Viral plant expression vector systems with expanded cargo space

Abstract

Aspects of the present disclosure relate to methods of engineering plant virus-derived expression vector systems for transient expression in plants, with expanded cargo capacity. Additional aspects provide plant virus expression vector systems, barley stripe mosaic virus expression vector systems, and tobravirus expression vector systems, as well as cDNAs and infectious RNAs of the same. Further aspects provide methods of using an engineered plant virus expression vector system for transient expression in planta.

Description

Attorney Docket No: 293082000140 VIRAL PLANT EXPRESSION VECTOR SYSTEMS WITH EXPANDED CARGO SPACE CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 632,782, filed April 11, 2024, to U.S. Provisional Patent Application No.63 / 650,824, filed May 22, 2024, and to U.S. Provisional Patent Application No.63 / 721,005, filed November 15, 2024, each of which is incorporated herein by reference in its entirety. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The content of the electronic sequence listing (293082000140seqlist.xml; Size: 770,708 bytes; and Date of Creation: April 4, 2025) is herein incorporated by reference in its entirety. FIELD

[0003] The present disclosure relates generally to the field of systems for transient gene expression in plants, and more specifically to engineering new plant virus-derived vector systems for transient gene expression in plants. BACKGROUND

[0004] Plant viruses are obligate parasites that can only replicate in host cells. Their genomes may consist of one or multiple RNA or DNA molecules (i.e., genomic partite). Most known plant viruses have a single-stranded RNA (ssRNA) genome, while others have a double- stranded RNA (dsRNA), single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA) genome. The ssRNAs may be of sense (positive) or antisense (negative). Some plant viruses pack their genome as one viral particle, and others pack each genomic partite into a virion which will infect the host, move within the host, and be transmitted between plants independently. Some plant viruses have a linear genome while others have a circular one.

[0005] Plant viruses can infect their host plants and propagate themselves and move within the infected host plants. They can replicate their genome, transcribe the RNAs, and translate the proteins they encode using the resources of the host cells. Some plant viruses can invade the host plants’ germline cells making seed transmission of the virus possible. These properties make plant viruses ideal candidates for being engineered as transient expression tools. In fact, numerous efforts have been made to engineer various plant viral vectors for studying gene functions (e.g., virus-induced gene silencing (VIGS) or virus-mediated overexpression (VOX) (see a review by Abrahamian et al., 2020)), expressing a foreign protein (Gleba et al.2005) or 1ny-2939083Attorney Docket No: 293082000140 delivering a guide RNA(s) or other CRISPR / Cas components needed for gene editing (see a review by Shen et al., 2024). For example, barley stripe mosaic virus (BSMV) and tobacco rattle virus (TRV) have been engineered as an expression vector system for VIGS (Holzberg et al., 2002; Zhuang et al., 2012) and VOX (Cheuk and Houde, 2018; Paudel et al., 2020), and for delivering guide RNAs for Cas9-based gene editing (Hu et al., 2019; Liu et al., 2022). However, these viral expression vectors only have limited cargo space(s) that limited their usage. Cheuk and Houde (2018) reported that the largest effective insert in their BSMV-γ2 expression vector was up to ~2.5 kb, while Burch-Smith et al. (2004) reported that their TRV RNA2-expression vector had a cargo space of up to ~1.5 kb. Beyond the stated limits, insert instability increased dramatically. It is well-known that the cargo spaces in current viral vectors are too small to carry the whole Cas9 expression package for gene editing. Therefore, novel solutions are required in order to provide genome editing molecules such as Cas9 for expression in plants. As such, there is a clear need for new viral expression vectors with expanded cargo capacity, and methods of creating such viral expression vectors. BRIEF SUMMARY OF THE INVENTION

[0006] In one aspect, provided herein is an expression vector system, including: (a) a vector comprising a Barley Strip Mosaic Virus (BSMV) RNAα, (b) a vector comprising a BSMV RNAβ1 derived from BSMV RNAβ, (c) a vector comprising a BSMV RNAβ2 derived from or consisting of BSMV RNAβ, and (d) at least one vector derived from BSMV RNAγ, wherein the expression vector system is capable of systemic infection and / or systemic expression in a plant. In some embodiments, the expression vector system comprises a BSMV RNAγ1 vector derived from BSMV RNAγ, and a BSMV RNAγ2 vector derived from BSMV RNAγ. In another aspect, provided herein is an expression vector system comprising: (a) a vector comprising a Barley Stripe Mosaic Virus (BSMV) RNAα, (b) at least one vector derived from BSMV RNAβ, (c) a vector comprising a BSMV RNAγ1 derived from BSMV RNAγ, comprising a functional subgenomic RNAγ (sgRNAγ) promoter, and (d) a vector comprising a BSMV RNAγ2 derived from BSMV RNAγ, comprising a functional subgenomic RNAγ (sgRNAγ) promoter, wherein the expression vector system is capable of systemic infection and / or systemic expression in a plant. In some embodiments, the expression vector system comprises a BSMV RNAβ1 vector derived from BSMV RNAβ, and a BSMV RNAβ2 vector derived from or comprising BSMV RNAβ. In some embodiments, both the RNAβ1 and RNAβ2 comprise a subgenomic RNAβ1 (sgRNAβ1) promoter. In some embodiments, the RNAβ1 comprises a βa gene and a cargo space, and / or wherein the RNAβ2 comprises a βb 2ny-2939083Attorney Docket No: 293082000140 gene, a βc gene, a βd gene, a βd’ gene, and a cargo space. In some embodiments, the RNAγ1 comprises a γa gene and a cargo space, and / or wherein the RNAγ2 comprises a cargo space and a γb gene. In some embodiments, the expression vector system comprises one, two, three, or four cargo spaces. In some embodiments, one or more of the cargo spaces are ligation- independent cloning (LIC) sites or restriction enzyme sites. In some embodiments, one or more cargo spaces comprise cargo nucleic acid. In some embodiments, RNAβ1 comprises a cargo nucleic acid, RNAβ2 comprises a cargo nucleic acid, RNAγ1 comprises a cargo nucleic acid, and / or RNAγ2 comprises a cargo nucleic acid. In some embodiments, one or more vectors is composed of cDNA. In some embodiments, the cDNA encoding the BSMV RNA or the modified BSMV RNA is operably linked to a promoter. In some embodiments, the promoter is for in vitro transcription by a DNA-dependent RNA polymerase. In some embodiments, the promoter is for in planta transcription. In some embodiments, the expression vector system includes a first cargo nucleic acid for transient expression in planta. In some embodiments, the first cargo nucleic acid is a genome editing component. In some embodiments, the first cargo nucleic acid encodes a Cas protein. In some embodiments, the expression vector system further comprising a second cargo nucleic acid for transient expression in planta. In some embodiments, the second cargo nucleic acid comprises one or more guide RNA(s) for the Cas protein. In some embodiments, the expression vector system further comprises a third cargo nucleic acid for transient expression in planta, wherein the third cargo nucleic acid encodes one or more guide RNA(s) for the Cas protein. In some embodiments, the expression vector system further comprises a fourth cargo nucleic acid for transient expression in planta, wherein the fourth cargo nucleic acid encodes a Cas protein. In some embodiments, the cDNA is cloned into a T-DNA-derived binary plasmid, wherein the T-DNA-derived binary plasmid does not comprise an IS3 mobile element, optionally wherein the IS3 mobile element is a Tn10 transposable element or an IS1 element. Also provided is an infectious RNA produced by in vitro transcription of a vector of the expression vector system of any one of the previous embodiments. Also provided is a liquid composition comprising infectious RNAs produced by in vitro transcription of each vector of the expression vector system of any one of the previous embodiments. Also provided is a method of transiently expressing an RNA or protein in a plant or plant cell, the method comprising: (a) providing the expression vector system of any one of the previous embodiments, wherein the expression vector system comprises at least one cargo nucleic acid sequence encoding an RNA or protein; (b) in vitro transcribing the vector system into infectious RNAs; and (c) inoculating a plant or plant cell with the infectious RNAs; wherein the RNA or protein encoded by the at least one cargo nucleic acid sequence is 3ny-2939083Attorney Docket No: 293082000140 transiently expressed in the plant or plant cell. Also provided is a method of transiently expressing an RNA or protein in a plant or plant cell, the method comprising: (a) cloning a nucleic acid sequence encoding an RNA or protein into the cargo space of the expression vector system any one of the previous embodiments; (b) in vitro transcribing the vector system into infectious RNAs; and (c) inoculating a plant or plant cell with the infectious RNAs; wherein the RNA or protein encoded by the nucleic acid sequence is transiently expressed in the plant or plant cell. Also provided herein is the plant or plant cell of the previous method embodiments.

[0007] In another aspect, provided herein is a method of engineering a novel viral system for expression in plants, the method comprising: (a) providing a plant virus nucleic acid; (b) determining a cutting site within the nucleic acid; (c) splitting the nucleic acid at the cutting site into a first engineered nucleic acid and a second engineered nucleic acid; and (d) introducing a cloning site on the first engineered nucleic acid and / or on the second engineered nucleic acid; thereby engineering a novel viral system for expression in plants comprising the first and / or the second engineered nucleic acid, optionally wherein the novel viral system is capable of systemic infection and / or systemic expression in a plant.

[0008] In one aspect, provided herein is a method of engineering a novel viral system for expression in plants, the method comprising: (a) providing a plant virus nucleic acid; (b) determining a cutting site within the nucleic acid; (c) splitting the nucleic acid at the cutting site into a first engineered nucleic acid and a second engineered nucleic acid; and (d) introducing a cloning site on the first engineered nucleic acid and / or on the second engineered nucleic acid; thereby engineering a novel viral system for expression in plants comprising the first and / or the second engineered nucleic acid. In one aspect, provided herein is a method of engineering a novel viral system for expression in plants, the method comprising: (a) providing a plant virus nucleic acid; (b) determining a cutting site within the nucleic acid, wherein the cutting site does not disrupt a replicase, moving protein (MP), cysteine-rich protein (CRP), or a sub-genomic promoter; (c) splitting the nucleic acid at the cutting site into a first engineered nucleic acid and a second engineered nucleic acid; and (d) introducing a cloning site on the first engineered nucleic acid and / or on the second engineered nucleic acid; thereby engineering a novel viral system for expression in plants comprising the first and / or the second engineered nucleic acid. In some embodiments, the novel viral system for expression in plants comprises the first engineered nucleic acid. In other embodiments, the novel viral system for expression in plants comprises the second engineered nucleic acid. In some embodiments, the novel viral system for expression in plants comprises the first engineered nucleic acid and the second engineered nucleic acid. In some embodiments, the novel viral system is capable of systemic 4ny-2939083Attorney Docket No: 293082000140 infection and / or systemic expression. In some embodiments, introducing the novel viral system into a plant results in systemic infection and / or systemic expression. In an additional aspect, provided herein is a method of engineering a novel viral system for expression in plants, the method comprising: (a) providing a plant virus nucleic acid; (b) identifying elements in the nucleic acid that promote viral function; (c) determining a cutting site within the nucleic acid, wherein the cutting site does not disrupt any of the elements identified in part (b); and (d) splitting the nucleic acid at the cutting site into a first engineered nucleic acid and a second engineered nucleic acid, and introducing a cloning site on the first engineered nucleic acid and / or on the second engineered nucleic acid; thereby engineering a novel viral system for expression in plants comprising the first and / or the second engineered nucleic acid. In some embodiments, the novel viral system for expression in plants comprises the first engineered nucleic acid. In other embodiments, the novel viral system for expression in plants comprises the second engineered nucleic acid. In some embodiments, the novel viral system for expression in plants comprises the first engineered nucleic acid and the second engineered nucleic acid. In some embodiments, the novel viral system is capable of systemic infection and / or systemic expression. In some embodiments, introducing the novel viral system into a plant results in systemic infection and / or systemic expression. In a further aspect, provided herein is a method of engineering a novel viral system for expression in plants, the method comprising: (a) providing a plant virus nucleic acid; (b) determining a cutting site within the nucleic acid; (c) splitting the nucleic acid at the cutting site into a first engineered nucleic acid and a second engineered nucleic acid; and (d) introducing a cloning site on the first engineered nucleic acid and / or on the second engineered nucleic acid; thereby engineering a novel viral system for expression in plants comprising the first and / or the second engineered nucleic acid, wherein the engineered plant viral system is capable of invading a plant germline cell. In some embodiments, the novel viral system for expression in plants comprises the first engineered nucleic acid. In other embodiments, the novel viral system for expression in plants comprises the second engineered nucleic acid. In some embodiments, the novel viral system for expression in plants comprises the first engineered nucleic acid and the second engineered nucleic acid. In some embodiments, the novel viral system is capable of systemic infection and / or systemic expression. In some embodiments, introducing the novel viral system into a plant results in systemic infection and / or systemic expression. In one embodiment of any of the above aspects, the plant virus nucleic acid is not a barley stripe mosaic virus (BSMV) RNA. In one embodiment of any of the above aspects, the plant virus nucleic acid is not a barley stripe mosaic virus (BSMV) RNAγ. In some embodiments of any of the above aspects, the 5ny-2939083Attorney Docket No: 293082000140 plant virus has a positive sense RNA genome. In a further embodiment, the engineered viral system is capable of invading a plant germline cell. In a further embodiment, introducing the engineered viral system into a plant results in the viral system invading a plant germline cell. In some embodiments, less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of endogenous plant virus nucleic acid sequence is shared between the first engineered nucleic acid and the second engineered nucleic acid. In some embodiments, minimizing the amount of overlapping endogenous plant virus nucleic acid sequence between the cargo site and an adjacent open reading frame comprises making a cutting site that is adjacent to a promoter, open reading frame, or other regulatory element that is not expressed elsewhere. In some embodiments, minimizing the amount of overlapping endogenous plant virus nucleic acid sequence between the cargo site and an adjacent open reading frame comprises making a cutting site that is 3’ to the 3’ end of a promoter that is not expressed elsewhere. In some embodiments, minimizing the amount of overlapping endogenous plant virus nucleic acid sequence between the cargo site and the nearest open reading frame comprises making a cutting site that is 3’ to the 3’ end of a unique open reading frame that is not expressed elsewhere. In some embodiments, minimizing the amount of overlapping endogenous plant virus nucleic acid sequence between the cargo site and the nearest open reading frame comprises making a cutting site that is 5’ to the 5’ end of a promoter that is not expressed elsewhere. In some embodiments, minimizing the amount of overlapping endogenous plant virus nucleic acid sequence between the cargo site and the nearest open reading frame comprises making a cutting site that is 5’ to the 5’ end of an open reading frame that is not expressed elsewhere. In some embodiments, the cutting site comprises all of the intergenic region. In some embodiments, the cutting site comprises all of the intergenic region and a portion of the nearest open reading frame. In some embodiments, the cutting site does not comprise all of the intergenic region. In some embodiments, the cutting site comprises at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more of the intergenic region. In another embodiment, step (a) further comprises identifying elements of the plant virus nucleic acid that promote viral function as an expression vector, and wherein the cutting site determined in step (b) does not disrupt an identified element that promotes viral function as an expression vector. In some embodiments, step (a) comprises identifying elements of the plant virus nucleic acid that promote viral function in viral induced genome editing (VIGE), and wherein the cutting site determined in step (b) does not disrupt an identified element that promotes viral function in VIGE. In an 6ny-2939083Attorney Docket No: 293082000140 additional embodiment, the elements that promote viral function comprise critical viral regulatory elements. In some embodiments, the elements that promote viral function comprise one or more elements selected from the group consisting of a replicase, a moving protein (MP), a cysteine-rich protein (CRP), and a sub-genomic promoter. In yet another embodiment, the elements that promote viral function comprise elements selected from the group consisting of sub-genomic DNA, sub-genomic RNA, enhancers, and cis-acting elements. In a still further embodiment, the viral function is selected from infection, movement in planta, replication, subgenomic expression, transcription, and translation. In another embodiment, the plant virus nucleic acid of step (a) comprises two or more viral open reading frames (ORFs), the first engineered nucleic acid comprises at least one ORF, and the second engineered nucleic acid comprises at least one ORF. In an additional embodiment, the plant virus nucleic acid of step (a) comprises two or more viral open reading frames (ORFs), and wherein the cutting site is located between two ORFs. In yet another embodiment, the first engineered nucleic acid and / or the second engineered nucleic acid comprise two or more ORFs, and wherein the method further comprises repeating steps (a)-(d) for the first engineered nucleic acid and / or the second engineered nucleic acid with two or more ORFs. In still another embodiment, the cloning site on the first engineered nucleic acid has a stable cargo capacity of approximately 120% the length of the second engineered nucleic acid before the introduction of a cloning site. In a further embodiment, the cloning site on the second engineered nucleic acid has a stable cargo capacity of approximately 120% the length of the first engineered nucleic acid before the introduction of a cloning site. In another embodiment, at least one cloning site is a ligation- independent cloning (LIC) site. In an additional embodiment, at least one cloning site is a restriction enzyme site. In a still further embodiment, the plant virus has an RNA genome. In an alternative embodiment, the plant virus has a DNA genome. In an additional embodiment, the plant virus has a single-stranded genome. In a further embodiment, the plant virus has a sense genome. In another embodiment, the plant virus has a positive-sense genome. In another embodiment, the plant virus has an anti-sense genome. In an additional embodiment, the plant virus has a negative sense genome. In yet another embodiment, the plant virus has a double- stranded genome. In a still further embodiment, the plant virus has a linear genome. In an alternative embodiment, the plant virus has a circular genome. In yet another embodiment, the plant virus is a monopartite viral system. In another embodiment, the plant virus is a multipartite viral system. In a further embodiment, the method further comprises repeating steps (a) – (d) for an additional partite within the multipartite viral system. In an additional embodiment, the plant virus is selected from the group consisting of a hordevirus, a tobravirus, 7ny-2939083Attorney Docket No: 293082000140 a geminivirus, a tobamovirus, a potexvirus, a potyvirus, a tombusvirus, a bromovirus, an alfamovirus, a cucumovirus, a rhabdovirus, an emaravirus, a tospovirus, an orthotospovirus, a benyvirus, and a comovirus. In another embodiment, the method further comprises producing a set of cDNAs that encode the engineered viral system for expression in plants. In yet another embodiment, each cDNA encodes a plant viral RNA or modified plant viral RNA that can be transcribed in vitro or in planta. In an additional embodiment, the method further comprises producing a set of plasmids comprising the cDNAs. In an additional embodiment, each cDNA comprises an endonuclease cutting site at the 3’ end of the cDNA. In a further embodiment, the endonuclease cutting site is an SpeI site, a BamHI site, or an MluI site. In another embodiment, each cDNA encoding the plant viral RNA or the modified plant viral RNA is operably linked to a promoter. In yet another embodiment, each cDNA encoding the plant viral RNA or the modified plant viral RNA is operably linked to a promoter for in vitro transcription by a DNA-dependent RNA polymerase. In an additional embodiment, wherein the promoter for in vitro transcription is a T7 promoter. In another embodiment, each cDNA encoding the plant viral RNA or the modified plant viral RNA is operably linked to a terminator. In a further embodiment, the terminator is a 35S terminator. In a still further embodiment, each cDNA encoding the plant viral RNA or the modified plant viral RNA is operably linked to a self- cleaving ribozyme. In another embodiment, each cDNA encoding the plant viral RNA or the modified plant viral RNA is operably linked to a promoter for in planta transcription. In an additional embodiment, the promoter is a CaMV 35S promoter or a 2X CaMV 35S promoter. In a further embodiment, the method further comprises inserting a first cargo insert into a first cloning site, wherein the first cargo insert comprises nucleic acid for transient expression in planta. In a further embodiment, the first cargo insert comprises nucleic acid encoding a genome editing component. In some embodiments, the first cargo insert comprises nucleic acid encoding a Cas protein. In an additional embodiment, the Cas protein is selected from the group consisting of a Cas9, a Cas12a, a Cas12b, a Cas12c, a Cas12d, a Cas12e, a Cas12f, a Cas12g, a Cas12h, a Cas12i, a Cas12j, a Cas12k, a Cas12l, and a Cas12m. In another embodiment, the Cas protein is a modified Cas protein, optionally wherein the Cas protein is a nickase or a nuclease-deficient Cas protein. In a further embodiment, the Cas protein is a nickase. In another embodiment, the Cas protein is a nuclease-deficient Cas protein. In yet another embodiment, the first cargo insert further comprises nucleic acid encoding one or more guide RNA(s) for the Cas protein. In a still further embodiment, the method further comprises cloning a second cargo insert into a second cloning site, wherein the second cargo insert comprises nucleic acid encoding: (a) one or more guide RNA(s) for the Cas protein, (b) a repair template with 8ny-2939083Attorney Docket No: 293082000140 homology arms for homology-directed repair (HDR), and / or (c) an expression cassette for CRISPR-based gene knockout, knock-in, or HDR. In a further embodiment, the method further comprises cloning a third cargo insert into a third cloning site, wherein the third cargo insert comprises nucleic acid encoding: (a) one or more guide RNA(s) for the Cas protein, (b) a repair template with homology arms for homology-directed repair (HDR), and / or (c) an expression cassette for CRISPR-based gene knockout, knock-in, or HDR. In another embodiment, the first cargo insert comprises nucleic acid encoding a base editor protein. In yet another embodiment, the first cargo insert further comprises nucleic acid encoding one or more guide RNA(s) for the base editor protein. In some embodiments, the method further comprises cloning a second cargo insert into a second cloning site, wherein the second cargo insert comprises nucleic acid encoding one or more guide RNA(s) for the base editor protein. In a further embodiment, the method further comprises cloning a third cargo insert into a third cloning site, wherein the third cargo insert comprises nucleic acid encoding one or more guide RNA(s) for the base editor protein. In some embodiments, the base editor is a guanine base editor. In some embodiments, the base editor is a cytosine base editor. In some embodiments, the base editor is an adenine base editor. In some embodiments, the base editor is a thymine base editor. In some embodiments, the base editor is fused to a uracil DNA glycosylase inhibitor (UGI). In a further embodiment, the first cargo insert comprises nucleic acid encoding a prime editor protein. In another embodiment, the first cargo insert further comprises nucleic acid encoding one or more prime editor guide RNA(s) (pegRNA(s)) for the prime editor protein. In an additional embodiment, the method further comprises cloning a second cargo insert into a second cloning site, wherein the second cargo insert comprises one or more prime editor guide RNA(s) (pegRNA(s)) for the prime editor. In yet another embodiment, the method further comprises cloning a third cargo insert into a third cloning site, wherein the third cargo insert comprises one or more prime editor guide RNA(s) (pegRNA(s)) for the prime editor. In a still further embodiment, the first cargo insert comprises nucleic acid encoding one or more zinc finger nuclease (ZFN) monomers. In a further embodiment, the method further comprises cloning a second cargo insert into a second cloning site, wherein the second cargo insert comprises nucleic acid encoding one or more zinc finger nuclease (ZFN) monomers. In a still further embodiment, the method further comprises cloning a third cargo insert into a third cloning site, wherein the third cargo insert comprises nucleic acid encoding one or more zinc finger nuclease (ZFN) monomers. In some embodiments, the first cargo insert comprises nucleic acid encoding one or more TALEN monomers. In a further embodiment, the method further comprises cloning a second cargo insert into a second cloning site, wherein the second cargo insert 9ny-2939083Attorney Docket No: 293082000140 comprises nucleic acid encoding one or more TALEN monomers. In yet another embodiment, the method further comprises cloning a third cargo insert into a third cloning site, wherein the third cargo insert comprises nucleic acid encoding one or more TALEN monomers. In some embodiments, the first cargo insert comprises nucleic acid encoding a homing meganuclease or a derivative of a homing meganuclease. In a further embodiment, the first cargo insert comprises nucleic acid encoding two or more homing meganucleases. In yet another embodiment, the method further comprises cloning a second cargo insert into a second cloning site, wherein the second cargo insert comprises nucleic acid encoding one or more homing meganucleases. In a still further embodiment, the method further comprises cloning a third cargo insert into a third cloning site, wherein the third cargo insert comprises nucleic acid encoding one or more homing meganucleases. In some embodiments, the method further comprises cloning the cDNA into a T-DNA-derived binary plasmid. In a further embodiment, the T-DNA-derived binary plasmid is a pCass4-Rz plasmid. In an additional embodiment, the binary plasmid does not comprise a mobile element of the IS3 family, optionally a Tn10 transposable element or IS1 element. In another embodiment, the binary plasmid does not comprise a sequence with at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 57 or SEQ ID NO: 58. In some embodiments, the method further comprises cloning the cDNA into a pZfl9U-derived plasmid. In some embodiments, the method further comprises producing infectious RNAs by in vitro transcribing each cDNA. In an additional embodiment, the method further comprises producing a liquid composition comprising the infectious RNAs. In some embodiments, the method further comprises producing an inoculant mixture comprising infectious RNAs produced by in vitro transcription of each cDNA. In another embodiment, at least one infectious RNA comprises a cargo space insert comprising nucleic acid. In an additional embodiment, at least one infectious RNA comprises a cargo nucleic acid encoding a genome editing component. Also provided herein is an expression vector system for transient expression in plants produced by the method of any of the previous embodiments. Also provide herein is an infectious RNA produced by the method of any of the previous embodiments. Also provided herein is a liquid composition or inoculant mixture produced by the method of any one of the previous embodiments.

[0009] In another aspect, provided herein is a method of providing a nucleic acid sequence to a plant or plant cell, the method comprising: (a) providing an expression vector system produced by the method of any one of the previous embodiments, wherein the expression vector system comprises at least one cargo space insert comprising nucleic acid; (b) in vitro 10ny-2939083Attorney Docket No: 293082000140 transcribing the vector system into infectious RNAs; and (c) inoculating a plant or plant cell with the infectious RNAs; wherein the at least one nucleic acid is provided to the plant or plant cell. In an additional aspect, provided herein is a method of transiently expressing an RNA or protein in a plant or plant cell, the method comprising: (a) providing an expression vector system produced by the method of any one of the previous embodiments, wherein the expression vector system comprises at least one cargo space insert comprising nucleic acid encoding an RNA or protein; (b) in vitro transcribing the vector system into infectious RNAs; and (c) inoculating a plant or plant cell with the infectious RNAs; wherein the RNA or protein(s) encoded by the at least one nucleic acid are transiently expressed in the plant or plant cell. In a further aspect, provided herein is a method of providing a nucleic acid sequence to a plant or plant cell, the method comprising: (a) cloning a nucleic acid sequence into the cargo space of an expression vector system produced by the method of any one of the previous embodiments; (b) in vitro transcribing the vector system into infectious RNAs; and (c) inoculating a plant or plant cell with the infectious RNAs; wherein the nucleic acid sequence is provided to the plant or plant cell. In another aspect, provided herein is a method of transiently expressing an RNA or protein in a plant or plant cell, the method comprising: (a) cloning a nucleic acid sequence encoding an RNA or protein into the cloning site of an expression vector system produced by the method of any one of the previous embodiments; (b) in vitro transcribing the vector system into infectious RNAs; and (c) inoculating a plant or plant cell with the infectious RNAs; wherein the RNA or protein encoded by the nucleic acid sequence is transiently expressed in the plant or plant cell. In an additional aspect, provided herein is a method of editing the genome of a plant or plant cell, the method comprising: (a) providing an expression vector system produced by the method of any one of the previous embodiments, wherein the expression vector system comprises a cargo space insert comprising nucleic acid encoding a genome editing component; (b) in vitro transcribing the vector system into infectious RNAs; and (c) inoculating a plant or plant cell with the infectious RNAs; wherein the genome editing component is provided to the plant or plant cell, and wherein the genome of the plant or plant cell is edited. In some embodiments, inoculating the plant comprises performing seed imbibition with the infectious RNAs. In other embodiments, inoculating the plant comprises rubbing the infectious RNAs or a composition comprising the infectious RNAs onto the surface of the plant. In a further aspect, provided herein is a method of providing a nucleic acid sequence to a plant, the method comprising: (a) providing an expression vector system produced by the method of any one of the previous embodiments, wherein the expression vector system comprises at least one cargo space insert comprising nucleic acid sequence; (b) 11ny-2939083Attorney Docket No: 293082000140 transforming the vector system into Agrobacterium; (c) providing the Agrobacterium to a first plant; (d) collecting leaf sap from the first plant; and (e) inoculating a second plant with the leaf sap; wherein the nucleic acid sequence is provided to the second plant. In another aspect, provided herein is a method of transiently expressing an RNA or protein in a plant, the method comprising: (a) providing an expression vector system produced by the method of any one of the previous embodiments, wherein the expression vector system comprises at least one cargo nucleic acid sequence encoding an RNA or protein; (b) transforming the vector system into Agrobacterium; (c) providing the Agrobacterium to a first plant; (d) collecting leaf sap from the first plant; and (e) inoculating a second plant with the leaf sap; wherein the RNA or protein encoded by the at least one cargo nucleic acid sequence is transiently expressed in the second plant. In an additional aspect, provided herein is a method of providing a nucleic acid sequence to a plant, the method comprising: (a) cloning a nucleic acid sequence into the cloning site of an expression vector system produced by the method of any one of the previous embodiments; (b) transforming the expression vector system into Agrobacterium; and (c) providing the Agrobacterium to a plant; wherein the nucleic acid sequence is provided to the plant. In a further aspect, provided herein is a method of providing a nucleic acid sequence to a plant, the method comprising: (a) cloning a nucleic acid sequence into the cloning site of an expression vector system produced by the method of any one of the previous embodiments; (b) transforming the expression vector system into Agrobacterium; (c) providing the Agrobacterium to a first plant; (d) collecting leaf sap from the first plant; and (e) inoculating a second plant with the leaf sap; wherein the nucleic acid sequence is provided to the second plant. In another aspect, provided herein is a method of transiently expressing an RNA or protein in a plant, the method comprising: (a) cloning a nucleic acid sequence into the cloning site of an expression vector system produced by the method of any one of the previous embodiments, wherein the expression vector system comprises at least one cargo nucleic acid sequence encoding an RNA or protein; (b) transforming the expression vector system into Agrobacterium; and (c) providing the Agrobacterium to a plant; wherein the RNA or protein encoded by the at least one cargo nucleic acid sequence is transiently expressed in the plant. In an additional aspect, provided herein is a method of transiently expressing an RNA or protein in a plant, the method comprising: (a) cloning a nucleic acid sequence into the cloning site of an expression vector system produced by the method of any one of the previous embodiments, wherein the expression vector system comprises at least one cargo nucleic acid sequence encoding an RNA or protein; (b) transforming the expression vector system into Agrobacterium; (c) providing 12ny-2939083Attorney Docket No: 293082000140 the Agrobacterium to a first plant; (d) collecting leaf sap from the first plant; and (e) inoculating a second plant with the leaf sap; wherein the RNA or protein encoded by the at least one cargo nucleic acid sequence is transiently expressed in the second plant. In a further aspect, provided herein is a method of editing the genome of a plant, the method comprising: (a) providing an expression vector system produced by the method of any one of the previous embodiments, wherein the expression vector system comprises a cargo space insert comprising nucleic acid encoding a genome editing component; (b) transforming the expression vector system into Agrobacterium; and (c) providing the Agrobacterium to a plant; wherein the genome editing component is provided to the plant, and wherein the genome of the plant is edited. In another aspect, provided herein is a method of editing the genome of a plant, the method comprising: (a) providing an expression vector system produced by the method of any one of the previous embodiments, wherein the expression vector system comprises a cargo space insert comprising nucleic acid encoding a genome editing component; (b) transforming the expression vector system into Agrobacterium; (c) providing the Agrobacterium to a first plant; (d) collecting leaf sap from the first plant; and (e) inoculating a second plant with the leaf sap; wherein the genome editing component is provided to the second plant, and wherein the genome of the second plant is edited. In some embodiments of any of the above aspects, the first plant is Nicotiana benthamiana. In some embodiments of any of the above aspects, the Agrobacterium are provided to the first plant or plant by leaf infiltration. In some embodiments of any of the above aspects, the method further comprises phenotyping and / or genotyping the plant or second plant for successful expression. In some embodiments of any of the above aspects, the method further comprises phenotyping and / or genotyping the plant or second plant for genome editing. In some embodiments of any of the above aspects, the method further comprises harvesting seed from the plant or second plant. In a further embodiment, the method further comprises germinating the seed, assaying the offspring for viral RNA, and selecting virus-free offspring. In an additional embodiment, assaying the offspring for viral RNA comprises an RT-PCR assay. In some embodiments of any of the above aspects, the plant, plant cell, or second plant is a monocot or monocot cell. In some embodiments, the monocot or monocot cell is selected from the group consisting of wheat, maize, barley, rice and Miscanthus, or a cell thereof. In some embodiments, the monocot or monocot cell is selected from the group consisting of Hordeum vulgare, Zea mays, Triticum aestivum, Triticum turgidum, Oryza spp., Secale cereale, Sorghum bicolor, Avena sativa, Saccharum spp., Brachypodium distachyon, Panicum virgatum, and Miscanthus spp. In some embodiments of any of the above aspects, the plant, plant cell, or second plant is a dicot or dicot cell. In some embodiments, the dicot or dicot cell is selected 13ny-2939083Attorney Docket No: 293082000140 from the group consisting of beans, beets, brassicas, peas, peppers, spinach, tobacco, soybean, tomato, cotton, Arabidopsis, rose, strawberry, chrysanthemum and tulip, or a cell thereof, optionally wherein the dicot or dicot cell is Arabidopsis or tobacco, or a cell thereof. In some embodiments, the dicot or dicot cell is Arabidopsis or tobacco, or a cell thereof. In some embodiments, the dicot or dicot cell is selected from the group consisting of Nicotiana spp., Arabidopsis, Glycine max, Solanum lycopersicum, Solanum tuberosum, Helianthus annuus, Gossypium spp., Fagopyrum esculentum, Trifolium pratense, Brassica oleracea, Pisum sativum, Nasturtium officinale, Medicago sativa, and Raphanus sativus or a cell thereof.

[0010] In an additional aspect, provided herein is a method of transiently expressing a nucleic acid in a plant, the method comprising: (a) providing an expression vector system, wherein the expression vector system comprises: (i) a first expression vector comprising a tobravirus RNA1-derived RNA, (ii) a second expression vector comprising a tobravirus RNA1-derived RNA, and (iii) a third expression vector comprising a tobravirus RNA2 or tobravirus RNA2- derived RNA, and (A) a nucleic acid encoding a tobravirus replicase, (B) a nucleic acid encoding a tobravirus moving protein, (C) a nucleic acid encoding a tobravirus cysteine-rich protein, (D) a nucleic acid encoding a tobravirus coat protein, and (E) one, two, three, four, five, or six cargo space inserts each comprising a nucleic acid sequence; (b) transforming the expression vector system into Agrobacterium; and (c) providing the Agrobacterium to a plant; wherein the one, two, three, four, five, or six cargo space inserts comprising the nucleic acid sequences are provided to the plant, and wherein the nucleic acid sequences are transiently expressed in the plant. In another aspect, provided herein is a method of transiently expressing a nucleic acid in a plant, the method comprising: (a) providing an expression vector system, wherein the expression vector system comprises: (i) a first expression vector comprising a tobravirus RNA1-derived RNA, (ii) a second expression vector comprising a tobravirus RNA1- derived RNA, and (iii) a third expression vector comprising a tobravirus RNA2 or tobravirus RNA2-derived RNA, and (A) a nucleic acid encoding a tobravirus replicase, (B) a nucleic acid encoding a tobravirus moving protein, (C) a nucleic acid encoding a tobravirus cysteine-rich protein, (D) a nucleic acid encoding a tobravirus coat protein, and (E) one, two, three, four, five, or six cargo space inserts each comprising a nucleic acid sequence; (b) in vitro transcribing the expression vector system to produce infectious RNAs; and (c) providing the infectious RNAs to a plant; wherein the one, two, three, four, five, or six cargo space inserts comprising nucleic acid sequences are provided to the plant, and wherein the nucleic acid sequences are transiently expressed in the plant. In an additional aspect, provided herein is a method of transiently expressing a nucleic acid in a plant, the method comprising: (a) providing an 14ny-2939083Attorney Docket No: 293082000140 expression vector system, wherein the expression vector system comprises: (i) a first expression vector comprising a tobravirus RNA1-derived RNA, and (ii) a second expression vector comprising a tobravirus RNA1-derived RNA, and (A) a nucleic acid encoding a tobravirus replicase, (B) a nucleic acid encoding a tobravirus moving protein, (C) a nucleic acid encoding a tobravirus cysteine-rich protein, and (D) one or more cargo space inserts each comprising a nucleic acid sequence; (b) transforming the expression vector system into Agrobacterium; and (c) providing the Agrobacterium to a plant; wherein the one or more cargo space inserts comprising the nucleic acid sequences are provided to the plant, and wherein the nucleic acid sequences are transiently expressed in the plant. In another aspect, provided herein is a method of transiently expressing a nucleic acid in a plant, the method comprising: (a) providing an expression vector system, wherein the expression vector system comprises: (i) a first expression vector comprising a tobravirus RNA1-derived RNA, and (ii) a second expression vector comprising a tobravirus RNA1-derived RNA, and (A) a nucleic acid encoding a tobravirus replicase, (B) a nucleic acid encoding a tobravirus moving protein, (C) a nucleic acid encoding a tobravirus cysteine-rich protein, and (D) one or more cargo space inserts each comprising a nucleic acid sequence; (b) in vitro transcribing the expression vector system to produce infectious RNAs; and (c) providing the infectious RNAs to a plant; wherein the one or more cargo space inserts comprising nucleic acid sequences are provided to the plant, and wherein the nucleic acid sequences are transiently expressed in the plant. In an additional aspect, provided herein is a plant modified by or subjected to the method of any embodiment of the above aspects. In some embodiments of any of the above aspects, (a) the cargo nucleic acid is delivered at a higher efficiency than a cargo nucleic acid delivered with a control expression vector system, (b) one or more of the vectors have increased stability in planta compared to a vector from a control expression vector system, (c) there is an increase in in vitro transcription of one or more of the vectors compared to a control expression vector system, (d) the expression vector system produces more γb protein compared to a control expression vector system, (e) the expression vector system results in improved efficiency of transient expression in planta compared to a control expression vector system, and / or (f) the expression vector system results in improved efficiency of cell-to-cell movement of the viral vectors in planta compared to a control expression vector system.

[0011] In another aspect, provided herein is a method of transiently expressing a nucleic acid in a plant, the method comprising: (a) providing: (i) a first expression vector comprising a BSMV RNAα, (ii) a second expression vector comprising a BSMV RNAβ, (iii) a third expression vector comprising a BSMV RNAγ1, wherein the BSMV RNAγ1 comprises a 15ny-2939083Attorney Docket No: 293082000140 subgenomic RNAγ (sgRNAγ) promotor, and (iv) a fourth expression vector comprising a BSMV RNAγ2, wherein the BSMV RNAγ2 comprises a sgRNAγ promotor, wherein one or more cargo nucleic acids is comprised within (i) the BSMV RNAβ vector, (ii) the BSMV RNAγ1 vector, and / or (iii) the BSMV RNAγ2 vector; (b) transforming the first, second, third, and fourth expression vectors into Agrobacterium; and (c) providing the Agrobacterium to a plant; wherein the one or more cargo nucleic acids are provided to the plant, and wherein the one or more cargo nucleic acids are transiently expressed in the plant. In another aspect, provided herein is a method of transiently expressing a nucleic acid in a plant, the method comprising: (a) providing an expression vector system, wherein the expression vector system comprises: (i) a first expression vector comprising a BSMV RNAα, (ii) a second expression vector comprising a BSMV RNAβ, (iii) a third expression vector comprising a BSMV RNAγ- derived RNAγ1, wherein the BSMV RNAγ1 comprises a subgenomic RNAγ (sgRNAγ) promotor, and (iv) a fourth expression vector comprising a BSMV RNAγ-derived RNAγ2, wherein the BSMV RNAγ2 comprises a sgRNAγ promotor, wherein one or more cargo nucleic acids is comprised within (i) the BSMV RNAβ vector, (ii) the BSMV RNAγ1 vector, and / or (iii) the BSMV RNAγ2 vector; (b) in vitro transcribing the expression vector system to produce infectious RNAs; and (c) providing the infectious RNAs to a plant; wherein the one or more cargo nucleic acids is provided to the plant, and wherein the one or more cargo nucleic acids are transiently expressed in the plant. In another aspect, provided herein is a method of transiently expressing a nucleic acid in a plant, the method comprising: (a) providing an expression vector system, wherein the expression vector system comprises: (i) a first expression vector comprising a BSMV RNAα, (ii) a second expression vector comprising a BSMV RNAβ-derived RNAβ1, (iii) a third expression vector comprising a BSMV RNAβ- derived RNAβ2, and (iv) a fourth expression vector comprising a BSMV RNAγ, wherein one or more cargo nucleic acids is comprised within (i) the BSMV RNAβ1 vector, (ii) the BSMV RNAβ2 vector, and / or (iii) the BSMV RNAγ vector; (b) in vitro transcribing the expression vector system to produce infectious RNAs; and (c) providing the infectious RNAs to a plant; wherein the one or more cargo nucleic acids is provided to the plant, and wherein the one or more cargo nucleic acids are transiently expressed in the plant.

[0012] In another aspect, provided herein is an expression vector system comprising: (a) a vector comprising a Barley Stripe Mosaic Virus (BSMV) RNAα, (b) a vector comprising a BSMV RNAβ1 derived from BSMV RNAβ, comprising an sgRNAβ1 promoter, (c) a vector comprising a BSMV RNAβ2 derived from BSMV RNAβ. comprising a sgRNAβ1 promoter, (d) a vector comprising a BSMV RNAγ1 derived from BSMV RNAγ, further comprising a 16ny-2939083Attorney Docket No: 293082000140 functional sgRNAγ promoter, and (e) a vector comprising a BSMV RNAγ2 derived from BSMV RNAγ, further comprising a functional sgRNAγ promoter. In another aspect, provided herein is an expression vector system comprising: (a) a vector comprising a Barley Stripe Mosaic Virus (BSMV) RNAα, (b) a vector comprising a BSMV RNAβ1 derived from BSMV RNAβ, comprising an sgRNAβ1 promoter, (c) a vector comprising a BSMV RNAβ2 derived from BSMV RNAβ, comprising a sgRNAβ1 promoter, (d) a vector comprising a BSMV RNAγ1 derived from BSMV RNAγ, further comprising a functional sgRNAγ promoter, and (e) a vector comprising a BSMV RNAγ2 derived from BSMV RNAγ, further comprising a functional sgRNAγ promoter, wherein the expression vector system does not comprise an IS3 family mobile element. In another aspect, provided herein is an expression vector system comprising: (a) a vector comprising a Barley Stripe Mosaic Virus (BSMV) RNAα, (b) a vector comprising a BSMV RNAβ1 derived from BSMV RNAβ, comprising an sgRNAβ1 promoter, (c) a vector comprising a BSMV RNAβ2 derived from BSMV RNAβ, comprising a sgRNAβ1 promoter, and (d) a vector comprising a BSMV RNAγ or a BSMV RNA derived from BSMV RNAγ.

[0013] In an additional aspect, provided herein is a method of transiently expressing a nucleic acid in a plant, the method comprising: (a) providing an expression vector system, wherein the expression vector system comprises: (i) a first expression vector comprising a BSMV RNAα, (ii) a second expression vector comprising a BSMV RNAβ1, (iii) a third expression vector comprising a BSMV RNAβ2, (iv) a fourth expression vector comprising a BSMV RNAγ1, wherein the BSMV RNAγ1 comprises a subgenomic RNAγ (sgRNAγ) promotor; and (v) a fifth expression vector comprising a BSMV RNAγ2, wherein the BSMV RNAγ2 comprises a sgRNAγ promotor, wherein one or more cargo nucleic acids is comprised within (i) the BSMV RNAβ1 vector, (ii) the BSMV RNAβ2 vector, (iii) the BSMV RNAγ1 vector, and / or (iv) the BSMV RNAγ2 vector; (b) transforming the first, second, third, fourth, and fifth expression vectorsAgrobacterium; and (c) providing the Agrobacterium to a plant; wherein the one or more cargo nucleic acids are provided to the plant, and wherein the one or more cargo nucleic acids are transiently expressed in the plant. In an additional aspect, provided herein is a method of transiently expressing a nucleic acid in a plant, the method comprising: (a) providing: (i) a first expression vector comprising a BSMV RNAα; (ii) a second expression vector comprising a BSMV RNAβ1; (iii) a third expression vector comprising a BSMV RNAβ2; (iv) a fourth expression vector comprising a BSMV RNAγ1, wherein the BSMV RNAγ1 comprises a subgenomic RNAγ (sgRNAγ) promotor; and (v) a fifth expression vector comprising a BSMV 17ny-2939083Attorney Docket No: 293082000140 RNAγ2, wherein the BSMV RNAγ2 comprises a sgRNAγ promotor, wherein one or more cargo nucleic acids is comprised within (i) the BSMV RNAβ1 vector, (ii) the BSMV RNAβ2 vector, (iii) the BSMV RNAγ1 vector, and / or (iv) the BSMV RNAγ2 vector; (b) in vitro transcribing the first, second, third, fourth, and fifth expression vectors to produce infectious RNAs; and (c) providing the infectious RNAs to a plant; wherein the one or more cargo nucleic acids are provided to the plant, and wherein the one or more cargo nucleic acids are transiently expressed in the plant.

[0014] An aspect of the disclosure includes a method of heritable, non-transgenic, tissue- culture-free editing of a genome in a seed or pollen of a plant, the method including: (a) providing an engineered viral vector system, wherein the engineered viral vector system comprises one or more genome editing components; and (b) introducing the engineered viral vector system to the plant, wherein the genome of a seed or pollen of the plant is edited, and wherein the plant is corn, soy, or wheat. In some embodiments, step (b) includes performing seed imbibition using a composition comprising the viral vector system. In some embodiments, the editing of the genome of the seed or pollen is non-transgenic. In some embodiments, the editing of the genome of the seed or pollen is heritable. In some embodiments, the editing of the genome of the seed or pollen is tissue-culture-free. In some embodiments, the expression of the genome editing components is transient. In some embodiments, a polynucleotide encoding the genome editing component is not integrated into the genome of the seed or pollen. In some embodiments, the composition is leaf sap collected from a host plant infiltrated with the viral vector system. In some embodiments, the host plant is Nicotiana benthamiana. In some embodiments, the one or more genome editing components include a guide RNA and a Cas protein. In some embodiments, the Cas protein is selected from the group consisting of a Cas9, a Cas12a, a Cas12b, a Cas12c, a Cas12d, a Cas12e, a Cas12f, a Cas12g, a Cas12h, a Cas12i, a Cas12j, a Cas12k, a Cas12l, and a Cas12m. In one embodiment, the plant is corn. In another embodiment, the plant is soy. In an additional embodiment, the plant is wheat. In some embodiments, the engineered viral vector system comprises an additional vector in comparison to a non-engineered viral vector system of the same viral species. In one embodiment, the engineered viral vector system is a BSMV system comprising a RNAβ1 and a RNAβ2. In some embodiments, the engineered viral vector system is a BSMV system comprising a RNAγ1 and a RNAγ2. In some embodiments, the engineered viral vector system is a tobravirus-derived system, wherein the expression vector system includes a first vector derived from a tobravirus RNA1 and a second vector derived from a tobravirus RNA1. 18ny-2939083Attorney Docket No: 293082000140

[0015] An aspect of the disclosure includes an expression vector system including a Barley Stripe Mosaic Virus (BSMV) with a split RNAβ, wherein the vector system includes an RNAβ1 and an RNAβ2. Another aspect of the disclosure includes an expression vector system including: (a) a vector including a Barley Strip Mosaic Virus (BSMV) RNAα, (b) a vector including a BSMV RNAβ1 derived from BSMV RNAβ, (c) a vector including a BSMV RNAβ2 derived from BSMV RNAβ, and (d) at least one vector derived from BSMV RNAγ. In some embodiments, the vector system is capable of systemic infection and / or systemic expression. In some embodiments, introducing the vector system into a plant results in systemic infection and / or systemic expression. In one embodiment of either of the above aspects, both the RNAβ1 and RNAβ2 include an sgRNAβ1 promoter. In a further embodiment, the sgRNAβ1 promoter includes SEQ ID NO: 49. In an additional embodiment, which may be combined with any of the preceding embodiments, the RNAβ1 includes a βa gene and a cargo space. In yet another embodiment, which may be combined with any of the preceding embodiments, the RNAβ2 includes a βb gene, a βc gene, a βd gene, a βd’ gene, and a cargo space. In an additional embodiment, which may be combined with any of the preceding embodiments, the expression vector system further includes one to four cloning sites. In still another embodiment, RNAβ1 includes a cloning site. In another embodiment, RNAβ2 includes a cloning site. In a further embodiment, RNAβ1 includes a cloning site and RNAβ2 includes a cloning site. In another embodiment, which may be combined with any of the preceding embodiments, the expression vector system further includes one to four cargo nucleic acids. In an additional embodiment, RNAβ1 includes a cargo nucleic acid. In another embodiment, RNAβ2 includes a cargo nucleic acid. In a further embodiment, RNAβ1 includes a cargo nucleic acid and RNAβ2 includes a cargo nucleic acid. In some embodiments, which may be combined with any of the preceding embodiments, the expression vector system includes a BSMV RNAγ1 vector derived from BSMV RNAγ, and a BSMV RNAγ2 vector derived from BSMV RNAγ. In a certain embodiment, the RNAγ1 includes a cloning site. In a further embodiment, the RNAγ2 includes a cloning site. In an additional embodiment, the RNAγ1 includes a cloning site and the RNAγ2 includes a cloning site. In a further embodiment, which may be combined with any of the preceding embodiments, at least one cloning site is a ligation- independent cloning (LIC) site. In another embodiment, which may be combined with any of the preceding embodiments, at least one cloning site is a restriction enzyme site. In one embodiment, the RNAγ1 includes a cargo nucleic acid. In a further embodiment, the RNAγ2 includes a cargo nucleic acid. In another embodiment, the RNAγ1 includes a cargo nucleic acid and the RNAγ2 includes a cargo nucleic acid. In an additional embodiment, which may be 19ny-2939083Attorney Docket No: 293082000140 combined with any of the preceding embodiments, one or more vectors is composed of cDNA. In one embodiment, the cDNA encodes a BSMV RNA or a modified BSMV RNA. In yet another embodiment, which may be combined with any of the preceding embodiments, the cDNA is included within a plasmid. In still another embodiment, which may be combined with any of the preceding embodiments, the cDNA includes an endonuclease cutting site at the 3’ end of the cDNA. In a further embodiment, the endonuclease cutting site is an SpeI site, a BamHI site, or an MluI site. In an additional embodiment, which may be combined with any of the preceding embodiments, the cDNA encoding the BSMV RNA or the modified BSMV RNA is operably linked to a promoter. In a further embodiment, which may be combined with any of the preceding embodiments, the cDNA encoding the BSMV RNA or the modified BSMV RNA is operably linked to a promoter for in vitro transcription by a DNA-dependent RNA polymerase. In an additional embodiment, the promoter is a T7 promoter. In another embodiment, which may be combined with any of the preceding embodiments, the cDNA encoding the BSMV RNA or the modified BSMV RNA is operably linked to a promoter for in planta transcription. In yet another embodiment, the promoter is a CaMV 35S promoter or a 2X CaMV 35S promoter.

[0016] In an additional embodiment, which may be combined with any of the preceding embodiments, the vector system includes a first cargo nucleic acid for transient expression in planta. In still another embodiment, the first cargo nucleic acid is a genome editing component. In a further embodiment, the first cargo nucleic acid encodes a Cas. In some embodiments, the first cargo nucleic acid encodes a Cas. In another embodiment, which may be combined with any of the preceding embodiments, the nucleic acid encoding the Cas is less than about 3 kb long. In an additional embodiment, the expression vector system further includes a second cargo nucleic acid including one or more guide RNA(s) for the Cas. In another embodiment, which may be combined with any of the preceding embodiments, the Cas is selected from CjCas9, a Cas12f, a Cas12j, a Cas12e, and a Cas12l. In one embodiment, which may be combined with any of the preceding embodiments, the Cas is Cas12f1. In still another embodiment, the expression vector system further includes a second cargo nucleic acid, wherein the first cargo nucleic acid and the second cargo nucleic acid together encode a split Cas. In a further embodiment, the split Cas is SaCas9N and SaCas9C. In an additional embodiment, which may be combined with any of the preceding embodiments, the expression vector system further includes a third cargo nucleic acid including one or more guide RNA(s) for the Cas; or a third cargo nucleic acid and a fourth cargo nucleic acid each including one or more guide RNA(s) for the Cas. In another embodiment, the first cargo nucleic acid encodes a 20ny-2939083Attorney Docket No: 293082000140 base editor. In a further embodiment, the expression vector system further includes a second cargo nucleic acid including one or more guide RNA(s) for the base editor. In an additional embodiment, which may be combined with any of the preceding embodiments, the base editor is a cytidine base editor. In another embodiment, which may be combined with any of the preceding embodiments, the base editor is an adenine base editor. In yet another embodiment, the first cargo nucleic acid encodes a prime editor. In a further embodiment, the expression vector system further includes a second cargo nucleic acid including one or more prime editor guide RNA(s) (pegRNA(s)) for the prime editor. In an additional embodiment, the first cargo nucleic acid encodes a first zinc finger nuclease (ZFN) monomer, and further including a second cargo nucleic acid encoding a second ZFN monomer. In yet another embodiment, the first cargo nucleic acid is on the β1 or γ2 vector, and wherein the first cargo nucleic acid encodes two ZFN monomers. In an additional embodiment, the expression vector system further includes a cargo nucleic acid encoding a third ZFN monomer, a cargo nucleic acid encoding a fourth ZFN monomer, a cargo nucleic acid encoding a fifth ZFN monomer, and / or a cargo nucleic acid encoding a sixth ZFN monomer. In another embodiment, the first cargo nucleic acid encodes a TALEN monomer. In yet another embodiment, the expression vector system further includes a second cargo nucleic acid encoding a second TALEN monomer. In an additional embodiment, the expression vector system further includes a cargo nucleic acid encoding a third TALEN monomer, and / or a cargo nucleic acid encoding a fourth TALEN monomer. In an additional embodiment, the first cargo nucleic acid encodes a homing meganuclease or a derivative of a homing meganuclease. In a further embodiment, the expression vector system includes two, three, four, five, or six homing meganucleases. In another embodiment, which may be combined with any of the preceding embodiments, the expression vector system includes a BSMV RNAβ1 cDNA including a sequence with 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 24. In yet another embodiment, which may be combined with any of the preceding embodiments, the expression vector system includes a BSMV RNAβ2 cDNA including a sequence with 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 25. In another embodiment, which may be combined with any of the preceding embodiments, the cDNA is cloned into a T-DNA-derived binary plasmid. In a further embodiment, the expression vector system includes an RNAβ1 T- DNA-derived plasmid including a sequence with 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 28. In still another embodiment, which may be combined with any of the preceding embodiments, the expression 21ny-2939083Attorney Docket No: 293082000140 vector system further includes an RNAβ2 T-DNA-derived plasmid including a sequence with 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 29. In yet another embodiment, which may be combined with any of the preceding embodiments, the cDNA is cloned into a pZfl9U-derived plasmid. In a further embodiment, the expression vector system includes an RNAβ1 pZfl9U-derived plasmid including a sequence with 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 32. In another embodiment, which may be combined with any of the preceding embodiments, the expression vector system further includes an RNAβ2 pZfl9U-derived plasmid including a sequence with 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 33.

[0017] An additional aspect of the disclosure provides an infectious RNA produced by in vitro transcription of a vector of the expression vector system of any one of the preceding embodiments.

[0018] A further aspect of the disclosure provides a liquid composition including infectious RNAs produced by in vitro transcription of each vector of the expression vector system of any one of the preceding embodiments.

[0019] Yet another aspect of the disclosure provides an inoculant mixture including infectious RNAs, wherein the mixture includes: (a) a Barley Stripe Mosaic Virus (BSMV) RNAα, (b) a BSMV RNAβ1 derived from BSMV RNAβ, (c) a BSMV RNAβ2 derived from BSMV RNAβ, and (c) at least one RNA derived from BSMV RNAγ, and wherein at least one RNA includes a cargo nucleic acid. In a further embodiment of this aspect, both the RNAβ1 and RNAβ2 include a subgenomic RNAβ1 (sgRNAβ1) promoter. In an additional embodiment of this aspect, which may be combined with any of the preceding embodiments of this aspect, the sgRNAβ1 promoter includes SEQ ID NO: 49. In yet another embodiment of this aspect, which may be combined with any of the preceding embodiments of this aspect, the RNAβ1 includes a βa gene and a cargo space. In still another embodiment of this aspect, which may be combined with any of the preceding embodiments of this aspect, the RNAβ2 includes a βb gene, a βc gene, a βd gene, a βd’ gene, and a cargo space. In an additional embodiment of this aspect, which may be combined with any of the preceding embodiments of this aspect, the RNAβ1 includes a cargo nucleic acid. In another embodiment of this aspect, which may be combined with any of the preceding embodiments of this aspect, the RNAβ2 includes a cargo nucleic acid. In an additional embodiment of this aspect, which may be combined with any of the preceding embodiments of this aspect, the RNAβ1 includes a cargo nucleic acid and the RNAβ2 includes a cargo nucleic acid. In a further embodiment of this aspect, which may be 22ny-2939083Attorney Docket No: 293082000140 combined with any of the preceding embodiments of this aspect, the mixture includes a BSMV RNAγ1 and a BSMV RNAγ2. In some embodiments of this aspect, the RNAγ1 includes a cargo nucleic acid. In some embodiments of this aspect, the RNAγ2 includes a cargo nucleic acid. In some embodiments of this aspect, the RNAγ1 includes a cargo nucleic acid and the RNAγ2 includes a cargo nucleic acid.

[0020] In certain embodiments of this aspect, which may be combined with any of the preceding embodiments of this aspect, at least one infectious RNA includes a cargo nucleic acid encoding a genome editing component. In a further embodiment of this aspect, at least one infectious RNA includes a cargo nucleic acid encoding a Cas. In another embodiment of this aspect, at least one infectious RNA includes a cargo nucleic acid encoding a Cas, wherein the nucleic acid encoding the Cas is less than about 3 kb long. In an additional embodiment of this aspect, at least one infectious RNA includes a cargo nucleic acid including one or more guide RNAs for the Cas. In another embodiment of this aspect, which may be combined with any of the preceding embodiments, the Cas is selected from CjCas9, a Cas12f, a Cas12j, a Cas12e, and a Cas12l. In yet another embodiment of this aspect, which may be combined with any of the preceding embodiments, two infectious RNAs include cargo nucleic acids that together encode a split Cas. In one embodiment of this aspect, which may be combined with any of the preceding embodiments of this aspect, the split Cas is SaCas9N and SaCas9C. In a further embodiment of this aspect, which may be combined with any of the preceding embodiments of this aspect, at least one infectious RNA includes a cargo nucleic acid encoding a base editor. In another embodiment of this aspect, which may be combined with any of the preceding embodiments, at least one infectious RNA includes a cargo nucleic acid including one or more guide RNAs for the base editor. In a still further embodiment of this aspect, which may be combined with any one of the preceding embodiments of this aspect, the base editor is a cytidine base editor. In another embodiment of this aspect, which may be combined with any one of the preceding embodiments of this aspect, the base editor is an adenine base editor. In yet another embodiment of this aspect, which may be combined with any of the preceding embodiments of this aspect, at least one infectious RNA includes a cargo nucleic acid encoding a prime editor. In an additional embodiment of this aspect, which may be combined with any of the preceding embodiments of this aspect, at least one infectious RNA includes a cargo nucleic acid including a prime editing gRNA (pegRNA) for the prime editor. In a further embodiment of this aspect, which may be combined with any of the preceding embodiments of this aspect, at least one infectious RNA includes a cargo nucleic acid encoding a zinc finger nuclease (ZFN) monomer. In yet another embodiment of this aspect, the mixture includes 23ny-2939083Attorney Docket No: 293082000140 infectious RNA including one or more cargo nucleic acids that encode a first ZFN monomer and a second ZFN monomer. In a further embodiment of this aspect, which may be combined with any of the preceding embodiments of this aspect, the mixture includes infectious RNA further including a third ZFN monomer, a fourth ZFN monomer, a fifth ZFN monomer, and / or a sixth ZFN monomer. In an additional embodiment of this aspect, which may be combined with any of the preceding embodiments of this aspect, at least one infectious RNA includes a cargo nucleic acid encoding a TALEN monomer. In an additional embodiment of this aspect, the inoculant mixture further includes a second cargo nucleic acid encoding a second TALEN monomer. In yet another embodiment of this aspect, the inoculant mixture further includes a cargo nucleic acid encoding a third TALEN monomer, and / or a cargo nucleic acid encoding a fourth TALEN monomer. In another embodiment of this aspect, at least one infectious RNA includes a cargo nucleic acid encoding a homing meganuclease or a derivative of a homing meganuclease. In one embodiment of this aspect, the mixture includes cargo nucleic acids encoding two, three, four, five, or six homing meganucleases or derivatives of homing meganucleases. In another embodiment of this aspect, which may be combined with any of the preceding embodiments of this aspect, both the BSMV RNAβ1 and BSMV RNAβ2 include a sgRNAβ1 promoter. In a further embodiment of this aspect, which may be combined with any of the preceding embodiments of this aspect, the sgRNAβ promoter includes SEQ ID NO: 49.

[0021] Another aspect of the present disclosure provides a method of providing a nucleic acid sequence to a plant or plant cell, the method including: (a) providing the inoculant mixture of any one of the preceding inoculant mixture embodiments including at least one cargo nucleic acid; and (b) inoculating a plant or plant cell with the inoculant mixture, wherein the at least one cargo nucleic acid is provided to the plant or plant cell. An additional aspect of the disclosure provides a method of providing a nucleic acid sequence to a plant or plant cell, the method including: (a) providing the expression vector system of any one of the preceding expression vector embodiments, wherein the expression vector system includes at least one cargo nucleic acid sequence; (b) in vitro transcribing the vector system into infectious RNAs; and (c) inoculating a plant or plant cell with the infectious RNAs; wherein the at least one cargo nucleic acid sequence is provided to the plant or plant cell. A further aspect of the disclosure provides a method of transiently expressing an RNA or protein in a plant or plant cell, the method including: (a) providing the expression vector system of any one of the preceding expression vector system embodiments, wherein the expression vector system includes at least one cargo nucleic acid sequence encoding an RNA or protein; (b) in vitro transcribing the vector system into infectious RNAs; and (c) inoculating a plant or plant cell with the 24ny-2939083Attorney Docket No: 293082000140 infectious RNAs; wherein the RNA or protein encoded by the at least one cargo nucleic acid sequence is transiently expressed in the plant or plant cell. Yet another aspect of the present disclosure provides a method of providing a nucleic acid sequence to a plant or plant cell, the method including: (a) cloning a nucleic acid sequence into the cloning site of the expression vector system any one of the preceding expression vector system embodiments; (b) in vitro transcribing the vector system into infectious RNAs; and (c) inoculating a plant or plant cell with the infectious RNAs; wherein the nucleic acid sequence is provided to the plant or plant cell. A still further aspect of the present disclosure provides a method of transiently expressing an RNA or protein in a plant or plant cell, the method including: (a) cloning a nucleic acid sequence encoding an RNA or protein into the cloning site of the expression vector system any one of the preceding expression vector system embodiments; (b) in vitro transcribing the vector system into infectious RNAs; and (c) inoculating a plant or plant cell with the infectious RNAs; wherein the RNA or protein encoded by the nucleic acid sequence is transiently expressed in the plant or plant cell. Still another aspect of the present disclosure provides a method of editing the genome of a plant or plant cell, the method including: (a) providing the expression vector system of any one of the preceding expression vector system embodiments; (b) in vitro transcribing the vector system into infectious RNAs; and (c) inoculating a plant or plant cell with the infectious RNAs; wherein the genome editing component is provided to the plant or plant cell, and wherein the genome of the plant or plant cell is edited. In some embodiments of any of the preceding method aspects, inoculating the plant includes performing seed imbibition with the infectious RNAs. In some embodiments of any of the preceding method aspects, inoculating the plant includes rubbing the infectious RNAs or a composition including the infectious RNAs into the surface of the plant. In some embodiments of any of the preceding method aspects, inoculating the plant cell includes providing the infectious RNAs to the plant cell in vitro.

[0022] An additional aspect of the present disclosure provides a method of providing a nucleic acid sequence to a plant, the method including: (a) providing the expression vector system of any one of the preceding expression vector system embodiments wherein the expression vector system includes at least one cargo nucleic acid sequence; (b) transforming the vector system into Agrobacterium; (c) providing the Agrobacterium to a first plant; (d) collecting leaf sap from the first plant; and (e) inoculating a second plant with the leaf sap; wherein the cargo nucleic acid sequence is provided to the second plant. Yet another aspect of the present disclosure provides a method of transiently expressing an RNA or protein in a plant, the method including: (a) providing the expression vector system of any one of the preceding 25ny-2939083Attorney Docket No: 293082000140 expression vector system embodiments, wherein the expression vector system includes at least one cargo nucleic acid sequence encoding an RNA or protein; (b) transforming the vector system into Agrobacterium; (c) providing the Agrobacterium to a first plant; (d) collecting leaf sap from the first plant; and (e) inoculating a second plant with the leaf sap; wherein the RNA or protein encoded by the at least one cargo nucleic acid sequence is transiently expressed in the second plant. A further aspect of the present disclosure provides a method of providing a nucleic acid sequence to a plant, the method including (a) cloning a nucleic acid sequence into the cloning site of the expression vector system any one of the preceding expression vector system embodiments; (b) transforming the expression vector system into Agrobacterium; and (c) providing the Agrobacterium to a plant; wherein the nucleic acid sequence is provided to the plant. Another aspect of the present disclosure provides a method of providing a nucleic acid sequence to a plant, the method including: (a) cloning a nucleic acid sequence into the cloning site of the expression vector system any one of the preceding expression vector system embodiments; (b) transforming the expression vector systemAgrobacterium; (c) providing the Agrobacterium to a first plant; (d) collecting leaf sap from the first plant; and (e) inoculating a second plant with the leaf sap; wherein the nucleic acid sequence is provided to the second plant. A further aspect of the present disclosure provides a method of transiently expressing an RNA or protein in a plant, the method including: (a) cloning a nucleic acid sequence into the cloning site of the expression vector system any one of the preceding expression vector system embodiments, wherein the expression vector system includes at least one cargo nucleic acid sequence encoding an RNA or protein; (b) transforming the expression vector system into Agrobacterium; and (c) providing the Agrobacterium to a plant; wherein the RNA or protein encoded by the at least one cargo nucleic acid sequence is transiently expressed in the plant. Still another aspect of the present disclosure provides a method of transiently expressing an RNA or protein in a plant, the method including: (a) cloning a nucleic acid sequence into the cloning site of the expression vector system any one of the preceding expression vector system embodiments, wherein the expression vector system includes at least one cargo nucleic acid sequence encoding an RNA or protein; (b) transforming the expression vector system into Agrobacterium; (c) providing the Agrobacterium to a first plant; (d) collecting leaf sap from the first plant; and (e) inoculating a second plant with the leaf sap; wherein the RNA or protein encoded by the at least one cargo nucleic acid sequence is transiently expressed in the second plant. An additional aspect of the present disclosure provides a method of editing the genome of a plant, the method including: (a) providing the expression vector system of any one of the preceding expression vector system embodiments; (b) transforming the expression vector 26ny-2939083Attorney Docket No: 293082000140 system into Agrobacterium; and (c) providing the Agrobacterium to a plant; wherein the genome editing component is provided to the plant, and wherein the genome of the plant is edited. Yet another aspect of the present disclosure provides a method of editing the genome of a plant, the method including: (a) providing the expression vector system of any one of the preceding expression vector system embodiments; (b) transforming the expression vector system into Agrobacterium; (c) providing the Agrobacterium to a first plant; (d) collecting leaf sap from the first plant; and (e) inoculating a second plant with the leaf sap; wherein the genome editing component is provided to the second plant, and wherein the genome of the second plant is edited. In some embodiments of any of the preceding method aspects, the first plant is Nicotiana benthamiana. In some embodiments of any of the preceding method aspects, the Agrobacterium are provided to the first plant or plant by leaf infiltration. In some embodiments of any of the preceding method aspects, the method further includes phenotyping and / or genotyping the plant or second plant for successful expression. In an additional embodiment of any of the preceding method aspects, which may be combined with any of the preceding embodiments, the method further includes phenotyping and / or genotyping the plant or second plant for genome editing. In yet another embodiment of any of the preceding method aspects, which may be combined with any of the preceding embodiments, the method further includes harvesting seed from the plant or second plant. In a further embodiment of any of the preceding method aspects, which may be combined with any of the preceding embodiments, the method further includes germinating the seed, assaying the offspring for viral RNA, and selecting virus- free offspring. In another embodiment of any of the preceding method aspects, assaying the offspring for viral RNA includes an RT-PCR assay. In yet another embodiment of any of the preceding method aspects, which may be combined with any of the preceding embodiments, the plant, plant cell, or second plant is a monocot or monocot cell. In some embodiments of any of the preceding method aspects, the monocot or monocot cell is selected from Hordeum vulgare, Zea mays, Triticum aestivum, Triticum turgidum, Oryza spp., Secale cereale, Sorghum bicolor, Avena sativa, Saccharum spp., Brachypodium distachyon, Panicum virgatum, and Miscanthus spp. In another embodiment of any of the preceding method aspects, which may be combined with any of the preceding embodiments, the plant, plant cell, or second plant is a dicot or dicot cell. In some embodiments of any of the preceding methods aspects, the dicot or dicot cell is selected from Nicotiana spp., Arabidopsis, Glycine max, Solanum lycopersicum, Solanum tuberosum, Helianthus annuus, Gossypium spp., Fagopyrum esculentum, Trifolium pratense, Brassica oleracea, Pisum sativum, Nasturtium officinale, Medicago sativa, and Raphanus sativus or a cell thereof. 27ny-2939083Attorney Docket No: 293082000140

[0023] In some aspects, herein is provided an improved expression vector system for inducing transient expression of a cargo construct in planta, including a Barley Stripe Mosaic Virus (BSMV) with a split RNAγ including a γ1 expression vector and a γ2 expression vector. The improved split-γ system does not contain a mobile element of the IS3 family, such as Tn10 or IS1, in the plasmid backbone, and includes functional subgenomic promotors for both the RNAγ1 and RNAγ2 vectors. In some aspects, the improved split-γ vector system has improved expression and improved genetic stability during replication compared to the previously available split-γ BSMV vector system. In some aspects, the improved split-γ vector system is suitable for use in a four-part vector system or a five-part vector system, allowing for expanded capabilities of multiplexing vector cargo for transient expression in planta. In some embodiments, the vector system is capable of systemic infection and / or systemic expression. In some embodiments, introducing the vector system into a plant results in systemic infection and / or systemic expression.

[0024] In some aspects, herein is provided an expression vector system including a Barley Stripe Mosaic Virus (BSMV) with a split RNAγ, wherein the expression vector system includes an RNAγ1 and an RNAγ2, wherein the RNAγ1 and the RNAγ2 each include a functional subgenomic RNAγ (sgRNAγ) promoter. In some aspects, herein is provided an expression vector system including: (a) a vector including a Barley Stripe Mosaic Virus (BSMV) RNAα, (b) at least one vector derived from BSMV RNAβ, (c) a vector including a BSMV RNAγ1 derived from BSMV RNAγ, and (d) a vector including a BSMV RNAγ2 derived from BSMV RNAγ, further including a functional sgRNAγ promoter. In some embodiments, the vector system is capable of systemic infection and / or systemic expression. In some embodiments, introducing the vector system into a plant results in systemic infection and / or systemic expression. In some embodiments, the sgRNAγ promoter includes SEQ ID NO: 56. In some embodiments, the RNAγ1 includes a γa gene and a cargo space. In some embodiments, the RNAγ2 includes a cargo space and a γb gene. In some embodiments, which may be combined with any of the preceding embodiments, herein is provided an expression vector system further including two cloning sites. In one embodiment, RNAγ1 includes a cloning site. In another embodiment, RNAγ2 includes a cloning site. In yet another embodiment, RNAγ1 includes a cloning site and RNAγ2 includes a cloning site. In another embodiment, which may be combined with any of the preceding embodiments, the expression vector system further includes one to four cargo nucleic acids. In one embodiment, RNAγ1 includes a cargo nucleic acid. In another embodiment, RNAγ2 includes a cargo nucleic acid. In yet another embodiment, RNAγ1 includes a cargo nucleic acid and RNAγ2 includes a cargo nucleic acid. In some 28ny-2939083Attorney Docket No: 293082000140 embodiments, which may be combined with any of the preceding embodiments, the expression vector system includes a BSMV RNAβ1 vector derived from BSMV RNAβ, and a BSMV RNAβ2 vector derived from BSMV RNAβ. In one embodiment, the RNAβ1 includes a cloning site. In another embodiment, the RNAβ2 includes a cloning site. In a further embodiment, the RNAβ1 includes a cloning site and the RNAβ2 includes a cloning site. In some embodiments, at least one cloning site is a ligation-independent cloning (LIC) site. In some embodiments, at least one cloning site is a restriction enzyme site. In one embodiment, the RNAβ1 includes a cargo nucleic acid. In another embodiment, the RNAβ2 includes a cargo nucleic acid. In yet another embodiment, the RNAβ1 includes a cargo nucleic acid and the RNAβ2 includes a cargo nucleic acid. In another embodiment, which may be combined with any of the preceding embodiments, one or more vectors is composed of cDNA. In some embodiments, the cDNA encodes a BSMV RNA or a modified BSMV RNA. In some embodiments, the cDNA is included within a plasmid. In some embodiments, the cDNA includes an endonuclease cutting site at the 3’ end of the cDNA. In some embodiments, the endonuclease cutting site is an SpeI site, a BamHI site, or an MluI site. In some embodiments, the cDNA encoding the BSMV RNA or the modified BSMV RNA is operably linked to a promoter. In some embodiments, the cDNA encoding the BSMV RNA or the modified BSMV RNA may be operably linked to a promoter for in vitro transcription by a DNA-dependent RNA polymerase. In some embodiments, the promoter is a T7 promoter. In some embodiments, the cDNA encoding the BSMV RNA or the modified BSMV RNA is operably linked to a promoter for in planta transcription. In some embodiments, the promoter is a CaMV 35S promoter or a 2X CaMV 35S promoter. In some embodiments, the vector system includes a first cargo nucleic acid for transient expression in planta. In some embodiments, the first cargo nucleic acid is a genome editing component. In some embodiments, the first cargo nucleic acid encodes a Cas. In some embodiments, the expression vector system further includes a second cargo nucleic acid including one or more guide RNA(s) for the Cas. In some embodiments, the nucleic acid encoding the Cas is less than about 3 kb long. In some embodiments, the Cas is selected from the group consisting of CjCas9, a Cas12f, a Cas12j, a Cas12e, and a Cas12l. In some embodiments, the expression vector system further includes a second cargo nucleic acid, wherein the first cargo nucleic acid and the second cargo nucleic acid together encode a split Cas. In some embodiments, the split Cas is SaCas9N and SaCas9C. In some embodiments, the expression vector system further includes a third cargo nucleic acid including one or more guide RNA(s) for the Cas; or a third cargo nucleic acid and a fourth cargo nucleic acid each including one or more guide RNA(s) for the Cas. In some embodiments, the first cargo nucleic 29ny-2939083Attorney Docket No: 293082000140 acid encodes a base editor. In some embodiments, the expression vector system further includes a second cargo nucleic acid including one or more guide RNA(s) for the base editor. In some embodiments, the base editor is a cytidine base editor. In some embodiments, the base editor is an adenine base editor. In some embodiments, the first cargo nucleic acid encodes a prime editor. In some embodiments, the expression vector system further includes a second cargo nucleic acid including one or more prime editor guide RNA(s) (pegRNA(s)) for the prime editor. In some embodiments, the first cargo nucleic acid encodes a first zinc finger nuclease (ZFN) monomer. In some embodiments, herein is provided an expression vector system wherein the first cargo nucleic acid encodes a first zinc finger nuclease (ZFN) monomer, and further including a second cargo nucleic acid encoding a second ZFN monomer. In some embodiments, the first cargo nucleic acid is on the β1 or γ2 vector, and the first cargo nucleic acid encodes two ZFN monomers. In some embodiments, the expression vector system further includes a cargo nucleic acid encoding a third ZFN monomer, a cargo nucleic acid encoding a fourth ZFN monomer, a cargo nucleic acid encoding a fifth ZFN monomer, and / or a cargo nucleic acid encoding a sixth ZFN monomer. In one embodiment, the first cargo nucleic acid encodes a TALEN monomer. In another embodiment, the expression vector system further includes a second cargo nucleic acid encoding a second TALEN monomer. In some embodiments, the expression vector system further includes a cargo nucleic acid encoding a third TALEN monomer, and / or a cargo nucleic acid encoding a fourth TALEN monomer. In some embodiments, the first cargo nucleic acid encodes a homing meganuclease or a derivative of a homing meganuclease. In some embodiments, the expression vector system includes two, three, four, five, or six homing meganucleases. In one embodiment, which may be combined with any of the preceding embodiments, the expression vector system includes a BSMV RNAγ1 cDNA including a sequence with at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 26. In another embodiment, which may be combined with any of the preceding embodiments, the expression vector system includes a BSMV RNAγ2 cDNA including a sequence with at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 27. In some embodiments, the cDNA is cloned into a T-DNA-derived binary plasmid. In some embodiments, the T-DNA-derived binary plasmid does not include a mobile element of the IS3 family. In some embodiments, the T-DNA-derived binary plasmid does not include a TN10 transposable element. In some embodiments, the T-DNA-derived binary plasmid does not include a sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 57. In some embodiments, the T-DNA-derived binary 30ny-2939083Attorney Docket No: 293082000140 plasmid does not include an IS1 element. In some embodiments, the T-DNA derived binary plasmid does not include a sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 58. In some embodiments, the T- DNA-derived binary plasmid is a pCass4-Rz plasmid. In some embodiments, the expression vector system includes an RNAγ1 T-DNA-derived plasmid including a sequence with at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 31. In some embodiments, the expression vector system includes an RNAγ2 T-DNA-derived plasmid including a sequence with at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 30. In some embodiments, the cDNA is cloned into a pZfl9U-derived plasmid. In some embodiments, the expression vector system includes an RNAγ1 pZfl9U-derived plasmid including a sequence with at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 35. In some embodiments, the expression vector system further includes an RNAγ2 pZfl9U-derived plasmid including a sequence with at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 34. In some embodiments, herein is provided an infectious RNA produced by in vitro transcription of a vector of the expression vector system of any one of the preceding embodiments. In some embodiments, herein is provided a liquid composition including infectious RNAs produced by in vitro transcription of each vector of the expression vector system of any one of the preceding embodiments. In some aspects, herein is provided an inoculant mixture including infectious RNAs, wherein the mixture includes: (a) a Barley Stripe Mosaic Virus (BSMV) RNAα, (b) at least one RNA derived from BSMV RNAβ, (c) a BSMV RNAγ1 derived from BSMV RNAγ, and (d) a BSMV RNAγ2 derived from BSMV RNAγ, further including a functional sgRNAγ promoter, and wherein at least one infectious RNA includes a cargo nucleic acid. In some embodiments, both the RNAγ1 and RNAγ2 include an sgRNAγ promoter. In some embodiments, the sgRNAγ promoter includes SEQ ID NO: 56. In one embodiment, the RNAγ1 includes a γa gene and a cargo space. In another embodiment, the RNAγ2 includes a cargo space and a γb gene. In a further embodiment, the RNAγ1 includes a cargo nucleic acid. In yet another embodiment, the RNAγ2 includes a cargo nucleic acid. In some embodiments, the RNAγ1 includes a cargo nucleic acid and the RNAγ2 includes a cargo nucleic acid. In some embodiments, the mixture includes a BSMV RNAβ1 and a BSMV RNAβ2. In one embodiment, the RNAβ1 includes a cargo nucleic acid. In another embodiment, the RNAβ2 includes a cargo nucleic acid. In yet another embodiment, the RNAβ1 includes a cargo nucleic acid and the RNAβ2 includes a cargo nucleic acid. In some embodiments, at least 31ny-2939083Attorney Docket No: 293082000140 one infectious RNA includes a cargo nucleic acid encoding a genome editing component. In some embodiments, at least one infectious RNA includes a cargo nucleic acid encoding a Cas. In some embodiments, at least one infectious RNA includes a cargo nucleic acid including one or more guide RNAs for the Cas. In some embodiments, the nucleic acid encoding the Cas is less than about 3 kb long. In some embodiments, the Cas is selected from the group consisting of CjCas9, a Cas12f, a Cas12j, a Cas12e, and a Cas12l. In some embodiments, two infectious RNAs include cargo nucleic acids that together encode a split Cas. In some embodiments, at least one infectious RNA includes a cargo nucleic acid including one or more guide RNAs for the split Cas. In some embodiments, the split Cas is SaCas9N and SaCas9C. In some embodiments, at least one infectious RNA includes a cargo nucleic acid encoding a base editor. In some embodiments, at least one infectious RNA includes a cargo nucleic acid including one or more guide RNAs for the base editor. In some embodiments, the base editor is a cytidine base editor. In some embodiments, the base editor is an adenine base editor. In some embodiments, at least one infectious RNA includes a cargo nucleic acid encoding a prime editor. In some embodiments, at least one infectious RNA includes a cargo nucleic acid including a prime editing gRNA (pegRNA) for the prime editor. In some embodiments, at least one infectious RNA includes a cargo nucleic acid encoding a zinc finger nuclease (ZFN) monomer. In some embodiments, the mixture includes infectious RNA including one or more cargo nucleic acids that encode a first ZFN monomer and a second ZFN monomer. In some embodiments, the mixture includes infectious RNA further including a third ZFN monomer, a fourth ZFN monomer, a fifth ZFN monomer, and / or a sixth ZFN monomer. In some embodiments, at least one infectious RNA includes a cargo nucleic acid encoding a TALEN monomer. In a further embodiment, the inoculant mixture further includes a second cargo nucleic acid encoding a second TALEN monomer. In yet another embodiment, the inoculant mixture further includes a cargo nucleic acid encoding a third TALEN monomer, and / or a cargo nucleic acid encoding a fourth TALEN monomer. In some embodiments, at least one infectious RNA includes a cargo nucleic acid encoding a homing meganuclease or a derivative of a homing meganuclease. In some embodiments, the mixture includes cargo nucleic acids encoding two, three, four, five, or six homing meganucleases or derivatives of homing meganucleases. In some embodiments, both the BSMV RNAβ1 and BSMV RNAβ2 include a sgRNAβ1 promoter. In some embodiments, the sgRNAβ promoter includes SEQ ID NO: 49.

[0025] In some aspects, herein is provided a T-DNA-derived binary plasmid, including: (a) a cDNA encoding a Barley Stripe Mosaic Virus (BSMV) RNAα, (b) a cDNA encoding a BSMV RNAβ, (c) a cDNA encoding a BSMV RNAβ1 derived from a BSMV RNAβ, (d) a cDNA 32ny-2939083Attorney Docket No: 293082000140 encoding a BSMV RNAβ2 derived from a BSMV RNAβ, (e) a cDNA encoding a BSMV RNAγ, (f) a cDNA encoding a BSMVγ1 derived from a BSMV RNAγ, or (g) a cDNA encoding a BSMVγ2 derived from a BSMV RNAγ, wherein the binary plasmid does not include a mobile element of the IS3 family. In some embodiments, the T-DNA-derived binary plasmid does not include a Tn10 transposable element. In some embodiments, the binary plasmid does not include a sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 57. In some embodiments, the T-DNA-derived binary plasmid does not include an IS1 element. In some embodiments, the T-DNA derived binary plasmid does not include a sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 58. In some embodiments, the binary plasmid is a pCass4-Rz plasmid. In some embodiments, the binary plasmid includes a cDNA encoding a Barley Stripe Mosaic Virus (BSMV) RNAα. In some embodiments, the binary plasmid includes a cDNA encoding a BSMV RNAβ. In some embodiments, the BSMV RNAβ includes a cargo nucleic acid. In some embodiments, the binary plasmid includes a cDNA encoding a BSMV RNAβ1 derived from a BSMV RNAβ. In some embodiments, the RNAβ1 includes a subgenomic RNAβ1 (sgRNAβ1) promoter. In some embodiments, the sgRNAβ1 promoter includes SEQ ID NO: 49. In some embodiments, the RNAβ1 includes a cargo nucleic acid. In some embodiments, the binary plasmid includes a cDNA encoding a BSMV RNAβ2 derived from a BSMV RNAβ. In some embodiments, the RNAβ2 includes a subgenomic RNAβ1 (sgRNAβ1) promoter. In some embodiments, the sgRNAβ1 promoter includes SEQ ID NO: 49. In some embodiments, the RNAβ2 includes a cargo nucleic acid. In some embodiments, the binary plasmid includes a cDNA encoding a BSMV RNAγ. In some embodiments, the BSMV RNAγ includes a subgenomic RNAγ (sgRNAγ) promoter. In some embodiments, the sgRNAγ promoter includes SEQ ID NO: 56. In some embodiments, the BSMV RNAγ includes a cargo nucleic acid. In some embodiments, the binary plasmid includes a cDNA encoding a BSMV RNAγ1 derived from a BSMV RNAγ. In some embodiments, the BSMV RNAγ1 includes a subgenomic RNAγ (sgRNAγ) promoter. In some embodiments, the sgRNAγ promoter includes SEQ ID NO: 56. In some embodiments, the BSMV RNAγ1 includes a cargo nucleic acid. In some embodiments, the binary plasmid includes a cDNA encoding a BSMV RNAγ2 derived from a BSMV RNAγ. In some embodiments, the BSMV RNAγ2 includes a subgenomic RNAγ (sgRNAγ) promoter. In some embodiments, the sgRNAγ promoter includes SEQ ID NO: 56. In some embodiments, the BSMV RNAγ2 includes a cargo nucleic acid. In some embodiments, the cDNA includes an endonuclease cutting site at the 3’ end of the cDNA. In some embodiments, the endonuclease cutting site is an SpeI site, a BamHI 33ny-2939083Attorney Docket No: 293082000140 site, or an MluI site. In some embodiments, the cDNA encoding the BSMV RNA or the modified BSMV RNA is operably linked to a promoter. In some embodiments, the cDNA encoding the BSMV RNA or the modified BSMV RNA is operably linked to a promoter for in vitro transcription by a DNA-dependent RNA polymerase. In some embodiments, the promoter is a T7 promoter. In some embodiments, the cDNA encoding the BSMV RNA or the modified BSMV RNA is operably linked to a promoter for in planta transcription. In some embodiments, the promoter is a CaMV 35S promoter or a 2X CaMV 35S promoter. In some embodiments, the cDNA includes a cargo nucleic acid for transient expression in planta. In some embodiments, the cargo nucleic acid encodes a genome editing component. In some embodiments, the genome editing component is selected from the group consisting of a Cas, one or more gRNAs for a Cas, an N-terminal piece of a split Cas, a C-terminal piece of a split Cas, one or more gRNAs for a split Cas, a base editor, one or more gRNAs for a base editor, a prime editor, a prime editor guide RNA (pegRNAs) for a prime editor, a zinc finger nuclease (ZFN) monomer, two ZFN monomers, a TALEN monomer, a homing meganuclease monomer or a derivative of a homing meganuclease monomer, a homing meganuclease dimer or a derivative of a homing meganuclease dimer, and a template nucleic acid. In some embodiments, the cargo nucleic acid encodes a Cas. In some embodiments, the cargo nucleic acid encoding the Cas is less than about 3 kb long. In some embodiments, the Cas is selected from the group consisting of CjCas9, a Cas12f, a Cas12j, a Cas12e, and a Cas12l. In some embodiments, the Cas is Cas12f1. In some embodiments, herein is provided an infectious RNA produced by in vitro transcription of the binary plasmid of any one of the preceding claims. In a further embodiment, herein is provided a liquid composition including the infectious RNA of the preceding embodiment, wherein the liquid composition includes: (a) BSMV RNAα, (b) BSMV RNAβ, or BSMV RNAβ1 and BSMV RNAβ2, and (c) BSMV RNAγ, or BSMV RNAγ1 and BSMV RNAγ2. In some aspects, herein is provided a method of providing a nucleic acid sequence to a plant or plant cell, the method including: (a) providing the inoculant mixture of any one of the preceding claims, or the liquid composition of the preceding claims, including at least one cargo nucleic acid; and (b) inoculating a plant or plant cell with the inoculant mixture, wherein the at least one cargo nucleic acid is provided to the plant or plant cell. In some embodiments, herein is provided a method of providing a nucleic acid sequence to a plant or plant cell, the method including: (a) providing the expression vector system of any one of the preceding embodiments, wherein the expression vector system includes at least one cargo nucleic acid sequence; (b) in vitro transcribing the vector system into infectious RNAs; and (c) inoculating a plant or plant cell with the infectious RNAs; wherein the at least one cargo nucleic 34ny-2939083Attorney Docket No: 293082000140 acid sequence is provided to the plant or plant cell. In some embodiments, herein is provided a method of transiently expressing an RNA or protein in a plant or plant cell, the method including: (a) providing the expression vector system of any one of the preceding embodiments, wherein the expression vector system includes at least one cargo nucleic acid sequence encoding an RNA or protein; (b) in vitro transcribing the vector system into infectious RNAs; and (c) inoculating a plant or plant cell with the infectious RNAs; wherein the RNA or protein encoded by the at least one cargo nucleic acid sequence is transiently expressed in the plant or plant cell. In another embodiment, herein is provided a method of providing a nucleic acid sequence to a plant or plant cell, the method including: (a) cloning a nucleic acid sequence into the cloning site of the expression vector system any one of the preceding embodiments; (b) in vitro transcribing the vector system into infectious RNAs; and (c) inoculating a plant or plant cell with the infectious RNAs; wherein the nucleic acid sequence is provided to the plant or plant cell. In yet another embodiment, herein is provided a method of transiently expressing an RNA or protein in a plant or plant cell, the method including: (a) cloning a nucleic acid sequence encoding an RNA or protein into the cloning site of the expression vector system of any one of the preceding claims; (b) in vitro transcribing the vector system into infectious RNAs; and (c) inoculating a plant or plant cell with the infectious RNAs; wherein the RNA or protein encoded by the nucleic acid sequence is transiently expressed in the plant or plant cell. In still another embodiment, herein is provided a method of editing the genome of a plant or plant cell, the method including: (a) providing the expression vector system of any one of the preceding embodiments; (b) in vitro transcribing the vector system into infectious RNAs; and (c) inoculating a plant or plant cell with the infectious RNAs; wherein the genome editing component is provided to the plant or plant cell, and wherein the genome of the plant or plant cell is edited. In some embodiments, inoculating the plant includes performing seed imbibition with the infectious RNAs. In some embodiments, inoculating the plant includes rubbing the infectious RNAs or a composition including the infectious RNAs into the surface of the plant. In some embodiments, inoculating the plant cell includes providing the infectious RNAs to the plant cell in vitro. In one embodiment, herein is provided a method of providing a nucleic acid sequence to a plant, the method including: (a) providing the expression vector system of any one of the preceding embodiments, wherein the expression vector system includes at least one cargo nucleic acid sequence; (b) transforming the vector system into Agrobacterium; (c) providing the Agrobacterium to a first plant; (d) collecting leaf sap from the first plant; and (e) inoculating a second plant with the leaf sap; wherein the cargo nucleic acid sequence is provided to the second plant. In another embodiment, herein is provided a method of transiently 35ny-2939083Attorney Docket No: 293082000140 expressing an RNA or protein in a plant, the method including: (a) providing the expression vector system of any one of the preceding embodiments, wherein the expression vector system includes at least one cargo nucleic acid sequence encoding an RNA or protein; (b) transforming the vector system into Agrobacterium; (c) providing the Agrobacterium to a first plant; (d) collecting leaf sap from the first plant; and (e) inoculating a second plant with the leaf sap; wherein the RNA or protein encoded by the at least one cargo nucleic acid sequence is transiently expressed in the second plant. In a further embodiment, herein is provided a method of providing a nucleic acid sequence to a plant, the method including: (a) cloning a nucleic acid sequence into the cloning site of the expression vector system any one of the preceding embodiments; (b) transforming the expression vector system into Agrobacterium; and (c) providing the Agrobacterium to a plant; wherein the nucleic acid sequence is provided to the plant. In another embodiment, herein is provided a method of providing a nucleic acid sequence to a plant, the method including: (a) cloning a nucleic acid sequence into the cloning site of the expression vector system any one of the preceding embodiments; (b) transforming the expression vector systemAgrobacterium; (c) providing the Agrobacterium to a first plant; (d) collecting leaf sap from the first plant; and (e) inoculating a second plant with the leaf sap; wherein the nucleic acid sequence is provided to the second plant. In another embodiment, herein is provided a method of transiently expressing an RNA or protein in a plant, the method including: (a) cloning a nucleic acid sequence into the cloning site of the expression vector system any one of the preceding claims, wherein the expression vector system includes at least one cargo nucleic acid sequence encoding an RNA or protein; (b) transforming the expression vector systemAgrobacterium; and (c) providing the Agrobacterium to a plant; wherein the RNA or protein encoded by the at least one cargo nucleic acid sequence is transiently expressed in the plant. In a further embodiment, herein is provided a method of transiently expressing an RNA or protein in a plant, the method including: (a) cloning a nucleic acid sequence into the cloning site of the expression vector system any one of the preceding embodiments, wherein the expression vector system includes at least one cargo nucleic acid sequence encoding an RNA or protein; (b) transforming the expression vector system into Agrobacterium; (c) providing the Agrobacterium to a first plant; (d) collecting leaf sap from the first plant; and (e) inoculating a second plant with the leaf sap; wherein the RNA or protein encoded by the at least one cargo nucleic acid sequence is transiently expressed in the second plant. In one embodiment, herein is provided a method of editing the genome of a plant, the method including: (a) providing the expression vector system of any one of the preceding embodiments; (b) transforming the expression vector system into Agrobacterium; and (c) providing the 36ny-2939083Attorney Docket No: 293082000140 Agrobacterium to a plant; wherein the genome editing component is provided to the plant, and wherein the genome of the plant is edited. In another embodiment, herein is provided a method of editing the genome of a plant, the method including: (a) providing the expression vector system of any one of the preceding embodiments; (b) transforming the expression vector system into Agrobacterium; (c) providing the Agrobacterium to a first plant; (d) collecting leaf sap from the first plant; and (e) inoculating a second plant with the leaf sap; wherein the genome editing component is provided to the second plant, and wherein the genome of the second plant is edited. In some embodiments, the first plant is Nicotiana benthamiana. In some embodiments, the Agrobacterium are provided to the first plant or plant by leaf infiltration. In an additional embodiment of any of the preceding method aspects, which may be combined with any of the preceding embodiments, the method further includes phenotyping and / or genotyping the plant or second plant for successful expression. In a further embodiment, which may be combined with any of the preceding embodiments, the method further includes phenotyping and / or genotyping the plant or second plant for genome editing. In another embodiment, which may be combined with any of the preceding embodiments, the method further includes harvesting seed from the plant or second plant. In some embodiments, herein is provided a method of any one of the preceding embodiments, further including germinating the seed, assaying the offspring for viral RNA, and selecting virus-free offspring. In some embodiments, assaying the offspring for viral RNA includes an RT-PCR assay. In yet another embodiment of any of the preceding method aspects, which may be combined with any of the preceding embodiments the plant, plant cell, or second plant is a monocot or monocot cell. In some embodiments, the monocot or monocot cell is selected from the group consisting of Hordeum vulgare, Zea mays, Triticum aestivum, Triticum turgidum, Oryza spp., Secale cereale, Sorghum bicolor, Avena sativa, Saccharum spp., Brachypodium distachyon, Panicum virgatum, and Miscanthus spp. In yet another embodiment of any of the preceding method aspects, which may be combined with any of the preceding embodiments, the plant, plant cell, or second plant is a dicot or dicot cell. In some embodiments, the dicot or dicot cell is selected from the group consisting of Nicotiana spp., Arabidopsis, Glycine max, Solanum lycopersicum, Solanum tuberosum, Helianthus annuus, Gossypium spp., Fagopyrum esculentum, Trifolium pratense, Brassica oleracea, Pisum sativum, Nasturtium officinale, Medicago sativa, and Raphanus sativus or a cell thereof. In some embodiments of any of the previous method aspects, which may be combined with any of the preceding embodiments, (a) the cargo nucleic acid is delivered at a higher efficiency than a cargo nucleic acid delivered with a control expression vector system, (b) one or more of the vectors have increased stability in planta compared to a 37ny-2939083Attorney Docket No: 293082000140 vector from a control expression vector system, (c) there is an increase in in vitro transcription one or more of the vectors compared to a control expression vector system, (d) the expression vector system produces more γb protein compared to a control expression vector system, (e) the expression vector system results in improved efficiency of transient expression in planta compared to a control expression vector system, and / or (f) the expression vector system results in improved efficiency of cell-to-cell movement of the viral vectors in planta compared to a control expression vector system. In some embodiments of any of the previous methods aspects, (a) the cargo nucleic acid is delivered at a higher efficiency than with a control expression vector system, (b) the vectors have increased stability in planta, and / or (c) there is an increase in in vitro transcription of the vectors. In some embodiments, the expression vector system produces more γb protein than a control expression vector system. In some embodiments, the expression vector system results in improved efficiency of transient expression in planta compared to a control expression vector system. In some embodiments, the expression vector system results in improved efficiency of cell-to-cell movement of the viral vectors in planta compared to a control expression vector system.

[0026] In one aspect, herein is provided an expression vector system including (a) a vector including a Barley Stripe Mosaic Virus (BSMV) RNAα, (b) a vector including a BSMV RNAβ, (c) a vector including a BSMV RNAγ1 derived from BSMV RNAγ, further including a functional sgRNAγ promoter, and (d) a vector including a BSMV RNAγ2 derived from BSMV RNAγ, further including a functional sgRNAγ promoter, wherein the expression vector system does not include a mobile element of the IS3 family. In some embodiments, the T-DNA- derived binary plasmid does not include a Tn10 transposable element. In some embodiments, the T-DNA-derived binary plasmid does not include an IS1 element. In some embodiments, the T-DNA derived binary plasmid does not include a sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 57 or SEQ ID NO: 58.

[0027] In one aspect, herein is provided a method of transiently expressing a nucleic acid in a plant, the method including: (a) providing: (i) a first expression vector including a BSMV RNAα, (ii) a second expression vector including a BSMV RNAβ, (iii) a third expression vector including a BSMV RNAγ1, wherein the BSMV RNAγ1 includes a subgenomic RNAγ (sgRNAγ) promotor, and (iv) a fourth expression vector including a BSMV RNAγ2, wherein the BSMV RNAγ2 includes a sgRNAγ promotor, wherein one or more cargo nucleic acids is included within (i) the BSMV RNAβ vector, (ii) the BSMV RNAγ1 vector, and / or (iii) the BSMV RNAγ2 vector; (b) transforming the first, second, third, and fourth expression vectors 38ny-2939083Attorney Docket No: 293082000140 into Agrobacterium; and (c) providing the Agrobacterium to a plant; wherein the one or more cargo nucleic acids are provided to the plant, and wherein the one or more cargo nucleic acids are transiently expressed in the plant.

[0028] In an additional aspect, herein is provided a method of transiently expressing a nucleic acid in a plant, the method including: (a) providing: (i) a first expression vector including a BSMV RNAα, (ii) a second expression vector including a BSMV RNAβ, (iii) a third expression vector including a BSMV RNAγ1, wherein the BSMV RNAγ1 includes a subgenomic RNAγ (sgRNAγ) promotor, and (iv) a fourth expression vector including a BSMV RNAγ2, wherein the BSMV RNAγ2 includes a sgRNAγ promotor, wherein one or more cargo nucleic acids is included within (i) the BSMV RNAβ vector, (ii) the BSMV RNAγ1 vector, and / or (iii) the BSMV RNAγ2 vector; (b) in vitro transcribing the first, second, third, and fourth expression vectors to produce infectious RNAs; and (c) providing the infectious RNAs to a plant; wherein the one or more cargo nucleic acids is provided to the plant, and wherein the one or more cargo nucleic acids are transiently expressed in the plant.

[0029] In an additional aspect, herein is provided an expression vector system including: (a) a vector including a Barley Stripe Mosaic Virus (BSMV) RNAα, (b) a vector including a BSMV RNAβ1 derived from BSMV RNAβ, (c) a vector including a BSMV RNAβ2 derived from BSMV RNAβ, (c) a vector including a BSMV RNAγ1 derived from BSMV RNAγ, further including a functional sgRNAγ promoter, and (d) a vector including a BSMV RNAγ2 derived from BSMV RNAγ, further including a functional sgRNAγ promoter, wherein the expression vector system does not include a mobile element of the IS3 family. In some embodiments, the T-DNA-derived binary plasmid does not include a Tn10 transposable element. In some embodiments, the T-DNA-derived binary plasmid does not include an IS1 element. In some embodiments, the T-DNA derived binary plasmid does not include a sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NOs: 57 or 58.

[0030] In another aspect, herein is provided a method of transiently expressing a nucleic acid in a plant, the method including: (a) providing: (i) a first expression vector including a BSMV RNAα, (ii) a second expression vector including a BSMV RNAβ1, (iii) a third expression vector including a BSMV RNAβ2, (iv) a fourth expression vector including a BSMV RNAγ1, wherein the BSMV RNAγ1 includes a subgenomic RNAγ (sgRNAγ) promotor; and (v) a fifth expression vector including a BSMV RNAγ2, wherein the BSMV RNAγ2 includes a sgRNAγ promotor, wherein one or more cargo nucleic acids is included within (i) the BSMV RNAβ1 vector, (ii) the BSMV RNAβ2 vector, (iii) the BSMV RNAγ1 vector, and / or (iv) the BSMV 39ny-2939083Attorney Docket No: 293082000140 RNAγ2 vector; (b) transforming the first, second, third, fourth, and fifth expression vectors into Agrobacterium; and (c) providing the Agrobacterium to a plant; wherein the one or more cargo nucleic acids are provided to the plant, and wherein the one or more cargo nucleic acids are transiently expressed in the plant.

[0031] In an additional aspect, herein is provided a method of transiently expressing a nucleic acid in a plant, the method including: (a) providing: (i) a first expression vector including a BSMV RNAα, (ii) a second expression vector including a BSMV RNAβ1, (iii) a third expression vector including a BSMV RNAβ2, (iv) a fourth expression vector including a BSMV RNAγ1, wherein the BSMV RNAγ1 includes a subgenomic RNAγ (sgRNAγ) promotor, and (v) a fifth expression vector including a BSMV RNAγ2, wherein the BSMV RNAγ2 includes a sgRNAγ promotor, wherein one or more cargo nucleic acids is included within (i) the BSMV RNAβ1 vector, (ii) the BSMV RNAβ2 vector, (iii) the BSMV RNAγ1 vector, and / or (iv) the BSMV RNAγ2 vector; (b) in vitro transcribing the first, second, third, fourth, and fifth expression vectors to produce infectious RNAs; and (c) providing the infectious RNAs to a plant; wherein the one or more cargo nucleic acids are provided to the plant, and wherein the one or more cargo nucleic acids are transiently expressed in the plant.

[0032] In one aspect, provided herein is a tobravirus-derived expression vector system for expression in plants, wherein the expression vector system includes a first vector derived from a tobravirus RNA1 and a second vector derived from a tobravirus RNA1. In some embodiments, the vector system is capable of systemic infection and / or systemic expression. In some embodiments, introducing the vector system into a plant results in systemic infection and / or systemic expression. In some embodiments, the first vector includes a cargo space or a cargo space insert. In another embodiment, the first vector includes a cargo space. In a further embodiment, the first vector includes a cargo space, wherein the cargo space is a ligation- independent cloning (LIC) site. In some embodiments, the LIC site includes SEQ ID NO: 156 or SEQ ID NO: 172. In one embodiment, the LIC site is SEQ ID NO: 156. In another embodiment, the LIC site is SEQ ID NO: 172. In an additional embodiment, the first vector includes a cargo space, wherein the cargo space is a restriction enzyme site. In yet another embodiment, the first vector includes a cargo space insert. In a further embodiment, the first vector cargo space insert is less than or equal to about 1.44kb, 840bp, or 600bp in length. In one embodiment, the first vector cargo space insert is less than or equal to about 1.44 kb in length. In one embodiment, the first vector cargo space insert is less than or equal to about 1.2 kb in length. In one embodiment, the first vector cargo space insert is less than or equal to about 840 bp in length. In one embodiment, the first vector cargo space insert is less than or equal to 40ny-2939083Attorney Docket No: 293082000140 about 700 bp in length. In one embodiment, the first vector cargo space insert is less than or equal to about 600 bp in length. In one embodiment, the first vector cargo space insert is less than or equal to about 500 bp in length. In some embodiments, which may be combined with any of the previous embodiments, the second vector includes a cargo space or a cargo space insert. In an additional embodiment, the second vector includes a cargo space. In a further embodiment, the second vector includes a cargo space, wherein the cargo space is a ligation- independent cloning (LIC) site. In some embodiments, the LIC site includes SEQ ID NO: 156 or SEQ ID NO: 172. In one embodiment, the LIC site is SEQ ID NO: 156. In another embodiment, the LIC site is SEQ ID NO: 172. In yet another embodiment, the second vector includes a cargo space, wherein the cargo space is a restriction enzyme site. In still another embodiment, the second vector includes a cargo space insert. In an additional embodiment, the second vector cargo space insert is less than or equal to about 7.2kb or 6.0kb in length. In one embodiment, the first vector cargo space insert is less than or equal to about 7.2 kb in length. In one embodiment, the first vector cargo space insert is less than or equal to about 6.0 kb in length. In one embodiment, the first vector cargo space insert is less than or equal to about 5.0 kb in length. In another embodiment, which may be combined with any of the previous embodiments, the first vector includes a replicase. In one embodiment, the first vector includes a tobravirus replicase. In some embodiments, which may be combined with any of the previous embodiments, the first vector includes a methyltransferase domain and a helicase domain of a tobravirus replicase, and the second vector includes an RNA-dependent RNA polymerase domain of a tobravirus replicase. In another embodiment, which may be combined with any of the previous embodiments, the first vector includes an RNA-dependent RNA polymerase domain of a tobravirus replicase, and the second vector includes a methyltransferase domain and a helicase domain of a tobravirus replicase. In an additional embodiment, which may be combined with any of the previous embodiments, the first vector includes a methyltransferase domain and a helicase domain of a tobravirus replicase, and the expression vector system includes a third vector derived from a tobravirus RNA1 including an RNA-dependent RNA polymerase domain of a tobravirus replicase. In yet another embodiment, which may be combined with any of the previous embodiments, the first vector includes an RNA-dependent RNA polymerase domain of a tobravirus replicase, and the expression vector system includes a third vector derived from a tobravirus RNA1 including a methyltransferase domain and a helicase domain of a tobravirus replicase. In a further embodiment, which may be combined with any of the previous embodiments, the first vector includes a viral moving protein. In one embodiment, the first vector includes a tobravirus moving protein. In a still further embodiment, 41ny-2939083Attorney Docket No: 293082000140 which may be combined with any of the previous embodiments, the second vector includes a viral moving protein. In one embodiment, the second vector includes a tobravirus moving protein. In a further embodiment, which may be combined with any of the previous embodiments, the first vector includes a viral cysteine-rich protein. In one embodiment, the first vector includes a tobravirus cysteine-rich protein. In a still further embodiment, which may be combined with any of the previous embodiments, the second vector includes a viral cysteine-rich protein. In one embodiment, the second vector includes a tobravirus cysteine-rich protein. In another embodiment, which may be combined with any of the previous embodiments, the first vector includes a subgenomic promoter operably linked to the cargo space or cargo space insert. In a further embodiment, the subgenomic promoter of the first vector is the tobravirus moving protein promoter. In an additional embodiment, the subgenomic promoter of the first vector is the pea early-browning virus coat protein promoter (PEBV CP promoter). In some embodiments, which may be combined with any of the previous embodiments, the first vector cargo space insert includes a subgenomic promoter operably linked to a nucleic acid for expression. In a further embodiment, the first vector cargo space insert includes a pea early-browning virus coat protein promoter (PEBV CP promoter). In yet another embodiment, which may be combined with any of the previous embodiments, the second vector includes a subgenomic promoter operably linked to the cargo space or cargo space insert. In one embodiment, the subgenomic promoter of the second vector is the tobravirus moving protein promoter. In another embodiment, the subgenomic promoter of the second vector is a pea early-browning virus coat protein promoter (PEBV CP promoter). In some embodiments, which may be combined with any of the previous embodiments, the second vector cargo space insert includes a subgenomic promoter operably linked to a nucleic acid for expression. In a further embodiment, the second vector cargo space insert includes a pea early- browning virus coat protein promoter (PEBV CP promoter). In another embodiment, which may be combined with any of the previous embodiments, the expression vector system further includes a first tobravirus RNA2 vector or a first vector derived from a tobravirus RNA2 vector. In an additional embodiment, the first tobravirus RNA2 vector or first tobravirus RNA2- derived vector includes a viral coat protein. In one embodiment, the first tobravirus RNA2 vector or first tobravirus RNA2-derived vector includes a tobravirus coat protein. In still another embodiment, the first tobravirus RNA2 vector or first tobravirus RNA2-derived vector includes a viral coat protein. In an additional embodiment, the first tobravirus RNA2 vector or first tobravirus RNA2-derived vector includes a viral moving protein. In one embodiment, the first tobravirus RNA2 vector or first tobravirus RNA2-derived vector includes a tobravirus 42ny-2939083Attorney Docket No: 293082000140 moving protein. In a further embodiment, the first tobravirus RNA2 vector or first tobravirus RNA2-derived vector includes a viral cysteine-rich protein. In one embodiment, the first tobravirus RNA2 vector or first tobravirus RNA2-derived vector includes a tobravirus cysteine- rich protein. In another embodiment, the first tobravirus RNA2 vector or first tobravirus RNA2- derived vector includes a cargo space or a cargo space insert. In a further embodiment, the first tobravirus RNA2 vector or first tobravirus RNA2-derived vector includes a cargo space. In a still further embodiment, the first tobravirus RNA2 vector or first tobravirus RNA2-derived vector includes a cargo space, wherein the cargo space is a ligation-independent cloning (LIC) site. In some embodiments, the LIC site includes SEQ ID NO: 156 or SEQ ID NO: 172. In one embodiment, the LIC site is SEQ ID NO: 156. In another embodiment, the LIC site is SEQ ID NO: 172. In yet another embodiment, the first tobravirus RNA2 vector or first tobravirus RNA2-derived vector includes a cargo space, wherein the cargo space is a restriction enzyme site. In an additional embodiment, the first tobravirus RNA2 vector or first tobravirus RNA2- derived vector includes a cargo space insert. In a further embodiment, the first tobravirus RNA2 vector or first tobravirus RNA2-derived vector cargo space insert is shorter than or equal to about 1.8kb or 1.2kb in length. In one embodiment, the first tobravirus RNA2 vector or first tobravirus RNA2-derived vector cargo space insert is shorter than or equal to about 1.8 kb in length. In one embodiment, the first tobravirus RNA2 vector or first tobravirus RNA2-derived vector cargo space insert is shorter than or equal to about 1.5 kb in length. In one embodiment, the first tobravirus RNA2 vector or first tobravirus RNA2-derived vector cargo space insert is shorter than or equal to about 1.2 kb in length. In one embodiment, the first tobravirus RNA2 vector or first tobravirus RNA2-derived vector cargo space insert is shorter than or equal to about 1.0 kb in length. In another embodiment, which may be combined with any of the previous embodiments, the first tobravirus RNA2 vector or first tobravirus RNA2-derived vector includes a subgenomic promoter operably linked to the cargo space or cargo space insert. In one embodiment, the subgenomic promoter of the first tobravirus RNA2 vector or first tobravirus RNA2-derived vector is the tobravirus ORF2b and ORF2c promoter. In one embodiment, the subgenomic promoter of the first tobravirus RNA2 vector or first tobravirus RNA2-derived vector is a pea early-browning virus coat protein promoter (PEBV CP promoter). In an additional embodiment, the first tobravirus RNA2 vector or first tobravirus RNA2-derived vector cargo space insert includes a subgenomic promoter operably linked to a nucleic acid for expression. In one embodiment, the first tobravirus RNA2 vector or first tobravirus RNA2-derived vector cargo space insert includes a pea early-browning virus coat protein promoter (PEBV CP promoter). In another embodiment, which may be combined with 43ny-2939083Attorney Docket No: 293082000140 any of the previous embodiments, the expression vector system further includes a second tobravirus RNA2 vector or a second vector derived from a tobravirus RNA2 vector. In an additional embodiment, the second tobravirus RNA2 vector or second tobravirus RNA2- derived vector includes a viral coat protein. In one embodiment, the second tobravirus RNA2 vector or second tobravirus RNA2-derived vector includes a tobravirus coat protein. In still another embodiment, the second tobravirus RNA2 vector or second tobravirus RNA2-derived vector includes a viral coat protein. In an additional embodiment, the second tobravirus RNA2 vector or second tobravirus RNA2-derived vector includes a viral moving protein. In one embodiment, the second tobravirus RNA2 vector or second tobravirus RNA2-derived vector includes a tobravirus moving protein. In a further embodiment, the second tobravirus RNA2 vector or second tobravirus RNA2-derived vector includes a viral cysteine-rich protein. In one embodiment, the second tobravirus RNA2 vector or second tobravirus RNA2-derived vector includes a tobravirus cysteine-rich protein. In another embodiment, the second tobravirus RNA2 vector or second tobravirus RNA2-derived vector includes a cargo space or a cargo space insert. In a further embodiment, the second tobravirus RNA2 vector or second tobravirus RNA2-derived vector includes a cargo space. In a still further embodiment, the second tobravirus RNA2 vector or second tobravirus RNA2-derived vector includes a cargo space, wherein the cargo space is a ligation-independent cloning (LIC) site. In some embodiments, the LIC site includes SEQ ID NO: 156 or SEQ ID NO: 172. In one embodiment, the LIC site is SEQ ID NO: 156. In another embodiment, the LIC site is SEQ ID NO: 172. In yet another embodiment, the second tobravirus RNA2 vector or second tobravirus RNA2-derived vector includes a cargo space, wherein the cargo space is a restriction enzyme site. In an additional embodiment, the second tobravirus RNA2 vector or second tobravirus RNA2-derived vector includes a cargo space insert. In a further embodiment, the second tobravirus RNA2 vector or second tobravirus RNA2-derived vector cargo space insert is shorter than or equal to about 1.8kb or 1.2kb in length. In one embodiment, the second tobravirus RNA2 vector or second tobravirus RNA2-derived vector cargo space insert is shorter than or equal to about 1.8 kb in length. In one embodiment, the second tobravirus RNA2 vector or second tobravirus RNA2- derived vector cargo space insert is shorter than or equal to about 1.5 kb in length. In one embodiment, the second tobravirus RNA2 vector or second tobravirus RNA2-derived vector cargo space insert is shorter than or equal to about 1.2 kb in length. In one embodiment, the second tobravirus RNA2 vector or second tobravirus RNA2-derived vector cargo space insert is shorter than or equal to about 1.0 kb in length. In another embodiment, which may be combined with any of the previous embodiments, the second tobravirus RNA2 vector or second 44ny-2939083Attorney Docket No: 293082000140 tobravirus RNA2-derived vector includes a subgenomic promoter operably linked to the cargo space or cargo space insert. In one embodiment, the subgenomic promoter of the second tobravirus RNA2 vector or second tobravirus RNA2-derived vector is the tobravirus ORF2b and ORF2c promoter. In one embodiment, the subgenomic promoter of the second tobravirus RNA2 vector or second tobravirus RNA2-derived vector is a pea early-browning virus coat protein promoter (PEBV CP promoter). In an additional embodiment, the second tobravirus RNA2 vector or second tobravirus RNA2-derived vector cargo space insert includes a subgenomic promoter operably linked to a nucleic acid for expression. In one embodiment, the second tobravirus RNA2 vector or second tobravirus RNA2-derived vector cargo space insert includes a pea early-browning virus coat protein promoter (PEBV CP promoter). In some embodiments, which may be combined with any of the previous embodiments, the first vector is derived from a Tobacco Rattle Virus (TRV) RNA1. In some embodiments, which may be combined with any of the previous embodiments, the first vector is derived from a Pea Early- Browning Virus (PEBV) RNA1. In some embodiments, which may be combined with any of the previous embodiments, the first vector is derived from a Pepper Ringspot Virus (PepRSV) RNA1. In some embodiments, which may be combined with any of the previous embodiments, the second vector is derived from a Tobacco Rattle Virus (TRV) RNA1. In some embodiments, which may be combined with any of the previous embodiments, the second vector is derived from a Pea Early-Browning Virus (PEBV) RNA1. In some embodiments, which may be combined with any of the previous embodiments, the second vector is derived from a Pepper Ringspot Virus (PepRSV) RNA1. In some embodiments, which may be combined with any of the previous embodiments, the first tobravirus RNA2 vector or first tobravirus RNA2-derived vector is derived from a TRV RNA2. In some embodiments, which may be combined with any of the previous embodiments, the first tobravirus RNA2 vector or first tobravirus RNA2- derived vector is derived from a PEBV RNA2. In some embodiments, which may be combined with any of the previous embodiments, the first tobravirus RNA2 vector or first tobravirus RNA2-derived vector is derived from a PepRSV RNA2. In some embodiments, which may be combined with any of the previous embodiments, the second tobravirus RNA2 vector or second tobravirus RNA2-derived vector is derived from a TRV RNA2. In some embodiments, which may be combined with any of the previous embodiments, the second tobravirus RNA2 vector or second tobravirus RNA2-derived vector is derived from a PEBV RNA2. In some embodiments, which may be combined with any of the previous embodiments, the second tobravirus RNA2 vector or second tobravirus RNA2-derived vector is derived from a PepRSV RNA2. In some embodiments, the first vector includes a nucleic acid encoding a tobravirus 45ny-2939083Attorney Docket No: 293082000140 replicase, and a cargo space or cargo space insert. In a further embodiment, the first vector includes, from 5’ to 3’, a nucleic acid encoding a tobravirus replicase, and a cargo space or cargo space insert. In an additional embodiment, the first vector includes a nucleic acid with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 59, SEQ ID NO: 78, or SEQ ID NO: 97. In a further embodiment, the first vector includes a nucleic acid encoding a tobravirus replicase, a nucleic acid encoding a tobravirus moving protein, and a cargo space or cargo space insert. In another embodiment, the first vector includes, from 5’ to 3’, a nucleic acid encoding a tobravirus replicase, and a cargo space or cargo space insert. In yet another embodiment, the first vector includes a nucleic acid with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 62, SEQ ID NO: 81, or SEQ ID NO: 100. In a still further embodiment, the first vector includes a nucleic acid encoding a tobravirus replicase, a cargo space or cargo space insert, and a nucleic acid encoding a tobravirus cysteine-rich protein. In another embodiment, the first vector includes, from 5’ to 3’, a nucleic acid encoding a tobravirus replicase, a cargo space or cargo space insert, and a nucleic acid encoding a tobravirus cysteine-rich protein. In a further embodiment, the first vector includes a nucleic acid with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 67, SEQ ID NO: 86, or SEQ ID NO: 105. In yet another embodiment, the second vector includes a cargo space or cargo space insert, and a nucleic acid encoding a tobravirus cysteine-rich protein. In a further embodiment, the second vector includes, from 5’ to 3’, a cargo space or cargo space insert, and a nucleic acid encoding a tobravirus cysteine-rich protein. In an additional embodiment, the second vector includes a nucleic acid with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 63, SEQ ID NO: 82, or SEQ ID NO: 101. In another embodiment, the second vector includes, from 5’ to 3’, a subgenomic promoter operably linked to a cargo space or cargo space insert, and a nucleic acid encoding a tobravirus cysteine-rich protein. In a further embodiment, the second vector includes a nucleic acid with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 64, SEQ ID NO: 83, or SEQ ID NO: 102. In yet another 46ny-2939083Attorney Docket No: 293082000140 embodiment, the second vector includes a cargo space or cargo space insert, and a nucleic acid encoding a tobravirus moving protein. In one embodiment, the second vector includes, from 5’ to 3’, a cargo space or cargo space insert, and a nucleic acid encoding a tobravirus moving protein. In a further embodiment, the second vector includes a nucleic acid with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 65, SEQ ID NO: 84, or SEQ ID NO: 103. In a still further embodiment, the second vector includes, from 5’ to 3’, a subgenomic promoter operably linked to a cargo space or cargo space insert, and a nucleic acid encoding a tobravirus moving protein. In yet another embodiment, the second vector includes a nucleic acid with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 66, SEQ ID NO: 85, or SEQ ID NO: 104. In an additional embodiment, the second vector includes a cargo space or cargo space insert, a nucleic acid encoding a tobravirus moving protein, and a nucleic acid encoding a tobravirus cysteine-rich protein. In a further embodiment, the second vector includes, from 5’ to 3’, a cargo space or cargo space insert, and a nucleic acid encoding a tobravirus cysteine-rich protein. In yet another embodiment, the second vector includes a nucleic acid with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 63, SEQ ID NO: 82, or SEQ ID NO: 101. In a still further embodiment, the second vector includes, from 5’ to 3’, a subgenomic promoter operably linked to a cargo space or cargo space insert, and a nucleic acid encoding a tobravirus cysteine-rich protein. In an additional embodiment, the second vector includes a nucleic acid with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 64, SEQ ID NO: 83, or SEQ ID NO: 102. In a further embodiment, the second vector includes a cargo space or cargo space insert, and a nucleic acid encoding a tobravirus moving protein. In an additional embodiment, the second vector includes, from 5’ to 3’, a cargo space or cargo space insert, and a nucleic acid encoding a tobravirus moving protein. In still another embodiment, the second vector includes a nucleic acid with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 65, SEQ ID NO: 84, or SEQ ID NO: 103. In an additional embodiment, the second vector includes, from 5’ to 3’, a subgenomic promoter 47ny-2939083Attorney Docket No: 293082000140 operably linked to a cargo space or cargo space insert, and a nucleic acid encoding a tobravirus moving protein. In a further embodiment, the second vector includes a nucleic acid with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 66, SEQ ID NO: 85, or SEQ ID NO: 104. In another embodiment, the second vector includes a cargo space or cargo space insert, a nucleic acid encoding a tobravirus moving protein, and a nucleic acid encoding a tobravirus cysteine-rich protein. In a further embodiment, the second vector includes, from 5’ to 3’, a cargo space or cargo space insert, a nucleic acid encoding a tobravirus moving protein, and a nucleic acid encoding a tobravirus cysteine-rich protein. In an additional embodiment, the second vector includes a nucleic acid with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 60, SEQ ID NO: 79, or SEQ ID NO: 98. In one embodiment, the expression vector system includes a vector derived from a tobravirus RNA1 with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 68, SEQ ID NO: 87, or SEQ ID NO: 106. In one embodiment, the expression vector system includes a vector derived from a tobravirus RNA1 with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 69, SEQ ID NO: 88, or SEQ ID NO: 107. In an additional embodiment, the expression vector system includes a vector derived from a tobravirus RNA1 with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 72, SEQ ID NO: 91, or SEQ ID NO: 110. In an additional embodiment, the expression vector system includes a vector derived from a tobravirus RNA1 with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 70, SEQ ID NO: 89, or SEQ ID NO: 108. In an additional embodiment, the expression vector system includes a vector derived from a tobravirus RNA1 with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 71, SEQ ID NO: 90, or SEQ ID NO: 109. In an additional embodiment, the expression vector system includes a vector derived from 48ny-2939083Attorney Docket No: 293082000140 a tobravirus RNA1 with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 73, SEQ ID NO: 92, or SEQ ID NO: 111. In still another embodiment, the second vector includes, from 5’ to 3’, a subgenomic promoter operably linked to a cargo space or cargo space insert, a nucleic acid encoding a tobravirus moving protein, and a nucleic acid encoding a tobravirus cysteine-rich protein. In a further embodiment, the second vector includes a nucleic acid with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 61, SEQ ID NO: 80, or SEQ ID NO: 99. In an additional embodiment, the first tobravirus RNA2 vector or first tobravirus RNA2-derived vector includes a nucleic acid encoding a tobravirus coat protein, a nucleic acid encoding a tobravirus moving protein, and a cargo space or cargo space insert. In a further embodiment, the first tobravirus RNA2 vector or first tobravirus RNA2-derived vector includes, from 5’ to 3’, a nucleic acid encoding a tobravirus coat protein, a nucleic acid encoding a tobravirus moving protein, and a cargo space or cargo space insert. In still another embodiment, the first tobravirus RNA2 vector or first tobravirus RNA2-derived vector includes a nucleic acid with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 76, SEQ ID NO: 95, or SEQ ID NO: 114. In an additional embodiment, the first tobravirus RNA2 vector or first tobravirus RNA2-derived vector includes, from 5’ to 3’, a nucleic acid encoding a tobravirus coat protein, a nucleic acid encoding a tobravirus moving protein, and a subgenomic promoter operably linked to a cargo space or cargo space insert. In yet another embodiment, the first tobravirus RNA2 vector or first tobravirus RNA2-derived vector includes a nucleic acid with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 77, SEQ ID NO: 96, or SEQ ID NO: 115. In a still further embodiment, the first tobravirus RNA2 vector or first tobravirus RNA2- derived vector includes a nucleic acid encoding a tobravirus coat protein, and a cargo space or cargo space insert. In yet another embodiment, the first tobravirus RNA2 vector or first tobravirus RNA2-derived vector includes, from 5’ to 3’, a nucleic acid encoding a tobravirus coat protein, and a cargo space or cargo space insert. In an additional embodiment, the first tobravirus RNA2 vector or first tobravirus RNA2-derived vector includes a nucleic acid with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, 49ny-2939083Attorney Docket No: 293082000140 at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 74, SEQ ID NO: 93, or SEQ ID NO: 112. In a further embodiment, the first tobravirus RNA2 vector or first tobravirus RNA2-derived vector includes, from 5’ to 3’, a nucleic acid encoding a tobravirus coat protein, and a subgenomic promoter operably linked to a cargo space or cargo space insert. In an additional embodiment, the first tobravirus RNA2 vector or first tobravirus RNA2-derived vector includes a nucleic acid with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 75, SEQ ID NO: 94, or SEQ ID NO: 113. In one embodiment, the expression system includes: (i) a first vector including a nucleic acid encoding a tobravirus replicase, and a cargo space or cargo space insert; (ii) a second vector including a cargo space or cargo space insert, and a nucleic acid encoding a tobravirus cysteine-rich protein; and (iii) a third vector including a nucleic acid encoding a tobravirus coat protein, a nucleic acid encoding a tobravirus moving protein, and a cargo space or cargo space insert. In one embodiment, the expression system includes: (i) a first vector including a nucleic acid encoding a tobravirus replicase, and a cargo space or cargo space insert; (ii) a second vector including a cargo space or cargo space insert, a nucleic acid encoding a tobravirus moving protein, and a nucleic acid encoding a tobravirus cysteine-rich protein; and (iii) a third vector including a nucleic acid encoding a tobravirus coat protein, and a cargo space or cargo space insert. In one embodiment, the expression system includes: (i) a first vector including a nucleic acid encoding a tobravirus replicase, a cargo space or cargo space insert, and a nucleic acid encoding a tobravirus cysteine- rich protein; (ii) a second vector including a cargo space or cargo space insert, and a nucleic acid encoding a tobravirus moving protein; and (iii) a third vector including a nucleic acid encoding a tobravirus coat protein, and a cargo space or cargo space insert. In one embodiment, the expression system includes: (i) a first vector comprising a nucleic acid encoding a tobravirus replicase, and a cargo space or cargo space insert; (ii) a second vector comprising a cargo space or cargo space insert, and a nucleic acid encoding a tobravirus cysteine-rich protein; (iii) a third vector comprising a cargo space or cargo space insert, and a nucleic acid encoding a tobravirus moving protein; and (iv) a fourth vector comprising a nucleic acid encoding a tobravirus coat protein, and a cargo space or cargo space insert. In one embodiment, the expression system includes: (i) a first vector comprising a nucleic acid encoding a methyltransferase domain and a helicase domain of a tobravirus, and a cargo space or cargo space insert; (ii) a second vector comprising a cargo space or a cargo space insert, and a nucleic acid encoding an RNA-dependent RNA polymerase domain of a tobravirus replicase; (iii) a 50ny-2939083Attorney Docket No: 293082000140 third vector comprising a cargo space or cargo space insert, and a nucleic acid encoding a tobravirus cysteine-rich protein; (iv) a fourth vector comprising a cargo space or cargo space insert, and a nucleic acid encoding a tobravirus moving protein; and (v) a fifth vector comprising a nucleic acid encoding a tobravirus coat protein, and a cargo space or cargo space insert.

[0033] In another aspect, provided herein is a tobravirus-derived expression vector system for expression in plants, wherein the expression vector system includes a first vector derived from a tobravirus RNA2 and a second vector derived from a tobravirus RNA2, wherein each of the first vector and second vector include a cloning site and an endogenous tobravirus open reading frame (ORF). In an additional embodiment, the first vector includes a viral coat protein. In one embodiment, the first vector includes a tobravirus coat protein. In still another embodiment, the first vector includes a viral coat protein. In an additional embodiment, the first vector includes a viral moving protein. In one embodiment, the first vector includes a tobravirus moving protein. In a further embodiment, the first vector includes a viral cysteine- rich protein. In one embodiment, the first vector includes a tobravirus cysteine-rich protein. In another embodiment, the first vector includes a cargo space or a cargo space insert. In a further embodiment, the first vector includes a cargo space. In a still further embodiment, the first tobravirus vector includes a cargo space, wherein the cargo space is a ligation-independent cloning (LIC) site. In some embodiments, the LIC site includes SEQ ID NO: 156 or SEQ ID NO: 172. In one embodiment, the LIC site is SEQ ID NO: 156. In another embodiment, the LIC site is SEQ ID NO: 172. In yet another embodiment, the first vector includes a cargo space, wherein the cargo space is a restriction enzyme site. In an additional embodiment, the first vector includes a cargo space insert. In a further embodiment, the first vector cargo space insert is shorter than or equal to about 1.8kb or 1.2kb in length. In one embodiment, the first vector cargo space insert is shorter than or equal to about 1.8 kb in length. In one embodiment, the first vector cargo space insert is shorter than or equal to about 1.5 kb in length. In one embodiment, the first vector cargo space insert is shorter than or equal to about 1.2 kb in length. In one embodiment, the first vector cargo space insert is shorter than or equal to about 1.0 kb in length. In another embodiment, which may be combined with any of the previous embodiments, the first vector includes a subgenomic promoter operably linked to the cargo space or cargo space insert. In one embodiment, the subgenomic promoter of the first vector is the tobravirus ORF2b and ORF2c promoter. In one embodiment, the subgenomic promoter of the first vector is a pea early-browning virus coat protein promoter (PEBV CP promoter). In an additional embodiment, the first vector cargo space insert includes a subgenomic promoter 51ny-2939083Attorney Docket No: 293082000140 operably linked to a nucleic acid for expression. In one embodiment, the first vector cargo space insert includes a pea early-browning virus coat protein promoter (PEBV CP promoter). In an additional embodiment, the second vector includes a viral coat protein. In one embodiment, the second vector includes a tobravirus coat protein. In still another embodiment, the second vector includes a viral coat protein. In an additional embodiment, the second vector includes a viral moving protein. In one embodiment, the second vector includes a tobravirus moving protein. In a further embodiment, the second vector includes a viral cysteine-rich protein. In one embodiment, the second vector includes a tobravirus cysteine-rich protein. In another embodiment, the second vector includes a cargo space or a cargo space insert. In a further embodiment, the second vector includes a cargo space. In a still further embodiment, the second vector includes a cargo space, wherein the cargo space is a ligation-independent cloning (LIC) site. In some embodiments, the LIC site includes SEQ ID NO: 156 or SEQ ID NO: 172. In one embodiment, the LIC site is SEQ ID NO: 156. In another embodiment, the LIC site is SEQ ID NO: 172. In yet another embodiment, the second vector includes a cargo space, wherein the cargo space is a restriction enzyme site. In an additional embodiment, the second vector includes a cargo space insert. In a further embodiment, the second vector cargo space insert is shorter than or equal to about 1.8kb or 1.2kb in length. In one embodiment, the second vector cargo space insert is shorter than or equal to about 1.8 kb in length. In one embodiment, the second vector cargo space insert is shorter than or equal to about 1.5 kb in length. In one embodiment, the second vector cargo space insert is shorter than or equal to about 1.2 kb in length. In one embodiment, the second vector cargo space insert is shorter than or equal to about 1.0 kb in length. In another embodiment, which may be combined with any of the previous embodiments, the second vector includes a subgenomic promoter operably linked to the cargo space or cargo space insert. In one embodiment, the subgenomic promoter of the second vector is the tobravirus ORF2b and ORF2c promoter. In one embodiment, the subgenomic promoter of the second vector is a pea early-browning virus coat protein promoter (PEBV CP promoter). In an additional embodiment, the second vector cargo space insert includes a subgenomic promoter operably linked to a nucleic acid for expression. In one embodiment, the second vector cargo space insert includes a pea early-browning virus coat protein promoter (PEBV CP promoter). In some embodiments, which may be combined with any of the previous embodiments, the first vector is derived from a Tobacco Rattle Virus (TRV) RNA2. In some embodiments, which may be combined with any of the previous embodiments, the first vector is derived from a Pea Early-Browning Virus (PEBV) RNA2. In some embodiments, which may be combined with any of the previous embodiments, the first vector 52ny-2939083Attorney Docket No: 293082000140 is derived from a Pepper Ringspot Virus (PepRSV) RNA2. In some embodiments, which may be combined with any of the previous embodiments, the second vector is derived from a Tobacco Rattle Virus (TRV) RNA2. In some embodiments, which may be combined with any of the previous embodiments, the second vector is derived from a Pea Early-Browning Virus (PEBV) RNA2. In some embodiments, which may be combined with any of the previous embodiments, the second vector is derived from a Pepper Ringspot Virus (PepRSV) RNA2. In some embodiments, which may be combined with any of the previous embodiments, the expression vector system further includes a first tobravirus RNA1 vector or first tobravirus RNA1-derived vector. In some embodiments, which may be combined with any of the previous embodiments, the expression vector system further includes a second tobravirus RNA1 vector or second tobravirus RNA1-derived vector. In some embodiments, the first tobravirus RNA1 vector or first tobravirus RNA1-derived vector includes a cargo space or a cargo space insert. In another embodiment, the first tobravirus RNA1 vector or first tobravirus RNA1-derived vector includes a cargo space. In a further embodiment, the first tobravirus RNA1 vector or first tobravirus RNA1-derived vector includes a cargo space, wherein the cargo space is a ligation-independent cloning (LIC) site. In some embodiments, the LIC site includes SEQ ID NO: 156 or SEQ ID NO: 172. In one embodiment, the LIC site is SEQ ID NO: 156. In another embodiment, the LIC site is SEQ ID NO: 172. In an additional embodiment, the first tobravirus RNA1 vector or first tobravirus RNA1-derived vector includes a cargo space, wherein the cargo space is a restriction enzyme site. In yet another embodiment, the first tobravirus RNA1 vector or first tobravirus RNA1-derived vector includes a cargo space insert. In a further embodiment, the first tobravirus RNA1 vector or first tobravirus RNA1-derived vector cargo space insert is less than or equal to about 1.44kb, 840bp, or 600bp in length. In one embodiment, the first tobravirus RNA1 vector or first tobravirus RNA1-derived vector cargo space insert is less than or equal to about 1.44 kb in length. In one embodiment, the first tobravirus RNA1 vector or first tobravirus RNA1-derived vector cargo space insert is less than or equal to about 1.2 kb in length. In one embodiment, the first tobravirus RNA1 vector or first tobravirus RNA1- derived vector cargo space insert is less than or equal to about 840 bp in length. In one embodiment, the first tobravirus RNA1 vector or first tobravirus RNA1-derived vector cargo space insert is less than or equal to about 700 bp in length. In one embodiment, the first tobravirus RNA1 vector or first tobravirus RNA1-derived vector cargo space insert is less than or equal to about 600 bp in length. In one embodiment, the first tobravirus RNA1 vector or first tobravirus RNA1-derived vector cargo space insert is less than or equal to about 500 bp in length. In some embodiments, which may be combined with any of the previous embodiments, 53ny-2939083Attorney Docket No: 293082000140 the second tobravirus RNA1 vector or first tobravirus RNA1-derived vector includes a cargo space or a cargo space insert. In an additional embodiment, the second tobravirus RNA1 vector or first tobravirus RNA1-derived vector includes a cargo space. In a further embodiment, the second tobravirus RNA1 vector or first tobravirus RNA1-derived vector includes a cargo space, wherein the cargo space is a ligation-independent cloning (LIC) site. In some embodiments, the LIC site includes SEQ ID NO: 156 or SEQ ID NO: 172. In one embodiment, the LIC site is SEQ ID NO: 156. In another embodiment, the LIC site is SEQ ID NO: 172. In yet another embodiment, the second tobravirus RNA1 vector or first tobravirus RNA1-derived vector includes a cargo space, wherein the cargo space is a restriction enzyme site. In still another embodiment, the second tobravirus RNA1 vector or first tobravirus RNA1-derived vector includes a cargo space insert. In an additional embodiment, the second tobravirus RNA1 vector or first tobravirus RNA1-derived vector cargo space insert is less than or equal to about 7.2kb or 6.0kb in length. In one embodiment, the first tobravirus RNA1 vector or first tobravirus RNA1-derived vector cargo space insert is less than or equal to about 7.2 kb in length. In one embodiment, the first tobravirus RNA1 vector or first tobravirus RNA1-derived vector cargo space insert is less than or equal to about 6.0 kb in length. In one embodiment, the first tobravirus RNA1 vector or first tobravirus RNA1-derived vector cargo space insert is less than or equal to about 5.0 kb in length. In another embodiment, which may be combined with any of the previous embodiments, the first tobravirus RNA1 vector or first tobravirus RNA1- derived vector includes a replicase. In one embodiment, the first tobravirus RNA1 vector or first tobravirus RNA1-derived vector includes a tobravirus replicase. In some embodiments, which may be combined with any of the previous embodiments, the first tobravirus RNA1 vector or first tobravirus RNA1-derived vector includes a methyltransferase domain and a helicase domain of a tobravirus replicase, and the second tobravirus RNA1 vector or first tobravirus RNA1-derived vector includes an RNA-dependent RNA polymerase domain of a tobravirus replicase. In another embodiment, which may be combined with any of the previous embodiments, the first tobravirus RNA1 vector or first tobravirus RNA1-derived vector includes an RNA-dependent RNA polymerase domain of a tobravirus replicase, and the second tobravirus RNA1 vector or first tobravirus RNA1-derived vector includes a methyltransferase domain and a helicase domain of a tobravirus replicase. In a further embodiment, which may be combined with any of the previous embodiments, the first tobravirus RNA1 vector or first tobravirus RNA1-derived vector includes a viral moving protein. In one embodiment, the first tobravirus RNA1 vector or first tobravirus RNA1-derived vector includes a tobravirus moving protein. In a still further embodiment, which may be combined with any of the previous 54ny-2939083Attorney Docket No: 293082000140 embodiments, the second tobravirus RNA1 vector or first tobravirus RNA1-derived vector includes a viral moving protein. In one embodiment, the second tobravirus RNA1 vector or first tobravirus RNA1-derived vector includes a tobravirus moving protein. In a further embodiment, which may be combined with any of the previous embodiments, the first tobravirus RNA1 vector or first tobravirus RNA1-derived vector includes a viral cysteine-rich protein. In one embodiment, the first tobravirus RNA1 vector or first tobravirus RNA1-derived vector includes a tobravirus cysteine-rich protein. In a still further embodiment, which may be combined with any of the previous embodiments, the second tobravirus RNA1 vector or first tobravirus RNA1-derived vector includes a viral cysteine-rich protein. In one embodiment, the second tobravirus RNA1 vector or first tobravirus RNA1-derived vector includes a tobravirus cysteine-rich protein.

[0034] In some embodiments of either of the previous aspects, which may be combined with any of the previous embodiments, one or more vectors is composed of cDNA. In a further embodiment, the cDNA encodes a tobravirus RNA or a modified tobravirus RNA that can be transcribed in vitro or in planta. In an additional embodiment, the cDNA is included within a plasmid. In another embodiment, the cDNA includes an endonuclease cutting site at the 3’ end of the cDNA. In yet another embodiment, the endonuclease cutting site is an SpeI site, a BamHI site, or an MluI site. In a further embodiment, the cDNA encoding the tobravirus RNA or the modified tobravirus RNA is operably linked to a promoter. In another embodiment, the cDNA encoding the tobravirus RNA or the modified tobravirus RNA is operably linked to a promoter for in vitro transcription by a DNA-dependent RNA polymerase. In yet another embodiment, the promoter for in vitro transcription is a T7 promoter. In an additional embodiment, the cDNA encoding the tobravirus RNA or the modified tobravirus RNA is operably linked to a terminator. In one embodiment, the terminator is a 35S terminator. In another embodiment, the cDNA encoding the tobravirus RNA or the modified tobravirus RNA is operably linked to a self- cleaving ribozyme. In an additional embodiment, the cDNA encoding the tobravirus RNA or the modified tobravirus RNA is operably linked to a promoter for in planta transcription. In one embodiment, the promoter is a CaMV 35S promoter or a 2X CaMV 35S promoter. In some embodiments, which may be combined with any of the previous embodiments, the expression vector system includes a first cargo insert including nucleic acid for transient expression in planta. In some embodiments, the first cargo insert includes nucleic acid encoding a genome editing component. In a further embodiment, the first cargo insert includes nucleic acid encoding a Cas protein, optionally wherein the first cargo insert is on the second vector. In another embodiment, the nucleic acid encoding the Cas protein is less than or equal to about 55ny-2939083Attorney Docket No: 293082000140 6kb in length. In an additional embodiment, the Cas protein is selected from the group consisting of Cas9, Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas12f, Cas12g, Cas12h, Cas12i, Cas12j, Cas12k, Cas12l, and Cas12m. In a further embodiment, the Cas protein is a modified Cas protein, optionally wherein the Cas protein is a nickase or a nuclease-deficient Cas protein. In yet another embodiment, the first cargo insert further includes nucleic acid encoding one or more guide RNA(s) for the Cas protein. In a still further embodiment, the vector system includes a second cargo insert including nucleic acid encoding: (a) one or more guide RNA(s) for the Cas protein, (b) a repair template with homology arms for homology- directed repair (HDR), and / or (c) an expression cassette for CRISPR-based gene knockout, knock-in, or HDR. In a further embodiment, the vector system includes a third cargo insert including nucleic acid encoding: (a) one or more guide RNA(s) for the Cas protein, (b) a repair template with homology arms for homology-directed repair (HDR), and / or (c) an expression cassette for CRISPR-based gene knockout, knock-in, or HDR. In an additional embodiment, the first cargo insert includes nucleic acid encoding a base editor protein, optionally wherein the first cargo insert is on the second vector. In a further embodiment, the first cargo insert further includes nucleic acid encoding one or more guide RNA(s) for the base editor protein. In an additional embodiment, the vector system includes a second cargo insert including nucleic acid encoding one or more guide RNA(s) for the base editor protein. In another embodiment, the vector system includes a third cargo insert including nucleic acid encoding one or more guide RNA(s) for the base editor protein. In one embodiment, the base editor is a guanine base editor. In one embodiment, the base editor is a cytosine base editor. In one embodiment, the base editor is an adenine base editor. In one embodiment, the base editor is a thymine base editor. In some embodiments, the base editor is fused to a uracil DNA glycosylase inhibitor (UGI). In an additional embodiment, the first cargo insert includes nucleic acid encoding a prime editor protein, optionally wherein the first cargo insert is on the second vector. In another embodiment, the first cargo insert further includes nucleic acid encoding one or more prime editor guide RNA(s) (pegRNA(s)) for the prime editor protein. In yet another embodiment, the expression vector system further includes a second cargo nucleic acid including one or more prime editor guide RNA(s) (pegRNA(s)) for the prime editor. In a still further embodiment, the expression vector system further includes a third cargo nucleic acid including one or more prime editor guide RNA(s) (pegRNA(s)) for the prime editor. In an additional embodiment, the first cargo insert includes nucleic acid encoding a first zinc finger nuclease (ZFN) monomer. In another embodiment, the first cargo insert includes nucleic acid encoding one, two, three, four, or five zinc finger nuclease (ZFN) monomers, optionally 56ny-2939083Attorney Docket No: 293082000140 wherein the first cargo insert is on the second vector. In a further embodiment, the expression vector system further includes a second cargo insert, wherein the second cargo insert includes nucleic acid encoding a zinc finger nuclease (ZFN) monomer, optionally wherein the second cargo insert is on the first vector. In an additional embodiment, the expression vector system further includes a third cargo insert, wherein the third cargo insert includes nucleic acid encoding one or two zinc finger nuclease (ZFN) monomers, optionally wherein the third cargo insert is on the third vector. In yet another embodiment, the first cargo insert includes nucleic acid encoding a TALEN monomer. In an additional embodiment, the first cargo insert includes nucleic acid encoding two long (L) TALEN monomers, optionally wherein the first cargo insert is on the second vector. In a further embodiment, the first cargo insert includes nucleic acid encoding a long (L) TALEN monomer and a short (S) TALEN monomer, optionally wherein the first cargo insert is on the second vector. In another embodiment, the expression vector system further includes a second cargo nucleic acid encoding a TALEN monomer. In an additional embodiment, the expression vector system further includes a second cargo nucleic acid encoding a short TALEN monomer. In yet another embodiment, the expression vector system further includes a third cargo nucleic acid encoding a short TALEN monomer. In a still further embodiment, the expression vector system further includes a second cargo nucleic acid encoding a short TALEN monomer. In an additional embodiment the expression vector system further includes a third cargo nucleic acid encoding a long TALEN monomer. In a further embodiment, the first cargo insert includes nucleic acid encoding a homing meganuclease or a derivative of a homing meganuclease. In an additional embodiment, the first cargo insert includes nucleic acid encoding one, two, three, four, or five homing meganucleases, optionally wherein the first cargo insert is on the second vector. In a still further embodiment, the expression vector system further includes a second cargo insert, wherein the second cargo insert includes nucleic acid encoding a homing meganuclease, optionally wherein the second cargo insert is on the first vector. In yet another embodiment, the expression vector system further includes a third cargo insert, wherein the third cargo insert includes nucleic acid encoding one or two homing meganucleases, optionally wherein the third cargo insert is on the third vector. In another embodiment, which may be combined with any of the previous embodiments, the cDNA is cloned into a T-DNA-derived binary plasmid. In one embodiment, the T-DNA-derived binary plasmid is a pCass4-Rz plasmid.

[0035] In another aspect, provided herein is an infectious RNA produced by in vitro transcription of a vector of the expression vector system of any one of the previous embodiments of the expression vector system aspects. 57ny-2939083Attorney Docket No: 293082000140

[0036] In another aspect, provided herein in a liquid composition including the infectious RNA of the preceding embodiment. Also provided herein is a liquid composition including infectious RNAs produced by in vitro transcription of each vector of the expression vector system of any one of the preceding embodiments of the expression vector system aspects. Also provided herein is a liquid composition including infectious RNAs produced by in vitro transcription of each vector of the expression vector system of any one of the preceding embodiments of the expression vector system aspects. Further provided herein is an inoculant mixture including infectious RNAs produced by in vitro transcription of each vector of the expression vector system of any one of the previous embodiments of the expression vector system aspects.

[0037] In a further aspect, provided herein is an inoculant mixture including infectious RNAs, wherein the mixture includes: (a) a first tobravirus RNA1-derived RNA, (b) a second tobravirus RNA1-derived RNA, and (c) a tobravirus RNA2 or tobravirus RNA2-derived RNA, and wherein at least one infectious RNA includes a cargo space insert including nucleic acid. In a further embodiment, at least one tobravirus RNA1-derived RNA includes a cargo space insert including nucleic acid. In yet another embodiment, the first tobravirus RNA1-derived RNA or the second tobravirus RNA1-derived RNA includes nucleic acid encoding a tobravirus replicase. In yet another embodiment, which may be combined with any of the previous embodiments, the first tobravirus RNA1-derived RNA and / or the second tobravirus RNA1- derived RNA includes a tobravirus moving protein. In a still further embodiment, which may be combined with any of the previous embodiments, the first tobravirus RNA1-derived RNA and / or the second tobravirus RNA1-derived RNA includes a tobravirus cysteine-rich protein. In an additional embodiment, which may be combined with any of the previous embodiments, the first tobravirus RNA1-derived RNA and / or the second tobravirus RNA1-derived RNA includes a cargo space insert including nucleic acid. In another embodiment, which may be combined with any of the previous embodiments, the tobravirus RNA2 or tobravirus RNA2- derived RNA includes a cargo space insert including nucleic acid. In some embodiments, which may be combined with any of the previous embodiments, at least one infectious RNA includes a cargo nucleic acid encoding a genome editing component.

[0038] In a further aspect, provided herein is a T-DNA-derived binary plasmid, including a cDNA of the expression vector of any one embodiment of a preceding expression vector system aspect. In one embodiment, the binary plasmid is a pCass4-Rz plasmid. In an additional embodiment, the cDNA includes a cargo space insert including nucleic acid. In a further embodiment, which may be combined with any of the previous embodiments, the cDNA 58ny-2939083Attorney Docket No: 293082000140 includes an endonuclease cutting site at the 3’ end of the cDNA. In a still further embodiment, the endonuclease cutting site is an SpeI site, a BamHI site, or an MluI site. In yet another embodiment, which may be combined with any of the previous embodiments, the cDNA is operably linked to a promoter. In another embodiment, which may be combined with any of the previous embodiments, the cDNA is operably linked to a promoter for in vitro transcription by a DNA-dependent RNA polymerase. In one embodiment, the promoter is a T7 promoter. In an additional embodiment, which may be combined with any of the previous embodiments, the cDNA encoding the expression vector is operably linked to a promoter for in planta transcription. In another embodiment, the promoter is a CaMV 35S promoter or a 2X CaMV 35S promoter. In some embodiments, which may be combined with any of the previous embodiments, the cDNA includes a cargo space insert including nucleic acid for transient expression in planta. In a further embodiment, the nucleic acid encodes or provides a genome editing component. In yet another embodiment, the genome editing component is selected from the group consisting of a Cas, one or more gRNAs for a Cas, a base editor, one or more gRNAs for a base editor, a prime editor, a prime editor guide RNA (pegRNAs) for a prime editor, a zinc finger nuclease (ZFN) monomer, two ZFN monomers, three ZFN monomers, four ZFN monomers, five ZFN monomers, a TALEN monomer, a long TALEN monomer, a short TALEN monomer, a pair of long TALEN monomers, a long and a short TALEN monomer, a homing meganuclease monomer, two homing meganuclease monomers, three homing meganuclease monomers, four homing meganuclease monomers, five homing meganuclease monomers, and a template nucleic acid. In some embodiments, the nucleic acid encodes a Cas. In some embodiments, the Cas is selected from the group consisting of Cas9, Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas12f, Cas12g, Cas12h, Cas12i, Cas12j, Cas12k, Cas12l, and Cas12m.

[0039] In an additional aspect, provided herein is an infectious RNA produced by in vitro transcription of the binary plasmid of any one embodiment of a preceding binary plasmid aspect.

[0040] In an additional aspect, provided herein is a liquid composition including the infectious RNA of any embodiment of a previous infectious RNA aspect, wherein the liquid composition includes: (a) a first tobravirus RNA1-derived RNA and a second tobravirus RNA1-derived RNA, and (b) a tobravirus RNA2 or tobravirus RNA2-derived RNA; wherein (a) and / or (b) include one or more RNAs encoding a tobravirus replicase, a tobravirus moving protein, a tobravirus cysteine-rich protein, and a tobravirus coat protein, and further including at least one cargo space insert including nucleic acid. 59ny-2939083Attorney Docket No: 293082000140

[0041] In one aspect, provided herein is a method of providing a nucleic acid sequence to a plant or plant cell, the method including: (a) providing the inoculant mixture of any one embodiment of the preceding inoculant mixture aspects, or the liquid composition of any one embodiment of the preceding liquid composition aspects, including at least one cargo nucleic acid including nucleic acid; and (b) inoculating a plant or plant cell with the inoculant mixture, wherein the at least one cargo space insert is provided to the plant or plant cell.

[0042] In one aspect, herein is provided a method of providing a nucleic acid sequence to a plant or plant cell, the method including: (a) providing the expression vector system of any one embodiment of the preceding expression vector aspects, wherein the expression vector system includes at least one cargo space insert including nucleic acid; (b) in vitro transcribing the vector system into infectious RNAs; and (c) inoculating a plant or plant cell with the infectious RNAs; wherein the at least one nucleic acid is provided to the plant or plant cell.

[0043] In an additional aspect, provided herein is a method of transiently expressing an RNA or protein in a plant or plant cell, the method including: (a) providing the expression vector system of any one embodiment of the preceding expression vector system aspects, wherein the expression vector system includes at least one cargo space insert including nucleic acid encoding an RNA or protein; (b) in vitro transcribing the vector system into infectious RNAs; and (c) inoculating a plant or plant cell with the infectious RNAs; wherein the RNA or protein(s) encoded by the at least one nucleic acid are transiently expressed in the plant or plant cell. In an additional aspect, provided herein is a method of providing a nucleic acid sequence to a plant or plant cell, the method including: (a) cloning a nucleic acid sequence into the cloning site of the expression vector system of any one embodiment of the preceding expression vector system aspects; (b) in vitro transcribing the vector system into infectious RNAs; and (c) inoculating a plant or plant cell with the infectious RNAs; wherein the nucleic acid sequence is provided to the plant or plant cell.

[0044] In yet another aspect, provided herein is a method of transiently expressing an RNA or protein in a plant or plant cell, the method including: (a) cloning a nucleic acid sequence encoding an RNA or protein into the cloning site of the expression vector system of any one embodiment of the preceding expression vector aspects; (b) in vitro transcribing the vector system into infectious RNAs; and (c) inoculating a plant or plant cell with the infectious RNAs; wherein the RNA or protein encoded by the nucleic acid sequence is transiently expressed in the plant or plant cell.

[0045] In a still further aspect, provided herein is a method of editing the genome of a plant or plant cell, the method including: (a) providing the expression vector system of any one 60ny-2939083Attorney Docket No: 293082000140 embodiment of the preceding expression vector aspects; (b) in vitro transcribing the vector system into infectious RNAs; and (c) inoculating a plant or plant cell with the infectious RNAs; wherein the genome editing component is provided to the plant or plant cell, and wherein the genome of the plant or plant cell is edited. In some embodiments, inoculating the plant includes performing seed imbibition with the infectious RNAs. In some embodiments, inoculating the plant includes rubbing the infectious RNAs or a composition including the infectious RNAs onto the surface of the plant. In some embodiments, inoculating the plant cell includes providing the infectious RNAs to the plant cell in vitro.

[0046] In an additional aspect, provided herein is a method of providing a nucleic acid sequence to a plant, the method including: (a) providing the expression vector system of any one embodiment of the preceding expression vector aspects, wherein the expression vector system includes at least one cargo space insert including nucleic acid sequence; (b) transforming the vector system into Agrobacterium; (c) providing the Agrobacterium to a first plant; (d) collecting leaf sap from the first plant; and (e) inoculating a second plant with the leaf sap; wherein the nucleic acid sequence is provided to the second plant.

[0047] In an additional aspect, provided herein is a method of transiently expressing an RNA or a protein in a plant, the method including: (a) providing the expression vector system of any one embodiment of the preceding expression vector aspects, wherein the expression vector system includes at least one cargo nucleic acid sequence encoding an RNA or protein; (b) transforming the vector system into Agrobacterium; (c) providing the Agrobacterium to a first plant; (d) collecting leaf sap from the first plant; and (e) inoculating a second plant with the leaf sap; wherein the RNA or protein encoded by the at least one cargo nucleic acid sequence is transiently expressed in the second plant.

[0048] In another aspect, provided herein is a method of providing a nucleic acid sequence to a plant, the method including: (a) cloning a nucleic acid sequence into the cloning site of the expression vector system of any one embodiment of the preceding expression vector aspects; (b) transforming the expression vector system into Agrobacterium; and (c) providing the Agrobacterium to a plant; wherein the nucleic acid sequence is provided to the plant.

[0049] In another aspect, provided herein is a method of providing a nucleic acid sequence to a plant, the method including: (a) cloning a nucleic acid sequence into the cloning site of the expression vector system of any one embodiment of the preceding expression vector aspects; (b) transforming the expression vector system into Agrobacterium; (c) providing the Agrobacterium to a first plant; (d) collecting leaf sap from the first plant; and (e) inoculating a 61ny-2939083Attorney Docket No: 293082000140 second plant with the leaf sap; wherein the nucleic acid sequence is provided to the second plant.

[0050] In another aspect, provided herein is a method of transiently expressing an RNA or protein in a plant, the method including: (a) cloning a nucleic acid sequence into the cloning site of the expression vector system of any one embodiment of the preceding expression vector aspects, wherein the expression vector system includes at least one cargo nucleic acid sequence encoding an RNA or protein; (b) transforming the expression vector system into Agrobacterium; and (c) providing the Agrobacterium to a plant; wherein the RNA or protein encoded by the at least one cargo nucleic acid sequence is transiently expressed in the plant.

[0051] In an additional aspect, provided herein is a method of transiently expressing an RNA or protein in a plant, the method including: (a) cloning a nucleic acid sequence into the cloning site of the expression vector system of any one embodiment of the preceding expression vector aspects, wherein the expression vector system includes at least one cargo nucleic acid sequence encoding an RNA or protein; (b) transforming the expression vector system into Agrobacterium; (c) providing the Agrobacterium to a first plant; (d) collecting leaf sap from the first plant; and (e) inoculating a second plant with the leaf sap; wherein the RNA or protein encoded by the at least one cargo nucleic acid sequence is transiently expressed in the second plant. In one embodiment, herein is provided a method of editing the genome of a plant, the method including: (a) providing the expression vector system of any one embodiment of the preceding expression vector aspects; (b) transforming the expression vector system into Agrobacterium; and (c) providing the Agrobacterium to a plant; wherein the genome editing component is provided to the plant, and wherein the genome of the plant is edited.

[0052] In another aspect, provided herein is a method of editing the genome of a plant, the method including: (a) providing the expression vector system of any one embodiment of the preceding expression vector aspects; (b) transforming the expression vector system into Agrobacterium; (c) providing the Agrobacterium to a first plant; (d) collecting leaf sap from the first plant; and (e) inoculating a second plant with the leaf sap; wherein the genome editing component is provided to the second plant, and wherein the genome of the second plant is edited. In some embodiments, the first plant is Nicotiana benthamiana. In some embodiments, the Agrobacterium are provided to the first plant or plant by leaf infiltration. In an additional embodiment of any of the preceding method aspects, which may be combined with any of the preceding embodiments, the method further includes phenotyping and / or genotyping the plant or second plant for successful expression. In a further embodiment, which may be combined with any of the preceding embodiments, the method further includes phenotyping and / or 62ny-2939083Attorney Docket No: 293082000140 genotyping the plant or second plant for genome editing. In another embodiment, which may be combined with any of the preceding embodiments, the method further includes harvesting seed from the plant or second plant. In some embodiments, herein is provided a method of any one of the preceding embodiments, further including germinating the seed, assaying the offspring for viral RNA, and selecting virus-free offspring. In some embodiments, assaying the offspring for viral RNA includes an RT-PCR assay.

[0053] In an additional aspect, provided herein is a method of transiently expressing a nucleic acid in a plant, the method including: (a) providing an expression vector system to the plant, wherein the expression vector system includes: (i) a first expression vector including a tobravirus RNA1-derived RNA, (ii) a second expression vector including a tobravirus RNA1- derived RNA, (iii) a third expression vector including a tobravirus RNA2 or tobravirus RNA2- derived RNA, and (A) a nucleic acid encoding a tobravirus replicase, (B) a nucleic acid encoding a tobravirus moving protein, (C) a nucleic acid encoding a tobravirus cysteine-rich protein, (D) a nucleic acid encoding a tobravirus coat protein, and (E) one, two, three, four, five, or six cargo space inserts each including a nucleic acid sequence; (b) transforming the expression vector system into Agrobacterium; and (c) providing the Agrobacterium to a plant; wherein the one, two, three, four, five, or six cargo space inserts including the nucleic acid sequences are provided to the plant, and wherein the nucleic acid sequences are transiently expressed in the plant.

[0054] In an additional aspect, provided herein is a method of transiently expressing a nucleic acid in a plant, the method including: (a) providing an expression vector system to the plant, wherein the expression vector system includes: (i) a first expression vector including a tobravirus RNA1-derived RNA, (ii) a second expression vector including a tobravirus RNA1- derived RNA, and (iii) a third expression vector including a tobravirus RNA2 or tobravirus RNA2-derived RNA, and (A) a nucleic acid encoding a tobravirus replicase, (B) a nucleic acid encoding a tobravirus moving protein, (C) a nucleic acid encoding a tobravirus cysteine-rich protein, (D) a nucleic acid encoding a tobravirus coat protein, and (E) one, two, three, four, five, or six cargo space inserts each including a nucleic acid sequence; (b) in vitro transcribing the expression vector system to produce infectious RNAs; and (c) providing the infectious RNAs to a plant; wherein the one, two, three, four, five, or six cargo space inserts including nucleic acid sequences are provided to the plant, and wherein the nucleic acid sequences are transiently expressed in the plant.

[0055] In an additional aspect, provided herein is a method of transiently expressing a nucleic acid in a plant, the method including: (a) providing an expression vector system to the plant, 63ny-2939083Attorney Docket No: 293082000140 wherein the expression vector system includes: (i) a first expression vector including a tobravirus RNA1-derived RNA, and (ii) a second expression vector including a tobravirus RNA1-derived RNA, and (A) a nucleic acid encoding a tobravirus replicase, (B) a nucleic acid encoding a tobravirus moving protein, (C) a nucleic acid encoding a tobravirus cysteine-rich protein, and (D) one or more cargo space inserts each including a nucleic acid sequence; (b) transforming the expression vector system into Agrobacterium; and (c) providing the Agrobacterium to a plant; wherein the one or more cargo space inserts including the nucleic acid sequences are provided to the plant, and wherein the nucleic acid sequences are transiently expressed in the plant.

[0056] In an additional aspect, provided herein is a method of transiently expressing a nucleic acid in a plant, the method including: (a) providing an expression vector system to the plant, wherein the expression vector system includes: (i) a first expression vector including a tobravirus RNA1-derived RNA, and (ii) a second expression vector including a tobravirus RNA1-derived RNA, and (A) a nucleic acid encoding a tobravirus replicase, (B) a nucleic acid encoding a tobravirus moving protein, (C) a nucleic acid encoding a tobravirus cysteine-rich protein, and (D) one or more cargo space inserts each including a nucleic acid sequence; (b) in vitro transcribing the expression vector system to produce infectious RNAs; and (c) providing the infectious RNAs to a plant; wherein the one or more cargo space inserts including nucleic acid sequences are provided to the plant, and wherein the nucleic acid sequences are transiently expressed in the plant.

[0057] In a further embodiment of any one of the preceding method aspects, the plant, plant cell, or second plant is a monocot or monocot cell. In an additional embodiment, the monocot or monocot cell is selected from the group consisting of wheat, maize, barley, rice and Miscanthus, or a cell thereof. In another embodiment of any one of the preceding method aspects, the plant, plant cell, or second plant is a dicot or dicot cell. In a still further embodiment, the dicot or dicot cell is selected from the group consisting of beans, beets, brassicas, peas, peppers, spinach, tobacco, soybean, tomato, cotton, Arabidopsis, rose, strawberry, chrysanthemum and tulip, or a cell thereof. In some embodiments, the dicot or dicot cell is selected from the group consisting of Arabidopsis and tobacco, or a cell thereof. In certain embodiments, which may be combined with any of the previous embodiments, the infectious RNAs enter the germline cells. In other embodiments, the infectious RNAs do not enter the germline cells. In certain embodiments, which may be combined with any of the previous embodiments, the one or more cargo space inserts including nucleic acid sequences enter the 64ny-2939083Attorney Docket No: 293082000140 germline cells. In other embodiments, the one or more cargo space inserts including nucleic acid sequences do not enter the germline cells.

[0058] Also provided herein is a plant modified by or subject to any one embodiment of the preceding method aspects. Also provided herein is a seed of the plant, a cell of the plant, or a commercial product derived from the plant. DESCRIPTION OF THE FIGURES

[0059] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0060] The present application can be understood by reference to the following description taken in conjunction with the accompanying figures.

[0061] FIG. 1A shows a negative stain electron micrograph of barley stripe mosaic virus (BSMV) strain ND18, adapted from Kendall et al. Virology 443:265-270, 2013; Scalebar: 500Å. FIG.1B shows the genome organization of BSMV strain ND18.

[0062] FIG.2 shows a schema of a five-part split β and split γ BSMV vector system.

[0063] FIG.3A shows a cartoon (not to scale) depicting creation of BSMV β1 (left) and β2 (right) vectors. Ligation-independent cloning (LIC) sequences are added to the 3’ and 5’ ends of primers used in the amplification. Arrowheads show primers used and their locations; PCR primer pairs are indicated by arrows of the same color (light grey pair versus dark grey pair for each construct). FIG.3B shows a cartoon (not to scale) depicting creation of the new BSMVγ1 (left) and γ2 (right) vectors. Ligation-independent cloning (LIC) sequences are added to the 3’ and 5’ ends of primers used in the amplification. Arrowheads show primers used and their locations; PCR primer pairs are indicated by arrows of the same color (light grey pair versus dark grey pair for each construct).

[0064] FIG.4 shows a schematic illustration of the general procedure for inserting a gene or cargo of interest (GOI) into one of the ligation-independent cloning (LIC) sites on the TRV- derived expression vectors for its expression in planta.

[0065] FIG.5 shows a BSMV vector system carrying Cas12f1 and a gRNA for Cas12f1.

[0066] FIG. 6 shows a five-component BSMV system for delivery / expression of Cas9 reagents.

[0067] FIG.7 shows a diagram of the architecture of a sequence encoding a fusion protein for the dCas12f1-based CBE system. 65ny-2939083Attorney Docket No: 293082000140

[0068] FIG.8 shows a diagram of the architecture of a sequence encoding a fusion protein for the dCas12f1-based adenine base editor (ABE) system.

[0069] FIG. 9 shows a diagram of the architecture of a sequence encoding a zinc finger nuclease monomer with, from left to right, a 3X FLAG tag, a SV40 NLS, five ZFs, and a FokI domain.

[0070] FIG.10 shows a BSMV vector system carrying six engineered homing endonucleases (HEs) to simultaneously target three genome regions.

[0071] FIG. 11 shows an example of a typical repeat domain and the structure of a pair of TALEN monomers with uneven length that fit into the cargo-space of the four-component BSMV vector system.

[0072] FIG.12A shows the first part of a sequence alignment of pCass4-RZ, an exemplary T-DNA-derived binary plasmid provided by the present application (top row of alignment, SEQ ID NO: 18), in comparison to previously existing pCaBS-γ (second row, SEQ ID NO: 19) and pCaBS-γ1 (third row, SEQ ID NO: 20), and a Tn10 transposable element (bottom row, SEQ ID NO: 57, Tb927.3.1050). FIG.12B shows the second part of a sequence alignment of the present pCass4-RZ (top row of alignment, SEQ ID NO: 18), in comparison to pCaBS-γ (second row, SEQ ID NO: 19), pCaBS-γ1 (third row, SEQ ID NO: 20), and a Tn10 transposable element (bottom row, SEQ ID NO: 57, Tb927.3.1050). FIG. 12C shows the third part of a sequence alignment of the present pCass4-RZ (top row of alignment, SEQ ID NO: 18), in comparison to pCaBS-γ (second row, SEQ ID NO: 19), pCaBS-γ1 (third row, SEQ ID NO: 20), and a Tn10 transposable element (bottom row, SEQ ID NO: 57, Tb927.3.1050).

[0073] FIG. 13 shows a sequence alignment of pCaBS-γ2 (top row, SEQ ID NO: 21), BSMVγ (middle row, SEQ ID NO: 22), and pNHA23 (bottom row, SEQ ID NO: 23). The subgenomic RNAγ (sgRNAγ) promoter is underlined (SEQ ID NO: 56).

[0074] FIG.14 shows the amplification plot from an RT-qPCR reaction assaying expression levels of BSMV γb RNA and β-actin control in Nb tobacco after infiltration with a previously existing four-part BSMV system (“CH”) or an improved four-part BSMV system with split RNAγ1 and RNAγ2 of the present application (“NHA”). Samples are indicated in the legend in order from least cycles to most cycles on average for exceeding the threshold. The threshold is set at 0.01, shown with the horizontal line through the amplification plot.

[0075] FIG. 15A shows a schematic of the organization (not to scale) of the infectious cDNAs of the wildtype tobacco rattle virus (TRV), cloned into a T-DNA derived plasmid. FIG. 15B shows a schematic of the organization (not to scale) of the infectious cDNAs of the engineered tobacco rattle virus (TRV), cloned into a T-DNA derived plasmid. The 66ny-2939083Attorney Docket No: 293082000140 TRV1b / TRV1bp vectors are interchangeable, and the TRV2 / TRV2p vectors are also interchangeable. In both of FIGS.15A-15B, LB & RB indicate left and right borders of the T- DNA, respectively; p35S indicates a CaMV 35S promoter; 5’UTR & 3’ UTR indicate the 5’ and the 3’ untranslated regions; MP indicates the 29 kDa moving protein; CRP indicates the 16 kDa cysteine-rich protein; Rz indicates the ribozyme; 35S terminator indicates the 35S terminator; LIC indicates the ligation-independent cloning site; CP indicates the virus coat protein; PEBV indicates the promoter for pea early-browning virus coat protein. The two 35S promoters can also be replaced by a T7 promoter for in vitro transcription of infectious RNAs for RNA-based applications.

[0076] FIG. 16A shows a schematic illustration of the method for constructing the TRV1a vector from TRV RNA1. FIG. 16B shows a schematic illustration of the method for constructing the TRV1b and TRV1bp vectors from TRV RNA1. FIG.16C shows a schematic illustration of the method for constructing the TRV2 and TRV2p vectors from TRV RNA2. In each of FIGS.16A-16C, MP indicates the moving protein; CRP indicates the 16 kDa cysteine- rich protein; PEBV indicates the promoter for the coat protein of pea early-browning virus; LIC indicates the ligation independent cloning site; Horizontal arrows indicate the positions of PCR primers, with primer pairs within each schematic labeled with the same number. The schematics are not to scale.

[0077] FIG. 17 shows a schematic for a TRV-derived vector system carrying CRISPR / Cas components. MP indicates the 29 kDa moving protein; CRP indicates the 16 kDa cysteine-rich protein; CP indicates the virus coat protein.

[0078] FIG.18 shows a diagram of the architecture of coding sequences encoding the fusion protein for the dCas12f1-based cytosine base editor (CBE, top), adenine base editor (ABE, middle), and guanine base editor (GBE, bottom) systems.

[0079] FIG.19 shows a schematic for a TRV-derived vector system carrying the components of a base editing system. MP indicates the 29 kDa moving protein; CRP indicates the 16 kDa cysteine-rich protein; CP indicates the virus coat protein; dCas-CBE cassette indicates the cassette with a dCas-cytosine base editor fusion protein; gRNAs indicates guide RNAs or guide RNA cassettes for the dCas.

[0080] FIG. 20 shows a diagram of the architecture of a coding sequence encoding a zinc finger nuclease (ZFN) monomer consisting of a 3X FLAG tag (first arrow), a SV40 nuclear localization signal (SV40 NLS, second arrow), five zinc fingers (third arrow), and a Fok1 cleavage domain (fourth arrow). 67ny-2939083Attorney Docket No: 293082000140

[0081] FIG. 21 shows a schematic for a TRV-derived vector system carrying eight engineered homing endonucleases (HEs) to simultaneously target four genome regions. MP indicates the 29 kDa moving protein; CRP indicates the 16 kDa cysteine-rich protein; CP indicates the virus coat protein; each HE indicates an engineered homing endonuclease.

[0082] FIG.22A shows an example of a typical repeat domain and the structure of a pair of TALEN monomers with uneven length that fit into the cargo spaces of the TRV-derived vector system. FIG. 22B shows a schematic for a TRV-derived vector system carrying a pair of TALEN monomers of uneven lengths that fit into the cargo space of the TRV vectors for multiplexing. MP indicates the 29 kDa moving protein; CRP indicates the 16 kDa cysteine- rich protein; CP indicates the virus coat protein; L monomer indicates a long TALEN monomer; S monomer indicates a short TALEN monomer.

[0083] FIG. 23A shows a schematic of the organization (not to scale) of the infectious cDNAs of a wildtype tobravirus, cloned into a T-DNA derived plasmid. FIG. 23B shows schematics of exemplary engineered infectious cDNAs derived from a tobravirus RNA1, each cloned into a T-DNA derived plasmid. From top to bottom, engineered cDNAs are referred to as: RNA1a, RNA1b, RNA1bp, RNA1c, RNA1d, RNA1dp, RNA1e, RNA1ep, and RNA1f. FIG.23C shows additional exemplary engineered infectious cDNAs derived from a tobravirus RNA1, each cloned into a T-DNA derived plasmid. From top to bottom, engineered cDNAs are referred to as: RNA1g, RNA1gp, RNA1h, RNA1hp, RNA1i, and RNA1ip. FIG. 23D shows schematics of exemplary engineered infectious cDNAs derived from a tobravirus RNA2, each cloned into a T-DNA derived plasmid. From top to bottom, engineered cDNAs are referred to as: RNA-MP, RNA-MPp, RNA2a, and RNA2ap. For each of FIGS.23A-23D, LB & RB indicate left and right borders of the T-DNA, respectively; p35S indicates a CaMV 35S promoter; 5’UTR & 3’ UTR indicate the 5’ and the 3’ untranslated regions; MP indicates the 29 kDa moving protein; CRP indicates the 16 kDa cysteine-rich protein; Rz indicates the ribozyme; 35S terminator indicates the 35S terminator; LIC indicates the ligation-independent cloning site; CP indicates the virus coat protein; PEBV indicates the promoter for pea early- browning virus coat protein. The two 35S promoters can also be replaced by a T7 promoter for in vitro transcription of infectious RNAs for RNA-based applications.

[0084] FIG. 24A shows a schematic illustration of the method for constructing the RNA1a vector, comprising a replicase gene and a cargo space, from a tobravirus RNA1. FIG. 24B shows a schematic illustration of the method for constructing the RNA1b vector (left) or the RNA1bp vector (right) from a tobravirus RNA1. The RNA1b and RNA1bp vectors each comprise a cargo space, a moving protein, and a cysteine-rich protein gene, with the RNA1bp 68ny-2939083Attorney Docket No: 293082000140 vector additionally comprising a subgenomic promoter operably linked to the cargo space. FIG. 24C shows a schematic illustration of the method for constructing the RNA1c vector, comprising a replicase gene, a moving protein, and a cargo space, from a tobravirus RNA1. FIG.24D shows a schematic illustration of the method for constructing the RNA1d vector (left) or the RNA1dp vector (right) from a tobravirus RNA1. The RNA1d and RNA1dp vectors each comprise a cargo space and a cysteine-rich protein gene, with the RNA1dp vector additionally comprising a subgenomic promoter operably linked to the cargo space. FIG. 24E shows a schematic illustration of the method for constructing the RNA1e vector, comprising a cargo space and a moving protein, from a tobravirus RNA1. FIG.24F shows a schematic illustration of the method for constructing the RNA1ep vector, comprising a subgenomic promoter operably linked to a cargo space, and a moving protein, from a tobravirus RNA1. FIG. 24G shows a schematic illustration of the method for constructing the RNA1f vector, comprising a replicase gene, a cargo space, and a cysteine-rich protein gene, from a tobravirus RNA1. FIG. 24H shows a schematic illustration of the method for constructing the RNA1g vector, comprising a first portion of a split replicase gene comprising the methyltransferase and helicase motifs, and a cargo space, from a tobravirus RNA1. FIG. 24I shows a schematic illustration of the method for constructing the RNA1gp vector, comprising a first portion of a split replicase gene comprising the methyltransferase and helicase motifs, and a subgenomic promoter operably linked to a cargo space, from a tobravirus RNA1. FIG. 24J shows a schematic illustration of the method for constructing the RNA1h vector (left) or the RNA1hp vector (right) from a tobravirus RNA1. The RNA1h and RNA1hp vectors each comprise a cargo space, a second portion of a split replicase gene comprising the RdRP motif, a moving protein, and a cysteine-rich protein gene, with the RNA1hp vector additionally comprising a subgenomic promoter operably linked to the cargo space. FIG. 24K shows a schematic illustration of the method for constructing the RNA1i vector, comprising a cargo space and a second portion of a split replicase gene comprising the RdRP motif, from a tobravirus RNA1. FIG. 24L shows a schematic illustration of the method for constructing the RNA1ip vector, comprising a subgenomic promoter operably linked to a cargo space, and a second portion of a split replicase gene comprising the RdRP motif, from a tobravirus RNA1. FIG.24M shows a schematic illustration of the method for constructing the RNA-MP vector (left) or the RNA- MPp vector (right) from a tobravirus RNA1 and tobravirus RNA2. The RNA-MP and RNA- MPp vectors each comprise a viral coat protein gene, a moving protein gene, and a cargo space, with the RNA-MPp vector additionally comprising a subgenomic promoter operably linked to the cargo space. FIG. 24N shows a schematic illustration of the method for constructing the 69ny-2939083Attorney Docket No: 293082000140 RNA2a vector (left) or the RNA2ap vector (right) from a tobravirus RNA2. The RNA2a and RNA2ap vectors each comprise a viral coat protein gene and a cargo space, with the RNA2ap vector additionally comprising a subgenomic promoter operably linked to the cargo space. In each of FIGS.24A-24N, MP indicates the moving protein; CRP indicates the 16 kDa cysteine- rich protein; PEBV indicates the promoter for the coat protein of pea early-browning virus; LIC indicates the ligation independent cloning site; MT-HEL indicates the first portion of a split replicase gene comprising the methyltransferase and helicase motifs; RdRP indicates the second portion of a split replicase gene comprising the RNA-dependent RNA polymerase motif; CP indicates a viral coat protein; Horizontal arrows indicate the positions of PCR primers. The schematics are not to scale.

[0085] FIG. 25A shows photos of albino patches or stripes on the leaves of maize plants edited by vCas12j2 with guide RNA ZPSY1-3 targeting the first exon of ZmPSY1 in maize variety B73. FIG.25B shows a photo showing change in endosperm color from yellow to white among the T1 kernels from a B73 maize plant edited by vCas12j2 with guide RNA ZPSY1-3.

[0086] FIG. 26 shows a cartoon schematic showing recombination between NHA21 and NHA22b, indicated by the X in the top, and the resulting vectors.

[0087] FIG.27A shows photos of T0 soybean plants grown from seeds imbibed with a five component system with α, β1:vCas12j2, β2, γ1:JGPDS8, and γ2:vCas12j2. Albino patches are visible along the veins of the leaves. FIG.27B shows photos of T1 soybean plants grown from T0 plants grown from seeds imbibed with the five component system, showing albino patches on leaves. DETAILED DESCRIPTION

[0088] The following description sets forth exemplary methods, parameters and the like. It should be recognized, however, that such description is not intended as a limitation on the scope of the present disclosure but is instead provided as a description of exemplary embodiments. I. Definitions

[0089] Before describing the invention in detail, it is to be understood that this invention is not limited to particular compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. 70ny-2939083Attorney Docket No: 293082000140

[0090] As used herein, the terms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Where a term is provided in the singular, the inventors also contemplate aspects of the invention described by the plural of that term.

[0091] As used here, the term “about” refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se.

[0092] As used herein, the term “and / or” is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and / or” as used in a phrase such as “A and / or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or B; A or C; B or C; A and B; A and C; B and C; A (alone); B (alone); and C (alone).

[0093] As used herein, the terms “comprise,” “comprises,” “comprising,” “include,” “includes,” and “including” can be interchanged and are to be construed as at least having the features to which they refer while not excluding any additional unspecified features.

[0094] As used herein, the term “cargo space” refers to a site in a vector into which a nucleic acid of interest can be or has been inserted, such that it is carried as “cargo” by the vector into a host cell. As non-limiting examples, a cloning site, a ligation-independent cloning site, and a restriction enzyme digest site can provide a cargo space. As used herein, the term “cargo space insert” or “cargo nucleic acid” refers to a nucleic acid of interest inserted into a cargo space. The capacity of a cargo space or the maximum size of a cargo space insert is defined as the maximum length of a DNA(s) that can be inserted into a cargo space of the vector and that can be delivered into and stably expressed in host plant cells. This limitation is not only defined by the physical space of the viral particle, but also by the instability of the insert itself in planta. It is well known that a sequence inserted into a plant viral vector is generally unstable because whole or partial deletion of the insert may occur during viral vector replication, with the likelihood that a deletion will occur increasing in correlation to increasing insert size (Pogue et al., 2002; Bruun-Rasmussen et al., 2007; Cakir and Scofield, 2008; Scofield, 2023).

[0095] Although the following description uses terms “first,” “second,” etc. to describe various elements, these elements should not be limited by the terms. These terms are only used to distinguish one element from another. For example, a first plant could be termed a second plant, and, similarly, a second plant could be termed a first plant, without departing from the 71ny-2939083Attorney Docket No: 293082000140 scope of the various described embodiments. The first plant and the second plant are both plants, but they are not the same plant.

[0096] As used herein, the terms “include,” “includes,” and “including” are to be construed as at least having the features to which they refer while not excluding any additional unspecified features.

[0097] As used herein, the term “plant” includes a whole plant and any descendant, cell, tissue, or part of a plant. The term “plant parts” include any part(s) of a plant, including, for example and without limitation: seed (including mature seed and immature seed); a plant cutting; a plant cell; a plant cell culture; or a plant organ (e.g., intact nodal bud, shoot apex or shoot apical meristem, root apex or root apical meristem, lateral meristem, intercalary meristem, zygotic embryo, somatic embryo, ovule, pollen, microspore, anther, tassel, ear shank, kernel, hypocotyl, cotyledon, leaf, petiole, stem, internode, tuber, root, flowers, fruits, shoots, and explants). A plant tissue or plant organ may be a seed, protoplast, callus, or any other group of plant cells that is organized into a structural or functional unit. A plant cell or tissue culture may be capable of regenerating a plant having the physiological and morphological characteristics of the plant from which the cell or tissue was obtained, and of regenerating a plant having substantially the same genotype as the plant. Regenerable cells in a plant cell or tissue culture may be embryos, protoplasts, meristematic cells, callus, pollen, leaves, anthers, roots, root tips, silk, flowers, kernels, ears, cobs, husks, or stalks. In contrast, some plant cells are not capable of being regenerated to produce plants and are referred to herein as “non- regenerable” plant cells.

[0098] As used herein, “systemic infection” refers to an infection of a plant by a virus or viral system such as a viral expression system, wherein the virus spreads from an initial infected tissue in the plant to at least a second tissue in the plant. In some embodiments, the initial infected tissue is a leaf. In some embodiments, the initial infected tissue is a seed. In some embodiments the viral system is detectable throughout the plant. In some embodiments the viral system is detectable in reproductive tissue. In some embodiments the viral system is detectable in both vegetative tissue to reproductive tissue. As used herein, “systemic expression” of a polynucleotide or polypeptide refers to expression within a plant of a polynucleotide or polypeptide carried by a virus or viral system such as a viral expression system, wherein expression of the polynucleotide or polypeptide is detectable in at least the initial infected tissue and a second tissue in the plant. In some embodiments, the initial infected tissue is a leaf. In some embodiments, the initial infected tissue is a seed. In some embodiments expression of the polynucleotide or polypeptide is detectable in a leaf that was not inoculated with the virus or 72ny-2939083Attorney Docket No: 293082000140 viral system. In some embodiments expression of the polynucleotide or polypeptide is detectable in reproductive tissue of the plant. In some embodiments, expression is detectable four days after inoculation (4 dpi). In some embodiments, expression is detectable twelve days after inoculation (12 dpi). In some embodiments, expression is detectable at 4 dpi, 5 dpi, 6 dpi, 7 dpi, 8 dpi, 9 dpi, 10 dpi, 11 dpi, 12 dpi, 13 dpi, 14 dpi, and / or 15 dpi. In some embodiments, expression is detectable by qPCR.

[0099] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. II. Methods of engineering novel viral systems for expression in plants

[0100] This application discloses a method to jointly engineer the splitting of a genomic polynucleotide of a plant virus into two or multiple plant expression vectors with increased cargo spaces and the preservation / enhancement of its full and / or critical functionality. This parent polynucleotide can be an RNA or a DNA; it can be single-stranded or double-stranded; it can be of sense or antisense; and it can be linear or circular. A candidate virus for this method should have some flexibility for variation in the number and length of polynucleotides in its genome. Further, before or during the engineering process, the virus should (1) have a fully sequenced genome, or minimally the sections targeted for cutting sequenced, and, for a RNA virus, reverse transcribed into cDNA clone(s), and (2) the boundaries of the ORFs and regulatory elements of the candidate virus should be identified and / or fully known.

[0101] The plant expression vectors so produced need to have the following characteristics in order to preserve critical function(s): 1) The splitting is done in such a way that all the critical regulatory elements (e.g., promoters for sub-genomic DNA / RNA, enhancers, cis-acting elements, etc.) for the expression of the ORF(s) and / or the cargo(s) will be physically preserved together with the ORF and / or the cargo(s) that replace the ORF(s); 2) Each expression vector should contain all the necessary elements for its replication so that each can be replicated in vivo independently or collectively; 3) Each expression vector should contain at least one unique ORF of the parent genomic polynucleotide and can also contain at least one cloning site for expression of a cargo(s); 4) Each expression vector should retain all the necessary regulatory elements for translation of the ORFs or the cargo replacing an ORF and, if needed, for transcription of any sub-genomic polynucleotides. Alternatively, these regulatory elements can be included in the inserted cargo; 5) Two or more expression vectors can be assembled as an expression system with the member vectors being functionally complementary to each other 73ny-2939083Attorney Docket No: 293082000140 so that the system retains all of the essential viral ORFs and regulatory elements, and thus all the functions of the parent virus for plant infection, virus replication, ORF expression, cargo translation, cargo transcription, and viral movement from cell to cell and within the host plant; 6) Duplication of an ORF in such an expression system is allowed if the redundancy will not interfere with the normal function of the duplicated ORF or those of other ORFs in the system.

[0102] Methods of determining ORF start and end boundaries are known in the art, including but not limited to reviewing the viral sequence for translation start and stop codons and comparing whether the predicted encoded protein matches the known protein sequence. Transcription start sites can also be estimated by the conserved sequence transcription initiation site GCAT(A / T).

[0103] Identification of promoters can be accomplished by methods known in the art, including but not limited to functional tests of deletions (i.e. “promoter bashing”).

[0104] Provided that all the above conditions are met, a candidate virus genome can be split at any point for creating expression vectors with desired cargo spaces. After one or more splitting points are determined, the split fragments can be amplified by PCR or synthesized by a vendor. Cloning sites are added during the PCR reactions or the fragment synthesis. The fragments so produced are then ligated together to make the expression vectors, as illustrated in FIGS.3A-3B, 16A-16C, and 24A-24N.

[0105] There has been an attempt at applying a similar method to generate an enhanced expression vector, involving splitting a viral vector but without identifying critical elements within the viral genome, and thus not without significant compromise to the viral vector’s functionality. Cheuk and Houde (2018) tried to apply the splitting strategy to BSMV, which has a tripartite genome consisting of RNAα, RNAβ and RNAγ, by developing RNAγ into two expression vectors, γ1 and γ2, such that γ1 carries the γa open reading frame (ORF) and a cargo space in the place of the γb ORF, while γ2 carries the γb ORF and a cargo space in the place of the γa ORF. In this way, they were partially successful in expanding the natural ~500-bp cargo space of one RNAγ vector into two RNAγ vectors with ~1 kb and a ~2.4 kb cargo spaces to deliver two cargos for their VIGS and VOX studies in a four-component vector system (i.e., an α vector, a β vector, a γ1 vector and a γ2 vector) (Cheuk and Houde, 2018; Bouard and Houde, 2022; Bouard et al., 2024). However, this did not preserve the full functionality or critical functionality of the BSMV vectors. In BSMV, the γb protein is responsible for the critical function of neutralizing the host RNA silencing that destroys BSMV RNAs, and γb is mainly translated from the sub-genomic RNAγb (Jackson et al., 2009). However, Cheuk and Houde (2018) unintentionally split RNAγ at a critical location for viral function, destroying the 74ny-2939083Attorney Docket No: 293082000140 subgenomic promoter and thus impairing the RNAγb transcription. There were two significant results of this. Firstly, the whole system became less effective in invading germline cells, due to a failure to counter the RNA silencing by the plant host that prevents a virus from entering germline cells (Li and Ding 2024). Since the γb protein is responsible for overcoming this obstacle, the γ2 vector engineered by the Cheuk and Houde method translated much less γb protein due to the lack of RNAγb transcription. Secondly, the lack of enough γb protein provides much less protection for BSMV RNAs than is needed in the host cells making the RNAs unstable. The overall value of the vector as a gene-editing tool for inheritable, stable gene-editing was, therefore, greatly compromised. By contrast, the methods provided herein not only expand cargo space, but also preserve critical and / or full functionality of multi- component plant viral expression vector systems.

[0106] As a nonlimiting example, barley stripe mosaic virus (BSMV) RNAβ can be split to form two complementary vectors, each containing at least one unique BSMV RNAβ and optionally also containing a cargo space. However, this splitting strategy can be applied to any virus that meet the prerequisites.

[0107] BSMV RNAβ has been sequenced and fully studied for its genomic structure, replication and functions (Gustafson and Armor, 1986; Zhou and Jackson, 1996a, 1996b; Jackson et al., 2009). RNAβ is responsible for infection and systemic movement (Petty and Jackson, 1990; Lawrence and Jackson, 2001a, 2001b). It has four ORFs (βa, βb, βc and βd) encoding five proteins (the βa, βb, βc, βd and βd’ proteins). The βa ORF resides between the 90nt and the 686nt positions; the βb ORF locates between the 804nt and the 2341nt positions; the βc ORF resides between the 2515nt and the 2982nt positions; and the βd ORF overlaps with the βb and βc ORFs residing between the 2313nt and the 2703nt positions. Therefore, the βb, βc and βd ORFs form a triple gene block (TGB) encoding TGB proteins that physically interact with each other enabling systemic viral movement (Lawrence DM, Jackson AO. 2001b).

[0108] In planta, the genomic RNAβ functions as the template for making the complementary negative RNA, which will, in turn, transcribe more copies of genomic RNAβ and, at the same time, transcribe two sub-genomic RNAs (RNAβ1 and RNAβ2). The core promoter for RNAβ1 transcription resides between the β and the βb ORFs overlapping the last four nucleotides with the helper sequence extending into the last 72 nucleotides of the βa ORF (Zhou et al., 1996a). The core promoter for RNAβ2 starts at the -64 nt from the transcription initiation point (the 2278nt). BMSV transcribes two separate sub-genomic RNAs for the TGB with the effect of 75ny-2939083Attorney Docket No: 293082000140 controlling expression levels of individual ORFs in the TGB, with the βb ORF expression level being about 100 fold higher than the other two TGB ORFs, βc and βd.

[0109] The genomic RNAβ functions as the mRNA for βa (coat) protein translation; RNAβ1 is the mRNA for the βb (TGB1) protein; RNAβ2 is the mRNA for the βc (TGB3) and βd (TGB2) proteins using a leaky scanning mechanism for the expression, and the βd’ (TGB2’) protein is a readthrough product of the amber stop codon of the βd ORF and is dispensable for viral functions (Zhou et al., 1996b). The first 89 nucleotides from the 5’ end are the 5’UTR. The last ~256 nucleotides are the 3’UTR. Both the 5’ and the 3’ UTRs and the intergenic region between the βa ORF and the βb ORF are required for RNAβ replication.

[0110] Therefore, BSMV RNAβ can be split to make at least two expression vectors with each having a unique ORF, a cargo, and all the necessary sequence elements for replication. As a non-limiting example, the β1 vector can be produced by replacing all or a part of the TGB with a cloning site. To gain the largest cargo space while preserving the βa ORF, a 2176-bp fragment from the first nucleotide of the βb ORF at the 804nt to near the end of the βc ORF at 2979nt is replaced with the cloning site, providing a cargo space of ~3 kb. The promoter for sub-genomic RNAβ1 is preserved to drive the transcription of a sub-genomic RNA from the cargo for its high-level expression. Alternatively, the split point can be anywhere within the 2176-bp fragment with varying cargo space. The expression vector β1 so produced will carry a functional βa ORF to produce the coat protein (CP). Since the CP is dispensable for virus infection, replication and movement, part of the βa ORF can also be deleted in addition to TGB to maximum the cargo space if the promoter for sub-genomic RNAβb is retained. However, CP functions in virion formation which eases host pathogenetic responses. A CP-deficient vector system will be more aggressive in pathogenicity and the disease phenotype will be more severe (Jackson et al., 2009).

[0111] To produce a β2 expression vector, splitting can occur at any point within the βa ORF for a cargo space of up to 700 bp as long as the core promoter for sub-genomic RNAβ1 is preserved. Since the βa ORF serves directly as the βa mRNA, a promoter and its terminator need to be in place, either incorporated into the vector itself or into the cargo, if RNA transcription from the cargo is desired.

[0112] The β1 and β2 plant expression vectors so produced can provide two cargo spaces. They may be assembled with RNAα, RNAγ1, and RNAγ2 vectors to form an expression system that provides four cargo spaces of ~7 kb in total, or can be assembled with RNAα and RNAγ to form an expression system that provides two cargo spaces of ~4 kb in total. 76ny-2939083Attorney Docket No: 293082000140 III. Engineered barley stripe mosaic virus (BSMV) systems for expression in plants

[0113] Barley Stripe Mosaic Virus (BSMV) is a rod-shaped virus in the Hordeivirus family with a rigid helical virion of ~25 nm in diameter (FIG.1). It can infect more than 250 monocot and dicot plant species including economically important crops such as wheat, barley, maize, rice, rye, oat, sorghum, pearl miller, alfalfa, cotton, tomato, tobacco, sunflower, pea, sugar beet, spinach, and radish (McKinney and Greeley, 1965; Jackson and Lane, 1981; Bragg et al., 2008; Cheuk and Houde, 2018; Chen et al., 2022; Chen et al., 2022). The virus infects plants through minor mechanical injuries on the surface of a host mesophyll or epidermal cell where it targets chloroplasts and nuclei (McKinney and Greeley, 1965). Once in the host plant, it replicates in the chloroplasts (Zhang et al., 2017) and systematically spreads by cell-to-cell movement and via the vascular system to other parts of the plant, including, most critically, into meristems and germline cells (Crowley, 1959; McKinney and Greeley, 1965; Jackson et al., 2009). BSMV is generally transmitted through minor mechanical injury by body contact or more commonly through seeds at a rate of ~60%, allowing selection of offspring with or without the virus. Different strains of BSMV may have different numbers of RNA partite in their viral genomes (Palomar et al., 1977), and individual BSMV RNA can move within the host without assembling a single virion with other BSMV RNAs (Jackson et al., 2009), which provides flexibility for manipulation of this virus.

[0114] Strains of BSMV are known in the art, as in McKinney and Greely, 1965; Timian, 1973; Jackson & Brakke, 1973; Lane, 1974; Jackson et al., 2009; and Hafez et al., 2014). Strains include: Latent strain, Mild strain VML, Argentina mild strain, Mild strain L1B, Moderate strain LSP, Type strain, Moderate strain LIT, Moderate strain LIT, Oklahoma moderate strain, Moderate strain LQ, Moderate strain YLM, Moderate strain LRM, Argentina moderate strain, Severe strain L4A, Wisconsin severe strain, Severe strain SOM, Mississippi severe strain, Argentina severe strain, Yellow-leaf strain, White-leaf strain, Lethal white-leaf strain, Albino strain, Coarse-blotch strain, Fleck-blotch strain, Argentina dwarf strain, Necrosis strain, Mild oat strain, California mild oat strain, Spindle-stripe oat strain, Eyespot oat strain, Moderate oat strain, California moderate oat strain, Lethal rosette oat strain, cereal virus (CV) CV12, CV16, CV17, CV20, CV21, CV22, CV24, CV26, CV28, CV29, CV31, CV32, CV40, CV41, CV42, CV52, CV54, CV55, CV65, CV66, CV67, BSMV China, BSMV Egyptian, Russian, Norwich, North Dakota 18 (ND18), and Rothamsted. In some embodiments, the expression vector system is modified from BSMV strain ND18. In some embodiments, the expression vector system is modified from a BSMV strain selected from Latent strain, Mild 77ny-2939083Attorney Docket No: 293082000140 strain VML, Argentina mild strain, Mild strain L1B, Moderate strain LSP, Type strain, Moderate strain LIT, Moderate strain LIT, Oklahoma moderate strain, Moderate strain LQ, Moderate strain YLM, Moderate strain LRM, Argentina moderate strain, Severe strain L4A, Wisconsin severe strain, Severe strain SOM, Mississippi severe strain, Argentina severe strain, Yellow-leaf strain, White-leaf strain, Lethal white-leaf strain, Albino strain, Coarse-blotch strain, Fleck-blotch strain, Argentina dwarf strain, Necrosis strain, Mild oat strain, California mild oat strain, Spindle-stripe oat strain, Eyespot oat strain, Moderate oat strain, California moderate oat strain, Lethal rosette oat strain, cereal virus (CV) CV12, CV16, CV17, CV20, CV21, CV22, CV24, CV26, CV28, CV29, CV31, CV32, CV40, CV41, CV42, CV52, CV54, CV55, CV65, CV66, CV67, BSMV China, BSMV Egyptian, Russian, Norwich, North Dakota 18 (ND18), and Rothamsted.

[0115] BSMV is known to infect over 250 species of monocots and dicots (Jackson and Lane, 1981), including but not limited to Nicotiana spp., Arabidopsis, Glycine max, Solanum lycopersicum, Solanum tuberosum, Helianthus annuus, Gossypium spp., Fagopyrum esculentum, Trifolium pratense, Brassica oleracea, Pisum sativum, Nasturtium officinale, Medicago sativa, and Raphanus sativus. This provides the possibility that the expression vector systems of the present disclosure may be functional for transient expression in over 250 species. In some embodiments, the expression vector systems provided herein are used to infect a plant or plant cell selected from Nicotiana spp., Arabidopsis, Glycine max, Solanum lycopersicum, Solanum tuberosum, Helianthus annuus, Gossypium spp., Fagopyrum esculentum, Trifolium pratense, Brassica oleracea, Pisum sativum, Nasturtium officinale, Medicago sativa, and Raphanus sativus, or a cell thereof. In some embodiments, the methods provided herein include infection of a plant or plant cell selected from Nicotiana spp., Arabidopsis, Glycine max, Solanum lycopersicum, Solanum tuberosum, Helianthus annuus, Gossypium spp., Fagopyrum esculentum, Trifolium pratense, Brassica oleracea, Pisum sativum, Nasturtium officinale, Medicago sativa, and Raphanus sativus, or cell thereof.

[0116] BSMV strain ND18 was engineered as a transient expression vector system that was used in many labs for decades (Petty et al., 1989; Scofield et al., 2005; Jackson et al., 2009; Yuan et al., 2011; Cheuk & Houde, 2018; Zhuang et al., 2013; Paudel et al., 2020). This strain has a tripartite positive single-stranded RNA genome consisting of RNAα, RNAβ, and RNAγ (FIG.1B). RNAγ and, in some cases, RNAβ was modified to carry an expression insert or two. The inserts were either placed at the 5’- or the 3’-end of the γb ORF (Scofield et al., 2005; Yuan et al., 2011; Zhuang et al., 2013), or replaced the βa ORF or the βb ORF partially (Joshi 78ny-2939083Attorney Docket No: 293082000140 et al., 1990; Holzberg et al., 2002) or the γa ORF and / or the γb ORF entirely (Cheuk and Houde, 2018; Paudel et al., 2020).

[0117] The original three-component pBSMV expression vectors were made by inserting a full-length cDNA of each BSMV RNA into pZfl9U plasmid (Petty et al., 1988). The pBSMV plasmids were used for in vitro transcription of infectious BSMV RNAs, which is controlled by a T7 promotor and linearized by an MluI (for the α and γ vectors) or an SpeI digestion (for the β vector). Because of the high cost of in vitro transcription, difficulty in handling RNA and a need for high throughput operation, Yuan et al. (2011) invented a new three-component vector system (pCaBS-α, pCaBS-β and pCaBS-γ) for DNA-based applications. In this system, each of the three full-length BSMV cDNAs was cloned into pCass4-Rz (a T-DNA-derived binary plasmid). In these three vectors, T7 promotor was replaced by a double CaMV 35S promotor. The MluI and the SpeI sites were also removed from this system. These changes enabled transcription of the BSMV RNAs in planta while disabling their transcription in vitro.

[0118] For DNA-based applications, there are two methods of delivery. The first way is to biolistically bombard the T-DNA-based plasmids directly into a plant of interest (Hu et al., 2019). This method requires special bombardment equipment, and the tissues to which it can be applied are very limited. More commonly, the three plasmids are individually transformed into Agrobacterium cells which are then mixed and co-infiltrated into Nicotiana benthamiana (Nb tobacco) leaves. The viruses produced in Nb leaves are then harvested to be used as the inoculant for infection of other plants of interest. The advantage of the DNA methods is the relative ease of handling the DNA.

[0119] For RNA-based applications, the in vitro transcribed infectious BSMV RNAs were manually rubbed into the surface of a plant of interest with fingers (Scofield and Brandt, 2017). A big advantage of the RNA-based application over a DNA-based application is its time saving feature since the usual steps of transforming Agrobacterium and agroinfiltration are omitted. Its big disadvantage, however, is the difficulty in handling RNA because RNA is very easily destroyed biologically, chemically, or physically. This method has been used to knock-down (Zhuang et al., 2013) and overexpress (Paudel et al., 2020) Fusarium head blight resistance gene WFhb1-1 in wheat.

[0120] Both RNA-based (pBSMVα, pBSMVβ, and pBSMVγ) and DNA-based (pCaBS-α, pCaBS-β and pCaBS-γ) three-component BSMV vector systems have very limited cargo space(s) (<500 bp). To expand cargo space, Cheuk and Houde (2018) reengineered the pCaBS- γ vector by splitting it into two (pCaBS-γ1 and pCaBS-γ2). The resultant four-component system (pCaBS-α, pCaBS-β, pCaBS-γ1 and pCaBS-γ2) can simultaneously express two inserts, 79ny-2939083Attorney Docket No: 293082000140 one by the γ1 vector (~1 kb) and one by the γ2 vector (~2.5 kb). Still, the carrying capacity of current BSMV expression systems remains a key limiting factor in their application in transient gene expression since many genes are greater in size than the cargo space. Limited by cargo space, most applications of the BSMV expression system in CRISPR / Cas gene editing are done by delivering gRNA(s) into a transgenic Cas9 plants (Hu et al., 2019; Li et. al., 2021; Chen et al., 2022b) or by splitting Cas9 into Cas9N and Cas9C and delivering them separately (Kaya et al., 2017; Chen et al., 2022a). This cargo space limitation also prohibits high-powered multiplex gene editing, in which many guide RNAs must be delivered. A. Engineered BSMV expression vector systems with split RNAβ

[0121] The present disclosure provides a new BSMV vector system for gene delivery and transient expression in plants. As illustrated in FIG. 2, this system includes two newly engineered BSMV vectors: β1 and β2, exemplified respectively in SEQ ID NOs: 24 and 25, and a vector with the native α; the system also includes either one vector with the native γ in a four-component system, or with two vectors γ1 and γ2 in a five-component system. The β1 is produced by replacing the portion of RNAβ containing the βb, βc, βd and βd’ ORFs with a ligation-independent cloning (LIC) site. The β2 is produced by replacing a large portion of the βa ORF with a LIC site. Creation of the β1 and the β2 vectors has at least doubled the cargo space of the vector system compared with the known four-component system (Cheuk and Houde, 2018).

[0122] The β1 vector can provide up to 3 kb of cargo space, and the β2 vector can host up to 1 kb of cargo. Depending on the need, these vectors can be used in a four-component system or a five-component system. Basically, the β1 and the β2 vectors can be used together with the native RNAα and the RNAγ vector to provide three LICs for gene expression (four-component; one LIC on each of the β1, β2, and γ vectors); or the β1, the β2, the γ1 and the γ2 vectors can work together with native RNAα to provide four LICs (five-component; one LIC on each of the β1, β2, γ1, and γ2 vectors).

[0123] For Agroinfiltration-based delivery, these two new BSMV vectors have been incorporated into pCass4-RZ (a T-DNA-derived plasmid) (SEQ ID NOs: 28 and 29). The plasmids are transformed into Agrobacterium for plant infiltration. In the plant cells, the cDNAs will be transcribed into viral RNAs.

[0124] For RNA-infection-based delivery, the same β1 and β2 vectors have been incorporated into pBSMV(3-7T) (a pZfl9U-derived plasmid) (SEQ ID NOs: 32 and 33). 80ny-2939083Attorney Docket No: 293082000140 Infectious RNA vectors are made in vitro by transcription from these cDNA clones and used for infection of plant tissues.

[0125] In plant tissue, the viral RNAs direct their own replication, as well as transcription and / or translation of their sequences. RNAα directs translation of αa protein. RNAβ1 directs translation of βa protein and transcription of a subgenomic RNA that encodes the cargo nucleotides. RNAβ2 directs translation of the cargo nucleotides and transcription of subgenomic RNAs encoding βb, βc, βd, and βd’ proteins. RNAγ directs translation of γa protein and transcription of a subgenomic RNA encoding γb proteins. In the five-component system in which γ1 and γ2 are used, γ1 directs translation of γa protein and transcription of a subgenomic RNA encoding the cargo, and γ2 directs the translation of a cargo and transcription of a subgenomic RNA encoding γb protein. Therefore, cargo nucleotides that are cloned into the β1 or the γ1 cloning site are encoded by the corresponding subgenomic RNA, and thus should have higher translation rates than cargo nucleotides cloned into the β2 or the γ2 cloning site.

[0126] This BSMV vector system can be adopted for use in gene editing efforts using CRISPR / Cas, for example, Cas12f1. Since the average length of current coding sequences are <2.4 kb for a dCas12f1-based base editor molecular reagent and <2 kb for a ZFN, TALEN or HE monomers, this viral expression system is compatible with these gene editing platforms. The coding sequences for the two ZFN, TALEN, or HE monomers can be carried with one in the β1 vector and one in the γ2 vector. Components of an editor like the split Cas9 developed by Zetche et al. (2015), Kaya et al. (2017) or Chen et al. (2022a) can also be delivered by two or more vectors of the present system, separately.

[0127] BSMV is known to transcribe two subgenomic (sg) RNAs, sgRNAβ1 (2.5 kb) and sgRNAβ2 (0.96 kb), from RNAβ in planta (Petty and Jackson, 1990; Zhou and Jackson, 1996). sgRNAβ1 serves as the mRNA for translating βb protein and sgRNAβ2 serves as the mRNA for βc, βd, and βd’ proteins (Zhou and Jackson, 1996; Jackson et al., 2009). Forming nucleoprotein movement complexes (MC) is required for BSMV’s cell-to-cell movement and βb, βc, βd and γb proteins are required in the forming of MCs (Jackson et al., 2009). The amount of these structural proteins needed for wrapping up newly replicated viral RNAs for their cell- to-cell movement are nevertheless relatively much larger than non-structural αa, βa, and γa proteins. Producing sgRNAβs is the route by which BSMV increases copies of these structural genes. In the presently disclosed β1 expression vector, the sequence for βb, βc and βd is replaced with a cloning site but the promoter for sgRNAβ1 (SEQ ID NO: 49) has been preserved. Therefore, the vector will transcribe a sgRNA that functions as the mRNA for 81ny-2939083Attorney Docket No: 293082000140 translating the nucleotides in the cargo, effectively increasing the copy number of these nucleotides and thus increasing their production in planta. The β2 vector will transcribe the sgRNAβ1 and sgRNAβ2 for translation of βb, βc, βd, and βd' proteins.

[0128] In some applications, a helper RNA sequence, such as one encoded by Flowering Locus T (FT) (Jackson et al., 2012; Notaguchi et al., 2015; Ellison et al.,2020), a tRNA (Ellison et al., 2020; Nagalakshmi et al., 2022; Zhang et al., 2016; Weiss et al., 2024) or a WUSCHEL (WUS) fragment (Ellison et al., 2020), is used to help systemic movement of an engineered viral vector within the host plant. Mobile helpers such as these can be incorporated into a cargo construct of a BSMV-derived vector if needed. B. Improved engineered BSMV expression vector systems with split RNAγ

[0129] Cheuk and Houde (2018) reengineered the pCaBS-γ vector by splitting it into two (pCaBS-γ1 and pCaBS-γ2). The resultant four-component system (pCaBS-α, pCaBS-β, pCaBS-γ1 and pCaBS-γ2) can simultaneously express two inserts, one by the γ1 vector (~1 kb) and one by the γ2 vector (~2.5 kb). However, this four-component split-γ system contains a Tn10 transposable element (SEQ ID NO: 57) that results in decreased genetic stability during replication. Further sequencing of the pCaBS-γ1:ccdB system revealed the presence of an IS1 element (SEQ ID NO: 58) in a similar location to the Tn10 element. Both IS1 and Tn10 elements are members of the IS3 family of mobile elements, which can contribute to decreased genetic stability during replication. Additionally, the γ2 vector contains an incomplete subgenomic RNAγ (sgRNAγ) promoter that inhibits translation of the γb protein, negatively affecting the efficiency of the system. As such, there exists a need for improved BSMV expression vectors system with split RNAγ. a. BSMV expression vector systems with improved RNAγ1 and RNAγ2 vectors

[0130] The present disclosure provides a new BSMV vector system for gene delivery and transient expression in plants. As illustrated in FIG. 2, this system includes two newly engineered BSMV vectors: an improved γ1 and γ2, exemplified respectively in SEQ ID NOs: 26 and 27, and a vector with the native α; the system also includes either one vector with the native β in a four-component system, or two vectors β1 and β2 derived from the native β for use in a five-component system. The β1 is produced by replacing the portion of RNAβ containing the βb, βc, βd and βd’ ORFs with a ligation-independent cloning (LIC) site. The β2 is produced by replacing a large portion of the βa ORF with a LIC site. Creation of the improved γ1 and γ2 vectors has increased the genetic stability of the vector system and increased 82ny-2939083Attorney Docket No: 293082000140 expression of the cargo nucleic acid(s) compared with the previously existing four-component split-γ vector system (Cheuk and Houde, 2018).

[0131] The γ1 vector can provide up to about 1 kb of cargo space, and the γ2 vector can host up to about 2.5-3 kb of cargo. Depending on the need, these vectors can be used in a four- component system or a five-component system. Basically, the γ1 and the γ2 vectors can be used together with the native RNAα and the RNAβ vector to provide two LICs for gene expression (four-component; one LIC on each of the γ1 and γ2 vectors); or the γ1, the γ2, the β1 and the β2 vectors can work together with native RNAα to provide four LICs (five- component; one LIC on each of the β1, β2, γ1, and γ2 vectors).

[0132] For Agroinfiltration-based delivery, these two new BSMV vectors have been incorporated into pCass4-RZ (a T-DNA-derived plasmid) (SEQ ID NOs: 30 and 31). The plasmids are transformed into Agrobacterium for plant infiltration. In the plant cells, the cDNAs will be transcribed into viral RNAs.

[0133] For RNA-infection-based delivery, the same γ1 and γ2 vectors have also been incorporated into a pZfl9U-derived plasmid (SEQ ID NOs: 34 and 35). Infectious RNA vectors are then made by in vitro transcription from these cDNA clones and are used for infection of plant tissues.

[0134] In plant tissue, the viral RNAs direct their own replication, as well as transcription and / or translation of their sequences, as follows. RNAα directs translation of αa protein. RNAβ1 directs translation of βa protein and transcription of a subgenomic RNA that encodes the cargo nucleotides. RNAβ2 directs translation of the cargo nucleotides and transcription of subgenomic RNAs encoding βb, βc, βd, and βd’ proteins. RNAγ directs translation of γa protein and transcription of a subgenomic RNA encoding γb proteins. In the five-component system in which γ1 and γ2 are used, γ1 directs translation of γa protein and transcription of a subgenomic RNA encoding the cargo, and γ2 directs the translation of a cargo and transcription of a subgenomic RNA encoding γb protein. Therefore, cargo nucleotides that are cloned into the β1 or the γ1 cloning site will be encoded by the corresponding subgenomic RNA, and thus should have higher translation rates than cargo nucleotides cloned into the β2 or the γ2 cloning site.

[0135] This BSMV vector system can be adopted for use in gene editing efforts using CRISPR / Cas, for example, Cas12f1. Since the average length of current coding sequences are <2.4 kb for a dCas12f1-based base editor molecular reagent and <2 kb for a ZFN, TALEN or HE monomers, this viral expression system is compatible with these gene editing platforms. The coding sequences for the two ZFN, TALEN, or HE monomers can be carried with one in 83ny-2939083Attorney Docket No: 293082000140 the β1 vector and one in the γ2 vector. Components of an editor like the split Cas9 developed by Zetche et al. (2015), Kaya et al. (2017), or Chen et al. (2022) can also be delivered by two or more vectors of the present system, separately.

[0136] BSMV is known to transcribe a subgenomic (sg) RNA from RNAγ in planta (Petty and Jackson, 1990); Zhou and Jackson, 1996). γb protein plays a key role in protecting the virus from destruction by suppressing host RNA defense mechanisms (Bragg and Jackson, 2004) and protecting viral replication (Torrance et al., 2006; Petty et al., 1990) in planta. The γb protein is also involved in systemic movement of BSMV in planta (Petty et al., 1990). Although RNAγ is a positive sense RNA from which γb protein can directly be translated, a sub-genomic RNAγ is transcribed from the 3’ portion of RNAγ in plant cells, from which mRNA for γb protein translation is generated (Jackson et al., 1983; Gustafson et al., 1987). In fact, γb protein is predominately translated from this mRNA, not from RNAγ directly, in planta (Gustafson et al., 1987).

[0137] Provided herein are BSMV expression vectors systems including an RNAγ1 with a functional sgRNAγ promoter, and an RNAγ2 with a functional sgRNAγ promoter. The introduction of a functional sgRNAγ on the RNAγ2 vector results in increased levels of γb protein. It is well known that the γb protein plays a key role in protecting the virus from destruction by suppressing host RNA defense mechanisms, such that the improved system has increased infection, increased viral levels, increased cell-to-cell motility, and thus an increase in transient expression of the nucleic acid cargo(s).

[0138] In some applications, a helper RNA sequence, such as one encoded by Flowering Locus T (FT) (Jackson et al., 2012; Notaguchi et al., 2015; Ellison et al.,2020), a tRNA (Ellison et al., 2020; Nagalakshmi et al., 2022; Zhang et al., 2016; Weiss et al., 2024) or a WUSCHEL (WUS) fragment (Ellison et al., 2020), is used to help systemic movement of an engineered viral vector within the host plant. Mobile helpers such as these can be incorporated into a cargo construct of the tobravirus-derived vector described in this disclosure if needed. b. BSMV expression vector systems with improved genetic stability

[0139] The present disclosure provides BSMV expression systems including T-DNA-derived plasmids with improved genetic stability, wherein the T-DNA-derived plasmids do not include a mobile element of the IS3 family. As shown in the Examples, existing T-DNA-derived BSMV vector systems were all found to harbor a Tn10 transposable element or an IS1 mobile element at least on the RNAγ, RNAγ1, and other RNAγ-derived vectors. Both IS1 and Tn10 elements are members of the IS3 family of mobile elements, which can contribute to decreased 84ny-2939083Attorney Docket No: 293082000140 genetic stability during replication. The systems provided herein that do not include a mobile element of the IS3 family have increased genetic stability, such that the viral vectors of the present application lose their cargo at lower rates than viral vectors of a control system. c. Control expression vector systems

[0140] In some embodiments, a vector system provided herein possesses one or more advantages over a control expression vector system. In some embodiments, the control expression vector system includes the original BSMV split-γ vector system (Cheuk & Houde, 2018), wherein the original BSMV split-γ vector system includes a mobile element of the IS3 family, such as a Tn10 transposable element or IS1 element, and wherein the RNAγ2 does not include an sgRNAγ promotor. In some embodiments, the control vector system includes a BSMV split-γ vector system in which the RNAγ2 does not include a sgRNAγ promotor. In some embodiments, the control vector system includes a BSMV split-γ vector system that includes an IS3 family mobile element, optionally a Tn10 transposable element or an IS1 element. In some embodiments, the control vector system includes a BSMV vector system that includes an IS3 family mobile element, optionally a Tn10 transposable element or an IS1 element. In some embodiments, the control vector system includes a BSMV split-γ vector system that includes an IS1 mobile element. In some embodiments, the control vector system includes a BSMV vector system that includes an IS1 mobile element. In some embodiments, the control expression vector system includes pCaBS-γ (SEQ ID NO: 19) or pCaBS-γ1 (SEQ ID NO: 20).

[0141] In some embodiments, the advantage or improvement includes increased genetic stability compared to a control expression vector system. In some embodiments, the advantage or improvement includes increased cargo expression levels compared to a control expression vector system. In some embodiments, the advantage or improvement includes increased production of the γb protein compared to a control expression vector system. In some embodiments, the advantage or improvement includes improved efficiency of transient expression in planta compared to a control expression vector system. In some embodiments, the advantage or improvement includes improved efficiency of cell-to-cell movement of the viral vectors in planta compared to a control expression vector system.

[0142] In some embodiments, a vector system provided herein is an improvement over a control expression vector system. In some embodiments, the vector system provided herein is an improvement over the original split-γ vector system (Cheuk & Houde, (2018) A new barley stripe mosaic virus allows large protein overexpression for rapid functional analysis. Plant 85ny-2939083Attorney Docket No: 293082000140 Physiol 176:1919-1931). In some embodiments, a vector system provided herein is an improvement over a BSMV split-γ vector system that does not include a sgRNAγ promotor. In some embodiments, a vector system provided herein is an improvement over a BSMV split-γ vector system that includes an IS3 family mobile element. In some embodiments, a vector system provided herein is an improvement over a BSMV split-γ vector system that includes a Tn10 transposable element. In some embodiments, the Tn10 transposable element includes SEQ ID NO: 57. In some embodiments, a vector system provided herein is an improvement over a BSMV split-γ vector system that includes an IS1 mobile element. In some embodiments, the Tn10 transposable element includes SEQ ID NO: 58. C. Engineered BSMV expression vector systems for genome editing

[0143] CRISPR / Cas, base editors (BEs) and prime editors (PEs) are all based on Cas endonucleases (e.g., Cas9, Cas12a, Cas12f, Cas12j, etc.). In a CRISPR / Cas system, a Cas- gRNA complex searches and binds on target DNA sequences specified by the gRNA. The Cas endonuclease then breaks one or both stands of the target DNA to make mutations. The Cas and the gRNA can be fused together for a single delivery or be delivered separately as two reagents.

[0144] BEs and PEs do not break any of the DNA strands for mutant creation. Instead, they have one or several functional domains fused to a catalytically impaired Cas endonuclease (dCas) (e.g., dCas9, dCas12a, dCas12f, dCas12j, etc.) and make change(s) within the sequence(s) targeted by the dCas-gRNA complex (Kantor et al., 2020). For BEs, an ssDNA- deaminase specific either for cytidine in CBE (cytidine base editing) or for adenine in ABE (adenine base editing) is fused to the N terminal of the dCas that removes the aminol group from the targeted C or A base changing it to uracil (U) or inosine (I), respectively. In CBE, a uracil DNA glycosylase inhibitor is optionally also added to the C-terminal of the dCas to inhibit uracil DNA glycosylase (UDG) in the cells which catalyzes removal of U from DNA during DNA repairing in cells. The smallest coding sequences for ABE and CBE are, respectively, ~1947 bp or ~2355 bp in length if a dCas12f1 is used or ~2862-3578 bp in length if a dCas12j2 is used. Base editors are described in the art, for example in US Patent 10,113,163, US Patent 10,167,457, and US Patent 11,820,990.

[0145] For prime editors (PEs), an engineered reverse transcriptase (RT) enzyme is fused to a dCas. In addition, the gRNA is extended to contain a primer binding site and a RT template sequence (thus called pegRNA). The pegRNA is, therefore, capable of identifying the target site and providing the new genetic information to replace the target DNA nucleotides by the 86ny-2939083Attorney Docket No: 293082000140 RT domain. The RT-dCas complex and the pegRNA can be delivered separately. Prime editors are described in the art, for example, in US Patent 11,447,770.

[0146] Zinc finger nucleases (ZFNs), Transcription activator-like effector nucleases (TALENs), and homing endonucleases (HEs) all usually function by forming a dimer at the binding site, with each monomer being a fusion protein of binding and cutting domains; however, monomer applications of these technologies exist and are known in the art. The binding domains recognize and bind a target DNA sequence which brings the cutting domains of the two monomers together, breaking the DNA double strands within the working window, resulting in erroneous repairs or corrective recombination. The binding domains are all engineered for targeting a particular double-stranded DNA sequence, and they have exquisite specificity mainly due to their long (~20-30 nt) binding site, a feature that becomes essential in some applications.

[0147] Using FokI endonuclease as the cutting domain is currently common for ZFNs and TALENs. For binding, ZFNs use designed zinc-finger domains and TALENs utilize domains derived from transcription activator-like effectors. Unlike ZFNs and TALENs, HEs use a homing nuclease like I-CreI as the cutting domain, and the binding domains in most cases are not clearly separated from the cutting domain. Use of zinc finger nucleases for genetic modification is known in the art, as in Maeder et al. (2008). D. Four-part BSMV vector systems

[0148] In one aspect of the current disclosure, here is provided a four-part BSMV expression control vector system. One aspect of the current disclosure provides an expression vector including: (a) a vector comprising a Barley Stripe Mosaic Virus (BSMV) RNAα, (b) a vector comprising a BSMV RNAβ1 derived from a BSMV RNAβ, (c) a vector comprising a BSMV RNAβ2 derived from BSMV RNAβ, and (d) a vector comprising a BSMV RNAγ.

[0149] In one aspect of the current disclosure, here is provided a four-part BSMV expression control vector system. One aspect of the current disclosure provides an expression vector including: (a) a vector comprising a Barley Stripe Mosaic Virus (BSMV) RNAα, (b) a vector comprising a BSMV RNAβ, (c) a vector comprising a BSMV RNAγ1 derived from BSMV RNAγ, further comprising a functional sgRNAγ promoter, and (d) a vector comprising a BSMV RNAγ2 derived from BSMV RNAγ, further comprising a functional sgRNAγ promoter, wherein the expression vector system does not comprise a mobile element of the IS3 family. In some embodiments, the four-part BSMV expression control vector system includes an α, a β, a γ1, and a γ2 vector, wherein the γ1 and the γ2 vectors are from the improved split-γ system. 87ny-2939083Attorney Docket No: 293082000140 In some embodiments, the four-part BSMV expression control vector system including the improved split-γ vectors has increased genetic stability. In some embodiments, the four-part BSMV expression control vector system including the improved split-γ vectors has increased expression levels of the γb protein. In some embodiments of any of the previous method aspects, which may be combined with any of the preceding embodiments, (a) the cargo nucleic acid is delivered at a higher efficiency than a cargo nucleic acid delivered with a control expression vector system, (b) one or more of the vectors have increased stability in planta compared to a vector from a control expression vector system, (c) there is an increase in in vitro transcription one or more of the vectors compared to a control expression vector system, (d) the expression vector system produces more γb protein compared to a control expression vector system, (e) the expression vector system results in improved efficiency of transient expression in planta compared to a control expression vector system, and / or (f) the expression vector system results in improved efficiency of cell-to-cell movement of the viral vectors in planta compared to a control expression vector system. In some embodiments of any of the previous methods aspects, (a) the cargo nucleic acid is delivered at a higher efficiency than with a control expression vector system, (b) the vectors have increased stability in planta, and / or (c) there is an increase in in vitro transcription of the vectors. In some embodiments, the expression vector system produces more γb protein than a control expression vector system. In some embodiments, the expression vector system results in improved efficiency of transient expression in planta compared to a control expression vector system. In some embodiments, the expression vector system results in improved efficiency of cell-to-cell movement of the viral vectors in planta compared to a control expression vector system.

[0150] Yet another aspect of the disclosure provides an inoculant mixture including infectious RNAs, wherein the mixture includes: (a) a Barley Stripe Mosaic Virus (BSMV) RNAα, (b) at least one RNA derived from BSMV RNAβ, (c) a BSMV RNAγ1 derived from BSMV RNAγ, wherein the BSMV RNAγ1 includes a subgenomic promotor, (d) a BSMV RNAγ2 derived from BSMV RNAγ, wherein the BSMV RNAγ2 includes a subgenomic promotor, and wherein at least one RNA includes a cargo nucleic acid. In an additional embodiment of this aspect, which may be combined with any of the preceding embodiments of this aspect, the sgRNAγ promoter includes SEQ ID NO: 56. In yet another embodiment of this aspect, which may be combined with any of the preceding embodiments of this aspect, the RNAγ1 includes a γa gene and a cargo space. In still another embodiment of this aspect, which may be combined with any of the preceding embodiments of this aspect, the RNAγ2 includes a γb gene and a cargo space. In an additional embodiment of this aspect, which may be 88ny-2939083Attorney Docket No: 293082000140 combined with any of the preceding embodiments of this aspect, the RNAγ1 includes a cargo nucleic acid. In another embodiment of this aspect, which may be combined with any of the preceding embodiments of this aspect, the RNAγ2 includes a cargo nucleic acid. In an additional embodiment of this aspect, which may be combined with any of the preceding embodiments of this aspect, the RNAγ1 includes a cargo nucleic acid and the RNAγ2 includes a cargo nucleic acid. In a further embodiment of this aspect, which may be combined with any of the preceding embodiments of this aspect, the mixture includes at least one RNA derived from BSMV RNAβ. In some embodiments of this aspect, the RNAβ includes a cargo nucleic acid. E. Five-part BSMV vector systems

[0151] In some aspects, herein is provided a five-part BSMV expression control vector system. One aspect of the current disclosure provides an expression vector including: (a) a vector comprising a Barley Stripe Mosaic Virus (BSMV) RNAα, (b) a vector comprising a BSMV RNAβ1 derived from BSMV RNAβ, comprising an sgRNAβ1 promoter, (c) a vector comprising a BSMV RNAβ2 derived from BSMV RNAβ, comprising a sgRNAβ1 promoter, (d) a vector comprising a BSMV RNAγ1 derived from BSMV RNAγ, further comprising a functional sgRNAγ promoter, and (e) a vector comprising a BSMV RNAγ2 derived from BSMV RNAγ, further comprising a functional sgRNAγ promoter. In some embodiments, the expression vector system does not comprise a mobile element of the IS3 family. In some embodiments, the five-part BSMV expression control vector system includes an α, a β1, a β2, a γ1, and a γ2 vector. In some embodiments, the five-part BSMV expression control vector including a split-β and an improved split-γ vector system has increased cargo space for multiplexing. In some embodiments, the five-part BSMV expression control vector system including the improved split-γ vectors has increased genetic stability. In some embodiments, the five-part BSMV expression control vector system including the improved split-γ vectors has increased expression levels of the γb protein.

[0152] Engineered BSMV vectors β1 and β2 are exemplified respectively in SEQ ID NOs: 24 and 25. The β1 is produced by replacing the portion of RNAβ containing the βb, βc, βd and βd’ ORFs with a ligation-independent cloning (LIC) site. The β2 is produced by replacing a large portion of the βa ORF with a LIC site. Use of the β1 and the β2 vectors has at least doubled the cargo space of the vector system compared with the known four-component system (Cheuk and Houde, 2018). 89ny-2939083Attorney Docket No: 293082000140

[0153] The γ1 vector can provide up to about 1 kb of cargo space, and the γ2 vector can host up to about 2.5-3 kb of cargo. The β1 vector can provide up to about 3 kb of cargo space, and the β2 vector can host up to about 1 kb of cargo. In total, the γ1, the γ2, the β1, and the β2 vectors can work together with native RNAα to provide four LICs / cargo spacers (a five- component system, with one LIC on each of the β1, β2, γ1, and γ2 vectors) with approximately 7 kb in cargo space. F. Methods of use of engineered BSMV expression vector systems

[0154] The general procedure to apply this BSMV vector system includes: 1) adding a 5’ and a 3’ LIC cloning adaptor to the corresponding end of a coding sequence of interest; 2) cloning the modified coding sequence into an LIC in either the β, the β1, the β2, the γ, the γ1, or the γ2 vector; 3a) for the RNA method, in vitro transcribing the infectious RNA vectors and mix all the components equally in volume as the inoculant for infecting a plant of interest; 3b) for the DNA method, mixing all the required Agrobacterium strains harboring the cDNA vectors equally, infiltrating them into the plant of interest if the plant can be infected by the Agrobacterium strains; 4) if the plant of interest cannot be infected by the Agrobacterium strains, infiltrating Nb tobacco leaves first and then collecting leaf sap as the inoculant to infect the plant of interest either by seed imbibition or by rubbing it into the surface of plant tissue / organ; 5) phenotyping and / or genotyping the treated plants for successful expression; 6) if the expression is for gene editing, selecting edited plants, harvesting seeds from them, and selecting virus-free offspring germinated from the harvested seeds with RT-PCR assay.

[0155] For DNA-based applications, there are two methods of delivery. The first way is to biolistically bombard the T-DNA-based plasmids directly into a plant of interest (Hu et al., 2019). This method requires special bombardment equipment and the tissues to which it can be applied are very limited. More commonly, the plasmids are individually transformed into Agrobacterium cells which are then mixed and co-infiltrated into Nicotiana benthamiana (Nb tobacco) leaves. The viruses produced in Nb leaves are then harvested to use as inoculant for infection of other plants of interest. The advantage of the DNA methods is the relative ease of handling the DNA.

[0156] For RNA-based applications, the in vitro transcribed infectious BSMV RNAs were manually rubbed into the surface of a plant of interest with fingers (Scofield and Brandt, 2017). A big advantage of the RNA-based application over a DNA-based application is its time saving feature since the usual steps of transforming Agrobacterium and agroinfiltration are omitted. Its big disadvantage, however, is the difficulty in handling RNA because RNA is very easily 90ny-2939083Attorney Docket No: 293082000140 destroyed biologically, chemically, or physically. This method has been used to knock-down (Zhuang et al., 2013) and overexpress (Paudel et al., 2020) Fusarium head blast resistance gene WFhb1-1 in wheat. G. Control Expression Vector Systems

[0157] In some embodiments, a vector system provided herein possesses one or more advantages over a control expression vector system. In some embodiments, the control expression vector system includes the original BSMV split-γ vector system (Cheuk & Houde, (2018) A new barley stripe mosaic virus allows large protein overexpression for rapid functional analysis. Plant Physiol 176:1919-1931), wherein the original BSMV split-γ vector system includes a mobile element of the IS3 family, such as a Tn10 transposable element or IS1 element, and wherein the RNAγ2 does not include an sgRNAγ promotor. In some embodiments, the control vector system includes a BSMV split-γ vector system in which the RNAγ2 does not include a sgRNAγ promotor. In some embodiments, the control vector system includes a BSMV split-γ vector system that includes an IS3 family mobile element, optionally a Tn10 transposable element or an IS1 element. In some embodiments, the control vector system includes a BSMV vector system that includes an IS3 family mobile element, optionally a Tn10 transposable element or an IS1 element. In some embodiments, the control vector system includes a BSMV split-γ vector system that includes an IS1 mobile element. In some embodiments, the control vector system includes a BSMV vector system that includes an IS1 mobile element. In some embodiments, the control expression vector system includes pCaBS- γ (SEQ ID NO: 19) or pCaBS-γ1 (SEQ ID NO: 20).

[0158] In some embodiments, the advantage or improvement includes increased genetic stability compared to a control expression vector system. In some embodiments, the advantage or improvement includes increased cargo expression levels compared to a control expression vector system. In some embodiments, the advantage or improvement includes increased production of the γb protein compared to a control expression vector system. In some embodiments, the advantage or improvement includes improved efficiency of transient expression in planta compared to a control expression vector system. In some embodiments, the advantage or improvement includes improved efficiency of cell-to-cell movement of the viral vectors in planta compared to a control expression vector system.

[0159] In some embodiments, a vector system provided herein is an improvement over a control expression vector system. In some embodiments, the vector system provided herein is an improvement over the original split-γ vector system (Cheuk & Houde, (2018) A new barley 91ny-2939083Attorney Docket No: 293082000140 stripe mosaic virus allows large protein overexpression for rapid functional analysis. Plant Physiol 176:1919-1931). In some embodiments, a vector system provided herein is an improvement over a BSMV split-γ vector system that does not include a sgRNAγ promotor. In some embodiments, a vector system provided herein is an improvement over a BSMV split-γ vector system that includes an IS3 family mobile element. In some embodiments, a vector system provided herein is an improvement over a BSMV split-γ vector system that includes a Tn10 transposable element. In some embodiments, the Tn10 transposable element includes SEQ ID NO: 57. In some embodiments, a vector system provided herein is an improvement over a BSMV split-γ vector system that includes an IS1 mobile element. In some embodiments, the Tn10 transposable element includes SEQ ID NO: 58. IV. Engineered tobravirus systems for expression in plants

[0160] Tobravirus is a viral genus that consists of TRV, pea early-browning virus (PEBV) and pepper ringspot virus (PepRSV) (Robinson and Harrison, 1989). Tobravirus has a wide range of host plant species, most of which are dicots (Harrison & Robinson, 1986), but also including monocots such as wheat, maize, and barley (Zhang et al., 2017). Tobravirus viruses are mainly transmitted via root-feeding nematodes, although seed transmission has been observed in some host plant species. Research has revealed that tobravirus viruses can enter somatic apical meristem tissues of N. benthamiana and Arabidopsis but is not transmitted through their seeds to next generation (Martín-Hernández & Baulcombe, 2008). This characteristic makes tobravirus viruses an excellent expression vector system that can deliver and express cargo in meristem tissues for gene editing, while the viral vectors themselves will be eliminated from the seeds the meristem tissues derive.

[0161] The genomes of all three tobravirus species are similarly organized and usually consist of two positive-sense, single-stranded RNAs (called RNA1 and RNA2 hereafter) (FIG.15A) (MacFarlane, 2021). The flexibility of RNA2 in length and content has made it attractive for engineering as expression vectors (Ratcliff et al.2001; Liu et al.2002; Weiss et al., 2024). As a result, almost all the tobravirus-derived expression vectors created so far are engineered from RNA2. Burch-Smith et al. (2004) reported that a TRV RNA2-derived expression vector can have a cargo space of up to 1.5 kb; beyond that, the insert instability increases dramatically. Therefore, the relatively small cargo space in a RNA2-derived expression vector limits its applications, particularly in delivery of gene editing reagents since Cas gene constructs used in gene editing applications are usually over 3.0 kb in length. Meanwhile, RNA1 has been barely touched for vector engineering. Guilford et al. (1991), Zieggler-Graff et al. (1991) and Deng et 92ny-2939083Attorney Docket No: 293082000140 al. (2013) developed expression vectors out of TRV RNA1 by replacing either the whole or partial sequence of MP or CRP with a cargo. They found that the deleted parts must be compensated by a functional counterpart in the same cells since they are indispensable to viral normal activities and functions. Besides, the cargo spaces created were very limited. No further effort to engineer or use a RNA1-derived expression vector has since been reported. Therefore, a need exists for improved tobravirus-derived expression systems with increased cargo capacity and / or delivery capabilities.

[0162] Strains of TRV are known in the art, including but not limited to SYM (Hamilton et al., 1987), ORY (Sudarshana & Berger, 1998), PSG (Cornelissen et al., 1986), PLM (Angenent et al., 1989b), TCM (Angenent et al., 1986), PpK20 (Hernández et al., 1995), Rostock (unpublished), TpO1 (MacFarlane et al., 1999), PaY4 (unpublished), and ON (Uhde et al., 1998). In some embodiments, the plant virus is a tobacco rattle virus isolate selected from the group consisting of SYM, ORY, PLM, PSG, TCM, PpK20, Rostock, TpO1, PaY4, and ON.

[0163] Strains of PEBV are known in the art, including but not limited to SP5 (MacFarlane et al., 1989), Algerian (Ferguson, 1999), TpA56 (MacFarlane & Brown, 1995), and E116 (Swanson & MacFarlane, 1999). In some embodiments, the plant virus is a pea early brown virus isolate selected from the group consisting of SP5, Algerian, TpA56, and E116.

[0164] Strains of PepRSV are known in the art, including but not limited to CAM (Bergh et al., 1985). In some embodiments, the plant virus is a pepper ringspot virus CAM isolate. A. Engineered tobravirus expression vector systems

[0165] Existing plant viral delivery systems do not have the cargo capacity to deliver many of the standard components used in gene editing, such as a 4 kb nucleic acid encoding a Cas9 cassette, let alone Cas fusion proteins that are useful in base editing or prime editing. While others have focused on engineering novel gene editing components that have smaller cargo requirements, such as RNA-guided TnpB (Weiss et al. 2024), a different approach is to engineer new expression vector systems that have increased cargo capacity.

[0166] Tobravirus is a viral genus that consists of tobacco rattle virus (TRV), pea early- browning virus (PEBV) and pepper ringspot virus (PepRSV) (Robinson and Harrison, 1989). Tobravirus has a wide range of host plant species, most of which are dicots (Harrison & Robinson, 1986), but also including monocots such as wheat, maize, and barley (Zhang et al., 2017). Tobravirus viruses are mainly transmitted via root-feeding nematodes, although seed transmission has been observed in some host plant species. Research has revealed that tobravirus viruses can enter somatic apical meristem tissues of N. benthamiana and 93ny-2939083Attorney Docket No: 293082000140 Arabidopsis but is not transmitted through their seeds to next generation (Martín-Hernández & Baulcombe, 2008). This characteristic makes tobravirus viruses an excellent expression vector system that can deliver and express cargo in meristem tissues for gene editing, but the viral vectors themselves will be eliminated from the seeds the meristem tissues derive.

[0167] The genomes of all three tobravirus species are similarly organized and usually consist of two positive-sense, single-stranded RNAs (called RNA1 and RNA2 hereafter) (FIG.15A) (MacFarlane, 2021). Each tobravirus RNA is individually encapsulated into a rod-shaped particle. RNA1 can infect plants alone without forming virus particles. RNA1 is about 6.8 kb in size and bears three open reading frames (ORFs) for all the functions necessary for virus replication and spreading, and in fact has been observed to move within a plant without RNA2—something once referred to as non-moving (NM) in TRV (MacFarlane, 1999). ORF1a is the most 5’ proximal ORF and directly translates a replicase, which can be a small or a large protein (134 kDa or 194 kDa in TRV) depending on whether or not a read-through translation occurs. The small protein contains a methyltransferase (MT) motif and a helicase (HEL) motif, and the large protein has an additional RNA-dependent RNA polymerase (RdRp) motif. ORF1b encodes a moving protein (MP). ORF1c encodes a cysteine-rich protein (CRP). Both MP and CRP are translated from their own sub-genomic RNAs (i.e., sgRNA1a and sgRNA1b) which are transcribed from genomic RNA1 in planta. Both MP and CRP contribute to the suppression of host RNA silencing defenses (Ghazala et al. 2008; Martín-Hernández & Baulcombe 2008; Martínez-Priego et al.2008; Deng et al.2013).

[0168] RNA2 bears ORFs 2a, 2b and 2c. However, the length of RNA2 varies considerably among isolates that have been sequenced so far (1.8-3.9 kb in TRV), where one or both of ORF2b and ORF2c may be missing. Some isolates may also encode additional transcripts at the most 5’ proximal region ahead of 2a (MacFarlane, 1999). In addition, the 3’ UTR of RNA2 may recombine with that of RNA1, introducing further variability. Nevertheless, RNA2 molecules always have ORF2a that encodes the viral coat protein (CP). ORFs 2b and 2c translate two non-structural proteins produced during infection that are not part of the final structure of the viral particle, that enable the tobravirus viruses to enter stubby-root nematodes, their transmitting agent. RNA2 genes are expressed from sub-genomic RNAs that are transcribed from the genomic RNA2 in planta. The flexibility of RNA2 in length and content has made it attractive for engineering as expression vectors (Ratcliff et al.2001; Liu et al.2002; Weiss et al., 2024). As a result, almost all the tobravirus-derived expression vectors created so far are engineered from RNA2. Burch-Smith et al. (2004) reported that a TRV RNA2-derived expression vector can have a cargo space of up to 1.5 kb; beyond that, the insert instability 94ny-2939083Attorney Docket No: 293082000140 increases dramatically. Therefore, the relatively small cargo space in a RNA2-derived expression vector limits its applications, particularly in delivery of gene editing reagents since Cas gene constructs used in gene editing applications are usually over 3.0 kb in length. Therefore, a need exists for improved tobravirus-derived expression systems with increased cargo capacity and / or delivery capabilities.

[0169] Because RNA2 has been extensively engineered as various expression vectors, little room has been left for further cargo expansion. Meanwhile, RNA1 has been barely touched for vector engineering. Guilford et al. (1991), Zieggler-Graff et al. (1991) and Deng et al. (2013) developed expression vectors out of TRV RNA1 by replacing either the whole or partial sequence of MP or CRP with a cargo. They found that the deleted parts must be compensated by a functional counterpart in the same cells since they are indispensable to viral normal activities and functions. Besides, the cargo spaces created were very limited. Therefore, no further effort to engineer or use a RNA1-derived expression vector has since been reported. On the other hand, deletion mutants of RNA1 were made for functional analyses (Guilford et al., 1991; Zieggler-Graff et al., 1991; Martin-Hernandez & Baulcombe, 2008). Zieggler-Graff et al. (1991) demonstrated that the deleted section can be functionally compensated by a wildtype counterpart that was co-inoculated with the deletion strain, which suggests that more than one RNA1-derived RNAs can co-exist and co-function in the same host cells. Nevertheless, there is great utility to be gained from a novel strategy to split RNA1 apart as expression vectors.

[0170] Provided herein are transient expression systems for plants engineered by splitting tobravirus RNA1 into two or more expression vectors. RNA1 normally has three ORFs that encode four different proteins: a small replicase protein with a methyltransferase motif and a helicase motif (MT-HEL), a large replicase protein with a methyltransferase motif, a helicase motif, and an RNA-dependent RNA polymerase motif (MT-HEL-RdRp), a moving protein (MP), and a cysteine-rich protein (CRP), which are necessary for viral infection, replication, and cell-to-cell movement. RNA1 vectors may function without the co-existence of RNA2, resulting in the primary loss of function of its transmissibility through its natural transmission vector, nematodes. To make this expression system work, each component vector must bear a unique coding sequence for at least one of the four proteins. In this way, component vectors in this system are functionally complementary to each other. In theory, more than fourteen kinds of engineered RNA1 vectors are possible, providing cargo spaces of various lengths, cloning sites of various number, and different position(s) where the cloning site(s) are incorporated into each vector (FIGS.23A-23C). 95ny-2939083Attorney Docket No: 293082000140

[0171] The RNA2 vector has also been engineered to accompany the RNA1-derived vectors, to provide the viral coat protein (CP) and an extra cargo space. An engineered RNA2 vector may also bear a coding sequence(s) for one or more of the RNA1 proteins. Exemplary RNA2 vectors are provided in FIG. 23D. In some embodiments, the tobravirus-derived expression vector system does not comprise an RNA2 vector or RNA2-derived vector. The inclusion of a RNA2-derived vector in this system is dependent on its functional complementarity in the system. Without being bound by theory, the coat protein delivered on RNA2 helps protect the viral RNAs from host defense systems and is associated with reduced viral pathogenicity, and the RNA2 ORF2b and ORF2c proteins aid the virus to enter the stubby-root nematode that acts as a transmission agent. If these functions are not necessary in the desired system embodiment, RNA2 may be dispensed with.

[0172] In some applications, an intuitive combination of vectors can be formed that contains a single copy of each of the necessary tobravirus genes for infection, replication, and movement from cell-to-cell in the host plant. These may contain two or more vectors. For instance, an RNA1a vector (e.g., TRV1a, PEBV1a, or PepRSV1a) comprising a replicase ORF (FIG.15B, top box) may accompany an RNA1b vector, which bears ORFs 1a and 1b containing MP and CRP respectively (FIG. 15B, middle box, top). This intuitive system can also have a third component, which can be an RNA1-derived vector, an RNA2 vector, or an RNA2-derived expression vector, such as but not limited to, a TRV2a or a TRV2ap vector (FIG.15C, bottom box). An example of an intuitive four-component system contains TRV1a (which contains a replicase), TRV1d (which contains CRP), TRV1e (which contains MP), and TRV2a (FIGS. 23B-23D). An intuitive five-component system could contain TRV1d (which contains CRP), TRV1e (which contains MP), TRV1g (which contains MT-HEL), TRV1i (which contains RdRp), and TRV2a (FIGS.23B-23D). An additional example contains TRV1a (which contains replicase), TRV1dp (which contains CRP), and TRV-MPp (which contains MP).

[0173] In some embodiments, non-intuitive combination of vectors can be formed, containing all necessary genes for infection, replication, and movement from cell-to-cell in the host plant along with duplicates or redundancy of genes. An example is a combination of an RNA1b or RNA1bp vector with an RNA1c vector, which would duplicate the MP (FIGS.23B).

[0174] These expression vectors are engineered out of the infectious cDNA clones of RNA1 and / or RNA2 (Hamilton & Baulcombe, 1989; Liu et al.2002). Each vector can be driven by a promoter (such as 2X CaMV 35S) and terminated by a self-cleaving ribozyme (Rz) and a corresponding terminator (e.g., 35S terminator) for Agrobacterium-based applications (DNA- based application hereafter) or by a T7 promoter and terminated by self-cleaving ribozyme (Rz) 96ny-2939083Attorney Docket No: 293082000140 and a corresponding terminator for infectious RNA-based applications (RNA-based application hereafter). Other promoters and their terminators can be used as well.

[0175] As illustrated by FIG.16A and FIG.24A, the RNA1a vectors are made by deleting the entire MP and CRP coding sequences (~1.2 kb) from RNA1. Nevertheless, the promoter for transcription of the sub-genomic RNA encoding MP is preserved. A ligation-independent cloning (LIC) site is inserted in the place between the promoter and the 3’ UTR of RNA1. As a result, the vector will transcribe a sub-genomic RNA encoding the cargo to effectively increase its copy number and thus produce more gene product(s) in planta. This vector may have a cargo space of at least 1.2 kb, which is estimated by the length of the deleted fragment. The RNA1c vectors (FIG.24C) are made by replacing the CRP ORF with an LIC site, while the RNA1d vectors (FIG. 24D) are made by replacing the entire replicase ORF and most of the MP ORF (without deleting the promoter for the sub-genomic CRP RNA) with an LIC cargo space. Since RNA1 functions as the mRNA for replicase, both the RNA1b and RNA1d vectors are suitable for expressing a heterologous protein directly from the cargo. The RNA1e vector (FIGS.24E-24F) is made by replacing most of the replicase ORF with a LIC site and deleting the CRP ORF. RNA1f (FIG.24G) is created by replacing the MP ORF with a LIC site. The RNA1g vectors (FIG.24H) are created by replacing the RdRp motif of the replicase, the MP ORF and the CRP ORF with a LIC site. The RNA1h vectors (FIG.24J) are made by replacing the MT and HEL motifs of the replicase with an LIC site. The RNA1i vectors (FIGS. 24K- 24L) are made by removing the MP and CRP ORFs and replacing the MT-HEL motif with an LIC site.

[0176] In some applications, an RNA needs to be transcribed from the cargo. For example, cargo that contains a sequence coding for guide RNA(s) for CRISPR / Cas gene editing must be transcribed as an RNA. This option is not available for RNA1b, RNA1d, RNA1e, RNA1g, RNA1h and RNA1i vectors because their LICs take partially or completely replace the replicase ORF, which uses genomic RNA1 as the mRNA for replicase translation. To solve this problem, a promoter for the coat protein of PEBV is added to the 5’ ends of the LIC sites. The derived vectors are, therefore, named as RNA1bp, RNA1dp, RNA1ep, RNA1gp, RNA1hp and RNA1ip and can be used as an alternative to RNA1b, RNA1d, RNA1e, RNA1g, RNA1h and RNA1i respectively. The PEBV-CP promoter enables transcription of a sub-genomic RNA encoding the cargo, which not only allows the cargos at the following LIC site to be transcribed as RNA but also increases the copies of the cargo for stronger expression. Alternatively, a promoter such as PEBV-CP can be incorporated into the cargo space insert (rather than the vector itself) for transcription of a sub-genomic RNA from the cargo for the same purpose. 97ny-2939083Attorney Docket No: 293082000140

[0177] RNA2a and RNA2ap vectors are made by replacing ORFs 2b and 2c on RNA2 with an LIC site. In the RNA2a vector, the promoter for the sub-genomic RNA encoding ORFs 2b and 2c is preserved. In the RNA2ap vector, the native promoter has been replaced by a PEBV- CP promoter, which performs better in certain host plant species. In some gene editing applications, the TRV RNA2 promoter for transcription of ORF2b sub-genomic RNA is replaced by the promoter for PEBV’s coat protein to drive the expression of the cargo (Ali et al., 2015; Aragones et al., 2022; Ellison et al., 2020; Ghoshal et al., 2020; MacFarlane and Popovich, 1999). This is because that PEBV-CP promoter has been found to perform better than the native promoter in some plant species in driving the cargo expression (Guo et al.2022). These two versions of RNA2-derived vectors (RNA2a and RNA2ap) are interchangeable in the system, and each may have a cargo space of approximately 1.5 kb in length as estimated by Burch-Smith et al. (2004).

[0178] Therefore, the tobravirus-derived expression systems may provide at least two, three, four, or five cargo spaces, by combining at least two, three, four, or five tobravirus-derived expression vectors. Table 1 lists the conservative estimated cargo space provided by each tobravirus expression vector, made on the basis of the fragment(s) of RNA1 and / or RNA2 that the cargo replace. The listed cargo spaces are reasonably expected to have a stable capacity of up to approximately 20% greater than the replaced length. Without being bound by theory, it is possible that the cargo spaces can hold longer nucleic acid inserts, but there would be a corresponding increase in the likelihood of the virus expelling the cargo during viral replication in planta. A typical Cas9 expression cassette is around 4 kb in length and thus should well fit into the approximately 5 kb or 6 kb cargo spaces in RNA1b / bp, RNA1d / dp, or RNA1e / ep, with additional cargo spaces available for multiple gRNAs for multiplex gene-editing either on the same vector or on the other vectors used in combination within this system. This is in stark contrast to other engineered plant virus delivery systems, which do not have the cargo capacity to carry a 4 kb insert. The engineered tobravirus systems therefore offer both an increased number of potential cargo spaces, as well as increased cargo capacity within certain cargo spaces. This will enable the delivery of larger cargo, allowing for expression of larger proteins or protein fusions in planta. Table 1. Estimated cargo space provided by each tobravirus expression vector. Estimations are .98ny-2939083Attorney Docket No: 293082000140

[0179] In some applications, a helper RNA sequence, such as one encoded by Flowering Locus T (FT) (Jackson et al., 2012; Notaguchi et al., 2015; Ellison et al.,2020), a tRNA (Ellison et al., 2020; Nagalakshmi et al., 2022; Zhang et al., 2016; Weiss et al., 2024) or a WUSCHEL (WUS) fragment (Ellison et al., 2020), is used to help systemic movement of an engineered viral vector within the host plant. Mobile helpers such as these can be incorporated into a cargo construct of the tobravirus-derived vector described in this disclosure if needed.

[0180] For DNA-based applications, there are two methods of delivery. The first way is to biolistically bombard the T-DNA-based plasmids directly into a plant of interest (Hu et al., 2019). This method requires special bombardment equipment, and the tissues to which it can be applied are very limited. More commonly, the plasmids are individually transformed into Agrobacterium cells which are then mixed and co-infiltrated into Nicotiana benthamiana (Nb tobacco) leaves. The viruses produced in Nb leaves are then harvested to be used as the inoculant for infection of other plants of interest. The advantage of the DNA methods is the relative ease of handling the DNA.

[0181] For RNA-based applications, the in vitro transcribed infectious tobravirus RNAs are manually rubbed into the surface of a plant of interest with fingers (Scofield and Brandt, 2017). A big advantage of the RNA-based application over a DNA-based application is its time saving feature since the usual steps of transforming Agrobacterium and agroinfiltration are omitted. Its big disadvantage, however, is the difficulty in handling RNA because RNA is very easily destroyed biologically, chemically, or physically.

[0182] The engineered plant vector systems can be used in a wide variety of host plants, based on the numerous plants that tobraviruses are known to infect. Many of the tobravirus host plants are dicots, such as but not limited to beans, beets, brassicas, peas, peppers, spinach, tobacco, soybean, tomato, cotton, Arabidopsis, rose, strawberry, chrysanthemum, and tulip, but tobraviruses are also known to infect a number of monocot species, such as but not limited to wheat, maize, barley, rice and Miscanthus. As such, the tobravirus-derived systems can be used 99ny-2939083Attorney Docket No: 293082000140 to infect a different and expanded set of host plants as other engineered plant viral systems, such as barley stripe mosaic virus, which predominantly infects monocots. In some embodiments, the dicot or dicot cell is selected from the group consisting of Arabidopsis and tobacco, or a cell thereof. In certain embodiments, the infectious RNAs enter the germline cells. In other embodiments, the infectious RNAs do not enter the germline cells. In certain embodiments, the one or more cargo space inserts including nucleic acid sequences enter the germline cells. In other embodiments, the one or more cargo space inserts including nucleic acid sequences do not enter the germline cells. B. Engineered tobravirus expression vector systems for genome editing

[0183] CRISPR / Cas, base editors (BEs) and prime editors (PEs) are all based on Cas endonucleases (e.g., Cas9, Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas12f, Cas12g, Cas12h, Cas12i, Cas12j, Cas12k, Cas12l and Cas12m etc.). In a CRISPR / Cas system, a Cas-gRNA complex searches and binds on target DNA sequences specified by the gRNA. The Cas endonuclease then breaks one or both stands of the target DNA to make mutations. The Cas and the gRNA can be complexed together for a single delivery or be delivered separately as two reagents.

[0184] BEs and PEs do not break any of the DNA strands for mutant creation. Instead, they have one or several functional domains fused to a catalytically impaired Cas endonuclease (dCas) (e.g., dCas9, dCas12a, dCas12f, dCas12j, etc.) and make change(s) within the sequence(s) targeted by the dCas-gRNA complex (Kantor et al., 2020). For BEs, an ssDNA- deaminase specific either for cytidine in CBE (cytidine base editing) or for adenine in ABE (adenine base editing) is fused to the N terminal of the dCas that removes the aminol group from the targeted C or A base changing it to uracil (U) or Inosine (I), respectively. In CBE, a uracil DNA glycosylase inhibitor is optionally also added to the C-terminal of the dCas to inhibit uracil DNA glycosylase (UDG) in the cells which catalyzes removal of U from DNA during DNA repairing in cells. The smallest coding sequences for ABE and CBE are, respectively, ~1947 bp or ~2355 bp in length if a dCas12f1 is used or ~2862-3578 bp in length if a dCas12j2 is used. Base editors are described in the art, for example in US Patent 10,113,163, US Patent 10,167,457, and US Patent 11,820,990.

[0185] For prime editors (PEs), an engineered reverse transcriptase (RT) enzyme is fused to a dCas. In addition, the gRNA is extended to contain a primer binding site and a RT template sequence (thus called pegRNA). The pegRNA is, therefore, capable of identifying the target site and providing the new genetic information to replace the target DNA nucleotides by the 100ny-2939083Attorney Docket No: 293082000140 RT domain. The RT-dCas complex and the pegRNA can be delivered separately. Prime editors are described in the art, for example, in US Patent 11,447,770.

[0186] Zinc finger nucleases (ZFNs), Transcription activator-like effector nucleases (TALENs), and homing endonuc...

Claims

Attorney Docket No: 293082000140 CLAIMS What is claimed is:

1. An expression vector system, comprising: (a) a vector comprising a Barley Strip Mosaic Virus (BSMV) RNAα, (b) a vector comprising a BSMV RNAβ1 derived from BSMV RNAβ, (c) a vector comprising a BSMV RNAβ2 derived from or consisting of BSMV RNAβ, and (d) at least one vector derived from BSMV RNAγ, wherein the expression vector system is capable of systemic infection and / or systemic expression in a plant.

2. The expression vector system of claim 1, wherein the expression vector system comprises a BSMV RNAγ1 vector derived from BSMV RNAγ, and a BSMV RNAγ2 vector derived from BSMV RNAγ.

3. An expression vector system comprising: (a) a vector comprising a Barley Stripe Mosaic Virus (BSMV) RNAα, (b) at least one vector derived from BSMV RNAβ, (c) a vector comprising a BSMV RNAγ1 derived from BSMV RNAγ, comprising a functional subgenomic RNAγ (sgRNAγ) promoter, and (d) a vector comprising a BSMV RNAγ2 derived from BSMV RNAγ, comprising a functional subgenomic RNAγ (sgRNAγ) promoter, wherein the expression vector system is capable of systemic infection and / or systemic expression in a plant.

4. The expression vector system of claim 3, wherein the expression vector system comprises a BSMV RNAβ1 vector derived from BSMV RNAβ, and a BSMV RNAβ2 vector derived from or comprising BSMV RNAβ.

5. The expression vector system of any one of claims 1, 2, and 4, wherein both the RNAβ1 and RNAβ2 comprise a subgenomic RNAβ1 (sgRNAβ1) promoter.

6. The expression vector system of any one of claims 1, 2, and 4, wherein the RNAβ1 comprises a βa gene and a cargo space, and / or wherein the RNAβ2 comprises a βb gene, a βc gene, a βd gene, a βd’ gene, and a cargo space.

7. The expression vector system of any one of claims 2-6, wherein the RNAγ1 comprises a γa gene and a cargo space, and / or wherein the RNAγ2 comprises a cargo space and a γb gene. 151ny-2939083Attorney Docket No: 293082000140 8. The expression vector system of any one of claims 1-7, comprising one, two, three, or four cargo spaces, optionally wherein one or more of the cargo spaces are ligation- independent cloning (LIC) sites or restriction enzyme sites, optionally wherein one or more cargo spaces comprise cargo nucleic acid.

9. The expression vector system of claim 8, wherein RNAβ1 comprises a cargo nucleic acid, RNAβ2 comprises a cargo nucleic acid, RNAγ1 comprises a cargo nucleic acid, and / or RNAγ2 comprises a cargo nucleic acid.

10. The expression vector system of any one of claims 1-9, wherein one or more vectors is composed of cDNA.

11. The expression vector system of claim 10, wherein the cDNA encoding the BSMV RNA or the modified BSMV RNA is operably linked to a promoter, optionally wherein the promoter is for in vitro transcription by a DNA-dependent RNA polymerase, or optionally wherein the promoter is for in planta transcription.

12. The expression vector system of any one of claims 1-11, comprising a first cargo nucleic acid for transient expression in planta, wherein the first cargo nucleic acid is a genome editing component.

13. The expression vector system of claim 12, wherein the first cargo nucleic acid encodes a Cas protein, and further comprising a second cargo nucleic acid for transient expression in planta, wherein the second cargo nucleic acid comprises one or more guide RNA(s) for the Cas protein.

14. The expression vector system of claim 13, further comprising a third cargo nucleic acid for transient expression in planta, wherein the third cargo nucleic acid encodes one or more guide RNA(s) for the Cas protein, and / or further comprising a fourth cargo nucleic acid for transient expression in planta, wherein the fourth cargo nucleic acid encodes a Cas protein.

15. The expression vector system of any one of claims 10-14, wherein the cDNA is cloned into a T-DNA-derived binary plasmid, wherein the T-DNA-derived binary plasmid does not comprise an IS3 mobile element, optionally wherein the IS3 mobile element is a Tn10 transposable element or an IS1 element.

16. An infectious RNA produced by in vitro transcription of a vector of the expression vector system of any one of claims 1-15. 152ny-2939083Attorney Docket No: 293082000140 17. A liquid composition comprising infectious RNAs produced by in vitro transcription of each vector of the expression vector system of any one of claims 10-15.

18. A method of transiently expressing an RNA or protein in a plant or plant cell, the method comprising: (a) providing the expression vector system of any one of claims 10-15, wherein the expression vector system comprises at least one cargo nucleic acid sequence encoding an RNA or protein; (b) in vitro transcribing the vector system into infectious RNAs; and (c) inoculating a plant or plant cell with the infectious RNAs; wherein the RNA or protein encoded by the at least one cargo nucleic acid sequence is transiently expressed in the plant or plant cell.

19. A method of transiently expressing an RNA or protein in a plant or plant cell, the method comprising: (a) cloning a nucleic acid sequence encoding an RNA or protein into the cargo space of the expression vector system any one of claims 6-15; (b) in vitro transcribing the vector system into infectious RNAs; and (c) inoculating a plant or plant cell with the infectious RNAs; wherein the RNA or protein encoded by the nucleic acid sequence is transiently expressed in the plant or plant cell.

20. The plant or plant cell of claim 17 or claim 18.

21. A method of engineering a novel viral system for expression in plants, the method comprising: (a) providing a plant virus nucleic acid; (b) determining a cutting site within the nucleic acid; (c) splitting the nucleic acid at the cutting site into a first engineered nucleic acid and a second engineered nucleic acid; and (d) introducing a cloning site on the first engineered nucleic acid and / or on the second engineered nucleic acid; thereby engineering a novel viral system for expression in plants comprising the first and / or the second engineered nucleic acid, optionally wherein the novel viral system is capable of systemic infection and / or systemic expression in a plant. 153ny-2939083

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