Artificial tombusvirus group satellite rnas

Abstract

Synthetic Tombusviridae satellite RNA molecules and satellite particles containing the same are disclosed. Also disclosed are synthetic Tombusviridae satellite RNA molecules containing internal heterologous RNA virus (HRV) amplicons. Methods of using the Tombusviridae satellite RNA molecules and satellite particles containing the same to alter plant phenotype, improve stress resistance of plants, and improve pest and pathogen resistance of plants are also disclosed.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to provisional application filed May 3, 2023, U.S. Serial No. 63 / 499,740, which is hereby incorporated by reference in its entirety.

[0003] Merging of sequence lists

[0004] This application includes a sequence list, which has been electronically submitted in XML file format and is hereby incorporated herein by reference in its entirety. The XML file, created on April 28, 2024, is named P14422WO00.xml and has a size of 1,455,916 bytes. An XML file, created on May 1, 2023, named P14422US00.xml, has a size of 1,028,856 bytes and was filed on May 3, 2023, in Provisional Application U.S. Serial No. 63 / 499,740, which is also incorporated herein by reference in its entirety. Background Technology

[0005] There is a need in the field for modified polynucleotides to improve the phenotype and genotype of organisms; particularly for agricultural applications to improve plants (e.g., crops).

[0006] Members of the Tomato Cluster Dwarf Virus Family are RNA viruses with a single-component (except for the Dianthovirus genus, which is two-component), linear, single-stranded genome of about 4 to 6 kilobases (kb). Summary of the Invention

[0007] This disclosure provides a recombinant RNA molecule comprising, from its 5' end to its 3' end: (a) a 5' RNA replication element recognized by Tomato dwarfvirus RNA-dependent RNA polymerase (RdRP); (b) a cargo RNA molecule (cargo RNA sequence); and (c) a 3' RNA replication element recognized by RdRP; wherein the 5' RNA replication element, the cargo RNA molecule, and the 3' RNA replication element are operatively linked, and wherein the cargo RNA molecule is heterologous to the 5' RNA replication element and the 3' RNA replication element. In some embodiments, (i) the 5' RNA replication element and the 3' RNA replication element are obtained from the same Tomato dwarfvirus family genome or from Tomato dwarfvirus family genomes that have at least 85%, 90%, 95%, 98%, or 99% sequence identity with each other and are optionally related; (ii) the 5' RNA replication element, the 3' RNA replication element, and the RdRP are obtained from the same Tomato dwarfvirus family genome or from Tomato dwarfvirus family genomes that have at least 85%, 90%, 95%, 98%, or 99% sequence identity with each other and are optionally related; or (iii) the 5' RNA replication element, the 3' RNA replication element, and / or the RdRP coding region are obtained from different Tomato dwarfvirus family genomes, and the members of each respective set of the 5' RNA replication element, the 3' RNA replication element, and / or the RdRP coding region have at least 85%, 90%, 95%, 98%, or 99% sequence identity with each other. In some embodiments, the RNA molecule comprises: at least one heterologous RNA virus (HRV) amplicon oriented with a sense or antisense relative to the first 5' RNA replication element, comprising: I. (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 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 operatively linked; or II. a heterologous RNA virus (HRV) subgenomic promoter operatively linked to the cargo RNA molecule; wherein the subgenomic promoter is recognized by a heterologous RNA virus RNA-dependent RNA polymerase (hrvRdRP). Agricultural formulations comprising the recombinant RNA disclosed herein, as well as bacterial, fungal, plant, insect, and invertebrate cells, are also provided.

[0008] An expression system is also provided, comprising: (a) an RNA molecule containing the recombinant RNA molecule disclosed herein; 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. In some embodiments, the cell further comprises one or more of the following: (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 effector; (iii) an RNA cleaving agent that cleaves the RNA molecule; (iv) a second RNA-dependent RNA polymerase (second RdRP) protein that recognizes the HRV 5' or 3' replication region and / or the subgenomic promoter in the RNA molecule; (v) a viral mobile protein (MP); (vi) a heterologous RNA virus (HRV); or (vii) hrvRdRP, optionally wherein the hrvRdRP recognizes the HRV 5' or 3' replication region and / or the subgenomic promoter.

[0009] A method for providing synthetic Tomato Cluster Dwarf Virus family satellite RNA to plants is also provided, the method comprising contacting the plant with any recombinant RNA molecule disclosed herein.

[0010] A method for constructing synthetic Tomato Cluster Dwarf Virus Family satellite RNA in plant cells is also provided, the method comprising: providing a plant cell with any recombinant RNA molecule disclosed herein, wherein the plant cell contains an RdRP protein that recognizes 5' RNA replication elements and 3' RNA replication elements, wherein the RNA molecule optionally contains a capsidization recognition element (ERE) and is, or may be, capsidated by a capsid protein, thereby wherein the RdRP protein catalyzes the synthesis of the synthetic Tomato Cluster Dwarf Virus Family satellite RNA from the recombinant RNA molecule.

[0011] A method for obtaining phenotypic changes in plants or plant cells is also provided, comprising: providing a plant or plant cell with any recombinant RNA molecule disclosed herein, wherein the cargo RNA molecule contains RNA that achieves the phenotypic change of the plant or plant cell compared to a plant or plant cell lacking the recombinant RNA, wherein the plant or plant cell contains an RdRP protein that recognizes the 5' RNA replication element and the 3' RNA replication element and catalyzes the synthesis of synthetic Tomato dwarf Virus RNA from the recombinant RNA molecule, and wherein the cargo RNA molecule achieves the phenotypic change. In some embodiments, the method further comprises providing the plant with hrvRdRP that recognizes the HRV 5' or 3' replication region and / or the subgenomic promoter in the synthetic Tomato dwarf Virus satellite RNA, optionally wherein the hrvRdRP is provided by introducing recombinant DNA or RNA encoding the hrvRdRP into the plant or a portion thereof.

[0012] A method for manufacturing synthetic Tomato Cluster Dwarf Virus family satellite particles is also provided, the method comprising combining any recombinant RNA molecule disclosed herein with a viral capsid protein, wherein the recombinant RNA molecule contains a capsidation recognition element (ERE), and wherein the ERE provides capsidation of the RNA by the viral capsid protein.

[0013] Plant propagules are also provided, which contain any recombinant RNA molecules disclosed herein and Tomato Cluster Dwarf Virus Family RdRPs, optionally wherein the plant propagules further contain heterologous RNA virus RdRPs that recognize the HRV5' or 3' replication region and / or subgenomic promoter in synthetic Tomato Cluster Dwarf Virus Family satellite RNA. In some embodiments, the plant propagule further comprises a heterologous RNA virus RdRP that recognizes the HRV 5' or 3' replication region and / or subgenomic promoter in the synthetic Tomato Cluster Dwarf Virus family satellite RNA, and said heterologous RNA virus RdRP is an RdRP from the genera Alphaflexivirus, Betaflexivirus, Celavirus, Bromovirus, Closterovirus, Comovirus, Potato Xvirus, Potato Yvirus, Tobamovirus, Tomato Cluster Dwarf Virus, Tospoviridae, Trivirinae, Tymovirus, Varicosavirus, or Secoviridae.

[0014] Plants containing any recombinant RNA molecules disclosed herein and RdRPs from the Tomato Cluster Dwarf Virus Family are also provided, optionally wherein the plant propagules further contain heterologous RNA virus RdRPs that recognize the HRV 5' or 3' replication region and / or subgenomic promoter in synthetic Tomato Cluster Dwarf Virus Family satellite RNA.

[0015] A self-replicating Tomato dwarfvirus satellite system that, when introduced into a plant or plant cell, comprises: (1) any recombinant Tomato dwarfvirus satellite RNA disclosed herein (e.g., a recombinant RNA molecule); and (2) an exogenous Tomato dwarfvirus capable of replicating in the plant or plant cell and encoding a Tomato dwarfvirus RdRP that recognizes 5' and 3' replicase recognition sequences in the recombinant Tomato dwarfvirus satellite RNA, optionally wherein the Tomato dwarfvirus satellite system further comprises a heterologous RNA virus RdRP that recognizes the HRV 5' or 3' replication region and / or subgenomic promoter in the synthetic Tomato dwarfvirus satellite RNA. Attached Figure Description

[0016] Figure 1 A non-limiting embodiment of the structure of a Tomato Cluster Dwarf Virus family satellite construct is shown. In this embodiment, the 5' RNA replication element is labeled "5'RRE," and the 3' RNA replication element is labeled "3'RRE."

[0017] Figure 2 A non-limiting embodiment of a Tomato Cluster Dwarf Virus family satellite construct containing a heterologous RNA virus (HRV) amplicon is shown, the amplicon comprising: (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). Both sense and antisense orientations of the HRV amplicon relative to the Tomato Cluster Dwarf Virus family 5' RNA replication element are shown. In some embodiments, at least one cleavable sequence is located at: (i) the 5' of the 5' RNA replication element or the 3' of 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 a siRNA or miRNA recognition site. In some embodiments, a subgenomic promoter and / or IRES is operatively linked to the cargo RNA.

[0018] Figure 3 A non-limiting embodiment of a Tomato Cluster Dwarf Virus Family satellite construct containing a heterologous RNA virus (HRV) amplicon comprising a heterologous RNA virus (HRV) subgenomic promoter (HRV sgp) recognized by a heterologous RNA virus RNA-dependent RNA polymerase (hrvRdRP) and operatively linked to a cargo RNA molecule is shown. An embodiment in which the HRV sgp and cargo RNA are oriented with respect to the Tomato Cluster Dwarf Virus Family 5'RRE is shown in a sense orientation and an antisense orientation, respectively.

[0019] Figure 4 A satellite construct of the Tomato Cluster Dwarf Virus family is described, comprising a heterologous RNA virus (HRV) subgenomic promoter (HRV sgp) wherein: (i) an HRV sgp is operatively linked to a cargo RNA; and (ii) an HRV sgp is operatively linked to an RNA encoding hrvRdRP (which recognizes both HRV sgp) (i.e., can drive the expression of the operatively linked hrvRdRP and cargo RNA). In some embodiments, an IRES is operatively linked to the cargo RNA and / or an IRES is operatively linked to the RNA encoding hrvRdRP.

[0020] Figure 5 Non-limiting embodiments of a Tomato Cluster Dwarf Virus Family satellite construct containing a heterologous RNA virus (HRV) amplicon comprising a heterologous RNA virus (HRV) subgenomic promoter (HRV sgp) recognized by a heterologous RNA virus RNA-dependent RNA polymerase (hrvRdRP) and operatively linked to a cargo RNA molecule flanked by HRV 5'RR and HRV 3'RR are depicted. Embodiments in which the HRV sgp and cargo RNA are oriented with or without sense relative to the Tomato Cluster Dwarf Virus Family 5'RR are shown. The HRV 5'RR and 3'RR flanked by the cargo RNA provide hrvRdRP-mediated RNA replication, the RNA comprising HRV 5'RR, cargo RNA, and HRV 3'RR from 5' to 3'. In some embodiments, the HRV 5'RR and 3'RR are flanked by ribozymes.

[0021] Figure 6 A symbiotic satellite carrying cargo RNA molecules was depicted, comprising an HRV (“HRV1”, e.g., tobacco mosaic virus, TMV) amplicon designed for amplification via an HRV (“HRV1”, e.g., TMV) RdRP that binds to a replication region or subgenomic promoter. This symbiotic satellite is a Tomato Cluster Dwarf Virus family satellite. The resulting transcript includes RNA encoding an HRVRdRP (“HRV1 RdRP”, solid square) (which can further amplify the HRV amplicon) and RNA encoding another cargo (solid circle). In the absence of the symbiotic virus, no amplification of the symbiotic satellite occurs.

[0022] Figure 7A symbiotic satellite with cargo RNA molecules is depicted, comprising an HRV (HRV1, e.g., tobacco mosaic virus (TMV)) amplicon, designed for amplification via an HRVRdRP (“HRV1 RdRP”, solid square) that binds to the HRV1 replication region, wherein the symbiotic satellite is a Tomato Cluster Dwarf Virus family satellite. The resulting transcript comprises RNA encoding an HRV(HRV1) RdRP that can further amplify the HRV amplicon. The HRV1 amplicon includes a sequence (indicated in italics) of an HRV2 amplicon that encodes and / or does not encode cargo (solid circle) and is designed for amplification in the presence of a second acute virus RdRP (“HRV2 RdRP”, hexagonal symbol), which can be provided, for example, by introducing a second acute virus (“HRV2”, e.g., cowpeamosaic virus (CPMV)) into the plant. In the absence of the symbiotic virus, no amplification of the symbiotic satellite occurs.

[0023] Figure 8 A symbiotic satellite carrying a cargo RNA molecule was depicted, comprising an HRV (“HRV1”, e.g., tobacco mosaic virus, TMV) amplicon designed for amplification via an HRV (“HRV1”, e.g., TMV) RdRP binding to either of two subgenomic promoters, wherein the symbiotic satellite is a Tomato Cluster Dwarf Virus family satellite. The resulting transcript comprises RNA encoding an HRV RdRP (“HRV1 RdRP”, a solid square) (which can further amplify the HRV amplicon) and RNA encoding a non-coding RNAi cargo, the sense and antisense strands of which are formed during the amplification process to yield a double-stranded RNA molecule (dsRNA) for silencing a target gene. In the absence of the symbiotic virus, no amplification of the symbiotic satellite occurs. Detailed Implementation

[0024] definition

[0025] As used herein, the term “and / or” should be considered as a specific disclosure of each of two specified features or components, with or without the other. Therefore, the term “and / or” as used in phrases such as “A and / or B” herein is intended to include “A and B”, “A or B”, “A” (alone), and “B” (alone). Similarly, the term “and / or” as used in phrases such as “A, B, and / or C” is intended to cover each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0026] As used in this article, the terms “F1”, “F2”, etc., refer to offspring plants or seeds of parent plants that have been self-pollinated or hybridized with another plant.

[0027] As used herein, when referring to a second element to describe a first element, the term "heterogeneous" means that the first and second elements do not exist in nature in the arrangement described. For example, a heterologous nucleic acid molecule or sequence is a nucleic acid molecule or sequence that: (a) is not natural for the cell expressing it; (b) is linked or fused to a nucleic acid molecule or sequence that is not linked or fused to in nature or not in the same manner as in nature; (c) has been artificially altered or mutated relative to its natural state; or (d) has had its expression altered compared to its natural expression level under similar conditions. For example, a "heterogeneous promoter" is used to drive transcription of a sequence not naturally transcribed by said promoter (e.g., a eukaryotic promoter used to drive transcription of a DNA molecule encoding a Tomato dwarfvirus RNA sequence); thus, a "heterogeneous promoter" sequence can be included in an expression construct using recombinant nucleic acid technology. In embodiments, recombinant polynucleotides (such as those provided in this disclosure) comprise genetic sequences of two or more different Tomato dwarfviruses that are "heterogeneous" because they do not naturally coexist. In some embodiments, “heterogeneous” refers to a molecule or discrete portion of a molecule; for example, it refers to a cargo RNA molecule (e.g., a nucleic acid, such as RNA encoding a protein, ssRNA, regulatory RNA, interfering RNA, or guide RNA), which may be part of a larger molecule, or to a structure that is not naturally present in Tomato dwarf virus family plants (e.g., including structures such as: promoters (e.g., for DNA-dependent RNA polymerases) or subgenomic promoters (e.g., for RNA-dependent RNA polymerases), RNA effectors, RNA cleavage agent recognition sites, or containing or encoding polynucleotides such as: expression-enhancing elements, capsid recognition elements (EREs), selectable or scoreable markers, DNA aptamers, RNA aptamers, transcription factor binding sites, internal ribosome entry sites (IRES), DNA spacers, RNA cleavage agent recognition sites, tRNA-like elements, or transcript-stabilized or transcript-destabilized RNA sequences).

[0028] As used herein, the terms “include,” “includes,” and “including” should be interpreted as having at least the features they refer to without excluding any additional unspecified features. The terms “comprise,” “comprises,” and “comprising” respectively mean “include,” “includes,” and “including.”

[0029] As used herein, the term "internal ribosome entry site" or "IRES" refers to a sequence (e.g., an RNA sequence) capable of recruiting ribosomes and translation machinery to initiate translation from an RNA sequence. IRES elements are typically between 100 and 800 nucleotides. Appropriate IRES can be obtained from plant and plant virus IRES sequences, such as the encephalocarditis virus IRES (ECMV), the corn hsp101 IRES 5'UTR, the tobacco mosaic virus crTMV CR-CP 148 IRES that infects cruciferous plants, the tobacco etch virus (TEV) IRES 5'UTR, and the hibiscus chlorotic ringspot virus (HCRSV) IRES. Furthermore, in the embodiments, the IRES sequence is derived from non-plant eukaryotic virus sequences, including but not limited to: acute bee paralysis virus (ABPV), classical swine fever virus (CSFV), Coxsackievirus B3 (CVB3), encephalomyocarditis virus (ECMV), enterovirus 71 (E71), hepatitis A virus (HAV), human rhinovirus (HRV2), human lymphotropic virus (HTLV), polyomavirus (PV), and zea mays (ZmHSP101). Examples of IRES sequences that can be used in the compositions and methods described herein are shown in Table 5.

[0030] As used herein, the phrase “operably linked” refers to a parallel relationship in which the components described in this way are in a relationship that allows them to function in their intended manner. For example, if a promoter provides transcription or expression of a coding sequence, then the promoter is operably linked to the coding sequence.

[0031] As used herein, the term “percentage of identity” refers to the percentage (%) of sequence identity relative to a reference polynucleotide or polypeptide sequence after alignment using standard techniques. Alignments for determining the percentage of identity of nucleic acid or amino acid sequences can be performed in various ways within the capabilities of those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, PSI-BLAST, or Megalign software. In some embodiments, the software is MUSCLE (Edgar, Nucleic Acids Res., 32(5): 1792-1797, 2004). Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required to achieve maximum alignment across the full length of the sequences being compared. For example, in this embodiment, the sequence alignment computer program BLAST is used to generate the percentage of identity values ​​(Altschul et al. (1990) J. Mol. Biol., 215: 403-410). As an illustration, the percentage of sequence identity of a given nucleic acid or amino acid sequence A with, compared to, or relative to a given nucleic acid or amino acid sequence B (which can be alternatively expressed as the phrase: a given nucleic acid or amino acid sequence A having a certain percentage of sequence identity with, compared to, or relative to a given nucleic acid or amino acid sequence B) is calculated as follows: 100 multiplied by (fraction X / Y), where X is the number of nucleotides or amino acids in A and B that are scored as the same match by the sequence alignment program (e.g., BLAST), and where Y is the total number of nucleotides or amino acids in B.

[0032] As used herein, the term "plant" includes the whole plant as well as any offspring, cells, tissues, or parts of the plant. The term "plant part" includes any one or more parts of the plant, including, for example, but not limited to: seeds (including mature and immature seeds); plant cuttings; plant cells; plant cell cultures; or plant organs (e.g., pollen, mature or immature embryos, flowers, fruits, buds, leaves, roots, stems, and explants). In embodiments, plant tissues or plant organs are or include seeds, protoplasts, callus, or any other group of plant cells organized into structural or functional units. In embodiments, plant cells or tissue cultures are capable of regenerating plants having the physiological and morphological characteristics of the plant from which said cells or tissues are derived, and are capable of regenerating plants having substantially the same genotype as said plant. Regenerable cells in plant cells or tissue cultures may include embryos, protoplasts, meristematic cells, callus, pollen, leaves, anthers, roots, root tips, flowers, and / or stems. In contrast, some plant cells cannot regenerate to produce plants and are referred to herein as "non-regenerable" plant cells.

[0033] As used in this article, the term "transcriptome" refers to the sum of all RNA molecules expressed in a cell. Such RNA molecules include mRNA, tRNA, ribosomal RNA, miRNA, viral RNA (genomic and subgenomic), and long non-coding RNA.

[0034] In the event of any inconsistency between the foregoing definition and the definition provided in any patent or non-patent reference incorporated herein by reference, any patent or non-patent reference cited herein, or any patent or non-patent reference found elsewhere, it shall be understood that the foregoing definition shall be used herein.

[0035] Unless otherwise stated, the nucleic acid sequences described herein are given in a 5' to 3' orientation when read from left to right. As specified, the nucleic acid sequences may be provided as DNA or RNA. Furthermore, due to known codon degeneracy, different nucleic acid sequences can encode the same polypeptide sequence, and such modified nucleic acid sequences (e.g., for codon optimization purposes for a given species) are within the scope of this disclosure. Where terms are provided in the singular, they also cover aspects of the invention described by the plural form of the terms.

[0036] This disclosure specifically provides recombinant polynucleotides (e.g., recombinant DNA, recombinant RNA, recombinant ssRNA, recombinant dsRNA, recombinant vectors, etc.) comprising one or more sequences of or derived from Tomato dwarfviruses; particularly 5' or 3' RNA replication elements recognized by Tomato dwarfvirus RNA-dependent RNA polymerase (RdRP). This disclosure further relates to methods for preparing and using such recombinant polynucleotides, for example, by expressing heterologous cargo sequences in plants using such recombinant polynucleotides, and optionally thereby modifying the expression of endogenous target sequences and / or the genotype or phenotype of the plant. In embodiments, Tomato dwarfviruses are symbiotic Tomato dwarfviruses, i.e., Tomato dwarfviruses are specific to or natural to a given eukaryotic host (e.g., host plant), do not cause significant negative effects on the host (i.e., are considered non-pathogenic), typically exist in persistent but low populations (i.e., low viral titers), and are typically vertically transmitted to the host's offspring.

[0037] In one aspect, this disclosure relates to a recombinant DNA molecule comprising a promoter that is functional in the cell and operatively linked to a DNA sequence encoding an RNA molecule. The RNA molecule comprises, in a 5' to 3' sequence: (a) a 5' RNA replication element capable of being recognized by Tomato dwarfvirus RNA-dependent RNA polymerase (RdRP); (b) a cargo RNA sequence; and (c) a 3' RNA replication element capable of being recognized by Tomato dwarfvirus RdRP. Figure 1Examples of general structures of DNA polynucleotides encoding Tomato dwarfvirus family satellites are shown, wherein in some embodiments, the 5' RNA replication element corresponds to the 5' untranslated region (UTR) of the Tomato dwarfvirus family virus, and wherein the 3' RNA replication element corresponds to the 3' untranslated region (UTR) of the Tomato dwarfvirus family virus. In some embodiments, the 5' RNA replication element and / or the 3' RNA replication element comprises nucleotides extending into the predicted coding sequence or open reading frame of the Tomato dwarfvirus family virus.

[0038] The recombinant DNA molecules provided herein may include promoters that are functional in cells (e.g., bacterial cells, plant cells, fungal cells, or animal cells) and are operatively linked to a DNA sequence encoding an RNA molecule (e.g., a 5' RNA replication element, a cargo RNA sequence; and a 3' RNA replication element; a ribozyme, an intron, or an RNA encoding a protein (e.g., a capsid protein, a mobile protein, RdRP, or an RdRP protein that recognizes the HRV 5' or 3' replication region and / or a subgenomic promoter).

[0039] In the embodiments, a functional promoter in plant cells provides systemic gene expression, or alternatively provides cell, tissue, or organ-specific gene expression, or expression that can be induced by external signals or agents (e.g., light, pathogens, wounds, stress, or hormones that can induce elements or chemical inducers) or elements that can regulate gene transcription in the cell cycle; such elements may be located in the 5' or 3' region of a natural gene or engineered into a polynucleotide.

[0040] Promoters include those from viruses, bacteria, fungi, animals, and plants. Suitable promoters can be used to drive expression via any RNA polymerase (e.g., RNA pol I, pol II, or pol III). Examples of promoters include those from cauliflower mosaic virus (e.g., p35S), bacteriophages (e.g., pT7), and plants (e.g., pATUBQ10). In some embodiments, the promoter is operatively linked to a nucleotide sequence encoding multiple guide RNAs, wherein the sequences encoding guide RNAs are separated by cleavage sites (such as nucleotide sequences encoding microRNA recognition / cleavage sites or self-cleaving ribozymes) (see, for example, Ferré-D'Amaré and Scott (2010) Cold Spring Harbor Perspectives Biol. [Cold Spring Harbor Perspectives in Biology], 2: a003574). In some embodiments, the promoter is a pol II promoter operatively linked to a nucleotide sequence encoding RNA. In some embodiments, the promoter operatively linked to one or more polynucleotides encoding elements of a genome editing system is a constitutive promoter driving DNA expression in plant cells. In some embodiments, the promoter drives DNA expression in the cell nucleus or organelles such as chloroplasts or mitochondria. Examples of constitutive promoters active in plant cells include the CaMV 35S promoter disclosed in U.S. Patent Nos. 5,858,742 and 5,322,938, the rice actin promoter disclosed in U.S. Patent No. 5,641,876, the maize chloroplast aldolase promoter disclosed in U.S. Patent No. 7,151,204, and cauliflower alkaloid synthase (NOS) and octopus alkaloid synthase (OCS) promoters from Agrobacterium tumefaciens. In some embodiments, the promoter operatively linked to one or more polynucleotides encoding elements of the genome editing system is a promoter from fig mosaic virus (FMV), the RUBISCO promoter, or a pyruvate-phosphate dual kinase (PDK) promoter (which is active in the chloroplasts of mesophyll cells). In some embodiments, the promoter is heterogeneous to the cell in which it functions and / or to other elements operatively connected to the promoter. All patent publications cited in this paragraph are incorporated herein by reference in their entirety.

[0041] The recombinant polynucleotides provided herein comprise or encode RNA molecules containing 5' and 3' RNA replication elements recognized by the Tomato Cluster Dwarf Virus Family RNA-dependent RNA polymerase (RdRP). In some embodiments, recognition of the Tomato Cluster Dwarf Virus Family RdRP is identified in an in vitro RdRP assay (e.g., adapted from Horiuchi et al., Plant Cell Physiol. 42(2): 197-203, 2001). In some embodiments, recognition of the Tomato Cluster Dwarf Virus Family RdRP is identified by an in vivo RdRP assay, wherein RNA containing 5' and 3' RNA replication elements is introduced into RdRP-containing cells, and RNA replication is measured (e.g., by RT-PCR assay or by assay of a reporter gene encoded by cargo RNA located in RNA containing 5' and 3' RNA replication elements). In some embodiments, RdRP-containing cells are engineered by introducing a gene or RNA molecule encoding RdRP into the cells. In other embodiments, the cells containing RdRP are cells containing a Tomato dwarfvirus expressing RdRP; in such embodiments, the Tomato dwarfvirus may be a virus that is natural to the cell or known to be naturally present in the cell, or it may be a non-natural Tomato dwarfvirus, or alternatively, a recombinant virus of any suitable virus family may be engineered to express Tomato dwarfvirus RdRP. In some embodiments, the recombinant polynucleotide (e.g., recombinant DNA or recombinant RNA) comprises 5' and 3' RNA replication elements derived from the same Tomato dwarfvirus genome or derived from Tomato dwarfvirus genomes having at least 85%, 90%, 95%, 98%, or 99% sequence identity with each other and optionally related. In embodiments, Tomato dwarfvirus genomes having at least 85%, 90%, 95%, 98%, or 99% sequence identity with each other are taxonomically related, for example, classified as belonging to the same genus, family, and / or target genome. In some embodiments, the recombinant polynucleotide (e.g., recombinant DNA or recombinant RNA) comprises a 5' RNA replication element, a 3' RNA replication element, and an RdRP derived from the same Tomato dwarfvirus family genome or from Tomato dwarfvirus family genomes that have at least 85%, 90%, 95%, 98%, or 99% sequence identity with each other and are optionally taxonomically related (e.g., classified as belonging to the same genus, family, and / or target genome). Non-limiting examples of 5' RNA replication elements and 3' RNA replication elements from the same Tomato dwarfvirus family genome, and the corresponding RdRP proteins that identify these 5' RNA replication elements and 3' RNA replication elements, include those shown in each row of Table 18.In some embodiments, the recombinant polynucleotide (e.g., recombinant DNA or recombinant RNA) comprises a 5' RNA replication element, a 3' RNA replication element, and / or an RdRP coding region derived from two Tomato dwarfvirus family genomes, wherein members of each pair of the 5' RNA replication element, 3' RNA replication element, and RdRP coding region of the two Tomato dwarfvirus family genomes have at least 85%, 90%, 95%, 98%, or 99% sequence identity with each other. In some embodiments, the recombinant polynucleotide (e.g., recombinant DNA or recombinant RNA) comprises a 5' RNA replication element and a 3' RNA replication element derived from different Tomato dwarfvirus family genomes. In some embodiments, the recombinant polynucleotide (e.g., recombinant DNA or recombinant RNA) comprises a 5' RNA replication element, a 3' RNA replication element, and an RdRP derived from different Tomato dwarfvirus family genomes. In some embodiments, the different Tomato dwarfvirus family genomes will have less than 85%, 80%, 75%, or 70% sequence identity with each other. In some embodiments, different Tomato Cluster Dwarf Virus family genomes will have sequence identity with each other of any one of 50%, 60%, or 65% to 70%, 75%, 80%, or 84%.

[0042] In some embodiments, combinations of 5' RNA replication elements, 3' RNA replication elements, and RdRPs shown in a single row of Table 18, or variants thereof having at least 85%, 90%, 95%, 98%, or 99% sequence identity with said 5' RNA replication elements, 3' RNA replication elements, and RdRPs, or variants thereof wherein the secondary structure of the RNA replication elements is conserved, are used together in the expression systems, plant cells, plant propagules, plants, or methods provided herein. In some embodiments, the 5' RNA replication elements and 3' RNA replication elements or variants thereof in a given row of Table 18 are operatively linked to cargo RNA and replicated by the corresponding RdRP or variant thereof in said row. In some embodiments, combinations of the 5' RNA replication element, the 3' RNA replication element, and RdRP shown in the given rows of Table 18 (i.e., SEQ ID NOs: 467, 468, and 469, respectively) or variants thereof disclosed herein or otherwise are used in the dicotyledonous cell-based expression systems, dicotyledonous cells, dicotyledonous propagules, dicotyledonous plants, or related dicotyledonous plant-based methods provided herein. In some embodiments, the aforementioned dicotyledonous plants are members of the following genera: Brassica, Capsicum, Citrus, Cucumis, Cucurbita, Cotton, Nicotiana, Olea, Solanum, or Glycine. In some embodiments, combinations of the 5' RNA replication element, the 3' RNA replication element, and RdRP shown in the given rows of Table 18 (i.e., SEQ ID NOs: 467, 468, 469, respectively) or variants thereof disclosed herein or otherwise are used in the monocotyledonous cell-based expression systems, monocotyledonous cells, monocotyledonous propagules, monocotyledonous plants, or related monocotyledonous plant-based methods provided herein. In some embodiments, the aforementioned monocotyledonous plants are members of the genera *Avena*, *Haloxylon*, *Oryza*, *Secale*, *Triticum*, *Sorghum*, or *Zea*.

[0043] Table 1 shows examples of DNA molecules encoding RNA molecules containing or having 5' and 3' RNA replication elements recognized by RdRP of the Tomato dwarf Virus Family. DNA molecules encoding RNA containing or having 5' RNA replication elements recognized by RdRP of the Tomato dwarf Virus Family include SEQ ID NO: 467. DNA molecules encoding RNA containing or having 3' RNA replication elements recognized by RdRP of the Tomato dwarf Virus Family include SEQ ID NO: 468.

[0044] Structural features identified in the 5' and 3' RNA replication elements of the Tomato dwarf Virus family (e.g., dsRNA hairpins and ssRNA loops) are shown in Table 1 using dotted bracket notation. The dotted bracket notation provided in Table 1 was generated using RNA Fold software, which is used to predict RNA secondary structures based on minimum free energy predictions of base pair probabilities. A dot '.' indicates an unpaired base, and brackets '(' or ')' indicate a paired base. The bracket notation is further described in Mattei et al., Nucleic Acids Research, 42(10): 6146-6157, 2014; Ramlan and Zauner: at the International Workshop on Computing With Biomolecules, E. Csuhaj-Varju, R. Freund, M. Oswald and K. Salomaa (eds.), 27 August 2008, Vienna, Austria, pp. 75-86, from: Austrian Computer Society, 2008; and Hofacker et al., Monatshefte für Chemie Chem. Monthly, 125: 167-188, 1994. The size of such structural features can range from 20, 30, or 40 to approximately 500 nucleotides (nt). These structural features can be used to design engineered polynucleotide sequences for use as RNA replication elements in the Tomato dwarf Virus family and / or to construct variants of the sequences shown in SEQ ID NO: 467-468 for use as 5' and 3' RNA replication elements. In some embodiments, one or more residues in the RNA secondary structure shown in Table 1 or in equivalent RNA are substituted with different nucleotides that maintain the RNA secondary structure (e.g., the presence or absence of base pairing). In some embodiments, the RNA secondary structure shown in Table 1 or in equivalent RNA is maintained by substitutions in the nucleotide sequence that do not result in positional changes of nucleotides that do not result in base pairing or non-base pairing. In some embodiments, the RNA secondary structure shown in Table 1 or in equivalent RNA is maintained by replacing non-base pairing nucleotides in the secondary structure with nucleotides that will not result in base pairing, and / or replacing base pairing nucleotides in the secondary structure with nucleotides that will result in base pairing. In this context, it should be understood that in some embodiments, the maintenance of the RNA secondary structure does not have to be absolute (e.g., the structure is partially maintained).In some embodiments, the dsRNA structure is partially maintained when one, two, three, or more nucleotides (particularly at the 5' and / or 3' ends of the hairpin-forming structure) are replaced by nucleotides that do not pair, thereby reducing the total length of the dsRNA in the structure. In some embodiments, the unpaired RNA structure is partially maintained when one, two, three, or more nucleotides (particularly at the 5' and / or 3' ends of the loop structure) are replaced by nucleotides that pair, thereby reducing the total length of the ssRNA in the loop structure. The ability of such partially maintained secondary structures to be recognized by the corresponding Tomato Cluster Dwarf Virus RdRP is monitored by in vitro or in vivo assays. Examples of Tomato Cluster Dwarf Virus satellite RNAs include those in which the 5' RNA replication element contains one or more of these 5' structural features and / or those in which the 3' RNA replication element contains one or more of these 3' structural features. In some embodiments, the 5' RNA replication element comprises RNA encoded by DNA having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 467, optionally wherein said encoded RNA maintains or partially maintains the corresponding structural features shown in Table 1. In some embodiments, the 3' RNA replication element comprises RNA encoded by DNA having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 468, wherein said encoded RNA optionally maintains or partially maintains the corresponding structural features shown in Table 1.

[0045] The recombinant polynucleotides (e.g., recombinant DNA, recombinant RNA, recombinant ssRNA, recombinant dsRNA, recombinant vectors, etc.) provided in this article may also contain or encode additional RNA elements.

[0046] Examples of additional RNA elements include RNA encoding the capsid or coat protein (CP) of the Tomato dwarfvirus family. Examples of DNA sequences encoding the Tomato dwarfvirus family CP include the corresponding sequences of the Tomato dwarfvirus family genome shown in Table 1. Examples of DNA sequences encoding the Tomato dwarfvirus family CP also include the sequence of SEQ ID NO: 472 and a DNA sequence having at least 85%, 90%, 95%, 98%, or 99% identity with it. Examples of DNA sequences encoding the Tomato dwarfvirus family CP also include the DNA sequence encoding CP disclosed in SEQ ID NO: 471 and a DNA sequence having at least 85%, 90%, 95%, 98%, or 99% identity with it. Examples of DNA sequences encoding the Tomato dwarfvirus family CP and Tomato dwarfvirus family CP sequences also include the sequences shown in Table 17 and DNA and protein sequences having at least 85%, 90%, 95%, 98%, or 99% sequence identity with them.

[0047] Examples of additional RNA elements include RNA encoding RdRPs of the Tomato Dwarf Virus Family. Examples of DNA sequences encoding RdRPs of the Tomato Dwarf Virus Family include the corresponding sequences of the Tomato Dwarf Virus Family genomes shown in Table 1. Examples of DNA sequences encoding RdRPs of the Tomato Dwarf Virus Family also include the sequences of SEQ ID NO: 470 and 588, and DNA sequences having at least 85%, 90%, 95%, 98%, or 99% identity with them. Examples of DNA sequences encoding RdRPs of the Tomato Dwarf Virus Family also include the DNA sequences encoding RdRPs of the Tomato Dwarf Virus Family disclosed in SEQ ID NO: 469 and 587, and DNA sequences having at least 85%, 90%, 95%, 98%, or 99% identity with them. Examples of DNA sequences and protein sequences encoding RdRPs of the Tomato Dwarf Virus Family also include the sequences shown in Table 17, and DNA and protein sequences having at least 85%, 90%, 95%, 98%, or 99% identity with them.

[0048] Other examples of RNA elements include RNA encoding viral mobile proteins (MPs). In some embodiments, the cargo RNA contains RNA encoding viral MPs. Without being bound by hypotheses or theories, it is thought that viral mobile proteins bind to RNA and facilitate its movement throughout the plant (and thus facilitate the movement of cargo RNA), for example, via plasmodesmata. Viral MPs include mobile proteins identified from tobacco mosaic virus (TMV), cowpea mosaic virus, potato leafroll virus, tomato spotted wilt virus, and tomato mosaic virus. MPs from various viruses are described in Table 3.

[0049] Examples of other RNA elements include tRNA-like sequences (TLS). TLS can trigger the mobility of otherwise immobile RNA, thereby facilitating the systematic delivery of RNA molecules. TLS includes tRNAs and tRNA-like sequences identified from other genetic elements, such as mRNA. The isoleucine-encoded tRNA by SEQ ID NO: 466 is an example of a useful tRNA-like sequence. Other mobile RNAs are described in Table 4, including TLSs identified in Arabidopsis that can be used to construct polynucleotides. In constructing Table 4, mobile mRNA sequences were downloaded from the Arabidopsis PLAMO database. tRNA “seed alignments” from the RFAM database were downloaded in Stockholm format (multiple sequence alignment + secondary structure). A covariance model for the tRNA Stockholm alignments was created using INFERNAL. PLAMO mRNA sequences were scanned for significant similarity to tRNAs based on primary and secondary structure characteristics. mRNA sequences with significant hits (E-val < 1) were then saved to a fasta file. In one embodiment, such a tRNA-like sequence comprises a tRNA-like sequence from Arabidopsis flowering time T(FT) mRNA. In some embodiments, the RNA molecule comprises at least one RNA encoding a viral MP, a tRNA-like sequence from Arabidopsis FT mRNA, and a capsid recognition element (ERE) comprising TMV-OAS. In some embodiments, the RNA molecule comprises 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 comprises a modified tRNA-like sequence having at least 90%, 95%, 98%, or 99% sequence identity with a scaffold tRNA-like sequence encoded by a DNA sequence selected from the group consisting of SEQ ID NOs: 76-123 and 466 and maintaining the secondary structure of the scaffold tRNA-like sequence.

[0050] Other examples of RNA elements include RNA encoding a viral capsid protein (CP). Such a capsid protein is sometimes also referred to as an outer coat protein, where both the capsid protein and the outer coat protein are referred to as "CP". In an example, the capsid protein is heterologous to a Tomato dwarf Virus family virus. In an example, the capsid protein is a Tomato dwarf Virus family capsid protein. In some embodiments, the cargo RNA contains RNA encoding a viral CP. CP can be provided, for example, by co-expression of a recombinant construct encoding CP or by natural expression of a virus that is endogenous to plant cells or introduced into plant cells. If the RNA molecule contains a capsidation recognition element (ERE) (e.g., an assembly origin sequence (OAS)), capsidation of the RNA molecule is achieved via CP. Table 2 describes several OAS and CP sequences from various viruses that can be used to engineer constructs that provide RNA capsidation. In an example, the OAS is located near the 3' end of the construct, for example, within the 3' region of the cargo RNA or at the 3' of the cargo RNA. For example, in some embodiments, the OAS is found at the 5' of a 3' RNA replication element (e.g., the 3' RNA replication element shown in Table 1). In the embodiment, the TMV-OAS located at the 3' end of the RNA molecule is recognized by the TMV capsid protein, resulting in the assembly of TMV virions around the RNA.

[0051] This document also provides examples of recombinant RNA complexed with RNA-binding proteins (RBPs). Examples of RBPs include RNA recognition motifs (RRMs), such as: (i) Lys / Arg-Gly-Phe / Tyr-Gly / Ala-Phe / Tyr-Val / Ile / Leu-X-Phe / Tyr, where X can be any amino acid (SEQ ID NO: 464); (ii) Ile / Val / Leu-Phe / Tyr-Ile / Val / Leu-X-Asn-Leu, where X can be any amino acid (SEQ ID NO: 465). Such RBPs and RRMs include those disclosed in Maris et al. 2005, doi.org / 10.1111 / j.1742-4658.2005.04653.x.

[0052] Other examples of RNA elements include at least one ribozyme. Ribozymes include self-cleaving ribozymes, ligand-responsive ribozymes (aptamers), trans-cleaving ribozymes designed to cleave target sequences (e.g., trans-cleaving hammerhead ribozymes (RNA encoded by SEQ ID NO: 421) designed to cleave the phytoene desaturase (PDS) sequence of pepper), hepatitis D virus (HDV) ribozymes (RNA encoded by SEQ ID NO: 423), or hammerhead ribozymes (RNA encoded by SEQ ID NO: 420). In various embodiments, the polynucleotide includes a variety of ribozymes. Useful ribozymes include twisted ribozymes, hammerhead ribozymes, hairpin ribozymes, and other ribozymes. Non-limiting examples of useful ribozymes include those provided in Table 14. In some embodiments, such ribozymes (e.g., self-cleaving ribozymes) are located at the 5' and / or 3' of the 5' RNA replication element in recombinant RNA. In some embodiments, such ribozymes (e.g., self-cleaving ribozymes) are located at the 5' and / or 3' of the HRV 5' RNA replication region in recombinant RNA containing an embedded heterologous RNA virus (HRV) amplicon. 3' of the RNA replication region.

[0053] Other embodiments of RNA elements include intron sequences. Table 6 describes examples of intron sequences that may be included in the recombinant polynucleotides provided herein. In some embodiments, the intron sequence is placed in the 5' UTR downstream of a promoter that drives recombinant RNA expression (e.g., a promoter active in plant cells). In some embodiments, the intron sequence is placed at the 5' of a 5' RNA replication element, in cargo RNA, or at the 3' of a 3' RNA replication element.

[0054] Examples of recombinant polynucleotides and other RNA elements include subgenomic promoters recognized by RNA-dependent RNA polymerase (RdRP) and / or RNA molecules encoding RdRP. Examples of such subgenomic promoters and RdRPs include the subgenomic promoter and RdRP of bromegranate mosaic virus (Siegal et al. 1998, doi: 10.1073 / pnas.95.20.11613), and the subgenomic promoters and RdRPs of barley yellow dwarf virus (BYDV) sgRNA1, sgRNA2, and sgRNA3 (Koev and Miller; J Virol. [Journal of Virology] July 2000; 74(13): 5988-96. Doi: 10.1128 / jvi.74.13.5988-5996.2000), Alternanthera mosaic virus (AltMV-MU) sgp1, sgp2 and sgp3 subgenomic promoters and RdRP (Putlyaev et al., Biochemistry (Mosc). [Biochemistry (Moscow)]; 80(8): 1039-46 DOI: 10.1134 / S000629791508009X). Further examples of subgenomic promoters are provided in Table 16. In some embodiments, such a subgenomic promoter is placed at the 5' and / or 3' of an RNA molecule containing a 5' RNA replication element, cargo RNA, and a 3' RNA replication element to allow the production of any one or both of the + and - strands of the RNA molecule upon provision of RdRP. In some embodiments, such a subgenomic promoter is operatively linked to a cargo RNA molecule and / or any additional RNA element to allow the production of the corresponding cargo and / or additional RNA upon provision of RdRP. In some embodiments, the subgenomic promoter is operatively linked to cargo RNA containing HRV repressive RNA or cargo RNA encoding proteins that inhibit HRV infection, movement, spread, and / or replication. In some embodiments, the subgenomic promoter is operatively linked to cargo RNA containing RNA having at least 20 consecutive nucleotides having sequences identical or complementary to segments of equal length in the genomic RNA of HRV. In some embodiments, the subgenomic promoter is operatively linked to a cargo RNA comprising an RNA having at least 20 consecutive nucleotides having a sequence that is identical or complementary to an isometric segment in the genomic RNA of HRV that does not encode hrvRdRP.

[0055] Examples of other optional elements in recombinant polynucleotides provided herein include: (a) discrete expression cassettes comprising a second promoter operatively linked to the DNA sequence to be transcribed, and optionally a terminator element (see, for example, the NOS or CaMV35S terminator); (b) expression-enhancing elements (e.g., DNA encoding expression-enhancing intron sequences); (c) DNA or RNA sequences encoding markers (e.g., selectable markers), such as DNA or RNA encoding antibiotic resistance or herbicide resistance sequences; DNA encoding scoring markers or detectable markers (e.g., β-glucuronidase, fluorescent proteins, luciferase, etc.); (d) DNA aptamers; (e) DNA or RNA sequences encoding RNA aptamers; and (f) T-DNA left and right boundary DNA sequences. (g) a spacer DNA sequence; (h) a DNA sequence encoding a transcription factor binding site; (i) a DNA sequence encoding a localization sequence (e.g., DNA encoding a target peptide such as a nuclear localization signal (NLS), mitochondrial localization signal, or plastid localization signal); or (j) a DNA sequence encoding at least one sequence-specific recombinase recognition site (SSRRS: e.g., a pair of sequence-specific recombinase recognition sites recognized by a given recombinase, such as a LOX site recognized by a CRE recombinase); and (k) a DNA sequence encoding a transcript stable or transcript destabilized sequence (see, for example, U.S. Patent Application Publication 2007 / 0011761, which is incorporated herein by reference in its entirety; Geisberg et al. (2014) Cell 156: 812-824).

[0056] This document provides recombinant polynucleotides comprising cargo RNA molecules or DNA encoding cargo RNA molecules. In some embodiments, the recombinant polynucleotide comprises a single cargo RNA molecule. In other embodiments, the recombinant polynucleotide comprises at least two cargo RNA molecules, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 cargo RNA molecules; in embodiments, the at least two cargo RNA molecules are identical (e.g., multiple copies of a non-coding RNA sequence or multiple copies of an RNA sequence encoding a polypeptide) or different (e.g., two or more different non-coding RNA sequences, or two or more different coding RNA sequences, or a combination of non-coding and coding cargo RNA sequences).

[0057] In some embodiments, the cargo RNA molecule is up to about 6 kilobases (kb) in length. The length of the cargo RNA molecule can range from any of about 20 nucleotides (nt), 100 nt, 200 nt, 300 nt, 400 nt, 500 nt, 600 nt, 700 nt, 800 nt, or 900 nt to any of 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 11 kb, 12 kb, 13 kb, or 14 kb. Other lengths of cargo RNA molecules less than or equal to 100 nucleotides (nt) can range from any of about 20 nt, 30 nt, or 40 nt to any of 50 nt, 60 nt, 70 nt, 80 nt, 90 nt, or 100 nt. In some embodiments, recombinant RNA containing cargo RNA up to about 6 kb in length can be capsidated with a Tomato dwarf Virus capsid protein. In such embodiments, the length of the cargo RNA can range from any of about 20 nt, 100 nt, 200 nt, 300 nt, 400 nt, 500 nt, 600 nt, 700 nt, 800 nt, or 900 nt to about 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, or 6 kb. In some embodiments, recombinant RNA containing cargo RNA up to about 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 11 kb, 12 kb, 13 kb, or 14 kb can be captopped with the heteroviral capsid proteins shown in Table 2. In some embodiments, recombinant RNA containing cargo RNA up to about 14 kb and captopped with heteroviral capsid proteins can contain the OAS elements shown in Table 2 and be captopped with the corresponding capsid proteins shown in Table 2. In some embodiments, the cargo RNA molecule is larger than 14 kb, for example, 15 kb, 16 kb, 17 kb, 18 kb, 19 kb, or even 20 kb. In embodiments, the cargo RNA molecule includes: (a) at least one coding sequence, (b) at least one non-coding sequence, or (c) both at least one coding sequence and at least one non-coding sequence. Such cargo RNA molecules include combinations of coding / non-coding sequences; multiple non-coding / coding sequences; and aptamers, ribozymes, and other elements described herein.In embodiments, the cargo RNA molecule includes (a) a coding sequence to be expressed in a plant, and (b) at least one non-coding sequence that modifies the expression or translation of said coding sequence, such as a recognition and cleavage sequence of siRNA or miRNA endogenously expressed in a plant (see, for example, U.S. Patent Nos. 8,334,430, 9,139,838, 9,976,152, 10,793,869, 10,876,126), wherein said recognition and cleavage sequence can bind to and cleave RNA transcripts containing said recognition and cleavage sequence; in such embodiments, spatial, temporal, or developmentally specific expression of the coding sequence can be achieved in a plant. In embodiments, the expression of the coding or non-coding cargo RNA in a plant can be optimized by methods including using codons that occur more frequently in plant genes, and / or eliminating polyadenylation sites in the cargo RNA (e.g., as described in at least U.S. Patent Nos. 5,380,831, 5,689,052, and 7,741,118, each of which is incorporated herein by reference in its entirety).

[0058] In embodiments, the cargo RNA molecule includes at least one coding sequence (e.g., a translatable sequence). Therefore, in some embodiments, the coding sequence is a protein or polypeptide, as described in the working examples of this disclosure. In some embodiments, the cargo RNA comprises selectable marker RNA encoding an antibiotic resistance or herbicide resistance polypeptide sequence or scoreable marker RNA encoding a scoreable marker protein (e.g., β-glucuronidase, fluorescent protein, luciferase, etc.). Examples of selectable marker / selector combinations include glyphosate resistance EPSPS enzyme and / or glyphosate oxidase / glyphosate, biammoniaphosphonate resistance (bar) or glufosinate-butylphosphonate acyltransferase (pat) / glufosinate, or neomycin phosphotransferase (npt) / neomycin or kanamycin. Examples of scoreable markers include β-glucuronidase (GUS), luciferase, and fluorescent proteins such as green fluorescent protein (GFP), yellow fluorescent protein (YFP), and cyan fluorescent protein (CFP). In embodiments, the cargo RNA sequence encodes at least one protein or polypeptide that provides a desired trait in plants expressing said protein or polypeptide. Non-limiting examples of polypeptides suitable for agricultural applications include, for example, bacteriocins, lysins, antimicrobial peptides, C-rich peptides from root nodules, and bacterial cell-regulating peptides. Such polypeptides can be used to alter the levels, activity, or metabolism of target microorganisms to increase the fitness of beneficial insects (such as bees and silkworms) or to reduce the fitness of harmful invertebrates (such as aphids, caterpillars, beetle larvae, and mites). Examples of agriculturally useful polypeptides include peptide toxins, such as those naturally produced by entomopathogenic bacteria (e.g., Bacillus thuringiensis, Photorhabdus luminescens, Serratia entomophila, or Xenorhabdus nematophila), as known in the art. Examples of agriculturally useful peptides include peptides (including small peptides such as cyclic dipeptides or diketopiperazine) for controlling agriculturally important pests or pathogens, such as antimicrobial or antifungal peptides for controlling plant diseases, or pest-killing peptides (e.g., insecticidal peptides and / or nematode-killing peptides) for controlling invertebrate pests (such as insects or nematodes). Examples of antimicrobial peptides include cathepsin inhibitors, cephalosporins, β-defensins, and amphibian antimicrobial peptides (e.g.,Examples of peptides useful in agriculture include esculentin, gaegurin, brevinin, ranatuerin, ranacyclin, uperin, oceanin, grahamin, nigrocin, temporin, maximin, enterococcin, ponicerins, megourins, apidaecins, abaecins, punicerins, bacteriocins, and lanofibrillarin, dermcidin, formacin, halocidins, lactobacillus, tachystatins, and some insecticidal toxins produced by spiders and scorpions. Examples of agriculturally useful peptides include antibodies, nanobodies, and fragments thereof, such as antibody or nanobodies fragments that retain at least some (e.g., at least 10%) of the specific binding activity of the complete antibody or nanobody. Examples of agriculturally useful peptides include transcription factors, such as plant transcription factors; see, for example, the AtTFDB database listing families of transcription factors identified in the model plant Arabidopsis thaliana, which is publicly available at agris-knowledgebase[dot]org / AtTFDB / . Examples of agriculturally useful peptides include nucleases, such as exonucleases or endonucleases (e.g., Cas nucleases, such as Cas9 or Cas12a). Examples of agriculturally useful peptides further include cell-penetrating peptides, enzymes (e.g., amylases, cellulases, peptidases, lipases, chitinases), and peptide pheromones (e.g., yeast or fungal mating pheromones, invertebrate reproductive and larval signaling pheromones, see, for example, Altstein (2004) Peptides, 25: 1373-1376). Examples of agriculturally useful peptides confer beneficial agronomic traits, such as herbicide tolerance, insect control, improved yield, increased resistance to fungal or oomycete diseases, increased viral resistance, increased nematode resistance, increased bacterial disease resistance, plant growth and development, improved starch yield, improved oil yield, high oil yield, improved fatty acid content, high protein yield, fruit ripening, increased animal and human nutrition, biopolymer yield, resistance to environmental stresses, medicinal peptides (e.g., hormones, enzymes, transcription factors, antigens, antibodies, or antibody fragments) and secretible peptides, improved processing traits, and improved digestibility (e.g., ...).Reduced levels of toxins or compounds with “anti-nutritional” properties (such as lignin, lectins, and phytates), enzyme production, flavor, nitrogen fixation, hybrid seed production, fiber production, and biofuel production. Non-limiting examples of agriculturally useful peptides include peptides that confer herbicide resistance (US Patent Nos. 6,803,501; 6,448,476; 6,248,876; 6,225,114; 6,107,549; 5,866,775; 5,804,425; 5,633,435; and 5,463,175), and increased yield (US Patent Nos. RE38,446; 6,716,474; 6,663,906; 6,476,295; 6,441,277; 6,423,828; 6,399,330; 6,372,211; 6,23...). 5,971; 6,222,098; and 5,716,837), Insect Control (US Patent Nos. 6,809,078; 6,713,063; 6,686,452; 6,657,046; 6,645,497; 6,642,030; 6,639,054; 6,620,988; 6,593,293; 6,555,655; 6,538,109; 6,537,756; 6,521,442; 6,501,009; 6,468,523; 6,326,351; 6,313,378; 6,284,949; 6,281 ,016;6,248,536;6,242,241;6,221,649;6,177,615;6,156,573;6,153,814;6,110,464;6,093,695;6,063,756;6,063,597;6,023,013;5,959,091;5,942,664;5,942,658、5,880,275;5,763,245;5,763,241;10,017,549;10,233,217;10,487,123;10,494,408;10, 494,409; 10,611,806; 10,612,037; 10,669,317; 10,827,755; 11,254,950; 11,267,849; 11,130,965; 11,136,593; and 11,180,774), fungal disease resistance (US Patent Nos. 6,653,280; 6,573,361; 6,506,962; 6,316,407; 6,215,048; 5,516,671; 5,773,696; 6,121,436; 6,316,407; and 6,506,962),Virus resistance (US Patent Nos. 6,617,496; 6,608,241; 6,015,940; 6,013,864; 5,850,023; and 5,304,730), nematode resistance (US Patent No. 6,228,992), bacterial disease resistance (US Patent No. 5,516,671), plant growth and development (US Patent Nos. 6,723,897 and 6,518,488), starch yield (US Patent Nos. 6,538,181; 6,538,179; 6,538,178; 5,750,876; 6,476,295), improved oil yield (US Patent No. 6,444). 876; 6,426,447; and 6,380,462), high oil yield (US Patent Nos. 6,495,739; 5,608,149; 6,483,008; and 6,476,295), improved fatty acid content (US Patent Nos. 6,828,475; 6,822,141; 6,770,465; 6,706,950; 6,660,849; 6,596,538; 6,589,767; 6,537,750; 6,489,461; and 6,459,018), high protein yield (US Patent No. 6,380,466), and fruit ripening (US Patent No. 5,51). 2,466), increased animal and human nutrition (US Patent Nos. 6,723,837; 6,653,530; 6,5412,59; 5,985,605; and 6,171,640), biopolymers (US Patent Nos. RE37,543; 6,228,623; 5,958,745; and 6,946,588), environmental stress resistance (US Patent No. 6,072,103), pharmaceutical peptides (e.g., hormones, enzymes, transcription factors, antigens, antibodies, or antibody fragments) and secretible peptides (US Patent Nos. 6,812,379; 6,774,283; 6,140,075; and 6,080,560), Improved processing properties (US Patent No. 6,476,295), improved digestibility (US Patent No. 6,531,648), low raffinose (US Patent No. 6,166,292), industrial enzyme yield (US Patent No. 5,543,576), improved flavor (US Patent No. 6,011,199), nitrogen fixation (US Patent No. 5,229,114), hybrid seed yield (US Patent No. 5,689,041), fiber yield (US Patent Nos. 6,576,818; 6,271,443; 5,981,834; and 5,869,720), and biofuel yield (US Patent No. 5,998,700). In some embodiments, the cargo RNA encodes one or more small signal transduction peptides (SSPs), also known as peptide hormones.Due to their small size (5-75 amino acids) and efficiency, SSPs are an attractive option for use as cargo in RNA symbiotic satellites. In some embodiments, SSPs are obtained by processing longer precursor peptides (derived from ORF regions). In other embodiments, SSPs are derived from a wider range of sources, including intergenic / intronic regions, long non-coding RNAs, pri-miRNAs, and the 5′ and 3′ UTRs of mRNAs. Non-limiting examples of SSPs include the peptides miPEP172c, miPEP171d, BomiPEP397a, AtmiPEP397a, BvmiPEP164b, and AtmiPEP164b shown in Table 13.

[0059] In some embodiments, the RNA molecule further includes an internal ribosome entry site (IRES) located at the 5' end and immediately adjacent to at least one coding sequence. In yet other embodiments, the cargo RNA molecule includes multiple coding sequences, and the RNA molecule further includes an IRES located at the 5' end and immediately adjacent to each coding sequence (e.g., an open translation reading frame encoding a target protein). Useful IRES sequences include those described in Table 5.

[0060] In some embodiments, the cargo RNA molecule includes non-coding sequences, such as those described in the working examples of this disclosure. Such non-coding sequences include hairpin RNA (hpRNA); RNA forming multiple stem loops; RNA pseudoknots; RNA sequences forming at least partially double-stranded RNA; small interfering RNA (siRNA) or siRNA precursors; microRNA (miRNA) or miRNA precursors; ribozymes; ligand-responsive ribozymes (aptases); RNA aptamers; or long non-coding RNA (lncRNA). In some embodiments, the cargo RNA includes selectable or scoreable RNA markers, such as RNA aptamers or regulatory RNAs, such as siRNA or siRNA precursors (see, for example, U.S. Patent Nos. 8,404,927, 8,455,716, 9,777,288, 10,378,012), miRNA or miRNA precursors (see, for example, U.S. Patent Nos. 8,410,334, 8,395,023, 9,708,620), trans-acting siRNA or trans-acting siRNA precursors (see, for example, U.S. Patent Nos. 8,030,473, 8,476,422, 8,816,061, 9,018,002), staged sRNA or staged sRNA. NA precursors (see, for example, U.S. Patent No. 8,404,928), siRNA or miRNA decoys (see, for example, U.S. Patent Nos. 8,946,511, 9,873,888), siRNA or miRNA cleavage inhibitors (see, for example, U.S. Patent No. 9,040,774), siRNA or miRNA recognition and cleavage sequences (see, for example, U.S. Patent Nos. 8,334,430, 9,139,838, 9,976,152, 10,793,869, 10,876,126), riboswitch (see, for example, U.S. Patent Application Publication No. 20130102651; U.S. Patent No. 6,630,306; U.S. Patent No. 6,949,379), or ribozymes. Suitable RNA aptamers include those that exhibit fluorescence after binding to the molecule. For example, a fluorescent RNA aptamer could be a broccoli RNA aptamer. Other fluorescent RNA aptamers that can be used include, but are not limited to, spinach, spinach 2, carrot, radish, corn, red broccoli, orange broccoli, and broccoli florets. Other useful RNA aptamers that can be used include those provided in Table 15. Suitable regulatory RNAs can be used to downregulate (i.e., silence) the expression of marker genes. For example, phytopenic lipase (PDS) is widely used as a marker gene because silencing this gene produces a photobleaching phenotype.Regulatory RNAs (such as decoys or cleavage blockers) can also be used to interfere with pathways regulated by endogenous small RNAs, thereby producing a visible phenotype; see, for example, U.S. Patent Nos. 8,946,511, 9,873,888, and 9,040,774.

[0061] This document provides antiviral cargo RNA, particularly antiviral cargo RNA targeting viral pathogens. In some embodiments, the antiviral cargo RNA comprises heterologous RNA virus (HRV) repressive RNA or encodes an HRV repressive protein, wherein the HRV repressive RNA or protein inhibits HRV infection, movement, transmission, and / or replication. Target viral pathogens include *Hypervira*, *Hypervira*, *Brassica napus*, *Celavirus*, *Leptovirus*, *Cowpea Mosaic Virus*, *Potatovirus X*, *Potatovirus Y*, *Tobacco Mosaic Virus*, *Tomato Cluster Dwarf Virus*, *Tomato Spotted Wilt Virus*, *Trigeneae*, *Turnip Yellow Mosaic Virus*, *Varicillium Vein Virus*, or *Associated Cowpea Mosaic Virus*. In some embodiments, the target viral pathogens are cucumber mosaic virus, wheatgrass mosaic virus, citrus degeneration virus, beet yellowing virus, cowpea mosaic virus, potato virus X; pepper mottle virus, bean yellow mosaic virus, barley stripe mosaic virus, wheat stripe mosaic virus, rice yellow mottle virus, maize dwarf mosaic virus, zucchini yellow mosaic virus, watermelon mosaic virus, sugarcane mosaic virus, tobacco mosaic virus, tomato mosaic virus, tomato brown wrinkled fruit virus, turnip vesicle virus, and pepper mild spot. Latent viruses include: turnip shrunken virus, tomato dwarf virus, tomato leaf spot virus, watermelon bud necrosis virus, turnip yellow mosaic virus, spinach latent virus, olive latent virus 2, citrus yellow vein virus, potato latent virus, apple stem groove virus, citrus leaf mottle virus, apple latent spherical virus, soybean latent spherical virus, celery latent virus, black forage leaf vein varices virus-like virus, corn suscal virus, horseradish latent virus, soybean latent virus, rice latent virus 1, or rice latent virus 2. In some embodiments, the targeted viral pathogens are heterologous RNA viruses disclosed in Table 7. In some embodiments, antiviral repressive RNA (RNAi sequences) used as cargo RNA is obtained for the selected viral pathogen target gene using siRNA / miRNA prediction tools (see, for example, at the World Wide Web website “zhaolab[dot]org / pssRNAit / ”). Other examples of non-coding RNA sequences with antiviral activity that can be used as antiviral cargo RNA (e.g., dsRNA molecules that produce miRNA or siRNA) include those disclosed in U.S. Patent No. 8,455,716, which is incorporated herein by reference in its entirety. Non-limiting examples of viral targets for antiviral cargo RNA molecules include viral genes and genomes provided in Table 7, as well as other variants of those viral sequences. In some embodiments, cargo RNA encoding antiviral proteins is provided. Non-limiting examples of antiviral proteins include the N protein (Whitham, S. et al., Cell 78, 1101-1115 (1994)) and endogenous plant virus resistance proteins provided in Table 8.

[0062] This document provides antifungal cargo RNAs, particularly those targeting plant fungal pathogens. Target fungal pathogens include species of the genera *Botrytis*, *Fusarium*, *Magnaporthe*, *Phytophthora*, *Rhizoctonia*, *Sclerotinia*, and *Verticillium* sp. In some embodiments, the antifungal cargo RNA comprises a non-coding RNA sequence with antifungal activity (e.g., a dsRNA molecule that produces miRNA or siRNA), particularly dsRNAs targeting fungal pathogen gene targets. In some embodiments, such antifungal cargo RNAs containing dsRNA-mediated fungal pathogen control are modeled using those described below: 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: 10.3389 / fmicb.2019.01662. Non-limiting examples of antifungal cargo RNAi targets are provided in Table 10. In some embodiments, antifungal repressive RNA (RNAi sequence) used as cargo RNA is obtained for selected fungal pathogen target genes (e.g., fungal pathogen genes shown in Table 10) using siRNA / miRNA prediction tools (see, for example, at the World Wide Web website “zhaolab[dot]org / pssRNAit / ”). In other embodiments, the antifungal cargo RNA encodes an antifungal protein.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), and antifungal peptides that inhibit conidial germination, including their homodimers, heterodimers, and fusions with signal peptides or cell-penetrating peptides (e.g., sequences provided in Tables 4 and 5 of WO 2023 / 004435); and various antifungal and antimicrobial peptides disclosed below: De Cesare et al. (2020) mBio[microorganism] 11: e02123-20; doi.org / 10.1128 / mBio.02123-20, including Table 1 (supplementary material, journals[dot]asm[dot]org / doi / suppl / 10.1128 / mbio.02123-20 / suppl_file / mbio.02123-20-st001[dot]pdf), and other antifungal proteins provided in Table 11.

[0063] This article provides insecticidal or insect-inhibiting cargo RNAs, particularly insecticidal or insect-inhibiting cargo RNAs targeting insects. Target insects include sucking insects (e.g., Heteropteran and Homopteran insects, including aphids, whiteflies, and mirid bugs), caterpillars (e.g., Lepidopteran insects, including fall armyworm, black rootworm, corn earworm, soybean looper, and bean hairy caterpillar), beetles (e.g., Coleopteran insects, including Colorado potato beetle and corn rootworm), and flies (e.g., Dipteran insects, including Mediterranean fruit fly (Ceratitis capitata)). The insecticidal or insect-inhibiting cargo RNAs provided herein can target insects at different stages of their development (e.g., embryonic, larval, pupa, or adult stages). In some embodiments, insecticidal or insect-inhibiting cargo RNAs comprise non-coding RNA sequences having insecticidal or insect-inhibiting activity (e.g., dsRNA molecules that produce miRNAs or siRNAs), particularly dsRNAs targeting insect target genes. In some embodiments, such insecticidal cargo RNAs comprising dsRNA-mediated insect control comprise those described in or modeled in U.S. Patent Nos. 11,091,770 and 11,186,837, each of which is incorporated herein by reference in its entirety. Non-limiting examples of insecticidal or insect-inhibiting cargo RNAi targets are provided in Table 9. Non-limiting examples of insecticidal cargo RNAi targets include insect actin genes, SNF7 genes, tyrosine hydroxylase genes, C002 genes, humpback genes, V-ATPase A subunit genes, COPI-coated β primary subunit genes, ribosomal protein L19 genes, and ubiquitin C genes. In some embodiments, insecticidal or insect repressive RNA (RNAi sequences) used as cargo are obtained for selected insect target genes (e.g., insect genes shown in Table 9 or U.S. Patent Nos. 11,091,770 and 11,186,837) using siRNA / miRNA prediction tools (see, for example, at the World Wide Web site “zhaolab[dot]org / pssRNAit / ”). In other embodiments, the insecticidal cargo RNA encodes insecticidal proteins. Useful insecticidal proteins encoded by the insecticidal cargo RNA include native and modified Bacillus thuringiensis Cry, vegetative insecticidal protein (VIP), and Cyt protein (Palma et al. 2014, doi: 10.3390 / toxins6123296; U.S. Patent No. 11,267,849, which are incorporated herein by reference in their entirety) and the insecticidal or insect repressive proteins provided in Table 9.

[0064] Cargo RNA can also encode “resistance” or “R” genes that confer resistance to certain arthropods, bacteria (such as species of *Pseudomonas*, *Xanthomonas*, and *Erwinia*) and fungal pathogens (including species of *Cochliobolus*, *Blumeria*, *Fusarium*, *Melampsora*, and *Pyrrosia*). Non-limiting examples of R genes encoded by cargo RNA include those provided in Table 12.

[0065] In some embodiments, the cargo RNA molecule contains CRISPR guide RNA, such as crRNA, gRNA, or sgRNA. CRISPR-associated endonucleases (such as Cas9, Cas12, and Cas13 endonucleases) are used as genome editing tools in various plants; see, for example, Wolter et al. (2019) BMC Plant Biol. [BMC Plant Biology], 19: 176-183; Aman et al. (2018) Genome Biol. [Genome Biology], 19: 1-10. CRISPR / Cas9 requires a two-component crRNA containing a target sequence (“CRISPR RNA” or “crRNA” sequence) and a Cas9 nuclease recruitment sequence (tracrRNA): tracrRNA “guide RNA” (“gRNA”). Efficient Cas9 gene editing has also been achieved using chimeric “single guide RNA” (“sgRNA”) (an engineered (synthetic) single RNA molecule that mimics the naturally occurring crRNA-tracrRNA complex and contains tracrRNA (for binding nucleases) and at least one crRNA (to guide the nuclease to the targeted sequence for editing); see, for example, Cong et al. (2013) Science, 339: 819-823; Xing et al. (2014) BMC Plant Biology, 14: 327-340. Chemically modified sgRNAs have been shown to be effective in genome editing; see, for example, Hendel et al. (2015) Nature Biotechnol, 985-991. Commercial manufacturers of CRISPR nucleases and guide RNAs offer algorithms for designing guide RNA sequences; see, for example, the guide design tool offered by Integrated DNA Technologies at www.idtdna.com / pages / products / crispr-genome-editing / alt-r-crispr-cas9-system. Some Cas nucleases (including Cas12a and Cas13) do not require tracrRNA.

[0066] This article also provides recombinant polynucleotides in which the 5' and 3' RNA replication elements of a Tomato Cluster Dwarf Virus are side-mounted with an internal sequence, wherein the cargo RNA is operatively linked to one or more elements of a heterologous RNA virus (HRV).

[0067] In some embodiments, cargo RNA is embedded within a heterologous RNA virus (HRV) amplicon comprising: (i) an 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 (i), (ii), and (iii) are operatively linked. An illustrative example of a Tomato Cluster Dwarf Virus family satellite construct having such an HRV amplicon is shown in Figure 2 middle. Figure 6The amplification of such HRV amplicons in plants containing Tomato Cluster Dwarf Virus family satellite constructs, Tomato Cluster Dwarf Virus family viruses, and HRV RdRP is demonstrated. Table 7 shows examples of the HRV 5' replication region (5'RR), 3' replication region (3'RR), and the corresponding HRV RNA-dependent RNA polymerase (RdRP) that recognizes such replication regions. In some embodiments where the cargo RNA encodes a protein, an internal ribosome entry site (IRES; e.g., IRES in Table 5) is typically operatively linked to the cargo RNA. In some embodiments, one or more self-cleaving or inducible ribozymes are operatively linked to the 5' end of the HRV 5'RR and the 3' end of the HRV 3'RR. In some embodiments, the HRV amplicons further include a subgenomic promoter operatively linked to the cargo RNA molecule. Examples of subgenomic promoters include the subgenomic promoters of HRV and / or bromegranate mosaic virus (Siegal et al. 1998, doi: 10.1073 / pnas.95.20.11613), and the subgenomic promoters of barley yellow dwarf virus (BYDV) sgRNA1, sgRNA2, and sgRNA3 (Koev and Miller; J Virol. [Journal of Virology] July 2000; 74(13): 5988-96, doi: 10.1128 / jvi.74.13.5988-5996.2000), Alternaria mosaic virus (AltMV-MU) sgp1, sgp2 or sgp3 subgenomic promoters (Putlyaev et al., Biochemistry (Mosc). [Biochemistry (Moscow)]; 80(8): 1039-46, doi: 10.1134 / S000629791508009X). Other examples of subgenomic promoters include those in Table 16. Such HRV amplicones can be positively or negatively oriented relative to the 5' RNA replication element of the Tomato dwarf Virus Family. When HRV amplicons are oriented in a sense orientation relative to the 5' RNA replication element of the Tomato dwarf Virus family, HRV 5'RR and 3'RR are present in a sense orientation in the recombinant RNA molecule, as in the corresponding sequences found in the positive (+) strand of the HRV genomic RNA. When HRV amplicons are oriented in an antisense orientation relative to the 5' RNA replication element of the Tomato dwarf Virus family, HRV 5'RR and 3'RR are present in an antisense orientation in the recombinant RNA molecule, as in the corresponding sequences found in the negative (-) strand of the HRV genomic RNA. Under certain conditions, in plant cells containing recombinant RNA with HRV amplicons or in plants provided with RNA-dependent RNA polymerase (hrvRdRP) that recognizes HRV 5'RR and 3'RR, HRV amplicons undergo amplification (e.g., hrvRdRP-mediated replication).Such hrvRdRP can be provided from sources including: (i) HRV infection; (ii) introduction of a polynucleotide encoding hrvRdRP via vector-mediated delivery (e.g., Agrobacterium-mediated delivery or viral vector-mediated delivery); or (iii) introduction of a nucleic acid encoding hrvRdRP. Non-limiting examples of recombinant satellite RNA are shown below. Figure 7 In this embodiment, the recombinant satellite RNA contains HRV 5'RR and 3'RR sequence pairs that are recognized by two distinct HRV RdRPs (“HRV1 RdRP” and “HRV2 RdRP”). ​​Amplification (e.g., an increase in the copy number of HRV amplicon) provides additional copies of the cargo RNA and an enhancement of the desired phenotype conferred by the cargo RNA (e.g., increased antiviral, antifungal, or insecticidal activity compared to control plants lacking amplified cargo RNA or lacking cargo RNA).

[0068] In other embodiments, the recombinant nucleotides provided herein comprise 5' and 3' RNA replication elements of a Tomato Cluster Dwarf Virus family side-linked heterologous RNA virus (HRV) subgenomic promoter operatively linked to a cargo RNA molecule; wherein the subgenomic promoter is recognized by a heterologous RNA virus RNA-dependent RNA polymerase (hrvRdRP). Figure 3 An illustrative example of a Tomato Cluster Dwarf Virus Family satellite construct with a subgenomic promoter having a positive or negative orientation relative to 5'RRE is shown. Figure 8Another illustrative example is shown in a plant containing a Tomato Cluster Dwarf Virus family satellite construct, where a subgenomic promoter drives the expression of HRV RdRP and dsRNA cargoes. In some embodiments where the cargo RNA encoded by the subgenomic promoter is typically operatively linked to the cargo RNA, an internal ribosome entry site (IRES; e.g., IRES in Table 5) is typically operatively linked to the cargo RNA. Examples of subgenomic promoters include subgenomic promoters of HRV and / or subgenomic promoters of barley yellow dwarf virus (BYDV) (Siegal et al. 1998, doi: 10.1073 / pnas.95.20.11613), and subgenomic promoters of barley yellow dwarf virus (BYDV) sgRNA1, sgRNA2, and sgRNA3 (Koev and Miller; J. Virol. [Journal of Virology] July 2000; 74(13): 5988-96. Doi: 10.1128 / jvi.74.13.5988-5996.2000), Alternaria mosaic virus (AltMV-MU) sgp1, sgp2 or sgp3 subgenomic promoters (Putlyaev et al., Biochemistry (Mosc); 80(8): 1039-46 DOI: 10.1134 / S000629791508009X). Other subgenomic promoters include those in Table 16. Such subgenomic promoters and operatively linked cargo RNAs can be positively or negatively oriented relative to the 5' RNA replication element of the Tomato dwarf Virus family. Expressing cargo RNA starting from a subgenomic promoter provides an additional copy of the cargo RNA and an enhancement of the desired phenotype conferred by the cargo RNA (e.g., increased antiviral, antifungal, or insecticidal activity compared to control plants lacking additional cargo RNA expression or lacking cargo RNA). When the subgenomic promoter and the operably linked cargo RNA are oriented in a sense orientation relative to the 5' RNA replication element of the Tomato dwarf Virus family, the subgenomic promoter and the operably linked cargo RNA exist as the sense strand in the recombinant RNA molecule, wherein the subgenomic promoter is recognized by hrvRdRP to produce the desired cargo RNA. When the subgenomic promoter and the operably linked cargo RNA are oriented in a sense orientation (positive strand) relative to the 5' RNA replication element of the Tomato dwarf Virus family in the recombinant RNA molecule, the subgenomic promoter can be recognized by hrvRdRP to produce the desired cargo RNA. When the HRV amplicon is oriented in an antisense orientation relative to the 5' RNA replication element of the Tomato dwarf Virus family in the recombinant RNA molecule, the subgenomic promoter cannot be recognized by hrvRdRP to produce the desired cargo RNA.However, the negative strand of the recombinant RNA molecule produced by RdRP from the Tomato dwarf Virus family will contain a positive-oriented subgenomic promoter and operatively linked cargo RNA, wherein the subgenomic promoter can be recognized by hrvRdRP to produce the desired cargo RNA. In some embodiments, the HRV amplicon further comprises HRV 5'RR and 3'RR side-linked cargo RNA and providing hrvRdRP-mediated RNA replication, wherein the RNA from 5' to 3' comprises HRV 5'RR, cargo RNA, and HRV 3'RR (e.g., as shown). Figure 5 (As shown in the non-limiting examples). Under certain conditions provided by a plant cell or plant containing a recombinant RNA comprising a subgenomic promoter and operably linked cargo RNA, an RNA encoding a cargo molecule (e.g., cargo RNA synthesis from a subgenomic promoter mediated by hrvRdRP) can be produced. Such hrvRdRP can be provided from sources including: (i) HRV infection; (ii) introduction via vector-mediated delivery (e.g., Agrobacterium-mediated delivery or viral vector-mediated delivery); (iii) introduction of nucleic acid encoding hrvRdRP; or (iv) inclusion of cargo RNA in recombinant nucleotides comprising 5' and 3' RNA replication elements of Tomato dwarfvirus. In one particular embodiment, the subgenomic promoter and operably linked cargo RNA are present as an antisense strand in the recombinant RNA molecule, and the cargo RNA encodes both hrvRdRP and a second coding or non-coding RNA, wherein both hrvRdRP and the second coding or non-coding RNA are operably linked to a subgenomic promoter recognized by hrvRdRP. In some embodiments, IRES is operatively linked to RNA encoding hrvRdRP. The generation of the negative strand of a recombinant nucleotide containing 5' and 3' RNA replication elements of the Tomato Cluster Dwarf Virus family yields RNA in which a subgenomic promoter recognized by hrvRdRP can drive the expression of HRV RdRP and a second coding or non-coding RNA. Figure 4 An illustrative example of a Tomato Cluster Dwarf Virus family satellite construct with a subgenomic promoter that is antisense-oriented relative to 5'RRE and drives the expression of both hrvRdRP, which recognizes the subgenomic promoter, and a second cargo RNA.

[0069] In a related aspect, the RNA molecule comprising at least one HRV amplicon is directly amplified by hrvRdRP (e.g., without initial or further amplification by Tomato dwarf Virus RdRP). In one embodiment, the HRV amplicon comprises, 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 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 operatively linked. In another embodiment, the HRV amplicon comprises, in 5' to 3' order, a subgenomic promoter operatively linked to (v) the cargo RNA molecule, wherein the subgenomic promoter is recognized by hrvRdRP. Such HRV amplicons may be provided in isolated form or in a composition. In embodiments, RNA molecules comprising HRV amplicons can be provided to plants, for example, through transcription from recombinant DNA molecules encoding RNA molecules transiently expressed or stably integrated into the plant genome, or through delivery of exogenous RNA molecules comprising HRV amplicons to the plant, for example, by contacting the plant surface with exogenous RNA molecules comprising HRV amplicons, or by introducing exogenous RNA molecules comprising HRV amplicons into the plant's vascular system (e.g., by injection, infusion, petiole uptake, root uptake). In embodiments, the cargo RNA molecule comprises at least one antiviral RNA (e.g., antiviral repressive RNA or RNA encoding an antiviral polypeptide) that provides the plant with resistance to at least one viral pathogen (in some cases, which may be the heterologous RNA virus itself). Such embodiments can be used as antiviral treatments for plants to prevent or reduce the severity of plant infection by viral pathogens.

[0070] In some embodiments, plants including citrus species (which contain satellite RNA of the Tomato Cluster Dwarf Virus Family containing HRV amplicon disclosed herein) can exhibit control over HRVs including: citrus descent virus, citrus vein spike virus, citrus bark cracking virus, hops dwarfing virus, citrus roughness virus, citrus scale virus, citrus yellow vein virus, citrus leaf curl virus, citrus dwarfing virus, citrus bark cracking virus, citrus yellow vein-associated virus, citrus wrinkled leaf virus, citrus variegated virus, citrus yellow mottle-associated virus, Indian citrus ringspot virus, citrus leaf mottle virus, citrus sudden death-associated virus, and citrus coguvirus. In other embodiments, plants of the genus Citrus (which contain satellite RNA of olive latent virus 1 containing the HRV amplicon disclosed herein) may exhibit control over HRVs including: Citrus Degeneration Virus, Citrus Vein Virus, Citrus Crack Virus, Hops Dwarf Virus, Citrus Rough Virus, Citrus Scaly Virus, Citrus Yellow Vein Virus, Citrus Curved Leaf Virus, Citrus Dwarf Virus, Citrus Bark Crack Virus, Citrus Yellow Vein-associated Virus, Citrus Wrinkled Leaf Virus, Citrus Variegated Virus, Citrus Yellow Mottle-associated Virus, Indian Citrus Ringspot Virus, Citrus Leaf Mottle Virus, Citrus Sudden Death-associated Virus, or Citrus coguvirus. In some embodiments, plants including olive (Osmanthus fragrans) (which contain satellite RNA of the Tomato Cluster Dwarf Virus Family containing the HRV amplicon disclosed herein) can exhibit control over HRVs including: olive latent virus 1, olive latent virus 2, olive latent virus 3, olive leaf yellowing-associated virus, olive mild mosaic virus, olive latent ringspot virus, tobacco mosaic virus, tobacco necrosis virus, cucumber mosaic virus, cherry leaf curl virus, mustard green mosaic virus, and strawberry latent ringspot virus. In other embodiments, plants such as olive (Osmanthus fragrans) (which contain satellite RNA of olive latent virus 1 containing the HRV amplicon disclosed herein) can exhibit control over HRVs including: olive latent virus 1, olive latent virus 2, olive latent virus 3, olive leaf yellowing-associated virus, olive mild mosaic virus, olive latent ringspot virus, tobacco mosaic virus, tobacco necrosis virus, cucumber mosaic virus, cherry leaf curl virus, mustard green mosaic virus, and strawberry latent ringspot virus. Without seeking to be limited by theory, RNA molecules containing HRV amplification sequences (such as HRV amplicon containing (1) a pair of HRV 5' and 3' RNA replication regions or (2) a subgenomic promoter recognized by hrvRdRP, as described in this paper) also potentially act as "sponges" or "decoys," reducing the efficiency of the corresponding hrvRdRP in recognizing and amplifying the HRV viral genome itself, thereby potentially reducing the harmful effects of pathogenic HRV on infected plants.

[0071] An RNA polynucleotide comprising at least one cleavable sequence is provided. In some embodiments, at least one cleavable sequence is located at: (i) the 5' end of the 5' RNA replication element or the 3' end of 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. In some embodiments, the cleavable sequence is a self-cleaving ribozyme (e.g., hammerhead ribozyme; Tang and Breaker. Proc Natl Acad Sci USA [Proceedings of the National Academy of Sciences of the United States of America]. May 23, 2000; 97(11): 5784-9. doi: 10.1073 / pnas.97.11.5784), a self-cleavage-inducible ribozyme, or a siRNA or miRNA recognition site.

[0072] In embodiments, the cargo RNA molecule integrated into the polynucleotide includes at least one CRISPR guide RNA; the release of the guide RNA is mediated by, for example, a side-attached DR sequence, a ribozyme sequence or other self-cleaving or trans-cleaving RNA, or by cleavage by an endogenous ribonuclease. The corresponding Cas nuclease can be provided by delivery alone or in parallel, for example, 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. For many Cas nucleases, the design of the guide sequence is limited by the requirement that the DNA target sequence (with which the crRNA is designed to be complementary) must be adjacent to the protospacer adjacent motif (“PAM”) sequence recognized by the specific Cas nuclease to be used. Cas nucleases recognize specific PAM sequences, and there are multiple nucleases and corresponding PAM sequences; see, for example, Smakov et al. (2017) Nature Reviews Microbiol. [Nature Microbiology Review], doi: 10.1038 / nrmicro.2016.184. For example, Cas9 nucleases cleave dsDNA requiring a GC-rich PAM sequence at the 3' end of the DNA target sequence targeted by the crRNA component of the guide RNA, and cleavage leaves blunt ends. Cas12a nucleases cleave dsDNA requiring a T-rich PAM sequence at the 5' end of the DNA target sequence targeted by the crRNA component of the guide RNA, and cleavage leaves staggered ends with 5' overhangs. Cas13 nucleases cleave single-stranded RNA and do not require a PAM sequence; instead, Cas13 nucleases are directed to their targets via a single crRNA with a directing repeat sequence (“DR”). In practice, the crRNA component of the guide RNA is typically designed to be between 17 and 24 nucleotides in length (usually 19, 20, or 21 nucleotides) and precisely complementary (i.e., perfectly base-paired) to the target gene or nucleic acid sequence of its neighboring PAM motif (when required by the Cas nuclease). You can use crRNA components that are less than 100% complementary to the target sequence (e.g., crRNAs that are 20 nucleotides long and have 1-4 mismatches with the target sequence), but this increases the likelihood of off-target effects.

[0073] Non-limiting examples of effective guide RNA design can be found, for example, in U.S. Patent Application Publications US 2019 / 0032131, 2015 / 0082478, and 2019 / 0352655, the full text of which is incorporated herein by reference. For gene editing purposes, CRISPR “arrays” can be designed to include one or more guide RNA sequences corresponding to one or more desired target DNA sequences; see, for example, Cong et al. (2013) Science, 339: 819-823; Ran et al. (2013) Nature Protocols, 8: 2281-2308.

[0074] In some embodiments, the 5' RNA replication element comprises a 5' UTR element of the Tomato dwarfviridae genome (e.g., the Tomato dwarfviridae genome comprising the 5' UTR of the Tomato dwarfviridae genome shown in Table 1). In embodiments, the 5' RNA replication element further comprises a Tomato dwarfviridae genome sequence naturally located at the 3' end of the 5' UTR sequence and optionally adjacent to or immediately adjacent to it. In embodiments, the 3' RNA replication element comprises a 3' UTR sequence of the Tomato dwarfviridae genome (e.g., the Tomato dwarfviridae genome comprising the 3' UTR of the Tomato dwarfviridae genome shown in Table 1). In embodiments, the 3' RNA replication element further comprises a Tomato dwarfviridae genome sequence naturally located at the 5' end of the 3' UTR sequence and optionally adjacent to or immediately adjacent to it.

[0075] In other embodiments, the RNA molecule further comprises at least one RNA molecule encoding a viral MP. In some embodiments, the at least one RNA molecule encoding the MP is located before (a) the cargo RNA molecule, (b) after the cargo RNA molecule, or (c) both before and after the cargo RNA molecule. In embodiments, the at least one RNA sequence encoding the MP comprises at least two RNA sequences encoding different MPs or a single RNA sequence encoding multiple copies of the MP.

[0076] In some embodiments, the recombinant DNA molecule further includes a discrete expression cassette comprising a second promoter that is functional in the cell and operatively linked to a DNA sequence encoding at least one viral mobile protein, and an optional terminator element.

[0077] In some embodiments, the RNA molecule further includes a capsid recognition element (ERE), wherein the ERE is located near or adjacent to a 3' RNA replication element, and optionally the 3' RNA replication element comprises a 3' UTR sequence of a Tomato Cluster Dwarf Virus. In embodiments, the ERE includes a viral OAS (such as Tobacco Mosaic Virus OAS (TMV-OAS) or an OAS shown in Table 2).

[0078] In some embodiments, the RNA molecule further comprises at least one tRNA-like sequence (TLS), and said at least one tRNA-like sequence comprises a tRNA-like sequence from Arabidopsis thaliana FT mRNA (e.g., TLS from Arabidopsis thaliana FT mRNA in Table 4). In some embodiments, the RNA molecule comprises a tRNA-like sequence encoded by a DNA sequence selected from the group consisting of SEQ ID NO: 76-123 and 466. In some embodiments, the RNA molecule comprises a modified tRNA-like sequence having at least 90% sequence identity with a scaffold tRNA-like sequence encoded by a DNA sequence selected from the group consisting of SEQ ID NO: 76-123 and 466 and maintaining the secondary structure of said scaffold tRNA-like sequence. In still other embodiments, the RNA molecule further comprises at least one RNA encoding a viral MP, a tRNA-like sequence from Arabidopsis thaliana FT mRNA, and a capsid recognition sequence including TMV-OAS.

[0079] In some embodiments, the cargo RNA molecule is up to about 6 kb in length. In embodiments, the cargo RNA molecule includes: (a) at least one coding sequence, (b) at least one non-coding sequence, or (c) both at least one coding sequence and at least one non-coding sequence. In other embodiments, the cargo RNA molecule includes at least one coding sequence, and wherein the RNA molecule further includes an internal ribosome entry site (IRES) located at the 5' end of said at least one coding sequence and immediately adjacent thereto. In other embodiments, the cargo RNA molecule includes multiple coding sequences, and wherein the RNA molecule further includes an IRES located at the 5' end of each coding sequence and immediately adjacent thereto.

[0080] In other embodiments, the cargo RNA molecule includes at least one non-coding sequence, and said at least one non-coding sequence is selected from the group consisting of: hairpin RNA (hpRNA); RNA forming multiple stem loops; RNA pseudoknots; RNA sequences forming at least partially double-stranded RNA; small interfering RNA (siRNA) or siRNA precursors; microRNA (miRNA) or miRNA precursors; ribozymes; ligand-responsive ribozymes (aptamers); RNA aptamers; and long non-coding RNA (lncRNA).

[0081] In one embodiment, a DNA sequence encoding at least one ribozyme is provided. In another embodiment, the at least one ribozyme is located at the 5' of a 5' RNA replication element or the 3' of a 3' RNA replication element. In another embodiment, a DNA sequence encoding at least one ligand-responsive ribozyme (aptamer enzyme) is provided. In another embodiment, the at least one ligand-responsive ribozyme is located at the 5' of a 5' RNA replication element or the 3' of a 3' RNA replication element.

[0082] This document also provides recombinant RNA molecules comprising the 5' RNA replication element disclosed above or otherwise, one or more cargo RNA molecules, and a 3' RNA replication element, as well as other elements disclosed above or otherwise. In some embodiments, the recombinant RNA molecules are generated from the recombinant DNA molecules provided herein. In some embodiments, the recombinant RNA molecules are generated by an in vivo or in vitro (e.g., cell-free) RNA replication process through the action of RdRP on: (i) the 5' and 3' RNA replication elements; and / or (ii) a subgenomic promoter.

[0083] Expression systems comprising recombinant polynucleotides are also provided. Such expression systems include both cell-based and cell-free expression systems. In some embodiments, a cell-free expression system may 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, it may further include at least one additional RNA or other element. Examples of additional elements include at least one RNA encoding a viral MP, at least one tRNA-like sequence, OAS, an RdRP protein recognizing the 5' and 3' RNA replication elements, a subgenomic promoter, and / or an RdRP recognizing the HRV 5' or 3' replication region and / or a subgenomic promoter. In some embodiments, the RdRP protein is provided by a Tomato dwarf Virus (e.g., an endogenous Tomato dwarf Virus for the cells in which expression is required). In some embodiments, a cell-based expression system may include (a) a recombinant DNA molecule comprising a heterologous promoter that is functional in the cell and operatively linked to a DNA sequence encoding an RNA molecule, the RNA molecule comprising, in a 5' to 3' order: (i) a 5' RNA replication element; (ii) a cargo RNA molecule; and (iii) a 3' RNA replication element; and optionally, it further comprises at least one additional RNA or other element. Examples of additional elements include at least one RNA encoding a viral MP, at least one tRNA-like sequence, an OAS, an RdRP protein recognizing the 5' and 3' RNA replication elements, a subgenomic promoter, and / or an RdRP recognizing the subgenomic promoter. In some embodiments, a cell-based expression system may include (a) a recombinant RNA molecule comprising, in a 5' to 3' order: (i) a 5' RNA replication element; (ii) a cargo RNA molecule; and (iii) a 3' RNA replication element; and optionally, it further comprises at least one additional RNA or other element. Other embodiments of RNA elements include at least one RNA encoding a viral MP, at least one tRNA-like sequence, OAS, an RdRP protein recognizing 5' and 3' RNA replication elements, a subgenomic promoter, and / or an RdRP recognizing the HRV 5' or 3' replication region and / or a subgenomic promoter. In some embodiments, the RdRP protein is provided by a Tomato dwarf Virus virus. In some embodiments, the RdRP recognizing the HRV 5' or 3' replication region and / or a subgenomic promoter is provided by a heterologous RNA virus (HRV) or another nucleic acid introduced into the cell (e.g., by a vector or other recombinant nucleic acid).In other embodiments, the 5' RNA replication element and the 3' RNA replication element are derived from the same Tomato dwarfviridae virus and / or from the same Tomato dwarfviridae genome (e.g., both derived from the same Tomato dwarfviridae capsid genome or both derived from the same Tomato dwarfviridae genome) or from Tomato dwarfviridae genomes having at least 85%, 90%, 95%, 98%, or 99% sequence identity with each other and optionally being related. In some embodiments, the cells used in the expression system are bacterial cells, plant cells, fungal cells, or animal cells (e.g., insect cells). In embodiments, the cells used in the expression system endogenously contain a Tomato dwarfviridae virus having a genome encoding RdRP, which recognizes the 5' and 3' RNA replication elements. In some embodiments, the expression system further includes a viral capsid protein that is recognized by a capsidation recognition element and capsids the RNA molecule. In some embodiments, the viral capsid protein: (a) is expressed by a recombinant DNA molecule in the cell (e.g., wherein the recombinant DNA molecule further includes a discrete expression cassette containing a second promoter operatively linked to the DNA sequence encoding the viral capsid protein, and optionally a terminator element), (b) is co-expressed by a second recombinant DNA molecule in the cell; (c) is exogenously provided to the cell; or (d) is expressed by a virus in the cell. In embodiments, the RdRP protein is heterologous to the cell. In embodiments, the RdRP protein is exogenously provided to the cell. In some embodiments, the RdRP protein recognizing 5' and 3' RNA replication elements is endogenously expressed in plant cells by a Tomato Cluster Dwarf Virus (e.g., when the Tomato Cluster Dwarf Virus is naturally present in the plant cell). In embodiments, the Tomato Cluster Dwarf Virus is natural or specific to the plant cell. In embodiments, the plant cell-specific Tomato Cluster Dwarf Virus is nonpathogenic. In embodiments, the plant cell-specific Tomato Cluster Dwarf Virus is nonpathogenic and symbiotic. In this embodiment, the Tomato Cluster Dwarf Virus is an exogenously introduced Tomato Cluster Dwarf Virus (i.e., not specific or natural to the host, but artificially introduced). For example, a Tomato Cluster Dwarf Virus naturally found in a plant species, variety, or germplasm can be introduced into different plant species, varieties, or germplasm with or without a corresponding recombinant Tomato Cluster Dwarf Virus satellite RNA.In an embodiment, a complete self-replicating Tomato dwarfvirus satellite system is introduced into a plant or plant cell, wherein the self-replicating Tomato dwarfvirus satellite system comprises: (1) a recombinant Tomato dwarfvirus satellite RNA comprising, from its 5' end to its 3' end: (a) a 5' RNA replication element recognized by Tomato dwarfvirus RNA-dependent RNA polymerase (RdRP); (b) a cargo RNA molecule; and (c) a 3' RNA replication element recognized by RdRP; wherein the 5' RNA replication element, the cargo RNA molecule, and the 3' RNA replication element are operatively 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) a foreign Tomato dwarfvirus virus (e.g., a Tomato dwarfvirus virus that is not specific to or natural to the plant or plant cell) capable of replicating and encoding the 5' and 3' replicase recognition sequences in the recombinant Tomato dwarfvirus satellite RNA that recognize the RdRP. In an embodiment, the Tomato Cluster Dwarf Virus RdRP comprises a protein having at least 85%, 90%, 95%, 97%, or 99% sequence identity with SEQ ID NO: 469 or 587. In an embodiment, the recombinant DNA molecule or recombinant RNA molecule further comprises at least one RNA encoding a viral MP, a tRNA-like sequence from Arabidopsis thaliana FT mRNA, and a capsid recognition sequence including TMV-OAS.

[0084] This document provides for cells comprising any of the recombinant polynucleotides (e.g., recombinant DNA, recombinant RNA, or vectors containing or encoding them) disclosed above or otherwise. Cells comprising recombinant polynucleotides include prokaryotic cells (e.g., bacteria, such as bacteria capable of transforming eukaryotic cells) or eukaryotic cells (e.g., plant cells, fungal cells, or animal cells (e.g., insect cells)). In some embodiments, the cells are bacterial cells capable of transforming plant cells (e.g., Agrobacterium species, Sinorhizobium sp., Mesorhizobium sp., Bradyrhizobium sp., Rhizobium sp., or Ensifer sp. cells). Bacterial cells capable of transforming plant cells suitable for use with the recombinant polynucleotides provided herein include Agrobacterium species, Rhizobium species, Meso- and Slow-growing Rhizobium species, Slow-growing Rhizobium species, Rhizobium species, or Cyclocarya species cells, disclosed in U.S. Patent Application Publications US 20170369898 and US 20180312854 (each incorporated herein by reference in its entirety).

[0085] This document also provides vectors suitable for maintaining, propagating, and / or expressing recombinant polynucleotides in the aforementioned prokaryotic or eukaryotic cells. Such vectors may comprise any of the recombinant polynucleotides, recombinant DNA molecules, and recombinant RNA molecules disclosed for the foregoing or otherwise, as well as those polynucleotide molecules described in the examples. In some embodiments, the bacteria mediating plant transformation are Agrobacterium species, Rhizobium species, Mesoprothiolane species, Slow-growing Rhizobium species, Rhizobium species, or Fibromyces species. In embodiments where Agrobacterium species are used, the vector comprises T-DNA (e.g., as described in U.S. Patent Application Publications US 20170369898 and US 20180312854, each incorporated herein by reference in its entirety) flanked by a recombinant DNA molecule encoding a recombinant RNA molecule. In some embodiments, the vector is contained within plant cells or bacterial cells (Agrobacterium species, Rhizobium species, Mesoprothiolane species, Slow-growing Rhizobium species, Rhizobium species, or Fibromyces species cells).

[0086] Viral particles comprising any of the recombinant RNA molecules disclosed for the foregoing or otherwise are also provided. In one embodiment, recombinant RNA is introduced into a host or production plant using Agrobacterium-mediated transformation polynucleotides comprising (5' to 3'): (i) a promoter and TLS element operatively linked to a viral MP coding sequence, flanking 5' and 3' RNA replication elements of the Tomato dwarf Virus Family; (ii) a promoter and TLS element operatively linked to a cargo RNA molecule, flanking 5' and 3' RNA replication elements of the Tomato dwarf Virus Family; (iii) a promoter operatively linked to an RdRP coding sequence; and (iv) a promoter operatively linked to a CP coding sequence. As described, the heterologous promoter independently drives the expression of the capsid protein and the cargo. The RNA expressed by the polynucleotide includes OAS. Transformation of the host plant produces synthetic Tomato dwarf Virus Family satellite RNA and satellite particles containing capsidized RNA. Subsequently, expressed and capsidated Tomato dwarfvirus satellite RNA is isolated from leaf material or other tissues of the host plant, purified (and, if necessary, formulated) for high-pressure spraying onto plants endogenously containing the corresponding Tomato dwarfvirus or recombinant (e.g., stably transformed or transiently expressed in the plant) Tomato dwarfvirus RdRP for subsequent expression and replication of the Tomato dwarfvirus satellite RNA and capsidated satellite particles containing it. In some embodiments, spraying with capsidated satellite particles containing certain cargo RNA molecules can be used to modify the plant as needed. The presence of mobile proteins and / or tRNA-like sequences facilitates systemic movement throughout the plant, which receives high-pressure sprayed satellite particles containing desired recombinant RNA molecules encoding MP and cargo RNA. In some embodiments, plants lacking systemic Tomato dwarfvirus providing Tomato dwarfvirus RdRP may further contain recombinant DNA or RNA molecules encoding and providing RdRP, for example, stably integrated into the plant genome or transiently expressed in the plant. Transgenic or transiently expressed RdRP of the Tomato Cluster Dwarf Virus Family can also be used to evaluate recombinant Tomato Cluster Dwarf Virus Family satellites in situ in plants.

[0087] Target plants and plant cells used as hosts for the synthetic Tomato dwarfvirus family satellite RNA (e.g., recombinant RNA) provided herein include monocotyledonous and dicotyledonous plants, as well as plant cells capable of supporting the replication of Tomato dwarfviruses. In embodiments, the Tomato dwarfvirus is endogenous to the plant or plant cell (specific or naturally present therein). In other embodiments, the Tomato dwarfvirus is introduced into and established in the plant or plant cell. Embodiments include interrow crops, fruit-producing plants and trees, vegetables, trees, and ornamental plants (including ornamental flowers, shrubs, trees, ground cover plants, and turfgrass). In some embodiments, host plants and plant cells for the synthetic Tomato dwarfvirus family satellite RNA include interrow crops, fruit-producing plants and trees, vegetables, trees, and ornamental plants (including ornamental flowers, shrubs, trees, ground cover plants, and turfgrass). In the embodiments, the host plants and plant cells used to synthesize the Tomato Cluster Dwarf Virus family satellite RNA include commercially important cultivated crops, trees, and plants, including: alfalfa, almond (Prunus dulcis), apple (Malus x domestica), apricot (Prunus armeniaca, P. brigantine, P. mandshurica, P. mume, P. sibirica), asparagus (Asparagus officinalis), banana (Musa spp.), barley (Hordeum vulgare), beans (Phaseolus spp.), blueberries, and cranberries (Vaccinium spp.). spp.), cocoa beans (Theobroma Cacao), canola and rapeseed or rapeseed (Brassica napus), Polish canola (Brassica rapa), and related cruciferous vegetables, including broccoli, kale, cabbage, and turnip (Ethiopian mustard (Brassica carinata), mustard greens (B. juncea), cabbage (B. oleracea), European rapeseed (B. napus), black mustard (B. nigra), and rutabaga (B. nigra).(rapa), and their hybrids), carnation (Dianthus caryophyllus), carrot (Daucus carota sativus), cassava (Manihot esculentum), cherry (Prunus avium), chickpea (Cicer arietinum), chicory (Cichorium intybus), red peppers and other peppers of the Capsicum genus (C. frutescens, C. chinense, C. pubescens, C. baccatum), chrysanthemum (Chrysanthemum species), coconut (Cocos nucifera), coffee (wild and cultivated Coffea species). spp.), including small-bean coffee (Coffea arabica), medium-bean coffee (Coffea canephora), and large-bean coffee (Coffea liberica), cotton (upland cotton (Gossypium hirsutum L.)), cowpea (Vigna unguiculata and other Vigna species (Vigna spp.)), fava bean (Vicia faba), cucumber (Cucumis sativus), red and currants (Ribes spp.), date (Phoenix dactylifera), duckweed (Lemnoideae), eggplant or dwarf gourd (Solanum melongena), eucalyptus (Eucalyptus spp.), flax (Linum usitatissumum L.), geranium (species of the genus Pelargomum spp.), grapefruit (species of the genus Citrus x paradisi), grape (species of the genus Vitus spp.).This includes wine grapes (Vitus vinifera and its hybrids), guava (Psidium guajava), hops (Humulus lupulus), ramie and hemp (Cannabis sativa and Cannabis spp.), iris (Iris spp.), lemon (Citrus limon), lettuce (Lactuca sativa), lime (Citrus spp.), maize (Zea mays L.), mango (Mangifera indica), mangosteen (Garcinia mangostana), and melon (Cucumis melon). melo), millet (Setarias pp., Echinochloa, Eleusine spp., Panicum spp., Penniisetum spp.), oat (Avena sativa), oil palm (Ellis quineensis), olive (Olea europaea), onion (Allium cepa) and other allium species (Allium spp.), orange (Citrus sinensis), papaya (Carica papaya), peach and nectarine (Prunus persica), pear (Pyrus spp.), pea (Pisa sativum), peanut (Arachis chinensis) Hypogaea), Peony (Paeonia spp.), Petunia (Petunia spp.), Pineapple (Ananascomosus), Plantain (Musa spp.), Plum (Prunus domestica), Poinsettia (Euphorbia pulcherrima), Poplar (Populus spp.), Potato (Solanum tuberosum), Pumpkin and Cucurbita pepo, Cucurbita maxima, Cucurbita maxima.moschata), rice (Oryza sativa L.), rose (Rosa spp.), rubber (Hevea brasiliensis), rye (Secalecereale), safflower (Carthamus tinctorius L.), sesame seeds (Sesameindium), sorghum (Sorghum bicolor), soybean (Glycine max L.), strawberry (Fragaria spp., Fragaria x ananassa), sugar beet (Beta vulgaris), sugarcane (Saccharum spp.), sunflower (Helianthus annuus), sweet potato (Ipomoea) Batas, oranges (Citrus tangerina), tea (Camellia sinensis), tobacco (Nicotiana tabacum L.), tomatoes (Solanum lycopersicum or Lycopersiconesculentum), tulips (Tulipa spp.), walnuts (Juglans spp. L.), watermelons (Citrullus lanatus), wheat (Triticumaestivum), and yams (Discorea spp.).Wild relatives of cultivated plants are also of interest. In some embodiments, the host plant or plant cell used for the synthesis of the Tomato Cluster Dwarf Virus Family satellite RNA is a dicotyledonous plant or plant cell selected from the genera *Brassica*, *Capsicum*, *Cucumis*, *Cucumis*, *Cotton*, *Tobacco*, *Solanum*, or *Soybean*. In some embodiments, the host plant or plant cell used for the synthesis of the Tomato Cluster Dwarf Virus Family satellite RNA is a monocotyledonous plant or plant cell selected from the genera *Oat*, *Barley*, *Oryza*, *Rye*, *Wheat*, *Sorghum*, or *Corn*. In some embodiments, the monocotyledonous target plant and plant cell used as the host for the synthesized Tomato Cluster Dwarf Virus Family satellite RNA (e.g., recombinant RNA) provided herein includes oat, barley, rice (*Oryza sativa*, *Oryza glaberrima*, *Oryza rufipogene*), rye, wheat (common wheat), sorghum, and corn plants and plant cells.

[0088] Methods of using or uses of any of the recombinant polynucleotides, expression systems, cells, and / or vectors disclosed for the foregoing or otherwise are also provided for: (i) providing synthetic Tomato dwarf virus family satellite RNA to plant cells; (ii) obtaining phenotypic changes in plants or plant cells; (iii) increasing plant resistance to pests or pathogens; (iv) increasing plant resistance to stress; (v) expressing polypeptides in plants or plant cells; and / or (v) manufacturing synthetic Tomato dwarf virus particles. In some embodiments of the methods, the recombinant RNA molecule or its preparation is provided by contacting the plant or plant cells with the recombinant RNA molecule or its preparation. In other embodiments, the recombinant RNA molecule is provided by expressing a DNA molecule or preparation encoding the recombinant RNA molecule in the plant or plant cells. In other embodiments, the recombinant RNA molecule is provided by contacting the plant or plant cells with a cell (such as a bacterial cell) containing a DNA molecule encoding the recombinant RNA molecule and capable of transforming the plant or plant cells. In other embodiments, the recombinant RNA molecule is provided by contacting the plant or plant cells with a satellite particle or its preparation containing a capsidized recombinant RNA molecule. In some embodiments, the 5' RNA replication element has a nucleotide sequence derived from or derived from a Tomato dwarfvirus family genome sequence; and / or the 3' RNA replication element has a nucleotide sequence derived from or derived from a Tomato dwarfvirus family genome sequence. In some embodiments, the 5' and / or 3' RNA replication elements can be obtained from a corresponding Tomato dwarfvirus family genome sequence by synthesizing or cloning a copy of the corresponding Tomato dwarfvirus family genome sequence. In some embodiments, the 5' and / or 3' RNA replication elements can be derived from a corresponding Tomato dwarfvirus family genome sequence by synthesizing one or more modified copies of the Tomato dwarfvirus family genome sequence. Such genomic sequence modifications of the Tomato Cluster Dwarf Virus Family present in the derived sequences include: (i) nucleotide substitutions maintaining RNA secondary structure; (ii) nucleotide substitutions based on consensual sequences obtained by alignment of 5' or 3' RNA replication elements; (iii) insertions, deletions, and / or substitutions of nucleotides that facilitate assembly and / or operative linkage with other elements in satellite RNA, including cargo RNA molecules, tRNA-like elements, capsid recognition elements (EREs), RNA encoding viral mobility proteins (MPs), IRES elements, HRV 5'RR, HRV 3'RR, and / or HRV subgenomic promoters; or (iv) any combination of (i), (ii), or (iii). In some embodiments, the plant cells comprise Tomato Cluster Dwarf Virus Family viruses, and the RdRP protein is provided to the plant cells via said Tomato Cluster Dwarf Virus Family viruses. In some embodiments, the Tomato Cluster Dwarf Virus Family viruses are specific to the plant cells. In some embodiments, the plant cell-specific Tomato Cluster Dwarf Virus Family viruses are non-pathogenic to the plant cells and / or symbiotic.In other embodiments, the Tomato Cluster Dwarf Virus is exogenously provided to the plant cells. In some embodiments, the RdRP protein is exogenously provided to the plant cells. In some embodiments, the recombinant RNA molecule is produced in a fermentation system. In some embodiments, the recombinant RNA molecule is provided to the plant cells by transcription of a recombinant DNA construct comprising a promoter that is functional in the plant cells and operatively linked to a DNA sequence encoding the recombinant RNA molecule. In some embodiments, the recombinant RNA molecule further comprises a capsidation recognition element (ERE), and the plant cells further comprise a viral capsid protein (CP) capable of capsidating the synthetic Tomato Cluster Dwarf Virus satellite RNA. In some embodiments, the viral capsid protein is exogenously provided to the plant cells. In other embodiments, the recombinant DNA construct further comprises a DNA sequence encoding the viral capsid protein. Still in other embodiments, the recombinant DNA construct further comprises a second promoter that is functional in the plant cells and operatively linked to a DNA sequence encoding the viral capsid protein. In some embodiments, the viral capsid protein is expressed in the plant cells and capsids the synthetic Tomato Cluster Dwarf Virus satellite RNA. In other embodiments, the plant cells comprise a Tomato Cluster Dwarf Virus family virus, and the Tomato Cluster Dwarf Virus family virus provides the plant cells with: (a) an RdRP protein, (b) a viral capsid protein, or (c) both the RdRP protein and the viral capsid protein. In some embodiments, the method may further comprise the first step of providing a plant population comprising plant cells containing: (i) a Tomato Cluster Dwarf Virus family virus providing RdRP; or (ii) a recombinant polynucleotide molecule encoding RdRP; and then providing the recombinant RNA molecule to the plant containing the plant cells. In some embodiments, the method may further comprise the step of determining whether the plant cells contain a Tomato Cluster Dwarf Virus family virus that can provide RdRP. In embodiments where it is determined that the plant cells contain a Tomato Cluster Dwarf Virus family virus that can provide RdRP, the Tomato Cluster Dwarf Virus family virus, the RdRP protein, and / or the recombinant polynucleotide encoding RdRP are optionally provided to the plant cells non-exogenously. In other embodiments where the plant cells are determined not to contain a Tomato Cluster Dwarf Virus Family virus that can provide RdRP and the Tomato Cluster Dwarf Virus Family virus is provided exogenously to the plant cells, the RdRP protein or a recombinant polynucleotide encoding RdRP is provided exogenously to the plant cells, or a combination of a Tomato Cluster Dwarf Virus Family virus, the RdRP protein, or a polynucleotide encoding RdRP is provided exogenously to the plant cells.In an embodiment, a complete self-replicating Tomato dwarfvirus satellite system is introduced into a plant or plant cell, wherein the self-replicating Tomato dwarfvirus satellite system comprises: (1) a recombinant Tomato dwarfvirus satellite RNA comprising, from its 5' end to its 3' end: (a) a 5' RNA replication element recognized by Tomato dwarfvirus RNA-dependent RNA polymerase (RdRP); (b) a cargo RNA molecule; and (c) a 3' RNA replication element recognized by RdRP; wherein the 5' RNA replication element, the cargo RNA molecule, and the 3' RNA replication element are operatively 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) a foreign Tomato dwarfvirus virus (e.g., a Tomato dwarfvirus virus that is not specific to or natural to the plant or plant cell) capable of replicating and encoding the 5' and 3' replicase recognition sequences in the recombinant Tomato dwarfvirus satellite RNA that recognize the RdRP. In this embodiment, the Tomato Cluster Dwarf Virus Family RdRP comprises a protein having at least 85%, 90%, 95%, 97%, or 99% sequence identity with SEQ ID NO: 469 or 587. The presence of Tomato Cluster Dwarf Virus Family viruses in a target plant can be determined by RNA detection assays (e.g., RT-PCR assays) using nucleic acid probes and / or primers that can detect any portion of the Tomato Cluster Dwarf Virus Family genome, including 5' RNA replication elements, CP and / or RdRP coding regions, and / or 3' RNA replication elements. Such probes and primers include those that detect any 5' or 3' RNA replication elements shown in Table 1 or those having significant sequence identity with them (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 of Tomato dwarfviruses in target plants can be determined by protein detection (e.g., immunoassay) targeting Tomato dwarfvirus CPs or RdRPs (e.g., CPs or RdRPs encoded by the Tomato dwarfvirus genome disclosed in Table 1 or CPs or RdRPs homologous to them). The target plants and plant cells used in the method include all of the aforementioned target plants and plant cell hosts containing synthetic Tomato dwarfvirus satellite RNAs (e.g., recombinant RNAs).

[0089] In some embodiments of any of the methods disclosed for the foregoing or otherwise, the recombinant RNA achieves: (i) a phenotypic change in a plant or plant cell; (ii) an increase in plant resistance to pests or pathogens; or (iii) an increase in plant resistance to stress, wherein the recombinant RNA may include RNA that regulates target gene expression relative to the expression of a target gene in a control plant or plant cell not provided with the recombinant RNA molecule, and the regulation results in a phenotypic change, increased resistance to pests or pathogens, or increased resistance to stress. In embodiments, the regulation is (a) an increase in target gene expression; or (b) a decrease in target gene expression. In some embodiments, the expression of the target gene is increased relative to a reference level (e.g., the level present in control plants or plant cells lacking recombinant RNA) 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%, 4 6%, 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%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% or more. In some embodiments, the expression of the target gene is increased by up to about 2, 3, 4, 5, 6, 8, 9, 10 or more relative to a reference level (e.g., the level present in control plants or plant cells lacking recombinant RNA molecules).In some embodiments, the expression of the target gene is reduced 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%, 4 6%, 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%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% or more. In some embodiments, the expression of the target gene is reduced by up to about 2, 3, 4, 5, 6, 8, 9, 10, or more times relative to a reference level (e.g., the level present in control plants or plant cells lacking recombinant RNA molecules). Regulation of target gene expression can be achieved by one or more RNAs used to modify the genome, epigenome, and / or transcriptome of the plant or plant cell. RNAs used to modify the genome include gRNAs recognized by CAS nucleases, RNAs encoding TALENs, or artificial zinc finger proteins (aZFNs). RNAs used to modify the epigenome include RNAs that provide RNA-guided DNA methylation, such as DNA methylation in the promoter region of the target gene (Matzke and Mosher (2014), doi: 10.1038 / nrg3683). Examples of RNAs used to modify the transcriptome include one or more RNAs containing any of the following: hairpin RNA (hpRNA); RNA forming multiple stem loops; RNA pseudoknots; RNA molecules forming at least partially double-stranded RNA; small interfering RNA (siRNA) or siRNA precursors; microRNA (miRNA) or miRNA precursors; staged siRNA or staged siRNA precursors (see, for example, U.S. Patent No. 8,404,928); ribozymes; ligand-responsive ribozymes (aptamers); RNA aptamers; or long non-coding RNA (lncRNA).

[0090] In any of the foregoing or otherwise disclosed methods in which phenotypic changes are obtained or achieved in plants or plant cells, the cargo RNA molecule may be included in the plant or plant cells that achieve phenotypic changes compared to plants or plant cells lacking recombinant RNA. In some embodiments, the altered phenotype includes developmental rate, growth rate, size, yield (e.g., intrinsic yield), vigor, photosynthetic capacity, flavor, starch yield, protein content, carbohydrate content, oil content, fatty acid content, lipid content, digestibility, biomass, shoot length, root length, root structure, seed setting rate, seed weight, seed quality (e.g., nutrient content), germination rate, fruit set rate, fruit maturity rate, biopolymer yield, fiber yield, biofuel yield, yield of medicinal peptides (e.g., hormones, enzymes, transcription factors, antigens, antibodies, or antibody fragments), yield of secretible peptides, enzyme yield, improved processing traits, or amount of harvestable product. In some embodiments, the altered phenotype includes the taste, appearance, or shelf life of products harvested from the plant. In other embodiments, the altered phenotypes include flower size, flower color, flower pattern, flower morphology (including the presence or absence of stamens), number of flowers, flower lifespan, flower fragrance, leaf size, leaf color, leaf pattern, leaf morphology, plant height, or plant structure.

[0091] In any of the foregoing or otherwise disclosed methods in which plant resistance to pests or pathogens is increased, the recombinant RNA may comprise RNA that inhibits gene expression of the pest or pathogen and / or inhibits genome replication of the pest or pathogen. In some embodiments, the pest or pathogen is selected from the group consisting of bacteria, viruses other than those of the Tomato Cluster Dwarf Virus family, fungi, oomycetes, and invertebrates (e.g., arthropods or nematodes). Other target viruses besides those from the Tomato Dwarf Virus family include: (i) positive-sense RNA viruses from the Bromoviridae, Closteroviridae, Luteoviridae, or PotatoY Viridae families; (ii) negative-sense RNA viruses from the Bunyaviridae and Rhabdoviridae families; (iii) dsDNA viruses from the Caulimoviridae family; and (iv) ssDNA viruses from the Geminiviridae family. Target arthropod pests include Coleoptera and Lepidoptera insects. Target fungal pathogens include species of the genera *Puccinia*, *Botrytis*, and *Puccinia* spp.; species of *Fusarium*, *Erythromyces*, *Mycosphaerella*, *Colletotrichum*, *Ustilago*, *Scattered Rust*, *Phakopsora*, *Phytophthora*, and *Rhizoctonia*. In the embodiments, the cargo RNA molecules increase the plant's resistance to pests or pathogens compared to plants without recombinant RNA molecules.

[0092] In any of the foregoing or otherwise disclosed methods in which plant resistance to stress is increased, the recombinant RNA may comprise RNA targeting plant genes that provide such resistance. In embodiments, the RNA that achieves increased plant resistance to stress in a plant or plant cell comprises RNA that regulates target gene expression relative to the expression of a target gene in a control plant or plant cell not provided with the recombinant RNA molecule, and wherein said regulation results in increased stress resistance. In embodiments, the regulation is (a) increased target gene expression; or (b) decreased target gene expression. In still other embodiments, the RNA that achieves increased plant resistance to stress in a plant or plant cell comprises messenger RNA encoding a protein that confers stress resistance. In still other embodiments, the messenger RNA comprises an RNA sequence absent in the transcriptome of a plant or plant cell lacking the recombinant RNA. In embodiments, stress includes at least one abiotic stress selected from the group consisting of: nutrient stress, light stress, drought stress, heat stress, and cold stress. In other embodiments, the stress includes at least one biological stress selected from the group consisting of crowding, shading, and allelopathic effects (e.g., caused by allelochemicals, including juglone produced by walnut trees).

[0093] In any of the foregoing or otherwise disclosed methods for expressing a foreign polypeptide (e.g., a foreign polypeptide) in a plant or plant cell, the cargo RNA may encode the foreign polypeptide. In examples, the polypeptide is isolated (e.g., separated from at least one other cellular component (e.g., carbohydrate, lipid, or another protein)) or purified.

[0094] In any of the foregoing or otherwise disclosed methods for manufacturing synthetic Tomato dwarf Virus satellite particles, such manufacturing can occur in a cell-based system or a cell-free system. A cell-based method for manufacturing synthetic Tomato dwarf Virus satellite particles may include: (a) providing the recombinant RNA molecule to plant cells, wherein the recombinant RNA molecule contains a capsidization recognition element (ERE), and wherein the plant cells contain an RdRP protein and a viral capsid protein that recognize the 5' RNA replication element and the 3' RNA replication element and catalyze the synthesis of synthetic Tomato dwarf Virus satellite RNA from the recombinant RNA molecule, wherein the ERE provides capsidization of the RNA by the viral capsid protein; and optionally isolating the synthetic Tomato dwarf Virus satellite particles from plant cells, plants containing the plant cells, or from the plant cells or a culture medium in which the plant is grown. A cell-free method for manufacturing synthetic Tomato dwarf Virus Family satellite particles includes a method of combining a recombinant RNA molecule with a viral capsid protein in a container, wherein the recombinant RNA molecule contains an ERE, and wherein the ERE provides capsidation of the RNA by the viral capsid protein in the container; optionally, the method further includes separating the synthetic Tomato dwarf Virus Family satellite particles from uncombined RNA and / or viral capsid proteins in the container. In some embodiments of cell-based or cell-free methods, the synthetic satellite particles are isolated (e.g., separated from at least one other cellular component (e.g., organelle, membrane, carbohydrate, lipid, or another protein)) or purified. In some embodiments, the method may further include formulating the synthetic Tomato dwarf Virus Family satellite particles.

[0095] Synthetic Tomato Cluster Dwarf Virus Family satellite particles containing recombinant RNA are also provided, including those prepared by the foregoing methods. A method for providing any of the foregoing synthetic Tomato Cluster Dwarf Virus Family satellite particles to a plant is also provided, the method comprising contacting the plant with the synthetic Tomato Cluster Dwarf Virus Family satellite particles or formulations thereof (e.g., spraying, dusting, injection, soaking, etc.).

[0096] The recombinant polynucleotides described herein, the cells containing them, and the synthetic Tomato dwarf Virus family satellite particles can be formulated in pure form (e.g., the composition contains only the recombinant polynucleotides) or with one or more additional formulation components to facilitate the application or delivery of the composition. In embodiments, additional formulation components include, for example, a carrier (i.e., a component that has an active role in delivering the active agent (e.g., the recombinant polynucleotide); for example, the carrier may encapsulate, covalently or non-covalently modify the active agent, or otherwise associate with the active agent in a manner that improves the delivery of the active agent; or excipients (e.g., delivery mediators, adjuvants, diluents, surfactants, stabilizers, or tensioners). In some embodiments, the composition is formulated for delivery to plants.

[0097] In some aspects, this disclosure provides formulations comprising any of the compositions described herein. In some embodiments, the formulation is a liquid, gel, or powder. In some embodiments, the formulation is configured to be sprayed onto a plant, injected into a plant (see, for example, U.S. Patent No. 11,844,318), or otherwise introduced into the vascular system of a plant, rubbed onto leaves, soaked into a plant, coated onto a plant, or coated onto seeds, or delivered by root uptake (e.g., in a hydroponic system or via soil).

[0098] Depending on the intended purpose and the prevailing circumstances, the composition may be formulated as an emulsifiable concentrate, a suspension concentrate, a direct-spray solution or a dilutable solution, a coating ointment, a diluted emulsion, a spray powder, a soluble powder, a dispersible powder, a wettable powder, a dust, granules, an encapsulator in a polymeric substance, a microcapsule, a foam, an aerosol, a carbon dioxide gas formulation, a tablet, a resin formulation, a paper formulation, a nonwoven fabric formulation, or a knitted or woven fabric formulation. In some cases, the composition is a liquid. In some cases, the composition is a solid. In some cases, the composition is an aerosol, such as in a pressurized aerosol can.

[0099] In some cases, the recombinant polynucleotide constitutes about 0.1% to about 100% of the composition, such as any one of the following: 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 the active ingredient (e.g., recombinant polynucleotide). In some cases, the composition comprises at least any one of 0.1%, 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more of the active ingredient (e.g., recombinant polynucleotide). In some cases, a concentrate is preferred as a commercial product, and end users typically use diluted formulations (with significantly lower concentrations of the active ingredient).

[0100] In some embodiments, the formulation is used for local delivery to plants. In some embodiments, local delivery is by spraying, wiping (e.g., with or without abrasives), soaking, coating (e.g., coating with microparticles or nanoparticles), or delivery via root uptake (e.g., in a hydroponic system or by root drenching).

[0101] In some embodiments, the composition further comprises a carrier and / or an excipient. In other embodiments, the composition does not contain a carrier or excipient, for example, it contains naked polynucleotides (e.g., naked RNA).

[0102] In some embodiments, the recombinant polynucleotide is delivered at a concentration of at least 0.1 grams per acre, such as at least 0.1, 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 grams per acre. In some embodiments, delivery is carried out at less than 120 liters per acre, such as less than 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 10, 5, or 2 liters per acre, or less than 1 liter per acre.

[0103] In some aspects, the formulation includes a carrier. In some embodiments, the formulation is an emulsion or reverse emulsion, liquid, or gel. In embodiments, the formulation includes a carrier that acts as a physical support (e.g., a solid or semi-solid surface or matrix, powder, or particles or nanoparticles). In embodiments, an active agent is encapsulated or encapsulated in, attached to, or compounded with a carrier, said carrier including liposomes, vesicles, micelles, or other fluid compartments. In embodiments, an active agent is encapsulated or encapsulated in, attached to, or compounded with a carrier, said carrier including naturally occurring or synthetic, branched or linear polymers (e.g., pectin, agarose, chitin, chitosan, DEAE-glucan, polyvinylpyrrolidone (“PVP”), or polyethyleneimine (“PEI”)). In embodiments, the carrier includes cationic or cationically charged components, such as cationic liposomes or cationic polymers such as polyamines (e.g., spermine, spermidine, putrescine). In the embodiments, the carrier includes peptides such as enzymes (e.g., cellulase, pectin lyase, dissociation enzyme, pectinase), cell-penetrating peptides, or pore-forming peptides (e.g., polylysine, polyarginine, or polyhomarginine peptides).

[0104] Non-limiting examples of carriers include cationic liposomes and polymeric nanoparticles, such as those reviewed in Zhang et al. (2007) J. Controlled Release, 123: 1-10, and cross-linked multilayer liposomes described in U.S. Patent Application Publication 2014 / 0356414 A1, which are incorporated herein by reference in their entirety. In embodiments, carriers include nanomaterials such as carbon or silica nanoparticles, carbon nanotubes, carbon nanofibers, or carbon quantum dots. Non-limiting examples of carriers include particles or nanoparticles of various sizes and shapes (e.g., particles or nanoparticles made of materials such as carbon, silicon, silicon carbide, gold, tungsten, polymers, or ceramics), magnetic particles or nanoparticles (e.g., silenceMag Magnetotransfection). TM Reagents, OZ Bioscience, San Diego, California; abrasives or scratching agents; needles or microneedles; matrix and grid.

[0105] In some embodiments, the microparticles and nanoparticles can be used to deliver polynucleotide compositions or nucleases or both. Useful microparticles and nanoparticles include those made of: metals (e.g., gold, silver, tungsten, iron, cerium), ceramics (e.g., alumina, silicon carbide, silicon nitride, tungsten carbide), polymers (e.g., polystyrene, polyacetylene, 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, nanocomposites, or nanotubes), and composite materials (e.g., polyvinylcarbazole / graphene, polystyrene / graphene, platinum / graphene, palladium / graphene nanocomposites). In some embodiments, such microparticles and nanoparticles are further covalently or non-covalently functionalized, or further comprise modifiers or crosslinking materials, such as polymers (e.g., linear or branched polyethyleneimine, polylysine), polynucleotides (e.g., DNA or RNA), polysaccharides, lipids, polyethylene glycols (e.g., polyethylene glycol, thiolated polyethylene glycol), peptides or proteins, and detectable markers (e.g., fluorophores, antigens, antibodies, or quantum dots). In various embodiments, such microparticles and nanoparticles are neutral, positively charged, or negatively charged.

[0106] Examples of compositions including microparticles include those formulated as liquids, colloids, dispersions, suspensions, aerosols, gels, and solids. Examples include nanoparticles immobilized to a surface or support, such as an array of carbon nanotubes vertically aligned on a silicon or copper wafer substrate. Examples include polynucleotide compositions comprising microparticles (e.g., gold or tungsten or magnetic particles) delivered via bioprojectile technology or utilizing magnetic force. Particle sizes used in bioprojectiles are typically in the “microparticle” range, for example, gold microcarriers in the size range of 0.6, 1.0, and 1.6 micrometers (see, for example, Gene gun system, Bio-Rad, Hercules, California, user manual; Randolph-Anderson et al. (2015) "Submicron gold particles are superior to larger particles for efficient Transformation of organelles and some cell types: Submicron gold particles are superior to larger particles, enabling highly efficient transformation of organelles and certain cell types. [Transformation], Bio-Rad Laboratories US / EG Report 2015), but successful delivery of biological projectiles using larger (40-48 nm) nanoparticles in cultured animal cells has been reported; see O'Brian and Lummis (2011) BMC Biotechnol. [BMC Biotechnology], 11: 66-71.

[0107] Other embodiments of useful microparticles are nanoparticles, which are typically in the nanometer (nm) size range or less than 1 micrometer, for example, with diameters 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. Specific, non-limiting embodiments of commercially available nanoparticles (all from Sigma-Aldrich Corp., St. Louis, Missouri) include gold nanoparticles with diameters of 5, 10, or 15 nm; silver nanoparticles with diameters of 10, 20, 40, 60, or 100 nm; palladium “nanopowder” with a diameter less than 25 nm; and single-walled, double-walled, and multi-walled carbon nanotubes, for example, with diameters of 0.7–1.1, 1.3–2.3, 0.7–0.9, or 0.7–1.3 nm, or nanotube bundle sizes of 2–10 nm x 1–5 μm, 6–9 nm x 5 μm, 7–15 nm x 0.5–10 μm, 7–12 nm x 0.5–10 μm, 110–170 nm x 5–9 μm, or 6–13 nm x 2.5–20 μm. Examples include polynucleotide compositions comprising materials such as gold, silicon, cerium, or carbon, such as gold or gold-plated nanoparticles, silicon carbide whiskers, corundum, porous silica nanoparticles, gelatin / silica nanoparticles, cerium dioxide nanoparticles (nanoceria) or cerium oxide nanoparticles (CNP), carbon nanotubes (CNTs) (such as single, double, or multi-walled carbon nanotubes and their chemically functionalized forms (e.g., carbon nanotubes partially functionalized with amides, amino groups, carboxylic acids, sulfonic acids, or polyethylene glycol)), and graphene or graphene oxide or graphene complexes; see, for example, Wong et al. (2016) Nano Lett., 16: 1161-1172; Giraldo et al. (2014) Nature Materials, 13: 400-409; Shen et al. (2012) Theranostics, 2: 283-294; Kim et al. (2011) Bioconjugate Chem. [Bioconjugate Chemistry], 22: 2558-2567; Wang et al. (2010) J. Am. Chem. Soc. Comm. [Journal of the American Chemical Society Communications], 132: 9274-9276; Zhao et al. (2016) Nanoscale Res. Lett. [Nanoscale Research Letters], 11: 195-203; and Choi et al. (2016) J. Controlled Release [Journal of Controlled Release], 235: 222-235.See also, for example, various types of particles and nanoparticles, their preparation and methods of use (e.g., in the delivery of polynucleotides and peptides into cells) disclosed in U.S. Patent Application Publications 2010 / 0311168, 2012 / 0023619, 2012 / 0244569, 2013 / 0145488, 2013 / 0185823, 2014 / 0096284, 2015 / 0040268, 2015 / 0047074 and 2015 / 0208663, all of which are incorporated herein by reference in their entirety.

[0108] In some aspects, the composition includes excipients, such as delivery mediators, adjuvants, diluents, surfactants, stabilizers, or tensioners, or combinations thereof. In some embodiments, the excipient is a crop oil concentrate, vegetable oil concentrate, modified vegetable oil, nitrogen source, sedimentation (drift control) and / or retention aid (with or without ammonium sulfate and / or defoamer), compatibilizer, buffer and / or acidifier, water conditioner, basic blend, spreader and / or extender, adjuvant plus foliar fertilizer, defoamer, foam marker, odorant or box cleaner and / or neutralizer. In some embodiments, the excipient is an adjuvant described in the Compendium of Herbicide Adjuvants (Young et al. (2016). Compendium of Herbicide Adjuvants (13th Edition), Purdue University).

[0109] Examples of delivery media and diluents include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, tragacanth gum, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, saline solution, syrup, methylcellulose, methylparaben and propylparaben, talc, magnesium stearate, and mineral oil. Further examples of delivery media include, but are not limited to, solid or liquid excipients, solvents, stabilizers, sustained-release excipients, colorants, and surfactants. In some cases, the excipient (e.g., the delivery media) is a stabilizing media. In embodiments, stabilizing media include, for example, epoxidized vegetable oils, defoamers (e.g., silicone oils), preservatives, viscosity modifiers, binders, or thickeners. In some cases, the stabilizing media is a buffer suitable for recombinant polynucleotides. In some cases, the composition is microencapsulated in a polymer bead delivery media. In some cases, the stabilizing medium protects the recombinant polynucleotide from UV and / or acidic conditions. In some cases, the delivery medium contains a pH buffer. In some cases, the composition is formulated to have a pH in the range of about 4.5 to about 9.0, including, for example, a pH in the range of about 5.0 to about 8.0, about 6.5 to about 7.5, or about 6.5 to about 7.0.

[0110] In some cases, the compositions described herein include adjuvants. Adjuvants are reagents that do not possess polynucleotide activity but impart beneficial properties to a formulation. For example, adjuvants are premixed in a formulation or added to a spray can to improve mixing, application, or performance. They are widely used in products designed for foliar application. Adjuvants can be used to tailor formulations to specific needs and to compensate for local conditions. Adjuvants can be designed to perform specific functions, including wetting, spreading, adhesion, reducing evaporation, reducing volatility, buffering, emulsifying, dispersing, reducing spray drift, and reducing foaming. No single adjuvant can perform all of these functions, but compatible adjuvants can often be combined to perform multiple functions simultaneously.

[0111] Non-limiting examples of adjuvants included in formulations include binders, dispersants, and stabilizers, particularly, for example, casein, gelatin, polysaccharides (e.g., starch, gum arabic, cellulose derivatives, alginate, etc.), lignin derivatives, bentonite, sugars, synthetic water-soluble polymers (e.g., polyvinyl alcohol, polyvinylpyrrolidone, polyacrylic acid, etc.), PAP (acid isopropyl phosphate), BHT (2,6-di-tert-butyl-4-methylphenol), BHA (a mixture of 2-tert-butyl-4-methoxyphenol and 3-tert-butyl-4-methoxyphenol), vegetable oils, mineral oils, fatty acids, and fatty acid esters.

[0112] In the embodiments, the compositions provided herein are in liquid formulations. Liquid formulations are typically mixed with water, but in some cases are used with crop oils, diesel fuels, kerosene, or other light oils as excipients. The amount of the active ingredient (e.g., recombinant polynucleotide) typically ranges from about 0.5% to about 80% by weight.

[0113] In embodiments, emulsifiable concentrate formulations contain a liquid active ingredient, one or more petroleum-based solvents, and a reagent that allows the formulation to mix with water to form an emulsion. Such concentrates can be used in agricultural, ornamental plant and lawn, forestry, construction, food processing, livestock, and public health pest formulations. In embodiments, these are suitable for application equipment ranging from small portable sprayers to hydraulic sprayers, small-volume ground sprayers, mist sprayers, and small-volume aircraft sprayers. Some active ingredients are readily soluble in liquid excipients. When mixed with excipients, they form solutions that do not precipitate or separate, such as homogeneous solutions. In embodiments, these types of formulations include an active ingredient, a carrier and / or excipients, and one or more other components. The solutions can be used with any type of indoor and outdoor sprayer.

[0114] In some cases, compositions are formulated as reverse emulsions. A reverse emulsion is a water-soluble active ingredient dispersed in an oil excipient. Reverse emulsions require an emulsifier that allows the active ingredient to mix with a significant amount of petroleum-based excipient (typically fuel oil). Reverse emulsions help reduce drift. With other formulations, some spray drift occurs when water droplets begin to evaporate before reaching the target surface; as a result, the droplets become very small and lightweight. Because oil evaporates more slowly than water, reverse emulsion droplets shrink less, and more active ingredient reaches the target. Oil further helps reduce runoff and improve rain resistance. It also acts as a spreader by improving surface coverage and absorption. Due to the relatively large and heavy droplets, thorough coverage on the underside of the leaf is difficult. Reverse emulsions are most commonly used along right-of-way areas (where drift into vulnerable non-target areas can be a problem).

[0115] Flowable or liquid formulations combine many features of emulsifiable concentrates and wettable powders. Manufacturers use these formulations when the active ingredient is a solid insoluble in water or oil. The active ingredient, impregnated in a substance such as clay, is ground into a very fine powder. The powder is then suspended in a small amount of liquid. The resulting liquid product is quite viscous. Flowable formulations and liquids share many characteristics of emulsifiable concentrates, and they also have similar drawbacks. They require moderate agitation to keep them suspended and leave visible residue, similar to those found in wettable powders.

[0116] Flowable animals / liquids are easy to handle and apply. Because they are liquids, they can spill and splash. They contain solid particles, which can promote wear on nozzles and pumps. Flowable animal and liquid suspensions tend to settle in their containers. Because flowable animal and liquid formulations tend to settle, packaging them in five-gallon or smaller containers makes remixing easier.

[0117] Aerosol formulations contain one or more active ingredients and solvents. Most aerosols contain a low percentage of active ingredients. There are two types of aerosol formulations—ready-to-use formulations that are typically used in pressurized, sealed containers, and those designed for electric or gasoline-powered aerosol generators (which release the formulation as smoke or mist).

[0118] Ready-to-use (RTU) aerosol formulations are typically small, self-contained units that release the formulation when the nozzle valve is triggered. Under pressure, an inert gas drives the formulation through a fine pore, creating tiny droplets. These products are used in greenhouses, small areas inside buildings, or localized outdoor areas. Commercial models containing five to five pounds of active ingredient are typically refillable.

[0119] Aerosol or mist formulations are not under pressure. They are used in machines that use a rapidly rotating disc or heated surface to break down liquid formulations into fine mists or fogs (aerosols).

[0120] In some embodiments, the composition comprises a liquid excipient. In embodiments, the liquid excipient includes, for example, aromatic or aliphatic hydrocarbons (e.g., xylene, toluene, alkylnaphthalene, phenylxylene ethane, kerosene, gas oil, hexane, cyclohexane, etc.), halogenated hydrocarbons (e.g., chlorobenzene, dichloromethane, dichloroethane, trichloroethane, etc.), alcohols (e.g., methanol, ethanol, isopropanol, 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.), and esters (e.g., The following are listed as ingredients: ethyl acetate, butyl acetate, etc.), ketones (e.g., acetone, methyl ethyl ketone, methyl isobutyl ketone, cyclohexanone, etc.), nitriles (e.g., acetonitrile, isobutyronitrile, etc.), sulfoxides (e.g., dimethyl sulfoxide, etc.), amides (e.g., N,N-dimethylformamide, N,N-dimethylacetamide), cyclic imides (e.g., N-methylpyrrolidone), alkyl carbonates (e.g., propylene carbonate, etc.), vegetable oils (e.g., soybean oil, cottonseed oil, etc.), essential vegetable oils (e.g., orange oil, hexamethylenetetramine oil, lemon oil, etc.), or water.

[0121] In some embodiments, the composition comprises a gaseous excipient. Gaseous excipients include, for example, butane, Freon, liquefied petroleum gas (LPG), dimethyl ether, and carbon dioxide.

[0122] In some embodiments, the composition is provided as a dry formulation. Dry formulations can be of two types: ready-to-use and concentrates that must be mixed with water for spray application. Most dust formulations are ready-to-use and contain a low percentage of active ingredient (less than about 10% by weight) plus very fine dry inert excipients (e.g., talc, chalk, clay, nut shells, or volcanic ash). The individual dust particles vary in size. Some dust formulations are concentrates and contain a high percentage of active ingredient. In some embodiments, these dust formulations are mixed with dry inert excipients prior to application. In some embodiments, the dust is used dry and may easily drift to non-target sites.

[0123] In some cases, the composition is formulated as a powder. In other cases, it is formulated as a wettable powder. Wettable powders are dry, finely ground formulations that resemble dust. They typically must be mixed with water for application as a spray. However, a few products can be applied as either dust or wettable powder—the choice depends on the applicant. Wettable powders contain approximately 1% to approximately 95% active ingredient by weight; in some cases, more than approximately 50%. The particles are insoluble in water. They quickly precipitate unless continuously stirred to keep them in suspension. They can be used for most pest problems and in most types of spray equipment where stirring is possible. Wettable powders exhibit excellent residual activity. Due to their physical properties, most formulations remain on the surface of treated porous materials such as concrete, plaster, and untreated wood. In such cases, only water permeates the material.

[0124] In some cases, compositions are formulated as soluble powders. Soluble powder formulations resemble wettable powders. However, when mixed with water, soluble powders readily dissolve and form true solutions. Once thoroughly mixed, no further stirring is required. The amount of active ingredient in soluble powders ranges from about 15% to about 95% by weight; in some cases, it exceeds about 50%. Soluble powders possess all the advantages of wettable powders and, except for the inhalation hazard during mixing, do not have any of the disadvantages of wettable powders.

[0125] In some cases, compositions are formulated as water-dispersible granules. Water-dispersible granules (also known as dry flowable granules) are similar to wettable powders (except they are not dust-like) in that they are formulated as small, easily measurable granules. Water-dispersible granules must be mixed with water to be applied. Once in water, the granules break down 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, typically up to 90% by weight. Water-dispersible granules share many of the same advantages and disadvantages as wettable powders, except that they are easier to measure and mix. Due to the lower dust content, there is less inhalation hazard to the applicator during handling.

[0126] In some embodiments, the composition comprises a solid excipient. Solid excipients include the following subcategories of powders or granules: clays (e.g., kaolin, diatomaceous earth, bentonite, Fubasami clay, acid clay, etc.), synthetic hydrated silica, talc, ceramics, other inorganic minerals (e.g., sericite, quartz, sulfur, activated carbon, calcium carbonate, hydrated silica, etc.), substances that can sublimate and are solid at room temperature (e.g., 2,4,6-triisopropyl-1,3,5-trioxane, naphthalene, p-dichlorobenzene, camphor, adamantane, etc.); wool; silk; cotton; hemp; pulp; synthetic resins (e.g., polyethylene resins, such as low-density polyethylene, linear low-density polyethylene, and high-density polyethylene; ethylene-vinyl ester copolymers, such as ethylene-vinyl acetate copolymers; ethylene-methacrylate copolymers, such as ethylene-methyl methacrylate copolymers and ethylene-ethyl methacrylate copolymers; ethylene-acrylate copolymers, such as ethylene-methyl acrylate copolymers and ethylene-ethyl acrylate copolymers; ethylene-vinyl carboxylic acid copolymers, such as ethylene-acrylic acid copolymers; ethylene... Polypropylene-tetracyclododecene copolymers; polypropylene resins, such as propylene homopolymers and propylene-ethylene copolymers; poly-4-methylpentene-1, polybutene-1, polybutadiene, polystyrene; acrylonitrile-styrene resins; styrene elastomers, such as acrylonitrile-butadiene-styrene resins, styrene-conjugated diene block copolymers, and styrene-conjugated diene block copolymer hydrides; fluoropolymers; acrylic resins, such as poly(methyl methacrylate); polyamide resins, such as nylon 6 and nylon 66; polyester resins, such as polyethylene... Phthalate, polyethylene naphthalate, polybutylene terephthalate, and polycyclohexyl dimethyl terephthalate; polycarbonate, polyacetal, polyacrylamide sulfone, polyarylate, polyacrylate, hydroxybenzoic acid polyester, polyetherimide, polyester carbonate, polyphenylene ether resin, polyvinyl chloride, polyvinylidene chloride, polyurethane, and porous resins (such as foamed polyurethane, foamed polypropylene, or foamed ethylene), glass, metal, ceramics, fibers, fabrics, knitted fabrics, sheets, paper, yarns, foams, porous materials, and multifilaments.

[0127] In some cases, the composition is provided in a microencapsulated formulation (e.g., nanocapsules). The microencapsulated formulation is mixed with water and sprayed in the same manner as other sprayable formulations. Upon spraying, the microencapsulation shell or coating ruptures and slowly releases the active ingredient.

[0128] In some cases, the composition is provided in liposomes. In other cases, the composition is provided in vesicles.

[0129] In some cases, the compositions described herein include surfactants. Surfactants (also known as wetting agents and spreading agents) physically alter the surface tension of spray droplets. For a formulation to function properly, the spray droplets must be able to wet the leaf and spread evenly on the leaf surface. Surfactants increase the coverage area of ​​the formulation, thereby increasing the exposure of the active agent. Surfactants are particularly important when the formulation is applied to waxy or hairy leaves. Without proper wetting and spreading, spray droplets often run off or fail to adequately cover the leaf surface. However, excessive surfactant can lead to excessive runoff and reduced efficacy.

[0130] Surfactants can be classified as anionic, cationic, or nonionic. The activity of a formulation in the presence of a nonionic surfactant can differ considerably from that in the presence of a cationic or anionic surfactant. Choosing the wrong surfactant can reduce product efficacy and harm target plants. For example, anionic surfactants are most effective when used with contact pesticides (pesticides that control pests through direct contact rather than systemic absorption). Cationic surfactants are typically not used as standalone surfactants because they are often phytotoxic.

[0131] Nonionic surfactants, often used with systemic pesticides, help the spray penetrate the plant cuticle. Nonionic surfactants are compatible with most pesticides, and most EPA-registered pesticides requiring surfactants recommend the use of nonionic surfactants. Adjuvants include, but are not limited to, adhesives, extenders, plant penetrants, compatibilizers, buffers or pH adjusters, drift control additives, defoamers, and thickeners.

[0132] Non-limiting examples of surfactants included in the compositions described herein include alkyl sulfate salts, alkyl sulfonates, alkyl aryl sulfonates, alkyl aryl ethers and their polyoxyethyleneized products, polyethylene glycol ethers, polyol esters, and sugar alcohol derivatives. In some embodiments, the surfactant is a nonionic surfactant, a surfactant with a nitrogen source, a silicone surfactant, or a high surfactant oil concentrate.

[0133] In formulations and forms of use prepared from such formulations, in the examples, the recombinant polynucleotide may be in mixture with other active compounds, such as pestidal agents (e.g., insecticides, fungicides, acaricides, nematicides, molluscicides, fungicides, attractants, growth regulators, or herbicides). As used herein, the term "pesticidal agent" means any substance or mixture of substances intended to prevent, destroy, suppress, or mitigate the effects of any pest. Pesticides can be chemical or biological agents used to combat pests, including insects, mollusks, pathogens, weeds, nematodes, and microorganisms that compete with humans for food, damage property, spread disease, or are unpleasant. The term "pesticidal agent" further encompasses other bioactive molecules, such as antibiotics, antiviral pesticides, antifungals, anti-helmintics, nutrients, pollen, sucrose, and / or agents that weaken or slow insect movement.

[0134] When applying recombinant polynucleotides to plants, it is also possible to mix them with other known compounds (such as herbicides, fertilizers, growth regulators, safeners, and information chemicals) or with agents used to improve plant characteristics.

[0135] In another aspect, this disclosure relates to a method for producing modified plant propagules comprising at least one plant cell containing a recombinant RNA molecule. The method generally includes the steps of isolating plant propagules from a mixed population of plant cells containing both plant cells containing the recombinant RNA molecule and plant cells lacking the recombinant RNA molecule. The recombinant RNA molecule comprises, in a 5' to 3' order, a 5' RNA replication element recognizable by the RdRP; a cargo RNA sequence; and a 3' RNA replication element recognizable by the RdRP. In some embodiments, the isolated plant propagules comprising at least one plant cell containing the recombinant RNA molecule will contain no or substantially no plant cells lacking the recombinant RNA. In some embodiments, such isolated plant propagules substantially free of plant cells lacking the recombinant RNA may comprise plant propagules in which at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the plant cells contain recombinant DNA molecules. In embodiments, the plant cell mixed population comprises a population of protoplasts or cells from callus, explants, plant parts, or the whole plant. In embodiments, the plant cell mixed population may comprise a population of plant cells in which less than 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or 1% of the plant cells contain recombinant RNA molecules. In some embodiments, the plant cell mixed population comprises plant cells containing RdRP of the Tomato Cluster Dwarf Virus Family and plant cells lacking RdRP of the Tomato Cluster Dwarf Virus Family. In some embodiments, plant cells lacking RdRP of the Tomato Cluster Dwarf Virus Family will also lack recombinant RNA. In other embodiments, the plant cell mixed population comprises plant cells containing RdRP of the Tomato Cluster Dwarf Virus Family. In some embodiments, plant cells containing RdRP of the Tomato Cluster Dwarf Virus Family may further contain recombinant RNA. In some embodiments, the cargo RNA sequence comprises RNA encoding selectable or scoreable markers. In embodiments, the plant cell mixed population is screened or selected for the presence of plant cells containing recombinant RNA molecules prior to isolating plant propagules. In such screening, a mixed population of cells or a portion thereof is subjected to the determination of a screenable marker for the presence of recombinant RNA molecules (e.g., an RNA sequence diagnosing the presence of recombinant RNA molecules or polypeptides encoded by recombinant RNA molecules) and separated from plant cells lacking recombinant RNA molecules. In some embodiments, separation includes selecting plant cells containing recombinant RNA molecules prior to separating plant propagules.In cases where the recombinant RNA encodes a selectable marker (e.g., a protein confers resistance to a selector such as a herbicide or antibiotic), examples of such selection may include exposing a mixed population of plant cells to the selector (e.g., a herbicide or antibiotic) and isolating plant cells that survive the selector exposure. Examples of selectable marker / selector combinations include glyphosate-resistant EPSPS enzymes and / or glyphosate oxidase / glyphosate, biammoniaphosphonate resistance (bar) or glufosinate-butylphosphonate acyltransferase (pat) / glufosinate, or neomycin phosphotransferase (npt) / neomycin or kanamycin. In some embodiments, the selectable or scoreable marker is an RNA aptamer (e.g., a broccoli aptamer) or a regulatory RNA (e.g., siRNA, siRNA precursor, miRNA, or miRNA precursor, or staged siRNA or staged siRNA precursor that downregulates the expression of endogenous genes in a plant to produce a detectable phenotype (e.g., bleaching caused by downregulation of pigment-producing genes). In embodiments, the mixed population is located within a plant or plant part. In some embodiments, plants or plant parts are screened or selected for the presence of recombinant RNA molecules before isolating plant propagules. In other embodiments, plants or plant parts are screened or selected for the systematic presence of recombinant RNA molecules before isolating plant propagules. In some embodiments, plant cells, plants, or plant parts in a mixed population or isolated from the plant lack DNA encoding recombinant RNA molecules. In embodiments, plant propagules containing recombinant RNA molecules are isolated by detecting RNA molecules in one or more plant cells containing said recombinant RNA molecules and separating one or more plant cells containing said recombinant RNA molecules from plant cells lacking said recombinant DNA molecules. In some embodiments, plant propagules are chimeras containing both plant cells containing recombinant RNA molecules and plant cells lacking recombinant RNA molecules. In other embodiments, modified plant propagules are chimeras containing both plant cells containing RdRP of the Tomato Cluster Dwarf Virus Family and plant cells lacking RdRP of the Tomato Cluster Dwarf Virus Family. In some embodiments, at least 99%, 98%, 95%, 90%, 85%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or 1% of the plant cells in the chimera may contain recombinant RNA molecules. In some embodiments, particularly advantageous for regulatory reasons, the plant propagule lacks DNA encoding the recombinant RNA molecule. In embodiments, the modified plant propagule comprises cells containing recombinant RNA molecules, or seeds, seedlings, ovules, embryos, pollen, roots, stems, leaves, buds, tubers, rhizomes, stolons, bulbs, explants, or callus tissues containing cells with recombinant RNA molecules. Plant propagules prepared by any of the foregoing methods and / or incorporating any of the foregoing characteristics are also provided herein.

[0136] In some embodiments, any of the foregoing methods may further include propagating cells, seeds, seedlings, ovules, embryos, pollen, roots, stems, leaves, buds, tubers, rhizomes, stolons, bulbs, explants, or callus to obtain progeny, wherein the progeny contains recombinant RNA molecules. In other embodiments, cell propagation consists of culturing multiple explants obtained from cells, seeds, seedlings, ovules, embryos, pollen, roots, stems, leaves, buds, tubers, rhizomes, stolons, bulbs, explants, or callus. In yet another embodiment, the isolated propagation body comprises cells, and the foregoing methods may further include regenerating from said cells plants, seedlings, ovules, embryos, pollen, roots, stems, leaves, buds, tubers, rhizomes, stolons, bulbs, explants, or callus containing recombinant RNA. In other embodiments, the isolated propagule comprises callus tissue, and the foregoing method may further include regenerating from the callus tissue a plant, seedling, ovule, embryo, pollen, root, stem, leaf, bud, tuber, rhizome, stolon, bulb, or explant containing recombinant RNA. In embodiments, the plant is regenerated, and the foregoing method may further include recovering from the plant F1 seeds or F1 progeny or cloned progeny containing recombinant RNA.

[0137] In another aspect, this disclosure relates to a method of providing a plant or plant part with synthetic Tomato Cluster Dwarf Virus family satellite RNA by grafting one plant part onto another. In some embodiments, the method may include grafting a scion onto a rootstock containing any of the recombinant DNA molecules and / or recombinant RNA molecules disclosed above or otherwise (e.g., recombinant RNA containing, in a 5' to 3' sequence, a 5' RNA replication element recognizable by Tomato Cluster Dwarf Virus family RNA-dependent RNA polymerase (RdRP); a cargo RNA sequence; and a 3' RNA replication element recognizable by Tomato Cluster Dwarf Virus family RdRP), wherein at least one cell of the rootstock and / or scion contains Tomato Cluster Dwarf Virus family RdRP. In some embodiments, the scion may contain plant buds, apical or other meristems, leaves attached to petioles, or other plant parts, and the rootstock may contain roots and above-ground parts of the plant (including the main stem, secondary stems, leaves, and / or reproductive structures of the plant). In embodiments, the scion and / or rootstock do not contain DNA encoding the recombinant RNA molecule. In this embodiment, the scion lacks recombinant RNA molecules prior to grafting. In this embodiment, the rootstock contains RdRP from the Tomato Cluster Dwarf Virus Family. In some embodiments, the RdRP is provided by a rootstock-specific Tomato Cluster Dwarf Virus Family virus (e.g., a non-pathogenic and / or symbiotic Tomato Cluster Dwarf Virus Family virus). In other embodiments, the RdRP is exogenously provided to the rootstock (e.g., via a DNA expression cassette integrated into the chromosomal or plastid DNA of the rootstock or via a recombinant viral vector containing DNA or RNA encoding the RdRP). In this embodiment, the scion contains RdRP from the Tomato Cluster Dwarf Virus Family. In some embodiments, the RdRP is provided by a scion-specific Tomato Cluster Dwarf Virus Family virus (e.g., a non-pathogenic and / or symbiotic Tomato Cluster Dwarf Virus Family virus). In other embodiments, the RdRP is exogenously provided to the scion (e.g., via a DNA expression cassette integrated into the chromosomal or plastid DNA of the scion or via a recombinant viral vector containing DNA or RNA encoding the RdRP). In embodiments, the Tomato Cluster Dwarf Virus RdRP comprises a protein having at least 85%, 90%, 95%, 97%, or 99% sequence identity with SEQ ID NO: 469 or 587. In embodiments, the rootstock and / or scion comprises a heteroviral capsid protein that can capsidate the recombinant RNA molecule. In some embodiments, the rootstock and / or scion comprises a recombinant RNA molecule capsidated by a heteroviral capsid protein.

[0138] In another aspect, this disclosure relates to a method of producing a grafted plant comprising a recombinant RNA molecule containing, in a 5' to 3' sequence, a 5' RNA replication element capable of being recognized by Tomato dwarfvirus RNA-dependent RNA polymerase (RdRP); a cargo RNA sequence; and a 3' RNA replication element capable of being recognized by Tomato dwarfvirus RdRP. In some embodiments, the recombinant RNA molecule is provided by contacting the scion, rootstock, or both the scion and rootstock with a composition containing the recombinant RNA molecule before grafting the scion onto the rootstock to produce the grafted plant. In some embodiments, at least one cell of the rootstock and / or scion contains Tomato dwarfvirus RdRP before contacting the scion, rootstock, or both the scion and rootstock with the composition. In embodiments, the rootstock contains Tomato dwarfvirus RdRP. In some embodiments, the Tomato dwarfvirus RdRP is provided by a Tomato dwarfvirus virus specific to the rootstock (e.g., a non-pathogenic and / or symbiotic Tomato dwarfvirus virus). In other embodiments, the Tomato Cluster Dwarf Virus Family RdRP is exogenously provided to the rootstock (e.g., via a DNA expression cassette integrated into the chromosomal or plastid DNA of the rootstock or via a recombinant viral vector containing DNA or RNA encoding the RdRP). In embodiments, the scion contains the Tomato Cluster Dwarf Virus Family RdRP. In some embodiments, the RdRP is provided by a Tomato Cluster Dwarf Virus Family virus specific to the scion (e.g., a non-pathogenic and / or symbiotic Tomato Cluster Dwarf Virus Family virus). In other embodiments, the RdRP is exogenously provided to the scion (e.g., via a DNA expression cassette integrated into the chromosomal or plastid DNA of the scion or via a recombinant viral vector containing DNA or RNA encoding the RdRP). In embodiments, the Tomato Cluster Dwarf Virus Family RdRP contains a protein having at least 85%, 90%, 95%, 97%, or 99% sequence identity with SEQ ID NO: 469 or 587. In embodiments, the DNA encoding the recombinant RNA molecule is not present in the scion, rootstock, and / or grafted plant. The composition may be provided to the scion, rootstock, or both the scion and rootstock according to any of the formulations disclosed herein. In some embodiments, the formulation is a liquid, gel, or powder. In some embodiments, the formulation is configured to be sprayed onto the scion, rootstock, or both the scion and rootstock; injected into the scion, rootstock, or both the scion and rootstock; soaked into the scion, rootstock, or both the scion and rootstock; or coated onto the scion, rootstock, or both the scion and rootstock. In some embodiments, contact includes immersing the scion, rootstock, or both the scion and rootstock in the composition prior to grafting.

[0139] In another aspect, this disclosure relates to a method for producing plants that deliver any of the recombinant RNA molecules provided herein or otherwise disclosed to progeny plants or seeds. In some embodiments, the method includes the steps of isolating F1 progeny plants or seeds from an F1 plant or seed population obtained from at least one parent plant containing the recombinant RNA molecule, comprising at least one cell containing a Tomato Cluster Dwarf Virus Family RNA-dependent RNA polymerase (RdRP) and the recombinant RNA molecule, wherein the recombinant RNA molecule comprises, in a 5' to 3' order, a 5' RNA replication element recognizable by the Tomato Cluster Dwarf Virus Family RdRP; a cargo RNA sequence; and a 3' RNA replication element recognizable by the Tomato Cluster Dwarf Virus Family RdRP. In embodiments, parent plants or portions thereof containing plant cells are screened or selected for the presence of the recombinant RNA molecule prior to the isolation of F1 progeny plants or seeds. In some embodiments, parent plants or one or more portions thereof are screened for the systematic presence of the recombinant RNA molecule prior to the isolation of F1 progeny plants. In other embodiments, the parent plant's floral tissues (e.g., whole flower or bud, sepals, calyx, or petals), male reproductive tissues (e.g., stamens, anthers, or pollen), or female reproductive tissues (e.g., whole fruit, ovary, pericarp, ovules, seed coat, endosperm, or embryo) are screened or selected for the presence of recombinant RNA molecules. In embodiments, F1 seeds are obtained from parent plants selected for the presence of recombinant RNA molecules in pericarp tissue. In embodiments, F1 progeny plants or seeds containing said cells are isolated by screening F1 plant or seed populations obtained from parent plants for the presence of recombinant RNA molecules, and propagating F1 progeny plants or seeds containing said recombinant RNA molecules. In such screening, the progeny plants or their seeds are subjected to determination of screenable markers for the presence of recombinant RNA (e.g., RNA sequences diagnosing the presence of recombinant RNA molecules or polypeptides encoded by recombinant RNA molecules) and separated from progeny plants and seeds lacking recombinant RNA. Such screening assays can be non-destructive, in which a portion of the progeny seed or plant is removed and measured without loss of viability or reproductive capacity of the seed or plant tissue containing the recombinant RNA. In some embodiments, F1 seeds of the parent plant are non-destructively screened for the presence of the recombinant RNA molecule. In other embodiments, F1 seeds of the parent plant are non-destructively screened for the presence of the recombinant RNA molecule in maternally derived tissue or endosperm tissue of the seed. Methods suitable for such screening using non-destructive assays of seeds or other plant tissues include, but are not limited to, those disclosed in U.S. Patent Applications US 20220221377 and US 20210259176, both of which are incorporated herein by reference in their entirety. In some embodiments, the cargo RNA sequence comprises RNA encoding selectable or scoreable markers.In some embodiments, the recombinant RNA molecule encodes a selectable marker, and F1 progeny plants or seeds containing the recombinant RNA molecule are isolated by selecting F1 progeny plants or seeds containing the recombinant RNA molecule in response to the presence of the selectable marker. In cases where the recombinant RNA encodes a selectable marker (e.g., a protein conferring resistance to a selector (such as a herbicide or antibiotic)), examples of such selection may include exposing progeny seeds or plants to the selector (e.g., a herbicide or antibiotic) and isolating progeny seeds or plants that survive the selector exposure. Examples of selectable marker / selector combinations include glyphosate-resistant EPSPS enzymes and / or glyphosate oxidase / glyphosate, biammoniaphosphonate resistance (bar) or glufosinate-butylphosphonate acyltransferase (pat) / glufosinate, and neomycin phosphotransferase (npt) / neomycin or kanamycin. In some embodiments, the selectable or scoreable marker is an RNA aptamer or regulatory RNA. In some embodiments, the F1 progeny plants or seeds lack DNA encoding the recombinant RNA molecule. In other embodiments, the parental plant lacks DNA encoding the recombinant RNA molecule. In embodiments, selected F1 progeny plants transfer the recombinant RNA molecule to at least the F2 progeny. In some embodiments, the F1 progeny plants or seed population are obtained from a parental plant used as a pollen recipient. In other embodiments, the F1 progeny plants or seed population are obtained from a parental plant used as a pollen donor. In some embodiments, the F1 progeny plants or seed population are obtained through self-pollination of the parental plants. In other embodiments, the F1 progeny plants or seed population are obtained from a sexual hybridization of two parental plants. In some embodiments, the parental plant containing the recombinant RNA molecule is the maternal plant. In other embodiments, the parental plant containing the recombinant RNA molecule is the paternal plant, and the recombinant RNA molecule is transferred in the pollen of the paternal plant.

[0140] In some embodiments, the method may further include introducing a recombinant RNA molecule or a polynucleotide encoding the recombinant RNA molecule into a plant cell and obtaining a parent plant containing the recombinant RNA molecule from the plant cell. In embodiments, 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 mobile protein (MP); (b) at least one tRNA-like sequence; and (c) an assembly origin sequence (OAS). In embodiments, the parent and / or plant contains a heterologous viral capsid protein capable of capsidating the recombinant RNA molecule. In some embodiments, the parent and / or progeny plant contains a recombinant RNA molecule capsidated by a heterologous viral capsid protein.

[0141] In another aspect, this disclosure relates to a method for barcoding plants, plant cells, their progeny, or portions thereof. The method includes providing a plant or plant cell with any of the recombinant RNA molecules provided herein or otherwise disclosed, wherein the cargo RNA of said recombinant RNA molecule comprises a barcoded RNA molecule, and wherein said plant or plant cell comprises RdRP of the Tomato Cluster Dwarf Virus Family. In embodiments, the barcoded RNA molecule comprises a sequence that uniquely identifies the plant, plant cell, its progeny, or portions thereof. In some embodiments, the barcoded RNA may be a randomly generated sequence. In some embodiments, the barcoded RNA molecule comprises a sequence not present in the genome and / or transcriptome of a wild-type plant of the same species, its pathogen, or its symbiont. In some embodiments, the barcoded RNA molecule comprises a forward primer binding site and a reverse primer binding site for detecting the barcoded RNA molecule. In embodiments, the barcoded RNA molecule comprises a non-protein-coding sequence. In some embodiments, the length of the barcoded RNA sequence is up to about 6 kb. In some embodiments, the length of the barcoded RNA is 10 to 6000 nucleotides, 20 to 1000 nucleotides, or 50 to 500 nucleotides. In some embodiments, the length of the barcoded RNA molecule is 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, or 6000 nucleotides. In embodiments, the plant transmits the recombinant RNA molecule containing the barcoded RNA to its offspring. In some embodiments, the plant, plant cells, its offspring, or portions thereof lack the DNA encoding the recombinant RNA molecule. In some embodiments, the method may further include isolating F1 progeny plants or seeds containing at least one cell containing Tomato Cluster Dwarf Virus Family RdRP and recombinant RNA molecules. In some embodiments, the F1 progeny plants or seeds are obtained from a plant used as a pollen recipient. In other embodiments, the F1 progeny plants or seeds are obtained from a plant used as a pollen donor. In embodiments, the F1 progeny plants or seeds are obtained through self-pollination of the parent plants. In embodiments, the method may further include propagating plants or plant cells to obtain plant parts or plant propagules containing barcode RNA molecules.

[0142] On the other hand, this disclosure relates to methods for identifying barcoded plants, plant parts, or plant cells. The methods include screening for the presence of barcoded RNA molecules in plants, plant parts, or plant cells, wherein said plants, plant parts, or plant cells contain any of the recombinant RNA molecules provided herein or otherwise disclosed, and wherein the cargo RNA of said recombinant RNA molecules contains barcoded RNA molecules. In some embodiments, the methods include obtaining a nucleic acid sample from a plant, plant part, or plant cell; and detecting the presence of barcoded RNA molecules in the sample. Assays for detecting barcoded RNA include RNA detection assays (e.g., RT-PCR assays) using nucleic acid probes and / or primers capable of detecting barcoded RNA and / or sequencing the barcoded RNA. Such screening assays can be non-destructive assays, wherein a portion of a seed or plant is removed and assayed without causing loss of viability or reproductive capacity of the seed or plant tissue containing the barcoded RNA. Nondestructive assays suitable for such screening of seeds or other plant tissues include, but are not limited to, those disclosed in U.S. Patent Applications US 20220221377 and US 20210259176, which are incorporated herein by reference in their entirety. In some embodiments, seeds of plants are nondestructively screened for the presence of barcoded RNA molecules. In other embodiments, seedlings, ovules, embryos, pollen, roots, stems, leaves, buds, tubers, rhizomes, stolons, bulbs, explants, or callus tissues are screened for the presence of barcoded RNA molecules.

[0143] In some optional embodiments, the methods disclosed herein are not processes for modifying the phylogenetic or genetic identity of humans. In some optional embodiments, the methods disclosed herein are not processes for modifying the genetic identity of animals, processes that may cause suffering to the animals without any substantial medical benefit to humans or animals, and do not involve animals produced by such processes. In some optional embodiments, the methods disclosed herein are not methods for treating a human or animal body by surgery or therapy. In some optional embodiments, the cells disclosed herein are not human embryos. In some optional embodiments, the cells disclosed herein are not human or parts thereof at different stages of their formation and development. In some optional embodiments provided herein, the plant cells, plant propagules (e.g., seeds, seedlings, ovules, embryos, pollen, roots, stems, leaves, buds, tubers, rhizomes, stolons, bulbs, explants, or callus tissues) and plants provided herein are produced not only through biological processes. In some of the optional embodiments provided herein, the methods provided herein for producing plant cells, plant propagules (e.g., seeds, seedlings, ovules, embryos, pollen, roots, stems, leaves, buds, tubers, rhizomes, stolons, bulbs, explants, or callus tissue) and plants are not merely biological processes.

[0144] Example

[0145] Various embodiments of the compositions, systems, and methods described herein are shown, for example, in the following set of numbered examples.

[0146] 1. A recombinant RNA molecule comprising, from its 5' end to its 3' end: (a) A 5' RNA replication element recognized by RdRP, an RNA-dependent RNA polymerase of the Tomato dwarf Virus family; (b) Cargo RNA molecules; and (c) 3' RNA replication element recognized by the RdRP; The 5' RNA replication element, the cargo RNA molecule, and the 3' RNA replication element are operatively linked, and 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 derived from the same Tomato dwarf Virus family genome or from a Tomato dwarf Virus family genome having at least 85%, 90%, 95%, 98%, or 99% sequence identity with each other and optionally being related; (ii) the 5' RNA replication element, the 3' RNA replication element, and the cargo RNA molecule are operatively linked. The 5' RNA replication element and the RdRP are obtained from the same Tomato dwarf Virus family genome or from Tomato dwarf Virus family genomes that have at least 85%, 90%, 95%, 98%, or 99% sequence identity with each other and are optionally related; or (iii) the 5' RNA replication element, the 3' RNA replication element, and / or the RdRP coding region are obtained from different Tomato dwarf Virus family genomes, and the members of each respective set of the 5' RNA replication element, the 3' RNA replication element, and / or the RdRP coding region have at least 85%, 90%, 95%, 98%, or 99% sequence identity with each other.

[0147] 2. The recombinant RNA molecule as described in Example 1, wherein: (a) The 5' RNA replication element comprises at least one RNA secondary structure provided in Table 1 or encoded by an RNA molecule of SEQ ID NO: 467; and / or (b) The 3' RNA replication element comprises at least one RNA secondary structure provided in Table 1 or encoded by an RNA molecule of SEQ ID NO: 468.

[0148] 3. The recombinant RNA molecule as described in Example 1, wherein: (a) The 5' RNA replication element comprises an RNA molecule encoded by SEQ ID NO: 467; a variant thereof encoded by a DNA molecule having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO: 467; or a variant thereof wherein one or more nucleotides in the RNA secondary structure are replaced by different nucleotides that maintain the RNA secondary structure; and / or (b) The 3' RNA replication element comprises an RNA molecule encoded by SEQ ID NO: 468, or a variant thereof encoded by a DNA molecule having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO: 468; or a variant thereof wherein one or more base-paired residues in the RNA secondary structure are replaced by different nucleotides that maintain the RNA secondary structure.

[0149] 4. The recombinant RNA molecule as described in Example 3, wherein the RNA secondary structure is maintained by replacing unpaired nucleotides in the secondary structure with nucleotides that will not pair bases, and / or replacing paired nucleotides in the secondary structure with nucleotides that will pair bases.

[0150] 5. The recombinant RNA molecule as described in any one of Examples 1 to 4, wherein: (a) The 5' RNA replication element comprises at least one segment of the 5' untranslated region (UTR) of the Tomato dwarf Virus family genome or a variant thereof having one or more nucleotide substitutions, insertions, and / or deletions, wherein the variant is recognized by the RdRP, and optionally wherein the 5' RNA replication element further comprises a genomic sequence of the Tomato dwarf Virus family virus naturally located at and adjacent to the 3' of the 5' UTR sequence; and / or (b) The 3' RNA replication element comprises at least one segment of the 3' UTR of the Tomato dwarf Virus family genome or a variant thereof having one or more nucleotide substitutions, insertions and / or deletions, wherein the variant is recognized by the RdRP, and optionally wherein the 3' RNA replication element further comprises a genome sequence of the Tomato dwarf Virus family virus naturally located at the 5' and adjacent to the 3' UTR sequence, and optionally wherein the Tomato dwarf Virus family genomes of (a) and (b) are identical.

[0151] 6. A recombinant RNA molecule as described in any one of Examples 1 to 5, wherein the RNA molecule further comprises at least one of the following: (i) a tRNA-like element, optionally wherein the tRNA-like element provides intercellular movement of the RNA, and optionally wherein the intercellular movement is mediated by a viral mobility protein (MP); (ii) a capsidation recognition element (ERE), optionally wherein the ERE provides capsidation of the RNA via a Tomato dwarf Virus capsid protein, or optionally wherein the ERE provides capsidation of the RNA via a non-Tomato dwarf Virus capsid protein; (iii) an RNA effector; and / or (iv) RNA encoding a viral mobility protein (MP), optionally wherein an internal ribosome entry site (IRES) is operatively linked to the MP-encoding RNA.

[0152] 7. The recombinant RNA molecule as described in Example 6, wherein the tRNA-like element comprises a tRNA-like molecule derived from Arabidopsis thaliana FT mRNA, or a tRNA-like sequence encoded by a DNA sequence selected from the group consisting of SEQ ID NO: 76-123 and 466, or a modified tRNA-like sequence having at least 90% sequence identity with a scaffold tRNA-like sequence encoded by a DNA sequence selected from the group consisting of SEQ ID NO: 76-123 and 466 and maintaining the secondary structure of the scaffold tRNA-like sequence, and / or wherein the ERE is tobacco mosaic virus (TMV) OAS.

[0153] 8. The recombinant RNA molecule as described in Example 1, wherein the length of the cargo RNA molecule is up to about 6 kb.

[0154] 9. The recombinant RNA molecule as described in Example 1, wherein the cargo RNA molecule comprises: (a) at least one coding sequence, optionally wherein the coding sequence encodes a selectable or scoreable marker; (b) at least one non-coding sequence; or (c) both at least one coding sequence and at least one non-coding sequence.

[0155] 10. The recombinant RNA molecule as described in Example 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) operatively linked to the at least one coding sequence, optionally wherein the operatively linked IRES is located at the 5' of and immediately adjacent to the coding sequence.

[0156] 11. The recombinant RNA molecule as described in Example 1, wherein the cargo RNA molecule comprises at least one non-coding sequence, and wherein the at least one non-coding sequence is a hairpin RNA (hpRNA); RNA forming multiple stem loops; RNA pseudoknots; RNA molecules forming at least partially double-stranded RNA; small interfering RNA (siRNA) or siRNA precursors; microRNA (miRNA) or miRNA precursors; staged RNA or staged RNA precursors; ribozymes; ligand-responsive ribozymes (aptases); RNA aptamers; or long non-coding RNA (lncRNA).

[0157] 12. The recombinant RNA molecule as described in Example 1, further comprising RNA encoding at least one ribozyme, optionally wherein the at least one ribozyme is located at the 5' of the 5' RNA replication element or the 3' of the 3' RNA replication element.

[0158] 13. The recombinant RNA molecule as described in Example 1, further comprising an RNA molecule containing at least one ligand-responsive ribozyme (aptamer), optionally wherein the at least one ligand-responsive ribozyme is located at the 5' of the 5' RNA replication element or the 3' of the 3' RNA replication element.

[0159] 14. The recombinant RNA molecule as described in Example 1, wherein: (i) the RNA further comprises at least one segment of its reverse complementary RNA molecule; and / or (ii) the recombinant RNA molecule is complexed with one or more RNA-binding proteins or capsidated by a viral capsid protein, optionally wherein the viral capsid protein is a Tomato dwarf Virus capsid protein, optionally wherein the RNA comprises an ERE providing capsidation of the RNA by the Tomato dwarf Virus capsid protein, and / or optionally wherein the RNA-binding protein comprises an RNA recognition motif.

[0160] 15. The recombinant RNA molecule as described in Example 1, wherein the RNA molecule comprises: At least one heterologous RNA virus (HRV) amplicon, which is positively or negatively oriented relative to the first 5' RNA replication element, contains: I. (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 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 operatively linked; or II. A heterologous RNA virus (HRV) subgenome promoter operatively linked to the cargo RNA molecule; wherein the subgenome promoter is recognized by the heterologous RNA virus RNA-dependent RNA polymerase (hrvRdRP).

[0161] 16. The recombinant RNA as described in Example 15, wherein the RNA molecule comprises, from the 5' end to the 3' end: (a) The 5' RNA replication element; (b) The HRV amplicon oriented antisense relative to the first 5' RNA replication element; optionally, the HRV amplicon further comprises: (i) an RNA molecule encoding an HRV RNA-dependent RNA polymerase (hrvRdRP) 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 HRV RNA-dependent RNA polymerase (hrvRdRP) operably linked to a subgenomic promoter recognized by said hrvRdRP; and (c) The 3' RNA replication element.

[0162] 17. The recombinant RNA as described in Example 15 or 16, wherein the HRV 5'RR, HRV 3'RR, and hrvRdRP comprise HRV 5'RR, HRV 3'RR, and hrvRdRP from viruses selected from the group consisting of: *Hypervira*, *Hypervira*, *Brassica napus*, *Celavirus*, *Leptovirus*, *Cowpea Mosaic Virus*, *Potato Virus X*, *Potato Virus Y*, *Tobacco Mosaic Virus*, *Tomato Cluster Dwarf Virus*, *Tomato Spotted Wilt Virus Family*, *Trigeneae* subfamily, *Turnip Yellow Mosaic Virus*, *Vein Varicose Vein Virus*, and *Associated Cowpea Mosaic Virus Family*.

[0163] 18. The recombinant RNA as described in Example 17, wherein: (i) the genus *Bombyx mottlevirus* is cucumber mosaic virus, spinach latent virus, olive latent virus, or *Bombyx mottlevirus*; (ii) the genus *Lycovirus* is citrus senescence virus or beet yellowing virus; (iii) the genus *Cowpea mosaic virus* is cowpea mosaic virus, apple latent spherical virus, or soybean latent spherical virus; (iv) the genus *PotatoXvirus* is potato X virus or citrus yellowing virus; (v) the genus *PotatoYvirus* is 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; (vi) the genus of tobacco mosaic virus is tobacco mosaic virus, tomato mosaic virus, tomato brown wrinkled fruit virus, turnip vein virus, or pepper mild mottle virus; (vii) the genus of tomato dwarf virus is turnip wrinkled virus or tomato dwarf virus; (iv) the family of tomato wilt virus is tomato wilt virus or watermelon bud necrosis virus; or (viii) the genus of turnip yellow mosaic virus is turnip yellow mosaic virus, citrus yellowing vein virus, potato latent virus, apple stem groove virus, or citrus leaf mottle virus.

[0164] 19. The recombinant RNA as described in any one of Examples 1 to 18, wherein the HRV 5'RR and the HRV 3'RR are derived from the same HRV genome.

[0165] 20. The recombinant RNA as described in any one of Examples 1 to 18, wherein the HRV 5'RR and the HRV 3'RR are derived from different HRV genomes.

[0166] 21. The recombinant RNA molecule as described in any one of Examples 1 to 20, wherein: a) The HRV 5'RR comprises at least one RNA secondary structure using RNA encoded by SEQ ID NO: 161 to 185 or 186, or comprises an RNA sequence encoded by SEQ ID NO: 161 to 185 or 186, or comprises at least 80%, 85%, 90%, or 95% of a continuous fragment of the complete sequence of 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 using RNA encoded by SEQ ID NO: 187 to 210 or 211, or an RNA sequence comprising RNA encoded by SEQ ID NO: 187 to 210 or 211, or a continuous fragment comprising at least 80%, 85%, 90% or 95% of the complete sequence of RNA encoded by SEQ ID NO: 187 to 210 or 211.

[0167] 22. The recombinant RNA molecule as described in any one of Examples 1 to 20, wherein: (a) The HRV 5'RR is encoded by: a DNA molecule comprising SEQ ID NO: 161 to 185 or 186; or a variant thereof comprising DNA having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO: 161 to 185 or 186; or a variant thereof wherein one or more residues in the RNA secondary structure are replaced by different nucleotides that maintain the RNA secondary structure; and / or (b) The HRV 3'RR is encoded by: a DNA molecule comprising SEQ ID NO: 187 to 210 or 211; or a variant thereof comprising DNA having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO: 187 to 210 or 211; or a variant thereof wherein one or more residues in the RNA secondary structure are replaced by different nucleotides that maintain the RNA secondary structure.

[0168] 23. The recombinant RNA molecule as described in Example 15, wherein: (a) The HRV 3'RR comprises at least one segment of the 3' untranslated region (UTR) of the HRV genome or an RNA molecule having one or more nucleotide substitutions, insertions and / or deletions thereof, wherein the HRV 3'RR or the variant is recognized by the hrvRdRP, and optionally the RNA comprising the HRV 3'RR further comprises the genomic sequence of the HRV naturally located at and adjacent to the 5' of the 3'UTR sequence; And / or (b) The HRV 5'RR comprises at least one segment of the 3' untranslated region (UTR) of the HRV genome or an RNA molecule having one or more nucleotide substitutions, insertions and / or deletions thereof, wherein the HRV 5'RR or the variant is recognized by the hrvRdRP, and optionally the RNA comprising the HRV 5'RR further comprises the genomic sequence of the HRV naturally located at and adjacent to the 3' of the 5'UTR sequence.

[0169] 24. The recombinant RNA molecule as described in any one of Examples 1 to 23, wherein the cargo RNA comprises HRV repressive RNA or encodes an HRV repressive protein, wherein the HRV repressive RNA or HRV repressive protein inhibits the infection, movement, spread and / or replication of the HRV.

[0170] 25. The recombinant RNA molecule as described in any one of Examples 1 to 24, wherein the cargo RNA comprises RNA having at least 20 consecutive nucleotides having a sequence that is the same as or complementary to an isolength segment in the genomic RNA of the HRV.

[0171] 26. The recombinant RNA molecule as described in any one of Examples 1 to 24, wherein the cargo RNA comprises RNA having at least 20 consecutive nucleotides having a sequence that is the same as or complementary to an isometric segment in the genomic RNA of the HRV that does not encode the hrvRdRP.

[0172] 27. The recombinant RNA molecule as described in any one of Examples 1 to 26, wherein: (i) The HRV is cucumber mosaic virus, and the HRV 5'RR contains RNA encoded by the cucumber mosaic virus 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95% or 99% sequence identity with the cucumber mosaic virus 5'RR DNA sequence in Table 7, and the HRV 3'RR contains RNA encoded by the cucumber mosaic virus 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95% or 99% sequence identity with the cucumber mosaic virus 3'RR DNA sequence in Table 7; (ii) The HRV is bromeliad mosaic virus, and the HRV 5'RR contains RNA encoded by the bromeliad mosaic virus 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the bromeliad mosaic virus 5'RR DNA sequence in Table 7, and the HRV 3'RR contains RNA encoded by the bromeliad mosaic virus 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the bromeliad mosaic virus 3'RR DNA sequence in Table 7; (iii) The HRV is a citrus degeneration virus, and the HRV 5'RR contains RNA encoded by the citrus degeneration virus 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the citrus degeneration virus 5'RR DNA sequence in Table 7; the HRV 3'RR contains RNA encoded by the citrus degeneration virus 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the citrus degeneration virus 3'RR DNA sequence in Table 7; and the RdRP contains a protein having at least 85%, 90%, 95%, or 99% sequence identity with the citrus degeneration virus RdRP sequence in Table 7. (iv) The HRV is beet yellow virus, and the HRV 5'RR contains RNA encoded by the beet yellow virus 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the beet yellow virus 5'RR DNA sequence in Table 7; the HRV 3'RR contains RNA encoded by the beet yellow virus 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the beet yellow virus 3'RR DNA sequence in Table 7; and the RdRP contains a protein having at least 85%, 90%, 95%, or 99% sequence identity with the beet yellow virus RdRP sequence in Table 7. (v) The HRV is cowpea mosaic virus, and the HRV 5'RR contains RNA encoded by the cowpea mosaic virus 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95% or 99% sequence identity with the cowpea mosaic virus 5'RR DNA sequence in Table 7. (vi) The HRV is Potato Virus X, and the HRV 5'RR contains RNA encoded by the Potato Virus X 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the Potato Virus X 5'RR DNA sequence in Table 7; the HRV 3'RR contains RNA encoded by the Potato Virus X 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the Potato Virus X 3'RR DNA sequence in Table 7; and the RdRP contains a protein having at least 85%, 90%, 95%, or 99% sequence identity with the Potato Virus X RdRP sequence in Table 7. (vii) The HRV is a pepper mottle virus, and the HRV 5'RR contains RNA encoded by the pepper mottle virus 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the pepper mottle virus 5'RR DNA sequence in Table 7; the HRV 3'RR contains RNA encoded by the pepper mottle virus 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the pepper mottle virus 3'RR DNA sequence in Table 7; and the RdRP contains a protein having at least 85%, 90%, 95%, or 99% sequence identity with the pepper mottle virus RdRP sequence in Table 7. (viii) The HRV is soybean yellow mosaic virus, and the HRV 5'RR contains RNA encoded by the soybean yellow mosaic virus 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the soybean yellow mosaic virus 5'RR DNA sequence in Table 7; the HRV 3'RR contains RNA encoded by the soybean yellow mosaic virus 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the soybean yellow mosaic virus 3'RR DNA sequence in Table 7; and the RdRP contains a protein having at least 85%, 90%, 95%, or 99% sequence identity with the soybean yellow mosaic virus RdRP sequence in Table 7. (ix) The HRV is barley stripe mosaic virus, and the HRV 5'RR contains RNA encoded by the barley stripe mosaic virus 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the barley stripe mosaic virus 5'RR DNA sequence in Table 7; the HRV 3'RR contains RNA encoded by the barley stripe mosaic virus 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the barley stripe mosaic virus 3'RR DNA sequence in Table 7. (x) The HRV is wheat stripe mosaic virus, and the HRV 5'RR contains RNA encoded by the wheat stripe mosaic virus 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the wheat stripe mosaic virus 5'RR DNA sequence in Table 7; the HRV 3'RR contains RNA encoded by the wheat stripe mosaic virus 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the wheat stripe mosaic virus 3'RR DNA sequence in Table 7; and the RdRP contains a protein having at least 85%, 90%, 95%, or 99% sequence identity with the wheat stripe mosaic virus RdRP sequence in Table 7. (xi) The HRV is rice yellow mottle virus, and the HRV 5'RR contains RNA encoded by the rice yellow mottle virus 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the rice yellow mottle virus 5'RR DNA sequence in Table 7; the HRV 3'RR contains RNA encoded by the rice yellow mottle virus 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the rice yellow mottle virus 3'RR DNA sequence in Table 7; and the RdRP contains a protein having at least 85%, 90%, 95%, or 99% sequence identity with the rice yellow mottle virus RdRP sequence in Table 7. (xii) The HRV is maize dwarf mosaic virus, and the HRV 5'RR contains RNA encoded by the maize dwarf mosaic virus 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the maize dwarf mosaic virus 5'RR DNA sequence in Table 7; the HRV 3'RR contains RNA encoded by the maize dwarf mosaic virus 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the maize dwarf mosaic virus 3'RR DNA sequence in Table 7; and the RdRP contains a protein having at least 85%, 90%, 95%, or 99% sequence identity with the maize dwarf mosaic virus RdRP sequence in Table 7. (xiii) The HRV is the zucchini yellow mosaic virus, and the HRV 5'RR contains RNA encoded by the zucchini yellow mosaic virus 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the zucchini yellow mosaic virus 5'RR DNA sequence in Table 7; the HRV 3'RR contains RNA encoded by the zucchini yellow mosaic virus 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the zucchini yellow mosaic virus 3'RR DNA sequence in Table 7; and the RdRP contains a protein having at least 85%, 90%, 95%, or 99% sequence identity with the zucchini yellow mosaic virus RdRP sequence in Table 7. (xiv) The HRV is watermelon mosaic virus, and the HRV 5'RR contains RNA encoded by the watermelon mosaic virus 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the watermelon mosaic virus 5'RR DNA sequence in Table 7; the HRV 3'RR contains RNA encoded by the watermelon mosaic virus 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the watermelon mosaic virus 3'RR DNA sequence in Table 7; and the RdRP contains a protein having at least 85%, 90%, 95%, or 99% sequence identity with the watermelon mosaic virus RdRP sequence in Table 7. (xv) The HRV is sugarcane mosaic virus, and the HRV 5'RR contains RNA encoded by the sugarcane mosaic virus 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the sugarcane mosaic virus 5'RR DNA sequence in Table 7; the HRV 3'RR contains RNA encoded by the sugarcane mosaic virus 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the sugarcane mosaic virus 3'RR DNA sequence in Table 7; and the RdRP contains a protein having at least 85%, 90%, 95%, or 99% sequence identity with the sugarcane mosaic virus RdRP sequence in Table 7. (xvi) The HRV is tobacco mosaic virus, and the HRV 5'RR contains RNA encoded by the tobacco mosaic virus 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the tobacco mosaic virus 5'RR DNA sequence in Table 7; the HRV 3'RR contains RNA encoded by the tobacco mosaic virus 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the tobacco mosaic virus 3'RR DNA sequence in Table 7; and the RdRP contains a protein having at least 85%, 90%, 95%, or 99% sequence identity with the tobacco mosaic virus RdRP sequence in Table 7. (xvii) The HRV is tomato mosaic virus, and the HRV 5'RR contains RNA encoded by the tomato mosaic virus 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the tomato mosaic virus 5'RR DNA sequence in Table 7; the HRV 3'RR contains RNA encoded by the tomato mosaic virus 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the tomato mosaic virus 3'RR DNA sequence in Table 7; and the RdRP contains a protein having at least 85%, 90%, 95%, or 99% sequence identity with the tomato mosaic virus RdRP sequence in Table 7. (xviii) The HRV is Tomato Brown Wrinkled Fruit Virus, and the HRV 5'RR contains RNA encoded by the Tomato Brown Wrinkled Fruit Virus 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the Tomato Brown Wrinkled Fruit Virus 5'RR DNA sequence in Table 7; the HRV 3'RR contains RNA encoded by the Tomato Brown Wrinkled Fruit Virus 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the Tomato Brown Wrinkled Fruit Virus 3'RR DNA sequence in Table 7; and the RdRP contains a protein having at least 85%, 90%, 95%, or 99% sequence identity with the Tomato Brown Wrinkled Fruit Virus RdRP sequence in Table 7. (xix) The HRV is var. turnip virus, and the HRV 5'RR contains RNA encoded by the var. turnip virus 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the var. turnip virus 5'RR DNA sequence in Table 7; the HRV 3'RR contains RNA encoded by the var. turnip virus 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the var. turnip virus 3'RR DNA sequence in Table 7; and the RdRP contains a protein having at least 85%, 90%, 95%, or 99% sequence identity with the var. turnip virus RdRP sequence in Table 7. (xx) The HRV is a mild mottle virus of pepper, and the HRV 5'RR contains RNA encoded by the 5'RR DNA sequence of the mild mottle virus of pepper in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95% or 99% sequence identity with the 5'RR DNA sequence of the mild mottle virus of pepper in Table 7; the HRV 3'RR contains RNA encoded by the 3'RR DNA sequence of the mild mottle virus of pepper in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95% or 99% sequence identity with the 3'RR DNA sequence of the mild mottle virus of pepper in Table 7; and the RdRP contains a protein having at least 85%, 90%, 95% or 99% sequence identity with the RdRP sequence of the mild mottle virus of pepper in Table 7. (xxi) The HRV is turnip shrub virus, and the HRV 5'RR contains RNA encoded by the turnip shrub virus 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the turnip shrub virus 5'RR DNA sequence in Table 7; the HRV 3'RR contains RNA encoded by the turnip shrub virus 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the turnip shrub virus 3'RR DNA sequence in Table 7; and the RdRP contains a protein having at least 85%, 90%, 95%, or 99% sequence identity with the turnip shrub virus RdRP sequence in Table 7. (xxii) The HRV is Tomato dwarf virus, and the HRV 5'RR contains RNA encoded by the Tomato dwarf virus 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the Tomato dwarf virus 5'RR DNA sequence in Table 7; the HRV 3'RR contains RNA encoded by the Tomato dwarf virus 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the Tomato dwarf virus 3'RR DNA sequence in Table 7; and the RdRP contains a protein having at least 85%, 90%, 95%, or 99% sequence identity with the Tomato dwarf virus RdRP sequence in Table 7. (xxiii) The HRV is tomato spotted wilt virus, and the HRV 5'RR contains RNA encoded by the tomato spotted wilt virus 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the tomato spotted wilt virus 5'RR DNA sequence in Table 7; the HRV 3'RR contains RNA encoded by the tomato spotted wilt virus 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the tomato spotted wilt virus 3'RR DNA sequence in Table 7; and the RdRP contains a protein having at least 85%, 90%, 95%, or 99% sequence identity with the tomato spotted wilt virus RdRP sequence in Table 7. (xxiv) The HRV is watermelon bud necrosis virus, and the HRV 5'RR contains RNA encoded by the watermelon bud necrosis virus 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the watermelon bud necrosis virus 5'RR DNA sequence in Table 7; the HRV 3'RR contains RNA encoded by the watermelon bud necrosis virus 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the watermelon bud necrosis virus 3'RR DNA sequence in Table 7; and the RdRP contains a protein having at least 85%, 90%, 95%, or 99% sequence identity with the watermelon bud necrosis virus RdRP sequence in Table 7. (xxv) The HRV is turnip yellow mosaic virus, and the HRV 5'RR contains RNA encoded by the turnip yellow mosaic virus 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the turnip yellow mosaic virus 5'RR DNA sequence in Table 7; the HRV 3'RR contains RNA encoded by the turnip yellow mosaic virus 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the turnip yellow mosaic virus 3'RR DNA sequence in Table 7; and the RdRP contains a protein having at least 85%, 90%, 95%, or 99% sequence identity with the turnip yellow mosaic virus RdRP sequence in Table 7. (xxvi) The HRV is spinach latent virus, and the HRV 5'RR contains RNA encoded by the spinach latent virus 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the spinach latent virus 5'RR DNA sequence in Table 7; the HRV 3'RR contains RNA encoded by the spinach latent virus 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the spinach latent virus 3'RR DNA sequence in Table 7; and the RdRP contains a protein having at least 85%, 90%, 95%, or 99% sequence identity with the spinach latent virus RdRP sequence of SEQ ID NO: 510. (xxvii) The HRV is spinach latent virus, and the HRV 5'RR contains RNA encoded by the spinach latent virus 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the spinach latent virus 5'RR DNA sequence in Table 7; the HRV 3'RR contains RNA encoded by the spinach latent virus 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the spinach latent virus 3'RR DNA sequence in Table 7; and the RdRP contains a protein having at least 85%, 90%, 95%, or 99% sequence identity with the spinach latent virus RdRP sequence of SEQ ID NO: 511. (xxviii) The HRV is olive latent virus 2, and the HRV 5'RR contains RNA encoded by the olive latent virus 2 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the olive latent virus 2 5'RR DNA sequence in Table 7; the HRV 3'RR contains RNA encoded by the olive latent virus 2 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the olive latent virus 2 3'RR DNA sequence in Table 7; and the RdRP contains a protein having at least 85%, 90%, 95%, or 99% sequence identity with the olive latent virus 2 RdRP sequence in Table 7. (xxix) The HRV is Citrus yellowing virus, and the HRV 5'RR contains RNA encoded by the Citrus yellowing virus 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the Citrus yellowing virus 5'RR DNA sequence in Table 7; the HRV 3'RR contains RNA encoded by the Citrus yellowing virus 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the Citrus yellowing virus 3'RR DNA sequence in Table 7; and the RdRP contains a protein having at least 85%, 90%, 95%, or 99% sequence identity with the Citrus yellowing virus RdRP sequence in Table 7. (xxx) The HRV is a potato latent virus, and the HRV 5'RR contains RNA encoded by the potato latent virus 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the potato latent virus 5'RR DNA sequence in Table 7; the HRV 3'RR contains RNA encoded by the potato latent virus 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the potato latent virus 3'RR DNA sequence in Table 7; and the RdRP contains a protein having at least 85%, 90%, 95%, or 99% sequence identity with the potato latent virus RdRP sequence in Table 7. (xxxi) The HRV is apple stem grooving virus, and the HRV 5'RR contains RNA encoded by the apple stem grooving virus 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the apple stem grooving virus 5'RR DNA sequence in Table 7; the HRV 3'RR contains RNA encoded by the apple stem grooving virus 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the apple stem grooving virus 3'RR DNA sequence in Table 7; and the RdRP contains a protein having at least 85%, 90%, 95%, or 99% sequence identity with the apple stem grooving virus RdRP sequence in Table 7. (xxxii) The HRV is a citrus leaf mottle virus, and the HRV 5'RR contains RNA encoded by the citrus leaf mottle virus 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the citrus leaf mottle virus 5'RR DNA sequence in Table 7; the HRV 3'RR contains RNA encoded by the citrus leaf mottle virus 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the citrus leaf mottle virus 3'RR DNA sequence in Table 7; and the RdRP contains a protein having at least 85%, 90%, 95%, or 99% sequence identity with the citrus leaf mottle virus RdRP sequence in Table 7. (xxxiii) The HRV is apple latent spheroid virus, and the HRV 5'RR contains RNA encoded by the apple latent spheroid virus 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the apple latent spheroid virus 5'RR DNA sequence in Table 7; the HRV 3'RR contains RNA encoded by the apple latent spheroid virus 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the apple latent spheroid virus 3'RR DNA sequence in Table 7; and the RdRP contains a protein having at least 85%, 90%, 95%, or 99% sequence identity with the apple latent spheroid virus RdRP sequence in Table 7. (xxxiv) The HRV is soybean latent spheroid virus, and the HRV 5'RR contains RNA encoded by the soybean latent spheroid virus 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the soybean latent spheroid virus 5'RR DNA sequence in Table 7; the HRV 3'RR contains RNA encoded by the soybean latent spheroid virus 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the soybean latent spheroid virus 3'RR DNA sequence in Table 7; and the RdRP contains a protein having at least 85%, 90%, 95%, or 99% sequence identity with the soybean latent spheroid virus RdRP sequence in Table 7. (xxxv) The HRV is celery latent virus, and the HRV 5'RR contains RNA encoded by the celery latent virus 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the celery latent virus 5'RR DNA sequence in Table 7; the HRV 3'RR contains RNA encoded by the celery latent virus 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the celery latent virus 3'RR DNA sequence in Table 7; and the RdRP contains a protein having at least 85%, 90%, 95%, or 99% sequence identity with the celery latent virus RdRP sequence in Table 7. (xxxvi) The HRV is a black grass varicose vein virus-like virus, and the HRV 5'RR contains RNA encoded by the black grass varicose vein virus-like virus 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the black grass varicose vein virus-like virus 5'RR DNA sequence in Table 7; the HRV 3'RR contains RNA encoded by the black grass varicose vein virus-like virus 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the black grass varicose vein virus-like virus 3'RR DNA sequence in Table 7; and the RdRP contains a protein having at least 85%, 90%, 95%, or 99% sequence identity with the black grass varicose vein virus-like virus sequence in Table 7; or (xxxvii) The HRV is a maize suscal virus, and the HRV 5'RR contains RNA encoded by the maize suscal virus 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the maize suscal virus 5'RR DNA sequence in Table 7; the HRV 3'RR contains RNA encoded by the maize suscal virus 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the maize suscal virus 3'RR DNA sequence in Table 7; and the RdRP contains a protein having at least 85%, 90%, 95%, or 99% sequence identity with the maize suscal virus sequence in Table 7.

[0173] 28. The recombinant RNA molecule of any one of Examples 1 to 27, further comprising RNA encoding at least one cleavable sequence, optionally wherein the at least one cleavable sequence is located at: (i) the 5' end of the 5' RNA replication element or the 3' end of 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, wherein the cleavable sequence is optionally a self-cleaving ribozyme, a self-cleaving inducible ribozyme, or a siRNA or miRNA recognition site.

[0174] 29. An agricultural formulation comprising a recombinant RNA molecule as described in any one of Examples 1 to 28.

[0175] 30. The agricultural formulation as described in Example 29, wherein the recombinant RNA molecule is complexed with one or more RNA-binding proteins or capsidated by a viral capsid protein, optionally wherein the viral capsid protein is a Tomato dwarf Virus capsid protein, and optionally wherein the RNA contains an ERE that provides capsidation of the RNA by the Tomato dwarf Virus capsid protein.

[0176] 31. The agricultural formulation as described in Example 30, wherein the RNA-binding protein comprises an RNA recognition motif.

[0177] 32. The agricultural formulation as described in Example 30 or 31, wherein the viral capsid protein is heterologous to the Tomato Cluster Dwarf Virus family virus.

[0178] 33. The agricultural formulation as described in any one of Examples 29 to 32, wherein the formulation comprises the recombinant RNA molecule as well as a carrier, excipient and / or adjuvant.

[0179] 34. A cell comprising a recombinant RNA molecule as described in any one of Examples 1 to 28, wherein the cell is a bacterial cell, fungal cell, plant cell, insect cell, or invertebrate cell.

[0180] 35. The cell as described in Example 34, wherein the cell is a plant cell and the cell does not contain DNA encoding the recombinant RNA molecule.

[0181] 36. The cell as described in Example 34, wherein the cell contains a recombinant DNA molecule encoding the recombinant RNA molecule.

[0182] 37. An expression system comprising: (a) An RNA molecule containing the recombinant RNA molecule as described in any one of Examples 1 to 28; and (b) Cells containing the recombinant RNA molecule and the RdRP protein that recognizes the 5' and 3' RNA replication elements of the recombinant RNA molecule.

[0183] 38. The expression system as described in Example 37, wherein the recombinant RNA molecule comprises an operatively linked capsidation recognition element (ERE) recognized by a viral capsid protein, and wherein the cell contains the viral capsid protein.

[0184] 39. The expression system as described in Example 37, wherein the capsid recognition element (ERE) is a Tomato dwarf virus family ERE, wherein the viral capsid protein in the cell is a Tomato dwarf virus family capsid protein, and wherein the RNA molecule is capsidated by the Tomato dwarf virus family capsid protein.

[0185] 40. The expression system as described in Examples 37, 38 or 39, further comprising the reverse complementary sequence of the recombinant RNA molecule.

[0186] 41. The expression system as described in any one of Examples 37 to 40, wherein the cell is a bacterial cell, plant cell, fungal cell, insect cell, or invertebrate cell.

[0187] 42. The expression system of any one of Examples 37 to 41, wherein the cell further comprises: (i) a viral capsid protein (CP); (ii) an RNA-binding protein (RBP) capable of binding to the RNA molecule, optionally wherein the RBP binds to an RNA effector; (iii) an RNA cleaving agent for cleaving the RNA molecule; (iv) a second RNA-dependent RNA polymerase (RdRP) protein (second RdRP) that recognizes the HRV 5' or 3' replication region and / or the subgenomic promoter in the RNA molecule; (v) a viral mobile protein (MP); (v) a heterologous RNA virus (HRV); or (vi) hrvRdRP, optionally wherein the hrvRdRP recognizes the HRV 5' or 3' replication region and / or the subgenomic promoter.

[0188] 43. The expression system as described in Example 42, wherein the CP, RBP, RdRP, second RdRP, hrvRdRP and / or the MP: (a) are expressed by a recombinant DNA molecule in the cell; (b) are exogenously provided to the cell; (c) are expressed by a recombinant RNA molecule in the cell; or (d) are expressed by a virus in the cell.

[0189] 44. The expression system as described in Example 42, wherein the RdRP, CP, RBP, second RdRP, hrvRdRP and / or the MP protein are heterologous to the cells.

[0190] 45. An expression system as described in Examples 42 or 44, wherein the RdRP, second RdRP, or hrvRdRP protein, or the polynucleotide encoding the RdRP, second RdRP, or hrvRdRP protein (a) is expressed by a recombinant DNA molecule in the cell; (b) is exogenously provided to the cell; (c) is expressed by a recombinant RNA molecule in the cell; or (d) is expressed by a virus in the cell.

[0191] 46. ​​The expression system as described in any one of Examples 37 to 45, wherein the cell is a plant cell.

[0192] 47. The expression system as described in Example 46, wherein the plant cells contain a Tomato Cluster Dwarf Virus expressing the RdRP protein, the RdRP protein recognizing the 5' RNA replication element and the 3' RNA replication element, and / or wherein the plant cells contain an HRV expressing the second RdRP or hrvRdRP protein.

[0193] 48. The expression system as described in Example 47, wherein the Tomato Cluster Dwarf Virus family virus is naturally present in the plant cells.

[0194] 49. A method for providing a plant with synthetic Tomato Cluster Dwarf Virus family satellite RNA, the method comprising contacting the plant with a recombinant RNA molecule as described in any one of Examples 1 to 28.

[0195] 50. The method as described in Example 49, wherein contact comprises spraying, dusting, injecting, or soaking the plant or a portion thereof with the recombinant RNA molecule or the preparation.

[0196] 51. The method as described in Example 49 or 50, further comprising providing the plant with an hrvRdRP that identifies the HRV 5' or 3' replication region and / or the subgenomic promoter, optionally wherein the hrvRdRP is provided by introducing recombinant DNA or RNA encoding the hrvRdRP into the plant or a portion thereof.

[0197] 52. A method for establishing a synthetic Tomato Cluster Dwarf Virus Family satellite RNA in plant cells, the method comprising: providing a plant cell with a recombinant RNA molecule as described in any one of Examples 1 to 28; wherein the plant cell contains an RdRP protein that recognizes the 5' RNA replication element and the 3' RNA replication element, wherein the RNA molecule optionally contains an ERE and is capsidated by a capsid protein, whereby the RdRP protein catalyzes the synthesis of the synthetic Tomato Cluster Dwarf Virus Family satellite RNA from the recombinant RNA molecule.

[0198] 53. The method as described in Example 52, wherein the plant cells contain a Tomato Cluster Dwarf Virus family virus, and wherein the RdRP protein is provided to the plant cells via the Tomato Cluster Dwarf Virus family virus.

[0199] 54. The method as described in Example 52 or 53, wherein the Tomato Cluster Dwarf Virus is specific to the plant cell, and optionally the Tomato Cluster Dwarf Virus specific to the plant cell is non-pathogenic and / or symbiotic.

[0200] 55. The method as described in Examples 52, 53 or 54, wherein the capsid protein comprises a Tomato dwarf virus family capsid protein, and the ERE is recognized by the Tomato dwarf virus family capsid protein, optionally wherein the recombinant RNA molecule comprises an operatively linked capsid recognition element (ERE) recognized by the viral capsid protein.

[0201] 56. The method of any one of Examples 52 to 55, further comprising providing the plant with hrvRdRP that recognizes the HRV 5' or 3' replication region and / or the subgenomic promoter in the synthetic Tomato Cluster Dwarf Virus family satellite RNA, optionally wherein the hrvRdRP is provided by introducing recombinant DNA or RNA encoding the hrvRdRP into the plant or a portion thereof.

[0202] 57. A method for obtaining phenotypic changes in plants or plant cells, the method comprising: providing a plant or plant cell with a recombinant RNA molecule as described in any one of Examples 1 to 28, wherein the cargo RNA molecule contains RNA that achieves the phenotypic changes of the plant or plant cell compared to a plant or plant cell lacking the recombinant RNA, wherein the plant or plant cell contains an RdRP protein that recognizes the 5' RNA replication element and the 3' RNA replication element and catalyzes the synthesis of synthetic Tomato dwarf Virus RNA from the recombinant RNA molecule, and wherein the cargo RNA molecule achieves the phenotypic changes.

[0203] 58. The method as described in Example 57, wherein the RNA that achieves the phenotypic change in the plant or plant cell comprises at least one RNA selected from siRNA or siRNA precursor, miRNA or miRNA precursor, and staged siRNA or staged siRNA precursor.

[0204] 59. The method as described in Example 57, wherein the RNA that achieves the phenotypic change in the plant or plant cell comprises messenger RNA.

[0205] 60. The method as described in Example 59, wherein the messenger RNA comprises an RNA molecule not present in the genome of the plant or plant cell.

[0206] 61. The method as described in Example 57, wherein the RNA that achieves the phenotypic change of the plant or plant cell comprises RNA for modifying the genome of the plant or plant cell.

[0207] 62. The method of Example 57, wherein the RNA that achieves the phenotypic change of the plant or plant cell comprises RNA for modifying the transcriptome and / or epigenome of the plant or plant cell, optionally wherein the RNA for modifying the epigenome targets an endogenous plant gene for RNA-induced transcriptional silencing.

[0208] 63. The method of any one of Examples 57 to 62, wherein the phenotypic change comprises an increase in the plant’s resistance to pests or pathogens, optionally wherein the pests or pathogens are selected from the group consisting of bacteria, viruses other than Tomato Cluster Dwarf Virus viruses, fungi, oomycetes, and invertebrates.

[0209] 64. The method as described in Example 63, wherein the pathogen is a heterologous RNA virus (HRV), optionally wherein the HRV is a virus selected from the group consisting of: *Hyperviridae*, *Hyperviridae*, *Brassica napus*, *Celavirus*, *Leptovirus*, *Cowpea Mosaic Virus*, *Potato Virus X*, *Potato Virus Y*, *Tobacco Mosaic Virus*, *Tomato Clump Dwarf Virus*, *Tomato Spotted Wilt Virus Family*, *Trigeneae* subfamily, *Turnip Yellow Mosaic Virus*, *Vein Varicose Vein Virus*, and *Associated Cowpea Mosaic Virus Family*.

[0210] 65. The method as described in Example 64, wherein: (i) the genus *Bombyx mottle virus* is cucumber mosaic virus, spinach latent virus, olive latent virus, or *Bombyx mottle virus*; (ii) the genus *Lycovirus* is citrus senescence virus or beet yellowing virus; (iii) the genus *Cowpea mosaic virus* is cowpea mosaic virus, apple latent spherical virus, or soybean latent spherical virus; (iv) the genus *Potato X virus* is potato X virus or citrus yellowing virus; (v) the genus *Potato Y virus* is pepper mottle virus, bean yellow mosaic virus, barley stripe mosaic virus, or wheat stripe mosaic virus. (vi) The genus of tobacco mosaic virus is tobacco mosaic virus, tomato mosaic virus, turnip mosaic virus or pepper mild mottle virus; (vii) The genus of tomato dwarf virus is turnip shrunken virus or tomato dwarf virus; (iv) The family of tomato wilt virus is tomato wilt virus or watermelon bud necrosis virus; or (viii) The genus of turnip yellow mosaic virus is turnip yellow mosaic virus, citrus yellowing mosaic virus, potato latent virus, apple stem groove virus or citrus leaf mottle virus.

[0211] 66. The method of any one of Examples 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 including nutrient stress, light stress, water stress, heat stress and / or cold stress, or optionally wherein the stress comprises at least one biotic stress including crowding, shading or allelopathy.

[0212] 67. The method of any one of Examples 57 to 66, wherein the recombinant RNA molecule is provided to the plant or plant cells in the form of RNA, capsidated RNA or a formulation thereof.

[0213] 68. The method as described in Example 67, wherein the capsidated RNA comprises synthetic Tomato Cluster Dwarf Virus Family satellite particles.

[0214] 69. The method of any one of Examples 57 to 68, wherein the provision comprises contacting the plant or plant cells with the RNA, capsidized RNA or a formulation thereof, optionally wherein the contact comprises spraying, dusting, injecting or soaking the plant or plant cells with the RNA, capsidized RNA or a formulation thereof.

[0215] 70. The method of any one of Examples 57 to 69, wherein the recombinant RNA further comprises its reverse complementary RNA molecule.

[0216] 71. The method of any one of Examples 57 to 70, further comprising providing the plant with hrvRdRP that recognizes the HRV 5' or 3' replication region and / or the subgenomic promoter in the synthetic Tomato Cluster Dwarf Virus family satellite RNA, optionally wherein the hrvRdRP is provided by introducing recombinant DNA or RNA encoding the hrvRdRP into the plant or a portion thereof.

[0217] 72. A method for manufacturing synthetic Tomato Dwarf Virus family satellite particles, the method comprising combining a recombinant RNA molecule as described in any one of Examples 1 to 28 with a viral capsid protein, wherein the recombinant RNA molecule includes a capsidation recognition element (ERE), and wherein the ERE provides capsidation of the RNA by the viral capsid protein.

[0218] 73. The method as described in Example 72, wherein the recombinant RNA molecule is combined with the viral capsid protein in a container.

[0219] 74. The method as described in Example 73, wherein the combination comprises: (a) Providing the recombinant RNA molecule to plant cells, wherein the recombinant RNA molecule contains a capsidation recognition element (ERE), and wherein the plant cells contain an RdRP protein and a viral capsid protein that recognize the 5' RNA replication element and the 3' RNA replication element and catalyze the synthesis of Tomato dwarf Virus satellite RNA from the recombinant RNA molecule, wherein the ERE provides capsidation of the RNA by the viral capsid protein; and optionally (b) Isolating the synthetic Tomato Cluster Dwarf Virus Family satellite particles from the plant cells, the plant containing the plant cells, or from the plant cells or the culture medium in which the plant grows.

[0220] 75. The method as described in Examples 72, 73 or 74, further comprising the step of formulating the synthetic Tomato Cluster Dwarf Virus Family satellite particles, wherein the formulation comprises combining the synthetic Tomato Cluster Dwarf Virus Family satellite particles with a carrier, excipient and / or adjuvant.

[0221] 76. A plant propagule comprising a recombinant RNA molecule as described in any one of Examples 1 to 28 and a Tomato Cluster Dwarf Virus RdRP, optionally wherein the plant propagule further comprises a heterologous RNA virus RdRP that recognizes the HRV 5' or 3' replication region in the synthetic Tomato Cluster Dwarf Virus satellite RNA and / or the subgenomic promoter.

[0222] 77. The plant propagation body as described in Example 76, wherein the plant propagation body is a seed, seedling, root, stem, leaf, bud, tuber, rhizome, stolon, bulb, explant, embryo, or callus.

[0223] 78. The plant propagule as described in Example 76 or 77, wherein the plant propagule is a chimera comprising both a plant cell containing the recombinant RNA molecule and a plant cell lacking the recombinant RNA molecule.

[0224] 79. The plant propagule as described in Examples 76, 77 or 78, wherein the plant propagule lacks DNA encoding the recombinant RNA molecule.

[0225] 80. A plant propagule as described in any one of Examples 76 to 79, wherein the plant propagule further comprises a heterologous RNA virus RdRP that recognizes the HRV 5' or 3' replication region and / or the subgenomic promoter in the synthetic Tomato Cluster Dwarf Virus Family satellite RNA, and the heterologous RNA virus RdRP is a RdRP from the genera *Hyperviridae*, *Hyperviridae*, *Brassica napus*, *Celavirus*, *Leptochloavirus*, *Cowpea Mosaic Virus*, *Potato Virus X*, *Potato Virus Y*, *Tobacco Mosaic Virus*, *Tomato Cluster Dwarf Virus*, Tomato Spotted Wilt Virus Family, Trigeneinae, *Turnip Yellow Mosaic Virus*, *Vein Varicose Vein Virus*, or associated Cowpea Mosaic Virus Family, or an RdRP shown in Table 7.

[0226] 81. A plant comprising a recombinant RNA molecule as described in any one of Examples 1 to 28 and a Tomato Cluster Dwarf Virus RdRP, optionally wherein the plant propagule further comprises a heterologous RNA virus RdRP that recognizes the HRV 5' or 3' replication region in the synthetic Tomato Cluster Dwarf Virus satellite RNA and / or the subgenomic promoter.

[0227] 82. The plant as described in Example 81, wherein the plant is a monocotyledonous plant or a dicotyledonous plant.

[0228] 83. The plant as described in Example 81, wherein the plant belongs to the Asteraceae, Cucurbitaceae, Fabaceae, Oleaceae, Poaceae, Rutaceae or Solanaceae families.

[0229] 84. The plant as described in Examples 81, 82 or 83, wherein the plant lacks DNA encoding the recombinant RNA molecule.

[0230] 85. A plant as described in any one of Examples 81 to 84, wherein the plant comprises a Tomato Cluster Dwarf Virus family virus, and wherein the Tomato Cluster Dwarf Virus family RdRP is provided to the plant cells via the Tomato Cluster Dwarf Virus family virus, optionally wherein the Tomato Cluster Dwarf Virus family RdRP comprises a protein having at least 85%, 90%, 95%, 97%, or 99% sequence identity with SEQ ID NO: 469 or 587.

[0231] 86. The plant as described in any one of Examples 81 to 85, wherein the Tomato Cluster Dwarf Virus family virus is specific to the plant, and optionally wherein the specific Tomato Cluster Dwarf Virus family virus is non-pathogenic and / or symbiotic.

[0232] 87. The plant as described in any one of Examples 81 to 86, wherein the Tomato Cluster Dwarf Virus Family RdRP, the 5' RNA replication element and / or the 3' RNA replication element are derived from a Tomato Cluster Dwarf Virus Family virus containing one or both of the Tomato Cluster Dwarf Virus Family RdRP, the 5' RNA replication element and / or the 3' RNA replication element.

[0233] 88. The plant as described in any one of Examples 81 to 87, wherein the RdRP has at least 85%, 90%, 95%, 97%, 98%, or 99% identity with SEQ ID NO: 469 or 587, wherein the 5' RNA replication element has at least 85%, 90%, 95%, 97%, 98%, or 99% identity with the RNA encoded by SEQ ID NO: 467, and / or wherein the 3' RNA replication element has at least 85%, 90%, 95%, 97%, 98%, or 99% identity with the RNA encoded by SEQ ID NO: 468.

[0234] 89. The plant as described in any one of Examples 81 to 88, wherein the plant is a grafted plant, and wherein the rootstock and / or scion of the grafted plant contains at least one cell containing the recombinant RNA and the RdRP of the Tomato Cluster Dwarf Virus Family.

[0235] 90. The plant as described in any one of Examples 81 to 89, wherein the plant is not produced by an inherently biological process.

[0236] 91. The plant as described in any one of Examples 81 to 90, wherein the plant further comprises a heterologous RNA virus (HRV) RdRP that recognizes the HRV 5' or 3' replication region and / or the subgenomic promoter in the synthetic Tomato Cluster Dwarf Virus Family satellite RNA, and the heterologous RNA virus RdRP is a HRV from the genera *Hyperviridae*, *Hyperviridae*, *Brassica napus*, *Celavirus*, *Leptochloavirus*, *Cowpea Mosaic Virus*, *Potatovirus X*, *Potatovirus Y*, *Tobacco Mosaic Virus*, *Tomato Cluster Dwarf Virus*, Tomato Spotted Wilt Virus Family, Trigeneinae, *Turnip Yellow Mosaic Virus*, *Vein Varicella Virus*, or associated Cowpea Mosaic Virus Family RdRP or an RdRP shown in Table 7.

[0237] 92. A Tomato Cluster Dwarf Virus Family satellite system capable of self-replication upon introduction into a plant or plant cell, said Tomato Cluster Dwarf Virus Family satellite system comprising: (a) a recombinant Tomato Cluster Dwarf Virus Family satellite RNA as described in any one of Examples 1 to 28; and (b) an exogenous Tomato Cluster Dwarf Virus Family virus capable of replicating in said plant or plant cell and encoding a Tomato Cluster Dwarf Virus Family RdRP that recognizes 5' and 3' replicase recognition sequences in said recombinant Tomato Cluster Dwarf Virus Family satellite RNA, optionally wherein said Tomato Cluster Dwarf Virus Family satellite system further comprises a heterologous RNA virus RdRP that recognizes the HRV 5' or 3' replication region and / or subgenomic promoter in said synthetic Tomato Cluster Dwarf Virus Family satellite RNA.

[0238] 93. A self-replicating Tomato Cluster Dwarf Virus Family satellite system as described in Example 92, wherein the exogenous Tomato Cluster Dwarf Virus Family virus is specific to or naturally occurring for different species, varieties, or germplasm of the plant.

[0239] 94. The self-replicating Tomato Cluster Dwarf Virus Family satellite system as described in Example 92, wherein the Tomato Cluster Dwarf Virus Family satellite system further comprises a heterologous RNA virus (HRV) RdRP that identifies the HRV 5' or 3' replication region and / or the subgenomic promoter in the synthetic Tomato Cluster Dwarf Virus Family satellite RNA, and the heterologous RNA virus RdRP is optionally a RdRP from the genera *Hyperviridae*, *Hyperviridae*, *Brassica napus*, *Celavirus*, *Leptovirus*, *Cowpea Mosaic Virus*, *Potato Virus X*, *Potato Virus Y*, *Tobacco Mosaic Virus*, *Tomato Cluster Dwarf Virus*, Tomato Spotted Wilt Virus Family, Trigeneinae, *Turnip Yellow Mosaic Virus*, *Vein Varicose Vein Virus*, or associated Cowpea Mosaic Virus Family, or an RdRP shown in Table 7.

[0240] 95. A recombinant DNA molecule comprising a first promoter operatively linked to DNA encoding an RNA molecule as described in any one of Examples 1-28.

[0241] 96. A cell comprising a recombinant DNA molecule as described in Example 95, wherein the cell is a bacterial cell, fungal cell, plant cell, insect cell, or invertebrate cell.

[0242] 97. A vector for bacterial-mediated plant transformation, said vector comprising a recombinant DNA molecule as described in Example 95.

[0243] 98. The vector as described in Example 97, wherein the bacteria mediating the transformation of the plant are Agrobacterium species, Rhizobium sinense species, Meso- and Slow-growing Rhizobium species, Slow-growing Rhizobium species, Rhizobium species, or Cyclocarya species, and the vector is suitable for transformation with said bacteria.

[0244] 99. The vector as described in Example 97 or 98, wherein the bacteria mediating the transformation of the plant is an Agrobacterium species, and wherein the vector further comprises T-DNA side-mounted to a DNA molecule encoding the recombinant RNA molecule.

[0245] 100. The carrier as described in Examples 97, 98 or 99, which is contained in bacterial or plant cells.

[0246] 101. An expression system comprising: (a) a recombinant DNA molecule as described in Example 95; and (b) a cell containing the recombinant DNA molecule and an RdRP protein that recognizes 5' and 3' RNA replication elements encoded by the DNA molecule.

[0247] 102. The expression system as described in Example 101, wherein the recombinant DNA molecule further comprises at least one additional element containing: (i) DNA encoding at least one RNA encoding a viral mobile protein (MP); (ii) DNA encoding at least one tRNA-like molecule; (iii) DNA encoding a capsid recognition element (ERE); (iv) DNA encoding RNA comprising, from 5' to 3', and operably linked to: a 5' RNA replication element recognized by a Tomato dwarf Virus RNA-dependent RNA polymerase (RdRP), the RNA of (i), and optionally operably linked to the RNA of (ii) and / or (iii), and a 3' RNA replication element; (v) DNA encoding an RNA promoter; (vi) DNA encoding an HRV recognition element. DNA of an RNA-dependent RNA polymerase (RdRP) for a 5' or 3' replication region and / or a subgenomic promoter; and / or (vii) a DNA molecule containing a promoter operatively linked to DNA encoding at least one of (i), (ii), (iii), (iv), (v), or (vi).

[0248] 103. The expression system as described in Example 101 or 102, wherein the cell is a bacterial cell, plant cell, fungal cell, insect cell, or invertebrate cell, optionally wherein the bacterial cell is a species of Agrobacterium, Rhizobium sinense, Rhizobium mesenterogenes, Rhizobium stenogenum, Rhizobium stenogenum, Rhizobium stenogenum, or Rhizobium stenogenum.

[0249] 104. The expression system of any one of Examples 101, 102, or 103, further comprising: (i) a viral capsid protein capable of capsidating an RNA molecule containing the capsidation recognition element (ERE); (ii) an RNA-binding protein (RBP) capable of binding to an RNA molecule encoded by the DNA molecule, optionally wherein the RBP binds to an RNA effector; (iii) an RNA cleaving agent for cleaving the RNA molecule; and / or (iv) an RNA promoter-dependent RNA polymerase (RdRP) protein that recognizes an HRV 5' or 3' replication region and / or subgenomic promoter in the RNA molecule.

[0250] 105. The expression system as described in Example 104, wherein the viral capsid protein is: (a) expressed by the recombinant DNA molecule in the cell, (b) co-expressed by a second recombinant DNA molecule in the cell, (c) exogenously provided to the cell, or (d) expressed by a virus in the cell.

[0251] 106. The expression system as described in Example 104 or 105, wherein: (i) the capsid protein, viral mobile protein (MP), RdRP protein and / or RdRP protein recognizing the HRV 5' or 3' replication region and / or subgenomic promoter is heterologous to the cell, and / or (ii) wherein the RdRP protein or the polynucleotide encoding the RdRP protein is exogenously provided to the cell.

[0252] 107. The expression system as described in any one of Examples 101 to 106, wherein the cell is a plant cell.

[0253] 108. The expression system of 107, wherein the plant cells contain a Tomato Cluster Dwarf Virus virus, the Tomato Cluster Dwarf Virus virus expressing the RdRP protein that recognizes the 5' RNA replication element and the 3' RNA replication element.

[0254] 109. The expression system as described in Example 108, wherein the Tomato Cluster Dwarf Virus is naturally present in the plant cells.

[0255] 110. The expression system as described in any one of Examples 101 to 109, wherein the recombinant DNA molecule further comprises at least one RNA encoding a viral MP, a tRNA-like molecule from Arabidopsis thaliana FT mRNA, and a capsid recognition element comprising TMV-OAS.

[0256] 111. An agricultural formulation comprising an expression system as described in any one of Examples 101 to 110.

[0257] 112. The agricultural formulation as described in Example 111, wherein the formulation comprises the expression system and a carrier, excipient and / or adjuvant.

[0258] 113. An agricultural formulation comprising a recombinant DNA molecule as described in Example 95.

[0259] 114. The agricultural formulation as described in Example 113, wherein the formulation comprises the recombinant DNA molecule as well as a carrier, excipient and / or adjuvant.

[0260] 115. A method for producing a foreign polypeptide in a plant or plant cell, comprising: providing a plant or plant cell containing a recombinant RNA molecule as described in any one of Examples 1 to 28 or a recombinant DNA molecule as described in Example 95, wherein the cargo RNA molecule encoded by the RNA or DNA molecule contains a translatable messenger RNA encoding the foreign polypeptide, wherein the plant or plant cell contains an RdRP protein that recognizes the 5' RNA replication element and the 3' RNA replication element of the recombinant RNA and catalyzes the synthesis of a Tomato dwarf Virus satellite RNA synthesized from the recombinant RNA molecule, and wherein the foreign polypeptide is translated from the translatable messenger RNA; optionally wherein the plant or plant cell further contains an HRV 5' or 3' replication region and / or subgenomic promoter in the RNA molecule, and a heterologous RNA virus (HRV) RNA promoter-dependent RNA polymerase (hrvRdRP) protein that recognizes the RNA promoter.

[0261] 116. The method of Example 115, further comprising the following first step: providing a plant population comprising the plant cells containing a Tomato Cluster Dwarf Virus virus providing the RdRP protein; and then providing the plant comprising the plant cells with the recombinant RNA or DNA molecule, the cells, the vector, the recombinant RNA or DNA molecule or a formulation thereof, and optionally further comprising providing the plant comprising the plant cells with the hrvRdRP.

[0262] 117. The method as described in Example 115 or 116, wherein the Tomato Cluster Dwarf Virus is exogenously provided to the plant cells, and / or wherein the HRV encoding the hrvRdRP is provided to the plant cells.

[0263] 118. The method as described in Examples 115, 116 or 117, wherein the RdRP and / or hrvRdRP proteins or recombinant polynucleotides encoding the RdRP and / or hrvRdRP proteins are exogenously provided to the plant cells.

[0264] 119. The method of any one of Examples 115 to 118, further comprising the following first step: providing a plant population containing the plant cells, the plant cells containing the RdRP protein or a recombinant polynucleotide encoding the RdRP; and then providing the recombinant RNA molecule to the plant containing the plant cells.

[0265] 120. The method of any one of Examples 115 to 119, wherein the recombinant RNA molecule has been produced in a fermentation system.

[0266] 121. The method of any one of Examples 115 to 120, further comprising the step of determining whether the plant cells contain a Tomato Cluster Dwarf Virus family virus that can provide the RdRP.

[0267] 122. A method for producing modified plant propagules comprising at least one plant cell containing a recombinant RNA molecule as described in Examples 1 to 28, the method comprising isolating plant propagules containing at least one plant cell containing the recombinant RNA molecule and a tomato dwarf virus family RNA-dependent RNA polymerase (RdRP) from a mixed population of plant cells containing both plant cells containing the recombinant RNA molecule and plant cells lacking the recombinant RNA molecule.

[0268] 123. The method as described in Example 122, wherein the plant cell mixed population comprises a population of protoplasts or a population of cells in callus or explant.

[0269] 124. The method as described in Example 122 or 123, wherein the mixed population of plant cells comprises plant cells containing the RdRP of the Tomato Cluster Dwarf Virus Family and plant cells lacking the RdRP of the Tomato Cluster Dwarf Virus Family.

[0270] 125. The method as described in Examples 122, 123 or 124, wherein the plant cell mixed population comprises plant cells containing the RdRP of the Tomato Cluster Dwarf Virus Family.

[0271] 126. The method of any one of Examples 122 to 125, wherein the plant cell population is screened or selected for the presence of plant cells containing the recombinant RNA molecule prior to the isolation of the plant propagules.

[0272] 127. The method of any one of Examples 122 to 126, wherein the separation comprises selecting plant cells containing the recombinant RNA molecule prior to separating the plant propagule.

[0273] 128. The method of any one of Examples 122 to 127, wherein the mixed population is located within a plant or plant part.

[0274] 129. The method of any one of Examples 122 to 128, wherein the plant or plant part is screened or selected for the presence of the recombinant RNA molecule prior to isolating the plant propagule.

[0275] 130. The method of any one of Examples 122 to 128, wherein the plant or plant part is screened or selected for the systematic presence of the recombinant RNA molecule prior to isolating the plant propagule.

[0276] 131. The method of any one of Examples 122 to 130, wherein the recombinant RNA molecule encodes a selectable marker, and plant propagules containing the recombinant RNA molecule are isolated by selecting for the presence or absence of the selectable marker.

[0277] 132. The method of Example 131, further comprising selecting plant propagules containing recombinant RNA molecules, wherein the selectable marker has been removed.

[0278] 133. The method of any one of Examples 122 to 132, wherein the plant propagule containing the recombinant RNA molecule is separated by: detecting the RNA molecule in one or more plant cells containing the recombinant RNA molecule, and separating the one or more plant cells containing the recombinant RNA molecule from plant cells lacking the recombinant DNA molecule.

[0279] 134. The method of any one of Examples 122 to 133, wherein the plant propagule is a chimera comprising both a plant cell containing the recombinant RNA molecule and a plant cell lacking the recombinant RNA molecule.

[0280] 135. The method of any one of Examples 122 to 134, wherein the plant propagule is a chimera comprising both plant cells containing the RdRP of the Tomato Cluster Dwarf Virus Family and plant cells lacking the RdRP of the Tomato Cluster Dwarf Virus Family.

[0281] 136. The method of any one of Examples 122 to 134, wherein the plant propagule lacks DNA encoding the recombinant RNA molecule.

[0282] 137. The method of any one of Examples 122 to 136, wherein the plant or plant part lacks DNA encoding the recombinant RNA molecule.

[0283] 138. The method of any one of Examples 122 to 137, wherein the plant propagule comprises the cell, or a seed, seedling, root, stem, leaf, bud, tuber, rhizome, stolon, bulb, explant, or callus containing the cell.

[0284] 139. The method of Example 138, further comprising propagating the cells, seeds, seedlings, roots, stems, leaves, buds, tubers, rhizomes, stolons, bulbs, explants or callus to obtain progeny, wherein the progeny contains the recombinant RNA molecule.

[0285] 140. The method as described in Example 139, wherein the propagation comprises culturing multiple explants obtained from the cells, seeds, seedlings, roots, stems, leaves, buds, tubers, rhizomes, stolons, bulbs, explants, or callus tissue.

[0286] 141. The method of any one of Examples 122 to 140, wherein the isolated propagule comprises the cell, and the method further comprises regenerating from the cell a plant, seedling, root, stem, leaf, bud, tuber, rhizome, stolon, bulb, explant, or callus containing the recombinant RNA.

[0287] 142. The method of any one of Examples 122 to 140, wherein the isolated propagule comprises callus tissue, and the method further comprises regenerating from the callus tissue a plant, seedling, root, stem, leaf, bud, tuber, rhizome, stolon, bulb, or explant containing the recombinant RNA.

[0288] 143. The method as described in Examples 141 or 142, wherein the plant is regenerated, and wherein the method further comprises recovering F1 seeds or F1 progeny containing the recombinant RNA from the plant.

[0289] 144. A method for providing a plant with synthetic Tomato Cluster Dwarf Virus Family satellite RNA, the method comprising: grafting a scion onto a rootstock containing a recombinant RNA molecule as described in any one of Examples 1 to 28, wherein at least one cell of the rootstock and / or the scion contains the Tomato Cluster Dwarf Virus Family RdRP.

[0290] 145. The method as described in Example 144, wherein the scion lacks the recombinant RNA molecule prior to grafting.

[0291] 146. The method as described in Examples 144 or 145, wherein the rootstock comprises the Tomato Cluster Dwarf Virus Family RdRP.

[0292] 147. The method of any one of Examples 144 to 146, wherein the RdRP is provided by a Tomato Cluster Dwarf Virus Family virus specific to the rootstock, optionally wherein the Tomato Cluster Dwarf Virus Family virus specific to the rootstock is non-pathogenic and / or symbiotic.

[0293] 148. The method of any one of Examples 144 to 146, wherein the RdRP is provided exogenously to the rootstock.

[0294] 149. The method of any one of Examples 144 to 148, wherein the scion comprises the Tomato Cluster Dwarf Virus Family RdRP.

[0295] 150. The method of any one of Examples 144 to 149, wherein the RdRP is provided by a Tomato Cluster Dwarf Virus Family virus specific to the scion, and / or wherein the RdRP is provided exogenously to the scion, optionally wherein the Tomato Cluster Dwarf Virus Family virus specific to the scion is non-pathogenic and / or symbiotic.

[0296] 151. A method for producing a plant that delivers a recombinant RNA molecule to offspring plants or seeds, the method comprising isolating an F1 offspring plant or seed population from an F1 plant or seed population obtained from a parent plant containing a recombinant RNA molecule as described in any one of Examples 1 to 28, comprising at least one cell containing a Tomato dwarf virus family RNA-dependent RNA polymerase (RdRP) and said recombinant RNA molecule.

[0297] 152. The method of Example 151, wherein the F1 progeny plants or seeds comprising the cells are isolated by: screening for the presence of the recombinant RNA molecule to obtain an F1 plant or seed population from the parent plant, and propagating F1 progeny plants or seeds containing the recombinant RNA molecule.

[0298] 153. The method as described in Example 151, wherein the recombinant RNA molecule encodes a selectable marker, and F1 progeny plants or seeds containing the recombinant RNA molecule are separated by selecting F1 progeny plants or seeds containing the recombinant RNA molecule based on the presence or absence of the selectable marker.

[0299] 154. The method of any one of Examples 151 to 153, wherein the F1 progeny plants or seeds lack DNA encoding the recombinant RNA molecule.

[0300] 155. The method of any one of Examples 151 to 154, wherein the parent plant lacks DNA encoding the recombinant RNA molecule.

[0301] 156. The method as described in Example 153, wherein the selected F1 progeny plants deliver the recombinant RNA molecule to at least the F2 progeny.

[0302] 157. The method of any one of Examples 151 to 156, wherein the F1 offspring plants or seed population are obtained from a parent plant used as a pollen receptor.

[0303] 158. The method of any one of Examples 151 to 156, wherein the F1 offspring plants or seed populations are obtained from parent plants used as pollen donors.

[0304] 159. The method of any one of Examples 151 to 156, wherein the F1 offspring plants or seed population are obtained by self-pollination of the parent plants.

[0305] 160. The method of any one of Examples 151 to 159, wherein the parent plant or a portion thereof containing the plant cells is screened or selected for the presence of the recombinant RNA molecule prior to the isolation of the F1 progeny plants or seeds.

[0306] 161. The method of any one of Examples 151 to 160, wherein the parent plant or one or more portions thereof are screened for the systematic presence of the recombinant RNA molecule prior to the isolation of the F1 progeny plants, optionally wherein the portions comprise floral tissue or male or female reproductive tissue.

[0307] 162. The method as described in any one of Examples 151 to 161, wherein the pericarp of the parent plant is screened or selected for the presence of the recombinant RNA molecule.

[0308] 163. The method of any one of Examples 151 to 162, wherein the F1 seeds are obtained from a parental plant selected based on the presence or absence of the recombinant RNA molecule in the pericarp tissue.

[0309] 164. The method of any one of Examples 151 to 163, wherein the F1 seeds of the parent plant are screened nondestructively for the presence of the recombinant RNA molecule.

[0310] 165. The method as described in Example 165, wherein the F1 seeds of the parent plant are nondestructively screened by determining the presence of the recombinant RNA molecule in the maternal-derived tissue or endosperm tissue of the seed.

[0311] 166. The method of any one of Examples 151 to 165, further comprising introducing the recombinant RNA molecule or the polynucleotide encoding the recombinant RNA molecule into a plant cell and obtaining a parent plant containing the recombinant RNA molecule from the plant cell.

[0312] 167. The method of any one of Examples 151 to 166, wherein the propagator, plant, plant part, scion and / or rootstock contains a heteroviral capsid protein capable of capsidating the recombinant RNA molecule and / or contains the recombinant RNA molecule capsidated by the heteroviral capsid protein.

[0313] 168. A method for barcoding a plant, plant cell, its progeny, or a portion thereof, the method comprising providing the plant or plant cell with a recombinant RNA molecule as described in any one of Examples 1 to 28, wherein the cargo RNA of the recombinant RNA molecule comprises a barcoded RNA molecule, and wherein the plant or plant cell comprises a Tomato dwarf virus family RNA-dependent RNA polymerase (RdRP).

[0314] 169. The method as described in Example 168, wherein the barcode RNA molecule contains a sequence that uniquely identifies the plant, plant cell, its progeny, or a portion thereof.

[0315] 170. The method as described in Examples 168 or 169, wherein the barcode RNA molecule comprises a sequence not present in the genome and / or transcriptome of a wild-type plant of the same species, its pathogen, or its symbiont.

[0316] 171. The method of any one of Examples 168 to 170, wherein the barcode RNA molecule comprises a random sequence.

[0317] 172. The method of any one of Examples 168 to 171, wherein the barcode RNA molecule comprises a forward primer binding site and a reverse primer binding site not present in the genome and / or transcriptome of a wild-type plant of the same species, its pathogen, or its symbiont.

[0318] 173. The method of any one of Examples 168 to 172, wherein the length of the barcode RNA molecule is up to about 6 kb.

[0319] 174. The method of any one of Examples 168 to 172, wherein the length of the barcode RNA molecule is 10 to 6000 nucleotides, 20 to 1000 nucleotides, or 50 to 500 nucleotides, optionally wherein the length of the barcode RNA molecule is 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 1... 50, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, or 6000 nucleotides.

[0320] 175. The method of any one of Examples 168 to 174, wherein the barcode RNA molecule comprises a non-protein-coding sequence.

[0321] 176. The method of any one of Examples 168 to 174, wherein the plant delivers the recombinant RNA molecule to its offspring.

[0322] 177. The method of any one of Examples 168 to 176, wherein the plant, plant cell, its offspring or a portion thereof lacks DNA encoding the recombinant RNA molecule.

[0323] 178. The method of any one of Examples 168 to 176, further comprising isolating F1 progeny plants or seeds containing at least one cell containing the Tomato Cluster Dwarf Virus RdRP and the recombinant RNA molecule.

[0324] 179. The method as described in Example 178, wherein the F1 offspring plants or seeds are obtained from plants used as pollen receptors.

[0325] 180. The method as described in Example 178, wherein the F1 offspring plants or seeds are obtained from plants used as pollen donors.

[0326] 181. The method as described in Example 178, wherein the F1 offspring plants or seeds are obtained by self-pollination of the plants.

[0327] 182. The method of any one of Examples 168 to 181, further comprising propagating the plant or plant cells to obtain a plant part or plant propagule containing the barcode RNA molecule, optionally wherein the propagule comprises callus, tuber and / or rootstock.

[0328] 183. A method for identifying a barcoded plant, plant part, or plant cell, the method comprising screening for the presence of a barcoded RNA molecule in the plant, plant part, or plant cell, wherein the plant, plant part, or plant cell contains a recombinant RNA molecule as described in any one of Examples 1 to 28, wherein the cargo RNA of the recombinant RNA molecule contains the barcoded RNA molecule.

[0329] Summary of the table Table 1. DNA coding sequences of 5' and 3' RNA replication elements in Tomato Clump Dwarf Virus Family

[0330] 1 An NCBI descriptor refers to the database access number of the National Center for Biotechnology Information (NCBI) for an entry in the World Wide Web Internet database “ncbi[dot]nlm[dot]nih.gov / nuccore”.

[0331] 2RNA equivalents of DNA sequences were also considered, and these equivalents can be obtained from the provided DNA sequences.

[0332] Table 2. Viral capsid proteins and assembly origin sequences

[0333]

[0334] 1 The descriptor “NC_XXXXXX.1” refers to the database access number of the National Center for Biotechnology Information (NCBI) in the World Wide Web Internet database “ncbi[dot]nlm[dot]nih.gov / nuccore”.

[0335] 2 RNA equivalents of DNA sequences were also considered, and these equivalents can be obtained from the provided DNA sequences.

[0336] Table 3. Mobile proteins.

[0337]

[0338] 1 The descriptor “NC_XXXXXX.1” refers to the database access number of the National Center for Biotechnology Information (NCBI) in the World Wide Web Internet database “ncbi[dot]nlm[dot]nih.gov / nuccore”.

[0339] Table 4. tRNA-like sequences

[0340]

[0341] 1 The descriptor “ATXXXXXXX.X” refers to the Arabidopsis Information Resource (TAIR) database accession number for the entry in the World Wide Web Internet database “arabidopsis.org”.

[0342] Table 5. IRES sequences

[0343] 1 The descriptor “NC_XXXXXX.1” refers to the database access number of the National Center for Biotechnology Information (NCBI) in the World Wide Web Internet database “ncbi[dot]nlm[dot]nih.gov / nuccore”.

[0344] 2RNA equivalents of DNA sequences were also considered, and these equivalents can be obtained from the provided DNA sequences.

[0345] Table 6. Intron Sequences

[0346] Table 7: Heterologous RNA Viruses (HRVs) and Target Virus Pathogens

[0347]

[0348]

[0349] 1 The descriptor “NC_XXXXXX.1” refers to the database access number of the National Center for Biotechnology Information (NCBI) in the World Wide Web Internet database “ncbi[dot]nlm[dot]nih.gov / nuccore”.

[0350] 2 RNA equivalents of DNA sequences were also considered, and these equivalents can be obtained from the provided DNA sequences.

[0351] Table 8. Antiviral cargo molecules

[0352]

[0353] 1 The descriptor “ATXGXXXXX” refers to the Arabidopsis Information Resource (TAIR) database accession number for the entry in the World Wide Web database “arabidopsis.org”. Other descriptors refer to the National Center for Biotechnology Information (NCBI) database accession numbers for the entries in the World Wide Web database “ncbi[dot]nlm[dot]nih.gov / nuccore”.

[0354] Table 9 Insecticidal or insect-inhibiting and nematode-inhibiting cargo molecules

[0355]

[0356]

[0357]

[0358]

[0359] 1 The descriptor refers to the database access number of the National Center for Biotechnology Information (NCBI) for an entry in the World Wide Web Internet database “ncbi[dot]nlm[dot]nih.gov / nuccore”.

[0360] Table 10 Antifungal and antibacterial cargo RNAi targets

[0361]

[0362]

[0363] 1 The descriptor refers to the database access number of the National Center for Biotechnology Information (NCBI) in the World Wide Web Internet database “ncbi[dot]nlm[dot]nih.gov / nuccore”.

[0364] Table 11. Antifungal and antibacterial cargo proteins

[0365]

[0366]

[0367] 1 The descriptor refers to the database access number of the National Center for Biotechnology Information (NCBI) in the World Wide Web Internet database “ncbi[dot]nlm[dot]nih.gov / nuccore”.

[0368] Table 12. resistance gene

[0369]

[0370]

[0371]

[0372]

[0373] 1The descriptor “ATXGXXXXX” refers to the Arabidopsis Information Resource (TAIR) database accession number for the entry in the World Wide Web database “arabidopsis.org”. Other descriptors refer to the National Center for Biotechnology Information (NCBI) database accession numbers for the entries in the World Wide Web database “ncbi[dot]nlm[dot]nih.gov / nuccore”.

[0374] Table 13 Bioactive plant peptides peptide name sequence Function miPEP172c SEQ ID NO: 408 Increases soybean nodulation (improves nitrogen fixation and yield) miPEP171d SEQ ID NO: 409 Increase the formation of adventitious roots in grapevines BomiPEP397a SEQ ID NO: 410 Increases leaf surface area (36%) and root development in cabbage. AtmiPEP397a SEQ ID NO: 411 Increase root development in Arabidopsis thaliana BvmiPEP164b SEQ ID NO: 412 Growth inhibition of Barbare vulgaris (weed) AtmiPEP164b SEQ ID NO: 413 Root growth inhibition in Arabidopsis

[0375] Table 14 Ribozyme

[0376]

[0377] 1 also considers RNA equivalents of DNA sequences, and said equivalents can be obtained from the provided DNA sequences.

[0378] Table 15 RNA aptamers RNA aptamers <![CDATA[Sequence 1 > chili SEQ ID NO: 439 broccoli SEQ ID NO: 440 Corn-SAM SEQ ID NO: 441 mango SEQ ID NO: 442 Mango II SEQ ID NO: 443 Mango III SEQ ID NO: 444 Mango III (A10U) SEQ ID NO: 445 Mango IV SEQ ID NO: 446 pumpkin SEQ ID NO: 447 chili SEQ ID NO: 448 spinach SEQ ID NO: 449 Spinach 2 SEQ ID NO: 450 Orange Broccoli SEQ ID NO: 451 DIR2s SEQ ID NO: 452

[0379] 1 DNA equivalents of RNA sequences were also considered, and these equivalents can be obtained from the provided RNA sequences.

[0380] Table 16 subgenome promoters subgenome promoters <![CDATA[Sequence 1 > Pea early brown virus CP SEQ ID NO: 453 Tobacco brittle virus strain TCM CP* SEQ ID NO: 454 Tobacco brittle virus strain PSG CP* SEQ ID NO: 455 Tobacco brittle virus strain PLB CP* SEQ ID NO: 456 Tobacco brittle virus strain PRV CP* SEQ ID NO: 457 Pea early brown virus 12K SEQ ID NO: 458 Pea early browning virus 29.6K SEQ ID NO: 459 Pea early brown virus 29K SEQ ID NO: 460 Tobacco mosaic virus CP SEQ ID NO: 461 Tobacco mosaic virus (MP) SEQ ID NO: 462 Cucumber mosaic virus CP SEQ ID NO: 463

[0381] 1 RNA equivalents of DNA sequences were also considered, and these equivalents can be obtained from the provided DNA sequences.

[0382] *Goulden et al. (1990) Nucleic Acids Res., 18: 4507-4512, DOI: 10.1093 / NAR / 18.15.4507.

[0383] Table 17. RdRP and CP sequences of Tomato Cluster Dwarf Virus Family

[0384] Table 18. 5' and 3' RNA replication element pairs and RdRP of the Tomato Cluster Dwarf Virus Family genome.

[0385] Example Example 1. Antiviral Tomato Cluster Dwarf Virus Family Satellite with Embedded HRV Amplicon and RNAi Cargo

[0386] The Tomato Cluster Dwarf Virus Family Satellite (COMSAT) carrying antiviral repressive RNA (RNAi) cargo is typically provided in the following 5' to 3' orientations: (i) a 5' replication element from a Tomato Cluster Dwarf Virus Family virus; (ii) a heterologous RNA virus (HRV) 5' replication region (HRV 5'RR); (iii) an antiviral RNA molecule that induces an RNAi response; (iv) a heterologous RNA virus (HRV) 3' RNA replication region (HRV 3'RR); and (v) a 3' RNA replication element from a Tomato Cluster Dwarf Virus Family virus.

[0387] COMSAT containing 5' and 3' RNA replication elements of the Tomato Cluster Dwarf Virus family, which are side-linked with TMV amplicons, is provided as SEQ ID NO: 473. The TMV amplicons contain cargo RNA containing a pepper mild mottle virus (PMMoV) RNAi inducible sequence (e.g., a PMMoV sequence that can form dsRNA). The satellite's elements are shown in Table 19.

[0388] Table 19. Genetic elements Nucleotide positions in SEQ ID NO: 473 5' RNA replication element of Tomato dwarf virus family 1-60 TMV 5' Copy Area 61-128 Antiviral RNAi cargo (PMMoV RNAi inducible sequence) 129-1106 TMV 3' Copy Area 1107-1310 ZmHSP101 IRES (Optional) 1311-1457 TMV MP (optional) 1458-2264 Ileu tRNA 2265-2346 3' RNA replication element of Tomato dwarf virus family 2347-2598

[0389] Example 2. Antiviral Tomato Dwarf Virus Family Satellite with Embedded HRV Amplicon and Antiviral Protein Cargo

[0390] The Tomato Cluster Dwarf Virus Family Satellite (COMSAT) with embedded HRV amplicon carrying antiviral protein cargo is typically provided in the following 5' to 3' orientations: (i) a 5' replication element from a Tomato Cluster Dwarf Virus Family virus; (ii) a heterologous RNA virus (HRV) 5' replication region (HRV 5'RR); (iii) antiviral protein cargo; (iv) a heterologous RNA virus (HRV) 3' RNA replication region (HRV 3'RR); and (v) a 3' RNA replication element from a Tomato Cluster Dwarf Virus Family virus.

[0391] A COMSAT containing 5' and 3' RNA replication elements of a Tomato Cluster Dwarf Virus (TMV) amplicon, comprising a tobacco mosaic virus (TMV) amplicon, is provided as SEQ ID NO: 474. The TMV amplicon contains an antiviral cargo protein encoding the N gene of tobacco. This satellite contains TMV 5' and 3' replication region sequences (HRV 5' and 3' RR sequences) to facilitate secondary amplification of TMV (a pathogenic tobacco mosaic virus). The components of this satellite are shown in Table 20. Similar antiviral Tomato Cluster Dwarf Virus (TCDV) satellites are designed for secondary amplification via Tomato Mosaic Virus (TMV) or Tomato Brown Curly Fruit Virus (TCBV), which are also pathogenic Tobacco Mosaic Virus (TBV). These satellites contain the N protein (SEQ ID NO: 254), L4 protein (SEQ ID NO: 256), or a combination of N and L4 proteins as antiviral cargo sequences, replacing the HRV 5' and 3' RR sequences of TMV with those of TMV or TCBV as provided in Table 7, to promote secondary amplification of TMV or TCBV, respectively. Solanaceae plants containing TMDV viruses (or obtaining appropriate TMDV RdRPs through transient or transgenic expression, etc.), and Solanaceae plants containing these antiviral TMDV virus satellites, are expected to exhibit resistance to Tobacco Mosaic Virus, Tomato Mosaic Virus, or Tomato Brown Curly Fruit Virus, respectively.

[0392] Table 20. Genetic elements Nucleotide positions in SEQ ID NO: 474 5' RNA replication element of Tomato dwarf virus family 1-60 TMV 5' Copy Area 61-128 PSVI IRES 129-324 Antiviral protein cargo (coding sequence of the N gene) 325-3759 TMV 3' Copy Area 3760-3963 ZmHSP101 IRES (Optional) 3964-4110 TMV MP (optional) 4111-4917 Ileu tRNA 4918-4999 3' RNA replication element of Tomato dwarf virus family 5000-5251

[0393] Example 3. Antiviral Tomato Cluster Dwarf Virus Family Satellite Carrying RNAi Cargo

[0394] The Tomato Cluster Dwarf Virus Family Satellite (COMSAT) carrying antiviral RNAi cargo is typically provided in the following 5' to 3' orientations: (i) a 5' replication element from a Tomato Cluster Dwarf Virus Family virus; (ii) an antiviral RNA molecule that induces an RNAi response; and (iii) a 3' RNA replication element from a Tomato Cluster Dwarf Virus Family virus.

[0395] COMSAT containing 5' and 3' RNA replication elements of Tomato Cluster Dwarf Virus family, which are laterally attached to cargo RNA, is provided as SEQ ID NO: 475. The cargo RNA contains a pepper light mottle virus (PMMoV) RNAi inducible sequence (e.g., a PMMoV sequence that can form dsRNA). The satellite elements are shown in Table 21.

[0396] Table 21. Genetic elements Nucleotide positions in SEQ ID NO: 475 5' RNA replication element of Tomato dwarf virus family 1-60 Antiviral RNAi cargo (PMMoV RNAi inducible sequence) 61-1038 ZmHSP101 IRES 1039-1185 TMV MP 1186-1992 Ileu tRNA 1993-2074 3' RNA replication element of Tomato dwarf virus family 2075-2326

[0397] Example 4. Antiviral Tomato Dwarf Virus Family Satellite Carrying Antiviral Proteins

[0398] The Tomato Cluster Dwarf Virus Family Satellite (COMSAT) carrying antiviral protein cargo is typically provided in the following 5' to 3' orientations: (i) a 5' replication element from a Tomato Cluster Dwarf Virus Family virus; (ii) the antiviral protein cargo; and (iii) a 3' RNA replication element from a Tomato Cluster Dwarf Virus Family virus.

[0399] COMSAT, comprising 5' and 3' RNA replication elements of the Tomato dwarf Virus family containing a side-mounted antiviral protein cargo, is provided as SEQ ID NO: 476. The antiviral protein cargo contains the coding sequence of the N gene of tobacco. The satellite's elements are shown in Table 22.

[0400] Table 22. Genetic elements Nucleotide positions in SEQ ID NO: 476 5' RNA replication element of Tomato dwarf virus family 1-60 PSVI IRES 61-256 Antiviral protein cargo (coding sequence of the N gene) 257-3691 ZmHSP101 IRES 3692-3838 TMV MP 3839-4645 Ileu tRNA 4646-4727 3' RNA replication element of Tomato dwarf virus family 4728-4979

[0401] Example 5. Determination of antiviral activity in plants containing antiviral Tomato Cluster Dwarf Virus family satellites with antiviral cargoes.

[0402] Antiviral COMSAT (e.g., Examples 1-4) or control RNA (e.g., RNA lacking antiviral cargo) is delivered to target host plants (e.g., pepper or tomato plants) via micro-bombardment (“biological projectile” delivery) using a gene gun or via bacterial-mediated (e.g., via Agrobacterium) transient expression. Host plants lacking suitable endogenous Tomato Dwarf Virus RdRP can be obtained, for example, via transient or transgenic expression. Plants (e.g., pepper and tomato plants) are grown in a growth chamber using a long photoperiod (16h light / 8h dark) with a light intensity of 100-150 μmol until seedlings are approximately 2 weeks old. Antiviral COMSAT is prepared as an in vitro transcribed (IVT) product (capped or uncapped) or as an Agrobacterium binary vector. For micro-bombardment, IVT products were coated onto the surface of gold nanoparticles (which precipitated on the inner surface of the bullet tube); these were accelerated at a helium pressure of 100 to 180 psi on the abaxial surface of seedling leaves. For Agrobacterium infiltration, clones carrying COMSAT in binary vectors were transformed into Agrobacterium GV 2260, and the transformed Agrobacterium were selected by growth at 28°C under appropriate antibiotic selection. Positive transformants were verified by PCR and then grown overnight at 28°C in liquid LB medium with appropriate antibiotics plus rifamycin to maintain Agrobacterium and prevent contamination. Agrobacterium cells were collected by centrifugation and resuspended in MMA buffer at 0.2 OD600, incubated on a shaker at 28°C for 2 hours, and then infiltrated onto the abaxial side of the leaves. Control plants were treated in the same manner, but using non-COMSAT control IVT transcripts or empty buffer. Tissues from systemic leaves (leaves distal to the treated leaves) were collected at different time points following COMSAT delivery for RNA extraction, followed by cDNA synthesis. COMSAT titers in systemic tissues were monitored by qRT-PCR using COMSAT-specific primers.

[0403] The efficacy of antiviral COMSAT can be tested by exciting COMSAT-treated plants with a target viral pathogen (e.g., cucumber mosaic virus CMV or tobacco mosaic virus TMV), for example, through mechanical infection. Infectious formulations of such acute viral pathogens are prepared in situ on plants (e.g., in *Nicotiana benthamiana*) or in binary or T7-based vectors as infectious clones with GFP fusions. Suitable viral inoculum can be prepared as infectious sap extracted from infected plants, *Agrobacterium*-based inoculum, or IVT products. The inoculum is introduced into the leaves of COMSAT-treated plants by friction inoculation (for infectious sap or IVT products), *Agrobacterium* infiltration (for infectious clones with GFP fusions in binary vectors), or micro-bombardment (for plasmids of infectious clones in IVT products or binary vectors). For friction inoculation, the infectious sap or IVT product was diluted 3-fold in 0.05 M phosphate buffer (pH 7.4) or diluted to 50 ng / µL and dropped onto the adaxial surface of leaves pre-powdered with an abrasive (such as carborundum or bentonite). The inoculum was gently spread onto the powdered leaf surface using gloved fingers or a cotton swab; after 30 seconds, the inoculated leaves were washed with water. Microbombardment of infectious clones was performed as previously described (see, for example, the delivery technique described in PCT / US22 / 78963; also see Bio-Rad Tech Note 2531, “Inoculation of Viral RNA and cDNA to Potato and Tobacco Plants Using the Helios”). TM Gene Gun [Utilizing Helios TM [Gene gun inoculates viral RNA and cDNA into potato and tobacco plants]. Inoculation of Agrobacterium-based viral pathogen inoculum is performed using a method similar to that used for COMSAT inoculation, but the Agrobacterium suspension is diluted to 0.1 OD600.

[0404] Typically, the symptoms and titers of acute viral pathogens in plants are monitored over time to confirm the progression or decline of viral infection. The effectiveness of antiviral COMSAT is evaluated by comparing the relative titers of systemically infected virus in plants treated with antiviral COMSAT with those in control plants. Tissues from inoculated leaves and systemically infected leaves (at the distal end of inoculated leaves) are collected, and total RNA is extracted, optionally followed by cDNA synthesis. Viral titers can be quantitatively measured by qRT-PCR using virus-specific primers. Viral titers or their presence can be measured by immunoassay methods; for example, using commercially available strip assays (for TMV, Agdia). Agdia, Inc. (Elkhart, Indiana, USA) routinely detects tobacco mosaic virus (TMV) in plants. Alternatively, viral titers are qualitatively assessed by viral disease symptoms or by alternative measurements (e.g., measuring GFP expressed by a viral construct fused with GFP). Typically, qRT-PCR measurements are normalized relative to endogenous reference gene controls (e.g., actin, tubulin, ubiquitin-3, GADPH, or translation elongation factor EF1a, used alone or preferably multiple, e.g., at least three reference genes or at least three δCt values). The viral titer (as a fold change relative to an endogenous reference gene) is compared to the viral titer of the control.

[0405] Example 6. Insect-inhibiting Tomato Cluster Dwarf Virus Satellites Carrying Insecticidal Proteins or RNAi Cargo

[0406] The Tomato Cluster Dwarf Virus Family Satellite (COMSAT) carrying insecticidal RNAi cargo is typically provided in the following 5' to 3' orientations: (i) a 5' replication element from a Tomato Cluster Dwarf Virus Family virus; (ii) an insecticidal RNA molecule that induces an RNAi response; and (iii) a 3' RNA replication element from a Tomato Cluster Dwarf Virus Family virus.

[0407] COMSAT, comprising 5' and 3' RNA replication elements of the Tomato Cluster Dwarf Virus family with side-attached cargo RNA, is provided as SEQ ID NO: 477. The cargo RNA contains an RNAi-inducible sequence targeting the Colorado potato beetle. The satellite's elements are shown in Table 23.

[0408] Table 23.

[0409] The Tomato Cluster Dwarf Virus Family Satellite (COMSAT) carrying insecticidal protein cargo is typically provided in the following 5' to 3' orientations: (i) a 5' replication element from a Tomato Cluster Dwarf Virus Family virus; (ii) the insecticidal protein cargo; and (iii) a 3' RNA replication element from a Tomato Cluster Dwarf Virus Family virus.

[0410] COMSAT, comprising 5' and 3' RNA replication elements of the Tomato dwarf Virus family containing a lateral insecticidal cargo protein, is provided as SEQ ID NO: 478, wherein the insecticidal cargo protein contains the coding sequence of the Vip3Aa gene. The satellite's elements are shown in Table 24.

[0411] Table 24. Genetic elements Nucleotide positions in SEQ ID NO: 478 5' RNA replication element of Tomato dwarf virus family 1-60 PSVI IRES 61-256 Insecticidal protein cargo (coding sequence of the Vip3Aa gene) 257-2626 ZmHSP101 IRES 2627-2773 TMV MP 2774-3580 Ileu tRNA 3581-3662 3' RNA replication element of Tomato dwarf virus family 3663-3914

[0412] The COMSATs in Tables 23 and 24 are established in host plants in a manner substantially as described in Example 5. Host plants lacking suitable endogenous Tomato Cluster Dwarf Virus (TCDV) viruses can obtain suitable TCDV RdRPs, for example, through transient or transgenic expression. The efficacy of insecticides or insect-suppressing COMSATs can be tested by stimulating COMSATs and control plants with target insects (e.g., Colorado potato beetle for plants treated with COMSAT in Table 23; fall armyworm, corn earworm, or black rootworm for plants treated with COMSAT in Table 24).

[0413] Example 7. Antifungal Tomato Cluster Dwarf Virus Satellite Carrying Antifungal Proteins or RNAi Cargo

[0414] The Tomato Cluster Dwarf Virus Family Satellite (COMSAT) carrying antifungal RNAi cargo is typically provided in the following 5' to 3' orientations: (i) a 5' replication element from a Tomato Cluster Dwarf Virus Family virus; (ii) an antifungal RNA molecule that induces an RNAi response; and (iii) a 3' RNA replication element from a Tomato Cluster Dwarf Virus Family virus.

[0415] COMSAT, comprising 5' and 3' RNA replication elements of the Tomato Cluster Dwarf Virus family containing side-joined cargo RNA, is provided as SEQ ID NO: 479. The cargo RNA contains an RNAi-induced sequence targeting the DCL gene of *Botrytis cinerea*. The satellite's elements are shown in Table 25.

[0416] Table 25.

[0417] The Tomato Cluster Dwarf Virus Family Satellite (COMSAT) carrying antifungal protein cargo is typically provided in the following 5' to 3' orientations: (i) a 5' replication element from a Tomato Cluster Dwarf Virus Family virus; (ii) the antifungal protein cargo; and (iii) a 3' RNA replication element from a Tomato Cluster Dwarf Virus Family virus.

[0418] COMSAT, comprising 5' and 3' RNA replication elements of the Tomato dwarf Virus family containing a side-linked antifungal cargo protein, is provided as SEQ ID NO: 480. The insecticidal cargo protein contains the coding sequence of the CaAMP1 gene. The satellite's elements are shown in Table 26.

[0419] Table 26. Genetic elements Nucleotide positions in SEQ ID NO: 480 5' RNA replication element of Tomato dwarf virus family 1-60 PSVI IRES 61-256 Antifungal cargo (coding sequence of the CaAMP1 gene) 257-817 ZmHSP101 IRES 818-964 TMV MP 965-1771 Ileu tRNA 1772-1853 3' RNA replication element of Tomato dwarf virus family 1854-2105

[0420] The COMSAT construct was built using Golden Gate assembly technology, employing materials including the synthesized cargo and other COMSAT components synthesized from a commercial supplier. The plasmid was used as a template for the PCR reaction to amplify the expression cassette containing the T7 promoter. Using this PCR product as a template, MEGAscript from Thermo Fisher Scientific was employed. TM In vitro transcription was performed using the T7 transcription kit. The in vitro synthesized RNA or protein cargo was then transformed into the leaves of the target crop using Bio-Rad gold nanoparticles, following the guidelines specified in the Helios Gene Gun manual or via Agrobacterium-mediated transformation. For host plants lacking suitable endogenous Tomato Dwarf Virus (RDV) viruses, suitable RdRPs could be obtained, for example, through transient or transgenic expression. Two weeks post-transformation, replication and persistence of the delivered RNA or protein expression in local leaves could be determined by RT-PCR on local tissues. Systemic RNA / protein movement in samples collected from distal untreated leaf samples at 1, 2, and 3 months post-transformation was determined by qRT-PCR / ELISA.

[0421] To verify the efficacy of COMSAT against fungi, in vitro and in vivo fungal bioassays were performed. In vitro assays of the antifungal agent were performed according to Duanis-Assaf et al. (Plant Biotechnol J., January 2022; 20(1): 226-237). In vivo assays of detached leaves were performed according to Li et al. (Molecular Plant Microbe Interact., December 2019; 32(12): 1649-1664). In COMSAT and control plants, disease phenotype was assessed by measuring the area of ​​lesions formed on inoculated leaves 3 and 6 days post-inoculation.

[0422] Example 8. Replication of COMSAT virus (family Tomato dwarf virus) in tomatoes

[0423] A DNA molecule encoding RNA containing the following molecules was cloned into the pUC19 plasmid back...

Claims

1. A recombinant RNA molecule comprising, from its 5' end to its 3' end: (a) A 5' RNA replication element recognized by RdRP, an RNA-dependent RNA polymerase of the Tomato dwarf Virus family; (b) Cargo RNA molecules; and (c) 3' RNA replication element recognized by the RdRP; The 5' RNA replication element, the cargo RNA molecule, and the 3' RNA replication element are operatively linked; the cargo RNA molecule is heterologous to the 5' RNA replication element and the 3' RNA replication element; and optionally, wherein: (i) the 5' RNA replication element and the 3' RNA replication element are derived from the same Tomato dwarfvirus family genome or from Tomato dwarfvirus family genomes that have at least 85%, 90%, 95%, 98%, or 99% sequence identity with each other; (ii) the 5' RNA replication element, the 3' RNA replication element, and the RdRP are derived from the same Tomato dwarfvirus family genome or from Tomato dwarfvirus family genomes that have at least 85%, 90%, 95%, 98%, or 99% sequence identity with each other; or (iii) the coding regions of the 5' RNA replication element, the 3' RNA replication element, and / or the RdRP are derived from different Tomato dwarfvirus family genomes, and the members of each corresponding set of the 5' RNA replication element, the 3' RNA replication element, and / or the RdRP coding region have at least 85%, 90%, 95%, 98%, or 99% sequence identity with each other.

2. The recombinant RNA molecule as described in claim 1, wherein: (a) The 5' RNA replication element comprises at least one RNA secondary structure provided in Table 1 or encoded by an RNA molecule of SEQ ID NO: 467; and / or (b) The 3' RNA replication element comprises at least one RNA secondary structure provided in Table 1 or encoded by an RNA molecule of SEQ ID NO:

468.

3. The recombinant RNA molecule as described in claim 1, wherein: (a) The 5' RNA replication element comprises an RNA molecule encoded by SEQ ID NO: 467; a variant thereof encoded by a DNA molecule having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO: 467; or a variant thereof wherein one or more nucleotides in the RNA secondary structure are replaced by different nucleotides that maintain the RNA secondary structure; and / or (b) The 3' RNA replication element comprises an RNA molecule encoded by SEQ ID NO: 468, or a variant thereof encoded by a DNA molecule having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO: 468; or a variant thereof wherein one or more base-paired residues in the RNA secondary structure are replaced by different nucleotides that maintain the RNA secondary structure.

4. The recombinant RNA molecule of claim 3, wherein the RNA secondary structure is maintained by replacing unpaired nucleotides in the secondary structure with nucleotides that will not pair bases, and / or replacing paired nucleotides in the secondary structure with nucleotides that will pair bases.

5. The recombinant RNA molecule as described in claim 1, wherein: (a) The 5' RNA replication element comprises at least one segment of the 5' untranslated region (UTR) of the Tomato dwarf Virus family genome or a variant thereof having one or more nucleotide substitutions, insertions, and / or deletions, wherein the variant is recognized by the RdRP, and optionally wherein the 5' RNA replication element further comprises a genomic sequence of the Tomato dwarf Virus family virus naturally located at and adjacent to the 3' of the 5' UTR sequence; and / or (b) The 3' RNA replication element comprises at least one segment of the 3' UTR of the Tomato dwarf Virus family genome or a variant thereof having one or more nucleotide substitutions, insertions and / or deletions, wherein the variant is recognized by the RdRP, and optionally wherein the 3' RNA replication element further comprises a genome sequence of the Tomato dwarf Virus family virus naturally located at the 5' and adjacent to the 3' UTR sequence, and optionally wherein the Tomato dwarf Virus family genomes of (a) and (b) are identical.

6. The recombinant RNA molecule of claim 1, wherein the RNA molecule further comprises at least one of the following: (i) a tRNA-like element, optionally wherein the tRNA-like element provides intercellular movement of the RNA, and optionally wherein the intercellular movement is mediated by a viral mobility protein (MP); (ii) a capsidation recognition element (ERE), optionally wherein the ERE provides capsidation of the RNA via a Tomato dwarf Virus capsid protein, or optionally wherein the ERE provides capsidation of the RNA via a non-Tomato dwarf Virus capsid protein; (iii) an RNA effector; and / or (iv) RNA encoding a viral mobility protein (MP), optionally wherein an internal ribosome entry site (IRES) is operatively linked to the MP-encoding RNA.

7. The recombinant RNA molecule of claim 6, wherein the tRNA-like element comprises a tRNA-like molecule derived from Arabidopsis thaliana FT mRNA, or a tRNA-like sequence encoded by a DNA sequence selected from the group consisting of SEQ ID NO: 76-123 and 466, or a modified tRNA-like sequence having at least 90% sequence identity with a scaffold tRNA-like sequence encoded by a DNA sequence selected from the group consisting of SEQ ID NO: 76-123 and 466 and maintaining the secondary structure of the scaffold tRNA-like sequence, and / or wherein the ERE is tobacco mosaic virus (TMV) OAS.

8. The recombinant RNA molecule of claim 1, wherein the length of the cargo RNA molecule is up to about 6 kb.

9. The recombinant RNA molecule of claim 1, wherein the cargo RNA molecule comprises: (a) at least one coding sequence, optionally wherein the coding sequence encodes a selectable or scoreable marker; (b) at least one non-coding sequence; or (c) both at least one coding sequence and at least one non-coding sequence.

10. The recombinant RNA molecule of claim 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) operatively linked to the at least one coding sequence, optionally wherein the operatively linked IRES is located at the 5' of and immediately adjacent to the coding sequence.

11. The recombinant RNA molecule of claim 1, wherein the cargo RNA molecule comprises at least one non-coding sequence, and wherein the at least one non-coding sequence is a hairpin RNA (hpRNA); RNA forming multiple stem loops; RNA pseudoknots; RNA molecule forming at least partially double-stranded RNA; small interfering RNA (siRNA) or siRNA precursor; microRNA (miRNA) or miRNA precursor; staged RNA or staged RNA precursor; ribozyme; ligand-responsive ribozyme (aptase); RNA aptamer; or long non-coding RNA (lncRNA).

12. The recombinant RNA molecule of claim 1, further comprising RNA encoding at least one ribozyme, optionally wherein the at least one ribozyme is located at the 5' of the 5' RNA replication element or the 3' of the 3' RNA replication element.

13. The recombinant RNA molecule of claim 1, further comprising an RNA molecule containing at least one ligand-responsive ribozyme (aptamer), optionally wherein the at least one ligand-responsive ribozyme is located at the 5' of the 5' RNA replication element or the 3' of the 3' RNA replication element.

14. The recombinant RNA molecule of claim 1, wherein: (i) the RNA further comprises at least one segment of its reverse complementary RNA molecule; and / or (ii) the recombinant RNA molecule is complexed with one or more RNA-binding proteins or capsidated by a viral capsid protein, optionally wherein the viral capsid protein is a Tomato dwarf Virus capsid protein, optionally wherein the RNA comprises an ERE that provides capsidation of the RNA by the Tomato dwarf Virus capsid protein, and / or optionally wherein the RNA-binding protein comprises an RNA recognition motif.

15. The recombinant RNA molecule of claim 1, wherein the RNA molecule comprises: At least one heterologous RNA virus (HRV) amplicon, which is positively or negatively oriented relative to the first 5' RNA replication element, contains: I. (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 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 operatively linked; or II. A heterologous RNA virus (HRV) subgenome promoter operatively linked to the cargo RNA molecule; wherein the subgenome promoter is recognized by the heterologous RNA virus RNA-dependent RNA polymerase (hrvRdRP).

16. The recombinant RNA of claim 15, wherein the RNA molecule comprises, from the 5' end to the 3' end: (a) The 5' RNA replication element; (b) The HRV amplicon oriented antisense relative to the first 5' RNA replication element; optionally, the HRV amplicon further comprises: (i) an RNA molecule encoding an HRV RNA-dependent RNA polymerase (hrvRdRP) 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 HRV RNA-dependent RNA polymerase (hrvRdRP) operably linked to a subgenomic promoter recognized by said hrvRdRP; and (c) The 3' RNA replication element.

17. The recombinant RNA of claim 15, wherein the HRV 5'RR, HRV 3'RR, and hrvRdRP comprise HRV 5'RR, HRV 3'RR, and hrvRdRP from viruses selected from the group consisting of: *Hypervira*, *Hypervira*, *Brassica napus*, *Celavirus*, *Leptovirus*, *Cowpea Mosaic Virus*, *Potato Virus X*, *Potato Virus Y*, *Tobacco Mosaic Virus*, *Tomato Cluster Dwarf Virus*, *Tomato Spotted Wilt Virus Family*, *Trigeneae* subfamily, *Turnip Yellow Mosaic Virus*, *Vein Varicose Vein Virus*, and *Associated Cowpea Mosaic Virus Family*.

18. The recombinant RNA of claim 17, wherein: (i) The genus *Bombyx mottle virus* refers to cucumber mosaic virus, spinach latent virus, olive latent virus, or *Bombyx mottle virus*; (ii) The genus *Lycovirus* refers to citrus senescence virus or beet yellowing virus; (iii) The genus *Cowpea mosaic virus* refers to cowpea mosaic virus, apple latent spherical virus, or soybean latent spherical virus; (iv) The genus *Potato X virus* refers to potato X virus or citrus yellowing virus; (v) The genus *Potato Y virus* refers to pepper mottle virus, bean yellow mosaic virus, barley stripe mosaic virus, wheat stripe mosaic virus, rice yellow mottle virus, and maize dwarf virus. (vi) The Tobacco Mosaic Virus genus is Tobacco Mosaic Virus, Tomato Mosaic Virus, Tomato Brown Wrinkled Fruit Virus, Turnip Vein Virus, or Pepper Mild Mottle Virus; (vii) The Tomato Cluster Dwarf Virus genus is Turnip Wrinkled Virus or Tomato Cluster Dwarf Virus; (iv) The Tomato Spotted Wilt Virus Family is Tomato Spotted Wilt Virus or Watermelon Bud Necrosis Virus; or (viii) The Turnip Yellow Mosaic Virus genus is Turnip Yellow Mosaic Virus, Citrus Yellowing Vein Virus, Potato Latent Virus, Apple Stem Groove Virus, or Citrus Leaf Mottle Virus.

19. The recombinant RNA of claim 15, wherein the HRV 5'RR and the HRV 3'RR are derived from the same HRV genome.

20. The recombinant RNA of claim 15, wherein the HRV 5'RR and the HRV 3'RR are derived from different HRV genomes.

21. The recombinant RNA molecule of claim 15, wherein: (a) The HRV 5'RR comprises at least one RNA secondary structure using RNA encoded by SEQ ID NO: 161 to 185 or 186, or comprises an RNA sequence encoded by SEQ ID NO: 161 to 185 or 186, or comprises at least 80%, 85%, 90%, or 95% of a continuous fragment of the complete sequence of 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 using RNA encoded by SEQ ID NO: 187 to 210 or 211, or comprises an RNA sequence encoded by SEQ ID NO: 187 to 210 or 211, or comprises at least 80%, 85%, 90% or 95% of the complete sequence of RNA encoded by SEQ ID NO: 187 to 210 or 211.

22. The recombinant RNA molecule of claim 15, wherein: (a) The HRV 5'RR is encoded by: a DNA molecule comprising SEQ ID NO: 161 to 185 or 186; or a variant thereof comprising DNA having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO: 161 to 185 or 186; or a variant thereof wherein one or more residues in the RNA secondary structure are replaced by different nucleotides that maintain the RNA secondary structure; and / or (b) The HRV 3'RR is encoded by: a DNA molecule comprising SEQ ID NO: 187 to 210 or 211; or a variant thereof comprising DNA having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO: 187 to 210 or 211; or a variant thereof wherein one or more residues in the RNA secondary structure are replaced by different nucleotides that maintain the RNA secondary structure.

23. The recombinant RNA molecule of claim 15, wherein: (a) The HRV 3'RR comprises at least one segment of the 3' untranslated region (UTR) of the HRV genome or an RNA molecule having one or more nucleotide substitutions, insertions and / or deletions thereof, wherein the HRV 3'RR or the variant is recognized by the hrvRdRP, and optionally the RNA comprising the HRV 3'RR further comprises the genomic sequence of the HRV naturally located at and adjacent to the 5' of the 3'UTR sequence; And / or (d) The HRV 5'RR comprises at least one segment of the 3' untranslated region (UTR) of the HRV genome or an RNA molecule having one or more nucleotide substitutions, insertions and / or deletions thereof, wherein the HRV 5'RR or the variant is recognized by the hrvRdRP, and optionally the RNA comprising the HRV 5'RR further comprises the genomic sequence of the HRV naturally located at the 3' of the 5'UTR sequence and adjacent to it.

24. The recombinant RNA molecule of claim 15, wherein the cargo RNA comprises HRV repressive RNA or encodes an HRV repressive protein, wherein the HRV repressive RNA or HRV repressive protein inhibits the infection, movement, spread and / or replication of the HRV.

25. The recombinant RNA molecule of claim 15, wherein the cargo RNA comprises RNA having at least 20 consecutive nucleotides having a sequence that is identical or complementary to an isolength segment in the genomic RNA of the HRV.

26. The recombinant RNA molecule of claim 15, wherein the cargo RNA comprises RNA having at least 20 consecutive nucleotides having a sequence that is identical or complementary to an isometric segment in the genomic RNA of the HRV that does not encode the hrvRdRP.

27. The recombinant RNA molecule of claim 15, wherein: (i) The HRV is cucumber mosaic virus, and the HRV 5'RR contains RNA encoded by the cucumber mosaic virus 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95% or 99% sequence identity with the cucumber mosaic virus 5'RR DNA sequence in Table 7, and the HRV 3'RR contains RNA encoded by the cucumber mosaic virus 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95% or 99% sequence identity with the cucumber mosaic virus 3'RR DNA sequence in Table 7; (ii) The HRV is bromeliad mosaic virus, and the HRV 5'RR contains RNA encoded by the bromeliad mosaic virus 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the bromeliad mosaic virus 5'RR DNA sequence in Table 7, and the HRV 3'RR contains RNA encoded by the bromeliad mosaic virus 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the bromeliad mosaic virus 3'RR DNA sequence in Table 7; (iii) The HRV is a citrus degeneration virus, and the HRV 5'RR contains RNA encoded by the citrus degeneration virus 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the citrus degeneration virus 5'RR DNA sequence in Table 7; the HRV 3'RR contains RNA encoded by the citrus degeneration virus 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the citrus degeneration virus 3'RR DNA sequence in Table 7; and the RdRP contains a protein having at least 85%, 90%, 95%, or 99% sequence identity with the citrus degeneration virus RdRP sequence in Table 7. (iv) The HRV is beet yellow virus, and the HRV 5'RR contains RNA encoded by the beet yellow virus 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the beet yellow virus 5'RR DNA sequence in Table 7; the HRV 3'RR contains RNA encoded by the beet yellow virus 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the beet yellow virus 3'RR DNA sequence in Table 7; and the RdRP contains a protein having at least 85%, 90%, 95%, or 99% sequence identity with the beet yellow virus RdRP sequence in Table 7. (v) The HRV is cowpea mosaic virus, and the HRV 5'RR contains RNA encoded by the cowpea mosaic virus 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95% or 99% sequence identity with the cowpea mosaic virus 5'RR DNA sequence in Table 7. (vi) The HRV is Potato Virus X, and the HRV 5'RR contains RNA encoded by the Potato Virus X 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the Potato Virus X 5'RR DNA sequence in Table 7; the HRV 3'RR contains RNA encoded by the Potato Virus X 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the Potato Virus X 3'RR DNA sequence in Table 7; and the RdRP contains a protein having at least 85%, 90%, 95%, or 99% sequence identity with the Potato Virus X RdRP sequence in Table 7. (vii) The HRV is a pepper mottle virus, and the HRV 5'RR contains RNA encoded by the pepper mottle virus 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the pepper mottle virus 5'RR DNA sequence in Table 7; the HRV 3'RR contains RNA encoded by the pepper mottle virus 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the pepper mottle virus 3'RR DNA sequence in Table 7; and the RdRP contains a protein having at least 85%, 90%, 95%, or 99% sequence identity with the pepper mottle virus RdRP sequence in Table 7. (viii) The HRV is soybean yellow mosaic virus, and the HRV 5'RR contains RNA encoded by the soybean yellow mosaic virus 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the soybean yellow mosaic virus 5'RR DNA sequence in Table 7; the HRV 3'RR contains RNA encoded by the soybean yellow mosaic virus 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the soybean yellow mosaic virus 3'RR DNA sequence in Table 7; and the RdRP contains a protein having at least 85%, 90%, 95%, or 99% sequence identity with the soybean yellow mosaic virus RdRP sequence in Table 7. (ix) The HRV is barley stripe mosaic virus, and the HRV 5'RR contains RNA encoded by the barley stripe mosaic virus 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the barley stripe mosaic virus 5'RR DNA sequence in Table 7; the HRV 3'RR contains RNA encoded by the barley stripe mosaic virus 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the barley stripe mosaic virus 3'RR DNA sequence in Table 7. (x) The HRV is wheat stripe mosaic virus, and the HRV 5'RR contains RNA encoded by the wheat stripe mosaic virus 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the wheat stripe mosaic virus 5'RR DNA sequence in Table 7; the HRV 3'RR contains RNA encoded by the wheat stripe mosaic virus 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the wheat stripe mosaic virus 3'RR DNA sequence in Table 7; and the RdRP contains a protein having at least 85%, 90%, 95%, or 99% sequence identity with the wheat stripe mosaic virus RdRP sequence in Table 7. (xi) The HRV is rice yellow mottle virus, and the HRV 5'RR contains RNA encoded by the rice yellow mottle virus 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the rice yellow mottle virus 5'RR DNA sequence in Table 7; the HRV 3'RR contains RNA encoded by the rice yellow mottle virus 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the rice yellow mottle virus 3'RR DNA sequence in Table 7; and the RdRP contains a protein having at least 85%, 90%, 95%, or 99% sequence identity with the rice yellow mottle virus RdRP sequence in Table 7. (xii) The HRV is maize dwarf mosaic virus, and the HRV 5'RR contains RNA encoded by the maize dwarf mosaic virus 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the maize dwarf mosaic virus 5'RR DNA sequence in Table 7; the HRV 3'RR contains RNA encoded by the maize dwarf mosaic virus 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the maize dwarf mosaic virus 3'RR DNA sequence in Table 7; and the RdRP contains a protein having at least 85%, 90%, 95%, or 99% sequence identity with the maize dwarf mosaic virus RdRP sequence in Table 7. (xiii) The HRV is the zucchini yellow mosaic virus, and the HRV 5'RR contains RNA encoded by the zucchini yellow mosaic virus 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the zucchini yellow mosaic virus 5'RR DNA sequence in Table 7; the HRV 3'RR contains RNA encoded by the zucchini yellow mosaic virus 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the zucchini yellow mosaic virus 3'RR DNA sequence in Table 7; and the RdRP contains a protein having at least 85%, 90%, 95%, or 99% sequence identity with the zucchini yellow mosaic virus RdRP sequence in Table 7. (xiv) The HRV is watermelon mosaic virus, and the HRV 5'RR contains RNA encoded by the watermelon mosaic virus 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the watermelon mosaic virus 5'RR DNA sequence in Table 7; the HRV 3'RR contains RNA encoded by the watermelon mosaic virus 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the watermelon mosaic virus 3'RR DNA sequence in Table 7; and the RdRP contains a protein having at least 85%, 90%, 95%, or 99% sequence identity with the watermelon mosaic virus RdRP sequence in Table 7. (xv) The HRV is sugarcane mosaic virus, and the HRV 5'RR contains RNA encoded by the sugarcane mosaic virus 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the sugarcane mosaic virus 5'RR DNA sequence in Table 7; the HRV 3'RR contains RNA encoded by the sugarcane mosaic virus 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the sugarcane mosaic virus 3'RR DNA sequence in Table 7; and the RdRP contains a protein having at least 85%, 90%, 95%, or 99% sequence identity with the sugarcane mosaic virus RdRP sequence in Table 7. (xvi) The HRV is tobacco mosaic virus, and the HRV 5'RR contains RNA encoded by the tobacco mosaic virus 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the tobacco mosaic virus 5'RR DNA sequence in Table 7; the HRV 3'RR contains RNA encoded by the tobacco mosaic virus 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the tobacco mosaic virus 3'RR DNA sequence in Table 7; and the RdRP contains a protein having at least 85%, 90%, 95%, or 99% sequence identity with the tobacco mosaic virus RdRP sequence in Table 7. (xvii) The HRV is tomato mosaic virus, and the HRV 5'RR contains RNA encoded by the tomato mosaic virus 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the tomato mosaic virus 5'RR DNA sequence in Table 7; the HRV 3'RR contains RNA encoded by the tomato mosaic virus 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the tomato mosaic virus 3'RR DNA sequence in Table 7; and the RdRP contains a protein having at least 85%, 90%, 95%, or 99% sequence identity with the tomato mosaic virus RdRP sequence in Table 7. (xviii) The HRV is Tomato Brown Wrinkled Fruit Virus, and the HRV 5'RR contains RNA encoded by the Tomato Brown Wrinkled Fruit Virus 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the Tomato Brown Wrinkled Fruit Virus 5'RR DNA sequence in Table 7; the HRV 3'RR contains RNA encoded by the Tomato Brown Wrinkled Fruit Virus 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the Tomato Brown Wrinkled Fruit Virus 3'RR DNA sequence in Table 7; and the RdRP contains a protein having at least 85%, 90%, 95%, or 99% sequence identity with the Tomato Brown Wrinkled Fruit Virus RdRP sequence in Table 7. (xix) The HRV is var. turnip virus, and the HRV 5'RR contains RNA encoded by the var. turnip virus 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the var. turnip virus 5'RR DNA sequence in Table 7; the HRV 3'RR contains RNA encoded by the var. turnip virus 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the var. turnip virus 3'RR DNA sequence in Table 7; and the RdRP contains a protein having at least 85%, 90%, 95%, or 99% sequence identity with the var. turnip virus RdRP sequence in Table 7. (xx) The HRV is a mild mottle virus of pepper, and the HRV 5'RR contains RNA encoded by the 5'RR DNA sequence of the mild mottle virus of pepper in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95% or 99% sequence identity with the 5'RR DNA sequence of the mild mottle virus of pepper in Table 7; the HRV 3'RR contains RNA encoded by the 3'RR DNA sequence of the mild mottle virus of pepper in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95% or 99% sequence identity with the 3'RR DNA sequence of the mild mottle virus of pepper in Table 7; and the RdRP contains a protein having at least 85%, 90%, 95% or 99% sequence identity with the RdRP sequence of the mild mottle virus of pepper in Table 7. (xxi) The HRV is turnip shrub virus, and the HRV 5'RR contains RNA encoded by the turnip shrub virus 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the turnip shrub virus 5'RR DNA sequence in Table 7; the HRV 3'RR contains RNA encoded by the turnip shrub virus 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the turnip shrub virus 3'RR DNA sequence in Table 7; and the RdRP contains a protein having at least 85%, 90%, 95%, or 99% sequence identity with the turnip shrub virus RdRP sequence in Table 7. (xxii) The HRV is Tomato dwarf virus, and the HRV 5'RR contains RNA encoded by the Tomato dwarf virus 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the Tomato dwarf virus 5'RR DNA sequence in Table 7; the HRV 3'RR contains RNA encoded by the Tomato dwarf virus 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the Tomato dwarf virus 3'RR DNA sequence in Table 7; and the RdRP contains a protein having at least 85%, 90%, 95%, or 99% sequence identity with the Tomato dwarf virus RdRP sequence in Table 7. (xxiii) The HRV is tomato spotted wilt virus, and the HRV 5'RR contains RNA encoded by the tomato spotted wilt virus 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the tomato spotted wilt virus 5'RR DNA sequence in Table 7; the HRV 3'RR contains RNA encoded by the tomato spotted wilt virus 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the tomato spotted wilt virus 3'RR DNA sequence in Table 7; and the RdRP contains a protein having at least 85%, 90%, 95%, or 99% sequence identity with the tomato spotted wilt virus RdRP sequence in Table 7. (xxiv) The HRV is watermelon bud necrosis virus, and the HRV 5'RR contains RNA encoded by the watermelon bud necrosis virus 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the watermelon bud necrosis virus 5'RR DNA sequence in Table 7; the HRV 3'RR contains RNA encoded by the watermelon bud necrosis virus 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the watermelon bud necrosis virus 3'RR DNA sequence in Table 7; and the RdRP contains a protein having at least 85%, 90%, 95%, or 99% sequence identity with the watermelon bud necrosis virus RdRP sequence in Table 7. (xxv) The HRV is turnip yellow mosaic virus, and the HRV 5'RR contains RNA encoded by the turnip yellow mosaic virus 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the turnip yellow mosaic virus 5'RR DNA sequence in Table 7; the HRV 3'RR contains RNA encoded by the turnip yellow mosaic virus 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the turnip yellow mosaic virus 3'RR DNA sequence in Table 7; and the RdRP contains a protein having at least 85%, 90%, 95%, or 99% sequence identity with the turnip yellow mosaic virus RdRP sequence in Table 7. (xxvi) The HRV is spinach latent virus, and the HRV 5'RR contains RNA encoded by the spinach latent virus 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the spinach latent virus 5'RR DNA sequence in Table 7; the HRV 3'RR contains RNA encoded by the spinach latent virus 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the spinach latent virus 3'RR DNA sequence in Table 7; and the RdRP contains a protein having at least 85%, 90%, 95%, or 99% sequence identity with the spinach latent virus RdRP sequence of SEQ ID NO:

510. (xxvii) The HRV is spinach latent virus, and the HRV 5'RR contains RNA encoded by the spinach latent virus 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the spinach latent virus 5'RR DNA sequence in Table 7; the HRV 3'RR contains RNA encoded by the spinach latent virus 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the spinach latent virus 3'RR DNA sequence in Table 7; and the RdRP contains a protein having at least 85%, 90%, 95%, or 99% sequence identity with the spinach latent virus RdRP sequence of SEQ ID NO:

511. (xxviii) The HRV is olive latent virus 2, and the HRV 5'RR contains RNA encoded by the olive latent virus 2 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the olive latent virus 2 5'RR DNA sequence in Table 7; the HRV 3'RR contains RNA encoded by the olive latent virus 2 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the olive latent virus 2 3'RR DNA sequence in Table 7; and the RdRP contains a protein having at least 85%, 90%, 95%, or 99% sequence identity with the olive latent virus 2 RdRP sequence in Table 7. (xxix) The HRV is Citrus yellowing virus, and the HRV 5'RR contains RNA encoded by the Citrus yellowing virus 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the Citrus yellowing virus 5'RR DNA sequence in Table 7; the HRV 3'RR contains RNA encoded by the Citrus yellowing virus 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the Citrus yellowing virus 3'RR DNA sequence in Table 7; and the RdRP contains a protein having at least 85%, 90%, 95%, or 99% sequence identity with the Citrus yellowing virus RdRP sequence in Table 7. (xxx) The HRV is a potato latent virus, and the HRV 5'RR contains RNA encoded by the potato latent virus 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the potato latent virus 5'RR DNA sequence in Table 7; the HRV 3'RR contains RNA encoded by the potato latent virus 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the potato latent virus 3'RR DNA sequence in Table 7; and the RdRP contains a protein having at least 85%, 90%, 95%, or 99% sequence identity with the potato latent virus RdRP sequence in Table 7. (xxxi) The HRV is apple stem grooving virus, and the HRV 5'RR contains RNA encoded by the apple stem grooving virus 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the apple stem grooving virus 5'RR DNA sequence in Table 7; the HRV 3'RR contains RNA encoded by the apple stem grooving virus 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the apple stem grooving virus 3'RR DNA sequence in Table 7; and the RdRP contains a protein having at least 85%, 90%, 95%, or 99% sequence identity with the apple stem grooving virus RdRP sequence in Table 7. (xxxii) The HRV is a citrus leaf mottle virus, and the HRV 5'RR contains RNA encoded by the citrus leaf mottle virus 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the citrus leaf mottle virus 5'RR DNA sequence in Table 7; the HRV 3'RR contains RNA encoded by the citrus leaf mottle virus 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the citrus leaf mottle virus 3'RR DNA sequence in Table 7; and the RdRP contains a protein having at least 85%, 90%, 95%, or 99% sequence identity with the citrus leaf mottle virus RdRP sequence in Table 7. (xxxiii) The HRV is apple latent spheroid virus, and the HRV 5'RR contains RNA encoded by the apple latent spheroid virus 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the apple latent spheroid virus 5'RR DNA sequence in Table 7; the HRV 3'RR contains RNA encoded by the apple latent spheroid virus 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the apple latent spheroid virus 3'RR DNA sequence in Table 7; and the RdRP contains a protein having at least 85%, 90%, 95%, or 99% sequence identity with the apple latent spheroid virus RdRP sequence in Table 7. (xxxiv) The HRV is soybean latent spheroid virus, and the HRV 5'RR contains RNA encoded by the soybean latent spheroid virus 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the soybean latent spheroid virus 5'RR DNA sequence in Table 7; the HRV 3'RR contains RNA encoded by the soybean latent spheroid virus 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the soybean latent spheroid virus 3'RR DNA sequence in Table 7; and the RdRP contains a protein having at least 85%, 90%, 95%, or 99% sequence identity with the soybean latent spheroid virus RdRP sequence in Table 7. (xxxv) The HRV is celery latent virus, and the HRV 5'RR contains RNA encoded by the celery latent virus 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the celery latent virus 5'RR DNA sequence in Table 7; the HRV 3'RR contains RNA encoded by the celery latent virus 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the celery latent virus 3'RR DNA sequence in Table 7; and the RdRP contains a protein having at least 85%, 90%, 95%, or 99% sequence identity with the celery latent virus RdRP sequence in Table 7. (xxxvi) The HRV is a black grass varicose vein virus-like virus, and the HRV 5'RR contains RNA encoded by the black grass varicose vein virus-like virus 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the black grass varicose vein virus-like virus 5'RR DNA sequence in Table 7; the HRV 3'RR contains RNA encoded by the black grass varicose vein virus-like virus 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the black grass varicose vein virus-like virus 3'RR DNA sequence in Table 7; and the RdRP contains a protein having at least 85%, 90%, 95%, or 99% sequence identity with the black grass varicose vein virus-like virus sequence in Table 7; or (xxxvii) The HRV is a maize suscal virus, and the HRV 5'RR contains RNA encoded by the maize suscal virus 5'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the maize suscal virus 5'RR DNA sequence in Table 7; the HRV 3'RR contains RNA encoded by the maize suscal virus 3'RR DNA sequence in Table 7, or RNA encoded by DNA having at least 85%, 90%, 95%, or 99% sequence identity with the maize suscal virus 3'RR DNA sequence in Table 7; and the RdRP contains a protein having at least 85%, 90%, 95%, or 99% sequence identity with the maize suscal virus sequence in Table 7.

28. The recombinant RNA molecule of claim 15, further comprising RNA encoding at least one cleavable sequence, optionally wherein the at least one cleavable sequence is located at: (i) the 5' end of the 5' RNA replication element or the 3' end of 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, wherein the cleavable sequence is optionally a self-cleaving ribozyme, a self-cleaving inducible ribozyme, or a siRNA or miRNA recognition site.

29. An agricultural formulation comprising a recombinant RNA molecule as described in any one of claims 1 to 28.

30. The agricultural formulation of claim 29, wherein the recombinant RNA molecule is complexed with one or more RNA-binding proteins or capsidated by a viral capsid protein, optionally wherein the viral capsid protein is a Tomato dwarf Virus capsid protein, and optionally wherein the RNA comprises an ERE that provides capsidation of the RNA by the Tomato dwarf Virus capsid protein.

31. The agricultural formulation of claim 30, wherein the RNA-binding protein comprises an RNA recognition motif.

32. The agricultural formulation of claim 30, wherein the viral capsid protein is heterologous to the Tomato Cluster Dwarf Virus family.

33. The agricultural formulation of claim 29, wherein the formulation comprises the recombinant RNA molecule as well as a carrier, excipient and / or adjuvant.

34. A cell comprising a recombinant RNA molecule as claimed in any one of claims 1 to 28, wherein the cell is a bacterial cell, fungal cell, plant cell, insect cell, or invertebrate cell.

35. The cell of claim 34, wherein the cell is a plant cell and the cell does not contain DNA encoding the recombinant RNA molecule.

36. The cell of claim 34, wherein the cell comprises a recombinant DNA molecule encoding the recombinant RNA molecule.

37. An expression system comprising: (a) An RNA molecule containing the recombinant RNA molecule as described in any one of claims 1 to 28; and (b) Cells containing the recombinant RNA molecule and the RdRP protein that recognizes the 5' and 3' RNA replication elements of the recombinant RNA molecule.

38. The expression system of claim 37, wherein the recombinant RNA molecule comprises an operatively linked capsidation recognition element (ERE) recognized by a viral capsid protein, and wherein the cell contains the viral capsid protein.

39. The expression system of claim 37, wherein the capsid recognition element (ERE) is a Tomato dwarf virus family ERE, wherein the viral capsid protein in the cell is a Tomato dwarf virus family capsid protein, and wherein the RNA molecule is capsidated by the Tomato dwarf virus family capsid protein.

40. The expression system of claim 37, further comprising the reverse complementary sequence of the recombinant RNA molecule.

41. The expression system of claim 37, wherein the cell is a bacterial cell, plant cell, fungal cell, insect cell, or invertebrate cell.

42. The expression system of claim 37, wherein the cell further comprises: (i) a viral capsid protein (CP); (ii) an RNA-binding protein (RBP) capable of binding to the RNA molecule, optionally wherein the RBP binds to an RNA effector; (iii) an RNA cleaving agent for cleaving the RNA molecule; (iv) a second RNA-dependent RNA polymerase (RdRP) protein (second RdRP) that recognizes the HRV 5' or 3' replication region and / or the subgenomic promoter in the RNA molecule; (v) a viral mobile protein (MP); (v) a heterologous RNA virus (HRV); or (vi) hrvRdRP, optionally wherein the hrvRdRP recognizes the HRV 5' or 3' replication region and / or the subgenomic promoter.

43. The expression system of claim 42, wherein the CP, RBP, RdRP, second RdRP, hrvRdRP and / or the MP: (a) are expressed by a recombinant DNA molecule in the cell; (b) are exogenously provided to the cell; (c) are expressed by a recombinant RNA molecule in the cell; or (d) are expressed by a virus in the cell.

44. The expression system of claim 42, wherein the RdRP, CP, RBP, second RdRP, hrvRdRP and / or the MP protein are heterologous to the cell.

45. The expression system of claim 42, wherein the RdRP, second RdRP, or hrvRdRP protein, or the polynucleotide encoding the RdRP, second RdRP, or hrvRdRP protein (a) is expressed by a recombinant DNA molecule in the cell; (b) is exogenously provided to the cell; (c) is expressed by a recombinant RNA molecule in the cell; or (d) is expressed by a virus in the cell.

46. ​​The expression system of claim 37, wherein the cell is a plant cell.

47. The expression system of claim 46, wherein the plant cell contains a Tomato dwarf virus expressing the RdRP protein, the RdRP protein recognizing the 5' RNA replication element and the 3' RNA replication element, and / or wherein the plant cell contains an HRV expressing the second RdRP or hrvRdRP protein.

48. The expression system of claim 47, wherein the Tomato Cluster Dwarf Virus is naturally present in the plant cells.

49. A method for providing a plant with synthetic Tomato Cluster Dwarf Virus family satellite RNA, the method comprising contacting the plant with a recombinant RNA molecule as claimed in any one of claims 1 to 28.

50. The method of claim 49, wherein contact comprises spraying, dusting, injecting, or soaking the plant or a portion thereof with the recombinant RNA molecule or the preparation.

51. The method of claim 49, further comprising providing the plant with hrvRdRP that identifies the HRV 5' or 3' replication region and / or the subgenomic promoter, optionally wherein the hrvRdRP is provided by introducing recombinant DNA or RNA encoding the hrvRdRP into the plant or a portion thereof.

52. A method for constructing synthetic satellite RNA of the Tomato Cluster Dwarf Virus family in plant cells, the method comprising: Provide plant cells with the recombinant RNA molecule as described in any one of claims 1 to 28; The plant cells described herein contain RdRP proteins that recognize the 5' and 3' RNA replication elements, wherein the RNA molecules optionally contain EREs and are capsidated by capsid proteins. Therefore, the RdRP protein catalyzes the synthesis of the synthetic Tomato Cluster Dwarf Virus family satellite RNA from the recombinant RNA molecule.

53. The method of claim 52, wherein the plant cell comprises a Tomato Cluster Dwarf Virus, and wherein the RdRP protein is provided to the plant cell via the Tomato Cluster Dwarf Virus.

54. The method of claim 52, wherein the Tomato Cluster Dwarf Virus is specific to the plant cell, and optionally wherein the plant cell-specific Tomato Cluster Dwarf Virus is non-pathogenic and / or symbiotic.

55. The method of claim 52, wherein the capsid protein comprises a Tomato dwarf virus family capsid protein, and the ERE is recognized by the Tomato dwarf virus family capsid protein, optionally wherein the recombinant RNA molecule comprises an operatively linked capsid recognition element (ERE) recognized by the viral capsid protein.

56. The method of claim 52, further comprising providing the plant with hrvRdRP that recognizes the HRV 5' or 3' replication region and / or the subgenomic promoter in the synthetic Tomato dwarf Virus satellite RNA, optionally wherein the hrvRdRP is provided by introducing recombinant DNA or RNA encoding the hrvRdRP into the plant or a portion thereof.

57. A method for obtaining phenotypic changes in plants or plant cells, the method comprising: Provide a plant or plant cell with a recombinant RNA molecule as described in any one of claims 1 to 28, wherein the cargo RNA molecule comprises RNA that achieves a phenotypic change in the plant or plant cell compared 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 the 3' RNA replication element and catalyzes the synthesis of synthetic Tomato dwarf Virus RNA from the recombinant RNA molecule, and wherein the cargo RNA molecule achieves the phenotypic change.

58. The method of claim 57, wherein the RNA that achieves the phenotypic change in the plant or plant cell comprises at least one RNA selected from siRNA or siRNA precursor, rniRNA or miRNA precursor, and staged siRNA or staged siRNA precursor.

59. The method of claim 57, wherein the RNA that achieves the phenotypic change in the plant or plant cell comprises messenger RNA.

60. The method of claim 59, wherein the messenger RNA comprises an RNA molecule not present in the genome of the plant or plant cell.

61. The method of claim 57, wherein the RNA that achieves the phenotypic change of the plant or plant cell comprises RNA for modifying the genome of the plant or plant cell.

62. The method of claim 57, wherein the RNA that achieves the phenotypic change in the plant or plant cell comprises RNA for modifying the transcriptome and / or epigenome of the plant or plant cell, optionally wherein the RNA for modifying the epigenome targets an endogenous plant gene for RNA-induced transcriptional silencing.

63. The method of claim 57, wherein the phenotypic change includes increased resistance of the plant to pests or pathogens, optionally wherein the pests or pathogens are selected from the group consisting of bacteria, viruses other than Tomato dwarf virus family viruses, fungi, oomycetes, and invertebrates.

64. The method of claim 63, wherein the pathogen is a heterologous RNA virus (HRV), optionally wherein the HRV is a virus selected from the group consisting of: *Hyperviridae*, *Hyperviridae*, *Brassica napus*, *Celavirus*, *Leptochloavirus*, *Cowpea Mosaic Virus*, *Potatovirus X*, *Potatovirus Y*, *Tobacco Mosaic Virus*, *Tomato Cluster Dwarf Virus*, *Tomato Spotted Wilt Virus Family*, *Trigeneae* subfamily, *Turnip Yellow Mosaic Virus*, *Vein Varicose Vein Virus*, and *Associated Cowpea Mosaic Virus Family*.

65. The method of claim 64, wherein: (i) The genus *Bombyx mottle virus* refers to cucumber mosaic virus, spinach latent virus, olive latent virus, or *Bombyx mottle virus*; (ii) The genus *Lycovirus* refers to citrus senescence virus or beet yellowing virus; (iii) The genus *Cowpea mosaic virus* refers to cowpea mosaic virus, apple latent spherical virus, or soybean latent spherical virus; (iv) The genus *Potato X virus* refers to potato X virus or citrus yellowing virus; (v) The genus *Potato Y virus* refers to pepper mottle virus, bean yellow mosaic virus, barley stripe mosaic virus, wheat stripe mosaic virus, rice yellow mottle virus, or maize dwarf mosaic virus. Leaf virus, zucchini yellow mosaic virus, watermelon mosaic virus or sugarcane mosaic virus; (vi) the genus of tobacco mosaic virus is tobacco mosaic virus, tomato mosaic virus, tomato brown wrinkled fruit virus, turnip vein virus or pepper mild mottle virus; (vii) the genus of tomato dwarf virus is turnip wrinkled virus or tomato dwarf virus; (iv) the family of tomato spotted wilt virus is tomato spotted wilt virus or watermelon bud necrosis virus or (viii) the genus of turnip yellow mosaic virus is turnip yellow mosaic virus, citrus yellowing vein virus, potato latent virus, apple stem groove virus or citrus leaf mottle virus.

66. The method of claim 57, wherein the phenotypic change includes an increase in the plant’s resistance to stress, optionally wherein the stress includes at least one abiotic stress comprising nutrient stress, light stress, water stress, heat stress and / or cold stress, or optionally wherein the stress includes at least one biotic stress comprising crowding, shading or allelopathy.

67. The method of claim 57, wherein the recombinant RNA molecule is provided to the plant or plant cells in the form of RNA, capsidated RNA or a formulation thereof.

68. The method of claim 67, wherein the capsidated RNA comprises synthetic Tomato dwarf virus family satellite particles.

69. The method of claim 57, wherein the provision comprises contacting the plant or plant cells with the RNA, capsidated RNA or a formulation thereof, optionally wherein contact comprises spraying, dusting, injecting or soaking the plant or plant cells with the RNA, capsidated RNA or a formulation thereof.

70. The method of claim 57, wherein the recombinant RNA further comprises its reverse complementary RNA molecule.

71. The method of claim 57, further comprising providing the plant with hrvRdRP that recognizes the HRV 5' or 3' replication region and / or the subgenomic promoter in the synthetic Tomato dwarf Virus satellite RNA, optionally wherein the hrvRdRP is provided by introducing recombinant DNA or RNA encoding the hrvRdRP into the plant or a portion thereof.

72. A method for manufacturing synthetic Tomato Dwarf Virus family satellite particles, the method comprising combining a recombinant RNA molecule as described in any one of claims 1 to 28 with a viral capsid protein, wherein the recombinant RNA molecule includes a capsidation recognition element (ERE), and wherein the ERE provides capsidation of the RNA by the viral capsid protein.

73. The method of claim 72, wherein the recombinant RNA molecule is combined with the viral capsid protein in a container.

74. The method of claim 73, wherein the combination comprises (a) Providing the recombinant RNA molecule to plant cells, wherein the recombinant RNA molecule contains a capsidation recognition element (ERE), and wherein the plant cells contain an RdRP protein and a viral capsid protein that recognize the 5' RNA replication element and the 3' RNA replication element and catalyze the synthesis of Tomato dwarf Virus satellite RNA from the recombinant RNA molecule, wherein the ERE provides capsidation of the RNA by the viral capsid protein; and optionally (b) Isolating the synthetic Tomato Cluster Dwarf Virus Family satellite particles from the plant cells, the plant containing the plant cells, or from the plant cells or the culture medium in which the plant grows.

75. The method of claim 72, further comprising the step of formulating the synthetic Tomato Cluster Dwarf Virus Family satellite particles, wherein the formulation comprises combining the synthetic Tomato Cluster Dwarf Virus Family satellite particles with a carrier, excipients and / or adjuvants.

76. A plant propagule comprising a recombinant RNA molecule as claimed in any one of claims 1 to 28 and a Tomato Cluster Dwarf Virus RdRP, optionally wherein the plant propagule further comprises a heterologous RNA virus RdRP that recognizes the HRV 5' or 3' replication region in the synthetic Tomato Cluster Dwarf Virus satellite RNA and / or the subgenomic promoter.

77. The plant propagation body of claim 76, wherein the plant propagation body is a seed, seedling, root, stem, leaf, bud, tuber, rhizome, stolon, bulb, explant, embryo, or callus.

78. The plant propagule of claim 76, wherein the plant propagule is a chimera comprising both a plant cell containing the recombinant RNA molecule and a plant cell lacking the recombinant RNA molecule.

79. The plant propagule of claim 76, wherein the plant propagule lacks DNA encoding the recombinant RNA molecule.

80. The plant propagule of claim 76, wherein the plant propagule further comprises a heterologous RNA virus RdRP that recognizes the HRV 5' or 3' replication region and / or the subgenomic promoter in the synthetic Tomato Cluster Dwarf Virus Family satellite RNA, and the heterologous RNA virus RdRP is a RdRP from the genera *Hyperviridae*, *Hyperviridae*, *Brassica napus*, *Celavirus*, *Leptochloavirus*, *Cowpea Mosaic Virus*, *Potatovirus X*, *Potatovirus Y*, *Tobacco Mosaic Virus*, *Tomato Cluster Dwarf Virus*, *Tomato Spotted Wilt Virus*, *Trigeneae*, *Turnip Yellow Mosaic Virus*, *Vein Varicella Virus*, or *Associated Cowpea Mosaic Virus*, or an RdRP from the family *Cowpea Mosaic Virus*, or an RdRP shown in Table 7.

81. A plant comprising a recombinant RNA molecule as claimed in any one of claims 1 to 28 and a Tomato Cluster Dwarf Virus RdRP, optionally wherein the plant propagule further comprises a heterologous RNA virus RdRP that recognizes the HRV 5' or 3' replication region in the synthetic Tomato Cluster Dwarf Virus satellite RNA and / or the subgenomic promoter.

82. The plant of claim 81, wherein the plant is a monocotyledonous plant or a dicotyledonous plant.

83. The plant of claim 81, wherein the plant belongs to the Asteraceae, Cucurbitaceae, Fabaceae, Oleaceae, Poaceae, Rutaceae or Solanaceae families.

84. The plant of claim 81, wherein the plant lacks DNA encoding the recombinant RNA molecule.

85. The plant of claim 81, wherein the plant comprises the Tomato Cluster Dwarf Virus family virus, and wherein the Tomato Cluster Dwarf Virus family RdRP is provided to the plant cells via the Tomato Cluster Dwarf Virus family virus, optionally wherein the Tomato Cluster Dwarf Virus family RdRP comprises a protein having at least 85%, 90%, 95%, 97%, or 99% sequence identity with SEQ ID NO: 469 or 587.

86. The plant of claim 81, wherein the Tomato Cluster Dwarf Virus family virus is specific to the plant, optionally wherein the specific Tomato Cluster Dwarf Virus family virus is non-pathogenic and / or symbiotic.

87. The plant of claim 81, wherein the Tomato Cluster Dwarf Virus Family RdRP, the 5' RNA replication element and / or the 3' RNA replication element are derived from a Tomato Cluster Dwarf Virus Family virus containing one or both of the Tomato Cluster Dwarf Virus Family RdRP, the 5' RNA replication element and / or the 3' RNA replication element.

88. The plant of claim 81, wherein the RdRP has at least 85%, 90%, 95%, 97%, 98%, or 99% identity with SEQ ID NO: 469 or 587, wherein the 5' RNA replication element has at least 85%, 90%, 95%, 97%, 98%, or 99% identity with the RNA encoded by SEQ ID NO: 467, and / or wherein the 3' RNA replication element has at least 85%, 90%, 95%, 97%, 98%, or 99% identity with the RNA encoded by SEQ ID NO:

468.

89. The plant of claim 81, wherein the plant is a grafted plant, and wherein the rootstock and / or scion of the grafted plant contains at least one cell containing the recombinant RNA and the RdRP of the Tomato Cluster Dwarf Virus Family.

90. The plant of claim 81, wherein the plant is not produced by an inherently biological process.

91. The plant of claim 81, wherein the plant further comprises a heterologous RNA virus (HRV) RdRP that recognizes the HRV 5' or 3' replication region and / or the subgenomic promoter in the synthetic Tomato Cluster Dwarf Virus Family satellite RNA, and the heterologous RNA virus RdRP is a HRV from the genera *Hyperviridae*, *Hyperviridae*, *Brassica napus*, *Celavirus*, *Leptochloavirus*, *Cowpea Mosaic Virus*, *Potatovirus X*, *Potatovirus Y*, *Tobacco Mosaic Virus*, *Tomato Cluster Dwarf Virus*, *Tomato Spotted Wilt Virus*, *Trigeneae*, *Turnip Yellow Mosaic Virus*, *Vein Varicella Virus*, or *Associated Cowpea Mosaic Virus*, or an RdRP listed in Table 7.

92. A Tomato Cluster Dwarf Virus Family satellite system capable of self-replication upon introduction into a plant or plant cell, said Tomato Cluster Dwarf Virus Family satellite system comprising: (a) a recombinant Tomato Cluster Dwarf Virus Family satellite RNA as claimed in any one of claims 1 to 28; and (b) an exogenous Tomato Cluster Dwarf Virus Family virus capable of replicating in said plant or plant cell and encoding a Tomato Cluster Dwarf Virus Family RdRP that recognizes 5' and 3' replicase recognition sequences in said recombinant Tomato Cluster Dwarf Virus Family satellite RNA, optionally wherein said Tomato Cluster Dwarf Virus Family satellite system further comprises a heterologous RNA virus RdRP that recognizes an HRV 5' or 3' replication region and / or subgenomic promoter in said synthetic Tomato Cluster Dwarf Virus Family satellite RNA.

93. The self-replicating Tomato Cluster Dwarf Virus Family satellite system as described in claim 92, wherein the exogenous Tomato Cluster Dwarf Virus Family virus is specific to or naturally occurring for different species, varieties, or germplasm of the plant.

94. The self-replicating Tomato Cluster Dwarf Virus Family satellite system of claim 92, wherein the Tomato Cluster Dwarf Virus Family satellite system further comprises a heterologous RNA virus (HRV) RdRP that identifies the HRV 5' or 3' replication region and / or the subgenomic promoter in the synthetic Tomato Cluster Dwarf Virus Family satellite RNA, and the heterologous RNA virus RdRP is optionally a RdRP from the genera *Hyperviridae*, *Hyperviridae*, *Brassica napus*, *Celavirus*, *Leptovirus*, *Cowpea Mosaic Virus*, *Potato Virus X*, *Potato Virus Y*, *Tobacco Mosaic Virus*, *Tomato Cluster Dwarf Virus*, *Tomato Spotted Wilt Virus*, *Trigeneae*, *Turnip Yellow Mosaic Virus*, *Vein Varicose Vein Virus*, or *Associated Cowpea Mosaic Virus*, or an RdRP shown in Table 7.

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