Protection of plants from pathogens by inducing systemic or local defense responses in plants.

Abstract

Compositions of RNAi molecules for targeting plant genes involved in the suppression of systemic or local defense response pathways for controlling pathogens (e.g., fungal pathogens belonging to the genera Fusarium or Phytophthora), and related methods for using the same are provided herein.

Description

[Technical Field]

[0001] Related applications This application claims priority under 35 U.S. SC § 119(e) to U.S. Provisional Application USSN 63 / 488,962, filed on 7 March 2023, which is incorporated herein by reference.

[0002] Reference to electronic sequence listings The contents of the electronic sequence listing (G083070045WO00-SEQ-MSB.xml; size: 534,002 bytes; and creation date: March 6, 2024) are incorporated herein by reference in their entirety. [Background technology]

[0003] background Plant crops are targets for attacks by pathogens (fungi, bacteria, etc.). According to the United Nations Food and Agriculture Organization, farmers worldwide lose 30-40 percent of their crops due to pests and diseases. Crop maintenance and health are essential for yield and quality, ultimately requiring long-term strategies to minimize pest and disease outbreaks. The annual cost of controlling crop pathogenic pests is estimated at tens of millions of dollars, and if left uncontrolled, the estimated annual cost of crop losses can reach billions of dollars. The most common current methods of protection against pests are the application of chemicals to crops that bind to essential proteins in the pests, thereby impairing the pests' ability to damage crops (e.g., killing the pests or reducing their spore formation, growth, or reproductive capacity). These chemical fungicides can also bind to similar proteins in beneficial organisms in the field, causing collateral damage locally where the peptides or other fungicides are sprayed, remotely through residual peptides or other fungicides entering waterways, or by air currents. These fungicides may also have varying levels of toxicity to humans. [Overview of the project]

[0004] overview This disclosure provides compositions, polynucleotides, gene constructs, uses, and methods for controlling plant infection by various pathogens (e.g., pathogens that cause damage to crop plants) by inhibiting plant genes involved in the suppression of systemic or local defense responses (one or more) to pathogens. These inventions represent a more targeted and environmentally friendly approach than standard pathogen protection methods. For example, aspects of this disclosure provide polynucleotides (e.g., isolated polynucleotides) that inhibit plant genes involved in the suppression of systemic or local defense responses (one or more) to pathogens. In some embodiments, the plant genes are involved in the inhibition of the systemic acquired resistance (SAR) pathway. In some embodiments, the plant genes are involved in the suppression of systemic or local defense responses to fungal pathogens.

[0005] The plant genes targeted in the present invention may be selected from the group consisting of MPK4, CPR5, MPK4a, RLK3, NPR3, RFC3, SNI1, CEV1, COI1, RAP, DMR6-like, ARR9, NPR4, NAC25, BRI1a, BRI1b, TAR2, YUC2a, ETR1, EIN4, ETR2, ACS1, CRY1b, CRY2, ACO4, CTR1, MPK6, RIN4b, RIN4a, RapTOR, TOR, GID1B, GID1C, DHS, GA3OX, HOS15, eIF_5A, CLH2, SPL12l, EIN5, WRKY33, WRKY11, BZR1, EDR1, EXA1, WRKY40, JAZ7, PW220, DMR6, RPLK3, JAZ, and PAM16.

[0006] The plant genes targeted in this invention are MPK4 (mitogen-activated protein kinase 4), CPR5 (constitutive expression of PR gene 5), MPK4a (mitogen-activated protein kinase 4a), RLK3 (receptor-like kinase / leucine-rich repeat receptor-like kinase), NPR3 (NPR-1-like protein 3 / salicylate receptor), RFC3 (replication factor C subunit 3), and SNI1 (NPR-11 inhibitor), CEV1 (constitutive expression of VSP1 / cellulose synthase family protein), COI1 (coronatin insensitivity 1), RAP (RNA binding domain), DMR6 (downy mildew resistance 6), DMR6-like (downy mildew resistance 6-like), ARR9 (response regulator 9 / two-component response regulator-like protein), NPR4 (NPR1-like protein 4 / salicylate receptor), NAC25 (encodes NAC domain-containing protein 25), BRI1a (brassinosteroid insensitivity 1a / leucine-rich receptor-like protein kinase), BRI1b (brassinosteroid insensitivity 1a / leucine-rich receptor-like protein kinase). (Lasinosteroid-insensitive 1b / leucine-rich receptor-like protein kinase), TAR2 (tryptophan aminotransferase-related 2), YUC2a (yucca / indole-3-pyruvate monooxygenase), ETR1 (ethylene response 1 / ethylene receptor histidine kinase), EIN4 (ethylene-insensitive 4 / ethylene receptor histidine kinase), ETR2 (ethylene response 2 / ethylene receptor histidine kinase), ACS1 (ACC synthase 1 / enzymatically inactive form of 1-aminocyclopropane-1-carboxylate synthesis) Enzymes), CRY1b (Cryptochrome 1 / flavin-type blue light receptor), CRY2 (Cryptochrome 2 / flavin-type blue light receptor), ACO4 (1-aminocyclopropane-1-carboxylate oxidase 4), CTR1 (Constitutive triple response 1 / serine / threonine protein kinase), MPK6 (Mitogen-activated protein kinase 6), RIN4b (RPM1 interacting protein 4b), RIN4a (RPM1 interacting protein 4), RapTOR (TOR regulation-related protein), TOR (encoding the target of rapamycin), GID1B (GA-insensitive dwarf 1B / gibberellin receptor, GID1C (GA-insensitive dwarf 1C / gibberellin receptor), DHS (deoxyhypsin synthase), GA3OX (gibberellin 3-oxidase), HOS15 (high expression of osmoregulatory gene 15 / WD-40 repeat protein), eIF_5A (eukaryotic translation initiation factor 5A), CLH2 (chlorophyllase 2), SPL12l (Squamosa promoter-like protein 12), EIN5 (ethylene-insensitive 5 / 5'→3' exoribonuclease), WRKY33 (WRKYThe following may be selected from the group consisting of DNA-binding protein 33 (DNA-binding transcription factor), WRKY11 (WRKY DNA-binding protein 11 / DNA-binding transcription factor), BZR1 (brassinazole resistance 1 / DNA-binding transcription repressor), EDR1 (enhanced disease resistance 1 / protein kinase), EXA1 (essential for Potexvirus accumulation 1), WRKY40 (WRKY DNA-binding protein 40 / DNA-binding transcription factor), JAZ7 (encoding jasmonic acid ZIM domain protein 7), PW220 (JAZ10 / wound-inducing promoter 220 / jasmonic acid ZIM domain protein 10), RPLK3 (receptor-like protein kinase 3-like / leucine-rich receptor-like protein kinase family protein / ortholog of AT5G65700), JAZ (jasmonic acid ZIM domain protein), and PAM16 (presequence translocase-associated motor 16).

[0007] The plant gene may be MPK4a, RLK3, RIN4a, CEV1, CPR5, COI1, DHS, ETR1, EIN4, ETR2, or WRKY40. In some embodiments, the plant gene is the CPR5 gene.

[0008] The plant genes may originate from Arabidopsis thaliana (At), Glycine max (Gm), Lactuca sativa (Ls), Nicotiana benthamiana (Nb), Solanum lycopersicum (Sl), or Spinacia oleracea (So). The plant genes may also originate from plants of the Solanaceae, Brassicaceae, Poaceae, Cucurbitaceae, Fobaceae, Apiaceae, Amaranthaceae, or Malvaceae families.

[0009] In some embodiments, the plant gene is selected from the group consisting of AtMPK4, AtCPR5, GmMPK4a, GmRLK3, GmCPR5, GmNPR3, GmRFC3, GmSNI1, GmCEV1, GmCOI1, GmRAP, GmDMR6-like, GmARR9, GmNPR4, GmNAC25, GmBRI1a, GmBRI1b, GmTAR2, GmYUC2a, GmETR1, GmEIN4, GmETR2, GmACS1, GmCRY1b, GmCRY2, GmACO4, GmCTR1, GmMPK6, GmRIN4b, GmRIN4a, GmRapTOR, GmTOR, GmGID1B, GmGID1C, GmDHS, GmGA3OX, GmHOS15, GmeIF_5A, GmCLH2, GmSPL12l, LsCPR5, LsCPR5, NbEIN5, SlEIN5, SlCPR5, SoWRKY33, SoWRKY11, SoHOS15, SoBZR1, SoEDR1, SoEXA1, SoWRKY40, SoJAZ7, SoPW220, SoMPK6, SoSPL12I, SoCEV1, SoCPR5, SoCOI1, SoDMR6, SoDHS, SoelF-5A, SoETR1, SoEIN4, SoETR2, SoCTR1, SoMPK4a, SoRPLK3, SoBZR1, SoWRKY40, SoJAZ, SoDHS, SomelF5-A, SoMPK4a, SoETR1, SoEIN4, SoETR2, SoCEV1, and SoCPR5.

[0010] In some embodiments, the polynucleotide (e.g., an isolated polynucleotide) is ribonucleic acid (RNA). In some embodiments, the polynucleotide (e.g., an isolated polynucleotide) is deoxyribonucleic acid (DNA). In some embodiments, the polynucleotide is double-stranded RNA (dsRNA). In some embodiments, the RNA is double-stranded RNA (dsRNA) comprising a first strand that is complementary to a segment of the coding region of messenger RNA (mRNA) encoded by a gene and a second strand that is complementary to the first strand. In some embodiments, the polynucleotide is single-stranded RNA (ssRNA).

[0011] In some embodiments, the gene comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with any one of sequence numbers 1-86 or 265-274. In some embodiments, the polynucleotide (e.g., isolated polynucleotide) is complementary to a segment of a plant gene (e.g., a plant gene comprising a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with any one of sequence numbers 1-86 or 265-274). In some embodiments, a polynucleotide (e.g., isolated polynucleotide) is complementary to a segment of a plant gene (e.g., a plant gene containing a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with any one of sequence numbers 1-86 or 265-274), where the segment of the plant gene contains at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 consecutive nucleotides of the plant gene. In some embodiments, a polynucleotide (e.g., isolated polynucleotide) contains a nucleic acid sequence identical or complementary (e.g., perfectly complementary) to at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutive nucleotides. In some embodiments, the polynucleotide is at least 70%, 80%, 85%, 90%, 95%, or 100% identical or complementary to at least 21 consecutive nucleotides of a plant gene.

[0012] In some embodiments, a polynucleotide (e.g., an isolated polynucleotide) comprises a strand having a nucleic acid sequence that has at least 70% identity with any one of SEQ ID NOs: 87-258 and 275-290. In some embodiments, the polynucleotide comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with any one of SEQ ID NOs: 87-258 and 275-290. In some embodiments, the polynucleotide comprises at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 consecutive nucleotides having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to a corresponding segment of any one of SEQ ID NOs: 87-258 and 275-290.

[0013] In some embodiments, a polynucleotide (e.g., an isolated polynucleotide) comprises a strand having a nucleic acid sequence that has at least 70% identity with any one of SEQ ID NOs: 173-258 or 283-290. In some embodiments, the polynucleotide comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with any one of SEQ ID NOs: 87-172 or 275-282.

[0014] In some embodiments, the polynucleotide is a dsRNA comprising a second strand complementary to a first strand. In some embodiments, the polynucleotide is a dsRNA comprising (a) a first strand having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with any one of sequence numbers 87-172 or 275-282; and (b) a first strand having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with any one of sequence numbers 173-258 or 283-290.

[0015] In some embodiments, the polynucleotide comprises at least 18, 19, 20, 21, 25, 50, 100, 150, 200, 250, 300, 400, or 450 consecutive nucleotides, which are complementary to a segment of DNA or a target gene having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-86 or 265-274, or which contain at least about 85%, at least 90%, at least 95%, at least about 98%, or about 100% sequence identity. In some embodiments, the polynucleotide comprises at least 18, 19, 20, 21, 25, 50, 100, 150, 200, 250, 300, 350, 400, or 450 consecutive nucleotides, which are complementary to a sequence segment selected from the group consisting of SEQ ID NOs. 87-258 or 275-290, or which contain at least about 85%, at least about 90%, at least about 95%, at least about 98%, about 100%, or 100% sequence identity.

[0016] In some embodiments, the polynucleotide is a dsRNA comprising a first strand containing at least 18, 19, 20, 21, 25, 50, 100, 150, 200, 250, 300, 350, 400, or 450 consecutive nucleotides, which are complementary to a sequence segment selected from the group consisting of SEQ ID NOs. 87-172 and 275-282, or contain at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 98%, approximately 100%, or 100% sequence identity. The first strand further comprises a second strand complementary to the first strand, the second strand comprising 18, 19, 20, 21, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 consecutive nucleotides, which are complementary to a corresponding reverse complementary sequence segment selected from the group consisting of SEQ ID NOs: 173-258, 283-290, or contain at least about 85%, at least about 90%, at least about 95%, at least about 98%, about 100%, or 100% sequence identity.

[0017] In some embodiments, the polynucleotide comprises at least about 200 consecutive nucleotides that are complementary to or share at least about 90% identity with a sequence segment selected from the group consisting of SEQ ID NOs. 87-258 or 275-290.

[0018] In some embodiments, the DNA or target gene has a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 5, 35, 36, 41, 42, 62, 85, and 86. In some embodiments, the polynucleotide contains at least 18, 19, 20, 21, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 consecutive nucleotides that are complementary to a segment of a sequence selected from the group consisting of SEQ ID NOs: 88, 91, 121, 122, 127, 128, 148, 171, 172, and 174, 177, 207, 208, 213, 214, 234, 257, and 285, or contain at least about 85%, at least about 90%, at least about 95%, at least about 98%, about 100%, or 100% sequence identity. In some embodiments, the polynucleotide comprises at least about 200 consecutive nucleotides that are complementary to or share at least about 90% identity with a sequence segment selected from the group consisting of SEQ ID NOs: 88, 91, 121, 122, 127, 128, 148, 171, 172, 174, 177, 207, 208, 213, 214, 234, 257, and 285.

[0019] In some embodiments, the DNA or target gene has a nucleotide sequence selected from the group consisting of SEQ ID NOs: 3, 74, 75, 76, and 82. In some embodiments, the polynucleotide contains at least 18, 19, 20, 21, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 consecutive nucleotides that are complementary to a segment of a sequence selected from the group consisting of SEQ ID NOs: 89, 160, 161, 162, 168, 175, 246, 247, 248, and 254, or contain at least about 85%, at least about 90%, at least about 95%, at least about 98%, about 100%, or 100% sequence identity. In some embodiments, the polynucleotide comprises at least about 200 consecutive nucleotides that are complementary to or share at least about 90% identity with a sequence segment selected from the group consisting of SEQ ID NOs: 89, 160, 161, 162, 168, 175, 246, 247, 248, and 254.

[0020] In some embodiments, the DNA or target gene has the nucleotide sequence of SEQ ID NO: 4. In some embodiments, the polynucleotide contains at least 18, 19, 20, 21, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 consecutive nucleotides that are complementary to a sequence segment selected from the group consisting of SEQ ID NOs: 90 and 176, or contain at least 90% sequence identity. In some embodiments, the polynucleotide contains at least 200 consecutive nucleotides that are complementary to a sequence segment selected from the group consisting of SEQ ID NOs: 90 and 176, or contain at least 90% identity.

[0021] In some embodiments, the DNA or target gene has the nucleotide sequence of SEQ ID NO: 25. In some embodiments, the polynucleotide contains at least 18, 19, 20, 21, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 consecutive nucleotides that are complementary to a sequence segment selected from the group consisting of SEQ ID NOs: 111 and 197, or contain at least 90% sequence identity. In some embodiments, the polynucleotide contains at least 200 consecutive nucleotides that are complementary to a sequence segment selected from the group consisting of SEQ ID NOs: 111 and 197, or contain at least 90% identity.

[0022] In some embodiments, the DNA or target gene has a nucleotide sequence selected from the group consisting of SEQ ID NOs: 9, 59, 60, 61, and 84. In some embodiments, the polynucleotide contains at least 18, 19, 20, 21, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 consecutive nucleotides that are complementary to a segment of a sequence selected from the group consisting of SEQ ID NOs: 95, 181, 145, 231, 146, 232, 147, 233, 170, and 256, or contain at least about 85%, at least about 90%, at least about 95%, at least about 98%, about 100%, or 100% sequence identity. In some embodiments, the polynucleotide comprises at least about 200 consecutive nucleotides that are complementary to or share at least about 90% identity with a sequence segment selected from the group consisting of SEQ ID NOs: 95, 181, 145, 231, 146, 232, 147, 233, 170, and 256.

[0023] In some embodiments, the DNA or target gene has a nucleotide sequence selected from the group consisting of SEQ ID NOs: 10 and 63. In some embodiments, the polynucleotide contains at least 18, 19, 20, 21, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 consecutive nucleotides that are complementary to a segment of the sequence selected from the group consisting of SEQ ID NOs: 96, 182, 149, and 235, or contain at least about 90% sequence identity. In some embodiments, the polynucleotide contains at least about 200 consecutive nucleotides that are complementary to a segment of the sequence selected from the group consisting of SEQ ID NOs: 96, 182, 149, and 235, or contain at least about 90% identity.

[0024] In some embodiments, the DNA or target gene has a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29, 65, 66, and 80. In some embodiments, the polynucleotide contains at least 18, 19, 20, 21, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 consecutive nucleotides that are complementary to a segment of the sequence selected from the group consisting of SEQ ID NOs: 115, 201, 151, 237, 152, 238, 166, and 252, or contain at least about 85%, at least about 90%, at least about 95%, at least about 98%, about 100%, or 100% sequence identity. In some embodiments, the polynucleotide comprises at least about 200 consecutive nucleotides that are complementary to or share at least about 90% identity with a sequence segment selected from the group consisting of SEQ ID NOs: 115, 201, 151, 237, 152, 238, 166, and 252.

[0025] In some embodiments, the DNA or target gene has a nucleotide sequence selected from the group consisting of SEQ ID NOs: 18 and 83. In some embodiments, the polynucleotide contains at least 18, 19, 20, 21, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 consecutive nucleotides that are complementary to a segment of the sequence selected from the group consisting of SEQ ID NOs: 104, 190, 169, and 255, or contain at least about 85%, at least about 90%, at least about 95%, at least about 98%, about 100%, or 100% sequence identity. In some embodiments, the polynucleotide contains at least about 200 consecutive nucleotides that are complementary to a segment of the sequence selected from the group consisting of SEQ ID NOs: 104, 190, 169, and 255, or contain at least about 90% identity.

[0026] In some embodiments, the DNA or target gene has a nucleotide sequence selected from the group consisting of SEQ ID NOs. 52, 53, and 78. In some embodiments, the polynucleotide contains at least 18, 19, 20, 21, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 consecutive nucleotides that are complementary to a segment of the sequence selected from the group consisting of SEQ ID NOs. 138, 224, 139, 225, 164, and 250, or contain at least about 85%, at least about 90%, at least about 95%, at least about 98%, about 100%, or 100% sequence identity. In some embodiments, the polynucleotide contains at least about 200 consecutive nucleotides that are complementary to a segment of the sequence selected from the group consisting of SEQ ID NOs. 138, 224, 139, 225, 164, and 250, or contain at least about 90% identity. In some embodiments, a polynucleotide (e.g., isolated polynucleotide) has a length of at least 18 to 600 nucleotides, at least 21 to 600 nucleotides, at least 50 to 600 nucleotides, at least 100 to 500 nucleotides, at least 200 to 500 nucleotides, or at least 300 to 450 nucleotides. In some embodiments, a polynucleotide (e.g., isolated polynucleotide) has a length of at least 18 nucleotides, at least 21 nucleotides, at least 50 nucleotides, at least 100 nucleotides, at least 200 nucleotides, or at least 300 nucleotides. Polynucleotides may inhibit 1, 2, 3, or 4 independent plant genes and their paralogs that are involved in the suppression of systemic or local responses to pathogens.The polynucleotide may be a chimeric polynucleotide containing at least two different nucleic acid sequences, each of which inhibits a different plant gene involved in the suppression of a systemic or local response to a pathogen.

[0027] In some embodiments, the polynucleotide comprises a first nucleic acid that inhibits ETR1, a second nucleic acid that inhibits EIN4, and a third nucleic acid that inhibits ETR2.

[0028] Several aspects of this disclosure provide compositions comprising the polynucleotides described herein. In some embodiments, the composition further comprises carbon quantum dots. In some embodiments, the composition further comprises polyethylene glycol (PEG) (PEG200, for example). In some embodiments, the composition further comprises one or more chemifungicides and / or biofungicides. In some embodiments, the use of the polynucleotides of the present invention enables lower doses of chemifungicides or biofungicides without loss of the efficacy of the chemifungicide or biofungicide.

[0029] In some embodiments, the composition further comprises a ligand. The ligand may be polyethylene glycol (e.g., PEG200, PEG1000, PEG2000, PEG5000, or PEG10000), a carbohydrate, a porphyrin conjugate, a peptide, or a lipid. In some embodiments, the composition further comprises a surfactant. In some embodiments, the surfactant is Pluronic F-127.

[0030] Some aspects of this disclosure provide methods for increasing resistance to pathogens in plants, which include activating a systemic or local defense response in the plant to a pathogen prior to the plant's exposure to the pathogen.

[0031] Some aspects of this disclosure provide methods for increasing resistance to pathogens in plants, which include increasing the activity of systemic or local defense responses to pathogens.

[0032] Some aspects of this disclosure provide methods for increasing the lifespan of a plant or the average lifespan of a plant population, or increasing the biomass of a plant population or increasing the yield of a plant population, which include activating a systemic defense response to a pathogen prior to the exposure of the plant or plant population to a pathogen.

[0033] Activating a systemic defense response in plants may include inhibiting one or more plant genes involved in the suppression of a systemic response, optionally one or more plant genes involved in a systemic defense response to fungal pathogens or other microorganisms, and optionally the genes involved in the inhibition of the systemic acquired resistance (SAR) pathway.

[0034] In some embodiments, inhibiting one or more plant genes involved in the suppression of systemic or local defense responses results in further increased expression of genes or gene groups involved in inducing or executing systemic or local defense responses. In some embodiments, genes involved in inducing or executing systemic or local defense responses encode antifungal proteins. In some embodiments, genes involved in inducing or executing systemic or local defense responses encode transcription factors. In some embodiments, genes involved in inducing or executing systemic or local defense responses encode plant hormone biosynthetic enzymes. In some embodiments, genes involved in inducing or executing systemic or local defense responses encode plant hormone receptors. In some embodiments, genes involved in inducing or executing systemic or local defense responses encode transcription factors. In some embodiments, genes involved in inducing or executing systemic or local defense responses encode phosphate relay signaling proteins. In some embodiments, genes involved in inducing or executing systemic or local defense responses encode biosynthetic enzymes responsible for the synthesis of one or more antifungal metabolites. In some embodiments, genes involved in inducing or executing systemic or local defense responses encode proteins or metabolites incorporated into the cell wall that are responsible for physically preventing the spread of pathogens. In some embodiments, genes involved in inducing or executing systemic or local defense responses encode enzymes for the biosynthesis of plant cell wall components. In some embodiments, genes involved in inducing or executing systemic or local defense responses encode enzymes for the digestive walls of fungi. In some embodiments, genes involved in inducing or executing systemic or local defense responses include one or more PR1, PR3, PR5, HARB, ZF, GT61, and bHLH.

[0035] In some embodiments, inhibiting one or more plant genes involved in the suppression of systemic or local defense responses includes delivering polynucleotides or proteins to the plant that inhibit the expression of one or more plant genes.

[0036] In some embodiments, polynucleotides that inhibit expression function via RNA interference (RNAi) mechanisms. In some embodiments, one or more plant genes are selected from the group consisting of MPK4, CPR5, MPK4a, RLK3, NPR3, RFC3, SNI1, CEV1, COI1, RAP, DMR6-like, ARR9, NPR4, NAC25, BRI1a, BRI1b, TAR2, YUC2a, ETR1, EIN4, ETR2, ACS1, CRY1b, CRY2, ACO4, CTR1, MPK6, RIN4b, RIN4a, RapTOR, TOR, GID1B, GID1C, DHS, GA3OX, HOS15, eIF_5A, CLH2, SPL12l, EIN5, WRKY33, WRKY11, BZR1, EDR1, EXA1, WRKY40, JAZ7, PW220, DMR6, RPLK3, JAZ, and PAM16.In some embodiments, one or more plant genes include AtMPK4, AtCPR5, GmMPK4a, GmRLK3, GmCPR5, GmNPR3, GmRFC3, GmSNI1, GmCEV1, GmCOI1, GmRAP, GmDMR6-like, GmARR9, GmNPR4, GmNAC25, GmBRI1a, GmBRI1b, GmTAR2, GmYUC2a, and GmET R1, EIN4, ETR2, GmACS1, GmCRY1b, GmCRY2, GmACO4, GmCTR1, GmMPK6, GmRIN4b, GmRIN4a, GmRapTOR, GmT OR, GmGID1B, GmGID1C, GmDHS, GmGA3OX, GmHOS15, GmeIF_5A, GmCLH2, GmSPL12l, LsCPR5, LsCPR5, LsHOS 15, LsEDR1, LsPAM16, LsEXA1, LsMPK6, LsWRKY33, LsBZR1, NbEIN5, SlEIN5, SlCPR5, SoWRKY33, SoWRKY 11, SoHOS15, SoBZR1, SoEDR1, SoEXA1, SoWRKY40, SoJAZ7, SoPW220, SoMPK6, SoSPL12I, SoCEV1, SoCPR5 The group is selected from SoCOI1, SoDMR6, SoDHS, SomelF-5A, SoETR1, SoEIN4, SoETR2, SoCTR1, SoMPK4a, SoRPLK3, SoBZR1, SoWRKY40, SoJAZ, SoDHS, SomelF5-A, SoMPK4a, SoETR1, SEIN4, SoETR2, SoCEV1, and SoCPR5.

[0037] In some embodiments, inhibiting one or more plant genes includes delivering one or more polynucleotides described herein, or one or more compositions described herein, to a plant.

[0038] In some embodiments, polynucleotides are delivered to plants at the seedling or seed stage of the plant life cycle. In some embodiments, polynucleotides are delivered to the leaves or roots of the plant. In some embodiments, polynucleotides are delivered to the seeds of the plant, and optionally, polynucleotides are delivered by immersion of the plant seeds.

[0039] In some embodiments, the polynucleotides comprise nucleic acid sequences complementary to paralogs, homologous, or functionally redundant plant genes involved in the suppression of systemic or local defense responses. In some embodiments, the homologous, paralogous, or functionally redundant plant genes originate from the same species.

[0040] The plants treated in the present invention may be any plant. In some embodiments, the plants may be Asteraceae plants, Solanaceae plants, Brassicaceae plants, Poaceae plants, Cucurbitaceae plants, Fabaceae plants, Apiaceae plants, Amaranthaceae plants, Malvaceae plants, Actinidiaceae plants, Amaryllidaceae plants, Anacardiaceae plants, Annonaceae plants, Boraginaceae plants, Bromeliaceae plants, Chenopodiaceae plants, Convolvulaceae plants, Ebenaceae plants, Er The plants are selected from the group consisting of icaceae plants, Fabaceae or Leguminosae plants, Grossulariaceae plants, Lamiaceae plants, Lauraceae plants, Lythraceae plants, Malvaceae plants, Moraceae plants, Musaceae plants, Myrthaceae plants, Oleaceae plants, Passifloraceae plants, Polygonaceae plants, Rosaceae plants, Rutaceae plants, Sapindaceae plants, Verbenaceae plants, Vitaceae plants, and Zingiberaceae plants. In some embodiments, the plants are vegetables, fruits, grains, or legumes. In some embodiments, the plants are row crops, flowering plants, or trees. In some embodiments, the plants are oilseed crops, vegetables, legumes, fiber crops, ornamental plants, and spices. The plants may be maize, grains, cotton, fruits, nuts, rice, or soybeans. In some aspects, the plant is soybean, spinach, lettuce, cauliflower, pea sprout, rice, or wheat.

[0041] In some aspects, the pathogen is a bacterial pathogen, a viral pathogen, or a fungal pathogen. In some aspects, the pathogen is a fungal pathogen. In some aspects, the fungal pathogen is a genera of Fusarium, Phytophthora, Rhizoctonia, Macrophomina, Septoria,Pythium It belongs to the genera Sclerotina, Colletotrichum, Cladosporium, Peronospora, Stemphylium, Bremia, or Erysiphe. In some aspects, the pathogen is a bacterial pathogen. In some aspects, the bacterial pathogen belongs to the genera Pseudomonas or Erwinia. In some aspects, the pathogen is a virus. In some embodiments, the viruses are beet curly top virus (BCTV), beet western yellowing virus (BWYV), cucumber mosaic virus (CMV), impatiens necrotizing spot virus (INSV), tobacco rattle virus (TRV), tomato yellow necrosis virus (TSWV), lettuce infectious yellow spot virus (LIYV), lettuce mosaic virus (LMV), beet western yellowing virus (BWYV), cucumber mosaic virus (CMV), alfalfa mosaic virus (AMV), big vein virus, tomato yellow necrosis virus, impatiens necrotizing spot virus, lettuce necrotizing dwarf virus, and tomato bushy dwarf virus.

[0042] The pathogen may be Fusarium spp., Phakospora spp., Rhizoctonia spp., Aspergillus spp., Gibberella spp., Pyricularia spp., Alternaria spp., and Phytophthora spp. In some embodiments, the pathogen is Rhizoctonia solani.

[0043] In some embodiments, the systemic or local protective response pathway is the systemic acquired resistance (SAR) pathway.

[0044] The method may further include determining the expression level of one or more systemic or local defense response pathway genes. The expression levels of PR genes (e.g., PR1, PR3, or PR5 genes) may be determined. The expression levels of HARB, ZF, GT61, and bHLH may also be determined. [Brief explanation of the drawing]

[0045] [Figure 1A] Figures 1A and 1B demonstrate that carbon quantum dots can deliver dsRNA to soybean plants, resulting in a visible phenotype (luminescence from carbon quantum dots) in the first true leaves 12 days after planting.

[0046] [Figure 1B] Figures 1A and 1B demonstrate that carbon quantum dots can deliver dsRNA to soybean plants, resulting in a visible phenotype (luminescence from carbon quantum dots) in the first true leaves 12 days after planting.

[0047] [Figure 2] Figure 2 shows that delivery of dsRNAs targeting the systemic acquired resistance (SAR) pathway genes can upregulate the SAR pathway in soybeans.

[0048] [Figure 3] Figure 3 shows that delivery of dsRNA targeting the SAR pathway gene can protect soybean seedlings from fungal diseases.

[0049] [Figure 4A] Figures 4A and 4B show that when a dsRNA (GS200) targeting GmMPK4a was applied as a seed treatment, the seedlings were protected from Rhizoctonia solani. [Figure 4B] Figures 4A and 4B show that when a dsRNA (GS200) targeting GmMPK4a was applied as a seed treatment, the seedlings were protected from Rhizoctonia solani.

[0050] [Figure 5] Figure 5 shows that a dsRNA (GS200) targeting GmMPK4a increased GmPR1 expression (a molecular marker for the systemic acquired resistance pathway) in soybean plants. [Figure 6]Figure 6 shows that a dsRNA (GS200) targeting GmMPK4a increased GmPR1 expression (a molecular marker for the systemic acquired resistance pathway) in soybean plants.

[0051] [Figure 7] Figure 7 shows that the dsRNA (GS201) targeting GmRLK3 increased GmPR5 expression in soybean plants. [Figure 8] Figure 8 shows that the dsRNA (GS201) targeting GmRLK3 increased GmPR5 expression in soybean plants.

[0052] [Figure 9] Figure 9 shows that the dsRNA (GS2110) targeting GmRIN4a increased GmPR1 expression in soybean plants.

[0053] [Figure 10] Figure 10 shows that the dsRNA (GS2109) targeting GmRIN4b increased GmPR1 expression in soybean plants.

[0054] [Figure 11] Figure 11 shows that the dsRNA (GS279) targeting GmCEV1 increased GmPR3 expression in soybean plants.

[0055] [Figure 12] Figure 12 shows that the dsRNA (GS280) targeting GmCOI1 increased GmPR1 expression in soybean plants.

[0056] [Figure 13] Figure 13 shows that the dsRNA (GS415) targeting GmDHS increased GmPR1 expression in soybean plants.

[0057] [Figure 14]Figure 14 shows that a chimeric dsRNA (GS289) targeting GmETR1, GmEIN4, and GmETR2 increased GmPR1 expression in soybean plants.

[0058] [Figure 15] Figure 15 shows that the dsRNA (GS275) targeting GmCPR5 increased GmPR1 expression in soybean plants.

[0059] [Figure 16A] Figure 16A shows the activation of natural plant SAR protection for seedlings.

[0060] [Figure 16B] Figure 16B shows that the dsRNA (GS6263) targeting SoWRKY33 increased the expression of defense-related genes in spinach seedlings.

[0061] [Figure 17A] Figures 17A and 17B show that the dsRNA (GS5461) targeting LsCPR5 improved the health of lettuce plants in a fusarium chamber assay. [Figure 17B] Figures 17A and 17B show that the dsRNA (GS5461) targeting LsCPR5 improved the health of lettuce plants in a fusarium chamber assay.

[0062] [Figure 18] Figure 18 shows that a dsRNA (GS5461) targeting LsCPR5 increased lettuce survival across three lettuce varieties and two field locations under field fusarium load.

[0063] [Figure 19]Figure 19 shows an exemplary chimeric dsRNA structure for targeting homologous or functionally redundant genes from the same species. For example, homologs A and B (from the same species) are closely related but not 100% identical (<95% identity). Depending on the level of identity, the chimeric dsRNA may or may not be a continuous full-length match for both target genes. When the percentage identity is low, the chimera is designed by stitching sequences from the target genes. Therefore, it will not be a full-length match for all targets.

[0064] [Figure 20] Figure 20 shows the percentage increase in healthy spinach plants when dsRNA targeting the defense suppressor gene (CPR5) is included, compared to standard seed priming treatment up to day 21 of chamber-grown Fusarium oxysporum loading. *p<0.12

[0065] [Figure 21] Figure 21 shows an increase in the percentage of healthy lettuce plants and a twofold reduction in plant mortality when including LsCPR5-targeting dsRNA (GS5461) compared to standard seed priming treatment up to day 35 of chamber-grown Fusarium oxysporum loading. *p<0.12, **p<0.02

[0066] [Figure 22] Figure 22 shows that in lettuce leaves, an increase in the immunogene LsGT61 (Panel A) corresponds to a decrease in the GS5461 target gene LsCPR5 (Panel B) *p<0.05. [Modes for carrying out the invention]

[0067] Detailed description According to several aspects of this disclosure, RNA interference (RNAi) molecules (e.g., dsRNA) that target plant genes involved in the suppression of systemic and / or local defense response pathways (e.g., systemic acquired resistance pathways) are effective in interfering with gene-encoded mRNA in plant cells, thereby reducing or eliminating mRNA translation (e.g., into its corresponding protein). Systemic and / or local defense response pathways (e.g., MPK4, CPR5, MPK4a, RLK3, NPR3, RFC3, SNI1, CEV1, COI1, RAP, DMR6-like, ARR9, NPR4, NAC25, BRI1a, BRI1b, TAR2, YUC2a, ETR1, EIN4, ETR2, ACS1, CRY1b, CRY2, ACO4, CTR1, MPK6, RIN4b, RIN4a, RapTOR, TOR, GID1B, GID1C, DH Inhibition of genes involved in the repression of genes such as S, GA3OX, HOS15, eIF_5A, CLH2, SPL12l, EIN5, WRKY33, WRKY11, BZR1, EDR1, EXA1, WRKY40, JAZ7, PW220, DMR6, RPLK3, JAZ, and PAM16 results in activation or increased activity of systemic and / or local defense response pathways, thereby increasing the resistance of plants to pathogens (e.g., fungal pathogens). Accordingly, in several aspects, this disclosure provides polynucleotides, compositions, uses, and methods for controlling pathogen infection in plants by contacting any part of the plant (e.g., seeds, roots, tubers, stems, branches, leaves, flowers, etc.) with RNAi molecules as provided herein. When used herein, RNAi molecules are RNA-based molecules that function via sequence-specific mechanisms that repress gene expression through translational or transcriptional repression. RNAi molecules may be double-stranded or single-stranded nucleic acids. In some embodiments, RNAi molecules comprise RNA and / or DNA nucleotides (i.e., ribonucleotides and / or deoxyribonucleotides).

[0068] Systemic and local responses by plants to pathogens involve reactions that occur within the plant for the purpose of defending against exogenous factors such as pathogens (e.g., fungal pathogens). Local responses occur in the plant organ that is attacked or otherwise threatened by the pathogen. For example, if a pathogen attacks the plant's root system, the plant's local response involves the activation of defense pathways and genes within the root system. Systemic responses occur throughout the entire plant (or at least one organ of the plant that was not initially threatened by the pathogen) when any region or organ of the plant is threatened by the pathogen.

[0069] In some cases, a systemic response is involved in the enhancement of resistance known as systemic acquired resistance (SAR). In the SAR state, the plant is primed (sensitized) to activate a defense response more rapidly and effectively upon a second encounter with a pathogen attack. SAR is induced by most pathogens that cause tissue necrosis, either as part of a hypersensitive response (HR) or as a symptom of disease. SAR provides activity against a broad range of pathogens, including viruses, bacteria, oomycetes, and fungi. In some embodiments, SAR provides persistent protection that can last for several weeks to a month. The core set of SAR pathway genes (SAR genes) may include genes encoding pathogenicity-associated (PR) proteins. Selected PR proteins have been identified as acid β-1,3-glucanase (BGL2) and chitinase (PR-3). In some embodiments, the compositions and methods of this disclosure are designated to target plant genes (MPK4, CPR5, MPK4a, RLK3, NPR3, RFC3, SNI1, CEV1, COI1, RAP, DMR6-like, ARR9, NPR4, NAC25, BRI1a, BRI1b, TAR2, YUC2a, ETR1, EIN4, ETR2, ACS1, CRY1b, CRY2, ACO4, CTR1, MPK6, RIN4b, RIN4a, RapTOR, TOR, GID1B, GID1C, DHS, GA3OX, HOS15, eIF_5A, CLH2, SPL12l, EIN5, WRKY33, WRKY11, BZR1, EDR1, EXA1, WRKY40, JAZ7, PW220, DMR6, RPLK3, JAZ, and PAM16, etc., suppress (or inhibit) the SAR pathway.

[0070] The selection method in this disclosure is one or more plant genes (MPK4, CPR5, MPK4a, RLK3, NPR3, RFC3, SNI1, CEV1, COI1, RAP, DMR6-like, ARR9, NPR4, NAC25, BRI1a, BRI1b, TAR2, YUC2a, ETR1, EIN4, ETR2, ACS1, CRY1b, CRY2, ACO4, CTR1, MPK6, RIN4b, RIN4a, RapT The method is involved in the inhibition of (such as OR, TOR, GID1B, GID1C, DHS, GA3OX, HOS15, eIF_5A, CLH2, SPL12l, EIN5, WRKY33, WRKY11, BZR1, EDR1, EXA1, WRKY40, JAZ7, PW220, DMR6, RPLK3, JAZ, and PAM16) and the subsequent determination of the expression level (one or more) of one or more SAR genes. In some embodiments, the one or more SAR genes observed in such a method include PR genes (e.g., PR1, PR3, PR5).

[0071] In some embodiments, the methods of the disclosure are involved in the inhibition of the MPK4a, RLK3, RIN4a, CEV1, CPR5, COI1, DHS, ETR1, EIN4, ETR2, or WRKY40 genes. In some embodiments, the methods of the disclosure are involved in the inhibition of the CPR5 gene.

[0072] Gene expression in cells (e.g., plant cells) is considered inhibited or reduced via contact with RNAi molecules if, for example, the levels of mRNA and / or proteins encoded by the gene are reduced by at least 5% (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%) in the cells compared to control cells not in contact with RNAi molecules. For example, delivery of RNAi molecules (e.g., dsRNA) targeting plant genes involved in the suppression of systemic or local defense response pathways to cells (e.g., contact between RNAi molecules and cells) may result in a reduction in the amount of RNA transcripts and / or proteins (e.g., encoded by plant genes) (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%) compared to cells not in contact with RNAi molecules targeting plant genes.

[0073] In some embodiments, the RNAi molecules of this disclosure specifically inhibit the expression of one or more plant genes involved in the repression of systemic or local defense response pathways (e.g., the SAR pathway) without any biologically relevant or biologically significant negative physiological effects (e.g., a significant decrease in plant growth or plant yield). In some embodiments, the RNAi molecules specifically inhibit (reduce or block) the translation of proteins encoded by plant genes by specifically inhibiting (e.g., degrading) the expression of protein-coding mRNA (e.g., mRNA encoded by a gene containing any one sequence of SEQ ID NOs: 1-86 or 265-274). Specific inhibition of plant genes includes either a measurable decrease in gene expression (e.g., mRNA expression and / or protein expression) or a detectable complete absence of gene expression (e.g., mRNA expression and / or protein expression). In some embodiments, inhibition of plant genes is associated with a decrease in the activity of the resulting protein. In some embodiments, inhibition of plant genes involved in the suppression of systemic or local defense response pathways further results in increased expression of genes or gene groups (such genes encoding proteins with antifungal and / or antimicrobial properties) involved in inducing or executing systemic or local defense response pathways.

[0074] In some embodiments, the RNAi molecules of this disclosure specifically inhibit the expression of one or more plant genes involved in the repression of systemic or local defense response pathways without biologically relevant or biologically significant off-target effects (without relevant or significant changes in the expression of non-target genes). In some embodiments, the RNAi molecules specifically inhibit the expression of proteins encoded by plant genes by specifically inhibiting protein-coding mRNA (e.g., by specifically inhibiting mRNA translation). Specific inhibition of plant genes involved in the repression of systemic or local defense response pathways results in a measurable decrease in plant gene expression (e.g., mRNA expression and / or protein expression) or a detectable complete absence of gene expression (e.g., mRNA expression and / or protein expression). In some embodiments, inhibition of plant genes results in a decrease in the activity of the resulting protein.

[0075] It should be understood that "RNAi molecules targeting plant genes involved in the suppression of systemic or local defense responses to pathogens" encompass "RNAi molecules targeting mRNA encoded by plant genes involved in the suppression of systemic or local defense responses to pathogens." RNAi molecules are considered to target a gene of interest (e.g., a plant gene involved in the suppression of systemic or local defense responses to pathogens) if the RNAi molecule binds to mRNA (e.g., transiently) and inhibits (reduces or blocks) mRNA translation, for example, due to the degradation of the mRNA. In some embodiments, RNAi molecules are double-stranded RNA molecules. In some embodiments, polynucleotides are also understood to be double-stranded RNA (e.g., dsRNA) that inhibits the expression of a gene coding region (e.g., a SAR pathway gene). In other embodiments, polynucleotides are DNA sequences encoding dsRNA. In other embodiments, polynucleotides are antisense RNA. It should be understood that the sequences disclosed herein as DNA sequences can be converted from DNA sequences to RNA sequences by substituting each thymine with uracil. Furthermore, RNA sequences can be converted back to DNA sequences by substituting each uracil with thymine.

[0076] composition RNAi molecules that target plant genes involved in the repression of systemic or local defense response pathways, as provided herein, are designed in some embodiments to be complementary (e.g., partially complementary or fully complementary) to the mRNA encoded by the plant gene. Examples of DNA sequences encoding plant genes involved in the repression of systemic or local defense response pathways are provided in sequence numbers 1-86 or 265-274, or in the orthologue or homolog sequences of either such gene. Examples of RNA molecules that target plant genes involved in the repression of systemic or local defense response pathways are provided in sequence numbers 87-258 and 275-290. The RNAi molecule is complementary to the mRNA if it is able to bind to the mRNA under physiological conditions. In some embodiments, the RNAi molecule is complementary to the mRNA if it forms base pairs with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the mRNA over the length of the RNAi molecule and / or the mRNA.

[0077] As used herein with respect to two polynucleotides or polypeptides, the terms “sequence identity” or “identity” refer to residues in the sequences of two molecules that are identical when aligned for maximum correspondence across a specified comparison window. Percentage identity is calculated by determining the number of positions where identical nucleotide or amino acid residues exist in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. A sequence that is identical at all positions to a reference sequence is said to be 100% identical to the reference sequence, and vice versa. Percentage identity of two nucleotide sequences may also be determined by comparing two optimally aligned sequences of a molecule (e.g., nucleic acid sequences or polypeptide sequences) across a comparison window, where the portion of the sequence in the comparison window may include additions or deletions (i.e., gaps) compared to a reference sequence (which does not include additions or deletions) for optimal alignment of the two sequences. The optimal alignment for comparing two or more sequences may be performed using local or global alignment via various available computer programs. The algorithm by Smith TF and Waterman MS (1981), Identification of common molecular subsequences, J.Mol.Biol., Vol. 147, No. 1: pp. 195-, PubMed:7265238[SVP]DOI:10.1016 / 0022-2836(81)90087-5, is a suitable local alignment strategy and is utilized by tools such as EMBOSS Water (https: / / www.ebi.ac.uk / Tools / psa / emboss_water / ).The algorithm by Needleman SB and Wunsch CD (1970), "A general method applicable to the search for similarities in the amino acid sequence of two proteins," J.Mol.Biol., Vol. 48, No. 3: pp. 443-453, PubMed: 5420325, DOI: 10.1016 / 0022-2836(70)90057-4, is a suitable global alignment strategy and is utilized by tools such as EMBOSS Needle (www.ebi.ac.uk / Tools / psa / emboss_needle / ). Depending on the sequences being compared and the parameters involved, a local or global alignment strategy may be more likely to find the optimal alignment, although both strategies may be used to determine the optimal alignment that gives the most accurate percentage identity.

[0078] In some embodiments, RNAi molecules target two or more genes involved in the suppression of systemic or local defense response pathways, for example, by using an RNAi molecule with a chimeric design. Such genes may include, for example, the genes of sequence numbers 1-86 or 265-274, or paralogs, orthologues, or homologs of any of them. The chimeric trigger may be, for example, a continuous full-length match of the target genes, depending on the level of identity. If the percentage identity is low, the chimera may be designed, for example, by stitching sequences from the target genes. Therefore, it may not be a full-length match for every target.

[0079] In some embodiments, an RNAi molecule (e.g., a chimeric dsRNA) comprises a first nucleic acid sequence that targets a first gene for inhibition and a second nucleic acid sequence that targets a second gene for inhibition. The first nucleic acid sequence and / or the second nucleic acid sequence are 18-1000, 18-950, 18-900, 18-850, 18-800, 18-750, 18-700, 18-650, 18-600, 18-500, 18-450, 18-400, 18-350, 18-300, 18-250, 18-200, 18-180, 18-100, 18-50, 19-1000, 19-950, 19-900, 19-850, 19-800, 19-750, 19-700, 19-650, 19-600, 19-500 , 19~450, 19~400, 19~350, 19~300, 19~250, 19~200, 19~190, 19~100, 19~50, 20~1000, 20~950, 20~900, 20~850, 20~800, 20~750, 20~700, 20~650, 20~600, 20~500, 20~450, 20~400, 20~350, 20~300, 20~250, 20~200, 20~200, 20~100, 20~50, 21~1000, 21~950, 21~900, 21~850, 21 ~800, 21~750, 21~700, 21~650, 21~600, 21~500, 21~450, 21~400, 21~350, 21~300, 21~250, 21~210, 21~210, 21~100, 21~50, 22~1000, 22~950, 22~900, 22~850, 22~800, 22~750, 22~700, 22~650, 22~600, 22~500, 22~450, 22~400, 22~350, 22~300, 22~250, 22~220, 22~220, 22~1 00, 22~50, 23~1000, 23~950, 23~900, 23~850, 23~800, 23~750, 23~700, 23~650, 23~600, 23~500, 23~450, 23~400, 23~350, 23~300, 23~250, 23~230, 23~230, 23~100, 23~50, 24~1000, 24~950, 24~900, 24~850, 24~800, 24~750, 24~700, 24~650, 24~600, 24~500, 24~450, 24~400,The sequence may contain 24-350, 24-300, 24-250, 24-240, 24-240, 24-100, 24-50, 25-1000, 25-950, 25-900, 25-850, 25-800, 25-750, 25-700, 25-650, 25-600, 25-500, 25-450, 25-400, 25-350, 25-300, 25-250, 25-250, 25-250, 25-100, or 25-50 nucleotides or nucleotide base pairs. In some embodiments, the first nucleic acid sequence is at least 70%, 80%, 90%, 95%, or 100% complementary to the segment of the first gene. In some embodiments, the second nucleic acid sequence is at least 70%, 80%, 90%, 95%, or 100% complementary to the segment of the second gene.

[0080] In some embodiments, the RNAi molecules that target plant genes provided herein include MPK4, CPR5, MPK4a, RLK3, CPR5, NPR3, RFC3, SNI1, CEV1, COI1, RAP, DMR6-like, ARR9, NPR4, NAC25, BRI1a, BRI1b, TAR2, YUC2a, ETR1, EIN4, ETR2, ACS1, CRY1b, CRY2, ACO4, CTR1, MPK6, RIN4b, RIN4a, RapTOR, TOR, GID1B, GID1C, DHS, GA3OX, HOS15, eIF_5A, CLH2, SPL12l, LsCPR5, L It is designed to be complementary to mRNA segments encoded by genes selected from the group consisting of sCPR5, NbEIN5, EIN5, CPR5, WRKY33, WRKY11, HOS15, BZR1, EDR1, EXA1, WRKY40, JAZ7, PW220, MPK6, SPL12I, CEV1, CPR5, COI1, DMR6, DHS, melF-5A, ETR1, EIN4, ETR2, CTR1, MPK4a, RPLK3, BZR1, WRKY40, JAZ, DHS, melF5-A, MPK4a, ETR1, SEIN4, ETR2, CEV1, and CPR5.

[0081] In some embodiments, the RNAi molecules that target plant genes provided herein include MPK4, CPR5, MPK4a, RLK3, NPR3, RFC3, SNI1, CEV1, COI1, RAP, DMR6-like, ARR9, NPR4, NAC25, BRI1a, BRI1b, TAR2, YUC2a, ETR1, EIN4, ETR2, ACS1, CRY1b, CRY2, ACO4, CTR1, MPK6, RIN4b, R The RNAi molecules provided herein that target plant genes are designed to be complementary to mRNA segments encoded by genes selected from the group consisting of IN4a, RapTOR, TOR, GID1B, GID1C, DHS, GA3OX, HOS15, eIF_5A, CLH2, SPL12l, EIN5, WRKY33, WRKY11, BZR1, EDR1, EXA1, WRKY40, JAZ7, PW220, DMR6, RPLK3, JAZ, and PAM16. In some embodiments, the plant gene-targeting RNAi molecules provided herein are designed to be complementary to mRNA segments encoded by the MPK4a, RLK3, RIN4a, CEV1, CPR5, COI1, DHS, ETR1, EIN4, ETR2, PR1, PR3, PR5, or WRKY40 genes. In some embodiments, the plant gene-targeting RNAi molecules provided herein are designed to be complementary to the mRNA segment encoded by the CPR5 gene.

[0082] The double-stranded RNA (dsRNA) of this disclosure is, in some embodiments, a plant gene described herein (e.g., MPK4, CPR5, MPK4a, RLK3, NPR3, RFC3, SNI1, CEV1, COI1, RAP, DMR6-like, ARR9, NPR4, NAC25, BRI1a, BRI1b, TAR2, YUC2a, ETR1, EIN4, ETR2, ACS1, CRY1b, CRY2, ACO4, CTR1, MPK6, RIN4b, RIN4a, RapTOR, TOR, GID1B, GID1C It comprises a first strand that binds to a segment of messenger RNA (mRNA) encoded by a gene selected from the group consisting of DHS, GA3OX, HOS15, eIF_5A, CLH2, SPL12l, EIN5, WRKY33, WRKY11, BZR1, EDR1, EXA1, WRKY40, JAZ7, PW220, DMR6, RPLK3, JAZ, and PAM16 (for example, at least partially complementary or entirely complementary), and a second strand that is complementary to the first strand.

[0083] The double-stranded RNA (dsRNA) of this disclosure is, in some embodiments, a plant gene described herein (e.g., MPK4, CPR5, MPK4a, RLK3, NPR3, RFC3, SNI1, CEV1, COI1, RAP, DMR6, DMR6-like, ARR9, NPR4, NAC25, BRI1a, BRI1b, TAR2, YUC2a, ETR1, EIN4, ETR2, ETR1 / EIN4 / ETR2, ACS1, CRY1b, CRY2, ACO4, CTR1, MPK6, RIN4b, RIN4a, RapTOR, TOR) The dsRNA comprises a first strand (for example, at least partially complementary or entirely complementary) that binds to a segment of messenger RNA (mRNA) encoded by a gene selected from the group consisting of GID1B, GID1C, DHS, GA3OX, HOS15, eIF_5A, CLH2, SPL12l, EIN5, WRKY33, WRKY11, BZR1, EDR1, EXA1, WRKY40, JAZ7, PW220, melF-5A, RPLK3, and JAZ, and a second strand complementary to the first strand. The dsRNA may include RNA strands of the same or different lengths. In some embodiments, the dsRNA includes a first strand (for example, an antisense strand) that is the same length as the second strand (for example, a sense strand). In some embodiments, the dsRNA includes a first strand (for example, an antisense strand) that is different in length from the second strand (for example, a sense strand). The first chain may be about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or more than 20% longer than the second chain. The first chain may be 1–5, 2–5, 2–10, 5–10, 5–15, 10–20, 15–20, or more than 20 nucleotides longer than the second chain.

[0084] dsRNA molecules can also be associated with a single oligonucleotide in a stem-loop structure (where the self-complementary sense and antisense regions of the RNA molecule are linked by one or more nucleic acid-based or non-nucleic acid-based linkers), as well as a circular single-stranded RNA having a stem containing two or more loop structures and self-complementary sense and antisense strands (where the circular RNA can be processed in vivo or in vitro to produce an active RNAi molecule capable of mediating RNAi). The RNAi molecule may contain a 3' overhang at one end of the molecule; the other end may be blunt-ended or have an overhang (5' or 3'). If the RNAi molecule contains overhangs at both ends of the molecule, the lengths of the overhangs may be the same or different.

[0085] The single-stranded RNAs disclosed herein are, in some aspects, plant genes (e.g., MPK4, CPR5, MPK4a, RLK3, CPR5, NPR3, RFC3, SNI1, CEV1, COI1, RAP, DMR6-like, ARR9, NPR4, NAC25, BRI1a, BRI1b, TAR2, YUC2a, ETR1, EIN4, ETR2, ACS1, CRY1b, CRY2, ACO4, CTR1, MPK6, RIN4b, RIN4a, RapTOR, TOR, GID1B, GID1C, DHS, GA3OX, HOS15, eIF_5A, CLH2, SPL12l, LsCPR5, LsCPR5, N It contains a strand that binds to a segment of mRNA encoded by mRNA encoded by a gene selected from the group consisting of bEIN5, EIN5, CPR5, WRKY33, WRKY11, HOS15, BZR1, EDR1, EXA1, WRKY40, JAZ7, PW220, MPK6, SPL12I, CEV1, CPR5, COI1, DMR6, DHS, melF-5A, ETR1, EIN4, ETR2, CTR1, MPK4a, RPLK3, BZR1, WRKY40, JAZ, DHS, melF5-A, MPK4a, ETR1, SEIN4, ETR2, CEV1, and CPR5.

[0086] The single-stranded RNAs disclosed herein are, in some embodiments, plant genes (e.g., MPK4, CPR5, MPK4a, RLK3, NPR3, RFC3, SNI1, CEV1, COI1, RAP, DMR6-like, ARR9, NPR4, NAC25, BRI1a, BRI1b, TAR2, YUC2a, ETR1, EIN4, ETR2, ACS1, CRY1b, CRY2, ACO4, CTR1, MPK6, RIN4b, RIN4a, Ra It contains a strand that binds to a segment of mRNA encoded by mRNA encoded by a gene selected from the group consisting of pTOR, TOR, GID1B, GID1C, DHS, GA3OX, HOS15, eIF_5A, CLH2, SPL12l, EIN5, WRKY33, WRKY11, BZR1, EDR1, EXA1, WRKY40, JAZ7, PW220, DMR6, RPLK3, JAZ, and PAM16.

[0087] The RNAi molecules provided herein may vary in length. In some embodiments, long RNA molecules (e.g., dsRNA or ssRNA) are applied to plants, but it should be understood that after entering the cells, the RNA is cleaved by Dicer enzymes into shorter RNA fragments having, for example, 15–25 nucleotides in length. Thus, the RNAi molecules of this disclosure may be delivered, for example, as fragments of 15–25 nucleotides, or they may be delivered as longer double-stranded polynucleotides (e.g., at least 100 nucleotides).

[0088] Therefore, in some embodiments, RNAi molecules that target plant genes involved in the suppression of systemic or local defense response pathways contain 15 to 1010 nucleotides (ssRNA) or nucleotide base pairs (dsRNA). For example, the RNAi molecules of this disclosure are 15-1000, 15-950, 15-900, 15-850, 15-800, 15-750, 15-700, 15-650, 15-600, 15-500, 15-450, 15-400, 15-350, 15-300, 15-250, 15-200, 15-150, 15-100, 15-50, 16-1000, 16-950, 16-900, 16-850, 16-800, 16-750, 16-700, 16-650, 16-600, 16-500, 16- 450, 16~400, 16~350, 16~300, 16~250, 16~200, 16~150, 16~100, 16~50, 17~1000, 17~950, 17~900, 17~850, 17~800, 17~750, 17~700, 17~650, 17~600, 17~500, 17~450, 17~400, 17~350, 17~300, 17~250, 17~200, 17~150, 17~100, 17~50, 18~1000, 18~950, 18~900, 18~850, 18 ~800, 18~750, 18~700, 18~650, 18~600, 18~500, 18~450, 18~400, 18~350, 18~300, 18~250, 18~200, 18~180, 18~100, 18~50, 19~1000, 19~950, 19~900, 19~850, 19~800, 19~750, 19~700, 19~650, 19~600, 19~500, 19~450, 19~400, 19~350, 19~300, 19~250, 19~200, 19~190, 1 9-100, 19-50, 20-1000, 20-950, 20-900, 20-850, 20-800, 20-750, 20-700, 20-650, 20-600, 20-500, 20-450, 20-400, 20-350, 20-300, 20-250, 20-200, 20-200, 20-100, 20-50, 21-1000, 21-950, 21-900, 21-850, 21-800, 21-750, 21-700, 21-650, 21-600, 21-500, 21-450,21-400, 21-350, 21-300, 21-250, 21-210, 21-210, 21-100, 21-50, 22-1000, 22-950, 22-900, 22-850, 22-800, 22-750, 22-700, 22-650, 22-600, 22-500, 22-450, 22-400, 22-350, 22-300, 22-250, 22-220, 22-220, 22-100, 22-50, 23-1000, 23-950, 23-900, 23-850, 23-800, 23-750, 23-700, 23-650, 23-600, 23-500, 23-450, 23-400, 23-350, 23-300, 23-250, 23-230, 23-230, 23-100, 23-50, 24-1000, 24-950, 24-900, 24-850, 24-800, 24-750, 24-700, 24-650, 24-600, 24-500, 24-450, 24-400, 24-350, 24-300, 24-250, 24-240, 24-240, 24-100, 24-50, 25-1000, It may contain 25-950, 25-900, 25-850, 25-800, 25-750, 25-700, 25-650, 25-600, 25-500, 25-450, 25-400, 25-350, 25-300, 25-250, 25-250, 25-250, 25-100, or 25-50 nucleotides or nucleotide base pairs. In some embodiments, an RNAi molecule contains or comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 25, at least 50, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, or at least 1000 nucleotides or nucleotide base pairs.

[0089] The full length of the RNAi molecule of the present invention may be 18 or more consecutive nucleotides. In some embodiments, the RNAi molecule (e.g., a polynucleotide as described herein) may include nucleotides in addition to nucleotides that target a plant gene (e.g., a plant gene containing a sequence selected from the group consisting of SEQ ID NOs. 1-86 or 265-274, or RNA transcribed therefrom). Similarly, the RNAi molecule of the present disclosure (e.g., a polynucleotide as described herein) may include one or more sequences that are about 75% to about 100% identical to 18 or more consecutive nucleotides of a sequence selected from the group consisting of SEQ ID NOs. 87-258 and 275-290.

[0090] In some embodiments, additional irrelevant sequences may also be included. In other words, the total length of the polynucleotide can be longer than the length of the polynucleotide segment or segment designed to repress one or more target genes. For example, the polynucleotide may have nucleotides adjacent to an "active" segment (for example, the "active" segment may be a sequence complementary to the segment of the target gene or the mRNA transcribed therefrom, or a sequence selected from SEQ ID NOs. 87-258 and 275-290 that repress the target gene, or may contain "spacer" nucleotides between active segments, or may have additional nucleotides at the 5' end, or at the 3' end, or at both the 5' and 3' ends). In one embodiment, the RNAi molecule may contain additional nucleotides that are not specifically related to (have complementary or non-identical) the sequences disclosed herein. For example, such an RNAi molecule may contain nucleotides to provide a stabilizing secondary structure or for convenience in cloning or manufacturing. Good. Or, they may be impurities found at the 5' and / or 3' ends of the RNA strand. In one embodiment, the RNAi molecule may include additional nucleotides located directly adjacent to the active segment. In one embodiment, the polynucleotide includes one such segment having an additional 5'G or an additional 3'C or both adjacent to the segment. In another embodiment, the polynucleotide is a double-stranded RNA containing additional nucleotides that form one or more overhangs. In yet another embodiment, the polynucleotide may include one or more active segments described herein, as well as additional segments that are active for other target genes. Thus, in various embodiments, the nucleotide sequence of the entire RNAi molecule may not be 100% identical or complementary to the trigger sequence disclosed herein, nor to the sequence of consecutive nucleotides in the target gene. In some embodiments, the polynucleotide is complementary to a segment of a plant gene (for example, a plant gene containing a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with any one of SEQ ID NOs: 1-86 or 265-274). In some embodiments, the polynucleotide is complementary to a segment of a plant gene (for example, a plant gene containing a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with any one of SEQ ID NOs: 1-86 or 265-274), where the segment of the plant gene contains at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 consecutive nucleotides of the plant gene.

[0091] In some embodiments, RNAi molecules targeting plant genes involved in the suppression of systemic or local defense response pathways include, or consist of, a sequence complementary to the mRNA or mRNA segment encoded by the plant gene. In some embodiments, RNAi molecules targeting plant genes involved in the suppression of systemic or local defense response pathways include, or consist of, a sequence complementary to the mRNA or mRNA segment encoded by any one of the DNA sequences SEQ ID NOs: 1-86 or 265-274. In some embodiments, RNAi molecules targeting plant genes involved in the suppression of systemic or local defense response pathways include, or consist of, a sequence complementary to the mRNA encoded by any one of the DNA sequences SEQ ID NOs: 1-86 or 265-274.

[0092] In some embodiments, RNAi molecules targeting plant genes involved in the suppression of systemic or local defense response pathways contain or consist of a sequence complementary to a segment of mRNA encoded by a region or segment of plant gene DNA. In some embodiments, the RNAi molecule targets mRNA encoded by a region of plant gene DNA that contains or may consist of any sequence encompassed by nucleotides 1-500, nucleotides 10-500, nucleotides 25-500, nucleotides 50-500, nucleotides 100-500, nucleotides 150-500, nucleotides 200-500, nucleotides 250-500, nucleotides 300-500, nucleotides 350-500, nucleotides 400-500, or nucleotides 450-500. In some embodiments, the RNAi molecule targets mRNA encoded by a region of plant gene DNA that contains, or may contain, any sequence encompassed by nucleotides 200-950, nucleotides 250-950, nucleotides 300-950, nucleotides 350-950, nucleotides 400-950, nucleotides 450-950, nucleotides 500-950, nucleotides 550-950, nucleotides 200-700, nucleotides 250-700, nucleotides 300-700, nucleotides 350-700, nucleotides 400-700, nucleotides 450-700, nucleotides 500-700, nucleotides 550-700, nucleotides 600-700, or nucleotides 650-700.

[0093] The term "gene" should be understood to encompass coding nucleic acids and non-coding nucleic acids. Therefore, in some embodiments, plant genes involved in the repression of systemic or local defense response pathway genes encode mRNA containing a 5' untranslated region, an open reading frame, and a 3' untranslated region. Thus, in some embodiments, RNAi molecules bind to the 5' untranslated region, the open reading frame, and / or the 3' untranslated region of mRNA.

[0094] In some embodiments, RNAi molecules that target plant genes involved in the suppression of systemic or local defense response pathways contain or consist of one RNA sequence from sequence numbers 87-258 and 275-290.

[0095] In some embodiments, the RNAi molecule comprises or consists of at least one continuous sequence having 70% to 100% identity (e.g., 70% to 100%, 75% to 100%, 80% to 100%, 85% to 100%, 90% to 100%, 95% to 100%, 96% to 100%, 97% to 100%, 98% to 100%, 99% to 100%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) with an RNA sequence encoded by a plant gene involved in the suppression of systemic or local defense response pathways. In some embodiments, the plant gene comprises one DNA sequence of sequence numbers 1 to 86 or 265 to 274. In some embodiments, an RNAi molecule comprises or consists of at least one continuous sequence having 70% to 100% identity (for example, 70% to 100%, 75% to 100%, 80% to 100%, 85% to 100%, 90% to 100%, 95% to 100%, 96% to 100%, 97% to 100%, 98% to 100%, 99% to 100%, 80%, 85%, 90%, 95%, 96%, 97%, 97%, 98%, 98%, 99%, or 100% identity) with one of the DNA sequences of sequence numbers 1 to 86 or 265 to 274, or with a gene containing a homolog, ortholog, or paralog of any of them.

[0096] In some embodiments, RNAi molecules targeting plant genes involved in the suppression of systemic or local defense response pathways include or consist of at least one continuous sequence that is 70% to 100% complementary to the RNA sequence encoded by the plant gene (e.g., 70% to 100%, 75% to 100%, 80% to 100%, 85% to 100%, 90% to 100%, 95% to 100%, 96% to 100%, 97% to 100%, 98% to 100%, 99% to 100%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% complementary). In some embodiments, the plant gene includes one DNA sequence of sequence numbers 1 to 86 or 265 to 274. In some embodiments, RNAi molecules targeting plant genes involved in the suppression of systemic or local defense response pathways include or consist of at least one continuous sequence that is 70% to 100% complementary (e.g., 70% to 100%, 75% to 100%, 80% to 100%, 85% to 100%, 90% to 100%, 95% to 100%, 96% to 100%, 97% to 100%, 98% to 100%, 99% to 100%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98%, 99%, or 100% identity) to one of the DNA sequences of sequence numbers 1 to 86 or 265 to 274, or to an RNA sequence encoded by a gene containing a homolog, ortholog, or paralog of any of these sequences.

[0097] In some embodiments, RNAi molecules targeting plant genes involved in the suppression of systemic or local defense response pathways have 70% to 100% identity (e.g., 70% to 100%, 75% to 100%, 80% to 100%, 85% to 100%, 90% to 100%, 95% to 100%, 96% to 100%, 97% to 100%, 98% to 100%, 99% to 100%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to one of the RNA sequences or segments of RNA sequences among sequence numbers 87 to 258 and 275 to 290, at least 15, at It comprises or consists of 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 25, at least 50, at least 75, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, at least 600, at least 650, at least 700, at least 750, at least 800, at least 850, at least 900, at least 950, or at least 1000 nucleotides or nucleotide base pairs.In some embodiments, the RNAi molecule has 70% to 100% identity (for example, 70% to 100%, 75% to 100%, 80% to 100%, 85% to 100%, 90% to 100%, 95% to 100%, 96% to 100%, 97% to 100%, 98% to 100%, 99% to 100%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to one RNA sequence or segment of an RNA sequence from sequence numbers 87 to 258 and 275 to 290, with at least 15, at least 16, at least 17, at least 18 , comprising or consisting of at least 19, at least 20, at least 21, at least 25, at least 50, at least 75, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, at least 600, at least 650, at least 700, at least 750, at least 800, at least 850, at least 900, at least 950, or at least 1000 nucleotides or nucleotide base pairs. In some embodiments, the RNAi molecule comprises one or more sequences identical or complementary to 18 or more consecutive nucleotides of a sequence selected from the group consisting of SEQ ID NOs. 87-258 and 275-290. In some embodiments, the RNAi molecule comprises one or more sequences that are at least 90% identical or complementary to 21 or more consecutive nucleotides of a sequence selected from the group consisting of SEQ ID NOs. 87-258 and 275-290. In some embodiments, the RNAi molecule contains one or more sequences that are identical or completely complementary to 21 or more consecutive nucleotides of sequences selected from the group consisting of SEQ ID NOs. 87-258 and 275-290.

[0098] In some embodiments, RNAi molecules targeting plant genes involved in the suppression of systemic or local defense response pathways are 70% to 100% complementary (e.g., 70% to 100%, 75% to 100%, 80% to 100%, 85% to 100%, 90% to 100%, 95% to 100%, 96% to 100%, 97% to 100%, 98% to 100%, 99% to 100%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% complementary) to one RNA sequence or segment of an RNA sequence from sequence numbers 87 to 258 and 275 to 290, It contains or consists of at least 17, at least 18, at least 19, at least 20, at least 21, at least 25, at least 50, at least 75, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, at least 600, at least 650, at least 700, at least 750, at least 800, at least 850, at least 900, at least 950, or at least 1000 nucleotides or nucleotide base pairs.In some embodiments, RNAi molecules targeting plant genes involved in the suppression of systemic or local defense response pathways are 70% to 100% complementary (e.g., 70% to 100%, 75% to 100%, 80% to 100%, 85% to 100%, 90% to 100%, 95% to 100%, 96% to 100%, 97% to 100%, 98% to 100%, 99% to 100%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% complementary) to one RNA sequence or segment of an RNA sequence, either sequence 1-86 or 265-274, with at least 15, at least 16, and a small number of RNAs. It contains or consists of at least 17, at least 18, at least 19, at least 20, at least 21, at least 25, at least 50, at least 75, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, at least 600, at least 650, at least 700, at least 750, at least 800, at least 850, at least 900, at least 950, or at least 1000 nucleotides or nucleotide base pairs.

[0099] In some embodiments, the RNAi molecule has 70% to 100% identity (for example, 70% to 100%, 75% to 100%, 80% to 100%, 85% to 100%, 90% to 100%, 95% to 100%, 96% to 100%, 97% to 100%, 98% to 100%, 99% to 100%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) with respect to one RNA sequence or segment of an RNA sequence from sequence numbers 87 to 258 and 275 to 290, with respect to 10 to 25, 10 to 24, 10 to 23, 10 to 22, 10 to 21, 10 to 20, 11 to 25, 11 to 24, 11 to 23, 11 to 22, 11 ~21, 11~20, 12~25, 12~24, 12~23, 12~22, 12~21, 12~20, 13~25, 13~24, 13~23, 13~22, 13~21, 13~20, 14~25, 14~24, 14~23, 14~22, 14~21, 14~20, 15~25, 15~24, 15~23, 15~22, 15~2 It contains or consists of 1, 15-20, 16-25, 16-24, 16-23, 16-22, 16-21, 16-20, 17-25, 17-24, 17-23, 17-22, 17-21, 17-20, 18-25, 18-24, 18-23, 18-22, 18-21, or 18-20 consecutive nucleotides.In some embodiments, RNAi molecules exhibit 70% to 100% identity (for example, 70% to 100%, 75% to 100%, 80% to 100%, 85% to 100%, 90% to 100%, 95% to 100%, 96% to 100%) to RNA sequences or segments of RNA sequences with sequence numbers 87 to 258 and 275 to 290. Having identity of %, 97%~100%, 98%~100%, 99%~100%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%, 10~25, 10~24, 10~23, 10~22, 10~21, 10~20, 11~25, 11~24, 11~23, 11~22, 11~21, 11-20, 12-25, 12-24, 12-23, 12-22, 12-21, 12-20, 13-25, 13-24, 13-23, 13-22, 13-21, 13-20, 14-25, 14-24, 14-23, 14-22, 14-21, 14-20, 15-25, 15-24, 15-23, 15-22, 15-21, It contains or consists of 15-20, 16-25, 16-24, 16-23, 16-22, 16-21, 16-20, 17-25, 17-24, 17-23, 17-22, 17-21, 17-20, 18-25, 18-24, 18-23, 18-22, 18-21, or 18-20 consecutive nucleotides.

[0100] In some embodiments, the RNAi molecule is 70% to 100% complementary to one RNA sequence or segment of an RNA sequence from sequence numbers 87-258 and 275-290 (for example, 70% to 100%, 75% to 100%, 80% to 100%, 85% to 100%, 90% to 100%, 95% to 100%, 9 6%~100%, 97%~100%, 98%~100%, 99%~100%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% complementary), 10~25, 10~24, 10~23, 10~22, 10~21, 10~20, 11~25, 11~24, 11~23, 11~22, 11~21 , 11-20, 12-25, 12-24, 12-23, 12-22, 12-21, 12-20, 13-25, 13-24, 13-23, 13-22, 13-21, 13-20, 14-25, 14-24, 14-23, 14-22, 14-21, 14-20, 15-25, 15-24, 15-23, 15-22, 15-21 It contains or consists of 15-20, 16-25, 16-24, 16-23, 16-22, 16-21, 16-20, 17-25, 17-24, 17-23, 17-22, 17-21, 17-20, 18-25, 18-24, 18-23, 18-22, 18-21, or 18-20 consecutive nucleotides.

[0101] In some embodiments, the RNAi molecule targets one of the genes described in Table 1. In some embodiments, the RNAi molecule targets one or more genes belonging to the species provided in Table 1. In some embodiments, the RNAi molecule contains or consists of one of the dsRNA sequences provided in Table 1. In some embodiments, the RNAi molecule contains or consists of one of the reverse complementary sequences provided in Table 2.

[0102] Unless otherwise specified, nucleic acid sequences in this specification are shown in the 5' to 3' direction when read from left to right. Those skilled in the art will recognize that a given DNA sequence is understood to define a corresponding RNA sequence that is identical to the DNA sequence except that the thymine (T) nucleotides of the DNA are replaced with uracil (U) nucleotides. Thus, providing a particular DNA sequence is understood to define an exact 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), and the second polynucleotide completely hybridizes to the first polynucleotide by forming Watson-Crick base pairs. In the case of a DNA:DNA double helix (hybridized strand), the base pairs are adenine:thymine or guanine:cytosine; in the case of a DNA:RNA double helix, the base pairs are adenine:uracil or thymine:adenine or guanine:cytosine. In the case of RNA:RNA double helix, the base pairs are adenine:uracil or guanine:cytosine. Thus, the nucleotide sequences of a perfectly hybridized blunt-ended double-stranded polynucleotide (where there is "100% complementarity" between the strands, or where they are "perfectly complementary") are clearly defined by providing the nucleotide sequence of one strand, whether given as DNA or RNA. To be “complementary” to a target gene, or a fragment of a target gene or mRNA, or the second strand of a dsRNA, means that a polynucleotide strand (or at least one strand of a double-stranded polynucleotide) is designed to hybridize to a target gene, or a fragment of a target gene, or a transcript of a target gene, or the other strand of a fragment of a target gene, or a dsRNA (generally under physiological conditions such as those found in plant or fungal cells); those skilled in the art will understand that such hybridization does not necessarily require 100% sequence identity or complementarity.In some embodiments, the trigger may be designed to be not 100% identical to the target gene sequence, but to remain complementary to the target gene sequence or the RNA transcribed therefrom. When a first nucleic acid sequence has a functional relationship with a second nucleic acid sequence, the first nucleic acid sequence is “operably” linked to or “linked” with the second nucleic acid sequence. For example, when a promoter provides transcription or expression of DNA, the promoter sequence is “operably linked” to the DNA. Generally, operably linked DNA sequences are contiguous.

[0103] The percentage identity of two nucleotide sequences may also be determined by comparing two optimally aligned sequences of a molecule (e.g., nucleic acid sequences or polypeptide sequences) across a comparison window, where the portion of the sequence in the comparison window may include additions or deletions (i.e., gaps) compared to a reference sequence (which does not contain additions or deletions) for optimal alignment of the two sequences. Optimal alignment for comparing two or more sequences may be performed using local or global alignment via various available computer programs. The algorithm by Smith TF and Waterman MS (1981), Identification of common molecular subsequences, J.Mol.Biol., Vol. 147, No. 1: pp. 195-197, PubMed: 7265238, DOI: 10.1016 / 0022-2836(81)90087-5, is a suitable local alignment strategy and is available through tools such as EMBOSS Water (www.ebi.ac.uk / Tools / psa / emboss_water / ). The algorithm by Needleman SB and Wunsch CD (1970), "A general method applicable to the search for similarities in the amino acid sequence of two proteins," J.Mol.Biol., Vol. 48, No. 3: pp. 443-53, PubMed: 5420325, DOI: 10.1016 / 0022-2836(70)90057-4, is a suitable global alignment strategy and is utilized by tools such as EMBOSS Needle (www.ebi.ac.uk / Tools / psa / emboss_needle / ). Depending on the sequences being compared and the parameters involved, a local or global alignment strategy may be more likely to find the optimal alignment, although both strategies may be used to determine the optimal alignment that gives the most accurate percentage identity.

[0104] The polynucleotides provided herein (such as RNAi molecules that target plant genes involved in the suppression of systemic or local defense response pathways) are, in some embodiments, designed to have at least one silencing factor complementary to a segment of the mRNA sequence (e.g., fully (100%) or partially (less than 100%, e.g., 90%–99%) complementary). In some embodiments, the polynucleotide contains at least one silencing factor that is essentially identical or essentially complementary to the plant mRNA. In some embodiments, the polynucleotide contains 2–5, ~10, 2–20, 2–20, 2–40, or 2–50 silencing factors. In some embodiments, the polynucleotide comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 21, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 silencing factors.

[0105] RNAi molecules provided herein that target plant genes involved in the suppression of systemic or local defense response pathways may be any form of RNA, encompassing single-stranded RNA (ssRNA) and double-stranded RNA (dsRNA). Non-exclusive examples of single-stranded RNA include mRNA, microRNA (miRNA) (e.g., artificial miRNA (amiRNA)), small interfering RNA (siRNA), piwi-binding RNA (piRNA), and antisense RNA. Double-stranded RNA includes fully double-stranded molecules that do not contain a single-stranded region (e.g., loops or overhangs), as well as partially double-stranded molecules that contain both double-stranded and single-stranded regions (e.g., loops or overhangs). Furthermore, RNAi molecules may also be single-stranded RNA molecules with secondary structures that include significant double-stranded features, such as hairpin RNA, although these are not limited to these. Thus, RNAi molecules may, in some embodiments, be short hairpin RNA (shRNA).

[0106] In some embodiments, RNAi molecules targeting plant genes involved in the repression of systemic or local defense response pathways include dsRNA, ssRNA, siRNA, miRNA (e.g., amirRNA), piRNA, mRNA, or shRNA. In some embodiments, RNAi molecules targeting plant genes involved in the repression of systemic or local defense response pathways include more than one form of RNA. For example, RNAi molecules targeting plant genes involved in the repression of systemic or local defense response pathways may include ssRNA and dsRNA. In some embodiments, RNAi molecules targeting plant genes involved in the repression of systemic or local defense response pathways include a hybrid of RNA and DNA. In some embodiments, RNAi molecules targeting plant genes involved in the repression of systemic or local defense response pathways include amiRNA processed from a long precursor transcript of non-protein-coding RNA, which is partially self-complementary to mediate the silencing of target mRNA. In some embodiments, amiRNAs are designed by replacing the mature 21-nucleotide miRNA sequence in a miRNA precursor with a 21-nucleotide fragment derived from a target gene (Frontiers in Plant Science, Sebastian et al., 2017). The amiRNA may have lengths of, for example, at least 18–500 nucleotides, at least 21–500 nucleotides, at least 50–500 nucleotides, at least 100–500 nucleotides, or at least 200–500 nucleotides.

[0107] RNAi molecules targeting plant genes involved in the repression of systemic or local defense response pathways may be provided as a mixture of RNAi molecules targeting one or more plant genes involved in the repression of systemic or local defense response pathways, for example, a mixture of RNAi molecules targeting sequences of different plant genes or homologous genes. Any number of distinct RNAi molecules targeting plant genes involved in the repression of systemic or local defense response pathways may be provided in a mixture of RNAi molecules targeting one or more genes. In some embodiments, a mixture of RNAi molecules targeting plant genes involved in the repression of systemic or local defense response pathways comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 distinct RNAi molecules (having different sequence / nucleotide compositions) targeting sequences of different plant genes or homologous genes.

[0108] In some embodiments, RNAi molecules targeting plant genes involved in the repression of systemic or local defense response pathways are provided as a mixture of RNAi molecules that are (whole or partially) complementary to different segments of mRNA encoded by the same plant gene. Any number of RNAi molecules targeting plant genes involved in the repression of systemic or local defense response pathways that are complementary to different segments of mRNA (for example, sequences encoded by a gene containing any one of sequence numbers 1-86 or 265-274) may be provided in the mixture of RNAi molecules. In some embodiments, the mixture of RNAi molecules contains at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 RNAi molecules that target plant genes involved in the repression of systemic or local defense response pathways. In some embodiments, the mixture of RNAi molecules contains 2-5 or 2-10 RNAi molecules that target the same plant gene.

[0109] In some embodiments, RNAi molecules provided herein that target plant genes involved in the repression of systemic or local defense response pathways may have one or more mismatches compared to the corresponding sequence of mRNA. Complementary regions within RNAi molecules that target plant genes involved in the repression of systemic or local defense response pathways may have up to one, two, three, or four mismatches, as long as they maintain the ability to form complementary base pairs with mRNA under appropriate hybridization conditions. Alternatively, complementary regions on RNAi molecules may have one, two, three, or four or fewer mismatches, as long as the RNAi molecule maintains the ability to form complementary base pairs with mRNA under appropriate hybridization conditions. Complementary regions within RNAi molecules may have up to one, two, three, or four mismatches per 50, 75, or 100 nucleotides. For example, if an RNAi has a 100-nucleotide complementary region with a target gene, the complementary region within the RNAi may have up to 1, 2, 3, or 4 mismatches every 20, 30, 40, or 50 nucleotides (for example, the complementary region within the RNAi may have up to 20 mismatches across the 100-nucleotide complementary region).

[0110] In some embodiments, if there are more than one mismatch in the complementarity region, they may be arranged consecutively (e.g., two, three, four, or more consecutively), or they may be scattered throughout the complementarity region, as long as RNAi molecules targeting plant genes involved in the suppression of systemic or local defense response pathways maintain the ability to form complementary base pairs with mRNA under appropriate hybridization conditions.

[0111] RNAi molecules targeting plant genes involved in the repression of systemic or local defense response pathways may be modified in various ways to improve or control their specificity, stability, delivery, bioavailability, degradation, resistance to nuclease degradation, base pairing properties, RNA distribution, and cellular uptake, as well as other characteristics related to their use. For example, see Bramsen et al., Nucleic Acids Res., 2009, Vol. 37, pp. 2867-2881; Bramsen and Kjems, Frontiers in Genetics, Vol. 3 (2012): pp. 1-22. Accordingly, in some embodiments, RNAi molecules targeting plant genes involved in the repression of systemic or local defense response pathways may include one or more (at least one) appropriate modifications. In some embodiments, modified RNAi molecules targeting plant genes involved in the repression of systemic or local defense response pathways have modifications in their bases, sugars (e.g., ribose, deoxyribose), or phosphate groups.

[0112] RNAi molecules that target plant genes involved in the suppression of systemic or local defense response pathways, produced by the methods provided herein, may be modified as described herein. In some embodiments, RNAi molecules are produced according to the methods described herein and then modified. In some embodiments, RNAi molecules are produced according to the methods described herein using modified starting materials. In some embodiments, the modified starting materials are modified nucleic acid bases. In some embodiments, the modified starting materials are modified nucleosides. In some embodiments, the modified starting materials are modified nucleotides.

[0113] In some embodiments, modified RNAi molecules targeting plant genes involved in the suppression of systemic or local defense response pathways include skeletal modifications. In some embodiments, skeletal modifications result in a longer half-life of RNA (e.g., nuclease-mediated degradation) due to reduced degradation. This results in a longer half-life. Suitable skeletal modifications include, but are not limited to, phosphorothioate modifications, phosphorodithioate modifications, p-ethoxy modifications, methylphosphonate modifications, methylphosphothioate modifications, alkyl- and aryl-phosphonates (charged phosphonate oxygen is substituted with an alkyl or aryl group), alkylphosphotriesters (charged oxygen moiety is alkylated), peptide nucleic acid (PNA) skeletal modifications, and locked nucleic acid (LNA) skeletal modifications. These modifications may be used in combination with each other and / or in combination with phosphodiester linkages.

[0114] Alternatively or additionally, RNAi molecules targeting plant genes involved in the suppression of systemic or local defense response pathways may include other modifications, including modifications at the base or sugar moiety. Examples include RNA with sugars covalently attached to low molecular weight organic groups other than the 3'-hydroxyl group and the 5'-phosphate group (e.g., 2'-O-alkylated ribose), or RNA with sugars such as arabinose instead of ribose. RNA also includes substituted purines and substituted pyrimidines, such as C-5 propine-modified bases (Wagner et al., Nature Biotechnology, Vol. 14: pp. 840-844, 1996). Other purines and pyrimidines, but not limited to these, include 5-methylcytosine, 2-aminopurine, 2-amino-6-chloropurine, 2,6-diaminopurine, and hypoxanthine. Other such modifications are well known to those skilled in the art.

[0115] RNAi molecules containing nucleotide sequences complementary to all or a segment of the target sequence can be designed and prepared using any appropriate method.

[0116] How to use

[0117] Aspects of this disclosure provide methods for controlling pathogen infection, in some embodiments, that involve delivering an effective amount of RNAi molecules (or compositions comprising RNAi molecules that target plant genes involved in the suppression of systemic or local defense response pathways) to a plant. In some embodiments, the delivery of the RNAi molecules described herein activates or increases the activity of systemic or local defense response pathways in the plant (for example, as described in Conrath U. Systemic acquired resistance. Plant Signal Behav. July 2006; Vol. 1, No. 4: pp. 179-184; the contents of which are incorporated by reference). In some embodiments, the systemic defense response pathway is the systemic acquired resistance pathway.

[0118] Further aspects of this disclosure provide methods for increasing resistance to pathogens in plants, including activating systemic or local defense responses, or systemic or local defense response pathways, to pathogens prior to exposure of the plants to the pathogens. In some embodiments, delivery of RNAi molecules described herein increases the resistance of plants to pathogens by at least 10%, 20%, 30%, 40%, 50%, 60%, or 70% compared to controls.

[0119] Further aspects of this disclosure provide methods for increasing the lifespan of plants or the average lifespan of plant populations, or for increasing the biomass of plant populations or for increasing the yield of plant populations. In some embodiments, delivery of the RNAi molecules described herein to plants or plant populations increases the lifespan of plants by at least 10%, 20%, 30%, 40%, 50%, 60%, or 70% compared to controls. In some embodiments, delivery of the RNAi molecules described herein to plant populations increases the biomass of plant populations by at least 10%, 20%, 30%, 40%, 50%, 60%, or 70% compared to controls. In some embodiments, delivery of the RNAi molecules described herein to plant populations increases the yield of plant populations by at least 10%, 20%, 30%, 40%, 50%, 60%, or 70% compared to controls.

[0120] In some embodiments, RNAi molecules are delivered to plants, for example by delivery to seeds, before pathogen infection, and function to suppress the expression of genes involved in the suppression of systemic or local defense responses against pathogens, thereby activating systemic or local defense responses before pathogen infection; RNAi molecules are delivered before or after pathogen infection, resulting in an increase in the plant's ability to control pathogens. In some embodiments, RNAi molecules function to suppress the expression of genes involved in the suppression of systemic or local defense responses against pathogens, thereby resulting in increased expression of genes involved in the activation or increased activity of systemic or local defense response systems, resulting in an increase in the plant's ability to control pathogens.

[0121] In this context, “control” or “controlling” includes, but is not limited to, increasing mortality, inhibiting growth, inhibiting the spread of a plant from one area to another, or reducing toxicity or pathogenicity, or reducing the pathogen’s ability to proliferate / regenerate (spore formation). In some aspects, the pathogen is a fungal pathogen. In some aspects, the fungal pathogen is a fungus belonging to the Fusarium or Phytophthora families.

[0122] In some aspects, fungal pathogens include Albugo spp. (white rust) in ornamental plants, vegetables (e.g., A. candida) and sunflowers (e.g., A. tragopogonis); Alternaria spp. (alternaria leaf spot) in vegetables, rapeseed (A. brassicola or brassicae), sugar beet (A. tenuis), fruits, rice, soybeans, potatoes (e.g., A. solani or A. alternata), tomatoes (e.g., A. solani or A. alternata) and wheat; Aphanomyces spp. in sugar beet and vegetables; Ascochyta spp. in cereals and vegetables, e.g., A. tritici (anthracnose) in wheat and A. hordei in barley; Bipolaris spp. and Drechslera spp. (teleomorph: Cochliobolus) (spp.), for example, sesame leaf spot (D. maydis) or maize sooty spot (B. zeicola) in maize, for example, leaf spot (B. sorokiniana) in cereals, and for example, B. oryzae in rice and turf; Blumeria (formerly Erysiphe) graminis (powdery mildew) in cereals (for example, wheat or barley); Botrytis cinerea (teleomorph: Botryotinia fuckeliana: gray mold) in fruits and berries (for example, strawberries), vegetables (for example, lettuce, carrots, celery and cabbage), rapeseed, flowers, grapes, forestry plants and wheat; Bremia in lettuce lactucae (downy mildew); Ceratocystis (syn. Ophiostoma) spp. (rot or wilt) in broad-leaved and evergreen trees, e.g., C. ulmi (elm wilt) in elm; corn (e.g., gray leaf spot: C. zeae-maydis), rice, sugar beet (e.g., C. beticola), sugarcane, vegetables, coffee, soybean (e.g., C. sojina or C. kikuchii) and Cercospora spp. (Cercospora leaf spot) in rice; tomato (e.g., C.Cladosporium (leaf mold) and in cereals spp., e.g., C. herbarum in wheat; ergot in cereals; Cochliobolus (asexual generation: Helmintosporium of the genus Vipolaris) spp. (leaf spot) in maize (C. carbonum), cereals (e.g., C. sativus, asexual generation: B. sorokinana), and rice (e.g., C. miyabeanus, asexual generation: H. oryzae); Colletatrichum (teleomorph: Glomerella) spp. (anthrax) in soft fruits, potatoes (e.g., C. coccodes, black spot), beans (e.g., C. lindemuthianum), and soybeans (e.g., C. truncatum or C. gleosporioides); Corticium spp., for example C. sasakii (sheath blight) in rice; Corynespora cassiicola (leaf spot) in soybeans and ornamental plants; Cycloconium spp., for example C. oleaginum in olive trees; and Cyclindrocarpon spp. in fruit trees, grapes (for example C. liriodendri, sexual generation: Neonectria liriodendri: blackfoot) and ornamental plants (for example fruit tree gall or young grape wilt, sexual generation: Nectria spp. or Neonectria spp.); Dematophora (sexual generation: Rosellinia) Necatris (root rot and stem rot) in soybeans; Diaporthe fungi, e.g., D. phaseolorum (damping-off) in soybeans; cereals such as maize, barley (e.g., D. teres, net spot disease) and wheat (e.g., D. tritici-repentis: yellowish-brown spot disease), rice, and turfgrass; Drechslera (syn. Helminthosporium, sexual generation: Pyrenophora) spp.; Formitiporia (syn. Phellinus) punctata, F.Erysiphe spp. (powdery mildew) in grapes, caused by mediterranea, Phaeomoniella chlamydospora (formerly known as Phaeoceramonium chlamydosporum), Phaeoceramonium aleophilum and / or Botryosphaeria obtusa; Elsinoe spp. in pome fruits (E. pyri), soft fruits (E. veneta: anthracnose) and grapes (E. ampelina: anthracnose); Entyloma oryzae (leaf spot disease) in rice; Epicoccum spp. (black mold) in wheat; Erysiphe spp. (powdery mildew) in vegetables such as sugar beet (E. betae), cucurbits (e.g., E. cichoracearum), cabbage, rapeseed (e.g., E. cruciferarum) (e.g., E. pisi); and Eutypa in fruit trees, grapes and ornamental woods. Fusarium (sexual generation: Gibberella) spp. (wilting, root rot, or stem rot) in various plants, including F. graminearum or F. culmorum (root rot, scab, or ear blight) in cereals (e.g., wheat or barley), F. oxysporum in tomatoes, F. solani (f. sp. glycine, now syn. F. virguliforme), F. tucumaniae, and F. brasiliense, which cause sudden death syndrome in soybeans, respectively, as well as F. verticillioides in maize; Gaeumannomyces in cereals (e.g., wheat or barley) and maize. Graminis (damping-off disease); Gibberella spp. in cereals (e.g., G. zeae) and rice (e.g., G. fujikuroi: bakanae disease).;Glomerella cingulata in grapes, pome fruits and other plants, and G. gossypii in cotton; cereal staining complex in rice; Guignardia bidwellii (black rot) in grapes and juniper; Gymnosporangium spp. in Rosaceae plants, e.g., G. sabinae (rust) in pears; Helminthosporium spp. (syn. Drechslera, sexual generation: Cochliobolus) in maize, cereals and rice; Hemileia spp., e.g., H. vastatrix (coffee leaf rust) in coffee; Isariopsis clavispora (syn. Cladosporium vitis) in grapes; Macrophomina in soybeans and cotton Phaseolina (syn. phaseoli) (root rot and stem rot); Microdochium (syn. Fusarium) nivale (red snow mold) in cereals (e.g., wheat or barley); Microsphaera diffusa (powdery mildew) in soybeans; Monilinia spp., e.g., M. laxa, M. fruticola and M. fructigena (flower leaf blight and branch leaf blight, brown rot) in drupes and other Rosaceae plants; Mycosphaerella spp., e.g., M. graminicola (asexual generation: Septoria) in cereals, bananas, soft fruits and peanuts, e.g., M. graminicola (asexual generation: Septoria) in wheat Examples include P. tritici (Septoria spot) or M. fijiensis (black sigatoka disease) in bananas; Peronospora spp. (downy mildew) in cabbage (e.g., P. brassicae), rapeseed (e.g., P. para-sitica), onion (e.g., P. destructor), tobacco (P. tabacina) and soybean (e.g., P. manshurica); Phakopsora pachyrhizi and P. meibomiae (soybean rust) in soybeans; for example, grapes (e.g., P. tracheiphila and P. tetraspora) and soybeans (e.g., P.Phytophthora in various plants such as Phialophora spp. in gregata (stem rot); Phoma lingam (root rot and stem rot) in rapeseed and cabbage, and P. betae (root rot, leaf spot, and damping-off) in sugar beet; Phhomopsis spp. in sunflowers, grapes (e.g., P. viticola: can and leaf spot) and soybeans (e.g., stem rot: P. phaseoli, sexual generation: Diaporthe phaseolorum); Physoderma maydis (brown spot) in maize; and Phytophthora in various plants such as paprika and cucurbits (e.g., P. capsici), soybeans (e.g., P. megasperma, syn. P. sojae), potatoes and tomatoes (e.g., P. infantans: leaf blight) and broad-leaved trees (e.g., P. ramorum: oak sudden death). spp. (wilting disease, roots, leaves, fruits and stem roots); Plasmodiophora brassicae (clubroot) in cabbage, rapeseed, radish and other plants; Plasmopara spp., e.g., P. viticola (grape downy mildew) in grapes and P. halstedii in sunflowers; Podosphaera spp. (powdery mildew) in rose plants, hops, pome fruits and soft fruits, e.g., P. leucotricha in apples; Polymyxa spp., e.g., in cereals such as barley and wheat (P. graminis) and sugar beet (P. betae), causing infectious viral diseases; Pseudocercosporella herpotrichoides (eye spot disease, sexual generation: Tapesia) in cereals. Pseudoperonospora (downy mildew) in various plants, for example P.cubensis in cucurbitaceae or P.humili in hops; Pseudopezicula tracheiphila (red spot disease or red burn disease, asexual generation: Phialophora) in grapes; Puccinia spp. (rust disease) in various plants, for example P. in cereals such as wheat, barley or rye.P. triticina (brown rust or leaf rust), P. striiformis (stripe rust or yellow rust), P. hordei (small rust), P. graminis (stem rust or black rust) or P. recondita (brown rust or leaf rust), P. kuehnii (orange rust) in sugarcane and P. asparagi in asparagus; Pyrenophora (asexual generation: Drechslera) tritici-repentis (yellowish-brown spot) in wheat or P. teres (net-like spot) in barley; Pyricularia spp., for example, P. oryzae (sexual generation: Magnaporthe) in rice. P. grisea (rice blight) and P. grisea in turfgrass and cereals; Pythium spp. (damping-off disease) in turfgrass, rice, maize, wheat, cotton, rapeseed, sunflower, soybean, sugar beet, vegetables and various other plants (e.g., P. ultimum or P. aphanidermatum); Ramularia spp., e.g. R. collo-cygni (Ramularia leaf spot disease, physiological leaf spot disease) in barley and R. beticola in sugar beet; Rhizoctonia spp. in cotton, rice, potato, turfgrass, maize, rapeseed, potato, sugar beet, vegetables and various other plants, e.g. R. solani (root rot and stem rot) in soybean, R. solani (sheath blight) in rice or R. cerealis (Rhizoctonia spring blight) in wheat or barley; Rhizopus in strawberries, carrots, cabbage, grapes and tomatoes Stolonifer (black mold, soft rot); Rhynchosporium secalis (scorched disease) in barley, rye, and rye; Sarocladium oryzae and S. attenuatum (leaf sheath rot) in rice; Sclerotinia spp. in vegetables and crops such as rapeseed, sunflower (e.g., S. sclerotiorum) and soybean (e.g., S. rolfsii or S. sclerotiorum).(Stem rot or white mold); Septoria spp. in various plants, e.g., S. glycines (brown spot) in soybeans, S. tritici (Septoria spot) in wheat, and S. (syn. Stagonospora) nodorum (Stagonospora spot) in cereals; Uncinula (syn. Erysiphe) necator (powdery mildew, asexual generation: Oidium tuckeri) in grapes; Setospaeria spp. (leaf blight) in maize (e.g., S. turcicum, syn. Helminthosporium turcicum) and turfgrass; Sphacelotheca spp. (smut) in maize, (e.g., S. reiliana: head smut), sorghum, and sugarcane; Sphaerotheca fuliginea (powdery mildew) in cucurbits; Spongospora in potatoes subterranea (powdered crust disease) and the infectious viral diseases caused by it; Stagonospora spp. in cereals, e.g., S. nodorum in wheat (Stagonospora spot disease, sexual generation: Leptosphaeria [syn. Phaeosphaeria] nodorum); Synchytrium endobioticum in potatoes (potato wart disease); Taphrina spp., e.g., T. deformans in peaches (leaf curl disease) and T. pruni in plums (plum pocket disease); Thielaviopsis spp. in tobacco, pomelo, vegetables, soybeans and cotton (black root rot), e.g., T. basicola (syn. Chalara elegans); Tilletia in cereals, e.g., T. tritici (syn. T caries, wheat baccata) and T. cantraversa (miniature baccata) in wheat. spp. (common smut or foul-smelling smut); Typhula incarnata (gray snow mold) in barley or wheat; Urocystis spp., e.g., U. occulta (stem smut) in rye; legumes (e.g., U. appendiculatus, syn. U.Uromyces spp. (rust) in vegetables such as phaseoll and sugar beet (e.g., U. betae); Ustilago spp. (naked smut) in cereals (e.g., U. nuda and U. avaenae), maize (e.g., U. maydis corn smut) and sugarcane; Venturia spp. (scab) in apples (e.g., V. inaequalis) and pears; and Verticillium spp. (wilting disease) in various plants such as fruit and ornamental plants, grapes, soft fruits, vegetables and crops, e.g., strawberries. This is V. dahliae, found in rapeseed, potatoes, and tomatoes.

[0123] In some embodiments, the delivery method involves applying a composition containing RNAi molecules to the surface of a plant. In some embodiments, compositions containing RNAi molecules that target plant genes involved in the suppression of systemic or local defense response pathways are solids, liquids (including homogeneous mixtures such as soluble liquid concentrates, as well as heterogeneous mixtures such as suspensions, colloids, micelles, and emulsions), powders, suspensions, emulsions, sprays, encapsulated or microencapsulated formulations, microbeads or other carrier particles, in or on films or coatings, on or within a matrix, or treatment of leaves, seeds, roots, or stems. In some embodiments, the surface is a plant leaf, flower, or fruit. In some embodiments, the compositions containing RNAi molecules described herein include carbon quantum dots. Carbon quantum dots are luminescent nanoparticles containing carbon that can impart chiral properties. Carbon quantum dots may have low cytotoxicity and be easily synthesized. In some embodiments, carbon quantum dots are as described in Hu et al., "High efficiency transport of quantum dots into plant roots with the aid of silwet L-77," Plant Physiology and Biochemistry, Vol. 48: pp. 703-709 (2010). Carbon quantum dots may be useful for the delivery of RNAi molecules to plants (e.g., to plant seeds or leaves). Carbon dots can be prepared by any method known in the art, including, for example, U.S. Patent Application Publication No. 2020 / 0385748 or Hendrix, B. et al. (2021), "Systemic GFP silencing is associated with high transgene expression in Nicotiana benthamiana," PLOS ONE, edited by KRDavis, Vol. 16, No. 3, p.e0245422.

[0124] In some embodiments, the composition further comprises a water-removing agent. In some embodiments, the composition comprises lipid nanoparticles. In some embodiments, the composition comprises polyethylene glycol (PEG) molecules. The PEG molecules may be PEG200, PEG500, PEG1000, PEG2500, PEG5000, or any other reasonable PEG molecule.

[0125] In some embodiments, the composition comprises formulation components described in International Publication No. 2022 / 235895, which are incorporated herein by reference. Such formulation components may, for example, play a role in increasing the storage stability of nucleic acids to inhibit systemic or local defense response pathways in plants.

[0126] Non-limiting examples include emulsions, concentrated solutions, low-concentration solutions, very low-volume concentrated solutions, water-soluble concentrated solutions, water-soluble liquid solutions, smoke, mist, reverse emulsions, flowables, aerosols, homogeneous and heterogeneous mixtures, suspensions (water and oil-based), dust, powders (wettable or soluble), granules (water-dispersible or dry-flowable), pellets, capsules, fumigants, encapsulated or microencapsulated formulations, or any combination thereof.

[0127] In some embodiments, compositions containing RNAi molecules may be applied as concentrates, sprays (after dilution or concentration), mist, infallow, seed treatment, seed coating, drenching, dripping, or any other form suitable for application between rows. RNAi molecules described herein, but not limited to, may be delivered to any part of the plant including leaves, stems, flowers, fruits, shoots, roots, seeds, tubers, anthers, stamens, and / or pollen. In some embodiments, RNAi is delivered mechanically via high-pressure spraying or sandblasting.

[0128] In some embodiments, RNAi molecules are delivered to seeds using seed immersion techniques. Seed immersion techniques may be as described in International Publication No. 2013175480; U.S. Patent Nos. 10,240,161; U.S. Patent Nos. 10,240,162; U.S. Patent No. 10,934,555; or U.S. Patent No. 10,683,505; the contents of each thereof are incorporated herein by reference. In some embodiments, seed immersion techniques are as described in Halmer, P., Methods to improve seed performance in the field, Handbook of Seed Physiology: Applications to Agriculture; The Haworth Press, New York (2004), Chapter 5; pp. 125-166; the contents of which are incorporated herein by reference. In some embodiments, seed immersion is carried out via methods described in the examples herein.

[0129] An exemplary seed immersion method involves treating healthy, uniform seeds with a test formulation diluted in water (for example, with approximately 30 seeds per 8 ml of diluted formulation, resulting in a working concentration of dsRNA of 0.2 mg / ml). The seeds are then incubated for a certain period (for example, about 12 to 24 hours) in the absence of light (and optionally with slow stirring or shaking). After treatment, the seeds may be placed in a dish, air-dried (for example, for 20 to 30 minutes), and then planted in soil.

[0130] In some embodiments, RNAi molecules are delivered to plants by spraying them onto above-ground plant parts, such as leaves, stems, fruits, vegetables, or flowers. Formulations known in the art that are suitable for spraying may be used. These include, for example, the formulations and methods described in U.S. Patent Application Publication 2020 / 0385748 or Hendrix, B. et al. (2021), "Systemic GFP silencing is associated with high transgene expression in Nicotiana benthamiana," PLOS ONE, edited by KRDavis, Vol. 16, No. 3, p.e0245422, available at https: / / doi.org / 10.1371 / journal.pone.0245422.

[0131] In some embodiments, the composition comprises an RNAi molecule and at least one additive selected from adjuvants, attractants, sterilizers, growth regulators, carriers or diluents, stabilizers, and / or pesticides (one or more) (e.g., insecticides, fungicides, and / or herbicides). Formulation components may also be used (e.g., adjuvants such as defoamers, buffers, compatibilizers, drift control additives, emulsifiers, fillers, inversion emulsifiers, plant penetration agents, toxicity mitigators, spreading agents, adhesives, surfactants, thickeners, and wetting agents).

[0132] In some embodiments, the composition comprises an RNAi molecule that targets plant genes involved in the suppression of systemic or local defense response pathways, and at least one of various fertilizers, pesticides, insecticides, acaricides, fungicides, pesticides and / or biochemical agents (e.g., microorganisms, plant-integrated protective agents (PIPs), and / or biochemical agents) (spiromesifen, spirodiclofen, spirotetramat, pyridaben, tebufenpyrad, tolfenpyrad, fenpyroximate, fluphenerim, pyrimidifen, phenazaquin, rotenone, cyenopyroximate). Rafen, hydramethylnon, acequinosyl, fluacrypyrim, aluminum phosphide, calcium phosphide, phosphine, zinc phosphide, cyanide, diafenthiuron, azocyclotin, cyhexatine, fenbutatine oxide, propargit, tetradiphon, bensultap, thiocyclam, thiosultap sodium, flonicamide, etoxazole, clofentezine, diflovidazine, hexythiazox, chlorfluazuron, bistriflurone, diflubenzuron, flucycloxuron, flufenoxuron, hexaflumuron, lufenu Ron, Novalon, Noviflumuron, Teflubenzuron, Triflumuron, Buprofezin, Cyromazine, Hydroprene, Quinoprene, Methoprene, Phenoxycarb, Pyriproxyfen, Pymetrozine, Pyrifluquinazone, Chlorfenapyr, Tralopyril, Methyl bromide and / or other alkyl halides, Chloropicrin, Sulfuryl fluoride, Bencrotiaz, Quinomethionate, Cryolite, Methylneodecanamide, Benzoximate, Simiazole, Fluensulfone, Azadirachtin, Bifenazate, Amidoflumet, Dicofor, Pry Phenat, Cyflumetofen, Pyridaryl, Beauveria Basiana GHA, Sulfoxaflor, Spinetoram, Spinosad, Spinosad, Emamectin Benzoate, Lepimectin, Milbemectin, Abamectin, Methoxyfenozide, Chromafenozide, Halofenozide, Tebufenozide, Amitraz, Chlorantraniliprole, Cyantraniliprole, Flubendiamide, Alpha-Endosulfan, Chlordane, Endosulfan, Fipronil, Acetoprole, Ethiprole, Pyrafluprole, Pyriprole, Indoxacarb,Metaflumizone, Acrinatrin, Arethrin, Arethrin-cis-trans, Arethrin-trans, Beta-cyfluthrin, Beta-cypermethrin, Bifenthrin, Bioarethrin, Bioarethrin S-cyclopentenyl, Bioresmethrin, Cycloprotrin, Cyfluthrin, Cyhalothrin, Cypermethrin, Cyphenothrin [(1R)-trans-isomer], Dimefluthrin, Empenthrin [(EZ)-(1R)-isomer], Esfenvalerate, Etofenprox, Fenpropathrin, Fenvalerate, Flucitrinate Flumethrin, gamma-cyhalothrin, lambda-cyhalothrin, meperfluthrin, metofluthrin, permethrin, phenothrin [(1R)-trans-isomer], prallethrin, profluthrin, protrefenbut, resmethrin, silafluofen, tau-fluvalinate, tefluthrin, tetramethrin, tetramethrin [(1R)-isomer], tetramethylfluthrin, teta-cypermethrin, tralomethrin, transfluthrin, zeta-cypermethrin, alpha-cypermethrin, deltamethrin, DDT, methoxychlor, Thiodicarb, alanicarb, aldicarb, bengiocarb, benfuracarb, butoxycarboxime, carbaryl, carbofuran, carbosulfan, ethiofencarb, phenobucarb, formethaneate, furathiocarb, isoprocarb, methiocarb, methomyl, metholcarb, oxamyl, pyrimicarb, propoxul, thiophanox, triazamate, trimetacarb, XMC, xylylcarb, chlorpyrifos, malathion, acephate, azamethifos, azinphosethyl, azinphosmethyl, kazusafos, chlorethoxyfos Chlorphenvinphos, Chlormephos, Chlorpyrifos-methyl, Coumaphos, Cyanophos, Demeton-S-methyl, Diazinon, Dichlorvos / DDVP, Diclotophos, Dimethoate, Dimethylvinphos, Disulfon, EPN, Ethion, Etoprophos, Famfur, Phenamiphos, Fenitrothion, Fenthion, Honophos, Fothiazate, Imisiaphos, Isofenphos-methyl, Mecarbam, Metamidophos, Methidathion, Mevinphos, Monoclotophos, Nared, Omethoate, Oxydemeton-methyl, Parathion, Parathion-methyl,Fentart, phorate, phosalon, phosmet, phosphamidone, foxim, pyrimiphosethyl, profenofos, propafos, propetamphos, prothiofos, pyraclofos, pyridaphenthion, quinalfos, sulfotep, tebupyrimfos, temefos, terbufos, tetrachlorvinfos, thiometon, triazofos, trichlorfon, bamidothion, imidacloprid, thiamethoxam, acetamiprid, clothianidin, dinotefuran, nitenpyram, nicotine, thiacloprid, cyantraniliprole, carbamate, ol Ganophosphate, cyclodiene organochlorin, phenylpyrazole (fiproles), pyrethroid, pyrethins, DDT methoxychlor, neonicotinoid, nicotine, sulfoxymine, butenolide, mesoionic compounds, spinosin, ivermectin, milbernycins, juvenile hormone analog, phenoxycarb, pyriproxyfen, alkyl halides, chloropicrin, fluoride, boric acid, tartaric acid, methyl isothiocyanate generator, pyridineazomethine inducer Body, pyropen, clofentidine, diflovidazine, hexythiazox, etoxazole, diafenthiuron, organotin acaricides, propargit, tetradiphon, pyrrole, dinitrophenol, sulfuramide, Nereis toxin analog, benzoylurea, buprofezin, ciromazine, diacylhydrazine, amitraz, hydramethylnon, acekinosyl, fluacrypyrim, bifenazate, METI acaricides and insecticides, rotenone, oxadiazine, semicarbazone, tetronic acid derivatives and tetramic acid derivatives, phosphides, cyanides, ba Taketonitrile derivatives, carboxanilide, diamide, flonicamide, metadiamide isoxazoline, granulovirus (GV), nuclear polyhedron virus (NPV), GS-omega / kappa HXTX-Hv1a peptide, azadirachtin, benzoximate, bromopropylate, quinomethionate, dicofol, lime sulfur mixture, mancozeb, pyridaryl, sulfur, benzimidazole, dicarboximide, pyridine, pyrimidine, triazole, acylalanine, pyridinecarboxamide, anilinopyrimidine, quinone external inhibitors (QoI-fungicides),Phenylpyrrole, quinoline, hydroxyanilide, toluamide, cyanoacetamidooxime, dinitrophenyl chloronate, phosphonate, carboxylic acid amide (CAA - fungicide), M1 inorganic, M2 inorganic, M3 dithiocarbamate, M4 phthalimide, paraffin oil, petroleum-based horticultural oil, palmitic acid oil, stearic acid oil, linoleic acid oil, oleic acid oil, canola oil, soybean oil, oregano oil, tagetes oil, balsa Mufā oil, thyme oil, black pepper oil, mint oil, cedarwood oil, fish oil, jojoba oil, lavandin oil, castor oil, eucalyptus oil, osimum oil, patchouli oil, citrus oil, mugwort oil, camphor oil, wintergreen oil, methyl eugenol oil, thymol oil, geranium oil, sesame oil, linseed oil, cottonseed oil, lemongrass oil, bergamot oil, mustard oil, orange oil, citronella oil, tea tree oil, neem oil, garlic oil, Bacillus sphaericus, Bacillus thuringiensis (for example, Bacillus thuringiensis var. aizawai, Bacillus thuringiensis var. israelensis, Bacillus thuringiensis var. kurstaki, Bacillus thuringiensis var. sphaericus, Bacillus thuringiensis var. This includes mixtures of tenebrionensis) and the insecticidal proteins they produce (for example, Cry1Ab, Cry1Ac, Cry1Fa, Cry1A.105, Cry2Ab, Vip3A, mCry3A, Cry3Ab, Cry3Bb, Cry34Ab1 / Cr35Ab1, and as further illustrated in Crickmore, N., Baum, J., Bravo, A., Lereclus, D., Narva, K., Sampson, K., Schnepf, E., Sun, M. and Zeigler, DR "Bacillus thuringiensis toxin nomenclature" (2018)). Paenibacillus popilliae, Serratia entomophila, nuclear polyhedron virus, granulosis virus, unconfined baculovirus, Beauveria spp, Metarhizium,Entomophaga, Zoopthora, Paecilomyces fumosoroseus, Normuraea, Lecanicillium lecanii, Nosema, Thelohania, Varimorpha, Steinernema spp, Heterorhabditis spp, or any combination thereof, including azinophosmethyl, acephate, isoxathion, isofenphos, ethione, etrimphos, oxydemetonmethyl, oxydeprophos, quinalphos, chlorpyrifos, chlorpyrifosmethyl, chlorfenvinphos, cyanophos, dioxabenzophos, dichlorvos, disulfone, dimethylvinphos, dimethoate, sulprophos, diazinon, thiometon, tetrachlorvinphos, temephos, tebupyrimphos, te Rubuphos, Nared, Bamidthion, Pyraclophos, Pyridafenthion, Pirimiphosmethyl, Fenitrothion, Fenthion, Fenthoate, Flupyrazophos, Prothiophos, Propaphos, Profenophos, Foxim, Hosalon, Fosmet, Formotion, Phorate, Malathion, Mecarbam, Mesulfenphos, Metamidophos, Methidathion, Parathion, Methylparathion, Monoclotophos, Trichloroulfone, EPN, Isazofos, Isamidophos, Kazusaphos Diamidaphos, diclofenthion, thionazine, phenamiphos, fostiazate, phostiethane, phosphocarb, DSP, etoprophos, alanicarb, aldicalb, isoprocarb, ethiofencarb, carbaryl, carbosulfan, xylylcarb, thiodicarb, pyrimicarb, phenobucarb, furathiocarb, propoxul, bengiocarb, benfuracarb, methomyl, metolcarb, XMC, carbofuran, aldoxycarb, oxamyl, acrin Thrin, Arethrin, Esfenvalerate, Empenthrin, Cycloprothrin, Cyhalothrin, Gamma-Cyhalothrin, Lambda-Cyhalothrin, Cyfluthrin, Beta-Cyfluthrin, Cypermethrin, Alpha-Cypermethrin, Zeta-Cypermethrin, Silafluofen, Tetramethrin, Tefluthrin, Deltamethrin, Tralomethrin, Bifenthrin, Phenothrin, Fenvalerate, Fenpropathrin, Flamethrin, Prallethrin, Flucitrinate,Fluvalinate, flubrocitrinate, permethrin, resmethrin, etofenprox, cartap, thiocyclam, bensultap, acetamiprid, imidacloprid, clothianidin, dinotefuran, thiacloprid, thiamethoxam, nitenpyram, chlorfluazurone, diflubenzuron, teflubenzuron, triflumulone, novaron, nobiflumulone, bistriflurone, fluazurone, flucycloxurone, flufenoxurone, hexaflumurone, lufenuron, chromafenozide, tebufenozide, halofenozide, methoxyfenozide, diofenolan, cyromazine, Pyriproxyfen, buprofezin, methoprene, hydroprene, quinoprene, triazamate, endosulfan, chlorfensone, chlorobenzylate, dicofor, bromopropylate, acetoprole, fipronil, ethiprole, pyrethrin, rotenone, nicotinic sulfate, spinosad, fipronil, spirotetramat, abamectin, acekinosyl, amidoflumet, amitraz, etoxazole, quinomethionate, clofentezine, fenbutatine oxide, dienochlor, cyhexatine, spirodiclofen, spiromesifen, tetradiphon, tebufenpyrad, bina, Pacryl, bifenazate, pyridaben, pyrimidifen, phenazaquin, phenothiocarb, fenpiroximate, fluacrypyrim, fluazinam, flufenzin, hexythiazox, propargit, polynactin complex, milbemectin, lufenuron, mecarbam, methiocarb, mevinphos, halfenprox, azadirachtin, diafenthiuron, indoxacarb, emamectin benzoate, potassium oleate, sodium oleate, chlorfenapyr, tolfenpyrad, pymetrozine, phenoxycarb, hydra Methylnon, hydroxypropyl starch, pyridaryl, fluphenelim, flubendiamide, flonicamide, metaflumizole, lepimectin, TPIC, albendazole, oxybendazole, oxfendazole, triclamide, fensulfothione, fenbendazole, levamisol hydrochloride, morantel tartrate, dazomet, metam sodium, triadimephon, hexaconazole, propiconazole, ipconazole, prochloraz, triflumizole, tebuconazole, epoxyconazole, difenoconazole, flusilazole, Liadimenol, cyproconazole, metconazole, fluquinconazole, vitertanol, tetraconazole, triticonazole, flutriafole, penconazole, diniconazole, fenbuconazole, bromconazole, imibenconazole, simeconazole, mycrobutanil, himexazole, imazalil, flametopyr, tifluzamide, etridiazole, oxpoconazole, oxpoconazole fumarate, pefurazoate, prothioconazole, pyrifenox, phenalimol, nualimol, bupirimate, mepanipyrim, cypro Dinyl, pyrimethanil, metalaxyl, mephenoxam, oxadixyl, benalaxyl, thiophanate, thiophanate-methyl, benomyl, carbendazim, fuberidazole, thiabendazole, mancozeb, propineb, zineb, methylam, maneb, dilam, thiram, chlorothalonil, etaboxam, oxycarboxyne, carboxyne, flutolanil, silthiofam, mepronil, dimethomorph, fenpropidine, fenpropimorph, spiloxamine, tridemorph, dodemorph, flumorph, azoxystrobin, kresoxim-methyl,Metminostrobin, Orysastrobin, Fluoxastrobin, Trifloxystrobin, Dimoxystrobin, Pyraclostrobin, Picoxystrobin, Iprodione, Procymidone, Vinclozoline, Clozolinate, Fursulfamide, Dazomet, Methylisothiocyanate, Chloropicrin, Metasulfocarb, Hydroxyisoxazole, Potassium Hydroxyisoxazole, Eclomazole, DD, Carbam, Basic Copper Chloride, Basic Copper Sulfate, Copper Nonylphenolsulfonate, Oxine Copper, DBEDC, Anhydrous Copper Sulfate, Copper Sulfate Pentahydrate, Cupric hydroxide, inorganic sulfur, hydrated sulfur, lime sulfur, zinc sulfate, fentin, sodium bicarbonate, potassium bicarbonate, sodium hypochlorite, silver, edifenphos, tolclophosmethyl, fosetyl, iprofenphos, dinocap, pyrazophos, carpropamide, fusalide, tricyclazole, pyroquilon, diclocimet, phenoxanil, kasugamycin, validamycin, polyoxin, blastosidine S, oxytetracycline, mildiomycin, streptomycin, rapeseed oil, machine oil, benthiavalicarb isopropyl, iprofalica Lube, propamocarb, diethofencarb, fluoroimide, fludioxanil, fenpiclonil, quinoxyfen, oxolinic acid, chlorothalonil, captan, forpet, probenazole, acibenzoral-S-methyl, thiadinil, cyflufenaamide, fenhexamide, diflumetrim, metraphenone, picobenzamide, proquinazide, famoxadone, cyazofamide, phenamide, zoxamide, boscalid, cymoxanil, dithianone, fluazinam, diclofluanid, triforine, isoprothiolane, felimzon, dik The active ingredient may further be selected from the group consisting of lomedine, tecrophthalam, pencyclon, quinomethionate, iminoctadine acetate, iminoctadine albesilate, ambam, polycarbamate, thiadiazine, chloroneb, nickel dimethyldithiocarbamate, guazatin, dodecylguanidine acetate, quintozen, tolfluanide, anirazine, nitrotar isopropyl, fenitropan, dimethylmol, bentazole, flumetova, mandipropamide, and penthiopyrad, or any combination thereof.

[0133] In some embodiments, RNAi molecules that target plant genes involved in the suppression of systemic or local defense response pathways may be applied topically to plants or seeds (e.g., via immersion, coating, dusting, or spraying), or plant cells may be manipulated to express RNAi molecules.

[0134] The plant may be any plant that is a common plant crop. In some aspects, the plant is a plant of the Solanaceae family (for example, the Solanaceae family). Examples of plants of the Solanaceae family include, but are not limited to, potato plants (Solanum tuberosum), black nightshade plants (Solanum rostratum), eggplant plants (Solanum melongena), tomato plants (Solanum lycopersicum), tobacco plants (Nicotiana tabacum), chili pepper plants (Capsicum annum), and woody nightshade plants (Solanum dulcamara). In some aspects, the plant is a vegetable, fruit, or legume. In some aspects, the plant is soybean, spinach, lettuce, cauliflower, pea, or rice.

[0135] In some embodiments, the plants are Abelmoschus esculentus, Abelmoschus manihot, Abutilon theophrasti, Acca sellowiana, Acer saccharum, Acmella oleracea, Actinidia arguta, Actinidia chinensis, Actinidia deliciosa, Actinidia kolomikta, Actinidia polygama, Adansonia digitata, Aeschynomene americana, Aframomum melegueta, Agave tequilana, Agropyron cristatum, Allium ampeloprasum, Allium cepa, Allium chinense, Allium fistulosum, Allium ledebourianum, Allium macrostemon, Allium ramosum, Allium sativum, Allium schoenoprasum, Allium scorodoprasum, Allium tuberosum, Alocasia cucullata, Alocasia macrorrhizos, Alpinia galanga, Alpinia globosa, Alpinia malaccensis, Alpinia officinarum, Amaranthus caudatus, Amaranthus cruentus, Amaranthus dubius, Amaranthus hybridus, Amaranthus hypochondriacus, Amaranthus tricolor, Amelanchier alnifolia, Amelanchier canadensis, Ammi majus, Amomum compactum, Amomum verum, Amorphophallus harmandii, Amorphophallus konjac, Amorphophallus paeoniifolius, Amphicarpaea bracteata, Anacardium occidentale, Ananas comosus, Ananasparguazensis, Anethum graveolens, Angelica archangelica, Angelica japonica, Annona cherimola, Annona macroprophyllata, Annona montana, Annona muricata, Annona purpurea, Annona reticulata, Annona scleroderma, Annona squamosa, Anredera baselloides, Anredera cordifolia, Anthoxanthum odoratum, Anthriscus cerefolium, Antidesma bunius, Apios americana, Apium carvi, Apium graveolens, Arachis glabrata, Arachis hypogaea, Arachis villosulicarpa, Aralia cordata, Archidendron bigeminum, Archidendron jiringa, Arctium lappa, Areca catechu, Arenga pinnata, Argania spinosa, Armoracia rusticana, Arracacia xanthorrhiza, Arrhenatherum elatius, Artocarpus altilis, Artocarpus camansi, Artocarpus heterophyllus, Artocarpus integer, Artocarpus lacucha, Artocarpus rigidus, Asclepias syriaca, Asimina triloba, Asparagus officinalis, Atriplex hortensis、Avena abyssinica、Avena sativa、Averrhoa bilimbi、Averrhoa carambola、Baccaurea dulcis、Baccaurea motleyana、Baccaurea racemosa、Baccaurea ramiflora、Bactris gasipaes、Balanites aegyptiaca、Bambusa bambos、Bamboohorsfieldii、Bambusa odashimae、Bambusa vulgaris、Barbarea verna、Barbarea vulgaris、Barringtonia procera、Basella alba、Bassia indica、Bertholletia excelsa、Beta palonga、Beta vulgaris、Bixa orellana、Blighia sapida、Boesenbergia rotunda、Bomarea edulis、Borago officinalis、Bouea macrophylla、Bouteloua curtipendula、Brachiaria deflexa、Brasenia schreberi;、Brassica carinata、Brassica juncea, Brassica napus, Brassica nigra, Brassica oleracea, Brassica rapa, Bromelia pinguin, Bromus catharticus, Bromus mango, Bromus marginatus, Bromus secalinus, Brosimum alicastrum, Broussonetia luzonica, Bunchosia armeniaca, Bunchosia costaricensis, Bunium bulbocastanum, Cajanus cajan, Calathea allouia, Calystegia sepium, Camellia japonica, Camellia sinensis、Campanula rapunculus、Campomanesia guaviroba, Campomanesia lineatifolia, Canarium album, Canarium indicum, Canarium ovatum, Canavalia cathartica, Canavalia ensiformis, Canavalia gladiata, Canavalia plagiosperma, Canna indica, Cannabis sativa, Capparis spinosa, Capsicum annuum, Capsicumbaccatum, Capsicum chinense, Capsicum pubescens, Cardiocrinum cordatum, Carica papaya, Carica pentagona, Carissa macrocarpa, Carthamus tinctorius, Carum carvi, Carum roxburghianum, Carya cathayensis, Carya illinoinensis, Carya ovata, Caryocar nuciferum, Casimiroa edulis, Castanea crenata, Castanea dentata, Castanea mollissima, Castanea pumila, Castanea sativa, Catha edulis, Celosia argentea, Centrosema pubescens, Ceratonia siliqua, Ceratotheca sesamoides, Cereus hexagonus, Chaenomeles sinensis, Chaerophyllum bulbosum, Chamaedorea tepejilote, Chenopodium album, Chenopodium berlandieri, Chenopodium bonus-henricus, Chenopodium capitatum, Chenopodium foliosum, Chenopodium pallidicaule, Chenopodium quinoa, Chionachne gigantea, Chrysanthemum morifolium, Chrysobalanus icaco, Chrysophyllum africanum, Chrysophyllum cainito, Cicer arietinum, Cichorium endivia, Cichorium intybus, Cinnamomum burmanni, Cinnamomum cassia, Cinnamomum loureiroi, Cinnamomum tamala, Cinnamomum verum, Citrullus lanatus, Citrus aurantiifolia, Citrus aurantium, Citrushystrix, Citrus jambhiri, Citrus japonica, Citrus limon, Citrus maxima, Citrus medica, Citrus mitis, Citrus paradisi, Citrus reticulata, Citrus sinensis, Clausena lansium, Claytonia perfoliata, Cnidoscolus aconitifolius, Cnidoscolus urens, Cocos nucifera, Coffea arabica, Coffea canephora, Coffea congensis, Coffea eugenioides, Coffea liberica, Cola nitida, Colocasia esculenta, Corchorus olitorius, Corchorus trilocularis, Cordia dodecandra, Cordyline fruticosa, Coriandrum sativum, Corylus avellana, Corylus chinensis, Corylus heterophylla, Corylus maxima, Corylus sieboldiana, Crambe hispanica, Crambe maritima, Crataegus azarolus, Crataegus hupehensis, Crataegus orientalis, Crescentia cujete, Crocus sativus, Crotalaria longirostrata, Croton tiglium, Cryptotaenia japonica, Cucumis melo, Cucumis metuliferus, Cucumis sativus, Cucurbita argyrosperma, Cucurbita ficifolia, Cucurbita maxima, Cucurbita moschata, Cucurbita pepo, Cuminum cyminum, Curcuma angustifolia, Curcuma heyneana, Curcuma longa, Curcuma pierreana, Curcuma zanthorrhiza, Cyamopsistetragonoloba, Cyclanthera pedata, Cydonia oblonga, Cynara cardunculus, Cyperus cyperoides, Cyperus esculentus, Cyphomandra betacea, Cyrtocarpa procera, Cyrtosperma merkusii, Dacryodes edulis, Daucus carota, Dendrocalamus asper, Dendrocalamus latiflorus, Desmodium discolor, Desmodium intortum, Desmodium uncinatum, Digitaria exilis, Digitaria iburua, Digitaria sanguinalis, Dimocarpus longan, Dioscorea alata, Dioscorea bulbifera, Dioscorea cayennensis, Dioscorea dumetorum, Dioscorea esculenta, Dioscorea flabellifolia, Dioscorea japonica, Dioscorea nummularia, Dioscorea oppositifolia, Dioscorea pentaphylla, Dioscorea piperifolia, Dioscorea quartiniana, Dioscorea trifida, Diospyros discolor, Diospyros ebenum, Diospyros kaki, Diospyros lotus, Diospyros major, Diospyros nigra, Diospyros virginiana、Dipteryx odorata、Dovyalis caffra、Dovyalis hebecarpa、Durio oxleyanus、Durio zibethinus、Dysphania ambrosioides、Echinochloa colona、Echinochloa crus-vonis、Echinochloa esculenta、Echinochloa frumentacea、Elaeagnus multiflora、Elaeagnuspungens, Elaeagnus umbellata, Elaeis guineensis, Elaeocarpus floribundus, Eleocharis dulcis, Elettaria cardamomum, Eleusine coracana, Elymus canadensis, Elymus caninus, Elymus hispidus, Elymus repens, Elymus smithii, Elymus spicatus, Ensete ventricosum, Enydra fluctuans, Eragrostis tef, Eriobotrya japonica, Eriochloa polystachya, Erioglossum rubiginosum, Eruca vesicaria, Etlingera elatior, Eugenia pyriformis, Eugenia uniflora, Euryale ferox, Euterpe oleracea, Eutrema japonicum, Fagopyrum esculentum, Feronia limonia, Ficus auriculata, Ficus carica, Ficus sycomorus, Flacourtia indica, Flacourtia rukam, Fragaria ananassa, Fragaria chiloensis, Fragaria moschata, Fragaria virginiana, Fragaria viridis, Galega officinalis、Garcinia cochinchinensis、Garcinia dulcis、Garcinia indica、Garcinia madruno、Garcinia mangostana、Garcinia multiflora、Garcinia pedunculata、Garcinia prainiana、Garcinia xanthochymus、Genipa americana、Gigantochloa ligulata、Glebions coronaria、Glebions segetum、Gleditsia triacanthos、Glycine max、Glycyrrhizaechinata, Glycyrrhiza glabra, Glycyrrhiza uralensis, Gossypium herbaceum, Gossypium hirsutum, Grewia asiatica, Guizotia abyssinica, Helianthus annuus, Helianthus tuberosus, Hemerocallis fulva, Hibiscus cannabinus, Hibiscus radiatus, Hibiscus sabdariffa, Hodgsonia macrocarpa, Hordeum vulgare, Houttuynia cordata, Hovenia dulcis, Humulus lupulus, Hydrolea zeylanica, Hylocereus undatus, Hyptis suaveolens, Ilex paraguariensis, Illicium verum, Inga edulis, Inga feuilleei, Inga laurina, Inocarpus fagifer, Ipomoea aquatica, Ipomoea batatas, Ipomoea eriocarpa, Ipomoea mammosa, Irvingia gabonensis, Iva annua, Jaltomata procumbens, Jasminum sambac, Jatropha curcas, Jatropha multifida, Juglans ailanthifolia, Juglans hindsii, Juglans honorei, Juglans nigra, Juglans regia, Kaempferia galanga, Kaempferia rotunda, Lablab purpureus, Lactuca indica, Lactuca sativa, Lagenaria siceraria, Lansium parasiticum, Lathyrus cicera, Lathyrus ochrus, Lathyrus sativus, Lathyrus sylvestris, Lathyrus tuberosus, Launaea taraxacifolia, Laurus nobilis, Lecythis zabucajo, Lensculinaris, Lepidium meyenii, Lepidium sativum, Leucaena leucocephala, Levisticum officinale, Lilium auratum, Lilium lancifolium, Lilium leichtlinii, Linum usitatissimum, Lippia adoensis, Litchi chinensis, Litsea calophylla, Lolium perenne, Luffa acutangula, Luffa cylindrica, Lupinus albus, Lupinus angustifolius, Lupinus cosentinii, Lupinus luteus, Lupinus mutabilis, Lupinus perennis, Lupinus tauris, Lycianthes moziniana, Lycium chinense, Macadamia integrifolia, Macrotyloma uniflorum, Malpighia emarginata, Malpighia glabra, Malpighia urens, Malus baccata, Malus domestica, Malus micromalus, Malus prunifolia, Malus sieboldii, Malus spectabilis, Malus turkmenorum, Malva verticillata, Mammea americana, Mangifera caesia, Mangifera foetida, Mangifera indica, Mangifera odorata, Manihot esculenta, Manilkara zapota, Maranta arundinacea, Medicago lupulina, Medicago sativa, Medicago truncatula, Melicoccus bijugatus, Melilotus macrorhizus, Mentha canadensis, Mentha spicata, Mentha suaveolens, Mesua ferrea, Metroxylon sagu, Momordica charantia, Monsteradeliciosa, Moringa oleifera, Morus alba, Morus nigra, Mucuna pachylobia, Mucuna pruriens, Muntingia calabura, Murraya koenigii, Musa acuminata, Musa balbisiana, Myrceugenia lanceolata, Myrica rubra, Myristica fragrans, Myrrhis odorata, Nasturtium officinale, Nelumbo nucifera, Nephelium lappaceum, Nephelium mutabile, Neptunia oleracea, Nigella sativa, Nopalea cochenillifera, Nopalea dejecta, Ocimum basilicum, Ocotea quixos, Oenanthe javanica, Olea europaea, Omphalea megacarpa, Opuntia boldinghii, Opuntia crystalenia, Opuntia elatior, Opuntia ficus-indica, Opuntia fusicaulis, Opuntia hyptiacantha, Opuntia leucotricha, Opuntia robusta, Opuntia undulata, Origanum majorana, Origanum vulgare, Oryza glaberrima, Oryza sativa、Osmanthus fragrans、Oxalis deppei、Oxalis tuberosa、Pachyrhizus ahipa、 Pachyrhizus erosus, Pachyrhizus tuberosus, Pandanus amaryllifolius, Pandanus brosimos, Pandanus tectorius, Pangium edule, Panicum antidotale, Panicum miliaceum, Panicum virgatum, Parkia biglobosa, Parkia speciosa, Parmentiera aculeata, Parmentiera cereifera, Paspalum dilatatum, Paspalum distichum, Paspalum notatum, Paspalum plicatulum, Paspalum scrobiculatum, Passiflora alata, Passiflora antioquiensis, Passiflora caerulea, Passiflora edulis, Passiflora laurifolia, Passiflora ligularis, Passiflora maliformis, Passiflora mollissima, Passiflora pinnatistipula, Passiflora quadrangularis, Pastinaca sativa, Paullinia cupana, Peltophorum pterocarpum, Pennisetum glaucum, Peperomia pellucida, Pereskia aculeata, Perilla frutescens, Persea americana, Persea schiedeana, Persicaria hydropiper, Petasites japonicus, Petroselinum crispum, Phacelia tanacetifolia, Phaseolus acutifolius, Phaseolus coccineus, Phaseolus dumosus, Phaseolus lunatus, Phaseolus ritensis, Phaseolus vulgaris, Phleum pratense, Phoenix atlantica, Phoenix dactylifera, Phoenixsylvestris, Phragmites australis, Phyllanthus distichus, Phyllanthus emblica, Phyllostachys dulcis, Physalis alkekengi, Physalis ixocarpa, Physalis peruviana, Physalis philadelphica, Physalis pruinosa, Physalis pubescens, Phytolacca acinosa, Pimenta dioica, Pimpinella anisum, Piper auritum, Piper guineense, Piper longum, Piper nigrum, Piper retrofractum, Pistacia vera, Pisum sativum, Pithecellobium dulce, Pithecellobium jiringa, Plantago major, Plectranthus amboinicus, Plectranthus esculentus, Plinia cauliflora, Plukenetia volubilis, Poa compressa, Poa nemoralis, Poa pratensis, Polygala butyracea, Polygonum maximowiczii, Polygonum odoratum, Polymnia sonchifolia, Polyscias fruticosa, Portulaca oleracea, Pouteria caimito, Pouteria campechiana, Pouteria lucuma, Pouteria macrophylla, Pouteria procera, Pouteria sapota, Pouteria viridis, Proboscidea louisianica, Prunus americana, Prunus angustifolia, Prunus armeniaca, Prunus avium, Prunus cerasifera, Prunus cerasus, Prunus domestica, Prunus dulcis, Prunus kansuensis, Prunus mira, Prunusmume, Prunus munsoniana, Prunus nigra, Prunus persica, Prunus pseudocerasus, Prunus salicina, Prunus simonii, Prunus tomentosa, Prunus ussuriensis, Psidium acutangulum, Psidium cattleianum, Psidium friedrichsthalianum, Psidium guajava, Psidium guineense, Psidium sartorianum, Psophocarpus tetragonolobus, Puccinellia nuttalliana, Pueraria montana, Pueraria phaseoloides, Punica granatum, Pyrus bretschneideri, Pyrus communis, Pyrus pyrifolia, Quararibea cordata, Raphanus raphanistrum, Rheum officinale, Rheum rhabarbarum, Rhodomyrtus tomentosa, Ribes aureum, Ribes cynosbati, Ribes longiracemosum, Ribes nigrum, Ribes petraeum, Ribes rubrum, Ribes spicatum, Ribes uva-crispa, Rollinia dolabripetala, Rollinia mucosa, Rolliniopsis discreta, Rorippa indica, Rubus brasiliensis, Rubus chamaemorus, Rubus glaucus, Rubus idaeus, Rubus illecebrosus, Rubus laciniatus, Rubus occidentalis, Rubus phoenicolasius, Rubus rosifolius, Rumex acetosa, Rumex alpinus, Rumex patientia, Rumex scutatus, Saccharum officinarum, Sagittaria sagittifolia, Salaccazalacca, Salsola komarovii, Salsola soda, Salvia hispanica, Salvia officinalis, Sambucus nigra, Sandoricum koetjape, Sanguisorba minor, Sanguisorba officinalis, Sauropus androgynus, Sclerocarya birrea, Secale cereale, Sechium edule, Sechium tacaco, Sedum rupestre, Semecarpus anacardium, Sesamum alatum, Sesamum indicum, Sesamum radiatum, Sesbania grandiflora, Setaria italica, Sida rhombifolia, Sinapis alba, Smallanthus sonchifolius, Solanum aethiopicum, Solanum anomalum, Solanum betaceum, Solanum chaucha, Solanum curtilobum, Solanum duplosinuatum, Solanum goniocalyx, Solanum lycopersicum, Solanum macrocarpon, Solanum melongena, Solanum muricatum, Solanum phureja, Solanum quitoense, Solanum scabrum, Solanum sessiliflorum, Solanum stenotomum, Solanum tuberosum, Solanum uporo, Solanum green, Sorbus domestica, Sorghum almum, Sorghum bicolor, Sorghum drummondii, Sphenostylis stenocarpa, Spilanthes acmella, Spinacia oleracea, Spondias dulcis, Spondias lakonensis, Spondias mombin, Spondias pinnata, Spondias purpurea, Stachysaffinis, Stelechocarpus burahol, Stenocereus griseus, Stenocereus pruinosus, Stenocereus queretaroensis, Stenocereus stellatus, Stevia rebaudiana, Suaeda glauca, Syzygium aromaticum, Syzygium cumini, Syzygium formosum, Syzygium jambos, Syzygium malaccense, Syzygium samarangense, Tacca leontopetaloides, Talinum fruticosum, Talinum paniculatum, Talinum portulacifolium, Talisia esculenta, Tamarindus indica, Tanacetum vulgare, Taraxacum campylodes, Terminalia catappa, Tetragonia tetragonioides, Theobroma bicolor, Theobroma cacao, Theobroma grandiflorum, Theobroma microcarpum, Thymus herba-barona, Thymus vulgaris, Trapa natans, Trichosanthes cucumerina, Trichosanthes japonica, Trichosanthes pilosa, Trifolium alexandrinum, Trifolium ambiguum, Trifolium fragiferum, Trifolium hybridum, Trifolium incarnatum, Trifolium pannonicum, Trifolium pratense, Trifolium repens, Trifolium resupinatum, Trifolium semipilosum, Trifolium subterraneum, Trigonella foenum-graecum, Triphasia trifolia, Tripsacum dactyloides, Triticum aestivum, Triticumboeoticum, Triticum carthlicum, Triticum dicoccon, Triticum durum, Triticum monococcum, Triticum spelta, Triticum sphaerococcum, Triticum timopheevii, Tropaeolum leptophyllum, Tropaeolum tuberosum, Tylosema esculentum, Typha latifolia, Ullucus tuberosus, Urena lobata, Vaccinium ashei, Vaccinium corymbosum, Vaccinium macrocarpon, Vaccinium oxycoccos, Valeriana sambucifolia, Valerianella locusta, Vangueria madagascariensis, Vanilla planifolia, Vanilla pompona, Vanilla tahitensis, Vasconcellea pubescens, Veronica anagallis, Vicia articulata, Vicia benghalensis, Vicia cracca, Vicia ervilia, Vicia faba, Vicia graminea, Vicia hirsuta, Vicia ludoviciana, Vicia monantha, Vicia narbonensis, Vicia pannonica, Vicia sativa, Vicia sepium, Vicia tetrasperma, Vicia unijuga, Vicia villosa、Vigna aconitifolia、Vigna angularis、Vigna cylindrica、Vigna mungo、Vigna radiata、Vigna subterranea、Vigna umbellata、Vigna unguiculata、Vitellaria paradoxastrumarium mioga, Zingiber officinale, Zizania aquatica, Zizania latifolia, Zizania palustris, and Ziziphus jujuba.

[0136] Therefore, in some embodiments, the method includes delivering an RNAi molecule to a plant (e.g., a soybean plant) in an amount effective to suppress plant infection and thereby control a pathogen (e.g., a fungal pathogen). In other embodiments, the method includes delivering an RNAi molecule described herein to a soybean plant in an amount effective to suppress plant infection by a pathogen, or to suppress the spread of a pathogen from one part of a plant to another, or to suppress the spread of a pathogen from one cell or cell type to another cell or cell type, or to suppress the spread of a pathogen from one tissue or tissue type to another tissue or tissue type. In other embodiments, the method includes, for example, delivering an RNAi molecule described herein to a lettuce plant in an amount effective for suppressing infection of a plant by a pathogen, or suppressing the spread of a pathogen from one part of a plant to another, or suppressing the spread of a pathogen from one cell or cell type to another cell or cell type, or suppressing the spread of a pathogen from one tissue or tissue type to another tissue or tissue type, or suppressing the spread of a pathogen from one part of a plant to another, or suppressing the spread of a pathogen from one cell or cell type to another cell or cell type, or suppressing the spread of a pathogen from one tissue or tissue type to another tissue or tissue type. In yet another embodiment, the method includes, for example, delivering an RNAi molecule described herein to a spinach plant in an amount effective to suppress infection of a plant by a pathogen, or to suppress the spread of a pathogen from one part of a plant to another, or to suppress the spread of a pathogen from one cell or cell type to another cell or cell type, or to suppress the spread of a pathogen from one tissue or tissue type to another tissue or tissue type, or to suppress the spread of a pathogen from one part of a plant to another, or to suppress the spread of a pathogen from one cell or cell type to another cell or cell type, or to suppress the spread of a pathogen from one tissue or tissue type to another tissue or tissue type.

[0137] The delivery of RNAi molecules to a plant (e.g., a part of a plant) may include, for example, the topical application (e.g., dipping, coating, or dusting) of RNAi molecules or compositions containing RNAi molecules to any part of the plant (e.g., roots, tubers, stems, branches, leaves, flowers, etc.) or the ground (e.g., soil, mud, grass, etc.). The delivery step may also include genetically engineering plant cells to express RNAi molecules.

[0138] The effective dose is the amount of RNAi molecules described herein required to provide a beneficial effect against infection by a pathogen, either alone or in combination with one or more other additives. Beneficial effects include, but are not limited to, increased mortality, inhibited growth, inhibited spread from one area of ​​a plant to another, or a reduction in the spread of toxicity or pathogenicity from one area of ​​a plant to another, or a reduction in the pathogen's ability to proliferate / regenerate (spore formation). Beneficial effects may be demonstrated by a reduction of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to a control in the affected surface area or plant part weight, plant part mass, or plant part volume (optionally, the plant part is a leaf). The plant part used to measure the beneficial effect may be, for example, a root, shoot, fruit, flower, leaf, or any marketable part of the plant. Beneficial effects may also be demonstrated by an increase in the functionality of plant parts compared to a control (e.g., an increase in leaf photosynthetic activity) or by an increase in the quality of the marketable parts of the plant. In some embodiments, the control is in the absence of the RNAi molecule of the present invention. In some embodiments, an effective amount of the RNAi molecule described herein completely eliminates pathogen infection. Beneficial effects also include a reduction in the expression of genes involved in the suppression of systemic or local defense response pathways (e.g., the systemic acquired resistance pathway).

[0139] The effective amount will vary depending on the particular plant, the severity of the infection, the duration of the infection, previous exposure to the pathogen, and similar factors within the knowledge and expertise of the skilled person. These factors are well known to the skilled person and can be addressed by routine experimentation only. In order to increase efficiency and reduce costs, it is generally preferred that a lower effective concentration, i.e., the lowest concentration that provides control of the pathogen, is used.

[0140] The effective amount of the RNAi molecules described herein may also vary depending on the delivery method.

[0141] In some embodiments, the effective amount of the RNAi molecule is in micrograms (μg) of RNAi molecule per square centimeter (cm 2 , 2 , 2 , 2 , 2 , 2 , 2 , 2 , 2 , 2 , 2 , 2 , 2 , 2 ) of the surface of the plant or the ground (by way of example, soil, mud, grass, etc.), i.e., μg / cm 2 Thus, in some embodiments, the effective amount of the RNAi molecule comprises from 0.001 μg / cm 2 to [10 μg / cm 2 In some embodiments, the effective amount of the RNAi molecule is from 0.001 μg / cm 2 to 9 μg / cm 2 , from 0.001 μg / cm 2 to 8 μg / cm 2 , from 0.001 μg / cm 2 to 7 μg / cm 2 , from 0.001 μg / cm 2 to 6 μg / cm 2 , from 0.001 μg / cm 2 to 5 μg / cm 2 , from 0.001 μg / cm 2 to 4 μg / cm 2 , from 0.001 μg / cm 2 to 3 μg / cm 2 , from 0. 2 2 to 2 μg / cm 2 , from 0.001 μg / cm 2 to 1 μg / cm 2 , from 0.001 μg / cm 2 to 0.1 μg / cm 2 or 0.001 μg / cm2 ~0.01 μg / cm³ 2 It includes. In some embodiments, the effective amount of RNAi molecules is 0.01 μg / cm³. 2 ~10 μg / cm³ 2 , 0.1 μg / cm³ 2 ~10 μg / cm³ 2 , 1 μg / cm³ 2 ~10 μg / cm³ 2 , 2 μg / cm³ 2 ~10 μg / cm³ 2 3 μg / cm³ 2 ~10 μg / cm³ 2 4 μg / cm³ 2 ~10 μg / cm³ 2 5 μg / cm³ 2 ~10 μg / cm³ 2 , 6 μg / cm³ 2 ~10 μg / cm³ 2 7 μg / cm³ 2 ~10 μg / cm³ 2 , 8 μg / cm³ 2 ~10 μg / cm³ 2 , or 9 μg / cm³ 2 ~10 μg / cm³ 2 Includes.

[0142] In some embodiments, the effective amount of RNAi molecules is expressed as grams (g) of RNAi molecules per acre (ac.) of plant or ground surface (e.g., soil, mud, grass, etc.), i.e., g / ac. Thus, in some embodiments, the effective amount of RNAi molecules includes 0.01 g / ac. to 100 g / ac. In some aspects, effective amounts of RNAi molecules include 0.01 g / ac. to 90 g / ac., 0.01 g / ac. to 80 g / ac., 0.01 g / ac. to 70 g / ac., 0.01 g / ac. to 60 g / ac., 0.01 g / ac. to 50 g / ac., 0.01 g / ac. to 40 g / ac., 0.01 g / ac. to 30 g / ac., 0.01 g / ac. to 20 g / ac., 0.01 g / ac. to 10 g / ac., 0.01 g / ac. to 1 g / ac., or 0.01 g / ac. to 0.1 g / ac. In some aspects, effective amounts of RNAi molecules include 0.1 g / ac. to 100 g / ac., 1 g / ac. to 100 g / ac., 10 g / ac. to 100 g / ac., 20 g / ac. to 100 g / ac., 30 g / ac. to 100 g / ac., 40 g / ac. to 100 g / ac., 50 g / ac. to 100 g / ac., 60 g / ac. to 100 g / ac., 70 g / ac. to 100 g / ac., 80 g / ac. to 100 g / ac., or 90 g / ac. to 100 g / ac.

[0143] In some aspects, an effective amount of RNAi molecules is expressed as grams of RNA per gram of seed. Therefore, in some aspects, an effective amount of RNAi molecules is approximately 0.001 μg of RNA / g seeds to approximately 10 mg of RNA / g seeds, approximately 0.1 mg of RNA / g seeds to approximately 1 mg of RNA / g seeds, approximately 0.1 mg of RNA / g seeds to approximately 0.5 g of RNA / g seeds, approximately 0.1 mg of RNA / g seeds to approximately 0.3 g of RNA / g seeds, approximately 0.15 g of RNA / g seeds to approximately 0.25 mg / g seeds, approximately 0.01 mg of RNA / g seeds to approximately 0.03 g of RNA / g seeds, and approximately 0.15 mg of RNA / g seeds. Contains RNA / g seeds ~ approximately 0.25g of RNA / g seeds, approximately 0.001μg of RNA / g seeds ~ approximately 10μg of RNA / g seeds; or approximately 0.001μg of dsRNA / g seeds ~ approximately 1μg of dsRNA / g seeds; or approximately 0.001μg of dsRNA / g seeds ~ approximately 0.1μg of dsRNA / g seeds; or approximately 0.001μg of dsRNA / g seeds ~ approximately 0.1μg of dsRNA / g seeds; or approximately 0.001μg of dsRNA / g seeds ~ approximately 0.01μg of dsRNA / g seeds. In some aspects, an effective amount of RNAi molecules is found in 10 mg dsRNA / g seeds, 5 g dsRNA / g seeds, 1 g dsRNA / g seeds, 0.95 mg dsRNA / g seeds, 0.9 mg dsRNA / g seeds, 0.85 mg dsRNA / g seeds, 0.8 mg dsRNA / g seeds, 0.75 mg dsRNA / g seeds, 0.7 mg dsRNA / g seeds, 0.65 mg dsRNA / g seeds, 0.65 mg dsRNA / g seeds, 0.5 mg dsRNA / g seeds, 0.45 mg dsRNA / g seeds, 0.4 mg dsRNA / g seeds. g seeds, 0.35 mg dsRNA / g seeds, 0.34 mg dsRNA / g seeds, 0.33 mg dsRNA / g seeds, 0.32 mg dsRNA / g seeds, 0.31 mg dsRNA / g seeds, 0.3 mg dsRNA / g seeds, 0.29 mg dsRNA / g seeds, 0.28 mg dsRNA / g seeds, 0.27 mg dsRNA / g seeds, 0.26 mg dsRNA / g seeds, 0.25 mg dsRNA / g seeds, 0.24 mg dsRNA / g seeds, 0.23 mg dsRNA / g seeds, 0.22 mg dsRNA / g seeds, 0.21 mg dsRNA / g seeds, 0.2mg dsRNA / g seeds, 0.19mg dsRNA / g seeds, 0.18mg dsRNA / g seeds, 0.17mg dsRNA / g seeds, 0.16mg dsRNA / g seeds, 0.15mg dsRNA / g seeds, 0.14mg dsRNA / g seeds, 0.13mg dsRNA / g seeds, 0.12mg dsRNA / g seeds, 0.11mg dsRNA / g seeds, 0.1mg dsRNA / g seeds, 0.09mg dsRNA / g seeds, 0.08mg dsRNA / g seeds, 0.07mg dsRNA / g seeds, 0.06mg g dsRNA / g seeds, 0.05 mg dsRNA / g seeds, 0.04 mg dsRNA / g seeds, 0.039 mg dsRNA / g seeds, 0.038 mg dsRNA / g seeds, 0.037 mg dsRNA / g seeds, 0.036 mg dsRNA / g seeds, 0.035 mg dsRNA / g seeds, 0.034 mg dsRNA / g seeds, 0.033 mg dsRNA / g seeds, 0.032 mg dsRNA / g seeds, 0.031 mg dsRNA / g seeds, 0.03 mg dsRNA / g seeds, 0.029 mg dsRNA / g seeds, 0.02 8mg dsRNA / g seeds 0.027mg dsRNA / g seeds 0.026mg dsRNA / g seeds 0.025mg dsRNA / g seeds 0.024mg dsRNA / g seeds 0.023mg dsRNA / g seeds 0.022mg dsRNA / g seeds, 0.021mg dsRNA / g seeds, 0.02mg dsRNA / g seeds, 0.019mg dsRNA / g seeds, 0.018mg dsRNA / g seeds 0.017mg dsRNA / g seeds 0.016mg dsRNA / g seeds 0.015mg dsRNA / g seeds 0. Contains 0.014 mg of dsRNA / g seeds, 0.013 mg of dsRNA / g seeds, 0.012 mg of dsRNA / g seeds, 0.011 mg of dsRNA / g seeds, 0.01 mg of dsRNA / g seeds, 10 μg of dsRNA / g seeds, 5 μg of dsRNA / g seeds, 1 μg of dsRNA / g seeds, 0.5 μg of dsRNA / g seeds, 0.1 μg of dsRNA / g seeds, 0.05 μg of dsRNA / g seeds, or 0.01 μg of dsRNA / g seeds, or 0.005 μg of dsRNA / g seeds, or 0.001 μg of dsRNA / g seeds.

[0144] In some embodiments, the effectiveness of RNAi molecules that control pathogens can be determined by the RNAi molecule's ability to slow the rate of infection by the pathogen or to increase the survival rate of plants.

[0145] In some embodiments, the effectiveness of an RNAi molecule can be determined by its ability to reduce percentage plant defoliation caused by a pathogen. Percentage plant defoliation refers to the percentage of plant material (e.g., soybean leaves) destroyed or otherwise affected by a pathogen. In some embodiments, an RNAi molecule causes a reduction of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 100% in the percentage plant portion surface area, plant portion mass, or plant portion volume affected by the pathogen (optionally, the plant portion is leaves). In some embodiments, an RNAi molecule causes a reduction of less than 40%, 30%, 25%, 20%, 15%, 10%, 5%, 3%, or 1% in the percentage plant portion surface area, plant portion mass, or plant portion volume (optionally, the plant portion is leaves) affected by the pathogen. In some embodiments, the percentage of plant defoliation remains below 40%, 30%, 25%, 20%, 15%, 10%, 5%, 3%, or 1% for at least 5, 6, 7, 8, 9, 10, 15, or 20 days after exposure of the plant to the pathogen. In some embodiments, the ability of an RNAi molecule to reduce the affected percentage of plant portion surface area, plant portion mass, or plant portion volume (optionally the plant portion being leaves) is compared to a control (e.g., a control molecule or untreated conditions). In some embodiments, the RNAi molecule causes the affected percentage of plant portion surface area, plant portion mass, or plant portion volume (optionally the plant portion being leaves) to decrease by 80%, 70%, 60%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 3%, or 1% compared to a control. In some embodiments, the affected percentage of plant surface area, plant mass, or plant volume (where the plant portion is optionally a leaf) is measured over time (for example, over the course of several days of exposure of the plant to the pathogen). In some embodiments, the affected percentage of plant surface area, plant mass, or plant volume (where the plant portion is optionally a leaf) is measured after 3, 4, 5, 6, 7, 8, 9, 10 days or more of exposure.

[0146] In some embodiments, RNAi molecules may be formulated in solution. In some embodiments, the effective amount of RNAi molecules in solution is expressed as nanograms (ng) or micrograms (μg) of RNAi molecules per milliliter (ml) of solution, i.e., ng / ml. Thus, in some embodiments, the solution contains RNAi molecules at concentrations of 10 ng / ml to 100 μg / ml. In some embodiments, the solution contains RNAi molecules at concentrations of 0.001 mg to 0.1 mg / ml, 10 ng / ml to 100 μg / ml, 100 ng / ml to 100 μg / ml, 250 ng / ml to 100 μg / ml, 750 ng / ml to 100 μg / ml, 1000 ng / ml to 100 μg / ml, 10 μg / ml to 100 μg / ml, 25 μg / ml to 100 μg / ml, 50 μg / ml to 100 μg / ml, or 75 μg / ml to 100 μg / ml. In some aspects, the solution contains RNAi molecules at concentrations of 10 ng / ml to 100 μg / ml, 10 ng / ml to 75 μg / ml, 10 ng / ml to 50 μg / ml, 10 ng / ml to 25 μg / ml, 10 ng / ml to 10 μg / ml, 10 ng / ml to 1000 ng / ml, 10 ng / ml to 1000 ng / ml, 10 ng / ml to 750 ng / ml, 10 ng / ml to 500 ng / ml, 10 ng / ml to 250 ng / ml, 10 ng / ml to 100 ng / ml, 10 ng / ml to 75 ng / ml, 10 ng / ml to 50 ng / ml, or 10 ng / ml to 25 ng / ml.

[0147] In some embodiments, the solution comprises RNAi molecules and at least one additional additive (e.g., a fungicide, surfactant, or other non-pesticide). In some embodiments, such mixture contains RNAi molecules at concentrations of 0.0001 μg / ml to 10 μg / ml (e.g., applied to the surface of plants and / or ground (e.g., soil, mud, grass, etc.)). In some aspects, such mixtures contain RNAi molecules at concentrations of 0.001 μg / ml to 10 μg / ml, 0.01 μg / ml to 10 μg / ml, 0.1 μg / ml to 10 μg / ml, 1 μg / ml to 10 μg / ml, 2 μg / ml to 10 μg / ml, 3 μg / ml to 10 μg / ml, 4 μg / ml to 10 μg / ml, 5 μg / ml to 10 μg / ml, 6 μg / ml to 10 μg / ml, 7 μg / ml to 10 μg / ml, 8 μg / ml to 10 μg / ml, or 9 μg / ml to 10 μg / ml. In some aspects, such mixtures contain RNAi molecules at concentrations of 0.0001 μg / ml to 9 μg / ml, 0.0001 μg / ml to 8 μg / ml, 0.0001 μg / ml to 7 μg / ml, 0.0001 μg / ml to 6 μg / ml, 0.0001 μg / ml to 5 μg / ml, 0.0001 μg / ml to 4 μg / ml, 0.0001 μg / ml to 3 μg / ml, 0.0001 μg / ml to 2 μg / ml, 0.0001 μg / ml to 1 μg / ml, 0.0001 μg / ml to 0.1 μg / ml, 0.0001 μg / ml to 0.01 μg / ml, or 0.0001 μg / ml to 0.001 μg / ml.

[0148] The process of delivering RNAi molecules to any part of a plant (e.g., roots, tubers, stems, branches, leaves, flowers, etc.) or the ground (e.g., soil, mud, grass, etc.) may include single application (single contact) or multiple applications (multiple contacts) of RNAi molecules to the plant (e.g., seeds, roots, stems, flowers, leaves, tubers) or the ground (e.g., soil, mud, grass, etc.). Delivery to a part of the plant may be in the form of a spray (e.g., pressurized / aerosolized spray, pump), a solid (e.g., powder, pellets, bait), or a liquid (e.g., homogeneous mixtures such as solutions and heterogeneous mixtures such as suspensions (water and oil-based), colloids, micelles, and emulsions). The contact period may vary. In some embodiments, delivery involves exposing a portion of a plant to an RNAi molecule for a sufficient period to regulate a target gene, thereby increasing mortality, inhibiting growth, inhibiting the spread from one region of the plant to another, or reducing toxicity or pathogenicity, or reducing the pathogen's ability to proliferate / regenerate (spore formation).

[0149] In some embodiments, delivery includes applying RNAi molecules to a portion of the plant surface. In some embodiments, application of RNAi molecules to a portion of the surface includes spraying, coating, and / or dusting the surface or a portion thereof. In some embodiments, application of RNAi molecules to a portion of the surface includes drenching or applying RNAi molecules as a granular or powdered formulation to soil adjacent to the plant roots.

[0150] In some embodiments, delivery involves contacting seeds with RNAi molecules. In some embodiments, contacting seeds with RNAi molecules can be achieved using any method known in the art that allows an effective amount of dsRNA to enter the seeds. These examples include, but are not limited to, immersion, spraying, priming, or coating seeds with powder, emulsion, suspension, or solution. In some embodiments, seed coating or seed treatment compositions include RNAi molecules and at least one plant enhancer, but is not limited to, an active substance intended to positively influence seed germination, plant emergence, plant growth, plant defense, plant development, and / or plant yield.

[0151] RNAi molecules may be applied to any part of a plant (e.g., roots, tubers, stems, branches, leaves, flowers, etc.). In some embodiments, RNAi molecules may be in contact with the above-ground parts of a plant (e.g., leaves) and / or the underground parts of a plant (e.g., roots) and may comprise at least one infall formulation selected from the group consisting of powder, granules, pellets, capsules, soluble liquid concentrates, sprays (after dilution or concentration), mist, infall, seed treatment, seed coating, drenching, drip irrigation, or any other form suitable for application between rows. The parts of a plant that may be in contact with the RNAi molecules described herein include, but are not limited to, leaves, stems, flowers, fruits, shoots, roots, seeds, tubers, anthers, stamens, or pollen. In some embodiments, RNAi is delivered mechanically via high-pressure spray or sandblasting.

[0152] The consequences of inhibition can be confirmed by any suitable assay for evaluating one or more characteristics of a pathogen, or by biochemical techniques for evaluating molecules (e.g., RNA, protein) that indicate expression. In some embodiments, the extent to which the RNAi molecule provided herein reduces the expression level of a target gene is evaluated by comparing the expression level (e.g., mRNA level or protein level) with the expression level of a suitable control (e.g., the level of expression in cells or cell populations to which the RNAi molecule was not delivered or a negative control was delivered). In some embodiments, the suitable control level of expression may be a predetermined level or value such that the control level does not need to be measured each time. The predetermined level or value may take various forms. In some embodiments, the predetermined level or value may be a single cutoff value such as a median or mean.

[0153] In some embodiments, delivery of RNAi molecules targeting plant genes involved in the suppression of systemic or local defense response pathways as described herein results in a decrease in expression levels in plant cells. In some embodiments, the decrease in expression levels of plant genes involved in the suppression of systemic or local defense response pathways may be at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% compared to a control level. In some embodiments, the control level is the expression level (or average level across cell populations) in similar plant cells not exposed to the RNAi molecule. In some embodiments, the control level is the expression level (or average level across cell populations) in similar plant cells exposed to an RNAi molecule targeting a gene not expressed by plant cells, e.g., green fluorescent protein (GFP) or an RNAi molecule not targeting any plant gene. In some embodiments, a decrease in the expression levels of plant genes involved in the suppression of systemic or local defense response pathways results in increased expression of one or more genes in the systemic or local defense response pathways.

[0154] In some embodiments, the effect of delivering RNAi molecules to cells or plants is evaluated after a finite period. For example, the level of a target gene may be determined in the cells or plants at least 4, 8, 12, 18, or 24 hours after delivery of the RNAi molecule; or at least 1, 2, 3, 4, 5, 6, 7, or 14 days after delivery. In another example (for instance, when an RNAi molecule is delivered to a seed), the level of target gene expression may be determined in the cell or plant at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 days after planting the seed.

[0155] In some embodiments, delivery of RNAi molecules as described herein results in an increase in mortality, inhibition of growth, inhibition of spread from one region of a plant to another, or a decrease in toxicity or pathogenicity, or a decrease in the pathogen's ability to proliferate / regenerate (spore formation). In some embodiments, the increase in mortality, inhibition of growth, inhibition of spread from one region of a plant to another, or a decrease in toxicity or pathogenicity, or a decrease in the pathogen's ability to proliferate / regenerate (spore formation), is, as required, a decrease or increase of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% compared to a control level. In some embodiments, the control level is the pathogen's mortality, growth, toxicity, pathogenicity, or ability to proliferate / regenerate (spore formation) in a plant not in contact with the RNAi molecule.

[0156] In some embodiments, delivery of RNAi molecules as described herein results in an increase in mortality or inhibition of growth, inhibition of spread from one region of a plant to another, a decrease in toxicity or pathogenicity, or a decrease in the pathogen's ability to proliferate / regenerate (spore formation). In some embodiments, the increase in the level of mortality may be at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% compared to a control. In some embodiments, the control is the mortality rate between pathogens on plants that have not been in contact with the RNAi molecule.

[0157] Aspects of this disclosure provide plants that express RNAi molecules as described herein. In some embodiments, the DNA encoding the RNAi molecules provided herein is provided to a plant (plant seed or cell) so that the plant expresses the RNAi molecules. In some embodiments, the DNA encoding the RNAi molecules is expressed in the plant by transgenic expression, for example, by stably incorporating the DNA encoding the RNAi molecules into the plant genome so that the plant expresses the RNAi molecules.

[0158] The pathogens are harmful nematodes (for example, cyst nematodes Heterodera spp., especially soybean cyst nematodes Heterodera glycines, root nodule nematodes Meloidogyne spp., lance nematodes Hopolaimus spp., stunt nematodes Tylenchorhynchus spp., spiral nematodes Helicotylenchus spp., root rot nematodes Pratylenchus spp., ring nematodes Criconema spp., and leaf nematodes Aphelenchus spp. or Aphelenchoides). Plant pathogens of interest, which may be classified as spp., encompass all pathogens to which plants have an innate ability to control through systemic or local defense responses. These include fungi (e.g., fungi that cause powdery mildew, gray mold, rust, leaf spot and leaf blight, damping-off, root rot, crown rot, cottonseed rot, stem gall, branch gall, vascular wilt, smut, or mold, but which include Fusarium spp., Phakospora spp., Rhizoctonia spp., Aspergillus spp., Gibberella spp., Pyricularia spp., Alternaria spp., and Phytophthora spp.), bacteria (e.g., leaf spotting, fireblight, crown gall, and bacterial wilt) This may include bacteria that cause wilt, molybacteria (e.g., Mycoplasma that causes yellowing disease, and Spiroplasma such as Spiroplasma kunkelii that causes maize dwarfism), and viruses (e.g., viruses that cause mosaic, leaf vein green banding, flecking, spotting, or abnormal growth).

[0159] Method for producing RNAi molecules

[0160] RNAi molecules as provided herein may be produced by any suitable method known in the art. Examples of methods for producing RNAi molecules include, but are not limited to, in vitro transcription (IVT), chemosynthesis, expression in organisms (e.g., plants), or expression in cell culture (e.g., plant cell culture), and microbial fermentation.

[0161] In some aspects, RNAi molecules may be produced according to the cell-free production methods described in international patent application publication WO2017 / 176963A1, published on October 12, 2017, entitled "Cell-Free Production of Ribonucleic Acid"; U.S. provisional patent application USSN62 / 571,071, filed on October 11, 2017, entitled "Methods and Compositions for Nucleoside Triphosphate and Ribonucleic Acid Production"; and international patent application publication WO2019 / 075167A1, published on April 18, 2019; each of these is incorporated herein by reference.

[0162] Any suitable DNA encoding an RNAi molecule as described herein may be used in the methods described herein. The DNA may be single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA). In some embodiments, the DNA comprises one or more DNA expression cassettes, which, upon transcription, produce single-stranded RNA (ssRNA) molecules (e.g., remaining single-stranded or folded into an RNA hairpin) or complementary ssRNA molecules that anneal to produce double-stranded RNA (dsRNA) molecules.

[0163] In some embodiments, the DNA comprises a promoter (e.g., an inductive promoter) operably ligated to a nucleotide sequence encoding RNA complementary to a segment of a plant gene, and optionally a terminator. In other embodiments, the DNA comprises a first promoter (e.g., an inductive promoter) operably ligated to a nucleotide sequence encoding RNA complementary to a segment of a plant gene, and optionally a terminator, and a second promoter (e.g., an inductive promoter) operably ligated to a nucleotide sequence encoding a second RNA complementary to the first RNA, and optionally a terminator. In other embodiments, the DNA comprises a promoter (e.g., an inductive promoter) operably ligated to a nucleotide sequence encoding a first region of RNA, followed by one or more nucleotides of a loop region, followed by a second region of RNA, and optionally a terminator, where the first region of RNA is complementary to a segment of a plant gene, and the second region is complementary to the first region. In yet another embodiment, the DNA comprises a first strand comprising a first promoter (e.g., an inducible promoter) and optionally a terminator operably ligated to a nucleotide sequence encoding a first RNA complementary to a segment of a plant gene, and a second strand comprising a second promoter (e.g., an inducible promoter) and optionally a terminator operably ligated to a nucleotide sequence encoding a second RNA complementary to the first RNA, wherein the first and second promoters are operably ligated to a nucleotide sequence encoding a desired targeting RNA, wherein bidirectional transcription of the nucleotide sequence encoding the desired targeting RNA results in complementary RNA molecules that anneal to form a dsRNA molecule.

[0164] The DNA is typically provided on a vector such as a plasmid, but other template formats may be used (e.g., linear DNA produced by polymerase chain reaction (PCR), chemical synthesis, or other means known in the art). In some embodiments, more than one DNA is used in the reaction mixture. In some embodiments, two, three, four, five, or more different DNAs are used in the reaction mixture. In some embodiments, the DNA is a construct as described in International Publication No. 2021 / 113774, which is incorporated herein by reference.

[0165] The promoter or terminator may be a naturally occurring sequence or a manipulated (e.g., synthetic) sequence. In some embodiments, the manipulated sequence is modified to enhance transcriptional activity. In some embodiments, the promoter is a naturally occurring sequence. In other embodiments, the promoter is a manipulated sequence. In some embodiments, the terminator is a naturally occurring sequence. In other embodiments, the terminator is a manipulated sequence.

[0166] In some embodiments, polynucleotides (e.g., dsRNA) are produced according to the method described in U.S. Patent No. 10,954,541; or according to U.S. Patent No. 10,858,385 (the entire contents of each of these are incorporated herein by reference).

[0167] example

[0168] Examples are provided below to allow for a more complete understanding of the inventions described herein. The examples described herein are provided to illustrate the methods, compositions, and systems provided herein and should not be construed as limiting their scope.

[0169] Example 1. Delivery of double-stranded RNA via carbon dots. Carbon quantum dots can deliver dsRNAs (LD045, GmMPK4a, GmRLK3) targeting plant genes involved in the suppression of systemic or local defense response pathways to soybean plants, resulting in a visible phenotype in the first true leaves 12 days after planting. The ability of carbon dots to deliver dsRNAs into the cytoplasm of soybean plants was compared to self-assembling lipid nanoparticle formulations by visual evaluation of leaf phenotypes associated with high levels of downregulation of gene targets GS200 and GS201. The specific carbon dots described herein (CQD3) were prepared by mixing 9 ml of carbowax PEG200 with 3 ml of water on a magnetic stirring plate. In a separate container, 100 mg of branched PEI, 800Da was weighed into a glass scintillation vial and 2 ml of water was added. The samples were mixed on a magnetic stirring plate, and then the PEG solution was poured on top. The samples were mixed until homogeneous. Next, the solution was transferred to a larger container and microwaved in a household microwave oven at 1350W for 3 minutes, resulting in an average temperature of 170°C. The resulting pale yellow solution was cooled to room temperature before being used in the formulation. Alternatively, the 3-minute 1350W household microwave step can be omitted to form a non-dot version involving simple complex formation of dsRNA with PEG200-stabilized PEI. Healthy, uniform seeds were hand-picked, washed with water for 2-3 hours at room temperature, and dried at 25°C-28°C for 4 hours to overnight. Seeds were treated with a test formulation (1.5 g / L) diluted with water to achieve a working concentration of dsRNA of 0.2 mg / ml, using approximately 30 seeds per 8 ml of diluted formulation. In this example, the seeds were incubated in the dark for 12 or 24 hours with gentle shaking. After treatment, the seeds were placed in a petri dish, air-dried for 20-30 minutes, and then planted in soil. The plants were grown in a greenhouse at latitude 36.05 and longitude -78.95 under long-day conditions, with a daytime temperature of 25°C (15 hours) and a nighttime temperature of 20°C (9 hours). The soil used was Fafard 2 mix, consisting of 75% Canadian sphagnum moss and peat moss, 20% perlite, 5% vermiculite, dolomite lime, a persistent wetting agent, and RESiLIENCE.The plants were fertilized once a week, on Thursdays with a half-concentration of approximately 150 ppm of Hoagland, and once a week, on Wednesdays with a full-concentration of approximately 300 ppm of Hoagland. Phenotype and germination rate were visually scored 12 days after planting.

[0170] Figure 1A is a table of results; carbon dots produced visible leaf phenotypes for GmMPK4a and GmRLK3, but not for water (negative control) and PformPB01 (seed immersion). Figure 1B is a representative image of the leaf phenotypes observed compared to the untargeted control dsRNA LD045 (which does not target SAR pathway repressor genes).

[0171] Example 2. dsRNA molecules that target genes involved in the suppression of pathogen defense pathways. dsRNA molecules targeting systemic plant genes involved in the suppression of systemic or local defense response pathways can upregulate pathogen defense pathways (e.g., the systemic acquired resistance pathway) in soybean plants. This regulation was associated with the suppression of the intended target gene. Delivery was by seed immersion as described in Example 1, but differed in that the dsRNA, formulated at 0.2 mg / ml, was immersed overnight (approximately 18 hours). Seeds were sown in soil pots and grown in a greenhouse at a daytime temperature of 21°C–25°C (15 hours) and a nighttime temperature of 20°C–21°C (9 hours). At 15 and 37 days, leaves were sampled by hole punch from the first three true leaves to test gene expression by qRT-PCR. Of the 19 targets tested, eight were found to possess this activity (MPK4a, RLK3, RIN4a, CEV1, CPR5, COI1, DHS, and chimeric dsRNA (targeting ETR1 / EIN4 / ETR2). The results are shown in Figure 2.

[0172] Example 3. dsRNAs that target genes involved in suppressing pathogen defense pathways protect plant seedlings from fungal pathogens. dsRNA molecules targeting putative SAR pathway suppressor genes can protect soybean seedlings from fungal diseases (Figure 3). Delivery was performed by seed immersion in dsRNA formulated at 0.2 mg / ml as described in Example 2. dsRNA-treated seeds were sown in the presence of fungal inoculant using vermiculite substrate instead of soil, and watered with 0.5 times the amount of hogland as needed. They were compared to a simulated treatment control with either a fungal pathogen belonging to the genera Fusarium (Fv) or Phytophthora (Ps). Two root metrics and two shoot metrics were disease score (determined by visual inspection) and dry tissue weight at 14 days postplanting. Statistical evaluation of seven replicates per treatment was performed using Tukey's HSD test, with p<0.05 considered significant. The fractions in the table indicate the number of significantly positive tests observed (numerator) out of the number of viable tests completed (denominator).

[0173] If either the visual score or the weight metric was positive, it was counted as a positive result for that histological type. Root influence was considered more relevant to Ps, while shoot metrics were considered more relevant to Fv.

[0174] Example 4. dsRNAs that target genes involved in suppressing pathogen defense pathways protect plant seedlings from fungal pathogens. A dsRNA targeting GmMPK4a (GS200) was applied to soybean seedlings as a seed treatment as described in Example 2, protecting the seedlings from Rhizoctonia solani infection (Figures 4A-4B). The methods for plant growth and evaluation were the same as in Example 3, except that Rhizoctonia solani fungal inoculum was used. The root weight of Rs-infected seedlings treated with GS200 via seed was comparable to that of commercially available chemical fungicides. Both the commercial treatment and the GS200 treatment resulted in heavier roots than control R. solani-infected seedlings. Nonspecific dsRNA control treatment did not result in greater root weight compared to the infected control.

[0175] Example 5. dsRNA targeting genes involved in the suppression of pathogen defense pathways Further tests were conducted to investigate whether various RNAi molecules with trigger sequences that downregulate the expression of response repressor genes correspondingly resulted in upregulation of defense response markers. These defense response markers include, for example, PR1, PR3, and PR5. These marker genes are expressed when the plant defense response pathway is activated. The tests were performed as described in Example 2, and the expression of target response repressor genes and defense response markers was measured.

[0176] Figure 5 shows the increase in GmPR1 expression for GS200 / GmMPK4a as a percentage increase compared to the control. Individual seedlings were labeled 1-8. The control was untargeted dsRNA, and the seed immersion method from Example 2 was used with the CQD3 formulation, with GmActin as the normalizer. Primary leaves, sample 72 was 37 days post-treatment. Sample 72 used a single control group.

[0177] Figure 6 shows the increase in GmPR1 expression for GS200 / GmMPK4a as a percentage increase compared to the control. Individual seedlings were labeled 1-8. The control was LD45, and the seed immersion method was used with the CQD3 formulation, with GmActin as the normalizer. Secondary leaves were observed; sample 72 was 37 days post-treatment, sample 74 was 18 days post-treatment, and sample 76 was 23 days post-treatment. Samples 72 and 74 used a single control group. Sample 76 used the mean of two control groups. The seed immersion method from Example 2 was used.

[0178] Figure 7 shows the increase in GmPR5 expression for GS201 / GmRLK3 as a percentage increase compared to the control. Individual seedlings were labeled 1-8. The control was LD45, and the seed immersion method from Example 2 was used with the CQD3 formulation, with GmActin as the normalizer. Primary leaves, sample 72 was 37 days post-treatment. Sample 72 used a single control group.

[0179] Figure 8 shows the increase in GmPR5 expression for GS201 / GmRLK3 as a percentage increase compared to the control. Individual seedlings were labeled 1-8. The control was LD45, and the seed immersion method was used with a formulation of CQD3 + bPEI800 (heated) and normalized GmActin. Secondary leaves; sample 72 was 37 days post-treatment, sample 74 was 18 days post-treatment, and sample 76 was 23 days post-treatment. Samples 72 and 74 used a single control group. Sample 76 used the mean of two control groups. The seed immersion method from Example 2 was used.

[0180] Figure 9 shows the increase in GmPR1 expression for GS2110 / GmRIN4a as a percentage increase compared to the control. The mean control LD45 between two independent samples A and B was used for comparison with sample 79, and a single pooled group was used for sample 93. The seed immersion method from Example 2 was used with the CQD3 formulation and normalizer GmActin. Secondary leaves, sample 79 was 21 days post-treatment, and sample 93 was 22 days post-treatment.

[0181] Figure 10 shows the increase in GmPR1 expression for GS2109 / GmRIN4b as a percentage increase compared to the control. The mean control LD45 between two independent samples A and B was used for comparison of sample 79, and a single pooled group was used for sample 93. The seed immersion method from Example 2 was used with the CQD3 formulation and normalizer GmActin. Secondary leaves, sample 79 was 21 days post-treatment, and sample 93 was 22 days post-treatment. Since no significant increase in GmPR1 was detected, the sample 93 group was not tested for RIN4b expression (nt).

[0182] Figure 11 shows the increase in GmPR3 expression for GS279 / GmCEV1 as a percentage increase compared to the control. The mean control LD45 between two independent samples A and B was used for comparison of sample 77, and a single pooled group was used for sample 93. The seed immersion method from Example 2 was used together with the CQD3 formulation and normalizer GmActin. Secondary leaves, sample 77 was 28 days post-treatment, and sample 93 was 22 days post-treatment.

[0183] Figure 12 shows the increase in GmPR1 expression for GS280 / GmCOI1 as a percentage increase compared to the control. The mean control LD45 between two independent samples A and B was used for comparison of sample 77, and a single pooled group was used for sample 93. The seed immersion method from Example 2 was used with the CQD3 formulation and normalizer GmActin. Secondary leaves, sample 77 was 28 days post-treatment, and sample 93 was 22 days post-treatment.

[0184] Figure 13 shows the increase in GmPR1 expression for GS415 / GmDHS as a percentage increase compared to the control. In addition to the seed immersion method from Example 2, used with the CQD3 formulation and normalizer GmActin, control LD45 was used. Secondary leaves, sample 96 was 19 days post-treatment and sample 105 was 15 days post-treatment.

[0185] Figure 14 shows the increase in GmPR1 expression for GS289 / GmETR1:GmEIN4:GmETR2 as a percentage increase compared to the control. In addition to seed immersion methods using CQD3 formulation and normalizer GmActin, control LD45 was used. Secondary leaves, sample 96 was 19 days post-treatment, and sample 105 was 15 days post-treatment.

[0186] Figure 15 shows the increase in GmPR1 expression for GS275 / GmCPR5 as a percentage increase compared to the control. The mean control LD45 between two independent samples A and B was used for comparison of sample 77, and a single pooled group was used for sample 93. The seed immersion method from Example 2 was used with the CQD3 formulation and normalizer GmActin. Secondary leaves, sample 77 was 28 days post-treatment, and sample 93 was 22 days post-treatment.

[0187] In summary, these data indicate that dsRNA molecules targeting SAR pathway repressor genes for inhibition benefit soybean plants through the induction of PR1 gene expression, which is induced in response to various pathogens. PR1 gene expression is a useful molecular marker for SAR response.

[0188] Example 6. Increase in defense genes in spinach plants due to dsRNA delivery. dsRNA molecules targeting plant genes involved in the suppression of systemic or local defense response pathways can increase the expression of spinach defense genes. A dsRNA targeting SoWRKY33 (GS6263) was delivered to spinach seedlings using a commercially available priming method, where each lot of spinach underwent a preliminary test to determine the amount of priming water required to prime the seeds without causing germination. A typical example may be holding the seeds at 33% moisture content at 20°C for 7 days. After priming in the presence of the dsRNA was achieved, the seeds were dried back to a typical seed transport moisture content, then planted in soil pots, and grown in a greenhouse for 16 days at 21°C–25°C (15 hours) during the day and 20°C–21°C (9 hours) at night. After 16 days, the mRNA expression levels of four spinach immunogenes and the target SoWRKY33 were determined by qRT-PCR. In these treated spinach plants, the relative expression of three of the four immunogenes increased, while the relative expression of SoWRKY33 decreased. See Figure 16B.

[0189] These data demonstrate that delivery of dsRNA molecules targeting plant genes involved in the suppression of systemic or local defense response pathways can increase gene expression in the defense pathways of spinach plants (for example, by decreasing the expression of target genes and ultimately increasing the expression of genes that protect against pathogens).

[0190] Example 7. Improvement of lettuce plant health through dsRNA delivery. dsRNA molecules targeting plant genes involved in the suppression of systemic or local defense response pathways can improve the health of lettuce plants. A dsRNA (GS5461) targeting LsCPR5 was delivered to lettuce seedlings using a commercially available seed priming method. Here, individual lot of lettuce seed was evaluated through preliminary testing to determine whether the seeds were photodormant or thermal dormant, and the results were used to mathematically determine the priming temperature, duration, and biological additives required to prime a specific lot and achieve a primed state. A typical example involves priming lettuce seeds at 15°C for 7 hours in a priming solution containing a formulation of a natural plant growth regulator while exposing the seeds to narrow-spectrum red light. After the primed state was achieved in the presence of the dsRNA, the seeds were dried and returned, planted in soil, and grown for 27 days in a Fusarium chamber (i.e., a fungal pathogen environment) at a day / night temperature of 28°C / 20°C and a photoperiod of 12 / 12. Fusarium oxysporum inoculum was prepared by taking discs from Fusarium oxysporum colonies on potato dextrose agar and creating a powdered inoculum characterized by CFU per gram. For lettuce soil inoculum, a target of 1000 CFU per gram of soil was established. A control group of lettuce seeds treated with standard pesticides was included. Plant biomass (Figure 17A) and growth vigor (Figure 17B) were determined on days 7, 14, 21, and 27.

[0191] dsRNA-treated plants were found to have improved biomass and growth potential up to 27 days after initial treatment. These data demonstrate that delivery of dsRNA molecules targeting plant genes involved in the suppression of systemic or local defense response pathways can improve the health of lettuce plants (for example, by suppressing gene expression, thereby inducing activation or increased activity of defense responses).

[0192] Example 8. Field trials of dsRNA dsRNA molecules targeting plant genes involved in the suppression of systemic or local defense response pathways can improve the health of lettuce plants. A dsRNA (GS5461) targeting LsCPR5 was delivered to lettuce seedlings (three different varieties) and grown in a randomized, fully partitioned plot design at one of two field locations in Yuma, Arizona. The dsRNA delivery method was as described in Example 7. The control group received standard priming treatment without fungicides (UTC). The lettuce plants were exposed to infection by naturally occurring Fusarium pathogens in the field.

[0193] At the final harvest day, 86 days after planting, the percentage survival rate of the plants was determined by manually counting plots (Figure 18). dsRNA-treated plants were found to have improved survival rates compared to plants treated with a standard priming method that did not include fungicides. These data demonstrate that delivery of dsRNA molecules targeting plant genes involved in the suppression of systemic or local defense response pathways can improve the health of lettuce plants in the field (e.g., by increasing the expression of pathogen-immune genes).

[0194] Example 9: dsRNAs that target genes involved in suppressing pathogen defense pathways protect plant seedlings from fungal pathogens. A dsRNA targeting SoCPR5 (GS5462) was applied to spinach seedlings as a seed treatment as described in Example 6 to protect the seedlings from Fusarium oxysporum infection (Figure 20). After priming was achieved in the presence of the dsRNA, the seeds were dried and replanted in soil and grown for 27 days in a Fusarium chamber (i.e., a fungal pathogen environment) at a day / night temperature of 28°C / 20°C and a photoperiod of 12 / 12. Fusarium oxysporum inoculum was prepared by taking discs from Fusarium oxysporum colonies on potato dextrose agar and preparing a powder inoculum characterized by CFU per gram. For spinach soil inoculation, a target of 500 CFU per gram of soil was created. A control group of spinach seeds treated with standard pesticides was included. On days 7, 14, and 21, the number of healthy plants was counted and the population percentage was determined. The results are shown in Figure 20. Treatment with GS5462 dsRNA resulted in comparable survival rates to spinach plants compared to standard treatment on day 14 under Fusrium loading, and demonstrated a significant improvement (*p<0.12) in survival rate at day 21. [Table A]

[0195] Table A shows the fold change in gene expression of target genes and four defense-related genes when a series of dsRNAs targeting specific genes are included in spinach seed priming treatment, compared to the inclusion of a non-targeted dsRNA control.

[0196] Example 10. dsRNAs that target genes involved in the suppression of pathogen defense pathways can affect a representative set of defense genes. A series of dsRNAs targeting genes that control plant defense responses were applied to spinach seeds as described in Example 6, resulting in significant changes in defense pathway gene expression. dsRNA delivery was the same as in Example 9. Plant growth was the same as in Example 6. Spinach leaf tissue was sampled 16 days after planting. Results are shown in Table 2. Bold numbers indicate statistical significance (p<0.1). Target gene expression for each trigger sequence was measured by qRT-PCR, and the fold change compared to the untargeted control dsRNA is shown in the target column. Several triggers resulted in statistically significant downregulation of their respective target genes. Significant increases in target gene expression may be due to a negative feedback loop of autoregulation that potentially manifests as a time-dependent oscillation wave over the 16 days of growth. Gene expression was also measured for markers of the defense response, specifically the PR1, HARB, ZF, and bHLH genes. The column labeled with each gene name shows the fold change compared to the untargeted control. If a dash is placed after the target gene name, it indicates a paralog of the target gene, and that the expression of that particular paralog was assayed by qRT-PCR.

[0197] Example 11. Improved health and reduced mortality in lettuce plants through dsRNA delivery. dsRNA molecules targeting plant genes involved in the suppression of systemic or local defense response pathways can improve the health of lettuce plants. A dsRNA targeting LsCPR5 (GS5461) was delivered to lettuce seedlings in the same manner as in Example 7. After priming was achieved in the presence of dsRNA containing formulation CQD3, the seeds were dried and rehydrated, planted in soil, and grown at 20°C / 20°C day / night until soil germination. Subsequently, they were grown in a Fusarium chamber (i.e., a fungal pathogen environment) at 28°C / 20°C day / night and a photoperiod of 12 / 12. Fusarium soil inoculation was performed in the same manner as in Example 7. Sixty plants in three sets per treatment were scored for germination, individual plant health, and weekly plant mortality over 35 days.

[0198] dsRNA-treated plants were found to have an improved percentage of healthy plants and reduced mortality by day 35. The results are shown in Figure 21. These results demonstrate that delivery of dsRNA molecules targeting the plant gene CPR5, which is involved in the suppression of systemic or local defense response pathways, improves the health and reduces mortality in lettuce plants (for example, by suppressing gene expression, thereby inducing activation or increased activity of the defense response).

[0199] Example 12: Increase in defense genes in lettuce plants due to dsRNA delivery. dsRNA molecules targeting plant genes involved in the suppression of systemic or local defense response pathways can increase the expression of lettuce defense genes. A dsRNA targeting LsCPR5 (GS5461) or a non-targeted control was delivered to lettuce seedlings using a commercially available priming method as described in Example 7 and grown in a greenhouse as described in Example 6. After 14 days, the mRNA expression levels of the lettuce immunogene LsGT61 and the targeted LsCPR5 were determined by qRT-PCR. Compared to the non-targeted dsRNA control, LsGT61 expression was found to be increased, while LsCPR5 expression was found to be decreased *p<0.05 (presumably due to direct inhibition by dsRNA). See Figure 22.

[0200] These data demonstrate that delivery of dsRNA molecules targeting plant genes involved in the suppression of systemic or local defense response pathways can increase gene expression in the defense pathways of lettuce plants (for example, by decreasing the expression of target genes and ultimately increasing the expression of genes that protect against pathogens). [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8]

[0201] Equivalents and range Unless otherwise indicated or otherwise obvious from the context, articles such as “a,” “an,” and “the” in a claim may mean one or more. Unless otherwise indicated or otherwise obvious from the context, a claim or statement containing “or” among one or more members of a group is considered satisfied if one member, more than one member, or all members of that group are present in, adopted into, or otherwise related to a given product or process. The present invention encompasses embodiments in which exactly one member of that group is present in, adopted into, or otherwise related to a given product or process. The present invention encompasses embodiments in which more than one or all of the members of that group are present in, adopted into, or otherwise related to a given product or process.

[0202] Furthermore, the present invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the enumerated claims are introduced into another claim. For example, any claim dependent on another claim may be modified to include one or more limitations found in any other claim dependent on the same basic claim. Where elements are presented, for example, as enumerated in Markush group form, each subgroup of the element is also disclosed, and any element(s) may be removed from the group. In general, where the present invention or an aspect of the present invention is considered to include certain elements and / or features, it should be understood that certain aspects of the present invention or certain aspects of the present invention consist of, or substantially consist of, such elements and / or features. For the sake of brevity, those aspects are not specifically expressed in this specification in haec verba.

[0203] It should also be noted that the terms “comprising” and “containing” are intended to be open and allow for the inclusion of additional elements or processes. Where a range is given, the endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and the understanding of those skilled in the art, a value expressed as a range may be assumed to be any specific value or a subrange of the range described in different aspects of the invention, up to one-tenth of the lower limit of the range, unless the context explicitly indicates otherwise.

[0204] This application references various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. In the event of any conflict between any of the incorporated references and this specification, this specification shall prevail. In addition, any particular aspect of the Invention that falls within the scope of prior art may be expressly excluded from any one or more of the claims. Such aspects may be excluded even if the exclusion is not expressly stated herein, as they would be considered known to those skilled in the art. Any particular aspect of the Invention may be excluded from any of the claims for any reason, whether or not it relates to the existence of prior art.

[0205] Those skilled in the art will recognize many equivalents to the specific embodiments described herein, or at best, can verify them using routine experiments. The scope of the embodiments described herein is not intended to be limited to the foregoing, but rather as described in the appended claims. Those skilled in the art will understand that various changes and modifications to this description may be made without departing from the spirit or scope of the invention, as defined in the following claims.

Claims

1. Polynucleotides that inhibit plant genes involved in suppressing systemic or local responses to pathogens.

2. The polynucleotide according to claim 1, wherein the plant gene is involved in the suppression of a systemic response to a pathogen.

3. The polynucleotide according to claim 1, wherein the plant gene is involved in suppressing localized responses to pathogens.

4. The polynucleotide according to any one of claims 1 to 3, wherein the plant gene is involved in the inhibition of the systemic acquired resistance (SAR) pathway.

5. The polynucleotide according to any one of claims 1 to 3, wherein the pathogen is a fungal pathogen.

6. The plant genes include MPK4 (mitogen-activated protein kinase 4), CPR5 (constitutive expression of PR gene 5), MPK4a (mitogen-activated protein kinase 4a), RLK3 (receptor-like kinase / leucine-rich repeat receptor-like kinase), NPR3 (NPR-1-like protein 3 / salicylate receptor), RFC3 (replication factor C subunit 3), and SNI1 (NPR-11 inhibitor), CEV1 (constitutive expression of VSP1 / cellulose synthase family protein), COI1 (coronatin insensitivity 1), RAP (RNA binding domain), DMR6 (downy mildew resistance 6), DMR6-like (downy mildew resistance 6-like), ARR9 (response regulator 9 / two-component response regulator-like protein), NPR4 (NPR1-like protein 4 / salicylate receptor), NAC25 (encodes NAC domain-containing protein 25), BRI1a (brassinosteroid insensitivity 1a / leucine-rich receptor-like protein kinase), BRI1b (brassinosteroid insensitivity 1a / leucine-rich receptor-like protein kinase). (Lasinosteroid-insensitive 1b / leucine-rich receptor-like protein kinase), TAR2 (tryptophan aminotransferase-related 2), YUC2a (yucca / indole-3-pyruvate monooxygenase), ETR1 (ethylene response 1 / ethylene receptor histidine kinase), EIN4 (ethylene-insensitive 4 / ethylene receptor histidine kinase), ETR2 (ethylene response 2 / ethylene receptor histidine kinase), ACS1 (ACC synthase 1 / enzymatically inactive form of 1-aminocyclopropane-1-carboxylate synthase) ), CRY1b (Cryptochrome 1 / flavin-type blue light receptor), CRY2 (Cryptochrome 2 / flavin-type blue light receptor), ACO4 (1-aminocyclopropane-1-carboxylate oxidase 4), CTR1 (Constitutive triple response 1 / serine / threonine protein kinase), MPK6 (Mitogen-activated protein kinase 6), RIN4b (RPM1 interacting protein 4b), RIN4a (RPM1 interacting protein 4), RapTOR (TOR regulation-related protein), TOR (encoding the target of rapamycin), GID1B ( GA-insensitive dwarf 1B / gibberellin receptor), GID1C (GA-insensitive dwarf 1C / gibberellin receptor), DHS (deoxyhypsin synthase), GA3OX (gibberellin 3-oxidase), HOS15 (high expression of osmotically responsive genes 15 / WD-40 repeat protein), eIF_5A (eukaryotic translation initiation factor 5A), CLH2 (chlorophyllase 2), SPL12l (squamosa promoter-like protein 12), EIN5 (ethylene-insensitive 5 / 5'→3' exoribonuclease), WRKY33 (WRKYA polynucleotide according to any one of claims 1 to 5, selected from the group consisting of DNA-binding protein 33 (DNA-binding transcription factor), WRKY11 (WRKY DNA-binding protein 11 (DNA-binding transcription factor)), BZR1 (brassinazole resistance 1 (DNA-binding transcription repressor)), EDR1 (enhanced disease resistance 1 (protein kinase)), EXA1 (essential for Potexvirus accumulation 1), WRKY40 (WRKY DNA-binding protein 40 (DNA-binding transcription factor)), JAZ7 (encoding jasmonic acid ZIM domain protein 7), PW220 (JAZ10 (wound-inducing promoter 220 (jasmonic acid ZIM domain protein 10)), RPLK3 (receptor-like protein kinase 3-like (leucine-rich receptor-like protein kinase family protein (AT5G65700) ortholog)), JAZ (jasmonic acid ZIM domain protein) and PAM16 (presequence translocase associated motor 16).

7. The polynucleotide according to any one of claims 1 to 6, wherein the plant genes are MPK4a, RLK3, RIN4a, CEV1, CPR5, COI1, DHS, ETR1, EIN4, ETR2, PR1, PR3, PR5, and WRKY40 genes.

8. The polynucleotide according to claim 7, wherein the plant gene is the CPR5 gene.

9. The polynucleotide according to any one of claims 1 to 8, wherein the plant gene is derived from Arabidopsis thaliana (At), Glycine max (Gm), Lactuca sativa (Ls), Nicotiana benthamiana (Nb), Solanum lycopersicum (Sl), or Spinacia oleracea (So).

10. The polynucleotide according to any one of claims 1 to 9, wherein the plant gene is derived from a Solanaceae plant, Brassicaceae plant, Poaceae plant, Cucurbitaceae plant, Fobaceae plant, Apiaceae plant, Amaranthaceae plant, or Malvaceae plant.

11. The plant genes are AtMPK4, AtCPR5, GmMPK4a, GmRLK3, GmCPR5, GmNPR3, GmRFC3, GmSNI1, GmCEV1, GmCOI1, GmRAP, GmDMR6-like, GmARR9, GmNPR4, GmNAC25, GmBRI1a, GmBRI1b, GmTAR2, GmYUC2a, GmETR1 / EIN4 / ET R2, GmACS1, GmCRY1b, GmCRY2, GmACO4, GmCTR1, GmMPK6, GmRIN4b, GmRIN4a, GmRapTOR, GmTOR, GmGID 1B, GmGID1C, GmDHS, GmGA3OX, GmHOS15, GmeIF_5A, GmCLH2, GmSPL12l, LsCPR5, LsCPR5, NbEIN5, SlE A polynucleotide according to any one of claims 1 to 10, selected from the group consisting of IN5, SlCPR5, SoWRKY33, SoWRKY11, SoHOS15, SoBZR1, SoEDR1, SoEXA1, SoWRKY40, SoJAZ7, SoPW220, SoMPK6, SoSPL12I, SoCEV1, SoCPR5, SoCOI1, SoDMR6, SoDHS, SomelF-5A, SoETR1, SoEIN4, SoETR2, SoCTR1, SoMPK4a, SoRPLK3, SoBZR1, SoWRKY40, SoJAZ, SoDHS, SomelF5-A, SoMPK4a, SoETR1, SoSEIN4, SoETR2, SoETR1 / EIN4 / ETR2, SoCEV1, and SoCPR5.

12. The polynucleotide according to any one of claims 1 to 11, wherein the polynucleotide is ribonucleic acid (RNA).

13. The polynucleotide according to any one of claims 1 to 12, wherein the polynucleotide is double-stranded RNA.

14. The polynucleotide according to claim 13, wherein the RNA is a double-stranded RNA (dsRNA) comprising a first strand complementary to a segment of the coding region of messenger RNA (mRNA) encoded by a plant gene, and a second strand complementary to the first strand.

15. The polynucleotide according to any one of claims 1 to 12, wherein the polynucleotide is single-stranded RNA.

16. The polynucleotide according to any one of claims 1 to 15, wherein the plant gene comprises a nucleic acid sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with any one of sequence numbers 1 to 86 or 265 to 274.

17. The polynucleotide according to any one of claims 1 to 16, wherein the polynucleotide comprises a nucleic acid sequence identical or fully complementary to at least 18 consecutive nucleotides of a plant gene and / or a nucleic acid sequence 90% identical or complementary to at least 21 consecutive nucleotides.

18. The polynucleotide according to any one of claims 1 to 17, wherein the polynucleotide comprises a nucleic acid sequence identical to or completely complementary to at least 21 consecutive nucleotides.

19. The polynucleotide according to any one of claims 1 to 18, wherein the polynucleotide comprises a nucleic acid sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity with any one of sequence numbers 87-172 or 275-282.

20. The polynucleotide according to any one of claims 1 to 19, wherein the polynucleotide comprises at least 18, 19, 20, 21, 25, 50, 100, 150, 200, 250, 300, 400, or 450 consecutive nucleotides, which are complementary to a segment of DNA or a target gene having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 86 or 265 to 274, or which contain at least about 85%, at least 90%, at least 95%, at least about 98%, or about 100% sequence identity.

21. The polynucleotide according to any one of claims 1 to 20, comprising at least 18, 19, 20, 21, 25, 50, 100, 150, 200, 250, 300, 350, 400, or 450 consecutive nucleotides, wherein the polynucleotide comprises at least 18, 19, 20, 21, 25, 50, 100, 150, 200, 250, 300, 350, 400, or 450, which are complementary to a sequence segment selected from the group consisting of SEQ ID NOs. 87–258 or 275–290, or which have at least about 85%, at least about 90%, at least about 95%, at least about 98%, about 100%, or 100% sequence identity.

22. A dsRNA comprising a first strand containing at least 18, 19, 20, 21, 25, 50, 100, 150, 200, 250, 300, 350, 400, or 450 consecutive nucleotides, wherein the polynucleotide is complementary to a sequence segment selected from the group consisting of SEQ ID NOs. 87–172 and 275–282, or contains at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 98%, approximately 100%, or 100% sequence identity; and a second strand complementary to the first strand. The polynucleotide according to claim 21, further comprising a second chain comprising 18, 19, 20, 21, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 consecutive nucleotides, which are complementary to a corresponding reverse complementary sequence segment selected from the group consisting of SEQ ID NOs. 173–258, 283–290, or which contain at least about 85%, at least about 90%, at least about 95%, at least about 98%, about 100%, or 100% sequence identity.

23. The polynucleotide according to claim 22, wherein the polynucleotide comprises at least about 200 consecutive nucleotides that are complementary to or have at least about 90% identity with a sequence segment selected from the group consisting of SEQ ID NOs. 87-258 or 275-290.

24. The polynucleotide according to claim 20, wherein the DNA or target gene has a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 5, 35, 36, 41, 42, 62, 85, and 86.

25. The polynucleotide according to claim 24, wherein the polynucleotide comprises at least 18, 19, 20, 21, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 consecutive nucleotides, which are complementary to a sequence segment selected from the group consisting of SEQ ID NOs. 88, 91, 121, 122, 127, 128, 148, 171, 172, and 174, 177, 207, 208, 213, 214, 234, 257, and 285, or which contain at least about 85%, at least about 90%, at least about 95%, at least about 98%, about 100%, or 100% sequence identity.

26. The polynucleotide according to claim 25, wherein the polynucleotide comprises at least about 200 consecutive nucleotides that are complementary to or have at least about 90% identity with a sequence segment selected from the group consisting of SEQ ID NOs: 88, 91, 121, 122, 127, 128, 148, 171, 172, 174, 177, 207, 208, 213, 214, 234, 257, and 285.

27. The polynucleotide according to claim 20, wherein the DNA or target gene has a nucleotide sequence selected from the group consisting of SEQ ID NOs: 3, 74, 75, 76, and 82.

28. The polynucleotide according to claim 27, wherein the polynucleotide comprises at least 18, 19, 20, 21, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 consecutive nucleotides, which are complementary to a sequence segment selected from the group consisting of SEQ ID NOs. 89, 160, 161, 162, 168, 175, 246, 247, 248, and 254, or which contain at least about 85%, at least about 90%, at least about 95%, at least about 98%, about 100%, or 100% sequence identity.

29. The polynucleotide according to claim 28, wherein the polynucleotide comprises at least about 200 consecutive nucleotides that are complementary to or have at least about 90% identity with a sequence segment selected from the group consisting of SEQ ID NOs: 89, 160, 161, 162, 168, 175, 246, 247, 248, and 254.

30. The polynucleotide according to claim 20, wherein the DNA or target gene has the nucleotide sequence of SEQ ID NO:

4.

31. The polynucleotide according to claim 30, comprising at least 18, 19, 20, 21, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 consecutive nucleotides, wherein the polynucleotide comprises

32. The polynucleotide according to claim 31, wherein the polynucleotide comprises at least about 200 consecutive nucleotides that are complementary to or have at least about 90% identity with a sequence segment selected from the group consisting of SEQ ID NOs. 90 and 176.

33. The polynucleotide according to claim 20, wherein the DNA or target gene has the nucleotide sequence of SEQ ID NO:

25.

34. The polynucleotide according to claim 33, comprising at least 18, 19, 20, 21, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 consecutive nucleotides, wherein the polynucleotide comprises which are complementary to a sequence segment selected from the group consisting of SEQ ID NOs: 111 and 197, or contain at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least 100%, or 100% sequence identity.

35. The polynucleotide according to claim 34, wherein the polynucleotide comprises at least about 200 consecutive nucleotides that are complementary to or have at least about 90% identity with a sequence segment selected from the group consisting of SEQ ID NOs. 111 and 197.

36. The polynucleotide according to claim 20, wherein the DNA or target gene has a nucleotide sequence selected from the group consisting of SEQ ID NOs: 9, 59, 60, 61, and 84.

37. The polynucleotide according to claim 36, wherein the polynucleotide comprises at least 18, 19, 20, 21, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 consecutive nucleotides, which are complementary to a sequence segment selected from the group consisting of SEQ ID NOs. 95, 181, 145, 231, 146, 232, 147, 233, 170, and 256, or which contain at least about 85%, at least about 90%, at least about 95%, at least about 98%, about 100%, or 100% sequence identity.

38. The polynucleotide according to claim 37, wherein the polynucleotide comprises at least about 200 consecutive nucleotides that are complementary to or have at least about 90% identity with a sequence segment selected from the group consisting of SEQ ID NOs: 95, 181, 145, 231, 146, 232, 147, 233, 170, and 256.

39. The polynucleotide according to claim 20, wherein the DNA or target gene has a nucleotide sequence selected from the group consisting of SEQ ID NOs: 10 and 63.

40. The polynucleotide according to claim 39, wherein the polynucleotide comprises at least 18, 19, 20, 21, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 consecutive nucleotides, the polynucleotide comprising a sequence segment selected from the group consisting of SEQ ID NOs. 96, 182, 149, and 235, or containing at least about 85%, at least about 90%, at least about 95%, at least about 98%, about 100%, or 100% sequence identity.

41. The polynucleotide according to claim 40, wherein the polynucleotide comprises at least about 200 consecutive nucleotides that are complementary to or have at least about 90% identity with a sequence segment selected from the group consisting of SEQ ID NOs. 96, 182, 149, and 235.

42. The polynucleotide according to claim 20, wherein the DNA or target gene has a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29, 65, 66, and 80.

43. The polynucleotide according to claim 42, wherein the polynucleotide comprises at least 18, 19, 20, 21, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 consecutive nucleotides, the polynucleotide comprising at least 18, 19, 20, 21, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 consecutive nucleotides, which are complementary to a sequence segment selected from the group consisting of SEQ ID NOs. 115, 201, 151, 237, 152, 238, 166, and 252, or which contain at least about 85%, at least about 90%, at least about 95%, at least about 98%, about 100%, or 100% sequence identity.

44. The polynucleotide according to claim 43, wherein the polynucleotide comprises at least about 200 consecutive nucleotides that are complementary to or have at least about 90% identity with a sequence segment selected from the group consisting of SEQ ID NOs: 115, 201, 151, 237, 152, 238, 166, and 252.

45. The polynucleotide according to claim 20, wherein the DNA or target gene has a nucleotide sequence selected from the group consisting of SEQ ID NOs: 18 and 83.

46. The polynucleotide according to claim 45, wherein the polynucleotide comprises at least 18, 19, 20, 21, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 consecutive nucleotides, the polynucleotide being complementary to a sequence segment selected from the group consisting of SEQ ID NOs. 104, 190, 169, and 255, or containing at least about 85%, at least about 90%, at least about 95%, at least about 98%, about 100%, or 100% sequence identity.

47. The polynucleotide according to claim 46, wherein the polynucleotide comprises at least about 200 consecutive nucleotides that are complementary to or have at least about 90% identity with a sequence segment selected from the group consisting of SEQ ID NOs: 104, 190, 169, and 255.

48. The polynucleotide according to claim 20, wherein the DNA or target gene has a nucleotide sequence selected from the group consisting of SEQ ID NOs. 52, 53, and 78.

49. The polynucleotide according to claim 48, wherein the polynucleotide comprises at least 18, 19, 20, 21, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 consecutive nucleotides, which are complementary to a sequence segment selected from the group consisting of SEQ ID NOs. 138, 224, 139, 225, 164, and 250, or which contain at least about 85%, at least about 90%, at least about 95%, at least about 98%, about 100%, or 100% sequence identity.

50. The polynucleotide according to claim 49, wherein the polynucleotide comprises at least about 200 consecutive nucleotides that are complementary to or have at least about 90% identity with a sequence segment selected from the group consisting of SEQ ID NOs: 138, 224, 139, 225, 164, and 250.

51. The polynucleotide according to any one of claims 1 to 50, wherein the polynucleotide has a length of 18 to 600 nucleotides, 21 to 600 nucleotides, 50 to 600 nucleotides, 100 to 600 nucleotides, or 200 to 600 nucleotides.

52. The polynucleotide according to any one of claims 1 to 51, wherein the polynucleotide inhibits at least two plant genes involved in the suppression of a systemic or local response to a pathogen.

53. The polynucleotide according to any one of claims 1 to 52, wherein the polynucleotide is a chimeric polynucleotide comprising at least two different nucleic acid sequences, each of which inhibits a different plant gene or paralog involved in the suppression of a systemic or local response to a pathogen.

54. The polynucleotide according to claim 52 or 53, wherein the polynucleotide comprises a first nucleic acid that inhibits ETR1, a second nucleic acid that inhibits EIN4, and a third nucleic acid that inhibits ETR2.

55. The polynucleotide according to any one of claims 1 to 54, wherein inhibiting a plant gene involved in the suppression of a systemic or local defense response further results in increased expression of one or more genes involved in inducing or performing a systemic or local defense response, wherein optionally, one or more genes are selected from the group consisting of the PR1 gene, PR3 gene, PR5 gene, HARB gene, ZF gene, GT61 gene, and bHLH gene.

56. A vector encoding a polynucleotide according to any one of claims 1 to 55.

57. The vector according to claim 56, wherein the vector is a plasmid or a viral vector.

58. A DNA construct comprising a heterologous promoter operably ligated to a sequence encoding a polynucleotide according to any one of claims 1 to 55.

59. DNA encoding a polynucleotide according to any one of claims 1 to 58.

60. A composition comprising a polynucleotide according to any one of claims 1 to 58.

61. The composition according to claim 60, further comprising carbon quantum dots.

62. The composition according to claim 60 or 61, further comprising a ligand.

63. The composition according to claim 62, wherein the ligand is polyethylene glycol (PEG), a carbohydrate, a porphyrin conjugate, a peptide, or a lipid.

64. The composition according to claim 63, wherein PEG is PEG200, PEG1000, PEG2000, PEG5000, or PEG10000.

65. Seeds that have been in contact with a polynucleotide or composition according to any one of claims 1 to 55.

66. The seeds according to claim 65, wherein the seeds are selected from the group consisting of Arabidopsis thaliana (At) seeds, Glycine max (Gm) seeds, Lactuca sativa (Ls) seeds, Nicotiana benthamiana (Nb) seeds, Solanum lycopersicum (Sl) seeds, and Spinacia oleracea (So) seeds.

67. Seeds according to any one of claims 65 to 66, wherein the seeds are in contact via seed immersion.

68. The seeds according to any one of claims 65 to 67, wherein the seeds have improved resistance to pathogen infection compared to control seeds that have not been contacted with polynucleotides or the composition.

69. The seed according to claim 68, wherein the improved resistance results in a reduction in the surface area of ​​the plant portion affected by the pathogen.

70. The seed according to claim 69, wherein the pathogen is a fungal pathogen.

71. The seed according to claim 69, wherein the plant part is a leaf.

72. A method for increasing resistance to pathogens in plants, comprising activating a systemic or local defense response in the plant to the pathogen prior to the plant's exposure to the pathogen.

73. A method for increasing resistance to pathogens in plants, comprising increasing the activity of a systemic or local defense response to the pathogen.

74. The method according to claim 72 or 73, comprising activating or increasing the activity of a systemic response to a pathogen.

75. The method according to claim 72 or 73, comprising activating or increasing the activity of a local response to a pathogen.

76. A method for increasing the lifespan of a plant or the average lifespan of a plant population, or for increasing the biomass of a plant population, or for increasing the yield of a plant population, or for reducing the surface area affected by infection, the method comprising activating a systemic defense response to a pathogen prior to the exposure of a plant or plant population to a pathogen.

77. The method according to any one of claims 72 to 76, wherein the step of activating a systemic defense response includes inhibiting one or more plant genes involved in the suppression of the systemic response in a plant.

78. A systemic defense response against fungal pathogens or other microorganisms, one or more plant genes, according to claim 77.

79. The method according to claim 77 or 78, wherein one or more plant genes are involved in the inhibition of the systemic acquired resistance (SAR) pathway.

80. The method according to claim 77 or 78, wherein the step of activating a systemic defense response further comprises increasing the expression of one or more genes involved in inducing or performing a systemic defense response, wherein the one or more genes are optionally selected from the group consisting of PR1, PR3, PR5, HARB, ZF, GT61, and bHLH genes.

81. The method according to any one of claims 77 to 80, comprising inhibiting one or more plant genes involved in the suppression of systemic or local defense responses, or delivering a polynucleotide or protein to a plant that inhibits the expression of one or more plant genes.

82. The method according to claim 81, wherein the polynucleotide that inhibits expression functions via an RNA interference (RNAi) mechanism.

83. One or more plant genes are MPK4 (mitogen-activated protein kinase 4), CPR5 (constitutive expression of PR gene 5), MPK4a (mitogen-activated protein kinase 4a), RLK3 (receptor-like kinase / leucine-rich repeat receptor-like kinase), NPR3 (NPR-1-like protein 3 / salicylate receptor), RFC3 (replication factor C subunit 3), SNI1 (NPR-11 inhibitor), CEV1 (constitutive expression of VSP1 / cellulose synthase family protein), COI1 (coronatin insensitivity 1), RAP (RNA binding domain), DMR6 (downy mildew resistance 6), DMR6-like (downy mildew resistance 6-like), ARR9 (response regulator 9 / two-component response regulator-like protein), NPR4 (NPR1-like protein 4 / salicylate receptor), NAC25 (encodes NAC domain-containing protein 25), BRI1a (brassinosteroid insensitivity 1a / leucine-rich receptor-like protein kinase), BRI1b (brassinosteroid insensitivity 1a / leucine-rich receptor-like protein kinase). (Lasinosteroid-insensitive 1b / leucine-rich receptor-like protein kinase), TAR2 (tryptophan aminotransferase-related 2), YUC2a (yucca / indole-3-pyruvate monooxygenase), ETR1 (ethylene response 1 / ethylene receptor histidine kinase), EIN4 (ethylene-insensitive 4 / ethylene receptor histidine kinase), ETR2 (ethylene response 2 / ethylene receptor histidine kinase), ACS1 (ACC synthase 1 / enzymatically inactive form of 1-aminocyclopropane-1-carboxylate synthase) ), CRY1b (Cryptochrome 1 / flavin-type blue light receptor), CRY2 (Cryptochrome 2 / flavin-type blue light receptor), ACO4 (1-aminocyclopropane-1-carboxylate oxidase 4), CTR1 (Constitutive triple response 1 / serine / threonine protein kinase), MPK6 (Mitogen-activated protein kinase 6), RIN4b (RPM1 interacting protein 4b), RIN4a (RPM1 interacting protein 4), RapTOR (TOR regulation-related protein), TOR (encoding the target of rapamycin), GID1B ( GA-insensitive dwarf 1B / gibberellin receptor), GID1C (GA-insensitive dwarf 1C / gibberellin receptor), DHS (deoxyhypsin synthase), GA3OX (gibberellin 3-oxidase), HOS15 (high expression of osmotically responsive genes 15 / WD-40 repeat protein), eIF_5A (eukaryotic translation initiation factor 5A), CLH2 (chlorophyllase 2), SPL12l (squamosa promoter-like protein 12), EIN5 (ethylene-insensitive 5 / 5'→3' exoribonuclease), WRKY33 (WRKYThe method according to any one of claims 77 to 82, selected from the group consisting of DNA-binding protein 33 (DNA-binding transcription factor), WRKY11 (WRKY DNA-binding protein 11 (DNA-binding transcription factor)), BZR1 (brassinazole resistance 1 (DNA-binding transcription repressor)), EDR1 (enhanced disease resistance 1 (protein kinase)), EXA1 (essential for Potexvirus accumulation 1), WRKY40 (WRKY DNA-binding protein 40 (DNA-binding transcription factor)), JAZ7 (encoding jasmonic acid ZIM domain protein 7), PW220 (JAZ10 (wound-inducing promoter 220 (jasmonic acid ZIM domain protein 10)), RPLK3 (receptor-like protein kinase 3-like (leucine-rich receptor-like protein kinase family protein (AT5G65700) ortholog)), JAZ (jasmonic acid ZIM domain protein) and PAM16 (presequence translocase associated motor 16).

84. One or more plant genes are AtMPK4, AtCPR5, GmMPK4a, GmRLK3, GmCPR5, GmNPR3, GmRFC3, GmSNI1, GmCEV1, GmCOI1, GmRAP, GmDMR6-like, GmARR9, GmNPR4, GmNAC25, GmBRI1a, GmBRI1b, GmTAR2, GmYUC2a, GmETR1, EIN4, ETR2, GmACS1, GmCRY1b, GmCRY2, GmACO4, GmCTR1, GmMPK6, GmRIN4b, GmRIN4a, GmRapTOR, GmTOR, GmGID1B, GmGID1C, GmDHS, GmGA3O X, GmHOS15, GmeIF_5A, GmCLH2, GmSPL12l, LsCPR5, LsCPR5, NbEIN5, SlEIN5, SlCPR5, SoWRKY33, SoWRKY11, SoHOS15, SoBZR1, SoEDR1, SoEXA1, SoWRKY40, SoJAZ7, SoP W220, SoMPK6, SoSPL12I, SoCEV1, SoCPR5, SoCOI1, SoDMR6, SoDHS, SomelF-5A, SoETR1, SoEIN4, SoETR2, SoCTR1, SoMPK4a, SoRPLK3, SoBZR1, SoWRKY40, SoJAZ, SoDHS The method according to any one of claims 77 to 82, selected from the group consisting of SomelF5-A, SoMPK4a, SoETR1, SEIN4, SoETR2, SoCEV1, and SoCPR5.

85. The method according to any one of claims 72 to 84, wherein inhibiting one or more plant genes is a means of delivering a polynucleotide according to any one of claims 1 to 55 or a composition according to any one of claims 60 to 64 to a plant.

86. The method according to any one of claims 81 to 85, wherein the polynucleotide is delivered to the plant at the seedling or seed stage of the plant life cycle.

87. The method according to claim 85, wherein the polynucleotide is delivered to the leaves or roots (one or more) or seeds of a plant.

88. The method according to claim 87, wherein the polynucleotide is delivered by immersing one or more plant seeds.

89. The method according to any one of claims 72 to 88, wherein the polynucleotide comprises a nucleic acid sequence complementary to homologous or functionally redundant plant genes involved in the suppression of systemic or local defense responses.

90. The method according to claim 89, wherein the polynucleotide and homologous or functionally redundant plant genes are derived from the same species.

91. The method according to any one of claims 72 to 90, wherein the plant is selected from the group consisting of Arabidopsis thaliana (At), Glycine max (Gm), Lactuca sativa (Ls), Nicotiana benthamiana (Nb), Solanum lycopersicum (Sl), and Spinacia oleracea (So).

92. The method according to any one of claims 72 to 91, wherein the plant is selected from the group consisting of Solanaceae plants, Brassicaceae plants, Poaceae plants, Cucurbitaceae plants, Fobaceae plants, Apiaceae plants, Amaranthaceae plants, and Malvaceae plants.

93. The method according to any one of claims 72 to 92, wherein the plant is a vegetable, fruit, grain, or legume.

94. The method according to any one of claims 72 to 92, wherein the plant is a row crop.

95. The method according to any one of claims 72 to 92, wherein the plant is corn, grains, cotton, fruits, nuts, rice, soybeans, oilseed crops, vegetables, legumes, fiber crops, ornamental plants, and spices.

96. The method according to any one of claims 72 to 92, wherein the plant is soybean, spinach, lettuce, cauliflower, pea sprout, rice, or wheat.

97. The method according to any one of claims 72 to 96, wherein the pathogen is a bacterial pathogen, a viral pathogen, or a fungal pathogen.

98. The method according to claim 97, wherein the pathogen is a fungal pathogen.

99. The method according to claim 98, wherein the pathogen is Fusarium spp., Phakospora spp., Rhizoctonia spp., Aspergillus spp., Gibberella spp., Pyricularia spp., Alternaria spp., and Phytophthora spp.

100. The method according to claim 99, wherein the pathogen is Rhizoctonia solani.

101. The method according to claim 99 or 100, wherein the fungal pathogen belongs to the genus Fusarium or Phytophthora.

102. The method according to claim 101, wherein the fungal pathogen is Fusarium oxysporum.

103. The method according to any one of claims 72 to 102, wherein the systemic or local protective response pathway is the systemic acquired resistance (SAR) pathway.

104. The method according to any one of claims 72 to 103, further comprising determining the expression level of one or more systemic or local defense response pathway genes.

105. The method according to claim 40, wherein the expression levels of the PR, HARB, ZF, or bHLH gene are determined.

106. The method according to claim 105, wherein the PR gene is PR1, PR3, or PR5.

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