AU2018202034A1 - Phased small rnas - Google Patents

Abstract

Abstract This invention discloses recombinant DNA constructs encoding phased small RNAs useful in regulating expression of one or more genes of interest. Also disclosed by this invention are transgenic plant cells, plants, and seeds containing a recombinant DNA construct of this invention

Description

invention discloses recombinant DNA constructs encoding phased small RNAs useful in regulating expression of one or more genes of interest. Also disclosed by this invention are transgenic plant cells, plants, and seeds containing a recombinant DNA construct of this invention

2018202034 22 Mar 2018

2018202034 22 Mar 2018

AUSTRALIA Patents Act 1990

Regulation 3.2

APPLICANT:

Invention Title:

Complete Specification

Standard Patent

Divisional

Monsanto Technology, LLC PHASED SMALL RNAS

The following statement is a full description of this invention, including the best method of performing it known to me:

PHASED SMALL RNAS

2018202034 22 Mar 2018

Cross-reference to Related Applications and Incorporation of Sequence Listings [0001] This application claims the benefit of priority of U. S. Provisional Patent Application 60/841,608, which was filed on 31 August 2006, and is a divisional of AU 2016204315, which is a divisional of AU 2013203734, which is a divisional of AU, 2007290367, ail of which are incorporated by reference in their entirety herein. The sequence listing contained in the file “38-2l(54702)A.ST25.txt” (file size of 21 KB in operating system MS-Windows, recorded on 31 August 2006, and filed with U. S. Provisional Patent Application 60/841,608 on 31 August 2006) is incorporated by reference in its entirety herein. The sequence listing contained in the file named “38-21(55702)B.txt”, which is 54 kilobytes (measured in operating system MS-Windows), recorded on 29 August 2007, and located in computer readable form on a compact disk (CD-R), is filed herewith and incorporated herein by reference.

Field of the Invention [0002] This invention discloses recombinant DNA constructs and phased small RNAs useful in regulating expression of one or more genes of interest. Also disclosed by this invention are non-natural transgenic plant cells, plants, and seeds containing a recombinant DNA construct of this invention. The reference to any prior art in this specification is not, and should not be taken as an acknowledgement or any form of suggestion that such art forms part of the common general knowledge in Australia.

Background of the Invention [0003] Methods of gene suppression include use of anti-sense, co-suppression, and RNA interference. Anti-sense gene suppression in plants is described by Shewmaker et al. in United States Patents 5,107,065, 5453,566, and 5,759,829. Gene suppression in bacteria using DNA which is complementary to mRNA encoding the gene to be suppressed is disclosed by Inouye et al. in United States Patents 5,190,931, 5,208,149, and 5,272,065. RNA interference or RNA-mediated gene suppression has been described by, e. g., Redenbaugh et al. in “Safety Assessment of Genetically Engineered Fruits and Vegetables”, CRC Press, 1992; Chuang et al. (2000) PNAS, 97:4985-4990; and Wesley et al. (2001) Plant J., 27:581-590.

[0004] Several cellular pathways involved in RNA-mediated gene suppression have been described, each distinguished by a characteristic pathway and specific components. See, for example, the reviews by Brodersen and Voinnet (2006), Trends Genetics, 22:268-280, and Tomari and Zamore (2005) Genes & Dev., 19:517-529. The siRNA pathway involves the nonphased cleavage of a double-stranded RNA to small interfering RNAs (“siRNAs”). The

WO 2008/027592

PCT/US2007/019283

2018202034 22 Mar 2018 microRNA pathway involves microRNAs (“miRNAs”), non-protein coding RNAs generally of between about 19 to about 25 nucleotides (commonly about 20 - 24 nucleotides in plants) that guide cleavage in trans of target transcripts, negatively regulating the expression of genes involved in various regulation and development pathways; see Ambros et a/. (2003) RNA, 9:277-279. Plant miRNAs have been defined by a set of characteristics including a paired stem-loop precursor that is processed by DCL1 to a single specific -21-nucleotide miRNA, expression of a single pair of miRNA and miRNA* species from the double-stranded RNA precursor with two-nucleotide 3’ overhangs, and silencing of specific targets in trans. See Bartel (2004) Cell, 116:281-297; Kim (2005) Nature Rev. Mol. Cell Biol., 6:376-385; JonesRhoades et al. (2006) Annu. Rev. Plant Biol., 57:19-53; Ambros et al. (2003) RNA, 9:277-279. In the trans-acting siRNA (“ta-siRNA”) pathway, miRNAs serve to guide in-phase processing of siRNA primary transcripts in a process that requires an RNA-dependent RNA polymerase for production of a double-stranded RNA precursor; trans-acting siRNAs are defined by lack of secondary structure, a miRNA target site that initiates production of double-stranded RNA, requirements of DCL4 and an RNA-dependent RNA polymerase (RDR6), and production of multiple perfectly phased ~21-nt small RNAs with perfectly matched duplexes with 2nucleotide 3’ overhangs (see Allen etal. (2005) Cell, 121:207-221).

[0005] This invention discloses a novel pathway for RNA-mediated gene suppression, based on an endogenous locus termed a “phased small RNA locus”, which transcribes to an RNA transcript forming a single foldback structure that is cleaved in phase in vivo into multiple small double-stranded RNAs (termed “phased small RNAs”) capable of suppressing a target gene. In contrast to siRNAs, a phased small RNA transcript is cleaved in phase. In contrast to miRNAs, a phased small RNA transcript is cleaved by DCL4 or a DCL4like orthologous ribonuclease (not DCL1) to multiple abundant small RNAs capable of silencing a target gene. In contrast to the ta-siRNA pathway, the phased small RNA locus transcribes to an RNA transcript that forms hybridized RNA independently of an RNAdependent RNA polymerase and without a miRNA target site that initiates production of double-stranded RNA. Novel recombinant DNA constructs that are designed based on a phased small RNA locus are useful for suppression of one or multiple target genes, without the use of miRNAs, ta-siRNAs, or expression vectors designed to form a hairpin structure for processing to siRNAs. Furthermore, the recognition sites corresponding to a phased small RNA are useful for suppression of a target sequence in a cell or tissue where the appropriate phased small RNA is expressed endogenously or as a transgene.

38-21(54702)B PCT

2018202034 22 Mar 2018

Summary of the Invention [0006] In one aspect, this invention provides a recombinant DNA construct encoding a transcript that folds into hybridized RNA that is cleaved in phase in vivo into multiple small double-stranded RNAs (“phased small RNAs”) for gene suppression, preferably wherein the hybridized RNA is produced independently of an RNA-dependent RNA polymerase and is cleaved in phase in vivo by DCL4 or a DCL4-like ribonuclease (such as a DCL4 orthologue from any monocot or dicot plant).

[0007] Another aspect of this invention provides a recombinant DNA construct including DNA that transcribes to: (a) a first series of contiguous RNA segments, and (b) a second series of contiguous RNA segments, wherein the first series of contiguous RNA segments hybridize in vivo to the second series of RNA segments to form hybridized RNA that is cleaved in phase in vivo into multiple small double-stranded RNAs (“phased small RNAs”) for gene suppression.

[0008] In a further aspect, this invention provides a recombinant DNA construct including a promoter operably linked to DNA that transcribes to RNA including: (a) at least one exogenous recognition site recognizable by a phased small RNA expressed in a specific ceil of a multicellular eukaryote, and (b) target RNA to be suppressed in the specific cell, wherein the target RNA is to be expressed in ceils of the multicellular eukaryote other than the specific ceil.

[0009] Other aspects of this invention are methods of use of the recombinant DNA constructs of this invention for providing protection to plants from pests or pathogens, as well as non-natural transgenic plant cells, plants, and seeds containing in their genome a recombinant DNA construct of this invention. Other specific embodiments of the invention are disclosed in the following detailed description. Throughout this specification and the claims, unless the context requires otherwise, the word “comprise” and its variations, such as “comprises” and “comprising,” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

Brief Description of the Drawings [0010] Figure 1 depicts a non-limiting hybridized RNA that is cleaved in phase in vivo into multiple small double-stranded RNAs (“phased small RNAs”) of this invention, as described in Example 1.

[0011] Figure 2 depicts a non-limiting example of a genomic DNA sequence (SEQ ID NO. 9) including the cDNA sequence, intronic sequence, and foldback arms which transcribe to hybridized RNA of this invention, as described in Example 1.

WO 2008/027592

PCT/US2007/019283

2018202034 22 Mar 2018 [0012] Figure 3 depicts results of gene silencing in maize embryos using a recombinant DNA construct including DNA that transcribes to RNA containing a recognition site corresponding to at least one phased small RNA of this invention, as described in Example

2. . · [0013] Figure 4 depicts the results of northern blot analysis of tissue from developing maize kernels using a single oligonucleotide probe with sequence complementary to an endogenous phased small RNA, as described in Example 2.

[0014] Figure 5 depicts the predicted secondary structure of a phased small RNA template sequence (SEQ ID NO. 17) based on an endogenous phased small RNA locus, and of an engineered gene suppression construct (SEQ ID NO. 32) that is designed to suppress multiple target genes, as described in Example 3.

[0015] Figure 6 depicts the RNA transcript (SEQ ID NO. 34) containing hybridized RNA in a single foldback structure predicted from an endogenous phased small RNA locus (LOC_Osl2g42380.1|11982.m08017) having the sequence of SEQ ID NO. 33, as described in Example 4. This locus was identified from rice mature grain and seedling, and contained the phased small RNAs listed in Table 4. The transcript (SEQ ID NO. 34) includes 5’ flanking sequence (SEQ ID NO. 66) and 3’ flanking sequence (SEQ ID NO. 67), and a spacer sequence (SEQ ID NO. 68), located between the 5’ and 3’ arms of the foldback structure. The 5’ and 3’ termini of the transcript are indicated at the top of the figure; at the bottom of the figure, the transcript is shown to have a compact turn or loop of only 3 nucleotides.

[0016] Figure 7A depicts the small RNA abundance in transcripts per quarter million sequences (“tpq”) along about 2 kilobases of the phased small RNA locus having the sequence of SEQ ID NO. 33, as described in Example 4. Figure 7B depicts an expanded view of this small RNA region and the 21-nucleotide phasing of the small RNA abundance.

[0017] Figure 8 depicts results of studies further characterizing the 0s06g21900 phased small RNA locus, as described in Example 5. Figure 8A depicts the transcript corresponding to a precursor cloned from the 0s06g21900 phased sRNA locus. This precursor contained the phased small RNAs distributed between two regions along the transcript. Figure 8B depicts the two exons (Exons 2 and 3, indicated by the shaded regions) contained within this locus and that form a long, imperfect foldback structure containing eight 21-nucleotide phased small RNAs, separated by an -1.2 kB intron. Figure 8C depicts results of Southern blot analysis confirming that expression of the phased small RNA from the 0s06g21900 phased sRNA locus is found in rice grain but not rice seedlings or in other plant species tested. Figure 8D depicts results of Southern blot analysis of transformed Arabidopsis thaliana Columbia (Col-0) ecotype and mutants dell-7 and dcl4-l\ the blot was analyzed with probes

WO 2008/027592

PCT/US2007/019283

2018202034 22 Mar 2018 corresponding to phased small RNAs “P7” (SEQ ID NO. 6), and “P5” (SEQ ID NO. 4), a canonical miRNA (miR173) and a frans-acting siRNA (ta-siR255). These results demonstrate that the 0s06g21900 phased small RNA locus was efficiently processed in a dicot plant and required DCL4, not DCL1, to be cleaved in phase.

[0018] Figure 9 depicts the hybridized RNA structures predicted from transcripts of a naturally occurring phased small RNA locus and a synthetic phased small RNA locus, as described in Example 7. Figure 9A depicts the foldback structure of the transcript of the endogenous Os06g21900 phased small RNA locus (SEQ ID NO. 69); Figure 9B depicts the foldback structure of the synthetic phased small RNA precursor encoded by SEQ ID NO. 77.

Detailed Description of the Invention [0019] Unless defined otherwise, all technical and scientific terms used have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used and the manufacture or laboratory procedures described below are well known and commonly employed in the art. Conventional methods are used for these procedures, such as those provided in the art and various general references. Unless otherwise stated, nucleic acid sequences in the text of this specification are given, when read from left to right, in the 5’ to 3’ direction. Nucleic acid sequences may be provided as DNA or as RNA, as specified; disclosure of one necessarily defines the other, as is known to one of ordinary skill in the art. Where a term is provided in the singular, the inventors also contemplate aspects of the invention described by the plural of that term. The nomenclature used and the laboratory procedures described below are those well known and commonly employed in the art. Where there are discrepancies in terms and definitions used in references that are incorporated by reference, the terms used in this application shall have the definitions given. Other technical terms used have their ordinary meaning in the art that they are used, as exemplified by a variety of technical dictionaries. The inventors do not intend to be limited to a mechanism or mode of action. Reference thereto is provided for illustrative purposes only.

Recombinant DNA Construct Encoding a Transcript that Folds into Hybridized RNA that is Cleaved in Phase in Vivo [0020] This invention provides a recombinant DNA construct encoding a transcript that folds into hybridized RNA that is cleaved in phase in vivo into multiple small doublestranded RNAs for gene suppression. By “hybridized RNA” is meant RNA that has undergone Watson-Crick base-pairing between strands of RNA that are substantially complementary, thus

WO 2008/027592

PCT/US2007/019283

2018202034 22 Mar 2018 forming RNA that is substantially, but preferably not completely, base-paired or annealed between strands; this is in contrast to conventional siRNA production from two perfectly or near-perfectly base-paired strands that form fully double-stranded RNA. In one preferred embodiment, “hybridized RNA” includes two single strands of RNA that are part of a single molecule, where the two single strands are substantially complementary and arranged antiparallel to each other, thus allowing the two single strands to base-pair to each other; formation of the hybridized RNA occurs by intramolecular base-pairing. In an alternative embodiment, “hybridized RNA” includes two single strands of RNA that each are part of a separate molecule; formation of the hybridized RNA occurs by intermolecular base-pairing. In a particularly preferred embodiment, the recombinant DNA encodes an RNA transcript that forms a foldback structure including two anti-parallel single-stranded arms, wherein one arm is substantially but not perfectly complementary with the other arm, and base-pairing between the two arms of the foldback structure results in substantially but not perfectly double-stranded RNA that includes mismatches; preferably the mismatches are distributed along the length of the hybridized RNA (such as at least one mismatch within a given segment of 21 contiguous nucleotides), such as is depicted in Figure 1, Figure 6, and Figure 8B. Thus, in one preferred embodiment of this invention, the hybridized RNA includes a structure derived from a transcript of a naturally occurring phased small RNA locus, such as a structure selected from the structures depicted in Figure 1, Figure 5, Figure 6, and Figure 8B.

[0021] The hybridized RNA is produced independently of an RNA-dependent RNA polymerase; this is in contrast to irans-siRNA production wherein double-stranded RNA is formed through the action of an RNA-dependent RNA polymerase that synthesizes a complementary strand using the original RNA transcript as a template. Preferably, the nucleotides that form the hybridized RNA are nucleotides of the original RNA transcript (the “plus” strand) transcribed from the recombinant DNA construct, and not nucleotides on a second RNA molecule such as one (the “minus” strand) formed by the action of an RNAdependent RNA polymerase on the original RNA transcript.

[0022] By “cleaved in phase” in vivo is meant that the hybridized RNA is enzymatically processed or “cut” in vivo into shorter segments wherein the site of cleavage is not randomly distributed along the hybridized RNA. The hybridized RNA is processed in vivo by a ribonuclease into multiple, shorter, substantially (but preferably not perfectly) doublestranded segments, that is to say, multiple small substantially double-stranded RNAs, which are arranged in a contiguous fashion, such as is depicted in Figure 1, Figure 6, and Figure 8B; this is in contrast to canonical microRNAs, wherein there is substantial accumulation of only a single mature microRNA from the precursor transcript. The ribonucleotide cleaves the

WO 2008/027592

PCT/US2007/019283

2018202034 22 Mar 2018 hybridized RNA non-randomly, resulting in a frequency distribution of the multiple small double-stranded RNAs that is phased; this is in contrast to conventional siRNA production wherein the site of cleavage does not result in a phased frequency distribution of siRNAs. Preferably the phasing of this frequency distribution is about 21 nucleotides, such as is depicted in Figure 7. Thus, the multiple small double-stranded RNAs produced from a recombinant construct of this invention are termed “phased small RNAs”. The term “phased small RNAs” is also applied to a single strand of the pair of strands that forms a given small double-stranded RNA produced from a recombinant construct of this invention; in a preferred embodiment, one strand of the pair accumulates to a greater level than the other.

[0023] In a preferred embodiment, the hybridized RNA is cleaved in phase in vivo by a ribonuclease other than a DCL1 ribonuclease. Iri a most preferred embodiment, the hybridized RNA is cleaved in phase in vivo by DCL4 or a DCL4-like ribonuclease (such as a DCL4 orthologue from any monocot or dicot plant, such as, but not limited to, plants of commercial or agricultural interest, such as crop plants (especially crop plants used for human food or animal feed), wood-, fiber-, pulp-, or cellulose-producing trees and plants, vegetable plants, fruit plants, and ornamental plants, such as those listed below under the heading “Making and Using Transgenic Plant Cells and Transgenic Plants”) into multiple small doublestranded RNAs (“phased small RNAs”) for gene suppression.

[0024] In a preferred embodiment hybridized RNA is cleaved in phase in vivo to at least three small double-stranded RNAs (“phased small RNA”). Various embodiments encompassed by this invention include recombinant DNA constructs that give rise to 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or even more phased small RNAs, respectively.

[0025] Each phased small RNA is contiguous to the next; non-limiting examples of this arrangement are depicted in Figure 1, Figure 6, and Figure 8B. Each phased small RNA includes two anti-parallel RNA segments, each of which contains from about 20 to about 27 nucleotides (e. g., 20, 21, 22, 23, 24, 25, 26, or 27 nucleotides). The base-pairing between the two anti-parallel RNA segments is sufficient for them to form a substantially double-stranded RNA. In a preferred embodiment, each small double-stranded RNA or phased small RNA includes at least one mismatch. A mismatch generally includes one or more non-base-paired nucleotides (on either or on both segments), forming a “bump” or “bulge” or “loop” within the otherwise base-paired double-stranded RNA. In a preferred embodiment, each phased small RNA includes one or more unpaired bases forming an overhang at one or at both ends; most preferably the overhang is a 2-nucleotide overhang such as is depicted in Figure 1, Figure 6, and Figure 8B.

WO 2008/027592

PCT/US2007/019283

2018202034 22 Mar 2018 [0026] Each phased small RNA preferably is capable of suppressing a target gene. In one embodiment, the gene suppression by the phased small RNAs is of one target gene; for example, each phased small RNA suppresses the same segment of a single target gene or different segments of a single target gene. In another embodiment, the gene suppression by the phased small RNAs is of multiple target genes. While the inventors do not limit themselves to any single mechanism of action, it is most preferred that one of the two anti-parallel strands that makes up each phased small RNA is substantially complementary, or near perfectly complementary, or even perfectly complementary, to a target gene or sequence, as described below under the heading “Target Genes”, which also provides a detailed discussion of suitable target genes.

’ [0027] In a preferred embodiment of this invention, the recombinant DNA construct includes a nucleotide sequence derived from a phased small RNA template sequence selected from the group consisting of SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 17, SEQ ID NO. 33, the DNA sequence encoding SEQ ID NO. 34, and SEQ ID NO. 69, wherein at least one segment of 21 contiguous nucleotides in the phased small RNA template sequence is modified to suppress a target gene (that is, modified to suppress a gene other than the native target of the native phased small RNA). In a preferred embodiment of this invention, the recombinant DNA construct includes a nucleotide sequence derived from a phased small RNA template sequence selected from the group consisting of SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 17, SEQ ID NO. 33, the DNA sequence encoding SEQ ID NO. 34, and SEQ ID NO. 69, wherein multiple segments of about 20 to about 28 (e. g., 20, 21, 22, 23, 24, 25, 26, 27, or 28) contiguous nucleotides are modified such that the transcript is cleaved in phase in vivo to multiple synthetic phased small RNAs that suppress a target gene.

[0028] In another preferred embodiment of this invention, the recombinant DNA construct includes at least one nucleotide sequence selected from the group consisting of SEQ ID NO. 18, SEQ ID NO. 23, SEQ ID NO. 27, the DNA sequence encoding SEQ ID NO. 66, the DNA sequence encoding SEQ ID NO. 67, and the DNA sequence encoding SEQ ID NO. 68.

A Recombinant DNA Construct Transcribing to a First and Second Series of Contiguous RNA Segments that Form Hybridized RNA that is Cleaved in Phase in Vivo [0029] Another aspect of this invention provides a recombinant DNA construct including DNA that transcribes to: (a) a first series of contiguous RNA segments, and (b) a second series of contiguous RNA segments, wherein the first series of contiguous RNA .

WO 2008/027592

PCT/US2007/019283

2018202034 22 Mar 2018 segments hybridize in vivo to the second series of RNA segments to form hybridized RNA that is cleaved in phase in vivo into multiple small double-stranded RNAs (“phased small RNAs”) for gene suppression. This recombinant DNA construct can include a naturally occurring phased small RNA locus, such as is described in this disclosure, or can include a non-naturally occurring, synthetic sequence. Most preferably, the hybridized RNA is cleaved in phase by DCL4 or a DCL4-like ribonuclease (such as a DCL4 orthologue from any monocot or dicot plant, such those listed below under the heading “Making and Using Transgenic Plant Cells and Transgenic Plants”).

[0030] In preferred embodiments of the recombinant DNA construct, each of the RNA segments consists of between 20 to 27 nucleotides. In a particularly preferred embodiment, the first and second series include RNA segments of 21 nucleotides. Each pair of small RNAs that make up a given small double-stranded RNA (“phased small RNA”) can be of the same length, or can be of different lengths, such as illustrated in Figure 8B and Table 4. In a preferred embodiment, the pair of small RNAs that make up a given small double-stranded RNA (“phased small RNA”) is a pair of 21-nucleotide small RNAs. In one embodiment, the contiguous RNA segments are all of the same size. In a preferred embodiment, the RNA segments all consist of 21 nucleotides. In another embodiment, the RNA segments vary in size; preferably they are sized such that when the first series and second series of contiguous RNA segments hybridize, the individual segments are aligned in a manner permitting cleavage in phase to the intended small double-stranded RNAs. In a preferred embodiment, the first and second series of contiguous RNA segments contain an equal number of RNA segments which are arranged such that when the first series and second series of contiguous RNA segments hybridize and form an RNA duplex (hybridized RNA), a RNA segment of a given size in the first series is hybridized to a corresponding RNA segment in the second series of equivalent size.

[0031] In a preferred embodiment of the recombinant DNA construct, strands of the hybridized RNA are located on a single molecule and the construct further includes DNA that transcribes to a spacer that links the first and second series of contiguous RNA segments. The spacer generally includes DNA that does not correspond to the target gene (although in some embodiments can include sense or anti-sense sequence of the target gene). The spacer includes at least about 4 nucleotides; in various embodiments, the spacer contains at least about 4, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 80, at least about 100, at least about 120, or at least about 150 nucleotides. In one embodiment, the spacer is a contiguous nucleotide sequence derived from the spacer of a transcript of a naturally occurring phased small RNA locus. Non-limiting

WO 2008/027592

PCT/US2007/019283

2018202034 22 Mar 2018 examples of spacers are provided by SEQ ID NO. 23 and SEQ ID NO. 68 as well as spacers having at least 90% sequence identity with either of SEQ ID NO. 23 and SEQ ID NO. 68.

[0032] In one embodiment, the spacer is designed to transcribed to single-stranded RNA or to at least partially double-stranded RNA (such as in a “kissing stem-loop” arrangement), or to an RNA that assumes a secondary structure or three-dimensional configuration (e. g., a large loop of anti-sense sequence of the target gene or an aptamer) that confers on the transcript an additional desired characteristic, such as increased stability, increased half-life in vivo, or cell or tissue specificity. In one example, the spacer is transcribed to a stabilizing loop that links the first and second series of contiguous RNA segments (see, for example, Di Giusto and King (2004) J. Biol. Chern., 279:46483-46489). In another example, the spacer transcribes to RNA including an RNA aptamer (e. g., an aptamer that binds to a cell-specific ligand) that allows cell- or tissue-specific targetting of the phased small RNAs.

[0033] In many embodiments, the recombinant DNA construct further includes DNA encoding 5’ flanking sequence (e. g., SEQ ID NO. 18 and SEQ ID NO. 66) and/or 3’ flanking sequence (e. g., SEQ ID NO. 27 and SEQ ID NO. 67) that are adjacent to the portion of the transcript that is the hybridized RNA. In other embodiments of the recombinant DNA construct, the strands of the hybridized RNA are located on separate molecules. Preferred embodiments of the invention include a recombinant DNA construct including at least one nucleotide sequence derived from a transcript of a naturally occurring phased small RNA locus selected from the group consisting of 5’ flanking sequence, 3’ flanking sequence, and spacer sequence. Non-limiting embodiments include a recombinant DNA construct including at least one nucleotide sequence selected from the group consisting of SEQ ID NO. 18, SEQ ID NO. 23, SEQ ID NO. 27, the DNA sequence encoding SEQ ID NO. 66, the DNA sequence encoding SEQ ID NO. 67, and the DNA sequence encoding SEQ ID NO. 68. Additional embodiments of the invention include a recombinant DNA construct including at least one nucleotide sequence having at least 90% sequence identity with any of SEQ ID NO. 18, SEQ ID NO. 23, SEQ ID NO. 27, the DNA sequence encoding SEQ ID NO. 66, the DNA sequence encoding SEQ ID NO. 67, and the DNA sequence encoding SEQ ID NO. 68.

[0034] Recombinant DNA constructs of this invention are designed to suppress one or more target genes as described below under the heading “Target Genes”. The construct is designed so that each RNA segment of the first series hybridizes to an RNA segment of the second series; the transcript transcribed from the construct is cleaved in phase in vivo, resulting in multiple small double-stranded RNAs (“phased small RNAs”) which correspond to the paired hybridized RNA segments. Preferably, each of the phased small RNAs contains at least

WO 2008/027592

PCT/US2007/019283

2018202034 22 Mar 2018 one mismatch. A mismatch generally includes one or more non-base-paired nucleotides (on either or on both segments), forming a “bump” or “bulge” or “loop” within the otherwise basepaired double-stranded RNA. In a preferred embodiment, each phased small RNA includes one or more unpaired bases forming an overhang at one or at both ends; most preferably the overhang is a 2-nucleotide overhang such as is depicted in Figure 1, Figure 6, and Figure 8B. In a preferred embodiment, at least one of the phased small RNAs is designed to suppress one or more target genes; that is, one of the two anti-parallel strands that makes up the phased small RNA is substantially complementary to a target gene.

[0035] The first and second series of contiguous RNA segments are designed so that when the first series and second series of contiguous RNA segments hybridize and form an RNA duplex (hybridized RNA), the small double-stranded RNAs resulting from cleavage inphase of the hybridized RNA silence the target gene or genes. Most preferably, the secondary structure of the transcript is maintained so as to be substantially similar to that of a naturally occurring phased small RNA locus (e. g., as depicted in Figure 1, Figure 6, and Figure 8B).

[0036] A general method for designing RNA segments corresponding to phased small RNAs for silencing a target gene, useful in making a recombinant DNA construct of this invention, includes the steps:

(a) Selecting a unique target sequence of at least 18 nucleotides specific to the target gene, e. g. by using sequence alignment tools such as BLAST (see, for example, Altschul et al. (1990) J. Mol. Biol., 215:403-410; Altschul et al. (1997) Nucleic Acids Res., 25:3389-3402), for example, of both maize cDNA and genomic DNA databases, to identify target transcript orthologues and any potential matches to unrelated genes, thereby avoiding unintentional silencing of non-target sequences.

(b) Analyzing the target gene for undesirable sequences (e. g., matches to sequences from non-target species, especially animals), and score each potential 19-mer segment for GC content, Reynolds score (see Reynolds et al. (2004) Nature Biotechnol., 22:326-330), and functional asymmetry characterized by a negative difference in free energy (“AAG”) (see Khvorova et al. (2003) Cell, 115:209-216). Preferably 19-mers are selected that have all or most of the following characteristics:

(1) a Reynolds score >4, (2) a GC content between about 40% to about 60%, (3) a negative AAG, (4) a terminal adenosine, (5) lack of a consecutive run of 4 or more of the same nucleotide; (6) a location near the 3’ terminus of the target gene; (7) minimal differences from the engineered phased siRNA precursor transcript. Preferably multiple (3 or more) 19-mers are selected for testing.

WO 2008/027592

PCT/US2007/019283

2018202034 22 Mar 2018 (c) Determining the reverse complement of the selected 19-mers to use in making a synthetic 21-mer phased small RNAs\; the additional nucleotide at position is preferably matched to the selected target sequence, and the nucleotide at position is preferably chosen to be unpaired to prevent spreading of silencing on the target transcript;

(d) Testing the synthetic phased small RNAs, for example, in an

Agrobacterium-mediated transient Nicotiana benthamiana assay for siRNA expression and target repression.

and (e) Cloning the most effective phased small RNAs into a construct for stable transformation of maize (see the sections under the headings “Making and Using Recombinant DNA Constructs” and “Making and Using Transgenic Plant Cells and Transgenic Plants”).

[0037] The recombinant DNA construct is made by commonly used techniques, such as those described under the heading “Making and Using Recombinant DNA Constructs” and illustrated in the working Examples. The recombinant DNA construct is particularly useful for making transgenic plant cells, transgenic plants, and transgenic seeds as discussed below under “Transgenic Plant Cells and Transgenic Plants”.

A Recombinant DNA Construct Including an Exogenous Phased Small RNA Recognition Site [0038] Another aspect of this invention includes a recombinant DNA construct including a promoter operably linked to DNA that transcribes to RNA including: (a) at least one exogenous recognition site recognizable by a phased small RNA expressed in a specific cell of a multicellular eukaryote, and (b) target RNA to be suppressed in the specific cell, wherein the target RNA is to be expressed in cells of the multicellular eukaryote other than the specific cell. The exogenous recognition site includes an RNA sequence that is substantially complementary, or near perfectly complementary, or even perfectly complementary, to a phased small RNA; the exogenous recognition site hybridizes to the phased small RNA, leading to suppression or silencing of the target RNA. The phased small RNA can be transcribed from a native or endogenous phased small RNA locus, or from a synthetic phased small RNA locus that is transgenically expressed. In various embodiments of the recombinant DNA construct, the at least one exogenous recognition site is located within at least one of: (a) the 5’ untranslated region of the target RNA; (b) the 3’ untranslated region of the target RNA; and (c) the target RNA.

WO 2008/027592

PCT/US2007/019283

2018202034 22 Mar 2018 [0039] In non-limiting embodiments, the exogenous recognition site is recognized by and silenced by a phased small RNA having a nucleotide sequence selected from the group consisting of SEQ ID NOS. 1, 2,3,4, 5,6,7,35,36, 37, 38, 39,40,41,42,43, 44,45,46,47, 48, 49, 50, 51, 52, 53,54, 55,56, 57, 58,59,60,61, 62, 63, 64, and 65. In preferred embodiments, the exogenous recognition site is recognized by and silenced by a phased small RNA having a nucleotide sequence selected from the group consisting of SEQ ID NOS. 1, 2, 3, 4, 5, 6, 7, 35, 37, 38, 39, 40, 41, 42, 43, 45, 46, 49, 51, 52, 53, 54, 55, 56, 60, 61, 62, 63, 64, and 65. In a particularly preferred embodiment, the exogenous recognition site is recognized by and silenced by a phased small RNA having a nucleotide sequence selected from the group consisting of SEQ ID NOS. 1, 2, 3, 4, 5, 6,7,37, 38, 40, 41, 46,61, 62,63, 64, and 65.

Non-Natural Transgenic Plant Cells and Transgenic Plants [0040] A further aspect of this invention provides a non-natural transgenic plant cell having in its genome any of the recombinant DNA constructs of this invention. Thus, the inventors claim a non-natural transgenic plant cell having in its genome a recombinant DNA construct encoding a transcript that folds into hybridized RNA that is cleaved in phase in vivo into multiple small double-stranded RNAs for gene suppression. The inventors also claim a non-natural transgenic plant cell having in its genome a recombinant DNA construct including DNA that transcribes to: (a) a first series of contiguous RNA segments, and (b) a second series of contiguous RNA segments, wherein the first series of contiguous RNA segments hybridize in vivo to the second series of RNA segments to form hybridized RNA that is cleaved in phase in vivo into multiple small double-stranded RNAs (“phased small RNAs”) for gene suppression. The inventors further claim a non-natural transgenic plant cell having in its genome a recombinant DNA construct including a promoter operably linked to DNA that transcribes to RNA including: (a) at least one exogenous recognition site recognizable by a phased small RNA expressed in a specific cell of a multicellular eukaryote, and (b) target RNA to be suppressed in the specific cell, wherein the target RNA is to be expressed in cells of the multicellular eukaryote other than the specific cell.

[0041] Also provided are a non-natural transgenic plant containing the non-natural transgenic plant cell of this invention, a non-natural transgenic plant grown from the nonnatural transgenic plant cell of this invention, and non-natural transgenic seed produced by the non-natural transgenic plants. The non-natural transgenic plant of this invention includes plants of any developmental stage, and includes a non-natural transgenic regenerated plant prepared from the non-natural transgenic plant cells disclosed herein, or a non-natural transgenic progeny plant (which can be an inbred or hybrid progeny plant) of the regenerated

WO 2008/027592

PCT/US2007/019283

2018202034 22 Mar 2018 plant, or non-natural transgenic seed of such a non-natural transgenic plant. Also provided and claimed is a non-natural transgenic seed having in its genome a recombinant DNA construct of this invention. The non-natural transgenic plant cells, non-natural transgenic plants, and nonnatural transgenic seeds of this invention are made by methods well-known in the art, as described below under the heading “Making and Using Transgenic Plant Cells and Transgenic Plants”.

[0042] The non-natural transgenic plant cell can include an isolated plant cell (e. g., individual plant cells or cells grown in or on an artificial culture medium), or can include a plant cell in undifferentiated tissue (e. g., callus or any aggregation of plant cells). The nonnatural transgenic plant cell can include a plant cell in at least one differentiated tissue selected from the group consisting of leaf (e. g., petiole and blade), root, stem (e. g., tuber, rhizome, stolon, bulb, and corm) stalk (e. g., xylem, phloem), wood, seed, fruit (e. g., nut, grain, fleshy fruits), and flower (e. g., stamen, filament, anther, pollen, carpel, pistil, ovary, ovules).

[0043] The non-natural transgenic plant cell or non-natural transgenic plant of the invention can be any suitable plant cell or plant of interest. Both transiently transformed and stably transformed plant cells are encompassed by this invention. Stably transformed transgenic plants are particularly preferred. In many preferred embodiments, the non-natural transgenic plant is a fertile transgenic plant from which seed can be harvested, and the invention further claims non-natural transgenic seed of such transgenic plants, wherein the seed preferably also contains the recombinant construct of this invention.

[0044] In some embodiments of this invention, the non-natural plant is a non-natural transgenic plant, and all cells (with the possible exception of haploid cells) and tissues of the plant contain the recombinant DNA construct of this invention. In other embodiments, the non-natural plant is not completely transgenic, but includes both non-natural transgenic cells or tissues and non-transgenic cells or tissues (for example, transgenic tissue grafted onto nontransgenic tissue). In a non-limiting embodiment, the plant includes a non-transgenic scion and a transgenic rootstock including the transgenic plant cell, wherein the non-transgenic scion and transgenic rootstock are grafted together. Such embodiments are particularly useful where the plant is one that is commonly vegetatively grown as a scion grafted onto a rootstock (wherein scion and rootstock can be of the same species or variety or of different species or variety); non-limiting examples include grapes (e. g., wine grapes and table grapes), apples, pears, quince, avocados, citrus, stone fruits (e. g., peaches, plums, nectarines, apricots, cherries), kiwifruit, roses, and other plants of agricultural or ornamental importance.

[0045] Also encompassed by this invention are non-natural plants that are not transgenic in the sense of having had recombinant DNA introduced into their genome, but are

WO 2008/027592

PCT/US2007/019283

2018202034 22 Mar 2018 non-natural plants having a genome that has been artificially modified by means other than recombinant DNA technology. Such artificial modifications of the native genomic sequence include insertions, deletions, substitutions, frame shifts, transpositions, duplications, and inversions. Artificial modification of a native genomic sequence is achieved by any means, including mutagenesis by chemicals (such as methane sulfonate, methyl methane sulfonate, diethylsulfate), nitrosoguanidine, and other alkylating agents, base analogues such as 5-bromodeoxyuridine, interchelating agents such as ethidium bromide, crosslinking agents such as platinum, and oxidating agents such as nitrous acid or reactive oxygen species) or mutagenesis by physical treatments (such as exposure to ultraviolet light, radioactive isotopes, or ionizing radiation). Such mutagenesis can be random or non-random (e. g., site-directed mutagenesis). Mutagenesis can be carried out with intact plants, plant tissues, or plant cells. One nonlimiting example of mutagenesis is treatment of maize pollen with an alkylating agent. Mutagenesis is generally carried out on a population, following screening of that population to allow selection of individuals having the desired property. These non-natural plants are useful in ways similar to those described below for transgenic plants; for example, they can be grown for production of seed or other harvestable parts, or used to grow progeny generations (including hybrid generations).

Target Genes [0046] Phased small RNAs of this invention and the recombinant DNA constructs encoding such can be designed to suppress any target gene or genes. The target gene can be translatable (coding) sequence, or can be non-coding sequence (such as non-coding regulatory sequence), or both, and can include at least one gene selected from the group consisting of a eukaryotic target gene, a non-eukaryotic target gene, a microRNA precursor DNA sequence, and a microRNA promoter. The target gene can be native or endogenous to the cell (e. g., a cell of a plant or animal) in which the recombinant DNA construct is transcribed, or can be native to a pest or pathogen of the plant or animal in which the recombinant DNA construct is transcribed. The target gene can be an exogenous gene, such as a transgene in a plant. A target gene can be a native gene targetted for suppression, with or without concurrent expression of an exogenous transgene, for example, by including a gene expression element in the recombinant DNA construct from which the phased small RNAs are transcribed, or in a separate recombinant DNA construct. For example, it can be desirable to replace a native gene with an exogenous transgene homologue.

[0047] The target gene can include a single gene or part of a single gene that is targetted for suppression, or can include, for example, multiple consecutive segments of a '15

WO 2008/027592

PCT/US2007/019283

2018202034 22 Mar 2018 target gene, multiple non-consecutive segments of a target gene, multiple alleles of a target gene, or multiple target genes from one or more species. A target gene can include any sequence from any species (including, but not limited to, non-eukaryotes such as bacteria, and viruses; fungi; plants, including monocots and di cots, such as crop plants, ornamental plants, and non-domesticated or wild plants; invertebrates such as arthropods, annelids, nematodes, and molluscs; and vertebrates such as amphibians, fish, birds, domestic or wild mammals, and even humans.

[0048] In one embodiment, the target gene is exogenous to the plant in which the recombinant DNA construct is to be transcribed, but endogenous to a pest or pathogen (e. g., viruses, bacteria, fungi, oomycetes, and invertebrates such as insects, nematodes, and ’ molluscs) of the plant. The target gene can include multiple target genes, or multiple segments of one or more genes. In one preferred embodiment, the target gene or genes is a gene or genes of an invertebrate pest or pathogen of the plant. These embodiments are particularly useful in providing transgenic plants having resistance to one or more plant pests or pathogens, for example, resistance to a nematode such as soybean cyst nematode or root knot nematode, or to a pest insect, or to at least one pathogenic virus, bacterium, or fungus.

[0049] The target gene can be translatable (coding) sequence, or can be non-coding sequence (such as non-coding regulatory sequence), or both. Non-limiting examples of a target gene include non-translatable (non-coding) sequence, such as, but not limited to, 5’ untranslated regions, promoters, enhancers, or other non-coding transcriptional regions, 3’ untranslated regions, terminators, and introns. Target genes include genes encoding microRNAs, small interfering RNAs, RNA components of ribosomes or ribozymes, small nucleolar RNAs, and other non-coding RNAs (see, for example, non-coding RNA sequences provided publicly at rfam.wustl.edu; Erdmann et al. (2001) Nucleic Acids Res., 29:189-193; Gottesman (2005) Trends Genet., 21:399-404; Griffiths-Jones et al. (2005) Nucleic Acids Res., 33:121-124). One specific example of a target gene includes a microRNA recognition site (that is, the site on an RNA strand to which a mature miRNA binds and induces cleavage). Another specific example of a target gene includes a microRNA precursor sequence native to a pest or pathogen of the transgenic plant, that is, the primary transcript encoding a microRNA, or the RNA intermediates processed from this primary transcript (e. g., a nuclear-limited primiRNA or a pre-miRNA which can be exported from the nucleus into the cytoplasm). See, for example, Lee et al. (2002) EMBO Journal, 21:4663-4670; Reinhart et al. (2002) Genes &

Dev., 16:161611626; Lund et al. (2004) Science, 303:95-98; and Millar and Waterhouse (2005) Funct. Integr Genomics, 5:129-135. Target genes can also include translatable (coding) sequence for genes encoding transcription factors and genes encoding enzymes involved in the

WO 2008/027592

PCT/US2007/019283

2018202034 22 Mar 2018 biosynthesis or catabolism of molecules of interest (such as, but not limited to, amino acids, fatty acids and other lipids, sugars and other carbohydrates, biological polymers, and secondary metabolites including alkaloids, terpenoids, polyketides, non-ribosomal peptides, and secondary metabolites of mixed biosynthetic origin).

[0050] In many preferred embodiments, the target gene is an essential gene of a plant pest or pathogen. Essential genes include genes that are required for development of the pest or pathogen to a fertile reproductive adult. Essential genes include genes that, when silenced or suppressed, result in the death of the organism (as an adult or at any developmental stage, including gametes) or in the organism’s inability to successfully reproduce (e. g., sterility in a male or female parent or lethality to the zygote, embryo, or larva). A description of nematode essential genes is found, e. g., in Kemphues, K. “Essential Genes” (December 24,2005), WormBook, ed. The C. elegans Research Community, WormBook, doi/10.1895/wormbook. 1.57.1, available on line at www.wormbook.org. Non-limiting examples of nematode essential genes include major sperm protein, RNA polymerase Π, and chitin synthase (see, e. g., U. S. Patent Application Publication US20040098761 Al); additional soybean cyst nematode essential genes are provided in U. S. Patent Application 11/360,355, filed 23 February 2006, incorporated by reference herein. A description of insect genes is publicly available at the Drosophila genome database (available on line at flybase.bio.indiana.edu/). The majority of predicted Drosophila genes have been analyzed for function by a cell culture-based RNA interference screen, resulting in 438 essential genes being identified; see Boutros et al. (2004) Science, 303:832-835, and supporting material available on line at www.sciencemag.org/cgi/content/full/303/5659/832/DCl. A description of bacterial and fungal essential genes is provided in the Database of Essential Genes (“DEG”, available on line at tubic.tju.edu.cn/deg/); see Zhang et a/. (2004) Nucleic Acids Res., 32:D271D272.

[0051] Plant pest invertebrates include, but are not limited to, pest nematodes, pest molluscs (slugs and snails), and pest insects. Plant pathogens of interest include fungi, oomycetes, bacteria (e. g., the bacteria that cause leaf spotting, fireblight, crown gall, and bacterial wilt), mollicutes, and viruses (e. g., the viruses that cause mosaics, vein banding, flecking, spotting, or abnormal growth). See also G. N. Agrios, “Plant Pathology” (Fourth Edition), Academic Press, San Diego, 1997, 635 pp., for descriptions of fungi, bacteria, mollicutes (including mycoplasmas and spiroplasmas), viruses, nematodes, parasitic higher plants, and flagellate protozoans, all of which are plant pests or pathogens of interest. See also the continually updated compilation of plant pests and pathogens and the diseases caused by such on the American Phytopathological Society’s “Common Names of Plant Diseases”,

WO 2008/027592

PCT/US2007/019283

2018202034 22 Mar 2018 compiled by the Committee on Standardization of Common Names for Plant Diseases of The American Phytopathological Society, 1978-2005, available online at www.apsnet.org/online/common/top.asp.

[0052] Non-limiting examples of fungal plant pathogens of particular interest include,

e. g., the fungi that cause powdery mildew, rust, leaf spot and blight, damping-off, root rot, crown rot, cotton boll rot, stem canker, twig canker, vascular wilt, smut, or mold, including, but not limited to, Fusarium spp., Phakospora spp., Rhizoctonia spp., Aspergillus spp., Gibberella spp., Pyricularia spp., and Alternaria spp.. Specific examples of fungal plant pathogens include Phakospora pachirhizi (Asian soy rust), Puccinia sorghi (com common rust), Puccinia polysora (com Southern rust), Fusarium oxysporum and other Fusarium spp., Alternaria spp., Penicillium spp., Rhizoctonia solani, Exserohilum turcicum (Northern com leaf blight), Bipolaris maydis (Southern com leaf blight), Ustilago maydis (com smut), Fusarium graminearum (Gibberella zeae), Fusarium verticilliodes (Gibberella moniliformis),

F. proliferatum (G. fujikuroi var. intermedia), F. subglutinans (G. subglutinans), Diplodia maydis, Sporisorium holci-sorghi, Colletotrichum graminicola, Setosphaeria turcica, Aureobasidium zeae, Sclerotinia sclerotiorum, and the numerous fungal species provided in Tables 4 and 5 of U. S. Patent 6,194,636, which is incorporated in its entirety by reference herein. Non-limiting examples of plant pathogens include pathogens previously classified as fungi but more recently classified as oomycetes. Specific examples of oomycete plant pathogens of particular interest include members of the genus Pythium (e. g., Pythium aphanidermatum) and Phytophthora (e. g., Phytophthora infestans, Phytophthora sojae,) and organisms that cause downy mildew (e. g., Peronospora farinosa).

[0053] Non-limiting examples of bacterial pathogens include the mycoplasmas that cause yellows disease and spiroplasmas such as Spiroplasma kunkelii, which causes com stunt, eubacteria such as Pseudomonas avenae, Pseudomonas andropogonis, Erwinia stewartii, Pseudomonas syringae pv. syringae, Xylella fastidiosa, and the numerous bacterial species listed in Table 3 of U. S. Patent 6,194,636, which is incorporated in its entirety by reference herein.

[0054] Non-limiting examples of viral plant pathogens of particular interest include maize dwarf mosaic vims (MDMV), sugarcane mosaic vims (SCMV, formerly MDMV strain B), wheat streak mosaic virus (WSMV), maize chlorotic dwarf virus (MCDV), barley yellow dwarf vims (BYDV), banana bunchy top vims (BBTV), and the numerous viruses listed in Example 7 below and in Table 2 of U. S. Patent 6,194,636, which is incorporated in its entirety by reference herein.

WO 2008/027592

PCT/US2007/019283

2018202034 22 Mar 2018 [0055] Non-limiting examples of invertebrate pests include cyst nematodes Heterodera spp. especially soybean cyst nematode Heterodera glycines, root knot nematodes Meloidogyne spp., lance nematodes Hoplolaimus spp., stunt nematodes Tylenchorhynchus spp., spiral nematodes Helicotylenchus spp., lesion nematodes Pratylenchus spp., ring nematodes Criconema spp., foliar nematodes Aphelenchus spp. or Aphelenchoides spp., com rootworms, Lygus spp., aphids and similar sap-sucking insects such as phylloxera (Daktulosphaira vitifoliae), com borers, cutworms, armyworms, leafhoppers, Japanese beetles, grasshoppers, and other pest coleopterans, dipterans, and lepidopterans. Specific examples of invertebrate pests include pests capable of infesting the root systems of crop plants, e. g., northern com rootworm (Diabrotica barber!), southern com rootworm (Diabrotica undecimpunctata), Western com rootworm (Diabrotica virgifera), com root aphid (Anuraphis maidiradicis), black cutworm (Agrotis ipsilon), glassy cutworm (Crymodes devastator), dingy cutworm (Feltia ducens), claybacked cutworm (Agrotis gladiaria), wireworm (Melanotus spp., Aeolus mellillus), wheat wireworm (Aeolus mancus), sand wireworm (Horistonotus uhlerii), maize billbug (Sphenophorus maidis), timothy billbug (Sphenophorus zeae), bluegrass billbug (Sphenophorus parvulus), southern com billbug (Sphenophorus callosus), white grubs (Phyllophaga spp.), seedcom maggot (Delia platura), grape colaspis (Colaspis brunnea), seedcom beetle (Stenolophus lecontei), and slender seedcom beetle (Clivinia impressifrons), as well as the parasitic nematodes listed in Table 6 of U. S. Patent 6,194,636, which is incorporated in its entirety by reference herein.

[0056] Invertebrate pests of particular interest, especially in but not limited to southern hemisphere regions (including South and Central America) include aphids, com rootworms, spodoptera, noctuideae, potato beetle, Lygus spp., any hemipteran, homopteran, or heteropteran, any lepidopteran, any coleopteran, nematodes, cutworms, earworms, armyworms, borers, leaf rollers, and others. Arthropod pests specifically encompassed by this invention include various cutworm species including cutworm (Agrotis repleta), black cutworm (Agrotis ipsilon), cutworm (Anicla ignicans), granulate cutworm (Feltia subterranea), “gusano dspero” (Agrotis malefida)·, Mediterranean flour moth (Anagasta kuehniella), square-necked grain beetle (Cathartus quadricollis), flea beetle (Chaetocnema spp), rice moth (Coreyra cephalonica), com rootworm or “vaquita de San Antonio” (Diabotica speciosa), sugarcane borer (Diatraea saccharalis), lesser cornstalk borer (Elasmopalpus lignosellus), brown stink bug (Euschistus spp.), com earworm (Helicoverpa zea), flat grain beetle (Laemophloeus minutus), grass looper moth (Mods latipes), sawtoothed grain beetle (Oryzaephilus surinamensis), meal moth (Pyralisfarinalis), Indian meal moth (Plodia interpunctella), com leaf aphid (Rhopalosiphum maidis), brown burrowing bug or “chinche

WO 2008/027592

PCT/US2007/019283

2018202034 22 Mar 2018 subterrdnea (Scaptocoris castanea), greenbug (Schizaphis graminum), grain weevil (Sitophilus zeamais), Angoumois grain moth (Sitotroga cerealella), fall armyworm (Spodoptera frugiperda), cadelle beetle (Tenebraides mauritanicus), two-spotted spider mite (Tetranychus urticae), red flour beetle (Triboleum castaneum), cotton leafworm (Alabama argillacea), boll weevil (Anthonomus grandis), cotton aphid (Aphis gossypii), sweet potato whitefly (Bemisia tabaci), various thrips species (Frankliniella spp.), cotton earworm (Helicoverpa zea), “oruga bolillera” (e. g., Helicoverpa geletopoeon), tobacco budworm (Heliothis virescens), stinkbug (Nezara viridula), pink boll worm (Pectinophora gossypiella), beet armyworm (Spodoptera exigua), spider mites (Tetranychus spp.), onion thrips (Thrips tabaci), greenhouse whitefly (Trialeurodes vaporarium), velvetbean caterpillar (Anticarsia gemmatalis), spotted maize beetle or “astilo moteado” (Astylus atromaculatus), “oruga de la alfalfa” (Colias lesbia), “chinche marron” or “chinche de los cuemos” (Dichelops furcatus), “alquiche chico” (Edessa miditabunda), blister beetles (Epicauta spp.), “barrenador del brote” (Epinotia aporema), “oruga verde del yuyo Colorado” (Loxostege bifidalis), rootknot nematodes (Meloidogyne spp.), “oruga cuarteadora” (Mods repanda), southern green stink bug (Nezara viridula), “chinche de la alfalfa” (Piezodorus guildinii), green cloverworm (Plathypena scabra), soybean looper (Pseudoplusia includens), looper moth “isoca medidora del girasol” (Rachiplusia nu), yellow woolybear (Spilosoma virginica), yellowstriped armyworm (Spodoptera omithogalli),various root weevils (family Curculionidae), various wireworms (family Elateridae), and various white grubs (family Scarabaeidae). Nematode pests specifically encompassed by this invention include nematode pests of maize (Belonolaimus spp., Trichodorus spp., Longidorus spp., Dolichodorus spp., Anguina spp., Pratylenchus spp., Meloidogyne spp., Heterodera spp.), soybean (Heterodera glycines, Meloidogyne spp., Belonolaimus spp.), bananas (Radopholus similis, Meloidogyne spp., Helicotylenchus spp.), sugarcane (Heterodera sacchari, Pratylenchus spp., Meloidogyne spp.), oranges (Tylenchulus spp., Radopholus spp., Belonolaimus spp., Pratylenchus spp., Xiphinema spp.), coffee (Meloidogyne spp., Pratylenchus spp.), coconut palm (Bursaphelenchus spp.), tomatoes (Meloidogyne spp., Belonolaimus spp., Nacobbus spp.), grapes (Meloidogyne spp., Xiphinema spp., Tylenchulus spp., Criconemella spp.), lemon and lime (Tylenchulus spp., Radopholus spp., Belonolaimus spp., Pratylenchus spp., Xiphinema spp.), cacao (Meloidogyne spp., Rotylenchulus reniformis), pineapple (Meloidogyne spp., Pratylenchus spp.,

Rotylenchulus reniformis), papaya (Meloidogyne spp., Rotylenchulus reniformis), grapefruit (Tylenchulus spp., Radopholus spp. Belonolaimus spp., Pratylenchus spp., Xiphinema spp., and broad beans (Meloidogyne spp.).

WO 2008/027592

PCT/US2007/019283

2018202034 22 Mar 2018 [0057] Target genes from pests can include invertebrate genes for major sperm protein, alpha tubulin, beta tubulin, vacuolar ATPase, glyceraldehyde-3-phosphate dehydrogenase, RNA polymerase Π, chitin synthase, cytochromes, miRNAs, miRNA precursor molecules, miRNA promoters, as well as other genes such as those disclosed in U. S. Patent Application Publication 2006/0021087 Al, PCT Patent Application PCT/US05/11816, and in Table Π of U. S. Patent Application Publication 2004/0098761 Al, which are incorporated by reference herein. Target genes from pathogens can include genes for viral translation initiation factors, viral replicases, miRNAs, miRNA precursor molecules, fungal tubulin, fungal vacuolar ATPase, fungal chitin synthase, fungal MAP kinases, fungal Pad Tyr/Thr phosphatase, enzymes involved in nutrient transport (e. g., amino acid transporters or sugar transporters), enzymes involved in fungal celi wall biosynthesis, cutinases, melanin biosynthetic enzymes, polygalacturonases, pectinases, pectin lyases, cellulases, proteases, genes that interact with plant avirulence genes, and other genes involved in invasion and replication of the pathogen in the infected plant. Thus, a target gene need not be endogenous to the plant in which the recombinant DNA construct is transcribed. A recombinant DNA construct encoding phased small RNAs of the invention can be transcribed in a plant and used to suppress a gene of a pathogen or pest that may infest the plant.

[0058] Specific, non-limiting examples of suitable target genes also include amino acid catabolic genes (such as, but not limited to, the maize LKR/SDH gene encoding lysineketoglutarate reductase (LKR) and saccharopine dehydrogenase (SDH), and its homologues), maize zein genes, genes involved in fatty acid synthesis (e. g., plant microsomal fatty acid desaturases and plant acyl-ACP thioesterases, such as, but not limited to, those disclosed in U.

S. Patent Numbers 6,426,448, 6,372,965, and 6,872,872), genes involved in multi-step biosynthesis pathways, where it may be of interest to regulate the level of one or more intermediates, such as genes encoding enzymes for polyhydroxyalkanoate biosynthesis (see, for example, U. S. Patent No. 5,750,848); and genes encoding cell-cycle control proteins, such as proteins with cyclin-dependent kinase (CDK) inhibitor-like activity (see, for example, genes disclosed in International Patent Application Publication Number WO 05007829A2). Target genes can include genes encoding undesirable proteins (e. g., allergens Or toxins) or the enzymes for the biosynthesis of undesirable compounds (e. g., undesirable flavor or odor components). Thus, one embodiment of the invention is a transgenic plant or tissue of such a plant that is improved by the suppression of allergenic proteins or toxins, e. g., a peanut, soybean, or wheat kernel with decreased allergenicity. Target genes can include genes involved in fruit ripening, such as polygalacturonase. Target genes can include genes where expression is preferably limited to a particular cell or tissue or developmental stage, or where

WO 2008/027592

PCT/US2007/019283

2018202034 22 Mar 2018 expression is preferably transient, that is to say, where constitutive or general suppression, or suppression that spreads through many tissues, is not necessarily desired. Thus, other examples of suitable target genes include genes encoding proteins that, when expressed in transgenic plants, make the transgenic plants resistant to pests or pathogens (see, for example, genes for cholesterol oxidase as disclosed in U. S. Patent No. 5,763,245); genes where expression is pest- or pathogen-induced; and genes which can induce or restore fertility (see, for example, the barstar/bamase genes described in U. S. Patent No. 6,759,575); all the publications and patents cited in this paragraph are incorporated by reference in their entirety herein.

[0059] The phased small RNAs can be designed to be more specifically suppress the target gene, by designing the phased small RNAs to include regions substantially non-identical to a non-target gene sequence. Non-target genes can include any gene not intended to be silenced or suppressed, either in a plant containing the recombinant DNA construct encoding the phased small RNAs or in organisms that may come into contact with the phased small RNAs. A non-target gene sequence can include any sequence from any species (including, but not limited to, non-eukaryotes such as bacteria, and viruses; fungi; plants, including monocots and dicots, such as crop plants, ornamental plants, and non-domesticated or wild plants; invertebrates such as arthropods, annelids, nematodes, and molluscs; and vertebrates such as amphibians, fish, birds, domestic or wild mammals, and even humans).

[0060] In one embodiment, the target gene is a gene endogenous to a given species, such as a given plant (such as, but not limited to, agriculturally or commercially important plants, including monocots and dicots), and the non-target gene can be, e. g., a gene of a nontarget species, such as another plant species or a gene of a virus, fungus, bacterium, invertebrate, or vertebrate, even a human. One non-limiting example is where the phased small RNAs are designed to suppress a target gene that is a gene endogenous to a single species (e. g., Western com rootworm, Diabrotica virgifera virgifera LeConte) but to not suppress a non-target gene such as genes from related, even closely related, species (e. g., Northern com rootworm, Diabrotica barberi Smith and Lawrence, or Southern com rootworm, Diabrotica undecimpunctata).

[0061] In other embodiments (e. g., where it is desirable to suppress a target gene across multiple species), it may be desirable to design the phased small RNAs to suppress a target gene sequence common to the multiple species in which the target gene is to be silenced. Thus, a recombinant DNA construct encoding phased small RNAs can be designed to be specific for one taxon (for example, specific to a genus, family, or even a larger taxon such as a phylum, e. g., viruses or arthropoda) but not for other taxa (e. g., plants or vertebrates or

WO 2008/027592

PCT/US2007/019283

2018202034 22 Mar 2018 mammals). In one non-limiting example of this embodiment, a recombinant DNA construct encoding phased small RNAs can be selected so as to target pathogenic fungi (e. g., a Fusarium spp.) but not target any gene sequence from beneficial fungi.

[0062] In another non-limiting example of this embodiment, phased small RNAs for gene silencing in com rootworm can be selected to be specific to all members of the genus Diabrotica. In a further example of this embodiment, such DkzZ/roizca-targetted phased small RNAs can be selected so as to not target any gene sequence from beneficial coleopterans (for example, predatory coccinellid beetles, commonly known as ladybugs or ladybirds) or other beneficial insect species.

[0063] The required degree of specificity of an RNA for silencing a target gene depends on various factors. Factors can include the size of the phased small RNAs that are expected to be produced by the action of a ribonuclease (preferably DCL4 or a DCL4 orthologue) on the hybridized RNA, and the relative importance of decreasing the phased small RNAs’ potential to suppress non-target genes. In a non-limiting example, where the phased small RNAs are expected to be 21 base pairs in size, one particularly preferred embodiment includes RNA for silencing a target gene that encodes regions substantially non-identical to a non-target gene sequence, such as regions within which every contiguous fragment including at least 21 nucleotides matches fewer than 21 (e. g., fewer than 21, or fewer than 20, or fewer than 19, or fewer than 18, or fewer than 17) out of 21 contiguous nucleotides of a non-target gene sequence. In another embodiment, regions substantially non-identical to a non-target gene sequence include regions within which every contiguous fragment including at least 19 nucleotides matches fewer than 19 (e. g., fewer than 19, or fewer than 18, or fewer than 17, or fewer than 16) out of 19 contiguous nucleotides of a non-target gene sequence.

[0064] In some embodiments, it may be desirable to design phased small RNAs for silencing a target gene to include regions predicted to not generate undesirable polypeptides, for example, by screening the recombinant DNA construct encoding the phased small RNAs or each component phased small RNA for sequences that may encode known undesirable polypeptides or close homologues of these. Undesirable polypeptides include, but are not limited to, polypeptides homologous to known allergenic polypeptides and polypeptides homologous to known polypeptide toxins. Publicly available sequences encoding such undesirable potentially allergenic peptides are available, for example, the Food Allergy Research and Resource Program (FARRP) allergen database (available at allergenonline.com) or the Biotechnology Information for Food Safety Databases (available at www.iit.edu/~sgendel/fa.htm) (see also, for example, Gendel (1998) Adv. FoodNutr. Res., 42:63-92). Undesirable sequences can also include, for example, those polypeptide sequences

WO 2008/027592

PCT/US2007/019283

2018202034 22 Mar 2018 annotated as known toxins or as potential or known allergens and contained in publicly available databases such as GenBank, EMBL, SwissProt, and others, which are searchable by the Entrez system (www.ncbi.nih.gov/Entrez). Non-limiting examples of undesirable, potentially allergenic peptide sequences include glycinin from soybean, oleosin and agglutinin from peanut, glutenins from wheat, casein, lactalbumin, and lactoglobulin from bovine milk, and tropomyosin from various shellfish (allergenonline.com). Non-limiting examples of undesirable, potentially toxic peptides include tetanus toxin tetA from Clostridium tetani, diarrheal toxins from Staphylococcus aureus, and venoms such as conotoxins from Conus spp. and neurotoxins from arthropods and reptiles (www.ncbi.nih.gov/Entrez).

[0065] In one non-limiting example, the recombinant DNA construct encoding the phased small RNAs or each component phased small RNA is screened to eliminate those transcribable sequences encoding polypeptides with perfect homology to a known allergen or toxin over 8 contiguous amino acids, or with at least 35% identity over at least 80 amino acids; such screens can be performed on any and all possible reading frames in both directions, on potential open reading frames that begin with AUG (ATG in the corresponding DNA), or on all possible reading frames, regardless of whether they start with an AUG (or ATG) or not. When a “hit” or match is made, that is, when a sequence that encodes a potential polypeptide with perfect homology to a known allergen or toxin over 8 contiguous amino acids (or at least about 35% identity over at least about 80 amino acids), is identified, the nucleic acid sequences corresponding to the hit can be avoided, eliminated, or modified when selecting sequences to be used in an RNA for silencing a target gene. In one embodiment the recombinant DNA construct encoding the phased small RNAs or each component phased small RNA is designed so no potential open reading frames that begin with AUG (ATG in the corresponding DNA) is included.

[0066] Avoiding, elimination of, or modification of, an undesired sequence can be achieved by any of a number of methods known to those skilled in the art. In some cases, the result can be novel sequences that are believed to not exist naturally. For example, avoiding certain sequences can be accomplished by joining together “clean” sequences into novel chimeric sequences to be used in the recombinant DNA construct encoding the phased small RNAs.

[0067] Applicants recognize that in some dsRNA-mediated gene silencing, it is possible for imperfectly matching dsRNA sequences to be effective at gene silencing. For example, it has been shown that mismatches near the center of a miRNA complementary site has stronger effects on the miRNA’s gene silencing than do more distally located mismatches. See, for example, Figure 4 in Mallory et al. (2004) EMBO J., 23:3356-3364. In another

WO 2008/027592

PCT/US2007/019283

2018202034 22 Mar 2018 example, it has been reported that, both the position of a mismatched base pair and the identity of the nucleotides forming the mismatch influence the ability of a given siRNA to silence a target gene, and that adenine-cytosine mismatches, in addition to the G:U wobble base pair, were well tolerated (see Du et al. (2005) Nucleic Acids Res., 33:1671-1677). Thus, each phased small RNA need not always have 100% sequence complementarity with the intended target gene, but generally would preferably have substantial complementarity with the intended target gene, such as about 95%, about 90%, about 85%, or about 80% complementarity with the intended target gene. One strand of the hybridized RNA (or each component phased small RNA or RNA segment) is preferably designed to have substantial complementarity to the intended target (e. g., a target messenger RNA or target non-coding RNA), such as about 95%, about 90%, about 85%, or about 80% complementarity to the intended target. In a nonlimiting example, in the case of a component phased small RNA consisting of two 21nucleotide strands, one of the two 21-nucleotide strands is substantially but not perfectly complementary to 21 contiguous nucleotides of a target RNA; preferably the nucleotide at position 21 is unpaired with the corresponding position in the target RNA to prevent transitivity.

[0068] One skilled in the art would be capable of judging the importance given to screening for regions predicted to be more highly specific to the target gene or predicted to not generate undesirable polypeptides, relative to the importance given to other criteria, such as, but not limited to, the percent sequence identity with the intended target gene or the predicted gene silencing efficiency of a given sequence. For example, it may be desirable for the phased small RNAs to be active across several species, and therefore one skilled in the art can determine that it is more important to include in the recombinant DNA construct encoding the phased small RNAs regions specific to the several species of interest, but less important to screen for regions predicted to have higher gene silencing efficiency or for regions predicted to generate undesirable polypeptides.

Combinations of Phased Small RNAs with Pesticidal Compositions [0069] Phased small RNAs of this invention and the recombinant DNA constructs encoding such phased small RNAs are useful in controlling pests and pathogens of plants, and thus this invention further claims methods of controlling pests and pathogens of plant wherein a phased small RNA of this invention is provided to at least one cell of the plant to be protected from the pest or pathogen. The phased small RNA is generally provided by in vivo transcription of a recombinant DNA construct of this invention in a cell of the plant.

WO 2008/027592

PCT/US2007/019283

2018202034 22 Mar 2018 [0070] Methods of controlling pests and pathogens of plant with a phased small RNA of this invention can be used in combination with other methods or compositions for controlling pests and pathogens of plants. Thus, this invention also provides combinations of methods and combinations of compositions for controlling pests and pathogens of plants. In a preferred embodiment, the methods to be combined operate by different mechanisms to provide protection to the plant from the pest or pathogen. For example, invertebrate pests can be controlled by providing an alternative gene suppression element (such as an engineered microRNA or an engineered siRNA produced by a conventional double-stranded RNA transcript transgenically expressed in the plant) designed to silence a gene of the invertebrate pest; see, for example, the various recombinant DNA constructs and methods for gene suppression disclosed in U. S. Patent Application Publication 2006/0200878, which are incorporated herein by reference.

[0071] One non-limiting example is a method for controlling insect pests of plants, including providing at least one phased small RNA designed to silence an insect pest gene and an insecticidal agent (e. g., a Bacillus thuringiensis or “Bt” insecticidal protein) to which the insect pest is susceptible. When Bt proteins are provided in the diet of insect pests (e. g., via topical application to the plant or by transgenic expression in the plant) a mode of action for controlling the insect pest is exhibited that is dramatically different from the mode of action of the methods and compositions using phased small RNAs. In preferred embodiments, the combination results in synergies that were not known previously in the art for controlling insect infestation. Transgenic plants that produce one or more phased small RNA molecules that inhibit some essential biological function in a target pest along with one or more Bt insecticidal proteins that are toxic to the target pest provide surprising synergies. One synergy is the reduction in the level of expression required for either the phased small RNA(s) or the Bt insecticidal protein(s). When combined together, a lower effective dose of each pest control agent is required.

[0072] Another non-limiting example is a method for controlling viral pests of plants, including providing at least one phased small RNA designed to silence an viral pathogen gene and a composition to induce viral coat protein-mediated resistance to the plant (e. g., by transgenic expression of the viral coat protein in a plant cell). In preferred embodiments, the combination results in a synergy between the two protective components, so that a lower effective dose of each pathogen control agent is achieved.

WO 2008/027592

PCT/US2007/019283

2018202034 22 Mar 2018

Making and Using Recombinant DNA Constructs [0073] The recombinant DNA constructs of this invention are made by any method suitable to the intended application, taking into account, for example, the type of expression desired and convenience of use in the plant in which the construct is to be transcribed. General methods for making and using DNA constructs and vectors are well known in the art and described in detail in, for example, handbooks and laboratory manuals including Sambrook and Russell, “Molecular Cloning: A Laboratory Manual” (third edition), Cold Spring Harbor Laboratory Press, NY, 2001. An example of useful technology for building DNA constructs and vectors for transformation is disclosed in U. S. Patent Application Publication 2004/0115642 Al, incorporated herein by reference. DNA constructs can also be built using the GATEWAY™ cloning technology (available from Invitrogen Life Technologies, Carlsbad, CA), which uses the site-specific recombinase LR cloning reaction of the Integrase/αΖί system from bacteriophage lambda vector construction, instead of restriction endonucleases and ligases. The LR cloning reaction is disclosed in U. S. Patents 5,888,732 and 6,277,608, and in U; S. Patent Application Publications 2001/283529,2001/282319 and 2002/0007051, all of which are incorporated herein by reference. The GATEWAY™ Cloning Technology Instruction Manual, which is also supplied by Invitrogen, provides concise directions for routine cloning of any desired DNA into a vector comprising operable plant expression elements. Another alternative vector fabrication method employs ligation-independent cloning as disclosed by Aslandis et al. (1990) Nucleic Acids Res., 18:6069-6074 and Rashtchian et al. (1992) Biochem., 206:91-97, where a DNA fragment with single-stranded 5’ and 3’ ends is annealed to complementary 5' and 3' single-stranded ends of at least one other DNA fragment to produce a desired vector which can then be ligated and amplified in vivo.

[0074] In certain embodiments, the DNA sequence of the recombinant DNA construct includes sequence that has been codon-optimized for the plant in which the recombinant DNA construct is to be expressed. For example, a recombinant DNA construct to be expressed in a plant can have all or parts of its sequence (e. g., the first gene suppression element or the gene expression element) codon-optimized for expression in a plant by methods known in the art. See, e. g., U. S. Patent 5,500,365, incorporated by reference, for a description of codon-optimization for plants; see also De Amicis and Marchetti (2000) Nucleic Acid Res., 28.3339-3346.

Making and Using Transgenic Plant Cells and Transgenic Plants [0075] Where a recombinant DNA construct of this invention is used to produce a non-natural transgenic plant cell, non-natural transgenic plant, or non-natural transgenic seed

WO 2008/027592

PCT/US2007/019283

2018202034 22 Mar 2018 of this invention, transformation can include any of the well-known and demonstrated methods and compositions. Suitable methods for plant transformation include virtually any method by which DNA can be introduced into a cell, such as by direct delivery of DNA (e. g., by PEGmediated transformation of protoplasts, by electroporation, by agitation with silicon carbide fibers, and by acceleration of DNA coated particles), by Agrobacterium-mediated transformation, by viral or other vectors, etc. One preferred method of plant transformation is microprojectile bombardment, for example, as illustrated in U.S. Patents 5,015,580 (soy), 5,550,318 (maize), 5,538,880 (maize), 6,153,812 (wheat), 6,160,208 (maize), 6,288,312 (rice) and 6,399,861 (maize), and 6,403,865 (maize), all of which are incorporated by reference.

[0076] Another preferred method of plant transformation is Agrobacterium-mediated transformation. In one preferred embodiment, the non-natural transgenic plant cell of this invention is obtained by transformation by means of Agrobacterium containing a binary Ti plasmid system, wherein the Agrobacterium carries a first Ti plasmid and a second, chimeric plasmid containing at least one T-DNA border of a wild-type Ti plasmid, a promoter functional in the transformed plant cell and operably linked to a gene suppression construct of the invention. See, for example, the binary system described in U. S. Patent 5,159,135, incorporated by reference. Also see De Framond (1983) Biotechnology, 1:262-269; and Hoekema et al., (1983) Nature, 303:179. In such a binary system, the smaller plasmid, containing the T-DNA border or borders, can be conveniently constructed and manipulated in a suitable alternative host, such as E. coli, and then transferred into Agrobacterium.

[0077] Detailed procedures for Agrobacterium-medaated transformation of plants, especially crop plants, include, for example, procedures disclosed in U. S. Patents 5,004,863, 5,159,135, 5,518,908, 5,846,797, and 6,624,344 (cotton); 5,416,011, 5,569,834, 5,824,877, 5,914,451 6,384,301, and 7,002,058 (soy); 5,591,616 5,981,840, and 7,060,876 (maize); 5,463,174 and 5,750,871 (brassicas, including rapeseed and canola), and in U. S. Patent Application Publications 2004/0244075 (maize), 2004/0087030 (cotton) and 2005/0005321 (soy), all of which are incorporated by reference. Additional procedures for Agrobacteriummediated transformation are disclosed in WO9506722 (maize). Similar methods have been reported for many plant species, both dicots and monocots, including, among others, peanut (Cheng et al. (1996) Plant Cell Rep., 15: 653); asparagus (Bytebier et al. (1987) Proc. Natl. Acad. Sci. U.S.A., 84:5345); barley (Wan and Lemaux (1994) Plant Physiol., 104:37); rice (Toriyama etal. (1988) Bio/Technology, 6:10; Zhang et al. (1988) Plant Cell Rep., 7:379; wheat (Vasil et al. (1992) Bio/Technology, 10:667; Becker et al. (1994) Plant J. , 5:299), alfalfa (Masoud et al. (1996) Transgen. Res., 5:313); brassicas (Radke et al. (1992) Plant Cell Rep., 11:499-505); and tomato (Sun et al. (2006) Plant Cell Physiol., 47:426-431). See also a

WO 2008/027592

PCT/US2007/019283

2018202034 22 Mar 2018 description of vectors, transformation methods, and production of transformed Arabidopsis thaliana plants where transcription factors are constitutively expressed by a CaMV35S promoter, in U. S. Patent Application Publication 2003/0167537 Al, incorporated by reference. Non-natural transgenic plant cells and transgenic plants can also be obtained by transformation with other vectors, such as, but not limited to, viral vectors (e. g., tobacco etch potyvirus (TEV), barley stripe mosaic virus (BSMV), and the viruses referenced in Edwardson and Christie, The Potyvirus Group: Monograph No. 16, 1991, Agric. Exp. Station, Univ. of Florida), plasmids, cosmids, YACs (yeast artificial chromosomes), BACs (bacterial artificial chromosomes) or any other suitable cloning vector, when used with an appropriate transformation protocol, e. g., bacterial infection (e.g., with Agrobacterium as described above), binary bacterial artificial chromosome constructs, direct delivery of DNA (e. g., via PEG-mediated transformation, desiccation/inhibition-mediated DNA uptake, electroporation, agitation with silicon carbide fibers, and microprojectile bombardment). It would be clear to one of ordinary skill in the art that various transformation methodologies can be used and modified for production of stable transgenic plants from any number of plant species of interest.

[0078] Transformation methods to provide non-natural transgenic plant cells and nonnatural transgenic plants containing stably integrated recombinant DNA are preferably practiced in tissue culture on media and in a controlled environment. “Media” refers to the numerous nutrient mixtures that are used to grow cells in vitro, that is, outside of the intact living organism. Recipient cell targets include, but are not limited to, meristem cells, callus, immature embryos or parts of embryos, and gametic cells such as microspores, pollen, sperm, and egg cells. Any cell from which a fertile plant can be regenerated is contemplated as a useful recipient cell for practice of the invention. Callus can be initiated from various tissue sources, including, but not limited to, immature embryos or parts of embryos, seedling apical meristems, microspores, and the like. Those cells which are capable of proliferating as callus can serve as recipient cells for genetic transformation. Practical transformation methods and materials for making transgenic plants of this invention (e. g., various media and recipient target cells, transformation of immature embryos, and subsequent regeneration of fertile transgenic plants) are disclosed, for example, in U. S. Patents 6,194,636 and 6,232,526 and U.

S. Patent Application Publication 2004/0216189, which are incorporated by reference.

[0079] In general transformation practice, DNA is introduced into only a small percentage of target cells in any one transformation experiment. Marker genes are generally used to provide an efficient system for identification of those cells that are stably transformed by receiving and integrating a transgenic DNA construct into their genomes. Preferred marker

WO 2008/027592

PCT/US2007/019283

2018202034 22 Mar 2018 genes provide selective markers which confer resistance to a selective agent, such as an antibiotic or herbicide. Any of the antibiotics or herbicides to which a plant cell may be resistant can be a useful agent for selection. Potentially transformed cells are exposed to the selective agent. In the population of surviving cells will be those cells where, generally, the resistance-conferring gene is integrated and expressed at sufficient levels to permit cell survival. Cells can be tested further to confirm stable integration of the recombinant DNA. Commonly used selective marker genes include those conferring resistance to antibiotics such as kanamycin or paromomycin (nptll), hygromycin B (aph IV) and gentamycin (aac3 and aacC4) or resistance to herbicides such as glufosinate (bar or pat), glyphosate (EPSPS), and dicamba. Examples of useful selective marker genes and selection agents are illustrated in U.

S. Patents 5,550,318, 5,633,435,5,780,708, and 6,118,047, all of which are incorporated by reference. A particularly preferred herbicide resistance gene is a glyphosate acetyl transferase, disclosed as SEQ ID NO. 68 in U. S. Patent Application Publication 2007/0079393 Al, which is specifically incorporated by reference. Screenable markers or reporters, such as markers that provide an ability to visually identify transformants can also be employed. Non-limiting examples of useful screenable markers include, for example, a gene expressing a protein that produces a detectable color by acting on a chromogenic substrate (e. g., 7>eia-glucuromdase (GUS) (uidA.) or luciferase (luc)) or that itself is detectable, such as green fluorescent protein (GFP) (gfp) or an immunogenic molecule. Those of skill in the art will recognize that many, other useful markers or reporters are available for use.

[0080] Detecting or measuring transcription of the recombinant DNA construct in the non-natural transgenic plant cell of the invention can be achieved by any suitable method, including protein detection methods (e. g., western blots, ELISAs, and other immunochemical methods), measurements of enzymatic activity, or nucleic acid detection methods (e. g., Southern blots, northern blots, PCR, RT-PCR, fluorescent in situ hybridization). Such methods are well known to those of ordinary skill in the art as evidenced by-the numerous handbooks available; see, for example, Joseph Sambrook and David W. Russell, “Molecular Cloning: A Laboratory Manual” (third edition), Cold Spring Harbor Laboratory Press, NY, 2001; Frederick M. Ausubel etal. (editors) “Short Protocols in Molecular Biology” (fifth edition), John Wiley and Sons, 2002; John M. Walker (editor) “Protein Protocols Handbook” (second edition), Humana Press, 2002; and Leandro Pena (editor) “Transgenic Plants:

Methods and Protocols”, Humana Press, 2004.

[0081] Other suitable methods for detecting or measuring transcription of the recombinant DNA construct in the non-natural transgenic plant cell of the invention include measurement of any other trait that is a direct or proxy indication of suppression of the target

WO 2008/027592

PCT/US2007/019283

2018202034 22 Mar 2018 gene in the transgenic plant cell in which the recombinant DNA construct is transcribed, relative to one in which the recombinant DNA is not transcribed, e. g., gross or microscopic morphological traits, growth rates, yield, reproductive or recruitment rates, resistance to pests or pathogens, or resistance to biotic or abiotic stress (e. g., water deficit stress, salt stress, nutrient stress, heat or cold stress). Such methods can use direct measurements of a phenotypic trait or proxy assays (e. g., in plants, these assays include plant part assays such as leaf or root assays to determine tolerance of abiotic stress). Non-limiting methods include direct measurements of resistance to the invertebrate pest (e. g., damage to plant tissues) or proxy assays (e. g., plant yield assays, or bioassays such as the Western com rootworm (Diabrotica virgifera virgifera LeConte) larval bioassay described in International Patent Application Publication W02005/110068 A2 and U. S. Patent Application Publication US 2006/0021087 Al, incorporated by reference, or the soybean cyst nematode bioassay described by Steeves et al. (2006) Funct. Plant Biol., 33:991-999, wherein cysts per plant, cysts per gram root, eggs per plant, eggs per gram root, and eggs per cyst are measured.

[0082] The recombinant DNA constructs of the invention can be stacked with other recombinant DNA for imparting additional traits (e. g., in the case of transformed plants, traits including herbicide resistance, pest resistance, cold germination tolerance, water deficit tolerance, and the like) for example, by expressing or suppressing other genes. Constructs for coordinated decrease and increase of gene expression are disclosed in U.S. Patent Application Publication 2004/0126845 Al, incorporated by reference.

[0083] Seeds of transgenic, fertile plants can be harvested and used to grow progeny generations, including hybrid generations, of non-natural transgenic plants of this invention that include the recombinant DNA construct in their genome. Thus, in addition to direct transformation of a plant with a recombinant DNA construct of this invention, non-natural transgenic plants of the invention can be prepared by crossing a first plant having the recombinant DNA with a second plant lacking the construct. For example, the recombinant DNA can be introduced into a plant line that is amenable to transformation to produce a transgenic plant, which can be crossed with a second plant line to introgress the recombinant DNA into the resulting progeny. A transgenic plant of the invention can be crossed with a plant line having other recombinant DNA that confers one or more additional trait(s) (such as, but not limited to, herbicide resistance, pest or disease resistance, environmental stress resistance, modified nutrient content, and yield improvement) to produce progeny plants having recombinant DNA that confers both the desired target sequence expression behavior and the additional trait(s).

WO 2008/027592

PCT/US2007/019283

2018202034 22 Mar 2018 [0084] Typically, in such breeding for combining traits the transgenic plant donating the additional trait is a male line and the transgenic plant carrying the base traits is the female line. The progeny of this cross segregate such that some of the plant will carry the DNA for both parental traits and some will carry DNA for one parental trait; such plants can be identified by markers associated with parental recombinant DNA Progeny plants carrying DNA for both parental traits can be crossed back into the female parent line multiple times, e. g., usually 6 to 8 generations, to produce a progeny plant with substantially the same genotype as one original transgenic parental line but for the recombinant DNA of the other transgenic parental line.

[0085] Yet another aspect of the invention is a non-natural transgenic plant grown from the non-natural transgenic seed of the invention. This invention contemplates transgenic plants grown directly from transgenic seed containing the recombinant DNA as well as progeny generations of plants, including inbred or hybrid plant lines, made by crossing a transgenic plant grown directly from transgenic seed to a second plant not grown from the same transgenic seed.

[0086] Crossing can include, for example, the following steps:

(a) plant seeds of the first parent plant (e. g., non-transgenic or a transgenic) and a second parent plant that is transgenic according to the invention;

(b) grow the seeds of the first and second parent plants into plants that bear flowers;

(c) pollinate a flower from the first parent with pollen from the second parent; and (d) harvest seeds produced on the parent plant bearing the fertilized flower.

[0087] It is often desirable to introgress recombinant DNA into elite varieties, e. g., by backcrossing, to transfer a specific desirable trait from one source to an inbred or other plant that lacks that trait. This can be accomplished, for example, by first crossing a superior inbred (“A”) (recurrent parent) to a donor inbred (“B”) (non-recurrent parent), which carries the appropriate gene(s) for the trait in question, for example, a construct prepared in accordance with the current invention. The progeny of this cross first are selected in the resultant progeny for the desired trait to be transferred from the non-recurrent parent “B”, and then the selected progeny are mated back to the superior recurrent parent “A”. After five or more backcross generations with selection for the desired trait, the progeny are hemizygous for loci controlling the characteristic being transferred, but are like the superior parent for most or almost all other genes. The last backcross generation would be selfed to give progeny which are pure breeding for the gene(s) being transferred, i. e., one or more transformation events.

[0088] Through a series of breeding manipulations, a selected DNA construct can be moved from one line into an entirely different line without the need for-further recombinant

WO 2008/027592

PCT/US2007/019283

2018202034 22 Mar 2018 manipulation. One can thus produce inbred plants which are true breeding for one or more DNA constructs. By crossing different inbred plants, one can produce a large number of different hybrids with different combinations of DNA constructs. In this way, plants can be produced which have the desirable agronomic properties frequently associated with hybrids (“hybrid vigor”), as well as the desirable characteristics imparted by one or more DNA constructs.

[0089] Genetic markers can be used to assist in the introgression of one or more DNA constructs of the invention from one genetic background into another. Marker assisted selection offers advantages relative to conventional breeding in that it can be used to avoid errors caused by phenotypic variations. Further, genetic markers can provide data regarding the relative degree of elite germplasm in the individual progeny of a particular cross. For example, when a plant with a desired trait which otherwise has a non-agronomically desirable genetic background is crossed to an elite parent, genetic markers can be used to select progeny which not only possess the trait of interest, but also have a relatively large proportion of the desired germplasm. In this way, the number of generations required to retrogress one or more traits into a particular genetic background is minimized. The usefulness of marker assisted selection in breeding non-natural transgenic plants of the current invention, as well as types of useful molecular markers, such as but not limited to SSRs and SNPs, are discussed in PCT Application Publication WO 02/062129 and U. S. Patent Application Publications Numbers 2002/0133852, 2003/0049612, and 2003/0005491, each of which is incorporated by reference in their entirety.

[0090] In certain non-natural transgenic plant cells and non-natural transgenic plants of the invention, it may be desirable to concurrently express (or suppress) a gene of interest while also regulating expression of a target gene. Thus, in some embodiments, the transgenic plant contains recombinant DNA including both a transgene transcription unit for expressing at least one gene of interest and a gene suppression element for suppressing a target gene.

[0091] Thus, as described herein, the non-natural transgenic plant cells or non-natural transgenic plants of the invention can be obtained by use of any appropriate transient or stable, integrative or non-integrative transformation method known in the art or presently disclosed. The recombinant DNA constructs can be transcribed in any plant cell or tissue or in a whole plant of any developmental stage. Transgenic plants can be derived from any monocot or dicot plant, such as, but not limited to, plants of commercial or agricultural interest, such as crop plants (especially crop plants used for human food or animal feed), wood-, fiber-, pulp-, or cellulose-producing trees and plants, vegetable plants, fruit plants, and ornamental plants. Non-limiting examples of plants of interest include grain crop plants (such as wheat, oat,

WO 2008/027592

PCT/US2007/019283

2018202034 22 Mar 2018 barley, maize, rye, triticale, rice, millet, sorghum, quinoa, amaranth, and buckwheat); forage crop plants (such as forage grasses and forage dicots including alfalfa, vetch, clover, and the like); oilseed crop plants (such as cotton, safflower, sunflower, soybean, canola, rapeseed, flax, peanuts, and oil palm); tree nuts (such as walnut, cashew, hazelnut, pecan, almond, macadamia, and the like); sugarcane, coconut, date palm, olive, sugarbeet, tea, and coffee; wood-, fiber-, pulp-, or cellulose-producing trees and plants (for example, cotton, flax, jute, ramie, sisal, kenaf, switchgrass, and bamboo); vegetable crop plants such as legumes (for example, beans, peas, lentils, alfalfa, peanut), lettuce, asparagus, artichoke, celery, carrot, radish, cassava, sweet potato, yam, cocoa, coffee, tea, the brassicas (for example, cabbages, kales, mustards, and other leafy brassicas, broccoli, cauliflower, Brussels sprouts, turnip, kohlrabi), edible cucurbits (for example, cucumbers, melons, summer squashes, winter squashes), edible alliums (for example, onions, garlic, leeks, shallots, chives), edible members of the Solanaceae (for example, tomatoes, eggplants, potatoes, peppers, groundcherries), and edible members of the Chenopodiaceae (for example, beet, chard, spinach, quinoa, amaranth); fruit crop plants such as apple, pear, citrus fruits (for example, orange, lime, lemon, grapefruit, and others), stone fruits (for example, apricot, peach, plum, nectarine), banana, pineapple, grape, kiwifruit, papaya, avocado, fig, mango, and berries; and ornamental plants including ornamental flowering plants, ornamental trees and shrubs, ornamental groundcovers, and ornamental grasses. Preferred dicot plants include, but are not limited to, canola, broccoli, cabbage, carrot, cauliflower, Chinese cabbage, cucumber, dry beans, eggplant, fennel, garden beans, gourds, lettuces, melons, okra, peas, peppers, pumpkin, radishes, spinach, squash, watermelon, cotton, potato, quinoa, amaranth, buckwheat, safflower, soybean, sugarbeet, and sunflower. Preferred monocots include, but are not limited to, wheat, oat, barley, maize (including sweet com and other varieties), rye, triticale, rice, ornamental and forage grasses, sorghum, millet, onions, leeks, and sugarcane, more preferably maize, wheat, and rice.

[0092] The ultimate goal in plant transformation is to produce plants which are useful to man. In this respect, non-natural transgenic plants of the invention can be used for virtually any purpose deemed of value to the grower or to the consumer. For example, one may wish to harvest the transgenic plant itself, or harvest transgenic seed of the transgenic plant for planting purposes, or products can be made from the transgenic plant or its seed such as oil, starch, ethanol or other fermentation products, animal feed or human food, pharmaceuticals, and various industrial products. For example, maize is used extensively in the food and feed industries, as well as in industrial applications. Further discussion of the uses of maize can be found, for example, in U. S. Patent Numbers 6,194,636, 6,207,879, 6,232,526, 6,426,446, 6,429,357, 6,433,252, 6,437,217, and 6,583,338, incorporated by reference, and PCT

WO 2008/027592

PCT/US2007/019283

2018202034 22 Mar 2018

Publications WO 95/06128 and WO 02/057471. Thus, this invention also provides commodity products produced from a non-natural transgenic plant cell, plant, or seed of this invention, including, but not limited to, harvested leaves, roots, shoots, tubers, stems, fruits, seeds, or other parts of a plant, meals, oils, extracts, fermentation or digestion products, crushed or whole grains or seeds of a plant, or any food or non-food product including such commodity products produced from a transgenic plant cell, plant, or seed of this invention. The detection of one or more of nucleic acid sequences of the recombinant DNA constructs of this invention in one or more commodity or commodity products contemplated herein is de facto evidence that the commodity or commodity product contains or is derived from a transgenic plant cell, plant, or seed of this invention.

f0093] In preferred embodiments, the non-natural transgenic plant prepared from the non-natural transgenic plant cell of this invention, i. e., a transgenic plant having in its genome a recombinant DNA construct of this invention has at least one additional altered trait, relative to a plant lacking the recombinant DNA construct, selected from the group of traits consisting of:

(a) improved abiotic stress tolerance;

(b) improved biotic stress tolerance;

(c) modified primary metabolite composition;

(d) modified secondary metabolite composition;

(e) modified trace element, carotenoid, or vitamin composition;

(f) improved yield;

(g) improved ability to use nitrogen or other nutrients;

(h) modified agronomic characteristics;

(i) modified growth or reproductive characteristics; and (j) improved harvest, storage, or processing quality.

[0094] In particularly preferred embodiments, the non-natural transgenic plant is characterized by: improved tolerance of abiotic stress (e. g., tolerance of water deficit or drought, heat, cold, non-optimal nutrient or salt levels, non-optimal light levels) or of biotic stress (e. g., crowding, allelopathy, or wounding); by a modified primary metabolite (e. g., fatty acid, oil, amino acid, protein, sugar, or carbohydrate) composition; a modified secondary metabolite (e. g., alkaloids, terpenoids, polyketides, non-ribosomal peptides, and secondary metabolites of mixed biosynthetic origin) composition; a modified trace element (e. g., iron, zinc), carotenoid (e. g., fcefa-carotene, lycopene, lutein, zeaxanthin, or other carotenoids and xanthophylls), or vitamin (e. g., tocopherols) composition; improved yield (e. g., improved yield under non-stress conditions or improved yield under biotic or abiotic stress); improved

WO 2008/027592

PCT/US2007/019283

2018202034 22 Mar 2018 ability to use nitrogen or other nutrients; modified agronomic characteristics (e. g., delayed ripening; delayed senescence; earlier or later maturity; improved shade tolerance; improved resistance to root or stalk lodging; improved resistance to “green snap” of stems; modified photoperiod response); modified growth or reproductive characteristics (e. g., intentional dwarfing; intentional male sterility, useful, e. g., in improved hybridization procedures; improved vegetative growth rate; improved germination; improved male or female fertility); improved harvest, storage, or processing quality (e. g., improved resistance to pests during storage, improved resistance to breakage, improved appeal to consumers); or any combination of these traits.

[0095] In one preferred embodiment, non-natural transgenic seed, or seed produced by the non-natural transgenic plant, has modified primary metabolite (e. g., fatty acid, oil, amino acid, protein, sugar, or carbohydrate) composition, a modified secondary metabolite (e. g., alkaloids, terpenoids, polyketides, non-ribosomal peptides, and secondary metabolites of mixed biosynthetic origin) composition, a modified trace element (e. g., iron, zinc, sulfur), organic phosphate (e. g, phytic acid), carotenoid (e. g., riefa-carotene, lycopene, lutein, zeaxanthin, or other carotenoids and xanthophylls), or vitamin (e. g., tocopherols,) composition, an improved harvest, storage, or processing quality, or a combination of these. For example, it can be desirable to modify the amino acid (e. g., lysine, methionine, tryptophan, or total protein), oil (e. g., fatty acid composition or total oil), carbohydrate (e. g., simple sugars or starches), trace element, carotenoid, or vitamin content of seeds of crop plants (e. g., canola, cotton, safflower, soybean, sugarbeet, sunflower, wheat, maize, or rice), preferably in combination with improved seed harvest, storage, or processing quality, and thus provide improved seed for use in animal feeds or human foods. In another example, it can be desirable to modify the quantity or quality of polysaccharides (e. g., starch, cellulose, or hemicellulose) in plant tissues for use in animal feeds or human foods or for fermentation or biofuel production. In another instance, it can be desirable to change levels of native components of the transgenic plant or seed of a transgenic plant, for example, to decrease levels of proteins with low levels of lysine, methionine, or tryptophan, or to increase the levels of a desired amino acid or fatty acid, or to decrease levels of an allergenic protein or glycoprotein (e. g., peanut allergens including ara h 1, wheat allergens including gliadins and glutenins, soy allergens including P34 allergen, globulins, glycinins, and conglycinins) or of a toxic metabolite (e. g., cyanogenic glycosides in cassava, solanum alkaloids in members of the Solanaceae).

WO 2008/027592

PCT/US2007/019283

2018202034 22 Mar 2018

Examples

Example 1 [0096] This example describes a non-limiting embodiment of DNA encoding a transcript that folds into hybridized RNA that is cleaved in phase in vivo into multiple small double-stranded RNAs for gene suppression. More specifically, this example provides nucleic acid sequences, obtained from monocot crop plants, that are useful in making a single recombinant DNA molecule encoding a transcript that folds into hybridized RNA that is cleaved in phase in vivo into multiple small double-stranded RNAs for gene suppression, independently of an RNA-dependent RNA polymerase.

[0097] Several RNA libraries were cloned from mature rice (Oryza sativa) grain and from various maize (Zea mays) tissues by high-throughput sequencing (Margulies et al. (2005) Nature, 437:376-380). Among the most abundant sequences cloned from mature rice grain and maize root, 32 DAP (days after pollinations) and 39 DAP kernels were seven 21 -mer RNAs (SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, and SEQ ID NO. 7), listed in Table 1.

Table 1

SEQ ID NO.	Clone number	DNA sequence	Species/Tissue*
1	141121	ATGCAAGTGATGTAGCGCCCC	rice grain, maize root, maize 32DAP and 39DAP kernel
2	297263	ATATAGGAGTCACTCAGGAAA	rice grain, maize 32 DAP kernel
3	1196700	TCTTTGCCCTCTTTAGTGCTT	rice grain, maize 32DAP kernel
4	880479	TATGGATGGGCACCATCTTCA	rice grain, maize 32 DAP kernel
5	1275002	TGGCCACCAACAACATCAGCA	rice grain, maize 32DAP kernel
6	1379342	TGCCCCACCAAGAGAACGCCG	rice grain, maize root, maize leaf, maize 32DAP and 39DAP kernel
7	544819	TGCCTGAGGAACACCACCAGG	rice grain, maize root, maize 32DAP and 39DAP kernel

*DAP, days after pollination [0098] These seven 21-mer RNAs, when aligned to the rice genome at locus Os6g21900, were located in two adjacent regions containing seven and six 21-nt siRNAs aligned end to end, respectively, and forming a single foldback structure depicted in Figure 1.

WO 2008/027592

PCT/US2007/019283

2018202034 22 Mar 2018

No siRNAs from this gene were present in other libraries, and only a very small number were sequenced from the putative loop region between arms of the foldback structure. Additional sequencing results indicated that this foldback structure contained at least three other potential 21-mer RNAs (in phase with and distal to the first seven 21-mers and loop), although small RNAs predicted to result from in vivo cleavage of these additional phased small doublestranded RNAs were cloned only at low abundances.

[0099] Although many variants of small RNAs were identified, only a single unique 21-nucleotide (21-nt) phase from the plus strand was supported by sequence information (Table 2). “Phase fullness” indicates how many 21-nt phases are occupied by sequenced small RNAs in both strands; for example, a fullness of 0.5 for frame 7.0 with a phase length of 8 indicates that all eight 21-nt frames are occupied in the plus strand, but none are occupied in the hypothetical minus strand. “Phase uniqueness” represents a probability score for phased small RNAs, which takes into account phase occupancy and abundance of small RNAs in each phase. Frame 16.1 and Frame 7.0 represent each side of the foldback structure (depicted in Figure 1, with the first seven phased small RNAs shown) and its abundant phased small RNAs; phasing is highly supported (uniqueness >0.97) for this structure, whereas all other potential small RNA phases were poorly supported by sequence data (uniqueness <0.005).

WO 2008/027592

PCT/US2007/019283

2018202034 22 Mar 2018

Table 2

Frame	Start (phase number)	Phase length	Phase fullness	Phase uniqueness	Small RNA abundance	Average copies
16.1	5099(1),5120(2), 5141(3),5162(4), 5183(5), 5204(6), 5246(8)	8	0.43	0.9963	344, 89484, 3393, 3121, 10455, 71, 31	15271
7.0	3620(1),3641(2), 3662(3), 3683(4), 3704(5),3725(6), 3746(7), 3767(8)	8	0.5	0.9717	67, 6875,1289, 3,151,7,67, 11619	2510
19.0	3611(1),-3630(2),3651(3),3653(3),3693(5), -3714(6), 3735(7), 3737(7),3756(8), 3758(8)	8	0.62	0.0049	27,1,1,59,1,3, 6,2,1, 1	10
16.0	3629(1),3650(2), 3671(3),-3711(5), 3713(5)	5	0.5	0.0013	1,5,2,2,2	2
4.0	3638(1), 3680(3), 3722(5),-3741(6), 3764(7)	7	0.35	0.0011	17,1,2,2, 1	5
18.0	-5120(1),5122(1),- 5141(2),5164(3),5185(4)	4	0.62	0.001	1, 15, 1,73, 17	21
6.0	3640(1), 3661(2), 3682(3), 3724(5), 3745(6), 3766(7)	7	0.42	0.0008	9,3,1,1,2,2	3
15.0	5140(1),5161(2), 5182(3),5203(4)	4	0.5	0.0007	1,71,4,4	20
14.0	3669(1),3711(3),3753(5)	5	0.3	0.0006	1,6, 1	3
11.0	5199(1), 5220(2), 5262(4)	4	0.37	0.0004	1,3, 1	2
20.0	3654(1),3675(2),- 3736(5),3780(7)	7	0.28	0.0003	2,2,1,2	2
3.0	3637(1), 3658(2), 3700(4), 3721(5), 3740(6)	6	0.41	0.0003	2,1.2,1,1	1

[00100] The genomic sequence and putative precursors for the Oryza sativa foldback structure are given in SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, and SEQ ID NO. 11. One genomic DNA sequence (SEQ ID NO. 9) was predicted to include the cDNA sequence, intrOnic sequence, and foldback arms as shown in Figure 2, and several alternatively spliced versions of this transcript were found in cDNA databases. One abundant alternatively spliced transcript (SEQ ID NO. 10) indicates removal of one half of the foldback structure, probably preventing small RNA production. An expressed sequence tag (SEQ ID NO. 11) was identified as representing the complementary sequence to SEQ ID NO. 10. A canonical TATA box (indicated by the boxed nucleotides in Figure 2) is located 34 bases upstream of the predicted transcription start site in SEQ ID NO. 8, evidence that this is the bona fide 5’ end of the transcript. A single foldback structure is thus transcribed from one promoter and forms, independently of an RNA-dependent RNA polymerase, the hybridized RNA that is cleaved in

WO 2008/027592

PCT/US2007/019283

2018202034 22 Mar 2018 phase in vivo into multiple small double-stranded RNAs for gene suppression. Alternatively, two (or more) splicing variants transcribed from the same promoter each contains one of each of the arms of the foldback structure, and come together in trans, independently of an RNAdependent RNA polymerase, to form the hybridized RNA that is cleaved in phase in vivo into multiple small double-stranded RNAs.

[00101] The evidence collected supports this locus as a novel type of RNA-mediated regulatory (suppression) element. Unlike frans-acting siRNAs, all of the multiple small double-stranded RNAs derive from the original RNA transcript or plus strand of the precursor, independently of an RNA-dependent RNA polymerase and without a miRNA target site that initiates production of double-stranded RNA. Unlike microRNAs, the locus is cleaved in vivo to multiple abundant phased small RNAs, and (as described below in Example 5), this process requires DCL4 (or a DCL4 orthologue) and not DCL1. The inventors therefore term this novel locus a “phased small RNA” locus.

[00102] Expression of this particular phased small RNA locus appears to be restricted primarily to mature grain in both maize and rice, indicating an endogenous function related to repression of genes involved in maturation or maintenance of the embryogenic state in mature grain. Putative targets were predicted for each of the seven phased small RNAs (SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, and SEQ ID NO. 7), following target prediction guidelines described in Allen et al. (2005) Cell, 121:207-221. Non-limiting examples of these targets, which include members of the HAK2 high affinity potassium transporter family, are given in Table 3; the corresponding maize loci from the public database, Maize Assembled Genomic Island (available on line at magi.plantgenomics.iastate.edu, see Fu et al. (2005) Proc. Natl. Acad. Sci. USA, 102:1228212287) are also provided.

WO 2008/027592

PCT/US2007/019283

2018202034 22 Mar 2018

Table 3

SEQ ID NO.	Clone number	DNA sequence	Target gene (rice)	Maize locus*	E value
3	1196700	TCTTTGCCCTCTTTAGTGCTT	0s01gl2300 Protein kinase domain	MAGI4_89402	3.00E-24
MAGI4 122217	7.00E-19
MAGI4 104144	1.00E-17
MAGI4 99444	1.00E-14
MAGI4 39748	2.00E-14
MAGI4 22926	3.00E-14
Osl2gl7310 myosin heavy chain	MAGI4 70672	0
MAGI4_141564	0
5	1275002	TGGCCACCAACAACATCAGCA	Os09g29660 ABC2 type transporter	MAGI4_27534	2.00E-22
0s01g70940 Potassium transporter 7	MAGI4_99444	0
Os09g27580 potassium uptake protein	MAGI4_99444	3.00E-68
Os08g38980 Chloride channel protein	MAGI4_25450	e-127

*publicly available at magi.plantgenomics.iastate.edu

Example 2 [00103] This example describes a non-limiting embodiment of a recombinant DNA construct including a promoter operably linked to DNA that transcribes to RNA including: (a) at least one exogenous recognition site recognizable by a phased small RNA expressed in a specific cell of a multicellular eukaryote, and (b) target RNA to be suppressed in the specific cell, wherein the target RNA is to be expressed in cells of the multicellular eukaryote other than the specific cell. More specifically, this example describes a recombinant DNA construct including DNA that transcribes to RNA containing an exogenous recognition site corresponding to at least one phased small RNA derived from an endogenous phased small RNA locus.

[00104] Recombinant DNA constructs were designed to include a gene expression element for expression of a gene of interest (in this non-limiting example, the reporter gene, fteta-glucoronidase, “GUS”), and a recognition site (target site) corresponding to at least one phased small RNA of this invention (e. g., any one or more of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ Π) NO. 6, and SEQ ID NO. 7 as described in Example 1). Similar recombinant DNA constructs can be designed, wherein a recognition site (target site) corresponding to at least one phased small RNA of this invention is included to regulate the expression of a gene expression element for the expression of a gene

WO 2008/027592

PCT/US2007/019283

2018202034 22 Mar 2018 of interest (which can be translatable or coding sequence or non-coding sequence, including regulatory sequence), e. g., those described under the heading “Target Genes”, or alternatively to regulate the expression of a gene suppression element (e. g., sense, anti-sense, combinations of sense and anti-sense, tandem repeats of sense or of anti-sense, microRNAs, siRNAs, and any other construct designed to reduce the expression of a target gene).

[00105] A control construct (pMON94320) with a 35S promoter driving expression of GUS and an Hspl7 terminator included the partial sequence SEQ ID NO. 12, with an insertion site (indicated in bold font in Figure 3B) located between the GUS coding sequence and the Hspl7 terminator. Three additional constructs based on this control construct were designed, each containing at least one recognition site corresponding to a phased small RNA of ’ this invention. The first construct (pMON100574) included the partial sequence SEQ ID NO. 13, which contained one of the 21-mer phased small RNAs (SEQ ID NO. 6) described in Example 1, incorporated in the sense orientation at the insertion site (Figure 3C). The second construct (pMON100575) included the partial sequence SEQ ID NO. 14, which contained one of the 21-mer phased small RNAs (SEQ ID NO. 6) described in Example 1, incorporated in the anti-sense orientation (i. e.·, as a recognition site corresponding to SEQ ID NO. 6 in the sense orientation) at the insertion site (Figure 3D). The third construct (pMONl 00576) included the partial sequence SEQ ID NO. 15, which contained two of the 21-mer phased small RNAs (SEQ ID NO. 5 and SEQ ID NO. 6) described in Example 1, both incorporated in the anti-sense orientation (i. e., as recognition sites corresponding to SEQ ID NO. 5 and SEQ ID NO. 6 in the sense orientation) at the insertion site (Figure 3E).

[00106] Maize tissue from developing kernels was analyzed by northern blot using a single probe with the sequence CGGCGTTCTCTTGGTGGGGCA (SEQ ID NO. 16, i. e., the anti-sense sequence of SEQ ID NO. 6). The results, depicted in Figure 4, indicated transcription of the endogenous maize phased small RNA locus, especially in developing embryo and to a lower extent in developing endosperm, further corroborating the cloning results given in Table 1.

[00107] Maize zygotic embryos (21—22 days after pollination) were transformed with the recombinant DNA constructs by particle bombardment, using about 0.5 micrograms DNA delivered with one shot of a helium particle gun. Bombarded tissue was incubated for 24 or 48 hours in a dark reach-in growth chamber at 26 degrees Celsius. The embryos were stained in 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid solution (24 hours at 37 degrees Celsius) followed by clearing of the stained tissue in 70% ethanol. Expression of the gene of interest (GUS) encoded by the gene expression element was indicated by the level of staining in the embryos; GUS expression was predicted to be silenced by the endogenous maize phased

WO 2008/027592

PCT/US2007/019283

2018202034 22 Mar 2018 small RNA locus. As predicted, GUS expression was silenced in the embryos transformed with the constructs (pMON100575 and pMON100576) containing at least one recognition site corresponding to a phased small RNA of this invention (Figure 3A). The silencing observed in the embryos transformed with pMON100574 was presumably due to endogenous anti-sense transcript present in low abundance as was observed in the cloned rice RNA libraries (see Table 2 for abundances of cloned small RNAs).

[00108] Recognition sites corresponding to phased small RNAs of this invention are useful for regulating expression of a transgene in a construct including at least one such recognition site. Thus, this invention provides a recombinant DNA construct including a promoter operably linked to DNA that transcribes to RNA including: (a) at least one exogenous recognition site recognizable by a phased small RNA expressed in a specific cell of a multicellular eukaryote, and (b) target RNA to be suppressed in the specific cell, wherein the target RNA is to be expressed in cells of the multicellular eukaryote other than the specific cell. The invention includes a recombinant DNA construct including a transgene and at least one recognition site that corresponds to one or more phased small RNAs of this invention, useful for expression of that transgene in tissues other than those in which the phased small RNAs are expressed, and suppression of the transgene in tissues where the phased small RNAs are expressed. For example, SEQ ID NO. 6 has been shown to be expressed in rice kernel (Example 1) and in com kernel (this example); a construct containing a transgene (e. g., an herbicide tolerance gene such as 5-enolpyruvylshikimate-3-phosphate synthase) and at least one recognition site corresponding to SEQ ID NO. 6 is useful for suppression of the transgene in at least rice or com kernel.

Example 3 [00109] This example describes a non-limiting embodiment of a recombinant DNA construct including DNA that transcribes to: (a) a first series of contiguous RNA segments, and (b) a second series of contiguous RNA segments, wherein the first series of contiguous RNA segments hybridize in vivo to the second series of RNA segments to form hybridized RNA that is cleaved in phase in vivo into multiple small double-stranded RNAs (“phased small RNAs”) for gene suppression. Preferably the hybridized RNA is produced independently of an RNA-dependent RNA polymerase. The recombinant DNA construct of this invention can include a synthetic phased small RNA locus (which can transcribe to a longer or shorter transcript than that transcribed from a naturally occurring phased small RNA locus), designed to be cleaved in vivo and in phase into any number of phased small RNAs for suppression of one or more target genes.

WO 2008/027592

PCT/US2007/019283

2018202034 22 Mar 2018 [00110] This example provides an embodiment of a recombinant DNA construct including nucleic acid sequences derived from monocot crop plants, that transcribes to an RNA containing a single foldback structure cleavable in vivo and in phase to multiple small doublestranded RNAs for gene suppression, independently of an RNA-dependent RNA polymerase.

A phased small RNA locus from monocot crops was found to have the single foldback structure depicted in Figure 1 (see Example 1). This locus includes at least seven 21-mer RNAs (SEQ ID NO. 1, SEQ ID NO. 2,. SEQ ID NO. 3, SEQ ED NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, and SEQ ID NO. 7), each and any of which can be engineered to suppress expression of one target gene or of multiple target genes in trans. In this non-limiting example, a recombinant DNA construct based on this locus was designed to transcribe to a single transcript including an imperfect foldback structure for suppressing multiple endogenous genes in maize: (1) the messenger RNA encoding the LKR region of the lysine ketoglutarate reductase/saccharopine dehydrogenase gene, LKR/SDH, and (2) the messenger RNA encoding the dominant Waxy gene, which encodes an enzyme for starch synthesis; a “waxy” (nonstarchy) mutant phenotype characterized by decreased amylose and increased amylopectin (branched starch) is typically seen in plants homozygous for the naturally occurring, recessive allele (wx/wx) and is useful as a visual marker of inheritance in maize breeding.

[00111] The recombinant DNA sequence was designed based on a 939-nucleotide starting sequence (SEQ ID NO. 17), which included, in order:

(1) a 5’leader sequence (SEQ ID NO. 18);

(2) the 5’ arm of the foldback structure, including a first series of contiguous RNA segments, i. e., seven contiguous 21-mers (SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, and SEQ ID NO. 7) in the order listed (5’ to 3’);

(3) spacer sequence (SEQ ID NO. 23) forming a loop joining the 5’ and 3’ arms of the foldback structure;

(4) the 3’ arm of the foldback structure, including a second series of contiguous RNA segments, i. e., seven contiguous 21-mers (SEQ ID NO. 24, SEQ ID NO. 6, SEQ ID NO. 5, SEQ ID NO. 4, SEQ ID NO. 3, SEQ ID NO. 25, and SEQ ID NO. 26) in the order listed (5’to 3’); and (5) a 3’ untranslated region and terminator (SEQ ID NO. 27) [00112] This starting sequence (SEQ ID NO. 17) is useful as a phased small RNA template on which a gene suppression construct is based; any one or more of the contiguous 21-mers (or the contiguous RNA segments in the corresponding RNA transcript) that form the foldback structure can be modified or engineered to silence a target gene, as described above

WO 2008/027592

PCT/US2007/019283

2018202034 22 Mar 2018 under the heading “A Recombinant DNA Construct Transcribing to a First and Second Series of Contiguous RNA Segments that Form Hybridized RNA that is Cleaved in Phase in vivo”.

In this non-limiting embodiment, the 21-mers engineered to silence one or more target genes are preferably selected from SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, and SEQ Π) NO. 7. Preferably, a spacer sequence (such as SEQ ID NO. 23) forming a loop joining the 5’ and 3’ arms of the foldback structure is maintained in the engineered gene suppression construct.

[00113] In one specific example, selected 21-mer sequences (synthetic phased small RNAs) were designed to target LKR (SEQ ID NO. 28, and SEQ ID NO. 29) or Waxy (SEQ ID NO. 30, and SEQ ID NO. 31), respectively. These are cloned into expressed phase locations of the template sequence to yield the gene suppression construct having the sequence aatcttattctacatatttctatcttatatagaacaactagcatagctctcgttgcccagccaggttgcccagccaggttgcctggtgcacaat gagagctggctagggcggactcattctgctgttggtgcccaacgatgctagctgctactcatactagtgaagcctgccatggttctgagaa atttttggatactccgctgcgtagatatgcactaaaagcttgtatgtttcgctgactacatactatggatatcacctgtttgacaagagaaggat tacataccacgatgaagatgaattggaacatgATGCAAGTGATGTAGCGCCCCATATAGGAGTCACTC AGGAAAGCGCaGCTCGCCAccGAGATGcGCCcAAGATGCAGGTGcATGCTGAcgctaTT

GGcGGCCtCGCATAGATCcCTTGATaTGACTTTGTgGATGCAGAaAGCGGTGcccacgg cgacgccaaaaaatgcaaagttggccaacacatagctcactgcatcgtcaagtagagctgcttaatcactgagggtatatacatttagttc gccttcttcagcgttgccatggacaCCGCTcTCTGCATCaACAAAGTGAcATCAAGtGATCTATGC

GtGGCCaCCAAcaacaTCAGCATaCACCTGCATCTTtGGCaCATCTCctTGGCGAGCg

GCGCttTCCGTAGTGATTCCTATACGGGGTGCTACTTCACTTGGATCAtgttacaatttatcttc atcgtgatatatgctccttctgttctcacataggtgatatcttaaaatgtatgaggcatatatactttctacctaatattataaagtatatgcctctat atagatcaaataaagcagaaaagtcattgttattaccaatcgtgtacttttgttctaaacatctcaactagtttaaagtatttgtctctcttga (SEQ ID NO. 32); the 5’ and 3’ arms of the foldback structure are indicated by underlined text, engineered 21-mers are shown in bold font, and intentionally mismatched nucleotides are indicated by lower case font. Additional sequence on each foldback arm was also modified in order to preserve the original secondary structure (including the location of mismatched bases) of the template sequence (SEQ ID NO. 17). Figure 5 depicts the predicted secondary structure of the RNA transcribed respectively from the template sequence (SEQ ID NO. 17) and from the engineered gene suppression construct (SEQ ID NO. 32). Expression of the engineered gene suppression construct is driven by an appropriate endosperm specific promoter, such as a maize zein or B32 promoter (nucleotides 848 through 1259 of GenBank accession number X70153, see also Hartings et al. (1990) Plant Mol. Biol., 14:1031-1040, which is incorporated herein by reference). Additional synthetic phased small RNA constructs are designed in a similar manner to silence multiple target genes, such as combinations of

WO 2008/027592

PCT/US2007/019283 endogenous genes (e. g., LKR/SDH, GLABRA1, DWARF4, and CLAVATA) or transgenes (e.

g., reporter genes such as GUS or GFP, or selectable markers such as a gene imparting antibiotic or herbicide tolerance).

2018202034 22 Mar 2018

Example 4 [00114] This example describes a non-limiting embodiment of an RNA transcript that folds into hybridized RNA that is cleaved in phase in vivo into multiple small double-stranded RNAs for gene suppression, wherein the hybridized RNA is produced independently of an RNA-dependent RNA polymerase. More specifically, this example provides nucleic acid sequences, obtained from monocot crop plants, that are useful in making a recombinant DNA construct encoding a transcript that folds into hybridized RNA that is cleaved in phase in vivo into multiple small double-stranded RNAs for gene suppression, independently of an RNAdependent RNA polymerase.

[00115] Following the methods described in detail in Example 1, a second “phased small RNA” locus was identified from rice (Oryza sativa) mature grain and seedling RNA libraries. This locus, LOC_Osl2g42380.1|ll982.m08017, had the DNA sequence GATTCTCCCCTGCGCCGCCGCCGCCGCCGCCGCCTCAATCGGGCGAAGCCGCCCTCGCCGC CGTCGCGGCGGCGGCGGCGAGGGCGAGCTCCTGCGAGAGATCCTCCGCCGCCTCATGCCT CGCGCGCGCGCTCCCGCTCCCGCTCrCGCCTGCAGTATTTGTTCCATTGCCGCGCACCACTT TCCGGTGGGCGGCGGGCAATGCTAGGGGTTAAGAGACCTTCTCTCCCCGAGATGGAGGCG CCGGGCGGCGCGGCGGGGGACGCGGAGGAGGAAGTTGATGCCCGGATCCGCTGGGTTCCA TGGTGGCTGCTATGGAATGGTGGAATTGCTTGGATGGCCACGAAGGGGATCGACGCCAAT TGTTTGGCGACCTCTACGATAGAATCGCGTCGAGTCGGGGTGTTCTTTCCTGTTATTACTAG AAGTAGTTGAATTTCGTGATTGAACACACAAGGAAGCTTGATATCGCGTCGGGGGTGTTCT TTCCTGTTATTACTAGATGTAGTrGGGTTTCGTGATTGAACACCTAAGGAAAGGAAGCTTG ATAAATGGAAGATAGTCCAGCAAGTTTTGAAGATGATAGAAAATTTGAGCGCGTCGTAGT AACTGTCGTCCACGATCACGTCCAGTGTTGTTCATGGCATGGGGGATGGAGTCAGGATCCT TGAGGCGTCTGCTCCTGTTGCACTGCTTCATGCCTTTCCTGCCTTCTAGGATGCTTAAGATG GTTGCGAAGTCAGGTGCTTGGGAGTTCATGAAGCGGTCATAATCAATTTCGCTCTCTGTAG TACTTTCTCTGGTGTCTTCCCCGTTGCTTCCTTrrGGAAGAAAAGCGTCCTTTAGAATCTCT tgagagAgtgcactttctccctctcctgccatcagtagtgcctttattttcgcttggtttcc GCATCATCAGGTGGCACTTATAGAAATTATTTTATGGAGGAAAAAGCATTGTATGGCATGA TAGAAATATCCTTATGGATAAAACTAGGACACTTGCAAGTGTTCAATGGGAGTCACCTTAC CTTTTTTGCCTACCTGTCTGGATTTCATGAATGGGATTCCTTCTCCTGCGCCGGTGCTGTCTT CTCAAATGGGAAATGGAGGCAAGCATCTGCCCTGTTCCATGGTGGCAGCCATGGAATGAT GGGATTTCTTTGATGGTCATAAAGGAGATCAAAACCAACGGTTGGCAATCTCTGCAGGGA

WO 2008/027592

PCT/US2007/019283

2018202034 22 Mar 2018

TGATGAACCAGGCTTGTAATATCTGTTGCTGATTTCTTTGGAAGACATAACGGCAAGCTTC

ATGGGGCAGGATGGATTTCAGATGGTTGCTTCAGCCATGTCTCAAGATTCAGTTGATGGAC

CTCAAGTTTCTGGGTGCAGTGCCACGAGTCTTGGTCAGCCCAAGAGTAAGCGCAGGACTG

GTGACAAGGCAAGAGGGGAGAAGAAGGCACTCAAAGTTAAGATTAACCTTGCCAGCGCG

GCCAAAAAAATTAAGAAAAGTAGCAAAAAGAAGGGCAAAAAGGGCACTGTTGCTGGCAG

GATAGGGAGAAAATGCACTCTCTCAAGAGATTCTAAAGGGCGCTTTCTTCCAAGAGAGAG

TAAGGGGGGAGACATCGGAGGAAATGCTACAGAGAGTGAAGTTGATTATGACCGCTTCAT

GAACTTTCAGGCACCTGACTTCGCTACCATCTTAAGTATTTTGAAAGGCTGGAAAGGCATG

AAGCAATGTAACAAGATCAGGCGCCTCAAGGATCCTGACTTCGTCCCTCTCATGAACGTCA

TGAGCAACACTGGATATGTGACCGAGGATGATGGTCACTATGATGTGCTGAAAGTCTTGAT

GCATGCAGATGGCTGGTCTGCATAGTGATTCAAGCTCTCAAATCAAAACATTCAGGCCTAT

GGCCTTGTTGCTAGAACAGTGGTTTCTTCTTTCACCTTTAAAACTTGATGGACTTTGTTCCA

TTTATCTTAGAAATTTTGTTGCCCTTGAGTCCGGTGGATATGTACTGGAGTATGCTATACTG

GGTGATTTAATGGTGATAATGTTAAATCTTGATACTAGTTCAAAAAAAAAAAAAAAAAAA

AAAA (SEQ ID NO. 33), and a phase uniqueness score of 0.959, highly supportive of the predicted RNA transcript having the sequence

AGUCCAGCAAGUUUUGAAGAUGAUAGAAAAUUUGAGCGCGUCGUAGUAACUGUCGUCC

ACGAUCACGUCCAGUGUUGUUCAUGGCAUGGGGGAUGGAGUCAGGAUCCUUGAGGCGU

CUGCUCCUGUUGCACUGCUUCAUGCCUUUCCUGCCUUCUAGGAUGCUUAAGAUGGUUGC

GAAGUCAGGUGCUUGGGAGUUCAUGAAGCGGUCAUAAUCAAUUUCGCUCUCUGUAGUA cuuucucuggugucuuccccguugcuuccuuuuggaagaaaagcGuccuuuagaaucu

CUUGAGAGAGUGCACUUUCUCCCUCUCCUGGCAUCAGUAGUGCCUUUAUUUUCGCUUGG

UUUCCGGCAAAAAGGGCACUGUUGCUGGCAGGAUAGGGAGAAAAUGCACUCUCUCAAG

AGAUUCUAAAGGGCGCUUUCUUCCAAGAGAGAGUAAGGGGGGAGACAUCGGAGGAAAU

GCUACAGAGAGUGAAGUUGAUUAUGACCGCUUCAUGAACUUUCAGGCACCUGACUUCG

CUACCAUCUUAAGUAUUUUGAAAGGCUGGAAAGGCAUGAAGCAAUGUAACAGAUCAGG

CGCCUCAAGGAUCCUGACUUCGUCCCUCUCAUGAACGUCAUGAGCAACACUGGAUAUGU

GACCGAGGAUGAUGGUCACUAUGAUGUGCUGAAA (SEQ ID NO. 34) and containing the sequence and single foldback structure as shown in Figure 6. Figure 7A depicts the siRNA abundance in transcripts per quarter million sequences (“tpq”) along the entire sequence (about kilobases), and Figure 7B depicts an expanded view of the siRNA region and the 21nucleotide phasing of the small RNA abundance from this locus.

[00116] As with the phased small RNA locus described in Example 1, the locus having SEQ ID NO. 33 was predicted to transcribe to RNA (SEQ ID NO. 34) forming hybridized RNA independently of an RNA-dependent RNA polymerase and to be cleavable in vivo in phase into multiple small double-stranded RNAs. Unlike frans-acting siRNAs, all of the multiple small double-stranded RNAs derive from the original RNA transcript or plus

WO 2008/027592

PCT/US2007/019283

2018202034 22 Mar 2018 strand of the precursor, independently of an RNA-dependent RNA polymerase and without a miRNA target site that initiates production of double-stranded RNA. Unlike microRNAs, the locus is cleaved in vivo to multiple abundant phased small RNAs, and (as described below in Example 5), this process requires DCL4 or a DCL4 orthologue and not DCL1.

[00117] Data on the phased small RNAs from this locus (SEQ ID NO. 33) are provided in Table 4. The majority of these phased small RNAs were cloned from the rice small RNA libraries, and several were also identified in maize (Zea mays) RNA libraries prepared from kernels (32 days after pollination and 39 days after pollination) and root (V9 stage), indicating that a similar phased small RNA locus exists in maize. The transcript (SEQ ID NO. 34) predicted from this locus (SEQ ID NO. 33) also includes 5’ flanking sequence AGUCCAGCAAGUUUUGAAGAUGAUAGAAAAUUUGAGCGCGUCGUAGUAACUGU CGUCCACGA (SEQ ID NO. 66) and 3’ flanking sequence

GAGGAUGAUGGUCACUAUGAUGUGCUGAAA (SEQ ID NO. 67) as well as a spacer sequence UUUAUUUUCGCUUGGUUUCCGGCAAAAAGGG (SEQ ID NO. 68) located between the 5’ and 3’ arms of the foldback structure, that includes a 3-nucleotide turn. Figure 6 depicts the relative position of each small RNA along the 5’ and 3’ arms of the hybridized RNA (foldback) structure (SEQ ID NO. 34) predicted from the rice locus (SEQ ID NO. 33). Most, but not all, of these small RNAs are 21-mers. The small RNA predicted to be encoded by SEQ ID NO. 59 contains 27 nucleotides, including a large bulge of 8 unpaired nucleotides; modification of this sequence so that this small RNA is closer to two helical turns (about 21 nucleotides) is predicted to result in processing of this small RNA.

WO 2008/027592

PCT/US2007/019283

2018202034 22 Mar 2018

maize V9

root

CM

4)

a.

Ή3

.sa <

B

Os

2

Q

4)

O

*

Os tn

Q.

a.

4)

4)

•H <

5

CM

co

CM

so

§

ce ε

Q cm co

u JS

co CM

Ο

CM

S

*

Λ

M

-<e

1:. rice

seedlin

o so

| 56.8

| 817.5

O wS

I 86.6

| 310.7

©

Ο

| 118.5

ο CM

Ο C*S

O C*S

p CM

I 13.9

p vS

O c*S

p

c

Os

to

T-t

CM

ο

.8

“3

SO CM

oo

o

co

s©

tT

SO

τΤ

00

cn

00

m

cn

Os

m

Os

u

CM

os

CM

rt

Os

©

OS

ο

Ο

o

O

o

oo

CM

CM

06

r-

CM

<

P

o

o

P

o

o

p

§

P

<

<

P

Ρ

Ρ

υ Ρ

to

r s

o

<

ρ

<

<

P §

o

P

P

o

p

υ

p

P

p

§

Ρ

Ρ

δ

<

s^

o

P

P

δ

kJ

P

kJ

<

o

p

5

<

δ

P

o

p

Ρ

EJ

δ

<

<3

£z

o

P

<

<

JD

2

<

<

o

p

<

p

5

P

o

P

p

<

Ρ

Ρ

ρ

ρ

ρ

tz

c5

2

p

u

<

P

<

<

P

p

p

□

p

o

P

P

o

Q

Ρ

Ρ

ο

<

Ρ

Π

o

P

p

o

P

o

o

p

p

p

p

p

P

P

P

δ

<

ρ

<

ο

Ρ

rh

<

<

p

o

o

o

kJ

P

o

o

o

o

δ

<

δ

P

δ

ο

ρ

ρ

ο

Ρ

S.7

o

P

<

c5

P

<

<

<

<

o

P

P

P

P

to

p

Ρ

ο

<

Ρ

ΓΠ

P

P <

P

<*

<

<z kJ

kJ

P

S

5

<3

g

<

t-J

< o

P

o

<

Ρ

f )

Ρ

ΓΊ

o

Q

5

u

<

<

P

<

B

o

δ

to

p

ό

o

δ

o

I—)

Ρ

<

ο

cb

Ρ

q

<

δ

o

δ

p

P kJ

kJ

o

S

p

to

o

p

δ

P

P

P

ό

5

Η

Ρ

ό

<;

o

<

<

o

P

o

δ

p

o

o·

o

<

kJ

o

<

< o

<

|_J

p

Ρ

Ρ

ο

Ρ

<

<3

p

<

o

p

&

<

kJ

Sri

P

P

b_J

P

Ρ

υ

Ρ

δ

o

δ

o

<

p

p

<3

<_>

P

(J

c5

o

δ

p

P

ρ

Ρ

ο

Ρ

δ

Ρ

<

C_>

<_>

u

p

o

co

P

<

p

o

<

P

to

δ

o

<

p

ρ

Ρ

<

δ

<?

u

§

P

o

<

p

ρ

rO

P

P <

P

cp

o

P

P

p

<

p

δ

ο

Ρ

Ρ

<*

<

<

ρ

o

<

<

JAUCAC

o

o

¢5

p

<

E )

<

P

p

ρ

ρ

ζί

U

ο

<ί

tz

o

o

u

P

p

P

<3

p

p

EJ

E5

cp

P

P

Ρ

δ

Ρ

Ρ

ο

7?

CJ

<

<

δ

u

P <

p

p

P

<

p

o

δ

δ

cp

<

<

ο

<]

5S

Ρ

ο

o <

p

o

o

o

u

o

ρ

δ

p

p

u

Lj

P

P

o

<3

P

Γ )

ο

υ

<

<

<

<

P

<

P

<

to

0

P

p

<

P

P

o

ρ

Ρ

Ρ

Ρ

ο

o

P

<

P

P

s_/

< o

o P

P

P

a

« « z—s

O' Ed

S .3 z ce

SO

CM so

co so

ττ so

in Ό

ζΛ

e

a

8

o Ed

ό

in

so

r*

oo

Os

o

CM

CO

ΜΤ

m

so

r*

00

Ο\

o

CM

co

in

SO

r*

00

Os

o

2;

X

cn

cn

co

co

co

•Β·

in

in

in

in

in

m

in

«η

in

in

so

(Λ

I sRNA

identifier

| 792014 1

« « »

| 118041 1

Γ 657519 1

Γ611711 |

I 1016358 |

I 577487 1

| 515019 |

| 803519 1

*

ί 459001 |

1 1119948 I

| 645846 |

*

| 1147125 1

| 73294 1

| 1002514 I

| 1160057 |

1287753 1

| 1396420 |

1 1125181 |

628491 1

so Os ID OO Os

*

«

1249464 |

e

Q

ε

£

£

ε

ε

£

ε

ε

£

£

£

£

£

£

£

£

£

ε

£

£

£

£

£

ε

B

£

1

B e

$

¢3

a

a

c3

c3

a

c3

«3

a

a

a

a

a

a

a

a

a

<3

a

a

a

a

a

e3

a

a

3

2

W-l

so

wn

V>

w->

sn

w->

t/>

so

•η

m

m

c*>

en

cn

m

cn

cn

cn

CO

co

CO

CO

* “tpq, transcripts per quarter million sequence reads (given as averages of three sequencing runs) ♦♦small RNA also cloned from maize ♦♦♦predicted from the phased sRNA locus sequence (SEQ ID NO. 33) but not cloned

WO 2008/027592

PCT/US2007/019283

2018202034 22 Mar 2018

Example 5 [00118] This example describes a non-limiting embodiment of DNA encoding a transcript that folds into hybridized RNA that is cleaved in phase in vivo into multiple small double-stranded RNAs (“phased small RNAs”) for gene suppression, wherein the hybridized RNA is produced independently of an RNA-dependent RNA polymerase. The 0s06g21900 phased sRNA locus (described in Example 1 and with the partial structure depicted in Figure 1) is processed in vivo to multiple phased small RNAs; all of the multiple small doublestranded RNAs derive from the plus strand of the precursor, which distinguishes them from trans-acting siRNAs. And, unlike microRNAs, the locus contains multiple abundant phased small RNAs. This example provides further characterization of a phased small RNA locus as clearly distinct from canonical microRNAs and irans-acting siRNAs.

[00119] The 0s06g21900 phased sRNA locus, located on rice Chromosome 6, was further characterized. A 898-nucleotide precursor that mapped to this locus was sequenced from library clone LIB4833-001-R1-N1-G10 and found to have the DNA sequence AATCTTATTCTACATATTTCTATCTTATATAGAACAACTAGCATAGCTCTCGTTGCCCAGCC AGGTTGCCCAGCCAGGTTGCCTGGTGCACAATGAGAGCTGGCTAGGGCGGACTCATTCTG CTGTTGGTGCCCAACGATGCTAGCTGCTACTCATACTAGTGAAGCCTGCCATGGTTCTGAG AAATTnTGGATACTCCGCTGCGTAGATATGCACTAAAAGCTTGTATGTTTCGCTGACTAC ATACTATGGATATCACCTGTTTGACAAGAGAAGGATTACATACCACGATGAAGATGAATT GGAACATGATGCAAGTGATGTAGCGCCCCATATAGGAGTCACTCAGGAAAGCACAGAAGA GGGAGAAGATGTAGACGGTGCCCATCCACATGCTGACGCTATTGGCGGCCTCGGCGTTCTC CTGGTGGAGCACCTGCCTGAGGAACACCACCAGGCCCACGGCGACGCCAAAAAATGCAAA GTTGGCCAACACATAGCTCACTGCATCGTCAAGTAGAGCTGCTTAATCACTGAGGTATATA CATTTAGTTCGCCTTCTTCAGCGTTGCCATGGACCTGGTGATGTTCTTCCGGCGGGTGCCCC ACCAAGAGAACGCCGTGGCCACCAACAACATCAGCATATGGATGGGCACCATCTTCATCT TTGCCCTCTTTAGTGCTTTCCGTAGTGATTCCTATACGGGGTGCTACTTCACTTGGATCATG TTACAATTTATCTTCATCGTGATATATGCTCCTTCTGTTCTCACATAGGTGATATCTTAAAA TGTATGAGGCATATATACTTTCTACCTAATATTATAAAGTATATGCCTCTATATAGATCAA ATAAAGCAGAAAAGTCATTGTTATTACAAAAAAAAAAAAAAAAAA (SEQ ID NO. 69); the corresponding transcript contained the phased small RNAs (see Example 1) distributed between two regions along the transcript (Figure 8A). This locus contains two exons (Exons 2 and 3, indicated by the shaded regions) that form a long, imperfect foldback structure containing eight 21-nucleotide phased small double-stranded RNAs, separated by an -1.2 kB intron (Figure 8B). No small RNAs were found that match Exon 1, nor was any miRNA target sequence that could initiate a trans-acting siRNA phasing identified (see Allen et al. (2005) Cell. 121:207-221; Vaucheret (2005) Sci. STKE, 2005, e43; and Yoshikawa et al.

WO 2008/027592

PCT/US2007/019283

2018202034 22 Mar 2018 (2005) Genes Dev., 19:2164-2175). RNA gel blot analysis of the most abundant phased small RNA confirmed that expression was specific to rice grain (Figure 8C). Neither of the two most abundant phased small RNAs (“P7”, SEQ ID NO. 6, and “P4”, SEQ ID NO. 3) was detected in rice seedlings or in other plant species tested.

[00120] The phased small RNAs form a novel class of regulatory small RNAs that differ from both canonical microRNAs (miRNAs) and frans-acting siRNAs. The phased small RNAs disclosed herein are to some extent reminiscent of miR163 in Arabidopsis in which two phases of siRNAs were sequenced with a single small RNA (miR163 itself) significantly accumulating; see Allen et al. (2004) Nat. Genet., 36:1282-1290; and Kurihara and Watanabe (2004) Proc. Natl. Acad. Sci. U. S. A., 101:12753-12758. However, the phased small RNAs clearly differ from miR163 in that multiple abundant phased small RNAs are processed and can be isolated from a single transcript. The phased small RNAs locus is a single, extended, imperfect foldback structure (for example, the loci depicted in Figure 1, Figure 6, or Figure 8B), and therefore is also clearly different from the iranj-acting siRNA loci identified in Arabidopsis, which require an RNA-dependent RNA polymerase (RDR6) to generate the double-stranded RNA from which the phased siRNAs are processed.

[00121] The extended foldback structure of the 0s06g21900 phased small RNA locus suggests that this precursor is not processed via the canonical miRNA pathway. The phased nature of the phased small RNAs further indicates that they are the result of processing by DCL4 or a DCL4 orthologue rather than by DCL1. To further confirm that the phased small RNAs disclosed herein are unique and distinct from both canonical microRNAs (miRNAs) and rrens-acting siRNAs, the full length cDNA from the 0s06g21900 phased small RNA locus was transformed into Arabidopsis thaliana Columbia (Col-0) ecotype and mutants dcll-7 (a DCL1 knock-out) and dcl4-l (a DCL4 knock-out). RNA was extracted and blots analyzed using probes corresponding to phased small RNAs “P7” (SEQ ID NO. 6), and “P5” (SEQ ID NO.

4), a canonical miRNA (miR173) and a zrans-acting siRNA (ta-siR255) (Figure 8D).

[00122] Phased 21-nucleotide small RNAs were highly expressed in transformation events from Col-0 and dcll-7, but in the dcl4-l mutant, 21-nucleotide phased sRNAs were absent, with only faint 24-nucleotide small RNAs observed (similar to what was observed for ta-siR255). These data are consistent with the function of DCL4 in processing small RNAs in phase, but, unlike trans-acting siRNAs, no miRNA initiation site was required in the case of the phased small RNA loci disclosed herein. These data also demonstrated that the phased small RNA locus from a monocot crop plant was efficiently processed in a dicot plant. Thus, phased sRNAs are processed through pathways distinct from those of both canonical microRNAs (miRNAs) and Zrans-acting siRNAs. As described in other Examples disclosed

WO 2008/027592

PCT/US2007/019283

2018202034 22 Mar 2018 herein, the phased small RNA locus is useful as a template for designing a recombinant DNA construct encoding a transcript that folds into hybridized RNA that is cleaved in phase in vivo into multiple small double-stranded RNAs for gene suppression, or alternatively as a template for designing a recombinant DNA construct including DNA that transcribes to: (a) a first series of contiguous RNA segments, and (b) a second series of contiguous RNA segments, wherein the first series of contiguous RNA segments hybridize in vivo to the second series of RNA segments to form hybridized RNA that is cleaved in phase in vivo into multiple small double-stranded RNAs (“phased small RNAs”) for gene suppression.

Example 6 [00*123] This example describes a non-limiting embodiment of a recombinant DNA construct including DNA that transcribes to: (a) a first series of contiguous RNA segments, and (b) a second series of contiguous RNA segments, wherein the first series of contiguous RNA segments hybridize in vivo to the second series of RNA segments to form hybridized RNA that is cleaved in phase in vivo into multiple small double-stranded RNAs (“phased small RNAs”) for gene suppression;

[00124] This example provides an embodiment of a recombinant DNA construct including nucleic acid sequences derived from monocot crop plants, that transcribes to an RNA containing a single foldback structure cleavable in vivo and in phase to multiple small doublestranded RNAs for gene suppression, independently of an RNA-dependent RNA polymerase. This example is a recombinant DNA construct designed to suppress multiple target genes. The 0s06g21900 phased small RNA locus (see Example 1) was modified to suppress three target genes as follows: nucleotides of the phased small RNAs with the identifiers 1196700 (SEQ ID NO. 3), 1379342 (SEQ ID NO. 6), and 544819 (SEQ ID NO. 7) were replaced, respectively, with nucleotides corresponding to a segment of the GL1, IDA, and LFY genes from Arabidopsis thaliana. The resulting sequence was

GGTACCAATCTTATTCTACATATTTCTATCTTATATAGAACAACTAGCATAGCTCTCGTTGC

CCAGCCAGGTTGCCCAGCCAGGTTGCCTGGTGCACAATGAGAGCTGGCTAGGGCGGACTC

ATTCTGCTGTTGGTGCCCAACGATGCTAGCTGCTACTCATACTAGTGAAGCCTGCCATGGT

TCTGAGAAATTTTTGGATACTGCGCTGCGTAGATATGCACTAAAAGCrTGTATGTTTCGCT

GACTACATACTATGGATATCACCTGTTTGACAAGAGAAGGATTACATACCACGATGAAGA

TGAATTGGAACATGATGCAAGTGATGTAGCGCCCCATATAGGAGTCACTCAGGACTCCAC

GGTCATTGTGTATCATGTAGACGGTGCCCATCCACATGCTGACGCTATTGGCGGCCTTGGT

CCTTCATAGAGACCCAACCTAACAGTGAACGTACTGTCGCCCCACGGCGACGCCAAAAAA

TGCAAAGTTGGCCAACACATAGCTCACTGCATCGTCAAGTAGAGCTGCTTAATCACTGAGG

TATATACATTTAGTTCGCCTTCTTCAGCGTTGCCATGGAGCGACAGAACGTTCACGGTTAG

WO 2008/027592

PCT/US2007/019283

2018202034 22 Mar 2018

GTTGTGTCTCTTTGAAGGACCATGGCCACCAACAACATCAGCATATGGATGGGCACCATCT TCATGATGAACAATGACGGTGGAGTCCGTAGTGATTCCTATACGGGGTGCTACTTCACTTG GATCATGTTACAATTTATCTTCATCGTGATATATGCTCCTTCTGTTCTCACATAGGTGATAT CTTAAAATGTATGAGGCATATATACTTTCTACCTAATATTATAAAGTATATGCCTCTATATA GATCAAATAAAGCAGAAAAGTCATTGTTATTACGTTAAC (SEQ ID NO. 70). This sequence was synthesized, subcloned into a dicot binary vector (pMON97890) including a glyphosate resistance selectable marker, and transformed into Arabidopsis thaliana using a floral dip technique as described by Clough and Bent (1998), Plant J,, 16:735-743. The resulting events are selected using glyphosate, and selected plants are screened for the expected phenotypes, i. e., loss of trichomes by GL1 suppression (Marks and Feldmann (1989) Plant Cell, 1:1043-1050), prevention of petal abscission by IDA suppression (Butenko et al. (2003) Plant Cell, 15:2296-2307), and flower to leaf conversion by LFY suppression (Schwab et al. (2006) ΡΖαηί CeZZ, 18:1121-1133).

Example 7 [00125] This example describes a non-limiting embodiment of a recombinant DNA construct including DNA that transcribes to: (a) a first series of contiguous RNA segments, and (b) a second series of contiguous RNA segments, wherein the first series of contiguous RNA segments hybridize in vivo to the second series of RNA segments to form hybridized RNA that is cleaved in phase in vivo into multiple small double-stranded RNAs (“phased small RNAs”) for gene suppression. More specifically, this example describes a recombinant DNA construct that transcribes to RNA that is cleaved in vivo in phase into phased small RNAs for gene suppression of multiple viruses in plants.

[00126] Phased small RNAs were designed to target highly homologous regions of economically important geminiviruses, tospoviruses, and a potexvirus that infect tomato.

These viruses include Tomato yellow leaf curl virus (Dominican Republic isolate), Tomato leaf curl New Delhi virus, Tomato severe leaf curl virus, Pepper huasteco yellow vein virus, Pepper golden mosaic virus, Pepino mosaic virus, Tomato spotted wilt virus, Groundnut bud necrosis virus, and Capsicum chlorosis virus. Homologous regions allow a limited set of phased small RNAs to control many viruses; additionally, these conserved regions are predicted to be less likely to evolve resistance due to base changes that would impede or prevent suppression by phased small RNAs. Reynolds score, functional asymmetry, and miRNA properties were considered when selecting target sequences for suppression. Multiple phased small RNAs are utilized to improve silencing and prevent resistance.

WO 2008/027592

PCT/US2007/019283

2018202034 22 Mar 2018 [00127] In this non-limiting example, nucleotide sequences for suppressing multiple viral targets were used to replace native sequences (i. e., segments each of 21 contiguous nucleotides) of the abundant phased small RNAs derived from a scaffold sequence (the 0s06g21900 cDNA, SEQ ID NO. 69), with additional nucleotides changed where necessary in order to preserve secondary structure as found in the native precursor transcript. The replacement 21-nucleotide segments included two sequences for suppressing geminiviruses, TGGTACAACGTCATTGATGAC (SEQ ID NO. 71) and TGGACCTTACATGGCCCTTCA (SEQ ID NO. 72), one sequence for suppressing potexviruses,

TAATTGTGCAGCTCATCACCC (SEQ ID NO. 73), and three sequences for suppressing tospoviruses (one for each segment of these tripartite viruses),

TAGATGGGAAATATAGATATC (SEQ ID NO. 74, targetting the tospovirus M segment), TGCTTATATGTATGTTCTGTA (SEQ ID NQ. 75, targetting the tospovirus L segment), and TCAAGAGTCTTTGAAAGAAAG (SEQ ID NO. 76, targetting the tospovirus S segment). The replacement segments were incorporated into a DNA sequence encoding a synthetic phased small RNA precursor (i. e., an RNA transcript that is cleaved in vivo in phase into phased small RNAs for gene suppression of multiple viruses in plants),

AATCTTATTCTACATATTTCTATCTTATATAGAACAACTAGCATAGCTCTCGTTGCC

CAGCCAGGTTGCCCAGCCAGGTTGCCTGGTGCACAATGAGAGCTGGCTAGGGCGG

ACTCATTCTGCTGTTGGTGCCCAAGGATGCTAGCTGCTAGTCATACTAGTGAAGCC

TGCCATGGTTCTGAGAAATTTTTGGATACTCCGCTGCGTAGATATGCACTAAAAGC

TTGTATGTTTCGCTGACTACATACTATGGATATCACCTGTTTGACAAGAGAAGGAT

TACATACCACGATGAAGATGAATTGGAACATGTGGACCTTACATGGCCCTTCAA

TATAGGAGTCACTCAGGAgtcatCcGtgacGttAtAccAgacAtcCatatttCccatcCActtTctCtt agagacCcttgTgGGtGaTgagtTGcacaGttaCCTGCTTATATGTATGTTCTGTACCCACG

GCGACGCCAAAAAATGCAAAGTTGGCCAACACATAGCTCAGTGCATCGTCAAGTA

GAGCTGCTTAATCACTGAGGTATATACATTTAGTTCGCCTTCTTCAGCGTTGCCATG

GAtacaGaaAatacaTatCaGCGGGTAATTGTGCAGCTCATCACCCTCAAGAGTCTTT

GAAAGAAAGTAGATGGGAAATATAGATATCTGGTACAACGTCATTGATGACT

CCGTAGTGATTCCTATACtGaagGgccacgtAaggtGcaCATGTTACAATTTATCTTCATC GTGATATATGCTCCTTCTGTTCTCACATAGGTGATATCTTAAAATGTATGAGGCAT ATATACTTTCTACCTAATATTATAAAGTATATGCCTCTATATAGATCAAATAAAGC AGAAAAGTCATTGTTATTAC (SEQ ID NO. 77), where underlined text indicates the location of the replacement 21-nucleotide segments (phased small RNAs) for suppressing viruses, bold text indicates nucleotides in the foldback structure, and lower-case font indicates nucleotides changed to preserve secondary structure as found in the native precursor transcript.

WO 2008/027592

PCT/US2007/019283

2018202034 22 Mar 2018

Figure 9A depicts the foldback structure of the transcript of the endogenous 0s06g21900 phased small RNA locus (SEQ ID NO. 69); Figure 9B depicts the foldback structure of the synthetic phased small RNA precursor encoded by SEQ ID NO. 77.

Example 8 [00128] This example describes identification of targets of an RNA that is cleaved in vivo in phase into phased small RNAs for gene suppression. More specifically, this example describes identification of targets of phased small RNAs produced from a native phased small RNA locus.

[00129] Putative target genes regulated by the phased small RNAs (see Table 4) produced from the locus having SEQ ID NO. 33 were predicted from plant cDNA databases using the miRSite algorithm. miRSite predicts miRNA targets by comparison of sequence similarity between the input miRNA and the target cDNA dataset. The miRNA:target pairs (or analogously, the phased small RNA:target pairs) were scored based on rules established from experimentally validated miRNA targets (Allen et al. (2005) Cell, 121:207-221). Briefly, mispairs and single nucleotide gaps were scored as 1, G:U pairs as 0.5, scores for mispairs from bases 2 to 13 doubled, and summed along the length of the target. The predicted targets were ranked according to their penalty scores, with scores less than 4.5 considered as putative targets. In the case of conserved miRNAs or phased small RNA, targets present in orthologous genes and locations were given preference. Table 5 provides non-limiting examples of target genes (and the recognition site identified in the target gene’s RNA transcript) predicted to be regulated by regulated by phased small RNAs from the locus having SEQ ID NO. 33; the alignment of the phased small RNA and the recognition site is also depicted.

WO 2008/027592

PCT/US2007/019283

2018202034 22 Mar 2018 in ©

ΓΏ

E ’g* in in en in »n c*S in in tj·

«2 3

.

ιη

λ

in

in

ιη

ιη

ιη

ιη

ιη

ιη

ιη

*.

.·

σ

p—

tO

ρ—

rO

Ρ> —

tO

ρ

(0

5 —

(0

kp ——

to

& · fcn

3) —

to

< —

Ρ

<

Ο

©

w-

U

ο—

Ο

Ο —

υ

ρ ——

tO

ο—

υ

5 -—

to

Ο-U

Ο —

υ

Ρ —

to

Ρ —

<0

< —

P

0

< —

Ρ

ο —

U

Q —

σ

Ο-

υ

Ο-U

Ο —

U

Ο —

□

Ο —

ο

~ ·

3—

υ

3 —

υ

Ο —

υ

ο —

σ>

< —

Ρ

U-

σ

ο — υ

Ο —

υ

Ο-

υ

Ο —

υ

•n

kC —

<—

Ρ

< —

Ρ

< —

Ρ

u-

σ

<-

Ρ

< — 3

< —

Ρ

Ρ —

to

Ρ —

<0

r©

o —

Ό

ο —

υ

ο

&

Q —

σ»

σ> —

(0

Ο

ο — υ

Ο-

υ

Ρ-

to

& —

(0

o +*

< —

0

< —

Ρ

<—

Ρ

£>-

to

ο—

υ

D —

to

Ο—<ο

?—1 -

tO

ο —

σ

Ο-

σ

u

£2—

υ

ο —

υ

ο —

υ

ο-

U

ο —

σ

U —

U

5 ·· ο

5 —

to

Ρ-

σ

Ρ-

σ

’cl

p

υ

Ρ—

rO

ο —

(0

CJ —

σ>

(0

Ο-

ο—ο

Ο —

σ

ο—

υ

ο ··

ρ

Φ

o—

υ

ο

rO

ο —

υ

Ρ —

to

5 —

<0

to

<-3

< —

Ρ

ί—

Ρ

Ρ

p—

Ο)

ο—

σι

ο —

σι

& ““

tO

ι

σ

5-

to

Ο— (0

D —

to

2 —

Ρ

ii-

Ρ

Φ

< —

ρ

<—

Ρ

< —

Ρ

ο

1

Ο -;

σι

ο-

σ

ο — υ

Ο —

υ

ο —

υ

3 —

U

p—

0)

ρ—

σ

a —

&

<3 —

Ρ

< -

ρ

<—

Ρ

*ί π)

< —

Ρ

Ρ

U

Ρ —

to

’«J

pp-

05

σ

ρ ··

&

σ

Ρ-

(0

ο

υ

•3— 3

3 —

Ρ

Ρ —

σ

ρ —

σ

fl

5 —

rO

Ρ

<0

ρ —-

to

ο —

a

ο —

υ

ο—

U

Ο- U

ο ··

Ρ

ο

< —

Ρ

.O

p-

rO

ρ —

ro

ρ ~—

to

ο —

0)

ο

Ρ

ο-

σ

ο— ο

ο

Ρ

ο ··

Ρ

ο

to

p—

Ο)

a —

σι

a —

σ

α —

σ>

ο

Ρ

ο-

σ

Ο -· 3

ο —

□

ο —

υ

ο-

υ

Έ

ρ—

(0

to

υ

—

(0

—

tO

<0

f—ί-

to

ο— υ

ο-

U

Ρ ··

σ

ρ

ο

&0

p—

Ο)

ο —

σ

ο

υ

5 —

to

r5

(0

·—i-

(0

ο ·· Οι

ΰ ··

σ

ο-

ο

ο-

ο

g

<_>

Ρ

ο —

σ

U

υ

& *·

σ*

5 —

<0

ρ —-

σ

υ — σι

a —

σ

Ρ —

σ

Ρ —

σ

O &

p—

Οϊ

ο —

σ

ο —

σ

Ο-

σι

ο-

σ

ο-

σ

<— 3

<

to

Ρ ··

σ

Ρ ·

σ

m

ΓΌ

m

m

ΡΊ

m

η

ΓΟ

ro

m

V)

JU

Ξ rt

I s ώ o u u 8

C T 8 .S *S 3 I c

*3 o

CL

E.

o

1/2

O

Ό

C

W

T3 ©

c3

J3 ² S' ex .S

S *3 &o o

CL

CL K I U . CU = E § c g = £·§-§? 3 | -3 8 § g § ?- -3 § c3 ©

CX a

•a

K a

o ’3 .5 E

G o « S CL CL

O

CX

Λ _ δ ·£ ι2 3 3 2 co o.

u s

ee

-a Ε >> a jc xi ω ό tza ii ex ω sS

W co

Ok roo «*1 io a S . •a 3 X ό S’SgZ

U r/>

4> ^J u

r~ ©

TJ· = < 2 · a § 2oS a 5 OS W ^z a in

Tj· in

TJ •Λ

TJ

ΓΟΟ

Ok kO oo tm t—· PO o

r* \O

CN

Tt

CN m

Ok n

Ok m

Ok

Ok

Tj·

Ok kO

Ok

Ok

PQ

Ok

PO

WO 2008/027592

PCT/US2007/019283

2018202034 22 Mar 2018 [00130] The technique known as RNA ligase-mediated rapid amplification of cDNA 5’ ends (“5’ RLM-RACE”) is used to experimentally validate predicted targets in plants (e. g., rice and maize); see, for example, Kasschau et al. (2003) Dev. Cell, 4:205-217, and Llave et al. (2002) Science, 297:2053-2056. This approach relies on ligation of an RNA adapter molecule to the 5’ end of the cleavage site and is dependent on the 5’ phosphate left by RNAase ΠΙ enzymes including Agol. The resulting PCR products are sequenced and the relative number of clones which align to the predicted miRNA (or phased small RNA) cleavage site between nucleotides 10 and 11 relative to the miRNA (or phased small RNA) 5’ end provide an estimate of miRNA (or phased small RNA) activity. Results from 5’ RLM-RACE assays are used to confirm cleavage of a predicted target by any of the phased small RNAs.

[00131] Identification and validation of endogenous genes regulated by phased small RNAs from a natively expressed phased small RNA locus is useful, e. g., to eliminate or modify a phased small RNA recognition site in an endogenous gene in order to decouple expression of that gene from regulation by the phased small RNA that natively regulates expression of the gene. For example, the number of mispairs involving bases at positions 2 to 13 (in a phased small RNA recognition site having contiguous 21 nucleotides) can be increased to prevent recognition and cleavage by the phased small RNA.

[00132] All of the materials and methods disclosed and claimed herein can be made and used without undue experimentation as instructed by the above disclosure. Although the materials and methods of this invention have been described in terms of preferred embodiments and illustrative examples, it will be apparent to those of skill in the art that variations can be applied to the materials and methods described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

2018202034 22 Mar 2018

Claims (9)

1. The claims defining the invention are as follows.

   1. A recombinant DNA construct comprising a promoter operably linked to DNA that transcribes to RNA comprising:

   (a) at least one exogenous recognition site complementary to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 35, 36, 37, 38, 39, 49, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, and 65 and recognizable by a phased small RNA expressed in a specific cell of a multicellular eukaryote, and (b) target RNA to be suppressed in said specific cell, wherein said at least one exogenous recognition site comprises an RNA sequence that hybridizes to said phased small RNA, wherein said target RNA is to be suppressed by said phased small RNA in said specific cell, thereby said target RNA is expressed in cells of said multicellular eukaryote other than said specific cell, wherein said phased small RNA has a nucleotide sequence that maps to the plus strand of a phased small RNA locus, and wherein said phased small RNA locus contains at least three contiguous phased small RNAs.
2. 2. The recombinant DNA construct of claim 1, wherein said at least one exogenous recognition site is located within at least one of:

   (a) the 5 ’ untranslated region of said target RNA;

   (b) the 3 ’ untranslated region of said target RNA; and (c) said target RNA.
3. 3. A non-natural transgenic plant cell having in its genome the recombinant DNA construct of claim 1 or 2.
4. 4. A non-natural transgenic plant comprising the non-natural transgenic plant cell of claim 3.

   2018202034 22 Mar 2018

   WO 2008/027592

   PCT/US2007/019283

   1/9

   FIGURE 1

   2018202034 22 Mar 2018
5. 5' 3'

   SEQ ID NO. 1

   SEQ ID NO. 2

   SEQ ID NO. 7

   SEQ ID NO. 3

   SEQ ID NO. 4

   SEQ ID NO. 5

   SEQ ID NO. 6

   WO 2008/027592

   PCT/US2007/019283

   2/9

   FIGURE 2

   2018202034 22 Mar 2018 aaaatttctaccatctcacttttgtaataataccataaatgctttgccatatgtaaaaccgttcgagtagcgacaacaccggttctataaaagttgttccctttcc acgtacttataagcttatctagtgtgcacgcattccctttcxacgtatttccacgtatttccattaaccttatcttgtgtgcacgcataaggtacatgggtaataac atgttcttggaagtggtocttacctacaccgc|teFafaaabcgacgcctctcattgcgacaccaccAATCTTATTCTACATATTTCTATCTT

   ATATAGAACAACTAGCATAGCTCTCGTTGCCCAGCCAGGTTGCCCAGCCAGGTTGCCTGGTGCACAA

   TGAGAGCTGGCTAGGGCGGACTCATTCTGCTGTTGGTGCCCAACGATGCTAGCTGCTACTCATACTAG

   TGAAGCCTGCCATGGTTCTGAGAAATTTTTGGATACTCCGCTGCGTAGATATGCACTAAAAGCTTGTAT

   GTTTCGCTGACTACATACTATGgtgagatcctaaagtattacctatttatttacctttttatagtttctatatattacttcagatgacgagatcattta gcacgcataaacaagtcacaaattattaagtaaaattctttcaagtttttgcagacctttgggtatctttctcactttttatgtcttttttaacctcaaaagtcacatgt acattcaactgcattcatgtccaaatctttctagaatgattgcttggtctttgtttcgttactacggtgatttttcagcatgcatataagttcctcttcgttcttcgtttctc ctttcaaggattgatctatetaggggagataggaatcaagcaaattgttcaccgtcccatgcactatatgctagatcccattgttttttctttttaaataatgccatt tcacgaggtaccattgctttagaaaaaaaatggtcagtaacgagattttaatatctagcctgtcttttataagatataccaggtgtcttctataagatataccag gtaaatatagtaccatagaattttctaacggttcaaccagggacaatttgttttcacctagctgcgtacacacccattttatccacgcgcttcttaaataggttaa aaaatataaaacaaattggtagcatagattttctttaaagagacgtaaaatatattatgatgacaaatacctcttgtccaaccaggctcaaattattactctaa actgttttacaccaaaacttcagcctaaacagaaacatgcctggttatacatgccaggagattgcttgttcggtttggagaggattgagggattccgcacca ctaaaggtgtgtaataaatcccctccaatctcacttcttgaggatcaatcgaacatcacattaaaagaaaaaacatgtttggtgcacgttcttaaataaagg cggagtacaaacgtctgtcgaacagcaccaatgcaaacaactgaatttaacagccatttcatgataattatatatatatatatatatatataaaagatgtatct agctagactatatatatgtggtcatcctgtggactggagctgatcccctccacctccggctcgatgcccttgaacaaccgcgcgaacacgatgaacacga cgaggtccacggcggacagcacggcgagggtgatgaaggagcggtcgaggtggccgcggtcgagctcggccaggatccaccccgccgtccctccg ccggtccgccgccgcgaggcgacgccgctgatggcgctcaccatcaccatgctggcgtagttccccagcgagatggacgccatgcacagcgagctcc ccaggctcttcaccccctccggcgactgcacgttgaagaactccagctgccccacgtacacgaacacctccgacgcgcccatcaccgcgtactgcggc gcctgccacagcacgctcatggcgcggccgccggcgccggatcggcggcggcggtggacctcgacgaccgccgcggcgaccatgccgagcagcg cgatcacgaggcccgcgcccatgcgcttgagctcgccgacgccgcgcgggttcttggtcagcctcgccgccgcgggcaccaggacgtagtgggaga aggcgagcgtggcgagcacgccggcgacgtcgaacaccgacatggacgcggccggcgcgttgaacaggcccaggatgtcggtgtccatggccgc gccttgctccacgaacaaggacgacatctgggtgaactccacggagtagacgatgctgcagatccagatgggcaccatgctcaccacgcacttggcct cctccacctgcgtcaccgtgcacagtctccacgggttcttggcgttcccgtcgtggtagtcctcctcggtcgccgtcgccgccttgtcaagaaacctgagctg gtcgctgtgggcgagcttgccgacgccacggatcgccgagccctcgccatcgacctcgtggaggtggtcgccgggcggcggcacgatgtgccgcttgc ggtacgcggcgacgaacacctgggcgatgcgggtgagcgggttgccggcaggtcggacccggcggtagcgcggcgtgccgaggagaaagagcg cgagcgcgagcgcggcggcggcggtggagacccagaagccggcgacccaccggcccctgtcctcgaagaacaccaggacggagttgtagaaga gggagccgacgttgagcgagaggtagaagaggcagaagaaggcctgcttgcgccgccgctcgccggggtcggcgtcgtcgaactggtcggcgccg aacgtcgccaccgacggctggtacccgccgttcccgaacgccgccatgtagatggacaggtagaacaccgcgacgccacgccgggacggcgccgc gcactgcctgagcccgccgccgtcgccgcaccccggcggctccaccagcaagaaccacgacaacagcgacaggagcatcaacccctgcatgcca aaacacacaagaaattaaacttgtctcatgcatcaactgctgacactcttaaccttataataactttaaacttataattcagttttgctattttcagtcgtctgaaat caaaattgcacatatgctctgttttttacataattaagattgtttaaaatgttgacacagtatttatgttgttatattttatactactgctgagtttatcctgatatctgact gcatatttttcagGATATCACCTGTTTGACAAGAGAAGGATTACATACCACGATGAAGATGAATTGGAACATG

   ATqcaaqtqatqtaqcqccccatataaaaqtcactcaqaaaaqcacaqaaqagqaaqaaqatqtaqacqgtqcccatccacatacfqacqcteZfa qcqqcctoqqcqttctcctqqtqaaqcacctqcctoaqqaacaccaccaqqcccacqqcqacqccaaaaaatqcaaaqttqqccaacacataactc actgcatcgtcaagtagagctgcttaatcactgaggtaaaataaatattttaatttcttttggatcaaaccactatatatgcccccattttgcattgcagtgttgttc aacactggttagtttatctctactatatatcttaaaagcacagtcatccttattcccattctatccataagaaacactagaaaaaactaaccaattgagagaaa aatatgggagaagagaaaaaaaaattaaaccacattcaccatatcacatccgtttgcaaggcacggtcctatgactagtattgtataaaatgatagattgt tctccacattatattggtataaatactggactattagtaaatcaaacactattaaccacgaaaaaaaagagagagttgggatgagattgtggggattaaattt ttaccaagaagtagtgccattgtcatctttcctcttgaagtcttcagttctgggcttccctggaaatgttgggtctgatctteagtgtgcacaaatgactcattgtat atcatggaattgcatggagagcatgatcccacagattcaacatcttccattggcttttaaaaaaaagtagttgaggaaaaaggtgtcacaactcacttacc actctactagaaagtaataaggatagactaaaaattttagagtttttattcttggtttgattaattcgccgacaaataataagtacaaacagaacaaatgattct gaagtgttacctatcatattcaattataatattcaacgtaacaagtagcaatctaaaggacatcatcttggggaggtacttaattggtacttcctccattccaaa atgtttgacgccgttgactttttaaaatatgtttgaccgtttgtcttattcaaaaaatttaagtaattattaattcttttectatcatttgatttattgttaaatatacttttatgt atatatatagttttatatatttcataaaagtttttgaataagacgaacggtcaaacatatttaaaaaagccaacggcgtcaaacatttaaggaaggagggag tataatataaaaagaatatgatgtttttaggttttgtcctcttcttgaagaggtatatgccttcttaccattttagaaatacctcgccataccggagatatcaaacta attgcataatttcacaaatcatatttataaatgttttttattttatttttaaactttgctaggtatatacatttagttcgccttcttcagcgttgccatggacctggtgatGT

   TCTTCCGGCGGGTGCCCCACCAAGAGAACGCCGTGGCCACCAACAACATCAGCATATGGATGGGCA

   CCATCTTCATCTTTGCCCTCTTTAGTGCTTTCCGTAGTGATTCCTATACGGGGTGCTACTTCACTTGGAT

   CATGTTACAATTTATCTTCATCGTGATATATGCTCCTTCTGTTCTCACATAGGTGATATCTTAAAATGTA

   TGAGGCATATATACTTTCTACCTAATATTATAAAGTATATGCCTCTATATAGAATCAAATAAAGCAGAA

   AAGTCATTGTTATTACCAATCGTGTACTTTTGTTCTAAACATCTCAACTAGTTTAAAGTATTTGTCTCTCT

   TGAgcaatgggtttaaacctctccacggatgggagagaacctctactatttgattgttccaacttttgacacaatagaaacacagatgatactgaaggtat gaaaggtaaatagttagttaaggttccaatcattcaaatgctggaaagtacatttacttctattttaaactattaaggggtaaaaaaaaacagatatacgctct tactctgatctcaaatgccatgatctctgcagatcccacggtgtcgggaaccttcaatacgaatatatatataaaaaagaaaagatcagtaaggaaatgttt gatctgctagccttagttttcatattattaaattttagaaaatacaagtaagattataaaattataagtttgctacaatatttatgtctgaacatagtataa

   WO 2008/027592

   PCT/US2007/019283

   3/9

   FIGURE 3

   2018202034 22 Mar 2018 pMON94320 pMONl 00574 pMONl 00575 pMONl 00576

   B pMON94320(control GUS) atcgtcggctacagcctcgggaattctctGcatgcgtttggacgtatgctcattcag c pMONl00574(sense ID1379342)

   CTACAGCCTCGGGAATTCTGCCCCACCAAGAGAACGCCGTCTGCATGCGTTT

   Q pMON100575 (antisense ID1379342)

   CTACAGCCTC GGGAATTCCGGCGTTCTCTTGGTGGGGCATCTGCATGCGTTT

   E pMONl00576 (antisense ID1379342+ ID1275002)

   CTACAGCCTCGGGAATT CTGCTGATGTTGTTGGTGGCC ACGG CGTTCTCTTGGTGGGGC A TCTGCATGCGTTT

   WO 2008/027592

   PCT/US2007/019283

   4/9

   FIGURE 4

   2018202034 22 Mar 2018

   Developing Maize Endosperm (DAP) 10 DAP are whole kernel

   10 21 27 33 NA 10 15 21 27 33 39

   Developing Maize Embryo (DAP)

   21 27 33 44 21 27 33 39 44

   PCT/US2007/019283

   5/9

   WO 2008/027592

   2018202034 22 Mar 2018

   Endogenous phased small RNA transcript (SEQ ID NO. 17)

   FIGURE 5

   Engineered LKR/waxy suppression transcript (SEQ ID NO. 32)

   WO 2008/027592

   PCT/US2007/019283
6. 6/9

   FIGURE 6

   2018202034 22 Mar 2018

   5’ flanking sequence (SEQ ID NO. 66)

   3’ flanking sequence (SEQ ID NO. 67)

   SEQ ID NO. 35

   SEQ ID NO. 36

   SEQ ID NO. 37

   SEQ ID NO. 38

   SEQ ID NO. 39

   SEQ ID NO. 40

   SEQ ID NO. 41

   SEQ ID NO. 42

   SEQ ID NO. 43

   SEQ ID NO. 44

   SEQ ID NO. 45

   SEQ ID NO. 46

   SEQ ID NO. 47 spacer sequence (SEQ ID NO. 68)

   SEQ ID NO. 60

   SEQ ID NO. 59

   SEQ ID NO. 58

   SEQ ID NO. 57

   SEQ ID NO. 56

   SEQ ID NO. 55

   SEQ ID NO. 54

   SEQ ID NO. 53

   SEQ ID NO. 52

   SEQ ID NO. 51

   SEQ ID NO. 50

   SEQ ID NO. 49

   SEQ ID NO. 48

   WO 2008/027592

   PCT/US2007/019283
7. 7/9

   FIGURE 7

   2018202034 22 Mar 2018

   200

   180

   160

   140

   120

   100

   602

   623

   644

   665

   J L

   686

   707

   V \a.

   728

   749 Grain + Seedling

   770

   791

   B

   WO 2008/027592

   PCT/US2007/019283
8. 8/9

   FIGURE 8

   2018202034 22 Mar 2018

   6000

   5000

   4000

   3000

   3000

   1000

   5'end position of siRNA

   ' . . .,1- ....... . ¹ .

   ε ί

   oc

   SEQID NO. 69

   Os06g2190C s

   ............

   y

   SEQ ID NO. 1

   2Z3 29279

   Pl P2 I C—-Γ a _t-o--v·

   Pl’ P2*

   11.0

   SEQID NO. 2

   SSSfi 03

   P3 P4* ι :—:

   53.1

   P5·

   Pl’ P4 23.9 331S.1

   PS

   1427.2

   Z0 21.9

   P6* P7’

   P6 P7

   1586.3 35084.5

   P8·

   SEQID NO. 7

   4780.2

   PA

   SEQID SEQID SEQID SEQID NO. 3 NO. 4 NO. 5 NO. 6

   120.2

   Rice

   Seedling Grain ______Maize_______ ______Arabidopsis

   Leaf Root Kernel Mature Leaf Young Leaf

   5S rRNA/tRNA

   WO 2008/027592

   PCT/US2007/019283
9. 9/9

   2018202034 22 Mar 2018 to <J — «J

   U —

   8 =

   PQ

   2018202034 22 Mar 2018

   <110> Allen, Edwards Guo, Liang Heisel, Sara E. Ivashuta, Sergey I. Zhang, Yuanji I. <120> Phased Small RNAs <130> 38-21(54702)B <150> <151> 60/841,608 2006-08-31 <160> 97 <210> <211> <212> <213> 1 21 RNA Oryza sativa <400> 1 augcaaguga uguagcgccc c 21 <210> <211> <212> <213> 2 21 RNA Oryza sativa <400> 2 auauaggagu cacucaggaa a 21 <210> <211> <212> <213> 3 21 RNA Oryza sativa <400> 3 ucuuugcccu cuuuagugcu u 21 <210> <211> <212> <213> 4 21 RNA Oryza sativa <400> 4 uauggauggg caccaucuuc a 21

   2018202034 22 Mar 2018

   <210> 5 <211> 21 <212> RNA <213> Oryza sativa <400> 5 uggccaccaa caacaucagc a 21

   <210> 6 <211> 21 <212> RNA <213> Oryza sativa <400> 6 ugccccacca agagaacgcc g 21

   <210> 7 <211> 21 <212> RNA <213> Oryza sativa <400> 7 ugccugagga acaccaccca g 21

   <210> 8 <211> 5521 <212> DNA <213> Oryza sativa <400> 8

   aaaatttcta ccatctcact tttgtaataa taccataaat gctttgccat atgtaaaacc 60 gttcgagtag cgacaacacc ggttctataa aagttgttcc ctttccacgt acttataagc 120 ttatctagtg tgcacgcatt ccctttccac gtatttccac gtatttccat taaccttatc 180 ttgtgtgcac gcataaggta catgggtaat aacatgttct tggaagtggt ccttacctac 240 accgctatat aaagcgacgc ctctcattgc gacaccacca atcttattct acatatttct 300 atcttatata gaacaactag catagctctc gttgcccagc caggttgccc agccaggttg 360 cctggtgcac aatgagagct ggctagggcg gactcattct gctgttggtg cccaacgatg 420 ctagctgcta ctcatactag tgaagcctgc catggttctg agaaattttt ggatactccg 480 ctgcgtagat atgcactaaa agcttgtatg tttcgctgac tacatactat ggtgagatcc 540 taaagtatta cctatttatt taccttttta tagtttctat atattacttc agatgacgag 600 atcatttagc acgcataaac aagtcacaaa ttattaagta aaattctttc aagtttttgc 660 agacctttgg gtatctttct cactttttat gtctttttta acctcaaaag tcacatgtac 720

   2018202034 22 Mar 2018

   attcaactgc attcatgtcc aaatctttct agaatgattg cttggtcttt gtttcgttac 780 tacggtgatt tttcagcatg catataagtt cctcttcgtt cttcgtttct cctttcaagg 840 attgatctat ctaggggaga taggaatcaa gcaaattgtt caccgtccca tgcactatat 900 gctagatccc attgtttttt ctttttaaat aatgccattt cacgaggtac cattgcttta 960 gaaaaaaaat ggtcagtaac gagattttaa tatctagcct gtcttttata agatatacca 1020 ggtgtcttct ataagatata ccaggtaaat atagtaccat agaattttct aacggttcaa 1080 ccagggacaa tttgttttca cctagctgcg tacacaccca ttttatccac gcgcttctta 1140 aataggttaa aaaatataaa acaaattggt agcatagatt ttctttaaag agacgtaaaa 1200 tatattatga tgacaaatac ctcttgtcca accaggctca aattattact ctaaactgtt 1260 ttacaccaaa acttcagcct aaacagaaac atgcctggtt atacatgcca ggagattgct 1320 tgttcggttt ggagaggatt gagggattcc gcaccactaa aggtgtgtaa taaatcccct 1380 ccaatctcac ttcttgagga tcaatcgaac atcacattaa aagaaaaaac atgtttggtg 1440 cacgttctta aataaaggcg gagtacaaac gtctgtcgaa cagcaccaat gcaaacaact 1500 gaatttaaca gccatttcat gataattata tatatatata tatatatata aaagatgtat 1560 ctagctagac tatatatatg tggtcatcct gtggactgga gctgatcccc tccacctccg 1620 gctcgatgcc cttgaacaac cgcgcgaaca cgatgaacac gacgaggtcc acggcggaca 1680 gcacggcgag ggtgatgaag gagcggtcga ggtggccgcg gtcgagctcg gccaggatcc 1740 accccgccgt ccctccgccg gtccgccgcc gcgaggcgac gccgctgatg gcgctcacca 1800 tcaccatgct ggcgtagttc cccagcgaga tggacgccat gcacagcgag ctccccaggc 1860 tcttcacccc ctccggcgac tgcacgttga agaactccag ctgccccacg tacacgaaca 1920 cctccgacgc gcccatcacc gcgtactgcg gcgcctgcca cagcacgctc atggcgcggc 1980 cgccggcgcc ggatcggcgg cggcggtgga cctcgacgac cgccgcggcg accatgccga 2040 gcagcgcgat cacgaggccc gcgcccatgc gcttgagctc gccgacgccg cgcgggttct 2100 tggtcagcct cgccgccgcg ggcaccagga cgtagtggga gaaggcgagc gtggcgagca 2160 cgccggcgac gtcgaacacc gacatggacg cggccggcgc gttgaacagg cccaggatgt 2220 cggtgtccat ggccgcgcct tgctccacga acaaggacga catctgggtg aactccacgg 2280 agtagacgat gctgcagatc cagatgggca ccatgctcac cacgcacttg gcctcctcca 2340 cctgcgtcac cgtgcacagt ctccacgggt tcttggcgtt cccgtcgtgg tagtcctcct 2400 cggtcgccgt cgccgccttg tcaagaaacc tgagctggtc gctgtgggcg agcttgccga 2460 cgccacggat cgccgagccc tcgccatcga cctcgtggag gtggtcgccg ggcggcggca 2520 cgatgtgccg cttgcggtac gcggcgacga acacctgggc 3 gatgcgggtg agcgggttgc 2580

   2018202034 22 Mar 2018

   cggcaggtcg gacccggcgg tagcgcggcg tgccgaggag aaagagcgcg agcgcgagcg 2640 cggcggcggc ggtggagacc cagaagccgg cgacccaccg gcccctgtcc tcgaagaaca 2700 ccaggacgga gttgtagaag agggagccga cgttgagcga gaggtagaag aggcagaaga 2760 aggcctgctt gcgccgccgc tcgccggggt cggcgtcgtc gaactggtcg gcgccgaacg 2820 tcgccaccga cggctggtac ccgccgttcc cgaacgccgc catgtagatg gacaggtaga 2880 acaccgcgac gccacgccgg gacggcgccg cgcactgcct gagcccgccg ccgtcgccgc 2940 accccggcgg ctccaccagc aagaaccacg acaacagcga caggagcatc aacccctgca 3000 tgccaaaaca cacaagaaat taaacttgtc tcatgcatca actgctgaca ctcttaacct 3060 tataataact ttaaacttat aattcagttt tgctattttc agtcgtctga aatcaaaatt 3120 gcacatatgc tctgtttttt acataattaa gattgtttaa aatgttgaca cagtatttat 3180 gttgttatat tttatactac tgctgagttt atcctgatat ctgactgcat atttttcagg 3240 atatcacctg tttgacaaga gaaggattac ataccacgat gaagatgaat tggaacatga 3300 tgcaagtgat gtagcgcccc atataggagt cactcaggaa agcacagaag agggagaaga 3360 tgtagacggt gcccatccac atgctgacgc tattggcggc ctcggcgttc tcctggtgga 3420 gcacctgcct gaggaacacc accaggccca cggcgacgcc aaaaaatgca aagttggcca 3480 acacatagct cactgcatcg tcaagtagag ctgcttaatc actgaggtaa aataaatatt 3540 ttaatttctt ttggatcaaa ccactatata tgcccccatt ttgcattgca gtgttgttca 3600 acactggtta gtttatctct actatatatc ttaaaagcac agtcatcctt attcccattc 3660 tatccataag aaacactaga aaaaactaac caattgagag aaaaatatgg gagaagagaa 3720 aaaaaaatta aaccacattc accatatcac atccgtttgc aaggcacggt cctatgacta 3780 gtattgtata aaatgataga ttgttctcca cattatattg gtataaatac tggactatta 3840 gtaaatcaaa cactattaac cacgaaaaaa aagagagagt tgggatgaga ttgtggggat 3900 taaattttta ccaagaagta gtgccattgt catctttcct cttgaagtct tcagttctgg 3960 gcttccctgg aaatgttggg tctgatcttc agtgtgcaca aatgactcat tgtatatcat 4020 ggaattgcat ggagagcatg atcccacaga ttcaacatct tccattggct tttaaaaaaa 4080 agtagttgag gaaaaaggtg tcacaactca cttaccactc tactagaaag taataaggat 4140 agactaaaaa ttttagagtt tttattcttg gtttgattaa ttcgccgaca aataataagt 4200 acaaacagaa caaatgattc tgaagtgtta cctatcatat tcaattataa tattcaacgt 4260 aacaagtagc aatctaaagg acatcatctt ggggaggtac ttaattggta cttcctccat 4320 tccaaaatgt ttgacgccgt tgacttttta aaatatgttt gaccgtttgt cttattcaaa 4380

   2018202034 22 Mar 2018

   aaatttaagt aattattaat tcttttccta tcatttgatt tattgttaaa tatactttta 4440 tgtatatata tagttttata tatttcataa aagtttttga ataagacgaa cggtcaaaca 4500 tatttaaaaa agccaacggc gtcaaacatt taaggaagga gggagtataa tataaaaaga 4560 atatgatgtt tttaggtttt gtcctcttct tgaagaggta tatgccttct taccatttta 4620 gaaatacctc gccataccgg agatatcaaa ctaattgcat aatttcacaa atcatattta 4680 taaatgtttt ttattttatt tttaaacttt gctaggtata tacatttagt tcgccttctt 4740 cagcgttgcc atggacctgg tgatgttctt ccggcgggtg ccccaccaag agaacgccgt 4800 ggccaccaac aacatcagca tatggatggg caccatcttc atctttgccc tctttagtgc 4860 tttccgtagt gattcctata cggggtgcta cttcacttgg atcatgttac aatttatctt 4920 catcgtgata tatgctcctt ctgttctcac ataggtgata tcttaaaatg tatgaggcat 4980 atatactttc tacctaatat tataaagtat atgcctctat atagaatcaa ataaagcaga 5040 aaagtcattg ttattaccaa tcgtgtactt ttgttctaaa catctcaact agtttaaagt 5100 atttgtctct cttgagcaat gggtttaaac ctctccacgg atgggagaga acctctacta 5160 tttgattgtt ccaacttttg acacaataga aacacagatg atactgaagg tatgaaaggt 5220 aaatagttag ttaaggttcc aatcattcaa atgctggaaa gtacatttac ttctatttta 5280 aactattaag gggtaaaaaa aaacagatat acgctcttac tctgatctca aatgccatga 5340 tctctgcaga tcccacggtg tcgggaacct tcaatacgaa tatatatata aaaaagaaaa 5400 gatcagtaag gaaatgtttg atctgctagc cttagttttc atattattaa attttagaaa 5460 atacaagtaa gattataaaa ttataagttt gctacaatat ttatgtctga acatagtata 5520 a 5521 <210> 9 <211> 2454 <212> DNA <213> Oryza sativa <220> <221> misc_feature <222> (1)..(2454) <223> N is A, T, G, or C <220> <221> unsure <222> (1)..(2454) <223> unsure at all n locations <400> 9 aatcttattc tacatatttc tatcttatat agaacaacta gcatagctct cgttgcccag 60 ccaggttgcc cagccaggtt gcctggtgca caatgagagc tggctagggc ggactcattc 120

   2018202034 22 Mar 2018

   tgctgttggt gcccaacgat gctagctgct actcatacta gtgaagcctg ccatggttct 180 gagaaatttt tggatactcc gctgcgtaga tatgcactaa aagcttgtat gtttcgctga 240 ctacatacta tgctagacta tatatatgtg gtcatcctgt ggactggagc tgatcccctc 300 cacctccggc tcgatgccct tgaacaaccg cgcgaacacg atgaacacga cgaggtccac 360 ggcggacagc acggcgaggg tgatgaagga gcggtcgagg tggccgcggt cgagctcggc 420 caggatccac cccgccgtcc ctccgccggt ccgccgccgc gaggcgacgc cgctgatggc 480 gctcaccatc accatgctgg cgtagttccc cagcgagatg gacgccatgc acagcgagct 540 ccccaggctc ttcaccccct ccggcgactg cacgttgaag aactccagct gccccacgta 600 cacgaacacc tccgacgcgc ccatcaccgc gtactgcggc gcctgccaca gcacgctcat 660 ggcgcggccg ccggcgccgg atcggcggcg gcggtggacc tcgacgaccg ccgcggcgac 720 catgccgagc agcgcgatca cgaggcccgc gcccatgcgc ttgagctcgc cgacgccgcg 780 cgggttcttg gtcagcctcg ccgccgcggg caccaggacg tagtgggaga aggcgagcgt 840 ggcgagcacg ccggcgacgt cgaacaccga catggacgcg gccggcgcgt tgaacaggcc 900 caggatgtcg gtgtccatgg ccgcgccttg ctccacgaac aaggacgaca tctgggtgaa 960 ctccacggag tagacgatgc tgcagatcca gatgggcacc atgctcacca cgcacttggc 1020 ctcctccacc tgcgtcaccg tgcacagtct ccacgggttc ttggcgttcc cgtcgtggta 1080 gtcctcctcg gtcgccgtcg ccgccttgtc aagaaacctg agctggtcgc tgtgggcgag 1140 cttgccgacg ccacggatcg ccgagccctc gccatcgacc tcgtggaggt ggtcgccggg 1200 cggcggcacg atgtgccgct ggggttagtc ngggacgaac acctgggcga tgcgggtgag 1260 cgggttgccg gcaggtcgga cccggcggta gcgcggcgtg ccgaggagaa agagcgcgag 1320 cgcgagcgcg gcggcggcgg tggagaccca gaagccggcg acccaccggc ccctgtcctc 1380 gaagaacacc aggacggagt tgtagaagag ggagccgacg ttgagcgaga ggtagaagag 1440 gcagaagaag gcctgcttgc gccgccgctc gccggggtcg gcgtcgtcga actggtcggc 1500 gccgaacgtc gccaccgacg gctggtaccc gccgttcccg aacgccgcca tgtagatgga 1560 caggtagaac accgcgacgc cacgccggga cggcgccgcg cactgcctga gcccgccgcc 1620 gtcgccgcac cccggcggct ccaccaccca caaccacgac aacagcgaca ggagcatcaa 1680 ccccacgatg aagatgaatt ggaacatgat gcaagtgatg tagcgcccca tataggagtc 1740 actcaggaaa gcacagaaga gggagaagat gtagacggtg cccatccaca tgctgacgct 1800 attggcggcc tcggcgttct cctggtggag cacctgcctg aggaacacca ccaggcccac 1860 ggcgacgcca aaaaatgcaa agttggccaa cacatagctc acaagaagta gtgccattgt 1920

   2018202034 22 Mar 2018

   catctttcct cttgaagtct tcagttctgg gcttccctgg aaatgttggg tctgatcttc 1980 agtgtgcaca aatgactcat tgtatatcat ggaattgcat ggagagcatg atcccacaga 2040 ttcaacatct tccattggca ttttagaaat acctcgccat accggagata tcaaactggt 2100 gatgttcttc cggcgggtgc cccaccaaga gaacgccgtg gccaccaaca acatcagcat 2160 atggatgggc accatcttca tctttgccct ctttagtgct ttccgtagtg attcctatac 2220 ggggtgctac ttcacttgga tcatgttaca atttatcttc atcgtgatat atgctccttc 2280 tgttctcaca taggtgatat cttaaaatgt atgaggcata tatactttct acctaatatt 2340 ataaagtata tgcctctata tagaatcaaa taaagcagaa aagtcattgt tattaccaat 2400 cgtgtacttt tgttctaaac atctcaacta gtttaaagta tttgtctctc ttga 2454 <210> 10 <211> 684 <212> DNA <213> Oryza sativa <400> 10 aatcttattc tacatatttc tatcttatat agaacaacta gcatagctct cgttgcccag 60 ccaggttgcc cagccaggtt gcctggtgca caatgagagc tggctagggc ggactcattc 120 tgctgttggt gcccaacgat gctagctgct actcatacta gtgaagcctg ccatggttct 180 gagaaatttt tggatactcc gctgcgtaga tatgcactaa aagcttgtat gtttcgctga 240 ctacatacta tggatatcac ctgtttgaca agagaaggat tacataccac gatgaagatg 300 aattggaaca tgatgttctt ccggcgggtg ccccaccaag agaacgccgt ggccaccaac 360 aacatcagca tatggatggg caccatcttc atctttgccc tctttagtgc tttccgtagt 420 gattcctata cggggtgcta cttcacttgg atcatgttac aatttatctt catcgtgata 480 tatgctcctt ctgttctcac ataggtgata tcttaaaatg tatgaggcat atatactttc 540 tacctaatat tataaagtat atgcctctat atagatcaaa taaagcagaa aagtcattgt 600 tattaccaat cgtgtacttt tgttctaaac atctcaacta gtttaaagta tttgtctctc 660 ttgaacaaaa aaaaaaaaaa aaaa 684

   <210> 11 <211> 564 <212> DNA <213> Oryza sativa <220> <221> misc_feature <222> (1)..(564) <223> N is A, T, G

   2018202034 22 Mar 2018 <220>

   <221> unsure <222> (1)..(564) <223> unsure at all n locations <400> 11

   aatcttattc tacatatttc tatcttatat agaacaacta gcatagctct cgttgcccag 60 ccaggttgcc cagccaggtt gcctggtgca caatgagagc tggctagggc ggactcattc 120 tgctgttggt gcccaacgat gctagctgct actcatacta gtgaagcctg ccatggttct 180 gagaaatttt tggatactcc gctgcgtaga tatgcactaa aagcttgtat gtttcgctga 240 ctacatacta tggatatcac ctgtttgaca agagaaggat tacataccac gatgaagatg 300 aattggaaca tgatgcaagt gatgtagcgc cccatatagg agtcactcag gaaagcacag 360 aagagggaga agatgtagac ggtgcccatc cacatgctga cgctattggc ggcctcggcg 420 ttctcctggt ggagcacctg cctgaggaac accaccaggc ccacggcgac gccaaaaaat 480 gcaaagntgg ccaacacata gctcactgca tcgncaagna gagctgcnta atcactgagg 540 gatatccatt tannntggnc ttct 564 <210> 12 <211> 57 <212> DNA <213> artificial sequence <220> <223> synthetic construct <400> 12 atcgtcggct acagcctcgg gaattctctg catgcgtttg gacgtatgct cattcag 57

   2018202034 22 Mar 2018

   <210> 13 <211> 78 <212> DNA <213> artificial sequence <220> <223> synthetic construct <400> 13 atcgtcggct acagcctcgg gaattctgcc ccaccaagag aacgccgtct gcatgcgttt 60 ggacgtatgc tcattcag 78 <210> 14 <211> 78 <212> DNA <213> artificial sequence <220> <223> synthetic construct <400> 14 atcgtcggct acagcctcgg gaattccggc gttctcttgg tggggcatct gcatgcgttt 60 ggacgtatgc tcattcag 78 <210> 15 <211> 99 <212> DNA <213> artificial sequence <220> <223> synthetic construct <400> 15 atcgtcggct acagcctcgg gaattctgct gatgttgttg gtggccacgg cgttctcttg 60 gtggggcatc tgcatgcgtt tggacgtatg ctcattcag 99 <210> 16 <211> 21 <212> DNA <213> artificial sequence <220> <223> synthetic construct <400> 16 cggcgttctc ttggtggggc a 21

   2018202034 22 Mar 2018 <210> 17 <211> 939 <212> DNA <213> artificial sequence <220>

   <223> synthetic construct

   <400> 17 aatcttattc tacatatttc tatcttatat agaacaacta gcatagctct cgttgcccag 60 ccaggttgcc cagccaggtt gcctggtgca caatgagagc tggctagggc ggactcattc 120 tgctgttggt gcccaacgat gctagctgct actcatacta gtgaagcctg ccatggttct 180 gagaaatttt tggatactcc gctgcgtaga tatgcactaa aagcttgtat gtttcgctga 240 ctacatacta tggatatcac ctgtttgaca agagaaggat tacataccac gatgaagatg 300 aattggaaca tgatgcaagt gatgtagcgc cccatatagg agtcactcag gaaagcacag 360 aagagggaga agatgtagac ggtgcccatc cacatgctga cgctattggc ggcctcggcg 420 ttctcctggt ggagcacctg cctgaggaac accaccaggc ccacggcgac gccaaaaaat 480 gcaaagttgg ccaacacata gctcactgca tcgtcaagta gagctgctta atcactgagg 540 gtatatacat ttagttcgcc ttcttcagcg ttgccatgga cctggtgatg ttcttccggc 600 gggtgcccca ccaagagaac gccgtggcca ccaacaacat cagcatatgg atgggcacca 660 tcttcatctt tgccctcttt agtgctttcc gtagtgattc ctatacgggg tgctacttca 720 cttggatcat gttacaattt atcttcatcg tgatatatgc tccttctgtt ctcacatagg 780 tgatatctta aaatgtatga ggcatatata ctttctacct aatattataa agtatatgcc 840 tctatataga tcaaataaag cagaaaagtc attgttatta ccaatcgtgt acttttgttc 900 taaacatctc aactagttta aagtatttgt ctctcttga 939 <210> 18 <211> 312 <212> DNA <213> artificial sequence <220> <223> synthetic construct <400> 18 aatcttattc tacatatttc tatcttatat agaacaacta gcatagctct cgttgcccag 60 ccaggttgcc cagccaggtt gcctggtgca caatgagagc tggctagggc ggactcattc 120 tgctgttggt gcccaacgat gctagctgct actcatacta gtgaagcctg ccatggttct 180 gagaaatttt tggatactcc gctgcgtaga tatgcactaa aagcttgtat gtttcgctga 240

   2018202034 22 Mar 2018 ctacatacta tggatatcac ctgtttgaca agagaaggat tacataccac gatgaagatg 300 aattggaaca tg 312

   2018202034 22 Mar 2018

   <210> 19 <211> 21 <212> DNA <213> artificial sequence <220> <223> synthetic construct <400> 19 gcacagaaga gggagaagat g

   <210> 20 <211> 21 <212> DNA <213> artificial sequence <220> <223> synthetic construct <400> 20 tagacggtgc ccatccacat g

   <210> 21 <211> 21 <212> DNA <213> artificial sequence <220> <223> synthetic construct <400> 21 ctgacgctat tggcggcctc g

   <210> 22 <211> 21 <212> DNA <213> artificial sequence <220> <223> synthetic construct <400> 22 gcgttctcct ggtggagcac c

   <210> <211> <212> <213> 23 123 DNA artificial sequence <220> <223> synthetic construct <400> 23

   2018202034 22 Mar 2018

   cccacggcga cgccaaaaaa tgcaaagttg gccaacacat agctcactgc atcgtcaagt 60 agagctgctt aatcactgag ggtatataca tttagttcgc cttcttcagc gttgccatgg 120 acc 123

   <210> 24 <211> 21 <212> DNA <213> artificial sequence <220> <223> synthetic construct <400> 24 tggtgatgtt cttccggcgg g

   <210> 25 <211> 21 <212> DNA <213> artificial sequence <220> <223> synthetic construct <400> 25 tccgtagtga ttcctatacg g

   <210> 26 <211> 21 <212> DNA <213> artificial sequence <220> <223> synthetic construct <400> 26 ggtgctactt cacttggatc a

   <210> 27 <211> 210 <212> DNA <213> artificial sequence <220> <223> synthetic construct <400> 27 tgttacaatt tatcttcatc gtgatatatg ctccttctgt tctcacatag gtgatatctt 60 aaaatgtatg aggcatatat actttctacc taatattata aagtatatgc ctctatatag 120

   2018202034 22 Mar 2018

   atcaaataaa gcagaaaagt cattgttatt accaatcgtg tacttttgtt ctaaacatct 180 caactagttt aaagtatttg tctctcttga 210 <210> <211> <212> <213> 28 21 DNA artificial sequence

   <220> <223> synthetic construct <400> 28 tgacatcaag tgatctatgc g 21 <210> <211> <212> <213> 29 21 DNA artificial sequence

   <220> <223> synthetic construct <400> 29 tacacctgca tctttggcac a 21 <210> <211> <212> <213> 30 21 DNA artificial sequence

   <220> <223> synthetic construct <400> 30 tctccttggc gagcggcgca t 21 <210> <211> <212> <213> 31 21 DNA artificial sequence

   <220> <223> synthetic construct <400> 31 ttgtggatgc agaaagcggt g 21 <210> <211> <212> <213> 32 939 DNA artificial sequence

   2018202034 22 Mar 2018

   <220> <223> synthetic construct <400> 32 aatcttattc tacatatttc tatcttatat agaacaacta gcatagctct cgttgcccag 60 ccaggttgcc cagccaggtt gcctggtgca caatgagagc tggctagggc ggactcattc 120 tgctgttggt gcccaacgat gctagctgct actcatacta gtgaagcctg ccatggttct 180 gagaaatttt tggatactcc gctgcgtaga tatgcactaa aagcttgtat gtttcgctga 240 ctacatacta tggatatcac ctgtttgaca agagaaggat tacataccac gatgaagatg 300 aattggaaca tgatgcaagt gatgtagcgc cccatatagg agtcactcag gaaagcgcag 360 ctcgccaccg agatgcgccc aagatgcagg tgcatgctga cgctattggc ggcctcgcat 420 agatcccttg atatcacttt gtggatgcag aaagcggtgc ccacggcgac gccaaaaaat 480 gcaaagttgg ccaacacata gctcactgca tcgtcaagta gagctgctta atcactgagg 540 gtatatacat ttagttcgcc ttcttcagcg ttgccatgga caccgctctc tgcatcaaca 600 aagtgacatc aagtgatcta tgcgtggcca ccaacaacat cagcatacac ctgcatcttt 660 ggcacatctc cttggcgagc ggcgctttcc gtagtgattc ctatacgggg tgctacttca 720 cttggatcat gttacaattt atcttcatcg tgatatatgc tccttctgtt ctcacatagg 780 tgatatctta aaatgtatga ggcatatata ctttctacct aatattataa agtatatgcc 840 tctatataga tcaaataaag cagaaaagtc attgttatta ccaatcgtgt acttttgttc 900 taaacatctc aactagttta aagtatttgt ctctcttga 939 <210> 33 <211> 2072 <212> DNA <213> Oryza sativa <400> 33 gattctcccc tgcgccgccg ccgccgccgc cgcctcaatc gggcgaagcc gccctcgccg 60 ccgtcgcggc ggcggcggcg agggcgagct cctgcgagag atcctccgcc gcctcatgcc 120 tcgcgcgcgc gctcccgctc ccgctctcgc ctgcagtatt tgttccattg ccgcgcacca 180 ctttccggtg ggcggcgggc aatgctaggg gttaagagac cttctctccc cgagatggag 240 gcgccgggcg gcgcggcggg ggacgcggag gaggaagttg atgcccggat ccgctgggtt 300 ccatggtggc tgctatggaa tggtggaatt gcttggatgg ccacgaaggg gatcgacgcc 360 aattgtttgg cgacctctac gatagaatcg cgtcgagtcg gggtgttctt tcctgttatt 420

   actagaagta gttgaatttc gtgattgaac acacaaggaa gcttgatatc gcgtcggggg 480

   2018202034 22 Mar 2018

   tgttctttcc tgttattact agatgtagtt gggtttcgtg attgaacacc taaggaaagg 540 aagcttgata aatggaagat agtccagcaa gttttgaaga tgatagaaaa tttgagcgcg 600 tcgtagtaac tgtcgtccac gatcacgtcc agtgttgttc atggcatggg ggatggagtc 660 aggatccttg aggcgtctgc tcctgttgca ctgcttcatg cctttcctgc cttctaggat 720 gcttaagatg gttgcgaagt caggtgcttg ggagttcatg aagcggtcat aatcaatttc 780 gctctctgta gtactttctc tggtgtcttc cccgttgctt ccttttggaa gaaaagcgtc 840 ctttagaatc tcttgagaga gtgcactttc tccctctcct gccatcagta gtgcctttat 900 tttcgcttgg tttccgcatc atcaggtggc acttatagaa attattttat ggaggaaaaa 960 gcattgtatg gcatgataga aatatcctta tggataaaac taggacactt gcaagtgttc 1020 aatgggagtc accttacctt ttttgcctac ctgtctgcat ttcatgaatg ggattccttc 1080 tcctgcgccg gtgctgtctt ctcaaatggg aaatggaggc aagcatctgc cctgttccat 1140 ggtggcagcc atggaatgat gggatttctt tgatggtcat aaaggagatc aaaaccaacg 1200 gttggcaatc tctgcaggga tgatgaacca ggcttgtaat atctgttgct gatttctttg 1260 gaagacataa cggcaagctt catggggcac gatggatttc agatggttgc ttcagccatg 1320 tctcaagatt cagttgatgg acctcaagtt tctgggtgca gtgccacgag tcttggtcag 1380 cccaagagta agcgcaggac tggtgacaag gcaagagggg agaagaaggc actcaaagtt 1440 aagattaacc ttgccagccc ggccaaaaaa attaagaaaa gtagcaaaaa gaagggcaaa 1500 aagggcactg ttgctggcag gatagggaga aaatgcactc tctcaagaga ttctaaaggg 1560 cgctttcttc caagagagag taagggggga gacatcggag gaaatgctac agagagtgaa 1620 gttgattatg accgcttcat gaactttcag gcacctgact tcgctaccat cttaagtatt 1680 ttgaaaggct ggaaaggcat gaagcaatgt aacaagatca ggcgcctcaa ggatcctgac 1740 ttcgtccctc tcatgaacgt catgagcaac actggatatg tgaccgagga tgatggtcac 1800 tatgatgtgc tgaaagtctt gatgcatgca gatggctggt ctgcatagtg attcaagctc 1860 tcaaatcaaa acattcaggc ctatggcctt gttgctagaa cagtggtttc ttctttcacc 1920 tttaaaactt gatggacttt gttccattta tcttagaaat tttgttgccc ttgagtccgg 1980 tggatatgta ctggagtatg ctatactggg tgatttaatg gtgataatgt taaatcttga 2040 tactagttca aaaaaaaaaa aaaaaaaaaa aa 2072

   <210> 34 <211> 675 <212> RNA <213> Oryza sativa

   2018202034 22 Mar 2018

   <400> 34 aguccagcaa guuuugaaga ugauagaaaa uuugagcgcg ucguaguaac ugucguccac 60 gaucacgucc aguguuguuc auggcauggg ggauggaguc aggauccuug aggcgucugc 120 uccuguugca cugcuucaug ccuuuccugc cuucuaggau gcuuaagaug guugcgaagu 180 caggugcuug ggaguucaug aagcggucau aaucaauuuc gcucucugua guacuuucuc 240 uggugucuuc cccguugcuu ccuuuuggaa gaaaagcguc cuuuagaauc ucuugagaga 300 gugcacuuuc ucccucuccu gccaucagua gugccuuuau uuucgcuugg uuuccggcaa 360 aaagggcacu guugcuggca ggauagggag aaaaugcacu cucucaagag auucuaaagg 420 gcgcuuucuu ccaagagaga guaagggggg agacaucgga ggaaaugcua cagagaguga 480 aguugauuau gaccgcuuca ugaacuuuca ggcaccugac uucgcuacca ucuuaaguau 540 uuugaaaggc uggaaaggca ugaagcaaug uaacagauca ggcgccucaa ggauccugac 600 uucgucccuc ucaugaacgu caugagcaac acuggauaug ugaccgagga ugauggucac 660 uaugaugugc ugaaa 675 <210> 35 <211> 21 <212> RNA <213> Oryza sativa <400> 35 ucacguccag uguuguucau ^g 21

   2018202034 22 Mar 2018

   <210> 36 <211> 21 <212> RNA <213> Oryza sativa <400> 36 gcauggggga uggagucagg a 21 <210> 37 <211> 21 <212> RNA <213> Oryza sativa <400> 37 uccuugaggc gucugcuccu g 21 <210> 38 <211> 21 <212> RNA <213> Oryza sativa <400> 38 uugcacugcu ucaugccuuu c 21 <210> 39 <211> 21 <212> RNA <213> Oryza sativa <400> 39 cugccuucua ggaugcuuaa g 21 <210> 40 <211> 21 <212> RNA <213> Oryza sativa <400> 40 augguugcga agucaggugc u 21 <210> 41 <211> 21 <212> RNA <213> Oryza sativa <400> 41 ugggaguuca ugaagcgguc a 21

   2018202034 22 Mar 2018

   <210> 42 <211> 21 <212> RNA <213> Oryza sativa <400> 42 uaaucaauuu cgcucucugu a 21

   <210> 43 <211> 21 <212> RNA <213> Oryza sativa <400> 43 guacuuucuc uggugucuuc c 21

   <210> 44 <211> 21 <212> RNA <213> Oryza sativa <400> 44 ccguugcuuc cuuuuggaag a 21

   <210> 45 <211> 21 <212> RNA <213> Oryza sativa <400> 45 aaagcguccu uuagaaucuc u 21

   <210> 46 <211> 21 <212> RNA <213> Oryza sativa <400> 46 ugagagagug cacuuucucc c 21

   <210> 47 <211> 21 <212> RNA <213> Oryza sativa <400> 47 ucuccugcca ucaguagugc c 21

   2018202034 22 Mar 2018

   <210> 48 <211> 21 <212> RNA <213> Oryza sativa <400> 48 cacuguugcu ggcaggauag g 21

   <210> 49 <211> 21 <212> RNA <213> Oryza sativa <400> 49 gagaaaaugc acucucucaa g 21

   <210> 50 <211> 20 <212> RNA <213> Oryza sativa <400> 50 agauucuaaa gggcgcuuuc 20

   <210> 51 <211> 21 <212> RNA <213> Oryza sativa <400> 51 uuccaagaga gaguaagggg g 21

   <210> 52 <211> 21 <212> RNA <213> Oryza sativa <400> 52 gagacaucgg aggaaaugcu a 21

   <210> 53 <211> 21 <212> RNA <213> Oryza sativa <400> 53 cagagaguga aguugauuau g 21

   2018202034 22 Mar 2018

   <210> 54 <211> 21 <212> RNA <213> Oryza sativa <400> 54 accgcuucau gaacuuucag g 21 <210> 55 <211> 21 <212> RNA <213> Oryza sativa <400> 55 caccugacuu cgcuaccauc u 21 <210> 56 <211> 21 <212> RNA <213> Oryza sativa <400> 56 uaaguauuuu gaaaggcugg a 21 <210> 57 <211> 21 <212> RNA <213> Oryza sativa <400> 57 aaggcaugaa gcaauguaac a 21 <210> 58 <211> 20 <212> RNA <213> Oryza sativa <400> 58 gaucaggcgc cucaaggauc 20 <210> 59 <211> 27 <212> RNA <213> Oryza sativa <400> 59 cugacuucgu cccucucaug aacguca 27

   2018202034 22 Mar 2018

   <210> 60 <211> 23 <212> RNA <213> Oryza sativa <400> 60 ugagcaacac uggauaugug acc 23 <210> 61 <211> 21 <212> RNA <213> Zea mays <400> 61 uccuugaggc gucugcuccu g 21 <210> 62 <211> 21 <212> RNA <213> Zea mays <400> 62 uugcacugcu ucaugccuuu c 21 <210> 63 <211> 21 <212> RNA <213> Zea mays <400> 63 ugggaguuca ugaagcgguc a 21 <210> 64 <211> 21 <212> RNA <213> Zea mays <400> 64 ugagagagug cacuuucucc c 21 <210> 65 <211> 21 <212> RNA <213> Zea mays <400> 65 caccugacuu cgcuaccauc u 21

   2018202034 22 Mar 2018

   <210> 66 <211> 62 <212> RNA <213> Oryza sativa <400> 66 aguccagcaa guuuugaaga ugauagaaaa uuugagcgcg ucguaguaac ugucguccac 60 ga 62

   <210> 67 <211> 30 <212> RNA <213> Oryza sativa <400> 67 gaggaugaug gucacuauga ugugcugaaa 30

   <210> 68 <211> 31 <212> RNA <213> Oryza sativa <400> 68 uuuauuuucg cuugguuucc ggcaaaaagg g 31

   <210> 69 <211> 898 <212> DNA <213> Oryza sativa <400> 69

   aatcttattc tacatatttc tatcttatat agaacaacta gcatagctct cgttgcccag 60 ccaggttgcc cagccaggtt gcctggtgca caatgagagc tggctagggc ggactcattc 120 tgctgttggt gcccaacgat gctagctgct actcatacta gtgaagcctg ccatggttct 180 gagaaatttt tggatactcc gctgcgtaga tatgcactaa aagcttgtat gtttcgctga 240 ctacatacta tggatatcac ctgtttgaca agagaaggat tacataccac gatgaagatg 300 aattggaaca tgatgcaagt gatgtagcgc cccatatagg agtcactcag gaaagcacag 360 aagagggaga agatgtagac ggtgcccatc cacatgctga cgctattggc ggcctcggcg 420 ttctcctggt ggagcacctg cctgaggaac accaccaggc ccacggcgac gccaaaaaat 480 gcaaagttgg ccaacacata gctcactgca tcgtcaagta gagctgctta atcactgagg 540 tatatacatt tagttcgcct tcttcagcgt tgccatggac ctggtgatgt tcttccggcg 600 ggtgccccac caagagaacg ccgtggccac caacaacatc agcatatgga tgggcaccat 660

   2018202034 22 Mar 2018 cttcatcttt ttggatcatg gatatcttaa ctatatagat <210>

   <211>

   <212>

   <213>

   <220>

   <223>

   <400>

   ggtaccaatc gcccagccag tcattctgct ggttctgaga cgctgactac aagatgaatt ccacggtcat ttggtccttc aaaaatgcaa ctgaggtata cggttaggtt caccatcttc cttcacttgg ataggtgata atgcctctat <210>

   <211>

   <212>

   <213>

   <220>

   <223>

   <400>

   tggtacaacg

   gccctcttta gtgctttccg tagtgattcc tatacggggt gctacttcac 720 ttacaattta tcttcatcgt gatatatgct ccttctgttc tcacataggt 780 aatgtatgag gcatatatac tttctaccta atattataaa gtatatgcct 840 caaataaagc agaaaagtca ttgttattac aaaaaaaaaa aaaaaaaa 898 70 892 DNA artificial sequence

   synthetic construct

   ttattctaca tatttctatc ttatatagaa caactagcat agctctcgtt 60 gttgcccagc caggttgcct ggtgcacaat gagagctggc tagggcggac 120 gttggtgccc aacgatgcta gctgctactc atactagtga agcctgccat 180 aatttttgga tactccgctg cgtagatatg cactaaaagc ttgtatgttt 240 atactatgga tatcacctgt ttgacaagag aaggattaca taccacgatg 300 ggaacatgat gcaagtgatg tagcgcccca tataggagtc actcaggact 360 tgtgtatcat gtagacggtg cccatccaca tgctgacgct attggcggcc 420 atagagaccc aacctaacag tgaacgtact gtcgccccac ggcgacgcca 480 agttggccaa cacatagctc actgcatcgt caagtagagc tgcttaatca 540 tacatttagt tcgccttctt cagcgttgcc atggagcgac agaacgttca 600 gtgtctcttt gaaggaccat ggccaccaac aacatcagca tatggatggg 660 atgatgaaca atgacggtgg agtccgtagt gattcctata cggggtgcta 720 atcatgttac aatttatctt catcgtgata tatgctcctt ctgttctcac 780 tcttaaaatg tatgaggcat atatactttc tacctaatat tataaagtat 840 atagatcaaa taaagcagaa aagtcattgt tattacgtta ac 892

   DNA artificial sequence synthetic construct tcattgatga c 21

   2018202034 22 Mar 2018

   <210> 72 <211> 21 <212> DNA <213> artificial sequence <220> <223> synthetic construct <400> 72 tggaccttac atggcccttc a

   2018202034 22 Mar 2018

   <210> 73 <211> 21 <212> DNA <213> artificial sequence <220> <223> synthetic construct <400> 73 taattgtgca gctcatcacc c

   <210> 74 <211> 21 <212> DNA <213> artificial sequence <220> <223> synthetic construct <400> 74 tagatgggaa atatagatat c

   <210> 75 <211> 21 <212> DNA <213> artificial sequence <220> <223> synthetic construct <400> 75 tgcttatatg tatgttctgt a

   <210> 76 <211> 21 <212> DNA <213> artificial sequence <220> <223> synthetic construct <400> 76 tcaagagtct ttgaaagaaa g

   <210> <211> <212> <213> 77 880 DNA artificial sequence <220> <223> synthetic construct <400> 77

   2018202034 22 Mar 2018

   aatcttattc tacatatttc tatcttatat agaacaacta gcatagctct cgttgcccag 60 ccaggttgcc cagccaggtt gcctggtgca caatgagagc tggctagggc ggactcattc 120 tgctgttggt gcccaacgat gctagctgct actcatacta gtgaagcctg ccatggttct 180 gagaaatttt tggatactcc gctgcgtaga tatgcactaa aagcttgtat gtttcgctga 240 ctacatacta tggatatcac ctgtttgaca agagaaggat tacataccac gatgaagatg 300 aattggaaca tgtggacctt acatggccct tcaatatagg agtcactcag gagtcatccg 360 tgacgttata ccagacatcc atatttccca tccactttct cttagagacc cttgtgggtg 420 atgagttgca cagttacctg cttatatgta tgttctgtac ccacggcgac gccaaaaaat 480 gcaaagttgg ccaacacata gctcactgca tcgtcaagta gagctgctta atcactgagg 540 tatatacatt tagttcgcct tcttcagcgt tgccatggat acagaaaata catatcagcg 600 ggtaattgtg cagctcatca ccctcaagag tctttgaaag aaagtagatg ggaaatatag 660 atatctggta caacgtcatt gatgactccg tagtgattcc tatactgaag ggccacgtaa 720 ggtgcacatg ttacaattta tcttcatcgt gatatatgct ccttctgttc tcacataggt 780 gatatcttaa aatgtatgag gcatatatac tttctaccta atattataaa gtatatgcct 840 ctatatagat caaataaagc agaaaagtca ttgttattac 880 <210> 78 <211> 21 <212> RNA <213> Oryza sativa <400> 78 gugagaaagu gcccucucuc a 21 <210> 79 <211> 2040 <212> DNA <213> Oryza sativa <400> 79 ccacgcgtcc gccaaaagtg aactgtgaac cggacgatcc aggcatccag ctaaccgctt 60 ccccctcgtc gcgctcgcgc cgcgccgcgc ctcgcctcca ccagctacgc cgtcacgcga 120 gctcacggcc cgggggcctc ggaagcacaa ccaccacgcg tccacgccga agccacgagg 180 agagcctgtc tctcctccgg ggattctatc gccggctggt ctcagcggcg ccacttggag 240 caggcggcac ccgctccgta ctgctgctga tttggtgagc gcgggcagcc gcgggatggg 300 agatctggtg gcgtgatggt gagcccgggc agtaaccggg gcggcctgtc gcgcgtatcg 360

   2018202034 22 Mar 2018

   acgcggggcg gcgtcgccgg gccggggagc ccgcgcgcct ctcctgccgc gaccgctttc 420 gcggcgctac ggcgcaggtg gcggtgggcg cccccgggct cgtcgacgct ggagcgcgcg 480 gcccgcgcgt tcctgctggc ctccgcagcg ctcgtgctct cctgcgcgct ctacctctac 540 gtgctgcgct acgtcggccg gggaggtcgc gccttcgccg ccgcgggctt cgtcggggac 600 gccgtcctgg gcctcggcgg cgagccgtgc gacgtgttcg acggcgcctg ggtgcccgac 660 gacaccggcc tccgcccgct ctacaatagc tccgggtgcc cgttcgctga gcgcgggttt 720 gactgcctcg ccaacgggcg gaacgacact gggtacctca agtggcggtg gaagccgcgc 780 cggtgcggcg tgccgcggtt tgcggcccgc accgcgctgg agcggctgcg cgggaagcgg 840 gtggtgttcg tgggggattc catgagccgc tcgcagtggg agtccttcat atgcatgctc 900 atggccggcg tggatgaccc caggacggtc ttcgaggtga acgggaacga gatcaccaag 960 acgatacgcc acctggcggt caggttcgcg tctcacggcc tcaccgtgga attcttccgg 1020 tccgtgttcc tcgtgcagga gcatcctgcc ccgcggcatg cccccaagag ggtcaaatcc 1080 actttgaggc ttgacaggat ggataatttc agccggaaat gggtcaattc ggacgtactg 1140 attttcaaca ctgggcattg gtggacaccg accaaattgt ttgatacggg ttgctatttt 1200 caggctggac gttctcttaa attaggtaca tccattgatg ctggtttcag gatggcactg 1260 gagacctggg cctcatgggt acaaaaaaga gttgatttaa accgaacaca tgtattcttt 1320 cgcacatatg agccatcgca ttggggggat acaagccaaa aggtgtgtga ggtaacagag 1380 cagccttcat cagaggccaa aggaaatgat aagagtgaat ttggggctat acttgctgat 1440 gttgtaacca acatgaaagt tcctatcaca gtactaaatg taactttaat gggatcgttt 1500 cgaagtgatg cacatgttgg cacttggagt tatcctccca ctatacttga ttgcagccac 1560 tggtgtcttc ctggagtccc tgatgcttgg aacgaactcg tgttttcgta ccttttgaca 1620 aatggttggc gaaacatggc gggctgaatt ttttggcagc acaatctacc cgcacctatt 1680 gcattgtgat atttgacatt accaggtata ctagagaatt aacatacgtc cgggacaagg 1740 cagctgcagc tgctaggtgg ttcactggac ttctacattc ttttcttctt tttgattttt 1800 gactcgtata cacggtgaag catgctacat gcaactagag ttgtatgtag ttggtaaggg 1860 attagaaggc cttggcattc gttctatttg ctcaatttac taacggttca ttttattatg 1920 ttgttaaaaa tcgagtttgt attgtaacct gtatgtacaa acatttactt gatacattgt 1980 gagaaagtgc cctctctcaa ttggattgat aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2040 <210> 80 <211> 21 <212> RNA <213> Oryza sativa

   2018202034 22 Mar 2018

   <400> 80 gggcgaaggu gaacucucuc a <210> 81 <211> 2523 <212> DNA <213> Oryza sativa <400> 81 cggacgcgtg gggcgtgcgt ggtctccttc tgcttacgtg cccagtcctg ttcgtccctt caaagtttgt cctgaactgt tcatcatggt ggtgtttcta gcgttcacaa caccttgcga cacatatcag caaggtgaag taataagggc agaactaccc ttcagttact acagccttcc gagtgctgaa aacttaggcg agcttctgat tttccgtgta aatgtcaacg aatctctgta taatgtgaag ctcctcaagc agcgtagcca caatcttcct gtgaggaggt tcacagagca tccagttggt tatattccag aaggcacttc taaggtcttg gtccataagt atgaaggagg aatggaagtg atctcagaga ctgacaaaga tgaagttgtc ccatgcagcg tgaagcgtga tgataaagtc gatcctgtga actgccctgt gaaagagaag attactttta cttatgaggt atcacggtgg gatgcatacc tgaagatgga gaactctttg atggtaattc tatttttggc agtgaggagg gacttgactc ggtatgagga tgaggagctc tctggttgga agcttgttgt gaagctgctc tgtgtcatga ttggcgatgg cattttcttt gccgcatttg gcttcatgtc gatgatagtc ttttatatgt tacttggaat gaggacttta aaaggaacgt ccgagggatg

   ctctcgtggc gacgaccgag cggccgtcgc 60 cgccggcgac ggccacgacc tggttatagg 120 ggaaggctgg gtattctctg ctttgttagt 180 gtcattctac ttgccaggta gttatatgca 240 gaaggtgaac tctctcactt ccattgagac 300 atactgtcgt cccagagatg gggttaagaa 360 gggtgatcaa atagataatt ctccgtaccg 420 tctgtgtact acaaccccac ttgacgaggc 480 tgatctatac caggtgaaca tgattcttga 540 gaatggaata accatccagt ggacaggcta 600 tgatgtctac atcatcaatc acctgaaatt 660 cgaagtaaag gtagttggga ctggggaagg 720 tgccaattct ggatatgaga ttgtgggatt 780 tcctgaatcc atattgaagc ttaatatgta 840 ggagttggaa aaatctcaat tggttaggga 900 tgaatttgta aacagtgata tcaggtggcc 960 gggttcgaag attcactggt tttcaattat 1020 tggcattgta tttgtcatat tcttgcgtac 1080 gttggataag gaggcccaag ctcagatgaa 1140 tggagatgtc ttcagagaac caacctcacc 1200 ggttcagatt ttgggtatgg caattgttac 1260 tcctgcatcg agaggaatgt tgttgacagg 1320 tgtgtctggg tatgctgctg tcaggctctg 1380 gaggtctgtc tcctggtcaa ctgcttgttt 1440

   2018202034 22 Mar 2018

   cttccctggc attgtcttca ttgtcctcac tgtgttaaac ttcatgctgt ggacaagaaa 1500 tagtactgga gcccttccca tctcactttt ctttggcctt ttgtccttgt ggttctgtgt 1560 ctccgtgcca cttacccttt taggtggttt ctttggcaca agggctgagc caatagaatt 1620 ccctgttcga accaatcaga taccaagaga aatccctacg aagaagtact cattgctctt 1680 catacttggt gctggaactc taccttttgg aacactcttc atcgagctct tcttcattct 1740 ttctagtatt tggcttggaa ggttctatta cgtgtttggc ttcctccttg tcgtgcttct 1800 tttgctgatt gtggtgtgtg ctgaggtatc agttgttctt acctacatgc atctctgcgc 1860 ggaggactgg aggtggtggt ggaaagcttt ctttgcttct ggaacagtgg ccctttatgt 1920 gttcctttac tctatcaact acttggtgtt tgatctcaga agcttgagtg ggccagtttc 1980 tgctattctc tacattggat actctttcgt tgtctccctt gccattatgc tagcgactgg 2040 taccgttggc ttcctgacgt cgttctcttt tgtccactac cttttctcat cagtcaagat 2100 tgattgaaga tccagggttg tctttacaca aaatcacctg tgagctcaaa tgatatgacc 2160 attgcatctt gaaggccttt cacagagcag tgctgtttgt aatgtagctt attaccgaga 2220 gtctgagact gctgtacctt gtaatgaata gtatatttca gcagatgtgt tttgaagttt 2280 gtcacacttt gctacagcat tttgttgacc tgccaatact gtaggaaaag tcttgcgttt 2340 attatcccat ggtgccattt tgttgtctgt ttctttctgc aagattggct tgcagctgga 2400 gaactatacg ttcttatggt ataatctaca tgtgcaaaat gtttcccatc caaaaaaaaa 2460 aaaaaaatca gttcagaagt caccttcttt cgtgaatgtt ttgattccct gaggctactt 2520 tat 2523

   <210> 82 <211> 21 <212> RNA <213> Oryza sativa <400> 82 gccagaaggu gcacugucuc a 21

   <210> 83 <211> 530 <212> DNA <213> Oryza sativa <400> 83 gcagatgata gacacgacta aagattacat tcaagcgctg agcattgtgc cgacaagaga 60 gttggccttg cagacgtctc agattttcat cgaagtttca aagcacttga aagcccgcgt 120

   gatggtgacc accggaggca cgaatttgaa ggacgacata atgcgtatat acgaaaacgt 180

   2018202034 22 Mar 2018

   tcacgttatc attgcgactc ccggtcgcat actcgatctg atggagaaga aggttgccaa 240 gatgaacaac tgtcaaatgc ttgttctcga cgaagccgac aaacttctgt ctcgggattt 300 ccaggggctc ctcgatcgag tcatctcgtt cttgccgcaa gaaagacaaa tccttctcta 360 ttcagctacg ttcccgatga ccgttgaaga attcatgcgc cgtcacctca agaacgccta 420 cgagatcaac cttatggagg agctcacact caagggagtg acacagtact acgctttcgt 480 acaggaacgc cagaaggtgc actgtctcaa cacgcttttc tcgaaacttc 530 <210> 84 <211> 20 <212> RNA <213> Oryza sativa <400> 84 ggaaggccua agcagugcaa 20 <210> 85 <211> 2229 <212> DNA <213> Oryza sativa <400> 85 atggcttcaa gggcggtcat cagaagaagg aagtatcttt tggatcatgt taacgcacct 60 accctctcat tgtccccctt ctctaccttc caacatggaa gatctggttc tgaggatgaa 120 tcaagaatcg gacagcgatt tcttgagcaa agctctgggg attccaaatg ggagcaaggg 180 cagtatggtg tgaaattgat aaagggagat ctactagccc ttggtaatgg gcttctgcgg 240 cgcccagccc atgggatttc tctacctgct tatggaattg gaaggaagga atttgggttg 300 cctatgggtg ctagacattt gctgcagtca gtccgcacag cctcaactgc aacagctggg 360 caacctaagt tggatattga agatgaacag agtgaggatc agaaacagaa caaaaggaaa 420 aaggaggcat ccccagaaga atgtgatcag gctgtggaag gcctaagcag tgcaaaagct 480 aaagccaaag ctaagcaggt acaagaatct gtaaaggctg gccaatcaat tgtacgaaaa 540 ttctgggcga ggcttctggg tattggtcct gctctccgag ctgttgcttc gatgagcaga 600 gctgattggg ctgcaaagct gaagcactgg aaggatgaat ttgtgtcaac gctgcagcat 660 tactggttag ggacaaagct actctgggca gatgtgagga tttcgtcaag attactggtg 720 aaacttgctg gtggaaagaa cctttcaaga agagagagac aacaactgac ccgtacaaca 780 gcagatatct tcaggctggt accttttgct gtgttcatca ttgttccatt catggagttc 840 ttacttccag tgttcctcaa gttatttcca aatatgcttc cctcaacttt ccaagacaag 900

   2018202034 22 Mar 2018

   atgaaagaag aggaagcgtt gaaaaggaaa ctgaaagcaa gaatggagta tgccaagttt 960 ttgcaagata ctgcaaaaga aatggcaaag gaagttcaaa catcacgtag tggagaaata 1020 aaacaaacag ccgaagatct tgatgaattt ttgaacaagg ttaggagagg tgaacatgtc 1080 tcaaatgatg aaatcttgaa cttcgcgaag ctgtttaatg atgagctgac tttggataac 1140 atgagcagac cacgcttggt aaacatgtgc aaatatatgg gtattcgacc tttcggtact 1200 gaccactact tgaggttcat gcttcgcaaa aaactgcaag acattaagaa tgacgataag 1260 atgattcaag ctgagggtgt tgagtctctc tctgaagagg aacttcggca agcctgtcgt 1320 gaacgtggtc acctaggttt gctgtcaaca gaagaaatgc gccaacagct ccgagattgg 1380 ttggatctct cacttaatca tgctgtgcca tcctctcttc tcatactttc aagagctttt 1440 accgtatctg ggaaaatgaa gcctgaggag gctgttgtag caaccttatc ttctctacca 1500 gatgaagttg tggatacagt tgggaccgta ttgccatctg aagattcggt ttctgagagg 1560 aggagaaaac tggaattcct tgagatgcag gaagaactta tcaaggagga agagaagaag 1620 aaagagaaag aagaaaaagc gaaacaagag aaagaagaaa aggccaaact caaagaacca 1680 aaggctgctg aagaagattt ggctttgaag gaaatgactg gtcctactgc tagggaagaa 1740 gaagaactga gagaagcaaa acagcacgat aaggaaaagc tctgtaattt tagtcgagca 1800 ctggctgtac tggcatccgc atcgtctgtt agcaaggagc gtcaagagtt cctcagcctt 1860 gtcaacaaag agattgaact gtataactct atgcttgaaa aggagggtac agaaggtgaa 1920 gaggaagcta agaaagctta catggctgct agagaagagt cggacaaggc tgctgaggtt 1980 gatgaagaag aaaaggtctc atcggcgctg attgagaagg ttgatgctat gctccagaaa 2040 ttagaaaagg agattgatga cgtggatgca caaattggaa accgatggca aattcttgat 2100 agggatcttg atggcaaggt gactcctgag gaggtagcgt cagcagcagc ttatctgaag 2160 gatacaatag gaaaggaagg cgtccaagag cttgtcagca acctctctaa agacaaaggt 2220 cctccctga 2229

   2018202034 22 Mar 2018

   <210> 86 <211> 22 <212> RNA <213> Oryza sativa <400> 86 gaaauucaug gaagcagugc ag 22 <210> 87 <211> 1521 <212> DNA <213> Oryza sativa <400> 87 atggccgcct cctcttcctt cctcgccgcc ggccgccgcc tgatccgcct cggctgcggc 60 aggctcctcc ccgccggcca cgcgcgatcc catggctcca cccctgccct cattcgagcc 120 gccgccgccg cctcctcccc cgcctctcct cgcggccaca gcggggggag gaagccggcg 180 cggcccccga gcctgcagtc cacgctgtgg ccgctgggcc acccgggcac gctcctggtg 240 ccggagatcg agcggtgggc ggccaagcca ggcaaccgcc tccgccacgt cgagctcgag 300 cgcatcgtca aggagctccg caagcgacgc cgccaccgcc aggccctcga ggtctctgaa 360 tggatgaatg ccaagggaca tgtaaaattt ctgccaaagg atcacgctgt tcacctggat 420 ttgattggtg aaattcatgg aagcagtgca gccgagactt acttcaacaa cctgccagat 480 aaagataaga cagaaaaacc ctatggtgca cttcttaact gctacacacg ggaactcctg 540 gttgaaaaat cgttggctca ttttcagaag atgaaagagt tgggttttgt gttttccaca 600 ctcccctaca acaacatcat gggtctgtat acgaacctag ggcagcatga aaaggttcct 660 tcagtaattg cagagatgaa aagcaatggt atcgttcctg acaatttcag ctacagaata 720 tgcattaact cttatggcac aagggctgat tttttcggga tggaaaacac ccttgaagag 780 atggagtgtg aacctaaaat cgttgttgat tggaacacgt atgctgtcgt ggcaagcaac 840 tacattaagg gcaacataag ggagaaagca ttctctgcct taaagaaagc agaagcaaaa 900 ataaatataa aagattcaga ttcctataac cacctgattt ccttgtatgg acatctgggg 960 gacaaatcag aggtcaatag gctgtgggcg ctccaaatgt cgaactgcaa taggcatatt 1020 aataaggatt acactacaat gcttgcagtg ctcgtgaaac ttaatgagat tgaagaagct 1080 gaagtgttgc tgaaagagtg ggagtcgagc ggaaatgcat ttgacttcca agttccaaat 1140 gtcctgctca ctggataccg ccagaaggac ttgctggaca aggctgaggc acttctggat 1200 gatttcttga agaagggaaa gatgcctcct tcaaccagct gggcaattgt ggcagctggc 1260 tatgcggaga aaggtgatgc tgcgaaagca tatgagctga caaagaatgc cctatgtgta 1320

   2018202034 22 Mar 2018

   tatgctccaa atactggttg gatccctagg cctgggatga ttgagatgat acttaagtat 1380 cttggagatg aaggtgatgt cgaggaggtt gaaattttcg ttgatctgct gaaagttgct 1440 gtgccactga actcagatat gactgacgct ttgtcaaggg ctcgaatgag agaagaaaag 1500 aaggttaaag atgcagtgta a 1521 <210> 88 <211> 20 <212> RNA <213> Oryza sativa <400> 88 ggaaggccug aagcaugcaa 20 <210> 89 <211> 1980 <212> DNA <213> Oryza sativa <400> 89 cgtgcctttt ccgtcgtccc attcgccagg ggggaacggc aaagggcatc gtcgcaaaca 60 atcaagttcc acaaactcgc atctcatctg cttcgactcc accgaggact cccttctttc 120 ctcccactcc catctgctct cctcgccgcc acctgcgccg tcagagcacg gagcagtccg 180 cgaccacgct ttcgctgccg ctctccgaca ggcgacggag ccgcagctcc agtcgcagtc 240 ggctcccctg aattcgggct cgccaaatac cctccaatcg tctgcgtccg tcgtccggga 300 cttccggtgc aactgaatcc ggcaccacct gtgcggcctg tcatggcact tggaagagga 360 gggaggaagg actcaagagg ttaaggtacg gtatttatag atttggctga agaaactggt 420 tgttgctatt atgaagacta agacgagtag gagcttacag aagtctggga gaggtaacca 480 tgtccaagga gaagggccaa actgggttct tgttgctggc ggggttttgc ttagcacgct 540 ttcagtcaag gttgtgtgca aactaaagca gttgttagac gggaagcagc aaaataatac 600 tttcgaagct aaaggaaggc ctgaagcatg caagctgcat tcagatctct accggctcag 660 tgaccaaact ggctgctact actgtatgtc agggcttgca aatggtggag tggaagtcaa 720 gcaagcacca gcaagtcctg tacccaaatc agttgaatcc tcacttccac ttgtcaagat 780 acccacacca gaatcaagca aagagaacag cggtgttatg tggatatcct cacctgatcg 840 gctgaaagat cctcgaaggc catttcagta ctctaacagt tctggctctc cctgtgtttc 900 agaatcagga tctgacattt atggcaaaag agaggtcata cagaagctaa gacagcacct 960 caagaaacgt gatgagatga tcatggagat gcaaactcag attgctgatc ttaagaactc 1020

   tcttaacatt caggtgacac agtccagcaa tctgcagtct caattggatg ctgccaatcg 1080

   2018202034 22 Mar 2018

   tgatctgttt gaatctgaac gagagattca gcatctaagg aagattattg cagatcattg 1140 tgtcacagaa gcactctctc atgataaacc tttgcaagct gcgcattggc agccagatgc 1200 cgcaaatggg cattctaatg gctatggtga tggttgtgtt gatgatgctg acctgcattg 1260 tattagcatc gagaagagga aggtagaagt agagagggtg gagatgctca agaaagaggt 1320 ggttgaactg aaggaagtca ttgagggaaa ggactttgtg cttcagagct acaaggaaca 1380 gaaggtggaa ctcttctcaa agatcaggga gttgcaggaa aagctctcag cacaagtgcc 1440 aatcatcttg taggatctat ctgtgatact ttttagaaga ttgaatctaa gcataatgtt 1500 gccatgtccc atgagcagca gagggggtcc cgcttcagtg aagattgcag aaggtcttgg 1560 catttggcaa tcgtcacgca tgccaaacac catgctagga tctttgtgga atgcttctct 1620 tttcctttga ggggagcttt gcataatgtt aggttgattt gtttctttct tggttgtcat 1680 aatgttaggt tggtttgctt tcttccttct tcaatatcta gcccttggtt gctcaaagtt 1740 tacaaaggga ttcttttttt cagttgctag gcctcaggta actcaattga acttcatact 1800 caagttgctg tacaggttct cagatttcag gagacacaga agtctgtact gtgcctctgc 1860 ctcctgtttc atgctttttt tttgttaagt gatctttgga atgttaggtc catgacattt 1920 ctactatgag atttgaagac tatggcattg cccttttttg tgaaaaaaaa aaaaaaaaaa 1980 <210> 90 <211> 21 <212> RNA <213> Oryza sativa <400> 90 uggcugcuac auggacuccc ^g 21 <210> 91 <211> 1125 <212> DNA <213> Oryza sativa <400> 91 atggccggag acacgacgaa gcccacggag tcagccatcg tcgggagcac ggtgaccggg 60 caccacctgc tccacatcga cggctactcc cacaccaagg accgcctccc caatggctgc 120 tacatggact cccgcccttt caccgtggga ggccatctat ggcgcatcgg atactacccc 180 aacggcgacg tcgccgacgc ctccgcgtac atggccgtct acccttccat cgacgagaac 240 gtcatcgtcg ccgtcaaggc ttttgccaag ttcagcttgt tcttcaacgg cgagcccacg 300 ccgccggcgt ttgtgcatac cacagagcca ttcgtgttca gcaggaaggg gatcgggtat 360

   2018202034 22 Mar 2018

   ggttttagca agtatgccga gagggagttg atggagggct cgatcgtgga cgacaagttc 420 accatcaggt gcgacgtcgg cgtctccacg gagctccgcg cggaggacag gccgccgtcg 480 gacttcgcgg cggtggtgcc gccgtccgac ctgcaccggc acctcggcga ccttctggac 540 tccaagcacg gcgccgacgt cacgttccag gtcggcggcg aggcgttccg cgcgcaccgg 600 tacgtcctcg cggcgcggtc gccggtcttc agggcggagc tgttcggcgc catgagggag 660 gccaccgccg cggccgccgc gtcgtcgtcg gactcggagg cgatccgcgt ggacgacatg 720 gaggcgccgg tgttctccgc tctgctccgc ttcgtgtaca ccgacgcgtt gccggcgccc 780 ggcggagcgg acgacggaca agcggcagga ggaggatcgt attcggagga ggccgccatg 840 gctcagcacc tgctcgtcgc ggcggacagg tacgacctga agaggctgaa gctgctctac 900 gaagacaagc tacgcaggca catcgaagcc gcctccgccg cctccatgct cgcgttggtt 960 gagcagcacc attgccgagg cctcaaggag gcgtgcttgg tgttcctcag ctcgccggcc 1020 aaccttcacg ccgccatggg aagcgatgga tttgagcatt tatccaggag ctgccccggt 1080 gtgatcaagg agctaatatc caaacttgtt ccacgttgtg attag 1125 <210> 92 <211> 21 <212> RNA <213> Oryza sativa <400> 92 aggccuuuuc augaacuccc a 21 <210> 93 <211> 578 <212> DNA <213> Oryza sativa <400> 93 tggcgcggac tcatggggaa cggtgacgcg cctcctgtct tgttttcctt tcatggcgcc 60 gaccctcgcc gacgggtgat cgacgtcgcc cctattcatc tgcttgatgg tcccgcccca 120 tacgccgatg ctcacgcacc tcctccttct atctccttac agacgcgggg acgctcgccc 180 acggcggcgg cgggagctga gtcttctgtc agcacgctcc tcgaggtgcg cggactcacc 240 gaatacgtga aggagactgg gcagctgaat cctagccggc gatcacctta ccatccgcga 300 gggcgagatt catgctatta tggggaagaa cggctgcggc aagagcaacc ctcacaaaag 360 gtctcactgg gcagtctcat tatgaggtga cgggtggcac cattctcgtg gagggggggg 420 acctggttga catggagcca tatgacagac ctctagcagg ccttttcatg aactcccaag 480

   cacctatgtg agattcctga atcaacaatt tcgattttgt gctatggctg cgaatgctcg 540

   2018202034 22 Mar 2018

   ctaagaaagg aatggtctac cagcattggg ggcccttg 578 <210> 94 <211> 21 <212> RNA <213> Oryza sativa <400> 94 ggcgcucgcc uucgcaacca u 21 <210> 95 <211> 1188 <212> DNA <213> Oryza sativa <400> 95 atgaggcaca agcagaagac ccctccctgc acagtgcaca cgatactagc tagcccgatg 60 gccccaacct ccagcgttca ccgcgaaggc ggcagcgccg ccatggacat ggccgagctc 120 atccccacgc tgccgctgga gacggggagc ccgccgttcc cgctccggca atacggcggc 180 tactggctgc cggagtgggt cctccctggg ctcgaggccg tgcacacgcg cttcgagccg 240 aggccatccg acgtcttcct cgccagcttc cccaagtccg gcaccacctg gctcaaggcg 300 ctcgccttcg caaccatcaa ccggaccacc tacccgccgt ccggcgacgc ccacccgctc 360 cgccatcgcg gcccgcacga ctgcgtcaag ttcttcgagt ccaccttcgc catctccggc 420 gagggcggcg gcggagacgt ggacgtgttc gccgccctcc cgtcgccgcg cgcggttgct 480 cgcccgggga cgtgttcgcc gccctcccgt cgccgcgcgc ggttgctcgc cactcacatc 540 ccctactccc tcctgccgga gcgcatcacg tcggcggcgg cggacgacgg cgactccggt 600 tgccggatcg tgtacgtctg ccgggacccc aaggacgcgt tcgtctccat gtggctgttc 660 accatgagca acatggtgaa gggtgtcaca acgaccacgg acgaacacca cccggcggcg 720 gcggcggcgg cgccatcgat cgagcaggtg ttcgacctgt tctgcgacgg gcggagcatc 780 gctgggccgc agtggcacca cgtccgcgag tactgggagg agagccggag gcggccggag 840 aaggtcctct tcctccggta cgaggagatg ctgcgcgagc cggcgcgcaa cgtggagagg 900 ctcgccgagt tcctgcggtg cccgttcacc gccggcgagg tggcggccgg ggtggtggac 960 gccatcgtcg acctatgcag catcgaccga ctcaggaacg tgcaggcgaa caagaccggg 1020 gtgaccgacc tggcggtgaa gaaggagagc ttcttccgga gaggggtggc cggcgactgg 1080 agcaaccaca tgtcgccgga gatggcgtcg cggctggaca gggtcgtcga ggacgcgctg 1140 cgaggctccg ggttcacctt tgccgccgct gccggcgact ccgaatga 1188

   2018202034 22 Mar 2018 <210>

   <211>

   <212>

   <213>

   <400>

   ggcccaugac <210>

   <211>

   <212>

   <213>

   <400>

   atggccgcgg gcggcggcgg ctcaccatcg gtggccacca gcggagatcg tacgacggac atcatcacgg ctcttcgccg cacctgtaca ccgctcggat gacctctacg gcgctctgga gcggaatgga ttgaaggtcg catgctcttg tctatgtcag gagcgcgatc caagaactca gcaaaggaaa tgcggtttgg

   RNA

   Oryza sativa uuugcaacca c 21

   1197

   DNA

   Oryza sativa

   ccgtcgtcct cctccgccgc ctgcgcggcg tcacggcggc gccccggcgc 60 cgctgcccct gaccacgagc gtccggggtg tctccgattc gacggagccg 120 agacctcggt cccctacaag tcccacatcg tggacccgcc cccgcgcgag 180 cggcgcgcga gctcgccacc ttcttccgcg acatgtccgc catgcgccgc 240 cggcggactc gctgtacaag gcgaagctga tccgcggctt ctgccacctc 300 aggaggccgt cgcggtgggc atggaggcgg ccaccacccg cgccgacgcc 360 cctaccgcga ccactgcgcc tacctcgccc gcggcggcga cctcgccgcc 420 agctcatggg ccgccgcggc gggtgctcca gggggaaagg cgggtcgatg 480 agaaggacgc caacttctat ggcggccatg gcatcgtggg cgcgcaggtg 540 gcggcctcgc gttcgcgcag aggtacagga aggaggccgc cgtcacgttc 600 gcgacggcgc cgccaaccag gggcagctgt tcgaggcgct caacatggcg 660 agctgcccgt cgtgctcgtc tgcgagaaca accactatgg gatggggacg 720 gggcatcgaa aagccccgca tactacaaac gcggcgacta tgtgccagga 780 atggtatgga tgttcttgca gtcaaacaag cttgtaaatt tgccaagcaa 840 aaaatggacc gattattctt gagatggaca cctacagata ccacggacac 900 atccagggag cacttaccgc accagagatg aaattgcagg cataagacag 960 caattgaaag ggttaggaag ctactactgg cccatgactt tgcaaccaca 1020 aggacatgga gaaagaaata aggaagcaag tcgacactgc catcgcgaaa 1080 gtccaatgcc cgatccatct gagctcttta caaatgtata tgttaatgac 1140 agtcatttgg tgtggacagg aaggtggtga gaactgtact tccctag 1197