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Invented by Edwards Allen, William P. Donovan, Gregory R. Heck, James K. Roberts, Virginia Ursin, Yuanji Zhang, Monsanto Technology LLC

The Monsanto Technology LLC invention works as follows

This invention provides plants with resistance to invertebrate parasites. This invention discloses, more specifically, a non-naturally transgenic plant cell that expresses at least one invertebrate microRNA in planta to suppress a target gene for an invertebrate insect pest or symbionts associated with the pest. This invention also provides recombinant constructs to express at least one invertebrate microRNA in planta. Also included are non-natural plants containing non-natural plant cells, non-natural plants grown from nonnatural plant cells, nonnatural transgenic seeds produced by nonnatural plant cells, and commodity products made from nonnatural plant cells, nonnatural plant seed, or nonnatural plant. This invention also provides a method for suppressing atleast one target gene from an invertebrate that is a pest to a plant, or a symbiont of the invertebrate. The method includes providing a non-natural plant cell of the invention, in which the invertebrate in question is the pest invertebrate.

Background for Invertebrate microRNAs

MicroRNAs are non-protein-coding RNAs that have a range of nucleotides between 19 and 25 (commonly 20-24 in plants). They guide the cleavage of target transcripts in trans, negatively regulating gene expression in pathways involved in regulation and developmental pathways (Bartel, 2004 Cell, 116, 281-297). In some cases miRNAs are used to guide the in-phase processing siRNA primary transcripts. (2005) Cell, 121:207-221).

Many microRNA genes (MIR genes) have been identified and made publicly available in a database (?miRBase?, available on line at microrna.sanger.ac.uk/sequences; also see Griffiths-Jones et al. (2003) Nucleic Acids Res., 31:439-441). In 2001, Lee and Ambros published Science 294:862-864. Since then, microRNAs have been found in other invertebrates. See, for instance, Lim et. al. (2003) Genes Dev., 17:991-1008; Stark et al. Genome Res. 17:1865-1879 (2007) MIR genes are reported to be found in intergenic region, isolated or in clusters, in the genome. They can also occur entirely or partially in introns of genes (both protein-coding genes and non-protein coding genes). Kim (2005) Nature Rev. provides a recent overview of miRNA biogenesis. Mol. Cell Biol. 6:376-385. In some cases, transcription of MIR genes may be controlled by the promoter of a MIR. The primary transcript (called a “pri-miRNA”) can be large (several thousand kilobases), polycistronic and contain one or more premiRNAs, which are fold-back structures with a stem-loop structure that is processed into the mature miRNA. ?cap? See, for example, FIG. See, for instance, FIG. 1 in Kim (2005) Nature Rev. Mol. Cell Biol. 6:376-385.

The maturation of a miRNA (from its pre-miRNAs or pri-miRNAs), differs between plants and animals. In plant cells, it’s believed that microRNA precursor molecules undergo a major transformation into mature miRNA in the nucleus. However, in animal cells the primiRNA transcript, which is produced by Drosha (an animal-specific enzyme), is first processed in the cell nucleus before being exported to the cytoplasm, where the mature miRNA is then further processed. In plants, mature miRNAs have a length of 21 nucleotides. In animals, the miRNAs tend to be 22 nucleotides long. Kim (2005) Nature Rev. provides a recent overview of miRNA biogenesis both in plants and animals. Mol. Cell Biol. 6:376-385. Bartel (2004) Cell 116:281-297, Murchison and Hannon (2003) Curr. Opin. Cell Biol. 16:223-229, and Dugas & Bartel (2004) Curr. Opin. Plant Biol., 7:512-520. In addition, although a recent report described a miRNA from Arabidopsis (miR854) that is also found in animals (Arteaga Vazquez and al. Plant Cell 18:3355 – 3369 (2006), miRNA conservation appears to be kingdom specific. Plant miRNAs are different in many ways from their animal counterparts. They have shorter fold-backs of miRNA precursors (90 nucleotides compared to 180 in plants), the mature miRNA sequence is usually found at the stem base, more mismatches in the foldback and they derive from polycistronic signals. While animal miRNAs anneal imperfectly with the 3? While animal miRNAs generally anneal imperfectly to the 3? (2002) Cell, 110:513-520; Jones-Rhoades et al. (2006) Annu. Rev. Plant Biol., 57:19-53. This significant difference between animal and plant miRNAs makes it unlikely that miRNAs would be processed and function in both kingdoms.

Transgenic expression of miRNAs can be used to regulate the expression of target genes of miRNAs. The inclusion of a miRNA-recognition site in a transcript transgenically expressed is useful for regulating the expression of the transcription; see, for instance, Parizotto and al. (2004) Genes Dev., 18:2237-2242. The recognition sites of miRNAs are found in all regions of mRNAs, including the 5′ untranslated region, coding region and 3? The recognition sites of miRNAs have been validated in all regions of an mRNA, including the 5? Untranslated region, showing that the position of miRNA target sites relative to coding sequences may not affect suppression. Mol. Cell, 14:787-799, Rhoades et al. (2002) Cell, 110:513-520, Allen et al. (2004) Nat. Sunkar and Zhu, (2004) Plant Cell 16:2001-2019). MiRNAs are essential regulatory elements for eukaryotes. Transgenic suppression of miRNAs can be used to manipulate biological pathways and responses. Finaly, MIR promoters can be expressed in very specific ways (e.g. cell-specific or tissue-specific expression, or temporally-specific expression) and are therefore useful for recombinant constructs that want to induce a specific transcription. The U.S. Patent Application Publication No. 2006/0200878A1 describes in detail the various utilities of miRNAs and their precursors. It also includes their recognition sites and promoters. This is incorporated herein by reference. These utilities can include, but are not limited to: “(1) the expression a native (native) miRNA sequence or miRNA pre-cursor sequence to suppress target gene. (2) the expression a engineered (nonnative), miRNA sequence or miRNA pre-cursor sequence to suppress target gene. (3) the expression a transgene containing a recognition site for miRNAs where the transgene will be suppressed by the mature miRNA. (4) the expression a transgene that is driven

Animal miRNAs were used as precursors for expressing specific miRNAs within animal cells. For example, the human precursor miR-30 was expressed in both its native sequence as well as as a modified miRNA (artificially or engineered). (2002) Mol. Cell, 9:1327-1333 and Zeng et. al. (2005) J. Biol. Chem., 280:27595-27603). One mature miRNA can be precisely produced from a specific precursor. This is why such “artificial” miRNAs are possible. Engineered miRNAs have an advantage over double stranded RNAs (dsRNAs) because only one specific miRNA is expressed. This limits the possibility of off-target effects. Animal miRNAs interact with imperfect targets in the 3′ region. Synthetic miRNAs that have perfect complementarity with their target sequences can also guide target cleavage. Zeng et. al. (2003) RNA 9:112-123. (2003) Proc. Natl. Acad. Sci. U.S.A, 100:9779-9784).

Small interfering RNAs and micro RNAs” (Valencia Sanchez et al.) have been found to regulate gene transcription in both plants and animals. (2006) Genes Dev., 20:515-524; Nelson et al. (2003) Trends Biochem. Sci., 28:534-540). The experimental alteration of the siRNA levels results in phenotypic changes in nematodes. (Timmons and Fire, Nature 395:8543, 1998). The transgenically-expressed siRNA complementary to 16D10 was found to confer resistance against four species of root knot nematodes in a plant (Huang and al. (2006) Proc. Natl. Acad. Sci. U.S.A, 103:14302-14306). This invention reveals the use of invertebrate recombinant miRNAs expressed planta to regulate expression in invertebrates that ingest the miRNAs.

This invention discloses recombinant dna constructs that encode invertebrate miRNAs mature and their miRNA pre-cursors. These constructs are designed to express in plants. In certain embodiments, invertebrate precursors of miRNAs are engineered in a way to express artificially miRNAs that suppress or silence specific genes in invertebrates. This confers on a plant expressing these miRNAs resistance against an invertebrate ingesting the miRNAs. In some cases, RNAi transcripts (siRNAs or miRNAs) that are designed to suppress an invertebrate are ingested as larger transcripts. That is, they are larger than the typical 21-to-24 nucleotide segments that result from in planta process. RNAi transcriptions designed for ingestion should be resistant to planta processing. “The recombinant miRNAs in this invention, in comparison with plant-derived miRNAs, are preferably resistant against the plant-specific miRNA processing. However they are readily recognized by invertebrate cell where they are converted to mature miRNA.

The invention describes a non-naturally resistant plant to an invertebrate that consumes RNA from a plant. This plant includes a cell transgenic with a recombinant genome that is translated into a recombinant microRNA precursor.

Another aspect of the invention is the recombinant dna construct that can be transcribed into a recombinant precursor miRNA in the transgenic non-natural plant cell. In many embodiments, the recombinant DNA construct further includes one or more elements selected from: (a) a promoter functional in a plant cell; (b) a transgene transcription unit; (c) a gene suppression element; and (d) a transcription regulatory/transcript stabilizing element.

This invention also provides a transgenic plant cell that is not natural, a transgenic plant grown from the transgenic plant cells of this invention, as well as non-naturally transgenic seed produced by the transgenic plants. A non-natural plant that contains the transgenic cell of the invention is also provided, as are non-natural plants grown from the cell.

The following detailed description discloses other specific embodiments of this invention.

Unless defined differently, all technical and science terms used have the meaning that is commonly understood by a person of ordinary skill in this art. The nomenclature and laboratory or manufacturing procedures described in this document are generally well-known and widely used. These procedures are carried out using conventional methods, as described in the art or various general references. The nucleic acids sequences are written in this specification, unless otherwise specified, from left to the right in 5? Unless otherwise stated, nucleic acid sequences in the text of this specification are given when read from left to right, in the 5? direction. As specified, nucleic acid sequences can be presented as DNA or RNA; a disclosure of either one defines the other as one is well-known to those with ordinary knowledge of the field. When a singular term is used, the inventors are also considering aspects of the invention that can be described with the plural form of that term. The laboratory procedures and nomenclature described in this document are well-known and widely used. In the event of discrepancies between the terms and definitions in the references that are incorporated, the terms in the application will be given the defined meaning. As exemplified in a variety technical dictionaries, other technical terms have their normal meanings within the field of application. The inventors are not limiting themselves to one mechanism or method of action. “Reference to the invention is for illustration purposes only.

Plants Resistant to Invertebrate Pests

The invention describes a non-naturally resistant plant to an invertebrate that consumes RNA from a plant. This plant includes a cell transgenic with a recombinant genome that is translated into a recombinant microRNA precursor.

The recombinant construct is made by techniques known in the art, such as those described under the heading?Making and Using RecombinantDNA Constructs? The recombinant DNA is created using techniques well-known in the art. For example, those described under ‘Making and using Recombinant DNAConstructs? The working Examples are illustrated. The recombinant construct of DNA is especially useful for creating transgenic plant seeds, transgenic plants and transgenic cells as discussed in the section below titled ‘Making Transgenic Plant Cells or Transgenic Plants’. The recombinant precursor miRNA is usually transcribed into a single strand RNA. This single strand RNA contains at least one stem loop that is equivalent to a pre-miRNA that occurs naturally, as it is processed into a mature miRNA. The stem-loop occurs when the single strand is folded back on itself, and enough base pairings occur to stabilize the folded structure. The stem-loop is made up of two regions, a stem and a loop, both within a single strand RNA. The stem region consists of a first segment followed by a second segment that are connected through the loop region. The loop region may include more complex structures than a single-stranded, simple loop. A non-limiting illustration of this is shown by mir286. Note that the loop region can include structures more complex than a simple single-stranded loop. A non-limiting example of this is illustrated by mir286 (SEQID NO. In FIG. 3. First segment contains at least 19 nucleotides contiguous for silencing the messenger RNA that encodes the target gene. The second segment has at least 19 nucleotides. The length of the first and second segments is generally similar (in terms number of nucleotides that make up each segment), but not necessarily identical. The loop region lies on the single-stranded strand that connects the first and second segment. First and second segments are hybridized to form partially double-stranded DNA, in which at least one of the nucleotides of the first is unpaired. The mismatch causes a bulge, loop or kink to appear in the stem region. The mismatch may be caused by, for instance, a nucleotide from the second segment which does not pair with the nucleotide questioned of the initial segment or if there is at least one extra nucleotide or one missing nucleotide at the position of the nucleotide questioned of the first segments. FIG. FIG.

The first segment in the stem region contains at least 19 nucleotides that are contiguous for silencing the messenger RNA that encodes the target gene. The mature miRNA is made from these 19 contiguous nucleotides. These at least nineteen contiguous RNAs have at least 70% complementarity with a segment equivalent in length (that’s to say, at least 70% complementarity with a segment that has about the same number contiguous RNAs) of the target mRNA. “For example, if the first segment of stem region is composed of exactly 19 nucleotides to silence the target, then these 19 nucleotides could include 13 nucleotides, (13/19=68% complementary), 14 nucleotides, (14/19=74% complementary), 15 nucleotides, (15/19=79% complementary), 16 nucleotides, (16/19=84% complimentarity), 17 or 18 nucleotides, (18/19=95% complimentary),

In preferred embodiments, at least 19 nucleotides are at least 75% or at about 80% or at about 85% or at about 90% complementary to a segment equivalent in length of the target mRNA. In one embodiment, at least 19 nucleotides are at least 95% complementary to a target mRNA segment of equal length. In a second, particularly preferred embodiment the at least 19 nucleotides are 100% complementary to a target mRNA segment of equal length.

A person skilled in the art can easily select the degree of complementarity of the first segment’s 19 nucleotides in the stem region to a segment with an equivalent length in the mRNA target. Base pairing between nucleotides near the 5? The complementarity of the mature miRNA’s 5? The complementarity between the 5? The mature miRNA’s end and the target can be mismatched to a greater degree if they are closer together. In a preferred embodiment the nucleotide of the first segment of the stem region is chosen so that the mature mRNA produced from the stem loop is complementary to the target at the 8?most nucleotides. In a second preferred embodiment, the sequence of nucleotides in the first segment of the stem region is chosen so that the mature microRNA produced from the stem loop is complementary to the target miRNA at the 8 nucleotides closest to 5?. In another preferred embodiment, the nucleotide sequence of the stem region’s first segment is designed so that mature miRNA processed from the stem-loop structure has few or no G:U wobble base pairs.” “In another preferred embodiment, at least 19 consecutive nucleotides in the first segment of the stem region are designed to ensure that the mature miRNA produced from the stem-loop structures has few or none G:U wobble bases pairs.

The loop region of a stem-loop is typically between 4 and 40 nucleotides. In certain preferred embodiments the loop region contains consecutive nucleotides from a native sequence of an invertebrate precursor miRNA loop. In some embodiments the loop region is identical with a native sequence of an invertebrate precursor miRNA.

In some embodiments, stem-loops are processed into mature miRNAs in transgenic plant cells. These mature miRNAs typically have a length of 21, 22, 24, 25, or 26, depending on the nucleotide. In some embodiments, stem-loops are left relatively intact in transgenic plants cells (i.e., not cleaved into smaller polynucleotides), but they are processed to mature miRNAs (typically 21, 22, 23, 24 25, or 26 nucleotides long) within the gut of the invertebrate ingesting RNA of the plant including the transgenic cell.

In one embodiment, a single stem loop is included in the single strand RNA. This stem loop is then processed into a mature miRNA. In other embodiments the single strand RNA contains multiple stem-loops which are then processed into mature miRNAs. Multiple stem-loops can be made up of multiples stem-loops or stem-loops that are different. In one preferred embodiment of the invention, a single strand RNA contains multiple stem-loops which correspond to a polycistronic group of 8 miRNAs natively transcribed by invertebrates. A single strand of DNA with multiple stem-loops corresponds to a polycistronic 8-miRNA group found on Chromosome 2R of Drosophila (SEQ ID No. “1. See Example 1.

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