Transcomplementary vector system for highly efficient heterologous gene expression in plants.

Description

1 / 65 Transcomplementary vector system for highly efficient heterologous gene expression in plants.

[001] The present invention relates to a transcomplementary system for heterologous gene expression in plants and comprises a producer vector and an auxiliary vector, wherein I) said producer vector is incapable of performing at least one function necessary to cause systemic infection in plants, for example, by cell-to-cell replication and / or long-distance movement, and II) the auxiliary vector completes the missing function. Certain producer and auxiliary vectors in a transcomplementary system are derived from the same virus, or certain producer and auxiliary vectors in a transcomplementary system are derived from different plant viruses. The present invention likewise provides methods of use, for example, to improve the yield of heterologous expressed proteins, particularly in cultivated plants, and the ease of use of said system. TECHNICAL FIELD AND INDUSTRIAL APPLICABILITY OF THE INVENTION

[002] This invention comprises virus expression vectors selected from the Tobravirus group and, in particular, virus-derived sequences, methods for modifying such vectors, increasing gene expression in plants and uses thereof. STATEMENT REGARDING RESEARCH SPONSORED BY THE FEDERAL GOVERNMENT

[003] This invention was not made with any government support and the government does not hold any rights to this invention. BACKGROUND OF THE INVENTION

[004] In the last three decades, plants have been increasingly used as expression hosts for the heterologous expression of various recombinant proteins. Thus, this expression Petition 870250089530, dated 10 / 01 / 2025, page 33 / 103 2 / 65 heterologous transformation can be performed by integrating the gene of interest (GOI) into the plant genome, thereby creating a stable or transgenic transformed plant that stably expresses the target protein, or by introducing the GOI into a plant vector that can be introduced into the host plant and maintained transiently in said plant cells, thereby preventing GOI replication.

[005] According to current opinion, transient gene expression systems technology in plant systems has considerable advantages over stable expression, namely: (I) it does not require regeneration of the transformed cell; (II) it has no effect on the stability of the host genome; and (III) it is independent of the positional effects of T-DNA integration sites.

[006] Transient expression is primarily enabled by agroinfiltration or agroinfection (Grimsley et al., 1985). In agroinfection, a functional plant promoter and RNA- or DNA-based viral cDNA are transferred as Agrobacterium T-DNA into plant cells. The T-DNA is transcribed in the plant to generate biologically active viral RNAs that can initiate self-replication.

[007] Plant virus-based vectors allow for this rapid and transient expression of target proteins in whole plants (Pogue et al. 2002, Scholthof et al. 2002, Yusibov et al. 1999, Grimsley et al. 1986, Turpen et al. 1993) and numerous publications and patents in the field describe systems based on DNA and RNA viral vectors (Kumagai et al. 1994, Mor et al. 2003, US5316931, US5589367, US58866785, US5491076, US5981236, EP1706497, US8951791, US8936937, WO1998054342, WO02088369, WO2009087391).

[008] Existing viral vector systems are generally restricted to a narrow range of hosts in terms of their optimal performance, and even the level of expression of these vectors in their Petition 870250089530, dated 10 / 01 / 2025, page 34 / 103 3 / 65 most favorable host is well below the upper biological limit of expression systems in plants.

[009] Recently, a TMV expression vector compatible with agroinfections was constructed through extensive modifications, such as multiple mutations, destruction of cryptic introns, and insertions of multiple plant gene introns into TMV cDNA sequences in a binary vector (Marillonnet et al. 2005) named MagnICON (Gleba et al. 2005, Marillonnet et al. 2004, EP1706497). While these modifications have improved the efficiency with which TMV vectors can be introduced into plants, the MagnICON vector approach does not readily adapt to a high-throughput workflow.

[0010] The magnICON system has been used to produce a wide variety of plant proteins, including plague antigens (Santi et al., 2006), virus-like particles (VLPs) of the hepatitis B virus core antigen (HBcAg) (Huang et al., 2006), and Norwalk virus VLPs (Santi et al., 2008). Antibody production was, for a time, more problematic. Soon after the development of the MagnICON system, it became evident that viral competition would hinder the co-expression of multiple replicons in the same cells, making it impossible to produce oligomultimeric proteins such as immunoglobulin G (IgG) (Gleba et al., 2005).

[0011] The solution to this problem was to express one strand of IgG using MagnICON and the other strand using a different non-competitive viral vector system based on potato virus X (PVX). This allowed the production of assembled IgG in N. bentamiana (Giritch et al., 2006). Since then, IgG have been produced using this non-competitive viral vector strategy in an industrial setting as candidate vaccines for n-Hodgkin lymphoma (Bendandi et al., 2010). Petition 870250089530, dated 10 / 01 / 2025, page 35 / 103 4 / 65

[0012] Following the initial reports on MagnICON, a single-module overexpression vector based on TMV was developed. The TRBO system (US8936937) is based on a 35S promoter-led replicon, based on the TMV genome, from which the CP gene, under the control of the native subgenomic promoter present in the MP gene, is replaced by a multiple cloning site into which the GOI is inserted (Lindbo, 2007). Like MagnICON, TRBO replicons cannot move systemically, but they can move from cell to cell within an infected leaf; therefore, agroinfiltration is crucial to initiate whole-plant infection.

[0013] The author demonstrated that co-expression of an exogenous gene silencing suppressor had no effect on transgene expression. However, this was not the case for versions of the expression vector that still contained the CP gene, leading the author to suggest that this sequence may be an inducer of gene silencing.

[0014] Furthermore, it has since been determined that the suppressor of silencing activity is already present in the TMV replicase protein (Wang et al., 2012). In addition, the initiation of primary replicons in agroinfected cells appeared to be as efficient as the enhanced MagnICON vectors, but without the need to insert numerous exogenous sequences, such as introns, into the tobamovirus genes. The author suggests that this may be due to the fact that TRBO is based on the U1 strain of TMV, while MagnICON is based on turnip vein clearing virus (TVCV) and the cruciferous TMV strain (cr-TMV). Therefore, it appears that a TRBO vector has all the advantages of a MagnICON vector, while being smaller and simpler in its design.

[0015] Other transient expression systems are based on a variety of single- or double-stranded plant viruses, such as po Petition 870250089530, dated 10 / 01 / 2025, page 36 / 103 5 / 65 texvirus (Baulcome et al. 1995, Santa Cruz et al. 1996, Smolenska et al. 1998, O'Brien et al. 2000, Lico et al. 2006, Mardanova et al. 2009, Larsen and Curtis, 2012), tobravirus (Constantin et al. 2004, Deng et al. 2013, MacFarlane, 2010, Yang et al. 2013, MacFarlane and Popovich, 2000, Swanson et al. 2002), geminivirus (Mor et al. 2003, Zhang and Mason, 2006, Dugdale et al. 2013, Huang et al. 2010, Sainsbury and Lomonossoff, 2008) and comovirus (Sainsbury et al. 2010, Porta et al. 1994, Usha et al.) 1993, Gopinath and another 2000, Sainsbury and Lomonossoff, 2014, Peyret and Lomonossoff, 2013).

[0016] Thus, based on previous observation, there is still a great unmet need for the development of improved systems for heterologous expression of transgenes in plants that: I) contain convenient cloning sites for GOIs; II) can be used to infect plants easily and economically; III) lead to efficient systemic infection of inoculated plants to obtain high yields of recombinant product; IV) minimize the risk of environmental contamination (e.g., viruses that can infect non-target plants).

[0017] Furthermore, as interest in proteomic, biochemical, and protein structure increases, there is a growing need for efficient and user-friendly recombinant protein expression systems. Enhancing transient expression vectors to make them easier to use, more cost-effective, and produce higher levels of target proteins in competitive large-scale manufacturing will be of great use. SUMMARY OF THE INVENTION

[0018] The present invention encompasses the recognition of the need to develop plant expression systems that present only a minimal risk of environmental contamination and provides methods and reagents for the expression of polynucleotides in plants. Petition 870250089530, dated 10 / 01 / 2025, page 37 / 103 6 / 65 with a reduced risk of widespread contamination.

[0019] Some particular definitions and embodiments of the present invention will now be described in more detail. Several objectives and advantages of this invention will become apparent to those skilled in the art from the following detailed description of preferred embodiments, when read in the light of the accompanying drawings and claims. This application refers to several patents, patent applications, and publications. The contents of all of them are incorporated herein by reference. In addition, the following publications are incorporated herein by reference: Current Protocols in Molecular Biology, Current Protocols in Immunology, Current Protocols in Protein Science, and Current Protocols in Cell Biology, all volume editions of John Wiley & Sons, NY, beginning in 2020.

[0020] The present invention will be described with respect to specific embodiments and with reference to certain drawings, but the invention is not limited to them, but only to the claims. The drawings described are only schematic and not limiting. In the drawings, the size of some of the elements may be exaggerated and not drawn to scale for illustrative purposes. The dimensions and relative dimensions do not correspond to actual reductions of the invention for practical use.

[0021] Furthermore, the terms first, second, and the like in the description and claims are used to distinguish between similar elements and not necessarily to describe a sequence, whether temporally, spatially, in order of classification, or otherwise. It should be understood that the terms thus used are interchangeable in appropriate circumstances and that the embodiments of the invention described herein are capable of operating on sequences other than those described or illustrated herein.

[0022] Notwithstanding the exemplary embodiments described below, Petition 870250089530, dated 10 / 01 / 2025, p. 38 / 103 7 / 65 The present invention is limited only by the appended claims. The appended claims are explicitly incorporated into this detailed description, in which each claim and each combination of claims, as permitted by the dependency structure defined by the claims, forms a separate embodiment of the present invention.

[0023] The reference, throughout this descriptive report, to an embodiment or an embodiment or embodiments means that a particular feature, structure or resource described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearance of the phrases in an embodiment or in an embodiment in several places throughout this descriptive report does not necessarily refer to the same embodiment, but may. Furthermore, the particular features, structures or resources may be combined in any suitable manner, as would be evident to one with ordinary skill in the art from this description, in one or more embodiments.

[0024] Similarly, it should be appreciated that, in the description of exemplary embodiments of the invention, several features of the invention are sometimes grouped into a single embodiment, figure or description thereof, for the purpose of simplifying the description and assisting in the understanding of one or more of the various inventive aspects. This method of description, however, should not be interpreted as reflecting the intention that the claimed invention requires more features than those expressly mentioned in each claim.

[0025] Instead, as the following claims reflect, the inventive aspects reside in less than all the features of a single embodiment described above. Thus, the claims that follow the detailed description are expressly Petition 870250089530, dated 10 / 01 / 2025, p. 39 / 103 8 / 65 incorporated into this detailed description, with each claim representing, by itself, a separate embodiment of this invention.

[0026] Furthermore, although some embodiments described herein include some, but not other, features included in other embodiments, combinations of features from different embodiments must be within the scope of the invention and form different embodiments, as would be understood by those skilled in the art. For example, in the following claims, any of the claimed embodiments can be used in any combination.

[0027] Non-essential improvements and adjustments made to the method described by the invention shall still be within the scope of protection of the invention. However, the raw materials used are not always described in detail. The raw materials may be commercially available products. The process steps or preparation methods that are not always described in detail are process steps or preparation methods known to those skilled in the art.

[0028] The raw materials used to obtain the present invention are commercially available products; the process steps or preparation method, which likewise are not always described in detail, are process steps or preparation methods known to those skilled in the art.

[0029] When used here, the term about refers to ± 10%.

[0030] The term consisting of means including and limited to.

[0031] The term essentially consisting of means that the composition, process or structure may contain additional components, steps and / or parts, but only if the additional components, steps and / or parts meet the basic and innovative properties of the claimed composition and therefore do not alter the material. Petition 870250089530, dated 10 / 01 / 2025, page 40 / 103 9 / 65 mentions the basic and innovative characteristics of the claimed composition and / or method or structure.

[0032] When used here, the singular forms a, an and the include plural references, unless the context clearly indicates otherwise. For example, the term a compound or at least one compound may encompass a variety of compounds, including mixtures thereof, which may be used in food, cosmetics, pharmaceuticals, industrial products, medical products, laboratory culture media and many other applications.

[0033] Throughout this application, various embodiments of this description may be presented in a range format. It should be understood that the range format description is merely for convenience and brevity and should not be interpreted as an inflexible limitation on the scope of the description. In this way, the description of a range should be considered as having specifically described all possible subintervals as well as individual numerical values ​​within that range. For example, the description of a range such as 1 to 6 should be considered as having specifically described subintervals such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within that range, e.g., 1, 2, 3, 4, 5, and 6. This applies regardless of the range width.

[0034] Whenever a numerical range is indicated here, it must include any quoted numeral (fractional or integral) within the indicated range. The expressions ranging / varying between a first indicative number and a second indicative number and ranging / varying from a first indicative number to a second indicative number are used here interchangeably and must include the first and second indicated numbers and all fractional and integral numerals between them.

[0035] When used here, the term method refers to manner Petition 870250089530, dated 10 / 01 / 2025, page 41 / 103 10 / 65 methods, means, techniques and procedures for carrying out a given task, including, but not limited to, methods, means, techniques and procedures known or easily developed from methods, means, techniques and procedures known to professionals in the chemical, pharmacological, biological, biochemical and medical fields.

[0036] In the description provided here, several specific details are presented. However, it should be understood that embodiments of the invention may be practiced without these specific details. In other cases, well-known methods, structures and techniques have not been shown in detail so as not to impair the understanding of this description.

[0037] Note that the term comprising, as used in the claims, should not be interpreted as being restricted to the means listed below; it does not exclude other elements or steps. Thus, it should be interpreted as specifying the presence of the stated features, integers, steps or components, but does not preclude the presence or addition of one or more other features, integers, steps or components, or groups thereof.

[0038] In the present invention described, several specific details are presented. Embodiments of the present invention may be carried out without these specific details. Furthermore, well-known features, elements and / or steps are not necessarily described in detail for the purposes of clarity and conciseness of the present description.

[0039] In one aspect, expression vectors modified by Tobacco Rattle Virus (TRV) and novel methods for modifying said TRV vectors are provided. The methods provide modifications that improve both the options for cloning genes in TRV and the ease and efficiency of infecting plants with vectors. Petition 870250089530, dated 10 / 01 / 2025, page 42 / 103 11 / 65 TRV.

[0040] The present invention comprises a system for replicating or for replicating and expressing a sequence of interest in a host plant, comprising: I) a producer vector that allows trans-cell-to-cell movement of the transcomplementary viral replicon; II) an auxiliary vector, wherein said auxiliary vector is i) capable of systemic movement in said host plant, both in the presence of said producer vector, and ii) capable of expressing a polynucleotide of interest in said host plant with the aid of one or more proteins necessary for systemic movement, whether by replication, cell-to-cell and / or long-distance movement with the aid of said producer vector; and III) an integrase vector that allows the assembly in plant of components i) and ii), being the producer and auxiliary vector, thus allowing the resulting components to be recombined within the plant cell and leading to the creation of a vector sequence within a fully functional infectious replicon.

[0041] Wherein said producer vector is capable of replication or replicating and expressing said GOI in said host plant, but incapable of moving systemically in said host plant in the absence of one or more proteins expressed by said auxiliary vector, as defined by claim 1.

[0042] The present invention further provides a process for replicating or a process for replicating and expressing a GOI in a host plant, comprising supplying the cells of said host plant with said producer vector and said auxiliary vector, as defined in the claims. In a specific embodiment, the present invention further provides a process for replicating or a process for replicating and expressing a GOI in a plant Petition 870250089530, dated 10 / 01 / 2025, p. 43 / 103 12 / 65 host plant, comprising supplying the host plant cells with said producer vector, said auxiliary vector and said integrase vector, as defined in the claims. The invention can be used to replicate a GOI in a host plant or to replicate and express said GOI, for example, for the production of said GOI in said host plant. Furthermore, the present invention provides a process for producing polypeptides in plant hosts or in host plant cells.

[0043] In certain embodiments of the present invention, the auxiliary vector is dysfunctional for performing replication. For example, the producer vector may include an RNA polymerase coding sequence (e.g., a replicase coding sequence) and a movement protein coding sequence, such that said producer vector is capable of performing cell-to-cell movement, but may not have a CP coding sequence, such that said producer vector is not capable of performing long-distance systemic movement.

[0044] Therefore, the present invention reduces or eliminates the risk of dissemination to non-target plants, accompanied by (recombinant) vectors, thus significantly improving the environmental safety of heterologous gene expression in host plants and, moreover, allows greater flexibility in the cultivation of recipient host plants.

[0045] The present invention similarly provides vector systems that can be used as components for other purposes. For example, the present invention offers those skilled in the art, for example, a researcher, the flexibility to design a plant expression system with qualities of more than one plant virus. For example, in specific embodiments, the producing vector desirably has the GOI positioned so that its expression Petition 870250089530, dated 10 / 01 / 2025, page 44 / 103 13 / 65 is controlled by the CP promoter. Therefore, in many cases, it is desirable to base the producing vector on a viral system with a strong CP promoter.

[0046] However, viruses with strong CP promoters can sometimes have limited specificity for the host plant, as they may be unable to infect, replicate within, and / or allow cell-to-cell or long-distance movement within certain plant hosts. Therefore, it may be desirable to base the auxiliary vector on a viral system with broad host plant specificity, thereby allowing the exploitation of host plants that would otherwise normally be inaccessible to that viral system.

[0047] Furthermore, the inventor found that, despite the increased protein expression of a specific system in embodiments of the present invention, host plants transformed with a transcomplementary TRV vector of said invention appeared healthier, i.e., exhibited less necrosis, compared to plants transformed with a known TRV vector (pYL192 vector (TRV1), addgene plasmid no. 1489968) (FIG. 19B). Plant health is essential in downstream purification of GOIs, as tannins released as a result of necrosis can interfere with protein purification (Chatterjee et al. 2012), thus contributing to increased operating expenses (COGs), which in turn results in higher cost of goods sold (COGS).

[0048] In another aspect, the cloning options in the present invention have been improved by the development of a simple, innovative and inexpensive method for cloning DNA inserts into transcomplementary vector systems compatible with agroinfection. The cloning method allows for efficient targeting of PCR products without the need for digestion with restriction enzymes. The Petition 870250089530, dated 10 / 01 / 2025, page 45 / 103 The 14 / 65 cloning method does not depend on expensive topoisomerase or recombinase enzymes, using enzymes that are commonly available and known to those skilled in the art. Therefore, it allows the use of these vectors in high-throughput experiments.

[0049] In one particular aspect, the present invention provides an improvement over known transient expression technology in the prior art. In particular, the present invention provides a system for enhancing transcomplementary transient expression vectors to very high levels of heterologous protein expression in plant hosts, wherein said vectors are efficiently delivered to plant cells by agroinfection. DEFINITIONS

[0050] For the purposes of the present invention, the following terms have their meanings as understood by those skilled in the art.

[0051] “GENE”: In general, a gene includes regulatory gene coding sequences, such as promoters, enhancers, and terminators, and / or intron sequences, in addition to coding sequences or open reading frames (ORFs). The definition of a gene may include nucleic acids that do not code for proteins but provide templates for the transcription of functional RNA molecules.

[0052] The inventor notes, for the sake of clarity, that when used in the present invention, the term “gene” generally refers to a nucleic acid that includes a portion that codes for a protein; the term may likewise encompass regulatory coding sequences such as promoters, enhancers, terminators, etc. This definition is not intended to exclude the application of the definition “gene” to non-protein-coding expression units, but rather to clarify that, in most cases, the term, when used in the present invention, refers to a protein-coding nucleic acid. Petition 870250089530, dated 10 / 01 / 2025, p. 46 / 103 15 / 65 ínas.

[0053] “GENE PRODUCT” or “EXPRESSION PRODUCT”: generally, an RNA transcript of the gene or a polypeptide encoded by an RNA transcript of the gene. The expression of a gene or polynucleotide refers to I) transcription of the RNA of the gene or polynucleotide, or II) translation of the RNA transcript of the gene or polynucleotide, or both.

[0054] “ISOLATED”: when referred to here, a compound that is separated from the components to which it is normally associated or produced or prepared by a process involving human action.

[0055] “NATIVE” or “NATURAL”: means “naturally occurring” and, when used here, refers to things or components that occur in their relevant form in nature. In contrast, “not naturally occurring” refers to things or components whose form results from human action.

[0056] “OPERATIONALLY LINKED” or “OPERABLE LIGAND”: when referred to herein, it means the relationship between two individual nucleic acids or coding sequences, wherein the expression of one of the coding sequences is controlled, regulated, modulated, etc. by the other coding sequence. Note that a single coding sequence can be operationally linked to multiple other coding sequences. For example, a single promoter coding sequence can direct the transcription of multiple RNAs.

[0057] GOI; when referred to herein, means gene of interest and refers to any target coding sequence to be expressed in a host plant. In various embodiments of the present invention, the GOI may be a coding sequence for a protein, but it may also be a coding sequence that provides a template for the transcription of a structural RNA or an active RNA, such as a ribozyme, interfering RNA, and the like. Frequently, Petition 870250089530, dated 10 / 01 / 2025, p. 47 / 103 16 / 65 The GOI will be a gene that is not expressed in nature in the relevant plant cell type (e.g., heterologous expression), or is not expressed at the level at which the GOI is expressed when expression is achieved by human intervention. Furthermore, the GOI is not naturally associated with the coding sequences of the vector of the present invention. The abbreviation “GOI” is used interchangeably with “nucleic acid” or “nucleic acid molecule” and is referred to herein as a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides, or analogues thereof. Polynucleotides may have any three-dimensional structure and perform any function, known or unknown.The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (sites) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, DNA isolated from any sequence, RNA isolated from any sequence, nucleic acid probes, and inductors. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be introduced before or after polymer assembly. The nucleotide sequence may be interrupted by non-nucleotide components. A polynucleotide may also be modified after polymerization, such as by conjugation with a labeling component.

[0058] “EXOGENOUS RNA SEGMENT”: when referred to herein, describes an RNA segment to be inserted into the RNA of the virus to be modified, the source of the exogenous RNA segment being different from the RNA virus itself. The source may be another virus, a living organism such as a plant, animal, bacterium or fungus. The exogenous RNA may Petition 870250089530, dated 10 / 01 / 2025, p. 48 / 103 17 / 65 It can be a chemically synthesized RNA or a combination of the above. The exogenous RNA segment can perform any appropriate function known to be performed by an RNA segment. Such functions include, but are not limited to, a coding function in which the RNA acts as a messenger RNA that encodes a sequence which, when translated by the host cell, results in the synthesis of a polypeptide or protein with useful or desired properties. The RNA segment can also be structural, as in ribosomal RNA, regulatory, as in small nuclear RNAs or antisense RNA, or catalytic. An exogenous RNA segment can be a complete or partial coding sequence.

[0059] AUTOREPLICATE or AUTOREPLICATION: when referred to here, it means a vector with the ability to self-copy and that carries sufficient information in its own genetic elements so that it does not depend on other genetic elements for its replication within a plant host cell.

[0060] “VECTOR”: refers to a nucleic acid molecule capable of carrying another nucleic acid molecule to which it has been attached and may include a plasmid, cosmid or viral vector.

[0061] “PLASMID”: refers to autonomous extrachromosomal genetic elements found in many types of bacteria. They consist of closed circular DNA. Some can integrate into the host chromosome and replicate with it. Some viruses that infect prokaryotes have properties similar to those of plasmids, in particular, of integrating into the host cell chromosome. The vectors preferred in the present invention are maintained extrachromosomally.

[0062] “COMPONENT”; as mentioned here, is intended to include both protein-coding and non-coding sequences Petition 870250089530, dated 10 / 01 / 2025, p. 49 / 103 18 / 65 tes, such as cis action sequences (e.g., promoters, origin of assembly, portions corresponding to UTRs in mRNA).

[0063] “PHENOTYPIC TRAIT”; when referred to herein, is an observable, measurable or detectable property resulting from the expression or suppression of a gene or genes. “Phenotype” includes readily observable characteristics and biochemical processes.

[0064] “PROMOTER”; when referred to herein, is the 5' flanking non-coding sequence substantially adjacent to a coding sequence that is involved in the beginning of the transcription of the coding sequence. DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THIS INVENTION INVENTIVE VECTORS

[0065] As duly noted above, the present invention describes systems for heterologous expression of GOIs in host plants. These systems may include one or more viral components and, as such, a multitude of plant viruses are known to infect a variety of plant species. These can be used for heterologous expression according to the preferred embodiments of the present invention. FIG. 1. Represents a schematic representation of families of viruses that infect plants. Additional information can be found, for example, in Virus Taxonomy, Ninth Report of the International Committee on Taxonomy of Viruses (Andrew MQ King, Elliot Lefkowitz et al., 2011), the full contents of which are incorporated herein by reference.

[0066] A virus is a collection of one or more nucleic acid template molecules, usually enclosed in one or more protective protein or lipoprotein coats, capable of organizing its own replication only within suitable host cells. Within these cells, viral replication occurs: I) dependent on Petition 870250089530, dated 10 / 01 / 2025, pp. 50 / 103 19 / 65 host protein synthesis machinery, II) organized from assemblies of required materials, rather than binary fission, III) located in sites that are not separated from the contents of the host cell by a lipoprotein bilayer membrane, and IV) continuously giving rise to variants through various types of alterations in the viral nucleic acid.

[0067] Plants (cultivated, medicinal or ornamental) can be infected by viruses. It can all start with an insect bite, a nematode, physical damage to the cell or by microorganisms such as bacteria, fungi or archaea.

[0068] The virus only needs to reach one cell to initiate infection. However, since viruses cannot do anything on their own, they need to hijack the mechanisms of the infected cell to produce copies of themselves. Therefore, first, plant viruses need to cross the cell wall, as well as the protective layers composed of waxes and pectins.

[0069] Some types of enveloped viruses fuse directly to the outer (plasma) membrane of the cell, while most are fully enveloped by endocytosis (mechanical rupture) or similar processes and then fuse their envelope with the membrane of the inner organelle that surrounds them (e.g., an endosome) to gain access to the interior of the cell. Eventually, the descendant viruses are released into neighboring cells and this cycle repeats continuously.

[0070] Therefore, the virus can reach the plant's vascular system (similar to the circulatory system of animals) and can spread over long distances from the initial point of infection, infecting everything from roots to young leaves. In the laboratory, viruses are typically administered to plant cells simply by placing them in contact (friction or forced entry) with the plant. Petition 870250089530, dated 10 / 01 / 2025, page 51 / 103 20 / 65

[0071] The structure and replication of viruses have the following characteristics: I) the infectious nucleic acid can be DNA or RNA (but never both) and can be single-stranded or double-stranded. If the nucleic acid is single-stranded, it can be positive-sense or negative-sense. (Positive-sense has the sequence that would be used as an mRNA for translation to produce a virus-encoded protein), II) the mature virus particle can contain polynucleotides different from the genomic nucleic acid, III) where the genetic material consists of more than one nucleic acid molecule, each can be housed in a separate particle or all can be located in one particle, IV) viral genomes vary widely in size, encoding between 1 and about 250 proteins. Plant viral genomes are at the smaller end of this range, mainly encoding between 1 and 12 proteins.Plant virus-encoded proteins can have functions in virus replication, cell-to-cell or long-distance movement, virus structure, and transmission by invertebrates or fungi. In general, many viruses contain genes that encode one or more proteins that participate in the replication process (referred to here as replication proteins or replicating proteins). For example, many plant RNA viruses encode an RNA polymerase. V) Viruses undergo genetic changes. Point mutations occur frequently as a result of nucleotide changes caused by errors in the copying process during genome replication. Other types of genetic changes can result from recombination, rearrangement of genome fragments, loss of genetic material, or acquisition of nucleotide sequences from unrelated viruses or the host genome.VI) Enzymes specified by the viral genome may be present in the viral particle. Most of these enzymes are related to nucleic acid synthesis. VII) The replication of many viruses occurs in structures... Petition 870250089530, dated 10 / 01 / 2025, pp. 52 / 103 21 / 65 distinct virus-induced mutations in the plant cell. VIII) Some viruses share with certain non-viral nucleic acid molecules the property of integration into host cell genomes and translocation from one integration site to another. IX) Some viruses require the presence of another virus for their replication.

[0072] In general, for a virus to spread from cell to cell, it requires a functional coat and movement protein, while systemic (long-distance) dissemination requires a functional coat protein and, more generally, a functional movement protein as well. Furthermore, viruses may also contain other components that are necessary for local or systemic dissemination or to facilitate such dissemination. These cis-acting regulatory components may be coding or non-coding components. For example, they may correspond to portions of a 3'UTR of a viral transcript.

[0073] Any virus that infects plants can be used to prepare a viral vector or vector system according to the present invention. Particularly preferred viruses possess a positive (+) strand genome, either single-stranded (ss) or double-stranded (ds). The use of techniques and reagents for manipulating the genetic material present in such viruses is well known to those skilled in the art. Generally, a DNA copy of the viral genome is prepared and cloned into a bacterial vector.

[0074] Certain ssDNA viruses, preferably Mastrevirus within Geminiviridae, are likewise of particular interest. It will be further appreciated that, in general, the viral vectors and genomes of the present invention can exist in RNA or DNA form.

[0075] Furthermore, when reference is made to a characteristic, such as the genome or part thereof, of an RNA virus present in a DNA vector, it will be understood by those skilled in the art that Petition 870250089530, dated 10 / 01 / 2025, pp. 53 / 103 22 / 65 the characteristic is present as the DNA copy of the RNA form.

[0076] As described in the preferred embodiments of the present invention, several different types of viruses can be used independently or in combination with each other. Preferred viruses from the Virgaviridae family include Tobacco Rattle Virus, Pea Early Yellowing Virus and Pepper Ringing Virus, with Tobacco Rattle Virus being most preferably used.The preferred viruses of the Comovirinae subfamily include Cowpea Mosaic Virus, Andean Potato Mosaic Virus, Bean Pod Spot Virus, Bean Rough Mosaic Virus, Broad Bean Spot Virus, Broad Bean True Mosaic Virus, Cowpea Severe Mosaic Virus, Soybean Mosaic Virus, Pea Green Mosaic Virus, Pea Mild Mosaic Virus, Pigeon Pea Mosaic Virus, Radish Mosaic Virus, Red Clover Mosaic Virus, Pumpkin Mosaic Virus, Ullucus C Virus, with Cowpea Mosaic Virus being the most preferred.Preferred viruses of the Potyviridae family include Tobacco Corrosion Virus, Barley Yellow Mosaic Virus, Bellflower Vein Mosaic Virus, Blackberry Y Virus, Maclura Mosaic Virus, Potato Y Virus, Rose Yellow Mosaic Virus, Ryegrass Mosaic Virus, Sweet Potato Mild Mosaic Virus, Wheat Mosaic Virus, Wheat Streaked Mosaic Virus, Agropyron Mosaic Virus, Algerian Watermelon Mosaic Virus, Alpinia Mosaic Virus, Alstroemeria Mosaic Virus, Alternanthera Mild Mosaic Virus, Amaranth Leaf Mottling Virus, Amazon Lily Mosaic Virus, Angelica Y Virus, Y Virus of Celery, Araucaria Mosaic Virus, Arracacha Mottling Virus, Latent Artichoke Virus, Asparagus Virus 1, Banana Bract Mosaic Virus, Barbacena Virus Y, Virus of. Petition 870250089530, dated 10 / 01 / 2025, page 54 / 103 23 / 65 Barley Mild Mosaic Virus, Basella Rough Mosaic Virus, Bean Common Mosaic Necrosis Virus, Bean Common Mosaic Virus, Bean Yellow Mosaic Virus, Beetroot Mosaic Virus, Bidens pilosa Mosaic Virus, Bidens Piosa Mottling Virus, Blue Hyacinth Virus A, Broadleaf Sorrel Virus A, Bromine Streaked Mosaic Virus, Brugmansia Mosaic Virus, Brugmansia suaveolens Mottling Virus, Butterfly Flower Mosaic Virus, Caladenia Virus A, Calanthe Mild Mosaic Virus, Calla Lily Latent Virus, Aster Mottling Virus, Canna Yellow Stripe Virus, Cardamom Mosaic Virus, Virus of Vein Mottling Virus of Carnation, Carrot Thin Leaf Virus, Carrot Virus Y, Cassava Brown Streak Virus, Catharanthus Mosaic Virus, Celery Mosaic Virus, Ceratobium Mosaic Virus, Pepper Ringleaf Virus, Pepper Vein Mottling Virus, Chinese Artichoke Mosaic VirusChinese Yam Necrotic Mosaic Virus, Clitoria Virus Y, Clover Yellow Vein Virus, Coccinia Mottling Virus, Goosegrass Streak Virus, Colombian Datura Virus, Commelina Mosaic Virus, Cowpea Aphid-Transmitted Mosaic Virus, Cucumber Vein Yellowing Virus, Swamp Cypress Virus Y, Cyrtanthus elatus Virus A, Daphne Mosaic Virus, Daphne Virus Y, Taro Mosaic Virus, Datura Ribbon Virus, Diuris Virus Y, Jegue Orchid Virus A, East Asian Passiflora Virus, Endive Necrotic Mosaic Virus, Ringed Virus of Euphorbia, Freesia Mosaic Virus, Fritillaria Y Virus, Gloriosa Striped Mosaic Virus, Habenaria Mosaic Virus, Hardenbergia Mosaic Virus, Mandrake Mosaic Virus, Hibbertia Y Virus, Hippeastrum Mosaic Virus, Hordeum Mosaic Virus, Hyacinth Mosaic Virus, Flower Break Virus, Petition 870250089530, dated 10 / 01 / 2025, p. 55 / 103 24 / 65 Impatiens, Iris fulva Mosaic Virus, Mild Iris Mosaic Virus, Severe Iris Mosaic Virus, Japanese Yam Mosaic Virus, Jasmine T Virus, Johnsongrass Mosaic Virus, Kalanchoe Mosaic Virus, Keunjorong Mosaic Virus, Konjac Mosaic Virus, Leek Yellow Band Virus, Italian Necrotic Virus of Lettuce, Lettuce Mosaic Virus, Lily Mosaic Virus, Lupinus Mosaic Virus, Mild Mottling Virus of Licorice, Dwarf Maize Mosaic Virus, Mallow Vein Sweeping Virus, Meadow Saffron Breakdown Virus, Moroccan Watermelon Mosaic Virus, Narcissus Degeneration Virus, Virus of End-of-Season Yellowing of Narcissus, Latent Narcissus Virus, Narcissus Yellow Band Virus, Nerine Yellow Band Virus, Nothoscordum Mosaic Virus, Oat Mosaic Virus, Oat Necrotic Mottling Virus, Onion Yellow Dwarf Virus, Ornithogalum Mosaic VirusOrnithogalum virus 2, Ornithogalum virus 3, Panax virus Y, Papaya Leaf Distortion Mosaic Virus, Papaya Ringleaf Virus, Parsnip Mosaic Virus, Passionflower Chlorosis Virus, Passion Fruit Lignification Virus, Pea Seed-Transmitted Mosaic Virus, Peanut Mottling Virus, Pecan Mosaic-Associated Virus, Goosegrass Mosaic Virus, Pepper Mottling Virus, Severe Pepper Mosaic Virus, Pepper Vein Mottling Virus, Yellow Pepper Mosaic Virus, Peruvian Tomato Mosaic Virus, Pfaffia Mosaic Virus, Pleione virus Y, Virus of Plum Pox, Knotweed Mosaic Virus, Potato Virus A, Potato Virus V, Buttercup Leaf Distortion Virus, Buttercup Mild Mosaic Virus, Buttercup Mosaic Virus, Rhopalanthe Virus Y, Rice Necrotic Mosaic Virus, Sarcochilus Virus Y, Chive Mosaic VirusShallot Yellowband Virus Petition 870250089530, dated 10 / 01 / 2025, pp. 56 / 103 25 / 65 Sorghum Mosaic Virus, Soybean Mosaic Virus, Spartina Mottling Virus, Spiranthes Mosaic Virus 3, Pumpkin Vein Yellowing Virus, Sugarcane Mosaic Virus, Sugarcane Streaked Mosaic Virus, Sunflower Chlorotic Mottling Virus, Sunflower Mosaic Virus, Sunflower Ringspot Virus, Sweet Potato Downy Mosaic Virus, Sweet Potato Latent Virus, Sweet Potato Mild Spot Virus, Sweet Potato Virus 2, Sweet Potato Virus C, Sweet Potato Virus G, Pampas Grass Mosaic Virus, Tamarillo Leaf Malformation Virus, Mosaic Virus of Telfairia, Telosma Mosaic Virus, Thunberg's Fritillaria Mosaic Virus, Tobacco Mottling Virus, Tobacco Vein Banding Mosaic Virus, Tobacco Vein Mottling Virus, Mild Tomato Mosaic Virus, Tomato Necrotic Dwarfism Virus, Tradescantia Mild Mosaic Virus, Tuberose Mild Mosaic VirusTuber Mild Mottling Virus, Tulip Rupture Virus, Tulip Mosaic Virus, Turnip Mosaic Virus, Twisted Foot Chlorotic Streak Virus, Ugandan Cassava Brown Streak Virus, Vallota Mosaic Virus, Vanilla Twisted Streak Mosaic Virus, Verbena Y Virus, Watermelon Leaf Mottling Virus, Watermelon Mosaic Virus, Eqlid Wheat Mosaic Virus, Wheat Fusiform Streak Mosaic Virus, Wheat Yellow Mosaic Virus, Asymptomatic Wild Onion Virus, Wild Potato Mosaic Virus, Wild Tomato Mosaic Virus, Wisteria Vein Mosaic Virus, Yam Chlorotic Mosaic Virus, Virus of Yam Mild Mosaic Virus, Yam Mosaic Virus, Bean-Yam Mosaic Virus, Oat Yellow Grass Mosaic Virus, Zantedeschia Mild Mosaic Virus, Corn Mosaic Virus, Zucchini Thread Virus, Zucchini Tiger Mosaic Virus, Yellow Spot Virus of, Petition 870250089530, dated 10 / 01 / 2025, page 57 / 103 26 / 65 Zucchini, Zucchini Yellow Mosaic Virus, although Tobacco Corrosion Virus is more preferably used.

[0077] To generate viral vectors to be used in accordance with the present invention, a variety of elements of the preferred plant viruses mentioned above are genetically modified according to techniques known to those skilled in the art (examples can be found in Molecular Cloning - A laboratory manual, 4th edition, by Michael R. Green and Joseph Sambrook, 2012, Plant Virology Protocols: From Virus Isolation to Transgenic Resistance, Methods in Molecular Biology, volume 81, by Gary Foster, etc.). According to the present invention, at least two vectors are employed, one or both dependent on each other to support the systemic functioning and heterologous expression of one or more GOIs, thus recognizing that the viral components used in the present invention can complement each other in trans-complementation to facilitate their functionality.

[0078] In a specific embodiment of the present invention, a producer vector is prepared, which includes a GOI that is placed under the control of regulatory coding sequences responsible for direct expression in a host plant. In preferred embodiments, a GOI is placed under the control of a viral promoter, for example, by inserting coding sequences containing a CP subgenomic RNA promoter. For example, when desired, the natural viral CP gene can be replaced by a GOI.

[0079] The producing vector may lack one or more components essential for systemic movement. For example, in preferred embodiments of the present invention, the producing vector does not contain coding sequences sufficient for the expression of a functional CP gene, but may include a coding sequence that allows cell-to-cell movement. The producing vector may contain Petition 870250089530, dated 10 / 01 / 2025, pp. 58 / 103 27 / 65 having one or more coding sequences as an assembly origin, which may be necessary in cis to allow the dissemination of the virus when present in cis. For example, the producer vector in certain embodiments of the present invention may contain an assembly origin necessary for – or that allows – the activity of a CP coding sequence, whether of the same type of virus as said producer virus or of another virus chosen from the preferred list mentioned above. Such elements may comprise a recognition site for a CP coding sequence. In other preferred embodiments of the present invention, the producer vector may not possess certain coding sequences that facilitate sufficient expression of functional MP (and / or replicase coding sequences). In these embodiments of the present invention, the producer vector may or may not possess sufficient coding sequences for the expression of a functional CP.

[0080] In another particular embodiment of the present invention, an auxiliary vector is similarly prepared and is intended to transcomplement the producer vector with its missing components, which are necessary to enable systemic infection of a host plant. For example, in certain preferred embodiments of the present invention, said auxiliary vectors may include a functional CP coding sequence component. These auxiliary vectors are suitable for transcomplementing a producer vector lacking a functional CP coding sequence component.

[0081] The auxiliary vector may lack one or more viral coding components that encode a replicase or movement protein essential for sufficient systemic infection of a host plant, when such viral coding component is not absent in the producer vector. The auxiliary vector may include a GOI that may be identical to or different from the GOI in the producer vector. In these cases Petition 870250089530, dated 10 / 01 / 2025, pp. 59 / 103 28 / 65 sos, it may be desirable to use a helper vector that is designed to be defective for systemic infection by lacking one or more essential cis-action coding sequences to minimize the spread of the helper vector to non-target plants.

[0082] According to embodiments of the present invention, the auxiliary vector may (but not necessarily) include a cell-to-cell motion coding sequence and / or may not possess one or more replicase coding sequences. In those particular embodiments of the present invention, in which the auxiliary vector does not include a cell-to-cell motion coding sequence, such coding sequence component must be included in the producer vector.

[0083] The complete inventive vectors of the present invention include all the essential components necessary for the successful systemic infection of a host plant and the heterologous expression of a GOI. Different vectors or elements may be derived from a particular plant virus or from multiple plant viruses. In fact, as part of the present invention, it may often be desirable to prepare said inventive vectors from elements of different plant viruses in order to take full advantage of the unique viral characteristics of the plant viruses used (e.g., host range, promoter activity level, virion dimensions, etc.).

[0084] It will be appreciated that this general principle can be applied to any viral vector system comprising transcomplementary vector systems. As will be appreciated by those skilled in the art, the vector system presented in this invention is suitable for use according to the present invention, provided that the vector set includes a producer vector incapable of systemic viral infection (i.e., lacking one or more protein-coding sequences of Petition 870250089530, dated 10 / 01 / 2025, pages 60 / 103 29 / 65 functional replication, MP coding sequences or CP coding sequences) and an auxiliary vector that facilitates the function or functions absent in the producer vector. It should be understood that, in all embodiments of the present invention, no individual vector, producer or auxiliary, is capable of facilitating systemic viral infection, however, as a set, one or both vectors are competent for such infection and expression of one or more GOIs.

[0085] The inventive vector sets of the present invention thereby reduce the risk of GOI expression in an unintended (non-target) host plant, regardless of whether the plant species is the same as the target plant. The probability that the vectors, producer and helper, will co-infect an unintended host plant is therefore greatly reduced.

[0086] The vectors of the invention used herein refer to a set of nucleic acids capable of directing the transient expression of an exogenous GOI. The expression vector may include a promoter operationally linked to the exogenous GOI, restriction endonuclease sites, nucleic acids encoding one or more selection markers, and other nucleic acids useful in the practice of recombinant technologies.Preferably, the expression vector used in step a) comprises: prokaryotic DNA elements encoding a bacterial origin of replication and an antibiotic resistance gene; at least one heterologous nucleotide sequence encoding the fusion proteins mentioned above, according to the invention, operationally linked to a strong promoter, preferably a 35S promoter; more preferably, a doubly boosted 35S promoter, a silencing inhibitor, preferably p19; and elements that control transcript processing, such as termination / polyadenylation sequences, preferably the Tnos sequence. Petition 870250089530, dated 10 / 01 / 2025, pp. 61 / 103 30 / 65

[0087] Numerous promoters and enhancers of functional expression in plants, which may be tissue-specific, developmentally specific, constitutive, or inducible, can be used in conjunction with the constructs of the present invention. When used herein in the descriptive report and the claims section that follows, the term plant promoter or promoter includes a promoter that can direct gene expression in plant cells (including DNA-containing organelles, more specifically the protoplast). Such a promoter may be derived from a plant, bacterial, viral, fungal, or animal origin.

[0088] Such a promoter may be constitutive, i.e., capable of directing a high level of gene expression in a plurality of plant tissues; tissue-specific, i.e., capable of directing gene expression in one or more specific plant tissues; inducible, i.e., capable of directing gene expression under a stimulus; or chimeric, i.e., formed by portions of at least two different promoters. The plant promoter employed may be a constitutive promoter, a tissue-specific promoter, an inducible promoter, or a chimeric promoter. Examples of constitutive plant promoters include, among others, the CaMV35S and CaMV19S promoters, the FMV34S promoter, the sugarcane bacilliform badnavirus promoter, the CsVMV promoter, the Arabidopsis ACT2 / ACT8 actin promoter, the Arabidopsis ubiquitin UBQ1 promoter, the barley leaf thionin BTH6 promoter, and the rice actin promoter.Examples of tissue-specific promoters include, but are not limited to, the bean phaseolin storage protein promoter, the DLEC promoter, the PHS promoter, the zein storage protein promoter, the soybean conglutin gamma promoter, the AT2S1 gene promoter, the Arabidopsis ACT 11 actin promoter, the Brassica napus napA promoter, and the patatin gene promoter. Petition 870250089530, dated 10 / 01 / 2025, pp. 62 / 103 31 / 65 of the potato.

[0089] In general, the inventive vectors described in the present invention are prepared by altering the genome of an existing plant virus, preserving viral function, but simplifying genetic manipulation, for example, by removing specific genes and / or interrupting or replacing specific sequences to inactivate or replace them. Similarly, when an inventive vector is found to affirmatively express a particular GOI or activity, it is not necessary for the relevant gene to be identical to the corresponding gene found in nature. For example, it is readily apparent that a CP can sometimes tolerate small deletions (Satyanarayana et al., 2014) and still facilitate systemic movement. Thus, as long as any specific protein is functional, it can be used according to the present invention. On the other hand, large deletions (e.g., more than 20 amino acids) are not favored and should be avoided.

[0090] Very high sequence identities with the corresponding native proteins are generally preferred. Typically, the coding sequences expressed in the present invention may exhibit at least 50%, preferably 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the corresponding native protein. Consequently, the viral proteins of the present invention should typically exhibit 100% identity with critical functional components of the relevant native viral protein. However, it is convenient to note that if it is possible to perform a series of amino acid alterations in any specific protein to improve its individual functionality or that of other proteins in the assembly complex, this can be done without significantly affecting the functional activity and / or various other properties of the protein, such as stability, etc. In particular, Petition 870250089530, dated 10 / 01 / 2025, pages 63 / 103 32 / 65 Many proteins tolerate conservative amino acid changes (i.e., conservative substitution) at many different positions without significant reduction in activity or stability. These substitutions are well known in the art and represent an approach to obtaining polypeptides with properties similar or substantially similar to those of a given polypeptide by altering the amino acid (AA) sequence. In amino acid sequences or variants thereof, as defined herein, amino acids are denoted by single-letter symbols.

[0091] These one-letter and three-letter symbols are well known to those skilled in the art and have the following meaning: A (Ala) is alanine, C (Cys) is cysteine, D (Asp) is aspartic acid, E (Glu) is glutamic acid, F (Phe) is phenylalanine, G (Gly) is glycine, H (His) is histidine, I (lie) is isoleucine, K (Lys) is lysine, L (Leu) is leucine, M (Met) is methionine, N (Asn) is asparagine, P (Pro) is proline, Q (Gin) is glutamine, R (Arg) is arginine, S (Ser) is serine, T (Thr) is threonine, V (Vai) is valine, W (Trp) is tryptophan, and Y (Tyr) is tyrosine.

[0092] In general, amino acids have been classified and divided into groups according to I) charge (positive, negative, or uncharged), II) volume and polarity, and III) Grantham physicochemical distance, and combinations thereof. For example, amino acids can be divided into 6 categories based on volume and polarity: special (C), neutral and small (A, G, P, S, T), polar and relatively small (N, D, Q, E), polar and relatively large (R, H, K), nonpolar and relatively small (I, L, M, V), and nonpolar and relatively large (F, W, Y). A conservative amino acid substitution can be defined as one that replaces an amino acid with an amino acid from the same group. Thus, an infinite number of functionally equivalent proteins can be derived by introducing one or more conservative amino acid substitutions into any given viral protein. Petition 870250089530, dated 10 / 01 / 2025, pp. 64 / 103 33 / 65

[0093] Preferably, the above expression constructors are present in a vector and preferably comprise border sequences that allow the transfer and integration of the expression cassette into the organism's genome. Preferably, the constructor is a plant binary vector. Preferably, the binary transformation vector is based on pPZP (Hajdukiewicz et al., 1994). Other exemplary constructors include pBinl9 (see Frisch, DA, LW Harris-Haller et al. (1995). Complete Sequence of the binary vector Bin 19. Plant Molecular Biology 27: 405-409).

[0094] As described herein, the invention can be implemented by moving an expression cassette with the necessary components into an existing pBin expression cassette or, in other embodiments, a directly cloned pBin expression vector can be used. Preferably, the binary vectors include the ColEI origin of replication, although plasmids containing other origins of replication that similarly produce high copy numbers (such as pRi-based plasmids, Lee and Gelvin, 2008) may likewise be preferred, especially for transient expression systems.

[0095] If desired, selectable genetic markers can be included in the constructor, such as those that confer selectable phenotypes, such as resistance to antibiotics or herbicides (e.g., kanamycin, hygromycin, phosphinothricin, chlorsulfurone, methotrexate, gentamicin, spectinomycin, imidazolinones, and glyphosate). HOST PLANTS

[0096] It should be understood that, in general, any plant susceptible to viral infection can be used according to the present invention. In general, it is desirable to use host plants that are adaptable to growing sufficient biomass under defined (and / or confined) conditions, for example, in greenhouses and / or aqueous systems. It may likewise be desirable to select plants that Petition 870250089530, dated 10 / 01 / 2025, pp. 65 / 103 34 / 65 are not typically consumed by humans or domesticated animals and / or animals that are not normally part of the human food chain, so they can be grown outdoors without the concern that the expressed GOI may be ingested unintentionally.

[0097] In other situations, on the other hand, the use of edible plants may be desirable. Frequently, the host plant is chosen according to the GOI to be expressed. When a GOI is desired to be produced in high yields (as will often be the case), it is desirable to select host plants with relatively high biomass (e.g., tobacco, which has the additional advantages of: I) being highly susceptible to viral infection, II) short growth periods, and III) not being in the human food chain.

[0098] It is generally understood that plant hosts means any unicellular or multicellular organism or a cell, tissue, organ, part or propagation material (such as seeds or fruits) thereof that is capable of photosynthesis. Included for the purpose of the invention are all genera and species of higher and lower plants of the Plant Kingdom. Annual, perennial, monocotyledonous and dicotyledonous plants are preferred.

[0099] The term includes mature plants, seeds, shoots and seedlings and their derived parts, propagation material (such as seeds or microspores), plant organs, tissues, protoplasts, calluses and other cultures, for example, cell cultures, and any other type of plant cell grouping that provides functional or structural units. Mature plants refer to plants at any desired stage of development beyond that of the seedling. Seedling refers to a young, immature plant at an early stage of development. Annual, biennial, monocotyledonous and dicotyledonous plants Petition 870250089530, dated 10 / 01 / 2025, pp. 66 / 103 35 / 65 are preferred host organisms for the generation of transgenic plants.

[00100] Gene expression is, moreover, advantageous in all ornamental plants, useful or ornamental trees, flowers, cut flowers, shrubs, or lawns. Plants that can be mentioned by way of example, but not in a limiting way, are angiosperms, bryophytes, such as, for example, Hepaticae (liverworts) and Musci (mosses); pteridophytes, such as ferns, horsetails, and clubmosses; gymnosperms, such as conifers, cycads, ginkgo, and Gnetatae; algae, such as Chlorophyceae, Phaeophyceae, Rhodophyceae, Myxophyceae, Xanthophyceae, Bacillariophyceae (diatoms), and Euglenophyceae.

[00101] The most preferred are plants that are not used for food or feed purposes, such as Arabidopsis thaliana, or preferably Nicotiana tabacum, or even more preferably Nicotiana benthamiana. Alternatively, plants that are used for food or feed purposes can also be used, such as the Legume family, such as peas, alfalfa and soybeans; grasses, such as rice, corn, wheat, barley, sorghum, millet, rye, triticale or oats; the Umbelliferae family, especially the genus Daucus, particularly the species carota (carrot) and Apium, particularly the species Graveolens dulce (celery) and many others; The Solanaceae family, especially the genus Lycopersicon, most notably the species esculentum (tomato) and the genus Solanum, most notably the species tuberosum (potato) and melongena (eggplant), and the genus Capsicum, most notably the species annuum (peppers) and many others;The legume family, especially the genus Glycine, most notably the species max (soybean), alfalfa, pea, alfalfa, bean or peanut, and many others; and the cruciferous family (Brassicacea), especially the genus Brassica, most notably the species napus (rapeseed); Petition 870250089530, dated 10 / 01 / 2025, pp. 67 / 103 36 / 65 campestris (beetroot), oleracea cv Tastie (cabbage), oleracea cv Snowball Y (cauliflower) and oleracea cv Emperor (broccoli); and the genus Arabidopsis, especially the species thaliana and many others; of the family Compositae, especially the genus Lactuca, especially the species sativa (lettuce) and many others; of the family Asteraceae, such as sunflower, Tagetes, lettuce or Calendula, and many others; of the family Cucurbitaceae, such as melon, pumpkin / squash or zucchini, and flaxseed. Also preferred are species of cotton, sugarcane, hemp, flax, peppers and various species of trees, nuts and wine.

[00102] The system of the present invention can be employed to infect and / or express a GOI in host plants at any stage of development, including mature plants, seedlings, shoots and seeds. The system can be used to infect any part of a plant (e.g., roots, leaves, stems, etc.). GOI

[00103] The embodiments of the present invention can be used to release and / or express in plant hosts any specific GOI, such as enzymes, antibodies, hormones, cytokines, regulatory factors, structural proteins or any other GOI. The encoded proteins can be naturally occurring proteins or can be engineered or modified proteins, including, for example, fusion proteins (e.g., fusion proteins incorporating part or all of a plant virus protein, such as MP or CP).

[00104] In certain embodiments of the invention, the polynucleotide of interest comprises a portion encoding a marker, for example, a 6x-His, GST, MBP, CBP, Intein-CBD marker, streptavidin / biotin-based markers, HA marker, Myc marker, FLAG marker, and the like. Such markers can conveniently simplify the isolation and / or purification of the protein in question and can be incorporated into the C-terminal ends or Petition 870250089530, dated 10 / 01 / 2025, pp. 68 / 103 37 / 65 N. In certain embodiments of the invention, the marker is cleavable (for example, a marker cleavable by a protease, such as thrombin), so that it can be easily removed after purification, resulting in a protein with a native sequence.

[00105] In some cases, it may be desirable to use the inventive system to express more than one GOI in the same host plant (for example, using two different producer vectors, inserting two different GOIs into a producer vector, or inserting one GOI into the producer vector and one GOI into the helper vector), for example, to produce a multimeric protein or to simultaneously produce two or more different proteins.

[00106] The system of the present invention can be used for the heterologous expression of protein-coding or non-protein GOIs of prokaryotic or eukaryotic origin and, in general, includes the following pharmaceutical proteins of interest, but not limited to: hormones (insulin, thyroid hormone, catecholamines, gonadotropins, trophic hormones, prolactin, oxytocin, dopamine, bovine somatotropin, leptins and the like), growth hormones (e.g., human growth hormone), growth factors (e.g., epidermal growth factor, nerve growth factor, insulin-like growth factor and the like), growth factor receptors, cytokines and immune system proteins (e.g., interleukins, colony-stimulating factor (CSF), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), erythropoietin, tumor necrosis factor (TNF), interferons,integrins, addressins, selectins, homing receptors, T-cell receptors, immunoglobulins, soluble major histocompatibility complex antigens, immunologically active antigens (such as bacterial, parasitic, or viral antigens or allergens), autoantigens, antibodies), Petition 870250089530, dated 10 / 01 / 2025, pp. 69 / 103 38 / 65 enzymes (tissue plasminogen activator, streptokinase, cholesterol (biosynthetic or degradative), steroidogenic enzymes, kinases, phosphodiesterases, methylases, demethylases, dehydrogenases, cellulases, proteases, lipases, phospholipases, aromatases, cytochromes, adenylate or guanylate cyclases, neuraminidases and the like), receptors (steroid hormone receptors, peptide receptors), binding proteins (steroid binding proteins, growth hormone or growth factor binding proteins and the like), transcription and translation factors, oncoproteins or proto-oncoproteins (e.g., cell cycle proteins), muscle proteins (myosin or tropomyosin and the like), myeloproteins, neuroactive proteins, tumor growth suppressor proteins (angiostatin or endostatin, both inhibit the angiogenesis), antiseptic proteins (protein that increases bactericidal permeability), structural scleroproteins (such as collagen,Fibroin, fibrinogen, elastin, tubulin, actin, and myosin), blood proteins (thrombin, serum albumin, factor VII, factor VIII, insulin, factor IX, factor X, tissue plasminogen activator, protein C, von Willebrand factor, antithrombin III, glucocerebrosidase, erythropoietin granulocyte colony-stimulating factor (GCSF) or modified factor VIII, anticoagulants such as protein S and huridine) and a variety of vaccine components that may be formulated into a vaccine, such as viral coat proteins, viral G proteins, microbial cell wall proteins, microbial toxin proteins, tumor-specific antigens, nucleoproteins, nucleocapsids, and the like.

[00107] Similarly, a large number of enzymes that modify protein or small molecule substrates (e.g., kinases, hydrolases, transferases, etc.), proteins for use in diagnostic or research reagents, nutritionally relevant products or total parenteral nutrition (TPN), are known in the art and po Petition 870250089530, dated 10 / 01 / 2025, pp. 70 / 103 39 / 65 can be expressed in plant hosts using the inventive systems described herein. Introduction of Vectors in Plants and Quantification of Transgenes

[00108] It should be understood that the vectors of the present invention can be introduced into a plant cell by means of one or more manual inoculations, in which drops of the preparation are placed on the surface of a leaf and gently rubbed in; mechanized plant inoculations, in which inoculations in seedbeds are carried out by spraying the vector solution onto the plants (which may have cut leaves); high-pressure spraying on individual leaves, which provides inoculations in individual plants by spraying the leaves with a narrow, directed jet; and vacuum infiltration, in which inoculations can be carried out by subjecting the plant host to a substantial vacuum pressure environment to facilitate infection. Transfer of the vectors of the present invention preferably occurs through the use of Agrobacterium, which refers to a Gram-negative, rod-shaped, soil-borne phytopathogenic bacterium that causes crown gall.The term Agrobacterium includes, but is not limited to, the strains Agrobacterium tumefaciens (which typically causes crown gall in infected plants) and Agrobacterium rhizogenes (which causes hairy root disease in infected host plants). Infection of a plant cell with Agrobacterium generally results in the production of opines (e.g., nopalin, agropine, octopine, etc.) by the infected cell. Thus, Agrobacterium strains that cause the production of nopalin (e.g., strains LBA4301, C58, A208) are called nopalin-type Agrobacterium; Agrobacterium strains that cause the production of octopine (e.g., strains LBA4404, Ach5, B6) are called octopine-type Agrobacterium; and strains of... Petition 870250089530, dated 10 / 01 / 2025, pp. 71 / 103 40 / 65 Agrobacterium that cause the production of agropin (e.g., strains EHA105, EHA101, A281) are called agropin-like Agrobacteria.

[00109] Several suitable assays can be used to determine transgene expression. For example, non-limiting examples include the detection and / or quantification of the presence of transcribed sense or antisense strands of the transgene by conventional hybridization assays known in the art (e.g., Northern Blot analysis), amplification procedures (e.g., RT-PCR), and array-based technologies. Transgene expression can similarly be determined by examining the protein product. Several techniques are available in the art for protein analysis. Non-limiting examples include radioimmunoassays, ELISA (enzyme-linked immunosorbent assays), sandwich immunoassays, immunoradiometric assays and in situ immunoassays (using, for example, colloidal gold, enzyme markers, or radioisotopes), Western Blot analysis, immunoprecipitation assays, immunofluorescence assays, and SDS-PAGE.

[00110] It is generally understood by those skilled in the art that the determination of protein level involves: I) providing a biological sample containing polypeptides; and II) measuring the amount of any immunospecific binding that occurs between an antibody reactive to the transgenic product and a component in the sample, wherein the amount of immunospecific binding indicates the level of proteins expressed. Antibodies that specifically recognize and bind to the protein products of the transgene are required for immunoassays. These can be acquired from commercial suppliers or generated and screened using methods well known in the art.

[00111] The test protein sample can be prepared by homogenizing the eukaryotic transformant (e.g., cells from a Petition 870250089530, dated 10 / 01 / 2025, pp. 72 / 103 41 / 65 host plant) or its progenies produced from it (in the case of stable transformation) and, optionally, solubilizing the test protein using any suitable detergent. The results obtained using any such assay on a sample of a transformed, transient or stable host are compared with those of an untransformed source as a control. ISOLATION AND / OR FORMULATION OF EXPRESSED GOI PRODUCTS

[00112] In many embodiments of the present invention, it will be desirable to isolate the polynucleotide expression products from the plant tissues that express them. It may likewise be desirable to formulate such isolated products for the intended use (e.g., as a pharmaceutical or diagnostic agent, or as a reagent, etc.). In other embodiments, it will be desirable to formulate the products together with some or all of the plant tissues that express them.

[00113] When it is desirable to isolate the expression product from some or all of the plant tissues that express it, any available purification techniques may be employed. Those with ordinary experience in the art are familiar with a wide range of fractionation and separation procedures. Frequently, it will be desirable to make the product more than about 50%, preferably more than about 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% pure.

[00114] When it is desirable to formulate the product together with the plant material (for example, an oral vaccine formulation when properly formulated), it will often be desirable to use a plant that is not toxic to the recipient in question (for example, a human or other animal). The relevant plant tissue (for example, leaves) can simply be harvested and processed according to techniques known in the art, with due consideration. Petition 870250089530, dated 10 / 01 / 2025, pp. 73 / 103 42 / 65 ration to maintain the activity of the expressed product. In certain embodiments of the invention, it is desirable that the GOI has been expressed in an edible plant (and, specifically, in edible portions of the plant) so that the material can be subsequently consumed.

[00115] When the polynucleotide encodes or produces a therapeutic agent, it can be formulated according to known techniques. For example, an effective amount of a pharmaceutically active product can be formulated together with one or more pharmaceutically suitable carrier materials, organic or inorganic, liquid or solid. A pharmaceutically active product produced according to the present invention can be employed in pharmaceutical forms such as tablets, capsules, dispersions, suspensions, solutions, creams, ointments, aerosols, powder sachets, liquid solutions, solvents, diluents, surfactants, isotonic agents, thickening or emulsifying agents, preservatives and solid binders, provided that the biological activity of the protein is not destroyed by such pharmaceutical form.

[00116] Materials that can serve as pharmaceutically acceptable vehicles include, but are not limited to, sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth gum; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline solution; Ringer's solution; ethyl alcohol and phosphate buffer solutions, as well as other compatible non-toxic lubricants such as sodium lauryl sulfate and Petition 870250089530, dated 10 / 01 / 2025, pp. 74 / 103 43 / 65 magnesium stearate, as well as colorants, release agents, coating agents, sweeteners, flavoring agents and fragrance agents, preservatives and antioxidants may likewise be present in the composition, according to the formulator's judgment and in a composition for use according to the embodiments of the present invention, the vehicle and / or excipient may comprise an encapsulating material to encapsulate, at least temporarily, the compound.

[00117] A composition for parenteral injection for use according to the embodiments may be desired in the form of (for example, may consist of) a sterile aqueous or non-aqueous solution, a dispersion, a suspension and / or an emulsion and / or a sterile powder for reconstitution into a sterile injectable solution or dispersion prior to use. Examples of suitable aqueous and non-aqueous vehicles, diluents, solvents or carriers include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Adequate flowability may be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions and by the use of surfactants.

[00118] A composition desired for use according to embodiments of the present invention may likewise contain adjuvants, such as, among others, preservatives, humectants, emulsifying agents and dispersing agents. A composition for use according to embodiments of the present invention may include isotonic agents, such as sugars, sodium chloride and the like.

[00119] When prolonged absorption in an injectable pharmaceutical form is desired, the inclusion of absorption-delaying agents, such as aluminum monostearate and gelatin, may be useful. Petition 870250089530, dated 10 / 01 / 2025, pp. 75 / 103 44 / 65 adapted according to the embodiments of the present invention. In cases where a prolonged effect of the composition is desired, delaying the absorption of the composition when injected subcutaneously or intramuscularly, the composition may comprise a liquid suspension of a crystalline or amorphous material with low water solubility. The rate of drug absorption may then depend on its rate of dissolution, which, in turn, may depend on the crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered pharmaceutical form may be achieved by dissolving or suspending the drug in an oily vehicle. Injectable depot forms may be made by forming microencapsulated matrices of the drug in biodegradable polymers, such as polylactide-polyglycolide. Depending on the drug-to-polymer ratio and the nature of the specific polymer employed, the drug release rate may be controlled.Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Injectable depot formulations are similarly prepared by encapsulating the drug in liposomes or microemulsions compatible with body tissues.

[00120] Injectable formulations can be sterilized, for example, by filtration through a bacterial retention filter or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved or dispersed in sterile water or other sterile injectable medium immediately before use.

[00121] The composition for use according to the embodiments of the present invention may be provided in a solid pharmaceutical form for oral administration, such as, among others, capsules, tablets, pills, powders and granules. In such solid dosage forms, the active compounds may be mixed with at least one excipient or vehicle, such as sodium citrate or dicalcium phosphate and / or a) fillers. Petition 870250089530, dated 10 / 01 / 2025, pp. 76 / 103 45 / 65 or extenders, such as starches, lactose, sucrose, glucose, mannitol and silicic acid, b) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia, c) humectants, such as glycerol, d) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate, e) solution retarders, such as paraffin, f) absorption accelerators, such as quaternary ammonium compounds, g) wetting agents, such as, for example, acetyl alcohol and glycerol monostearate, h) absorbents, such as kaolin and bentonite clay, ei) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate and mixtures thereof. In the case of capsules, tablets and pills, the pharmaceutical form may also include buffering agents.Solid compositions of a similar type can likewise be employed as fillers in soft and hard gelatin capsules, using excipients such as lactose or milk sugar, as well as high molecular weight polyethylene glycols and the like.

[00122] Solid pharmaceutical forms of tablets, dragees, capsules, pills, and granules may be prepared with coatings and shells, such as enteric coatings and other coatings, such as extended-release, prolonged-release, delayed-release, and immediate-release coatings, well known in the pharmaceutical formulation art. Optionally, they may contain opacifying agents and may likewise be of a composition that releases the active ingredient(s) only, or preferably, in a specific part of the intestinal tract, optionally in a delayed manner. Examples of incorporation compositions that may be used include polymeric substances and waxes. The active compounds may likewise be in microencapsulated form, if appropriate, with one or more of the excipients mentioned above. Petition 870250089530, dated 10 / 01 / 2025, pp. 77 / 103 46 / 65

[00123] Liquid dosage forms for oral administration may include, but are not limited to, pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compounds, liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing and emulsifying agents such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, peanut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols, and sorbitan fatty acid esters, and mixtures thereof. In addition to inert diluents, oral compositions may also include adjuvants such as humectants, emulsifiers and suspending agents, sweeteners, flavorings and fragrances.In addition to the active compounds, suspensions may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum meta-hydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

[00124] Those skilled in the art will likewise recognize that a particularly preferred method for obtaining the desired pharmaceutically active products is by extraction. Infected plants can be extracted to remove the desired products from the residual biomass, thereby increasing the concentration and purity of the product. The plants can likewise be extracted in a buffered solution. For example, freshly harvested plants can be transferred to a quantity of ice water in a one-to-one weight ratio, buffered with, for example, phosphate buffer. Protease inhibitors can likewise be added when necessary. The plants can be broken up by vigorous mixing or Petition 870250089530, dated 10 / 01 / 2025, pp. 78 / 103 47 / 65 grinding while suspended in buffer solution, and the extracted biomass can be removed by filtration or centrifugation. The transgenic product transported in solution can be purified by additional steps or converted into dry powder by freeze-drying or precipitation. Extraction can similarly be carried out by pressing. Live plants can similarly be extracted by pressing in a press or by grinding as they pass through nearby rollers. The fluids extracted from the ground plants are collected and processed according to methods well known in the art. Extraction by pressing allows the release of the products in a more concentrated form. However, the overall product yield may be lower than if the product were extracted in solution.

[00125] The description of all references cited herein, insofar as they may be used by those skilled in the art for carrying out the present invention, is specifically incorporated herein by cross-reference. EXEMPLARY EXPERIMENTS AND RESULTS OF THE VECTOR SYSTEM OF THE INVENTION

[00126] The inventor has developed a transcomplementary vector system that can easily infect a wide range of host plants and yet present little or no risk of infectious dissemination. The vector system of the present invention, as exemplified, includes components of a single plant virus or components of different plant viruses. Construction of a transcomplementary TRV vector expressing GFP

[00127] This transcomplementary system includes components of the Tobacco Rattle Virus (TRV). Figure 2 shows the genome organization of TRV RNA 1 and RNA 2. TRV is a type species of the genus Tobravirus, family Virgaviridae (Carstens, 2010; Robinson, Petition 870250089530, dated 10 / 01 / 2025, pp. 79 / 103 48 / 65 (2005), and is a single-stranded, bipartite, positive-sense RNA virus composed of linear tubular particles of two predominant lengths, the larger one approximately 190 nm and the smaller one 50 to 115 nm, and a genome composed of RNA-1 and RNA-2. RNA-1 encodes two replicases and a cysteine-enriched protein, which is sufficient for replication and movement within the plant, even without RNA-2. A capsid protein (CP) and two non-structural proteins encoded by RNA-2 allow for virion formation and nematode-mediated transmission through plants (Ratcliff et al., 1999; Liu et al., 2002).

[00128] TRV has a particularly wide host range, infecting over 100 species in nature and over 400 species when tested in a greenhouse, although not all of these infections are systemic (SA MacFarlane, in Encyclopedia of Virology (Third Edition), 2008).

[00129] FIG. 3 illustrates a schematic diagram of a replication-competent, GFP-expressing transcomplementary TRV-vector system. After agroinfiltration of plants, sequences between the left edge (LB) and right edge (RB) of the plasmid vectors mediate gene transfer from the agrobacterium to a chromosome of a host plant cell. Instead of providing the complete vector as a linear DNA molecule, we use different agrobacteria to release various modules of the viral vector and the gene of interest. These components are assembled within a plant cell with the help of a site-specific recombinase (Bxb1 serine integrase). The resulting DNA is transcribed and spliced, thereby removing unwanted elements such as recombination sites, and leading to the creation of a perfect gene / vector sequence within a fully functional infectious replicon. Bxb1 Int-mediated recombination uses a minimum binding site of 48 bp attP Petition 870250089530, dated 10 / 01 / 2025, pp. 80 / 103 49 / 65 (binding phage) and a 38 bp cognate site attB (binding bacteria) between the donor and recipient DNAs. Binding sites B and P represent four half-sites. After Bxb1 recombination, one half-site of each remains, producing two 43 bp sites (attL and attR, Left and Right) flanking the inserted DNA. Bxb1 Int does not require other secondary factors and, under these conditions, provides irreversible unidirectional integration of donor DNAs.

[00130] The 5' producing vector consists of a modified version of the TRV RNA-1 genome (WD Hamilton, 1987). The resulting producer vector contains, in sequential order, a 754 bp chimeric constitutive promoter with a duplicated CaMV 35S enhancer element (Plant Mol Biol. 21:415, 1993; Biotechnol Lett. 29:1793, 2007), a 5121 bp RNA-dependent RNA polymerase (RdRp) encoding helicase (134 kDa), polymerase (194 kDa), both collectively known as replicase (Uniprot: P05080, 194K_TRVSY - release number 2014_04), a 756 bp movement protein (MP) (Uniprot: P69473, V29K_TRVSY - release number 2014_04), a 516 bp RNA silencing suppressor (P19) that replaces the native, but weaker, 16 kDa TRV silencing suppressor (Uniprot: P11690, P19_TBSVC - release number 2013_10), a 63 bp apoplast targeting signaling peptide derived from Arabidobsis thaliana 1 seed storage protein 2S (Uniprot: P15457,2SS1_ARATH - release number 2014_04), a 190 bp intron sequence of castor bean catalase (Uniprot: Q01297 CATA1_RICCO - release number 2014_02), a 48 bp attP(Bxbl) minimal binding site as one of the two target sites of serine integrase Bxb1, a 43 bp autocleavable hammerhead ribozyme to increase virus infectivity, and finally, the 254 bp nopalin synthase terminator (T-nos) and the poly(A) signal of Agrobacterium tumefaciens (A. tumefaciens). Petition 870250089530, dated 10 / 01 / 2025, pp. 81 / 103 50 / 65

[00131] The 3' auxiliary vector consists of a modified version of the TRV RNA-2 genome (WD Hamilton, 1987). The resulting auxiliary vector contains, in sequential order, a 38 bp cognate attB(Bxbl) site as one of the two target sites of the serine integrase Bxb1, a 190 bp intron sequence of castor oil catalase, the 726 bp GOI coding sequence “GFP” (Uniprot: P42212, GFP_AEQVI - release number 2014_04) which is N-terminally fused with the endoplasmic reticulum (ER) retention signal (KDEL), the 615 bp TRV RNA-2 coat protein (Uniprot: P05072, COAT_TRVTC - release number 2013_10), flanked by the 708 bp 3' untranslated region (3'UTR) of TRV RNA-2 to increase mRNA stability and regulate translation efficiency, and finally, the T us of 254 bp of A. tumefaciens.

[00132] The integrase vectors contain, in sequential order, a 754 bp chimeric constitutive promoter with a duplicated CaMV 35S enhancer element (Plant Mol Biol. 21:415, 1993; Biotechnol Lett. 29:1793, 2007), a 1500 bp Bxb1 serine integrase coding sequence (Uniprot: Q9B086, Q9B086_BPMB1 - publication number 2014_05), a 45 bp nuclear localization signal derived from mitogen-activated NPK1 protein kinase (Uniprot: Q40541, NPK1_TOBAC - publication number 2014_04), and finally, the 254 bp Tnos from A. tumefaciens.

[00133] The producing vector lacks long-distance mobility and, therefore, if plants are inoculated only with this vector, their infection is limited to local tissues (i.e., cells within the initially infected leaf). This replication-competent producing vector is administered together with a separate auxiliary vector containing a functional CP coding sequence. Preferably, the transcripts of these two vectors are mixed together and mechanically applied to the plant leaves. Replication of the auxiliary vector of Petition 870250089530, dated 10 / 01 / 2025, pp. 82 / 103 51 / 65 TRV RNA2 provides CP, which supports the long-distance movement of both vectors to the inoculated leaves. Both the producer and helper vectors are mixed with the integrase vector, and thus the components of the producer and helper vectors are assembled within a plant cell in trans with the help of a site-specific recombinase (Bxb1 serine integrase).

[00134] The resulting DNA is transcribed and processed, thereby removing unwanted elements, such as recombination sites, and leading to the creation of a perfect gene / vector sequence within a fully functional infectious replicon.

[00135] The two genes involved in the transmission of TRV RNA-2 (2b and 2c) to nematodes have been removed. Preferably, the integration of vectors into the host genome is avoided, so that transgenic plants are not produced and the risk of genetic alterations being introduced into the environment is minimized.

[00136] The producer, helper, and integrase fragments were cloned between the left and right edges of the T-DNA of an enhanced Agrobacterium binary vector, named pBINPLUS, based on the popular pBIN19 vector. Improvements over pBIN19 include the location of the selectable marker gene on the left edge of the TDNA, a higher copy number in E. coli, and two rare restriction sites around the multiple cloning site to facilitate cloning and analysis of T-DNA insertions in plant genomes. SEQ IP No. 1: T-DNA region of the producing vector (FIG. 3), where the sequence segments have the following function: Nucleotides 1 to 25 Left edge repeat of T-DNA Nucleotides 26 to 779 CaMV 35S double Nucleotides 780 to 5900 TRV RNA-1 RdRP 5' end Nucleotides 5901 to 6659 Movement protein TRV RNA-1 Nucleotides 6660 to 7176 Suppressor silencer P19 Petition 870250089530, dated 10 / 01 / 2025, pages 83 / 103 52 / 65 Nucleotides 7177 to 7240 Apoplast-directed signal peptide Nucleotides 7241 to 7421 Castor bean catalase intron Nucleotides 7422 to 7470 AttP (Bxb1 serine integrase) recombination site Nucleotides 7471 to 7514 Hammerhead ribozyme Nucleotides 7515 to 7769 Nos terminator Nucleotides 7770 to 7795 Right-edge T-DNA repeat ACCGTCCTAT ATAACACCAC ATTTGGCCAA CATGGTGGAG CACGACACTC TCGTCTACTC 121 181 241 301 361 421 481 541 601 661 721 781 841 901 961 1021 1081 1141 1201 1261 1321 1381 1441 1501 1561 1621 1681 1741 1801 1861 1921 CAAGAATATC AAAGATACAG TCTCAGAAGA CCAAAGGGCT ATTGAGACTT TTCAACAAAG GGTAATATCG GGAAACCTCC TCGGATTCCA TTGCCCAGCT ATCTGTCACT TCATCAAAAG GACAGTAGAA AAGGAAGGTG GCACCTACAA ATGCCATCAT TGCGATAAAG GAAAGGCTAT CGTTCAAGAT GCCTCTGCCG ACAGTGGTCC CAAAGATGGA CCCCCACCCA CGAGGAGCAT CGTGGAAAAA GAAGACGTTC CAACCACGTC TTCAAAGCAA GTGGATTGAT GTGAACATGG TGGAGCACGA CACTCTCGTC TACTCCAAGA ATATCAAAGA TACAGTCTCA GAAGACCAA GGGCTATTGA GACTTTTCAA CAAAGGGTAA TATCGGGAAA CCTCCTCGGA TTCCATTGCC CAGCTATCTG TCACTTCATC AAAAGGACAG TAGAAAAGGA AGGTGGCACC TACAAATGCC ATCATTGCGA TAAAAGGAAAG GCTATCGTTC AAGATGCCTC TGCCGACAGT GGTCCCAAAG ATGGACCCCC ACCCACGAGG AGCATCGTGG AAAAAGAAGA CGTTCCAACC ACGTCTTCAA AGCAAGTGGA TTGATGTGAT ATCTCCACTG ACGTAAGGGA TGACGCACAA TCCCACTATC CTTCGCAAGA CCCTTCCTCT ATATAAGGAA GTTCATTTCA TTTGGAGAGG ACACGCTGAA TGGCTAATGG AAATTTTAAG CTTTCTCAAC TTCTTAATGT TGATGAAATG TCTGCTGAAC AAAGATCTCA TTTTTTGAT CTTATGCTTA CTAGCCAGA TTGTGAAATT GGACAAATGA TGCAAAGAGT TGTTGTTGAT AAGGTTGATG ATATGATTAG AGAAAAAG ACTAAGGATC CAGTTATTGTTCATGAAGTT CTTTCTCAAA AGGAACAAAA TAAGCTTATG GAAATTTATC CAGAATTAA TATTGTTTTT AAGGATGATA AGAATGGT TCATGGATTT GCTGCTGCTG AAAGAAAGCT TCAAGCTCTT CTTCTTCTTG ATAGAGTTGATCC AGATTCATTGAA TTTTGGGTTA CTAGAGGAGA AAAGAGAATT CATTCTTGTT GTCCAAATCT TGATATTAGA GATGATCAAA GAGAAATTTC TAGACAAATT TTTCTTACTG CTATTGGAGA TCAAGCTAGA TCTGGAAAGA GACAAATGTC TGAAATGAATGT ATTGATTGTCGAT CAAATGCTGT TAGATGTAAT AATACTTATC AAGGATGTAC TTGTAGAGGA TTTTCTGATG GAAAGAAA GGGAGCTCAA TATGCTATTG CTCTTCATTC TCTTTATGAT TTTAAGCTTA AGGATCTTAT GGCTACTATG GTTGAAACTATT T AGTCATCACTAG ATCTATGCTT GTTGATGAAG GACCACTTCC ATCTGTTGAT GGATATTATA TGAAAGAA TGGAAAGATT TATTTTGGAT TTGAAAAGGA TCCATCTTTT TCTTATATTC ATGATTGGGA AGAATATAAG AAGTATCTTC TTAGTTTCATTATTAGTT CAAGTT TGAACCATGG CAAGTTAGAG GAGATACTAT GCTTTTTTCT ATTTATAGAA TTGCTGGAGT TCCAAAGA TCTCTTTCTT CTCAAGAATA TTATAGAAGA ATTTATTT CTAGATGGGA AAATATGGTT GTTGTTCCCAA TTTGATGATCT TGTTACTAGAGAAC TTGTTAAGAA GGATCTTTTT GTTGAAAAGC AATTTATGGA TAAGTGTCTT GATTATATTG CTAGACTTTC TGATCAACAA CTTACTATTT Petition 870250089530, of 01 / 10 / 2025, p. 84 / 103 53 / 65 CTAATGTTAA TTAAGAATAA TTAAGGAACA CTAAGCCAAT AATTTGCTAA TTTATCCAAA ATGAAAATTC GAATTACTCT TTGATATTAA TTAATGATTT ATCTTTCTGA ATGAATTTCT CATCTGTTGT TTACTCAAGC TTCTTTCTGG CTGAATGTGA CAGATTTGA ACTGAGTTGA TTGCTCAAAA CTGGAGGACA GTATTAGACC ATTGGTCTGG CTGTTGAAAG GACTTATTCT TTGTTGATGG GTGTTGATGT CTTCTAAGGG TGCATTGTGT CTCATGCTGG AAGGAGATCA CTTCTCTTGT CTGATGTTGC CTGCTTAGTTATGTTTAGTTATGTTGTT TTATTGTTGC ATGTTTCTAA TTGTTACTGA AAGGACCATG CTCTTTTTCC CTGATTGTAA TTGCTGAAAA GTCTTATCTT TCTTCTAATA ATTGGGTTCT TTTTATTAAT GCAATCTGTT GATTCTAGAG ATCTTCAACT TCTTGCTCAA AGTTGCTAGA CCAGTTATGA GAGAACTTAG AGAAGCTATT TACTTCTCTT ACTGATGTTC TTGGACTTAT TTCTAGAAAG TAAGATTGCT GTTGGAGAT TTGTTGGAAT GGTTGGAACT GAAGGTTCTT ACTTGGGCTA AGGATACTCC AAATGGACCA TCATAAGACT AAGGTTATTG TTTTTCTTTC TGTTGTTTAT REPEAT REPEAT ATGGACTTGT REPEATGCTT GAGAGGAGCT CATGTTCTTG ATGTTGAAAA TCCATGTAGA TTTTTCTTCT CTTTATTCTG CTTCTGAATC TGGAAACT AGTTAAGGCT AAGTCTGATA AGCTTCTTCA ACAAAAGAAG TTCTGCTAAG TTTTCTAATT ATTCTGGATC TTCTGTTAGA TGGATCTTCT AGATCTGGAC TTGGACTTCT TCTTGAAGAT TAGAGTTGGA GTTTCTAGAA AGGTTGATGA TGAAGAAGATGATTGATTGATTGATTGATTGATTGA AGTTGTTTCT AAGAGGAGAA ACTTCTGGAA CTAAGGTTCT TTGTAAGCCA AAATGTTCTT CCAGCTGTTA AGCCACTTGT TTCTAAGGGA TTATTTTCAA GTTATGGGAG GAGAAAGACT TCCAAAGAGA TTCTGTTGAT GCTAGAAGAG AATTTCCTTATGTTTTTTGATT ATAGAGATTA TTCTAGAGGA AAATTATCCA CATGGACTTG GAGTTTGGGA TGTTGAAATG AGTTGTTACT GAACATGCTT ATGTTTTTCA ACCAGATAAG ATATCTTGAA GTTGCTGTTT GGGAAAGAGG AATGCTTGTT AATGTCTGAT TATGTTAGTGATTGATTTTTTGA TTATTGATTTTCA TCTGCTCTTG ATCTTGGACC AGTTAATTGT AGTTCCAGGA TGTGGAAAGT CTACTATGAT TGTTAATTCT TGTTCTTTCT ACTGGAAGAG CTGCTACTGA TGATCTTATT ATTTCCATGT AAGCTTAAGA GAAGAGTTAA GACTGTTGAT TGATGGATTACTGACTGATTGTTGA AATGGTTTAT TTTTGTGCTC AAATTGCTGG AGCTAAGAGA AAATCAAATT TCTTTTAAGC CAAGAGTTTC TCAAGTTGAT TGGAAAGTTT GATATTGTTA CTGAAAAGAG AGAAACTTAT TGCTGTTCTT AATAAGTATT ATACTGGAGATGTTAGAACT TATGACTGTT AGAAAGATTG TTTCTAAGGA ACAAGTTTCT TATTACTTTT CTTCAATCTG AAAAGAAGGA ACTTGTTAAT TGCTGCTAAG GTTTCTACTG TTCATGAATC TCAAGGAGAA TGTTAGAACT AAGCCAACTG ATGATTCTAT TGCTAGAGGA TCTTTCTAGA CATACTCAAT CTCTTGTTTA TGAAACTGTT GGAAATTAGA GAATCTGCTG CTCTTACTAA GGCTGCTCTT AACTGTTCTT NNNAGATTTA GATCTAGATT TGAATGTTTTT TGCTGTTCCA GATTCTGGAA CTATTACTGA TCTTGAAATG AGGAAATTCT CTTAGAGATT CTTCTCTTGA TGGATATCTT TCTTAGACTT GATAATGTTA CTATTAAGTC TGGAAATTGG GGAAACTTTT CTTAAGCCAG TTATTAGCAAC TGCTATGCCA GGAGCTGCTG ACTCTTCTTG CTTACTAAA CTTTGGAAGC CTTATTGGAT GAACTTTGTT GCTATTGGAG TGTGATATGT TATTATGAAA GTTCTTCCAG GAAATTGCTG ACTTTCCAC TCTAATGTTC ATGGAACAAT GCTTTTTCTG CTTACTCCAC AAGACTGTTA CCAGTTGTTT GCTGAAAGAG GTTATTAGAA AAGAATTGGT AGAATGGATG AATGATTTTTG TGTAATAATA TCTTTTGAAC GCTAATCCAT GAAAGATTTG TCTTTTCTTA GCTCTTATGG TGTATTTGTC CTTAGATTTT AGATCTCCAG CATAATGCTA CTTAAGCCAG CTTCTTGCTC ACTTTTAAGG AGAGAATATC AAGGAAGATG GCTAGATTTT AGACATCATG TGGTATGATG GTTGCTACTA AAGGATAAGT GATAAGAGAA Petition 870250089530, dated 10 / 01 / 2025, pp. 85 / 103 54 / 65 AGACTACTCA ACTTGAATCT CTTCTTGCTC TTCAAAAGAG AAATCAAGCT GCTCCAGATC TTCAAAAA TGTTCATGCT ACTGTTCTTA TTGAAAAC TATGAGAAG CTTAAGTCTG TTGTTTATGA TGTTGGAAAG ATTAGAGCTG ATGAGTAATTG ATCCAGAATTG AAATCAATCT ACTGCTGTTC AAGCTAAGGT TGTTGCTGAT GTTAGAGAAC TTCATGAATTA TGATTATTCT TCTTATATGT ATATGATTAA GTCTGATGTT AAGCCAAGA CTGATCTTAC TCCACAATTT GAATATTCTG CTCTTCGAATTAC CTTGATTAATTAT CCAATTTTTA AGGAATTTAA TGAAAGAAAG CTTGATGCTA TGCAACCACA TTTTGTTTTT AATACTAGAA TGACTTCTTC TGATCTTAAT GATAGAGTTA AGTTTCTTAA TACTGAAGCT GCTTATGATT TTGTTATGAAT TGATATGTCT A TCAAGTTCTTGATA AACTTGAAAT TTATAGACTT TTTGGACTTG ATGAATGGGC TGCTTTTCTT TGGGAAGTTT CTCATACTCA AACTACTGTT AGAGATATTC AAAATGGAAT GATGGCTCAT ATTTGGTATC AACAAAAGTC TGGAGATGCT TTGATAGTACTATTATC TTCTTTCTGA ACTTCCACTT GAAAAGGCTG TTATGGTTAC TTATGGAGGA GATGATTCTC TTATTGCTTT TCCAAGAGGA ACTCAATTTG TTGATCCATG TCCAAAGCTT GCTACTAAGT GGAATTTTGA ATGTAAGATT TTTAAGTCAATG ATGTTCCAATGGTTTTGTGGA AAGTTTCTTC TTAAGACTTC TTCTTGTTAT GAATTTGTTC CAGATCCAGT TAAGGTTCTT ACTAAGCTTG GAAAGAAGTC TATTAAGGAT GTTCAACATC TTGCTGAAAT TTATATTTCT CTTAATGATT CTAATAGAGC TCTTGGAATTTGTTGCTGCTGCTGTT GTTTCTGATA GATATCTTTA TAAGGGAGAT TCTGTTCATG CTCTTTGTGC TCTTTGGAAG CATATTAAGT CTTTTACTGC TCTTTGTACT CTTTTTAGAG ATGAAAATGA TAAGGAACTT AATCCAGCTA AGGTTGATTG GAAGAAGGCT CAAAGAGCTG TTTAATTTGATTGATTGATGTTG AGTCTCTTGT TACTCTTAAG AAGAAGACTT TTGAAGTTTC TAAGTTTTCT AATCTTGGAG CTATTGAACT TTTTGTTGAT GGAAGAAGAA AGAGACCAAA GTATTTTCAT AGAAGAAGAG AAACTGTTCT TAATCATGTT GGAGGAAAGA AGTCTGAACCATAGTTAGTTATTAGTT TAAGATGATT AAGTCTTATG CTTTTCTTAA GATTGTTGGA GTTCAACTTG TTGTTACTTC TCATCTTCCA GCTGATACTC CAGGATTTAT TCAAATTGAT CTTCTTGATT CTAGACTTAC TGAAAAGAGA AAGAAGGGAA AGACTATTCA AGATTTAGATTGCTGTTGCTGTTGTTG CAATATAAGG TTGAATATTC TATTTCTACT CAAGAAAATG TTCTTGATGT TTGGAAGGTT GGATGTATTT CTGAAGGAGT TCCAGTTTGT GATGGAACTT ATCCATTTTC TATTGAAGTT TCTCTTATTT GGGTTGCTAC TGATTCTACTAGAAGACTTA ATGTTGAAGA ACTTAATTCT TCTGATTATA TTGAAGGAGA TTTTACTGAT CAAGAAGTTT TTGGAGAATT TATGTCTCTT AAGCAAGTTG AAATGAAGAC TATTGAAGCT AAGTATGATG GACCATATAG ACCAGCTACT ACTAGACCAA AGTCTCTTCT TTCTTCTGAA GATGTTAAGA GAGCTTCTAA TAAGAAGAAT TCTTCTATGG AAAGAGCTAT TCAAGGAAAT GATGCTAGAG AACAAGCTAA TTCTGAAAGA TGGGATGGAG GATCTGGAGG AACTACTTCT CCATTTAAGC TTCCAGATGA ATCTCCATCT TGGACTGAAT GGAGACTTCA TAATGATGAA ACTAATTCTA ATCAAGATAA TCCACTTGGA TTTAAGGAAT CTTGGGGATT TGGAAAGGTT GTTTTTAAGA GATATCTTAG ATATGATAGA ACTGAAGCTT CTCTTCATAG AGTTCTTGGA TCTTGGACTG GAGATTCTGT TAATTATGCT GCTTCTAGAT TTTTTGGATT TGATCAAATT GGATGTACT ATTCTATTAG ATTTAGAGGA GTTTCTATTA CTGTTTCTGG AGGATCTAGA ACTCTTCAAC ATCTTTGTGA AATGGCTATT AGATCTAAGC AAGAACTTCT TCAACTTGCT CCAATTGAAG TTGAATCTAA TGTTTCTAGA GGATGTCCAG AAGGAACTGA AACTTTTGAA AAGGAATCTG AAATGGCTAA TAAGCTTTTT CTTGTTTGTG CTGCTCTTGC TCTTTGTTTT CTTCTTACTA ATGCTCATTT AAAGATCAAA AAGAGGAAG Petition 870250089530, de 01 / 10 / 2025, pág. 86 / 103 55 / 65 7261 AAAAGAACCA ATCCTGGGAA AAGAGAAAAA TAAAAAAACT CGAAACTAGA AAGAAATTTG 7321 ACTAGATAAA AAATTAACTA ACCAATACCA CATTTATAAT GTATCGAAAT TGACTATTAG 7381 ACTAATGAAA TAAAGCACAC AGATACTACT ACTACTATCA ATGTCGGTTT GTCTGGTCAA 7441 CCACCGCGGT CTCAGTGGTG TACGGTACAA ACCGGCATGC TGATGAGTCC GTGAGGACGA 7501 AACGAGTAAG CTCGTCGATC GTTCAAACAT TTGGCAATAA AGTTTCTTAA GATTGAATCC 7561 TGTTGCCGGT CTTGCGATGA TTATTCATATA ATTTCTGTTG AATTACGTTA AGCATGTAAT 7621 AATTAACATG TAATGCATGA CGTTATTTAT GAGATGGGTT TTTATGATTA GAGTCCCGCA 7681 ATTATACATT TAATACGCGA TAGAAAACAA AATATAGCGC GCAAACTAGG ATAAATTATC 7741 GCGCGCGGTG TCATCTATGT TACTAGATCG ACTGTCCTAT ATAACCGCCC ATTTG SEQ ID No. 2: T-DNA region of the auxiliary vector (FIG. 3), wherein sequence segments have the following function: Nucleotides 1 to 25 Left edge repeat of T-DNA Nucleotides 26 to 63 Attp recombination site (Bxb1 serine integrase) Nucleotides 64 to 253 Castor oil catalase intron Nucleotides 254 to 979 N-terminally fused GFP KDEL Nucleotides 980 to 1594 Coating protein TRV RNA-2 Nucleotides 1595 to 2302 TRV RNA-2 3'UTR Nucleotides 2303 to 2556 Em terminator Nucleotides 2557 to 2581 Right edge repeat of T-DNA ACCGTCCTAT ATAACACCAC ATTTGGGCTT GTCGACGACG GCGGTCTCCG TCGTCAGGAT CATCATTTAA AGATCAAAAA GAGGAAGTAA AAGAACCAAT CCTGGGAAAA GAGAAAAATA 121 AAAAAACTCG AAACTAGAAA GAAATTTGAC TAGATAAAAA ATTAACTAAC CAATACCACA 181 TTTATAATGT ATCGAAATTG ACTATTAGAC TAATGAAATA AAGCACACAG ATACTACTAC 241 TACTATCAAT GTCAAGGATG AACTTATGTC TAAGGGAGAA GAACTTTTTA CTGGAGTTGT 301 TCCAATTCTT GTTGAACTTG ATGGAGATGT TAATGGACAT AAGTTTTCTG TTTCTGGAGA 361 AGGAGAAGGA GATGCTACTT ATGGAAAGCT TACTCTTAAG TTTATTTGTA CTACTGGAAA 421 GCTTCCAGTT CCATGGCCAA CTCTTGTTAC TACTTTTTCT TATGGAGTTC AATGTTTTTC 481 TAGATATCCA GATCATATGA AGCAACATGA TTTTTTTAAG TCTGCTATGC AGCAACATGA 541 TGTTCAAGATA AGAACTATTT TTTTAGGA TGATGGAAT AGAACTA QAGCTAGAGT 601 TAAGTTTGAA GGAGATACTC TTGTTAATAG AATTGAACTT AAGGGAATTG ATTTTAAGGA 661 AGATGGAAAT ATTCTTGGAC ATAAGCTTGA ATATATTAT AATTTCCATA ATGTTTATAT 721 TATGGCTGAT AAGCAAAGA ATGGAATTA GGTTATTTT AAGATTATTGA CATTATTGA 781 NATIONAL GATGATCT GTTCAACTTG CTGATCATTA TCAACAAAAT ACTCCAATTG GAGATGGACC 841 AGTTCTTCTT CCAGATAATC ATTATCTTTC TACTCAATCT GCTCTTTCTA AGGATCCAAA 901 TGAAAAGAGA GATCATATGG TTCTTCTTGA ATTTGTTACT GCTGCTGGAA TTACTCATGG 961 AATGGATGAA CTTTATAAGA TGGCTGGATC TTATGGAGAA ACTTTTGATG GAAAGATTCT 1021 TGATGATCTT TCTGGAGCTT GGGTTGAAAA GCATAATTGG TCTGATATTC TTAGAAGACT 1081 TACTAAGATT AAGTTTGCTC TTCAAGCTGA TAGAGATATG ATTCCAGGAA TTGTTGAAGA Petition 870250089530, of 01 / 10 / 2025, p. 87 / 103 56 / 65 1141 TCTTTCTACT GAAATTCCAG TTGATGAAAA TACTAGATTT CCATCTGGAA AGGTTTATCA 1201 TCTTCTTACT AAGGAAATGC TTATGGCTAT TGAAGCTATT CATGCTGCTT CTTCTTTTAA 1261 GAGAAGAGCT GAAGAAAAGA ATAATGTTAA TCCAAGACAA AGATTTGAAG CTGAATCTTC 1321 TTCTTCTCAA CTTCCATCTG GAGGACTTGT TGTTAGACCA GCTGCTGGAT CTGGAGATTC 1381 TTCTCTTGGA GAAGATCTTT TTTCTAATTC TAAGCTTGAT GATGCTTCTA CTGCTTTTCA 1441 TAAGTCTCTT GCTACTCTTA AGGGATCTAG ACCAAAGGCT GTTGTCCAAA GAACTTTTGA 1501 AAAGGAATAT TCTCTTAGAT GGACTGCTGC TGCTCCAGTT GCTGCTGCTG GAGGAACTCC 1561 ACCAGGAGGA AGATCTTGGA CTTGGAATCT TGTTTTCCGG GACCATCTTG ATATTTACAA 1621 CTTAACTTGT TCTCCCCCTT TCCTGGTGAG CAGTGCGGTT GTAGATGGAC ACTACGCACG 1681 GGATAAGTTC GTTAGTTTTC AAGGTGTATG TGGGTTCAAT CCGATGTTTC CAGACGTGAA 1741 TGGTTTGAAG AGTAGTTGGT CTTTAGGTCG TCAACTGGAT GATATACGGT CTCAAAAGAA 1801 AGAGGTTTCT GGAACAAACC AGGAGCCAAA CTATTACTAC GATGGCGACA CACTAAAACC 1861 GATCGGTTCA GGTGCGAGCG TTGTAGGAGA GAGAAGGCCG GGATGGAGGT GAATGTCCCG 1921 AAGACATTAA ACTACGTTCT TTAAGTAGAT CCGTGCCTGA AGTTTTAGGT TCAATTTAAA 1981 CCTACGAGAT TGACATTCTC GACTGATCTT GATTGATCGG TAAGTCTTTT GTAATTTAAT 2041 TTTCTTTTTG ATTTTATTTT AAATTGTTAT CTGTTTCTGT GTATAGACTG TTTGAGATCG 2101 GCGTTTAGGC TCATTGTCTT ACCATAGGGG AACGGACTTT GTTTGTGTTG TTATTTTATT 2161 TGTATTTTAT TAAAATTCTC AACGATCTGA AAAAGCCTCG CGCTAAGAGA TTGTTGGGGG 2221 GTGAGTAAGT ACTTTTAAAG TGATGATGGT TACAAAGGCA AAAGGGGTAA AACCCCTCGC 2281 CTACGTAAGC GTTATTACGC CCGATCGTTC AAACATTTGG CAATAAAGTT TCTTAAGATT 2341 GAATCCTGTT GCCGGTCTTG CGATGATTAT CATATAATTT CTGTTGAATT ACGTTAAGCA 2401 TGTAATAATT AACATGTAAT GCATGACGTT ATTTATGAGA TGGGTTTTTA TGATTAGAGT 2461 CCCGCAATTA TACATTTAAT ACGCGATAGA AAACAAAATA TAGCGCGCAA ACTAGGATAA 2521 ATTATCGCGC GCGGTGTCAT CTATGTTACT AGATCGACTG TCCTATATAA CCGCCCATTT 2581 G SEQ ID No. 3: T-DNA region of the integrase vector (FIG. 3), in which the sequence segments have the following function: Nucleotides 1 to 25 Left edge repeat of T-DNA Nucleotides 26 to 779 CaMV 35S Double Nucleotides 779 to 2278 Bxb1 serine integrase Nucleotides 2279 to 2323 Nuclear localization signal Nucleotides 2324 to 2578 Terminator Nucleotides 2579 to 2603 Right edge repeat of T-DNA ACCGTCCTAT ATAACACCAC ATTTGGCCAA CATGGTGGAG CACGACACTC TCGTCTACTC CAAGAATATC AAAGATACAG TTCCAGAAGA CCAAAGGGCT ATTGAGACTT TTCAACAAAG 121 GGTAATATCG GGAAACCTCC TCGGATTCCA TTGCCCAGCT ATCTGTCACT TCATCAAAAG 181 GACAGTAGAA AAGGAAGGTG GCACCTACAA ATGCCATCAT TGCGATAAAG GAAAGGCTAT 241 CGTTCAAGAT GCCTCTGCCG ACAGTGGTCC CAAAGATGGA CCCCCACCCA CGAGGAGCAT 301 CGTGGAAAAA GAAGACGTTC CAACCACGTC TTCAAAGCAA GTGGATTGAT GTGAACATGG 361 TGGAGCACGA CACTCTCGTC TACTCCAAGA ATATCAAAGA TACAGTCTCA GAAGACCAAA Petition 870250089530, dated 10 / 01 / 2025, pages 88 / 103 57 / 65 421 GGGCTATTGA GACTTTTCAA CAAAGGGTAA TATCGGGAAA CCTCCTCGGA TTCCATTGCC 481 CAGCTATCTG TCACTTCATC AAAAGGACAG TAGAAAAGGA AGGTGGCACC TACAAATGCC 541 ATCATTGCGA TAAAGGAAAG GCTATCGTTC AAGATGCCTC TGCCGACAGT GGTCCCAAAG 601 ATGGACCCCC ACCCACGAGG AGCATCGTGG AAAAAGAAGA CGTTCCAACC ACGTCTTCAA 661 AGCAAGTGGA TTGATGTGAT ATCTCCACTG ACGTAAGGGA TGACGCACAA TCCCACTATC 721 CTTCGCAAGA CCCTTCCTCT ATATAAGGAA GTTCATTTCA TTTGGAGAGG ACACGCTGAA 781 TGAGAGCTCT TGTTGTTATT AGACTTTCTA GAGTTACTGA TGCTACTACT TCTCCAGAAA 841 GACAACTTGA ATCTTGTCAA CAACTTTGTG CTCAAAGAGG ATGGGATGTT GTTGGAGTTG 901 CTGAAGATCT TGATGTTTCT GGAGCTGTTG ATCCATTTGA TAGAAAGAGA AGACCAAATC 961 TTGCTAGATG GCTTGCTTTT GAAGAACAAC CATTTGATGT TATTGTTGCT TATAGAGTTG 1021 ATAGACTTAC TAGATCTATT AGACATCTTC AACAACTTGT TCATTGGGCT GAAGATCATA 1081 AGAAGCTTGT TGTTTGACTGCTGCT ATTTTGCTATTA TACTACTCCA TTTGCTGCTG 1141 TTGTTATTGC TCTTATGGGA ACTGTTGCTC AAATGGAACT TGAAGCTATT AAGGAAAGAA 1201 ATAGATCTGC TGCTCATTTT AATATTAGAG CTGGAAAGTA TAGGGATCT CTTCCACCAT1GTCCACTGACTGTCGACT6 GTTGATGGAG AATGGAGACT TGTTCCAGAT CCAGTTCAAA 1321 GAGAAAGAAT TCTTGAAGTT TATCATAGAG TTGTTGATAA TCATGAACCA CTTCATCTTG 1381 TTGCTCATGA TCTTAATAGA AGAGGAGTTC TTTCTCCAAA GGATCATTAG GCTGATTAG14GATTGATT ACCACAAGGA AGAGAATGGT CTGCTACTGC TCTTAAGAGA TCTATGATTT 1501 CTGAAGCTAT GCTTGGATAT GCTACTCTTA ATGGAAAGAC TGTTAGAGAT GATGATGGAG 1561 CTCCACTTGT TAGAGCTGAA CCAATTACTTA CAGTTAGCTGACTGACTG 1621 AACTTGTTAA GACTTCTAGA GCTAAGCCAG CTGTTTCTAC TCCATCTCTT CTTCTTAGAG 1681 TTCTTTTTTG TGCTGTTTGT GGAGAACCAG CTTATAAGTT TGCTGGAGGA GGAAGAAAGC 1741 ATCCAAGATA TAGATGTAGAGA TCTGAGCAGAGAT TTTGAGGAGAT GGAACTGTTG 1801CTATGGCTGA ATGGGATGCT TTTTGTGAAG AACAAGTTCT TGATCTTCTTT GGAGATGCTG 1861 AAAGACTTGA AAAGGTTTGG GTTGCTGGAT CTGATTCTGC TGTTGAACTT GCTGAAGTTA 1921 ATGCTGAACT TGTTGATCTT ACTTCTCTTA TTGGATCTCC AGCTTATAGA GCTGGATCTC 1981 CACAAAGAGA AGCTCTTGAT GCTAGAATTG CTGCTCTGGC TGCTAGACAA GAAGAACTTG 2041 AAGGACTTGA AGCTAGACCA TCTGGATGGG AATGGAGAGA AACTGGACAA AGATTTGGAG 2101 ATTGGTGGAG AGAACAAG ACTGCTGCTA AGAATACTTG GCTTAGATCT ATGAATGTTA 2161 GACTTACTTT TGATGTTAGA GGAGGACTTA CTAGAACTAT TGATTTTGGA GATCTTCAAG 2221 AATATGAACA ACATCTTAGA CTTGGATCTG TTGTTGAAAG ACTTCATACT GGAATGTCTA 2281 GAAGATGGAA GGAAGAACTT GATGAAGAAC TTCAAAGAAA GAGAGATCGT TCAAACATTT 2341 GGCAATAAAG TTTCTTAAGA TTGAATCCTG TTGCCGGTCT TGCGATGATT ATCATATAAT 2401 TTCTGTTGAA TTACGTTAAG CATGTAATAA TTAACATGTA ATGCATGACG TTATTTATGA 2461 GATGGGTTTT TATGATTAGA GTCCCGCAAT TATACATTTA ATACGCGATA GAAAAAAAAA 2521 TATAGCGCGC AAACTAGGAT AAATTATCGC GCGCGGTGTC ATCTATGTTA CTAGATCGAC 2581 TGTCCTATAT AACCGCCCAT TTG Agroinfiltration procedure in N· benthamiana

[00137] The 3 resulting binary vectors were introduced separately into Agrobacterium tumefaciens GV3101, LBA4404 or LBA4301 strains by electroporation. The resulting agrobacteria were verified by restriction digestion or PCR of plasmin DNA. Petition 870250089530, dated 10 / 01 / 2025, pp. 89 / 103 58 / 65 mídio bacteria, cultured overnight at 30°C, were used to infiltrate leaves of 5-week-old N. benthamiana plants maintained at 2325°C. Briefly, the bacteria were pelleted by centrifugation for 5 minutes at 5,000 e, then resuspended in infiltration buffer [10 mM 2-(N-morpholino)ethanesulfonic acid (MES), pH 5.5 and 10 mM MgSO4] to OD600 = 0.2. The resulting bacterial suspensions were injected into the leaves with a needleless syringe via a small puncture. Plant tissue was harvested on 4 DPI unless otherwise noted. RT-PCR procedure

[00138] First-strand cDNAs were synthesized from 5 pg of total RNA treated with DNasel using the SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen, Thermo Fisher Scientific). PCR amplification was performed using the following protocol: initial denaturation at 94°C for 3 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 65°C for 30 s, elongation at 72°C for 1 min, and finally, a 3 min extension at 72°C. Isolation of protoplasts from the N. benthamiana mesophyll

[00139] Protoplasts were released from leaf tissue by incubation for 12–14 ha at 25 °C in the dark in a solution containing 0.5% Onozuka R-10 Cellulase (Duchefa Biochemie), 0.3% R-10 Macerozyme (Duchefa Biochemie), 0.2% Pectolyase Y-23 (Duchefa Biochemie), 0.5 M sucrose, 7 mM CaCl2, 90 mM glycine, and a protease inhibitor cocktail (Roche Life Science). The released protoplasts were filtered through a trilayer consisting of gauze (top layer), miracloth (intermediate layer), and a nylon mesh (bottom layer) with a pore diameter of 50 µm, resuspended in modified W5m solution, and centrifuged at 179 χ² g for purification. For the modified W5m solution, Ca(NO3)2 was used. Petition 870250089530, dated 10 / 01 / 2025, pages 90 / 103 59 / 65 of the solution was used instead of CaCl2 to avoid vacuolar acidification and cell expansion. The total protoplast yield reached 6.04 χ¹⁰⁷ protoplasts g⁻¹ fresh weight (FW), and protoplast viability was up to 95%. GFP visualization

[00140] Leaves expressing GFP were visualized under UV illumination generated by a B-100AP / R lamp (labortechnik). Protoplasts were visualized with a ZEISS Primovert inverted microscope with fluorescence filter sets for GFP (Techspec, excitation filter, 470 / 40 nm; barrier filter, 525 / 50 nm). Protoplasts were similarly visualized with an Axioplan 2 microscope (Zeiss) using a GFP filter block. Results Producer, helper, and integrase T-DNAs can be efficiently released to the same cell from different agrobacteria.

[00141] N. benthamiana, infiltration of leaf tissue with an Agrobacterium suspension resulted in transient expression in nearly 100% of mesophyll cells. To quantify the efficiency of co-release of producer, helper, and integrase vectors in the same cell, N. benthamiana leaves were co-infiltrated with a mixture of three different Agrobacterium cultures (producer vector with GV3101, helper vector with LBA4404, and integrase vector with LBA4301), and it was found that >95% of protoplasts isolated from infected areas expressed GFP (FIG. 4A). Subsequent studies showed that, excluding the integrase construct, no GFP expression was detected (FIG. 4B), and thus the integration of the integrase vector is mandatory for the functionality of the transcomplementary vector system.

[00142] Transiently expressed T-DNAs recombine efficiently in plant cells and can assemble in the plant.

[00143] It is known that T-DNAs can recombine after being Petition 870250089530, dated 10 / 01 / 2025, pp. 91 / 103 60 / 65 released into a plant cell (Vergunst, AC, Jansen, LE & Hooykaas, PJ, 1998). However, to be useful in gene assembly in the plant, site-specific recombination of T-DNAs needs to be very efficient. To measure the ability of T-DNAs to recombine under the control of the serine integrase Bxb1, we tested their ability by co-infiltration of the integrase vector (FIG. 3) with the producer and helper vector, or without it. Co-infiltration with the integrase vector resulted in clearly visible GFP staining throughout the infiltrated area (FIG. 5A), while no GFP activity could be detected in the absence of the integrase vector in the control infiltration, thus demonstrating that site-specific recombination of T-DNAs, assembly in the plant, and viral amplification occur efficiently in most infiltrated cells by co-infiltration of the integrase vector alone (FIG. 5B). Replacing the K16 protein of TRV RNA-1 with P19 improved the functionality of the PTGS suppressor.

[00144] RNA silencing is a host defense mechanism that limits the accumulation and spread of viruses in infected plants. Similarly, plant viruses encode silencing suppressors. In the positive-strand RNA virus Tobacco Rattle Virus (TRV), the silencing suppressor is a 16 kDa (16 K) protein encoded by RNA1. The suppressive action of the 16K protein is transient and weaker than that of the P19 suppressor, encoded by Tomato Bush Dwarf Virus (TBSV). Two transcomplementary vector systems were tested against each other for their ability to suppress PTGS and improve viral amplification and dissemination. By replacing the original 16K protein of TRV with the P19 suppressor gene of TBSV, PTGS was significantly more suppressed compared to the original vector system containing K16, significantly improving GFP expression (FIG. 6). Petition 870250089530, dated 10 / 01 / 2025, pp. 92 / 103 61 / 65 Replacing the K16 protein of TRV RNA-1 with the P19 protein improved the functionality of the PTGS suppressor.

[00145] RNA silencing is a host defense mechanism that limits the accumulation and spread of viruses in infected plants. Similarly, plant viruses encode silencing suppressors. In the positive-strand RNA virus Tobacco Rattle Virus (TRV), the silencing suppressor is a 16 kDa (16 K) protein encoded by RNA1. The suppressive action of the 16K protein is transient and weaker than that of the P19 suppressor, encoded by Tomato Bush Dwarf Virus (TBSV). Two transcomplementary vector systems were tested against each other for their ability to suppress PTGS and improve viral amplification and dissemination. By replacing the original 16K protein of TRV in the producing vector with the P19 suppressor gene of TBSV, PTGS was significantly more suppressed compared to the original vector system containing K16, thus significantly improving GFP expression (FIG. 6).

[00146] Intron flanking of Attp / Attb recombination sites improves GFP expression rates.

[00147] Splicing is the process by which introns, the non-coding regions of genes, are excised from the primary messenger RNA transcript, and exons (i.e., coding regions) are joined to generate mature messenger RNA. The latter serves as a template for the synthesis of a specific protein. Introns can increase transcription levels by affecting the transcription rate, nuclear export, and transcriptional stability. Furthermore, introns can similarly increase the efficiency of mRNA translation. One of the inventor's goals in designing this system was to improve viral expression rates and amplification by increasing the opportunity to remove unwanted sequences through splicing. Therefore, the sequences Petition 870250089530, dated 10 / 01 / 2025, pages 93 / 103 62 / 65 of the Attp recombination site in the 5' producer vector and Attb in the 3' helper vector were flanked by intron sequences. Three criteria were used to choose an ideal intron: (i) a size close to the average size of plant introns (100-250 bp), (ii) a low GC content, and (iii) splicing site sequences close to consensus. The first criterion is easy to meet, as numerous introns of all sizes are present in genomic databases. Therefore, we chose a 190 nt intron from castor bean catalase, which has an average GC distribution content of 13% (FIG. 7A). To meet the second criterion, we chose very T-rich sequences to flank the recombination sequences and ensured that no cryptic splicing sites were present in the intron near the GC-rich sequences of the recombination site.Finally, the sequences of the splice sites were modified to precisely match the consensus for dicotyledonous plants.

[00148] A set of producer and helper vectors containing castor catalase intron sequences flanking the Attp / Attb recombination sites, and a set of producer and helper vectors without the introduction sequences were designed to study the ability of intron inclusion to increase expression rates. Inclusion of castor catalase sequences flanking the recombination sites significantly improved expression rates (FIG. 7B and 7C).

[00149] The TRV RNA-2 helper vector complements the TRV RNA-1 producer vector, providing GFP expression under CP control in trans, enabling long-distance movement.

[00150] There are two types of viral movement: i) slow and local movement, in which the virus moves from one cell to neighboring cells, and ii) Petition 870250089530, dated 10 / 01 / 2025, pages 94 / 103 63 / 65 rapid and systemic movement, in which the virus moves from a site of infection to distant parts of the plant, hitching a ride on the plant's own supply lines (the veins). TRV does not require CP and viral particles for long-distance viral movement in plants and, consequently, systemic infection with RNA1 alone can occur (Swanson et al., 2002). Therefore, the vector system was designed so that the producer vector is functionalized only by i) supplying GOI and CP to enhance systemic infection in trans by the auxiliary vector and ii) supplying the integrase vector to allow the assembly of the producer and auxiliary vectors in the plant.

[00151] An expression experiment was conducted where the inventor clearly demonstrated that trans delivery of CP via the auxiliary vector allows for long-distance systemic infection of plant material. An auxiliary vector in which the CP coding sequence was deleted clearly demonstrated that only local infection was achieved. The experiment was performed by local agroinfiltration with a single-point syringe in both plant groups. Quantification of transient GFP expression in plants from both vector systems was analyzed. Top. Individual leaves of N. benthamiana infiltrated with (A) A. tumefaciens suspensions containing the vector system without the CP coding sequence in the auxiliary vector, or (B) A. tumefaciens suspensions containing the vector system with the CP coding sequence in the auxiliary vector. (C) Uninfiltrated control leaf. Leaves photographed (4 days post-infiltration, DPI) under UV light for GFP visualization. Bottom.Micrograms of GFP produced per gram of infiltrated tissue, as estimated by the GFP fluorescence assay. Labeling identical to that at the top of the figure. Plant extracts prepared 4-5 DPI were analyzed with a GFP fluorescence assay on plates. Three plants from each treatment were analyzed. Samples were analyzed in triplicate and values ​​were calculated. Petition 870250089530, dated 10 / 01 / 2025, pages 95 / 103 64 / 65 Purified GFP labeled with His-6 was used to generate a standard curve (FIG. 8A and 8B). Equivalents

[00152] Those skilled in the art will recognize, or be able to determine, using only routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above description, but rather as mentioned in the following claims. BRIEF DESCRIPTION OF THE FIGURES

[00153] FIG. 1 represents families and genera of DNA and RNA viruses that infect plants.

[00154] FIG. 2 represents the organization of the TRV genome.

[00155] FIG. 3 represents the construction of a transcomplementary TRV vector expressing GFP.

[00156] FIG. 4 represents a TRV transcomplementary system showing the expression of GFP in N. benthamia protoplasts.

[00157] FIG. 5 represents the successful transient expression of GFP using a TRV transcomplementary system that recombines efficiently in plant cells with the integrase vector.

[00158] FIG. 6 represents the enhanced suppression of PTGS by replacing the original k16 protein of TRV with the p19 protein of TBSV.

[00159] FIG 7 represents enhanced viral amplification and expression of GFP, flanking recombination sites with castor catalase intron sequences.

[00160] FIG 8 represents the TRV RNA-2 auxiliary vector complementing the TRV RNA-1 producer vector, providing GFP expression under in-trans CP control to enable long-distance movement.

[00161] FIG 9A represents a comparison of N. ben plants Petition 870250089530, dated 10 / 01 / 2025, pages 96 / 103 65 / 65 healthy and infected thamiana, cultured at 21 °C. Uninfected (left panel), infected with the producing vector containing the TBSV P19 silencing inhibitor (center), and infected with the producing vector containing the TRV native K19 silencing inhibitor (right panel).

[00162] FIG 9B illustrates the health of the plant after 10 days of inoculation with the vector of the invention versus the known TRV pyl 192 vector. Here, the control indicates the buffer control (0.01 M phosphate buffer).

[00163] FIG 10 shows the analysis of GFP in N. benthamiana based on GFP and western blot assays. (A) GFP image at 3 dpi in N. benthamiana infiltrated with the transcomplementary vector (TCTRV) with native ο K16 without intron flanking, the TCTRV vector with the P19 silencing suppressor without intron flanking, or the TC-TRV vector with the P19 silencing suppressor and with intron flanking. (B) Analysis of the GFP protein in infiltrated N. benthamiana as in (A). GFP expression for the TC-TRV vectors was monitored daily from 3 to 7 days post-infiltration (dpi) with Mock (M) and 00 (control Agrobacterium expressing an empty TRV vector) collected at 7 dpi. In each case, the top panels are western blots for GFP detection, while the bottom panels show the corresponding Coomassie Brilliant Blue (CB) staining of the gels for charge comparison.The red (S) size marker on the left of the western blots is 25 kDa, while the intense CB band size of a host protein is about 55 kDa, as inferred from the size marker on the left. Petition 870250089530, dated 10 / 01 / 2025, pp. 97 / 103

Claims

1 / 5 CLAIMS 1. A method for the high-level heterologous expression of a polynucleotide of interest in a host plant, characterized in that it comprises the following steps: (A) transforming said host plant with one or more suspension(s) of Agrobacterium, wherein said one or more suspension(s) of Agrobacterium contain in T-DNA a heterologous DNA having sequences encoding transcomplementary viral replicons operationally linked to a transcriptional promoter, wherein said transcomplementary viral replicons contain: (I) coding sequences that have viral replicon functions of a plant RNA viral replicon, and (II) a polynucleotide of interest, wherein said viral replicon functions of a plant RNA viral replicon, derived from a Tobacco Rattle Virus (TRV), are: (i) a producer vector that allows trans-cell-to-cell movement of trans-complementary viral replicon;(ii) an auxiliary vector, wherein said auxiliary vector is (i) capable of systemic movement in said host plant, both in the presence of said producer vector, and (ii) capable of expressing a polynucleotide of interest in said host plant with the aid of one or more proteins necessary for systemic movement, whether by replication, cell-to-cell and / or long-distance movement, or with the aid of said producer vector; and (iii) an integrase vector that allows in planta assembly of components (i) and (ii), being the producer and auxiliary vector, thus allowing the resulting components to be recombined within the plant cell and leading to the creation of a vector sequence within a fully functional infectious replicon;(B) maintain the plant under conditions and for a sufficient time to allow the producer vector, the helper vector, and the integrase vector to recombine within the plant cell, and the producer vector to complement the helper vector, so that systemic infection is achieved in the plant; and (C) maintain the plant under conditions and for a sufficient time so that the polynucleotide of interest is expressed in at least some plant cells.

2. Method, according to claim 1, characterized in that the native K16 suppressor of the TRV RNA-1 silencing protein is replaced with P19 from the Tomato Bush Dwarf Virus, resulting in improved suppression of RNA silencing.

3. A method according to claim 1 or 2, characterized in that the producer vector comprises an AttP recombination site and the auxiliary vector comprises the corresponding AttB recombination site, such that the integrase vector can provide in planta recombination of said producer and auxiliary vectors.

4. A method, according to any one of claims 1 to 3, characterized in that the integrase vector comprises an integrase coding sequence of a serine integrase, more specifically a Bxb1 serine integrase.

5. Method, according to any one of claims 1 to 4, characterized in that the producing vector comprises an apoplast targeting sequence, in particular an apoplast targeting sequence derived from the 2S seed storage protein 1 of Arabidobsis thaliana.

6. Method, according to any one of claims 1 to 5, characterized in that both the producing vector and the auxiliary vector comprise recombination sites flanked by intron elements to improve mRNA translation efficiency, more specifically, the intron elements are low GC content and derived from castor oil catalase.

7. A method, according to any one of claims 1 to 6, characterized in that the producing vector is functionalized with a unique self-cleaving hammerhead ribozyme to enhance viral infectivity.

8. Method, according to any one of claims 1 to 7, characterized in that the auxiliary vector comprises a non-coding portion comprising a 3' untranslated region of TRV RNA-2.

9. A method, according to any one of claims 1 to 8, characterized in that the Agrobacterium suspension has a cell concentration of said Agrobacterium corresponding to an optical density calculated at 600 nm of at most 0.001, wherein said calculated optical density is defined by a dilution of at least 1000 times of a suspension of said Agrobacterium with an OD at 600 nm of 1.

0.

10. A method, according to any one of claims 1 to 9, which is a process for transiently expressing said sequence of interest, characterized in that it comprises the transient transformation of said host plant, such as a plant, plant part or plant cell culture, with a nucleic acid molecule containing said heterologous DNA.

11. Method, according to any one of claims 1 to 10, characterized in that said transformation is carried out by infiltrating said host plant with said Agrobacterium suspension.

12. System for replication or for replicating and expressing Petition 870250089530, dated 01 / 10 / 2025, page.13 / 103 4 / 5 a sequence of interest in a host plant, characterized in that it comprises: (I) a producer vector that allows trans-cell-to-cell movement of the transcomplementary viral replicon; (II) an auxiliary vector, wherein said auxiliary vector is (i) capable of systemic movement in said host plant, both in the presence of said producer vector, and (ii) capable of expressing a polynucleotide of interest in said host plant with the aid of one or more proteins necessary for systemic movement, whether by replication, cell-to-cell and / or long-distance movement with the aid of said producer vector; and (i) an integrase vector that allows in planta assembly of components (i) and (ii), being the producer and auxiliary vector, thereby allowing the resulting components to be recombined within the plant cell and leading to the creation of a vector sequence within a fully functional infectious replicon.

13. A replication process or a process for replicating and expressing a GOI in a host plant, characterized in that it comprises providing the cells of said host plant with said producer vector and said auxiliary vector, as defined in any of the preceding claims.

14. Invention, characterized by any form of its embodiments or by any applicable category of claim encompassed by the matter initially described, disclosed or illustrated in this patent application or in examples presented herein; and / or (A) transforming said host plant with one or more suspension(s) of Agrobacterium, wherein said one or more suspension(s) of Agrobacterium contain in T-DNA a heterologous DNA exhibiting sequences encoding transviral replicons. Petition 870250089530, dated 01 / 10 / 2025, p.14 / 103 5 / 5 complementary vectors operationally linked to a transcriptional promoter; (B) maintaining the plant under conditions and for a sufficient time to allow the producer vector, the helper vector, and the integrase vector to recombine within the plant cell, and the producer vector to complement the helper vector, so that systemic infection is achieved in the plant; and (C) maintaining the plant under conditions and for a sufficient time for the polynucleotide of interest to be expressed in at least some plant cells; and / or Use of the vector system, as defined in any of the preceding claims, as components for other purposes. Petition 870250089530, dated 10 / 01 / 2025, p. 15 / 103.

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