EP4712771A2 - Compositions et procédés de gestion de la résistance aux pesticides - Google Patents

Compositions et procédés de gestion de la résistance aux pesticides

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Publication number
EP4712771A2
EP4712771A2 EP24808135.8A EP24808135A EP4712771A2 EP 4712771 A2 EP4712771 A2 EP 4712771A2 EP 24808135 A EP24808135 A EP 24808135A EP 4712771 A2 EP4712771 A2 EP 4712771A2
Authority
EP
European Patent Office
Prior art keywords
minicell
pesticide
agricultural composition
agricultural
gene
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP24808135.8A
Other languages
German (de)
English (en)
Inventor
Ameer Hamza SHAKEEL
Joseph Frank
Sepehr ZOMORODI
Payam POURTAHERI
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Agrospheres Inc
Original Assignee
Agrospheres Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Agrospheres Inc filed Critical Agrospheres Inc
Publication of EP4712771A2 publication Critical patent/EP4712771A2/fr
Pending legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/60—Isolated nucleic acids
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/48—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with two nitrogen atoms as the only ring hetero atoms
    • A01N43/56—1,2-Diazoles; Hydrogenated 1,2-diazoles
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • A01N63/22—Bacillus
    • A01N63/23—B. thuringiensis
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P3/00—Fungicides
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P7/00—Arthropodicides
    • A01P7/04—Insecticides

Definitions

  • the present disclosure generally relates to systems, compositions, and methods for managing pesticide resistance.
  • the present disclosure relates to use of minicell-based RNA for managing pesticide resistance and restoring one or more pests’ susceptibility to pesticides.
  • the present disclosure also relates to systems, compositions, and methods for delaying development of resistance to pesticides using minicell-based RNA.
  • Pesticides largely including insecticides, fungicides, herbicides, molluscicides, avicides, rodenticides, and pediculicides, have been globally utilized to control pests by both chemical and biological methods. Repeated use of the same pesticides can trigger undesirable genetic changes in a pest, which leads to another form of artificial selection, that is pesticide resistance. Pesticide resistance develops due to continuous use of chemical pesticides with the decreased susceptibility of a pest population to a pesticide that was previously effective at controlling the pest.
  • the present disclosure provides an agricultural composition comprising a first minicell encapsulating a nucleic acid that is capable of inducing RNA interference in an agricultural pest.
  • the agricultural composition further comprises a second minicell encapsulating the pesticide that is capable of killing or controlling the agricultural pest.
  • the nucleic acid reduces resistance to or tolerance of a pesticide in the agricultural pest.
  • the nucleic acid is capable of recovering the agricultural pest’s sensitivity or susceptibility to the pesticide. In some embodiments, the nucleic acid is capable of altering expression of a gene responsible for pesticide resistance or tolerance. In some embodiments, the expression of the gene responsible for pesticide resistance or tolerance is downregulated or upregulated. [0009] In some embodiments, the gene responsible for pesticide resistance or tolerance is an ion channel gene, a detoxification gene, a target site resistance gene, or a transporter gene. In some embodiments, the ion channel gene is a gene encoding Ryanodine receptor (RyR) or Voltage-gated sodium channel (VGSC).
  • RyR Ryanodine receptor
  • VGSC Voltage-gated sodium channel
  • the detoxification gene is selected from the group consisting of a gene encoding UDP-glycosyltransferase (UGT), Cytochrome P450 monooxygenase, Esterase, Carboxylesterase (CarE), and Glutathione S- transferase (GST).
  • the target site resistance gene is selected from the group consisting of a gene encoding Acetylcholinesterase (AChE), Voltage-gated sodium channel (VGSC), Gamma-aminobutyric acid (GABA) receptor, Nicotinic acetylcholine receptor (nAChR), and Glutamate-gated chloride channel (GluCl).
  • the transporter gene is selected from the group consisting of a gene encoding ATP-binding cassette (ABC) transporter, Solute carrier (SLC) transporter, Major facilitator superfamily (MFS) transporter, and P-glycoprotein.
  • ABSC ATP-binding cassette
  • SLC Solute carrier
  • MFS Major facilitator superfamily
  • P-glycoprotein P-glycoprotein.
  • the agricultural pest is resistant to or tolerant of the pesticide.
  • the agricultural pest is selected from the group consisting of insect, weed, fungus, algae, bacterium, rodent, larvae, virus, mite, tick, nematode, mollusca, lice, fish, and bird.
  • the pesticide is selected from the group consisting of an insecticide, a herbicide, a fungicide, an algaecide, a bactericide, a rodenticide, a larvicide, a repellent, a virucide, an ovicide, an acaricide, a nematicide, a molluscicide, a pediculicide, a piscicide, and an avicide.
  • the pesticide is a chemical pesticide or a biological pesticide.
  • the biological pesticide is a protein toxin.
  • the nucleic acid is capable of inducing RNA interference in at least one member from an order selected from the group consisting of: Coleoptera, Diptera, Hymenoptera, Lepidoptera, Homoptera, Hemiptera, Orthoptera, Thysanoptera, Dermaptera, Siphonaptera, and Trichoptera.
  • the nucleic acid is capable of inducing RNA interference in a member of the order Lepidoptera.
  • the nucleic acid is capable of inducing RNA interference in a member of the genus Plutella.
  • the nucleic acid is capable of inducing RNA interference in a member of the genus Spodoptera.
  • the nucleic acid is a RNA molecule.
  • the nucleic acid is at least one selected from the group consisting of: a double-stranded RNA (dsRNA) a short-hairpin RNA (shRNA), a small-interfering RNA (siRNA), and a microRNA (miRNA).
  • dsRNA double-stranded RNA
  • shRNA short-hairpin RNA
  • siRNA small-interfering RNA
  • miRNA microRNA
  • the minicell is ribonuclease deficient.
  • the minicell comprises at least one fusion protein.
  • the minicell comprises at least one fusion protein expressed on the surface of the minicell.
  • the minicell comprises at least one fusion protein expressed on the surface of the minicell, said fusion protein comprising at least one target cell adhesion moiety. In some embodiments, the minicell comprises at least one fusion protein expressed on the surface of the minicell, said fusion protein comprising a carbohydrate binding molecule.
  • the agricultural composition further comprises a solid, dry, or liquid carrier. In some embodiments, said solid carrier is in a form of granule or pellet and is selected from the group consisting of: diatomaceous earth, loam, silica, alginate, clay, bentonite, vermiculite, and combinations thereof.
  • said dry carrier in a form of powder and is selected from the group consisting of: peat, wheat, bran, vermiculite, clay mineral, calcium carbonate, dolomite, gypsum, bentonite, rock phosphate, phosphorous compound, titanium dioxide, humus, talc, alginate, activated charcoal, and combinations thereof.
  • said liquid carrier is in a form of liquid or emulsion, and is selected from the group consisting of a surfactant, an emulsifier, a crop oil concentrate, a penetrant, and combinations thereof.
  • the present disclosure provides an agricultural composition, comprising: a minicell encapsulating (i) a nucleic acid capable of inducing RNA interference in an agricultural pest and (ii) a pesticide capable of killing or controlling the agricultural pest, wherein the nucleic acid reduces resistance to or tolerance of the pesticide in the agricultural pest.
  • the present disclosure provides a method of reducing or suppressing pesticide resistance in an agricultural pest, the method comprising: applying an agricultural composition taught herein to an agricultural pest, wherein resistance to a pesticide in the agricultural pest is reduced or suppressed after the application of the agricultural composition.
  • the resistance to the pesticide is reduced at least 10% in the agricultural pest applied with the agricultural composition when comparing to an agricultural pest unapplied with the agricultural composition.
  • the present disclosure provides a method of restoring susceptibility of an agricultural pest to a pesticide, the method comprising: applying an agricultural composition of claim 1 or 36 to an agricultural pest, wherein the agricultural pest is restored to be susceptible to a pesticide after the application of the agricultural composition.
  • the susceptibility to the pesticide is restored at least 10% in the agricultural pest applied with the agricultural composition when comparing to an agricultural pest unapplied with the agricultural composition.
  • the agricultural pest applied with the agricultural composition is more sensitive to the pesticide than an agricultural pest unapplied with the agricultural composition.
  • Fig. 1 illustrates two separate minicells encapsulating pesticides and functional RNAs, respectively, and combinational use of the pesticides-encapsulated minicells and the functional RNAs-encapsulated minicells.
  • Fig.2 illustrates development of a minicell encapsulating both pesticides and RNAs.
  • a parental cell produces functional RNAs (such double-stranded RNAs; dsRNAs) and a minicell containing RNAs is derived from the parental cell. Then, the RNAs-encapsulated minicell is loaded with pesticides of interest, which ends up with an isolated minicell encapsulating both pesticides and RNAs.
  • Fig. 3 presents a bar graph showing efficacy of minicell-mediated RNA on control of diamondback moth (Plutella xylostella) with resistance to Cry1Ac protoxin. (i.e. no-QAGE strain). 4 303137700 [0022] Fig.4 shows actual efficacy test results of minicell-mediated RNA interference (RNAi), which are quantified and presented in Fig. 3. [0023] Fig. 5 presents a bar graph showing efficacy of minicell mediated RNA on control of protoxin-resistant diamondback moth and protoxin-susceptible diamondback moth, depending on fast and slow release of the minicell mediated RNA.
  • RNAi minicell-mediated RNA interference
  • Fig.6 shows actual efficacy test results of minicell-mediated RNA interference (RNAi), which are quantified and presented in Fig. 5.
  • Fig. 7A presents relative fold change ryanodine receptor 44F-like gene transcript determined by RT-qPCR analysis on diamondback moth treated with minicell mediated dsRNA.
  • Fig. 7B presents relative fold change of UDP-glucuronosyltransferase receptor gene transcript determined by RT-qPCR analysis on diamondback moth treated with minicell mediated dsRNA.
  • Figs. 8A-8H show natural infestation field study on cabbages treated with different combination of insecticides: Fig. 8A – untreated control; Fig.
  • Fig.9 shows results of efficacy of minicell-mediated RNA on control of fungi (Botrytis cinerea) with resistance to succinate dehydrogenase inhibitor (SDHI). DESCRIPTION OF THE INVENTION [0028]
  • the present disclosure provides application of a minicell platform formulated to deliver RNAs for delaying development of pesticide resistance, for restoring one or more pests’ susceptibility to pesticides before pesticide resistance has developed, and for reducing the developed pesticide resistance.
  • the present disclosure also provides co-application of a minicell-mediated RNA molecules with pesticides, thereby killing and controlling even pests that have developed pesticide resistance.
  • the present disclosure is generally directed to an agricultural composition comprising a pesticide (or a plurality of pesticides), a nucleic acid (i.e. functional RNAs for RNA interference), or a combination of both the pesticide and the nucleic acid within a minicell platform.
  • an agricultural composition and/or formulation comprising a 5 303137700 minicell comprising a pesticide or a plurality of pesticides, a minicell comprising a nucleic acid including dsRNA, siRNA, miRNA, and antisense RNA, and a minicell composing both a pesticide and a nucleic acid for RNAi.
  • a minicell comprising a nucleic acid including dsRNA, siRNA, miRNA, and antisense RNA
  • a minicell composing both a pesticide and a nucleic acid for RNAi.
  • agricultural composition or compound refers to a substance or compound used in agriculture, such as pesticides, herbicides, fertilizers, growth regulators, animal feeds, animal supplements, or veterinary medicines, but not for human uses. In some embodiments, agricultural activities encompass a wide range of practices, including crop production, livestock farming, forestry, aquaculture, and agroforestry.
  • biologically active indicates that a composition or compound itself has a biological effect, or that it modifies, causes, promotes, enhances, blocks, reduces, limits the production or activity of, or reacts with or binds to an endogenous molecule that has a biological effect.
  • a “biological effect” may be but is not limited to one that impacts a biological process in an plant; one that impacts a biological process in a pest, pathogen or parasite; one that generates or causes to be generated a detectable signal; and the like.
  • Biologically active agents, compositions, complexes or compounds may be used in agricultural applications and compositions. Biologically active agents, compositions, complexes or compounds act to cause or stimulate a desired effect upon a plant, an insect, a worm, bacteria, fungi, or virus.
  • Non-limiting examples of desired effects include, for example, preventing, treating or curing a disease or condition in a host suffering therefrom; limiting the growth of or killing a pest, a pathogen or a parasite that infects a host; augmenting the 6 303137700 phenotype or genotype of a host; stimulating a positive response in a plant to germinate, grow vegetatively, bloom, fertilize, produce fruits and/or seeds, and harvest; and controlling a pest to cause a disease or disorder.
  • biologically active compounds encompass a nucleic acid such as RNA biomolecule including antisense nucleic acid, dsRNA, shRNA, siRNA, miRNA, ribozyme, and aptamer.
  • biologically active indicates that the composition, complex or compound has an activity that impacts vegetative and reproductive growth of a plant in a positive sense, impacts a plant suffering from a disease or disorder in a positive sense and/or impacts a pest, pathogen or parasite in a negative sense.
  • a biologically active composition, complex or compound may cause or promote a biological or biochemical activity within a plant that is detrimental to the growth and/or maintenance of a pest, pathogen or parasite; or of cells, tissues or organs of a plant that have abnormal growth or biochemical characteristics and/or a pest, a pathogen or a parasite that causes a disease or disorder within a host such as a plant.
  • biocontrol or “biological control” refers to control of pests by interference with their ecological status, as by introducing a natural enemy or a pathogen into the environment. “Biocontrols” are interchangeably used with ‘biocontrol agents” and “biological control agents”, which are most often referred to as antagonists.
  • biocontrol refers to a compound or composition which originates in a biological matter and is effective in the treatment, prevention, amelioration, inhibition, elimination or delaying the onset of at least one of bacterial, fungal, viral, insect, or any other pest infections or infestations and inhibition of spore germination and hyphae growth. It is appreciated that any biocontrol agent is environmentally safe, that it, it is detrimental to the target species, but does not substantially damage other species in a non-specific manner. Furthermore, it is understood that the term “biocontrol agent” or “biocontrol compound” also encompasses the term “biochemical control agent” or “biochemical control compound”.
  • biostimulant refers to any microorganism or substance based on natural resources, in the form in which it is supplied to the user, applied to plants, seeds or the root environment soil and any other substrate with the intention to stimulate natural processes of plants to benefit their nutrient use efficiency and/or their tolerance to stress, regardless of its nutrients content, or any combination of such substances and/or microorganisms intended for this use.
  • biostimulants refer to biologically active compounds a polypeptide, a metabolite, a semiochemical, a hormone, a pheromone, a micronutrient and a nucleic acid such as RNA biomolecule including antisense nucleic acid, dsRNA, shRNA, siRNA, miRNA, ribozyme, and aptamer.
  • biological pesticides or “biopesticides” are substances derived from natural materials such as animals, plants, bacteria, and certain minerals. Unlike chemical pesticides, which are synthetically manufactured, biological pesticides leverage the inherent properties of living organisms or their byproducts to control pests.
  • biopesticide or “biopesticides” also refers to a substance or mixture of substances intended for preventing, destroying or controlling any pest. Specifically, the term relates to substances or mixtures which are effective for treating, preventing, ameliorating, inhibiting, eliminating or delaying the onset of bacterial, fungal, viral, insect- or other pest-related infection or infestation, spore germination and hyphae growth. Also used as substances applied to crops either before or after harvest to protect the commodity from deterioration during storage and transport. As a contraction of 'biological pesticides', biopesticides include several types of pest management intervention through predatory, parasitic, or chemical relationships. The term has been associated historically with biological control – and by implication – the manipulation of living organisms.
  • biopesticides refer to biologically active compounds a polypeptide, a metabolite, a semiochemical, a hormone, a pheromone, a macronutrient, a micronutrient and a nucleic acid such as RNA biomolecule including antisense nucleic acid, dsRNA, shRNA, siRNA, miRNA, ribozyme, and aptamer.
  • the biological pesticide is a protein toxin, that is a protoxin.
  • “chemical pesticides” are substances that are used to control, repel, or eliminate pests such as insects, weeds, fungi, and rodents that can harm crops or livestock, but not humans.
  • pesticides are formulated using various chemicals, including synthetic compounds, to target specific pests or pest categories. Chemical pesticides work by interfering with the pest's physiology, behavior, or reproductive system, ultimately reducing their population or preventing damage to crops or property.
  • the term “pest” is defined herein as encompassing vectors of plant, humans or livestock disease, unwanted species of bacteria, fungi, viruses, insects, nematodes mites, ticks or any organism causing harm during or otherwise interfering with the production, processing, storage, transport or marketing of food, agricultural commodities, wood and wood products or animal feedstuffs.
  • Insect pests include insects selected from the orders Coleoptera, Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera Orthroptera, Thysanoptera, 8 303137700 Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., particularly Lepidoptera and Coleoptera.
  • insects selected from the orders Coleoptera, Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera Orthroptera, Thysanoptera, 8 303137700 Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., particularly Lepidoptera and Coleoptera.
  • Those skilled in the art will recognize that not all compounds are equally effective against all pests.
  • Compounds of the embodiments display activity against insect pests, which may include economically important agronomic, forest, greenhouse, nursery ornamentals
  • cellular organism “microorganism” or “microbe” should be taken broadly. These terms are used interchangeably and include, but are not limited to, the two prokaryotic domains, Bacteria and Archaea, as well as certain eukaryotic fungi and protists.
  • prokaryotes is art recognized and refers to cells that contain no nucleus or other cell organelles. The prokaryotes are generally classified in one of two domains, the Bacteria and the Archaea. The definitive difference between organisms of the Archaea and Bacteria domains is based on fundamental differences in the nucleotide base sequence in the 16S ribosomal RNA.
  • the term “Archaea” refers to a categorization of organisms of the division Mendosicutes, typically found in unusual environments and distinguished from the rest of the prokaryotes by several criteria, including the number of ribosomal proteins and the lack of muramic acid in cell walls.
  • the Archaea consist of two phylogenetically-distinct groups: Crenarchaeota and Euryarchaeota.
  • the Archaea can be organized into three types: methanogens (prokaryotes that produce methane); extreme halophiles (prokaryotes that live at very high concentrations of salt (NaCl); and extreme (hyper) thermophilus (prokaryotes that live at very high temperatures).
  • methanogens prokaryotes that produce methane
  • extreme halophiles prokaryotes that live at very high concentrations of salt (NaCl)
  • extreme (hyper) thermophilus prokaryotes that live at very high temperatures.
  • these prokaryotes exhibit unique structural or biochemical attributes which adapt them to their particular habitats.
  • the Crenarchaeota consists mainly of hyperthermophilic sulfur-dependent prokaryotes and the Euryarchaeota contains the methanogens and extreme halophiles.
  • Bacteria or “eubacteria” refers to a domain of prokaryotic organisms.
  • Bacteria include at least 11 distinct groups as follows: (1) Gram-positive (gram+) bacteria, of which there are two major subdivisions: (1) high G+C group (Actinomycetes, Mycobacteria, Micrococcus, others) (2) low G+C group (Bacillus, Clostridia, Lactobacillus, Staphylococci, Streptococci, Mycoplasmas); (2) Proteobacteria, e.g., Purple photosynthetic+non- photosynthetic Gram-negative bacteria (includes most “common” Gram-negative bacteria); (3) 9 303137700 Cyanobacteria, e.g., oxygenic phototrophs; (4) Spirochetes and related species; (5) Planctomyces; (6) Bacteroides, Flavobacteria; (7) Chlamydia; (8) Green sulfur bacteria; (9) Green non-sulfur bacteria (also anaerobic phototrophs); (10) Radioresistant micrococci and relatives; (11) Thermoto
  • a “eukaryote” is any organism whose cells contain a nucleus and other organelles enclosed within membranes. Eukaryotes belong to the taxon Eukarya or Eukaryota. The defining feature that sets eukaryotic cells apart from prokaryotic cells (the aforementioned Bacteria and Archaea) is that they have membrane-bound organelles, especially the nucleus, which contains the genetic material, and is enclosed by the nuclear envelope. [0047] The terms “genetically modified host cell,” “recombinant host cell,” and “recombinant strain” are used interchangeably herein and refer to host cells that have been genetically modified by the cloning and transformation methods of the present disclosure.
  • wild-type microorganism or “wild-type host cell” describes a cell that occurs in nature, i.e. a cell that has not been genetically modified.
  • wild type strain or “wild strain” or “wild type cell line” refers to a cell strain/line that can produce minicells.
  • wild type bacterial strains and/or cell lines such as E. coli strain p678-54 and B. subtilis strain CU403 can make miniature cells deficient in DNA. Methods for producing such minicells are known in the art. See, for example, Adler et al., 1967, Proc. Natl. Acad. Sci. USA 57:321-326; Hansburg J, 1970 J.
  • phenotype refers to the observable characteristics of an individual cell, cell culture, organism, or group of organisms which results from the interaction between that individual’s genetic makeup (i.e., genotype) and the environment.
  • chimeric or “recombinant” when describing a nucleic acid sequence or a protein sequence refers to a nucleic acid, or a protein sequence, that links at least two heterologous polynucleotides, or two heterologous polypeptides, into a single macromolecule, or that rearranges one or more elements of at least one natural nucleic acid or protein sequence.
  • recombinant can refer to an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis or by the manipulation of isolated segments of nucleic acids by genetic engineering techniques.
  • a “synthetic nucleotide sequence” or “synthetic polynucleotide sequence” is a nucleotide sequence that is not known to occur in nature or that is not naturally occurring. Generally, such a synthetic nucleotide sequence will comprise at least one nucleotide difference when compared to any other naturally occurring nucleotide sequence.
  • a “synthetic amino acid sequence” or “synthetic peptide” or “synthetic protein” is an amino acid sequence that is not known to occur in nature or that is not naturally occurring. Generally, such a synthetic protein sequence will comprise at least one amino acid difference when compared to any other naturally occurring protein sequence.
  • nucleic acid refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides, or analogs thereof. This term refers to the primary structure of the molecule, and thus includes double- and single-stranded DNA, as well as double- and single-stranded RNA.
  • nucleic acid refers to any segment of DNA associated with a biological function.
  • genes include, but are not limited to, coding sequences and/or the regulatory sequences required for their expression.
  • Genes can also include non-expressed DNA 11 303137700 segments that, for example, form recognition sequences for other proteins.
  • Genes can be obtained from a variety of sources, including cloning from a source of interest or synthesizing from known or predicted sequence information, and may include sequences designed to have desired parameters.
  • the term “homologous” or “homologue” or “ortholog” is known in the art and refers to related sequences that share a common ancestor or family member and are determined based on the degree of sequence identity.
  • the terms “homology,” “homologous,” “substantially similar” and “corresponding substantially” are used interchangeably herein. They refer to nucleic acid fragments wherein changes in one or more nucleotide bases do not affect the ability of the nucleic acid fragment to mediate gene expression or produce a certain phenotype.
  • a functional relationship may be indicated in any one of a number of ways, including, but not limited to: (a) degree of sequence identity and/or (b) the same or similar biological function. Preferably, both (a) and (b) are indicated.
  • Homology can be determined using software programs readily available in the art, such as those discussed in Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1987) Supplement 30, section 7.718, Table 7.71. [0060]
  • endogenous or endogenous gene refers to the naturally occurring gene, in the location in which it is naturally found within the host cell genome.
  • operably linking a heterologous promoter to an endogenous gene means genetically inserting a heterologous promoter sequence in front of an existing gene, in the location where that gene is naturally present.
  • An endogenous gene as described herein can include alleles of naturally occurring genes that have been mutated according to any of the methods of the present disclosure.
  • the term “exogenous” is used interchangeably with the term “heterologous,” and refers to a substance coming from some source other than its native source.
  • the terms “exogenous protein,” or “exogenous gene” refer to a protein or gene 12 303137700 from a non-native source or location, and that have been artificially supplied to a biological system.
  • nucleotide change refers to, e.g., nucleotide substitution, deletion, and/or insertion, as is well understood in the art.
  • mutations contain alterations that produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded protein or how the proteins are made.
  • protein modification refers to, e.g., amino acid substitution, amino acid modification, deletion, and/or insertion, as is well understood in the art.
  • the term “at least a portion” or “fragment” of a nucleic acid or polypeptide means a portion having the minimal size characteristics of such sequences, or any larger fragment of the full length molecule, up to and including the full length molecule.
  • a fragment of a polynucleotide of the disclosure may encode an enzymatically active portion of a genetic regulatory element.
  • An enzymatically active portion of a genetic regulatory element can be prepared by isolating a portion of one of the polynucleotides of the disclosure that comprises the genetic regulatory element and assessing activity as described herein.
  • a portion of a polypeptide may be 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, and so on, going up to the full length polypeptide.
  • the length of the portion to be used will depend on the particular application.
  • a portion of a nucleic acid useful as a hybridization probe may be as short as 12 nucleotides; in some embodiments, it is 20 nucleotides.
  • a portion of a polypeptide useful as an epitope may be as short as 4 amino acids.
  • a portion of a polypeptide that performs the function of the full-length polypeptide would generally be longer than 4 amino acids.
  • Variant polynucleotides also encompass sequences derived from a mutagenic and recombinogenic procedure such as DNA shuffling.
  • Strategies for such DNA shuffling are known in the art. See, for example, Stemmer (1994) PNAS 91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameri et al.(1997) Nature Biotech. 15:436-438; Moore et al.(1997) J. Mol. Biol.272:336-347; Zhang et al.(1997) PNAS 94:4504-4509; Crameri et al.(1998) Nature 391:288-291; and U.S. Patent Nos.
  • PCR Protocols A Guide to 13 303137700 Methods and Applications (Academic Press, New York); Innis and Gelfand, eds. (1995) PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds. (1999) PCR Methods Manual (Academic Press, New York).
  • Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially-mismatched primers, and the like.
  • recombinant construct As used herein, the phrases “recombinant construct”, “expression construct”, “chimeric construct”, “construct”, and “recombinant DNA construct” are used interchangeably herein. Also, “construct”, “vector”, and “plasmid” are used interchangeably herein.
  • a recombinant construct comprises an artificial combination of nucleic acid fragments, e.g., 14 303137700 regulatory and coding sequences that are not found together in nature.
  • a chimeric construct may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. Such construct may be used by itself or may be used in conjunction with a vector.
  • a vector is used then the choice of vector is dependent upon the method that will be used to transform host cells.
  • a plasmid vector can be used.
  • the skilled artisan is well aware of the genetic elements that must be present on the vector in order to successfully transform, select and propagate host cells comprising any of the isolated nucleic acid fragments of the disclosure.
  • the skilled artisan will also recognize that different independent transformation events will result in different levels and patterns of expression (Jones et al., (1985) EMBO J. 4:2411-2418; De Almeida et al., (1989) Mol. Gen. Genetics 218:78-86), and thus that multiple events must be screened in order to obtain lines displaying the desired expression level and pattern.
  • a vector can also be a naked RNA polynucleotide, a naked DNA polynucleotide, a polynucleotide composed of both DNA and RNA within the same strand, a poly-lysine- conjugated DNA or RNA, a peptide-conjugated DNA or RNA, a liposome-conjugated DNA, or the like, that is not autonomously replicating.
  • expression refers to the production of a functional end-product e.g., an mRNA or a protein (precursor or mature).
  • the displayed polypeptide may be a protein or a protein domain which is either expressed on the minicell membrane or is associated with the minicell membrane such that the extracellular domain or domain of interest is exposed on the outer surface of the minicell (expressed and displayed on the surface of the minicell or expressed in the parental cell to be displayed on the surface of the segregated/budded minicell).
  • the "displayed" protein or protein domain is available for interaction with extracellular components.
  • a membrane-associated protein may 15 303137700 have more than one extracellular domain, and a minicell of the disclosure may display more than one membrane-associated protein.
  • polypeptide As used herein, the terms “polypeptide”, “protein” and “protein domain” refer to a macromolecule made up of a single chain of amino acids joined by peptide bonds. Polypeptides of the invention may comprise naturally occurring amino acids, synthetic amino acids, genetically encoded amino acids, non-genetically encoded amino acids, and combinations thereof. Polypeptides may include both L-form and D-form amino acids. [0073] As used herein, the term “enzymatically active polypeptide” refers to a polypeptide which encodes an enzymatically functional protein. The term “enzymatically active polypeptide” includes but not limited to fusion proteins which perform a biological function.
  • Exemplary enzymatically active polypeptides include but not limited to enzymes/enzyme moiety (e.g. wild type, variants, or engineered variants) that specifically bind to certain receptors or biological/chemical substrates to effect a biological function such as biological signal transduction or chemical inactivation.
  • enzymes/enzyme moiety e.g. wild type, variants, or engineered variants
  • protease deficiency can be created by deleting, removing, knock-out, silencing, suppressing, or otherwise downregulating at lease on endogenous protease.
  • Said proteases can include catastrophic proteases.
  • ribonuclease deficiency can be created by deleting, removing, knock-out, silencing, suppressing, or otherwise downregulating at lease on endogenous ribonuclease.
  • Said ribonuclease can include ribonuclease III.
  • HT115 E. coli strain is deficient in RNase III.
  • a ribonuclease-deficient strain is unable to and/or has a reduced capability of recognizing dsRNA and cleaving it at specific targeted locations. “Ribonuclease-deficient” can be interchangeably used as “ribonuclease-free” in the present disclosure.
  • sequence similarity or “similarity.” Means for making this adjustment are well-known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity.
  • a conservative substitution is given a score between zero and 1.
  • the scoring of conservative substitutions is calculated, e.g., according to the algorithm of Meyers and Miller, Computer Applic. Biol. Sci., 4:11-17 (1988).
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • An example of a local alignment algorithm utilized for the comparison of sequences is the NCBI Basic Local Alignment Search Tool (BLAST®) (Altschul et al. 1990 J. Mol. Biol.
  • NLM National Library of Medicine
  • binding site means a molecular structure or compound, such as a protein, a polypeptide, a polysaccharide, a glycoprotein, a lipoprotein, a fatty acid, a lipid or a nucleic acid or a particular region in such molecular structure or compound or a particular conformation of such molecular structure or compound, or a combination or complex of such molecular structures or compounds.
  • minicells can express and display both surface expressed binding proteins and biologically active compound such as polypeptide and/or proteins on their surface.
  • the surface expressed binding proteins are as a carbohydrate binding module (CBM) described above.
  • CBM carbohydrate binding module
  • the biologically/enzymatically active polypeptide/proteins, which are surface-expressed, comprise cell stimulation moiety and/or cell degradation moiety.
  • Non-limiting examples of such proteins include, but are not limited to ACC-deaminase, cellulase, phytase, chitinase, protease, phosphatase, nucleases, lipases, glucanases, xylanases, amylases, peptidases, peroxidases, ligninases, pectinases, hemicellulases, and keratinases.
  • the protein is lipase used as a biocontrol compound. In other embodiments, the protein is lipase used as a biostimulant compound.
  • the minicell-producing bacteria is a Gram-negative bacteria.
  • the Gram-negative bacteria includes, but is not limited to, Escherichia coli, Salmonella spp. including Salmonella typhimurium, Shigella spp. including Shigella flexneri, Pseudomonas aeruginosa, Agrobacterium, Campylobacter jejuni, Lactobacillus spp., Neisseria gonorrhoeae, and Legionella pneumophila,.
  • the minicell-producing Extremophilic bacteria can produce minicells naturally caused by endogenous or exogenous mutation(s) associated with cell division and/or chromosomal partitioning.
  • the minicell-producing Extremophilic bacteria comprises endogenous or exogenous gene(s) that is involved in cell division and/or chromosomal partitioning, where the gene is genetically modified such as by homologous recombination, compared to a corresponding wild-type gene.
  • the minicell-producing Extremophilic bacteria is deficient in protease and/or its activity naturally and/or by genetic engineering techniques disclosed herein.
  • the eukaryotic minicells can be produced from yeast cells, such as Saccharomyces cerevisiae, Pichia pastoris and/or Schizosaccharomyces pombe. 30 303137700 [00129] As one example, mutations in the yeast genes encoding TRF topoisomerases result in the production of minicells, and a human homolog of yeast TRF genes has been stated to exist (Castano et al., 1996, Nucleic Acids Res 24:2404-10).
  • An endophyte is an endosymbiont, often a bacterium or fungus, that lives within a plant for at least part of its life cycle.
  • the endophyte can transport itself from the environment to internal organs of plants.
  • Non-limiting examples of endophytes include Acidovorax facilis, Bradyrhizobium, Rhizobium, Rhodococcus rhodochrous, Colletotrichum, Curvularia, Epichlo ⁇ , Fusarium, Mycosphaerella, Neotyphodium, Piriformospora, and Serendipita.
  • minicells do not contain chromosomal DNA, the ability of plasmids, RNA, native and/or recombinantly expressed proteins, and other metabolites have all been shown to segregate into minicells. Some methods of construction of minicell-producing bacterial strains are discussed in detail in U.S. patent application Ser. No.10/154,951(US Publication No. US/2003/0194798 A1), which is hereby incorporated by reference in its entirety. [00142] Disruptions in the coordination between chromosome replication and cell division lead to minicell formation from the polar region of most rod-shaped prokaryotes.
  • minicell production can be achieved by the overexpression or mutation of genes involved in the segregation of nascent chromosomes into daughter cells. For example, mutations in the parC or mukB loci of E. coli have been demonstrated to produce 34 303137700 minicells.
  • coli results in the inability for this ring to be spatially restricted to the midsection of the cell, thus resulting in production of minicells upon cell division. Because the overproduction of FtsZ can create minicells, it can be overexpressed using a plasmid based system. [00145] The same can be demonstrated in the mutation-based minicell producing bacterial strains. For example, deletion of the Min locus in any of bacterial strains results in minicell production.
  • Cell division genes in which mutation can lead to minicell formation include but are not limited to the min genes (such as minC, minD, and minE).
  • E. coli rely on the min system in order to ensure proper replication of parent cells into daughter cells.
  • MinB operon This min system (known as the minB operon) consists of 3 parts, minD, minC, and minE. These genes work together in order to control the placement of the Z-ring which is comprised of polymerized FtsZ protein.
  • MinC consists of two distinct domains, both of which interact directly with the FtsZ protein in order to inhibit polymerization (Z-ring formation).
  • MinD is a protein that is associated with the membrane that forms at one of the cell’s poles and polymerizes toward the cell’s mid-point. It binds MinC which is distributed throughout the cytoplasm.
  • MinE is a protein that binds to MinD as well and releases MinC. It polymerizes into a ring like shape and oscillates from pole to pole in the cell.
  • the present disclosure teaches the use of gene editing technologies such as ZFNs, TALENS, CRISPR or homing endonucleases, to selectively edit target DNA regions.
  • the targeted DNA regions is ftsZ, minC, minD, minC/D, and minE.
  • Engineered nucleases such as zinc finger nucleases (ZFNs), Transcription Activator Like Effector Nucleases (TALENs), engineered homing endonucleases, and RNA or DNA guided endonucleases, such as CRISPR/Cas such as Cas9 or CPF1, are particularly appropriate to carry out some of the methods of the present disclosure.
  • the systems and methods disclosed herein can be used with the wild type Cas9 protein having double-stranded nuclease activity, Cas9 mutants that act as single stranded nickases, deactivated Cas9 (dCas9) that has no nuclease activity, or other mutants with modified nuclease activity.
  • dCas9 deactivated Cas9
  • a Type II nuclease may be catalytically dead (e.g. dCas9, “dead Cas9,” “deactivated Cas9”) such that it binds to a target sequence, but does not cleave.
  • dCAS9 is a variant of the CAS9 protein (CRISPR) that has had its active site altered to no longer be able to edit genomes, but can still bind to highly specific segments of the genome using a guide RNA. This protein can stop transcription of the gene if bound.
  • the dCAS9 gene can be placed under inducible control so that its expression would be controlled.
  • the guide RNA corresponding to the knockout within the Min system could be included on a plasmid or cut into the genome and placed under inducible control. Upon induction with this system, the guide RNA would direct the dCAS9 protein to the gene within the Min system in order to stop its expression.
  • the present disclosure teaches uses of the genetic manipulation technique using Lambda-Red recombination system in order to edit genome integrated with exogenous expression cassette such as an selectable marker such as antibiotic resistant gene.
  • an selectable marker such as antibiotic resistant gene is integrated into the host genome (e.g. bacteria) in order to knockout minC/D/CD gene for inducing minicell production.
  • the flippase can be used to remove the integrated antibiotic resistant gene cassette from the host genome.
  • a fragment of linear DNA is inserted into the genome directed by that fragment homology to the genome. This can be used to knock in genes of interest or to knockout genes of interest by replacing them with an antibiotic resistance cassette such as Chloramphenicol- resistant gene, kanamycin-resistant gene, spectinomycin-resistant gene, streptomycin-resistant gene, ampicillin-resistant gene, tetracycline-resistant gene, erythromycin-resistant gene, bleomycin-resistant gene, and bleomycin-resistant gene.
  • a E. coli P678-54 strain is obtained from Coli Genetic Stock Center (CGSC), and is used to produce minicells (Adler et al., 1967, Proc. Natl. Acad. Sci. USA 57:321-326; Hansburg J, 1970 J. Bacteriol.102(3):642-647; Frazer 1975, Curr. Topics Microbiol. Immunol.69:1-84).
  • CGSC Coli Genetic Stock Center
  • minicells is produced from a P678-54 E. coli parental strain.
  • the minicell produced from P678-54 parental bacterial strain is used as an anucleated cell- based platform (i.e.
  • minicell platform and/or an agricultural composition for the encapsulation and delivery of biologically active compounds.
  • Protease-deficient bacterial strains [00157] The present disclosure provides the production of minicells from B strains using genetically-engineering techniques including B strains including BL21, BL21 (DE3), and BL21-AI are deficient in Lon protease (cytoplasm) and OmpT protease (outer membrane). Accordingly, B strains as protease-deficient strains can be utilized to create protease-deficient and/or protease-deficient minicells.
  • the DE3 designation means that respective strains contain WKH ⁇ '( ⁇ O ⁇ VRJHQ ⁇ WKDW ⁇ FDUULHV ⁇ WKH ⁇ JHQH ⁇ IRU ⁇ 7 ⁇ 51$ ⁇ SRO ⁇ PHUDVH ⁇ XQder control of the lacUV5 promoter.
  • IPTG is required to maximally induce expression of the T7 RNA polymerase in order to express recombinant genes cloned downstream of a T7 promoter.
  • BL21(DE3) is suitable for expression from a T7 or T7-lac promoter or promoters recognized by the E.coli RNA polymerase: e.g. lac, tac, trc, ParaBAD, PrhaBAD and also the T5 promoter.
  • BL21-AI E. coli contains a chromosomal insertion of the gene encoding T7 RNA polymerase (RNAP) into the araB locus of the araBAD operon, placing regulation of T7 RNAP under the control of the arabinose-inducible araBAD promoter.
  • RNAP RNA polymerase
  • the BL21-AI strain does not contain the Ion protease and is deficient in the outer membrane protease, OmpT.
  • the genotype of BL21-AI is F- ompT hsdS B (r B - m B -) gal dcm araB::T7RNAP-tetA.
  • the BL21-AI has an arabinose promoter that controls the production T7 RNA Polymerase, while the BL21 (DE3) has a lac promoter that controls the production of the T7 RNA Polymerase.
  • LPS Lipopolysaccharide modified BL21 (DE3) cells
  • the LPS of the E. Coli is modified to be significantly less toxic.
  • This LPS modified BL21 (DE3) cells if necessary. This could also be branched out to other gram- negative bacterial cells. Safe usage of gram-negative cells can be beneficial for minicell and/or an agricultural composition.
  • ClearColi® BL21(DE3) cells are the commercially available competent cells with a modified LPS (Lipid IVA) that does not trigger the endotoxic response in diverse cells.
  • ClearColi cells lack outer membrane agonists for hTLR4/MD-2 activation; therefore, activation of hTLR4/MD-2 signaling by ClearColi® is several orders of magnitude lower as compared with E. coli wild-type cells.
  • Heterologous proteins prepared from ClearColi® are virtually free of endotoxic activity. After minimal purification from ClearColi cells, proteins or plasmids (which may contain Lipid IVA) can be used in most applications without eliciting an endotoxic response in human cells.
  • protease-deficient minicells disclosed herein are produced from protease-deficient parental strains including, but are not limited to, BL21 (DE3), BL21- AI and LPS-modified BL21 (DE3).
  • BL21 (DE3), BL21-AI and LPS- modified BL21 (DE3) strains are genetically engineered by deleting, mutating, knocking out, or disrupting minC, minD, and/or minC and minD gene(s) to induce minicell production.
  • BL21 (DE3), BL21-AI and LPS-modified BL21 (DE3) strains are genetically engineered by overexpressing ftsZ and/or minE genes to induce minicell production.
  • the present disclosure provides a new minicell-producing strain named as B8. This strain is the protease-deficient minicell-producing strain without the 39 303137700 T7 RNA Polymerase.
  • This minicell strain is produced from the BL21 (DE3) strain. While knocking out minC/D/CD, the T7 RNA Polymerase was silenced due to the homology of the introduced knockout via Lambda Red Transformation. This strain can be used for a need of a protease-deficient minicell, but not having the T7 RNA Polymerase. In some embodiments, minicells displayed an enzymatically active polypeptide such as complicated or toxic proteins on their surface, need to be more controlled and slower expression of the desired but complicated or toxic proteins.
  • the present disclosure teaches genotypes of newly-generated protease-deficient minicell strains comprising i) minC-deleted BL21(DE3); IKX$ ⁇ >ORQ@ ⁇ RPS7 ⁇ JDO ⁇ '( ⁇ >GFP@ ⁇ ⁇ KVG6 ⁇ '( ⁇ ⁇ V%DP+,R ⁇ ⁇ (FR5,-B int::(lacI::PlacUV5::T7 JHQH ⁇ L ⁇ ⁇ QLQ ⁇ ⁇ PLQ& ⁇ ii) minD-deleted BL21(DE3); IKX$ ⁇ >ORQ@ ⁇ RPS7 ⁇ JDO ⁇ '( ⁇ >GFP@ ⁇ ⁇ KVG6 ⁇ '( ⁇ ⁇ V%DP+,R ⁇ ⁇ (FR5,-B int::(lacI::PlacUV5::T7 JHQH ⁇ L ⁇ ⁇ QLQ ⁇ ⁇ PLQ' ⁇ LLL ⁇ minC/D-deleted BL21(DE3); IKX$ ⁇
  • minicells are protease-deficient because the parent cells are protease-deficient strains. Although chromosomes do not segregate into minicells, extrachromosomal and/or episomal genetic expression elements may segregate, or may be introduced into minicells after segregation from parent cells. In some embodiments, the disclosure is drawn to protease-deficient minicells comprising an expression element, 40 303137700 which may be an inducible expression element.
  • the inducible expression element such as an inducible promoter can be introduced to a recombinant plasmid used for homologous recombination to knock out and/or delete gene(s) involved to cell division and/or chromosomal partitioning such as minC, minD, and minC/D, a recombinant expression vector to overexpress gene(s) involved to cell division and/or chromosomal partitioning such as ftsZ and minE, and a recombinant expression vector for expressing an enzymatically active polypeptide including a protein of interest disclosed herein.
  • the inducible expression element comprises expression sequences operably linked to an open reading frame (ORF) that encodes proteins of interest disclosed herein.
  • an inducing agent is provided in order to induce expression of an ORF that encodes proteins of interest disclosed herein.
  • the disclosure teaches methods of making a protease-deficient bacterial minicell comprising a recombinant fusion protein that is not naturally found in parental cells.
  • the disclosure teaches method of preparing protease- deficient minicells from the host cells.
  • the present disclosure teaches production of protease-deficient minicells from B.
  • subtilis strains such as CU403 DIVIVA, CU403,DIVIVB,SPO-, CU403,DIVIVB and CU403,DIVIVB1 using by deleting, mutating, knocking out, or disrupting gene encoding WprA protease.
  • B. subtilis genetic manipulations work slightly differently than genetic manipulations in E. coli.
  • B. subtilis is known to readily undergo homologous recombination if DNA containing homology to the existing genome is inserted. This is unlike E. coli; E. coli has mechanisms in place to degrade any non-natural linear DNA present.
  • B. subtilis strains including, but are not limited to CU403 DIVIVA (BGSC No. 1A196), CU403,DIVIVB,SPO- (BGSC No. 1A197), CU403,DIVIVB (BGSC No. 1A292), CU403,DIVIVB1 (BGSC No. 1A513), KO7 can be used as parental bacterial cells to produce minicells.
  • B. subtilis strains including, but are not limited to CU403 DIVIVA (BGSC No. 1A196), CU403,DIVIVB,SPO- (BGSC No. 1A197), CU403,DIVIVB (BGSC No. 1A292), CU403,DIVIVB1 (BGSC No. 1A513), KO7 can be used as parental bacterial cells to produce minicells.
  • Bacillus Subtilis stains including, but are not limited to CU403 DIVIVA, CU403,DIVIVB,SPO-, CU403,DIVIVB and CU403,DIVIVB1 can be genetically modified by knocking out gene encoding WprA Protease in these strains.
  • WprA protease is known as one of the harshest proteases.
  • the pUC18 WprA-CamR vector is used.
  • This vector has the homologous arms corresponding to the gene coding for WprA cell wall protease that naturally occurs in B. subtilis which is undesirable for protein surface expression. These homologous arms flank a chloramphenicol resistance cassette in order to allow for selection.
  • the WprA-encoding nucleotide except the homologous arm is replaced with the chloramphenicol selection marker gene.
  • This plasmid can replicate within E. coli due to its origin of replication, thus when transformed into B.
  • subtilis it cannot replicate. After transformation, colonies are selected for using chloramphenicol in order to isolate the colonies in which the knockout of WprA successfully occurs. Because the plasmid cannot replicate in B. subtilis, only the cells can survive against the presence of chloramphenicol if the recombinant cassette having the chloramphenicol resistant marker gene is integrated to the genome of the B. subtilis cell by homologous recombination. [00171] B. subtilis secretes no fewer than seven proteases during vegetative growth and stationary phase. Strains in which multiple protease genes have been inactivated have proved to be superior to wild type strains in production of foreign proteins.
  • KO7 is prototrophic, free of secreted proteases, and have marker-free deletions in PY79 genetic background.
  • This KO7 is available from the BGSC as accession number 1A1133.
  • KO7 Genotype ⁇ QSU( ⁇ DSU( ⁇ HSU ⁇ PSU ⁇ QSU% ⁇ YSU ⁇ ESU ⁇
  • B. subtilis KO7 can be used for B. subtilis minicell production by knocking out DIV-IVA and DIV-IVB using genetic engineering techniques described in the present disclosure.
  • a minicell is produced from a P678-54 E. coli wild strain.
  • a minicell is produced from a protease-deficient E. coli strain including BL21, BL21(DE3), BL21-AI, LPS-modified BL21 (DE3) and B8.
  • a minicell is produced from a parental bacterial cell deficient in WprA protease.
  • a minicell is produced from a protease deficient B. subtilis parental bacterial cell.
  • a minicell is produced from produced from a protease deficient KO7 B. subtilis parental bacterial cell.
  • a minicell is produced from a 42 303137700 protease deficient B.
  • subtilis parental bacterial cell selected from the group consisting of: (1) CU403,DIVIVA; (2) CU403,DIVIVB,SPO-; (3) CU403,DIVIVB; and (4) CU403,DIVIVB1, wherein at least one protease encoding gene has been repressed, deleted, or silenced.
  • a minicell is produced from an eukaryotic cell.
  • the minicell produced as described above is used as an anucleated cell-based platform and/or an agricultural composition for the encapsulation and delivery of biologically active compounds.
  • minicells taught in the present disclosure is protease deficient or ribonuclease deficient.
  • said minicell is protease deficient. In some embodiments, said minicell is ribonuclease deficient. In some embodiments, said minicell is protease deficient and ribonuclease deficient. (ii) Ribonuclease-deficient bacterial strains [00175]
  • the present disclosure provides the production of minicells from HT115 (DE3) using genetically-engineering techniques.
  • HT115 (DE3) is a RNAi Feeding strain, which is an Rnase III-deficient E. coli strain with IPTG-inducible T7 Polymerase activity.
  • HT115 bacteria is grown on special RNAi NGM feeding plates that contain IPTG and the ampicillin analog carbenicillin. Carbenicillin is preferred over ampicillin because it tends to be more stable. Accordingly, HT115 strain as a ribonuclease- deficient strains can be utilized to create ribonuclease-deficient and/or ribonuclease-free PLQLFHOOV ⁇ 7KH ⁇ '( ⁇ GHVLJQDWLRQ ⁇ PHDQV ⁇ WKDW ⁇ UHVSHFWLYH ⁇ VWUDLQV ⁇ FRQWDLQ ⁇ WKH ⁇ '( ⁇ O ⁇ VRJHQ ⁇ WKDW ⁇ carries the gene for T7 RNA polymerase under control of the lacUV5 promoter.
  • IPTG is required to maximally induce expression of the T7 RNA polymerase in order to express recombinant genes cloned downstream of a T7 promoter.
  • HT115 (DE3) is suitable for expression from a T7 or T7-lac promoter or promoters recognized by the E.coli RNA polymerase: e.g. lac, tac, trc, ParaBAD, PrhaBAD and also the T5 promoter.
  • HT115 The genotype of HT115 (DE3) is: F-, mcrA, mcrB, IN(rrnD-rrnE)1, rnc14::Tn10(DE3 lysogen: lavUV5 promoter -T7 polymerase) (IPTG-inducible T7 polymerase) (RNAse III minus).
  • This strain grows on LB or 2XYT plates. This strain is tetracycline resistant.
  • researchers using this strain can test for expression by transforming in one of the plasmids from the Fire Vector Kit (1999) (pLT76, e.g.) using standard CaCl 2 transformation techniques. This strain is resistant to tetracycline, and can be cultivated at ⁇ LB, and aerobic.
  • ribonuclease-deficient minicells disclosed herein are produced from ribonuclease-deficient parental strains including, but are not limited to, HT115 (DE3).
  • HT115 (DE3) strain is genetically engineered by deleting, 43 303137700 mutating, knocking out, or disrupting minC, minD, and/or minC and minD gene(s) to induce minicell production.
  • HT115 (DE3) strain is genetically engineered by overexpressing ftsZ and/or minE genes to induce minicell production.
  • ribonuclease-deficient minicells disclosed herein can be produced from protease-deficient parental strains including, but are not limited to, BL21 (DE3), BL21-AI and LPS-modified BL21 (DE3), genetically engineered by deleting, mutating, knocking out, or disrupting gene(s) encoding ribonuclease III.
  • BL21 (DE3), BL21-AI and LPS-modified BL21 (DE3) strains in which ribonuclease III expression is suppressed, disrupted and/or nullified, are further genetically engineered by deleting, mutating, knocking out, or disrupting minC, minD, and/or minC and minD gene(s) to induce minicell production.
  • BL21 (DE3), BL21-AI and LPS-modified BL21 (DE3) strains, in which ribonuclease III expression is suppressed, disrupted and/or nullified are also genetically engineered by overexpressing ftsZ and/or minE genes to induce minicell production.
  • the present disclosure teaches genotypes of newly-generated ribonuclease-deficient minicell strains comprising i) minC-deleted and ribonuclease III-deleted BL21(DE3); fhuA2 >ORQ@ ⁇ RPS7 ⁇ JDO ⁇ ⁇ '( ⁇ >GFP@ ⁇ ⁇ KVG6 ⁇ '( ⁇ ⁇ ⁇ V%DP+,R ⁇ ⁇ (FR5,-B int::(lacI::PlacUV5::T7 JHQH ⁇ L ⁇ ⁇ QLQ ⁇ ⁇ PLQ& rnc14::Tn10, ii) minD-deleted and ribonuclease III-deleted BL21(DE3); IKX$ ⁇ >ORQ@ ⁇ RPS7 ⁇ JDO ⁇ '( ⁇ >GFP@ ⁇ ⁇ KVG6 ⁇ '( ⁇ ⁇ sBamHIo ⁇ (FR5,-B int::(lacI:
  • minicells are ribonuclease-deficient because the parent cells are ribonuclease-deficient strains.
  • chromosomes do not segregate into minicells, extrachromosomal and/or episomal genetic expression elements may segregate, or may be introduced into minicells after segregation from parent cells.
  • the disclosure is drawn to ribonuclease-deficient minicells comprising an expression element, which may be an inducible expression element.
  • the inducible expression element such as an inducible promoter can be introduced to a recombinant plasmid used for homologous recombination to knock out and/or delete gene(s) involved to cell division and/or chromosomal partitioning such as minC, minD, and minC/D, a recombinant expression vector to overexpress gene(s) involved to cell division and/or chromosomal partitioning such as ftsZ and minE, and a recombinant expression vector for expressing an enzymatically active polypeptide including a protein of interest disclosed herein.
  • the inducible expression element comprises expression sequences operably linked to an open reading frame (ORF) that encodes proteins of interest disclosed herein.
  • an inducing agent is provided in order to induce expression of an ORF that encodes proteins of interest disclosed herein.
  • the disclosure teaches methods of making a ribonuclease- deficient bacterial minicell comprising a recombinant fusion protein that is not naturally found in parental cells.
  • the disclosure teaches method of preparing ribonuclease-deficient minicells from the host cells. 45 303137700 [00181]
  • a minicell is produced from an eukaryotic cell.
  • minicells taught in the present disclosure is protease deficient or ribonuclease deficient. In some embodiments, said minicell is protease deficient. In some embodiments, said minicell is ribonuclease deficient. In some embodiments, said minicell is protease deficient and ribonuclease deficient. In some embodiments, said minicell is ribonuclease-deficient, and wherein said biologically active compound is a nucleic acid.
  • said biologically active compound is said nucleic acid is selected from the group consisting of an antisense nucleic acid, a double-stranded RNA (dsRNA), a short-hairpin RNA (shRNA), a small-interfering RNA (siRNA), a microRNA (miRNA), an antisense RNA, a ribozyme, an aptamer, and combination thereof.
  • dsRNA double-stranded RNA
  • shRNA short-hairpin RNA
  • siRNA small-interfering RNA
  • miRNA microRNA
  • an antisense RNA a ribozyme
  • aptamer aptamer
  • minicells are separated from parent cells glass-fiber filtration (Christen et al., Gene 23:195-198, 1983), and differential and zonal centrifugation (Barker et al., J. Gen. Microbiol.111:387-396, 1979), size-exclusion chromatography, e.g. gel- filtration, differential sonication (Reeve, J. N., and N. H. Mendelson.1973. Biochem. Biophys. Res. Commun. 53:1325-1330), and UV-irradiation (Tankersley, W. G., and J. M. Woodward. 1973.
  • minicells may be purified by the double sucrose gradient purification technique described by Frazer and Curtiss, Curr. Topics Microbiol. Immunol.69:1-84, 1975.
  • Other physical methods may also be used to remove parent cells from minicell preparations.
  • mixtures of parent cells and minicells are frozen WR ⁇ & ⁇ DQG ⁇ WKHQ ⁇ WKDZHG ⁇ VORZO ⁇ Frazer and Curtiss, Curr. Topics Microbiol.
  • agents may be used to prevent division of parental cells.
  • agents may include azide.
  • Azide is a reversible inhibitor of electron transport, and thus prevents cell division.
  • D-cycloserine or phage MS2 lysis protein may also serve as a biochemical approach to eliminate or inhibit dividing parental cells. (Markiewicz et al., FEMS Microbiol. Lett.70:119- 123, 1992). Khachatourians (U.S. Pat.
  • No.4,311,797 states that it may be desirable to incubate PLQLFHOO ⁇ SDUHQW ⁇ FHOO ⁇ PL[WXUHV ⁇ LQ ⁇ EUDLQ ⁇ KHDUW ⁇ LQIXVLRQ ⁇ EURWK ⁇ DW ⁇ ⁇ & ⁇ WR ⁇ ⁇ & ⁇ SULRU ⁇ WR ⁇ WKH ⁇ addition of penicillin G and further incubations.
  • (iii) Genetic Separation of Minicells from Parent Cells [00189] Alternatively or additionally, various techniques may be used to selectively kill, preferably lyse, parent cells.
  • minicells can internally retain M13 phage in the plasmid stage of the M13 life cycle, they are refractory to infection and lysis by M13 phage (Staudenbauer et al., Mol. Gen. Genet.138:203-212, 1975).
  • parent cells are infected and lysed by M13 and are thus selectively removed from a mixture comprising parent cells and minicells.
  • a chromosome of a parent cell may include a conditionally lethal gene. The induction of the chromosomal lethal gene will result in the destruction of parent cells, but will not affect minicells as they lack the chromosome harboring the conditionally lethal gene.
  • a parent cell may contain a chromosomal integrated bacteriophage comprising a conditionally lethal gene.
  • a bacteriophage is an integrated 47 303137700 lambda phage that has a temperature sensitive repressor gene (e.g., lambda cI857). Induction of this phage, which results in the destruction of the parent cells but not of the achromosomal minicells, is achieved by simply raising the temperature of the growth media.
  • a preferred bacteriophage to be used in this method is one that kills and/or lyses the parent cells but does not produce infective particles.
  • phage is one that lyses a cell but which has been engineered to as to not produce capsid proteins that are surround and protect phage DNA in infective particles. That is, capsid proteins are required for the production of infective particles.
  • toxic proteins may be expressed that lead to parental cell lysis.
  • these inducible constructs may employ a system to control the expression of a phage holing gene. Holin genes fall with in at least 35 different families with no detectable orthologous relationships (Grundling, A., et al. 2001. Proc. Natl. Acad. Sci.
  • compositions comprising the minicells is at least about 99.9%, about 99.8%, about 99.7%, about 99.6%, about 99.5%, about 99.4%, about 99.3%, about 99.2%, about 99.1%, about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, about 90%, about 89%, about 88%, about 87%, about 86%, about 85%, about 84%, about 83%, about 82%, about 81%, about 80%, about 79%, about 78%, about 77%, about 76%, about 75%, about 74%, about 73%, about 72%, about 71%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25% or about 20% free of minicell-producing parent cells.
  • Encapsulation is a process of enclosing the substances within an inert material, which protects from environment as well as control release of active compounds. Two type of encapsulation has been well studies; 1) Nanoencapsulation that is the coating of various substances within another material at sizes on the nano scale, and 2) Microencapsulation that is similar to nanoencapsulation aside from it involving larger particles and having been done for a greater period of time than nanoencapsulation. Encapsulation is a new technology that has wide applications in pharmaceutical industries, agrochemical, food industries and cosmetics. In some embodiments, at least one biologically active compound described herein is inert to a cell other than a cell of a target.
  • Invert suspension is an oil sub-category providing either a suspension of water encapsulated within an oil shell or water surrounded by an oil coating used to minimize the creation of driftable fines (sub 105 microns) after being sprayed through a nozzle tip. This technology works on reducing driftable fines for the active ingredients.
  • Polymer-based nanomaterials consist of a polymer that has nanoparticles or nanofillers dispersed within the polymer matrix. Typically, the polymers are contrasting (one hydrophobic, one hydrophilic) to sustain amphiphilic properties. Either synthetic or natural polymers (guar gum) act to increase the viscosity of the spray solution and affect the rheological profile by producing larger spray particles.
  • a minicell and/or an agricultural composition comprising the minicell has advantages in cost and biodegradability.
  • the minicell platforms are easily scaled through common, industrial fermentation practices. Once scaled, they can be purified through a series of centrifugation and/or filtration steps.
  • an anucleated cell-based minicell platform is advantageous compared to other encapsulation technologies in terms of biocompatibility for plant and environmental use; this 50 303137700 is because the anucleated cell-based minicell platform is derived by safe, commonly found microbes that are native to the applied areas and can safely biodegrade to be reused by the ecosystem. This platform suitable for scalable, non-toxic delivery can play an significant role in the field of agriculture.
  • the present disclosure provides a minicell and/or an agricultural composition for the encapsulation and delivery of biologically active compounds aims to protect the bioactivity from external factors until the compounds are applied to a target and to be slowly released to the intended target.
  • the various mechanisms by which biologically active compounds are typically lost to the environment are averted using the disclosed minicell-based encapsulation and delivery platform. This is because the lipid-bilayer of the minicell acts as an effective layer of protection against harsh environmental conditions. Specifically, the internalization of the active inside of the minicell protects the compounds against sharp changes in temperature, pH, or strong exposure to light.
  • the minicell protects the compounds against volatilization, photolytic degradation, and hydrolysis. Therefore, the biologically active compounds can remain protected from adverse external factors and is allowed for gradual and/or controlled release to intended targets via minicell-based platform that encapsulates the biologically active compound of interest.
  • the other benefit of the present disclosure provides a minicell and/or an agricultural composition for the encapsulation and delivery of biologically active compounds is that this platform offers the improved and enhanced targeting capability to the plant and its microenvironment.
  • the inherent surface chemistry of the outer membrane of the minicell- based bioparticle naturally mimics that of bacteria.
  • minicell-based platform biological membrane of the minicell has natural adherence to the various surfaces of plants.
  • This feature allows for delivering encapsulated biologically-active compounds including biocontrols and biostimulants in the minicell chassis that is targeted to adhere to plant surfaces and the soil microenvironment around the plant’s root system as well as to other targets such as pests, insects, bugs, weeds, worms, bacteria, viruses, pathogens, and parasites.
  • the present disclosure teaches uses of genetic engineering to give rise to surface-expressing moiety fused with specific 51 303137700 binding domain on the membrane of the minicell. In this way its ability to target the plant or the pest is significantly enhanced.
  • the present disclosure provides the genetic engineering techniques to make minicell-based platform with binding domains/motifs that functionalize the surface of the minicell. Proteins including specific binding domains and/or motifs are expressed on the surface of the minicells and specifically target binding sites that are present on the surface of plants or pests.
  • minicell-based platform can be functionalized by proteins with carbohydrate binding modules (CBMs) that can target and bind to carbohydrates such as cellulose, xylan, chitin, and lignin, which are important and ubiquitous structural components of plant cell walls. Because CBMs can recognize their binding site present on a subject such as a plant or a pest, the minicell-based platform comprising the functionalized binding domain allows for targeting with high specificity.
  • CBMs is not limited to agriculture uses. CBMs can be used for the purification of active ingredients or biomolecules through the means of cellulose columns.
  • composition useful for these embodiments may include at least one member selected from the group consisting of a tackifier, a microbial stabilizer, a fungicide, an antibacterial agent, a preservative, a stabilizer, a surfactant, an anti-complex agent, an herbicide, a nematicide, an insecticide, a plant growth regulator, a fertilizer, a rodenticide, a desiccant, a bactericide, a nutrient, or any combination thereof.
  • compositions may be shelf-stable.
  • any of the compositions described herein can include an agriculturally acceptable carrier (e.g., one or more of a fertilizer such as a non-naturally occurring fertilizer, an adhesion agent such as a non- naturally occurring adhesion agent, and a pesticide such as a non-naturally occurring pesticide).
  • an agriculturally acceptable carrier e.g., one or more of a fertilizer such as a non-naturally occurring fertilizer, an adhesion agent such as a non- naturally occurring adhesion agent, and a pesticide such as a non-naturally occurring pesticide.
  • a non-naturally occurring adhesion agent can be, for example, a polymer, 52 303137700 copolymer, or synthetic wax.
  • any of the coated seeds, seedlings, or plants described herein can contain such an agriculturally acceptable carrier in the seed coating.
  • Wetting agents such as natural or synthetic surfactants, which can be nonionic or ionic surfactants, or a combination thereof can be included in the composition.
  • Water-in-oil emulsions can also be used to formulate a composition that includes the isolated bacteria (see, for example, U.S. Patent No. 7,485,451).
  • Suitable formulations that may be prepared include wettable powders, granules, gels, agar strips or pellets, thickeners, liquids such as aqueous flowables, aqueous suspensions, water-in-oil emulsions, etc.
  • the formulation may include grain or legume products, for example, ground grain or beans, broth or flour derived from grain or beans, starch, sugar, or oil.
  • the agricultural carrier may be soil or a plant growth medium.
  • Other agricultural carriers that may be used include water, fertilizers, plant-based oils, humectants, or combinations thereof.
  • the agricultural carrier may be a solid, such as diatomaceous earth, loam, silica, alginate, clay, bentonite, vermiculite, seed cases, other plant and animal products, or combinations, including granules, pellets, or suspensions. Mixtures of any of the aforementioned ingredients are also contemplated as carriers, such as but not limited to, pesta (flour and kaolin clay), agar or flour-based pellets in loam, sand, or clay, etc.
  • Formulations may include food sources for the bacteria, such as barley, rice, or other biological materials such as seed, plant parts, sugar cane bagasse, hulls or stalks from grain processing, ground plant material or wood from building site refuse, sawdust or small fibers from recycling of paper, fabric, or wood.
  • Additional examples of agriculturally acceptable carriers include dispersants (e.g., polyvinylpyrrolidone/vinyl acetate PVPIVA S-630), surfactants, binders, and filler agents.
  • the present disclosure teaches that substances (including nucleic acids, RNA molecules, or agrochemicals) is retained within the minicell and be released over time.
  • the disclosure teaches a high value, low volume product of a minicell encapsulating at least one biologically active compounds and/or expressing a fusion protein.
  • the fusion protein has at least one surface expressing moiety and at least one cell adhesion moiety.
  • the fusion protein has at least one surface expressing moiety and at least one cell stimulation moiety.
  • the fusion protein has at least one surface expressing moiety and at least one cell degrading moiety.
  • the anucleated cell-based product can be sprayed much less than other commercially available agrochemical products and also retain the desired effects of the active compounds over a longer period of time.
  • controlled release means that one or more substances (including nucleic acids, RNA molecules, or agrochemicals) encapsulated by a minicell described in the present disclosed is released over time in a controlled manner.
  • the controlled release is meant for purposes of the present disclosure that, once the biologically active compound is released from the formulation, it is released at a controlled rate such that levels and/or concentrations of the compounds are sustained and/or delayed over an extended period of time from the start of compound release, e.g., providing a release over a time period with a prolonged interval.
  • Current controlled release mechanism is based mainly on fully encapsulation of fertilizer (e.g. Agrium, ICL, Kingenta and Ekompany) or pesticides (e.g. Adama, Syngenta, Bayer). Fully encapsulation of fertilizer is usually based on resins (e.g. polyurethanes) or sulfur base mixture. Pesticides are loaded into micro polymeric capsules.
  • the present disclosure teaches that biologically active compounds encapsulated by minicells disclosed herein can be released in a controlled manner.
  • the controlled release of the compounds are determined by a treatment of an agent such as glutaraldehyde, formaldehyde, as well as natural compounds, such as genipin, and epigallocatechin gallat, derivatives of ethylene glycol di(meth)acrylate, derivatives of methylenebisacrylamide, and formaldehyde-free crosslinking agent DVB (Divinyl Benzene).
  • a varying concentration of the agent e.g. glutaraldehyde
  • the agent includes, but is not limited to glutaraldehyde, formaldehyde, as well as natural compounds, such as genipin, and epigallocatechin gallat, derivatives of ethylene glycol di(meth)acrylate, derivatives of methylenebisacrylamide, and formaldehyde-free crosslinking agent DVB (Divinyl Benzene).
  • glutaraldehyde formaldehyde
  • natural compounds such as genipin, and epigallocatechin gallat
  • derivatives of ethylene glycol di(meth)acrylate derivatives of methylenebisacrylamide
  • DVB formaldehyde-free crosslinking agent
  • biologically active compounds encapsulated by minicells disclosed herein can be released at a rate of 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%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of a desired minicell unit/input per day.
  • an amount of the desired minicell unit/input accounts for encapsulated biologically active compounds.
  • Encapsulation amount of biologically active compounds can calculate encapsulation fraction and mass fraction, which determines the desired minicell unit and/or input per day.
  • minicells without treatment of an agent e.g. glutaraldehyde
  • glutaraldehyde which give rise to a controlled release of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of the desired input per day.
  • a varying concentration of the agent e.g. glutaraldehyde
  • solvents include, but are not limited to, CaCl 2 solution, ethanol, DMSO, polyethylene glycol, and glycerol. Not only can these solvents be used to increase the solubility of certain active compounds, but they may be used to increase the diffusion of the active compounds into the cell through certain protein channels or through the lipid bilayer of the outer membrane.
  • certain fixatives, preservatives, and cross-linking agents can be used to trap the active ingredient within the membrane of the minicell, cross-link certain active compounds to the minicell itself, and improve the stability of the minicell.
  • minicells described herein are treated with a solvent, agent, fixative, preservative, or cross-linking agent for better solubility, increased stability, or enhanced integrity.
  • said minicell exhibits a controlled release rate of said biologically active compound, wherein the release can be a steady release or an initial burst followed by steady release.
  • minicells can show their innate and modified stability and can withstand various environmental conditions and changes in temperature, pH, and/or shear stress.
  • the present disclosure teaches that the minicell can be derived from ribonuclease-deficient cell strains and/or protease-deficient cell strains.
  • the minicell can be generated from cell strains genetically engineered to disrupt structure/function of ribonuclease and/or protease.
  • the ribonuclease-deficient minicell can capture and deliver dsRNAs to a target disclosed herein.
  • the present disclosure teaches expression of dsRNA binding protein internally and/or externally. Once the dsRNA binding protein recognizes and binds to the dsRNA within the minicells, the dsRNA cannot flow back across the membrane. Also, the dsRNA binding protein can aid in dsRNA encapsulation and retention as well as protect dsRNA from degradation by RNase. On the other hand, the protease-deficient minicell can better encapsulate and retain dsRNA within the minicells when the dsRNA binding protein is expressed to protect dsRNA from RNase activity. RNase cannot have an easy access to the dsRNA bound to the dsRNA binding protein for degradation.
  • the dsRNA binding protein can also be expressed in conjunction with internal dsRNA production to ensure better retention.
  • the minicell expresses a polypeptide within the cell, and wherein the polypeptide binds to said at least one biologically active compound such as dsRNA within the cell.
  • said at least one biologically active compound is a dsRNA and wherein said polypeptide is a dsRNA binding protein.
  • the dsRNA binding protein increases stability of said dsRNA and protects said dsRNA from degradation.
  • the dsRNA binding protein is DRB4 protein.
  • an agricultural formulation comprises a polypeptide within minicells, wherein said polypeptide is expressed within said minicell, wherein said polypeptide binds to said nucleic acid.
  • said polypeptide is a dsRNA binding protein, and wherein said dsRNA binding protein increases loading and enhances the stability of dsRNA.
  • Invasive Delivery [00229] The present disclosure teaches an invasive delivery method of biologically active compounds into a target cell, which is not a mammalian cell by application of an agent that can help improve penetration of the minicell into targets such as plants, pests, insects, bugs, worms, pathogens and parasites.
  • the minicells encapsulating the biologically active compounds described herein is applied to a target cell with an agent.
  • the agent is an adjuvant for improving penetration of the minicell into the target cell and invasively delivering the biologically active compounds within the target cell.
  • the agent is a surfactant, an emulsifier, a crop oil concentrate, a penetrant, a salt or combination thereof.
  • the agent are methylated seed oil, N,N-dimethyldecanamide, and N-decyl-N- methly formamide.
  • a method of delivering at least one biologically active compound comprising: applying said minicell to said target cell with an agent, wherein said agent is an adjuvant for improving penetration of minicells into a target cell.
  • said agent is a surfactant, an emulsifier, a crop oil concentrate, a penetrant, a salt or combination thereof .
  • Various surfactants and other formulation additives can be used to enhance the uptake/invasiveness of nanoparticles or compounds into plants through the roots and leaves. Silicone surfactants can enhance the uptake of compounds and nanoparticles through the stomata, cuticle, and root system.
  • Lipid-based liquid crystalline nanoparticles can be used as a surfactant to improve delivery of biologically active compounds through the cuticle layer.
  • the present disclosure teaches an invasive delivery method of biologically active compounds into a target cell by expressing proteins that improve penetration of plant surface or increase uptake through the roots or stomata.
  • the minicells express at least one fusion protein comprises at least one surface expressing moiety and at least one target cell degradation moiety.
  • the target cell degradation moiety comprises an cutinase and cellulose, which can facilitate minicells to pass through plant surface and deliver biologically active compounds into a target cell, tissue or organ.
  • the intact minicell has a cutinase on its surface that facilitate said minicell to penetrate through a plant cuticle into the target cell.
  • the intact minicell expresses a heterologous cutinase that is displayed on its surface.
  • the intact minicell has a 58 303137700 cellulase on its surface that breaks down a target cell wall and facilitate said minicell to penetrate into the target cell.
  • the intact minicell has a heterologous cellulase that is displayed on its surface.
  • the present disclosure teaches an invasive delivery method of biologically active compounds into a target cell, which is not a mammalian cell, by generating minicells from plant invasive species such as Agrobacterium and Endophytes.
  • plant invasive species such as Agrobacterium and Endophytes.
  • the present disclosure provides compositions and methods of producing minicells from plant pathogenic bacteria and fungi such as endophytes.
  • the bacterial and/or yeast species has mechanisms to transport itself from the environment to the cells, internal tissues or organs of target plants.
  • minicells from these bacterial and yeast endophytes are produced.
  • the endophytes used for minicell production include, but are not limited to Acidovorax facilis, Bradyrhizobium, Rhizobium, Rhodococcus rhodochrous, Colletotrichum, Curvularia, Epichlo ⁇ , Fusarium, Mycosphaerella, Neotyphodium, Piriformospora, Serendipita.
  • the minicells derived from endophytes can encapsulate biologically active compounds described herein and deliver them into the internal parts of target plants by invasion/penetration mechanisms. [00235] There are several pathways by which biologically active compounds or particles are able to be uptaken through the leaf.
  • the ability for uptake through the stomata varies for each plant species, but the stomata has generally shown to have a high transport velocity into the leaf, especially for particles or compounds less than 10 nm. However, it is also the case that larger nanoparticles have been able to enter the plant through stomata openings. Foliar application of nanoparticles 59 303137700 has been shown to lead to translocation of nanoparticle from stomatal cavities to plant tissues, the vasculature, and roots cuticle (Schwab et al., J of Nanotoxicology 10(3):257-278, 2016).
  • minicells disclosed herein can be uptaken to target plants and translocated to target cells when the minicells encapsulating biologically active compounds are applied to leaves of target plants.
  • Agricultural applications of nanoparticles in soil can be very effective since nanoparticles generally accumulate in the first few meters or centimeters of the soil and therefore, interact closely with the rhizosphere.
  • minicells disclosed herein can be uptaken to target plants and translocated to target cells when the minicells encapsulating biologically active compounds are applied to soil and/or roots of target plants.
  • nanoparticles up to 500 nm and regardless of charge, can enter the plant cell through endocytosis.
  • Alternative pathways for nanoparticles and other compounds into plant cells are through the permeable pathways of the cell membrane themselves.
  • aquaporins allows for non-ionic, solutes to be non-selectively be uptaken into plant cells.
  • At least one biologically active compound is delivered into a target cell, which is not a mammalian cell, when the minicell described herein is applied by endocytosis.
  • minicells descried herein are applied to a target and delivered into a cell of a target by endocytosis.
  • Target is intended to include any target surface to which a compound, a minicell, an agricultural composition or a minicell of the present disclosure may be applied to a plant or a pest.
  • exemplary crops include but not limited to Row crops, specialty crops, commodity crops, and ornamental crops.
  • Examples of row crops include sunflower, potato, canola, dry bean, field pea, flax, safflower, buckwheat, cotton, maize, soybeans, and sugar beets.
  • Examples of commodity crops include maize, soybean and cotton.
  • Examples of ornamental crops include boxwood, christmas trees, greenhouse grown decorative plants [00242]
  • the present disclosure also teaches exemplary crops as a target, according to certain embodiments of the present disclosure, including vegetables such as broccoli, cauliflower, globe artichoke, peas, beans, kale, collard greens, spinach, arugula, beet greens, bok choy, chard, choi sum, turnip greens, endive, lettuce, mustard, greens, watercress, garlic chives, gai lan, leeks, Brussels sprouts, capers, kohlrabi, celery, rhubarb, cardoon, Chinese celery, lemon gass, asparagus, bamboo shoots, galangal, ginger, soybean, mung
  • the present disclosure also teaches exemplary aquaculture targets including fish, shrimp, shellfish, and crustacean.
  • the target can be viruses that cause diseases.
  • a target cell comprises a plant cell, an insect cell, a worm cell, a bacterial cell, a fungal cell, a virus and a cell of an aquatic animal, wherein said aquatic animal comprises a fish, a shellfish, and a crustacean.
  • the minicell and/or agricultural formulation as described herein is particularly useful within the fishing and aquaculture industries, primarily by causing a reduction in the harmful effects of microbial organisms exerted on shellfish, cartilaginous fish, fin fish or aquatic mammals.
  • Shellfish may comprise the group of filter-feeding bivalves such as e.g. clams, oysters, scallops and mussels, and may in addition comprise lobsters, crabs and shrimps.
  • Finfish include, but are not limited to the salmonid species including Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss).
  • Further aquatic animal is a fish including a gadid species including Gadus callarias, sea trout (Salmo trutta) and sea bass (Dicentrarchus labrax) and cod, eel as well as fresh water finfish and carp.
  • an aquatic animal may be a dolphin or a whale.
  • Aquatic animals further encompass any of the broadly known ornamental fish widely used throughout the hobby of fish tank maintenance. Ornamental hobby fish include both fresh water and salt water fish. Representative species of ornamental fish are well known to enthusiasts of the hobby.
  • the aquatic animal is an animal farmed in an aquaculture. The aquatic animal may be in an early developmental stage e.g., such as larvae and juvenile animals, or a later developmental stage subsequent to the juvenile stage.
  • the anucleated cell-based platform and/or agricultural formulation as described herein is targeted to a plant, an insect, a worm, a bacterium, a fungus, a virus and an aquatic animal, wherein said aquatic animal comprises a fish, a shellfish, and a crustacean.
  • the target is agricultural pests such as mites, aphids, whiteflies and thrips among the agricultural pests.
  • Examples of other agricultural insect pests than the mites, aphids, whiteflies and thrips include diamondback moth (Plutella xylostella), cabbage armyworm (Mamestra brassicae), common cutworm (Spodoptera litura), codlingmoth (Cydia pomonella), bollworm (Heliothis zea), tobacco budworm (Heliothis virescens), gypsy moth (Lymantria dispar), rice leafroller (Cnaphalocrocis medinalis), smaller tea tortrix 62 303137700 (Adoxophyes sp.), Colorado potato beetle (Leptinotarsa decemlineata), cucurbit leaf beetle (Aulacophora femoralis), boll weevil (Anthonomus grandis), planthoppers, leafhoppers, scales, bugs, grasshoppers, anthomyiid flies, scarabs, black cutworm (A
  • examples of other agricultural pests include soil pests, such as plant parasitic nematodes such as root-knot nematodes (Meloidogynidae), cyst nematodes (Heteroderidae), root-lesion nematodes (Pratylenchidae), white-tip nematode (Aphelenchoi desbesseyi), strawberry bud nematode (Nothotylenchus acris) and pine wood nematode (Bursaphelenchus xylophilus); gastropods such as slugs and snails; and isopods such as pill bugs (Armadillidium vulgare) and pill bugs (Porcellio scaber).
  • plant parasitic nematodes such as root-knot nematodes (Meloidogynidae), cyst nematodes (Heteroderidae), root-lesion nematodes (Pratylen
  • Examples of other insect pests include hygienic insect pests such as tropical rat mite (Ornithonyssus bacoti), cockroaches, housefly (Musca domestica) and house mosquito (Culex pipiens pallens); stored grain insect such as angoumois grain moth (Sitotroga cerealella), adzuki bean weevil (Callosobruchus chinensis), red flour beetle (Tribolium castaneum) and mealworms; clothes insect pests such as casemaking clothes moth (Tinea translucens) and black carpet beetle (Attagenus unicolor japonicus); house and household insect pests such as subterranean termites; domestic mites such a mold mite (Tyrophaqus putrescentiae), Dermatophagoides farinae and Chelacaropsis moorei; and hygienic insect pests such as tropical rat mite (Ornithonys
  • insects include insects selected from the orders Coleoptera, Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthoptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera, etc.
  • the insects are selected from cotton bollworm, native budworm, green mirids, aphids, green vegetable bugs, apple dimpling bugs, thrips (plaque thrips, tobacco thrips, onion thrips, western flower thrips), white flies and two spotted mites.
  • the insect pests of animals include fleas, lice, mosquitoes, flies, tsetse flies, ants, ticks, mites, silverfish and chiggers.
  • the above agricultural pests and insect pests are described, for example, in U.S. Patent Application Nos. 2012/0016022 and 2016/0174571, which are incorporated by reference herein in their entirety.
  • Delivery Amounts [00253]
  • biologically active compounds are encapsulated within the minicells described herein and delivered to a desired target. Amounts of an biologically active compound of interest are provided herein with percent weight proportions of the various 63 303137700 components used in the preparation of the minicell for the encapsulation and deliver of biologically active compounds.
  • the percent weight proportions of the various components used in the preparation of the minicell for the encapsulation and deliver of biologically active compounds can be varied as required to achieve optimal results.
  • the biologically active compounds including, but are not limited to a nucleic acid, a polypeptide, a metabolite, a semiochemical and a micronutrient polypeptide, are present in an amount of about 0.1 to about 90% by weight, is present in an amount of about 0.5 to about 80% by weight, 1 to about 70% by weight, 2 to about 60% by weight, 3 to about 55% by weight, 5 to about 50% by weight, 10 to about 45% by weight, and 15 to about 40% by weight, based on the total weight of the minicell within which an active compound of interest is encapsulated.
  • a polymer When a polymer is used in the preparation of the minicell disclosed herein, according to one embodiment it is present in an amount of about 0.01 to about 10% by weight based on the total weight of the minicell disclosed herein.
  • a co-solvent When a co-solvent is used in the preparation of the minicell disclosed herein, according to one embodiment it is present in an amount of about 0.1 to about 30% by weight based on the total weight of the minicell disclosed herein. Alternate percent weight proportions are also envisioned.
  • the biologically active compound of interest can be present in an amount of up to about 50% by weight; the solvent can be present in an amount of up to about 70% by weight; the surfactant can be present in an amount of up to about 40% by weight and the water can be present in an amount of from about 1 to about 90% by weight, based on the total weight of the minicell disclosed herein.
  • a minicell in the form of encapsulation of an biologically active compound of interest at least about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%, by weight of the biologically active compound within the minicell.
  • the biologically active compound within the minicell is present in an amount of at least about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 64 303137700 16, about 17, about 18, about 19, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 100 g/L.
  • the biologically active compound of interest and the minicell are present in compositions of the disclosure in a weight ratio of at least 1:200, 1:195, 1:190, 1:185, 1:180, 1:175, 1:170, 1:165, 1:160, 1:155, 1:150, 1:145, 1:140, 1:135, 1:130, 1:125, 1:120, 1:115, 1:110, 1:105, 1:100, 1:95, 1:90, 1:85, 1:80, 1:75, 1:70, 1:65, 1:60, 1:55, 1:50, 1:45, 1:40, 1:35, 1:30, 1:25, 1:20, 1:15, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1,7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:
  • the biologically active compound of interest and the minicell are present in a weight ratio of from about 1:50 to about 50:1, from about 1:40 to about 40:1, from about 1:30 to about 30:1, from about 1:20 to about 20:1, from about 1:10 to about 10:1, or from about 1:5 to about 5:1.
  • the density of the formulation of the minicell encapsulating the biologically active compound is least 0.01, at least 0.02, at least 0.03, at least 0.04, at least 0.05, at least 0.06, at least 0.07, at least 0.08, at least 0.09, at least 0.1, at least about 0.2, at least about 0.3, at least about 0.4, at least about 0.5, at least about 0.6, at least about 0.7, at least about 0.8, at least about 0.9, at least about 1.0, at least 1.1, at least about 1.2, at least about 1.3, at least about 1.4, at least about 1.5, at least about 1.6, at least about 1.7, at least about 1.8, at least about 1.9, at least about 2.0, at least 2.1, at least about 2.2, at least about 2.3, at least about 2.4, at least about 2.5, at least about 2.6, at least about 2.7, at least about 2.8, at least about 2.9, at least about 3.0, at least 3.1, at least about
  • an biologically active compound of interest for example, is present in at least about 20% of the total mass of the formulated product. In further embodiments, about 20 to 40% of the total mass of the formulated product is provided for the biologically active compound disclosed herein and the remaining about 60 to 80% of the mass is from the minicell. 65 303137700 [00260] In some embodiments, more than one non-expressed biologically active compounds can be encapsulated within the minicell.
  • the formulated product comprises two biologically active compounds that are present in compositions of the disclosure in a weight ratio of at least 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2,1:1, 2:1, 3:1, 4:1, 5:1, 6:1,7:1, 8:1, 9:1, or 10:1.
  • amounts of the biologically active compound about a concentration of about 0.01-20, about 0.1-15, about 0.2-10, about 0.3-9, about 0.3-8, about 0.5-5, about 1-3 g/L is provided for the formulated product.
  • the targeted delivery and controlled release disclosed herein can improve efficacy of the biologically active compounds so that the amounts of the biologically active compound can be used less.
  • the formulation of the minicell can be in a liquid or solid form.
  • the formulated product is a liquid form such as a solution.
  • the formulated product is a solid form such as a powder.
  • the agricultural formulation further comprises an agricultural chemical that is useful for promoting plant growth, reducing weeds, or reducing pests.
  • the formulations can be suitable for treating plants or plant propagation material, such as seeds, in accordance with the present disclosure, e.g., in a carrier.
  • Suitable additives include buffering agents, wetting agents, coating agents, polysaccharides, and abrading agents.
  • Exemplary carriers include water, aqueous solutions, slurries, solids and dry powders (e.g., peat, wheat, bran, vermiculite, clay, pasteurized soil, many forms of calcium carbonate, dolomite, various grades of gypsum, bentonite and other clay minerals, rock phosphates and other phosphorous compounds, titanium dioxide, humus, talc, alginate and activated charcoal.
  • Insecticides are substances used to kill insects and some other arthropods (mites, ticks, spiders, etc.) or to prevent them from causing damage. Insecticides include ovicides and larvicides used against insect eggs and larvae, respectively. Insecticides can be classified into two major groups: systemic insecticides, which have residual or long term activity; and contact insecticides, which have no residual activity. [00269] The mode of action describes how the pesticide kills or inactivates a pest. Many insecticides act at specific sites in the insect's nervous system. These usually provide very quick knockdown of insects that may ultimately die from dehydration or starvation.
  • xylostella RyR mRNA using RNAi (such as micro RNAs; miRNAs) can restore the toxicity of chlorantraniliprole against the fourth instar larvae from the resistant diamondback moth population (Li, X et al., Sci Rep 5, 14095 (2015).
  • RNAi such as micro RNAs; miRNAs
  • the expression of PxRyR can be regulated by two miRNAs, miR-7a and miR-8519 in P. xylostella.
  • EcR ecdysone receptor
  • miRNA-281 the expression of ecdysone receptor
  • nAChR nicotinic acetylcholine receptor
  • Insects including fall armyworm can develop resistance to many chemical insecticides: chlorpyriphos, permethrin, flubendamide, chlorantraniliprole, methomyl, thiodicarb, permethrin, chlorpyriphos, zeta-cypermethrin, deltamethrin, triflumuron, spinetoram, spinosad, emamectin benzoate and abamectin.
  • the minicell technology for delivery of RNAi molecules to a target with insecticides can be used to manage resistance of the fall armyworm to chemical insecticides as well as transgenic Bt corns.
  • Monera et al. J Econ Entomol actions 2019 Mar 21;112(2):792-802 reports that the resistance levels of fall armyworm to insecticides of different modes of action in fall armyworm populations from Puerto Rico and several Mexican states with different insecticide use patterns.
  • Mexican populations that expressed higher resistance ratios (RR50) were: Sonora (20-fold to chlorpyriphos), Oaxaca (19-fold to permethrin), and Sinaloa (10-fold to flubendamide).
  • the Puerto Rico population exhibited a remarkable field-evolved resistance to many pesticides.
  • the RR50 to the insecticides tested were: flubendiamide (500-fold), chlorantraniliprole (160-fold), methomyl (223-fold), thiodicarb (124-fold), permethrin (48-fold), chlorpyriphos (47-fold), zeta-cypermethrin (35- fold), deltamethrin (25-fold), triflumuron (20-fold), spinetoram (14-fold). Spinosad (eightfold), emamectin benzoate and abamectin (sevenfold) displayed lower resistance ratio. These compounds are still effective to manage fall armyworm resistance in Puerto Rico.
  • exemplary insecticides include, but are not limited to, thiamethoxam, imidacloprid, clothianidin, lamda-cyhalothrin, tefluthrin, beta-cyfluthrin, permethrin, abamectin, fipronil, cyanotraniliprole, chlorantraniliprole, and spinosad.
  • Fungicides are used both in agriculture and to fight fungal infections in animals. Chemicals used to control oomycetes, which are not fungi, are also referred to as fungicides, as oomycetes use the same mechanisms as fungi to infect plants.
  • Exemplary lists of fungicides are as follows: Azoxystrobin, Boscalid, BYF 14182, Carbendazim, Carboxin, Chlorothalonil, Fenamidone, Fludioxonil, Fluopicolide, Fluoxastrobin, Fluquinconazole, Flutriafol, Ipconazole, Iprodione, Isotianil, Mancozeb, Mefenoxam, Metalaxyl, Myclobutanil, Pencycuron, Prochloraz, Propiconazole, Prothioconazole, Pyraclostrobin, Pyrimethanil, Silthiopham, Tebuconazole, Thiophanate- methyl, Thiram, Tolylfluanid, Triadimenol, Triazoxide, Trifloxystrobin, Triflumuron, Triticonazole.
  • additional exemplary fungicides include, but are not limited to, sedaxane, fludioxonil, penthiopyrad, prothioconazole, flutriafol, difenoconazole, azoxystrobin, captan, cyproconazole, cyprodinil, boscalid, diniconazole, epoxiconazole, fluoxastrobin, trifloxystrobin, metalaxyl, metalaxyl-M (mefenoxam), fluquinconazole, fenarimol, nuarimol, pyrifenox, pyraclostrobin, thiabendazole, tebuconazole, triadimenol, benalaxyl, benalaxyl-M, benomyl, carbendazim, carboxin, flutolanil, fuberizadole, guazatine, myclobutanil
  • further exemplary fungicides include, but are not limited to, Cyprodinil ((4-cyclopropyl-6-methyl-pyrimidin-2-yl)-phenyl-amine), Dodine, Chlorothalonil, Folpet, Prothioconazole, Boscalid, Proquinazid, Dithianon, Fluazinam, Ipconazole, and Metrafenone.
  • Cyprodinil ((4-cyclopropyl-6-methyl-pyrimidin-2-yl)-phenyl-amine)
  • Dodine Chlorothalonil
  • Folpet Prothioconazole
  • Boscalid Proquinazid
  • Dithianon Fluazinam
  • Ipconazole and Metrafenone.
  • other exemplary fungicides includes, but are not limited to, fludioxonil, metalaxyl and a strobilurin fungicide, or a mixture thereof.
  • the strobilurin fungicide is azoxystrobin, picoxystrobin, kresoxim-methyl, or trifloxystorbin.
  • the agricultural chemical formulation comprises an effective amount of one or more of an insecticide selected from a phenylpyrazole and a neonicotinoid.
  • the phenylpyrazole is fipronil and the neonicotinoid is selected from 70 303137700 thiamethoxam, imidacloprid, thiacloprid, clothianidin, nitenpyram and acetamiprid.
  • the above compounds are described, for example, in U.S. Pat. No. 7,071,188, which is incorporated by reference herein in its entirety.
  • Botrytis cinerea is a major plant pathogen, causing gray mold rot in a variety of cultures. Repeated fungicide applications can result in the development of fungal populations with resistance to one or more fungicides.
  • RNAi can be utilized to negatively regulate expression of genes associated with fungicide resistance.
  • azoles are one of the main treatments in the management of Aspergillus diseases caused by ubiquitous fungi, such as Aspergillus fumigatus.
  • the main resistance mechanism is a combination of alterations in the gene cyp51A (TR34/L98H) (Berger S et al., Front Microbiol. 2017 Jun 7;8:1024).
  • Herbicides are often synthetic mimics of natural plant hormones which interfere with growth of the target plants. 71 303137700 [00286] Due to herbicide resistance, a number of products combine herbicides with different means of action. Integrated pest management may use herbicides alongside other pest control methods. However, these approaches have led to the evolution and widespread distribution of herbicide-resistant weeds, which has become a challenge for crop producers and land managers After commercialization of glyphosate-tolerant soybean in 1996 and corn in 1997, glyphosate has been used extensively for weed control and resulted in the evolution of glyphosate-resistant weeds. Even the weed species with multiple-resistance has acquired resistance against herbicides belonging to different chemistries.
  • exemplary herbicides includes, but are not limited to, paraquat, mesotrione, sulcotrione, clomazone, fentrazamide, mefenacet, oxaziclomefone, indanofan, glyphosate, prosulfocarb, molinate, triasulfuron, halosulfuron-methyl, pretilachlor, topramezone, tembotrione, isoxaflutole, fomesafen, clodinafop-propargyl, fluazifop-P-butyl, dicamba, 2,4-D (2,4-Dichlorophenoxyacetic acid), dicamba, atrazine, paraquat, S-metolachlor, glufos
  • RNA interference is a biological process that regulates the expression of genes in cells. It involves the silencing of specific genes through the use of small RNA molecules.
  • RNA interference involves with the introduction of double-stranded RNA (dsRNA) into the cell. This dsRNA is cleaved by an enzyme called Dicer into small interfering RNAs (siRNAs) or microRNAs (miRNAs). These small RNAs then bind to a protein complex called the RNA-induced silencing complex (RISC).
  • RISC RNA-induced silencing complex
  • RNAi is used as a promising tool for pest control, offering a targeted and environmentally friendly approach to managing insect pests.
  • RNAi is utilized as a biopesticide by identify specific genes essential for the survival or development of the target insect pest. These genes could be involved in vital physiological processes such as metabolism, development, or reproduction.
  • RNAi-based biopesticides offer advantages over traditional chemical pesticides. They are highly specific, targeting only the desired insect pests while leaving beneficial organisms unharmed. Additionally, they are environmentally friendly, as they degrade rapidly in the environment and reduce the risk of pesticide residues in food and water.
  • a dsRNA can target more than one target gene if the dsRNA sequence is processed into small RNAs that can recognize and/or bind to more than one target gene transcript based on its sequence homology.
  • a dsRNA can target 1 target gene, 2 target genes, 3 target genes, 4 target genes, 5 target genes, 6 target genes, 7 target genes, 8 target genes, 9 target genes, 10 target genes, or more than 10 target genes for degradation of target gene transcripts and/or inhibition of translation of target gene transcripts.
  • minicell encapsulates dsRNA that will be process into small RNAs or the processed small RNAs for RNAi in order to regulate the expression of target genes in cells.
  • minicells 73 303137700 (which is interchangeably used with minicell-encapsulated dsRNA) that confers protection and stabilization of dsRNAs by a barrier (that is, membranes of minicells).
  • minicells provide a protective barrier around the biological ingredients/compounds or chemical compounds (such as agrochemicals), shielding them from degradation due to environmental factors such as UV radiation, temperature fluctuations, moisture, and microbial activity. This helps maintain the viability and efficacy of the biologicals or chemicals during storage and application.
  • minicells can provide targeted delivery.
  • minicell-encapsulated biologicals such as dsRNA or small RNA
  • minicell-encapsulated agrochemicals can be designed to release their active ingredients slowly and steadily over time, allowing for controlled and targeted delivery to specific sites, such as pests, insects, plants or soil zones where they are needed most. This ensures optimal utilization of the biologicals and/or chemicals and reduces wastage.
  • minicells can provide enhanced persistence.
  • minicells help prolong the persistence of biologicals or agrochemicals in the environment by preventing rapid degradation or wash-off. This extends their effectiveness and reduces the frequency of application, leading to cost savings and environmental benefits.
  • minicells can provide improved handling and application. Minicell formulations can be easier to handle and apply compared to their liquid or powder counterparts. They can be formulated into various forms such as granules, pellets, or coated seeds, which are convenient to transport, store, and apply using standard agricultural equipment. [00298] In some embodiments, a minicell can encapsulate 1 dsRNA, 2 dsRNAs, 3 dsRNAs, 4 dsRNAs, 5 dsRNAs, 6 dsRNAs, 7 dsRNAs, 8 dsRNAs, 9 dsRNAs, 10 dsRNAs, or more than 10 dsRNAs, each of which targets a different target gene transcript.
  • SDHI (succinate dehydrogenase inhibitor) fungicides are a class of fungicides that target the succinate dehydrogenase enzyme complex in fungi, disrupting cellular respiration and leading to fungal death.
  • some examples of SDHI fungicides Boscalid, Flutolanil, Isopyrazam, Bixafen, Fluopyram, and Isofetamid.
  • Protein toxins also known as protoxins produced by bacteria or plants represent potent cytotoxic agents that may be coupled to specific carrier ligands used for cellular targeting. Current targeted toxins are comprised of fusion proteins that contain a potent toxin engineered in bacteria, along with a carrier ligand.
  • protoxins includes, but are not limited to, (i) insecticides, which are neonicotinoids, pyrethroids, Cry and Cyt toxins derived from Bacillus thuringiensis (Bt); (ii) fungicides, which are SDHI fungicides (Succinate Dehydrogenase Inhibitors), triazoles, strobilurins; (iii) herbicides, which are glyphosate, ALS inhibitors (Acetolactate Synthase Inhibitors), and photosystem II inhibitors (such as atrazine and diuron).
  • insecticides which are neonicotinoids, pyrethroids, Cry and Cyt toxins derived from Bacillus thuringiensis (Bt);
  • fungicides which are SDHI fungicides (Succinate Dehydrogenase Inhibitors), triazoles, strobilurins;
  • herbicides which are glyphosate, ALS inhibitors (Acetolactate Syn
  • Bacillus thuringiensis toxin refers to a group of protein toxins produced by the bacterium Bacillus thuringiensis (Bt). These toxins are commonly used as biopesticides to control insect pests in agriculture and forestry. Bt toxins are insecticidal proteins that are selectively toxic to certain insect species while being harmless to humans, animals, and most beneficial insects. They function by disrupting the gut lining of susceptible insect larvae, leading to paralysis, starvation, and eventual death. There are several types of Bt toxins, each targeting specific groups of insect pests.
  • Exemplary types of Bt toxins used in pest control include, but are not limited to, Cry toxins as pore-forming toxins that create pores in the gut epithelial cells of susceptible insects (Cry1A, Cry2A, Cry3, Cry4, Cry5, and others), .Cyt toxins as cytolytic toxins that disrupt the cell membranes of insect gut cells and often act synergistically with Cry toxins to enhance insecticidal activity, and Vip toxins (Vegetative 75 303137700 insecticidal protein toxins) that target the midgut epithelium of susceptible insects and disrupt cellular functions.
  • the disclosure provides a plurality of minicells. In some embodiments, the disclosure provides a plurality of minicells comprising at least one biologically active compound within said minicells. In other embodiments, the disclosure provides a plurality of minicells comprising at least one biocontrol within said minicells.
  • the disclosure provides a plurality of minicells comprising at least one nucleic acid such as RNA molecule including antisense nucleic acid, dsRNA, shRNA, siRNA, miRNA, ribozyme, and aptamer within said minicells.
  • nucleic acid such as RNA molecule including antisense nucleic acid, dsRNA, shRNA, siRNA, miRNA, ribozyme, and aptamer within said minicells.
  • the disclosure provides a plurality of minicells comprising at least one pesticide selected from herbicide, insecticides (which may include insect growth regulators, termiticides, etc.), a herbicide, a fungicide, an algaecide, a bactericide, a rodenticide, a larvicide, a repellent, a virucide, an ovicide, an acaricide, a nematicide, a molluscicide, a pediculicide, a piscicide, an avicide, and combinations thereof.
  • the disclosure provides a plurality of minicells comprising at least one herbicide.
  • the disclosure provides a plurality of minicells comprising at least one fungicide. In some embodiments, the disclosure provides a plurality of minicells comprising at least one antifungal. In some embodiments, the disclosure provides a plurality of minicells comprising at least one bactericide. In some embodiments, the disclosure provides a plurality of minicells comprising at least one pesticides within said minicells. In some embodiments, the disclosure provides a plurality of minicells comprising at least one pesticides within said minicells. In some embodiments, the disclosure provides a plurality of minicells comprising at least one pesticides within said minicells.
  • the disclosure provides a composition comprising a plurality of intact, bacterially-derived minicells. In some embodiments, the disclosure provides a composition comprising a plurality of intact, bacterially-derived minicells comprising at least one biologically active compound within said minicells.
  • 76 303137700 The present disclosure provides an agricultural composition, comprising a minicell encapsulating a nucleic acid that is capable of inducing RNA interference in an agricultural pest. In some embodiments, the nucleic acid reduces resistance to or tolerance of the pesticide in the agricultural pest. In some embodiments, the agricultural pest is resistant to or tolerant of the pesticide.
  • the pesticide is a chemical pesticide or a biological pesticide, which is an insecticide, a herbicide, a fungicide, an algaecide, a bactericide, a rodenticide, a larvicide, a repellent, a virucide, an ovicide, an acaricide, a nematicide, a molluscicide, a pediculicide, a piscicide, or an avicide.
  • the agricultural pest is insect, weed, fungus, algae, bacterium, rodent, larvae, virus, mite, tick, nematode, mollusca, lice, fish, or bird.
  • the nucleic acid is capable of recovering the agricultural pest’s sensitivity or susceptibility to the pesticide. In some embodiments, the nucleic acid is capable of altering expression of a gene responsible for pesticide resistance or tolerance. In some embodiments, the expression of the gene responsible for pesticide resistance or tolerance is downregulated. In other embodiments, the expression of the gene responsible for pesticide resistance or tolerance is upregulated. [00311] In some embodiments, the gene responsible for pesticide resistance or tolerance is an ion channel gene, a detoxification gene, a target site resistance gene, or a transporter gene. In some embodiments, the ion channel gene is a gene encoding Ryanodine receptor (RyR) or Voltage-gated sodium channel (VGSC).
  • RyR Ryanodine receptor
  • VGSC Voltage-gated sodium channel
  • the detoxification gene is a gene encoding UDP-glycosyltransferase (UGT), Cytochrome P450 monooxygenase, Esterase, Carboxylesterase (CarE), or Glutathione S-transferase (GST).
  • the target site resistance gene is a gene encoding Acetylcholinesterase (AChE), Voltage-gated sodium channel (VGSC), Gamma-aminobutyric acid (GABA) receptor, Nicotinic acetylcholine receptor (nAChR), or Glutamate-gated chloride channel (GluCl).
  • the transporter gene is a gene encoding ATP-binding cassette (ABC) transporter, Solute carrier (SLC) transporter, Major facilitator superfamily (MFS) transporter, or P-glycoprotein.
  • the nucleic acid is capable of inducing RNA interference in at least one member from an order selected from the group consisting of: Coleoptera, Diptera, Hymenoptera, Lepidoptera, Homoptera, Hemiptera, Orthoptera, Thysanoptera, Dermaptera, Siphonaptera, and Trichoptera.
  • the nucleic acid is capable of inducing RNA interference in a member of the order Lepidoptera.
  • the nucleic acid is capable of inducing RNA interference in a member of the genus Plutella. In some 77 303137700 embodiments, the nucleic acid is capable of inducing RNA interference in a member of the genus Spodoptera. [00313] In some embodiments, the nucleic acid is a RNA molecule, which is a double-stranded RNA (dsRNA), a short-hairpin RNA (shRNA), a small-interfering RNA (siRNA), a microRNA (miRNA), or an antisense RNA,. [00314] In some embodiments, the minicell is ribonuclease deficient.
  • dsRNA double-stranded RNA
  • shRNA short-hairpin RNA
  • siRNA small-interfering RNA
  • miRNA microRNA
  • the minicell is ribonuclease deficient.
  • the minicell comprises at least one fusion protein. In some embodiments, the minicell comprises at least one fusion protein expressed on the surface of the minicell. In some embodiments, the minicell comprises at least one fusion protein expressed on the surface of the minicell, said fusion protein comprising at least one target cell adhesion moiety. In some embodiments, the minicell comprises at least one fusion protein expressed on the surface of the minicell, said fusion protein comprising a carbohydrate binding molecule.
  • the present disclosure provides the agricultural composition comprises a minicell- encapsulated dsRNA (ME-dsRNA) and an agricultural chemical or biological pesticide. That is, the minicell-mediated RNA molecule can be co-applied with an agricultural chemical or biological pesticide.
  • ME-dsRNA minicell-encapsulated dsRNA
  • the pesticide can be applied exogenously along with the minicell-mediated RNA molecule.
  • the pesticide is encapsulated by another second minicell and applied with a first minicell encapsulating the nucleic acid capable of inducing RNAi.
  • the agricultural composition comprises the first minicell encapsulating a nucleic acid that is capable of inducing RNA interference and the second minicell encapsulating the pesticide.
  • the agricultural composition of the present disclosure further comprises an agricultural suitable additive or adjuvant.
  • minicell-encapsulated or minicell-mediated RNA molecule works as biopesticide to reduce a pest (such as insects and fungi) that is resistant to or tolerant of a chemical or biological pesticide.
  • a pest such as insects and fungi
  • at minicell- encapsulated or minicell-mediated RNA molecule can kill or control a pest (such as insects and fungi) that is resistant to or tolerant of a chemical or biological pesticide to reduce a number of pesticide-resistant pest population.
  • At minicell-encapsulated or minicell- mediated RNA molecule can kill or control a pest (such as insects and fungi) have developed resistance to or tolerance of a chemical or biological pesticide to reduce a number of pesticide- resistant pest population.
  • a pest such as insects and fungi
  • the present disclosure provides minicell-encapsulated or minicell- mediated RNA molecule prevents pests from developing resistance to pesticides.
  • the disclosure teaches (i) minicells encapsulated both RNA molecules (such as dsRNA, siRNA, miRNA, and antisense RNA) and chemical pesticides, (ii) minicell encapsulated RNAs paired with unencapsulated chemical pesticides, and (iii) a first minicell encapsulated RNAs paired with a second minicell encapsulated pesticides.
  • RNA molecules such as dsRNA, siRNA, miRNA, and antisense RNA
  • chemical pesticides such as dsRNA, siRNA, miRNA, and antisense RNA
  • minicell encapsulated RNAs paired with unencapsulated chemical pesticides such as dsRNA, siRNA, miRNA, and antisense RNA
  • minicell encapsulated RNAs paired with unencapsulated chemical pesticides such as dsRNA, siRNA, miRNA, and antisense RNA
  • an agricultural composition comprises a first minicell encapsulating a nucleic acid that is capable of inducing RNA interference in an agricultural pest and a second minicell encapsulating the pesticide that is capable of killing or controlling the agricultural pest, wherein the nucleic acid reduces resistance to or tolerance of a pesticide in the agricultural pest.
  • an agricultural composition comprises a minicell encapsulating (i) a nucleic acid capable of inducing RNA interference in an agricultural pest and (ii) a pesticide capable of killing or controlling the agricultural pest, wherein the nucleic acid reduces resistance to or tolerance of a pesticide in the agricultural pest.
  • an agricultural composition comprises a first minicell encapsulating a single-stranded nucleic acid (e.g., an antisense RNA) and a second minicell encapsulating the pesticide that is capable of killing or controlling the agricultural pest, wherein the single-stranded nucleic acid reduces resistance to or tolerance of a pesticide in an agricultural pest.
  • agricultural composition comprising: a minicell encapsulating (i) a single-stranded nucleic acid and (ii) a pesticide capable of killing or controlling an agricultural pest, wherein the single-stranded nucleic acid reduces resistance to or tolerance of a pesticide in the agricultural pest.
  • the present disclosure provides that this novel approach using a minicell platform for delivering a nucleic acid capable of inducing RNAi to a target along with chemical pesticides.
  • the present disclosure also provides that this novel approach using a minicell platform for delivering a nucleic acid capable of inducing RNAi to a target along with biopesticides such as protoxins.
  • Pests can introduce mutations into genes, which confer pesticide resistance to pesticides.
  • the RNA molecule encapsulated by minicell targets and downregulates expression of genes that are responsible for pesticide resistance.
  • the RNA molecule encapsulated by minicell targets and downregulates expression of mutated genes conferring pesticide resistance.
  • the RNA molecule restores sensitivity or susceptibility to a chemical or biological pesticide in an insect or fungal population by upregulating expression of a target gene via RNA-activation mechanism. 79 303137700 Co-application of RNAi with Pesticides Using Minicell Technology [00325] RNAi is a post-transcription gene regulation mechanism that is present in all known eukaryotes.
  • RNAi machinery The cellular RNAi machinery is initiated by dsRNAs that are initially processed into small interfering RNAs (siRNAs) by Dicer-like (DCL) proteins and eventually leads to the degradation of target mRNAs through the action of the gene silencing complex (RISC).
  • RISC gene silencing complex
  • RNAi-based genetic transformation technology has widely been utilized to control several insect pests, and diseases, in what is collectively coined as ‘host-induced gene silencing’ (HIGS) (Fire et al 1998; Baulcombe et al 2015). For instance, the expression of dsRNAs targeting dcl1/2 or target of the rapamycin (TOR) genes of B.
  • Targets of RNAi include but are not limited to genes encoding proteins and/or receptors associated with development of pesticide resistance in a pest or multiple herbicide- resistant weeds/plants.
  • dsRNA, antisense RNA, miRNA or siRNA for RNAi are loaded into the minicell platform.
  • the advantage of the minicell platform is that the encapsulation capsule and biomolecule of interest, in this case dsRNA, can both be produced in one fermentation batch. Once the dsRNA is produced and encapsulated in the minicell, the dsRNA is significantly more stable than dsRNA on its own.
  • the minicell platform has proven to significantly enhance the stability of dsRNA.
  • the present disclosure also describes a dsRNA bioproduction platform that is based on bacterial minicell carrier systems.
  • the present disclosure provides the development and applicability of minicell-based RNAi technology in agriculture, in combination with treatment of chemical or biological pesticides.
  • the disclosure presents a robust, scalable platform for producing minicell- encapsulated dsRNAs (ME-dsRNAs) and/or producing minicell-encapsulated pesticides.
  • ME-dsRNAs are treated with an exogenous pesticide or minicell- encapsulated pesticides.
  • the present disclosure provides the 80 303137700 development and applicability of minicell encapsulated or loaded with both dsRNAs and pesticides.
  • the minicell platform for delivering dsRNAs and/or pesticides has high stability, efficacy and scalability. [00331] In some embodiments, this minicell platform is incorporated into Integrated Pest Management (IPM) programs which reduce reliance on chemical control and rescue pests’ susceptibility to pesticide resistance. [00332] The present disclosure teaches the use of minicell-based RNAi technology in integrated pest/disease management programs for controlling pests, viruses, and other fungal pathogens and preventing them from development of pesticide resistance, in combination of use of synthetic chemicals or biocontrols.
  • IPM Integrated Pest Management
  • Escherichia coli derived anucleated minicells can be utilized as a cost-effective, scalable platform for dsRNA production and encapsulation.
  • minicell-encapsulated dsRNA (ME-dsRNA) is shielded from RNase degradation.
  • ME-dsRNAs selectively target genes encoding proteins and/or receptors associated with development of pesticide resistance in a pest or multiple herbicide-resistant weeds/plants, which would lead to the delaying of the development of resistance to transgenic insecticidal crops and/or chemical pesticides, and the rescuing of one or more pests' susceptibility to transgenic insecticidal crops and/or chemical pesticides.
  • the potential of ME-dsRNAs to enable the commercial application of RNAi based species-specific biocontrols along with application of conventional chemicals such as chemical insecticides, fungicides, herbicides, etc.
  • conventional chemicals such as chemical insecticides, fungicides, herbicides, etc.
  • the potential of ME-dsRNAs to enable the commercial application of RNAi based species-specific biocontrols along with application of biological chemicals such as protein toxins.
  • the present disclosure teaches methods of suppressing development of pesticide resistance in at least one pest or plants of one or more species, the method comprising: applying with at least one pesticide an agricultural formulation comprising a first minicell comprising at least one biologically active compound (e.g., RNA molecule that is a nucleic acid capable of inducing RNAi).
  • a biologically active compound e.g., RNA molecule that is a nucleic acid capable of inducing RNAi.
  • the present disclosure also teaches methods of restoring susceptibility of at least one pest or plants of one or more species to at least one pesticide, the method comprising: the method comprising: applying with at least one pesticide an agricultural formulation comprising a first minicell comprising at least one biologically active compound (e.g., RNA molecule, which is a nucleic acid capable of inducing RNAi or an antisense RNA).
  • RNA molecule which is a nucleic acid capable of inducing RNAi or an antisense RNA.
  • the at least one biologically active compound is a nucleic acid which is capable of inducing RNAi.
  • the at least one biologically active compound is a single-stranded nucleic acid, which is an antisense RNA or an antisense oligonucleotide. In some embodiments, the at least one biologically active compound is a nucleic acid which recognizes a transcript encoding a polypeptide within a cell of a target. In some embodiments, said at least one pesticide is applied exogenously. In other embodiments, said at least one pesticide is loaded into the first minicell comprising the at least one biologically active compound. In further embodiments, said at least one pesticide is loaded into a second minicell and the second minicell is applied to a target pest together with the first minicell comprising a RNA molecule.
  • said at least one pesticide is an insecticide, a herbicide, a fungicide, an algaecide, a bactericide, a rodenticide, a larvicide, a repellent, a virucide, an ovicide, an acaricide, a nematicide, a molluscicide, a pediculicide, a piscicide, and an avicide, or an antimicrobial.
  • said target comprises a plant, an insect, a worm, a bacterium, a fungus, a virus, a nematode, a snail, or a slug.
  • said nucleic acid is selected from the group consisting of an antisense nucleic acid, a double-stranded RNA (dsRNA), a short-hairpin RNA (shRNA), a small-interfering RNA (siRNA), a microRNA (miRNA), an antisense RNA, a ribozyme, an aptamer, and combination thereof.
  • dsRNA double-stranded RNA
  • shRNA short-hairpin RNA
  • siRNA small-interfering RNA
  • miRNA microRNA
  • an antisense RNA a ribozyme
  • aptamer aptamer
  • the RNA specifically acts on a pesticide-resistant pest to reduce, control, or counteract a built-up resistance, thereby keeping pesticide-resistant pests low in number and maintain a population of pesticide-resistant pests less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, and less than 1% among total pest population composed of pesticide-resistant pests and pesticide-susceptible pests.
  • minicell-encapsulated RNA molecule is applied with a chemical or biological insecticide sequentially or concurrently.
  • the RNA specifically acts on an insecticide-resistant pest to reduce, control, or counteract a built-up resistance, thereby keeping insecticide-resistant pests low in number and maintain a population of insecticide- resistant pests less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, and less than 1% 82 303137700 among total pest population composed of insecticide-resistant pests and insecticide-susceptible pests.
  • minicell-encapsulated RNA molecule is applied with a chemical or biological fungicide sequentially or concurrently.
  • the RNA specifically acts on an fungicide-resistant pest to reduce, control, or counteract a built-up resistance, thereby keeping fungicide-resistant pests low in number and maintain a population of fungicide-resistant pests less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, and less than 1% among total pest population composed of fungicide-resistant pests and fungicide-susceptible pests.
  • minicell-encapsulated RNA molecule is applied with a chemical or biological herbicide sequentially or concurrently.
  • the RNA specifically acts on an herbicide-resistant pest to reduce, control, or counteract a built-up resistance, thereby keeping herbicide-resistant pests low in number and maintain a population of herbicide-resistant pests less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, and less than 1% among total pest population composed of herbicide-resistant pests and herbicide-susceptible pests.
  • the present disclosure provides a method of reducing pesticide resistance in an agricultural pest, the method comprising applying an agricultural composition of the present disclosure (such as a minicell encapsulating a nucleic acid capable of inducing RNAi and/or a pesticide, and a minicell encapsulating an antisense RNA and/or a pesticide) to an agricultural pest that are resistant to or tolerant of a pesticide.
  • an agricultural composition of the present disclosure such as a minicell encapsulating a nucleic acid capable of inducing RNAi and/or a pesticide, and a minicell encapsulating an antisense RNA and/or a pesticide
  • the pesticide resistance in the agricultural pest is reduced or suppressed after the application of the agricultural composition.
  • pesticide resistance is reduced at least 1%, 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 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 80%, or at least 100% in the agricultural pest applied with the agricultural composition when comparing to an agricultural pest unapplied with the agricultural composition.
  • the present disclosure provides a method of restoring susceptibility of an agricultural pest to a pesticide, the method comprising applying an agricultural composition of the present 83 303137700 disclosure (such as a minicell encapsulating a nucleic acid capable of inducing RNAi and/or a pesticide, and a minicell encapsulating an antisense RNA and/or a pesticide) to an agricultural pest that are resistant to or tolerant of a pesticide, wherein the agricultural pest is restored to be susceptible to a pesticide after the application of the agricultural composition.
  • an agricultural composition of the present 83 303137700 disclosure such as a minicell encapsulating a nucleic acid capable of inducing RNAi and/or a pesticide, and a minicell encapsulating an antisense RNA and/or a pesticide
  • the susceptibility to the pesticide is restored at least 1%, 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 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 80%, or at least 100% in the agricultural pest applied with the agricultural composition when comparing to an agricultural pest unapplied with the agricultural composition.
  • the agricultural pest applied with the agricultural composition is more sensitive to the pesticide than an agricultural pest unapplied with the agricultural composition.
  • the pesticide is a chemical pesticide or a biological pesticide.
  • the pesticide is selected from the group consisting of an insecticide, a herbicide, a fungicide, an algaecide, a bactericide, a rodenticide, a larvicide, a repellent, a virucide, an ovicide, an acaricide, a nematicide, a molluscicide, a pediculicide, a piscicide, and an avicide.
  • the agricultural pest is selected from the group consisting of insect, weed, fungus, algae, bacterium, rodent, larvae, virus, mite, tick, nematode, mollusca, lice, fish, and bird.
  • RNA molecule i.e., dsRNA
  • Bt-resistant Diamondback Moth BBM
  • NO-QAGE strain obtained from Benzon Research was used as Bt-resistant DBM population for experiments. This Bt-resistant P. xylostella It was derived from crossing the susceptible "Geneva" strain with a population of field selected P.
  • xylostella 84 303137700 (evolved Bt resistance) originally collected in Hawaii.
  • the strain maintains a level of resistance to various Cry proteins that is approximately 500-1,000 times higher than the susceptible DBM strain.
  • the second instar (L 2 ) larvae of Bt-resistant DBM strain were treated with (1) two separate minicells encapsulating each of target dsRNAs used as bioinsecticides: i) ODBMhB_dsRNA (“ODBMhB”) and ii) ODBMhG_dsRNA (“ODBMhG”); (2) one empty minicell control not encapsulating any dsRNA (“P6NC”); (3) another minicell control encapsulating nontarget-dsRNA (“OBotN”); and (4) Untreated Water Control (“Water”).
  • ODBMhB_dsRNA refers to dsRNA encapsulated by type I of minicell having regular membrane designed for fast release of dsRNA.
  • ODBMhG_dsRNA refers to dsRNA encapsulated by type II of minicell having stronger membrane (treated with glutaraldehyde) designed for controlled, slow release of dsRNA.
  • Figs. 3-4 show percentage of dead DBM larvae in response to feeding on Canola leaf discs coated with ODBMhB_dsRNA and ODBMhG_dsRNA, and two controls (P6NC and OBotN) after 72 hours.
  • Example 2
  • RNA molecule i.e., dsRNA
  • DBM Diamondback Moth
  • CM Chiang Mai
  • KB Kanchanaburi
  • Diamide insecticides resistance of DBM populations [00354] To assess efficacy of minicell-encapsulated dsRNA on controlling diamide-resistant DBM populations, three diamide-resistant DBM populations listed in Table 1 were treated with (1) three separate minicells encapsulating each of target dsRNAs: i) AGS-dsRNA1, ii) AGS- dsRNA2, and iii) AGS-dsRNA3; (2) one empty minicell without dsRNA encapsulated (AGS- NC); (3) untreated control.
  • Table 2 show numbers of dead DBM larvae in response to feeding on three separate minicells each of which encapsulates dsRNAs (1-3), respectively, in comparison to minicell not encapsulating dsRNAs.
  • dsRNAs 1-3
  • AGS-dsRNA2 minicell-mediated dsRNAs
  • AGS-dsRNA3 minicell-mediated dsRNAs
  • R1G-Control 100ng (slow dsRNA release formulation) and R1B-control 100ng (fast dsRNA release formulation).
  • R1G_Control refers to an empty minicell having regular membrane, but no dsRNA encapsulated as control.
  • R1B_Control refers to an empty minicell having stronger membrane (treated with glutaraldehyde), but no dsRNA encapsulated as control.
  • Figs.5-6 show percentage of dead DBM larvae in response to feeding on Canola leaf discs coated with 100-125 ng of empty minicell lines and minicell encapsulated dsRNAs after 72 hours (72 hr).
  • Fig.5 shows that dsRNA1 encapsulated by slow release minicell formulation (R1ODBMhG-dsRNA1) led to about 60% DBM larvae death (60% mortality), while the fast release minicell formulation (R1ODBMhB-dsRNA1) had about 20% DBM larvae death (20% mortality) at 72 hours after treatment.
  • Fig.5 shows that dsRNA2 encapsulated by slow release minicell formulation (R1ODBMhNPG-dsRNA2) led to about 68% DBM larvae death (68% mortality), while the fast release minicell formulation (R1ODBMhNPB-dsRNA2) had about 28% DBM larvae death (28% mortality) at 72 hours after treatment.
  • Fig.5 shows that dsRNA3 encapsulated by slow release minicell formulation (R12AG_ dsRNA3) led to about 18% DBM larvae death (18% mortality), while the fast release minicell formulation (R12AB_ dsRNA3) had about 20% DBM larvae death (20% mortality) at 72 hours after treatment.
  • Fig.5 shows that dsRNA2 encapsulated by slow release minicell formulation (R1ODBMhNPG-dsRNA2) led to about 68% DBM larvae death (68% mortality), while the fast release minicell formulation (R1ODBMhNPB-dsRNA2) had about 28% DBM larvae death (28% mortality) at 72
  • dsRNA4 encapsulated by slow release minicell formulation led to about 66% DBM larvae death (66% mortality), while the fast release minicell formulation (R12BB_dsRNA4) had about 62% DBM larvae death (62% mortality) at 72 hours after treatment.
  • the response of Bt-resistant (left bars) and Bt-susceptible (right bars) 87 303137700 DBM larvae is presented in Fig. 5.
  • R1NC one empty minicell encapsulating no dsRNA
  • the target transcript i.e., ryanodine receptor 44F-like gene transcript
  • Figs.7A and 7B indicate that 100 ng of ODBMhG dsRNA caused about 4-8 fold down regulation of target transcripts by 72-hour post ingestion.
  • Foliar applied minicell-mediated dsRNA (ODBMhG_dsRNA) downregulates target transcripts to confer insecticidal activity.
  • ryanodine receptor 44F-like gene and UDP-glucuronosyltransferase receptor gene play key roles in developing pesticide/insecticide resistance in pests.
  • Ryanodine receptor is the target binding site for diamide chemicals. Overexpression of Ryanodine receptor or introduced mutation in the P.
  • xylostella ryanodine receptor e.g., G4946E
  • xylostella ryanodine receptor are linked to the high level of resistance to diamides in populations.
  • First, targeting DBM populations with polymorphisms that confer resistance is a mechanism of prevent those genotypes from proliferating and maintain a higher level of susceptible genotypes in the population.
  • Second, downregulating Ryanodine receptor expression is another mechanism of counteracting high levels of Ryanodine receptor expression.
  • UDP-glucuronosyltransferase receptor has a role in directly detoxifying chemical insecticides.
  • UDP-glucuronosyltransferase receptor gene By downregulating expression of UDP-glucuronosyltransferase receptor gene and similar detoxification genes (such as cytochrome P450 monooxygenase (P450), carboxylesterase (CarE) and glutathione S-transferase (GSTs) , pests can lose their sensitivity to pesticides including insecticides, Thus, sensitivity is enhanced in pest populations by downregulating or repressing these genes such as ryanodine receptor 44F-like gene and UDP- glucuronosyltransferase receptor gene.
  • Similar detoxification genes such as cytochrome P450 monooxygenase (P450), carboxylesterase (CarE) and glutathione S-transferase (GSTs)
  • P450 cytochrome P450 monooxygenase
  • CarE carboxylesterase
  • GSTs glutathione S-transferase
  • Minicell-dsRNA-B refers to dsRNA molecule encapsulated by minicell having regular membrane designed for fast release.
  • Minicell-dsRNA-G refers to refers to dsRNA molecule encapsulated by minicell having glutaraldehyde-treated membrane designed for slow release.
  • Naked RNA refers to RNA molecule not protected and/or encapsulated by minicell.
  • Treatment No.10 demonstrates the highest dead rate (7.8 out of 10 DBMs) among other treatments, indicating synergistic effect of co-application of minicell- mediated RNA and chemical insecticide at low dose on controlling insects (e.g., DBM).
  • Treatment No. 15 in Table 3 presents that 5 ng of minicell-RNA + Coragen® 3ul/l were co-applied twice at 0 and 24 hours, and then DBM mortality was measured at 48 hours from the initial treatment.
  • this co-application in treatment No. 15 demonstrates superior effects (at least 2 fold higher than) over treatment No. 16. Even the DBM mortality of treatment No.
  • Example 15 is comparable to a very high dose of Coragen® (25.0ul/l). This data also indicates synergistic effect of co-application of minicell-mediated RNA and chemical insecticide at low dose on controlling insects (e.g., DBM). 0.25% Dyne-Amic surfactant was added to each treatment. 90 303137700 Table 3. Mean number of dead DBM larvae at each 24-hour interval.
  • Cabbage cultivar used for the field experiments was 'Blue Dynasty'. Each treatment was replicated four times in a randomized block design. Insecticide treatment was made Day 1 (1 st day of week 1), Day 8 (1 st day of week 2), Day 15 (1 st day of week 3), and Day 22 (1 st day of week 4) at 1-week interval. Treatments applied with a CO 2 sprayer using three hollow cone 91 303137700 nozzles (overhead and on each side of the plant at 45 psi which deliver 1200 mls per treatment). Data on percent plant defoliation and insect counts were taken from five plants per plot on each sampling date. 0.25% Dyne-Amic surfactant was added to each treatment.
  • Minicell-dsRNA- HP refers to dsRNA-HP (dsRNA hairpin) encapsulated by minicell of the present disclosure.
  • Tables 4-8 present percentage of defoliation at Weeks 1-5 on 3 and 6 day after treatment (DAT) based on combinations of applications using minicell-dsRNA along with chemical insecticide (Coragen®) and or biological insecticide (Xentari®). Table 4. Percentage of defoliation at Week 1 on 3 and 6 day after treatment (DAT) Table 5. Percentage of defoliation at Week 2 on 3 and 6 day after treatment (DAT) Table 6. Percentage of defoliation at Week 3 on 3 and 6 day after treatment (DAT) 92 303137700 Table 7.
  • Figs. 8A-8H show cabbage defoliation at eight treatments as presented in Table 8; (1) Untreated Check (Fig.8A) showing about 11% defoliation by week 5, (2) Coragen® + Dyne- Amic (Fig. 8B) showing no sign of defoliation by week 5, (3) Xentari® + Dyne-Amic (Fig. 8C) showing ⁇ 1% defoliation by week 5, (4) Minicell-dsRNA HP-High + Dyne-Amic (Fig.
  • Minicell-dsRNA treatment in integrated pest management (IPM) rotations can reduce use of chemical insecticide (e.g., Coragen®, Xentari®) and/or biological insecticide (e.g., Bt toxin, Cry toxin) applied by 75%, while maintaining ⁇ 1% defoliation.
  • chemical insecticide e.g., Coragen®, Xentari®
  • biological insecticide e.g., Bt toxin, Cry toxin
  • Defoliation data from diamondback pressure was interpreted carefully due to confounding effects of concurrent fall armyworm, yellowstriped armyworm and imported cabbageworm infestations.
  • Rotational Xentari (1/4 rate) with Minicell-dsRNA-HP low performed better than the full rate of Xentari and statistically comparable to Coragen 8 fl/oz acre.
  • Minicell-dsRNA-HP formulation/product has synergy with Xentari and can perform similar to the synthetic standard.2) Tank mix with Xentari (1/2 rate) with Minicell- dsRNA-HP performs similar to full rate of Coragen 8 fl/oz acre. This suggests that the Minicell- dsRNA formulation/product can be used as a tank mix in conjunction with Xentari. 3) Rotational with Coragen (4 fl/oz) and Minicell-dsRNA-HP formulation/product performs similar to full rate of Coragen 8 fl/oz acre. This suggests that the Minicell-dsRNA-HP formulation/product can be used in conjunction with Coragen to reduce the development of resistance with this valuable synthetic chemical.
  • inventive concepts may be embodied as one or more methods, of which examples have been provided.
  • the acts performed as part of the methods may be ordered in any suitable way. Accordingly, embodiments may be constructed in which acts are performed in an order different than illustrated, which may include performing some acts simultaneously, even though shown as sequential acts in illustrative embodiments.
  • Nucleic acid that is capable of inducing RNA interference 1.
  • An agricultural composition comprising: a. a first minicell encapsulating a nucleic acid that is capable of inducing RNA interference in an agricultural pest, wherein the nucleic acid reduces resistance to or tolerance of a pesticide in the agricultural pest. 2.
  • the agricultural composition of embodiment 1 or 2 wherein the pesticide is a chemical pesticide or a biological pesticide. 4.
  • any one of embodiments 1-3 wherein the pesticide is selected from the group consisting of an insecticide, a herbicide, a fungicide, an algaecide, a bactericide, a rodenticide, a larvicide, a repellent, a virucide, an ovicide, an acaricide, a nematicide, a molluscicide, a pediculicide, a piscicide, and an avicide.
  • the agricultural pest is selected from the group consisting of insect, weed, fungus, algae, bacterium, rodent, larvae, virus, mite, tick, nematode, mollusca, lice, fish, and bird. 6.
  • the agricultural composition of embodiment 1, wherein the nucleic acid is capable of recovering the agricultural pest’s sensitivity or susceptibility to the pesticide. 7.
  • the agricultural composition of embodiment 1, wherein the nucleic acid is capable of altering expression of a gene responsible for pesticide resistance or tolerance.
  • the agricultural composition of embodiment 7, wherein the expression of the gene responsible for pesticide resistance or tolerance is downregulated.
  • the agricultural composition of embodiment 7, wherein the expression of the gene responsible for pesticide resistance or tolerance is upregulated.
  • the gene responsible for pesticide resistance or tolerance is an ion channel gene, a detoxification gene, a target site resistance gene, or a transporter gene. 11.
  • the agricultural composition of embodiment 10, wherein the ion channel gene is a gene encoding Ryanodine receptor (RyR) or Voltage-gated sodium channel (VGSC).
  • the detoxification gene is selected from the group consisting of a gene encoding UDP-glycosyltransferase (UGT), Cytochrome P450 monooxygenase, Esterase, Carboxylesterase (CarE), and Glutathione S- transferase (GST). 13.
  • the target site resistance gene is selected from the group consisting of a gene encoding Acetylcholinesterase (AChE), Voltage-gated sodium channel (VGSC), Gamma-aminobutyric acid (GABA) receptor, Nicotinic acetylcholine receptor (nAChR), and Glutamate-gated chloride channel (GluCl).
  • the transporter gene is selected from the group consisting of a gene encoding ATP-binding cassette (ABC) transporter, Solute carrier (SLC) transporter, Major facilitator superfamily (MFS) transporter, and P- glycoprotein.
  • ABS ATP-binding cassette
  • SLC Solute carrier
  • MFS Major facilitator superfamily
  • nucleic acid is capable of inducing RNA interference in at least one member from an order selected from the group consisting of: Coleoptera, Diptera, Hymenoptera, Lepidoptera, Homoptera, Hemiptera, Orthoptera, Thysanoptera, Dermaptera, Siphonaptera, and Trichoptera. 16.
  • nucleic acid is capable of inducing RNA interference in a member of the genus Plutella. 18.
  • nucleic acid is capable of inducing RNA interference in a member of the genus Spodoptera. 19.
  • nucleic acid is at least one selected from the group consisting of: a double-stranded RNA (dsRNA) a short- hairpin RNA (shRNA), a small-interfering RNA (siRNA), and a microRNA (miRNA). 21.
  • dsRNA double-stranded RNA
  • shRNA short- hairpin RNA
  • siRNA small-interfering RNA
  • miRNA microRNA
  • the agricultural composition of any one of embodiments 1-20, wherein the nucleic acid is shRNA. 23. The agricultural composition of any one of embodiments 1-20, wherein the nucleic acid is siRNA. 24. The agricultural composition of any one of embodiments 1-20, wherein the nucleic acid is miRNA. 25. The agricultural composition of any one of embodiments 1-24, wherein the minicell is ribonuclease deficient. 26. The agricultural composition of any one of embodiments 1-25, wherein the minicell comprises at least one fusion protein. 27. The agricultural composition of any one of embodiments 1-26, wherein the minicell comprises at least one fusion protein expressed on the surface of the minicell. 28.
  • the biological pesticide is a protein toxin.
  • a solid, dry, or liquid carrier is in a form of granule or pellet and is selected from the group consisting of: diatomaceous earth, loam, silica, alginate, clay, bentonite, vermiculite, and combinations thereof. 34.
  • the agricultural composition of embodiment 32 wherein said dry carrier in a form of powder and is selected from the group consisting of: peat, wheat, bran, vermiculite, clay mineral, calcium carbonate, dolomite, gypsum, bentonite, rock phosphate, phosphorous compound, titanium dioxide, humus, talc, alginate, activated charcoal, and combinations thereof.
  • said liquid carrier is in a form of liquid or emulsion, and is selected from the group consisting of a surfactant, an emulsifier, a crop oil concentrate, a penetrant, and combinations thereof.
  • An agricultural composition comprising: a minicell encapsulating (i) a nucleic acid capable of inducing RNA interference in an agricultural pest and (ii) a pesticide capable of killing or controlling the agricultural pest, wherein the nucleic acid reduces resistance to or tolerance of a pesticide in the agricultural pest.
  • a minicell encapsulating (i) a nucleic acid capable of inducing RNA interference in an agricultural pest and (ii) a pesticide capable of killing or controlling the agricultural pest, wherein the nucleic acid reduces resistance to or tolerance of a pesticide in the agricultural pest.
  • the agricultural pest is resistant to or tolerant of the pesticide.
  • the pesticide is a chemical pesticide or a biological pesticide.
  • any one of embodiments 36-38 wherein the pesticide is selected from the group consisting of an insecticide, a herbicide, a fungicide, an algaecide, a bactericide, a rodenticide, a larvicide, a repellent, a virucide, an ovicide, an acaricide, a nematicide, a molluscicide, a pediculicide, a piscicide, and an avicide.
  • the pesticide is selected from the group consisting of an insecticide, a herbicide, a fungicide, an algaecide, a bactericide, a rodenticide, a larvicide, a repellent, a virucide, an ovicide, an acaricide, a nematicide, a molluscicide, a pediculicide, a piscicide, and an avicide.
  • the pesticide is selected from the group consisting of an insecticide, a herbicide, a
  • any one of embodiments 36-39, wherein the agricultural pest is selected from the group consisting of insect, weed, fungus, algae, bacterium, rodent, larvae, virus, mite, tick, nematode, mollusca, lice, fish, and bird.
  • the nucleic acid is capable of recovering the agricultural pest’s sensitivity or susceptibility to the pesticide.
  • the agricultural composition of embodiment 36, wherein the nucleic acid is capable of altering expression of a gene responsible for pesticide resistance or tolerance.
  • the agricultural composition of embodiment 42 wherein the expression of the gene responsible for pesticide resistance or tolerance is upregulated.
  • the gene responsible for pesticide resistance or tolerance is an ion channel gene, a detoxification gene, a target site resistance gene, or a transporter gene.
  • the ion channel gene is a gene encoding Ryanodine receptor (RyR) or Voltage-gated sodium channel (VGSC). 47.
  • the agricultural composition of embodiment 45 wherein the detoxification gene is selected from the group consisting of a gene encoding UDP-glycosyltransferase (UGT), Cytochrome P450 monooxygenase, Esterase, Carboxylesterase (CarE), and Glutathione S- transferase (GST).
  • the target site resistance gene is selected from the group consisting of a gene encoding Acetylcholinesterase (AChE), Voltage-gated sodium channel (VGSC), Gamma-aminobutyric acid (GABA) receptor, Nicotinic acetylcholine receptor (nAChR), and Glutamate-gated chloride channel (GluCl).
  • the agricultural composition of embodiment 45 wherein the transporter gene is selected from the group consisting of a gene encoding ATP-binding cassette (ABC) transporter, Solute carrier (SLC) transporter, Major facilitator superfamily (MFS) transporter, and P- glycoprotein.
  • the nucleic acid is capable of inducing RNA interference in at least one member from an order selected from the group consisting of: Coleoptera, Diptera, Hymenoptera, Lepidoptera, Homoptera, Hemiptera, Orthoptera, Thysanoptera, Dermaptera, Siphonaptera, and Trichoptera. 51.
  • the agricultural composition of any one of embodiments 36-50, wherein the nucleic acid is capable of inducing RNA interference in a member of the genus Spodoptera.
  • the agricultural composition of any one of embodiments 36-53, wherein the nucleic acid is a RNA molecule. 55.
  • nucleic acid is at least one selected from the group consisting of: a double-stranded RNA (dsRNA), a short-hairpin RNA (shRNA), a small-interfering RNA (siRNA), and a microRNA (miRNA).
  • dsRNA double-stranded RNA
  • shRNA short-hairpin RNA
  • siRNA small-interfering RNA
  • miRNA microRNA
  • the agricultural composition of any one of embodiments 36-59, wherein the minicell is ribonuclease deficient. 61. The agricultural composition of any one of embodiments 36-60, wherein the minicell comprises at least one fusion protein. 62. The agricultural composition of any one of embodiments 36-61, wherein the minicell comprises at least one fusion protein expressed on the surface of the minicell. 63. The agricultural composition of any one of embodiments 36-62, wherein the minicell comprises at least one fusion protein expressed on the surface of the minicell, said fusion protein comprising at least one target cell adhesion moiety. 64.
  • 66. The agricultural composition of any one of embodiments 36-65, further comprising a solid, dry, or liquid carrier. 100 303137700 67.
  • said solid carrier is in a form of granule or pellet and is selected from the group consisting of: diatomaceous earth, loam, silica, alginate, clay, bentonite, vermiculite, and combinations thereof. 68.
  • the agricultural composition of embodiment 66 wherein said dry carrier in a form of powder and is selected from the group consisting of: peat, wheat, bran, vermiculite, clay mineral, calcium carbonate, dolomite, gypsum, bentonite, rock phosphate, phosphorous compound, titanium dioxide, humus, talc, alginate, activated charcoal, and combinations thereof.
  • said liquid carrier is in a form of liquid or emulsion, and is selected from the group consisting of a surfactant, an emulsifier, a crop oil concentrate, a penetrant, and combinations thereof.
  • a method of reducing or suppressing pesticide resistance in an agricultural pest comprising: applying an agricultural composition of embodiment 1 or 36 to an agricultural pest, wherein resistance to a pesticide in the agricultural pest is reduced or suppressed after the application of the agricultural composition.
  • the method of embodiment 70 wherein the agricultural pest is resistant to or tolerant of the pesticide.
  • 72. The method of embodiment 70, wherein the resistance to the pesticide is reduced at least 10% in the agricultural pest applied with the agricultural composition when comparing to an agricultural pest unapplied with the agricultural composition.
  • the pesticide is a chemical pesticide or a biological pesticide.
  • the biological pesticide is a protein toxin. 75.
  • the pesticide is selected from the group consisting of an insecticide, a herbicide, a fungicide, an algaecide, a bactericide, a rodenticide, a larvicide, a repellent, a virucide, an ovicide, an acaricide, a nematicide, a molluscicide, a pediculicide, a piscicide, and an avicide.
  • the agricultural pest is selected from the group consisting of insect, weed, fungus, algae, bacterium, rodent, larvae, virus, mite, tick, nematode, mollusca, lice, fish, and bird.
  • a method of restoring susceptibility of an agricultural pest to a pesticide comprising: applying an agricultural composition of embodiment 1 or 36 to an agricultural pest, wherein the agricultural pest is restored to be susceptible to a pesticide after the application of the agricultural composition.
  • the susceptibility to the pesticide is restored at least 10% in the agricultural pest applied with the agricultural composition when comparing to an agricultural pest unapplied with the agricultural composition.
  • the pesticide is a chemical pesticide or a biological pesticide.
  • the biological pesticide is a protein toxin.
  • the pesticide is selected from the group consisting of an insecticide, a herbicide, a fungicide, an algaecide, a bactericide, a rodenticide, a larvicide, a repellent, a virucide, an ovicide, an acaricide, a nematicide, a molluscicide, a pediculicide, a piscicide, and an avicide. 84.
  • the method of embodiment 77 wherein the agricultural pest is selected from the group consisting of insect, weed, fungus, algae, bacterium, rodent, larvae, virus, mite, tick, nematode, mollusca, lice, fish, and bird.
  • Single-stranded antisense oligonucleotide 1.
  • An agricultural composition comprising: a. a first minicell encapsulating a single-stranded nucleic acid, wherein the nucleic acid reduces resistance to or tolerance of a pesticide in an agricultural pest. 2.
  • the agricultural composition of embodiment 1, wherein the agricultural pest is resistant to or tolerant of the pesticide.
  • the agricultural composition of embodiment 1 or 2 wherein the pesticide is a chemical pesticide or a biological pesticide. 4.
  • any one of embodiments 1-3 wherein the pesticide is selected from the group consisting of an insecticide, a herbicide, a fungicide, an algaecide, a bactericide, a rodenticide, a larvicide, a repellent, a virucide, an ovicide, an acaricide, a nematicide, a molluscicide, a pediculicide, a piscicide, and an avicide.
  • the pesticide is selected from the group consisting of an insecticide, a herbicide, a fungicide, an algaecide, a bactericide, a rodenticide, a larvicide, a repellent, a virucide, an ovicide, an acaricide, a nematicide, a molluscicide, a pediculicide, a piscicide, and an avicide.
  • the pesticide is selected from the group consisting of an insecticide, a herbicide, a
  • the agricultural composition of embodiment 1 or 2 wherein the agricultural pest is selected from the group consisting of insect, weed, fungus, algae, bacterium, rodent, larvae, virus, mite, tick, nematode, mollusca, lice, fish, and bird. 6.
  • the agricultural composition of embodiment 1, wherein the nucleic acid is capable of recovering the agricultural pest’s sensitivity or susceptibility to the pesticide.
  • the agricultural composition of embodiment 1, wherein the nucleic acid is capable of altering expression of a gene responsible for pesticide resistance or tolerance.
  • the expression of the gene responsible for pesticide resistance or tolerance is downregulated. 9.
  • the gene responsible for pesticide resistance or tolerance is an ion channel gene, a detoxification gene, a target site resistance gene, or a transporter gene.
  • the ion channel gene is a gene encoding Ryanodine receptor (RyR) or Voltage-gated sodium channel (VGSC).
  • the detoxification gene is selected from the group consisting of a gene encoding UDP-glycosyltransferase (UGT), Cytochrome P450 monooxygenase, Esterase, Carboxylesterase (CarE), and Glutathione S- transferase (GST). 12.
  • the target site resistance gene is selected from the group consisting of a gene encoding Acetylcholinesterase (AChE), Voltage-gated sodium channel (VGSC), Gamma-aminobutyric acid (GABA) receptor, Nicotinic acetylcholine receptor (nAChR), and Glutamate-gated chloride channel (GluCl).
  • the transporter gene is selected from the group consisting of a gene encoding ATP-binding cassette (ABC) transporter, Solute carrier (SLC) transporter, Major facilitator superfamily (MFS) transporter, and P- glycoprotein.
  • nucleic acid is capable of inducing RNA interference in at least one member from an order selected from the group consisting of: Coleoptera, Diptera, Hymenoptera, Lepidoptera, Homoptera, Hemiptera, Orthoptera, Thysanoptera, Dermaptera, Siphonaptera, and Trichoptera. 15. The agricultural composition of any one of embodiments 1-14, wherein the nucleic acid is capable of inducing RNA interference in a member of the order Lepidoptera. 16. The agricultural composition of any one of embodiments 1-14, wherein the nucleic acid is capable of inducing RNA interference in a member of the genus Plutella.
  • the agricultural composition of any one of embodiments 1-14, wherein the nucleic acid is capable of inducing RNA interference in a member of the genus Spodoptera. 18.
  • the agricultural composition of any one of embodiments 1-17, wherein the nucleic acid is a RNA molecule.
  • the agricultural composition of any one of embodiments 1-18, wherein the nucleic acid is an antisense RNA.
  • 20. The agricultural composition of any one of embodiments 1-19, wherein the minicell is ribonuclease deficient.
  • the agricultural composition of embodiment 3, wherein the biological pesticide is a protein toxin. 26.
  • a second minicell encapsulating the pesticide that is capable of killing or controlling the agricultural pest is capable of killing or controlling the agricultural pest.
  • 27. The agricultural composition of any one of embodiments 1-26, further comprising a solid, dry, or liquid carrier.
  • said solid carrier is in a form of granule or pellet and is selected from the group consisting of: diatomaceous earth, loam, silica, alginate, clay, bentonite, vermiculite, and combinations thereof.
  • said liquid carrier is in a form of liquid or emulsion, and is selected from the group consisting of a surfactant, an emulsifier, a crop oil concentrate, a penetrant, and combinations thereof.
  • An agricultural composition comprising: a minicell encapsulating (i) a single-stranded nucleic acid and (ii) a pesticide capable of killing or controlling an agricultural pest, wherein the single-stranded nucleic acid reduces resistance to or tolerance of a pesticide in the agricultural pest.
  • a minicell encapsulating (i) a single-stranded nucleic acid and (ii) a pesticide capable of killing or controlling an agricultural pest, wherein the single-stranded nucleic acid reduces resistance to or tolerance of a pesticide in the agricultural pest.
  • the agricultural pest is resistant to or tolerant of the pesticide.
  • the pesticide is a chemical pesticide or a biological pesticide.
  • any one of embodiments 31-33 wherein the pesticide is selected from the group consisting of an insecticide, a herbicide, a fungicide, an algaecide, a bactericide, a rodenticide, a larvicide, a repellent, a virucide, an ovicide, an acaricide, a nematicide, a molluscicide, a pediculicide, a piscicide, and an avicide. 35.
  • any one of embodiments 31-34 wherein the agricultural pest is selected from the group consisting of insect, weed, fungus, algae, bacterium, rodent, larvae, virus, mite, tick, nematode, mollusca, lice, fish, and bird.
  • the nucleic acid is capable of recovering the agricultural pest’s sensitivity or susceptibility to the pesticide.
  • the nucleic acid is capable of altering expression of a gene responsible for pesticide resistance or tolerance.
  • the agricultural composition of embodiment 38, wherein the gene responsible for pesticide resistance or tolerance is an ion channel gene, a detoxification gene, a target site resistance gene, or a transporter gene.
  • the ion channel gene is a gene encoding Ryanodine receptor (RyR) or Voltage-gated sodium channel (VGSC).
  • the detoxification gene is selected from the group consisting of a gene encoding UDP-glycosyltransferase (UGT), Cytochrome P450 monooxygenase, Esterase, Carboxylesterase (CarE), and Glutathione S- transferase (GST). 105 303137700 42.
  • the target site resistance gene is selected from the group consisting of a gene encoding Acetylcholinesterase (AChE), Voltage-gated sodium channel (VGSC), Gamma-aminobutyric acid (GABA) receptor, Nicotinic acetylcholine receptor (nAChR), and Glutamate-gated chloride channel (GluCl).
  • the transporter gene is selected from the group consisting of a gene encoding ATP-binding cassette (ABC) transporter, Solute carrier (SLC) transporter, Major facilitator superfamily (MFS) transporter, and P- glycoprotein.
  • the nucleic acid is capable of inducing RNA interference in a member of the order Lepidoptera.
  • the nucleic acid is capable of inducing RNA interference in a member of the genus Plutella.
  • the agricultural composition of any one of embodiments 31-44, wherein the nucleic acid is capable of inducing RNA interference in a member of the genus Spodoptera.
  • the agricultural composition of any one of embodiments 31-47, wherein the nucleic acid is a RNA molecule.
  • the agricultural composition of any one of embodiments 31-48, wherein the nucleic acid is an antisense RNA.
  • the agricultural composition of any one of embodiments 31-49, wherein the minicell is ribonuclease deficient.
  • the agricultural composition of any one of embodiments 31-50, wherein the minicell comprises at least one fusion protein. 52.
  • a solid, dry, or liquid carrier is in a form of granule or pellet and is selected from the group consisting of: diatomaceous earth, loam, silica, alginate, clay, bentonite, vermiculite, and combinations thereof.
  • the agricultural composition of embodiment 56 wherein said dry carrier in a form of powder and is selected from the group consisting of: peat, wheat, bran, vermiculite, clay mineral, calcium carbonate, dolomite, gypsum, bentonite, rock phosphate, phosphorous compound, titanium dioxide, humus, talc, alginate, activated charcoal, and combinations thereof.
  • said liquid carrier is in a form of liquid or emulsion, and is selected from the group consisting of a surfactant, an emulsifier, a crop oil concentrate, a penetrant, and combinations thereof.
  • a method of reducing or suppressing pesticide resistance in an agricultural pest comprising: applying an agricultural composition of embodiment 1 or 31 to an agricultural pest, wherein resistance to a pesticide in the agricultural pest is reduced or suppressed after the application of the agricultural composition.
  • the agricultural pest is resistant to or tolerant of the pesticide.
  • the resistance to the pesticide is reduced at least 10% in the agricultural pest applied with the agricultural composition when comparing to an agricultural pest unapplied with the agricultural composition.
  • the pesticide is a chemical pesticide or a biological pesticide.
  • the pesticide is selected from the group consisting of an insecticide, a herbicide, a fungicide, an algaecide, a bactericide, a rodenticide, a larvicide, a repellent, a virucide, an ovicide, an acaricide, a nematicide, a molluscicide, a pediculicide, a piscicide, and an avicide.
  • the agricultural pest is selected from the group consisting of insect, weed, fungus, algae, bacterium, rodent, larvae, virus, mite, tick, nematode, mollusca, lice, fish, and bird.
  • a method of restoring susceptibility of an agricultural pest to a pesticide comprising: applying an agricultural composition of embodiment 1 or 31 to an agricultural pest, wherein the agricultural pest is restored to be susceptible to a pesticide after the application of the agricultural composition.
  • the agricultural pest is resistant to or tolerant of the pesticide.
  • the susceptibility to the pesticide is restored at least 10% in the agricultural pest applied with the agricultural composition when comparing to an agricultural pest unapplied with the agricultural composition.
  • the pesticide is a chemical pesticide or a biological pesticide.
  • the biological pesticide is a protein toxin.
  • the pesticide is selected from the group consisting of an insecticide, a herbicide, a fungicide, an algaecide, a bactericide, a rodenticide, a larvicide, a repellent, a virucide, an ovicide, an acaricide, a nematicide, a molluscicide, a pediculicide, a piscicide, and an avicide.
  • the pesticide is selected from the group consisting of an insecticide, a herbicide, a fungicide, an algaecide, a bactericide, a rodenticide, a larvicide, a repellent, a virucide, an ovicide, an acaricide, a nematicide, a molluscicide, a pediculicide, a piscicide, and an avicide.

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Abstract

La présente divulgation propose des systèmes, des compositions et des procédés pour restaurer la sensibilité d'un ou plusieurs organismes nuisibles aux pesticides et retarder le développement de la résistance aux pesticides. La présente divulgation propose une composition comprenant une minicellule comprenant (i) un acide nucléique qui cible un transcrit codant pour un polypeptide et/ou (ii) un pesticide chimique ou biologique. La présente divulgation propose également un procédé de gestion de la résistance aux pesticides qui peut être développé dans des organismes nuisibles exposés en continu ou de manière répétée à des pesticides.
EP24808135.8A 2023-05-16 2024-05-16 Compositions et procédés de gestion de la résistance aux pesticides Pending EP4712771A2 (fr)

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