. However, with specific expression in this invention is meant preferable expression in one or a few plant tissues. Expression patterns of a set of promoters are said to be complementary when the expression pattern of one promoter shows little overlap with the expression pattern of the other promoter. This measurement is indirect since the concentration of the reporter mRNA is dependent not only on its synthesis rate, but also on the rate with which the mRNA is degraded.

Therefore, the steady state level is the product of synthesis rates and degradation rates. The rate of degradation can however be considered to proceed at a fixed rate when the transcribed sequences are identical, and thus this value can serve as a measure of synthesis rates.

This list of techniques in no way represents all available techniques, but rather describes commonly used procedures used to analyze transcription activity and expression levels of mRNA. The analysis of transcription start points in practically all promoters has revealed that there is usually no single base at which transcription starts, but rather a more or less clustered set of initiation sites, each of which accounts for some start points of the mRNA.

Since this distribution varies from promoter to promoter the sequences of the reporter mRNA in each of the populations would differ from each other. Since each mRNA species is more or less prone to degradation, no single degradation rate can be expected for different reporter mRNAs.

This includes also part of the transcribed sequences. The direct fusion of promoter to reporter sequences would therefore lead to suboptimal levels of transcription. In principle, however, many more proteins are suitable for this purpose, provided the protein does not interfere with essential plant functions.

For quantification and determination of localization a number of tools are suited. Detection systems can readily be created or are available which are based on, e.

Protein levels can be determined in plant tissue extracts or in intact tissue using in situ analysis of protein expression. Generally, individual transformed lines with one chimeric promoter reporter construct will vary in their levels of expression of the reporter gene. Also frequently observed is the phenomenon that such transformants do not express any detectable product RNA or protein.

Likewise, leaf, and stem expression levels, are determined using whole extracts from leaves and stems. It is acknowledged however, that within each of the plant parts just described, cells with variable functions may exist, in which promoter activity may vary. Gene silencing may be transcriptional, when the suppression is due to decreased transcription of the affected genes, or post-transcriptional, when the suppression is due to increased turnover degradation of RNA species homologous to the affected genes English et al.

Gene silencing includes virus-induced gene silencing Ruiz et al. Silencing suppressor genes may be of plant, non-plant, or viral origin. Thus, a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified through, for example, the use of DNA shuffling. The terms also include non-naturally occurring multiple copies of a naturally occurring DNA sequence. Thus, the terms refer to a DNA segment that is foreign or heterologous to the cell, or homologous to the cell but in a position within the host cell nucleic acid in which the element is not ordinarily found.

Exogenous DNA segments are expressed to yield exogenous polypeptides. For example, altered nucleotide sequences which simply reflect the degeneracy of the genetic code but nonetheless encode amino acid sequences that are identical to a particular amino acid sequence are substantially similar to the particular sequences.

Modifications that result in equivalent nucleotide or amino acid sequences are well within the routine skill in the art. The target gene is not essential for replicon replication. Additionally, target genes may comprise native non-viral genes inserted into a non-native organism, or chimeric genes, and will be under the control of suitable regulatory sequences.

Thus, the regulatory sequences in the target gene may come from any source, including the virus. Target genes may include coding sequences that are either heterologous or homologous to the genes of a particular plant to be transformed. However, target genes do not include native viral genes. Typical target genes include, but are not limited to genes encoding a structural protein, a seed storage protein, a protein that conveys herbicide resistance, and a protein that conveys insect resistance.

The expression of a target gene in a plant will typically produce an altered plant trait. In addition to the ORF of the replication protein, the replication gene may also contain other overlapping or non-overlapping ORF s , as are found in viral sequences in nature.

Such chimeric genes also include insertion of the known sites of replication protein binding between the promoter and the transcription start site that attenuate transcription of viral replication protein gene.

It excludes young, growing tissue consisting of germline, meristematic, and not-fully-differentiated cells. Examples of methods of transformation of plants and plant cells include Agrobacterium -mediated transformation De Blaere et al.

Whole plants may be regenerated from transgenic cells by methods well known to the skilled artisan see, for example, Fromm et al. The nucleic acid molecule can be stably integrated into the genome generally known in the art and are disclosed in Sambrook et al.

See also Innis et al. Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially mismatched primers, and the like. They may be derived by self-fertilization of primary or secondary transformants or crosses of primary or secondary transformants with other transformed or untransformed plants.

Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides.

Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof e.

In higher plants, deoxyribonucleic acid DNA is the genetic material while ribonucleic acid RNA is involved in the transfer of information contained within DNA into proteins. The invention encompasses isolated or substantially purified nucleic acid or protein compositions. An isolated DNA molecule or polypeptide may exist in a purified form or may exist in a non-native environment such as, for example, a transgenic host cell.

For example, in various embodiments, the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0. The nucleotide sequences of the invention include both the naturally occurring sequences as well as mutant variant forms.

Such variants will continue to possess the desired activity, i. For nucleotide sequences comprising an open reading frame, variants include those sequences that, because of the degeneracy of the genetic code, encode the identical amino acid sequence of the native protein. Naturally occurring allelic variants such as these can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction PCR and hybridization techniques.

Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis and for open reading frames, encode the native protein, as well as those that encode a polypeptide having amino acid substitutions relative to the native protein.

Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given polypeptide. Thus, at every position where an arginine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded protein.

One of skill will recognize that each codon in a nucleic acid except ATG, which is ordinarily the only codon for methionine can be modified to yield a functionally identical molecule by standard techniques. In this manner, the open reading frames in genes or gene fragments can be synthesized utilizing plant-preferred codons. See, for example, Campbell and Gowri, for a discussion of host-preferred codon usage. Thus, the nucleotide sequences can be optimized for expression in any plant.

It is recognized that all or any part of the gene sequence may be optimized or synthetic. That is, synthetic or partially optimized sequences may also be used. Variant nucleotide sequences and proteins also encompass sequences and protein derived from a mutagenic and recombinogenic procedure such as DNA shuffling. With such a procedure, one or more different coding sequences can be manipulated to create a new polypeptide possessing the desired properties.

In this manner, libraries of recombinant polynucleotides are generated from a population of related sequence polynucleotides comprising sequence regions that have substantial sequence identity and can be homologously recombined in vitro or in vivo. Strategies for such DNA shuffling are known in the art. See, for example, Stemmer, ; Stemmer, ; Crameri et al. Such variants may result from, for example, genetic polymorphism or from human manipulation. Methods for such manipulations are generally known in the art.

Thus, the polypeptides may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. For example, amino acid sequence variants of the polypeptides can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art.

See, for example, Kunkel, ; Kunkel et al. Guidance as to appropriate amino acid substitutions that do not affect biological activity of the protein of interest may be found in the model of Dayhoff et al.

Conservative substitutions, such as exchanging one amino acid with another having similar properties, are preferred. Conservative substitution tables providing functionally similar amino acids are well known in the art. The following five groups each contain amino acids that are conservative substitutions for one another: Methionine M , Cysteine C ; Basic: See also, Creighton, It also typically comprises sequences required for proper translation of the nucleotide sequence.

The coding region usually codes for a protein of interest but may also code for a functional RNA of interest, for example antisense RNA or a nontranslated RNA, in the sense or antisense direction. The expression cassette comprising the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components.

The expression cassette may also be one which is naturally occurring but has been obtained in a recombinant form useful for heterologous expression. The expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or of an inducible promoter which initiates transcription only when the host cell is exposed to some particular external stimulus. In the case of a multicellular organism, the promoter can also be specific to a particular tissue or organ or stage of development.

Specifically included are shuttle vectors by which is meant a DNA vehicle capable, naturally or by design, of replication in two different host organisms, which may be selected from actinomycetes and related species, bacteria and eukaryotic e.

Preferably the nucleic acid in the vector is under the control of, and operably linked to, an appropriate promoter or other regulatory elements for transcription in a host cell such as a microbial, e. The vector may be a bi-functional expression vector which functions in multiple hosts. In the case of genomic DNA, this may contain its own promoter or other regulatory elements and in the case of cDNA this may be under the control of an appropriate promoter or other regulatory elements for expression in the host cell.

Marker genes typically include genes that provide tetracycline resistance, hygromycin resistance or ampicillin resistance. The plant tissue may be in plants or in organ, tissue or cell culture. The following terms are used to describe the sequence relationships between two or more nucleic acids or polynucleotides: A reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full length cDNA or gene sequence, or the complete cDNA or gene sequence.

Generally, the comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50, , or longer. Those of skill in the art understand that to avoid a high similarity to a reference sequence due to inclusion of gaps in the polynucleotide sequence a gap penalty is typically introduced and is subtracted from the number of matches. Methods of alignment of sequences for comparison are well known in the art.

Thus, the determination of percent identity between any two sequences can be accomplished using a mathematical algorithm. Preferred, non-limiting examples of such mathematical algorithms are the algorithm of Myers and Miller, ; the local homology algorithm of Smith et al. Computer implementations of these mathematical algorithms can be utilized for comparison of sequences to determine sequence identity. Such implementations include, but are not limited to: Alignments using these programs can be performed using the default parameters.

This algorithm involves first identifying high scoring sequence pairs HSPs by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence.

T is referred to as the neighborhood word score threshold Altschul et al. These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. For amino acid sequences, a scoring matrix is used to calculate the cumulative score.

Extension of the word hits in each direction are halted when the cumulative alignment score falls off by the quantity X from its maximum achieved value, the cumulative score goes to zero or below due to the accumulation of one or more negative-scoring residue alignments, or the end of either sequence is reached.

In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences see, e. One measure of similarity provided by the BLAST algorithm is the smallest sum probability P N , which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.

For example, a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid sequence to the reference nucleic acid sequence is less than about 0. See Altschul et al. Alignment may also be performed manually by inspection. For purposes of the present invention, comparison of nucleotide sequences for determination of percent sequence identity to the promoter sequences disclosed herein is preferably made using the BlastN program version 1.

When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties e. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution.

Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity.

Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.

The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by to yield the percentage of sequence identity.

One of skill in the art will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like. Another indication that nucleotide sequences are substantially identical is if two molecules hybridize to each other under stringent conditions see below.

Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides they encode are substantially identical. This may occur, e.

One indication that two nucleic acid sequences are substantially identical is when the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the polypeptide encoded by the second nucleic acid. Preferably, optimal alignment is conducted using the homology alignment algorithm of Needleman and Wunsch An indication that two peptide sequences are substantially identical is that one peptide is immunologically reactive with antibodies raised against the second peptide.

Thus, a peptide is substantially identical to a second peptide, for example, where the two peptides differ only by a conservative substitution. For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence s relative to the reference sequence, based on the designated program parameters.

As noted above, another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions. Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution.

An extensive guide to the hybridization of nucleic acids is found in Tijssen, An example of highly stringent wash conditions is 0. An example of stringent wash conditions is a 0. Often, a high stringency wash is preceded by a low stringency wash to remove background probe signal. An example medium stringency wash for a duplex of, e. An example low stringency wash for a duplex of, e.

will come across many

Thus, the transgenic plants according to the invention can be used for the breeding of improved plant lines that for example increase the effectiveness of conventional methods such as herbicide or pesticide treatment, or allow to dispense with said methods, due to their modified genetic properties.

Another aspect of the invention provides a method of altering gene expression that in turn may alter the phenotype of a plant by expression of a transcription factor or combination of transcription factors of the present invention. In this aspect, a recombinant expression cassette comprising a promoter operably linked to a polynucleotide encoding a transcription factor of the present invention, or a functional fragment thereof, is introduced into a plant using any method known in the art.

A variety of promoters can be used including those described herein, for example, constitutive, inducible, tissue specific, or developmentally regulated promoters. In this method, the expressed transcription factor binds to a gene in the plant resulting in altered, e. This change in gene expression can ultimately lead to a desirable change in the phenotype of the plant.

The gene upon which the transcription factor acts, can be an endogenous gene or an introduced gene or transgene. In one embodiment, transcription factors directed to particular pathways are used, for example, to provide resistance or tolerance of a stress in a plant.

The overexpression may be constitutive, or it may be desirable to express the desired gene s , i. Another aspect of the invention provides plants wherein the cells, genome, or components there of have been augmented with the polynucleotides of the present invention. In an alternative embodiment, the plant is transformed with a construct, the expression of which produces a product that interacts with the transcription factor encoded by any one of SEQ ED NO.: A further aspect provides a polynucleotide of at least 5, at least 10, at least 15, at least 25, at least 50 or at least nucleotides in length that is complementary to one of SEQ ED NO.: Thus, either a denatured test or nucleic acid molecule of the invention is first bound to a support and hybridization is effected for a specified period of time at a temperature of, e.

Depending upon the degree of stringency required, such reduced concentration buffers are typically single strength SC containing 0. Such polynucleotides can be used to identify orthologs of the transcription factors of the present invention especially when such polynucleotides are used in expression analysis to identify nucleotide sequences which share sequence homology and expression profiles similar to the transcription factors of the present invention.

A method to shuffle the nucleic acids of the invention is also provided. This method allows for the production of polypeptides having altered activity relative to the native form of the polypeptide. Accordingly, the invention provides cells and transgenic plants containing nucleic acid segments, produced through shuffling, that encode polypeptides having altered activity relative to the corresponding native polypeptide.

A computer readable medium containing the nucleic acid sequences of the invention as well as methods of use for the computer readable medium are provided. The invention further provides a method for marker-assisted breeding to select for plants having advantages associated with expression of particular transcription factors disclosed herein. The method involves contacting plant DNA or cDNA with a probe corresponding to a nucleic acid sequence in the sequence listing, or the orthologs thereof, and the corresponding genes, or a portion thereof which hybridizes under moderate stringency conditions to a gene corresponding to one of SEQ ED NO.: The amount or presence of the duplex is indicative of the presence or expression of particular transcription factors.

Such host cells can be bacterial cells, animal cells, yeast cells or plant cells. The following detailed description is provided to aid those skilled in the art in practicing the present invention.

Even so, this detailed description should not be construed to unduly limit the present invention as modifications and variations in the embodiments discussed herein can be made by those of ordinary skill in the art without departing from the spirit or scope of the present inventive discovery.

All publications, patents, patent applications, public databases, public database entries, and other references cited in this application are herein incorporated by reference in their entirety as if each individual publication, patent, patent application, public database, public database entries, or other reference were specifically and individually indicated to be incorporated by reference.

The term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, composed of monomers nucleotides containing a sugar, phosphate and a base that is either a purine or pyrimidine.

Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof e.

A "nucleic acid fragment" is a fraction of a given nucleic acid molecule. In higher plants, deoxyribonucleic acid DNA is the genetic material while ribonucleic acid RNA is involved in the transfer of information contained within DNA into proteins. The term "gene" is used broadly to refer to any segment of nucleic acid associated with a biological function.

For example, gene refers to a nucleic acid fragment that expresses mRNA or functional RNA, or encodes a specific protein, and which includes regulatory sequences. Genes also include nonexpressed DNA segments that, for example, form recognition sequences for other proteins. Genes can be obtained from a variety of sources, including cloning from a source of interest or synthesizing from known or predicted sequence information, and may include sequences designed to have desired parameters.

The term "native" or "wild type" gene refers to a gene that is present in the genome of an untransformed cell. A "marker gene" encodes a selectable or screenable trait.

The term "chimeric gene" refers to any gene that contains 1 DNA sequences, including regulatory and coding sequences, that are not found together in nature, or 2 sequences encoding parts of proteins not naturally adjoined, or. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or comprise regulatory sequences and coding sequences derived from the same source, but arranged in a manner different from that found in nature.

A "transgene" refers to a gene that has been introduced into the genome by transformation and is stably maintained. Transgenes may include, for example, genes that are either heterologous or homologous to the genes of a particular plant to be transformed. Additionally, transgenes may comprise native genes inserted into a non-native organism, or chimeric genes.

The term "endogenous gene" refers to a native gene in its natural location in the genome of an organism. A "foreign" gene refers to a gene not normally found in the host organism but that is introduced by gene transfer. Examples include the 3' non- regulatory regions of genes encoding nopaline synthase and the small subunit of ribulose bisphosphate carboxylase.

Such chimeric genes also include insertion of the known sites of replication protein binding between the promoter and the transcription start site that attenuates transcription of viral replication protein genes. Gene silencing may be transcriptional, when the suppression is due to decreased transcription of the affected genes, or post-transcriptional, when the suppression is due to increased turnover degradation of RNA species homologous to the affected genes English et al.

Gene silencing includes virus-induced gene silencing Ruiz et al. Silencing suppressor genes may be of plant, non-plant, or viral origin. In addition to the ORF of the replication protein, the replication gene may also contain other overlapping or non-overlapping ORF s , as are found in viral sequences in nature. Where genes are not "chromosomally integrated" they may be "transiently expressed.

The terms "heterologous DNA sequence," "exogenous DNA segment" or "heterologous nucleic acid," as used herein, each refer to a sequence that originates from a source foreign to the particular host cell or, if from the same source, is modified from its original form. Thus, a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified through, for example, the use of DNA shuffling.

The terms also include non-naturally occurring multiple copies of a naturally occurring DNA sequence. Thus, the terms refer to a DNA segment that is foreign or heterologous to the cell, or homologous to the cell but in a position within the host cell nucleic acid in which the element is not ordinarily found. Exogenous DNA segments are expressed to yield exogenous polypeptides. A "homologous" DNA sequence is a DNA sequence that is naturally associated with a host cell into which it is introduced.

Estimates of such homo logy are provided by either. For example, altered nucleotide sequences which simply reflect the degeneracy of the genetic code but nonetheless encode amino acid sequences that are identical to a particular amino acid sequence are substantially similar to the particular sequences.

Modifications that result in equivalent nucleotide or amino acid sequences are well within the routine skill in the art. An "oligonucleotide" corresponding to a nucleotide sequence of the invention, e. Generally specific primers are upwards of 14 nucleotides in length. For optimum specificity and cost effectiveness, primers of 16 to 24 nucleotides in length are useful. Those skilled in the art are well versed in the design of primers for use processes such as PCR. If required, probing can be done with entire restriction fragments of the gene disclosed herein which may be 's or even 's of nucleotides in length.

It may constitute an "uninterrupted coding sequence", i. An "intron" is a sequence of RNA which is contained in the primary transcript but which is removed through cleavage and re-ligation of the RNA within the cell to create the mature mRNA that can be translated into a protein.

The terms "open reading frame" and "ORF" refer to the amino acid sequence encoded between translation initiation and termination codons of a coding sequence.

The terms "initiation codon" and "termination codon" refer to a unit of three adjacent nucleotides 'codon' in a coding sequence that specifies initiation and chain termination, respectively, of protein synthesis mRNA translation. The nucleotide sequences of the invention can be introduced into any plant. The genes to be introduced can be conveniently used in expression cassettes for introduction and expression in any plant of interest.

Such expression cassettes will comprise the transcriptional initiation region of the invention linked to a nucleotide sequence of interest. Such an expression cassette may be provided with a plurality of restriction sites for insertion of the gene of interest to be under the transcriptional regulation of the regulatory regions. The expression cassette may additionally contain selectable marker genes. The cassette will include in the 5'-3' direction of transcription, a transcriptional and translational initiation region, a DNA sequence of interest, and a transcriptional and translational termination region functional in plants.

The termination region may be native with the transcriptional initiation region, may be native with the DNA sequence of interest, or may be derived from another source. Convenient termination regions are available from the Ti- plasmid of A. See also, Guerineau et al. When the RNA transcript is a perfect complementary copy of the DNA sequence, it is referred to as the primary transcript or it may be a RNA sequence derived from posttranscriptional processing of the primary transcript and is referred to as the mature RNA.

Regulatory sequences include enhancers, promoters, translation leader sequences, introns, and polyadenylation signal sequences. They include natural and synthetic sequences as well as sequences which may be a combination of synthetic and natural sequences. As is noted above, the term "suitable regulatory sequences" is not limited to promoters. It is present in the fully processed mRNA upstream of the initiation codon and may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency Turner et al.

The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor. The use of different 3' non-coding sequences is exemplified by Ingelbrecht et al.

The term "translation leader sequence" refers to that DNA sequence portion of a gene between the promoter and coding sequence that is transcribed into RNA and is present in the fully processed mRNA upstream 5' of the translation start codon. The translation leader sequence may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency. The term "intracellular localization sequence" refers to a nucleotide sequence that encodes an intracellular targeting signal.

An "intracellular targeting signal" is an amino acid sequence that is translated in conjunction with a protein and directs it to a particular sub- cellular compartment. The terms "protein," "peptide" and "polypeptide" are used interchangeably herein. The term "mature" protein refers to a post-translationally processed polypeptide without its signal peptide. The term "signal sequence" refers to a nucleotide sequence that encodes the signal peptide.

This type of promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. Accordingly, an "enhancer" is a DNA sequence which can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue specificity of a promoter.

It is capable of operating in both orientations normal or flipped , and is capable of functioning even when moved either upstream or downstream from the promoter. Both enhancers and other upstream promoter elements bind sequence-specific DNA-binding proteins that mediate their effects. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even be comprised of synthetic DNA segments.

A promoter may also contain DNA sequences that are involved in the binding of protein factors which control the effectiveness of transcription initiation in response to physiological or developmental conditions. With respect to this site all other sequences of the gene and its controlling regions are numbered.

Typically downstream sequences i. Promoter elements, particularly a TATA element, that are inactive or that have greatly reduced promoter activity in the absence of upstream activation are referred to as "minimal or core promoters.

A "minimal or core promoter" thus consists only of all basal elements needed for transcription initiation, e. For example, a regulatory DNA sequence is said to be "operably linked to" or "associated with" a DNA sequence that codes for an RNA or a polypeptide if the two sequences are situated such that the regulatory DNA sequence affects expression of the coding DNA sequence i. Coding sequences can be operably-linked to regulatory sequences in sense or antisense orientation.

The term "transformation" refers to the transfer of a nucleic acid fragment into the genome of a host cell, resulting in genetically stable inheritance.

Host cells containing the transformed nucleic acid fragments are referred to as "transgenic" cells, and organisms comprising transgenic cells are referred to as "transgenic organisms". Examples of methods of transformation of plants and plant cells include Agrobacterium-mediated transformation De Blaere et al. Whole plants may be regenerated from transgenic cells by methods well known to the skilled artisan see, for example, Fromm et al.

For example, "transformed," "transformant," and "transgenic" plants or calli have been through the transformation process and contain a foreign gene integrated into their chromosome. The term "untransformed" refers to normal plants that have not been through the transformation process. They may be derived by self-fertilization of primary or secondary transformants or crosses of primary or secondary transformants with other transformed or untransformed plants. It includes natural and synthetic sequences as well as sequences which may be a combination of synthetic and natural sequences.

Different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. New promoters of various types useful in plant cells are constantly being discovered, numerous examples may be found in the compilation by Okamuro et al.

Typical regulated promoters useful in plants include but are not limited to safener-inducible promoters, promoters derived from the tetracycline-inducible system, promoters derived from salicylate-inducible systems, promoters derived from alcohol-inducible systems, promoters derived from glucocorticoid- inducible system, promoters derived from pathogen-inducible systems, and promoters derived from ecdysome-inducible systems.

These also include promoters that are temporally regulated, such as in early or late embryogenesis, during fruit ripening in developing seeds or fruit, in fully differentiated leaf, or at the onset of senescence. ORF or portion thereof, or a transgene in plants. For example, in the case of antisense constructs, expression may refer to the transcription of the antisense DNA only.

Expression may also refer to the production of protein. It is acknowledged that a true specificity exists: This phenomenon is known as leaky expression. However, with specific expression in this invention is meant preferable expression in one or a few plant tissues.

The term "average expression" is used here as the average level of expression found in all lines that do express detectable amounts of reporter gene, so leaving out of the analysis plants that do not express any detectable reporter mRNA or protein. The "expression pattern" of a promoter with or without enhancer is the pattern of expression levels which shows where in the plant and in what developmental stage transcription is initiated by said promoter.

Expression patterns of a set of promoters are said to be complementary when the expression pattern of one promoter shows little overlap with the expression pattern of the other promoter. The level of expression of a promoter can be determined by measuring the 'steady state' concentration of a standard transcribed reporter mRNA. This measurement is indirect since the concentration of the reporter mRNA is dependent not only on its synthesis rate, but also on the rate with which the mRNA is degraded.

Therefore, the steady state level is the product of synthesis rates and degradation rates. The rate of degradation can, however, be considered to proceed at a fixed rate when the transcribed sequences are identical, and thus this value can serve as a measure of synthesis rates. This list of techniques in no way represents all available techniques, but rather describes commonly used procedures used to analyze transcription activity and expression levels of mRNA.

A commonly used procedure to analyze expression patterns and levels is through determination of the 'steady state' level of protein accumulation in a cell by using a reporter gene. In principle, however, many proteins are suitable for this purpose, provided the protein does not interfere with essential plant functions. Two principal methods for the control of expression are known, viz.: Overexpression can be achieved by insertion of one or more than one extra copy of the selected gene.

It is, however, not unknown for plants or their progeny, originally transformed with one or more than one extra copy of a nucleotide sequence, to exhibit the effects of underexpression as well as overexpression. For underexpression there are two principle methods which are commonly referred to in the art as "antisense downregulation" and "sense downregulation" sense downregulation is also referred to as "cosuppression".

Generically these processes are referred to as "gene silencing". Both of these methods lead to an inhibition of expression of the target gene.

Obtaining sufficient levels of transgene expression in the appropriate plant tissues is an important aspect in the production of genetically engineered crops. Expression of heterologous DNA sequences in a plant host is dependent upon the presence of an operably linked promoter that is functional within the plant host.

Choice of the promoter sequence will determine when and where within the organism the heterologous DNA sequence is expressed.

Furthermore, it is contemplated that promoters combining elements from more than one promoter may be useful. Thus, the elements from the promoters disclosed herein may be combined with elements from other promoters.

Promoters that are useful for plant transgene expression include those that are inducible, viral, synthetic, constitutive Odell et al. Where expression in specific tissues or organs is desired, tissue-specific promoters may be used. In contrast, where gene expression in response to a stimulus is desired, inducible promoters are the regulatory elements of choice. Where continuous expression is desired throughout the cells of a plant, constitutive promoters are utilized.

The choice of promoter will vary depending on the temporal and spatial requirements for expression, and also depending on the target species. In some cases, expression in multiple tissues is desirable. While in others, tissue-specific, e. Although many promoters from dicotyledons have been shown to be operational in monocotyledons and vice versa, ideally dicotyledonous promoters are selected for expression in dicotyledons, and monocotyledonous promoters for expression in monocotyledons.

However, there is no restriction to the provenance of selected promoters; it is sufficient that they are operational in driving the expression of the nucleotide sequences in the desired cell. These promoters include, but are not limited to, constitutive, inducible, temporally regulated, developmentally regulated, spatially-regulated, chemically regulated, stress- responsive, tissue-specific, viral and synthetic promoters.

Promoter sequences are known to be strong or weak. A strong promoter provides for a high level of gene expression, whereas a weak promoter provides for a very low level of gene expression. An inducible promoter is a promoter that provides for the turning on and off of gene expression in response to an exogenously added agent, or to an environmental or developmental stimulus.

A bacterial promoter such as the P tac promoter can be induced to varying levels of gene expression depending on the level of isothiopropylgalactoside added to the transformed bacterial cells. An isolated promoter sequence that is a strong promoter for heterologous nucleic acid is advantageous because it provides for a sufficient level of gene expression to allow for easy detection and selection of transformed cells and provides for a high level of gene expression when desired.

Within a plant promoter region there are several domains that are necessary for full function of the promoter. The first of these domains lies immediately upstream of the structural gene and forms the "core promoter region" containing consensus sequences, normally 70 base pairs immediately upstream of the gene. The core promoter region contains the characteristic CAAT and TATA boxes plus surrounding sequences, and represents a transcription initiation sequence that defines the transcription start point for the structural gene.

The presence of the core promoter region defines a sequence as being a promoter: Furthermore, the core promoter region is insufficient to provide full promoter activity. A series of regulatory sequences upstream of the core constitute the remainder of the promoter. The regulatory sequences determine expression level, the spatial and temporal pattern of expression and, for an important subset of promoters, expression under inductive conditions regulation by external factors such as light, temperature, chemicals, hormones.

A range of naturally-occurring promoters are known to be operative in plants and have been used to drive the expression of heterologous both foreign and endogenous genes in plants: The nucleotide sequences of this invention can also be expressed under the regulation of promoters that are chemically regulated. This enables the nucleic acid sequence or encoded polypeptide to be synthesized only when the crop plants are treated with the inducing chemicals.

A useful promoter for chemical induction is the tobacco PR- la promoter. Examples of some constitutive promoters which have been described include the rice actin 1 Wang et al. CaMV 19S Lawton et al. Examples of tissue specific promoters which have been described include the lectin Vodkin, ; Lindstrom et al.

Tissue specific enhancers are described in Fromm et al. Inducible promoters that have been described include the ABA- and turgor-inducible promoters, the promoter of the auxin-binding protein gene Schwob et al.

These include genes encoding the seed storage proteins such as napin, cruciferin, beta-conglycinin, and phaseolin zein or oil body proteins such as oleosin , or genes involved in fatty acid biosynthesis including acyl carrier protein, stearoyl-ACP desaturase.

And fatty acid desaturases fad , and other genes expressed during embryo development such as Bce4, see, for example, EP and Kridl et al. Particularly useful for seed-specific expression is the pea vicilin promoter Czako et al. Other useful promoters for expression in mature leaves are those that are switched on at the onset of senescence, such as the SAG promoter from Arabidopsis Gan et al.

A class of fruit-specific promoters expressed at or during antithesis through fruit development, at least until the beginning of ripening, is discussed in U. The promoter for polygalacturonase gene is active in fruit ripening. The polygalacturonase gene is described in U. Other examples of tissue-specific promoters include those that direct expression in leaf cells following damage to the leaf for example, from chewing insects , in tubers for example, patatin gene promoter , and in fiber cells an example of a developmentally- regulated fiber cell protein is E6 John et al.

The E6 gene is most active in fiber, although low levels of transcripts are found in leaf, ovule and flower. The tissue-specificity of some "tissue-specific" promoters may not be absolute and may be tested by one skilled in the art using the diphtheria toxin sequence.

One can also achieve tissue-specific expression with "leaky" expression by a combination of different tissue-specific promoters Beals et al. Other tissue-specific promoters can be isolated by one skilled in the art see U. Several inducible promoters "gene switches" have been reported. Many are described in the review by Gatz and Gatz These include tetracycline repressor system, Lac repressor system, copper-inducible systems, salicylate-inducible systems such as the PRla system , glucocorticoid- Aoyama et al.

Also included are the benzene sulphonamide- U. Other studies have focused on genes inducibly regulated in response to environmental stress or stimuli such as increased salinity. Drought, pathogen and wounding. Accumulation of metallocarboxypeptidase-inhibitor protein has been reported in leaves of wounded potato plants Graham et al. Other plant genes have been reported to be induced methyl jasmonate, elicitors, heat-shock, anaerobic stress, or herbicide safeners.

Regulated expression of the chimeric transacting viral replication protein can be further regulated by other genetic strategies. For example, Cre-mediated gene activation as described by Odell et al. Thus, a DNA fragment containing 3' regulatory sequence bound by lox sites between the promoter and the replication protein coding sequence that blocks the expression of a chimeric replication gene from the promoter can be removed by Cre-mediated excision and result in the expression of the trans-acting replication gene.

In this case, the chimeric Cre gene, the chimeric trans-acting replication gene, or both can be under the control of tissue- and developmental- specific or inducible promoters. An alternate genetic strategy is the use of tRNA suppressor gene. For example, the regulated expression of a tRNA suppressor gene can conditionally control expression of a trans-acting replication protein coding sequence containing an appropriate termination codon as described by Ulmasov et al.

Again, either the chimeric tRNA suppressor gene, the chimeric transacting replication gene, or both can be under the control of tissue- and developmental- specific or inducible promoters. Frequently it is desirable to have continuous or inducible expression of a DNA sequence throughout the cells of an organism in a tissue-independent manner.

For example, increased resistance of a plant to infection by soil- and airborne-pathogens might be accomplished by genetic manipulation of the plant's genome to comprise a continuous promoter operably linked to a heterologous pathogen-resistance gene such that pathogen- resistance proteins are continuously expressed throughout the plant's tissues.

Alternatively, it might be desirable to inhibit expression of a native DNA sequence within a plant's tissues to achieve a desired phenotype. In this case, such inhibition might be accomplished with transformation of the plant to comprise a constitutive, tissue-independent promoter operably linked to an antisense nucleotide sequence, such that constitutive expression of the antisense sequence produces an RNA transcript that interferes with translation of the mRNA of the native DNA sequence.

To define a minimal promoter region, a DNA segment representing the promoter region is removed from the 5' region of the gene of interest and operably linked to the coding sequence of a marker reporter gene by recombinant DNA techniques well known to the art.

The reporter gene is operably linked downstream of the promoter, so that transcripts initiating at the promoter proceed through the reporter gene. Reporter genes generally encode proteins which are easily measured, including, but not limited to, chloramphenicol acetyl transferase CAT , beta-glucuronidase GUS , green fluorescent protein GFP , beta-galactosidase beta- GAL , and luciferase.

The construct containing the reporter gene under the control of the promoter is then introduced into an appropriate cell type by transfection techniques well known to the art. To assay for the reporter protein, cell lysates are prepared and appropriate assays, which are well known in the art, for the reporter protein are performed.

For example, if CAT were the reporter gene of choice, the lysates from cells transfected with constructs containing CAT under the control of a promoter under study are mixed with isotopically labeled chloramphenicol and acetyl-coenzyme A acetyl-CoA. The CAT enzyme transfers the acetyl group from acetyl-CoA to the 2- or 3-position of chloramphenicol. The reaction is monitored by thin-layer chromatography, which separates acetylated chloramphenicol from unreacted material.

The reaction products are then visualized by autoradiography. The level of enzyme activity corresponds to the amount of enzyme that was made, which in turn reveals the level of expression from the promoter of interest. This level of expression can be compared to other promoters to determine the relative strength of the promoter under study.

In order to be sure that the level of expression is determined by the promoter, rather than by the stability of the mRNA, the level of the reporter mRNA can be measured directly, such as by Northern blot analysis.

These constructs are then introduced to cells and their activity determined. In addition to promoters, a variety of 5' and 3' transcriptional regulatory sequences are also available for use in the present invention.

Transcriptional terminators are responsible for the termination of transcription and correct mRNA polyadenylation. The 3' nontranslated regulatory DNA sequence may include from about 50 to about 1,, or about to about 1,, nucleotide base pairs and contains plant transcriptional and translational termination sequences.

Appropriate transcriptional terminators and those which are known to function in plants include the CaMV 35S terminator, the tml terminator, the nopaline synthase terminator, the pea rbcS E9 terminator, the terminator for the T7 transcript from the octopine synthase gene of Agrobacterium tumefaciens, and the 3' end of the protease inhibitor I or II genes from potato or tomato, although other 3 ' elements known to those of skill in the art can also be employed.

Alternatively, one also could use a gamma coixin, oleosin 3 or other terminator from the genus Coix. Useful 3' elements include those from the nopaline synthase gene of Agrobacterium tumefaciens Bevan et al. Other sequences that have been found to enhance gene expression in transgenic plants include intron sequences e. For example, a number of non- translated leader sequences derived from viruses are known to enhance expression. Other leaders known in the art include but are not limited to: See also, Della-Cioppa et al.

As the DNA sequence between the transcription initiation site and the start of the coding sequence, i. Preferred leader sequences are contemplated to include those that include sequences predicted to direct optimum expression of the attached gene, i. The choice of such sequences will be known to those of skill in the art in light of the present disclosure.

Sequences that are derived from genes that are highly expressed in plants will be most useful. Regulatory elements such as Adh intron 1 Callis et al. Examples of enhancers include elements from the CaMV 35S promoter, octopine synthase genes Ellis el al. Vectors for use in accordance with the present invention may be constructed to include the octopine synthase ocs enhancer element. This element was first identified as a 16 bp palindromic enhancer from the ocs gene of ultilane Ellis et al.

The use of an enhancer element, such as the ocs element and particularly multiple copies of the element, will act to increase the level of transcription from adjacent promoters when applied in the context of monocot transformation. Vectors for use in tissue-specific targeting of genes in transgenic plants will typically include tissue-specific promoters and may also include other tissue-specific control elements such as enhancer sequences.

Promoters which direct specific or enhanced expression in certain plant tissues will be known to those of skill in the art in light of the present disclosure. It is particularly contemplated that one may advantageously use the 16 bp ocs enhancer element from the octopine synthase ocs gene Ellis et al. Tissue specific expression may be functionally accomplished by introducing a constitutively expressed gene construct all tissues in combination with a transcription factor of the present invention that represses transcription of the introduced construct and that is expressed only in those tissues where the gene product is not desired.

Alternatively, tissue specific expression can be achieved by introducing a first construct expression of which is normally repressed in combination with a second construct containing a transcription factor of the present invention which induces expression of the first construct operably linked to a tissue specific promoter.

Expression of some genes in transgenic plants will be desired only under specified conditions. For example, it is proposed that expression of certain transcription factors of the present invention that confer resistance to environmental stress factors such as drought will be desired only under actual stress conditions. It is contemplated that expression of such genes throughout a plants development may have detrimental effects. It is known that a large number of genes exist that respond to the environment.

For example, expression of some genes such as rbcS, encoding the small subunit of ribulose bisphosphate carboxylase, is regulated by light as mediated through phytochrome. Other genes are induced by secondary stimuli. For example, synthesis of abscisic acid ABA is induced by certain environmental factors, including but not limited to water stress. It is also anticipated that expression of transcription factor genes conferring resistance to insect predation would be desired only under conditions of actual insect infestation.

Therefore, for some desired traits inducible expression of genes in transgenic plants will be desired. Expression of a gene in a transgenic plant may be desired only in a certain time period during the development of the plant.

Developmental timing is frequently correlated with tissue specific gene expression. For example, expression of zein storage proteins is initiated in the endosperm about 15 days after pollination. For quantification and determination of localization of expression within a plant, i.

Detection systems can readily be created or are available which are based on, e. Protein levels can be determined in plant tissue extracts or in intact tissue using in situ analysis of protein expression.

Generally, individual transformed lines with one chimeric promoter reporter construct will vary in their levels of expression of an included reporter gene. Also frequently observed is the phenomenon that such transformants do not express any detectable product RNA or protein.

The variability in expression is commonly ascribed to 'position effects', although the molecular mechanisms underlying this inactivity are usually not clear. In the context of the present invention, an "isolated" or "purified" DNA molecule or an "isolated" or "purified" polypeptide is a DNA molecule or polypeptide that, by the hand of man, exists apart from its native environment and is therefore not a product of nature. An isolated DNA molecule or polypeptide may exist in a purified form or may exist in a non-native environment such as, for example, a transgenic host cell.

For example, an "isolated" or "purified" nucleic acid molecule or protein, or biologically active portion thereof, is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.

An "isolated" nucleic acid is free of sequences preferably protein encoding sequences that naturally flank the nucleic acid i. For example, in various embodiments, the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0. The nucleotide sequences of the invention include both the naturally occurring sequences as well as mutant variant forms.

Such variants will continue to possess the desired activity, i. Thus, by "variants" is intended substantially similar sequences. For nucleotide sequences comprising an open reading frame, variants include those sequences that, because of the degeneracy of the genetic code, encode the identical amino acid sequence of the native protein. Naturally occurring allelic variants such as these can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction PCR and hybridization techniques.

Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis and for open reading frames, encode the native protein, as well as those that encode a polypeptide having amino acid substitutions relative to the native protein.

The following terms are used to describe the sequence relationships between two or more nucleic acids or polynucleotides: A reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full length cDNA or gene sequence, or the complete cDNA or gene sequence. Generally, the comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50, , or longer.

Those of skill in the art understand that to avoid a high similarity to a reference sequence due to inclusion of gaps in the polynucleotide sequence, a gap penalty is typically introduced and is subtracted from the number of matches. Methods of alignment of sequences for comparison are well known in the art. Thus, the determination of percent identity between any two sequences can be accomplished using a mathematical algorithm.

Preferred, non-limiting examples of such mathematical algorithms are the algorithm of Myers and Miller, ; the local homology algorithm of Smith et al. In one embodiment, optimal alignment is conducted using the homology alignment algorithm of Needleman and Wunsch An indication that two peptide sequences are substantially identical is that one peptide is immunologically reactive with antibodies raised against the second peptide. Thus, a peptide is substantially identical to a second peptide, for example, where the two peptides differ only by a conservative substitution.

Computer implementations of these mathematical algorithms can be utilized for comparison of sequences to determine sequence identity. Such implementations include, but are not limited to: Alignments using these programs can be performed using the default parameters.

This algorithm involves first identifying high scoring sequence pairs HSPs by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence.

T is referred to as the neighborhood word score threshold Altschul et al. These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them.

The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when the cumulative alignment score falls off by the quantity X from its maximum achieved value, the cumulative score goes to zero or below due to the accumulation of one or more negative-scoring residue alignments, or the end of either sequence is reached.

In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences see, e. One measure of similarity provided by the BLAST algorithm is the smallest sum probability P N , which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.

For example, a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid sequence to the reference nucleic acid sequence is less than about 0. See Altschul et al. Alignment may also be performed manually by inspection.

For purposes of the present invention, comparison of nucleotide sequences for determination of percent sequence identity to the promoter sequences disclosed herein is preferably made using the BlastN program version 1.

By "equivalent program" is intended any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by the preferred program. For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared.

When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence s relative to the reference sequence, based on the designated program parameters.

When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties e.

When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity.

Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by to yield the percentage of sequence identity.

One of skill in the art will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like.

Another indication that nucleotide sequences are substantially identical is if two molecules hybridize to each other under moderate or stringent conditions see below. The phrase "hybridize to" or "specifically hybridizing to" refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under moderate to stringent conditions, with respect to specificity, when that sequence is present in a complex mixture e.

Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. An extensive guide to the hybridization of nucleic acids is found in Tijssen, An example of highly stringent wash conditions is 0.

An example of stringent wash conditions is a 0. Often, a high stringency wash is preceded by a low stringency wash to remove background probe signal. An example medium stringency wash for a duplex of, e. An example low stringency wash for a duplex of, e. For short probes e. Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.

In general, a signal to noise ratio of 2X or higher than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization. Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the proteins that they encode are substantially identical.

Very stringent conditions are selected to be equal to the T m for a particular probe. Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides they encode are substantially identical. This may occur, e. One indication that two nucleic acid sequences are substantially identical is when the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the polypeptide encoded by the second nucleic acid.

Such variants may result from, for example, genetic polymorphism or from human manipulation. Methods for such manipulations are generally known in the art. Thus, the polypeptides may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. For example, amino acid sequence variants of the polypeptides can be prepared by mutations in the DNA.

Methods for mutagenesis and nucleotide sequence alterations are well known in the art. Although "protein" is often used in reference to relatively large polypeptides, and "peptide" is often used in reference to small polypeptides, usage of these terms in the art overlaps and varies. The term "polypeptide" as used herein refers to peptides, polypeptides and proteins, unless otherwise noted. As used herein, the terms "protein", "polypeptide" and "peptide" are used interchangeably herein when referring to a gene product.

The term "polypeptide" encompasses proteins of all functions, including enzymes. Thus, exemplary polypeptides include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments, and other equivalents, variants and analogs of the foregoing. The terms "polypeptide fragment" or "fragment", when used in reference to a reference polypeptide, refers to a polypeptide in which amino acid residues are deleted as compared to the reference polypeptide itself, but where the remaining amino acid sequence is usually identical to the corresponding positions in the reference polypeptide.

Such deletions can occur at the amino-terminus or carboxy-terminus of the reference polypeptide, or alternatively both. Fragments typically are at least 5, 6, 8 or 10 amino acids long, at least 14 amino acids long, at least 20, 30, 40 or 50 amino acids long, at least 75 amino acids long, or at least , , , , or more amino acids long. A fragment can retain one or more of the biological activities of the reference polypeptide.

In certain embodiments, a fragment can comprise a domain or feature, and optionally additional amino acids on one or both sides of the domain or feature, which additional amino acids can number from 5, 10, 15, 20, 30, 40, 50, or up to or more residues. Further, fragments can include a sub-fragment of a specific region, which sub-fragment retains a function of the region from which it is derived.

In another embodiment, a fragment can have immunogenic properties. As used herein, the term "pre-polypeptide" refers to a polypeptide that is normally targeted to a cellular organelle, such as a chloroplast, and still comprises a transit peptide.

As used herein, the term "primer" refers to a sequence comprising in one embodiment two or more deoxyribonucleotides or ribonucleotides, in another embodiment more than three, in another embodiment more than eight, and in yet another embodiment at least about 20 nucleotides of an exonic or intronic region.

Such oligonucleotides are in one embodiment between ten and thirty bases in length. The term "promoter" or "promoter region" each refers to a nucleotide sequence within a gene that is positioned 5' to a coding sequence and functions to direct transcription of the coding sequence. The promoter region comprises a transcriptional start site, and can additionally include one or more transcriptional regulatory elements.

A "minimal promoter" is a nucleotide sequence that has the minimal elements required to enable basal level transcription to occur. As such, minimal promoters are not complete promoters but rather are subsequences of promoters that are capable of directing a basal level of transcription of a reporter construct in an experimental system.

Minimal promoters include but are not limited to the CMV minimal promoter, the HSV-tk minimal promoter, the simian virus 40 SV40 minimal promoter, the human b-actin minimal promoter, the human EF2 minimal promoter, the adenovirus E1 B minimal promoter, and the heat shock protein hsp 70 minimal promoter. Minimal promoters are often augmented with one or more transcriptional regulatory elements to influence the transcription of an operatively linked gene. For example, cell-type-specific or tissue-specific transcriptional regulatory elements can be added to minimal promoters to create recombinant promoters that direct transcription of an operatively linked nucleotide sequence in a cell-type-specific or tissue-specific manner.

Different promoters have different combinations of transcriptional regulatory elements. Whether or not a gene is expressed in a cell is dependent on a combination of the particular transcriptional regulatory elements that make up the gene's promoter and the different transcription factors that are present within the nucleus of the cell.

As such, promoters are often classified as "constitutive", "tissue-specific", "cell-type-specific", or "inducible", depending on their functional activities in vivo or in vitro. For example, a constitutive promoter is one that is capable of directing transcription of a gene in a variety of cell types. Exemplary constitutive promoters include the promoters for the following genes which encode certain constitutive or "housekeeping" functions: Exemplary tissue-specific promoters include those promoters described in more detail hereinbelow, as well as other tissue- specific and cell-type specific promoters known to those of skill in the art.

When used in the context of a promoter, the term "linked" as used herein refers to a physical proximity of promoter elements such that they function together to direct transcription of an operatively linked nucleotide sequence. The term "transcriptional regulatory sequence" or "transcriptional regulatory element", as used herein, each refers to a nucleotide sequence within the promoter region that enables responsiveness to a regulatory transcription factor.

Responsiveness can encompass a decrease or an increase in transcriptional output and is mediated by binding of the transcription factor to the DNA molecule comprising the transcriptional regulatory element. In one embodiment, a transcriptional regulatory sequence is a transcription termination sequence, alternatively referred to herein as a transcription termination signal.

The term "transcription factor" generally refers to a protein that modulates gene expression by interaction with the transcriptional regulatory element and cellular components for transcription, including RNA. Polymerase, Transcription Associated Factors TAFs , chromatin-remodeling proteins, and any other relevant protein that impacts gene transcription.

As used herein, "significance" or "significant" relates to a statistical analysis of the probability that there is a non-random association between two or more entities. To determine whether or not a relationship is "significant" or has "significance", statistical manipulations of the data can be performed to calculate a probability, expressed as a "p-value".

Those p- values that fall below a user-defined cutoff point are regarded as significant. In one example, a p-value less than or equal to 0. The term "purified" refers to an object species that is the predominant species present i.

A "purified fraction" is a composition wherein the object species comprises at least about 50 percent on a molar basis of all species present. In making the determination of the purity of a species in solution or dispersion, the solvent or matrix in which the species is dissolved or dispersed is usually not included in such determination; instead, only the species including the one of interest dissolved or dispersed are taken into account.

The object species can be purified to essential homogeneity contaminant species cannot be detected in the composition by conventional detection methods wherein the composition consists essentially of a single species. A skilled artisan can purify a polypeptide of the presently disclosed subject matter using standard techniques for protein purification in light of the teachings herein.

Purity of a polypeptide can be determined by a number of methods known to those of skill in the art, including for example, amino-terminal amino acid sequence analysis, gel electrophoresis, and mass-spectrometry analysis. A "reference sequence" is a defined sequence used as a basis for a sequence comparison.

A reference sequence can be a subset of a larger sequence, for example, as a segment of a full-length nucleotide or amino acid sequence, or can comprise a complete sequence. Generally, when used to refer to a nucleotide sequence, a reference sequence is at least , or nucleotides in length, frequently at least nucleotides in length, and often at least nucleotides in length.

Because two proteins can each 1 comprise a sequence i. The term "regulatory sequence" is a generic term used throughout the specification to refer to polynucleotide sequences, such as initiation signals, enhancers, regulators, promoters, and termination sequences, which are necessary or desirable to affect the expression of coding and non-coding sequences to which they are operatively linked.

Exemplary regulatory sequences are described in Goeddel, , and include, for example, the early and late promoters of simian virus 40 SV40 , adenovirus or cytomegalovirus immediate early promoter, the lac system, the trp system, the TAC or TRC system, T7 promoter whose expression is directed by T7 RNA polymerase, the major operator and promoter regions of phage lambda, the control regions for fd coat protein, the promoter for 3- phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e.

The nature and use of such control sequences can differ depending upon the host organism. In prokaryotes, such regulatory sequences generally include promoter, ribosomal binding site, and transcription termination sequences.

The term "regulatory sequence" is intended to include, at a minimum, components whose presence can influence expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.

In certain embodiments, transcription of a polynucleotide sequence is under the control of a promoter sequence or other regulatory sequence that controls the expression of the polynucleotide in a cell-type in which expression is intended. It will also be understood that the polynucleotide can be under the control of regulatory sequences that are the same or different from those sequences which control expression of the naturally occurring form of the polynucleotide.

The term "reporter gene" refers to a nucleic acid comprising a nucleotide sequence encoding a protein that is readily detectable either by its presence or activity, including, but not limited to, luciferase, fluorescent protein e. Generally, a reporter gene encodes a polypeptide not otherwise produced by the host cell, which is detectable by analysis of the cell s , e. In certain instances, a reporter gene encodes an enzyme, which produces a change in fluorometric properties of the host cell, which is detectable by qualitative, quantitative, or semiquantitative function or transcriptional activation.

Exemplary enzymes include esterases,? As used herein, the term "sequencing" refers to determining the ordered linear sequence of nucleic acids or amino acids of a DNA or protein target sample, using conventional manual or automated laboratory techniques. As used herein, the term "substantially pure" refers to that the polynucleotide or polypeptide is substantially free of the sequences and molecules with which it is associated in its natural state, and those molecules used in the isolation procedure.

As used herein, the term "target cell" refers to a cell, into which it is desired to insert a nucleic acid sequence or polypeptide, or to otherwise effect a modification from conditions known to be standard in the unmodified cell. A nucleic acid sequence introduced into a target cell can be of variable length. Additionally, a nucleic acid sequence can enter a target cell as a component of a plasmid or other vector or as a naked sequence. As used herein, the term "transcription" refers to a cellular process involving the interaction of an RNA polymerase with a gene that directs the expression as RNA of the structural information present in the coding sequences of the gene.

The process includes, but is not limited to, the following steps: As used herein, the term "transcription factor" refers to a cytoplasmic or nuclear protein which binds to a gene, or binds to an RNA transcript of a gene, or binds to another protein which binds to a gene or an RNA transcript or another protein which in turn binds to a gene or an RNA transcript, so as to thereby modulate expression of the gene.

Such modulation can additionally be achieved by other mechanisms; the essence of a "transcription factor for a gene" pertains to a factor that alters the level of transcription of the gene in some way. The term "transfection" refers to the introduction of a nucleic acid, e.

The term "transformation" refers to a process in which a cell's genotype is changed as a result of the cellular uptake of exogenous nucleic acid. For example, a transformed cell can express a recombinant form of a polypeptide of the presently disclosed subject matter or antisense expression can occur from the transferred gene so that the expression of a naturally occurring form of the gene is disrupted. The term "vector" refers to a nucleic acid capable of transporting another nucleic acid to which it has been linked.

One type of vector that can be used in accord with the presently disclosed subject matter is an episome, i. Other vectors include those capable of autonomous replication and expression of nucleic acids to which they are linked.

Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as "expression vectors". In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.

In the present specification, "plasmid" and "vector" are used interchangeably as the plasmid is the most commonly used form of vector. However, the presently disclosed subject matter is intended to include such other forms of expression vectors which serve equivalent functions and which become known in the art subsequently hereto.

The term "expression vector" as used herein refers to a DNA sequence capable of directing expression of a particular nucleotide sequence in an appropriate host cell, comprising a promoter operatively linked to the nucleotide sequence of interest which is operatively linked to transcription termination sequences. The construct comprising the nucleotide sequence of interest can be chimeric. The construct can also be one that is naturally occurring but has been obtained in a recombinant form useful for heterologous expression.

The nucleotide sequence of interest, including any additional sequences designed to effect proper expression of the nucleotide sequences, can also be referred to as an "expression cassette". The terms "heterologous gene", "heterologous DNA sequence", "heterologous nucleotide sequence", "exogenous nucleic acid molecule", or "exogenous DNA segment", as used herein, each refer to a sequence that originates from a source foreign to an intended host cell or, if from the same source, is modified from its original form.

Thus, a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified, for example by mutagenesis or by isolation from native transcriptional regulatory sequences. The terms also include non-naturally occurring multiple copies of a naturally occurring nucleotide sequence.

Thus, the terms refer to a DNA segment that is foreign or heterologous to the cell, or homologous to the cell but in a position within the host cell nucleic acid wherein the element is not ordinarily found. Two nucleic acids are "recombined" when sequences from each of the two nucleic acids are combined in a progeny nucleic acid. Two sequences are "directly" recombined when both of the nucleic acids are substrates for recombination.

Two sequences are "indirectly recombined" when the sequences are recombined using an intermediate such as a cross over oligonucleotide. For indirect recombination, no more than one of the sequences is an actual substrate for recombination, and in some cases, neither sequence is a substrate for recombination. As used herein, the term "regulatory elements" refers to nucleotide sequences involved in controlling the expression of a nucleotide sequence. Regulatory elements can comprise a promoter operatively linked to the nucleotide sequence of interest and termination signals.

Regulatory sequences also include enhancers and silencers. They also typically encompass sequences required for proper translation of the nucleotide sequence. As used herein, the term "significant increase" refers to an increase in activity for example, enzymatic activity that is larger than the margin of error inherent in the measurement technique, in one embodiment an increase by about 2 fold or greater over a baseline activity for example, the activity of the wild type enzyme in the presence of the inhibitor , in another embodiment an increase by about 5 fold or greater, and in still another embodiment an increase by about 10 fold or greater.

As used herein, the terms "significantly less" and "significantly reduced" refer to a result for example, an amount of a product of an enzymatic reaction that is reduced by more than the margin of error inherent in the measurement technique, in one embodiment a decrease by about 2 fold or greater with respect to a baseline activity for example, the activity of the wild type enzyme in the absence of the inhibitor , in another embodiment, a decrease by about 5 fold or greater, and in still another embodiment a decrease by about 10 fold or greater.

As used herein, the terms "specific binding" and "immunological cross-reactivity" refer to an indicator that two molecules are substantially similar. An indication that two nucleic acid sequences or polypeptides are substantially similar is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with, or specifically binds to, the polypeptide encoded by the second nucleic acid. Thus, a polypeptide is typically substantially similar to a second polypeptide, for example, where the two polypeptides differ only by conservative substitutions.

The phrase "specifically or selectively binds to an antibody," or "specifically or selectively immunoreactive with," when referring to a polypeptide or peptide, refers to a binding reaction which is determinative of the presence of the polypeptide in the presence of a heterogeneous population of polypeptides and other biologies.

Thus, under designated immunoassay conditions, the specified antibodies bind to a particular polypeptide and do not bind in a significant amount to other polypeptides present in the sample. Specific binding to an antibody under such conditions can require an antibody that is selected for its specificity for a particular polypeptide. For example, antibodies raised to the polypeptide with the amino acid sequence encoded by any of the nucleic acid sequences of the presently disclosed subject matter can be selected to obtain antibodies specifically immunoreactive with that polypeptide and not with other polypeptides except for polymorphic variants.

A variety of immunoassay formats can be used to select antibodies specifically immunoreactive with a particular polypeptide. For example, solid phase ELISA immunoassays, Western blots, or immunohistochemistry are routinely used to select monoclonal antibodies specifically immunoreactive with a polypeptide. Typically a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 to times background.

As used herein, the term "subsequence" refers to a sequence of nucleic acids or amino acids that comprises a part of a longer sequence of nucleic acids or amino acids e. As used herein, the term "substrate" refers to a molecule that an enzyme naturally recognizes and converts to a product in the biochemical pathway in which the enzyme naturally carries out its function; or is a modified version of the molecule, which is also recognized by the enzyme and is converted by the enzyme to a product in an enzymatic reaction similar to the naturally-occurring reaction.

As used herein, the term "suitable growth conditions" refers to growth conditions that are suitable for a certain desired outcome, for example, the production of a recombinant polypeptide or the expression of a nucleic acid molecule. As used herein, the term "transformation" refers to a process for introducing heterologous DNA into a plant 1 cell, plant tissue, or plant. Transformed plant cells, plant tissue, or plants are understood to encompass not only the end product of a transformation process, but also transgenic progeny thereof.

As used herein, the terms "transformed", "transgenic", and "recombinant" refer to a host organism such as a bacterium or a plant into which a heterologous nucleic acid molecule has been introduced.

The nucleic acid molecule can be stably integrated into the genome of the host or the nucleic acid molecule can also be present as an extrachromosomal molecule. Such an extrachromosomal molecule can be auto-replicating. Transformed cells, tissues, or plants are understood to encompass not only the end product of a transformation process, but also transgenic progeny thereof. A "non-transformed," "non-transgenic", or "non-recombinant" host refers to a wild-type organism, e.

As used herein, the term "viability" refers to a fitness parameter of a plant. Plants are assayed for their homozygous performance of plant development, indicating which polypeptides are essential for plant growth.

In certain embodiments, the isolated nucleic acid molecule is derived from rice i. As used herein, the phrase "cell proliferation-related polypeptide" refers to a protein or polypeptide note that these two terms are used interchangeably throughout that is involved in cell proliferation, particularly plant cell proliferation. Such a polypeptide can be involved in an increase in cell proliferation; conversely, such a polypeptide can be involved in the abrogation or inhibition of cell proliferation.

Moreover, the polypeptide can be involved in cell proliferation only, for example, when the cell is exposed to a stress e.

In addition, the polypeptide can be involved in cell proliferation only when the cell is differentiating or developing.

A "cell proliferation-related polypeptide" of the presently disclosed subject matter is identified by the ability of an increase or decrease in the level of expression of such a polypeptide in a cell to modulate the rate of that cell's proliferation, whether alone or together with some other stimuli e.

As used herein, term "binds" means that a cell proliferation-related polypeptide preferentially interacts with a stated target molecule. In some embodiments, that interaction allows a biological read-out e. In some embodiments, that interaction is measurable e. Disclosed herein are rice O.

All of the cell proliferation-related proteins of the invention are related, and many interact with one another. Figures are schematic representations showing the interrelatedness of the different cell proliferation-related proteins of the invention. In certain embodiments, the presently disclosed subject matter provides an isolated nucleic acid molecule comprising a nucleotide sequence substantially similar to the nucleotide sequence of the nucleic acid molecule encoding a cell proliferation-related polypeptide disclosed herein.

In a broad sense, the term "substantially similar", as used herein with respect to a nucleotide sequence, refers to a nucleotide sequence corresponding to a reference nucleotide sequence i. In some embodiments, the substantially similar nucleotide sequence encodes the polypeptide encoded by the reference nucleotide sequence i. The term "substantially similar" is specifically intended to include nucleotide sequences wherein the sequence has been modified to optimize expression in particular cells.

The term "substantially similar", when used herein with respect to a protein or polypeptide, refers to a protein or polypeptide corresponding to a reference protein i. In one embodiment, the polypeptide is involved in a function such as abiotic stress tolerance, disease resistance, enhanced yield or nutritional quality or composition. In one embodiment, the polypeptide is involved in drought resistance.

Additionally, one skilled in the art will recognize that individual substitutions, deletions, or additions to a nucleic acid, peptide, polypeptide, or polypeptide sequence that alters, adds, or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservative modification" where the modification results in the substitution of an amino acid with a chemically similar amino acid. Conservative modified variants provide similar biological activity as the unmodified polypeptide.

Conservative substitution tables listing functionally similar amino acids are known in the art. The term "conservatively modified variant" also refers to a peptide having an amino acid residue sequence substantially similar to a sequence of a polypeptide of the presently disclosed subject matter in which one or more residues have been conservatively substituted with a functionally similar residue.

Examples of conservative substitutions include the substitution of one non-polar hydrophobic residue such as isoleucine, valine, leucine or methionine for another; the substitution of one polar hydrophilic residue for another such as between arginine and lysine, between glutamine and asparagine, between glycine and serine; the substitution of one basic residue such as lysine, arginine or histidine for another; or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another.

Amino acid substitutions, such as those which might be employed in modifying the polypeptides described herein, are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. An analysis of the size, shape and type of the amino acid side-chain substituents reveals that arginine, lysine and histidine are all positively charged residues; that alanine, glycine and serine are all of similar size; and that phenylalanine, tryptophan and tyrosine all have a generally similar shape.

Therefore, based upon these considerations, arginine, lysine and histidine; alanine, glycine and serine; and phenylalanine, tryptophan and tyrosine; are defined herein as biologically functional equivalents. Other biologically functionally equivalent changes will be appreciated by those of skill in the art. In making biologically functional equivalent amino acid substitutions, the hydropathic index of amino acids can be considered. Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics, these are: The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art.

It is known that certain amino acids can be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent protein.

As detailed in U. While discussion has focused on functionally equivalent polypeptides arising from amino acid changes, it will be appreciated that these changes can be effected by alteration of the encoding DNA, taking into consideration also that the genetic code is degenerate and that two or more codons can code for the same amino acid.

In one embodiment, the polypeptide is expressed in a specific location or tissue of a plant. In one embodiment, the location or tissue includes, but is not limited to, epidermis, vascular tissue, meristem, cambium, cortex, or pith. In another embodiment, the location or tissue is leaf or sheath, root, flower, and developing ovule or seed. In another embodiment, the location or tissue can be, for example, epidermis, root, vascular tissue, meristem, cambium, cortex, pith, leaf, or flower.

In yet another embodiment, the location or tissue is a seed. The polypeptides of the presently disclosed subject matter, fragments thereof, or variants thereof, can comprise any number of contiguous amino acid residues from a polypeptide of the presently disclosed subject matter, wherein the number of residues is selected from the group of integers consisting of from 10 to the number of residues in a full-length polypeptide of the presently disclosed subject matter.

In one embodiment, the portion or fragment of the polypeptide is a functional polypeptide. Methods of assaying and quantifying measures of activity and substrate specificity are well known to those of skill in the art. The isolated polypeptides of the presently disclosed subject matter can elicit production of an antibody specifically reactive to a polypeptide of the presently disclosed subject matter when presented as an immunogen.

Therefore, the polypeptides of the presently disclosed subject matter can be employed as immunogens for constructing antibodies immunoreactive to a polypeptide of the presently disclosed subject matter for such purposes including, but not limited to, immunoassays or polypeptide purification techniques. Immunoassays for determining binding are well known to those of skill in the art and include, but are not limited to, enzyme-linked immunosorbent assays ELISAs and competitive immunoassays.

The Yeast Two-Hybrid System The yeast two-hybrid system is a well known system which is based on the finding that most eukaryotic transcription activators are modular see e. The yeast two-hybrid system uses: In all of the Examples described below, an automated, high- throughput yeast two-hybrid assay technology provided by Myriad Genetics Inc.

Briefly, the target protein e. C, pAS such that an in-frame fusion between the Ga14p and target protein sequences was created. The resulting construct, the target gene construct, was introduced by transformation into a haploid yeast strain. A screening protocol was then used to search the individual baits against two activation domain libraries of assorted peptide motifs of greater than five million cDNA clones.

The libraries were derived from RNA isolated from leaves, stems, and roots of rice plants grown in normal conditions, plus tissues from plants exposed to various stresses input trait library , and from various seed stages, callus, and early and late panicle output trait library. To screen, a library of activation domain fusions i. The yeast strain that carried the activation domain constructs contained one or more Ga14p- responsive reporter genes, the expression of which can be monitored.

Yeast carrying the target gene construct was combined with yeast carrying the activation domain library. The two yeast strains mated to form diploid yeast and were plated on media that selected for expression of one or more Ga14p-responsive reporter genes. Thus, both hybrid proteins i. Colonies that arose after incubation were selected for further characterization.

The activation domain plasmid was isolated from each colony obtained in the two-hybrid search. The sequence of the insert in this construct was obtained by sequence analysis e. Thus, the identity of positives obtained from these searches was determined by sequence analysis against proprietary and public e.

Interaction of the activation domain fusion with the target protein was confirmed by testing for the specificity of the interaction. The activation domain construct was co-transformed into a yeast reporter strain with either the original target protein construct or a variety of other DNA-binding domain constructs. Expression of the reporter genes in the presence of the target protein but not with other test proteins indicated that the interaction was genuine.

The rice genome array contained mer oligonucleotide probes with sequences corresponding to the 3' ends of 21 , predicted open reading frames found in approximately 42, contigs that make up the rice genome map see Goff et al.

Sixteen different probes were used to measure the expression level of each nucleic acid. The sequences of the probes are available at http: The calculated expression value was determined based on the observed expression level minus the noise background associated with each probe. Experiments included evaluating the differential gene expression from various plant tissues comprising seed, root, leaf and stem, panicle, and pollen. Gene expression was also measured in plants exposed to environmental cold i.

Many of the cell proliferation-related proteins of the presently disclosed subject matter interact with one another. One aspect of the presently disclosed subject matter provides compositions and methods for modulating i. In particular, the nucleic acid molecules and polypeptides of the presently disclosed subject matter are expressed constitutively, temporally, or spatially e.

Therefore, the presently disclosed subject matter provides utility in such exemplary applications as altering the specified characteristics identified above. The isolated nucleic acid molecules of the presently disclosed subject matter are useful for expressing a polypeptide of the presently disclosed subject matter in a recombinantly engineered cell such as a bacterial, yeast, insect, mammalian, or plant cell.

Expressing cells can produce the polypeptide in a non-natural condition e. Those skilled in the art are knowledgeable in the numerous expression systems available for expression of nucleic acids encoding a polypeptide of the presently disclosed subject matter. In another aspect, the presently disclosed subject matter features a cell proliferation-related polypeptide encoded by a nucleic acid molecule disclosed herein. In certain embodiments, the cell proliferation-related polypeptide is isolated.

The presently disclosed subject matter further provides a method for modifying i. The method comprises introducing into a plant cell an expression cassette comprising a nucleic acid molecule of the presently disclosed subject matter as disclosed above to obtain a transformed plant cell or tissue, and culturing the transformed plant cell or tissue. The nucleic acid molecule can be under the regulation of a constitutive or inducible promoter. A plant or plant part having modified expression of a nucleic acid molecule of the presently disclosed subject matter can be analyzed and selected using methods known to those skilled in the art including, but not limited to, Southern blotting, DNA sequencing, or PCR analysis using primers specific to the nucleic acid molecule and detecting amplicons produced therefrom.

Modulation of Expression of Nucleic Acid Molecules. The compositions ,of the presently disclosed subject matter include plant nucleic acid molecules, and the amino acid sequences of the polypeptides or partial-length polypeptides encoded by nucleic acid molecules comprising an open reading frame. These sequences can be employed to alter the expression of a particular gene corresponding to the open reading frame by decreasing or eliminating expression of that plant gene or by overexpressing a particular gene product.

Methods of this embodiment of the presently disclosed subject matter include stably transforming a plant with a nucleic acid molecule of the presently disclosed subject matter that includes an open reading frame operatively linked to a promoter capable of driving expression of that open reading frame sense or antisense in a plant cell. By "portion" or "fragment", as it relates to a nucleic acid molecule that comprises an open reading frame or a fragment thereof encoding a partial-length polypeptide having the activity of the full length polypeptide, is meant a sequence having in one embodiment at least 80 nucleotides, in another embodiment at least nucleotides, and in still another embodiment at least nucleotides.

If not employed for expression, a "portion" or "fragment" means in representative embodiments at least 9, or 12, or 15, or at least 20, consecutive nucleotides e. Thus, to express a particular gene product, the method comprises introducing into a plant, plant cell, or plant tissue an expression cassette comprising a promoter operatively linked to an open reading frame so as to yield a transformed differentiated plant, transformed cell, or transformed tissue.

Transformed cells or tissue can be regenerated to provide a transformed differentiated plant. The transformed differentiated plant or cells thereof can express the open reading frame in an amount that alters the amount of the gene product in the plant or cells thereof, which product is encoded by the open reading frame. The presently disclosed subject matter also provides a transformed plant prepared by the methodsa disclosed herein, as well as progeny and seed thereof.

The presently disclosed subject matter further includes a nucleotide sequence that is complementary to one hereinafter "test" sequence that hybridizes under stringent conditions to a nucleic acid molecule of the presently disclosed subject matter, as well as an RNA molecule that is transcribed from the nucleic acid molecule. When hybridization is performed under stringent conditions, either the test or nucleic acid molecule of presently disclosed subject matter can be present on a support: Depending upon the degree of stringency required, such reduced concentration buffers are typically 1X SSC containing 0.

The plant cell is transformed with at least one such expression vector wherein the plant host cell can be used to regenerate plant tissue or an entire plant, or seed there from, in which the effects of expression, including overexpression and underexpression, of the introduced sequence or sequences can be measured in vitro or in planta.

In some embodiments, the presently disclosed subject matter features an isolated polypeptide comprising or consisting of an amino acid sequence substantially similar to the amino acid sequence of an isolated cell proliferation-related polypeptide of the presently disclosed subject matter. Because the proteins of the presently disclosed subject matter have a roll in cell proliferation, in certain embodiments, a cell introduced with a nucleic acid molecule of the presently disclosed subject matter has a different cell proliferation rate as compared to a cell not introduced with the nucleic acid molecule.

As discussed herein, all of the cell proliferation-related proteins described herein affect cell proliferation, either under normal conditions, under adverse conditions e. Accordingly, by changing the amount of a cell proliferation-related protein of the presently disclosed subject matter in a plant cell, the proliferation of that plant cell can be modulated. In some situations, increasing expression of a cell proliferation-related protein of the presently disclosed subject matter in a cell will cause that cell to increase its rate of proliferation, either alone or in response to some stimulus e.

In other situations, increasing expression of a cell proliferation-related protein of the presently disclosed subject matter in a cell causes that cell to reduce its rate of proliferation.

Similarly, decreasing the expression of a cell proliferation-related protein of the presently disclosed subject matter in a cell can increase or decrease that cell's rate of proliferation. What is relevant is that the rate of proliferation of the cell changes if the level of expression of a cell proliferation-related For example, overexpression of the protein can be accomplished by transforming the cell with a nucleic acid molecule encoding the protein according to standard methods such as those described above.

Reducing the level of expression of a cell proliferation-related protein of the presently disclosed subject matter in a cell is likewise simply 0 accomplished using standard methods. Alteration of the expression of a nucleotide sequence of the presently 0 disclosed subject matter, in one embodiment reduction of its expression, is obtained by "sense" suppression referenced in e. In this case, the entirety or a portion of a nucleotide sequence of the presently disclosed subject matter is comprised in a DNA molecule.

The nucleotide sequence is inserted in the DNA molecule in the "sense orientation", meaning that the coding strand of the nucleotide sequence can be transcribed. In one embodiment, the nucleotide sequence is fully 0 translatable and all the genetic information comprised in the nucleotide In another embodiment, the nucleotide sequence is partially translatable and a short peptide is translated. In another embodiment, the nucleotide sequence is transcribed but no translation product is made.

This is usually achieved by removing the start codon, i. In a further embodiment, the DNA molecule comprising the nucleotide sequence, or a portion thereof, is stably integrated 0 in the genome of the plant cell. In another embodiment, the DNA molecule comprising the nucleotide sequence, or a portion thereof, is comprised in an extrachromosomally replicating molecule.

The entirety or a portion of a nucleotide sequence of the presently disclosed subject matter is comprised in a DNA molecule. The DNA molecule can be operatively linked to a promoter functional in a plant cell, and introduced in a plant cell, in which the nucleotide sequence is expressible.

The nucleotide 0 sequence is inserted in the DNA molecule in the "antisense orientation", meaning that the reverse complement also called sometimes non-coding strand of the nucleotide sequence can be transcribed.

In one embodiment, the DNA molecule comprising the nucleotide sequence, or a portion thereof, is stably integrated in the genome of the plant cell.

Several publications describing this approach are cited for further illustration Green et al. Homologous Recombination 0 In another embodiment, at least one genomic copy corresponding to a nucleotide sequence of the presently disclosed subject matter is modified in the genome of the plant by homologous recombination as further illustrated in Paszkowski et al.

Homologous recombination can occur between the chromosomal copy of a nucleotide sequence in a cell and an incoming copy of the nucleotide sequence introduced in the cell by transformation. Specific modifications are thus accurately introduced in the chromosomal 0 copy of the nucleotide sequence. In one embodiment, the regulatory elements of the nucleotide sequence of the presently disclosed subject matter are modified.

Such regulatory elements are easily obtainable by screening a genomic library using the nucleotide sequence of the presently disclosed subject matter, or a portion thereof, as a probe.

The existing regulatory elements are replaced by different regulatory elements, thus altering expression of the nucleotide sequence, or they are mutated or deleted, thus abolishing the expression of the nucleotide sequence. In another embodiment, the nucleotide sequence is modified by deletion of a part of the nucleotide sequence or the entire nucleotide sequence, or by mutation. Expression of a mutated polypeptide in a plant cell is also provided in the presently disclosed subject matter.

Recent refinements of this technique to disrupt endogenous plant genes have been disclosed Kempin et al. In one embodiment, a mutation in the chromosomal copy of a nucleotide sequence is introduced by transforming a cell with a chimeric oligonucleotide composed of a contiguous stretch of RNA and DNA residues in a duplex conformation with double hairpin caps on the ends.

An additional feature of the oligonucleotide is for example the presence of 2'-O- methylation at the RNA residues. For example, this technique is further illustrated in U. The ribozyme is expressed in transgenic plants and results in reduced amounts of RNA coding for the polypeptide of the presently disclosed subject matter in plant cells, thus leading to reduced amounts of polypeptide accumulated in the cells.

This method is further illustrated in U. This is achieved by expression of dominant negative mutants of the polypeptides in transgenic plants, leading to the loss of activity of the endogenous polypeptide. The partitioned nucleic acids are amplified to yield a ligand-enriched mixture.

After several iterations a nucleic 0 acid with optimal affinity to the polypeptide is obtained and is used for expression in transgenic plants. In alternative embodiments, transcription of the nucleotide sequence is reduced or increased. Zinc finger polypeptides are disclosed in, for example, Beerli et al.

In one embodiment, the alteration of the expression of a nucleotide sequence of the presently disclosed subject matter, in one embodiment the reduction of its expression, is obtained by dsRNA interference. The entirety, or in one embodiment a portion, of a nucleotide 0 sequence of the presently disclosed subject matter, can be comprised in a DNA molecule. The size of the DNA molecule is in one embodiment from to nucleotides or more; the optimal size to be determined empirically.

In one embodiment, the first copy of the DNA molecule is the reverse complement also known as the non-coding strand and the second copy is the coding strand; in another embodiment, the first copy is the coding strand, and the second copy is the reverse complement. The size of the spacer DNA molecule is in one embodiment to 10, 0 nucleotides, in another embodiment to nucleotides, and in yet another embodiment to nucleotides in length.

The two copies of the DNA molecule separated by the spacer are operatively linked to a promoter functional in a plant cell, and introduced in a plant cell in which the nucleotide sequence is expressible. In one embodiment, the DNA molecule comprising the nucleotide sequence, or a portion thereof, is stably integrated 0 in the genome of the plant cell.

Several publications describing this approach are cited for further illustration Waterhouse et al. As used herein, the terms "RNA interference" and "post-transcriptional gene silencing" are used interchangeably and refer to a 0 process of sequence-specific modulation of gene expression mediated by a small interfering RNA siRNA; see generally Fire et al.

Thus, because described herein are nucleotide sequences encoding the cell proliferation-related proteins of the presently disclosed subject matter, RNAi can be readily designed. Indeed, 6 constructs encoding an RNAi molecule have been developed which continuously synthesize an RNAi molecule, resulting in prolonged repression of expression of the targeted gene Brummelkamp et al.

In transgenic plants containing one of the DNA molecules disclosed immediately above, the expression of the nucleotide sequence 0 corresponding to the nucleotide sequence comprised in the DNA molecule is in one embodiment reduced. Methods of insertional mutagenesis using T-DNA, transposons, oligonucleotides, or other methods known to those skilled in the art are also encompassed. In yet another embodiment, a mutation of a nucleic acid molecule of the presently disclosed subject matter is created in the genomic copy of the sequence in the cell or plant by deletion of a portion of the nucleotide 6 sequence or regulator sequence.

Methods of deletion mutagenesis are known to those skilled in the art. In yet another embodiment, a deletion is created at random in a large population of plants by chemical mutagenesis or irradiation and a plant with a deletion in a gene of the presently disclosed subject matter is isolated by 0 forward or reverse genetics.

Irradiation with fast neutrons or gamma rays is known to cause deletion mutations in plants Silverstone et al. Deletion mutations in a gene of the presently disclosed subject matter can be recovered in a reverse genetics strategy using PCR with pooled sets of genomic DNAs as has been 6 shown in C.

A forward genetics strategy involves mutagenesis of a line bearing a trait of interest followed by screening the M2 progeny for the absence of the trait.

Among these mutants would be expected to be some that disrupt a gene of the presently disclosed subject matter. This could be assessed by Southern blotting or PCR using primers designed for a gene of the presently disclosed subject matter with genomic DNA from these mutants.

Examples of nucleic acid molecules and expression cassettes for over- expression of a nucleic acid molecule of the presently disclosed subject matter are disclosed above. Methods known to those skilled in the art of over-expression of nucleic acid molecules are also encompassed by the 0 presently disclosed subject matter.

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