[go: up one dir, main page]

WO2024229385A1 - Arn satellite a base de ghabrivirales artificielles - Google Patents

Arn satellite a base de ghabrivirales artificielles Download PDF

Info

Publication number
WO2024229385A1
WO2024229385A1 PCT/US2024/027737 US2024027737W WO2024229385A1 WO 2024229385 A1 WO2024229385 A1 WO 2024229385A1 US 2024027737 W US2024027737 W US 2024027737W WO 2024229385 A1 WO2024229385 A1 WO 2024229385A1
Authority
WO
WIPO (PCT)
Prior art keywords
rna
virus
hrv
sequence
dna
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
PCT/US2024/027737
Other languages
English (en)
Inventor
David George BLUMSACK
James Conrad CHAMNESS
Fu Chyun CHU
Elizabeth Jane Antonelli DENNIS
Jean-Pierre Baptiste Séhi GLOUZON
Mehmet Ali HALAC
Yumeng HAO
Arjun Devang KHAKHAR
Kevin KLICKI
James Michael KREMER
Jayashree Kumar
Katherine Michelle LATOURRETTE
Barry Andrew Martin
Shankar Raj PANT
Derek Thomas ROTHENHEBER
Michka Gabrielle SHARPE
Aditya Sushil Kumar SINGH
Arjun Subedi
Phu Tri Tran
Paveena VICHYAVICHIEN
Reid Evan William WARSABA
Kaixi ZHAO
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.)
Flagship Pioneering Innovations VII Inc
Original Assignee
Flagship Pioneering Innovations VII 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 Flagship Pioneering Innovations VII Inc filed Critical Flagship Pioneering Innovations VII Inc
Publication of WO2024229385A1 publication Critical patent/WO2024229385A1/fr
Anticipated expiration legal-status Critical
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C12N15/8203—Virus mediated transformation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield

Definitions

  • Chrysoviridae typically have a multi- partite dsRNA genome of about 12.5 kb in length. Totiviridae typically have a monopartite dsRNA genome of about 4.6 to about 6.7 kb in length.
  • This disclosure provides recombinant RNA molecules comprising from 5’ terminus to 3’ terminus: (a) a 5’ RNA replication element recognized by a Ghabrivirales RNA-dependent RNA polymerase (RdRP); (b) a cargo RNA molecule (cargo RNA sequence); and (c) a 3’ RNA replication element recognized by the RdRP; wherein the 5’ RNA replication element, the cargo RNA molecule, and the 3’ RNA replication element are operably linked, and wherein the cargo RNA molecule is heterologous to the 5’ RNA replication element and the 3’ RNA replication element.
  • RdRP Ghabrivirales RNA-dependent RNA polymerase
  • cargo molecule cargo molecule
  • 3’ RNA replication element
  • the 5’ RNA replication element and the 3’ RNA replication element are obtained from the same Ghabrivirales genome or from Ghabrivirales genomes having at least 85%, 90%, 95%, 98%, or 99% sequence identity to one another and which are optionally related;
  • the 5’ RNA replication element, the 3’ RNA replication element, and the RdRP are obtained from the same Ghabrivirales genome or from Ghabrivirales genomes having at least 85%, 90%, 95%, 98%, or 99% sequence identity to one another and which are optionally related; or
  • the 5’ RNA replication element, the 3’ RNA replication element, and/or the RdRP coding region are obtained from different Ghabrivirales genomes, and the members of each respective set of the 5’ RNA replication elements, 3’ RNA replication elements, and/or RdRP coding regions have at least 85%, 90%, 95%, 98%, or 99% sequence identity to one another.
  • the RNA molecule comprises: at least one heterologous RNA virus (HRV) amplicon in sense or antisense orientation to the first 5’ RNA replication element comprising: I. Agent Ref: P14421WO00 - 2 - (i) a heterologous RNA virus (HRV) 5’ replication region (HRV 5’RR); (ii) the cargo RNA molecule; and (iii) the heterologous RNA virus (HRV) 3’ RNA replication region (HRV 3’RR); wherein the HRV 5’ RR and HRV 3’ RR HRV are recognized by a heterologous RNA virus RNA-dependent RNA polymerase (hrvRdRP); and wherein the HRV 5’RR, cargo RNA molecule, and HRV 3’RR are operably linked; or II.
  • HRV heterologous RNA virus
  • heterologous RNA virus (HRV) subgenomic promoter operably linked to the cargo RNA molecule; wherein the subgenomic promoter is recognized by a heterologous RNA virus RNA-dependent RNA polymerase (hrvRdRP).
  • HRV heterologous RNA virus RNA-dependent RNA polymerase
  • Agricultural formulations as well as bacterial, fungal, plant, insect, and invertebrate animal cells comprising the herein disclosed recombinant RNAs are also provided.
  • RNA molecules comprising the herein disclosed recombinant RNA molecules; and a cell containing the recombinant RNA molecule and an RdRP protein that recognizes the 5’ and 3’ RNA replication elements of the recombinant RNA molecule.
  • the cell further comprises one or more of: (i) a viral capsid protein (CP); (ii) an RNA-binding protein (RBP) that binds to the RNA molecule, optionally wherein the RBP binds to an RNA effecter; (iii) an RNA cleavage agent that cleaves the RNA molecule; (iv) a second RNA-dependent RNA polymerase (2 nd RdRP) protein that recognizes an HRV 5’ or 3’ replication region and/or a subgenomic promoter in the RNA molecule; (v) a viral movement protein (MP); (vi) a heterologous RNA virus (HRV); or (vii) an hrvRdRP, optionally wherein the hrvRdRP recognizes the HRV 5’ or 3’ replication region and/or the subgenomic promoter.
  • CP a viral capsid protein
  • RBP RNA-binding protein
  • RBP RNA-binding protein
  • methods of establishing a synthetic Ghabrivirales satellite RNA in a plant cell comprising: providing to a plant cell any of the herein disclosed recombinant RNA molecules, wherein the plant cell comprises an RdRP protein that recognizes the 5’ RNA replication element and 3’ RNA replication element, wherein the RNA molecule optionally comprises an encapsidation recognition element (ERE) and is or can be encapsidated by a capsid protein, whereby the RdRP protein catalyzes synthesis of the synthetic Ghabrivirales satellite RNA from the recombinant RNA molecule.
  • ERP encapsidation recognition element
  • Also provided are methods of obtaining a phenotypic change in a plant or plant cell comprising: providing to a plant or plant cell any of the herein disclosed recombinant RNA molecules, wherein the cargo RNA molecule comprises RNA that effects a phenotypic change in the plant or plant cell in comparison to a plant or plant cell lacking the recombinant RNA, wherein the plant or plant cell comprises an RdRP protein that recognizes the 5’ RNA replication element and 3’ RNA replication element and catalyzes synthesis of a synthetic Ghabrivirales RNA from the recombinant RNA molecule, and wherein and the cargo RNA molecule effects the phenotypic change.
  • the methods further comprise providing an hrvRdRP to the plant which recognizes the HRV 5’ or 3’ replication region and/or the subgenomic promoter in the synthetic Ghabrivirales satellite RNA, optionally wherein the hrvRdRP is provided by introducing a recombinant DNA or RNA encoding the hrvRdRP into the plant or a part thereof.
  • Agent Ref P14421WO00 - 3 - [0010] Also provided are methods of manufacturing a synthetic Ghabrivirales satellite particle, comprising combining any of the herein disclosed recombinant RNA molecules with a viral capsid protein, wherein the recombinant RNA molecule comprises an encapsidation recognition element (ERE), and wherein the ERE provides for encapsidation of the RNA by the viral capsid protein.
  • EEE encapsidation recognition element
  • plant propagules comprising any of the herein disclosed recombinant RNA molecules and a Ghabrivirales RdRP, optionally wherein the plant propagule further comprises a heterologous RNA virus RdRP which recognizes an HRV 5’ or 3’ replication region and/or a subgenomic promoter in the synthetic Ghabrivirales satellite RNA.
  • the plant propagule further comprises a heterologous RNA virus RdRP which recognizes an HRV 5’ or 3’ replication region and/or a subgenomic promoter in the synthetic Ghabrivirales satellite RNA
  • the heterologous RNA virus RdRP is an Alphaflexivirus, Betaflexivirus, Bromovirus, Celavirus, Closterovirus, Comovirus, Potexvirus, Potyvirus, Tobamovirus, Tombusvirus, Tospoviridae, Trivirinae, Tymovirus, Varicosavirus, or Secoviridae RdRP.
  • plants comprising any of the herein disclosed recombinant RNA molecules and a Ghabrivirales RdRP, optionally wherein the plant propagule further comprises a heterologous RNA virus RdRP which recognizes an HRV 5’ or 3’ replication region and/or a subgenomic promoter in the synthetic Ghabrivirales satellite RNA.
  • Ghabrivirales satellite systems that are self-replicating when introduced into a plant or plant cell, comprising: (1) any of the herein disclosed recombinant Ghabrivirales satellite RNAs (e.g., recombinant RNA molecules); and (2) an exogenous Ghabrivirales that is capable of replication in the plant or plant cells and that encodes the Ghabrivirales RdRP that recognizes the 5’ and 3’ RNA replication element sequences in the recombinant Ghabrivirales satellite RNA, optionally wherein the Ghabrivirales satellite system further comprises a heterologous RNA virus RdRP which recognizes an HRV 5’ or 3’ replication region and/or a subgenomic promoter in the synthetic Ghabrivirales satellite RNA.
  • RNAs e.g., recombinant RNA molecules
  • an exogenous Ghabrivirales that is capable of replication in the plant or plant cells and that encodes the Ghabrivirales RdRP that recognizes the 5’ and 3’
  • Figure 1 shows a non-limiting embodiment of a structure of a Ghabrivirales satellite construct.
  • the 5’ RNA replication element is labelled “5’ RRE” and the 3’ RNA replication element is labeled “3’ RRE.”
  • Figure 2 shows non-limiting embodiments of a Ghabrivirales satellite construct containing a heterologous RNA virus (HRV) amplicon comprising: (i) a heterologous RNA virus (HRV) 5’ replication region (HRV 5’RR); (ii) the cargo RNA molecule; and (iii) the heterologous RNA virus (HRV) 3’ RNA replication region (HRV 3’RR).
  • HRV heterologous RNA virus
  • cleavable sequence is located: (i) 5’ to the 5’ RNA replication element or 3’ to the 3’ RNA replication element; and/or (ii) between the 3’ end of the 5’ RNA replication element and the HRV amplicon and/or between the HRV amplicon and the 5’ end of the 3’ RNA replication element.
  • the cleavable sequence is optionally a self-cleaving ribozyme, a self-cleaving inducible ribozyme, or an Agent Ref: P14421WO00 - 4 - siRNA or miRNA recognition site.
  • a subgenomic promoter and/or an IRES is/are operably linked to the cargo RNA.
  • Figure 3 shows non-limiting embodiments of a Ghabrivirales satellite construct containing a heterologous RNA virus (HRV) amplicon comprising a heterologous RNA virus (HRV) subgenomic promoter (HRV sgp) which is recognized by a heterologous RNA virus RNA-dependent RNA polymerase (hrvRdRP) and which is operably linked to the cargo RNA molecule.
  • HRV sgp and cargo RNA are in sense and antisense orientation relative to the Ghabrivirales 5’ RRE are shown.
  • Figure 4 depicts a Ghabrivirales satellite construct comprising heterologous RNA virus (HRV) subgenomic promoters (HRV sgp) with: (i) one HRV sgp operably linked to a cargo RNA; and (ii) one HRV sgp operably linked to RNA encoding an hrvRdRP which recognizes both of the HRV sgp (i.e., can drive expression of the operably linked hrvRdRP and cargo RNA).
  • HRV sgp heterologous RNA virus
  • HRV sgp heterologous RNA virus subgenomic promoters
  • an IRES is operably linked to the cargo RNA and/or an IRES is operably linked to the RNA encoding the hrvRdRP.
  • FIG. 5 depicts non-limiting embodiments of a Ghabrivirales satellite construct containing a heterologous RNA virus (HRV) amplicon comprising a heterologous RNA virus (HRV) subgenomic promoter (HRV sgp) which is recognized by a heterologous RNA virus RNA-dependent RNA polymerase (hrvRdRP) and which is operably linked to the cargo RNA molecule flanked by HRV 5’RR and HRV 3’ RR.
  • HRV sgp and cargo RNA are in sense or antisense orientation relative to the Ghabrivirales 5’ RRE are shown.
  • the HRV 5’ RR and 3’ RR which flank the cargo RNA provide for hrvRdRP-mediated replication of an RNA comprising from 5’ to 3’ the HRV 5’ RR, cargo RNA, and HRV 3’ RR.
  • the HRV 5’ RR and 3’ RR are flanked by ribozymes.
  • Figure 6 depicts a commensal satellite with a cargo RNA molecule including an HRV (“HRV1”, e.g., tobacco mosaic virus, TMV) amplicon designed to be amplified by the HRV (“HRV1”, e.g., TMV) RdRP binding to either the replication regions or to the subgenomic promoter, where the commensal satellite is a Ghabrivirales satellite.
  • HRV tobacco mosaic virus
  • RdRP RNA encoding the HRV RdRP
  • solid squares which can further amplify the HRV amplicon, as well as RNA encoding another cargo (solid circles).
  • no amplification of the commensal satellite occurs.
  • Figure 7 depicts a commensal satellite with a cargo RNA molecule including an HRV (HRV1, e.g., tobacco mosaic virus, TMV) amplicon designed to be amplified by the HRV RdRP (“HRV1 RdRP”, solid squares) binding to the HRV1 replication regions, where the commensal satellite is a Ghabrivirales satellite.
  • HRV tobacco mosaic virus
  • the resulting transcripts include RNA encoding the HRV (HRV1) RdRP which can further amplify the HRV amplicon.
  • the HRV1 amplicon includes sequence for a HRV2 amplicon (indicated in italicized text), encoding a coding and/or noncoding cargo (solid circles) and designed to be amplified in the presence of a second acute viral RdRP (“HRV2 RdRP”, hexagonal symbol), which can be provided, e.g., by introduction of a second acute virus (“HRV2”, e.g., cowpea mosaic virus, CPMV) into the plant. In the absence of the commensal virus, no amplification of the commensal satellite occurs.
  • HRV2 RdRP hexagonal symbol
  • Figure 8 depicts a commensal satellite with a cargo RNA molecule including an HRV (“HRV1”, e.g., tobacco mosaic virus, TMV) amplicon designed to be amplified by the HRV (“HRV1”, e.g., TMV) RdRP binding to either of two subgenomic promoters, where the commensal satellite is a Ghabrivirales satellite.
  • HRV1 tobacco mosaic virus
  • TMV tobacco mosaic virus
  • the resulting transcripts include RNA encoding the HRV RdRP (“HRV1 RdRP”, solid squares), which can further amplify the HRV amplicon, as well as RNA encoding a noncoding RNAi cargo, the sense and antisense strands of which are formed during the amplification process to yield a double-stranded RNA molecule (dsRNA) for silencing of a target gene.
  • HRV1 RdRP solid squares
  • RNA encoding a noncoding RNAi cargo the sense and antisense strands of which are formed during the amplification process to yield a double-stranded RNA molecule (dsRNA) for silencing of a target gene.
  • dsRNA double-stranded RNA molecule
  • the term “and/or” as used in a phrase such as “A and/or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone).
  • the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).
  • F 1 plants or seed obtained from a parent plant which has been selfed or that has been crossed to another plant.
  • Ghabrivirales is used to refer to viruses in the families Chrysoviridae and Totiviridae.
  • heterologous when used to describe a first element in reference to a second element means that the first element and second element do not exist in nature disposed as described.
  • a heterologous nucleic acid molecule or sequence is a nucleic acid molecule or sequence that (a) is not native to a cell in which it is expressed, (b) is linked or fused to a nucleic acid molecule or sequence with which it is not linked to or fused to in nature, or with which it is not linked to or fused to in nature in the same way, (c) has been altered or mutated by the hand of man relative to its native state, or (d) has altered expression as compared to its native expression levels under similar conditions.
  • a “heterologous promoter” is used to drive transcription of a sequence that is not one that is natively transcribed by that promoter (e.g., a eukaryote promoter used to drive transcription of a DNA molecule encoding a Ghabrivirales RNA sequence); thus, a “heterologous promoter” sequence can be included in an expression construct by a recombinant nucleic acid technique.
  • a recombinant polynucleotide such as those provided by this disclosure includes genetic sequences of two or more different Ghabrivirales, which genetic sequences are “heterologous” in that they would not naturally occur together.
  • heterologous refers to a molecule or to a discrete part of a molecule; for example, referring to a cargo RNA molecule (e.g., a nucleic acid such as a protein-encoding RNA, an ssRNA, a regulatory RNA, an interfering RNA, or a guide RNA), which can be part of a larger molecule, or referring to a structure (e.g., structures including a promoter (e.g., for a Agent Ref: P14421WO00 - 6 - DNA dependent RNA polymerase) or subgenomic promoter (e.g., for an RNA-dependent RNA polymerase), an RNA effecter, RNA cleavage agent recognition site, or a polynucleotide comprising or encoding an expression-enhancing element, encapsidation recognition element (ERE), selectable or scoreable marker, DNA aptamer, RNA aptamer; a transcription factor binding
  • a structure e
  • IRES internal ribosome entry site
  • An IRES element is generally between 100-800 nucleotides.
  • IRES encephalomyocarditis virus IRES
  • ECMV encephalomyocarditis virus
  • maize hsp101 IRES 5’UTR crucifer infecting tobamovirus crTMV CR-CP 148 IRES
  • tobacco etch virus (TEV) IRES 5’UTR hibiscus chlorotic ringspot virus (HCRSV) IRES.
  • HCRSV hibiscus chlorotic ringspot virus
  • an IRES sequence is derived from non-plant eukaryotic virus sequences that include but are not limited to: acute bee paralysis virus (ABPV), classical swine fever virus (CSFV), coxsackievirus B3 virus (CVB3), encephalomyocarditis virus (ECMV), enterovirus 71 (E71), hepatitis A virus (HAV), human rhinovirus (HRV2), human rhinovirus (HRV2), human lymphotropic virus (HTLV), polyoma virus (PV), and Zea mays (ZmHSP101).
  • a virus A virus
  • HRV2 human rhinovirus
  • HRV2 human rhinovirus
  • HTLV human lymphotropic virus
  • PV polyoma virus
  • ZmHSP101 Zea mays
  • the phrase “operably linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner.
  • a promoter is operably linked to a coding sequence if the promoter provides for transcription or expression of the coding sequence.
  • percent identity refers to percent (%) sequence identity with respect to a reference polynucleotide or polypeptide sequence following alignment by standard techniques. Alignment for purposes of determining percent nucleic acid or amino acid sequence identity can be achieved in various ways that are within the capabilities of one of skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, PSI-BLAST, or Megalign software.
  • the software is MUSCLE (Edgar, Nucleic Acids Res., 32(5): 1792-1797, 2004).
  • MUSCLE Nucleic Acids Res., 32(5): 1792-1797, 2004.
  • percent sequence identity values are generated using the sequence comparison computer program BLAST (Altschul et al. (1990) J. Mol. Biol., 215:403-410).
  • the percent sequence identity of a given nucleic acid or amino acid sequence, A, to, with, or against a given nucleic acid or amino acid sequence, B, is calculated as follows: 100 multiplied by (the fraction X/Y), where X is the number of nucleotides or amino acids scored as identical matches by a sequence alignment program (e.g., BLAST) in that program’s alignment of A and B, and where Y is the total number of nucleotides or amino acids in B.
  • the term “plant” includes a whole plant and any descendant, cell, tissue, or part of a plant.
  • plant parts include any part(s) of a plant, including, for example and without limitation: seed (including mature seed and immature seed); a plant cutting; a plant cell; a plant cell culture; or a plant organ (e.g., pollen, mature or immature embryos, flowers, fruits, shoots, leaves, roots, stems, and explants).
  • a plant tissue or plant organ is or includes a seed, protoplast, callus, or any other group of plant cells that is organized into a structural or functional unit.
  • a plant cell or tissue culture is capable of regenerating a plant having the physiological and morphological characteristics of the plant from which the cell or tissue was obtained, and of regenerating a plant having substantially the same genotype as the plant.
  • Regenerable cells in a plant cell or tissue culture can include embryos, protoplasts, meristematic cells, callus, pollen, leaves, anthers, roots, root tips, flowers, and/or stalks.
  • some plant cells are not capable of being regenerated to produce plants and are referred to herein as “non-regenerable” plant cells.
  • the term “transcriptome” refers to the sum total of all RNA molecules expressed in a cell.
  • RNA molecules include mRNAs, tRNAs, ribosomal RNAs, miRNAs, viral RNAs (both genomic and sub-genomic), and long non-coding RNAs.
  • RNA molecules include mRNAs, tRNAs, ribosomal RNAs, miRNAs, viral RNAs (both genomic and sub-genomic), and long non-coding RNAs.
  • any of the preceding definitions is inconsistent with definitions provided in any patent or non-patent reference incorporated herein by reference, any patent or non-patent reference cited herein, or in any patent or non-patent reference found elsewhere, it is understood that the preceding definition will be used herein.
  • nucleic acid sequences described herein are given, when read from left to right, in the 5’ to 3’ direction. Nucleic acid sequences may be provided as DNA or as RNA, as specified.
  • nucleic acid sequences can encode the same polypeptide sequence, and such modified nucleic acid sequences (e.g., for the purposes of codon optimization for a given species) are within the scope of the present disclosure.
  • modified nucleic acid sequences e.g., for the purposes of codon optimization for a given species
  • recombinant polynucleotides e.g., recombinant DNA, recombinant RNA, recombinant ssRNAs, recombinant dsRNAs, recombinant vectors, etc.
  • recombinant polynucleotides including one or more sequences of or derived from a Ghabrivirales (GV); in particular, a 5’ or 3’ RNA replication element that is recognized by a Ghabrivirales RNA-dependent RNA polymerase (RdRP).
  • This disclosure is further related to methods of making and using such recombinant polynucleotides, for example, by Agent Ref: P14421WO00 - 8 - employing such recombinant polynucleotides to express a heterologous cargo sequence in a plant and optionally thereby modifying expression of an endogenous target sequence and/or genotype or phenotype of the plant.
  • the Ghabrivirales is a commensal Ghabrivirales, that is, a Ghabrivirales that is endemic or native to a given eukaryote host (such as a host plant) without causing apparent negative effects on the host (i.e., is considered non-pathogenic), is often present at a persistent but low population (i.e., low viral titer), and is often vertically transmitted to succeeding generations of the host.
  • this disclosure is related to a recombinant DNA molecule that includes a promoter that is functional in a cell and is operably linked to a DNA sequence encoding an RNA molecule.
  • the RNA molecule includes, in 5’ to 3’ order: (a) a 5’ RNA replication element that is capable of being recognized by a Ghabrivirales RNA-dependent RNA polymerase (RdRP); (b) a cargo RNA sequence; and (c) a 3’ RNA replication element that is capable of being recognized by the Ghabrivirales RdRP.
  • RdRP RNA-dependent RNA polymerase
  • Figure 1 shows an embodiment of a generalized structure of a DNA polynucleotide encoding a Ghabrivirales satellite, where in certain embodiments the 5’ RNA replication element corresponds to the 5’ untranslated region (UTR) of a Ghabrivirales and where the 3’ RNA replication element corresponds to the 3’ untranslated region (UTR) of a Ghabrivirales.
  • the 5’ RNA replication element and/or the 3’ RNA replication element include nucleotides that extend into the predicted coding sequence or open reading frame of the Ghabrivirales.
  • Recombinant DNA molecules provided herein can include a promoter that is functional in a cell (e.g., a bacterial cell, a plant cell, a fungal cell, an insect cell, a vertebrate animal cell, or an invertebrate animal cell) and is operably linked to a DNA sequence encoding an RNA molecule (e.g. a 5’ RNA replication element, a cargo RNA sequence; and a 3’ RNA replication element; a ribozyme, an intron, or a RNA encoding a protein (e.g., a capsid, movement, RdRP, or an RdRP protein that recognizes an HRV 5’ or 3’ replication region and/or a subgenomic promoter).
  • a promoter that is functional in a cell (e.g., a bacterial cell, a plant cell, a fungal cell, an insect cell, a vertebrate animal cell, or an invertebrate animal cell) and is operably linked to a
  • a promoter functional in a plant cell provides for systemic gene expression, or alternatively for cell-, tissue-, or organ-specific gene expression, or expression that is inducible by external signals or agents (for example, light-, pathogen-, wound-, stress-, or hormone-inducible elements, or chemical inducers) or elements that are capable of cell-cycle regulated gene transcription; such elements may be located in the 5’ or 3’ regions of the native gene or engineered into a polynucleotide.
  • Promoters include those from viruses, bacteria, fungi, animals, and plants.
  • RNA polymerase e.g., RNA pol I, pol II, or pol III
  • RNA polymerase e.g., RNA pol I, pol II, or pol III
  • Embodiments of promoters include those from cauliflower mosaic virus (e.g., p35S), bacteriophage (e.g., pT7), and plants (e.g., pATUBQ10).
  • the promoter is operably linked to nucleotide sequences encoding multiple guide RNAs, wherein the sequences encoding guide RNAs are separated by a cleavage site such as a nucleotide sequence encoding a microRNA recognition/cleavage site or a self-cleaving ribozyme (see, e.g., Ferré-D’Amaré and Scott (2010) Cold Spring Harbor Perspectives Biol., 2:a003574).
  • the promoter is a pol II promoter operably linked Agent Ref: P14421WO00 - 9 - to a nucleotide sequence encoding the RNA.
  • the promoter operably linked to one or more polynucleotides encoding elements of a genome-editing system is a constitutive promoter that drives DNA expression in plant cells.
  • the promoter drives DNA expression in the nucleus or in an organelle such as a chloroplast or mitochondrion.
  • constitutive promoters active in plant cells include a CaMV 35S promoter as disclosed in U.S. Pat. Nos.5,858,742 and 5,322,938, a rice actin promoter as disclosed in U.S. Pat. No.5,641,876, a maize chloroplast aldolase promoter as disclosed in U.S. Pat.
  • the promoter operably linked to one or more polynucleotides encoding elements of a genome-editing system is a promoter from figwort mosaic virus (FMV), a RUBISCO promoter, or a pyruvate phosphate dikinase (PDK) promoter, which is active in the chloroplasts of mesophyll cells.
  • FMV figwort mosaic virus
  • RUBISCO RUBISCO promoter
  • PDK pyruvate phosphate dikinase
  • the promoter is heterologous to the cell it is functional in and/or to the other elements to which the promoter is operably linked.
  • Embodiments of recombinant polynucleotides provided herein comprise or encode RNA molecules containing 5’ and 3’ RNA replication elements recognized by a Ghabrivirales RNA-dependent RNA polymerase (RdRP).
  • RdRP RNA-dependent RNA polymerase
  • recognition by a Ghabrivirales RdRP is identified in an in vitro RdRP assay (e.g., an assay adapted from Horiuchi et al. Plant Cell Physiol.42(2):197-203, 2001).
  • recognition by a Ghabrivirales RdRP is identified by an in vivo RdRP assay wherein an RNA comprising 5’ and 3’ RNA replication elements is introduced into a cell comprising the RdRP, and replication of the RNA is assayed (e.g., by an RT-PCR assay or an assay for a reporter gene encoded by a cargo RNA located in the RNA comprising 5’ and 3’ RNA replication elements).
  • cells comprising the RdRP are engineered by introducing a gene or RNA molecule encoding the RdRP into the cell.
  • the cell comprising the RdRP is a cell which contains a Ghabrivirales which expresses the RdRP; in such embodiments the Ghabrivirales virus can be one that is native to or is known to naturally occur in the cell, or it can be a non-native Ghabrivirales virus, or alternatively a recombinant virus of any suitable viral family that is engineered to express the Ghabrivirales RdRP.
  • the recombinant polynucleotides comprise a 5’ RNA replication element and a 3’ RNA replication element obtained from the same Ghabrivirales genome or from Ghabrivirales genomes having at least 85%, 90%, 95%, 98%, or 99% sequence identity to one another and which are optionally related.
  • the Ghabrivirales genomes having at least 85%, 90%, 95%, 98%, or 99% sequence identity to one another are taxonomically related, e.g., genomes classified as belonging to the same genus, family, and/or order.
  • the recombinant polynucleotides comprise a 5’ RNA replication element, a 3’ RNA replication element, and an RdRP are obtained from the same Ghabrivirales genome or from Ghabrivirales genomes having at least 85%, 90%, 95%, 98%, or 99% sequence identity to one another and which are optionally Agent Ref: P14421WO00 - 10 - taxonomically related, e.g., genomes classified as belonging to the same genus, family, and/or order.
  • Non-limiting examples of a 5’ RNA replication element and a 3’ RNA replication element from the same Ghabrivirales protein genome include those set forth in each row of Table 18.
  • Non-limiting examples of a 5’ RNA replication element and a 3’ RNA replication element from the same Ghabrivirales genome and the corresponding RdRP protein that recognizes that 5’ RNA replication element and 3’ RNA replication element include those set forth in each row of Table 18.
  • the RdRPs set forth in Table 18 also recognize the corresponding 5’ RNA replication element and a 3’ RNA replication element from the Ghabrivirales genome corresponding to the same Ghabrivirales (i.e., a Ghabrivirales having the genome of Table 18).
  • the recombinant polynucleotides comprise a 5’ RNA replication element, a 3’ RNA replication element, and/or an RdRP coding region are obtained from two Ghabrivirales genomes wherein the members of each pair of the 5’ RNA replication elements, 3’ RNA replication elements, and RdRP coding regions of the two Ghabrivirales genomes have at least 85%, 90%, 95%, 98%, or 99% sequence identity to one another.
  • the recombinant polynucleotides comprise a 5’ RNA replication element and a 3’ RNA replication element obtained from distinct Ghabrivirales genomes.
  • the recombinant polynucleotides e.g., recombinant DNAs or recombinant RNAs
  • the distinct Ghabrivirales genomes will have less than 85%, 80%, 75%, or 70% sequence identity to one another.
  • the distinct Ghabrivirales genomes will have 50%, 60%, or 65% to any one of 70%, 75%, 80%, or 84% sequence identity to one another.
  • the combination of 5’ RNA replication elements, 3’ RNA replication elements, and RdRP set forth in a single row of Table 18, or variants thereof having at least 85%, 90%, 95%, 98%, or 99% sequence identity to the 5’ RNA replication element, 3’ RNA replication elements, and RdRP, or variants thereof wherein the secondary structures of the RNA replication elements are conserved, are used together in an expression system, plant cell, plant propagule, plant, or method provided herein.
  • the 5’ RNA replication elements and 3’ RNA replication elements in a given row of Table 18 or variants thereof are operably linked to a cargo RNA and replicated by the corresponding RdRP or variant thereof in the row.
  • the combination of 5’ RNA replication elements, 3’ RNA replication elements, and RdRP set forth in any one of rows 1 to 3 of Table 18 or aforementioned or otherwise disclosed variants thereof are used in a dicot plant cell-based expression system, dicot plant cell, dicot plant propagule, dicot plant, or related dicot plant-based method provided herein.
  • the aforementioned dicot is a member of the genus Brassica, Capsicum, Cucumis, Cucurbita, Gossypium, Nicotiana, Solanum, or Glycine.
  • the combination of 5’ RNA replication elements, 3’ RNA replication elements, and RdRP set forth in rows 4 to 6 or aforementioned or otherwise disclosed variants thereof are used in a monocot plant cell-based expression system, monocot plant cell, monocot plant propagule, monocot Agent Ref: P14421WO00 - 11 - plant, or related monocot plant-based method provided herein.
  • the aforementioned monocot is a member of the genus Avena, Hordeum, Oryza, Secale, Triticum, Sorghum, or Zea.
  • Examples of DNA molecules which encode RNA molecules comprising or containing 5’ and 3’ RNA replication elements (RRE) recognized by a Ghabrivirales RdRP are set forth in Table 1.
  • DNA molecules which encode RNAs comprising or containing 5’ RNA replication elements recognized by a Ghabrivirales RdRP include SEQ ID NO: 467, 468, 469, 470, 471, 472, 473, 474, 549 to 552, or 557 to 559.
  • DNA molecules which encode RNAs comprising or containing 3’ RNA replication elements recognized by a Ghabrivirales RdRP include SEQ ID NO: 475, 476, 477, 478, 479, 480, 481, 482, 553 to 556, or 560 to 562.
  • Structural features e.g., dsRNA hairpins and ssRNA loops
  • the dot bracket notation provided in Table 1 was generated using RNA Fold software for predicting RNA secondary structure based on minimum free energy predictions of base pair probabilities.
  • a dot ‘.’ signifies an unpaired base and a bracket ‘(‘ or ‘)’ represents a paired base.
  • Dot bracket notation is further described in Mattei et al., Nucleic Acids Research, 42(10): 6146-6157, 2014; Ramlan and Zauner In International Workshop on Computing With Biomolecules, E. Csuhaj-Varju, R. Freund, M. Oswald and K. Salomaa (Eds.), 27 August 2008, Wien, Austria, pp.75–86, From: Austrian Computer Society, 2008; and Hofacker et al., Monatshefte für Chemie Chem. Monthly, 125: 167–188, 1994.
  • Such structural features can range in size from 20, 30, or 40 to about 500 nucleotides (nt). These structural features are useful for designing engineered polynucleotide sequences that function as Ghabrivirales RNA replication elements and/or for constructing variants of the sequences set forth in SEQ ID NO: 467 to 482 and 549 to 562 that function as 5’ and 3’ RNA replication elements. In certain embodiments, one of more residues in the RNA secondary structure set forth in Table 1 or in equivalent RNAs are substituted with distinct nucleotides which maintain the RNA secondary structure (e.g., presence or absence of base pairing).
  • the RNA secondary structure set forth in Table 1 or in equivalent RNAs is maintained by making substitutions in the nucleotide sequence that result in no changes in the position of base-paired nucleotides or non-base-paired nucleotides.
  • the RNA secondary structure set forth in Table 1 or in equivalent RNAs is maintained by substituting nucleotides in the secondary structure which are not base paired with nucleotides which will not base pair, and/or by substituting nucleotides in the secondary structure which are base paired with nucleotides which will base pair.
  • maintaining the RNA secondary structure need not be absolute (e.g., the structure is partially maintained).
  • a dsRNA structure is partially maintained when one, two, three or more nucleotides, particularly at the 5’ end and/or 3’ end of a hairpin-forming structure are substituted with nucleotides which do not base pair and thus reduce the total length of dsRNA in the structure.
  • an unpaired RNA structure is partially maintained when one, two, three or more Agent Ref: P14421WO00 - 12 - nucleotides, particularly at the 5’ end and/or 3’ end of a loop structure are substituted with nucleotides which base pair and thus reduce the total length of ssRNA in the loop structure.
  • Embodiments of Ghabrivirales satellite RNAs include those where the 5’ RNA replication element includes one or more of these 5’ structural features and/or wherein the 3’ RNA replication element includes one or more of these 3’ structural features.
  • the 5’ RNA replication elements comprise an RNA encoded by a DNA having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 467, 468, 469, 470, 471, 472, 473, 474, 549 to 552, or 557 to 559, optionally wherein the encoded RNA maintains or partially maintains a corresponding structural feature set forth in Table 1.
  • the 3’ RNA replication elements comprise an RNA encoded by a DNA having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 475, 476, 477, 478, 479, 480, 481, 482, 553 to 556, or 560 to 562, wherein the encoded RNA optionally maintains or partially maintains a corresponding structural feature set forth in Table 1.
  • Recombinant polynucleotides e.g., recombinant DNA, recombinant RNA, recombinant ssRNAs, recombinant dsRNAs, recombinant vectors, etc.
  • additional RNA elements include RNAs encoding a Ghabrivirales capsid or coat protein (CP).
  • Examples of DNA sequences encoding Ghabrivirales CP include the corresponding sequences of GV genomes set forth in Table 1 under descriptors “NC_XXXXX” which refer to the National Center for Biotechnology Information database accession number for entries in the world wide web internet database “ncbi[dot]nlm[dot]nih.gov/nuccore”.
  • Embodiments of additional RNA elements include RNAs encoding a Ghabrivirales RdRP.
  • Examples of DNA sequences encoding a Ghabrivirales RdRP include the corresponding sequences of GV genomes set forth in Table 1 under descriptors “NC_XXXXX” which refer to the National Center for Biotechnology Information database accession number for entries in the world wide web internet database “ncbi[dot]nlm[dot]nih.gov/nuccore”. Examples of DNA sequences encoding Ghabrivirales RdRP also include the sequence of the DNA encoding the GV RdRP disclosed in SEQ ID NO: 483, 484, 485, 486, 487, 488, 489, or 490 as well as DNA sequences having at least 85%, 90%, 95%, 98%, or 99% identity thereto.
  • RNAs encoding Ghabrivirales RdRP and Ghabrivirales RdRP protein sequences also include the sequences set forth in Table 17 as well as the DNA and protein sequences having at least 85%, 90%, 95%, 98%, or 99% identity thereto.
  • Embodiments of additional RNA elements include RNAs encoding a viral movement protein (MP).
  • the cargo RNA comprises an RNA encoding a viral MP.
  • the viral movement protein is believed to bind to the RNA and to assist its movement (and thus the movement of the cargo RNA) throughout the plant, e.g., via the plasmodesmata.
  • Viral MPs include movement proteins identified from tobacco mosaic virus (TMV), cowpea mosaic Agent Ref: P14421WO00 - 13 - virus, potato leafroll virus, tomato spotted wilt virus, and tomato mosaic virus. MPs from a variety of viruses are described in Table 3.
  • Embodiments of additional RNA elements include tRNA-like sequences (TLS). TLS can trigger mobility of otherwise nonmobile RNAs, assisting to increase systemic delivery of the RNA molecule. TLS include tRNAs and tRNA-like sequences identified from other genetic elements, e.g., mRNAs. An isoleucine tRNA encoded by SEQ ID NO: 466 is an example of a useful tRNA-like sequence.
  • RNA sequences including TLS identified in Arabidopsis which are useful for building polynucleotides are described in Table 4.
  • Table 4 mobile mRNA sequences were downloaded from the PLAMOM database for Arabidopsis.
  • the tRNA “seed alignment” from the RFAM database was downloaded in stockholm format (multiple sequence alignment + secondary structure).
  • a covariance model was created with INFERNAL for the tRNA stockholm alignment.
  • PLAMOM mRNA sequences were scanned for significant similarity to tRNAs based on primary and secondary structure features. mRNA sequences with significant hits (E-val ⁇ 1) were then saved to a fasta file.
  • such a tRNA-like sequence includes a tRNA-like sequence from an Arabidopsis Flowering Time T (FT) mRNA.
  • the RNA molecule includes at least one RNA encoding a viral MP, a tRNA-like sequence from an Arabidopsis FT mRNA, and an encapsidation recognition element (ERE) comprising TMV-OAS.
  • the RNA molecule includes a tRNA-like sequence encoded by a DNA sequence selected from the group consisting of SEQ ID NOs: 76-123, and 466.
  • the RNA molecule includes a modified tRNA-like sequence that has at least 90%, 95%, 98%, or 99% sequence identity to a scaffold tRNA-like sequence encoded by a DNA sequence selected from the group consisting of SEQ ID NOs: 76-123, and 466 and that maintains the secondary structure of the scaffold tRNA-like sequence.
  • RNAs encoding a viral capsid protein include RNAs encoding a viral capsid protein (CP).
  • CP viral capsid protein
  • Such capsid proteins are also sometimes referred to as coat proteins, with both capsid and coat proteins being referred to as “CP.”
  • the capsid protein is heterologous to the Ghabrivirales.
  • the cargo RNA comprises an RNA encoding a viral CP.
  • CP can be provided, e.g., by co-expression of a recombinant construct encoding the CP or by native expression by a virus endogenous to or introduced into a plant cell. Encapsidation of an RNA molecule by the CP is achieved provided it contains an encapsidation recognition element (ERE), e.g., an origin of assembly sequence (OAS).
  • ERP encapsidation recognition element
  • OAS origin of assembly sequence Table 2 describes several OAS and CP sequences from a variety of viruses useful in engineering constructs which provide for RNA encapsidation.
  • the OAS is positioned near the 3’ end of a construct, e.g., within the 3’ region of a cargo RNA or 3’ to a cargo RNA.
  • the OAS is found 5’ to the 3’ RNA replication elements (e.g., the 3’ RNA replication elements set forth in Table 1).
  • a TMV-OAS positioned at the 3’ end of the RNA molecule is recognized by the TMV capsid protein, leading to assembly of a TMV virion around the RNA.
  • Agent Ref P14421WO00 - 14 -
  • Embodiments wherein the recombinant RNAs are complexed with RNA binding proteins (RBPs) are also provided herein.
  • Embodiments of RBPs include RNA recognition motifs (RRMs) such as: (i) Lys/Arg-Gly-Phe/Tyr-Gly/Ala-Phe/Tyr-Val/Ile/Leu-X-Phe/Tyr, where X can be any amino acid (SEQ ID NO: 464); (ii) Ile/Val/Leu-Phe/Tyr-Ile/Val/Leu-X-Asn-Leu, where X can be any amino acid (SEQ ID NO: 465).
  • RBP and RRM include those disclosed in Maris et al.2005, doi.org/10.1111/j.1742-4658.2005.04653.x.
  • Embodiments of additional RNA elements include at least one ribozyme.
  • Ribozymes include self-cleaving ribozyme, a ligand-responsive ribozyme (aptazyme), a trans-cleaving ribozyme designed to cleave a target sequence (e.g., a trans-cleaving hammerhead ribozyme designed to cleave the pepper phytoene desaturase (PDS) sequence (the RNA encoded by SEQ ID NO: 421), a hepatitis delta virus (HDV) ribozyme (the RNA encoded by SEQ ID NO: 423), or a hammerhead ribozyme (the RNA encoded by SEQ ID NO: 420).
  • PDS pepper phytoene desaturase
  • HDV hepatitis delta virus
  • multiple ribozymes are included in a polynucleotide.
  • Useful ribozymes include Twister, Hammerhead, Hairpin, and other ribozymes.
  • Non- limiting examples of useful ribozymes include those provided in Table 14.
  • such a ribozyme e.g., a self-cleaving ribozyme
  • such a ribozyme e.g., a self-cleaving ribozyme
  • a ribozyme is located 5’ to the HRV 5’ RNA replication region and/or 3’ to the HRV 3’ RNA replication region in a recombinant RNA comprising an imbedded heterologous RNA virus (HRV) amplicon.
  • HRV heterologous RNA virus
  • intronic sequences are placed in a 5’UTR downstream of a promoter (e.g., a promoter active in plant cells) used to drive expression of a recombinant RNA.
  • a promoter e.g., a promoter active in plant cells
  • intronic sequences are placed 5’ to a 5’ RNA replication element, in a cargo RNA, or 3’ to a 3’ RNA replication element.
  • Embodiments of recombinant polynucleotides and additional RNA elements include subgenomic promoters recognized by an RNA-dependent RNA polymerase (RdRP) and/or RNA molecules encoding an RNA-dependent RNA polymerase (RdRP).
  • RdRP RNA-dependent RNA polymerase
  • RdRP RNA-dependent RNA polymerase
  • Examples of such subgenomic promoters and RdRP include a Brome Mosaic Virus subgenomic promoter and RdRP (Siegal et al.1998, doi: 10.1073/pnas.95.20.11613), barley yellow dwarf virus (BYDV) sgRNA1, sgRNA2, and sgRNA3 subgenomic promoters and RdRP (Koev and Miller; J Virol.2000 Jul;74(13):5988-96.
  • Brome Mosaic Virus subgenomic promoter and RdRP (Siegal et al.1998, doi: 10.1073/pnas.95.20.11613), barley yellow dwarf virus (BYDV) sgRNA1, sgRNA2, and sgRNA3 subgenomic promoters and RdRP (Koev and Miller; J Virol.2000 Jul;74(13):5988-96.
  • such subgenomic promoters are placed either 5’ and/or 3’ to an RNA molecule comprising a 5’ RNA replication element, a cargo RNA, and a 3’ RNA replication element to permit production of either or both + and – strands of the RNA molecule when the RdRP is provided.
  • RNA molecule comprising a 5’ RNA replication element, a cargo RNA, and a 3’ RNA replication element to permit production of either or both + and – strands of the RNA molecule when the RdRP is provided.
  • such subgenomic promoters are operably linked to a cargo RNA molecule and/or to any additional RNA element to permit production of the corresponding cargo and/or additional RNA when the RdRP is provided.
  • the subgenomic promoters are operably linked to a cargo RNA comprising an HRV-inhibitory RNA or to a cargo RNA that encodes a protein which inhibits infection, movement, transmission, and/or replication of the HRV.
  • the subgenomic promoters are operably linked to a cargo RNA comprising an RNA having at least 20 (e.g., 20, 21, 22, 23, 24, or 25) contiguous nucleotides having an identical or complementary sequence to a segment of equivalent length of the genomic RNA of the HRV.
  • the subgenomic promoters are operably linked to a cargo RNA comprising an RNA having at least 20 (e.g., 20, 21, 22, 23, 24, or 25) contiguous nucleotides having an identical or complementary sequence to a segment of equivalent length of the genomic RNA of the HRV which does not encode the hrvRdRP.
  • Embodiments of other optional elements in the recombinant polynucleotides provided herein include: a) a discrete expression cassette including a second promoter operably linked to a DNA sequence to be transcribed, and optionally a terminator element (see, e.g., a NOS or CaMV35S terminator); (b) an expression-enhancing element (e.g., a DNA encoding an expression-enhancing intronic sequence); (c) a DNA or RNA sequence encoding a marker (e.g., a selectable marker such as DNA or RNA encoding an antibiotic resistance or herbicide resistance sequence; DNA encoding a scorable marker or detectable label (e.g., a beta-glucuronidase, fluorescent protein, luciferase, etc.); (d) a DNA aptamer; (e) a DNA or RNA sequence encoding an RNA aptamer; (f) T-DNA left and right border DNA sequences; (g) a
  • recombinant polynucleotides comprising a cargo RNA molecule or comprising DNA encoding a cargo RNA molecule.
  • the recombinant polynucleotide includes a single cargo RNA molecule.
  • the recombinant polynucleotide includes at least two cargo RNA molecules, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 cargo RNA molecules; in embodiments, the at least two cargo RNA molecules are the same (e.g., multiple copies of a non-coding RNA sequence or multiple copies of a RNA sequence encoding a polypeptide) or are different (e.g., two or more different non-coding RNA sequences, or two or more different coding RNA sequences, or combinations of non-coding and coding cargo RNA sequences).
  • the at least two cargo RNA molecules are the same (e.g., multiple copies of a non-coding RNA sequence or multiple copies of a RNA sequence encoding a polypeptide) or are different (e.g., two or more different non-coding RNA sequences, or two or more different coding RNA sequences, or combinations of non-coding and coding cargo RNA sequences).
  • a cargo RNA molecule is up to about 4.6 kilobases (kb) in length (Totiviridae) or up to about 12.5 kilobases (kb) in length (Chrysoviridae).
  • Cargo RNA molecules can range in length from any one of about 20 nucleotides (nt), 100nt, 200nt, 300nt, 400nt, 500nt, 600nt, 700nt, 800nt, or 900nt to any one of about 1kb, 2kb, 3kb, 4kb, 5kb, 6kb, 8kb, 10kb, 12kb, or 12.5kb in length.
  • RNAs comprising a cargo RNA of up to about 4.6 kilobases (kb) in length (Totiviridae) or up to about 12.5 kilobases (kb) in length (Chrysoviridae) in length can in certain embodiments be encapsidated by a GV capsid protein.
  • cargo RNAs can range in length from any one of about 20nt, 100nt, 200nt, 300nt, 400nt, 500nt, 600nt, 700nt, 800nt, or 900nt to any one of about 1kb, 2kb, 3kb, 3.2kb, 4kb, 5kb, 6kb, 7kb, 8kb, 9kb, 10kb, 11kb, 12kb, 13kb, or 14 kb in length.
  • Recombinant RNAs comprising a cargo RNA of up to about 3.2kb, 4kb, 5kb, 6kb, 7kb, 8kb, 9kb, 10kb, 11kb, 12kb, 13kb, or 14kb in length can in certain embodiments be encapsidated by a heterologous viral capsid protein set forth in Table 2.
  • recombinant RNAs comprising a cargo RNA of up to about 14kb and encapsidated by a heterologous viral capsid protein can comprise an OAS element set forth in Table 2 and be encapsidated by a corresponding capsid protein set forth in Table 2.
  • the cargo RNA molecule is greater than 14kb, for example, 15kb, 16kb, 17kb, 18kb, 19kb, or even 20kb.
  • the cargo RNA molecule includes: (a) at least one coding sequence, (b) at least one non-coding sequence, or (c) both at least one coding sequence and at least one non-coding sequence.
  • Such cargo RNA molecules include combinations of coding/non-coding sequence; multiple non-coding/coding sequences; as well as aptamers, ribozymes, and other elements as is described herein.
  • the cargo RNA molecule includes: (a) a coding sequence to be expressed in a plant, and (b) at least one non-coding sequence that modifies expression or translation of the coding sequence, such as a recognition and cleavage sequence for an siRNA or miRNA that is endogenously expressed in the plant (see, e.g., US Patent Nos.8,334,430, 9,139,838, 9,976,152, 10,793,869, 10,876,126) and can bind to and cleave an RNA transcript containing the recognition and cleavage sequence; in such embodiments, it is possible to achieve spatially or temporally or developmentally specific expression of the coding sequence in the plant.
  • a coding or non-coding cargo RNA can be optimized for expression in plants by methods which include using codons which occur more frequently in plant genes and/or by eliminating polyadenylation sites in the cargo RNA (e.g., as described in at least US Pat. Nos.5380831, 5689052, and 7741118, which are each incorporated herein by reference in their entireties).
  • the cargo RNA molecule includes at least one coding sequence (e.g., a translatable sequence).
  • the coding sequence is accordingly a protein or a polypeptide such as those described in this disclosure’s working examples.
  • a cargo RNA comprises a selectable marker RNA encoding an antibiotic resistance or herbicide resistance polypeptide sequence or a scorable marker RNA encoding a scorable marker protein (e.g., a beta- Agent Ref: P14421WO00 - 17 - glucuronidase, fluorescent protein, luciferase, etc.).
  • a scorable marker protein e.g., a beta- Agent Ref: P14421WO00 - 17 - glucuronidase, fluorescent protein, luciferase, etc.
  • selectable marker/selection agent combinations include glyphosate-resistant EPSPS enzymes and/or glyphosate oxidases/glyphosate, a bialaphos resistance (bar) or phosphinothricin acyl transferase (pat) enzyme/glufosinate, or a neomycin phosphotransferase (npt)/neomycin or kanamycin.
  • scorable markers include ⁇ -glucuronidase (GUS), luciferase, and fluorescent proteins such as green fluorescent protein (GFP), yellow fluorescent protein (YFP), and cyan fluorescent protein (CFP).
  • the cargo RNA sequence encodes at least one protein or polypeptide that provides a desirable trait in a plant in which the protein or polypeptide is expressed.
  • polypeptides useful in agricultural applications include, for example, bacteriocins, lysins, antimicrobial peptides, nodule C-rich peptides, and bacteriocyte regulatory peptides.
  • Such polypeptides can be used to alter the level, activity, or metabolism of target microorganisms for increasing the fitness of beneficial insects (such as honeybees and silkworms) or for decreasing the fitness of pest invertebrates (such as aphids, caterpillars, beetle larvae, and mites).
  • Embodiments of agriculturally useful polypeptides include peptide toxins, such as those naturally produced by entomopathogenic bacteria (e.g., Bacillus thuringiensis, Photorhabdus luminescens, Serratia entomophila, or Xenorhabdus nematophila), as is known in the art.
  • entomopathogenic bacteria e.g., Bacillus thuringiensis, Photorhabdus luminescens, Serratia entomophila, or Xenorhabdus nematophila
  • Embodiments of agriculturally useful polypeptides include polypeptides (including small peptides such as cyclodipeptides or diketopiperazines) for controlling agriculturally important pests or pathogens, e.g., antimicrobial polypeptides or antifungal polypeptides for controlling diseases in plants, or pesticidal polypeptides (e.g., insecticidal polypeptides and/or nematicidal polypeptides) for controlling invertebrate pests such as insects or nematodes.
  • polypeptides including small peptides such as cyclodipeptides or diketopiperazines
  • antimicrobial polypeptides or antifungal polypeptides for controlling diseases in plants
  • pesticidal polypeptides e.g., insecticidal polypeptides and/or nematicidal polypeptides
  • invertebrate pests such as insects or nematodes.
  • Embodiments of antimicrobial polypeptides include cathelicidins, cecropins, beta-defensins, amphibian antimicrobial peptides (e.g., aurein-like peptides, esculentin, gaegurin, brevinin, rugosin, ranatuerin, ranacyclin, uperin, ocellatin, grahamin, nigrocin, dermoseptin, temporin, bombinin, maximin), enterocins, ponicerins, megourins, apidaecins, abaecins, attacin, bacteriocins and lantibiotics, dermcidin, formaecin, halocidins, lactocin, tachystatins, and some insecticidal toxins produced by spiders and scorpions.
  • amphibian antimicrobial peptides e.g., aurein-like peptides, esculentin
  • Embodiments of agriculturally useful polypeptides include antibodies, nanobodies, and fragments thereof, e.g., antibody or nanobody fragments that retain at least some (e.g., at least 10%) of the specific binding activity of the intact antibody or nanobody.
  • Embodiments of agriculturally useful polypeptides include transcription factors, e.g., plant transcription factors; see., e.g., the “AtTFDB” database listing the transcription factor families identified in the model plant Arabidopsis thaliana), publicly available at agris- knowledgebase[dot]org/AtTFDB/.
  • Embodiments of agriculturally useful polypeptides include nucleases, for example, exonucleases or endonucleases (e.g., Cas nucleases such as Cas9 or Cas12a).
  • Embodiments of agriculturally useful polypeptides further include cell-penetrating peptides, enzymes (e.g., amylases, cellulases, peptidases, lipases, chitinases), peptide pheromones (for example, yeast or fungal mating pheromones, invertebrate reproductive and larval signaling pheromones, see, e.g., Altstein (2004) Peptides, 25:1373–1376).
  • enzymes e.g., amylases, cellulases, peptidases, lipases, chitinases
  • peptide pheromones for example, yeast or fungal mating pheromones, invertebrate reproductive and
  • Embodiments of agriculturally useful polypeptides confer a beneficial agronomic trait, e.g., herbicide tolerance, insect control, modified yield, increased fungal or oomycte Agent Ref: P14421WO00 - 18 - disease resistance, increased virus resistance, increased nematode resistance, increased bacterial disease resistance, plant growth and development, modified starch production, modified oils production, high oil production, modified fatty acid content, high protein production, fruit ripening, enhanced animal and human nutrition, production of biopolymers, environmental stress resistance, pharmaceutical peptides (e.g., hormones, enzymes, transcription factors, antigens, antibodies, or antibody fragments) and secretable peptides, improved processing traits, improved digestibility (e.g., reduced levels of toxins or reduced levels of compounds with “anti-nutritive” qualities such as lignins, lectins, and phytates), enzyme production, flavor, nitrogen fixation, hybrid seed production, fiber production, and biofuel production.
  • beneficial agronomic trait
  • Non-limiting examples of agriculturally useful polypeptides include polypeptides that confer herbicide resistance (US Patent Nos.6,803,501; 6,448,476; 6,248,876; 6,225,114; 6,107,549; 5,866,775; 5,804,425; 5,633,435; and 5,463,175), increased yield (US Patent Nos.
  • the cargo RNA encodes one or more small signaling peptides (SSPs), also called peptide hormones, which are an attractive option for use as cargoes in RNA commensal satellites due to their small size (5-75 amino acids) and potency.
  • SSPs small signaling peptides
  • SSPs result from processing longer precursor polypeptides (derived from ORF regions).
  • SSPs originate from a wider range of sources including intergenic/intronic regions, long non-coding RNAs, pri-miRNAs, and 5′ and 3′ UTRs of mRNAs.
  • Non-limiting examples of SSPs include miPEP172c, miPEP171d, BomiPEP397a, AtmiPEP397a, BvmiPEP164b, and AtmiPEP164b peptides set forth in Table 13.
  • the RNA molecule further includes an internal ribosome entry site (IRES) located 5’ and immediately adjacent to the at least one coding sequence.
  • IRS internal ribosome entry site
  • the cargo RNA molecule includes multiple coding sequences, and the RNA molecule further includes an IRES located 5’ and immediately adjacent to each of the coding sequences (e.g., open translational reading frames encoding a protein of interest.
  • IRES sequences include those depicted in Table 5.
  • the cargo RNA molecule includes a non-coding sequence such as those described in this disclosure’s working examples.
  • non-coding sequences include a hairpin RNA (hpRNA); an RNA that forms multiple stem-loops; an RNA pseudoknot; an RNA sequence that forms at least partially double-stranded RNA; a small interfering RNA (siRNA) or siRNA precursor; a microRNA (miRNA) or miRNA precursor; a ribozyme; a ligand-responsive ribozyme (aptazyme); an RNA aptamer; or a long noncoding RNA (lncRNA).
  • hpRNA hairpin RNA
  • siRNA small interfering RNA
  • miRNA microRNA
  • aptazyme a ligand-responsive ribozyme
  • RNA aptamer or a long noncoding RNA (lncRNA).
  • the cargo RNA includes a selectable or scorable RNA marker, such as an RNA aptamer or a regulatory RNA, such as an siRNA or siRNA precursor (see, e.g., US Patent No.8,404,927, 8,455,716, 9,777,288, 10,378,012), a miRNA or a miRNA precursor (see, e.g., US Patent Nos.8,410,334, 8,395,023, 9,708,620), a trans-acting siRNA or trans-acting siRNA precursor (see, e.g., US Patent Nos.8,030,473, 8,476,422, 8,816,061, 9,018,002), a phased sRNA or phased sRNA precursor (see, e.g., US Patent No.8,404,928), an siRNA or miRNA decoy (see, e.g., US Patent Nos.8,946,511, 9,873,888), an siRNA or miRNA cleavage blocker (see, e
  • RNA aptamers include those that exhibit fluorescence upon binding a molecule.
  • the fluorescent RNA aptamer can be the Broccoli RNA aptamer.
  • RNA aptamers that can be used include, but are not limited to, Spinach, Spinach2, Carrot, Radish, Corn, Red Broccoli, Orange Broccoli, and Broccoli Fluorets.
  • Other useful RNA aptamers that can be used include those provided in Table 15.
  • Suitable regulatory RNAs can be used to down-regulate (i.e., silence) the expression of a marker gene.
  • PDS phytoene desaturase
  • silencing of the gene yields a photobleached phenotype is widely used as a marker gene because silencing of the gene yields a photobleached phenotype.
  • RNAs such as decoys or cleavage blockers can also be used to interfere with endogenous small RNA-regulated pathways, resulting in a visible phenotype; see, e.g., US Patent Nos.8,946,511, 9,873,888, 9,040,774).
  • Antiviral cargo RNAs, and in particular antiviral cargo RNAs directed against viral pathogens are provided herein.
  • the antiviral cargo RNAs comprise a heterologous RNA Virus (HRV)-inhibitory RNA or encode an HRV-inhibitory protein, wherein the HRV-inhibitory RNA or protein inhibits infection, movement, transmission, and/or replication of the HRV.
  • HRV RNA Virus
  • Target viral pathogens include an Alphaflexivirus, Betaflexivirus, Bromovirus, Celavirus, a Closterovirus, a Comovirus, a Potexvirus, Potyvirus, Tobamovirus, Tombusvirus, Tospoviridae, Trivirinae, Tymovirus, Varicosavirus, or Secoviridae.
  • the target viral pathogen is Cucumber Mosaic Virus, Brome mosaic virus, Citrus tristeza virus, Beet yellows virus, Cowpea mosaic virus, Potato virus X; Pepper mottle virus, Bean yellow mosaic virus, Barley stripe mosaic virus, Wheat stripe mosaic virus, Rice yellow mottle virus, Maize dwarf mosaic virus, zucchini yellow mosaic virus, watermelon mosaic virus, sugarcane mosaic virus, Tobacco mosaic virus, Tomato mosaic virus, Tomato brown rugose fruit virus, Turnip vein-clearing virus, Pepper mild mottle virus, Turnip crinkle virus, Tomato bushy stunt virus, Tomato spotted wilt virus, watermelon bud necrosis virus, Turnip yellow mosaic virus, Spinach latent virus, Olive latent virus 2, Citrus yellow vein clearing virus, Potato latent virus, Apple stem grooving virus, Citrus leaf blotch virus, Apple latent spherical virus, or Soybean latent spherical virus.
  • the targeted viral pathogen is a heterologous RNA virus disclosed in Table 7.
  • antiviral inhibitory RNAs (RNAi sequences) used as cargo RNAs are obtained for a chosen target gene of a viral pathogen using siRNA/miRNA prediction tools (see, e.g., on the world wide web internet site “zhaolab[dot]org/pssRNAit/).
  • siRNA/miRNA prediction tools see, e.g., on the world wide web internet site “zhaolab[dot]org/pssRNAit/).
  • Other examples of non-coding RNA sequences having antiviral activity e.g., dsRNA molecules which produce miRNA or siRNA
  • examples include those disclosed in US Patent No.8,455,716, which is incorporated herein by reference in its entirety.
  • Non-limiting examples of viral targets for antiviral cargo RNA molecules include the viral genes and genomes provided in Table 7 as well as other variants of those viral sequences.
  • cargo RNAs encoding antiviral proteins are provided.
  • Non-limiting examples of antiviral proteins include the N protein (Whitham, S. et al. Cell 78, 1101–1115 (1994)) and endogenous plant viral resistance proteins provided in Table 8, [0060]
  • Antifungal cargo RNAs, and in particular antifungal cargo RNAs directed against plant fungal pathogens, are provided herein.
  • Target fungal pathogens include Botrytis, Fusarium, Magnaporthe, Phytophthora, Rhizoctonia, Sclerotinia, and Verticillium sp.
  • the antifungal cargo RNA comprises a non-coding RNA sequence having antifungal activity (e.g., dsRNA molecules which produce miRNA or siRNA) and in particular a dsRNA directed against a fungal pathogen target gene.
  • such antifungal cargo RNAs comprising dsRNA-mediated control of fungal pathogens are modeled after those described in Qiao et al., 2021, doi: 10.1111/pbi.13589; Duanis-Assaf, et al., 2022, DOI: 10.1111/pbi.13708; Yang et al., 2022, doi: 10.3389/fmicb.2021.660976; Sundaresha et al., doi: 10.20944/preprints202102.0280.v1; and Gaffar et al., 2019, doi: 10.3389/fmicb.2019.01662.
  • Non-limiting examples of antifungal cargo RNAi targets are provided in Table 10.
  • antifungal inhibitory RNAs (RNAi sequences) used as cargo RNAs are obtained for a Agent Ref: P14421WO00 - 21 - chosen target gene of a fungal pathogen (e.g., a fungal pathogen gene set forth in Table 10) using siRNA/miRNA prediction tools (see, e.g., on the world wide web internet site “zhaolab[dot]org/pssRNAit/).
  • antifungal cargo RNAs encode antifungal proteins.
  • Useful antifungal proteins include nodule-specific cysteine-rich antimicrobial peptides (Vellivelli et al., 2020, doi: 10.1073/pnas.2003526117), defensins (Asano et al., 2013, doi: 10.1371/journal.ppat.1003581), the conidial germination-inhibiting antifungal peptides disclosed in International Patent Application publication WO2023/004435, which is incorporated by reference herein, including their homodimers, heterodimers, and fusions with signal or cell-penetrating peptides (e.g., the sequences provided in Tables 4 and 5 of WO2023/004435); the various antifungal antimicrobial peptides disclosed in De Cesare et al.
  • Insecticidal cargo RNAs and in particular insecticidal or insect inhibitory cargo RNAs directed against insects are provided herein.
  • Target insects include sucking insects (e.g., heteropteran and homopteran insects including aphids, whiteflies, and plant bugs), caterpillars (e.g., lepidopteran insects including fall army, black cutworm, corn earworm, soybean looper, and velvetbean caterpillar), beetles (e.g., coleopteran insects including Colorado Potato Beetle and corn rootworms), and flies (e.g., dipteran insects including Ceratitis capitata).
  • Insecticidal or insect inhibitory cargo RNAs provided herein can be directed against insects at various stages of their development (e.g., embryonic, larval, pupal, or adult stages).
  • the insecticidal or insect inhibitory cargo RNA comprises a non-coding RNA sequence having insecticidal or insect inhibitory activity (e.g., dsRNA molecules which produce miRNA or siRNA) and in particular a dsRNA directed against an insect target gene.
  • insecticidal cargo RNAs comprising dsRNA-mediated control of insects comprise or are modeled after those described in US Patent Nos.11,091,770 and 11,186,837, which are each incorporated herein by reference in their entireties.
  • Non-limiting examples of insecticidal or insect- inhibitory cargo RNAi targets are provided in Table 9.
  • Non-limiting examples of insecticidal cargo RNAi targets include insect Actin, SNF7, Tyrosine hydroxylase, C002, Hunchback, V-ATPase subunit A, COPI coatomer beta prime subunit, ribosomal protein L19, and ubiquitin C genes.
  • insecticidal or insect inhibitory RNAs (RNAi sequences) used as cargo are obtained for a chosen target gene of an insect (e.g., an insect gene set forth in Table 9 or US Patent Nos.11,091,770 and 11,186,837) using siRNA/miRNA prediction tools (see, e.g., on the world wide web internet site “zhaolab[dot]org/pssRNAit/).
  • insecticidal cargo RNAs encode insecticidal proteins.
  • Useful insecticidal proteins encoded by insecticidal cargo RNAs include native and modified Bacillus thuringiensis Cry, vegetative insecticidal proteins (VIP), and Cyt proteins (Palma et al.2014, doi: 10.3390/toxins6123296; US Patent No.11,267,849, incorporated herein by reference in its entirety) as well as insecticidal or insect-inhibitory proteins provided in Table 9.
  • Cargo RNAs can also encode “resistance” or “R” genes which confer resistance to certain arthropods, bacteria such as Pseudomonas sp., Xanthomonas sp., and Erwinia sp., and fungal pathogens including Cochliobolus, Blumeria, Fusarium, Melampsora, and Magnaporthe sp.
  • R genes encoded by cargo RNAs include those provided in Table 12.
  • the cargo RNA molecule comprises a CRISPR guide RNA.
  • CRISPR-associated endonucleases such as Cas9, Cas12, and Cas13 endonucleases are used as genome editing tools in different plants; see, e.g., Wolter et al. (2019) BMC Plant Biol., 19:176-183); Aman et al. (2016) Genome Biol., 19:1-10.
  • CRISPR/Cas9 requires a two-component crRNA:tracrRNA “guide RNA” (“gRNA”) that contains a targeting sequence (the “CRISPR RNA” or “crRNA” sequence) and a Cas9 nuclease-recruiting sequence (tracrRNA).
  • gRNA guide RNA
  • sgRNA single guide RNA
  • sgRNA single guide RNA
  • tracrRNA for binding the nuclease
  • crRNA to guide the nuclease to the sequence targeted for editing
  • CRISPR nucleases and guide RNAs provide algorithms for designing guide RNA sequences; see, e.g., guide design tools provided by Integrated DNA Technologies at www[dot]idtdna[dot]com/pages/products/crispr-genome-editing/alt-r- crispr-cas9-system.
  • RNAs are imbedded within a heterologous RNA virus (HRV) amplicon comprising; (i) an HRV 5’ replication region (HRV 5’ RR); (ii) the cargo RNA molecule; and (iii) the heterologous RNA virus (HRV) 3’ RNA replication region (HRV 3’RR), wherein (i), (ii), and (iii) are operably linked.
  • HRV heterologous RNA virus
  • FIG. 1 An illustrative example of a Ghabrivirales satellite construct with such an HRV amplicon is shown in Figure 2.
  • Amplification of such an HRV amplicon in plants comprising a Ghabrivirales satellite construct, the Ghabrivirales, and an HRV RdRP is illustrated in Figure 6.
  • Examples of HRV 5’ replication regions (5’RR), 3’ replication regions (3’RR), and corresponding HRV RNA-dependent RNA Polymerases (RdRP) that recognize such replication regions are set forth in Table 7.
  • an internal ribosome entry site IRES; e.g. an IRES in Table 5
  • IRES internal ribosome entry site
  • one or more self-cleaving or inducible ribozymes are operably linked to the 5’ end of the HRV 5’ RR and to the 3’ end of the HRV 3’ RR.
  • the HRV amplicon further comprises a subgenomic promoter which is operably linked to the cargo RNA molecule.
  • subgenomic promoters include a subgenomic promoter of the HRV and/or a Brome Mosaic Virus subgenomic promoter (Siegal et al.1998, doi: 10.1073/pnas.95.20.11613), barley yellow dwarf virus Agent Ref: P14421WO00 - 23 - (BYDV) sgRNA1, sgRNA2, and sgRNA3 subgenomic promoters (Koev and Miller; J Virol.2000 Jul;74(13):5988-96, doi: 10.1128/jvi.74.13.5988-5996.2000), and an Alternanthera mosaic virus (AltMV-MU) sgp1, sgp2, or sgp3 subgenomic promoter (Putlyaev et al., Biochemistry (Mosc).;80(8):1039-46, DOI: 10.1134/S000629791508009X).
  • HRV amplicons can be in the sense or antisense orientation with respect to the Ghabrivirales 5’ RNA replication element.
  • HRV amplicon When the HRV amplicon is oriented in the sense orientation relative to the Ghabrivirales 5’ RNA replication element, the HRV 5’ RR and 3’ RR are present in the recombinant RNA molecule in the sense orientation, as in the corresponding sequences found in the plus (+) strand of the HRV genomic RNA.
  • the HRV 5’ RR and 3’ RR are present in the recombinant RNA molecule in antisense orientation, as in the corresponding sequences found in the negative (-) strand of the HRV genomic RNA.
  • a plant cell or plant containing the recombinant RNA containing the HRV amplicon are provided with an RNA- dependent RNA polymerase that recognizes the HRV 5’ RR and 3’RR (hrvRdRP)
  • the HRV amplicon undergoes amplification (e.g., hrvRdRP-mediated replication).
  • Such hrvRdRP can be provided by sources that include: (i) infection by the HRV; (ii) introduction by vector-mediated delivery of a polynucleotide encoding the hrvRdRP (e.g., Agrobacterium-mediated delivery or viral vector mediated delivery); or (iii) introduction of a nucleic acid encoding the hrvRdRP.
  • sources that include: (i) infection by the HRV; (ii) introduction by vector-mediated delivery of a polynucleotide encoding the hrvRdRP (e.g., Agrobacterium-mediated delivery or viral vector mediated delivery); or (iii) introduction of a nucleic acid encoding the hrvRdRP.
  • HRV1 RdRP and “HRV2 RdRP”
  • Amplification e.g., an increase in copy number of the HRV amplicon
  • the recombinant nucleotides provided herein comprise Ghabrivirales 5’ and 3’ RNA replication elements flanking a heterologous RNA virus (HRV) subgenomic promoter operably linked to the cargo RNA molecule; wherein the subgenomic promoter is recognized by a heterologous RNA virus RNA-dependent RNA polymerase (hrvRdRP).
  • HRV heterologous RNA virus
  • hrvRdRP heterologous RNA virus RNA-dependent RNA polymerase
  • FIG. 8 Another illustrative example where subgenomic promoters drive expression of an HRV RdDp and a dsRNA cargo in plants comprising a Ghabrivirales satellite construct is shown in Figure 8.
  • an internal ribosome entry site IRES; e.g. an IRES in Table 5
  • IRES internal ribosome entry site
  • Embodiments of subgenomic promoters include a subgenomic promoter of the HRV and/or a Brome Mosaic Virus subgenomic promoter (Siegal et al.1998, doi: 10.1073/pnas.95.20.11613), barley yellow dwarf virus (BYDV) sgRNA1, sgRNA2, and sgRNA3 subgenomic promoters (Koev and Miller; J Virol.2000 Jul;74(13):5988-96.
  • a subgenomic promoter of the HRV and/or a Brome Mosaic Virus subgenomic promoter (Siegal et al.1998, doi: 10.1073/pnas.95.20.11613), barley yellow dwarf virus (BYDV) sgRNA1, sgRNA2, and sgRNA3 subgenomic promoters (Koev and Miller; J Virol.2000 Jul;74(13):5988-96.
  • BYDV barley yellow dwarf
  • RNAs from subgenomic promoters provides for additional copies of the cargo RNA and an enhancement of desirable phenotypes conferred by the cargo RNA (e.g., increased antiviral, antifungal, or insecticidal activity in comparison to control plants lacking the additional expressed cargo RNA or lacking the cargo RNA).
  • the subgenomic promoters and operably linked cargo RNAs are oriented in the sense orientation relative to the Ghabrivirales 5’ RNA replication element, the subgenomic promoters and operably linked cargo RNAs are present in the recombinant RNA molecule as a sense strand where the subgenomic promoter is recognized by the hrvRdRP to produce the desired cargo RNA.
  • the subgenomic promoters and operably linked cargo RNAs are oriented in the sense orientation relative to the Ghabrivirales 5’ RNA replication element in the recombinant RNA molecule (positive strand)
  • the subgenomic promoter can be recognized by the hrvRdRP to produce the desired cargo RNA.
  • the HRV amplicon is oriented in the antisense orientation relative to the Ghabrivirales 5’ RNA replication element in the recombinant RNA molecule, the subgenomic promoter cannot be recognized by the hrvRdRP to produce the desired cargo RNA.
  • the negative strand of the recombinant RNA molecule produced by the Ghabrivirales RdRP would contain the subgenomic promoters and operably linked cargo RNA in a sense orientation where the subgenomic promoter can be recognized by the hrvRdRP to produce the desired cargo RNA.
  • the HRV amplicons further comprise a HRV 5’ RR and 3’ RR which flank the cargo RNA and provide for hrvRdRP-mediated replication of an RNA comprising from 5’ to 3’ the HRV 5’ RR, cargo RNA, and HRV 3’ RR (e.g., as illustrated in the non-limiting example of Figure 5).
  • an RNA encoding the cargo molecule can be produced (e.g., via hrvRdRP-mediated synthesis of the cargo RNA from the subgenomic promoter).
  • Such hrvRdRP can be provided by sources that include: (i) infection by the HRV; (ii) introduction by vector-mediated delivery (e.g., Agrobacterium-mediated delivery or viral vector mediated delivery); (iii) introduction of a nucleic acid encoding the hrvRdRP; or (iv) inclusion of a cargo RNA in the recombinant nucleotides comprising Ghabrivirales 5’ and 3’ RNA replication elements.
  • vector-mediated delivery e.g., Agrobacterium-mediated delivery or viral vector mediated delivery
  • nucleic acid encoding the hrvRdRP e.g., Agrobacterium-mediated delivery or viral vector mediated delivery
  • inclusion of a cargo RNA in the recombinant nucleotides comprising Ghabrivirales 5’ and 3’ RNA replication elements.
  • the subgenomic promoter and operably linked cargo RNA are present in the recombinant RNA molecule as the antisense strand, and the cargo RNA encodes both an hrvRdRP and a second coding or non-coding RNA where both the hrvRdRP and a second coding or non-coding RNA are operably linked to a subgenomic promoter recognized by the hrvRdRP.
  • an IRES is operably linked to the RNA encoding the hrvRdRP.
  • RNA Agent Ref P14421WO00 - 25 - where the subgenomic promoters recognized by the hrvRdRP can drive expression of the HRV RdRp and a second coding or non-coding RNA.
  • An illustrative example of an Ghabrivirales satellite construct with subgenomic promoters in antisense orientation relative to the 5’ RRE and driving expression of both an hrvRdRP that recognizes the subgenomic promoters and a second cargo RNA is shown in Figure 4.
  • an RNA molecule including at least one HRV amplicon is amplified directly by the hrvRdRP (e.g., without initial or further amplification by the commensal viral RdRP).
  • the HRV amplicon includes, in 5’ to 3’ order, (i) a heterologous RNA virus (HRV) 5’ replication region (HRV 5’RR); (ii) a cargo RNA molecule; and (iii) a heterologous RNA virus (HRV) 3’ RNA replication region (HRV 3’RR); wherein the HRV 5’ RR and HRV 3’ RR HRV are recognized by a heterologous RNA virus RNA-dependent RNA polymerase (hrvRdRP); and wherein the HRV 5’RR, cargo RNA molecule, and HRV 3’RR are operably linked.
  • the HRV amplicon includes, in 5’ to 3’ order, a subgenomic promoter that is operably linked to (v) a cargo RNA molecule, wherein the subgenomic promoter is recognized by the hrvRdRP.
  • HRV amplicons can be provided in either an isolated form or in a composition.
  • the RNA molecule including the HRV amplicon can be provided to a plant, for example, by transcription in the plant from a recombinant DNA molecule encoding the RNA molecule that is transiently expressed in the plant or that is stably integrated into the plant’s genome, or by delivery to the plant of an exogenous RNA molecule including the HRV amplicon, for example, by contacting a surface of the plant with the exogenous RNA molecule including the HRV amplicon, or by introducing the exogenous RNA molecule including the HRV amplicon into the plant’s vascular system (e.g., by injection, infusion, petiole uptake, root uptake).
  • a plant for example, by transcription in the plant from a recombinant DNA molecule encoding the RNA molecule that is transiently expressed in the plant or that is stably integrated into the plant’s genome, or by delivery to the plant of an exogenous RNA molecule including the HRV amplicon, for example, by
  • RNA molecules are delivered by injection into the vascular system of plants, such as perennial or woody plants.
  • Examples of devices and methods for delivery via injection into plant vascular systems include those described in US Patent No.11844318; examples of delivery via injection into olive trees are described in PCT published application WO 2023/161802 and Grandi et al. (2023) Front. Plant Sci., 14:2023; doi.org/10.3389/fpls.2023.1180632; and examples of delivery via injection into citrus trees are described in PCT published application WO 2023/240208 and Al-Rimawi et al. (2019) Antibiotics, 8, 196; doi:10.3390/antibiotics8040196.
  • the cargo RNA molecule includes at least one antiviral RNA (e.g., an antiviral inhibitory RNA or an RNA encoding an antiviral polypeptide) that provides the plant with resistance to at least one viral pathogen (which in some instances can be the heterologous RNA virus itself).
  • antiviral RNA e.g., an antiviral inhibitory RNA or an RNA encoding an antiviral polypeptide
  • Such embodiments are useful as antiviral treatments for plants, to prevent or decrease the severity of infection of a plant by a viral pathogen.
  • plants including Brassicaceae, Graminaceae, and other plants that comprise Ghabrivirales satellite RNAs (e.g.
  • RNAs disclosed herein can exhibit control of various HRV.
  • Brassica spp. plants including Brassica campestris, Brassica napus, Brassica rapa, Brassica juncea, Brassica oleracea, and Brassica carinata that contain partitivirus Agent Ref: P14421WO00 - 26 - satellite RNA’s with 5’ RRE and 3’RRE from Brassica campestris chrysovirus (e.g., Table 1) and containing an HRV amplicon with suitable antiviral cargo can exhibit control of HRV that include Arabidopsis thaliana Art1 virus, Arabidopsis thaliana Athila virus, Arabidopsis thaliana AtRE1 virus, Arabidopsis thaliana Endovir virus, Arabidopsis thaliana evelknievel virus, Arabidopsis thaliana Ta1 virus, Arab
  • graminaceous plants including Oryza spp., Hordeum vulgare, Triticum aestivum, and Zea mays that contain partitivirus satellite RNA’s with 5’ RRE and 3’RRE from Zea mays chrysovirus (e.g., Table 1) and containing an HRV amplicon with suitable antiviral cargo can exhibit control of HRV that include Brome mosaic virus, Maize aumaivirus, Maize chlorotic dwarf virus, Maize chlorotic mottle virus, Maize dwarf mosaic virus, Maize necrotic streak virus, Maize rayado fino virus, Maize rough dwarf virus, Maize stripe tenuivirus, Maize white line mosaic virus, Maize yellow dwarf virus, and Maize yellow mosaic virus.
  • RNA molecules that contain HRV amplification sequences (such as the HRV amplicons described herein which contain either (1) a pair of HRV 5’ and 3’ RNA replication regions, or (2) a subgenomic promoter that is recognized by the hrvRdRP) potentially also serve as a “sponge” or “decoy” that reduces the corresponding hrvRdRP’s efficiency in recognizing and amplifying the HRV viral genome itself, thus potentially decreasing a pathogenic HRV’s deleterious effects on an infected plant.
  • HRV amplification sequences such as the HRV amplicons described herein which contain either (1) a pair of HRV 5’ and 3’ RNA replication regions, or (2) a subgenomic promoter that is recognized by the hrvRdRP
  • RNA polynucleotides comprising at least one cleavable sequence are provided.
  • the at least one cleavable sequence is located: (i) 5’ to the 5’ RNA replication element or 3’ to the 3’ RNA replication element; and/or (ii) between the 3’ end of the 5’ RNA replication element and the HRV amplicon and/or between the HRV amplicon and the 5’ end of the 3’ RNA replication element.
  • the cleavable sequence is a self-cleaving ribozyme (e.g., a hammerhead ribozyme; Tang and Breaker. Proc Natl Acad Sci USA.2000 May 23;97(11):5784-9.
  • a cargo RNA molecule that is integrated into a polynucleotide includes at least one CRISPR guide RNA; release of the guide RNA is mediated, e.g., by flanking DR sequences, ribozyme sequences, or other self-cleaving or trans-cleaving RNAs, or by cleavage by an endogenous ribonuclease.
  • the corresponding Cas nuclease can be provided by separate or concurrent delivery, e.g., by co-delivery with a vector or polynucleotide, or by transient or stable expression of the corresponding Cas nuclease in the cell to which the polynucleotide is delivered.
  • guide sequence designs are constrained by the requirement that the DNA target sequence (to which the crRNA is designed to be complementary) must be adjacent to a proto-spacer adjacent motif (“PAM”) sequence that is recognized by the specific Cas nuclease to be employed.
  • PAM proto-spacer adjacent motif
  • Cas nucleases recognize specific PAM sequences and there is a diversity of nucleases and corresponding PAM sequences; see, e.g., Smakov et al. (2017) Nature Agent Ref: P14421WO00 - 27 - Reviews Microbiol., doi:10.1038/nrmicro.2016.184.
  • Cas9 nucleases cleave dsDNA, require a GC-rich PAM sequence located 3’ to the DNA target sequence to be targeted by the crRNA component of the guide RNA, and cleave leaving blunt ends.
  • Cas12a nucleases cleave dsDNA require a T-rich PAM sequence located 5’ to the DNA target sequence to be targeted by the crRNA component of the guide RNA, and cleave leaving staggered ends with a 5’ overhang.
  • Cas13 nucleases cleave single-stranded RNAs and do not require a PAM sequence; instead, Cas13 nuclease are guided to their targets by a single crRNA with a direct repeat (“DR”).
  • DR direct repeat
  • the crRNA component of a guide RNA is generally designed to have a length of between 17 – 24 nucleotides (frequently 19, 20, or 21 nucleotides) and exact complementarity (i.
  • Non-limiting examples of effective guide RNA design are found, e.g., in US Patent Application Publications US 2019/0032131, 2015/0082478, and 2019/0352655, which are each incorporated by reference in their entirety herein.
  • CRISPR “arrays” can be designed to include one or multiple guide RNA sequences corresponding to one or more desired target DNA sequence(s); see, for example, Cong et al. (2013) Science, 339:819-823; Ran et al. (2013) Nature Protocols, 8:2281-2308.
  • the 5’ RNA replication element includes a 5’ UTR element of a Ghabrivirales genome (e.g., either a CP or RdRP GV genome including a GV 5’ UTR set forth in Table 1).
  • the 5’ RNA replication element further includes a genomic sequence of the Ghabrivirales that is natively located 3’ to and optionally adjacent or immediately adjacent to the 5’ UTR sequence.
  • the 3’ RNA replication element includes a 3’ UTR sequence of a Ghabrivirales genome (e.g., either a CP or RdRP GV genome including a GV 3’ UTR set forth in Table 1).
  • the 3’ RNA replication element further includes a genomic sequence of the Ghabrivirales that is natively located 5’ to and optionally adjacent or immediately adjacent to the 3’ UTR sequence.
  • the RNA molecule further includes at least one RNA molecule encoding a viral MP.
  • the at least one RNA molecule encoding an MP is located (a) before the cargo RNA molecule, (b) after the cargo RNA molecule, or (c) both before and after the cargo RNA molecule.
  • the at least one RNA sequence encoding an MP includes at least two RNA sequences encoding different MPs or a single RNA sequence encoding multiple copies of MPs.
  • the recombinant DNA molecule further includes a discrete expression cassette including a second promoter that is functional in the cell and is operably linked to a DNA sequence encoding at least one viral movement protein, and optionally a terminator element.
  • the RNA molecule further includes an encapsidation recognition element (ERE), where the ERE is located close to or adjacent to the 3’ RNA replication element, and Agent Ref: P14421WO00 - 28 - optionally wherein the 3’ RNA replication element includes a 3’ UTR sequence of the Ghabrivirales.
  • the ERE includes a viral OAS such as a tobacco mosaic virus OAS (TMV-OAS) or an OAS set forth in Table 2.
  • the RNA molecule further includes at least one tRNA-like sequence (TLS), and wherein the at least one tRNA-like sequence includes a tRNA-like sequence from an Arabidopsis FT mRNA (e.g. a TLS in an Arabidopsis FT mRNA of Table 4).
  • the RNA molecule includes a tRNA-like sequence encoded by a DNA sequence selected from the group consisting of SEQ ID NOs: 76-123, and 466.
  • the RNA molecule includes a modified tRNA-like sequence that has at least 90% sequence identity to a scaffold tRNA-like sequence encoded by a DNA sequence selected from the group consisting of SEQ ID NOs: 76-123, and 466and that maintains the secondary structure of the scaffold tRNA-like sequence.
  • the RNA molecule further includes at least one RNA encoding a viral MP, a tRNA-like sequence from an Arabidopsis FT mRNA, and an encapsidation recognition sequence including TMV-OAS.
  • the cargo RNA molecule is up to about 4.6 kilobases (kb) in length (Totiviridae) or up to about 12.5 kilobases (kb) in length (Chrysoviridae).
  • the cargo RNA molecule includes: (a) at least one coding sequence, (b) at least one non-coding sequence, or (c) both at least one coding sequence and at least one non-coding sequence.
  • the cargo RNA molecule includes at least one coding sequence, and wherein the RNA molecule further includes an internal ribosome entry site (IRES) located 5’ and immediately adjacent to the at least one coding sequence.
  • IRS internal ribosome entry site
  • the cargo RNA molecule includes multiple coding sequences, and wherein the RNA molecule further includes an IRES located 5’ and immediately adjacent to each of the coding sequences.
  • the cargo RNA molecule includes at least one non-coding sequence, and wherein the at least one non-coding sequence is selected from the group consisting of a hairpin RNA (hpRNA); an RNA that forms multiple stem-loops; an RNA pseudoknot; an RNA sequence that forms at least partially double-stranded RNA; a small interfering RNA (siRNA) or siRNA precursor; a microRNA (miRNA) or miRNA precursor; a ribozyme; a ligand-responsive ribozyme (aptazyme); an RNA aptamer; and a long noncoding RNA (lncRNA).
  • hpRNA hairpin RNA
  • siRNA small interfering RNA
  • miRNA microRNA
  • aptazyme a ligand-responsive ribozyme
  • a DNA sequence encoding at least one ribozyme is provided. In embodiments, the at least one ribozyme is located 5’ to the 5’ RNA replication element or 3’ to the 3’ RNA replication element. In embodiments, a DNA sequence encoding at least one ligand-responsive ribozyme (aptazyme) is provided. In embodiments, the at least one ligand-responsive ribozyme is located 5’ to the 5’ RNA replication element or 3’ to the 3’ RNA replication element.
  • RNA molecules comprising the aforementioned or otherwise disclosed 5’ RNA replication elements, a cargo RNA molecule(s), and 3’ RNA replication elements, as well as additional aforementioned or otherwise disclosed elements are also provided herein.
  • the recombinant RNA molecules are produced by a recombinant DNA molecule provided herein.
  • the recombinant RNA molecules are produced by an in vivo or in vitro (e.g., cell free) RNA replication process through the action of a RdRP acting on: (i) 5’ and 3’ RNA replication elements; and/or (ii) a subgenomic promoter.
  • Expression systems comprising the recombinant polynucleotides are also provided. Such expression systems include both cell-based and cell free expression systems.
  • cell-free expression system can include (a) an RNA molecule comprising, in 5’ to 3’ order: (i) a 5’ RNA replication element; (ii) a cargo RNA molecule; and (iii) a 3’ RNA replication element; and, optionally, further comprising at least one additional RNA or other element.
  • Embodiments of additional elements include at least one RNA encoding a viral MP, at least one tRNA-like sequence, an OAS, an RdRP protein that recognizes the 5’ and 3’ RNA replication elements, a subgenomic promoter, and/or an RdRP that recognizes an HRV 5’ or 3’ replication region and/or a subgenomic promoter.
  • the RdRP protein is provided by a Ghabrivirales, e.g. a Ghabrivirales that is endogenous to a cell in which expression is desired.
  • cell-based expression system can include (a) a recombinant DNA molecule including a heterologous promoter that is functional in a cell and is operably linked to a DNA sequence encoding an RNA molecule comprising, in 5’ to 3’ order: (i) a 5’ RNA replication element; (ii) a cargo RNA molecule; and (iii) a 3’ RNA replication element; and, optionally, further comprising at least one additional RNA or other element.
  • Embodiments of additional elements include at least one RNA encoding a viral MP, at least one tRNA-like sequence, an OAS, an RdRP protein that recognizes the 5’ and 3’ RNA replication elements, a subgenomic promoter, and/or an RdRP that recognizes the subgenomic promoter.
  • cell-based expression system can include (a) a recombinant RNA molecule comprising, in 5’ to 3’ order: (i) a 5’ RNA replication element; (ii) a cargo RNA molecule; and (iii) a 3’ RNA replication element; and, optionally, further comprising at least one additional RNA or other element.
  • Embodiments of additional RNA elements include at least one RNA encoding a viral MP, at least one tRNA-like sequence, an OAS, an RdRP protein that recognizes the 5’ and 3’ RNA replication elements, a subgenomic promoter, and/or an RdRP that recognizes an HRV 5’ or 3’ replication region and/or a subgenomic promoter.
  • the RdRP protein is provided by a Ghabrivirales.
  • an RdRP that recognizes an HRV 5’ or 3’ replication region and/or a subgenomic promoter is provided by a heterologous RNA virus (HRV) or by another nucleic acid introduced into the cell (e.g., by a vector or other recombinant nucleic acid).
  • HRV heterologous RNA virus
  • the 5’ RNA replication element and the 3’ RNA replication element are obtained from the same Ghabrivirales and/or from the same Ghabrivirales genome (e.g., both obtained from the same GV genome) or from Ghabrivirales genomes having at least 85%, 90%, 95%, 98%, or 99% sequence identity to one another and which are optionally related.
  • a cell used in the expression system is a bacterial cell, a plant cell, a fungal cell, a vertebrate animal cell (e.g., a mammalian or non-human mammalian cell) or an invertebrate animal cell (e.g., an insect cell).
  • a cell used in the expression system endogenously contains a Ghabrivirales having a genome that encodes an RdRP that recognizes the 5’ and 3’ RNA replication elements.
  • the expression system further includes a viral capsid protein that is recognized by the encapsidation recognition element and encapsidates the RNA molecule.
  • the viral capsid protein is: (a) expressed by the recombinant DNA molecule in the cell (e.g., where the recombinant DNA molecule further includes a discrete expression cassette comprising a second promoter operably linked to a DNA sequence encoding the viral capsid protein, and optionally a terminator element), (b) co-expressed by a second recombinant DNA molecule in the cell; (c) provided exogenously to the cell; or (d) expressed by a virus in the cell.
  • the RdRP protein is heterologous to the cell. In embodiments, the RdRP protein is provided exogenously to the cell.
  • the RdRP protein that recognizes the 5’ and 3’ RNA replication elements is endogenously expressed in the plant cell by the Ghabrivirales virus (e.g., where the Ghabrivirales virus occurs naturally in the plant cell).
  • the Ghabrivirales is native to or endemic to the plant cell.
  • the Ghabrivirales that is endemic to the plant cell is non-pathogenic.
  • the Ghabrivirales that is endemic to the plant cell is non-pathogenic and commensal.
  • the Ghabrivirales is an exogenously introduced Ghabrivirales (i.e., not endemic or native to the host, but artificially introduced).
  • a Ghabrivirales natively found in one plant species, variety, or germplasm can be introduced, with or without a corresponding recombinant Ghabrivirales satellite RNA, into a different plant species, variety, or germplasm.
  • a complete self-replicating Ghabrivirales satellite system is introduced into a plant or plant cells, wherein the self-replicating Ghabrivirales satellite system includes: (1) a recombinant Ghabrivirales satellite RNA comprising, from 5’ terminus to 3’ terminus: (a) a 5’ RNA replication element recognized by a Ghabrivirales RNA-dependent RNA polymerase (RdRP); (b) a cargo RNA molecule; and (c) a 3’ RNA replication element recognized by the RdRP; wherein the 5’ RNA replication element, the cargo RNA molecule, and the 3’ RNA replication element are operably linked, and wherein the promoter and the cargo RNA molecule are heterologous to the 5’ RNA replication
  • the Ghabrivirales RdRP comprises a protein having at least 85%, 90%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 491, 492, 493, 494, 495, or 496.
  • the recombinant DNA molecule or recombinant RNA molecule further comprises at least one RNA encoding a viral MP, a tRNA-like sequence from an Arabidopsis FT mRNA, and an encapsidation recognition sequence including a TMV- OAS.
  • Cells comprising any of the aforementioned or otherwise disclosed recombinant polynucleotides are provided herein.
  • Cells comprising the recombinant polynucleotides include prokaryotic (e.g., a bacterium, such as a bacterium capable of transforming a eukaryotic cell) or eukaryotic (e.g., a plant cell, fungal cell, or animal cell such as an insect cell) cells.
  • the cells are bacterial cells Agent Ref: P14421WO00 - 31 - capable of transforming a plant cell (e.g., an Agrobacterium sp., a Sinorhizobium sp., a Mesorhizobium sp., Bradyrhizobium sp., Rhizobium sp., or an Ensifer sp. cell.
  • a plant cell e.g., an Agrobacterium sp., a Sinorhizobium sp., a Mesorhizobium sp., Bradyrhizobium sp., Rhizobium sp., or an Ensifer sp. cell.
  • Bacterial cells capable of transforming a plant cell suitable for use with the recombinant polynucleotides provided herein include Agrobacterium sp., a Sinorhizobium sp., a Mesorhizobium sp., Bradyrhizobium sp., Rhizobium sp., or an Ensifer sp. cell are disclosed in US patent application publications US20170369898 and US20180312854, each incorporated herein by reference in their entireties.
  • Vectors suitable for maintenance, propagation, and/or expression of the recombinant polynucleotides in the aforementioned prokaryotic or eukaryotic cells are also provided herein.
  • Such vectors can comprise any of the aforementioned or otherwise disclosed recombinant polynucleotides, recombinant DNA molecules, and recombinant RNA molecules as well as those polynucleotides molecules described in the Examples.
  • the bacterium that mediates the plant transformation is an Agrobacterium sp., a Sinorhizobium sp., a Mesorhizobium sp., Bradyrhizobium sp., Rhizobium sp., or an Ensifer sp. In embodiments where an Agrobacterium sp.
  • the vector includes T-DNAs flanking the recombinant DNA molecule encoding the recombinant RNA molecule (e.g., as described in US patent application publications US20170369898 and US20180312854, each incorporated herein by reference in their entireties).
  • the vector is contained within a plant cell or within a bacterial cell (Agrobacterium sp., a Sinorhizobium sp., a Mesorhizobium sp., Bradyrhizobium sp., Rhizobium sp., or an Ensifer sp. cell).
  • Agrobacterium sp. a Sinorhizobium sp., a Mesorhizobium sp., Bradyrhizobium sp., Rhizobium sp., or an Ensifer sp. cell.
  • Viral particles comprising any of the aforementioned or otherwise disclosed recombinant RNA molecules are also provided.
  • the recombinant RNA is introduced into a host or production plant by using Agrobacterium-mediated transformation with a polynucleotide comprising (5’ to 3’): (i) a promoter which is operably linked to a viral MP coding sequence and a TLS element, flanked by Ghabrivirales 5’ and 3’ RNA replication elements; (ii) a promoter which is operably linked to a cargo RNA molecule and a TLS element, flanked by Ghabrivirales 5’ and 3’ RNA replication elements; (iii) a promoter operably linked to an RdRP coding sequence; and (iv) a promoter operably linked to a CP encoding sequence.
  • a polynucleotide comprising (5’ to 3’): (i) a promoter which is operably linked to a viral MP coding sequence and a TLS element, flanked by Ghabrivirales 5’ and 3’ RNA replication elements; (ii) a promote
  • Heterologous promoters independently drive expression of the capsid protein and the cargo as depicted.
  • the RNA expressed from the polynucleotide includes an OAS.
  • a host plant is transformed for production of the synthetic Ghabrivirales satellite RNA and satellite particles comprising the encapsidated RNA.
  • the expressed and encapsidated Ghabrivirales satellite RNA is subsequently isolated from leaf material or other tissue of the host plant, purified (and, if desired, formulated) for high pressure spraying onto plants that endogenously contain the corresponding Ghabrivirales or a recombinant source of the GV RdRP for subsequent expression and replication of the Ghabrivirales satellite RNA and satellite particles comprising the same in encapsidated form.
  • spraying with the encapsidated satellite particles with certain cargo RNA molecules can be used to modify the plant as desired.
  • the presence of a movement protein and/or tRNA-like sequences facilitates systemic movement throughout the plant receiving the high-pressure spray satellite particles comprising the desired recombinant RNA molecules comprising the encoded MP and cargo RNA.
  • plants without a systemic Ghabrivirales which provides the GV RdRP can further comprise a recombinant DNA or RNA molecule which encodes and provides the RdRP.
  • Target plants and plant cells used as hosts for synthetic Ghabrivirales satellite RNAs include both monocot and dicot plants and plant cells which can support Ghabrivirales replication.
  • the Ghabrivirales is endogenous to (endemic to or natively found in) the plant or plant cell.
  • the Ghabrivirales is introduced to and becomes established in the plant or plant cell.
  • Embodiments include row crop plants, fruit-producing plants and trees, vegetables, trees, and ornamental plants including ornamental flowers, shrubs, trees, groundcovers, and turf grasses.
  • the host plants and plant cells for synthetic Ghabrivirales satellite RNAs include row crop plants, fruit-producing plants and trees, vegetables, trees, and ornamental plants including ornamental flowers, shrubs, trees, groundcovers, and turf grasses.
  • the host plants and plant cells for synthetic Ghabrivirales satellite RNAs include commercially important cultivated crops, trees, and plants, including: alfalfa (Medicago sativa), almonds (Prunus dulcis), apples (Malus x domestica), apricots (Prunus armeniaca, P. brigantine, P. mandshurica, P. mume, P.
  • sibirica asparagus (Asparagus officinalis), bananas (Musa spp.), barley (Hordeum vulgare), beans (Phaseolus spp.), blueberries and cranberries (Vaccinium spp.), cacao (Theobroma cacao), canola and rapeseed or oilseed rape, (Brassica napus), Polish canola (Brassica rapa), and related cruciferous vegetables including broccoli, kale, cabbage, and turnips (Brassica carinata, B. juncea, B. oleracea, B. napus, B. nigra, and B.
  • Coffea arabica, Coffea canephora, and Coffea liberica cotton (Gossypium hirsutum L.), cowpea (Vigna unguiculata and other Vigna spp.), fava beans (Vicia faba), cucumber (Cucumis sativus), currants and gooseberries (Ribes spp.), date (Phoenix dactylifera), duckweeds (family Lemnoideae), eggplant or aubergine (Solanum melongena), eucalyptus (Eucalyptus spp.), flax (Linum usitatissumum L.), geraniums (Pelargonium spp.), grapefruit (Citrus x paradisi), grapes (Vitus spp.) including wine grapes (Vitus vinifera and hybrids thereof), guava (Psidium guajava), hops (Humulus lupulus), hemp and cannabis
  • the host plant or plant cells for synthetic Ghabrivirales satellite RNAs is a dicot plant or plant cell selected from the genera Brassica, Capsicum, Cucumis, Cucurbita, Gossypium, Nicotiana, Solanum, or Glycine.
  • the host plant or plant cells for synthetic Ghabrivirales satellite RNAs is a monocot plant or plant cell selected from the genera Avena, Hordeum, Oryza, Secale, Triticum, Sorghum, or Zea.
  • monocot target plants and plant cells used as hosts for synthetic Ghabrivirales satellite RNAs include oats (Avena sativa), barley (Hordeum vulgare), rice (Oryza sativa, Oryza glaberrima, Oryza rufipogen), rye (Secale cereale), wheat (Triticum aestivum), sorghum (Sorghum bicolor), and maize (Zea mays) plants and plant cells.
  • the recombinant RNA molecule or a formulation thereof is provided by contacting the plant or plant cell with the recombinant RNA molecule or formulation thereof.
  • the recombinant RNA molecule is provided by expressing in the plant or plant cell a DNA molecule that encodes the recombinant RNA molecule or a formulation.
  • the recombinant RNA molecule is provided by contacting the plant or plant cell with cells (such as bacterial cells) which comprise a DNA molecule that encodes the recombinant RNA molecule and are capable of transforming the plant or plant cell.
  • the recombinant RNA molecule is provided by contacting the plant or plant cell with a satellite particle comprising an encapsidated recombinant RNA molecule or a formulation thereof.
  • the 5’ RNA replication element has a nucleotide sequence obtained or derived from a Ghabrivirales genomic sequence (e.g., a Chrysovirus or Totivirus genome); and/or the 3’ RNA replication element has a nucleotide sequence obtained or derived from a Ghabrivirales genomic sequence (e.g., a Chrysovirus or Totivirus genome).
  • the 5’ and/or 3’ RNA replication element can be obtained from the corresponding Ghabrivirales genomic sequence by synthesizing or cloning a copy of the corresponding Ghabrivirales genomic sequence. In certain embodiments, the 5’ and/or 3’ RNA replication element can be derived from the corresponding Ghabrivirales genomic sequence by synthesizing a copy of a modified Agent Ref: P14421WO00 - 34 - Ghabrivirales genomic sequence or sequences.
  • Such modifications of Ghabrivirales genomic sequences present in a derived sequence include: (i) substitutions of nucleotides which maintain the RNA secondary structure; (ii) substitution of nucleotides based on a consensus obtained by alignment of 5’ or 3’ RNA replication elements; (iii) insertions, deletions, and/or substitution of nucleotides to facilitate assembly and/or operable linkage to other elements in the satellite RNA which include cargo RNA molecules, tRNA-like elements, encapsidation recognition element (ERE), RNA encoding a viral movement protein (MP), IRES elements, an HRV 5’RR, HRV 3’RR, and/or HRV subgenomic promoter; or (iv) any combination of (i), (ii), or (iii).
  • the plant cell includes the Ghabrivirales virus, and the RdRP protein is provided to the plant cell by the Ghabrivirales virus.
  • the Ghabrivirales is endemic to the plant cell.
  • the Ghabrivirales that is endemic to the plant cell is non-pathogenic and/or commensal to the plant cell.
  • the Ghabrivirales is exogenously provided to the plant cell.
  • the RdRP protein is exogenously provided to the plant cell.
  • the recombinant RNA molecule is produced in a fermentation system.
  • the recombinant RNA molecule is provided to the plant cell by transcribing in the plant cell a recombinant DNA construct including a promoter functional in the plant cell and operably linked to a DNA sequence encoding the recombinant RNA molecule.
  • the recombinant RNA molecule further includes an encapsidation recognition element (ERE), and the plant cell further includes a viral capsid protein (CP) capable of encapsidating the synthetic Ghabrivirales satellite RNA.
  • the viral capsid protein is exogenously provided to the plant cell.
  • the recombinant DNA construct further includes a DNA sequence encoding a viral capsid protein.
  • the recombinant DNA construct further includes a second promoter functional in the plant cell and operably linked to the DNA sequence encoding the viral capsid protein.
  • the viral capsid protein is expressed in the plant cell and encapsidates the synthetic Ghabrivirales satellite RNA.
  • the plant cell includes the Ghabrivirales, and the Ghabrivirales provides to the plant cell: (a) the RdRP protein, (b) the viral capsid protein, or (c) both the RdRP protein and the viral capsid protein.
  • the methods can further comprise a first step of providing a population of plants comprising the plant cells comprising: (i) the Ghabrivirales which provides the RdRP; or (ii) recombinant polynucleotide molecule that encodes the RdRP; and then providing the recombinant RNA molecule to the plants comprising the plant cells.
  • the methods can further comprise the step of determining if the plant cell comprises a Ghabrivirales which can provide the RdRP.
  • the plant cell comprises the Ghabrivirales which can provide the RdRP, the Ghabrivirales, the RdRP protein, and/or the recombinant polynucleotide encoding the RdRP is optionally not exogenously provided to the plant cell.
  • the RdRP protein or the recombinant polynucleotide encoding the RdRP is exogenously provided to the plant cell, or a combination of the Ghabrivirales, RdRP protein, or polynucleotide encoding the RdRP is Agent Ref: P14421WO00 - 35 - exogenously provided to the plant cell.
  • a complete self-replicating Ghabrivirales satellite system is introduced into a plant or plant cells, wherein the self-replicating Ghabrivirales satellite system includes: (1) a recombinant Ghabrivirales satellite RNA comprising, from 5’ terminus to 3’ terminus: (a) a 5’ RNA replication element recognized by a Ghabrivirales RNA-dependent RNA polymerase (RdRP); (b) a cargo RNA molecule; and (c) a 3’ RNA replication element recognized by the RdRP; wherein the 5’ RNA replication element, the cargo RNA molecule, and the 3’ RNA replication element are operably linked, and wherein the promoter and the cargo RNA molecule are heterologous to the 5’ RNA replication element and the 3’ RNA replication element; and (2) an exogenous Ghabrivirales (e.g., a Ghabrivirales that is not endemic or native to the plant or plant cells) that is capable of replication in the plant or plant cells and
  • the Ghabrivirales RdRP comprises a protein having at least 85%, 90%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 491, 492, 493, 494, 495, or 496.
  • the presence or absence of a Ghabrivirales in a target plant can be determined by an RNA detection assay (e.g., an RT-PCR assay) using nucleic acid probes and/or primers which can detect any part of a Ghabrivirales genome including a 5’ RNA replication element, a CP and/or RdRP coding region, and/or a 3’ RNA replication element.
  • Such probes and primers include those which detect any of the 5’ or 3’ RNA replication elements set forth in Table 1 or having significant sequence identity thereto (e.g., at least about 80%, 85%, 90%, 95%, 98%, or 99% of a length of at least about 18, 20, 30, 40 or 50 nt).
  • the presence or absence of a Ghabrivirales in a target plant can be determined by a protein detection assay (e.g., an immunoassay) directed to a GV CP or RdRP (e.g., a CP or RdRP encoded by or homologous to a CP or RdRP encoded by a GV genome disclosed in Table 1).
  • Target plants and plant cells used in the methods include all aforementioned target plants and plant cell hosts for synthetic Ghabrivirales satellite RNAs (e.g., recombinant RNAs).
  • the recombinant RNA that effects: (i) a phenotypic change in the plant or plant cell; (ii) increases a plant’s resistance to a pest or pathogen; or (iii) increases a plant’s resistance to stress can include an RNA for modulating a target gene’s expression relative to the target gene’s expression in a control plant or plant cell not provided with the recombinant RNA molecule, and the phenotypic change, increased resistance to the pest or pathogen, or increased resistance to stress is a result of the modulation.
  • the modulation is (a) an increase of the target gene’s expression; or (b) a decrease of the target gene’s expression.
  • expression of the target gene is increased by up to about 1%, 2%, 3%, %, 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%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%
  • expression of the target gene is increased by up to about 2-, 3-, 4-, 5-, 6-, 8-, 9-, 10-fold, or more relative to a reference level (e.g., a level found in a control plant or plant cell lacking the recombinant RNA molecule).
  • a reference level e.g., a level found in a control plant or plant cell lacking the recombinant RNA molecule.
  • expression of the target gene is decreased by at least about 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%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%
  • RNAs for modifying the genome include gRNAs recognized by CAS nucleases, RNAs encoding TALENs or artificial zinc finger proteins (aZFN).
  • RNAs for modifying the epigenome include RNAs which provide RNA directed DNA methylation such as in promoter regions of target genes (Matzke and Mosher (2014); doi: 10.1038/nrg3683).
  • Embodiments of an RNA for modifying the transcriptome include one or more RNAs that comprise any of: a hairpin RNA (hpRNA); an RNA that forms multiple stem-loops; an RNA pseudoknot; an RNA molecule that forms at least partially double-stranded RNA; a small interfering RNA (siRNA) or siRNA precursor; a microRNA (miRNA) or miRNA precursor; a phased siRNA or phased siRNA precursor (see, e.g., US Patent No.8,404,928); a ribozyme; a ligand-responsive ribozyme (aptazyme); an RNA aptamer; or a long noncoding RNA (lncRNA).
  • hpRNA hairpin RNA
  • the cargo RNA molecule can comprise an RNA that effects a phenotypic change in the plant or plant cell in comparison to a plant or plant cell lacking the recombinant RNA.
  • phenotypes that are changed include developmental rate, growth rate, size, yield (e.g., intrinsic yield), vigor, photosynthetic capability, flavor, starch production, protein content, carbohydrate content, oil content, fatty acid content, lipid content, digestibility, biomass, shoot length, root length, root architecture, seed set, seed weight, seed quality (e.g., nutritional content), germination, fruit set, rate of fruit ripening, production of biopolymers, production of fibers, production of biofuels, production of pharmaceutical peptides (e.g., hormones, enzymes, transcription factors, antigens, antibodies, or antibody fragments), production of secretable peptides, enzyme production, improved processing traits, or amount of harvestable produce.
  • developmental rate e.g., growth rate, size, yield (e.g., intrinsic yield), vigor, photosynthetic capability, flavor, starch production, protein content, carbohydrate content, oil content, fatty acid content, lipid content, digestibility, biomass, shoot length, root length
  • phenotypes that are changed include taste, appearance, or shelf-life of a product harvested from the plant.
  • phenotypes that are changed include flower size, flower color, flower patterning, flower Agent Ref: P14421WO00 - 37 - morphology including presence or absence of stamens, flower number, flower longevity, flower fragrance, leaf size, leaf color, leaf patterning, leaf morphology, plant height, or plant architecture.
  • the recombinant RNA can comprise an RNA that inhibits expression of a gene of the pest or pathogen and/or inhibits replication of the genome of the pest or pathogen.
  • the pest or pathogen is selected from the group comprising: a bacterium, a virus other than a Ghabrivirales, a fungus, an oomycete, and an invertebrate (e.g., an arthropod or a nematode).
  • Target viruses other than a Ghabrivirales include; (i) positive-strand RNA viruses in the Bromoviridae, Closteroviridae, Luteoviridae, or Potyviridae family; (ii) negative strand RNA viruses in the Bunyaviridae and Rhabdoviridae family; (iii) dsDNA viruses in the family Caulimoviridae; and (iv) ssDNA viruses in the family Geminiviridae.
  • Target arthropods pests include coleopteran and lepidopteran insects.
  • Target fungal pathogens include Magnaporthe spp., Botrytis spp., Puccinia spp.; Fusarium spp., Blumeria spp., Mycosphaerella spp., Colletotrichum spp., Ustilago spp., Melampsora spp., Phakopsora spp., Phytophthora spp., and Rhizoctonia spp.
  • the cargo RNA molecule effects an increase in the plant’s resistance to a pest or pathogen, relative to that in a plant not provided with the recombinant RNA molecule.
  • the recombinant RNA can comprise an RNA that targets a plant gene which provides such resistance.
  • the RNA that effects an increase in the plant’s resistance to stress in the plant or plant cell includes an RNA for modulating the target gene’s expression relative to the target gene’s expression in a control plant or plant cell not provided with the recombinant RNA molecule, and wherein the increase stress resistance is a result of the modulation.
  • the modulation is (a) an increase of the target gene’s expression; or (b) a decrease of the target gene’s expression.
  • the RNA that effects an increase in the plant’s resistance to stress in the plant or plant cell comprises a messenger RNA encoding a protein which confers the stress resistance.
  • the messenger RNA includes an RNA sequence absent in the transcriptome of the plant or plant cell lacking the recombinant RNA.
  • the stress includes at least one abiotic stress selected from the group including: nutrient stress, light stress, drought stress, heat stress, and cold stress.
  • the stress includes at least one biotic stress selected from the group including: crowding, shading, and allelopathy (e.g., resulting from allelopathic chemicals including a juglone produced by walnut trees).
  • the cargo RNA can encode the exogenous polypeptide.
  • the polypeptide is isolated (e.g., separated from at least one other cellular component such as a carbohydrate, a lipid, or another protein) or polypeptide is purified.
  • manufacture can occur in either a cell-based system or a cell-free system.
  • Cell-based methods of manufacturing a synthetic Ghabrivirales satellite particle can comprise: (a) providing to a plant cell the recombinant RNA molecule, wherein the recombinant RNA molecule comprises an encapsidation recognition element (ERE), and wherein the plant cell comprises an RdRP protein that recognizes the 5’ RNA replication element and 3’ RNA replication element catalyzes synthesis of a synthetic Ghabrivirales satellite RNA from the recombinant RNA molecule and a viral capsid protein, wherein the ERE provides for encapsidation of the RNA by the viral capsid protein; and optionally isolating the synthetic Ghabrivirales satellite particle from the plant cell, a plant comprising the plant cell, or from media in which the plant cell or the plant had been grown.
  • ERP encapsidation recognition element
  • Cell-free methods of manufacturing a synthetic Ghabrivirales satellite particle include methods where the recombinant RNA molecule is combined with a viral capsid protein in a vessel, wherein the recombinant RNA molecule comprises an ERE, and wherein the ERE provides for encapsidation of the RNA by the viral capsid protein in the vessel; optionally wherein the method further comprises isolating the synthetic Ghabrivirales satellite particle from uncombined RNA and/or viral capsid protein in the vessel.
  • the synthetic satellite particle is isolated (e.g., separated from at least one other cellular component such as an organelle, a membrane, a carbohydrate, a lipid, or another protein) or is purified.
  • the methods can further comprise formulating the synthetic Ghabrivirales satellite particle.
  • Synthetic Ghabrivirales satellite particles comprising the recombinant RNA, including those made by the aforementioned methods are also provided.
  • Methods of providing any of the aforementioned synthetic Ghabrivirales satellite particles to a plant including contacting (e.g., spraying, dusting, injecting, soaking, etc.) the plant with the synthetic Ghabrivirales satellite particle or a formulation thereof are also provided.
  • the recombinant polynucleotides, cells comprising the same, and synthetic Ghabrivirales satellite particles described herein can be formulated either in pure form (e.g., the composition contains only the recombinant polynucleotide) or together with one or more additional formulation components to facilitate application or delivery of the compositions.
  • the additional formulation component includes, e.g., a carrier (i.e., a component that has an active role in delivering the active agent (e.g., recombinant polynucleotide); for example, a carrier can encapsulate, covalently or non-covalently modify, or otherwise associate with the active agent in a manner that improves delivery of the active agent) or an excipient (e.g., a delivery vehicle, adjuvant, diluent, surfactant, stabilizer, or tonicity agent).
  • the composition is formulated for delivery to a plant. [0099]
  • the disclosure provides a formulation comprising any of the compositions described herein.
  • the formulation is a liquid, a gel, or a powder.
  • the formulation is configured to be sprayed on plants, to be injected into plants(see, e.g., US Patent No.11,844,318), or otherwise introduced into the vascular system of a plant, to be rubbed on Agent Ref: P14421WO00 - 39 - leaves, to be soaked into plants, to be coated onto plants, or be coated on seeds, or to be delivered through root uptake (e.g., in a hydroponic system or by a root drench ).
  • the composition can be formulated into emulsifiable concentrates, suspension concentrates, directly sprayable or dilutable solutions, coatable pastes, diluted emulsions, spray powders, soluble powders, dispersible powders, wettable powders, dusts, granules, encapsulations in polymeric substances, microcapsules, foams, aerosols, carbon dioxide gas preparations, or tablets.
  • the composition is a liquid.
  • the composition is a solid.
  • the composition is an aerosol, such as in a pressurized aerosol can.
  • the recombinant polynucleotide makes up about 0.1% to about 100% of the composition, such as any one of about 0.01% to about 100%, about 1% to about 99.9%, about 0.1% to about 10%, about 1% to about 25%, about 10% to about 50%, about 50% to about 99%, or about 0.1% to about 90% of active ingredients (e.g., recombinant polynucleotides).
  • the composition includes at least any of 0.1%, 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more active ingredients (e.g., recombinant polynucleotides).
  • the concentrated agents are preferred as commercial products, the final user normally uses diluted agents, which have a substantially lower concentration of active ingredient.
  • the composition is formulated for topical delivery to a plant.
  • the topical delivery is spraying, leaf rubbing (e.g., with or without an abrasive), soaking, coating (e.g., coating using micro-particulates or nano-particulates), or delivery through root uptake (e.g., delivery in a hydroponic system).
  • the composition further comprises a carrier and/or an excipient.
  • the composition does not comprise a carrier or excipient, e.g., comprises a naked polynucleotide (e.g., a naked RNA).
  • the recombinant polynucleotide is delivered at a concentration of at least 0.1 grams per acre, e.g., at least 0.1, 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 grams per acre. In some embodiments, less than 120 liters per acre is delivered, e.g., less than 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 10, 5, or 2 liters per acre or less than 1 liter per acre.
  • the formulation comprises a carrier.
  • the formulation is an emulsion or a reverse emulsion, a liquid, or a gel.
  • the formulation includes a carrier that serves as a physical support (e.g., solid or semi-solid surfaces or matrices, powders, or particles or nanoparticles).
  • the active agent is encapsulated or enclosed in or attached to or complexed with a carrier including a liposome, vesicle, micelle, or other fluid compartment.
  • the active agent is encapsulated or enclosed in or attached to or complexed with a carrier including a naturally occurring or synthetic, branched or linear polymer (e.g., pectin, agarose, chitin, chitosan, DEAE-dextran, polyvinylpyrrolidone (“PVP”), or polyethylenimine (“PEI”).
  • a carrier including a naturally occurring or synthetic, branched or linear polymer (e.g., pectin, agarose, chitin, chitosan, DEAE-dextran, polyvinylpyrrolidone (“PVP”), or polyethylenimine (“PEI”).
  • PVP polyvinylpyrrolidone
  • PEI polyethylenimine
  • the carrier includes cations or a cationic charge, such as cationic liposomes or cationic polymers such as Agent Ref: P14421WO00 - 40 - polyamines (e.g., sper
  • the carrier includes a polypeptide such as an enzyme, (e.g., cellulase, pectolyase, maceroenzyme, pectinase), a cell penetrating or pore- forming peptide (e.g., poly-lysine, poly-arginine, or polyhomoarginine peptides).
  • an enzyme e.g., cellulase, pectolyase, maceroenzyme, pectinase
  • a cell penetrating or pore- forming peptide e.g., poly-lysine, poly-arginine, or polyhomoarginine peptides.
  • Non-limiting examples of carriers include cationic liposomes and polymer nanoparticles such as those reviewed by Zhang et al. (2007) J. Controlled Release, 123:1 - 10, and the cross-linked multilamellar liposomes described in US Patent Application Publication 2014/035
  • the carrier includes a nanomaterial, such as carbon or silica nanoparticles, carbon nanotubes, carbon nanofibers, or carbon quantum dots.
  • a nanomaterial such as carbon or silica nanoparticles, carbon nanotubes, carbon nanofibers, or carbon quantum dots.
  • Non-limiting examples of carriers include particles or nanoparticles (e.g., particles or nanoparticles made of materials such as carbon, silicon, silicon carbide, gold, tungsten, polymers, or ceramics) in various size ranges and shapes, magnetic particles or nanoparticles (e.g., silenceMag Magnetotransfection TM agent, OZ Biosciences, San Diego, CA), abrasive or scarifying agents, needles or microneedles, matrices, and grids.
  • particles or nanoparticles e.g., particles or nanoparticles made of materials such as carbon, silicon, silicon carbide, gold, tungsten, polymers, or ceramics
  • magnetic particles or nanoparticles e.g., silenceMag Magneto
  • particulates and nanoparticulates are useful in delivery of the polynucleotide composition or the nuclease or both.
  • Useful particulates and nanoparticles include those made of metals (e.g., gold, silver, tungsten, iron, cerium), ceramics (e.g., aluminum oxide, silicon carbide, silicon nitride, tungsten carbide), polymers (e.g., polystyrene, polydiacetylene, and poly(3,4- ethylenedioxythiophene) hydrate), semiconductors (e.g., quantum dots), silicon (e.g., silicon carbide), carbon (e.g., graphite, graphene, graphene oxide, or carbon nanosheets, nanocomplexes, or nanotubes), and composites (e.g., polyvinylcarbazole/graphene, polystyrene/graphene, platinum/graphene, palladium/graphene nanocomposites
  • such particulates and nanoparticulates are further covalently or non-covalently functionalized, or further include modifiers or cross-linked materials such as polymers (e.g., linear or branched polyethylenimine, poly-lysine), polynucleotides (e.g., DNA or RNA), polysaccharides, lipids, polyglycols (e.g., polyethylene glycol, thiolated polyethylene glycol), polypeptides or proteins, and detectable labels (e.g., a fluorophore, an antigen, an antibody, or a quantum dot).
  • polymers e.g., linear or branched polyethylenimine, poly-lysine
  • polynucleotides e.g., DNA or RNA
  • polysaccharides e.g., DNA or RNA
  • lipids lipids
  • polyglycols e.g., polyethylene glycol, thiolated polyethylene glycol
  • Embodiments of compositions including particulates include those formulated, e.g., as liquids, colloids, dispersions, suspensions, aerosols, gels, and solids.
  • Embodiments include nanoparticles affixed to a surface or support, e.g., an array of carbon nanotubes vertically aligned on a silicon or copper wafer substrate.
  • Embodiments include polynucleotide compositions including particulates (e.g., gold or tungsten or magnetic particles) delivered by a Biolistic-type technique or with magnetic force.
  • the size of the particles used in Biolistics is generally in the “microparticle” range, for example, gold microcarriers in the 0.6, 1.0, and 1.6 micrometer size ranges (see, e.g., instruction manual for the Helios® Gene Gun System, Bio-Rad, Hercules, CA; Randolph-Anderson et al.
  • nanoparticles which are generally in the nanometer (nm) size range or less than 1 micrometer, e.g., with a diameter of less than about 1 nm, less than about 3 nm, less than about 5 nm, less than about 10 nm, less than about 20 nm, less than about 40 nm, less than about 60 nm, less than about 80 nm, and less than about 100 nm.
  • nanoparticles commercially available (all from Sigma-Aldrich Corp., St.
  • Louis, MO include gold nanoparticles with diameters of 5, 10, or 15 nm; silver nanoparticles with particle sizes of 10, 20, 40, 60, or 100 nm; palladium “nanopowder” of less than 25 nm particle size; single-, double-, and multi-walled carbon nanotubes, e.g., with diameters of 0.7 - 1.1, 1.3 - 2.3, 0.7 - 0.9, or 0.7 - 1.3 nm, or with nano tube bundle dimensions of 2 - 10 nm by 1- 5 micrometers, 6 - 9 nm by 5 micrometers, 7 - 15 nm by 0.5 - 10 micrometers, 7 - 12 nm by 0.5 - 10 micrometers, 110 - 170 nm by 5 - 9 micrometers, 6 - 13 nm by 2.5 - 20 micrometers.
  • Embodiments include polynucleotide compositions including materials such as gold, silicon, cerium, or carbon, e.g., gold or gold-coated nanoparticles, silicon carbide whiskers, carborundum, porous silica nanoparticles, gelatin/silica nanoparticles, nanoceria or cerium oxide nanoparticles (CNPs), carbon nanotubes (CNTs) such as single-, double-, or multi-walled carbon nanotubes and their chemically functionalized versions (e.g., carbon nanotubes functionalized with amide, amino, carboxylic acid, sulfonic acid, or polyethylene glycol moieties), and graphene or graphene oxide or graphene complexes; see, for example, Wong et al.
  • materials such as gold, silicon, cerium, or carbon, e.g., gold or gold-coated nanoparticles, silicon carbide whiskers, carborundum, porous silica nanoparticles, gelatin/silica nanoparticle
  • the composition includes an excipient, e.g., a delivery vehicle, adjuvant, diluent, surfactant, stabilizer, or tonicity agent or a combination thereof.
  • the excipient is a crop oil concentrate, a vegetable oil concentrate, a modified vegetable oil, a nitrogen source, a deposition (drift control) and/or retention agent (with or without ammonium sulfate and/or defoamer), a compatibility agent, a buffering agent and/or acidifier, a water conditioning agent, a basic blend, a spreader-sticker and/or extender, an adjuvant plus foliar fertilizer, an antifoam agent, a foam marker, a scent, or a tank cleaner and/or neutralizer.
  • the excipient is an adjuvant described in the Compendium of Herbicide Adjuvants (Young et al. (2016).
  • delivery vehicles and diluents include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, saline solution, syrup, methylcellulose, methyl- and propylhydroxybenzoates, talc, magnesium stearate, and mineral oil.
  • delivery vehicles include, but are not limited to, solid or liquid excipient materials, solvents, stabilizers, slow-release excipients, colorings, and surface-active substances (surfactants).
  • the excipient e.g., delivery vehicle
  • the stabilizing vehicle includes, e.g., an epoxidized vegetable oil, an antifoaming agent, e.g. silicone oil, a preservative, a viscosity regulator, a binding agent, or a tackifier.
  • the stabilizing vehicle is a buffer suitable for the recombinant polynucleotide.
  • the composition is microencapsulated in a polymer bead delivery vehicle.
  • the stabilizing vehicle protects the recombinant polynucleotide against UV and/or acidic conditions.
  • the delivery vehicle contains a pH buffer.
  • the composition is formulated to have a pH in the range of about 4.5 to about 9.0, including for example pH ranges of about any one of 5.0 to about 8.0, about 6.5 to about 7.5, or about 6.5 to about 7.0.
  • the composition provided herein includes an adjuvant.
  • Adjuvants are agents that do not possess the polynucleotide activity, but impart beneficial properties to a formulation. For example, adjuvants are either pre-mixed in the formulation or added to a spray tank to improve mixing or application or to enhance performance.
  • Adjuvants can be used to customize the formulation to specific needs and compensate for local conditions. Adjuvants can be designed to perform specific functions, including wetting, spreading, sticking, reducing evaporation, reducing volatilization, buffering, emulsifying, dispersing, reducing spray drift, and reducing foaming. No single adjuvant can perform all these functions, but compatible adjuvants often can be combined to perform multiple functions simultaneously.
  • adjuvants included in the formulation are binders, dispersants and stabilizers, specifically, for example, casein, gelatin, polysaccharides (e.g., starch, gum arabic, cellulose derivatives, alginic acid, etc.), lignin derivatives, bentonite, sugars, synthetic water-soluble polymers (e.g., polyvinyl alcohol, polyvinylpyrrolidone, polyacrylic acid, etc.), PAP (acidic isopropyl phosphate), BHT (2,6-di-t-butyl-4-methylphenol), BHA (a mixture of 2-t-butyl-4-methoxyphenol and 3- t-butyl-4-methoxyphenol), vegetable oils, mineral oils, fatty acids and fatty acid esters.
  • binders specifically, for example, casein, gelatin, polysaccharides (e.g., starch, gum arabic, cellulose derivatives, alginic acid, etc.), lignin derivatives, benton
  • the compositions provided herein are in a liquid formulation.
  • Liquid formulations are generally mixed with water, but in some instances are used with crop oil, diesel fuel, kerosene, or other light oil as an excipient.
  • the amount of active ingredient e.g., recombinant polynucleotides
  • an emulsifiable concentrate formulation contains a liquid active ingredient, one or more petroleum-based solvents, and an agent that allows the formulation to be mixed with water to form an emulsion.
  • Such concentrates can be used in agricultural, ornamental and turf, forestry, Agent Ref: P14421WO00 - 43 - structural, food processing, livestock, and public health pest formulations.
  • these are adaptable to application equipment from small portable sprayers to hydraulic sprayers, low-volume ground sprayers, mist blowers, and low-volume aircraft sprayers.
  • Some active ingredients readily dissolve in a liquid excipient. When mixed with an excipient, they form a solution that does not settle out or separate, e.g., a homogenous solution.
  • formulations of these types include an active ingredient, a carrier and/or an excipient, and one or more other ingredients. Solutions can be used in any type of sprayer, indoors and outdoors.
  • the composition is formulated as an invert emulsion.
  • An invert emulsion is a water-soluble active ingredient dispersed in an oil excipient.
  • Invert emulsions require an emulsifier that allows the active ingredient to be mixed with a large volume of petroleum-based excipient, usually fuel oil.
  • Invert emulsions aid in reducing drift. With other formulations, some spray drift results when water droplets begin to evaporate before reaching target surfaces; as a result, the droplets become very small and lightweight. Because oil evaporates more slowly than water, invert emulsion droplets shrink less, and more active ingredient reaches the target. Oil further helps to reduce runoff and improve rain resistance.
  • a flowable or liquid formulation combines many of the characteristics of emulsifiable concentrates and wettable powders. Manufacturers use these formulations when the active ingredient is a solid that does not dissolve in either water or oil. The active ingredient, impregnated on a substance such as clay, is ground to a very fine powder. The powder is then suspended in a small amount of liquid. The resulting liquid product is quite thick.
  • Flowables and liquids share many of the features of emulsifiable concentrates, and they have similar disadvantages. They require moderate agitation to keep them in suspension and leave visible residues, similar to those of wettable powders.
  • Flowables/liquids are easy to handle and apply. Because they are liquids, they are subject to spilling and splashing. They contain solid particles, so they contribute to abrasive wear of nozzles and pumps. Flowable and liquid suspensions settle out in their containers. Because flowable and liquid formulations tend to settle, packaging in containers of five gallons or less makes remixing easier.
  • Aerosol formulations contain one or more active ingredients and a solvent. Most aerosols contain a low percentage of active ingredients.
  • Ready to use aerosol formulations are usually small, self-contained units that release the formulation when the nozzle valve is triggered. The formulation is driven through a fine opening by an inert gas under pressure, creating fine droplets. These products are used in greenhouses, in small areas Agent Ref: P14421WO00 - 44 - inside buildings, or in localized outdoor areas. Commercial models, which hold five to 5 pounds of active ingredient, are usually refillable.
  • Smoke or fog aerosol formulations are not under pressure.
  • the composition comprises a liquid excipient.
  • a liquid excipient includes, for example, aromatic or aliphatic hydrocarbons (e.g., xylene, toluene, alkylnaphthalene, phenylxylylethane, kerosene, gas oil, hexane, cyclohexane, etc.), halogenated hydrocarbons (e.g., chlorobenzene, dichloromethane, dichloroethane, trichloroethane, etc.), alcohols (e.g., methanol, ethanol, isopropyl alcohol, butanol, hexanol, benzyl alcohol, ethylene glycol, etc.), ethers (e.g., diethyl ether, ethylene glycol
  • the composition comprises a gaseous excipient.
  • Gaseous excipients include, for example, butane gas, floron gas, liquefied petroleum gas (LPG), dimethyl ether, and carbon dioxide gas.
  • LPG liquefied petroleum gas
  • the compositions are provided as a dry formulation.
  • Dry formulations can be divided into two types: ready-to-use and concentrates that must be mixed with water to be applied as a spray. Most dust formulations are ready to use and contain a low percentage of active ingredients (less than about 10 percent by weight), plus a very fine, dry inert excipient (e.g., talc, chalk, clay, nut hulls, or volcanic ash). The size of individual dust particles varies. A few dust formulations are concentrates and contain a high percentage of active ingredients. In some embodiments, these are mixed with dry inert excipients before applying. In some embodiments, dusts are used dry and can easily drift to non-target sites. [0125] In some instances, the composition is formulated as a powder.
  • the composition is formulated as a wettable powder.
  • Wettable powders are dry, finely ground formulations that look like dusts. They usually must be mixed with water for application as a spray. A few products, however, can be applied either as a dust or as a wettable powder—the choice is left to the applicator. Wettable powders have about 1 to about 95 percent active ingredient by weight; in some cases, more than about 50 percent. The particles do not dissolve in water. They settle out quickly unless constantly agitated to keep them suspended. They can be used for most pest problems and in most types of spray equipment where agitation is possible. Wettable powders have excellent residual activity.
  • the composition is formulated as a soluble powder.
  • Soluble powder formulations look like wettable powders. However, when mixed with water, soluble powders dissolve readily and form a true solution. After they are mixed thoroughly, no additional agitation is necessary.
  • the amount of active ingredient in soluble powders ranges from about 15 to about 95 percent by weight; in some cases, more than about 50 percent. Soluble powders have all the advantages of wettable powders and none of the disadvantages, except the inhalation hazard during mixing.
  • the composition is formulated as a water-dispersible granule.
  • Water- dispersible granules also known as dry flowables, are like wettable powders, except instead of being dust-like, they are formulated as small, easily measured granules.
  • Water-dispersible granules must be mixed with water to be applied. Once in water, the granules break apart into fine particles similar to wettable powders. The formulation requires constant agitation to keep it suspended in water. The percentage of active ingredient is high, often as much as 90 percent by weight. Water-dispersible granules share many of the same advantages and disadvantages of wettable powders, except they are more easily measured and mixed.
  • the composition comprises a solid excipient.
  • Solid excipients include finely-divided powder or granules of clay (e.g.
  • kaolin clay diatomaceous earth, bentonite, Fubasami clay, acid clay, etc.
  • synthetic hydrated silicon oxide talc, ceramics, other inorganic minerals (e.g., sericite, quartz, sulfur, activated carbon, calcium carbonate, hydrated silica, etc.), a substance which can be sublimated and is in the solid form at room temperature (e.g., 2,4,6-triisopropyl-1,3,5-trioxane, naphthalene, p-dichlorobenzene, camphor, adamantan, etc.); wool; silk; cotton; hemp; pulp; synthetic resins (e.g., polyethylene resins such as low-density polyethylene, straight low-density polyethylene and high-density polyethylene; ethylene-vinyl ester copolymers such as ethylene-vinyl acetate copolymers; ethylene-methacrylic acid ester copolymers such as ethylene-methyl methacrylate copo
  • the composition is provided in a microencapsulated formulation (e.g., a nanocapsule). Microencapsulated formulations are mixed with water and sprayed in the same manner as other sprayable formulations. After spraying, the encapsulation shell or coating breaks down and slowly releases the active ingredient. [0130] In some instances, the composition is provided in a liposome. In some instances, the composition is provided in a vesicle. [0131] In some instances, a composition provided herein includes a surfactant. Surfactants, also called wetting agents and spreaders, physically alter the surface tension of a spray droplet.
  • surfactants also called wetting agents and spreaders
  • Surfactants For a formulation to perform its function properly, a spray droplet must be able to wet the foliage and spread out evenly over a leaf. Surfactants enlarge the area of formulation coverage, thereby increasing exposure to the active agent. Surfactants are particularly important when applying a formulation to waxy or hairy leaves. Without proper wetting and spreading, spray droplets often run off or fail to cover leaf surfaces adequately. Too much surfactant, however, can cause excessive runoff and reduce efficacy. [0132] Surfactants can be classified as anionic, cationic, or nonionic. Formulation activity in the presence of a nonionic surfactant can be quite different from activity in the presence of a cationic or anionic surfactant.
  • anionic surfactants are most effective when used with contact pesticides (pesticides that control a pest by direct contact rather than being absorbed systemically).
  • Cationic surfactants are not typically used as stand-alone surfactants because they usually are phytotoxic.
  • Nonionic surfactants often used with systemic pesticides, help sprays to penetrate plant cuticles.
  • Nonionic surfactants are compatible with most pesticides, and most EPA-registered pesticides that require a surfactant recommend a nonionic type.
  • Adjuvants include, but are not limited to, stickers, extenders, plant penetrants, compatibility agents, buffers or pH modifiers, drift control additives, defoaming agents, and thickeners.
  • surfactants included in the compositions described herein are alkyl sulfate ester salts, alkyl sulfonates, alkyl aryl sulfonates, alkyl aryl ethers and polyoxyethylenated products thereof, polyethylene glycol ethers, polyvalent alcohol esters and sugar alcohol derivatives.
  • the surfactant is a nonionic surfactant, a surfactant plus nitrogen source, an organo- silicone surfactant, or a high surfactant oil concentrate.
  • the recombinant polynucleotide can, in embodiments, be in a mixture with other active compounds, such as pesticidal agents (e.g., insecticides, sterilants, acaricides, nematicides, molluscicides, or fungicides, attractants, growth-regulating substances, or herbicides).
  • pesticidal agents e.g., insecticides, sterilants, acaricides, nematicides, molluscicides, or fungicides, attractants, growth-regulating substances, or herbicides.
  • pesticide refers to any substance or mixture of substances intended for preventing, destroying, repelling, or mitigating any pest.
  • a pesticide can be a chemical substance or biological agent used against pests including insects, mollusks, pathogens, weeds, nematodes, and microbes that compete with humans for food, destroy property, spread disease, or are a nuisance.
  • the term “pesticidal agent” further encompasses other bioactive molecules such as antibiotics, antivirals pesticides, antifungals, antihelminthics, nutrients, pollen, sucrose, and/or agents that stun or slow insect movement.
  • this disclosure is related to a method of producing a modified plant propagule that comprises at least one plant cell comprising a recombinant RNA molecule.
  • the method includes the steps of: isolating a plant propagule comprising at least one plant cell comprising a recombinant RNA molecule and a Ghabrivirales RNA-dependent RNA polymerase (RdRP) from a mixed population of plant cells comprising both plant cells comprising the recombinant RNA molecule and plant cells lacking the recombinant RNA molecule, wherein the recombinant RNA molecule comprises, in 5’ to 3’ order, a 5’ RNA replication element that is capable of being recognized by the Ghabrivirales RdRP; a cargo RNA sequence; and a 3’ RNA replication element that is capable of being recognized by the Ghabrivirales RdRP.
  • RdRP RNA-dependent RNA polymerase
  • the isolated plant propagule comprising at least one plant cell comprising a recombinant RNA molecule will be free or substantially free of plant cells lacking the recombinant RNA.
  • Such isolated plant propagules which are substantially free of plant cells lacking the recombinant RNA can in certain embodiments comprise plant propagules where at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the plant cells in the plant propagule contain the recombinant DNA molecule.
  • the mixed population of plant cells comprise a population of protoplasts or a population of cells in callus, an explant, a plant part, or whole plant.
  • the mixed population of plant cells can comprise a population of plant cells where less than 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or 1% of the plant cells in the population contain the recombinant RNA molecule.
  • the mixed population of plant cells comprise plant cells comprising the Ghabrivirales RdRP and plant cells lacking the Ghabrivirales RdRP.
  • the plant cells lacking the Ghabrivirales RdRP will also lack the recombinant RNA.
  • the mixed population of plant cells comprise plant cells comprising the Ghabrivirales RdRP.
  • the plant cells comprising the Ghabrivirales RdRP can further comprise the recombinant RNA.
  • the cargo RNA sequence comprises RNA that encodes a selectable or scorable marker.
  • the mixed population of plant cells is screened or selected for the presence of the plant cell comprising the recombinant RNA molecule prior to isolating the plant propagule. In such screens, the mixed population of cells or a portion thereof are subjected to an assay for a screenable marker for the presence of the recombinant RNA molecule (e.g., an RNA sequence diagnostic for presence of the recombinant RNA molecule or a polypeptide encoded by the recombinant RNA molecule) and separated from plant cells lacking the recombinant RNA molecule.
  • an assay for a screenable marker for the presence of the recombinant RNA molecule e.g., an RNA sequence diagnostic for presence of the recombinant RNA molecule or a polypeptide encoded by the recombinant RNA molecule
  • the isolation comprises selecting for the plant cell comprising the recombinant RNA molecule prior to isolating the plant propagule.
  • selectable marker e.g., a protein which confers resistance to a selection agent such as an herbicide or antibiotic
  • the isolation agent can comprise exposing the mixed population of plant cells to a selection agent (e.g., an herbicide or antibiotic) and isolating plant cells which survive exposure to the selection agent.
  • selectable marker/selection agent combinations include glyphosate-resistant EPSPS enzymes and/or glyphosate oxidases/glyphosate, a bialaphos resistance (bar) or phosphinothricin acyl transferase (pat) enzyme/glufosinate, or a neomycin phosphotransferase (npt)/neomycin or kanamycin.
  • the selectable or scorable marker is an RNA aptamer (e.g., a Broccoli aptamer) or a regulatory RNA (e.g., an siRNA, siRNA precursor, miRNA, or miRNA precursor, or a phased siRNA or phased siRNA precursor that downregulates expression of an endogenous gene in the plant, resulting in a detectable phenotype, e.g., bleaching caused by downregulation of a pigment- producing gene).
  • the mixed population is located within a plant or a plant part.
  • the plant or plant part is screened or selected for presence of the recombinant RNA molecule prior to isolating the plant propagule.
  • the plant or plant part is screened or selected for systemic presence of the recombinant RNA molecule prior to isolating the plant propagule.
  • the plant cells, plant, or plant part in the mixed population or that are isolated lack DNA that encodes the recombinant RNA molecule.
  • the plant propagule comprising the recombinant RNA molecule is isolated by detecting the RNA molecule in one or more plant cells comprising the recombinant RNA molecule and separating the one or more plant cells comprising the recombinant RNA molecule from the plant cells lacking the recombinant DNA molecule.
  • the plant propagule is a mosaic comprising both plant cells comprising the recombinant RNA molecule and plant cells lacking the recombinant RNA molecule.
  • the modified plant propagule is a mosaic comprising both plant cells comprising the Ghabrivirales RdRP and plant cells lacking the Ghabrivirales RdRP.
  • at least 99%, 98%, 95%, 90%, 85%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or 1% of the plant cells in the mosaic can comprise the recombinant RNA molecule.
  • the plant propagule lacks DNA that encodes the recombinant RNA molecule.
  • the modified plant propagule comprises the cell comprising the recombinant RNA molecule, or a seed, seedling, ovule, embryo, pollen, root, stem, leaf, shoot, tuber, rhizome, stolon, bulb, explant, or callus comprising the cell comprising the recombinant RNA molecule.
  • Plant propagules made by any of the aforementioned methods and/or incorporating any of the aforementioned features are also provided herein.
  • any of the aforementioned methods can further comprise multiplying the cell, seed, seedling, ovule, embryo, pollen, root, stem, leaf, shoot, tuber, rhizome, stolon, bulb, explant, or callus to obtain progeny, wherein the progeny comprise the recombinant RNA molecule.
  • the multiplying of the cells consists of culturing a plurality of explants obtained from Agent Ref: P14421WO00 - 49 - the cell, seed, seedling, ovule, embryo, pollen, root, stem, leaf, shoot, tuber, rhizome, stolon, bulb, explant, or callus.
  • the isolated propagule comprises the cell and the aforementioned methods can further comprise regenerating a plant, seedling, ovule, embryo, pollen, root, stem, leaf, shoot, tuber, rhizome, stolon, bulb, explant, or callus comprising the recombinant RNA from the cell.
  • the isolated propagule comprises callus and the aforementioned methods can further comprise regenerating a plant, seedling, ovule, embryo, pollen, root, stem, leaf, shoot, tuber, rhizome, stolon, bulb, or explant comprising the recombinant RNA from said callus.
  • a plant is regenerated and the aforementioned methods can further comprise recovering F1 seed or F1 progeny or clonal progeny comprising the recombinant RNA from the plant.
  • this disclosure is related to a method of providing a synthetic Ghabrivirales satellite RNA to a plant or plant part by grafting one plant part to another plant part.
  • the methods can comprise grafting a scion onto a rootstock comprising any of the aforementioned or otherwise disclosed recombinant DNA molecules and/or recombinant RNA molecules (e.g., a recombinant RNA comprising, in 5’ to 3’ order, a 5’ RNA replication element that is capable of being recognized by a Ghabrivirales RNA-dependent RNA polymerase (RdRP); a cargo RNA sequence; and a 3’ RNA replication element that is capable of being recognized by the Ghabrivirales RdRP), wherein at least one cell of the rootstock and/or the scion comprises the Ghabrivirales RdRP.
  • a recombinant RNA comprising, in 5’ to 3’ order, a 5’ RNA replication element that is capable of being recognized by a Ghabrivirales RNA-dependent RNA polymerase (RdRP); a cargo RNA sequence; and a 3’ RNA replication element that is capable of being recognized by the Ghabri
  • the scion can comprise a plant shoot, an apical or other meristem, a leaf attached to a petiole, or other plant part and the rootstock can comprise roots and aerial portions of the plant including the main stem, secondary stems, leaves, and/or reproductive structures of the plant,
  • DNA that encodes the recombinant RNA molecule is absent in the scion and/or the rootstock.
  • the scion lacks the recombinant RNA molecule prior to grafting.
  • the rootstock comprises the Ghabrivirales RdRP.
  • the Ghabrivirales RdRP is provided by a Ghabrivirales endemic to the rootstock (e.g., a Ghabrivirales which is non-pathogenic and/or commensal).
  • the Ghabrivirales RdRP is exogenously provided to the rootstock (e.g., via DNA expression cassette which is integrated into the chromosomal or plastidic DNA of the rootstock or via a recombinant viral vector comprising DNA or RNA encoding the RdRP).
  • the scion comprises the Ghabrivirales RdRP.
  • the RdRP is provided by a Ghabrivirales endemic to the scion (e.g., a Ghabrivirales which is non-pathogenic and/or commensal).
  • the RdRP is exogenously provided to the scion (e.g., via DNA expression cassette which is integrated into the chromosomal or plastidic DNA of the scion or via a recombinant viral vector comprising DNA or RNA encoding the RdRP).
  • the Ghabrivirales RdRP comprises a protein having at least 85%, 90%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 491, 492, 493, 494, 495, or 496.
  • the rootstock and/or the scion comprises a heterologous viral coat protein which can encapsidate the recombinant RNA molecule. In some embodiments, the rootstock and/or the scion comprises the recombinant RNA molecule encapsidated by a heterologous viral coat protein.
  • this disclosure is related to a method of producing a grafted plant comprising a recombinant RNA molecule comprising, in 5’ to 3’ order, a 5’ RNA replication element that is capable of being recognized by a Ghabrivirales RNA-dependent RNA polymerase (RdRP); a cargo RNA sequence; and a 3’ RNA replication element that is capable of being recognized by an the Ghabrivirales RdRP.
  • RdRP Ghabrivirales RNA-dependent RNA polymerase
  • the recombinant RNA molecule is provided by contacting the scion, the rootstock, or both the scion and the rootstock with a composition comprising the recombinant RNA molecule prior to grafting the scion onto the rootstock to produce the grafted plant.
  • at least one cell of the rootstock and/or the scion comprises a Ghabrivirales RdRP prior to contacting the scion, the rootstock, or both the scion and the rootstock with the composition.
  • the rootstock comprises the Ghabrivirales RdRP.
  • the Ghabrivirales RdRP is provided by a Ghabrivirales endemic to the rootstock (e.g., a Ghabrivirales which is non- pathogenic and/or commensal).
  • the Ghabrivirales RdRP is exogenously provided to the rootstock (e.g., via DNA expression cassette which is integrated into the chromosomal or plastidic DNA of the rootstock or via a recombinant viral vector comprising DNA or RNA encoding the RdRP).
  • the scion comprises the Ghabrivirales RdRP.
  • the RdRP is provided by a Ghabrivirales endemic to the scion (e.g., a Ghabrivirales which is non-pathogenic and/or commensal).
  • the RdRP is exogenously provided to the scion (e.g., via DNA expression cassette which is integrated into the chromosomal or plastidic DNA of the scion or via a recombinant viral vector comprising DNA or RNA encoding the RdRP).
  • the Ghabrivirales RdRP comprises a protein having at least 85%, 90%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 491, 492, 493, 494, 495, or 496.
  • DNA that encodes the recombinant RNA molecule is absent in the scion, the rootstock, and/or the grafted plant.
  • the composition can be provided to the scion, the rootstock, or both the scion and the rootstock according to any of the formulations disclosed herein.
  • the formulation is a liquid, a gel, or a powder.
  • the formulation is configured to be sprayed on to the scion, the rootstock, or both the scion and the rootstock; to be injected into the scion, the rootstock, or both the scion and the rootstock; to be soaked into the scion, the rootstock, or both the scion and the rootstock; or to be coated onto the scion, the rootstock, or both the scion and the rootstock.
  • the contacting comprises dipping the scion, the rootstock, or both the scion and the rootstock into the composition prior to grafting.
  • this disclosure is related to a method for producing a plant that transmits any of the aforementioned or otherwise disclosed recombinant RNA molecules provided herein to progeny plants or seed.
  • the methods include the steps of: isolating an F 1 progeny plant or seed comprising at least one cell comprising a Ghabrivirales RNA-dependent RNA polymerase (RdRP) and the recombinant RNA molecule comprising, in 5’ to 3’ order, a 5’ RNA replication element that is capable of being recognized by the Ghabrivirales RdRP); a cargo RNA sequence; and a 3’ RNA replication element that is capable of being recognized by the Ghabrivirales RdRP from a population of F 1 plants or seed obtained from at least one parent plant comprising the recombinant RNA molecule.
  • RdRP Ghabrivirales RNA-dependent RNA polymerase
  • the parent plant or a part thereof comprising the plant cell is screened or selected for presence of the recombinant RNA molecule prior to isolating the F 1 progeny plant or seed.
  • the parent plant or one or more parts thereof are screened for systemic presence of the recombinant RNA molecule prior to isolating the F 1 progeny plants.
  • floral tissue e.g., whole flowers or buds, sepal, calyx, or petal
  • male reproductive tissue e.g., stamen, anther, or pollen
  • female reproductive tissue e.g., whole fruit, ovary, pericarp, ovule, seed coat, endosperm, or embryo
  • F 1 seeds are obtained from a parent plant selected for presence of the recombinant RNA molecule in pericarp tissue.
  • the F 1 progeny plant or seed comprising the cell is isolated by screening the population of F 1 plants or seed obtained from a parent plant for the presence of the recombinant RNA molecule and propagating the F 1 progeny plant or seed comprising the recombinant RNA molecule.
  • the progeny plants or seed thereof are subjected to an assay for a screenable marker for the presence of the recombinant RNA (e.g., an RNA sequence diagnostic for presence of the recombinant RNA molecule or a polypeptide encoded by the recombinant RNA molecule) and separated from progeny plants and seed lacking the recombinant RNA progeny plants and seed lacking the recombinant RNA.
  • a screenable marker for the presence of the recombinant RNA e.g., an RNA sequence diagnostic for presence of the recombinant RNA molecule or a polypeptide encoded by the recombinant RNA molecule
  • Such screening assays can be non-destructive assays wherein a portion of the progeny seed or plant is removed and assayed without loss of the viability or ability to propagate the seed or plant tissue comprising the recombinant RNA.
  • an F 1 seed of the parent plant is non-destructively screened for presence of the recombinant RNA molecule.
  • the F 1 seed of the parent plant is non-destructively screened by assaying maternally derived or endosperm tissue of the seed for the presence of the recombinant RNA molecule.
  • the cargo RNA sequence comprises RNA that encodes a selectable or scorable marker.
  • the recombinant RNA molecule encodes a selectable marker and the F 1 progeny plant or seed comprising the recombinant RNA molecule is isolated by selecting the F 1 progeny plant or seed comprising the recombinant RNA molecule for presence of the selectable marker.
  • Examples of such selections in instances where the recombinant RNA encodes a selectable marker can comprise exposing the progeny seeds or plants to a selection agent (e.g., an herbicide or antibiotic) and isolating progeny seeds or plants which survive exposure to the selection agent.
  • a selectable marker e.g., a protein which confers resistance to a selection agent such as an herbicide or antibiotic
  • selectable marker/selection agent combinations include glyphosate-resistant EPSPS enzymes and/or glyphosate oxidases/glyphosate, a bialaphos resistance (bar) or phosphinothricin acyl transferase (pat) enzymes/glufosinate, and neomycin phosphotransferase (npt)/neomycin or kanamycin.
  • the selectable or scorable marker is an RNA aptamer or a regulatory RNA.
  • the F 1 progeny plant or seed lacks DNA that encodes the recombinant RNA molecule.
  • the parent plant lacks DNA that encodes the recombinant RNA Agent Ref: P14421WO00 - 52 - molecule.
  • the selected F 1 progeny plant transmits the recombinant RNA molecule to at least F 2 progeny.
  • the F 1 progeny plant or seed population is obtained from a parent plant used as a pollen recipient.
  • the F 1 progeny plant or seed population is obtained from a parent plant used as a pollen donor.
  • the F 1 progeny plant or seed population is obtained by selfing the parent plant.
  • the F 1 progeny plant or seed population is obtained from the sexual crossing of two parent plants.
  • the parent plant that comprises the recombinant RNA molecule is the female parent plant. In other embodiments, the parent plant that comprises the recombinant RNA molecule is the male parent plant, and the recombinant RNA molecule is transmitted in pollen of the male parent plant. [0142] In certain embodiments, the methods can further comprise introducing the recombinant RNA molecule or a polynucleotide encoding the recombinant RNA molecule into a plant cell and obtaining the parent plant comprising the recombinant RNA molecule from the plant cell.
  • the recombinant RNA molecule further comprises at least one additional element selected from the group consisting of: (a) at least one RNA encoding a viral movement protein (MP); (b) at least one tRNA-like sequence; and c) an origin-of-assembly sequence (OAS).
  • a parent and/or plant comprises a heterologous viral coat protein which can encapsidate the recombinant RNA molecule.
  • the parent and/or progeny plant comprises the recombinant RNA molecule encapsidated by a heterologous viral coat protein.
  • this disclosure is related to a method of barcoding a plant, plant cell, progeny thereof, or part thereof.
  • the methods comprise providing to the plant or plant cell any of the aforementioned or otherwise disclosed recombinant RNA molecules provided herein, wherein the cargo RNA of the recombinant RNA molecule comprises a barcode RNA molecule, and wherein the plant or plant cell comprises a Ghabrivirales RdRP.
  • the barcode RNA molecule comprises a sequence that uniquely identifies the plant, plant cell, progeny thereof, or part thereof.
  • the barcode RNA can be a randomly generated sequence.
  • the barcode RNA molecule comprises a sequence that is not present in the genome and/or transcriptome of a wild-type plant of the same species, a pathogen thereof, or a symbiont thereof. In some embodiments, the barcode RNA molecule comprises a forward primer binding site and a reverse primer binding site for detection of the barcode RNA molecule. In embodiments, the barcode RNA molecule comprises a non- protein coding sequence. In some embodiments, the barcode RNA sequence is up to about 4.6 kilobases (kb) in length (Totiviridae-based sattelites) or up to about 12.5 kilobases (kb) in length (Chrysoviridae- based sattelites).
  • the barcode RNA has a length of 10 to 5000 nucleotides, 20 to 1000 nucleotides, or 50 to 500 nucleotides. In certain embodiments, the barcode RNA molecule has a length of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, or 5000 nucleotides.
  • the plant transmits the recombinant RNA molecule comprising the barcode RNA to progeny.
  • the plant, plant cell, progeny Agent Ref: P14421WO00 - 53 - thereof, or part thereof lacks DNA that encodes the recombinant RNA molecule.
  • the methods can further comprise isolating an F 1 progeny plant or seed comprising at least one cell comprising the Ghabrivirales RdRP and the recombinant RNA molecule.
  • the F 1 progeny plant or seed is obtained from the plant used as a pollen recipient.
  • the F 1 progeny plant or seed is obtained from the plant used as a pollen donor.
  • the F 1 progeny plant or seed is obtained by selfing the parent plant.
  • the methods can further comprise propagating the plant or plant cell to obtain a plant part or a plant propagule comprising the barcode RNA molecule.
  • this disclosure is related to a method of identifying a barcoded plant, plant part, or plant cell. The methods comprise screening for the presence of a barcode RNA molecule in the plant, plant part, or plant cell, wherein the plant, plant part, or plant cell comprises any of the aforementioned or otherwise disclosed recombinant RNA molecules provided herein, and wherein the cargo RNA of the recombinant RNA molecule comprises the barcode RNA molecule.
  • the methods comprise obtaining a nucleic acid sample from the plant, plant part, or plant cell; and detecting the presence of the barcode RNA molecule in the sample.
  • Assays for detection of a barcode RNA include RNA detection assays (e.g., an RT-PCR assay) using nucleic acid probes and/or primers which can detect the barcode RNA and/or sequencing of the barcode RNA.
  • Such screening assays can be non-destructive assays wherein a portion of the seed or plant is removed and assayed without loss of the viability or ability to propagate the seed or plant tissue comprising the barcode RNA.
  • a seed of the plant is non-destructively screened for presence of the barcode RNA molecule.
  • a seedling, ovule, embryo, pollen, root, stem, leaf, shoot, tuber, rhizome, stolon, bulb, explant, or callus is screened for the presence of the barcode RNA molecule.
  • the methods disclosed herein are not processes for modifying the germ line or genetic identity of human beings.
  • the methods disclosed herein are not processes for modifying the genetic identity of animals which are likely to cause them suffering without any substantial medical benefit to man or animal, and are also not drawn to animals resulting from such processes. In certain optional embodiments, the methods disclosed herein are not methods for treatment of the human or animal body by surgery or therapy. In certain optional embodiments, the cells disclosed herein are not human embryos. In certain optional embodiments, the cells disclosed herein are not the human body or its parts, at the various stages of its formation and development.
  • the plant cells, plant propagules e.g., a seed, seedling, ovule, embryo, pollen, root, stem, leaf, shoot, tuber, rhizome, stolon, bulb, explant, or callus
  • plants provided herein are not produced by an exclusively biological process.
  • the methods for producing plant cells, plant propagules e.g., a Agent Ref: P14421WO00 - 54 - seed, seedling, ovule, embryo, pollen, root, stem, leaf, shoot, tuber, rhizome, stolon, bulb, explant, or callus
  • plants provided herein are not exclusively biological processes.
  • RNA molecule comprising from 5’ terminus to 3’ terminus: (a) a 5’ RNA replication element recognized by a Ghabrivirales RNA-dependent RNA polymerase (RdRP); (b) a cargo RNA molecule; and (c) a 3’ RNA replication element recognized by the RdRP; wherein the 5’ RNA replication element, the cargo RNA molecule, and the 3’ RNA replication element are operably linked and wherein the cargo RNA molecule is heterologous to the 5’ RNA replication element and the 3’ RNA replication element, optionally wherein: (i) the 5’ RNA replication element and the 3’ RNA replication element are obtained from the same Ghabrivirales genome or from Ghabrivirales genomes having at least 85%, 90%, 95%, 98%, or 99% sequence identity to one another and which
  • RNA replication element comprises at least one RNA secondary structure provided in Table 1 or adopted by an RNA molecule encoded by SEQ ID NO: 467, 468, 469, 470, 471, 472, 473, 474, 549 to 552, or 557 to 559; and/or (b) the 3’ RNA replication element (3’ RRE) comprises at least one RNA secondary structure provided in Table 1 or adopted by an RNA molecule encoded by SEQ ID NO: 475, 476, 477, 478, 479, 480, 481, 482, 553 to 556, or 560 to 562. [0149] 3.
  • RNA replication element comprises an RNA molecule encoded by SEQ ID NO: 467, 468, 469, 470, 471, 472, 473, 474, 549 to 552, or 557 to 559; a variant thereof encoded by a DNA molecule having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 467, 468, 469, 470, 471, 472, 473, 474, 549 to 552, or 557 to 559; or a variant thereof wherein one or more nucleotides in the RNA secondary structure are substituted with distinct nucleotides which maintain the RNA secondary structure; and/or Agent Ref: P14421WO00 - 55 - (b) the 3’ RNA replication element comprises an RNA molecule encoded by SEQ ID NO: 475, 476, 477, 478, 479, 480, 481, 482,
  • RNA secondary structure is maintained by substituting nucleotides in the secondary structure which are not base paired with nucleotides which will not base pair, and/or by substituting nucleotides in the secondary structure which are base paired with nucleotides which will base pair. [0151] 5.
  • the 5’ RNA replication element comprises at least a segment of the 5’ untranslated region (UTR) of the Ghabrivirales genome or a variant thereof having one or more nucleotide substitutions, insertions, and/or deletions, wherein the variant is recognized by the RdRP, optionally wherein the 5’ RNA replication element further comprises a genomic sequence of the Ghabrivirales that is natively located 3’ to and adjacent to the 5’ UTR sequence; and/or (b) the 3’ RNA replication element comprises at least a segment of the 3’ UTR of the Ghabrivirales genome or a variant thereof having one or more nucleotide substitutions, insertions, and/or deletions, wherein the variant is recognized by the RdRP, optionally wherein the 3’ RNA replication element further comprises a genomic sequence of the Ghabrivirales that is natively located 5’ to and adjacent to the 3’ UTR sequence, and optional
  • RNA molecule of any one of embodiments 1 to 5, wherein the RNA molecule further comprises at least one of: (i) a tRNA-like element, optionally wherein the tRNA-like element provides for intercellular movement of the RNA and optionally wherein the intercellular movement is mediated by a viral movement protein (MP); (ii) an encapsidation recognition element (ERE), optionally wherein the ERE provides for encapsidation of the RNA by a Ghabrivirales capsid protein or optionally wherein the ERE provides for encapsidation of the RNA by a non-Ghabrivirales capsid protein; (iii) an RNA effecter; and/or (iv) RNA encoding a viral movement protein (MP), optionally wherein an internal ribosome entry site (IRES) is operably linked to the RNA encoding the MP.
  • MP viral movement protein
  • IVS internal ribosome entry site
  • RNA-like element comprises a tRNA-like molecule from an Arabidopsis FT mRNA or is a tRNA-like sequence encoded by a DNA sequence selected from the group consisting of SEQ ID NOs: 76-123, and 466 or is a modified tRNA-like sequence that has at least 90% sequence identity to a scaffold tRNA-like sequence encoded by a DNA sequence selected from the group consisting of SEQ ID NOs: 76-123, and 466 and that maintains Agent Ref: P14421WO00 - 56 - the secondary structure of the scaffold tRNA-like sequence, and/or wherein the ERE is a tobacco mosaic virus (TMV) OAS.
  • TMV tobacco mosaic virus
  • RNA molecule of embodiment 1 wherein: (i) the cargo RNA molecule is up to about 4.6 kilobases (kb) in length and the 5’ RRE and 3’ RRE are obtained or derived from a Totivirus; or (ii) up to about 12.5 kilobases (kb) in length and the RRE and 3’ RRE are obtained or derived from a Chrysovirus . [0155] 9.
  • the cargo RNA molecule comprises at least one coding sequence, and wherein the RNA molecule further comprises an internal ribosome entry site (IRES) which is operably linked to at least one coding sequence, optionally wherein the operably linked IRES is located 5’ and immediately adjacent to the coding sequence.
  • IRES internal ribosome entry site
  • hpRNA hairpin RNA
  • RNA hairpin RNA
  • siRNA small interfering RNA
  • miRNA microRNA
  • miRNA miRNA
  • aptazyme a ligand-responsive ribozyme
  • RNA aptamer or a long noncoding RNA (ln
  • RNA binding proteins comprise an RNA recognition motif.
  • HRV heterologous RNA virus
  • a heterologous RNA virus (HRV) 5’ replication region HRV 5’RR
  • the cargo RNA molecule RNA molecule
  • the heterologous RNA virus (HRV) 3’ RNA replication region HRV 3’RR
  • the HRV 5’ RR and HRV 3’ RR HRV are recognized by a heterologous RNA virus RNA-dependent RNA polymerase (hrvRdRP); and wherein the HRV 5’RR, cargo RNA molecule, and HRV 3’RR are operably linked; or II.
  • heterologous RNA virus (HRV) subgenomic promoter operably linked to the cargo RNA molecule; wherein the subgenomic promoter is recognized by a heterologous RNA virus RNA-dependent RNA polymerase (hrvRdRP).
  • RNA molecule comprises from 5’ terminus to 3’ terminus: (a) the 5’ RNA replication element; (b) the HRV amplicon in antisense orientation to the first 5’ RNA replication element; optionally wherein the HRV amplicon further comprises: (i) an RNA molecule encoding an HRV RNA-dependent RNA polymerase (hrvRdRP) which is operably linked to the HRV 5’RR and HRV 3’RR, wherein the RNA molecule encoding the HRV RNA- dependent RNA polymerase (hrvRdRP) is optionally operably linked to a subgenomic promoter recognized by the hrvRdRP; or (ii) an RNA molecule encoding an HRV RNA-dependent RNA polymerase (hrvRdRP) which is operably linked to linked to a subgenomic promoter recognized by the hrvRdRP; and (c) the 3’ RNA replication element
  • a virus selected from the group consisting of an Alphaflexivirus, Betaflexivirus, Bromovirus, Celavirus, a Closterovirus, a Comovirus, a Potexvirus, Potyvirus, Tobamovirus, Tombusvirus, Tospoviridae, Trivirinae, Tymovirus, Varicosavirus, and Secoviridae.
  • the HRV 5’ RR comprises at least one RNA secondary structure adopted by an RNA encoded by SEQ ID NO: 161 to 185, or 186, or comprises the RNA sequence encoded by SEQ ID NO: 161 to 185, or 186, or comprises a contiguous fragment of at least 80%, 85%, 90%, or 95% of the full sequence of the RNA encoded by SEQ ID NO: 161 to 185, or 186; and/or (b) the HRV 3’ RR comprises at least one RNA secondary structure adopted by an RNA encoded by SEQ ID NO: 187 to 210, or 211, or comprises the RNA sequence of the RNA encoded by SEQ ID NO: 187 to 210, or 211, or comprises a contiguous fragment of at least 80%, 85%, 90%, or 95% of the full sequence of the RNA encoded by SEQ ID NO: 187 to 210, or 211.
  • the HRV 3’ RR comprises an RNA molecule containing at least a segment of the 3’ untranslated region (UTR) of the HRV genome, or a variant thereof having one or more nucleotide substitutions, insertions, and/or deletions, wherein the HRV 3’ RR or the variant is recognized by the hrvRdRP, optionally wherein the RNA comprising the HRV 3’ RR further comprises a genomic sequence of the HRV that is natively located 5’ to and adjacent to the 3’ UTR sequence; and/or (b) the HRV 5’ RR comprises an RNA molecule containing at least a segment of the 3’ untranslated region (UTR) of the HRV genome, or a variant thereof having one or more nucleotide substitutions, insertions, and/or deletions, wherein the HRV 5’ RR or the variant is recognized by the hrvRdRP, optionally
  • Agent Ref: P14421WO00 - 59 - [0170] 24 The recombinant RNA molecule of any one of embodiments 1 to 23, wherein the cargo RNA comprises an HRV-inhibitory RNA or encodes an HRV-inhibitory protein, wherein the HRV- inhibitory RNA or HRV-inhibitory protein inhibits infection, movement, transmission, and/or replication of the HRV. [0171] 25. The recombinant RNA molecule of any one of embodiments 1 to 24, wherein the cargo RNA comprises an RNA having at least 20 to 25 contiguous nucleotides having an identical or complementary sequence to a segment of equivalent length of the genomic RNA of the HRV. [0172] 26.
  • RNA comprises an RNA having at least 20 to 25 contiguous nucleotides having an identical or complementary sequence to a segment of equivalent length of the genomic RNA of the HRV which does not encode the hrvRdRP.
  • the cleavable sequence is optionally a self-cleaving ribozyme, a self-cleaving inducible ribozyme, or an siRNA or miRNA recognition site.
  • 33. The agricultural formulation of anyone of embodiments 29 to 32, wherein the formulation comprises the recombinant RNA molecule and a carrier, an excipient, and/or an adjuvant.
  • 34. A cell comprising the recombinant RNA molecule of any one of embodiments 1 to 28, wherein the cell is a bacterial cell, a fungal cell, a plant cell, an insect cell, or an invertebrate animal cell.
  • 35 The cell of embodiment 34, wherein the cell is a plant cell and DNA which encodes the recombinant RNA molecule is absent from the cell.
  • the cell of embodiment 34 wherein the cell comprises a recombinant DNA molecule which encodes the recombinant RNA molecule.
  • An expression system comprising: Agent Ref: P14421WO00 - 67 - (a) an RNA molecule comprising the recombinant RNA molecule of any one of embodiments 1 to 28; and (b) a cell containing the recombinant RNA molecule and an RdRP protein that recognizes the 5’ and 3’ RNA replication elements of the recombinant RNA molecule.
  • RNA molecule comprises an operably linked encapsidation recognition element (ERE) recognized by a viral capsid protein, and wherein the cell contains the viral capsid protein.
  • EEE operably linked encapsidation recognition element
  • 39 The expression system of embodiment 37, wherein the encapsidation recognition element (ERE) is a Ghabrivirales ERE, wherein the viral capsid protein in the cell is a Ghabrivirales capsid protein, and wherein the RNA molecule is encapsidated by the Ghabrivirales capsid protein.
  • ERE operably linked encapsidation recognition element
  • the viral capsid protein in the cell is a Ghabrivirales capsid protein
  • the RNA molecule is encapsidated by the Ghabrivirales capsid protein.
  • the cell is a bacterial cell, a plant cell, a fungal cell, an insect cell, or an invertebrate animal cell.
  • the cell further comprises: (i) a viral capsid protein (CP), (ii) an RNA-binding protein (RBP) that can bind to the RNA molecule, optionally wherein the RBP binds to an RNA effecter; (iii) an RNA cleavage agent that cleaves the RNA molecule; (iv) a second RNA-dependent RNA polymerase (RdRP) protein that recognizes an HRV 5’ or 3’ replication region and/or a subgenomic promoter in the RNA molecule (2 nd RdRP); (v) a viral movement protein (MP); (v) a heterologous RNA virus (HRV); or (vi) an hrvR
  • the expression system of embodiment 42 or 44 wherein the RdRP, 2 nd RdRP, or hrvRdRP protein or a polynucleotide encoding the RdRP, 2 nd RdRP, or hrvRdRP protein is (a) expressed by a recombinant DNA molecule in the cell; (b) provided exogenously to the cell; (c) expressed by a recombinant RNA molecule in the cell; or (d) expressed by a virus in the cell. [0192] 46. The expression system of any one of embodiments 37 to 45, wherein the cell is a plant cell. [0193] 47.
  • the expression system of embodiment 46 wherein the plant cell contains a Ghabrivirales which expresses the RdRP protein that recognizes the 5’ RNA replication element and the 3’ RNA replication element and/or wherein the plant cell contains an HRV that expresses the 2 nd RdRP or hrvRdRP protein.
  • a method of providing a synthetic Ghabrivirales satellite RNA to a plant comprising contacting the plant with the recombinant RNA molecule of any one of embodiments 1 to 28. [0196] 50.
  • contacting comprises spraying, dusting, injecting, or soaking the plant or a part thereof with the recombinant RNA molecule or the formulation.
  • 51. The method of embodiment 49 or 50, further comprising providing an hrvRdRP to the plant which recognizes the HRV 5’ or 3’ replication region and/or the subgenomic promoter, optionally wherein the hrvRdRP is provided by introducing a recombinant DNA or RNA encoding the hrvRdRP into the plant or a part thereof.
  • a method of establishing a synthetic Ghabrivirales satellite RNA in a plant cell comprising: providing to a plant cell the recombinant RNA molecule of any one of embodiments 1 to 28; wherein the plant cell comprises an RdRP protein that recognizes the 5’ RNA replication element and 3’ RNA replication element, wherein the RNA molecule is optionally comprises an ERE and is encapsidated by a capsid protein, whereby the RdRP protein catalyzes synthesis of the synthetic Ghabrivirales satellite RNA from the recombinant RNA molecule. [0199] 53.
  • the capsid protein comprises a Ghabrivirales capsid protein and the ERE is recognized by the Ghabrivirales capsid protein, optionally wherein the recombinant RNA molecule comprises an operably linked encapsidation recognition element (ERE) recognized by a viral capsid protein.
  • ERE operably linked encapsidation recognition element
  • any one of embodiments 52 to 55 further comprising providing an hrvRdRP to the plant which recognizes the HRV 5’ or 3’ replication region and/or the subgenomic promoter in the synthetic Ghabrivirales satellite RNA, optionally wherein the hrvRdRP is provided by introducing a recombinant DNA or RNA encoding the hrvRdRP into the plant or a part thereof. [0203] 57.
  • a method of obtaining a phenotypic change in a plant or plant cell comprising: providing to a plant or plant cell a recombinant RNA molecule of any one of embodiments 1 to 28, wherein the cargo RNA molecule comprises RNA that effects a phenotypic change in the plant or plant cell in comparison to a plant or plant cell lacking the recombinant RNA, wherein the plant or plant cell comprises an RdRP protein that recognizes the 5’ RNA replication element and 3’ RNA replication element and catalyzes synthesis of a synthetic Ghabrivirales RNA from the recombinant RNA molecule, and wherein the cargo RNA molecule effects the phenotypic change.
  • RNA that effects a phenotypic change in the plant or plant cell comprises at least one RNA selected from an siRNA or siRNA precursor, a miRNA or miRNA precursor, and a phased siRNA or phased siRNA precursor.
  • RNA that effects a phenotypic change in the plant or plant cell comprises a messenger RNA.
  • the messenger RNA comprises an RNA molecule absent in the genome of the plant or plant cell.
  • RNA that effects a phenotypic change in the plant or plant cell comprises an RNA for modifying the genome of the plant or plant cell.
  • RNA that effects a phenotypic change in the plant or plant cell comprises an RNA for modifying the transcriptome and/or epigenome of the plant or plant cell, optionally wherein the RNA for modifying the epigenome targets an endogenous plant gene for RNA-induced transcriptional silencing.
  • RNA for modifying the epigenome targets an endogenous plant gene for RNA-induced transcriptional silencing.
  • phenotypic change comprises an increase in the plant’s resistance to a pest or pathogen, optionally wherein the pest or pathogen is selected from the group comprising a bacterium, a virus other than a Ghabrivirales, a fungus, an oomycete, and an invertebrate.
  • the pest or pathogen is selected from the group comprising a bacterium, a virus other than a Ghabrivirales, a fungus, an oomycete, and an invertebrate.
  • the pathogen is a heterologous RNA virus (HRV), optionally wherein the HRV is a virus selected from the group consisting of an Alphaflexivirus, Betaflexivirus, Bromovirus, Celavirus, Closterovirus, Comovirus, Potexvirus, Potyvirus, Tobamovirus, Tombusvirus, Tospoviridae, Trivirinae, Tymovirus, Varicosavirus, and Secoviridae.
  • HRV heterologous RNA virus
  • the Bromovirus is a Cucumber Mosaic Virus, Spinach latent virus, Olive latent virus, or Brome mosaic virus;
  • the Closterovirus is a Citrus tristeza virus or Beet yellows virus;
  • the Comovirus is Cowpea mosaic virus, Apple latent spherical virus, or Soybean latent spherical virus;
  • the Potexvirus is Potato virus X or Citrus yellow vein clearing virus;
  • the Potyvirus is a Pepper mottle virus, Bean yellow mosaic virus, Barley stripe mosaic virus, Wheat stripe mosaic virus, Rice yellow mottle virus, Maize dwarf mosaic virus, zucchini yellow mosaic virus, watermelon mosaic virus, or sugarcane mosaic virus;
  • the Tobamovirus is a Tobacco mosaic virus, Tomato mosaic virus, Tomato brown rugose fruit virus, Turnip vein-clearing virus, or Pepper mild mottle virus;
  • the Tombusvirus is a Turn
  • phenotypic change comprises an increase in the plant’s resistance to stress
  • the stress comprises at least one abiotic stress comprising nutrient stress, light stress, water stress, heat stress, and/or cold stress, or Agent Ref: P14421WO00 - 70 - optionally wherein the stress comprises at least one biotic stress comprising crowding, shading, or allelopathy.
  • the recombinant RNA molecule is provided to the plant of plant cell in the form of an RNA, an encapsidated RNA, or a formulation thereof.
  • RNA comprises a synthetic Ghabrivirales satellite particle.
  • the providing comprises contacting the plant or plant cell with the RNA, encapsidated RNA, or formulation thereof, optionally wherein contacting comprises spraying, dusting, injecting, or soaking the plant or plant cell with the RNA, encapsidated RNA, or formulation thereof.
  • the recombinant RNA further comprises its reverse complementary RNA molecule.
  • any one of embodiments 57 to 70 further comprising providing an hrvRdRP to the plant which recognizes the HRV 5’ or 3’ replication region and/or the subgenomic promoter in the synthetic Ghabrivirales satellite RNA, optionally wherein the hrvRdRP is provided by introducing a recombinant DNA or RNA encoding the hrvRdRP into the plant or a part thereof. [0218] 72.
  • a method of manufacturing a synthetic Ghabrivirales satellite particle comprising combining the recombinant RNA molecule of any one of embodiments 1 to 28 with a viral capsid protein, wherein the recombinant RNA molecule comprises an encapsidation recognition element (ERE), and wherein the ERE provides for encapsidation of the RNA by the viral capsid protein. .
  • ERE encapsidation recognition element
  • the combining comprises (a) providing to a plant cell the recombinant RNA molecule, wherein the recombinant RNA molecule comprises an encapsidation recognition element (ERE), and wherein the plant cell comprises an RdRP protein that recognizes the 5’ RNA replication element and 3’ RNA replication element catalyzes synthesis of a synthetic Ghabrivirales satellite RNA from the recombinant RNA molecule and a viral capsid protein, wherein the ERE provides for encapsidation of the RNA by the viral capsid protein; and optionally (b) isolating the synthetic Ghabrivirales satellite particle from the plant cell, a plant comprising the plant cell, or from media in which the plant cell or the plant had been grown.
  • ERE encapsidation recognition element
  • [0221] 75 The method of embodiment 72, 73, or 74, further comprising the step of formulating the synthetic Ghabrivirales satellite particle wherein the formulating comprises combining the synthetic Ghabrivirales satellite particle with a carrier, an excipient, and/or an adjuvant.
  • 76 A plant propagule comprising the recombinant RNA molecule of any one of embodiments 1 to 28 and a Ghabrivirales RdRP, optionally wherein the plant propagule further comprises a Agent Ref: P14421WO00 - 71 - heterologous RNA virus RdRP which recognizes an HRV 5’ or 3’ replication region and/or the subgenomic promoter in the synthetic Ghabrivirales satellite RNA.
  • a heterologous RNA virus RdRP which recognizes an HRV 5’ or 3’ replication region and/or the subgenomic promoter in the synthetic Ghabrivirales satellite RNA
  • the heterologous RNA virus RdRP is an Alphaflexivirus, Betaflexivirus, Bromovirus
  • a plant comprising the recombinant RNA molecule of any one of embodiments 1 to 28 and a Ghabrivirales RdRP, optionally wherein the plant propagule further comprises a heterologous RNA virus RdRP which recognizes an HRV 5’ or 3’ replication region and/or the subgenomic promoter in the synthetic Ghabrivirales satellite RNA.
  • a heterologous RNA virus RdRP which recognizes an HRV 5’ or 3’ replication region and/or the subgenomic promoter in the synthetic Ghabrivirales satellite RNA.
  • 85. The plant of any one of embodiments 81 to 84, wherein the plant comprises a Ghabrivirales, and wherein the Ghabrivirales RdRP is provided to the plant cell by the Ghabrivirales, optionally wherein the Ghabrivirales RdRP comprises a protein having at least 85%, 90%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 491, 492, 493, 494, 495, or 496. [0232] 86.
  • RNA replication element has at least 85%, 90%, 95%, 97%, 98%, or 99% identity to the RNA encoded by SEQ Agent Ref: P14421WO00 - 72 - ID NO: 467, 468, 469, 470, 471, 472, 473, 474, 549 to 552, or 557 to 559, and/or wherein the 3’ RNA replication element has at least 85%, 90%, 95%, 97%, 98%, or 99% identity to the RNA encoded by SEQ ID NO: 475, 476, 477, 478, 479, 480, 481, 482, 553 to 556, or 560 to 562.
  • RNA virus RdRP which recognizes an HRV 5’ or 3’ replication region and/or the subgenomic promoter in the synthetic Ghabrivirales satellite RNA
  • the heterologous RNA virus RdRP is an Alphaflexivirus, Betaflexivirus, Bromovirus, Celavirus, Closterovirus, Comovirus, Potexvirus, Potyvirus, Tobamovirus, Tombusvirus, Tospoviridae, Trivirinae, Tymovirus, Varicosavirus, or Secoviridae RdRP or an RdRP set forth in Table 7. [0238] 92.
  • a Ghabrivirales satellite system that is self-replicating when introduced into a plant or plant cell, comprising: (1) a recombinant Ghabrivirales satellite RNA of any one of embodiments 1 to 28;and (2) an exogenous Ghabrivirales that is capable of replication in the plant or plant cells and that encodes the Ghabrivirales RdRP that recognizes the 5’ and 3’ RNA replication element sequences in the recombinant Ghabrivirales satellite RNA, optionally wherein the Ghabrivirales satellite system further comprises a heterologous RNA virus RdRP which recognizes an HRV 5’ or 3’ replication region and/or the subgenomic promoter in the synthetic Ghabrivirales satellite RNA. [0239] 93.
  • the self-replicating Ghabrivirales satellite system of embodiment 92 wherein the exogenous Ghabrivirales is endemic or native to a different species, variety, or germplasm of plant.
  • the Ghabrivirales satellite system further comprises a heterologous RNA virus (HRV) RdRP which recognizes an HRV 5’ or 3’ replication region and/or the subgenomic promoter in the synthetic Ghabrivirales satellite RNA, and the heterologous RNA virus RdRP is optionally an Alphaflexivirus, Betaflexivirus, Bromovirus, Celavirus, Closterovirus, Comovirus, Potexvirus, Potyvirus, Tobamovirus, Tombusvirus, Tospoviridae, Trivirinae, Tymovirus, Varicosavirus, or Secoviridae RdRP or an RdRP set forth in Table 7.
  • HRV heterologous RNA virus
  • a recombinant DNA molecule comprising a first promoter which is operably linked to DNA encoding the RNA molecule of any one of embodiments 1-28.
  • 96. A cell comprising the recombinant DNA molecule of embodiment 95, wherein the cell is a bacterial cell, a fungal cell, a plant cell, an insect cell, or an invertebrate animal cell.
  • 97. A vector for bacterially mediated plant transformation, comprising the recombinant DNA molecule of embodiment 95.
  • the vector of embodiment 97 wherein the bacterium that mediates the plant transformation is an Agrobacterium sp., a Sinorhizobium sp., a Mesorhizobium sp., a Bradyrhizobium sp., Rhizobium sp., or an Ensifer sp. and the vector is adapted for transformation with the bacterium. [0245] 99.
  • the vector of embodiment 97 or 98, wherein the bacterium that mediates the plant transformation is an Agrobacterium sp., and wherein the vector further comprises T-DNAs flanking the DNA molecule encoding the recombinant RNA molecule. [0246] 100.
  • An expression system comprising: (a) the recombinant DNA molecule of embodiment 95; and(b) a cell containing the recombinant DNA molecule and an RdRP protein that recognizes the 5’ and 3’ RNA replication elements encoded by the DNA molecule.
  • the recombinant DNA molecule further comprises at least one additional element comprising: (i) DNA encoding at least one RNA encoding a viral movement protein (MP); (ii) DNA encoding at least one tRNA-like molecule; (iii) DNA encoding an encapsidation recognition element (ERE); (iv) DNA encoding an RNA comprising, from 5’ to 3’ and operably linked, a 5’ RNA replication element recognized by a Ghabrivirales RNA-dependent RNA polymerase (RdRP), the RNA of (i) and optionally an operably linked RNA of (ii) and/or (iii), and a 3’ RNA replication element; (v) DNA encoding an RNA promoter; (vi) DNA encoding an RNA- dependent RNA polymerase (RdRP) that recognizes an HRV 5’ or 3’ replication region and/or a subgenomic promoter; and/or (vii)
  • 103 The expression system of embodiments 101 or 102, wherein the cell is a bacterial cell, a plant cell, a fungal cell, an insect cell, a non-human animal cell, a vertebrate animal cell, or an invertebrate animal cell, optionally wherein the bacterial cell is an Agrobacterium sp., a Sinorhizobium sp., a Mesorhizobium sp., Bradyrhizobium sp., Rhizobium sp., or an Ensifer sp. cell. [0250] 104.
  • RNA-binding protein that can bind to the RNA molecule encoded by the DNA molecule, optionally wherein the RBP binds to an RNA effecter;
  • RBP RNA-binding protein
  • an RNA cleavage agent that cleaves the RNA molecule; and/or
  • an RNA promoter dependent RNA polymerase (RdRP) protein that recognizes an HRV 5’ or 3’ replication region and/or a subgenomic promoter recognizes an RNA promoter in the RNA molecule.
  • invention 104 or 105 wherein: (i) the capsid protein, viral movement protein (MP), RdRP protein, and/or the RdRP protein that recognizes an HRV 5’ or 3’ Agent Ref: P14421WO00 - 74 - replication region and/or a subgenomic promoter is heterologous to the cell and/or (ii) wherein the RdRP protein or a polynucleotide encoding the RdRP protein is provided exogenously to the cell. [0253] 107. The expression system of any one of embodiments 101 to 106, wherein the cell is a plant cell. [0254] 108.
  • the expression system of 107 wherein the plant cell contains a Ghabrivirales which expresses the RdRP protein that recognizes the 5’ RNA replication element and the 3’ RNA replication element.
  • 109 The expression system of embodiment 108, wherein the Ghabrivirales occurs naturally in the plant cell.
  • 110 The expression system of any one of embodiments 101 to 109, wherein the recombinant DNA molecule further comprises at least one RNA encoding a viral MP, a tRNA-like molecule from an Arabidopsis FT mRNA, and an encapsidation recognition element comprising a TMV- OAS.
  • An agricultural formulation comprising the expression system of any one of embodiments 101 to 110.
  • a method of producing an exogenous polypeptide in a plant or plant cell comprising: providing a plant or plant cell comprising the recombinant RNA molecule of any one of embodiments 1 to 28 or the recombinant DNA molecule of embodiment 95, wherein the cargo RNA molecule encoded by the RNA or DNA molecule comprises a translatable messenger RNA encoding the exogenous polypeptide, wherein the plant or plant cell comprises an RdRP protein that recognizes the 5’ RNA replication element and the 3’ RNA replication element of the recombinant RNA and that catalyzes synthesis of a synthetic Ghabrivirales satellite RNA from the recombinant RNA molecule, and wherein the exogenous polypeptide is translated from the translatable messenger; optionally wherein the plant or plant cell further comprises a heterologous RNA virus (HRV) RNA promoter dependent RNA polymerase (hrvRdRP) protein that recognizes an HRV 5’ or 3’ replication region and/or a sub
  • the method of any one of embodiments 115 to 118 further comprising a first step of providing a population of plants comprising the plant cells comprising the RdRP protein or the recombinant polynucleotide encoding the RdRP; and then providing the recombinant RNA molecule to the plants comprising the plant cells.
  • 120 The method of any one of embodiments 115 to 119, wherein the recombinant RNA molecule has been produced in a fermentation system.
  • 121 The method of any one of embodiments 115 to 120, further comprising the step of determining if the plant cell comprises a Ghabrivirales which can provide the RdRP.
  • 122 The method of any one of embodiments 115 to 120, further comprising the step of determining if the plant cell comprises a Ghabrivirales which can provide the RdRP.
  • RdRP Ghabrivirales RNA- dependent RNA polymerase
  • the method of embodiment 122, 123, or 124, wherein the mixed population of plant cells comprise plant cells comprising the Ghabrivirales RdRP and plant cells lacking the Ghabrivirales RdRP.
  • 125 The method of embodiment 122, 123, or 124, wherein the mixed population of plant cells comprise plant cells comprising the Ghabrivirales RdRP.
  • 126 The method of any one of embodiments 122 to 125, wherein the mixed population of plant cells is screened or selected for the presence of the plant cell comprising the recombinant RNA molecule prior to isolating the plant propagule.
  • 127 is screened or selected for the presence of the plant cell comprising the recombinant RNA molecule prior to isolating the plant propagule.
  • the method of any one of embodiments 122 to 126, wherein the isolation comprises selecting for the plant cell comprising the recombinant RNA molecule prior to isolating the plant propagule.
  • Agent Ref P14421WO00 - 76 - [0276] 130.
  • any one of embodiments 122 to 132 wherein the plant propagule comprising the recombinant RNA molecule is isolated by detecting the RNA molecule in one or more plant cells comprising the recombinant RNA molecule and separating the one or more plant cells comprising the recombinant RNA molecule from the plant cells lacking the recombinant DNA molecule.
  • the plant propagule is a mosaic comprising both plant cells comprising the recombinant RNA molecule and plant cells lacking the recombinant RNA molecule.
  • any one of embodiments 122 to 140 wherein the isolated propagule comprises callus and the method further comprises regenerating a plant, seedling, root, stem, leaf, shoot, tuber, rhizome, stolon, bulb, or explant comprising the recombinant RNA from said callus.
  • a method of providing a synthetic Ghabrivirales satellite RNA to a plant comprising: grafting a scion onto a rootstock comprising recombinant RNA molecule of any one of embodiments 1 to 28, wherein at least one cell of the rootstock and/or the scion comprises the Ghabrivirales RdRP.
  • a method of providing a synthetic Ghabrivirales satellite RNA to a plant comprising: grafting a scion onto a rootstock comprising recombinant RNA molecule of any one of embodiments 1 to 28, wherein at least one cell of the rootstock and/or the scion comprises the Ghabrivirales RdRP.
  • 145 The method of embodiment 144, wherein the scion lacks the recombinant RNA molecule prior to grafting.
  • the rootstock comprises the Ghabrivirales RdRP.
  • a method for producing a plant that transmits a recombinant RNA molecule to progeny plants or seed comprising isolating an F 1 progeny plant or seed comprising at least one cell comprising a Ghabrivirales RNA-dependent RNA polymerase (RdRP) and the recombinant RNA molecule of any one of embodiments 1 to 28 from a population of F 1 plants or seed obtained from a parent plant comprising the recombinant RNA molecule.
  • RdRP RNA-dependent RNA polymerase
  • the method of embodiment 151, wherein the F 1 progeny plant or seed comprising the cell is isolated by screening the population of F 1 plants or seed obtained from a parent plant for the presence of the recombinant RNA molecule and propagating the F 1 progeny plant or seed comprising the recombinant RNA molecule.
  • the recombinant RNA molecule encodes a selectable marker and the F 1 progeny plant or seed comprising the recombinant RNA molecule is isolated by selecting the F 1 progeny plant or seed comprising the recombinant RNA molecule for presence of the selectable marker.
  • any one of embodiments 151 to 160 wherein the parent plant or one or more parts thereof are screened for systemic presence of the recombinant RNA molecule prior to isolating the F 1 progeny plants, optionally wherein the part comprises floral tissue or male or female reproductive tissue.
  • 162 The method of any one of embodiments 151 to 161, wherein the pericarp of the parent plant is screened or selected for presence of the recombinant RNA molecule.
  • 163 The method of any one of embodiments 151 to 162, wherein F 1 seeds are obtained from a parent plant selected for presence of the recombinant RNA molecule in pericarp tissue.
  • any one of embodiments 151 to 165 further comprising introducing the recombinant RNA molecule or a polynucleotide encoding the recombinant RNA molecule into a plant cell and obtaining the parent plant comprising the recombinant RNA molecule from the plant cell.
  • 167 The method of any one of embodiments 151 to 166, wherein the propagule, plant, plant part, scion, and/or rootstock comprises a heterologous viral coat protein which can encapsidate the recombinant RNA molecule and/or comprises the recombinant RNA molecule encapsidated by a heterologous viral coat protein.
  • the propagule, plant, plant part, scion, and/or rootstock comprises a heterologous viral coat protein which can encapsidate the recombinant RNA molecule and/or comprises the recombinant RNA molecule encapsidated by a heterologous viral coat protein.
  • a method of barcoding a plant, plant cell, progeny thereof, or part thereof comprising providing to the plant or plant cell the recombinant RNA molecule of any one of embodiments 1 to 28, wherein the cargo RNA of the recombinant RNA molecule comprises a barcode RNA molecule, and wherein the plant or plant cell comprises a Ghabrivirales RNA-dependent RNA polymerase (RdRP).
  • RdRP Ghabrivirales RNA-dependent RNA polymerase
  • the barcode RNA molecule comprises a sequence that is not present in the genome and/or transcriptome of a wild-type plant of the same species, a pathogen thereof, or a symbiont thereof. [0317] 171. The method of any one of embodiments 168 to 170, wherein the barcode RNA molecule comprises a random sequence. [0318] 172. The method of any one of embodiments 168 to 171, wherein the barcode RNA molecule comprises a forward primer binding site and a reverse primer binding site that is not present in the genome and/or transcriptome of a wild-type plant of the same species, a pathogen thereof, or a symbiont thereof. [0319] 173.
  • any one of embodiments 168 to 172 wherein the barcode RNA molecule is: (i) up to about 4.6 kilobases (kb) in length and the 5’ RRE and 3’ RRE are obtained or derived from a Totivirus; or (ii) up to about 12.5 kilobases (kb) in length and the RRE and 3’ RRE are obtained or derived from a Chrysovirus. [0320] 174.
  • RNA molecule comprises a non-protein coding sequence.
  • 176 The method of any one of embodiments 168 to 175, wherein the plant transmits the recombinant RNA molecule to progeny.
  • 177 The method of any one of embodiments 168 to 176, wherein the plant, plant cell, progeny thereof, or part thereof lacks DNA that encodes the recombinant RNA molecule.
  • 178 The method of any one of embodiments 168 to 174, wherein the barcode RNA molecule comprises a non-protein coding sequence.
  • any one of embodiments 168 to 177 further comprising isolating an F 1 progeny plant or seed comprising at least one cell comprising the Ghabrivirales RdRP and the recombinant RNA molecule.
  • 179 The method of embodiment 178, wherein the F 1 progeny plant or seed is obtained from the plant used as a pollen recipient.
  • 180 The method of embodiment 178, wherein the F 1 progeny plant or seed is obtained from the plant used as a pollen donor.
  • 181 The method of embodiment 178, wherein the F 1 progeny plant or seed is obtained by selfing the plant. [0328] 182.
  • a method of identifying a barcoded plant, plant part, or plant cell comprising screening for the presence of a barcode RNA molecule in the plant, plant part, or plant cell, wherein the plant, plant part, or plant cell comprises the recombinant RNA molecule of any one of embodiments 1 to 28, wherein the cargo RNA of the recombinant RNA molecule comprises the barcode RNA molecule.
  • RNA replication element DNA Coding Sequences SEQ Description 1 ID Dot-Bracket structure of encoded RNA NO 2 )) ... )) (( )) Agent Ref: P14421WO00 - 81 - SEQ Description 1 ID Dot-Bracket structure of encoded RNA NO 2 )) )) )) .) ... ... (( ). Agent Ref: P14421WO00 - 82 - SEQ Description 1 ID Dot-Bracket structure of encoded RNA NO 2 .( ... ... ).
  • RNA equivalents of the DNA sequences are also contemplated and can be obtained from the DNA sequences provided Table 2.
  • Viral capsid protein and origin of assembly sequences Origin of assembly Virus 1 sequence 2 Capsid Protein Agent Ref: P14421WO00 - 83 - Origin of assembly Virus 1 sequence 2 Capsid Protein database accession number for entries in the world wide web internet database “ncbi[dot]nlm[dot]nih.gov/nuccore.” 2 RNA equivalents of the DNA sequences are also contemplated and can be obtained from the DNA sequences provided.
  • Agent Ref P14421WO00 - 84 - Table 3. Movement proteins. Movement protein Virus (MP) Agent Ref: P14421WO00 - 85 - Movement protein Virus (MP) “ncbi[dot]nlm[dot]nih.gov/nuccore.” Table 4.
  • tRNA-like sequences Name 1 of Arabidopsis gene containing tRNA-like s equence that can confer cell-to-cell mobility Sequence2 Agent Ref: P14421WO00 - 86 - Name 1 of Arabidopsis gene containing tRNA-like S 2 s equence that can confer cell-to-cell mobility equence 1
  • IRES sequences IRES source NCBI Accession ID 1 Sequence 2 Agent Ref: P14421WO00 - 87 - IRES source NCBI Accession ID 1 Sequence 2 Solenopsis invicta virus NC_006559 SEQ ID NO: 130 T i i C 003 83 SE ID O 131 y Information database accession number for entries in the world wide web internet database “ncbi[dot]nlm[dot]nih.gov/nuccore.” 2 RNA equivalents of the DNA sequences are also contemplated and can be obtained from the DNA sequences provided. Table 6.
  • Intron sequences Intron number Sequence Agent Ref: P14421WO00 - 88 - Table 7: Heterologous RNA viruses (HRVs) and Target Viral Pathogens RDRP or RDRP or 5’ RR 3’ RR Replicase Replicase HRV name or Target NCBI Sequence 2 Sequence 2 NT AA Agent Ref: P14421WO00 - 89 - RDRP or RDRP or 5’ RR 3’ RR Replicase Replicase HRV name or Target NCBI Sequence 2 Sequence 2 NT AA Agent Ref: P14421WO00 - 90 - RDRP or RDRP or 5’ RR 3’ RR Replicase Replicase HRV name or Target NCBI Sequence 2 Sequence 2 NT AA ase “ncbi[dot]nlm[dot]nih.gov/nuccore.” 2 RNA equivalents of the DNA sequences are also contemplated and can be obtained from the DNA sequences provided.
  • e oter descriptors refer to the National Center for Biotechnology Information database accession number for entries in the world wide web internet database “ncbi[dot]nlm[dot]nih.gov/nuccore.” Table 9. Insecticidal or Insect Inhibitory and Nematocidal or Nematode Inhibitory Cargo Molecules NT AA NCBI Se uence Se uence Exam les Agent Ref: P14421WO00 - 92 - NT AA NCBI Sequence Sequence Examples Accession (SEQ ID (SEQ ID Cargo of Target Agent Ref: P14421WO00 - 93 - NT AA NCBI Sequence Sequence Examples Accession (SEQ ID (SEQ ID Cargo of Target Agent Ref: P14421WO00 - 94 - NT AA NCBI Sequence Sequence Examples Accession (SEQ ID (SEQ ID Cargo of Target n n er for entries in the world wide web internet database “ncbi[d
  • RNAi Targets Cargo (RNAi Examples of Target Gene) NCBI Accession ID 1 Sequence Target pathogen target crop Bc Chitin SEQ ID NO: bell pepper a a a ze ze ze Agent Ref: P14421WO00 - 96 - Cargo (RNAi Examples of Target Gene) NCBI Accession ID 1 Sequence Target pathogen target crop SEQ ID NO: Fusarium wheat soybean a a a er for entries in the world wide web internet database “ncbi[dot]nlm[dot]nih.gov/nuccore.” Agent Ref: P14421WO00 - 97 - Table 11.
  • RNA Aptamers RNA aptamer Sequence 1 Pepper SEQ ID NO: 439 Broccoli SEQ ID NO: 440 plated and can be obtained from the RNA Table 16 Subgenomic promoters Subgenomic promoter Sequence 1 Pea early-browning virus CP SEQ ID NO: 453 ed and can be obtained from the DNA sequences provided. *See Goulden et al. (1990) Nucleic Acids Res., 18:4507 – 4512, DOI:10.1093/NAR/18.15.4507. Table 17.
  • RNA replication RdRP DNA RdRP Protein l t C di S S Agent Ref: P14421WO00 - 106 - 5’ RNA replication RdRP DNA RdRP Protein element sequence Coding Sequence Sequence GV Genome or source 1 SEQ ID NO: SEQ ID NO: SEQ ID NO: Agent Ref: P14421WO00 - 107 - 5’ RNA replication RdRP DNA RdRP Protein element sequence Coding Sequence Sequence GV Genome or source 1 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: Agent Ref: P14421WO00 - 108 - 5’ RNA replication RdRP DNA RdRP Protein element sequence Coding Sequence Sequence GV Genome or source 1 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: base accession number for entries in the world wide web internet database “ncbi[dot]nlm[dot]nih.gov/nuccore.” Table 18.
  • a Ghabrivirales satellite (COMSAT) that carries an antiviral inhibitory RNA (RNAi) cargo is generally provided in 5’ to 3’ orientation as follows: (i) 5’ RNA replication element from the Ghabrivirales virus; (ii) a heterologous RNA virus (HRV) 5’ replication region (HRV 5’RR); (iii) an antiviral RNA molecule which induces an RNAi response; (iv) the heterologous RNA virus (HRV) 3’ RNA replication region (HRV 3’RR); and (v) and the 3’ RNA replication element from the Ghabrivirales virus.
  • COMSATs comprising Ghabrivirales 5’ and 3’ RNA replication elements from Brassica campestris chrysovirus 1 flanking a TMV amplicon containing a cargo RNA containing a Pepper mild mottle virus (PMMoV) RNAi inducing sequence (e.g., a PMMoV sequence which can form a dsRNA) are provided as SEQ ID NO: 497, 498, and 499.
  • the elements of the satellite are set forth in Tables 19 to 21. Table 19. Genetic element Nucleotide position in SEQ ID NO: 497 Agent Ref: P14421WO00 - 111 - Table 20.
  • a Ghabrivirales satellite (COMSAT) with an imbedded HRV amplicon that carries an antiviral protein cargo is generally provided in 5’ to 3’ orientation as follows: (i) 5’ RNA replication element from the Ghabrivirales virus; (ii) a heterologous RNA virus (HRV) 5’ replication region (HRV 5’RR); (iii) an antiviral protein cargo; (iv) the heterologous RNA virus (HRV) 3’ RNA replication region (HRV 3’RR); and (v) the 3’ RNA replication element from the Ghabrivirales virus.
  • COMSAT Ghabrivirales satellite
  • COMSATs comprising Ghabrivirales 5’ and 3’ RNA replication elements from Brassica campestris chrysovirus 1 flanking a TMV amplicon containing an antiviral cargo protein containing the coding sequence of the N gene of tobacco are provided as SEQ ID NO: 500, 501, and 502.
  • the elements of the satellite are set forth in Tables 22, 23, and 24.
  • a Ghabrivirales satellite (COMSAT) that carries an antiviral RNAi cargo is generally provided in 5’ to 3’ orientation as follows: (i) 5’ RNA replication element from the Ghabrivirales virus; (ii) an antiviral RNA molecule which induces an RNAi response; and (iii) the 3’ RNA replication element from the Ghabrivirales virus.
  • COMSATs comprising Ghabrivirales 5’ and 3’ RNA replication elements from Brassica campestris chrysovirus 1 flanking a cargo RNA containing a Pepper mild mottle virus (PMMoV) RNAi inducing sequence (e.g., a PMMoV sequence which can form a dsRNA) are provided as SEQ ID NO: 503, 504, and 505.
  • PMMoV Pepper mild mottle virus
  • a Ghabrivirales satellite (COMSAT) that carries an antiviral protein cargo is generally provided in 5’ to 3’ orientation as follows: (i) 5’ RNA replication element from the Ghabrivirales virus; (ii) an antiviral protein cargo; and (iii) the 3’ RNA replication element from the Ghabrivirales virus.
  • COMSAT Ghabrivirales satellite
  • COMSATs comprising Ghabrivirales 5’ and 3’ RNA replication elements flanking an antiviral protein cargo containing the coding sequence of the N gene of tobacco is provided as SEQ ID NO: 506, 507, and 508.
  • the elements of the satellite are set forth in Tables 28, 29, and 30. Table 28. Genetic element Nucleotide position in SEQ ID NO: 506 Ghabrivirales 5’ RNA replication element 1-101 a e . Genetic element Nucleotide position in SEQ ID NO: 507 ’ Table 30.
  • Antiviral COMSATs e.g., of Examples 1 to 4
  • control RNAs e.g., RNAs lacking the antiviral cargo
  • target host plants e.g., Arabidopsis thaliana or other brassicaceous plants (family Brassicaceae), maize (Zea mays) or other family Poaceae plants, or Nicotiana benthamiana, Petunia, pepper, tomato or other family Solanaceae plants
  • micro-bombardment biolistic” delivery
  • bacterially mediated e.g., by Agrobacterium
  • Host plants lacking the appropriate endogenous Ghabrivirales virus can otherwise be provided with the appropriate Ghabrivirales RdRP, e.g., through transient or transgenic expression.
  • Plants, e.g., pepper and tomato plants are grown in growth chambers using a long-day photoperiod (16 h light/8 h dark) with light intensity 100-150 micromole, until seedlings are about 2 weeks old.
  • Antiviral COMSATs are prepared either as in vitro transcribed (IVT) products (capped or uncapped), or as Agrobacterium binary vectors.
  • the IVT product is coated onto the surface of gold nanoparticles which are precipitated on inner surface of bullet tubes; these are accelerated at the abaxial surface of the seedlings’ leaves with helium pressure from 100 to 180 psi.
  • COMSAT-carrying clones in binary vector are transformed into Agrobacterium GV 2260, and the transformed Agrobacterium is selected by growing at 28°C under appropriate antibiotic selection. The positive transformants are verified by PCR and then grown at 28°C overnight in liquid LB medium with appropriate antibiotics plus rifamycin to maintain the Agrobacterium and prevent contamination.
  • Agrobacterium cells are collected by centrifugation and resuspended in MMA buffer at an OD600 of 0.2, incubated at 28°C on a shaker for 2 hours, and then infiltrated into the abaxial sides of the leaves.
  • Control plants are treated in the same way but with non-COMSAT control IVT transcripts, or empty buffers.
  • tissues from systemic leaves (leaves distal to the treated leaves) are collected and subjected to RNA extraction, followed by cDNA syntheses.
  • COMSAT titers are monitored in systemic tissue by qRT-PCR with COMSAT-specific primers.
  • the efficacy of an antiviral COMSAT can be tested by challenging the COMSAT-treated plants with the viral pathogen of interest (e.g., cucumber mosaic virus, CMV, or tobacco mosaic virus, TMV), for example, by mechanical infection.
  • the viral pathogen of interest e.g., cucumber mosaic virus, CMV, or tobacco mosaic virus, TMV
  • Infectious preparations of such acute viral pathogens are Agent Ref: P14421WO00 - 116 - prepared in planta (e.g., in Nicotiana benthamiana) or in the form of GFP-fused infectious clones in a binary vector or T7-based vector.
  • Suitable viral inoculums can be prepared as infectious sap extracted from an infected plant, as an Agrobacterium-based inoculum, or as IVT products.
  • the inoculum is introduced into leaves of the COMSAT-treated plants by rub-inoculation (for infectious sap or IVT product), by agroinfiltration (for GFP-fused infectious clone in binary vector), or by micro-bombardment (for IVT product or plasmid of infectious clone in binary vector).
  • the infectious sap or IVT products are respectively diluted 3 times or to 50ng/microliter in phosphate buffer 0.05M, pH 7.4 and dropped onto leaf adaxial surfaces which is pre-dusted with an abrasive such as carborundum or bentonite.
  • the inoculum is gently spread on the abrasive-dusted leaf surface by gloved fingers or cotton buds; after 30 seconds the inoculated leaf is washed with water.
  • Micro-bombardment of infectious clones is conducted following previously described methods (see, e.g., delivery techniques described in PCT/US22/78963; see also Bio-Rad Tech Note 2531 “Inoculation of Viral RNA and cDNA to Potato and Tobacco Plants Using the HeliosTM Gene Gun”).
  • Infiltration with an Agrobacterium-based viral pathogen inoculum is performed using methods similar to that used for COMSAT infiltration, but the Agrobacterium suspension is diluted at OD600 of 0.1.
  • Symptoms and titre of the acute viral pathogens are monitored in the plants, typically over time to confirm progress or decline of viral infection. Effectiveness of antiviral COMSATs is evaluated by comparing relative titres of systemic infected viruses in antiviral COMSAT-treated plants and the control plants. Tissues from inoculated leaves and systemic leaves (distal to the inoculated leaves) are collected, total RNA is extracted, optionally followed by cDNA synthesis. The viral titre can be quantitatively measured by qRT-PCR with virus-specific primers.
  • Viral titre or presence can be measured by immunoassay methods; for example, Tobacco mosaic virus (TMV) is routinely detected in plants using a commercially available strip assay (Agdia ImmunoStrip ® for TMV Agdia, Inc., Elkhart, IN, USA).
  • TMV Tobacco mosaic virus
  • strip assay Agdia ImmunoStrip ® for TMV Agdia, Inc., Elkhart, IN, USA.
  • viral titre is qualitatively evaluated by viral disease symptoms, or by proxy measurement (e.g., measuring GFP expressed by GFP-fused viral constructs).
  • qRT-PCR measurements are normalized to endogenous reference gene controls (e.g., actin, tubulin, ubiquitin-3, GADPH, or translation elongation factor EF1a, used individually or preferably in multiples, e.g., at least 3 reference genes or at least three deltaCt values).
  • endogenous reference gene controls e.g., actin, tubulin, ubiquitin-3, GADPH, or translation elongation factor EF1a, used individually or preferably in multiples, e.g., at least 3 reference genes or at least three deltaCt values.
  • the viral titre is compared to that of controls.
  • a Ghabrivirales satellite (COMSAT) that carries an insecticidal RNAi cargo is generally provided in 5’ to 3’ orientation as follows: (i) 5’ RNA replication element from the Ghabrivirales virus; (ii) an insecticidal RNA molecule which induces an RNAi response; and (iii) the 3’ RNA replication element from the Ghabrivirales virus.
  • COMSATs comprising Ghabrivirales 5’ and 3’ RNA replication elements from Brassica campestris chrysovirus 1 flanking a cargo RNA containing an RNAi inducing sequence that targets Agent Ref: P14421WO00 - 117 - Colorado potato beetle is provided as SEQ ID NO: 509, 510, and 511.
  • the elements of the satellite are set forth in Table 31, 32, and 33.
  • a Ghabrivirales satellite (COMSAT) that carries an insecticidal protein cargo is generally provided in 5’ to 3’ orientation as follows: (i) 5’ RNA replication element from the Ghabrivirales virus; (ii) an insecticidal protein cargo; and (iii) the 3’ RNA replication element from the Ghabrivirales virus.
  • COMSAT Ghabrivirales satellite
  • COMSATs comprising Ghabrivirales 5’ and 3’ RNA replication elements flanking an insecticidal cargo protein containing the coding sequence of the Vip3Aa gene is provided as SEQ ID NO: 512, 513, and 514.
  • the elements of the satellites are set forth in Table 34, 35, and 36.
  • COMSATS of Tables 30 to 35 are established in host plants essentially as set forth in Example 5. Host plants lacking the appropriate endogenous Ghabrivirales virus can otherwise be provided with the appropriate Ghabrivirales RdRP, e.g., through transient or transgenic expression.
  • an insecticidal or insect inhibitory COMSAT can be tested by challenging the COMSAT and control treated plants with the insect of interest (e.g., Colorado Potato Beetle for plants treated with the Table 31-33 COMSAT or Fall Armyworm, Corn Earworm, or Black Cutworm for plants treated with the Table 34-36 COMSAT.
  • insect of interest e.g., Colorado Potato Beetle for plants treated with the Table 31-33 COMSAT or Fall Armyworm, Corn Earworm, or Black Cutworm for plants treated with the Table 34-36 COMSAT.
  • a Ghabrivirales satellite (COMSAT) that carries an antifungal RNAi cargo is generally provided in 5’ to 3’ orientation as follows: (i) 5’ RNA replication element from the Ghabrivirales virus; (ii) an antifungal RNA molecule which induces an RNAi response; and (iii) the 3’ RNA replication element from the Ghabrivirales virus.
  • COMSATs comprising Ghabrivirales 5’ and 3’ RNA replication elements from Brassica campestris chrysovirus 1 flanking a cargo RNA containing an RNAi inducing sequence that targets a DCL gene of Botrytis cinerea are provided as SEQ ID NO: 515, 516, and 517.
  • the elements of the satellites are set forth in Table 37, 38, and 39.
  • COMSATs comprising Ghabrivirales 5’ and 3’ RNA replication elements flanking an antifungal cargo protein containing the coding sequence of the CaAMP1 gene is provided as SEQ ID NO: 518, 519, and 529.
  • the elements of the satellite are set forth in Table 40, 41, and 42.
  • the plasmid is used as a template for a PCR reaction to amplify the expression cassette with a T7 promoter. This PCR product is used as the template for an in vitro transcription reaction using a MEGAscriptTM T7 Transcription Kit from Thermo Fisher Scientific.
  • RNA or protein cargo is then transformed into leaves of the target crop or host plant using gold nanoparticles from Bio-Rad according to the guidelines specified in the Helios gene gun manual or via Agrobacterium mediated transformation.
  • Host plants lacking the appropriate endogenous Ghabrivirales virus can otherwise be provided with the appropriate Ghabrivirales RdRP, e.g., through transient or transgenic expression.
  • Replication and persistence of delivered RNA or expression of protein in local leaves can be determined by RT-PCR from local tissues at 2-weeks post transformation. Systemic movement of RNA/protein is determined by qRT-PCR/ELISA in the sample collected from distal untreated leaf samples after 1-, 2-, and 3-months post transformation.
  • RNA replication element sequence from Ghabrivirales is cloned into a pUC19 plasmid backbone and transformed into E. coli DH5a (New England Biolabs; C2987I). The plasmid is extracted, and PCR is used to add a T7 promotor sequence.
  • RNA quantity is measured using Agent Ref: P14421WO00 - 122 - a Nanodrop spectrophotometer (Thermo Fisher Scientific; 840274200) and the intactness is quality controlled through gel electrophoresis.
  • the synthesized and quality-controlled RNA molecule is precipitated onto gold nanoparticles and fired into 4-week-old Solanum lycopersicum ‘Early Girl’ leaves using a gene gun.
  • Alternative host plants include, e.g., Arabidopsis thaliana or other brassicaceous plants (family Brassicaceae), maize (Zea mays) or other family Poaceae plants, or Nicotiana benthamiana, Petunia, pepper, tomato or other family Solanaceae plants).
  • Host plants lacking the appropriate endogenous Ghabrivirales virus can otherwise be provided with the appropriate Ghabrivirales RdRP, e.g., through transient or transgenic expression.
  • the following leaf tissue samples are collected at 2-week and 4-week timepoints: fired leaf # 1; fired leaf # 2; nearest adjacent leaflet; opposite leaf, terminal leaflet; and apical leaf growth.
  • RNA is extracted with the MagMax Plant RNA Isolation kit (Thermo Fisher Scientific; A33899) and quality controlled through Nanodrop spectrophotometer (Thermo Fisher Scientific; 840274200) and RNA ScreenTape Analysis (Agilent; 5067-5576). Quality controlled RNA is used for cDNA synthesis and qRT-PCR is used to amplify the Dicer-like 1 and Dicer- like 2 fragments and reference gene fragment RPL2. Delta-delta Ct analysis is applied to determine LogFold change of the Ghabrivirales COMSAT in the tissues compared to non-Ghabrivirales COMSAT containing Solanum lycopersicum ‘Early Girl’ plant tissues.
  • Example 9 Example 9
  • Ghabrivirales COMSAT with cDNA-based cargo expression amplification A DNA molecule encoding an RNA molecule containing a 5' RNA replication element sequence from Ghabrivirales; Cricket paralysis virus IRES; Avian leukosis virus reverse transcriptase; tRNA primer binding site (tRNA-trp); CaMV 35s promoter; a protein cargo (Superfolder GFP); ZmHSP101 IRES; Tobacco mosaic virus movement protein; and a 3' RNA replication element site from Ghabrivirales is cloned into a pUC19 plasmid backbone and is transformed into E. coli DH5a (New England Biolabs; C2987I).
  • RNA quantity is measured using a Nanodrop spectrophotometer (Thermo Fisher Scientific; 840274200) and the intactness is quality controlled through gel electrophoresis.
  • the synthesized and quality-controlled RNA molecule is precipitated onto gold nanoparticles and fired into the 4-week-old Solanum lycopersicum ‘Early Girl’ leaves using a gene gun.
  • Alternative host plants include, e.g., Arabidopsis thaliana or other brassicaceous plants (family Brassicaceae), maize (Zea mays) or other family Poaceae plants, or Nicotiana benthamiana, Petunia, pepper, tomato or other family Solanaceae plants).
  • Host plants lacking the appropriate endogenous Ghabrivirales virus can otherwise be provided with the appropriate Ghabrivirales RdRP, e.g., through transient or transgenic expression.
  • the following leaf tissue samples are collected at 2-week and 4-week timepoints: fired leaf # 1; fired leaf # 2; nearest adjacent leaflet; opposite leaf, terminal leaflet; and apical leaf growth.
  • Protein (GFP) expression is quantified.
  • Total protein is extracted from collected tissue samples using the Pierce Plant Total Protein Extraction Kit (Thermo Fisher Scientific; A44056) according to manufacturer specifications, and quality controlled by Agent Ref: P14421WO00 - 123 - Nanodrop spectrophotometry paired with SDS-PAGE to verify extraction yield and integrity.
  • GFP expression is quantified relative to other protein expressing COMSAT designs as well as non-transfected tissue using the Abcam GFP ELISA Kit (ab171581) according to manufacturer specifications.
  • Example 10 Example 10.
  • Zea mays chrysovirus 1 nucleotide and amino acid sequence divergence This example illustrates nucleotide and amino acid sequence divergence among various isolates of a maize chrysovirus, Zea mays chrysovirus 1. Although there is substantial sequence divergence among these genomes, the RdRP sequences analyzed had the same length and shared greater than 90% sequence similarity. [0355] A maximum-likelihood phylogenetic tree with 100 bootstrap replicates was made in order to visualize divergence between publicly available Zea mays chrysovirus 1 nucleotide sequences obtained from NCBI. A “high quality” tree was made using only those NCBI sequences that are at least 90% full length, with less than 10% unknown characters.
  • the tree also included the sequence of a newly identified Zea mays chrysovirus 1 isolate, “ZmCV2.”
  • a radish chrysovirus (NC_043657.1/NC_043656.1/NC_043658.1 Raphanus sativas chrysovirus 1) was used as an outgroup.
  • Another tree was made of all NCBI Zea mays chrysovirus 1 sequences that are at least 50% full length, with less than 10% unknown characters, to further help determine the degree of divergence of Zea mays chrysovirus 1.
  • the RdRP protein sequences YP_0095516, AYD75728.1, AYD75725.1, AYD75736.1, AYD75731.1, AYD75753.1, and “ZmCV2” (SEQ ID NO: 536) were aligned. All had a protein length of 1090 amino acids and no gaps.
  • the “ZmCV2” RdRP sequence is 92.39% similar to YP_009551655.1, 92.39% similar to YP_009551655.1, 92.39% similar to AYD75728.1, 92.48% similar to AYD75725.1, 92.39% similar to AYD75736.1, 92.20% similar to AYD75731.1, and 91.93% similar to AYD75753.1.
  • the missing amino acids in the AYD75727.1 sequence are the first three amino acids, possibly a sequencing error.
  • the “ZmCV2” CP sequence (SEQ ID NO: 543) is 80.63% similar to QLM02964.1, 80.63% similar to YP_009551654.1, 80.21% similar to AYD75727.1, and 79.80% similar to AYD75724.1.
  • the “ZmCV2” P3 sequence is 82.48% similar to YP_009551656.1 with 4 gaps and 95% query coverage, 85.02% similar to AYD75737.1 with 4 gaps and 97% query coverage, 84.94% similar to AYD75732.1 with 4 gaps and 95% query coverage, 84.94% similar to AYD75729.1 with 4 gaps and 95% query coverage, and 84.65% similar to AYD75726.1 with 4 gaps and 95% query coverage.
  • An aspect of the invention is related to an in planta self-replicating commensal virus (e.g., a chrysovirus) satellite system including a plant or a plant cell that contains: (a) a recombinant commensal satellite RNA (or DNA encoding the recombinant commensal satellite RNA), and (b) a replicase capable of replicating the recombinant commensal satellite RNA, which in many cases is the RdRP that corresponds to the replicase recognition sequences in the commensal satellite RNA.
  • a self-replicating commensal virus e.g., a chrysovirus
  • a chrysovirus satellite (COMSAT) containing a cargo sequence generally includes, in 5’ to 3’ orientation: (i) a 5’ RNA replication element recognized by the chrysovirus RdRP; (ii) cargo RNA sequence (e.g., non-coding RNA or coding RNA or a combination of both non-coding RNA and coding RNA); and (iii) a 3’ RNA replication element recognized by the chrysovirus RdRP (see Table 17 and 18 for suitable chrysovirus 5’ and 3’ RNA replication elements and RdRP sequences).
  • the chrysovirus viral RdRP can be supplied in planta by the commensal virus itself (which can be endogenous or exogenously provided to the plant), or alternatively can be supplied as a heterologous molecule (e.g., as recombinant DNA or RNA encoding the RdRP, to be expressed in the plant).
  • chrysovirus satellite system An embodiment of such a self-replicating chrysovirus satellite system is illustrated in this example by a plant that is provided with (a) DNA encoding a recombinant chrysovirus satellite, and (b) recombinant DNA encoding the chrysovirus RdRP; analogous self- replicating chrysovirus satellite systems include an RdRP provided by the corresponding chrysovirus itself, e.g., exogenously introduced into the plant.
  • a thale cress (Arabidopsis thaliana) plant expresses a self- replicating Brassica campestris chrysovirus 1 (BrcCV1) system that includes (a) recombinant DNA encoding a BrcCV1 satellite RNA, and (b) recombinant DNA encoding a BrcCV1 RdRP.
  • a transgenic Arabidopsis thaliana plant heterologously expressing the Brassica campestris chrysovirus 1 (BrcCV1) RNA-dependent RNA polymerase (RdRP) is generated by floral dip transformation with a T-DNA construct.
  • the T-DNA construct includes an expression cassette for the BrcCV1 RdRP.
  • the T-DNA construct further includes an expression cassette for the BrcCV1 coat protein (CP).
  • the T-DNA construct also includes an expression cassette for the BrcCV1 uncharacterized protein (P3), which is a putative protease; see Zhang et al. (2017) Arch. Virol., 162:1107-1111, doi:10.1007/s00705-016-3196-x.
  • P3 BrcCV1 uncharacterized protein
  • a BrcCV1 COMSAT includes BrcCV15’ and 3’ RNA replication elements flanking a protein-coding sequence of interest (e.g., one or more cargo sequences from tables 7-13).
  • the BrcCV1 COMSAT further includes the BrcCV1 CP and/or P3 coding sequence(s), where these sequences are not provided as a protein-coding expression cassette in the T-DNA outside of the BrcCV1 COMSAT cassette.
  • the BrcCV1 COMSAT is prepared as an in vitro transcription (IVT) product comprising linear, positive-sense, single-stranded RNA, using a PCR template amplified from a plasmid template, and the T7 HiScribe Kit from NEB (E2040S, New England Biolabs, Ipswich, MA).
  • IVT product is prepared for biolistic delivery using the Helios Gene Gun system (#1652411, BioRad, Waltham, MA), and fired into the transgenic (BrcCV1 RdRP-expressing) Arabidopsis thaliana plant.
  • Replication of the BrcCV1 COMSAT in the transgenic (BrcCV1 RdRP- expressing) Arabidopsis thaliana plant is detectable by a negative-strand-specific taqman probe directed to a selected sequence of the BrcCV1 COMSAT.
  • This assay evinces replication because the biolistic delivery introduces only the positive-sense strand form of the BrcCV1 COMSAT, and the negative-sense strand of the BrcCV1 COMSAT can only be produced by replication.
  • a transgenic canola (Brassica napus) plant is generated by Agrobacterium-mediated transformation, e.g., following a protocol similar to that described by Cardoza & Stewart (2006), “Canola (Brassica napus L.).” In: Wang, K. (ed.) Agrobacterium Protocols. Methods in Molecular Biology, Vol.343. Humana Press, doi:10.1385/1-59745-130-4:257.
  • a maize (Zea mays) plant expresses a self-replicating Zea mays chrysovirus 1 (ZmCV1) system that includes (a) recombinant DNA encoding a ZmCV1 satellite RNA (e.g., comprising a Zea mays chrysovirus 15’RRE and 3’ RRE set forth in Table 1, 17, and 18), and (b) recombinant DNA encoding a ZmCV1 RdRP.
  • other family Poaceae plants e.g., Oryza spp., Sorghum spp. are used as the host.
  • the maize plant expressing the self-replicating ZmCV1 satellite system is generated by Agrobacterium-mediated transformation of immature embryos with a T-DNA construct following a protocol similar to that described by Frame et al. (2011). “Genetic Transformation Using Maize Immature Zygotic Embryos.” In: Thorpe, T., Yeung, E. (eds) Plant Embryo Culture. Methods in Molecular Biology, vol 710. Humana Press; doi 10.1007/978-1-61737-988-8_22.
  • the T-DNA construct includes an expression cassette for a ZmCV1 satellite (COMSAT), producing a positive-sense RNA transcript which is recognized and replicated by the RdRP expressed in trans.
  • COMSAT ZmCV1 satellite
  • the ZmCV1 satellite COMSAT includes, in 5’ to 3’ orientation: (i) a 5’ replicase recognition sequence (RNA replication element) recognized by the ZmCV1 RdRP; (ii) cargo RNA sequence (e.g., non-coding RNA or coding RNA or a combination of both non-coding RNA and coding RNA provided in Tables 7 to 13), e.g., RNA encoding one or more amino acid sequences provided in Tables 8 to 13; and (iii) a 3’ replicase recognition sequence (RNA replication element) recognized by the ZmCV1 RdRP.
  • RNA replication element recognized by the ZmCV1 RdRP
  • cargo RNA sequence e.g., non-coding RNA or coding RNA or a combination of both non-coding RNA and coding RNA provided in Tables 7 to 13
  • RNA encoding one or more amino acid sequences provided in Tables 8 to 13
  • a 3’ replicase recognition sequence RNA replication element
  • the T-DNA construct further includes an expression cassette for the Zea mays chrysovirus 1 (ZmCV1) RNA-dependent RNA polymerase (RdRP).
  • the T-DNA construct further includes an expression cassette for the ZmCV1 coat protein (CP) and/or an expression cassette for the ZmCV1 uncharacterized protein (P3), which is a putative protease (see Peyambari et al. (2016) J. Virol., 93:10.1128/jvi.01188-18, doi:10.1128/jvi.01188-18).
  • Replication of the ZmCV1 COMSAT in the transgenic plant is detectable by a negative-strand-specific taqman probe directed to the ZmCV1 COMSAT sequence.
  • the ZmCV1 COMSAT further incorporates the ZmCV1 only the positive-sense strand form of the ZmCV1 COMSAT, and the negative-sense CP and/or P3 coding sequences, where the CP and/or P3 coding sequence is not provided as a protein-coding expression cassette in the T-DNA outside of the ZmCV1 COMSAT cassette.
  • a maize (Zea mays) plant expresses a self-replicating Zea mays chrysovirus ZmCV2 system that includes (a) recombinant DNA encoding a ZmCV2 satellite RNA (e.g. comprising 5’RRE and 3’ RRE set forth in a rows of Table 18 which include SEQ ID NO: 552 and 556 or SEQ ID NO: 559 and 562, or sequences having at least 95% or 99% sequence identity respectively thereto), and optionally (b) recombinant DNA encoding a ZmCV2 (SEQ ID NO: 536) RdRP or sequences having at least 95% or 99% sequence identity thereto.
  • a ZmCV2 satellite RNA e.g. comprising 5’RRE and 3’ RRE set forth in a rows of Table 18 which include SEQ ID NO: 552 and 556 or SEQ ID NO: 559 and 562, or sequences having at least 95% or 99% sequence identity respectively thereto
  • a maize plant expressing an endogenous (e.g., from an endogenous or native ZmCV2 virus) or exogenous (e.g., transgenically or transiently expressed) ZmCV2 RdRP (SEQ ID NO:536) is provided with a ZmCV2 satellite RNA (or DNA encoding the same) that includes the corresponding 5’ RRE (SEQ ID NO:552), a cargo RNA of interest, and the corresponding 3’ RRE (SEQ ID NO:556).
  • other family Poaceae plants e.g., Oryza spp., Sorghum spp.
  • the T-DNA construct includes an expression cassette for a ZmCV2 satellite (COMSAT), producing a positive-sense RNA transcript which is recognized and replicated by the RdRP expressed in trans.
  • the ZmCV2 satellite COMSAT includes, in 5’ to 3’ orientation: (i) a 5’ replicase recognition sequence (RNA replication element) recognized by the ZmCV2 RdRP; (ii) cargo RNA sequence (e.g., non-coding RNA or coding RNA or a combination of both non-coding RNA and coding RNA provided in Tables 7 to 13), e.g., RNA encoding one or more amino acid sequences provided in Tables 8 to 13; and (iii) a 3’ replicase recognition sequence (RNA replication element) Agent Ref: P14421WO00 - 127 - recognized by the ZmCV2 RdRP.
  • the T-DNA construct further includes an expression cassette for the Zea mays chrysovirus 2 (ZmCV2) RNA-dependent RNA polymerase (RdRP).
  • the T-DNA construct further includes an expression cassette for the ZmCV2 coat protein (CP) and/or an expression cassette for the ZmCV2 uncharacterized protein (P3), which is a putative protease (see Peyambari et al. (2016) J. Virol., 93:10.1128/jvi.01188-18, doi:10.1128/jvi.01188-18).
  • Replication of the ZmCV2 COMSAT in the transgenic plant is detectable by a negative-strand-specific taqman probe directed to the ZmCV2 COMSAT sequence.
  • This assay evinces replication because transcription from the T-DNA cassette introduces a negative strand of the ZmCV2 COMSAT which can only be produced by replication.
  • the ZmCV2 COMSAT further incorporates the only the positive-sense strand form of the ZmCV2 COMSAT, and the negative-sense CP and/or P3 coding sequences, where the CP and/or P3 coding sequence is not provided as a protein-coding expression cassette in the T-DNA outside of the ZmCV2 COMSAT cassette.
  • a transgenic rice (Oryza sativa) plant is generated by Agrobacterium-mediated transformation of embryogenic callus induced from mature using a similar T-DNA construct containing an appropriate selectable marker and following known protocols; see Nishimura et al. (2006) Nature Protocols, 1:2796-2802, doi:10.1038/nprot.2006.469.
  • An analogous approach is used in wheat, sorghum, and millet. Example 12.
  • Satellite RNA Negative-Sense Strand where only the positive-sense strand of a satellite RNA is provided to a plant or plant cell (e.g., by delivery of the RNA itself or by delivery of DNA encoding the satellite RNA’s positive-sense strand), replication of the satellite RNA in a plant or plant cell can be verified by detection of the satellite RNA’s negative-sense strand, for example, by using a negative-sense-strand-specific Taqman® assay.
  • a bell pepper endornavirus (BPEV) satellite of 2357 nucleotides was constructed with the following elements in 5’ to 3’ orientation: (1) BPEV 5’ RNA replication element; (2) a first IRES sequence (PSVI IRES, with the addition of spacer nucleotides on the 5’ end); (3) RNA encoding a reporter protein (GFP); (4) a second IRES sequence (ZmHSP IRES); (5) a viral movement protein; (6) a zebrafish sequence (as a non-plant heterologous sequence for detection purposes); (7) an isoleucine tRNA sequence; (8) BPEV 3’ RNA replication element.
  • PSVI IRES first IRES sequence
  • GFP reporter protein
  • ZmHSP IRES ZmHSP IRES
  • Similar satellites can be designed and constructed using alternative genetic elements such as those provided elsewhere in this specification, such as other pairs of 5’ and 3’ RNA replication elements, other pairs of HRV 5’ and 3’ replication region sequences, and/or other cargo sequences.
  • IVTT in vitro transcription
  • Samples were collected 4 weeks post gene gun firing from a local leaf.
  • RNA extraction, library prep, and Illumina® sequencing using a stranded kit (NEBNext® UltraTM II Directional RNA Library Prep Kit for Illumina®, catalogue numbers E7760S or E7760L, New England Biolabs, Ipswich, Agent Ref: P14421WO00 - 128 - MA) with added DMSO treatment and plant rRNA depletion.
  • Reads were mapped against the BPEV satellite RNA’s sequence and negative-sense reads were identified using the Integrative Genomics Viewer (IGV) browser (Robinson et al., Bioinformatics, 39(1) (2023), btac830).
  • IGV Integrative Genomics Viewer
  • Negative-sense reads mapping to the BPEV satellite were present, confirming that replication of the BPEV satellite had occurred.
  • BPEV negative-sense reads were also identified in BPEV infected plants that served as a positive control for the analysis.
  • the above-described negative-strand detection method can be adapted for use in the detection of negative-strand RNA-produced Ghabrivirales satellite RNAs disclosed herein. Example 13.
  • RNA Negative-Sense Strand in a Tomato Plant where only the positive-sense strand of a satellite RNA is provided to a plant or plant cell (e.g., by delivery of the RNA itself or by delivery of DNA encoding the satellite RNA’s positive-sense strand), replication of the satellite RNA in a plant or plant cell can be verified by detection of the satellite RNA’s negative-sense strand, for example, by using a negative-sense-strand-specific Taqman® assay.
  • Negative sense GFP produced by a satellite RNA containing a GFP protein encoding cargo sequences is detected using Taqman® qPCR.
  • the reference sequence that can be used is an endogenous tomato gene, “Tom06” (NCBI Gene ID 101249087, ubiquitin-conjugating enzyme E2-17 kDa, from Solanum lycopersicum).
  • Taqman® qPCR primers and probes were ordered from Integrated DNA Technology, Coralville, IA.
  • the negative sense GFP probe is labeled with FAM520 dye with ZEN/Iowa Black FQ quencher.
  • the Tom06 probe is labeled with SUN5544 with ZEN/Iowa Black FQ quencher. Primers and probes are used at a 2:1 ratio.
  • cDNA is synthesized via reverse transcription (30 minutes at 60 degrees C, then 10 minutes at 85 degrees C) using Maxima H Minus First Strand cDNA synthesis Kit (K1652, Thermo Fisher Scientific, Waltham MA) using the RT primers in Table 26. This was followed by treatment (30 minutes at 37 degrees C, then 1 minute at 80 degrees C) with thermolabile exonuclease I (E1050 New England Biolabs, Ipswich MA) to remove excess RT primers. The synthesized cDNA was cleaned with RNAClean XP beads (A63987 Beckman Coulter, Brea CA).
  • Taqman® qPCR is carried out using Taqman® Advanced Fast MasterMix (444965, Thermo Fisher Scientific, Waltham MA) and the primer and probes in Table 27.
  • Other embodiments are within the numbered embodiments and claims.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Cell Biology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Virology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne des molécules synthétiques d'ARN satellite Ghabrivirales et des particules satellites contenant ces molécules. La présente invention porte également sur des molécules synthétiques d'ARN satellite Ghabrivirales contenant des amplicons internes de virus à ARN hétérologue (HRV). La présente invention concerne également des procédés d'utilisation des molécules d'ARN satellite Ghabrivirales et des particules satellites les contenant pour modifier les phénotypes des plantes, améliorer la résistance des plantes au stress et améliorer la résistance des plantes aux ravageurs et aux agents pathogènes.
PCT/US2024/027737 2023-05-03 2024-05-03 Arn satellite a base de ghabrivirales artificielles Pending WO2024229385A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202363499771P 2023-05-03 2023-05-03
US63/499,771 2023-05-03

Publications (1)

Publication Number Publication Date
WO2024229385A1 true WO2024229385A1 (fr) 2024-11-07

Family

ID=91193560

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2024/027737 Pending WO2024229385A1 (fr) 2023-05-03 2024-05-03 Arn satellite a base de ghabrivirales artificielles

Country Status (2)

Country Link
AR (1) AR132592A1 (fr)
WO (1) WO2024229385A1 (fr)

Citations (148)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5229114A (en) 1987-08-20 1993-07-20 The United States Of America As Represented By The Secretary Of Agriculture Approaches useful for the control of root nodulation of leguminous plants
US5304730A (en) 1991-09-03 1994-04-19 Monsanto Company Virus resistant plants and method therefore
US5322938A (en) 1987-01-13 1994-06-21 Monsanto Company DNA sequence for enhancing the efficiency of transcription
US5380831A (en) 1986-04-04 1995-01-10 Mycogen Plant Science, Inc. Synthetic insecticidal crystal protein gene
US5463175A (en) 1990-06-25 1995-10-31 Monsanto Company Glyphosate tolerant plants
US5512466A (en) 1990-12-26 1996-04-30 Monsanto Company Control of fruit ripening and senescence in plants
US5516671A (en) 1993-11-24 1996-05-14 Monsanto Company Method of controlling plant pathogens
US5543576A (en) 1990-03-23 1996-08-06 Mogen International Production of enzymes in seeds and their use
US5608149A (en) 1990-06-18 1997-03-04 Monsanto Company Enhanced starch biosynthesis in tomatoes
US5633435A (en) 1990-08-31 1997-05-27 Monsanto Company Glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthases
US5641876A (en) 1990-01-05 1997-06-24 Cornell Research Foundation, Inc. Rice actin gene and promoter
US5689041A (en) 1989-08-10 1997-11-18 Plant Gentic Systems N.V. Plants modified with barstar for fertility restoration
US5689052A (en) 1993-12-22 1997-11-18 Monsanto Company Synthetic DNA sequences having enhanced expression in monocotyledonous plants and method for preparation thereof
US5716837A (en) 1995-02-10 1998-02-10 Monsanto Company Expression of sucrose phosphorylase in plants
US5750876A (en) 1994-07-28 1998-05-12 Monsanto Company Isoamylase gene, compositions containing it, and methods of using isoamylases
US5763245A (en) 1991-09-23 1998-06-09 Monsanto Company Method of controlling insects
US5763241A (en) 1987-04-29 1998-06-09 Monsanto Company Insect resistant plants
US5773696A (en) 1996-03-29 1998-06-30 Monsanto Company Antifungal polypeptide and methods for controlling plant pathogenic fungi
US5850023A (en) 1992-11-30 1998-12-15 Monsanto Company Modified plant viral replicase genes
US5858742A (en) 1983-01-17 1999-01-12 Monsanto Company Chimeric genes for transforming plant cells using viral promoters
US5866775A (en) 1990-09-28 1999-02-02 Monsanto Company Glyphosate-tolerant 5-enolpyruvyl-3-phosphoshikimate synthases
US5869720A (en) 1993-09-30 1999-02-09 Monsanto Company Transgenic cotton plants producing heterologous peroxidase
US5880275A (en) 1989-02-24 1999-03-09 Monsanto Company Synthetic plant genes from BT kurstaki and method for preparation
US5942658A (en) 1993-07-29 1999-08-24 Monsanto Company Transformed plant with Bacillus thuringiensis toxin gene
US5942664A (en) 1996-11-27 1999-08-24 Ecogen, Inc. Bacillus thuringiensis Cry1C compositions toxic to lepidopteran insects and methods for making Cry1C mutants
US5958745A (en) 1996-03-13 1999-09-28 Monsanto Company Methods of optimizing substrate pools and biosynthesis of poly-β-hydroxybutyrate-co-poly-β-hydroxyvalerate in bacteria and plants
US5959091A (en) 1984-12-10 1999-09-28 Monsanto Company Truncated gene of Bacillus thuringiensis encoding a polypeptide toxin
US5981834A (en) 1988-10-04 1999-11-09 Monsanto Company Genetically engineering cotton plants for altered fiber
US5985605A (en) 1996-06-14 1999-11-16 Her Majesty The Queen In Right Of Canada, As Represented By The Dept. Of Agriculture & Agri-Food Canada DNA sequences encoding phytases of ruminal microorganisms
US5998700A (en) 1996-07-02 1999-12-07 The Board Of Trustees Of Southern Illinois University Plants containing a bacterial Gdha gene and methods of use thereof
US6011199A (en) 1992-12-15 2000-01-04 Commonwealth Scientific Method for producing fruiting plants with improved fruit flavour
US6013864A (en) 1993-02-03 2000-01-11 Monsanto Company Plants resistant to infection by luteoviruses
US6015940A (en) 1992-04-07 2000-01-18 Monsanto Company Virus resistant potato plants
US6023013A (en) 1997-12-18 2000-02-08 Monsanto Company Insect-resistant transgenic plants
US6063756A (en) 1996-09-24 2000-05-16 Monsanto Company Bacillus thuringiensis cryET33 and cryET34 compositions and uses therefor
US6063597A (en) 1997-12-18 2000-05-16 Monsanto Company Polypeptide compositions toxic to coleopteran insects
US6072103A (en) 1997-11-21 2000-06-06 Calgene Llc Pathogen and stress-responsive promoter for gene expression
US6080560A (en) 1994-07-25 2000-06-27 Monsanto Company Method for producing antibodies in plant cells
US6093695A (en) 1996-09-26 2000-07-25 Monsanto Company Bacillus thuringiensis CryET29 compositions toxic to coleopteran insects and ctenocephalides SPP
US6107549A (en) 1998-03-10 2000-08-22 Monsanto Company Genetically engineered plant resistance to thiazopyr and other pyridine herbicides
US6110464A (en) 1996-11-20 2000-08-29 Monsanto Company Broad-spectrum δ-endotoxins
US6121436A (en) 1996-12-13 2000-09-19 Monsanto Company Antifungal polypeptide and methods for controlling plant pathogenic fungi
US6140075A (en) 1994-07-25 2000-10-31 Monsanto Company Method for producing antibodies and protein toxins in plant cells
US6166292A (en) 1996-04-26 2000-12-26 Ajinomoto Co., Inc. Raffinose synthetase gene, method of producing raffinose and transgenic plant
US6171640B1 (en) 1997-04-04 2001-01-09 Monsanto Company High beta-conglycinin products and their use
US6228623B1 (en) 1996-03-13 2001-05-08 Monsanto Company Polyhydroxyalkanoates of narrow molecular weight distribution prepared in transgenic plants
US6228992B1 (en) 1998-05-18 2001-05-08 Pioneer Hi-Bred International, Inc. Proteins for control of nematodes in plants
US6242241B1 (en) 1996-11-20 2001-06-05 Monsanto Company Polynucleotide compositions encoding broad-spectrum δ-endotoxins
US6271443B1 (en) 1996-10-29 2001-08-07 Calgene Llc Cotton and rice cellulose synthase DNA sequences
USRE37543E1 (en) 1996-08-13 2002-02-05 Monsanto Company DNA sequence useful for the production of polyhydroxyalkanoates
US6372211B1 (en) 1997-04-21 2002-04-16 Monsanto Technolgy Llc Methods and compositions for controlling insects
US6380462B1 (en) 1998-08-14 2002-04-30 Calgene Llc Method for increasing stearate content in soybean oil
US6380466B1 (en) 1997-05-08 2002-04-30 Calgene Llc Production of improved rapeseed exhibiting yellow-seed coat
US6426447B1 (en) 1990-11-14 2002-07-30 Monsanto Technology Llc Plant seed oils
US6441277B1 (en) 1997-06-17 2002-08-27 Monsanto Technology Llc Expression of fructose 1,6 bisphosphate aldolase in transgenic plants
US6444876B1 (en) 1998-06-05 2002-09-03 Calgene Llc Acyl CoA: cholesterol acyltransferase related nucleic acid sequences
US6448476B1 (en) 1998-11-17 2002-09-10 Monsanto Technology Llc Plants and plant cells transformation to express an AMPA-N-acetyltransferase
US6459018B1 (en) 1998-06-12 2002-10-01 Monsanto Technology Llc Polyunsaturated fatty acids in plants
US6468523B1 (en) 1998-11-02 2002-10-22 Monsanto Technology Llc Polypeptide compositions toxic to diabrotic insects, and methods of use
US6483008B1 (en) 1990-08-15 2002-11-19 Calgene Llc Methods for producing plants with elevated oleic acid content
US6489461B1 (en) 1999-06-08 2002-12-03 Calgene Llc Nucleic acid sequences encoding proteins involved in fatty acid beta-oxidation and methods of use
US6495739B1 (en) 1998-07-24 2002-12-17 Calgene Llc Plant phosphatidic acid phosphatases
US6501009B1 (en) 1999-08-19 2002-12-31 Monsanto Technology Llc Expression of Cry3B insecticidal protein in plants
US6506962B1 (en) 1999-05-13 2003-01-14 Monsanto Technology Llc Acquired resistance genes in plants
US6518488B1 (en) 2000-07-21 2003-02-11 Monsanto Technology Llc Nucleic acid molecules and other molecules associated with the β-oxidation pathway
US6531648B1 (en) 1998-12-17 2003-03-11 Syngenta Participations Ag Grain processing method and transgenic plants useful therein
US6537750B1 (en) 1998-08-04 2003-03-25 Cargill Incorporated Plant fatty acid desaturase promoters
US6538178B1 (en) 1990-06-18 2003-03-25 Monsanto Technology Llc Increased starch content in plants
US6538181B1 (en) 1990-06-11 2003-03-25 Calgene Llc Glycogen biosynthetic enzymes in plants
US6541259B1 (en) 1999-04-15 2003-04-01 Calgene Llc Nucleic acid sequences to proteins involved in isoprenoid synthesis
US6555655B1 (en) 1999-05-04 2003-04-29 Monsanto Technology, Llc Coleopteran-toxic polypeptide compositions and insect-resistant transgenic plants
US6573361B1 (en) 1999-12-06 2003-06-03 Monsanto Technology Llc Antifungal proteins and methods for their use
US6589767B1 (en) 1997-04-11 2003-07-08 Abbott Laboratories Methods and compositions for synthesis of long chain polyunsaturated fatty acids
US6593293B1 (en) 1999-09-15 2003-07-15 Monsanto Technology, Llc Lepidopteran-active Bacillus thuringiensis δ-endotoxin compositions and methods of use
US6596538B1 (en) 1997-06-05 2003-07-22 Calgene Llc Fatty acyl-CoA: fatty alcohol acyltransferases
US6608241B1 (en) 1985-10-29 2003-08-19 Monsanto Technology Llc Protection of plants against viral infection
US6617496B1 (en) 1985-10-16 2003-09-09 Monsanto Company Effecting virus resistance in plants through the use of negative strand RNAs
US6620988B1 (en) 1997-12-18 2003-09-16 Monsanto Technology, Llc Coleopteran-resistant transgenic plants and methods of their production using modified Bacillus thuringiensis Cry3Bb nucleic acids
US6630306B1 (en) 1996-12-19 2003-10-07 Yale University Bioreactive allosteric polynucleotides
US6639054B1 (en) 2000-01-06 2003-10-28 Monsanto Technology Llc Preparation of deallergenized proteins and permuteins
US6653530B1 (en) 1998-02-13 2003-11-25 Calgene Llc Methods for producing carotenoid compounds, tocopherol compounds, and specialty oils in plant seeds
US6657046B1 (en) 2000-01-06 2003-12-02 Monsanto Technology Llc Insect inhibitory lipid acyl hydrolases
US6660849B1 (en) 1997-04-11 2003-12-09 Calgene Llc Plant fatty acid synthases and use in improved methods for production of medium-chain fatty acids
US6663906B2 (en) 1997-06-17 2003-12-16 Monsanto Technology Llc Expression of fructose 1,6 bisphosphate aldolase in transgenic plants
DE10225066A1 (de) * 2002-06-06 2003-12-18 Basf Plant Science Gmbh Neue Expressionssysteme für Pflanzen
USRE38446E1 (en) 1990-07-20 2004-02-24 Calgene, Llc. Sucrose phosphate synthase (SPS), its process for preparation its cDNA, and utilization of cDNA to modify the expression of SPS in plant cells
US6706950B2 (en) 2000-07-25 2004-03-16 Calgene Llc Nucleic acid sequences encoding β-ketoacyl-ACP synthase and uses thereof
US6713063B1 (en) 1996-11-20 2004-03-30 Monsanto Technology, Llc Broad-spectrum δ-endotoxins
US6723897B2 (en) 1998-08-10 2004-04-20 Monsanto Technology, Llc Methods for controlling gibberellin levels
US6723837B1 (en) 1999-07-12 2004-04-20 Monsanto Technology Llc Nucleic acid molecule and encoded protein associated with sterol synthesis and metabolism
US6770465B1 (en) 1999-06-09 2004-08-03 Calgene Llc Engineering B-ketoacyl ACP synthase for novel substrate specificity
US6774283B1 (en) 1985-07-29 2004-08-10 Calgene Llc Molecular farming
US6803501B2 (en) 2000-03-09 2004-10-12 Monsanto Technology, Llc Methods for making plants tolerant to glyphosate and compositions thereof using a DNA encoding an EPSPS enzyme from Eleusine indica
US6812379B2 (en) 1998-07-10 2004-11-02 Calgene Llc Expression of eukaryotic peptides in plant plastids
US6822141B2 (en) 1998-07-02 2004-11-23 Calgene Llc Diacylglycerol acyl transferase proteins
US6828475B1 (en) 1994-06-23 2004-12-07 Calgene Llc Nucleic acid sequences encoding a plant cytoplasmic protein involved in fatty acyl-CoA metabolism
WO2005049839A2 (fr) * 2003-11-10 2005-06-02 Icon Genetics Ag Systeme d'expression de vegetaux derive du virus arn
US6946588B2 (en) 1996-03-13 2005-09-20 Monsanto Technology Llc Nucleic acid encoding a modified threonine deaminase and methods of use
US6949379B2 (en) 2000-10-20 2005-09-27 Canji, Inc. Aptamer-mediated regulation of gene expression
US7151204B2 (en) 2001-01-09 2006-12-19 Monsanto Technology Llc Maize chloroplast aldolase promoter compositions and methods for use thereof
US20070011761A1 (en) 2005-05-19 2007-01-11 Monsanto Technology, L.L.C. Post-transcriptional regulation of gene expression
US20100311168A1 (en) 2009-04-07 2010-12-09 Dow Agrosciences Llc Nanoparticle mediated delivery of sequence specific nucleases
US8030473B2 (en) 2005-01-07 2011-10-04 State Of Oregon Acting By And Through The State Board Of Higher Education On Behalf Of Oregon State University Method to trigger RNA interference
US20120023619A1 (en) 2010-07-07 2012-01-26 Dow Agrosciences Llc Linear dna molecule delivery using pegylated quantum dots for stable trasformation in plants
US20120244569A1 (en) 2011-03-23 2012-09-27 Dow Agrosciences Llc Quantum dot carrier peptide conjugates suitable for imaging and delivery applications in plants
US8334430B2 (en) 2005-10-13 2012-12-18 Monsanto Technology Llc Methods for producing hybrid seed
US8395023B2 (en) 2004-12-21 2013-03-12 Monsanto Technology Llc Recombinant DNA constructs and methods for controlling gene expression
US8404928B2 (en) 2006-08-31 2013-03-26 Monsanto Technology Llc Phased small RNAs
US8404927B2 (en) 2004-12-21 2013-03-26 Monsanto Technology Llc Double-stranded RNA stabilized in planta
US8410334B2 (en) 2007-02-20 2013-04-02 Monsanto Technology Llc Invertebrate microRNAs
US20130102651A1 (en) 2007-08-28 2013-04-25 California Institute Of Technology General composition framework for ligand-controlled rna regulatory systems
US8455716B2 (en) 2009-04-20 2013-06-04 Monsanto Technology Llc Multiple virus resistance in plants
US20130145488A1 (en) 2011-12-06 2013-06-06 Iowa State University Research Foundation, Inc. Mesoporous silica nanoparticles suitable for co-delivery
US20130185823A1 (en) 2012-01-16 2013-07-18 Academia Sinica Mesoporous silica nanoparticle-mediated delivery of dna into arabidopsis root
US20140096284A1 (en) 2012-10-01 2014-04-03 Iowa State University Research Foundation, Inc. Method for the delivery of molecules lyophilized onto microparticles to plant tissues
US20140356414A1 (en) 2013-06-03 2014-12-04 University Of Southern California Targeted Crosslinked Multilamellar Liposomes
US8946511B2 (en) 2006-10-12 2015-02-03 Monsanto Technology Llc Plant microRNAs and methods of use thereof
US20150040268A1 (en) 2013-04-25 2015-02-05 Morflora Israel Ltd Methods and compositions for the delivery of nucleic acids to seeds
US20150047074A1 (en) 2013-08-09 2015-02-12 Massachusetts Institute Of Technology Nanobionic engineering of organelles and photosynthetic organisms
US20150082478A1 (en) 2013-08-22 2015-03-19 E I Du Pont De Nemours And Company Plant genome modification using guide rna/cas endonuclease systems and methods of use
US9040774B2 (en) 2008-07-01 2015-05-26 Monsanto Technology Llc Recombinant DNA constructs encoding ribonuclease cleavage blockers and methods for modulating expression of a target gene
US20150208663A1 (en) 2013-10-15 2015-07-30 Board Of Trustees Of The University Of Arkansas Method of delivery of bio-active agents to plant cells by using nano-sized materials as carriers
US9139838B2 (en) 2011-07-01 2015-09-22 Monsanto Technology Llc Methods and compositions for selective regulation of protein expression
US9777288B2 (en) 2013-07-19 2017-10-03 Monsanto Technology Llc Compositions and methods for controlling leptinotarsa
US20170369898A1 (en) 2014-12-19 2017-12-28 AgBiome, Inc. Sequences to facilitate incorporation of dna into the genome of an organism
US9873888B2 (en) 2009-10-23 2018-01-23 Monsanto Technology Llc Transgenic soybean plants and chromosomes
US9976152B2 (en) 2007-06-26 2018-05-22 Monsanto Technology Llc Temporal regulation of gene expression by microRNAs
US10017549B2 (en) 2008-08-29 2018-07-10 Monsanto Technology Llc Hemipteran and coleopteran active toxin proteins from Bacillus thuringiensis
US20180312854A1 (en) 2006-05-16 2018-11-01 Monsanto Technology Llc Use of non-agrobacterium bacterial species for plant transformation
US20190032131A1 (en) 2016-12-12 2019-01-31 Integrated Dna Technologies, Inc. Genome editing detection
US10233217B2 (en) 2014-10-16 2019-03-19 Monsanto Technology Llc Chimeric insecticidal proteins toxic or inhibitory to Lepidopteran pests
US10378012B2 (en) 2014-07-29 2019-08-13 Monsanto Technology Llc Compositions and methods for controlling insect pests
US20190352655A1 (en) 2017-01-28 2019-11-21 Inari Agriculture, Inc. Novel plant cells, plants, and seeds
US10487123B2 (en) 2014-10-16 2019-11-26 Monsanto Technology Llc Chimeric insecticidal proteins toxic or inhibitory to lepidopteran pests
US10612037B2 (en) 2016-06-20 2020-04-07 Monsanto Technology Llc Insecticidal proteins toxic or inhibitory to hemipteran pests
US10827755B2 (en) 2015-11-18 2020-11-10 Monsanto Technology Llc Insecticidal compositions and methods
US11091770B2 (en) 2014-04-01 2021-08-17 Monsanto Technology Llc Compositions and methods for controlling insect pests
US20210259176A1 (en) 2004-08-26 2021-08-26 Monsanto Technology Llc Methods of Seed Breeding Using High Throughput Nondestructive Seed Sampling
US11130965B2 (en) 2016-10-27 2021-09-28 Syngenta Participations Ag Insecticidal proteins
US11136593B2 (en) 2016-09-09 2021-10-05 Syngenta Participations Ag Insecticidal proteins
US11180774B2 (en) 2017-01-12 2021-11-23 Syngenta Participations Ag Insecticidal proteins
US11186837B2 (en) 2004-04-09 2021-11-30 Monsanto Technology Llc Compositions and methods for controlling Diabrotica
WO2021257987A2 (fr) * 2020-06-20 2021-12-23 Halo-Bio Rnai Therapeutics, Inc. Procédés et compositions de nanostructures d'arn pour la réplication et l'expression sous-génomique par l'arn polymérase dirigée par arn
US20220221377A1 (en) 2006-03-02 2022-07-14 Monsanto Technology Llc Automated Contamination-Free Seed Sampler And Methods Of Sampling, Testing And Bulking Seeds
WO2023004435A1 (fr) 2021-07-23 2023-01-26 Flagship Pioneering Innovations Vii, Llc Compositions de lutte contre les champignons et méthodes associées
WO2023161802A1 (fr) 2022-02-22 2023-08-31 Invaio Sciences International Gmbh Procédés et systèmes de lutte contre xylella fastidiosa dans les oliviers
WO2023240208A1 (fr) 2022-06-09 2023-12-14 Invaio Sciences, Inc. Lutte contre le verdissement des agrumes dans les plants d'agrumes à l'aide d'oxytétracycline
US11844318B2 (en) 2018-07-25 2023-12-19 Invaio Sciences International Gmbh Injection systems, injection tools and methods for same

Patent Citations (191)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5858742A (en) 1983-01-17 1999-01-12 Monsanto Company Chimeric genes for transforming plant cells using viral promoters
US5959091A (en) 1984-12-10 1999-09-28 Monsanto Company Truncated gene of Bacillus thuringiensis encoding a polypeptide toxin
US6774283B1 (en) 1985-07-29 2004-08-10 Calgene Llc Molecular farming
US6617496B1 (en) 1985-10-16 2003-09-09 Monsanto Company Effecting virus resistance in plants through the use of negative strand RNAs
US6608241B1 (en) 1985-10-29 2003-08-19 Monsanto Technology Llc Protection of plants against viral infection
US5380831A (en) 1986-04-04 1995-01-10 Mycogen Plant Science, Inc. Synthetic insecticidal crystal protein gene
US5322938A (en) 1987-01-13 1994-06-21 Monsanto Company DNA sequence for enhancing the efficiency of transcription
US6284949B1 (en) 1987-04-29 2001-09-04 Monsanto Company Insect-resistant plants comprising a Bacillus thuringiensis gene
US5763241A (en) 1987-04-29 1998-06-09 Monsanto Company Insect resistant plants
US5229114A (en) 1987-08-20 1993-07-20 The United States Of America As Represented By The Secretary Of Agriculture Approaches useful for the control of root nodulation of leguminous plants
US5981834A (en) 1988-10-04 1999-11-09 Monsanto Company Genetically engineering cotton plants for altered fiber
US5880275A (en) 1989-02-24 1999-03-09 Monsanto Company Synthetic plant genes from BT kurstaki and method for preparation
US7741118B1 (en) 1989-02-24 2010-06-22 Monsanto Technology Llc Synthetic plant genes and method for preparation
US5689041A (en) 1989-08-10 1997-11-18 Plant Gentic Systems N.V. Plants modified with barstar for fertility restoration
US5641876A (en) 1990-01-05 1997-06-24 Cornell Research Foundation, Inc. Rice actin gene and promoter
US5543576A (en) 1990-03-23 1996-08-06 Mogen International Production of enzymes in seeds and their use
US6538181B1 (en) 1990-06-11 2003-03-25 Calgene Llc Glycogen biosynthetic enzymes in plants
US5608149A (en) 1990-06-18 1997-03-04 Monsanto Company Enhanced starch biosynthesis in tomatoes
US6538178B1 (en) 1990-06-18 2003-03-25 Monsanto Technology Llc Increased starch content in plants
US6538179B1 (en) 1990-06-18 2003-03-25 Monsanto Technology Llc Enhanced starch biosynthesis in seeds
US5463175A (en) 1990-06-25 1995-10-31 Monsanto Company Glyphosate tolerant plants
USRE38446E1 (en) 1990-07-20 2004-02-24 Calgene, Llc. Sucrose phosphate synthase (SPS), its process for preparation its cDNA, and utilization of cDNA to modify the expression of SPS in plant cells
US6483008B1 (en) 1990-08-15 2002-11-19 Calgene Llc Methods for producing plants with elevated oleic acid content
US6248876B1 (en) 1990-08-31 2001-06-19 Monsanto Company Glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthases
US5633435A (en) 1990-08-31 1997-05-27 Monsanto Company Glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthases
US5804425A (en) 1990-08-31 1998-09-08 Monsanto Company Glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthases
US6225114B1 (en) 1990-09-28 2001-05-01 Monsanto Company Modified gene encoding glyphosate-tolerant 5-enolpruvyl-3-phosphoshikimate synthase
US5866775A (en) 1990-09-28 1999-02-02 Monsanto Company Glyphosate-tolerant 5-enolpyruvyl-3-phosphoshikimate synthases
US6426447B1 (en) 1990-11-14 2002-07-30 Monsanto Technology Llc Plant seed oils
US5512466A (en) 1990-12-26 1996-04-30 Monsanto Company Control of fruit ripening and senescence in plants
US5304730A (en) 1991-09-03 1994-04-19 Monsanto Company Virus resistant plants and method therefore
US5763245A (en) 1991-09-23 1998-06-09 Monsanto Company Method of controlling insects
US6015940A (en) 1992-04-07 2000-01-18 Monsanto Company Virus resistant potato plants
US5850023A (en) 1992-11-30 1998-12-15 Monsanto Company Modified plant viral replicase genes
US6011199A (en) 1992-12-15 2000-01-04 Commonwealth Scientific Method for producing fruiting plants with improved fruit flavour
US6013864A (en) 1993-02-03 2000-01-11 Monsanto Company Plants resistant to infection by luteoviruses
US5942658A (en) 1993-07-29 1999-08-24 Monsanto Company Transformed plant with Bacillus thuringiensis toxin gene
US5869720A (en) 1993-09-30 1999-02-09 Monsanto Company Transgenic cotton plants producing heterologous peroxidase
US5516671A (en) 1993-11-24 1996-05-14 Monsanto Company Method of controlling plant pathogens
US5689052A (en) 1993-12-22 1997-11-18 Monsanto Company Synthetic DNA sequences having enhanced expression in monocotyledonous plants and method for preparation thereof
US6828475B1 (en) 1994-06-23 2004-12-07 Calgene Llc Nucleic acid sequences encoding a plant cytoplasmic protein involved in fatty acyl-CoA metabolism
US6080560A (en) 1994-07-25 2000-06-27 Monsanto Company Method for producing antibodies in plant cells
US6140075A (en) 1994-07-25 2000-10-31 Monsanto Company Method for producing antibodies and protein toxins in plant cells
US5750876A (en) 1994-07-28 1998-05-12 Monsanto Company Isoamylase gene, compositions containing it, and methods of using isoamylases
US6235971B1 (en) 1995-02-10 2001-05-22 Monsanto Company Expression of sucrose phoshorylase in plants
US6222098B1 (en) 1995-02-10 2001-04-24 Monsanto Company Expression of sucrose phosphorylase in plants
US6476295B2 (en) 1995-02-10 2002-11-05 Monsanto Technology, Llc Expression of sucrose phosphorylase in plants
US5716837A (en) 1995-02-10 1998-02-10 Monsanto Company Expression of sucrose phosphorylase in plants
US6946588B2 (en) 1996-03-13 2005-09-20 Monsanto Technology Llc Nucleic acid encoding a modified threonine deaminase and methods of use
US5958745A (en) 1996-03-13 1999-09-28 Monsanto Company Methods of optimizing substrate pools and biosynthesis of poly-β-hydroxybutyrate-co-poly-β-hydroxyvalerate in bacteria and plants
US6228623B1 (en) 1996-03-13 2001-05-08 Monsanto Company Polyhydroxyalkanoates of narrow molecular weight distribution prepared in transgenic plants
US6215048B1 (en) 1996-03-29 2001-04-10 Monsanto Company Nucleic acid sequences encoding an antifungal polypeptide, aly AFP from alyssum and methods for their use
US6653280B2 (en) 1996-03-29 2003-11-25 Monsanto Technology Llc Antifungal polypeptide AlyAFP from Alyssum and methods for controlling plant pathogenic fungi
US5773696A (en) 1996-03-29 1998-06-30 Monsanto Company Antifungal polypeptide and methods for controlling plant pathogenic fungi
US6166292A (en) 1996-04-26 2000-12-26 Ajinomoto Co., Inc. Raffinose synthetase gene, method of producing raffinose and transgenic plant
US5985605A (en) 1996-06-14 1999-11-16 Her Majesty The Queen In Right Of Canada, As Represented By The Dept. Of Agriculture & Agri-Food Canada DNA sequences encoding phytases of ruminal microorganisms
US5998700A (en) 1996-07-02 1999-12-07 The Board Of Trustees Of Southern Illinois University Plants containing a bacterial Gdha gene and methods of use thereof
USRE37543E1 (en) 1996-08-13 2002-02-05 Monsanto Company DNA sequence useful for the production of polyhydroxyalkanoates
US6063756A (en) 1996-09-24 2000-05-16 Monsanto Company Bacillus thuringiensis cryET33 and cryET34 compositions and uses therefor
US6248536B1 (en) 1996-09-24 2001-06-19 Monsanto Company Bacillus thuringiensis CryET33 and CryET34 compositions and uses thereof
US6399330B1 (en) 1996-09-24 2002-06-04 Monsanto Technology Llc Bacillus thuringiensis cryet33 and cryet34 compositions and uses thereof
US6326351B1 (en) 1996-09-24 2001-12-04 Monsanto Technology Llc Bacillus thuringiensis CryET33 and CryET34 compositions and uses therefor
US6537756B1 (en) 1996-09-26 2003-03-25 Monsanto Technology, Llc Bacillus thuringiensis CryET29 compositions toxic to coleopteran insects and Ctenocephalides SPP
US6093695A (en) 1996-09-26 2000-07-25 Monsanto Company Bacillus thuringiensis CryET29 compositions toxic to coleopteran insects and ctenocephalides SPP
US6686452B2 (en) 1996-09-26 2004-02-03 Monsanto Technology Llc Bacillus thuringiensis CryET29 compositions toxic to coleopteran insects and ctenocephalides SPP
US6271443B1 (en) 1996-10-29 2001-08-07 Calgene Llc Cotton and rice cellulose synthase DNA sequences
US6576818B1 (en) 1996-10-29 2003-06-10 Calgene Llc Plant cellulose synthase and promoter sequences
US6156573A (en) 1996-11-20 2000-12-05 Monsanto Company Hybrid Bacillus thuringiensis δ-endotoxins with novel broad-spectrum insecticidal activity
US6538109B2 (en) 1996-11-20 2003-03-25 Monsanto Technology, Llc Polynucleotide compositions encoding broad spectrum delta-endotoxins
US6110464A (en) 1996-11-20 2000-08-29 Monsanto Company Broad-spectrum δ-endotoxins
US6281016B1 (en) 1996-11-20 2001-08-28 Monsanto Company Broad-spectrum insect resistant transgenic plants
US6242241B1 (en) 1996-11-20 2001-06-05 Monsanto Company Polynucleotide compositions encoding broad-spectrum δ-endotoxins
US6713063B1 (en) 1996-11-20 2004-03-30 Monsanto Technology, Llc Broad-spectrum δ-endotoxins
US6521442B2 (en) 1996-11-20 2003-02-18 Monsanto Technology Llc Polynucleotide compositions encoding broad spectrum δ-endotoxins
US6645497B2 (en) 1996-11-20 2003-11-11 Monsanto Technology, Llc Polynucleotide compositions encoding broad-spectrum δ endotoxins
US6221649B1 (en) 1996-11-20 2001-04-24 Monsanto Company Chimeric bacillus thuringiensis-endotoxins and host cells expressing same
US6809078B2 (en) 1996-11-27 2004-10-26 Monsanto Technology Llc Compositions encoding lepidopteran-toxic polypeptides and methods of use
US6177615B1 (en) 1996-11-27 2001-01-23 Monsanto Company Lepidopteran-toxic polypeptide and polynucleotide compositions and methods for making and using same
US6423828B1 (en) 1996-11-27 2002-07-23 Monsanto Technology Llc Nuclei acid and polypeptide compositions encoding lepidopteran-toxic polypeptides
US6153814A (en) 1996-11-27 2000-11-28 Monsanto Company Polypeptide compositions toxic to lepidopteran insects and methods for making same
US5942664A (en) 1996-11-27 1999-08-24 Ecogen, Inc. Bacillus thuringiensis Cry1C compositions toxic to lepidopteran insects and methods for making Cry1C mutants
US6313378B1 (en) 1996-11-27 2001-11-06 Monsanto Technology Llc Lepidopteran-resistent transgenic plants
US6316407B1 (en) 1996-12-13 2001-11-13 Monsanto Company Antifungal polypeptide from alfalfa and methods for controlling plant pathogenic fungi
US6121436A (en) 1996-12-13 2000-09-19 Monsanto Company Antifungal polypeptide and methods for controlling plant pathogenic fungi
US6630306B1 (en) 1996-12-19 2003-10-07 Yale University Bioreactive allosteric polynucleotides
US6171640B1 (en) 1997-04-04 2001-01-09 Monsanto Company High beta-conglycinin products and their use
US6660849B1 (en) 1997-04-11 2003-12-09 Calgene Llc Plant fatty acid synthases and use in improved methods for production of medium-chain fatty acids
US6589767B1 (en) 1997-04-11 2003-07-08 Abbott Laboratories Methods and compositions for synthesis of long chain polyunsaturated fatty acids
US6372211B1 (en) 1997-04-21 2002-04-16 Monsanto Technolgy Llc Methods and compositions for controlling insects
US6380466B1 (en) 1997-05-08 2002-04-30 Calgene Llc Production of improved rapeseed exhibiting yellow-seed coat
US6596538B1 (en) 1997-06-05 2003-07-22 Calgene Llc Fatty acyl-CoA: fatty alcohol acyltransferases
US6441277B1 (en) 1997-06-17 2002-08-27 Monsanto Technology Llc Expression of fructose 1,6 bisphosphate aldolase in transgenic plants
US6716474B2 (en) 1997-06-17 2004-04-06 Monsanto Technology Llc Expression of fructose 1,6 bisphosphate aldolase in transgenic plants
US6663906B2 (en) 1997-06-17 2003-12-16 Monsanto Technology Llc Expression of fructose 1,6 bisphosphate aldolase in transgenic plants
US6072103A (en) 1997-11-21 2000-06-06 Calgene Llc Pathogen and stress-responsive promoter for gene expression
US6642030B1 (en) 1997-12-18 2003-11-04 Monsanto Technology, Llc Nucleic acid compositions encoding modified Bacillus thuringiensis coleopteran-toxic crystal proteins
US6023013A (en) 1997-12-18 2000-02-08 Monsanto Company Insect-resistant transgenic plants
US6620988B1 (en) 1997-12-18 2003-09-16 Monsanto Technology, Llc Coleopteran-resistant transgenic plants and methods of their production using modified Bacillus thuringiensis Cry3Bb nucleic acids
US6063597A (en) 1997-12-18 2000-05-16 Monsanto Company Polypeptide compositions toxic to coleopteran insects
US6653530B1 (en) 1998-02-13 2003-11-25 Calgene Llc Methods for producing carotenoid compounds, tocopherol compounds, and specialty oils in plant seeds
US6107549A (en) 1998-03-10 2000-08-22 Monsanto Company Genetically engineered plant resistance to thiazopyr and other pyridine herbicides
US6228992B1 (en) 1998-05-18 2001-05-08 Pioneer Hi-Bred International, Inc. Proteins for control of nematodes in plants
US6444876B1 (en) 1998-06-05 2002-09-03 Calgene Llc Acyl CoA: cholesterol acyltransferase related nucleic acid sequences
US6459018B1 (en) 1998-06-12 2002-10-01 Monsanto Technology Llc Polyunsaturated fatty acids in plants
US6822141B2 (en) 1998-07-02 2004-11-23 Calgene Llc Diacylglycerol acyl transferase proteins
US6812379B2 (en) 1998-07-10 2004-11-02 Calgene Llc Expression of eukaryotic peptides in plant plastids
US6495739B1 (en) 1998-07-24 2002-12-17 Calgene Llc Plant phosphatidic acid phosphatases
US6537750B1 (en) 1998-08-04 2003-03-25 Cargill Incorporated Plant fatty acid desaturase promoters
US6723897B2 (en) 1998-08-10 2004-04-20 Monsanto Technology, Llc Methods for controlling gibberellin levels
US6380462B1 (en) 1998-08-14 2002-04-30 Calgene Llc Method for increasing stearate content in soybean oil
US6468523B1 (en) 1998-11-02 2002-10-22 Monsanto Technology Llc Polypeptide compositions toxic to diabrotic insects, and methods of use
US6448476B1 (en) 1998-11-17 2002-09-10 Monsanto Technology Llc Plants and plant cells transformation to express an AMPA-N-acetyltransferase
US6531648B1 (en) 1998-12-17 2003-03-11 Syngenta Participations Ag Grain processing method and transgenic plants useful therein
US6541259B1 (en) 1999-04-15 2003-04-01 Calgene Llc Nucleic acid sequences to proteins involved in isoprenoid synthesis
US6555655B1 (en) 1999-05-04 2003-04-29 Monsanto Technology, Llc Coleopteran-toxic polypeptide compositions and insect-resistant transgenic plants
US6506962B1 (en) 1999-05-13 2003-01-14 Monsanto Technology Llc Acquired resistance genes in plants
US6489461B1 (en) 1999-06-08 2002-12-03 Calgene Llc Nucleic acid sequences encoding proteins involved in fatty acid beta-oxidation and methods of use
US6770465B1 (en) 1999-06-09 2004-08-03 Calgene Llc Engineering B-ketoacyl ACP synthase for novel substrate specificity
US6723837B1 (en) 1999-07-12 2004-04-20 Monsanto Technology Llc Nucleic acid molecule and encoded protein associated with sterol synthesis and metabolism
US6501009B1 (en) 1999-08-19 2002-12-31 Monsanto Technology Llc Expression of Cry3B insecticidal protein in plants
US6593293B1 (en) 1999-09-15 2003-07-15 Monsanto Technology, Llc Lepidopteran-active Bacillus thuringiensis δ-endotoxin compositions and methods of use
US6573361B1 (en) 1999-12-06 2003-06-03 Monsanto Technology Llc Antifungal proteins and methods for their use
US6639054B1 (en) 2000-01-06 2003-10-28 Monsanto Technology Llc Preparation of deallergenized proteins and permuteins
US6657046B1 (en) 2000-01-06 2003-12-02 Monsanto Technology Llc Insect inhibitory lipid acyl hydrolases
US6803501B2 (en) 2000-03-09 2004-10-12 Monsanto Technology, Llc Methods for making plants tolerant to glyphosate and compositions thereof using a DNA encoding an EPSPS enzyme from Eleusine indica
US6518488B1 (en) 2000-07-21 2003-02-11 Monsanto Technology Llc Nucleic acid molecules and other molecules associated with the β-oxidation pathway
US6706950B2 (en) 2000-07-25 2004-03-16 Calgene Llc Nucleic acid sequences encoding β-ketoacyl-ACP synthase and uses thereof
US6949379B2 (en) 2000-10-20 2005-09-27 Canji, Inc. Aptamer-mediated regulation of gene expression
US7151204B2 (en) 2001-01-09 2006-12-19 Monsanto Technology Llc Maize chloroplast aldolase promoter compositions and methods for use thereof
DE10225066A1 (de) * 2002-06-06 2003-12-18 Basf Plant Science Gmbh Neue Expressionssysteme für Pflanzen
WO2005049839A2 (fr) * 2003-11-10 2005-06-02 Icon Genetics Ag Systeme d'expression de vegetaux derive du virus arn
US11186837B2 (en) 2004-04-09 2021-11-30 Monsanto Technology Llc Compositions and methods for controlling Diabrotica
US20210259176A1 (en) 2004-08-26 2021-08-26 Monsanto Technology Llc Methods of Seed Breeding Using High Throughput Nondestructive Seed Sampling
US10793869B2 (en) 2004-12-21 2020-10-06 Monsanto Technology Llc Recombinant DNA constructs and methods for controlling gene expression
US8395023B2 (en) 2004-12-21 2013-03-12 Monsanto Technology Llc Recombinant DNA constructs and methods for controlling gene expression
US9708620B2 (en) 2004-12-21 2017-07-18 Monsanto Technology Llc Recombinant DNA constructs and methods for controlling gene expression
US8404927B2 (en) 2004-12-21 2013-03-26 Monsanto Technology Llc Double-stranded RNA stabilized in planta
US8030473B2 (en) 2005-01-07 2011-10-04 State Of Oregon Acting By And Through The State Board Of Higher Education On Behalf Of Oregon State University Method to trigger RNA interference
US9018002B2 (en) 2005-01-07 2015-04-28 State Of Oregon Acting By And Through The State Board Of Higher Education On Behalf Of Oregon State University Method to trigger RNA interference
US8816061B2 (en) 2005-01-07 2014-08-26 State Of Oregon Acting By And Through The State Board Of Higher Education On Behalf Of Oregon State University Method to trigger RNA interference
US8476422B2 (en) 2005-01-07 2013-07-02 State of Oregon acting by and through the State Board of Higher Eduction on behalf of Oregon State University Method to trigger RNA interference
US20070011761A1 (en) 2005-05-19 2007-01-11 Monsanto Technology, L.L.C. Post-transcriptional regulation of gene expression
US10876126B2 (en) 2005-10-13 2020-12-29 Monsanto Technology Llc Methods for producing hybrid seed
US8334430B2 (en) 2005-10-13 2012-12-18 Monsanto Technology Llc Methods for producing hybrid seed
US20220221377A1 (en) 2006-03-02 2022-07-14 Monsanto Technology Llc Automated Contamination-Free Seed Sampler And Methods Of Sampling, Testing And Bulking Seeds
US20180312854A1 (en) 2006-05-16 2018-11-01 Monsanto Technology Llc Use of non-agrobacterium bacterial species for plant transformation
US8404928B2 (en) 2006-08-31 2013-03-26 Monsanto Technology Llc Phased small RNAs
US8946511B2 (en) 2006-10-12 2015-02-03 Monsanto Technology Llc Plant microRNAs and methods of use thereof
US8410334B2 (en) 2007-02-20 2013-04-02 Monsanto Technology Llc Invertebrate microRNAs
US9976152B2 (en) 2007-06-26 2018-05-22 Monsanto Technology Llc Temporal regulation of gene expression by microRNAs
US20130102651A1 (en) 2007-08-28 2013-04-25 California Institute Of Technology General composition framework for ligand-controlled rna regulatory systems
US9040774B2 (en) 2008-07-01 2015-05-26 Monsanto Technology Llc Recombinant DNA constructs encoding ribonuclease cleavage blockers and methods for modulating expression of a target gene
US10017549B2 (en) 2008-08-29 2018-07-10 Monsanto Technology Llc Hemipteran and coleopteran active toxin proteins from Bacillus thuringiensis
US20100311168A1 (en) 2009-04-07 2010-12-09 Dow Agrosciences Llc Nanoparticle mediated delivery of sequence specific nucleases
US8455716B2 (en) 2009-04-20 2013-06-04 Monsanto Technology Llc Multiple virus resistance in plants
US9873888B2 (en) 2009-10-23 2018-01-23 Monsanto Technology Llc Transgenic soybean plants and chromosomes
US20120023619A1 (en) 2010-07-07 2012-01-26 Dow Agrosciences Llc Linear dna molecule delivery using pegylated quantum dots for stable trasformation in plants
US20120244569A1 (en) 2011-03-23 2012-09-27 Dow Agrosciences Llc Quantum dot carrier peptide conjugates suitable for imaging and delivery applications in plants
US9139838B2 (en) 2011-07-01 2015-09-22 Monsanto Technology Llc Methods and compositions for selective regulation of protein expression
US20130145488A1 (en) 2011-12-06 2013-06-06 Iowa State University Research Foundation, Inc. Mesoporous silica nanoparticles suitable for co-delivery
US20130185823A1 (en) 2012-01-16 2013-07-18 Academia Sinica Mesoporous silica nanoparticle-mediated delivery of dna into arabidopsis root
US20140096284A1 (en) 2012-10-01 2014-04-03 Iowa State University Research Foundation, Inc. Method for the delivery of molecules lyophilized onto microparticles to plant tissues
US20150040268A1 (en) 2013-04-25 2015-02-05 Morflora Israel Ltd Methods and compositions for the delivery of nucleic acids to seeds
US20140356414A1 (en) 2013-06-03 2014-12-04 University Of Southern California Targeted Crosslinked Multilamellar Liposomes
US9777288B2 (en) 2013-07-19 2017-10-03 Monsanto Technology Llc Compositions and methods for controlling leptinotarsa
US20150047074A1 (en) 2013-08-09 2015-02-12 Massachusetts Institute Of Technology Nanobionic engineering of organelles and photosynthetic organisms
US20150082478A1 (en) 2013-08-22 2015-03-19 E I Du Pont De Nemours And Company Plant genome modification using guide rna/cas endonuclease systems and methods of use
US20150208663A1 (en) 2013-10-15 2015-07-30 Board Of Trustees Of The University Of Arkansas Method of delivery of bio-active agents to plant cells by using nano-sized materials as carriers
US11091770B2 (en) 2014-04-01 2021-08-17 Monsanto Technology Llc Compositions and methods for controlling insect pests
US10378012B2 (en) 2014-07-29 2019-08-13 Monsanto Technology Llc Compositions and methods for controlling insect pests
US10669317B2 (en) 2014-10-16 2020-06-02 Monsanto Technology Llc Chimeric insecticidal proteins toxic or inhibitory to lepidopteran pests
US10233217B2 (en) 2014-10-16 2019-03-19 Monsanto Technology Llc Chimeric insecticidal proteins toxic or inhibitory to Lepidopteran pests
US10611806B2 (en) 2014-10-16 2020-04-07 Monsanto Technology Llc Chimeric insecticidal proteins toxic or inhibitory to Lepidopteran pests
US10494409B2 (en) 2014-10-16 2019-12-03 Monsanto Technology Llc Chimeric insecticidal proteins toxic or inhibitory to lepidopteran pests
US10494408B2 (en) 2014-10-16 2019-12-03 Monsanto Technology Llc Chimeric insecticidal proteins toxic or inhibitory to lepidopteran pests
US11267849B2 (en) 2014-10-16 2022-03-08 Monsanto Technology Llc Chimeric insecticidal proteins toxic or inhibitory to lepidopteran pests
US10487123B2 (en) 2014-10-16 2019-11-26 Monsanto Technology Llc Chimeric insecticidal proteins toxic or inhibitory to lepidopteran pests
US20170369898A1 (en) 2014-12-19 2017-12-28 AgBiome, Inc. Sequences to facilitate incorporation of dna into the genome of an organism
US10827755B2 (en) 2015-11-18 2020-11-10 Monsanto Technology Llc Insecticidal compositions and methods
US11254950B2 (en) 2016-06-20 2022-02-22 Monsanto Technology Llc Insecticidal proteins toxic or inhibitory to hemtpteran pests
US10612037B2 (en) 2016-06-20 2020-04-07 Monsanto Technology Llc Insecticidal proteins toxic or inhibitory to hemipteran pests
US11136593B2 (en) 2016-09-09 2021-10-05 Syngenta Participations Ag Insecticidal proteins
US11130965B2 (en) 2016-10-27 2021-09-28 Syngenta Participations Ag Insecticidal proteins
US20190032131A1 (en) 2016-12-12 2019-01-31 Integrated Dna Technologies, Inc. Genome editing detection
US11180774B2 (en) 2017-01-12 2021-11-23 Syngenta Participations Ag Insecticidal proteins
US20190352655A1 (en) 2017-01-28 2019-11-21 Inari Agriculture, Inc. Novel plant cells, plants, and seeds
US11844318B2 (en) 2018-07-25 2023-12-19 Invaio Sciences International Gmbh Injection systems, injection tools and methods for same
WO2021257987A2 (fr) * 2020-06-20 2021-12-23 Halo-Bio Rnai Therapeutics, Inc. Procédés et compositions de nanostructures d'arn pour la réplication et l'expression sous-génomique par l'arn polymérase dirigée par arn
WO2023004435A1 (fr) 2021-07-23 2023-01-26 Flagship Pioneering Innovations Vii, Llc Compositions de lutte contre les champignons et méthodes associées
WO2023161802A1 (fr) 2022-02-22 2023-08-31 Invaio Sciences International Gmbh Procédés et systèmes de lutte contre xylella fastidiosa dans les oliviers
WO2023240208A1 (fr) 2022-06-09 2023-12-14 Invaio Sciences, Inc. Lutte contre le verdissement des agrumes dans les plants d'agrumes à l'aide d'oxytétracycline

Non-Patent Citations (44)

* Cited by examiner, † Cited by third party
Title
ABRAHAMIAN PETER ET AL: "Annual Review of Virology Plant Virus-Derived Vectors: Applications in Agricultural and Medical Biotechnology", ANNU. REV. VIROL, vol. 7, 10 June 2020 (2020-06-10), pages 513 - 535, XP093047962, Retrieved from the Internet <URL:https://www.annualreviews.org/doi/suppl/10.1146/annurev-virology-010720-054958> DOI: 10.1146/annurev-virology-010720- *
AL-RIMAWI ET AL., ANTIBIOTICS, no. 8, 2019, pages 196
ALTSCHUL ET AL., J. MOL. BIOL., vol. 215, 1990, pages 403 - 410
ALTSTEIN, PEPTIDES, vol. 25, 2004, pages 1373 - 1376
AMAN ET AL., GENOME BIOL, vol. 19, 2018, pages 1 - 10
ASIIMWE ET AL., PLANT PATHOLOGY, vol. 69, 2020, pages 585 - 597
CHOI ET AL., J. CONTROLLED RELEASE, vol. 235, 2016, pages 222 - 235
CONG ET AL., SCIENCE, vol. 339, 2013, pages 819 - 823
DE CESARE, MBIO, vol. 11, 2020, pages 02123 - 20
DUANIS-ASSAF, PLANT BIOTECHNOL J, vol. 20, no. 1, January 2022 (2022-01-01), pages 226 - 237
EDGAR, NUCLEIC ACIDS RES., vol. 32, no. 5, 2004, pages 1792 - 1797
FRAME ET AL.: "Plant Embryo Culture. Methods in Molecular Biology", vol. 710, 2011, HUMANA PRESS, article "Genetic Transformation Using Maize Immature Zygotic Embryos"
GEISBERG ET AL., CELL, vol. 156, 2014, pages 812 - 824
GIRALDO ET AL., NATURE MATERIALS, vol. 13, pages 400 - 409
GRANDI ET AL., FRONT. PLANT SCI, vol. 14, 2023, pages 2023
HENDEL ET AL., NATURE BIOTECHNOL., 2015, pages 985 - 991
HOFACKER ET AL., MONATSHEFTE FOR CHEMIE CHEM. MONTHLY, vol. 125, 1994, pages 167 - 188
HORIUCHI ET AL., PLANT CELL PHYSIOL., vol. 42, no. 2, 2001, pages 197 - 203
KHAKHAR ARJUN ET AL: "RNA Viral Vectors for Accelerating Plant Synthetic Biology", FRONTIERS IN PLANT SCIENCE, vol. 12, 23 June 2021 (2021-06-23), XP093047125, Retrieved from the Internet <URL:https://www.frontiersin.org/articles/10.3389/fpls.2021.668580/full> DOI: 10.3389/fpls.2021.668580 *
KIM, BIOCONJUGATE CHEM, vol. 22, 2011, pages 2558 - 2567
KOEVMILLER, J VIROL, vol. 74, no. 13, July 2000 (2000-07-01), pages 5988 - 96
LI, MOL PLANT MICROBE INTERACT, vol. 32, no. 12, December 2019 (2019-12-01), pages 1649 - 1664
MATTEI ET AL., NUCLEIC ACIDS RESEARCH, vol. 42, no. 10, 2014, pages 6146 - 6157
NISHIMURA ET AL., NATURE PROTOCOLS, vol. 343, 2006, pages 2796 - 2802
O'BRIANLTMIMIS, BMC BIOTECHNOL, vol. 11, 2011, pages 66 - 71
PEYAMBARI ET AL., J. VIROL, vol. 93, 2018, pages 93
PUTLYAEV ET AL., BIOCHEMISTRY (MOSC)., vol. 80, no. 8, pages 1039
PUTLYAEV ET AL., BIOCHEMISTRY (MOSC, vol. 80, no. 8, pages 1039 - 46
RAMLANZAUNER: "International Workshop on Computing With Biomolecules", 27 August 2008, AUSTRIAN COMPUTER SOCIETY, pages: 75 - 86
RAN ET AL., NATURE PROTOCOLS, vol. 8, 2013, pages 2281 - 2308
RANDOLPH-ANDERSON ET AL.: "Submicron gold particles are superior to larger particles for efficient Biolistic® transformation of organelles and some cell types", BIO-RAD US/EG BULLETIN 2015, 2015
ROBINSON ET AL., BIOINFORMATICS, vol. 39, no. 1, 2023, pages btac830
SHEN ET AL., THERANOSTICS, vol. 2, 2012, pages 283 - 294
TANGBREAKER, PROC NATL ACAD SCI USA., vol. 97, no. 11, 23 May 2000 (2000-05-23), pages 5784 - 9
WANG ET AL., J. AM. CHEM. SOC. COMM., vol. 132, 2010, pages 9274 - 9276
WHITLIAM, S. ET AL., CELL, vol. 78, 1994, pages 1101 - 1115
WOLTER ET AL., BMC PLANT BIOL, vol. 19, 2019, pages 176 - 183
WONG ET AL., NANO LETT, vol. 16, 2016, pages 1161 - 1172
XING ET AL., BMC PLANT BIOL, vol. 14, 2014, pages 327 - 340
YOUNG ET AL.: "Compendium of Herbicide Adjuvants", 2016, PURDUE UNIVERSITY
ZHANG ET AL., J. CONTROLLED RELEASE, vol. 123, 2007, pages 1 - 10
ZHANG JUN ET AL: "The genome sequence of Brassica campestris chrysovirus 1, a novel putative plant-infecting tripartite chrysovirus", ARCHIVES OF VIROLOGY, vol. 162, no. 4, 1 April 2017 (2017-04-01), AT, pages 1107 - 1111, XP093188204, ISSN: 0304-8608, Retrieved from the Internet <URL:https://link.springer.com/content/pdf/10.1007/s00705-016-3196-x.pdf> DOI: 10.1007/s00705-016-3196-x *
ZHANG, ARCH. VIROL, vol. 162, 2017, pages 1107 - 1111
ZHAO ET AL., NANOSCALE RES. LETT, vol. 11, 2016, pages 195 - 203

Also Published As

Publication number Publication date
AR132592A1 (es) 2025-07-16

Similar Documents

Publication Publication Date Title
US12102091B2 (en) Control of plant pests using RNA molecules
US12077763B2 (en) RNA-based control of powdery mildew
US20250270582A1 (en) Methods for modifying plants with a recombinant rna molecule
US20250230452A1 (en) Polynucleotides for modifying organisms
US11578338B2 (en) Hemipteran active insecticidal protein
US20150203866A1 (en) Methods and compositions for plant pest control
CN118556127A (zh) 用于修饰生物体的多核苷酸
WO2024229385A1 (fr) Arn satellite a base de ghabrivirales artificielles
WO2024229398A1 (fr) Arn satellite d&#39;amalgavirus artificiel
WO2024229356A2 (fr) Arn satellites de solemoviridae artificiels
WO2024229359A2 (fr) Arn satellite de tymovirales artificiels
WO2024229351A1 (fr) Arn satellite de secoviridae artificiels
WO2024229362A1 (fr) Arn satellites martellivirales artificiels
WO2024229403A1 (fr) Systèmes d&#39;amplification d&#39;arn satellite endornaviral pour plantes
WO2024229395A1 (fr) Systèmes d&#39;amplification d&#39;arn satellite de partitivirus pour plantes
WO2024229347A1 (fr) Arn satellite de tombusviridae artificiel
US20230189818A1 (en) Rna-based control of botrytis
CN118922549A (zh) 用于修饰生物体的多核苷酸
MX2007004883A (en) Methods and materials for conferring resistance to pests and pathogens of plants

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24727989

Country of ref document: EP

Kind code of ref document: A1

REG Reference to national code

Ref country code: BR

Ref legal event code: B01A

Ref document number: 112025023882

Country of ref document: BR

WWE Wipo information: entry into national phase

Ref document number: 2024727989

Country of ref document: EP