System and methods for the biocontrol of plant pathogens
The inventive technology relates to novel systems, methods, and strategies for the control of plant pathogens and herbivores. The inventive technology may specifically include novel compositions, systems, and methods configured to deliver inhibitory RNA molecules to a pathogen-infected plant. In a preferred embodiment, the invention may include genetically engineered endophytic bacteria configured to deliver one or more inhibitory RNA molecules to a pathogen-infected plant. One preferred embodiment may include a novel trans-kingdom delivery of hairpin RNA targeting viral encoded proteins resulting in the reduction of viral protein accumulation levels.
1. A method for the bio-control of a plant pathogen comprising the steps of:
providing a genetically modified endophyte bacteria that is an RNaseIII mutant having suppressed RNaseIII activity relative to a wild type endophyte bacteria, wherein said genetically modified endophyte bacteria is also transformed with a nucleic acid construct comprising a polynucleotide sequence operably linked to a promoter encoding at least one small inhibitory ribonucleic acid (siRNA) that forms a double-stranded RNA (dsRNA) or a hairpin (hpRNA) directed to at least one target sequence in a plant pathogen, wherein said genetically modified endophyte bacteria is Bacillus subtilis or Bacillus cereus ; and
introducing said genetically modified endophyte bacteria to a plant,
wherein said genetically modified endophyte bacteria colonizes or lives in the leaves, stem, or roots of said plant and stably expresses said dsRNA or hpRNA in said plant for a time sufficient to induce an RNA interference (RNAi) mechanism in said plant that downregulates said at least one target sequence in a plant pathogen.
2. The method of claim 1 , wherein said step of introducing said genetically modified endophyte bacteria to a plant comprises a method selected from the group consisting of: soaking, spraying, injecting, feeding, introducing to through an aerosolized disbursement, introducing to through an environmental aerosolized disbursement, introducing to through an environmental aerosolized disbursement in water sources, brushing, dressing, dripping, and coating the mosquito and wherein during the introduction the bacteria is lyophilized, freeze-dried, microencapsulated, desiccated, in an aqueous carrier or in a solution.
3. The method of claim 2 , wherein said plant is a monocot or a dicot.
4. The method of claim 3 , wherein said plant is selected from the group consisting of: grains, corn, wheat, rice, barley, oats, sorghum, millet, sunflower, safflower, cotton, soy, canola, alfalfa, Arabidopsis, cannabis , potato, Brassica , peanut, tobacco, tropical fruits and flowers, banana, duckweed, gladiolus , sugar cane, pineapples, dates, onions, pineapple, cashews, pistachios, flowers, ornamentals, conifers, deciduous, grapes, citrus, roses, apples, peaches, strawberries, almonds, coffee, oaks, beans, legumes, watermelon, squashes, cabbage, turnip, mustard, cacti, pecans, flax, sweet potato, soybean, coconut, avocado, beets, cantaloupe and vegetables.
5. The method of claim 4 , wherein said plant pathogen comprises a plant pathogen selected from the group consisting of: a plant virus; a plant viroid; a fungus; a pest, a herbivore, and a mold.
6. The method of claim 5 , wherein said plant virus is selected from the group consisting of: Tobacco mosaic virus (TMV); Tomato spotted wilt virus (TSWV); Tomato yellow leaf curl virus (TYLCV); Cucumber mosaic virus (CMV); Potato virus Y (PVY); Cauliflower mosaic virus (CaMV); African cassava mosaic virus (ACMV); Plum pox virus (PPV); Brome mosaic virus (BMV); Potato virus X (PVX); Citrus tristeza virus, Barley yellow dwarf virus, Potato leafroll virus and Tomato and bushy stunt virus.
7. The method of claim 6 , wherein said target sequence is selected from the group consisting of a sequence of a critical region of a virus, a conserved sequence of a family of viruses, and a conserved sequence among members of different viral families.
8. The method of claim 6 , wherein said target sequence which is suppressed is selected from the group consisting of: replicase gene, movement protein, coat protein in said Tobacco mosaic virus; glycoproteins (GPs), NSm protein, NSs protein, virus RNA genomic segment L, M, S in said Tomato spotted wilt virus; V1, V2, C1, C2, C3, C4, V1 protein in said Tomato yellow leaf curl virus; 1a, 2a2b protein, 3a, coat protein in said Cucumber mosaic virus; P1, HC-Pro, P3, 6k1, CI, 6k2, NIa, Nib, CP in said Potato virus Y; Movement protein, two aphid transmission factors (P2 and/or P3), the precursor of the capsid proteins (P4), polyprotein precursor of proteinase, P5, P6 protein in said Cauliflower mosaic virus; AV1, AV2, AC1, AC2, AC3, AC4, BC1, BV1 in said African cassava mosaic virus; P1, HC-Pro, P3, 6k1, CI, 6k2, NIa, Nib, CP in said Plum pox virus; 1a, 2a, movement protein, coat protein in said Brome mosaic virus; replicase, TGB1, TGB2, TGB3, coat protein in said Potato virus X.
9. The method of claim 8 , wherein said target sequence is selected from the group consisting of a sequence of a critical region of a fungus essential gene, a conserved sequence of a family of essential genes, and a conserved essential genes among members of different viral families.
10. The method of claim 9 , wherein said target sequence is selected from the group consisting of: a non-coding region of RNA, a coding region of RNA; the target sequence containing a splice site of RNA.
11. The method of claim 1 , wherein the nucleic acid construct is a plasmid.
12. The method of claim 11 , wherein said plasmid is modified to include the target sequence from the group of plasmids consisting of: pAD-WRKY-GHY1; pAD-ADH-GHY2; pOXB-WRKY-GHY3; pOXB-ADH-GHY4; pAD-WRKY-GHY5; hpRNA pAD-ADH-GHY6; and hpRNA pAD-WRKY-GHY7.
13. The method of claim 1 , wherein said promoter comprises a promoter selected from the group consisting of: a non-constitutive promotor; an inducible promotor, a pathogen-inducible promoter; a tissue-preferred; a tissue-specific promotor, a plant-specific promotor, or a constitutive promotor.
14. The method of claim 1 , wherein said siRNA migrates throughout the plant.
15. The method of claim 1 , wherein said genetically modified endophyte bacteria continually expresses said nucleic acid construct thereby initiating a sustained RNAi mechanism in said plant, wherein said plant has a greater than wildtype resistance to said plant pathogen infection.
16. The method of claim 1 , wherein said genetically modified endophyte bacteria further co-expresses in said plant a nucleotide sequence operably linked to a promoter encoding at least one helper gene.
17. The method of claim 1 , wherein said genetically modified endophyte bacteria further co-expresses at least one additional siRNA molecule in said plant directed to at least one different target sequence in a plant pathogen.
18. The method of claim 16 , wherein said helper gene is selected from the group consisting of: VrrA; SID1; SID2; AGO1; AGO2; AGO7; YmdB; YmdB; Staufen; RDE-4; HlyA; Sec- and Tat-secretory signal peptides; one or more cell-penetrating peptides (CPPs); Tat; Antennapedia; ACC deaminase; one or more phloem RNA transporters; PP2-A1; PSRP1; DRB1; DRB4; HEN1 and STV1.
19. The method of claim 1 , wherein said siRNA is configured to have one or more specific RNA motifs incorporated into said dsRNA to facilitate its transmission through phloem of the plant.
20. The method of claim 1 , wherein said polynucleotide sequence encoding said at least one small inhibitory ribonucleic acid (siRNA) is selected from the group consisting of SEQ ID NOs. 30, 31, 32, 33, 34, 35 and 36.