RNA-based control of deoxynivalenol production of fusarium genus

Abstract

The present disclosure relates to a method of using dsRNA to reduce or eliminate DON production of and / or control of Fusarium graminearum or other fungal pathogens of the genus Fusarium. The present disclosure relates to a method of using dsRNA to reduce or eliminate DON production of and / or control of Fusarium graminearum or other fungal pathogens of the genus Fusarium. In particular embodiments, methods and compositions are described for controlling DON production and / or controlling fungal pathogens by causing a reduction in death, growth inhibition, virulence or pathogenicity or a reduction in reproductive / reproductive capacity (sporogenesis) by using exogenous dsRNA application to a plant, the plants are infected or infected with Fusarium graminearum or another member of the Fusarium genus that produces DON or is otherwise associated with Fusarium complexes that cause gibberellic disease.

Description

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

[0002] References to Electronic Sequence Listings

[0003] This application contains a sequence listing that has been submitted electronically in .XML format and is hereby incorporated 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 part of the specification and is hereby incorporated by reference in its entirety. Background Art

[0004] Fusarium head blight (FHB) is a scab disease of wheat, barley and other small cereals caused by a complex of Fusarium species. Fusarium graminearum is one of the most widespread and economically important species of this complex due to its ability to produce high levels of mycotoxins. Infection with Fusarium species can lead to significant yield losses and reduced grain quality, as well as the production of mycotoxins after ingestion by animals and humans (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 caused by fungal infection to wheat and / or barley head, but more specifically the associated mycotoxin (e.g., deoxynivalenol or "DON") contamination, which often leads to reduced grain quality, thus affecting pricing and distribution to terminal markets (Foroud et al., 2019). It was estimated that the cost of FHB in the United States exceeded $1 billion in 1993 and $500 million in 1994. More recently, the cost of epidemic FHB in the United States between 1993 and 2001 was estimated at $7.67 billion (Mielniczuk and -Bednarz, 2020). It is estimated that in China, FHB could affect up to 7 million hectares of land in an epidemic year and that 2.5 million tonnes of grain could be lost. Illnesses in humans due to mycotoxin ingestion have been reported in China, India and Japan, while illnesses in animals due to mycotoxin ingestion have been reported in many parts of the world (Dubin et al., 1997).

[0005] Infection occurs during the flowering period through the anthers, followed by colonization of other parts of the floret, and then entering the vessels and pith of the rachis and spreading from there up and down the bulb (reviewed by Trail et al., 2005). Many Fusarium species in the FHB complex produce trichothecenes. For example, F. graminearum and F. culmorum may produce deoxynivalenol (DON) and nivalenol (NIV), and F. sporotrichioides may produce T-2 toxin. Many Fusarium species have been described as being able to biosynthesize other mycotoxins, such as fumonisins and zearalenone (ZEA) (Desjardins, 2006), including both F. graminearum and F. culmorum that produce ZEA (Mielniczuk and -Bednarz, 2020). The first key step in the trichothecene biosynthetic pathway is the conversion of farnesyl pyrophosphate to trichothecenes catalyzed by the trichothecene synthase encoded by TRI5 (Hohn and Beremand, 1989). The expression of TRI5 and other genes in the gene cluster is associated with the production of trichothecenes both in planta and in vitro culture (Brown et al., 2004; Kimura et al., 2003).

[0006] In addition to the role of trichothecenes in disease processes, their presence in cereals has important implications for human and animal health. In Europe and the United States, the concentrations of these toxins in cereals are under increasing scrutiny, and legal limits for contamination of cereals and foods with trichothecenes such as DON are currently being prepared (Anonymous, 2005; van Egmond et al., 2007). In mammalian systems, DON has been shown to be a potent inhibitor of conversion and causes acute symptoms (e.g., vomiting and feed refusal) as well as chronic symptoms such as growth retardation, ovarian failure, and immunosuppression in farm animals such as pigs (Bennett and Klich, 2003; Rocha et al., 2005).

[0007] To address these problems, the present invention relates, inter alia, to the topical application of double-stranded RNA ("dsRNA") that targets fungal genes associated with the DON production pathway for RNA interference and thereby reduces or eliminates DON production by Fusarium graminearum or other fungal pathogens that cause Fusarium head blight in plants or plant parts intended for human or animal consumption. Summary of the invention

[0008] RNA interference (RNAi) technology has been shown to be a highly selective biological treatment that silences the gene expression of pests and pathogens through internal biological processes. Exogenous application of double-stranded RNA (dsRNA) that initiates RNAi has been used to effectively control certain plant pests and pathogen species. The present disclosure relates to a method for reducing or eliminating the DON production of the fungal pathogen Fusarium graminearum or other fungal pathogens of the genus Fusarium using dsRNA and / or controlling the Fusarium graminearum or other fungal pathogens of the genus Fusarium. In a specific embodiment, it is described to control the production of DON and / or control the method and composition of fungal pathogens by causing death, growth inhibition, reduction of toxicity or pathogenicity or reduction of reproduction / reproductive capacity (spore formation) by using exogenous dsRNA applied to plants, the plant is infected or can be infected with Fusarium graminearum or another member of the genus Fusarium that produces DON or is otherwise associated with the Fusarium complex that causes head blight. Such plants can include, for example, corn and small grains, such as wheat, barley, flax, buckwheat, rye and oats.

[0009] The compositions and methods described herein include recombinant polynucleotide molecules, such as single-stranded or double-stranded DNA or RNA molecules, referred to herein as "triggers", which can be used to reduce or eliminate DON production by Fusarium graminearum or related species; or recombinant DNA constructs, which are used to prepare such RNA molecules or to prepare transgenic plants expressing such RNA molecules. In some embodiments, polynucleotide triggers are provided as locally applied agents for controlling or preventing DON production by Fusarium graminearum on plants and / or causing death, growth inhibition, reduction in virulence or pathogenicity, or reduction in reproduction / reproductive capacity (spore formation) of Fusarium graminearum. In some embodiments, the plant is corn or a small grain (e.g., wheat, barley, oats, flax, rye, or buckwheat) that has increased resistance to infection by Fusarium graminearum and / or has increased resistance to the effects of DON produced by Fusarium graminearum, such as transgenic plants (including seeds and fertile parts) expressing polynucleotide triggers are provided. In some embodiments, plants (including seeds or fertile parts) that have been topically treated with a composition comprising a polynucleotide trigger are provided (e.g., plants that have been sprayed with a solution of dsRNA molecules). Also provided are polynucleotide-containing compositions for topical application to Fusarium graminearum or to plants, plant parts or seeds infected or potentially infected with Fusarium graminearum.

[0010] Several embodiments relate to inhibiting target genes in Fusarium graminearum by polynucleotide triggers. Such target genes may include any gene of Fusarium associated with DON production pathways. The nucleotide sequences referred to herein as "target gene sequence groups" or "target gene sequences" of such target genes are provided herein, and the nucleotide sequences relate to SEQ ID No: 1-16, 65-70 and 87-151. Certain embodiments of the present invention relate to polynucleotides (e.g., dsRNA) designed to hybridize with RNA transcripts of target genes to produce RNAi. The nucleotide sequences referred to herein as "trigger sequence groups" or "trigger sequences" are also provided, and the nucleotide sequences relate to SEQ ID NO: 17-32, 75-78. "RNA trigger sequence groups" or "RNA trigger sequences" are further provided herein, and they relate to SEQ ID NO: 33-48, 79-82 and 153-225. Also provided herein are reverse complements for the RNA trigger sequence set, referred to as "RNA trigger sequence reverse complement set" or "RNA trigger sequence reverse complement", which reverse complements relate to SEQ ID No: 49-64, 83-86 and 227-299. The RNA trigger sequence reverse complement set is a perfect complement of the sequence of the 5' to 3' read segment in the RNA trigger sequence set. The RNA trigger sequence set is designed based on the corresponding mRNA transcript of the target gene sequence to affect RNAi on such transcripts, thereby preventing or reducing the translation of related proteins. By reducing the translation of related proteins, the trigger sequence of the present invention disrupts the DON pathway in Fusarium, thereby reducing the production of DON in Fusarium. Tables 1 and 2 provided herein match various target gene sequences with their corresponding trigger sequences, RNA trigger sequences, and RNA trigger sequence reverse complement sequences. SEQ ID NO is related to the sequence provided in the SEQ ID list. It is noteworthy that the SEQ ID list submitted with this paper shows that SEQ ID NO:33-64,79-86,153-225 and 227-299 are DNA, which is due to the limitation of the new ST26 format. For the purpose of this disclosure, SEQ ID NO:33-64,79-86,153-225 and 227-299 are RNA sequences, in which the thymine indicated is uracil. SEQ ID NO:75-78 is the DNA version of SEQ ID NO:79-82. It will be appreciated by those of ordinary skill in the art that by replacing any uracil with thymine, any one of the RNA sequences herein can be read as a DNA sequence, and the DNA template for such RNA sequences will include the DNA base complement of a given RNA sequence, and the RNA sequence with the identity of the target gene DNA sequence quoted will replace thymine with uracil, and the RNA with complementarity with the target gene DNA sequence quoted will include the RNA base complement of a given DNA sequence.

[0011] In one aspect, a method for reducing or eliminating DON production by a species of the genus Fusarium (e.g., Fusarium graminearum) on a plant, the method comprising contacting Fusarium with a polynucleotide comprising at least one segment of 18 or more consecutive nucleotides having a sequence having about 95% to about 100% identity (e.g., would include a segment of 21 consecutive nucleotides having a sequence having 100% identity) to a DNA, or its DNA complement, or a corresponding fragment of any gene identified in Table 1 or Table 2, or a homolog thereof, the DNA having a sequence selected from the group consisting of a target gene sequence group. In one embodiment, the method for reducing or eliminating DON production by Fusarium graminearum on a plant comprises contacting Fusarium graminearum with a polynucleotide comprising a nucleotide sequence complementary to at least 18 consecutive nucleotides of a target gene or RNA transcribed from the target gene, the target gene having a nucleotide sequence selected from the group consisting of a target gene sequence group. In some embodiments, the polynucleotide comprises at least 18 consecutive nucleotides complementary to a sequence selected from a group consisting of a RNA trigger sequence group or a reverse complement group of an RNA trigger sequence or a sequence that is about 95% to about 100% identical. In some embodiments, 18 or more consecutive nucleotides are 21 or more consecutive nucleotides, 50 or more consecutive nucleotides, 150 or more consecutive nucleotides, 200 or more consecutive nucleotides, 250 or more consecutive nucleotides, 300 or more consecutive nucleotides, 350 or more consecutive nucleotides, 400 or more consecutive nucleotides, 450 or more consecutive nucleotides, 500 or more consecutive nucleotides, 550 or more consecutive nucleotides or 600 or more consecutive nucleotides. In some embodiments, the polynucleotide is designed to have complementarity with 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 a RNA trigger sequence group or a reverse complement group of an RNA trigger sequence. In some embodiments, contact with the polynucleotide is achieved by applying the polynucleotide or a composition or solution containing the polynucleotide directly to Fusarium graminearum or a surface or substrate (e.g., plant or soil) in contact with Fusarium graminearum (e.g., by spraying or dusting or soaking). 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, bulbs, stems, ears, seeds, roots or other plant parts infected or potentially infected with Fusarium graminearum. In some embodiments, contact with the polynucleotide is achieved by providing a transgenic plant expressing a sequence that reduces or eliminates DON production by Fusarium graminearum.In some embodiments, the polynucleotide comprises two or more regions targeting two or more target genes associated with DON production, including targeting one or more DON genes in Fusarium graminearum and / or targeting one or more homologs of such DON genes in another species of Fusarium that produces DON, thereby reducing DON production in Fusarium graminearum, and reducing DON production in one or more additional species of Fusarium. In some cases, the percent identity between the gene target region of Fusarium graminearum and the homolog region present in one or more additional species of Fusarium (such as Fusarium luteum) will be high enough so that the same region in the polynucleotide will cause RNAi in the one or more species, thereby reducing DON production in the one or more species.

[0012] Several embodiments relate to a method for reducing or eliminating DON production by Fusarium graminearum by exposing Fusarium graminearum to a composition comprising one or more formulations or delivery agents and a polynucleotide that produces 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 its DNA complement, and wherein the agent acts upon contact or ingestion (e.g., internal absorption / transfection) of Fusarium graminearum to inhibit biological functions within Fusarium graminearum, thereby reducing or eliminating DON production by Fusarium graminearum and / or otherwise controlling Fusarium graminearum. In some embodiments, the polynucleotide comprises one or more nucleotide sequences selected from the group of RNA trigger sequences or the reverse complement of RNA trigger sequences, 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 the group of RNA trigger sequences or the reverse complement of RNA trigger sequences. In some embodiments, the polynucleotide is RNA, and in some embodiments, the RNA is double-stranded RNA.

[0013] In some embodiments, the agent comprises the following:

[0014] (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 consecutive nucleotides that are 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 or a segment of RNA transcribed from the target gene, the target gene having a nucleotide sequence selected from the group consisting of a target gene sequence;

[0015] (b) an effective amount of at least one polynucleotide comprising at least one silencing element that is 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, 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 the target gene sequence;

[0016] (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, the target gene having a nucleotide sequence selected from the group consisting of the target gene sequence; or

[0017] (d) an RNA molecule that reduces or eliminates DON production in Fusarium graminearum when transfected into or contacted with the Fusarium 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 consecutive nucleotides that are 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 having a nucleotide sequence selected from the group consisting of the target gene sequence, or a segment of an RNA transcribed from the target gene. or (e) a double-stranded RNA molecule that reduces or eliminates DON production in Fusarium graminearum when transfected into or contacted with the Fusarium 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 consecutive nucleotides that are substantially complementary to, or comprise at least 85%, 90%, 95%, 98%, or 100% sequence identity with, a target gene or a segment of RNA transcribed from the target gene, wherein the target gene has a nucleotide sequence selected from the group consisting of the target gene sequence; or

[0018] (f) an effective amount of at least one double-stranded RNA, the at least one double-stranded RNA comprising at least one strand comprising a sequence selected from the group consisting of, 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: an RNA trigger sequence and the reverse complement of the RNA trigger sequence; or

[0019] (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 consecutive nucleotides of a nucleotide sequence selected from the group consisting of: an RNA trigger sequence and the reverse complement of the RNA trigger sequence, 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: an RNA trigger sequence and the reverse complement of the RNA trigger sequence; or

[0020] (h) 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 nucleotide sequence selected from the group consisting of: an RNA trigger sequence and the reverse complement of the RNA trigger sequence; or

[0021] (i) an RNA molecule that reduces or eliminates DON production by the Fusarium graminearum when transfected into or contacted with the Fusarium 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 consecutive nucleotides that are substantially 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 segment of a nucleotide sequence selected from the group consisting of: the target gene sequence, the RNA trigger sequence, and the reverse complement of the RNA trigger sequence; or

[0022] (j) a double-stranded RNA molecule that, when transfected or contacted with Fusarium graminearum, reduces or eliminates DON production by the Fusarium 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 consecutive nucleotides that are 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 segment of a nucleotide sequence selected from the group consisting of: an RNA trigger sequence and the reverse complement of the RNA trigger sequence;

[0023] (k) a double-stranded RNA molecule that reduces or eliminates DON production by Fusarium graminearum when transfected or contacted with the Fusarium 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 a reverse complement of the RNA trigger sequence.

[0024] In certain embodiments, compositions containing polynucleotides are formulated for application to plant fields, for example in the form of a sprayable solution or emulsion, a tank mix, or a 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 applied to plant fields are known in the art, and the polynucleotides of the present invention may be formulated in any suitable composition. An example is a composition described in PCT / US / 2022 / 027816, disclosed as WO2022 / 0235895 on November 10, 2022, which is incorporated herein by reference in its entirety.

[0025] Any suitable DNA encoding RNAi molecules targeting the target genes 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 cassettes that, when transcribed, produce single-stranded RNA (ssRNA) molecules (e.g., remain single-stranded or fold into RNA hairpins) or complementary ssRNA molecules that bond to produce double-stranded RNA (dsRNA) molecules.

[0026] Various methods for preparing RNA are known in the art, and the RNA of the present invention can be prepared 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 disclosed in U.S. Patent No. 10,858,385 (disclosed in US2019 / 0144489) on May 16, 2019 and disclosed in US Patent No. 10,954,541 (disclosed in US2017 / 0292138) on October 12, 2017, each of which is incorporated herein by reference. Examples of endogenously delivered RNAi molecules for use in the present invention include, but are not limited to, those described in: U.S. Patent No. 11,142,768, published on May 14, 2020 (Publication No. US2020 / 0149044), U.S. Patent No. 11,185,079, published on March 26, 2020 (Publication No. US2020 / 0093138), PCT / US / 2021 / 032334, published on November 18, 2021 (International Publication No. WO 2021 / 231791), all of which are incorporated herein by reference.

[0027] Several embodiments relate to a method of providing a plant with increased resistance to DON production by Fusarium graminearum, the method comprising topically applying to the plant a composition comprising at least one polynucleotide that prevents or reduces DON production by RNAi against a gene in the DON production pathway (ty), the at least one polynucleotide comprising a gene identified in Table 1 or Table 2, or a DNA complement thereof, or having a sequence selected from the group consisting of a target gene sequence group. In one embodiment, the method of providing a plant with increased resistance to infection by Fusarium graminearum comprises topically applying to the plant a composition comprising at least one polynucleotide comprising a nucleotide sequence complementary to at least 18 consecutive nucleotides of a target gene or RNA transcribed from the target gene, the target gene having a nucleotide sequence selected from the group consisting of a target gene sequence. In some embodiments, the at least one polynucleotide comprises a sequence selected from the group consisting of an RNA trigger sequence or the reverse complement of an RNA trigger sequence, 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 an RNA trigger sequence or the reverse complement of an RNA trigger sequence. 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 the reverse complement of an RNA trigger sequence. In one embodiment, a method of providing a plant having increased resistance to DON production by Fusarium graminearum comprises topically applying a composition comprising at least one polynucleotide to a plant in such a manner that an effective amount of the polynucleotide is transfected into or contacted with Fusarium graminearum infecting the plant, the polynucleotide comprising at least 18 consecutive nucleotides complementary to a target gene or a portion of an RNA transcribed from a target gene, the target gene having a nucleotide sequence selected from the group consisting of a target gene sequence group. In some embodiments, the polynucleotide comprises one or more nucleotide sequences selected from the group of RNA trigger sequences or the reverse complement of RNA trigger sequences, 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 the reverse complement of an RNA trigger sequence. 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 the reverse complement of an RNA trigger sequence. In some embodiments, the polynucleotide is a dsRNA. Several embodiments relate to compositions comprising polynucleotides formulated for application to a plant field, for example in the form of a sprayable solution or emulsion, a tank mix, or a powder.

[0028] Several embodiments relate to a composition for reducing or eliminating DON production by Fusarium graminearum, the composition comprising an effective amount of at least one polynucleotide molecule, the at least one polynucleotide molecule comprising at least one segment of 18 or more consecutive nucleotides, the consecutive nucleotides being substantially identical or complementary to a corresponding fragment of a DNA or its DNA complement (e.g., a segment of 21 consecutive nucleotides having a sequence with 100% identity or complementarity thereto), the DNA having a sequence selected from the group consisting of a target gene sequence group. In some embodiments, the polynucleotide molecule comprises at least 18 consecutive nucleotides, the consecutive nucleotides being complementary to a target gene or a portion of an RNA transcribed from a target gene, the target gene having a nucleotide sequence selected from the group consisting of a target gene sequence group. In some embodiments, the polynucleotide comprises one or more nucleotide sequences selected from the group of RNA trigger sequences or the reverse complement of an RNA trigger sequence, or comprises a nucleotide sequence complementary to a nucleotide sequence selected from an RNA trigger sequence or the reverse complement of an RNA trigger sequence 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. 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 the reverse complement of an RNA trigger sequence. 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, for example in the form of a sprayable solution or emulsion, a tank mix or a powder, and optionally comprising one or more additional components such as a carrier, a surfactant, an organosilicon, an organosilicon 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.

[0029] Several embodiments relate to a method of providing a plant with increased resistance to DON production by Fusarium graminearum, the method comprising expressing in a plant at least one polynucleotide comprising at least one segment of 18 or more consecutive nucleotides that are substantially identical or complementary to a corresponding segment of a DNA or its complement of DNA (e.g., a segment of 21 consecutive nucleotides having a sequence that is 100% identical or complementary thereto), the DNA having a sequence selected from the group consisting of a target gene sequence group. In some embodiments, the polynucleotide comprises one or more nucleotide sequences selected from the group of trigger sequences, the group of RNA trigger sequences, or the group of reverse complements of RNA trigger sequences, 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 the group of RNA trigger sequences or reverse complements of RNA trigger sequences. In some embodiments, the polynucleotide is a dsRNA comprising one or more sequences selected from the group of RNA trigger sequences and a corresponding sequence selected from the reverse complement of RNA trigger sequences.

[0030] Several embodiments relate to a recombinant DNA construct comprising a heterologous promoter operably linked to a DNA element comprising a sequence for producing an RNA described herein that can be used to prevent or reduce DON production in a species of the genus Fusarium. 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 the group of RNA trigger sequences or the reverse complement of an RNA trigger sequence. Related embodiments include a plant chromosome or plastid or a recombinant plant viral vector or a recombinant baculovirus vector comprising a recombinant DNA construct, or comprising a DNA element that does not contain a heterologous promoter.

[0031] Several embodiments relate to a transgenic plant cell having in its genome a recombinant DNA encoding an RNA that inhibits the expression of a target gene in Fusarium graminearum contacted with or transfected with the RNA, thereby eliminating or reducing DON production in Fusarium graminearum. In some embodiments, the target gene is a target gene identified in Table 1 or Table 2. A specific embodiment is a transgenic plant cell having in its genome a recombinant DNA encoding an RNA for silencing one or more target genes selected from a target gene sequence group. In some embodiments, the RNA comprises one or more nucleotide sequences selected from a RNA trigger sequence group or a reverse complement of an RNA trigger sequence, 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 an RNA trigger sequence or a reverse complement of an RNA trigger sequence.

[0032] Several embodiments relate to an isolated recombinant RNA molecule that reduces or eliminates DON production by Fusarium graminearum when transfected with or contacted with Fusarium graminearum, wherein the recombinant RNA molecule comprises at least one segment of 18 or more consecutive nucleotides that are substantially complementary to a corresponding segment of a DNA or its DNA complement (e.g., a segment of 21 consecutive nucleotides having a sequence that is 100% complementary thereto), the DNA having a sequence selected from the group consisting of a target gene sequence group. In some embodiments, the recombinant RNA molecule is a double-stranded RNA. Specific embodiments include an isolated recombinant double-stranded RNA molecule whose strand has a sequence selected from the group consisting of an RNA trigger sequence group, an RNA trigger sequence reverse complement group, or a combination thereof. Other embodiments relate 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.

[0033] Several embodiments relate to a method of providing a plant with increased resistance to DON production by Fusarium graminearum, the method comprising providing to the plant at least one polynucleotide comprising at least one segment of 18 or more consecutive nucleotides that are substantially identical or complementary to a corresponding fragment of a target gene selected from a target gene sequence group (e.g., a segment of 21 consecutive nucleotides having a sequence that is 100% identical or complementary thereto). In one embodiment, the method of providing a plant with DON production by Fusarium graminearum comprises providing to the plant at least one polynucleotide comprising at least one segment that is identical or complementary to at least 18 consecutive nucleotides of a 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 the group of RNA trigger sequences or the group of reverse complements of RNA trigger sequences, 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 the reverse complement of an RNA trigger sequence. In some embodiments, the polynucleotide is a dsRNA. In some embodiments, the dsRNA comprises: a first strand comprising one or more sequences selected from an RNA trigger sequence; and a second strand comprising one or more corresponding sequences selected from the reverse complement of an RNA trigger sequence.

[0034] Several embodiments relate to a method of reducing or eliminating DON production by Fusarium graminearum by contacting Fusarium graminearum with a polynucleotide comprising at least one segment of 18 or more contiguous nucleotides that is substantially identical or complementary to a corresponding segment of an equivalent length of a portion of a 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 of a sequence having 100% identity or complementarity thereto). In some embodiments, the polynucleotide is a double-stranded RNA.

[0035] Several embodiments relate to artificial compositions comprising at least one polynucleotide as described herein. In some embodiments, formulations are provided that can be used for topical application to plants or materials that need to be protected from the effects of DON production by Fusarium graminearum. In some embodiments, recombinant constructs and vectors that can be used to prepare transgenic plant cells and transgenic plants are provided. In some embodiments, formulations and coatings for treating plants, plant seeds, or fertile parts are provided. In some embodiments, commercial products and foods (particularly commercial products and foods with detectable amounts of polynucleotides as described herein) produced by such plants, seeds, or fertile parts treated with or containing polynucleotides as described herein are provided. Several embodiments relate to polyclonal or monoclonal antibodies that bind to proteins encoded by sequences or fragments of sequences selected from the target gene sequence group. On the other hand, polyclonal or monoclonal antibodies are provided that bind to proteins encoded by sequences or fragments of sequences selected from the RNA trigger sequence group or its complement. Such antibodies are prepared by conventional methods known to those of ordinary skill in the art.

[0036] Other aspects and specific embodiments of the invention are disclosed in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Graphs showing the reduction of DON by application of the trigger sequences disclosed herein are provided.

[0038] Figure 2 Graphs are provided that illustrate how specific trigger sequence embodiments reduce DON production.

[0039] Figure 3 Graphs are provided that show how certain trigger sequence embodiments reduce the expression of their corresponding target genes.

[0040] Figure 4 Graphs showing the reduction of DON by application of the trigger sequences disclosed herein are provided. DETAILED DESCRIPTION

[0041] I. Definitions

[0042] 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 the invention belongs. Where a term is provided in the singular, the inventors of the present invention also contemplate various aspects of the invention described in the plural form of the term. In the event of differences in the terms and definitions used in the references incorporated by reference, the terms used in this application shall have the definitions given herein. Other technical terms used have their ordinary meaning in the field in which they are used, as exemplified by various field-specific dictionaries, such as the American "Scientific Dictionary" (Editors of American Heritage Dictionaries, 2011, Houghton Mifflin Harcourt, Boston and New York), "McGraw-Hill Dictionary of Scientific and Technical Terms" (6th edition, 2002, McGraw-Hill, New York), or "Oxford Dictionary of Biology" (6th edition, 2008, Oxford and Oxford University Press, Oxford and New York). The inventors do not intend to be limited to the mechanism of action or mode of action. References thereto are provided for illustrative purposes only.

[0043] Unless otherwise indicated, when reading from left to right, the nucleic acid sequence in the text of this specification is given in 5' to 3' direction. It will be appreciated by those skilled in the art that it will be understood that a given DNA sequence defines a corresponding RNA sequence, which is identical to a DNA sequence except that thymine (T) nucleotides in DNA are replaced with uracil (U) nucleotides. Therefore, providing a specific DNA sequence can be understood as defining an accurate RNA equivalent. A given first polynucleotide sequence, whether DNA or RNA, further defines the sequence of its exact complement (which may be DNA or RNA), the exact complement being the second polynucleotide that is fully hybridized with the first polynucleotide by forming a Watson-Crick base pair (Watson-Crick base-pair). For DNA: DNA duplexes (hybridized chains), base pairs are adenine: thymine or guanine: cytosine; for DNA: RNA duplexes, base pairs are adenine: uracil or guanine: cytosine. Thus, the nucleotide sequence of a completely hybridized blunt-ended double-stranded polynucleotide ("100% complementarity" between the strands or the strands are "complementary") can be unambiguously defined as long as the nucleotide sequence of one strand (whether DNA or RNA) is provided. By "substantially identical" or "substantially complementary" to a target gene or a fragment of a target gene, it is meant that the 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 in a plant or fungal cell); one skilled in the art will appreciate that such hybridization does not necessarily require 100% sequence identity or complementarity. In some embodiments, a trigger may be designed to be not 100% identical to a target gene sequence, but still complementary to the sequence of a target gene or RNA transcribed therefrom. A first nucleic acid sequence is "operably" linked or "connected" to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, if a promoter provides transcription or expression of a DNA, then the promoter sequence is "operably linked" to the DNA. Typically, operably linked DNA sequences are contiguous.

[0044] 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 or more nucleotides. Polynucleotides also include molecules containing a plurality of nucleotides, including atypical 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 DsiRNA", 2011 (Integrated DNA Technologies Coralville, Iowa). Typically, a polynucleotide as described herein, whether DNA or RNA or both, and whether single-stranded or double-stranded, includes at least one stretch of 18 or more consecutive nucleotides (or, in the case of a double-stranded polynucleotide, at least 18 consecutive base pairs) that are substantially identical or complementary to an equivalent size fragment of the DNA of the target gene or the RNA transcript of the target gene. Throughout this disclosure, "at least 18 consecutive" means "about 18 to about 10,000, including every integer point therebetween".Thus, embodiments of the invention include oligonucleotides of 18-25 nucleotides in length (18-mers, 19-mers, 20-mers, 21-mers, 22-mers, 23-mers, 24-mers, or 25-mers), or intermediate length polynucleotides of 26 or more nucleotides in length (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 9, 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 about 300 nucleotides in length), or a long polynucleotide of greater than about 300 nucleotides in length (e.g., between about 300 and about 400 nucleotides, between about 300 and about 700, between about 400 and about 500 nucleotides, between about 500 and about 600 nucleotides, between about 600 and about 700 nucleotides, between about 700 and about 800 nucleotides, between about 800 and about 900 nucleotides, between about 900 and about 1000 nucleotides, between about 1000 and about 1100 nucleotides, between about 1100 and about 1200 nucleotides, between about 1200 and about 1300 nucleotides, between about 1300 and about 1400 nucleotides, between about 1500 and about 1600 nucleotides, between about 1600 and about 1700 nucleotides, between about 1700 and about 1800 nucleotides, between about 1800 and about 1900 nucleotides, between about 1900 and about 2000 nucleotides, between about 1900 and about 2100 nucleotides, between about 1900 and about 2200 nucleotides, between about 1900 and about 2300 nucleotides, between about 1900 and about 2400 nucleotides, between about 1900 and about 2500 nucleotides, between about 1900 and about 2600 nucleotides, between about 1900 and about 2700 nucleotides In some embodiments, the polynucleotide may be between about 100 and about 1000 nucleotides, between about 300 and about 500 nucleotides, between about 300 and about 600 nucleotides, between about 300 and about 700 nucleotides, between about 300 and about 800 nucleotides, between about 300 and about 900 nucleotides, or about 1000 nucleotides, or even greater than about 1000 nucleotides in length, such as a polynucleotide up to the entire length of the target gene (including the coding portion or the non-coding portion or both the coding portion and the non-coding portion of the target gene). When the polynucleotide is double-stranded, its length can similarly be described in terms of base pairs.

[0045] The polynucleotides described herein can be single-stranded (ss) or double-stranded (ds). "Double-stranded" refers to base pairing that occurs between fully complementary antiparallel nucleic acid chains, usually under physiologically relevant conditions, to form a double-stranded nucleic acid structure. Embodiments include polynucleotides 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 hybrids, antisense ssDNA or antisense ssRNA; A mixture of polynucleotides of any of these types can be used. In some embodiments, the polynucleotide is a double-stranded RNA having a length greater than the typical length of naturally occurring regulatory small RNA (such as endogenously produced siRNA and mature miRNA). In some embodiments, the polynucleotide is a double-stranded RNA having a length of at least about 30 continuous base pairs. In some embodiments, the polynucleotide is a double-stranded RNA having a length between about 50 and about 600 base pairs. In some embodiments, the polynucleotide may include components other than standard ribonucleotides, for example, an embodiment is an RNA comprising terminal deoxyribonucleotides.

[0046] In various embodiments, the polynucleotides described herein include naturally occurring nucleotides, such as nucleotides present in DNA and RNA. In certain embodiments, the polynucleotide is a combination of ribonucleotides and deoxyribonucleotides, for example, a synthetic polynucleotide consisting mainly of ribonucleotides but having one or more terminal deoxyribonucleotides or one or more terminal dideoxyribonucleotides, or a synthetic polynucleotide consisting mainly of deoxyribonucleotides but having one or more terminal dideoxynucleotides. In certain embodiments, the polynucleotide includes atypical 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 2011 / 0171287, U.S. Patent Publication 2011 / 0171176, U.S. Patent Publication 2011 / 0152353, U.S. Patent Publication 2011 / 0152346 and U.S. Patent Publication 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 can be partially or fully modified with phosphorothioate, phosphorodithioate, or methylphosphonate internucleotide linkage modifications, modified nucleobases or modified sugars can be used in oligonucleotide or polynucleotide synthesis, and an oligonucleotide or polynucleotide can be labeled with a fluorescent moiety (e.g., fluorescein or rhodamine) or other label (e.g., biotin).

[0047] Several embodiments relate to a polynucleotide comprising at least one segment of 18 or more consecutive nucleotides having a sequence having about 95% to about 100% identity to a fragment of equivalent length of a target gene selected from the group consisting of 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 thereof, or a DNA or RNA complement of any of the foregoing. In some embodiments, the number of consecutive nucleotides is at least 18, such as at least 21, between 18-24, between 20-30, between 20-50, between 20-100, between 50-100, between 50-600, between 100-250, between 250-600, between 400-600, between 200-1000 or between 500-2000 or even more. In some embodiments, the number of consecutive nucleotides is greater than 18, e.g., 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or greater 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 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 consecutive nucleotides. In some embodiments, the consecutive nucleotides comprise about the same number of nucleotides as any one of the sequences of the set of RNA trigger sequences. 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 means that any of these lower limits of the group can be individualized) consecutive nucleotides having a sequence having about 100% identity to a fragment of an equivalent length of: a target gene selected from the group consisting of the genes identified in Table 1 or Table 2, or a homolog thereof, or a DNA of a target gene having a sequence selected from the target gene sequence group, or a DNA complement of any of the sequences of the RNA trigger sequence group, or a reverse complement group of RNA 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 polynucleotide is a double-stranded nucleic acid (e.g., dsRNA), one strand of which comprises at least one segment of at least 18, 19, 20, 21, 50, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or 650 consecutive nucleotides having about 100% identity to an equivalent length of a target gene selected from the group consisting of the genes identified in Table 1 or Table 2, or a DNA of a target gene having a sequence selected from the target gene sequence group, or any one of the RNA trigger sequence group. DNA complement, or RNA trigger sequence reverse complement set, or any RNA transcript thereof, or DNA or RNA complement of any of the foregoing; expressed in base pairs, such double-stranded nucleic acid comprises at least one segment of at least 18 consecutive perfectly matched base pairs, which corresponds to the equivalent length of the following fragments: a target gene selected from the group consisting of genes identified in the target gene sequence group, a DNA of a target gene having a sequence selected from the target gene sequence group, or a DNA complement of an RNA trigger sequence group, or a reverse complement set of RNA trigger sequences, or any RNA transcript thereof, or DNA or RNA complement of any of the foregoing. In some embodiments, the length of each segment contained in the polynucleotide is greater than the typical length of a naturally occurring regulatory small RNA, for example, the length of each segment is at least about 30 consecutive nucleotides (or base pairs). 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 between about 300 and about 650 nucleotides (for single-stranded polynucleotides) or base pairs (for double-stranded polynucleotides). In some embodiments, the polynucleotide is a dsRNA of between about 300 and about 650 base pairs, such as a dsRNA of the length of any of the RNA trigger sequences disclosed in Table 1 or Table 2, or any of the figures below.

[0048] Several embodiments relate to polynucleotides designed to regulate expression by inducing regulation or inhibiting one or more Fusarium target genes associated with 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 specific embodiments, are 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 substantially identical or substantially complementary to the nucleotide sequence of a segment of a Fusarium graminearum target gene or cDNA (e.g., a target gene sequence group), or to the sequence of an RNA transcribed from a Fusarium 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," "trigger or triggers."

[0049] Effective polynucleotides of any size 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 prepare a composition (e.g., a composition for topical application, or a recombinant DNA construct that can be used to prepare dsRNA or transgenic plants). In other embodiments, a mixture or pool of different polynucleotide triggers is used; in this case, the polynucleotide trigger can be directed to a single target gene or multiple target genes. In some embodiments, a single polynucleotide can target more than one target gene associated with DON production, including genes from the same Fusarium and / or genomes of more than one related Fusarium.

[0050] As used herein, the term "isolated" refers to separating a molecule from other molecules that are usually associated with it in its natural or native state. Thus, the term "isolated" may refer to a DNA molecule that has been separated from other DNA molecules that are usually combined with the DNA molecule in its natural or native state. Such DNA molecules may exist in a recombinant state, such as a recombinant DNA molecule. Thus, a DNA molecule fused to a regulatory or coding sequence that is not usually associated with it, for example as a result of recombinant technology, is considered to be isolated even if it is integrated into a cell chromosome as a transgene or exists with other DNA molecules.

[0051] Several embodiments relate to a polynucleotide designed to inhibit one or more genes ("target genes"). The term "gene" refers to any part of a nucleic acid that provides transcript expression or encodes a transcript. A "gene" may include, but is not limited to, a promoter region, a 5' untranslated region, a transcript coding region that may include an intron region, a 3' untranslated region, or a combination of these regions. In some embodiments, the target gene may include a coding or non-coding sequence or both. In other embodiments, the target gene has a sequence that is identical or complementary to a messenger RNA, for example, in some embodiments, the target gene is represented by its corresponding cDNA. In a specific embodiment, the polynucleotide is designed to inhibit one or more target genes, wherein each target gene is selected from the group consisting of the genes identified in Table 1 or Table 2, or is encoded by a DNA sequence selected from a target gene sequence group, or in a specific embodiment, is selected from the group consisting of: FGP1, ELP3, SPT7, MAF1, MVD1, HEP1, SET1, and FKPB12. In various embodiments, the polynucleotide is designed to inhibit or downregulate one or more target genes, wherein each target gene is selected from the group consisting of genes identified in Table 1 and Table 2, or is encoded by a sequence selected from the target gene sequence group, and can be designed to inhibit 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 consecutive nucleotides, and the consecutive nucleotides have 100% identity with the equivalent length of the following fragments: genes identified in Table 1 and Table 2 or homologs thereof, DNA of a target gene having a sequence selected from the target gene sequence group, or its DNA complement. In this case, each segment may be the same or different in size or sequence, and may be sense or antisense relative to the target gene. For example, in one embodiment, the polynucleotide comprises a plurality of segments arranged in tandem or repeats, wherein each segment comprises 21 or more consecutive nucleotides having a sequence having 100% identity with a fragment of equivalent length of the following: a gene identified in Table 1 or Table 2 or a homolog thereof, a target gene sequence group, a DNA having a sequence selected from a target gene sequence group, or a target gene, 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 the reverse complements of the RNA trigger sequences for the target genes disclosed in Tables 1 and 2. In some embodiments, one or more of the segments correspond to one or more segments of 21 or more consecutive nucleotides, which are selected from the sequences of the RNA trigger sequences or the reverse complements of the RNA trigger sequences for the target genes disclosed in Tables 1 and 2. In some embodiments, the segments may be from different regions of the target gene, for example, the segments may correspond to the exon regions of the target gene. In some embodiments, "spacer" nucleotides that do not correspond to the target gene may be optionally used between segments or adjacent segments.

[0052] As used herein, unless the context clearly indicates otherwise, the term "plant" refers to a plant susceptible to infection by Fusarium, i.e., Fusarium graminearum. Examples of plants susceptible to infection by Fusarium include corn and small grains, such as wheat, barley, flax, buckwheat, rye, and oats. In a specific embodiment, the plant is wheat.

[0053] Additional definitions are provided in the following sections.

[0054] II. Polynucleotides for Controlling DON Production by Fusarium graminearum

[0055] The polynucleotides of the present disclosure can be used to reduce DON production by fungal pathogens of the genus Fusarium on plants through RNAi, and effectively control or prevent Fusarium infection of plants. According to some aspects of the present disclosure, the polynucleotides effectively interfere with mRNA encoded by one or more Fusarium graminearum target genes associated with DON production.

[0056] In some embodiments, the polynucleotide comprises 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, or between about 200 and 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 consecutive nucleotides, the consecutive nucleotide sequence 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 100% identity or completely 100% identity with the corresponding fragment of equivalent length below: DNA of a target gene having a sequence selected from the group consisting of a target gene sequence group, or its DNA complement or RNA transcribed therefrom. In some embodiments, the target gene is a gene identified in Table 1 or Table 2 or a homolog thereof. In specific embodiments, the target gene is selected from the group consisting of: FGP1, ELP3, SPT7, MAF1, MVD1, HEP1, SET1, FKPB12, or in more specific embodiments comprises FGP1.In one embodiment, the polynucleotide comprises a nucleotide sequence that overlaps with 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 750, at least 800, at least 800, at least 900, at least 1000, at least 1250, at least 1500, at least 2000, at least 2500, at least 3000, at least 4000, at least 5000, at least 6000, at least 7500, at least 8000, at least 8000, at least 9000, at least 10000, at least 10000, at least 10000 In some embodiments, the one or more target genes are substantially complementary to at least 600, at least 700, at least 800, at least 900, or at least 1,000, or between about 200 and about 400, or about 300 and about 500, or about 300 and about 600, or about 300 and about 650, or about 400 and about 700 consecutive nucleotides, the one or more target genes are identified in Table 1 or Table 2, or have a nucleotide sequence selected from the group consisting of the target gene sequence group, or in a specific embodiment selected from the group consisting of: SEQ ID NO: 16, 68-70, 108, 114, 120, 139 and 146, or in another specific embodiment comprises SEQ ID NO: 16. In some embodiments, the one or more target genes comprise one or more genes identified in Table 1 or Table 2 or aa homologs thereof. In specific embodiments, the one or more target genes comprise one or more genes selected from the group consisting of: FGP1, ELP3, SPT7, MAF1, MVD1, HEP1, SET1, FKPB12, or in more specific embodiments, FGP1.

[0057] In some embodiments, the polynucleotide comprises at least 21 consecutive nucleotides, which are substantially complementary to the corresponding fragments of equivalent length: the target gene identified in Table 1 or Table 2 or its homologue, or a target gene having a DNA sequence selected from the group consisting of the target gene sequence group, or its DNA complement or RNA transcribed therefrom. In some embodiments, the polynucleotide comprises at least 300 consecutive nucleotides, which are substantially complementary to the corresponding fragments of equivalent length: the target gene identified in Table 1 or Table 2 or its homologue, or a target gene having a DNA sequence selected from the group consisting of the target gene sequence group, or its DNA complement or RNA transcribed therefrom. In a specific embodiment, the target gene is selected from the group consisting of: FGP1, ELP3, SPT7, MAF1, MVD1, HEP1, SET1, FKPB12, or in a more specific embodiment, comprises FGP1. In some embodiments, the polynucleotide is designed to have complementarity with the mRNA encoded by the target gene. In some embodiments, the polynucleotide is a double-stranded RNA. And in some embodiments, the double-stranded RNA comprises one chain and a second chain complementary thereto, wherein the one chain comprises a sequence selected from the group consisting of: SEQ ID NO:48, 80-82, 182, 188, 194, 213 and 220, or in a specific embodiment, comprises one chain and a second chain complementary thereto, wherein the one chain comprises a sequence selected from the group consisting of: SEQ ID NO:32-38, 79-82 and 268, or in a more specific embodiment, comprises one chain and a second chain complementary thereto, wherein the one chain comprises the sequence of SEQ ID NO:32.

[0058] In some embodiments, the polynucleotide comprises a sequence of contiguous nucleotides that is substantially complementary or completely (100%) identical to a fragment of an equivalent length of a target gene identified in Table 1 or Table 2, or a homolog thereof, or a target gene having the following sequence, or its DNA complement, or RNA transcribed therefrom, the sequence being selected from the group of target gene sequences, or in a specific embodiment, from the group consisting of: SEQ ID NO: SEQ ID NO: 16, 68-70, 108, 114, 120, 139, and 146, or in another specific embodiment comprising SEQ ID NO: 16. 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 an equivalent length of a target gene identified in Table 1 or Table 2, or a DNA of a target gene having a sequence selected from the group of target gene sequences, or its DNA complement, or RNA transcribed therefrom. In some embodiments, the number of consecutive nucleotides is greater than 18, e.g., 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or greater 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. In some embodiments, the consecutive nucleotides are between about 200 and about 400, or about 300 and about 500, or about 300 and about 600, or about 300 and about 750, or about 300 and about 900. In some embodiments, the polynucleotide comprises at least one segment of at least 18, 19, 20 or 21 (references to at least 18, 19, 20, 21 as used throughout the text mean that any of these lower limits of the group can be individualized) consecutive nucleotides having a sequence with about 100% identity to a fragment of an equivalent length of: DNA of a target gene having a sequence selected from the target gene sequence group, or its DNA complement, or DNA of a target gene identified in Table 1 or Table 2 or a homolog thereof.

[0059] In one embodiment, the polynucleotide comprises at least one segment of 21 consecutive nucleotides that are substantially complementary to or have 100% identity with a target gene, or a DNA complement thereof, or a corresponding fragment of RNA transcribed therefrom, the target gene having a DNA sequence selected from the group consisting of: SEQ ID NO: 16, 68-70, 108, 114, 120, 139, and 146. In some embodiments, in addition to the one or more segments of 21 consecutive nucleotides that have 100% identity with the corresponding fragment of the target gene, the polynucleotide comprises one or more "neutral" sequences (sequences that have no sequence identity or complementarity with the target gene), and thus the polynucleotide as a whole has a much lower overall sequence identity with the target gene.

[0060] 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 with one or more target genes or homologs thereof, or their DNA complements, or corresponding fragments of RNA transcribed therefrom, the one or more target genes identified in Table 1 or Table 2, or having a DNA sequence selected from the group of target gene sequences, or in specific embodiments, selected from the group consisting of: SEQ ID NO: 16, 68-70, 108, 114, 120, 139 and 146. In some embodiments, in addition to the one or more segments of 21 contiguous nucleotides that have 100% identity with the corresponding fragments of ≥1 target gene, the polynucleotide comprises one or more "neutral" sequences (sequences that have no sequence identity or complementarity with the target gene), and thus the polynucleotide as a whole is given a lower overall sequence identity with the target gene. In one embodiment, the polynucleotide comprises a combination of 21 or more consecutive nucleotides or longer segments, the consecutive nucleotides are complementary to or have 100% identity with the corresponding fragments distributed over the entire length of the target gene, or its DNA complement, or the RNA transcribed therefrom, the target gene having a DNA sequence selected from the target gene sequence group. In some embodiments, in addition to one or more segments of 21 consecutive nucleotides with 100% identity with the corresponding fragments distributed over the entire length of the target gene, the polynucleotide comprises one or more "neutral" sequences (sequences that have no sequence identity or complementarity with the target gene), and thus the polynucleotide as a whole is given a lower overall sequence identity with the target gene.

[0061] In some embodiments, the polynucleotide comprises a sequence that is substantially 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 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, at least 500, at least 600, or at least 700 consecutive nucleotides of the following sequence, wherein the following sequence is selected from the group consisting of the group of RNA trigger sequences or the group of reverse complements of RNA trigger sequences, or in specific embodiments, is selected from the group consisting of: SEQ ID NO: 48, 64, 80-82, 84-86, 182, 188, 194, 213, 220, 256, 262, 268, 287 and 294.

[0062] In some embodiments, the polynucleotide comprises 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 the following sequence, wherein the following sequence is selected from the RNA trigger sequence or the reverse complement of the RNA trigger sequence, or in some specific embodiments, is selected from the group consisting of: SEQ ID NO:48, 64, 80-82, 84-86, 182, 188, 194, 213, 220, 256, 262, 268, 287 and 294.

[0063] Several embodiments relate to a polynucleotide comprising a sequence having about 95% to about 100% identity with a sequence selected from the group consisting of a set of RNA trigger sequences or a set of reverse complements of RNA trigger sequences. Several embodiments relate to a polynucleotide comprising at least one segment of 18 or more consecutive nucleotides having a sequence having about 95% to about 100% identity with a portion of a sequence selected from the group consisting of a set of RNA trigger sequences or a set of reverse complements of RNA trigger sequences. In some embodiments, the number of consecutive nucleotides is at least 18, for example, between 18-24, or between 18-28, or between 20-30, or between 20-50, or between 20-100, or between 50-100, or between 50-500, or between 100-250, or between 100-500, or between 200-1,000, or between 500-700, or even more. In some embodiments, the number of consecutive nucleotides is greater than 18, e.g., 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or greater 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 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 310, about 320, about 330, about 340, about 350 about 30, 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 consecutive nucleotides. 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 means that any of these lower limits of the group can be individualized) consecutive nucleotides having a sequence with about 100% identity to a fragment of equivalent length found in the following sequence, wherein the following sequence is selected from the group consisting of a group of RNA trigger sequences and a group of reverse complements of RNA trigger sequences.In some embodiments, the polynucleotide comprises at least one segment of at least 200, 300, 400, 500, 600 or 700 consecutive nucleotides having a sequence that is at least 85% identical to a fragment of equivalent length found in the following sequences, wherein the following sequences are selected from the group consisting of RNA trigger sequences or the reverse complement of RNA trigger sequences, or in some more specific embodiments, selected from the group consisting of: SEQ ID No: 48, 64, 80-82, 84-86, 182, 188, 194, 213, 220, 256, 262, 268, 287 and 294, or in a specific embodiment, selected from SEQ ID No: 48.

[0064] In some embodiments, the polynucleotide is a double-stranded nucleic acid (e.g., dsRNA), one strand of which comprises at least 18, 19, 20, 21, 22, 23, 24, 50, 75, 100, 150, 200, 250, 300, 400, 500, or 600 or 700 consecutive nucleotides having a sequence having about 95% to 100% identity with the DNA of a target gene, or its DNA complement, or a fragment of an equivalent length of a gene identified in Table 1 or Table 2, or a homolog thereof, the target gene having a sequence selected from the group of target gene sequences. In certain embodiments, such a target gene is selected from the group consisting of FGP1, ELP3, SPT7, MAF1, MVD1, HEP1, SET1, FKPB12, or in more specific embodiments comprises FGP1. In base pairs, such double-stranded nucleic acids include at least 18, 19, 20, 21, 22, 23, 24, 50, 75, 100, 150, 200, 250, 300, 400, 500 or 600 or 700 consecutive perfectly matched base pairs of at least one segment, the base pairs corresponding to the DNA of the target gene, or its DNA complement, or a fragment of the equivalent length of the gene identified in Table 1 or Table 2 or its homolog, the target gene having a sequence selected from the target gene sequence group. In some embodiments, the length of each segment contained in the polynucleotide is greater than the typical length of the naturally occurring regulatory small RNA, for example, the length of each segment is at least about 30 consecutive nucleotides (or base pairs). In some embodiments, the total length of the polynucleotide or the length of each segment contained in the polynucleotide is less than the total sequence group length of the DNA or target gene having a sequence selected from the target gene sequence group. In some embodiments, the total length of the polynucleotide is between about 50 and about 750 nucleotides (for single-stranded polynucleotides) or base pairs (for double-stranded polynucleotides). In some embodiments, the polynucleotide is a dsRNA of between about 200 and about 750 base pairs, such as a dsRNA of the length of any one of the RNA trigger sequences disclosed in the Figures and Tables. In some embodiments, the dsRNA comprises a strand comprising a sequence selected from the group consisting of SEQ ID NO: 48, 80-82, 182, 188, 194, 213, and 220. In some embodiments, the dsRNA comprises a strand comprising at least one segment of at least 200, 300, 400, 500, 600, or 700 consecutive nucleotides having a sequence having at least 85% identity to a fragment of equivalent length found in the following sequence, the following sequence selected from the group consisting of the group of RNA trigger sequences and the group of reverse complements of RNA trigger sequences.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 are at least 85% identical to a fragment of an equivalent length found in the following sequences selected from the group consisting of: SEQ ID NO:48, 64, 80-82, 84-86, 182, 188, 194, 213, 220, 256, 262, 268, 287, and 294.

[0065] In some embodiments, the polynucleotide is designed to have complementarity with 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 is bound to (e.g., substantially 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 of 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 different in length from 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 nucleotides or more than 20 nucleotides longer than the second strand. dsRNA molecules can also be assembled by: a single oligonucleotide in a stem-loop structure, wherein the self-complementary sense region and antisense region of the RNA molecule are connected by a nucleic acid-based or non-nucleic acid-based joint; and a circular single-stranded RNA joint having two or more ring structures and a stem comprising a self-complementary sense strand and an antisense strand, wherein the circular RNA can be processed in vivo or in vitro to produce an active RNAi molecule capable of mediating RNAi. The RNAi molecule can include a 3' overhang at one end of the molecule; the other end can be a flat end, or it can 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 can be the same or different.

[0066] In some embodiments, the polynucleotide is designed to have complementarity with a region of a Fusarium graminearum gene associated with the DON pathway, and downregulation of the Fusarium graminearum gene reduces or eliminates DON production in Fusarium graminearum and increases mortality, causing growth inhibition or a reduction in reproductive capacity. In another embodiment, the polynucleotide comprises one or more sequences described herein for reducing DON production in Fusarium graminearum when transfected into or contacted with Fusarium graminearum, and one or more sequences that cause mortality, growth inhibition, reduction in virulence or pathogenicity, or reduction in reproduction / reproductive capacity (spore formation) of Fusarium graminearum when transfected into or contacted with Fusarium graminearum. Other embodiments target genes associated with the DON pathway of another Fusarium, such as Fusarium luteum or other species in the Fusarium genus that produce DON and / or cause Fusarium head blight in plants.

[0067] In some embodiments, the dsRNA comprises a strand comprising one or more nucleotide sequences, the one or more nucleotide sequences being 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 completely 100% identical to a sequence selected from an RNA trigger sequence or a reverse complement of an RNA trigger sequence. 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 consecutive nucleotides, the consecutive nucleotides being about 95% to about 100% identical to an equivalent portion of a sequence selected from a group of RNA trigger sequences or a group of reverse complements of RNA trigger sequences. Such dsRNA may further comprise a second strand complementary to the first strand. In some embodiments, the dsRNA comprises a first strand and a second strand, wherein the first strand comprises a nucleotide sequence selected from an RNA trigger sequence, and the second strand is selected from the reverse complement of the corresponding RNA trigger sequence. Specific embodiments include those in which the polynucleotide is a dsRNA, wherein the dsRNA comprises a first strand and a second strand, wherein the first strand comprises a nucleotide sequence selected from the group consisting of: SEQ ID NO: 48, 64, 80-82, 84-86, 182, 188, 194, 213, 220, 256, 262, 268, 287 and 294, and the second strand comprises nucleotides complementary to the first strand.

[0068] III. Length of the polynucleotide.

[0069] As provided herein, the length of the RNAi molecule of the target gene can vary. It should be understood that in some embodiments, when long RNA (e.g., dsRNA or ssRNA) molecules are applied (e.g., applied to plants), after entering the cell of the target fungus (e.g., Fusarium graminearum), the dsRNA is cut by the Dicer enzyme into a shorter double-stranded RNA fragment of, for example, 15 to 25 nucleotides in length. Therefore, the RNAi molecule of the present disclosure can be delivered as, for example, 15 to 25 nucleotide fragments, or can be delivered as a longer double-stranded nucleic acid (e.g., at least 100 nucleotides).

[0070] The total length of the polynucleotide of the present invention may be greater than or equal to 18 consecutive nucleotides, and may include nucleotides other than consecutive nucleotides having a sequence having about 75% to about 100% identity with a target gene identified in Table 1 or Table 2 or a target gene having a sequence selected from the group consisting of a target gene sequence group, or a fragment of an equivalent length of its DNA or its DNA complement or RNA transcribed therefrom. Similarly, the polynucleotide of the present invention may include one or more sequences that are about 75% to about 100% identical to 18 or more consecutive nucleotides of the following sequence, the following sequence being selected from the group consisting of an RNA trigger sequence group or an RNA trigger sequence reverse complement group, and may additionally include additional unrelated sequences. In other words, the total length of the polynucleotide may be greater than the length of a portion or segment of a polynucleotide designed to inhibit one or more target genes.

[0071] For example, the polynucleotide may have nucleotides flanking an "active" segment that inhibits a target gene (e.g., an "active" segment may be a sequence that is substantially complementary to a segment of a target gene or mRNA transcribed therefrom, or may be a sequence selected from a group of RNA trigger sequences or a group of reverse complements of RNA trigger sequences), or include "spacer" nucleotides between active segments, or may have additional nucleotides at the 5' end, at the 3' end, or at both the 5' end and the 3' end. In one embodiment, the polynucleotide may include additional nucleotides that are not specifically related to the sequences disclosed herein for controlling powdery mildew (having non-complementary or non-identical sequences). For example, such polynucleotides may contain nucleotides that provide a stable secondary structure or facilitate cloning or manufacturing. In one embodiment, the polynucleotide may include additional nucleotides positioned adjacent to the active segment. In one embodiment, the polynucleotide comprises one such segment, adjacent to which is an additional 5'G or an additional 3'C or both. In another embodiment, the polynucleotide is a double-stranded RNA comprising additional nucleotides to form one or more overhangs, for example, a dsRNA comprising two deoxyribonucleotides to form a 3' overhang. In other embodiments, the polynucleotide may comprise one or more active segments described herein, as well as additional segments that are active against other target genes of Fusarium graminearum or against another fungus of the genus Fusarium, such as another Fusarium that produces DON or is related to the Fusarium complex that causes Fusarium head blight in plants.

[0072] Thus, in various embodiments, the nucleotide sequence of the entire polynucleotide is not 100% identical or complementary to the set of RNA trigger sequences or the set of reverse complements of RNA trigger sequences, and is not 100% identical and complementary to the sequence of consecutive nucleotides in the following DNA: a target gene or homolog thereof identified in Table 1 or Table 2, or a target gene or its DNA complement having a sequence selected from the group consisting of the target gene sequence set. For example, in some embodiments, the polynucleotide comprises at least two segments each having 21 consecutive nucleotides, wherein the consecutive nucleotides have a sequence that is 100% identical to a fragment of a DNA or its DNA complement having a sequence selected from the group consisting of the target gene sequence set, wherein (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 segments appear in the DNA or its DNA complement having a sequence selected from the group consisting of the target gene sequence set.

[0073] Several embodiments relate to polynucleotides designed to regulate expression by inducing downregulation or inhibition of Fusarium graminearum target genes. In some embodiments, the polynucleotides are designed to have a nucleotide sequence that is substantially identical or substantially complementary to the nucleotide sequence of a Fusarium graminearum target gene or cDNA (e.g., a target gene sequence group), or to the sequence of an RNA transcribed from a Fusarium graminearum target gene, which may be a coding sequence or a non-coding sequence. These effective polynucleotide molecules for regulating expression may be referred to herein as "polynucleotides," "polynucleotide triggers," "triggers," or "triggers." Examples of such embodiments include polynucleotides comprising one or more sequences selected from an RNA trigger sequence group and an RNA trigger sequence reverse complement group. Additional examples include a polynucleotide comprising at least one segment of 18 or more consecutive nucleotides having a sequence having about 95% to about 100% identity with a portion of a sequence selected from an RNA trigger sequence group or an RNA trigger sequence reverse complement group.

[0074] Effective polynucleotides of any size 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 prepare a composition (e.g., a composition for topical application, or a recombinant DNA construct that can be used to prepare a transgenic plant). In other embodiments, a mixture or pool of different polynucleotide triggers is used; in this case, the polynucleotide trigger can be directed to a single target gene or multiple target genes.

[0075] IV. Permitted Mismatches

[0076] As used herein, "substantially identical" or "substantially complementary" means that a polynucleotide (or at least one strand of a double-stranded polynucleotide) has sufficient identity or complementarity with a target gene or an RNA transcribed from a target gene (e.g., a transcript) to inhibit the expression of a target gene (e.g., to affect a decrease in the level or activity of a target gene transcript and / or an encoded protein). A polynucleotide as described herein does not need to have 100% identity or complementarity with a target gene or an RNA transcribed from a target gene to inhibit the expression of a target gene (e.g., to affect a decrease in the level or activity of a target gene transcript or an encoded protein, or to provide control of DON production by Fusarium graminearum). In some embodiments, a polynucleotide or a portion thereof is designed to be substantially identical or substantially complementary to a sequence of at least 18 or 19 consecutive nucleotides in a target gene or an RNA transcribed from a 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 consecutive nucleotides in a target gene or an RNA transcribed from a target gene. In certain embodiments, "substantially identical" polynucleotides have 100% sequence identity or at least about 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity when compared to a sequence of 18 or more consecutive nucleotides in an endogenous target gene or RNA transcribed from a target gene. In certain embodiments, "substantially complementary" polynucleotides have 100% sequence complementarity or at least about 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence complementarity when compared to a sequence of 18 or more consecutive nucleotides in a target gene or RNA transcribed from a target gene.

[0077] Sequence identity: As used herein in the context of two polynucleotides or polypeptides, the terms "sequence identity" or "identity" refer to the residues in the sequences of two molecules that are the same when aligned for maximum correspondence over a specified comparison window.

[0078] The percent identity is calculated by determining the number of positions at which the same nucleotide or amino acid residue occurs in the two sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percent identity of the sequence. A sequence that is identical at every position 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 (e.g., nucleic acid sequences or polypeptide sequences) of the molecule over a comparison window, wherein the sequence portion in the comparison window may contain additions or deletions (i.e., gaps) compared to the reference sequence (not containing additions or deletions) to achieve optimal alignment of the two sequences. Optimal alignment of two or more sequences can be performed using local or global alignments by various available computer programs. 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 It is a suitable 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", Journal of Molecular Biology (J.Mol.Biol.) 48(3):443-53 PubMed:5420325 DOI:10.1016 / 0022-2836(70)90057-4 is a suitable 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 being compared and the relevant parameters, a local or global alignment strategy may be more likely to find the best alignment, but either strategy can be used to confirm the best alignment and thus provide the most accurate percent identity.

[0079] Regarding the numerical value of sequence length, the term "about" means a + / - variation of up to 1%-5% of the value. For example, about 30 consecutive nucleotides means a range of 27-33 consecutive nucleotides, or any range therebetween. Regarding the numerical value of the percentage of sequence identity, the term "about" means a + / - variation of up to 1%-3% of the percentage value 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%. Therefore, about 98% sequence identity means a range of 95-100%.

[0080] Polynucleotides containing mismatches with target genes or transcripts can be used for certain embodiments of compositions and methods described herein. In certain embodiments, variants provided herein contain randomly placed mutations, wherein for a given mutation, four nucleotides (A, U, G, C) are selected with approximately equal probability. In certain embodiments, these mutations may be distributed in a small region of the sequence, or may be widely distributed in the sequence length. In certain embodiments, polynucleotides include at least 18, or at least 19, or at least 21 continuous nucleotides, which are substantially identical or substantially complementary to the sections of equivalent lengths in the transcripts of target genes or target genes. In certain embodiments, polynucleotides of 18, 19, 20, or 21 or more continuous nucleotides substantially identical or substantially complementary to the sections of equivalent lengths of the transcripts of target genes or target genes can have 1 or 2 mismatches with the target genes or transcripts (i.e., there are 1 or 2 mismatches between the 21 continuous nucleotides of the polynucleotides and the sections of equivalent lengths of the transcripts of the target genes or target genes). 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 18, 19, 20, or 21 or more consecutive nucleotides that are identical or complementary to a target gene or a segment of an equivalent length in a transcript of a target gene may have 1 or 2 or more mismatches with the target gene or transcript.

[0081] When designing polynucleotides with mismatches with endogenous target genes or RNA transcribed by target genes, mismatches more likely to be tolerated of certain types and certain positions can be used. In certain embodiments, mismatches formed between adenine and cytosine or guanosine and uracil residues are used, as described in Du et al., (2005), "Nucleic Acids Res.", 33: 1671-1677. In certain embodiments, mismatches in 19 base pair overlaps are located at low tolerance positions 5, 7, 8 or 11 (starting from the 5' end of 19 nucleotide targets), medium tolerance positions 3, 4 and 12-17 (starting from the 5' end of 19 nucleotide targets) and / or high tolerance positions at either end of the complementary region, i.e., positions 1, 2, 18 and 19 (starting from the 5' end of 19 nucleotide targets), as described in Du et al., (2005), "Nucleic Acids Res.", 33: 1671-1677. In routine assays, the tolerable mismatch can be determined empirically.

[0082] V. Embedding Silencing Elements in Neutral Sequences

[0083] In some embodiments, the silencing element comprising a sequence corresponding to the target gene and responsible for the observed suppression of the target gene is embedded in a "neutral" sequence, i.e., inserted into another nucleotide that has no sequence identity or complementarity with the target gene. Neutral sequences may be desirable, for example, to increase the overall length of the polynucleotide or to impart a desired property, such as increased binding to the silencing complex. For example, it may be desirable for polynucleotides to have a specific size for reasons of stability, manufacturing cost-effectiveness, or effective biological activity (such as silencing efficiency). In some embodiments, neutral sequences can also be used to form favorable secondary structures, such as loops in hairpin triggers or as spacers between trigger regions.

[0084] Thus, in one embodiment, a 21 base pair dsRNA silencing element is embedded in an additional 39 base pair neutral sequence, thus forming a polynucleotide of about 60 base pairs, the silencing element corresponding to a target gene identified in Table 1 or Table 2 or a homolog thereof, or a target gene having a DNA sequence selected from the target gene sequence group and found to control DON production in Fusarium graminearum. In some embodiments, the dsRNA trigger comprises a neutral sequence between about 60 and about 500 or between 100 and about 450 base pairs, in which at least one segment of 21 consecutive nucleotides is embedded, the consecutive nucleotides having a sequence having 100% identity or 100% complementarity to a fragment of an equivalent length of a target gene having a sequence selected from the target gene sequence group. In another embodiment, a single 21 base pair silencing element having a sequence that is 100% identical or 100% complementary to a fragment of equivalent length of a target gene was found to be effective when embedded in a larger portion of a neutral sequence, for example, in the case where the total length of the polynucleotide 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 a total length of 210 base pairs, containing a single 21 base pair trigger (having 100% identity or complementarity to a 21 nucleotide fragment of a target gene) embedded in an additional 189 base pairs of neutral sequence, will have about 10% overall sequence identity with the target gene.

[0085] VI. Related Technologies

[0086] Embodiments of polynucleotides and nucleic acid molecules as described herein may include additional elements, such as promoters, transcription initiation elements, transcription extension elements, transcription termination elements, small RNA recognition sites, aptamers or ribozymes, additional and additional expression cassettes for expressing coding sequences (e.g., to express transgenes, such as fungicidal proteins or selectable markers) or non-coding sequences (e.g., to express additional repressive elements). For example, one aspect of the invention provides a recombinant DNA construct comprising a heterologous promoter having a transcription initiation sequence, operably linked to a DNA comprising at least one segment of 18 or more consecutive nucleotides having a sequence having about 95% to about 100% identity to a fragment of an equivalent length of a DNA having a sequence selected from the target gene sequence group or its DNA complement. Another aspect of the present invention provides a recombinant DNA construct comprising a heterologous promoter having a transcription initiation sequence, the heterologous promoter being operably linked to a DNA encoding an RNA hairpin having an antisense region having a sequence or a fragment of a sequence selected from the group selected from the group of RNA trigger sequences and the group of reverse complements of RNA trigger sequences. In another embodiment, a recombinant DNA construct comprising a promoter operably linked to a DNA is stably integrated into a plant genome in which an RNA transcript comprising an RNA aptamer and an RNA silencing element is expressed in a plant cell, the DNA encoding: (a) an RNA silencing element for inhibiting a target gene selected from the group of target gene sequences, and (b) an aptamer; the aptamer is used to guide the RNA silencing element to a desired location in a cell. In another embodiment, the inclusion of one or more recognition sites bound and cleaved by a small RNA (e.g., by a miRNA or siRNA expressed only in a specific cell or tissue) allows for a more precise expression pattern in a plant, wherein expression of the recombinant DNA construct is inhibited where the small RNA is expressed. Such additional elements are described below.

[0087] VII. Controlling DON production by Fusarium graminearum by contacting with polynucleotides.

[0088] Provided herein is a method for reducing or eliminating DON production of Fusarium graminearum or other fungal pathogens in the genus Fusarium that produces DON and / or causes Fusarium head blight. Such methods include contacting Fusarium graminearum with any polynucleotide and other compositions described herein. Some embodiments relate to methods for reducing or eliminating DON production of Fusarium graminearum on plants by contacting plants with any polynucleotide or other compositions described in, for example, Section II or Section VIII or elsewhere herein. Some embodiments relate to methods for reducing or eliminating DON production of Fusarium graminearum by contacting Fusarium graminearum on plants with polynucleotides, the polynucleotides comprising at least one segment of 18 or more continuous nucleotides, the continuous nucleotides and the corresponding fragments of the DNA of the target gene of the group consisting of another gene associated with DON production of Fusarium genus selected from the group consisting of genes identified in Table 1 or Table 2 or genes associated with DON production of Fusarium genus have about 95% to about 100% identity or complementarity. In one embodiment, a method for reducing or eliminating DON production by Fusarium graminearum on a plant by contacting the Fusarium graminearum with a polynucleotide comprising at least 18 consecutive nucleotides, the consecutive nucleotides having 100% identity with a target gene having a DNA sequence of SEQ ID NO: 16 or a corresponding fragment of its DNA complement. In other embodiments, a method for reducing or eliminating DON production by Fusarium graminearum on a plant by contacting the Fusarium graminearum with a polynucleotide comprising at least 18 consecutive nucleotides, the consecutive nucleotides having 100% identity with 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 a corresponding fragment of its DNA complement. In a specific embodiment, the target gene is selected from the group consisting of FGP1, ELP3, SPT7, MAF1, MVD1, HEP1, SET1, FKPB12 or homologs thereof of Fusarium graminearum, or in a more specific embodiment, FGP1 or a homolog thereof.

[0089] In some embodiments, the polynucleotide is a double-stranded RNA. In some embodiments, the polynucleotide (e.g., double-stranded RNA) is chemically or enzymatically synthesized, or is produced by expression in a microorganism or by expression in a plant cell. Embodiments include embodiments in which the polynucleotide is a dsRNA, the dsRNA comprising a chain having a sequence selected from an RNA trigger sequence group or an RNA trigger sequence reverse complement group. Embodiments further include embodiments in which the polynucleotide comprises at least one segment of 18 or more consecutive nucleotides, the consecutive nucleotides having a sequence having about 95% to about 100% identity with a portion of a sequence selected from an RNA trigger sequence group or an RNA trigger sequence reverse complement group. The polynucleotide used in the method can be designed for a plurality of target genes. Related aspects of the invention include using isolated polynucleotides in the method. Specific embodiments include those wherein the polynucleotide is a dsRNA comprising the sequence of SEQ ID NO:48, 64, 80-82, 84-86, 182, 188, 194, 213, 220, 256, 262, 268, 287, and 294, or its complement.

[0090] In some embodiments, the contiguous nucleotides have a sequence with about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identity to a fragment of an equivalent length of DNA or its DNA complement of a target gene identified in Table 1 or Table 2, or a target gene having a sequence selected from the target gene sequence group. In some embodiments, the contiguous nucleotides are completely (100%) identical to a fragment of an equivalent length of DNA or its DNA complement of a target gene identified in Table 1 or Table 2, or a target gene having a sequence selected from the target gene sequence group. In some embodiments, the polynucleotide has an overall sequence with about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identity to a fragment of an equivalent length of DNA or its DNA complement of a target gene identified in Table 1 or Table 2, or a target gene having a sequence selected from the target gene sequence group. In some specific embodiments, the target gene is selected from the group consisting of: FGP1, ELP3, SPT7, MAF1, MVD1, HEP1, SET1, FKPB12, or in more specific embodiments comprises FGP1.

[0091] In one embodiment, the polynucleotide comprises at least one segment of 21 consecutive nucleotides that are 100% identical to a corresponding fragment of a target gene or its DNA complement, the target gene having a DNA sequence selected from the group consisting of: SEQ ID NO: 16, 68-70, 108, 114, 120, 139 and 146. In some embodiments, in addition to the one or more segments of 21 consecutive nucleotides that are 100% identical to the corresponding fragment of the target gene, the polynucleotide comprises a "neutral" sequence (a sequence that has no sequence identity or complementarity to the target gene), and thus the polynucleotide as a whole has a much lower overall sequence identity to the target gene.

[0092] In certain embodiments, the polynucleotides used for this method are provided with isolated DNA or RNA fragments. In certain embodiments, the polynucleotides used for this method are not a part of an expression construct, and lack other elements (such as promoter or terminator sequences). Such polynucleotides can be relatively short, such as between about 18 and about 300 or between about 50 and about 750 nucleotides (for single-stranded polynucleotides) or between about 18 and about 300 or between about 50 and about 750 base pairs (for double-stranded polynucleotides) between the single-stranded or double-stranded polynucleotides. In certain embodiments, the polynucleotides are dsRNAs between about 100 and about 750 base pairs, such as SEQ ID NO:48,64,80-82,84-86,182,188,194,213,220,256,262,268,287 and 294 dsRNA triggers of any length of dsRNA. Alternatively, the polynucleotide can be provided in a more complex construct, for example as part of a recombinant expression construct, or included in a recombinant vector, for example 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 fungicidal protein).

[0093] Several embodiments relate to a method for reducing or eliminating DON production by Fusarium graminearum on a plant by contacting the Fusarium graminearum with a polynucleotide comprising at least one segment of 18 or more contiguous nucleotides that are substantially identical or complementary to a fragment of an 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 a dsRNA having a strand having a sequence selected from the group consisting of the RNA trigger sequence group. In some embodiments, the present invention provides a method for reducing or eliminating DON production by Fusarium graminearum on a plant by contacting the Fusarium graminearum with an effective amount of a solution comprising double-stranded RNA from the RNA trigger sequence group, and the solution further comprising an organosilicone surfactant.

[0094] In various embodiments of the methods, the contacting comprises applying a suitable composition comprising any polynucleotide 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 infected or potentially infected with Fusarium graminearum; such compositions can be provided, for example, in the form of a solid, liquid (including homogeneous mixtures, such as soluble liquid concentrates and heterogeneous mixtures, such as suspensions, colloids, micelles, and emulsions), a powder, a suspension, an emulsion, a spray, an encapsulated or microencapsulated formulation in or on microbeads or other carrier particles, in a film or coating, or on or within a substrate, or as a foliar, seed, root, or stem treatment. Compositions useful for facilitating the application of dsRNA to plants to contact insecticide (including dsRNA insecticide) formulations of pests or pathogens are known in the art, and any suitable formulation can be used with the polynucleotides of the invention. In one embodiment, the surface is a leaf, bulb, stem, ear, flower, or fruit of a plant. In such embodiments, application can be achieved by spraying the leaves, bulbs, stems, spikes, flowers or fruits of the plant. Contact can also be in the form of a seed treatment agent. Suitable adhesives, inert carriers, surfactants, etc. can be optionally included in the composition, as known to those skilled in the art of formulations of pesticides and seed treatment agents. In certain embodiments, contact is included in providing a polynucleotide in the composition, and the composition further comprises one or more carriers and / or one or more surfactants (for example, organosilicones, organosilicon surfactants), non-polynucleotide fungicides, polynucleotide herbicidal molecules, polynucleotide insecticides, non-polynucleotide insecticides, non-polynucleotide herbicidal molecules, non-polynucleotide insecticides, polynucleotide insecticides, safeners and pathogen growth regulators. In one embodiment, contact is included in providing a polynucleotide in the composition, and the polynucleotide can be transfected into the fungus of Fusarium or absorbed by its interior.

[0095] VIII. Double-stranded RNA molecules

[0096] Another aspect of the present invention provides a double-stranded RNA molecule, wherein when the double-stranded RNA molecule is transfected into or contacted with one or more fungal pathogens of the genus Fusarium on a plant, DON production of the one or more fungal pathogens is reduced or eliminated. Such dsRNA molecules comprise the nucleotide sequence of any polynucleotide described in Section II above or elsewhere herein as being useful for controlling DON production of Fusarium graminearum. In another embodiment, the dsRNA may further comprise one or more sequences that, when transfected into or contacted with Fusarium graminearum, cause death, growth inhibition, reduction in toxicity or pathogenicity, or reduction in reproduction / reproductive capacity (spore formation) of the Fusarium graminearum. Certain embodiments of the present invention provide a double-stranded RNA molecule that reduces or eliminates DON production by Fusarium graminearum when transfected into or contacted with Fusarium graminearum on a plant, wherein the double-stranded RNA molecule comprises at least one segment of 18 or more consecutive nucleotides, the at least one segment being substantially identical or substantially complementary to a target gene identified in Table 1 or Table 2 or a segment of an equivalent length of a target gene having a sequence selected from the group of target gene sequences. In specific embodiments, the target gene is selected from the group consisting of: FGP1, ELP3, SPT7, MAF1, MVD1, HEP1, SET1, FKPB12, or in more specific embodiments comprises FGP1. In some embodiments, the dsRNA comprises a first strand comprising one or more sequences selected from the group of RNA trigger sequences or the group of reverse complements of RNA trigger sequences. In some embodiments, the dsRNA comprises a first strand comprising a sequence that is substantially 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 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, at least 500, at least 600, or at least 700 consecutive nucleotides of: the following sequence is selected from the group consisting of an RNA trigger sequence or the reverse complement of an RNA trigger sequence, or in specific embodiments, is selected from the group consisting of: SEQ ID NO: 1 NO: 48, 64, 80-82, 84-86, 182, 188, 194, 213, 220, 256, 262, 268, 287 and 294.In some embodiments, the dsRNA comprises at least one segment of 18 or more consecutive nucleotides, and the consecutive nucleotides have about 95% to about 100% identity with a portion of a sequence selected from the group consisting of a RNA trigger sequence group or a reverse complement of an RNA trigger sequence group. In some embodiments, the dsRNA comprises a first strand, and the first strand comprises a sequence selected from an RNA trigger sequence or a reverse complement of an RNA trigger 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 completely 100% identical. In some embodiments, the dsRNA comprises a nucleotide sequence selected from the group consisting of: SEQ ID NO: 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 reverse complement of the corresponding RNA trigger sequence. In some embodiments, the dsRNA comprises a first strand comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 48, 80-82, 182, 188, 194, 213, and 220, and further comprises a second strand comprising a sequence selected from the group consisting of a corresponding complementary sequence, the corresponding complementary sequence selected from the group consisting of SEQ ID NO: 48, 64, 80-82, 84-86, 182, 188, 194, 213, 220, 256, 262, 268, 287, and 294.

[0097] The total length of one strand of the dsRNA can be greater than or equal to 18 consecutive nucleotides, and can include nucleotides other than consecutive nucleotides having a sequence of about 95% to about 100% with a portion of a sequence selected from the group of RNA trigger sequences or the reverse complement of the RNA trigger sequence or selected from the group consisting of: SEQ ID NO: 48, 64, 80-82, 84-86, 182, 188, 194, 213, 220, 256, 262, 268, 287 and 294. The dsRNA comprising a nucleotide sequence selected from the group of RNA trigger sequences and the reverse complement of the RNA trigger sequence can include nucleotides other than nucleotides of a sequence selected from the group of RNA trigger sequences and the reverse complement of the RNA trigger sequence. In other words, the total length of the dsRNA strand can be greater than the length of a sequence selected from the group of RNA trigger sequences or the reverse complement of the RNA trigger sequence or a portion of the sequence. For example, the dsRNA may have nucleotides flanking the "active" segment that inhibits the target gene, or include "spacer" nucleotides between the active segments, or may have additional nucleotides at the 5' end or at the 3' end or at both the 5' end and the 3' end. In one embodiment, the dsRNA may include additional nucleotides that are non-specifically associated with the target gene targeted by a given trigger (having a sequence that is not complementary or identical to the target gene), for example, nucleotides that provide a stable secondary structure or facilitate cloning or manufacturing. In one embodiment, the dsRNA may include additional nucleotides positioned adjacent to a sequence or a portion of the sequence selected from the RNA trigger sequence group or the RNA trigger sequence reverse complement group. In one embodiment, the dsRNA comprises one such segment, and adjacent to the segment is an additional 5'G or an additional 3'C or both. In another embodiment, the dsRNA further comprises additional nucleotides to form an overhang, for example, a dsRNA comprising 2 deoxyribonucleotides to form a 3' overhang. Thus, in various embodiments, the nucleotide sequence of the complete dsRNA is not 100% identical or complementary to the RNA trigger sequence or the reverse complement sequence of the RNA trigger sequence. For example, in some embodiments, the dsRNA comprises at least two segments each having 21 consecutive nucleotides, wherein the consecutive nucleotides have a sequence that is 100% identical to a portion of a sequence selected from the RNA trigger sequence or the reverse complement of the RNA trigger sequence, wherein (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 appear in the target gene.

[0098] In some embodiments, the length of the double-stranded RNA molecule is between about 50 and about 750 base pairs. In some embodiments, the double-stranded RNA molecule comprises a plurality of segments of 18 or more consecutive nucleotides, the consecutive nucleotides being substantially identical or substantially complementary to a segment of an equivalent length of a target gene identified in Table 1 or Table 2 or a homologue thereof or a target gene having a sequence selected from the target gene sequence group, and optionally comprising at least a second segment of 18 or more consecutive nucleotides, the consecutive nucleotides being substantially identical or substantially complementary to a segment of an equivalent length of a second target gene identified in Table 1 or Table 2 or a homologue thereof or a target gene having a sequence selected from the target gene sequence group. In some embodiments, the double-stranded RNA molecule comprises a plurality of segments of 18 or more consecutive nucleotides, the consecutive nucleotides being substantially identical or substantially complementary to a segment of an equivalent length of a target gene, wherein the segments are 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 be optionally used between segments or in adjacent segments), or from different target genes. In some embodiments, the double-stranded RNA molecule comprises a plurality of segments of 18 or more consecutive nucleotides, the consecutive nucleotides being substantially identical or substantially complementary to a segment of an equivalent length of a target gene identified in Table 1 or Table 2 or a target gene having a sequence selected from the target gene sequence group, wherein the segments are from different regions of the target gene and are 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 each having 18 consecutive nucleotides, the consecutive nucleotides being 100% identical or 100% complementary to a segment of an equivalent length of a target gene identified in Table 1 or Table 2 or a target gene having a sequence selected from the target gene sequence group, wherein the segments are from different regions of the target gene and are 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 comprising a sequence selected from the group consisting of an RNA trigger sequence group or its complement.

[0099] The double-stranded RNA molecule can be topically applied to plants to reduce or eliminate DON production in Fusarium graminearum or another fungus in the genus Fusarium that produces DON and / or causes Fusarium head blight. The double-stranded RNA molecule can be provided in a form suitable for transfection or direct contact with Fusarium graminearum, such as in the form of a spray or powder. Other methods and suitable compositions for providing double-stranded RNA molecules are similar to those described in the preceding paragraphs of other aspects of the invention.

[0100] Several embodiments relate to tank mixtures comprising one or more polynucleotides and water or other solvents (optionally including an organosilicon surfactant). Compositions suitable for topical application of dsRNA and other polynucleotides to plants to protect plants from pests or pathogens are well known in the art. Such compositions include any composition described in PCT / US / 2022 / 027816. Embodiments include tank mixture formulations of polynucleotides and optionally at least one pesticide. Embodiments of such compositions include embodiments in which one or more polynucleotides are provided in living or dead microorganisms such as bacteria or fungi or yeast cells, or embodiments provided as microbial fermentation products, or embodiments provided in living or dead plant cells, or embodiments provided as synthetic recombinant polynucleotides. In one embodiment, the composition includes a non-pathogenic strain of a microorganism containing a polynucleotide as described herein; the ingestion of the microorganism reduces or eliminates DON production by Fusarium graminearum and / or causes growth inhibition, reduction in toxicity or pathogenicity, or reduction in reproduction / reproductive capacity (spore formation) or death of the Fusarium graminearum; non-limiting examples of suitable microorganisms include E. coli, B. thuringiensis, Pseudomonas sp., Photorhabdus sp., Xenorhabdus sp., Serratia entomophila, and related Serratia species. 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; treatment of a Fusarium graminearum infection on a plant with the plant viral vector causes growth inhibition, mortality, reduction in toxicity or pathogenicity, or reduction in reproductive / reproductive capacity (spore formation) of the Fusarium graminearum. In one embodiment, the composition comprises a baculovirus vector comprising a polynucleotide as described herein; ingestion of the vector reduces or eliminates DON production by the Fusarium graminearum and / or causes growth inhibition, mortality, reduction in toxicity or pathogenicity, or reduction in reproductive / reproductive capacity (spore formation) of the Fusarium graminearum.In one embodiment, a polynucleotide as described herein is encapsulated in a synthetic matrix (such as a polymer), or attached to microparticles, and topically applied to the surface of a plant; infection of Fusarium graminearum on the topically treated plant reduces or eliminates DON production by Fusarium graminearum, causes growth inhibition, mortality, or a reduction in toxicity or pathogenicity, or a reduction in reproductive / reproductive capacity (spore formation). In one embodiment, a polynucleotide as described herein is provided in the form of a plant cell (e.g., a transgenic plant cell of the invention) expressing the polynucleotide; infection of the plant cell or the contents of the plant cell by Fusarium graminearum reduces or eliminates DON production by Fusarium graminearum.

[0101] In some embodiments, one or more polynucleotides as described herein are provided together with suitable adhesives and wetting agents required for efficient foliar coverage, and UV protectants to protect polynucleotides such as dsRNA from UV damage. In some embodiments, one or more polynucleotides as described herein are further provided together with: carriers, surfactants, organosilicones, organosilicon surfactants, non-polynucleotide fungicides, polynucleotide herbicidal molecules, non-polynucleotide herbicidal molecules, non-polynucleotide insecticides, polynucleotide insecticides, non-polynucleotide insecticides, safeners, and pathogen growth regulators. In some embodiments, the composition further includes at least one insecticide or fungicide.

[0102] Such compositions are applied in any convenient manner, for example, by spraying or dusting the Fusarium graminearum directly, or by spraying or dusting the plants (including, for example, the leaves, stems or leaf balls of the plants) or the environment in which it is desired to control DON production by Fusarium graminearum, 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 a soil drench around the roots of the plants in which it is desired to control DON production by Fusarium graminearum.

[0103] The effective amount of a polynucleotide as described herein is sufficient to reduce or eliminate the amount of DON produced by fungal pathogens of the genus Fusarium, such as Fusarium graminearum; The effective amount of a polynucleotide is determined using conventional assays. Although there is no upper limit to the concentration and dosage of the polynucleotides that can be used in the methods and compositions provided herein, lower effective concentrations and dosages are generally sought for efficiency and economy. Non-limiting examples of the effective amount of a polynucleotide include polynucleotides in the range of about 10 nanograms per milliliter to about 100 micrograms per milliliter sprayed on plants in liquid form, or polynucleotides in the range of about 10 milligrams per acre to about 100 grams per acre applied to plant fields. In the case of local application of polynucleotides as described herein to plants, the volume of sprays or treatment agents applied to plant leaves or leaf balls or other plant part surfaces such as petals, stems, fruits, anthers, pollen, leaves, leaf balls, spikes, roots or seeds can be considered to adjust the concentration. In one embodiment, a useful treatment agent for herbaceous plants using a 25-mer polynucleotide as described herein is about 1 nanomole (nmol) of polynucleotide per plant, such as about 0.05 to 1 nmol of polynucleotide per plant. Other embodiments of herbaceous plants include useful ranges of polynucleotides from about 0.05 to about 100 nmol per plant, or from about 0.1 to about 20 nmol, or from about 1 nmol to about 10 nmol. In certain embodiments, about 40 to about 50 nmol of ssDNA polynucleotides are 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 mg / ml, or about 0.14 mg / ml of dsRNA or ssDNA (21 polymers) is applied. In certain embodiments, about 0.5 to about 1.5 mg / ml of the present invention is applied. Compositions of dsRNA polynucleotides having about 50 to about 200 or more nucleotides are applied. In certain embodiments, about 1 nmol to about 5 nmol of the present invention dsRNA is applied to plants. In certain embodiments, the polynucleotide composition applied topically to plants contains at least one polynucleotide of the present invention at a concentration of about 0.01 to about 10 mg / ml, or about 0.05 to about 2 mg / ml, or about 0.1 to about 2 mg / ml. In some embodiments, very large plants, trees or vines that are applied at a concentration of about 5 g to about 100 g of polynucleotide active ingredient per hectare may require a correspondingly larger amount of polynucleotide. When using long dsRNA molecules of the present invention that can be processed into multiple oligonucleotides (e.g., multiple triggers encoded by a single recombinant DNA molecule of the present invention), lower concentrations can be used. Non-limiting examples of effective polynucleotide treatment regimens include treatments of about 0.1 to about 1 nmol of polynucleotide molecules per plant, or between about 1 nmol and about 10 nmol of polynucleotide molecules per plant, or between about 10 nmol to about 100 nmol of polynucleotide molecules per plant.

[0104] When the compositions and polynucleotides of the invention are topically applied to plants, such as in the form of a spray, the application can be carried out at any suitable stage of plant growth. Exemplary applications to cereals can be carried out at heading stage (Feekes 10.1-10.5) and / or flowering stage (Feekes 10.5.1-10.5.3), and if additional applications are used, can be applied as late as Feekes 10.5.4.

[0105] In some embodiments, one or more polynucleotides are provided with a "transfer agent", which is an agent that enables a topically applied polynucleotide to enter an organism's cell. Such a transfer agent can be incorporated as part of a composition comprising a polynucleotide as described herein, or can be applied before, simultaneously with, or after the polynucleotide is applied. In some embodiments, a transfer agent is an agent that increases the uptake of the polynucleotides of the present invention by Fusarium graminearum. In some embodiments, a transfer agent is an agent that regulates the surface of a plant tissue (e.g., a seed, leaf, bulb, spike, stem, root, flower, or fruit) to allow the polynucleotide to penetrate into a plant cell. In some embodiments, a transfer agent enables a polynucleotide to enter a plant cell through a cuticle wax barrier, stomata, and / or a cell wall or membrane barrier.

[0106] Suitable transfer agents include agents that increase the permeability outside the organism or increase the permeability of the cells of the organism to the polynucleotide. Suitable transfer agents include chemical agents, physical agents or combinations thereof. Chemical agents for regulating or transferring include (a) surfactants, (b) organic solvents or aqueous solutions or aqueous mixtures of organic solvents, (c) oxidants, (d) acids, (e) bases, (f) oils, (g) enzymes, or any combination thereof. In certain embodiments, the application of polynucleotides and transfer agents optionally includes an incubation step, a neutralization step (e.g., to neutralize acid, base or oxidant, or to inactivate enzymes), a flushing step or a combination thereof. Suitable transfer agents can be in the form of emulsions, reverse emulsions, liposomes or other micellar compositions, or polynucleotides can be made to take the form of emulsions, reverse emulsions, liposomes or other micellar compositions. Embodiments of transfer agents include counterions or other molecules known to associate with nucleic acid molecules, such as inorganic ammonium ions, alkylammonium ions, lithium ions, polyamines such as spermine, spermidine or putrescine, and other cations. Examples 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 nucleotide phosphates in non-aqueous systems (such as solvents used in synthetic reactions). Examples of transfer agents include naturally derived or synthetic oils with or without surfactants or emulsifiers, such as oils of plant origin, crop oils (such as those listed in the 9th edition of the catalog of Herbicide Adjuvants, publicly available online at herbicide.adjuvants.com), paraffin oils, polyol fatty acid esters, or oils with short chain molecules modified with amides or polyamines such as polyethyleneimine or N-pyrrolidine.

[0107] Examples of transfer agents include organosilicon formulations. For example, a suitable transfer agent is an organosilicon formulation commercially available as: SILWET Brand surfactant (having CAS No. 27306-78-1 and EPA No.: CAL.REG. NO. 5905-50073-AA) and currently available from Momentive Performance Materials, Albany, NY. BREAK-THRU S240 brand polyether-modified polysiloxane (CASRN proprietary) surfactant, currently available from Goldschmidt Chemical Corporation, Hopewell, VA. BREAK-THRU S279 end-capped polyether trisiloxane surfactant, components of which are listed on the following chemical inventories: EINECS, TSCA, ENCS, AICS, ECL, PICCSCHINA, NDSL. INDUCE brand adjuvant NMFC, Item 42652, Class 60, currently available from Helena Chemical Company, Collierville, TN. and Brand surfactant (having CA REG No. 34704-50065, currently available from Loveland Products, Inc. Greely, CO.). One embodiment includes a composition comprising a polynucleotide and BREAK-thru301. One embodiment includes a composition comprising a polynucleotide and a transfer agent, the transfer agent included in the range of about 0.015 to about 2 weight percent (wt %) (e.g., about 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.04 5, 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 %) of an organosilicon preparation such as Silwet L-77, Break-thru S240, Break-thru S279, Induce or Franchise. One embodiment includes a composition comprising a polynucleotide of the invention and a transfer agent comprising SILWET in a range of about 0.3 to about 1 weight percent (wt%) or about 0.5 to about 1 weight percent (wt%). BREAK-THRU S240, BREAK-THRU S279, Induce, or Franchise brand surfactants.

[0108] The organosilicon compounds that can be used as transfer agents for use in the present invention include, but are not limited to, compounds comprising: (a) a covalently linked trisiloxane head group, (b) a covalently linked alkyl linker, including but not limited to a n-propyl linker, (c) a covalently linked polyethylene glycol chain, and (d) a terminal group. The trisiloxane head group of such organosilicon compounds includes, but is not limited to, heptamethyltrisiloxane. The alkyl linker may include, but is not limited to, an n-propyl linker. The polyethylene glycol chain includes, but is not limited to, polyethylene glycol or polypropylene glycol. The polyethylene glycol chain may include a mixture that provides an average chain length "n" of about "7.5". In certain embodiments, the average chain length "n" may vary between about 5 and about 14. The terminal group may include, but is not limited to, an alkyl group, such as a methyl group. The organosilicon compounds that can be used 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:

[0109]

[0110] (Compound I: polyalkylene oxide heptamethyltrisiloxane, average n=7.5).

[0111] Organosilicone compounds that can be used as transfer agents, for example, freshly prepared organosilicone compounds are used in concentrations ranging from about 0.015 to about 2 weight percent (wt %) (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 %).

[0112] Embodiments of transfer agents 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, 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, a composition comprising a polynucleotide includes an ammonium salt at a concentration greater than or equal to 300 millimolar. In certain embodiments, a composition comprising a polynucleotide includes an organosilicone transfer agent at a concentration of about 0.015 to about 2 weight percent (wt%) and ammonium sulfate at a concentration of about 80 to about 1200 mM or about 150 mM to about 600 mM.

[0113] Embodiments of transfer agents include phosphates. Phosphates that can be used for compositions comprising polynucleotides include, but are not limited to, calcium phosphates, magnesium phosphates, potassium phosphates, or sodium phosphates. In certain embodiments, compositions comprising polynucleotides include phosphates having a concentration of at least about 5 millimoles, at least about 10 millimoles, or at least about 20 millimoles. In certain embodiments, compositions comprising polynucleotides include phosphates in the range of about 1mM to about 25mM or in the range of about 5mM to about 25mM. In certain embodiments, compositions comprising polynucleotides include sodium phosphates in the range of at least about 5 millimoles, at least about 10 millimoles, or at least about 20 millimoles. In certain embodiments, compositions comprising polynucleotides include sodium phosphates in the range of about 5 millimoles, about 10 millimoles, or about 20 millimoles. In certain embodiments, compositions comprising polynucleotides include sodium phosphates in the range of about 1mM to about 25mM or in the range of about 5mM to about 25mM. In certain embodiments, the composition comprising the 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. In certain embodiments, the composition comprising the polynucleotide comprises a sodium phosphate buffer at a pH of about 6.8.

[0114] Embodiments of transfer agents include surfactants and / or effective molecules contained therein. Surfactants and / or effective molecules contained therein include, but are not limited to, sodium or lithium salts of fatty acids (such as tallow or tallow fatty amine or phospholipids) and organosilicone surfactants. In certain embodiments, compositions comprising polynucleotides are 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 polynucleotides are formulated with non-polynucleotide herbicides, such as glyphosate, auxin-like benzoic acid herbicides including dicamba, chloramben and TBA, glufosinate, auxin-like herbicides including phenoxycarboxylic acid herbicides, pyridinecarboxylic acid herbicides, quinolinecarboxylic acid herbicides, pyrimidinecarboxylic acid herbicides and benazoline-ethyl herbicides, sulfonylureas, imidazolinones, bromoxynil, dalapon, cyclohexanedione, protoporphyrinogen oxidase inhibitors, and 4-hydroxyphenyl-pyruvate-dioxygenase inhibiting herbicides.

[0115] IX. Compositions for controlling DON production by Fusarium graminearum

[0116] Another aspect of the present invention provides a composition for reducing or eliminating DON production of Fusarium graminearum, the composition comprising an effective amount of at least one RNA. Such RNA can be any RNA described in Section II, Section VIII or elsewhere herein. In one embodiment, the RNA comprises 18 or more, or 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, 1000 or more, 1250 or more, 1500 or more, 2000 or more, 2500 or more, 3000 or more, 4000 or more, 5000 or more, 6000 or more, 7000 or more, 8000 or more, 9000 or more, 10 ... or more or 1,000 or more contiguous nucleotides having 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 completely 100% identity to a target gene identified in Table 1 or Table 2, or a homolog thereof, or a target gene having a sequence selected from the group consisting of a target gene sequence group, or a corresponding fragment of a DNA, or a DNA complement thereof, or an RNA transcribed therefrom. In an embodiment, the RNA comprises a nucleotide sequence that is substantially 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 consecutive 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.

[0117] In some embodiments, the RNA comprises a sequence that is substantially 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 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. In some embodiments, the polynucleotide comprises a sequence that is substantially 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 the reverse complement of an RNA trigger sequence. In some embodiments, the polynucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 48, 80-82, 182, 188, 194, 213, and 220.

[0118] "Effective amount" means the amount of an agent that effectively induces a biological change in a fungal pathogen, thereby eliminating or reducing the DON production of the fungal pathogen; in some embodiments, applying an effective amount of RNA to a plant improves the plant's resistance to damage from Fusarium graminearum. The RNA may be longer than one or more segments it contains (i.e., the RNA may contain additional nucleotides 3' and / or 5' of the segment), but each segment and the corresponding fragment of the target gene have an equivalent length. The RNA used in the method can be designed for multiple target genes. Embodiments include embodiments in which the composition comprises: an effective amount of a polynucleotide comprising at least 18, 19, 20, or 21 consecutive nucleotides complementary to 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 target gene sequence group, or a portion of an RNA transcribed from such a target gene; or an effective amount of at least one polynucleotide comprising at least one silencing element, the at least one silencing element being substantially complementary or substantially identical to at least 21 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 target gene sequence group; or an effective amount of at least one RNA embodiment comprising at least one segment, the at least one segment being complementary to a target gene having SEQ ID NO: 16 or at least 18, 19, 20 or 21 consecutive nucleotides of a target gene of the nucleotide sequence of the target gene or an RNA transcribed from the target gene are identical or complementary; or an RNA molecule that reduces or eliminates DON production by Fusarium graminearum when transfected into Fusarium graminearum on a plant or contacted with the Fusarium graminearum, wherein the RNA molecule comprises a residue selected from SEQ ID NO: 16, 68-70, 108, 114, 120, 139 and 146 of the nucleotide sequence of at least 18, 19, 20 or 21 consecutive nucleotides complementary to a target gene or a portion of RNA transcribed from the target gene; or an effective double-stranded RNA molecule that reduces or eliminates DON production by Fusarium graminearum when transfected into or contacted with the Fusarium graminearum, wherein at least one strand of the effective double-stranded RNA molecule comprises 21 consecutive nucleotides complementary to a portion of the target gene or RNA transcribed from the target gene, wherein the target gene has a sequence selected from the group consisting of target gene sequences; or an effective amount of at least one double-stranded RNA, wherein the at least one double-stranded RNA comprises a sequence selected from the group of RNA trigger sequences. In some embodiments, the polynucleotide is a 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 the group consisting of an RNA trigger sequence, a reverse complement of an RNA trigger sequence.

[0119] In various embodiments, the composition for eliminating or reducing DON production in Fusarium graminearum is in the form of at least one selected from the group consisting of solid, liquid (including homogeneous mixtures such as soluble liquid concentrates and heterogeneous mixtures such as suspensions, colloids, micelles and emulsions), powders, suspensions, emulsions, aerosols, encapsulated or microencapsulated formulations, in or on microbeads or other carrier particles, in films or coatings, or on or in substrates, or as leaf, seed, root, bulb, ear or stem treatments. Suitable adhesives, inert carriers, surfactants, and the like may optionally be included in the composition containing polynucleotides, as known to those skilled in the art of formulations of fungicides and seed, stem, ear, bulb, fruit or foliar treatments. Such compositions may include any of the compositions described in PCT / US / 2022 / 027816, published as WO2022 / 0235895 on November 10, 2022, which is incorporated herein by reference in its entirety. In some embodiments, the composition is at least one implantable formulation selected from the group consisting of microparticles, granules or capsules implanted in a plant; in such embodiments, the method comprises implanting the implantable formulation in a plant. In one embodiment, the composition can be transfected by or otherwise internally absorbed by Fusarium graminearum. In some embodiments, the composition further comprises one or more components selected from the group consisting of: a carrier, a surfactant, an organosilicone, an organosilicone surfactant, a polynucleotide herbicidal molecule, a non-polynucleotide herbicidal molecule, a non-polynucleotide pesticide, a polynucleotide pesticide and a safener, a pathogen growth regulator. In one embodiment, the composition further comprises a non-ionic organosilicone surfactant, such as Brand surfactants, such as SILWET Brand surfactant, having CAS No. 27306-78-1 and EPA No.: CAL.REG.NO.5905-50073-AA, currently available from Momentive Performance Materials Group, Albany, New York. One embodiment includes a composition further comprising BREAK-THRU 301. Other surfactants include, for example, BREAK-THRU S240 brand polyether-modified polysiloxane (CASRN proprietary) surfactant, currently available from Goldschmidt Chemical Company, Hopewell, Virginia; BREAK-THRUS 279 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 adjuvant NMFC Item 42652, Class 60, currently available from Helena Chemical Company, Collierville, Tennessee. With LECI- Brand surfactant (having CA REG No. 34704-50065, currently available from Loveland Products, Inc., Greeley, Colorado. Alternatively, the plants are topically treated with the composition and with a separate (before, after or simultaneously) application of a material that improves the efficacy of the composition. For example, the plants may be topically treated with a first topical application of a nonionic organosilicone surfactant (such as Brand surfactants, such as SILWET BREAK-THRU S24, BREAK-THRU S279, INDUCE or FRANCHISE brand surfactants) followed by a second topical application of the composition to spray the plants, or vice versa.

[0120] It is expected that certain RNAs (e.g., dsRNA triggers described in the working examples) used in this method in combination with one or more fungicidal polynucleotides and / or one or more non-polynucleotide fungicides will result in enhanced plant protection when compared to the DON reduction or elimination effects obtained using the RNA and fungicidal RNA and / or non-polynucleotide fungicides alone.

[0121] In various embodiments, the composition comprises a microbial cell or is produced in a microorganism. For example, the composition may include a bacterial or yeast cell or may be produced in the bacterial or yeast cell. In similar embodiments, the composition comprises a transgenic plant cell or is produced in a plant cell (e.g., a plant cell transiently expressing a polynucleotide); such plant cells may be cells in a plant or cells grown in tissue culture or cell suspension.

[0122] In one embodiment, the composition is provided in the form of any plant infected by Fusarium graminearum, wherein the RNA is contained in or on the plant. Such plants can be stable transgenic plants expressing RNA, or non-transgenic plants transiently expressing RNA, or non-transgenic plants that have been treated with RNA (e.g., by spraying or coating). Stable transgenic plants typically contain a recombinant construct encoding RNA integrated into their genome.

[0123] The RNA used in the composition can be single-stranded (ss) or double-stranded (ds). Embodiments include embodiments 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); mixtures of RNA of any of these types can be used. In one embodiment, double-stranded DNA / RNA hybrids are used. The RNA can include components other than standard ribonucleotides, for example, one embodiment is an RNA containing terminal deoxyribonucleotides.

[0124] The RNA in the composition has at least one segment of 18 or more consecutive nucleotides having a sequence having about 95% to about 100% identity with a target gene identified in Table 1 or Table 2 or a homolog thereof, or a target gene having a sequence selected from the target gene sequence group, or a fragment of an equivalent length of its DNA complement. In one embodiment, the RNA comprises at least one segment of 18 or more consecutive nucleotides, which are substantially identical or complementary to a target gene identified in Table 1 or Table 2 or a homolog thereof, or a fragment of an equivalent length of a target gene having a sequence selected from the group consisting of the target gene sequence group. In some embodiments, the consecutive nucleotides have a sequence having about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identity with a DNA of a target gene identified in Table 1 or Table 2 or a fragment of a DNA having a sequence selected from the target gene sequence group, or a fragment of its DNA complement. In some embodiments, the contiguous nucleotides are completely (100%) identical to a fragment of an equivalent length of a DNA of a target gene or homolog thereof identified in Table 1 or Table 2, or a DNA having a sequence selected from the target gene sequence group, or a fragment of its DNA complement. In some embodiments, the RNA has an overall sequence having about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to a DNA of a target gene or homolog thereof identified in Table 1 or Table 2, or a DNA having a sequence selected from the target gene sequence group, or a fragment of its DNA complement.

[0125] The RNA in the composition comprises at least one segment of 18 or more consecutive nucleotides having a sequence having about 95% to about 100% identity with a target gene identified in Table 1 or Table 2, or a homolog thereof, or a fragment of an equivalent length of a DNA or its DNA complement having a sequence selected from the target gene sequence group. In some embodiments, the RNA comprises at least one segment of 18 or more, for example, between 18-24, or between 18-28, or between 20-30, or between 20-50, or between 20-100, or between 50-100, or between 50-500, or between 100-250, or between 100-500, or between 200-1000, or between 500-2000, or even more consecutive nucleotides. In some embodiments, the segment comprises 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, 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 310, about 320, about 330, about 340, about 350 In certain embodiments, the RNA comprises at least one segment of at least 18, 19, 20, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 350, 400, 450, 500, 550, 650, 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 with 100% identity to a target gene identified in Table 1 or Table 2, or a homologue thereof, or a fragment of an equivalent length of a target gene or its DNA complement having a sequence selected from the target gene sequence group. In specific embodiments, the RNA is a double-stranded nucleic acid (e.g., dsRNA), wherein one strand comprises at least one segment of at least 18, 19, 20 or 21 consecutive nucleotides having a sequence that is 100% identical to a DNA having a sequence selected from the target gene sequence group or a fragment of an equivalent length of a target gene or its DNA complement; expressed in base pairs, such a double-stranded nucleic acid comprises at least one segment of at least 18, 19, 20 or 21 consecutive fully matched base pairs corresponding to a DNA having a sequence selected from the target gene sequence group or a fragment of an equivalent length of a target gene or its DNA complement.In certain embodiments, the length of each segment contained in the RNA is greater than the typical length of naturally occurring regulatory small RNAs, for example, the length of each segment is at least about 30 consecutive nucleotides (or base pairs). In certain embodiments, the total length of the RNA or the length of each segment contained in the RNA is less than the total length of the sequence of interest (DNA or target gene having a sequence selected from a group consisting of a target gene sequence group). In certain embodiments, the total length of the RNA is between about 50 and about 500 nucleotides (for single-stranded RNA) or base pairs (for double-stranded RNA). In certain embodiments, the RNA comprises at least one RNA chain between about 50 and about 750 nucleotides in length.

[0126] The RNA in the composition is generally designed to inhibit one or more genes ("target genes"). Such target genes may include coding sequences or non-coding sequences or both. In a specific embodiment, RNA is designed to inhibit one or more target genes, wherein each target gene has a DNA sequence selected from a group consisting of a target gene sequence group. In various embodiments, RNA is designed to inhibit one or more genes, wherein each target gene is selected from a target gene identified in Table 1 or Table 2 or a homologue thereof or a sequence selected from a group consisting of a target gene sequence group, and can be designed to inhibit multiple genes from these groups, or target different regions of one or more genes in these genes. In one embodiment, RNA includes multiple parts or segments, each of which includes at least one segment of 21 consecutive nucleotides, and the consecutive nucleotides have a sequence with 100% identity to a target gene identified in Table 1 or Table 2 or a homologue thereof or a fragment of an equivalent length of a DNA or its DNA complement having a sequence selected from a target gene sequence group. In such cases, each part may be identical or different in size or in sequence, and may be sense or antisense relative to the target gene. For example, in one embodiment, the RNA may include multiple parts arranged in tandem or repeats, wherein each part comprises at least one segment of 21 consecutive nucleotides, the consecutive nucleotides having a sequence that is 100% identical to a fragment of an equivalent length of a DNA or its DNA complement having a sequence selected from the target gene sequence group; the segments may be from different regions of the target gene, for example, 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 segments or in adjacent segments.

[0127] The total length of the RNA in the composition may be greater than 18 consecutive nucleotides, and may include nucleotides other than consecutive nucleotides having a sequence having about 95% to about 100% identity with a target gene identified in Table 1 or Table 2 or a homolog thereof, or a fragment of an equivalent length of a DNA or its DNA complement having a sequence selected from the target gene sequence group. In other words, the total length of the RNA may be greater than the length of a portion or segment of the RNA designed to inhibit one or more target genes, wherein each target gene is selected from the group consisting of the target genes identified in Table 1 or Table 2 or a homolog thereof, or has a DNA sequence selected from the group consisting of the target gene sequence group. For example, the RNA may have nucleotides flanking an "active" segment of at least one segment of 18 or more consecutive nucleotides that inhibit a target gene, or include "spacer" nucleotides between active segments, or may have additional nucleotides at the 5' end or at the 3' end or at both the 5' end and the 3' end. In one embodiment, the RNA comprises additional nucleotides that are non-specifically associated with the DNA of the target gene (having a sequence that is not complementary or identical thereto), for example, nucleotides that provide a stable secondary structure or facilitate cloning or manufacturing. In one embodiment, the RNA comprises additional nucleotides positioned adjacent to one or more segments of 18 or more consecutive nucleotides, the one or more segments having a sequence with a DNA having a sequence selected from a group consisting of a target gene sequence group or a fragment of an equivalent length of a target gene having about 95% to about 100% identity or complementarity. In one embodiment, the RNA comprises one such segment, adjacent to the segment is an additional 5'G or an additional 3'C or both. In another embodiment, the RNA is a double-stranded RNA comprising additional nucleotides to form an overhang, for example, a dsRNA comprising 2 deoxyribonucleotides to form a 3' overhang. Therefore, in various embodiments, the nucleotide sequence of the complete RNA is not 100% identical or complementary to the fragment of the consecutive nucleotides in the target gene. For example, in some embodiments, the RNA comprises at least two segments each having 21 consecutive nucleotides having a sequence that is 100% identical to a fragment of a DNA having a sequence selected from the target gene sequence group or its DNA complement, wherein (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 appear in the DNA having a sequence selected from the target gene sequence group or its DNA complement.

[0128] In various embodiments, the RNA in the composition comprises naturally occurring ribonucleotides. Embodiments include, for example, synthetic RNAs that are composed entirely of ribonucleotides or that are composed primarily of ribonucleotides but that have one or more terminal deoxyribonucleotides or one or more terminal dideoxyribonucleotides. In certain embodiments, the RNA comprises atypical 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 suitable methods known to those skilled in the art.

[0129] In certain embodiments, RNA is provided as the RNA of separation that is not a part of expression construct.In certain embodiments, RNA is provided as the RNA of separation lacking other elements such as promoter or terminator sequence.Such RNA can be relatively short, such as between about 18 and about 300 or between about 50 and about 750 nucleotides (for single-stranded RNA) or between about 18 and about 300 or between about 50 and about 750 base pairs (for double-stranded RNA) single-stranded or double-stranded RNA.Alternately, RNA can be provided in more complicated construct, such as as a part of recombinant expression construct, or be included in recombinant vector, such as be included in recombinant plant virus vector or recombinant baculovirus vector.In certain embodiments, such recombinant expression construct or carrier are designed to include other elements, such as including other RNA of encoding fit or ribozyme or for expressing the expression cassette of gene (for example, fungicidal protein) paid attention to.

[0130] X. Methods for providing plants with improved resistance to DON production by Fusarium graminearum, and plants provided thereby Plants, plant parts and seeds

[0131] Several embodiments relate to methods of providing plants with improved resistance to DON production by Fusarium graminearum, the methods comprising providing to the plant at least one polynucleotide described herein that is effective in reducing or eliminating DON production by Fusarium graminearum, including, for example, a polynucleotide described in Section II or Section VIII or a composition described herein.

[0132] In a related aspect, the present invention relates to plants with improved resistance to DON production by Fusarium graminearum, wherein the improved resistance is provided by expressing in the plant at least one polynucleotide described herein that is effective in reducing or eliminating DON production by Fusarium graminearum (including, for example, a polynucleotide described in Section II or Section VIII).

[0133] In yet another aspect, the present invention relates to seeds or fertile parts (especially transgenic progeny seeds or fertile parts) produced by plants with improved resistance to DON production by Fusarium graminearum as provided by the methods described. Also contemplated are commercial products produced by plants with improved resistance to DON production by Fusarium graminearum as provided by the methods described, and commercial products produced by transgenic progeny seeds or fertile parts of such plants.

[0134] The polynucleotides used in the methods can be designed for a variety of target genes. Embodiments include embodiments where the composition comprises a dsRNA with a strand having a sequence selected from the group consisting of a group of RNA trigger sequences. Related aspects of the invention include compositions for topical application and isolated polynucleotides used in the methods, as well as plants with improved resistance to DON production in Fusarium graminearum provided by the methods.

[0135] As used throughout this article, "topical application" means application to the surface or exterior of an object, such as the surface or exterior of a plant, such as application to the surface of a plant part (such as a leaf, stem, ear, bulb, flower, fruit, bud, root, stem, seed, flower, anther or pollen), or to the entire plant, or to the above-ground or underground parts of a plant. Topical application can be performed on a non-living surface, such as application to soil, or to a surface or matrix through which Fusarium graminearum can contact the polynucleotide. In various embodiments of the method, a composition comprising at least one polynucleotide is topically applied to a plant in a suitable form, such as a solid, liquid (including homogeneous mixtures, such as solutions and heterogeneous mixtures, such as suspensions, colloids, micelles and emulsions), powders, suspensions, emulsions, sprays, encapsulated or microencapsulated formulations, provided in or on microbeads or other carrier particles, in a film or coating, or on or in a matrix, or provided as a leaf, seed, root, ear, bulb or stem treatment. In some embodiments of the methods, the polynucleotide-containing composition is topically applied to the above-ground parts of the plant, such as by spraying or dusting the leaves, ears, bulbs, stems, and flowering parts of the plant.

[0136] Embodiments of the methods include topical application of foliar sprays (e.g., spraying a liquid composition containing polynucleotides on the leaves of a plant) or foliar dusting (e.g., dusting a plant with a composition containing polynucleotides in powder form or in the form of carrier particles). In other embodiments, the composition containing polynucleotides is topically applied to underground parts of a plant, such as to roots, for example, by soil immersion. In other embodiments, the composition containing polynucleotides is topically applied to seeds growing into a plant. Topical application can be in the form of topical treatment of plant fruits or seeds of plant fruits. Suitable adhesives, inert carriers, surfactants, etc. can be optionally included in the composition containing polynucleotides, as known to those skilled in the art of formulations of fungicides and seed or stem treatments.

[0137] In some embodiments, the polynucleotide-containing composition is at least one topical implant formulation selected from the group consisting of particles, pellets, or capsules that are topically implanted in a plant; in such embodiments, the method comprises topically implanting the topical implant formulation in a plant. In one embodiment, the polynucleotide-containing composition can be transfected by or otherwise internally absorbed by Fusarium graminearum. In some embodiments, the polynucleotide-containing composition further comprises a carrier and / or a surfactant (e.g., a nonionic surfactant). Examples of nonionic organosilicone surfactants include Brand surfactants BREAK THRU S240, BREAK THRU S279, BREAK THRU 301, nduce and franchise, such as SILWET A first topical application of a surfactant may be followed by a second topical application of a polynucleotide-containing composition, or vice versa. In some embodiments, plants are topically treated with a polynucleotide-containing composition and with a separate (before, after, or simultaneously) application of a substance that improves the efficacy of the polynucleotide-containing composition. For example, a first topical application of a surfactant containing a nonionic organosilicone surfactant (such as Brand surfactants, such as Brand surfactants BREAK THRU S240, BREAKTHRU S279, BREAK THRU 301, Induce and Franchise, such as SILWET brand surfactant) solution, followed by a second topical application of the polynucleotide-containing composition to spray the plants, or vice versa.

[0138] It is expected that certain polynucleotides useful in polynucleotide-containing compositions (e.g., the polynucleotide triggers described in the working examples) in combination with one or more non-polynucleotide agents will result in enhanced improvements in reducing or eliminating DON production by Fusarium when compared to the effects obtained with either a polynucleotide alone or a non-polynucleotide agent alone.

[0139] In certain embodiments, the polynucleotides in the compositions containing polynucleotides are provided as separated DNA or RNA fragments. In certain embodiments, the polynucleotides in the compositions containing polynucleotides are not a part of expression constructs, and lack other elements (such as promoter or terminator sequences). Such polynucleotides can be relatively short, such as between about 18 and about 300 or between about 50 and about 700 nucleotides (for single-stranded polynucleotides) or between about 18 and about 300 or between about 50 and about 700 base pairs (for double-stranded polynucleotides) between single-stranded or double-stranded polynucleotides. In certain embodiments, polynucleotides are dsRNAs between about 100 and about 700 base pairs, such as the dsRNAs of any length in the dsRNA triggers disclosed in the figures and tables. Alternatively, polynucleotides can be provided in more complex constructs, such as as a part of a recombinant expression construct, or included in a recombinant vector, such as included in a recombinant plant virus vector or a recombinant baculovirus vector. Such recombinant expression constructs or vectors can be designed to include additional elements, such as an expression cassette for expression of a gene of interest (eg, a fungicidal protein).

[0140] XII. Recombinant DNA constructs for controlling DON production by Fusarium graminearum.

[0141] 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 in Fusarium graminearum, including, for example, a DNA element comprising at least one segment of 18 or more consecutive nucleotides having a sequence having about 95% to about 100% identity with a DNA having a sequence selected from the group of target gene sequences, or a DNA complement thereof, or a fragment of a 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 complementary to at least 18, 19, 20, 21, 50, 150, 250, 300, 400, 500, 600, or 650 consecutive nucleotides of a target gene or RNA transcribed from the target gene, the target gene having a sequence selected from the group consisting of the target gene sequence group, or in a more specific embodiment, having a sequence selected from the group consisting of: SEQ ID NO: 16, 68-70, 108, 114, 120, 139 or 146; or (b) a DNA comprising 18, 19, 20, 21, 50, 150, 250, 300, 400, 500, 600 or 650 or more consecutive nucleotides, wherein the consecutive nucleotides have 100% identity with a fragment of an equivalent length of a DNA or its DNA complement having a sequence selected from the group consisting of: a target gene sequence group or in a more specific embodiment, SEQ ID NO: 16, 68-70, 108, 114, 120, 139 or 146; or (b) a DNA comprising 18, 19, 20, 21, 50, 150, 250, 300, 400, 500, 600 or 650 or more consecutive nucleotides, wherein the consecutive nucleotides have 100% identity with a DNA having a sequence selected from the group consisting of: a target gene sequence group or in a more specific embodiment, SEQ ID NO:16, 68-70, 108, 114, 120, 139 and 146; or (c) a DNA encoding at least one silencing element, wherein the at least one silencing element 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 the target gene, wherein the target gene has a sequence selected from the group consisting of: a target gene sequence group, or in a more specific embodiment, SEQ ID NO:16, 68-70, 108, 114, 120, 139 and 146; or (d) DNA encoding at least one silencing element, wherein the at least one silencing element comprises at least 18, 19, 20, 21, 50, 150, 250, 300, 400, 500, 600 or 650 consecutive nucleotides, wherein the consecutive nucleotides are complementary to a target gene or a portion of an RNA transcribed from the target gene, wherein the target gene is selected from the genes identified in Table 1 or Table 2 or homologs thereof, or in more specific embodiments, selected from FGP1, ELP3, SPT7, MAF1, MVD1, HEP1, SET1, FKPB12,;or (e) a DNA encoding an RNA comprising at least 18, 19, 20, 21, 50, 150, 250, 300, 400, 500, 600 or 650 consecutive nucleotides, wherein the consecutive nucleotides are complementary to a nucleotide sequence selected from the group of RNA trigger sequences or its complement or a homologous nucleotide sequence from a species of Fusarium, wherein the homologous nucleotide sequence has at least 95% sequence identity with the nucleotide sequence selected from the group of RNA trigger sequences, wherein the sequence identity percentage is calculated over the same length; or (f) a DNA encoding an RNA comprising at least one double-stranded RNA region, wherein the at least one double-stranded RNA At least one strand of region A comprises at least 18, 19, 20, 21, 50, 150, 250, 300, 400, 500, 600 or 650 consecutive nucleotides complementary to a nucleotide sequence selected from the group consisting of RNA trigger sequences or its complement or a homologous nucleotide sequence from a species of Fusarium, wherein the homologous nucleotide sequence has at least 95% sequence identity with a nucleotide sequence selected from the group consisting of RNA trigger sequences, wherein the sequence identity percentage is calculated over the same length; or (g) a DNA encoding an RNA comprising a nucleotide sequence selected from the group consisting of RNA trigger sequences or its complement. Embodiments include a recombinant DNA construct comprising a heterologous promoter operably linked to a DNA element encoding an RNA having a sequence selected from the group consisting of SEQ ID NO: 48, 80-82, 182, 188, 194, 213 and 220 or a combination thereof, or a complement thereof. ;

[0142] Embodiments include a recombinant DNA construct comprising a heterologous promoter operably linked to a DNA encoding a dsRNA having a sequence selected from the group consisting of a group of RNA trigger sequences. The recombinant DNA construct can be used, for example, to provide plants with improved resistance to DON production in Fusarium graminearum by expressing transcripts of such recombinant DNA constructs in plants. The recombinant DNA construct can also be used to prepare polynucleotides that can be used to prepare compositions that can be applied to plants, seeds, fertile plant parts, soil or fields or surfaces that need protection from DON. Related aspects of the invention include: a composition comprising a recombinant DNA construct; a plant chromosome or plasmid or a recombinant plant virus vector or a recombinant baculovirus vector comprising a recombinant DNA construct; a transgenic plant cell having a recombinant DNA construct in its genome, and a transgenic plant comprising such a transgenic plant cell or a fruit, seed or fertile part of the transgenic plant; and plants with improved resistance to DON production by fungi of the genus Fusarium (e.g., Fusarium graminearum), and plants with pest or pathogen resistance (the resistance is provided by expression of a recombinant DNA construct or RNA encoded therein or treatment with a recombinant DNA construct or RNA encoded therein).

[0143] The recombinant DNA construct comprises a heterologous promoter operably linked to a DNA comprising at least one segment of 18 or more consecutive nucleotides having a sequence having about 95% to about 100% identity with a DNA of a target gene or homolog thereof identified in Table 1 or Table 2 or a fragment of an equivalent length of a DNA or its DNA complement having a sequence selected from the target gene sequence group. In some embodiments, the segment of 18 or more consecutive nucleotides has a sequence having about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identity with a fragment of a DNA or its DNA complement having a sequence selected from the target gene sequence group. In some embodiments, the consecutive nucleotides are completely (100%) identical to a fragment of an equivalent length of a DNA or its DNA complement having a sequence selected from the target gene sequence group. In some embodiments, the DNA has an overall sequence having about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identity with a DNA or its DNA complement having a sequence selected from the target gene sequence group.

[0144] Therefore, the recombinant DNA construct comprises a heterologous promoter operably connected to a DNA, and the DNA comprises at least one section of 18 or more continuous nucleotides, and the DNA is designed to suppress the expression of a target gene or its DNA complement with a sequence selected from the target gene sequence group. In certain embodiments, the DNA comprises 18 or more, for example, between 18-24, or between 18-28, or between 20-30, or between 20-50, or between 20-100, or between 50-100, or between 50-500, or between 100-250, or between 100-500, or between 200-650, 500-1000, or between 500-2000, or even at least one section of more continuous nucleotides. In some embodiments, the segment comprises 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, 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 310, about 320, about 330, about 340, about 350 In certain embodiments, the DNA encodes an RNA containing at least one segment of at least 18, 19, 20, or 21 consecutive nucleotides having a sequence that is 100% identical to a target gene having a sequence selected from the group of target gene sequences, or a fragment of an equivalent length of a DNA of a target gene or a homolog thereof identified in Table 1 or Table 2. In specific embodiments, the DNA encodes a double-stranded nucleic acid (e.g., dsRNA) wherein one strand comprises at least one segment of at least 18, 19, 20 or 21 consecutive nucleotides having a sequence that is 100% identical to a DNA having a sequence selected from the target gene sequence group or a fragment of an equivalent length of a target gene or its DNA complement; expressed in base pairs, such a double-stranded nucleic acid comprises at least one segment of at least 18, 19, 20 or 21 consecutive fully matched base pairs corresponding to a DNA having a sequence selected from the target gene sequence group or a fragment of an equivalent length of a target gene or its DNA complement.In certain embodiments, the length of each segment contained in the DNA is greater than the typical length of naturally occurring regulatory small RNAs. In some embodiments, the length of each segment is at least about 30 consecutive nucleotides (or base pairs). In some embodiments, the total length of the DNA or the length of each segment contained in the polynucleotide is less than the total length of the sequence of interest (DNA or target gene having a sequence selected from the group consisting of the target gene sequence group). In some embodiments, the total length of the DNA is between about 50 and 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.

[0145] The recombinant DNA construct comprises a heterologous promoter operably connected to a DNA, and the DNA is generally designed to suppress one or more genes ("target genes"). Such target genes may include coding sequences or non-coding sequences or both. In a specific embodiment, the recombinant DNA construct is designed to suppress one or more target genes, wherein each target gene has a DNA sequence selected from a group consisting of a target gene sequence group. In various embodiments, the recombinant DNA construct is designed to suppress one or more genes, wherein each gene has a sequence selected from a group consisting of a target gene sequence group, and may be designed to suppress multiple genes from this group, or target one or more different regions of these genes. In one embodiment, the recombinant DNA construct comprises a heterologous promoter operably connected to a plurality of parts or segments, each of which comprises at least one segment of 21 continuous nucleotides, and the continuous nucleotides have a sequence with a fragment of an equivalent length of a DNA or its DNA complement having a sequence selected from the target gene sequence group having 100% identity. In such cases, each part may be identical or different in size or in sequence, and may be sense or antisense relative to the target gene. For example, in one embodiment, a recombinant DNA construct may include a heterologous promoter operably linked to multiple parts arranged in tandem or repeats, wherein each part comprises at least one segment of 21 consecutive nucleotides having a sequence that is 100% identical to a fragment of an equivalent length of a DNA or its DNA complement having a sequence selected from the target gene sequence group; the segments may be from different regions of the target gene, for example, 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 segments or in adjacent segments.

[0146] The recombinant DNA construct comprises a heterologous promoter operably linked to a DNA, the total length of which may be greater than 18 consecutive nucleotides, and may include nucleotides other than a segment of at least one segment of 18 or more consecutive nucleotides, the consecutive nucleotides having a sequence having about 95% to about 100% identity with a fragment of an equivalent length of a DNA or its DNA complement having a sequence selected from a target gene sequence group. In other words, the total length of the DNA may be greater than the length of the segment of the DNA designed to inhibit one or more target genes, wherein each target gene has a DNA sequence selected from a group consisting of a target gene sequence group or selected from a homolog thereof. For example, the DNA may have nucleotides flanking an "active" segment of at least one segment of 18 or more consecutive nucleotides that inhibit a target gene, or include "spacer" nucleotides between active segments, or may have additional nucleotides at the 5' end or at the 3' end or at both the 5' end and the 3' end. In one embodiment, the heterologous promoter is operably linked to a DNA comprising additional nucleotides that are non-specifically associated with (have sequences that are not complementary or identical to) a DNA or a target gene having a sequence selected from the group of target gene sequences, or its DNA complement, for example, nucleotides that provide a stable secondary structure or facilitate cloning or manufacturing). In one embodiment, the heterologous promoter is operably linked to a DNA comprising additional nucleotides positioned adjacent to one or more segments of 18 or more consecutive nucleotides, the one or more segments having a sequence that is about 95% to about 100% identical or complementary to a DNA or a fragment of an equivalent length of a target gene having a sequence selected from the group consisting of the group of target gene sequences. In one embodiment, the heterologous promoter is operably linked to a DNA comprising one such segment, adjacent to the segment is an additional 5'G or an additional 3'C or both. In another embodiment, the heterologous promoter is operably linked to a DNA encoding a double-stranded RNA comprising 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 DNA having a sequence selected from the group consisting of the target gene sequence group or a fragment of consecutive nucleotides in the target gene. For example, in some embodiments, the heterologous promoter is operably linked to a DNA comprising at least two segments each having 21 consecutive nucleotides having a sequence that is 100% identical to a fragment of a DNA having a sequence selected from the group consisting of the target gene sequence group or its DNA complement, wherein (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 appear in the DNA having a sequence selected from the group consisting of the target gene sequence group or its DNA complement.

[0147] In the recombinant DNA construct, the heterologous promoter is operably linked to DNA encoding a transcript, which may be single-stranded (ss) or double-stranded (ds) or a combination of both. Embodiments of the methods include embodiments in which the DNA encodes a transcript comprising sense single-stranded RNA (ssRNA), antisense ssRNA or double-stranded RNA (dsRNA) or a combination of any of these.

[0148] The recombinant DNA construct is provided by suitable means known to those skilled in the art. Embodiments include embodiments in which the recombinant DNA construct is synthesized in vitro, produced by expression in a microorganism or cell culture (such as a plant cell grown in culture), produced by expression in a plant cell or produced by microbial fermentation.

[0149] The heterologous promoter used 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".

[0150] In certain embodiments, the recombinant DNA construct comprises a second promoter that is also operably connected to the DNA. For example, the DNA comprising at least one section of 18 or more continuous nucleotides can have two promoters on the side, and the two promoters are arranged so that the promoters are transcribed in opposite directions and in a convergent manner, producing DNA that is complementary to each other and can hybridize to form the opposite strand transcript of double-stranded RNA. In one embodiment, the DNA is positioned between two root-specific promoters, which enables the DNA to be transcribed in opposite directions, causing the formation of dsRNA.

[0151] In some embodiments, in addition to the heterologous promoter, the recombinant DNA construct further comprises other DNA elements, the heterologous promoter is operably linked to the DNA of at least one segment comprising 18 or more consecutive nucleotides, and the embodiment of the transcript has a sequence having about 95% to about 100% identity with a fragment of equivalent length of a DNA or its DNA complement having a sequence selected from the target gene sequence group. Such DNA elements are known in the art and include, but are not limited to, introns, recombinase recognition sites, aptamers or ribozymes, and additional expression cassettes for expressing coding sequences (e.g., to express transgenic proteins such as fungicidal proteins or selectable markers) or non-coding sequences (e.g., to express additional inhibitory elements). Including one or more recognition sites for binding and cleavage by small RNAs (e.g., by miRNAs or siRNAs expressed only in specific cells or tissues) allows for more precise expression patterns in plants, where the expression of the recombinant DNA construct is inhibited where the small RNA is expressed.

[0152] In some embodiments, the recombinant DNA construct is provided in a recombinant vector. "Recombinant vector" means a recombinant polynucleotide molecule used to transfer genetic information from one cell to another. Embodiments suitable for 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 containing DNA elements but without heterologous promoters. Examples of plasmids used with the present invention include, for example, PCT / US2020 / 063490, disclosed as WO2021 / 113774 on June 10, 2021, which is incorporated herein by reference in its entirety.

[0153] In certain embodiments, the recombinant DNA construct is provided in a plant chromosome or plastid, for example, provided in a transgenic plant cell or a transgenic plant. Therefore, the present invention also encompasses transgenic plant cells having a recombinant DNA construct in its genome, and transgenic plants or partially transgenic plants including such transgenic plant cells. Partially transgenic plants include, for example, non-transgenic scions grafted onto transgenic rootstocks including transgenic plant cells. Embodiments include transgenic tomato rootstocks including transgenic plant cells. Plants can be any plants infected by Fusarium graminearum. Embodiments include embodiments in which plants are ungerminated plant seeds, plants in a vegetative stage, or plants in a reproductive stage. On the other hand, the present invention relates to seeds (especially transgenic progeny seeds) produced by transgenic plants having a recombinant DNA construct as described herein in its genome. It is also contemplated that commodity products produced by such transgenic plants, and commodity products produced by transgenic progeny seeds of such transgenic plants.

[0154] The recombinant DNA construct can be provided in the form of a composition for topical application to the surface of a plant or plant seed, root or stem, or for topical application to any material that needs to be protected from the DON produced by Fusarium graminearum. Similarly, the recombinant DNA construct can be provided in a composition for topical application to Fusarium graminearum, or provided in a composition for internal absorption (e.g., transfection) by Fusarium graminearum. In various embodiments, such compositions containing the recombinant DNA construct are provided in the form of at least one of the following groups selected from: solid, liquid (including homogeneous mixtures, such as solutions and heterogeneous mixtures, such as suspensions, colloids, micelles and emulsions), powders, suspensions, emulsions, sprays, encapsulation or microencapsulation formulations, provided in or on microbeads or other carrier particles, in films or coatings, or on or in a matrix, or provided as a leaf, seed, root or stem treatment agent. Topical application can be in the form of topical treatment of plant fruits or seeds of plant fruits. Suitable binders, inert carriers, surfactants, and the like may be included in the composition containing the recombinant DNA construct, such as those known to those skilled in the art of formulations of pesticides and seed treatments. In some embodiments, the composition for topical application containing the recombinant DNA construct is at least one topical implantable formulation, the at least one topical implantable formulation being selected from the group consisting of microparticles, granules, or capsules that are topically implanted in a plant; in such embodiments, the method comprises implanting a topical implantable formulation in a plant. In one embodiment, the composition containing the recombinant DNA construct for topical application can be internally absorbed by Fusarium graminearum (e.g., transfected). In some embodiments, the composition containing the recombinant DNA construct further comprises one or more components selected from the group consisting of: carriers, 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 non-ionic organosilicon surfactant, such as Brand surfactants, such as SILWET Brand surfactant, with CAS No. 27306-78-1 and EPA No.: CAL.REG.NO.5905-50073-AA, currently available from Momentive Performance Materials Corporation in Albany, New York. BREAK-THRU S240 brand is a polyether modified polysiloxane (CASRN proprietary) surfactant, currently available from Goldschmidt Chemical Company in Hopewell, Virginia. BREAK-THRU S279 is a capped polyether trisiloxane surfactant, the components of which are listed on the following chemical inventories: EINECS, TSCA, ENCS, AICS, ECL, PICCS CHINA, NDSL. INDUCE brand adjuvant NMFC, Item 42652, Class 60, currently available from Helena Chemical Company in Collierville, Tennessee. With LECI- Brand surfactant (having CA REG No. 34704-50065, currently available from Loveland Products, Inc., Greeley, Colorado. One embodiment includes a composition further comprising BREAK-thru 301.

[0155] It is expected that the combination of certain recombinant DNA constructs as described herein (e.g., recombinant DNA constructs comprising polynucleotide triggers as described in the working examples) (whether transgenic or topically applied) with one or more non-polynucleotide insecticides (whether transgenic or topically applied) will result in enhanced improvements in reducing or eliminating DON production and pest / pathogen infestation by Fusarium graminearum when compared to the effects obtained with a recombinant DNA construct alone or a non-polynucleotide insecticide alone. In one embodiment, a recombinant DNA construct for expressing one or more polynucleotides and one or more genes encoding non-polynucleotide insecticides was found to provide improved resistance to DON production and pest / pathogen infestation by Fusarium graminearum 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 insecticide.

[0156] 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 include a bacterium or yeast cell or may be produced in the bacterium or yeast cell. In similar embodiments, the composition containing the recombinant DNA construct comprises a transgenic plant cell or is produced in a plant cell (e.g., a plant cell transiently expressing the recombinant DNA construct); such plant cells may be cells in a plant or cells grown in tissue culture or cell suspensions.

[0157] XIII. Transgenic Plant Cells

[0158] Several embodiments relate to transgenic plant cells expressing polynucleotides that can be used in the methods and compositions described herein for inhibiting the expression of a target gene in Fusarium graminearum or other species of the genus Fusarium, or for reducing or eliminating DON production in Fusarium graminearum or other species of the genus Fusarium. In one aspect, the present invention provides a transgenic plant cell having in its genome a recombinant DNA encoding an RNA comprising at least one segment of 18 or more consecutive nucleotides having a sequence having from about 95% to about 100% identity with a fragment of a DNA or its DNA complement having a sequence selected from the group of target gene sequences. In one aspect, the present invention provides a transgenic plant cell having in its genome a recombinant DNA encoding an RNA or its DNA complement comprising at least one silencing element that is substantially identical or substantially complementary to a fragment of a target gene sequence of Fusarium graminearum, wherein the target gene sequence is selected from the group of target gene sequences. In one aspect, the present invention provides a transgenic plant cell having in its genome a recombinant DNA encoding RNA that inhibits the expression of a target gene in Fusarium graminearum that contacts or internally absorbs the RNA, wherein the RNA comprises at least one silencing element having at least one segment of 18 or more consecutive nucleotides complementary to a fragment of the target gene, and wherein the target gene is selected from the group consisting of genes in the target gene sequence group. A specific embodiment is a transgenic plant cell having in its genome a recombinant DNA encoding RNA that inhibits the expression of a target gene in Fusarium graminearum that contacts or internally absorbs the RNA, wherein the RNA comprises at least one silencing element having at least one segment of 18 or more consecutive nucleotides complementary to a fragment of one or more target gene sequence groups. In one aspect, the present invention provides a transgenic plant cell having in its genome a recombinant DNA encoding RNA having a sequence selected from the trigger sequence group. Such transgenic plant cells can be used to provide transgenic plants with improved resistance to DON production by Fusarium graminearum when compared to control plants lacking such plant cells. The transgenic plant cell can be an isolated transgenic plant cell, or a transgenic plant cell grown in culture, or a transgenic cell of any transgenic plant infected with Fusarium graminearum.

[0159] In one embodiment, the recombinant DNA is stably integrated into the genome of the transgenic plant, whereby it can be expressed in one or more cells of the transgenic plant. Methods for providing stably transformed plants are provided in the section entitled "Making and Using Transgenic Plant Cells and Transgenic Plants".

[0160] Several embodiments relate to a transgenic plant cell having in its genome a recombinant DNA encoding an RNA or its complement that inhibits expression of a target gene in Fusarium graminearum that contacts or internally absorbs the RNA, wherein the RNA comprises at least one silencing element complementary to the target gene, and wherein the target gene sequence is selected from the group of target gene sequences. In some embodiments, the silencing element comprises at least one 18 or more consecutive nucleotides having a sequence having about 95% to about 100% complementarity to a fragment of an equivalent length of DNA having a sequence selected from the group consisting of the group of target gene sequences. In some embodiments, the silencing element comprises at least one 18 or more consecutive nucleotides capable of hybridizing in vivo or under physiological conditions (e.g., physiological conditions as typically found in cells of Fusarium graminearum) to a fragment of an equivalent length of DNA having a sequence selected from the group consisting of the group of target gene sequences. The number of consecutive nucleotides is at least 18, for example, between 18-24, or between 18-28, or between 20-30, or between 20-50, or between 20-100, or between 50-100, or between 50-500, or between 100-250, or between 100-500, or between 200-1000, or between 500-2000, or even more. In some embodiments, the number of consecutive nucleotides is greater 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 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 310, about 320, about 330, about 340, about 350 In some embodiments, the silencing element comprises at least one segment of at least 18, 19, 20, or 21 consecutive nucleotides having a sequence that is 100% identical to a DNA having a sequence selected from the group of target gene sequences, or a DNA complement thereof, or a fragment of an equivalent length of a target gene.In a specific embodiment, the RNA is a double-stranded nucleic acid (e.g., dsRNA), wherein one chain comprises at least one segment of at least 18, 19, 20 or 21 consecutive nucleotides, the consecutive nucleotides having a sequence with 100% identity to a DNA having a sequence selected from the target gene sequence group or a fragment of an equivalent length of a target gene or its DNA complement; expressed in base pairs, such double-stranded nucleic acids comprise at least one segment of at least 18, 19, 20 or 21 consecutive fully matched base pairs, the base pairs corresponding to a fragment of an equivalent length of a DNA having a sequence selected from the target gene sequence group or a target gene or its DNA complement. In a specific embodiment, the length of each silencing element contained in the RNA is greater than the typical length of a naturally occurring regulatory small RNA. In some embodiments, the length of each segment is at least about 30 consecutive nucleotides (or base pairs). In a specific embodiment, the length of the RNA is between about 50 and about 500 nucleotides. In a specific embodiment, the RNA has a sequence selected from the RNA trigger sequence group.

[0161] In some embodiments, the transgenic plant cell is further capable of expressing an additional heterologous DNA sequence. In a specific embodiment, the transgenic plant cell has stably integrated into 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.

[0162] In a related aspect, the invention relates to a transgenic plant, comprising a transgenic plant cell, a commercial product produced by the transgenic plant and transgenic progeny, a plant seed, or a transgenic fertile part of a transgenic plant. Commercial products produced by transgenic plants, and commercial products produced by transgenic progeny seeds of such transgenic plants are also contemplated.

[0163] XIV. Methods for Producing Polynucleotides Targeting RNAi

[0164] The polynucleotides of the claimed methods and compositions can be produced by any suitable method known in the art. Examples of methods for producing RNA molecules of the present disclosure include, but are not limited to, in vitro transcription (IVT) (e.g., transcription using T7 polymerase or other polymerases), chemical synthesis, expression in an organism (e.g., a plant or microorganism), or expression in a cell culture (e.g., a plant cell culture), and microbial fermentation. In some embodiments, the RNA described herein is prepared by any of the methods 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.

[0165] XV. Promoter

[0166] The promoter used in the present invention works in the cell in which the construct is intended to be transcribed. Usually, these promoters are heterologous promoters, such as those used in the recombinant construct, i.e., they are not found in nature to be operably connected with other nucleic acid elements used in the construct described herein. In various embodiments, the promoter is selected from the group consisting of: constitutive promoters, spatial specific promoters, temporal specific promoters, development-specific promoters and inducible promoters. In many embodiments, the promoter is a promoter that works in plants, such as pol II promoter, pol III promoter, pol IV promoter or pol V promoter.

[0167] Non-constitutive promoters suitable for use with recombinant DNA constructs of the present invention include space-specific promoters, time-specific promoters and inducible promoters. Space-specific promoters can include organelle-specific promoters, cell-specific promoters, tissue-specific promoters or organ-specific promoters (for example, respectively for plastid-specific promoters, root-specific promoters, pollen-specific promoters or seed-specific promoters expressed in plastids, roots, pollen or seeds). In many cases, seed-specific promoters, embryo-specific promoters, aleurone-specific promoters or endosperm-specific promoters are particularly useful. Time-specific promoters can include promoters intended to promote expression during certain developmental stages in the growth cycle of plants, or during different times of day or night, or in different seasons of the year. Inducible promoters include promoters induced by chemicals or by environmental conditions, such as but not limited to biological or abiotic stress (for example, water shortage or drought, heat, cold, high or low nutrition or salt level, high or low light level or pest or pathogen infection). MicroRNA promoters are useful, especially those with temporal specificity, spatial specificity or inducible expression patterns; examples of miRNA promoters, and methods for identifying miRNA promoters with specific expression patterns are provided in U.S. Patent Application Publications 2006 / 0200878, 2007 / 0199095 and 2007 / 0300329, which are incorporated herein by reference in particular. Expression-specific promoters can also include promoters that are generally constitutively expressed but differ in degree or "strength" of expression, including promoters that are generally considered to be "strong promoters" or "weak promoters".

[0168] Promoters of particular interest include the following examples: the protein nopaline synthase promoter isolated from the T-DNA of Agrobacterium; the cauliflower mosaic virus (CaMV) 35S promoter; enhanced promoter elements or chimeric promoter elements, such as the enhanced CaMV promoter linked to an enhancer element (intron from heat shock protein 70 of Zea mays). 35S promoter; root-specific promoters, such as those disclosed in U.S. Pat. Nos. 5,837,848, 6,437,217, and 6,426,446; the maize L3 oleosin promoter, disclosed in U.S. Pat. No. 6,433,252; promoters of plant nuclear genes encoding plastid-localized aldolases, disclosed in U.S. Patent Application Publication 2004 / 0216189; cold-inducible promoters, disclosed in U.S. Pat. No. 6,084,089; salt-inducible promoters, disclosed in U.S. Pat. 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-deficiency-inducible promoters, disclosed in U.S. Patent Application Publication 2004 / 0123347A1. All of the above patents and patent publications disclosing promoters and their use, particularly in recombinant DNA constructs that function in plants, are incorporated herein by reference.

[0169] Plant vascular or phloem-specific promoters of interest include, for example, the rolC or rolA promoter of Agrobacterium rhizogenes, the promoter of T-DNA gene 5 of Agrobacterium tumefaciens, the promoter of rice sucrose synthase RSs1 gene, the Commelina yellow mottled bacilliform DNA virus promoter, the coconut leaf rot virus promoter, the rice tungro bacilliform virus promoter, the virus promoter), the promoter of the pea glutamine synthase GS3A gene, the invCD111 and invCD141 promoters of the potato invertase gene, a promoter isolated from Arabidopsis (according to Kertbundit et al., (1991) Proc. Natl. Acad. Sci. USA., 88:5212-5216, which was shown to have phloem-specific expression in tobacco), the VAHOX1 promoter region, the pea cell wall invertase gene promoter, the acid invertase gene promoter from carrot, 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.

[0170] Promoters suitable for use with the recombinant DNA constructs or polynucleotides of the present invention may include polymerase II ("pol II") promoters and polymerase III ("pol III") promoters. RNA polymerase II transcribes structural or catalytic RNAs that are typically shorter than 400 nucleotides in length and recognizes a simple run of T residues as a termination signal; the RNA polymerase II has been used to transcribe siRNA duplexes (see, e.g., Lu et al., (2004) Nucleic Acids Research, 32: e171). Thus, in certain embodiments, the Pol II promoter is a promoter in which short RNA transcripts are produced from the recombinant DNA constructs of the present invention. In one embodiment, the recombinant DNA construct comprises a pol II promoter to express RNA transcripts flanked by self-cleaving ribozyme sequences (e.g., self-cleaving hammerhead ribozymes), resulting in a processed RNA (e.g., a single-stranded RNA that binds to a transcript of a target gene of Fusarium graminearum) having defined 5' and 3' ends without potentially interfering flanking sequences. Alternative methods use pol III promoters to produce 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 that produce 2 base pair overhangs (UU) in the transcribed RNA; this is useful for, for example, the expression of siRNA-type constructs. The use of pol III promoters to drive the expression of siRNA constructs has been reported; see van deWetering 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 are commercially available; see, e.g., Invitrogen, "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).

[0171] Promoter elements can include nucleic acid sequences that are not naturally occurring promoters or promoter elements or homologs thereof but can regulate the expression of a gene. Examples of such "gene-independent" regulatory sequences include naturally occurring or artificially designed RNA sequences (which include ligand binding regions or aptamers (see "aptamers" below) and regulatory regions (which can be cis-acting)). See, e.g., Isaacs et al., (2004), Nature Biotechnology, 22:841-847, Bayer and Smolke (2005), Nature Biotechnology, 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. Struct.Mol.Biol.), 11:29-35. Such "ribose regulators" can be selected or designed for specific spatial or temporal specificity, for example, to regulate the translation of DNA encoding a silencing element for inhibiting a target gene of Fusarium graminearum only in the presence (or absence) of a given concentration of an appropriate ligand. An example is a ribose regulator that responds to an endogenous ligand (e.g., jasmonic acid or salicylic acid) produced by a plant when under stress (e.g., abiotic stress such as water, temperature or nutrient stress, or biotic stress such as attachment by pests or pathogens); under stress, the level of the endogenous ligand increases to a level sufficient to cause the ribose regulator to begin transcription of DNA encoding a silencing element for inhibiting a target gene of Fusarium graminearum.

[0172] XVI. Recombinase Sites

[0173] In certain 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, wherein the site-specific recombination of the DNA between the loxP sites is mediated by the Cre recombinase. The position and relative orientation of the loxP sites are selected to achieve the desired recombination; for example, when the loxP sequences are in the same direction, the DNA between the loxP sites is excised in a circular form. In another embodiment, the recombinant DNA construct comprises DNA encoding a loxP site; in the presence of the Cre recombinase and another DNA with the loxP site, the two DNAs are recombined.

[0174] XVII. Transgenic Transcription Unit

[0175] In some embodiments, the recombinant DNA construct or polynucleotide of the present invention comprises a transgenic transcription unit. The transgenic transcription unit comprises a DNA sequence encoding a gene of interest, such as a native protein or a heterologous protein. The gene of interest can be any coding sequence or non-coding sequence from any species (including but not limited to non-eukaryotic organisms, such as bacteria and viral fungi, protists, plants, invertebrates and vertebrates). The transgenic transcription unit may further include a 5' or 3' sequence or both required for transcription of the transgene.

[0176] XVIII. Introns

[0177] In some embodiments, the recombinant DNA construct or polynucleotide of the present invention comprises DNA encoding a spliceable intron. "Intron" generally means a segment of DNA (or RNA transcribed from such segment) located between exons (protein coding segments of DNA or corresponding transcribed RNA), wherein during the maturation of the messenger RNA, the intron present is enzymatically "spliced" out or removed from the RNA chain by a cleavage / ligation process occurring in the nucleus of a eukaryotic organism. The term "intron" also applies to non-coding DNA sequences that are transcribed into RNA segments that can be spliced ​​out from mature RNA transcripts, but are not introns present between protein coding exons. One example of these is a spliceable sequence that has the ability to enhance expression of downstream coding sequences in plants (in some cases, especially in monocots); these spliceable sequences are naturally located in the 5' untranslated region of some plant genes and in some viral genes (e.g., the tobacco mosaic virus 5' leader sequence or "omega" leader sequence described by Gallie and Walbot (1992), Nucleic Acids Research, 20: 4631-4638 as enhancing expression in plant genes). These spliceable sequences or "expression-enhancing introns" can be artificially inserted in the 5' untranslated region of a plant gene, between promoters, but before any protein-coding exons. Examples of such expression-enhancing introns include, but are not limited to, the corn alcohol dehydrogenase (Zm-Adh1), the corn Bronze-1 expression-enhancing intron, the rice actin 1 (Os-Act1) intron, the Shrunken-1 (Sh-1) intron, the corn sucrose synthase intron, the 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 for improving recombinant DNA constructs for plants by including an expression-enhancing intron derived from the 70 kilodalton corn heat shock protein (hsp70) in a non-translated leader positioned 3' from the gene promoter and 5' from the first protein-coding exon.

[0178] XIX. Ribozymes

[0179] In some embodiments, the recombinant DNA construct or polynucleotide of the 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 for cleaving transcribed RNA to provide a defined segment of RNA, such as a silencing element for inhibiting a target gene of Fusarium graminearum.

[0180] XX. Gene suppression elements

[0181] In some embodiments, the recombinant DNA construct or polynucleotide of the present invention comprises DNA encoding an additional gene suppression element for suppressing a target gene in the DON pathway other than the Fusarium graminearum target gene, such as a target gene required for another essential metabolic function of Fusarium graminearum or a target gene of another fungus of the genus Fusarium. The target gene to be suppressed may include coding sequences or non-coding sequences or both.

[0182] Suitable gene suppression elements are described in detail in U.S. Patent Application Publication 2006 / 0200878 (, the disclosure of which is specifically incorporated herein by reference, and include one or more of the following:

[0183] o(a) a DNA comprising at least one antisense DNA segment that is antisense to at least one segment of a gene to be inhibited;

[0184] o(b) DNA comprising multiple copies of at least one antisense DNA segment that is antisense to at least one segment of a gene to be inhibited;

[0185] o(c) a DNA comprising at least one sense DNA segment, the at least one sense DNA segment being at least one segment of a gene to be suppressed;

[0186] o(d) DNA comprising multiple copies of at least one sense DNA segment, the at least one sense DNA segment being at least one segment of a gene to be suppressed;

[0187] o(e) DNA which is transcribed into RNA to inhibit a gene to be inhibited by forming a double-stranded RNA, and comprises at least one antisense DNA segment which is antisense to at least one segment of the gene to be inhibited and at least one sense DNA segment which is at least one segment of the gene to be inhibited;

[0188] o(f) DNA which is transcribed into RNA to inhibit a gene to be inhibited by forming a single double-stranded RNA and comprises a plurality of tandem antisense DNA segments which are antisense to at least one segment of the gene to be inhibited and a plurality of tandem sense DNA segments which are antisense to at least one segment of the gene to be inhibited;

[0189] o(g) DNA, which is transcribed into RNA by forming multiple double strands of RNA to inhibit a gene to be inhibited, and comprises a plurality of antisense DNA segments that are antisense to at least one segment of the gene to be inhibited and a plurality of sense DNA segments that are at least one segment of the gene to be inhibited, and wherein the plurality of antisense DNA segments and the plurality of sense DNA segments are arranged as a series of inverted repeat sequences;

[0190] o(h) DNA comprising nucleotides derived from plant miRNA;

[0191] o(i) DNA containing nucleotides of siRNA;

[0192] o(j) DNA that is transcribed into an RNA aptamer capable of binding to a ligand; and

[0193] o(k) is transcribed into DNA of an RNA aptamer capable of binding to a ligand, and into DNA of a regulatory RNA capable of regulating the expression of a gene to be inhibited, wherein the regulation depends on the conformation of the regulatory RNA, and the conformation of the regulatory RNA is allosterically affected by the binding state of the RNA aptamer.

[0194] In some embodiments, introns are used to deliver gene suppression elements in the absence of any protein coding exons (coding sequences). In one example, introns are interrupted by embedding gene suppression elements within the introns, such as expression enhancing introns, wherein upon transcription, the gene suppression elements are excised from the introns. Thus, protein coding exons are not required to provide the gene suppression function of the recombinant DNA constructs disclosed herein.

[0195] XXI. Transcriptional regulatory elements

[0196] In certain embodiments, the recombinant DNA construct of the present invention or polynucleotides include DNA encoding transcriptional regulatory elements. Transcriptional regulatory elements include elements (relative to their expression in the absence of such regulatory elements) that regulate the expression level of the recombinant DNA construct of the present invention. Examples of suitable transcriptional regulatory elements include riboswitches (cis or trans-acting), transcriptional stabilizing sequences, transcriptional initiation sites, transcriptional extension sequences, transcriptional termination elements, and miRNA recognition sites, as described in detail in U.S. Patent Application Publication 2006 / 0200878, which is incorporated herein by reference in particular.

[0197] XXII. Preparation and Use of Transgenic Plant Cells and Transgenic Plants

[0198] The transformation of plants can include any of several well-known methods and compositions. Suitable methods for plant transformation include virtually any method by which DNA can be introduced into cells. One plant transformation method is microprojectile bombardment, for example, as shown below: U.S. Patent No. 5,015,580 (soybean), U.S. Patent No. 5,538,880 (corn), U.S. Patent No. 5,550,318 (corn), U.S. Patent No. 5,914,451 (soybean), U.S. Patent No. 6,153,812 (wheat), U.S. Patent No. 6,160,208 (corn), U.S. Patent No. 6,288,312 (rice), U.S. Patent No. 6,365,807 (rice), and U.S. Patent No. 6,399,861 (corn) and U.S. Patent No. 6,403,865 (corn), all of which are incorporated by reference to enable the generation of transgenic plants.

[0199] Another useful plant transformation method is Agrobacterium-mediated, carried out by Agrobacterium containing a binary Ti plasmid system, wherein the Agrobacterium carries a first Ti plasmid (often disarmed) and a second chimeric plasmid containing at least one T-DNA border of a 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, smaller plasmids containing one or more T-DNA borders can be conveniently constructed and manipulated in a suitable alternative host such as Escherichia coli, and then transferred to Agrobacterium.

[0200] Detailed procedures for Agrobacterium-mediated transformation of plants, particularly crop plants, include those disclosed in U.S. Pat. Nos. 5,004,863, 5,159,135, and 5,518,908 (cotton); U.S. Pat. Nos. 5,416,011, 5,569,834, 5,824,877, and 6,384,301 (soybean); U.S. Pat. Nos. 5,591,616 and 5,594,617 (cowberry); , 981,840 (corn); 5,463,174 (brassicas, including rapeseed), 7,026,528 (wheat) and 6,329,571 (rice), and U.S. Patent Application Publication 2004 / 0244075 (corn) and 2001 / 0042257A1 (sugar beet), all of which are hereby incorporated by reference for enabling the production of transgenic plants. U.S. Patent Application Publication 2011 / 0296555 discloses transformation vectors (including vector sequences) and detailed protocols for transforming corn, soybeans, rapeseed, cotton, and sugarcane in Example 5, and is specifically incorporated by reference for enabling the production of transgenic plants. Similar methods have been reported for many plant species (both dicots and monocots), including 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. J., 5:299), alfalfa (Masoud et al., (1996) Transgen. Res., 5:313); and tomato (Sun et al., (2006) Plant Cell Physiol., 47:426-431). See also U.S. Patent Application Publication 2003 / 0167537A1 for a description of vectors, transformation methods, and production of transformed Arabidopsis thaliana plants in which transcription factors are constitutively expressed by the CaMV35S promoter, which is incorporated by reference. Transformation methods particularly useful for plants are well known in the art.See, e.g., publicly described transformation methods for 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: Basic Protocols". World: Essential Protocols", Ian S. Curtis (ed.), Springer, 2004)) and pepper (Li et al., (2003) Plant Cell Physiology, 21:785-788). Stable transgenic potato, tomato and eggplant have been commercially introduced into 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 GM Crop Database for a large number of publicly available commercial genetically modified crops; 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 transformation methods for other plant species are well known in the art, see, for example, the encyclopedic 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), which describes transformation procedures for cereals and forages (rice, maize, wheat, barley, oats, sorghum, pearl millet, finger millet, cool-season forage grasses, and barley grass), oilseed crops (soybean, oilseed brassica, sunflower, peanut, flax, sesame, and safflower), legume cereals and forages (common bean, cowpea, pea, bean, lentil, runner bean, Asian bean, pigeon pea, vetch, chickpea, lupin, alfalfa, and clover), temperate fruits and nuts (apples, pears, peaches, plums, berry crops, cherries, grapes, olives, almonds and Persian walnuts), tropical and subtropical fruits and nuts (citrus, grapefruit, bananas and plantains, pineapples, papayas, mangoes, avocados, kiwis, passion fruit and persimmons), vegetable crops (tomatoes, eggplants, peppers, cabbage, radishes, carrots, gourds, garlic, asparagus and leafy vegetables), sugar, tuber and fiber crops (sugarcane, sugar beets, stevia, potatoes, sweet potatoes, cassava and cotton), plantation crops, ornamentals and turf grasses (tobacco, coffee, cocoa, tea, rubber trees, medicinal plants, ornamentals and turf grasses) and forest tree species.

[0201] The transgenic plant cell containing the recombinant DNA of stable integration and the transformation method of transgenic plant are provided, preferably in the tissue culture on culture medium and in controlled environment, implement." culture medium " refers to be used for in vitro, promptly makes the multiple nutrient mixture of cell growth outside complete living organism.Recipient cell target includes but is not limited to meristem cell, callus, immature embryo or each part of embryo, and gamete cell, such as microspore, pollen, sperm and egg cell.Can be from there regenerated any cell of fertile plant be envisioned as useful receptor cell for implementing the present invention.Callus can be started from various tissue sources, includes but is not limited to each part of immature embryo or embryo, seedling top meristem, microspore etc.Those cells that can be used as callus proliferation can be used as receptor cells for genetic transformation. Useful transformation methods and materials for preparing the transgenic plants of the present invention (e.g., various culture 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 hereby incorporated by reference.

[0202] In general transformation practice, in any one transformation experiment, DNA is introduced into only a small portion of target cells. Marker genes are generally used to provide a kind of efficient system, for identifying those cells that are stably transformed by receiving and integrating the transgenic DNA construct into the genome of the cell. Preferred marker genes provide selective markers, and selective markers confer resistance to selective agents such as antibiotics or herbicides. Any antibiotic or herbicide to which plant cells are resistant may be an agent that can be used for selection. Potentially transformed cells are exposed to the selective agent corresponding to the marker. In the colony of surviving cells, it is usually those cells that have integrated the gene (selective marker) that confer resistance and are expressed at a level that is enough to allow cells to survive in the presence of selective agents. Cells can be further tested to confirm the stable integration of recombinant DNA. Commonly used selective marker genes include those that confer resistance to antibiotics, such as kanamycin or paromomycin (nptll), hygromycin B (aph IV) and gentamycin (gentamycin) (aac3 and aacC4) or to herbicides, such as glufosinate (bar or pat) and glyphosate (EPSPS). Examples of useful selective marker genes and selection agents are described in U.S. Patents 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 reporter genes, such as markers that provide the ability to visually identify transformants, can also be used. Examples of useful screenable markers include, for example, genes that express a protein that produces a detectable color by acting on a chromogenic substrate (e.g., beta-glucuronidase (GUS) (uidA) or luciferase (luc), or genes that are themselves detectable, such as green fluorescent protein (GFP) (gfp) or immunogenic molecules. Those skilled in the art will recognize that many other useful markers or reporter genes are available for use.

[0203] Detecting or measuring transcription of a recombinant DNA construct in transgenic plant cells can be achieved by any suitable method, including protein detection methods (e.g., Western blot, ELISA and other immunochemical methods), measurement of enzymatic activity, or nucleic acid detection methods (e.g., Southern blot, northern blot, PCR, RT-PCR, fluorescence in situ hybridization).

[0204] Other suitable methods for detecting or measuring transcription of a recombinant polynucleotide targeting a Fusarium graminearum target gene of the present invention in a plant cell include measuring any other trait that is a direct or surrogate indicator of the expression level of the target gene in Fusarium graminearum relative to the expression level observed in the absence of the recombinant polynucleotide (e.g., growth rate, mortality, or reproductive or recruitment rate of Fusarium graminearum) or measuring damage (e.g., root damage) or yield loss in plants or plant fields infected by Fusarium graminearum. In general, suitable methods for detecting or measuring transcription of a recombinant polynucleotide of interest in a plant cell include, for example, gross or microscopic morphological traits, growth rate, yield, reproductive or recruitment rate, resistance to pests or pathogens, or resistance to biological or abiotic stresses (e.g., water stress, salt stress, nutritional stress, heat or cold stress). Such methods may use direct measurements of phenotypic traits or surrogate assays (e.g., in plants, these assays include plant part assays, such as leaf or root assays, to determine tolerance to abiotic stresses). Such methods include direct measurements of resistance to Fusarium graminearum (eg, damage to plant tissue) or alternative assays (eg, plant yield assays or bioassays).

[0205] The recombinant DNA constructs of the present invention can be stacked with other recombinant DNAs for imparting additional traits, such as by expressing or suppressing other genes (e.g., in the case of transformed plants, traits include 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 2004 / 0126845A1, which is specifically incorporated by reference.

[0206] Can gather the seed of fertile transgenic plant and be used to grow the filial generation of transgenic plant of the present invention, comprise hybrid offspring, described transgenic plant comprises recombinant DNA construct in its genome.Therefore, except with recombinant DNA construct directly transformed plant of the present invention, transgenic plant of the present invention can be prepared by the first plant with recombinant DNA and the second plant hybridization that lacks described construct.For example, recombinant DNA can be introduced into the plant line that is suitable for conversion to produce transgenic plant, described transgenic plant can be hybridized with the second plant line so that recombinant DNA is infiltrated in the filial generation of gained.Transgenic plant of the present invention can be hybridized with the plant line with other recombinant DNA that gives one or more other proterties (such as but not limited to herbicide resistance, pest or disease resistance, environmental stress resistance, nutrient content and output improvement), to produce the filial generation plant with the recombinant DNA that gives the target sequence expression behavior of expectation and other proterties.

[0207] In such breeding for combined traits, the transgenic plant providing the additional trait can be the male line (pollinator), and the transgenic plant carrying the basic trait can be the female line. The progeny of this hybridization is separated so that some plants will carry the DNA of two parental traits, and some will carry the DNA of one parental trait; such plants can be identified by markers associated with the parental recombinant DNA. Progeny plants carrying the DNA of two parental traits can be crossed back to the maternal line multiple times, for example, usually 6 to 8 generations, to produce homozygous progeny plants with substantially the same genotype as the recombinant DNA of one original transgenic parental line and another transgenic parental line.

[0208] Yet another aspect of the invention is a transgenic plant grown from the transgenic seeds of the invention. The invention contemplates transgenic plants grown directly from transgenic seeds containing recombinant DNA, as well as progeny of plants produced by crossing a transgenic plant grown directly from a transgenic seed with a second plant not grown from the same transgenic seed, including inbred plant lines or hybrid plant lines. Crossing may include, for example, the following steps:

[0209] o(a) plant seeds or stem cuttings of a first parent plant (eg, non-transgenic or transgenic) and a second parent plant transgenic according to the present invention;

[0210] o(b) growing seeds or stem cuttings of the first parent plant and the second parent plant into flowering plants;

[0211] o(c) pollinating flowers from the first parent with pollen from the second parent; and

[0212] o(d) Collecting seeds produced on the parent plant carrying the fertilized flower.

[0213] It is often desirable to infiltrate recombinant DNA into superior varieties, such as by backcrossing, to transfer a specific desired trait from one source to an inbred plant or other plant lacking the trait. This can be accomplished, for example, by first hybridizing an excellent inbred line ("A") (recurrent parent) with a donor inbred line ("B") (non-recurrent parent), the donor inbred line carrying the appropriate gene for the trait in question, such as a construct prepared according to the invention. The progeny of this hybridization are first selected among the resulting progeny to transfer the desired trait from the non-recurrent parent "B", and then the selected progeny are mated back to the excellent recurrent parent "A". After five or more backcross generations in which the desired trait is selected, the progeny may be substantially hemizygous for the loci controlling the transferred properties, but the progeny are similar to the superior parent for most or nearly all other genes. The final backcross generation will be selfed to produce progeny that are pure breeding for the transferred genes (e.g., one or more transformation events).

[0214] By a series of breeding operations, selected DNA construct can be moved in completely different systems from a system, and does not need further recombinant manipulation.Therefore, can produce the inbred plant that is real breeding for one or more DNA constructs.By hybridizing different inbred plants, can produce a large number of different hybrids with the combination of different DNA constructs.In this way, can produce the plant of the characteristic of the agronomic characteristics (" hybrid vigor ") with the expectation usually relevant to hybrid and the expectation given by one or more DNA constructs.

[0215] In certain transgenic plant cells and transgenic plants of the present invention, it is sometimes desirable to simultaneously express a gene of interest while also modulating the expression of a Fusarium graminearum target gene. Thus, in some embodiments, the transgenic plant contains a recombinant DNA further comprising a gene expression element for expressing at least one gene of interest, and the transcription of the recombinant DNA construct of the present invention is affected by the simultaneous transcription of the gene expression element.

[0216] The present invention also provides commodity products produced by the transgenic plant cells, plants or seeds of the present invention, including but not limited to collected leaves, heads, ears, roots, buds, stems, fruits, seeds or other parts of plants, oils, extracts, fermentation or digestion products, or any food or non-food products, including such commodity products produced by the transgenic plant cells, plants or seeds of the present invention. Detection of one or more nucleic acid sequences of the recombinant DNA construct of the present invention in one or more commodities or commodity products contemplated herein is evidence of the fact that the commodity or commodity product contains or is derived from the transgenic plant cells, plants or seeds of the present invention.

[0217] Typically, 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 caused by infection with Fusarium graminearum. In various embodiments, for example, where a transgenic plant expresses a recombinant DNA construct of the present invention (which is stacked with other recombinant DNAs to confer additional traits), the transgenic plant has at least one additional altered trait relative to a plant lacking the recombinant DNA construct, the additional altered trait being selected from the group consisting of:

[0218] o(a) Improved abiotic stress tolerance;

[0219] o(b) improved tolerance to biotic stress;

[0220] o(c) Improved primary metabolite composition;

[0221] o(d) Improved secondary metabolite composition;

[0222] o(e) Improved trace element, carotenoid or vitamin composition;

[0223] o(f) increased production;

[0224] o(g) Improved ability to utilize nitrogen, phosphorus or other nutrients;

[0225] o(h) modified agronomic characteristics;

[0226] o(i) modified growth or reproductive characteristics; and

[0227] o(j) Improved harvesting, storage or processing quality.

[0228] In some embodiments, the transgenic plant is characterized by: improved tolerance to abiotic stress (e.g., tolerance to water shortage or drought, heat, cold, non-optimal nutrient or salt levels, non-optimal light levels) or improved tolerance to biotic stress (e.g., crowding, phytochemical antagonism or injury); improved primary metabolite (e.g., fatty acids, oils, amino acids, proteins, sugars or carbohydrates) composition; improved secondary metabolite (e.g., alkaloids, terpenoids, polyketides, non-ribosomal peptides and secondary metabolites of mixed biosynthetic origin) composition; improved trace element (e.g., iron, zinc), carotenoid (e.g., β-carotene, lycopene, lutein, zeaxanthin or other carotenoids and xanthophylls) or vitamin (e.g., tocopherol) composition; increased production; improved yield (e.g., increased yield under non-stress conditions or increased yield under biotic or abiotic stress); improved ability to use nitrogen, phosphorus or other nutrients; modified agronomic characteristics (e.g., delayed maturity; delayed senescence; earlier or later maturity; improved shade tolerance; improved resistance to root or stalk lodging; improved resistance to "green tips" of stems; improved photoperiod response); modified growth or reproductive characteristics (e.g., intentional dwarfing; intentional male sterility, which can be used, for example, in improved hybridization programs); improved vegetative growth rate; improved germination; improved male or female fertility); improved collection, storage or processing quality (e.g., improved resistance to pests during storage, improved resistance to breakage, improved consumer appeal); or any combination of these traits.

[0229] In another embodiment, the transgenic seed or the seed produced by the transgenic plant has an improved primary metabolite (e.g., fatty acid, oil, amino acid, protein, sugar or carbohydrate) composition, an improved secondary metabolite composition, an improved trace element, carotenoid or vitamin composition, improved collection, storage or processing quality, or a combination of these. In another embodiment, it may be desirable to alter the level of a natural component of the transgenic plant or the seed of the transgenic plant, for example, to reduce the level of an allergenic protein or glycoprotein or a toxic metabolite.

[0230] Usually, the colony of the transgenic plant regenerated from the transgenic plant cell is screened to identify the transgenic plant cell that develops into the transgenic plant with the desired proterties.Measure transgenic plant to detect enhanced proterties, such as enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein and enhanced seed oil.Screening method is included in the direct screening proterties in greenhouse or field test, or screens alternative proterties.This type of analysis relates to the change of chemical composition, biomass, physiological properties or morphology of detection plant.By analyzing the content of seed composition and protein, free amino acid, oil, free fatty acid, starch, tocopherol or other nutrients, the change of chemical composition can be detected.By measuring plant height, stem diameter, internode length, root and branch and leaf dry weight, detect the change of growth or biomass characteristics.By assessing the response to stress conditions, for example, the mensuration under the stress conditions imposed (such as lack of water, nitrogen or phosphorus, cold or hot growth conditions, pathogen or insect attack, light lack or plant density increase) identifies the change of physiological properties. Other selection characteristics include days to flowering, days to pollen drop, days to fruit maturity, fruit quality or yield, leaf extension rate, chlorophyll content, leaf temperature, stand, seedling vigor, internode length, plant height, leaf number, leaf area, tillering, brace roots, stay green, stem lodging, root lodging, plant health, fertility, green tips, and pest resistance. In addition, the phenotypic characteristics of the collected fruits or seeds can be evaluated; for example, in plants, this can include the total number or weight of the collected fruits, or the color, acidity, sugar content, or taste of such fruits.

[0231] The following examples are presented for purposes of illustration and should not be construed as limiting.

[0232] Examples

[0233] summary

[0234] The present examples are intended to highlight the development of exogenous application of dsRNA to control DON production by Fusarium graminearum on plants.

[0235] Introduction

[0236] RNA interference (RNAi) is a naturally occurring cell defense system mediated by double-stranded RNA (dsRNA). The first component of the RNAi mechanism in response to dsRNA is the RNase III endonuclease Dicer-2, which cuts dsRNA into short (usually 19-21 nucleotides long) interfering RNA (siRNA). Dicer-2 promotes the transfer of siRNA to the RNA-induced silencing complex with the help of dsRNA binding proteins. RNAi promotes gene silencing, thereby affecting the translation of host genetic material. Because RNAi is a sequence-specific method for suppressing the expression of targeted genes, and because each species is defined by the uniqueness of its gene sequence, RNAi can be designed in a species-specific manner. By targeting the genes necessary for the production of DON by pathogens, RNAi can be selectively used to control the production of DON in Fusarium graminearum and Fusarium graminearum, without adversely affecting non-targeted beneficial species. Tables 1 and 2 show lists of internal reference numbers, relevant starting sequence accession numbers, and target gene sequences, trigger sequences, and trigger RNA reverse complements.

[0237] Table 1

[0238]

[0239]

[0240] Table 2

[0241]

[0242]

[0243]

[0244] Example 1

[0245] DON reduction:

[0246] In vitro 96-well plates with DON medium (nutrient-rich M9 medium plus DON inducer Agmatine) were inoculated with Fusarium graminearum spores and then treated with dsRNA. After the plates were incubated 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 with the untreated control (UTC). Error bars indicate one standard deviation between three independent runs. Results are shown in Figure 1 and Figure 4In, it is shown that each test sequence shows a reduction in DON compared to the untreated control. Tables 3 and 4 show other results, indicating the average DON reduction % and standard deviation of multiple runs. In Tables 3 and 4, the average DON reduction % of the sequences scored as ++ is greater than a standard deviation of the negative control (GS5797), and the standard deviation is less than 20%. The sequences scored as + show that the average DON reduction % is greater than the negative control.

[0247] Table 3

[0248]

[0249] Table 4

[0250]

[0251]

[0252]

[0253]

[0254] Example 2

[0255] Average DON production by Fusarium graminearum on isolated wheat bulbs:

[0256] Inoculate wheat bulbs in vitro with Fusarium graminearum spores and then treat with dsRNA. After 12 days, wheat bulbs were collected and analyzed using liquid chromatography-mass spectrometry (LC-MS) to quantify total deoxynivalenol (DON and ADON) mycotoxins. Average DON is plotted as parts per million (ppm) of the dry weight of wheat bulbs. Error bars indicate one standard deviation between two independent runs. Results are shown in Figure 2 In the Figures, the reduction in DON production for each tested sequence compared to the negative control is shown.

[0257] Example 3

[0258] Relative expression of target genes

[0259] Mycelia from Fusarium graminearum samples tested as described in Example 1 were collected and RNA was extracted using Quick RNA Miniprep from Zymo Research. RNA was reverse transcribed with oligonucleotide dT primers, and gene expression was estimated by relative quantification relative to Fusarium housekeeping genes using qPCR. The fold change of target genes was calculated using the ΔΔCt method and plotted relative to the corresponding negative control treatment. Statistical significance was determined using a nonparametric two-sided T test. Error bars indicate 95% confidence intervals. Results are shown in Figure 3 , indicating that the tested sequence significantly reduced the expression of the relevant target gene compared with the negative control.

Claims

1. A composition for controlling DON production in Fusarium graminearum, the composition 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 consecutive nucleotides that are substantially 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 or a segment of RNA transcribed from the target gene, the target gene having a nucleotide sequence selected from the group consisting of: SEQ ID NOs: 1-16, 65-70 and 87-151; or (b) an effective amount of at least one polynucleotide, wherein the at least one polynucleotide comprises at least one silencing element, wherein the at least one silencing element is bound to at least 18, 19, 20, 21, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600 or 650 consecutive nucleotides are substantially complementary to, or comprise at least about 85%, at least about 90%, at least about or 95% sequence identity thereto, wherein the 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 substantially 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, or a segment of an RNA transcribed from the target gene, the target gene having a nucleotide sequence selected from the group consisting of: SEQ ID NOs: 1-16, 65-70, and 87-151; or (d) an RNA molecule that reduces or eliminates DON production in Fusarium graminearum when transfected into or contacted with the Fusarium graminearum, wherein the RNA molecule comprises at least 18, 19, 20, 21, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or 650 consecutive nucleotides that are substantially 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 the target gene, the 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 reduces or eliminates DON production in Fusarium graminearum when transfected or contacted thereto, 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 consecutive nucleotides that are substantially 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 the target gene, wherein the 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, the 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 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 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: SEQ ID NOs: 33-64, 79-86, 153-225, and 226-299; or (h) an effective amount of at least one RNA comprising at least one segment that is substantially 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 Fusarium graminearum on a plant, reduces or eliminates DON production by the Fusarium 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 substantially 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 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 or contacted with Fusarium graminearum on a plant, reduces or eliminates DON production by the Fusarium 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 consecutive nucleotides that are substantially 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 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 double-stranded RNA molecule that reduces or eliminates DON production by Fusarium graminearum when transfected or contacted to Fusarium graminearum on 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. The composition of claim 1, wherein the composition is in at least one form selected from the group consisting of solid, liquid, powder, suspension, emulsion, spray, encapsulate, microbead, carrier microparticle, film, matrix, seed treatment, soil drench and implantable formulation.

3. The composition of any one of claims 1 or 2, further comprising at least one component selected from the group consisting of a carrier, 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 comprises a double-stranded RNA molecule that reduces or eliminates DON production by Fusarium graminearum when transfected into or contacted with the Fusarium graminearum, wherein the double-stranded RNA molecule comprises at least one segment that is substantially complementary to, or comprises at least 95% sequence identity to, at least 21 consecutive nucleotides of a target gene or RNA transcribed from the target gene, the target gene having a sequence selected from the group consisting of: SEQ ID NOs: 1-16, 65-70 and 87-151, and wherein the double-stranded RNA molecule is at least 300 base pairs in length or between about 350 and about 600 base pairs in length.

5. The composition of any one of claims 1, 2, 3 or 4, wherein the polynucleotide, or the RNA or the dsRNA comprises a dsRNA comprising a first strand comprising a nucleotide sequence that is 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. The composition of claim 5, wherein the first strand comprises a nucleotide sequence that is about at least 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 any one of claims 5 or 6, wherein the polynucleotide, or the RNA or the dsRNA further comprises a second strand complementary to the first strand.

8. A composition according to any one of claims 1 to 7, wherein the nucleotide sequence of (f), (g), (h), (i), (j) or (k) is selected from the group consisting of: SEQ ID NO:48, 64, 80-82, 84-86, 182, 188, 194, 213, 220, 256, 262, 268, 287 and 294.

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 according to any one of claims 1 to 7, wherein the target gene recited in (a)-(e) has a nucleotide sequence selected from the group consisting of: SEQ ID NO: 16, 68-70, 108, 114, 120, 139 and 146.

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 reduces or eliminates 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 the expression of a Fusarium graminearum target gene, wherein the first strand of the dsRNA comprises an RNA sequence of at least 300 nucleotides in length and 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. The dsRNA of claim 12, wherein the second strand of the dsRNA is complementary to the first strand.

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 95% identical, at least about 98% identical, about 100% identical, or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 48, 64, 80-82, 84-86, 182, 188, 194, 213, 220, 256, 262, 268, 287, and 294.

15. The dsRNA of claim 14, wherein the RNA sequence comprises a nucleotide sequence at least about 98% identical to a nucleotide sequence selected from the group consisting of SEQ ID NO: 48, 64, 80-82, 84-86, 182, 188, 194, 213, 220, 256, 262, 268, 287, and 294.

16. The dsRNA according to any one of claims 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 of one or more additional species of Fusarium, optionally wherein the one or more additional species of Fusarium comprises Fusarium fulveum.

18. A dsRNA that inhibits expression of a Fusarium graminearum target gene, wherein the 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 95% identical, at least about 98% identical, about 100% identical, or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 48, 64, 80-82, 84-86, 182, 188, 194, 213, 220, 256, 262, 268, 287, and 294. The dsRNA of claim 18 , further comprising a second strand complementary to the first strand.

20. The dsRNA of any one of claims 18 and 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. 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. The composition of claim 22, further comprising one or more additional components selected from the group consisting of a carrier, a surfactant, an organosilicone, an organosilicone surfactant, a polynucleotide herbicidal molecule, a non-polynucleotide herbicidal molecule, a polynucleotide insecticide, a non-polynucleotide insecticide, a polynucleotide fungicide, a non-polynucleotide fungicide, a polynucleotide insecticide, a non-polynucleotide insecticide, a safener, and a pathogen growth regulator.

24. The composition of any one of claims 1 to 11, wherein the at least 18 consecutive nucleotides recited in (a)-(e), (g)-(j) are at least 300 consecutive nucleotides.

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

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. The method of any one of claims 25 or 26, wherein the contacting comprises applying the composition to the surface of a plant that is or can be infected by the fungus.

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

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

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

31. The method of any one of claims 25 to 30, wherein the composition comprises any one 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 comprises Fusarium graminearum, optionally wherein the one or more fungi of the genus Fusarium further comprises one or more additional fungal species, optionally wherein the one or more additional fungal species comprises Fusarium graminearum.

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

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

35. The method of claim 34, wherein the topical application comprises spraying the composition onto the leaves, bulbs or ears 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. The method of claim 36, wherein the plant is a cereal.

38. The method of any one of claims 33 to 37, wherein RNA interference is induced and DON production by Fusarium graminearum is reduced or eliminated.

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

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

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

42. The method of any one 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 NO: 48, 64, 80-82, 84-86, 182, 188, 194, 213, 220, 256, 262, 268, 287 and 294.

43. The method of any one 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 of claim 39(d) or (e) is selected from the group consisting of SEQ ID NO:48, 64, 80-82, 84-86, 182, 188, 194, 213, 220, 256, 262, 268, 287 and 294.

45. according to the method described in any one of claims 39 to 44, wherein said method comprises topically applying to said plant a composition comprising at least one polynucleotide, said at least one polynucleotide comprising a nucleotide sequence that is substantially complementary to at least 18, 19, 20 or 21 consecutive nucleotides of SEQ ID NO:48, or comprising at least 85%, 90% or 95% sequence identity thereto.

46. ​​The method of any one of claims 39 to 45, wherein the method comprises contacting the Fusarium graminearum with an effective amount of a solution comprising double-stranded RNA, wherein at least one strand of the double-stranded RNA is substantially 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, and DON production by the Fusarium graminearum is reduced or eliminated.

47. The method of claim 46, wherein at least one strand of the double-stranded RNA comprises a sequence that is substantially complementary to, or comprises at least 85%, 90% or 95% sequence identity to, at least 200, 300, 400, 500 or 600 consecutive nucleotides of a nucleotide sequence selected from the group consisting of SEQ ID NO:48, 64, 80-82, 84-86, 182, 188, 194, 213, 220, 256, 262, 268, 287 and 294.

48. The method of claim 46 or 47, wherein the solution further comprises one or more components selected from the group consisting of an organosilicone surfactant, a carrier, an organosilicone, an organosilicone surfactant, a polynucleotide herbicidal molecule, a non-polynucleotide herbicidal molecule, a polynucleotide pesticide, a non-polynucleotide pesticide, a safener, and a pathogen growth regulator.

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

50. The method of any one of claims 39 to 49, wherein the at least 18 consecutive nucleotides recited in (a)-(e) are at least 400 consecutive nucleotides.

51. A plant or a fruit, seed or propagable part thereof having increased resistance to DON production by Fusarium graminearum provided by the method of any one of claims 39 to 50.

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

53. A recombinant DNA construct comprising a heterologous promoter operably linked to: (a) a DNA comprising a nucleotide sequence that is substantially complementary to, or comprises at least 85%, 90% or 95% sequence identity to, at least 18 consecutive nucleotides of a target gene or an RNA transcribed from the target gene, wherein the target gene has a sequence selected from the group consisting of: SEQ ID NOs: 1-16, 65-70 and 87-151; or (b) a DNA comprising 18 or more consecutive nucleotides that are at least 85%, 90% or 95% identical to a fragment of an equivalent length of a DNA or its DNA complement, wherein the DNA has a sequence selected from the group consisting of: SEQ ID NOs: 1-16, 65-70 and 87-151; or (c) a DNA encoding at least one silencing element that is substantially complementary to at least 18 consecutive nucleotides of a target gene or RNA transcribed from the target gene, or comprises at least 85%, 90% or 95% sequence identity thereto, wherein the target gene has a sequence selected from the group consisting of: SEQ ID NO: 1-16, 65-70 and 87-151; or (d) a DNA encoding an RNA comprising a sequence selected from the group consisting of SEQ ID NOs: 33-64, 79-86 and 153-225, or a sequence having at least 85%, 90% or 95% sequence identity thereto. 227-299。 54. A plant chromosome or plasmid or recombinant plant virus vector or recombinant baculovirus vector comprising the recombinant DNA construct according to claim 53.

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

56. A transgenic plant or a fruit, seed or fertile part thereof, wherein the transgenic plant comprises the transgenic plant cell according to claim 55.

57. The plant of any one of claims 55 or 56, wherein the at least 18 consecutive nucleotides recited in (a)-(d) are at least 18, 19, 20 or 21 consecutive nucleotides.

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

59. according to the recombinant DNA construct described in claim 53, wherein the described at least 18 consecutive nucleotides recited in (a)-(d) are at least 300 consecutive nucleotides.

60. The plant chromosome or plastid or recombinant plant viral vector or recombinant baculoviral vector of claim 54, wherein the at least 18 consecutive nucleotides recited in (a)-(d) are at least 300 consecutive nucleotides.

61. The transgenic plant cell of claim 55, wherein the at least 18 consecutive nucleotides recited in (a)-(d) are at least 300 consecutive nucleotides.

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