WO2017044372A1 - Chloroplast transit peptides and methods of their use - Google Patents

Abstract

Methods and compositions are provided for targeting a polypeptide of interest to a chloroplast. Recombinant polynucleotides comprising a nucleotide sequence encoding a chloroplast transit peptide (CTP) operably linked to a heterologous polynucleotide of interest are provided. Recombinant polypeptides encoding the same, as well as, cells, plant cells, plants and seeds are further provided which comprise the recombinant polynucleotides. Methods of use of the various sequences are also provided.

Description

CHLOROPLAST TRANSIT PEPTIDES AND METHODS OF ΤΗΕΠΙ USE

FIELD OF THE INVENTION

[0001] This invention is in the field of molecular biology. More specifically, this invention pertains to targeting sequences of interest to a chloroplast by employing novel chloroplast transit peptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 62/217,337, filed September 11, 2015, the contents of this application is herein incorporated by reference in their entirety.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB

[0003] The official copy of the sequence listing is submitted electronically via EFS- Web as an ASCII formatted sequence listing with a file named AgB014seq_listing.txt, created on August 26, 2016, and having a size of 13,271 MB and is filed concurrently with the specification. The sequence listing contained in this ASCII formatted document is part of the specification and is herein incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

[0004] Plastids are a heterogeneous family of organelles found ubiquitously in plants and algal cells. Most prominent are the chloroplasts, which carry out such essential processes as photosynthesis and the biosynthesis of fatty acids as well as of amino acids. Chloroplasts are complex organelles composed of six distinct suborganellar

compartments: three different membranes (the two envelope membranes and the internal thylakoid membranes) and three compartments (the innermembrane space of the envelope, the stroma and the thylakoid lumen.) More than 98% of all plastid proteins are translated on cytosolic ribosomes. Such proteins are posttranslationally targeted to and imported into the organelle. For a review, see, Jarvis et al. (2008) New Phytologist 179:257-285. Such translocation is mediated by multiprotein complexes in the outer and inner envelope membranes called TOC (Translocon at the Outer envelope membrane of Chloroplasts) and TIC (Translocon at the Inner envelope membrane of Chloroplasts). See, Soil et al. (2004) Nature Reviews. Molecular Cell Biology 5: 198-208, Bedard et al. (2005) Journal oj Experimental Botany 56:2287-2320, Kessler et al. (2006) Traffic I'.lA -lSl, and Smith et al. (2006) Canadian Journal of Botany 84:531-542. Once the chloroplast precursor enters the stroma, the transit peptide is cleaved off, leaving the remaining part of the protein to take on its final conformation or engage one of a number of different sorting pathways. See, Keegstra et al. (1999) Plant Cell 11 : 557-570, Jarvis et al. (2004) and Gutensohn et al. (2006) Journal of Plant Physiology 163:333-347.

[0005] Methods and compositions are needed to allow heterologous polypeptides to be targeted to the chloroplast.

BRIEF SUMMARY OF THE INVENTION

[0006] Methods and compositions are provided for targeting a polypeptide of interest to a chloroplast. Recombinant polynucleotides comprising a nucleotide sequence encoding a chloroplast transit peptide (CTP) operably linked to a heterologous polynucleotide of interest are provided. Recombinant polypeptides encoding the same, as well as, cells, plant cells, plants and seeds are further provided which comprise the recombinant polynucleotides. Methods of use of the various sequences are also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1 aligns several EPSPS proteins from different species against CP4, an herbicide-resistant EPSPS enzyme coded by Agrobacterium (SEQ ID NO: 14). The sequences are identified in Example 1.

DETAILED DESCRIPTION OF THE INVENTION

[0008] The present inventions now will be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the inventions are shown. Indeed, these inventions may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Like numbers refer to like elements throughout.

[0009] Many modifications and other embodiments of the inventions set forth herein will come to mind to one skilled in the art to which these inventions pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the inventions are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

I. Compositions

A. Overview

[0010] As used herein, a "plastid" refers to an organelle present in plant cells that stores and manufactures chemical compounds used by the cell, such as starch, fatty acids, terpenes, and that has been derived from a proplastid. Thus, plastids of plants typically have the same genetic content. Plastids include chloroplasts, which are responsible for photosynthesis, amyloplasts, chromoplasts, statoliths, leucoplasts, elaioplasts, and proteinoplasts. Plastids contain photosynthetic machinery and many additional biosynthetic enzymes including those leading to the production of fatty acids, amino acids, carotenoids, terpenoids, and starch. Thus, there is a need for the ability to target polypeptides of interest to plastids to modulate or alter the physiological processes that occur within these organelles. In addition, some polypeptides are toxic when expressed recombinantly in the cytoplasm. Because plastids are subcompartments, it is possible to target polypeptides of interest to the plastids to sequester them from the cytoplasm, and thus allow for higher expression levels. Furthermore, expression of recombinant polypeptides in plastids may facilitate isolation of the polypeptide for various

applications. As discussed in further detail herein, novel chloroplast transit peptides are provided which can be used in plastid targeting. [0011] The compositions provided herein include recombinant polynucleotides comprising a nucleotide sequence encoding a novel chloroplast transit peptide (CTP) operably linked to a heterologous nucleotide sequence encoding a polypeptide of interest. The CTP-encoding sequences disclosed herein, when assembled within a DNA construct such that the CTP-encoding sequence is operably linked to a nucleotide sequence encoding the polypeptide of interest, facilitate co-translational or post-translational transport of the peptide of interest to the chloroplast of a plant cell.

B. Chloroplast Transit Peptides

[0012] Chloroplasts are organelles found in plant cells and eukaryotic algae that conduct photosynthesis. The chloroplast is a complex cellular organelle composed of three membranes: the inner envelope membrane, the outer envelope membrane, and the thylakoid membrane. The membranes together enclose three aqueous compartments termed the intermediate space, the stroma, and the thylakoid lumen. While chloroplasts contain their own circular genome, many constituent chloroplast proteins are encoded by the nuclear genes and are cytoplasmically-synthesized as precursor forms which contain N-terminal extensions known as chloroplast transit peptides (CTPs). As used herein, the term "chloroplast transit peptide" or "CTP" refers to the N-terminal portion of a chloroplast precursor protein and influences the recognition of the chloroplast surface and mediates the post-translational translocation of pre-proteins across the chloroplast envelope and into the various subcompartments within the chloroplast (e.g., stroma, thylakoid and thylakoid membrane). Thus, as used herein, a polypeptide having "CTP activity" comprises a polypeptide which when operably linked to the N-terminal region of a protein of interest facilitates translocation of the polypeptide of interest to the chloroplast.

[0013] Assays to determine the efficiency by which the CTP sequences provided herein target a protein of interest to a chloroplast include, for example, Mishkind et al. (1985) J of Cell Biol 100:226-234, which is herein incorporated by reference in its entirety. A reporter gene such as glucuronidase (GUS), chloramphenicol acetyl transferase (CAT), or green fluorescent protein (GFP) is operably linked to the CTP sequence. This fusion is placed behind the control of a suitable promoter, ligated into a transformation vector, and transformed into a plant or plant cell. Following an adequate period of time for expression and localization into the chloroplast, the chloroplast fraction is extracted and reporter activity assayed. The ability of the CTP sequences to target and deliver the reporter protein to the chloroplast can be compared to other known CTP sequences. See, de Castro Silva Filho et al. (1996) Plant Mol. Biol. 30: 769-780. Protein import can also be verified in vitro through the addition of proteases to the isolated chloroplast fraction. Proteins which were successfully imported into the chloroplast are resistant to the externally added proteases whereas proteins that remain in the cytosol are susceptible to digestion. Protein import can also be verified by the presence of functional protein in the chloroplast using standard molecular techniques for detection, by evaluating the phenotype resulting from expression of a chloroplast targeted protein, or by microscopy.

/^'. Other components of the CTPs Provided Herein

[0014] It is recognized that the various CTPs disclosed herein can be modified to improve and/or alter the translocation of the polypeptide of interest into the chloroplast. For example, the various CTPs disclosed herein can further comprise additional sequences which modulate the final location of the polypeptide of interest in the chloroplast. For example, the various CTPs disclosed herein could further comprise a thylakoid lumen targeting domain. Proteins to be targeted to the thylakoid lumen comprise an additional cleavable targeting signal, which like the transit peptide, is removed once translocation is complete. The luminal targeting peptides are extremely similar to the signal peptides that mediate inner membrane transport in bacteria. See, for example, Keegstra et al. (1999) Plant Cell 11 :557-570, Jarvis (2004) Current Biology 14: R1064-R1077, Gutensohn et al. (2006) Journal of Plant Physiology 163:333-347, and Jarvis (2008) New Phytologist 179:257-285, all of which are incorporated by reference in their entirety, which discuss the various sorting pathways in a chloroplast. Such regions which modulate the location of the polypeptide of interest in a chloroplast may be native (derived from a region of the same chloroplast targeted polypeptide as the CTP) or heterologous to the operably linked CTP provided herein.

[0015] The term "chloroplast transit peptide cleavage site" refers to a site between two amino acids in a chloroplast-targeting sequence at which the chloroplast processing protease acts. CTPs target the desired protein to the chloroplast and can facilitate the protein's translocation into the organelle. This is accompanied by the cleavage of the transit peptide from the mature polypeptide or protein at the appropriate transit peptide cleavage site by a chloroplast processing protease. Accordingly, a CTP can further comprise a suitable cleavage site for the correct processing of the pre-protein to the mature polypeptide contained within the chloroplast. As discussed above, the sequences beyond the cleaved fragments may be important for localization/transport efficiency and be employed with any of the CTPs disclosed herein. ii. Polynucleotide and Polypeptide Fragments and Variants of CTPs

[0016] Fragments and variants of the CTP-sequences {i.e. SEQ ID NOS: 1, 2, and 3 (see Table 1 below) and the polynucleotides encoding the same) are also encompassed herein. By "fragment" is intended a portion of the polynucleotide or a portion of the amino acid sequence and hence protein encoded thereby. Fragments of a polynucleotide may encode protein fragments that retain CTP activity when reconstituted in a CTP and are thus capable of facilitating the translocation of a polypeptide of interest into the chloroplast of a plant. Alternatively, fragments of a polynucleotide that are useful as a hybridization probe generally do not encode fragment proteins retaining biological activity. Thus, fragments of a nucleotide sequence may range from at least about 10, 20, 30, 40, 50, 60, 70, 80 nucleotides or up to the full length CTP.

[0017] A fragment of a polynucleotide that encodes a biologically active portion of a CTP-polypeptide will encode at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 contiguous amino acids, or up to the total number of amino acids present in any one of SEQ ID NOS: 1, 2, or 3. Fragments of a CTP-encoding sequence that are useful as hybridization probes or PCR primers generally need not encode a biologically active portion of a CTP.

[0018] "Variant" CTP is intended to mean a protein derived from the CTP {i.e. SEQ ID NOS: 1, 2 and/or 3) by deletion {i.e., truncation at the 5' and/or 3' end) and/or a deletion or addition of one or more amino acids at one or more internal sites in the CTP and/or substitution of one or more amino acids at one or more sites in the CTP. Variant proteins encompassed are biologically active, that is they continue to possess the desired biological activity of the CTP, that is, have CTP activity when reconstituted in a CTP. Such variants may result from, for example, genetic polymorphism or from human manipulation.

[0019] For polynucleotides encoding a CTP, a variant comprises a polynucleotide having a deletion (i.e., truncations) at the 5' and/or 3' end and/or a deletion and/or addition of one or more nucleotides at one or more internal sites within the

polynucleotide and/or a substitution of one or more nucleotides at one or more sites in the polynucleotide. Variant polynucleotides also include synthetically derived

polynucleotides, such as those generated, for example, by using site-directed mutagenesis or gene synthesis but which still encode a CTP.

[0020] Biologically active variants of a CTP provided herein (and the polynucleotide encoding the same) will have at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the polypeptide of any one of SEQ ID NO: 1, 2, or 3 as set forth in Table 1 below.

[0021] The CTP-sequences and the active variants and fragments thereof may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. For example, amino acid sequence variants and fragments of the CTPs can be prepared by mutations in the DNA. Methods for mutagenesis and polynucleotide alterations are well known in the art. See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods in Enzymol. 154:367-382; U.S.

Patent No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York) and the references cited therein. 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. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.), herein incorporated by reference. Conservative substitutions, such as exchanging one amino acid with another having similar properties, may be optimal. [0022] Obviously, the mutations that will be made in the DNA encoding the variant must not place the sequence out of reading frame and optimally will not create complementary regions that could produce secondary mRNA structure. See, EP Patent Application Publication No. 75,444.

[0023] Variant polynucleotides and proteins also encompass sequences and proteins derived from a mutagenic and recombinogenic procedure such as DNA shuffling. With such a procedure, one or more different CTP-sequences can be manipulated to create a new CTP 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. See, for example, Stemmer (1994) Proc. Natl. Acad. Sci. USA 91 : 10747-10751; Stemmer (1994) Nature 370:389-391; Crameri et al. (1997) Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol. Biol. 272:336-347; Zhang et al. (1997) Proc. Natl. Acad. Sci. USA 94:4504-4509; Crameri et al. (1998) Nature 391 :288-291 ; and U.S. Patent Nos. 5,605,793 and 5,837,458.

/^'/^'/^'. Sequence Comparisons

[0024] As used herein, the term "identity" or "percent identity" when used with respect to a particular pair of aligned amino acid sequences, refers to the percent amino acid sequence identity that is obtained by counting the number of identical matches in the alignment and dividing such number of identical matches by the length of the aligned sequences. As used herein, the term "similarity" or "percent similarity" when used with respect to a particular pair of aligned amino acid sequences, refers to the sum of the scores that are obtained from a scoring matrix for each amino acid pair in the alignment divided by the length of the aligned sequences.

[0025] Unless otherwise stated, identity and similarity will be calculated by the Needleman-Wunsch global alignment and scoring algorithms (Needleman and Wunsch (1970) J. Mol. Biol. 48(3):443-453) as implemented by the "needle" program, distributed as part of the EMBOSS software package (Rice,P. Longden,! and Bleasby,A., EMBOSS: The European Molecular Biology Open Software Suite, 2000, Trends in Genetics 16, (6) pp276— 277, versions 6.3.1 available from EMBnet at embnet.org/resource/emboss and emboss.sourceforge.net, among other sources) using default gap penalties and scoring matrices (EBLOSUM62 for protein and EDNAFULL for DNA). Equivalent programs may also be used. By "equivalent program" is intended any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by needle from EMBOSS version 6.3.1.

[0026] Additional mathematical algorithms are known in the art and can be utilized for the comparison of two sequences. See, for example, the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the BLAST programs of Altschul et al. (1990) J. Mol. Biol. 215:403. BLAST nucleotide searches can be performed with the BLASTN program, to obtain nucleotide sequences homologous to pesticidal-like nucleic acid molecules of the invention. BLAST protein searches can be performed with the BLASTP program to obtain amino acid sequences homologous to the CTP molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-Blast can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs {e.g., BLASTX and

BLASTN) can be used. Alignment may also be performed manually by inspection.

[0027] Two sequences are "optimally aligned" when they are aligned for similarity scoring using a defined amino acid substitution matrix {e.g., BLOSUM62), gap existence penalty and gap extension penalty so as to arrive at the highest score possible for that pair of sequences. Amino acid substitution matrices and their use in quantifying the similarity between two sequences are well-known in the art and described, e.g., in Dayhoff et al. (1978) "A model of evolutionary change in proteins." In "Atlas of Protein Sequence and Structure," Vol. 5, Suppl. 3 (ed. M. O. Dayhoff), pp. 345-352. Natl. Biomed. Res.

Found., Washington, D.C. and Henikoff et al. (1992) Proc. Natl. Acad. Sci. USA

89: 10915-10919. The BLOSUM62 matrix is often used as a default scoring substitution matrix in sequence alignment protocols. The gap existence penalty is imposed for the introduction of a single amino acid gap in one of the aligned sequences, and the gap extension penalty is imposed for each additional empty amino acid position inserted into an already opened gap. The alignment is defined by the amino acids positions of each sequence at which the alignment begins and ends, and optionally by the insertion of a gap or multiple gaps in one or both sequences, so as to arrive at the highest possible score. While optimal alignment and scoring can be accomplished manually, the process is facilitated by the use of a computer-implemented alignment algorithm, e.g., gapped BLAST 2.0, described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402, and made available to the public at the National Center for Biotechnology Information Website (www.ncbi.nlm.nih.gov). Optimal alignments, including multiple alignments, can be prepared using, e.g., PSI-BLAST, available through www.ncbi.nlm.nih.gov and described by Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402.

[0028] With respect to an amino acid sequence that is optimally aligned with a reference sequence, an amino acid residue "corresponds to" the position in the reference sequence with which the residue is paired in the alignment. The "position" is denoted by a number that sequentially identifies each amino acid in the reference sequence based on its position relative to the N-terminus. For example, if in SEQ ID NO: X position 1 is M, position 2 is A, position 3 is N, etc. When a test sequence is optimally aligned with SEQ ID NO: X, a residue in the test sequence that aligns with the N at position 3 is said to "correspond to position 3" of SEQ ID NO: X. Owing to deletions, insertion, truncations, fusions, etc., that must be taken into account when determining an optimal alignment, in general the amino acid residue number in a test sequence as determined by simply counting from the N-terminal will not necessarily be the same as the number of its corresponding position in the reference sequence. For example, in a case where there is a deletion in an aligned test sequence, there will be no amino acid that corresponds to a position in the reference sequence at the site of deletion. Where there is an insertion in an aligned reference sequence, that insertion will not correspond to any amino acid position in the reference sequence. In the case of truncations or fusions there can be stretches of amino acids in either the reference or aligned sequence that do not correspond to any amino acid in the corresponding sequence. C. Polynucleotides/Polypeptides of Interest

[0029] Any heterologous polynucleotide of interest {i.e., the "polypeptide of interest") may be used with the CTP-encoding sequences disclosed herein {i.e. SEQ ID NOS: 1, 2, 3 (shown in Table 1 below), or active variants or fragments thereof). It is recognized that any polypeptides of interest can be operably linked to the CTP-encoding sequences provided herein and expressed in a plant. Any polypeptide of interest can be operably linked to the CTP-encoding sequence. For example, polypeptides of interest operably linked to the CTP-encoding sequence can include a polypeptide that has an increase in folding capacity in the chloroplast environment and also polypeptides that are toxic to the plant cell if present in the cytoplasm.

[0030] Such polynucleotides/polypeptides of interest include, but are not limited to, herbicide-tolerance coding sequences, insecticidal coding sequences, nematicidal coding sequences, antimicrobial coding sequences, antifungal coding sequences, antiviral coding sequences, abiotic and biotic stress tolerance coding sequences, or sequences modifying plant traits such as yield, grain quality, nutrient content, starch quality and quantity, nitrogen fixation and/or utilization, and oil content and/or composition. More specific polynucleotides of interest include, but are not limited to, genes that improve crop yield, polypeptides that improve desirability of crops, genes encoding proteins conferring resistance to abiotic stress, such as drought, nitrogen, temperature, salinity, toxic metals or trace elements, or those conferring resistance to toxins such as pesticides and herbicides, or to biotic stress, such as attacks by fungi, viruses, bacteria, insects, and nematodes, and development of diseases associated with these organisms.

[0031] In addition, the polypeptide can include an insecticidal polypeptide who's targeting to the chloroplast is desired. Such insecticidal polypeptides, include, for example, Cry, Cyt, BIN, Mtx toxins, Vips, BinA/BinB, Phosphoinositide phospholipase C proteins and PI-PLC toxin class proteins.

[0032] An "herbicide resistance protein" or a protein resulting from expression of an "herbicide resistance-encoding nucleic acid molecule" includes proteins that confer upon a cell the ability to tolerate a higher concentration of an herbicide than cells that do not express the protein, or to tolerate a certain concentration of an herbicide for a longer period of time than cells that do not express the protein. Herbicide resistance traits may be introduced into plants by genes coding for resistance to herbicides that act to inhibit the action of acetolactate synthase (ALS), genes coding for resistance to herbicides that act to inhibit the action of glutamine synthase, such as phosphinothricin or basta (e.g., the bar gene), HPPD, glyphosate (e.g., the EPSP synthase gene) or other such genes known in the art.

[0033] The polynucleotide sequences of interest may encode proteins involved in providing disease or pest resistance. By "disease resistance" or "pest resistance" is intended that the plants avoid the harmful symptoms that are the outcome of the plant- pathogen interactions. Disease resistance and insect resistance genes such as lysozymes or cecropins for antibacterial protection, or proteins such as defensins, glucanases or chitinases for antifungal protection, or Bacillus thuringiensis endotoxins, protease inhibitors, collagenases, lectins, or glycosidases for controlling nematodes or insects are all examples of useful gene products.

[0034] It is recognized that any polypeptide of interest may be modified to comprise, for example, a short linker sequence or a reporter gene in order to allow detection of the protein in the chloroplast.

D. Plants

[0035] Compositions comprising a cell, a transgenic plant cell, a transgenic plant, transgenic plant parts and seeds, plant explants and grain having the recombinant polynucleotide encoding a CTP operably linked to a heterologous polynucleotide encoding a polypeptide of interest are further provided. In one embodiment, a cell, a plant cell, a plant, plant parts and seeds, plant explants and grain comprise at least one polynucleotide encoding a CTP provided herein {i.e. SEQ ID NOS: 1, 2, 3 shown in Table 1 below, or active variants or fragments thereof) operably linked to a polypeptide of interest.

[0036] As used herein, the term plant includes whole plants, plant organs, plant tissues, seeds and plant cells and progeny of the same, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps, and plant cells that are intact in plants or parts of plants such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips, anthers, and the like. Grain is intended to mean the mature seed produced by commercial growers for purposes other than growing or reproducing the species. Progeny, variants, and mutants of the regenerated plants are also included, provided that these parts comprise the introduced recombinant polynucleotides.

[0037] A transformed plant or transformed plant cell provided herein is one in which genetic alteration, such as transformation, has been affected as to a gene of interest, or is a plant or plant cell which is descended from a plant or cell so altered and which comprises the alteration. A "transgene" is a gene that has been introduced into the genome by a transformation procedure. Accordingly, a "transgenic plant" is a plant that contains a transgene, whether the transgene was introduced into that particular plant by transformation or by breeding; thus, descendants of an originally -transformed plant are encompassed by the definition. A "subject plant or plant cell" is one in which genetic alteration, such as transformation, has been affected as to a gene of interest, or is a plant or plant cell which is descended from a plant or cell so altered and which comprises the alteration. A "control" or "control plant" or "control plant cell" provides a reference point for measuring changes in phenotype of the subject plant or plant cell. A control plant or plant cell may comprise, for example: (a) a wild-type plant or cell, i.e., of the same genotype as the starting material for the genetic alteration which resulted in the subject plant or cell; (b) a plant or plant cell of the same genotype as the starting material but which has been transformed with a null construct {i.e., with a construct which does not express the CTP operably linked to a polypeptide of interest, such as a construct comprising a marker gene); (c) a plant or plant cell which is a non- transformed segregant among progeny of a subject plant or plant cell; (d) a plant or plant cell genetically identical to the subject plant or plant cell but which is not exposed to conditions or stimuli that would induce expression of the recombinant polynucleotide; or (e) the subject plant or plant cell itself, under conditions in which the recombinant polynucleotide is not expressed.

[0038] Plant cells that have been transformed to have a recombinant polynucleotide encoding a CTP operably linked to a polypeptide of interest provided herein can be grown into whole plants. The regeneration, development, and cultivation of plants from single plant protoplast transformants or from various transformed explants is well known in the art. See, for example, McCormick et al. (1986) Plant Cell Reports 5:81-84;

Weissbach and Weissbach, In: Methods for Plant Molecular Biology, (Eds.), Academic Press, Inc. San Diego, Calif, (1988). This regeneration and growth process typically includes the steps of selection of transformed cells, culturing those individualized cells through the usual stages of embryonic development through the rooted plantlet stage. Transgenic embryos and seeds are similarly regenerated. The resulting transgenic rooted shoots are thereafter planted in an appropriate plant growth medium such as soil. The regenerated plants can be self-pollinated to provide homozygous transgenic plants.

Otherwise, pollen obtained from the regenerated plants is crossed to seed-grown plants of agronomically important lines. Conversely, pollen from plants of these important lines is used to pollinate regenerated plants. Two or more generations may be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure expression of the desired phenotypic characteristic has been achieved. In this manner, the compositions presented herein provide transformed seed (also referred to as "transgenic seed") having a polynucleotide provided herein, for example, a recombinant polynucleotide encoding a CTP operably linked to a polypeptide of interest, stably incorporated into their genome.

[0039] The recombinant polynucleotides disclosed herein may be used for

transformation of any plant species, including, but not limited to, monocots (e.g., maize, sugarcane, wheat, rice, barley, sorghum, or rye) and dicots (e.g., soybean, Brassica, sunflower, cotton, or alfalfa). Examples of plant species of interest include, but are not limited to, corn (Zea mays), Brassica sp. (e.g. , B. napus, B. rapa, B. juncea), particularly those Brassica species useful as sources of seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana)), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.), oats, barley, vegetables, ornamentals, and conifers.

[0040] Vegetables include, but not limited to, tomatoes (Lycopersicon esculentum), lettuce (e.g., Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Lathyrus spp.), and members of the genus Cucumis such as cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon (C. melo). Ornamentals include, but not limited to, azalea (Rhododendron spp.), hydrangea (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils

(Narcissus spp.), petunias (Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia (Euphorbia pulcherrima), and chrysanthemum.

[0041] Conifers that may be employed in practicing the present invention include, for example, pines such as loblolly pine (Pinus taeda), slash pine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contoria), and Monterey pine (Pinus radiata); Douglas-fir (Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitka spruce (Picea glauca); redwood (Sequoia sempervirens); true firs such as silver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedars such as Western red cedar (Thuja plicata) and Alaska yellow-cedar (Chamaecyparis nootkatensis), and Poplar and

Eucalyptus. In specific embodiments, plants of the present invention are crop plants (for example, corn, alfalfa, sunflower, Brassica, soybean, cotton, safflower, peanut, sorghum, wheat, millet, tobacco, etc.). In other embodiments, corn and soybean plants are optimal, and in yet other embodiments corn plants are optimal.

[0042] Other plants of interest include grain plants that provide seeds of interest, oilseed plants, and leguminous plants. Seeds of interest include grain seeds, such as corn, wheat, barley, rice, sorghum, rye, etc. Oil-seed plants include cotton, soybean, safflower, sunflower, Brassica, maize, alfalfa, palm, coconut, etc. Leguminous plants include beans and peas. Beans include guar, locust bean, fenugreek, soybean, garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea, etc.

[0043] Depending on the polypeptide of interest, the transgenic plants, plant cells or seeds expressing a recombinant polynucleotide provided herein may have a change in phenotype, including but not limited to, an altered pathogen or insect defense mechanism, an increased resistance to one or more herbicides, an increased ability to withstand stressful environmental conditions, and the like.

E. Polynucleotide Constructs

[0044] Also provided are isolated or recombinant polynucleotides and nucleic acid constructs that encode the CTPs disclosed herein {i.e. SEQ ID NOS: 1, 2, 3 (see Table 1 below) or active variants or fragments thereof) operably linked to a polynucleotide encoding a polypeptide of interest. As used herein, "encodes" or "encoding" refers to a DNA sequence which can be processed to generate an RNA and/or polypeptide.

[0045] The terms "polynucleotide," "polynucleotide sequence," "nucleic acid sequence," and "nucleic acid fragment" are used interchangeably herein. These terms encompass nucleotide sequences and the like. A polynucleotide may be a polymer of RNA or DNA that is single- or double-stranded, that optionally contains synthetic, non- natural or altered nucleotide bases. A polynucleotide in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA, synthetic DNA, or mixtures thereof. The use of the term "polynucleotide" is not intended to limit the present invention to polynucleotides comprising DNA. Those of ordinary skill in the art will recognize that polynucleotides, can comprise ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and

ribonucleotides include both naturally occurring molecules and synthetic analogues. The polynucleotides provided herein also encompass all forms of sequences including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-and-loop structures, and the like.

[0046] The compositions provided herein can comprise an isolated or substantially purified polynucleotide. An "isolated" or "purified" polynucleotide is substantially or essentially free from components that normally accompany or interact with the polynucleotide as found in its naturally occurring environment. Thus, an isolated or purified polynucleotide 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. Optimally, an "isolated"

polynucleotide is free of sequences (optimally protein encoding sequences) that naturally flank the polynucleotide (i.e., sequences located at the 5' and 3' ends of the

polynucleotide) in the genomic DNA of the organism from which the polynucleotide is derived. For example, in various embodiments, the isolated polynucleotide can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequence that naturally flank the polynucleotide in genomic DNA of the cell from which the polynucleotide is derived.

[0047] Further provided are recombinant polynucleotides comprising the CTP sequences and polynucleotide sequences encoding the polypeptides of interest. The terms "recombinant polynucleotide" and "recombinant DNA construct" are used interchangeably herein. A recombinant construct comprises an artificial or heterologous combination of nucleic acid sequences, e.g., regulatory and coding sequences that are not found together in nature. For example, a recombinant polynucleotide can comprise a CTP operably linked to a heterologous polynucleotide encoding a polypeptide of interest. In other embodiments, a recombinant construct may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. Such a construct may be used by itself or may be used in conjunction with a vector. If a vector is used, then the choice of vector is dependent upon the method that will be used to transform host cells as is well known to those skilled in the art. For example, a plasmid vector can be used. The skilled artisan is well aware of the genetic elements that must be present on the vector in order to successfully transform, select and propagate host cells comprising any of the isolated nucleic acid fragments of the invention.

[0048] The recombinant polynucleotides disclosed herein can be provided in expression cassettes for expression in a plant or other organism or cell type of interest. The cassette can include 5' and 3' regulatory sequences operably linked to the recombinant polynucleotide or active variant or fragment thereof. "Operably linked" is intended to mean a functional linkage between two or more elements. For example, an operable linkage between a polynucleotide of interest and a regulatory sequence (i.e., a promoter) is a functional link that allows for expression of the polynucleotide of interest. Operably linked elements may be contiguous or non- contiguous. When used to refer to the joining of two protein coding regions, by operably linked is intended that the coding regions are in the same reading frame. The cassette may additionally contain at least one additional gene to be cotransformed into the organism. Alternatively, the additional gene(s) can be provided on multiple expression cassettes. Such an expression cassette is provided with a plurality of restriction sites and/or recombination sites for insertion of the recombinant polynucleotide or active variant or fragment thereof to be under the transcriptional regulation of the regulatory regions. The expression cassette may additionally contain selectable marker genes.

[0049] The expression cassette can include in the 5 '-3' direction of transcription, a transcriptional and translational initiation region (i.e., a promoter), a CTP-encoding sequence or active variant or fragment thereof operably linked to a polynucleotide encoding a polypeptide of interest and a transcriptional and translational termination region (i.e., termination region) functional in plants. The regulatory regions (i.e., promoters, transcriptional regulatory regions, and translational termination regions) and/or the CTP-encoding sequence and/or the polynucleotide encoding the polypeptide of interest may be native/analogous to the host cell or to each other. Alternatively, the regulatory regions and/or the CTP-encoding sequence and/or the polynucleotide encoding the polypeptide of interest may be heterologous to the host cell or to each other. In specific embodiments, the CTP-encoding sequence is operably linked to the 5' end of the polynucleotide of interest, such that, in the resulting recombinant polypeptide, the CTP is operably linked to the N-terminal region of the polypeptide of interest.

[0050] As used herein, "heterologous" in reference to a sequence is a sequence that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention. For example, a CTP operably linked heterologous polynucleotide of interest will not naturally be linked to the polynucleotide in nature. In another example, a promoter operably linked to a heterologous polynucleotide is from a species different from the species from which the polynucleotide was derived, or, if from the

same/analogous species, one or both are substantially modified from their original form and/or genomic locus, or the promoter is not the native promoter for the operably linked polynucleotide.

[0051] The termination region may be native with the transcriptional initiation region, may be native with the operably linked polynucleotide sequence of interest, may be native with the plant host, or may be derived from another source (i.e., foreign or heterologous) to the promoter, the CTP, the polynucleotide sequence of interest, the plant host, or any combination thereof.

[0052] In preparing the expression cassette, the various DNA fragments may be manipulated, so as to provide for the DNA sequences in the proper orientation. Toward this end, adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions, may be involved.

[0053] Where appropriate, the polynucleotides may be optimized for increased expression in the transformed plant. That is, the polynucleotides can be synthesized using plant-preferred codons for improved expression. See, for example, Campbell and Gowri (1990) Plant Physiol. 92: 1-11 for a discussion of host-preferred codon usage. Methods are available in the art for synthesizing plant-preferred genes. See, for example, U.S. Patent Nos. 5,380,831, and 5,436,391, and Murray et al. (1989) Nucleic Acids Res. 17:477-498, herein incorporated by reference.

[0054] The expression cassettes may additionally contain 5' leader sequences. Such leader sequences can act to enhance translation. Translation leaders include: picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5' noncoding region) (Elroy-Stein et al. (1989) Proc. Natl. Acad. Sci. USA 86:6126-6130); potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Gallie et al. (1995) Gene 165(2):233- 238), MDMV leader (Maize Dwarf Mosaic Virus) (Virology 154:9-20), and human immunoglobulin heavy-chain binding protein (BiP) (Macejak et al. (1991) Nature 353:90-94); untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling et al. (1987) Nature 325:622-625); tobacco mosaic virus leader (TMV) (Gallie et al. (1989) in Molecular Biology of RNA, ed. Cech (Liss, New York), pp. 237-256); and maize chlorotic mottle virus leader (MCMV) (Lommel et al. (1991) Virology 81 :382-385. See also, Della-Cioppa et al. (1987) Plant Physiol. 84:965-968.

[0055] A number of promoters can be used to express the recombinant

polynucleotides provided herein. The promoters can be selected based on the desired outcome. It is recognized that different applications can be enhanced by the use of different promoters in the expression constructs to modulate the timing, location and/or level of expression of the recombinant polynucleotide. Such expression constructs may also contain, if desired, a promoter regulatory region (e.g., one conferring inducible, constitutive, environmentally- or developmentally regulated, or cell- or tissue- specific/selective expression), a transcription initiation start site, a ribosome binding site, an RNA processing signal, a transcription termination site, and/or a polyadenylation signal, including the native promoter of the polynucleotide sequence of interest.

[0056] In some embodiments, an expression construct provided herein can be combined with constitutive, tissue-preferred, or other promoters for expression in plants. Examples of constitutive promoters include, for example, the cauliflower mosaic virus (CaMV) 35S transcription initiation region, the - or 2'-promoter derived from T-DNA of Agrobacterium tumefaciens, the ubiquitin 1 promoter, the Smas promoter, the cinnamyl alcohol dehydrogenase promoter (U.S. Pat. No. 5,683,439), the Nos promoter, the pEmu promoter, the rubisco promoter, the GRPl-8 promoter and other transcription initiation regions from various plant genes known to those of skill. If low level expression is desired, weak promoter(s) may be used. Weak constitutive promoters include, for example, the core promoter of the Rsyn7 promoter (WO 99/43838 and U.S. Pat. No. 6,072,050), the core 35S CaMV promoter, and the like. Other constitutive promoters include, for example, U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121;

5,569,597; 5,466,785; 5,399,680; 5,268,463; and 5,608,142. See also, U.S. Pat. No. 6,177,611, herein incorporated by reference. [0057] Examples of inducible promoters are the Adhl promoter which is inducible by hypoxia or cold stress, the Hsp70 promoter which is inducible by heat stress, the PPDK promoter and the pepcarboxylase promoter which are both inducible by light. Also useful are promoters which are chemically inducible, such as the In2-2 promoter which is safener induced (U.S. Pat. No. 5,364,780), the ERE promoter which is estrogen induced, and the Axigl promoter which is auxin induced and tapetum specific but also active in callus (PCT US01/22169).

[0058] Examples of promoters under developmental control include promoters that initiate transcription preferentially in certain tissues, such as leaves, roots, fruit, seeds, or flowers. An exemplary promoter is the anther specific promoter 5126 (U.S. Pat. Nos. 5,689,049 and 5,689,051). Examples of seed-preferred promoters include, but are not limited to, 27 kD gamma zein promoter and waxy promoter, Boronat, A. et al. (1986) Plant Sci. 47:95-102; Reina, M. et al. Nucl. Acids Res. 18(21):6426; and Kloesgen, R. B. et al. (1986) Mol. Gen. Genet. 203:237-244. Promoters that express in the embryo, pericarp, and endosperm are disclosed in U.S. Pat. No. 6,225,529 and PCT publication WO 00/12733. The disclosures for each of these are incorporated herein by reference in their entirety.

[0059] Chemical-regulated promoters can be used to modulate the expression of a gene in a plant through the application of an exogenous chemical regulator. Depending upon the objective, the promoter may be a chemical-inducible promoter, where application of the chemical induces gene expression, or a chemical-repressible promoter, where application of the chemical represses gene expression. Chemical-inducible promoters are known in the art and include, but are not limited to, the maize In2-2 promoter, which is activated by benzenesulfonamide herbicide safeners, the maize GST promoter, which is activated by hydrophobic electrophilic compounds that are used as pre-emergent herbicides, and the tobacco PR- la promoter, which is activated by salicylic acid. Other chemical-regulated promoters of interest include steroid-responsive promoters (see, for example, the glucocorticoid-inducible promoter in Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88: 10421-10425 and McNeills et al. (1998) Plant J. 14(2):247-257) and tetracycline- inducible and tetracycline-repressible promoters (see, for example, Gatz et al. (1991) Mo/. Gen. Genet. 227:229-237, and U.S. Pat. Nos. 5,814,618 and 5,789,156), herein incorporated by reference.

[0060] Tissue-preferred promoters can be utilized to target enhanced expression or a recombinant polynucleotide within a particular plant tissue. Tissue-preferred promoters are known in the art. See, for example, Yamamoto et al. (1997) Plant J. 12(2):255-265; Kawamata et al. (1997) Plant Cell Physiol. 38(7):792-803; Hansen et al. (1997) Mo/. Gen Genet. 254(3):337-343; Russell et al. (1997) Transgenic Res. 6(2): 157-168; Rinehart et al. (1996) Plant Physiol. 112(3): 1331-1341 ; Van Camp et al. (1996) Plant Physiol. 112(2): 525-535; Canevascini et al. (1996) Plant Physiol. 1 12(2):513-524; Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778; Lam (1994) Results Probl. Cell Differ. 20: 181-196; Orozco et al. (1993) Plant Mol Biol. 23(6): 1 129-1138; Matsuoka et al. (1993) Proc Natl. Acad. Sci. USA 90(20): 9586-9590; and Guevara-Garcia et al. (1993) Plant J. 4(3):495-505. Such promoters can be modified, if necessary, for weak expression.

[0061] Leaf-preferred promoters are known in the art. See, for example, Yamamoto et al. (199Ί) Plant J. 12(2)255-265; Kwon et al. (1994) Plant Physiol. 105:357-67; Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778; Gotor et al. (1993) Plant J. 3: 509-18; Orozco et al. (1993) Plant Mol. Biol. 23(6): 1129-1138; and Matsuoka et al. (1993) Proc. Natl. Acad. Sci. USA 90(20): 9586-9590. In addition, the promoters of cab and rubisco can also be used. See, for example, Simpson et al. (1958) EMBO J 4:2723- 2729 and Timko et al. (1988) Nature 318:57-58.

[0062] The expression cassette can also comprise a selectable marker gene for the selection of transformed cells. Selectable marker genes are utilized for the selection of transformed cells or tissues.

II. Methods of Introducing

[0063] The methods provided herein comprise introducing into a cell, plant cell, plant or seed a recombinant polynucleotide or nucleic acid construct encoding a CTP provided herein {i.e. Any of the CTPs provided herein and/or SEQ ID NOS: 1 , 2, 3 (see Table 1 below), or active variants or fragments thereof) operably linked to a heterologous polynucleotide encoding a polypeptide of interest. [0064] The methods provided herein do not depend on a particular method for introducing a sequence into the host cell, only that the polynucleotide gains access to the interior of a least one cell of the host. Methods for introducing polynucleotides into host cells (i.e. plants) include, but are not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.

[0065] The terms "introducing" and "introduced" are intended to mean providing a nucleic acid (e.g., recombinant polynucleotide) or protein into a cell. Introduced includes reference to the incorporation of a nucleic acid into a eukaryotic or prokaryotic cell where the nucleic acid may be incorporated into the genome of the cell, and includes reference to the transient provision of a nucleic acid or protein to the cell. Introduced includes reference to stable or transient transformation methods, as well as sexually crossing. Thus, "introduced" in the context of inserting a nucleic acid fragment (e.g., a

recombinant polynucleotide) into a cell, means "transfection" or "transformation" or "transduction" and includes reference to the incorporation of a nucleic acid fragment into a eukaryotic or prokaryotic cell where the nucleic acid fragment may be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).

[0066] "Stable transformation" is intended to mean that the nucleotide construct introduced into a host (i.e., a plant) integrates into the genome of the plant and is capable of being inherited by the progeny thereof. "Transient transformation" is intended to mean that a polynucleotide is introduced into the host (i.e., a plant) and expressed temporally.

[0067] Transformation protocols as well as protocols for introducing polynucleotide sequences into plants may vary depending on the type of plant or plant cell, i.e., monocot or dicot, targeted for transformation. Suitable methods of introducing polynucleotides into plant cells include microinjection (Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606,

Agrobacterium-mediated transformation (Townsend et al, U.S. Patent No. 5,563,055; Zhao et al, U.S. Patent No. 5,981,840), direct gene transfer (Paszkowski et al. (1984) EMBO J. 3:2717-2722), and ballistic particle acceleration (see, for example, Sanford et al, U.S. Patent No. 4,945,050; Tomes et al, U.S. Patent No. 5,879,918; Tomes et al, U.S. Patent No. 5,886,244; Bidney et al, U.S. Patent No. 5,932,782; Tomes et al (1995) "Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment," in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips (Springer- Verlag, Berlin); McCabe et al (1988) Biotechnology 6:923-926); and Lecl transformation (WO 00/28058). Also see Weissinger et al (1988) _^ ««. Rev. Genet. 22:421-477; Sanford et al. (1987) Particulate Science and Technology 5:27-37 (onion); Christou e/ a/. (1988) Plant Physiol. 87:671-674 (soybean); McCabe et al. (1988) Bio/Technology 6:923-926 (soybean); Finer and McMullen (1991) In Vitro Cell Dev. Biol. 27P: 175-182 (soybean); Singh et al. (1998) Theor. Appl. Genet. 96:319-324 (soybean); Datta et al. (1990) Biotechnology 8:736-740 (rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309 (maize); Klein et al (1988) Biotechnology 6:559-563 (maize); Tomes, U.S. Patent No. 5,240,855; Buising et al, U.S. Patent Nos. 5,322,783 and 5,324,646; Tomes et al. (1995) "Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment," in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg (Springer- Verlag, Berlin) (maize); Klein et al. (1988) Plant Physiol. 91 :440-444 (maize); Fromm et al (1990) Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren et al. (1984) Nature London) 311 :763-764; Bowen et al, U.S. Patent No. 5,736,369 (cereals); Bytebier et al (1987) Proc. Natl. Acad. Sci. USA

84:5345-5349 (Liliaceae); De Wet et al. (1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman et al. (Longman, New York), pp. 197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports 9:415-418 and Kaeppler et al. (1992) Theor. Appl. Genet. 84:560-566 (whisker-mediated transformation); D'Halluin et al. (1992) Plant Cell 4: 1495-1505 (electroporation); Li e/ al. (1993) Plant Cell Reports 12:250-255 and Christou and Ford (1995) Annals of Botany 75:407-413 (rice); Osjoda et al (1996) Nature Biotechnology 14:745-750 (maize via Agrobacterium tumefaciens); all of which are herein incorporated by reference. In one non-limiting embodiment, a transformation vector disclosed in U.S. Provisional Application 62/094,782, herein incorporated by reference, is employed. [0068] In specific embodiments, the recombinant polynucleotides disclosed herein can be provided to a plant using a variety of transient transformation methods. Such transient transformation methods include, but are not limited to, the introduction of the recombinant polynucleotide or variants thereof directly into the plant. Such methods include, for example, microinjection or particle bombardment. See, for example, Crossway et al. (1986) Mol Gen. Genet. 202: 179-185; Nomura et al. (1986) Plant Sci. 44:53-58; Hepler ei a/. (1994) Proc. Natl. Acad. Sci. 91: 2176-2180 and Hush et al.

(1994) The Journal of Cell Science 107:775-784, all of which are herein incorporated by reference. Alternatively, the polynucleotides can be transiently transformed into the plant using techniques known in the art. Such techniques include viral vector system and the precipitation of the polynucleotide in a manner that precludes subsequent release of the DNA. Thus, the transcription from the particle-bound DNA can occur, but the frequency with which it is released to become integrated into the genome is greatly reduced. Such methods include the use of particles coated with polyethylimine (PEI; Sigma #P3143).

[0069] In other embodiments, recombinant polynucleotides disclosed herein may be introduced into plants by contacting plants with a virus or viral nucleic acids. Generally, such methods involve incorporating a nucleotide construct provided herein within a viral DNA or RNA molecule. Methods for introducing polynucleotides into plants and expressing a protein encoded therein, involving viral DNA or RNA molecules, are known in the art. See, for example, U.S. Patent Nos. 5,889,191, 5,889,190, 5,866,785,

5,589,367, 5,316,931, and Porta et al. (1996) Molecular Biotechnology 5:209-221 ; herein incorporated by reference.

[0070] The cells that have been transformed may be grown into plants in accordance with conventional ways. See, for example, McCormick et al. (1986) Plant Cell Reports 5:81-84. These plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting progeny having constitutive expression of the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure expression of the desired phenotypic characteristic has been achieved. In this manner, transformed seed (also referred to as "transgenic seed") having a recombinant polynucleotide disclosed herein, for example, an expression cassette provided herein, stably incorporated into their genome is provided.

III. Methods of Use

[0071] Provided herein is a method of targeting a polypeptide of interest to a chloroplast comprising expressing a recombinant polynucleotide encoding a CTP provided herein {i.e. SEQ ID NOS: 1, 2, 3 (see Table 1 below), or active variants or fragments thereof) operably linked to a heterologous polynucleotide encoding a polypeptide of interest in a cell, plant cell, plant, plant part or seed.

[0072] Methods of the present invention are directed to the proper expression, translocation, and processing of chloroplast-targeted sequences in plants and plant cells under the control of the CTP sequences disclosed herein. For the purposes of the present invention, a "processed" chloroplast targeted protein is one in which the CTP has been removed. At the time of translocation of a chloroplast targeted protein into the chloroplast of a plant cell, the CTP is removed from the targeted protein by cleavage at a particular "cleavage site" between the CTP and the mature protein.

[0073] Depending on the polypeptide of interest targeted to the chloroplast, the transgenic plants may have a change in phenotype, including, but not limited to, an altered pathogen or insect defense mechanism, an increased resistance to one or more herbicides, an increased ability to withstand stressful environmental conditions, a modified ability to produce starch, a modified level of starch production, a modified oil content and/or composition, a modified ability to utilize, partition and/or store nitrogen, and the like. These results can be achieved through the expression and targeting of a polypeptide of interest to chloroplasts in plants, wherein the polypeptide of interest functions in the chloroplast. The CTP sequences provided herein are useful for targeting native sequences as well as heterologous (non-native) sequences in plants.

[0074] Non-limiting examples of methods and compositions disclosed herein are as follows:

1. A recombinant polynucleotide encoding a chloroplast transit peptide (CTP) operably linked to a heterologous polynucleotide encoding a polypeptide of interest, wherein the CTP comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1, 2, or 3 (shown in Table 1), wherein the amino acid sequence has CTP activity.

2. The recombinant polynucleotide of embodiment 1, wherein the CTP comprises the amino acid sequence as set forth in SEQ ID NO: 1, 2, or 3 shown in Table 1 herein.

3. The recombinant polynucleotide of embodiment 1 or 2, wherein said polynucleotide is operably linked to a promoter active in a plant cell.

4. The recombinant polynucleotide of embodiment 1, 2, or 3, wherein the polypeptide of interest confers herbicide resistance or pesticidal resistance to a cell.

5. A vector comprising the recombinant polynucleotide of any one of embodiments 1-4.

6. A cell comprising at least one recombinant polynucleotide of any of embodiments 1-4 or the vector of embodiment 5.

7. The cell of embodiment 6, wherein said cell is a plant cell.

8. The cell of embodiment 7, wherein the recombinant polynucleotide or vector is stably incorporated into the genome of said plant cell.

9. The cell of embodiment 7 or 8, wherein said plant cell is from a monocot.

10. The cell of embodiment 9, wherein said monocot is maize, wheat, rice, barley, sorghum, sugarcane, or rye.

11. The cell of embodiment 7 or 8, wherein said plant cell is from a dicot.

12. The cell of embodiment 11, wherein the dicot is soybean, Brassica, sunflower, cotton, or alfalfa.

13. A plant comprising at least one plant cell of any one of embodiments 7-12.

14. A plant explant comprising at least one plant cell of any one of embodiments 7-12.

15. A transgenic seed produced by the plant of embodiment 13, wherein said seed comprises said recombinant polynucleotide.

16. A recombinant polypeptide encoded by the polynucleotide of any one of embodiments 1-4. 17. A method of targeting a polypeptide of interest to a chloroplast comprising expressing the recombinant polynucleotide of any one of embodiments 1-4 in a plant cell.

18. A method of targeting a polypeptide of interest to a chloroplast comprising introducing the recombinant polynucleotide of any one of embodiments 1-4 into a plant cell and expressing said recombinant polynucleotide in the plant cell.

19. The method of embodiment 17 or 18, wherein said method further comprises regenerating a transgenic plant from said plant cell.

20. The method of any one of embodiments 17-19, wherein said plant cell is from a monocot.

21. The method of embodiment 20, wherein said monocot is maize, wheat, rice, barley, sorghum, sugarcane or rye.

22. The method of any one of embodiments 17-19, wherein said plant cell is from a dicot.

23. The method of embodiment 22, wherein said dicot is soybean, Brassica, sunflower, cotton, or alfalfa.

24. The method of any one of embodiments 17-23, wherein said polypeptide of interest comprises an insecticidal protein and expression of said polypeptide controls a pest.

EXAMPLES

[0075] The following examples are offered to illustrate, but not to limit, the claimed invention. It is understood that the examples and embodiments described herein are for illustrative purposes only, and persons skilled in the art will recognize various reagents or parameters that can be altered without departing from the spirit of the invention or the scope of the appended claims.

Example 1

[0076] Agriculture relies more and more on new cultivars of crops and also on use of new traits—genes/proteins that provide certain beneficial characteristics to a particular crop—to continue to provide improved yield and financial benefits to farmers. The most important and sought-after traits include resistance to herbicides that kill weeds, resistance towards insect pests, including Lepidopteran, Coleopteran, and Hemipteran insects, resistance to diseases pests or Disease. Some proteins, for example some of those that provide herbicide resistance, express better or have a target they interact with in an intracellular compartment rather than in the cytosol. In these cases the proteins need to be expressed in or translocated into a different plant organelles. Chloroplasts and plastids are organelles of choice for some of the proteins for delivery. For a nuclear-encoded protein, where the gene is encoded by plant genomic DNA (the usual result of plant

transformation when making a genetically modified crop) to enter the chloroplast requires the presence of a Chloroplast Transit Peptide (CTP) on the protein's N- terminus. There is a certain specificity for recognition of the host plant species and functionality of CTPs; some of the monocot- originated CTPs don't function well in dicot plants, while some of the dicot CTPs do not work well in monocot crops. The functioning of algal CTPs in higher plants is also unpredictable. Here we report the use of three CTPs for directing heterologous proteins into chloroplasts of monocot plants, such as corn, wheat and rice, and dicot plants, such as soybean and cotton. One of the DNA sequences encoding a CTP originates from the 5 -enol-pyruvyl-shikimate-3 -phosphate synthase (EPSPS) gene of the green alga Volvox carteri, where it directs the encoded protein to the chloroplast. The other two CTPs originate from brown Diatom algae (Diatomophyceae) and brown algae Ectocarpus siliculosus.

[0077] Sequences of several EPSPS proteins were analyzed by aligning them against CP4 (an herbicide-resistant EPSPS enzyme coded by Agrobacterium). See Figure 1. The sequences listed in Figure 1 are listed the following table:

[0078] Arbitrarily, Gly20 of CP4 were selected as the beginning of the core EPSPS. All of the other analyzed sequences, including ones from Volvox, Diatom, and

Ectocarpus, had a predicted peptide at least 59 amino acids in length located N-terminal to the chosen conserved Gly amino acid terminus. These sequences were chosen as CTPs (listed in Table 2) and used to direct CP4 to chloroplasts of transgenic corn. Transformed corn is selected on the media containing herbicide glyphosate.

Table 1. Summary of Chloroplast Transit Peptide (CTP) Sequences

[0079] The article "a" and "an" are used herein to refer to one or more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one or more element.

[0080] All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0081] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

Claims

THAT WHICH IS CLAIMED:

1. A recombinant polynucleotide encoding a chloroplast transit peptide (CTP) operably linked to a heterologous polynucleotide encoding a polypeptide of interest, wherein the CTP comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1, 2, or 3, and wherein the amino acid sequence has CTP activity.

2. The recombinant polynucleotide of claim 1, wherein the CTP comprises the amino acid sequence as set forth in SEQ ID NO: 1, 2, or 3.

3. The recombinant polynucleotide of claim 1 or 2, wherein said

polynucleotide is operably linked to a promoter active in a plant cell.

4. The recombinant polynucleotide of claim 1, 2, or 3, wherein the polypeptide of interest confers herbicide resistance or pesticidal resistance to a cell.

5. A vector comprising the recombinant polynucleotide of any one of claims

1-4.

6. A cell comprising at least one recombinant polynucleotide of any of claims 1-4 or the vector of claim 5.

7. The cell of claim 6, wherein said cell is a plant cell.

8. The cell of claim 7, wherein the recombinant polynucleotide or vector is stably incorporated into the genome of said plant cell.

9. The cell of claim 7 or 8, wherein said plant cell is from a monocot.

10. The cell of claim 9, wherein said monocot is maize, wheat, rice, barley, sorghum, sugarcane, or rye.

11. The cell of claim 7 or 8, wherein said plant cell is from a dicot.

12. The cell of claim 11, wherein the dicot is soybean, Brassica, sunflower, cotton, or alfalfa.

13. A plant comprising at least one plant cell of any one of claims 7-12.

14. A plant explant comprising at least one plant cell of any one of claims 7-

12.

15. A transgenic seed produced by the plant of claim 13, wherein said seed comprises said recombinant polynucleotide.

16. A recombinant polypeptide encoded by the recombinant polynucleotide of any one of claims 1-4.

17. A method of targeting a polypeptide of interest to a chloroplast comprising expressing the recombinant polynucleotide of any one of claims 1-4 in a plant cell.

18. A method of targeting a polypeptide of interest to a chloroplast comprising introducing the recombinant polynucleotide of any one of claims 1-4 into a plant cell and expressing said recombinant polynucleotide in the plant cell.

19. The method of claim 17 or 18, wherein said method further comprises regenerating a transgenic plant from said plant cell.

20. The method of any one of claims 17-19, wherein said plant cell is from a monocot.

21. The method of claim 20, wherein said monocot is maize, wheat, rice, barley, sorghum, sugarcane, or rye.

22. The method of any one of claims 17-19, wherein said plant cell is from a dicot.

23. The method of claim 22, wherein said dicot is soybean, Brassica, sunflower, cotton, or alfalfa.

24. The method of any one of claims 17-23, wherein said polypeptide of interest comprises an insecticidal protein and expression of said polypeptide controls a pest.