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

• Agent Ref P14354WO00 - 1 - PARTITIVIRAL SATELLITE RNA AMPLIFICATION SYSTEMS FOR PLANTS CROSS-REFERENCE TO RELATED APPLICATIONS
• This application claims priority to provisional application U.S. Serial No.63/499,724 filed May 3, 2023, which is incorporated herein by reference in its entirety. INCORPORATION OF SEQUENCE LISTING [0002]
• the instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety.
• the XML file, created on May 1, 2024, is named P14354WO00.xml, and is 4,889,046 bytes in size.
• Partitiviruses are dsRNA viruses having a bipartite genome which is separately encapsidated.
• One PV genome comprises a dsRNA encoding an RNA-dependent RNA polymerase (RdRP) and the other PV genome comprises a dsRNA encoding a capsid protein (Roossinck, M. doi/10.1111/nph.15364).
• RdRP RNA-dependent RNA polymerase
• RNA molecules comprising from 5’ terminus to 3’ terminus: (a) a 5’ RNA replication element (5’ RRE) recognized by a Partitivirus RNA-dependent RNA polymerase (RdRP); (b) a cargo RNA molecule (cargo RNA sequence); and (c) a 3’ RNA replication element (3’ RRE) 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.
• 5’ RRE 5’ RNA replication element
• RdRP Partitivirus RNA-dependent RNA polymerase
• the 5’ RNA replication element and the 3’ RNA replication element are obtained from the same partitivirus genome or from partitivirus 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 partitivirus genome or from partitivirus 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 partitivirus 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. (i) a heterologous Agent Ref: P14354WO00 - 2 - 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 partitivirus 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 partitivirus 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 partitivirus 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 partitivirus 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 P14354WO00 - 3 - [0010] Also provided are methods of manufacturing a synthetic partitivirus 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.
• ERE encapsidation recognition element
• plant propagules comprising any of the herein disclosed recombinant RNA molecules and a partitivirus 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 partitivirus 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 partitivirus 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 RdRP set forth in Table 7.
• plants comprising any of the herein disclosed recombinant RNA molecules and a partitivirus 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 partitivirus satellite RNA.
• Partitivirus satellite systems that are self-replicating when introduced into a plant or plant cell, comprising: (1) any of the herein disclosed recombinant partitivirus satellite RNAs (e.g., recombinant RNA molecules); and (2) an exogenous partitivirus that is capable of replication in the plant or plant cells and that encodes the partitivirus RdRP that recognizes the 5’ and 3’ replicase recognition sequences in the recombinant partitivirus satellite RNA, optionally wherein the partitivirus 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 partitivirus satellite RNA.
• recombinant partitivirus satellite RNAs e.g., recombinant RNA molecules
• an exogenous partitivirus that is capable of replication in the plant or plant cells and that encodes the partitivirus RdRP that recognizes the 5’ and 3’ replicase recognition sequences in the re
• Figure 1 shows a non-limiting embodiment of a structure of a partitivirus 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 partitivirus 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: P14354WO00 - 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 partitivirus 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 partitivirus 5’ RRE are shown.
• Figure 4 depicts a partitivirus 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.
• Figure 5 depicts non-limiting embodiments of a partitivirus 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 heterologous RNA virus subgenomic promoter
• hrvRdRP heterologous RNA virus RNA-dependent RNA polymerase
• Embodiments where the HRV sgp and cargo RNA are in sense or antisense orientation relative to the partitivirus 5’ RRE are shown.
• 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 partitivirus satellite.
• HRV1 tobacco mosaic virus
• HRV1 tobacco mosaic virus
• 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.
• 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.
• 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., 2021, doi: 10.20944/preprints202102.0280.v1; and Gaffar et al., 2019, doi: Agent Ref: P14354WO00 - 21 - 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 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, 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.
• 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, Agent Ref: P14354WO00 - 22 - 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 partitivirus satellite construct with such an HRV amplicon is shown in Figure 2.
• Amplification of such an HRV amplicon in plants comprising a partitivirus satellite construct, the partitivirus, 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 Agent Ref: P14354WO00 - 23 - 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, 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 partitivirus 5’ RNA replication element.
• HRV amplicon When the HRV amplicon is oriented in the sense orientation relative to the partitivirus 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”
• the recombinant nucleotides provided herein comprise partitivirus 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
• FIG. 3 An illustrative example of a partitivirus satellite construct with subgenomic promoters in sense or antisense orientation relative to the 5’ RRE is shown in Figure 3.
• FIG 8. Another illustrative example where subgenomic promoters drive expression of an HRV RdDp and a dsRNA cargo in plants comprising a COMMENSAL 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 Agent Ref: P14354WO00 - 24 - 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 Agent Ref: P14354WO00 -
• 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 partitivirus 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 partitivirus 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 partitivirus 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 partitivirus 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 partitivirus 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 partitivirus 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 replication elements results in an RNA 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 a partitivirus 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
• 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 legumes (Fabaceae), Chenopodiaceae, Apiaceae, Rosaceae, Cannabaceae, solanaceous plants (Solanaceae), Caryophyllaceae, Brassicaceae, and Graminaceae plants that comprise partitivirus satellite RNAs containing HRV amplicons disclosed herein can exhibit control of a wide variety of HRV.
• leguminous plants including Medicago sativa, vetches, and Trifolium spp.
• partitivirus satellite RNA’s with 5’ RRE and 3’RRE from Vicia cryptic virus, White clover cryptic virus 1, Red clover cryptic virus 2, White clover cryptic virus 2, Crimson clover cryptic virus 2, or White clover cryptic virus 3 (e.g., Table 1) and containing an HRV amplicon with suitable antiviral cargo can exhibit control of HRV that include Bean common mosaic necrosis virus, Bean common mosaic virus, Bean leafroll virus, Bean mild mosaic virus, Bean necrotic mosaic orthotospovirus, Bean pod mottle virus, Bean rugose mosaic virus, Bean yellow disorder virus, Bean Agent Ref: P14354WO00 - 26 - yellow mosaic virus, Broad bean mottle virus, Broad bean necrosis virus, Broad bean stain virus, Broad bean true mosaic virus, Broad bean wilt virus 1, Broad bean wilt virus 2, Faba bean polerovirus 1, Southern bean mosaic virus, Southern cowpea mosaic virus, Soybean dwarf virus, Soybean latent spherical virus,
• Chenopodiaceae plants such as Beta spp. (beets), Chenopodium spp., and the like, that comprise partitivirus satellite RNAs with 5’ RRE and 3’RRE from Beet cryptic virus 1, Beet cryptic virus 2, or Beet cryptic virus 3 (e.g., Table 1) and containing an HRV amplicon with suitable antiviral cargo can exhibit control of HRV that include Amaranthus leaf mottle virus, Beet black scorch virus, Beet chlorosis virus, Beet mild yellowing virus, Beet mosaic virus, Beet necrotic yellow vein virus, Beet pseudoyellows virus, Beet ringspot virus, Beet soil-borne mosaic virus, Beet soil-borne virus, Beet virus Q, Beet western yellows virus, Beet yellow stunt virus, Beet yellows virus, Spinach amalgavirus 1, Spinach latent virus, Spinach temperate virus.
• Apiaceae plants such as Daucus carota, Anethum graveolens, and the like, that comprise partitivirus satellite RNAs with 5’ RRE and 3’RRE from Carrot cryptic virus, Carrot temperate virus 1, Carrot temperate virus 2, Carrot temperate virus 3, Carrot temperate virus 4, and Dill cryptic virus 2 (e.g., Table 1) and containing an HRV amplicon with suitable antiviral cargo can exhibit control of HRV that include Carrot Ch virus 1, Carrot ch virus 2, Carrot closterovirus 1, Carrot cryptic virus, Carrot mottle mimic virus, Carrot necrotic dieback virus, Carrot red leaf virus, Carrot temperate viruses, Carrot thin leaf virus, Carrot torradovirus 1, Carrot virus Y, and Carrot yellow leaf virus.
• HRV that include Carrot Ch virus 1, Carrot ch virus 2, Carrot closterovirus 1, Carrot cryptic virus, Carrot mottle mimic virus, Carrot necrotic dieback virus, Carrot red leaf virus, Car
• Rosaceae plants such as Prunus spp., Malus spp., Rubus spp., and the like, that comprise partitivirus satellite RNAs with Cherry chlorotic rusty spot associated partitivirus 5’ RRE and 3’RRE (e.g., Table 1) that contain an HRV amplicon with suitable antiviral cargo can exhibit control of HRV that include Prune dwarf virus, Prune virus CRSaV-1, Prunus necrotic ringspot virus, Prunus virus F, Prunus virus T, Asian prunus virus 1, Asian prunus virus 2, Apricot latent ringspot virus, Apricot latent virus, Apricot pseudo-chlorotic leaf spot virus, Apricot vein clearing associated virus, Cherry chlorotic rusty spot associated partitivirus, Cherry green ring mottle virus, Cherry leaf roll virus, Cherry mottle leaf virus, Cherry necrotic rusty mottle virus, Cherry rasp leaf virus, Cherry rusty mottle associated virus, Cherry twisted leaf associated virus, Cherry virus A, Peach chlorotic
• Cannabaceae plants such as Cannabis sativa and Humulus lupulus that comprise partitivirus satellite RNAs with 5’ RRE and 3’RRE from Cannabis cryptic virus, Hop trefoil cryptic virus 1, Hop trefoil cryptic virus 2, and Hop trefoil cryptic virus 3 (e.g., Table 1) that contain an HRV amplicon with suitable antiviral cargo can exhibit control of HRV that include Lettuce chlorosis virus, Hop latent viroid, Hop stunt viroid, Tobacco mosaic virus, Beet curly top virus, and Cannabis cryptic virus.
• solanaceous plants including Capsicum spp., Solanum spp., and the like, that comprise partitivirus satellite RNAs with 5’ RRE and 3’RRE from Pepper cryptic virus 1 or Pepper cryptic virus 2 (e.g., Table 1) that contain an HRV amplicon with suitable antiviral cargo
• HRV that include Tomato apical stunt viroid, Tomato aspermy virus, Tomato black ring virus, Tomato blistering mosaic tymovirus, Tomato bushy stunt virus, Tomato chlorosis virus, Tomato chlorotic dwarf viroid, Tomato chlorotic spot orthotospovirus, Tomato fruit blotch virus, Tomato infectious chlorosis virus, Tomato mild mottle virus, Tomato mosaic virus, Tomato necrotic streak virus, Tomato necrotic stunt virus, Tomato planta macho viroid, Tomato spotted wilt orthotospovirus, Tomato torrado virus, Tomato yellow ring orthotospovirus, Tomato
• Dianthus spp. plants that comprise partitivirus satellite RNAs with 5’ RRE and 3’RRE from Carnation cryptic virus 1 (e.g., Table 1) that contain an HRV amplicon with suitable Agent Ref: P14354WO00 - 28 - antiviral cargo can exhibit control of HRV that include Carnation cryptic virus 1, Carnation Italian ringspot virus, Carnation mottle virus, Carnation necrotic fleck virus, and Carnation ringspot virus.
• Brassicaceae plants such as Raphanus spp.
• partitivirus satellite RNAs with Radish yellow edge virus 5’ RRE and 3’RRE e.g., Table 1
• RRE and 3’RRE e.g., Table 1
• HRV partitivirus satellite RNAs with Radish yellow edge virus 5’ RRE and 3’RRE (e.g., Table 1) that contain an HRV amplicon with suitable antiviral cargo
• 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, Arabidopsis thaliana Tat4 virus, Brassica oleracea Melmoth virus, Turnip crinkle virus, Turnip mosaic virus, Turnip rosette virus, Turnip vein-clearing virus, Turnip yellow mosaic virus, Turnip yellows virus,
• Graminaceae plants such as Lolium spp., Zea spp., Oryza spp., Triticum spp., and the like, that comprise partitivirus satellite RNAs with Ryegrass cryptic virus 5’ RRE and 3’RRE (e.g., Table 1) that contain an HRV amplicon with suitable antiviral cargo can exhibit control of HRV that include Barley yellow dwarf viruses, Barley stripe mosaic virus, Barley mild mosaic virus, Barley yellow mosaic virus, 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, Maize yellow mosaic virus, Oat blue dwarf virus, Oat chlorotic stunt virus, Oat golden stripe virus, Oat mosaic virus, Oat necrotic mottle virus, Rice black streaked
• 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 Agent Ref: P14354WO00 - 29 - 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 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 partitivirus genome (e.g., either a CP or RdRP PV genome including a PV 5’ UTR set forth in Table 1).
• the 5’ RNA replication element further includes a genomic sequence of the partitivirus 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 partitivirus genome (e.g., either a CP or RdRP PV genome including a PV 3’ UTR set forth in Table 1).
• the 3’ RNA replication element further includes a genomic sequence of the partitivirus 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 optionally wherein the 3’ RNA replication element includes a 3’ UTR sequence of the partitivirus.
• the ERE includes a viral OAS such as a tobacco mosaic virus OAS (TMV-OAS) or an OAS set forth in Table 2.
• TLS tobacco mosaic virus OAS
• 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.
• 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. In some embodiments, 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 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.
• 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.
• 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 partitivirus, e.g. a partitivirus 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 Agent Ref: P14354WO00 - 32 - 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 partitivirus.
• 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 partitivirus and/or from the same partitivirus genome (e.g., both obtained from the same PV capsid genome or both obtained from the same PV RdRP genome) or from partitivirus 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 partitivirus 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 partitivirus virus (e.g., where the partitivirus virus occurs naturally in the plant cell).
• the partitivirus is native to or endemic to the plant cell.
• the partitivirus that is endemic to the plant cell is non-pathogenic.
• the partitivirus that is endemic to the plant cell is non-pathogenic and commensal.
• the partitivirus is an exogenously introduced partitivirus (i.e., not endemic or native to the host, but artificially introduced).
• a partitivirus natively found in one plant species, variety, or germplasm can be introduced, with or without a corresponding recombinant partitivirus satellite RNA, into a different plant species, variety, or germplasm.
• a complete self-replicating partitivirus satellite system is introduced into a plant or plant cells, wherein the self-replicating partitivirus satellite system includes: (1) a recombinant partitivirus satellite RNA comprising, from 5’ terminus to 3’ terminus: (a) a 5’ RNA replication element recognized by a partitivirus 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)
• the partitivirus RdRP comprises a protein having at least 85%, 90%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 592-610, 704, or 710.
• 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 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.
• 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.
• 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.
• 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).
• a bacterial 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 partitivirus 5’ and 3’ RNA replication elements; (ii) a promoter which is operably linked to a cargo RNA molecule and a TLS element, flanked by partitivirus 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 Agent Ref: P14354WO00 - 34 - 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 partitivirus 5’ and 3’ RNA replication elements
• 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 partitivirus satellite RNA and satellite particles comprising the encapsidated RNA.
• the expressed and encapsidated partitivirus 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 partitivirus or a recombinant source of the PV RdRP for subsequent expression and replication of the partitivirus 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 partitivirus which provides the PV 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 partitivirus satellite RNAs include both monocot and dicot plants and plant cells which can support partitivirus replication.
• the partitivirus is endogenous to (endemic to or natively found in) the plant or plant cell.
• the partitivirus 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 partitivirus 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 partitivirus 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), Agent Ref: P14354WO00 - 35 - 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), hop
• the host plant or plant cells for synthetic partitivirus 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 partitivirus 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 partitivirus 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 Agent Ref: P14354WO00 - 36 - 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 partitivirus genomic sequence; and/or the 3’ RNA replication element has a nucleotide sequence obtained or derived from a partitivirus genomic sequence.
• the 5’ and/or 3’ RNA replication element can be obtained from the corresponding partitivirus genomic sequence by synthesizing or cloning a copy of the corresponding partitivirus genomic sequence.
• the 5’ and/or 3’ RNA replication element can be derived from the corresponding partitivirus genomic sequence by synthesizing a copy of a modified partitivirus genomic sequence or sequences.
• modifications of partitivirus 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 partitivirus virus, and the RdRP protein is provided to the plant cell by the partitivirus virus.
• the partitivirus is endemic to the plant cell.
• the partitivirus that is endemic to the plant cell is non-pathogenic and/or commensal to the plant cell.
• the partitivirus 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 partitivirus 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 partitivirus satellite RNA.
• the plant cell includes the partitivirus, and the partitivirus 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 Agent Ref: P14354WO00 - 37 - first step of providing a population of plants comprising the plant cells comprising: (i) the partitivirus 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 partitivirus which can provide the RdRP.
• the plant cell comprises the partitivirus which can provide the RdRP, the partitivirus, the RdRP protein, and/or the recombinant polynucleotide encoding the RdRP is optionally not exogenously provided to the plant cell.
• the plant cell does not comprise the partitivirus which can provide the RdRP and the partitivirus is exogenously provided to the cell
• the RdRP protein or the recombinant polynucleotide encoding the RdRP is exogenously provided to the plant cell, or a combination of the partitivirus, RdRP protein, or polynucleotide encoding the RdRP is exogenously provided to the plant cell.
• a complete self-replicating partitivirus satellite system is introduced into a plant or plant cells, wherein the self-replicating partitivirus satellite system includes: (1) a recombinant partitivirus satellite RNA comprising, from 5’ terminus to 3’ terminus: (a) a 5’ RNA replication element recognized by a partitivirus 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 partitivirus (e.g., a partitivirus that is not endemic or native to the plant or plant cells) that is capable of replication in the plant or plant cells and that encodes the partitivirus RdRP that recognize
• the partitivirus RdRP comprises a protein having at least 85%, 90%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 592-610, 704, or 710.
• the presence or absence of a partitivirus 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 partitivirus 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 partitivirus in a target plant can be determined by a protein detection assay (e.g., an immunoassay) directed to a PV CP or RdRP (e.g., a CP or RdRP encoded by or homologous to a CP or RdRP encoded by a PV 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 partitivirus 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 Agent Ref: P14354WO00 - 38 - 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 Agent Ref: P14354WO00 - 39 - 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 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 partitivirus, a fungus, an oomycete, and an invertebrate (e.g., an arthropod or a nematode).
• Target viruses other than a partitivirus 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 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 Agent Ref: P14354WO00 - 40 - 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-free methods of manufacturing a synthetic partitivirus 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 partitivirus 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 partitivirus satellite particle.
• Synthetic partitivirus satellite particles comprising the recombinant RNA, including those made by the aforementioned methods are also provided. Methods of providing any of the aforementioned synthetic partitivirus satellite particles to a plant, including contacting (e.g., spraying, dusting, injecting, soaking, etc.) the plant with the synthetic partitivirus satellite particle or a formulation thereof are also provided.
• 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.
• 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 or otherwise introduced into the vascular system of a plant, to be rubbed on 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 via soil).
• 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 or by a root drench).
• 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).
• 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.
• 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 carrier includes cations or a cationic charge, such as cationic liposomes or cationic polymers such as polyamines (e.g., spermine, spermidine, putrescine).
• 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 poly
• the carrier includes 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.
• 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).
• metals e.g., gold, silver, tungsten, iron, cerium
• ceramics e.g., aluminum oxide, silicon carbide,
• 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. (2015) "Submicron gold particles are superior to larger particles for efficient Biolistic® transformation of organelles and some cell types", Bio-Rad US/EG Bulletin 2015), but successful Biolistics delivery using larger (40 - 48 nanometer) nanoparticles has been reported in cultured animal cells; see O'Brian and Lummis (2011) BMC Biotechnol., 11 :66 - 71.
• 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.
• 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).
• Examples of 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 Agent Ref: P14354WO00 - 45 - 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
• 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, 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. It further serves as a sticker-spreader by improving surface coverage and absorption. Because droplets are relatively large and heavy, it is difficult to get thorough coverage on the undersides of foliage.
• 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.
• Aerosol formulations contain one or more active ingredients and a solvent. Most aerosols contain a low percentage of active ingredients. There are two types of aerosol formulations—the ready- to-use type commonly available in pressurized sealed containers and those products used in electrical or gasoline-powered aerosol generators that release the formulation as a smoke or fog.
• 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 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. They are used in machines that break the liquid formulation into a fine mist or fog (aerosol) using a rapidly whirling disk or heated surface.
• 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 dimethyl ether, diethylene glycol monomethyl ether, diethylene glycol monoethyl ether, propylene glycol monomethyl ether, tetrahydrofuran, dioxane, etc.), esters
• 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.
• 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. Because of their physical properties, most of the formulation remains on the surface of treated porous materials such as concrete, plaster, and untreated wood. In such cases, only the water penetrates the material. [0131] In some instances, 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. [0135] In some instances, the composition is provided in a liposome. In some instances, the composition is provided in a vesicle. [0136] 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. [0137] 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 Agent Ref: P14354WO00 - 49 - 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.
• the term “pesticidal agent” 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 partitivirus 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 partitivirus RdRP; a cargo RNA sequence; and a 3’ RNA replication element that is capable of being recognized by the partitivirus RdRP.
• RdRP partitivirus 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 partitivirus RdRP and plant cells lacking the partitivirus RdRP.
• the plant cells lacking the partitivirus RdRP will also lack the recombinant RNA.
• the mixed population of plant cells comprise plant cells comprising the partitivirus RdRP.
• the plant cells Agent Ref: P14354WO00 - 50 - comprising the partitivirus RdRP can further comprise the recombinant RNA.
• 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 modified plant propagule comprises the cell comprising the recombinant RNA molecule, or a seed, seedling, ovule, Agent Ref: P14354WO00 - 51 - 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 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.
• 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.
• 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 Agent Ref: P14354WO00 - 54 - selection agent.
• a selectable marker e.g., a protein which confers resistance to a selection agent such as an herbicide or antibiotic
• a selection agent e.g., an herbicide or antibiotic
• 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 partitivirus 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.
• 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 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 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 partitivirus 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 partitivirus genome or from partitivirus genomes having at least 85%, 90%, 95%, 98%, or 99% sequence identity to one another and which are optionally related; (i) the 5’ RNA replication element and the 3’ RNA replication element are obtained from the same partitivirus genome or from partitivirus genomes having at least 85%, 90%, 95%, 98%,
• the 5’ RNA replication element comprises at least a segment of the 5’ untranslated region (UTR) of the partitivirus 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 partitivirus 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 partitivirus 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 partitivirus that is natively located 5’ to and adjacent to the 3’ UTR sequence, and optionally wherein the partitivirus genome
• 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: P14354WO00 - 58 - 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 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.
• 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 a Bromovirus, a Closterovirus, a Comovirus, a Potexvirus, Potyvirus, Tobamovirus, Tombusvirus, Tospoviridae, and Tymovirus.
• 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: 161 to 185, or 186; and/or (b) the HRV 3’ RR comprises at least one RNA secondary structure adopted by an
• 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
• 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.
• Agent Ref P14354WO00 - 61 -
• 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. [0177] 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.
• cleavable sequence is optionally a self-cleaving ribozyme, a self-cleaving inducible ribozyme, or an siRNA or miRNA recognition site.
• 29. An agricultural formulation comprising the recombinant RNA molecule of any one of embodiments 1 to 28.
• RNA binding proteins comprise an RNA recognition motif.
• viral capsid protein is heterologous to the partitivirus.
• 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. [0186] 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.
• 36 The cell of embodiment 34, wherein the cell comprises a recombinant DNA molecule which encodes the recombinant RNA molecule.
• 37 An expression system comprising: (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. [0189] 38.
• 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
• the encapsidation recognition element (ERE) is a partitivirus ERE, wherein the viral capsid protein in the cell is a partitivirus capsid protein, and wherein the RNA molecule is encapsidated by the partitivirus capsid protein.
• invention 37, 38, or 39 further comprising the reverse complement of the recombinant RNA molecule.
• 41 The expression system of any one of embodiments 37 to 40, wherein the cell is a bacterial cell, a plant cell, a fungal cell, an insect cell, or an invertebrate animal cell.
• 42 The expression system of any one of embodiments 37 to 40, wherein 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 hrvRdRP, optionally wherein the hrvRdRP recognizes the HRV 5’ or 3’ replication region and/or the subgenomic promoter.
• CP viral capsid protein
• RBP RNA-binding protein
• RBP RNA-binding protein
• 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. [0197] 46. The expression system of any one of embodiments 37 to 45, wherein the cell is a plant cell. [0198] 47.
• the expression system of embodiment 46 wherein the plant cell contains a partitivirus 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.
• 48 The expression system of embodiment 47, wherein the partitivirus occurs naturally in the plant cell.
• 49 A method of providing a synthetic partitivirus satellite RNA to a plant, comprising contacting the plant with the recombinant RNA molecule of any one of embodiments 1 to 28. [0201] 50.
• contacting comprises spraying, dusting, injecting, or soaking the plant or a part thereof with the recombinant RNA molecule or the formulation.
• 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.
• Agent Ref P14354WO00 - 70 - [0203] 52.
• a method of establishing a synthetic partitivirus 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 partitivirus satellite RNA from the recombinant RNA molecule.
• the plant cell comprises a partitivirus and wherein the RdRP protein is provided to the plant cell by the partitivirus.
• the capsid protein comprises a partitivirus capsid protein and the ERE is recognized by the partitivirus 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 partitivirus 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. [0208] 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 partitivirus RNA from the recombinant RNA molecule, and wherein the cargo RNA molecule effects the phenotypic change. [0209] 58.
• 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 Agent Ref: P14354WO00 - 71 - plant cell, optionally wherein the 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 partitivirus, a fungus, an oomycete, and an invertebrate.
• the pest or pathogen is selected from the group comprising a bacterium, a virus other than a partitivirus, 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
• 66 The method of any one of embodiments 57 to 62, wherein the phenotypic change comprises an increase in the plant’s resistance to stress, optionally wherein the stress comprises at least one abiotic stress comprising nutrient stress, light stress, water stress, heat stress, and/or cold stress, or optionally wherein the stress comprises at least one biotic stress comprising crowding, shading, or allelopathy.
• the stress comprises at least one abiotic stress comprising nutrient stress, light stress, water stress, heat stress, and/or cold stress, or optionally wherein the stress comprises at least one biotic stress comprising crowding, shading, or allelopathy.
• 67 The method of any one of embodiments 57 to 66, wherein 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 partitivirus 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 method of any one of embodiments 57 to 69, wherein the recombinant RNA further comprises its reverse complementary RNA molecule. [0222] 71.
• 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 partitivirus 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. [0223] 72.
• a method of manufacturing a synthetic partitivirus 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 partitivirus 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 partitivirus 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
• the plant propagule of embodiment 76 wherein the plant propagule is a seed, a seedling, root, stem, leaf, shoot, tuber, rhizome, stolon, bulb, explant, embryo, or callus.
• 78. The plant propagule of embodiment 76 or 77, wherein the plant propagule is a mosaic comprising both plant cells comprising the recombinant RNA molecule and plant cells lacking the recombinant RNA molecule.
• 79 The plant propagule of embodiment 76, 77, or 78, wherein the plant propagule lacks DNA encoding the recombinant RNA molecule.
• a plant comprising the recombinant RNA molecule of any one of embodiments 1 to 28 and a partitivirus 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 partitivirus satellite RNA.
• a heterologous RNA virus RdRP which recognizes an HRV 5’ or 3’ replication region and/or the subgenomic promoter in the synthetic partitivirus satellite RNA.
• the plant of embodiment 81 wherein the plant is of the family Asteraceae, Brassicaceae, Cannabaceae, Caryophyllaceae, Cucurbitaceae, Fabaceae, Poaceae, Rosaceae, or Solanaceae.
• the partitivirus RdRP comprises a protein having at least 85%, 90%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 592-610, 704, or 710.
• partitivirus RdRP the partitivirus RdRP, the 5’ RNA replication element, and/or the 3’ RNA replication element are derived from a partitivirus comprising one or both of the partitivirus RdRP, 5’ RNA replication element, and/or 3’ RNA replication elements.
• RNA virus RdRP which recognizes an HRV 5’ or 3’ replication region and/or the subgenomic promoter in the synthetic partitivirus satellite RNA
• the heterologous RNA virus RdRP is an Alphaflexivirus, Betaflexivirus, Bromovirus, Celavirus, Closterovirus, Comovirus, Potexvirus, Agent Ref: P14354WO00 - 74 - Potyvirus, Tobamovirus, Tombusvirus, Tospoviridae, Trivirinae, Tymovirus, Varicosavirus, or Secoviridae RdRP set forth in Table 7. [0243] 92.
• a partitivirus satellite system that is self-replicating when introduced into a plant or plant cell, comprising: (1) a recombinant partitivirus satellite RNA of any one of embodiments 1 to 28;and (2) an exogenous partitivirus that is capable of replication in the plant or plant cells and that encodes the partitivirus RdRP that recognizes the 5’ and 3’ replicase recognition sequences in the recombinant partitivirus satellite RNA, optionally wherein the partitivirus 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 partitivirus satellite RNA. [0244] 93.
• the self-replicating partitivirus satellite system of embodiment 92 wherein the exogenous partitivirus is endemic or native to a different species, variety, or germplasm of plant.
• the partitivirus 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 partitivirus satellite RNA, and the heterologous RNA virus RdRP is optionally an Alphaflexivirus, Betaflexivirus, Bromovirus, Closterovirus, Comovirus, Potexvirus, Potyvirus, Tobamovirus, Tombusvirus, Tospoviridae, Trivirinae, Tymovirus, 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.
• 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. [0251] 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 Agent Ref: P14354WO00 - 75 - operably linked, a 5’ RNA replication element recognized by a partitivirus 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 subgen
• 103 The expression system of embodiments 101 or 102, wherein the cell is a bacterial cell, a plant cell, a fungal cell, or an 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. [0255] 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’ 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.
• MP viral movement protein
• RdRP protein and/or the RdRP protein that recognizes an HRV 5’ or 3’ 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.
• 107 The expression system of any one of embodiments 101 to 106, wherein the cell is a plant cell.
• 108 The expression system of any one of embodiments 101 to 106, wherein the cell is a
• the expression system of 107 wherein the plant cell contains a partitivirus 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 partitivirus 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 partitivirus 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 subgeno
• RdRP partitivirus RNA- Agent Ref
• 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.
• 128 The method of any one of embodiments 122 to 127, wherein the mixed population is located within a plant or a plant part.
• 129 The method of any one of embodiments 122 to 128, wherein the plant or plant part is screened or selected for presence of the recombinant RNA molecule prior to isolating the plant propagule.
• 130 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.
• 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.
• the method further comprises recovering F 1 seed or F 1 progeny comprising the recombinant RNA from the plant.
• 144 The method of 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 partitivirus 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 partitivirus RdRP.
• a method of providing a synthetic partitivirus 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 partitivirus 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 partitivirus 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 partitivirus 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.
• 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.
• Agent Ref P14354WO00 - 80 -
• 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 partitivirus RNA-dependent RNA polymerase (RdRP).
• RdRP partitivirus 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. [0322] 171. The method of any one of embodiments 168 to 170, wherein the barcode RNA molecule comprises a random sequence. [0323] 172. The method of any one of embodiments 168 to 170, 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. [0324] 173.
• any one of embodiments 168 to 172, wherein the barcode RNA molecule is up to about 3.2 kb in length. [0325] 174.
• 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.
• any one of embodiments 168 to 177 further comprising isolating an F 1 progeny plant or seed comprising at least one cell comprising the partitivirus 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. [0333] 182.
• a method of identifying a barcoded plant, plant part, or plant cell the method 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. Summary of Tables Table 1.
• RNA Replication Element DNA Coding Sequences SEQ Description 1 ID Dot-Bracket structure of encoded RNA ... Agent Ref: P14354WO00 - 82 - SEQ Description 1 ID Dot-Bracket structure of encoded RNA NO 2 ..) ... ). . .. ) ) ). ... Agent Ref: P14354WO00 - 83 - SEQ Description 1 ID Dot-Bracket structure of encoded RNA NO 2 (( ).) Agent Ref: P14354WO00 - 84 - SEQ Description 1 ID Dot-Bracket structure of encoded RNA NO 2 ...
• Agent Ref: P14354WO00 - 139 - SEQ Description 1 ID Dot-Bracket structure of encoded RNA NO 2 ).
• Agent Ref P14354WO00 - 158 - SEQ Description 1 ID
• tRNA-like sequences Name 1 of Arabidopsis gene containing tRNA-like Sequence2 Agent Ref: P14354WO00 - 162 - Name 1 of Arabidopsis gene containing tRNA-like a n confer cell-to-cell mobility Sequen 2 s equence that c ce Agent Ref: P14354WO00 - 163 - Name 1 of Arabidopsis gene containing tRNA-like e that can confer cell-to-cell mobility S 2 s equenc equence esource (TAIR) database opsis.org.” Table 5.
• TAIR equenc equence esource
• IRES sequences IRES source NCBI Accession ID 1 Sequence 2 Cardiovirus A SEQ ID NO: 124 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. Agent Ref: P14354WO00 - 164 - Table 6.
• Agent Ref P14354WO00 - 169 - NT AA Seq. Seq. NCBI SE (SEQ ID C Agent Ref: P14354WO00 - 170 - NT AA Seq. Seq. NCBI SE (SEQ ID C s, s, , , , s s, s, er or entres n t e word wde web nternet database ncb[dot]nm[dot]n .gov/nuccore.
• RNAi NCBI Accession Examples of target Tar et Gene ID 1 Se uence Taret athoen cro Agent Ref: P14354WO00 - 171 - Cargo (RNAi NCBI Accession Examples of target Target Gene) ID 1 Sequence Target pathogen crop SEQ ID Agent Ref: P14354WO00 - 172 - Cargo (RNAi NCBI Accession Examples of target Target Gene) ID 1 Sequence Target pathogen crop Polygalacturonase SEQ ID tomato wheat er o e es e wo w e we e e aaase c o o .gov uccoe. Table 11.
• Agent Ref: P14354WO00 - 179 - NCBI or TAIR Gene Source Effector Accession NT AA 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 13 Bioactive Plant peptides Peptide name Sequence Function SEQ ID NO: Increase nodulation in soybean (improved nitrogen fixation, a e Ribozymes Ribozyme NCBI Accession ID Rfam ID Sequence 1 KV7674721 Agent Ref: P14354WO00 - 180 - Ribozyme NCBI Accession ID Rfam ID Sequence 1 Hammerhead targeting pepper Table 15 RNA Aptamers RNA aptamer Sequence 1 Pe er SEQ ID NO: 439 Agent Ref: P14354WO00 - 181 - 1 DNA equivalents of the RNA sequences are also contemplated and can be obtained from the RNA sequences
• Subgenomic promoter Sequence 1 Pea early-browning virus CP SEQ ID NO: 453 T b l i i TCM CP* SE ID NO 454 ed and can be obtained from the DNA *See Goulden et al. (1990) Nucleic Acids Res., 18:4507 – 4512, DOI:10.1093/NAR/18.15.4507.
• a partitivirus 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 partitivirus 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 partitivirus virus.
• COMSAT partitivirus satellite
• RNAi antiviral inhibitory RNA
• Two COMSATs comprising partitivirus 5’ and 3’ RNA replication elements 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: 713 and 714.
• the elements of the satellites are set forth in Tables 21 and 22. Table 21. Genetic element Nucleotide position in SEQ ID NO: 713 Agent Ref: P14354WO00 - 200 - Table 22.
• a partitivirus 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 partitivirus 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 partitivirus virus.
• COMSAT partitivirus satellite
• Two COMSATs comprising partitivirus 5’ and 3’ RNA replication elements 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: 711 to 712.
• the elements of the satellites are set forth in Table 23 and 24.
• Antiviral COMSATs e.g., of Examples 1 to 2
• control RNAs e.g., RNAs lacking the antiviral cargo
• target host plants such as pepper or tomato plants via micro- bombardment (“biolistic” delivery) using a “gene gun” or by bacterially mediated (e.g., by Agrobacterium) transient expression.
• Antiviral COMSATs are prepared either as in vitro transcribed (IVT) products (capped or uncapped), or as Agrobacterium binary vectors.
• IVT in vitro transcribed
• 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 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, Agent Ref: P14354WO00 - 202 - TMV), for example, by mechanical infection.
• the viral pathogen of interest e.g., cucumber mosaic virus, CMV, or tobacco mosaic virus, Agent Ref: P14354WO00 - 202 - TMV
• Infectious preparations of such acute viral pathogens are 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.
• 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 titers 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
• viral titre is qualitatively evaluated by viral disease symptoms, or by proxy measurement (e.g., measuring GFP expressed by GFP-fused viral constructs).
• 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).
• the viral titre (as fold changed, relative to an endogenous reference gene) is compared to that of controls.
• Example 4. Replication of PCV1 in bell pepper plants.
• Agrobacterium strains having the T-DNAs encoding the RDRP and CP genomes of PCV1 set forth in SEQ ID NOs: 575 and 576 were infiltrated into bell pepper (Capsicum annuum) leaves.
• Table 25 Genetic element Nucleotide position in SEQ ID Agent Ref: P14354WO00 - 203 - Genetic element Nucleotide position in SEQ ID NO: 575 PCV1 RDRP 5’UTR 8390 to 8481 Genetic element Nucleotide position in SEQ ID NO: 576 ter ve ays, was co ecte rom t e s te o n ltration.

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