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(12) Patent Application Publication (10) Pub. No.: US 2013/0205441 A1 Lira Et Al

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(12) Patent Application Publication (10) Pub. No.: US 2013/0205441 A1 Lira Et Al US 20130205441A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2013/0205441 A1 Lira et al. (43) Pub. Date: Aug. 8, 2013 (54) CHLOROPLAST TRANSIT PEPTIDE Publication Classification (71) Applicant: Dow Agrosciences LLC, Indianapolis, (51) Int. Cl. IN (US) C07K I4/45 (2006.01) (52) U.S. Cl. (72) Inventors: Justin M. Lira, Zionsville, IN (US); CPC .................................... C07K 14/415 (2013.01) Robert M. Cicchillo, Zionsville, IN USPC .......... 800/278; 536/23.6:530/324; 435/189: (US); Carla N. Yerkes, Crawfordsville, 435/227; 435/232; 435/193; 435/320.1; IN (US); Andrew E. Robinson, 435/419: 800/298; 800/306; 800/314: 800/307; Brownsburg, IN (US) 800/317: 800/305; 800/309: 800/317.1: 800/317.2: 800/313; 800/312:800/322; 800/317.3; 800/317.4: 800/308; 800/320.1; (73) Assignee: DOWAGROSCIENCES LLC, 800/320.2: 800/320:800/320.3: 435/468; Indianapolis, IN (US) 435/418; 800/300; 800/301; 800/302 (57) ABSTRACT (21) Appl. No.: 13/757.456 This disclosure concerns compositions and methods for tar geting peptides, polypeptides, and proteins to plastids of plas tid-containing cells. In some embodiments, the disclosure (22) Filed: Feb. 1, 2013 concerns chloroplast transit peptides that may direct a polypeptide to a plastid, and nucleic acid molecules encoding O O the same. In some embodiments, the disclosure concerns Related U.S. Application Data methods for producing a transgenic plant material (e.g., a (60) Provisional application No. 61/593,555, filed on Feb. transgenic plant) comprising a chloroplast transit peptide, as 1, 2012, provisional application No. 61/625.222, filed well as plant materials produced by Such methods, and plant on Apr. 17, 2012. commodity products produced therefrom. Patent Application Publication Aug. 8, 2013 Sheet 1 of 4 US 2013/0205441 A1 Patent Application Publication Aug. 8, 2013 Sheet 2 of 4 US 2013/0205441 A1 FG, 2 SEOI DNO: 1. ). Salina GPDH SECDNC); 2 D. Salina. EPSPS D. salira GPDH (EU 6.24 4 O 6) D. Salina. EPSPS (AMBM 68 632) FG, 3. SEQCO: A C. r SEQ NC 5 O. C, reinhardtii E. Salina EPS FIG. 4 ZmPer53' UTR TraP14 v2 GFP ... / TraP14 v2. ( . attl2 Zinn Ubi intron pDAB109902 6083 bp ZUbi? promoter pUC origin Patent Application Publication Aug. 8, 2013 Sheet 3 of 4 US 2013/0205441 A1 F.G. 5 F.G. 6 Overdrive T-DNA Border B SpnR Atubio promoter v2 Atubi? 0 promoter intron v. 1 TraP14 v2 DAB107532 DG-32 W 3 trfA p 11456bp AtuORF233' UTR v 1 oriT CSWMV Promoter V 2 T-DNA Border A . .1 . N DSM2 W2 T-DNA Border A, AtuORF1 3' UTR v 6 T-DNA Border A Patent Application Publication Aug. 8, 2013 Sheet 4 of 4 US 2013/0205441 A1 F.G. 7 Overdrive T-DNA Border B SpnR - Atubit 0 promoter v2 Attu bit 0 promoter intron v. 1 TraP24 v2 DGT-33 v3 trfA pDAB107534 11444bp AtuORF233' UTR v oriT 8. CsVMV Promoter v2 ... | y DSN2 V2 T-DNA Border A / \ AtuORF13' UTR v6 TDNA Border A TDNA Border A US 2013/0205441 A1 Aug. 8, 2013 CHLOROPLAST TRANSIT PEPTIDE instance, triazine-derived herbicides inhibit photosynthesis by displacing a plastoquinone molecule from its binding site CROSS-REFERENCE TO RELATED in the 32 kD polypeptide of the photosystem II. This 32 kD APPLICATIONS polypeptide is encoded in the chloroplast genome and Syn 0001. This application claims the benefit of U.S. Provi thesized by the organelle machinery. Mutant plants have been sional Patent Application Ser. No. 61/593,555 filed Feb. 1, obtained which are resistant to triazine herbicides. These 2012, and also to U.S. Provisional Patent Application Ser. No. plants contain a mutant 32 kD polypeptide from which the 61/625.222, filed Apr. 17, 2012, the disclosure of each of plastoquinone can no longer be displaced by triazine herbi which is hereby incorporated herein in its entirety by this cides. Sulfonylureas inhibit acetolactate synthase in the chlo reference. roplast. Acetolactate synthase is involved in isoleucine and valine synthesis. Glyphosate inhibits the function of 5-enol STATEMENT ACCORDING TO 37 C.F.R. pyruvyl-3-phosphoshikimate synthase (EPSPS), which is an S1.821(c) or (e) SEQUENCE LISTING enzyme involved in the synthesis of aromatic amino acids. All SUBMITTED AS ASCII TEXT FILE these enzymes are encoded by the nuclear genome, but they are translocated into the chloroplast where the actual amino 0002 Pursuant to 37 C.F.R. S1.821(c) or (e), a file con acid synthesis takes place. taining an ASCII text version of the Sequence Listing has 0007 Most chloroplast proteins are encoded in the been Submitted concomitant with this application, the con nucleus of the plant cell, synthesized as larger precursor pro tents of which are hereby incorporated by reference. teins in the cytosol, and post-translationally imported into the chloroplast. Import across the outer and inner envelope mem FIELD OF THE DISCLOSURE branes into the stroma is the major means for entry of proteins 0003. This disclosure relates to compositions and methods destined for the stroma, the thylakoid membrane, and the for genetically encoding and expressing polypeptides that are thylakoid lumen. Localization of imported precursor proteins targeted to chloroplasts of higher plants. In certain embodi to the thylakoid membrane and thylakoid lumen is accom ments, the disclosure relates to amino acid sequences that plished by four distinct mechanisms, including two that are target polypeptides to chloroplasts, and/or nucleic acid mol homologous to bacterial protein transport systems. Thus, ecules encoding the same. In certain embodiments, the dis mechanisms for protein localization in the chloroplast are in closure relates to chimeric polypeptides comprising an amino part derived from the prokaryotic endosymbiont. Cline and acid sequence that control the transit of the chimeric polypep Henry (1996), Annu. Rev. Cell. Dev. Biol. 12:1-26. tides to chloroplasts, and/or nucleic acid molecules encoding 0008 Precursor proteins destined for chloroplastic the same. expression contain N-terminal extensions known as chloro plast transit peptides (CTPs). The transit peptide is instru BACKGROUND mental for specific recognition of the chloroplast Surface and 0004 Plant cells contain distinct subcellular organelles, in mediating the post-translational translocation of pre-pro referred to generally as “plastids.' that are delimited by char teins across the chloroplastic envelope and thence to the Vari acteristic membrane systems and perform specialized func ous Sub-compartments within the chloroplast (e.g., stroma, tions within the cell. Particular plastids are responsible for thylakoid, and thylakoid membrane). These N-terminal tran photosynthesis, as well as the synthesis and storage of certain sit peptide sequences contain all the information necessary chemical compounds. All plastids are derived from proplas for the import of the chloroplast protein into plastids; the tids that are present in the meristematic regions of the plant. transit peptide sequences are necessary and Sufficient for Proplastids may develop into, for example: chloroplasts, etio plastid import. plasts, chromoplasts, gerontoplasts, leucoplasts, amylo 0009 Plant genes reported to have naturally-encoded tran plasts, elaioplasts, and proteinoplasts. Plastids existina semi sit peptide sequences at their N-terminus include the chloro autonomous fashion within the cell, containing their own plast small subunit of ribulose-1,5-bisphosphate caroxylase genetic system and protein synthesis machinery, but relying (RuBisCo) (de Castro Silva-Filho et al. (1996), Plant Mol. upon a close cooperation with the nucleo-cytoplasmic system Biol. 30:769-80; Schnell et al. (1991), J. Biol. Chem. 266: in their development and biosynthetic activities. 3335-42); EPSPS (see, e.g., Archer et al. (1990), J. Bioenerg. 0005. In photosynthetic leaf cells of higher plants the most and Biomemb. 22:789-810 and U.S. Pat. Nos. 6,867,293, conspicuous plastids are the chloroplasts. The most essential 7,045,684, and Re. 36,449); tryptophan synthase (Zhao et al. function of chloroplasts is the performance of the light-driven (1995), J. Biol. Chem. 270:6081-7); plastocyanin (Lawrence reactions of photosynthesis. But, chloroplasts also carry out et al. (1997), J. Biol. Chem. 272:20357-63); chorismate syn many other biosynthetic processes of importance to the plant thase (Schmidt et al. (1993), J. Biol. Chem. 268:27447-57): cell. For example, all of the cell's fatty acids are made by the light harvesting chlorophyll afb binding protein (LHBP) enzymes located in the chloroplast Stroma, using the ATP, (Lamppa et al. (1988), J. Biol. Chem. 263:14996-14999); and NAOPH, and carbohydrates readily available there. More chloroplast protein of Arabidopsis thaliana (Lee et al. (2008), over, the reducing power of light-activated electrons drives Plant Cell 20:1603-22). United States Patent Publication No. the reduction of nitrite (NO) to ammonia (NH) in the US 2010/007 1090 provides certain chloroplast targeting pep chloroplast; this ammonia provides the plant with nitrogen tides from Chlamydomonas sp. required for the synthesis of amino acids and nucleotides. 0010. However, the structural requirements for the infor 0006. The chloroplast also takes part in processes of par mation encoded by chloroplast targeting peptides remains ticular importance in the agrochemical industry. For example, elusive, due to their high level of sequence diversity and lack it is known that many herbicides act by blocking functions of common or consensus sequence motifs, though it is pos which are performed within the chloroplast. Recent studies sible that there are distinct subgroups of chloroplast targeting have identified the specific target of several herbicides. For peptides with independent structural motifs. Lee et al. (2008), US 2013/0205441 A1 Aug. 8, 2013 Supra. Further, not all of these sequences have been useful in chloroplast transit peptide operably linked to a nucleotide the heterologous expression of chloroplast-targeted proteins sequence of interest.
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