RNA-based regulation of deoxynivalenol production by Fusarium.

Abstract

The present disclosure relates to approaches using dsRNA to reduce or eliminate the production of DON by fungal pathogens of the genus Fusarium, F. graminearum, or other fungal pathogens, and / or to control F. graminearum or other fungal pathogens of the genus Fusarium. In certain embodiments, methods and compositions are described for controlling DON production and / or controlling fungal pathogens by causing mortality, reduced growth, reduced virulence or pathogenicity, or reduced reproduction / fertility (sporulation) by using the application of exogenous dsRNA administered to plants that are infected or can be infected with F. graminearum or another member of the Fusarium species complex that produces DON or otherwise causes blight disease.

Description

[Technical field]

[0001] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 328,216, filed April 6, 2022, and U.S. Provisional Application No. 63 / 488,689, filed March 6, 2023, the contents of which are incorporated by reference in their entireties herein.

[0002] Reference to Electronic Sequence Listing This application contains a Sequence Listing that has been submitted electronically in .XML format and is incorporated herein by reference in its entirety. The .XML copy, created on April 6, 2023, is named "16206022PC0_SequenceListing.xml" and is 518,000 bytes in size. The Sequence Listing contained in this .XML file is a part of this specification and is incorporated herein by reference in its entirety. [Background technology]

[0003] Fusarium head blight (FHB) is a scab disease of wheat, barley, and other small cereals, caused by the Fusarium species complex. Fusarium graminearum is one of the most widespread and economically impactful species of this complex due to its ability to produce high levels of mycotoxins. Infection with Fusarium can cause significant yield losses and grain quality losses, as well as mycotoxinosis in animals and humans if ingested (Ireta and Gilchrist, 1994; Baht et al., 1989; Luo, 1988; Snidjers, 1989; Marasas et al., 1988). Of particular importance is not only the necrotic damage to wheat and / or barley heads caused by fungal infection, but more specifically the associated mycotoxin (e.g., deoxynivalenol or "DON") contamination, which often results in reduced grain quality that affects pricing and distribution to final markets (Foroud et al., 2019). Losses from FHB in the United States were estimated to exceed US$1 billion in 1993 and US$500 million in 1994. More recently, losses from epidemic FHB outbreaks in the United States from 1993 to 2001 were estimated at US$7.67 billion (Mielniczuk and Skwarylo-Bednarz, 2020). In China, it has been estimated that FHB may affect up to 7 million hectares and that 2.5 million tons of grain may be lost in epidemic years. Diseases associated with mycotoxin ingestion in humans have been reported in China, India, and Japan, while diseases associated with mycotoxin ingestion in animals have been reported in many parts of the world (Dubin et al., 1997).

[0004] Infection occurs through the anthers at anthesis, followed by colonization of other parts of the floret, then enters the vascular bundles and the pith of the rachis, from where it spreads up and down the head (reviewed in Trail et al., 2005). Many Fusarium spp. in the FHB complex produce trichothecene toxins. For example, deoxynivalenol (DON) and nivalenol (NIV) can be produced by F. graminearum and F. culmorum, and T-2 toxin can be produced by F. sporotrichioides. The biosynthesis of other mycotoxins, such as fumonisins and zeralenone (ZEA), has been described in several Fusarium spp. (Desjardins, 2006), including F. graminearum and F. culmorum, both of which produce ZEA (Mielniczuk and Skwarylo-Bednarz, 2020). The first defined step in the trichothecene biosynthetic pathway is the conversion of farnesyl pyrophosphate to trichodiene, catalyzed by trichodiene synthase encoded by TRI5 (Hohn and Beremand, 1989). Expression of TRI5 and other genes in the gene cluster correlates with the production of trichothecenes in both plants and in vitro cultures (Brown et al., 2004; Kimura et al., 2003).

[0005] In addition to the role of trichothecenes in disease processes, the presence of trichothecenes in grains has important consequences for human and animal health. The concentrations of these toxins in grains are under increasing scrutiny in both Europe and the United States, where legally enforceable limits on contamination with trichothecenes such as DON in grains and foods are now in place (Anonymous, 2005; van Egmond et al., 2007). In mammalian systems, DON has been shown to be a potent translation inhibitor, causing acute symptoms such as vomiting and feed refusal, as well as chronic symptoms such as growth retardation, reduced ovarian function, and immunosuppression in livestock such as pigs (Bennett and Klich, 2003; Rocha et al., 2005).

[0006] To address these problems, the present invention relates to locally applied double-stranded RNA ("dsRNA") that targets, inter alia, fungal genes involved in the pathway of DON production by RNA interference, thereby reducing or eliminating the production of DON by Fusarium head blight-causing F. graminearum or other fungal pathogens in plants or plant parts used for human or animal consumption. Summary of the Invention

[0007] RNA interference (RNAi) technology has been shown to be a highly selective biological treatment that silences gene expression of pests and pathogens through internal biological processes. Exogenous application of double-stranded RNA (dsRNA) to initiate RNAi has been used to effectively control certain plant pest and pathogen species. The present disclosure relates to an approach using dsRNA to reduce or eliminate the production of DON by fungal pathogens of the genus Fusarium, F. graminearum, or other fungal pathogens, and / or to control F. graminearum or other fungal pathogens of the genus Fusarium. In certain embodiments, methods and compositions are described for controlling DON production and / or fungal pathogens by causing mortality, growth inhibition, reduced virulence or pathogenicity, or reduced reproduction / fertility (sporulation) by using the application of exogenous dsRNA administered to plants infected or capable of being infected with F. graminearum or another member of the Fusarium genus, which is involved in the Fusarium species complex that produces DON or otherwise causes blight disease. Such plants may include, for example, corn and small grains such as wheat, barley, flax, buckwheat, rye, and oats.

[0008] Compositions and methods described herein include recombinant polynucleotide molecules, such as single- or double-stranded DNA or RNA molecules, referred to herein as "triggers," that are useful for reducing or eliminating the production of DON by F. graminearum or related species, or recombinant DNA constructs for making such RNA molecules or for making transgenic plants expressing such RNA molecules. In some embodiments, a polynucleotide trigger is provided as a topically applied agent to control or prevent the production of DON on a plant by F. graminearum and / or to cause death, reduced growth, reduced virulence or pathogenicity, or reduced reproduction / fertility (sporulation) of F. graminearum. In some embodiments, a plant is provided that is a corn or small grain (e.g., wheat, barley, oats, flax, rye, or buckwheat) with improved resistance to infection by F. graminearum and / or resistance to the effects of DON produced by F. graminearum, such as a transgenic plant (including seeds or reproductive parts) expressing a polynucleotide trigger. In some embodiments, plants (including seeds or propagules) are provided that are topically treated with a composition comprising a polynucleotide trigger (e.g., a plant sprayed with a solution of dsRNA molecules). Polynucleotide-containing compositions are also provided that are topically applied to F. graminearum, or to plants, plant parts, or seeds that are infected or capable of being infected by F. graminearum.

[0009] Some embodiments relate to the suppression of target genes in F. graminearum by polynucleotide triggers. Such target genes may include any gene of Fusarium species involved in the DON production pathway. Provided herein are nucleotide sequences of such target genes, referred to herein as "target gene sequence groups" or "target gene sequences", which relate to SEQ ID NOs: 1-16, 65-70, and 87-151. Certain embodiments of the invention relate to polynucleotides (e.g., dsRNA) designed to hybridize to RNA transcripts of target genes resulting in RNAi. Also provided herein are nucleotide sequences, referred to herein as "trigger sequence groups" or "trigger sequences", which relate to SEQ ID NOs: 17-32, 75-78. Further provided herein are "RNA trigger sequence groups" or "RNA trigger sequences", which relate to SEQ ID NOs: 33-48, 79-82, and 153-225. Reverse complements to the RNA trigger sequences are also provided herein and are referred to as "RNA trigger sequence reverse complements" or RNA trigger sequence reverse complements, which relate to SEQ ID NOs: 49-64, 83-86, and 227-299. The RNA trigger sequence reverse complements are the perfect complements to sequences in the RNA trigger sequence read 5' to 3'. The RNA trigger sequences are designed to affect RNAi on the corresponding mRNA transcripts of the target gene sequences, preventing or reducing translation of associated proteins. By reducing translation of associated proteins, the trigger sequences of the present invention disrupt the DON pathway in Fusarium, resulting in reduced production of DON by Fusarium. Tables 1 and 2 provided herein match various target gene sequences to their corresponding trigger sequences, RNA trigger sequences, and RNA trigger sequence reverse complement sequences. The SEQ ID NOs relate to the sequences provided in the SEQ ID list. Please note that the SEQ ID listing submitted herewith indicates that SEQ ID NOs: 33-64, 79-86, 153-225, and 227-299 are DNA due to limitations in the new ST26 format.For purposes of this disclosure, SEQ ID NOs: 33-64, 79-86, 153-225, and 227-299 are RNA sequences in which the indicated thymines are uracils. SEQ ID NOs: 75-78 are DNA versions of SEQ ID NOs: 79-82. One of skill in the art will understand that any of the RNA sequences herein can be read as a DNA sequence by replacing any uracils with thymines, that DNA templates of such RNA sequences include DNA base complements to the given RNA sequence, that RNA sequences with identity to the cited target gene DNA sequences replace thymines with uracils, and that RNAs with complementarity to the cited target gene DNA sequences include RNA base complements to the given DNA sequence.

[0010] In one aspect, a method of reducing or eliminating DON production by a species of the genus Fusarium (e.g., F. graminearum) on a plant comprises contacting Fusarium with a polynucleotide comprising at least one segment of 18 or more contiguous nucleotides having a sequence of about 95% to about 100% identity (e.g., including a segment of 21 contiguous nucleotides having a sequence of 100% identity) to a corresponding fragment of DNA having a sequence selected from the group consisting of a target gene group or its DNA complement, or any of the genes identified in Table 1 or Table 2. In one embodiment, a method of reducing or eliminating DON production by F. graminearum on a plant comprises contacting F. graminearum with a polynucleotide comprising a nucleotide sequence that is complementary to at least 18 contiguous nucleotides of a target gene having a nucleotide sequence selected from the group consisting of target gene sequences or an RNA transcribed from the target gene. In some embodiments, the polynucleotide comprises a sequence complementary to or about 95% to about 100% identical to at least 18 contiguous nucleotides of a sequence selected from the group consisting of RNA trigger sequences or RNA trigger sequence reverse complements. In some embodiments, the 18 or more contiguous nucleotides are 21 or more contiguous nucleotides, 50 or more contiguous nucleotides, 150 or more contiguous nucleotides, 200 or more contiguous nucleotides, 250 or more contiguous nucleotides, 300 or more contiguous nucleotides, 350 or more contiguous nucleotides, 400 or more contiguous nucleotides, 450 or more contiguous nucleotides, 500 or more contiguous nucleotides, 550 or more contiguous nucleotides, or 600 or more contiguous nucleotides. In some embodiments, the polynucleotide is designed to have complementarity to an mRNA encoded by a target gene. In some embodiments, the polynucleotide is a double-stranded RNA (dsRNA). In some embodiments, the polynucleotide comprises one or more nucleotide sequences selected from the group consisting of RNA trigger sequences or RNA trigger sequence reverse complements.In some embodiments, contact with the polynucleotide is achieved by topical application (e.g., by spraying, dusting, or immersion) of the polynucleotide, or a composition or solution containing the polynucleotide, directly to F. graminearum or to a surface or matrix (e.g., plant or soil) that will be contacted by F. graminearum. In some embodiments, topical application of the polynucleotide, or a composition or solution containing the polynucleotide, is achieved by spraying the polynucleotide, or a composition or solution containing the polynucleotide, onto leaves, heads, stems, ears, seeds, roots, or other plant parts that are infected or capable of being infected by F. graminearum. In some embodiments, contact with the polynucleotide is achieved by providing a transgenic plant that expresses a sequence that reduces or eliminates DON production by F. graminearum. In some embodiments, the polynucleotide comprises two or more regions targeting two or more target genes involved in the production of DON, including targeting one or more DON genes in F. graminearum and / or targeting one or more homologues of such DON genes in another species of the genus Fusarium that produces DON, thereby reducing the production of DON by F. graminearum and reducing the production of DON by one or more additional species of the genus Fusarium. In some examples, the percent identity between the regions of the gene targets of F. graminearum and the regions of homologues found in one or more additional species of the genus Fusarium, such as F. culmorum, will be sufficiently high so that the same region in the polynucleotide will cause RNAi in one or more species, thereby resulting in reduced DON production by one or more species.

[0011] Some embodiments relate to methods of reducing or eliminating DON production by F. graminearum by providing exposure of F. graminearum to a composition comprising one or more formulations or delivery agents and a polynucleotide that induces RNAi against one or more of the gene targets disclosed in Table 1 or Table 2, or a gene target having a sequence selected from the group consisting of a target gene sequence group or a DNA complement thereof, wherein the agent functions upon contact or ingestion (e.g., internal absorption / transfection) by F. graminearum to inhibit a biological function within F. graminearum, reduce or eliminate DON production by F. graminearum, and / or otherwise control F. graminearum. In some embodiments, the polynucleotide comprises one or more nucleotide sequences selected from a group of RNA trigger sequences or a reverse complement of an RNA trigger sequence, or the polynucleotide comprises one or more nucleotide sequences that are about 95% to about 100% identical to one or more nucleotide sequences selected from a group of RNA trigger sequences or a reverse complement of an RNA trigger sequence. In some embodiments, the polynucleotide is RNA, and in some embodiments, the RNA is double-stranded RNA.

[0012] In some embodiments, the agent is (a) an effective amount of a polynucleotide comprising at least 18, 19, 20, 21, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 contiguous nucleotides that are complementary to or have at least about 85%, at least about 90%, at least about 95%, at least about 98%, about 100%, or 100% sequence identity to a target gene or a segment of RNA transcribed from the target gene having a nucleotide sequence selected from the group consisting of target gene sequences; (b) an effective amount of at least one polynucleotide comprising at least one silencing element that is complementary to or has at least about 85%, at least about 90%, at least about 95% sequence identity with at least 18, 19, 20, 21, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 consecutive nucleotides of a target gene or an RNA transcribed from the target gene, wherein the target gene has a nucleotide sequence selected from the group consisting of target gene sequences; (c) an effective amount of at least one RNA comprising at least one segment that is complementary to, or comprises at least about 85%, at least about 90%, at least about 95%, at least about 98%, about 100%, or 100% sequence identity to, at least 18, 19, 20, 21, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 consecutive nucleotides of a target gene or a segment of an RNA transcribed from the target gene having a nucleotide sequence selected from the group consisting of target gene sequences; or (d) an RNA molecule that, when transfected into or contacted with said F. graminearum, causes a reduction or elimination of DON production in F. graminearum, wherein the RNA molecule comprises at least 18, 19, 20, 21, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 contiguous nucleotides that are complementary to or have at least about 85%, at least about 90%, at least about 95%, at least about 98%, or about 100%, or 100% sequence identity to a target gene or a segment of RNA transcribed from said target gene having a nucleotide sequence selected from the group consisting of target gene sequences; or (e) a double-stranded RNA molecule that, when transfected into or contacted with said F. graminearum, causes a reduction or elimination of DON production in F. graminearum, wherein at least one strand of the double-stranded RNA molecule is complementary to, or comprises at least 85%, 90%, 95%, 98%, or 100% sequence identity to, a target gene or a segment of RNA transcribed from said target gene, and comprises at least 18, 19, 20, 21, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 contiguous nucleotides, and wherein the target gene has a sequence selected from the group consisting of target gene sequences; or (f) an effective amount of at least one double-stranded RNA, the at least one strand of which comprises a sequence selected from the group consisting of an RNA trigger sequence and an RNA trigger sequence reverse complement, or a sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98%, about 100%, or 100% sequence identity thereto; or (g) an effective amount of a polynucleotide comprising at least 18, 19, 20, 21, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 contiguous nucleotides of a nucleotide sequence selected from the group consisting of an RNA trigger sequence and an RNA trigger sequence reverse complement, or a sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98%, or about 100% sequence identity thereto; or (h) an effective amount of at least one RNA comprising at least one segment complementary to, or comprising at least about 85%, at least about 90%, at least about 95%, at least about 98%, about 100%, or 100% sequence identity to, at least 18, 19, 20, 21, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 consecutive nucleotides of a nucleotide sequence selected from the group consisting of an RNA trigger sequence and an RNA trigger sequence reverse complement; or (i) an RNA molecule that, when transfected into or contacted with said F. graminearum, causes a reduction or elimination of DON production by F. graminearum, wherein the RNA molecule comprises at least 18, 19, 20, 21, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 contiguous nucleotides that are complementary to or have at least about 85%, at least about 90%, at least about 95%, at least about 98%, about 100%, or 100% sequence identity to a segment of a nucleotide sequence selected from the group consisting of a target gene sequence, an RNA trigger sequence, and an RNA trigger sequence reverse complement; or (j) a double-stranded RNA molecule that, when transfected into or contacted with said F. graminearum, causes a reduction or elimination of DON production by F. graminearum, wherein at least one strand of the double-stranded RNA molecule comprises at least 18, 19, 20, 21, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 contiguous nucleotides that are complementary to or have at least about 85%, at least about 90%, at least about 95%, at least about 98%, about 100%, or 100% sequence identity to a segment of a nucleotide sequence selected from the group consisting of an RNA trigger sequence and an RNA trigger sequence reverse complement; (k) a double-stranded RNA molecule that, when transfected into or contacted with the F. graminearum, causes a reduction or elimination of DON production by the F. graminearum, wherein at least one strand of the double-stranded RNA molecule comprises at least about 85%, at least about 90%, at least about 95%, at least about 98%, about 100%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of an RNA trigger sequence and an RNA trigger sequence reverse complement.

[0013] In certain embodiments, the composition containing the polynucleotide is formulated for application to the plant field, for example, as a sprayable solution or emulsion, tank mix, or powder. In some embodiments, the agent is biologically produced, for example, in the form of a microbial fermentation product, or expressed in a transgenic plant cell. Various methods and compositions for formulating polynucleotides for application to the plant field are known in the art, and the polynucleotides of the present invention can be formulated in any suitable composition. Examples are the compositions described in PCT / US / 2022 / 027816, published on November 10, 2022 as WO2022 / 0235895, which is incorporated herein by reference in its entirety.

[0014] Any suitable DNA encoding an RNAi molecule that targets a target gene described herein can be used in the compositions and methods described herein. The DNA can be single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA). In some embodiments, the DNA comprises one or more DNA expression cassette(s) that, when transcribed, produces a single-stranded RNA (ssRNA) molecule (e.g., remains single-stranded or folds into an RNA hairpin), or a complementary ssRNA molecule that anneals to produce a double-stranded RNA (dsRNA) molecule.

[0015] Various methods for making RNA are known in the art, and the RNA of the present invention can be produced by any suitable method known in the art. Examples of methods for producing RNA include, but are not limited to, in vitro transcription (IVT), chemical synthesis, microbial fermentation, or cell-free methods, such as those described in U.S. Patent No. 10,858,385 (U.S. Patent Publication No. 2019 / 0144489), published May 16, 2019, and U.S. Patent No. 10,954,541 (U.S. Patent Publication No. 2017 / 0292138), published October 12, 2017, each of which is incorporated herein by reference. Examples of RNAi molecules for endogenous delivery for use with the present invention include, but are not limited to, those described in U.S. Pat. No. 11,142,768 published on May 14, 2020 (U.S. Patent Publication No. 2020 / 0149044), U.S. Pat. No. 11,185,079 published on March 26, 2020 (U.S. Patent Publication No. 2020 / 0093138), and PCT / US / 2021 / 032334 published on November 18, 2021 (International Publication No. WO 2021 / 231791), all of which are incorporated by reference herein.

[0016] Some embodiments relate to a method of providing a plant with improved resistance to DON production by F. graminearum, comprising topically applying to the plant a composition comprising at least one polynucleotide that prevents or reduces the production of DON via RNAi against a gene in the DON production pathway, ty), comprising a gene identified in Table 1 or Table 2, or having a sequence selected from the group consisting of target gene sequences or DNA complements thereof. In one embodiment, the method of providing a plant with improved resistance to F. graminearum infection comprises topically applying to the plant a composition comprising at least one polynucleotide that comprises a nucleotide sequence selected from the group consisting of target gene sequences, or a nucleotide sequence that is complementary to at least 18 contiguous nucleotides of a target gene having RNA transcribed from the target gene. In some embodiments, the at least one polynucleotide comprises a sequence selected from the group consisting of an RNA trigger sequence or an RNA trigger sequence reverse complement, or comprises a nucleotide sequence at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 98%, or about 100%, or 100% identical to the RNA trigger sequence or the RNA trigger sequence reverse complement. In some embodiments, the polynucleotide is a dsRNA comprising one or more sequences selected from an RNA trigger sequence and a corresponding sequence selected from an RNA trigger sequence reverse complement. In one embodiment, a method for providing a plant with improved resistance to DON production by F. graminearum comprises topical application to the plant of a composition comprising at least one polynucleotide in such a manner that an effective amount of the polynucleotide is transfected by or contacted with the F. graminearum infected plant, the polynucleotide comprising at least 18 consecutive nucleotides complementary to a target gene having a nucleotide sequence selected from the group consisting of target gene sequences, or a portion of an RNA transcribed from the target gene.In some embodiments, the polynucleotide comprises one or more nucleotide sequences selected from the RNA trigger sequence group or the RNA trigger sequence reverse complement group, or comprises a nucleotide sequence that is at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 98% identical to the RNA trigger sequence or the RNA trigger sequence reverse complement. In some embodiments, the polynucleotide is a dsRNA that comprises one or more sequences selected from the RNA trigger sequence and a corresponding sequence selected from the RNA trigger sequence reverse complement. In some embodiments, the polynucleotide is a dsRNA. Some embodiments relate to compositions comprising the polynucleotide formulated for application to plant fields, for example, as a sprayable solution or emulsion, tank mix, or dust.

[0017] Some embodiments relate to compositions for reducing or eliminating DON production by F. graminearum, comprising an effective amount of at least one polynucleotide molecule comprising at least one segment of 18 or more contiguous nucleotides that are essentially identical or complementary to a corresponding fragment of DNA having a sequence selected from the group consisting of a target gene sequence group or its DNA complement (e.g., a segment of 21 contiguous nucleotides having a sequence of 100% identity or complementarity). In some embodiments, the polynucleotide molecule comprises at least 18 contiguous nucleotides that are complementary to a target gene having a nucleotide sequence selected from the group consisting of a target gene sequence group or a portion of an RNA transcribed from a target gene. In some embodiments, the polynucleotide comprises one or more nucleotide sequences selected from an RNA trigger sequence group or an RNA trigger sequence reverse complement trigger, or comprises a nucleotide sequence that is complementary to, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 98%, or about 100%, or 100% identical to, a nucleotide sequence selected from an RNA trigger sequence or an RNA trigger sequence reverse complement. In some embodiments, the polynucleotide is a dsRNA comprising one or more sequences selected from an RNA trigger sequence and a corresponding sequence selected from an RNA trigger sequence reverse complement. In some embodiments, the polynucleotide molecule is a recombinant polynucleotide. In some embodiments, the polynucleotide molecule is RNA. In some embodiments, the polynucleotide molecule is a dsRNA.Related embodiments include compositions comprising polynucleotide molecules formulated for application to a field of plants, e.g., as a sprayable solution or emulsion, tank mix, or dust, and optionally comprising one or more additional components, such as a carrier agent, a surfactant, an organosilicone, an organosilicone surfactant, a polynucleotide herbicidal molecule, a non-polynucleotide herbicidal molecule, a polynucleotide pesticide, a non-polynucleotide pesticide, a polynucleotide fungicide, a non-polynucleotide fungicide, a polynucleotide insecticide, a non-polynucleotide insecticide, a safener, and a pathogen growth regulator.

[0018] Some embodiments relate to a method of providing a plant with improved resistance to DON production by F. graminearum, comprising expressing in the plant at least one polynucleotide comprising at least one segment of 18 or more contiguous nucleotides that is essentially identical or complementary to a corresponding fragment of DNA having a sequence selected from the group consisting of a target gene sequence group or its DNA complement (e.g., a segment of 21 contiguous nucleotides having a sequence of 100% identity or complementarity). In some embodiments, the polynucleotide comprises one or more nucleotide sequences selected from a trigger sequence group, an RNA trigger sequence group, or an RNA trigger sequence reverse complement group, or comprises a nucleotide sequence that is at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 98% identical to a sequence selected from an RNA trigger sequence or an RNA trigger sequence reverse complement. In some embodiments, the polynucleotide is a dsRNA comprising one or more sequences selected from an RNA trigger sequence and a corresponding sequence selected from an RNA trigger sequence reverse complement.

[0019] Some embodiments relate to recombinant DNA constructs comprising a heterologous promoter operably linked to a DNA element comprising a sequence for the production of an RNA as described herein, such that it is useful for preventing or reducing the production of DON by Fusarium species. In some embodiments, the DNA element encodes a double-stranded RNA. In some embodiments, the double-stranded RNA comprises one or more nucleotide sequences selected from an RNA trigger sequence group or an RNA trigger sequence reverse complement. Related embodiments include a plant chromosome or plastid or a recombinant plant virus vector or recombinant baculovirus vector comprising the recombinant DNA construct or comprising a DNA element that does not comprise a heterologous promoter.

[0020] Some embodiments relate to a transgenic plant cell having in its genome a recombinant DNA encoding an RNA that suppresses expression of a target gene in F. graminearum that is contacted with or transfected with an RNA that results in the elimination or reduction of the production of DON by F. graminearum. In some embodiments, the target gene is a target gene identified in Table 1 or Table 2. Particular embodiments are transgenic plant cells having in their genome a recombinant DNA encoding an RNA for silencing one or more target genes selected from a group of target gene sequences. In some embodiments, the RNA comprises one or more nucleotide sequences selected from a group of RNA trigger sequences or a group of RNA trigger sequence reverse complements, or comprises a nucleotide sequence that is at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 98%, or about 100%, or 100% identical to a sequence selected from a group of RNA trigger sequences or a group of RNA trigger sequence reverse complements.

[0021] Some embodiments relate to an isolated recombinant RNA molecule that, when transfected or contacted with F. graminearum, causes a reduction or elimination of DON production by F. graminearum, where the recombinant RNA molecule comprises at least one segment of 18 or more contiguous nucleotides that is essentially complementary to a corresponding fragment of DNA having a sequence selected from the group consisting of a target gene sequence or its DNA complement (e.g., a segment of 21 contiguous nucleotides having a sequence of 100% complementarity). In some embodiments, the recombinant RNA molecule is double-stranded RNA. Certain embodiments include an isolated recombinant double-stranded RNA molecule having a strand with a sequence selected from the group consisting of an RNA trigger sequence, an RNA trigger sequence reverse complement, or a combination thereof. Another embodiment relates to an isolated recombinant double-stranded RNA molecule comprising a first strand having a sequence selected from the group consisting of SEQ ID NOs: 48, 64, 80-82, 84-86, 182, 188, 194, 213, 220, 256, 262, 268, 287, and 294, and a second strand complementary to the first strand.

[0022] Some embodiments relate to a method of providing a plant with improved resistance to DON production by F. graminearum, comprising providing to the plant at least one polynucleotide comprising at least one segment of 18 or more contiguous nucleotides that is essentially identical or complementary to a corresponding fragment of a target gene selected from a group of target gene sequences (e.g., a segment of 21 contiguous nucleotides having a sequence of 100% identity or complementarity). In one embodiment, the method of providing a plant with DON production by F. graminearum comprises providing to the plant at least one polynucleotide comprising at least one segment that is identical or complementary to at least 18 contiguous nucleotides of the target gene or RNA transcribed from the target gene, wherein the target gene is selected from the group consisting of the genes identified in Table 1 or Table 2. In some embodiments, the polynucleotide comprises one or more nucleotide sequences selected from a group of RNA trigger sequences or a group of RNA trigger sequence reverse complements, or comprises a nucleotide sequence that is at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 98% identical to an RNA trigger sequence or an RNA trigger sequence reverse complement. In some embodiments, the polynucleotide is a dsRNA. In some embodiments, the dsRNA comprises a first strand that comprises one or more sequences selected from an RNA trigger sequence and a second strand that comprises one or more corresponding sequences selected from an RNA trigger sequence reverse complement.

[0023] Some embodiments relate to methods of reducing or eliminating DON production by F. graminearum by contacting F. graminearum with at least one segment of 18 or more contiguous nucleotides that is essentially identical or complementary to a corresponding fragment of equivalent length of a portion of the DNA sequence of a target gene selected from the genes identified in Table 1 or Table 2 (e.g., a segment of 21 contiguous nucleotides with 100% identity or complementarity). In some embodiments, the polynucleotide is double-stranded RNA.

[0024] Some embodiments relate to artificial compositions comprising at least one polynucleotide as described herein. Some embodiments provide formulations useful for topical application to plants or materials in need of protection from DON production by F. graminearum. Some embodiments provide recombinant constructs and vectors useful for making transgenic plant cells and transgenic plants. Some embodiments provide formulations and coatings useful for treating plants, plant seeds or reproductive parts. Some embodiments provide commercial products and foods produced from such plants, seeds or reproductive parts treated with or containing the polynucleotides described herein, particularly commercial products and foods having detectable amounts of the polynucleotides described herein. Some embodiments relate to polyclonal or monoclonal antibodies that bind to proteins encoded by sequences or fragments of sequences selected from the target gene sequences. Another aspect relates to polyclonal or monoclonal antibodies that bind to proteins encoded by sequences or fragments of sequences selected from the RNA trigger sequences or their complements. Such antibodies are produced by routine methods known to those skilled in the art.

[0025] Other aspects and specific embodiments of the present invention are disclosed in the following detailed description. [Brief description of the drawings]

[0026] [Figure 1] 1 provides a graph showing DON reduction by application of the trigger sequences disclosed herein. [Diagram 2] 1 provides graphs showing how certain trigger sequence embodiments reduce DON production. [Diagram 3] Graphs are provided that show how certain trigger sequence embodiments reduce expression of their corresponding target genes. [Figure 4] 1 provides a graph showing DON reduction by application of the trigger sequences disclosed herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] I. Definition Unless otherwise defined, 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. Where a term is described in the singular, the inventors also contemplate aspects of the invention described by the plural of that term. In the event of a discrepancy in terms and definitions used in references incorporated by reference, the terms used in this application shall have the definitions given herein. Other technical terms used may be found in various dictionaries specialized in the field, such as "The American Heritage® Science Dictionary" (Editors of the American Heritage Dictionaries, 2011, Houghton Mifflin Harcourt, Boston and New York), "McGraw-Hill Dictionary of Scientific and Technical Terms" (6 th edition, 2002, McGraw-Hill, New York), or the “Oxford Dictionary of Biology” (6 th"Terms and expressions have their ordinary meaning in the art in which they are used, as exemplified by "Terms and Expression in the Art of Drug Delivery," 2008 edition, Oxford University Press, Oxford and New York. The inventors do not intend to be limited to a particular mechanism or mode of action. References thereto are provided for illustrative purposes only.

[0028] Unless otherwise stated, nucleic acid sequences in the body of this specification are presented in a 5' to 3' direction when read from left to right. Those skilled in the art will recognize that a given DNA sequence is understood to define a corresponding RNA sequence that is identical to the DNA sequence, except for the replacement of thymine (T) nucleotides in the DNA with uracil (U) nucleotides. Thus, providing a particular DNA sequence is understood to define the exact RNA equivalent. A given first polynucleotide sequence further defines the sequence of a second polynucleotide, whether DNA or RNA, that is its exact complement (which may be DNA or RNA), by forming Watson-Crick base pairs that perfectly hybridize to the first polynucleotide. In the case of a DNA:DNA duplex (hybridized strand), the base pairs are adenine:thymine or guanine:cytosine, and in the case of a DNA:RNA duplex, the base pairs are adenine:uracil or guanine:cytosine. Thus, the nucleotide sequence of a fully hybridized blunt-ended double-stranded polynucleotide (where there is "100% complementarity" between the strands or where the strands are "complementary") is clearly defined by providing the nucleotide sequence of one strand, whether given as DNA or RNA. "Essentially identical" or "essentially complementary" to a target gene or a fragment of a target gene means that a polynucleotide strand (or at least one strand of a double-stranded polynucleotide) is designed to hybridize to a target gene or a fragment of a target gene, or a transcript of a target gene or a fragment of a target gene (generally under physiological conditions such as those found in a plant or fungal cell), and one of skill in the art will understand that such hybridization does not necessarily require 100% sequence identity or complementarity. In some embodiments, a trigger may be designed to be complementary to a sequence of a target gene or an RNA transcribed therefrom, but not 100% identical to the sequence of the target gene. A first nucleic acid sequence is "operably" connected or "linked" with a second nucleic acid sequence when the first nucleic acid sequence is placed into a functional relationship with the second nucleic acid sequence.For example, a promoter sequence is "operably linked" to DNA if the promoter provides for transcription or expression of the DNA. Generally, operably linked DNA sequences are contiguous.

[0029] The term "polynucleotide" generally refers to a DNA or RNA molecule containing multiple nucleotides, and generally refers to both "oligonucleotides" (polynucleotide molecules of 18-25 nucleotides in length) and longer polynucleotides of 26 nucleotides or more. Polynucleotides also include molecules containing multiple nucleotides, including non-canonical nucleotides or chemically modified nucleotides, as commonly practiced in the art. See, for example, the chemical modifications disclosed in the technical manual "RNA Interference (RNAi) and DsiRNAs", 2011 (Integrated DNA Technologies Coralville, Iowa). Generally, the polynucleotides described herein, whether DNA or RNA, or both, and whether single-stranded or double-stranded, contain at least one segment of 18 or more contiguous nucleotides (or at least 18 contiguous base pairs in the case of double-stranded polynucleotides), that are essentially identical or complementary to a comparable sized fragment of the DNA of the target gene or the RNA transcript of the target gene. Throughout this disclosure, "at least 18 contiguous" means "from about 18 to about 10,000, including all integer points therebetween."Thus, embodiments of the present invention include oligonucleotides having a length of 18 to 25 nucleotides (18mer, 19mer, 20mer, 21mer, 22mer, 23mer, 24mer, or 25mer), or intermediate length polynucleotides having a length of 26 nucleotides or more (26, 27, 28, 29, 30, 31, 32, 33, 34, 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, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 120, 121, 122, 123, 124, 125, 126, 3, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, or polynucleotides of about 300 nucleotides), or longer nucleotides having a length of more than 300 nucleotides (e.g., polynucleotides of about 300 to about 400 nucleotides, about 300 to about 700, about 400 to about 500 nucleotides, about 500 to about 600 nucleotides, about 600 to about 700 nucleotides, about 700 to about 800 nucleotides, about 800 to about 900 nucleotides, about 900 to about 1000 nucleotides, about 300 to about 500 nucleotides, about 300 to about 600 nucleotides, about 300 to about 700 nucleotides, about 300 to about 800 nucleotides, about 300 to about 900 nucleotides, or about 1000 nucleotides, or even longer than about 1000 nucleotides, for example, up to the full length of the target gene, including the coding or non-coding, or both coding and non-coding, portions of the target gene). When the polynucleotide is double-stranded, its length can be described in terms of base pairs as well.

[0030] The polynucleotides described herein can be single-stranded (ss) or double-stranded (ds). "Double-stranded" generally refers to base pairing that occurs between antiparallel nucleic acid strands that are sufficiently complementary to form a double-stranded nucleic acid structure under physiologically relevant conditions. Embodiments include those in which the polynucleotide is selected from the group consisting of sense single-stranded DNA (ssDNA), sense single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), double-stranded DNA (dsDNA), double-stranded DNA / RNA hybrid, antisense ssDNA, or antisense ssRNA, and mixtures of polynucleotides of any of these types may be used. In some embodiments, the polynucleotide is a double-stranded RNA that is longer than is typical of naturally occurring small regulatory RNAs (e.g., endogenously produced siRNAs and mature miRNAs). In some embodiments, the polynucleotide is a double-stranded RNA that is at least about 30 contiguous base pairs in length. In some embodiments, the polynucleotide is a double-stranded RNA that has a length of about 50 to about 600 base pairs. In some embodiments, a polynucleotide can include components other than standard ribonucleotides, for example, one embodiment is RNA that includes terminal deoxyribonucleotides.

[0031] In various embodiments, the polynucleotides described herein include naturally occurring nucleotides, such as those found in DNA and RNA. In certain embodiments, the polynucleotide is a combination of ribonucleotides and deoxyribonucleotides, such as a synthetic polynucleotide that is primarily composed of ribonucleotides but has one or more terminal deoxyribonucleotides or one or more terminal dideoxyribonucleotides, or a synthetic polynucleotide that is primarily composed of deoxyribonucleotides but has one or more terminal dideoxyribonucleotides. In certain embodiments, the polynucleotide includes non-canonical nucleotides, such as inosine, thiouridine, or pseudouridine. In certain embodiments, the polynucleotide includes chemically modified nucleotides. Examples of chemically modified oligonucleotides or polynucleotides are well known in the art, see, for example, U.S. Patent Publication Nos. 2011 / 0171287, 2011 / 0171176, 2011 / 0152353, 2011 / 0152346, and 2011 / 0160082, which are incorporated herein by reference. Illustrative examples include, but are not limited to, the naturally occurring phosphodiester backbone of an oligonucleotide or polynucleotide, which may be partially or fully modified with phosphorothioate, phosphorodithioate, or methylphosphonate internucleotide linkage modifications, modified nucleoside bases or modified sugars may be used in oligonucleotide or polynucleotide synthesis, and oligonucleotides or polynucleotides may be labeled with fluorescent moieties (e.g., fluorescein or rhodamine) or other labels (e.g., biotin).

[0032] Some embodiments relate to a polynucleotide comprising at least one segment of 18 or more contiguous nucleotides having a sequence of about 95% to about 100% identity to a fragment of a target gene selected from the group consisting of the genes identified in Table 1 or Table 2, a DNA of a target gene having a sequence selected from the group of target gene sequences or the group of trigger sequences, or an RNA transcript of any of them, or a DNA or RNA complement of any of the foregoing. In some embodiments, the contiguous nucleotides are at least 18, e.g., at least 21, 18-24, 20-30, 20-50, 20-100, 50-100, 50-600, 100-250, 250-600, 400-600, 200-1000, or 500-2000, or even more. In some embodiments, the contiguous nucleotides are more than 18, e.g., 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30, e.g., about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 110, about 120, about The number of contiguous nucleotides is about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, or more than 500. In some embodiments, the contiguous nucleotides include about the same number of nucleotides as any of the sequences in the RNA trigger sequence group.In some embodiments, the polynucleotide comprises at least one segment of at least 18, 19, 20, or 21 (as used throughout, reference to at least 18, 19, 20, or 21 is intended to mean that any of these lower limits of the group may be individualized) contiguous nucleotides having a comparable length fragment of a target gene selected from the group consisting of the genes identified in Table 1 or Table 2 or homologs thereof, or the DNA of a target gene having a sequence selected from the group of target gene sequences, or the DNA complement of any of the sequences of the group of RNA trigger sequences or the group of RNA trigger sequence reverse complements, or the RNA transcript of any of them, or a sequence of 100% identity to the DNA or RNA complement of any of the foregoing. In some embodiments, the polynucleotide is a fragment of at least 18, 19, 20, 21, 50, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or 650 contiguous nucleotides that have 100% identity to a fragment of equivalent length of a target gene selected from the group consisting of the genes identified in Table 1 or Table 2, the DNA of a target gene having a sequence selected from the group of target gene sequences, or the DNA complement of any of the group of RNA trigger sequences or the group of RNA trigger sequence reverse complements, or the RNA transcript of any of the foregoing. A double-stranded nucleic acid (e.g., dsRNA) having one strand containing at least one segment of a base pair, such double-stranded nucleic acid, when expressed as base pairs, comprises at least one segment of at least 18 contiguous perfectly matched base pairs corresponding to a fragment of equivalent length of a target gene selected from the group consisting of the genes identified in the target gene sequence group, the DNA of a target gene having a sequence selected from the target gene sequence group, or the DNA complement of an RNA trigger sequence group or an RNA trigger sequence reverse complement group, or an RNA transcript of any of them, or the DNA or RNA complement of any of the foregoing.In some embodiments, each segment contained in the polynucleotide is longer than that typical of naturally occurring regulatory small RNAs, e.g., each segment is at least about 30 contiguous nucleotides (or base pairs) in length. In some embodiments, the total length of the polynucleotide, or the length of each segment contained in the polynucleotide, is less than the total length of the DNA or target gene. In some embodiments, the total length of the polynucleotide is about 300 to about 650 nucleotides (for single-stranded polynucleotides) or base pairs (for double-stranded polynucleotides). In some embodiments, the polynucleotide is a dsRNA of about 300 to about 650 base pairs, such as a dsRNA of any of the lengths of the RNA trigger sequences disclosed in Table 1 or Table 2 or any of the following figures.

[0033] Some embodiments relate to polynucleotides designed to regulate expression by inducing regulation or repression of one or more Fusarium target genes involved in DON production. In some embodiments, the one or more target genes are selected from the group consisting of genes identified in Table 1 or Table 2 or homologs thereof, or in particular embodiments, selected from the group consisting of FGP1, ELP3, SPT7, MAF1, MVD1, HEP1, SET1, and FKPB12. In some embodiments, the polynucleotides are designed to have a nucleotide sequence that is essentially identical or essentially complementary to the nucleotide sequence of an F. graminearum target gene or a segment of a cDNA (e.g., a group of target gene sequences) or the sequence of an RNA transcribed from an F. graminearum target gene, which may be a coding sequence or a non-coding sequence. These effective polynucleotide molecules that regulate expression may be referred to herein as "polynucleotides," "polynucleotide triggers," "triggers," or "triggers."

[0034] Any size of effective polynucleotide can be used alone or in combination in the various methods and compositions described herein. In some embodiments, a single polynucleotide trigger is used to generate a composition (e.g., a composition for topical application, or a recombinant DNA construct useful for generating dsRNA or transgenic plants). In other embodiments, a mixture or pool of different polynucleotide triggers is used, in which case the polynucleotide trigger can be for a single target gene or for multiple target genes. In some embodiments, a single polynucleotide can target two or more target genes involved in the production of DON, including genes from the same Fusarium species and / or genes from two or more related Fusarium species.

[0035] As used herein, the term "isolated" refers to the separation of a molecule from other molecules that are normally associated with it in its native or natural state. Thus, the term "isolated" may refer to a DNA molecule that is separated from other DNA molecule(s) that are normally associated with it in its native or natural state. Such DNA molecules may be present in a recombinant state, such as recombinant DNA molecules. Thus, for example, DNA molecules that are fused to regulatory or coding sequences with which they are not normally associated, as a result of recombinant techniques, are considered to be isolated even when integrated into a chromosome of a cell as a transgene or when present together with other DNA molecules.

[0036] Some embodiments relate to polynucleotides designed to suppress one or more genes ("target genes"). The term "gene" refers to any portion of a nucleic acid that provides expression of a transcript or that encodes a transcript. A "gene" may include, but is not limited to, a promoter region, a 5' untranslated region, a transcribed coding region that may include an intron region, a 3' untranslated region, or a combination of these regions. In some embodiments, the target gene(s) may include coding or non-coding sequences, or both. In other embodiments, the target gene has a sequence identical or complementary to a messenger RNA, for example, in some embodiments, the target gene is represented by its corresponding cDNA. In certain embodiments, the polynucleotide is designed to suppress one or more target genes, each target gene being selected from the group consisting of genes identified in Table 1 or Table 2, or encoded by a DNA sequence selected from the group of target gene sequences, or in certain embodiments, selected from the group consisting of FGP1, ELP3, SPT7, MAF1, MVD1, HEP1, SET1, and FKPB12. In various embodiments, the polynucleotide is designed to suppress or downregulate one or more target genes, each target gene being selected from the group consisting of genes identified in Tables 1 and 2, or encoded by a sequence selected from the group of target gene sequences, and may be designed to suppress multiple target genes or target different regions of one or more of these target genes. In one embodiment, the polynucleotide comprises multiple segments of 21 or more contiguous nucleotides having 100% identity to the DNA of a target gene having a sequence selected from the group of target gene sequences or DNA complements thereof, a fragment of equivalent length of a gene identified in Tables 1 and 2 or a homolog thereof. In such cases, each segment may be identical or different in size or sequence, and may be sense or antisense to the target gene.For example, in one embodiment, the polynucleotide comprises multiple segments in a tandem or repeat arrangement, each segment comprising 21 or more consecutive nucleotides having a sequence of 100% identity to a fragment of comparable length of a gene or homolog thereof identified in Table 1 or Table 2, a group of target gene sequences, a DNA or target gene having a sequence selected from a group of target gene sequences, or a DNA complement of any of the foregoing. In some embodiments, one or more of the segments correspond to one or more of the RNA trigger sequences or reverse complements of the RNA trigger sequences of the target genes disclosed in Table 1 and Table 2. In some embodiments, one or more of the segments correspond to one or more segments of 21 or more consecutive nucleotides from a sequence selected from the RNA trigger sequences or reverse complements of the RNA trigger sequences of the target genes disclosed in Table 1 and Table 2. In some embodiments, the segments may be from different regions of the target gene, e.g., the segments may correspond to different exon regions of the target gene. In some embodiments, "spacer" nucleotides that do not correspond to the target gene may be optionally used between or adjacent to the segments.

[0037] The term "plant" as used herein, unless otherwise clearly indicated in the context of the present specification, refers to a plant that is susceptible to Fusarium infection, i.e., Fusarium graminearum infection. Examples of plants that are susceptible to Fusarium infection include corn and small grains such as wheat, barley, flax, buckwheat, rye, and oats. In a specific embodiment, the plant is wheat.

[0038] Further definitions are provided in the following sections.

[0039] II. Polynucleotides for the control of DON production by F. graminearum The polynucleotides of the present disclosure are useful for reducing DON production by fungal pathogens of the genus Fusarium on plants via RNAi and are effective in controlling or preventing Fusarium infection in plants. According to some embodiments of the present disclosure, the polynucleotides are effective in interfering with mRNA encoded by one or more F. graminearum target genes involved in the production of DON.

[0040] In some embodiments, the polynucleotide has a sequence of about 75% to about 100% identity, about 80% to about 100% identity, about 85% to about 100% identity, about 90% to about 100% identity, about 95% to about 100% identity, about 98% to about 100% identity, about 100% identity, or exactly 100% identity to a corresponding fragment of equivalent length of DNA of a target gene having a sequence selected from the group consisting of target gene sequences or their DNA complements or RNA transcribed therefrom. The target gene comprises at least one segment of 3 or more, 24 or more, 25 or more, 30 or more, 50 or more, 75 or more, 100 or more, 125 or more, 150 or more, 200 or more, 250 or more, 300 or more, 400 or more, 500 or more, 600 or more, 700 or more, 800 or more, 900 or more, or 1,000 or more, or about 200 to about 400, or about 300 to about 700, or about 300 to about 500, or about 300 to about 600, or about 300 to about 650, or about 400 to about 700 contiguous nucleotides. In some embodiments, the target gene is a gene identified in Table 1 or Table 2, or a homolog thereof. In particular embodiments, the target gene is selected from the group consisting of FGP1, ELP3, SPT7, MAF1, MVD1, HEP1, SET1, FKPB12, or in more particular embodiments, comprises FGP1.In one embodiment, the polynucleotide is selected from the group consisting of one or more target genes or homologs thereof, or target gene sequences identified in Table 1 or Table 2, or in a particular embodiment, selected from the group consisting of SEQ ID NO: 16, 68-70, 108, 114, 120, 139, and 146, or in another particular embodiment, having a nucleotide sequence comprising SEQ ID NO: 16, or a DNA complement thereof, or at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 25, at least 30, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, at least 68, at least 69, at least 70, at least 71, at least 72, at least 73, at least 74, at least 75, at least 76, at least 77, at least 78, at least 79, at least 80, at least 81, at least 82, at least 83, at least 84, at least 85, at least 86, at least 87, at least 88, at least 89, at least 90, at least 91, at 4, at least 25, at least 30, at least 50, at least 75, at least 100, at least 125, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, or at least 1,000, or about 200 to about 400, or about 300 to about 500, or about 300 to about 600, or about 300 to about 650, or about 400 to about 700 contiguous nucleotides. In some embodiments, the one or more target genes include one or more genes identified in Table 1 or Table 2, or aa homologs thereof. In particular embodiments, the one or more target genes include one or more genes selected from the group consisting of FGP1, ELP3, SPT7, MAF1, MVD1, HEP1, SET1, FKPB12, or in more particular embodiments, FGP1.

[0041] In some embodiments, the polynucleotide comprises at least 21 contiguous nucleotides that are essentially complementary to a target gene having a DNA sequence selected from the group consisting of a target gene or a homolog thereof, or a group of target gene sequences or a DNA complement thereof, identified in Table 1 or Table 2, or a corresponding fragment of equivalent length of the RNA transcribed therefrom. In some embodiments, the polynucleotide comprises at least 300 contiguous nucleotides that are essentially complementary to a target gene having a DNA sequence selected from the group consisting of a target gene or a homolog thereof, or a group of target gene sequences or a DNA complement thereof, identified in Table 1 or Table 2, or a corresponding fragment of equivalent length of the RNA transcribed therefrom. In particular embodiments, the target gene is selected from the group consisting of FGP1, ELP3, SPT7, MAF1, MVD1, HEP1, SET1, FKPB12, or in more particular embodiments, FGP1. In some embodiments, the polynucleotide is designed to have complementarity to the mRNA encoded by the target gene. In some embodiments, the polynucleotide is a double-stranded RNA. In some embodiments, the double-stranded RNA comprises one strand comprising a sequence selected from the group consisting of SEQ ID NOs: 48, 80-82, 182, 188, 194, 213, and 220, and a second strand complementary thereto, or in particular embodiments, one strand comprising a sequence selected from the group consisting of SEQ ID NOs: 32-38, 79-82, and 268, and a second strand complementary thereto, or in more particular embodiments, one strand comprising the sequence of SEW ID NO:32, and a second strand complementary thereto.

[0042] In some embodiments, the polynucleotide comprises a sequence of contiguous nucleotides that is essentially complementary or exactly (100%) identical to a target gene or a homolog thereof, or a group of target gene sequences identified in Table 1 or Table 2, or in certain embodiments, a sequence selected from the group consisting of SEQ ID NO: 16, 68-70, 108, 114, 120, 139, and 146, or in other specific embodiments, a fragment of equivalent length of DNA of a target gene having a sequence comprising SEQ ID NO: 16, or its DNA complement, or RNA transcribed therefrom. In some embodiments, the polynucleotide has an overall sequence identity of about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% to a fragment of equivalent length of DNA of a target gene having a sequence selected from a group of target gene sequences or its DNA complement, or RNA transcribed therefrom, of a target gene or a homolog thereof, or a group of target gene sequences identified in Table 1 or Table 2. In some embodiments, the contiguous nucleotides are more than 18, e.g., 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30, e.g., about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 110, about 120, about 130, about The number of consecutive nucleotides is about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, or more than 750. In some embodiments, the consecutive nucleotides are about 200 to about 400, or about 300 to about 500, or about 300 to about 600, or about 300 to about 750, or about 300 to about 900.In some embodiments, the polynucleotide comprises at least one segment of at least 18, 19, 20, or 21 (as used throughout, reference to at least 18, 19, 20, 21, etc. is intended to mean that any of these lower limits of the group may be individualized) contiguous nucleotides having a sequence that is 100% identical to the DNA of a target gene having a sequence selected from a group of target gene sequences or its DNA complement, or to a fragment of equivalent length of the DNA of a target gene or a homolog thereof identified in Table 1 or Table 2.

[0043] In one embodiment, the polynucleotide comprises at least one segment of 21 contiguous nucleotides that is essentially complementary to, or has 100% identity to, a corresponding fragment of a target gene having a DNA sequence selected from the group consisting of SEQ ID NOs: 16, 68-70, 108, 114, 120, 139, and 146, or its DNA complement, or RNA transcribed therefrom. In some embodiments, the polynucleotide comprises one or more segments of 21 contiguous nucleotides that have 100% identity to the corresponding fragment of the target gene, as well as one or more "neutral" sequences (sequences that have no sequence identity or complementarity to the target gene), such that the polynucleotide as a whole has much less overall sequence identity to the target gene.

[0044] In one embodiment, the polynucleotide comprises a combination of multiple segments of 21 or more contiguous nucleotides that are complementary to or have 100% identity to one or more of the target genes or homologs thereof, or target gene sequences identified in Tables 1 or 2, or corresponding fragments having a DNA sequence selected from the group consisting of SEQ ID NOs: 16, 68-70, 108, 114, 120, 139, and 146, or DNA complements thereof, or RNA transcribed therefrom. In some embodiments, the polynucleotide comprises one or more segments of 21 contiguous nucleotides that have 100% identity to the corresponding fragments of one or more target genes, plus one or more "neutral" sequences (sequences that have no sequence identity or complementarity to the target genes), such that the polynucleotide as a whole has much less overall sequence identity to a given target gene. In one embodiment, the polynucleotide comprises a combination of multiple segments of 21 or more contiguous nucleotides or longer that are complementary to or have 100% identity with corresponding fragments positionally distributed throughout the length of the target gene having a DNA sequence selected from the group of target gene sequences or their DNA complements, or RNA transcribed therefrom. In some embodiments, the polynucleotide comprises one or more segments of 21 contiguous nucleotides that have 100% identity with corresponding fragments positionally distributed throughout the length of the target gene, in addition to one or more "neutral" sequences (sequences that have no sequence identity or complementarity with the target gene), such that the polynucleotide as a whole has much less overall sequence identity with a given target gene.

[0045] In some embodiments, the polynucleotide is selected from the group consisting of RNA trigger sequences or reverse complements of RNA trigger sequences, or in certain embodiments, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 50, or at least 51 of a sequence selected from SEQ ID NOs: 48, 64, 80-82, 84-86, 182, 188, 194, 213, 220, 256, 262, 268, 287, and 294. The present invention includes a sequence that is essentially complementary to, or is about 75% to about 100%, about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, 95% to about 100%, about 98% to about 100%, about 100%, or 100% identical to, 25, at least 50, at least 75, at least 100, at least 150, at least 200, at least 300, at least 400, at least 500, at least 600, or at least 700 consecutive nucleotides.

[0046] In some embodiments, the polynucleotide comprises an RNA trigger sequence or an RNA trigger sequence reverse complement, or in some specific embodiments, a sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, about 100%, or exactly 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 48, 64, 80-82, 84-86, 182, 188, 194, 213, 220, 256, 262, 268, 287, and 294.

[0047] Some embodiments relate to polynucleotides comprising a sequence of about 95% to about 100% identity to a sequence selected from the group consisting of RNA trigger sequences or reverse complements of RNA trigger sequences. Some embodiments relate to polynucleotides comprising at least one segment of 18 or more contiguous nucleotides having a sequence of about 95% to about 100% identity to a portion of a sequence selected from the group consisting of RNA trigger sequences or reverse complements of RNA trigger sequences. In some embodiments, the contiguous nucleotides are at least 18, e.g., 18 to 24, or 18 to 28, or 20 to 30, or 20 to 50, or 20 to 100, or 50 to 100, or 50 to 500, or 100 to 250, or 100 to 500, or 200 to 1,000, or 500 to 700, or even more. In some embodiments, the contiguous nucleotides are more than 18, e.g., 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30, e.g., about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 110, about 120, about 130, or more than 140, e.g., about 150, about 160, about 170, about 180, about 190, about 200, or more than 200, e.g., about 250, about 260, about 270, about 280, about 290, or more than 30, e.g., about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 110, about 120, about 13 ... The number of consecutive nucleotides is 0, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, or more than 700. In some embodiments, the polynucleotide comprises at least one segment of at least 18, 19, 20, or 21 (as used throughout, reference to at least 18, 19, 20, 21, etc. is intended to mean that any of these lower limits of the group may be individualized) contiguous nucleotides having a sequence that is 100% identical to a fragment of equivalent length found in a sequence selected from the group consisting of RNA trigger sequences or RNA trigger sequence reverse complements.In some embodiments, the polynucleotide is selected from the group consisting of RNA trigger sequences and RNA trigger sequence reverse complements, or in some more particular embodiments, selected from the group consisting of SEQ ID NOs: 48, 64, 80-82, 84-86, 182, 188, 194, 213, 220, 256, 262, 268, 287, and 294, or in particular embodiments, comprises at least one segment of at least 200, 300, 400, 500, 600, or 700 contiguous nucleotides having a sequence at least 85% identical to a fragment of equivalent length found in the sequence of SEQ ID NO: 48.

[0048] In some embodiments, the polynucleotide is a double-stranded nucleic acid (e.g., dsRNA) having one strand comprising at least one segment of at least 18, 19, 20, 21, 22, 23, 24, 50, 75, 100, 150, 200, 250, 300, 400, 500, or 600, or 700 contiguous nucleotides having about 95%-100% identity to a target gene having a sequence selected from the group of target gene sequences or its DNA complement, or to a fragment of equivalent length of DNA of a gene or homolog thereof identified in Table 1 or Table 2. In certain specific embodiments, such a target gene is selected from the group consisting of FGP1, ELP3, SPT7, MAF1, MVD1, HEP1, SET1, FKPB12, or in more particular embodiments, comprises FGP1. When expressed as base pairs, such double-stranded nucleic acids contain at least one segment of at least 18, 19, 20, 21, 22, 23, 24, 50, 75, 100, 150, 200, 250, 300, 400, 500, or 600, or 700 contiguous perfectly matched base pairs corresponding to a target gene having a sequence selected from the target gene sequence group or its DNA complement, or a fragment of equivalent length of DNA of a gene identified in Table 1 or Table 2 or a homolog thereof. In some embodiments, each segment contained in the polynucleotide is of a length longer than that typical of naturally occurring small regulatory RNAs, e.g., each segment is at least about 30 contiguous nucleotides (or base pairs) in length. In some embodiments, the total length of the polynucleotide, or the length of each segment contained in the polynucleotide, is less than the total length of the target gene or DNA having a sequence selected from the target gene sequence group. In some embodiments, the total length of the polynucleotide is about 50 to about 750 nucleotides (for single-stranded polynucleotides) or base pairs (for double-stranded polynucleotides). In some embodiments, the polynucleotide is a dsRNA of about 200 to about 750 base pairs, such as a dsRNA of any of the lengths of the RNA trigger sequences disclosed in the figures and tables.In some embodiments, the dsRNA comprises one strand comprising a sequence selected from the group consisting of SEQ ID NOs: 48, 80-82, 182, 188, 194, 213, and 220. In some embodiments, the dsRNA comprises one strand comprising at least one segment of at least 200, 300, 400, 500, 600, or 700 contiguous nucleotides having a sequence at least 85% identical to a fragment of equivalent length found in a sequence selected from the group consisting of RNA trigger sequences and RNA trigger sequence reverse complements. In some embodiments, the dsRNA comprises a strand comprising at least one segment of at least 200, 300, 400, 500, 600, or 700 contiguous nucleotides that is at least 85% identical to a fragment of equivalent length found in a sequence selected from the group consisting of SEQ ID NOs: 48, 64, 80-82, 84-86, 182, 188, 194, 213, 220, 256, 262, 268, 287, and 294.

[0049] In some embodiments, the polynucleotide is designed to have complementarity to the mRNA encoded by the target gene. In some embodiments, the polynucleotide is a dsRNA. In some embodiments, the dsRNA comprises a first strand that binds to (e.g., is essentially complementary to) the mRNA encoded by the target gene, and a second strand that is complementary to the first strand. The dsRNA may comprise RNA strands that are the same length or different lengths. In some embodiments, the dsRNA comprises a first strand (e.g., an antisense strand) that is the same length as the second strand (e.g., a sense strand). In some embodiments, the dsRNA comprises a first strand (e.g., an antisense strand) that is a different length than the second strand (e.g., a sense strand). The first strand may be about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or more than 20% longer than the second strand. The first strand may be 1-5, 2-5, 2-10, 5-10, 5-15, 10-20, 15-20, or 20 more nucleotides longer than the second strand. dsRNA molecules may also be assembled from a single oligonucleotide in a stem-loop structure, where the self-complementary sense and antisense regions of the RNA molecule are linked by a stem comprising a nucleic acid-based or non-nucleic acid-based linker(s), as well as a circular single-stranded RNA with two or more loop structures, and the self-complementary sense and antisense strands, and the circular RNA may be processed either in vivo or in vitro to generate an active RNAi molecule capable of mediating RNAi. The RNAi molecule may include a 3' overhang at one end of the molecule, and the other end may be blunt-ended or may also have an overhang (5' or 3'). When the RNAi molecule includes an overhang at both ends of the molecule, the length of the overhang may be the same or different.

[0050] In some embodiments, the polynucleotide is designed to have complementarity to a region of an F. graminearum gene involved in the DON pathway, the downregulation of which causes both a reduction or elimination of DON production and an increase in mortality, a reduction in growth, or a reduction in reproductive capacity in F. graminearum. In another embodiment, the polynucleotide comprises one or more sequences described herein for a reduction in DON production in F. graminearum, and one or more sequences that, when transfected into or contacted with F. graminearum, cause mortality, a reduction in growth, a reduction in virulence or pathogenicity, or a reduction in reproduction / fertility (sporulation) in F. graminearum. Other embodiments target genes involved in the DON pathway in another Fusarium species, such as Fusarium culmorum, or other species of the Fusarium genus, that produce DON and / or contribute to Fusarium head blight in plants.

[0051] In some embodiments, the dsRNA comprises one strand comprising one or more nucleotide sequences that are at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, about 100%, or exactly 100% identical to a sequence selected from the RNA trigger sequence or the RNA trigger sequence reverse complement. In some embodiments, the dsRNA comprises at least one segment of 18, 19, 20, 21, 22, 23, 24, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, or 700 or more contiguous nucleotides that have about 95% to about 100% identity to the equivalent portion of a sequence selected from the RNA trigger sequence group or the RNA trigger sequence reverse complement group. Such a dsRNA may further comprise a second strand that is complementary to the first strand. In some embodiments, the dsRNA comprises a first strand comprising a nucleotide sequence selected from an RNA trigger sequence and a second strand selected from the corresponding reverse complement of the RNA trigger sequence. Particular embodiments include those in which the polynucleotide is a dsRNA comprising a first strand comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 48, 64, 80-82, 84-86, 182, 188, 194, 213, 220, 256, 262, 268, 287, and 294, and a second strand complementary to the first strand.

[0052] III. Polynucleotide Length. The RNAi molecules targeted to the target genes provided herein may vary in length. In some embodiments, long RNA (e.g., dsRNA or ssRNA) molecules are applied (e.g., to plants), but it should be understood that after entering the cells of the target fungus, e.g., F. graminearum, the dsRNA is cleaved by the Dicer enzyme into shorter double-stranded RNA fragments, e.g., having a length of 15-25 nucleotides. Thus, the RNAi molecules of the present disclosure may be delivered as, e.g., 15-25 nucleotide fragments, or they may be delivered as longer double-stranded nucleic acids (e.g., at least 100 nucleotides).

[0053] The total length of the polynucleotide of the invention may be 18 or more contiguous nucleotides, in addition to contiguous nucleotides having about 75% to about 100% identity to a fragment of equivalent length of the DNA of the target gene having a sequence selected from the group consisting of the target gene or target gene sequences or their DNA complements identified in Table 1 or Table 2, or to the RNA transcribed therefrom. Similarly, the polynucleotide of the invention may include one or more sequences that are about 75% to about 100% identical to 18 or more contiguous nucleotides of a sequence selected from the group consisting of the RNA trigger sequence or the RNA trigger sequence reverse complement, in addition to additional unrelated sequences. In other words, the total length of the polynucleotide may be longer than the length of the section or segment of the polynucleotide designed to suppress one or more target genes.

[0054] For example, a polynucleotide may have nucleotides adjacent to an "active" segment that represses a target gene (e.g., an "active" segment may be a sequence that is essentially complementary to a segment of the target gene or mRNA transcribed therefrom, or may be a sequence selected from a group of RNA trigger sequences or a group of RNA trigger sequence reverse complements), or may include "spacer" nucleotides between active segments, or may have additional nucleotides at the 5' end, or the 3' end, or at both the 5' and 3' ends. In one embodiment, a polynucleotide may include additional nucleotides that are not specifically related (have a non-complementary or non-identical sequence) to the sequences disclosed herein for controlling powdery mildew. For example, such polynucleotides may contain nucleotides to provide a stabilizing secondary structure or for convenience of cloning or manufacturing. In one embodiment, a polynucleotide may include additional nucleotides located immediately adjacent to an active segment. In one embodiment, a polynucleotide includes one such segment with an additional 5'G, or an additional 3'C, or both, adjacent to the segment. In another embodiment, the polynucleotide is a double-stranded RNA that includes additional nucleotides to form one or more overhangs, e.g., a dsRNA that includes two deoxyribonucleotides to form a 3' overhang. In other embodiments, the polynucleotide may include one or more active segments listed herein as well as additional segments that are active against other target genes of F. graminearum, or against other fungi of the genus Fusarium, such as other Fusarium species involved in the Fusarium species complex that produce DON or cause Fusarium head blight in plants.

[0055] Thus, in various embodiments, the nucleotide sequence of the entire polynucleotide is not 100% identical or complementary to an RNA trigger sequence or an RNA trigger sequence reverse complement, and is not 100% identical or complementary to a sequence of contiguous nucleotides in the DNA of a target gene having a sequence selected from the group consisting of a target gene or a homolog thereof identified in Table 1 or Table 2, or a target gene sequence or a DNA complement thereof. For example, in some embodiments, the polynucleotide comprises at least two segments of 21 contiguous nucleotides each having a sequence of 100% identity to a fragment of DNA having a sequence selected from the group consisting of a target gene sequence or a DNA complement thereof, where (1) the at least two segments are separated by one or more spacer nucleotides, or (2) the at least two segments are arranged in an order different from the order in which the corresponding fragment occurs in a DNA having a sequence selected from the group consisting of a target gene sequence or a DNA complement thereof.

[0056] Some embodiments relate to polynucleotides designed to regulate expression by inducing downregulation or repression of F. graminearum target genes. In some embodiments, polynucleotides are designed to have a nucleotide sequence that is essentially identical or essentially complementary to a nucleotide sequence of an F. graminearum target gene or cDNA (e.g., a group of target gene sequences) or a sequence of an RNA transcribed from an F. graminearum target gene, which may be a coding sequence or a non-coding sequence. These effective polynucleotide molecules that regulate expression may be referred to herein as "polynucleotides," "polynucleotide triggers," "triggers," or "triggers." Examples of such embodiments include polynucleotides that include one or more sequences selected from the group of RNA trigger sequences and the group of RNA trigger sequence reverse complements. Further examples include polynucleotides that include at least one segment of 18 or more contiguous nucleotides having a sequence of about 95% to about 100% identity to a portion of a sequence selected from the group of RNA trigger sequences or the group of RNA trigger sequence reverse complements.

[0057] Any size of effective polynucleotide can be used alone or in combination in the various methods and compositions described herein. In some embodiments, a single polynucleotide trigger is used to generate a composition (e.g., a composition for topical application, or a recombinant DNA construct useful for generating transgenic plants). In other embodiments, a mixture or pool of different polynucleotide triggers is used, in which case the polynucleotide trigger can be for a single target gene or for multiple target genes.

[0058] IV. Permitted Mismatches "Essentially identical" or "essentially complementary" as used herein means that a polynucleotide (or at least one strand of a double-stranded polynucleotide) has sufficient identity or complementarity to a target gene or RNA transcribed from a target gene (e.g., a transcript) to suppress expression of the target gene (e.g., affecting a reduction in the level or activity of the target gene transcript and / or the encoded protein). The polynucleotides described herein do not need to have 100 percent identity or complementarity to a target gene or RNA transcribed from a target gene to suppress expression of a target gene (e.g., affecting a reduction in the level or activity of the target gene transcript or encoded protein, or to provide control of DON production by F. graminearum). In some embodiments, a polynucleotide or a portion thereof is designed to be essentially identical or essentially complementary to a sequence of at least 18 or 19 contiguous nucleotides in either the target gene or the RNA transcribed from the target gene. In some embodiments, a polynucleotide or a portion thereof is designed to be 100% identical or 100% complementary to one or more sequences of 21 contiguous nucleotides in either the target gene or the RNA transcribed from the target gene. In certain embodiments, an "essentially identical" polynucleotide has 100 percent sequence identity or at least about 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent sequence identity when compared to a sequence of 18 or more contiguous nucleotides in either the endogenous target gene or the RNA transcribed from the target gene. In certain embodiments, an "essentially complementary" polynucleotide has 100 percent sequence complementarity or at least about 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent sequence complementarity when compared to a sequence of 18 or more contiguous nucleotides in either the endogenous target gene or the RNA transcribed from the target gene.

[0059] Sequence identity: As used herein in the context of two polynucleotides or polypeptides, the term "sequence identity" or "identity" refers to the residues in the sequences of the two molecules that are the same when aligned for maximum correspondence over a specified comparison window.

[0060] The percentage of identity is calculated by determining the number of positions where the same nucleotide or amino acid residue occurs in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. A sequence that is identical at all positions compared to a reference sequence is said to be 100% identical to the reference sequence, and vice versa. The percent identity of two nucleotide sequences can be determined by comparing two optimally aligned sequences of molecules (e.g., nucleic acid sequences or polypeptide sequences) over a comparison window, and the portion of the sequence in the comparison window may contain additions or deletions (i.e., gaps) compared to the reference sequence (which does not contain additions or deletions) due to optimal alignment of the two sequences. Optimal alignment for comparing two or more sequences can be performed using local or global alignment via various available computer programs. The algorithm of Smith TF and Waterman MS (1981) Identification of common molecular subsequences J. Mol. Biol. 147(1):195-7 PubMed:7265238 DOI:10.1016 / 0022-2836(81)90087-5 is the preferred local alignment strategy and is used by tools such as EMBOSS Water (https: / / www.ebi.ac.uk / Tools / psa / emboss_water / ).The algorithm of Needleman SB and Wunsch CD (1970) A general method applicable to the search for similarities in the amino acid sequence of two proteins J. Mol. Biol. 48 (3): 443-53 PubMed: 5420325 DOI: 10.1016 / 0022-2836 (70) 90057-4 is a preferred global alignment strategy and is used by tools such as EMBOSS Needle (https: / / www.ebi.ac.uk / Tools / psa / emboss_needle / ). Depending on the sequences to be compared and the associated parameters, local or global alignment strategies may be more likely to find an optimal alignment, but both strategies can be used to ascertain the optimal alignment that gives the most accurate percent identity.

[0061] The term "about" with respect to numerical values ​​of sequence length means the stated value with a + / - variance of up to 1-5%. For example, about 30 contiguous nucleotides means a range of 27-33 contiguous nucleotides, or any range therebetween. The term "about" with respect to numerical values ​​of sequence identity percentage means the stated percentage value with a + / - variance of up to 1-3 percent, rounded to the nearest integer. For example, about 90% sequence identity means a range of 87-93%. However, the percentage of sequence identity cannot exceed 100 percent. Thus, about 98% sequence identity means a range of 95-100%.

[0062] Polynucleotides containing mismatches to target genes or transcripts may be used in certain embodiments of the compositions and methods described herein. The variants provided herein contain randomly placed mutations, in some embodiments, with four nucleotides (A, U, G, C) selected with approximately equal probability for a given mutation. In some embodiments, these mutations may be distributed over a small region of the sequence or may be distributed broadly over the length of the sequence. In some embodiments, the polynucleotide comprises at least 18 or at least 19 or at least 21 contiguous nucleotides that are essentially identical or essentially complementary to a segment of equivalent length in the target gene or transcript of the target gene. In certain embodiments, a polynucleotide of 18, 19, 20, or 21 or more contiguous nucleotides that are essentially identical or essentially complementary to a segment of equivalent length in the target gene or transcript of the target gene may have one or two mismatches to the target gene or transcript (i.e., one or two mismatches between the 21 contiguous nucleotides of the polynucleotide and the segment of equivalent length in the target gene or transcript of the target gene). In certain embodiments, a polynucleotide of about 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700 or more nucleotides containing a stretch of 18, 19, 20, or 21 or more contiguous nucleotides of identity or complementarity to a segment of equivalent length in a target gene or a transcript of a target gene, which may have one or more mismatches to the target gene or transcript.

[0063] In designing a polynucleotide with mismatches to an endogenous target gene or to an RNA transcribed from a target gene, certain types of mismatches and mismatches at certain positions that are more likely to be tolerated may be used. In certain embodiments, mismatches formed between adenine and cytosine or between guanosine and uracil residues are used, as described in Du et al. (2005) Nucleic Acids Res., 33:1671-1677. In some embodiments, mismatches in the 19 base pair overlap region are located at the low tolerated positions 5, 7, 8, or 11 (from the 5' end of the 19 nucleotide target), the moderately tolerated positions 3, 4, and 12-17 (from the 5' end of the 19 nucleotide target), and / or the highly tolerated positions at either end of the complementary region, i.e., positions 1, 2, 18, and 19 (from the 5' end of the 19 nucleotide target), as described in Du et al. (2005) Nucleic Acids Res., 33:1671-1677. The tolerated mismatches can be determined empirically in routine assays.

[0064] V. Embedding silencing elements in neutral sequences In some embodiments, the silencing element that corresponds to the target gene and contains the sequence responsible for the observed suppression of the target gene is embedded in a "neutral" sequence, i.e., inserted with additional nucleotides that do not have sequence identity or complementarity to the target gene. Neutral sequences may be desirable, for example, to increase the overall length of the polynucleotide or to impart desired properties, such as increased binding to the silencing complex. For example, it may be desirable for the polynucleotide to be of a certain size for reasons of stability, cost-effectiveness in production, or effective biological activity, such as silencing efficiency. In some embodiments, neutral sequences are also useful in forming preferred secondary structures, such as loops in hairpin triggers, or as spacers between trigger regions.

[0065] Thus, in one embodiment, a 21 base pair dsRNA silencing element corresponding to a target gene or a homolog thereof identified in Table 1 or Table 2, or a target gene having a DNA sequence selected from a group of target gene sequences found to provide control of DON production in F. graminearum, is embedded in an additional 39 base pair neutral sequence, thus forming a polynucleotide of about 60 base pairs. In some embodiments, the dsRNA trigger comprises a neutral sequence of about 60 to about 500, or 100 to about 450 base pairs, within which is embedded at least one segment of 21 contiguous nucleotides having a sequence of 100% identity or 100% complementarity to a fragment of equivalent length of a target gene having a sequence selected from a group of target gene sequences. In another embodiment, a single 21 base pair silencing element having a sequence of 100% identity or 100% complementarity to a comparable length fragment of the target gene is found to be effective when embedded in a larger section of neutral sequence, for example, where the total polynucleotide length is about 60 to about 300 base pairs. In embodiments where the polynucleotide includes a region of neutral sequence, the polynucleotide will have a relatively low overall sequence identity compared to the target gene. For example, a dsRNA having an overall length of 210 base pairs containing a single 21 base pair trigger (of 100% identity or complementarity to a 21 nucleotide fragment of the target gene) embedded in an additional 189 base pairs of neutral sequence will have an overall sequence identity with the target gene of about 10%.

[0066] VI. Related Technologies The polynucleotide and nucleic acid molecule embodiments described herein may include additional elements such as promoters, transcription initiation elements, transcription elongation elements, transcription termination elements, small RNA recognition sites, aptamers or ribozymes, additional and additional expression cassettes for expressing coding sequences (e.g., for expressing a transgene such as a bactericidal protein or a selectable marker), or non-coding sequences (e.g., for expressing additional inhibitory elements). For example, aspects of the invention provide recombinant DNA constructs comprising a heterologous promoter having a transcription initiation sequence operably linked to DNA comprising at least one segment of 18 or more contiguous nucleotides having a sequence of about 95% to about 100% identity to a fragment of equivalent length of DNA having a sequence selected from a group of target gene sequences or a DNA complement thereof. Another aspect of the invention provides recombinant DNA constructs comprising a heterologous promoter having a transcription initiation sequence operably linked to DNA encoding an RNA hairpin having an antisense region having a sequence or a fragment of a sequence selected from a group of RNA trigger sequences and a group of RNA trigger sequence reverse complements. In another embodiment, a recombinant DNA construct comprising (a) an RNA silencing element for suppressing a target gene selected from a group of target gene sequences, and (b) a promoter operably linked to DNA encoding an aptamer is stably integrated into the genome of a plant, from which an RNA transcript comprising an RNA aptamer and an RNA silencing element is expressed in the plant's cells, and the aptamer serves to guide the RNA silencing element to a desired location in the cell. In another embodiment, the inclusion of one or more recognition sites for binding and cleavage by small RNA (e.g., miRNA or siRNA that are expressed only in specific cells or tissues) allows for a more precise expression pattern in the plant, and the expression of the recombinant DNA construct is suppressed when the small RNA is expressed. Such additional elements are described below.

[0067] VII. Control of DON production by Fusarium Graminearum by contact with polynucleotides. Provided herein are methods for reducing or eliminating DON production by F. graminearum or other fungal pathogens of the genus Fusarium that produce DON and / or contribute to Fusarium head blight. Such methods include contacting F. graminearum with any of the polynucleotides and other compositions described herein. Some embodiments relate to methods of reducing or eliminating DON production by F. graminearum on a plant by contacting the plant with, for example, any of the polynucleotides or other compositions described in Section II or Section VIII or elsewhere herein. Some embodiments relate to methods of reducing or eliminating DON production by F. graminearum on a plant by contacting F. graminearum with a polynucleotide that includes at least one segment of 18 or more contiguous nucleotides having about 95% to about 100% identity or complementarity to a corresponding fragment of DNA of a target gene selected from the group consisting of a gene identified in Table 1 or Table 2, or another gene involved in the production of DON by Fusarium. In one embodiment, a method of reducing or eliminating DON production by F. graminearum on a plant by contacting F. graminearum with a polynucleotide comprising at least 18 contiguous nucleotides having 100% identity to a corresponding fragment of a target gene having a DNA sequence selected from the group consisting of SEQ ID NO: 16, 68-70, 108, 114, 120, 139, and 146, or DNA complements thereof. In a particular embodiment, the target gene is selected from the group consisting of FGP1, ELP3, SPT7, MAF1, MVD1, HEP1, SET1, FKPB12, or homologs thereof of F. graminearum, or in a more particular embodiment, FGP1 or a homolog thereof.

[0068] In some embodiments, the polynucleotide is double-stranded RNA. In some embodiments, the polynucleotide (e.g., double-stranded RNA) is chemically or enzymatically synthesized or produced by expression in a microorganism or by expression in a plant cell. Embodiments include those in which the polynucleotide is a dsRNA comprising a strand having a sequence selected from the RNA trigger sequence group or the RNA trigger sequence reverse complement group. Embodiments further include those in which the polynucleotide comprises at least one segment of 18 or more contiguous nucleotides having a sequence of about 95% to about 100% identity to a portion of a sequence selected from the RNA trigger sequence group or the RNA trigger sequence reverse complement group. The polynucleotides for use in the methods may be designed against multiple target genes. Related aspects of the invention include isolated polynucleotides for use in the methods. Particular embodiments include those in which the polynucleotide is a dsRNA comprising the sequence of SEQ ID NOs: 48, 64, 80-82, 84-86, 182, 188, 194, 213, 220, 256, 262, 268, 287, and 294, or a complement thereof.

[0069] In some embodiments, the contiguous nucleotides have a sequence that is about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to a fragment of equivalent length of DNA of a target gene having a sequence selected from a target gene sequence group or a DNA complement thereof identified in Table 1 or Table 2. In some embodiments, the contiguous nucleotides are exactly (100%) identical to a fragment of equivalent length of DNA of a target gene having a sequence selected from a target gene sequence group or a DNA complement thereof identified in Table 1 or Table 2. In some embodiments, the polynucleotide has an overall sequence that is about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to a fragment of equivalent length of DNA of a target gene having a sequence selected from a target gene sequence group or a DNA complement thereof identified in Table 1 or Table 2. In some embodiments, the target gene is selected from the group consisting of FGP1, ELP3, SPT7, MAF1, MVD1, HEP1, SET1, FKPB12, or in more particular embodiments, comprises FGP1.

[0070] In one embodiment, the polynucleotide comprises at least one segment of 21 contiguous nucleotides that has 100% identity to a corresponding fragment of a target gene having a DNA sequence selected from the group consisting of SEQ ID NOs: 16, 68-70, 108, 114, 120, 139, and 146, or a DNA complement thereof. In some embodiments, the polynucleotide comprises "neutral" sequences (sequences that have no sequence identity or complementarity to the target gene) in addition to one or more segments of 21 contiguous nucleotides that have 100% identity to a corresponding fragment of the target gene, such that the polynucleotide as a whole has much less overall sequence identity to the target gene.

[0071] In some embodiments, the polynucleotides of use in the methods are provided as isolated DNA or RNA fragments. In some embodiments, the polynucleotides of use in the methods are not part of an expression construct and lack additional elements such as promoter or terminator sequences). Such polynucleotides may be relatively short, such as single- or double-stranded polynucleotides of about 18 to about 300, or about 50 to about 750 nucleotides (for single-stranded polynucleotides), or about 18 to about 300, or about 50 to about 750 base pairs (for double-stranded polynucleotides). In some embodiments, the polynucleotide is a dsRNA of about 100 to about 750 base pairs, such as a dsRNA of any of the lengths of the dsRNA triggers of SEQ ID NOs: 48, 64, 80-82, 84-86, 182, 188, 194, 213, 220, 256, 262, 268, 287, and 294. Alternatively, the polynucleotide may be provided as part of a more complex construct, such as a recombinant expression construct, or may be included in a recombinant vector, such as a recombinant plant virus vector or a recombinant baculovirus vector. In some embodiments, such recombinant expression constructs or vectors are designed to include additional elements, such as an expression cassette for expressing a gene of interest (e.g., a bactericidal protein).

[0072] Some embodiments relate to methods of reducing or eliminating DON production by F. graminearum on a plant by contacting the F. graminearum with a polynucleotide comprising at least one segment of 18 or more contiguous nucleotides that is essentially identical or complementary to a fragment of equivalent length of DNA of a target gene selected from the group consisting of the genes identified in Table 1 or Table 2. In some embodiments, the polynucleotide comprises dsRNA having a strand with a sequence selected from the group consisting of RNA trigger sequences. In some embodiments, the invention provides methods for reducing or eliminating DON production by F. graminearum on a plant by contacting the F. graminearum with an effective amount of a solution comprising double-stranded RNA from the group of RNA trigger sequences, the solution further comprising an organosilicone surfactant.

[0073] In various embodiments of the method, contacting includes application of a suitable composition comprising any of the polynucleotides described herein (e.g., a polynucleotide described in Section II, a dsRNA described in Section VIII, or a composition described in Section IX or elsewhere herein) to the surface of a plant that is infected or capable of being infected with F. graminearum, such compositions may be provided, for example, as a solid, liquid (including homogenous mixtures such as soluble liquid concentrates, and non-homogeneous mixtures such as suspensions, colloids, micelles, and emulsions), powder, suspension, emulsion, spray, encapsulated or microencapsulated formulation, in or on microbeads or other carrier particles, in a film or coating, or on or within a matrix, or as a leaf, seed, root, or stem treatment. Compositions of pesticide formulations, including dsRNA pesticides, useful for facilitating application of dsRNA to plants for the purpose of contacting pests or pathogens are known in the art, and any suitable formulation may be used with the polynucleotides of the invention. In one embodiment, the surface is a plant leaf, head, stem, panicle, flower, or fruit. In one such embodiment, application may be accomplished by spraying the leaves, heads, stems, ears, flowers, or fruits of the plant. The contacting may also be in the form of a seed treatment. Suitable binders, inert carriers, surfactants, etc. may optionally be included in the composition as known to those skilled in the art of pesticide and seed treatment formulation. In some embodiments, the contacting comprises providing a polynucleotide in a composition that further comprises one or more carrier agents and / or one or more surfactants (e.g., organosilicon, organosilicon surfactants), non-polynucleotide fungicides, polynucleotide herbicide molecules, polynucleotide insecticides, non-polynucleotide insecticides, non-polynucleotide herbicide molecules, non-polynucleotide pesticides, polynucleotide pesticides, safeners, and pathogen growth regulators. In one embodiment, the contacting comprises providing a polynucleotide in a composition that can be transfected into or otherwise internally absorbed by fungi of the genus Fusarium.

[0074] VIII. Double-stranded RNA molecules Another aspect of the invention provides double-stranded RNA molecules that, when transfected into or contacted with the fungal pathogen, reduce or eliminate DON production by one or more fungal pathogens of the genus Fusarium (e.g., F. graminearum) on a plant. Such dsRNA molecules comprise the nucleotide sequence of any of the polynucleotides described in Section II above or elsewhere herein as useful for controlling DON production by F. graminearum. In another embodiment, the dsRNA may further comprise one or more sequences that, when transfected into or contacted with F. graminearum, cause death, reduced growth, reduced virulence or pathogenicity, or reduced reproduction / fertility (sporulation) in F. graminearum. Certain embodiments of the present invention provide double-stranded RNA molecules that, when transfected or contacted with F. graminearum, cause the reduction or elimination of DON production by F. graminearum on a plant, the double-stranded RNA molecule comprising at least one segment of 18 or more contiguous nucleotides that is essentially identical to or essentially complementary to a target gene identified in Table 1 or Table 2, or a segment of equivalent length of a target gene having a sequence selected from a group of target gene sequences. In certain embodiments, the target gene is selected from the group consisting of FGP1, ELP3, SPT7, MAF1, MVD1, HEP1, SET1, FKPB12, or in more particular embodiments, FGP1. In some embodiments, the dsRNA comprises a first strand comprising one or more sequences selected from a group of RNA trigger sequences, or a group of RNA trigger sequence reverse complements.In some embodiments, the dsRNA is selected from the group consisting of an RNA trigger sequence or an RNA trigger sequence reverse complement, or in certain embodiments, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, or at least 60 of a sequence selected from SEQ ID NOs: 48, 64, 80-82, 84-86, 182, 188, 194, 213, 220, 256, 262, 268, 287, and 294. The first strand comprises a sequence that is essentially complementary to, or about 75% to about 100%, about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, 95% to about 100%, about 98% to about 100%, about 100%, or 100% identical to, at least 50, at least 75, at least 100, at least 150, at least 200, at least 300, at least 400, at least 500, at least 600, or at least 700 contiguous nucleotides. In some embodiments, the dsRNA comprises at least one segment of 18 or more contiguous nucleotides having about 95% to about 100% identity to a portion of a sequence selected from the group consisting of RNA trigger sequences or RNA trigger sequence reverse complements. In some embodiments, the dsRNA comprises a first strand comprising a sequence at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, about 100%, or exactly 100% identical to a sequence selected from an RNA trigger sequence or an RNA trigger sequence reverse complement. In some embodiments, the dsRNA comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 48, 64, 80-82, 84-86, 182, 188, 194, 213, 220, 256, 262, 268, 287, and 294. In some embodiments, the dsRNA further comprises a second strand complementary to the first strand. In some embodiments, the dsRNA comprises a first strand comprising a nucleotide sequence selected from an RNA trigger sequence and further comprises a second strand comprising a sequence selected from the corresponding RNA trigger sequence reverse complement.In some embodiments, the dsRNA comprises a first strand comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 48, 80-82, 182, 188, 194, 213, and 220, and further comprises a second strand comprising a sequence selected from the corresponding complementary sequence selected from the group consisting of SEQ ID NOs: 48, 64, 80-82, 84-86, 182, 188, 194, 213, 220, 256, 262, 268, 287, and 294.

[0075] The total length of one strand of the dsRNA may be longer than or equal to 18 consecutive nucleotides and may include nucleotides in addition to consecutive nucleotides having a sequence of about 95% to about 100% of a sequence selected from the group of RNA trigger sequences or the group of RNA trigger sequence reverse complements, or a portion of a sequence selected from the group consisting of SEQ ID NOs: 48, 64, 80-82, 84-86, 182, 188, 194, 213, 220, 256, 262, 268, 287, and 294. A dsRNA containing a nucleotide sequence selected from the group of RNA trigger sequences and the group of RNA trigger sequence reverse complements may include nucleotides in addition to the nucleotides of a sequence selected from the group of RNA trigger sequences and the group of RNA trigger sequence reverse complements. In other words, the total length of the dsRNA strand may be longer than the length of a sequence or a portion of a sequence selected from the group of RNA trigger sequences or the group of RNA trigger sequence reverse complements. For example, the dsRNA may have nucleotides adjacent to the "active" segments that suppress the target gene, or may include "spacer" nucleotides between the active segments, or may have additional nucleotides at the 5' end, or the 3' end, or at both the 5' and 3' ends. In one embodiment, the dsRNA may include additional nucleotides that are not specifically related (have non-complementary or non-identical sequences) to the target gene targeted by a given trigger, such as nucleotides that provide a stabilizing secondary structure or for convenience of cloning or manufacturing. In one embodiment, the dsRNA may include additional nucleotides located immediately adjacent to a sequence or a portion of a sequence selected from the RNA trigger sequence group or the RNA trigger sequence reverse complement group. In one embodiment, the dsRNA includes one such segment with an additional 5'G or an additional 3'C, or both, adjacent to the segment. In another embodiment, the dsRNA further includes additional nucleotides to form an overhang, such as two deoxyribonucleotides to form a 3' overhang. Thus, in various embodiments, the nucleotide sequence of the entire dsRNA is not 100% identical or complementary to the RNA trigger sequence or the RNA trigger sequence reverse complement.For example, in some embodiments, the dsRNA comprises at least two segments of 21 contiguous nucleotides each having 100% sequence identity to a portion of a sequence selected from an RNA trigger sequence or an RNA trigger sequence reverse complement, and (1) the at least two segments are separated by one or more spacer nucleotides, or (2) the at least two segments are arranged in an order different from the order in which the corresponding fragments occur in the target gene.

[0076] In some embodiments, the double-stranded RNA molecule is about 50 to about 750 base pairs in length. In some embodiments, the double-stranded RNA molecule comprises a plurality of segments of 18 or more contiguous nucleotides that are essentially identical or essentially complementary to a segment of equivalent length of a target gene having a sequence selected from a target gene sequence group or a homolog thereof identified in Table 1 or Table 2, and optionally comprises at least a second segment of 18 or more contiguous nucleotides that are essentially identical or essentially complementary to a segment of equivalent length of a target gene having a sequence selected from a second target gene having a sequence selected from a target gene sequence group or a homolog thereof identified in Table 1 or Table 2. In some embodiments, the double-stranded RNA molecule comprises a plurality of segments of 18 or more contiguous nucleotides that are essentially identical or essentially complementary to a segment of equivalent length of a target gene, the segments being derived from different regions of the target gene (e.g., the segments may correspond to different exon regions of the target gene, and "spacer" nucleotides that do not correspond to the target gene may optionally be used between or adjacent to the segments) or derived from different target genes. In some embodiments, the double-stranded RNA molecule comprises a plurality of segments of 18 or more contiguous nucleotides that are essentially identical or essentially complementary to a segment of equivalent length of a target gene or a target gene or a group of target gene sequences identified in Table 1 or Table 2, the segments being derived from different regions of the target gene and arranged in the double-stranded RNA molecule in an order different from the order in which the segments naturally occur in the target gene. In some embodiments, the double-stranded RNA molecule comprises a plurality of segments of 18 contiguous nucleotides each having a sequence that is 100% identical or 100% complementary to a segment of equivalent length of a target gene or a target gene or a group of target gene sequences identified in Table 1 or Table 2, the segments being derived from different regions of the target gene and arranged in the double-stranded RNA molecule in an order different from the order in which the segments naturally occur in the target gene.In some embodiments, the double-stranded RNA molecule comprises one strand that comprises a sequence selected from the group consisting of RNA trigger sequences or their complements.

[0077] The double-stranded RNA molecule may be applied topically to plants to reduce or eliminate DON production by F. graminearum or other fungal pathogens of the genus Fusarium that produce DON and / or contribute to Fusarium head blight. The double-stranded RNA molecule may be provided in a form suitable for transfection or direct contact with F. graminearum, for example, in the form of a spray or powder. Other methods and suitable compositions for providing the double-stranded RNA molecule are similar to those described in the preceding paragraphs for other aspects of the invention.

[0078] Some embodiments relate to tank mixes comprising one or more polynucleotides and water or other solvents, optionally including organosilicone surfactants. Compositions suitable for topical application of dsRNA and other polynucleotides to plants to protect the plants from pests or pathogens are well known in the art. Such compositions include any of the compositions described in PCT / US / 2022 / 027816. Embodiments include tank mix formulations of polynucleotides and optionally at least one pesticide. Embodiments of such compositions include those in which one or more polynucleotides are provided to a living or dead microorganism, such as a bacteria or fungus or yeast cell, or provided as a microbial fermentation product, or provided to a living or dead plant cell, or provided as a synthetic recombinant polynucleotide. In one embodiment, the composition comprises a non-pathogenic strain of a microorganism containing a polynucleotide as described herein, and ingestion of the microorganism results in reduced or eliminated DON production by F. graminearum and / or results in suppression of growth, reduced virulence or pathogenicity, or reduced reproduction / fertility (sporulation), or death of F. graminearum. Non-limiting examples of suitable microorganisms include E. coli, B. thuringiensis, Pseudomonas sp., Photorhabdus sp., Xenorhabdus sp., Serratia entomophila and related Serratia sp., B. sphaericus, B. cereus, B. laterosporus, B. popilliae, Clostridium bifermentans and other Clostridium species, or other spore-forming Gram-positive bacteria. In one embodiment, the composition comprises a plant viral vector comprising a polynucleotide as described herein. Infection of a plant treated with the plant viral vector by F. graminearum results in reduced growth, mortality, reduced virulence or pathogenicity, or reduced reproduction / fertility (sporulation) of F. graminearum. In one embodiment, the composition comprises a baculoviral vector comprising a polynucleotide as described herein.Ingestion of the vector results in the reduction or elimination of DON production by F. graminearum, and / or the inhibition of growth, death, or reduced virulence or pathogenicity, or reduced reproduction / fertility (sporulation) of F. graminearum. In one embodiment, the polynucleotides described herein are encapsulated in a synthetic matrix, such as a polymer, or bound to a particle and applied topically to the surface of the plant. Infection of the topically treated plant with F. graminearum results in the reduction or elimination of DON production by F. graminearum, which is inhibited in growth, death, or reduced virulence or pathogenicity, or reduced reproduction / fertility (sporulation). In one embodiment, the polynucleotides described herein are provided in the form of a plant cell (e.g., a transgenic plant cell of the invention) expressing the polynucleotide, and infection of the plant's cells or the contents of the plant's cells with F. graminearum results in the reduction or elimination of DON production by F. graminearum.

[0079] In some embodiments, one or more polynucleotides described herein are provided with suitable stickers and wetting agents necessary for efficient foliar coverage, as well as UV protectants to protect polynucleotides such as dsRNA from UV damage. In some embodiments, one or more polynucleotides described herein are provided with carrier agents, surfactants, organosilicones, organosilicon surfactants, non-polynucleotide fungicides, polynucleotide herbicidal molecules, non-polynucleotide herbicidal molecules, non-polynucleotide pesticides, polynucleotide pesticides, non-polynucleotide insecticides, safeners, and pathogen growth regulators. In some embodiments, the composition further comprises at least one pesticide or fungicide.

[0080] Such compositions are applied in any convenient manner, for example, by spraying or dusting directly onto F. graminearum, or by spraying or dusting onto plants (including, for example, the leaves, stems, or heads of the plants) or the environment in which control of DON production by F. graminearum is desired, or by applying a coating to the surface of the plants, or by applying a coating to seeds in preparation for planting, or by applying to the soil around the roots of plants in which control of DON production by F. graminearum is desired.

[0081] An effective amount of the polynucleotides described herein is an amount sufficient to reduce or eliminate DON production by fungal pathogens of the Fusarium genus, e.g., F. graminearum, and the determination of the effective amount of the polynucleotide is performed using routine assays. There is no upper limit to the concentration and dosage of the polynucleotide that may be useful in the methods and compositions provided herein, but generally lower effective concentrations and dosages are required for efficiency and economy. Non-limiting embodiments of effective amounts of the polynucleotide include a range of about 10 nanograms per milliliter to about 100 micrograms per milliliter of the polynucleotide in liquid form sprayed on the plant, or a range of about 10 milligrams per acre to about 100 grams per acre of the polynucleotide applied to a field of the plant. When the polynucleotides described herein are applied topically to the plant, the concentration can be adjusted to take into account the volume of the spray or treatment applied to the surface of the leaves or heads or other plant parts of the plant, such as petals, stems, fruits, anthers, pollen, leaves, heads, panicles, roots, or seeds. In one embodiment, a useful treatment for herbaceous plants using the 25mer polynucleotides described herein is about 1 nanomole (nmol) of polynucleotide per plant, e.g., about 0.05 to 1 nmol of polynucleotide per plant. Other embodiments of herbaceous plants include useful ranges of about 0.05 to about 100 nmol, or about 0.1 to about 20 nmol, or about 1 nmol to about 10 nmol of polynucleotide per plant. In certain embodiments, about 40 to about 50 nmol of ssDNA polynucleotide is applied. In certain embodiments, about 0.5 nmol to about 2 nmol of dsRNA is applied. In certain embodiments, a composition containing about 0.5 to about 2.0 milligrams per milliliter, or about 0.14 milligrams per milliliter of dsRNA or ssDNA (21mer) is applied. In certain embodiments, about 0.5 to about 1.5 milligrams per milliliter of a composition of dsRNA polynucleotides of the invention of about 50 to about 200 or more nucleotides is applied. In certain embodiments, about 1 nmol to about 5 nmol of a dsRNA of the invention is applied to the plant.In certain embodiments, polynucleotide compositions applied topically to plants contain at least one polynucleotide of the invention at a concentration of about 0.01 to about 10 milligrams per milliliter, or about 0.05 to about 2 milligrams per milliliter, or about 0.1 to about 2 milligrams per milliliter. In some embodiments, a concentration of about 5 g to about 100 g of polynucleotide active ingredient per hectare is applied, with very large plants, trees, or vines potentially requiring correspondingly larger amounts of polynucleotide. Lower concentrations may be used when using long dsRNA molecules of the invention that can be processed into multiple oligonucleotides (e.g., multiple triggers encoded by a single recombinant DNA molecule of the invention). Non-limiting examples of effective polynucleotide treatment regimes include treatments of about 0.1 to about 1 nmol of polynucleotide molecule per plant, or about 1 nmol to about 10 nmol of polynucleotide molecule per plant, or about 10 nmol to about 100 nmol of polynucleotide molecule per plant.

[0082] When the compositions and polynucleotides of the present invention are applied locally to plants, such as in the form of a spray, application can be made at any suitable stage of plant development. Exemplary applications to cereal crops can be made at heading (Feekes 10.1-10.5) and / or flowering (Feekes 10.5.1-10.5.3), and if further applications are used, they can be applied as late as Feekes 10.5.4.

[0083] In some embodiments, one or more polynucleotides are provided with a "transfer agent," which is an agent that allows the topically applied polynucleotide to enter the cells of an organism. Such a transfer agent may be incorporated as part of a composition comprising the polynucleotide described herein, or may be applied prior to, simultaneously with, or subsequent to application of the polynucleotide. In some embodiments, the transfer agent is an agent that improves uptake of the polynucleotide of the invention by F. graminearum. In some embodiments, the transfer agent is an agent that modifies the surface of a plant tissue, such as a seed, leaf, head, ear, stem, root, flower, or fruit, to allow the polynucleotide to penetrate into the plant cell. In some embodiments, the transfer agent allows passage of the polynucleotide through the epidermal wax barrier, stomata, and / or cell wall or membrane barrier into the plant cell.

[0084] Suitable transfer agents include agents that increase the permeability of the exterior of the organism or increase the permeability of the cells of the organism to the polynucleotide. Suitable transfer agents include chemical agents, or physical agents, or combinations thereof. Chemical agents for conditioning or transfer include (a) detergents, (b) organic solvents or aqueous solutions or mixtures of organic solvents, (c) oxidizing agents, (d) acids, (e) bases, (f) oils, (g) enzymes, or any combination thereof. In some embodiments, application of the polynucleotide and transfer agent optionally includes an incubation step, a neutralization step (e.g., to neutralize the acid, base, or oxidizing agent or to inactivate the enzyme), a rinsing step, or a combination thereof. Suitable transfer agents may be in the form of an emulsion, inverse emulsion, liposomes, or other micellar composition, or may be made into the polynucleotide in the form of an emulsion, inverse emulsion, liposomes, or other micellar composition. Embodiments of transfer agents include counterions or other molecules known to associate with nucleic acid molecules, e.g., inorganic ammonium ions, alkyl ammonium ions, lithium ions, polyamines such as spermine, spermidine, or putrescine, and other cations. Embodiments of transfer agents include organic solvents such as DMSO, DMF, pyridine, N-pyrrolidine, hexamethylphosphoramide, acetonitrile, dioxane, polypropylene glycol, or other solvents that are miscible with water or that dissolve phosphonucleotides in non-aqueous systems (such as those used in synthesis reactions). Embodiments of transfer agents include naturally derived or synthetic oils, with or without surfactants or emulsifiers, e.g., plant derived oils, crop oils (e.g., the 9 th These include those listed in the Compendium of Herbicide Adjuvants, such as those published at herbicide.adjuvants.com), paraffin oils, polyol fatty acid esters, or oils having short chain molecules modified with amides or polyamines such as polyethyleneimine or N-pyrrolidine.

[0085] The embodiment of the transfer agent includes organosilicone preparations. For example, suitable transfer agents are: an organosilicone formulation commercially available as SILWET L-77® brand surfactant with CAS number 27306-78-1 and EPA number CAL.REG.NO.5905-50073-AA, currently available from Momentive Performance Materials, Albany, NY; BREAK-THRU S 240 brand polyether modified polysiloxane (CASRN proprietary) surfactant, currently available from Goldschmidt Chemical Corporation, Hopewell, VA; BREAK-THRU S 279 end-capped polyether trisiloxane surfactant, whose components are listed in the following chemical inventories: EINECS, TSCA, ENCS, AICS, ECL, PICCS CHINA, NDSL; INDUCE brand auxiliary NMFC item 42652, class 60, currently available from Helena Chemical Company, Collierville, TN. FRANCHISE® with LECI-TECH® brand surfactants having CA REG No. 34704-50065, currently available from Loveland Products, Inc. Greely, Colo. One embodiment includes a composition comprising a polynucleotide and BREAK-thru 301. In one embodiment, the polynucleotide is combined with Silwet L-77, Break-thru 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9 ...4, 3.5, 3.7, 3.8, 3.9, 3.1, 3.2, 3.5, 3.6, 3.7, 3.9, 3.1, 3.3, 3.4, 3.5, 3.8, 3.9, 3.1, 3.2, 3.3, 3.5, 3.6, 3.7, 3.8, 3.9, 3.1, 3.2, 3.3, 3.4, 3.5, 3.8, 3.9, 3.1, 3.2, 3.4, 3.5, 3.9, 3.1, 3.3, 3.5, 3.6, 3.7, 3.8, 3.9, 3.1, 3.2, 3.3, 3.4, 3.5, 3.8, 3.9, 3.1, 3.2, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 3.1, 3.2, 3.3, 3.4, 3.5, 3.7, 3.8, 3. and a transfer agent comprising an organosilicone preparation such as S240, Break-thru S279, Induce, or Franchise.One embodiment includes a composition comprising a polynucleotide of the invention and a transfer agent comprising SILWET L-77®, BREAK-THRU S240, BREAK-THRU S279, Induce, or Franchise brand surfactant in the range of about 0.3 to about 1 weight percent (wt percent), or about 0.5 to about 1 weight percent (wt percent).

[0086] Organosilicone compounds useful as transfer agents for use in the present invention include, but are not limited to, compounds that include: (a) a covalently attached trisiloxane head group; (b) a covalently attached alkyl linker, including, but not limited to, an n-propyl linker; (c) a covalently attached polyglycol chain; and (d) an end group. Trisiloxane head groups of such organosilicone compounds include, but are not limited to, heptamethyltrisiloxane. Alkyl linkers can include, but are not limited to, an n-propyl linker. Polyglycol chains can include, but are not limited to, polyethylene glycol or polypropylene glycol. Polyglycol chains can include a mixture that provides an average chain length "n" of about "7.5". In certain embodiments, the average chain length "n" can vary from about 5 to about 14. End groups can include, but are not limited to, alkyl groups such as methyl groups. Organosilicone compounds useful as transfer agents include, but are not limited to, trisiloxane ethoxylate surfactants or polyalkylene oxide modified heptamethyltrisiloxane. An example of a transfer agent for use in the present invention is Compound I: [ka] (Compound I: polyalkylene oxide heptamethyltrisiloxane, average n=7.5). Organosilicon compounds useful as transfer agents are used as freshly made concentrations ranging, for example, from about 0.015 to about 2 weight percent (wt percent) (e.g., about 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.5 wt percent).

[0087] Embodiments of the transfer agent include one or more salts, such as ammonium chloride, tetrabutylphosphonium bromide, and ammonium sulfate, provided in or used with a composition comprising a polynucleotide. In some embodiments, the ammonium chloride, tetrabutylphosphonium bromide, and / or ammonium sulfate are used at a concentration of about 0.5% to about 5% (w / v), or about 1% to about 3% (w / v), or about 2% (w / v). In certain embodiments, the composition comprising a polynucleotide includes an ammonium salt at a concentration of 300 millimolar or greater. In certain embodiments, the composition comprising a polynucleotide includes an organosilicone transfer agent at a concentration of about 0.015 to about 2 weight percent (wt percent), and ammonium sulfate at a concentration of about 80 to about 1200 mM, or about 150 mM to about 600 mM.

[0088] An embodiment of the transfer agent comprises a phosphate salt. Phosphate salts useful in compositions comprising polynucleotides include, but are not limited to, calcium phosphate, magnesium phosphate, potassium phosphate, or sodium phosphate salt. In certain embodiments, compositions comprising polynucleotides comprise a phosphate salt at a concentration of at least about 5 millimolar, at least about 10 millimolar, or at least about 20 millimolar. In certain embodiments, compositions comprise polynucleotide phosphate salts in the range of about 1 mM to about 25 mM, or in the range of about 5 mM to about 25 mM. In certain embodiments, compositions comprise polynucleotide sodium phosphate salts at a concentration of at least about 5 millimolar, at least about 10 millimolar, or at least about 20 millimolar. In certain embodiments, compositions comprising polynucleotides comprise sodium phosphate salts at a concentration of about 5 millimolar, about 10 millimolar, or about 20 millimolar. In certain embodiments, compositions comprising polynucleotides comprise sodium phosphate salts in the range of about 1 mM to about 25 mM, or in the range of about 5 mM to about 25 mM. In certain embodiments, a composition comprising a polynucleotide comprises a sodium phosphate salt in the range of about 10 mM to about 160 mM, or in the range of about 20 mM to about 40 mM, hi certain embodiments, a composition comprising a polynucleotide comprises a sodium phosphate buffer at a pH of about 6.8.

[0089] The transfer agent embodiment includes a surfactant and / or an effective molecule contained therein. The surfactant and / or the effective molecule contained therein includes, but is not limited to, sodium or lithium salts of fatty acids (such as tallow or tallow amines or phospholipids) and organosilicone surfactants. In certain embodiments, the composition containing the polynucleotide is formulated with counterions or other molecules known to associate with nucleic acid molecules. Non-limiting examples include tetraalkylammonium ions, trialkylammonium ions, sulfonium ions, lithium ions, and polyamines such as polyethyleneimine, spermine, spermidine, or putrescine. In certain embodiments, compositions comprising a polynucleotide are formulated with a non-polynucleotide herbicide, for example, an auxin-like herbicide including glyphosate, dicamba, chloramben, and TBA, glufosinate, phenoxycarboxylic acid herbicides, pyridine carboxylic acid herbicides, quinoline carboxylic acid herbicides, pyrimidine carboxylic acid herbicides, and benazolin ethyl herbicides, sulfonylureas, imidazolinones, bromoxynil, dalapon, cyclohexandione, protoporphyrinogen oxidase inhibitors, and 4-hydroxyphenylpyruvate dioxygenase inhibiting herbicides.

[0090] IX. Compositions for controlling DON production by F. graminearum Another aspect of the invention provides a composition for reducing or eliminating DON production by F. graminearum comprising an effective amount of at least one RNA. Such RNA can be any of the RNAs described in Section II, Section VIII, or elsewhere herein. In one embodiment, the RNA has about 75% to about 100% identity, about 80% to about 100% identity, about 85% to about 100% identity, about 90% to about 100% identity, about 95% to about 100% identity, about 98% to about 100% identity, about 10 ... 100% identity or exactly 100% identity of at least one segment of 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, 25 or more, 30 or more, 50 or more, 75 or more, 100 or more, 125 or more, 150 or more, 200 or more, 250 or more, 300 or more, 400 or more, 500 or more, 600 or more, 700 or more, 800 or more, 900 or more, or 1,000 or more contiguous nucleotides. In one embodiment, the RNA comprises a nucleotide sequence that is essentially complementary to at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 50, at least 75, at least 100, at least 125, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, or at least 1,000 contiguous nucleotides of a target gene identified in Table 1 or Table 2 or a homolog thereof, or a target gene having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 16, 68-70, 108, 114, 120, 139, and 146 or a DNA complement thereof, or an RNA transcribed from such a target gene.

[0091] In some embodiments, the RNA comprises at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 50, at least 75, at least 100, at least 150, at least 200, at least 300, at least 400, or at least 500 consecutive nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 48, 64, 80-82, 84-86, 182, 188, 194, 213, 220, 256, 262, 268, 287, and 294. The sequence is essentially complementary to, or is about 75% to about 100%, about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, 95% to about 100%, about 98% to about 100%, about 100%, or 100% identical to, the sequence. In some embodiments, the polynucleotide comprises a sequence essentially complementary to, or at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, about 100%, or exactly 100% identical to, a sequence selected from an RNA trigger sequence or an RNA trigger sequence reverse complement. In some embodiments, the polynucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 48, 80-82, 182, 188, 194, 213, and 220.

[0092] By "effective amount" is meant an amount of agent effective to induce a biological change in a fungal pathogen that results in the elimination or reduction of production of DON by the fungal pathogen. In some embodiments, applying an effective amount of RNA to a plant improves the plant's resistance to damage by F. graminearum. The RNA may be longer than the segment(s) it contains (i.e., the RNA may contain additional nucleotides 3' and / or 5' of the segment), but each segment and corresponding fragment of the target gene are of comparable length. RNA for use in this method may be designed against multiple target genes.In an embodiment, the composition comprises an effective amount of a polynucleotide comprising at least 18, 19, 20, or 21 consecutive nucleotides that are complementary to a portion of a target gene or a homolog thereof identified in Table 1 or Table 2, or a target gene having a nucleotide sequence selected from a group of target gene sequences, or an RNA transcribed from the target gene, or an effective amount of at least one polynucleotide comprising at least one silencing element that is essentially complementary or essentially identical to at least 21 consecutive nucleotides of the target gene or the RNA transcribed from the target gene, wherein the target gene has a nucleotide sequence selected from a group of target gene sequences, or an effective amount of at least one RNA comprising at least one segment that is identical or complementary to at least 18, 19, 20, or 21 consecutive nucleotides of the target gene or the RNA transcribed from the target gene having the nucleotide sequence of SEQ ID NO: 16, or or an effective double-stranded RNA that, when transfected into or contacted with F. graminearum, causes a reduction or elimination of DON production by F. graminearum on a plant, wherein the RNA molecule comprises at least 18, 19, 20, or 21 contiguous nucleotides that are complementary to a target gene or a portion of the RNA transcribed from the target gene having a nucleotide sequence selected from SEQ ID NOs: 16, 68-70, 108, 114, 120, 139, and 146; or an effective double-stranded RNA that, when transfected into or contacted with F. graminearum, causes a reduction or elimination of DON production by F. graminearum on a plant, wherein at least one strand of the effective double-stranded RNA comprises 21 contiguous nucleotides that are complementary to a target gene or a portion of the RNA transcribed from the target gene, and the target gene has a sequence selected from the group consisting of a target gene sequence, or an effective amount of at least one double-stranded RNA that comprises a sequence selected from the group of RNA trigger sequences. In some embodiments, the polynucleotide is double-stranded RNA.In some embodiments, the polynucleotide (e.g., double-stranded RNA) is chemically or enzymatically synthesized, or produced by expression in a microorganism, or by expression in a plant cell.Embodiments include compositions comprising a dsRNA having a sequence selected from an RNA trigger sequence, an RNA trigger sequence reverse complement.

[0093] In various embodiments, the composition for eliminating or reducing DON production in F. graminearum is in at least one form selected from the group consisting of a solid, a liquid (including homogeneous mixtures such as soluble liquid concentrates, and non-homogeneous mixtures such as suspensions, colloids, micelles, and emulsions), a powder, a suspension, an emulsion, an aerosol, an encapsulated or microencapsulated formulation, in or on a microbead or other carrier particle, in a film or coating, or on or within a matrix, or as a leaf, seed, root, head, ear, or stem treatment. Suitable binders, inert carriers, surfactants, and the like may optionally be included in the polynucleotide-containing composition, which are known to those skilled in the art of fungicides and seed, stem, ear, head, fruit, or foliar treatment formulations. Such compositions may include any of the compositions described in PCT / US / 2022 / 027816, published November 10, 2022 as WO2022 / 0235895, the entire contents of which are incorporated herein by reference. In some embodiments, the composition is at least one implantable formulation selected from the group consisting of particles, pellets, or capsules implanted in a plant, and in such embodiments, the method includes implanting the implantable formulation in a plant. In one embodiment, the composition can be transfected or otherwise internally absorbed by F. graminearum. In some embodiments, the composition further comprises at least one component selected from the group consisting of carrier agents, surfactants, organosilicones, organosilicon surfactants, polynucleotide herbicidal molecules, non-polynucleotide herbicidal molecules, non-polynucleotide pesticides, polynucleotide pesticides, and safeners, pathogen growth regulators. In one embodiment, the composition further comprises a nonionic organosilicon surfactant, such as SILWET® brand surfactant, e.g., SILWET L-77® brand surfactant, which has CAS No. 27306-78-1 and EPA No.: CAL.REG.NO.5905-50073-AA and is currently available from Momentive Performance Materials, Albany, NY.One embodiment includes compositions further comprising BREAK-thru 301. Other surfactants include, for example: BREAK-THRU S 240 brand polyether modified polysiloxane (CASRN proprietary) surfactant, currently available from Goldschmidt Chemical Corporation, Hopewell, VA; BREAK-THRU S 279 endcapped polyether trisiloxane surfactant, the components of which are listed in the following chemical inventories: EINECS, TSCA, ENCS, AICS, ECL, PICCS CHINA, NDSL; INDUCE brand auxiliary NMFC item 42652, class 60, currently available from Helena Chemical Company, Collierville, TN; FRANCHISE® with LECI-TECH® brand surfactant, having CA REG No. 34704-50065, currently available from Loveland Products, Inc. Greely, CO. Alternatively, the plant is topically treated with the composition and a separate (preceding, subsequent, or simultaneous) application of a substance that improves the effectiveness of the composition.For example, the plant can be sprayed with a first topical application of a solution containing a nonionic organosilicone surfactant such as SILWET brand surfactant, for example, SILWET L-77, BREAK-THRU S24, BREAK-THRU S279, INDUCE, or FRANCHISE brand surfactant, followed by a second topical application of the composition, or vice versa.

[0094] The combination of a particular RNA for use in this method (e.g., a dsRNA trigger as described in the Examples) with one or more bactericidal polynucleotides and / or one or more non-polynucleotide fungicides is expected to provide enhanced protection of plants when compared to the effects obtained with the DON-reducing or -eliminating RNA alone, and the bactericidal RNA and / or non-polynucleotide fungicide alone.

[0095] In various embodiments, the composition comprises or is produced in a microbial cell. For example, the composition may comprise or be produced in a bacterial or yeast cell. In similar embodiments, the composition comprises or is produced in a transgenic plant cell (e.g., a plant cell that transiently expresses the polynucleotide). Such a plant cell may be a cell within a plant or a cell grown in tissue culture or cell suspension.

[0096] In one embodiment, the composition is provided in the form of any plant subject to infection by F. graminearum, and the RNA is contained in or on the plant. Such plants can be stable transgenic plants that express the RNA, or non-transgenic plants that transiently express the RNA or are treated with the RNA, for example, by spraying or coating. Stable transgenic plants generally contain a recombinant construct that encodes the RNA integrated into their genome.

[0097] The RNA useful in the composition can be single-stranded (ss) or double-stranded (ds). Embodiments include those in which the RNA is at least one selected from the group consisting of sense single-stranded RNA (ssRNA), antisense single-stranded (ssRNA), or double-stranded RNA (dsRNA), and mixtures of any of these types of RNA can be used. In one embodiment, a double-stranded DNA / RNA hybrid is used. The RNA polynucleotide can include components other than standard ribonucleotides, for example, one embodiment is an RNA that includes terminal deoxyribonucleotides.

[0098] The RNA in the composition has at least one segment of 18 or more contiguous nucleotides having a sequence of about 95% to about 100% identity to a fragment of equivalent length of a target gene having a sequence selected from a gene or a homolog thereof or a group of target gene sequences or a DNA complement thereof identified in Table 1 or Table 2. In one embodiment, the RNA comprises at least one segment of 18 or more contiguous nucleotides that is essentially identical or complementary to a fragment of equivalent length of a target gene having a sequence selected from a group consisting of a target gene or a homolog thereof or a group of target gene sequences identified in Table 1 or Table 2. In some embodiments, the contiguous nucleotides have a sequence of about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to a fragment of DNA having a sequence selected from a target gene or a homolog thereof or a group of target gene sequences or a DNA complement thereof identified in Table 1 or Table 2. In some embodiments, the contiguous nucleotides are exactly (100%) identical to a fragment of equivalent length of DNA having a sequence selected from the DNA of a target gene or a homolog thereof identified in Table 1 or Table 2, or a group of target gene sequences or their DNA complements. In some embodiments, the RNA has an overall sequence of about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to the DNA of a target gene or a homolog thereof identified in Table 1 or Table 2, or a fragment of DNA having a sequence selected from the group of target gene sequences or their DNA complements.

[0099] The RNA in the composition comprises at least one segment of 18 or more contiguous nucleotides having a sequence of about 95% to about 100% identity to a fragment of equivalent length of DNA having a sequence selected from a gene or a homolog thereof or a target gene sequence group or a DNA complement thereof identified in Table 1 or Table 2. In some embodiments, the RNA comprises at least one segment of 18 or more contiguous nucleotides, e.g., 18-24, or 18-28, or 20-30, or 20-50, or 20-100, or 50-100, or 50-500, or 100-250, or 100-500, or 200-1000, or 500-2000, or even more. In some embodiments, a segment is more than 18 contiguous nucleotides, e.g., 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30, e.g., about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 110, about 120, about 130, or more than 140, e.g., about 15, about 16, about 17, about 18, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 2 The RNA comprises 20, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, about 300, about 350, about 400, about 450, about 500, about 550, about 650, about 700, or more than 700 consecutive nucleotides. In certain embodiments, the RNA comprises at least one segment of at least 18, 19, 20, or 21 consecutive nucleotides having a sequence of 100% identity to a fragment of equivalent length of a target gene having a sequence selected from a gene identified in Table 1 or Table 2 or a homolog thereof or a group of target gene sequences or DNA complements thereof.In certain embodiments, the RNA is a double-stranded nucleic acid (e.g., dsRNA) having one strand containing at least one segment of at least 18, 19, 20, or 21 consecutive nucleotides with a sequence of 100% identity to a fragment of equivalent length of DNA having a sequence selected from the target gene sequence group or its DNA complement, and when expressed as base pairs, such a double-stranded nucleic acid contains at least one segment of at least 18, 19, 20, or 21 consecutive perfectly matched base pairs corresponding to a fragment of equivalent length of DNA or target gene having a sequence selected from the target gene sequence group or its DNA complement. In certain embodiments, each segment contained in the RNA is of a length longer than that typical of naturally occurring small regulatory RNA, for example, each segment is at least about 30 consecutive nucleotides (or base pairs) in length. In some embodiments, the total length of the RNA or the length of each segment contained in the RNA is shorter than the total length of the sequence of interest (DNA or target gene having a sequence selected from the target gene sequence group). In some embodiments, the total length of the RNA is about 50 to about 500 nucleotides (for single-stranded RNA) or base pairs (for double-stranded RNA). In some embodiments, the RNA comprises at least one RNA strand that is about 50 to about 750 nucleotides in length.

[0100] The RNA in the composition is generally designed to inhibit one or more genes ("target genes"). Such target genes may include coding or non-coding sequences, or both. In certain embodiments, the RNA is designed to inhibit one or more target genes, each of which has a DNA sequence selected from the group consisting of target gene sequences. In various embodiments, the RNA is designed to inhibit one or more genes, each of which is selected from the target genes or their homologs identified in Table 1 or Table 2, or a sequence selected from the group consisting of target gene sequences, and may be designed to inhibit multiple genes from these groups, or to target different regions of one or more of these genes. In one embodiment, the RNA comprises multiple sections or segments, each of which comprises at least one segment of 21 contiguous nucleotides having a sequence of 100% identity to a fragment of equivalent length of DNA having a sequence selected from the genes or their homologs identified in Table 1 or Table 2, or the target gene sequences, or their DNA complements. In such cases, each section may be identical or different in size or sequence, and may be sense or antisense to the target gene. For example, in one embodiment, the RNA may contain multiple sections in tandem or repeated sequences, each section containing at least one segment of 21 consecutive nucleotides with a sequence of 100% identity with a fragment of equivalent length of DNA having a sequence selected from the target gene sequence group or its DNA complement, and the segments may be from different regions of the target gene, e.g., the segments may correspond to different exon regions of the target gene, and "spacer" nucleotides that do not correspond to the target gene may optionally be used between or adjacent to the segments.

[0101] The total length of the RNA in the composition may be more than 18 contiguous nucleotides and may include nucleotides in addition to contiguous nucleotides having a sequence of about 95% to about 100% identity to a fragment of equivalent length of DNA having a sequence selected from a gene or homolog thereof or a group of target gene sequences or DNA complements thereof identified in Table 1 or Table 2. In other words, the total length of the RNA may be longer than the length of a section or segment of the RNA designed to inhibit one or more target genes, each target gene being selected from the group consisting of a target gene or homolog thereof identified in Table 1 or Table 2, or having a DNA sequence selected from the group consisting of a group of target gene sequences. For example, the RNA may have nucleotides adjacent to the "active" segment of at least one segment of 18 or more contiguous nucleotides that inhibits a target gene, or may include "spacer" nucleotides between active segments, or may have additional nucleotides at the 5' end, or the 3' end, or at both the 5' and 3' ends. In one embodiment, the RNA comprises additional nucleotides not specifically related (having a sequence that is not complementary or identical) to the DNA of the target gene, e.g., nucleotides to provide a stabilizing secondary structure or for convenience of cloning or manufacturing. In one embodiment, the RNA comprises additional nucleotides located immediately adjacent to one or more segments of 18 or more contiguous nucleotides having a sequence of about 95% to about 100% identity or complementarity to a DNA having a sequence selected from the group consisting of target gene sequences or a fragment of equivalent length of the target gene. In one embodiment, the RNA comprises one such segment having an additional 5'G or an additional 3'C, or both, adjacent to the segment. In another embodiment, the RNA is a double-stranded RNA that includes additional nucleotides to form an overhang, e.g., a dsRNA that includes two deoxyribonucleotides to form a 3' overhang. Thus, in various embodiments, the nucleotide sequence of the entire RNA is not 100% identical or complementary to a fragment of contiguous nucleotides in the target gene.For example, in some embodiments, the RNA comprises at least two segments of 21 contiguous nucleotides each having a sequence of 100% identity to a fragment of DNA having a sequence selected from the target gene sequence group or its DNA complement, and (1) the at least two segments are separated by one or more spacer nucleotides, or (2) the at least two segments are arranged in an order different from the order in which corresponding fragments occur in DNA having a sequence selected from the target gene sequence group or its DNA complement.

[0102] In various embodiments, the RNA in the composition is composed of naturally occurring ribonucleotides.Embodiments include, for example, synthetic RNA that is entirely or mainly composed of ribonucleotides, but has one or more terminal deoxyribonucleotides or one or more terminal dideoxyribonucleotides.In certain embodiments, the RNA comprises non-canonical nucleotides, such as inosine, thiouridine, or pseudouridine.In certain embodiments, the RNA comprises chemically modified nucleotides.The RNA in the composition can be provided by any suitable means known to those skilled in the art.

[0103] In some embodiments, the RNA is provided as an isolated RNA that is not part of an expression construct. In some embodiments, the RNA is provided as an isolated RNA that lacks additional elements such as promoter or terminator sequences. Such RNA may be relatively short, such as, for example, a single-stranded or double-stranded RNA of about 18 to about 300, or about 50 to about 750 nucleotides (in the case of single-stranded RNA), or about 18 to about 300, or about 50 to about 750 base pairs (in the case of double-stranded RNA). Alternatively, the RNA may be provided as part of a more complex construct, such as a recombinant expression construct, or may be included in a recombinant vector, such as a recombinant plant virus vector or a recombinant baculovirus vector. In some embodiments, such recombinant expression constructs or vectors are designed to include additional elements, such as additional RNA encoding an aptamer or ribozyme, or including an expression cassette for expressing a gene of interest (e.g., a bactericidal protein).

[0104] Methods for Providing Plants with Improved Resistance to DON Production by XF Graminearum, and Plants, Plant Parts, and Seeds So Provided - Patent application Some embodiments relate to methods of providing a plant with improved resistance to DON production by F. graminearum, comprising providing to the plant at least one polynucleotide described herein, such that the polynucleotide is effective to reduce or eliminate DON production by F. graminearum, including, for example, those described in Section II or Section VIII, or compositions described herein.

[0105] In a related aspect, the invention relates to plants with improved resistance to DON production by F. graminearum, provided by expressing in the plant at least one polynucleotide described herein, such as those described in Section II or Section VIII, effective to reduce or eliminate DON production by F. graminearum.

[0106] In yet another aspect, the invention relates to seeds or propagules (particularly transgenic progeny seeds or propagules) produced by the plants having improved resistance to DON production by F. graminearum provided by the method. Also contemplated are commercial products produced by the plants having improved resistance to DON production by F. graminearum provided by the method, and commercial products produced from transgenic progeny seeds or propagules of such plants.

[0107] The polynucleotides used in the method can be designed against multiple target genes.Embodiments include those in which the composition comprises a dsRNA having a strand with a sequence selected from the group consisting of RNA trigger sequences.Related aspects of the invention include the compositions and isolated polynucleotides for topical application used in the method, and the plants with improved resistance to DON production by F. graminearum provided by the method.

[0108] As used throughout the specification, "topical application" refers to application to the surface of a plant part such as a leaf, stem, ear, head, flower, fruit, bud, root, stem, seed, flower, anther, or pollen, or to the surface or exterior of a plant, such as application to the entire plant or above-ground or below-ground parts of a plant. Topical application can be performed on a non-living surface, such as application to the soil, or to a surface or matrix where F. graminearum can encounter the polynucleotide. In various embodiments of the method, a composition comprising at least one polynucleotide is applied locally to a plant in a suitable form, for example, a solid, liquid (including homogeneous mixtures such as solutions, and non-homogeneous mixtures such as suspensions, colloids, micelles, and emulsions), powder, suspension, emulsion, spray, encapsulated or microencapsulated formulation, in or on microbeads or other carrier particles, in a film or coating, or on or within a matrix, or as a leaf, seed, root, ear, head, or stem treatment. In some embodiments of the method, the polynucleotide-containing composition is applied topically to the above-ground parts of the plant, for example, sprayed or dusted onto the leaves, panicles, heads, stems, and flowering parts of the plant.

[0109] Embodiments of the method include topical application of a foliar spray (e.g., spraying the leaves of a plant with a liquid polynucleotide-containing composition) or foliar dusting (e.g., dusting the plant with a polynucleotide-containing composition in the form of a powder or on carrier particles). In other embodiments, the polynucleotide-containing composition is applied locally to below-ground portions of the plant, such as the roots, for example, by soil drainage. In other embodiments, the polynucleotide-containing composition is applied locally to the seeds that grow into the plant. Topical application can be in the form of a topical treatment of the fruit of the plant or the seeds from the fruit of the plant. Suitable binders, inert carriers, surfactants, and the like can optionally be included in the polynucleotide-containing composition, as are known to those skilled in the art of fungicide and seed or stem treatment formulations.

[0110] In some embodiments, the polynucleotide-containing composition is at least one locally implantable formulation selected from the group consisting of particles, pellets, or capsules locally implanted in the plant, and in such embodiments, the method comprises locally implanting the locally implantable formulation in the plant. In one embodiment, the polynucleotide-containing composition can be transfected or otherwise internally absorbed by F. graminearum. In some embodiments, the polynucleotide-containing composition further comprises a carrier agent and / or a surfactant (e.g., a non-ionic surfactant). Examples of non-ionic organosilicone surfactants include SILWET® brand surfactants BREAK THRU S240, BREAK THRU S279, BREAK THRU 301, nduce, and Franchise, such as SILWET L-77® brand surfactant. A first topical application of the surfactant can be followed by a second topical application of the polynucleotide-containing composition, or vice versa. In some embodiments, plants are topically treated with a polynucleotide-containing composition and a separate (preceding, subsequent, or simultaneous) application of a substance that improves the effectiveness of the polynucleotide-containing composition. For example, plants can be sprayed with a first topical application of a solution containing a nonionic organosilicone surfactant, such as SILWET® brand surfactants, BREAK THRU S240, BREAK-THRU S279, BREAK-THRU 301, Induce, and Franchise, e.g., SILWET L-77® brand surfactant, followed by a second topical application of a polynucleotide-containing composition, or vice versa.

[0111] It is expected that the combination of certain polynucleotides useful in the polynucleotide-containing compositions (e.g., the polynucleotide triggers described in the Examples) with one or more non-polynucleotide agents will result in enhanced improvements in the reduction or elimination of DON production by Fusarium when compared to the effects obtained with the polynucleotide alone or the non-polynucleotide agent alone.

[0112] In some embodiments, polynucleotides useful in polynucleotide-containing compositions are provided as isolated DNA or RNA fragments. In some embodiments, polynucleotides useful in polynucleotide-containing compositions are not part of an expression construct and lack additional elements such as promoter or terminator sequences). Such polynucleotides may be relatively short, such as single- or double-stranded polynucleotides of about 18 to about 300, or about 50 to about 700 nucleotides (for single-stranded polynucleotides), or about 18 to about 300, or about 50 to about 700 base pairs (for double-stranded polynucleotides). In some embodiments, the polynucleotide is a dsRNA of about 100 to about 700 base pairs, such as a dsRNA of any of the lengths of the dsRNA triggers disclosed in the figures and tables. Alternatively, the polynucleotide may be provided as part of a more complex construct, such as a recombinant expression construct, or may be included in a recombinant vector, such as a recombinant plant virus vector or a recombinant baculovirus vector. Such recombinant expression constructs or vectors can be designed to contain additional elements, such as an expression cassette for expressing a gene of interest (eg, a bactericidal protein).

[0113] XII. Recombinant DNA constructs for controlling DON production by F. graminearum. Another aspect of the present invention provides a recombinant DNA construct comprising a heterologous promoter operably linked to a DNA element comprising a sequence corresponding to a sequence described herein that is effective for controlling DON production by F. graminearum, e.g., a fragment of DNA having a sequence selected from a group of target gene sequences or their DNA complements, or a DNA element comprising at least one segment of 18 or more contiguous nucleotides having a sequence that is about 95% to about 100% identical to a fragment of DNA of a target gene identified in Table 1 or Table 2. In some embodiments, the recombinant DNA construct comprises a heterologous promoter operably linked to: (a) a DNA comprising a nucleotide sequence that is complementary to at least 18, 19, 20, 21, 50, 150, 250, 300, 400, 500, 600, or 650 consecutive nucleotides of a target gene having a sequence selected from the group consisting of SEQ ID NOs: 16, 68-70, 108, 114, 120, 139, and 146, or, in more specific embodiments, SEQ ID NOs: 16, 68-70, 108, 114, 120, 139, and 146, or a DNA complement thereof; or (b) a DNA comprising a nucleotide sequence that is complementary to at least 18, 19, 20, 21, 50, 150, 250, 300, 400, 500, 600, or 650 consecutive nucleotides of a target gene having a sequence selected from the group consisting of SEQ ID NOs: 16, 68-70, 108, 114, 120, 139, and 146, or, in more specific embodiments, ... or (c) a DNA encoding at least one silencing element that is complementary to at least 18, 19, 20, 21, 50, 150, 250, 300, 400, 500, 600, or 650 consecutive nucleotides of a target gene or an RNA transcribed from a target gene, wherein the target gene has a sequence selected from the group consisting of a target gene sequence group, or in more specific embodiments, SEQ ID NOs: 16, 68-70, 108, 114, 120, 139, and 146; or (d) a gene identified in Table 1 or Table 2 or a homolog thereof, or in more specific embodiments, a target gene selected from FGP1, ELP3, SPT7, MAF1, MVD1, HEP1, SET1, FKPB12;or (e) a DNA encoding at least one silencing element comprising at least 18, 19, 20, 21, 50, 150, 250, 300, 400, 500, 600, or 650 consecutive nucleotides that are complementary to a portion of an RNA transcribed from a target gene; or (f) a DNA encoding an RNA comprising at least 18, 19, 20, 21, 50, 150, 250, 300, 400, 500, 600, or 650 consecutive nucleotides that are complementary to a nucleotide sequence selected from the RNA trigger sequence group or a complement thereof, or a homologous nucleotide sequence from a Fusarium species, wherein the homologous nucleotide sequence has at least 95% sequence identity with a nucleotide sequence selected from the RNA trigger sequence group, and the sequence identity percentage is greater than or equal to 18, 19, 20, 21, 50, 150, 250, 300, 400, 500, 600, or 650 consecutive nucleotides that are complementary to a nucleotide sequence selected from the RNA trigger sequence group or a complement thereof, or (f) DNA encoding an RNA comprising at least one double-stranded RNA region, at least one strand of which comprises at least 18, 19, 20, 21, 50, 150, 250, 300, 400, 500, 600, or 650 contiguous nucleotides that are complementary to a nucleotide sequence selected from the RNA trigger sequence group or its complement, or a homologous nucleotide sequence from a Fusarium species, wherein the homologous nucleotide sequence has at least 95% sequence identity with a nucleotide sequence selected from the RNA trigger sequence group, the sequence identity percentage being calculated over the same length; or (g) DNA encoding an RNA comprising a nucleotide sequence selected from the RNA trigger sequence group or its complement. Embodiments include recombinant DNA constructs comprising a heterologous promoter operably linked to a DNA element encoding an RNA having a sequence selected from the group consisting of SEQ ID NOs: 48, 80-82, 182, 188, 194, 213, and 220, or combinations thereof, or complements thereof. ,

[0114] An embodiment includes a recombinant DNA construct comprising a heterologous promoter operably linked to DNA encoding a dsRNA having a strand with a sequence selected from the group consisting of RNA trigger sequences. The recombinant DNA construct is useful, for example, for providing plants with improved resistance to DON production by F. graminearum by expressing the transcript of such a recombinant DNA construct in the plant. The recombinant DNA construct is also useful for producing polynucleotides useful for making compositions that can be applied to plants, seeds, propagable plant parts, soil or fields, or surfaces in need of protection from DON. Related aspects of the invention include compositions comprising a recombinant DNA construct; a plant chromosome or plastid or a recombinant plant viral vector or a recombinant baculoviral vector comprising the recombinant DNA construct; a transgenic plant cell having a recombinant DNA construct in its genome, a transgenic plant comprising such a transgenic plant cell, or a fruit, seed, or propagule of a transgenic plant; a plant having improved resistance to DON production by fungi of the genus Fusarium, e.g., F. graminearum, and pest or pathogen resistance provided by expression of or treatment with a recombinant DNA construct or RNA encoded therein.

[0115] The recombinant DNA construct comprises a heterologous promoter operably linked to DNA comprising at least one segment of 18 or more contiguous nucleotides having a sequence of about 95% to about 100% identity to the DNA of a gene identified in Table 1 or Table 2 or a homolog thereof, or a fragment of equivalent length of DNA having a sequence selected from the target gene sequence group or its DNA complement. In some embodiments, the segment of 18 or more contiguous nucleotides has a sequence that is about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to a fragment of DNA having a sequence selected from the target gene sequence group or its DNA complement. In some embodiments, the contiguous nucleotides are exactly (100%) identical to a fragment of equivalent length of DNA having a sequence selected from the target gene sequence group or its DNA complement. In some embodiments, the DNA has an overall sequence identity of about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% to a DNA having a sequence selected from a group of target gene sequences or their DNA complements.

[0116] Thus, the recombinant DNA construct comprises a heterologous promoter operably linked to DNA comprising at least one segment of 18 or more contiguous nucleotides designed to suppress expression of a target gene having a sequence selected from a group of target gene sequences or its DNA complement. In some embodiments, the DNA comprises at least one segment of 18 or more contiguous nucleotides, e.g., 18-24, or 18-28, or 20-30, or 20-50, or 20-100, or 50-100, or 50-500, or 100-250, or 100-500, or 200-650, 500-1000, or 500-2000, or even more. In some embodiments, a segment is more than 18 contiguous nucleotides, e.g., 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30, e.g., about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 110, about 120, about 130, or more than 140, e.g., about 15, about 16, about 17, about 18, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 2 The DNA comprises 20, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, or more than 650 consecutive nucleotides. In certain embodiments, the DNA encodes a target gene having a sequence selected from a group of target gene sequences or a DNA complement thereof, or an RNA comprising at least one segment of at least 18, 19, 20, or 21 consecutive nucleotides having a sequence of 100% identity to a comparable length fragment of the DNA of a target gene or a homolog thereof identified in Table 1 or Table 2.In certain embodiments, the DNA encodes a double-stranded nucleic acid (e.g., dsRNA) having one strand containing at least one segment of at least 18, 19, 20, or 21 contiguous nucleotides with a sequence of 100% identity to a fragment of equivalent length of DNA having a sequence selected from the target gene sequence group or its DNA complement, and when expressed as base pairs, such double-stranded nucleic acid contains at least one segment of at least 18, 19, 20, or 21 contiguous perfectly matched base pairs corresponding to a fragment of equivalent length of DNA having a sequence selected from the target gene sequence group or its DNA complement. In certain embodiments, each segment contained in the DNA is of a length longer than that typical of naturally occurring small regulatory RNA. In some embodiments, each segment is at least about 30 contiguous nucleotides (or base pairs) in length. In some embodiments, the total length of the DNA or each segment contained in the polynucleotide is shorter than the total length of the sequence of interest (DNA having a sequence selected from the target gene sequence group or a target gene). In some embodiments, the total length of the DNA is about 50 to about 650. In some embodiments, the DNA encodes an RNA having a sequence selected from the group consisting of an RNA trigger sequence or an RNA trigger sequence reverse complement, or a combination thereof, or a complement thereof.

[0117] A recombinant DNA construct generally comprises a heterologous promoter operably linked to DNA designed to repress one or more genes ("target genes"). Such target genes may comprise coding or non-coding sequences, or both. In certain embodiments, a recombinant DNA construct is designed to repress one or more target genes, each target gene having a DNA sequence selected from the group consisting of a target gene sequence group. In various embodiments, a recombinant DNA construct is designed to repress one or more genes, each gene having a sequence selected from the group consisting of a target gene sequence group, and may be designed to repress multiple genes from this group, or to target different regions of one or more of these genes. In one embodiment, a recombinant DNA construct comprises a heterologous promoter operably linked to multiple sections or segments, each of which comprises at least one segment of 21 contiguous nucleotides having a sequence of 100% identity to a fragment of equivalent length of DNA having a sequence selected from the target gene sequence group or its DNA complement. In such cases, each section may be identical or different in size or sequence and may be sense or antisense to the target gene. For example, in one embodiment, a recombinant DNA construct may comprise a heterologous promoter operably linked in tandem or repeat sequences to multiple sections, each section comprising at least one segment of 21 contiguous nucleotides having a sequence of 100% identity to a fragment of equivalent length of DNA having a sequence selected from a group of target gene sequences or its DNA complement, and the segments may be from different regions of the target gene, e.g., the segments may correspond to different exon regions of the target gene, and "spacer" nucleotides that do not correspond to the target gene may optionally be used between or adjacent to the segments.

[0118] The recombinant DNA construct comprises a heterologous promoter operably linked to DNA that may have a total length greater than 18 contiguous nucleotides, and may contain nucleotides in addition to at least one segment of 18 or more contiguous nucleotides having a sequence of about 95% to about 100% identity to a fragment of equivalent length of DNA having a sequence selected from the group of target gene sequences or their DNA complements. In other words, the total length of the DNA may be longer than the length of the segment of DNA designed to repress one or more target genes, each target gene having a DNA sequence selected from the group consisting of the group of target gene sequences or their homologs. For example, the DNA may have nucleotides adjacent to the "active" segment of at least one segment of 18 or more contiguous nucleotides that represses the target gene, or may include "spacer" nucleotides between the active segments, or may have additional nucleotides at the 5' end, or the 3' end, or at both the 5' and 3' ends. In one embodiment, the heterologous promoter is operably linked to DNA having a sequence selected from the group consisting of target gene sequences or its DNA complement, or DNA containing additional nucleotides not specifically related (having a sequence that is not complementary or identical) to the target gene, e.g., nucleotides to provide a stabilizing secondary structure or for convenience of cloning or manufacturing. In one embodiment, the heterologous promoter is operably linked to DNA having a sequence selected from the group consisting of target gene sequences or DNA containing additional nucleotides located immediately adjacent to one or more segments of 18 or more contiguous nucleotides having a sequence of about 95% to about 100% identity to or complementary to a fragment of equivalent length of the target gene. In one embodiment, the heterologous promoter is operably linked to DNA containing one such segment, with an additional 5'G or an additional 3'C, or both, adjacent to the segment. In another embodiment, the heterologous promoter is operably linked to DNA encoding a double-stranded RNA containing additional nucleotides to form an overhang.Thus, in various embodiments, the nucleotide sequence of the entire DNA operably linked to the heterologous promoter is not 100% identical or complementary to a segment of contiguous nucleotides in a target gene or DNA having a sequence selected from the group consisting of target gene sequences. For example, in some embodiments, a heterologous promoter is operably linked to a DNA that comprises at least two segments of 21 contiguous nucleotides each having a sequence of 100% identity to a segment of DNA having a sequence selected from the group of target gene sequences, or its DNA complement, where (1) the at least two segments are separated by one or more spacer nucleotides, or (2) the at least two segments are arranged in an order different from the order in which the corresponding fragment occurs in a DNA having a sequence selected from the group of target gene sequences, or its DNA complement.

[0119] In the recombinant DNA construct, a heterologous promoter is operably linked to DNA encoding a transcript that may be single-stranded (ss) or double-stranded (ds), or a combination of both. Embodiments of the method include those in which the DNA encodes a transcript that comprises a sense single-stranded RNA (ssRNA), an antisense ssRNA, or a double-stranded RNA (dsRNA), or any combination thereof.

[0120] The recombinant DNA construct is provided by any suitable means known to those of skill in the art. Embodiments include those in which the recombinant DNA construct is synthesized in vitro, produced by expression in a microbial or cell culture (such as a plant cell grown in culture), produced by expression in a plant cell, or produced by microbial fermentation.

[0121] The heterologous promoter of use in the recombinant DNA construct is selected from the group consisting of a promoter that functions in plants, a promoter that functions in prokaryotes, a promoter that functions in fungal cells, and a baculovirus promoter. Non-limiting examples of promoters are described in the section entitled "Promoters."

[0122] In some embodiments, the recombinant DNA construct also includes a second promoter operably linked to the DNA. For example, the DNA containing at least one segment of 18 or more consecutive nucleotides can be flanked by two promoters arranged such that the promoters transcribe convergently in opposite directions, resulting in opposite strand transcripts of the DNA that are complementary to each other and can hybridize to each other to form double-stranded RNA. In one embodiment, the DNA is located between two root-specific promoters, which allows transcription of the DNA in opposite directions, resulting in the formation of dsRNA.

[0123] In some embodiments, the recombinant DNA construct comprises other DNA elements in addition to a heterologous promoter operably linked to DNA comprising at least one segment of 18 or more contiguous nucleotides having a sequence of about 95% to about 100% identity to a fragment of equivalent length of DNA having a sequence selected from the target gene sequence group or its DNA complement. Such DNA elements are known in the art and include, but are not limited to, introns, recombinase recognition sites, aptamers or ribozymes, as well as additional expression cassettes for expressing coding sequences (e.g., cassettes for expressing transgenes such as bactericidal proteins or selection markers) or non-coding sequences (e.g., for expressing additional inhibitory elements). The inclusion of one or more recognition sites for binding and cleavage by small RNAs (e.g., miRNAs or siRNAs that are expressed only in specific cells or tissues) allows for a more precise expression pattern in plants, and expression of the recombinant DNA construct is inhibited when the small RNA is expressed.

[0124] In some embodiments, the recombinant DNA construct is provided in a recombinant vector. By "recombinant vector" is meant a recombinant polynucleotide molecule used to transfer genetic information from one cell to another. Suitable embodiments of the present invention include, but are not limited to, recombinant plasmids, recombinant cosmids, artificial chromosomes, and recombinant viral vectors, such as recombinant plant viral vectors and recombinant baculovirus vectors. Alternative embodiments include recombinant plasmids, recombinant cosmids, artificial chromosomes, and recombinant viral vectors, such as recombinant plant viral vectors and recombinant baculovirus vectors, that contain DNA elements without a heterologous promoter. Examples of plasmids for use with the present invention include, for example, those described in PCT / US2020 / 063490, published on June 10, 2021 as WO2021 / 113774, which is incorporated herein by reference in its entirety.

[0125] In some embodiments, the recombinant DNA construct is provided in a plant chromosome or plastid, e.g., a transgenic plant cell or a transgenic plant. Thus, transgenic plant cells having a recombinant DNA construct in their genome, as well as transgenic or partially transgenic plants comprising such transgenic plant cells, are also encompassed by the present invention. Partial transgenic plants include, for example, non-transgenic scions grafted onto transgenic rootstocks comprising transgenic plant cells. An embodiment includes a transgenic tomato rootstock comprising transgenic plant cells. The plant may be any plant subject to infection by F. graminearum. An embodiment includes those in which the plant is an ungerminated plant seed, a plant in a vegetative stage, or a plant in a reproductive stage. In yet another aspect, the present invention relates to seeds (particularly transgenic progeny seeds) produced by transgenic plants having in their genome a recombinant DNA construct as described herein. Also contemplated are commercial products produced by such transgenic plants, and commercial products produced from transgenic progeny seeds of such transgenic plants.

[0126] The recombinant DNA construct may be provided in a composition for topical application to the surface of a plant or the seeds, roots, or stems of a plant, or for topical application to any substrate requiring protection from DON produced by F. graminearum. Similarly, the recombinant DNA construct may be provided in a composition for topical application to F. graminearum, or for internal absorption (e.g., transfection) by F. graminearum. In various embodiments, such compositions containing the recombinant DNA construct are provided in at least one form selected from the group consisting of solids, liquids (including homogeneous mixtures such as solutions, and non-homogeneous mixtures such as suspensions, colloids, micelles, and emulsions), powders, suspensions, emulsions, sprays, encapsulated or microencapsulated formulations, in or on microbeads or other carrier particles, in films or coatings, or on or within matrices, or as leaf, seed, root, or stem treatments. Topical application may be in the form of topical treatment of the fruit of the plant or seeds from the fruit of the plant. Suitable binders, inert carriers, surfactants, etc. may be included in the composition containing the recombinant DNA construct, which are known to those skilled in the art of pesticide and seed treatment formulations. In some embodiments, the composition for topical application containing the recombinant DNA construct is at least one topically implantable formulation selected from the group consisting of particles, pellets, or capsules that are topically implanted in the plant, and in such embodiments, the method includes topically implanting the topically implantable formulation in the plant. In one embodiment, the composition for topical application containing the recombinant DNA construct may be internally absorbed (e.g., transfected) by F. graminearum. In some embodiments, the composition containing the recombinant DNA construct further comprises at least one component selected from the group consisting of carrier agents, surfactants, organosilicones, organosilicon surfactants, polynucleotide herbicidal molecules, non-polynucleotide herbicidal molecules, non-polynucleotide pesticides, polynucleotide pesticides, safeners, and pathogen growth regulators.In one embodiment, the composition containing the recombinant DNA construct further comprises a nonionic organosilicone surfactant, such as SILWET® brand surfactant, e.g., SILWET L-77® brand surfactant, having CAS number 27306-78-1 and EPA number: CAL.REG.NO.5905-50073-AA, currently available from Momentive Performance Materials, Albany, NY. BREAK-THRU S 240 brand is a polyether modified polysiloxane (CASRN proprietary) surfactant, currently available from Goldschmidt Chemical Corporation, Hopewell, VA. BREAK-THRU S 279 is an end-capped polyether trisiloxane surfactant, the components of which are listed in the following chemical inventories: EINECS, TSCA, ENCS, AICS, ECL, PICCS CHINA, NDSL. INDUCE brand supplement NMFC item 42652, class 60, currently available from Helena Chemical Company, Collierville, TN. FRANCHISE® with LECI-TECH® brand surfactants having CA REG No. 34704-50065, currently available from Loveland Products, Inc. Greely, Colo. One embodiment includes a composition further comprising BREAK-thru 301.

[0127] It is expected that combining certain recombinant DNA constructs described herein (e.g., recombinant DNA constructs comprising a polynucleotide trigger as described in the Examples), whether transgenically expressed or topically applied, with one or more non-polynucleotide pesticides, whether transgenically expressed or topically applied, will result in enhanced improvement in the reduction or elimination of DON production by F. graminearum and pest / pathogen infestation, when compared to the effect obtained with the recombinant DNA construct alone or the non-polynucleotide pesticide alone. In one embodiment, a recombinant DNA construct for expressing one or more polynucleotides and one or more genes encoding a non-polynucleotide pesticide is found to provide improved resistance to DON production by F. graminearum and pest / pathogen infestation in plants expressing the recombinant DNA construct. One embodiment relates to a recombinant DNA construct for expressing an RNA comprising a segment having a sequence selected from the group of trigger sequences and one or more genes encoding a non-polynucleotide pesticide.

[0128] In various embodiments, the composition containing the recombinant DNA construct comprises a microbial cell or is produced in a microorganism. For example, the composition for containing the recombinant DNA construct may comprise or be produced in a bacterial or yeast cell. In similar embodiments, the composition containing the recombinant DNA construct comprises or is produced in a transgenic plant cell (e.g., a plant cell that transiently expresses the recombinant DNA construct). Such a plant cell may be a cell within a plant or a cell grown in tissue culture or cell suspension.

[0129] XIII. Transgenic Plant Cells Some embodiments relate to transgenic plant cells expressing polynucleotides useful in the methods and compositions described herein for suppressing expression of a target gene in F. graminearum or other Fusarium species or for reducing or eliminating DON production by F. graminearum or other Fusarium species. In one aspect, the invention provides a transgenic plant cell having in its genome recombinant DNA encoding an RNA comprising at least one segment of 18 or more contiguous nucleotides having a sequence of about 95% to about 100% identity to a fragment of DNA having a sequence selected from a group of target gene sequences or a DNA complement thereof. In one aspect, the invention provides a transgenic plant cell having in its genome recombinant DNA encoding an RNA comprising at least one silencing element essentially identical or essentially complementary to a fragment of a target gene sequence of F. graminearum, the target gene sequence being selected from a group of target gene sequences or a DNA complement thereof. In one aspect, the invention provides a transgenic plant cell having in its genome a recombinant DNA encoding an RNA that suppresses expression of a target gene in F. graminearum that contacts or internalizes the RNA, the RNA comprising at least one silencing element having at least one segment of 18 or more contiguous nucleotides that is complementary to a fragment of the target gene, the target gene being selected from the group consisting of genes of a group of target gene sequences. A particular embodiment is a transgenic plant cell having in its genome a recombinant DNA encoding an RNA that suppresses expression of a target gene in F. graminearum that contacts or internalizes the RNA, the RNA comprising at least one silencing element having at least one segment of 18 or more contiguous nucleotides that is complementary to a fragment of one or more of the group of target gene sequences. In one aspect, the invention provides a transgenic plant cell having in its genome a recombinant DNA encoding an RNA having a sequence selected from the group of trigger sequences.Such transgenic plant cells are useful for providing transgenic plants having improved resistance to DON production due to F. graminearum infection when compared to control plants lacking such plant cells. The transgenic plant cells can be isolated transgenic plant cells, or transgenic plant cells grown in culture, or transgenic cells of any transgenic plant that are subject to infection by F. graminearum.

[0130] In one embodiment, the recombinant DNA can be stably integrated into the genome of the transgenic plant and expressed in a cell(s) of the transgenic plant. Methods for providing stably transformed plants are provided in the section "Making and using transgenic plant cells and transgenic plants."

[0131] Some embodiments relate to a transgenic plant cell having in its genome a recombinant DNA encoding an RNA that suppresses expression of a target gene in F. graminearum that contacts or internalizes the RNA, the RNA comprising at least one silencing element complementary to the target gene, the target gene sequence being selected from the group consisting of target gene sequences or complements thereof. In some embodiments, the silencing element comprises at least one 18 or more contiguous nucleotides having a sequence that is about 95% to about 100% complementary to a fragment of equivalent length of DNA having a sequence selected from the group consisting of target gene sequences. In some embodiments, the silencing element comprises at least one 18 or more contiguous nucleotides that can hybridize in vivo or under physiological conditions (e.g., physiological conditions normally found in cells of F. graminearum) to a fragment of equivalent length of DNA having a sequence selected from the group consisting of target gene sequences. The contiguous nucleotides are at least 18, e.g., 18 to 24, or 18 to 28, or 20 to 30, or 20 to 50, or 20 to 100, or 50 to 100, or 50 to 500, or 100 to 250, or 100 to 500, or 200 to 1000, or 500 to 2000, or even more. In some embodiments, the contiguous nucleotides are more than 18, e.g., 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30, e.g., about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 11, or more. The number of consecutive nucleotides is 0, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, about 300, about 350, about 400, about 450, about 500, or more than 500 consecutive nucleotides.In certain embodiments, the silencing element comprises at least one segment of at least 18, 19, 20, or 21 consecutive nucleotides with a sequence of 100% identity with a fragment of equivalent length of DNA or a target gene having a sequence selected from the target gene sequence group or its DNA complement. In certain embodiments, the RNA is a double-stranded nucleic acid (e.g., dsRNA) having one strand comprising at least one segment of at least 18, 19, 20, or 21 consecutive nucleotides with a sequence of 100% identity with a fragment of equivalent length of DNA or its DNA complement having a sequence selected from the target gene sequence group, and when expressed as base pairs, such double-stranded nucleic acid comprises at least one segment of at least 18, 19, 20, or 21 consecutive perfectly matched base pairs corresponding to a fragment of equivalent length of DNA or its DNA complement having a sequence selected from the target gene sequence group. In certain embodiments, each silencing element contained in the RNA is of a length longer than that typical of naturally occurring regulatory small RNA. In some embodiments, each segment is at least about 30 contiguous nucleotides (or base pairs) in length. In certain embodiments, the RNA is about 50 to about 500 nucleotides in length. In certain embodiments, the RNA has a sequence selected from a group of RNA trigger sequences.

[0132] In some embodiments, the transgenic plant cell can further express additional heterologous DNA sequences. In certain embodiments, the transgenic plant cell stably integrates in its genome (i) a recombinant DNA encoding at least one RNA having a sequence selected from the group of RNA trigger sequences, and (ii) a DNA encoding at least one fungicide.

[0133] In related aspects, the invention is directed to transgenic plants, including transgenic plant cells, commercial products produced from the transgenic plants, and transgenic progeny, plant seeds, or transgenic propagable parts of the transgenic plants. Also contemplated are commercial products produced by the transgenic plants, and commercial products produced from transgenic progeny seeds of such transgenic plants.

[0134] XIV. METHODS OF PRODUCING POLYNUCLEOTIDES FOR RNAi The polynucleotides of the claimed methods and compositions can be produced by any suitable method known in the art. Examples of methods for producing the RNA molecules of the present disclosure include, but are not limited to, in vitro transcription (IVT) (such as transcription using T7 polymerase or other polymerases), chemical synthesis, expression in an organism (e.g., a plant or a microorganism) or in a cell culture (e.g., a plant cell culture), and microbial fermentation. In some embodiments, the RNA described herein is produced via any one of the processes for cell-free production of RNA described in U.S. Pat. No. 10,858,385 or U.S. Pat. No. 10,954,541, both of which are incorporated herein by reference.

[0135] XV. Promoter The promoters used in the present invention function in the cells in which the construct is intended to be transcribed. Generally, these promoters are heterologous promoters used in the recombinant constructs, i.e., they are not found essentially operably linked to other nuclear elements used in the constructs described herein. In various embodiments, the promoter is selected from the group consisting of constitutive promoters, spatially specific promoters, time specific promoters, development specific promoters, and inducible promoters. In many embodiments, the promoter is a promoter that functions in plants, such as a pol II promoter, a pol III promoter, a pol IV promoter, or a pol V promoter.

[0136] Non-constitutive promoters suitable for use with the recombinant DNA constructs of the present invention include spatially specific promoters, time specific promoters, and inducible promoters. Spatial specific promoters may include organelle-, cell-, tissue-, or organ-specific promoters (e.g., plastid-, root-, pollen-, or seed-specific promoters for expression in plastids, roots, pollen, or seeds, respectively). Often, seed-, embryo-, aleurone-, or endosperm-specific promoters are particularly useful. Time specific promoters may include promoters that tend to promote expression at specific developmental stages of the plant's growth cycle, or at different times of the day and night, or during different seasons of the year. Inducible promoters include promoters that are induced by chemicals or by environmental conditions, such as, but not limited to, biotic or abiotic stresses (e.g., lack of water or drought, heat, cold, high or low nutrient or salt levels, high or low light levels, or pest or pathogen infection). MicroRNA promoters are particularly useful that have time-specific, spatially specific, or inducible expression patterns, and examples of miRNA promoters, as well as methods for identifying miRNA promoters with specific expression patterns, are provided in U.S. Patent Application Publication Nos. 2006 / 0200878, 2007 / 0199095, and 2007 / 0300329, which are specifically incorporated herein by reference. Expression-specific promoters can also include promoters that are generally constitutively expressed but with different degrees or "strengths" of expression, including promoters that are commonly considered to be "strong promoters" or "weak promoters."

[0137] Promoters of particular interest include, for example, the opaline synthase promoter isolated from the Agrobacterium T-DNA; the cauliflower mosaic virus (CaMV) 35S promoter; enhanced promoter elements such as the enhanced CaMV 35S promoter linked to an enhancer element or chimeric promoter elements (intron from maize heat shock protein 70); root specific promoters such as those disclosed in U.S. Pat. Nos. 5,837,848, 6,437,217, and 6,426,446; and the ribozyme promoter disclosed in U.S. Pat. No. 6,433,252. No. 6,140,078; light inducible promoters disclosed in U.S. Pat. No. 6,294,714; pathogen inducible promoters disclosed in U.S. Pat. No. 6,252,138; and water deficit inducible promoters disclosed in U.S. Pat. App. No. 2004 / 0123347A1. All of the above patents and patent publications that disclose promoters and their use in recombinant DNA constructs that function specifically in plants are incorporated herein by reference.

[0138] Examples of plant vascular or phloem specific promoters of interest include the rolC or rolA promoter of Agrobacterium rhizogenes, the promoter of A. tumefaciens T-DNA gene 5, the rice sucrose synthase RSs1 gene promoter, the Commelina yellow spot badna virus promoter, the coconut leaf rot virus promoter, the rice tungro bacilliform virus promoter, the promoter of the pea glutamine synthetase GS3A gene, the invCD111 and invCD141 promoters of the potato invertase gene, the Kertbundit et al. Examples of such promoters include a promoter isolated from Arabidopsis, which was shown to express specifically in the phloem of tobacco by G. et al. (1991) Proc. Natl. Acad. Sci. USA., 88:5212-5216, the VAHOX1 promoter region, the pea cell wall invertase gene promoter, the carrot-derived acid invertase gene promoter, the promoter of the sulfate transporter gene Sultr1, the promoter of the plant sucrose synthase gene, and the promoter of the plant sucrose transporter gene.

[0139] Suitable promoters for use with the recombinant DNA constructs or polynucleotides of the invention may include polymerase II ("pol II") and polymerase III ("pol III") promoters. RNA polymerase II transcribes structural or catalytic RNAs that are usually shorter than 400 nucleotides in length and recognizes a simple run of T residues as a termination signal. It has been used to transcribe siRNA duplexes (see, e.g., Lu et al. (2004) Nucleic Acids Res., 32:e171). Thus, pol II promoters are a particular embodiment for producing short RNA transcripts from the recombinant DNA constructs of the invention. In one embodiment, the recombinant DNA construct comprises a pol II promoter for expressing an RNA transcript flanked by self-cleaving ribozyme sequences (e.g., self-cleaving hammerhead ribozymes), resulting in a processed RNA, such as a single-stranded RNA that binds to the transcript of the F. graminearum target gene, with defined 5' and 3' ends that are free of potentially interfering flanking sequences. An alternative approach uses pol III promoters to generate transcripts with relatively defined 5' and 3' ends, i.e., transcribe RNA with minimal 5' and 3' flanking sequences. In some embodiments, pol III promoters (e.g., U6 or H1 promoters) are used to add short AT-rich transcription termination sites resulting in a two base pair overhang (UU) to the transcribed RNA, which is useful, for example, for the expression of siRNA type constructs. The use of pol III promoters to drive the expression of siRNA constructs has been reported. See van de Wetering et al. (2003) EMBO Rep., 4:609-615, and Tuschl (2002) Nature Biotechnol., 20:446-448. Baculovirus promoters, such as the baculovirus polyhedrin and p10 promoters, are known in the art and commercially available.See, e.g., Invitrogen's "Guide to Baculovirus Expression Vector Systems (BEVS) and Insect Cell Culture Techniques", 2002 (Life Technologies, Carlsbad, Calif.) and FJ Haines et al. "Baculovirus Expression Vectors", undated (Oxford Expression Technologies, Oxford, UK).

[0140] A promoter element may comprise a nucleic acid sequence that is not a naturally occurring promoter or promoter element or a homolog thereof, but is capable of regulating expression of a gene. Examples of such "gene-independent" regulatory sequences include naturally occurring or artificially designed RNA sequences that contain ligand-binding regions or aptamers (see below "aptamers") and regulatory regions (capable of cis-acting). See, e.g., Isaacs et al. (2004) Nat. Biotechnol., 22:841-847; Bayer and Smolke (2005) Nature Biotechnol., 23:337-343; Mandal and Breaker (2004) Nature Rev. Mol. Cell Biol., 5:451-463; Davidson and Ellington (2005) Trends Biotechnol., 23:109-112; Winkler et al. (2002) Nature, 419:952-956; Sudarsan et al. (2003) RNA, 9:644-647; and Mandal and Breaker (2004) Nature Struct. Mol. Biol., 11:29-35. Such "riboregulators" can be selected or designed for a particular spatial or temporal specificity, for example, to regulate the translation of DNA encoding a silencing element for suppressing F. graminearum target genes only in the presence (or absence) of a given concentration of the appropriate ligand. One example is a riboregulator that is responsive to an endogenous ligand (e.g., jasmonic acid or salicylic acid) produced by the plant under stress (e.g., abiotic stress such as water, temperature, or nutrient stress, or biotic stress such as infestation by pests or pathogens). Under stress, the level of the endogenous ligand increases to a level sufficient for the riboregulator to initiate transcription of DNA encoding a silencing element for suppressing F. graminearum target genes.

[0141] XVI. Recombinase Sites In some embodiments, the recombinant DNA construct or polynucleotide of the present invention comprises DNA encoding one or more site-specific recombinase recognition sites. In one embodiment, the recombinant DNA construct comprises at least one pair of loxP sites, and the site-specific recombination of DNA between the loxP sites is mediated by Cre recombinase. The position and relative orientation of the loxP sites are selected to achieve the desired recombination, for example, if the loxP sites are in the same orientation, the DNA between the loxP sites is excised in a circular fashion. In another embodiment, the recombinant DNA construct comprises DNA encoding one loxP site, and recombines the two DNAs in the presence of Cre recombinase and another DNA with a loxP site.

[0142] XVII. Transgene Transcription Unit In some embodiments, the recombinant DNA construct or polynucleotide of the invention comprises a transgene transcription unit. The transgene transcription unit comprises a DNA sequence encoding a gene of interest, e.g., a native or heterologous protein. The gene of interest can be any coding or non-coding sequence from any species, including but not limited to bacteria, and non-eukaryotic organisms such as viruses fungi, protists, plants, invertebrates, and vertebrates. The transgene transcription unit may further comprise 5' or 3' sequences, or both, required for transcription of the transgene.

[0143] XVIII. Intron In some embodiments, a recombinant DNA construct or polynucleotide of the invention comprises DNA encoding a spliceable intron. "Intron" generally refers to a segment of DNA (or RNA transcribed from such a segment) located between exons (protein-coding segments of DNA or the corresponding transcribed RNA); during maturation of messenger RNA, present introns are enzymatically "spliced ​​out" or removed from the RNA strand by a cleavage / ligation process that occurs in the eukaryotic nucleus. The term "intron" also applies to non-coding DNA sequences that can be spliced ​​out of the mature RNA transcript, but are transcribed into an RNA segment that is not an intron found between protein-coding exons. One example of these are splicable sequences that have the ability to enhance expression in plants (in some cases, particularly monocotyledons) of downstream coding sequences; these splicable sequences are naturally located in the 5' untranslated regions of some plant and some viral genes (e.g., the Tobacco Mosaic Virus 5' leader sequence or "omega" leader described by Gallie and Walbot (1992) Nucleic Acids Res., 20:4631-4638 as enhancing expression in plant genes). These splicable sequences or "expression-enhancing introns" can be artificially inserted into the 5' untranslated regions of plant genes between the promoters but before any protein-coding exons. Examples of such expression-enhancing introns include, but are not limited to, the maize alcohol dehydrogenase (Zm-Adh1), maize Bronze-1 expression-enhancing intron, rice actin 1 (Os-Act1) intron, Shrunken-1 (Sh-1) intron, maize sucrose synthase intron, heat shock protein 18 (hsp18) intron, and the 82 kilodalton heat shock protein (hsp82) intron.U.S. Pat. Nos. 5,593,874 and 5,859,347, specifically incorporated herein by reference, describe methods of improving recombinant DNA constructs for use in plants by including an expression-enhancing intron derived from the 70 kilodalton maize heat shock protein (hsp70) in the untranslated leader located 3' from the gene promoter and 5' from the first protein-coding exon.

[0144] XIX. Ribozymes In some embodiments, the recombinant DNA construct or polynucleotide of the present invention comprises DNA encoding one or more ribozymes. Ribozymes of particular interest include self-cleaving ribozymes, hammerhead ribozymes, or hairpin ribozymes. In one embodiment, the recombinant DNA construct comprises DNA encoding one or more ribozymes that serve to cleave transcribed RNA to provide a defined segment of RNA, such as a silencing element for suppressing F. graminearum target genes.

[0145] XX. Gene suppression elements In some embodiments, a recombinant DNA construct or polynucleotide of the invention comprises DNA encoding additional gene suppression elements to suppress a target gene other than the F. graminearum target gene in the DON pathway, such as a target gene essential for another essential metabolic function in F. graminearum, or a target gene in another fungus of the genus Fusarium. The target gene to be suppressed may include coding or non-coding sequences, or both.

[0146] Suitable gene suppression elements are described in detail in U.S. Patent Application Publication No. 2006 / 0200878, the disclosure of which is specifically incorporated herein by reference, and include one or more of the following: (a) DNA comprising at least one antisense DNA segment that is antisense to at least one segment of the gene to be suppressed; (b) DNA comprising multiple copies of at least one antisense DNA segment that is antisense to at least one segment of the gene to be silenced; (c) DNA comprising at least one sense DNA segment which is at least one segment of the gene to be silenced; (d) DNA comprising multiple copies of at least one sense DNA segment which is at least one segment of the gene to be silenced; (e) a DNA that is transcribed into RNA to suppress a gene to be suppressed by forming a double-stranded RNA, the DNA comprising at least one antisense DNA segment that is antisense to at least one segment of the gene to be suppressed and at least one sense DNA segment that is at least one segment of the gene to be suppressed, (f) a DNA that is transcribed into RNA for suppressing a gene to be suppressed by forming a single double-stranded RNA, the DNA comprising a plurality of contiguous antisense DNA segments that are antisense to at least one segment of the gene to be suppressed and a plurality of contiguous sense DNA segments that are at least one segment of the gene to be suppressed; (g) a DNA for transcribing into RNA for suppressing a gene to be suppressed by forming a plurality of RNA duplexes, the DNA comprising a plurality of antisense DNA segments that are antisense to at least one segment of the gene to be suppressed and a plurality of sense DNA segments that are at least one segment of the gene to be suppressed, the plurality of antisense DNA segments and the plurality of sense DNA segments being arranged in a series of inverted repeats; (h) DNA containing nucleotides derived from a plant miRNA; (i) DNA comprising siRNA nucleotides; (j) DNA that is transcribed into an RNA aptamer capable of binding to a ligand, and (k) DNA that transcribes into an RNA aptamer capable of binding to a ligand, and DNA that transcribes into a regulatory RNA capable of regulating expression of a gene to be silenced, wherein regulation is dependent on the conformation of the regulatory RNA, and wherein the conformation of the regulatory RNA is allosterically affected by the binding state of the RNA aptamer.

[0147] In some embodiments, introns are used to deliver gene suppression elements in the absence of any protein-coding exons (coding sequences).In one example, an intron, such as an expression-enhancing intron, is interrupted by embedding a gene suppression element within the intron, and upon transcription, the gene suppression element is excised from the intron.Therefore, protein-coding exons are not required to provide the gene suppression function of the recombinant DNA construct disclosed herein.

[0148] XXI. Transcriptional Regulatory Elements In some embodiments, the recombinant DNA construct or polynucleotide of the present invention comprises DNA encoding a transcriptional regulatory element. The transcriptional regulatory element includes an element that regulates the expression level of the recombinant DNA construct of the present invention (relative to its expression in the absence of such regulatory element). Examples of suitable transcriptional regulatory elements include riboswitches (cis or trans acting), transcription stabilizing sequences, transcription initiation sites, transcription elongation sequences, transcription termination elements, and miRNA recognition sites, as described in detail in U.S. Patent Application Publication No. 2006 / 0200878, which is specifically incorporated herein by reference.

[0149] XXII. Production and Uses of Transgenic Plant Cells and Transgenic Plants Plant transformation can include any of several well-known methods and compositions. Suitable methods for plant transformation include virtually any method that can introduce DNA into cells. One method of plant transformation is particle bombardment, as shown, for example, in U.S. Pat. No. 5,015,580 (soybean), U.S. Pat. No. 5,538,880 (corn), U.S. Pat. No. 5,550,318 (corn), U.S. Pat. No. 5,914,451 (soybean), U.S. Pat. No. 6,153,812 (wheat), U.S. Pat. No. 6,160,208 (corn), U.S. Pat. No. 6,288,312 (rice), U.S. Pat. No. 6,365,807 (rice), and U.S. Pat. No. 6,399,861 (corn), and U.S. Pat. No. 6,403,865 (corn), all of which are incorporated by reference to allow the production of transgenic plants.

[0150] Another useful method of plant transformation is Agrobacterium-mediated by Agrobacterium containing a dual Ti plasmid system, which carries a first Ti plasmid (often non-disabled) and a second chimeric plasmid containing at least one T-DNA border of the wild-type Ti plasmid, a promoter that functions in transformed plant cells and is operably linked to a polynucleotide or recombinant DNA construct of the present invention. See, for example, the binary system described in U.S. Pat. No. 5,159,135, which is incorporated by reference. See also De Framond (1983) Biotechnology, 1:262-269, and Hoekema et al., (1983) Nature, 303:179. In such a binary system, the T-DNA border or a smaller plasmid containing the border can be suitably constructed and engineered in a suitable alternative host such as E. coli and then transferred to Agrobacterium.

[0151] Detailed procedures for Agrobacterium-mediated transformation of plants, particularly crop plants, are described in U.S. Pat. Nos. 5,004,863, 5,159,135, and 5,518,908 (cotton), 5,416,011, 5,569,834, 5,824,877, and 6,384,301 (soybean), 5,591,616 and 5,981,840 (corn), These include procedures disclosed in U.S. Patent Nos. 5,463,174 (Brassicas, including canola), 7,026,528 (wheat), and 6,329,571 (rice), as well as U.S. Patent Application Publication Nos. 2004 / 0244075 (corn) and 2001 / 0042257A1 (sugar beet), all of which are specifically incorporated by reference to enable the production of transgenic plants. U.S. Patent Application Publication No. 2011 / 0296555 discloses transformation vectors (including vector sequences) and detailed protocols for transforming corn, soybean, canola, cotton, and sugarcane in Example 5, which are specifically incorporated by reference to enable the production of transgenic plants. Similar methods have been used to characterize peanut (Cheng et al. (1996) Plant Cell Rep., 15:653), asparagus (Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA, 84:5345), barley (Wan and Lemaux (1994) Plant Physiol., 104:37), rice (Toriyama et al. (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) Plant Cell Rep., 15:653), among others. It has been reported in many plant species, both dicotyledonous and monocotyledonous, including peanut (Sun et al. (1996) Transgen. Res., 5:313), and tomato (Sun et al. (2006) Plant Cell Physiol., 47:426-431).See also the description of vectors, transformation methods, and production of transformed Arabidopsis plants in which transcription factors are constitutively expressed by the CaMV35S promoter in U.S. Patent Application Publication No. 2003 / 0167537A1, which is incorporated by reference. Transformation methods particularly useful for plants are well known in the art. For example, tomato (Sharma et al. (2009), J. Biosci., 34: 423-433), eggplant (Arpaia et al. (1997) Theor. Appl. Genet., 95: 329-334), potato (Bannerjee et al. (2006) Plant Sci., 170: 732-738, Chakravarty et al. (2007) Amer. J. Potato Res., 84: 301-311, S. Millam “Agrobacterium-mediated transformation of potato.” Chapter 19 (pp. 257-270), “Transgenic Crops of the World: Essential Protocols”, Ian S. Curtis (editor), Springer, 2004)), and pepper (Li et al. (2003) Plant Cell Reports, 21:785-788). Stable transgenic potatoes, tomatoes, and eggplants have been commercially introduced in various regions. See, for example, K. Redenbaugh et al. "Safety Assessment of Genetically Engineered Fruits and Vegetables: A Case Study of the FLAVR SAVR Tomato", CRC Press, Boca Raton, 1992, and the extensive published documentation of commercial genetically modified crops in the GM Crop Database.See CERA. (2012). GM Crop Database. Center for Environmental Risk Assessment (CERA), ILSI Research Foundation, Washington DC, (available electronically at cera-gmc.org / ?action=gm_crop_database). Various methods of transformation of other plant species are well known in the art, see, for example, the encyclopedia reference "Compendium of Transgenic Crop Plants", edited by Chittaranjan Kole and Timothy C. Hall, Blackwell Publishing Ltd., 2008; ISBN 978-1-405-16924-0 (available electronically at mrw.interscience.wiley.com / emrw / 9781405181099 / hpt / toc). This includes cereals and forage grasses (rice, corn, wheat, barley, oats, sorghum, pearl millet, finger millet, cool season forage grasses, and bahia grass), oil crops (soybean, oilseed crucifers, sunflower, peanut, flax, sesame, and safflower), legume grains and forages (bean, cowpea, pea, broad bean, lentil, tepary bean, Asian bean, pigeon pea, vetch, chickpea, lupin, alfalfa, and clover), temperate fruits and nuts (apple, pear, pea, plum, berries, cherries, grapes, olives, almonds, Persian walnuts), tropical and subtropical The authors describe transformation procedures for fruits and nuts (citrus, grapefruit, bananas and plantains, pineapple, papaya, mango, avocado, kiwifruit, passion fruit, and persimmon), vegetable crops (tomato, eggplant, bell pepper, cruciferous vegetables, radish, carrot, cucurbits, leeks, asparagus, leafy vegetables), sugar, tuber, fiber crops (sugarcane, sugar beet, stevia, potato, sweet potato, cassava, and cotton), plantation crops, ornamental, and turfgrass (tobacco, coffee, cocoa, tea, rubber tree, medicinal plants, ornamentals, turfgrass), and forest tree species.

[0152] The transformation method to provide transgenic plant cells and transgenic plants containing stably integrated recombinant DNA is preferably carried out in tissue culture on medium and in a controlled environment. "Medium" refers to a number of nutrient mixtures used to grow cells in vitro, i.e., outside an intact organism. Recipient cell targets include, but are not limited to, meristematic cells, callus, immature embryos or parts of embryos, and gamete cells such as microspores, pollen, sperm, and eggs. Any cell capable of regenerating a fertile plant is contemplated as a useful recipient cell for the practice of the present invention. Callus can be initiated from a variety of tissue sources, including, but not limited to, immature embryos or parts of embryos, apical meristems of seedlings, microspores, and the like. Those cells capable of growing as callus can serve as recipient cells for genetic transformation. Practical transformation methods and materials for producing the transgenic plants of the 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. Pat. Nos. 6,194,636 and 6,232,526, and U.S. Patent Application Publication No. 2004 / 0216189, which are specifically incorporated by reference.

[0153] In common 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 identifying those cells that are stably transformed by receiving and integrating the transgenic DNA construct into their genome. A preferred marker gene provides a selective marker that confers resistance to a selective agent, such as an antibiotic or herbicide. Any of the antibiotics or herbicides to which the plant cells are resistant can be useful agents for selection. Potentially transformed cells are exposed to the selective agent that corresponds to the marker. In the population of surviving cells, there will generally be cells in which the gene that confers resistance (selective marker) is integrated and expressed at a sufficient level to allow cell survival in the presence of the selective agent. The cells can be further tested to confirm stable integration of the recombinant DNA. Commonly used selectable marker genes include those that confer resistance to antibiotics such as kanamycin or paromycin (nptll), hygromycin B (aph IV) and gentamicin (aac3 and aacC4), or to herbicides such as glufosinate (bar or pat) and glyphosate (EPSPS). Examples of useful selectable marker genes and selection agents are provided in U.S. Patent Nos. 5,550,318, 5,633,435, 5,780,708, and 6,118,047, all of which are specifically incorporated by reference. Screenable markers or reporters, such as markers that provide the ability to visually identify transformants, may also be used. Examples of useful screenable markers include, for example, genes that express proteins that produce a detectable color by acting on a chromogenic substrate (e.g., beta-glucuronidase (GUS) (uidA) or luciferase (luc), or something that is itself detectable, such as green fluorescent protein (GFP) (gfp) or an immunogenic molecule. One of skill in the art will recognize that many other useful markers or reporters are available.

[0154] Detection or measurement of transcription of the recombinant DNA construct in the transgenic plant cells can be accomplished by any suitable method, including protein detection methods (e.g., Western blot, ELISA, and other immunochemical methods), measurement of enzyme activity, or nucleic acid detection methods (e.g., Southern blot, Northern blot, PCR, RT-PCR, fluorescent in situ hybridization).

[0155] Other suitable methods for detecting or measuring transcription in plant cells of recombinant polynucleotides of the invention targeting F. graminearum target genes include measuring any other trait that is a direct or surrogate indicator of the expression level of the target gene in F. graminearum relative to the expression level observed in the absence of the recombinant polynucleotide, such as growth rate, mortality, or reproduction or recruitment rate of F. graminearum, or measuring injury (e.g., root injury) or yield loss in plants or fields of plants infected with F. graminearum. In general, suitable methods for detecting or measuring transcription in plant cells of a recombinant polynucleotide of interest include, for example, gross or microscopic morphological traits, growth rate, yield, reproduction or recruitment rate, 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 measurement of phenotypic traits 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). Such methods include direct measurements of resistance to F. graminearum (eg, damage to plant tissue) or proxy assays (eg, plant yield assays, or bioassays).

[0156] The recombinant DNA constructs of the invention can be stacked with other recombinant DNA to confer additional traits, for example, by expressing or suppressing other genes (e.g., in the case of transformed plants, traits including herbicide resistance, pest resistance, cold germination tolerance, water deficit tolerance, etc.). Constructs for the coordinated reduction and increase of gene expression are disclosed in U.S. Patent Application Publication No. 2004 / 0126845A1, which is specifically incorporated by reference.

[0157] Seeds of fertile transgenic plants can be collected and used to grow progeny generations, including hybrid generations of transgenic plants of the invention that contain the recombinant DNA construct in their genome. Thus, in addition to direct transformation of plants with the recombinant DNA construct of the invention, transgenic plants of the invention can be prepared by crossing a first plant carrying the recombinant DNA with a second plant that lacks the construct. For example, 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 infiltrate the recombinant DNA into the resulting progeny. Transgenic plants of the invention can be crossed with plant lines carrying other recombinant DNA that confers one or more additional traits (including but not limited to herbicide resistance, pest or disease resistance, environmental stress resistance, modified nutrient content, and improved yield) to produce progeny plants carrying recombinant DNA that confers both the desired target sequence expression behavior and the additional trait(s).

[0158] In such breeding to combine traits, the transgenic plants providing the additional traits can be male (pollinator) and the transgenic plants carrying the base trait can be female. The progeny of this cross will segregate such that some plants carry DNA for both parental traits and some carry DNA for one parent trait. Such plants can be identified by markers associated with the parent recombinant DNA progeny plants carrying DNA for both parental traits and crossed multiple times, e.g., usually six to eight generations, to the female parent line to produce homozygous progeny plants having substantially the same genotype as one original transgenic parent line, as well as the recombinant DNA of the other transgenic parent line.

[0159] Yet another aspect of the present invention is a transgenic plant grown from a transgenic seed of the present invention. The present invention contemplates transgenic plants grown directly from a transgenic seed containing recombinant DNA, as well as progeny generations of plants including inbred or hybrid plant lines produced by crossing a transgenic plant grown directly from a transgenic seed with a second plant not grown from the same transgenic seed. Crossing can include, for example, the following steps: (a) planting seeds or stem cuttings of a first parent plant (e.g., non-transgenic or transgenic) and a second parent plant, which is transgenic according to the invention; (b) growing seeds or stem cuttings of the first and second parent plants into flowering plants; (c) pollinating flowers from the first parent with pollen from the second parent; (d) Harvest the seeds produced by the parent plant with fertilized flowers.

[0160] In many cases, it is desirable to introduce recombinant DNA into an elite variety to transfer a particular desired trait from one source to an inbred or other plant lacking that trait, for example, by backcrossing. This can be accomplished, for example, by first crossing a superior inbred line ("A") (recurrent parent) to a donor inbred line ("B") (non-recurrent parent) carrying the appropriate gene(s) for the trait in question, e.g., a construct prepared according to the present invention. The progeny of this cross are selected within the initially obtained progeny for the desired trait to be transferred from the non-recurrent parent "B", and the selected progeny are then crossed to the superior recurrent parent "A". After five or more generations of backcrossing with selection for the desired trait, the progeny may be essentially hemizygous for the locus controlling the transferred characteristic, but are superior parents for most or nearly all other genes. The final backcross generation will be selfed to give progeny that are pure breeding for the gene(s) to be transferred, e.g., one or more transformation events.

[0161] Through a series of breeding operations, a selected DNA construct can be transferred from one line to a completely different line without the need for further recombinant operations. Thus, inbred plants can be produced that are true breeding for one or more DNA constructs. By crossing various inbred plants, a large number of different hybrids can be produced that have various combinations of DNA constructs. In this way, plants can be produced that have the desired agronomic characteristics ("hybrid vigor") that are frequently associated with hybrids, as well as the desired characteristics imparted by one or more DNA constructs.

[0162] In certain transgenic plant cells and transgenic plants of the invention, it may be desirable to regulate the expression of a F. graminearum target gene while simultaneously expressing a gene of interest. Thus, in some embodiments, the transgenic plant comprises a recombinant DNA that further comprises a gene expression element for expressing at least one gene of interest, and transcription of the recombinant DNA construct of the invention is affected by the simultaneous transcription of the gene expression element.

[0163] The present invention also provides commercial products produced from the transgenic plant cells, plants, or seeds of the present invention, including, but not limited to, harvested leaves, heads, ears, roots, shoots, stems, fruits, seeds, or other parts of the plant, oils, extracts, fermentation or digestion products, or any food or non-food product containing such commercial products produced from the transgenic plant cells, plants, or seeds of the present invention. Detection of one or more nucleic acid sequences of a recombinant DNA construct of the present invention in one or more of the commercial products or articles of commerce contemplated herein is factual evidence that the commercial product or article of commerce contains or is derived from a transgenic plant cell, plant, or seed of the present invention.

[0164] Generally, the genome of a transgenic plant carrying a recombinant DNA construct of the present invention, or a portion thereof, exhibits increased resistance to DON production due to F. graminearum infection. In various embodiments, for example, when a transgenic plant expresses a recombinant DNA construct of the present invention stacked with other recombinant DNA to confer additional traits, the transgenic plant 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 biological stress tolerance; (c) an altered primary metabolite composition; (d) altered secondary metabolite composition; (e) modified trace element, carotenoid, or vitamin compositions; (f) improved yield; (g) improving the ability to use nitrogen, phosphate, or other nutrients; (h) modified agronomic characteristics; (i) Altered growth or reproductive characteristics; and (j) Improving the quality of harvesting, storage, or processing.

[0165] In some embodiments, the transgenic plant exhibits improved tolerance of abiotic stress (e.g., tolerance of water deficit or drought, heat, cold, non-optimal nutrient or salt levels, non-optimum light levels) or biotic stress (e.g., crowding, allelopathy, or injury), altered primary metabolite (e.g., fatty acids, oils, amino acids, proteins, sugars, or carbohydrates) composition, altered secondary metabolite (e.g., alkaloids, terpenoids, polyketides, non-ribosomal peptides, and secondary metabolites of mixed biosynthetic origin) composition, altered trace element (e.g., iron, zinc), carotenoid (e.g., beta-carotene, lycopene, lutein, zeaxanthin, or other carotenoids and xanthophylls), or vitamin (e.g., tocopherol) composition, improved yield (e.g., yield under non-stress conditions), or improved plant function (e.g., improved plant physiology, plant nutrition, and plant health). improved quantity, or improved yield under biotic or abiotic stress), improved ability to use nitrogen, phosphate, or other nutrients, altered agronomic characteristics (e.g., delayed ripening, delayed senescence, early or delayed maturation, improved shade tolerance, improved resistance to root or stem lodging, improved resistance to stem "green snap", altered photoperiod response), altered growth or reproductive characteristics (e.g., intentional dwarfing, intentional male sterility, useful, for example, in improved hybridization procedures, improved vegetative growth rate, improved germination; improved male or female fertility), improved harvest, storage, or processing qualities (e.g., improved resistance to pests during storage, improved resistance to spoilage, improved consumer appeal), or any combination of these traits.

[0166] In another embodiment, the transgenic seed, or the seed produced by the transgenic plant, has an altered primary metabolite (e.g., fatty acid, oil, amino acid, protein, sugar, or carbohydrate) composition, an altered secondary metabolite composition, an altered trace element, carotenoid, or vitamin composition, improved harvest, storage, or processing qualities, or a combination thereof. In another embodiment, it may be desirable to alter the levels of natural components of the transgenic plant or the seed of the transgenic plant, for example, to reduce the levels of allergenic proteins or glycoproteins, or of toxic metabolites.

[0167] Generally, a population of transgenic plants, each regenerated from a transgenic plant cell, is screened to identify transgenic plant cells that develop into transgenic plants with desired traits. The transgenic plants are assayed to detect enhanced traits, such as enhanced water use efficiency, enhanced cold tolerance, enhanced yield, enhanced nitrogen use efficiency, enhanced seed protein, and enhanced seed oil. Screening methods include direct screening of traits in greenhouse or field trials, or screening of alternative traits. Such analyses are directed to detecting changes in the chemical composition, biomass, physiological properties, or morphology of the plants. Changes in chemical composition can be detected by analysis of seed composition and content of protein, free amino acids, oil, free fatty acids, starch, tocopherol, or other nutrients. Changes in growth or biomass characteristics are detected by measuring plant height, stem diameter, internode length, root and shoot dry weight. Changes in physiological traits are identified by assessing the response to stress conditions, e.g., assays under imposed stress conditions such as water deficiency, nitrogen or phosphate deficiency, low or high growth conditions, pathogen or insect attack, light deficiency, or increased plant density. Other selection traits include days to flowering, days to pollen dispersal, days to fruit maturity, fruit quality or yield, leaf elongation rate, chlorophyll content, leaf temperature, standing position, seedling vigor, internode length, plant height, leaf number, leaf area, tillering, brace root, greening retention, stem lodging, root lodging, plant health, fertility, green snap, and pest resistance. Additionally, phenotypic traits of harvested fruit or seeds can be assessed, e.g., in plants, this can include the total number or weight of harvested fruit, or the color, acidity, sugar content, or flavor of such fruit.

[0168] The following examples are presented for purposes of illustration and should not be construed as limiting. EXAMPLES

[0169] summary This example aims to highlight the development of exogenous application of dsRNA to control DON production by F. graminearum in plants.

[0170] Introduction RNA interference (RNAi) is a naturally occurring cellular defense system mediated by double-stranded RNA (dsRNA). The first component of the RNAi machinery that responds to dsRNA is the RNase III endonuclease Dicer-2, which cleaves dsRNA into short (typically 19-21 nucleotides long) interfering RNAs (siRNAs). With the help of dsRNA-binding proteins, Dicer-2 facilitates the transfer of siRNAs into the RNA-induced silencing complex. RNAi promotes gene silencing that affects the translation of host genetic material. RNAi is a sequence-specific method of suppressing expression of target genes, and because each species is defined by the uniqueness of the sequence of its genes, RNAi can be designed in a species-specific manner. By targeting genes essential for the production of DON by the pathogen, RNAi can be selectively used to control the production of DON by F. graminearum and F. graminearum without adversely affecting non-target beneficial species. Tables 1 and 2 provide a list of internal reference numbers, associated start sequence accession numbers, and target gene sequences, trigger sequences, and trigger RNA reverse complements. [Table 1] [Table 2-1] [Table 2-2] [Table 2-3]

[0171] Example 1 DON Reduction: In vitro 96-well plates with DON medium (nutrient-rich M9 medium + DON inducer agmatine) were inoculated with Fusarium graminearum spores and then treated with dsRNA. After incubating the plates for 5 days, liquid cultures from each well were collected and analyzed using liquid chromatography-mass spectrometry (LC-MS) to quantify total deoxynivalenol (DON and ADON) mycotoxins. The average DON reduction percentage was calculated by comparing the total DON in each treatment to the untreated control (UTC). Error bars indicate one standard deviation among three independent runs. Results are shown in Figures 1 and 4. Each test sequence showed a reduction in DON compared to the untreated control. Further results are shown in Tables 3 and 4, showing the average DON reduction % and standard deviation across several runs. In Tables 3 and 4, sequences scored as ++ have an average DON reduction % of more than one standard deviation from the negative control (GS5797) and a standard deviation of less than 20%. Sequences scored as + indicate a mean % reduction in DON greater than the negative control. [Table 3] [Table 4-1] [Table 4-2] [Table 4-3]

[0172] Example 2 Average DON production by F. graminearum on detached wheat heads: Detached wheat heads were inoculated with Fusarium graminearum spores and then treated with dsRNA. After 12 days, wheat heads were collected and analyzed using liquid chromatography-mass spectrometry (LC-MS) to quantify total deoxynivalenol (DON and ADON) mycotoxins. Mean DON is plotted in parts per million (ppm) of the dry weight of the wheat head. Error bars indicate one standard deviation between two independent runs. The results are shown in Figure 2, where each test sequence showed reduced DON production compared to the negative control.

[0173] Example 3 Relative expression of target genes Mycelium from F. graminearum samples tested as described in Example 1 was collected and RNA was extracted using Zymo Research's Quick RNA Miniprep. RNA was reverse transcribed with oligo-dT primers and gene expression was estimated using qPCR by relative quantification to Fusarium housekeeping genes. Fold changes of target genes were calculated using the delta-delta Ct method and plotted against the respective negative control treatments. Statistical significance was determined using a non-parametric two-tailed T-test. Error bars indicate 95% confidence intervals. Results are shown in Figure 3 and demonstrate that the test sequences significantly reduced expression of the relevant target genes compared to the negative control.

Claims

1. A composition for controlling DON production in F. graminearum, comprising: (a) an effective amount of a polynucleotide comprising at least 18, 19, 20, 21, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or 650 contiguous nucleotides that are essentially complementary to, or comprise at least about 85%, at least about 90%, at least about 95%, at least about 98%, about 100%, or 100% sequence identity to, a target gene having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-16, 65-70, and 87-151, or a segment of RNA transcribed from said target gene; or (b) an effective amount of at least one polynucleotide comprising at least one silencing element that is essentially complementary to, or comprises at least about 85%, at least about 90%, at least about 95% sequence identity to, at least 18, 19, 20, 21, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or 650 consecutive nucleotides of a target gene or an RNA transcribed from said target gene, wherein said target gene has a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-16, 65-70, and 87-151; or (c) an effective amount of at least one RNA comprising at least one segment that is essentially complementary to, or comprises at least about 85%, at least about 90%, at least about 95%, at least about 98%, about 100%, or 100% sequence identity to, at least 18, 19, 20, 21, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or 650 consecutive nucleotides of a target gene having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-16, 65-70, and 87-151, or a segment of an RNA transcribed from said target gene; or (d) an RNA molecule that, when transfected into or contacted with F. graminearum, causes a reduction or elimination of DON production in said F. graminearum, wherein said RNA molecule comprises at least 18, 19, 20, 21, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or 650 contiguous nucleotides that are essentially complementary to, or comprise at least about 85%, at least about 90%, at least about 95%, at least about 98%, or about 100%, or 100% sequence identity to, a target gene or a segment of RNA transcribed from said target gene having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-16, 65-70, and 87-151; or (e) a double-stranded RNA molecule that, when transfected into or contacted with F. graminearum, causes a reduction or elimination of DON production in said F. graminearum, wherein at least one strand of the double-stranded RNA molecule comprises at least 18, 19, 20, 21, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or 650 contiguous nucleotides that are essentially complementary to, or comprise at least 85%, 90%, 95%, 98%, or 100% sequence identity to, a target gene or a segment of RNA transcribed from said target gene, and wherein said target gene has a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-16, 65-70, and 87-151; or (f) an effective amount of at least one double-stranded RNA comprising at least one strand comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 33-64, 79-86, 153-225, and 226-299, or a sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98%, about 100%, or 100% sequence identity thereto; or (g) an effective amount of a polynucleotide comprising at least 18, 19, 20, 21, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, or 600 contiguous nucleotides of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 33-64, 79-86, 153-225, and 226-299, or a sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98%, about 100%, or 100% sequence identity thereto; or (h) an effective amount of at least one RNA comprising at least one segment that is essentially complementary to, or comprises at least about 85%, at least about 90%, at least about 95%, at least about 98%, about 100%, or 100% sequence identity to, at least 18, 19, 20, 21, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 consecutive nucleotides of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 33-64, 79-86, 153-225, and 226-299; or (i) an RNA molecule that, when transfected into or contacted with F. graminearum, causes a reduction or elimination of DON production by said F. graminearum in a plant, wherein said RNA molecule comprises at least 18, 19, 20, 21, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 contiguous nucleotides that are essentially complementary to, or have at least about 85%, at least about 90%, at least about 95%, at least about 98%, about 100%, or 100% sequence identity to, a segment of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 33-64, 79-86, 153-225, and 226-299; or (j) a double-stranded RNA molecule that, when transfected into or contacted with F. graminearum, causes a reduction or elimination of DON production by said F. graminearum in a plant, wherein at least one strand of the double-stranded RNA molecule comprises at least 18, 19, 20, 21, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 contiguous nucleotides that are essentially complementary to or have at least about 85%, at least about 90%, at least about 95%, at least about 98%, about 100%, or 100% sequence identity to a segment of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 33-64, 79-86, 153-225, and 226-299; or (k) A composition comprising a double-stranded RNA molecule that, when transfected into or contacted with F. graminearum, causes a reduction or elimination of DON production by said F. graminearum in a plant, wherein at least one strand of the double-stranded RNA molecule comprises at least about 85%, at least about 90%, at least about 95%, at least about 98%, about 100%, or 100% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 33-64, 79-86, 153-225, and 226-299.

2. 10. The composition of claim 1, wherein the composition is in at least one form selected from the group consisting of a solid, a liquid, a powder, a suspension, an emulsion, a spray, an encapsulation, a microbead, a carrier particle, a film, a matrix, a seed treatment, a soil drench, and a transplantable formulation.

3. 3. The composition of claim 1 or 2, further comprising at least one component selected from the group consisting of a carrier agent, a surfactant, an organosilicone, an organosilicone surfactant, a polynucleotide herbicidal molecule, a non-polynucleotide herbicidal molecule, a non-polynucleotide pesticide, a polynucleotide pesticide, a safener, and a pathogen growth regulator.

4. The composition of any one of claims 1, 2, or 3, wherein the polynucleotide, or the RNA, or the dsRNA, when transfected into or contacted with F. graminearum, causes a reduction or elimination of DON production by the F. graminearum, and the double-stranded RNA molecule comprises at least one segment that is essentially complementary to or has at least 95% sequence identity with at least 21 consecutive nucleotides of a target gene or RNA transcribed from the target gene having a sequence selected from the group consisting of SEQ ID NOs: 1-16, 65-70, and 87-151, and the double-stranded RNA molecule is at least 300 base pairs in length, or is about 350 to about 600 base pairs in length.

5. 5. The composition of any one of claims 1, 2, 3, or 4, wherein the polynucleotide, or RNA, or dsRNA comprises a dsRNA comprising a first strand comprising a nucleotide sequence at least about 85%, at least about 90%, at least about 95%, at least about 98%, about 100%, or 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 33-64, 79-86, 153-225, and 226-299.

6. 6. The composition of claim 5, wherein the first strand comprises a nucleotide sequence that is at least about 98% identical to a sequence selected from the group consisting of SEQ ID NOs: 33-64, 79-86, 153-225, and 226-299.

7. The composition of claim 5 or 6, wherein the polynucleotide, or the RNA, or the dsRNA further comprises a second strand that is complementary to the first strand.

8. 8. The composition of any one of claims 1-7, wherein the nucleotide sequence of (f), (g), (h), (i), (j), or (k) is selected from the group consisting of SEQ ID NOs: 48, 64, 80-82, 84-86, 182, 188, 194, 213, 220, 256, 262, 268, 287, and 294.

9. 9. The composition of any one of claims 1 to 8, wherein the nucleotide sequence of (f), (g), (h), (i), (j), or (k) comprises SEQ ID NO:

48.

10. The composition of any one of claims 1 to 7, wherein the target genes listed in (a) to (e) have a nucleotide sequence selected from the group consisting of SEQ ID NOs: 16, 68-70, 108, 114, 120, 139, and 146.

11. 11. The composition of any one of claims 1 to 10, wherein the RNA molecule of (d) or (i), or the dsRNA molecule of (e), (j), or (k) further causes a reduction or elimination of DON production in one or more additional fungal pathogens of the genus Fusarium, optionally wherein the one or more additional fungal pathogens comprises F. culmorum.

12. A dsRNA that inhibits expression of a F. graminearum target gene, wherein a first strand of the dsRNA is at least 300 nucleotides in length and comprises an RNA sequence that is 85% to 100% complementary to an RNA encoded by a sequence selected from the group consisting of SEQ ID NOs: 16, 68-70, 108, 114, 120, 139, and 146.

13. 13. The dsRNA of claim 12, wherein a second strand of the dsRNA is complementary to the first strand.

14. 14. The dsRNA of claim 12 or 13, wherein the RNA sequence comprises a nucleotide sequence that is at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, about 100% identical, or 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 48, 64, 80-82, 84-86, 182, 188, 194, 213, 220, 256, 262, 268, 287, and 294.

15. 15. The dsRNA of claim 14, wherein the RNA sequence comprises a nucleotide sequence that is at least about 98% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 48, 64, 80-82, 84-86, 182, 188, 194, 213, 220, 256, 262, 268, 287, and 294.

16. The dsRNA of claim 14 or 15, wherein the nucleotide sequence is SEQ ID NO:

48.

17. The dsRNA of any one of claims 12 to 16, wherein the dsRNA further inhibits expression of a target gene in one or more additional species of the genus Fusarium, and optionally, the one or more additional species of the genus Fusarium comprises F. culmorum.

18. A dsRNA that inhibits expression of a F. graminearum target gene, wherein a first strand of the dsRNA comprises an RNA sequence that is at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, about 100% identical, or 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 48, 64, 80-82, 84-86, 182, 188, 194, 213, 220, 256, 262, 268, 287, and 294.

19. 20. The dsRNA of claim 18, further comprising a second strand that is complementary to the first strand.

20. 20. The dsRNA of claim 18 or 19, wherein the first strand of the dsRNA comprises an RNA sequence at least about 98% identical to a sequence selected from the group consisting of 48, 64, 80-82, 84-86, 182, 188, 194, 213, 220, 256, 262, 268, 287, and 294.

21. 21. The dsRNA of claim 20, wherein the first strand of the dsRNA comprises an RNA sequence that is at least about 98% identical to SEQ ID NO:

48.

22. A composition comprising the dsRNA of any one of claims 12 to 21 formulated for application in a form selected from the group consisting of a sprayable solution, an emulsion, a tank mix, and a powder.

23. 23. The composition of claim 22, further comprising one or more additional components selected from the group consisting of carrier agents, surfactants, organosilicones, organosilicon surfactants, polynucleotide herbicidal molecules, non-polynucleotide herbicidal molecules, polynucleotide pesticides, non-polynucleotide pesticides, polynucleotide fungicides, non-polynucleotide fungicides, polynucleotide insecticides, non-polynucleotide insecticides, safeners, and pathogen growth regulators.

24. 12. The composition of any one of claims 1 to 11, wherein the at least 18 contiguous nucleotides listed in (a)-(e), (g)-(j) is at least 300 contiguous nucleotides.

25. A method for reducing or eliminating DON production by one or more fungi of the genus Fusarium, comprising contacting the one or more fungi with a composition comprising a polynucleotide that causes RNA interference against one or more genes involved in the production of DON, resulting in reduced DON production by the fungi.

26. 26. The method of claim 25, wherein the one or more genes are selected from the group consisting of FGP1, ELP3, SPT7, MAF1, MVD1, HEP1, SET1, FKPB12.

27. 27. The method of claim 25 or 26, wherein said contacting comprises application of said composition to a surface of a plant that is infected or capable of being infected with said fungus.

28. 28. The method of claim 27, wherein said applying comprises spraying said composition onto the leaves, heads, stems, panicles, flowers, or fruits of said plant.

29. The method according to any one of claims 25 to 27, wherein the plant is selected from the group consisting of maize and small grains.

30. 30. The method of claim 29, wherein the plant is wheat.

31. The method of any of claims 25 to 30, wherein the composition comprises any of the compositions of claims 1 to 23.

32. The method of any one of claims 25 to 31, wherein the one or more fungi of the genus Fusarium include F. graminearum, and optionally the one or more fungi of the genus Fusarium further include one or more additional fungal species, and optionally the one or more additional fungal species include F. culmorum.

33. 22. A method for reducing or eliminating DON production by F. graminearum on a plant, comprising topically applying to said plant any of the compositions of claims 1-21.

34. 34. The method of claim 33, wherein the topical application comprises spraying the composition onto the leaves, heads, flowers, stems, ears, or fruits of the plant.

35. 35. The method of claim 34, wherein the topical application comprises spraying the composition onto the leaves, heads, or panicles of the plant.

36. The method according to any one of claims 33 to 35, wherein the plant is selected from the group consisting of wheat, barley, oats, and corn.

37. 37. The method of claim 36, wherein the plant is a cereal crop.

38. 38. The method of any of claims 33-37, wherein RNA interference is induced, resulting in the reduction or elimination of DON production by F. graminearum.

39. 1. A method for reducing or eliminating DON production by F. graminearum on a plant, comprising: (a) contacting said F. graminearum with at least one polynucleotide comprising a nucleotide sequence that is essentially complementary to, or comprises at least 85%, 90%, or 95% sequence identity to, at least 18 consecutive nucleotides of a target gene having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-16, 65-70, and 87-151, or an RNA transcribed from said target gene; or (b) topically applying to said plant a composition comprising at least one polynucleotide comprising a nucleotide sequence that is essentially complementary to, or comprises at least 85%, 90%, or 95% sequence identity to, at least 18 consecutive nucleotides of a target gene having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-16, 65-70, and 87-151, or an RNA transcribed from said target gene; or (c) expressing in said plant at least one polynucleotide comprising at least one segment essentially complementary to, or comprising at least 85%, 90%, or 95% sequence identity to, at least 18 consecutive nucleotides of a DNA having a sequence selected from the group consisting of SEQ ID NOs: 1-16, 65-70, and 87-151; (d) contacting the F. graminearum with an effective amount of double-stranded RNA, at least one strand of which comprises a segment that is essentially complementary to, or has at least 85%, 90%, or 95% sequence identity to, at least 18 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 33-64, 79-86, 153-225, and 227-299; or (e) locally applying to said plant an effective amount of double-stranded RNA, at least one strand of which comprises a segment that is essentially complementary to, or comprises at least 85%, 90%, or 95% sequence identity to, at least 18 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 33-64, 79-86, 153-225, and 227-299.

40. 40. The method of claim 39, wherein the polynucleotide is double-stranded RNA.

41. 41. The method of claim 39 or 40, wherein the double-stranded RNA is chemically or enzymatically synthesized, or produced by expression in a microbial organism, or by expression in a plant cell.

42. 42. The method of any of claims 39, 40, or 41, wherein the double-stranded RNA comprises a strand comprising a nucleotide sequence comprising at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 48, 64, 80-82, 84-86, 182, 188, 194, 213, 220, 256, 262, 268, 287, and 294.

43. The method of any of claims 39 to 42, wherein the double-stranded RNA comprises a strand comprising at least 21 consecutive nucleotides of SEQ ID NO:

48.

44. The method of any one of claims 39 to 43, wherein the nucleotide sequence according to claim 39 (d) or (e) is selected from the group consisting of SEQ ID NOs: 48, 64, 80-82, 84-86, 182, 188, 194, 213, 220, 256, 262, 268, 287, and 294.

45. 45. The method of any of claims 39-44, wherein the method comprises topically applying to the plant a composition comprising at least one polynucleotide comprising a nucleotide sequence that is essentially complementary to, or comprises at least 85%, 90%, or 95% sequence identity to, at least 18, 19, 20, or 21 consecutive nucleotides of SEQ ID NO:

48.

46. 46. ​​The method of any one of claims 39-45, wherein the method comprises contacting the F. graminearum with a solution comprising an effective amount of double-stranded RNA, wherein at least one strand of the double-stranded RNA is essentially complementary to, or comprises at least 85%, 90%, or 95% sequence identity to, at least 200, 300, 400, 500, or 600 contiguous nucleotides of a gene having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-16, 65-70, 87-151, and wherein RNA interference is induced, resulting in the reduction or elimination of DON production by the F. graminearum.

47. 47. The method of claim 46, wherein said at least one strand of said double stranded RNA comprises a sequence that is essentially complementary to, or comprises at least 85%, 90%, or 95% sequence identity to, at least 200, 300, 400, 500, or 600 contiguous nucleotides of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 48, 64, 80-82, 84-86, 182, 188, 194, 213, 220, 256, 262, 268, 287, and 294.

48. 48. The method of claim 46 or 47, wherein the solution further comprises one or more components selected from the group consisting of organosilicone surfactants, carrier agents, organosilicones, organosilicone surfactants, polynucleotide herbicidal molecules, non-polynucleotide herbicidal molecules, polynucleotide pesticides, non-polynucleotide pesticides, safeners, and pathogen growth regulators.

49. 49. The method of any one of claims 39 to 48, wherein the plant is selected from the group consisting of wheat, barley, oats, and corn.

50. 50. The method of any one of claims 39-49, wherein the at least 18 contiguous nucleotides listed in (a) through (e) is at least 400 contiguous nucleotides.

51. 51. A plant having improved resistant DON production of F. graminearum, or a fruit, seed, or propagule of said plant, provided by the method of any of claims 39-50.

52. 52. The plant of claim 51, wherein the plant is selected from the group consisting of wheat, barley, oats, and corn.

53. 1. A recombinant DNA construct comprising: (a) a DNA comprising a nucleotide sequence that is essentially complementary to, or has at least 85%, 90%, or 95% sequence identity with, at least 18 consecutive nucleotides of a target gene having a sequence selected from the group consisting of SEQ ID NOs: 1-16, 65-70, and 87-151, or an RNA transcribed from said target gene; or (b) a DNA comprising 18 or more consecutive nucleotides having at least 85%, 90%, or 95% identity to a fragment of equivalent length of a DNA having a sequence selected from the group consisting of SEQ ID NOs: 1-16, 65-70, and 87-151, or the DNA complements thereof; or (c) DNA encoding at least one silencing element that is essentially complementary to or has at least 85%, 90%, or 95% sequence identity with at least 18 consecutive nucleotides of a target gene or an RNA transcribed from said target gene, wherein said target gene has a sequence selected from the group consisting of SEQ ID NOs: 1-16, 65-70, and 87-151; or (d) A recombinant DNA construct comprising a heterologous promoter operably linked to DNA encoding an RNA comprising a sequence selected from the group consisting of SEQ ID NOs: 33-64, 79-86, and 153-225, 227-299, or a sequence having at least 85%, 90%, or 95% sequence identity thereto.

54. 54. A plant chromosome or plastid or a recombinant plant virus vector or a recombinant baculovirus vector comprising the recombinant DNA construct of claim 53.

55. A transgenic plant cell having in its genome the recombinant DNA construct described in claim 53.

56. 56. A transgenic plant comprising the transgenic plant cell of claim 55, or a fruit, seed, or propagule of said transgenic plant.

57. 57. The plant of claim 55 or 56, wherein the at least 18 contiguous nucleotides listed in (a) to (d) are at least 18, 19, 20, or 21 contiguous nucleotides.

58. A DNA encoding the polynucleotide, RNA, or dsRNA according to any one of claims 1 to 52.

59. 54. The recombinant DNA construct of claim 53, wherein the at least 18 contiguous nucleotides listed in (a) through (d) are at least 300 contiguous nucleotides.

60. 55. The plant chromosome or plastid, or recombinant plant virus vector or recombinant baculovirus vector of claim 54, wherein the at least 18 contiguous nucleotides listed in (a) to (d) are at least 300 contiguous nucleotides.

61. 56. The transgenic plant cell of claim 55, wherein the at least 18 contiguous nucleotides listed in (a) through (d) are at least 300 contiguous nucleotides.

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