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(12) Patent Application Publication (10) Pub. No.: US 2013/0205440 A1 Lira Et Al

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(12) Patent Application Publication (10) Pub. No.: US 2013/0205440 A1 Lira Et Al US 20130205440A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2013/0205440 A1 Lira et al. (43) Pub. Date: Aug. 8, 2013 (54) SYNTHETIC BRASSICA-DERIVED Publication Classification CHLOROPLAST TRANSIT PEPTIDES (51) Int. Cl. (71) Applicant: Dow AgroSciences LLC, Indianapolis, C07K I4/45 (2006.01) IN (US) (52) U.S. Cl. CPC .................................... C07K 14/415 (2013.01) (72) Inventors: Justin M. Lira, Zionsville, IN (US); USPC ......... 800/278:536/23.6:536/23.4:530/370; Robert M. Cicchillo, Zionsville, IN 435/189: 536/23.2:435/232; 435/183; (US); Carla N. Yerkes, Crawfordsville, 435/320.1; 435/419: 800/298; 800/306; IN (US); Andrew E. Robinson, 800/313; 800/314: 800/307; 800/317: 800/305; Brownsburg, IN (US) 800/309: 800/317.1: 800/317.2: 800/312: 800/322; 800/317.3; 800/3174: 800/308; 800/320.1; 800/320.2: 800/320:800/320.3: (73) Assignee: DOWAGROSCIENCES LLC, 435/411; 435/412: 435/.414; 435/416: 435/468; Indianapolis, IN (US) 800/300: 800/301; 800/302:435/415 (57) ABSTRACT (21) Appl. No.: 13/757,435 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 16 US 2013/0205440 A1 FIG. 1 mRNA molecule comprising a Brassica-derived chloroplast transit peptidc-encoding scquence Brassica-derived CTP Nucleotide sequence of interest 53 NOptional nucleotide scquences FIG. 2 Alignment of the predicted chloroplast transit peptides for the EPSPS protein from Brassica napus (SEQ ID NO:2) and Arabidopsis thaliana (SEQ ID NO:4). The asterisk indicates where the sequences were spit and recombined to form TraP12 and Trap 13. Brassica rapus (SEC) D NO: 2) Arabidopsis thaliana (SEC D NO: 4) Bria S8iCa rap us (SEC) D NO : 2) Arabidcps is thaliana (SEC D NO: 4) 3IIPRAYPI: Brassica rapus (SEO ID NO: 2) ArabidCps is thali and (SEC D NO: 4) Patent Application Publication Aug. 8, 2013 Sheet 2 of 16 US 2013/0205440 A1 FIG. 3 Plasmid map pDAB101981 TDNA BOrder B SpnRoverdrive Atubi? 0 promoter N At Ubi? 0 promoter intron 3. Trap 12 pDAB101981 AtuORF23 3' UTR 10955bp CSVMV promoter PAT OriT S. w AtuoRF1 3' UTR s TDNA BOrder A TDNA BOrder A TDNA BOrder A Patent Application Publication Aug. 8, 2013 Sheet 3 of 16 US 2013/0205440 A1 FIG. 4 Plasmid map pDAB 101989 OVerdrive / SpnR S. TDNA BOrder B Atubi? 0 promoter At Jbil O promoter intron Trap 13 pDAB101989 1O937 bp i-- AtuORF233' UTR CSVMV promoter u- Y OriTf N X PAT AtuORF13' UTR TDNA BOrder A/ r TDN A BOrder A TDNA BOrder A Patent Application Publication Aug. 8, 2013 Sheet 4 of 16 US 2013/0205440 A1 FIGS Plasmid map of pDAB 101908 TDNA BOrder B w Overdrive \ Š. Atubi? 0 promoter Š ------ Atubifo promoter intron STLS1 intron YFP pDAB101908 AtuORF23 3' UTR 1O931 bp CsVMV promoter a PAT oriT AtuORF13' UTR N \ TDNA BOrder A \ T-DNA Border A TDNA BOrder A Patent Application Publication Aug. 8, 2013 Sheet 5 of 16 US 2013/0205440 A1 FIG. 6 TraP12-YFP infiltrated into tobacco leaf tissue was translocated into the chloroplasts of the tobacco leaf tissue FIG. 7 Trap 13-YFP infiltrated into tobacco leaf tissue was translocated into the chloroplasts of the tobacco leaf tissue. Patent Application Publication Aug. 8, 2013 Sheet 6 of 16 US 2013/0205440 A1 FIG. 8 Non-targeted YFP controls that were infiltrated into tobacco leaf tissue were not incorporated into the chloroplasts of the tobacco leaf tissue. FIG. 9 TraP12-YFP transformed into maize protoplasts was translocated into the chloroplasts of the maize protoplast. Patent Application Publication Aug. 8, 2013 Sheet 7 of 16 US 2013/0205440 A1 FIG 10 Plasmid map pDAB 105528 TDNA BOrder B Overdrive \ SpnR Atubi10 promoter Atubi10 promoter intron Tap 12 W2 pDAB105528 11564 bp S s AtuORF233' UTR \ CSWMV Promoter oriT SS DSM2 Š N s AtuORF1 3' UTR TDNA BOrder A/ r TDNA BOrder A TDNA BOrder A Patent Application Publication Aug. 8, 2013 Sheet 8 of 16 US 2013/0205440 A1 FIG 11 Plasmid map pDAB105529 TDNA BOrder B Overdriv SpnR N AtUbi?10 promoter X Š --- S Atubi?0 promoter intron Trap 13 W2 DGT14 pDAB105529 115466 bp iS - AtuORF233' UTR CsVMV Promoter OriT xx DSM2 AtuORF13' UTR TDNA BOrder A TDNA BOrder A TDNA BOrder A Patent Application Publication Aug. 8, 2013 Sheet 9 of 16 US 2013/0205440 A1 FIG. 12 Plasmid map pDAB 107686 OV erdrive T-DNA Border B SpnR ZmUbi1 promoter ZmUbi1 intron TraP12 v 4 pDAB107686 TraP12 Cry2Aa 14349bp oriT TDNA BOrder A ZmLip 3' UTR -> T-DNA Border A--- N.s , T-DNA Border A \ ZmLip3' UTR I X. SCBW promoter Msv leader Adh 1 intron 6 Patent Application Publication Aug. 8, 2013 Sheet 10 of 16 US 2013/0205440 A1 FIG. 13 Plasmid map pDAB 107687 Overdriv e T-DNA Border B SpnR Zmubi1 promoter ^ Zmubi1 intron Cry2Aa pDAB107687 14127 bp s S oriT . SX ZmLip 3' UTR T-DNA Border A s & s --- & as ------ T-DNA Border AuŠ to TDNA Border A / / SCBV promoter ZmLip 3' UTRw / MSV leader \ AAD-1 Adh1 intron 6 Patent Application Publication Aug. 8, 2013 Sheet 11 of 16 US 2013/0205440 A1 F.G. 14 Plasmid map of pDAB 112711 OW erdrive T-DNA BOrder B / Zmubi1 promoter pDAB112711 Trap 12 Wip3Ab1 v6 oriT 14831 bp TDNA BOrder A is s^ is T-DNA Border A s SN ZmPer5 3' UTR TDNA Border A × / --- ZmLip3' UTR v 1 x SCBW promoter AAD-1 usy leader Adh1 intron 6 Patent Application Publication Aug. 8, 2013 Sheet 12 of 16 US 2013/0205440 A1 FIG. 1S Plasmid map of pDAB11479 Overdrive T-DNA BOrder B SpnR ^ Zmubi1 promoter Zm Ubi?1 intron pDAB111479 Wip3Ab1 v6 14606bp oriT T-DNA Border A T-DNA Border A. M. N. ZmPer53' UTR TDNA BOrder ZmLip 3' UTR SCBV promoter AAD-1 & Msv leader Adh1 intron 6 Patent Application Publication Aug. 8, 2013 Sheet 13 of 16 US 2013/0205440 A1 Plasmid map of pDAB 112712 Overdrive TDNA BOrder B s Zmubi1 promoter SS Zm Ubi?1 intron trfA : pDAB112712 TraP12 Wip3Ab1 v 7 14831 bp oriT as T-DNA Border A TDNA Border A- / S. ZmPer5 3' UTR TDNA BOrder A ZmLip3' UTR SCBW promoter MSW leader Adh 1 intron 6 Patent Application Publication Aug. 8, 2013 Sheet 14 of 16 US 2013/0205440 A1 FIG.17 Plasmid map of pDAB 112710 Overdriv e T-DNA Border B SpnR x × Zmubi1 promoter Zm Ubi1 intron p DAB11271 O u Wip3Ab1 v7 14606bp oriT T-DNA Border A T-DNA Border ASM S N ZmPer53' UTR TDNA BOrder ---a S / ZmLip 3' UTR SCBV promoter y AAD-1 Msv leader Adh1 intron 6 Patent Application Publication Aug. 8, 2013 Sheet 15 of 16 US 2013/0205440 A1 FIG. 18 Plasmid map of pDAB017540 Overdrive T-DNA BOrder B SpnR ^ Atubil O promoter Atubi10 promoter intron TraP12 v 3 Y pDAB107540 Crv2Ary2Aa 12113 bp -- oriT ? TDNA BOrder A Patent Application Publication Aug. 8, 2013 Sheet 16 of 16 US 2013/0205440 A1 FIG. 19 Plasmid map of pDAB 107617 Overdrive T-DNA Border B SpnR ^ - Atubil 0 promoter Atubi10 promoter intron s trfA Cry2Aa v3 pDAB107617 12O78bp AtuORF233' UTR OriT assassa & CSVMV Promoter T-DNA Border A -S y \ DSM2 //I T-DNA Border A / AtuORF13' UTR ? TDNA BOrder A US 2013/0205440 A1 Aug. 8, 2013 SYNTHETIC BRASSCA-DERVED have identified the specific target of several herbicides. For CHLOROPLAST TRANSIT PEPTIDES 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.
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