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WO 2014/160383 Al 2 October 2014 (02.10.2014) P O P C T (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2014/160383 Al 2 October 2014 (02.10.2014) P O P C T (51) International Patent Classification: BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, C12N 15/82 (2006.01) A01H 5/00 (2006.01) DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR, (21) International Application Number: KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, ME, PCT/US20 14/026448 MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, (22) International Filing Date: OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, 13 March 2014 (13.03.2014) SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, (25) Filing Language: English ZW. (26) Publication Language: English (84) Designated States (unless otherwise indicated, for every (30) Priority Data: kind of regional protection available): ARIPO (BW, GH, 61/779,840 13 March 2013 (13.03.2013) US GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, SZ, TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, (71) Applicant: E. I. DUPONT DE NEMOURS & COM¬ TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, PANY [US/US]; 1007 Market Street, Wilmington, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, Delaware 19898 (US). MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, (72) Inventor; and KM, ML, MR, NE, SN, TD, TG). (71) Applicant : MCGONIGLE, Brian [US/US]; c/o Pioneer Hi-Bred International, Inc., 7250 N.W. 62nd Avenue, Declarations under Rule 4.17 : Johnston, Iowa 5013 1-0552 (US). — as to applicant's entitlement to apply for and be granted a (74) Agent: BAUER, S. Christoper; Pioneer Hi-Bred Interna patent (Rule 4.1 7(H)) tional, Inc., 7250 N.W. 62nd Avenue, Johnston, Iowa Published: 5013 1-0552 (US). — with international search report (Art. 21(3)) (81) Designated States (unless otherwise indicated, for every kind of national protection available): AE, AG, AL, AM, — with sequence listing part of description (Rule 5.2(a)) AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, o o (54) Title: PRODUCTION OF SMALL INTERFERING RNAS IN PLANTA (57) Abstract: Disclosed herein are methods for reducing the expression of target nucleotide sequences using microRNAs. Also dis - closed herein are recombinant DNA constructs comprising nucleotide sequences encoding such microRNAs. Plants or plant part thereof comprising these recombinant DNA constructs and microRNAs are also provided. PRODUCTION OF SMALL INTERFERING RNAS IN PLANTA REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY The official copy of the sequence listing is submitted electronically as an ASCII formatted sequence listing with a file named "BB2224WOPCT_Sequence_Listing.TXT" 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. TECHNICAL FIELD This disclosure relates generally to the field of plant molecular biology. More specifically, it relates to constructs and methods to reduce the level of expression of a target sequence in a target organism. BACKGROUND A wide variety of eukaryotic organisms, including plants, animals, and fungi, have evolved several RNA-silencing pathways to protect their cells and genomes against invading nucleic acids, such as viruses or transposons, and to regulate gene expression during development or in response to external stimuli (for review, see Baulcombe (2005) Trends Biochem. Sci. 30:290-93; Meins et al. (2005) Annu. Rev. Cell Dev. Biol. 2 1:297- 318). In plants, RNA-silencing pathways have been shown to control a variety of developmental processes including flowering time, leaf morphology, organ polarity, floral morphology, and root development (reviewed by Mallory and Vaucheret (2006) Nat. Genet. 38:S31-36). All RNA-silencing systems involve the processing of double-stranded RNA (dsRNA) into small RNAs of 2 1 to 25 nucleotides (nt) by an RNaselll-like enzyme known as Dicer or Dicer-like in plants (Bernstein et al. (2001) Nature 409:363-66; Xie et al. (2004) PLOS Biol. 2 E 104:0642-52; Xie et al. (2005) Proc. Natl. Acad. Sci. USA 102:12984-89; Dunoyer et al. (2005) Nat. Genet. 37:1356-60). These small RNAs are incorporated into silencing effector complexes containing an Argonaute protein (for review, see Meister and Tuschl (2004) Nature 431 :343-49). Artificial microRNAs (amiRNAs) have been described in Arabidopsis targeting viral mRNA sequences (Niu et al. (2006) Nat. Biotechnol. 24:1420-1428) or endogenous genes (Schwab et al. (2006) Plant Cell 18:1 121-1 133). The amiRNA construct can be expressed under different promoters in order to change the spatial pattern of silencing (Schwab et al. (2006) Plant Cell 18:1 121-1 133). Artificial miRNAs replace the microRNA and its complementary star sequence in a miRNA precursor backbone and substitute sequences that target an mRNA to be silenced. Silencing by endogenous miRNAs can be found in a variety of spatial, temporal, and developmental expression patterns (Parizotto et al. (2007) Genes Dev. 18:2237-2242; Alvarez et al. (2006) Plant Cell 18:1 134-51). Traditionally, the primary method for impacting pest populations is the application of broad-spectrum chemical pesticides. However, consumers and government regulators alike are becoming increasingly concerned with the environmental hazards associated with the production and use of synthetic chemical pesticides. Because of such concerns, regulators have banned or limited the use of some of the more hazardous pesticides. Thus, there is substantial interest in developing alternative pesticides. SUMMARY Applicant has solved the problem through development of recombinant DNA constructs comprising 1) a precursor miRNAs that, when fully processed, yield 22 nucleotide mature miRNAs that is capable of triggering the production of secondary siRNAs in planta targeted to at least one exogenous gene, and 2) a polynucleotide sequence that includes at least one exogenous target site that can be cleaved by the 22 nucleotide miRNA. These siRNAs can, when ingested by a target organism, result in gene silencing of the targeted gene(s). One aspect is for a method for reducing expression of at least one target sequence, said method comprising: (a) expressing in a plant a recombinant DNA construct comprising: (i) a first polynucleotide sequence comprising a plant-specific promoter operably linked to a nucleotide sequence encoding a pre-miRNA, wherein said pre-miRNA comprises a 22 nucleotide mature miRNA; and (ii) a second polynucleotide sequence comprising at least one target sequence that can be cleaved by the mature mi-RNA processed by the pre-miRNA of (i), wherein said plant processes said pre-miRNA into mature miRNA and; (b) eliciting production of of secondary siRNAs in planta by the mature miRNA; wherein exposing a target organism to said plant comprising the secondary siRNAs of step (b), reduces expression of at least one target sequence in said target organism. Another aspect is for a recombinant DNA construct comprising: (a) a first polynucleotide sequence comprising a plant-specific promoter operably linked to a nucleotide sequence encoding a pre-miRNA, wherein said pre-miRNA comprises a 22 nucleotide mature miRNA; and (b) a second polynucleotide sequence comprising at least one exogenous target sequence that can be cleaved by the mature mi-RNA processed by the pre-miRNA of (a); wherein the mature miRNA elicits the production of secondary siRNAs. A further aspect is for a plant or a plant part thereof comprising: (a) a first recombinant DNA construct comprising a first plant-specific promoter operably linked to a polynucleotide encoding a first portion of a pre-miRNA, said first portion of a pre-miRNA comprising a first polynucleotide segment of 22 nucleotides; and (b) a second recombinant DNA construct comprising a second plant-specific promoter operably linked to a polynucleotide encoding a second portion of a pre-miRNA, said second portion of a pre- miRNA comprising a second polynucleotide segment complementary to said first polynucleotide segment; wherein said first polynucleotide segment has sufficient sequence complementary to at least one target sequence whose level of RNA is to be reduced but does not have sufficient sequence complementary to any RNAs of a plant expressing the recombinant DNA constructs; and further wherein said first polynucleotide sequence, when processed into a mature miRNA, elicits the production of secondary siRNAs. An additional aspect is for a plant or plant part thereof comprising: (a) a first polynucleotide comprising a plant-specific promoter operably linked to a nucleotide sequence encoding a pre-miRNA, wherein said pre-miRNA comprises a 22 nucleotide mature miRNA; and (b) a second polynucleotide sequence comprising at least one exogenous target sequence that can be cleaved by the mature mi-RNA processed from the pre-miRNA of (a); wherein said mature miRNA, elicits the production of secondary siRNAs. BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows the plasmid BB2224-1 . Figure 2 shows the plasmid BB2224-2. Figure 3 shows the plasmid BB2224-3. BRIEF DESCRIPTION OF THE BIOLOGICAL SEQUENCES The following sequences comply with 37 C.F.R. §§ 1.821-1 .825 ("Requirements for Patent Applications Containing Nucleotide Sequences and/or Amino Acid Sequence Disclosures - the Sequence Rules") and are consistent with World Intellectual Property Organization (WIPO) Standard ST.25 (2009) and the sequence listing requirements of the European Patent Convention (EPC) and the Patent Cooperation Treaty (PCT) Rules 5.2 and 49.5(a-bis), and Section 208 and Annex C of the Administrative Instructions.
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